Hyperthermia In Cancer Treatment: A Primer (Medical Intelligence Unit)

MEDICAL INTELUGENCE UNIT

Hyperthermia in Cancer Treatment: A Primer Gian Franco Baronzio, M.D. Radiotherapy Unit Policlinico di Monza Monza, Italy

E. Dieter Hager, M.D., Ph.D., D.Sc. Department of Hyperthermia BioMed-Klinik GmbH Bad Bergzabern, Germany

L A N D E S B I O S C I E N C E / EUREKAH.COM

GEORGETOWN, TEXAS

U.S.A.

SPRINGER SCIENCE+BUSINESS M E D I A

NEW YORK, NEW YORK

USA

HYPERTHERMIA IN CANCER TREATMENT: A PRIMER Medical Intelligence Unit Landes Bioscience / Eurekah.com Springer Science+Business Media, LLC

ISBN: 0-387-33440-8

Printed on acid-free paper.

Copyright ©2006 Landes Bioscience and Springer Science+Business Media, LLC All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher, except for brief excerpts in connection with reviews or scholarly analysis. Use in conneaion with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in the publication of trade names, trademarks, service marks and similar terms even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to careftilly review and evaluate the information provided herein. Springer Science+Business Media, LLC, 233 Spring Street, New York, New York 10013, U.SA. http://www.springer.com Please address all inquiries to the Publishers: Landes Bioscience / Eurekah.com, 810 South Church Street, Georgetown, Texas 78626, U.SJ\. Phone: 512/ 863 7762; FAX: 512/ 863 0081 http://www.eurekah.com http://www.landesbioscience.com Printed in the United States of America. 9 8 7 6 5 4 3 2 1

Library of Congress Cataloging-in-Publication Data A C L P . Catalogue record for this book is available from the Library of Congress.

This Book Is Dedicated to: My tender wife, Anna, to Attilio, Paul Junior and Miriam, my dear children who unselfishly endured my work. My father, Paul Senior, and my mother, Louise, who inspired my life and without whom I would never have achieved what I have. Peter M. GuUino, Gian Luigi Monticelli and Fulvio Pezza, my teachers who helped me to mature from a naiVe scientist to a more mature physician and who share their knowledge, experience and friendship. Gian Franco Baronzio My wife Claudia, to my dear children Marsha, Jonas and Simon, to my teachers in hyperthermia, and to all the scientists doing tough research in the emerging field of hyperthermia. Erich Dieter Hager

CONTENTS Preface Introduction Section I: Physical Aspects of Hyperthermia 1. Hyperthermia, Physics, Vector Potential, Electromagnetic Heating: A Primer Ugo Cerchiari Direct Heating Heating by Ultrasounds Heating by Electromagnetic Methods 2. Thermometry: Clinical Aspects and Perspectives Barbara Baiotto and Piergiorgio Marini Calculation of the Produced Power Deposition Calculation of the Resulting Temperature Profile: The "Bio-Heat Equation" Experimental Measurement of Temperature Invasive Methods Non-Invasive Methods 3. Physical Background and Technical Realizations of Hyperthermia Andras SzasZy Oliver Szasz and Nora Szasz Characterization Demand Control-Parameters Technical Challenges Electromagnetic Heating Processes Comparison of the Methods New Directions in Electromagnetic Oncologic Hyperthermia 4. Thermotherapy and Nanomedicine: Between Vision and Reality Andreas Jordan Section II: Biological Aspects of Hyperthermia 5. Influence of Tumor Microenvironment on Thermoresponse: Biologic and CHnical ImpUcations Gian Franco Baronzio, Alberto Gramaglia, Attilio Baronzio and Isabel Freitas Hypoxia, HIF-1 and Angiogenesis Tumor Bioenergetic Status, Hypoxia, pH Hyperthermia Effects on Tumor Blood Flow and Endothelium Clinical Methods for Improving Thermoresponse Tumor Blood Flow (TBF) Modulation Microenvironment Modification Heat Delivery Methods

xv xvii 1 3 5 5 12 19 19 21 23 23 24 27 29 34 37 40 50 52 60

65

67

(cH 73 79 81 82 83 85

6. Hyperthermia and Angiogenesis: Results and Perspectives Cristina Roca andLuca Primo Bloodvessels, Blood Flow and Microenvironment Tumor Angiogenesis Molecular Mechanisms of Angiogenesis Inhibition by H T Perspectives 7. Vascular Effects of Localized Hyperthermia Debra K. Kelleher and Peter Vaupel Vascular Effects of Localized Hyperthermia Vascular Effects of Combined Modalities 8. On the Biochemical Basis of Tumour Damage by Hyperthermia Paola Pietrangeli and Bruno Mondovl Glycolysis and Respiration Hyperthermia and DNA, RNA and Protein Synthesis Tumour Membranes and Hyperthermia Hyperthermia and Liposomes Hyperthermia and Immune Response Heat Shock Proteins Hyperthermia and Oxygen Free Radicals Hyperthermia and Amine Oxidases 9. Results of Hyperthermia Alone or with Radiation Therapy and/or Chemotherapy Pietro Gahriele and Cristina Roca Hyperthermia Alone Hyperthermia and Radiotherapy Interstitial Thermo-Brachytherapy Intracavitary Hyperthermia Hyperthermia Radiation and Chemotherapy Prognostic Factors Affecting the Response to Hyperthermia Perspectives

92 93 94 95 96 99 100 102 110 Ill Ill 112 112 113 114 115 115

119 120 121 122 123 123 123 124

10. Thermo-Chemo-Radiotherapy Association: Biological Rationale, Preliminary Observations on Its Use on Malignant Brain Tumors 128 Gian Franco Baronzio, Vincenzo Cerreta, Attilio Baronzio, Isabel Freitas, Marco Mapelli and Alberto Gramaglia Radiotherapy and Hyperthermia Interaction 128 Chemotherapy and Hyperthermia Interaction 135 Effects of Hyperthermia on Drugs Uptake and Targeting 137 Methods for Enhancing Thermal Sensitivity 138 Thermo-Chemo-Radiotherapy for Malignant Brain Tumors 143 Experimental Studies 145 Results 148

11. A Step Deep on Hyperthermia, Hypoxia and Chemotherapy Interactions Giammaria Fiorentiniy Ugo De Giorgiy Maurizio CantorCy Andrea Mamhrini andStefano Guadagni The Problem of Tumor Hypoxia in Anticancer Therapy Hypoxia and Chemotherapy Hyperthermia and Chemotherapy Hyperthermia and Antineoplastic Drugs Selection

156 157 159 159

Section III: Clinical Aspects of Hyperthermia

165

12. Locoregional Hyperthermia E. Dieter Hager Clinical Trials on Hyperthermia Non-Thermal Effects

167

13. Hyperthermia and Radiotherapy in the Management of Prostate Cancer Sergio Villa Hyperthermia and Radiotherapy: Toxicity

156

170 176

183 185

14. Tumor Ablation Using Radiofrequency Energy: Technical Methods and Application on Liver Tumors 190 Johannes-Marcus Hdnslery Luigi Solhiatiy E. Dieter Hagery Tiziana leracey Luca Cova and Gian Franco Baronzio Technical Development 191 Treatment 194 15. Cytoreduction, Peritonectomy and Hyperthermic Antiblastic Peritoneal Perfusion for the Treatment of Peritoneal Carcinomatosis Michele De Simone and Marco Vaira Generalities and Indications Surgical Techniques Semiclosed HAPP Technique Clinical Experience and Results 16. Hyperthermic Isolated Limb Perfusion Michele De Simone and Marco Vaira Introduction and Indications of Limb Perfusion Surgical Procedure Clinical Experiences and Results

199 199 201 205 206 208 208 209 215

17. Intracavitary Hyperthermic Perfusion E. Dieter Hager Clinical Experience 18. Whole Body Hyperthermia at 43.5-44**C: Dream or Reality? Alexey V. Suvemev, Georgy V. Ivanov, Anatoly V. Efremov and Roman Tchervov Necessity of High-Level Whole Body Hyperthermia Risk Factors of Whole Body Hyperthermia Over 43 °C and Pathogenetic Substantiation of Their Overcoming What Are Possible Ways to Suppress the Activity of Trypsin during Hyperthermia? Technological and Anaesthetic Features of Whole Body Severe Hyperthermia 19. Extreme Whole-Body Hyperthermia with Water-Filtered Infirared-A Radiation Alexander von Ardenne andHolger Wehner Technical Realisation of Water-Filtered Infrared-A Radiation Tolerance of Extreme Whole-Body Hyperthermia w^ith Water-Filtered Infrared-A Radiation First Clinical Results

218 220 227

227 228 230 232

237 239 240 242

20. Effects of Local and Whole Body Hyperthermia on Immunity 247 Gian Franco Baronzio, Roberta Delia Seta, Mario DAmico, Attilio Baronzioy Isabel FreitaSy Giorgio Forzenigo, Alberto Gramaglia andE. Dieter Hager Tumor Immunity 247 Therapeutic Modalities 250 Effects of Thermal Component of Fever on Immune System 254 Effects of Induced Thermal Elevation (Hyperthermia) on Immunity 256 Specific Effects of Hyperthermia on Immune Therapeutic Modalities 259 Effects of Hyperthermia on Lymphocyte Homing 261 The Danger Model and Hyperthermia-Effects on Dendritic Cell Maturation and Stimulation of Innate-Adaptive Immunity 261 Effects of Hyperthermia on Metastatic Process 264

21.

Fever, Pyrogens and Cancer Ralf Kleefand E. Dieter Hager History and Background Rationale Epidemiology The Immunological Basis of Endo- and Exotoxin-Induced Tumor Regression Proposed Mechanism of Action Toxicity

22. Future Perspectives of Interstitial and Perfusional Hyperthermia Gian Franco Baronzioy Michele De Simone, Gianmaria Fiorentiniy Salvatore D*Angelay Giovanni Visconti andE. Dieter Hager Radiofrequency Ablation (RFA) Liver Cancer Renal Cell Carcinoma Breast Cancer Lung Tumors Bone Tumors Miscellaneous Tumors Treated with RFA Perfusional Treatment Addendum: Radiofrequency Thermal Ablation in the Treatment of Lung Malignancies Cosimo Gadaletay Anna Catino and Vittorio Mattioli Patients and Methods Results Discussion Index

276 276 277 279 286 299 308 338

340 342 342 343 343 344 344 344 352 353 354 356 361

EDITORS Gian Franco Baronzio Radiotherapy Unit Policlinico di Monza Monza, Italy Email: [email protected] Chapters 5, 10, 14, 20, 22

E. Dieter Hager Department of Hyperthermia BioMed-KlinikGmbH Bad Bergzabern, Germany Email: [email protected] Chapters 12, 14, 17. 20, 21, 22

CONTRIBUTORS Barbara Baiotto Medical Physics Unit Institute for Cancer Research and Treatment (IRCC) Turin, Italy Chapter 2

Ugo Cerchiari National Cancer Institute, Milan Milano, Italy Email: [email protected] Chapter 1

Attilio Baronzio University of Novara Pharmacology DPT Novara, Italy Chapters 5. 10,20

Vincenzo Cerreta Radiotherapy Unit Policlinico Di Monza Monza, Italy Chapter 10

Maurizio Cantore Department of Oncology City Hospital Carrara Massa-Carrara, Italy Chapter 11

Luca Cova Interventional Radiology Unit Busto Arsizio Hospital Busto A (VA), Italy Chapter 14

Anna Catino Interventional Radiology Operative Unit Critical Area Department Oncology Institute Bari, Italy Chapter 22

Mario D'Amico Hematology and Oncology Unit Carrara Hospital Carrara, Italy Chapter 20

Salvatore D'Angelo Maffucci Liver Unit Moscati Hospital Avellino, Italy Chapter 22 Ugo De Giorgi Medical Oncology Unit "S. Giuseppe" Hospital Empoli, Florence, Italy Chapter 11 Michele De Simone Department of Surgical Oncology "S. Giuseppe" Hospital Empoli, Florence, Italy Email: m.desimone@usll 1 .tos.it Chapters 15> 16, 22 Roberta Delia Seta Hematology and Oncology Unit Carrara Hospital Carrara, Italy Chapter 20 Anatoly V. Efremov International Health Center Novosibirsk, Russia Chapter 18 Gianmaria Fiorentini Department of Oncology "S. Giuseppe" Hospital Empoli, Florence, Italy Email: oncologiaempoli@usl 11. toscana. it Chapters 11, 22 Giorgio Forzenigo Gynecologic Oncology Unit Gallarate Hospital Gallarate (VA), Italy Chapter 20

Isabel Freitas Department of Animal Biology CNR Institute of Molecular Genetics Section of Histochemistry and Cytometry University of Pavia Pavia, Italy Chapters 5, 10, 20 Cosimo Gadaleta Interventional Radiology Operative Unit Critical Area Department Oncology Institute Bari, Italy Email: [email protected] Chapter 22 Pietro Gabriele Radiotherapy Unit Institute for Cancer Research and Treatment Candiolo, Turin, Italy Email: [email protected] Chapter 9 Alberto Gramaglia Radiotherapy Unit Policlinico di Monza Monza, Italy Chapters 5, 10, 20 Stefano Guadagni Department of Surgical Sciences University of L'Aquila L^Aquila, Italy Chapter 11 Johannes-Marcus Hansler Department of Internal Medicine I University of Erlangen-Nuremberg Erlangen, Germany Email: j ohannes. haensler@ med 1 .imed. uni-erlangen.de Chapter 14

Tiziana lerace Interventional Radiology Unit Busto Arsizio Hospital Busto A (VA), Italy Chapter 14 Georgy V. Ivanov International Health Center Novosibirsk, Russia Chapter 18 Andreas Jordan MagForce Nanotechnologies GmbH and Center of Biomedical Nanotechnology (CBN) Department of Radiology Charit^ - University Medicine Berlin Berlin, Germany Email: [email protected] Chapter 4 Debra K. Kelleher Institute of Physiology and Pathophysiology University of Mainz Mainz, Germany Email: [email protected] Chapter 7

RalfKleef Institute for Hyperthermia and Immunotherapy Vienna, Austria Email: [email protected] Chapter 21 Andrea Mambrini Department of Oncology City Hospital Carrara Massa-Carrara, Italy Chapter 11

Marco Mapelli Radiotherapy Unit Physics Department Policlinico di Monza Monza, Italy Chapter 10 Piergiorgio Marini Medical Physics Unit Institute for Cancer Research and Treatment (IRCC) Turin, Italy Email: [email protected] Chapter 2 Vittorio Mattioli Interventional Radiology Operative Unit Critical Area Department Oncology Institute Bari, Italy Chapter 22 Bruno Mondovl Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and C.N.R. Centre of Molecular Biology University "La Sapienza" Roma, Italia Email: [email protected] Chapter 8 Paola Pietrangeli Department of Biochemical Sciences "A. Rossi Fanelli" and C.N.R. Centre of Molecular Biology "La Sapienza" University Rome, Italy Chapter 8 Luca Primo Division of Molecular Angiogenesis Institute for Cancer Research and Treatment Candiolo, Italy Email: [email protected] Chapter 6

Cristina Roca Division of Molecular Angiogenesis Institute for Cancer Research and Treatment (IRCC) Candiolo, Turin, Italy Email: [email protected] Chapters 6, 9 Luigi Solbiati Interventional Radiology Unit Busto Arsizio Hospital BustoA(VA),Italy Chapter 14 Alexey V. Suvernev International Health Center Novosibirsk, Russia Chapter 18 Andras Szasz Biotechnics Department Faculty of Engineering Szent Istvan University Budapest, Hungary Email: [email protected] Chapter 3 Nora Szasz Biotechnics Department Faculty of Engineering Szent Istvan University Budapest, Hungary Chapter 3 Oliver Szasz Biotechnics Department Faculty of Engineering Szent Istvan University Budapest, Hungary Chapter 3

Roman Tchervov Siberian Institute of Hyperthermia Iskitim, Russia Email: [email protected] Chapter 18 Marco Vaira Department of Surgical Oncology "S. Giuseppe" Hospital Empoli, Florence, Italy Chapters 15, 16 Peter Vaupel Institute of Physiology and Pathophysiology University of Mainz Mainz, Germany Chapter 7 Sergio Villa National Cancer Institute Milan Radiotherapy Service Milano, Italy Email: [email protected] Chapter 13 Giovanni Visconti Endocrinologist Ferno (VA), Italy Chapter 22 Alexander von Ardenne Von Ardenne Institute Dresden, Germany Email: [email protected] Chapter 19 Holger Wehner Wilhelmshaven, Germany Chapter 19

PREFACE Although remarkable progress has been made in cancer therapy, many cancers, particularly solid cancers, are still untreatable by conventional therapies such as radiation, immunotherapy, surgery or chemotherapy. This creates the need to improve cancer treatment. Hyperthermia for its synergistic action with the aforementioned modalities may be considered the fifth modality of treatment. Hyperthermia is defined as a therapy in which tumor temperature is raised to values between 4 r C and 45**C by external means. It can be applied locally/ regionally or to the whole body depending from the stage of the cancer patients. For decades hyperthermia has been an area of laboratory investigation with moments of enthusiasm and disappointment, but now there is renewed interest. Its effectiveness as a cancer treatment has been demonstrated by many trials in Europe. These trials have highlighted that hyperthermia improves cancer treatment results while decreasing the side effects of conventional therapies. Following overviews on hyperthermia physics, this book comprehensively describes the biological rationale for associating hyperthermia with radiation and chemotherapy and the biological and clinical effects of heat on cancerous and normal tissues. Chapters are arranged in three main sections (physical and methodological studies, biologic principles, clinical studies). The first part devoted to the physical principles underlying heat generation in tumor tissue has been kept to a minimum, so as not to put off clinicians or students. An entire chapter regards thermometry since temperature measurements, or better, thermal dose calculations are clinically critical. Currendy no simple methods of temperature measurement inside tumor mass are available. The advent of noninvasive thermometry is warranted; some attempts have been made using ultrasound and magnetic resonance. Unfortunately, these measurements call for skilled teams composed of medical physicists and clinicians. Nanotherapy application with heat is reviewed by an expert in the field. The interactions of hyperthermia with tumor metabolism and its environment, particularly the effects on angiogenesis and vasculature, are discussed broadly and in depth in the second section. A chapter describes the tumor microenvironment and its manipulation in order to increase thermoresponse. A specific chapter is devoted to clinical trials with chemotherapy and radiotherapy, offering the opportunity to understand the therapeutic gain of heat. Aspects of tumor biology relevant to this kind of treatment and especially to brain tumors are described with particular attention to their clinical relevance. The third section of the book deals with the clinical applications of radiofrequency and perfusion hyperthermia; other methods for generating heat such as microwaves or ultrasound have been avoided. Interstitial hyperthermia applications on the liver and antiblastic limb perfiision applications are described by experts in the field.

Whole body hyperthermia treatment is described by two groups of authors that use different modalities of heating. Taking into account the side effects of various cancer therapies on immunity, we thought it appropriate to evaluate the various therapeutic approaches and interactions of this kind of therapy with immunity. A chapter on fever therapy has been also added, and the reader can understand the specific benefits of the thermal component of fever on the immune response. The main purpose of the book is clinical, and it must be considered a primer or an update for experts on the matter. One of our purposes is to provide physicists and engineers with information on the biological effects of heat on tumor tissue, aspects not deeply discussed in bioengineering curricula. We hope this book will be of interest to internists, oncologists and to all physicians involved in the management of cancer patients. Gian Franco Baronzio Erich Dieter Hager

= = =

INTRODUCTION

= = =

Introduction and Brief Historical Notes on Hyperthermia Ifknowledge can create problems, it is not through ignorance that we can solve them. —Isaac Asimov The art of medicine consists in amusing the patient while nature cures the disease. —Voltaire (1694-1778)

According to the National Institutes of Health (NIH) hyperthermia (also called thermal therapy or thermotherapy) is defined as a type of cancer treatment in which body tissue is exposed to high temperatures (up to 106*'F), to damage and kill cancer cells, or to make cancer cells more sensitive to the effects of radiation and certain anticancer drugs. Hyperthermia (HT) is used as an adjunct therapy to radiotherapy and/or chemotherapy to increase their effectiveness,^'^ but hyperthermia alone exhibits both antineoblastic and immunological effects. For decades hyperthermia has been an area of investigation with moments of excitement and disappointment, but now encouraging clinical results have renewed interest in its clinical application.^ Historically, hyperthermia was used many centuries ago by Romans, Greeks and Egyptians to cure breast masses.^ Indian ayurvedic physicians practiced local and whole body hyperthermia in 3000 BC. The method consisted of at least five stages of oleation, dietary regimens and purgation with locally or whole body heat application, e.g., a poultice of cotton wool or heated stones employed to treat the liver. Whole body hyperthermia (WBHT) was obtained using vapor produced by sprinkling liquids over heated stones, bricks or metal blocks. Many recommendations and simple methods for estimating the quantity of heat delivered have been described.^ In 1868 Busch in Germany concluded that fever induced by certain bacteria as erysipelas can cause tumor regression or cure cancer. This was concluded after the observations of a patient with a soft tissue sarcoma of the neck infected by erysipelas.^ The causative agent (streptococcus) at that time was not identified. Subsequently, in 1891, a young American surgeon named Coley, unaware of Buschs observation, observed a regression of a soft tissue sarcoma in a patient infected by erysipelas. Stunned by the finding, he searched the medical literature and found many publications confirming the same observation.^'^ Coley initially prepared a culture of streptococci injected at the tumor site with encouraging results. He even noted that the presence of Serratia marcenscens could enhance the virulence of streptococci and that a remote injection from the tumor could result equally in tumor regression. After these observations Coley incorporated Serratia marcenscens

into the streptococcal vaccine, forming the "Coley s toxin"/ The intravenous route was the most effective, and a dose of the toxin was considered sufficient only if accompanied by fever (39-40**C). Sustained pyrexia was considered the critical point in tumor regression.^'^ It was also observed that those who developed the highest fever were most often the ones with the longest survival. Other antitumoral effects not linked to fever are now recognized.^ The fever and pyrexia inducers are now recognized to be caused by tumor necrosis factor-alpha (TNF-a) and other cytokines.^ In the last decade numerous randomized or nonrandomized clinical trials have been conducted.^'^ Most studies were done in combination with radiotherapy (RT) and chemotherapy, in different order, to obtain a better loco-regional control of superficial tumors. Among these, recurrent and primary breast lesions, head and neck neoplasms and melanoma have been treated with radiotherapy in association with hyperthermia. The thermal enhancement ratio was increased for all cases from 1.4 to about 2. The most important prognostic factors for a complete response were radiation dose, tumor size, minimum thermal dose and temperature. The total number of heat fractionations delivered do not appear to be important, provided that adequate heat is delivered in at least one or two sessions. ^° Although there are positive clinical trials, oncologists are skeptical about hyperthermia even if hyperthermia is the only therapy able to exploit the unfavorable tumor microenvironment. In fact, within tumors, regions with reduced blood supply, with active anaerobic metabolism and low pH, are the most sensitive to the cytotoxic effects of hyperthermia as compared to radiotherapy or chemotherapy. For larger tumors, acidic-hypoxic environments are the rule. Furthermore, local hyperthermia (LHT) has been demonstrated to induce radiosensitization at temperatures < 42**C, and to increase oxygenation, an issue which would partially explain its radiosensitization effect.^ Some trials on esophageal cancers have been done using (triple modality) radiotherapy, hyperthermia, chemotherapy [RT-HT-CH]). The results are encouraging, depending on the disease stage, as two-year survival rates have been observed in the range of 20-30%. Despite this, progression of the disease and relapse are common. In the case of stomach and pancreatic cancers, combined therapy HT + chemotherapy (mitomycin-C, 5-fluorouracil) have been performed with positive effects on survival and on objective complaints. ^^ Interstitial hyperthermia (thermal ablation with RF or laser), has reached good therapeutic targets in terms of clinical results, side effects, limitations and costs. On the wave of these positive results, interstitial hyperthermia is now gaining new fields of applications, e.g., in liver, breast, kidney, bone and lung tumors.

Patients with peritoneal carcinomatosis or sarcomatosis have a poor prognosis even when the disease is confined within the abdominal cavity.^^'^^ Different therapies have been proposed, rangingfi*omsurgery to intracavitary chemotherapy. Regional perfiision chemotherapy achieves a high intraperitoneal concentration, minimizing systemic toxicity. However, an intraperitoneal route cannot guarantee adequate drug penetration into larger tumors. To overcome this problem, debulking surgery combined with chemotherapy has been proposed. ^"^ Despite this procedure, the combined treatment has not improved the clinical outcome markedly. Heat has been demonstrated to boost the activity of some antineoblastic drugs (cisplatinum, doxorubicin, mitomycin-C) suggesting that the combination of debulking surgery + chemo-hyperthermia (HIIC) can maximize the antitumor cytotoxic effect. ^^ Phase III studies are still in progress. Phase II studies were positive for overall and disease-free survival rates; however randomized clinical trials are necessary to provide a definitive response.^^'^^ On the basis of peritoneal chemo-hyperthermia experience, other perfiisional hyperthermia techniques have been developed for treating life threatening tumors confined to liver, lung, pleura and limbs. Isolated organ perfusion systems were developed by Creech. Subsequently, the method was adapted for treating various organs with hyperthermia by different authors including Cavaliere in Italy.^^ Perfusional techniques have become standardized and complications have been reduced to a minimum. Approximately 8 to 10% of primary melanomas involve extremities. In this case all the extremities are at risk, but amputation does not cure the disease. To try a definitive cure isolated limb perfusion (ILP) has become the choice. Initially melphalan was identified as the best agent to use with ILP, subsequently it was demonstrated that increased activity was achieved in combination with hyperthermia. Cytokines have also been approved for combination. Regional hyperthermia + ILP resulted in a response rate between 78 to 8 3 % for melanomas. Based on these results the method was applied to soft tissue sarcomas; melphalan or doxorubicin were used, reaching response rates between 4 5 % and 60%.^^ Recendy European investigators have shown that it is possible to reach a complete response rate near the 90% and a longer duration response by adding to melphalan tumor necrosis factor-alpha (TNF-a).^^' ^^ Different trials in Europe and overseas are in progress with TNF-a, and other biological response modifiers (BRMs) such as interleukin-2 (IL-2) and interferon gamma (INF-y). Notwithstanding these positive results, Takahashi clearly illustrated the problems that must be resolved to consider hyperthermia clinically relevant. They are: specific apparatus to maintain a stable temperature distribution, cost of therapy, long hospital stay, and clinical know-how to avoid complications. Furthermore the author concluded that "For clinicians to accept hyperthermia as conventional

therapy, more evidence must be drawn from prospective studies, and a definitive evaluation of the prognostic factors is needed". ^^ Insight from earUer cHnical trials with hyperthermia indicates: 1. For establishing a hyperthermia clinical trial, quahty criteria are needed; these have been collected separately by Overgaard and Nielsen.^^'^^ 2. Temperature measurements, or better, thermal dose calculations are critical. This confirms the in vitro studies that have established that thermal cytotoxicity is a function of both temperature and time. The measurement of the thermal dose is the great challenge for the future. Actually no simple cUnical methods of temperature acquisition inside tumor mass are available. The advent of noninvasive thermometry is warranted; some attempts have been made using ultrasound and magnetic resonance. Unfortunately, these measurements require skilled teams, composed of physician and clinicians. 3. How many hyperthermic applications are necessary to obtain good clinical results? Animal studies indicate that sometimes only one or two sessions of HT are sufficient. Clinical trials indicate that a single treatment once a week for at least five weeks is necessary but any definitive response has not yet been obtained. The role of the biological effects of hyperthermia like immunotoxicity, antiangiogenesis, and proteasome inhibition have to be further elucidated. Synergistic effects with antibody targeted therapy are also new, promising aspects in hyperthermia treatment. In conclusion, better use of the biological basis of hyperthermia, associated with better thermal dosimetry, will permit hyperthermia to become more than an unfulfilled promise. References 1. Van der Zee J. Heating the patient: A promising approach? Ann Oncol 2002; 13:1173-1184. 2. Urano M, Kuroda M, Nishimura Y. For the clinical application of thermochemotherapy given at mild temperature. Int J Hyperthermia 1999; 15:79-102. 3. Falk MH, Issels RD. Hyperthermia in oncology. Invited review. Int J Hyperthermia 2001; 17:1-18 4. Singh BB. Hyperthermia: an ancient science in India. Int J Hyperthermia 1991; 7:1-6. 5. Busch W.Verhandlungen artzlicher gesellschaften. Berl Klein Wochenschr 1868; 5:137-138. 6. Coley WB. The treatment of malignant tumors by repeated inoculations of erysipelas with a report on ten original cases: Am J Med Sci 1893; 105:487-511.

7. Bickels I, Kollender Y, Merinsky O et al. Coley's toxin: Historical perspective 2002; IMAJ; 4:471-472. 8. Hobohm U. Fever and cancer. Cancer Immunol Immunother 2001; 50:391-396. 9. Dinarello CA. Cytokines as Endogenous pyrogens. In: Macckowiak PA, ed. Fever: Basic mechanisms and management. 2 ed. Lippincott-Raven Publishers, Philadelphia. 1997: 7:87-117. 10. Engin K, Leeper DB, Tupchong L et al: Thermoradiotherapy in the management of superficial malignant tumors. Clin Cancer Res 1995; 1:139-145. 11. Takahashi I, Emi Y, Hasuda S et al. Clinical application of hyperthermia combined with anticancer drugs for the treatment of solid tumors. Surgery 2002; 131:578-84. 12. Rossi C, Foletto M, Pilati P et al. Hyperthermic intraoperative intraperitoneal chemotherapy with cisplatin and doxorubicin in patients who undergo cytoreductive surgery for peritoneal Carcinomatosis and Sarcomatosis. Phase 1 Study. Cancer 2002; 94:492-499. 13. Bozzetti F, Vaglini M, Deraco M. Intraperitoneal hyperthermic chemotherapy in gastric cancer: Rationale for a new approach. Tumori 1998; 84:483-488. 14. Begossi G, Gonzales-Moreno, Ortega-Perez G et al. Cytoreduction and intraperitoneal chemotherapy for the management of peritoneal Carcinomatosis, Sarcomatosis and mesothelioma. E JSO2002; 28:80-87. 15. Speyer JL, Collins JM, Dederick RL et al. Phase 1 and pharmacological studies of 5-Fluouracil administered intraperitoneally. Cancer Res 1980; 40:567-572. 16. Elias DM, Ouellet JF. Intraperitoneal chemohyperthermia: Surgical Oncology Clinics of North America 2001; 10:915-933. 17. Omlor GH. Introduction historical review. In: Omlor,Vaupel, Alexander, eds. Isolated hyperthermic limb perfusion. Austin: R.G. Landes; 1995:1-9. 18. Van der Zee J, Kroon BBR, Nieweg OE et al. Rationale for different approaches to combined melphalan and hyperthermia in regional isolated perfusion. Eur J Cancer 1997; 33:1546-1550. 19. Fraker DL. Hyperthermic regional perfusion for melanoma and sarcoma of the limbs. Curr Prob Surg 1999; 36:844-901. 20. Hokenberger P, Kettelhack C. Clinical management and current research in isolated limb perfusion for sarcoma and melanoma. Oncology 1998; 55:89-102. 21. Overgaard J. The rationale for clinical trials in hyperthermia. In: Field SB, Hand JW, eds. An introduction to the practical aspects of clinical hyperthermia. New York: Taylor&Francis Publishers; 1990:213-242. 22. Nielsen OS, Munro AJ, Warde PR. Assessment of palliative response in hyperthermia. Int J Hyperthermia 1992; 8:11-21.

Acknowledgements I would like to express my sincere gratitude to everyone involved in writing this book. To Francesco Miramonti for his help in realizing many of the figures present in this book, and to Isabel Freitas for her care in ameliorating my limited knowledge. Gian Franco Baronzio In this book Gian Franco Baronzio and I have acknowledged some of the researchers who have contributed to hyperthermia research. This should be a primer with the most relevant research and clinical applications in hyperthermia to date. I want to give a special thanks to all the authors who contributed to this book as a milestone in hyperthermia. Especially I want to thank Gian Franco who initiated this book and who has done so much work and spent so much time preparing this book over the last three years. I think he must have been living with this task day and night all this time. In addition, I would like to thank all colleagues and friends who have contributed to this book and given ideas about interesting, relevant aspects of hyperthermia. Many thanks to my teachers and pioneers in hyperthermia: Josef Issels, Paolo Pontiggia, Harry Le Veen, Manfred von Ardenne, Peter Vaupel, Sergej Osinsky, Sam P. Yarmonenko, Shigeru Fujimoto and Francois Gilly. Erich Dieter Hager Finally, we would like to thank Sara Lord from Landes Bioscience for her qualified assistance in revising this book.

SECTION I

Physical Aspects of Hyperthermia

CHAPTER 1

Hyperthermia, Physics, Vector Potential, Electromagnetic Heating: A Primer Ugo Cerchiari* Abstract

H

eating methodologies of restricted and specific body volumes as means to treat cancer are critically examined from the physical point of view. Difficulties in the application of heating methodologies are considered in relation to the different means giving more space to the means more suitable for modelling. Since the usual approach of electromagnetic heating and of its modelling is difficult, the use of vector potential is suggested and some simple calculations and considerations are presented for electromagnetic field modelling.

Introduction Heat is a mechanical energy of incoherent nature of small volumes of matter typically of atomic or molecular dimensions. Incoherent here means that velocities of near atoms are randomly directed and in solids represent vibration of those small portions of matter with random amplitudes and random phases. "Random" means that all possible movements (degrees of freedom) are present and share almost same energy. This energy is continuously exchanged among them. Temperature T is a parameter that is linked to the energy Q by a coefficient C called specific heat Q = CT. Thus in a homogeneous sample of matter temperature is a "measure" of heat. If the temperature of a volume of biological content increases, as a consequence biochemical reactions increase their speed. Initially this speeds up "life," as can be observed in reptiles or also in many species of mammalians, considering temperature and activity. Yet all functions must increase proportionately. In case of an insufficient supply, substrates are quickly consumed, and essential reactions enter in shortage of biological energy and substances and reject metabolites accumulate. These consequences can lead to essential impairment of functions including repair and transport.Thus, in case of stressed biology, heat may be sufficient to increase death rate in cells. These effects become apparent as temperature goes over a definite threshold (42.5°C). Increasing temperature (beyond 45°C) biological molecules, due to the vibrations of their atoms start to suffer new chemical reactions leading to unwanted products. Those modifications in nature of biological molecules (denaturation) further impair the ability of cells and tissues in doing what they normally do performing their functions. Finally a necrosis is induced. These last thermal effects damage both normal tissues as well as cancer. Thus heat may be used to treat cancer if this energy in mainly distributed to tumours, possibly in *Ugo Cerchiari—National Cancer Institute, Milan, Via G. Venezian 1, 20133 Milano, Italy. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

Hyperthermia in Cancer Treatment: A Primer conjunction with drugs and /or radiotherapy and, if raise in temperature is in a limited interval, to avoid death of normal tissue. Cells killing by hyperthermia have to be evaluated by a special kind of dosimetry. Heat deposition measured by power per gram (SAR) has no significance since only thermal damaging effects due to temperature exposition should be taken into consideration. Dose as "the measure of thermal effects" is a function of temperature, of time and of thermal sensitivity of target tissue that may depend on the presence of substrates and of drugs or previous damage. Since even thermometry is not easy at the moment, only simple practical rules can be given, derived by very rough considerations. Chemical reactions starts effectively as thermal energy reaches an activation threshold and increase their velocity as temperature rises (roughly doubling their rate every 10°C) but biological chemistry requires that only a class of reactions should be allowed by limiting the temperature of activation of unwanted reactions. Thus useful reactions are supported by complex mechanisms of transport and catalysis as well as feedback regulation to allow working in a limited temperature range. Equally effective as temperature is shortage of substrates, oxygen especially, as it is well known. So it must be expected that this could be the primary parameter to control beyond temperature. Yet usually temperature is "the" parameter which is controlled and clinical practice su^ests that a temperature of 42.5°C should be kept for 4 hours to obtain the same effect of 45°C for ten minutes. In between of the two a working temperature and a practical time of treatment has to be clinically decided mainly in relation of the weight of the region to be heated and blood perfusion. Various methods have been developed to release heat in tumours having different features: 1. Direct heating by conduction by contact with a heat source: a. localized transcutaneous (superficial) b. invasive, interstitial and intracavitary c. extracorporeal circulation of hot blood in an organ d. heating generalized to whole body by conduction from a thermostatic bath 2. Indirect heating by deposition of coherent energy relaxing locally to incoherent energy (heat): a. mechanical vibration (ultrasound waves) b. low or high frequency electromagnetic fields All these methods face great difficulties deriving mainly from the complex nature of organs and tissues as regards their not uniform physical properties, geometry and blood flow. Since the temperature threshold of biological damage and the increase of the damage with temperature are critical it is immediately obvious that to predict or control the temperature distribution in a body region is mandatory. Unfortunately this is a difficult task. Accepted thermometry requirements are: 1. Overall accuracy < 0.2°C 2. Response time < 4 seconds 3. Sensor size < 2 mm 4. Possibly immunity or no substantial interference with the heating technique Since energy deposition in tissues as well as cooling by blood flow are difficult to model, a good thermometry control with few exceptions is always needed in clinical practice. Unfortunately not invasive thermometry, that in principle coiJd be attained by microwave radiometry, MRI temperature-dependent signal or electrical impedance tomography, still do not meet the requirements for clinical application. Thermometry will be treated in more details elsewhere in this book. As starting sources of Hyperthermia physics we suggest the articles in references 1-4.

HyperthermiUy Physics, Vector PotentiaU Electromagnetic Heating: A Primer

Direct Heating Direct heating is efficiently attained in a range of 10 mm in depth from a heat source covering the lesion with a border in excess of 20 mm. This condition can be met for cutaneous or intracavitary lesions. Interstitial treatments can be planned with linear heat sources spaced no more than 15 mm apart. Sources are tubes 1.6-2 mm in diameter with turbulent hot water flow or electrically heated probes. Normal tissue can be partly preserved by thermally insulating probes with low heat conducting coating in case of tubes or by electrical heated probes of suitable length. Temperature control poses no problem with any kind of invasive thermometry combined with sources. Unfortunately these devices are not commercially available as dedicated systems. Direct heating as a whole body technique as been used in the past by pyrogenic toxins or heat bath (water and/or air) but it is obviously limited in temperature and was usefully used in combination with other agents. Now could be used as a background temperature control in local heating. Yet the experience suggest that it is a risky treatment. Regional perfusion by external circulation of hot blood has also been attempted and also this technique has to be considered of difficult execution.

Heating by Ultrasounds Mechanical oscillations propagate in media and vibration, in contiguous portions of matter, induce a stress due to a difference in phase of local oscillations. During the cycle of stress and relaxation the medium transforms part of the mechanical coherent energy of oscillation (sound) into random movement thus absorbing "sound energy " and transforming it into heat. The portion of coherent energy transformed into heat is called absorbed energy and the loss in coherent energy is called attenuation.The velocity of sound in soft tissues is around 1500 m/s while in lung is significantly less (1000-600 m/s strongly dependent on inflation) and in bone considerably higher (2000-3500 m/s) .Temperature does not affect velocities to much. Mechanical waves are commonly induced in tissues by discs of piezoelectric materials. Piezoelectric discs have two electrodes on each face and if a voltage is applied across the crystals then mechanical compression or dilatation occurs. A piezoelectric disc (transducer) in contact with a medium acts as an oscillating piston if an oscillating voltage is applied. Thus a compression wave with oscillation in forward and backward direction (longitudinal in respect to the thickness of the disc) is generated. This in principle, what really happens is different, since piezoelectric crystals are sufficiently rigid to have their own oscillating frequencies and modes that are excited by mechanical stress making front face oscillate with not uniform amplitude. These not uniform amplitudes could generate small hot spots in front of crystals. To mitigate this problem the exciting frequency is modulated to mix secondary excitement modes and a bolus of water is placed between the disc and the skin to attenuate secondary (higher) frequencies. Transducers have dimensions between 3 and 12 cm in diameters driven by a frequency (v) in the range from 0.5 to 3 MHz (ultrasound US). At these frequencies the wavelength (k) in soft tissues is between 0.5 to 5 mm generating almost plane waves with litde diffiaction. Power densities in front of transducers is in mean 0.5 to 2 W/cm^. At higher power densities oscillation amplitudes can induce "boiling" (cavitation) in the water bolus and bubbles can induce scattering of waves. To avoid cavitation it is necessary to degas the water. Water in front of the disc firsdy heated by US can circulate in a cooler and thus provide cooling in the first centimetre of skin. Let us examine ultrasound waves in a uniform medium to understand some basic properties. A volume, of uniform medium, is excited by a plane transducer T at the left (Fig. 1). In front of T pressure waves are generated in the medium with fronts parallel to the surface of T. A small volume element of cylindrical shape is displaced by the wave from its equilibrium position X of a quantity A.

5

Hyperthermia in Cancer Treatment: A Primer

1r

A* •

A1 da

"

1

W

!

; ^

' • w

^

=;

;

1

dA/dx dx

A _w

^

J

^ 'W"

Figure 1. A cylindrical microscopic volume whose base at equilibrium has position x is displaced by pressure of an amount A. The opposite base is displaced ofA'. Cylinder lenght dx is reduced by an amount dA/dx dx. Displacement is not uniform along x in the volume since pressure wave strains the medium. As a consequence the volume element changes in length of an amount which is: dl = dA/dx dx This changes the original volume dK= dx da by the quantity:

8K=dlda = aA/axdxda Change in volume modifies internal pressure by an amount dp depending on a coefficient K, characteristic of the meditun, called bulk modulus, representing the change of pressure due to the fractional change in volume 8V/dV: dp = K 8V/dV= K aA/ax dx da/(dx da) = K dA/dx Since the strains before and after the volume element are not equal, pressures as well are not equal, thus moving the volume element. This difference in pressure, indicated by d p, depending on the second derivative of A is: d^p = Ka^A/ax2dx The force acting on the volume element, and effectively moving the element, is the pressure across the area da:

df = d^p da = K a^A/ax^ da dx The mass dm of the volume element is related to density p of the medium by: dm = p d a dx Acceleration of the volume element is dl^A/d^ i.e., the second time derivative of the displacement.

HyperthermiUy Physics, Vector PotentiaU Electromagnetic Heating: A Primer

7

Neglecting at the moment the attenuation of the motion due to the frictional force we may write the equation of motion (f = ma) for the volume element:

K a^A/ax^ da dx = p da dx a^A/at^ or a^A/ax^ - p/K a^A/at^ = o

(i)

This equation has the following solution : A = Aocos(27i;(vt-x/X))

(2)

where V is the frequency i.e., the number of oscillation made in one seconds at a fixed point and X is the wave length i.e., the space spanned by an oscillation at a fixed time and AQ represents the maximum displacement due to the vibration of the transducer. It is easily seen, substituting expression (2) into eq. (1), that A-V must be equal to (K/p)^^"^. The solution of eq. (1) represent a simple harmonic wave whose phases (i.e., the argument of the cosine) are constant along planes orthogonal to the direction of propagation x. The crests, where cos(27C(vt -x/A ) = 1 i.e., where the phase is equal to zero, are located where Vt -x/X=0 and thus move at velocity c = x/t = Xv = (K/p)^^^. Sound is only slighdy attenuated in tissues since it travels many X, s. Disc diameter is considerably larger than \ thus oscillations in the medium can be modelled by a simple exponentially attenuated plane wave whose amplitude of oscillation A is given by A=Ao e^-^^ ^ cos(27C(vt -x/X))

(3)

This expression is a solution of an equation slightly more complex than (1), taking in to account that, besides the elastic force K a^A/ax^,during the movement the volume element is also subject to a frictional force proportional to the velocity dA/dx and opposite to it i.e., -T|aA/ at. The coefficient T| is called viscosity. The new attenuated wave equation is :

Ka^A/ax^ - ^^A/^t - p a^A/at^ = o

(4)

The meaning of equation (3) is simply that the amplitude of oscillation decreases exponentially A(x)=Ao e^'^^ giving at a depth x a reduced oscillating amplitude. Substituting expression (3) into eq. (4) it is easily found that the conditions that allow to use (3) as a solution of eq. (4) are: K(|Li2 - {2%IXf) + p(27CV)2 = 0

and

\i = TivWK = r\dK

(5)

Since the frictional coefficient T| is very little, in relation to K, it may be considered zero in the second condition (5). This gives |X = 0, i.e., the exponential factor e^'^^^ is constant and equal to 1 as in eq. (2). The first condition (5) becomes K(27c/A,)^ = p(27CV)'^ corresponding to the previous condition Xv = (K/p) "^ for the velocity. For this reason, and for the great approximations implied in treating biological samples, the velocity c is considered c = {KJp) . Parameters in relations (5) depend on physics and technology: V is chosen by technology, K is very high and T| is experimentally found dependent on V as follows r| = r|tV" with n in the range 1 to 2. The exponent n and the coefficient T|t depend on tissue. Usually |X is of greater practical use then viscosity T|, thus the previous discussion leads to the consideration that: |I = |Xt v"

where |Xt = Tjtc/K.

The usefulness of equation (3) depends only on the fact that it gives the evaluation of attenuation in the medium but is not useful in reasoning locally where attenuation may be neglected and local values of energy, amplitudes and power may be considered to depend on local amplitude of oscillation given by A(x)=Ao e^'^^

Hyperthermia in Cancer Treatment: A Primer For instance local energy may be easily calculated from the fact that the oscillation energy of a volume element is all in kinetic form when velocity is at maximum and since, from eq. (2), velocity is: dkldi = -27CV A(x)sin (27c(vt -x/X)) the maximum volume velocity is 27CV A(x). Kinetic energy of the unit volume element (energy density) is one half of the product of the mass by the square of velocity. Thus local energy density is: E = p/2 (27CV A(x))2=2p7C^r2 Ao^ e^'^M^^

(6)

From eq. (6) it easily seen that energy density decreases with depth by a factor of e^'^^^ For this reason the energy absorbed in the volume in the unit time (Absorbed Power Density) is the difference of energy between two near points i.e., (the x differential of (6) divided by propagation time dt = dx/c (the time that energy takes to travel dx.) i.e.,: APD = dE/dx c/dx = -4 c^p7c\^ Ao^ e^'^M^^ = A c^ipTC^^ A(x)2

(7)

Firsdy equations (6) and (7) show that local energy and APD are proportional to the square of local amplitude. APD is the power density absorbed in the unit volume, that is only a fraction of the power (which is called intensity) transmitted to the unit volume and there present. Another quantity, often used, related to the APD is the Specific Absorption Rate (SAR) that refers the absorbed power to the mass of the unit volume and thus is related to APD by: SAR = APD/p

(8)

Considering eq. (3) and eq. (7) it is evident that what firsdy matters in relation to energy deposition are the exponential factors e^'^^ and e^'^^^ When the depth x equals the length l/|l, the wave has an ampUtude A reduced of a factor 1/ e (i.e., one third or 36.8%) of the input amplitude AQ. Usually p, is quoted in Np (Nepers) by metre yet to be intuitive it is interesting to consider that the value of l/p, ranges between 12 cm and 1.2 cm in soft tissues if frequency varies between 0.5 to 5 MHz. Attenuation in bone is much higher i.e., 3 - 0.1 cm for V in the same range between 0.5 to 5 MHz. As may be suspected by the reduction by a factor of 10 of 1/(1 related to a rise in frequency of factor of 10, attenuation increases roughly linearly with frequency in soft tissues and with the square of frequency in bone. Passing from a medium to another waves change velocity, direction and amplitude and can be pardy reflected at the interface. To understand what happens it is easier not to consider the interaction at the interface but to examine the behaviour of waves few wave lengths from it. Frequency of oscillation can not change but wave velocity is different in the two media. To statt and to clarify some ideas it is useful to do some simple considerations regarding wave fronts. A preview of wave evolution may be attained according to Huygens principle: considering each point of the medium as a source of a spherical wave inflating at the velocity of the perturbation, the envelope of the spherical waves gives the new wave front with the appropriate phase. By this principle it is possible to model the transmitted and reflected wave fronts considering every point at the interface as generating a spherical wave at the instant that it is interested by the impinging wave front The velocities vi of incoming and reflected waves are equal since the medium is the same. The transmitted wave in medium 2 has a velocity V2. For this reason the transmitted wave front changes direction (V2 > vi in Fig. 2). In Figure 2 it is represented the evolution of a wave front at time ti and at time t2 > ti.

Hyperthermia, Physics, Vector Potential, Electromagnetic Heating: A Primer

Otr =

«!

sin(a,) / sin(a,) ^ VjM (Snell's law)

o

\ a,

«r

/

ti

^^^

inc. wave front

reil. wave front at tj t2 residual incident wave front

/ • ^ ^V?v

\

A \ V2 \

B /

t2 trans. Wave front Ot

\

0 Figure 2. The incident wave front at time ti with incidence angle ttj travels against the interface I between two media. At time t2 wave front is partly reflected with reflection angle CLj and partly transmitted with transmission angle (Xj. Transmission angle and incidence angle are related by Snell's law since wave fronts move with different velocities in the two media. Reflection angle (Xr is equal to ttj since in the same medium velocity is the same.

At time t2 the wave front is represented with a reflected and a transmitted front at an interface I and a residual incident front yet to be split. As the intersection of the front of the wave with the interface I moves from A to B, the wave front splits into one reflected and one transmitted wave front. If the intersection takes a unit of time to travel the length AB then vi = AB sin((Xi) and V2 = AB sin(at). Thus sin(ai)/sin(at) = vi/v2 If at= nil then sin(at)=l and sin((Xi) = vi/v2, thus if the angle OA > arcsin(vi/v2) there is no transmitted wave since "also" the transmitted wave is reflected back. Changing medium, waves change velocity, amplitude, direction and, keeping frequency obviously unaltered, change wavelength. The easiest way to understand these changes is to consider those parameters that remain unchanged i.e., momentum and energy. Mechanical momentum of impinging wave is conserved and thus must be equal to the sum of the two momenta of reflected and transmitted waves. Momentum is the product of mass by velocity of a small volume. This quantity is partly transmitted and partly reflected. The velocity of the volume element is given by the time derivative 3A/3t in each medium. Neglecting attenuation, as not interesting in the short range, and thus using solution (2) we have:

10

Hyperthermia in Cancer Treatment: A Primer dAJdt = -27CV A sin (27c(vt -x/Xi)

where the index i has value 1 or 2 according to the medium. This velocity, that incidentally is not the phase velocity of the waves but is the velocity of the volume element of the oscillating material, is different in each medium. The velocity of the volume changes also in ampUtude and direction (sign) during time since the factor sin (27C(vt - x/Xi) changes as time elapses. Yet the balance of the momentum conservation must hold instantaneously during the oscillation. This fact has two important consequences. The first is that oscillation at the interface in the two media must be in phase and the second is that the only thing that matters is the instantaneous relation among Ai^ific> Ai^refj A2,trasm i«c, the amplitudes of oscillation in media 1 and 2 for the incident, reflected and transmitted waves. The factor sin (27C(vt - x/X,) may be neglected since all waves are in phase. The mass to be taken into account during the exchange of momentum between the two media is obviously the mass of a volume of length proportional to the wavelength. In fact, during a period, a volume of length Xi is interested by the exchange of momentum at the interface. Thus we may write the following momentum conservation equation: vA^ipi d o dt Ai,inc = vA^ipi d o dt Ai,ref + VX2P2 dO dt A2,trasm

where vXiPi do dt = vi Pi do dt is the mass of a volume of density pi of length vi dt and section do.The As are the amplitudes that we have seen are proportional to the velocities. Simplifying and taking into account that the product Vj Pi is called acoustic impedance and it is indicated by Zj we have: ^ 1 Ai,inc = Zi Ai,ref + ^2 ^2,tnsm

(9)

(incidentally note that Z = v p do dt is also the mass of the interested volumes) This is a vector equation since the displacement A is a vector. Thus considering a wave impinging orthogonally on the interface line (wave front parallel to it) we obtain for the amplitudes: Zl Alpine = - Zi Aj^ref + Z2 A2,trasm

Now to conclude the evaluation of transmitted and reflected amplitudes we may use energy conservation. The kinetic energy of the same volume element is conserved as kinetic energy of the two volume element receiving reflected and transmitted energy. Since kinetic energy is mv^/2 and Zi,Z2 are the masses of these volumes and amplitudes are proportional to velocities we may write: Zl A\i,J2=

Zl A^Lref/2 + Z2

A\„^J2

these relations after some simple calculations lead to the following relations for the absolute amplitudes : Al,ref=(Z2-Zi)/(Z2+Z,)Ai.i„c

(10')

A2,trasm = 2 Z l / (Z2+ Zi) Ai,i„c

(10")

Or calling F the reflection coefBcient ((Z2- Zi)/ (Z2+ Zi))'^ Aucl=r"^Aunc

(10')

A2.,ra.m=(l-r^")Ai.i„c

(10")

Hyperthermia, Physics, Vector Potential, Electromagnetic Heating: A Primer

Figure 3. A) A spherical transducer can focus waves reducing problems related to scattering and diffusion. B) An array of transducers, acted with suitable phases, can do the same and can also slightly scan the treatment volume Since intensities are proportional to the square of the amplitudes, reflected energy is proportional to r . If we consider the following values of density and velocity we can evaluate the percent of power reflected for waves at the interface from soft tissues to bone or lung: Density of soft tissue will be considered 1 g/cm^ and sound velocity 1.5 10^ cm/s thus we have: Density Sound Velocity r % Cortical bone 2 g/cm^ 3 10^ cm/s 30% Lung 0.3g/cm^ 0.5 10^ cm/s 80% From these evaluations follows that lung is practically not penetrable as well as gas bubbles in the bowel bone also presents great difFiculties. Ultrasomid waves can be focused if a spherical wave front is created as can be easily understood applying repeatedly the Huygens principle. By this means an imploding wave can be focused nearly in a volume of dimension of a wavelength if the medium is homogeneous (Fig. 3A). The trick is based on the arrival in phase of all portions of the wave front to the same point F. An imploding spherical wave may be obtained distributing a set of transducers on a spherical surface and acting them in phase. This is a very effective mean to obtain a disruptive force at F as it is used to destroy renal calculi. Fortunately this approach is not necessary for hyperthermia since is not easy to drive transducers to obtain an arrival in phase at F through different paths in different media. To obtain hyperthermia at the point F, leaving surrounding tissues relatively cold, it is sufficient to send waves through different paths controlling that F is in the path for each beam and, preferably to avoid interference, acting each transducer during different time intervals. A spherical imploding wave front may be useful to reduce attenuation due to diffraction and scattering. Since direction and penetration of energy depends on the shape of wave fronts is useful to produce wave fronts with suitable shapes. The most effective way to obtain a wave front with a variable shape is to assemble a set of transducers acting them with designed phases. This possibility is illustrated in Figure 3B where a plane array is excited adding a suitable time delay to the central elements in respect to the

11

12

Hyperthermia in Cancer Treatment: A Primer

peripheral ones. By this trick according to Huygens principle the wave front detaches firsdy from the external set of arrays producing a spherical wave. By the same mean the focal spot of the wave can be driven to scan a volume and in principle also the different path length along each ray from the transducer to the focus may be corrected to take into account physical properties of different tissues and at least reduce diffraction. Since temperature control requires the insertion of probes in the volume to be treated, echoes from these probes could be used to assure that the volume of interest is in the ultrasound beam.

Heating by Electromagnetic Methods Heating by electromagnetic fields is mainly attained using time varying fields in a practical range of frequencies between 10 MHz and 2500 Mhz. Reaction of biological tissue to electromagnetic fields depends strongly on frequencies since at low frequency the wave behaviour of EM field may be completely n^lected. The wavelength of an EM field in tissues at a frequency between 10 and lOOMHz is in fact in the order of several decimetres. In this case the field may be considered quasi static and heating effects derive mainly by Ohmic considerations. At higher frequencies wavelength are shorter (few centimetres) and propagation behaviour as diffraction and interference cannot be neglected.

Heating by Low Frequencies There are two essentially different way to use low frequency fields to heat: capacitive heating and magnetic induction heating. Capacitive Heating If we put tissues between two plates belonging to a oscillating circuit, closing by this interposition an electric circuit, the charges contained in the tissues are put in motion. Charges in motion react electrically with the others of the same or opposite sign and put in motion atoms and molecules that are essentially charged entities. This effect (Joule effect) is easily locally modelled by the Ohm law: J = Eo

(11)

where E is the electricfieldJ is the local current density (the current in the unit volume) and a is the conductivity (the inverse of specific resistance) and by the Joule law: A P D = W = E2O.

(12)

From these two relations it follows that the power released to the unit volume is proportional to the square of the field. This result is very general and similar to the one already seen for ultrasound. The electric field may be modelled by line of force along which charges are put in motion according to the local difference of potential. Obviously conductivities of various tissues and their geometrical distributions are very influential in determining both electric field distribution and final currents. To understand the effects of currents on tissues let us consider the simple case of two layers of fat and muscle interposed between two plates at a difference of potential AV (Fig. 4). We must consider the value of conductivities for muscle and fat tissue at low frequency that are respectively of about Om = 0.6 Ohm'Vm and Of = 0.01 to 0.05 Ohm'Vm for fat tissue. The field is orthogonal to the plates and thus currents flow in parallel channels from one plate to the other. Current in the channel of section s is constant along the channel and is proportional to AV and to the global conductivity of the channel. Global conductivity is the inverse of resistance x^ of the channel i.e., the sum of the two resistances tf and rmof fat portion and muscle portion of the channel.

Hyperthermia, Physics, Vector Potential, Electromagnetic Heating: A Primer

13

•-m

^<-

fiiii^

AVr

AV„

-•<-

Figure 4. Two slabs of different materials (fat and muscle) interposed between two parallel plates at different electric potential AV are traversed by an uniform distribution of lines of current. Yet if the conductivities of the two materials are different, also in the case that the thickness Lf is equal to L^, the difference of potential AV is not equally distributed and it is higher where the conductivity is lower (AVf > AVm). Thus the power released to fat is higher. Tc = Ff + Tm = Lf/(af S) + Lm/(ani s) = (Lf Gm + L ^ Of)/ Of Gm S

(13)

By the Ohm law the current in the channel is ic = AV/ tc and since re depends on the complete path in the channel also the powers in each portion of the channel depend on the influence of the two tissues in building up the overall resistance of the channel. The powers Wf and W^ in fat and muscle by Joule law are: Wf=ic^rfandWf=ic^rf

(14)

The ratio of these powers is Wf^Wn, = rf/rn^ = (Lf^(Gf s))/(W(Gm s)) = Lf Gm/U Gf The ratio of powers per unit length Gm/Gf i.e., varies from 0.6/0.05 = 10 to 0.6/0.01 = 60. This simply means that easily burns may occur in fat since the power is much more released in fat than in muscle being the muscle a "short circuit" in respect to fat if the overall resistance in the channel is sufficiently low.

Hyperthermia in Cancer Treatment: A Primer

14

^o Pi

r^^^^ 1 ^' ^ ~ ~ ^ ^ ^

AV ^

w

Figure 5. A slab of material interposed between two parallel plates of different areas and at different electric potential AV are traversed by a not uniform distribution of lines of current. Power per unit volume is higher where lines of current are denser i.e., where current is higher. If a significant power has to be sent to muscle-like tissues through a subcutaneous fat layer an efficient cooling of the skin must be put in effect. In any case much care must be taken since tissue heat conductivity is low and cooling is not eflFeaive at a depth beyond 10 mm from the cooling surface. Another interesting simple case is the following. Suppose we have a couple of conductive plates, Pi and P2 of different areas and some conductive tissue T between the two. Currents start from plate Pi and end to P2 (Fig. 5). Lines of current increase in density approaching to the smaller plate. Since the global current is the same, the current density J (which is inversely proportional to the areas) is higher on the smaller plate and much higher the power density W = ]^l(5 which depends on the square of J. Capacitive heating is generally applied only for superficial treatments since in depth distribution of conductivities is not known. Conductivities as seen before are the principal tissue features to be taken into account. Another difficulty comes from electrodes positioning since electric field distribution depends on the relative position of electrodes. In fact electric field is much higher in the region where plates are nearer. If plates have different areas or are not

Hyperthermia, Physics, Vector Potential, Electromagnetic Heating: A Primer

15

completely coupled to the skin the electric lines of force and thus currents concentrate on the reduced surfaces delivering there a higher power density. We could in principle profit of this behaviour using plates composed of multiple electrodes and using a complex multiplexing scheme of power on them. Impedance measures between electrodes of the array could be used to map internal conductivities. Interstitial Low Frequency Hyperthermia Since heating by low frequency is essentially due to Joule effect, a further application of low frequency is by implanted electrodes. This interstitial hyperthermia uses frequencies instead of continuous currents essentially to avoid direct electrical stimulation of nerve and muscles. Stimulation is avoided if frequencies are higher than few hundred kHz. Once an invasive approach is not a problem multiple implantation of electrodes and thermometric sensors is possible and a sufficient control in temperature is attained by power multiplexing over electrodes. Performances of this type of implantation is in theory superior to the one obtained by high frequencies electrodes fed by microwaves that need not to be used in couples. Heating by Magnetic Induction Magnetic induction is usually explained using the model of Faraday s law that essentially models how charges are put in motion considering the electromotive force generated in a circuit by the variation of the flux of the magnetic field chained to the same circuit. This law in its integral form is much useful in physical circuits of uniform material as coils etc. but is not suitable to explain local interaction of electromagnetic field with charges in low conducting materials. Magnetic field and Faraday law in itself is not the best instrument to understand this interaction even in differential form. For this purpose the electromagnetic field may be represented by a more intuitive vector field called vector potential A. This field is usually introduced as the field that can be thought to generate the magnetic field B (B = rot(A)) but with the limitation that, being A a potential, it is not completely defined. This introduction is awkward and confuses the simple nature of A. Thus it is useful for our purposes to introduce A without ambiguities as follows. If we consider a wire of highly conducting material in which flows a slowly varying current of intensity i, in each element dl of the wire we may represent the current as a vector i whose direction is the direction of the current in the wire. In any point P in the space around the wire the influence of this element of current may be represented by a vector i/r parallel to the current element and of intensity reduced by a factor 1/r where r is the distance between the point and the current element. The field A may be defined as: A = 47c/cLrei/rdl

(15)

That is, adding up at the point P all the contributions from all current elements of the wire we obtain A at the point P. In the following we will leave aside the coefficient Anic This definition ofA is particularly useful since can be shown that the electric field E at point P is proportional to -dAJdx i.e., simply to the time derivative of A. If at the point P there are electric charged particles, a local current density J is generated by the field E as usual J = Ea, where O is the local conductivity of the material. The power instandy delivered to the volume element is i.e., it is proportional to (3A/3t) G. What is more important in this case it is that the electromotive force is locally imposed by -3A/3t by an external (to the body) current distribution. In case of low conductivity and non magnetic materials, as is the case of tissues, currents put in motion are low and alter weakly the

16

Hyperthermia in Cancer Treatment: A Primer

A X = h tan(a)

/ A dx = h/cos^(a) da

r= h / c o s ( a )

/VV dA = i /r dx 1=3

N

dA = ( i / ( h / c o s ( a ) ) ) ( h / c o s ^ ( a ) ) da

A X2

Figure 6. The vector potenial Ap due to a quasi static current iflowingin afiniterectilinear segment from X2 to xi can be calculated adding up at point P all the infinitesimal vectors i = i dx reduced by a factor 1/r i.e., the inverse of the distance between the point and the current element. field A of the external currents. The electric field in this case does not propagate fi-om (far) charges and is slightly modified by local conductivities but is locally generated by the variation of A which in turn is weakly modified by biological tissues. This fact allows an easier calculation and planning of power distribution. It is usefiil to consider some simple circuit and the simple consequences in relation to the generated field A. Let us consider a finite rectilinear wire (Fig. 6) in which a current i flows (ignoring for the moment that a finite rectilinear wire is not a closed circuit). To calculate the field A at point P at a distance h fi-om the wire we must add (as parallel vectors) all dA = (i/r)dx contributions of all the line elements of length dx fi-om position xi to position X2 Thus A = Jl^ i/r dx = / ^ ' i(cos(a)/h) {\ilcos\a))

d a = / ^ ' i /cos(a) d a

= i log((l + sin(a2))(l - sin(ai))/ ((1 - sin(a2))(l - sin( aO))

(16)

As it is evident from this result the influence at the point P of each straight wire does not depend on the distance h of the point from the wire, but only on the angles under which the element is seen from the point.

Hyperthermiay Physics, Vector PotentiaU Electromagnetic Heating: A Primer

17

Figure 7. A) Represents in arbitrary units the modulus of the field A due to a constant current flowing in a coil of shape (B). Along the segment joining the two straight parts of the circuit the field is high and "collimated".

As a consequence two wires that are seen from P under the same angle have the same influence if the current i is the same (and the wire are parallel). On the contrary if the currents have equal intensity but opposite sense the total eff^ect of the two wires is zero at the point. Obviously currents are "closed" so coils are to be designed considering the complete and closed circuit of currents (and in principle also 3E/3t currents if charges accumulate generating an electric field from bare charges). In case of relatively low frequencies the wave length is lager then the dimensions of the coils. For this reason coils of good conducting material may be considered electrically neutral and thus at the same potential in relation to free charges. In this case currents are only those due to the masked charge currents in the wire. It is easy to deduce from simple symmetry considerations that A at the centre of a circular coil is zero, and so it is for -3A/3t, while close to the wires it is very high. So it is not appropriate to put a body inside a solenoid to heat deep volumes and neither to put a body between circular coils since the region in the centre will not be heated. As mentioned before, in this kind of design of coils and consequent field calculation, care must be given to the fact that electric field due to a bare charge distribution must be as low as possible. The existence of this fields in fact puts in motion charges in conductive materials that in their turn alter the quasi static electric field distribution from the far charges generating the problems mentioned in capacitive heating. As an application to the previous discussion the modulus of the A field of a C shaped coil (Fig. 7B) has been calculated and is here presented in arbitrary units. Figure 7A shows the modulus of A. The direction of A in the region of interest, i.e., at the center of the C coil between the two straight wires, is parallel to the two wires. This coil works essentially as if only the two linear opposite parts were present since the two semicircular parts in which the current flow in opposite directions give a roughly zero field at the C centre along the straight segments. This field is well localized in a "channel" between the two straight segments. The field distribution could be further modified and/or moved to deliver power in a localized volume. Usually other coil shapes are used to apply magnetic induction at relatively low frequencies.

18

Hyperthermia in Cancer Treatment: A Primer

Planar parallel currents and planar spiral currents have been applied but since field distribution are difficult to calculate with traditional approach in tissues, power distribution is essentially a matter of fact resolved by experiment.

Heating by High Frequencies Heating by high electromagnetic frequency occurs when wavelength are smaller than dimensions of volumes to be heated. Frequencies are in the order of GHz but even in this range wavelength is in the order of centimetres and for this reason it is difficult to deliver energy in small volumes. A wave model much like the one used for ultrasound (changing the meaning of physical parameters) may be used also in this case. Unfortunately plane wave approximation is no longer possible (due to Huygens principle) and beam divergence is highly significant as the source of the wave decreases in size. Since conductivities (including dielectric currents) increase for frequencies higher than 1 GHz and Absorbed Power Density (APD = E^o) is proportional to the conductivity. Even in the optimistic plane wave approximation attenuation is very high and reduces penetration to a depth less than 2 cm. High frequency applicators (or wave guides) are usually shaped as rectangular hollow cavities with a closed end. A single feed antenna is positioned within the cavity to excite the proper oscillating mode of the cavity with electric field parallel to the plane of the aperture. Since wavelength depend on the medium to reduce wavelength (and thus dimensions of the applicators) the hollow cavity is filled with high dielectric material. Coupling with the skin in usually obtained using a distilled water bolus. Power distribution on the plane of the opening is roughly uniform only in the middle (1/3 of the aperture). The only way to reach slighdy deeper points is to use a phased array source as discussed for ultrasound. Since this effect is, in this case, essentially an interference effect, dimensions of heated volumes are of the order of the wavelength in tissue. All these problems have lead to the attempt to inject radio frequency by implanted electrodes, that could be called more appropriately antennas, as mentioned for low frequency implants. Yet in this case the power distribution is much more dependent on the shape and properties of implanted antennas and in general less suitable to be controlled in shape in respect to the volume to be treated compared to a low frequency many-electrodes array.

References 1. Rocmer RB. Engineering aspects of hyperthermia therapy. Annu Rev Biomed Eng 1989; 1:347-376. 2. Cheung AY, Al-Atrash J. Microwave hyperthermia for cancer therapy. lEE proceedings 1987; 134:493-522. 3. Cheung AY, Neyzari A. Deep local hyperthermia for cancer therapy: External electromagnetic and ultrasound techniques. Cancer Res 1984; 44s:4736s-4744s. 4. Gelvich EA, Mazokhin VN. Technical aspects of electromagnetic hyperthermia in medicine. Critl Rev Biomed Eng 2001; 29:77-97.

CHAPTER 2

Thermometry: Clinical Aspects and Perspectives Barbara Baiotto and Piergiorgio Marini* Abstract

I

nadequate thermal dose received by the tumor can cause failures in hyperthermic treatments. In order to compare different treatments and to correlate the treatment data with the clinical results, it is mandatory to know what temperatures are reached in the target volume. From the total three-dimensional temperature, it is possible to deduce the "thermal dose", which is defined as what part of the body had which temperature for how long during a treatment. The actual temperature/heat-dose distribution in the tissue is one of the most important factors which determines the effectiveness of hyperthermic treatment. In order to calculate the resulting temperature profile given a spatial power deposition, we need a thermal model and a method to solve the heat transfer equation. In living tissue the heat transfer equation includes not only the conductive term, but also the convective and the metabolic ones. There are no practical methods to evaluate the total temperature distribution in the target volume during treatment. In the clinical practice thermocouples are inserted inside the treated volume to generate a temperature sampling that represents the total temperature profile. Methods of magnetic resonance imaging can be used to obtain a complete temperature profile.

Calculation of the Produced Power Deposition There are different models available for bioelectromagnetic dosimetry and numerical assessment of electromagnetic fields coupled to biological bodies. Values of interest in these assessments include induced current and specific absorption rate (SAR): AW F^ «MJ? = - — = < T - — ( W k g - ' ) Am 2p which represents a measure of the absorbed power DWdue to the electric field ^ i n a tissue with unit mass Aw, conducibility Cand density p. There are explicit and implicit methods to obtain the value of electromagnetic field components, depending on the number of variables included in the equation. We present two widely applied methods.

*Piergiorgio Marini—Medical Physics Unit, Institute for Cancer Research and Treatment (IRCC), Str. Prov. 142, Km 3.95, Candiolo, Turin 10060, Italy. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer^ edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

Hyperthermia in Cancer Treatment: A Primer

20

^

i"

X

/

f^

1 H,

x

'X

-<

>

Figure 1. The cell of the FDTD lattice.

Explicit Method: Finite-Difference Time-Domain Method (FDTD) The formulation of a continuous equation in a set of equations at the "Finite-Difference" (FDE), that express an unknown in terms of known values, is called "explicit".^ In each equation, only one variable is involved. The FDTD algorithm is simple and efficient and can easily model a wide variety of sources coupled to the body. The FDTD method has been used for applications over a large range of frequencies, from 60 Hz through 6 GHz. This method is a direct solution of the differential form of Maxwell's laws:

Vx^

-If]

where ^ and /fare the elearic and magnetic fields respectively and 8,^12^t the relative medium permettivity and the magnetic permeability, assumed isotropic, frequency-independent and constant over the region where the equation is being solved. This equations can be divided into six partial differential equations along x, j/, and z axis in three orthogonal component of ^ and H. In fact, the model space is divided into a lattice of a discrete unit cells (Fig. 1), and a space point in the lattice is: (x, yy £) = (/Ax, j^y, kAz) and any function of space and time is defined as:

F"{iJ, k) = FiiAxJAy, kAz, nAt) where Ax, Ay, Az are the lattice space resolutions in x, y, z directions. At is the time increment and i,j, k and n are integers. The Maxwell's laws are converted into a difference equations using the central difference approximations. The steps in the FDTD solution are: 1. define model value of the constant (such as e^ O", /X^.) at each location i,j, k, 2. assume initial conditions, 3. for each time step n, specify fields at source, calculate E" and }P^^'-^ for all locations, and 4. stop when the solution has converged.

Thermometry: Clinical Aspects and Perspectives

21

Figure 2. Crank-Nicolson grid. In the explicit solution method the approximation is called "forward in time central in space", because the unknown at the time {n+1/2) can be derived from its value at the time «, in different positions on the 3D lattice, using proper initial and boundary conditions. For broad-band simulations, because the electrical properties of biological tissues vary significantly with frequency, the limitations of the FDTD method are overcome by adding a differential equation relating the electric flux density D to the electric field E and by solving this new equation simultaneously with the standard FDTD equations. This approach is called Frequency-Dependent FDTD Method ((FDfTD).^ Since the FDTD and (FD)^TD methods are time-domain methods, when frequency-domain information is required some method of conversion must be used. An example of a frequency parameter which is calculated from field magnitudes is the SAR. There are several methods which historically have been used to transfer from sampled time domain to frequency domain data for bioelectromagnetic applications, such as peak detection methods, Fourier transform methods and a direct calculation method. The Fourier method is the most widely used and is highly accurate.

Implicit Method: Crank-Nicolson A formulation which involves more than one variable in each FDE is named "implicit method".^ In this formulation, we use an approximation "backward in time central in space". In this way, the spatial term is substituted by the mean of the central differences at the time n and (n+1). The points grid for the method is shown in Figure 2. This method offers stability and then a larger time step can be used for the resolution.

Calculation of the Resulting Temperature Profile: The "Bio-Heat Equation" The FDTD or the Crank Nicolson methods calculate the time-domain vector E and H at every location inside and outside of the body. These can be converted to frequency domain fields and obtain values commonly of interest in bioelectromagnetic simulations, such as SAR. Given a power deposition or a description of a conductive system together with a thermal characterisation of the treatment volume, the three dimensional temperature profile can be calculated. At the heart of the thermal model is the conduction process given by Fourier's heat conduction law that represents the heat balance: r)T ( W . m-^)

p^.cr^

= K,AT

where A is the Laplace operator, p^ Ct and Kf are the tissue density, specific heat and thermal conductivity respectively.

22

Hyperthermia in Cancer Treatment: A Primer

It is not difficult to calculate temperature distributions in stationary phantoms and use the previously described methods to solve this partial differential equation. In biological tissues however, the heat transfer is not a purely conductive process but the local blood flow introduces a convective heat transport mechanism. In fact, blood flow represents the primary mode of heat dissipation during local hyperthermia and any changes in blood flow induced during heating would have a significant impact on temperature distribution.^'^ Different models have been proposed in hterature in order to include the blood flow description in the heat dissipation.^' The first equation including the blood flow contribution in heat dissipation is the "Pennes formidation'' of the conventional "bio-heat equation**:^ dT p,'C,'—---K,'ST^SAR^M^B (W-m"^) ot where SAR is the specific absorption rate, M the metabolic heat production and B a term accounting for blood perfusion in a small tissue volume element. This term B or "heat-sink" term describing convection: B = w^c^{T-T,^) = w^Cf,{T-57)

(W • m"^)

is proportional, by mean of volumetric perfusion rate w/, and specific heat ci, of the blood, to difference between tissue temperature (7) and arterial blood temperature (Tan)- Usually the local arterial temperature is not known and one global value is chosen for 7"^^ the temperature of the in-flowing arterial blood, the body core temperature. In fact, the convective heat transfer is assumed to occur at the capillary level with blood arriving at the arteriolar connection at 37 °C and leaving the venular connection at the tissue temperature T. Weinbaum and Jiji^'^^ proposed the "A^modeF based on studies of peripheral muscle tissue, in which the total thermal effect of the blood flow was included in an effective conductivity iJQ^that accounts also for counter-current heat exchange between thermally significant artery-vein pairs in the microcirculation: K^^KlUawh)

(W.m-^°C)

where K^ is the conductivity due to the conduction, Wb is the perfusion rate and a SL constant accounting for the vessel size and the blood density. ^^ Previous continuous models are not exhaustive in presence of large vessels, that can entail local inhomogeneities in temperature distribution. In the "discrete vasculature" model small vessels can be described with a Kg^ but large vessels are separately represented. ^^'^^ In fact, vessels with diameter greater than 500 |Xm and flow velocity greater than 0.5 cm/s show a temperature not equal to the surrounding tissue temperature and produce cold regions, because of the its long equilibrium thermal length. A theoretical approach which allows the incorporation of these vessels in the finite difference model will be used. All these analytical models need boundary conditions to be set i.e. conditions that the variable have to satisfy. There are different boundary conditions:^ • Dirichlet conditions: the value of the variable is provided along the boundary, • Neumann conditions: the normal gradient of the variable is provided along the boundary, • Robin conditions: is a linear combination of the first ones, and • Mixed conditions: the condition is like Dirichlet along a portion at the boundary, like Neumann along another portion. From a computer model point of view these formulations offer an ideal test site: after setting the computer boundary conditions to match the ones used in analytical model, the computed temperature distributions can be compared with the analytical solution and with the measured one, when it is possible. The next step is the possibility to measure with invasive or not invasive methods the temperature in patients tissue during hyperthermic treatment.

Thermometry: Clinical Aspects and Perspectives

1 1

1 Ti ^ ^

A

^-U*. 1 B

1

\

K^^ T- 1 1 U

23

1

1 < \

A

1

^ 1 >

Figure 3. Thermocouples scheme. Reprinted with permission from ref. 14.

Experimental Measurement of Temperature Execute a temperature measuring into the patient can not been a simple procedure. In the previous section has been clear that knowledge of temperature in discrete points on temperature distribution is a essential condition for hyperthermic treatment; many methodologies or algorithm has been developed to realise this measurements. We can consider the methods of temperature monitoring in two principal classes: 1. Invasive methods 2. Non-Invasive methods In the first group, there are methods that need one or more probes to insert into the patient, for example thermocouples or laser probes. This kind of measure is a discrete method, we can know the temperature only in discrete points in the space and not a temperature distribution into the volume. Subsequently has been introduced some algorithms to calculate temperature distribution starting from a discrete number of temperature measurements. In the second group has been considered methods that not need to introduce probes into the patient. Technologies working in this class are Magnetic Resonance (MR), Ultrasounds (US), Radiometry or superficial measurements of temperature with algorithm to estimate temperature distribution into the patient. The differences about the two class are very impressive, in the first class we ca obtained a good accuracy on temperature monitoring (0.1 -^ 0.5 "C), but only in few points and with the handicap of invasive. The second class methods gives a distribution temperature (2D and with simple algorithm, 3D) in the target, with low accuracy respect to the first methods, high cost and a technology in evolution.

Invasive Methods Thermocouples Thermocouples are active transducer and they give a voltage through thermoelectric effect. In a circuit with two different metals A and B, their junctions are at different temperatures 7/ and T2 with a electric current I (Fig. 3).^ If we open the circuit, we find a potential different e that it depends on the different of the junctions temperatures, with a proportional coefficient, called Seebeck*s coefficient. e = SAB{TrT^ The Seebecks coefficient SAB is function of the two metals, and reality, it is not constant but it is even function of temperature. Conunercial thermocouples are classified with ANSI definition, like show in Table 1. The Costantana is a mix of 60% Copper and 40% Nichel; the Cromel is a mix of 90% Copper and 10% Nichel, while Alumel is a mix ok Nichel (until 5%) with Alluminium, Manganese and Silicio. We can notice from table that even for high temperature, the output voltage is very low. For example, the thermocouples used in Hyperthermia Oncology are T type (Costantana-Copper); if we consider a thermometric excursion of 37-43°C (typically

24

Hyperthermia in Cancer Treatment: A Primer

Table 1. Commercial thermocouples and ANSI denomination Type

Metal (Positive-Negative)

Seebeck's Coefficient (fiV/°C)

NIST Range (°C)

B E J K T

Platinum (30% Rh)-Platinum (6% Rh) Cromel-Costantana Iron-Costantana Cromel-Alumel Copper-Costantana

5.96 at 600°C 58.67 at 0°C 50.38 at 0°C 39.45 at 0°C 38.75 at OT

0 + 1820 -270 + 1000 -210 + 1200 -270 +1372 -270 + 400

hyperthermic temperature), the output voltage, approximately, will be 0.23 mV, with non linearity component negligible. If we want to obtain a precision of 0.1°C, the resolution and the accuracy of a voltmeter must be in order of 2 mV. The signal from thermocouples must be amplified by a dedicated amplifier, accounting for electric-related noise, that is important for this small voltage.

Non-Invasive Methods Magnetic Resonance Magnetic Resonance (MR) is a technology recendy used for thermometry monitoring. During heating, spatial temperature distributions were obtained using Proton Resonance Frequency (PRF) mediod.^5'^^ Experimentally, it has been demonstrated that temperature causes proton resonance frequency to shift by -0.001 ppm/'C. Temperature distributions can be obtained observing phase differences produce by heat directed into the target. ^^ We consider the change of water proton resonance frequency (A w/w) induced by temperature increase.

Where k has been estimated as -0.001 ppm/°C for water. In a static magnetic field this relatioship changes: A(» = y^.Ar.75o To measure the different phase of water proton, we must combine a gradient magnetic field with echo time (TE): AO = y^.Ar.75o- TE The variation of phase difference related to the temperature gives the temperature sensibility, it depends on the static magnetic field and the echo time TE of image sequence. The dependency of static magnetic field can be explain if we consider that for a 0.2 T magnet the time of scan is 8 times longer than that needed at 1.5 T. Consequendy the time of scan is reduced at the same matter like static magnetic field (Fig. 4). Phase changes were calculating by subtracting images acquired during heating from a reference image acquired immediately prior to heating. Another step to apply is the correction for phase drift by measuring phase change across an unheated region of the tissue over time (Fig. 5).

Ultrasounds Ultrasound is another technology utilised to measure temperature non-invasively. Many studies have been conducted in the last 20 years in this way because it is non-invasive and its cost is low.

Thermometry: Clinical Aspects and Perspectives

25

RF

nil""— itf^:

m

Slice SeiectJ

Readout

i/

— i^

Phase Encode -TR-

Echo

Echo delayed

-TEeff

Figure 4. Example of sequence to monitor in vivo thermal distribution. Reprinted with permission from

ref. 17. 035

OJS

o

\

c -a 2

1

4 ^

«L3 a2S

Of)

§ SI (U

<>-<5

^

« o'

i3

a>

iji»2«^

0.1

C3

^ 5

0.15

OOS 10

tS

20

25

Temperature change ("C)

A

30

35

0 ^ >

<

5

10

15

20

25

Temperature change (°C)

B

Figure 5. Phase and temperature changes in phantom and ex vivo bovine liver experiments. Reprinted with permission from ref 17.

Some study was based on the variation of the wave propagation velocity and of the wave attenuation but, in vivo implementation they showed high technologies limits. Changes in the resonance of the back-scattered radiofrequency signal from the tissue sample has been shown to be linearly related to the corresponding changes in temperature. Other studies are based on the variation of the US back scattering from heated tissues; ^^ in fact back scattering intensity of the ultrasound image is related to the temperature of the heated volume. Guiot et al showed a clear relationship between the US back scattering with temperature. The back scatter cross section depends on the difference in density and compressibility of the back scatter with respect to its background. Gas microbubbles are known to exhibit a compressibility about 20,000 times greater than liquid drops. Gas compressibility is related to the temperature; a better performance in temperature monitoring is obtained when gas microbubbles are used.

26

Hyperthermia in Cancer Treatment: A Primer

A serious problem is the stability of the microbubble as a result of the decreasing concentration of the scatters due to gas solubilitation and bubble destruction by the acoustic pressure. To evaluate the temperature, images are acquired and analysed by selecting a ROI (Region of Interest) with its grey level value. The MGL (Mean Grey Level) is computed by averaging the numerical values from the ROI. Mean values and uncertainty of MGL are estimated as average and standard deviation on same different images obtained from the same region at any given temperature. The sensitivity function is defined as: bMGUMGL s = •

AT References 1. Hoffmann KA. Introduction to computational fluid dynamics for engineers. Washington; 1992. 2. Furse CM, Chen JY, Gandhi OP. The use of the frequency-dependent finite-difference time-domain method for induced current and SAR calculations for heterogeneous model of the human body. IEEE Transactions Electromagnetic CompatibiHty 1994; 36:128-33. 3. Lj^endijk JJW, Schellekens M, Schipper J et al. A three-dimensional description of heating patterns in vascularised tissues during hyperthermic treatment. Phys Med Biol 1984; 29:495-507. 4. Jain RK. Determinants of tumor blood flow: A review. Cancer Res 1988; 48:2641-2658. 5. Waterman FM, Nerlinger RE, Moylan III DJ et al. Response of human tumor blood flow to local hyperthermia. Intl J Rad One Phys 1987; 13:75-82. 6. Crezee J, Mooibroek J, Lagendijk JJW et al. The theoretical and experimental evaluation of the heat transfer balance in perfused tissue. Phys Med Biol 1994; 39:813-832. 7. Pennes HH. Analysis of tissue and arterial blood temperatures in the resting human forearm. J Apphed Physiol 1948; 1:93-122. 8. Weinbaum S, Jiji LM. A new simpUfied bioheat equation for the effect of blood flow on local average tissue temperature. J Biomech Eng 1985; 107:131-139. 9. Weinbaum S, Jiji LM, Lemons DE. Theory and experiment for the effect of vascular microstructure on surface tissue heat transfer. J Biomech Eng 1984; 106:321-330. 10. Zhu L, Lemons DE, Weinbaum S. A new approach for predicting the enhancement in the effective conductivity of perfused muscle tissue due to hyperthermia. Ann Biomed Eng 1995; 23:1-12. 11. Charny CK, Weinbaum S, Levin RL. An evaluation of the Weinbaum-Jiji bioheat equation for normal and hyperthermic conditions. J Biomech Eng 1990; 112:80-87. 12. Lagendijk JJW. The influence of blood flow in large vessels on the temperature distribution in hyperthermia. Phys Med Biol 1982; 27:1301-1311. 13. Crezee J, Lagendijk JJW. Experimental verification of bioheat transfer theories: measurement of temperature profiles around large artificial vessels in perfused tissue. Phys Med Biol 1990; 35:905-23. 14. Locci N. Complementi di Misure. Cagliari: University di Cagliari-Dipartimento di Ingegneria Elettrica ed Elettronica; 2002:1-5. 15. Chopra R, Luginbuhl C, Weymouth AJ et al. Interstitial ultrasound heating for MR-giuded thermal therapy Phys Med Biol 2001; 46:3133-3145. 16. Raaymakers BW, Van Vulpen M, Lagendijk JJW et al. Determination and validation of the actual 3D temperature distribution during interstitial hyperthermia of prostate carcinoma. Phys Med Biol 2001; 46:3115-3131. 17. Chung YC, Duerk JL, Shankaranarayanan A et al. Temperature measurement using echo-shifted FLASH at low field for interventional MRI. J Mag Reson Imaging 1999; 9:138-145. 18. Guiot C, CavalH R, Gaglioti P et al. Temperature monitoring using ultrasound contrast agents: In vitro investigation on thermal stability. Ultrasonics 2004; 42(l-9):927-30.

CHAPTER 3

Physical Bac^round and Technical Realizations of Hyperthennia Andras Szasz,* Oliver Szasz and Nora Szasz Abstract

H

yperthermia is a medical heat-treatment, widely used in various medical fields and has a well-recognized effect in oncology. It is an ancient treatment. However, when making hyperthermia we are limited by numerous biological, physical/technical and physiological problems. The word hyperthermia means increased temperature by heating of tumors. This relatively simple, physical-physiological method has a phoenix-like history with some bright successes and many deep disappointments. Why is this enigma? What do we have in hand? Answers lie in the applied techniques.

Introduction Cancer and its treatment have been one of the greatest challenges in the medical science for centuries. Nowadays, enormous economic and human resources are involved in this field, but according to the epidemic data the solution shall still be awaited for. Sure, the cancer is not the first and probably not the last one among the diseases which despite of the exceptional human efforts have not had any cure for a long time. The development of the medical knowledge in most of the cases follows some critical situations and crises, preparing the medical science to avoid the next crisis of the same nature. The medieval medicine insisted basically on the ancient rule of "no cure"^ for cancer, but the diagnostics and categorization had been further developed."^ However, the treatment of patients suffering in cancer was put in practice only in the second half of the 19th century. Definitely, the main idea was always to concentrate on the drastic elimination of the tumor by surgical way, but they improved the therapies by drugs and diets as well.^ At the beginning of the 20th century the basis of the modern oncology had been established by a distinguished branch of scientists: Schleiden, Schwann, Virchow, Halstedt, Wertheim, Billroth and others.^ Nowadays, the oncology became one of the most interdisciplinary research fields: including the biology, biophysics, biochemistry, genetics, environmental sciences, epidemiology, immunology, microbiology, pathology, physiology, pharmacology, psychology, virology, etc. The modern oncology applies highly effective methods and treatments, but their side-effects and, in consequence, the impairment of the quality of life are also remarkable. In general, patients are treated with chemo- and radiotherapy to their toxicity limits in order to achieve maximal tumor destruction. However, these treatments are often not enough. In general, the tolerable toxic level limits the applications: the actually expected tumor destruction would request higher doses than it is tolerable as regards the accepted level of side effects (Fig. lA). The applied •Corresponding Author: Andras Szasz—Biotechnics Department, Faculty of Engineering, Szent Istvan University, Budapest, Hungary. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

Hyperthermia in Cancer Treatment: A Primer

28

s

1 i

Critical toxicity (tolerance level)

1 1

J

A

$

\

J*

i i

i

1 ^

Modified demand for iurttier tumor-destnicbon

time

B

ft t T Ti'-

Figure 1. A) There is a therapeutic gap between the toxic tolerance and the desired destruction. B) Treatments modify but do not eUminate the gap. Rationale of hyperthermia: surmount the therapeutic gap. therapies might drastically reduce the actual demand of the further tumor destruction, but unfortunately the acceptable toxic tolerance is also reduced and the therapeutic gap is reestablished in most of the cases (Fig. IB). The gap between the toxic tolerance and the desirable destruction has to be bridged by a method, such as the hyperthermia: based on physical and physiological effects, its stress has no chemical origin or serious toxicity. Fiyperthermia is an ideal combination therapy. It has low toxicity, mild side effects, and has been shown to provide synergies with many of the traditional treatment modalities. Besides the limited toxicity, the developed resistance against the actually applied treatments could also limit the efBcacy of the methods. While the first treatment is able to suppress the tumor under its detectability, but it is not the sure outcome, as some malignant cells remained behind keep the possibility of relapse. The observed and hopeful complete remission in most of the cases makes only a temporary success. Parallel with this a more serious problem arises than this: some of the nonvanished malignant cells might become resistant to the actual treatment, so its next application coidd not be that successful as it was before, and in the successive steps we lose this treatment facility. Hyperthermia can be helpful in these cases as well because it may resensitize the malignant cells and enables them to be destructed. Heat therapy (hyperthermia) is an aboriginal, traditional healing method. Even the first known, more than 5000 years old, written medical report from the ancient Egypt mentions hyperthermia. The use of hyperthermia for cancer therapy was first documented by Hypocrites for the treatment of breast tumor. His approach of course was mainly supported by the Greek philosophy, where the fire (heat) had the highest level of abilities and freedom. Hyperthermia was also mentioned throughout the Middle Ages,^ but due to the strict Galenus* school and the inadequate heating techniques, the treatment has never became a standard in the oncology practice. Among the first modern curative applications in oncology, Busch^ and Coley^ were successful at the end of the 19th century with artificial fever generated by infection and toxins, respectively. These systemic applications soon were followed by local and regional heating. The leading German surgeon in that time, Bauer KH opinion in his monograph "Das Krebsproblem'' about the oncologic hyperthermia is typical: "All of these methods impress the patient very much, they do not impress their cancer at all." However, very early, in 1912, a controlled Phase II clinical study was published on 100 patients showing the benefit of the thermo-radiation therapy. ^^ At the end of the last century, energy delivery by electromagnetic fields became possible; nevertheless, its use for hyperthermia only b^an about 30 years ago. The first symposium on oncological hyperthermia was held in Washington DC, USA in 1975; and the second one in Essen, Germany in 1977. Both conferences were supported by the local scientific

Physical Background and Technical Realizations ofHyperthermia

29

communities. We may reckon with the born of the modern oncological hyperthermia from this time on as a strong candidate and a member of the acknowledged tumor therapies. Hyperthermia today, like many early-stage therapies, lacks adequate treatment experience and long-range, comprehensive statistics that could help us optimize its use for all indications. Nowadays, the lack of acceptance of the oncological hyperthermia has not only statistical reasons; but the technical solutions are not adequate enough and the quality assurance, the control and standardization of the method itself have not been solved yet satisfactorily. Nevertheless, we will present a wealth of information about the mechanisms and effects of hyperthermia from the scientific literature and our own experience in the hope of proving hyperthermias worth for further research. Many of the researchers evaluating the capabilities of oncological hyperthermia share the opinion of the editorial comment oi European Journal of Cancer in 2001: the biological effects are impressive, but physically the heat delivery is problematic. The hectic results are repulsive for the medical community. The opinion, to blame the "physics" (means technical insufficiency) for inadequate treatments is general in the field of oncological hyperthermia, formulating: "The biology is with us, the physics are against us".^^ In the latest oncological hyperthermia consensus meeting the physics was less problematic. However, in accordance with the many complex physiological effects a modification was proposed: "The biology and the physics are with us, but the physiology is against us'*.^ The present situation apparendy supports the above opinions. The opinion, to blame the "physics" (means technical insufficiency) for inadequate treatments is general in the field of oncological hyperthermia. Is the modern technique unable to meet the demands, indeed? There is a definite group of physicians who submit that hyperthermia has a strong curative force in oncology; however, another group exists believing the opposite. Sure, both the positive and negative believers are not helpful to clarify the situation. We need interdisciplinary scientific analyses and hypotheses to go ahead with the topic. The state of oncological hyperthermia today is similar to that of radiology at its infancy. When ionizing radiation was first discovered, many hypothesized its usefulness in oncology, yet its exact techniques, dose, contraindications, limits, and the conditions of optimal treatment were determined only several decades later. This is a natural process: every beginning shows these "symptoms". However, the baby normally has to leave behind the teething-brash after a definite period. To remain a baby afterwards is abnormal. We think oncological hyperthermia has to get beyond the babyhood, it has to grow up in more definite manner; it has to cast off the infantile period! Our present paper tries to explain the problem of the technical solutions. We would like to promote the harmony of the disciplines, and this interdisciplinary approach might be "with us" to win the war against the cancerous diseases.

Characterization Demand Hyperthermia by its definition is the overheating of the selected tissues. Heat dosing and treatment standardization at hyperthermia are still significant problems. Technically, the dosing and control of the deep heat transfer is very difficult, requesting the same reproducible heat dose for each treatment within the target tissue. A "success parameter" has to take into account the efficiency of focusing, the heat conduction within the body and many other physiologic conditions before reliable protocols can be worked out.

Temperature as a Control Parameter There is a considerable discussion over the relevant treatment parameters, controls, and treatment optimization. Discussions are primarily centered on the role of temperature and the effects initialized by temperature. Various technical solutions are in use at oncological hyperthermia. The reached temperature as a control parameter is compared. At the clinical results the technical differences are ignored, therefore subjective comparison gains ground. In most of

30

Hyperthermia in Cancer Treatment: A Primer

the hyperthermia pubUcations the temperature and the heat as the definite concepts are equally and interchangeably used. By the definition, hyperthermia has to have a heating dose, which is, in most of the medical practices, immediately identified by the temperature. Furthermore, the missing consensus in the explanation of the underlying mechanisms hampers perspicacity. Consequendy, the selection of the proper technical quality control parameter is determined only by the actual technical solution and not by the desired effects. Discussions are primarily centered on the role of temperature and the effects initialized by temperature. However, it is physically incorrect. The temperature and heat are two different and definitely not exchangeable quantities in physics. The heat is a kind of energy, which may be generally characterized by the specific absorption rate (SAR) (integrative view) and, microscopically, by the selective energy depletion mechanisms. This physical quantity is an extensive thermodynamical parameter; which means that the thermal energy is proportional to the mass/ volume/part of the targeted material. The temperature is an intensive thermodynamic parameter; which characterizes the actual state irrespective of its mass/volume/part. If we apply the temperature as a characteristic feature of volume, than every sub-volume has the same temperature, and the volume is characterized in quasi-equilibrium. In most of the real cases we pump heat (energy) into the targeted system to change its chemical bonds and reactions. This is the case by the hyperthermia as well; our definite aim is to destroy the malignant cells/tissue. If the energy transfer is correct, than all the pumped in energy is devoted to make this job: changes the chemical bonds and destroys the actual biochemical processes in malignant tissue. If we were able to destroy the malignancy by changing only a single chemical bond, then our job would be simple: to deliver the corresponding braking energy (resonant frequency), break the bond, and no any temperature change would be requested. But the situation is not that simple. By pumping in a specific energy the temperature increases, and we get an average energization without targeting any specified structures. To show the difference let us show a simple example: the body temperature of healthy humans is fairly constant (deviation is less than ± 1°C, approx. 0.3%), while our energy consumption (heat equivalent) varies in a wide range individually (deviation can be far more than 100%), even the same individual can have very hectic energy intake depending on the complex conditions with no mentionable change in his body temperature. However, a phase transition (sudden structural/chemical change) at definite temperature could solve the contradiction in hyperthermia, this might justify the temperature concept. Indeed, a cellular phase transition observed around Al.yCp and also the surprisingly accurate fit of the Arrhenius plot to experimental results^^'^''^ supports well the idea. However, Arrhenius kink changes by the chemotherapies,^^ and furthermore, the phase-transition temperamre strongly depends on the heating dynamism (slow and rapid) ^^ and on the preheating conditions.^^ Other clinical observations also lead to recent doubts about this concept.^ ^ Interesting also that the thermal enhancement ratio (TER) increases linearly with the activation energy, so the step down heating based on the phase-transition concepr^ is broken. The healthy living state is in a dynamic equilibrium (homeostasis). The malignant tissue is not in equilibrium, its progress is permanent. The physiology deeply changes the conditions and modifies the situation. The physiological conditions are so effective that the drastically increased forwarded power (from 450 W to 900 W) in fact does not improve the measured temperature, and varies considerably patient by patient.^^ Due to the phvsiological factors, the heat-treatment success depends on the dynamism of the heat delivery. Measurements show that the tumor temperature is not significant when applying pretreatments and/or incubations.^^ The isothermal curves seem to be unscientific dreams in the living tissue, because its heat flow is uncontrolled, and it is not a homogeneous media regarding any of its relevant thermodynamical parameters. Disturbing factors in the various stage of malignancies are mainly the blood-flow, the possible liquids and the heat-conductivity differences. Additionally, we expect the main energy-consumption is taken on the chemical effects. This is manifested dominandy in the cellular distortions and does not increase the temperature.

Physical Background and Technical Realizations of Hyperthermia

31

The doubts increase when we observe that the uniform and high temperature in the tumor (near the physiological threshold of 42°C) caused by extreme whole body hyperthermia (WBH) does not show better results than the locoregional heating with lower temperature average and with very heterogeneous temperature distribution. Application of lower temperatures (mild WBH) for treatments of longer periods (keeping the dose) also showed surprisingly good efficacy.^ The delivered heat dose^^ (absorbed energy) or applied field"^ (electromagnetic influence) could determine the treatment efficacy. The incompleteness of the temperature only idea is well shown also by the necessity of two other characteristic parameters: the time (how long we are able to keep the given temperature, generally defined as a proportion of the actual treatment time) and the information on the temperature distribution (or quantities describing which portion of the tumor was over the given temperature, or measurement of the minimum and maximum temperature in the lesion) as well. When we talk about time-dependence, it means dynamism and nonequilibrium concept. By involving the time factor, the dynamics and the nonequilibrium (dynamic equilibrium, homeostasis) are automatically involved. The time by power is the delivered energy; but the time by temperature—as it is used in the hyperthermia practice—has not any physical meaning! Unfortunately, the temperature as an intensive thermostatic parameter is not enough for the proper control, even it may mislead the user. Recently, numerous scientific theories have also started to concentrate on the significance of thermally induced nonthermal effects,"^^ such as heat-shock protein (HSP) production.^^ The relevance of tumor temperature, heat dose, nonequilibrium, and nonthermal effects is apparent, and leads us to the conclusion that clinical outcome cannot be determined purely on the basis of any of these factors alone.

Thertnodynatnical

Approach

Heat put into a system {AQ) plus work done on a system {AW) is equal to the increase in internal energy of the system {AU)y (first law of thermodynamics'^^): AU = A(l + AW.

(1)

Sign A means a small change. During this small change the system does not alter (quasi-static approach). In a more rigorous description a differential calculus has to be used. Eq. (1) shows that the internal energy U is determined by the energy exchange. This exchange has various forms, all the available interactions (denote their number by n) have to be calculated. These terms can be easily defined by the pair products of intensive {Yi) and extensive (A^) parameters: AU=j^YiAXi,

(2)

For example some of the terms are: k

AU = TAS-pAV + 0Ae + [E^AP) + {H^AM)+Y,^i^^j+OlAs-^fi^

+ •^^ (3)

;=i

where T, S, p, V, O, e^ Ey P, H, M, OCy s, fi ^ are the absolute temperature, entropy, pressure, volume, electric potential, electric charge, electric field, electric polarization, magnetic field, magnetization, surface tension, surface area, linear force, length, respectively; while Nj and ^j are the number/mass and chemical potential of the various {k) particles (molecules, ions, clusters, etc.) in the system. Many other pair interactions (all the energetic terms) may be included in this energy balance. All the pairs have special biological meanings: TAS is the absorbed heat, pAV\s the work of pressure (volume changes), 0Ae is the work of the moving electric charges, (E-AP) and (H-AM) are the work of the electric and magnetic fields, ^ANj are the terms of the chemical reaction energies of various (j=l,2,3,...,k) species, aAs can be the energy of the surface (membrane) changes, JA i could be the work of the muscle-fibers, etc. Only one of the

52

Hyperthermia in Cancer Treatment: A Primer

terms, namely, the heat energy has temperature, all the other terms are other kinds of energy consumptions. Definitely, if there are not any structural, chemical etc. interactions in the system, only the heat is absorbed, than (1) has no ^ term: AU = mcAT = AQ_^AT

= —AQ mc

T,T.A^\-T.*SK \mc )

Vpc

(4)

(5)

where 7J, is the original body temperature [C*], c is the specific heat [/^kg/K\ showing that how much energy is required to heat up / kg tissue hy I K, p, Kand m are the density [kg/rr^], the mass [kg] and the volume [rr^] of the heated tissue, respectively. ^IQis the energy delivered [/] into the heated tissue. This picture could be the basic of the misleading interchange of the heat dose and temperature change; in (5) they are proportional. Do not forget that (5) is only valid if there is not any other interaction than the heat absorption. Of course, this is not the case at all in hyperthermia, where our definit goal is to change the structure and the biochemical constituents. In the study of (5) it is also important, that the time (dynamic effects) is not included in these static considerations. However, if the energy balance is time-dependent (the heat delivery and heat sink have power like relations, [//j]), than the temperature becomes also time-dependent. Have we any reason why hyperthermia uses intensive thermodynamic parameter (temperature) regarding the quasi-equilibrium of the treatment? No, of course, we have not. It is only the historical "bad habit". This bad characterization is mosdy responsible for the contradictory results, for the loss of comparability of the results, for the blame of physics/physiology!

Bio'Heat Equation The thermodynamical energy balance defines the actual heat exchange; the energy change in time (dU/dt), its flow in space is: grad(Iu)=^Iu/dxi)y and its sources qu=^e+^m+^hy where /(/ is the energy current density, qg, q^ and qi, are the energy source/sink from electric, metabolic and blood perfiision origins, respectively. The well known balance equation^^ is: dU at

4. diu i^Y dxi

f..

As it is well known from reference 31, the current density of the heat flow is proportional to the temperatiu*e gradient (heat diffusion):

/^=-;igrad(r) = - A X ^ ,

(7)

By substituting (7) into (6) and by using (4) we obtain the so-called bio-heat (Pennes-) equation^^ which serves for the description of tissue heating and internal energy transport. It has the form of dT

^ d^T

P^-^ = ^ £ T T + P^^^^^(^^ -T) + q,-^qm. ^

(8)

/=i dXi

where 7" is the temperature of tissue; p and c are the mass density and the specific heat of tissue, respectively; A is the coefficient of thermal conduction; PbCbVb is the blood perfusion; 7i is the blood temperature (it is in fact a negative value at the locoregional treatment, cooling term, due to Tb < T; while in the whole body treatment it will be positive).

Physical Background and Technical Realizations ofHyperthermia

9080 -

• Data 95% Confidence (Data)

y = 3019.5X + 2.8424 95% Confidence (Line)

i

f 7 70-

E

1 60-

• • •

t$ 5 0 0

33

^ - - • ^

40 '

• ^-"^"* 1 ^^ %'^"

• '^ • # •

1 30 » ^ 2 0 10 0 "i

c

0.006

'

0.01

'

0.015

'

0.02

'

0.025

•

0.03

1/t [t/day]

Figure 2. Metabolic heat production of breast cancer lesions (tumor size: 0.6-4.0 cm), shown as a function of the reciprocal value of volume doubling time. The rapid procedures (large 1/t values) are more intensive heat producers. The i]ir„ term is determined by the metabolic rate (A/) and the mass density: q^ = P^- The metabolic rate grows by the temperature gain AT: q^-^lA^ ;^^ therefore, in the case of 6°C increase the amount of growth will be 1.8-times. The metabolic heat production of tumor depends on the doubling time of its volume (ref. 34 and Fig. 2), so it is determined by the speed of the development. Defining qg is more complicated, because it needs an approximation of the energy absorbed from the electromagnetic fields. In real situation the bio-electromagnetic interactions are more complex, and the measurement of the absorbed energy is necessary. The energy absorption is characterized by the specific absorption rate (SAR, [W/kg]), identifying the absorbed energy in unit mass:

SAR = 4 ^ 1 = 4 ^ 1

(9)

The simplest case is the current flow through the given tissue, when the well-known Joule-heat is developed. This value (taking into account the well-known Ohm-law) is:

?'=7^^iLi=PSAR,

(10)

where Eiocd is the locally presented electric field in the tissue and s is the electric conductivity. The solution of the partial differential equation (bio-heat equation) (8) should be reasonably given numerically because of the following arguments: • The exact geometry of tissues is unknown; • The equation parameters are not available in their exact form, the values change by tissues, tumors and individuals, as well as those are nonhomogenic in the tumor-tissue; • A certain part of equation parameters—for example perfusion rate, metabolic thermal power, electric thermal power—can be expressed as a function of temperature, therefore the equation may be nonlinear as well; • The treating electric field and for this reason the electric thermal power can be expressed as a function of position, and because of the skin effect it depends also on the temperature;

34

Hyperthermia in Cancer Treatment: A Primer

• The parameters of the transitional zone between the tumorus and healthy tissues are unknown; therefore the exact definition of boundary conditions is almost impossible. The unanimity conditions of the bio-heat equation (8) are the initial data and boundary values. The initial data can be easily specified by the actual initial body temperature {T^, assuming its homogeneity in the malignant area and its surrounding. (Note: sometimes it is not the case because of the higher tumor temperature due to its increased metabolic rate.) However, the boundary conditions (which are essential to solve (8) at least numerically) are more complicated to determine. Three types of boundary conditions may be considered: • The boundary surface of the targeted area has a constant temperature. This assumption is supported by the observation that thefiinctioningof tissue is normal outside the area, thus the increase in temperature augments the perfusion rate and—as a result—keeps the healthy tissue section on constant temperature. • A boundary layer is formed on the surface of the targeted area. This could be physiologically constructed by a transient tissue between the malignant and healthy areas. The considerable increase of perfusion in the healthy tissue section close to the boundary surface presents an other problem, as the extent of this should be known as well. • The transition of conductive heat-flux density and temperature is continuous on the boundary surface of target area. The consideration of this boundary condition means that the bio-heat equation (8) shall be solved both for the target area and for the area outside thereof, and the two solutions shall be matched at the boundary of the two areas in consideration. This is a very complicated task, mainly in the case where the surroundings can not be considered as infinitely large. Therefore, a newer boundary condition is needed. These conditions—^with the additional problem of knowing the SAR distribution in-situ— clearly show that the solution of bio-heat equation does not realistic for practical use. However, note the actual driving forces in the equation, which are always the temperature changes (gradients in space and time), and no assumption of the homogenic temperature could be valid. (In the case of homogenic temperature the bio-heat equation (8) does not exists.)

Control-Parameters Classical Approach Thefirststandard ever for the nonionizing radiation^^ declared the maximal radiation limit Pmax =10 mW/cm^ as a danger-threshold for humans, supposing 0.1 °C increase of human body temperature by this energy absorption. Based on this concept any bio-application—in most of the cases—is controlled by the actual equilibrium temperature (considering the normal body temperature as a basis); and this is the starting point of the classical hyperthermia control as well. However, there are doubts in the scientific conmiunity on the issue if only the equilibrium temperature determines every biologically relevant processes. Nowadays, the delivered heat (absorbed energy dose) or appliedfield(electromagnetic influence) are also considered as important effects. However, serious arguments could be presented to support the presence of the thermally induced but basically nonthermal effects.^^ These involve the thermally and nonthermally generated chaperone proteins, which are heat-shock proteins (HSP)^^ for the most part. HSP proteins modify the stress tolerance to electromagnetic interaaions as well.^ Definitely, the most commonly used control parameter is the temperature. However, the measurement of the temperature in the malignant area is not a simple task: it is rather nonhomogeneous site by site, even on diffierent size scales (in cellular level the metabolic rate, in cluster level the necrotization, in tissue level the vasolidation makes the distribution inhomogeneous). In this manner the temperature measurement in a definite point may not give general information; it depends gready on the actual sensor position, the actual tumor status and physiology. Consequently, the usual contact temperature measurement can give realistic control if the average of some of them is observed. This disadvantage comes from the problems of the invasivity (possible infections, ulcers, metastases, pain, discomfort, etc.)

Physical Background and Technical Realizations ofHyperthermia

35

and the problem of the electromagnetic sensitivity of the sensors themselves. However, some noninvasive methods have been developed as well, but all of t h e m have some serious problems by restringing the wide range of reliable applications. T h e most popular noninvasive temperature measuring methods and their problems are: • Infrared image—It measures only the surface temperatures. • MR-image—Time-shifts are measured, which first of all depend on the chemical bonds. The calibration made on the phantom, where the chemical changes are reversible and different from the living target tissue, and where our primary goal is to change the chemical bonds into irreversible one. Without the proper calibration, the temperature from the time-shifts is not calculable. • Thermo-radiometry—(passive radar techniques). It is not accurate enough and only the multi-frequency application can give information about the depth distribution. Technically, these devices with satisfactory number of frequencies do not exist yet. • Impedance measurements—It gives overall information on the tumor, no vertical resolution available and the lateral resolution is also not satisfactory, and measures only the averages. T h e development of science and technics is rapid, and we expect to have more accurate and reliable noninvasive temperature measurements in the future. However, the question remains: do we force the optimal way for the control? Due to the missing phase transition, due to the principally incomplete approach, due to the problematic measurements we do not support the temperature as an overall control parameter. In fact, the search for the qualitative extensive parametrization is not a surprise in the medical treatment characterization. All the medical treatments are quantitatively characterized by extensive (never by intensive) thermodynamic parameters. An example only from the oncology: the radiotherapy uses the gray [Gy] (=10'^ rod), which is the absorbed energy pro mass (energy-density, [//^^]); the chemotherapy uses the chemo-dose, mass of the active agent pro mass of the target/[body-mass/body-surface]); surgery uses the volume and/or the mass; etc. Except the hyperthermia there is not any other medical application, which uses intensive parameter to control any processes! It is definitely because of the dynamical actions: the medical processes can not be characterized in quasi-thermostatic way.

Proposed

Characterization

In spite of its inadequate character, the temperature became gradually the base of the quality assurance and treatment control. To change it and to choose a heat-dose (energy-dependent) characterization are desirable. To find an extensive parameter to characterize the quantity of the effect like in other treatment modalities is mandatory. To measure the prompt effects during the treatment is really a quantitative factor, but to measure the conditions for the effective distortions afterwards, needs assumptions. T h e changes supposed to make the postponed actions (ischemia/hypoxia, stress factors, acidosis, etc.) are also measurable in situ. T h e temperature concept had oversimplified the situation: the suitably high temperature makes the jobs, prompt and postponed effects are expected, and it is supposed that the requested thermal dose has got into the target. As we see from (3), the temperaturedependent term (TAS) in the internal energy {AU) is the only one from many. T h e main factor of the real desired action: to have cellular distortion and chemical reactions. If the bio-system undergoes chemical reactions then the nontemperature parts of the internal energy become important. ^ We have to characterize the heat delivery, the treatment-effect in-situ in tumor-areas, as well as the actual quality of the hyperthermia treatments in order to keep proper control and to carry out the possible comparison of the various treatment techniques. There are various possibilities for the accomplishment of the relevant characterizations where biological/physiological parameter has to be applied for the characterization of treatment status. T h e actually used in-situ (or immediate off-situ) application of M R I check could be one of the possibilities in the selection of a qualitative parameter (first of all the spectroscopic one) without any force in

36

Hyperthermia in Cancer Treatment: A Primer

regard to an unrealistic temperature approximation. However, the solution is simpler, as the physiologically and physically well studied electrolyte environment of the tissue offers an optimal possibility. The actual electrolytes in the tissue (mainly, the extracellular matrix (ECM)) depends on the metabolic rate, on the chemical reactions and on the structural changes as well. (Sure, it is also temperaturedependent under these extensives.) The ionic density and the structural changes can be followed well by the complex impedance (its imaginary and real parts), which is—in this meaning—a perfect candidate in the control of the energy processes. Electric bio-impedance is a well known method and a simple electrical measurement technique. It measures the tissue's specific electric field distribution. The measurement uses the special frequency dispersion of the actual tissue. As early as 1940 both the whole-body electrolyte status and the local changes (ECG)^^ were studied by the method. Nowadays, it is widely applied, and received the FDA approval for breast tumor diagnostics. Which effects could be used for characterizing? • The prompt necrotic effect is trivially measurable by the impedance. During the capacitive hyperthermia most of the RF-current flows into the extracellular electrolyte, the cells are electronically capsulated (shielded) by their membrane by more than 1,000,000 Vim field strength. When the membrane is damaged, this (until that excluded) area becomes the part of the conductive process and the conductivity drastically changes.^^ (Before this, the impedance grows because the cells swell and the extracellular volume shrinks) " .^"^ The cellular volume and the forming edema are also measurable in situ."^^ The destructive phase of hyperthermia is well measurable, the necrotic and even the mitochondrial damage, as well as the cellular histolysis are observable."^ The process of the observed characteristic cellular response: cellular swelling, progressive membrane damage, cellular shrinkage and subsequent progressive histolysis. At the measurements, the histological changes'^ and the induced coagulative necrosis in human xenografts'^^ are reflected. Special self-similar structure and the hierarchical circuit are also assumed,^ which well agrees with the fractal physiology approaches and the connected dynamism (noises)."^^ • The intensive heat transfer intensifies the metabolic activity, which changes the ionic motility and conductivity. It is measurable by the temperaturedependence of the impedance."^^'^ • The impedance measures selectively: differentiates between the cancerous and healthy tissue, and is able to distinguish the extra- and intracellular electrolyte as well. It is clinically proven that the cancerous and healthy tissue of the hepatic tumors are significantly different,"^^ as well as the VX-2 carcinoma can be also measured. ^^ Well applicable is the impedance method for breast cancer biopsies,^^ for breast cancer diagnostics and prophylactics.^^'^^ Also clinically advanced lung cancer could be followed by the impedance observations,^^ as well as, it has been successfiiUy tried out in the skin-cancer diagnosis. ^^ There are commercially available devices for the impedance tomography for mamographical use.^^ The impedance spectroscopy is effective in early tumor stages^^ and single-cell characteristics^^ too. The method is cell-selective.^^ • Nowadays, the largest commercial application of the impedance method is the cosmetics. It can selectively and systemically measure the body electrolytes and fatty tissues^^ in the whole body.^^'^^ The volume of extracellular electrolyte can be determined.^^ • It is temperaturedependent and the hyperthermia can be monitored^^ (but it should be eliminated later for the distortion measurement"^-^). Arrhenius activation energy is also measured by impedance.^ • Numerous important physiologic parameters, like the apoptosis^^ and the ischemia^'^^ are controllable. • The impedance measurement is usefiil for the control of other treatment modalities. It adequately measures the distortion made by irradiation,^^ and the drug-effect can also be controlled.^® Such usual practice, like to follow the wound healing, is also objectively traceable.^^ • In some hyperthermia cases the impedance measurement becomes the control of the treatment quality. It is widely applied for RF ablation/interstitial technics without any control of the temperature. ^^'^^

Physical Background and Technical Realizations ofHyperthermia

37

Recently, scientists have started to realize that hyperthermia induced temperature gradients (driving force of the dynamic effects) could have significant biological role. A new branch of hyperthermia known as extracellular hyperthermia or electro-hyperthermia has been developed around this concept. Although this new technique recognizes the benefits of increased tissue temperature and its biological consequences it also argues that nonequilibrium thermal effects are partially responsible for the observed clinical deviations from the purely temperature based treatment theory. T h e applied impedance control facilitates to keep in hand the treatment and its effects, to characterize the actual phase of the process and to control the technology.

Technical Challenges In the early days, simple heat diffusion was applied by using hot water or wax baths and heated objects.^^ Today, focused and unfocused energy delivery are applied by using electromagnetic fields. To heat up the malignant tissues is a relatively simple demand but doing it selectively (focused on the malignancy only) up to the desired temperature (over 42 °C) is not a simple technical task. This complication was the basis of the editorial: the physics are against us.^^ T h e main technical problems: • Deepness—achieving a deep energy delivery, • Focusing—proper focusing (selection) on the malignant area, • Reproducibility—conducting the treatment in the reproducible way, • Control—having proper control of the process by keeping the parameters of the treatment, • Personalization—making an in-situ tuning to fit to the actual situation to the best. Regarding the technical and biophysical side of hyperthermia there are some problems. T h e most important ones are as follows: • The heat shock protein (HSP) concentration of tumorous tissues is high, as a matter of coarse, without any external influence and is further increased by the different inventions (apart form their modes). • The heat effect not suitably provided or focused may increase the oxygen supply of tumour, and therefore may intitiate its quick growth. The misfocused heat input involves the risk of necrocytosis of healthy tissues, and therefore may step up the formation of metastasis. • Technically, the reproduction and stability of heat input is difficult to control, therefore there is not any such technical "success parameter" the maintenance and monitoring of which can be considered as a reliable indicator of the success of treatment. The treatment of complex metamorphosis requires complex parameter ensemble, in this way, it is not to be expected that the succesful treatment process can be controlled by one or two parameters. • Technically, the main problem is the appropriate selection of the heat input, as well as, the selection of heat input technics and the formation of actual applicators are of vital importance. The applied eletromagnetic effect may cause in itself HSP increase and undesired tolerance formation without any heat input and temperature increase,^^ and may have some effect on the reactions of immune system as well.-^^

Overview of the Existing Methods Because of the huge difficulties in the proper technical realization, numerous and quite different technical solutions have been developed. Some categorizing points of the available techniques are collected in Table 1. T h e form of heat (energy) delivery has also considerable varieties (Tables 2, 3) applied in practice. Their invasivity and selectivity are categorized in Tables 4 and 5. Sure, the most modern heating technics are connected to the electromagnetic interactions. Their increasing n u m ber of applications could be observed in different categories of frequency (Table 6).

38

Hyperthermia in Cancer Treatment: A Primer

n

_ 11 ^ CD

u P

(D

nn o

>- 9-

§:

J^ 0) ^ 0^ • £ Q_ (- - ^

0)

O •U

y il

(D W) r T3

Q . (/)

t3

s

-
o u 0> c

o

03 DO

O

b o c _y

>

X

c

c_

03 W)

03 u.

(i; 03

^

E

TT

"5^

(D t/5

o u 0) ^c

SJ *E Q.

U <

5 c

CO

03 C

**-

E 6

lo^

u .3^ *" c E o a> 'fS D

u

>>

v«—

I

>

Ol 0)

>

>

c

(0

13

• >+=

•o (D

».^^^

:^i

v2 u O <

03

iZ

>

E DO 03 o E ^ 1-

c 2

£ E

CD

o 0) 00

_c

> 03

2

y jy .^ 'c o tn

"5

C

CD ^ _C

.y-6 t n _D

J^ ^ U H

0 OJ

«^ E

-

c: E

130 OJ O -^

.2 § c

p ^

(D

u ^

c

DO C

"TO

I 1

u

o

Si c

uo

i5 T^ "ocCD _3 ^ E O

C

S

Q,

0)

cj

O

a; -2

a;

E U

c

I

~~-' (D

o 03 CD

> s

o3

03 U

OJ

Q. 03

03

13 i ^C 3 - _(u C

03

Ql

Qj ^ Q-.>

s>

o

-C

o

L. tyi

T3


u

^

03 CD _C

(D -i«i C_ 03 -Q t/5

"u c

a;

o U

^^ u

03

y QL

-Si ID

03

c

CD Q. O

"8

(D

o

>^_C

J£? o3

E o

o; o

_n3 W) c

o

E 'c

E co CD

O

13 .E O 2

U

03 1 ^ c c ^ T:

> c>

c I

-Si

QJ

u *2- DO C

o j;^ I i .=^ X

<3

CD

u

t/5

.5

I is

E u E

to

c (D

^_, Si O

"S c § O § g

I

(/> >

V|JL

> >

c

C 03 _C

OS

c

> s

>

03 D

D

0)

^

.t-

E o u

(D

u

> V

0)

1

0

00 nj

03 U

c

c

c o

c

o

c

.^ >^ .y g

CJ

r

03 i«

tn

U

03

c

-^u^

-8

D

,-^ — _> b

E

o

_c

X 03

u c

(D X

o ^ r E oi CD

_o 03 U

O

0) «/i

T3 C

39

Physical Background and Technical Realizations of Hyperthermia

-56 Q . 0) U

^ c3

i>.

-a o c 'ob.E u (U c o <1) 3

O

^ 0

<1^ >

o

Q.

^

f8

"D •Q3


E o 13 -Si m

i:

C

C O

U

>>

O t/>

•5G

>> 2

C 03

03

•^1 cDO

on O

c

.9 ^-

c c

^

rOJ

0)

0)

g a; u

• ;

_c DO a; u >s u cc >s

.2 "§ 2

> > m m 03

i Is^ 1 8 C

-^

'-^ - Q Q - o3

: c W)

3 a» u ^ OJ

^I ^ to

Q.


03

03 " D

O O >

o

z

"^ -^ "^ .E a.E

c

^

y ^ E .E

CLB 03 U

C 03

DO

rs

•TD

a; to D U

o 03

1

o

r a; GJD

r

'175

> v

.2

Q.

"5 E

a; _c

*> 03 U 03

^^

^C

0^ QJ 03 "O (U

*j 03 U

CU

B

'3 vt—

E o n>^ 03

^

0)

u

i2 -o

a; 0 Q.

1 V §

5

03

>

01

y

"ti flj m V •—

I

c o U c o

u

O O g 03

.y

03

c

03:1

Q . (D 03 £

3 ^

O O O ^ _C J=

•8 o

S

•a

3>

E

I

§ p

•§

I

I

03

3

3

F

E

t/5

t/i

1^ u u

I

W)

u

.n u

>- a; E

o; L<^

Si.

3

0)


> > > u u u 03

03

I

{)

0)

03

0

T3 3

•Si

0

-a

u

u "O

a>

"0

CU

c C DO DO 03 03

•

-Q

^

>- "ad) E E c0 >> 3

Q. fC

8

U

^ •^ •^
U

y *c

^

1

^3

c

-C

E

c c u a; OJ i Q.

-iQ

"O

DO

8

03 3

03

DO r o OJ C

-Q 03

C

>^

_5

0 c OS

E a;

^

>

C

>

CD

DO u c^ DO C C a; 1^ 1^ 0 Q . Q . Q. c E 3 3 .0 ca a) 0 0

0

03

DO

CO 0)

U

U "D

CU

0

> > XJ 3

03 C DO

3

"3

•

c

?^

03

0

0

C

3

u 2 C

_c

Q-

0

s _c

U

X CD

Hyperthermia in Cancer Treatment: A Primer

40

Tables. Selectivity of hyperthermia methods Conduction methods

Selective by the placement of the heatsource. In many applications the selectivity is not the case (e.g., systemic heating)

Convective methods

Selective by the localizability of the fluid convection. In most of the applications it is used non-selectively.

Radiation methods

Most of them (except systemic applications) are selective. Two main categories: artificial selectivity (focus arrangement) our self-selectivity (e.g., impedance selection).

Bioactive methods

Non-selective in most of the cases. Some stimulations (e.g., galvanic) could be used locally, and offers selectivity.

Table 6. Applied frequency ranges for hyperthermia

Frequency Range [Hz] From

To

From

To

0 10,000 1,000

100 500,000 1,000,000 45,000,000 200,000,000 2,400,000,000

1 5 50 50 150 50

5 50 500 800 2000 2000

Electromagnetic Method Galvano- treatment (Hz region) Impedance heating (kHz region) Inductive heating (kHz region) Capacitive heating (MHz region) Antenna (phase) array (100 MHz region) Microwave radiation (over 70 MHz)

Typical Forwarded Energy* [W]

1,000,000 60,000,000 70,000,000

•The absorbed energy could be substantially smaller. Thi;> depends on the arrangement of the applicators and the system tuning.

Electromagnetic Heating Processes Basic Effects The electromagnetic fields (magnetic induction B and electric field strength E) interact with the material and change its state. The interacting electromagnetic field is described by the Maxwellian equations adapted in the actual medium: roxB = floEo^—'^Jtffy ^^^ at

' dt

, divE = pff,

divB = 0, (11)

jco £[(36;r)"' lO"'], n„ =[(4;r)l0-^] where the notations rot and div are the derivatives by the space variables (field changes in the space), as well as e^ and ^o are the absolute permittivity and magnetic permeability, respectively, p^and/^are the material-dependent sources of fields, that is, the effective charge and current

Physical Background and Technical Realizations ofHyperthermia

41

density, respectively. Their values depend on the free charge density (p) and their current densities (/), as well as the polarization {P) and magnetization {M) vectors of the material: Peff =—-

div/'

(12)

jeff = jLLoj + " ^ + ^OXM.

Assuming that the polarization depends only on the external electric field and the magnetization by the external magnetic field: P^EoacE

M =

oc„ -B l + a„

(13)

where OCe and am are the electric and magnetic susceptibilities, respectively, creating the relative electric permittivity and relative magnetic permeability £r= I + ag and jU^= 1 + a^, respectively. The energy density (pw) of the electromagnetic radiation^^ is: P ^ = £ £ £ L £ 2 ^ _ ^ ^ 2

(14)

In the case of radiofrequency currents the energy source (10) of bio-heat equation (8) mainly derives from the dielectric loss, so: qc = iTzfEo \m{er )EI^

= PSARRF .

(15)

The temperature gain by the absorbed energy (if the physiologic modifications are neglected) is shown in Figure 3. The complex amplitude of the energy delivery (energy radiation, e.g., Poynting vector (5) expressed in W/m ) can be calculated as (16)

S= —ExB. ^0

/Zy^^yy^

^^/^2?

^ -^ - 1 0 ^g^.

,^^^^y

^ ^ ^ ^ ^

i^>S^2^^5^VW^^^^^

w^^^^^^^^^^g

^^!^x''^x\S?C\.A ^^^^^^^^*AX3a

4Cwii

time [JtJiB]

^^l\r"

r-15

Figure 3. Result of a model calculation of the temperature gain in homogeneous media with stabilized surface temperature (constant environment).

Hyperthermia in Cancer Treatment: A Primer

42

Transmissbn direction

\7 Transmitter material Zj8^, ^ , a^, (ct^, % y^, 5^, XJ)] Layer-boundaries

Zl[si,^ Pji, o J, (a^, p^, Y^^, 8^^ X^)] ^ ^^^P H^ ^2> (^2> P2> ^2> ^2> ^ ) ]

da

^

Layers

Zj[83, ji^, 03, (a3, P3, Y3, 83, X3)]

^i+ll%l' M-fl-ls ^'i+l? (^H-P Hi+1» Yt+l'^+P V l ) j Nf

^ - l t % - p >%•.!> %-1> (%-!> PN-1> YN-P ^N-l^ ^ - 1 ) ] ZITI%> %> <^N» ( % ' ft^» YN' ^ '

^)]

^H-1

v

Figure 4. Layered structure of the treated body part. The layers represent different tissues (e.g., skin and its structures, adipose tissues, muscle tissue, tumor tissue, etc., noted by numeration), characterized by their "d" thicknesses and "Z" impedances, which depends on the "F" reflexion coefficient, V dielearic constant, "jl" magnetic permeability, "a" conduaion, "a" attenuation constant, "p" phase-constant, "7" propagation constant, "5" penetration depth, "X" wavelength in the medium. The electromagnetic energy absorbed by the treated part and converted into heat equals to the surface (A) integral of the vectors average value calculated for the boundary surface of the treated part. Therefore,

/> = iRef5-^.

(17)

The schematic structure of the complex conditions of the treated bio-matter is show^n in Figure 4. The multilayered structure is the most common from the point of view of the chosen locoregional treatment. The layers represent the various tissues, e.g., skin epidermis, skin capillary bed, fatty tissue, muscle tissue, tumor tissue, etc. It may include nonlayered structures like blood-vessels, bone structures, lymph nodes, etc. The tissue (and the transmitter material, which is in most of the cases air or water) is characterized from electromagnetic point of view by the complex impedance function, which depends on the basic material constants, such as, on the dielectric permittivity (e), the magnetic permeability (^) and the conduction (o) of the tissue. All the other parameters—like the attenuation constant (a), the phase-constant (j3), the propagation constant (}^, the penetration depth (5) and the aaual wavelength in the tissue (A), as well as the reflection ratio (/)—can be calculated^^ from these basic material constants f; \i and c(f\s the frequency of the applied treatment):

Physical Background and Technical Realizations ofHyperthermia

43

1/2

\lii^i

111

, i8 = fi)

^\£i

Yi=ai+jpi. [-^'"^l'

(18) -1/2

^ 1

,

2;r

1

2

[w = 27tE]

and the reflection and the impedances may be calculated^^'^^ in the ith layer as:

(19) [g-i = thYi+idi]

.

The interaction description is complicated enough, but it is definitely much more complex in the reality, where the perpendicularity, homogeneity, radiation condition, source geometry etc. might modify the results. This is the reason why at the calculation of real cases only numerical approximations can be considered. A minor possible simplification is that in the natural situation (without artificially added materials) the bio-material has no magnetic properties, as its relative magnetic permeability equals to one with high accuracy. We have further complications when trying to keep in hand both the rather complex bioelectric interactions and the highly sophisticated mathematical apparatus: if our aim is to accomplish a deep heating condition we face many problems physically and physiologically as well. The main problematic points are: 1. The incident energy has a frequency-dependent exponential decay in the depth (Fig, 5). The penetration is defined by the depth where the field intensity decreases by 1/e-part (about 36%) of the incident beam. (The absorbed energy is even less: about 14% of the incident energy.) The penetration of the field into the targeted material depends significantly on the applied antenna arrangement. The planar waves at the applied—relatively low—frequency generated by capacitive coupled antennas penetrate into the bio-systems by 14-20 cm-^^'^^ depending on the actual electric conductivity of the bio-tissue. The penetration depth for the planar waves is shown on Figure 6. 2. The incident energy beam reflects on the surfaces and internal boundaries as well. Generally, we must note that the reflection at the air-skin boundary is very high (it is more than 0.8 at 100 MHz and about 0.6 at 10 GHz82). 3. The subcutaneous capillary bed tries to balance the local homeostasis, cools the skin by intensive blood perfusion. Also the deeper layers are blood-perfunded delivering rapidly the heat away from the targeted area. 4. The physical/electrodynamical parameters guide the heat absorption, which could prefer other selectivity than the desired one (e.g., the bone treatment could be problematic). Hot-spots/ layers/areas could be generated in unwanted locations involving risks for the healthy tissue. The hot-spot formation depends on many physical and physiological factors, such as: • The local RF-current density increases by the decreased cross section of flow. • The reflected waves at the internal tissue boundaries are summed. • The tissues are nonhomogenic, their conduction and permittivity may change drastically by places. • The body cavities could serve as hollow-space resonators caused by standing waves and their local field maxima.

Hyperthermia in Cancer Treatment: A Primer

44

Figure 5. Schematic representation ofthe basic electromagnetic methods for an oscillating circuit. The three basic effects (elearic field, magnetic field and elearomagnetic radiation) are interconneaed, their aaual domination in one of the circuit elements decides about the effect.

24 •

1^

?18

C \

\

^ " " " - - - . . . . ^ ^ l O MHz

t

v

1 ^"

1" 2 0= 1 s/rT'^'^^'rrrtTrrr?-

0 10

0.6

1000

100

1,0

0.8

1.2

1.4

16

Conductivity (o) (S/mj

Frequency [MHz]

1 1

•

"J

.£€ '-•^^^'^'^-^--C-'' . > ^ & * ' ' . ' - . ' . ' ; " :--/'--'->"-.-'"^^i

..-

-"''>>"' Figure 6. Penetration depth of planar waves into the tissue versus the frequency and the conductivity. The depth is approximated from simplified conditions of the imperfect conductor. (The shaded area represents the most common values for muscle tissue.)

Physical Background and Technical Realizations of

Hyperthermia

45

Figure 7. T h e possible risk of surface burn originates from the perpendicular gradient of the field absorption, the low dielectric constant of the skin and subcutan fatty tissues, as well as from the possible lateral (surface) currents. All of these problems can be eliminated by technical improvements.

The dangerous energy absorption and the consequent hot-spot on the surface are created not only by the maximal incident energy but by the low relative dielectric constant (large voltage drop) and the possible lateral currents (Fig. 7).

Technical Solutions The electromagnetic, "deep-thermal" (not convective and not conductive) heating can be considered as significantly better than the convective and conductive methods. While the convective and conductive methods are limited by the thermodynamic heat conduction, the efficacy of the electromagnetic methods is mainly determined by the extensive interactions determined microscopically. The utilized energy can be uncoupled at any essential position of oscillatory circuit producing the given frequency. Therefore, this fact determines the radiation (Poyntings vector), the magnetic (inductive) and electric (capacitive) treatment techniques (Fig. 8). The techniques are basically different in their dominating electromagnetic effects, and also differ in their actual technical solution (Fig. 9). Main characteristics of the methods are collected in Figure 10.

Electromagnetic radiation equal for the tissues, uses simple absorption

Antenna Magnetic field

Electric field

Magnetic field

Electric field

Selective by niagrietic permeability

selective by the polarisation (rcpolaiisiition tieiit)

(rein«gnetis»tfoii-i»««it)

.jCondensor (capacity)

Coil ^ , / (inductivity)

"X" Figure 8. Schematic representation of the basic electromagnetic methods for an oscillating circuit. T h e three basic effects (electric field, magnetic field and electromagnetic radiation) are interconnected, and their actual domination in one of the circuit elements decides about the effect.

46

Hyperthermia in Cancer Treatment: A Primer

Electric field

•

(capacldve coupling)

Patient is in the ^^^cond

y •

B

R a d i a t i v e (antenna array) Patient is in the antenna array

Magnetic field i(lnductive coupling)

^

Figure 9. Basic arrangements ofthe electromagnetic methods: A) Capacitive coupling, usingthe electric field in a capacitor, B) Magnetic (inductive) coupling, using the magnetic field of a coil, C) Radiative coupling, using the radiated field of an antenna-array. Generally, the transmitting medium is water in the capacitive coupling and antenna array, while in the case of magnetic coupling it is simple the ambient air.

RF supply

A

Magnetic coil

Figure 10. Basic electromagnetic methods: A) inductive (magnetic), B) capacitive (elearic), C) antenna-array (radiative).

Radiative Coupling T h e radiative coupling by antenna array has two distinguishing categories in accordance with the relation of the wavelength (frequency) and the source-target distance (Fig. 11). If the target distance measured from the source is smaller than the applied wavelength, then the wave can not be considered as radiaton, it acts by the change of the fields (E and B), and the Poynting vector (S) has no definite role. T h e two radiative methods are mainly different in their penetration depth (the far -field is more shallow) and the focusing ability (the interference can be modified by the phase-frequency characteristics, the attenuation only by the orientation of the beam). Basic disadvantage of the microwave treatments of higher frequency and shorter wavelength lies in its low penetration depth. (From this respect it slightly differs from the diffusion heating.) Other disadvantage is the possibility of destniaion during the heating process, namely, similarly to the ionization radiation this method has harmful influence on the healthy tissues because of the high specific energy input and the selective energy absorption regarding the hydrogen oxide molecides. As the usage of kitchen microwave oven has also demonstrated, the open microwave source may entrain serious health problems. A part of the near-field treatments uses significantly higher frequencies (up to the limit, which determines the wavelength and the target-distance), and uses interference focusing. T h e applied antennas (dipoles) work in the capacitive regime, however their optimal tuning is far away from the direct capacitive coupling; an array is harmonized to reach the interferences in the desired depth and place. D u e to its higher frequency the penetration depth significantly decreases, which is partly compensated by the additional effects of the multi-antenna array.

Physical Background and Technical Realizations ofHyperthermia

Radiative

47

(antenna array)

Figure 11. The radiative coupling is well described by the radiation alone (Poynting veaor) when the wavelength is small (frequency is high). In this case only the energy attenuation acts. If the wavelength is longer than the source-target distance, interference occurs. T h e SAR values depend on the situation (far- or near-field), and are reduced considerably in near cases. T h e empirical ratio^^ observed: 7SAR =

SARn SARf^

1+

c.

U^/2)

1+

a (^/2)

(20)

where f^ and C,h are frequency-dependent parameters, while Xy and A/, are the wavelength in air at the target place; the indexes denote the vertical and horizontal wave-positions. T h e ^^ and C^h parameters were measured^ in the 10-915 M H z region. Using these data, the actual values of )^AR are always smaller than 1, measured also by others.^^ For 13.56 MFiz and 135.6 MFiz the values are 0.95 and 0.22, respectively; so the low frequencies in fact have no differences between the near- and far-field applications, while the SAR decreases in near fields at high frequency by more than 4-times. T h e inductive (magnetic) and capacitive (electric) couplings are always near field applications due to their low frequency and the field effects.

Radio-Frequency Waves in Near-Field

Radiation

T h e radio-wave treatments belong to the lower frequency group of nonionizing radiations, covering the electromagnetic range approximately from 1 M H z to 30 M H z . W i t h the reduction of frequency the risky and uncertain effects typical of microwave decrease, while the penetration depth significantly increases, and the specific delivered energy shows more homogenous distribution. Magnetic Field A possibility of energy decoupling induced fundamentally by magnetic field may signify deep energy absorption. T h e penetration depth of magnetic field is extremely high in bio-systems, because of the very week interaction between the magnetic field and bio-material. For this reason, the energy absorbed by the tissues—from the energy transported by the magnetic field— manifests itself in the Eddy current or induced current part, namely, it is proportional to the

48

Hyperthermia in Cancer Treatment: A Primer

conductivity of material. In this manner the decoupling with magnetic field is weak, its control is difficult and its selectivity can be assured only as a function of conductivity. The magnetic coupling can be enhanced significandy, if a magnetic material is placed into the targeted area. Generally, nano- or microparticle suspensions, seed, grain or rod magnetic pieces are inserted invasively. In more sophisticated cases the magnetic material s Curie-temperature (ferro-/paramagnetic phase transition) is set to a desired temperature in order to control the heat-delivery and fix the maximal temperature of the magnetic material (the ferromagnetic material absorbs, the paramagnetic does not absorb the magnetic field). Electric Field The energy depletion is effectuated dominandy by electric field anyway. Its advantage, namely, the strong interaction with the highly organized bio-matter (which is typically good dielectrics) also could be a disadvantage, though the penetration depth decreases as compared to the magnetic field. Consequently, the capacitive arrangement offers other advantages (the absorbed energy can be significandy increased and extra selectivity factors can be included in the system) and sets other problems (low penetration and possible surface or subcutaneous burn). Next, we are going to discuss the case when the treated, layered part (for example a kernel and its pellicle and surface interface) is between the electrodes of the condenser supplied by radio-frequency power supply (Fig. 12, a simplified model of Fig. 4). Now, we are going to determine the electric power transformed into heat in the treated area with some simplifying assumptions. (1) The dielectric materials pursuant to Figure 12 are homogenous, namely, the Er relative permeability and the (T electric conductivity are constant by layer (2). The problem is symmetric from geometric and electric point of view, namely, the thickness of the appropriate layers and their electric parameters are identical; the treated area is spherical and is to be found in the geometrical centre of the arrangement (3). The extension of the area where the treated part can be found is large as compared to the extension of the treated part. This later assumption assures that a homogenous field is produced even in the tissue part including the treated area. Its advantage is that the field to be matched in accordance with the boundary

Figure 12. Principle of dielectric heating (Layers represent the actual structure).

Physical Background and Technical Realizations ofHyperthermia

49

electrodes

<::

—--^•^

f \< c—'

L

^

s"

3U-i)l

l\

) electric ^^- field lines \

ma gnetic tleld lines

i

1

Figure 13. Physical model of dielectric heating.

condition can be determined by using an arrangement not including the treated area. It should be noted that this restriction is not too strong as the dipole field induced by the treated area is weak, and—as we will see—decreases in inverse ratio to the third power of the distance. The absorbed power which is transformed to heat in bio-matter can be calculated as follows: M^ a + jcoe ((Ti -\-j(oex)[c2-^j(oe2) (Jo + jOXp (cT + fiXgaRee) (21) , = €u:hhioo + joxo) + /2/o(cTi + jcDEi) + lilo{a2 + jcoej) ^ +2 Go + JQ)eQ

^

where U^ is the effective value of the generator voltage. The most important characteristic feature of the dielectric heating is that the order of magnitudes of the conducting and displacement (capacitive) currents is identical. At the same time this means that the magnitude order of dissipative (exothermal) process attached to the two types of current can be identical as well. The model of the one-layer dielectric heating can be seen in Figure 13. The field between the electrodes is excited by the high-frequency voltage impressed on the electrodes. The electromagnetic field can be expressed by using the Maxwell equation of electromagnetic field. As in the examined model the space charge equals to zero, and the living material is paramagnetic from magnetic point of view. We suppose that the living material between the electrodes can be considered homogenous—in the first approach—with linear behaviour at the given field strength. As the field strength is known, the electric thermal power can be calculated:

U{j(o)

MP4

U' (a +fi>eotgaRe fr) = —j|/o(/>r)| [c + 6>eotga Re e^)

2d

(22)

where we utilized that in the case of sinusoidal feeding \] = U exp(/a>f) and Jo(pr) is the 0th order Bessel function.^"^ The SAR can then be approximately calculated in the form of -\2 SAR = —Rel

pR

1

-^-du ^ . F ^ - J^zr - ^» J *" ip^)J' (p^)

cmo

(23)

Hyperthermia in Cancer Treatment: A Primer

50

Figure 14. Focusing arrangement by artificial focusing (radiative and magnetic approaches).

Radiative approach

f

ji MMA^ f^^^K^^^^^^KB^^^' 1

' i^ii^^WHBiM^^plF:-'/"

^VHIA /

o^-^ o Figure 15. Focusing arrangement by capacitive arrangement (self-selective technics).

Comparison of the Methods The typical characteristics of these methods are summarized in Table 7. Note that these values are only orienting, covering the mainstream of the electromagnetic heating; however, the actual applications could be rather different. In the foUowings, due to its dominant presence and practical importance, only the near-field applications are discussed. A significant difference in application of the various methods lies in the focusing mechanisms. The artificial focusing (radiative and magnetic approaches) directs the energy absorption into the area where the malignancy is located, and tries to heat up the area homogeneously, which is in most of the cases not homogeneous at all. In the case of multi-centered liaisons the covered area includes both the healthy and malignant areas (Fig. 14). In the capacitive case the actual differences in complex impedance and conductivity determine the area of the heat absorption irrespective of its size or multiplicity (Fig. 15). The advantage is the self-supporting nature, and it has a positive feedback by the growing temperature when the selective differences between the malignant and healthy areas become even more pronounced.

Physical Background and Technical Realizations of Hyperthermia

51 (U

0

y C

O

0 _C

•^ ro 9 T3

CU

>-

• c Jo o .to "O

P Q- . u GU

e-2 .^ 3

OS

2

E-g

^

P

.y -t:


Q-

O ."^ '€ -^J rs| n3 oJ
DO^^

O CU o 3 "D

-J N

9-c I o Q^ 03

Q;

^

^ DO

vO iS

^

^ .^

H

^ .y .y E

I

^

u cr

(D O

QJ

•—

r^

O

Q;

O T3 00 03

03

^

.3:^E

t

as

E^

Si i§.

Z

a^ 03 ^

m

-c

-Si •- c 5 <" c •" 2 '^

^ 5d « I S.E-? i J$ a; " &

I

"^

2

iS

o

II

3,

"Q3 t/5

o

O

0) Q^ OJ

.^

u

t)

0)

m *u

'P

H £ £ 5

(D " D •TO

1/)

.o LD

3 DO

u

(/5

O U

4-1

C 03

C OJ i-

u (11

^

cOJ

(0) (1) tn U

F o

O

0

O

V

o3

^ -z^ '^ 9V 'c

0)

in

cu c n

O)

DO-O

LO

v2 ^

^

-D 0)

o c a;

03

iI

V

3 U

3

I

P

E

O

03

-n

(U '-'

u i3 _c O _ : 2 u *^ •t;i n3

«/i

c

.E5 a3

E

03

E cu c

03

CD

a; u 03

CU 03

b u

c 03

Q.

o

cDO

.2

n3 ^ D 2 'F

J o

no

E

S -Z O nJ I

T3 CD <];

^

t E E o ^ •E'B

bO 03

E

03

^ Sb 00

c ^ ^ o ^

•Si

I

C

o

B^ .y o) cf ^ J

u

C

CU _>;"o

•Z

() a;

o

DO t^ CU

S ^ C _

(U

N (U o S

^

Si

5 2

K -Si o

u

u

^ -I

E

^

co

cu

^ **o>> CJ

ro en

a; E u O Li_

a; c ^

5 c ^

4-1

u

C

O

Q_

Li-

(D

*

03 tn

.55 bii

03

Ei|I-i i 5I J * 03 S CU

LU

CU

h-

^

OJ -O .b

LU

CU Q. 03

C Q. T3

52

Hyperthermia in Cancer Treatment: A Primer

New Directions in Electromagnetic Oncologic Hyperthermia Our main aim—namely to destroy selectively the malignant cells on microscopic level— needs a modification in our fixed ideas, because the applied hyperthermic effects are not selective enough, and their demand to the high temperatures introduces a new risk of toxicity: the deep burn. Recendy, scientists have started to realize that the thermostatical thinking (the temperature has to be raised up as homogeneously as possible in the malignant area) has to be replaced by a more adequate thermodynamical one (the properly conducted temperature differences could be effective in destruction). Now we see that the hyperthermia-induced temperature gradients could have significant biological effects. A new branch of hyperthermia known as extracellular hyperthermia^"^ or electro-hyperthermia^^ has been developed around this concept. Although this new technique recognizes the benefits of increased tissue temperature and its biological consequences, it also argues that nonequilibrium thermal effects are partially responsible for the observed clinical deviations from the purely temperature based treatment theory. Electro-hyperthermia is based on a capacitively coupled energy transfer applied at a frequency that is primarily absorbed in the extracellular matrix due to its inability to penetrate the cell membrane.^ Although these temperature gradients typically relax within a few milliseconds, a constant energy delivery can maintain this gradient for extended periods of time, and may initiate special transports through the cellidar membrane. The gradient-constructed heatflowmight be significandy large and damage the fu*st step of the membrane. This heat flow can produce extra ionic currents through the membrane, which depolarizes and therefore destabilizes the membrane. This requires ATP, resulting in ftirther heat production at the membrane. The membrane permeability of water is much higher than in the case of ions, therefore, it is the main transported component in the thermodynamic coupling. Therefore, this thermal flux builds up an intracellular pressure, which is about 30% above the normal.^ Since malignant cells typically have relatively rigid membranes due to increased phospholipids concentrations,^^ an increase in pressure will selectively destroy the malignant cells before it affects the healthy ones. These effeas allow the application of hyperthermia not only in a more effective way, but also for the areas which have been mosdy contraindicated for this method: the lung, liver, pancreas and brjiin treatments. Its preliminary clinical trials show the reality of the expectations: the malignancie^ of the liver,^^ brain^^ and pancreas^ are exceptionally well respond to such type of treatment.

Cellular Effects Some biophysical effects on cellular level might modify the technical applications. These microscopic effects mainly depend on the electric field, and in low frequency ranges may considerably influence the actual hyperthermia effects.^"* Absorption in Cellular Level The effective electric field acts by the high polarization (large dielectric constant) of the bio-systems. From dielectric point of view, the simplest approach is to study the cellular structure in its three rather different parts (Fig. 16). As^ calculated the frequency-dispersion energy absorption of the characteristic cellidar structures (Fig. 17). Changes of the Membrane Potential The cell-membrane potential as a fiinction of temperature may be calculated from the Goldman-Hodgkin-Katz (GHK) equation.^^ ^, _ kT ^^ CONaC^^a + (OKC^ + (Ogd^}

^^^^

where k is the Boltzmann-constant, e is the electron charge and Q denotes the concentrations in different ions {Na, K, C/subscripts) and different locations (extra- {e) and intracellidar (i) superscript), (Oi denotes the membrane permeability for the /th ionic components. (The GHK equation wasfirsdyproven on the nerve cells, but this describes the basic dynamical phenomena of the

Physical Background and Technical Realizations ofHyperthermia

53

ExtiaceNular electrolyte a^= 1.2 S/m; E^- 6w4-1
Figure 16. Cellular structure by its dielectric properties. 100

kHz

MHz

GHz

Figure 17. Frequency-dependence of the relative absorbed energy for the cellular parts.

membrane stabilization for every eukaryote system. The static part of membrane stability is described by the Donan-approach.) The temperature dependence of the membrane potential is trivially linear. Substituting the real values of the membrane potential Umembr. = -70.2 mV, the slope of its temperaturedependence equals to -0.23 mV/K, which is definitely not a large effect, and we may conclude that the cellular membrane will not be effected by the temperature alone. ie) _ . <e) _ . <e) _ , -^0) _ C^ C^^=150, O' -CI =% ^ci =125, CV=\% (OK : (ONa :COa=l: 0.04 :0.45, T = 36°C -ii) C)^i = 145, CV=%

(25)

Consequently, the membrane potential increases, and the cell is hyperpolarized under increasing temperature.

54

Hyperthermia in Cancer Treatment: A Primer

Temperature Gradient on the Membrane When the frequenq^ is low enough to the selective energy-absorption (see Fig. 17), then the temperature gradient has essential role in the membrane destruction. The actual energy balance is: pfiVidTIdt) = Iq + Pm^ where pici is the specific heat capacity of the cell, V is the cell-volume, Pm is the metabolic heat-power. The /^ heat-current contains a metabolic and an elearic term: Iq = I^^ + /^^ where Iq^ = -P^ ~ const., so Iqe = PiCiV{dTldi). Hence the heat-current density is^^^ = piCf{V/A){dTldt)y where ^ is the surface area of the cell. Its numerical value at IK/s heating rate equals to jqg = 140( W7w^, which is significant.'' The temperature difference between the two sides of cell membrane can be calculated by the expression AT = (jJcx) « 0.007K. Consequendy, the actual gradient on the 7 nm thick membrane amounts to 10 K/m, which is a remarkable large value! It is well above the natural heat flow induced by metabolism, which is only jqceU= 1.5-10'^(W7w^, thus causes negligible (AT = 7.5-10'^K) temperature difference at the cell membrane. Membrane Damage by Constrained Ion-Currents According to the Onsager's nonequilibrium thermodynamic approach, all the various transport processes are tighdy coupled, so in our present case, heat-transport is accompanied by electric current and mass transport. The Onsager-relation for heat and electric transport coupling takes the form of

Jq - -Lqq —^ + V "y"'

> ~ ~ V ^T + ^« ^

^ '

where 0 is the electric potential, and the Lik values are the transport coefficients. The observed values are one order of magnitude larger than the coupled one,^^ so we use the LqJLqq «10'^ approximation. Hence:y^«/^ {LqJLq^ « 1,510'^ {Alnt^. The usual data^^ ^^JNa = 1.26-10''^ (A/nr) 2jLidjjc= 4.53-10''^ {AJm^ for Na^ and K^ ions, respectively. In consequence, the forced current density is significandy larger than the natural one. This forced current is mainly Na^ influx. It depolarizes and therefore destabilizes the membrane, and stimulates the Na^/K^ pump activity which results in ATP transformations and further heating at the membrane. Membrane Damage by Increasing Pressure According to the Onsager's theorem, the heat flow is coupled to the volume (mass) transport as well. (The entire complex phenomenon is simplified on the intensive pairs only.) The relevant Onsager-relations are: JV = LwAp + Lvq — ,

Jq = LqvAp + Lqq —

.

(27)

The membrane permeability is much higher for water than for ions, so the main transported component in this coupling is water. The dynamic equilibrium hydrostatic pressure can be determined by the pressure equality on both sides of the membrane,^^ so:

^

=-£1

(28)

AT VTi where Q* is the transport heat and V is the molecular volume of the water. The transport-heat value for 1 liter water at normal conditions (room temperature, normal pressure) equals to Q^20 = 7\,2kJ, consequendy: ^ = -1.32.10^^ = - 1 3 2 ^ (29) AT K K This means that the 217"= 0.01K temperature difference generates 4 ^ = 1.32 bar =1.32-1 Or Pa pressure, which is a large value. Consequendy, the lateral tensile stress rises due to the lateral

Physical Background and Technical Realizations ofHyperthermia

55

dilatation generated by the radial mechanical pressure from the electric field, and the hydrostatic pressure difference tries to balance it. The lateral tensile stress calculated from the above nonequilibrium transports using Z) = 10 jim cell-diameter and B, = lOnm membrane thickness results in G„y = (DAp/ix) = 1.32(10 ^m/4l0 nm) « 3-lO^P^ = 30 MPa, which is a huge value! Comparing this value to the regular conditions: the induced scalar pressure (from the Maxwell stress-tensor ) is 0> = ££" « 3-10 Pay which is too small to be relevant and negligible in relation to the transport pressure. The maximal tolerable lateral tensile stress amounts to CTmax = (2 20)'10^Pa,^ which is smaller by two order of magnitudes than the temperature induced stress. The increase of the osmotic pressure also contributes as an additional factor to the damage of cellular membrane. The increase of the ECM temperature boosts the electro-chemical potentials in the ECM electrolyte. The chemical potentials are: Ji = IJ^io + V ip + RTlnyci + ZiFU, where V\ is the molar-volume, ^lo is the initial chemical potential, p is the pressure, / i s the activity coefficient, ci is the concentration and Zi is the ionizing state of the /-th component. F is the Faraday number and U\s the membrane potential. With the increasingy^j,, both^j^and jci decrease, therefore, in stationary membrane state the intracellular concentrations Ck (k = Na^, K^, CI') also grow. This process increases the osmotic pressure in the intracellular liquid, while decreases it in ECM. The above theoretical considerations can be used in many applications. Unfortunately, the complications in the analytical way force us to use different numerical calculations. The numerically calculated results for kernels are in good correspondence with the observations, and allow explaining some seed stimulation effects. Also the nondirected effect of the power lines in the plough-lands could be studied in this frame.

Summaiy In our opinion, the hyperthermia (definitely, it is a heat-dose treatment) is a temperaturedependent but not temperature-determined process. The temperature concept is not bad as long as the physiological factors (blood flow/vascularization, metabolism, chaperone-protein production, dissemination, apoptotic action, etc.) are included, and the tissue can be regarded as homogenic, semi-isolated from the surroundings. Unfortunately, these conditions are not common, so sometimes the temperature gives statistically significant, sometimes random results. This is the reason why some trials check the patients before, and divide them on the "beatable*' and "not-heatable" groups,^^ and randomize for trial only the previous group. For this group the anyway scientifically incorrect and assumed equivalence of the heat dose to the temperature is an acceptable approach. On the other hand, this preselection excludes a large number of patients receiving hyperthermia; however, this treatment could be a help for them as well. The exclusion was made on an insufficient characterization of the method. A relevant characterization of oncological hyperthermia for quality guidelines has to be started from the aims: it is to destroy the malignant cells! This demand contains some more precise requests: selectivity and blockage of fiirther proliferation and dissemination. Distortion could be promptly direct (the cells become necrotic during the treatment) or indirect (tune killing conditions; the cells become necrotic or apoptotic after the treatment). The demands actually do not contain any temperature request. Hyperthermia is an emerging effective treatment method in oncology. It has shown significant improvements in tumor response rates and patient morbidity in combination with other treatment methods, such as surgery, chemotherapy, radiation therapy and gene therapy or applied as a monotherapy. Nevertheless, hyperthermia is still in its infancy. The thermal dose quantification is likely to remain of practical importance. We have to characterize the hyperthermia by thermal dose and not by temperature. Thermal dose changes many energetic processes in the tissue, and in their physiology. Most of the changes (structural and chemical changes) are out of modification of the temperature, only the entropy changes. The nonequilibrium thermodynamics describes how the absorbed heat could excite various (e.g., diffiisional, electric, chemical, etc.) processes. The dynamism of the absorption determines the

56

Hyperthermia in Cancer Treatment: A Primer

dynamism of the processes (e.g., reaction rates, diffusion dynamism, etc.) and, by these, drives the efBcacy of the processes as well. These phenomena (which are anyway in the focus of the tumor destruction) are completely out of the possibility of temperature characterization. Hyperthermia suffers from a lack of standards and a lack of scientific consensus about its effects on malignant and healthy tissues. In order that hyperthermia shall gain widespread approval and clinical use, the technique requires extensive further research and standardization. For this we need an open mind and have to outgrow the dogmatic habits. The hyperthermia is an interdisciplinary approach. We have to use the historic roots, the scientific achievements on other areas. The hyperthermic oncology is in the wave of the demands: nontoxic, excellent in any combinations with other treatments, with minor contraindications. Hyperthermia has been considered a remarkably developing form of tumorous tissue overheating and tumour treatment. This method is based on the higher heat sensitivity of tumorous tissues and the totality of physiological processes resulting from the effect of heat.

Conclusions and Perspectives What / who is against the oncologic hyperthermia? Nothing / nobody from outside. We are against ourselves by constraining the temperature concept over all. The physics, biology and physiology are with us if we make a correct approach. We are convinced that the perspectives of hyperthermia in oncology are very bright and promising. What we have in hand is a practically non toxic effect with huge potential and advantages. However, we have to clarify the technical issues to make this capable method technically comparable, and provide for a control which is safe enough in terms of modern medical demands.

References I.Jones S, Henry W. Hippocrates. Cambridge: Harvard University Press, 1959. 2. Vesalius A. Dehumani corporis fabrica iibri septem, 1543. 3. Warren JC. Surgical observations on Tumors with cases and operations, 1837, (Ref: Pollay M: The first American book on tumors. Thesis, University of Madison, Wisconsin, 1955). 4. Seegenschmiedt MH, Vernon CC. A Historical perspective on hyperthermia in oncology. In: Seegenschmiedt MH, Fessenden P, Vernon CC, eds. Thermoradiotherapy and Thermochemotherapy. Vol 1. Berlin Heidelberg: Springer/Verlag, 1995. 5. Smith E. Egyptian surgical papyrus dated around 3000 B.C. (cited by: van der Zee J: Heating the patient: a promising approach? Ann Oncol 2002; 13:1173-1184). 6. Medieval literature—1. Medieval Turkish Surgical manuscript firom Charaf ed-Din, 1465 (Paris, BibHotheque Nationaie), 2. Armamentarium chirurgicum of Johann Schultes, Amsterdam 1672 (Paris, Bibliotheque de Faculte Medicine), cited by: Seegenschmiedt MH, Vernon CC. A historical perspective on hyperthermia in oncology. In: Seegenschmiedt MH, Fessenden P, Vernon CC, eds. Thermoradiotherapy and Thermochemotherapy. Vol 1. BerUn Heidelberg: Springer/Verlag, 1995. 7. Busch W. Uber den Einfluss welche heftigere Erysipeln zuweilig auf organisierte Neubildungenausuben, Vrh. Naturhist. Preuss Rhein Westphal 1866; 23:28-30. 8. Coley WB. The treatment of malignant tumors by repeated inoculationsof erysipelas, with a report often original cases. Am J Med Sci 1893; 105:488-511. 9. Westermark F. Uber die Behandlung des ulcerierenden Cervixcarcinoms mittels konstanter Warme. Zentralbl Gynaekol 1898; 22:1335-1337. 10. Westermark N. The effect of heat on rat tumors. Skand Arch Physiol 1927; 52:257-322. ll.Overgaard K. Uber Warmeterapie bosartiger Tumoren. Acta Radiol [Ther.] (Stockholm) 1934; 15:89-99. 12. MuUer C. Therapeutische Erfahrungen an 100 mit kombination von Rontgenstrahlen un Hochfrequenz, resp. Diathermic behandeleten bosartigen Neubildungen. Munchener Medizinische Wochenschrift 1912; 28:1546-1549. 13. Nielsen OS, Horsman M, Overgaard J. A future of hyperthermia in cancer treatment? Eur J Cane 2001; 37:1587-1589, (Editorial comment). 14. Osinsky S, Ganul V, Protsyk V et al. Local and regional hyperthermia in combined treatment of malignant tumors: 20 years experience in Ukraine. Awaji Japan: The Kadota Fund International Forum 2004.

Physical Background and Technical Realizations of Hyperthermia

57

15. Dewey W C , Hopwood LE, Sapareto SA et al. Cellular response to combination of hyperthermia and radiation. Radiology 1977; 123:463-474. 16. Lindholm C-E. Hyperthermia and Radiotherapy. Ph.D. Sweden: Thesis, Lund University, Malmo, 1992. 17. Hafstrom L, Rudenstam C M , Blomquist E et al. Regional hyperthermic perfusion with melphalan after surgery for recurrent malignant melanoma of the extremities. J Clin Oncol 1991; 9:2091-2094. 18. Urano M. Thermochemotherapy: From in vitro and in vivo experiments to potential clinical application. In: U r a n o M , D o u p l e E, eds. H y p e r t h e r m i a and Oncology. U t r e c h t - T o k y o : VSP 1994:4:169-204. 19. Hasegawa T, Gu Y-H, Takahashi T et al. Enhancement of hyperthermic effects using rapid heatin. In: Kosaka M, Sugahara T, Schmidt KL et al, eds. Thermotherapy for Neoplasia, Inflammation, and Pain. Tokyo-Berlin: Springer Verlag, 2001:439-444. 20. Lindegaard JC. Thermosensitization induced by step-down heating. Int J Hyperthermia 1992; 8:561-582. 2 1 . Wust P, Hildebrandt B, Sreenivasa G et al. Hyperthermia in combined treatment of cancer. T h e Lancet Oncol 2002; 3:487-497. 22. Hayashi S, Kano E, Hatashita M et al. Fundamental aspects of hyperthermia on cellular and molecular level. In: Kosaka M, Sugahara T, Schmidt KL et al, eds. Thermotherapy for Neoplasia, Inflammation, and Pain. Tokyo-BerUn: Springer Verlag, 2001:335-345. 2 3 . Karasawa K, Muta N , Nakagawa K et al. Thermoradiotherapy in the treatment of locally advanced Nonsmall cell lung cancer. Int J Rad Oncol Biol Phys 1994; 30:1171-1177. 24. Kraybill W , Olenki T. A phase I study of fever-range whole body hyperthermia (FR-WBH) in patients with advanced solid tumors: Correlation with mouse models. Int J Hyperthermia 2002; 18:3, (253-266 and Burd R, Dziedzic ST. Tumor cell apoptosis, lymphocyte recruitment and tumor vascular changes are induced by low temperature, long duration (feverlike) whole body hyperthermiia. J Cellular Physiology 1998; 177:137-147). 25. Field SB. Biological aspects of hyperthermia. Physics and Technology of Hyperthermia. In: Field SB, Franconi C, eds. N A T O ASI Series, E: Applied Sciences, N o . 127. Dordrecht/Boston: Martinus Nijhoff Publ, 1987:19-53. 26. Szasz A, Szasz O, Szasz N . Electrohyperthermia: A new paradigm in cancer therapy. Wissenschaft and Forschung, Deutsche Zeitschrift fiir Onkologie 2001; 33:91-99. 27. de Pomarai D, Daniels C, David H et al. Nonthermal heat-shock response to microwaves. Nature 2000; 405:417-418. 28. Bukau B, Horwich AL. The HSP70 and HSP60 chaperone machines. Cell 1998; 92:351-366. 29. Feynman PR, Leighton RB, Sands M. The feynman lectures on physics. Reading and Caltech, MA and CA, USA: Addison-Wesley Publ Co.. 1963. 30. Katchalsky A, Curran PF. Nonequilibrium thermodynamics in biophysics. Cambridge: Harvard University Press, 1967. 3 1 . Lupis C H P . Chemical Thermodynamics of Materials. NewYork, Amsterdam, Oxford, North Holland: 1983. 32. Pennes H H . J AppUed Physiology 1948; 1:93-122. 33. Matay G, Zombory L. Physiological effects of radiofrequency radiation and their application for medical biology, [in Hungarian], Muegyetemi Kiado, Budapest, 2000:80. 34. Gautherie M. Temperature and blood-flow patterns in breast cancer during natural evolution and following radiotherapy. In: Alan R Liss, ed. Biomedical Thermology. New York, 1982:21-24. 35. ANSI C95.1-1966 (H. Schwan, Pennsylvania USA). 36. Lin H , Head M, Blank M et al. Myc-Mediated transactivation of HSP70 expression following explosure to magnetic fields. J Cell Biochem 1998; 69:181-188. 37. Goodman R, Blank M. Insights into electromagnetic interaction mechanisms. J Cellular Physiology 192:16-22. 38. Lin H , Blank M, Goodman R. A magnetic field-responsive domain in the human HSP70 promoter. J Cell Biochem 1999; 75:170-176. 39. Scholz B, Anderson R. O n Electrical impedance scanning—principles and simulations. Electromedica 68 - Onco 2000:35-44. 40. Barnett A. Electrical method for studying water metabolism and transaction in body segments. Proc Soc Exp Biol Med 1940; 44:142-147. 4 1 . Nyboer J, Bango S, Barnett A et al. Radiocardiograms - the electrical impedance changes of the heart in relation to electrocardiorganms and heart sounds. J Clin Invest 1940; 19:963-966. 42. McRae DA, Esrick MA, Mueller SC. Noninvasive, in-vivo electrical impedance of E M T - 6 tumors during hyperthermia: Correlation with morphology and tumour-growth delay. Int J Hyperthermia 1997; 13:1-20.

58

Hyperthermia in Cancer Treatment: A Primer

43. Esrick MA, McRae DA. The effect of hyperthermia induced tissue conductivity changes on electrical impedance temperature mapping. Phys Med Biol 1994; 39:133-144. 44. McRae DA, Esrick MA. The dielectric parameters of excised EMT-6 tumours and their change during hyperthermia. Phys Med Biol 1992; 37:2045-2058. 45. McRae DA, Esrick MA, Mueller SC. Changes in the noninvasive, in vivo electrical impedance of the xenograpfts during the necrotic cell-response sequence. Int J Radiat Oncol Biol Phys 1999; 43:849-857. 46. Dissado LA, Alison JM, Hill RM et al. Dynamic scaling in the dielectric response of excised EMT-6 tumours undergoing hyperthermia. Phys Med Biol 1995; 40:1067-1084. 47. Szendro P, Vincze G, Szasz A. Bio-response on white-noise excitation. Electro-and Magnetobiology 2001; 20:215-229. 48. Gersing E. Monitoring temperature induced changes in tissue during hyperthermia by impedance methods. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: Applications to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:13-20. 49. Haemmerich D, Staelin ST, Tsai JZ et al. In vivo electrical conductivity of hepatic tumors. Physiol Meas 2003; 24:251-260. 50. Smith SR, Foster KR, Wolf GL. IEEE Trans Biomed Eng BME 1986; 33:522-525. 51. Jossinet J. The impedivity of freshly excised human breast tissue. Physiol Meas 1998; 19:61-75. 52. Jossinet J, Schmitt M. A review parameters for the bioelectrical characterization of breast tissue. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: Applications to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:30-41. 53. Chauveau N, Hamzaoui L, Rochaix P et al. Ex vivo discrimination between normal and pathological tissues in human breast surgical biopsies using bioimpedance spectroscopy. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: AppHcations to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:42-50. 54. Tosso S, Piccoli A, Gusella M et al. Nutrition. 2000; 16:120-124. 55. Glickman YA, Filo O, David M et al. Electrical impedance scanning: A new approach to skin cancer diagnosis. Skin Res Techn 2003; 9(3):262. 56. TransCan TS2000, Transcan Medical Ltd. Migdal Ha'Emek, Israel, distributed by Siemens AG, Germany. 57. Skourou C, Hoopes PJ, Strawbridge RR et al. Feasibility studies of electrical impedance spectroscopy for early tumour detection in rats. Physiol Meas 2004; 25:335-346. 58. Chillcott TC, Coster HGL. Electrical impedance tomography study of biological processes in a single cell. In: Riu P, Rosell J, Bragos R et al, eds. The data Electrical Bioimpedance Methods: Apphcations to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:269-286. 59. McRae, Esrick MA. Deconvolved electrical impedance spectra track distick cell morphology changes. IEEE Trans Biomed Eng 1996; 43:607-618. 60. Bioelectric impedance analysis in body composition measurement. National Institute of Health, USA: Technology Assessment Conference Statement, 1994:12-14. 61. Bowen WD, Beck CA, Iverson SJ. Bioelectrical Impedance analysis as a means of estimating total body water in grey seals. Can J Zool 1999; 77:418-422. 62. Talluri T, Lietdke RJ, Evangelisti A et al. Fat-free mass qualitative assessment with bioelectric impedance analysis. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: Apphcations to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:94-98. 63. Goovaerts HG, Faes THJC, DeValk-DeRoo GW et al. Estimation of extracellular volume by two frequency measurement. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: Applications to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:99-104. 64. McRae DA, Esrick MA. Changes in electrical impedance of skeletal muscle measured during hyperthermia. Int J Hyperthermia 1993; 9:247-261. 65. Shchepotin IB, McRae DA, Shabahang M et al. Hyperthermia and verapamil inhibit the growth of human colon cancer xenografts in vivo through apoptosis. Anticancer Res 1997; 17:2213-2216. GG. Casas O, Bragos R, Riu PJ et al. In vivo and in situ ischemic tissue characterization using electrical impedance spectroscopy. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: Apphcations to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:51-58. G7. Schafer M, Kirlum H-J, Schlegel C et al. Dielectric properties of scaletal muscle during ischemia in the frequency-range from 50Hz to 200 MHz. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: Applications to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:59-64. 68. Gheorghiu M, Gersing E, Gheorghiu E. Quantitative analysis of impedance spectra of organs during ischemia. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: Applications to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:65-71.

Physical Background and Technical Realizations of Hyperthermia

59

69. Osterman KS, Paulsen KD, Hoopes PJ. Application of linear circuit models to impedance spectra in irradiated muscle. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: Applications to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:21-29. 70. Santini M T , Cametti C, Zimatore G et al. A dielectric relaxation study on the effects of the antitumor drugs Lomidamineand Rhein on the membrane electrical properties of Erlich ascites tumour cells. Anticancer Res 1995; 15:29-36. 7 1 . Keese CR, Wegener J, Walker SR et al. Electrical wound-healing assay for cells in vitro, PNAS, Proceedings. Nat Acad Sci USA 2004; 101:1554-1559. 72. Avitall B, Mughal K, Hare J et al. The effects of electrode-tissue contact on radiofrequency lesion generation. Pacing Chn Electrophysiol 1997; 20:2899-2910. 73. Schmidt D , Trubenbach J, Konig C W et al. Radiofrequency ablation ex vivo: Comparison of the efficacy impedance control mode versus manual control mode by using internally cooled clustered electrode. Rofo Fortschr Geb Rontgenstr Neuen Bildgeb Verfahr 2003; 175:967-972. 74. Szasz A, Vincze Gy, Szasz O et al. An energy analysis of extracellular hyperthermia. Magneto- and electro-biology 2003; 22:103-115. 75. Seegenschmiedt M H , Vernon C C . A Historical perspective on hyperthermia in oncology. In: Seegenschmiedt M H , Fessenden P, Vernon C C , eds. Thermoradiotherapy and Thermochemotherapy. Berlin: Clinical Applications, Springer Verlag, 1995:2:3-46. 7Ct. Blank M. Coupling of AC electric fields to cellular processes. First International Symposium on Nonthermal Medical/Biological Treatments Using Electromagnetic Fields and Ionized Gases, ElectroMed'99, Norfolk VA, USA, Symposium Record Abstracts, 1999:23. 77. Young RA. Stress proteins and immunology. Ann Rev Immunology 1990; 8:401-420. 78. Jackson J D . Classical Electrodynamics. New York: John Wiley and Sons Inc., 1999. 79. Rao N N . Elements of engineering electromagnetics. London: Prentice Hall International, 1994. 80. Matay G, Zombory L. Physiological effects of radiofrequency radiation and their application for medical biology, [in Hungarian], Muegyetemi Kiado, Budapest, 2000:71. 8 1 . Polk C, Postow E. Handbook of biological effects of electromagnetic fields. New York, London, Tokyo: C R C Press, 1996:15. 82. Tremoliers J. Effects biologiques des champs electromametiques non ionisants. Electron Appl 1978; 7:71-77. 83. Chatterjee I, Hagmann M, Gandhi O . An empirical realtionship for electromagnetic energy absorption in man for near-field exposure condition. IEEE Trans O n Microwave Theory and Techniques, M T T - 2 9 , 1981; 11:1235-1238. 84. Chain C. A theoretical basis for microwave and RF field effects on excitable cellular membranes. IEEE Trans O n Microwave Theory and Techniques, M T T - 2 9 , 1980; 2:142-147. 85. Iskander M, Olson S, MacCalmont J. Near-field absorption characteristics of models in the resonance frequency range. IEEE Trans O n Microwave Theory and Techniques, M T T - 3 5 , 1987; 8:776-779. 86. Kotnik T, Miklavcic D . Theoretical evaluation of the distributed power dissipation in biological cells exposed to electric field. Bioelectromagnetics 2000; 21:385-394. 87. Galeotti T, Borrello S, Minotti G et al. Membrane alterations in cancer cells: The role of oxy radicals. In: Bianchi G, Carafoli E, Scarpa A, eds. Membrane Pathology. Ann New York Acad Sci 1986:488:468-480. 88. Hager ED, Dziambor H, Hohmann D et al. Deep hyperthermiawith radiofrequencies in patients with liver metastases from colorectal cancer. Anticancer Res 1999; 19:3403-3408. 89. Hager D, Dziambor H , App EM et al. ASCO 2003 Meeting, Chicago USA, 2003:470. 90. Dani A, Varkonyi A, Nyiro I et al. Clinical experience of elecctro-hyperthermia for advanced pancreatic tumors. E S H O 2003 Conference, Munich 2003:41. 9 1 . Hodgkin AL, Huxley A. A quantitative description of membrane current and its application to conduction and excitation of nerve. J Physiol 1952; 117:500-544. 92. Weiss TF. Cellular Biophysics. Cambridge, MA, USA: A Bradford Book, The M I T Press, 1996. 93. Spanner D C . Symp Soc Exptl Biol 1954; 8:76. 94. Sackmann E. Physical basis of self-organisation and function of membranes: Physics of vesicles. In: Lipowsky, Sackmann E, Elsevier Science BV. eds. Handbook of Biological Physics 1995:1. 95. Vujaskovic Z, Jones EL, Oleson JR et al. A Randomized trial of hyperthermia and radiation for superficial tumors. Awaji Yumebutai, Japan: Presentation and Abstracts for The Kadota Fund International Forum, 2004.

CHAPTER 4

Thermotherapy and Nanomedicine: Between Vision and Reality Andreas Jordan* Summary

A

lthough nanoparticles have been already applied on patients in clinical trials, generally nanotechnology in medicine is regarded rather a vision than a realistic option. Progress in this field arises particularly from the combination of molecular biology and nano(bio) technology. From the viewpoint of entrepreneurs nanotechnology is only a tool to develop new products, however nanotechnology itself is not a product. We developed a new cancer treatment platform technology termed MagForce Nanotherapy, in which nanotechnology has the potential to cause a revolution in tumor therapy.

Introduction The idea of traveling through the vessels of a body in a "nanobot"* to heal diseases from "the inside", as shown in the Oscar-awarded movie "Fantastic Voyage" (Stephen Boyd, 1966), is indeed quite attractive, but unfortunately it also inherits general difficulties, especially in combating cancer. Theoretically, an ingeniously built nano-vehicle, controlled from outside the body, coiJd move through vessels. Nevertheless, the fantasy story would end very fast, because the inmiune system of the human body would quickly destroy the submarine, as it does enduringly with bacteria, viruses and other foreign particles. But even if this problem could be solved, the submarine woidd still not know how to destroy cancer cells selectively while sparing normal cells. Hereto defined molecules on the surface of tumor and normal cells ("targets") have to be identified for distinguishing between these cells. A solid tumor consists of a number of different sub-populations, whose genomes are different from each other expressing those targets or not. Therefore not all cells of the tumor are recognized by their specific target molecules, which build the source of recurrent, often multi-resistant tumor growth. Even the mixture of different target recognizing molecules is not a guarantee that all tumor cells are affected. This general problem cannot be solved by any "nanobot" approach. How we get along with this new knowledge without "nanobots",fixturewill tell. Answers to these problems may rather derive from research in the fields of molecular biology, where certain success has already been obtained concerning different tumor entities. Boundaries between nanotechnology and molecular biology blur. It is of general acceptance, that in the future even single molecules and atoms are to be controlled, and then nanotechnology will probably gain the same importance that molecular biology has today. As one of the first applications of nanotechnology in medicine the group of the authors developed a worldwide new Nano-cancer-therapy in more than 15 years of fundamental •Andreas Jordan—MagForce Nanotechnologies GmbH and Center of Biomedical Nanotechnology (CBN), Department of Radiology, Charite - University Medicine Berlin, Spandauer Damm 130, 14050 Berlin, Germany. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

Thermotherapy and Nanomedicine

61

research at the Charitd - University Medicine Berlin. Via foundation of the MagForce® Nanotechnologies GmbH in Berlin, research lead to products, which have already been tested in clinical trials and which are already requested by numerous cancer clinics, even before their approval. The principle of the method termed MagForce Nanotherapy is simple: Iron-oxide nanoparticles are directly injected into the tumor and release heat after inductively induced activation by an alternating magnetic field. Despite this simple-sounding approach, it was nanotechnology to be the key for realizing this new cancer-therapy: • Only nanoparticles extract high energy per applied mass from a magnetic field • Due to their enormous surface only nanoparticles are able to carry a huge number of binding sites for cancer cells /target molecules • Only nanoparticles are able to intrude deeply into tumor tissue • Only nanoparticles with special coatings • are recognized delayed by the immune system and thus reach their targets • can be ingested in great quantities by tumor cells • form a homogeneous fluid of low viscosity in water • remain in the tumor tissue even after interstitial application for a long time and are not being washed out So far, the MagForce Nanotherapy is, in a first step, a new form of local thermotherapy of deep-seated tumors. Clinical trials in this field done so far demonstrated a good feasibility and tolerability of the new technique. Later the nanoparticles are supposed to function as transport vehicles for medical agents, isotopes or genes. MagForce is doing research in this field for years now, predominantly in cooperation with the Leibniz-Institute for New Materials (INM), Saarbriicken, Germany and different departments of the Charit^ - University Medicine, Berlin, Germany. The MagForce Nanotherapy offers the possibility of repeated heat treatments of basically every region of the body very precisely without repeated application of the particles. Intratumoral temperatures can be varied according to clinical requirements between hyperthermia (up to 45°C for supporting radiochemotherapy) and thermoablation with temperatures of up to 70°C. The method is based on the defined power transfer to biocompatible iron-oxide nanoparticles in an alternating magnetic field. The patented nanotechnological design of the MagForce nanoparticle shell leads to preferred intracellular absorption into proliferating cells like tumor cells (Fig. 1). The particles function as "Trojan horses", thus destroying tumor cells, whereas healthy tissue is spared. Particles generate heat by relaxation processes of the particle core and emit it into the surrounding tissue.^ Thermotherapy is performed in a specially designed magnetic field applicator (Fig. 2). Due to its construction and safety standards, the system can be applied on diff^erent tumor entities in every region of the human body. To date, the MagForce Nanotherapy is being investigated only at the Charit^ - University Medicine Berlin, Germany. Treatment modalities are shown in Figure 2. In early 2007 the new method is supposed to be available for all clinics in Europe. From March 2003 to July 2004, the worldwide first feasibility study on thermotherapy using magnetic nanoparticles was performed on 14 patients with glioblastoma multiforme. In Germany, more than 2000 patients (Incidence: 3/100,000) die on this aggressive tumor every year. Median overall survival after first-line therapy does not exceed 12-15 months and no significant increase has been achieved over the last decade, despite modern diagnostics and treatments with surgery, radiotherapy and chemotherapy. ' The therapy was tolerated well by all patients, and in all cases intratumoral temperatures of 42-45°C could be achieved, even in deep-seated tumors. Signs of local efficacy could be observed in all patients. Detailed results will be published soon.

62

Hyperthermia in Cancer Treatment: A Primer

Figure 1. Intracellular uptake of MagForce Nanoparticles in a human mammary carcinoma cell-line. Tumor cells marked like this can later be identified by MRI scans^ (electron micrograph from ref. 2).

Figure 2. A patient suffering from glioblastoma multiform during treatment in the magnetic field applicator (MFH® 300F, MagForce Nanotechnologies, Berlin, Germany).

Thermotherapy and Nanomedicine

63

Subsequent to this trial, an efficaq^ study with 65 patients, suffering from recurrences of glioblastoma multiforme or anaplastic astrocytoma started in January 2005. Another trial started in February 2004 with 25 patients suffering from local tumor recurrences without distant metastases (e.g., sarcoma, cancer of the rectum, prostate-, ovarian- and cervix carcinoma) and who had received all kinds of conventional therapies before. These patients received repeated thermotherapies in combination with radiotherapy (afterloading method). Also in this study, positive results were obtained concerning feasibility and tolerability of the technique. Desired intratumoral temperatures were obtained and clear signs of local efficacy could be observed. Beginning in May 2004 a feasibility study with patients suffering from local recurrences of prostate carcinoma is now nearing completion. Other trials are in preparation.

Further Information http: //www. magforce. de

Acknowledgements The Center of Biomedical Nanotechnology (CBN) at the Charit^ Berlin, Germany is cofinanced by the European Community, European Fund for Regional Development (EFRE)Project NanoMed. The Federal Ministry of Education and Science (BMBF), Projekttrager Jiilich (PTJ), and Verein deutscher Ingenieure (VDI) within the framework of the support program Nanobiotechnology (Project MNC, TAN) granted project support.

References 1. Jordan A, Wust P, Fahling H et al. Inductive heating of ferrimagnetic particles and magnetic fluids: Physical evaluation of their potential for hyperthermia. Int J Hyperthermia 1993; 9:51-68. 2. Davis FG, Freels S, Grutsch J et al. Survival rates in patients with primary malignant brain tumors stratified by patient age and tumor histological type: An analysis based on Surveillance, Epidemiology, and End Results (SEER) data, 1973-1991. J Neurosurg 1998; 88:1-10. 3. Stupp R, Mason W P , Van Den Bent MJ et al. Concomitant and adjuvant temozolomide (TMZ) and radiotherapy (RT) for newly diagnosed glioblastoma multiforme (GBM). Conclusive results of a randomized phase III trial by the E O R T C Brain & RT Groups and N C I C Clinical Trials Group. J CHn Oncol, 2004 A S C O Annual Meeting Proceedings (Post-Meeting Edition) 2004; 22:2. 4. Pinkernelle J, Teichgraeber U, Neumann F et al. Imaging of single human carcinoma cells in vitro using a clinical whole body mr scanner at 3.0T. Magn Reson Med. 2005 May;53(5):l 187-92. 5. Stupp R, Mason W P , van den Bent MJ et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005; 352:987-996.

SECTION II

Biological Aspects ofHyperthemiia

CHAPTER 5

Influence of Tumor Microenvironment on Thermoresponse: Biologic and Clinical Implications Gian Franco Baronzio,* Alberto Gramaglia, Attilio Baronzio and Isabel Freitas Abstract

S

olid tumours tend to have a more acidic and hypoxic microenvironment than normal tissue. This hostile microenvironment results from a disparity between oxygen supply and demand of the tumor tissue. Overcoming hypoxia tumor induces a new vascular supply. This new vasculature is however inefficient and chaotic. It perpetuates the factors that have stimulated its induction. This review focuses on these processes and peculiarly on angiogenesis, tumor vascular morphology, hypoxia, pH, and the metabolic-vascular events induced or following tumour tissue heating. The various mechanisms that either modulate tumor microenvironments or blood perfusion during hyperthermia are described, providing also the many clinical modalities that may enhance or sensitize cancer cells to heat.

Introduction: Tumor Microenvironment Human solid neoplasia should be regarded as an intricate, yet poorly organized "organoid", whose function is maintained by a dynamic interplay between neoplastic and host cells. ^'^ This interplay constitutes the tumour metabolic microenvironment, defined by Vaupel as a complex pathophysiological entity resulting by the interactions of self-influencing factors, which go hand in hand: tumor perfusion, tumor oxygenation status, pH distribution and metabolic bioenergetic status.

Hypoxia, HIF-1 and Angiogenesis Hypoxioy HIF'l The growth of tumours beyond a critical mass >l-2 mm^ (10^ cells) is dependent on adequate blood supply. '^ Up to a distance from host vessel of 100-200 |im the initial foci of neoplastic cells receive their nutrients and oxygen by diffiision. Beyond this distance, hypoxia occurs and the need of adequate blood supply is crucial. ^'^ However, the establishment of this neovascular supply in the attempt to overcome hypoxia is inefficient and irregular. It may not occur at the same rate as the proliferation of the tumour. The result is the persistence within the tumour mass of •Corresponding Author: Gian Franco Baronzio—Family Medicine Area, ASL-01 Legnano; Radiotherapy Unit, Policlinico di Monza, Via Amati 11, 20052 Monza (Mi), Italy. Office address: P.O.B. 5, 20029 Turbigo (Mi), Italy. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer^ edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

68

Hyperthermia in Cancer Treatment: A Primer

Figure 1. Photomicrograph of a section of mammary tumor, developed by a 5 month old female transgenic MMTV-neu (erbB2) mouse. T: tumor cord; N = necrosis; Hyp: hypoxic, perinecrotic, rim of tumor cells; C: capillary. Hematoxylin and Eosin. 40x objective. heterogeneous microregions of nonproliferating hypoxic cells, which are surrounded by vital well nourished and proliferating cells (Fig. \).^ Several methods to measure hypoxia are currendy available and have demonstrated the presence of hypoxic cells both in experimental and in human tumours.^ The hypoxic microenvironments are characterized by low oxygen tension, low extracellular pHe, high interstitial fluid pressure, glucose deficiency, multidrug resistance, increased extracellular lactate concentration and tendency to metastasization. The formine of new blood vessels depends on a balance between angiogenesis inhibitors and promoters. '^ Tumor regional hypoxia and hypoglycaemia are the principal stimulators for the expression of local proangiogenic cytokines, especially vascular endothelial growth factor (VEGF) (Fig. 2).^'^ ^ The early response gene that produces hypoxia inducible factor-1 (HIF-1) and its subunits (HIF-1 a and -Ip) regulate VEGF expression. HIF-1 is a protein of 120 KDa, member of the basic helix-loop-heltx superfamilv transcription factors, and its expression is very sensitive to oxygen concentration (1% 02). ^'^^ The adaptation to hypoxia by earlier proliferating neoplastic cells results in the induction of genes that regulate the anaerobic metabolism, nitric oxide synthase and the angiogenesis process.^'^^ Recent studies have shown that HIF-1 and VEGF transcripts are overexpressed by several human neoplastic cells including breast, prostate, gastric, colon, lung, bladder and endometrium and they are more active in hypoxic and necrotic areas. ^ ^^ VEGF is correlated to vascular density, especially in brain tmnors and it is associated with bad prognosis. ^^ VEGF or vascular permeabilin^ factor (VPF), is a 32 to 44-KDa multifunctional potent stimulator of endothelial cells. ^^' It becomes active by binding to three high afFmity tyrosine kinase receptors [VEGFR-l(flt-l), VEGFR-2(KDR/Fik-1) andVEGFR-3(Flt-4)], diat are highly expressed

Influence ofTumorMicroenvironmenton Thermoresponse

69

OROWTH FACTOR & CYTOKINES (FDGF, TGf^, iL1^,TNFa, iLe4t8,IL10) '"

l,weHE«2tori^(fMHi)}

^

TUMOR BLOOD FLOW

INFLAMMATORY REACTION

PERITUMORAL INFLAMMATORY REACTION

j BLOOD STASIS

Platelet aggregation leukocyte sticking

, , i::::: Figure 2. In this figure the structural and functional eflFects of Hypoxia, HIF-1 and VEGF on tumor microcirculation, cancer metabolism and therapies are illustrated. The vicious circles that occur are also shown. (Modified with permission fi-om: Baronzio et al. Anticancer Res 1994; 14:1145-1154.)

on endothelial tumour vessels but not on mature vessels. They exert different effects on endothelial cells (ECs). VEGFR-1 mediates cell motility of ECs, whereas VEGFR-2 regulates vascular permeability and VEGFR-3 lymphoangiogenesis.^^'^ '^^ Hypoxia appears to be the principal stimulus for the production and the stabilization of VEGF and VEGF mRNA, but recent evidences suggest that VEGF expression has increased in many tumours, as well in absence of hypoxia. This increase results from at least three factors: (A) loss of function of some tumour suppressor genes (such as the von Hippel-Lindau (VHL), p53, pl6); (B) activation of oncogenes (including raf, ras,FiER2/erb2 (neu) and src '^^"^'^) (C) excessive quantity of growth factors, produced by tumour cells and their supporting network

70

Hyperthermia in Cancer Treatment: A Primer

Table 1. Structural and functional abnormalities of tumor vasculature Decreased vascular density Heterogeneous distribution of microvessels Increased presence of sinusoids, dead ends and arterio-venous anastomosis Lack of arteriolar vessels Decreased perfusional pressure and increased geometric resistance absence of lymphatics, increased TIF, hemoconcentration, increased viscosity Incomplete basement membrane and absence of smooth muscle Decreased stabilization for absence of pericytes and mural cells Decreased leukocyte-endothelium adhesion Absence of innervation and regulation according to metabolic demand

Tumor Neovascularization The acquisition of new in-growing vessels may occur by different mechanisms. ECs are normally quiescent and tightly regulated by a delicate balance between proangiogenic and antiangiogenic molecules. '^^ In presence of an excessive secretion of angiogenic molecules by tumours, ECs are stimulated and they organize themselves in vessel structure through multistep sequential and distinct processes, depending on tumour type and anatomic localization. These processes include vessel Cooption, vasculogenesis and angiogenesis. Vessel-Cooption

It is the process in which tumours take up preexisting normal blood vessels and use them for their initial growrth. As just described, this is a limiting process and irrelevant in the great majority of solid tumours; in fact, cancer cells grow until oxygen demand exceeds supply and the distance from host vessels is lower than 100-200 jlm. Vasculogenesis

It is the mechanism in which precursors endothelial ECs from bone marrow are recruited by the tumor and aggregate to form new blood vessels. Recent studies have demonstrated this process in experimental animal tumours, but its relevance in human neoplasia is not fully elucidated.2^-^5 Angiogenesis Upon adequate stimulus, endothelial cells begin to sprout from preexisting capillaries and after the degradation of the extracellular matrix (ECM) by matrix metalloproteases, and the expression of adhesion molecules such as avp3 integrin, migrate and organize themselves in capUlary tube formation and ultimately in a vascular network '^

Tumor Vascular Morphology-Perjusion and Hypoxia Blood vessels associated with the tumor tend to be significantly different in architecture from the surrounding normal tissue (see Table 1)^^'^^ and they show, in presence of VEGF and other cytokines, a decreased expression of leukocyte-endothelial cell adhesion molecules (ICAM-1,VCAM-1, E-selectin ) and an enhanced expression of CD 44?^'^^ The decreased expression of adhesion molecules reduce significantly leukocytes and natural killer cells (NKs) recruitment, partially contributing to the phenomenon of immune-evasion,^^'^^ whereas the enhanced expression of CD44 may confer a growth advantage on many neoplastic cells. Moreover, VEGF induces tumor neovessels to become leakier and to lose a large quantity of fluids (proteins and other circulating macromolecules) towards the interstitium. Fluid accumulated inside the tumor interstitium, knovm as tumor interstitial fluid (TIF), occupies from 30% to 60% of the tumour volume and compared to normal fluids it has a different biochemical nature.^ '^^ TIF retention causes an increase in tumor interstitial pressure (TIFP)

Influence of Tumor Microenvironment on Thermoresponse

71

(Fig. 2).^^"^^ TIFP increase goes from tumour centre towards periphery, reaching the value of 50 mm Hg, as Jain and coworkers measured in human and experimental tumours on colon, breast, head-neck carcinomas and metastases specimens from lung, liver and lymph nodes. °" "^ Tumor blood flow (TBF) is determined by arteriovenous pressure difference (A Pa-v) divided by a factor T| for Z, where Tj expresses the flow resistance or blood viscosity and Z the geometric resistance.^ TBF=APa-v/TlZ

(1)

Normally in tumors, A Pa-v is lower than in normal microcirculation. This is caused by the increased number of arteriovenous fistulae present at tumor periphery, that creates a low resistance pathway at tumour surface and diverts blood from entering the tumour mass. Indicator dilution methods, angiography and experimental studies have confirmed this anomalous behaviour.^"^'^^ Associated to low perfusional tumour supply, Sevick and Jain have found that viscosity parameters Z and T| were abnormally high on tissue isolated mammary Adenocarcinoma (R3230AC) and on 22 carcinosarcoma. The geometric resistance Z is a complex function of vascular morphology (i.e., number of blood vessels, branching pattern, diameter, length and volume), and increases according to tumor size, length and vessel tortuosity. ^ Tumor neovessels are tortuous, heterogeneous, inefficient and devoid of hierarchisation, as confirmed by different Authors using biochemical, corrosion cast and electronic microscopic studies.^^'"^^ Tumor blood viscosity T| has been found elevated and correlated with the shear rate, hematocrit and circulating blood cells deformability. ' Normally capillary diameter ranges from 3.5 to 20 |xm, so at some point along their travel in the capillaries, red blood cells (RBCs) as well as white cells (WBCs) have to undergo deformation to pass through. By contrast in the tumor microvasculature, various factors can modify RBCs and WBCs deformability. Particularly the low oxygen partial pressure, the acidic pH, and the increased concentration of fibrinogen tend to make red blood cells and leucocytes less deformable, and more sticky, thus easily trapped intravascularly and blocking TBF.^ A further modifier factor of viscosity is the local increase of the hematocrit. Several authors^ '^^''^' have demonstrated that this phenomenon is to be ascribed to the fluid loss operating in tumor capillaries with a consequent local hemoconcentration and further worsening of blood flow. Blood flow measurements in human cancer show heterogeneous values going from highly vascularized organs, such as brain, and poorly vascularized, such as adipose tissue. Perfusion flow at tumour level is higher or lower than in the tissue of origin, depending on the physiological state of the latter. ' ^ It is higher at the tumour periphery than in the central zone and generally primary tumours are better supplied than metastatic lesions. In most studies, perfusion on rodent tumours decreases with tumour size when compared to normal tissue. However, in the minority of experiments the decreased flow was not confirmed even in a similar tumor type. Several pathophysiological mechanisms have been proposed to explain this difference such as transplantation site, stage of tumor growth, flow registration and recording methods.^ TBF is not regulated according to metabolic demand as in the case of normal tissues. This decreased metabolic adaptability to cells associated to an irregular blood availability (perfusion) produce a clusters of cells, lacking nutrients and oxygen (hypoxic cells). Two kinds of hypoxic or clusters of cells in situation of low energy state have been recognized: (A) Diffusion-limited or chronic hypoxic cells; (B) Transient or acute hypoxic cells. The two types of hypoxia have different origin and coexist together in a well-perfused zone of the tumour mass too, causing a functional disturbance of macro and microflow. '^ A more realistic vision shows that these two situations change continuously, because tumour blood flow is time fluctuating."^^"^^ Chronic hypoxia is the result of availability of nutrients and oxygen towards the tumoral tissue, the diffusion in the extracellular space and the respiration rate of cancer cells. ' It has been calculated and observed that when cancer cells are > 100-200 jim away from functional blood supply become hypoxic and suffering (Figs. 1,3).

72

Hyperthermia in Cancer Treatment: A Primer

Figure 3. Photomicrograph of a section of mammary tumor, developed by a 5 month old female transgenic MMTV-neu (erbB2) mouse. T: tumor cord; N: necrosis; L: lymphatic vessel; C: capillary. Hematoxylin and Eosin. 20x objective. Cells adjacent to capillaries displayed a mean oxygen concentration of 2%, located at 200 P-m displayed a mean oxygen concentration of 0.2%.^ The distance from the nutritive vessels, the haemoglobin concentration and the blood flow crossing that tumour area are the only parameters responsible for chronic hypoxia. In fact, the removal of O2 by tumour cells, has been calculated to be efficient or better than the one of normal tissues, revealing that neoplastic cells in vivo do not have impaired ability to utilize oxygen as proposed in the past. Acute hypoxia is the restdt of intermittent opening and reopening of tumor blood vessels. Among the various factors responsible for this temporarily blood flow stop, two of them seem the most plausible: A. TIFP combined with the irregular expansion of tumour mass, whose three dimensional growth is subjected to a continuous remodelling in a confined space and it causes a temporary compression or occlusion of some tumour capillaries. ^'^^'^^ B. transient stop of tumor bloodflowor supply by platelets plug (see Fig. 4).5''^7 In our opinion, this intravascular thrombosis deserves to be taken into much higher account than usually done. ^ In fact, the majority of cancer patients have coagidation abnormalities associated to hypoxia. ^ Recently, it has been demonstrated that hypoxia not only induces VEGF but also stimulates endothelial cells to over express tissue factor (TF) and Plasminogen activator inhibitor (PAI-I). These factors induce endothelium to become prothrombotic and causefibrinformation and platelet activation.^^ Furthermore, VEGF binds tofibrinogenand fibrin by stimidating endothelial cell proliferation.^^ Fibrin has been demonstrated to be essential for supporting endothelial cells spreading and migration.^ The haemostatic system, in a certain sense, becomes a regulator of angiogenesis and it can partially explain acute hypoxia and its regional appearance and disappearance.^ Concluding hypoxia becomes a

Influence ofTumorMicroenvironmenton Thermoresponse

73

Figure 4. Photomicrograph of a platelet thrombus in a peripheral blood smear of a mouse bearing an Ehrlich carcinoma implanted on the hind leg. MGG staining. X 63 objective. Differential interference contrast.

self-perpetuating mechanism able to trigger angiogenesis, intratumoral fluid accumulation and thrombosis (Figs. 2, 4)."^^

Tumor Bioenergetic Status, Hypoxia, pH Normally cancer cells display many altered metabolic abnormalities including an increased capacity to metabolise carbohydrates mainly by anaerobic glycolysis even under aerobic conditions. This metabolic behaviour results from the induction of many enzymes* involved in the intermediate reactions of glycolysis by HIF-1 genes. ^"^'^^ The relevance of these alterations is that oxidation of glucose stops at the stage of pyruvic acid and proceeds anaerobically producing for the same ATP amount six times more lactic acid and H^ than normal cells. The excessive lactic acid and H^ accumulation in tumour milieu, matched with the compromised interstitial fluid transport, causes the decrease of the external tumor pH (pHe).55-5^ Recently studies indicate that both local glucose-glutamine and oxygen availabilities affect tumour acidity independently. In the authors' opinion these findings have significant implications for cancer treatment.^^ Most pH estimations in tumour tissues were obtained by the insertion of microelectrodes.^^' They demonstrated the value of pHe in a range between 5.6 to 7.G (normal tissue pHe is 6.8) and they pointed out that tumors grow in an acidic environment. However for many years microelectrodes measurements were inaccurate and the calculated pHi was thought to be acidic. Recently the advent of ^^ P magnetic resonance spectroscopy, noninvasive method

*Enzymes HIP induced: [GLUT-1, type I exokinase, Aldolase A, Lactate dehydrogenase, Phosphofructokinase L, Posphoglycerate kinase 1 and pyruvate kinase M].

74

Hyperthermia in Cancer Treatment: A Primer

Table 2. pH regulatorsand inhibitors pH Regulators

pH Inhibitors

1) V-ATPase (Vacuolar proton pump)

1A) IB) 2A) 2B) 2C) 3A) 3B) 4A) 4B)

2) Lactate/H'^Symport [MCT]

3) NAVH-'exchanger [NHE] 4) CL/HCOa-[BCT]

Bafilomycin Oximidine a-cyano-4-hydroxylcinnamic acid [CNCN] Lonidamine Quercetin Amiloride Cariporide Cariporide 4,4'-diisocyanatostil-bene-2, 2'-disulfonic acid [DIDS]

Mitochondrial Metabolic Inhibitors 1) Lonidamine 2) Metaiodobenzylguanidine [MIBG] of measurement, has permitted to measure pHi and p H e simultaneously more accurately. Associated to these measures other parameters of interest can be obtained and are: tissue perfusion and vessel permeability. These studies have shown that in a majority of animal and human tumors p H e is lower than pHi. In fact, the tumor pHi resulted, similar to that normal tissue or near neutrality (i.e., ± 0 . 1 to 0.2 p H units) whereas p H e obtained from different human tumors were 0.41 ± 0.27 units lower than the normal tissue one.^^'^^ T h e neutral p H i and p H gradient with the acidification of tumor milieu can be explained by the following factors: A. In normal tissue the lactic acid, accumulated in the interstitial fluid, is rapidly removed through lymphatic drainage, whereas in TIF is not so easily removed (due to a compromised vasculature and a absent lymphatic drainage).^^ B. Cancer cells, as normal cells, express a number of pH regulatory mechanisms to maintain a cytosol pH near neutrality (pHi = 7 A). The mechanisms underlying regulation of intracellular pH have been identified as their inhibitors and are illustrated and listed in Table 2 and Figure 5.^^ A brief description of the most important exchanger mechanisms active in cancer cells and their inhibitors is reported: 1. Vacuolar-type H* ATP-ase is a ion transporter regulated by ATP-dependent mechanism. 2. H+-lactate cotransport Na ^ / H * exchanger [MCT],is an exchanger mechanism revers ibly blocked by Amiloride and quercetin. 3. Na^dependent C 1 7 H C 0 3 ' exchanger [BCT], is blocked by disulfonic stilbene derivative 4,4'-diisothiocyanostilbene -2,2' disulfonic acid [DIDS] 4. Sodium-proton exchanger [NHE]. The N H E family of ion exchangers includes six isoforms (NHE1-NHE6) that function in an electroneutral exchange of intracellular H+ for extracellular Na^ They are blocked by Amiloride and its derivative Cariporide. 5,6. electrogenic N a ^ - H C 0 3 ' cotransport T h e lactate efflux and formation can be also blocked by metabolic inhibitors such as: lonidamine or by meta-Iodio-benzylguanidine (MIBG) or alpha-cyano-4-hydroxy-cinnamic acid ( C N C n ) . These drugs are active on mitochondrial generation of lactic acid by blocking Krebs cycle) (Table 2 and Fig. 5).

Influence of Tumor Microenvironment on Thermoresponse

75

GLUCOSE 10% Lonidaminie Quercetin CNCn

KREBS CYCLE

AMILORIDE CARIPORIDE

/5^

4i3 DiDS

fv-ATPaseJ

pHi>7

ypHe<6.8

Na-»I t HC03Na

Figure 5. In this diagram the pH regulatory mechanisms and the inhibitors used to acidify in a acute way the intracellular environment are illustrated. 1) vacuolar -type H^ ATP-ase; 2) H*- lactate cotransport Na^/ H exchanger [MCT], 3) Na^-dependent C17HC03' exchanger [BCT], 4) Sodium- proton exchanger [NHE], 5,6) electrogenic Na^-HC03'cotransport.

Effects of Hyperthermia on Metabolism Recent studies have emphasized that metabolic alterations often follow hyperthermia. Mueller-Klieser et al, using bioluminescence and photon counting methods ftjr imaging in situ the metabolites used by tumour tissues, have shown an enhanced glycolytic activity ft)llowed by an increase in lactate levels upon heating application. The higher glucose and lactate concentrations may be the result of a temporarily increase in blood flow and an expansion of interstitial tumour compartment. Furthermore, the intensified acidosis with the enlargement of hypoxic tumor areas that follows the transient decrease in tumor perfusion application sensitize and facilitate tumor cell destruction by hyperthermia (Fig. G).^^

Effects ofpH on Hyperthermia Since Grays studies in the fifties (1955), it has been demonstrated that hypoxic and acidic regions in solid human tumors are common. These cells, chronically exposed to low extracellular pH, are relatively resistant to ionizing radiation but they tend to be markedly sensitive to the thermal damage. '^^ Earlier studies, in vitro conditions, ascribed this effect to the low p H of tumor milieu (pHe) and showed that thermosensitization was more evident at mild temperatures (e.g., < 42°C) rather than at higher temperatures. Gerweck and coll have demonstrated that environmental parameters such as hypoxia and acidosis, can strongly modify the cellular hyperthermic response. The Chinese Hamster Ovary (CHO) cancer cells survival curves

76

Hyperthermia in Cancer Treatment: A Primer

INITfAL VASODILATATION IN TUMOR VESSELS

of »fidoth»Hum

' ,

f \

Va^Bttiar

|

pemwabitlty j

Fluid leakage

I

TIFP TfF

k f

ANAEROBIC ^GLYCOLYSIS

INDIRECT EFFECT DIRECT EFFECT

Figure 6. In this figure the effects of HT on hypoxic cells, on tumor microcirculation and on surrounding normal tissue are illustrated. In bold black are shown the direct effeas of HT, whereas the indirect effects are shown in gray. (Modified with permissionfrom:Baronzio et al. Anticancer Res 1994; 14:1145-1154.) in vitro have clearly evidenced that the oxygen enhancement ratio (OER) for hyperthermic cell kill is « 1 in contrast with 2.5 to 3.0 for radiation inactivation (Fig. 1)1^ From these studies the authors concluded that hypoxic cells appeared to be slightly more sensitive to hyperthermia than oxygenated cells. As hypoxic cells live in acidic environments, these authors studied the same C H O cells in vitro at different pH of medium (see Fig. 7). They concluded, in accordance with other authors, that exposure of tumor cells to hyperthermia at conditions of pH below 7.4 enhanced cell lethality. Such increased cell sensitivity, often larger by a factor

Influence of Tumor Microenvironment on Thermoresponse

1.0 ^^5:%^,^^ ^^Sw 0.37

77

PE = AEROBIC = 0.77 PE = HYPOXIC = 0.92

0.10 NA^AEROBIC 0.037 HYPOXIC-A

\

0.01 CO

0.037 0.001 0.00037 0.00011 0

1

1

4

7

t

t

10 13

t

1

16

19

1

1

MIN OF HEAT TREATMENT Figure 7. OER curves of Chinese hamster ovary ceils (CHO) treated with hyperthermia. Hypoxia was induced 10-20 min prior to treatment. (Suit an Gerweck, Cancer Res 1979; 39:2290-2298. Reprinted form re£ 70 with permission.)

of lO"^ at some dose level, continued even during the development of thermotolerance.^^'^^ For clinical purposes, Gerweck and Richards^ ^ have introduced the concept of "pH enhancement ratio" that is the ratio of the inactivation rates at pFie G.7 vs. pH 7 A. This pH range has been calculated in vitro on different tumor cell lines and according to these authors, it is the only value among which pH sensitisation takes place.'^^ These authors refer that the pH sensitising effect is manifest over a pH range which is observed in tumor tissue, i.e., pH G.G to 7.0 (Fig. g\ 68.70.71

However, heat sensitivity of cancer cells seems to be limited when they are exposed to a chronic low pHe-^"^ In fact, Hahn and Shiu,^^ after studying CHO cells in vitro, maintained in acidic medium for different time before the exposition to heat treatment, have demonstrated that cells exposed to a low pH for prolonged periods were less sensitive to heat than cells exposed to an acidic medium shortly before heating. Other authors have confirmed these observations and have shown that cancer cells exposed for long period to low pH milieu, are less responsible to heat treatment and have a higher pHi compared to a brief period adapted one.^^'^^'^^ Other experimental studies have demonstrated that it is sufficient an acute reduction of pHe below 7.0-7.2 to increase the hyperthermia damage and to decrease thermotolerance. These studies in vitro have been performed under conditions where pHe was rapidly altered, a condition that does not correspond to one in vivo conditions where low pHe is gradually achieved and the cells are exposed to an acidic environment for more prolonged periods, the so-called chronic hypoxic cells. These phenomena can explain the less dramatic effects on hypoxic cells obtained on patients after a treatment of hyperthermia as reported by van der Berg.'^'^ Recent studies have tried to differentiate the pHe sensitising effect in vitro and in vivo. Rhee et al studied the effectiveness of low extracellular pH in sensitising cells to heat using SCK mammary carcinoma cells of A/J mice. They have demonstrated a different thermosensitivity and thermotolerance following hyperthermia, at different pH

Hyperthermia in Cancer Treatment: A Primer

78

60

520

^dO

MINUTES AT

4rC

240

60

120

180

240

300

MINUTES AT 42^C

Figure 8. CHO cells cultured at different pH under aerobic conditions and heated during the midportion ofpH exposure conditions. (Suit an Gerweck, Cancer Res 1979; 39:2290-2298. Reprintedfromref 70 with permission.) values (pH 7.2, G.d, 5.5) on in vivo and in vitro derived cells. For any heating temperature tested the sensitising effects of pFi was much smaller on in vivo derived cells that on in vitro derived cells. Furthermore, this reduction of pFi effect was observed for cells derived from larger tumors as well as tumors at an early stage of growth in which the internal milieu was not acidic. This indicates that cellular adaptation to low intratumor pH was not the sensitising factor and might be related to other factors than pH, such as nutrients deprivation and decreased blood perfiision.^^'^^ Although recent experiments proved that thermosensitivity is more dependent upon pHi rather than pHe^^'^^ pHe value alone has demonstrated to be a useful prognostic indicator and that the reduction in pHg induces a decrease in the intracellular pH. Since that extracellular pHe is the result of lactic acid accumulation, von Ardenne tried to further lower the tumor milieu pFi through the administration of supraphysiological levels of glucose. Earlier animal and human experiments indeed demonstrated that intraperitoneal or endovenous injection of glucose reduced intratumor pHe and increased the response to thermotherapy.^ However, recent studies have evidenced that the change of pH is induced by tumor blood flow reduction or nutrition deprivation rather than by the increased glycolysis

(Fig. 9)7''" Although the experimental evidence has demonstrated that hyperglycaemia is a useful method for enhancing the response to thermoradiotherapy, its clinical application is not yet of routine. This is partly due to a fear of inducing uncontrollable physiologic alterations and to an absence of a standardized protocol.^^'^^ The clinical application and administration of hyperglycaemia will be discussed later.

Influence ofTumorMicroenvironmenton Thermoresponse

79

GLUCOSE 10%

500CC X 45'

i

-^*

EXTRACELLULAR LACTIC ACID CONCENTRATION

SHD

CARDJAC OUTPUT

• RBC DEFORMABILITY

•

LEUCOCYTES PLATELETS INTERACTION BLOOD

'VISCOSITY

• TBF • HEAT SENSITIVITY

Figure 9. In this diagram the direct effects of glucose administration (bold white) on tumor blood flow (TBF) and on tumor extracellular/intracellular pH are illustrated. Associated are shown the indirect effect of hyperglycaemia on cardiac output (bold gray) with the consequent decrease in TBF.

Hyperthermia Effects on Tumor Blood Flow and Endothelium Introduction Hyperthermia has as principal goal that of destroying tumor tissue (vasculature included) by achieving a temperature that exceeds the cytotoxic threshold (42.5**C) and induces cell death in tumor tissue with a selective sparing of normal surrounding tissue.^"^ Although cancer cells are destroyed by hyperthermia alone, many factors, including the cell type and blood perfusion, influence its success. In fact, it has been shown experimentally and theoretically that heat transferred away from a tissue is the result of the rate and the volume of blood flowing through that tissue (perfusion).^^'^^ Since the importance of blood perfusion in heat dissipation, a brief description of heat effects on tumor blood flow [TBF] and on endothelium is useful.

Effects on Tumor Blood Flow (TBF) and on Normal Circulation The relationship between temperature rise and perfusion has been demonstrated by Jain et al^^ to be inversely correlated: as the perfusion rate decreases the tumor temperature increases. The gap in temperature obtainable between normal and tumor tissue is due to the differences in conduction and convection characteristics between the two tissues. Tumor blood flow and distribution are different from normal tissue and show regional variations in the same tumor itself as quantified by GuUino and Grantham. Blood flow in human cancer showed a greater variability as compared to animals, however a different flow between normal and tumor tissue exists. Tumor blood flow appears to be inferior than that of normal tissue and to have a decreased adaptability to metabolic demands and physical stress

80

Hyperthermia in Cancer Treatment: A Primer

reasons are to be ascribed to the absence of innervation. ' The vasodilatation that happens after heat application to normal tissues is not present at the same extent in tumor vasculature. ' This determines a decreased convection and permits to entrap heat in the tumor area, rising the temperature in that target area.^'^'^^ In fact, different authors heating deep seated human tumors by radiofrequency (13.56 MHz) therapv, have reported that tumor temperature was higher than that of surrounding normal tissue.^ After heat exposition macroscopic blood flow measurements in normal tissue such as skin or muscle showed a rapid and a dynamic vasodilatation with increased permeability of the vascular wall (Fig. 6). Song ' and Vaupel^^ demonstrated that the degree of alteration was temperature and treatment duration dependent. The heating changes in the tumor were slighdy absent or increased at the beginning of treatment, if the treatment time was prolonged a decrease with a stasis of blood flow took place.^^'^^ Similar conclusions on blood flow behaviour during hyperthermia have been reached by other authors using microscopic measurement such as RBC velocity, laser doppler flowmetry and hydrogen clearance methods.^^'^^ Jain et al^^ studied RBC velocity and vessel lumen diameter in mature granulation tissue and in neoplastic tissue (VX2 carcinoma). They found and confirmed the above-mentioned observations, but noted that stasis occurred in normal and tumor tissue both. The difference in stasis was dependent on temperature. In fact, the stasis in normal tissue occurred later and at higher temperature (47*C) whereas stasis in tumor tissue was reached before and at a lower temperature (4l,00°C).^^ As for stasis, Li^^ founded that the recovery kinetics of tumor blood flow after heating was temperature-dependent, i.e., blood stasis occurred in the range 3-5 hr after heating, remaining low for 24 hr and partially recovering after 48 hr. Aside the vasodilatation a complexity of other events follow heat application. They are different biochemical and microcirculatory changes, such as: acidosis, RBC stiffening and aggregation, degenerative changes of endothelium, increased vascular permeability, platelet aggregation, leukocyte sticking and intravascular clotting (Fig. 6).^^'^^ These phenomena worsen the tumor microenvironment further and explain why neoplastic cells are damaged more easily by temperature (42-45 °C) than normal cells.^^

Effects on Endothelium Different in vivo and in vitro studies have shown that endothelial cells [ECs] and in particular those proliferating ones can be damaged by heat. The damages can regard the integrity of endothelium or its vulnerability to heat. Histological methods have revealed that after hyperthermia, a rapid reduction and rearrangement of F-actin stressfibersfollows. Thesefibersmaintain the junctional integrity among the cells. Their lack determines an increase in vascular permeability, a phenomenon usually observed after HT.^^^'^ The effects of hyperthermia are not however similar in all tumor types. This difference has been demonstrated by Nishimiu-a^^^ and it is dependent on the quantity of connective tissues present in the vascular architecture. Furthermore Hyperthermia r^ulates positively different adhesion molecules on endothelium surfaces providing an increased recruitment of T cells to tumors. This effect, associated to the increased vascidar permeability and extravasation, partially explains the escape of leukocytes from the vasculature and the peritumoral inflammatory reaction. As described, tumor blood vessels are more vulnerable to heat than normal surrounding blood vessels, probably for their structurally inunaturity. '^'^^ Furthermore, tumor vessels, as neoplastic cells have shown to acquire thermal adaptation (Thermotolerance) to reheating.^ This phenomenon, referred by Song as vascular Thermotolerance (VT), differs as regards neoplastic cells Thermotolerance (NT). NT refers generally to a resistance to heat induced cell killing through the production of Heat shock proteins (HSP), while VT is a thermal adaptation developed by tumor vessels that initially respond to the stress of reheating by increasing blood flow instead of reducing it. The reasons for this behaviour are not completely known and remain actually only speculative.^^

Influence of Tumor Microenvironment on Thermoresponse

Table 3. Clinical methods for improving

thermoresponse

Tumor Blood Flow Modification

Tumor Microenvironment Modification

A) TBF reduction or deprivation: clamping of nutritive vessels chemoembolization B) Drugs able to modify TBF: Vasodilators: [hydralazine, calcium blocking agents, verapamil, flunarizine, serotonin and its analogues]

A) Hyperglycaemia

Vascular targeting agents (VTA): [FAA, DMXXA, CA4DP]

81

B) Drugs active on tumor metabolism: [amiloride, lonidamine, stilbene derivatives (DIDS), MIBG, Quercetin, 2 Deoxy Glucose (2 DC)] C) Modifiers of thermal sensitivity: [Anesthetics, Lonidamine, Calcium antagonists, EFAs, COX2, Betulinic Acid, aldehydes. Vitamins, Quercetin] D) Heat delivery methods - Rapid heating - M i l d hyperthermia

Antiangiogenetic Effect of Hyperthermia Hyperthermia has demonstrated to kill tumor cells by a direct and an indirect mechanisms. The indirect killing mechanism has been recognized as an inhibitory effect on angiogenesis. In the 60s the inhibition of angiogenesis was ascribed to ischaemia with a consequent obstruction and destruction of tumor blood vessels followed by an inability to form new vessels.^^'^® Recently a biochemical mechanism has been recognized. ^ ^^ These Authors have clearly demonstrated that heat application can inhibit angiogenesis by activation of a Plasminogen Activator Inhibitor I-dependent mechanism (PAI-I). The effect has been investigated and found operating in vivo and in vitro with a reduction in the number of microvessels. Endothelial cells viability was not affected. ^^'^

Clinical Methods for Improving Thermoresponse Introduction Tumor vascular supply is extremely important for maintaining tumor growth, progression and metastasization. Furthermore, tumor perfusion can affect related micro environmental parameters, such as oxygenation status, pH distribution, bioenergetic status, nutrient supply and sensitivity of cancer cells to anticancer treatments, hyperthermia included.^^'^^ Blood flow is the major determinant of heat dissipation and it is responsible for the selective and uniform heating increase in tumor target area respect the normal counterpart. The relationship between blood flow and the effective tumor conductivity has been studied by Jain et al,^^ who found that temperature rise in tumor mass is inversely and linked to the effective thermal conductivity. Since hypoxic cells and preferentially cells exposed, to an acute acidification have shown an increased thermosensitivity (Fig. 7), many investigators have developed different methodologies with the aim of increasing thermoresponse as the heat induced cell damage.^^ Clinically, a modification of tumor thermoresponse could be obtained or modulating tumor blood flow, tumor microenvironment or trying to ameliorate the heat deposition into the tumour tissue (Table 3, and Fig. 10).^^'^^'^°^'^°^ Otherwise expressed the clinical attempt to increase the heat response can be reached or by inducing hypoxia through blood flow manipulation or trying to render tumor cells more vulnerable to heat.

Hyperthermia in Cancer Treatment: A Primer

82

TBF MODIFICATION

HYPERTHERMIA APOPTOSISfflECROSIS Querc«tln

VTAJ-^

METABOLIC INHIBITORS Amiioride

0

OXYGENATED CELLS HYPOXIC CELLS

Radiotherapy Chemotherapy

MICROMILIEAU MODIFICATION

MEMBRANE MODIFIERS

Figure 10. In thisfigurethe interactions of hyperthermia with the chemotherapy and radiotherapy are illustrated together the principal points of tumor microenvironment modification [TBF (tumor blood flow); micromilieu, membranes, metabolism] for improving the therapeutic response to heat. An attempt has been done by Authors to depia the various points of intervention of drugs in order to obtain a clinical and intelligible method of treatment.

Tumor Blood Flow (TBF) Modulation Blood Flow Reduction or Deprivation Cutting off the tumors blood supply by physical clamping produces 100% radiobiological hypoxia^^ that increases cancer cells death by apoptosis. Field^ has demonstrated drastic changes in the isoefFect relationship when deprivation of blood supply by clamping was applied upon hyperthermia. Firstly, tumor tissue becomes more sensitive, equivalent to a factor near two in heating time; secondly the transition at 42.5°C is eliminated, showing a reduction or an abolishment of thermo tolerance. Clamping of Nutritive Vessels Blood flow deprivation by clamping blood supply has been demonstrated in vivo to make tumor cells totally hypoxic and to enhance significantly thermal sensitivity on animals. Thermal enhancement had a ratio of 1.8-2.6 and was dependent on time. The proportion of tumor controlled by hyperthermia increased alone from 33% to 83%, depending on whether the clamp was applied immediately before heating or 60' before heating. No cures were observed for heat applied immediately before clamping, or immediately after the release of the clamps ^^ Other authors have demonstrated that vascidar occlusion by clamping followed by glucose load can decrease tumor pH ftirther and consequently enhance thermoresponse.^^ Chemoembolization Since blood flow reduction by clamping is not clinically attainable, embolization has been used as a method of blood flow stoppage. Embolization of liver tumors can be indicated for the treatment of colorectal metastases, hepatocellular carcinoma (HCC), and metastases from other parts of the body. Chemoembolization is a combination of two effective therapies with

Influence of Tumor Microenvironment on Thermoresponse

83

the aim of improving both. One is the high concentration drug delivery to tumor mass, the second is the production of hypoxia for inhibiting the active efflux of the administred drug.^^^ As previously reported hypoxia improves the response to heat. This does chemoemboiization an efficient method of treatment in association with thermotherapy for treating Uver. Liver has two main blood supplies which keeps it alive and functioning. The portal vein supplies 75% of the blood entering the liver and the hepatic arteries supply the remaining 25%, although they are the ones that provide nearly 100% of the blood that feeds primitive and secondary liver tumors.^ ^^ The seal of hepatic arteries concentrates the level of chemotherapy, 10 to 25 times higher, than that of standard chemotherapy. To underfed humans liver tumors hepatic arteries are blocked or embolized by different methods. The most used is an oil-based mixture associated to chemotherapy. This embolization consists in lipiodol (ethiodizedoil) or starch microspheres (DSM) alone or associated with chemotherapy. DSM (Spherex, Pharmacia, Sweden) are particles of cross-linked starch, measuring 20-70 [xm, degraded by amylases, and able to block hepatic artery transiently and reversibly. Tanaka et al^^ blocked the hepatic artery, injecting a mixture of Lipiodol / or DSM plus anticancer drugs [e.g.. Mitomycin C (10-20 mg) or 5-FU (500-750 mg)]. Forty eight hours after this block they performed hyperthermia twice a week for a total of 4-6 treatments. Results of this HT schedule were evaluated by CT images and angiograms. The mean maximal temperature(TiviAx) reached was of the order of 41.5°C and the response rate was of 40%.

Drugs AbU to Modify Tumor Blood FhuP^'^'"^'^'^'^ The goal of TBF modulation by drugs is to make the tumor sufficiently hypoxic/or underfed and consequently more thermo sensible, similarly to clamping or Chemoemboiization. Drugs, which are of course less cumbersome of the preceding methods of treatment, are of two types: vasodilatators and agents that attack vascular endothelium (VTA). Vasodilatators starves the tumor through a steal phenomenon, while VTA get tumor hypoxic by disrupting and decreasing the nutritive vessels. A. Vasodilatators: Hydralazine, calcium blocking agents (verapamil,flunarizine),serotonin and its analogues.102.103,108 B. Vascular targeting agents (VTA): FAA, DMXXA, CA4DP, TNF; IL.L76.i03,ii4,ii5

Microenvironment Modification Hyperglycaemia and Drugs Acting on Tumor Metabolism^^*^^*^^*^^*^^ Several animal studies have demonstrated that hyperglycaemia can sensitise mammalian cells to heat.^^ '^^^ Initially the effect was thought to be metabolic induced (pH drop, due to excessive lactic acid production), but recent studies by Ward-Hartley and Jain have demonstrated that the hypoxia and pH reductions are secondary to the tumor blood flow reduction. ^^^ Calderwood and Dickson reported that intraperitoneal injection of glucose reduced tumor blood flow by more than 90% for a few hours. ^^^ Furthermore, these authors have demonstrated that blood flow inhibition and pH reduction are related to the serum concentration of glucose level. ^^^ Nagata^^^ reported similar effects on 25 cancer patients who received intravenous injection of 500 ml of 10% glucose. Tanaka^^"^ has clearly demonstrated that patients treated with hyperglycaemia were more responsive to thermoradiotherapy than a control group treated by thermoradiotherapy alone. The mechanisms responsible for blood flow reduction in tumors during hyperglycaemia are multiple and have been explained by Ward and Jain.^^ The reduction is a consequence of systemic and local effects both. The systemic effects were ascribed to a significant cardiac output redistribution that consequently reduced tumour blood flow by steal phenomena (see Fig. 9). The local mechanism that contributed to reduce tumor blood flow was linked to red blood cell (RBC) deformability decrease. Initially this increase in rigidity was postulated to be pH-dependent,^^ but Crandall et al^^^ have demonstrated that it was necessary a long exposure time to a low pH for obtaining a RBC rigidity.

84

Hyperthermia in Cancer Treatment: A Primer

as CONTROL ACNU (20HG/KGI < Q.U

ACNU (20 MG/KG)* GLUCOSE ACNU I10M6/KG)

BT/An

~\ ^

1/ '

»••—-• HYPERTHERMIA

^

ACNU (10HG/KG) •HYPERTHERMIA

0.2

ACNU (10MG/KG) •HYPERTHERMIA •GLUCOSE

B

12

16

20

2U

28

32

DAYS AFTER TREATMENT

Figure 11. Growth curves of BT4 An tumors after ACNU 20 or 10 mg/kg combined with hyperthermia with or without hyupertonic gluc»se 6 g/Kg i.p. two hours before treatment. It is interesting to note the decreased tumor regrowth after glucose-chemotherapy-HT administration. (Shem BC, Dahl O. J Neurooncology 1991; 10:247-251.) Traykov and Jain questioned diis lag period and demonstrated that RBC deformability followed almost inmiediately the infusion of glucose and galactose, ^^^ This rapid local phenomena associated to the steal effect of cardiac output are responsible for the initial blood flow reduction. The metabolic interaction with pH decrease can perpetuate the reduction of blood flow for many hours after glucose administration. Studies by Hasegawa^^^ have shown that a difference in pH drop exists between tumor and normal tissue. In fact, 30-60 min after glucose administration the pH rapidly dropped of 0.3 to 0.6 units in tumor, as compared to a decrease by only 0.1 unit of normal tissue. The complete recovery to baseline was also different in the two tissues. Animal studies have demonstrated that the administration of elucose prior to hyperthermia can modify tumor regrowth and thermotolerance (i.e.. Fig. 11). ^^ Tumor regrowth delay was greater in the group treated with glucose as compared to the group not treated. Thermotolerance disappeared 12 h after heat treatment in the group with glucose administration, whereas in the untreated group thermotolerance disappeared only after 72 h.^^^ The majority of experimental studies have been performed in vitro and in animals. Recendy, after the increased use of hyperthermia in association with radiotherapy, many htunan studies have been performed. Dickson Calderwood demonstrated that serum glucose levels in the range of 1000 mg/dl can cause a complete cessation of tumor blood flow. Similar levels of glucose in humans are however not attainable without side effects. Levels of 400mg/dl have been demonstrated by Lippmann et al^^^ the maximum attainable for long periods (24h) with no modifications of blood count and acid base equilibrium. Krag et al^^ with the goal of achieving a steady level of 400 mg/dl for short periods of time (3h), intravenously administered a glucose loading dose of 77 mg/m^ to three patients with advanced metastatic cancer. After 20 minutes, levels over 400 mg/dl were obtained and maintained for 3 h with no apparent side effects and rebound hypoglycaemia. Levels beyond 700 mg/dl were attempted, but could not be maintained without side effects. Other authors have studied and compared the effects on tumor blood

Influence of Tumor Microenvironment on Thermoresponse

85

Table 4. Treatment schedule clinically used by our group Ten minutes after radiotherapy an infusion of Glucose 10% 500 cc is initiated and continued for 90'. Thirty minutes after radiotherapy, Hyperthermia is initiated and for brain tumors last 60', whereas for solid tumors located in other sides 90'. To increase the intracel lular pH, a week before the treatment, we use oral ly quercetin (1200 mg/dai ly) and Moduretic® (Amiloride, 5 mg/daily). Quercetin plus Celebrex® (Celecoxib 200-400 mg/daily) is used to reduce thermotolerance, too. In certain situation we insert Lonidamine as anticancer and metabolic inhibitor.

flow, pHe and the clinical response of human patients submitted to a glucose load.^'^'^'^^^ Nagata et al demonstrated that a glucose administration of 500 ml of 10% glucose by intravenous route reduced the tumour blood flow, measured by laser Doppler flowmetry, to 66% of the baseline level at 30 min after the beginning of infusion. A complete tumor response (CR) of 30% was obtained on glucose treated patients compared to a group treated only by Radiotherapy and hyperthermia.^^^ Leeper et al^^ have determined whether intravenous (i.v.) or combined intravenous plus oral glucose administration were more effective in inducing acute tumor extracellular acidification. They concluded that the effects of hyperglycaemia induced by i.v. + oral administration were similar and i.v. exhibited an acidification of 0.14 ± 0.002 pH unit after 91 ± 7 min of infusion. Engin and coU.^^ have evaluated the importance of extracellular pH as prognostic indicator of tumor response to thermoradiotherapy. The authors measured the tumor pHg of 26 human tumors with a needle microelectrode of 2.5 cm of length. They reported that the difference in pHe exhibited by complete responding (CR) patients and non completely responding (NCR) 6.88 ± 0.09 versus 7.24 ± 0.09 was statistically significant (p>0.08). On these grounds, they suggest that extracellular pHg measurement may be a useful prognostic indicator of tumor response to thermotherapy. Although, a pHg reduction of «0.2 units is easily obtainable by glucose load, greater reduction >0.5 units are necessary for inducing acute intracellular acidosis. ^^^ The degree of reduction of pH; that accompanies acute extracellular acidification is the critical factor for sensitizing cells to hyperthermia and for abrogating the heat shock proteins induction. Recently various Authors have demonstrated that for obtaining such pHedrop in melanoma, metabolic inhibitors such as meta-Iodio-benzylguanidine (MIBG) or alpha-cyano-4-hydroxy-cinnamic acid (CNCn) must be added.^'^^ For understanding, the biochemical points of action of these inhibitors (see Fig. 5). In conclusion, the concomitant administration of glucose together with MIBG increases the tumour magnitude and duration of acidification and the oxygen tension. ^^^'^^^ This association has the potential to improve response to radiation therapy and to hyperthermia itself The protocol used in our laboratory was similar to that used by Nagata (Table 4).^^^ Following these studies, we used 500 cc of Glucose at 10% obtaining blood glucose value of 300-400 mg/dl without side effects (unpublished observations). Recently, following the suggestions of Leeper group we have added to the hyperglycemia two metabolic inhibitors such as quercetin and Amiloride (Moduretic®) (Table 4).

Modifiers of Thermal Sensitivily Lidocaine and anesthetics,^^^'^ Calcium antagonists,^^^ polyunsaturated fatty acids.^^^ cycloxygenase inhibitors,^^^ betulinic acid,^^^ aldehydes,^^^ vitamins and bioflavonoids.^^^'^ "^

Heat Delivery Methods Tumor cell killing curves by heat show a shape that it is both time and temperature-dependent and not dissimilar from those obtained for X-rays. The data in vitro are consistent with results in vivo and show that relatively small changes in temperature can have a large effect on cell killing^ ^'^ The critical temperature has been demonstrated to be between 42.5°C and 43°C.

86

Hyperthermia in Cancer Treatment: A Primer

Unfortunately the hyperthermia devices now in use are not able to keep this range of temperature for enough time uniformly. This justifies the attempt done by different authors to change or to modify the treatment application.

Rapid Heating Rapid heating is a method developed by Hasegawa group^^^ in the attempt to shorten hyperthermic treatment still reaching temperature sufficient to kill tumour cells and to change tumor blood perfusion. The experiments have been made on C3H mice inoculated with SCC-VII tumor in the thigh, heated with warm water bath and RF heating devices. C3H mice were divided in two treatment groups and compared, the former in which the heating temperature was increased to the target temperature in 1 min, and the latter group in which the heating temperature was gradually increased. The following parameters were studied: changes in blood flow in tumour and normal tissue, tumour growth rate, cancer cells apoptosis. Changes in blood flow were not observed in the slow heating group before or after the hyperthermic treatment, whereas in the rapid heating group a significant increase in blood flow was observed in the normal tissue followed by a significant decrease after heat treatment in the tumour tissue. Tumor growth delay was more evident in the RF rapid heating group compared with warm water heating group. Apoptosis and cytokinetic activity modifications were favorable to the rapid heating group, revealing that a vascular injury was effectively obtained with a shortage in treatment time in this group. ^ Clinical studies on this methodology are warranted.

Mild Hyperthermia and Oxygenation As previous described, solid tumors contain regions of low extracellular pH and oxygen that may affect treatment outcome. Laboratory and clinical data confirm that hyperthermia may enhance the therapeutic index of ionizing radiation.^ ^ Several mechanisms have been found and are summarized in the recent reviews of Kampinga and Vujaskovic.^ Among these mechanisms, tumor oxygenation improvement after mild hyperthermia (HT with temperature between 39-42.5°C) is now considered to be of the utmost importance. As determined by Song et al,^^ normal and tumor tissue show a different behavior following heat deposition. Blood tumor vessels respond markedly different to a second heat application showing a greater vulnerability and vasodilatation to heat than normal surrounding blood vessels. This phenomena, referred by Song as vascular thermotolerance (VT), appears to account for the improvement in the tumour blood flow observed after the reheating at 42.5"C. As the blood flow increase, an improvement in tumor oxygenation follows which may last for as long as 24-48 j^ 94,95 Tumor oxygenation by mild HT has been found to be more effective than carbogen breathing in increasing the radiation response of experimental tumors.^ '^ Clinical studies on 18 patients with locally advanced breast cancer treated with thermo-chemo-radiotherapy have confirmed these experimental results. Tumour oxygenation improvement appeared to be temperature-dependent and associated with the lower thermal doses.

Sununaiy and Conclusions Tumour hypoxia is a problem that makes tumors more resistant to ionizing radiation and chemotherapeutic drugs. Hyperthermia represents a possibility in its overcoming; overall in association with other therapies such as Radiotherapy and chemo-immunotherapy.^ Moreover the effect of mild HT on oxygenation is of great relevance in fact, temperature of 39-39.5''C is more easily obtainable in clinic than killing temperature of 42.5°C.

Acknowledgements We thank for her secretarial assistance N. Tortolone and L. Scappini (Novara University Medical Library).

Influence of Tumor Microenvironment

on Thermoresponse

87

References 1. Ruiter D, Bogenreider T, Herlyn M. Melanoma-stroma interactions: Structural and functional aspects: Lancet Oncol 2002; 3:35-42. 2. Delpech B. Le stroma des cancers. Bull Cancer 1991; 78:869-900. 3. Singh S, Ross SR, Acena M et al. Stroma is critical for preventing or permitting immunological destruction of antigenic cancer cells. J Exp Med 1992; 175:139-146. 4. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply and metabolic microenvironment of human tumors, a review. Cancer Res 1989; 49:6449-6465. 5. Freitas I, Baronzio GF. Tumor hypoxia, reoxygenation and oxygenation strategies: Possible role in photodynamic therapy. J Photochem Photobiol B Biol 1991; 11:3-30. 6. Folkman J. T u m o r Angiogenesis: Therapeutic implications. N Engl J Med 1971; 285:1182-1186. 7. Berges G, Benjamin L. Tumorigenesis and the angiogenic switch. Nat Rev Cancer 2002; 3:401-410. 8. Mc Donald D M , Choyke PL. Imaging of angiogenesis: From microscope to clinic. N a t Med 2003; 9:713-725. 9. Pugh C W , Ratcliffe PJ. Regulation of angiogenesis by hypoxia role of the HIF system. Nat Med 2003; 9:677-684. 10. Semenza GL. HIF-1 and human disease: One highly involved factor. Gene Dev 2000; 14:1983-1991. 11. Semenza GL. Homeostatic regulation by Hypoxia -inducible factorl. Science and Medicine 2002; 8:338-347. 12. Dachs G U , Tozer G M . Hypoxia modulated gene expression: Angiogenesis, metastasis and therapeutic exploitation. Eur J Cancer 2000; 36:1649-1660. 13. Ferrara N , Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997; 18:4-25. 14. Papetti M, Herman I. Mechanisms of normal and tumor-derived angiogenesis. Am J Physiol Cell Physiol 2002; 282:c947-c970. 15. Griffioen AW, Molema G. Angiogenesis:Potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation. Pharmacol Rev 2000; 52:238-268. 16. Kurebayashi J, Osuki T, Kunishe H et al. Expression of Vascular endothelial growth factor (VEGF) family members in breast cancer. Jpn J Cancer Res 1999; 90:977-981. 17. Balbay M D , Pettaway CA, Kuniyasu H et al. Highly metastatic human prostate cancer growing within prostate of athymic mice overexpresses vascular endothelial growth factor. Clin Cancer Res 1999; 5:783-789. 18. L Hlatky P, Hahnfeldt C, Tsionou C. Coleman: Vascular endothelial growth factor: Environmental controls and effects in angiogenesis. Br J Cancer 1996; (Suppl XII):sl51-sl56. 19. Pepper MS. Lymphangiogenesis and tumor metastasis: Myth or reality? Clin Cancer Res 2001; 7:462-468. 20. Polverini PJ. How the extracellular matrix and macrophages contribute to angiogenesis-dependent diseases. Eur J Cancer 32A; 14:2430-2437. 2 1 . Chiford SC, Maher ER. Von Hippel-Lindau disease. Adv Cancer Res 2001; 82:85-105. 22. Grugel S, Finkezeller G, Weindel K et al. Both v-Ha-ras and v-raf stimulate expression of Vascular endothelial growth factor in N I H 3T3 cells. J Biol Chem 1995; 270:25915-9. 23. Takakura N , Watanabe T, Suenobu S et al. A role for Hematopoietic stem cells in promoting angiogenesis. Cell 2000; 102:199-209. 24. Lyden D, Hattori K, Dias S et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks t u m o r angiogenesis and growth. N a t u r e M e d 2 0 0 1 ; 7(11):1194-1201. 25. Abramsson A, Berlin O , Papayan H et al. Analysis of mural cell recruitment to tumor vessels. Circulation 2002; 105:112-117. 26. Dewirst M W , Tso CY, Oliver R et al. Morphologic and hemodynamics comparison of tumor and healing normal tissue microvasculature. It J Radiat Oncol Biol Phys 1989; 17:91-99. 27. Konerding MA, Malkusch W, Klaptor B et al. Evidence for characteristic vascular patterns in solid tumors: Quantitative studies using corrosion casts. Br J cancer 1999; 80:724-32. 28. Konerding MA, Steiberg F, van Ackern C et al. Vascular patterns of tumors: Scanning and trasmission electron microscopic studies on humam xenografts. Strahlentherapie un Onkologie 1992; 168:444-452. 29. Hirshi KK, D'Amore PA. Control of angiogenesis by pericytes: Molecular mechanism and significance. In: Goldberg ID, Rosen EM, eds. Regulation of Angiogenesis: Birkhauser Press, 1997:419-428. 30. Griffioen AW, Tromp SC, Hillen HFP. Angiogenesis modulates the tumor immune response. Int J Exp Path 1998; 79:363-368. 3 1 . Griffioen AW, Coenen M J H , Damen CA et al. C D 4 4 is involved in tumor angiogenesis; an activation antigen on human endothelial cells. Blood 1997; 90:1150-59.

88

Hyperthermia in Cancer Treatment: A Primer

32. Carlos TM. Leucocyte recruitment at sites of tumor: Dissonant orchestration. J Leucoc Biol 2001; 70:171-184. 33. Sevick EM, Jain RK. Measurement of capillary filtration coefficient in a solid tumor. Cancer Res 1991; 51:1352-1355. 34. Jain RK. Determinants of tumor blood flow: A review. Cancer Res 1988; 48:2641-2658. 35. GuUino PM. The internal Milieu of Tumors. Prog Exp Tum Res 1966; 8:1-25. 36. Gullino PM. Extracellular compartments of solid tumors. In: Beckert FB, ed. Cancer, a Comprehensive Treatise. Vol 3, Biology of Tumors: Cellular Biology and Growth. New York: Plenum, 1975:327-354. 37. Freitas I, Baronzio GF, Bono B et al. Tumor interstitial Fluid: Misconsidered component of internal milieu of a solid tumor. Anticancer Res 1997; 17:165-172. 38. Nagy JA, Herzberg KT, Dvorak JM et al. Pathogenesis of malignant ascites formation: Initiating events that lead to fluid accumulation. Cancer Res 1991; 53:2631-2643. 39. Steen RG. Oedema and tumor perfusion: Characterization by quantitative IH MR imaging. Am J Radiol 1992; 158:259-264. 40. Boucher Y, Less JR, Posner MC et al. Interstitial hypertension in human primary and metastatic tumors. In: Chapman JD, Dewey WC, Withmore GF, eds. Radiation Research: A Twentieth Century Perspective. Vol 1. San Diego: Academic Press, 1991:465. 41. Gutmann R, Leuning M, Feyh J et al Interstitial Hypertension in head and neck tumors in patients. Correlation with tumor size. Cancer Res 1992; 52:1993-1995. 42. Sevick EM, Jain RK. Geometric resistance to blood flow in solid tumours perfused ex vivo: Effects of tumor size and perfusion pressure. Cancer Res 1989; 49:3506-3512. 43. Sevick EM, Jain RK. Viscous resistance to blood flow in soHd tumors: Effect of hematocrit and intratumor viscosity. Cancer Res 1989; 49:3513-3519. 44. Peterson HI. Modification of tumor blood flow, a review. Int J Radiat Biol 1991; 60:201-210. 45. Vaupel P. Tumor blood flow. In: Molls M, Vaupel P, eds. Blood Perfusion and microenvironment of human tumors. Berlin Heidelberg, New York: SpringerVerlag, 2000:41-46. 46. Durand RE. Intermittent blood flow in soUd tumours an under appreciated source of "drug resistance". Cancer Metastasis Rev 2001; 20:57-61. 47. Baronzio GF, Freitas I, Kwann H. Tumor microenvironment (hypoxia-interstitial Fluid) and haemorheologic abnormalities. Semin Thromb Hemost 2003; 29:489-497. 48. Denko NC, Giaccia AJ. Tumor hypoxia, the physiological link between Trousseau's Syndrome (carcinoma -induced Coagulopaty) and metastasis. Cancer Res 2001; 61:795-798. 49. Verheul HMW, Hoekman JK, Broxterman HJ et al. Vascular endothelial factor-stimulated endothelial cells promote adhesion and activation of platelets. Blood 2000; 96:4216-4221. 50. Sahni A, Francis CW. Vascular endotheUal growth factor binds to fibrinogen and fibrin stimulates endotheUal cell proHferation. Blood 2000; 96:3772-3778. 51. Browder T, Folkman J, Pirie-Sheperd S. The hemostatic system as regulator of angiogenesis. J Biochem 2000; 275:1521-1524. 52. Dang CV, Semenza GL. Oncogenic alterations of metabolism. TIBS 1999; 24:68-72. 53. Shapot VS. Biochemical aspects of tumor growth. MIR Publishers, 1980. 54. Behrooz A, Ismail-Beigi F. Stimulation of glucose transport by Hypoxia: Signals and mechanisms. New Physiol Sci 1999; 14(6):105-110. 55. Younes M, Lechago LV, Somano JR et al. Wide expression of the human erythrocyte glucose transporter Glut 1 in human cancers. Cancer Res 1996; 56:1164-1167. 56. Gullino PM, Grantham FH, Smith SH et al. Modification of the acid base status of the internal miheu of tumors. J Nad Cancer Inst 1965; 34:857-869. 57. Asby BS, Cantab MB. pH studies in human malignant tumours. Lancet 1996; 2:312-315. 58. Griffiths JR, Mclntyre DJ, Howe FA et al. Why are cancers acidic? A carrier mediated diffusion model for H^transport in the interstitial fluid. In: Goodie JA, Chadwick DJ, eds. The Tumor Microenvironment: Causes and Consequences of Hypoxia and Acidity. Novartis Foundation Symposium. Chichester, New York: John Wiley, 2001:46-67. 59. Helmlinger G, Sckell A, Dellian M et al. Acid production in glycolysis-impaired tumors provides new insights into tumour metabolism. Clin Cancer Res 2002; 8:1284-1291. 60. Stubbs M, McSheehy PMJ, Griffiths JR et al. Causes and consequences of tumor acidity and implications for treatment. Mol Med Today 2000; 6:15-19. 61. Newell K, Tannock. Regulation of intracellular pH and viability of tumors cells. Funktionsanalyse Biologischer Systeme 1991; 20:219-234. 62. Izumi H, Torigoe T, Ishiguchi H et al. Cellular pH regulators: Potentially promising molecular targets for cancer chemotherapy. Cancer Treat Rev 2003; 29:541-549. 63. Vaupel P. Pathophysiological effects of hyperthermia in solid tumors and their clinical impHcation. In: Georg H Omlor, Peter Vaupel, Cristof Alexander RG, eds. Isolated Hyperthermic Limb Perfusion. Georgetown: Landes Bioscience, 1995:9-45.

Influence of Tumor Microenvironment

on Thermoresponse

89

64. Mueller-Klieser W, Walenta S, Paschen W et al. Metabolic imaging in microregions of tumors and normal tissues with bioluminescence and photon counting. J N C I 1988; 80:842-848. 65. Vaupel P. Pathophysiological mechanisms of hyperthermia in cancer therapy. In: Gautherie M, ed. Biological Basis of Oncologic Thermotherapy. Berlin, Heidelberg, New York: Springer-Verlag, 1990:73-134. GG. Gerweck LE. Tumor p H : Implication for treatment and novel drug design. Semin Radiat Oncol 1998; 8(3):176-182. G7. Hetzel FW. Biological rationale for hyperthermia. Radiol Clin North Am 1989; 27:499-508. 68. Dudar TE, Jain RK. Differential response of normal and tumor microenvironment to hyperthermia. Cancer Res 1984; 44:605-612. 69. Hall EJ. Hyperthermia. In: Hall EJ, ed. Radiobiology for the Radiologist. Lippincott: Wilkins Williams, 2000:495-520. 70. Suit H D , Gerweck LE. Potential for hyperthermia and radiation therapy. Cancer Res 1979; 39:2290-2298. 7 1 . Gerwek LE, Richards B. Influence of p H on thermal sensitivity of cultured human glioblastoma cells. Cancer Res 1981; 41:4019-4024. 72. Gerwek LE. Modifiers of Thermal effects. In: Urano M, Douple E, eds. Hyperthermia and Oncology. Vol 1. The Netherlands: VSP, 1988:83-98. . 73. Hahn G M , Shiu E. Adaptation to low p H modifies thermal and thermochemical response of mammalian cells. Int J Hyperthermia 1986; 2:379-387. 74. Gerwek LE, Richards B. Influence of p H on thermal sensitivity of cultured human gHoblastoma cells. Cancer Res 1981; 41:4019-4024. 75. Lyons JC, Kim G, Song CW. Modification of intracellular p H and Thermosensitivity. Radiation Research 1992; 129:79-87. 7G. Song CW, Park H, Griffin RJ. Theoretical and experimental basis of Hyperthermia. In: Kosaka M, Sugahara T, Schmidt KL et al, eds. Thermotherapy for neoplasia, inflammation, and pain. Tokyo: Springer Verlag, 2001:394-407. 77. Van der Berg A, Wike-Hooley JL, Broekmayer-Reurink et al. The relationship between the unmodified initial tissue p H of human tumors and the response to combined radiotherapy and local hyperthermia treatment. Eur J Cancer Clin Oncol 1989; 25:73-78. 78. Rhee JC, Eddy HA, Salazar O M et al. A differential low ph effect on tumour cells grown in vivo and in vitro when treated with hyperthermia. Int J Hyperthermia 1991; 7:75-84. 79. Hall EJ. Hyperthermia. In: Hall EJ, ed. Radiobiology for the radiologist. Lippincott: WilUams Wilkins, 2000:495-520. 80. Gerwek LE, Dahlberg WK, Greco B. Effect of p H on single or fractionated heat treatment at Al-AyC. Cancer Res 1983; 43:1163-1167. 8 1 . Nielsen O S , Overgaard J. Effect of extracellular p H on Thermotolerance and recovery of hyperthermic damage in vitro. Cancer Res 1979; 38:2772-2778. 82. Han JS, Storck C W , Wachsberger PR et al. Acute extracellular acidification increases nuclear associated protein levels in human melanoma cells during 42 degrees C hyperthermia and enhances cell killing. Int J Hyperthermia 2002; 18:404-415. 83. Field SB. In vivo aspects of hyperthermic oncology. In: Field SB, Hand JW, eds. An Introduction to the Practical Aspects of Clinical Hyperthermia. London, New York, Philadelphia: Taylor & Francis, 1990:55-68. 84. Van De Merwe SA, Van Den Berg Block E, Kroon BBR et al. Temporary vascular occlusion and glucose: Effects on tumour and normal tissue PH in animal experiments. Int J Hyperthermia 1995; 11:829-839. 85. Engin K, Leeper DB, Thistlethwaite AJ et al. Tumor extracellular p H as a prognostic factor in thermoradiotherapy. Int J Radiation Oncology Biol Phys 1994; 29:125-132. 86. Von Ardenne. Selective multiphase cancer therapy. Conceptual aspects and experimental basis. Adv Pharmacol Chemother 1972; 10:339-380. 87. Dixon JA, Calderwood SK. Effect of hyperglycaemia and hyperthermia on the, glycolsis p H and respiration of the Yoshida sarcoma in vivo. J Natl Cancer Inst 1979; 63:1371-1381. 88. MuUer-Klieser W , W a l e n t a S, Kellher D K et al. T u m o u r growth i n h i b i t i o n by i n d u c e d hyperglycaemia / hyperlactatacidaemia and localized hyperthermia. Int J Hyperthermia 1996; 12:501-11. 89. Lippmann H G , Graichen D. Glucose and K* balance during high dosage intravenous glucose infusion. Infusionsther Klin Ernahr 1977; 4:166-178. 90. van Den Berg AP, van Den Berg AE, Kal H B et al. A moderate elevation of blood glucose level increases the effectiveness of thermoradiotherapy in a rat tumor modelll. Improved tumor control at clinically achievable temperatures. Int J Radiat Oncol Biol Phys 2001; 50:793-801.

90

Hyperthermia in Cancer Treatment: A Primer

91. Krag DN, Storm FK, Morton DL. Induction of transient hyperglycaemia in cancer patients. Int J Hyperthermia 1990; 6:741-744. 92. Ward KA, Jain RK. Response of tumours to hyperglycaemia: Characterization, significance and role in hyperthermia. Int J Hyperthermia 1988; 4:223-250. 93. Gullino PM, Grantham FH. Studies on the exchange of fluids between host and tumor,II. The blood flow of hepatomas and other tumors in rats and mice. J Nad Cancer Inst 1961; 27:1465-1491. 94. Song CW, Chelstrom LM, Sung JH. Effects of a second heating on tumor blood flow. Radiat Res 1990; 122:66-71. 95. Song CW. Effect of local hyperthermia on blood flow and microenvironment: A review. Cancer Res 1984; 44(suppl):4721-4730. 96. Li GC. Thermal biology and physiology in clinical hyperthermia: Current status and future needs. Cancer Res 1984; 44(suppl):4886s-4893s. 97. Vaupel P, KalUnowski F. Physiological effects of hyperthermia. Recent Results in Cancer 1987; 104:71-109. 98. Reinhold HS, Endrich B. Tumor microcirculation as a target for hyperthermia. Int J Hyperthermia 1986; 2:11-137. 99. LeVeen HH, Wapnick S, Piccione V et al. Tumor eradication by radiofrequency therapy. Response in 21 patients. J Am Med Assoc 1976; 235:2198-2200. 100. Kim JH, Hahn EW, Tokita N et al. Local tumor Hyperthermia in combination with radiation therapy. 1. Malignant cutaneous lesions. Cancer 1977; 40:161-69. 101. Hiraoka M, Shiken JO, Keizo A et al. Radiofrequency capacitive Hyperthermia for deep-seated tumors, 1. Studies on Thermometry Cancer 1987; 121-127. 102. Tanaka Y. Thermal response of microcirculation and modification of tumor blood flow in treating the tumors. In: Kosaka M, Sugahara T, Schmidt KL et al, eds. Theoretical and experimental basis of Hyperthermia. In Thermotherapy for neoplasia, inflammation, and pain. Tokyo: Springer Verlag, 2001:408-419. 103. Jain RK, Ward-Hardey K. Tumor bloodflow-Characterization,modifications and role in hyperthermia. IEEE Transactions on Sonics and Ultrasonic 1984; 31:504-526. 104. Fajardo LF, Prionas SD. EndotheUal cells and hyperthermia. Int J Hyperthermia 1994; 3:347-353. 105. Nishimura Y, Hiraoka M, Jo S et al. Microangiographic and histologic analysis of the effects of hyperthermia on murine tumor vasculature. Int J Radiat Oncol Biol Phys 1988; 15:411-420. 106. Evans SS, Frey M, Scheider DM et al. Regulation of leukocyte-endothelial cell interaction in tumor immunity. In: Mihich and Croce, eds. Biology of Tumors. Plenum Press, 1998:(Ch 20):273-286. 107. Roca C, Primo L, Valdembri D et al. Hyperthermia inhbits angiogenesis by a Plasminogen Activator Inhibitor -I dependent mechanism. Cancer Res 2003; 63:1500-1507. 108. Jirtle RL. Chemical modifications of tumor blood flow. Int J Hyperthermia 1988; 4:355-371. 109. Suit H, Shalek RJ. Response of spontaneous mammary carcinoma of the C3H mouse to X-irradiation given under conditions of local tissue anoxia. J Nat Cancer Inst 1963; 31:497-509. 110. Hill SA, Denekamp J. The effect of vascular occlusion on the thermal sensitisation of a mouse tumour. Br J Radiol 1978; 51:997-1002. 111. Stuart K- Chemoembolization in the management of liver tumors. The Oncologist 2003; 8:425-437. 112. Tanaka Y, Yamamoto K, Nagata K. Effects of multimodal treatment and hyperthermia on hepatic tumors. Cancer Chemother Pharmacol Suppl 1998; 1:111-114. 113. Hirst DG, Hirst VX, Shaffi KM et al. The influence of vasoactive agents on the perfusion of tumors growing in three sites in the mouse. Int J Radiat Oncol Biol Phys 1991; 60:211-218. 114. Baguley BC, Wilson WR. Potential of DMXAA combination therapy for solid tumors. Expert Rev Anticancer Ther 2002; 2:593-603. 115. Horsman MR, Murata R. Combination of vascular targeting agents with thermal or radiation therapy. Int J Radiat Oncol Biol Phys 2002; 54:1518-1523. 116. Calderwood SK, Dickson JA. Effect of hyperthermia on blood flow, pH and response to hyperthermia(42°C) of the Yoshida sarcoma in the rat. Anticancer Res 1980; 40:4728-4733. 117. Dickson JA, Calderwood SK. Thermosensitivity of neoplastic tissues in vivo. In: Storm FK, ed. Hyperthermia in Cancer Therapy. Boston, GK: Hall, 1983:63-140. 118. Nagata K, Tanaka Y, Akagi K et al. Enhancement of thermoradiotherapy by glucose administration for superficial malignant tumors. J Radiat Res 1998; 14:157-167. 119. Crandall E, Crtz A, Osher A et al. Influence of pH on elastic deformability of the human erythrocyte membrane. Am J Physiol 1978; 235:c269-c278. 120. Traykov TT, Jain RK. Effect of glucose and galactose on red blood cell membrane deformability. Int J Microcirc CUn Exp 1987; 6:35-44. 121. Hasegawa T, Gu Y-H, Takahashi T et al. Effects of hyperthermia-induced changes in pH value on tumor response and thermotolerance. In: Kosaka M, Sugahara T, Schmidt KL et al, eds. Thermotherapy for Neoplasia, Inflammation, and Pain.Tokyo: Springer Verlag, 2001:431-438.

Influence of Tumor Microenvironment

on Thermoresponse

91

122. Shem BC, Dahl O . Thermal enhancement of A C N U and potentiation of thermochemotherapy with A C N U by hypertonic glucose in the BT4 A rat glioma. J Neuroncol 1991; 10:247-252. 123. Lippmann H G , Graichen D. Glucose and K* balance during high dosage intravenous glucose infusion. Infusionsther Klin Ernahr 1977; 4:166-178. 124. V o n A r d e n n e M . In vivo T h e o r i e zum glykolytishen Stoffwechsel der T u m o r e n ihrer Cbersauerbarkeit dutch Hyperglykamie. In: Hippokates Verlag Stuttgart, ed. Systemidche Krebs-Mehrschritt-Therapie. 1997:35-45. 125. Nagata K, Murata T, Shiga T et al. Enhancement of thermoradiotherapy by glucose administration for superficial malignant tumours. Int J Hyperthermia 1998; 14:157-167. 126. Leeper DB, Engin K, Wang J-H et al. Effect of I.V. glucose versus combined I.V. plus oral glucose on human tumour extracellular p H for potential sensitisation to Thermotherapy. Int J Hyperthermia 1998; 257-269. 127. Engin K, Leeper DB, Cater JR et al. Extracellular p H distribution in human tumors. Int J Hyperthermia 1995; 11:211-216. 128. Han JS, StorcK CW, Wachsberger PR et al. Acute extracellular acidification increases nuclear associated protein levels in human melanoma cells during 42°Chyperthermia and enhances cell killing. Int J Hyperthermia 2002; 18:404-415 129. Coss R, Storck CW, Daskalaqkis C et al. Intracellularacidification abrogates theheat shock response andcompromises survival of human melanoma cells. Mol Cancer Therapeut 2003; 383-388. 130. Burd R, Wachsberger PR, Biaglow JE et al. Absence of Crabtree effect in human melanoma cells adapted to growth at low p H : Reversal by respiratory inhibitors. Cancer Res 2001; 61:5630-5635. 131. Zhou R, Bansal N , Leeper D R et al. Enhancement of hyperglycemia-induced acidification of human melanoma xenografts with inhibitors of respiration and ion transport. Acad Radiol 2001; 8:571-582. 132. Zhou R, Bansal N , Leeper D R et al. Intracellular acidification of human melanoma xenografts by respiratory inhibitor m-Iodio-benzylguanidine plus hyperglycemia: A^^ P Magnetic resonance spectroscopy study. Cancer Res 2000; 61:3532-3536. 133. Hahn G M . Thermal Enhancement of the actions of anticancer agents. In: Hahn G M , ed. Hyperthermia and Cancer. New York, London: Plenum Press, 1982:55-85. 134. Sensiterra GA, Lepock JR. Thermal destabilization of transmembrane proteins by local anesthetics. Int J Hyperthermia 2000; 16:1-17. 135. Kameda K, Kondo T, Tanabe K et al. The role of intracellular Ca 2+ in apoptosis induced hyperthermia and ist enhancement by verapamil in U937 cells. Int J Radiat Oncol Biol Phys 2001; 49:1369-1379. 136. Kokura S, Yoshikawa T, Kaneko T et al. Efficacy of hyperthermia and polyunsaturated fatty acids on experimental carcinoma. Cancer Res 1997; 57:2200-2202. 137. Asea A, Mallick R, Lechpammer S et al. Cycloxygenase inhibitors are potent sensitizers of prostate tumours to hyperthermia and radiation. Int J Hyperthermia 2001; 17:401-414. 138. Wachsberger PR, Burd R, Wahl ML et al. Betulinic acid sensitization of low p H adapted human melanoma cells to hyperthermia. Int J Hyperthermia 2002; 18:153-164. 139. Kim J H . Modification of thermal effects: Chemical modifiers. In: Urano M, Douple E, eds. Hyperthermia and Oncology. Vol 1. The Netherlands: VSP, 1988:83-119. 140. Prasad K, Kumar B, Yan X-D et al. a-tocopheryl succinate, the most effective form of Vit. E for adjuvant cancer treatment: A review. J Am Coll N u t r 2003; 22:108-117. 141. Callari D , Sinatra F, Paravizzini GL et al. All trans retionic acid sensitizes colon cancer cells to hyperthermia cytotoxic effects. Int J Oncol 2003; 23:181-188. 142. Wachsberger PR, Burd R, Bhala SB et al. Quercetin sensitizes cells to hyperthermia. Int J Hyperthermia 2003; 19:507-519. 143. Van der Zee J. Heating the patient: A promising approach? Annals of Oncology 2002; 13:1173-1184. 144. Kampinga KH, Dikomey E. Hyperthermia radiosensitization: Mode of action and clinical relevance. Int J Radiat Biol 2001; 77:399-408. 145. Hasegawa T, Gu YH, Takahashi T et al. Enhancement of hyperthermic effects using rapid heating. In: Kosaka M, Sugahara T, Schmidt KL et al, eds. Thermotherapy for neoplasia, inflammation, and pain. Tokyo: Springer Verlag, 2001:439-444. 146. Vujaskovic Z, Song CW. Physiological mechanisms underlying heat-induced radiosensitization. Int J Hyperthermia 2004; 20:163-174. 147. Song CW, Park H, Griffin RJ. Improvement of tumour oxygenation by mild hyperthermia. Radiat Res 2001; 155:515-528. 148. Jones EL, Prosnitz LR, Dewhirst M W et al. Thermochemoradiotherapy improves oxygenation in locally advanced breast cancer. Clin Cancer Res 2004; 10:4287-4293. 149. Pontiggia P, Mc Laren JR, Baronzio GF et al. T h e biological responses to heat. Adv Exp Med Biol 1990; 267:271-291.

CHAPTER 6

Hyperthertnia and Angiogenesis: Results and Perspectives Cristina Roca and Luca Primo* Abstract

H

yperthermia (HT) is a promising method for cancer treatment when combined with radiotherapy or chemotherapy. The molecular mechanisms of anti-tumoral efficacy of HT are not well imderstood. Besides its direct cytotoxic effect on tumor cells, HT injures the normal microvasculature and, in particular, tumor vessels. This effect on microvasculature represents an important mechanism of tumor growth inhibition exerted by HT. Recendy, many studies have been made to understand the effects of HT on tumor vessels and, in particular, on new vessel formation that occurs during tumor progression. The tumor vasculature develops in a process known as angiogenesis that consists of the formation of new blood vessels from preexisting ones. Angiogenesis is essential for tumor progression and, without blood vessels, tumors can not grow beyond a critical size or metastatize to another organ. HT above 42°C inhibits endothelial cell (EC) differentiation on capillary-like structures both in vitro and in vivo. At least three distinct mechanisms have been described to be involved in angiogenesis inhibition by HT: direct cytotoxicity on proliferating ECs, down-modulation of vascular endothelial growth factor (VEGF) production by tumor cells and induction of the plasminogen activator inhibitor-1 (PAI-1) expression. These data indicate that inhibition of angiogenesis exerted by heat shock could represent an important mechanism of tumor control in clinical HT and coidd suggest a new rationale for a combined cancer therapy based on HT associated with anti-angiogenic molecules.

Introduction The scientific discipline of HT biology emerged in the 1970s largely from laboratories engaged in research in radiobiology. However, unequivocal identification of the mechanisms leading to favourable clinical research of HT have not yet identified for various reasons. ^"^ Although a large number of preclinical studies are available on different aspeas of HT action, the cellular and molecular pathways underlying this beneficial outcome of patients are still poorly understood. In in vitro studies and in animal experiments, HT exhibited a direct cell killing effect at temperatures ranging from 42°C to 45°C. Furthermore, acute and chronic heating of cells and tissue induces a variety of changes, including alterations of nuclear and cytoskeletal struaures, metabolic pathways and intracellular signals? HT affects fluidity and stability of cellular membranes and impedes the function of transmembrane transport proteins and cell surface receptors. Increasedfluidityof cell membranes was observed in thermosensitive, but not thermotolerant

•Corresponding Author: Luca Primo—Division of Molecular Angiogenesis, Institute for Cancer Research and Treatment, Str. Prov. 142 Km.3.95, Candiolo, TO 10100, Italy. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

Hyperthermia and Angiogenesis: Results and Perspectives

cells. As a consequence of these effects, the heated cells undergo changes in membrane potential, elevated intracellular sodium and calcium content, as well as an elevation of potassium efflux. Besides, HT has been demonstrated to induce various changes of cytoskeletal organization (cell shape, mitotic apparatus, intracytoplasmatic membranes such as endoplasmatic reticulum and lysosomes), but there was no clear correlation found between these phenomenological changes and thermosensitivity of various cell lines. '^ Studies of the influence of HT on nucleic acid synthesis indicate that intracellular de novo synthesis and polymerization of both RNA and DNA molecules is rapidly and markedly inhibited after exposure to heat.^ Whereas RNA synthesis recovers rapidly after termination of heat exposure, DNA synthesis is inhibited for a longer period. ^^'^ Heat shock also induces a^regation of denaturated proteins at the nuclear matrix. This is mainly due to insolubility of cellular proteins after heat-induced protein unfolding, entailing enhancement of the nuclear protein concentration. On the trascriptional and translational level, heat shock induces the expression of a unique set of genes, the heat shock genes, that encode for of a number of proteins, called the heat shock proteins (HSP). HSP represent a heterogeneous group of molecular chaperones consisting of at least five subgroups with different molecular mass and partially varying biologic function. HSP are usually divided into small HSP (molecular mass < 40 KDa), and the HSP 60, HSP 70, HSP 90 and HSP 100 proteins families. All HSP families share their chaperoning function, i.e., they unselectively bind to hydrophobic protein sequences liberated by denaturation. In HT, HSPs are thought to be involved in the protection of cells against heat damage. One of the most interesting aspects of thermal biology is the response of heated cells to a heat challenge. Mammalian cells, when exposed to a nonlethal heat shock, have the ability to acquire a transient resistance to one or more subsequent exposures to elevated temperatures. This phenomenon has been termed thermotolerance and it has been suggested that HSPs are involved in the development of transient thermotolerance, in the acquisition of the permanent heat resistance and in the protection of cells from thermal stress. Moreover, HT may be able to cause damage preferentially in tumors relative to normal tissues because the structure and function of the vasculature and related microenvironment in tumors are rather different from those in normal tissues. In this chapter we will face several aspects of the effects of HT on the angiogenic process in light of the knowledge of some molecular aspects involved in angiogenesis inhibition by HT.

Blood Vessels, Blood Flow and Microenvironment One of the most important effects of HT on normal and tumor tissue is the modification of blood flow, and, as a consequence, the regulation of oxygen and nutrient supply. Moderate HT (< 42 °C) has been demonstrated to improve blood flow especially when applied on tumor tissue, which are normally characterized by reduced blood flow and hypoxic microenvironment. The increase of tumor oxygenation following HT improves the effectiveness of radiotherapy.^^ In addition, the delivery of anti-neoplastic drugs can be favored by increased tumor blood flow. Opposite effects on tissue microenvironment have been observed when temperatures were above 42 °C. HT at these temperatures has been shown to decrease blood flow and an excess exposure of tissue to heat results in a breakdown of vasculature followed by necrosis of the tissue.^ The microenvironment of malignant tumors is characterized by a reduction of blood flow and blood vessels density that favors hypoxia, acidosis and energy deprivation.^'^ Tumor vessels often show increased permeability and may have gaps between adjacent Ecs.^^ Unlike vessels of normal tissue, which respond to heat by dilation and increase in blood flow (and thus increase in heat loss by convention), tumor vessels, which lack appropriate innervation, do not dilate and heat diffusion is poor. Therefore, the heat dissipation by blood flow in tumor is slower than in normal tissue and the acidic and hypoxic environment increases the thermosensitivity of tumor cells. Moreover the tumor vasculature can be also severelv damaged at temperatures which may alter but not damage the vasculature of normal tissue.^ The alteration in tumor blood flow and microvasculature induced by HT can partially explain the increased thermal sensitivity of tumor compared to normal tissues. On the other

93

94

Hyperthermia in Cancer Treatment: A Primer

hand, the preferential damage of tumor vessels as opposed to quiescent vessels is difficult to explain. Although some evidences support the hypothesis that ECs are more thermosensitive than other stromal cells, these data do not clarify whether HT is more cytotoxic on tumor ECs than on normal Ecs.^^'^^

Tumor Angiogenesis Angiogenesis, the formation of new blood vessels from preexisting ones, occurs primarily during embryonic development, although it also participates in many adult physiological processes such as the female reproductive cycle and wound healing. In adults, the vascular network is quiescent and angiogenesis is normally tri^ered only locally and transiendy. Angiogenesis is essential for tumor progression. During the premalignant stages of tumor development, cancer cells activate the quiescent vascidature to produce new blood vessels through an angiogenic switch. ^^ Without blood vessels, tumors can not grow beyond a critical size or metastatize to another organ. The control of tumor angiogenesis is separate from that of cancer cell proliferation. This raises the possibility that anti-angiogenic drugs can offer a treatment that is complementary to traditional chemotherapy and radiotherapy that directly target tumor cells. A balance between pro- and anti-angiogenic molecules rebates the process of angiogenesis. It is now widely accepted that the "angiogenic switch" is "off" when the effect of pro-angiogenic molecules is balanced by the one exerted by anti-anriogenic molecules, whereas is "on" when the net balance is tipped in favor of angiogenesis. Pro- and anti-angiogenic molecules can emanate from cancer cells, ECs, stromal cells, blood and the extracellidar matrix. Among such molecides, there are various soluble factors inducing angiogenesis and some endogenous inhibitors. The members of both families of VEGFs and angiopoietins have a predominant role in the new vessel formation, in the stimulation of proliferation, migration and differentiation of Ecs.^^ Tumor vessels develop with two distinct mechanisms: by sprouting or by inmssusception. The first type of angiogenesis involves sprouting of capillaries from preexisting blood vessels. In this case ECs degrade the extracellular matrix, migrate and proliferate, allowing the formation of coherent extension from the primary vessel.^^ The mechanism of intussusception involves the splitting of preexisting vessels by proliferation of ECs within a vessel.^^ This proliferation results in the formation of a large lumen that is then split by insertion of tissue colunms. Moreover, tumor cells can also grow around an existing vessel to form a perivascidar cuff or recruit circulating endothelial precursors.^^ VEGFs and angiopoietins are activators of all these angiogenesis mechanisms but their excessive tumor production causes the formation of vessels struaurally and frmctionally abnormal. Tumor vasculature is highly disorganized compared to normal vessels, with tortuous and dilated vessels, and excessive branching and shunts. Consequendy, blood flow is chaotic and vessels are leaky.^ Indeed, angiogenesis enhances the entry of tumor cells into the circulation by providing an increased density of immature, highly permeable blood vessels that have litde basement membrane and fewer intercellular junctional complexes than normal mature vessels. Various anti-angiogenic approaches to treat tumors are already in clinical trials, alone or in combination with conventional therapies.^^"^^ Strategies to inhibit angiogenesis includes the use of molecules that: (i) interfere with angiogenic ligands or their receptors (neutralizing antibodies to VEGF, synthetic inhibitors of VEGF receptor signaling), (ii) increase the level of endogenous inhibitors (angiostatin, endostatin), (iii) target tumor vasculature with different mechanism (TNP-470, Thalidomide, Combrestatin A-4) (iiii) inhibit matrix metalloproteinases (Marimastat, AG3340).^^ Endothelial cell-specific endogenous inhibitors, such as endostatin and angiostatin, appear particularly promising in preclinical studies and are actually in phase I of clinical trials.^ However, the most effective strategy will most likely be to combine angiogenesis inhibitors with traditional cancer therapies. Recent reports testify the power of such combination approaches: the tumor treatment with radiation combined with angiostatin or antibody anti-VEGF greatiy enhances the effects of radiation alone.^^'^^

Hyperthermia and Angiogenesis: Results and Perspectives

95

Figure 1. Effects of different temperatures of heating (37, 39, 41, 43°C) on in vitro angiogenesis. A) ECs plated on three-dimensional extracellular matrix preparation at the temperature of 43''C, spontaneously differentiate into multicellular capillary-like structures. B-D) a significant inhibition of morphogenesis is evident at 4 r C (C) and 43°C (D), while at 39''C there was only a slight reduction of tube formation (B).

Molecular Mechanisms of Angiogenesis Inhibition by HT Inhibition of angiogenesis has been suggested to play a role in tumor regression activity exerted by HT.^^ Since the 1960s a numbers of investigators have described and categorized the effects of H T on microvessels in vivo and on cultured ECs in vitro. Both ECs and microvessels can be lethally damaged by the H T doses used in antineoplastic therapy. Some studies have shown that the thermal sensitivity of ECs is dose-dependent within the therapeutic range of 42-45°C/30 min.^ Furthermore, proliferating capillary ECs are clearly more thermosensitive than nonproliferating cells. Since neoplasms contain a larger proportion of proliferating EC than normal tissues do,^ H T might damage preferentially the neoplastic microvasculature over the adjacent normal vasculature. Even though Fajardo et al demonstrated that H T inhibits angiogenesis in a dose-related manner,^^ the exact mechanisms by which H T exerts its anti-angiogenic activity are not clearly understood. Obviously it must be due to interference with one or more of the midtiple steps in the process of angiogenesis, from endothelial cell migration through EC replication to remodelling.^ '^^ Recently, some studies have been made to better address the molecidar aspects of this biological effect.^^'^^ H T at 42°C suppresses the gene expression and thus the production of VEGF in human fibrosarcoma HT-1080 cells and inhibits VEGF in vitro angiogenic action on human umbilical vein endothelial cells (HUVEC). These results suggest that H T acts as an anti-angiogenic tool by suppressing the expression of tumor-derived VEGF production thereby inhibiting endothelial-cell proliferation and extracellular matrix remodelling in blood vessels. Moreover, H T at 43°C for 1 hour is able to completely block the in vitro differentiation of ECs into capillary-like structures, without affecting EC survival and proliferation rate (Fig. 1).^^ This effect is not caused by direct cytotoxicity, but is dependent on modulation of

96

Hyperthermia in Cancer Treatment: A Primer

20 18 16

I• I I II Vehicle

VEGF-A,65

VEGF.A,65 WW

VEGF-A,65 fAbaPAI-1

VEGF.A,65 fHlf AbaPAI-1

Figure 2. Effects of FiT on angiogenic response to VEGF-A165 of heat-treated CAM. In this assay gelatin sponges, adsorbed with VEGF-A165 or vehicle alone, are implanted on the top of growing chorioallantoic membranes (CAMs). After two days of stimulation, VEGF-A165 induces a strong angiogenic response in the CAM tissue, as shown in thefigureabove by the number of new vessels formed after stimulation with the angiogenic factor. Two hours of heat treatment at 43°C is sufficient to inhibit the VEGF-Ai65-induced angiogenesis in CAM, while the addition of a neutralizing antibody against PAI-1 is able to abrogate the inhibitory effect of FIT on angiogenesis. angiogenesis-involved genes. The gene profile analysis performed on heated ECs clearly shows that FiT activates a specific gene response that involves the transcription of the human plasminogen activator inhibitor-1, PAI-1, the key regulator of the plasminogen activation pathway. This is a proteolytic cascade implicated in many physiological and pathological processes, including vascular thrombosis, metastasis diffusion, inflammation and angiogenesis. During angiogenesis ECs secrete many extracellular matrix proteases, such as human urokinase plasminogen activator (u-PA) and matrix metalloproteases, (MMPs) that allow EC extravasation, invasion of the stromal space and basement membrane remodelling. After FiT, elevated levels of endogenous PAI-1 (the inhibitor of the above-mentioned proteases) in ECs, are sufficient to inhibit angiogenesis in vitro and in vivo. The neutralization of PAI-1 activity (with neutralizing anti PAI-1 antibody or in PAI-1 knock -out mice) is sufficient to partially block the anti-angiogenic effect of FIT both in vitro and in vivo angiogenic models (Fig. 2). These results indicate that the heat-mediated PAI-1 induction is an important pathway by which FIT exerts its anti-tumor activity thereby representing a rationale for a combined cancer therapy based on FiT associated with anti-angiogenic molecules.

Perspectives Clinically, FiT alone has no role to play in the curative treatment of tumors, but significant benefit has been reported in a number of clinical studies when FiT and radiation are combined."^^ Unfortunately, temperatures of around 42.5-43°C are required to efficiently enhance radiation damage, and these temperatures are difficult to obtain clinically. The failure to heat tumors to effective temperatures reduces the benefits of combined treatment. Growing evidence shows that the combination of cytotoxic drugs targeting vasctdature with FiT elicits a significant tumor response. In particidar, combretastatin A-4 and vinblastine enhance the antivascular activity of local FiT and induce a prolonged tumor growth delay. ' Moreover, the pretreatment with vascular target agents enhances the efficacy of FiT, performed at 40.5-41.5°C, alone or combined with radiation therapy."^ The combined antitumor activity of

Hyperthermia and Angiogenesis: Results and Perspectives

97

vinblastin, combretastatin and HT may be related to both their antiangiogenic activity and to direct disruption of tumor blood vessels. In a similar manner, HT, above 42 °C exhibits antivascular and antiangiogenic activity in in vitro experiments and in murine tumor models. The recent evidence that angiogenic vasculature is a target of HT tumor treatment, opens new approaches for combined therapies with angiogenesis inhibitors and HT. Blocking tumor neovascularization is a promising strategy to inhibit tumor growth, and various antiangiogenic drugs are actually in clinical trials. In spite of this, the failure of some angiogenesis inhibitors in advanced clinical trials suggests that they might be combined with other conventional drugs to be effective.^^ Likewise, recent studies have demonstrated that combination of radiation with anti-angiogenic drugs increases the anti-tumor effects of radiation and that VEGF-A expression is induced in tumor cells in vitro and in vivo after exposure to ionizing radiation. These last observations suggest that radiation-induced VEGF-A specifically protects tumor capillaries from the toxic effects of radiation.^^' ' ^ Thus, it is tempting to speculate that a treatment schedide including HT, radiation therapy and anti-angiogenic compounds could overcome the problem of ineffective heating of tumor, improve the efficacy of radiotherapy in tumor killing and enhance the anti-tumor activity of angiogenesis inhibitors.

References 1. Hildebrandt B, Wust P, Ahlers O et al. The cellular and molecular basis of H T . Crit Rev Oncol Hematol 2002; 43(l):33-56. 2. Gerweck LE. H T in cancer therapy: The biological basis and unresolved questions. Cancer Res 1985; 45:3408-14. 3. Li GC, Mivechi N F , Weitzel G. Heat shock proteins, thermotolerance, and their relevance to clinical H T . Int J H T 1995; ll(4):459-88. 4. Coss R, Linnemanns W . The effects of H T on the cytoskeleton, a review. IntJ H T 1996; 12(2): 173-96. 5. Hahn G. H T and Cancer. NewYork: Plenum, 1982. 6. Overgaard J, Poulsen HS. Effect of H T and environmental acidity on the proteolytic activity in murine ascites tumor cells. J Natl Cancer Inst 1977; 58(4):1159-6l. 7. Streffer C. Aspects of metabolic change after H T . Recent Results Cancer Res 1988; 107:7-16. 8. Henle KJ, Leeper DB. Effects of H T (45 degrees) on macromolecular synthesis in Chinese hamster ovary cells. Cancer Res 1979; 39(7 Pt l):2665-74. 9. Song CW. Effect of local H T on blood flow and microenvironment: A review. Cancer Res 1984; 44(10 Suppl):4721s-30s. 10. Song CW, Park H, Griffin RJ. Improvement of tumor oxygenation by mild H T . Radiat Res 2001; 155(4):515-28. 11. Griffin RJ, Okajima K, Barrios B et al. Mild temperature H T combined with carbogen breathing increases t u m o r partial pressure of oxygen ( p 0 2 ) and radiosensitivity. Cancer Res 1996; 56(24):5590-3. 12. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: A review. Cancer Res 1989; 49(23):6449-65. 13. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000; 407(6801):249-57. 14. Brown SL, H u n t JW, Hill RP. Differential thermal sensitivity of tumour and normal tissue microvascular response during H T . Int J H T 1992; 8(4):501-14. 15. Fajardo LF, Prionas SD. Endothelial cells and H T . Int J H T 1994; 10(3):347-53. 16. Fajardo LF, Schreiber AB, Kelly NI et al. Thermal sensitivity of endothelial cells. Radiat Res 1985; 103(2):276-85. 17. Hanahan D , Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996; 86:353-64. 18. Folkman J. Clinical applications of research on angiogenesis. N Engl J Med 1995; 333:1757-63. 19. Hanahan D , Weinberg RA. The hallmarks of cancer. Cell 2000; 100(l):57-70. 20. Yancopoulos G D , Klagsbrun M, Folkman J. Vasculogenesis, angiogenesis, and growth factors: Ephrins enter the fray at the border. Cell 1998; 93(5) :661-4. 2 1 . Bussolino F, Mantovani A, Persico G. Molecular mechanisms of blood vessel formation. TiBS 1997; 22:251-56. 22. Patan S, M u n n LL, Jain RK. Intussusceptive microvascular growth in a human colon adenocarcinoma xenograft: A novel mechanism of tumor angiogenesis. Microvasc Res 1996; 51(2):260-72. 23. Holash J, Maisonpierre PC, Compton D et al. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science 1999; 284(5422): 1994-8.

98

Hyperthermia in Cancer Treatment: A Primer

24. Jain RK. Determinants of tumor blood flow: A review. Cancer Res 1988; 48(10):264l-58. 25. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995; 1:27-31. 26. Ferrara N, Alitalo K. Clinical applications of angiogenic growth factors and their inhibitors. Nat Med 1999; 5(12):1359-64. 27. Kerbel R, Folkman J. Clinical translation of angiogenesis inhibitors. Nat Rev Cancer 2002; 2(10):727-39. 28. O'Reilly MS, Holmgren L, Shing Y et al. Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 1994; 79:315-28. 29. Eder Jr JP, Supko JG, Clark JW et al. Phase I clinical trial of recombinant human endostatin administered as a short intravenous infusion repeated daily. J Clin Oncol 2002; 20(18):3772-84. 30. O'Reilly MS, Boehm T, Shing Y et al. Endostatin: An endogenous inhibitor of angiogenesis and tumor growth. Cell 1997; 88(2):277-85. 31. Mauceri HJ, Hanna NN, Beckett MA et al. Combined effects of angiostatin and ionizing radiation in antitumour therapy. Nature 1998; 394(6690):287-91. 32. Gorski DH, Mauceri HJ, Salloum RM et al. Potentiation of the antitumor effect of ionizing radiation by brief concomitant exposures to angiostatin. Cancer Res 1998; 58(24):5686-9. 33. Hobson B, Denekamp J. Endothelial proliferation in tumours and normal tissues: Continuous labeUing studies. Br J Cancer 1984; 49(4):405-13. 34. Denekamp J, Hobson B. Endothelial-cell proliferation in experimental tumors. British J Cancer 1982; 46:711-20. 35. Fajardo LF, Prionas SD, Kowalski J et al. HT inhibits angiogenesis. Radiat Res 1988; ll4(2):297-306. 36. Furcht LT. Critical factors controUing angiogenesis: Cell products, cell matrix, and growth factors. Lab Invest 1986; 55(5):505-9. 37. Folkman J. Angiogenesis: Initiation and control. Ann NY Acad Sci 1982; 401:212-27. 38. Roca C, Primo L, Valdembri D et al. HT inhibits angiogenesis by a plasminogen activator inhibitor-1 dependent mechanism. Cancer Res 2003; 0:0. 39. Sawaji Y, Sato T, Takeuchi A et al. Anti-angiogenic action of HT by suppressing gene expression and production of tumour-derived vascular endothelial growth factor in vivo and in vitro. Br J Cancer 2002; 86(10): 1597-603. 40. Falk MH, Issels RD. HT in oncology. Int J HT 2001; 17(1):1-18. 41. Nielsen OS, Horsman M, Overgaard J. A future for HT in cancer treatment? Eur J Cancer 2001; 37(13):1587-9. 42. Horsman MR, Murata R. Combination of vascular targeting agents with thermal or radiation therapy. Int J Radiat Oncol Biol Phys 2002; 54(5):1518-23. 43. Wust P, Hildebrandt B, Sreenivasa G et al. HT in combined treatment of cancer. Lancet Oncol 2002; 3(8):487-97. 44. Eikesdal HP, Bjerkvig R, Dahl O. Vinblastine and HT target the neovasculature in BT(4)AN rat gUomas: Therapeutic implications of the vascular phenotype. Int J Radiat Oncol Biol Phys 2001; 51(2):535-44. 45. Murata R, Overgaard J, Horsman MR. Combretastatin A-4 disodium phosphate: A vascular targeting agent that improves that improves the anti-tumor effects of HT, radiation, and mild thermoradiotherapy. Int J Radiat Oncol Biol Phys 2001; 51(4):1018-24. 46. Gorski DH, Beckett MA, Jaskowiak NT et al. Blockage of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation. Cancer Res 1999; 59(l4):3374-8. 47. Geng L, Donnelly E, McMahon G et al. Inhibition of vascular endothelial growth factor receptor signaling leads to reversal of tumor resistance to radiotherapy. Cancer Res 2001; 6l(6):24l3-9.

CHAPTER 7

Vascular Effects of Localized Hyperthermia Debra K. Kelleher* and Peter Vaupel Abstract

W

hen hyperthermia is applied in vitro, no fundamental differences can be seen between the response to normal and tumor cells. In vivo however, selective damage of tumor cells can be achieved and this phenomenon can be largely attributed to a number of characteristic properties of the blood vessels within solid tumors. Changes in blood flow induced by hyperthermia can influence the response of a tumor to heat either by affecting the delivery of heat through changes in heat dissipation or by a modulation of the tumor microenvironment which may in turn affect the thermosensitivity of tumor cells. Studies in experimental and human tumors suggest however that an accurate prediction of changes in blood flow during heating is not possible so that such changes cannot be used as a basis for the combination of hyperthermia with other therapy modalities. Even so, when the underlying mechanisms responsible for the antitumor effects of a combination of hyperthermia with either radiotherapy or chemotherapy are considered, it becomes evident that either an increase or a decrease in blood flow could potentially contribute to the cytotoxic effect. A further interesting approach is in the use of antivascular drugs or vascular-targeted photodynamic therapy in order to specifically reduce tumor blood flow prior to or during hyperthermia treatment. Experimental data suggest that a considerable enhancement of the antitumor effect can be achieved with this approach. By reducing heat dissipation, such an approach may in future also be of use in overcoming problems related to insufficient temperature increases frequently seen in the clinical setting.

Introduction The impact of tumor blood flow on the success of different forms of cancer therapy is already well established.^ When hyperthermia is considered, blood flow changes induced by this therapy form can influence the tumor response to heat in two ways. Firstly, changes in tumor blood flow will affect the delivery of heat to the tumor mass by causing changes in heat dissipation away from the tumor. Secondly, changes in blood flow will affect the metabolic microenvironment and are thus capable of modulating the thermosensitivity of tumor cells. With this in mind, numerous investigations have been undertaken to assess changes in blood flow in tumor and normal tissues during hyperthermia. These are summarized below. At the same time, blood flow changes occurring when hyperthermia is combined with other therapy modalities are examined.

*Corresponding Author: Debra K. Kelleher—Institute of Physiology and Pathophysiology, University of Mainz, Duesbergweg 6, 55128 Mainz, Germany. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer^ edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

100

Hyperthermia in Cancer Treatment: A Primer

Vascular ££Fects of Localized Hyperthermia When the effects of hyperthermia on normal and tumor cells are assessed under in vitro conditions, no fundamental differences are found with respect to thermosensitivity, The therapeutic benefits of hyperthermia cannot therefore be attributed to a greater susceptibility of tumor cells to heat. In vivo however, a quasi-selective damage of tumor cells can be achieved when the tissue is heated to temperatures between 40 and 44°C. A number of characteristic properties of blood vessels in solid tumors appear to be responsible for this "selectivity". Microscopic examination reveals a number of structural and functional features of tumor blood vessels including tortuosity, excessive branching, blind endings, lack of smooth muscle in the vessel walls together with a lack of pericytes, interrupted endothelial linings and basement membranes. Additionally, a hierarchical organization is missing and significant arterio-venous shimt perfusion and temporal variations in blood flow (including temporary stasis) have been shown to occur.^' The competence of the vasculature to regulate flow in response to changes in a tissues demands is limited in tumor tissue compared to normal tissue. Subsequendy, an insufficient nutrient and oxygen supply in tumor tissue results in the development of hypoxic tissue areas, acidosis and energy depletion.^ When cells in vitro are artificially exposed to hypoxic conditions, low pH or low intracellular ATP levels, they are found to be more susceptible to hyperthermia, so that the development of such conditions within tumors as found in vivo can at least partially explain the enhanced sensitivity of tumors to hyperthermia. The relative importance and the individual role of hypoxia, low pH and depleted energy levels is difficult to decipher since they are closely interrelated, so that alterations in any one of these parameters may affect the others. When the physiological response of nonnal tissue to being heated up to temperatures between 4l°C and 45°C is investigated, an increase in perfusion is typically seen since this is the major route by which heat is normally dissipated away from tissues such that a deleterious heat load can be avoided. Numerous investigators have attempted to quantify this response in terms of the increase in blood flow seen, and these investigations have been summarized in reviews by Vaupel and Song et al,^ where increases in skin perfusion and skeletal muscle perfusion of up to a factor of 15 and 10 respectively were found, although the magnitude of flow increase appears to vary considerably depending on the species and tissue investigated, the technique used for heating and the heating protocol. Comparable measurements in tumor tissue (both in experimental and human tumors^'^) have revealed pronounced tumor-to-mmor variation, with increases, decreases and lack of change in tumor perfusion having been reported. While some of this variability may again be explained by factors such as different heating-up rates, heating duration, thermal doses, temperature monitoring and heating systems, the use of tumors with different histology, implantation or growth sites and different tumor volumes together with the variability in the response to hyperthermia of different areas within the same tumor need to additionally be considered. When the available data are taken together, it appears that the change in blood flow upon heating is generally much greater in normal tissue than in tumors. Where increases in tumor perfusion occur, these are usually no greater than 1.5-2.0 fold.^'^ Within a single tumor entity, the changes seen depend upon the degree of heating applied, as exemplified by Song and colleagues who carried out investigations in SCK tumors in mice.^ This study showed a significant increase in blood flow following heating to 42.5°C for 1 h followed by a decline thereafter with recovery to the control level within 5 h. Heating to 43.5°C induced an initial increase in perfusion over the first 30 min followed by a pronounced reduction from which the tumor had not completely recovered even 24 h after heating. Upon 44.5°C hyperthermia, only a decrease in blood flow occurred, which became even more pronounced after completion of heating. Similar temperature-related effects were also seen in the R3230 adenocarcinoma growing in rats.^^ The variability in the response to heating within a single tumor model has also been assessed in a study of the micro regional physiology of rat DS-sarcomas during hyperthermia.^^ Here, tumors of different sizes were heated to 44°C for 60 min and micro regional perfusion was assessed

Vascular Effects ofLocalized Hyperthermia

101

250 tumor periphery 1 tumor periphery 2 tumor center

200

£

150

LL

O 100

50 '^'^CJCXD

^..._..j •10

0

,

^ 10

^

1 20

^

1 30

,

^ 40

^

^ 50

^

1 60

,

^ , 70

.^ 80

.^

p__, 90

I

,

100

I 110

time (min)

-stumor periphery 1 tumor periphery 2 tumor center

-^ - , _ _ ^ _ _

•10

0

10

20

30

_ ^ — ^ _ _ — , — J — , — ^ — , — ^ — , . . _ . ^ — , . — ,

40 50 60 time (min)

70

80

90

100

110

Figure 1. Relative red blood cell flux (rel. LDF) during hyperdiermia in DS-sarcomas. Measurements were made simultaneously from two different sites within each tumor. The upper panel shows results from a tumor with a volume of 0.9 ml and the lower panel from a tumor with a volume of 2 ml. The shaded area indicates the time over which heating was applied. Adapted from reference 11.

continuously and simultaneously at multiple sites within the tumor using the laser Doppler technique. The data obtained indicated substantial inter-site variability which occurred in all tumor size ranges studied and was independent of the measurement site and of the actual temperature reached at individual measurement sites. Examples of measurements performed in individual tumors are shown in Figure 1. In light of these findings, it seems probable that the variations attributed to inter-tumor differences in other studies are partly caused by intra-tumor variations in the response to hyperthermia. At the same time, it would appear that measurements made at individual sites within a given tumor may not necessarily be representative of changes taking place at other sites within the same tumor. It is therefore questionable whether

102

Hyperthermia in Cancer Treatment: A Primer

Table 1. Major mechanisms involved in the shutdown of tumor blood flow upon hyperthermia Intravascular events "sludging" erythrocyte aggregation platelet aggregation thrombus formation leukocyte adhesion high blood viscosity/viscous resistance to flow erythrocyte crenation intensified acidosis leading to erythrocyte rigidity formation of fibrinogen gel Vessel wall alterations endothelial swelling endothelial degeneration vessel wall rupture vasoconstriction in larger pre-existing arterioles at tumor periphery increased geometric resistance to flow Extravascular events interstitial edema hemorrhage into interstitial space Opposing host tissue/tumor mechanisms reactive hyperemia in adjacent normal tissue "steal" phenomenon (diversion of blood flow from tumor to normal tissue)

such measurements can provide information which is of relevance for an assessment of average changes in perfusion v^dthin a tumor, and highlights the difficulties encountered in accurately predicting the biological behavior of a tumor undergoing hyperthermia on the basis of such single site measurements. Nevertheless, when mean values from several sites within individual tumors were calculated in the latter study, the extent of the decrease in perfusion at the end of the measurement period was found to increase with enlarging tumor size. Since, in the clinical setting, hyperthermia is usually applied during a number of sessions, the effects of multiple heatings on tumor blood flow are of particular significance. Here again however, no clear-cut effects can be elucidated from the available literature, with contradictory results being obtained. Whereas Nah and colleagues^^ showed that heating the rat R3230 tumor to 42.5°C for 1 h induced vascular thermal adaptation so that when a second heating was applied, much greater increases in blood flow were seen, others saw an enhanced shutdown of the tumor microcirculation following sequential heat treatment. ^^ When a shutdown of tumor blood flow occurs upon hyperthermia, a wide range of mechanisms appear to be involved. These include both intra- and extravascular events, effects on the vessel wall and mechanisms in which opposing actions between host tissue and tumor tissue play a role. Table 1 shows a summary of such mechanisms which have been discussed in detail by Vaupel.

Vascular E£Fects of Combined Modalities Combined Irradiation and Hyperthermia Hyperthermia appears to be one of the most potent radiosensitizers known. ^ The complementary effect seen when radiotherapy and hyperthermia are combined occurs for a number of reasons. Firsdy, cells whose radiosensitivity is compromised by the presence of hypoxia and low pH areas are patticularly sensitive to heat. Secondly, cells in the late S-phase are resistant to

Vascular Effects ofLocalized Hyperthermia

103

irradiation but show an increased susceptibility to hyperthermia.^^ Furthermore, although studies in which blood flow changes during hyperthermic treatment of human tumors have been assessed have delivered inconclusive results with both increases and decreases being reported, hyperthermia-related changes in tumor blood flow, whether increases or decreases, may have an impact on the therapeutic efficacy. When tumor blood flow increases, an accompanying increase in oxygen delivery will occur and this should in turn result in increased radiosensitivity. Decreases in tumor blood flow on the other hand will reduce the tumor's oxygen supply, leading to hypoxia, making the tumor more resistant to standard radiotherapy, but at the same time rendering it highly susceptible to hyperthermia. A further important mechanism is that hyperthermia inhibits—probably via an effect on cellular proteins—the cellular repair of radiation-induced DNA damage. A number of animal studies have attempted to examine blood flow changes when irradiation and hyperthermia are applied. These investigations have generally shown that the greatest anti-tumor effect could be found when the two treatment components were applied simultaneously or when irradiation was followed closely by hyperthermia treatment. With increasing intervals between the application of irradiation and hyperthermia, the antitumor effect was seen to be reduced. In the latter of these studies. Song and colleagues attempted to assess whether vascular changes might be involved in this effect and found that when tumors were first irradiated (20 Gy, single dose) and subsequendy heated (43"C, 1 h), an increase in blood flow occurred which was generally greater than that seen upon irradiation or heat alone and persisted for approximately 4 weeks post treatment. These combined effects on blood flow diminished with increasing inter-treatment intervals. When these experimental data are extrapolated to the clinical setting it should however be remembered that although combination treatments involving irradiation and hyperthermia may show maximal cytotoxicitv when the two components are applied simultaneously, this will rarely be logistically possible. ^

Combined Chemotherapy and

Hyperthermia

The capability of hyperthermia to enhance the effects of chemotherapy in vitro or in vivo has been extensively reviewed by Dahl. The effects of a number of drugs, most prominendy the alkylating agents, can be potentiated by combination with hyperthermia. The main mechanisms involved appear to be increased drug uptake into the tumor cells, enhanced DNA damage, and—^where an increase in blood flow concurrently occurs—increased drug delivery (i.e., improved pharmacokinetics) to the tumor. Additionally, an impact on the pharmocodynamics of anticancer drugs in tumor cells undergoing heating needs to be considered. As with combined radiotherapy and hyperthermia, maximal effects were found when chemotherapy and hyperthermia were applied simultaneously.

Combined Photodynamic

Therapy and

Hyperthermia

Whereas the enhancement of tumor response in vivo is now clinically well-established for the combination of hyperthermia with radiotherapy or chemotherapy, the possibility of combining photodynamic therapy and hyperthermia has received less attention, despite the fact that there is considerable evidence from in vitro studies that such an application may result in a synergistic effect. ^^'^^ Investigations in vivo also showed promising effects. Henderson et al used a combination of Photofrin-based photodynamic therapy and microwave-induced hyperthermia for treating experimental fibrosarcomas in mice and showed that the combination of these modalities led to a potentiated cytotoxic effect with 45% of animals showing long-term tumor control in comparison to less than 10% with either hyperthermia or photodynamic therapy alone. In a similar study of rats bearing rhabdomyosarcomas treated with photofrin-based photodynamic therapy and a radiofrequency heating method for interstitial hyperthermia, Levendag et al obtained a 41 % cure rate, whereas no animals were cured when either of the treatment components were applied alone. Likewise, synergistic effects were reported for this therapy combination in the chick chorioallantoic membrane model.'^ Some

104

Hyperthermia in Cancer Treatment: A Primer

studies, while not providing a thorough assessment of the optimum sequencing of the therapy components in vivo, did at least attempt to address this question. For example, in two studies,^ '^^ the sequence of application of the therapy components was found to have a dramatic effect on treatment outcome, with potentiation occurring when photodynamic therapy was applied before hyperthermia, whereas a reversal of the sequence produced only an additive effect. This dependency of treatment order on the extent of tumor response was also found in the in vitro studies discussed above. In general, the efficacy of a simultaneous application of the two components was however not investigated in any of these studies, primarily due to the technical difficulties of simultaneously inducing tissue heating and of delivering light for activation of the photosensitizer, despite the fact that a therapy combination which could be easily applied in a single session, would nevertheless, from a clinical point of view, be favorable. As far as mechanisms are concerned, traditionally in photodynamic therapy, it was thought that a photosensitizer—in order to be effective—^should primarily target the tumor cell. Effects on the vasculature, although considered to be important in the tumor eradication process, were believed to be detrimental to the photodynamic effect if they occurred during treatment, since even partial vascidar shutdown might lead to a decrease in oxygen delivery.^ As is the case with radiotherapy and some forms of chemotherapy, the photodynamic effect is inherently oxygen-dependent. ^Thus, an induction of oxygen deprivation was thought to be unfavorable since it might result in a "self-limitation" of the treatment effect. More recendy, several investigators have nevertheless examined a number of antitumor approaches targeted primarily at the tumor vasculature. These have included photodynamic therapy protocols aimed at inducing primarily a vascular effect rather than a direct cytotoxic effect.^ '^^ Such an approach is also now being clinically applied in verteporfin-based photodynamic treatment of age-related macular degeneration, where the blood vessels are the sole treatment target.^^ The residts of studies in tumors using second generation photosensitizers such as bacteriochlorophyll-serine or palladium-pheophorbide as photosensitizers suggest that these agents have a primarily anti-vascular action. Zilberstein et al, for example, found that the maximum antitumor effect was achieved when illumination coincided with the highest concentration of bacteriochlorophyll-serine in blood (i.e., direcdy after drug injection), and that this treatment resulted in extensive vascidar damage.^ ^ Palladium-pheophorbide appears to have a similar mode of action with extensive vascular damage having been found in rat C6 glioma xenografts'^ and in human prostatic small cell carcinoma xenografts,^' and changes suggestive of a primarily anti-vascular effect being seen during laser Doppler studies in rat tumors' and with blood oxygenation level-dependent (BOLD) contrast magnetic resonance imaging (MRI) in experimental melanoma.'^ On the basis of these effects the possibility of combining an antivascular photodynamic treatment approach with hyperthermia has now also been considered.' ''^ In these studies, bacteriochlorophyll-serine was used as the photosensitizer and an important aspect of the methodology employed was a simultaneous application of photodynamic therapy and heat using a single irradiator. Pronounced inhibitions of tumor growth were seen with this simultaneous combined treatment, with the effects being considerably greater than with either photodynamic therapy or hyperthermia alone. The probability of the tumors not reaching the target volume within 90 days following combined treatment was 78%, whereas with photodynamic therapy alone it was 36% and with hyperthermia alone 15%. The effects of the combination therapy using bacteriochlorophyll serine on tumor perfusion and oxygenation were subsequendy also evaluated. When hyperthermia alone was applied, the tumor perfusion was found to steadily increase reaching levels 80% greater than initial values, with this effect remaining for the duration of the treatment period. Corresponding to this, an increase (approximately 50%) in oxygenation also occurred. In contrast, under combined hyperthermia and photodynamic therapy, a dramatic decrease in tumor perfusion of approximately 90% was seen, with tumor oxygenation concurrendy reaching levels of anoxia (Fig. 2). In light of these effects, it is a paradox that the issue of hyperthermia was seen as a "problem** in the application of photodynamic therapy in the past. Early photodynamic studies using high fluence rates

Vascular Effects ofLocalized Hyperthermia

105

250 n

200

Bchl-ser 0 250

200

^

150 CM

O

I 100 4

f'/VC^W"^''^ \

-10

0

10

20

30

40

50

'

I

60

70

80

time (min) Figure 2. Relative red blood cell (RBC) flux (upper panel) and tumor oxygen tension (p02; lower panel) as a function of time during localized hyperthermia (43"C, 60 min; filled circles) or combined bacteriochlorophyll-serine(Bchl-ser)-based photodynamic therapy and hyperthermia (open circles) treatment. Data points indicate mean values ± SEM for at least five tumors. Adapted from reference 37.

106

Hyperthermia in Cancer Treatment: A Primer

unavoidably resulted in uncontrolled (and generally unmonitored) tumor heating. As pointed out by Kinsey and coUeagues,^^ initial reports of cell killing solely due to a photodynamic process were presumably only partially correct since tissue heating will most certainly have contributed to the antitumor effect. A retrospective evaluation of the role of hyperthermia in these early studies on photodynamic therapy is not possible without detailed information on the tissue temperatures achieved due to the fact that the extent of tissue heating occurring during photodynamic therapy is dependent on a range of variables including light absorption and scattering, exposure time and tissue properties such as thermal conduction and perfusion, and is therefore difficult to predict.^^ At the same time, uncontrolled and immonitored tissue heating appears not to be a reasonable option for enhancing the photodynamic effect since the temperature window for this enhancement is apparendy narrow, lying between approximately 40 and 44.5°C. ^' The simultaneous application of hyperthermia and photodynamic therapy as described by Kelleher et al^ '^^ and outlined above involved a controlled hyperthermia treatment. It is also of interest that a potentiation of the effects of hyperthermia and photodynamic therapy could additionally be achieved using 5-aminolaevulinic acid, a photosensitizer requiring cellular uptake and conversion to a photoactive substance, which appears to rely predominandy on a direct cytotoxic effect on tumor cells rather than on vascular effects. The underlying mechanisms of the effects seen when hyperthermia is combined with either a "vascular'* or "nonvascular** targeted photosensitizer are of considerable interest and appear to be numerous. Since the photodynamic process has been shown to be inherendy temperature-dependent, ^ photodynamic therapy should show an increased cytotoxic effect whenever it is performed in heated tissues, irrespective of which photosensitizer is used. Additionally, in vitro studies have indicated that photodynamic therapy and hyperthermia can have a concerted effect on certain molecules or supramolecidar structiyes^^ and that photodynamic therapy-induced repairable lesions may be converted to irreparable ones when hyperthermia is additionally applied. Modification of the metabolic microenvironment upon therapy may also influence the efficacy of the treatment components since increased lactic acid formation was seen upon treatment which may result in a lower pH in tumor tissue.^^'"^^ When bacteriochlorophyll-serine-based photodynamic therapy was applied in combination with hyperthermia, injection of the photosensitizer close to the time of illumination was necessary due to the short biological half-life of this substance. ^ In the protocol developed for the combined therapy, bacteriochlorophyll-serine was injeaed 10 minutes after conmiencement of hyperthermia, at a time when the tumor temperature had reached approximately 40 °C and was still rising. The rationale for the choice of this timing was based on the findings of the laser Doppler studies which indicated that there was an approximately 10% increase in tumor perftision at this time, which could therefore be exploited to enhance the delivery of the photosensitizer to the tumor tissue. The selection of a vascular-targeted photodynamic therapy for combination with hyperthermia was also considered to be of interest in terms of achieving adequate tissue heating since, in the clinical setting, a major obstacle to an effective hyperthermia treatment is the failure of the protocol used to attain a therapeutically relevant temperature elevation in the tumor tissue. While this may pardy be due to limitations of the heating equipment employed, the relatively high perftision rates which have been found in human tumors may also lead to an undesirable dissipation of heat away from the malignancy.^ If the tumor perftision, and thus heat dissipation, is restricted by a vascular-based photodynamic therapy, then a more effective heating should be possible, an aspect which could be of particular relevance in the clinical setting.

Combination ofHyperthermia tuith Antivascular Drugs A further approach to enhancing the antitumor effects of hyperthermia is the combination with agents able to preferentially destroy an established tumor vessel network (vascular targeted therapy). This approach differs to antiangiogenic therapies since it attempts to exploit the differences between the vessels found in normal and tumor tissues in order to disrupt the

Vascular Effects of Localized Hyperthermia

107

existing tumor vasculature, rather than inhibiting the growth of new vessels. Experimental studies have shown that the use of vascular disrupting agents such as combretastatin, flavone acetic acid and DMXAA to reduce tumor blood flow can improve the response of tumors to hyperthermia. '^^ Where combretastatin was applied, the greatest response enhancement was seen when this agent was administered 2 to 6 hours prior to hyperthermia, corresponding to the time when the greatest reduction of tumor perfusion was seen. An attempt to assess the specificity of these effects of combretastatin showed that some enhancement of the susceptibility to heat occurred in normal tissues but this was generally not as pronounced as the effects occurring in tumor tissue. ^

Conclusions Despite many years of basic research on the physiological and pathophysiological actions of hyperthermia and the slowly increasing acceptance of hyperthermia as a clinical therapy option, an accurate prediction of the changes occurring in tumor blood flow during heating to therapeutically relevant temperatures is not possible. This is due to the large number of confounding variables (related both to treatment protocol and inherent tumor properties) which can influence tumor perfusion during hyperthermia. The assumption of reproducible and predictable heating-induced changes in tumor blood flow can therefore not form the premise for a combination of hyperthermia with other tumor modalities. However, conditions enhancing the effects of ionizing radiation typically reduce the effects of hyperthermia. Thus, low blood flow tumor areas will be more resistant to ionizing radiation but more susceptible to hyperthermia, whereas high blood flow areas will prove more difficult to heat but show higher radiosensitivity. In light of this complementary action, effects of hyperthermia on blood flow—in the form of either an increase or a decrease—should not prove deleterious for the effects of thermoradiotherapy. This should also be the case in instances where hyperthermia is combined with chemotherapy. A further approach, which has also been presented here is the use of photodynamic therapy or antivascular drugs to reduce tumor blood flow during or prior to hyperthermia treatment. Experimental data have confirmed that such methods can considerably enhance the antitumor effect so that clinical data will hopefully also show promise when they become available.

References 1. In: Vaupel P, Jain R, eds. Tumor Blood Supply and Metabolic Microenvironment. Stuttgart: Gustav Fischer, 1991. 2. StrefFer C. Molecular and cellular mechanisms of hyperthermia. In: Seegenschmiedt M H , Fessenden P, Vernon C C , eds. Thermoradiotherapy and Thermochemotherapy. Biology, Physiology, and Physics. Vol 1. Berlin: Springer, 1995:47-74. 3. Konerding MA, Malkusch W, Klapthor B et al. Evidence for characteristic vascular patterns in solid tumours: Quantitative studies using corrosion casts. Br J Cancer 1999; 80:724-732. 4. Vaupel P. Tumor microenvironmental physiology and its implications for radiation oncology. Semin Radiat Oncol 2004; 14:198-206. 5. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: A review. Cancer Res 1989; 49:6449-6465. 6. Vaupel P. Pathophysiological mechanisms of hyperthermia in cancer therapy. In: Gautherie M, ed. Biological Basis of Oncologic Thermotherapy. Berlin: Springer, 1990:73-134. 7. Song CW, Choi IB, Nah BS et al. Microvasculature and perfusion in normal tissues and tumors. In: Seegenschmiedt M H , Fessenden P, Vernon C C , eds. Thermoradiotherapy and Thermochemotherapy. Biology, Physiology, and Physics. Vol 1. Berlin: Springer, 1995:139-156. 8. Vaupel PW, Kelleher DK. Metabolic status and reaction to heat of normal and tumor tissue. In: Seegenschmiedt M H , Fessenden P, Vernon C C , eds. Thermoradiotherapy and Thermochemotherapy. Biology, Physiology, and Physics. Vol 1. BerUn: Springer, 1995:157-176. 9. Song C W , Lin JC, Chelstrom LM et al. T h e kinetics of vascular thermotolerance in SCK tumors of A/J mice. Int J Radiat Oncol Biol Phys 1989; 17:799-802. 10. Shakil A, Osborn JL, Song CW. Changes in oxygenation status and blood flow in a rat tumor model by mild temperature hyperthermia. Int J Radiat Oncol Biol Phys 1999; 43:859-865.

108

Hyperthermia in Cancer Treatment: A Primer

11. Kelleher DK, Engel T, Vaupel PW. Changes in microregional perfusion, oxygenation, ATP and lactate distribution in subcutaneous rat tumours upon water-filtered IR-A hyperthermia. Int J Hyperthermia 1995; 11:241-255. 12. Nah BS, Choi IB, Oh WY et al. Vascular thermal adaptation in tumors and normal tissue in rats. Int J Radiat Oncol Biol Phys 1996; 35:95-101. 13. Eddy HA, Chmielewski G. Effect of hyperthermia, radiation and adriamycin combinations on tumor vascular function. Int J Radiat Oncol Biol Phys 1982; 8:1167-1175. 14. Kampinga HH, Dikomey E. Hyperthermic radiosensitization: Mode of action and cUnical relevance. Int J Radiat Biol 2001; 77:399-408. 15. Streffer C. Biological basis of thermotherapy. In: Gautherie M, ed. Biological Basis of Oncologic Thermotherapy. BerUn: Springer, 1990:1-71. 16. Song CW, Kim JH, Rhee JG et al. Effect of X irradiation and hyperthermia on vascular function in skin and muscle. Radiat Res 1983; 94:404-415. 17. Wust P, Hildebrandt B, Sreenivasa G et al. Hyperthermia in combined treatment of cancer. Lancet Oncol 2002; 3:487-497. 18. Dahl O. Interaction of heat and drugs in vitro and in vivo. In: Seegenschmiedt MH, Fessenden P, Vernon CC, eds. Thermoradiotherapy and Thermochemotherapy. Biology, Physiology, and Physics. Vol 1. Berlin: Springer, 1995:103-121. 19. Christensen T, Wahl A, Smedshammer L. Effects of haematoporphyrin derivative and light in combination with hyperthermia on cells in culture. Br J Cancer 1984; 50:85-89. 20. Mang TS, Dougherty TJ. Time and sequence-dependent influence of in vitro photodynamic therapy (PDT) survival by hyperthermia. Photochem Photobiol 1985; 42:533-540. 21. Rasch MH, Tijssen K, Vansteveninck J et al. Synergistic interaction of photodynamic therapy with the sensitizer aluminum phthalocyanine and hyperthermia on loss of clonogenicity of CHO cells. Photochem Photobiol 1996; 64:586-593. 22. Henderson BW, Waldow SM, Potter WR et al. Interaction of photodynamic therapy and hyperthermia: Tumor response and cell survival studies after treatment of mice in vivo. Cancer Res 1985; 45:6071-6077. 23. Levendag PC, Marijnissen HPA, De Ru VJ et al. Interaction of interstitial photodynamic therapy and interstitial hyperthermia in a rat rhabdomyosarcoma—a pilot study. Int J Radiat Oncol Biol Phys 1988; 14:139-145. 24. Kimel S, Svaasand LO, Hammer-Wilson M et al. Demonstration of synergistic effects of hyperthermia and photodynamic therapy using the chick chorioallantoic membrane model. Laser Surg Med 1992; 12:432-440. 25. Chen Q, Chen H, Shapiro H et al. Sequencing of combined hyperthermia and photodynamic therapy. Radiat Res 1996; 146:293-297. 26. Henderson BW, Fingar VH. Oxygen limitation of direct tumor cell kill during photodynamic treatment of a murine tumor model. Photochem Photobiol 1989; 49:299-304. 27. Hockel M, Vaupel P. Tumor hypoxia: Definitions and current clinical, biologic, and molecular aspects. J Nad Cancer Inst 2001; 93:266-276. 28. Fingar VH. Vascular effects of photodynamic therapy. J CHn Laser Med Surg 1996; 14:323-328. 29. Fingar VH, Kik PK, Haydon PS et al. Analysis of acute vascular damage after photodynamic therapy using benzoporphyrin derivative (BPD). Br J Cancer 1999; 79:1702-1708. 30. Dougherty TJ. An update on photodynamic therapy applications. J Clin Laser Med Surg 2002; 20:3-7. 31. Zilberstein J, Schreiber S, Bloemers MC et al. Antivascular treatment of soHd melanoma tumors with bacteriochlorophyll-serine-based photodynamic therapy. Photochem Photobiol 2001; 73:257-266. 32. Schreiber S, Gross S, Brandis A et al. Local photodynamic therapy (PDT) of rat C6 glioma xenografts with Pd-bacteriopheophorbide leads to decreased metastases and increase of animal cure compared with surgery. Int J Cancer 2002; 99:279-285. 33. Koudinova NV, Pinthus JH, Brandis A et al. Photodynamic therapy with Pd-bacteriopheophorbide (TOOKAD): Successful in vivo treatment of human prostatic small cell carcinoma xenografts. Int J Cancer 2003; 104:782-789. 34. Kelleher DK, Thews O, Scherz A et al. Perfusion, oxygenation status and growth of experimental tumors upon photodynamic therapy with Pd-bacteriopheophorbide. Int J Oncol 2004; 24:1505-1511. 35. Gross S, Gilead A, Scherz A et al. Monitoring photodynamic therapy of solid tumors online by bold-contrast MRI. Nat Med 2003; 9:1327-1331. 36. Kelleher DK, Thews O, Rzeznik J et al. Water-filtered infrared-A radiation: A novel technique for localized hyperthermia in combination with bacteriochlorophyll-based photodynamic therapy. Int J Hyperthermia 1999; 15:467-474.

Vascular Effects of Localized Hyperthermia

109

37. Kelleher DK, Thews O , Scherz A et al. Combined hyperthermia and chlorophyll-based photodynamic therapy: Tumour growth and metabolic microenvironment. Br J Cancer 2003; 89:2333-2339. 38. Kinsey J H , Cortese DA, Neel H B . Thermal considerations in murine tumor killing using hematoporphyrin derivative phototherapy. Cancer Res 1983; 43:1562-1567. 39. Svaasand L O , Doiron DR, Dougherty TJ. Temperature rise during photoradiation therapy of malignant tumors. Med Phys 1983; 10:10-17. 40. Waldow SM, Henderson BW, Dougherty TJ. Enhanced tumor control following sequential treatments of photodynamic therapy (PDT) and localized microwave hyperthermia in vivo. Laser Surg Med 1984; 4:79-85. 4 1 . Kelleher DK, Bastian J, Thews O et al. Enhanced effects of aminolaevulinic acid-based photodynamic therapy through local hyperthermia in rat tumours. Br J Cancer 2003; 89:405-411. 42. Gottfried V, Kimel S. Temperature effects on photosensitized processes. J Photochem Photobiol B 1991; 8:419-430. 43. Prinsze C, D u b b e l m a n T M , Van Steveninck J. P o t e n t i a t i o n of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenase by photodynamic treatment. A possible model for the synergistic interaction between photodynamic therapy and hyperthermia. Biochem J 1 9 9 1 ; 276:357-362. 44. Chen B, Xu Y, Agostinis P et al. Synergistic effect of photodynamic therapy with hypericin in combination with hyperthermia on loss of clonogenicity of RIF-1 cells. Int J Oncol 2 0 0 1 ; 18:1279-1285. 45. Rosenbach-Belkin V, Chen L, Fiedor L et al. Serine conjugates of chlorophyll and bacteriochlorophyll: Photocytotoxicity in vitro and tissue distribution in mice bearing melanoma tumors. Photochem Photobiol 1996; 64:174-181. 46. Siemann D W , Bibby M C , Dark GG et al. Differentiation and definition of vascular-targeted therapies. Clin Cancer Res 2005; 11:416-420. 47. Murata R, Overgaard J, Horsman MR. Combretastatin A-4 disodium phosphate: A vascular targeting agent that improves the anti-tumor effects of hyperthermia, radiation, and mild thermoradiotherapy. Int J Radiat Oncol Biol Phys 2001; 51:1018-1024. 48. Horsman MR, Murata R. Combination of vascular targeting agents with thermal or radiation therapy. Int J Radiat Oncol Biol Phys 2002; 54:1518-1523. 49. Eikesdal H P , Schem B-C, Mella O et al. The new tubulin-inhibitor combretastatin A-4 enhances thermal damage in the BT4An rat glioma. Int J Radiat Oncol Biol Phys 2000; 46:645-652. 50. Eikesdal H P , Bjerkvig R, Dahl O . Vinblastine and hyperthermia target the neovasculature in BT(4)AN rat gliomas: Therapeutic implications of the vascular phenotype. Int J Radiat Oncol Biol Phys 2001; 51:535-544. 51. Eikesdal H P , Bjerkvig R, Mella O et al. Combretastatin A-4 and hyperthermia; A potent combination for the treatment of solid tumors. Radiother Oncol 2001; 60:147-154.

CHAPTER 8

On the Biochemical Basis of Tumour Damage by Hyperthermia Paola Pietrangeli and Bruno Mondovi* Abstract

T

umour cells are selectively inhibited by hyperthermia (41-42.5°C) in the same conditions where normal cells are not damaged. At higher temperature, also normal cells are injured. In spite of the large number of reports on the cytotoxic effect of hyperthermia the mechanisms of heat cytotoxicity are yet unclear. It appears plausible that concomitant phenomena, triggered by heat and related each other, may be involved. The major points on this subject are the following: i. DNA, RNA synthesis, DNA repair mechanism and cell respiration are affected; ii. Tumour cell membranes are damaged as it is demonstrated by alteration of their permeability and the effect of empty liposomes; iii. DNA polymerase-P, a key enzyme in muld-step repair system, should be involved; iv. Dilation of mitochondria cristae and dissociation of poliribosomes were observed; V. Heat shock proteins should be involved; vi. Heat appears to increase the flux of oxygen free radicals mediating in part the cytotoxicity.

Introduction First of all hyperthermia refers to temperatures above about 5 centigrades the normal body temperature of the animal being studied. In vitro experiments have demonstrated that neoplastic cells are more sensitive to heat than normal cells of the some histological type, even if the latter divide at a faster rate than the tmnour cells. ^ For man, the range is between 40 and 43 °C, where only tumour cells are damaged while at higher temperatiue also normal cells are injured. Therefore we can consider a selective heat sensitivity of tiunour cells for man in this range of temperature. Neoplastic cells acquired the thermosensitivity together with the malignant transformation. About one and half century ago Bush.^ observed that temperature above the physiological value appears to damage cancer cells. For over 200 years dramatic "spontaneous" regression of various types of cancer were observed and in last century a large number of authors reported their individual observations of the beneficial effects of infections, bacterial vaccines, inflammation, fever or incomplete surgery. It was found in all types of neoplastic disease the majority of spontaneous regression occurred following streptococcal infections (like erysipelas). The complete regression of melanomas was described in a patient affected by erysipelas after several days of fever over 40°C.5 Coley, in die end of 19th century suggested to induce fever by inoculating bacterial toxins for the treatment of tumour. Regression of sarcomas in patients receiving erysipelas infections was reported.^'^ •Corresponding Author: Bruno Mondovl—Dipartimento di Scienze Biochimiche "A. Rossi Panel I i", and C.N.R. Centre of Molecular Biology, Universita "La Sapienza", P.Le Aldo Mro, 5-00185 Roma, Italia. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

On the Biochemical Basis of Tumour Damage by Hyperthermia

111

Systematic biochemical and clinical studies were performed by Rome and Madison groups'''^ in order to use hyperthermia for cancer therapy demonstrating that the treatment of cancer patients with hypertermia as such or together with chemical and/or radiation can give encouraging results in tumour cancer therapy. In this chapter a briefly summary of these researches, started on 1960 and some major biochemical questions of the thermosensitivity of tumour cells will be discussed.

Glycolysis and Respiration Westermark^ found that glycolysis, both aerobic and anaerobic, was rapidly inhibited in two experimental rat tumours while the Rome-Madison group ' published that glycolysis was unaffected by exposure up to 44°C in a large number of several animal and human malignant tumours. In some cases, where the rate of lactate production seemed to decline upon prolonged thermal exposure, it was subsequently ascertained that the inhibition observed could be attributed to an acidification of the medium. Von Ardenne et al^^ reported an inhibition of glycolysis in Ehrlich ascites mouse carcinoma cells only at very last event. A much more reliable parameter was oxygen consumption which underwent a severe inhibition upon thermal exposure of the neoplastic cells. The oxygen uptake of Novikoff hepatoma cells at 38 and 42°C were measured manometrically in the conventional way with air as the gas phase. After one hour the endogenous respiration at 42°C plateaued rapidly and was considerably less than that at 38 °C; similar effects were observed with the addition of glucose. ^° Whether the cells were incubated for 2 hours or more at 42°C and then returned at 38°C, oxygen uptake was irreversibly inhibited. Partial inhibition was observed when the cells were incubated at 42-44°C for 30 min then by measuring the respiration at 38°C. Also in the case of Ehrlich ascite carcinoma and in a human melanoma cells an inhibition of oxygen uptake in hyperthermic conditions was demonstrated. No effect of heat on respiration was found with normal and regenerating rat liver cells. When a minimal deviation hepatoma 5123 cell suspension was incubated at 38, 42 and 43°C the variation was irrelevant. However, whether hepatoma 5123 cells were incubated for 3.5 hours at 43°C and then returned at 38°C in the presence of glucose and succinate, oxygen uptake was lower. Conversely, no inhibition of oxygen uptake was observed in regenerating liver cells treated in identical conditions or for 3.5 hours also at 44°C.^^

Hyperthermia and DNA, RNA and Protein Synthesis A different set of parameters which are much more affected by thermal exposure concern the synthesis of macromolecules necessary to the cells: proteins and nucleic acids. In cells from Novikoff rat hepatoma, as well as from Morris 5123 rat hepatoma and from a human osteosarcoma, the DNA, RNA and protein synthesis were all strongly inhibited while regenerating liver cells were not affected. A prominent role of rRNA synthesis in the genesis of heat induced cell damage was found by investigations on the simultaneous action of heat and of some specific metabolic inhibitors of DNA and protein synthesis that decrease the thermal sensitivity of HeLa cells, while agents interfering with RNA synthesis act as enhancer of hyperthermia.^ The mechanism by which exposure of ascites tumour cells to supranormal temperatures causes an irreversible inhibition of uridine incorporation into RNA was investigated. Heat-treated cells were still able to incorporate labeled nucleotides, and even nucleosides, into RNA, if these precursors were added at sufficiently high concentrations. The passive permeability of the cell membrane increased exponentially with temperature, but this increase was fully reversible. At variance with the results obtained with agents which increase cell permeability, or which inhibit nucleoside permeation, heat treatment of Ehrlich ascites cells did not modify the ability of labeled uridine to be metabolised by these cells. The incubation of Chinese hamster cells, grown at confluent monolayer cultures, at 42.5°C, significantly inhibits the DNA repair synthesis which follows exposure of the cells to UV irradiation: 3 hours preincubation at 42.5°C prevents almost completely the repair of DNA damage produced to a single UV dose of 10 ]lrc^.

112

Hyperthermia in Cancer Treatment: A Primer

Whether hyperthermia follows the radiative treatment, the decline to DNA repair synthesis appears after 60 min incubation. DNA polymerase-p,^^ a key enzyme in multistep repair system, could be considered as a possible target because of its well known heat sensitivity.

Tumour Membranes and Hyperthermia The hypothesis of a primary lesion of the plasma membrane of the tumour cell is worth special consideration. In this context, it should be pointed out that heat-induced cell damage, as measured by oxygen uptake and by thymidine incorporation into DNA, is abolished upon disruption of the neoplastic cells. ^^' Polyenic antibiotic filipine and ethanoP^'^^ showing a marked selectivity for the lipid components of cell membrane, show a marked synergism with thermal exposure, acting as sensitisers for subsequent action of heat and inhibit the incorporation of labeled nucleic acids precursors into tumour cells at concentrations which did not significandy affect regenerating liver cells. These results suggest that the inhibition of DNA, RNA and protein synthesis may be related to a primary alteration of cellular membranes of tumours which are probably different from that of normal cells. In this context, the effect of temperature in potassium-dependent stimulation transcellular migration in normal and neoplastic cells was studied. ^'^ A shift from 38°C and 42°C, or even at 40°C, irreversibly modified, in Novikoff hepatoma cells but not in their counterpart (i.e., in normal or regenerating liver cells), the potassium-dependent transcellular migration of glutamate was observed. Ultrastructural changes induced by hyperthermia in Chinese hamster V79fibroblastwas demonstrated by Arancia et al.^^ In particular, the plasma membrane, mitochondria, ribosomes and nuclear envelope were studied compared to control cells maintained at 37°C, the heated cells, processed for electron microscopy under identical conditions, showed remarkable ultrastructural changes, depending on both the temperature employed and duration of treatment. In the heat-treated cells the plasma membrane shows small interruption after 1 hour of treatment at 42"C. This alteration becomes progressively more evident with the increase of both length and temperature of treatment. After 3 hours at 43°C the discontinuity of the membrane is clearly evident. Finally, treatment at 45*'C produces loss of large segments of the plasma membrane. Very interesting results were obtained on mitochondria: at 37''C cells they display a cylindrical shape with well preserved and parallel cristae in a dense matrix. After 1 hour at 42°C the mitochondria still have a dense matrix but some cristae appear to be dilated or vesicular. These changes are even more evident after 1 hour of treatment at 43°C: the mitochondria appear to be swollen and the intracristal spaces enlarged. At 45°C for 1 hour all mitochondria are aggregated in a perinuclear area and exhibit very irregular and dilated cristae in a matrix with decreased density. These alterations probably reflea changes in their functions: verv similar alterations were observed after exposure to uncouplers of oxidative phosphorylation. In the heated samples the polyribosomes appear dissociated, and the single ribosomal unites are free in a cytoplasmic matrix increasingly extracted with longer periods of treatment and increasing temperature. After 6 hours of heating an irregular dilation of the nuclear envelope was observed. In cells treated at 43°C the nuclear membranes are not well defined and after 1 hour at 45°C they are not parallel, gready dilated and several foldings of the outer membrane are present. ^^

Hyperthermia and Liposomes The possible involvement of cell membranes target of hyperthermia is further confirmed by the effect of the enhancement of hyperthermic damage of tumour cells by liposome treatment. In this context it should be pointed out that cell membrane modifications have been obtained by means of liposome-cell interaction^^ which could be of particular interest because of the well characterised difference in membrane composition and fluidity of tumour cells with respect to normal ones.^^ Cell survival experiments performed on the relatively thermoresistant human melanoma cell line Ml4^^ showed that cell pretreatment with L-a-dipalmitoylphosphatidylcholine-containing liposomes enhances the cell killing induced by hyperthermia.

On the Biochemical Basis of Tumour Damage by Hyperthermia

113

although Uposomes per se did not appear toxic when their concentration was below 1000 nmol phospholipid/2.5 x 10 cells, while liposome treatment of cells before heat exposure determined a marked damaging effect at 100 nmol phospholipid/2.5 x 10 cells. The observation by electron microscopy seem to demonstrate that, multilamellar vesicles can either fuse with the plasma membrane or be taken up by the cell. In particular, the outer lipid bilayer of the liposomes appears to be involved in the fusion event leading to the release of the inner layers into the cell. Probably the internalised vesicles are capable of fusing with the intracellular membranes. Thus, the lipid components of the liposomes would be inserted in both plasma and intracellular membranes upon the interaction of multilamellar vesicles with M14 cells. For that reason, the cell membranes could be a site at which liposome pretreatment of cell enhance the hyperthermic damage. The proximity of multilamellar liposomes and nuclear envelope, observed by electron microscopy,^^ suggested a possible interaction between liposomes and nuclear membranes. Electron spin resonance experiments demonstrated a membrane fluidity decreasing after liposome treatment. It seem likely that the insertion of lipid domains of different organisation into plasma membrane following the incorporation of the liposomes, as evidenced by electron microscopy results, could lead to impaired ftinctioning of some membrane proteins. The interaction of multilamellar liposomes and cultured cells was also studied at mild hyperthermia, i.e., 37 and 4l.5°C for 2 hours. A dose-dependent impairment of cell survival was observed as a function of liposomes concentration. An enhancement of the cytotoxic effect was observed at 4l.5°C. This effect went on even after 24 hours from the end of treatment. Probably hyperthermia acts at the initial stage of treatment by accelerating the kinetics of the membrane-liposome interaction. Since in recovered cells numerous vesicles could be detected inside the cytoplasm, it may argued that the internalised liposomes continue to exert their effects on intracellular membranes, and that these membranes may be considered more critical than the plasma membrane for liposome cytotoxic action. Probably when the liposome treatment is carried out under mild hyperthermia, the ftision of the outer layers of multilamellar liposomes with the plasma membrane, and then the entry of the inner layers inside the cells are favoured and accelerated. We can therefore concluded that the liposome administration with mild hyperthermia appears to have a synergistic effect on cultured melanoma cells. Although the role of the cell membranes in the cytotoxic effect of hyperthermia has not yet been established, a key role of the membrane protein components was proposed.^ '^^ It has been suggested that lipid (both cholesterol and phospholipid):protein weight ratios correlate with increasing resistance of cells to an elevation in temperature. The major membrane components can influence and perhaps predict cellular survival to hyperthermia.^

Hyperthermia and Immune Response The possibility that plasma membranes should be the primary site damaged by exposure to supranormal temperatures appears to be also confirmed by the results obtained on the increased immunogenicity of Ehrlich ascites cells after heat treatment. ^ It was in fact demonstrated that inoculation of 10 viable, untreated cells to swiss mice resulted in 100% tumour take, the mean survival time of the animals being about 18-20 days. Exposure to 38**C for 2, 3 or 6 hours caused little modification of these parameters; after 1 hour at 42°C, tumour taken declined sensibly and, after 3 hours or more at this temperature, there was 100% survival for more than 3 months. Immunisation was performed by two intraperitoneal inoculation of heat heated 10'^ cells, at 20 day interval. Two schedules of heat treatment were used comparatively, with exposure time at 42°C being 3 or 6 hours. Only the group immunised with cells exposed for 3 hours at 42°C had more than 50% survival after 35 days when challenged with 10'^ viable cells. Non immunised animals had, after the same challenged dose, 100% of tumour take. A plausible hypothesis of these results seem to be the unmasking or modification of some antigenic determinant cell surface during the initial phase of exposure at high temperature leading to increased immunogenicity. Upon further heat treatment, some damage to these surface determinants may occur, so that the immunity evoked by the heat-treated cells is either lower

114

Hyperthermia in Cancer Treatment: A Primer

or less effective toward viable cells. A protection against spontaneous mouse manunary adenocarcinoma by inoculation of heat-treated syngeneic manmiary tumour cells was also observed.^^ These results support the possibility of the inunune defence mechanism may play a role in contributing to the regression after hyperthermic perftision of otherwise untreatable tumours.

Heat Shock Proteins The problem of the relationship between enhancement of immunogenicity after hyperthermia appears to be related on the heat shock proteins (hsp).^^The induction of hsp is a general phenomenon observed in almost all organisms and cultured cell lines after exposure to subletal temperature, as well as to the stimidi which are injurious to the cells.^ In eukaryotic cells the hsp, identified by SDS polyacrilamide gel electrophoresis, may be classified in four major groups designed 16-28, 69-70, 80-90, 110, based on their respective molecular weights expressed in kilodalton. Hsps of the same site are so similar each other in different cell types and organisms that antibodies against a particular hsp from one specie usually cross-reacts with similar proteins from phylogenetically distant species. In particular, the hsp with molecular weight around 70 and 90 KD seem to be the most highly conserved in nature. Each specific temperature value reached during the heating process induces the expression of a specific hsp pattern. In vitro conditions, the pattern of hsps induced in tumour cells has been reported to change in dependence of the different cell lines, culture conditions, protocol of hyperthermic treatment. Hsps appear to be related to the heat effect on tumour cells essentially by different mechanisms: i. Correlation between induction of hsps and development of tolerance to subsequent thermal shock, ii. Correlation between cytotoxicity and heat-induced increase in nuclear matrix associated proteins, iii. Use of hsps in immunotherapy. These facts are apparendy in contrast each other, but, taking into account their different mechanisms of action, a possible concatenation can exist. Initially, the role of hsps appeared to involve a thermotolerance mechanism: brief sublethal heat exposure of cells induced a quick expression of hsps that conferred total protection against a subsequent but lethal exposure to hyperthermia. Then, it was shown that hsps were able to protect cells from different sources of stress such as oxidant radicals or endotoxin.^^'^^ In this context, Frossard ^^ demonstrated that hsp70 prolongs survival in rats exposed to hyperthermia. In addition, hsp25 overexpressed in L929 cells were shown have increased expression of the manganese superoxide dismutase gene and its enzyme activity.^^ It shoidd be taking into account that acute heat shock might residt in a redistribution of critical cell proteins and their absorption on the cell of nucleoskeleton.'^' The subsequent functional sequentiation of these proteins results in cytotoxicity. This speculation is supported by the observation that a specific set of phospholipids with molecular mass similar to the HeLa hsps have been observed to become tighdy associated with the cell s nucleoskeleton during acute thermal shock^^ To investigate the usefulness of hsp promoter for heat cancer gene therapy, hyperthermia and HSV thimidine kinase (tk) suicide gene combination therapy was checked with mouse mammalian cancer cell line FM3A. Hsp promoter activity was markedly increased after heat shock (4l-45°C) with maximum activation at 3 hr. The suppression of heat-induced accumulation of hsp72 by bleomicin appears to contribute to enhance cytotoxicity of the simultaneous treatment of 40'*C hyperthermia and bleomicin.^^ A direct relationship between hsp and cancer inmiunotherapy was in recent years developed. Tumour derived hsp-peptide complexes (particularly hsp70 and grp94/gp96) have been demonstrated to serve as effective vaccines producing antitumour inmiune responses in animals and man.^^ In this context, it should be pointed out that the realisation of significance of hsp come from the observation that tumour-cell derived hsps could immunise against tumours.

On the Biochemical Basis of Tumour Damage by Hyperthermia

115

although there were no structural difference between hsps, from normal cells and cancer cells. Studies of this puzzle have uncovered two unique immunological properties of hsps. One is their ability to associate and present antigenic fmgerprints/peptides of cells to MHC antigens. The other is their ability to activate dendritic cells, which are the most efficient antigen- presenting cells. These surprising immunological attributes of hsps are now the basis for a number of already completed chemical trials for cancer immunotherapy.^^

Hyperthermia and Oxygen Free Radicals Thermotolerance in same way could be correlated with other biochemical mechanisms causing the heat damage of cancer cells. In CHO and ovarian carcinoma cells, a rise in Cu,Zn-SOD activity was reported after heating to induce thermotolerance.^^ The increase of the steady state concentration of the superoxide radical O2', H2O2 and the more reactive hydroxyl radical O H (by H2O2 and O2' in Haber-Weiss reaction catalysed by transition metal ions), could be responsible for the cellular damage after heat treatment. It is well known that oxygen free radicals (ROS) are imputable for inactivation of enzymes, degradation of DNA and polysaccarides and induction of lipid peroxidation. Christiansen et al^^ observed that oxidative phosphorylation of mouse liver mitochondria is uncoupled after treatment at 4l-45°C. In cloned normal mouse embryo cells and their Simian virus 40-transferred derivatives, a correlation between low levels of antioxidant enzymes and thermosensitivity have been demonstrated. In this context, it is important to focus that transferred cells containing undetectable Mn-superoxide dismutase (Mn-SOD) and markedly low level of Cu,Zn-SOD, catalase and glutathione peroxidase activities, are selectively killed by exposure to hyperthermia, whereas the normal cells, having significantly higher enzyme activities, showed to be resistant to heat treatment. In addition, in both cell types the hyperthermic effect appears to be enhanced after a pretreatment with diethyldithiocarbamate, a well known inhibitor of Cu,Zn-SOD. The role of production of ROS can also be affected by heat at the level of oxidases. In this regard, a special emphasis should be given to the oxidative degradation of hypoxanthine to xanthine, and xanthine to urate by xanthine oxidase. In fact, as a consequence of insufficient ATP production, imputable to decreased respiration in hyperthermic conditions, an increase of cytosolic Ca^^ may occur, which in turn activates a protease capable of converting xanthine dehydrogenase to the oxidase. ^ A human melanoma cell line (Ml4) was enriched in superoxide dismutase (SOD) activity by treatment with enzyme-containing liposomes. The effect of hyperthermia on SOD-liposome enriched cells was tested by evaluating cell survival and measuring the incorporation of labelled L-leucine. A balance of some protective and lethal effect imputable to SOD and liposome respectively was su^ested. '^ An accelerated rate of production of ROS could also a consequence of the increased rate of enzymatic activity of amine oxidases on biogenic amines.

Hyperthermia and Amine Oxidases Immobilised pig kidney diamine oxidase (DAO) injected into the peritoneal cavity of swiss mice 24 hr after the viable intraperitoneal transplantation of Ehrlich ascite cells remarkably inhibited tumour growth. ^ These results suggest a possible use of AOs in cancer therapy. It is interesting to consider that an inflexion point on the dependence of swine kidney diamine oxidase activity upon the temperature was found at 40-43 °C, exacdy the temperature used for hyperthermic treatment of tumours. Probably, this result is imputable to a conformational transition of this enzyme as a function of temperature. The activation energies with putrescine as substrate calculated from the Arrhenius plot were 38,23 Kcal/mol for the temperature interval 25-40°C and only 15,14 Kcal/mol for the range 45-60°C. These values suggest two different conformations, one corresponding to the interval below 40°C and another one between 43-60 °C, with intermediate transitory form corresponding to the inflexion point at 40-43"C.

116

Hyperthermia in Cancer Treatment: A Primer

In order to have more informations on the mechanism involved in effect of amine oxidases on tumours, and to study a possible use of amine oxidases as adjuvants in hyperthermia, the effect of bovine serum amine oxidase were studied in cidtured cells. It was demonstrated ' that bovine serum amine oxidase (BSAO), in the presence of exogenous spermine, caused cytotoxicity in Chinese hamster ovary cells (CHO). Cytotoxicity occurred when cells were exposed to BSAO (0.06-16 jlg/ml) in the presence of spermine. BSAO and spermine alone were not toxic at these concentrations. This cytotoxicity was accelerated at 42°C relative to 37°C. As reported above, BSAO seems to have more then one conformation as a function of temperature. Kinetic analysis of the enzymatic reaction, as a function of spermine concentration, show a Michaelis-Menten saturation kinetics. The apparent Vmax increased from 19.1 ± 0.4 nM min'^ at 37'C to 23.0 ± 0.3 ^iM min'^ at 42'C. The apparent K^ decreased from 25.5 ± 2.6 |xM at 37°C to 17.7 ±1.3 |xM at 42°C. It was observed that heat increased the cytotoxicity of both exogenous H2O2 and exogenous aldehyde acrolein, thus, both these species could contribute to the thermal enhancement of cytotoxicity caused by BSAO and spermine. ^ The effect of temperature was observed in the presence of exogenous catalase, therefore this cytotoxicity was attributed also to aldehydes. The involvement of aldehydes in cytotoxicity at 42°C was also confirmed by complete inhibition of cytotoxicity with both exogenous aldehyde dehydrogenase and exogenous catalase added in the incubation mixture. A particular interesting finding was that by incubating the cells in the presence of BSAO, 50 |lM spermine and exogenous catalase which were not toxic at 37°C, contributed to cytotoxicity at 42°C and therefore the oxidation products of amines resemble to be thermosensitiser."^^ The thermosensitizing activity of aldehyde(s) produced in the BSAO-catalyzed oxidation of spermine has potential value for improving the therapeutic effects of hyperthermia and could be considered for future application in cancer therapy. Amine oxidases, in turn, can be internalised into the cells. ' In fact, cultured hepatocytes express binding sites for BSAO on their membrane surfaces evaluated at the electron microscope level by using enzyme-gold complexes. Hepatocytes show binding sites as small clusters of gold granules, not bound in a specialised region of the plasma membrane. The binding competition of enzyme-gold ligand to cells was achieved by preincubation with uncoupled BSAO. In addition, enrichment of a human leukemia cell line (K562) with a plant diamine oxidase was recendy obtained. ^ These results suggest the possible use of amine oxidases in physiological and hyperthermic conditions as potential antineoplastic drugs^^ taking in account, also their involvement in apoptotic phenomena. In fact, as demonstrated by Malorni et al^^ both pargyline and clorgyline, classical inhibitors of mitochondrial mono amine oxidases (MAO) are capable of protecting cells from apoptosis induced by serum starvation. In addition, Marcocci et al demonstrated that the structure and function of the mitochondrial membrane is modulated by the activity of MAO A and MAO B at low concentrations of benzylamine and octopamine, probably through the enzymatic production of H2O2. As mentioned above in this chapter, mitochondria are particularly sensitive to hyperthermia. Therefore, by considering the importance of apoptotic phenomena, in the regulation of human cells growing the simultaneous use of hyperthermia and enhancement of toxic products for cells, like H2O2 and aldehydes as metabolic products of amine oxidases, could be helpful in cancer therapy. As general conclusion, more then one biochemical systems appears to be involved in the hyperthermic damage, but so far it is not possible to describe a specific biochemical target of the hyperthermic damage, occurring most probably a simultaneous concurrence of different causes.

Acknowledgement This paper was supported by C.N.R. grant n° G002FD1 Agenzia 2000 and MURST

On the Biochemical Basis of Tumour Damage by Hyperthermia

117

References 1. Giovanella BC, Morgan AC, Stehlin JS et al. Selective lethal effect of supranormal temperatures on mouse sarcoma cells. Cancer Res 1973; 33:2568-2578. 2. Chen T T , Heidelberger C. Quantitative studies on the malignant transformation of mouse prostate cells by carcinogenic hydrocarbons in vitro. Int J Cancer 1969; 4:166-178. 3. Bush W. Ober den Einfluss, welchen heftigere Erysipeln zuweilen auf organisierte Neubildungen ausuben: Verhandl. Naturhist Preuss Rhein Westphal 1866; 23:28-37. 4. Nauts H C . The beneficial effects of bacterial infections on host resistance to cancer end results in 449 cases. A Study and abstracts of reports in the world medical literature (1775-1980) and personal communications. Monograph n" 8. 2nd ed. 1980. 5. Burns P. Die Heilwirkung des Erysipels auf Geschwulste. Beitr Klin Chir 1887; 3:443-453. 6. Coley W B . The treatment of malignant tumors by repeated inoculations of erysipelas with a report of ten original cases. Am J Med Sci 1893; 105:487-495. 7. Marcocci L, Mondovl B. Biochemical and ultrastructural changes in the hyperthermic treatment of tumor cells: An outline. Consensus on hyperthermia for the 1990s. In: Bicher H I , McLaren JR, Pighucci C M , eds. CHnical pratice in cancer treatment. New York: Plenum Press, 1990:99-120. 8. Giovanella BC, Mondovl B. Recent results in cancer research. In: Rossi-Fanelli A, Cavaliere R, Mondovl B et al, eds. Selective Sensitivity of Cancer Cells. 1977. 9. Westermark N . The effect of heat upon rat tumors. Scand Arch Physiol 1927; 52:257-266. 10. Cavaliere R, Cioccatto EC, Giovannella BC et al. Selective heat sensitivity of cancer cells cancer. Biochemical and clinical studies. Cancer 1967; 20:1351-1381. 11. Mondovl B, Strom R, Rotilio G et al. The biochemical mechanism of selective heat sensitivity of cancer cells. I. Studies on cellular respiration. Europ J Cancer 1969; 5:129-136. 12. Von Ardenne M, Reitnauer PG, Reiger F. Zur Erkundung therapeutischer Angriffspunktenim intermediaren Stofifwechsel der Krebszelle. In: Von Ardenne M, ed. Theoretische und experimentelle Grundlagen der Krebs-Mehrschritt-Therapie. Berlin: VEB Verlag Volk u n d G e s u n d h e i t , 1967:267-277. 13. Mondovl B, Finazzi-Agr6 A, Rotilio G et al. The biochemical mechanism of selective heat sensitivity of cancer cells. II. Studies on nucleic acids and protein synthesis. Europ J Cancer 1969; 5:137-146. 14. Strom R, Santoro AS, Crifo' C et al. The biochemical mechanism of selective heat sensitivity of cancer cells. IV. Inhibition of RNA synthesis. Eur J Cancer 1973; 9:103-112. 15. Dube DK, Seal G, Loeb LA. Differential heat sensitivity of mammalian D N A polymerases. Biochem Biophys Res Commun 1976; 76:483-487. 16. Emmelot P, Bos CJ. Studies on plasma membranes. VI. Differences in the effect of temperature on the ATPase and (Na*-K*)-ATPase activities of plasma membranes isolated from rat liver and hepatoma. Biochim Biophys Acta 1968; 150:354-363. 17. Strom R, Caiafa P, Mondovl B et al. Effect of temperature on potassium-dependent stimulation of transcellular migration in normal and neoplastic cells. Febs Letters 1969; 3:343-350. 18.Arancia G, Crateri Trovalusci P, Mariutti G et al. Ultrastructural changes induced by hyperthermia in Chinese hamster V79 fibroblasts. Int J Hyperthermia 1989; 5:341-350. 19. Buffa P, Guarriera-Bobyleva V, Muscatello U et al. Conformational changes of mitochondria associated with uncoupling of oxidative phosphorylation in vivo and in vitro. Nature 1970; 226:272-274. 20. Margolis LB. Cell interaction with model membranes probing, modification and simulation of cell surface functions. Biochim Biophys Acta 1984; 779:161-189. 2 1 . Shinitzki M. Membrane fluidity in malignancy adversative and recuperative. Biochim Biophys Acta 1984; 738:251-261. 22. Laudonio N , Marcocci L, Arancia G et al. Enhancement of hyperthermic damage on M l 4 melanoma cells by Uposome pretreatment. Cancer Res 1990; 50:5119-5126. 23. Arancia G, Calcabrini A, Matarrese P et al. Effects of incubation with liposomes at different temperatures on cultured melanoma cells (M14). Int J Hyperthermia 1994; 10:101-114. 24. Lepock JR. Involvement of membranes in cellular responses to hyperthermia. Radiat Res 1982; 92:433-438. 25. Lepock JR, Cheng KH, Al-Qysi H et al. Thermotropic lipid and protein transitions in chinese hamster lung cell membranes: Relationship to hyperthermic cell killing. Can J Biochem Cell Biol 1983; 61:421-427. 26. Cress AE, Culver PS, Moon T E et al. Correlation between amounts of cellular membrane components and sensitivity to hyperthermia in a variety of mammalian cell lines in culture. Cancer Res 1982; 42:1716-1721. 27. Mondovi B, Santoro AS, Strom R et al. Increased immunogenicity of Ehrlich ascites cells after heat treatment. Cancer 1972; 30:885-888.

118

Hyperthermia in Cancer Treatment: A Primer

28. Check JH, Childs TC, Brady LW et al. Protection against spontaneous mouse mammary adenocarcinoma by inoculation of heat-treated syngeneic mammary tumor cells. Int J Cancer 1971; 7:403-408. 29. Manjili MH, Wang XY, Park J et al. Immunotherapy of cancer using heat shock proteins. Front Biosci 2002; 7:d43-52. 30. Hard FU. Molecular chaperones in cellular protein folding. Nature 1996; 381:571-579. 31. Kiang JG, Tsokos GC. Heat shock protein 70 kDa: Molecular biology, biochemistry, and physiology. Pharmacol Ther 1998; 80:183-201. 32. Frossard JL. Heat shock protein 70 (HSP70) prolongs survival in rats exposed to hyperthermia. Eur J Clin Invest 1999; 29:561-562. 33. Yi MJ, Park SH, Cho HN et al. Heat-shock protein 25 (Hspbl) regulates manganese superoxide dismutase through activation of Nfkb (NF-kappaB). Radiat Res 2002; 158:641-649. 34. Warters RL, Brizgys LM, Sharma R et al. Heat shock (45 degrees C) results in an increase of nuclear matrix protein mass in HeLa cells. Int J Radiat Biol Relat Stud Phys Chem Med 1986; 50:253-268. 35. Welch WJ, Feramisco JR. Purification of the major mammalian heat shock proteins. J Biol Chem 1982; 257:14949-14959. 36. Jin ZH, Shioura H, Kano E et al. Efi^ects of combined treatment with 40°C hyperthermia and bleomycin on the accumulation of heat shock protein in murine L cells. Int J Oncology 2002; 20:137-142. 37. Liu B, De Filippo M, Zihal L. Overcoming immune tolerance to cancer by heat shock protein vaccines. Molecular Cancer Therapeutics 2002; 1:1147-1151. 38. Lx)ven DP, Leeper DL, Oberley LW. Superoxide dismutase levels in Chinese hamster ovary cells and ovarian carcinoma cells after hyperthermia or exposure to cycloheximide. Cancer Res 1985; 45:3029-3033. 39. Christiansen EN, Kvamme E. Effect of thermal treatment on mitochondria of brain, liver ascites cells. Acta Physiol Scand 1969; 76:472-484. 40. Omar RA, Yano S, Kikkawa Y. Antioxidant enzymes and survival of normal and Simian Virus 40-transformed mouse embryo cells after hyperthermia. Cancer Res 1987; 47:3473-3476. 41. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. New Engl J Med 1985; 312:159-163. 42. Bozzi A, Laudonio N, Zupi G et al. Interaction of superoxide dismutase-containing liposomes with a human melanoma cell line in hyperthermic conditions. Intern J Hyperthermia 1987; 3:553. 43. Mondovl B, Gerosa P, Cavaliere R. Studies on the effect of polyamines and their products on EhrUch ascites tumours. Agents and Actions 1982; 12:450-451. 44. Mondovl B, Agostinelli E, Przybytkowski F et al. Amine oxidases as a possible antineoplastic drugs. In: Alberghina L, Frontali L, Sensi P, eds. Proceedings of the 6th European Congress on Biotechnology. Elsevier Sciences BV, 1994:775-778. 45. Mondovl B, Befani O, Gerosa P et al. Specific temperature dependence of diamine oxidase activity and its thermal stability in the presence of polyvinylalcohol. Agents Actions 1992; 37:220-226. 46. Averill-Bates D, Agostinelli, Przybytkowski E et al. Cytotoxicity and kinetic analysis of purified bovine serum amine oxidase in the presence of spermine in Chinese Hamster ovary cells. Arch Biochem Biophys 1993; 300:75-79. 47. Agostinelli E, Przybytkowski E, Mondovl B et al. Heat enhancement of cytotoxicity induced by oxidation products of spermine in Chinese Hamster ovary cells. Biochem Pharmacol 1994; 48:1181-1186. 48. Dini L, AgostineUi E, Mondovl B. Cultured hepatocytes bind and internalize bovine serum amine oxidase-gold complexes. Biochem Biophys Res Commun 1991; 179:1169-1174. 49. Marcocci L, Nocera S, Roig MB et al. Enrichment of a human leukemia cell line (K562) with a plant histaminase. Inflamm Res 2001; 50:S134-S135. 50. Pietrangeli P, Mondovl B. Amine oxidases and tumors. NeuroToxicology 2004; 25:317-324. 51. Malorni W, GiammarioH AM, Matarrese P et al. Protection againsts apoptosis by monoamine oxidase A inhibitors. Febs Letters 1998; 426:155-159. 52. Marcocci L, De Marchi U, Milella ZG et al. Role of monoamine oxidases on rat liver mitochondrial ftinction. Inflamm Res 2001; 50:S132-S133.

CHAPTER 9

Results of Hyperthermia Alone or with Radiation Therapy and/or Chemotherapy Pietro Gabriele and Cristina Roca* Abstract

T

he interest in clinical hyperthermia (HT) was maximum in eighties and decreases during nineties of the last century but now, thank to possibility of heat deeply and to measure the temperature not invasively the interest is growing another time. On the other hand the biology of HT, clearly established during seventies, is now discussed with particular attention to the molecular pattern. Heat alone can be used as a cytotoxic agent. Results from 14 studies about lesions treated with HT alone reported complete response (CR) rate of 13% and overall response (OR) rate of 51%; the response time was short. HT alone can obtain results similar to some drugs employed as monochemotherapy. The result depends strictly from the possibility to obtain a good quality geometrical heat and to prescribe a sufficient number of heat session. The recommendation of the International Consensus meeting on HT held in 1989 was that results of heat alone should be used as a reference for such combinations. We performed from 1983 to 1996 some studies concerning the association of HT and radiations (RT). The most important results were the 90% and 75% of OR and CR for chest wall recurrences (the majority of them pretreated with radiotherapy); the data obtained were similar to residts of multicentric Italian data obtained in 212 lesions treated in 10 radiotherapy centres. In the Overgaard, Myerson and van der Zee reviews about numerous studies the therapeutic enhancement ratio for patients treated with the association versus patients treated with radiotherapy alone is for the great majority of studies between 1.5 and 2.0 From 22 randomized studies we found in 15 a statistically significant advantage for patients treated with the association of HT and RT or radiochemotherapy versus patients treated with RT or chemotherapy alone. We would emphasise that the two American studies by RTOG (Radiation Therapy Oncology Group) are inconclusive because of sub-optimal technical way of HT. The use of interstitial HT permits heat delivery to a well-defined volume which is frequently inaccessible to external local or deep HT. Interstitial HT uses placement into the treatment-planned volume of multiple microwave or radiofrequency antennas. Intracavitary HT associated with radiation therapy and/or chemotherapy is under study from about 20 years, in particular for the carcinoma of the oesophagus. Several hundred patients have been treated in phase I-II studies in the far East: all reports showed good treatment *Corresponding Author: Cristina Roca—Division of Molecular Angiogenesis, Institute for Cancer Research and Treatment (IRCC), Strada Provinciale 142, Candiolo, Turin 10060, Italy. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

120

Hyperthermia in Cancer Treatment: A Primer

Tabic 1. Results of HT alone Author

Year

Pts. N.

HT Method

Prescribed T

Corry^^ Gabriele^'' Kim8 Luk^7 Marmor^^ Perez^^

1982 1990 1979 1981 1982 1983 1980

28 60 19 11 44 6 6

US MW RF inductive MW US MW MW

43-50 °C 42-44 °C 41-43.5°C 42.5 °C 43-45 °C 41-43 °C 42-44 °C

U20

N. Treatments 6-12 2-12 2-9 5-12 6 6 2-9

CR (%)

OR

18 15 21 18 11 16.5 0

39 32 8 32 33 50

tolerance and a lack of significant late complications but most of the reports are based on small number of patients and not provide sufficient information. A strong biological rationale exists for the use of local HT and systemic chemotherapy in patients with superficial tumors. Superficial metastases are oft:en associated with additional occult distant metastases that warrant systemic treatment. Preliminary results employing cisplatinum and bleomycin with local HT revealed high response rate even in tumors located in previously irradiated sites: the better results were obtained in the treatment of breast carcinoma, head and neck and malignant melanoma. The most important prognostic factors affecting the response to HT are RT or heat dose: some of them may be more important than others in the clinical application, e.g., the temperature and total heating time, and, when HT is done in association with RT, radiation dose. The great challenge for HT in the next future is to provide adequate heating to the full tumor volume, in particular for deep seated tumors. Radiation Therapy Oncology Group (RTOG) studies demonstrated that 42°C minimum temperature not were obtained for most tumors; now some devices will ultimately lead to better minimum temperature not only for superficial tumors but also for deep seated lesions. Another way to ameliorate HT in clinical setting will be the possibility to measure the temperature not invasively by means of magnetic resonance (MR) or ultrasound (US).

Introduction From the first International Congress on Clinical HT in Washington in 1975, HT has increased in clinical oncology. The interest in clinical HT was maximum in eighties {W International Congress in Aahrus in 1984) and decreases during nineties of the last century but now, thank to possibility of heat deeply and to measure the temperature not invasively, the interest is growing another time. On the other hand the biology of HT, clearly established during seventies, ' is now discussed and (reviewed refs. 3-6) with particular attention to the molecidar pattern'^'^ and vascular targets. ^°'^^

Hyperthermia Alone Heat alone can be used as a cytotoxic agent. Results from 14 studies about lesions treated with HT alone including a total of 343 patients, reported in the Consensus Conference of 1989,^^ the CR rate was 13% (0-40%) and the OR rate was 51%; the response time (time to recurrence) was very short. In a personal experience regarding 60 superficial lesions, recurrent to surgery, radiotherapy and/or chemotherapy, treated with microwave (915 and 434 MHz) in a mean of 6 heat sessions, the complete response rate was 15%.^"^'^^ In Table 1 are summarized the results of some studies of HT alone with the technical detailed note, temperature and treatments number.

Results ofHyperthermia Alone or with Radiation Therapy and/or Chemotherapy

121

Table 2, Results of HT and radiotherapy (University of Turin, 1983-1996) Site/Tumor

Pts. N.

% CR

% O.R.

Head and neck recurrent (SCC)^^'^^ Head and neck advanced (SCC)^^'^^ Parotid region (Adenocarcinoma like)^^'^^ Chest wall recurrences^^'^^ Adenocarcinoma vaginal recurrences^^ Perineal recurrences^^'^"^ Malignant melanoma^^ Recurrent Sarcoma^"^ Lymphoma^^

38 33 20 62.5

75 80 80 92

30 50 100 58

7 48 (86) 33

28 42

—

% L.C.

% Survival

— 62 60

75

84 87

—

— —

—

—

62

CR: complete response; OR: overall responsei; LC: local control

In conclusion H T alone can obtain results similar to some drugs employed as monochemotherapy. The result depends strictly from the possibility to obtain a good quality geometrical heat and to prescribe a sufficient number of heat session (mean 6). The recommendation of the International Consensus Meeting on HT is that data on heat alone should be used as a reference for such combinations.^^

Hyperthermia and Radiotherapy Not Randomised Studies We performed in a period of 13 years (1983-1996) some studies concerning the association of HT and radiations. Generally HT was done immediately before radiotherapy for 6 (range: 2-10) heat sessions of half-one hour. The temperature, controlled by means of invasive thermometry with fibre optic thermometers, was between 42 and 44°C in the measured points.^^'^^ In Table 2 are reported the results obtained in the various sites treated in our personal experience; the results are reported in terms of CR, OR (WHO criteria), LC (2/3 years minimum follow-up) and 3/5 years survival. The most important results were the 90% and 75% of OR and CR for chest wall recurrences (the majority of them pretreated with radiotherapy). The data obtained were similar to results of multicentric Italian data obtained in 212 lesions treated in 10 radiotherapy centres (Aviano, Catania, Genoa, Messina, Padoa, Ravenna, Rome, San Giovanni Rotondo, Trento and Turin).^^ Radiotherapy dose rarely exceeded 40 Gy in the treatment of recurrences whereas the dose for untreated advanced cases (parotid, head and neck and intact breast) was 60 Gy; for malignant melanoma large fraction of 4-9 Gy were employed for a total dose of 27-40 Gy^^ The complications rate varied from 4% in recurrent head and neck to only 1% in the treatment of parotid adenocarcinoma like and malignant melanoma. ^^ The most frequent complications were local burns, soft tissue necrosis and general symptoms like fever. We performed also some studies about H T in not cancerous diseases, in association or not with radiation therapy.^^'^^

Randomized Studies In the Overgaard,^^ Myerson ^ and van der Zee '^ reviews about numerous studies the therapeutic enhancement ratio for patients treated with the association versus patients treated with radiotherapy alone is for the great majority of studies between 1.5 and 2.0 (Table 3). From 22 randomized studies we found in 15 a statistically significant advantage for patients treated with the association of HT done with MW, RF or US and /radio or radiochemotherapy

122

Hyperthermia in Cancer Treatment: A Primer

Table 3. Clinical results of the randomised studies of HT and raidiotherapy Author

Year

Pts. Number

RT

RT+HT

TER

Arcangeli"*^

1984 1984 1990 1986 1989 1989 1987 1986 2001 1984 1986 1988 1982 1987 1987 1987 1983 1987 1986 1994 1998 1998 1998 1993

163 45 65 86 92 24 65 46 40 33 66 238 124 87 313 101 154 185 90 78 41 70 306 122

38 9 46 50 63 63 86 33 50 25 35 39 29 25 25 39 41 33 31 36 41 35 41 5

73 42 66 60 82 83 100 50 85 71 72 72 54 46 63 62 69 64 65 73 83 62 59 23

1.95 4.67 1.43 1.20 1.28 1.32 1.16 1.52 1.70 2.84 2.06 1.85 1.86 1.84 2.52 1.59 1.68 1.94 2.10 2.03 2.02 1.78 1.45 4.60

Bey Datta^^ Dunlop^^ Egawa Fuwa^^ Goldobenko'^^ Gonzales^° Harima^^ Hiraoka" Hornback^^ Kim«

Li Lindholm^'^ Murakthodzaev Overgaard^^ Perez^^ Shidnia Steeves Valdagni^^ Van der Zee^^ Van der Zee^'' Van der Zee ^^

You

versus patients treated with radio or chemotherapy alone. We would emphasise that the two American studies by RTOG are inconclusive because of sub-optimal technical way of HT. In Table 3 are reported the most important studies of HT and radiotherapy and the results in terms of response/control and the TER.

Interstitial Thermo-Brachytherapy The use of interstitial HT permits heat delivery to a well-defined volume, which is frequendy inaccessible to external local or deep HT. Interstitial HT uses placement into the treatment-planned volume of multiple microwave or radiofrequency antennas.^^

Table 4. Clinical

results of the studies of interstitial

thermobrachytherapy

Author

Year

Pts Number

Follow-up

CR

PR

NR

Oleson^^ Cosset^^ Puthawala Emami^^ Petrovich^"^

1984 1985 1985 1987 1989

52 29 43 44 44

3-18m

39% 80% 86% 60% 65%

42% 20% 14% 26% 32%

9%

2m 6m 6-60m 6-30m

CR: complete response; PR: partial response; NR: no response

14% 3%

Results ofHyperthermia Alone or with Radiation Therapy and/or Chemotherapy

123

Table 5. Clinical results of the studies ofHT and chemotherapy (and radiotherapy) Author

Year

Pts N.

Chemo

Chemo+HT

RT+HT

Arcangell^° Emami^^ Gabriele''^ Kitamura^^ Orecchia''^ Steindorfer''^ Zhang

1983 1989 1991 1995 1991 1987 1988

43 21 10 66 20 22 28

95% 49% 10% 6% 10% 8% 41%

45% (67% OR) (50% OR) 25% (70%) 80% (85% OR)

p<0.01

p<0.01

In Table 4 are summarized some results reported in the literature.

Intracavitary Hyperthermia Intracavitary HT associated with radiation therapy and/or chemotherapy is under study from about 20 years, in particular for the carcinoma of the oesophagus. ^' Several hundred patients have been treated in phase I-II studies in China, Japan and Russia: all reports showed a good treatment tolerance and a lack of significant late complications but most of the reports are based on small number of patients and not provide sufficient informations on technical parameters, lack of intratumoral temperature measurement, and insufficient clinical data on important parameters of the treatment. In fact some data are uninterpretable results.

Hyperthermia Radiation and Chemotherapy A strong biological rationale exists for the use of local HT and systemic chemotherapy in patients with superficial and deep sited tumors.^^ Superficial metastases are often associated with additional occult distant metastases that warrant systemic treatment. Preliminary results employing cisplatinum and bleomycin with local HT revealed high response rate even in tumors located in previously irradiated sites: the better results were obtained in the treatment of breast carcinoma (Cruciani^^), head and neck and malignant melanoma. Amichetti, ^ following the previous papers by Trento group about radioHT, has published about triple association of radioHT and concomitant chemotherapy. In Table 5 are summarized the most important results published in literature about chemo-HT with or without the triple association with radiation therapy.

Prognostic Factors Affecting the Response to Hyperthermia The most important prognostic factor affecting the response to HT are listed in Table 6: some of them may be more important than others in the clinical application, e.g., the temperature and total heating time, and, when HT is done in association with radiotherapy, radiation

Table 6. Prognostic factors Temperature Time of heating Rate of heating and temporal fluctuation of the temperature Spatial distribution of the temperature Combination with radiation therapy (radiotherapy dose, dose per fraction, timing) Combination with chemotherapy (drug dose, timing) Environnamental factors (pH, nutrients level) Intrinsic sensivity

124

Hyperthermia in Cancer Treatment: A Primer

dose.^^'^ In some particular cases, e.g., for malignant melanoma, radiation dose per fraction is the most important prognostic factor."^'^^'^^

Perspectives The potential role of HT is not only limited to its interaction with traditional treatment modalities. There are a number of exciting areas in which it may have a new role to play, such as gene therapy, where the targeting of therapeutic genes was performed by heat via the HSP (heat shock protein) promoter. One of the major challenges in the current development of gene therapy strat^ies is the ability to regulate the expression of therapeutic genes to adequate levels.7'^ Another new area where HT seems to be useful is in combination with vascular targeting therapies. Numerous clinical trials are currendy in progress investigating the potential of anti-cancer agents that can either inhibit the formation of new vessels by angiogenesis, or damage the already established tumor blood supply. More recent studies have shown that the anti-tumor activity of such vascular targeting therapies can be significandy enhanced by combination widi HT.^^'^^

Conclusion The great challenge for HT in the next future is to provide adequate heating to the full tumor volume, in particular for deep seated tumors. Moreover, the amelioration of HT treatment plan could improve the correct use of HT in clinical setting. ^^ RTOG studies demonstrated that 42°C minimum temperature not were obtained for most tumors; now some devices will ultimately lead to better minimum temperature not only for superficial tumors but also for deep seated lesions. Another way to ameliorate HT in clinical setting will be the possibility to measure the temperature with minimal invasively methods^ or not invasively by means of Magnetic Resonance or Ultrasound.^^' ^

References 1. Stewart FA, Denekamp J. The therapeutic advantage of combined heat and X rays on a mouse fibrosarcoma. Br J Radiol 1978; 51(604):307-16. 2. Hahn G. Hyperthermia and Cancer. NewYork: Plenum, 1982. 3. Hildebrandt B, Wust P, Ahlers O et al. The cellular and molecular basis of hyperthermia. Crit Rev Oncol Hematol 2002; 43(l):33-56. 4. Urano M, Kuroda M, Nishimura Y. For the clinical application of thermochemotherapy given at mild temperatures. Int J Hyperthermia 1999; 15(2):79-107. 5. Kampinga HH, Dikomey E. Hyperthermic radiosensitization: Mode of action and clinical relevance. Int J Radiat Biol 2001; 77(4):399-408. 6. Ito A, Shinkai M, Honda H et al. Augmentation of MHC class I antigen presentation via heat shock protein expression by hyperthermia. Cancer Immunol Immunother 2001; 50(10):515-22. 7. Gerner EW, Hersh EM, Pennington M et al. Heat-inducible vectors for use in gene therapy. Int J Hyperthermia 2000; 16(2):171-81. 8. Kim JH, Hahn EW, Ahmed SA. Combination hyperthermia and radiation therapy for malignant melanoma. Cancer 1982; 50(3):478-82. 9. Lohr F, Hu K, Huang Q et al. Enhancement of radiotherapy by hyperthermia-regulated gene therapy. Int J Radiat Oncol Biol Phys 2000; 48(5):1513-8. 10. Eikesdal HP, Bjerkvig R, Mella O et al. Combretastatin A-4 and hyperthermia; a potent combination for the treatment of soHd tumors. Radiother Oncol 2001; 60(2): 147-54. 11. Guiot C, Madon E, Allegro D et al. Perfusion and thermal field during hyperthermia. Experimental measurements and modelling in recurrent breast cancer. Phys Med Biol 1998; 43(10):2831-43. 12. Horsman MR, Murata R. Combination of vascular targeting agents with thermal or radiation therapy. Int J Radiat Oncol Biol Phys 2002; 54(5):1518-23. 13. Valdagni R. International Consensus meeting on Hyperthermia: Final report. Int J Hyperthermia 1990; 6(5):839-77 discussion 79-80. 14. Gabriele P, Orecchia R, Ragona R et al. Hyperthermia alone in the treatment of recurrences of malignant tumors. Experience with 60 lesions. Cancer 1990; 66(10):2191-5. 15. Sannazzari GL, Gabriele P, Orecchia R et al. Head and neck tumors: Hyperthermia alone. Hyperthermic Oncology 1989; 2:438-41.

Results of Hyperthermia Alone or with Radiation Therapy and/or Chemotherapy

125

16. Corry PM, Spanos WJ, Tilchen EJ et al. Combined ultrasound and radiation therapy treatment of human superficial tumors. Radiology 1982; 145(1): 165-9. 17. Luk KH, Purser PR, Castro JR et al. Clinical experiences with local microwave hyperthermia. Int J Radiat Oncol Biol Phys 1981; 7(5):6l5-9. 18. Marmor JB, Pounds D , Hahn G M . Clinical studies with ultrasound-induced hyperthermia. Natl Cancer Inst Monogr 1982; 61:333-7. 19. Perez CA, Nussbaum G, Emami B et al. Clinical results of irradiation combined with local hyperthermia. Cancer 1983; 52(9): 1597-603. 20. U R, Noell KT, Woodward KT et al. Microwave-induced local hyperthermia in combination with radiotherapy of human malignant tumors. Cancer 1980; 45(4):638-46. 2 1 . Gabriele P, Orecchia R, Fillini C et al. Clinical hyperthermia, alone or with radiation therapy: Results of a preUminary study on recurrences of cancers. Arch Geschwulstforsch 1989; 59(3): 177-81. 22. Gabriele P, Ozzello F, Orecchia R et al. First experience on man treated by microwave and RF hyperthermia. Abstract 4th International Symposium of Hyperthermic Oncology 1984. 23. Gabriele P, Orecchia R, Ozzello F et al. Hyperthermia alone or combined with radiotherapy in the treatment of recurrences of pretreated tumors. Int J Hyperthermia 1987; 3(6):562-63. 24. Sannazzari GL, Gabriele P, Orecchia R. Thermal dose and fractionation in clinical hyperthermia. Radiol Med (Torino) 1990; 79(6):6l4-21. 25. Orecchia R, Gabriele P, Tseroni V et al. Analysis of prognostic factors in a series of 171 recurrent tumors treated by hyperthermia (HT) alone or combined with irradiation ( H T + R T ) . Hyperthermic Oncology 1989; 619-20. 26. Sannazzari GL, Gabriele P, Orecchia R et al. Radiologic hyperthermia with microwaves and radiofrequencies. II. Results in 57 neoplastic recurrences. Radiol Med (Torino) 1986; 72(7-8):573-8. 27. Gabriele P, Amichetti M, Orecchia R et al. Hyperthermia and radiation therapy for inoperable or recurrent parotid carcinoma. A phase I/II study. Cancer 1995; 75(4):908-13. 28. Gabriele P, Ozzello F, Tseroni V et al. A case of acinic cell carcinoma of the parotid gland treated with radiotherapy combined with hyperthermia and Doridamine. Radiol Med (Torino) 1990; 80(5):756-8. 29. Gabriele P, Ruo Redda M G , Nassisi D et al. Results and prognostic variables of recurrentn breast cancer treated by radiotherapy and hyperthermia. Hyperthermic Oncology 1996; 292-94. 30. Gabriele P, Orecchia R, Ruo Redda M G et al. Hyperthermia and radiations in chest wall recurrences. Hyperthermic Oncology 1992; I 380. 3 1 . Sannazzari GL, Gabriele P, Orecchia R et al. Results of hyperthermia, alone or combined with irradiation, in chest wall recurrences of breast cancer. Tumori 1989; 75(3):284-8. 32. Orecchia R, Gabriele P, Madon E et al. Vaginal recurrences of ovarian carcinoma: Treatment by intracavitary hyperthermia and low-dose radiation therapy. E S H O Meeting 1990; 527. 33. Gabriele P, Orecchia R, Sannazzari GL. Hyperthermia alone as third line therapy for recurrent adenocarcinoma. ESHO Meeting 1990; 516. 34. Gabriele P, Ruo Redda M G , Verna R. Hyperthermia and radiotherapy in the treatment of perineal recurrences. E S H O Meeting 1994. 35. Gabriele P, Ruo Redda M G , Nassisi D. Hyperthermia of malignant melanoma. Panminerva Medica 1996; 38:18-19. 36. Donato V, Zurlo A, Nappa M et al. Multicentre experience with combined hyperthermia and radiation therapy in the treatment of superficially located nonHodgkin's lymphomas. J Exp Clin Cancer Res 1997; l6(l):87-90. 37. Gabriele P, Malinverni G, Delmastro E et al. Radiotherapy associated with hyperthermia in degenerative and inflammatory disorders: A prospective randomized trial. Radiotherapy Oncology 1999; 1:108 53 supplement. 38. Gabriele P, Malinverni G, Rosmino C et al. A prospective randomized trial in orthovoltage therapy and hyperthermia associated treatments for degenerative and inflammatory disorders of the shoulders joint. Acta International Society Hyperthermic Oncology 2000; 2 3 1 . 39. Giombini A, Di Cesare A, Casciello G et al. Hyperthermia at 434 M H z in the treatment of overuse sport tendinopathies: A randomised controlled cUnical trial. Int J Sports Med 2002; 23(3):207-ll. 40. Overgaard J. The current and potential role of hyperthermia in radiotherapy. Int J Radiat Oncol Biol Phys 1989; l6(3):535-49. 4 1 . Myerson R, Moros E, Roti Roti J. Principles and practice of radiation oncology. Lippincott Pub 1997; 637-83. 42. van der Zee J, Wust P. Hyperthermia and radiotherapy. Progress in radio-oncology VII 2002; 6 5 3 . 43. Harari P, Hynynen K, Poemer R. Scanned focused ultrasound hyperthermia. Clinical response evaluation. Int J Radiat Biol Phys 1991; 21:831.

126

Hyperthermia in Cancer Treatment: A Primer

44. Perez CA, Pajak T, Emami B et al. Rajidomized phase III study comparing irradiation and hyperthermia with irradiation alone in superficial measurable tumors. Final report by the Radiation Therapy Oncology Group. Am J Clin Oncol 1991; 14(2): 133-41. 45. Arcangeli G, Nervi C, Cividalli A et al. Problem of sequence and fi-actionation in the clinical application of combined heat and radiation. Cancer Res 1984; 44(10 Suppl):4857s-63s. 46. Datta NR, Bose AK, Kapoor HK et al. Head and neck cancers: Results of thermoradiotherapy versus radiotherapy. Int J Hyperthermia 1990; 6(3):479-86. 47. Dunlop PR, Hand JW, Dickinson RJ et al. An assessment of local hyperthermia in clinical practice. Int J Hyperthermia 1986; 2(l):39-50. 48. Fuwa N, Morita K, Kimura C et al. Clinical experience of 8MHz RF hyperthermia with radiotherapy of cancer of the uterine cervix. Gan No Rinsho 1987; 33(7):799-806. 49. Goldobenko GV, Durnov LA, Knysh VI et al. Experience with thermoradiotherapy of malignant tumors. Med Radiol (Mosk) 1987; 32(l):36-8. 50. Gonzalez Gonzalez D, van Dijk JD, Blank LE et al. Combined treatment with radiation and hyperthermia in metastatic malignant melanoma. Radiother Oncol 1986; 6(2): 105-13. 51. Harima Y, Nagata K, Harima K et al. A randomized clinical trial of radiation therapy versus thermoradiotherapy in stage IIIB cervical carcinoma. Int J Hyperthermia 2001; 17(2):97-105. 52. Hiraoka M, Jo S, Dodo Y et al. Clinical results of radiofrequency hyperthermia combined with radiation in the treatment of radioresistant cancers. Cancer 1984; 54(12):2898-904. 53. Hornback NB, Shupe RE, Shidnia H et al. Advanced stage IIIB cancer of the cervix treatment by hyperthermia and radiation. Gynecol Oncol 1986; 23(2): 160-7. 54. Lindholm CE, Kjellen E, Nilsson P et al. Microwave-induced hyperthermia and radiotherapy in human superficial tumours: Clinical results with a comparative study of combined treatment versus radiotherapy alone. Int J Hyperthermia 1987; 3(5):393-4ll. 55. Overgaard J. Hyperthermia as an adjuvant to radiotherapy. Review of the randomized multicenter studies of the European Society for Hyperthermic Oncology. Strahlenther Onkol 1987; 163(7):453-7. 56. Valdagni R, Amichetti M. Report of long-term follow-up in a randomized trial comparing radiation therapy and radiation therapy plus hyperthermia to metastatic lymph nodes in stage IV head and neck patients. Int J Radiat Oncol Biol Phys 1994; 28(1): 163-9. 57. van der Zee J, Gionzales Gonzales D, Vernon CC. Therapeutic gain by hyperthermia added to radiotherapy. Progress in radio-oncology 1998; VI: 137-45. 58. Gabriele P, Ozello F, Tseroni V et al. Interstitial hyperthermia (IHT): Technical problems and methodology. Adv Exp Med Biol 1990; 267:121-7. 59. Horsman M, Overgaard J. Simultaneous and sequential treatment with radiation and hyperthermia: A comparative assessment. Interstitial hyperthermia 1992; 11-33. 60. Orecchia R, Gabriele P, Munoz F et al. The clinical effect of hyperthermia alone for recurrent tumors. ESHO Meeting 1989; C-06. 61. Oleson JR, Calderwood SK, Coughlin CT et al. Biological and clinical aspects of hyperthermia in cancer therapy. Am J Clin Oncol 1988; ll(3):368-80. 62. Cosset JM, Dutreix J, Haie C et al. Interstitial thermoradiotherapy: A technical and clinical study of 29 implantations performed at the Institut Gustave-Roussy. Int J Hyperthermia 1985; 1(1):3-13. 63. Emami B, Scott C, Perez CA et al. Phase III study of interstitial thermoradiotherapy compared with interstitial radiotherapy alone in the treatment of recurrent or persistent human tumors. A prospectively controlled randomized study by the Radiation Therapy Group. Int J Radiat Oncol Biol Phys 1996; 34(5):1097-104. 64. Petrovich Z, Langholz B, Lam K et al. Interstitial microwave hyperthermia combined with iridium-192 radiotherapy for recurrent tumors. Am J Clin Oncol 1989; 12(3):264-8. 65. Rigotti E, Sannazzari GL, Gabriele P et al. Treatment of tumors by local hyperthermia in urology and gynecology (author's transl). J Radiol 1979; 60(ll):681-4. Gd. Hamazoe R, Maeta M, Kaibara N. Intraperitoneal thermochemotherapy for prevention of peritoneal recurrence of gastric cancer. Final results of a randomized controlled study. Cancer 1994; 73(8):2048-52. ^1. Emami B, Myerson R, Pilepich R. Treatment of recurrent superficial tumors with hyperthermia and bleomycin. Proc DC Annual Meeting NAHG 1989. 68. Cruciani G, Molinari AL, Marangolo M et al. Applicability of local hyperthermia as adjuvant to systemic chemotherapy. Tumori 1987; 73(6):629-33. 69. Amichetti M, Graiff C, Fellin G et al. Cisplatin, hyperthermia, and radiation (trimodal therapy) in patients with locally advanced head and neck tumors: A phase I-II study. Int J Radiat Oncol Biol Phys 1993; 26(5):801-7.

Results of Hyperthermia

Alone or with Radiation

Therapy and/or Chemotherapy

127

70. Arcangeli G, Cividalli A, Nervi C et al. Tumor control and therapeutic gain with different schedules of combined radiotherapy and local external hyperthermia in human cancer. Int J Radiat Oncol Biol Phys 1983; 9(8):1125-34. 7 1 . Gabriele P, Orecchia R. Radiotherapy of laryngeal carcinoma ( T l a / b excluding glottic cancer). Critical review of the literature and personal experience. Radiol Med (Torino) 1991; 81(3):320-6. 72. Kitamura K, Kuwano H , Watanabe M et al. Prospective randomized study of hyperthermia combined with chemoradiotherapy for esophageal carcinoma. J Surg Oncol 1995; 60(l):55-8. 7 3 . Steindorfer P, Jakse R, Germann R et al. Hyperthermia as an adjuvant to radiation- and/or chemotherapy in far advanced recurrences of the head and neck region. Strahlenther Onkol 1987; l63(7):449-52. 74. Kapp DS, Cox RS. Thermal treatment parameters are most predictive of outcome in patients with single tumor nodules per treatment field in recurrent adenocarcinoma of the breast. Int J Radiat Oncol Biol Phys 1995; 33(4):887-99. 75. Overgaard J, Gonzalez Gonzalez D, Hulshof M C et al. Randomised trial of hyperthermia as adjuvant to radiotherapy for recurrent or metastatic malignant melanoma. European Society for Hyperthermic Oncology. Lancet 1995; 345(8949):540-3. 76. Overgaard J, Gonzalez D , Hulshof M C et al. Hyperthermia as an adjuvant to radiation therapy of recurrent or metastatic malignant melanoma. A multicentre randomized trial by the European Society for Hyperthermic Oncology. Int J Hyperthermia 1996; 12(l):3-20. 77. Huang Q , H u JK, Lohr F et al. Heat-induced gene expression as a novel targeted cancer gene therapy strategy. Cancer Res 2000; 60(13):3435-9. 78. Kanamori S, Nishimura Y, Okuno Y et al. Induction of vascular endothelial growth factor (VEGF) by hyperthermia and/or an angiogenesis inhibitor. Int J Hyperthermia 1999; 15(4):267-78. 79. Horsman MR, Murata R, Overgaard J. Improving local tumor control by combining vascular targeting drugs, mild hyperthermia and radiation. Acta Oncol 2001; 40(4):497-503. 80. Lagendijk JJ. Hyperthermia treatment planning. Phys Med Biol 2000; 45(5):R6l-76. 8 1 . Dario P, Toro M, Carrozza C et al. A new minimally invasive microprobe (500 micron) for microwave and RF hyperthermia. Hyperthermic Oncology 1996:439-41. 82. Wust P, Seebass M. Successfull implementation of non invasive magnetic resonance monitoring part-body hyperthermia. Proc E S H O 2002:31-39. 83. Cavalli R, Musacchio C, Guiot C et al. Measuring the temperature by portraiting the tumor volume with US: Preliminary in vitro investigations. Proc E S H O 2002:33-36

CHAPTER 10

Thermo-Chemo-Radiotherapy Association: Biological Rationale, Preliminary Observations on Its Use on Malignant Brain Tumors Gian Franco Ban>iizio,*\^cetizo Ceneta, Atdlio Baronzio, Isabel Freitas, Marco Mapelli and Alberto Gramaglia Everything should be made as simple as possible, but not simpler.—A. Einstein

Abstract

T

his chapter focuses on the biological rationale and the advantages for combining hyperthermia radiotherapy and chemotherapy. Other clinical aspects such as sequence of administration, effects on drug uptake and methods used to improve the efficacy of HT are also discussed. The actual applications and effects of HT on brain are presented. Furthermore, the preliminary clinical results obtained, by our group, on malignant brain tumors using this triple combination of cure are illustrated. The survival curves of brain tumors in total and of glioblastomas, treated with HT, were compared to the single standard application of conformational radiotherapy. The life quality and survival results are favorable to HT, however the number of patients treated is limited. In every case we surest its use for better clinical control of the disease.

Radiotherapy and Hyperthermia Interaction Hyperthermia Killing Curves (Arrhenius Relationship) Heat cell killing occurs exponentially as a function of time and dose and its shape is not dissimilar from those obtained for X-rays (Fig. lA). The data in vitro are consistent with results in vivo and they show that a relatively small changes in temperature can have a large effect on cell killing. ^'^ Another way to describe the kinetics of tumor cell killing is to use the Arrhenius relationship. This analysis relates time and temperature and it is the basis for the calculation of thermal activation energy."^ Arrhenius plot (Fig. IB) shows that the reciprocal of DQ values plotted versus reciprocal of the absolute temperature 1/T results in a straight line and it permits to calculate the activation energy required to obtain thermal damage. The dramatic change of the slope of the curve occurring over 43°C, called the break point, means that the activation energy is different below and above this point, reflecting a different mechanism of cell killing. Above 43 °C the activation energy for heat toxicity is similar to that for protein denaturation suggesting that the target is a protein (chromosomal proteins, nuclear matrix repair enzymes. •Corresponding Author: Gian Franco Baronzio—Family Medicine Area, ASL-01 Legnano; Radiotherapy Unit, Policlinico di Monza, Via Amati 11, 20052 Monza (Mi), Italy. Office address: P.O.B. 5, 20029 Turbigo (Mi), Italy. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

Thermo- Chemo-Radiotherapy Association

129

-lljS

12^

43^5

44.5

4i^.S

A^^

AH = 148Kcal/mol«

. ^-• \6'\r

^ ^ Break Point (43*C)

^S^\

AH= 3 6 5 KcQi/mole • A

\(A

3ia EXPOSURE TO HYPERTHERMIA (minutes)

B

317

3t« I/T

3i5

A$YNCHi
314

3i3

(X 10*)

Figure 1. Survival curves of Chinese hamster ovary ceils (CHO) heated at different temperatures for varying lengths of time. B) A typical Arrhenius plot calculated from slopes of (A) and the break point developing at 43''C are shown. (Dewey WC, Hopwood LE Sapareto SA et al. Cellular responses to combinations of hyperthermia and radiation. Radiology 1977; 123:463-474. Reprinted with permission from re£ 8.) membrane components);^'^ below the break point thermotolerance can develop gradually during the heating suggesting a cell adaptability (see Thermotolerance paragraph). The Arrhenius plot can be modified by different factors such as pH, step-down heating, some chemotherapeutic drugs, ATP status, cell cycle phase, Bioflavonoids and CoX2 inhibitors.'^'^'^'^'^^

Hypoxia, pH Blood perfusion in large solid tumors is generally poorer than that in normal tissue. ^^ The vascular beds in tumors are chaotic and poorly organized resulting in temporal and spatial unbalanced blood supply.^^Therefore, many regions within tumors result hypoxic / acidic and resistant to radiation and chemotherapy. The chronic or transient deficiency in tumor perfusion can generate state of chronic or acute hypoxia.^ Chronic hypoxia develops when cancer growth outstrips its blood supply, reaching a critical mass > 1-2 mm^ (10 cells) and a distance from host nutritive vessel > of 100-200 |im (Fig. 2). Regions of transient hypoxia can develop within tumor mass following a temporary interruption of blood flow. Transient perfusional hypoxia is caused by various mechanisms. The most plausible ones seem to be: a. Irregular expansion of tumour mass, whose three dimensional growth is subjected to a continuous remodelling, in a confined space, causes a temporary compression or occlusion of some tumour capillaries (Fig, 5)}^'^^ b. Transient stop of tumor blood flow or supply by platelets plug.^^'^^ In our opinion, this intravascular thrombosis deserves to be taken into much higher account than usually done.^^ In fact, the majority of cancer patients has coagulation abnormalities associated to hypoxia. ^^'^^ Recendy, it has been demonstrated that hypoxia not only induces VEGF but also stimulates endothelial cells to over express tissue factor (TF) and plasminogen activator inhibitor (PAI-I). These factors induce endothelium to become prothrombotic and cause fibrin formation and platelet activation. Furthermore, VEGF binds to fibrinogen and fibrin by stimulating

Hyperthermia in Cancer Treatment: A Primer

130

Figure 2. Photomicrograph of a section of mammary tumor, developed by a 5 month old female transgenic MMTV-neu (erbB2) mouse. T: tumor cord; N: necrosis; Hyp: hypoxic, perinecrotic, rim of tumor cells; C: capillary. Hematoxylin and Eosin. 40x objective. endothelial cell proliferation.^^ Fibrin has been demonstrated to be essential to supporting endothelial cells spreading and migration. ^^ The haemostatic system becomes, in a certain sense, a regulator of angiogenesis and can partially explain acute hypoxia and its regional appearance and disappearance. In conclusion hypoxia becomes a self perpetuating mechanism able to trigger angiogenesis, intratumoral fluid accumulation and thrombosis (Fig. 3)16-18

As just described, the two types of hypoxia have different origin and coexist together in a well-perflised zone of the tumor mass, causing a functional disturbance of macro and microflow. A more realistic vision shows that these two situations change continuously, because tumor blood flow is time fluctuating. Furthermore in this low flow r^ions associated but occurring independently of hypoxia an acidic environment is present. ^ ^ ' ' ' ^ ^ Hypoxia alone is the single strongest prognostic indicator of radiotherapy outcome. In fact, it has been observed that larger tumor have a greater proportion of hypoxic cells and respond less to radiotherapy.^^'^ '^^ The precise nature by which hypoxia exerts its adverse effects on cells radiosensitivity is not completely understood.^ It is believed that oxygen enhance the efficacy of radiotherapy by inducing free radicals production in the tumor area causing DNA damage.^'^^'^^

Oxygen Enhancement Ratio (OER) To better understanding the importance of oxygen on radiotherapy, survival curves of oxygenated and hypoxic cells treated with radiotherapy are generally compared. The ratio of hypoxic to aerated dose needed to achieve the same biologic effect is called oxygen enhancement ratio (OER) and is defined by the Equation 1.^^

Thermo- Chemo-Radiotherapy Association

131

GROWTH FACTOR & CYTOKiNES (PDGF. TGF^, IL1p,TNFo, IL6.IU,lt10)"

PERiTUMORAL INFLAMMATORY REACTION

NO LYMPHATIC L J NETWORK

Figure 3. In this figure the structural and functional effects of hypoxia, HIF-1 and VEGF on tumor microcirculation, cancer metabolism and therapies, are illustrated. (Modified with permission from: Baronzio et al. Anticancer Res 1994; 14:1145-1154.) OER = Dose in hypoxic conditions/ Dose in aerated conditions (to achieve same cell survival fraction) (Eq. 1) Gerweck et al,^ using the same methodology, compared the biological effect of hyperthermia on hypoxic and aerobic cells. The OER found by these authors and other confirmed that hypoxic cells sensitivity to hyperthermia were equal or greater than oxygenated cells (OER «; < 1 for HT; OER > 2.5 -3 for RT),^^'"^^ as shown in Figure 4. Acute and chronic hypoxic cells, submitted to heat, behave similarly. In fact, acute hypoxic cells have an OER near 1, whereas chronically hypoxic cells have an OER slighdy less. Furthermore, cells cultured at lower pH have a low extracellular pH, that is 0.2,0.4 units lower than blood.^'^'^^ These cells are more responsive to H T as shown in Figure 5, however the effect seems not completely dependent

132

Hyperthermia in Cancer Treatment: A Primer

1.0 ••^8;;;5w^ ^ ^ V 0.37

PE = AEROBIC = 0.77 PE = HYPOXIC = 0.92

0.10 \\^AEROBIC 0.037 HYPOXIC-\\

-J

i D CO

0.01 0.037 0.001 0.00037 0.0001 C1

t

1

4

7

1

10

1

i

i

13

16

19

MIN OF HEAT TREATMENT

Figure 4. OER curves of Chinese hamster ovary ceils (CHO) treated with hyperthermia. Hypoxia was induced 10-20 min prior to treatment. (Suit an Gerweck, Cancer Res 1979; 39:2290-2298. Reprinted with permission from ref 1.) from hypoxia. In fact cancer cells chronically deprived of nutrients, peculiarly of serum, are extremely sensitive to heat as demonstrated by Hahn.^

Cells Cycle Stage Beyond hypoxia, the reasons for combining hyperthermia and ionizing radiation are based on the following considerations: A. Heat is effective against S phase cells a relatively radio resistant phase. In fact RT effect is maximal, in G2 phase.^"^'-^^ B. Heat can interact with radiation and potentiates its cellular action by retarding the repair of radiation induced DNA damage;-^^ C. HT can induce a cytotoxic killing effect by apoptosis an important mechanism of death not essentially determined by radiation.-^^'^^

Thermal Enhancement Ratio (TER) - Therapeutic Gain Factor - Heat Radiotherapy Sequence The Radiosensitization effect of hyperthermia was quantified using the thermal enhancement ratio (TER). TER represents the ratio between the minimal dose of radiation to induce a biological effect and the dose required when radiotherapy and hyperthermia are used combined, [Eq. 2].i'2 TER = Radiation dose alone / Radiation dose with heat (to achieve same end point)

(Eq.2)

Thermo- Chemo-Radiotherapy Association

60

<20 MINUTES

^80

240

133

300

AT 41*C

60

120 MINUTES

i80

240

AT 4B*C

Figure 5. CHO cells cultured at different pH under aerobic conditions and heated during the midportion of pH exposure conditions. (Suit an Gerweck, Cancer Res 1979; 39:2290-2298. Reprinted with permission from ref. 1.) TGF = TER (Tumor)/ TER (Normal Tissues)

(Eq. 3)

A supplementary way to express the biologic effect of heat and radiation is the therapeutic gain factor (TGF) that is defined as the ratio of the TER in tumor to the TER in normal tissues, [Eq. 3]. Fiowever, this comparison is not obtained easily, because the tumor and normal tissues are not equally heated. In fact, a greater heat washout is present in normal tissue compared to tumor tissue.'^' A practical aspect in order to obtain the maximum effect by combining radiation and hyperthermia is the time and the sequence of their application. Studies by Overgaard have shown that an advantageous clinical TER was obtained when FiT and RT are delivered concomitantly; however, for inherent clinical difficultness to operate synchronously; a satisfactory TER is obtainable delivering FiT and RT within a short period. Some investigators use a time interval of 2 - 4 hs between radiation and heat to obtain a satisfactory TER (Fig. 6). Interaction between RT and FiT can be additive or super-additive. RT survival curves are influenced by FiT addition. Their behaviour has been studied on cells cultures and in animal experiments evidencing that temperature is critical for the determination of their sequence and clinical uselfuness. Brief exposure to temperatures of about 45 °C can steepen the X ray survival curve reducing the DQ. The predominant effect in this case is radiosensitization and the change of X-rays survival curve is minimal. For temperature more clinically obtainable (< 43°C) (not involved in cell killing) the principal effect is the removal of shoulder effect from X rays survival curve: this result proves that heat treatment after irradiation is the best sequence. The different effect obtained by higher (> 45°C) or lower temperatures (< 43°C) may reflect different critical target or induced thermotolerance. Recently Song et al ' have explained another enhancing factor that justifies the effectiveness of FiT and RT combination.

134

Hyperthermia in Cancer Treatment: A Primer

ao

4 2 5 % . 60 min RAOUTION i f F O t f HfAT

HiAT iirOKE iAOtATlON

T16 Simuftsn^ous

z <

S^qumtttmi

kU

ThBftpmutic Gmm Fmctoe^

<

f-^

I 1.0

as^24

6

JL.

JL.

J-

j;.

-L-

J~

4

2

0

2

4

6

24

HOURS BETWEEN TREATMENTS

Figure 6. Thermal enhancement ratio (TER) as a fimaion of time interval and sequence between hyperthermia and radiation treatment of a C3H mammary carcinoma and its surrounding skin. (Overggard J. Int J Radiation Oncology Biol Phys 1989; 16:535-549. Reprinted with permissionfromref 27.) These authors in accordance with others^^'^^ have reported that there is abundant evidence that oxygenation improves after moderate heating (42 °C). The improvement in blood flow and thereby of oxygen content may increase the effectiveness of radiotherapy and chemotherapy. In every case, the clinical evidences concerning this moderate blood flow enhancement are not sure and need further investigations.^^

Clinical Thermal Dosimetry: CEM One of major impediments to use clinically hyperthermia is the lack of a standardized thermal dosimetry. This is not a simple task, it is due to different factors. The two most important ones are: a non invasive method of temperature determination and the incapacity to have a 3D dimensional dose. Actually temperature measurements are invasive and still limited to few points measurement within a tumor. The hyperthermic treatment is usually delivered as a multiple sequenced treatment and its biological effect is time temperature-dependent. This implies that time temperature relationship varies in the same patient and from patient to patient. Thus a clinical useful unit of thermal dose could take into consideration the two variables. With the goal of reaching a uniform target temperature-time combination and a clinical method of utility, Sapareto and Dewey^ proposed the use of CEM 43 °C (Cumulative equivalent minutes) as a formulation for comparing and normalizing thermal data from various HT treatments (Time- temperature combination) [Eqs. 4 and 5]. CEM43''C = tR^^^-'^

(Eq.4)

CEM43°C = StR^^^-'^"^

(Eq.5)

Equation 4 CEM 43°C = cumulative equivalent minutes at 43°C (temperature suggested for normalization), t = time of treatment in minutes, T = average temperature during desired interval of heating, R is a constant = 0.5 when above break point and = 0.25 when below break

Thermo-Chemo-Radiotherapy Association

135

point. Equation 4, for complex time-temperature sequence the heating profile is divided into intervals of time and CEM is calculated summing the average temperature Tavg for the entire heating regimen.

Thermotolerance This phenomenon refers to a development of resistance following prior hyperthermia treatment. Chronic heating (multiple fractions) at low temperatures, below the break point, (40-42 °C), allows the development of thermotolerance. Thermotolerance occurs in almost mammalian cells in vitro and in vivo, and it is characterized by an appearance of a resistance plateau in the survival curve. Mammalian cells respond to environmental stress by activating heat shock transcription factors (HSFl) that regulate increased synthesis of heat shock proteins (HSPs). Heat shock proteins (HSPs) comprise several different families of proteins that are induced in response to a wide variety of physiological and environmental insults. Two of them (HSP 70 and HSP27) have been demonstrated to inactivate apoptosis. Hyperthermia has a killing effect on tumor cells or by inducing necrosis (thermal ablation) or programmed cell death (apoptosis), depending on the temperature used [Apoptosis for temperature < 42.5°C; necrosis for temperature > 45°C]. Recendy the demonstrated importance covered by HSPs and Prostaglandins (PGs) in the inactivation of apoptosis opens new therapeutically opportunities in sensitizing tumor cells to hyperthermia.^' ^ Furthermore, other faaors have been found to modify thermotolerance ant heat response such as pH, nutrients and oxygen status. Thermotolerance can also modify the degree of thermosensitization to radiation and chemotherapeutic drugs.^'^^ Although the nature of thermotolerance has yet to be clarified, its understanding and manipulation is critical in association with radiation, in a fractionated treatment schedule.

Chemotherapy and Hyperthermia Interaction Introduction

Biological Aspects

Several in vitro and in vivo animal studies have demonstrated that the combination of hyperthermia and cytostatic drugs can be employed synergistically.^^ However not all chemotherapeutic agents behave similarly at elevated temperature. Studies conducted by Urano et in vivo on animal tumor systems, demonstrated that some drugs, such as cisplatinum and bleomycin, have an increase in the activation energy in range of temperatures between 40°C and 45°C. For these drugs a gain in cytotoxiciy at elevated temperature is obtained, for other drugs this effect has not been demonstrated.^^ This result has permitted to classify the interaction between anticancer drugs and hyperthermia as supraddictive, additive and independent

(Table \)?^'^^ Additive means that drugs increase linearly their cytotoxic activity with increasing temperature i.e., 5FU, methotrexate, vincristine. Supraddictive means that drugs have a threshold behavior: no increase of cytotoxicity at lower temperature, marked increase above a distinct threshold temperature (i.e., bleomycin, BCNU, CDDP, Mitomycin). Independent behavior means no effect of temperature on drug activity (i.e., Ara-c, topotecan). As outlined by Dahl and Mella,^"^ the increased effect of some drugs at temperature < 42°C may be related to altered drug pharmacokinetic or pharmacodynamics parameters.

Pharmacokinetics Parameters (Drug Uptake, pH, and

Metabolism)

The aqueous solubility of some drugs (i.e., BCNU, CCNU) increases with increasing temperature, whereas others (i.e., nitosureas, methotrexate, cisplatin and chlorambucil) tend to be reduced at higher temperature. Generally the drug uptake of alkilating agents, cisplatin, doxorubicin, bleomycin, 5FU increases with the temperature, whereas the uptake of methotrexate seems not to be affected. These data have been obtained in vitro and in vivo.^^'^^ As known by pharmacology, the drugs behave like a weak base or acid, according to the pH of tumor environment. Ionization decreases lipid solubility and diffusibility across cancer

136

Hyperthermia in Cancer Treatment: A Primer

Table 1. Activity of antitumor agents, vascular targeting agents, natural substances and various drugs in presence of hyperthermia, hypoxia and pHe

Drugs

Hyperthermia

ADRIAMYCIN BLEOMYCIN MITOMYCIN BCNU CARBOPLATIN MISONIDAZOLE MITOXANTRONE LONIDAMINE MELPHALAN VINCRISTINE METHOTREXATE 5FU METABOLIC INHIBITORS CA4DP DMXXA POLYAMINES EFAs ANAESTETICS VIT.E BETULINICACID QUERCETIN RETINOICACID FAA COX2 INHIBITORS

Tt

Oxgentated Cells

tt tt ttt t t t t

n Tt tt tt tt tt t tt t

t t

t t* t. t. t. t. t. t. t. t* t* t.

Hypoxic Cells

pHe < 7.4

References

ND

ND

ttt

ttt ttt tt tt tt tt t ND ND

30,31,33 33,35 33,35,42 108,109,110 30,32,34 30 33,34 32,33,34 32,41 32,34

t*

26,30,33

t.

79,81,82,84 79,80 90 92,93 30,61,87,88 94 91 96,97 95 69,70,71,72 10

tttt tt ttt tt t

ND ND

T: additive effect with HT; t » : enhancing effect in presence of HT; t t : Supradditive effect in presence of HT; CA-4DP: Combretastatin-A4; DMXXA: 5,6-dimeithylxanthenone-4-acetic acid; EFAs: essential fatty acids; FAA: flavone acetic acid; ND: no data.

cells membranes. The effects of acidic TIF on various anticancer drugs have been studied and a remarkable influence on their activity has been found.^'^^^^ CCNU, cyclophosphamide, bleomycin, ifosfamide, melphalan and cisplatin have shown an increase activity in acidic environment and an additive effect in presence of heat (Table 1).^^ Hyperthermia may alter hepatic - renal metabolism and excretion. For example, during WBHT a slight decrease in renal elimination of carboplatin associated to moderate increase in its nephrotoxicity has been detected.^^' This example outlines an important consideration on the different effects exerted by local hyperthermia (LHT) and WBHT. In the first case, the single organ is treated and the metabolism and drug distribution can interest that single organ, whereas for WBHT the long lasting exposure and the influence exerted on different organs (especially by circidation) may influence the pharmacokinetics of different drugs used synchronously.

Pharmacodynamics

Parameters

The mechanisms responsible for the effect of potentiation of cell killing by hyperthermia have not been completely explained.^^ In the case of melphalan it has been ascribed to an increase influx leading to higher intracellular accumulation and alkylations; in the case of cisplatinum and derivatives to an a enhanced formation of DNA-platinum adducts. ^ Mito-

Thermo-Chemo-Radiotherapy Association

137

mycin C is a quinone that under hypoxic and low pH conditions is subjected to one or two electron reduction. This reduction leads to the formation of unstable reactive molecules (free radicals) which determine irreversible DNA interstrand cross-links. Mitomycin C can be classed as hypoxia activated drugs and has a clinical relevance as their use in many clinical trials. ' Starting from these studies, a new class of molecules, the bioreductive drugs, which exert their anticancer activity in hypoxic and acidic environment, have been created. ^' ^ Tirazapamine (TPZ) is one of these new molecules. It exerts its effects by producing, like Mitomycin, free radicals and removing hydrogen atoms from macromolecules. This effect produces a damaging of DNA through the formation of both single - double strand breaks. TPZ is selectively metabolized by hypoxic cells. However, TPZ alone does not exert any antitumor effect, whereas combined with radiotherapy and HT, in experimental tumors and clinical trials, has shown to be highly effective.

Drug Heat Sequence Studies in cell culture have shown that the maximal cytotoxicity was reached when the drug was scheduled simultaneously with HT. When drugs were delivered 2 to 3 hs post H T the supraddictive effect was lost. The effects of timing and sequence between drugs and HT have been evaluated in vivo models. These studies have observed the maximum efficacy when drugs were delivered just before heating.^ '^^'^^ However not all drugs behave similarly; for example the antimetabolite gemcitabicine must be delivered 24 hs before heat application to obtain the maximum effect as demonstrated in vitro in rat model. VP-l6etoposide decreases its activity when combined with local HT, whereas it shows a different behavior when combined with WBH.^2

Trimodality

Therapy

Herman et al^^ proposed in the late 1988 to combine HT RT and chemotherapy (trimodality therapy) in order to obtain a better local control of the disease. These and other authors^ ^'^^'^ reported that the combination of anticancer drugs, such as cisplatin, radiation and heat was better than the two separate modalities. These authors in a recent review have summarized many combinations of this trimodality treatment and they concluded that chemotherapy is to be delivered following a right schedule to obtain the maximum effect.^^'^ Herman et al suggest to obtain a long lasting local control of tumor by using the maximal tolerated radiation dose followed by adjuvant chemotherapy and hyperthermia. Radiation was used at the maximum dose because it is the single most effective method of treatment.^^'^ Clinical examples of trimodality association are treatments of head and neck tumors, esophageal ^ and brain tu48

mors.

Effects of Hyperthermia o n Drugs Uptake and Targeting Different anatomical and physiological barriers may limit drug penetration into tumor tissue.^ ^'^^ One of the main barriers is interstitial penetration and transport. In solid tumor, interstitial fluid pressure (IFP) is significantly increased compared to normal tissue, and it is responsible for an outward pressure gradient that reduces tumor drug penetration. ' ' Another barrier is cellular targeting. The goal of cellular targeting is to increase exposure of tumor to drug improving response reducing antineoplastic agents toxicity. Hyperthermia may improve drug concentration in target tissue by different mechanisms and can be utilized with new drug delivery approaches for better targeting anticancer drugs.

Influence ofHTon Permeability

Interstitial Fluid Pressure (IFP) and

Microvascular

Elevated interstitial fluid pressure in the tumor mass may limit the delivery and distribution of therapeutic agents.^^'^^ Jain and coworkers ^ have demonstrated that HT induces a significant decrease in IFP ameliorating in this sense the drug uptake. Furthermore, H T increases

138

Hyperthermia in Cancer Treatment: A Primer

drug extravasation and uptake by altering tumor microvascular permeability as demonstrated byNUsen^OandLefor.^^

Influence ofHTon Drug Accumulation and Resistance Hahn et al^^ have also shown an increased cellular uptake of chemotherapeutic drugs, such as adriamycin at elevated temperatures. This increased uptake can partially explaining the decreased resistance in presence of HT, as demonstrated in preclinical and clinical studies on mitomycin C, cisplatin, B C N U and anthracyclines.^^

Influence ofHT on Drug Retention As previous reported, the better time of drug administration is concomitandy or just prior HT. In fact H T determines for the first 20-25 minutes a vasodilatation, that is more evident in the tumor microcirculation compared to the normal counterpart. After this vasodilatation a decreased perfiision/or a blood flow stopping happens. This events can obtain an initial enhancement of drug retention in tumors followed by their entrapping in the tumor area.

Interaction ofHT with Drug Delivery Technologies (Liposomes, Magnetic DrugSy Nanoparticles, Degradable Microspheres)fi)rImproving Drug Targeting Drug targeting is a method for distributing drug in the body in such way that its major fraction interacts exclusively with the target tissue (tumor cells) at the cellular level. H T can enhance drug delivery and efficacy when combined with appropriate drug delivering methodologies, such as: a. Liposomes: Liposomes are small unilamellar lipid vesicles designed to have specific temperature-dependent phase transitions point. Many drugs such as methotrexate and doxorubicin can be included in these lipid vesicles and released at the point of phase transition temperature, avoiding systemic side-effects and increasing tumour cell killing.^^'^^ Maekawa et al55 have demonstrated a survival prolongation in rats receiving temperaturesensitive liposomes containing bleomycin compared to groups receiving hyperthermia or bleomycin alone, or a combination of both. b. Magnetic drugs: Recently a new class of liposomes (magnetic cationic liposomes [MCL])has been developed. These liposomes consist in cationic liposomes containing 10 nm of magnetite nanoparticles obtained by sonication. Preliminary experimental results on hamster osteosarcoma in association with hyperthermia (obtained by the application of a magnetic field with a frequency of 118 KHz), has shown a complete regression in the group using this methodology compared to a control group. ^^ c. Nanoparticles: are particulate system with a size between 500 nm and 1 mm. They are used since 1970 to carry vaccine or anticancer drugs.^^ Kong et al^^ have investigated their behavior during HT and have demonstrated that their extravasation was temperature-dependent and lasting 6 h post heat application. d. Degradable starch microspheres: Starch microspherex DSM (Spherex, Pharmacia, Sweden) are particles of cross-linked starch, measuring 20-70 jXm, degraded by amylases, and able to block transiendy and reversibly microcirculation. This method can achieve a larger increase in intratumoral temperature when administered before hyperthermia (for a decreased washout effect of heat), or can trap drugs within the heated area if used after drug administration.^^

Methods for Enhancing Thermal Sensitivity The special nature of tumor blood flow and its consequences hypoxia and intra tumoral pH reduction, previous described, does not simply produce a special hostile microenvironment but offer a method to enhance thermal sensitivity. In other words, the most important aspect of heat deposition into a tumor mass is to obtain intratumor temperature sufficient high (> 42.5°C) and homogeneous to achieve the maximum tumor kill. Clinically this energy distribution can

Thermo-Chemo-Radiotherapy Association

139

TBF MODIFICATION

HYPERTHERMIA

[mK

APOPTOSIS/NECROSiS Quercetin

METABOLIC INHIBITORS Amiloride

OXYGENATED CELLS

Radiotherapy Chemotherapy

HYPOXIC CELLS

MICROMILIEAU MODIFICATION

MEMBRANE MODIFIERS

Figure 7. The interactions of hyperthermia with chemotherapy, radiotherapy are illustrated together the principal points of tumor microenvironment modulation [TBF, micromilieu, membranes, metabolism] for improving the therapeutic response to heat. Some drugs points of intervention are depicted in order to obtain a clinical and intelligible method of treatment (TBF: tumor blood flow; VTA; vascular targeting agents).

be influenced by many pathophysiological factors such as: (a) the depth of tumor mass; (b) its 3D conformation; (c) the quantity of body fat; (d) the interstitial pH (pHe)or the intracellular pH (pHi); (e) the tumor membranes composition; (f) the tumor tissue perfusion, (g) the time of exposure to heat. Although some of them (tumor mass depth, 3D conformation and body fat composition) are not modifiable, tumor perfusion, oxygenation, extracellular pH (pHg) and membranes composition can be manipulated to improve clinically Fiyperthermia response (Fig.7).5'-«'

Metabolic and Micromilieau

Modification

Extracellular acidification of tumors has demonstrated to enhance the effect of hyperthermia, to inhibit thermotolerance in cultured tumor cells and to enhance the cytotoxicity of certain chemotherapeutic drugs. ^''^^'^^ Tumors generally exhibit high levels of glycolysis even under aerobic conditions. The excessive production of lactate from pyruvate and the decreased wash out in the tumor microenvironment carry to tumor extracellular acidification. However, this acidification would not achieve if the tumor exhibit a high level of oxidative metabolism, which competes with lactate for the available pyruvate. '^' ^ For a sensitization to heat, reduction of intracellular pH (pHi) is more important than the reduction of extracellular pH (pHg) as reviewed by Ward and Jain. However, Ward and Jain outlined that the effect of glucose load is not to be ascribed to a reduction of pH but to a decreased tumor perfusion and consequently to a deprivation of nutrients. Earlier experiments have demonstrated that i.v or i.p injection of glucose is able to reduce intratumoral pH, ' but the possible competition of lactated for pyruvate has prodded some researchers to use various metabolic inhibitors to enhance the effect of glucose load and tumor intracellular acidification further (Fig. 8). '^' ^' ^' When metabolic inhibitors such as: substitutes of glucose (D-Glucose, 5-thio-d-glucose), metabolic inhibitors (lonidamide, metaiodobenzylguanidine [MIBG]), or inhibitors of Na / H

Hyperthermia in Cancer Treatment: A Primer

140 GLUCOSE 10%

Lonidaminie Quercetin CNCn

500CC X 45'

^

pHi>7

ypHe<6.8

rV-ATPaseJ

Figure 8. In this diagram the pH regulatory mechanisms and the inhibitors (—|) used to acidify in an acute way the intracellular environment are illustrated. 1) vacuolar-type H* ATP-ase; 2) H*-lactate cotransport Na^/H exchanger [MCT]; 3) Na^-dependent C17HC03' exchanger [BCT]; 4) Sodium -proton exchanger [NHE]; 5,6) electrogenic Na^-HC03'cotransport. and HCO37 CI' antiporters (cariporide, 4,4,-diisothiocyanatostilbene -2,2,disulfonic acid [DIDS], were added to the combined treatment of heat and chemotherapy, the tumor growth was more delayed than the singidar combined treatment.^^'^^' ' '

Tumor Blood Flow (TBF) Modulation The goal of TBF modulation is to make tumor sufficiently hypoxic or underfed so to determine an increased thermal sensitivity. This biologic effect can be obtained or by stopping TBF or by modifying the tumor / stroma vasculature. TBF Reduction or Deprivation With clamping or chemoembolization the tumor becomes totally or partially hypoxic and very sensitive to heat.^^'^^ Drugs Able to Modify Tumor Microcirculation TBF /stroma microcirculatory modification can underfed tumor indirectly (Steal Phenomenon) or directly destroying tumor vasculature (VTAs). Steal phenomenon is a biologic effect obtained by vasodilatators. They divert blood flow towards normal tissues decreasing, so, TBF. This diversion occurs because tumor vasculature lacks of innervation and metabolic adaptability.11.12.59

Thermo-Chemo-Radiotherapy Association

141

Vasodilatators Hydralazine It is an arteriolar vasodilatator currently used as anthyipertensive agents. Its association with hyperthermia has been tested on C3H/Tif mammary carcinoma with a clear enhancement of heat damage. This effect was independent of the hydralazine doses used, as well as the time and temperature of heating. The hypoxia, that follows for a steal phenomena, is the major responsible for the consequent enhanced response to heat. Calcium Blocking Agents (Verapamil, Flunarizine) They are vasodilator drugs able to alter smooth muscle contraction by blocking entry of Ca^^ into myocytes. They can increase experimentally tumor blood flow and intratumoral drug retention however the experimental conditions were not clinically relevant. In fact, in many studies, tumor blood flow decreased probably for a steal phenomenon, demonstrating that tumor vasculature heterogeneity is not simply predictable.^ Serotonin and its Analogues 5-hydroxytryptamine [5-HT] (Serotonin) stimulates endothelial cells to release nitric oxyde (NO). NO causes the relaxation of endothelial smooth muscle cells producing a vasodilatation. In fact, Jirde^^ has demonstrated that the decline in tumor blood flow produced by Serotonin is secondary to a vasodilatation in surrounding normal tissues and to a local blood pressure decrease. Vascular Targeting Agents (VTAs) Recently in a variety of transplanted and spontaneous murine solid tumors VTAs have demonstrated to induce tumor necrosis, if the reduction of tumor blood flow is kept for sufficient period. Despite these effects tumor inhibition has been always short, suggesting their use in combination with other therapies. Hyperthermia and radiotherapy are the most likely to benefit from combination with VTAs. The rationale for using VTAs in association with HT is founded on the blood flow reduction induced by VTAs and on the increased degree of hypoxia that follows, with the consequent enhancement of heat application effectiveness. With RT a benefit should be possible because the use of VTAs after RT can cause an extensive necrosis of tumor central core leaving an outer shell of viable and oxygenated cells, very responsive to radiotherapy. Horsman and coll. have studied the response of C3H mouse mammary carcinoma to heat in combination with various VTAs. They have demonstrated a linear relationship between the time of heating and tumor growth time delay.^^ DMXXA: 5,6-dimethyLxanthenone-4-acetic acid and Flavone acetic acids (FAA) have shown a greater effect compared to Combretastatin-A4 (CA4DP). Their effect was maximal when heating was starting after drug administration. Flavone Acetic Flavone Acetic [FAA] is a synthetic bioflavonoid, able to induce in vivo a rapid and marked reduction in tumor blood flow. FAA exerts in addition to tumor blood flow decline a drop in platelets numbers and in the blood clotting time.^ Hill et al'^^ in a series of experiments have concluded that the antitumor activity of FAA involves besides tumor vasculature also a tumor-host factor. This factor has been identified as an immune mechanism and it consists in an increased local production of TNF-a and natural killer activity.^^'^"^ This involvement of TNF-a is supported by the fact that tumor shutdown by FAA is inhibited by antibodies to TNF-a.^^'^^ Different animal studies, in vivo, have demonstrated that the association between FAA and localized hyperthermia determines an important reduction of tumor blood flow.^^ Furthermore, FAA increases the effect of hydralazine and the cytotoxicity of Mitomycin C as demonstrated by Yamamoto'^^ and Takeuchi.^ Notwithstanding these positive results in animal experiments,^^ FAA has demonstrated to be of any clinical utility as shown by Kerr.^^ This Author,^^ in a phase 2 trial, reported that the administration of FAA by 6 h intravenous infusion to 19 patients with advanced colorectal carcinoma and 15 patients with advanced

142

Hyperthermia in Cancer Treatment: A Primer

malignant melanoma, was associated with no toxicity, but in contrast with the results predicted from the phase I study, the clinical response was generally mild in either disease conditions. 596-Dimetliylxanthenone-4-Acetic Acid [DMXXA] It is a new investigational flavonoid with an antivascular activity greater than FAA. DMXXA has been studied in a wide variety of tumors including transplanted and spontaneous rodent tumors and human tumor xenografts in nude mice. The reduction of tumor blood flow is obtained without significant effect on normal tissue.^^ The mechanism of action, like FAA, seems to be indirect and to involve the in situ production of vascular mediators such as nitric oxide (NO), serotonin, von Willebrand s factor and TNF-a. These substances have been studied and found elevated in s.c murine Colon 38 carcinomas growing in normal or tumor necrosis factor receptor -1 knockout mice. The finding that nitric oxide (NO), serotonin and TNF-a are released after DMXXA suggests that these substances contribute to the antivascular effect of the drug.^^ Different clinical trials are in progress, also in association with hyperthermia.^^ Combretastatm-A4 [CA4DP] It is an agent member of a family of chemicals obtained from the bark of South African willow. It is a prodrug, that binds to tubuUn microtubules of proliferating capillary endothelial cells, partially sparing normal nonproliferating endothelial cells.^^ This mechanism of action determines a prolonged and extensive shutdown of blood flow earring to necrosis the central mass of the tumor but lets a rim of viable cells adjacent to normal tissue.^^'^^ These effects have been demonstrated in a variety of tumor models including transplanted and spontaneous rodent tumors.^^'^^'^^ The persistence of viable cells permits a rapid tumor regrowth. Various strategies have been attempted in order to delay tumor regrowth such as drug readministration. This strategy has been demonstrated to improve tumor response but was unable to completely eliminate clinically the rim of viable tumor cells. A different approach to overcome this factor is to use CA4DP in a combined modality with conventional anticancer therapy. Recent studies have demonstrated that the combination of this drug with radiation and hyperthermia is feasible. The concept of combining these therapies together is double. In fact the cells let viable by CA4AP are generally at tumor periphery and well oxygenated, hence responsive to radiation.^^'^ The best sequence of CA4DP administration in this case is after radiotherapy. In the case of hyperthermia, studies done by Horsman and Murata ^ indicate that better results were obtainable when CA4DP were administered before hyperthermia. An interesting aspect is that the enhancement occurred at mild temperature from 40.5°C to 41°. These temperatures are easily obtainable in humans. Such combinations merit to be clinically tested. TNF(x,IL-la TNFaand IL-la are a cytokines with pleiotropic biologic activities that have demonstrated in vivo an antivascular activity. Both can decrease tumor blood supply and are able to induce hypoxia, as demonstrated by Kluge.^^ An enhanced antitumor effect has been obtained combining Interleukin 1-a with hyperthermia.^^ Furthermore TNFa has been demonstrated to be induced by antivascular agent such as DMXAA and FAA partially explaining their activity.^^'^^

Role ofBiological Membranes in Heat Response Heat has demonstrated to induce morphological and physiological alterations on cell membranes. ^' Membranes consist of a lipid bilayer composed of phospholipids and cholesterol with proteins embedded in this bilayer. Its lipid component can deeply modify their normal function of barrier.^^ The weight ratio of cholesterol-phospholipids/ protein and the degree of saturation can increase or decrease the membrane fluidity and consequentiy the membrane permeability to different ions.^^ After hyperthermia an increased inward accumulation of Ca ^ may occur. Calcium is biologically very active and take part in many cellular process such as apoptosis. Beyond intracellular calcium increase, hyperthermia can also alter the rate of

Thermo- Chemo-Radiotherapy Association diflFusion of other ions and the function of membrane kinase enzymes. Different authors have outlined that cellular membranes composition can alter the hyperthermia response and a reciprocal interaction between the two is present. Many substances belonging to drugs or natural substances can alter biological membranes and sensitise tumour cells to hyperthermia.

Drugs a. Lidocaine and anesthetics^®'^'^^ b. Calcium antagonists^^ c. Cycloxygenase inhibitors'^ Natural Substances a. Anaesthetics and alcohols^®'^^'^^ b. Polyamines aldehydes^® c. Betulinic acid^' d. Polyunsaturated fatty acids^^'^^ e. Vitamins and bioflavonoids^®'^'^^^ Anaesthetics (lidocaine, procaine, and dibucaine) have shown to interact with membranes and to increase thermal sensitization depending on concentration and temperature.^^ Alcohols, such as ethanol, have been shown to be thermal sensitizers and to have effects similar to the heat. In fact alcohols increase membrane fluidity and affect protein structure and function.^'^'^^ Other factors acting directly or indirectly on membranes such as calcium antagonist drugs (verapamil),^^ COX2 prostaglandin inhibitors,^^ bleomycin,^^ polyamines-aldehydes and essential fatty acids (EFAs)^'^ have been demonstrated to be useful in conjunction with heat. Our group^^ suggested the use of EFAs of marine origin for treating hepatocarcinoma in association with radiofrequency hyperthermia. Kokura et al, on experimental basis, have confirmed this suggestion and proved that EFAs administration carries to a gain of 30% in association with HT.^^ Among natural substances, retinoids^^ vit. E and bioflavonoids^"^'^^ differently exert a thermal sensitization. According to Prasad a-Tocopheryl succinate represents the most active form of vit. E. It association with heat is justified by its capacity to reduce currently temperature used to obtain the same biological effect. Trans retinoic acid sensitises colon adenocarcinoma cell line HT29 to HT decreasing spreading activity of these cells.^^ Quercetin sensitises cells suppressing thermotolerance development by inhibiting the synthesis of HSP70; '^^ furthermore recent studies have shown an enhancing effect at low pH tumoral environment.

Thermo-Chemo-Radiotherapy for Malignant Brain Tumors Introduction Glioblastoma is relatively frequent and represents the most malignant form of primary brain tumors. Despite the major advances in treatment such as gamma knife and radiosurgerv the prognosis is poor and generally is fatal within 1 to 2 years after the onset of symptoms. ' The resistance of gliomas to treatment with radiation and antineoplastic drugs may result in part from the effects of the extensive, severe hypoxia that is present in these tumors. ^^^'^^^ Beyond hypoxia, malignant glioma tends to infiltrate and to be replicative, so, hyperthermia represents a method to overcome hypoxia and to permit a greater local control.

Effects of Hyperthermia on Gliomas: In Vitro and in Vivo Studies Different studies in vitro have demonstrated that heat at 4l-44''C is effective and cytotoxic against glioma cells in association with RT.^^^ Fuse et al have recently tried to understand the direct effect of heat on human glioblastoma cell line A172.^°^ The cells were treated for 1 h at 43-44°C in the growing phase. Heat treatment induced cell death in a time and temperature-dependent manners. Using Hoechst 33342, the authors have demonstrated morphological nuclear changes that are consistent with apoptosis, such as accumulation of p53 protein, bax proteins and mRNA.^^^

145

144

Hyperthermia in Cancer Treatment: A Primer s 5

^ ^

,'f|10)

U

>^^ 3

^ j ^ ^ f^^^

UJ

1^ QL D O

'•*'

1

T ^^^M I D

y^

A

X^'"''^ ^''

1 '

> i ! 11)

^^ •

_j

o >

.--^no) ^.-T'MO}

§ as h-

LU 0-5

'

\ \

> H-

<

0.A

•k

_

LU

v

\

BTiAn 0.2

I/

\\

i

A

i 0

i/ /

\

"* 1 J2

i 16

a • h ii

/

i 20

t Ik

28

a • A, ^

rnwTPrJI OUNI r\VjL ACNU (20MG/KG) ACNU (20 MG/KG)--GLUCOSE

a

o ACNU (lOMG/KG)

•• 0 X

• HYPERTHERMIA 0 ACNU (10MG/KG)*HYPERTHERMIA X ACNU (lOMG/KG) •HYPERTHERMIA * GLUCOSE

32

DAYS AFTER TREATMENT

Figure 9. Growth curves of BT4An tumors after ACNU 20 or 10 mg/kg combined with hyperthermia with or without hypertonic glucose 6 g/Kg i.p. 2 hours before treatment (Shem BC, Dahl O. J Neuroncology 1991; 10:247-251 with permissionfromref 107). Tanaka et al^^"^ have studied the combined effects of heat and chemotherapy on human glioblastoma cell line (SKMGl) and on 3 rat malignant brain tumor cell lines (T9,EB 679, TR 481). Treatments included heat alone (42'C for 1 h), drug alone (42"C for Ih), and HT (42°C for 1 h) before, after or concomitantly to Nimustine (ACNU), Cisplatin or Aclarubin (ACR) exposures. The greatest cytotoxic effect was obtained using simultaneously HT plus ACNU, ACR or cisplatin. Different works have been performed in animals in vivo. Kobayashi^°5 andTanaka^^ obtained a significant prolongation of survival on two different animal models using magnetic induction heating (45°C for 1 h) through a ferromagnetic implant. Furthermore, Tanaka group demonstrated a longest survival when implanted HT was used combined with Chemotherapy (ACNU). Shem and Dahl^^^ conducted an interesting work on animals in vivo using HT associated to chemotherapy on glioblastoma. This methodology is the biological basis for our approach of Trimodality treatment (RT + HT + BCNU and glucose load) on glioblastoma and astrocytomas. They used Carmustine (BCNU) as chemotherapeutic agent for its demonstrated efficacy on gliomas. ^°^'^^^ Furthermore, associated to HT, nitrosureas (BCNU) showed an interesting behavior that consisted in a higher cytotoxicity when used at ph 6.5-7.0 at 43*'C compared to that at normal pH and temperature.^^° Studies by Watanabe have also demonstrated that Nitrosureas had similar cytotoxicity under euoxic and hypoxic conditions.^^^ As previous, reported the majority of human tumors have a hypoxic core and gliomas are not an exception. Hypertonic glucose has demonstrated to induce pH extracellular change by increasing lactic acid production during HT increasing so the effect of Nitrostu'eas at high temperature. Moreover, this work has analyzed the sequence of administration of the various therapies.^ As shown in Figure 9, ACNU followed by HT + glucose is the best sequence. The growth rate of tumors decreased after this sequence of therapy and the effect lasted for 2 weeks at least (Fig. 9).^^^ Another interesting aspect of use of HT in gliomas is the thermotolerance behavior. In

Thermo-Chemo-Radiotherapy Association

145

fact gliomas, unlike other cells, do not develop thermotolerance when treated for long time at mild temperature at 39-42°C for up to 48 hrs. Thermotolerance develops at temperature above 42 °C and its decay is temperature-dependent.

Clinical Human Trials ofHT^

RT on Brain

Tumors

Different brain heating methods have been used and may be classified as external or interstitial. All methods have advantages and disadvantages; however, the principal discriminator between them is the dimensional target control of the power deposition. Different clinical trials on human have been collected in two recent reviews. The authors conclude that HT is a feasible treatment and has an effective approach to brain tumors. Important selection criteria are tumor size (> 6 cm) and tumor location. The majority of studies have been conducted with interstitial hyperthermia as adjuvant to convectional radiotherapy^ ^ '^ ^ and have demonstrated an increased survival. An example is the work of Sneed et al^ that have treated a considerable number of patients (112 pts) with a significant median survival increase for the groups treated with heat compared to non heat-treated patients.

Toxicity

ofHT

Seegenschmiedt^ in his review of 1995 affirmed that treatment toxicity is relatively low and long-term side effects are similar to that observed with RT alone. Ikeda et al^^^ studied the toxicity of radiofrequency interstitial HT in dog and found alteration of blood brain barrier (BBB). Other authors outlined that the maximum tolerated heat dose of central nervous system lies in the range of 40-60 min at 42-42.5°C or 10-30 min at 43°C.^^^ A recent review by Sharma and Hoopes^^^ has reported that HT produces specific alterations in the mammalian CNS that may have long term behavioral, physiological and pathological consequences. The morphological alterations for temperature in the range 40°C to 42 "C for 4 hrs regard the axons, the nerve cells, the glial cells and the vascular endothelium. Sneed randomized study had demonstrated that HT has an acceptable toxicity, in fact no grade 5 toxicity was found outside 4 patients on 112 (3.5%) with grade 2 and j}^^ In conclusion we may say that HT treatment of brain, once believed to be nontoxic, must be delivered under the observation of skillful staff to avoid serious side effects.

Experimental Studies Conformational

Radiotherapy

Administration

Conformal radiotherapy (CFRT) or Linac radiosurgery is now a mainstay of treatment for patients with primary and metastatic brain cancers. ^^^'^'^^ It allows delivering a larger dose of radiation on the lesion sparing normal tissues surrounding the tumor area. Radiation oncologists using computerized tomography scans (CT) or Magnetic resonance (MRI) scans, or both, can determine the cancer size and shape in 3 dimensions (3D) (Fig. 10). In this way precisely focused, high dose, radiation beams can be delivered to cancer mass (usually 3 cm or less in diameter) in a single or multiple treatment sessions. Brain cancers treated with these techniques are generally considered inoperable. Prior to radiation patients are fitted with a head frame, meantime CT and MRI scans are performed to determine treatment planning. After the acquisition of these informations, patients are positioned on a sliding bed around the linear accelerator circles (Fig. II). The linear accelerator directs arcs of radioactive photon beams to tumor. ^"^^ The pattern of the arc is computer-matched to the tumor shape using specific multileaf collimators.

Hyperthermia

Device

Characteristics

Synchrotherm radiofrequency (RF) clinical hyperthermia unit was developed by DUER®, Vigevano, Italy. It consists of following components: (1) a RF generator (13.56MHz), (2) a pair of mobile plates or electrodes with independent superficial cooling system, (3) a heat exchanger, (4) a computerized control console. Special characteristic of this device compared to

146

Hyperthermia in Cancer Treatment: A Primer

Figure 10. Example of RT isodose calculation for brain tumors. similar on commerce is the complete automatic coupling of power deposition with minimum human engagement as described in a previous work of our group. ^ The choice of plates/electrodes available in diameters of 10,15,20,25,30 cm and the appropriate application on opposite sides of the brain to better focalize the power deposition depended principally from the volume and the depth from the skull of cancer mass. The thermal profiles to obtain a probable deposition of the energy were obtained by heating patterns produced in a static phantoms under various conditions. Cylindrical phantoms with diameters of 30 cm and various thicknesses were made of 4% agar gel containing 0.2% NaCl. The isotherms were monitored and reconstructed through computerizations of images obtained by a special film sensible to temperature (Fig. 12). A flexible vinyl sheet, forming a space filled with 0.4% NaCl solution, covered the surfaces of the electrodes. The saline solution circulated between the electrode and the heat exchangers. Differently to other cooling system, the two electrodes were independently controlled and were simply adaptable to the contour of the brain patients, thanks to their flexibility (Fig. 13). These plates are coupled to opposite side of the patient *s brain and kept in place thanks to a girdle permitting a better contact over the irre^arity of the skull contour.

Patients Profile and Treatment Schedule Thirteen patients (4 female, 9 male; median age 37 ± 11 years old) with astrocytomas of various degree, were treated combining chemotherapy, radiotherapy and hyperthermia with the following sequence. HT was applied after 2 hrs from CRT administration, and the patients used orally 120 mg of BCNU two hs prior HT. BCNU was administered once every cycle of HT, generally at the first application. A complete cycle of HT consisted in five applications, applied every 48 hrs (Table 2). Four mg of e.v. dexamethasone was started 1/2 h before HT associated to the hypertonic solution of glucose 10% 500 cc that lasted for all the treatment period (60') (Table 2). Dexamethasone was preferred for its relatively little mineralcorticoid

Thermo- Chemo-Radiotherapy Association

147

Figure 11. Example of patient's positioning before conformational radiotherapy treatment. Plate 2C cm cooiing 10*

p £^^

Plate 10 cm cooling 10'

Plate 12 cm cooling 10* ^

38*C 1 39*C 1 4II*C

• 41'C 1 U42-C 1 1

Plate 20 cm

Plate 20 cm

Plate 26 cm

Figure 12. Thermal distribution in an agar phantom (30 cm x 30 cm) heated with a pair of different electrodes. The saline bolus (cooling) between agar phantom and electrodes was kept at 10°C. The focusing effect and the possibilities on using plates of different sizes in order to heat the tumor at different depth are also illustrated. (The thermal distribution ofthe agar phantom were kindly supplied by DUER® Vigevano.)

148

Hyperthermia in Cancer Treatment: A Primer

Figure 13. Example of electrode contour adaptability.

Table 2. Treatment schedule cimically used by our group for treating glioblastoma Ten minutes after conformational radiotherapy an infusion of glucose 10% 500 cc is initiated and continued for 90'. Thirty minutes after radiotherapy, hyperthermia is initiated and last 60 minutes. The HT treatment is performed twice weekly for three/or four weeks. To increase the intracellular pH, a week before the treatment, we use orally quercetin (1200 mg/daily) and Moduretic® (Amiloride, 5mg/ daily). Quercetin is used to reduce thermotolerance. Celebrex® (Celecoxib 200-400 mg/daily) is used for its antiangiogenicity and growth delay activity.

activity and fluid retention. Patients were treated orally with a standard dose of 100 mg of anticonvulsants to control and avoid seizures. They received a supplementation of bioflavonoids and ome^a three fatty acids, following the standard demonstrated to be effeaive by our group/^^ to avoid radiation damage. The principal end point of this preliminary work has been the overall calculated survival, according the Kaplan-Meyer method starting on the first day of conformational radiotherapy (CRT). The control group was constituted by 17 patients with Astrocytomas of III and IV stage (8 F; 9 M median age 41 ±14) treated with CRT alone. The significance was posed as p < 0.05 and the follow up was achieved at 45 day interval with CT / MR or positron emission tomography (PET) with 82 Rb and 18 F-FDG.

Results The survival curves of the groups were compared, according to the Kaplan-Meyer method and log Rank test. Hyperthermia group survival was compared in total with the group of brain tumors consisting of glioblastoma, with the best response according to the standard therapy of our institution. The first survival curve (Fig. 14) represents all patients treated with HT versus CRT. The significance between the two groups favors the HT treated group (p = 0.003). To avoid bias, the 2nd survival curve were compared, excluding from the HT group 4 pts, with low malignancy tumors. Even in this 2nd comparison (Fig. 15) the HT group survival was evidently increased (p = 0.04). Four patients are actually alive and with more or less remarkable quality of life. Our patients did not suffer of side effects out of that induced by CRT.

Thermo-Chemo-Radiotherapy Association

149

P=0.003

1 O

L i Glucose + BCNU +HT

0,6-

\

a™

CRT

CO

0.2-

\ 12 2

6

10

20

16 14

18

24 22

28 26

32 30

36 34

40 38

44 42

46

MONTHS Figure 14. Survival curves of patients aflPected by glioblastoma treated with CRT alone and patients with brain tumors of different histologies treated with HT + CRT.

C

1-

P= 0,040

O CO CO

0. 8-

>

E 0.6-

Glucose + BCNU + HT

\

V

CRT

0.4-

n.

0.2-

\ 12 2

6

10

20

16 14

18

24 22

28 26

32 30

40

36 34

38

44 42

46

Months Figure 15. Survival curves of patients affected by glioblastoma treated with CRT alone and with CRT + HT.

150

Hyperthermia in Cancer Treatment: A Primer

Figure 16. Magnetic resonance (MR) scans of a 37 year old man with a vast mid brain-temporal astrocytoma inoperable. A regression of the tumor is shown at 1 years follow-up after 2 cycle of RT+ HT. The patient at 5 year follow-up is still alive and has resumed his work.

Conclusions and Comments Actually a strong biological base for associating hyperthermia to radiotherapy and to chemotherapy exists. On the other part, the confirmation of the potential addition of hyperthermia and radiotherapy in several phase III trials oblige the oncologists to take in greater consideration this association, and to consider the combination CRT, chemotherapy and glucose load an attractive approach at least for non responsive tiunor such as glioblastomas. Preliminary studies combining local hyperthermia chemotherapy and glucose load in the treatment of primary brain tumors have been demonstrated by our group feasible and useful. It has been demonstrated by the increased survival of these kind of patients and by the reduction of great intracranial tumor mass (Fig. 16), however many aspects are to be clarified, for example the methodology and the validity of the treatment. In fact, for a definitive approval of this approach, future patients will be randomized and treated with different sequence of HT-CRT. In this preliminary study HT chemotherapy followed CRT however a different sequence such as HT chemotherapy before CRT must be proved and compared.

Thermo-Chemo-Radiotherapy

Association

151

References 1. Suit H D , Gerweck LE. Potential for hyperthermia and radiation therapy. Cancer Res 1979; 39:2290-2298. 2. Hall EJ. Hyperthermia. In: Hall EJ, ed. Radiobiology for the Radiologist. Lippincott Williams Wilkins, 2000:495-520. 3. Sapareto SA, Dewey W C . Thermal dose determination in cancer therapy. Int J Radiat Oncol Biol Phys 1984; 10:787-800. 4. Lepock JR. Cellular efifects of hyperthermia: Relevance to the minimum dose for thermal damage. Int J Hyperthermia 2003; 19:252-266. 5. Oleson JR. Hyperthermia. In: Mauch PM, Loffler JS, eds. Radiation Oncology - Technology and Biology. W B Saunders, 1994:276-299. 6. Dewhirst M W , Viglianti BL, Lora-Michels M et al. Basic principles of thermal dosimetry and thermal thresholds for tissue damage from hyperthermia. Int J Hyperthermia 2003; 19:267-294. 7. Dewey W C . Arrhenius relantionship from molecule and cell to the cUnic. Int J Hyperthermia 1994; 10:457-483. 8. Dewey W C , Hopwood LE Sapareto SA et al. Cellular responses to combinations of hyperthermia and radiation. Radiology 1977; 123:463-474. 9. Calderwood SK, Asea A. Targeting HSP70-induced thermotolerance for design of thermal sensitizers. Int J Hyperthermia 2002; 18:597-608. 10. Asea A, Mallick R, Lechpammer S et al. Cyclooxygenase inhibitors are potent sensitizers of prostate tumours to hyperthermia and radiation. Int J Hyperthermia 2 0 0 1 ; 17:401-414. 11. Jain RK. Determinants of tumor blood flow: A review. Cancer Res 1988; 48:2641-2658. 12. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply and metabolic microenvironment of human tumors, a review. Cancer Res 1989; 49:6449-6465. 13. Freitas I, Baronzio GF. Tumor hypoxia, reoxygenation and oxygenation strategies: Possible role in photodynamic therapy. J Photochem Photobiol B Biol 1991; 11:3-30. 14. Gullino PM. The internal milieu of tumors. Prog Exp T u m Res 1966; 8:1-25. 15. Vaupel P. Tumor blood flow. In: Molls M, Vaupel P, eds. Blood Perfusion and microenvironment of human tumors. Berlin Heidelberg New York: SpringerVerlag, 2000:41-46. 16. Durand RE. Intermittent blood flow in solid tumours an under -appreciated source of "drug resistance". Cancer Metastasis Rev 2001; 20:57-61. 17. Baronzio GF, Freitas I, Kwann H. T u m o r microenvironment (hypoxia -interstitial Fluid) and haemorheologic abnormalities. Semin Thromb Hemost 2003; 29:489-497. 18. Denko N C , Giaccia AJ. Tumor hypoxia, the physiological link between Trousseau's Syndrome (carcinoma -induced Coagulopaty) and metastasis. Cancer Res 2001; 61:795-798. 19. Hall EJ. T h e oxygen eff^ect and reoxygenation. In: Hall EJ, ed. Radiobiology for the radiologist. Lippincott Williams Wilkins, 2000:91-111. 20. Gerwek LE, Richards B. Influence of p H on thermal sensitivity of cultured human glioblastoma cells. Cancer Res 1981; 41:4019-4024. 2 1 . Gerwek LE, Dahlberg WK, Greco B. Effect of p H on single or fractionated heat treatment at AlAyC. Cancer Res 1983; 43:1163-1167. 22. Asby BS, Cantab MB. p H studies in human malignant tumours. Lancet 1996; 2:312-315. 23. Stubbs M , McSheehy PMJ, Griffiths JR et al. Causes and consequences of tumor acidity and imphcations for treatment. Mol Med Today 2000; 6:15-19. 24. Hahn G M . Metabolic aspects of the role of hyperthermia in mammalian cell inactivation and their possible relevance to cancer treatment. Cancer Res 1974; 34:3117-3123. 25. Kamping H H , Dikomey E. Hyperthermic radiosensitization: Mode of action and clinical relevance. Int J Radiat Biol 2001; 77:399-408. 26. Song C W , Park H , Griffin RJ, Theoretical and experimental basis of Hyperthermia. In: Kosaka M , Sugahara T, Schmidt KL et al, eds. Thermotherapy for neoplasia, inflammation, and pain. Springer Verlag Tokyo, 2001:394-407. 27. Overgaard J. The current and potential role of hyperthermia in radiotherapy. Int J Radiation O n cology Biol Phys 1989; 16:535-549. 28. Song C W , Park H, Griffin RJ. Improvement of tumor oxygenation by mild hyperthermia. Radiation Research 2001; 155:515-528. 29. Bicher H I , Hetzel FW, Sandhu TS et al. Effects of hyperthermia on normal and tumor microenvironment. Radiology 1980; 137:523-30. 30. Hahn G M . Thermal Enhancement of the actions of anticancer agents. In: Hahn G M , ed. Hyperthermia and Cancer. New York, London: Plenum Press, 1982:55-85. 31. U r a n o M , K u r o d a M , N i s h i m u r a Y. I n v i t e d Review. For T h e clinical a p p l i c a t i o n of thermochemotherapy given at mild temperatures. Int J Hyperthermia 1999; 18:79-107.

152

Hyperthermia in Cancer Treatment: A Primer

32. Dahl O, Mella D. Hyperthermia and chemotherapeutic agents. In: Field SB, Hand JW, eds. An introduction to the practical aspects of clinical hyperthermia. London, New York, Philadelphia: Taylor and Francis, 1990:108-142. 33. Herman TS, Teicher BA, Jochelson M et al. Rationale for use of local hyperthermia with radiation therapy and selected anticancer drugs in locally advanced human malignancies. Int J Hyperthermia 1988; 4:143-158. 34. Teicher BA, Herman ST. Trimodality therapy: Antitumor chemotherapy with hyperthermia and radiation. In: Mauch PM, Loeffler Jay S, eds. Radiation Oncology Technology and biology. Philadelphia, London, Toronto, Montreal, Sydney, Tokyo: WB Saunders, 1994:316-347. 35. Raaphorst GP. Fundamental aspects of hyperthermic biology. In: Field SB, Hand JW, eds. An introduction to the practical aspects of clinical hyperthermia. London, New York, Philadelphia: Taylor and Francis, 1990:108-142. 36. Gerweck L, Tumor PH. Implications for treatment and novel drug design. Seminars in Radiation Oncology 1998; 8:176-182. 37. Douple EB, Strohbehn JW, de Sieyes DC et al. Therapeutic potentiation of cis-dichlorodiammineplatinum (II) and radiation by interstitial microwave hyperthermia in a mouse tumor. National Cancer Institute Monographs 1982; 61:259-262. 38. Herman TS, Teicher BA. Sunmmary of studies adding systemic chemotherapy to local hyperthermia and radiation. Int J Hyperthermia 1994; 10:443-449. 39. Vanakoski J, Seppala T. Heat exposure and drugs. ClinPharmacokinet 1998; 34:311-322. 40. Gerke P, Filejski W, Robins HI et al. Nephrotoxicity of ifofosfamide, carboplatin and etoposide (ICE) alone or combined with extracorporeal or radiant-heat-induced whole-body hyperthermia. J Cancer Res Clin Oncol 2000; 126:173-177. 41. Bates DA, MacKillop WJ. Effect of hyperthermia on the uptake and cytotoxicity of melphalan in Chinese hamster ovary cells. Int J Radiat Oncol Biol Phys 1998; 16:187-191. 42. Zaffaroni N, Villa R, Orlandi L et al. Effect of hyperthermia on the formation and removal of DNA interstrand cross-Unks induced by melphalan in primary cultures of human malignant melanoma. Int J Hyperthermia 1992; 8:341-349. 43. Rienbroek RC, van de Vaart PJ, Haveman J et al. Hyperthermia enhances the cytotoxicity activity and platinum -DNA adduct formation of lobaplatin and oxiplatin in cultured SW 1573 cells. J Cancer Res Clin Oncol 1977; 1213:6-12. 44. Denny WA. The role of hypoxia - activated prodrugs in cancer therapy. Lancet Oncol 2000; 1:25-29. 45. Masunaga S-I, Ono K, Nishimura Y et al. Combined effects of tirapazamine and mild hyperthermia on anti-angiogenic agent (TNP-470) treated tumors-reference to the effect on intratumor quiescent cells. Int J Radiat Oncol Biol Phys 2000; 47:799-807. 46. Arcangeli G, Cividalli A, Lovisolo G et al. Effectiveness of Local Hyperthermia in association with radiotherapy or chemotherapy. In ArcangeU G, Mauro F, eds. Comparison of multimodality treatments on multiple neck node metastases. Proceedings of the 1st meeting of European group of hyperthermia in radiation oncology, Milan, Masson Italia Editori: 1980:257-265. 47. Nozoe T, Saeki H, Ito S et al. Preoperative hyperthermochemoradiotherapy for esophageal carcinoma. Surgery 2002; 131:s35-s38. 48. Sneed PK, Stea B. Thermoradiotherapy for brain tumors. In: Seegenschmiedt MH, Fessenden P, Vernon CC, eds. Thermoradiotherapy and Thermochemotherapy. BerHn, Heidelberg, New York: Springer Verlag, 1995:159-175. 49. Leunig M, Goetz AE, Dellian M et al. Interstitial fluid pressure in solid tumors following hyperthermia: Possible correlation with therapeutic response. Cancer Res 1992; 52:487-490. 50. Nilsen NO. Endothelial changes and microvascular leakage due to hyperthermia in chick embryos. Wirchows Arch B Cell Pathol Incl Mol Pathol 1984; 46:165-174. 51. Lefor AT, MaKohon S, Ackerman NB. The effects hyperthermia on vascular permeabiUty in experimental liver metastasis. J Surg Oncol 1985; 28:297-300. 52. Towle LR. Hyperthermia and Drug Resistance. In: Urano, Douple eds. Hyperthermia and Oncology. VSP, 1994:4:91-113. 53. Drummond DC, Meyer O, Hong K et al. Optimizing Hposomes for delivery of chemotherapeutic stents to solid tumors. Pharmacological Reviews 1999; 51:692-743. 54. Sandip BT, Udupa N, Rao BSS et al. Thermosensitive liposomes and localised hyperthermia - An effective biomodality approach for tumor management. Indian J Pharmacol 2000; 32:214-220. 55. Maekawa S, Sugimachi K, Kitamura Y et al. Selective treatment of metastatic lymph nodes with combination of local hyperthermia and temperaturesensitive liposomes containing bleomycin. Cancer Treat Rep 1987; 71:1053-1059. 56. Matsuoka F, Shinkai M, Honda H et al. Hyperthermia using magnetite cationic liposomes for hamster osteosarcoma. Biomagn Res Tech 2004; 2:3.

Thermo-Chemo-Radiotherapy

Association

153

57. Moghini SM, Hunter AC, Murray JC. Long-circulating and target-specific nanoparticles: Theory and practice. Pharmacology Rev 2001; 53:283-318. 58. Kong G, Braun R D , Dewhirst W. Characterization of the effect of hyperthermia on nanoparticles extravasation from tumor vasculature. Cancer Res 2 0 0 1 ; 61:3027-3032. 59. Jirtle RL. Chemical modification of tumor blood flow. Int J Hyperthermia 1988; 4:355-371. 60. Yatvin M W , Cramp WA. Role of cellular membranes in hyperthermia: Some observations and theories reviewed. Int J Hyperthermia 1993; 9:165-185. 6 1 . Sensiterra GA, Lepock JR. Thermal destabilization of transmembrane proteins by local anesthetics. Int J Hyperthermia 2000; 16:1-17. 62. Zhou R, Bansal N , Leeper DB et al. Enhancement of hyperglycemia-induced acidification of human melanoma xenografts with inhibitors of respiration and ion transport. Acad Radiol 2 0 0 1 ; 8:571-582. 63. Zhou R, Bansal N , Leeper DB et al. Intracellular acidification of human melanoma xenografts by the respiratory inhibitor m-iodobenzylguanidine plus hyperglycemia: A^^ P magnetic resonance spectroscopy study. Cancer Res 2000; 60:3532-3536. GA. Ward KA, Jain RK. Response of tumours to hyperglycaemia: Characterization, significance and role in hyperthermia. Int J Hyperthermia 1988; 4:223-250. 65. Song C W , Lyons J C , Makepeace C et al. Effects of H M A , an analog of amiloride, o n thermosensitivity of tumors in vivo. Int J Radiation Oncology Biol Phys 1994; 30:133-139. 66. Lyons JC, Kim GE, Song C W . Modification of intracellular p H and thermosensitivity. Radiat Res 1992; 129:79-87. G7. Horsman MR, Christensen KL, Overgaard J. Hydralazine -induced enhancement of hyperthermic damage in a C 3 H mammary carcinoma in vivo. Int J Hyperthermia 1989; 5:123-136. 68. Vaupel P, Menke H. Effects of various calcium antagonists on blood flow and red blood cell flux in malignant tumors. Prog Appl Microcirc 1989; 14:88-103. 69. Horsman MR, Murata R. Combination of vascular targeting agents with thermal or radiation therapy. Int J Radiat Oncol Biol Phys 2002; 54:1518-1523. 70. Yanamoto M, Kusumoto T, Endo K et al. Vasoacting agents Flavone acetic acid and Hydralazine given in combination enhance antitumor effects under condition of hyperthermia. Oncology 1996; 53:147-152. 7 1 . Hill SA, Williams KB, Denekamp J. Studies with a panel of tumors having a variable sensitivity to FAA, to investigate the mechanisms of action. Int J Radiat Oncol Biol Phys 1991; 60:379-384. 72. Baguley BC, Calveley SB, Crowe KK et al. Comparison of the effects of Flavone acetic acid, fostriecin, homoharringtonine and tumor necrosis factor alpha on colon 38 tumors in mice. Eur J Cancer Clin Oncol 1989; 25:263-269. 73. Chin L-M, Baguley BC. Induction of natural killer cell activity by the antitumor compound flavone acetic (NSC 347512). Eur J Cancer CHn Oncol 1987; 23:1047-1050. 74. Mahadevan V, MaliK STA, Meager A et al. Role of tumor necrosis factor in Flavone acetic acid induced tumor shutdown. Cancer Res 1990; 50:5537-5542. 75. Horsman MR, Samponson LE, Chaplin DJ et al. T h e in vivo interaction between flavone acetic acid and Hyperthermia. Int J Hyperthermia 1996; 12:779-789. 7G. Takeuchi H , Baba H , Maehara Y et al. Flavone acetic acid increases the cytotoxicity of Mitomycin c when combined with hyperthermia. Cancer Chemother Pharmacol 1996; 38:1-8. 77. Mc Bibby, Double JA. Flavone acetic acid - from laboratory to clinic and back. Anticancer Drugs 1993; 4:3-17. 78. Kerr DJ, Maughan T, Newlands E et al. Phase two trials of Flavone acetic acid in advanced malignant melanoma and colorectal cancer. Br J Cancer 1989; 60:104-106. 79. Baguley BC, Wilson WR. Potential of DMXAA combination therapy for solid tumors. Expert Rev Anticancer Ther 2002; 2:593-603. 80. Baguley BC. Small molecule cytokine inducers causing tumor necrosis. Curr Opin Invest Drugs 2001; 2:967-975. 8 1 . Nihei Y, Suzuki M , Okamo A et al. Evaluation of antivascular and antimitotic effects of tubulin binding agents in solid tumour therapy. Jpn J Cancer Res 1999; 90:1387-1396. 82. Grosios K, Holwell SE, McGown et al. In vivo and n vitro evaluation of combretastatin A-4 and its sodium phosphate prodrug. Br J Cancer 1999; 81:1318-1327. 83. Beauregard DA, Hill SA, Chaplin DJ et al. T h e susceptibility of tumors to the antivascular drug combretastatin A4 phosphate correlates with vascular permeabiHty. Cancer Res 2 0 0 1 ; 61:6811-6815. 84. Tozer G M , Kanthou C, Parkins CS et al. The biology of the combretastatin as tumor vascular agents. International J Exp Pathol 2002; 83:21-38.

154

Hyperthermia in Cancer Treatment: A Primer

85. Kluge M, Elger B, Engel T et al. Acute effects of tumor necrosis a or lymphotoxin on global blood flow, Laser Doppler flux, and bioenergetic status of subcutaneous rodent tumors. Cancer Res 1992; 52:2161-2173. 86. Song CW, Lin J-C, Lyons JC. Antitumor effect of interleukin 1-a in combination with hyperthermia. Cancer Res 1993; 53:324-328. 87. Kim JH. Modification of thermal effects: Chemical modifiers. In: Urano M, Douple E, eds. Hyperthermia and Oncology. The Netherlands: VSP, 1988:1:99-119. 88. Marcocci L, Mondovl B. Biochemical and ultrastructural changes in the hyperthermic treatment of tumor cells: An outline. Consensus On Hyperthermia for 1990s. Adv Exp Med Biol 1990; 267:99-120. 89. Kameda K, Kondo T, Tanabe K et al. The role of intracellular Ca 2+ in apoptosis induced hyperthermia and ist enhancement by verapamil in U937 cells. Int J Radiat Oncol Biol Phys 2001; 49:1369-1379. 90. Agostinelli E, Arancia G, Calcabrini A et al. Hyperthermia-induced biochemical and ultrastructural modifications in cultured cells. Exp Oncol 1995; 17:269-276. 91. Wachsberger PR, Burd R, Wahl ML et al. BetuHnic acid sensitization of low pH adapted human melanoma cells to hyperthermia. Int J Hyperthermia 2002; 18:153-164. 92. Baronzio GF, Solbiati L, lerace T et al. Adjuvant therapy with essential fatty acids (EFAs) for primary liver tumors: Some hypotheses. Med Hypotheses 1995; 44:149-154. 93. Kokura S, Yoshikawa T, Kaneko T et al. Efficacy of hyperthermia and polyunsaturated fatty acids on experimental carcinoma. Cancer Res 1997; 57:2200-2202. 94. Prasad K, Kumar B, Yan X-D et al. a-tocopheryl succinate, the most effective form of Vit. E for adjuvant cancer treatment: A review. J Am Coll Nutr 2003; 22:108-117. 95. Callari D, Sinatra F, Paravizzini GL et al. All trans retionic acid sensitizes colon cancer cells to hyperthermia cytotoxic effects. Ind J Oncol 2003; 23:181-188. 96. Wachsberger PR, Burd R, Bhala SB et al. Quercetin sensitizes cells to hyperthermia. Int J Hyperthermia 2003; 19:507-519. 97. Middleton E, Kandaswami C, Theoharis C et al. The effects of plant flavonoids on mammalian cells: Implications for inflammation, heart disease, and cancer. Pharmacol Rev 2000; 52:673-751. 98. Harsh IVth GF. Management of recurrent gliomas. In: Berger Michel S, Wilson Charles B, eds. The Gliomas. Philadelphia, London, Toronto, Miami, Sydney, Tokyo: WB Saunders Publisher, 1999:649-659. 99. Shapiro WR, Shapiro JR, Walker RW. Central nervous system. In: Abeloff MD, Armitage JO, Lichter AS et al, eds. Clinical Oncology. New York, Edinburgh, London, Madrid, Melbourne, San Francisco, Tokyo: Churchill Livingstone, 2000:1103-1192. 100. Knisely JPS, Rockwell S. Importance of hypoxia in the biology and treatment of brain tumors. Neuroimag Clin N Am 2002; 12:525-536. 101. Brat DJ, Mapstone TB. Malignant glioma physiology: Cellular response to hypoxia and its role in tumor progression. Ann Intern Med 2003; 138:659-668. 102. Gerweck LE, Richards B. Influence of pH on the thermal sensitivity of cultured human glioblastoma. Cancer Res 1981; 41:845-849. 103. Fuse T, Yoon KW, Kato T et al. Heat-induced apoptosis in human glioblastoma cell line A172. Neurosurgery 1998; 42:843-849. 104. Tanaka T, Kobayashi T, Kida Y et al. The effect of hyperthermia and antitumor drugs on brain tumor cells Hnes. Can To Kagaku Ryoho 1986; 13:2993-2997. 105. Kobayashi T, Kida Y. Magnetic induction hyperthermia for brain tumor using ferromagnetic implant with low Curie temperature. Experimental studies J Neurooncol 1986; 4:175-181. 106. Tanaka T, Kobayashi, Takahashi T et al. Effect of combined treatment with magnetic induction hyperthermia and chemotherapy in a rabbit brain tumor model. Neurol Med Chir (Tokyo) 1989; 29:377-381. 107. Shem BC, Dahl O. Thermal enhancement of ACNU and potentiation of thermochemotherapy with ACNU by hypertonic glucose in the BT4 A rat glioma. J Neuroncol 1991; 10:247-252. 108. Raaphorst GP, Cheng ENG, Shahine B. Comparison of Radiosensitization by 4 r C hyperthermia during low dose rate irradiation and during pulsed simulated low dose rate irradiation in human glioma cells. Int J Radiation Oncology Biol Phys 1999; 44:185-188. 109. Papavero L, Loew F, Jaskshe H. Intracarotid infusion of ACNU and BCNU as adjuvant therapy of malignant gliomas. Acta Neurochir 1987; 85:128-137. 110. Hahn GM, Shiu EC. Effect of pH and elevated temperature on the cytotoxicity of some chemotherapeutic agents on Chinese hamster cells in vitro. Cancer Res 1983; 43:5789-5791. 111. Watanabe M, Tanaka R, Rondo H et al. Effects of antineoplastic agents and hyperthermia on cytotoxicity toward chronically hypoxic glioma cells. Int J Hyperthermia 1992; 8:131-138.

Thermo- Chemo-Radiotherapy

Association

155

112. Raaphorst GP, Mao J, N G CE. Thermotolerance in human glioma cells. Int J Hyperthermia 1995; 11:523-529. 113. Sneed PK, Stea B. Thermoradiotherapy for brain tumors. In: Seegenschmiedt M H , Fessenden P, Vernon C C , eds. Thermoradiotherapy and Thermochemotherapy. Berlin, Heidelberg, New York: Springer Verlag, 1995:159-175. 114. Talcahashi H, Suda T, Motoyama H et al. Radiofrequency interstitial hyperthermia on malignant brain tumors: Development of heating system. Experimental Oncology 2000; 22:186-190. 115. Seegenschmiedt M H , Feldmann HJ, Wust P et al. Hyperthermia- Its actual role in Radiation Oncology, part IV Thermoradiotherapy for malignant brain tumors. Strhlentherapie und Onkologie 1995; 171:560-572. 116. Sneed P, Stauffer PR, McDermott M W et al. Survival benefit of hyperthermia in a prospective randomized trial of brachytherapy boost ± hyperthermia for glioblastoma multiforme. Int J Radiation Oncology Biol Phys 1998; 40:287-295. 117. Ikeda N , Hayashida O, BCameda H et al. Experimental study on thermal damage to dog normal brain. Int J Hyperthermia 1994; 10:553-561. 118. Sminia P, Van Der Zee J, Wondergem J et al. Effect of hyperthermia on central nervous system: A review. Int J Hyperthermia 1994; 10:1-30. 119. Sharma H S , Hoopes PJ. Hyperthermia induced pathophysiology of the central nervous system. Int J Hyperthermia 2003; 19:325-354. 120. Larson D , Shreve D C , Gutin P. Radiosurgery. In: Berger Michel S, Wilson Charles B, eds. T h e Gliomas. Philadelphia, London, T o r o n t o , Miami, Sydney, Tokyo: W B Saunders Publisher, 1999:511-518. 121. Purdy JA. Three dimensional conformal radiation therapy: Physics, treatment planning, and clinical aspects. In: Perez CA, Brady LW, Halperin EC et al, eds. Principles and practice of Radiation Oncology. 4th ed. Philadelphia, Baltimore, New York, London: Linpiccott Williams and Wilkins, 2004:283-313. 122. Gramaglia A, Loi GF, Mongioj V et al. Increased survival in brain metastatic patients treated with Stereotactic radiotherapy, omega three fatty acids and bioflavonoids. Anticancer Res 1999; 19:5583-5586. 123. Baronzio GF, Gramaglia A, Scorsetti M et al. Biosynchrotherm: New capacitive hyperthermia device: Preliminary clinical reports. Abs XXIII meeting of International Clinical hyperthermia Society- Lyon France 2000.

CHAPTER 11

A Step Deep on Hyperthermia, Hypoxia and Chemotherapy Interactions Giammaria Horendni,* Ugo De Giofg^, Mauiizio Cantore, Andrea Mambrini andStefmo Guadagni Introduction

C

ancer physiology can be a new significant target for therapy. Nonsurgical approaches to cancer treatment, primarily radiation therapy and chemotherapy, are almost exclusively based on agents that kill cells. The main problem with these current treatments, however, is that they do not have specificity for cancer cells. In the case of antineoplastic drugs, it is primarily the rapid proliferation of many of the cancer cells that makes them more sensitive to cell killing than their normal cellular counterparts, for radiation therapy, a degree of specificity is achieved by localizing the radiation to the tumour and its immediate surrounding normal tissue. However, both treatments are limited by their toxic effects on normal cells. To achieve greater efficacy many researchers are attempting to stress differences between normal and malignant cells at the cellular milieu and biomolecular properties. The physiology of solid tumours at the microenvironmental level is sufficiendy different from that of the normal tissues from which they arise to provide a unique and selective target for cancer treatment.

The Problem of Tumor Hypoxia in Anticancer Therapy Tumour hypoxia is a very important factor in oncology since it contributes to tumour progression by the activation of genes associated with those promoting angiogenesis. Moreover it has profound effects on therapy: oxygen helps to stabilise radiation damage in DNA, while hypoxic cells show considerable (about five-fold) resistance to radiotherapy, this is considered the major cause for the failure of radiotherapy in some tumours. Attempts to overcome this effect include the use of hyperthermia, oxygen-mimetic "radiosensiters" and multifractional radiotherapy to allow reoxygenation of tumour tissue. Radiosensiters are drugs designed to act similarly to oxygen in fixing radiation damage in DNA, but they are less rapidly metabolised, and are therefore more widely distributed in tumour tissues. There are also good reasons why, and considerable evidence to show how, hypoxic cells in tumours limit the efficacy of anticancer drugs. The pioneering work of Gray ^ demonstrated that the sensitivity to radiation damage of cells and tissues depends on the presence of oxygen at the time of irradiation. The histological studies on human lung adenocarcinomas by Thomlinson and Gray^ provided an explanation of the mechanism by which cells could become hypoxic in tumours. They postulated that, because of their unrestrained growth, tumour cells would be forced away from vessels, beyond

•Corresponding Author: Gianmaria Fiorentini—Department of Oncology, "S. Giuseppe" Hospital, Empoli (Fl), Italy. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

A Step Deep on Hyperthermia, Hypoxia and Chemotherapy Interactions

157

the effective diffusion distance of oxygen in respiring tissue, thereby becoming hypoxic and eventually necrotic. There are two important further consequences of reducing oxygen concentration: (a) the fraction of proliferating cells and/or the rate of cell proliferation decreases as a function of distance from the vascular supply, a phenomenon that is largely the result of decreasing oxygen levels.^'^ An important consequence of this hypoxia-induced inhibition of proliferation is that, because most anticancer drugs are primarily effective against rapidly dividing cells, their effectiveness would be expected to fall off as a function of distance from blood vessels. This has been shown experimentally^ "^ and (b) since hypoxic cells are the ones most distant from blood vessels, they will be exposed to lower concentrations of drug than those adjacent to blood vessels, primarily as a result of the metabolism of such agents through successive cellular layers. Hypoxia in solid tumours, however, has an important consequence in addition to conferring a direct resistance to radiation and chemotherapy. ^^ Graeber showed that low oxygen levels caused apoptosis in minimally transformed mouse embryo fibroblasts and that this apoptosis depended to a large extent on wild-type p53 genotype. They further showed, using these same cells growing as solid tumours in immune-deprived mice, that apoptosis colocalized with hypoxic regions in tumours derived from p53 wild-type mice. In tumours derived from p53 -/-cells, there was much less apoptosis and no colocalization with tumour hypoxia. These findings provide evidence that hypoxia, by selecting for mutant/>53, might predispose tumours to a more malignant phenotype. Clinical data support this conclusion. Studies on both soft tissue sarcomas and on carcinomas of the cervix have shown that hypoxic tumours are more likely to be metastatic. However, others have proposed that tumour hypoxia can occur in a second way, by temporary obstruction or cessation of tumour blood flow, the so called acute hypoxia model. Definitive evidence for this type of acute hypoxia arising from fluctuating blood flow, has come from elegant studies with transplanted tumours in mice using diffusion limited fluorescent dyes. Because fluctuating blood flow has also been demonstrated in human tumours, it is likely that this type of hypoxia is also present in human tumours. The consequences of acute hypoxia will be similar to those of the diffusion-limited hypoxia. Any cells surrounding a closed blood vessel will be resistant to radiation killing because of their lack of oxygen at the time of radiation and will be exposed to lower levels of anticancer drugs than those surrounding blood vessels with a normalflow.This would be expected to lead to differences in response to anticancer agents, as has been observed in experimental tumours. The low oxygen levels in tumours can be probably turned from a disadvantage to an advantage in cancer treatment. Such a possibility was proposed 20 years ago by Lin, who reasoned that compounds based on the quinone structure of mitomycin C might be more active in hypoxic tumours. It was known that mitomycin C required metabolic reduction of the benzoquinone ring to produce the cytotoxic bifunctional alkylating agent. Lin reasoned that a lower oxidation reduction (redox) potential for tumour tissue, relative to most normal tissues, could increase reductive activation of these quinone derivatives in tumours. Although this was not the correct mechanism for the increased cytotoxicity of mitomycin C and certain analogues toward hypoxic cells (much lower levels of hypoxia are needed to change cellular redox potential), these studies were important in su^esting the potential of hypoxia-activated drugs and led to the concept of selectively killing the hypoxic cells in solid tumours.

Hypoxia and Chemotherapy There are presently three different classes of hypoxia-specific drugs that are in use clinically or are being developed for clinical use. They are the quinone antibiotics, the nitroimidazoles, and the benzotriazine di-N-oxides. In the quinone class, the three principal agents of current clinical interest are mitomycin C, porfiromycin and E09. All are structurally similar and require reductive metabolism for activity. Each of them is converted by reductive metabolism to a bifunctional alkylating agent and probably produces its major cytotoxic activity through the formation of DNA interstrand cross-links.

158

Hyperthermia in Cancer Treatment: A Primer

Mitomycin C, considered to be the prototype bioreductive drug, was introduced into clinical use in 1958 and has demonstrated efficacy towards a number of different tumours, in combination with other selective drugs whose toxicity towards hypoxic cells is modest, with values for hypoxic cytotoxicity ratios (the ratio of drug concentration to produce equal cell kill for aerobic and hypoxic cells) of one (no preferential toxicity) to approximately £IYC. However, based on this activity, mitomycin C has been combined with radiotherapy in two randomized trials of head and neck cancer, the pooled restdts of which gave a statistically significant disease-free survival benefif^'^ The third drug in this series, E09, is a much more efficient substrate for DT-diaphorase than either mitomycin C or porfiromycin and shows high toxicity to both aerobic and hypoxic cells with high DT-diaphorase levels. Cells with low DT-diaphorase levels are much less susceptible to killing by E09 under aerobic conditions, but this drug shows a high, up to 50-fold, preferential toxicity toward hypoxic cells. However, the pharmacokinetics of this agent work against its clinical utility, and phase I clinical studies have shown litde activity of this drug. A second class of bioreductive agents is that of the nitroimidazoles, the first two of which, metronidazole and misonidazole, have been extensively tested as hypoxic radiosensitizing agents. Further drug development by Adams^ produced a compound, RSU1069, which has been shown to be a highly efficient cytotoxic agent with activity both in vitro and in vivo. RSU1069 has an hypoxic cytotoxicity ratio of some 10-100 for different cell lines in vitro, and it, or its prodrug, RB6145, has shown excellent activity with mouse tumour models when combined with irradiation or agents that induce hypoxia. Unfortunately, however, clinical testing of RB6145 has been aborted due to irreversible cytotoxicity toward retinal cells. Tirapazamine (TPZ) is the first, and thus far, only representative of the third class of hypoxia-selective cytotoxins. The mechanism for the preferential toxicity of TPZ towards hypoxic cells is the result of an enzymatic reduction that adds an electron to the TPZ molecide, forming a highly reactive radical. This radical is able to cause celi killing by producing DNA damage leading to chromosome aberrations. Moreover, DNA damage occurs only from TPZ metabolism within the nucleus. TPZ produces specific potentiation of celi kill by radiation and cisplatinum. Specifically, the synergistic cytotoxic interaction observed when TPZ and cisplatinum are given in sequence depends on the TPZ exposure being under hypoxic conditions. In fact, there is no interaction when TPZ is given under aerobic conditions. It has also been demonstrated that the cytotoxic activity of TPZ under hypoxia is independent of p53 gene status of tumour cells. This drug has 100-fold differential toxicity toward hypoxic vs. aerobic cells. Based on experimental studies that evaluated the responsiveness of tumour cells under aerobic and hypoxic conditions, Teicher^^ classified chemotherapeutic agents into three groups: (1) preferentially toxic in aerobic conditions (bleomycin, procarbazine, streptonigril, actinomycin D, vincristine and melphalan); (2) preferentially toxic under hypoxic conditions (mitomycin C and adriamycin); (3) no major preferential toxicity to oxygenation (cisplatinum, 5-fluorouracil and methotrexate).

Hypoxia and Gene Therapy The newest direction for exploiting tumour physiology is aimed toward the evolving field of gene therapy. In this novel approach to anticancer therapy, genetic material is transferred into cells with the idtimate goal of selectively killing cancer cells and sparing normal cells. Recent studies have regarded the possibility of using the hypoxia-signalling pathway to selectively activate gene expression.^ ^' Hypoxia induces the expression of a number of genes, principally via the stabilization of members of the bHLH/PAS family of transcription factors that bind to a consensus DNA sequence, the hypoxia response element (HRE). Physiologically regulated expression vector systems, containing HRE sequences, are now under development, to target therapeutic gene expression to tumour cells characterized by low oxygen tension.

A Step Deep on Hyperthermia, Hypoxia and Chemotherapy Interactions

159

From a clinical point of view the combination of hyperthermia and hypoxia seems to add activity to intra-arterial chemotherapy.^'^ At the same lime the exposure of body regions, such as pelvis or limbs, to a locally high dose of bioreductive agent such as mitomycin C, in hypoxic conditions shows activity in refractory cancers. ^^"^^ Following the study of genes we understood that there are other ways in which hypoxia might contribute to drug resistance. One is through the amplification of genes, such as dihydrofolate reductase, conferring various glucose-regulated proteins that appear to be responsible for resistance to doxorubicin, etoposide and camptothecin.

Hyperthermia and Chemotherapy Preclinical thermo-chemotherapy studies have given valuable information on the schedule of the cytotoxic interaction between the different agents and on the molecular mechanisms responsible for the potentiating effect. Several studies have demonstrated that the cytotoxic activity of various chemotherapeutic agents is enhanced by mild or moderate hyperthermia (40.5-43°C).^^ In these investigations the effect of scheduling on the cytotoxic interaction between hyperthermia and drugs has also been investigated in in vitro experimental systems. There are data regarding doxorubicin, the platinum compounds cisplatin and carboplatin, the bifunctional alkylating agent melphalan and the antimetabolite methotrexate which indicate that in each case the maximal cytotoxicity occurs when the drug is administered simultaneously with hyperthermia. ^ '^ The mechanisms responsible for the effect of hyperthermia on cell killing by anticancer drugs are not entirely understood. For example, for melphalan, which is widely used in experimental and clinical thermo-chemotherapy studies, different putative mechanisms of potentiation have been suggested including: an increase in melphalan influx leading to a higher intracellular drug accumulation.^° The alteration of the DNA quaternary structure, which favours alkylation; the interference with drug-DNA adduct metabolism and inhibition of repair^ ^ the stabilization of drug-induced G2 phase cell accumulation^^ through the inhibition of p32^ kinase activity.^'^'^^ As regards cisplatin, it has been demonstrated that the cytotoxic activity of this compound, as well as that of the platinum derivatives lobaplatin and oxaliplatin, is increased under hyperthermic conditions as the consequence of an enhanced formation of DNA-platinum adducts."^ Preclinical studies have also significantly contributed to the proposition of potential cellular determinants of response to individual and combined treatments. The relevance of cell kinetic and DNA ploidy characteristics as indicators of thermoresponsiveness has been determined in primary cultures of human melanoma. Results from this study showed that the median 3H-thymidine labelling index of sensitive tumours was four-fold that of resistant tumours. Moreover, thermosensitivity was found more frequently in tumours with a diploid nuclear DNA content than in those with DNA aneuploidy. Since heat and drug sensitivity may be related to the ability of tumour cells to mount a stress response, the relationship between constitutive (and inducible) levels of heat shock proteins (HSPs) and thermosensitivity has been evaluated in testes and bladder cancer cell lines. No correlation between constitutive levels of HSP90 or HSP72/73 and cellular thermoresponsiveness was found. However, results suggest that low HSP27 expression might contribute to heat sensitivity.

Hyperthermia and Antineoplastic Drugs Selection The most effective agent(s) at elevated temperatures have yet to be determined. Some studies suggest that the drug of choice at elevated temperatures may be different from that at the physiological temperature, and that the alkylating agents may be most effective at elevated temperatures. To further investigate these possibilities, the effect of chemotherapeutic agents were compared by Takemoto. He studied these agents: cyclophosphamide, ifosfamide, melphalan, cisplatin, 5-fluorouracil, mitomycin C and bleomycin. Three tumours (mammary

160

Hyperthermia in Cancer Treatment: A Primer

carcinoma, osteosarcoma and squamous cell carcinoma) were used. They were transplanted into the feet of C3H/He mice. When tumours reached 65 mm, a test agent was injected intraperitoneally. Tumours were inmiediately heated at 41.5°C for 30 min, and the tumour growth (TG) time was studied for each tumour. Using theTG times, theTG-50 (the time required for one-half of the total number of the treated tumours to reach the volume of 800 mm from 65 mm was calculated. Subsequendy, the tumour growth delay time (GDT) and the thermal enhancement ratio (TER) were obtained. The GDT was the difference between theTG-50 of treated tumours and that of nontreated control tumours. The TER was the ratio of the GDT of a group treated with an agent at 4l.5*'C to that of a group treated with the agent at room temperature. Results showed that the top three effective agents tested at 41.5°C were solely alkylating agents cyclofosphamide, ifosfamide and melphalan for each kind of tumour. A GDT of cisplatin was smaller than those of the alkylating agents. The smallest TER, 1.1, was observed for 5-fluorouracil, which was given for manmiary carcinoma, and for mitomycin C, which was given for squamous cell carcinoma. It could be concluded that the alkylating agents at elevated temperatures might be the drugs of choice for many types of tumours.

Alkylating Agents and Oxaliplatin Urano^^ studied the effects of various agents on animal tumours with different histopathology at elevated temperatures. His studies indicated that alkylating agents were most effective to all tumours at a moderately elevated temperature. Cisplatin was also effective to all tumours, but its effectiveness at 41.5°C was less than that of alkylating agents. To quantitatively study these findings, the magnitude of thermal enhancement of melphalan, an alkylating agent, and that of oxaliplatin, a new platinum compound, were studied by this author, at 37-44.5°C by the colony formation assay. The dose of each agent was kept constant, and cell survival was determined as a function of treatment time. The cell survival curve was exponentially related with treatment time at all test temperatures, and theT(O) (the time to reduce survival from 1 to 0.37) decreased with an increasing temperature. These results suggested that the cytotoxic effect of these agents occurred with a constant rate at 37°C, and the rate was facilitated with an increasing temperature. This suggests that heat can accelerate the cytotoxic chemical reaction, leading to substantial thermal enhancement. The thermal enhancement ratio (TER, the ratio of the T(0) at 37°C to the T(0) at an elevated temperature) increased with an increase in the temperature. The activation energy for melphalan at moderately elevated temperatures was largest among the agents tested in the laboratory and that for oxaliplatin was approximately half of the melphalan activation energy. This suggests that the thermal enhancement for the cytotoxicity of melphalan or alkylating agents might be the greatest.

Docetaxel Recent studies suggest that docetaxel may show improved response at elevated temperatures. Factors that may modify the thermal enhancement of docetaxel were studied by Mohamed to optimize its clinical use with hyperthermia. The tumor studied was an early-generation isotransplant of a spontaneous C3Hf/Sed mouse fibrosarcoma, Fsa-II. Docetaxel was given as a single intraperitoneal injection. Hyperthermia was achieved by immersing the tumor-bearing foot into a constant temperature water bath. Four factors were studied: duration of hyperthermia, sequencing of hyperthermia with docetaxel, intensity of hyperthermia, and tumor size. To study duration of hyperthermia tmnors were treated at 41.5 °C for 30 or 90 min inunediately after intraperitoneal administration of docetaxel. For sequencing of hyperthermia and docetaxel, animals received hyperthermia treatment of tumors for 30 min at 41.5°C immediately after drug administration, hyperthermia both immediately and 3 hr after docetaxel administration and hyperthermia given only at 3 hr after administration of docetaxel. Intensity of hyperthermia was studied using heat treatment of tumors for 30 min at 41.5 or 43.5°C immediately following docetaxel administration. Effect of tumor size was studied by delaying

A Step Deep on Hyperthermia, Hypoxia and Chemotherapy Interactions

161

experiments until three times the tumor volume was observed. Treatment of tumors lasted for 30 min at 41.5°C immediately following drug administration. Tumor response was studied using the mean tumor growth time. Hyperthermia in the absence of docetaxel had a small but significant effect on tumor growth time at 43.5°C but not at 4l.5°C. Hyperthermia at 4l.5°C for 90 min immediately after docetaxel administration significantly increased mean tumor growth time (P = 0.0435) when compared to tumors treated with docetaxel at room temperature. Treatment for 30 min had no effect. Application of hyperthermia immediately and immediately plus 3 hr following docetaxel was effective in delaying tumor growth. Treatment at 3 hr only had no effect. No significant difference in mean tumor growth time was observed with docetaxel and one half hour of hyperthermia at 41.5 or 43.5°C. For larger tumors, hyperthermia alone caused a significant delay in tumor growth time. Docetaxel at 41.5°C for 30 min did not significantly increase mean tumor growth time compared to large tumors treated with docetaxel at room temperature. Docetaxel shows a moderate increase in anti-tumor activity with hyperthermia. At 41.5°C the thermal enhancement of docetaxel is time-dependent if hyperthermia is applied immediately following drug administration. With large tumors docetaxel alone or docetaxel plus hyperthemia showed the greatest delays in tumor growth time in the experiments.

Hyperthermia and Gene Therapy Li reported the activity of adenovirus-mediated heat-activeted antisense Ku70 expression radiosensitizers tumor cells in vitro and in vivo. Ku70 is one component of a protein complex, Ku70 and Ku80, that functions as a heterodimer to bind DNA double-strand breaks and activates DNA-dependent protein kinase. The previous study of this group with Ku70-/- and Ku80-/- mice, and cell lines has shown that Ku70- and Ku80-deficiency compromises the ability of cells to repair DNA double-strand breaks, increases radiosensitivity of cells, and enhances radiation-induced apoptosis. In this study, Li examined the feasibility of using adenovirus-mediated, heat-activated expression of antisense Ku70 RNA as a gene therapy paradigm to sensitize cells and tumors to ionizing radiation. First, they performed experiments to test the heat inducibility of heat shock protein (hsp) 70 promoter and the efficiency of adenovirus-mediated gene transfer in rodent and human cells. Replication-defective adenovirus vectors were used to introduce a recombinant DNA construct, containing the enhanced green fluorescent protein (EGFP) under the control of an inducible hsp70 promoter, into exponentially growing cells. At 24 h after infection, cells were exposed to heat treatment, and heat-induced EGFP expression at different times was determined by flow cytometry. The data by Li clearly show that heat shock at 42°C, 43"C, or 44°C appears to be equally effective in activating the hsp70 promoter-driven EGFP expression (>300-fold) in various tumor cells. Second, the authors have generated adenovirus vectors containing antisense Ku70 under the control of an inducible hsp70 promoter. Exponentially growing cells were infected with the adenovirus vector, heat shocked 24 h later, and the radiosensitivity determined 12 h after heat shock. Our data show that heat shock induces antisense Ku70 RNA, reduces the endogenous Ku70 level, and significantly increases the radiosensitivity of the cells. Third, the author has performed studies to test whether Ku70 protein level can be down-regulated in a solid mouse tumor (FSa-II), and whether this results in enhanced radiosensitivity in vivo, as assessed by in vivo/in vitro colony formation and by the tumor growth delay. Their data demonstrate that heat-shock-induced expression of antisense Ku70 RNA attenuates Ku70 protein expression in FSa-II tumors, and significantly sensitizes the FSa-II tumors to ionizing radiation. Taken together, these interesting results suggest that adenovirus-mediated, heat-activated antisense Ku70 expression may provide a novel approach to radiosensitize human tumors in combination with hyperthermia.

162

Hyperthermia in Cancer Treatment: A Primer

Conclusions The comprehension, about 50 years ago, that hypoxic cells are resistant to X-rays led to the concept that cancers might be resistant to radiotherapy and chemotherapy because of their poor oxygen supply and subsequent hypoxia. Now tumour hypoxia is seen as a mechanism of resistance to many antineoplastic drugs, as well as a predisposing factor toward increased malignancy and metastases. However tumour hypoxia is a unique target for hyperthermia and cancer bioreductive therapy that could be exploited for therapeutic use. A hypoxic cell is unable to have a stable pH; this increases the permeability of the cell membrane so that antineoplastic agents can easily move through the membrane improving the global concentration of the drug both inside and outside the cell. Hyperthermia seems the best opportunity to enhance these phenomena.

References 1. Gray LH, Conger AD, Ebert M et al. Concentration oi oxygen dissolved in tissue at the time of irradiation as a factor in radiotherapy. Br J Radiol 1953; 26:638-48. 2. Thomlinson RH, Gray LH. The histological structure of some human lung cancers and the possible implications for radiotherapy. Br J Cancer 1955; 9:539-49. 3. Moulder JE, Rockwell S. Tumor hypoxia: Its impact on cancer therapy. Cancer Metastasis Rev 1987; 5:313-41. 4. Brown JM. The hypoxic cell: A target for selective cancer therapy. Cancer Res 1999; 59:5863-70. 5. Rauth AM, Melo T, Misra V. Bioreductive therapies: An overview of drugs and their mechanis m of action. Int J Radiat Oncol Biol Phys 1998; 42:755-62. 6. Wouters BG, Wang LH, Brown JM. Tirapazamine: A new drug producing tumor specific enhancement of platinum-based chemotherapy in non small cell lung cancer. Ann Oncol 1999; 10(suppl 5):S29-S33. 7. Graeber TG, Osmanian C, Jacks T. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature 1996; 379:88-91. 8. Lin A, Cosby L, Shansky C et al. Potential bioreductive alkylating j^ents. 1. Benzoquinone derivates. J Med Chem 1972; 15:1247-52. 9. Adams GE, Stratford IJ. Bioreductive drugs for cancer therapy: The search for tumour specificity. Int J Radiat Oncol Biol Phys 1994; 29:231-8. 10. Teicher BA, Holden SA, Al-Achi A et al. Classification of antineoplastic treatments by their differential toxicity toward putative oxygenated and hypoxic tumor subpopulation in vivo in the FSallC murinefibrosarcoma.Cancer Res 1990; 50:3339-44. 11. Binley L, Iqball S, Kingsman S et al. An adenoviral vector regulated by hypoxia for the treatment of ischaemic disease and cancer. Gene Ther 1999; 6:1721-7. 12. Zaffaroni N, Fiorentini G, De Giorgi U. Hyperthermia and hypoxia: New developments in anticancer chemotherapy. Eur J Surg Oncol 2001; 27:340-2. 13. Guadagni S, Fiorentini G, Palumbo G et al. Hypoxic pelvic perfiision with mitomycin C using a simpHfied ballon-occlusion technique in the treatment of patients with unresecuble locally recurrent rectal cancer. Arch Surg 2001; 136(1): 105-12. 14. Guadagni S, Russo F, Rossi CR et al. Deliberate hypoxic pelvic and limb chemoperfiision in the treatment of recurrent melanoma. Am J Sur 2002; 183:28-36. 15. Fiorentini G, Poddie D, Graziani G et al. Hypoxic isolated limb perfiision with mitomycin C in locally recurrent melanoma and sarcoma: Results of a phase II study. Reg Cancer Treat 1995; 8:135-9. 16. Brown JM, Giacca AJ. The unique physiology of solid tumore: Opportunities (and problems) for cancer therapy. Cancer Res 1998; 58:1408-16. 17. Urano M, Kuroda M, Nishimura Y. For the clinical application of thermochemotherapy given at mild temperatures. Int J Hyperther 1999; 2:79-107. 18. Zaffaroni N, Villa R, Daidone MG et al. Antitumor activity of hyperthermia alone or in combination with cisplatin and melphalan in primary cultures of human malignant melanoma. Int J Cell Cloning 1989; 7:385-94. 19. Kusumoto T, Holden SA, Ara G et al. Hyperthermia and platinum complexes: Time between treatments and synergy in vitro and in vivo. Int J Hyperther 1995; 11:575-86. 20. Bates DA, Mackillop WJ. Effect of hyperthermia on the uptake and cytotoxicity of melphalan in Chinese hamster ovary cells. Int ] Radiat Onco/ Biol Phys 1998; 16:187-91.

A Step Deep on Hyperthermiay Hypoxia and Chemotherapy Interactions

163

2 1 . Zaffaroni N , Villa R, Orlandi L et al. Effect of hyperthrmia on the formation and removal of DNA interstrand cross-links induced by melphalan in primary cultures of human malignant melanoma. Int J Hyperther 1992; 8:341-9. 22. Orlandi L, Zaffaroni N , Bearzatto A et al. Effect of melphalan and hyperthermia on cell cycle progression and cyclin Bl expression in human melanoma cells. Cell ProUf 1995; 28:617-30. 23. Orlandi L, Zaffaroni N , Bearzatto A et al. Effect of melphalan and hyperthermia on p34cdc2 kinase activity in human melanoma cells. Br J Cancer 1996; 74:1924-8. 24. Rietbroek RC, van de Vaart PJ, Haveman J et al. Hyperthermia enhances the cytotoxic activity and platinum-DNA adduct formation of lobaplatin and oxaliplatin in cultured SW 1573 cells. J Cancer Res Clin Oncol 1997; 123:6-12. 25. Orlandi L, Costa A, Zaffaroni N et al. Relevance of cell kinetic and ploidy characteristics for the thermal response of malignant melanoma primary cultures. Int J Oncol 1993; 2:523-6. 26. Richards E H , Hickman JA, Masters JR. Heat shock protein expression in testis and bladder cancer ceh lines exhibiting differential sensitivity to heat. Br J Cancer 1995; 72:620-6. 27. Takemoto M, Kuroda M, Urano M et al. Effect of various chemotherapeutic agents given with mild hyperthermia on different types of tumours. Int J Hyperthermia 2003; 19(2): 193-203. 28. Urano M , Ling C C . Thermal enhancement of melphalan and oxaliplatin cytotoxicity in vitro. Int J Hyperthermia 2002; 18(4):307-15. 29. Mohamed F, Stuart OA, Glehen O et al. Docetaxel and hyperthermia: Factors that modify thermal enhancement. J Surg Oncol 2004; 8 8 ( l ) : l 4 - 2 0 . 30. Li G C , H e F, Shao X et al. Adenovirus-mediated heat-activated antisense Ku70 expression radiosensitizes tumor cells in vitro and in vivo. Cancer Res 2003; 63(12):3268-74.

SECTION III

Clinical Aspects of Hyperthermia

CHAPTER 12

Locoregional Hyperthermia E. Dieter Hager* Abstract

L

ocoregional hyperthermia can be differentiated into external, interstitial and endocavitary hyperthermia. Different heat delivery systems are available: antennae array, capacitive coupled, and inductive devices. Depending on localization and size of the tumour different methods and techniques can be applied: superficial, intratumoral (thermoablation), deep hyperthermia, endocavitary, and part-body hyperthermia. Randomized clinical trials have been performed mosdy with electromagnetic applicators for superficial hyperthermia in combination with radiotherapy, deep hyperthermia with and without radiation, and endocavitary hyperthermia in combination with chemotherapy and radiotherapy. In randomized clinical trials it could be demonstrated, that loco-regional deep hyperthermia with antennae array or capacitive coupled hyperthermia devices may increase response rate, disease free survival and overall survival of patients with cancer in combination with radiotherapy or chemotherapy without increasing the toxicity of standard therapies.

Introduction Hyperthermia is one of the most promising new multidisciplinary approaches to cancer therapy. The rationale for raising temperature in tumor tissue is based on a direct cell-killing effect at temperatures above 4l-42°C and a synergistic interaction between heat and radiation as well as various antineoplastic agents. The thermal dose-response depends also on microenvironmental factors such as pH, and p02 in the tumor tissue. Depending on the physical characteristics of the energy field applied, also other mechanisms of tumor destruction or growth retardation may be relevant. Tissue-specific electromagnetic interactions may be possible, depending on frequency and applicator technique used, due to inhomogeneities in the relative dielectric permittivity, relative magnetic permeability, specific conductivity, and ion distribution in cancer tissue compared to normal tissue. The effects of hyperthermia on the host and cancer tissue are pleio tropic and depend mainly on the temperature and the physical techniques applied. The biological and molecular mechanisms of these effects are changes in the membrane, ^'^ the cytoskeleton, the ion-gradient and membrane potential, svnthesis of macromolecules and DNA-replication,^^ intra- and extra-cellular pH (acidosis ) and decrease in intracellular ATP. ^^ Genes can be up-regidated or down-regulated by heat, for example the heat-shock proteins (HSP).^^ Synergistic effects by interactions with antineoplastic agents, radiation and heat can be several powers of ten even at moderate temperatures. In addition, reduced chemotherapy resistancy, possibly due to increased tissue penetration, increased membrane permeability, and activated metabolism, has been observed. *E. Dieter Hager—Department of Hyperthermia, BioMed-Klinik GmbH, Tischberger Str. 5-8, D-76887 Bad Bergzabern, Germany. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

168

Hyperthermia in Cancer Treatment: A Primer

Immunological effects of hyperthermia may play an additional role in cancer therapy such as immunological effects on cellular effector cells (emigration, migration and activation), induction of cytokines, chemokines and heat shock proteins (chaperones), and modulation of cell adhesion molecules. The induction of heat-shock proteins might increase specific immune responses to cancer cells. Locoregional hyperthermia can be differentiated into A. External hyperthermia • Local hyperthermia (short waves/radiofrequencies (SW/RF), microwaves (MW)) • Regional deep hyperthermia (RF, MW, ultrasound (US)) • Part-body hyperthermia (RF, MW, infrared (IR), heat perfusion) B. Interstitial hyperthermia with • RF electrodes (i.e., needles) • HF or MW antennas • Laser fibres • Ultrasound transducers • Magnetic rods/seeds and fluid C. Endocavitary hyperthermia (sy: intraluminal) • RF electrodes (i.e., coils) • Radiative (IR, laser) • Heat sources (hot fluid perfusion, extracorporal perfiision) depending on the method of the external heating devices and the area treated with hyperthermia (Fig. 1). With RF capacitive heating devices delivering 8-27 MHz and annidar phased-array systems delivering 60-430 MHz electromagnetic waves local and regional deep hyperthermia (DHT) can be applied for superficial and deep seated tumors. As a general physical rule: the higher the frequency of the electromagnetic field the less deep the penetration depth will be. Therefore lower frequencies are used more frequendy for deep seated tumors and higher frequencies for superficial tumors. Molecules with dipoles, like water, can be excited in such alternating electromagnetic fields which will be measured as heat. With capacitively-coupled electrodes and perfusion with heated fluid larger anatomical areas like the peritoneum, the bladder, the pleural cavity and the whole liver and lung or extremities can be heated up, which is called part-body hyperthermia (PBHT). Depending on the frequencies applied and with new applicator techniques and with sufficient monitoring PBHT is also possible with dipole antennae devices. Interstitial hyperthermia delivers the heat direcdy at the site of the tumor. For interstitial hyperthermia high frequency needle electrodes at 375 kHz (i.e., high frequency-induced thermotherapy; HiTT), microwave antennas, ultrasound transducers, laser fiber optic conductors (laser-induced thermotherapy; LiTT), or ferromagnetic rods, seeds or fluids (magnetic fluid hyperthermia (i.e., with nanoparticles), MFH) are injected or implanted into the tumor. In some cases the interstitial hyperthermia is combined with a brachytherapy by an afterloading method. With these applicators a heat can be applied high enough to induce in tumors thermonecrosis at a distance of 1 to 2 cm around the hot source. This technique is suitable for 1-5 tumors less than 5 cm in diameter. Insertion of antennas or electrodes into lumens of the human body such as the oesophagus, rectum, bladder, urethra, vagina and the uterine cervix are used for endocavitary hyperthermia. With this technique larger applicators than for interstitial hyperthermia with higher penetration depth can be applied. Perfusional hyperthermia with fluids (water, blood) is used to deliver heat withfluidsinto cavities like the peritoneum, the pleural space, or the bkdder. The perfusate is combined with antineoplastic agents or cytokines, like TNF-a (see Chapter entided "Perfiisional Peritoneal Hyperthermia"). Extracorporal heat exchange is commonly used to heat up blood for the perfusion of extremities. Deep hyperthermia (DHT) is referred to the induction of heat in deep seated tumors, e.g., of the pelvis, abdomen, liver, lung, or brain-by external energy applicators. The

Locoregional Hyperthermia

169

Figure 1. Technical devices for deep hyperthermia: A) high frequency induced thermo-dierapy; B) RF capacitively-coupied electrodes; C) multi-antenna applicator (dipole pairs).

170

Hyperthermia in Cancer Treatment: A Primer

Table /. Different heat delivery methods Heat Delivery Methods

Examples

Conductive Radiative Mechanical Antennas Capacitive Inductive Bioactive

Cavitational water-heating; extra-corporal blood heating; RF needles Infrared light (IR-A, -B, -C) Ultrasound Multi-antenna-dipole applicators Condenser Ferromagnetic rods/seeds/fluids Pyrogens, cytokines

technical features for the treatment of deep seated tumors are interstitial applicators (i.e., conductive), electromagnetic antenna-dipole arrangements, capacitive-coupled electrodes, ultrasound, and magnetic fields (see Table 1). The technique used, will restrict the application to certain body areas. The different electromagnetic techniques used for transferring energy in regional deep hyperthermia are: • radiofrequencies (RF-DHT) between 5-27 MHz • highfrequencies(HF-DHT) between 60-430 MHz (decimetre waves) and • microwaves (MW-DHT) atfrequencieslarger than or equal 1 GHz (centimetre waves). The absorption of the electromagnetic field (EMF) is depending from physical properties of the penetrated tissue, like conductivity and dielectricity which may cause focusing effects and electromagnetic coupling. The distribution of the temperature within tumor tissue is inhomogeneous due to intra- and extratumoral perfusion r^ulations, electric characteristics of the tissues and thermal conductivity, and ranges between 39 and 43°C. In addition to the thermal effects, frequency dependent non-thermal effects may play an essential role. Physical aspects (impedance and interaction with dipoles) let expect a special role for EMF in the radiofrequency range between 1-30 MHz. First experimental and clinical trials have been performed in the 1960s with radiofrequencies in the range between 8 and 27 MHz (LeVeen). This technique is now most frequently used in Japan and Russia. In Japan most clinical research has been performed with RF-technique at 8 MHz."^^ In Europe, especially the Netherlands and Germany, most frequently high frequency technique systems with dipole antennae operating at frequencies of 60 to 120 MHz (BSD-2000) are used in clinical research. Since the end of the eighties 13.56 MHz RF capacitive heating devices are available also for superficial and deep hyperthermia in Europe, especially in Germany and Italy.

Clinical Trials on Hyperthermia Superficial Hyperthermia Superficial tumors can be heated by (a) waveguide applicator, (b) spiral applicator, (c) current sheet applicator, (d) ultrasonic applicator, (e) RF-needles and (f) infrared sources. Electromagnetic applicators for superficial hyperthermia have a typical frequency of 150-430 MHz. Most convenient for local hyperthermia are water-filtered infrared sources. The therapeutic depths with these applicators is about 3 cm. By Medline database research up to October 2003, six randomized prospective phase III trials (RCT) on radiotherapy alone compared with radiotherapy combined with hyperthermia could be identified (Table 2). In all of these trials the combination radiotherapy plus hyperthermia showed better response rates. Overall survival benefit was only noted in one RCT trial.

Locoregtonal Hyperthermia

Table 2, Randomized

controlled

171

trials on superficial

hyperthermia

Tumor Site

Experimental

No. Control of Pts

Primary Endpoints

HT Better

Survival Benefit

Ref.

Head and neck (primary)

RT + sHT

RT

65

Response at 8 v^eeks

Yes

No

39

Melanoma (metastatic or recurrent)

RT + sHT

RT

68

Complete response

Yes

No

40

Superficial RT + sHT (head and neck, breast, miscellaneous)

RT

245

Initial response

possibly

No

41

Head and neck (N3 primary) (2-6 times)

RT + sHT

RT

44

Response

Yes

Yes

42

Breast (advanced primary or recurrent)

RT + sHT

RT

307

Initial response

Yes

No

43

Head and neck, breast, sarcoma melanoma

RT + 2X sHT

RT+ 1xsHT

173

Response

No

No

44

Abbreviations: RT: radiotherapy; sHT: superficial hyperthermia

Interstitial

Hyperthermia

For direct thermal ablation of tumors by interstitial hyperthermia most frequently ferromagnetic rods or seeds are implanted into the tumor and excited by an alternating external magnetic field. For the treatment of glioblastoma this treatment modality has been shown to improve overall survival (Table 3). The percutaneous, minimal invasive interstitial thermal ablation by means of laser or high frequency current (radiofrequency or microwave fields) which are introduced through a fibre optic conductor (LiTT) or special HF needle electrodes (HiTT), is a new therapeutic modality

Table 3. Randomized

controlled

trials with interstitial

hyperthermia

Control

No. of Pts

Primary Endpoints

HT Better

Survival Benefit

Ref.

Head and neck, iRF + iHT breast, melanoma, others

iRT

184

Response

No

No

45

Glioblastoma

RT+iRT

79

2-year survival

Yes

Yes

46

Tumor Site

Experimental

RT+iRT+iHT

Abbreviations: iRF: interstitial radiofrequency; RT: radiotherapy; iRT: interstitial radiotherapy; iHT: interstitial hyperthermy

172

Hyperthermia in Cancer Treatment: A Primer

for palliative and potentially curative therapy of primary liver tumors and liver metastases, especially if surgery is not acceptable or the tumors are not resectable. For RF thermo ablation multiple array needle electrodes (LeVeen needle) or hollow needle electrodes which can be perfused with physiological saline solution (Bechtold) are used. T h e needles are heated up with high frequency alternating current. The laser-induced thermotherapy was applied for the first time by Hashimoto et al^^ for the treatment of hepatic tumors and in the last years further developed by Vogel et al.^^ In a non-randomized trial Vogel et al, could show that in a total of 646 patients with 1.829 liver metastases up to 5 cm in diameter, mainly from colorectal (n = 1.126 metastases) and breast (n = 294 metastases) carcinoma by LiTT a local timior control rate of 97.3% after six months follow-up could be achieved.^^ The median siu^val rate of 39.8 months for colorectal liver metastases and 55.4 months for liver metastases of the breast are comparable with data from literature on surgical tumor resection. First results of the RF needle technique are comparable with LiTT or tumor resection.^ '^^ These methods for the non-surgical treatment of tumor patients, preferably for inoperable malignant nodules of the liver (hepatocellular carcinoma and metastases) is highly promising. Also other tumors from the brain, breast, thyroid, parathyroid, lung and bone, and malignant lymphomas can be treated by this method. T h e advantages of these methods are that they can be applied: • if surgery is not acceptable or the tumors are not respectable, • with low risk compared to surgery, • at different times repeatedly, and • on an outpatient basis and at lower costs. T h e perfused needle electrodes have advantages compared to other techniques: • increased thermolesion up to 40 to 50 mm diameter compared to 10 to 15 mm by increased conductivity around the needle • single needle system instead of multi array antennae systems • thin needles with about 2 mm diameters • ultrasound-guided application and • lower costs for the needles. In the future, magnetic fluid (f e, ferromagnetic nanopanicles) will be added to the therapeutic arsenal, which can be heated up by an external alternating magnetic field (magnetic field hyperthermia, MFH).^^ Very promising phase I/II studies have been closed.

Endocavitary Hyperthermia Via intraluminal placed antennas heat can be applied in organs such as the oesophagus, rectum, urethra (prostate), vagina, and the uterine cervix. Radiofrequencies and microwaves are most frequendy used for the endocavitary hyperthermia (Table 4). A survival benefit could be shown in most clinical studies. Regional

Deep

Hyperthermia

D e e p Hyperthermia w i t h Multi-Anteima Applicator Systems Tumours in the abdominal area can be heated up by arrays of antennas, which are arranged as dipole antenna pairs in a ring around the patient. T h e Sigma-60 applicator of the BSD-2000 system is a widely used applicator, which consists of four dipole antenna pairs. T h e novel multi-antenna applicator Sigma-Eye consists of 12 dipole pairs. Each antenna pair can be controlled in phase, amplitude, frequency and electric field to focus the heat in the area of the tumor. Frequencies in the range of 60-150 M H z are used for this technique. Two randomized phase III trials with multi-antenna applicators have been published up to the end of 2 0 0 3 and two trials are ongoing (Table 5). In two of these trials external radiotherapy was compared with combined radiotherapy and regional deep hyperthermia in the treatment of patients with primary cervix uteri (stage III) and primary or recurrent pelvic

173

Locore^onal Hyperthermia

Table 4. Randomized controlled and observational trials with endocavitary hyperthermia

Tumor Site

Experimental

Control

No. of Pts

OR [%] Control

OR [%] with HT

Survival Benefit Remarks

Ref.

Oesophagus

CT+HT

CT

40

19

41

No

RCT

28

Oesophagus

RT+HT

53

8

70

RCT

29

Oesophagus

RT + CT + HT

53

8

27

RCT

30

Oesophagus

RT + CT + HT

Rt + CT

66

59

81,2

Yes

RCT

47

Oesophagus

Ext. RT + MW+HT

ext. RT

66

Yes

OT

32

Rectum

RT+HT

RT

115

Yes

RCT

48

Yes

OT

52

Yes

RCT

53

Rectum

RT + CT + HT

RT + CT 36

Bladder, neoadj.

M W + CT

CT

52

CR:22

Bladder, adj.

M W + CT

CT

58

Rec: 64

Bladder, recurrent

hyperthermic perfusion + CT

10

CR:66 Rec: 15

Yes

RCT

54

90

Yes

OT

55

Abbreviations: RT: radiotherapy; CT: chemotherapy; MW: microwaves; RCT: randomized controlled trial; OT: open-label observational study; CR: complete response; Rec: recurrence after adjuvant treatment; neoadj.: neoadjuvant; adj.: adjuvant.

Table 5. Randomized trials on regional deep hyperthermia with antenna applicator systems

Tumor Site

Experimental Control

No. of Pts

Primary Endpoints

HT Better

Survival Benefit

Ref.

Cervix uteri (primary, stage III)

RT + DHT

RT

40

CR

Yes

No

18

Primary or recurrent pelvic (cervix, rectum, bladder)

RT + DHT

RT

361

CR, survival

Yes

Yes

19

Rectum (uT3/4)

RT + CT + DHT

RT + CT

>150

Disease-free survival

ongoing

Soft-tissue sarcoma (high risk)

CT + DHT

CT

>150

Disease-free survival

ongoing

Abbreviations: RT: radiotherapy; CT: chemotherapy; [DHT: deep hyperthlermia; CR: complete response

tumors. The number of complete response rates could be improved in both clinical studies and a survival benefit was demonstrated in one trial.

174

Hyperthermia in Cancer Treatment: A Primer

Table 6. Randomized trials with RF capacitive coupled heating devices

Tumor Site

Experimental

Control

No. of Pts

OR[%] Control

OR[%] with HT

Survival Benefit

Cervix Cervix Cervix Cervix Colorectal Gastric Colorectal Bladder

RT+HT RT+HT RT+HT RT+HT RT+HT RT+HT RT+HT RT+HT

RT RT RT RT RT RT HT HT

65 66 37 40 24 293 71 49

46 35 53 50 10 35,5 36 48

66 72 83 85 43 57,6 54 83

n.d. n.d. n.d. Yes n.d Yes Yes

Ref. 57 58 59 60 26 33 61 62

Abbreviations: RT: radiotherapy; CT: chemotherapy; HT: hyperthermia; OR: overall response; Obs: open-label observational study; RCT: randomized controlled trial; n.d.: not defined

Regional Deep Hyperthermia with Radiofrequency Capacitiye-Coupled Electrodes Deep seated tumors can be heated by RF capacitive-coupled electrodes. For these systems mostly radiofrequencies in the range between 8 and 27 MHz are used. In the 1960s Le Veen developed a machine for induction of hyperthermia in tissue with radiofrequencies by capacitively-coupling of electromagnetic fields (EMF) at 13.56 MHz. It has been shown that RF capacitive heating devices can effectively raise the temperature of lung and liver tumors in humans (see for review ref 49), though van Rhoon failed to raise the temperature with capacitive plate applicators at 13.56 MHz in tumors of the pelvic area of patients above 40.9°C.^ This technique can even be applied for the treatment of brain tumors^^ RF Hyperthermia without Combination with Radio- or Chemotherapy Clinical trials with hyperthermia mostly have been performed in combination with radiation or antineoplastic agents (Tables 6, 7). But some first results from hyperthermia trials with capacitive coupled radiowaves with 13.56 MHz in the treatment of patients with primary tumors or metastases in the liver, lung, pancreas and brain without combination with radio- or chemotherapy are promising and should be validated with randomized trials. Lung Cancer In a prospective open-label observational study 63 patients with histological proven small cell lung cancer (n = 10) and non-small cell Itmg cancer (n = 53) at far advanced stage of disease have been treated with regional deep hyperthermia (DHT) with RF capacitive coupled short waves of 13.56 MHz.^^M patients were inoperable, refractory or at stage of relapse after prior surgery (30%), chemotherapy (46%), and/or radiotherapy (46%). Eighty-six percent of the patients presented with restrictive disorder of pulmonary ventilation. The median time between first diagnosis of inoperabel cancer or relapse (local and distant progression) and beginning of DHT was 3.9 months. Only 2 patients were treated vnth palliative chemotherapy 8.4 and 28.5 months after the start of DHT due to tumor-associated symptoms (e.g., pain). The median overall survival time (MST) of all patients was 14.0 months from first diagnosis of advanced lung cancer. From relapse after surgery or first diagnosis of inoperable stage of disease the MST was 10.3 months. The one- and two-year survival rates from progression of disease were 37% and 18%, respectively.

Locore^onal Hyperthermia

175

Table 7. Non-randomized clinical trials with RF capacitive coupled heating devices Tumor Site

Experimental

Cervix Cervix Breast Breast Breast Colorectal Colorectal Colorectal Colorectal Gastric Gastric, adv. Gliomas "III, IV Liver (HCC) Liver (Met) Liver (HCC) Liver (Met) Lung (SCLC, SCLC) Lung (NSCLC) Lung (SCLN, NSCLC) Oesophagus Pancreas Pancreas Pancreas Sarcoma

C T + HT RT+HT RT+HT RT+HT RT+ HT RT+HT RT+HT RT+HT RT+HT RT + CT + HT CT+HT HT CT+HT HT + CT HT HT HT RT+HT RT+HT RT+ HT CT+HT HT HT RT+HT

Control

_ RT RT RT RT RT RT RT

CT CT

RT

-

No. of Pts 23 40 9 24 13 48 117 101 14 21 33 36 48 80 73 45 63 20 25 313 22 20 46 31

OR [%] with HT

Survival Better

-

52 80 100 83 92 11 69 71 100 89 39

43

56

-

-

n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

OR [%] Control

_ 50 63 84 0 33 55 20

25

-

31 (SD51) 31 (SD27)

75 80 63 36

74

Yes Yes Possibly

Ref. 63 64 20 21 65 22 23 24 25 35 25 38 27 37 66 67 36 67 90 31 25 68 69 70

Abbreviations: HCC: hepatocellular carcinoma; Met: metastases: SCLC: small cell lung cancer; NSCLC: non-small cell lung cancer; HT: hyperthermia; RT: radiotherapy; CT: chemotherapy; adv: advanced; n.d.: not defined

Liver Metastases from Colorectal Cancer Patients at advanced stage of colorectal cancer with liver metastases have been treated with deep hyperthermia alone or in combination with chemotherapy (5-FU + FA). RF capacitive coupled electrodes with a radiofrequency of 13.56 MHz (RF-DHT) was applied.^^ Median total survival time of all 80 patients from first diagnosis of disease was 34.4 months, and from first diagnosis of progression (metastases or relapse) 24.5 months, and from beginning of first RF-DHT alone (n = 50) 16 months. Patients who received RF-DHT followed by chemotherapy in combination with hyperthermia (n = 30) survived at a median of 11 months. Survival rates of all patients (n = 80) from first diagnosis of progression (metastases or relapse) were 91 ± 3 % , 51 ± 6% and 31 ± 6 % at 1, 2 and 3 years, respectively. Pancreas Cancer In a retrospective analysis of the treatment of 20 patients with inoperable or relapsed cancer of the pancreas the treatment with RF-DHT (13.56 MHz) resulted in a median survival time of 12months.^^ In a prospective open trial with AG patients with far advanced (non-resectable, relapsed or metastatized) pancreatic carcinoma were treated with RF capacitive heating at 13.56 MHz. Median age at study entry was 62 years (range 38-82), median Karnofskys index 50% (range 30-90). Six patients suffered from jaundice and 10 showed ascites at study entry. The

176

Hyperthermia in Cancer Treatment: A Primer

multimodal non-toxic treatment consisted of regional RF-deep hyperthermia (13.56 MHz, Synchrotherm, Italy) combined with complementary therapies (prteolytic enzymes, antihormonal therapy, etc.). The median overall survival of the patients was 10.5 months (range 2-76, mean 18 months) fromfirstdiagnosis of disease and 5 months from the beginning of the multimodal treatment. Most patients experienced essential improvement in quality of life (68% freedom from pain, 24% marked pain relief); 64% improved appetite (thereof 24% normal appetite) over a relatively long period of time, and reduction of jaundice and ascites. Gliomas The primary aim of this study was to define the feasibility of RF-DHT deep hyperthermia (RF-DHT) in the treatment of patients with progressive gliomas afi:er standard therapy and to estimate the effect on survival.^^ Between 09/97 and 09/02, 36 patients with gliomas (9 patients with anaplastic astrocytome WHO grade III, and 27 patients with glioblastoma multiforme WHO grade IV were treated with RF-DHT and Boswellia caterii, an inhibitor of leukotriene synthesis for inhibition of peritoneal edema. DHT was performed with a 13.56 MHz capacitive coupled RF-device. Patients with inoperable or subtotally resected and recurrent gliomas (WHO grade III and IV) with progression after radio- and/or chemotherapy and a Karnofsky Performance Score of > 50% were included. The study was designed as a prospective open-label, single-arm, mono-centred observational phase II trial. Primary endpoints were median survival time and survival-rate (Kaplan-Meier estimation). The survival was calculated on the basis of an intention-to-treat-analysis. Deep hyperthermia of brain tumors with RF capacitive hyperthermia at 13.56 MHz is feasible and without severe side effects. The RF-DHT-treatment is well tolerated and even patients at far advanced stages of disease can be treated. Complete and partial remission or retardation of tumor growth could be observed (Fig. 2). Prolongation of MST compared to historical controls and improvement of quality of live (EORTC QLQ-C30 questionnaires) is clinically significant. The median overall survival time of patients with anaplastic astrocytoma (WHO grade III) was 106±47 months [95% conficence intervall 14 to 197 months] and for patients with glioblastoma multiforme (WHO grade IV) 20±5 months [95% confidence interval 10 to 31 months]. The survival rates are listed in Tables 8 and 9.

Non-Thermal Effects The differences in the relative dielectric permittivity and magnetic permeability, the electric conductivity and the different ion distribution between normal and malignant tissue may explain different physical and physiological behaviour of the cells in an electric or magnetic field. It is possible that especially electromagnetic fields in the range between 1 and 30 MHz exhibit non-thermal antineoplastic effects on cancer cells by direct electromagnetic coupling, i.e., with the cell membrane, receptors or ion channels. Tumour growth inhibition has been shown also for interactions with alternating magnetic fields.^^ The application of low power electric fields (<5W) has also found to be effective against cells and tumors without increasing the temperature.^^'^^ Yet few studies discuss the biological mechanisms involved in the mechanisms involved with the interactions between EMF and tissue. In his book. Exploring Biological Closed Electric Circuits (BCEC) Nordenstrom from the Karolinska Institute in Stockholm^^ describes different circulatory system pathways for which any serious disruption in the flow of energy and material can produce error, malfiinctions, disruptions and disease. O'clock from Minnesota State University could demonstrate a proliferation suppression of malignant cells (retinoblastoma cells) by direct electrical current within a 10 to 15 MA range.^^ Non-equilibrium thermal effects might be at least partially-responsible for antineoplastic effects in tumor tissue. Capacitively-coupled energy transfer in the frequency range between 8 and 27 MHz may not penetrate the cell membrane and will be absorbed primarily in the extracellular space. A constant energy delivery may maintain over time a temperature gradient

Locoregional Hyperthermia

177

Figure 2. Complete remission from anaplastic astrocytoma (WHO grade III) with RF-DHT at 13.56 MHz lasting more than 3+ years after recurrence following surgery, radiotherapy and chemotherapy.

between the extra- and intracellular space, causing ionic currents through the membrane which depolarizes and therefore destabilizes the membrane. An increased transmembraneous water influx by the thermal flux can increase the intracellular pressure, which is about 30% above the normal.^ Since malignant cells typically have relatively more rigid membranes than normal cells due to increased phospholipid concentrations,^^ an increase in pressure will selectively destroy more malignant cells. These effects might be the reasons why RF capacitively coupled hyperthermia may be used for the treatment of areas which have been contra indicated for other methods of hyperthermia, such as of the liver, lung, pancreas and brain.

Conclusions Locoregional hyperthermia may contribute to therapeutic improvements in the treatment of cancer patients. Randomised controlled phase III trials have shown that these methods increase at least at several indications the response rate, disease free and overall survival of patients with cancer without increasing the toxicity of other combinational treatments. Nevertheless,

178

Hyperthermia in Cancer Treatment: A Primer

Time From

1 -Year ± s.e.

2-Year ± s.e.

3-Year ± s.e.

4-Year ± s.e.

5-Year ± s.e.

1st Diagnosis Progression

100 100

75±15 75±15

75±15 60±18

56±20 40±20

56±20 40±20

1st Hyperthermia

78±14

65±17

65±17

43±21

43±21

Censored: 5 (56%); events: 4; s.e.: standard error

Table 9. Survival probability: glioblastoma multiforme WHO grade IV (n=27) 1 -Year ± s.e.

2-Year ± s.e.

3-Year ± s.e.

4-Year ± s.e.

5-Year ± s.e.

1 st Diagnosis

70±9

30±9

9±6

9±6

4±4

Progression

55±10

26±9

7±6

7±6

0

1st Hyperthermia

39±10

13±7

7±6

0

0

Time From

censored: 3 (11%); events: 24; s.e. : standard error

the different methods are associated with systemic and local side-effects. For three types of tumors, the locally advanced cervical cancer, advanced head and neck tumors and glioblastoma, a survival benefit has been shown in randomized controlled trials. In other tumors, such as local recurrent breast cancer and recurrent melanoma an increase in local response but no positive effect on recurrence-free or overall survival has been demonstrated. The recurrence rate of carcinoma of the bladder can be reduced markedly by hyperthermic perfusion. Patients with peritoneal metastases from ovarian cancer respond much better to hyperthermic perfusion chemotherapy compared to systematic chemotherapy, especially after first line therapy. The superficial, interstitial and perfusional hyperthermic methods provide at the time the most effective hyperthermic methods with significant improvements in clinical outcome in oncology, as quality of life and overall survival. Further technical improvements are desired to optimize the therapeutic outcome. The optimal technique, i.e., applied frequency, maximal temperature, time of exposure, time interval with other antineoplastic modalities, has still to be defined. Non-invasive techniques for the measurement of the intratumoral temperature distribution may overcome the present burdened and risky invasive measurements. Non-thermal effects may also play a role by direct interactions of electromagnetic and ultrasonic waves in cancer tissue, on subcellular and molecular levels. There are some interesting hints, showing that deep hyperthermia with radiofrequencies may have some different effects and may exhibit antineoplastic activity without radio- or chemotherapy. Marked improvements in quality of life, pain relief and prolongation of survival could be observed in first observational studies. These encouraging results deserve to be confirmed in randomized clinical trials. But, with respea to evidence-based gradings of clinical trials it should be mentioned that K. Benson et al^^ and J. Concato et al^^ could show in meta-analysis from 235 clinical studies that well-designed observational studies do not systematically overestimate the magnitude of the effects of treatment as compared with those in randomized, controlled trials on the same topic.

Locoregional Hyperthermia

179

Acknowledgement I thank Mrs. M. Riese for the literature search and manuscript assistance. References 1. Heilbrunn LV. The colloid chemistry of protoplasm. Am J Physiol 1924; 69:190-199. 2. Yatvin M B , Dennis W H . Membrane lipid composition and sensivity to killing by hyperthermia, Procaine and Radiation, In: StrefFer C, van Beuningen D , Dietzel F et al, eds. Cancer Therapy by Hyperthermia and Radiation. Baltimore/Munich: Urban & Schwarzenberg, 1978:157-159. 3. Streffer C. Biological basis of thermotherapy (with special reference to Oncology). In: Gautherie M, ed. Biological Basis of Oncologic Thermotherapy. Berlin: Springer Verlag, 1990:1-72. 4. Bowler K, Duncan CJ, Gladwell RT et al. Cellular heat injury. Comp Biochem Physiol 1973; 45A:44l-450. 5. Belehradek J. Physiological aspects of heat and cold. Am Rev Physiol 1957; 19:59-82. 6. Wallach D F H . Action of Hyperthermia and Ionizing radiation on plasma membranes. In: Streffer C, van Beuningen D , Dietzel F et al, eds. Cancer Therapy by Hyperthermia and Radiation. Baltimore/Munich: Urban & Schwarzenberg, 1978:19-28. 7. Nishida T, Akagi K, Tanaka Y. Correlation between cell killing effect and cellmembrane potential after heat treatment: analysis using fluorescent dye and flow cytometry. Int J Hyperthermia 1997; 13:227-234. 8. Weiss TF. Cellular Biophysics, Vol. 2. Electrical Properties. Cambridge: M I T Press, 1996. 9. Mikkelsen RB, Verma SP, Wallach D F H . Hyperthermia and the membrane potential of erythrocyte membranes as studied by Raman Spectroscopy. In: Streffer C, van Beuningen D , Dietzel F et al, eds. C a n c e r T h e r a p y by H y p e r t h e r m i a and R a d i a t i o n . B a l t i m o r e / M u n i c h : U r b a n & Schwarzenberg, 1978:160-162. 10. Hahn C M . The heat-shock response: Effects before, during and after Gene activation. In: Gautherie M, ed. Biological Basis of Oncologic Thermotherapy. Berlin: Springer Verlag, 1990:135-159. 11. Hodgkin AL, Katz B. The effect of temperature on the electrical activity of the giant axon of squid. J Physiol 1949; 108:37-77. 12. Keszler G, Csapo Z, Spasokoutskaja T et al. Hyperthermy increase the phosporylation of deoxycytidine in the membrane phospholipid precursors and decrease its incorporation into D N A . Adv Exper Med Biol 2000; 486:33-337. 13. Dikomey E, Franzke J. Effect of heat on induction and repair of D N A strand breaks in X-irradiated C H O cells. Int J Radiat Biol 1992; 61:221-234. 14. Yutaka Okumura, Makoto Ihara et al. Heat Inactivation of DANN-Dependent Protein Kinase: Possible Mechanism of Hyperthermic Radio-sensitization. In: Kosaka M, Sugahara T, Schmidt KL, et al, eds. Thermotherapy for Neoplasia, Inflammation, and Pain. Tokyo: Springer Verlag, 2001:420-423. 15. Weiss TF. Cellular Biophysics, Vol. 1. Transport. Cambridge: M I T Press, 1996. 16. Dewhirst M W , Ozimek EJ, Gross J et al. Will hyperthermia conquer the elusive hypoxic cell? Radiology 1980; 137:811-817. 17. Vaupel PW, Kelleher DK. Metabolic status and reaction to heat of Normal and tumor tisuue. In: Seegenschmiedt M H , Fesseden P, Vernon C C , eds. Thermoradiotherapy and Thermochemotherapy, Vol. 1. Biology, physiology and physics. Berlin/Heidelberg: Springer Verlag, 1996:157-176. 18. Li G C , Mivechi N F , Weitzel G. Heat shock proteins, thermotolerance, and their relevance for clinical hyperthermia. Int J Hyperthermia 1995; 11:459-88. 19 Stein U, Rau B, Wust P et al. Hyperthermia for treatment of rectal cancer: evaluation for induction of multidrug resistance (mdrl) expression. Int J Cancer 1999; 80:5-12. 20. Raymond U, Hiraoka M, Takahashi M et al. Thermoradiotherapy of refractory malignant tumors: and experience with microwave and RF capacitive hyperthermia. Medical Instrumentation 1984; 18:181-186 2 1 . Fuwa N , Morita K, Kimura C et al. Combined treatment of radio-therapy and local hyperthermia using 8 M H z RF-wave for advanced carcinoma of the breast. In: Onoyama Y, ed. Hyperthermic Oncology '86 in Japan. Proceedings of the 3rd annual meeting of the Japanese Societey of Hyperthermic Oncology 1986:337-338. 22. Goldobenko GV, Durnov LA, Knysh VI et al. Experience of the use of thermoradiotherapy of malignant tumors. Med Radiol (Russian) 1987; 32:36-37. 23. Tsyb AF, Berdov BA. The use of local hyperthermia for therapy of cancer patients. Med Radiol (Russian) 1987; 32:25-29. 24. Savchenco N E , Zhakov IG, Fradkin SZ et al. The use of hyperthermia in oncology. Med Radiol (Russian) 1987; 32:19-24.

180

Hyperthermia in Cancer Treatment: A Primer

25. Hamazoe R, Maeta M, Murakami A et al. Heating efficiency of radiofrequency capacitive hyperthermia for treatment of deep-seated tumors in the peritoneal cavity. J Surg Oncol 1991; 48:176-179. 26. Hiraoka M, Jo S, Dodo Y et al. Clinical results of radiofrequency hayperthermia combined with radiation in the treatment of radioresistant cancers. Cancer 1984; 54:2898-2904. 27. Kondo M, Oyamada H, Yoshikawa T. Therapeutic effects of chemoembolization using degradable starch microspheres and regional hyperthermia on unresectable hepatocellular carcinoma. In: Matsuda T, ed. Cancer treatment by hyperthermia and drugs. London/Washington DC: Taylor & Francis, 1993:317-327. 28. Sugimachi K, Kuwano H, Ide H et al. Chemotherapy combined with or without hyperthermia for patients with oesophageal carcinoma: a prospective randomized trial. Int J Hyperthermia 1994; 4:485-493. 29. Sugimachi K, Kitamura K, Baba K. Hyperthermia combined with chemotherapy and irradiation for patients with carcinoma of the oesophagus: a prospective randomized trial. Int J Hyperthermia 1992; 8:289-295. 30. Sugimachi K, Kitamura K, Baba K et al. Hyperthermia combined with chemotherapy and irradiation for patients with carcinoma of the oesophagus-A prospective randomized trial. Int J Hyperthermia 1992; 8:289-295. 31. Muratkhozhaev NK, Svetitsky PV, Kochegarov AA et al. Hyperthermia in therapy of cancer patients. Med Radiol (Russian) 1987; 32:30-36. 32. Wang J, Li D, Chen N. Intracavitary microwave hyperthermia combined with external irradiation in the treatment of esophageal cancer [Article in Chinese] Zhonhua Zhong Liu Za Zhi 1996; 18(l):51-54. 33. Shchepotin IB, Evans SR, Chorny V et al. Intensive pre-operative radiotherapy with local hyperthermia for the treatment of gastric carcinoma. Surg Oncol 1994; 1:37-44. 34. Kakehi M, Ueda K, Mukojima T et al. Multi-institutional clinical studies on hyperthermia combined with radiotherapy of chemotherapy in advanced cancer of deep-seated organs. Int J Hyperthermia 1990; 6:619-640. 35. Nagata Y, Hiraola M, Nishimura Y et al. Radiofrequency hyperthermia for advanced gastric cancer. In: Gerner EW, ed. Hyperthermic Oncology. Tucson: Arizona Board of Regents, 1992:407-412. 36. Hager ED, Krautgartner I, Popa C et al. Deep Hyperthermia with short waves of patients with advanced stage lung cancer. Hyperthermia in clinical practice. XXII Meeting of the International Chnical Hyperthermia Society, 1999. 37. Hager ED, Dziambor H, Hohmann D et al. Deep hyperthermia with radiofrequencies in patients with liver metastases from colorectal cancer. Anticancer Res 1999; 19:3403-3408. 38. Hager ED, Dziambor H, App EM et al. The treatment of patients woth high-grade malignant ghomas with RF-hyperthermia. Proc ASCO 2003; 22(470): 118. 39. Datta NR, Bose Ak, Kapoor HK et al. Head and nech cancers: results of thermoradiotherapy versus radiotherapy. Int J Hyperthermia 1990; 6:479-86. 40. Overgaard J, Gonzalez Gonzalez D et al. Randomised trial of hyperthermia as adjuvant to radiotherapy for recurrent or metastatic malignant melanoma. Lancet 1995; 345:540-43. 41. Perez CA, Pajak T, Emami B et al. Randomized phase III study comparing irradiation and hyperthermia with irradiation alin in superficial measurable tumors: final report by the Radiation Therapy Oncology Group. Am J Clin Oncol 1991; 14:133-41. 42. Valdagni R, Amichetti M. Report of a long-term follow-up in a randomized trial comparing radiation therapy and radiation plus hyperthermia to metastatic lymph nodes in stage IV head and neck cancer patients. Int J Radiat Oncol 1993; 28:163-69. 43. Vernon C, Hand JW, Field SB et al. Radiotherapy with or without hyperthermia in the treatment of superficial localized breast cancer: results from five randomized collected trials. Int J Radiat Oncol Biol Phys 1996; 35:731-44. 44. Emami B, Myerson RJ, Cardenes H et al. Combined hyperthermia and irradiation in the treatment of superficial tumors: results of a prospective randomized trial of hyperthermia fractionation (1/wk vs 2/wk). Int Radiat Oncol Biol Phys 1992; 24:1451-52. 45. Emami B, Scott C, Perez CA et. Al. Phase III study of interstitial thermoradiotherapy compared with interstitial radiotherapy alone in the treatment of recurrent or persistant human tumors. A prospectively controlled randomized study by the Radiation Therapy Group. Int J Oncol Biol Phys 1996; 34:1097-104. 46. Sneed PK, Stauffer PR, Mc Dermott MW et al. Survival benefit of hyperthermia in a prospective randomized trial of brachy-therapy boost +/- haperthermia for glibostoma multiforme. Int J Radiat Oncol Biol Phys 1998; 40:287-95. 47. Kitamura K, Kuwano H, Watanabe M et al. Prospective randomized study of hyperthermia combined with chemotherapy for esophageal carcinoma. J Surg Oncol 1995; 60:55-58.

Locoregional Hyperthermia

181

48. Berdov BA, Menteshashvili G Z . Thermoradiotherapy of patients with locally advanced carcinoma of the rectum. Int J Hyperthermia 1990; 6:881-90. 49. Hiraoka M , Mitsumori M, Nagata Y et al. Current status of clinical hyperthermic oncology in Japan. 50. Benson K, Hartz AJ. A comparison of observational studies and randomized, controlled trials. N Engl J Med 2000; 342: 1878-86. 51. Concato J, Shah N , Horwitz RI. Randomized, controlled trials, observational studies, and the hierarchy of research designs. N Engl J Med 2000; 342:1887-92. 52. O h n o S, Tomoda M, Tomisaki S et al. Improved surgical results after combining preoperative hyperthermia with chemotherapy and radiotherapy for patients with carcinoma of the rectum. Dis Colon Rectum 1997; 40(4) :401-406. 53. Colombo R, Pozzo LF, Lev A et al. Neoadjuvant combined microwave induced local hyperthermia and tropical chemotherapy versus chemotherapy alone for superficial bladder cancer. J Urol 1996; 155:1227-1232. 54. Colombo R, Pozzo LF, Lev A et al. Adjuvant microwave hyperthermia and Mitomycin C versus Mitomycin C alone for superficial bladder cancer. Europ Urol 1999; 35(suppl 2). 55. Hager E D , Strama H, Hohmann D et al. Prevention of cystectomy of recurrent bladder carcinoma by intravesical hyperthermic perfusion chemotherapy (IVHP). Antic Res 1998; 18:4807-5006. 56. Van Rhoon G, van der Zee J, Broekmeyer-Reurik M P et al. Radiofrequency capacitive heating of deep-seated tumors using pre-cooling of the subcutaneous tissues: results on thermometry in Dutch patients. Int J Hyperthermia 1992; 8:843-854. 57. Datta NR, Bose AK, Kapoor HK. Thermoradiotherapy in the management of carcinoma cervix (IIIB): a controlled clinical study. Indian Med. Gazette 1987; 121:68-71. 58. Hornbach N B , Shupe RE, Shidnia H et al. Advanced stage IIIB cancer of the cervix treatment by hyperthermia and radiation. Gynecol Oncol 1986; 23:160-167. 59. Harima Y, Nagata K, Harima K et al. A randomized clinical trial of radiation therapy versus thermoradiotherapy in stage IIIB cervical carcinoma. Int J Hyperthermia 2001; 17(2):97-105. 60. Harima Y, Nagata K, Harima K et al. A randomized clinical trial of radiation therapy versus thermoradiotherapy in stage Illb cervical carcinoma. Int J Hyperthermia 2001; 17:97-105. 6 1 . Nishimura Y, Hiraoka M, Akuta K et al. Hyperthermia combined with radiation therapy for primary unresectable and recurrent colorectal cancer. Int J Radiat Oncol Biol Phys 1992; 23:759-768. 62. Masunaga S, Hiraoka M, Akuta K et al. The phase I/II trial of preoperative thermoradiotherapy in the treatment of urinary bladder cancer. Int J Hyperthermia 1994; 10:31-40. 63. Rietbroek RC, Schiltuis MS, Bakker PM et al. Phase II trial of weekly locoregional hyperthermia and cisplatin in patients with a previously irradiated recurrent carcinoma of the uterine cervix. Cancer 1997; 79:935-942. 64. Harima Y, Nagata K, Harima K et al. Bax and Bcl-2 protein expression following radiation therapy versus radiation plus thermotherapy in stage IIIB cervical carcinoma. Cancer 2000; 88:132-138. 65. Masunaga S, Hiraoka M, Takahashi M et al. Clinical results of thermradiotherapy for locally advanced and/or resurrent breast cancer-comparison of results with radiotherapy alone. Int J Hyperthermia 1990; 6:487-497. GG. Nagata Y, Hiraoka M, Nishimura Y et al. Clinical results of radiofrequency hyperthermia for malignant liver tumors. Int J Radiat Oncol Biol Phys; 1997; 38(2):359-365. 67. Hiraoka M, Masunaga S, Nishimura Y et al. Regional hyperthermia combined with radiotherapy in the treatment of lung cancer. Int J Radiat Oncol Biol Phys 1992; 22:1009-1014. 68. Hager ED, Siifie B, Popa C et al. Complex therapy of the not in sano respectable carcinoma of the pancreas-a pilot study. J Cancer Res Clin Oncol 1994; 120(Suppl.):R47,P1.04.15. 69. Hager ED, Dziambor H, Hoehmann D. Survival and quality of life patients with advanced pancreatic cancer. Proc ASCO 2002; 21(2357):136b. 70. Hiraoka M , Nishimura Y, Masunaga S et al. Clinical results of thermoradiotherapy of soft tissue tumors. Int J Hyperthermia 1995; 11:365-377. 7 1 . O'clock C D . Effects of magnetic fields on health and disease. Dtsch Zschr Onkol 2003; 35:15-23. 72. Watson BW. Reappraisal: The treatment of tumors with direct electric current. Med Sci Rec 1991; 19:103-105.. 73. Samuelsson L, Jonsson L, Stahl E. Percutaneous treatment of pulmonary tumors by electrolysis. Radiologie 1983; 23:284-287. 74. Miklavcic D , Sersa G, Kryzanowski M. Tumor treatment by direct electric current, tumor temperature and p H , electrode materials and configuration. Bioelectr Bioeng 1993; 30:209-211. 75. Katzberg AA. The induction of cellular orientation by low-level electrical currents. Ann New York Acad Sci 1974; 238:445-450. 7G. Szasz A, Vincze GY, Szasz O et al. An energy analysis of extracellular hyperthermia, accepted for publication in Magneto- and electro-biology. 2003; in print.

182

Hyperthermia in Cancer Treatment: A Primer

77. Kotnik T, Miklavcic D. Theoretical evaluation of the distributed power dissipation in biological cells exposed to electric field. Bioelectromagnetics 2000; 21:385-394. 78. Galeotti T, Borrello S, Minotti L. Membrane alterations in cancer cells: the role of oxy radicals. An New York Acad Sci Vol 488. Membrane Pathology, Bianchi G, Carafoli E, Scarpa A, eds. 1986:468-480. 79. Nordenstrom, BEW. Biological Closed Electric Circuits: Clinical, Experimental and theoretical evidence for an additional circulatory system. Nordic Medical Publications. Stockholm, 1983. 80. O'clock CD, Leonhard T. In Vitro Response of retino-blastoma, lymphoma and non-malignant cells to direct current: therapeutic implications. Dtsch Zschr Onkol 2001; 33:85-90. 81. Hashimoto D, Takami M, Idezuki Y. In depth radiation therapy by YAG laser for malignant tumors in the liver under ultrasonic imaging. Gastroenterology 1985; 88:1663. 82. Vogl J, Mack MG, Straub R et al. Percutaneous MRI-guided laser-induced thermotherapy fpr hepatic metastases for colorectal cancer. Lancet 1997; 350:29. 83. Vogl J, Mack MG, Roggan A. Magnetresonanztomographisch gesteuerte laserinduzierte Thermotherapie von Lebermetastasen. Dtsch Arzteblatt 2000; 37:2039ff. 84. Becker D, Hansler JM, Strobel D et al. Percutaneous ethanol injection and radio-frequency ablation for the treatment of nonresectable colorectal liver metastases-techniques and results. Langenbeck's Arch Surg 1999; 384:339-343. 85. Hansler J, Becker D, Miiller W et al. Ultraschallgesteuerte Interstitielle Hochfrequenz-Thermotherapie (HFTT)-In-vitro-Untersuchung an der Rinderleber. Ultraschall in Med 1998; 19:59-63. 86. Kettenbach J, Kosder W, Riicklinger E et al. Percutaneous Salin-Enhanced Radiofrequency Ablation of Unresectable Hepatic Tumors: Initial Experience in 26 Patients. AJR 2003; 180:1537. 87. Pearson AS, Izzo F, Fleming RY et al. et al. Intraoperative radiofrequency ablation or cryoablation for hepatic malignancies. Am J Surg 1999; 178:592-599. 88. Wood TF, Rose DM, Chung M et al. Radiofrequency ablation of 231 unresectable hepatic tumors: indications, limitations, and complications. Ann Surg Oncol 2000; 7(8):593-600. 89. Jordan A, Scholz R, Maier-Hauff K et al. Presentation of a new magnetic field therapy system for the treatment of human solid tumors with magnetic fluid hyperthermia. J Magnetism Magn Mat 2001; 225:118-126. 90. Kakehi M, Ueda K, Mukomojima M et al. Multi-institutional clinical studies on hyperthermia combined with radiotherapy or chemotherapy in advanced cancer of deep-seated organs. Int J Hyperthermia 1996; 6(4):719-740.

CHAPTER 13

Hyperthermia and Radiotherapy in the Management of Prostate Gmcer Sergio Villa* Abstract

C

arcinoma of the prostate has been included in the top five big killer neoplasms. The management of advanced disease is still a problem in oncology. Starting from the beginning of the eighties, hyperthermia has been associated to radiotherapy with the aim to increase the local control. This issue summarizes the indication, technical modality and results of the combined treatment, both as radical and salvage therapy.

Introduction In 1995, prostate cancer was the third most common cancer in men in Europe, with some 156,000 new cases.^ Incidence rates in Northern and Western areas of the continent result higher than in Southern or Eastern countries but the geographical variations were probably due to the different medical practices between countries rather than to any real variation in the underlying risk. In fact, the increase of new diagnoses is strongly related to the diffusion of haematic dosage of prostate specific antigen (PSA) in the daily clinical practice."^^ In Italy, in the same period, the age-standardized incidence was 39.4/100,000 whilst the mortality was 19.1/100,000.^ The gold standard in the management of clinically localized prostate adenocarcinoma is debated. At to day, prostatectomy and radiotherapy (RT) represent the most common approach forTl-T3,NX-0,M0 patients but both these modalities of treatment have showed its therapeutic limitations in the local control of bulky tumors (T2b-T3).^^' ^ In these cases, external beams radiotherapy (ERT) is common preferred to surgery, but both clinical and radiobiological evidence indicate that prostate cancer cells are relatively resistant to radiation. On the other hand, the date of literature suggests that improving of local control may potentially impact favorably on the survival of these subsets of patients.^^'^^ With the aim to obtain an increase of local control,^^'^^'"^^ starting from the beginning of eighties hyperthermia (HT) has been used both as salvage treatment in the patients with clinically confirmed post-prostatectomy or radiation recurrence of disease,'7.1837 and as concomitant boost in local advanced tumors.l'1530.35,42,44

Rationale In vivo, mild HT seems to induce no significant effects in the prostatic tumor cells. Strohmaier et al^^ found hyperemic alterations of the prostatic stroma and a diffuse oedema with interstitial •Corresponding Author: Sergio Villa— National Cancer Institute Milan, Radiotherapy Service, Via G. Venezian 1, Milano 20133, Italy. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

184

Hyperthermia in Cancer Treatment: A Primer

lymphoplasmacellular infiltration without definitive signs of tumor cells necrosis, in twenty untreated patients undergone to four sessions of local HT (42-43°C). On the contrary, in several studies performed in vitro on human tumor cell lines to test the effect of thermotherapy, prostatic carcinoma cells of human origin were found to be more sensitive to mild HT than other human cancer cells. ^'^'^^'^'^'^^ Some investigators have suggested that HT may temporarily alter cell-mediated immunity. Stawarz et al^ measured T-lymphocyte subset ratios, mitogenic responses to phytohemaglutinin and concanavalin A, T-cell suppressor activity, and natural killer cell cytotoxic activity in a group of 15 patients, bearing local advanced or metastatic prostate cancer, managed with palliative transrectal microwave HT only. Starting from the end of HT, they observed a significant improvement in the monitored immune parameters over pretreatment status, which peaked at two months and returned to near pretreatment levels by six months. On the other hand, Li and Franklin^^ found that apoptosis represents an important mode of death in heated prostate tumor cells but it is insignificant in the irradiated cells. Therefore, HT may have a really efficacy in the control of radioresistant hypoxic cells. Nevertheless, the reason why HT at moderate temperatures may provide a therapeutic advantage in prostate cancer patients undergone concomitant RT is not known at today.

Hyperthermia: Technical Modalities The goal of HT technical modalities is to elevate the temperature in the prostatic area that may have tumor cells. When HT is performed as a concomitant boost during a cycle of radical RT, the temperature must be elevated to 42-43°C for 30-60 min. On the contrary, in the patients imdergone to exclusive rescue HT for clinically post-surgery or RT recurrence, the tissues must be heated over 55°C to have a cytotoxic effect.^^'^ Achieving optimal temperatures onto the prostate gland is the most important condition to perform a really therapeutic treatment, even if it remains a formidable technical problem.^ Actually, three technical approach are used to perform thermotherapy in the patients bearing prostate cancer: regional hyperthermia, intraluminal (transrectal or transurethral) hyperthermia and interstitial hyperthermia. Regional hyperthermia (RHT) is noninvasive technique performed with a coaxial system. This system uses a circumferential antenna around the pelvic region. Temperatures are measured intraluminally in the bladder, urethra and rectum to obtain an impression of tumor temperature. Furthermore, temperature is measured orally or in esophagus to determine body temperature. Because the whole pelvic region is heated, the average body temperature rises over than 38°C during the session of treatment. In the daily clinical practice, it is difficult to produce high temperatures using noninvasive device, such as radiofrequency phased arrays, due to acute toxicity, particularly pain, which limits the power, which can be applied to the patients.^ The reasons for this inability could be explained by the anatomic location of the gland. In fact, prostate is shielded anteriorly by bone and fat, both of which are low conductivity materials. Transrectal hyperthermia (TRHT) is performed as follows: under sterile conditions, thermometer probes are placed into the prostate gland. Subsequendy, simulation fdms are taken to verify the placement of thermocouples relative to skeletal landmarks and to verify the depth and location of the transrectal ultrasound (US) appUcator with respect to the thermocouple probes. Patient is placed in the left lateral decubitus and the hyperthermia applicator is placed onto the rectal vault. At this time, baseline measurements are taken and the treatment starts. The US power is increased gradually until the therapeutic temperature is reached. Once treatment is completed both the applicator and the thermocouples are removed. The patients scheduled for two HT treatments, have got the fractions separated by at least one week. Interstitial hyperthermia (IHT) technique is an invasive modality of heating of the prostate region. IHT is performed with the patients under epidural or general anesthesia by placing a mean of 12 catheters in the prostate through a template under transrectal ultrasonography guidance, with the same technology used for brachytherapy prostate implants. In each catheter are inserted a probe with two electrodes and thermocouple with sensors to measure the temperature profile along the catheter track. Additional thermometry catheters are located onto

Hyperthermia and Radiotherapy in the Management ofProstate Cancer

185

the gland and in the rectum and in the urethra to monitor the temperature during the treatment. Recently, Tucker et al have proposed a new system to perform IHT. The technique is thermal ablation of the prostate by permanently implanted biocompatible rods. An array of rods (a ferromagnetic alloy 7% cobalt and 93% palladium by weight) is placed percutaneously onto the gland under transrectal ultrasound and fluoroscopic guidance. The patient is then placed in a coil system that heats the array of rods by an extracorporel alternating magnetic field. The produced heat causes necrosis of the normal glandular tissue, as well as the cancer, within the array of rods. Actually, adequate HT is proved difficult to realize both with regional radio frequency^ and intraluminal or interstitial technology. The hyperthermia devices used must ensure a proper distribution of heat to achieve a sufficient therapeutic effect in the tumor tissue. Intraluminal techniques in the rectum and/or urethra are minimally invasive but the heat distribution is highly nonuniform.^'^O'i^'^1

Radiotherapy: Technical Modalities At today, external beams radiotherapy (ERT) and brachytherapy (BRT) are usually used in the management of prostate cancer and both these modality of irradiation are tested concomitandy to HT. ERT is delivered in one fraction day, 5 days a week, for 7-8 weeks using high-energy photon beams of linear accelerators. The target is represented by prostate lodge (gland ± seminal vesicles). Prophylactic irradiation of pelvic lymph nodes is debated. Actually, a total dose higher than 70 Gy, delivered with three-dimensional conformal radiotherapy (3DCRT), is considered the gold standard as a radical treatment for prostate cancer. BRT is performed as follows: with the patients under epidural or general anesthesia, a large number of seeds are placed, under US guidance, within the prostate gland using a template fixed to the perineum. Preimplant and post-implant dosimetry evaluation, with three dimensional treatment planning system, are performed to verify the effective dose distribution both in the neoplastic target, represented to prostate gland alone, and in critical organs as anterior rectum wall or urethra.

Hyperthermia and Radiotherapy: Toxicity The most common acute side effects of this treatment regimen are urinary-related (increasing frequency and dysuria) or bowel-related (more frequent bowel movements and diarrhea). In added, the majority of patients also develop perineal discomfort after HT procedure, secondary to the placement of hyperthermia probes or devices. A limited number of patients show hypotension during HT session. The men, bearing benign prostatic hypertrophy (BPH), with a positive history of obstructive urinary symptoms show a higher risk to develop severe urinary retention and spasm. Finally, myonecrosis and peripheral neuropathy are referred as rare complications observed among the patients included in combined RT-HT phase I-II studies.^ However, the most important critical organ in the combined treatment is the rectal wall. Nevertheless, there are limited published findings on the impact of HT directed to the prostate on rectal toxicity. Reports describing the relationship of thermal parameters with rectal toxicity have used tumor or generalized normal tissue temperature profiles. To date, the association of tissue-specific parameters for rectal wall temperature with increased rectal toxicity has not been defined. In a final report of a Phase I trial using the transrectal ultrasound device used in our Phase II study Algan et al^ noted no added long-term toxicities with the addition of H T to radiation treatment (68 Gy) of prostate cancer in 26 patients with median follow-up of 71 months. The thermal treatment goal in this study was obtainment of temperatures of 42.5°C within the gland for 30 min for either one or two HT treatments. The authors do not report the rectal wall temperatures associated with this favorable toxicity profile. In treatment of prostate cancer with BRT and interstitial HT, Prionas et al^^ reported no acute GI toxicity. The percentage of mapped temperatures exceeding 41 °C, 42°C, and 43°C were 67%, 46% and 27% within tumor and 26%, 11% and 4% in normal surrounding tissues. No specific attempt

Hyperthermia in Cancer Treatment: A Primer

186

Table 1. Hyperthermia with concurrent radiotherapy in the management of prostate cancer patients

References

Year

Ptsn.

Stage

Hyperthermia

Yerushalmi Prionas Anscher Algan Hurwitz van Vulpen

'81-83 '87-92 '87-94 '90-93 '97-00 '99-01

20 31 18 26 30 12

C-D1 B-C T2b-T4 C2-D1 T2b-T4 T3

RHT IHT RHT TRUSH TRUSH IHT

Radiotherapy EBRT Gy BRT 60-65 ,^192

65-70 67-70 70 70

was made, however, to define rectal wall temperatures. Hurwitz et al have found a direct correlation of rectal wall toxicity to thermal dose parameters for thirty patients undergoing transrectal ultrasound HT combined with external beams radiation for treatment of locally advanced (T2^-T3^) prostate cancer. The rate of acute grade 2, according to RTOG/EORTC criteria, proctitis was greater for patients with an allowable rectal wall temperature of > 40°C.

Hyperthermia and Radiotherapy: Results and Discussion Tables 1 and 2 summarize some of the most important series referred to patients managed by HT ± RT. Some remarks are needed. At the first, the number of patients enrolled in these series is small and extremely heterogeneous. In added, a large part of these date are referred to phase I/II studies, hence, the time of the follow up is too short to evaluate the real therapeutic impact of the treatments. Herein the results of HT, both as boost in locally advanced prostate tumor and as salvage treatment for recurrent disease after prostatectomy or radical RT, are reviewed and debated. HT Plus RT as Radical Treatment There are a few reports of deep hyperthermia and radiation in patients with untreated prostate cancer. In the Duke University series, Anscher et al^ refer the date of 18 patients with newly diagnosed stage T3-T4 prostate cancer treated with definitive ERT and deep HT. The 3-vear local control and distant failure free survival were 93% and 68% respectively. Algan et al in a series of twenty-six patients undergone to HT and concomitant ERT for local advanced prostate cancer reported a 5-years overall and b-NED survival of 88% and 35% respectively. These data do not shown an effective increase in therapeutic efficacy if compared to ^NED survival rates observed in the series of patients, with the same local extension of disease.

Table 2. Hyperthermia with or without concurrent radiotherapy in the management of the patients bearing local recurrences of prostate cancer

References

Year

Ptsn.

First Therapeutic Local Approach

Hyperthermia

Kaplan Prionas Anscher Kalapurakal Sherar

Not indicated '87-92 '87-94 '97-99 '98-00

4 5 3 3 25

BRT prostatectomy prostatectomy EBRT EBRT

Exclusive RHT IHT RHT RHT Exclusive IHT

Radiotherapy EBRT Gy BRT

65-70 30-50

Hyperthermia and Radiotherapy in the Management ofProstate Cancer

187

managed by 3DCRT exclusive in recent dose escalation studies. Hanks et al^'^ in a dose escalation study, performed at Philadelphia Fox Chase Cancer Center, have found ^NED 5-year rates of 32% and 53% in a subset of patients with unfavorable prognostic factors undergone to 3DCRT at 75 and 81 Gy respectively. Fiveash et al^ have reported the results of a multi-institutional review focused to the outcome of high grade prostate cancer patients managed by ERT. The authors referred a 5-years ^NED rate of 44.8% in seventy-five T3-T4 patients with 8-10 Gleason Pattern Score (GPS). Pollack et al"^^ have reported 78% of freedom from failure rate at 6-year in 60 patients, bearing T3 prostate tumor, managed with 3DCRT at the total dose of 78 Gy. In added, 3DCRT seems to have an effective dramatic impact on local control at the dose nearly 80 Gy. In fact, Zelefsky et al found only 1/15 positive biopsy in a subset of patients undergone to 3DCRT at the total dose of 81 Gy. It is noteworthy that the results above have been obtained without a concomitant increase of acute or deferred toxicity. The investigators ^'^'^^' reported a significant (> grade 2) long-term gastrointestinal and/or urinary toxicities in less than 15% of irradiated patients. HT as Salvage Treatment The overall incidence of biochemical progression following treatment with radical prostatectomy, radiation therapy or radiation plus hormone treatment is 15-40%.^'^^'^^ Patients with recurrent prostate cancer after RT are commonly retreated with anti-androgens or orchiectomy."^^ The anti-tumor effects of such hormonal treatment last for approximately 1-2 years, after which time the tumor become hormone refractory. ^^ Prostate cancer has a long natural history, and patients with early biochemical failure following radiation are expected to have an average survival of 5-10 years.^^ As a consequence of the long survival and lack of treatment options for hormone refractory disease, some of these patients develop symptoms due to disease progression in the pelvis. Tumor invasion into the urethra and urinary bladder causes urinary outlet obstruction. Tumor invasion onto the rectum may result in bleeding and rectal obstruction. Tumor invasion onto the pelvic lymph nodes can cause lymphedema in the external genitals and/or extremities. At last, tumor invasion onto the pelvis nerves and/or bones may result in intractable pelvic and perineal pain as well as pathological fracture. ^^ At today, the treatment options available for the management of locally advanced, previously irradiated, hormone refractory prostate cancer include palliative surgical procedures such as trans-urethral-prostatectomy (TURP), ureteric stenting in case of hydronephrosis, cystoscopic tumor fulguration to limit urinary bleeding and colostomy/urinary diversion to overcome rectal and/or bladder obstruction. Chemotherapy has been used in hormone refractory prostate cancer, but the tumor response rate are low and short lived. Pienta et al have treated 42 patients with oral etoposide and estramustine in a phase II study. The overall response rate reported to the investigators was 36% and the average duration of serum PSA decline was 8 weeks among responders. Hence, hyperthermia has been tested as salvage treatment in the subset of irradiated patients with biochemical or local clinically evidence of recurrent disease. On the other hand, the number of patients enrolled in HT series is strongly limited. Kaplan and Bagshaw^^ have used RHT plus ERT (60 Gy in split course) in four patients previously undergone to BRT with IT. ^^^ None of the patients experienced severe rectal or bladder reactions and 3/4 patients achieved complete clinical response at 7-24 months after retreatment. Kalapurakal et al undergone to combined rescue treatment three men with symptomatic, locally advanced, previously irradiated and hormone refractory prostate cancer. All the patients had RHT and ERT (30.6-50 Gy). Two patients showed tumor control at 12 and 24 months. A patient developed local recurrence 17 months after the end of salvage treatment. Prionas^^ and Anscher^ included in their series some cases of rescue treatment, but the outcomes of these patients have not carried out. Recently, Sherar et al^ have published the date referred to a group of twenty-five patients, bearing post-ERT local recurrence prostate carcinoma, managed by exclusive IHT. The negative biopsy rate at 24 weeks has been GA%. Nevertheless, only the 52% of these patients have had a nadir PSA value less than 0.5 ng/ml and these results suggests that the disease was not

188

Hyperthermia in Cancer Treatment: A Primer

local alone but also regional or systemic. Longer term follow-up of PSA and biopsy results is required in larger groups of patients to confirm the potential of salvage thermal therapy.

Conclusion Review of the literature shows that HT does not significandy improve the therapeutic effect of RT. Hence, it must not be considered as an effective therapeutic option in the management of local advanced prostate cancer. On the otherhand, microwave thermoablation techniques, as intraluminal or interstitial hyperthermia, may have a potential role in the therapeutic approach of local recurrences. Even if the use of H T in patients with biochemical and/or clinical failures after RT cannot be considered adequate treatment because the incidence of subclinical regional or systemic metastases is very high, it could defer the start of hormonal manipulation and, consequendy, enhance patient survival. However, further technologic developments and clinical trials are needed to evaluate the effective role of hyperthermia in the management of prostatic neoplasms.

References 1. Algan O, Fosmire H, Hynynen K et al. External beam radiotherapy and hyperthermia in the treatment of patients with locally advanced prostate carcinoma. Cancer 2000; 89:399-403. 2. Anscher MS, Samulski TV, Leopold KA et al. Phase I/II study of external radio frequency phased array hyperthermia and external radiotherapy in the treatment of prostate cancer. Technique and results of intraprostatic temperature measurements. Int J Radiat Oncol Biol Phys 1992; 24:489-495. 3. Anscher MS, Samulski TV, Dodge R et al. Combined external beam irradiation and external regional hyperthermia for locally advanced adenocarcinoma of the prostate. Int J Radiat Oncol Biol Phys 1997; 37(5):1059-1065. 4. Astrahan MA, Ameye F, Oyen R et al. Interstitial temperature measurements during transurethral microwave hyperthermia. J Urol 1991; 145:304-308. 5. Ben-Yosef R, Sullivan DM, Kapp DS. Peripheral neuropathy and myonecrosis following hyperthermia and radiation therapy for recurrent prostatic cancer: Correlation of damage with predicted SAR pattern. Int J Hyperthermia 1992; 8:173-185. 6. BoUa M, CoUette L, Blank L et al. Long-term results with immediate androgen suppression and external irradiation in patients with locally advanced prostate cancer (an EORTC study): A phase III randomized trial. Lancet 2002; 360:103-6. 7. Bray F, Sankila R, Ferlay J et al. Estimates of cancer incidence and mortality in Europe in 1995. Eur J Cancer 2002; 38:99-166. 8. Emami B, Scott C, Perez CA et al. Phase III study of interstitial thermotherapy compared with interstitial radiotherapy alone in the treatment of recurrent or persistent human tumors: A prospectively controlled randomized study by the Radiation Therapy Group. Int J Radiat Oncol Biol Phys 1996; 34:1097-1104. 9. Fiveash JB, Hanks GE, Roach III M et al. 3D conformal radiation therapy (3DCRT) for high grade prostate cancer: A multi institutional review. Int J Radiat Oncol Biol Phys 2000; 47:335-342. 10. Fosmire H, Hynynen K, Drach GW et al. Feasibility and toxicity of transrectal ultrasound hyperthermia in the treatment of locally advanced adenocarcinoma of the prostate. Int J Radiat Oncol Biol Phys 1993; 26:253-259. 11. Gottheb CF, Seibert GB, Block NL. Interaction of irradiation and microwave-induced hyperthermia in the Dunning R3327G prostatic adenocarcinoma mode. Radiology 1988; 169:243-247. 12. Hanks GE, Hanlon AL, Schultheiss TE et al. Dose escalation with 3D conformal treatment: Five years outcomes, treatment optimization, and future directions. Int J Radiat Oncol Biol Phys 1998; 4:501-510. 13. Holaman M, Carlton Jr CE, Scardino PT. The frequency and morbidity of local tumor recurrence after definitive radiotherapy for stage C prostate cancer. J Urol 1991; 146:1578-1582. 14. Hurwitz MD, Kaplan ID, Svensson GK et al. Feasibility and patient tolerance of a novel transrectal ultrasound hyperthermia system for treatment of prostate cancer. Int J Hyperthermia 2001; 17:31-37. 15. Hurwitz MD, Kaplan I, Hansen JL et al. Association of rectal toxicity with thermal dose parameters in treatment of locally advanced prostate cancer with radiation and hyperthermia. Int J Radiat Oncol Biol Phys 2002; 53:913-918. 16. Kalapurakal JA, Mittal BB, Sathiaseelan V. Reirradiation and external hyperthermia in locally advanced, radiation recurrent, hormone refractory prostate cancer: A preliminary report. Br J Radiol 2001; 74:745-751. 17. Kaplan ID, Cox RS, Bagshaw MA. Radiotherapy for prostate cancer: Patients selection and impact of local control. Urology 1994; 43:634-639.

Hyperthermia

and Radiotherapy

in the Management

of Prostate Cancer

189

18. Kaver I, Ware JL, Wilson J D et al. Effect of radiation combined with hyperthermia on human prostatic carcinoma cell lines in culture. Urology 1991; 38:88-92. 19. Krumholtz JS, Andriole GL. The surgery of prostate cancer: An update of contemporary radical prostatectomy and brachytherapy series. Semin Surg Oncol 1999; 17:213-218. 20. Kuban DA, El-Mahdi AM, Schellhammer PF. Potential benefit of improved local tumor control in patients with prostatic carcinoma. Cancer 1995; 75:2373-2381. 2 1 . Lancaster C , Toi A, Trachtenberg J. Interstitial microwave thermoablation for localized prostate cancer. Urology 1999; 53:828-831. 22. Li WX, Franklin WA. Radiation- and heat-induced apoptosis in PC-3 prostate cancer cells. Radiat Res 1998; 150:190-194. 23. Mendecki J, Friedenthal E, Botstein C et al. Microwave applicators for localized hyperthermia treatment of cancer of the prostate. Int J Radiat Oncol Biol Phys 1980; 6:1583-1588. 24. Myerson RJ, Scott CB, Emami B et al. Phase I/II study to evaluate radiation therapy and hyperthermia for deep seeped tumors: A report of R T O G 89-08. Int J Hypertherm 1996; 12:449-459. 25. Parker C C , Dearnaley D P . The management of PSA failure after radiotherapy for localized prostate cancer. Radiother Oncol 1998; 49:103-110. 26. Peschke P, H a h n EW, Wenz F et al. Differential sensitivity of three sublines of the rat dunning prostate tumor system R3327 to radiation and/or local tumor hyperthermia. Radiat Res 1998; 150:423-430. 27. Pienta KJ, Redman B, Hussain M et al. Phase II evaluation of oral estramustine and oral etoposide in hormone refractory adenocarcinoma of the prostate. J Clin Oncol 1994; 12:2005-2012. 28. Pollack A, Zagars GK, Starkschall G et al. Prostate cancer radiation dose response: Results of the M . D . Anderson Phase III randomized trial. Int J Radiat Oncol Biol Phys 2002; 53:1097-1105. 29. Potosky AL, Miller BA, Albertsen PC et al. The role of increasing detection in the rising incidence of prostate cancer. JAMA 1995; 273(7):548-552. 30. Prionas SD, Kapp DS, Goffinet D R et al. Thermometry of interstitial hyperthermia given as an adjuvant to brachytherapy for the treatment of carcinoma of the prostate. Int J Radiat Oncol Biol Phys 1994; 28(0:151-162. 3 1 . Prostate Cancer Trialists's Collaborative Group. Maximum androgen blokade in advanced prostate cancer: An overview of randomised trials. Lancet 2000; 355:1491-1498. 32. Ryu S, Brown SL, Kim SH et al. Preferential radiosensitization of human prostatic carcinoma cells by mild hyperthermia. Int J Radiat Oncol Biol Phys 1996; 34:133-138. 33. Sandler H M , D u n n RL, McLaughlin P W et al. Overall survival after prostate-specificantigen-detected recurrence following conformal radiation-therapy. Int J Radiat Oncol Biol Phys 2000; 48:629-633. 34. Scheiblich J, Petrowicz O . Radiofrequency-induced hyperthermia in the prostate. J Microw Power 1982; 17:203-209. 35. Servadio C, Leib Z. Hyperthermia in the treatment of prostate cancer. Prostate 1984; 5:205-211. 36. Sherar M D , Gertner MR, Clarence KK et al. Interstitial microwave thermal therapy for prostate cancer: Method of treatment and results of a phase I/II trial. J Urol 2001; 166:1707-1714. 37. Shipley W U , Thames H , Sandler H M et al. Radiation therapy for clinically localized prostate cancer: A multi-institutional pooled analysis. JAMA 199; 281:1598-1604. 38. Stawarz B, Zielinski H, Szmigielski S et al. Transrectal hyperthermia as palliative treatment for advanced adenocarcinoma of prostate and studies of cell-mediated immunity. Urology 1993; 41:548-553. 39. Strohmaier WL, Bichler R H , Bocking A et al. Histological effects of local microwave hyperthermia in prostatic cancer. Int J Hyperthermia 1991; 7:27-33. 40. Tucker R D , Platz CE, Huidobro C et al. Interstitial thermal therapy in patients with localized prostate cancer: Histologic analysis. Urology 2002; 60:166-169. 4 1 . Venn SN, Hughes SW, Montgomery BSI et al. Heating characteristics of a 434 M H z transurethral system for the treatment of BPH and interstitial thermometry. Int J Hyperthermia 1996; 12:271-278. 42. van Vulpen M , Raaymakers BW, Lagendijk JJW et al. Three-dimensional controlled interstitial hyperthermia combined with radiotherapy for locally advanced prostate carcinoma. A feasibility study. Int J Radiat Oncol Biol Phys 2002; 53:116-126. 43. Yerushalmi A, Servadio C, Leib Z et al. Local hyperthermia for treatment of carcinoma of the prostate: A preliminary report. Prostate 1982; 3:623-630. 44. Yerushalmi A, Shani A, Fishelovitz Y et al. Local microwave hyperthermia in the treatment of carcinoma of the prostate. Oncology 1986; 43:299-305. 45. Zagars GK, Pollack A, Smith LG. Conventional external beam radiation therapy alone or with androgen ablation for clinical stage III (T3,>DC/0,M0) adenocarcinoma of the prostate. Int J Radiat Oncol Biol Phys 1999; 44:809-819. 46. Zelefsky MJ, Leibel SA, Gaudin PB et al. Dose escalation with tree-dimensional conformal radiation therapy affects the outcome in prostate cancer. Int J Radiat Oncol Biol Phys 1998; 491-500.

CHAPTER 14

Tumor Ablation Using Radiofrequency Energy: Technical Methods and AppHcation on Liver Tumors Johannes-Marcus Hansler,* Luigi Solbiati, £• Dieter Hager> Tiziana lerace, Luca Cova and Gian Franco Baronzio Abstract

R

adiofrequency ablation is used for die treatment of a variety of neoplasms including: osteoid osteoma, hepatocellular carcinoma, renal cell carcinoma, bronchopulmonary carcinoma, parathyroid adenoma;^ hepatic and retroperitoneal metastases from a variety of primary tumors. The size of the coagulation zone is a crucial factor, as only a complete coagulation of the tumor including a sufficient safety zone inhibits local recurrence. Thus many efforts have been made to enlarge the coagulation zone using multiprobe arrays, saline perfiision, internal cooling, bipolar technique, pulsed application or a combination of these mentioned techniques. Tumors up to 5 cm can now be effectively treated, taking inclusion and exclusion criteria into account. Lately published data su^ests that RF ablation is far more than an electro-physical tool to generate a thermal tumor destruction, it also induces a significant activation of tumor-specific T lymphocytes. Percutaneous, image-guided, tumor ablation using thermal energy sources such as radiofrequency (RF) have received increasing attention as promising techniques for the treatment of focal malignant diseases. Often these therapies can still be used when more invasive surgical techniques are no longer feasible due to concomitant disease or tumor localisation. Several studies showed an impressive long term survival for patients with primary and secondary malignant tumors of the liver^ comparable with the data published for surgical resection.^ Unfortunately large prospective, randomised studies are missing. Potential benefits of percutaneous tumor ablation include: decreased cost and morbidity; the possibility of performing the procedure on outpatients and, the possibility of treating patients who would not be considered candidates for surgery due to age, comorbidity or disease spread. Additionally, recent studies support the idea that RFA induces a tumorspecific T-cell activation."^ An important limitation of RF tumor ablative techniques was the extent of coagidation that could be produced with a single RF application, i.e., the tumor size which coidd be practically treated in a single session.^ As many tumors show an advanced size at the time of their detectioUi either the use of multiple treatment probes, multiple treatment sessions, or both was required. A major focus of research has therefore been on the development of techniques to achieve single session, large-volume tissue necrosis in a safe and readily accomplished manner. •Corresponding Author: Johannes-Marcus Handler—Department of Internal Medicine I, University of Eriangen-Nuremberg, Ulmenweg 18, D-91054 Eriangen, Germany. Email: johannes.haensler@med1 .imed.uni-erlangen.de

Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

Tumor Ablation Using Radiofrequency Energy

191

Figure 1. CT scan of a liver colorectal carcinoma metastases before treatment.

After preliminary animal studies,^'^° radiofrequency ablation has been used for the treatment of a variety of neoplasms including: osteoid osteoma, hepatocellular carcinoma, renal cell carcinoma, bronchopulmonary carcinoma, parathyroid adenoma; hepatic and retroperitoneal metastases from a variety of primary tumors. The procedures are generally performed using thin (14-21 gauge), partially insulated electrodes which are placed under imaging guidance (CT, MRI, or ultrasound) into the tumor to be ablated (see Fig. 1). When attached to an appropriate radiofrequency generator, the RF current flows from the exposed tip of the RF needle, through the bio tissue of the human body, either to a neutral grounding pad (monopolar application) or to a second inserted RF needle (bipolar technique). Using monopolar technique, a large dispersive electrode (grounding pad) is usually placed on the patient s back, belly or thigh. A second needle electrode is used, instead of the grounding pad, for bipolar ablation. Current passing through tissue leads to ion agitation, which is converted into heat by friction. The process of cellular heating induces cellular damage. The amount of damage is a function of temperature and time. For example, a coagulation necrosis is achieved applying 70°C for less than 1 second or 50°C for about 200 seconds. Several approaches have been made to increase the diameter of coagulation necrosis achieved by RF ablation techniques. ^^ These include: (1) the use of multiprobe, hooked, and bipolar needle arrays; (2) intraparenchymal injection/infusion of saline prior to and/or during RF application; (3) internally cooled RF electrodes; and (4) algorithms for current application which maximize energy deposition but avoid tissue boiling, charring or cavitation.

Technical Development Monopolar Electrode Techniques The initial use of RF ablation techniques was for neurosurgical and cardiac applications such as the treatment of benign hyperactive neurologic foci or aberrant intracardiac conductive pathways. For these indications, precision rather than coagulation size was the key objective.

192

Hyperthermia in Cancer Treatment: A Primer

Fig^re 2. Some metastases as on Figure 1 after treatment with RFA (multiprobe arrays). the lack of coagulation size imposed by the use of conventional RF needle electrodes (i.e., maximum size of 1.6 cm) did not play any role or was even welcomed. When scientists started to assess RFA for tumor ablation it became immediately clear that coagulation size would be the limiting factor. Using basically a sharpened wire as an active electrode resulted unavoidably in small coagulation sizes due to desiccation processes. In order to enlarge coagulation zones they came up with several technical attempts, which are to be discussed in the following.

Saline Instillation during RF Ablation Several investigators have attempted to increase the RF coagulation necrosis by injecting isotonic or hypertonic saline into the tissue either before or diuing RF, or both (see Fig. 2)P'^^'^^'^ The authors showed a significant increase in coagulation size. Three potential effects of saline injection may explain the observed significant increase in volume: (1) enlargement of effective electrode surface area via augmented tissue ionicity; (2) improved tolerance of sustained high generator output due to tissue cooling, decreased tissue impedance, or both; and, (3) direct effects of heated saline which subsequendy diffuses into tissue. However, it was noted that the resulting areas of coagulation necrosis were quite irregular in shape, and the size of tissue necrosis was sometimes difficult to predict. Recent research seems to have succeeded in overcoming the problem of irregular coagulation zones and difficult size prediction by using bipolar application. Other advantages of saline infused RF are the reports that this technique can quite safely be used in the vicinity of vessels and the gallbladder.^^

Internally Cooled Electrodes Another approach in radiofrequency energy delivery was the development of internally cooled RF electrodes. These electrodes have an internal lumen through which chilled perfusate is circtdated during RF application. By cooling the electrode tip during the application of radiofrequency energy, it is possible to increase generator output, while at the same time

Tumor Ablation Using Radiofrequency Energy

193

avoiding tissue boiling and cavitation immediately adjacent to the needle tip. Thus the cooling counteracts the rise of impedance, resulting in greater tissue necrosis compared to a "hot wire" needle. The sizes obtained by a saline-infiised single needle cannot quite be reached. Therefore the coagulation size seems to be less irregular. By using generators with more power (150 Watt - 2000 mA) volumes of necroses ranging in diameter between 3-3.5 cm for a single electrode have been attained.

Pulsed RF Application Each attempt to increase the coagulation zone seeks to balance the need to apply more energy against the deleterious effects of tissue charring and cavitation which occurs when too much energy is applied too rapidly. As described above, once tissue charring occurs, circuit impedance increases to the point where energy can no longer be applied to the system. In order to explore alternative methods for delivering energy to RF electrodes, Goldberg et al investigated the possibility of applying radiofrequency in a pulsed, rather than continuous manner (manuscript in review). The goal of these experiments was to increase current density surrounding the RF electrodes, while allowing brief periods for heat to dissipate from the tissues immediately adjacent to the electrodes to prevent tissue cavitation. RF was applied in ex-vivo liver using internally cooled electrodes with peak currents of 1500-2000 mA. Peak currents were maintained for approximately 15 seconds, and alternated with periods during which RF current was reduced to 500 mA for approximately 15 seconds. These experiments demonstrated that greater tissue necrosis could be produced using this pulsed current technique compared with otherwise similar experiments in which maximal RF current was applied to the same electrodes. Remote thermometry demonstrated both more rapid increases and higher sustained tissue temperatures with the pulsed RF technique. Further work remains to be done in order to optimize the algorithms for applying RF energy to treatment electrodes, however it appears at this time that pulsed application of radiofrequency energy will increase the volume of tissue necrosis which can be achieved in a single treatment session. ^

Multiprobe Arrays Despite all the effort that has been made modifying needle applicators, the coagulation size which is now limited to 3.5 cm using a single probe is still dissatisfying, even though a substantial advancement has been made. Thus, in order to further increase the diameter of coagulation necrosis, multiple parallel placed needle electrodes were tested. ^^ Probe configuration and spacing were varied. The coagulation volume obtained with simultaneous RF application was compared with sequentially applied RF to each individual probe within the array. In all cases, the volume of coagulation necrosis obtained with simultaneous RF application was greater than that resulting from sequential application. However, spacing of probes more than 1.5 cm apart resulted in discontinuous coagulation in central areas between the probes.

Hooked Needles Another variation of the multiprobe array system are "hooked needles" or "umbrella" electrodes. ^^''^^ They consist of a thick trocar, which is placed into the target area, then multiple prongs are deployed. By enlarging the surface of the active electrode the energy density per square millimetre of the active electrode is reduced, resulting in less desiccation. Furthermore, the prongs can be gradually deployed, advancing the hooks from coagulated and desiccated tissue slowly to uncoagulated tissue, steadily increasing the coagulation zone. With these electrodes volumes of necrosis of diameters between 3.5-6 cm can be produced. The drawback is the quite exorbitant price.

Bipolar Arrays Most radiofrequency ablation has been performed using monopolar needles. Another possibility of applying RF energy is the bipolar technique, replacing the neutral grounding pad by a second needle applicator inserted at a spacing of about 2-3 cm from the other needle,'^^ or

194

Hyperthermia in Cancer Treatment: A Primer

bipolarity within the same needle. The alternating current now creates two (overlapping) coagulation zones. The electric resistance within the tissue is reduced as the distance between the bipolar needles is much shorter compared to the distance between a monopolar needle and the grounding pad. The electric field lines run much more condensed and predictable compared to the monopolar technique. Interestingly^ the shape of the coagulation changes in relation to the needle applicator. On the monopolar setting the needle is more or less in the center of the coagulation while using bipolar technique the coagulation zone mosdy forms between both needle applicators. The resulting coagulation is significandy larger compared to the monopolar technique. However, the shape of the coagulation necrosis tends to be elliptical rather than spherical. To overcome this three needles can be inserted which are sequentially connected in bipolar technique. The University of Erlangen is working with this new technique with great success. Combining Techniques Merging internal cooling electrodes and multiple probes led to an electrode with three internally cooled needles 0.5 cm apart, called cluster electrode able to obtain a coagidation necrosis of 4,7-6.0 cm in diameter. The use of cooled cluster electrodes offers the potential to significandy increase the volume of coagulation necrosis compared to a single treatment session. Another combination actually in use is a multiprobe application using bipolar technique and constant saline inftision resulting in a mean coagulation size of 7.5 cm in ex vivo trials (manuscript in review) and about 6 cm in clinical use. Also the combination of "hooked needles" with deployable prongs and constant saline infusion, is reported to result in impressive coagulation zones, occasionally exceeding 6 cm.

Treatment A carefiil clinical evaluation of the patient is necessary to establish the indication for RF ablation and should include history and fidl clinical and laboratory assessments. Selection of patients eligible for RFA is crucial for treatment success. The number of lesions to be treated (HCC, or metastases) should be limited to four. Usually the tumor size shoidd not exceed 5 cm as the local recurrence rate increases drastically in larger tumors due to incomplete treatment and the increasing presence of satellite metastases, though there are incidental reports of larger tumors being successfidly treated using RF, especially using multiple needle insertions, occlusion of tumor blood supply or the intraoperative approach.^^'^^ Liver ftinction is another important factor especially in the treatment of HCC, as Child Pugh C patients do not profit from RF ablation as they mosdy do not suffer a tumor associated death but die from impaired liver function or associated diseases. Prothrombin time ratio should be greater than 50% and a platelet count higher than 50,000/mL. In patients with hepatic metastases, the indication for RF ablation depends mainly on the histology and resection of the primary tumor. The rationale is based on the demonstration that, in certain types of malignancies, surgical removal of hepatic tumor burden improved survival.^ '^^ The best indication for percutaneous RF treatment is represented by metachronous from gastrointestinal malignancies, especially colorectal adenocarcinoma^ and endocrine tumors. In selected cases, patients with liver metastases from other primary neoplasms, such as breast adenocarcinoma, can be considered for RF ablation in the setting of a multidisciplinary approach. Treatment of lesions located in the vicinity of hepatic vessels is relatively safe as the vascular wall is protecting it from thermal damage by the cooling effect of the blood perfusion. On the other handi the same effect seems to be responsible for incomplete treatment adjacent to larger vessels due to the heat loss. Treatment of lesions adjacent to the gallbladder or the hepatic hilum has been reported to be possible,^"^ though thermal injury to the biliary tract cannot be completely excluded. Lesions located along the surface of the liver are associated with a higher risk of complications, as

Tumor Ablation Using Radiofrequency Energy

195

Figure 3. Triple needle application with saline perfused neddle applicators. adjacent structures can be damaged (a) by the needle itself and (b) by the dispersing coagulation zone. Especially in the latter case severe complications can occur, especially when the gut is involved. A perforation with consequential peritonitis may occur with a possible delay of as much as two weeks after treatment. Treatment of tumors in such difficult locations should be referred to experienced centres. RF ablation can be safely performed under general anaesthesia using endotracheal intubation and mechanical ventilation or conscious sedation. The RF ablation can be controlled by CT, MRI or US. The ablation may take place in the CT-, MRI-, US-room, or in the operation theatre. A variety of RF needles and generators are commercially available (in alphabetical order: Berchtold (now Integra), Celon, Radionics, Radiotherapeutics, RITA). Usually percutaneous RF ablation is performed under (contrast-enhanced) ultrasound guidance. In some patients CT guidance is used when sonographic targeting is not possible, with significantly increased procedure time (see Figs. 3, 4). The choice of the electrodes is based on the size of the target nodule: for lesions smaller than 2 cm, a single insertion of a single electrode suffices. Lesions measuring 2-3 cm can be treated with a single insertion of a cluster electrode, multi-array or hooked needle or with multiple insertions of a single electrode. Lesions exceeding 3 cm can only be treated using a cluster electrode, multi-array or hooked needle. Each RF application lasts 10 to 15 minutes and the total procedure time ranges from 15 minutes for small solitary lesions to 60 minutes and more for large and/or multiple ablations. B-mode and colour/power Doppler ultrasound are not reliable to assess the size and completeness of induced coagulation necrosis at the end of ablation; furthermore, additional repositioning of the electrode is usually hampered by the hyperechogenic "cloud'' appearing around the distal probe. Therefore, contrast-enhanced ultrasound performed at the presumed end of the treatment—^with the patient still under anaesthesia—enables the assessment of the extent of achieved coagulation, revealing also viable tumor portions requiring additional 27 treatment.

196

Hyperthermia in Cancer Treatment: A Primer

Figure 4. RFA schema. In our experience, patients usually experience mild to moderate pain in the right abdomen lasting for two to five days which is particularly severe in patients with subcapsular lesions. A long-acting anaesthetic drug (ropivacaine) can be injected pericapsular and subcapsular at the end of the treatment to reduce postoperative pain. Also standard analgesics do help in these cases. Fever may be present for the first 2-3 days after treatment and recedes with antipyretic drugs (e.g. acetaminophen or metamizol). No impairment has been observed in liver function tests. Liver enzymes rise regularly afi:er treatment and stay elevated for up to one month. Thus liver enzymes should not be controlled after RF on a regular basis. Initial cross-sectional examinations serve to assess completeness of treatment and provide a basis for imaging follow-up. However, given the resolution and accuracy of current imaging techniques, residual microscopic foci of malignancy at the periphery of a treated lesion can go undetected and cause local recurrence. Contrast-enhanced helical CT and MRI are used in the long-term assessment of therapeutic response, allowing a certain discrimination between ablated and residual viable tumor^ in the long term follow up, while the initial CT or MRT performed shordy after RF is often unable to differentiate tumor tissue from peripheral inflammation. Cross-sectional imaging studies obtained at 3-4 month intervals are correlated with the development of tumor marker levels (if available) to detect local or distant recurrences. CEUS has proved to be valuable, also in the follow-up, for the detection or confirmation of local recurrences and new lesions and for contrast-guided retreatment.'^^ FDG-labelled positron emission tomography (PET) offers great potential in the evaluation of treatment response for liver metastases and is synergic, perhaps superior, to cross-sectional imaging modalities in the surveillance of treated patients. According to reports areas of abnormal FDG uptake following ablative procedures represent a disease relapse or residual viable tumor following ablation with a high d^ree of sensitivity.^^

Tumor Ablation

Using Radiofrequency Energy

197

Table 1. One and five-year survival after RFA Selected Publications: Author Tumor

Number of Pat.

One-Year Survival

Five-Year Survival

Rossi 1996^^ Buscarini 2001^2 Raut 2005^3 Solbiati 2003^^ Gillams20053^

39 88 194 89 167

94% 89% 84% 62%

40% 33% 55% 22% 30%

HCC HCC HCC CRC CRC

-

HCC: hepatocellular carcinoma; CRC: liver metastases of a colorectal carcinoma

Conclusion In conclusion, RF treatment is a new, minimally invasive procedure for patients with malignancies that can achieve effective and reproducible tumor ablation with lower associated morbidity and cost than other interventions. In Table 1 we show the survival percent at 1 and five years obtained by different authors. We predict that RF ablation will become more and more important in the management of patients with primary or secondary hepatic malignancies. In support of this see Table 1 where we report the survival percent at 1 and five years obtained by different authors. Surgical resection has traditionally been considered the only potentially curative option for patients with liver cancer, but there is currently a consensus that, with continued improvements in technology and increasing clinical experience, percutaneous thermal ablation may soon challenge partial hepatectomy as the treatment of choice for patients with limited hepatic tumors. Lately published data suggests that RF ablation is far more than an electro-physical tool to generate a thermal tumor destruction, it induces a significant activation of tumor-specific T lymphocytes.

References 1. Hansler J, Harsch I, Strobel D et al. Treatment of a solitary parathyroidal adenoms with percutaneous RF ablation. Ultraschall in der Medizin 2002; 23:202-206. 2. Vogel TJ, Mack M G , Straub R et al. Percutaneous thermoablation of malignant liver tumors, Fortschr Rontgenstr 2000; 172:12-22. 3. Nordlinger B, Guiguet M, Vaillant J C et al. Surgical Resection of Colorectal Carcinoma Metastases to the Liver. Cancer 2000; 1254-1261. 4. Wissniowski Th T, Hansler J, Neureiter D et al. Activation of tumor-specific T lymphocytes by radio-frequency ablation of a VX2 hepatoma in rabbit. Cancer Res 2003; 63(19):6496-500. Erratum in: Cancer Res 2003; 63(21):7543. 5. Livraghi T, Goldberg SN, Solbiati L et al. Percutaneous radiofrequency ablation of liver metastases from breast cancer: initial experience in 24 patients. Radiology 2001; 220:145-9 6. M C Gahan JP, Browing P D , Tesluk H. Hepatic ablation using radiofrequency electrocautery. Invest Radiol 1990; 25:267-270. 7. Rossi S, Fornari F, Pathies C et al. Thermal lesion induced by 480 kHz localized current field in guinea pig and pig liver. Tumori 1990; 76:54-57. 8. Sanchez R, Van Sonnenberg E, D'Agostino H et al. Percutaneous tissue ablation by radiofrequency thermal energy as prelim to tumor ablation. Minimally Invasive Therapy 1993; 2:299-305. 9. Hansler J, Neureiter D, Wasserburger M et al. Percutaneous ultrasound-guided radio-frequency thermo-ablation (RFTA) using perfused needle applicators - improved survival inVX2 tumor model in the rabbit. Radiology 2004; 230(1): 169-174. 10. Hansler J, Neureiter D, Strobel D et al. Cellular and vascular reactions in the liver to radio-frequency thermo-ablation with wet needle applicators. Study on juvenile domestic pigs. Eur Surg Res 2002; 34:357-363.

198

Hyperthermia in Cancer Treatment: A Primer

11. Goldberg SN, Gazelle GS, Dawson SL et al. Tissue ablation with radiofrequency: effect of probe size, gauge, duration, and temperature on lesion volume. Acad Radiol 1995; 2:399-404. 12. Livrj^hi T, Goldberg SN, Monti F et al. Saline-enhanced radiofrequency tissue ablation in the treatment of liver metastases. Radiology 1997; 202:205-210. 13. Hansler J, Frieser M, Schaber S et al. Radiofrequency ablation (RFA) of hepatocellular carcinoma with a saline perfused device: a pilot study. J Vase Interv Radiol 2003; 14:575-580. 14. Hansler J, Witte A, Strobel D et al. Radiofrequency ablation with perfused needle applicators for the treatment of primary and secondary tumors of the liver, Ultraschall Med 2003; 24:27-33. 15. Goldberg SN, Gazelle GS, Solbiati L et al. Radiofrequency tissue ablation: increased lesion diameter with a perfusion electrode. Acad Radiol 1996; 3:636-644. 16. Solbiati L, Goldberg SN, lerace T et al. Hepatic metastases: percutaneous radio-frequency ablation with cooled-tip electrodes. Radiology 1997; 205:367-373. 17. Goldberg SN, Gazelle GS. Radiofrequency tissue ablation: physical principles and techniques for increasing coagulation necrosis. Hepatogastroenterology 2001; 48(38):359-67. 18. Goldberg SN, Gazelle GS, Dawson SL et al. Tissue ablation with radiofrequency using multi-probe arrays. Acad Radiol 1995; 2:670-674. 19. Leveen RF. Laser hyperthermia and radiofrequency ablation of hepatic lesions. Semin Interv Radiol 1997; 14:313-324. 20. Siperstein AE, Rogers SJ, Hansen PD et al. Laparoscopic thermal ablation of hepatic neuroendocrine tumor metastases. Siu-gery 1997; 122(6):1147-1155. 21. McGahan JP, Wei-Zhong G, Brock JM et al. Hepatic ablation using bipolar radiofrequency electrocautery. Acad Radiol 1996; 3:418-422. 22. Goldberg SN, Solbiati L, Hahn PF et al. Radiofrequency tumor ablation using a clustered electrode technique: results in animals and patients with liver metastases. Radiology 1998; 209:371-379. 23. Rossi S, Garbagnati F, Lcncioni R et al. Percutaneous radiofrequency thermal ablation of nonresecable hepatocellular carcinoma after occlusion of tumor blood supply. Radiology 2000; 217:119-126. 24. Scheele J, Stangl R, Altendorf-Hofmann A. Hepatic metastases from colorectal carcinoma: impact of surgical resection on the natural history. Br J Surg 1990; 77:1241-1246. 25. Bolton JS, Fuhrman GM. Survival after resection of multiple bilobular hepatic metastases from colorectal carcinoma. Ann Surg 2000; 231:743-51. 26. Solbiati L, Livraghi T, Goldberg SN et al. Percutaneous radiofrequency ablation of hepatic metastases from colorectal cancer: long-term results in 117 patients. Radiology 2001; 221:159-66. 27. Solbiati L, Tonolini M, Cova L et al. The role of contrast-enhanced ultrasound in the detection of focal liver lesions. European Radiology 2001; 11:15-26. 28. Rhim H, Goldberg SN, Dodd GD 3rd et al. Essential techniques for successful radio-frequency thermal ablation of malignant hepatic tumors. Radiographics 2001; 21:17-35. 29. Solbiati L, Goldberg SN, lerace T et al. Radiofrequency ablation of hepatic metastases: post procedural assessment with a US microbubble contrast agent, early experience. Radiology 1999; 211:643-9. 30. Anderson GS, Brinkmann F, Soulen MC et al. FDG positron emission tomography in the surveillance of hepatic tiunors treated with radiofrequency ablation. Clin Nucl Med 2003; 28:192-7. 31. Rossi S, Di Stasi M, Buscarini E et al. Percutaneous RF interstitial thermal ablation in the treatment of hepatic cancer. AJR Am J Roentgenol 1996; l67(3):759-68. 32. Buscarini L, Buscarini E, Di Stasi M et al. Percutaneous radiofrequency ablation of small hepatocellular carcinoma: long-term results. Eur Radiol 2001; 11(6):914-21. 33. Raut CP, Izzo F, Marra P et al. Significant long-term survival after radiofrequency ablation of unresectable hepatocellular carcinoma in patients with cirrhosis. Ann Surg Oncol 2005; 12(8):616-28. 34. Sobiati L. Euroson 2003, Lecture. 35. Gillams AR, Lees WR. Radio-frequency ablation of colorectal liver metastases in 167 patients. Eur Radiol 2004; l4(12):2261-7.

CHAPTER 15

Cytoreduction, Peritonectomy and Hyperthermic Antiblastic Peritoneal Perfusion for die Treatment of Peritoneal Qrcinomatosis Michele De Simone* and Marco Vaira Generalities and Indications

P

eritoneal carcinomatosis may present along with gastrointestinal or female genital tumors (including pseudomyxoma peritonei, a variable malignancy myxoid tumor, arising from the appendix). It is also the common way of presentation of primitive peritoneal tumors (like peritoneal mesothelioma). Peritoneal carcinomatosis has been considered nearly impossible to treat with surgery until a few years ago. Moreover, the results obtained with systemic chemotherapy were poor. In the *80s, some authors developed and improved a new combined technique to manage peritoneal carcinomatosis, consisting in cytoreduction of neoplastic lesions, peritonectomy (removal of peritoneum macroscopically affected from tumor) and hyperthermic antiblastic peritoneal perfusion (HAPP). Carcinomatosis nodes usually affects peritoneum in preferential sites as the pelvis, the ileum-caecal angle, the right diaphragm and retrohaepatic space, the lesser omentum, the left diaphragm and paracolic spaces. In fact, in these regions the peristalsis is less effective, there are the points of peritoneal fluid absorbtion and, finally, these areas are particularly anfractuous, with virtual spaces, so the circulation of fluids is slow and neoplastic cells may easily lodge. At least, cells may deposit under action of the force of gravity. For these reasons, peritonectomy is particularly indicated in these regions. The rationale of combining hyperthermia and chemotherapy has been described in previous chapters. The peritoneal cavity can be considered a "pharmacological sanctuary" for the presence of the peritoneal-plasmatic barrier, that is independent from mesothelial layer, and preserves the leakage to systemic circulation of high molecular weight drugs as cisplatinum, mitomycin C, doxorubicin, oxaliplatinum. So it*s possible the use of an high concentration of cytostatic drugs into the tumor area, with mild side-effects. The HAPP has maximal efficacy when all the macroscopic disease has been removed with cytoreduction and peritonectomy. There is loss of efficacy on nodules bigger than 3 mm, because this is the maximal depth that the drugs can reach, even in hyperthermic condition. The techniques described to perform the HAPP are three: "closed", "open" and "semiclosed" one. In "closed" technique, the perfusion is performed when the operation is completed and •Corresponding Author: Michele De Simone—Department of Surgical Oncology, "S. Giuseppe'' Hospital, Empoli, Florence, Italy. Email: [email protected] / m.desimone@usl11 .tos.it

Hyperthermia in Cancer Treatment: A Primer^ edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

200

Hyperthermia in Cancer Treatment: A Primer

Figure 1. Semiclosed HAPP: the skin is hanged widi Backaus forceps and self-blocking strings to the self-retainig retraaor. The surgeon s hand mixes the perfusate through the PVC sheetfixedto the edge of the incision, in order to distribute the drug and guarantee the homogeneous distribution of temperature. the abdomen is closed, using tubes placed during the surgical time. The most important problem described with this technique is the difficulty to reach homogeneous distribution of temperature and drugs into the abdominal cavity. Moreover, a visual and manual control of the abdomen is not possible during the perfusion. The "open" technique has been described by P.H. Sugarbaker, it consists in perform the perfusion when the abdomen is still open, hanging all the abdominal wound to an autostatic retraaor placed upon the incisional area (Coliseum technique). This way, during perfusion, the surgeon may check the abdominal cavity, and stir the peritoneal solution, to obtain an homogeneous distribution of drugs and temperature. The criticism is related both to the large loss of temperature from the wide abdominal incision, and to the possibility of leakage of drugs from the abdomen. In the original "semiclosed" technique, the abdominal wall is partially closed and hanged to the autostatic retractor, only the central part of the incision remains open (Fig. 1). Through this small opening, the surgeon can mix the perfusate solution and check what happens in the abdomen. Those contrivances consent both the homogeneous distribution of temperature and drugs and low risk of drug leakage from the abdominal cavity. In our opinion, this kind of procedure collects the advantages of the other two techniques and reduces their drawbacks. Briefly, the semiclosed technique permits: (1) manual distribution of temperature and perfusate; (2) rapid reaching of ideal temperature for HAPP; (3) to perform anastomosis after HAPP, so all the bowel surfaces are exposed to drugs; (4) reduction of drugs leakage from wound margins, with major safety for the operatory room-staff.

Treatment ofPeritoneal Carcinomatosis

201

Figure 2. The access to the peritoneal cavity is lateral and not on the midline, in order to remove the epigastric peritoneum and the eventual previous surgical scars. In the picture the previous scar is kept with the forceps.

Surgical Techniques Peritoneal perfusion is really effective only if preceded by aggressive cytoreductive surgery. So surgery becomes mandatory in treatment of peritoneal carcinomatosis. Some techniques of "centripetal'* aggression of tumor have been codified. They are known with the name of peritonectomy, and they allow to remove both visceral lesions and the peritoneum infiltrated by the carcinosis. Six different techniques of peritonectomy are described: (1) Epigastric peritonectomy consists in the "en bloc" removal of previous scar, round and falciform haepatic ligaments (Fig. 2); (2) Central peritonectomy consists in greater omentectomy with removal of the superficial layer of transverse mesocolon (Fig. 3). Sometimes it's necessary the removal of the spleen "en bloc" with the omentum, associated to peritoneal stripping of right and left abdominal wall; (3) Right diaphragmatic peritonectomy (Fig. 4), with liver mobilization and removal of the right diaphragmatic peritoneum and the peritoneum covering the right adrenal gland, right kidney and duodenum (Fig. 5); (4) Left diaphragmatic peritonectomy, with removal of peritoneum covering the left adrenal gland and left kidney (Fig. 6); (5) Lesser omentum peritonecomy, consisting in cholecistectomy, peritoneal removal from porta haepatis, removal of lesser omentum and of the tumor near the caudate lobe (Fig. 7); Pelvic peritonectomy with "en bloc" removal of rectum and sigmoid colon, uterus and adnexa, and removal of vescical peritoneum (Fig. 8). Peritonectomies are performed when an highly aggressive surgery is indicated (for example in the Pseudomyxoma Peritonei, a tumor characterized by large diffusion of mucinous carcinosis, with low malignancy rate). Peritonectomies are varying combined with resections of viscera involved in the neoplastic invasion. Resection of rectum and sigmoid colon, hystero-oophorectomy, splenectomy, omentectomy and colecistectomy are common procedures in the treatment of peritoneal

202

Hyperthermia in Cancer Treatment: A Primer

Figure 3. When omentectomy in performed, it is possible to see the greater curvature after peritonectomy. In the low part of the piaure the cx)lon is held with the forceps.

Figure 4. Right diaphragmatic peritonectomy.

Treatment ofPeritoneal Carcinomatosis

203

Figure 5. Right diaphragmatic peritonectomy completed. It is possible to see the diaphragmatic muscle, the lower surface of the liver, and the right anterolateral part of caval vein after peritonectomy.

Figure 6. Diaphragmatic peritoneum with massive carcinosis from pseudomyxoma peritonei. The peritoneum is removed from the posterior fascia of the rectiun muscle and it is held with forceps.

204

Figure 7. Lesser omentum peritoneaomy is completed.

Figure 8. Pelvic peritoneaomy is completed.

Hyperthermia in Cancer Treatment: A Primer

Treatment ofPeritoneal Carcinomatosis

OUTFLOW TUBES ^

205

OUTFLOW ^ TUBE INFLOW TUBES

Figure 9. Scheme of position of the tubes in the abdominal cavity to perform the HAPP. carcinomatosis. Total gastrectomy is rarely performed. The "en bloc" visceral resection with adjacent peritoneum maybe required, because of massive neoplastic infiltration. The approach is completely extra-peritoneal (the routine approach for the pelvis).

Semiclosed HAPP Technique When the surgical time is completed, the HAPP is performed. The "semi-closed" method consists in placing five drains in the abdominal cavity: the inflow drains (two) and have multiple holes. They are Y-shaped and present 4 diffusion lines for the homogeneous distribution of drugs into the abdominal cavity (one subdiaphragmatic branch, one sovramesocolic branch, one among the ileal loops, one in the pelvis). Three outflow tubes are placed respectively in the pelvis and in the subdiaphragmatic spaces (Fig. 9). Backaus forceps are used to close the cranial and caudal portion of abdominal wound. The skin is then suspended to self-retainig retractor (placed at more or less 15 cm from abdomen) by plastic self-blocking strings. This kind of placement creates the virtual cavity needed to perform the HAPP The central portion of the wound is suspended to the retractor too and covered with a PVC sheet that presents a hole in the middle (Fig. 1). The drains are connected to a perfusion system formed by two pumps and a heat exchanger to heat the perfusion liquid. The two pumps (inflow and outflow), are connected through a reservoir, so it s possible to reach a continuous circulation of the perfusate at the speed of 1 L/min. The pumps are checked by a computed system, that controls the flows and the heat exchanger. Three intraperitoneal temperatures are checked by probes, moreover, it's important the measure of inflow, outflow and patient s esophageal temperature. Temperature is a critical factor; when it's higher than 44°C, the recovery of intestinal anastomosis can be delayed, and generally the risk of postoperative intestinal perforations is increased. On the

206

Hyperthermia in Cancer Treatment: A Primer

other hand, when temperatures are too low, the effectiveness of drugs in strongly decreased. The amount of the circulating perfusate (solution for peritoneal dialysis) is calculated considering the patient body surface. The drugs employed are different and choosen on neoplasias hystological type. Currendy, the drugs of choice are CDDP, Mitomycin-C, Doxorubicin, Oxaliplatinum, differendy combined and associated. During perfusion, the surgeon mixes the perfusate using his hand through the hole in the PVC sheet. When intraperitoneal temperature of 41.5°C is reached, the drugs are added to the circuit and the HAPP is performed for 60 minutes. During the perfusion, an iced helmet is placed on patient s head, and cold Hquids are infused i.v., to prevent systemic hyperthermia. A diuresis higher than 120 ml/lOminutes is suitable to prevent renal complications.

Clinical Experience and Results From October 1995 to January 2005 we performed 235 operations for peritoneal carcinomatosis. In 70 cases we only performed surgery (28 explorative laparatomies and 42 debulking and peritonectomies without HAPP); in 165 cases we performed cytoreduction and HAPP The peritoneal carcinomatosis arose from: ovarian cancer in 44 cases (two protocols have been activated for ovarian cancer: in 19 cases the disease was at the first relapse, with carcinomatosis, after one line of chemotherapy, with a disease free interval of 3 months at least. In 25 cases the disease was plurirelapsing); colorectal cancer in 32 cases; pseudomyxoma peritonei (PMP) in 37 cases; peritoneal mesothelioma in 26 cases; abdominal sarcoma in 14 cases; gastric cancer in 6 cases; other cancers in 6 cases (endometrial carcinoma, small cell desmoplastic tumor, etc.). In the patients who underwent cytoreduction + HAPP, morbidity was 18,8% (31/165) and perioperative mortality was 4.8% (8/165). Most common complications were sepsis, postoperative haemorrhage (even after 72/96 hours from the operations), dehiscence of intestinal anastomosis, mild acute renal failure, temporary medullary aplasia, A.R.D.S.

Classification ofResults per Pathology PMP: all complete cytoreduction; 32 patients are alive without evidence of disease (the longest follow-up is 8 years), 4 patients are alive with disease, 1 patient died of disease with relapse after only 6 months from operation. Colonic Cancer: 23 patients have been treated with a protocol using CDDP and mitomycin-C, without very satisfactory results, with a median survival time of 14.5 months; the last 9 patients have been treated with a new protocol (Elias is the author proposing this treatment) providing a careful selection of patients and the employment i.v. of 5-FU and folinic acid, followed by HAPP with oxaliplatinum. Although the results are still too recent for a statistical evaluation, the first patient has been operated more than 2 years ago and he is not yet presenting relapse of disease. Ovarian Cancer: in 19 patients at the first relapse after 1 line of chemotherapy and DFI > 3 months, the median survival time is 726 days. For the 25 patients with plurirelapsing disease, the median survival time is definitely shorter: 427 days. Mesothelioma: in the patients who underwent cytoreduction and HAPP morbidity was 21%; 3 patients presented ARDS. The median survival time is 40 months. Sarcoma: the residts are not statistically valuable. Gastric Carcinoma: results in our casuistry were very poor, with a median survival time of about 6 months. Those results, combined to aggressiveness of the treatment brought us to quit this technique for patients with peritoneal carcinomatosis from gastric cancer. Anyway there are some Authors reporting good residts in the treatment with neo-adjuvant intent of gastric cancer with serosal invasion, without evident peritoneal carcinomatosis at the moment of operation.

Treatment of Peritoneal Carcinomatosis

207

References 1. Averbach A M , Sugarbaker PH. Methodologic considerations in treatment using intraperitoneal chemotherapy. Cancer Treat Res 1996; 82:289-309. 2. Cavaliere F, Perri P, Di Filippo F et al. Treatment of peritoneal carcinomatosis with intent to cure. J Surg Oncol 2000; 7 4 ( l ) : 4 l - 4 . 3. De Simone M, Barone R, Vaira M et al. Semi-closed hyperthermic-antiblastic peritoneal perfusion (HAPP) in the treatment of peritoneal carcinosis. J Surg Oncol 2003; 82(2): 138-40. 4. Elias D, Antoun S, Raynard B et al. Treatment of peritoneal carcinomatosis using complete excision and intraperitoneal chemohyperthermia. A phase I-II study defining the best technical procedures] Chirurgie 1999; 124(4):380-9. 5. Gilly FN, Carry PY, Sayag AC et al. Regional chemotherapy (with mitomycin C) and intra-operative hyperthermia for digestive cancers with peritoneal carcinomatosis. Hepatogastroenterology 1994; 4l(2):124-9. 6. Glehen O , Sugarbaker PH, Elias D et al. Cytoreductive surgery combined with perioperative intraperitoneal chemotherapy for the management of peritoneal carcinomatosis from colorectal cancer. A multi-institutional study for 506 patients. J Clin Oncol 2004; 22:3284 7. Loggie BW, Fleming RA, McQuellon RP et al. Cytoreductive surgery with intraperitoneal hyperthermic chemotherapy for disseminated peritoneal cancer of gastrointestinal origin. Am Surg 2000 Jun;66(6):561-8. 8. Sugarbaker PH. Peritonectomy procedures. Ann Surg 1995; 221(l):29-42. 9. Sugarbaker P H , Ronnett BM, Archer A et al. Pseudomyxoma peritonei syndrome. Adv Surg 1996; 30:233-80. 10. Sugarbaker P H , Chang D. Results of treatment of 385 patients with peritoneal surface spread of appendiceal malignancy. Ann Surg Oncol 1999; 6(8):727-31.

CHAPTER 16

Hyperthermic Isolated limb Perfusion Michele De Simone* and Marco Vaira Abstract

I

n this overview we describe surgical procedures and hyperdierniic-isolated limb perfusion techniques for the treatment of in transit metastases from melanoma and sarcoma of the Umbs. We also briefly analyze the rationale of limb perfusion. The procedures are divided, for teaching piuposes, in three phases (surgical procedure, perfiision time, reconstructive phase). Finally we present a brief summary of our results obtained in the treatment of sarcoma and melanoma. We have performed 91 limb perftisions on 86 patients (5 patients have been treated twice). We obtained an objective response on 93.6% of patients with in-transit metastases from melanoma (45.5% presented a complete response and 48.1% a partial response). About sarcoma of limbs, we reached an objective response on 80% of patients. Side effects have been mild and not life threatening (e.g., edema of the limb, leukopenia and a compartment syndrome)

Introduction and Indications of Limb Perfusion The principles underlying the synergistic effects of cytostatic drugs and hyperthermia have been extensively described in previous chapters; briefly, we must emphasize that isolated limb perfusion offers two main pharmacokinetic advantages compared to systemic neoplastic treatment: (a) high drug concentration in the tumor area and (b) low systemic toxicity. Those important eff^ects have been clinically applied initially for in-transit metastases from melanoma. In this condition, melanoma disseminates through the whole limb from the initial site to the regional lymph nodes. This situation is not easily managed with conventional surgical or chemotherapeutic treatments but is still a locally-advanced disease. Isolated limb perfusion achieves good survival and quality of life results without the toxic effects of systemic chemotherapy. Limb perfusion consists of three phases: 1. Surgical ablative phase (node dissection and vessel preparation) 2. Perfusion phase 3. Reconstructive phase Basically, the treatment consists in isolating the limb from systemic circulation and perform limb perfusion with cytostatic drugs for 60 minutes with extracorporeal circulation (ECC) (Fig. 1). At the beginning of the perfusion phase, blood in the limb is heated; when the tumor site and whole limb reach an homogeneous temperature of 41.3-41.5°C, the drugs are injected into the perfusion circuit at high concentration with low side effects for the rest of the body. The pharmacological benefit is linked not only to high concentration but also to a continuous circulation of drugs in the limb that increases the cytostatic uptake from tumor. As previously *Corresponding Author: Michele De Simone—Department of Surgical Oncology "S. Giuseppe" Hospital, Empoli, Florence, Italy. Email: [email protected] / [email protected]

Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

Hyperthermic Isolated Limb Perfusion

209

Esophageal temperaturej detector T* = 36*-37n

\

i I

J ' i

IVescical temperature detector T* = 36° 37/

-2.

Venous line

•

Anticancer Drug

A

Abductors J ' ' = m a x 41,8\

^

pump (40-80 mi/l Vol. arto)

Oxygen 95% Heat

^^Ox^Q^n^ior

exchanger T* » m a x '

mm Thermostatic Sink with pump

Figure 1. Circuit scheme of isolated limb perfusion. described, hyperdiermia interacts with anticancer drugs at multiple levels. O n e direct effect is selective damage of tumor cells due to the decreased adaptability of tumor vessels to elevated tempeerature. This effect manifests as increased heat-entrapment in tumor areas compared to normal tissue, rendering hyperthermia a selective therapy. Another effect is related to increased permeability of tumor-cell membrane, induced by heat, that allows accumulation of drugs inside cancer cells. T h e intracellular concentration of drugs combined with heat shock causes impairment of D N A repair and disrupts D N A duplication. This action is especcially marked in tumor cells which have a high proliferative index. Hyperthermic isolated limb perfusion is indicated in following disease stages: • treatment of in transit metastases from melanoma (Figs. 2, 3, 4) • palliative treatment for melanoma when residual life quality is the target. • curative treatment of soft tissue sarcomas • limb-saving procedure and • neoadiuvant treatment to reduce bulky tumors and allow successive conservative surgery.

Surgical Procedure Ablative Phase Lower Limbs Iliac access is preferred for lower limbs. This access allows both iliac lymph node dissection and inguinal lymph no perfusion. Iliac node dissection is mandatory because in a non-negligible percentage of patients (30% in some studies) iliac-obturator lymph node disease can be present although the inguinal lymph nodes appear uninvolved. In selected cases (second perfusion or previous iliac lymphadenectomy), femoral access has to be utilizedd. Iliac access requires external iliac and obturator lymph node dissection with isolation of internal and external iliac vessels. Illiac perfusion, when groin lymphatic metastases are present, requires inguinal-crural and iliac-obturator nodes en-bloc dissection. In this casee, wide incision must be d o n e with section and reconstruction of the inguinal ligament. This perfusion access is accompanied by high morbidity (diastasis of w o u n d margins, lymphorrhhea, w o u n d

210

Hyperthermia in Cancer Treatment: A Primer

Figure 2. Lower limb after limb perfusion.

Figure 3. Same limb of Figure 2. Thirty days after perftision.

Figure 4. Same limb of Figure 2. Sixty days after perftision.

Hyperthermic Isolated Limb Perfusion

211

Figure 5. Iliac vessels after lymph node dissection.

infection). Recently, to reduce this morbidity, we combine laparoscopic iliac obturator node dissection with an "open" crural dissection. After this procedure we cannulate the femoral vessels for perfusion. Going back to iliac access, when iliac vessels are isolated, we proceed towards the femoral vessels, with isolation, ligature and division of circumflex, epigastric and other main collateral vessels. This manoeuvre reduces drug leakage into the systemic circulation. In fact leakage is responsible both for systemic toxicity and ineffective treatment due to systemic hemodilution of the drug. After the isolation of collateral branches of iliac vessels, radical iliac vessel node dissection is performed to the common iliac biftircation (Fig. 5). Then follows isolation on elastic drain of the obturator nerve to preserve it from iatrogenic injury, and obturator fossa dissection (Figs. 6,7,8). In our experience, lymphadenectomy is easier if performed with an electrosurgical knife with bipolar forceps; moreover, the application of metal clips assures hemostasis and postoperative control of lymphorrhea due to lymph node dissection. When iliac access is not feasable or indicated, femoral access is done. We perform a longitudinal elliptical incision 2 cm medial and 2 cm caudal to the iliac anterior superior spine, that extends caudally to the apex of Scarpa's triangle. This is followed by a subcutaneous flap 1 cm in thickness extending to the muscular sheath which is removed en bloc with subcutaneous flap, lymph nodes and perivascular tissue. During preparation of the sheath and the lymphadenectomy, the saphenous vein and the deep circumflex and epigastric vessels are tied and dissected. Upper Limbs The patient is positioned with limbs abducted; we incise from the sternoclavicular joint to the insertion of the large pectoral muscle on the humerus. Then we lance the large pectoral sheath and separate the muscle fibers to reach the cephalic vein, that is tied at its origin. The following step is division of the small pectoral muscle. After this we isolate and tie collateral vessels of the axillary artery and vein to reduce drug leakage. When vessels are isolated, the node dissection is performed.

212

Hyperthermia in Cancer Treatment: A Primer

Figure 6. Obturator nerve isolated on elastic string (blue string).

Figure 7. Obturator fossa nodes dissection.

Figure 8. Obturator nodes dissection completed.

Hyperthermic Isolated Limb Perfusion

213

Figure 9. Operating room, limb heater linked to double-sheet drape.

Perfusion Time During the preparatory phase, different temperature probes are positioned on the limb (skin, tumor site, adductor muscles, pretibial muscles) for monitoring the temperature of whole limb during perfusion. To keep temperature uniformity along the treated region, the limb is covered with a double-sheet drape linked to an air-heater which is activated at the beginning of the operation (Fig. 9). When the iliac-obturator dissection has been completed, we isolate on a tourniquet the internal and external iliac vessels. If internal vein isolation is difficult or dangerous, we isolate the common iliac vein. When vessels are completely isolated and prepared for cannulation, we heparinize thee patient (300 i.u. of heparin/kg); then we clamp external and internal iliac vessels (or common iliac vein) with tourniquets. On external vessels we perform an incision and place vascular catheters into iliac vessels. The position of catheters is very important: the tips of the catheters must be placed before the bifurcation of common femoral vessels, just under the inguinal ligament (Fig. 10). In femoral access, the femoral vessels are cannulated with the most cranial incision that can be done. The catheters are linked to the extracorporeal circulation system, consisting of an oxygenator, a heat exchanger, and a peristaltic pump. The extracorporeal circulation starts at a minimum flow that gradually increases until it reaches flow appropriate to limb volume (usually a flow between 50 and 70 ml/liter of limb). After some minutes of circulation, the superficial blood circulation of the limb is blocked by a rubber drain twisted around the root of the limb and anchored to the operating table (Fig. 11). When circulation is started, an important target is a constant level of solution inside the oxygenator; this level is directly related to a balance between systemic and isolated limb circulation. This is accomplished by equilibrating arterial and venous flow. Venous flow is determined by gravity so the critical flow that we can regulate is arterialflow.All these contrivances, combined with ligation of collateral vessels and proper balance between systemic circulation and limb flow of perfusion, limits drug leakage. Fieat comes both from the heat exchanger and the double-sheet drape wrapped around the limb until an homogeneous temperature of 41.5°C is reached. When we achieve optimal temperature and perfusion flow balance, we

214

Hyperthermia in Cancer Treatment: A Primer

Figure 10. Cannulation of iliac vessels.

Figure 11. During perfusion, rubber tourniquet on the root of the limb. inject the drug (melphalan 10 mg/liter tissues for lower limb and 12 mg/liter for upper limb) into the circuit. The drug dose is divided in three parts administered in successive periods of time with an interval of 5 minutes. Perfusion is maintained for 60 minutes after drug injection. Leakage Monitoring During hyperthermic isolated limb perftision, the control of drug leakage into the systemic circidation is of vital importance in order to reduce systemic toxicity and maintain maximum drug concentration in the tumor. We measure leakage infusing in the ECC circuit with human serum albumin labelled with ^^'"Tc. The ^^"^Tc-labelled albumin is traced on a Geiger-Midler counter placed over the liver. Systemic leakage is quantitatively expressed as a percentage, whereby a 100% leak is considered to give a homogeneous distribution of the tracer in the body (Fig. 12). We consider a value of leakage < 5% acceptable. It is important not to overcome a threshold value of 10%.

Hyperthermic Isolated Limb Perfusion

215

Figure 12. Geiger-Muller counter for leakage measurement. The detection probe is positioned on the projection of the liver on the abdominal wall.

Reconstruction Time Once the perfusion phase is finished, the vessels are uncannulated and sutured. Heparin is neutralized by protamine solfate. When hemostasis is achiieved and, two drains are placed (Fig. 13). In iliac access, one drain is positioned near the vessels that underwent lymphadenectomy and the other in the subcutaneous space. In femoral access, transposition of the sartorius muscle on the femoral vessels is performed.This procedure is accomplished by separating the cranial head of the muscle from its bone insertion and transposing it to adjacent vessels. The transposed muscle is fixed on the inguinal ligament with nonresorbable suture. This surgical procedure supports subcutaneous tissu. It prevents dehiscence of the femoral vessels due to infection of the surgical wound and, at the very least, prevents inguinal hernia that may follow this kind of operation.

Clinical Experiences and Results From 1995 to December 2004, 79 patients with metastases from melanoma and 12 with sarcoma were treated with 91 limb perfusions; in fact, 5 patients with melanoma have been treated twice. The main features of the patients were: • Median age 65 years (35-81 range) • 69 patients were at stage III (26 on stage Ilia; 43 on stage IIIAB) • 10 patients at stage rV • 76 lower limbs; 3 upper limbs • All patients, except 5, were previously treated with surgical removal of metastases and/or with lymph node dissection • In 22 cases, more than 10 in-transit metastases were present. The follow-up and statistical elaboration of results confirmed the eff^ectiveness of isolated limb perfusion. N o perioperative mortality was observed.

216

Hyperthermia in Cancer Treatment: A Primer

Figure 13. At the end of perfusion: iliac vessels sutured. Major morbidity as follows: • Edema of perfused limb (not only a complication, but an indirect sign of well-done perfusion), more or less, in each patient treated • 8 cases of transitory leukopenia and platelet disorder • 1 case of retroperitoneal hemorrhage treated conservatively Late complications inccluded a wound diastasis and a chronic ischemia. Our results are summarized in Table 1 and are comparable to those reported in the literature. The median survival has been evaluated in 48 months for patients at stage III and in 43 months for stage III AB. It is remarkable that even for patients in stages III and IV that demonstrated a partial response, we obtained adequate control of bulky disease and good quality of life until the death which generally occurred from systemic spread. Of those patients with sarcomas, 12 patients have been treated with isolated limb perfusion. There was no perioperative mortality and the side effects were: • edema of the limb • leukopenia (1 case) • a compression syndrome treated with decompressive fasciotomy.

Table 1. Clinical results in melanoma A) Complete response B) Partial response (Lesions Necrosis > 50%) C) Objective response (A+B)

36 (45.5%) 38(48.1%) 74 (93.6%)

Hyperthermic Isolated Limb Perfusion

217

For this kind of disease we obtained an objective response in 80% of treated cases, and we must emphasize that for almost all of them, limb salvage was possible, with improvement of quality of life.

References 1. Cavaliere R, Di Filippo F, Cavaliere F et al. Medical radiology, thermoradiotherapy and thermochemotherapy. In: Seegenschmiedt M H , ed. Clinical practice of hyperthermic extremity perfusion in combination with radiotherapy and chemotherapy. Springer-Verlag 1996; 2:323-345. 2. Krementz ET. Regional perfusion. Current sophistication, what next? Cancer 1986; 57:416. 3. SchrafFordt H , Koops BBR, Kroon FJ et al. Management of local recurrence, satellites and in transit metastases of the limbs with isolation perfusion. In: Lejeune FJ, ed. Malignant Melanoma. Pub McGraw-Hill 1994; 221-231 4. Hahn G C . Hyperthermia and cancer. Plenum Press, New York: 1982 5. McBride M , M c M u r t r e y MJ, Copeland EM et al. Regional chemotherapy by isolation-perfusion. Int Adv Surg Oncol 1978; 1:1-9. 6. Autier P. Epidemiology of Melanoma. In: Lejeune FJ, ed. Malignant Melanoma. Pub McGraw-Hill 1994; 1-7 7. Fraker DL, Coit D G . Isolated perfusion of extremity tumor. In: Lotze M T , ed. Regional Therapy of Advanced Cancer. Philadelphia: Pub. Lippincott-Raven, 1997; 333-349 8. Cavaliere F, Di Filippo, CavaUere F et al. Clinical practice of hyperthermic extremity perfusion in combination with radiotherapy and chemotherapy. In: Seegenschmiedt M H , ed. Medical Radiology, Thermoradiotherapy and Thermochemotherapy. Springer-Verlag, 1996; 2:323-345. 9. Clark AJ, Grabs PG, Parsons et al. Melphalan uptake, hyperthermic synergism and drug resistance in a human cell culture model for the isolated limb perfusion of melanoma. Melanoma Res 1994; 4(6):365-370. 10. Di Fihppo, Calabro A, GiannareUi D et al. Prognostic variables in recurrent limb melanoma treated with hyperthermic antiblastic perfusion. Cancer 1989; 63:2551-2561. 11. Bowers J, Copeland EM. Surgical limb perfusion for extremity melanoma. Surg Oncol 1994; 3:91-102.

CHAPTER 17

Intracavitary Hyperthermic Perfiision E. Dieter Hager* Introduction

D

irect intraperitoneal (IP) installation of anticancer agents for the treatment of patients with peritoneal carcinomatosis or sarcomatosis has pharmacological advantages compared to intravenous systemic therapy in terms of local drug concentrations (Table 1). The ratio of antineoplastic agent in the dialysate compared to the levels in the blood is 18-1,000 times greater, depending on the drug (Table 2).^ In a pharmacological study comparing intravenous versus intraperitoneal infusion of carboplatin the 24 hr platinum AUG in the peritoneal cavity was 280 times higher when carboplatin was administered with IP route.^ In addition, pharmacological studies showed, that IP infusion is a pharmacologically more reasonable route for systemic chemotherapy of carboplatin. The peritoneal space/plasma barrier provides dose-intensive therapy. The increase of local concentrations of antineoplastic drugs leads to improved response rates and significant increased recurrence-free survival in patients with peritoneal carcinosis. Recent studies showed in woman with stage III epithelial ovarian cancer, that integrated intraperitoneal chemotherapy into front-line therapy reported promising results with median survival times of 49-63 months and 2-year survival rates of 70-80%,^ compared to median survival of 41-52 months and 2-year survival of 65-70% in women who undergone optimal debulking surgery (<1 cm residual) followed by intravenous chemotherapy only. Additional modifications to increase response rates and reduce chemotherapy resistance may improve these results. Hyperthermia in combination with chemotherapy may play a significant role in improving the outcome (Table 3). Direa cytostatic and cytotoxic effects of hyperthermia starts at 40-41 °C and at temperatures above 43°C exponential time and temperature-dependent inactivation is observed. The biophysical and biochemical effects of hyperthermia are partially understood, and include denaturation of macromolecules, increased neovasailar permeabiUty, and perturbation of multmolecular complexes such as receptors. Induction of apoptosis and antiangiogenic effeas are observed. In addition, synergistic antineoplastic effects between hyperthermia and some anticancer agents have been experimentally and clinically demonstrated. The increase is explained by an increase in cell membrane permeability, altered active drug transport and altered cell metabolism. '^ The penetration of antineoplastic agents into tissues is improved by thermal processes and drug resistance can be reduced by heat as long as heat shock proteins are not involved. The administration of mitomycin to hypoxic tumor cells at 43 °C results in a 40-fold increase in cell killing compared with 37°C.^ Thermal enhancement has been demonstrated also for platinum compounds like cisplatin, carboplatin, and oxaliplatin.^'^ In addition, both in animal experiments and humans, cisplatin has been shown to penetrate much deeper under *E. Dieter Hager—Department of Hyperthermia, BioMed-Klinik GmbH, Tischberger Str. 5-8 D-76887 Bad Bergzabern, Germany. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer^ edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

Intracavitary Hyperthermic Perfusion

219

Table /. Rationale for intraperitoneal therapy with antineoplastic agents of patients with peritoneal carcinomatosis or sarcomatosis 1. Spread of epithelial and stromal type of cancer is restricted to the abdominal cavitiy 2. High risk due to peritoneal carcinomatosis or sarcomatosis (e.g., ascites) 3. High concentrations of antineoplastic drugs within the peritoneal cavity following i.p. drug administration compared to i.v. application. 4. Increased exposure time to antineoplastic agents due to the peritoneal space/plasma barrier. 5. Reduction of toxicity following i.p. application owing to lower systemic oncentrations of cytotoxic agents. 6. Systemic effects especially on regional lymph nodes and liver metastases.

Table 2, Intraperitoneal/plasma concentrations of antineoplastic agents^ Agent Carboplatinum Cisplatinum Mitomycin C 5-Fluorouracil Doxorubicin Mitoxantrone Paclitaxel

i.p./Plasma-Relation 18 20 71 298 474 620 1,000

Table 3. Rationale for intraperitoneal hyperthermic perfusion chemotherapy 1. 2. 3. 4. 5.

Induction of apoptosis (direct heat-related cytotoxic effect) Induction of antioangiogenic effects Synergistic interactions of heat with selected antineoplastic agents Reduction of drug resistance (due to increased membrane permeability and metabolism) Immunologic effects on cellular effector cells (emigration, migration, chemotaxis, and activation) 6. induction of cytokines (IL-1, -2, -6, -12, TNF-a, NO, CSFs) and chemokines 7. Induction of heat-shock-proteins with presentation of tumor-associated antigens 8. Modulation of cell adhesion molecules

hyperthermic conditions. ' Increased response rates and survival may therefore be expected from intraperitoneal hyperthermic perfusion chemotherapy (IPHC) compared to 37°C. Other drugs that enhance the antineoplastic effect of chemotherapeutic drugs include bleomycin/^ doxorubicin,^^ carboplatin/ irinotecan,^'^ ifosfamid,^^ gemcitabine and vinblastine.^^ Immunologic effects on cellular effector cells and the secretion of cytokines, induced by heat, may also contribute to synergism. The antitumoral effects of tumor necrosis factor a and IL-la^^ is enhanced by hyperthermia. Intraperitoneal hyperthermic perfusion chemotherapy may be performed: (a) perioperatively prior to surgery (induction therapy), during or after cytoreductive surgery, or (b) postoperatively or after relapse as a percutaneous (closed) intraperitoneal perfusion therapy. The latter can be performed repeatedly (long-term IPHC). Indications for the intraperitoneal hyperthermic

220

Hyperthermia in Cancer Treatment: A Primer

Table 4. Indications for intraperitoneal hyperthermic perfusion chemotherapy Primary

Secondary

-

Mesothelioma Sarcoma Desmoplastic tumors Ovarian cancer Stomac cancer Gastrointestinal cancer Sarcomatosis Pseudomyxoma peritonei

perfusion chemotherapy are listed in Table 4. Feasibility, tolerability, pharmacokinetics, and efficacy of intraperitoneal hyperthermic perfusion with chemotherapy was studied in phase I-III clinical trials.

Clinical Experience Methods and Technique The first intraperitoneal application of hyperthermia by perfusion was first described in a clinical situation in 1980 by Spratt et al.^^ Since that time, a growing number of centres in the USA, Europe and Asia have reported use of the technique, mostly from surgical departments (Table 5). The technique of closed or partially closed continuous hyperthermic perfusion is performed by one or more Tenkhoflf-type inflow catheters and some multiperforated outflow drains. Perioperatively perfusion may be performed with a temporary closed abdomen, or with an open abdomen technique. In a different percutaneous technique, developed by Pontiggia and Hager et al^^ repeated intraperitoneal hyperthermic chemoperfusion is performed initially via a Verres needle and, after the abdomen is flooded with dialysis solution, with a perforated catheter (e.g., Periocart catheter) by a continuous perfusion with a single needle intermittent pump system (e.g., Bellco). Antineoplastic agents, mostly platinum derivates or mitomycin, are added to the perfusate once the temperature of 4l-43°C is reached inside the abdomen. A target temperature of at least 42-43 "C should be reached. The treatment time is between 60 and 120 minutes. Perioperative Intraperitoneal Hyperthermic Chemoperfusion After cytoreductive surgery IPHC can be applied to reduce residual cells in the abdominal cavity. Spratt et al described in 1980 a clinical delivery system for intraperitoneal hyperthermic chemotherapy. They reported that a case of recurrent pancreatic cancer spreading to the splenic hilus, omentum, transverse mesocolon, and peritoneal cavity was effectively treated by extensive tumor resection plus intraperitoneal hyperthermic perfusion. Gastric Cancer Koga et al^° and Fujimura et al^^ reported in 1988 and 1990, respectively, that continuous peritoneal hyperthermic perfusion of mitomycin C is effective for prevention or treatment of peritoneal recurrence of gastric cancer. In a prospective phase II trial Fujimoto et al^^ demonstrated that the prognosis of patients with far advanced refractory gastric cancer with large serosal invasion and/or peritoneal dissemination could be improved by intraperitoneal hyperthermochemotherapy in combination with mitomycin C. In a randomized study with 141 patients with gastric carcinoma with macroscopic serosal invasion Fujimoto et al could confirm that the IPHC-group had a significant higher survival rate and better prognosis than the control group with surgery alone.^^

Intracavitary Hyperthermic Perfusion

Table 5. Clinical

trials on intraperitoneal

221

hyperthermic

perfusion

chemotherapy

Survival No. of Patients

Authors (Year) [Ref. No.]

Cancer Indications Year When (Peritoneal Carcinomatosis) Measured

%

Fujimoto et al, 1991^^

61

Refractory Gastric

2

64 vs 1 7

Sugarbaker et al, 1995^^

181

CRC/Appendiceal

3 3 3 3

99 (G 1) 65 (G II) 66 (Gill) 20 (G IV)

Sugarbaker et al, 1998^^

572

Appendix (n=400) Colorectal (n=45) Gastric IV (n=13) Sarcomatosis (n=50) Mesothhelioma etc. (n=48 Malignant Ascites (n=16)

3 3 3 3 3 3

83 41 31 43 27 0

Fujimoto et al, 1999^^

141

Advanced Gastric

4

76 vs 58

Fujimuraetal, 1999^^

14

Peritoneal Carcinomatosis

3

22

Beaujard et al, 2000^°

27

Peritoneal Carcinomatosis

1

50

Cavaliereetal, 2000"*^

35

Peritoneal Carcinomatosis

2

55*

Pestiau and Sugarbaker, 2000"^^

104

Colon

3

29

Loggie et al, 2000"*^

79

Appendix (n=22 Stomach (n=19) Large Intestine (n=38)

3 3 3

52 14 24

Hageretal, 2001^^

36

Ovarian

5

35

Ellas e t a l , 2001 "^^

64

Colorectal

5

27**

W i t k a m e t a l , 2001"*^

29

Colorectal

3

23

C u l l i f o r d e t a l , 2001^7

54

Colon/Appendiceal

5

28* 55

D e r a c o e t a l , 2001 ^"^

27

Ovarian

2

Verwaair et al, 2002^^

48

Colorectal

2

63

G l e h e n e t a l , 2003^^

56

Peritoneal Carcinomatosis

2

79 (RO) 45 (R2)

Shen et al, 2003^^

40

Gastrointestinal

3

25

Scuderi S et al, 2003^°

132

Pseudomyxoma peritonei

5

20 NED 1 RFC

Rossi et al, 2003^^

13

Gastric

1

71

Pilati et al, 2 0 0 3 "

34

Colon

2

31

Elias and Pocard, 2 0 0 3 "

34

Colorectal

3

65**

Cavaliereetal, 2003^"*

69

Colorectal

4

45**

Rossi et al, 2004^^

29

Sarcomatosis

5

37

G l e h e n e t a l , 2004^^

506

Colorectal

2

43 vs 16

Summary

n = 2311

*Appendiceal malignancies included; **Complete cytoreductive surgery. >Abbreviations: NFD: no evidence of disease; RFC: recurrence; vs: versus; R: residual

222

Hyperthermia in Cancer Treatment: A Primer

Ovarian Cancer Epithelial ovarian cancer spread primarily by continuous extension of exfoliating cells into the peritoneal cavity and rarely systemically. Therefore, regional intraperitoneal chemotherapy might benefit these patients. Because antineoplastic agents can penetrate into tumor tissue in combination with heat much deeper than with normothermic perfusion, hyperthermia may have additional advantages. Several phase I and II trials of intraperitoneal hyperthermic chemoperfusion with platinum compounds after cytoreductive surgery have been reported or initiated. Wether the pharmacokinetic advantage of intraperitoneal hyperthermic chemoperfiision will translate into a clinical benefit has yet to be confirmed in randomized phase III trials. Colorectal Cancer After curative colorectal cancer resection, peritoneal carcinomatosis is the second most common site for recurrence. Intraperitoneal hyperthermic perfusion in the treatment of patients with peritoneal carcinomatosis with mitomycin C or cisplatin has been reported in numerous phase I and II trials.^^'^^ A prospective phase III trial comparing intravenous chemotherapy plus cytoreduction plus intraperitoneal hyperthermic chemoperfusion with intravenous therapy alone, in patients with peritoneal carcinomatosis of colorectal cancer, was initiated at the Netherlands Cancer Institute. The preliminary residts are promising. ^^ Mesothelioma^ Pseudomyxoma and Sarcoma Mesothelioma, pseudomyxoma, and sarcoma are relatively unresponsive to systemic chemotherapy. Intraperitoneal normothermic chemotherapy increases response rate, but early relapse is conunon in most patients. With a two-year survival rate of 80% recent results of a trial with intraperitoneal hyperthermic chemoperfusion of patients with peritoneal mesothelioma reported from the National Cancer Institute in the USA demonstrate superior antineoplastic activity and gives hope for new treatment options.^^ Case reports from Gilly et al Sugarbaker and Hager about good response on IPHC-treatment of patients with locally aggressive pseudomyxoma peritonei are very promising. Percutaneous Intraperitoneal Hyperthermic Chemoperfusion This conservative technique can be performed without abdominal surgery and can be repeated oftenly. Long-term treatment is possible by this method which is a great advantage compared to the periopertive I PHC. Therefore, this technique can be applied not only in patients with peritoneal carcinomatosis inunediately after cytoreductive surgery but also in patients after relapse, in chemotherapy resistant or refractory cases, far advanced stages of peritoneal carcinomatosis or sarcomatosis and in patients with recurrent malignant ascites. Ovarian Cancer Hager et al demonstrated a 65%, 39% and 16% survival rate of patients with progredient far advanced and chemotherapy resistant or refractory peritoneal disseminated epithelial and stromal ovarian cancer with primary stage III and IV disease from first IPHC-treatment at one-year, two-years and five-years, respectively.^^ The patients have been treated before entry to the trial with platinum- and/or taxane-based regimens at an average of 12.5 cycles. The overall median survival of these patients from first diagnosis of disease was 49 months. Considering that the expected 1-year survival rate of these patients would be smaller than 2%, this prospective, open-label trial gives evidence for a new very effective treatment option for patients with peritoneal disseminated disease. The advantage of this technique is, that it can be repeated as long as perfusion of the abdomen is possible. Also patients with stromal cancer respond to this percutaneous IPHC.

Intracavitary Hyperthermic Perfusion

225

Gastrointestinal Cancer Also patients with peritoneal disseminated colorectal and gastric carcinoma can be treated in combination with mitomycin C, 5-FU, and/or oxaliplatin repeatedly safe and effectively with this treatment modality. The response in the case of colorectal cancer is less than in patients with ovarian or gastric cancer. Conclusion: Further trials have to proof evidence of efficacy of IPHC with this indications. Cervical Uterine Cancer Patients with peritoneal disseminated cervical or uterus carcinoma can be treated with intraperitoneal hyperthermic perfusion in combination with platinum compounds (cisplatin, carboplatin, oxaliplatin) or mitomycin (personal experiences). Conclusion: Preliminary case reports of the treatment of patients with chemotherapy resistant peritoneal disseminated cervical cancer are indicating good responses. Bladder Cancer Depending on stage and grade of the tumors 40-80% of patients with superficial bladder cancer will have recurrent tumors after intravesicular chemo- or immunotherapy with MMC, ADM, BCG, or IFN-a. Repeated intravesicular hyperthermic chemotherapy with perfiision technique via a catheter or by microwaves can be used to treat patients with recurrent superficial or muscle invading bladder cancer. In a neoadjuvant intravesicular hyperthermic chemotherapy Colombo et al^^ demonstrated pathological complete responses in GG% of the cases compared with 22% of the cases in the conventional with a normothermic intravesicular chemotherapy treated control group using a 915 MHz microwave source that directly heats the bladder walls within a temperature range of 42.5 to 45.5°C. The expected high recurrence rate of superficial bladder cancer after TUR of the bladder and normothermic intravesical chemotherapy with MMC could be reduced from 64% in the control group to 15.5% in the adjuvant combined hyperthermia with MMC treated group, as could be shown by Colombo et al in a randomized, multicentric study. Intravesicular hyperthermic chemoperfusion with MMC at temperatures above 42°C may prevent cystectomy in advanced, inoperable patients with bladder cancer as has been shown by Hager et al.^^ The recurrence rate and -frequency could be reduced substantially. Conclusion: Intravesical hyperthermic chemotherapy is safe and widely more effective than chemotherapy alone in ablating superficial or recurrent bladder cancer. It can be used also as an organ-sparing treatment of muscle-invasive bladder cancer.

Toxicity Perioperative Intraperitoneal Hyperthermic Chemoperfusion Postoperartive morbidity and mortality after intraperitoneal hyperthermic chemoperfiision (IHCP) relate mainly to the extent and duration of surgery, and not to hyperthermic perfusion itself Extensive cytoreductive surgery followed by intraperitoneal hyperthermic chemoperfiision is associated with considerable rates of morbidity and mortality. In a study of morbidity and mortality rates following IPHC with MMC after intensive cytoreductive surgery of patients with colorectal cancer the postoperative mortality rate was 5% and complications were noted in 35% of patients.^ Mainly digestive fistula, prolonged ileus, and pleurisy have been reported."^^ In some studies comparable side effects have been described as with cytoreductive surgery alone. More experience may reduce the risks of treatment. Percutaneous Intraperitoneal Hyperthermic Chemoperfusion Percutaneous intraperitoneal or intravesicular hyperthermic chemotherapy is feasible, safe and exhibits much less toxicity than systemic chemotherapy.^"^ In 1.8% of the treatments, peritoneal dismrbances with symptoms of subileus were observed and in 5% peritoneal irritations.

224

Hyperthermia in Cancer Treatment: A Primer

depending on dosage of antineoplastic agents. Most adverse effects were due to W H O grade 1 and 2 nausea and vomiting. This treatment can be repeated ofteniy without remarkable increased toxicity. Up to 30 treatments could have been performed in patients with peritoneal carcinomatosis and sarcomatosis.^^ Limitations for this method are extended adhesions or massive tumor burden.

Conclusions Excellent survival in patients with peritoneal carcinomatosis and sarcomatosis can be achieved by intraperitoneal hyperthermic chemoperfusion with or without optimal debulked cancer. Hyperthermia may increase response rates, reduce resistance to antineoplastic agents and increase quality of Ufe and survival. Most of the studies are phase I and II trials. Some of these studies suffer from methodological flaws with respect to sample size, inclusion of different tumor types, absence of clearly defined endpoints and statistical estimations. Some of the studies have been performed as randomized controlled phase III trials and some phase III trials are ongoing. IPHC is one of the most promising new cancer treatment modalities at this time. Well designed randomized studies should be performed to establish the role of this relatively well tolerated technique in thefixturetreatment of peritoneal disseminated malignant diseases.

References 1. Markmann M. The role of intraperitoneal chemotherapy in ovarian cancer. Cancer Treat Res 1994; 70:73-82. 2. Miyagi Y, Fujiwara K, Fujiwara M et al. Intraperitoneal (IP) infiision is a pharmacologically more reasonable route for systemic chemotherapy of carboplatin. A comparative pharmacolokinetic ananlysis of platinum using a new mathematical model after IP vs. IV infiision of carboplatin. A Sankai Gynecology Study Group (SGSG) study. Proc Am Soc CHn Oncol 2002; 21:2167. 3. Rothenberg ML, Liu PY, Wilcznyski S et al. Excellent 2-year survival in w o m e n with optimally-debulked ovarian cancer treated with intraperitoneal and intravenous chemotherapy: A S W O G - E C O G - N C I C study (S9619). Proc Am Soc CHn Oncol 2002; 21:809. 4. Hahn G M , Shiu EC. Effect of p H and elevated temperatures on the cytotoxicity of some chemotherapeutic agents on Chinese hamster cells in vitro. Cancer Res 1983; 43:5789-5791. 5. Hahn G M . Potential for therapy of drugs and hyperthermia. Cancer Res 1979; 39:2264-2268. 6. Teicher BA, Kowal C D , Kennedy KA et al. Enhancement by hyperthermia of the in vitro cytotoxicity of mitomycin C toward hypoxic tumor cells. Cancer Res 1981; 41:1096-1099. 7. O h n o S, Siddik Z H , Baba H et al. Effect of carboplatin combined with whole body hyperthermia on normal tissue and tumors in rats. Cancer Res 1991; 51:2994-3000. 8. Rietbroek RC, van de Vaart PJM, Haveman J et al. Hyperthermia enhances the cytotoxicity and platinum-DNA adduct formation of lobaplatin and oxaliplatin in cultured SW 1573 cells. J Cancer Res CHn Oncol 1997; 123:6-12. 9. Zakris EL, Dewhirst M W , Riviere JE et al. Pharmacokinetics and toxicity of intraperitoneal cisplatin combined with regional hyperthermia. J CHn Oncol 1987; 5:1613-1620. 10. Dogramatzis D, Nishikawa K, Newman RA. Interaction of hyperthermia with bleomycin and liblomycin: Effects on C H O cells in vitro. Anticancer Res 1991; 11:1359-1364. 11. Jacquet P, Averbach A, Stuart OA et al. Hyperthermic intraperitoneal doxorubicin: Pharmacokinetics, metabolism, and tissue distribution in a rat model. Cancer Chemother Pharmacol 1998; 41:147-154. 12. Katschinski D M , Robins HI. Hyperthermic modulation of SN-38-induced topoisomerase I D N A cross-linking and SN-38 cytotoxicity through altered topoisomerase I activity. Int J Cancer 1999; 80:104-109. 13. Kutz ME, Mulkerin DL, Wiedemann GJ et al. In vitro studies of the hyperthermic enhancement of activated ifosfamide (4-hydroperoxy-ifosfamide) and glucose isophosphoramide mustard. Cancer Chemother Pharmacol 1997; 40:167-171. 14. Haveman J, Rietbroek RC, Geerdink A et al. Effect of hyperthermia on the cytotoxicity of 2'-2'-difluorodeoxycytidine (gemicitabine) in cultured SW1573 cells. Int J Cancer 1995; 62:627-630. 15. Dumontet C, Bodin F, Michal Y. Potential interactions between antitubulin agents and temperature: Implications for modulation of multidrug resistance. Clin Cancer Res 1998; 4:1563-1566.

Intracavitary Hyperthermic

Perfusion

225

16. Yamauchi N , Watanabe N , Maeda M et al. Mechanism of synergistic cytotoxic effect between tumor necrosis factor and hyperthermia. Jpn J Cancer Res 1992; 83:540-545. 17. Song C W , Lin JC, Lyions JC. Antitumor effect of interleukin l a in combination with hyperthermia. Cancer Res 1993; 4:34-39. 18. Hager E D , Dziambor H, Strama H et al. Intraperitoneal hyperthermic perfusion (IPHP) chemotherapy of patients with peritoneal disseminated drug resistano ovarian cancer. Southern Med J 1996; 89:143. 19. Spratt JS, Adcock RA, Muscovin M et al. Clinical delivery system for intraperitoneal hyperthermic chemotherapy. Cancer Res 1980; 40:256-260. 20. Koga S, Hamazoe R, Maeta M et al. Prophylactic therapy for peritoneal recurrence of gastric cancer by continuous hyperthermic peritoneal perfusion with mitmycin C. Cancer 1988; 61:232-237. 2 1 . Fujimura T , Yonemura Y, Fushida S et al. Coninuous hyperthermic peritoneal perfusion for the treatment of peritoneal dissemination in gastric cancers and subsequent second-look operation. Cancer 1990; 65:65-71. 22. Fujimoto S, Takahashi M, Okui K. A prospective study on combined treatment of intraperitoneal hyperthermic and surgery for patients with refractory gastric cancer. In: Taguchi T, Aigner KR, eds. Mitomycin C in Cancer Chemotherapy Today. Excerpta Medica, 1991. 23. Fujimoto S, Takahashi M, Mutou t et al. Successful intraperitoneal hyperthermic chemoperftision for the prevention of postoperative peritoneal recurrence in patients with advanced gastric carcinoma. Cancer 1999; 85:529-534. 24. Deraco M , Rossi CR, Pennacchioli E et al. Cytoreductive surgery followed by intraperitoneal hyperthermic perfusion in the treatment of recurrent epithelial ovarian cancer: A phase II clinical study. Tumori 2001; 87:120-126. 25. Sugarbaker P H , Gianola FJ, Speyer JL et al. Prospective randomized trial of intravenous vs. intraperitoneal 5-FU in patients with advanced primary colon or rectal cancer. Semin Oncol 1995; 12:101-111. 26. Sugarbaker P H . Treatment of peritoneal carcinomatosis from colon or appendiceal cancer with induction intraperitoneal chemotherapy. Cancer Treat Res 1996; 82:317-325. 27. Loggie BW, Fleming RA, McQuellon RP et al. Cytoreductive surgery with intraperitoneal hyperthermic chemotherapy for disseminated peritoneal cancer of gastrointestinal origin. Am Surg 2000; 66:561-568. 28. De Simone M, Aimone M, Izzo G et al. Peritonectomy and hyperthermic antiblastic peritoneal perfusion (HAT) for peritoneal carcinomatosis. J Exp Clin Cancer Res 1997; 16:356-357. 29. Glehen O , Mithieux F, Osinsky D et al. Surgery combined with peritonectomy procedures and intraperitoneal chemohpyerthermia in abdominal cancers with peritoneal carcinomatosis: A phase II study. J Clin Oncol 2003; 21:799-806. 30. Zoetmulder FA, van der Vange N , Witkamp AJ et al. Hyperthermia intra-peritoneal chemotherapy (HIPEC) in patients with peritoneal pseudomyxoma or peritoneal metastases of colorectal carcinoma; good preliminary results from the Netherlands Cancer Institute. Ned Tijdschr Geneesk 1999; 143:1863-1868. 3 1 . Park BJ, Alexander HR, Libutti SK et al. Treatment of primary peritoneal mesotherlioma by continuous hyperthermic peritoneal perfusion (CHPP). Ann Surg Oncol 1999; 6:582-590. 32. Hager ED, Dziambor H, Hohmann D et al. Intraperitoneal hyperthermic perfusion chemotherapy of patients with chemotherapy-reistant peritoneal disseminated ovarian cancer. Int J Gynecol Cancer 2001; l l ( S u p p l l):57-63. 33. Colombo R, Da Pozzo LF, Lev A et al. Neoadjuvant combined microwave induced local hyperthermia and topical chemotherapy versus chemotherapy alone for superficial bladder cancer. J Urol 1996; 155(4): 1227-3. 34. Colombo R, Da Pozzo LF, Lev a et al. Adjuvant microwave hyperthermia and mitomycin C versus mitomycin C alone for superficial bladder cancer. Eur Urol 1999; 35(Suppl 2). 35. Hager ED, Strama H , Hohmann D et al. Prevention of cystectomy of recurrent bladder cancer by intravesical hyperthermic perfusion chemotherapy (IVHP). Anticancer Res 1998; 18:4807-5006. 36. Jacquet P, Stephens A D , Averbach AM et al. Analysis of morbidity and mortality in 60 patients with peritoneal carcinomatosis treated by cytoreductive surgery and heated intraoperative intraperitoneal chemotherapy. Cancer 1996; 77:1037-1042. 37. McQuellon RP, Loggie BW, Fleming RA et al. Quality of life after intraperitoneal hyperthermic chemotherapy (IPHC) for peritoneal carcinomatosis. Eur J Surg Oncol 2001; 27:65-73. 38. Sugarbaker et al. Management of Peritoneal surface malignancy using intraperitoneal chemotherapy and cytoreductive surgery. Ludann Company, USA, 1998. 39. Fujimura T, Yonemura Y, Fujita H et al. Chemohyperthermic peritoneal perfusion for peritoneal dissemination in various intrabdominal malignancies. Int Surg 1999; 84:60-66.

226

Hyperthermia in Cancer Treatment: A Primer

40. Beaujard AC, Glehen O, Caillot JL et al. Intraperitoneal chemohyperthermia with mitomycin C for digestive tract cancer patients with peritoneal carcinomatis. Cancer 2000; 88:2512-2519. 41. Cavaliere F, Di Filippo F, Botti C et al. Peritonectomy and hyperthermic antiblastic perfusion in the treatment of peritoneal carcinomatosis. Eur J Surg Oncol 2000; 26(5):486-491. 42. Pestieau SR, Sugarbaker PH. Treatment of primary colon cancer with peritoneal carcinomatosis: Comparison of concomitant vs. delayed management. Dis Colon Rectum 2000; 43:1341-1346. 43. Loggie BW, Fleming RA et al. Cytoreductive Surgery with Intraperitoneal Hyperthermic Chemotherapy for Disseminated Peritoneal Cancer of Gastrointestinal Origin. Am Surg 2000; 66:561-568. 44. Hager ED, Dziambor H, Hohmann et al. Intraperitoneal hyperthermic perfusion chemotherapy of patients with chemotherapy-resistant peritoneal disseminated ovarian cancer. Int J Gyn Cancer 2001; ll(Suppl l):57-63. 45. Elias D, Blot F, El Otmany A et al. Curative treatment of peritoneal carcinomatosis arising from colorectal cancer by complete resection and intraperitoneal chemotherapy. Cancer 2001; 92:71-76. 46. Witkamp AJ, de Bree E, Kaag MM et al. Extensive cytoreductive surgery followed by intra-operative hyperthermic intraperitoneal chemotherapy with Mitomycin-C in patients with peritoneal carcinomatosis of colorectal origin. Eur J Cancer 2001; 37:979-984. 47. CuUiford Att, Brooks Ad, Sharma S et al. Surgical debulking and intraperioneal chemotherapy for established peritoneal metastases from colon and appendix cancer. Ann Surg Oncol 2001; 8:787-795. 48. Verwaal V, Ruth S, Bree E et al. Randomized trial of cytoreduction and hyperthermic intraperitoneal chemotherapy versus systemic chemotherapy and palliative surgery in patients with peritoneal carcinomatosis of colorectal cancer. J Clin Oncol 2003; 21:3737-3743. 49. Shen P, Levine EA, Hall J et al. Factors predicting survival after intraperitoneal hyperthermic chemotherapy with Mitomycin C after cytoreductive surgery for patients with peritonad carcinomatosis. Arch Surg 2003; 138:26-33. 50. Scuderi S, Costamagna D, Vaira M et al. Treatment of pseudomyxoma peritonei using cytoreduction and intraperitoneal hyperthermic chemotherapy. Tumori 2003; 89(Suppl 4):43-45. 51. Rossi CR, Pilati P, Mocellin S et al. Hyperthermic intraperitoneal intraoperative chemotherapy for peritoneal carcinomatosis arising from gastric adenocarcinoma. Tumori 2003; 2(5):S54-S57. 52. Pilati P, Mocellin S, Rossi RC et al. Cytoreducitve Surgery combined with hyperthermic intraperitoneal intrapoperative Chemotherapy for peritoneal carcinomatosis arising from colon adenocarcinoma. Ann Surg Oncol 2003; 10(5):508-513. 53. Elias D, Pocard M. Treatment and prevention of peritoneal carcinomatosis from colorectal cancer. Surg Oncol N Am 2003; 12:543-559. 54. Cavaliere F, Peri P, Rossi CR et al. Indications for integrated surgical treatment of peritoneal carcinomatosis of colorectal origin: Experience of the Italian Society of Locoregional Integrated Therapy in Oncology, Tumori 2003; 89:21-23. 55. Rossi RC, Deraco M, de Simone M et al. Hyperthermic intraperitoneal intraoperative chemotherapy ager cytoreducitve surgery for the treatment of abdominal sarcomatosis. Cancer 2004; 100(9):1943-1950. 56. Glehen O, Kwiatkowski PH, Sugarbaker D et al. Cytoreducitve surgery combined with perioperative intraperitoneal chemotherapy for the management of peritoneal carcinomatosis from colorectal cancer: A multi-institutional study. J CHn Oncol 2004; 22(16):3284-3292.

CHAPTER 18

Whole Body Hyperthemiia at 43.5-44''C: Dream or Reality? Alexey V. Suvernev, Georgy V. Ivanov, Anatoly V. E£reinov and Roman Tchervov* Abstract

A

high level of body temperature (43°C) is needed for effective use of whole body hyperthermia. Such a high level hyperthermia can only be safely used taking into account a theory of developing post-aggressive hyperproteolysis.^'^ Besides the control of proteolysis, it is also necessary to apply total phentanyl anesthesia, high-frequency lung ventilation and a high rate of heating.^ Clinical application of this method allows inducing the apoptosis of malignant cells, decreasing the viral load in HIV and HCV-infected patients and also causing a general sanitary effect. Use of water immersion makes the technology noninvasive and "physiological**. Application of this whole body hyperthermia technology reduces ventilation time and complications.

Introduction It is known, that therapeutic opportunities of the thermal factor are used since ancient times. However, in the 20th century scientists and physicians have reached essential results in application of hyperthermia in treatment of oncological, immunological, viral and other diseases. Necessity of development and improvement of hyperthermic technologies was predetermined because of insufficient efficacy of commonly used surgical, pharmacological, immunobiological and other methods of treatment of dangerous (oncological, viral, immunological) diseases and detection of unknown clinical effects of a hyperthermia. Those clinical significant effects are: 1. Destruction of malignant cells due to induction of necrobiosis and apoptosis by the thermal factor. 2. Elimination of tolerance of malignant cells to chemotherapy 3. Potentiation of medical effects of chemotherapy in combination with hyperthermia. It allows to reduce dosages of chemotherapy, decreasing damaging effect to the healthy tissues and keeping antineoplastic activity. 4. Activation of immunomodulating effects of hyperthermia, increase of protective forces of the patient, which important and never occurs after chemo- and radiotherapy.

Necessity of High-Level Whole Body Hyperthemiia It is known that there are no universal solutions for technical realization of artificial hyperthermia and for choosing its rational temperature range. Different variants of local and whole •Corresponding Author: Roman Tchervov—Siberian Institute of Hyperthermia, Nagornaya 14a, lskitim-5, 633205, Russia. Email: [email protected]. Hyperthermia in Cancer Treatment: A Primer^ edited by Gian Franco Baronzio and E. Dieter Hager. © 2 0 0 6 Landes Bioscience and Springer Science+Business Media.

228

Hyperthermia in Cancer Treatment: A Primer

body hyperthermia (wave, immersion and perfusional) should not be opposed to each other. It is necessary to take into account the concrete cHnical situations to achieve the maximal medical effect. Below we summarize arguments which were taken into account at studying and applying a high level (extreme) hyperthermia (up to 44.0°C). First of all, it is important to reply: why is it necessary to achieve WBH above 43 "C? The answer to this question is obvious not only for oncology, but also for those areas of medical practice where a selective cell damaging effect of heat is required. In particular, it is actual for oncological, virological and allergological practice, when it is necessary to initiate a necrobiosis and apoptosis of malignant cells, to suppress the HlV-infection or to destroy para-proteins and pathological antibodies. Some inspiring results in this field have been already received. So E. Kano (1987) has established, that "... energy of activation of heat cell killing at temperatures from above 43.0''C and below 43.0 "C is equal to 150 kcal l[i and560 kcal l\i accordingly..." Thus, on the one hand it is possible "to examine" an organism of the cancer patient protractedly on its nonspecific resistance to temperature up to 4 r C during 1-3 hours per hope that cancer cells in his organism appear less steady against the increased temperature. On the other hand, in conditions of adequate anesthetic protection, rapidly provide the 43.0-43.5-44.0°C level of hyperthermia and to start the biological mechanism of apoptosis in cancer cells. We shall note, that doctor Kano with colleagues within the next 10 years repeatedly confirmed reliability of the phenomenon registered by them certainly accepted by us in attention. Also it is necessary to note, that Mathe G. on XXIVth Congress in Rome (September 2001) also has confirmed, that"... apoptosis ofcancer cells is started only at achievement oftemperature in 43.0''C..." The authority of these and other known scientists allows to consider that high level hyperthermia (above 43 °C) is basic for oncological practice. Moreover, application of whole body hyperthermia in an interval of 40-42"C is fraught with a potential danger of dissemination of malignant cells and stimulation of their growth.

Risk Factors of Whole Body Hyperthermia Over 43**C and Pathogenetic Substantiation of Their Overcoming It is known, that homoiothermic organisms ".. .are sheltered right at the threshold of thermal death..." Artificial realization of whole body hyperthermia even in an interval of 41.8-42.0°C is bound to the risk of development of dangerous complications. Those are: • Thermal shock • Brain edema • Acute circulatory insufficiency • Hepato-renal syndrome • Acute respiratory distress syndrome (ARDS) • Disseminated intravascular coagulation The probability of occurrence of the specified complications is especially great in patients with oncological pathology; in elderly and senile age, when hyperthermia application is compelled on a background of multiple organ failure and the general bad state of health. In this connection at the 32nd Congress in Okayama (1994)^^ it was noticed, that whole body hyperthermia up to 43"C is desired for clinical practice, but mortality reaches a level of 17%. However our experience in whole body hyperthermia over 43°C with more than 500 patients, who successftdly and repeatedly undergone this procedure with no complications and multiple organ failure, is a basis to assert about a basic opportunity of safe extreme hyperthermia. Below there are some pathogenetic positions by which we were guided during development and perfection of high level whole body hyperthermia (43.5-44.0°C).

Whole Body Hyperthermia at 43.5-44 "C: Dream or Reality?

37°C

Normal protein

229

43°C

Partial denaturation

37°C

Normal protein

Figure 1. The process of reversible temperature disintegration of protein structure.

It is known, that".. .during cell reactions to damaging agents, plasmic proteins undergo the reversible structural changes keeping nuclear composition invariable..." ^ Such changes are happen by turns of nuclear groups around the single bonds. The essence of conformational changes of proteins is in redistribution of binding energy which can result in breaks of existing and establishment of weak bonds supporting secondary, tertiary and quaternary structure of a protein molecule. ^"^ It is known, that the spatial structure of a protein molecule is supported by various forces. Hydrogen bridges are established between oxygen of carbonyl groups of one amino acid with the imine nitrogen of another (N-H 0=C). They basically support the secondary structure of protein molecules. Covalent disulfide bonds (-S-S-) can participate in architectonics of tertiary structure. Influence of hydrophilic and hydrophobic sites of a protein molecule on an arrangement of water in their nearest environment creates quaternary structure determining stability of a protein macromolecule. It is also known, that the temperature maximum of stability of the most of proteins is much lower than a temperature of their vital optimum. Hence, the rise of temperature over an optimum should reduce stability of the protein molecules. And, the rise of temperature is more intensively destabilizing the periphery, the most reactive parts of macromolecules which are remote from an internal hydrophobic nucleus globule. Process of denaturation is more or less a complete destruction of quaternary, tertiary and secondary structure without hydrolytic splitting of peptide bonds. Increased temperature, in comparison with others well-known denaturation agents (urea, alcohol, acetamide) is the most universal "destructor" of macromolecular structures. Certainly, increasing a temperature, depending on its level and time of exposure, macromolecule changes occur step-by-step: from light disturbances of a stereochemical configuration to the formation of more or less chaotic clews of polypeptide bonds with a complete loss of function (Fig. 1). If the disturbances in macromolecular structure are incomplete, the termination of heating and decrease of temperature to an initial level enables renaturation. Restoration of initial, native structure and renewal of function is considerably longer than damage and depends on both a depth of reversible denaturation and the type of protein macromolecule. In our experience, after heating the patient up to 44°C, a process of functional restoration of various protein structures lasts from 2 until 8-16 hours. In this period, meeting the certain conditions, the structure and function of protein macromolecule are gradually restored. However, the given pattern has an exception relevant to the unique enzyme trypsin. It is known, that this enzyme with rather simple structure and small molecular weight, can completely

Hyperthermia in Cancer Treatment: A Primer

230

37°C

43X

37X

Products of proteolytjc destruction of proteins causing severe intoxication

Trypsin

Figure 2. Participation of trypsin in hyperthermic proteolysis. restore its structure and ftinction within 10-15 min. after heating up to 43-44°C! Thus, a dangerous pathogenetic situation appears. A huge amount of partially denaturated proteins (substratum) and an increase in active, nonspecific exo-endoproteinase (trypsin). Not without reason, Lehninger^^ and Szent-Gyorgyi compared it to a "spring" or a "zipper". Proteinases also are denaturising agents. They destruct not only complicated structures but also even peptide bonds, breaking a primary structure of protein. The high activity of proteolysis in an overheated organism causes accumulation of a plenty of oligopeptides inducing endotoxemia. In other words, for many protein structures: the high temperature causes partial denaturation, and proteolysis destroys macromolecules (Fig. 2). In our opinion, a pathogenetic pattern mentioned above, is the one of key positions that should be taken into account for safe performance of extreme whole body hyperthermia. We established, that the phenomenon of hypertrypsinemia is a typical, nonspecific reaction of an organism to any stress and shock (Fig. 3). The data given in Figure 3, shows that high trypsin activity in blood of experimental animals was registered within 1 hour after any stress. We revealed, that the source of trypsinemia are the zymogenic granules of the pancreas, from which the shock enzyme "evades" into the bloodstream and causes a hyperproteolysis and endotoxemia. Hence, at anaesthetic management of high-level artificial hyperthermia is necessary to prevent hypertrypsinemia and to control proteolysis.

What Are Possible Ways to Suppress the Activity of Trypsin during Hyperthermia? It appears to be a problem because polyvalent proteinase inhibitors, commonly used in clinical practice, have a protein nature and denaturise at high temperature. In other words, the target was an alternative way of proteinase inhibition. Searching for a pharmacological preparation, capable to inhibit the activity of trypsin, the attention was drawn to clinical application of formaldehyde, described in V.V. Kovanov's works (1980-1982).^"^ It was established later, that formaldehyde blocks the action of proteolytic enzymes of trypsin type and can essentially reduce a proteolytic activity of the blood. As intravenous introduction of formaldehyde is not permitted by the pharmacopoeia, an alternative variant of its application was found. It lies in the application of hexamethylenetetramine which is commonly used in medical practice. This preparation introduced to the blood can split into formaldehyde and anmionia, on condition that liver and kidneys are ftmctioning normally.

Whole Body Hyperthermia at 43.5-44 "C: Dream or Reality?

231

Blood level of trypsine in rats (MED) 0.0

2.0

4.0

6.0

8.0

10.0

12.0

Physical training Anaphylaxia Imnrtobilisation Hyperthermia Ishaemlareperfusion stress 30* occlusion of V, Portae

Haemorrage Burn Pyrogenal Trauma Adrenaline

Figure 3. Activity of trypsin in blood of rats at 1 hour after aggression.

Considering the above arguments, hexamethylenetetramine can be validly used in patients with a hyperactivity of trypsin. Using this preparation a relevant decrease of trypsin activity from levels of 2.34-9.72 MED to 0.2-0.3 MED was noted. The optimum doze of hexamethylenetetramine (80 mg/kg) and its half-time excretion (4-6 hours) were found empirically using recommendations of the pharmacopoeia. It is a very important point of a problem, taking into account an essential circumstance: most frequently antiproteinase preparations used in medical practice (Trasilol, Contrical, Gordox etc.) are ineffective in trypsinemia and absolutely not effective at high temperature.^^ It happens because the specified polyvalent trypsin inhibitors have protein structure and are subjected to disintegration by temperature. On the contrary, the formaldehyde formed in blood at hydrolysis of hexamethylenetetramine, keeps the antiproteinase properties even at 44°C. Thus, one of the basic conditions of a safe whole body extreme hyperthermia (> 43 °C) was met by a maximal suppression of proteolytic activity in blood. This became possible in view of

252

Hyperthermia in Cancer Treatment: A Primer

Figure 4. A general view of aaive immersion-cx)nveaional heating of the patient. the concept about a negative role of postaggressive trypsinemia and the finding of an alternative way to inhibit the trypsin activity.

Technological and Anaesthetic Features of Whole Body Severe Hyperthermia To achieve a condition of controlled whole body extreme hyperthermia, we use a simple variant of immersion-convectional for physical heating of a patient s body. Patient is under general anaesthesia, heating with a water, warmed up to 44.0-47.0°C. The process of active physical heating is carried out in a special bath, for example "CHIRANA'' (Fig. 4). The patient's body and extremities immersed in warmed water except the head. No cranio-cerebral hypothermia is performed during the process of heating. Process of active heating should be fast enough (0.2-0.5''C/min.). Registration of the body temperature of the patient is made with thermal sensors, installed in a gullet. Process of heat transfer is reached by the relevant peripheral vasodilatation and hyperkinetic reaction of circulatory system. That provides equalization of a temperatm-e gradient between peripheral and the central organs. After achievement of a necessary level of hyperthermia, the process of heating stops and the patient is takenfiroma bath. The further process of normothermia restoration occurs passively and usually lasts 35-45 minutes. The specified method of whole body hyperthermia is achieved under conditions of special anaesthetic protection, whose goal is prevention of probable complications. To avoid such complications it is necessary to meet a number of indispensable conditions: 1. Special preprocedural preparation 2. Total intravenous anaesthesia

Whole Body Hyperthermia at 45.5-44"C: Dream or Reality^

235

Figure 5. Disappearance of multiple melanoma metastases in liver. 3. Control of proteolysis 4. Application of high-frequency artificial lung ventilation 5. Maintenance of high rate of active heating 6. Special monitoring of homeostasis. Clinically relevant effects of whole body hyperthermia technology (43-44 "C), can be illustrated by the following facts: 1. Disappearance of multiple melanoma metastases in liver (Fig. 5). 2. Fast decrease of HIV-1 RNA plasma concentration in HIV-infected patient (Fig. 6). 3. Removal of abstinence syndrome and total elimination of physical addiction in patients with drug abuse (Heroin, Methadone). 4. Elimination of an allergen (chloropicrin) and circulating immune complexes from the plasma of a patient with atopic bronchial asthma (Fig. 7). 5. Normalization of lung function parameters in a patient with atopic bronchial asthma (Fig. 8).

500 000,

Figure 6. Fast decrease of HIV-1 RNA plasma concentration in HIV-infected patient.

Hyperthermia in Cancer Treatment: A Primer

254

19,0

I before WBH I after WBH

15,0

10,0

5,0

A

0,0

300.0 before WBH I after WBH

E

200,0

•5

100,0

i

Figure 7. Elimination of an allergen (chloropicrin) and circulating immune complexesfromthe plasma of a patient with atopic bronchial asthma. Fast decrease of HIV-1 RNA in FIFV-infected patient after WBH (Fig. 6) gave us a good reason forftirtherinvestigations in thisfield.We conducted a small pilot study to reveal HIV-1 RNA dynamics after hyperthermia and it brought interesting resiJts.

Method The group of investigation included 10 patients with progressive viral load. Whole body hyperthermia with general anesthesia used to achieve patients core temperature of 43.0-43.7'*C. Each patient had four procedures of hyperthermia within 12 months. All patients were

Whole Body Hyperthermia at 45.5-44"C: Dream or Reality^

255

W before WBH 11 after WBH

Figure 8. Normalization of lung function parameters in a patient with atopic bronchial asthma. observed not less than 12 months after the first procedure. Three of those patients observed over 15 months. One patient observed 33 months.

Results Within 12 months viral load in all patients decreased to less than 10% of initial level. Four patients with longer follow-up had viral load less than 7% of initial level at the moment of last observation. One patient with 33 month observation had viral load 0.9% of initial level (Fig. 9). None of the patients used known antiretroviral chemotherapy.

Discussion Such stable long-term depression of viral load in investigation group brought up a number of questions which have no answers yet. It s still unclear why viral load is not rising when absolute C D 4 count increases to normal values. At the same time stable persistence of minimal viral load is also not understood. There is an impression that those patients are quite similar to true long-term "nonprogressors*'. If it will be proven, there will be a reason to conclude that hyperthermic treatment with high level whole body hyperthermia (over 43'C) converts HIV-progressors to nonprogressors.

Conclusion 1. Until now the relevant negative activity of proteolysis wasn't taken into account when dealing with the problem of endotoxication caused by extreme thermal exposure. Developing a trypsin inhibition method during thermal exposure can prevent endotoxication and significantly decrease hyperthermic complications. 2. Premature conclusions about inefficiency of hyperthermia in HIV-infection stated in 90s formally "closed" research work in that direction. Development of intensive and safe hyperthermia can possibly resurrect interest in thermomedicine for HIV/AIDS.

236

Hyperthermia in Cancer Treatment: A Primer

Figure 9. Long-term dynamics ofHIV-1 RNA after hyperthermia. Decrease ofviral load after extreme whole body hypenJiermia.

References 1. Suvernev AV, Meshalkin EN, Sergievskii VS et al. Trypsinemia in stress reactions of an organism. Novosibirsk, Nauka: Siberian branch 1982. 2. Suvernev AV. Primary prophylaxis allows the portabiUty of general hyperthermia up to 44°C in dogs. Human adaptation and primary prophylaxis. Novosibirsk, 1986:1:117-118. 3. Suvernev AV, Gleim GK. Pancreatogeneous intravascular hyperproteolysis. New methods of diagnostics, treatment and prevention of diseases. Novosibirsk, 1987:200-201. 4. Suvernev AV, Rodionov SU, Plyaskin KP et al. Application of whole body hyperthermia in treatment of oncological pathology Organization of palliative care and treatment of severe forms of malignancies. Moscow, 1995:113-114. 5. Suvernev AV. An experience of whole body hyperthermia in treatment of oncological pathology. Visceral Tumors. Tomsk, 1995:97-99. 6. Suvernev AV, Gleim GK, Takach GL et al. Problems of whole body hyperthermia over 42°C. Hyperthermia in Oncology. Minsk, 1990:115. 7. Suvernev AV, Patoka AV, Gleim GK. Experience of whole-body hyperthermia for treatment of oncological patients. Materials of the 2nd Far-eastern International Congress of Multimodal Cancer Treatment. Vladivistok, 1994:45. 8. Kano E. Fundamentals of thermochemotherapy of cancer. Can No Rinsho 1987; 33(13):l657-63. 9. Mathe G. From mechanisms of action to relation indications of hyperthermia at 40°C of 43°C in cancer treatment. XXIV International Congress on CHnical Hyperthermia. Rome, 2001. 10. Proc. 32nd Annual Congress of Japan Society of Cancer Therapy Meeting, Japan: Okayama, 1994. 11. Nasonov DN et al. Biological reaction on external influence. Moscow-Leningrad, 1940. 12. Koshland DE. Federat. Proc 1964; 23(3 pt l):719-726. 13. Lehninger L. Biochemistry Moscow, 1974. 14. Kovanov W et al. Proc. of the 1st Moscow Medical Institute Moscow, 1982. 15. Litvinov IV. Choosing the method of ventilation at whole body hyperthermia in cancer patients. Novosibirsk, 1998:19, (Ref Type: Dissertation). 16. Stebbing J, Gazzard B, Kim L et al. The heat-shock protein receptor CD91 is up-regulated in monocytes of HIV-1-infected "true" long-term nonprogressors. Blood 2003; 101(10):4000-4.

CHAPTER 19

Extreme Whole-Body Hyperthemiia with Water-Filtered Infrared-A Radiation Alexander von Ardenne* and Holger Wehner Abstract

T

he testing of various methods to realise extreme whole-body hyperthermia (eWBH) finally led to the utilisation of radiative systems. Among these the application of water-filtered infrared-A radiation (wIRA) distinguished itself by its high penetration, all the way into the capillary bed of the skin. With wIRA the interfering infrared-B and the infrared-C is eliminated from the heat radiation. Thus a clearly higher radiation power can be applied at a tolerable level than by applying unfiltered heat radiation. In two independent phase I clinical studies the high tolerance of eWBH (approx. 42°C/60 min) was proven in the scope of the so-called systemic cancer multistep therapy (sCMT) while applying wIRA. First proof of the retardation of tumour progression could be carried out by a retrospective observation study of over 490 sCMT treatment courses of cancer patients with various different tumour entities at an advanced stage. A phase I/II clinical study on the treatment of 19 patients with metastatic colorectal cancer with sCMT and wIRA in combination with chemotherapy suggests that sCMT may enhance the effect of chemotherapy. In a prior study on the treatment of patients with metastasised adenocarcinomas only 3/19 patients remained in progression.

Introduction Lamps with water-filtered infrared radiation for therapeutic applications had already been produced at the beginning of the 20th century.^ In 1931 A. Bachem^ could already prove that skin only featured a high transmission depth or penetration for heat radiation from the spectral region of infrared-A (760 nm ... 1,400 nm). In the spectral regions of infrared-B (1,400 nm ... 3,000 nm) and the close infrared-C (3,000 nm. 1 mm) on the other hand, the transmission is very slight which leads, at an application of a high radiation power, to an overburdening of the skin (Fig. 1). As standard electric light bulbs and halogen lamps radiate approx. half of their energy in the spectral region of infrared-B and -C, this share of radiation has to be eliminated. This is possible with a water layer, which placed in the ray path, acts like an edge filter and eliminates the infrared-B and -C share from the heat radiation (Fig. 2). If a water layer of appropriate thickness is placed, for example, in front of a halogen lamp with approx. 2600°K filament temperature, this results in the spectral radiation distribution displayed in Figure 3. In this way heat radiation is generated with a spectral distribution, the focus of which is in the area of highest transmission of the skin. This is defined as water-filtered infrared-A radiation (wIRA). *Corresponding Author: Alexander von Ardenne—Von Ardenne Institute, Zeppelinstr. 7, D-01324 Dresden, Germany. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer^ edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

Hyperthermia in Cancer Treatment: A Primer

238

.9 1.0-

'liilWilifiltft' ^ * "

Visua)

E 0.6go.4So.2300

Skin

f

to;

1 « | ; 1500 1800 2100 2400 2700 3000nm wavelength

Figure 1. Transmission of electromagnetic waves in the spectral region of infrared-A, -B and -C through a human skin layer of a thickness of 1.4 mm. U. Henschke^ provided an early quantitative substantiation for the tolerance of water-fdtered infrared-A radiation. He publicised that for long-term application the maximum toleration of radiation intensity for water-filtered heat radiation was double as high as for standard bulb radiation. Unfortunately this knowledge did not lead to a wider range of application in the following decades in clinics. For patients with advanced and metastasised malignant tumours, only a systemic therapeutic approach is adequate. Such a prerequisite is, among others, offered by the extreme whole-body hyperthermia (eWBH) with target temperatures around 42°C, which is usually combined with adapted chemotherapy today. After ten years of research and development in the field of radio frequency hyperthermia at 27 MHz to bring about a noncontact whole-body hyperthermia in combination with an increase of temperature locally^ (Fig. 4), M. von Ardenne 1985 recognised the limits of the application at that time: The development of hot spots, impossibility of temperature mapping, high demand made on technology and shielding. A way out of this dilemma was offered by the idea of thermal treatment of the outer body shell. As even according to the law of thermo-dynamics, it is only a question of time until the innermost point of an entity that is surrounded by an isothermal outer body shell is at the same temperature level as the outside body shell itself. To achieve this, a thermal source is required, the radiation of which only penetrates the millimetre area of the skin and is then almost completely absorbed. If this is successful, simple two-step temperature monitoring is possible by which the temperature of the skin (outside body shell) and, on the other hand, the body core

Water \

!i500

I

\

I

I

I

1800 2100 2400 2700 3000 nm wavelength

Figure 2. Transmission of electromagnetic waves in the spectral region of infrared-A, -B and -C through a water layer of defined thickness.

Extreme Whole-Body Hyperthermia with Water-Filtered Infrared-A Radiation

^ a> 1.0

259

Visual

Halogen Lamp + Water Filter T 1 1 r 1500 1800 2100 2400 2700 3000 nm wavelength good penetrallon of sidn + absorption of H20«specHic frequencies

Figure 3. Radiation spectrum of a halogen lamp after passing a water filter (water-filtered infrared-A radiation). temperature, is observed during hyperthermia treatment by inserting a temperature sensor into a body opening, e.g., the rectum.

Technical Realisation of Water-Filtered Infrared-A Radiation Inspired by E. Braun, in 1985 M. von Ardenne once again took up the principle of water-filtered infrared-A radiation whereby he was able to implement the idea of heating up the body shell. The water-filtered infrared-A radiation penetrates into the capillary bed of the corium, where the direct transmission of heat into the blood circulation system takes place (Fig. 5) and thus an equalisation of the temperature takes place in the whole organism.

Figure 4. Radio frequency hyperthermia with a 27 MHz-SELECTOTHERM system for the noncontact combined whole-body and local hyperthermia with an applicator that can be focused on the tumour (1987).

Hyperthermia in Cancer Treatment: A Primer

240

1.0 <0

Visual

0.8

c 0)

E 0,6 CO
g 0.4

1

stratum germjnativum *0.25.„0.5mm 0.2 H cohum * 0.5...2.0 mm subcutis^2.0...2.5mm 600

1000

1500

2000

wavelength [nm]

water-filtered infrared-A radiation Figure 5. Absorption of the infrared-A radiation in the various skin and tissue layers by superficial radiation (ace. to Borchert and Jubitz ) below: Intensity distribution of water-filtered infrared-A radiation A system for extreme whole-body hyperthermia with water-filtered infrared-A radiation was developed in several generations in close collaboration with the photo dermatology department of the University Clinic Charit^, Berlin.^ Such a system of the third generation is depicted in Figure 6. The patient lies on a knot-free net and is heated from top and from bottom by five radiator groups with water-filtered infrared-A radiation. The distribution of radiation intensity at patient level is adjusted manually, the temperatures are displayed and recorded without interruption. An efficient system with water-fdtered infrared-A radiation for whole-body hyperthermia was at disposal for the first time with this hyperthermia system. It is characterised by its open equipment design, good possibilities to observe the patient and good controllability of body temperature with the help of four temperature sensors. Despite the open equipment design a quick increase in temperature is possible. Independent of the developer, this medical technology was tested under clinical conditions and its suitability for whole-body hyperthermia was confirmed.^ Figure 7 shows a temperaturetime chart with the course of body core temperature (rectum/rekt) and three temperatures close to the surface (axilla/axil, hypogastrium/ubm, lumbar spine/lws).

Tolerance of Extreme Whole-Body Hyperthermia with Water-Filtered In£rared-A Radiation The extreme whole-body hyperthermia is generally carried out in the form of systemic cancer multistep therapy (sCMT).^ The sCMT developed by M. von Ardenne consists of the three main steps, extreme whole-body hyperthermia, induced hyperglycaemia and relative hyperoxaemia which are usually combined with a chemotherapy protocol adapted to the respective tumour entity. ^^ In the meantime it has become general consensus that an extreme whole-body hyperthermia already has to be concomitant as supportive measure for

Extreme Whole-Body Hyperthermia with Water-Filtered Infrared-A Radiation

241

Figure 6. Whole-body hyperthermia system for extreme whole-body hyperthermia with water-filtered infi-ared-A radiation of the type IRATHERM 2000 (Producer: Von Ardenne Institut fur Angewandte Medizinische Forschung GmbH Germany). the effective supply of the healthy tissue at an increased metabolic rate with a high blood sugar level and an increased oxygen rate. The "good systemic tolerability" of extreme whole-body hyperthermia in the form of sCMT with water-filtered infrared-A radiation with only "minimal side effects" had already been proven for the first time in 1994 on 103 patients in the scope of a phase I clinical study. ^ ^ The cancer

SB *

§§

IJ Figure 7. Temperature-time chart with the course of 4 body temperatures (Therapy No. 880, Clinic for systemic Cancer Multistep Therapy Dresden, Germany).

242

Hyperthermia in Cancer Treatment: A Primer

patients with metastasised and/or recurrent primary tumours received conventional pretreatment and were in a stage of progression. During the hyperthermia phase the patients were sedated by modified neuroleptic analgesia in a state of spontaneous respiration. This result was confirmed 5 years later by a phase I/II clinical study of another workgroup which sedated the patients by intubated anaesthesia (total intravenous anaesthesia).^^ It confirmed that sCMT does not lead to any serious or sustained organ dysfiinction and can therefore be regarded as a "safe therapy". The side effects displayed in Table 1 represent a therapy period of approximately 15 months which had been preceded by approximately 600 therapies with extreme whole-body hyperthermia.^^ In this respect a routine can be accepted in the therapy method. All the cancer patients had metastatic cancer and were in a reduced general condition before treatment began.

First Clinical Results So far only few clinical studies on the therapy of cancer patients with extreme whole-body hyperthermia using water-filtered infrared-A radiation have been publicised. Most of the cancer patients were in an advanced stage where there was a progression that was no longer controllable by the conventional methods of oncology. In the scope of a retrospective observation study all the sCMTs carried out in a clinic in the period 12/1990 to 12/1995 were evaluated ace. to UICC criteria usually in combination with adapted chemotherapies (ChT). 490 sCMT-one time therapies of random cancer patients (0 55 years) with different tumour entities were evaluated. The average body core temperature in the 60 min plateau was 41. 9 ± 0.3°C, the maximum blood sugar concentration in the temperature plateau was 27.0 ± 3.7 mmol/1. Figure 8 displays tumour entities evaluated depending on UICC criteria of which at least 15 patients received therapy. In a considerable share of patients the progression of the disease (PD) could be brought to a temporary standstill (NC) or even turned into a response (MR, PR, CR). The statistics of findings was made in a continued observation period of at least three months by two restagings at intervals of at least 4 weeks. Furthermore, in the scope of a prospective phase I/II study at the University Clinic Virchow, Berlin, patients with metastatic colorectal cancer were treated with sCMT in combination with ChT.^^ The extreme whole-body hyperthermia was also implemented applying water-filtered infrared-A radiation at 41.8°C body core temperature for 60 min. Furthermore, an induced hyperglycaemia of approx. 22 nmiol/l and a relative hyperoxaemia by the inhalation of an air mixture enriched by 50% oxygen was set. 8/19 patients responded to three therapy courses ChT (folinic acid + 5-FU + mitomycin C) (PR) and obtained three further courses of ChT alone (control group). 10/19 patients who had not responded to ChT (NC, PD) received three additional courses of ChT combined with sCMT (test group). One patient who did not respond to initial ChT (NC) declined sCMT therapy. The result of the therapy showed that a PR was induced in 3/10 patients and in a further 6/10 patients a PD could be prevented. Furthermore, despite the negative selection of the patients in the test group, a fairly similar course of progression-free survival was observed in the test and control group (Fig. 9). In the scope of a pilot study 19 patients with metastasised adenocarcinomas (breast n = 7, ovarian n = 5, colorectal n = 7) received therapy with the above- mentioned procedure of sCMT in combination with various ChT. All patients were refractory to standard ChT and with progressive disease. As a result of the therapy 9/19 patients showed a PR, 7/19 an NC. 3/19 patients showed further tumour progression. The treatment of 2/2 patients with refractory germ cell tumours by the above mentioned procedure of sCMT combined with ChT (ifosfamide, carboplatin, etoposide) displayed a PR in both cases after three courses. ^^ This result and further observations of the course taken were encouragement enough to carry out a phase II study, which has not been concluded yet. With extreme whole-body hyperthermia, but without making use of water-filtered infrared radiation, seven phase II studies and four phase III studies are being carried out in Germany (2004) apart from the named clinical studies. ^^

Extreme Whole-Body Hyperthermia with Water-Filtered Infrared-A Radiation

243

Table 1. Side effects in 112 sCMT therapies of cancer patients in the stage of conventionally uncontrollable progression from 09/1995 to 12/1996 (figures = number of therapies) 1 1

Toxicity 4 - 6 Weeks after sCMT

Maximum Toxicity Posttherapeutic 1

2

De gree of V^/HOTox city 1 2

1 ^

1 ^

Haematoiogic Toxicity Leucopenia

11

20

11

7

Thrombopenia

6

10

10

1

Hb

8

7

Hemorrhages

1

5

10

2

1

1

4

1

3

1

1 ^

2

1

1

Gastrointestlnai Toxicity Nausea/Vomiting

3

1

Diarrhoea Obstipation Stomatitis

8

Renal Toxicity Creatinine

3

Urine

1

2

3

1

Laboratory Bilirubin

3

1

1 3

Gamma-GT Alkaline Phosphatase

1 1

2

Neurological Toxicity Peripheral Toxicity

5

6

5

2

1

1

others Skin Toxicity Irradiation Field

13

13

3

1

Caused by Bedding

21

22

6

1

1

1 1

1

1

27

42

1

2

Phlebitis Hair Loss Fever

1

Infections

3

4

1

Pains

3

7

9

Pulmonary Toxicity

1

1

1

Cardiac Toxicity Cardiac Rhythm 1

Cardiac Function Arterial Hypotension Allergic Reaction Arthralgia

1

2

1

4

1

244

Hyperthermia in Cancer Treatment: A Primer

n= 16

35

16

20

101

21

103

22

17

24

41

100

% 80

ffl ffl

60

QNC 40 I MR, PR, CR

20

Figure 8. Individual evaluations related to diagnosis according to UICC criteria in tumour entities with > 15 patients; 5% confidence interval for nonPD-PD limit.

chemotherapy (6 cycles) + SCMT

100

uiu

80H

k

> (0

60H

o

40H

Log-Rank: p=0.44 Wilcoxon: p=0.87 chemotherapy + sCMT

chemotherapy alone

2

20i

•~T-

10

20

30

40

50 weeks

60

—r~ 70

80

90

Figure 9. Progression-firee survival in test and control group. Note: The graph does not represent a comparison of similar patient groups: all patients treated with sCMT plus ChT previously had not responded to three courses of conventional ChT. All patients treated with ChT alone had a PR after three courses. (From Hildebrandtetal.^5)

Extreme Whole-Body Hyperthermia

with Water-Filtered Infrared-A

Radiation

245

5- I

8 &S

8^i

Figure 10. Temperature/pulse - time chart of a therapy with water-filtered infrared-A radiation between fever range hyperthermia and extreme whole-body hyperthermia. Hyperthermia equipment I R A T H E R M 1000 (Therapy No. 2, Clinic Eubios Griinhain, Germany).

Even though this contribution primarily deals with whole-body hyperthermia, it should not remain unmentioned that the water-filtered infrared-A radiation is also suitable for the implementation of moderate hyperthermia or fever range hyperthermia. In this case the patients are treated with a "temperature dose" of 39°C/3 h up to 40°C/6 h. Currently research groups around J. Bull, and W. Kraybill are working in this field with unfiltered heat radiation, whereby the patients are heated in a chamber. The water-filtered infrared-A radiation in an open equipment design, on the other hand, allows for the one-sided heating of the patient - fever range hyperthermia. Nevertheless, a quick increase in temperature at good tolerance and controllability is given of the temperature level aimed at. The temperature/pulse time chart with a fever range hyperthermia above the aimed at temperature displayed in Figure 10, shows the good controllability of the hyperthermia with water-filtered infrared-A radiation. Furthermore, the heat radiation concentrated on the patient can be individually adjusted to spot and intensity.

Conclusions The tolerance of the water-filtered infrared-A radiation to realise a whole-body hyperthermia in the form of sCMT in clinical routine could be proven by phase I studies. Initial clinical results allow for the assumption that this method could intensify the effect of ChT, as could be displayed in the example of patients with metastatic colorectal cancer and adenocarcinomas refractory germ cell tumours with small case numbers. The still unsatisfactory situation of available data for the proof of effectiveness and first positive results are an encouragement to carry out higher-grade studies. It is still absolutely unanswered as to which extent this method could decrease the chance of formation of metastases if, for instance, implemented post-operatively.

References 1. Malten H . Die Lichttherapie. Munchen: Bergmann, 1926:40-60. 2. Bachem A, Reed CI. The penetration of light through human skin. Amer J Physiol 1931; 97:86-91. 3. Henschke U. Biologische und physikalische Grundlagen der Rot- und Ultrarotstrahlentherapie. Strahlentherapiel939; 66:646-662. 4 Wust P, Hildebrandt B, Sreenivasa G et al. Hyperthermia in combined treatment of cancer. Lancet Oncol 2002; 3:494. 5. Ardenne Mvon, Kriiger W. Combined whole-body and local hyperthermia for cancer treatment: C M T selectotherm technique. In: Gautheria M, Ernest Albert U, eds. Proc Sympos Biomed Thermology, Strasbourg 1981. New York: Allan Liss, 1982:705-713.

246

Hyperthermia in Cancer Treatment: A Primer

6. Borchert R, Jubitz W. Infrarottechnik. Berlin: Verl Technik,1958. 7. MefFert H, Hecht HC, Gunther H et al. Biophysikalische Ergebnisse des Klinischen Tests der IRA-Therm-Hyperthermietechnik der 2. Generation ThermoMed 1990; 6:71-78. 8. Wust P, Riess H, Hildebrandt B et al. Feasibility and analysis of thermal parameters for the whole-body hyperthermia system IRATHERM 2000. Intl J Hyperthermia 2000; 4:325-339. 9. Ardenne Mvon. Principles and Concept 1993 of the systemic Cancer Multistep Therapy (sCMT). Strahlenther. Onkol 1994; 170:581-589. 10. Ardenne Mvon. Systemische Krebs-Mehrschritt-Therapie. Stuttgart: Hippokrates Verls^, 1997. 11. Steinhausen D, Mayer WK, Ardenne Mvon. Evaluation of systemic tolerance of 42.0''C infrared-A whole-body hyperthermia in combination with hyperglycemia and hyperoxemia: A phase-I study. Strahlenther Onkol 1994; 170:322-334. 12. Kerner T, Deja M, Ahlers O et al. Whole-body hyperthermia: A secure procedure for patients with various malignancies? Intensive Care Med 1999; 25:959-965. 13. loccit. 10: 219. 14. loccit. 10: 195 ff. 15. Hildebrandt B, Drager J, Kerner T et al. Whole-body hyperthermia in the scope of von Ardenne's systemic cancer multistep therapy (sCMT) combined with chemotherapy in patients with metastatic colorectal cancer: A phase I/II study. Intl J Hyperthermia 2004; 3:317-333. 16. Bremer K, Meyer A, Lohmann R. Pilot study of whole-body hyperthermia combined with chemotherapy in patients with metastasised pretreated progressive breast, ovarian and colorectal carcinomas. Tumordiagnostik and Therapie 2001; 22:115-120. 17. Hildebrandt B, Wust P, Loffel J et al. Treatment of patients with refractory germ cell tumors with whole-body hyperthermia and chemotherapy. In: ESHO 1999 September 1-4. Rotterdam, 1999:66. 18. web site of "Interdisziplinare Arbeitsgruppe Hyperthermie / lAH" (sub organisation of German Cancer Society), 2004. www.hyperthermie.org. Zentren / Suche nach Karzinomen. 19. Bull JMC, Nagle VL, Scott G et al. A phase I study of optimally-timed Gemcitabine + Cisplatin/ Interferon-a combined with long-duration, low-temperature whole-body hyperthermia. In: ESHO 2001 May 30 - June 2. Session V Verona, 2001:67. 20. Bull JMC, Glenna LS, Strebel FR et al. Update of a phase I clinical trial using fever-range whole-body hyperthermia + Cisplatin + Gemcitabine + Metronomic, low-dose Interferon-a. In: 9th ICHO April 20-24. St Louis Missouri 2004:68. 21. Kraybill WG, Olenki T, Evans SS et al. A phase I study of fever-range whole body hyperthermia in patients with advanced solid tumours: Correlation with mouse models. Intl J Hyperthermia 2002; 3:253-266.

CHAPTER 20

Meets of Local and Whole Body Hyperthermia on Immunity Gian Franco Baronzio,* Roberta Delia Seta, Mario D'Amico, Attilio Baronzio, Isabel Freitas, Giorgio Forzenigo, Alberto Gramaglia and E. Dieter Hager Around every tumor there is a patient—Blacky Natl Cancer Inst Mono^ 1972; 35:276.

Abstract

I

n this review, we summarized the historical and experimental basis of cancer immunity and the role of fever and of artificial elevation of temperature on immunity. The interactions of heat in vitro and in vivo on cytotoxicity of immune competent cells are discussed as their positive contribution on the various cancer immunotherapeutic strategies. Furthermore we have described the link existing among heat shock proteins, Toll like receptors and innate immunity justifying the use of temperature elevation for treating cancer. The disputed and life-threatening effect of local and whole body hyperthermia on metastasis is also reviewed.

Introduction The role of the immune system in eradicating malignant cells is not yet clarified; however spontaneous regression of some cancers has been demonstrated to be associated to the induction of fever and activation of immunity. ^'^ The crucial importance of fever in these regressions justifies the attempt to induce artificial thermal elevation of body temperature (hyperthermia) for mimicking natural fever effects on cancer.^'^

Tumor Immunity Background Tumor regression in vivo is mediated by a complex interplay between two main mechanisms: innate and adaptive immune response (Tables 1,2), that is involved with the immune recognition of cancer cells. Innate mechanism [involving soluble and cellular components, (Table 1)^'^^ may trigger inflammatory events in the tumor microenvironment and in presence of a local adequate cytokine combination (IL-2, IL-12, IL-18, IL-23), stimulate dendritic cells (DCs),^ the most specialized antigen presenting cells (APCs), to react against tumor specific surface antigens (TAAs).^"^' After engulfment by DCs, TAAs are presented to naive T cells associated to Major Histocompatibility complex (MHC).^^ Naive T cells activation occurs when the antigenic peptide-MHC complex interacts with the T Cell Receptor (TCR). However TCR receptor binding is not sufficient for a full activation ofT cells unless *Corresponding Author: Gian Franco Baronzio—Family Medicine Area, ASL-01 Legnano; Radiotherapy Unit, Policlinico di Monza, Via Amati 11, 20052 Monza (Mi), Italy. Office address: P.O.B. 5, 20029 Turbigo (Mi), Italy. Email: [email protected] Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio

and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

248

Hyperthermia in Cancer Treatment: A Primer

Table /. Principal cellular and soluble antitumoral components of innate immune system • • • • • •

Dendritic cells Neutrophils Macrophages NK cells Cytokines Chemokines

• Complement • Fever • Defensins • Interferon producing cells • B cells • 7 6 ! cells

costimulatory molecules interact with the specific ligands on the surface of APC. The presence or absence of costimulatory signals like B7-1(CD80), B7-2(CD86) and CD40/CD40L), determines whether immune response becomes anergic or tolerant. CD40/CD40L is expressed transiently following TCR activation on the surface of CD4^ cells and it is a key molecule in mediating the activation of B cells and in controlling CDS T cells. Antigens can be associated to M H C I or MHCII class complexes and are presented by DCs to TCR of CD8^ and CD4^ T cells, respectively associated to the proper costimulatory molecules (Fig. 1). Both CD4^ and CDS"^ cells after activation and costimulation produce a series of cytokines that differentiate T- Helper (CD4+) lymphocytes in two subpopulations (TH 1, T H 2 cells). T H 1 cells produce IL-2, IFN-y, TNF-a and granulocyte macrophage colony stimulating factor (GM-CSF) that increase the activity of macrophages, and the expression of MHC class 1 molecules on the surfaces of CD8+ cells. T H 2 secretes another group of cytokines IL-4, IL-5 and IL-10, that induces naive B cells to produce antibodies. The shifting towards T H 2 pattern has recently been associated with an increased tumor metastasisation and a decreased survival in many human and animal neoplasia (Fig. 4). 12,14,17.18 CD8+, cytotoxic T cells (CTLs) are the major effectors of tumor regression; ^^ however CD4+T cells collaborate to their activation. Once activated CTLs do not need costimulation, since MHC-1-bound antigen is sufficient. For eliminating target cells (neoplastic cells) CTLs use three effector molecules: Perforins, Granzyme and Fas ligand. Associated to these killing mechanisms, CTLs secrete specific cytokines, such as: IFN-y, TNF-a andTNF-p. This pattern of cytokines plays an important role in the activation of macrophages which can exert a direct tumor cytotoxic or, conversely, stimulate tumor progression, depending on the tumor microenvironment.^^ Other cells morphologically and fiinctionally distinct, such as natural killer cells (NKs), macrophages and neutrophils, use pattern-recognition receptors and other cell-surface molecules to detect tumor cells directly. '^° Differently from T cells, NKs inhibit tumor growth in a MHC-non restricted manner. Frequently tumor cells (like stressed cells) express on their surfaces different glycoproteins (MICA and MICB) which ftmction as ligands for NKG2D receptors on NK cells. Once activated, these receptors stimulate NK cell activity.^^ By contrast, DCs use CD36 and (XvP5 int^rin to recognize and phagocytize apoptotic tumor cells. Apoptotic tumor cells in turn release heat shock proteins that, after specific interaction with CD91 receptors on DCs, induce their maturation: thus providing a tailored inunune response (see Figs. 1,4).^^

Table 2. Principal antitumoral components of adaptive immune system • • •

Dendritic cells Neutrophils Macrophages

• B Cells • CD4^ • CDB^

Effects ofLocal and Whole Body Hyperthermia on Immunity

249

INNATE ADAPTIVE IMMUNITY INTERACTIONS AND T-CELLS MEDIATED IMMUNE RESPONSE

iNNATE IMMUNITV'

ADAPTIVE IMMUNITY

» Inhibitory effect > Stimulatory effect

Figure 1. In this diagram the various mechanisms eUcited by stress for stimulating innate and adaptive immunity against cancer are illustrated. DCs: dendritic cells; CTL: cytotoxic T lymphocytes; TCR: T-cell receptor; MHC: major histocompatibility complex; Abs: antibodies; FRs: free radicals; TGFB: transforming growth factor-P; PGE2: prostaglandins of E2 type.

Tumor Microenvirontnent as Regulator of Cancer Immunity and Immune Evasion The switch to an angiogenic phenotype is a fundamental determinant of neoplastic growth and tumor progression. This occurs following the local secretion of specific angiogenic cytokines, especially vascular endothelial growth factor (VEGF).^^'^^ Recently VEGF has been demonstrated not only to promote angiogenesis but to suppress anti-tumor immune response principally by hampering leukocyte recruitment^^ and by inhibiting CD34'^ cell differentiation into dendritic cells."^^' Tumor immunotherapy success or failure is strongly dependent from leukocyte migration into tumor area.^'^^'^^ In fact, leukocytes and macrophages, before reaching the target tissue (tumor site), undergo a series of sequential steps during extravasation from blood into tissues: tethering, rolling, adhesion and diapedesis. Among these steps leukocytes adhesion to tumor endothelium is critical and it occurs through the expression of specific adhesion molecules, such as: L-selectin ligands, alpha-4beta-7 integrin adhesion receptors (a4b7) and mucosal addressin cell adhesion molecule-1 (CAM-1). Several animal experiments have shown that in presence of VEGF a significant decreased expression of these adhesion molecules occurs, determining a decline in leukocyte infiltration into the tumors mass.'^^''^^'^ Besides, this impaired tumor infiltration by immune competent cells, tumor environment (hypoxia, acidic pH) itself unfavourably modifies T lymphocytes, NK cells and macrophages activity.^^ In fact, this kind of tumor environment alters the pattern of secreted cytokines towards an immunosuppressive T H 2 pattern, permitting tumor escape from immune surveillance.'^^'^'^

250

Hyperthermia in Cancer Treatment: A Primer

Heat Shock Proteins (HSPs), Their Role in Antigen Presentation and in Cancer Immunity When cells are submitted to a variety of stressful events (e.g., heat, hypoxia, glucose deprivation), there is a rapid and coordinated increase in the expression of a group of proteins, the so-called heat shock proteins (HSPs).^ HSPs are one of the most conserved groups of proteins throughout evolution and are classified into several families according to their molecular weight in kilodaltons (e.g., HSPs 100, 90, 70, s60, s40) and their compartmentalization inside the cell (cytosol or endoplasmatic reticulum, mitochondria).^^'^^^® HSPs fulfil different important intracellular processes, such as protein synthesis, folding and they are activated by a specific set of genes induced by different physical stress such as elevated temperature, hypoxia, glucose deprivation and oxidative reagents.^^ Linearly at molecular level, heat stress increases the synthesis of HSP 70 until a certain threshold temperature that varies according to cell type. Beyond this threshold temperature their synthesis is inhibited and an exponential cell death follows. ^' Initially, the role of HSPs, peculiarly of HSP70, appeared to be implicated in the thermotolerance."^ ^ Recendy, they have been recognized to activate the immune system becoming a specialized carriers of antigenic peptides in vivo as well."^^' In fact, Srivastava et al have established that HSPs are not immunogenic per se, but when they are complexed with antigenic peptides, become powerful immunogens. In fact it has been found that cancer derived HSPs are highly specific and this specificity is associated with the agglomerate HSP/peptide. Once HSP-complexes (MCHl/MCH2+HSPs) are exposed at the outer surface of cancer cells, they interaa with macrophages and dendritic cells through specific surface receptors.^' ^ HSP70 binds to the surface of monocytes through the Cluster of Differentiation (CD) 14 (CD 14) receptor, whereas gp96 binds with the a-2 macroglobulin/LDL receptor related protein or CD91. Furthermore HSP60 has been demonstrated to be a ligand for the Toll-like receptors 4 (TLR4 and TLR2) complex on macrophages."^'"^ These data support the data that APCs (macrophages, dendritic cells) have evolved receptors for detecting danger signals (HSPs complexes) released during neoplasia. The exposure of the HSP chaperoned peptides by the MCHl and MCH2 molecules to macrophages or dendritic cells triggers a secretion of inflanunatory cytokines and costimulatory molecules, such as: IL-6, IL-12; TNF-a, B7 that induce the maturation of DCs towards theTn 1 phenotype. '^' ^ Their association, with a broad array of peptides generated within cells, make HSPs a good candidate for cancer vaccines."^^ In fact HSP-peptide complexes, isolated from a patients tumor, can be utilized as tailored patient specific antigens, which would avoid the search for specific epitopes. HSPs, isolated from cancer cells but not those derived from normal cells, can generate an immune response, as observed by Tamura and Srivastava. The potentiality of HSPs in tumor eradication has been validated in more than ten types of tumor models of different histologies and in different animal species, demonstrating that: (a) microgram quantities of HSPs are sufficient to generate substantial inunune response; ' (b) the immunogenicity of antigens expressed by dying cells occurs via necrosis or apoptosis. Among the two dying mechanisms only necrotic cells or heat stressed cells have been demonstrated to be able to elicit a tumor-specific inmiunity.^^'^^ Several non randomised clinical trials with heat shock protein-peptide complexes on human cancers are actually in progress.

Therapeutic Modalities Immunotherapy may be either active or passive, specific or nonspecific depending on the process of host immune system stimulation (see Table 3).

Passive Nonspecific Immunotherapy (LAK Cells, TIL) Passive adoptive immunotherapy involves the transfer of immune cells into patients from an external source. This cellular adoptive transfer consists in the generation of cells with antitumor activity obtained in vitro in presence of IL-2. According to their derivation they

Effects ofLocal and Whole Body Hyperthermia on Immunity

Table 3. Classification

of cancer

Specific

251

immunotherapy Nonspecific

Active Tumor cells based vaccines Tumor antigen vaccines DCs vaccine

Bacille Calmette-Guerin (BCG) Corynebacterium parvum Detox Viral vectors: Levamisole Retroviruses Coley's Toxins [CTs] Adenoassociated Cytokines: Viruses (AAV) Interferon-a (I FN-a) Gene therapy Non viral vectors lnterleukin-2 (iL-2) Naked DNA Tumor necrosis factor-a (TNF-a) Liposomes GM-CSF (Cationic lipids [lipoplexxes] and Pegylated Liposomes) HSPs Passive Monoclonal antibodies LAK cells (Lymphokine-activated natural killers) Tumor-infiltrating lymphocytes TIL (tumor infiltrating lymphocytes) Activated T cells Allogenic stem cell trabsplantation cytokines : Interferon-a (I FN-a) lnterleukin-2 (IL-2) GM-CSF Tumor necrosis factor-a (TNF-a) Flt3-L

can generate Lymphokine-Activated Killer Cells (LAK cells) or Tumor Infiltrating Lymphocytes (TIL). LAK cells are generated by culturing in vitro patient peripheral blood leukocytes with IL-2. These LAK cells are injected back into the patient with IL-2. Adoptive LAK therapy has been applied to advanced cases of renal carcinoma and melanomas with variable efficacy. TIL cells are generated fi-om mononuclear cells obtained firom infiltrates around the tumor after surgical resection. This approach, even if more specific, has yielded only limited success. ^^

Active and Passive Nonspecific Immunotherapy (Cytokines, IL-ly INF-CXy GM-CSF) IL-2 has been the first cytokine used alone or in combination with LAK cells for the treatment of different types of metastatic cancers. Patients with metastatic renal cell carcinoma and melanoma receiving IL-2+LAK had a higher rate of complete response. IL-2 has been administered with different regimes and doses demonstrating an elevated toxicity. ^"^'^"^ Numerous other cytokines have been identified and tried clinically, however only Interferon-a (INF-a) and Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) are currently used given the high toxicity of Interferon-y and IL-12.^'^'^'

Active Specific Immunotherapy (Vaccine, Gene Therapy, Heat Shock Proteins) Serological identification of antigens by recombinant expression cloning technique (SEREX) has recently permitted to detect more than 1500 TAAs holding a specific antitumor activity. ^^'^

252

Hyperthermia in Cancer Treatment: A Primer

Tumor vaccination (TV) is a therapeutic form of therapy involving patients with detectable disease and it is used for triggering a robust, appropriate and specific immune response towards a well characterized TAAs, avoiding immune-tolerance and providing a long lasting immune response. ^'^^ The first tumor vaccines were obtained by irradiating tumor cells. The obtained results were not enthusiastic, inducing several authors to combine tumor vaccines with nonspecific immune modulators, such as BCG, New Casde Disease virus (NDV) and Detox; however the survival rate was comparable to the group treated with chemotherapy. To ameliorate the results, newer vaccines, including allogenic or autologous tumor cells, were genetically manipulated in order to produce a stronger inmiune response. Tumor cells, were encoded with different cytokines, however only those engineered to produce GM-CSF proved to induce tumor rejection.^^ A large number of clinical trials, involving different strategies, are actually being conducted and have been reviewed elsewere. '^^ Some of them will however be discussed for a positive interaction with hyperthermia treatment (see Table 7). These are: dendritic cells, gene therapy and heat shock proteins. Dendritic Cells Dendritic cells (DCs) are a distinct population of leukocytes that play a crucial role as antigen presenting cells and as initiator of antitumor immunity.^^ DCs reside mainly in peripheral nonlymphoid tissue, after antigen uptake; however they migrate to the draining lymph node to present antigens to T cells. Preclinical studies have demonstrated that antigen pulsed DCs can generate a potent antitumor activity. DCs are artificially loaded with antigens by different techniques and can stimulate both innate and adaptive immune system.^'^ Many clinical trials, testing dendritic cell-based vaccines are in progress and have proved a partial clinical efficacy. ' In every case, preclinical studies have also demonstrated that the direct intratumoral injection of ex vivo-generated DCs can avoid the need for a tumor antigen loading ex vivo by improving antigen presentation or migration to lymph nodes. A required condition is that injected DCs must be genetically modified in vitro to produce cytokines and chemokines, such as: IL-2, IL-7, IL-12, CD40L, GM-CSF, lymphotactin or secondary lymphoid tissue chemokine (SLC).^^'^^ The presence of these cytokines may render DCs more able to capture and process TAAs. This overcomes the increased immune suppressive factors (IL-10, VEGF andTCFp PGE2) present in the tumor medium.69-^2 Gene Therapy Gene therapy is defined as the method of delivering genetic material into a cell, by altering the cellular phenotype permanendy or transiendy.^^ The efficient and precise targeting of a gene to specific cells or tissues remains the technical hurdle of gene therapy. The delivering methods consist in use of viral or nonviral vectors and in vivo or ex vivo gene therapy approach.^"^ The ex vivo approach involves the removal of patient's own cells and the readministration after genetic modification. By contrary, in vivo, therapy involves the direct injection of genetic material into patients. The most promising designed vectors for in vivo therapy are viral vectors. The viral vectors enter into cells by receptor mediated endocytosis and escape from endosomes after DNA delivery to nuclei. They have some disadvantages such as: (a) patient inmiune recognition of viral proteins with a consequent immune attack and destruction of infected cells, (b) restriction in size and quantity of transgene, (c) inability of repeated viral administration.^^'^ Most clinical studies have been carried out using retroviruses or adenoviruses as transfer vector with various advantages and disadvantages.^'^^ The targets of viral therapy can be: inactivation of oncogenes (i.e., ras, c-myc, c-erB-2, abl, bcl-2);'^'^ genes involved in tumor progression (i.e.. Triple-helix formation); immunomodulatory genes encoding cytokines (TNF-a, IL-2; GM-CSF, INF-y) costimulatory molecules (i.e., MCH molecules, CD40ligand, B7, Cd28); angiogenic factors (i.e., VEGF)^°'^5 ^ j tumor associated antigen genes.

Effects ofLocal and Whole Body Hyperthermia on Immunity

253

Non viral vectors (naked DNA, lipids, liposome) make use of physical mediods of gene transfer. They are free of the side effects of viral vectors, but they suffer of specificity/^ Ex vivo gene delivery is the best system for such vectors. Naked DNA in the form of plasmid can be injected into muscle tissues or conjugated with gold particles and bombarded into the tissues. Muscle tissue injection is the most effective delivery method of administration. Repeated injections determine an improvement in therapeutic gene expression but the effect does not last for long.'^^'^'^ Another route of DNA administration is the use of lipid vehicles. Two classes of lipid aggregates have been used: (1) cationic lipids [lipoplexes]; (2) stabilized liposomes. Lipoplexes are positively charged and interact with negatively charged DNA forming a stable complex. As outlined by Clark,^^ cationic lipids have many benefits, such as: easy and inexpensive way of production, non toxicity and potential of delivering large quantity of polynucleotides into cells. However, in order to develop commercially these vector systems, several barriers must be overcome, especially formulation and stability of manufacture.^^ The presence of an excessive number of particles positively charged favour precipitation and aggregation. To circumvent this problem of flocculation, hydrophilic polymers like polyethylene glycol polymers (PEG) have been created. ^^ These sterically stabilized liposomes (PEG liposomes) allow efficient encapsulation of DNA and oligonucletides. They also permit to prepare well-defined and uniform particles suitable for drug carrier.^^'^^ Furthermore PEG liposomes show an improvement in half life time due to a reduced renal and cellular cleareance. Also they show an enhanced protection from proteolysis with reduction of toxicity.^ ^'^'^ Other types of liposomes based on polyethyieneimine (PEI) have proved to be stable to nebulization and to be efficiently delivered as aerosol DNA plasmids to lung parenchyma,^^'^ exhibiting a greater concentration into lungs when compared to other route of administration such as intravenous route.^'^^ Concluding, vaccine therapy is a tailored specific therapy. However, critical issues to overcome are the choice of the delivery system and the identification of singular epitopes. Heat Shock Proteins Heat shock proteins meet the request for searching specific epitopes. In fact, they have been implicated at multiple points in the immune response, including initiation of proinflammatory cytokine production, antigen recognition and processing, and phenotypic maturation of DCs. ^ A preliminary clinical study on colorectal tumor metastasised to liver treated with autologous HSP-96, has clearly demonstrated important points.^^ Namely, HSP-complex induced a significant increase in committed lymphocytes, as response patients have a better clinical outcome as overall survival and as disease free survival and no toxicity was observed. '^^ The importance of hyperthermia in generating HSPs with the aim of immunization will be described later.

Active Nonspecific Immune Therapies: Coley Toxins (CTs) In 1868 W. Busch in Germany concluded that fever induced by certain bacteria from erysipelas can cause tumor regression or cure cancer. This was suggested after the observations on a patient with a soft tissue sarcoma of the neck infected by erysipelas. The causative agent (streptococcus) at that time was not identified. Subsequendy, in 1892, a young American surgeon W B. Coley, unaware of Busch's findings, observed a regression of soft tissue sarcomas in a patient infected by erysipelas. Stunned by that finding, he searched the medical literature and found many publications confirming his observation. '^ Coley prepared initially a culture of streptococci that he injected at the tumor site with encouraging results. He even noted that the presence oiSerratia marcenscens could enhance the virulence of streptococci and an injection remote from the tumor site coidd equally result in tumor regression. After these observations Coley incorporated Serratia marcenscens into the streptococcal vaccine just obtaining the so called "Coley s toxin" or "Mixed Bacterial Vaccine" (MBV). The intravenous route was the most effective; the toxin dose was considered sufficient only if accompanied by fever (39-40°C). Fever and the sustained pyrexia, now recognized to be elicited by tumor necrosis alpha (TNF-a) and by other cytokines, ^'^'^ were considered the critical points in the tumor regression. ' It was also observed that those who developed the highest fever were most often the ones with the longest survival.

254

Hyperthermia in Cancer Treatment: A Primer

Table 4, Effects of thermal component of fever on innate and adaptive immunity Immune Response

<39°C

Chemotaxis and emigration into inflamed or tumor tissue PMN numbers

T

Phagocytosis (free radicals++)

> 40°C

Animal Studies

T-cells CD4 CD4/CD8

t T

X

X

t(t++)

X

X X X X X X X X X X X

t

B cells DCs HSPs

U U CD3«i

»

r

» =4. » T t

Ref. 97,129

(T++) NK cells

Human Studies

tt

X X

X

97 133 97129 132 112 116 107 107 121 107 121 107 186,194 223

t : increased response; i: decreased response; PMN: polymorphonuclear leucocytes; **Nottrue for all bacterial species; and antibodies secretion is increased for a beneficial effect on T helper cells; t + : means increased response in presence of a biological response modifier; «: no variations.

Coley should be considered the first scientist to apply induced hyperthermia (fever) as immune treatment. ^'^ He noted that tumor regression was obtained only in presence of fever. Since then the interaction between hyperthermia (in whatever way obtained) and inmiunity has not been completely elucidated.^ To our opinion, a better comprehension of the effects of heat on inmiunity is obtained by understanding the effects of thermal component of fever on immunity (see Table 4).

EfFects of Thennal Component of Fever on Immune System Often in the presence of tumor and microrganisms, the host responds by increasing body temperature.^'^ Fever is a complex neuroendocrine adaptive response due to an increase in the set-point temperature regulator found in hypothalamic area. After their entry into organism, bacteria or viruses induce macrophages to produce a series of proinflammatory cytokines such as: interleukins (ILs) -1,2,6, tumor necrosis factor alpha (TNF-a), Interferon-a (IFN-a), IFN-y. These cytokines, with an additional mediator [prostaglandin E2 (PG E2) cyclooxygenase 2 prostaglandins derivate (COX-2)], act on the thermoregulatory area and reset it to a higher level of temperature, producing the febrile response.^^'^^ Temperature elevation has been suggested to be beneficial for the host.^^'^^ It is unlikely that evolution has maintained such an expensive defensive metabolic mechanism, without a role.^'^ However, the role of temperature on immune defence is not completely understood. Different studies support the idea that temperature rise has a beneficial role, such as an improved efficiency of macrophage killing activity and an increase on survival in mice infected by herpes virus or with rabies.^ ^ Leukocyte adhesion to endothelium and emigration to the site of inflammation are positively affected by heat as the antigen-non specific defence systems (chemotaxis, phagocytosis.

Ejfects ofLocal and Whole Body Hyperthermia on Immunity

255

120i

100

^

80 H

!t

60

2 40

\

20

'

'

0

'

I'"''

2

I '

'

4

1

I

6

8

24

32

48

Hours after Fever Induction with MBV

Figure 2. Time-dependent exemplary induction of IL-1 in a patient after induction of fever up to 39.8°C with a biological pyrogen (Vaccineurin®). complement haemolysis) (see Figs. 2,3). Even antigen-specific activation, proliferation, cytokine expression diflFerentiation and antibody secretion by lymphocytes are affected by temperature. T cell responsiveness to mitogens, IL-1, IL-2 and antigens increase linearly until a temperature of 39°C; beyond this limit a decline is observed.^ Furthermore, it has been shown that antibody secretion in vitro is temperature-dependent and this effect declines in the absence of T-helper

Leucocytes 16000 1 14000 12000 " 10000 cells .^^^

/ul 8°°^ 6000 4000 • 2000 -

0

2

4

6

8

24

32

48

168

Hours after Fever induction with MBV

Figure 3. Emigration, homing, proliferation and activation ofleukocytes in a patient after induction of fever up to 39.8°C with a biological pyrogen (Vaccineurin®) depending on time.

256

Hyperthermia in Cancer Treatment: A Primer

Table 5. Effects of WBHT, and LHT on immune response Immune Response

WBHT

LHT

Endothelial T[L-Selectin,a ^P7] t(±IFN-^ adhesion molecules [ICAM-1]

t T T

Lymphocyte infiltration into tumor

t i(RT)«

t T T t TGCSF

t(IL-2, PHA)

t leukocytes

(40T-12h) (4rC-12h) (43X • 2 h) (42.5-44°-1/2 h) (45°C • 1/2 h)

THS I I I

Human Studies

Animal Studies X X

X X X X

t(PMCT)

Antigen presentation Neutrophils increase

TH

(39.8°C • 6-8 h) (42.5°C • 6 h) (42.5°C-3h) (43°C • 3 h) (39.5°C • 2 h) (38-39.5T • 2 h) (43°C • 3/4 h) (39.5°C • 2 h) (38-39.5°C • 2 h (41.8° • 2 h) (39.5-40°C • 6 h)

X X X X X X X X X X

Ref. 117 131 136 129 130 135 173 137 138 140 107 132 157 107 132 134 134

TH: time of exposure to hyperthermia and heating degree in "C; PHA: phytohemmagglutinin; GCSF: granulocyte colony stimulating factor, PMCT: percutaneous microwave coagula tion therapy; RT: radiotherapy; LL: lowtemperature long duration WBH; HS: High temperature short duration WBH;0: macrophages.

A summary of temperature effect on specific and non specific inmiunity is presented in Table 4. From it you can conclude, that temperature on the order of fever range (39-40°C) is the most favourable ones for the immune response.^"^'^^'^^ The effects of hyperthermia on the immune system are complex and pleiotropic and are dependent on temperature, time and microenvironment.

Effects of Induced Thermal Elevation (Hyperthermia) on Immunity Temperature application above the physiologic range (> 42.5°C) has been demonstrated to induce various effects on immune system (see Tables 5, 6).^'^'^^'^^^

In Vitro Studies NK and LAK cell cytotoxicity was determined in a standard 4 h chromium releasing assay using K-562 human erythroleukemic cells as target. Cell viability was measured by esclusion of Trypan Blue dye.^^ Some studies have analysed the leukocyte function in presence of temperature associated with IL-2, TNF-cx or Interferon.^®^'^^^ The purpose was to verify the possible abrogation of the immunosuppressive effects of temperature and the rescue on their activity by these cytokines.^^^'^^^ Fuggetta et al^^^ have evaluated in vitro the influence of hyperthermia (HT) (1 h, 42°C) on the cytotoxicity of IL-2 activated NK cells. Hyperthermia reduced the lytic activity of NK cells profoundly. The inhibition of this lytic activity has been demonstrated to be transient and not due to an aptosis but rather to an induced reduction of the effector cells. ^^^ Furthermore these authors, in agreement with others have observed that the heat treatment of target cells alone (K 562 and Daudi cells) did not alter their sensitivity to lysis. ^ ^"^

Ejfects ofLocal and Whole Body Hyperthermia on Immunity

257

few IT—

(NCNrsi'^cNCNrMvDrsi

00

i§

v D - ^ L n v D K O O O ^ O r - r s i r O L O O ^ rsiLoi-ni-nLnLni-ovD*^vO>^'^'^

rsi 3

0)

to CJ (0 S)

X X X

= 5

X

•T

t

•-

Q.

.9 -^ Q_ O

T-

T— - C

fsi rsi rsj

-C

t

1—

X u : U X X U p^ U U 00

u'

LO O


O

00 00 y

y

u

.

u uu 0

0

u uu

0

«? t T— O^

r o O^ m "^ ro "^

cT) r^ T— ^ ^ ^

S4 Z

_j

z

B

Ll_

t t T-:

^1 B •ro

..

dJ ~ =! c /r.O

o

b O ' ^ OS CU as - = "D Q. ^

>

< +

+ T

Q_

"o^

T

Ll_

^ LO

-^ m -"^

I

I-

-

I

W)_g . 1

'^^^ ^ "S c

•gis

c/) y-

oik

"IfE .. o

^ ^^ ^CJ -^ S^ V QLg

Ll_ ^

Q^

LL_ ,

H- U a i I -

I

z

-

z

U

Irsi CN

_ i o " I7 —' o "

+

00

^ ? c

ao-.i

aa O) I— ns (N O) 5£J —

^"^^ O -D ^ 3:

0

i

13 0) t/i

^ '^% •- o ^

o

1/5 Q. 9 c a; uL ^ a; Z t/) *Cy ir

-Si

s.

-^ ^ S ^ .>^"° :su .

DJD t

c/i t a; 9 -

2

^^ "^

y o

rt/i

258

Hyperthermia in Cancer Treatment: A Primer

LAK cell cytotoxicity has been studied by Shen et al.^ ^^ They have demonstrated that LAK cell induced cytotoxicity was temperature-dependent. It enhanced in presence of febrile temperature (< 40°C) but decreased by exposure to 1 h at 42°C.^^^ In accordance, investigations by Singh et al have shown that LAK cell cytotoxicity, as with NK cell induced cytotoxicity, decreased at temperature above 38.5°C. TNF mediated cytotoxicity however was significantly enhanced at 40°C.^^^ By contrast, Fritz et al have obtained a certain variability on LAK cell mediated lysis in presence of heat and INF-y.^^^ From the various studies analysed it appears that the immune suppressive effects of heat on NK and LAK cells become evident at temperatures above 39°C. The response of NK and LAK cells to hyperthermia is temperature and dose-dependent and not related to cell viability or to the absolute number of cells. ^^^'^^^'^^^ In fact, several authors have noted litde loss of cell viability at 39°C,^^'^^'^^^ whereas a decrease was demonstrated at temperature above 42°C.^^ The lytic function is more heat sensitive than the recognition and binding functions. The extent of recovery of activity after exposure to heat is inversely correlated with the temperature and the recovery time can be complete.^ ^^'^^^ Human and animal cells show a different behaviour during heat exposure. Human and animal NK cells show similar inhibitory behaviour after heat and seem the most sensitive among the immune competent cells. On the contrary, B cells are less affected by heat while murine lymphocytes, compared to the human counterpart, appear to be more heat sensitive.

In Vivo Studies Conditions in vitro cannot simulate those in vivo; therefore Kappel et al^^ investigated the behaviour of NKs and other immune competent cells of healthy volunteers during whole body hyperthermia (WBHT) at 39.5°C for 2 h in vivo conditions. NK cells cytotoxicity increased in temperature-dependent way. Cells incubated with IL-2 or INF-a had an enhancement in cytotoxicity and nmnber compared with control values. At temperature above 39.5°C a slow decrease in number of CD3^ was evident with no variations regarding CD 19"^ cells (B cells).^° More recendy, Atanackovic et al^'^^ have examined the following inmiunological parameters during WBH treatment: (A) behavior of CTLs with a panel of Cluster of Differentiation (CD) activation markers; (B) serum cytokines; (C) intracellular cytokine levels (D) capacity of these cells to proliferate.^^ They have classified the effects of WBH on patients immunity in different arbitrary phase defined by post treatment time. Immediately after WBH, a drastic increase of NK cells and CD56 CTLs was noted in peripheral blood. This phenomenon was transient and followed, after 3 h post WBH, by a short period of reduced T cell activity, indicated by diminished serum levels of soluble interleukins 2 receptors (sIL-2R). In this first phase, a short-lived increase (for the first 5 hs following treatment) in the serum concentration levels of IL-6 was found with a recovery to normal values after 24h. TNF-a increased significandy during the first 24 h, associated to a marked increase in the peripheral percentage of CTLs and CD56. CTLs, CD56, sIL-2R and lymphocytes expressing CD 69 markers reached their maximal concentration 48 h post WBH. CD69 is an antigen expressed early in the activation of lymphoid cells, and it is considered restricted to activated lymphocytes and undetectable on resting lymphocytes. CD 69 is normally induced, after stimulation, with mitogens or cytokines like INF-a, INF-y or TNF-a. In agreement, the intracellular concentrations in CDS cells and in serum of INF-y and TNF-a were found elevated 24 h post WBH in the 80% of the patients. Other authors have studied the production of other cytokines in the serum of patients treated with local hyperthermia (LHT) and WBHT or perfiisional hyperthermia (PHT). The studies are summarized in Table 6. During LHT no detectable increase in IL-1, IL-6 or TNF-a was observed^^^ whereas after WBHT serum levels of IL-1 and IL-6 were increased. ^^^ Robins et al^^"^'^^^ investigated why, during WBHT at 4l.8°C, an enhancement of radiation or chemotherapy response was found without a concomitant increase in myelosuppression. For this reason, they studied an expanded panel of serum cytokines in different patients and found an increase of IL-lp,IL-6, IL-8, IL-10, G-CSF and TNF-a within hours after WBHT.^^'^^^'^^^'^^^ Moreover, they reported that bone marrow cells stimulated the production of IL-1 p^ IL-6 and

Effects ofLocal and Whole Body Hyperthermia on Immunity

259

that TNF-a increased further their plasma levels. This interaction between tumor cells and cytokines, such as interleukin IL-6 resulted in a secondary induction in the bone marrow (BM) of IL-3 and GM-CSF. These factors are produced by BM stromal cells and byT lymphocytes. The plasma levels of the cytokine panel increased 1 h following WBH and diminished after every WBH application reaching the minimun concentration after 4 cycles. ^^^ Alonso et al reported a similar cytokine increase with the addiction of IL-2, TGF-p, INF-y and INF-a in patients undergoing extracorporal perfusion. ^^® A disagreement between the two groups about the TGF-p production has not been completely clarified. Some other aspects of immune response are affected by WBHT and LHT and are illustrated in Table 5. The concentration of some adhesion molecules such as ICAM-1, L-selectin and a P? have been demonstrated to be induced and increased both by LHT and WBHT, augmenting the homing effect into the tumor area of lymphocytes, leukocytes, neutrophils and macSeveral authors have tried to verify the possible modification of tumor cell immunogenicity after exposure in vitro to hyperthermia treatment. ^^'^'^^^ Dickson concluded that immunization and survival of mice with Ehrlich ascites did not increase after HT.^^'^ By contrast Mondovl obtained an increase in the immunogenicity and found that heated cells had an higher immunogenicity compared to cells treated with irradiation. The phenomenon was dependent on the length of treatment and the choice of temperature was extremely important. ^^'^^^ Recent studies on ratT-9 glioma cells by Ito et al^"^ seem to confirm Mondovis experiments. In fact, these authors have shown a significant increase in MHC class 1 antigen on the surface of heated cells 24 h after heating associated to an increased expression of HSP70. No modifications of other surface immunologic mediators, such as ICAM-1 or MHC class II antigen were noted. The in vivo growth of T-9 glioma cells, using immtmocompetent syngeneic rats (F344) was significandy inhibited and associate to a an increased cytotoxic specificity.

Specific Effects of Hyperthermia on Immune Therapeutic Modalities Several recent studies, suggest that hyperthermia is suitable as a complementary therapeutic modality for immunotherapy (see Tables 6, 7).

Radioimmunotherapy and Monoclonal Antibody Therapy An enhancement of therapeutic outcome of radio-immunotherapy and monoclonal antibody (MAbs) activity in combination with WBH and LHT respectively has been demonstrated. The tumor uptake of MAbs against different experimental tumors (colon cancer, ^ glioma, breast and prostate carcinomas^ ) increased from 12% in non heated tumors to 42% in tumors heated with local hyperthermia (41.8°C x 4 h) and persisted in the heated tumors over 48 and 96 h. The increased uptake of MAbs obtained using local hyperthermia, seems not so be induced by a decrease in tumor interstitial fluid or modification of kinetic parameters, but rather to an increased extravasation.^

Gene Therapy The use of gene therapy as potential therapeutic method is increasing. Different authors have demonstrated that HT enhances the effect of viral gene vectors encoding IL-12 and the treatment was effective, devoid of systemic side effects and associated to a substantial tumor growth delay, as compared to animals treated without HT.^"^^'^^^ Mutated p53 genes are found in over 50% of all cancers and they are responsible for the decreased HT induced apoptosis. Adenovirus transfer of p53 associated with HT seems attractive for overcoming this resistance. In fact glioma and human salivary gland adenocarcinoma treated with this combination demonstrated a higher sensitivity to hyperthermia and radiotherapy. Other useful methods to obtain an efficient gene transduction is the use of liposomes. Several authors have demonstrated that the transduction of DNA plasmid complex (lipoplex) was more efficient under conditions of hyperthermia than at 37°C. An amelioration of this

Hyperthermia in Cancer Treatment: A Primer

260

Table 7. Effects of WBHT and LHT on different immune treatment modalities Therapeutic Modalities

Human Studies

WBHT

LHT

TH

MAbs Activity Vaccine Therapy

TRI

t

T

T T

HSPs

THSP110 TtHSP70, 90

T

(40°C X 3-6 h) (41.8°Cx4h) (39.5°C X 2 h) (42.5°Cx3 h) (41-45° X 1/2-1 h) (39.5°C X 1 h) X (41.8°C X 1 h)

DCs

T

T(HSP 70)

X X X X X X X

t(HSP 70) Gene Therapy

Animal Studies

X

T T

X X

(42°C X 3/4 h) (42°Cx1/2h) (43°C X 3/4 h; THSG 44°Cx1/3 h) T U251 glioma (43-44°C x 1 h) T N I H 3T3

(4rCx1 h)

tA549

(41°Cx1 h)

X X X

Ref. 142 143 120 138 151 120 197 47 194 195 148 149 150 151 153 153

TH: time of exposure to hyperthermia and heating degree in "C; HSG: human salivary gland adenocarcinoma cell line; DCs: dendritic cells; MAbs: monoclonal antibodies; NIH3T3: murine fibroblast; A549: human lung cell line; Rl: radioimmunotherapy.

effect has been demonstrated using thermosensitive liposomes or magnetic cationic liposomes, recently developed. ^^^'^^^'^^^

Cytokine Therapy The treatment of tumors with cytokines such as IL-2, TNF-a, INF-a and GM-CSF, has been disappointing. This has induced many researchers to use IL-2, GM-CSF, Interferons and TNF-a with WBHT and LHT^^"^^^^ Generally, an additive effect has been demonstrated without an increase in toxicity. The majority of studies have been conducted on animals using IL-2 and TNF-a. ^^^'^^"^ Human studies have been conducted, primarily with WBH and perfusional hyperthermia (PH) associated to TNF-a. ^^^'^^'^ IL-2 administered before local hyperthermia treatment has demonstrated to be additive and useful to treat mice with lung metastases.^ ' The same additive effect has been demonstrated by Geehan regarding melanoma and sarcoma. The response was obtained using IL-2 simultaneously to WBHT application. The doses and toxicity were lower than those usually reported. ^^^'^^ Fritz et al have demonstrated that the potentiation of IL-2 combined wdth HT is mediated by TNF-a induction. In fact, the effect of IL-2 was abrogated by anti-TNF-a antibodies. ^^® Whole body hyperthermia or local hyperthermia combined with low-dose IL-2 was more effective on reducing tumor growth than each modality alone, and the response was more pronounced for macroscopic tumor than for microscopic one. Geehan et al surest that this phenomena is to be ascribed by two factors: (A) selective increase in permeability in tumor vessels, as compared to normal tissues with a consequent accumidation of drug, (B) an augmented expression by HT of intercellidar adhesion molecide-1 (ICAM-1) followed by an increased homing into tumor tissue of LAK cells.^^^'^^°'^^^

Ejfects ofLocal and Whole Body Hyperthermia on Immunity We refer to the review of Klostergaard et al^^^ for studies on combination of hyperthermia with TNF-a and INF-a. In brief in vitro and in vivo studies on human and animal tumors indicate a sensitization of TNF-a effect combined with heat. Sensitization was greater when tumor targets were treated with TNF-a prior heating treatment and the effect in vivo can be reached with lower dosage and with less toxicity. It appears that the effect of TNF-a in vivo is partially due to an increase on plasma membrane receptor expression or affinity and on tumor vasculature. Klostergaard et al^"^^ reported similar effect using INF-a and INF-P both in vitro and in vivo. The maximum effect was observed with intratumoral administration and the time of administration with HT was less important. A combination of TNF and INF is possible with additive effect and decreased dosage. As for TNF-a the antiproliferative effect seems to be ascribed to a direct effect on plasma membrane receptor expression or affinity. Recent studies are more oriented to use TNF-a combined with chemotherapy and WBHT or with limb or organ isolated perfusional HT. As reported by many authors a synergism among hyperthermia, melphalan (L-PAM) and TNF-a in the clinical setting of limb perfusion for malignant melanoma and sarcoma has been demonstrated. ^^^ A TNF-a concentration superior to that achieved by bolus administration (10-20 )lg/ml) can be given locallv (1-2 ^ig/ml) associated only to mild toxicity (grade lor 2) in 25% of patients treated.^^^''^^^ A similar combination of therapeutic regimen for treatment of unresectable liver malignancies (confined to liver) by using isolated hepatic perfusion (IHP) has been studied by Alexander et al. According to a critical evaluation by these authors IHP with L-PAM and TNF-a is the best combination regimen for obtaining a good response rate as compared to other chemotherapeutic regimens using drugs such as FUDR.

Effects of Hyperthermia on Lymphocyte Homing As previous mentioned, leukocyte infdtration into tumor mass is mediated by the expression of various adhesion molecules and cytokines. ^'^^'^'^ Among these adhesion molecules, ICAM-1 and a4p7 integrin are pivotal in r^ulation of the migration of leukocytes from the blood vessels.^^ Their expression has been demonstrated to decrease in presence of VEGF, ' to increase in presence of febrile range temperature (38-40°C) and INF-a. ^^^'^^"^ In fact, there is an increasing evidence that local and whole body hyperthermia can enhance both L-selectin lymphocyte-endothelial cell adhesion. ^^^'^^^'^'^^ Regarding increase of adhesion molecules it appears that hyperthermia differs compared to other therapies. In fact the increase has been detected only on tumors microvasculature and on peritumor lymphatic not on surrounding normal vasculature. ^^^ The mechanism underlying HT control of L-selectin have revealed that febrile temperature do not increase lymphocyte L-selectin surface density or L-selectin dependent recognition of soluble carbohydrates but the avidity of preexisting adhesion molecules for physiologic ligands.^ This up regulation of adhesion process su^ests the use of LHT and WBHT in clinical settings for delivering selectively cytotoxic T-cells or gene armed lymphocytes only into the tumor area.

The Danger Model and Hyperthermia-Effects on Dendritic Cell Maturation and Stimulation of Innate-Adaptive Immunity During the last decade, we have learned a great deal about the molecular mechanisms responsible for the modulation of tumor immunity; however, litde advances have been made clinically so far. The reasons for this failure may be the result of several mechanisms: L not complete control of tumor microenvironment^^^ 2. non appropriate presentation of antigens^^^ 3. innate immunity not adequately stimulated or suppressed^^^ 4. tumor immune evasion. Hyperthermia has a role in controlling the first three mechanisms. Inside a tumor mass, it exists at least partially a stressful hypoxic and acidic micro milieu which hampers antigen presentation or immune effectorsftinction."^^'^^^On one hand, this kind of stressful envi-

261

Hyperthermia in Cancer Treatment: A Primer

262

CELL DEATH IMMUNITY

ANTITUMOR IMMUNITY

MACROPHAGE PHAGOCYTOSIS

/•-\'

A.

cytokines

DANGER SIGNAL

i"

MACROrMAOeS LYMPNOCTTES RBCRUiTMeNT

INNATE IMMUNITY

ADAPTIVE IMMUNITY

Figure 4. After heat stress the HSPs can induce tumor immunity by two mechanisms: Increasing Antigen presentation through CD91 receptors, or by TLRs receptors. ronment can modify the response of lymphocytes [in vitro] to synthesis and release of mitogens and cytokines during HT treatment,^^^ on the other part it is the more suitable for killing tumor cells by heat, and for the generation of the "Danger Signal". ^^^

Danger Model According to Matzingers danger model,^^^'^^^ tissue damage in general provides a stimulus for initiating protective inmiune response. Data by Feng et al^^ indicate that heat stressed tumor cells are capable of providing the necessary danger signals, likely through increasing surface expression of heat shock proteins (HSPs), '^^^ resulting in activation and for maturation of dendritic and natural killer cells. ^^^'^^"^ Heat stressed apoptotic 12B1-D1 cells, compared to not heat stressed cells, were more effective in stimulating dendritic cells to secrete interleukin-12 (IL-12) and in enhancing their immunostimulatory functions in mixed leukocyte reaaions. Thus, DCs are able to distinguish between stressed and non stressed cells undergoing programmed cell death. In conclusion, a tumor tissue that undergoes a stress response (i.e., heat) and goes in apoptosis increases the synthesis of stress proteins on their surfaces or releases products during tissue damage that are recognized by tumor-infiltrating lymphocytes as not-self This may generate potent antitumor T-cell responses. ^^^ In contrast, non stressed apoptotic tumor cells are recognized by the immune system as a physiologic process, critical to normal development and able to elicit only a non inflanmiatory/ or even tolerigenic bland danger signal (Fig. 4). Furthermore, HT stimulates DCs migration,^^^'^^^ lymphocyte homing^^^ and HSPs synthesis. ^^^'^^^ DCs emigration from peripheral organs to lymph organs, is crucial for their maturation and their shifting from an antigen-capturing mode to a T cell-sensitising mode. In this sense, Ostberg et aT^^'^^^ have observed an enhancement of antigen-dependent immune responses at skin level and the stimulation and emigration of epidermal DCs to draining

Effects of Local and Whole Body Hyperthermia on Immunity

263

lymph nodes following WBHT. Concluding, cell deadi occurring with a concomitant production of HSPs, such as during HT treatment, is highly immunogenic. Tumor immunogenicity is also enhanced in cancer cells over-expressing HSP70 and these cells induce a marked T H l type immune response compared to cells dying via apoptotic mechanism. ^^^'^^^ In Figure 4, the various mechanisms of cell death and the consequences on tumor immunity are illustrated. It is important to note that apoptosis after macrophage phagocytosis induce the production of the immunosuppressive cytokine IL-10.^^^ This can permit tumor tolerance.

Innate Immunity Heat Shock Proteins Link Hyperthermic treatment can induce and elicit a variety of innate inmiune responses. Innate immune responses have been shown to contribute to the control of tumor in mice and there is indirect evidence that it contributes to the control of cancer in humans too. This response is dependent on the stimulation of TLRs expressed on DCs surface as confirmed by recent studies by Hilf^° and Valubas.^^ In fact they have demonstrated that the *ER-resident chaperone Gp96 (*ER: endoplasmaticum retictdum) is a potent tumor vaccine in animal models, and it induces both innate and specific immunity in a high efficient way interacting with TLRs pathway. ^^ Intriguingly, many other HSPs and substances from nonmicrobial sources have been recendy shown to signal through TLRs. There are ten members of the TLR family identified to date,^'^^^ in particular TLR4 is involved in activation by HSP 60, TLR2/4 by HSP 70 and GP96, and TLR2 by necrotic cells.^^^ Furthermore, recent findings show that TLRs are expressed in a multitude of different cell types, such as APCs, macrophages and DCs. The best characterized regulator of TLR signalling is the NF-KB transcription factor, which controls the expression of many genes involved in the inflammatory response. ^^^'^ This engagement triggers the induction of proinflammatory cytokines and chemokines which contribute to the maturation of DCs and the activation of naive T cells (see Fig. 4). Between the cytokines and chemokines released IL-12, IL-18 and TNF-a have a crucial role in directing THl response.^^ IL12 stimulates, also, NK cells that have been demonstrated to be required for obtaining an adequate antitumor activity by HSPs. Recent studies by Tournier et al have confirmed that fever plays a role in activation of DCs through the release of different temperature sensitive cytokines. Another confirmation comes from a recent work by Basu et al^^ that have demonstrated that elevated temperature in the range of 39.5°C-4l°C causes immature DCs to mature, specifically through elevation of intracellular levels of HSP 90. The HSPs released from cells imdergoing necrotic death cause translocation of NFkB into the nucleus and maturation of DCs. Concomitandy, the elevations of the aforementioned cytokines (IL12 and TNFa) contributes further to DC-maturation. Additional study has outlined the importance of DCs maturation as an essential prerequisite for a successful vaccination. ^^^ Studies by Wang et al^"^^ have shown that fever, as wells as, HT combined with tumor derived HSP (HSPs 110 and 70) significandy enhanced the vaccine efficiency on mole-to-mole basis and reduced tumor volume. Okamoto^^^ and Schuller^^'^ reported that LHT induced tumor-specific CTL response on colon carcinoma and necrosis of hepatocellular carcinoma cells. Heat shocked activated DCs are more able to stimulate T cells than non heat shocked DCs, indicating that temperature elevation can be exploited to generate a powerful immune-activity. ' Hyperthermia stimulates also neutrophils and macrophages recruitment. '^^^ Two cells subset of innate immunity that have demonstrated to play an important role in host defence against tumors. ^^^ As demonstrated by Gough et al,^^ macrophages can distinguish between tumor cells dying through classical apoptosis or cells engineered to die through non apoptotic mechanisms. In a certain sense, the immune system is able to read in which way tumor cells die and to react consequently. Additionally the presence of HSP 70 acts as one component of a bimodal alarm signal that activates macrophages, in the presence of stressful-immunogenic tumor cell killing to become activated in an antitumoral way. Different studies have demonstrated that tumor immunogenicity is enhanced in cancer cells over-expressing HSP70 and these cells induce a marked T H l type immune response compared to cells dying via apoptotic mechanism (Fig. 4,)}^'^'^^^ The danger signal and theTRL activation can work in concert and

264

Hyperthermia in Cancer Treatment: A Primer

can augment the tumor immune response. In every case, this mechanisms are to be completely elucidated and clinical demonstrated.

Streptococcal Preparation OK'432 (Picibani^)-Corynebacterium Parvum Following the abandonment of Coleys Toxin, different authors, unaware of the importance of innate immunity in tumor rejection, demonstrated that cell wall bacteria products such as streptococcal preparation OK432 (Picibanil®) and Corynebacterium Parvum, can strengthen tumor lysis cells by lymphocytes when used with hyperthermia."^^"^^^ OK-432 is a lyophilized streptococcal preparation made by penicillin treatment of the Su-strain of A-group streptococcus and it has been used in Japan since 1975 as cancer agent. Picibanil® associated with hyperthermia has demonstrated in different animal strain to induce NK cell activity and to enhance tumor tissue response to heat.^^^'^^^ Corynebacterium Parvum is an aspecific immimotherapeutic agent constituted by bacterial cell wall glycoproteic and lipid fractions. In presence of hyperthermia it has demonstrated similar immune and thermal effects than OK-432.^^^'^ For Picibanil an involvement of TLR receptor 4 in its antitumor effect has been recendy found demonstrating a link between innate immunity and anticancer activity.^ At the light of these studies, heat can be considered as an aspecific innate immunity booster. The use of bacterial product extracts increase further this stimidation.

££Fects of Hyperthermia on Metastatic Process Metastatic process is the most fearsome aspect of cancer. The formation of metastases is a complex multistep process influenced by the host and by selection and resistance induced by the different therapeutic approaches used.^°^'^°^ Shah oudined in animal experiments that a sublethal heating (< 42.5°C) of tumors with a non-complete destruction of all cancer cells, may enhance metastases formation, in a way not dissimilar to chemo- and radiotherapy. In 1974, studies by Dickson and Ellis^^° opened a great controversy on the efficacy of hyperthermia treatment, in fact they reported an increased metastasization to the liver following heating of a Yoshida sarcoma in rats. Further studies reported by Shah^^^ showed an increased metastasization preferentially to lymph nodes and lungs following a non appropriate heating. A curative treatment with appropriate temperature (> 42.5°C) was associated to regression of primary tumor and was accompanied by increased host immunocompetence. An increase on metastization has been reported by some authors and attributed to a decreased activity of NK cells.6.7ao6 Recent studies using WBHT in humans and animals"^^ ^ did not found a decreased activity of NKs and CD4 cells in vivo, contradicting old studies in vitro. ^^^ Urano,^^^ Dickson and Shah^ observed that WBHT may induce metastases at a frequency higher than LHT. Urano et al revealed that the size of the tumor and not its immunogenicity was critical for metastasization during WBHT.^^^ To reduce this incidence Oda et al^^^ suggest the use of anticancer drugs associated to WBHT. Dickson and Shah^ reported the abrogation of the effects on immune response generated by curative local heating providing that WBHT was followed or was associated with local hyperthermia. More recent clinical studies on animals with melanoma, treated with Radiation alone or combined with LHT, have demonstrated no significant difference in metastasization between the two groups, though local recurrence was a common event and was associated with metastases appearance.^^ '^^^ Similar phase III trial on human melanomas treated using similar design has shown an improved local control and a reduced metastasization with combined RT+LHT.^^^ A recent randomised study on soft tissue osteosarcoma has demonstrated that the rate of metastasization with thermoradiotherapy is similar to that seen with preoperative radiation therapy alone.^ Similar conclusions about dissemination of malignant cells during WBHT in combination with chemotherapy for epithelial malignancies have been reached by Hegewisch-Becker and collaborators. In order to obtain a maximum benefit from tumor heating a functioning host immune response seems necessary. In fact, studies conducted in the late 1990 by Ponti^ia et al"^^^ have clearly demonstrated that patients with higher values of inflanmiatory tests {al glycoprotein > 1.0 g/1; high sedimentation rate > 50/Ih, and with a lymphocytes count < 500/mc, and a ratio

Effects ofLocal and Whole Body Hyperthermia on Immunity CD4/CD8 < 1.2) failed to obtain remission. The same authors outlined that patients responsive to BRM (i.e., INF-a) show an increase in NK cell number and responders have a better prognosis as compared to non responders.^^^ Another important phenomena to take into consideration is the abscopal response. It consists in a regression of tumor at other anatomic sites following curative heating of primary tumor. This phenomena has been described in animals and humans.^^'^^^ Disappearance of primary tumor and regression of distant metastases after hyperthermic limb perfiision in both sarcoma and melanoma has been ascribed to a non specific immune reaction, such as inflammatory reaction with an increased macrophage infiltrate."^^'^^'^^'^^^'^^ Clinical studies by Shah^^^ and Dickson^ ^® have not demonstrated distant metastases regression after primary tumor treatment with LHT. Abscopal phenomena seems not pertinent to WBH treatment.

Conclusions and Comments Tumor regression of some type of human tumors following HT has been reported. The evidence of an involvement of tumor inmiune response in this regression due to temperature elevation is increasing step by step. The effects of hyperthermia on the inmiune system are pleiotropic.^ First of all, the compartment shifting and homing of immune competent cells (T-lymphocytes) and neutrophils to tumor area is increased whereas cytotoxicity is only transitorily affected. ^^^ The increase of temperature correlates positively with an increased phagocytosis of leukocytes and macrophages within fever range temperatures. ^^^'^^^ B-cells are activated by heat which brings about an increase of the production of immunoglobulins. On the contrary NK cells cytotoxicity is suppressed and their activity more affected in yitro.^ '^^^'^^^'^'^^ This suppression is however temporary. In the range of temperature beyond 39° after a period of stunning a complete recovery follows. Some authors have also demonstrated that recovery can be augmented or cytotoxicity partially spared by using interferons^"^^'^^^ or antioxidants^ ^^ such as superoxide dismutase. A summary of the effects of LHT and WBH on LAK cell and NK cells cytotoxicity can be found in Table 8. From these studies some aspects become evident: 1. temperature is crucial 2. WBH affects less the cytoxicity activity of both immunocompetent cells than LHT 3. times of exposure to heat in vitro studies are not clinical comparable. In fact different parameters can influence lymphocyte reaction in vitro compared to in vivo situation. They are: (a) treatment time that is too long (3-4 h; 18 h) or too short (1/2 h), (b) the different lytic E:T ratio, (c) the nonuniform distribution of heat easily obtainable in vitro but not in vivo and (d) the pH of the medium. Studies by Skeen et al have tried to reproduce tumor pH microenvironment and its effect on lymphocytes. They have demonstrated that the pH of the medium (obtained adding lactic acid) had no effect at 37°C but showed a synergistic impairment with heat at 41, 42 and 43°C.^^^ These examples indicate experiments are to be conducted with appropriate methods for assessing in vitro and in vivo cellular immune response. We also suggest to employ experimental conditions well defined, standardized and nearer the clinical simation for better understanding HT effect on immunity. In particular: pH of medium, cytokine assay in situ, activation and impairment of cellular response by DCs use. Comprehensive clinical studies on W ^ H T are to our opinion those of Robins and Atanactovic.^^^'^^ ,123,125,127 CD4+ / CD8+ lymphocytes ratio does not change in vivo, whereas activation and maturation of dendritic cells is positively affected.^^ In fact, in animals experiments, febrile range temperature elevation (39.5-4l°C) have demonstrated to elicit DCs maturation through HSPs induction. Furthermore, for this order of heating WBHT induces a panel of cytokines similar to that induced during fever;"^^^'^^^ on the contrary LHT is not able to elicit any cytokines production (see Table 6).^^^ The induction of HSPs is rapid and occurs immediately following exposure to only a few degrees above normal physiological temperature stimulation.'^'^^ HSPs induction occurs during

265

266

Hyperthermia in Cancer Treatment: A Primer

tn

O 00 vD O^

a> T- O) o r - (N r - fN ^ T— rsl fSI

o >^

0)

CD

c

ry a;

•D 0)

-C

0) •u

•D

>

u c;

u ^ u

(Tl in

= 2

> s

o

Jl^ D

8._QJ1 O

Q . UL. —1

z

i|

X X X

ITt _ 1 Q_ _J T : CD C

X XX X

Z^

,o nS U

Q. t/i

«-H-

o >

T3 O

X XX X x x x x x x x x x x

O 1-

E oZ

c/^

^ ^ > 't^ > . j :

03 "D


(\)»u >c

m no

o c JS

U

a;

II

•o

z

^

-C _C _C

Z fN ^ "^

o > E

Al

.1 •2

00 -^ ^ O

o

03 •D

-

z -

C2 ^ Yl S ^«^<—>

n rt n nnr^'s' o 2 o CS CO ^ rsi 5: ^ ^ ^ ^ -^ ^1 '^->^-^^^< ><

S^ -c 0 ) CU • D DO

o

U

00 .E

o'

-^ >->^

1^ o

^^

u

U

^ z

O

Q U

€ ^

%

°,•.E X

^ » fid Q

' U

o ^

c o c

< - CL , - r QU 03

lis

"^

u

-

;^ ^ ;::: 1- ^ V V -

o

V|i«

u

"O

CN 7 ci ^ ;E -

U

8

OJ N—

u

I I

o

OJ 03 03 Of

u 00

^ <-

o r^ E

^2^ O

C "D

X-'E

Uj

00

a>

-5i

c 3 b E

'^ Q^

*t:

IrJ

p >

(J c/) 0) . .

o o; Q_ (D ^ 3

i

u 2

1^

^ o

^^, Q

z u

x ^OOCO

1-

Effects ofLocal and Whole Body Hyperthermia on Immunity

267

Table 9. Positive and negative effects of hyperthermia on immunity Positive Effects

Negative Effects

• increased antigen presentation

• no increase in cytokines production by local HT • difference in cytokines production by fever and WBH • transient decreased activity of lymphocytes and NK cells in vitro • possible increase in metastasization ? • not agreement on rescue activity of NK cells

• increased lymphocyte recruitment to tumor • amelioretion of activity of dendritic cells • positive interaction v^ith gene therapy • increased activity of IL-2 and TNFa and interferons + HT • production of HSPs • innate immunity stimulation

• no appropiate animal models • inappropiate human studies

hyperthermia treatment and represents a simple method for stimulating innate immunity^' and for harness a highly tailored cancer immunotherapy eliciting the maturation of dendritic cells and their recruitment into the tumor area.^^^'^^^' '^^^ In fact, tumor antigens presentation by HSPs isolated from cancer cells has been shown to induce a specific and individualized protective anti tumor response.^^ The tissue specificity of HSPs relies on the fact that they can bind antigenic peptides and improve endocytosis of the chaperoned peptides by APCs, enhancing their ability to stimulate peptide-specific T cells.^^ '^ Regarding the effects of hyperthermia on metastatic dissemination the conclusions are still controversial and many bias are linked to in vitro and in vivo assays for monitoring cancer immune response. However cUnical studies on moderate (fever range) and extreme WBHT seem to provide the evidence for a non significant metastatic spread. Concluding, positive and negative effects on immunity are produced by HT treatment and are summarized in Table 9. A positive association between hyperthermia and the various therapeutic strategies has been demonstrated, suggesting a use in conjunction with a stimulation of a tumor immunity such as : cytokines, monoclonal antibodies, gene therapy, dendritic cells administration (see Tables 6-8). The recent recognized involvement in innate immune response, increases further the interest of hyperthermia, but increase the r^ret for the time missed abandoning Coley s Toxin and Mixed Bacterial Vaccine too.

References 1. Papac RJ. Spontaneous regression of cancer. Cancer Treat Rev 1996; 22:395-423. 2. Abel U. Spontanremissionen und fieberhafte Erkrantungen. In: Heim ME, Schwarz R, eds. "Spontanremissionen in der Onkologie". 1998:68-75. 3. Hobohm U. Fever and cancer. Cancer Immunol Immunother 2001; 50:391-396. 4. Bickels J. Coley toxin: Historical perspective. IMAJ 2002; 4:471-472. 5. Coley-Nauts H, McLaren J. Coley toxins-the first century. "Consensus On Hyperthermia for 1990s". Adv Exp Med Biol 1990; 267:483-500. 6. Dickson JA, Shah SA. Immunologic aspects of hyperthermia. In: Storm FK, ed. Hyperthermia in Cancer Therapy Boston: G K Hall Pubhshers, 1983:487-543. 7. Dickson JA, Shah SA. Hyperthermia and the immune response in cancer therapy. Cancer Immunol Immunother 1980; 9:1-10. 8. Dranoff G. Cytokines in cancer pathogenesis and cancer therapy. Nat Rev Cancer 2004; 4:11-22. 9. Diefenbach A, Raulet DH. The innate immune response to tumors and 1st role in the induction of T-cell immunity. Immunol Rev 2002; 188:9-21. 10. Hilf N, Singh-Jasuja H, Schild H. The heat shock protein Gp96 links innate and specific immunity. Int J Hyperthermia 2002; 18:521-533. 11. Palucka K, Fay J, Banchereau J. Dendritic cells and tumour immunity. Curr Opin Oncol Endocr Metab Invesdg Drugs 1999; 1:282-290.

268

Hyperthermia in Cancer Treatment: A Primer

12. Bremes AJA, Parmiani G. Immunology and immunotherapy of human cancer: Present concepts and clinical developments. Grit Rev Oncol Hematol 2000; 34:1-25. 13. Ben-Efraim S. One hundred years of cancer immunotherapy: A critical appraisal. Tumor Biol 1999; 20:1-24. 14. Davis ID, JefFord M, Parente F et al. Rational approaches to human cancer immunotherapy. J Leukoc Biol 2003; 73:3-29. 15. Schirrmacher V, Freuerer M, Fournier P et al. T-cell priming in bone marrow: The potential for long-lasting protective anti-tumor immunity. Trends Mol Med 2003; 9:526-534. 16. Lord EM, Frelinger JG. Tumor immunotherapy: Gytoldnes and antigen presentation. Gancer Immunol Immunother 1998; 46:75-81. 17. Shurin MR, Lu L, Kalinsky P et al. TH1/TH2 in cancer, transplantation and pregnancy. Springer Semin Immunopathol 1999; 21:339-359. 18. Nishimura T, Iwakabe K, Sekimoto M et al. Distinct role of antigen specific T Helper type 1 (Thl) and Th2 cells in tumor eradication in vivo. J Exp Med 1999; 190:617-627. 19. Titu LV, Monson JRT, Greenman J. The role of GD8+ T cells in immune responses to colorectal cancer. Gancer Immunol Immunother 2002; 51:235-247. 20. Garlos TM. Leukocyte recruitment at sites of tumour: Dissonant orchestration. J Leukoc Biol 2001; 70:171-184. 21. Fauriat G, Marcenaro E, Sivori S et al. Natural Killer Gell-Triggering receptors in patients with acute leukaemia. Leuk Lymphoma 2003; 44:1683-1689. 22. Hoos A, Levey DL. Vaccination with heat shock protein-peptide complexes: From basic science to cHnical applications. Exp Rev Vaccines 2003; 2:369-379. 23. O' Byrne KJ, Dalgeish AG, Browning MJ et al. The relationship between angiogenesis and the immune response in carcinogenesis and progression of malignant disease. Eur J Gancer 2000; 36:151-169. 24. Vaupel P, Kallinoski F, Okunieff P. Blood flow, oxygen and nutrients supply, and metabolic microenvironment of human tumors, a review. Gancer Res 1989; 49:6449-6465. 25. Folkman J. Tumour angiogenesis: Therapeutic implications. N Engl J Med 1971; 285:1182-1186. 26. Freitas I, Baronzio GF. Tumour hypoxia, reoxygenation and oxygenation strategies: Possible role in photodynamic therapy. J Photochem Photobiol B Biol 1991; 11:3-30. 27. Berges G, Benjamin L. Tumorigenesis and the angiogenic switch. Nat Rev Gancer 2002; 3:401-410. 28. Griffioen AW, Molena G. Angiogenesis: Potentials for pharmacological intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation. Pharmacol Rev 2000; 52:238-268. 29. Griffioen AW, Selma G, Tromp SC et al. Angiogenesis modulates the tumour immune response. Int J Exp Patiiol 1998; 79:363-368. 30. Papetti M, Herman I. Mechanisms of normal and tumour-derived angiogenesis. Am J Physiol Gell Physiol 2002; 282:c947-c970. 31. Alexandrofif AB, Mclntyre GA, Porter JG et al. Sticky and smelly issues: Lessons on tumour cell and leukocyte trafficking, gene and immunotherapy of cancer. Br J Gancer 1998; 77:1806-1811. 32. Garlos TM, Harlan JM. Leukocyte-endothelial adhesion molecules. Blood 1994; 84:2068. 33. Kitayama J, Nagawa H, Yasuhara H et al. Suppressive effect of basic fibroblast growth factor on trans endotheHal emigration of GD4(+) T-lymphocyte. Gancer Res 1994; 54:4729. 34. Barleon B, Sozzani S, ZhouD et al. Migration of human monocytes in response to Vascular endotheHal growth factor (VEGF) is mediated via die VEGF receptor flt-1. Blood 1996; 87:3336-3343. 35. Borgstrom PG, Hughes GK, Hansell P et al. Leukocyte adhesion in angiogenic blood vessels. J Glin Invest 1997; 99:2246-2253. 36. Griffioen AW, Damen GA, Martinotti S. Endothelial IGAM-1 expression is suppressed in human malignancies; role of angiogenic factors. Gancer Res 1996; 56:1111. 37. Mantovani A, Sozzani S, Locati M et al. Macrophage polarization: Tumor associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trend Immunol 2002; 23:549-555. 38. Hightower LE. Heat shock, stress proteins, chaperones and proteotoxicity. Gell 66:191-197. 39. Smith DF, Whitesell L, Katsanis E. Molecular chaperones: Biology and prospects for pharmacological intervention. Pharm Rev 1998; 50:494-513. 40. ManjiU MH, Wang XY, Park J et al. Immunotherapy of cancer using Heat shock proteins. Front Biosci 2002; 7:d43-d52. 41. Li GG, Mivechi NF, Weitzel G. Heat shock proteins, thermotolerance and their relevance to clinical hyperthermia. Int J Hyperthermia 1995; 11:459-488. 42. Fortin A, Raybaud-Diog^ne H, T^tu B et al. Overexpression of the 27 KDA heat shoch protein is associated with thermotolerance and chemoresistance but not with radioresistance. Int J Radiation Oncol Biol Phys 2000; 46:1259-1266.

Effects ofLocal and Whole Body Hyperthermia on Immunity

269

43. Wallin RPA, Limdqvist A, More SH et al. Heat shock proteins as activators of the innate immune system. Trends Immunol 2002; 23:130-135. 44. Przepiorka D, Srivastava PK. Heat shock protein—peptide complexes as immunotherapy for human cancer. Mol Med Today 1998; 4(ll):478-84. 45. Manjili MH, Wang XY, Park J et al. Cancer immunotherapy: Stress proteins and hyperthermia. Int J Hyperthermia 2002; 18:506-520. 46. Wells A, MalKovsky M. Heat shock proteins, tumor immunogenicity and antigen presentation: An integrated view. Immunol Today 2001; 21:129-132. 47. Milani Y, Noessner E, Ghose S et al. Heat shock protein 70 in antigen presentation and immune stimulation. Int J Hyperthermia 2002; 18:563-575. 48. Flohe SB, Bruggemann J, Lendemans S et al. Human heat shock protein 60 induces maturation of dendritic cells versus a THl-promoting phenotype. J Immunol 2003; 170:2340-2348. 49. Liu B, DeFilippo AM, Li Z. Overcoming immune tolerance to cancer by heat shock protein vaccines. Mol Cancer Ther 2002; 1:1147-1151. 50. Tamura Y, Peng P, Lui K et al. Immunotherapy of tumors with autologous tumor - derived heat shock protein preparations. Science 1995; 269:117-120. 51. Srivastava P. Purification of heat shock protein-peptide complexes for use in vaccination against cancers and intracellular pathogens. Methods 1997; 12:165-171. 52. Basu S, Binder RJ, Suto R et al. Necrotic not apoptotic cell death release heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-kB pathway. Int Immunol 2000; 12:1539-1546. 53. Feng H, Zeng Y, Whitesell L et al. Stressed apoptotic tumor cells express heat shock proteins and elicit tumor specific immunity. Blood 2001; 197:3505-3512. 54. Horizons in Cancer Therapeutics. From Bench to bedsides 2001; 2(n. 1). 55. Rosenberg SA. Progress in the development of immunotherapy for the treatment of patients with cancer. J Intern Med 2001; 250:462-475. 56. Sahin U, Tiireci 0 , Pfreudschuh M. Serological identification of human tumor antigens. Curr Opin Immunol 1997; 9:709-716. 57. Bitton RJ, Guthmann MD, Gabri MR et al. Cancer Vaccines: An update with special focuses on ganghosides antigens (Review). Oncol Rep 2002; 9:267-276. 58. Ockert D, Schmitz M, Hampl M et al. Advances in cancer immunity. Immunol Today 1999; 20:63-65. 59. Espinoza-Delgado I: Cancer vaccine. Oncologist 2002; 7(suppl 3):s20-s33. 60. Ribas A, Butterfield LH, Glaspy JA et al. Current development in cancer vaccines and cellular immunotherapy. J Clin Oncol 2003; 21:2415-2432. 61. Osanto S. Vaccine trials for the cUnician: Prospects for tumor antigens. Oncologist 1997; 2:284-299. 62. Parmiani G, Pilla L, Castelli C et al. Vaccination of patients with solid tumours. Ann Oncol 2003; 14:817-824. 63. Fernandez NC, Lozier A, Flament C et al. Dendritic cells directly trigger NK cell fiinctions: Cross-talk relevant in innate antitumor immune response. Nat Med 1999; 5:405-411. 64. Celluzzi CM, MayordromoJI, Storkus WJ et al. Peptide pulsed dendritic Cells induce antigen -specific CTL-mediated protective antitumor activity. J Exp Med 1996; 183:283-287. 65. Ribas A, Butterfield LH, Glaspy JA et al. Cancer immunotherapy using gene- modified dendritic cells. Curr Gene Ther 2002; 2:57-78. GG. Weber J, Fong L. Clinical trials of Dendritic Cells in cancer. In: Lotze M, Thomson AW, eds. Dendritic Cells. San Diego, San Francisco: Academic Press, 2001:561-571. G7. Haupt K, Roggendorf M, Mann K. The potential of DNA vaccination against tumour-associated antigens for antitumor therapy. Exp Biol Med 2002; 227:227-237. 68. Pawelec G, Rees R, Kiessling R et al. Cells and Cytokines in immunotherapy and gene therapy of cancer. Crit Rev Oncog 1999; 10:83-127. 69. Baar J. Clinical appHcations of dendritic cell cancer vaccines. Oncologist 1999; 4:140-144. 70. Gabrilovich DI, Chen HL, Girgis KR et al. Production of vascular endothelial factor by tumour inhibits the fimctional maturation of dendritic cells. Nat Med 1996; 2:1096-1103. 71. Yamaguchi Y, Tsumura H, Miwa M et al. Contrasting effects of TGF-pi and TNF-a on the development og dendritic cells from progenitors in mouse bone marrow. Stem Cells 1997; 15:144-153. 72. Sharma S, Stolina M, Yang SC et al. Tumor cycloxygenase 2 -dependent suppression of dendritic cell fiinction. Clin Cancer Res 2003; 9:961-968. 73. Romano G. Gene transfer in experimental medecine. Drugs New Perspect 2003; 16:267-276. 74. Peng K-W. Strategies for targeting therapeutic gene delivery. Mol Med Today 1999; 5:448-453.

270

Hyperthermia in Cancer Treatment: A Primer

75. Romano G, Pacilio C, Giordano A. Gene transfer technology in therapy. Current applications and future goals. Stem Cells 1999; 17:191-202. IG. Navarro JG, Curiel DT, Douglas JT. Gene therapy of cancer. Eur J Cancer 1999; 35:867-885. 71. Kufe DW, Advani S, Weichselbaum R. Principles of gene therapy. In: Bast, Kufe, Pollock, Weichselbaum, Holland, Frei, eds. Cancer Medicine. Hamilton, Ontario: BC Decker Publisher, 2000:876-890. 78. Rochlitz CF. Gene therapy of cancer. Swiss Med WKLY 2001; 131:4-9. 79. Farzaneh F, Trefzer U, Sterry W et al. Gene therapy of cancer. Immunol Today 1998; 19:294-296. 80. Clark RP, Hersh EM. Cationic Hpid-mediated gene transfer: Current concepts. Curr Opin Mol Ther 1999; 1:158-76. 81. Davis ME. Non -viral gene delivery systems. Curr Opin Biothecnol 2002; 13:128-131. 82. Maurer N, Fenske DB, Cullis PR. Development in liposomal drug delivery systems. Expert Opin Biol Ther 2001; 1:923-947. 83. Boulikas T. Liposome DNA delivery and uptake by cells. Oncol Rep 1996; 3:989-005. 84. Koshkina NV, Agoulnik I, Melton SL et al. Biodistribution and pharmacokinetics of aerosol and intravenously administered DNA-polyethyleneimine complexes: Optimisation of pulmonary delivery and retention. Mol Ther 2003; 8:249-254. 85. Densmore CL. The reemergence of aerosol gene delivery: A viable approach to lung cancer therapy. Curr Cancer Drug Targets 2003; 3:273-284. 86. Singh-Jasuja H, Hilf N, Scherer HU et al. The heat shock protein gp96: A receptor-targeted cross-priming carrier and activator of dendritic cells. Cell Stress Chaperones 2000; 5(5):462-70. 87. Mazzaferro V, Coppa J, Carraba M et al. Vaccination with autologous derived Heat shock protein gp96 after liver resection for metastatic colorectal cancer. Clin Cancer Res 2003; 9:3235-3245. 88. Saper CB, Breder CD. The neurologic basis of fever. N Eng J Med 1994; 30:1880-1886. 89. Dinarello CA. Cytokines as endogenous pyrogens. J Infect Dis 1999; 179(suppl 2):s294-s304. 90. Netea MG, Kulberg BJ. Circulating cytokines as mediators of fever. J Infect Dis 1999; 179(suppl 2):sl78-sl84. 91. Blatteis CM, Sehic E. Cytokines and fever. Ann NY Acad Sci 1998; 840:608-618. 92. Ahlers O, Boehnke T, Kerner T et al. Induced hyperthermia causes significant changes in lymphocytes. Crit Care 1998; 2(suppl l):abstract p002. 93. Jampel HD, Duff GW, Gershon RK et al. Fever and immuneregulation. J Exp Med 1983; 157:1229-1238. 94. Hanson DF. Fever, temperature, and the immune rersponse. Ann NY Acad Sci 1997; 813:453-464. 95. Bell JF, Moore GF. Effects of high ambient temperature on various stage of rabies virus infection in mice. Infect Immun 1974; 10:510-515. 96. Hasday JD. The influence of temperature on host defenses. In: Mackowiak PA, ed. Fever: Basic Mechanisms and Management. 2nd ed. Philadelphia: Lippincott-Raven Publishers, 1977:177-196. 97. Roberts NJ. Impact of temperature elevation on immunologic defences. RID 1991; 13:452-472. 98. Lederman HM, Brill CR, Murphy PA. Interleukin-1 driven secretion of interleukin-2 is highly temperature-dependent. J Immunol 1987; 138:3808-3811. 99. Gautherie M. Hyperthermia and the immune system. In: Gautherie M, ed. Whole Body Hyperthermia: Biological and Clinical Aspects. Berlin-Heidelberg-New York: Springer-Verlag, 1992:6-15. 100. Dickson JA, Shah SA. Hyperthermia: The immune response and Tumour metastasis. Nad Cancer Inst Monogr 1982; 61:183-192. 101. Skeen MJ, McLaren JR, Olkowski ZL. Influences of hyperthermia on immunological functions. In: Anghileri L, Robert J eds. "Hyperthermia in Cancer Treatment". CRC Press, 1986:93-105. 102. Repasky E, Issels R. Physiological consequences of hyperthermia: Heat, heat shock proteins and the immune response. Int J Hyperthermia 2002; 18:486-488. 103. Bull JM, Lees DE, Schuettc WH et al. Immunological and physiological responses to whole-body hyperthermia. Nad Cancer Inst Monogr 1982; 61:177-181. 104. Roberts NJ, Lu ST, Michaelson SM. Hyperthermia and human leucocyte functions: DNA, RNA, and total protein synthesis after exposure to < 41° or >42.5° hyperthermia. Cancer Res 1985; 45:3076-3082. 105. Knox JD, Mitchel RE, Brown DL. Effects of hyperthermia on microtubule organization and cytolytic activity of murine cytotoxic T lymphocytes. Exp Cell Res 1991; 194:275-283. 106. Lamon EW, Parrish J, Marshall G et al. Synergistic induction of thermotolerance in murine natural killer cells by interferon a and mild heat shock. Radiat Res 1994; 139:364-369. 107. Kappel M, Stadeager C, Tvede N et al. Effects of in vivo hyperthermia on natural killer cell activity, in vitro proliferative responses and blood mononuclear cell subpopulations. Clin Exp Immunol 1991; 84:175-180.

Effects ofLocal and Whole Body Hyperthermia on Immunity

271

108. Yang H, Mitchel RE. Hyperthermic inactivation, recovery and induced Thermotolerance of human natural killer cell lytic function. Int J Hyperthermia 1991; 7:35-49. 109. Huang YH, Haegerstrand A, Frostergard. Effects of in vitro hyperthermia on proliferative responses and lymphocyte activity. Clin Exp Immunol 1996; 103:61-66. 110. Singh VK, Biswas S, Pandey CM et al. Effect of elevated temperature on cytotoxic effector cells. Pathobiology 1996; 64:150-155. 111. Azokar J, Yunis EJ, Essex M. Sensitivity of human natural killer cells to hyperthermia. Lancet 1982; i:16-17. 112. Kalland T, Dahlquist I. Effects of in vitro hyperthermia on human natural killer cells. Cancer Res 1983; 43:1842-1846. 113. Kraybill WG, Olenki T, Evans S et al. A phase 1 study of fever-range whole body hyperthermia (FR-WBH) in patients with advanced solid tumours: Correlation with mouse models. Int J Hyperthermia 2002; 18:253-266. 114. Yoshioka A, Miyachi Y, Toda K et al. Effects of local hyperthermia on natural killer activity in mice. Int J Hyperthermia 1990; 6:261-267. 115. Fuggetta MP, Alvino E, Tricarico M et al. In vitro effect of hyperthermia on natural cell-mediated cytotoxicity. Anticancer Res 2000; 20:1667-1672. 116. Shen R-N, Li Lu, Young P et al. Influence of elevated temperature on natural killer cell activity, lymphokine-activated killer cell activity and lectin-dependent cytotoxicity of human umbilical cord blood and adult blood cells. Int J Radiation Oncology Biol Phys 1994; 29:821-826. 117. Evans SS, Bain MD, Wang WC. Fever -range hyperthermia stimulates a4p7 integrin-dependent lymphocyte-endothelial adhesion. Int J Hyperthermia 200; 16:45-59. 118. Fritz KL, Koziol BS, Fabian DF et al. Variable modulation of LAK cell mediated cytotoxicity and ICAM-1 expression by interferon-gamma and hyperthermia in human melanoma cell lines. Electronic J Oncol 1999; 2:89-110. 119. Dinarello CA, Dempsey RA, Allegretta M et al. Inhibitory effects of elevated temperature on human cytokine production and Natural Killer activity. Cancer Res 1986; 46:6236-6241. 120. Wang X-Y, Kazim EA, Repasky A et al. Characterization of hsp 110 and glucose regulated protein 170 as cancer vaccine and the effect of fever -range hyperthermia on vaccine activity. J Immunol 2001; 166:490-497. 121. Atanackovic D, Nierhaus A, Neumeier et al. 41.8 °C whole body hyperthermia as an adjunct to chemotherapy induces prolonged T cell activation in patients with various malignant diseases. Cancer Immunol Immunother 2002; 51:603-613. 122. Schell SR, Wessels FJ, Abouhamze A. Pro-and Antinflammatory cytokine production after Radiofrequency ablation of unresectable tumors. J Am Coll Surg 2002; 195:774-781. 123. Haveman J, Geerdink AG, Rodermond HM. Cytokine production after whole body and localized hyperthermia. Int J Hyperthermia 1996; 12:791-800. 124. Robins HI, Kurz M, Wiedemann GJ et al. Cytokine induction by 41.8 degree °C whole body hyperthermia. Cancer Lett 1995; 97:195-201. 125. D'Oleire F, Schmitt CL, Robins HI. Cytokine induction in humans by 41,8 °C whole body hyperthermia. JNCI 1993; 85:833-8434. 126. Robins HI, Grosen E, Katschinski DM et al. Whole body hyperthermia induction of soluble tumor necrosis factor receptors: Implications for rheumatoid disease. J Rheumatol 1999; 26:2513-2516. 127. Katschinski DM, Wiedemann GJ, Robins HI et al. Whole body hyperthermia cytokine induction; a review and unifying hypothesis for myeloprotection in the setting of cytotoxic therapy. Cytokine Growth Factor Rev 1999; 10:93-97. 128. Alonso K. Cytokine levels in adult patients with solid tumors undergoing whole body hyperthermia (WBH). Int J Hyperthermia 1997; 13:559.562. 129. Morita M, Kuwano H, Arakri K et al. Prognostic significance of lymphocyte infiltration following preoperative chemoradiotherapy and hyperthermia for esophageal cancer. Int J Radiation Oncol Biol Phys 2001; 49:1259-1266. 130. Midis GP, Fabian DF, Lefor A. Lymphocyte migration to tumors after hyperthermia and immunotherapy. J Surg Res 1992; 52:530-536. 131. Lefor AT, Foster CE, Sartor W et al. Hyperthermia increases intercellular adhesion molecule-1 expression and lymphocyte adhesion to endotheHal cells. Surgery 1994; 116:214-221. 132. Kappel M, Khazami A, Nielsen H et al. Modulation of the counts and functions of neutrophils and monocytes under in vivo hyperthermia conditions. Int J Hyperthermia 1994; 10:165-173. 133. Hasday JD, Garrison A, Singh IS et al. Febrile-range hyperthermia augments pulmonary neutrophil recruitment and ampHfies pulmonary oxygen toxicity. Am J Pathol 2003; 162:2005-2017. 134. Ostberg JR, Repasky EA. Comparison of the effects of two different whole body hyperthermia protocols on the distribution of murine leukocyte populations. Int J Hyperthermia 2000; 16:29-43.

272

Hyperthermia in Cancer Treatment: A Primer

135. Dong BW, Zhang J, Liang P et al. Sequential pathological and immunologic analysis of percutaneous microwave coagulation therapy of hepatocellular carcinoma. Int J Hyperthermia 2003; 19:119-133. 136. Shah A, Ungerb E, Bain MD et al. Cytokines and adhesion molecule expression in primary human endothelial cells stimulated with fever - range hyperthermia. Int J Hyperthermia 2002; 18:534-551. 137. Dickson JA, Jasiewicz ML, Simpson AC. Immunogenicity of ascites tumor cells following in vitro hyperthermia. Nad Cancer Inst Monogr 1982; 61:235-238. 138. Mondovl B, Santoro SA, Strom R et al. Increased immunogenicity of Ehrlich Ascites cells after heat treatment. Cancer 1972; 30:885-888. 139. Moricca G, Cavaliere R, Caputo A. Hyperthermic treatment of tumors: Experimental and cUnical observations. In: Rentchnick P et al, eds. Recent Results in Cancer Research. Berlin, Heidelberg, New York: Springer, 1977:59:112-152. 140. Ito A, Masashige S, Honda H et al. Augmentation of MHC class I antigen presentation via heat shock protein expression by hyperthermia. Cancer Immunol Immunother 2001; 50:515-522. 141. Shen RN, Hornback NB, Shidnia H et al. Whole-body hyperthermia decreases lung metastases in chimeric monoclonal antibody in a subcutaneous xenograft model. CHn Cancer Res 1997; 3:63. 142. Saga T, Sakahara H, Nakamoto Y et al. Enhancement of the therapeutic outcome of radioimmunotherapy by combination with whole - body mild hyperthermia. Eur J Cancer 2001; 37:1429-1434. 143. Hauck ML, Dewhirst MW, Bigner DD et al. Local hyperthermia improves uptake of a chimeric monoclonal antibody in a subcutaneous Xenograft model. CHn Cancer Res 1997; 3:63-70. 144. Hauck ML, Zalutsky MR. The effects of local hyperthermia on the catabolism of a radioiodinated chimeric monoclonal antibody. Clin Cancer Res 1998; 4:2071-2077. 145. Kong G, Braun RD, Dewhirst MW. Characterization of the effect of hyperthermia on nanoparticle extravasation from tumor vasculature. Cancer Res 2001; 61:3027-3032. 146. Hauck ML, Coffin DO, Dodge RK et al. A local hyperthermia treatment which enhances antibody uptake in glioma xenograft model does not affec tumour interstitial fluid pressure. Int J Hyperthermia 1997; 13:307-316. 147. Hauck ML, Dewhirst MW, Zalusky MR. The effects of clinically relevant hyperthermic temperatures on the kinetic binding parameters of a monoclonal antibody. Nucl Med Biol 1996; 23:551-557. 148. Lohr F, Kang H, Huang Q et al. Enhancement of radiotherapy by hyperthermia-regulated gene therapy. Int J Radiation Oncol Biol Phys 2000; 48:1513-1518. 149. Huang Q, Hu JK, Lohr F et al. Heat induced gene expression as a novel targeted cancer gene dierapy strategy. Cancer Res 2000; 60:3435-3439. 150. Asaumi JI, Higuchi Y, Murakami J et al. Thermoradiotherapy combined with p53 therapy of human salivary gland adenocarcinoma cell line. Oncol Rep 2003; 10:71-74. 151. Okamoto K, Shinoura N, Egawa N et al. Adenovirus-mediated transfer of P53 augments hyperthermia-induced apoptosis in U251 glioma cells. Int J Radiation Oncol Biol Phys 2001; 50:525-531. 152. Yanase M, Shinkai M, Honda H et al. Antitumor immunity induction by intracellular hyperthermia using magnetite cationic Uposomes. Jpn J Cancer Res 1998; 89:775-782. 153. Mushiake H, Aoe M, Washio K et al. Enhancement of gene transduction efficiency in cancer cells using cationic liposomes with hyperthermia. Acta Med Okayama 2002; 56:35-42. 154. Yamauchi N, Watanabe N, Maeda M et al. Mechanism of synergistic Cytotoxic effect between tumour necrosis factor and hyperthermia. Jpn J Cancer Res 1992; 83:540-545. 155. Lin JC, Park J, Song W. Combined treatment of IL-la and TNF- a potentiates the antitumor effect of hyperthermia. Int J Hyperthermia 1996; 12:335-344. 156. Payne J. Mild hyperthermia modulates biological activity of interferons. Int J Hyperthermia 2000; 16:492-507. 157. Takada Y, Sato EF, Nakajima T et al. Granulocyte-colony stimulating factor enhances anti-tumor effect of hyperthermia. Int J Hyperthermia 2000; 16:275-286. 158. Lans TE, Bardett DL, Libutti SK et al. Role of tumor necrosis factor on toxicity and cytokine production after isolated hepatic perfusion. Clin Cancer Res 2001; 7:784-790. 159. Shen R-N, Lu L, Wu B et al. Effects of interleukin 2 treatment combined with local hyperthermia in mice inoculated with lewis lung carcinoma cells. Cancer Res 1990; 50:5027-5030. 160. Fritz KL, Koziol S, Dagmar FF et al. Tumor necrosis factor a mediates the antitumor effect of combined Interleukin-2 and whole body hyperthermia. J Surgical Res 1996; 60:55-60. 161. Geehan D, Fabian D, Lefor A. Immunotherapy and whole body hyperthermia as combined modality treatment of a subcutaneous murine sarcoma. J Surg Oncol 1993; 53:180-183. 162. Van der Zee J, Van den Aardweg GJMJ, Van Rhoon GC et al. Thermal enhancement of both tumor necrosis factor alpha-induced systemic toxicity and tumour cure in rats. Br J Cancer 1995; 71:1158-1162.

Effects ofLocal and Whole Body Hyperthermia on Immunity

273

163. Robins HI, Katschinski DM, Longo W et al. A pilot study of melphalan, tumour necrosis-a and 41.8°C whole-body hyperthermia. Cancer Chemother Pharmacol 1999; 43:409-414. 164. Robins HI, D'Oleire F, Kutz M et al. Cytotoxic interactions of tumor necrosis factor, melphalan and 41.8°C hyperthermia. Cancer Lett 1995; 89:55-62. 165. Eggermont AMM, Koops HS, Klausner JM et al. Isolated limb perfusion with tumour necrosis factor alpha and chemotherapy for advanced extremity soft tissue sarcomas. Semin Oncol 1997; 24:547-555. 166. Alexander HR, Bardett DL, Libutti SK. Current status of isolated perfusion with or without necrosis factor for the treatment of unresectable cancers confined to liver. Oncologist 2000; 5:416-424. 167. Sumida M, Isawa E, Kobayashi K et al. TNF-a and endotoxin levels in cancer patients undergoing intraperitoneal hyperthermic perftision. Int J Hyperthermia 1996; 12:607-615. 168. Strauch ED, Fabian DF, Turner J et al. Combined hyperthermia and immunotherapy treatment of multiple pulmonary metastases in mice. Surg Oncol 1994; 3:45-52. 169. Geehan D, Fabian D, Lefor A. Combined local hyperthermia and immunotherapy treatment of an experimental subcutaneous murine melanoma. J Surg Oncol 1995; 59:35-39. 170. Klostergaard J, Tomasovic SP. Hyperthermia and biological response modifiers. In: Urano M, Douple E, eds. Hyperthermia and Oncology. The Netherlands: VSP, 1994:4:219-258. 171. Van Der Veen AH, Ten Hagen TLM, DeWilt JHW et al. An overview on the use of TNF-a: Our experience with regional administration and developments towards new opportunities for systemic appHcation. Anticancer Res 2000; 20:3467-3474. 172. Sartor WM, Kyprianou N, Fabian DF et al. Enhanced expression ICAM-1 in a murine fibrosarcoma reduces tumour growth rate. J Surg Res 1995; 59:66-74. 173. Burd R, Dziedzic TS, Xu Y et al. Tumour cell apoptosis, lymphocyte recruitment and tumor vascular changes are induced by low temperature, long duration (Fever -like) whole body hyperthermia. J Cell Physiol 1998; 177:137-147. 174. Evans SS, Wang WC, Bain MD et al. Fever range hyperthermia dynamically regulates lymphocyte delivery to high endothelial venules. Blood 2001; 97:2727-2733. 175. Evans SS, Frey M, Scheider DM et al. Regulation of leukocyte-endotheHal cell interaction in tumor immunity. In: Mihich and Croce, eds. Biology of Tumors. Plenum Press, 1998:273-286. 176. Menoret A, Srivastava PK. The cancer microenvironment and its impact on immune response to cancer. In: Liu Y, ed. Molecular Approaches to Tumor Immunotherapy. Singapore, New Jersey, London, Hong Kong: Word Scientific Press, 1998:109-121. 177. Forsdyke DR. Heat shock proteins as mediators of aggregation-induced "danger" signals: Implications of the slow evolutionary fine-tuning of sequence for the antigenicity of cancer cells. Cell Stress Chaperones 1999; 4:205-210. 178. Seya T, Akazawa T, Uehori J et al. Role of Toll-like receptors and their adaptors in adjuvant immunotherapy for cancer. Anticancer Res 2003; 23:4369-4376. 179. Wojtowicz-Praga S. Reversal of tumour -induced immunosuppression: A new approach to cancer therapy. J Immunother 1997; 20:165-177. 180. Matzinger P. The danger model: A renewed sense of self. Science 2002; 296:301-305. 181. Matzinger P. Essay 1: The danger model historical context. Scand J Immunol 2001; 54:4-9. 182. Multhoff^ G, Boltzer C, Jennen L et al. A stress inducible 72KDa heat shock protein (HSP72) is expressed on the surface of human tumor cells, but not on normal cells. Int J Cancer 1995; 61:272-279. 183. Multhofif G. Activation of natural killer cells by heat shock protein 70. Int J Hyperthermia 2002; 18:576-583. 184. de Vries LJM, Lesterhuis J, Scharenborg Nm et al. Maturation of dendritic cells is a prerequisite for inducing immune response in advanced melanoma patients. Clin Cancer Res 2003; 9:5091-5100. 185. Ostberg JR, Patel R, Repasky EA. Regulation of immune activity by mild (fever-range) whole body hyperthermia: Effects on epidermal Langherans cells. Cell Stress Chaperones 2000; 5:458-461. 186. Ostberg JR, Kabingu E, Repasky EA. Thermal regulation of dendritic cell activation and migration from skin explants. Int J Hyperthermia 2003; 19:520-533. 187. Todryk SM, Cough MJ, Pockeley AG. Facets of heat shock protein 70 show immunotherapeutic potential. Immunology 2003; 110:1-9. 188. Todryk SM, Melcher AA, Dalgleish AG et al. Heat shock protein refine the danger theory. Immunology 2000; 99:334-337. 189. VoU RE, Hermann M, Roth EA et al. Immunosuppressive effects of apoptotic cells. Nature 1997; 350. 190. Valubas RM, Braedel S, Hilf N et al. The endoplasmatic reticulum-resident Heat Shock Protein Gp96 activates dendritic cells via the Toll Like Receptor 2/4 pathway. J Biol Chem 2002; 277:20847-20853.

274

Hyperthermia in Cancer Treatment: A Primer

191. Medzhitov R. Toil-like receptors and Innate immunity. Nature Rev 2001; 1:135-145. 192. Beg AA. Endogenous ligands of Toll-like receptors: Implications for regulating inflammatory and immune responses. Trends Immunol 2002; 23:509-512. 193. Liwu L. Regulation of innate immunity signalling and its connection with human diseases. Curr Drug Targets Inflamm Allergy 2004; 3:81-86. 194. Tournier JN, Hellman AQ, Lesca G et al. Fever-like thermal conditions regulate the activation of maturing dendritic cells. J Leukoc Biol 2003; 73:493-501. 195. Basu S, Srivastava PK. Fever-Uke temperature induces maturation of dendritic cells through induction of hsp 90. Int Immunol 2003; 15:1053-1061. 196. Okamoto M, Tazawa K, Kawagoshi M et al. The combined effect against colon-26 cells of heat treatment and immunization with heat treated colon-26 tumor cell extract. Int J Hyperthermia 2000; 16:263-273. 197. Schueller G, Paolini P, Friedl J et al. Heat treatment of hepatocellular carcinoma cells: Increased levels of heat shock proteins 70 and 90 correlate with cellular necrosis. Anticancer Res 2001; 21:295-300. 198. Di Carlo E, Forni G, LoUini PL et al. The intriguing role of polymorphonuclear neutrophils in antitumor reactions. Blood 2001; 97:339-345. 199. Gough MJ, Melcher AA, Ahmed A et al. Macrophages orchestrate the inmmune response to tumor cell deadi. Cancer Res 2001; 61:7240-7247. 200. Urano M, Taradi M, Taradi SK. Enhancement of the thermal response of murine tumour and normal tissues by a streptococcal preparation, OK-432 (Picibanil). Int J Hyperthermia 1991; 7:113-23. 201. Urano M, Yamashita T, Suit HD et al. Enhancement of thermal response of normal and malignant tissues by Corynebacterium parvum. Cancer Res 1984; 44:2341-2347. 202. Taradi M, Urano M, Taradi SK et al. Augmentation of mouse natural killer cell activity by combined hyperthermia and streptococcal preparation, OK-432 (Picibanil) treatment. Int J Hyperthermia 1991; 7:653-65. 203. Urano M, Overgaard M, Suit H et al. Enhancement by Corynebacterium parvum of the normal and tumor tissue response to hyperthermia. Cancer Res 1978; 38:862-4. 204. Shah SA. Enhanced thermal response of a rat sarcoma by Corynebacterium parvum. Cancer Lett 1985; 26:235-40. 205. Gridley DS, Nutter RL, Kettering JD et al. Mouse neoplasia and immunity: Effects of radiation, hyperthermia, 2-deoxy-D-glucose, and Corynebacterium parvum. Oncology 1985; 42:391-8. 206. Okamoto M, Oshikawa T, Tano T et al. Involvement of toll-like receptor 4 signalling in interferon- Y production and antitumor effect by Streptococcal agent OK-432. J Natl Cancer Inst 2003; 19:316-326. 207. Fidler IJ. The organ microenvironment and cancer metastasis. Differentiation 2002; 70:498-505. 208. Heppner GH, Miller BE. Therapeutic implications of tumor heterogeneity. Semin Oncol 1989; 2:91-105. 209. Shah S. Metastasis and hyperthermia. In: Anghileri L, Robert J, eds. Hyperthermia in Cancer Treatment. CRC Press, 1986:191-227. 210. Dickson JA, Ellis HA. Stimulation of tumor cell dissemination by raised temperature (42°C) in rats with transplanted Yoshida tumours. Nature 1974; 248:354. 211. Shen R, Hornback NB, Shidnia H et al. Whole body hyperthermia decreases lung metastases in lung tumor-bearing mice, possibly via a mechanism involving natural killer cells. J Clin Immunol 1987; 7:246-253. 212. Urano M, Rice L, Epstein R et al. Effect of whole -body hyperthermia on cell survival, metastases frequency, and host immunity in moderately and weakly immunogenic murine tumors. Cancer Res 1983; 43:1039-43. 213. Oda M, Koga S, Maeta M. Effects of total-body hyperthermia on metastases from experimental mouse tumors. Cancer Res 1985; 45:1532-1535. 214. Dewhirst MW, Sim Da, Forsyt K et al. Local control and distant metastases in primary canine malignant melanomas treated with hyperthermia /or radiotherapy. Int J Hyperthermia 1985; 1:219-234. 215. Prostnitz LR, Maguire P, Anderson JM et al. The treatment of high grade soft tissue sarcomas with preoperative thermoradiotherapy. Int J Radiat Oncol Biol Physis 1999; 45:941-949. 216. Hegewisch-Beecker S, Braun K, Otte M et al. Effects of whole body hyperthermia (4l.8°C) on the frequency of tumor cells in the peripheral blood of patients with advanced malignancies. Clinical Cancer Res 2003; 9:2079-2084. 217. Pontiggia P, Mc Laren JR, Baronzio GF et al. The biological response to heat. "Consensus On Hyperthermia for 1990s". Adv Exp Med Biol 1990; 267:271-291.

Effects ofLocal and Whole Body Hyperthermia on Immunity

275

218. Sitnicka EW, Olszewski WL, Lukomska B. Influence of whole body hyperthermia on natural cytotoxicity of liver blood- borne sinusoidal cells. Int J Hyperthermia 1993; 9:731-743. 219. Yang M, Lauzon W, Lemaire I. Effects of hyperthermia on natural killer cells: Inhibition of lytic function and microtubule organization. Int J Hyperthermia 1992; 8:87-97. 220. Zanker KS, Lange J. Whole body hyperthermia and natural killer activity. Lancet 1982; 1:1079-1080. 221. Kappel M, Tvede N, Hansen MB et al. Cytokine production ex vivo: Effect of raised body temperature. Int J Hyperthermia 1995; 11:329-335. 222. Quinn TD, Polk HC, Edwards MJ. Hyperthermic isolated limb perfusion increases circulating levels of inflammatory cytokines. Cancer Immunol Immunother 1995; 40:272-275. 223. Ostberg JR, Kaplan KC, Repasky EA. Induction of stress proteins in a panel of mouse tissues by fever -range whole body hyperthermia. Int J Hyperthermia 2002; 18:552-562. 224. Li Z, Menoret A, Srivastava P. Role of heat-shock proteins in antigen presentation and cross presentation. Curr Opin Immunol 2002; 14:45-51. 225. Kubista B, TriebK, Blahovec H et al. Hyperthermia increases the susceptibility of chondro-and osteosarcoma cells to natural killer cell-mediated lysis. Anticancer Res 2002; 22:789-792.

CHAPTER 21

Fever, Pyrogens and Cancer Ralf KleeP and K Dieter Hager Abstract

T

he observation that cancer patients who experienced a feverish period after surgery survived significandy longer than patients without fever and the fact that spontaneous tumor remission was observed mostly after a fever period was the rationale for the artificial induction of fever ("fever therapy'). The history and rationale for fever therapy are presented and the inmiunological basis for endo- and exotoxin-induced tumor regression are discussed on the basis of nearly 800 citations of research literature. The effects and clinical research of different biological inductors of hyperthermia like Coley's Toxin (MBV), Propioni Bacteria, Corynebacterium parvum, Bacillus Calmette Guerin (BCG), OK-432, Staphylococcus protein A and Streptokinase are described. Though the biological effects of fever on tumors are well characterized and interesting biological and immune ological results are obtained, and some clinical observational studies and small randomized trials show very promising results, larger controlled GCP-conform trials are still lacking. In combination of moderate and extreme whole body hyperthermia with chemotherapy, radiotherapy or immunotherapy with monoclonal antibodies significant improvement in outcome of the treatment of cancer patients is to be expected. The toxicities of active "fever therapy" or passive "fever-range whole body hyperthermia" are tolerable.

History and Background The history of fever therapy begun with the heroic induction of fever in the mid-19th century by the German physicians Busch,^ Fehleisen,^ and Richter^ by subcutaneous injection of toxins from erysipelas to treat cancer patients. The rational for this therapeutic approach was the observation, that cancer patients who experienced a feverish period after surgery survived significandy longer than patients without fever."^ The history of Coley s Toxin, a pyrogenic bacterial lysate from Serratia marcescens and Streptococcus pyogenes began at the turn of the 19th century at Memorial Sloan-Kettering Cancer Center in New York (Coley^'^^). William B. Coley, M.D., active career 1891-1936 using a bacterial vaccine to treat primarily inoperable sarcoma, accomplished a cure rate greater than 10%. After controlling for lapse time, the time from disease onset until start of treatment with Coley toxins, significandy higher cumulative survival was found for the Coley treatment in three subgroups: (1) Ovarian cancer, distant disease - higher survival in years 2-10 (10 year follow-up); (2) Breast cancer, premenopausal distant disease- higher survival in years 2-3 and 5-8 (8 year follow-up); and (3) breast, post-menopausal regional disease-higher survival in all 5 years of follow-up. Coley's interest in the subject developed when he lost his first cancer patient, a young girl from the Rockefeller family, with a sarcoma in her right arm. In spite of radical surgery, she •Corresponding Author: Ralf Kleef—Institute for Hyperthermia and Immunotherapy, Windmijhlgasse 30/7, A-1060 Vienna, Austria. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

Fever, Pyrogens and Cancer

277

later died of metastatic cancer. In the course of his work, the physician noted that patients who developed bacterial infections after sarcoma surgery faired much better than those who did not develop postoperative infections. Specifically, Coley studied the medical records of a patient with four instances of large recurrent inoperable sarcoma of the neck and noted that the patient experienced regression under the influence o^ erysipelas (a superficial streptococcal infection of the skin). To fiirther his research, Coley deliberately injected erysipelas into some of his cancer patients. Due to initial complications with the formtda, the formula was later changed to a combination of gram-positive heat killed streptococcus plus gram negative heat killed bacteria {Streptococcus pyogenes and Serratia marcescens) called Coley s Toxins or 'Mixed Bacterial Vaccine (MBV). Since then various researchers all over the world have used different bacterial products for the treatment of cancer patients to raise an unspecified immune response in the hope to stimulate humoral and cellular antitumor activities. Later, his daughter Helen Coley Nauts should found the Cancer Research Institute in New York, which has since pioneered the field. Coley s Toxins, consisting of the two bacteria Serratia marcescens and Streptococcus pyogenes, have been used in a variety of preparations and indications. They also came known as "fever therapy" or endogenous hyperthermia. Progress in immunology and the discovery of cytokines has led to a better understanding of the mechanisms involved. Coley Toxins and related approaches will be reviewed in respect to the variety of preparations, immunological and cUnical results. Coley is credited with pioneering the field of immunotherapy. In the mid-20th century Issels and Windstofier continued to treat cancer patients with MBV on an empirical basis. Hager and Abel^'^ stimulated in the eighties the clinical research and therapeutic use and basic and clinical research of endogenous, active hyperthermia ("fever therapy') with bacterial vaccines (MBV, Vaccinetuin ) and passive *Fever-Range Whole Body Hyperthermia* (FR-WBH) with infrared radiation by critical analysis and sunmiary of the available literature. Heckel^^ and von Ardenne^^^ developed heating devices for passive hyperthermia with infrared radiation. It has been su^ested^^ that an important prerequisition for the successftil eUcitation of an immunological response and induction of tumor cytotoxicity in the host following bacterial toxin exposure is the preactivation of the host. At Coley's time this has been the preexposure of large groups of the population to BCG. Importandy, in 1975 the group of Old detected TNF in a BCG-primed mouse. The inmiunologic preactivation has been confirmed by a large number of in vitro and in vivo studies as an important factor not only in respect to an effective immune response but also for the development of tolerance and toxicity. Interestingly, the epidemiology of cancer incidence and the incidence of febrile infections have been shown to have an inverse correlation and additionally, spontaneous remissions repeatedly have been reported to be associated with febrile infections (reviewed in ref 20). The evidence for these observations will be reviewed.

Rationale Emerging evidence has developed that cancer is an aberrant regulatory process in which the tumor is in a dynamic disequilibrium with the host. The inmiunological implications of this evidence are widely accepted in the scientific conmiunity but have not found their way into applied clinical practice yet. Immunological fiinctions not only are associated with the expression of oncogenes^^ but also with prognostic factors.^^ Moreover, immunosuppressive factors in cancer patients have been frequendy described, as demonstrated by in vitro reactions (reviewed in ref 32). Additionally, the exposure to endotoxins has been demonstrated to act as a powerfid inunune enhancer not only in immunocompromised cancer patients but also in other patient groups as demonstrated in numerous studies (i.e., reversal of virally mediated inmiunosuppression: Friedman et al^^). Furthermore, cytotoxic therapies have been shown to exert often long lasting immunologic depression with the subsequent risk of secondary malignancies. '^

278

Hyperthermia in Cancer Treatment: A Primer

Attempts to reintroduce differentiation and apoptosis is one line of research as has been shown with the successful differentiation therapies in acute promyelocytic leukemia or the clinical reversibility of MALT (gastric lymphomas) after successful eradication oi Helicobacter pylori infection.^^ Inununotherapy on the other hand aims to target the body s immune system to attack the cancer cells and has gained popularity as a treatment modality for malignant diseases in the 1960s. A large number of trials, using tumor vaccines, immunopotentiators, interferons, cytokines and "biological response modifiers", demonstrated antitumor effects in several malignancies, and, to date, immunotherapy plays a major role in the treatment of advanced renal cell carcinoma and superficial bladder carcinoma. Interferon or interleukin-2, which became available for large-scale clinical trials with the development of bioengineering, however, were shown to be not as effective in human trials as initially expected from animal models. The attempt of unspecific immunotherapy by raising a host response through the application of bacterially derived vaccines is a rational design opposed to the application of single cytokines. Together with our increasing knowledge of the complex immunological network this attempt, based on basic and clinical research should provide progress in treatment of human malignancies. The rationale for the further study of endo- and exotoxin based cancer therapies will be justified as follows: 1. Established cancer therapy has yet to be improved. ^^'^^ 2. A new paradigm in cancer treatment is warranted.^^'^^ 3. There is an inverse correlation between the incidence of infectious diseases and cancer risL20 4. During and immediately after febrile infections remissions of malignancies have been observed.^^ 5. Pyrogenic substances have been successfully administered in palliative and curative treatment protocols of metastatic cancer 6. Cytokine secretions such as IL-1, IL-6, GM-CSF, G-CSF, IL-12 Interferons, and TNF-a mediate the immunological reactions to the administration of Coley s Toxin. Their induction and the shift from typeTH 1 to type TH2 cytokines can be individually monitored and the therapy adjusted accordingly. With some exception, i.e., in leukemias, the application of single or combination of cytokines has not contributed to a major breakthrough in the continuing search for a cure for cancer. Otherwise, the imitation of a phylogenetic protection mechanism as old as fever may be safely exploited in association with the powerful diagnostic tools of molecular biology, which may allow the therapist to fine-tune the immunologic response to the given challenge. 7. Side effects of the treatment are manageable. 8. New preparation may be more effective than preparations such as Vaccineurin® and Novo-Pyrexal® or Picibanil® which have been used for this purpose in the past in Europe and Japan, respectively. New preparations are available."^^^ 9. The Office of Complementary and Alternative Medicine at the National Institutes of Health in the USA as well the German Ministry for Research list Coley s Toxins as a treatment approach with high priority for research. ^^'^^ 10. Due to the growth in "publicity" of unconventional cancer treatments, the rigorous scientific evaluation of this treatment approach may serve the public, medical providers and third party payers. More importantly, carefully designed studies according to GLP and GCP will lend credibility to this approach and will promote only the best available treatment and bacterial products and hopefully prevent the exploitation by less scientifically qualified providers. As will be discussed, a close immunologic monitoring of the patient is paramount to prevent enforcement of immunologic blocking mechanisms. 11 .This study may add benefits to developing new methods in cancer treatments.

Fever, Pyrogens and Cancer

279

Epidemiology Incidence of Malignancies and Missing History ofFever Clinical oncologists repeatedly report that cancer patients stress in their anamnesis that they were never ill before. As a result of this observation a number of epidemiological studies have been conducted which shall be briefly reviewed here. Already in 1854 Laurence acknowledges the fact that cancer patients have a "remarkable disease-free history". Schmidt^^ corroborates these findings in stating an "afebrile diathesis** in the history of cancer patients in a study of 241 subjects followed by EngeP^' ^ who compared 300 cancer with 300 noncancer patients. Engels' studies demonstrate a cancer risk for people who never experienced an infectious disease calculated with odds ratio (OR) of 2.5 to 46.2. Sinek finds similar results in 232 cancer patients, which he compared with 2.444 controls. More recent studies confirm the earlier work: Witzel^^ obtained anamnestic data from 150 cancer patients and 150 controls. In this study cancer patients exhibited significantly less visits to their physicians, had fewer secondary illnesses and fewer in-patient hospital referrals. Also, in thefiveyears preceding the diagnosis only two cancer patients developed fever compared to 20 subjects in the control group. Newhouse et al^^ found in a study of 300 women with cancer of the ovary amongst sociological factors like fewer marriages, fewer incidences of mumps, measles, or rubella compared to an age-matched control group. Remy et al found an increased cancer risk with an odds ratio of 2.6 for missing history of infectious organ diseases, 5.7 for missing history of common colds and 15.1 for missing history of fever. Grufferman et al studied environmental factors in the etiology of rhabdomyosarcoma in childhood. It is the only paper that finds an insignificant correlation with fewer immunizations and a higher rate of preventable infections associated with cancer risk. Ronne^^ could associate a missing history of measles in childhood with increased cancer risk for a variety of tumors in a historical prospective study. Out of 353 individuals with a n^ative history of measles 21 developed cancer versus only 1 case out of 230 controls with a positive history of measles (p < 0.001). Van Steensel-Moll et al^^ reported evidence of a lower frequency of infections in the first year of life for children with leukemia; in this register-based case-control study common colds, periods of fever, and primary childhood infections showed relative risks (RR) of 0.8, 0.9, and 0.8 respectively. The authors argue that stimulation of the immune system in early life may play a protective role in the development of leukemia. Chilvers et al performed a retrospective study in which not the absence of fever or infectious diseases but the absence of common cold or a positive history of allergies was tested for their impact on cancer risk. In this study for missing history of common cold and positive history of allergies no association with increased cancer risk could be established. Remy et al, Abel et al and Schlehofer et al^^ contradicted this study. Abel et al^^ established in case-control studies with 255 cancer patients compared with 230 controls the highest risk for patients with a low "Infection-Index". Schlehofer et al^^ investigated in a popidation-based-case-control study the medical risk factors of 226 patients with primary brain tumors and 418 controls. She stated a decreased RR for the development of brain tumors for those individuals who had had allergic diseases (RR 0.7; 95% confidence interval (CI) 0.5 to 1.0), diabetes (RR 0.7; 95% CI 0.3 to 1.8) and infections and cold (RR 0.3; 95% CI 0.1 to 0.8). Melanoma patients had fewer atopic symptoms than subjects did in the control group (p less than 0.05). Grossarth-Maticek et al^^ performed a ten-year prospective cohort study of 1353 persons. He concludes "episodes of high fever during the entire life span in the case of an acute illness as a typical reaction are inversely related to later cancer incidence when the subjective reporting of fever is accepted as valid evidence". Kolmel and Compagnone investigated the role of fever and atopy among melanoma patients. There were fewer feverish infections, while patients with atopy had more feverish complications of their symptoms. Finally Kolmel et al^^ demonstrated at 271 controls versus 139 melanoma patients an inverse relation between number of febrile infections and incidence of malignant mela-

280

Hyperthermia in Cancer Treatment: A Primer

The correlation between missing history of fever and cancer risk could not be confirmed for acute adult leukemia and ALL in a recent study by Cooper et al7^ The data of 624 patients with acute myeloid leukemia, 124 patients with acute lymphoblastic leukemia (AML) matched with 637 healthy population controls did not support a protective effect from antigenic stimulation in relation to the risk for acute leukemia in adults. While discussing the incidence of malignancies and missing history of fever the same question can be asked in relation to immunosuppressive drugs. There is considerable evidence that there is a higher cancer rate after the introduction of immunosuppressive methods accompanying transplantation surgery (Cole ''^'^). Data for increased incidence of neoplasms following therapeutic immunosuppression exist for lung carcinoma,^ lymphoma/^'^^ bladder tumors,^ next to several reviews on miscellaneous tumors.^^'^ These data deserve fiirther studies. Possible mechanisms for increased incidence of malignancies have not been elucidated yet and there consists even a controversy whether immunosuppressive or inmiunostimulatory events are mediating increased carcinogenesis. Furthermore the immunosuppressive effects of chemotherapy are well known and there has been association between chemotherapy and secondary malignancies.^3-^5.37-39,44

Spontaneous Remissions and Feverish Infections It is of obvious interest for this review to analyze the literature on reports of spontaneous remissions in cancer following infections with or without fever. 0*R^an and Hirshberg^'^ give an extensive overview of the field. The older literature consists mainly of the reports of Coley, meticulously documented in eighteen monographs mainly by his daughter Helen Coley Nauts.^^'^®^ Analysis of the literature^^ reveals leukemia with > 22% being the magnitude of cases where infection was associated with remission, followed by bone and connective tissue cancers with > 15%, melanoma with > 11% and lymphoma with > 7%. Spontaneous tumor remissions during or following feverish infections have been reported already in the beginning 19th century (Vautier,^^® see Nowotny^^^). There are several older reviews^^^'^^^ which report spontaneous remissions next to more recent studies by Everson and Cole,^ Stephenson et al,^^^ Cole,^^^'^^^ Nauts.^^^ Remissions of leukemia following systemic infections have been noted throughout the century.^^^'^^^ Stephenson et al^^^ reported in their analysis that an infection or persistent fever preceded 224 cases of spontaneous remissions. Additionally, febrile infections have been shown to increase the survival expectancy of cancer patients.^ "^^^ Nowacki and Szymendera^^^ state a highly unfavorable prognostic significance for postoperative fever and/or septic complications in colorectal cancer patients. Fucini et al^^^ disagree with this statement and show in a retrospective analysis no significant prognostic influence of postoperative fever and/or septic complications in this patient group. Treon and Broitman^^^ described post-transfusional hepatitis as a common complication in patients with acute myelogenous leukemia (AML) which "paradoxically" prolonged survival. They identified the impaired hepatic endotoxin (LPS) clearance in patients with acute viral hepatitis as the reason for endotoxemia and elevated TNF-a release, a mechanism referred to as endothelial translocation (see Translocation). They also observed virally induced IFN-y secretion, which in turn acts in synergy with TNF-a anti-proliferative and as a mechanism inducing differentiation. Finally, in a recent monograph on spontaneous remissions of malignant melanoma Maurer and Kolmel^ list 21 cases of the world literature, where febrile infections have been associated with spontaneous regression of metastatic melanoma. These authors state further "the connection of febrile infection and tumor regression is the most frequent association found in the literature". The following list contains the described and additional references in most of which spontaneous infection and/or fever had been associated to remission of neoplastic disease. Some articles refer to assumed mechanisms of spontaneous regression and some articles are review papers.

Fever, Pyrogens and Cancer

281

Table 1. Spontaneous remission listed under tumor types Types

References

Bone Tumors

Levin/^^ Cole/^^ Callan et al/^^ Copeland/^^ Eisenbud et al/^^ Collignon etaP'^^ Margolis and West/^^ Kapp/^° Rao et aP^^ Bluming and Ziegler^"*^ Nowacki and Szymendera/^^ Fucini et aP^^ Rebollo et al/^^ Zambrana et aP^^ Friedrichjr^^^ Temesrekasi/^^Woods^^^ Chien et al/"^^ Grossmann et al/^° Markovic et al,^^^ Tarazov^^^ H a r t / " Dock/23 Dreyfus/^^ Bassen etal/^^ Pelner et al/^^ Paolino and Sartoris/^^ Vladimirskaia/^^ Hardisty,^^^ Burgess and de Gruchy/^^ Wyszkowski et al/^°Matzker and Steinberg,^^^ Wiernik/^^ Barton et al/^^ Conrad and Barton/" Foon et al/^^ Vinogradova and Ivanina/" Sanz and Sanz/^^ Zhu and Quian, Kizaki et alJ-^^ Maekawa et alJ^^ Treon and BroitmanJ^^ Frick

Brain Tumors Burkitts Lymphoma Colorectal Cancer Gastric Cancer Gynecological Head and Neck Cancer Hepatocellular Cancer Leukemia

Lung Cancer I 180

Lymphoma and Non Hodgkin Lymphoma

Suggested Mechanisms

Melanoma

Multiple Myeloma Prostrate Cancer Renal Cell Cancer Retinoblastoma Reviews

Sarcoma

Qazl,'"-' Wolt,'°° Sureda et a!/"' De Berker et al,'"'^ Sawada et al^ HeinzlefetaP^° 193 Wolfenheim/^^ Tsubura et al/^^ Bagshawe et al/^^ Muckle et al, Schwartz et al/^^ Cho-Chung and Gullino/^^'^^^ Remy et al/^^ 203 Berendt/^^'200pedersenetal,2°^ Bolande,^^^ Nowotny/^^ Baker/' Stone,204 Stone,204 Jarpe jarpe et et al^^o^ al^^o^ Seachrist^o^ Seachrist^o^ Halliday Halliday et et apo^ apo^ Gunale and Tucker/°^ Wormald and Harper,^°^ al Harper,^^^ Wagner et al,^^^ Cook,2ii Grafton,2^2 Haliday et al^^o^ Motofei,^^^ Maurer and KolmeP^^ London^^^ Schurmanset aP^^ Katz and Schapira,^^^ Mangiapan et al,^^^ Edwards et aP^® Hunter,^^^ Verhoeff,^^^ Jain and Singh^^^ Bruns/^^ Eschweiler/^^ Rohdenburg/^"* Dobson and Dickey,^^^ Everson/^^-^^^Huth/2^ Stephenson etal/20cole/2^'^22sindelar/223 Nauts/°^ Chains and Stam,^^^ Seacrist,^"^ Kaiser^^^ Watson,^^^ Shore,^^^ Penner,^^^ Berner and Laub/^^ Nauts and Fowler/°3 Weintraub,^^^ Mizuno et al,^^^ Lei et aP^^

Spontaneous Remission Listed Under Tumor Types (Please see Table 1)

Fever and the Immune Response Fever as the imminent sign of infectious diseases has been used as a diagnostic indicator since ancient times."^^^ It is one of the oldest nonspecific responses to infection, both in vertebrates and invertebrates.^^ Temperature rise during fever establishes a cascade of host defense mechanisms that increases host survival and induces T cell proliferation and differentiation,

Hyperthermia in Cancer Treatment: A Primer

282

Leucocytes

cells

4

6 8 24 32 Hours after Fever induction with MBV

Figure 1. Time-dependent exemplary induaion of IL-1 in a patient after induction of fever up to 39.8°C with a biological pyrogen (Vaccineurin®). secretion of interferons (IFNs), antibodies and neutrophil migration.^^^'^ Fever as a part of the acute-phase reaction and the role of cytokines in thermoregulation have been reviewed recently by Dinarello.^^^'^^^ The interest in fever as a therapeutic tool is dating back to Parmenides (ca. 540-480 B.C.) who stated: "Give me the power to induce fever and I will cure all diseases'*. And in the seventeens century the English physician Sydenham (1624-1689) described the reaction of the organism to pyrogenic substances: "Fever is a mighty engine, which nature brings into the world for the conquest of her enemies'*. Ever since Burnet^^^"^ has postulated his theory of immunological surveillance and the first limitations of aggressive cancer treatments became obvious, research has focussed on the possible role of the immune system in cancer incidence and prognosis. As it has been shown and will be discussed further fever, as an innate and phylogenetic very old mechanism, deserves the best of our scientific attention as a powerful tool in the ongoing search for the cure of cancer. Cytokine research has elucidated the immunological response underlying fever. Direct primary endogenous pyrogens are IL-1 alpha, IL-1 beta, TNF-alpha, TNF-beta (lymphotoxin-alpha), IL-6, macrophage inflammatory protein 1, and IFN-alpha.^^^*^ ^ Indirect inducers are considered to be IL-2 and IFN-gamma. ^^ Exogenous pyrogens are considered to be the lipopolysaccharides of the cell wall of gramnegative bacteria such as Serratia marcescens and the exotoxins of grampositive bacteria such as streptococcus and staphylococcus, which are also called bacterial superantigens. Fever-induced temperature changes have been shown to augment immunological defense mechanisms in vivo and in yitro.^ ' Increased temperatures stimulate the proliferation but not cytotoxicity of cytotoxic T lymphocytes (CTL) which then can perform their effector function at all physiological temperatures in the body."^ ^'"^ ^ It has been shown that binding of bivalent antibody can neutralize picornaviruses by irreversibly neutralizing the virus at temperatures that are higher than physiological by disrupting the virion, leading to ejection of the RNA. Fever enhances this process in vivo, confirming the popular belief in the virtues of fever.^^^ Not all researchers report enhancement of immunity: Incubating temperatures of 39°C have been shown to suppress natural killer cell activity in vitro in the presence of IL-1 or interferon-alpha.^^° But the immunological effects are not only depending on temperature but also on time (Figs. 1-7).

Fever, Pyrogens and Cancer

283

Lymphocytes 4000

3000

cells 2000 /ul

1000

4

6 8 24 32 Hours after Fever induction with MBV

Figure 2. Emigration, homing, proliferation and activation of leukocytes in a patient after induction of fever up to 39.8°C with a biological pyrogen (Vaccineurin®) depending on time.

B-Lymphocytes

500

400

300

cells /ul 200

100

4

6

8

24

32

Hours after Fever induction with MBV

Figure 3. Emigration, homing, proliferation and activation of total lymphocytes in a patient after induction of fever up to 39.8°C with a biological pyrogen (Vaccineurin®) depending on time.

284

Hyperthermia in Cancer Treatment: A Primer

T-Lymphocytes 2000

1500

cells 1000 /ul

500

4 6 8 24 32 Hours after Fever induction with MBV

Figure 4. Emigration, homing, proliferation and activation of B-lymphocytes in a patient after induction of fever up to 39.8°C with a biological pyrogen (Vaccineurin®) depending on time.

NK-Cells

cell s

4 6 8 24 32 Hours after Fever induction with MBV Figure 5. Emigration, homing, proliferation and activation of T-lymphocytes in a patient after induction of fever up to 39.8°C with a biological pyrogen (Vaccineurin®) depending on time.

Fever, Pyrogens and Cancer

285

LAK-Ceils 500

400

300 ceils \ui 200

100

4 6 8 24 32 Hours after Fever induction with MBV

Figures 6. Emigration, homing, proliferation and activation of NK-cells in a patient after induction of fever up to 39.8°C with a biological pyrogen (Vaccineurin®) depending on time.

Figures 7. Emigration, homing, proliferation and activation of LAK-cells in a patient after induction of fever up to 39.8°C with a biological pyrogen (Vaccineurin®) depending on time.

286

Hyperthermia in Cancer Treatment: A Primer

Glucocorticoids inhibit various components of the acute phase response, particularly the increase in body temperature induced by endotoxins. Endogenous glucocorticoids function as part of an inhibitory feedback system involved in the modulation of fever by decreasing plasma IL-6, CSF, PGE2, and PGF2 alpha concentrations.^^^

The Immunological Basis of Endo- and Exotoxin-Induced Tumor Regression The Shwartzman Phenomenon The Phenomenon of Local Skin Reactivity to Various Microorganisms Shwartzman^ was the first to describe the phenomenon of local tissue reactivity, later referred as the Shwartzman phenomenon (SP). Because of the importance to the subject it shall be briefly described here: Shwartzman injected a single intradermal cultural filtrate from B. typhosus free of exotoxins into rabbits, which was followed 24 hours later by a single intravenous injection of the same filtrate. Only four hours after the intravenous injection Shwartzman observed severe hemorrhagic necrosis at the site of skin injection. Furthermore it was shown that the second dose of the filtrate had to be given intravenously since repeated intradermal injections did not elicit the SP. Interestingly the SP induced cross-reactivity since it could also be provoked when using intravenous injections from filtrates derived from biologically and serologically unrelated microorganisms. These mdxxAtA Meningococcusy B. typhosus, B. paratyphosus, B. coli, b. jriedlaendevy B. dysenteriae, B. prodigiosus (later known as Serratia marcescens), B. lepisepticus, B. pestis, B. influenzOy B. pertussis, and Vibrio cholera. Additionally ^c^rw lumbricoidis elicited the SP whereas yeast, ricin and diphtheria toxin did not show strong responses. The same phenomenon of different bacterial species sharing similar mechanisms of sensitization has been observed for the induction of endotoxin tolerance, as will be described below. Timing played a crucial role. The appropriate time between the initial skin injection and the subsequent intravenous injection for the intravenous injection ranged from eight to thirty-two hours after initial skin injection with an optimum incubation period of twenty-four hours. Outside this range no SP coidd be elicited. The pretreatment of a large amount of different microorganisms revealed considerable heat resistance which, though, differed widely between different strains and even within the same strain. Shwartzman observed fluctuations of the potency of various preparations in refrigerated storage. There was increase as well as decrease of potency upon storage of several months. It was hypothesized that fluctuations in potency of filtrates are accompanied by the formation of "toxoids'*, which retain their power to combine with neutralizing antibodies. Interestingly, SP could be elicited in rabbits, guinea pigs, goats, and horses but not in mice and rats. But, as will be discussed later, murine animals bearing sarcoma, again showed a marked SP in their tumor. Later, it became clear that interferon-gamma (IFN-ganuna) plays a critical role in eliciting the SP, since monoclonal antibodies to IFN-gamma could completely prevent the SP. Also, IFN-alpha and IFN-beta had a desensitizing effect.^^^ Reactivity of Malignant Neoplasms to the Phenomenon of Local Skin Reactivity Applying the same technique of sensitizing animals with intradermal injections of bacterial filtrates Shwartzman observed upon subsequent intravenous injection of bacterial filtrates into tumor bearing animals severe hemorrhagic necrosis and remissions of tumors. This observation referred to transplantable and spontaneous tumors. Comments on the antagonism between tuberculosis,^ malaria and tick fever^ and the development of carcinoma built the early epidemiological hypothesis about the protective mechanism of infections against cancer. Gratia and Linz^ continued Coley s early work in

FeveVy Pyrogens and Cancer

287

liposarcoma-bearing guinea pigs by combined intratumoral and intraperitoneal injection of 5. coli. Instead of the skin these animals were sensitized direcdy at the tumor site. In later experiments the authors only choose the intraperitoneal route without previous injection at the skin site and still could elicit severe hemorrhagic necrosis of tumors. N o hemorrhagic lesions were observed in other visceral sites. While the SP in nontumor bearing animals was restricted to nonmurine species, tumor-bearing rats and mice showed a marked SP in their tumors. Mouse sarcoma 180 inoculated by Shwartzman and Michailovsky^^^ were treated with intravenous injection o£Meningococcus 44B. Hemorrhagic tumor necrosis and complete regression of tumors were observed in mice receiving repeated intravenous and intraperitoneal injection of the bacterial filtrate as early as one hour later. Further experiments revealed "positively (1) and negatively (2) reacting tumors": 1. Positively responding tumors were sarcoma S/37, sarcoma 180, adenosarcoma M/63, Twort adenocarcinoma and Walker sarcoma. While **.. .very young, perfectly healthy tumors often gave no reaction, larger tumors gave practically 100 per cent positive results".-^^^ This observation is in consistency with the literature which postulates the necessity of an immune response to develop gradually as noted later by Berendt,"^^'-^^^'-^^^ and described by Wiemann and Starnes^^ as window in time. Obviously immunity can not be developed in hosts bearing very young tumors. 2. Negatively responding tumors were not further specified slow growing spontaneous or transplantable malignant tumors, which rarely or never regress, heterologous grafts of rapidly growing malignant tumors, which eventually regress, and benign, rapidly developing granulomas or embryomas, which eventually regress. Already in his time Shwartzman hypothesizes that the newly formed and highly fragile tumor vessels may have been one of the target mechanisms of endotoxin induced tumor necrosis. This observation is most interesting in the era of substances blocking VEGF and other mechanisms of nevascularization. Shear^ performed experiments with 2000 mice. He produced profound hemorrhagic necrosis and in some cases complete regression of malignant tumors following intravenous administration o^ Meningococcus. It further can be stated from this work and the experiments of Shwartzman and his contemporaries, that there is a direct correlation between the ability of a fdtrate from a given microorganism to prevent the development of sarcoma 180 in mice and to elicit the SP in rabbits. Much later, in 1985 Aoki and Mori^^^ described a local SP (LSP) confined to the tumor and a generalized SP (GSP) spreading to different visceral sites such as kidneys, liver, spleen and lung. Using E. coli endotoxin in Vx-2 carcinoma bearing cottontail rabbits they produced a GSP additionally to the hemorrhagic necrosis of tumors (LSP). The proposed mechanism of action for this phenomenon is disseminated intravascular coagulation (DIG), resulting in fulminate hepatitis and other organ changes. While GSP and DIG have not routinely been observed in patients undergoing mixed bacterial vaccine therapy for immunotherapy of cancer, those observations are important to keep in mind.

Reflections on Immunotherapy of Cancer with Bacterial Lipopolysaccharide (LPS) Gratia and Linz^^^ showed in guinea pigs the hemorrhagic necrosis of transplanted liposarcoma if the animals were treated with E. coli filtrates. Shwartzman and Michailovsky^ treating mice with the Sarcoma 180 with parenteral application oi Meningococcus culture filtrates observed hemorrhagic tumor necrosis and eventually complete remissions. Shear after isolating an endotoxin later defined as LPS as the active component of gramnegative bacteria, subsequendy induced necrosis in primary and experimental tumors.^ The discovery of LPS as the active compound of bacterial filtrates led to efforts to isolate and synthesize LPS. The group of Westphal at the Max Planck Institute in Freiburg pioneered this field (for a review see ref. 263). The endotoxin-induced necrosis is being initiated rather quickly: 4-8 hours following exposure to endotoxin the tumor tissue becomes inflamed; after additional 10-20 hours the center of the

288

Hyperthermia in Cancer Treatment: A Primer

tumor necrotises. Additionally, numerous endotoxin induced effects upon the immune system have been observed: Stimulation of the reticulo-endothelial system (RES), activation of macrophages,^^^ stimulation of B cell mitogenity,^^^'^^^ increased antibody synthesis,^^^'^^^ induction of interferons, fever, leukopenia followed by leukocytosis. (For reviews of molecular mechanisms see i.e., refs. 270-275.) It also should be mentioned that in vitro macrophage responsiveness to endotoxin does not necessarily indicate high in vivo sensitivity to endotoxin challenge.^^^ The immunological response to exposure of a variety of viruses and lipopolvsaccharides (endotoxins) has been clearly corresponding with antineoplastic effects. ''^^^^'^^^'^^'^^'^^ Moore et al^^^ showed that post-endotoxin sera (C parvum and S. abortus equi-Novo Pyrexal) contain high levels of myeloid colony-stimulating factor(s) (GM-CSF) and factors capable of inducing terminal granulocyte and macrophage differentiation of the murine myelomonocytic leukemic cell line WEHI-3. Also, exposure to endotoxins at work has been associated with decreased cancer risk.^^^ Kearney and Harrop^^^*^^^ argue that exposure to endotoxin might enhance tumor growth. They legitimately point to the importance of excluding endotoxin from solutions used in studies of experimental tumors. Other authors fear exposure to infections may lead to a process labeled "inflammatory oncotaxis".^^"^'^^^ Recendy, this phenomenon has been described further by researchers identifying cvtokines, leucocytes and macrophages in long standing cancers as promoters of tumor growth. '^^^ These infiltrations are being compared to chronic infections whereas the attempt to induce an immune response to cancer by artificially induced fever may be compared to an acute phase reaction observed in acute infections. The literature discussed in this review does not lend itself to suggest enhancement of tumor growth following antigenic exposure to spontaneous occurring tumors in human and animal models. Chun and Hoffmann^^^ reported that application of low doses of LPS could substantially increase the efficacy of TNF against murine cancers. What might be even more important is their observation that the blockage of two n^ative feedback responses occurring as a response to LPS treatment, namely the production of prostaglandin E (PGE2) and the generation of CD8^ suppressor T-lymphocytes (CD3^ CD 16/56-), dramatically increases the ability of mice to reject tumor transplants. In humans Otto et al^^^ achieved only one complete remission (CR) with intravenous endotoxins from Salmonella abortus equi in 27 patients with colorectal (1 CR, 2 PR) and 15 patients with nonsmall cell lung cancer (NR). While this group clearly coidd not come close to the results achieved by Coley, the group has performed numerous studies with the same strain oi SalmonelU^^^'^^^'^^^ (see Salmonella abortus equt).

Morita et al^ achieved dose-dependent inhibition of tumor growth with a synthetic lipid A analogue in a hamster pancreatic carcinoma model. Interestingly, endogenous tumor necrosis factor (TNF) activities were significandy greater in tumor than in serum, spleen and liver. Another group did not observe any antitumor effects with an isolated lipid A from Salmonella typhimurium and Salmonella minnesota?^^ TNF production by macrophages stimulated with lipid A after culture was much greater when the culture was performed in the presence of hamster pancreatic carcinoma cells (no cell-to-cell contact). Anti-TNF neutralizing antibodies inhibited the cytotoxic activity ofTNF secreted by macrophages. The authors hypothesize that lipid A displays antitumor effects by stimulating production of endogenous TNF in tumor macrophages, through activation and production of soluble macrophage-stimulating factors in cancer cells. Goto et al^°^ administered LPS intradermally in animal and human tumor models together with Cyclophosphamide, known for its synergistic effect with LPS (see: prostaglandins). After completion of dose escalation, the treatment was continued for at least 4 months, and it was found that 1800 ng/kg LPS was well tolerated. A significant level of cytokines was observed in the sera for at least 8 h. These results indicate higher tolerable doses and remarkably more continuous induction of the cytokines than were reported in a previous study by others using intravenous administration. Three of the five evaluable tumors showed a significant response to therapy.

Fever, Pyrogens and Cancer

289

Jimbo et al ^ showed that intravenous administration of a synthetic Upid A derivative significandy inhibited the growth of transplanted tumors in the liver of rabbits. These results suggest that systemic administration of lipid A induced selective tumor microcirculatory blood flow reduction via local endogenous TNF production. In contrast, local administration of human recombinant TNF alpha through the hepatic artery induced blood flow reduction not only in the tumor region but also in nontumorous liver tissue. Nowicki et aP°^ treated C57B1/6 mice bearing transplantable Lewis lung cancer (nonmetastatic subline) implanted either subcutaneously or intraperitoneally with macrophage colony stimulating factor (M-CSF), Escherichia coli lipopolysaccharide or both. LPS administered daily once a day for up to 30 days impaired both subcutaneous and intraperitoneal tumor growth and prolonged survival of tumor bearing mice. Macrophage colony stimulating factor administered daily, inhibited only subcutaneous tumor growth, both when administered alone and in combination with lipopolysaccharide, and had no effect on intraperitoneal tumors. Moreover, it did not prolong survival of tumor bearing mice, when administered alone, and nullified the effects of lipopolysaccharide when administered concomitantly. These data suggest that macrophage colony-stimidating factor, at least in this tumor model and in this dose schedule, offers little benefit. In contrast, the present data confirm earlier suggestions on the therapeutic usefulness of bacterial lipopolysaccharides in neoplastic disease. It has to be noted that hemorrhagic necrosis of tumors is to be distinguished from tumor regression.^^ Endotoxin-induced tumor necrosis takes place in the center of solid tumors, ofiien leaving a ring of viable tumor cells behind which eventually will lead to further cancer progression. Endotoxin-induced hemorrhagic necrosis always precedes tumor regression but is by itself only rarely followed by complete regression, i.e., tumor necrosis and tumor r^ression are mechanistically two separate events. It ftirther has been shown that endotoxin-induced tumor regression requires a state of T cell mediated immune response that is only induced in response to immunogenic tumors as classically defined. ^^"^^'^^^ Human tumor rejection antigens, which are recognized by T cells, may play an important role in the unspecific as well as specific immunotherapy for cancer.^^^'^^^ The hemorrhagic necrosis is thought to create conditions within the tumor, that are facilitating the entry and fiinctioning of effector T cells, an observation in accordance with the abilities of endo- and exotoxins to induce the expression of cell adhesion molecules^^^'^^^ and of TNF to induce capillary leaks.^^^'^^^ This T cell response again needs as an activation signal a prestimulation with antigenic substances such as BCG, corynebacterium parvum^^^'^ or the tumor antigens itself.^ The prestimulation has been associated with a highly activated macrophage system, which elicits the release of TNF and plays an important role in removing tumor cell debris. It is important to realize that the acquisition of concomitant inmiunity precedes endotoxin susceptibility. This process is generated over time following successive tumor growth and has been described as "window in time" when mice become susceptible to subsequent endotoxin challenge. ^^ In stunmary the following endogenous mediators have been identified which are relevant to endotoxin-induced tumor necrosis: TNF, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, granulocyte-macrophage differentiation factor (GM-DF), colony-stimulating-factor-1 (CSF-1), granulocyte-macrophage (GM-CSF), granulocyte-stimulating-factor (G-CSF), interferon-beta, and interferon-ganmia (for a review see ref 321). Moreover, the IL-12 mediated balance between T H l and TH2 cytokines on the one hand and the fimctional balance between prostaglandins and IL-1 mediated effects on the other side determines the type of immune response. Structural requirements of endo toxic reactions can be summarized as follows. (1) Lipid A structures proved to be the carrier of the toxic properties of endotoxin. (2) Conversely, beneficial reactions can be initiated not only by the complete structures but also by structural remains, which are no longer toxic. (3) Some of the split products in the lipid-free and polysaccharide-rich preparations can induce beneficial reactions. (4) Gram-negative bacteria can produce endotoxin-unrelated and beneficial compounds. Conventional endotoxin preparations are heterogeneous and often contain some of these unrelated substances.

290

Hyperthermia in Cancer Treatment: A Primer

Coley Toxins Used in the Treatment of Cancer Helen Coley Nauts, in an admirable eflfort, has compiled the work of her fadier William B. Coley in numerous articles and 18 monographs (Nauts HC: Monographs #87-108). The following critical points shall be mentioned from the analysis of Helen Coley Nauts work: Variability of the Preparations Used For the period between 1891 and 1953 Nauts reported the use of 14 different preparations of Coley Toxins (Nauts^^^) (see Table 2). The variability of the preparations used makes it desirable to determine which preparation reveals the bluest benefit in which cancer type. The importance of this fact has been stressed by Nowotny et al.^^^'^^^ Also, it is important to know which cytokine pattern is being induced by various techniques of growth and preparation. This research has been taking place in the nineties at the laboratories of Memorial Sloan-Kettering Cancer Center (personal communication: Nauts 1997) and awaits publication. Mikolasek^^^ demonstrated rejection of tumor allografts in mice treated with enzymes of (grampositive) Streptococcus pyogenes (streptolysine, streptokinase, streptodornase and hyaluronidase). He observed high antistreptolysine (ASLO) titers in the serum of mice and strong inhibition of subcutaneously implanted cystic adenocarcinoma following exposure to antigens. In addition, the author describes a complete spontaneous remission of a human adenocarcinoma of the uterus surviving 19 years since a Wertheim operation. Interestingly, this patient had had a high ASLO titer (595 lU/ml serum) which leads the author to speculate that an intercurrent Streptococcus pyogenes infection had taken place resulting in functional mitral valve impairment and induaion of inmiunity against metastatic disease. Although, as it is pointed out, the mouse tumor was allogenic and human tumors are autologous, the association between his observations appears to be valid. It might be interesting to look at epidemiological and clinical data to compare cancer incidence and ASLO titers in humans. Techniques and Timing of Administration Site and dosage of application of the Toxins are of considerable interest. Whereas Coley choose the intratumoral approach in his early years beginning in 1892 it was not until 1925 that Coley used the intravenous approach, which elicited stronger febrile reactions with smaller dosages. He also used intramuscular and subcutaneous administration, some of which have been questioned to be effective due to poor resorption. ^^^ It might be advisable to test small amounts of the toxins subcutaneously to rule out hypersensitivity reactions (personal observation). Of special interest is the intraperitoneal application since several cases of dramatic tumor regression in ovarian cancer have been reported following this route of administration.^ Application other than the intravenous route bears the advantage of slower release of endotoxins and more continuous stimulation of the host inmiune system. ^^^ Frequency and duration of injections of Coley Toxins obviously play another crucial part in the outcome of therapy. Daily or every other day injeaions often produced the best results. However, the general condition of the patient, compliance issues, the phenomenon of tolerance (see "Tolerance'' section) and hypersensitivity (see "Toxicity" section) suggest at least a 48-hour interval between injections. Coley suggested a six-month period during which treatment should be continued often performed by the attending family physician even after remission might have occurred. Only in later years Coley attributed treatment failures after initial tumor r^ression to a too early stop of therapy. ^^^' °^ Recent experiments confirm the importance of timing of toxine administration in experimental animal models. ^^ In this study it appeared that early intravesical BCG toxine therapy of bladder tumors, initiated after tumor inoculation resulted in slower progression rate than treatment initiation after a longer waiting period. Moreover, single injections of endotoxins in tumor bearing animals have been shown to induce periodically changing periods of enhanced beneficial effects, followed by phases of responsiveness below normal towards the inmiunological challenge.^^^ Further Nowotny et al^ recendy showed that the time intervals between

Fever, Pyrogens and Cancer

291

Table 2. Mechanisms following stimulation of humoral and cellular defense Type I Type II Type II Type IV

Type V Type VI Type VII Type VIII Type IX

Type X

Type XI

Type XII Type XH F Type XIII

Type IX

Living cultures o/"streptococcus erysipelatis (1891). Erysipelas toxins contained heat inactivated (100°C) erysipelas toxins (1892). Erysipelas toxins sterilized by passage through a Kitasato filter and not subjected to heat sterilization (1892). Mixed filtered toxins were the first mixed filtered toxins. Always freshly prepared, not-heated filtrate containing the soluble toxic products of streptococcus pyogenes and serratia marcescens (1892-1894). (Buxton's) Mixed filtered toxin. Buxton grew streptococcus pyogenes and serratia marcescens together in the same broth and filtered through a Kitasato filter (1894). (Buxton's) Mixed unfiltered toxin followed the procedure in type V but instead of being filtered they were heated for one hour at SOT - 6 0 T (1894-1907). Coley's mixed serum contained serum from streptococcus pyogenes and serratia marcescens prepared in the same way as diphtheria antitoxin (1894). Mixed toxins, filtered and unfiltered. Prepared by Lister Institute in London these toxins were similar to type IV, V and VI (1894-1943). Mixed unfiltered toxins prepared by Parke Davis & Co were the first commercially available preparations of streptococcus pyogenes and serratia marcescens (1899-1906). Mixed unfiltered toxins of streptococcus pyogenes and serratia marcescens prepared by Dr. Martha Tracy (1906-1907). ("The two organisms were grown separately and heated to 75°C for one hour {15°C higher than Type VI}. The amount of prodigiosus {serratia marcescens} was 5 mg per cc of the mixed toxins, determined by Kjeldahl's method of nitrogen determination. After mixing and bottling the toxins were again sterilized for two hours at 75°C. These were the most powerful of all, according to Coley.^^ They proved to be too toxic, due to large amounts of bacillus prodrigiosus"; cit. after Nauts^°^). (1906-1907). (Tracy) Mixed, unfiltered toxins of streptococcus pyogenes and serratia marcescens were prepared like Type X, with a lesser amount of serratia marcescens. ("This product appears to have been used in the largest number of successful results (Nauts etar°^); cit. after Nauts^°^). (1907-1922). (Tracy) Mixed filtered toxins. Similar to type XI but filtered. (Parke Davis) filtrate. Similar to type XII but stated to have been weaker (1906-1915). (Parke Davis) Mixed, unfiltered toxins were similar to type XII but Nauts assumed that..."although this product was more potent than type XII, it is now known that the active toxins and enzymes of streptococci are more thermolabile than those of bacillus prodigiosus, and therefore it would appear that heating the organism for 2 1/ 2 hours must have destroyed much of the streptococcus toxins"; (cit. after Nauts^°^). (1915-1951). (Sloan-Kettering) Mixed, unfiltered toxins prepared after Tracy XI formula with new strains of from streptococcus pyogenes and serratia marcescens (1946-1953).

endotoxin treatment and tumor challenge are of utmost importance to the capability of animals to reject a subsequent tumor challenge. While previous experiments used allogenic tumors and the elicited inmiune response was based on allogenic recognition and destruction of these tumors,^^ in this study endotoxin-induced rejection of less immunogenic tumors also was shown to be possible. However, there was a small time frame (-5 days until +1 days) when endotoxin inoculation elicited protection against subsequent tumor challenge. Later inoculation did not protect animals and higher doses even showed reduced inmiunity against leukemia L1210 cells. Conclusions from these observations for the therapy of established syngeneic tumors only can be speculative.

292

Hyperthermia in Cancer Treatment: A Primer

Stage of Disease The inverse relation between tumor load and immunological function is well established. ^ Chasseing et al^^^ coidd demonstrate that the immunosuppression associated with later stages of tumor development might be due to direct effects on monocytes, by down regulating IL-1 production. Also, in this study an increase in the levels of prostaglandin E2 and serum immune complexes could be detected. Related studies of the prognostic significance of circulating immime complexes (CIC) in malignant tumours of head and neck revealed a correlation between the level of CIC and stage of disease in head and neck cancer patients: Seropositivity for CIC increased quantitatively with stage of disease. ^^^ However, CIC containing MUC-1 encoded polymorphic epithelial mucin (PEM.CIC) was decreased in advanced breast cancer, i.e., there was an inverse correlation between positivity for PEM.CIC and extent of disease. Mucins, encoded by the MUCl gene, and CD43 (leukosialin) as the core protein, secreted or expressed in the plasma membrane of cancer cells could interfere with NK cell-mediated lysis in a dose-response-dependent way.^^^ The CTL response against differentiation antigens of the melanocyte lineage correlated inversely with antigen expression (Melan A/MART-1).^^^ Here, metastases increasing in size over time showed a loss of Melan A/MART-1 expression in the presence of CTL. Studies on natural killer (NK) cell activity showed a significandy lower cytotoxic activity in patients with laryngeal carcinoma who had histologically confirmed nodal involvement.^^ The study of serum inmiunoglobulins correlated with tumor load, while the estimation of CIC and blocking effect of cancer sera on normal lymphocytes was of diagnostic and prognostic significance.^^^ Wiltschke et al^^ showed reduced mitogenic stimulation of peripheral blood mononuclear cells as a prognostic parameter for the course of breast cancer in correlation to tumor size and axillary lymph node involvement. Thus, it is desirable to decrease the tumor burden prior to initiation of immunological therapies. If this attempt includes the use of chemotherapy, treatment related changes in the phenotype of target cells should be considered.^^ Radiation and Toxin Therapy Before the era of chemotherapy started in the 1950, irradiation was the treatment of choice for many inoperable tumors. The review of Coley s work shows that a large number of his patients received concomitant radiation therapy.^^'^^^'^^^'^^'^'^^^ Already in 1942 Shoulders^ ^ noted beneficial effects by combining the toxin therapy with irradiation in a series of far-advanced malignancies. Interestingly, toxins protected animals from otherwise lethal total body irradiation. Donaldson et al^ ^ observed the effect of Coley s Toxins and irradiation on the A. melanoma # 3 tumor in the golden hamster. She concludes: (1) toxin therapy does not affect survival; (2) toxin pretreatment potentiates X-ray therapy, (3) metastases are not affected; (4) normal tissues do not show increased radiosensitivity; (5) toxins plus X-ray therapy do not affect the prognosis or survival of the host; (6) toxins plus X-ray therapy show a synergistic effect**. Nauts several times points to the radiation-sensitizing effect of the toxins while normal tissue was better protected from side effects of radiation. Chandler^ achieved beneficial results in six out of eight patients with rhabdomyosarcoma, melanoma, and sarcoma. These tumors were usually considered radio resistant. The radioprotective effect later has been described by Behling and Nowotny,^^"^ and Nowotny.^^^'^"^^ Tang et al^'^ used Mixed Bacterial Vaccine (MBV) in the multi-modality treatment of hepatocellular carcinoma (HCC). Patients undergoing palliative resection and cisplatin therapy and radiotherapy, which were randomized to receive MBV, had an improved one and two year survival. In addition, MBV improved the "second look** resection rate to 40% as compared to 17% in the control. MBV could also prevent decrease of macrophage activity caused by radiotherapy. Kempin et al^^^'^^^ demonstrated improved remission rate and duration in nodular nonHodgkin lymphoma (NNHL) and advanced Nodular Lymphoma (NL) with the use of mixed bacterial vaccine (MBV) in combination with radiotherapy.

Fever, Pyrogens and Cancer

293

Other Toxins, Bacterial and Viral Products Used in the Treatment of Cancer Toxins and bacterial products in the treatment of cancer shall only be briefly mentioned because it would be beyond the scope of this review, which shall mosdy focus on Coley toxins (for reviews see refs. 347-354). Bacillus Calmette Guerin Old et al^ were the first to report upon beneficial effects of treating tumor bearing mice with bacillus calmette-guerin in the USA. Howard et al^^ confirmed earlier studies of the effect of BCG infection on the sensitization of mice to bacterial endotoxin and salmonella enteritidis infection. They found that mice infected two weeks previously with BCG were extremely susceptible to the lethal action of endotoxin. On the other hand mice were more resistant than normal to infection with Salmonella enteritidis. Without BCG administration the phenomenon of endotoxin tolerance would have occurred (discussed under "Tolerance" section). Ruddle and Waksman^^'^ demonstrated increased lymphocytic cytotoxicity after sensitization with tuberculoprotein. Schwartz et al^^"^ inhibited murine sarcoma virus oncogenesis with living BCG. Bluming and Ziegler^^^ and Mastrangelo^^^ successfully treated melanoma patients but observed different immunological effects of BCG depending on the route of administration. Hakim^^^'^ points to the possibility of enhanced tumor growth by BCG; he assumes that the serum from BCG-treated sarcoma-bearing animals blocks the spleen lymphocyte-mediated cytotoxic activities direaed against proliferation and growth of the sarcoma. Remissions of skin melanoma metastases following BCG injection have been shown by Remy et al.^^^ Vosika^ gives a comprehensive review of clinical immunotherapy trials of bacterial components derived from Mycobacteria and Nocardia: Preparations of isolated mycobacterial cell wall or cell wall skeleton attached to oil or to trehalose dimycolate have been favored over crude extracts and caused regression of disease and established tumor-specific immunity. Hence, BCG has been approved for the treatment of superficial bladder cancer.^^^'^^ Recently upregulation of Intercelluar-cell-adhesion-molecule-1 (ICAM-1) expression as an important mechanism of action of this treatment has been described on bladder tumours.^^^'^ The ICAM-1-GDI la pathway can render bladder tumour cells vulnerable to nonantigen specific cytotoxicity mediated by activated lymphocytes. Recent results of local BCG treatment forTl G3 bladder cancer, after TUR-B, showed a reduced risk of recurrence and mortality. Conti et al^^^ and Jackson et al^^^ could further show that BCG potentiates monocyte responses to lipopolysaccharide-induced tumor necrosis factor, soluble tumour necrosis factor receptors and interleukin-1, but not interleukin-6 in bladder cancer patients. Also, exposure to BCG has been shown to enhance interleukin-8 release in macrophages, a major inflammatory cytokine associated with enhancement of the immune response.^ It should be mentioned here that the group of Old discovered TNF in a BCG primed mouse.^^^ Propioni Bacteria Propioni bacteria (PB) (Synonym: Corynebacterium parvum) (CP) are amongst the potent immunomodulators stimulating cell populations involved in nonspecific resistance. Generally, the activated immune system provides protection from infectious pathogens and malignancies via mechanisms of recognition and elimination. Accordingly, administration of Propionibacteria could be shown to be of benefit in the treatment of neoplastic and infectious diseases. Thus, it may be recommended for further clinical investigations (for reviews see refs. 367-369). In vitro research showed induction of lymphokine-activated killer (LAK)-like cells capable of killing both natural killer (NK)-sensitive and NK-resistant tumor cells as well as syngeneic macrophages (M phi) (Chen et al^^°). Anti-interleukin-2 (IL-2) or anti-interferon (IFN) alpha, beta antibody significandy inhibited this induction of LAK-like activity by CP, suggesting that the generation of killer cells by CP was dependent on IL-2 and IFN(s) produced in the culture.

294

Hyperthermia in Cancer Treatment: A Primer

Bursuker et al^^^ showed, in accordance widi Keller et al,^^^ diat CP could render a murine nonimmunogenic tumor (Ml09) immunogenic. This immunity was tumor-specific and T-cell-dependent. T cells from mice whose progressive Ml09 tumors had been excised were capable, on passive transfer, of inhibiting adoptive immunotherapy of T-cell-deficient recipients by spleen cells from mice immunized with an admixture of Ml09 cells and CP. The authors argue, that the lack of anti-tumor immunity in this tumor model was not due to the absence of tumor-associated antigens but, instead, due to a shift of the balance from effector and suppressor arms of the immune response. Shifting the balance in favor of the effector arm by means of CP resulted in a measurable inmiune response to a noninmiunogenic tumor. Karashima et al^^^ showed that modification of NK cell activity is a possible basis for modulation of anti-metastatic activity by CP. Administration of CP showed a biphasic change in NK activity of the spleen cells and the peritoneal exsudate cells (PEC) in mice. Initially after administration of CP, the NK activity of the spleen cells and PEC was significandy augmented. At a later phase (14 days) after CP administration, the NK activity was deeply depressed. Further animal experiments with Propionibacterium /^rww-metabolites revealed stimulation of proliferation, maturation and emigration of thymocytes and lymphocytes,^ and inhibition of experimental lung metastasis of murine sarcoma L-1 in BALB/c-mice.^^^ Pulverer et al^^ further demonstrated a protective effect of combined treatment (CP and liver lectin blocking by D-galactose administration) on the liver colonization of RAW 117-HlO lymphosarcoma in BALB/c-mice. Both, immunomodulation with CP as well as liver lectin blocking by D-galaaose treatment significandy decreased the number of liver tumor colonies in this experimental model. The authors favor the combination of CP and D-galactose, which proved superior to each monotherapy since the liver colonization by RAW 117-H 10 lymphosarcoma could be completely inhibited. Lipton et al^^ demonstrated CP versus BCG as adjuvant immunotherapy of stage II malignant melanoma as superior over BCG when measuring disease-free and survival times. Foresti^ showed beneficial results in treating malignant pleural effusions with intrapleural instillations of CP. Of 20 patients with malignant pleural efftisions (MPE) treated with intrapleural CP, 18 (90%) had a CR and 2 patients (10%) had a PR. Preoperative immunostimidation by Propionibaaerium granulosum KP-45 in colorectal cancer resulted in resistance to the spread of cancer during operation.^^^ In this study PB was administered intravenously between the seventh and third day prior to surgical treatment for colorectal cancer. For stage I carcinoma the survival rates, were 9 1 % in the treated and 63% in the control group respectively. For stage II carcinoma the survival rate was 90% for the treated group with distant spread in 1 case and 45% in the control group where the rate of recurrence was 55%. For stages III and IV there was no statistically significant difference in survival between the treated and the control groups. Raica^^® studied 96 patients with superficial bladder tumors treated by transurethral resection in order to investigate the value of intravesical CP to prevent recurrences. Patients were studied in a 3-year follow-up. Recurrences were observed in 21.1% of cases in the CP treated group and in 54.5% of cases in the untreated group. Chronic lymphocyte infiltrates appeared to be the mediating event for the action of CP as an adjuvant therapy in urinary bladder cancer. These observations are in contrast with the recendy elucidated tumor growth promotion by immune components;^^^*^^^ thus the stage of the disease may be a critical factor since intravesical tumor cells only would recur in situ. Salmonella Abortus Equi Salmonella abortus equi has been studied for its antineoplastic effect by many authors in murine and human models^^^'^^^'^^^ (for reviews see refs. 263,275,321,381). Lipid A as the active compound has been isolated from the cell wall oi Salmonella abortus equi.^^^'^^^ The isolated product was known in Germany as Novo-Pyrexal . Engelhardt et al^^ determined dose-limiting toxicities including chills and fever (WHO grade III) following intravenous application at 1.0 ng/kg of body weight (maximal tolerated dose-1, MTD-1). The interesting finding was that endotoxin could be administered intravenously using 4,0 ng/kg body

Fever, Pyrogens and Cancer

295

weight when the patient was protected by 1,600 mg ibuprofen. The induction of high amounts of circulating tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-8 (IL-8), granulocyte colony-stimulating factor (G-CSF), and macrophage colony-stimulating factor (M-CSF) was not influenced by ibuprofen administration. Conversely, repeated injections of LPS at daily intervals residted in marked downregulation of the cytokine induction (see: Tolerance) with the exception of IL-1 beta and G-CSF.^^^ Interestingly, cancer patients pretreated with 50 |ig Interferon-y 12 hours prior to endotoxin administration exhibited not only prevention of downregulation of endogenous cytokines (IL-6, IL-8, G-CSF, TNF alpha) normally observed after repeated endotoxin application, but showed enhanced secretion of these cytokines to levels even higher than those achieved after the first LPS challenge.^^ As mentioned earlier, only moderate antitumor activity was observed in different tri^g 283,284,286;287,290 Pm^^^ results of Studies with Salmonella abortus equi will be discussed in the cytokine and tolerance section of this paper. OK-432 OK-432 is the benzylpenicillin-treated lyophilized powder oi Streptococcus pyogenes group A cell wall extract. The product is known in Japan as Picibanil®, Chugai Pharmaceutical Company, Tokyo, Japan. The TNF inducing properties of OK-432 are well known^^ (for reviews see: refs. 385-392). OK-432 has been widely tested in at least 18 randomized clinical trials. Caution in regard to validity and eeneralizability of these trials are indicated in reference to several aspect according to Abel:^ "(1) the overwhelming majority of trials has been conducted in the same geographical region, Japan; (2) the intention to treat was violated in most studies; (3) case numbers in most studies were very small; (4) careful statistical analysis revealed a null hypothesis in most studies; (5) randomization was conducted by the participating clinicians". Nonetheless, the antitumor effects of the substance are well established and warrant further careful designed prospective, randomized studies. Studies have been performed for cervical cancer,353-3« bladder cancer.^'^'^^ gastric carcinoma^'^^^ ^^^ cancer,'^*^'" liver cancer,^' '•'"^ malignant gliomas, chylothorax following esophageal cancer. In basic research and animal models Nio et al^^^ demonstrated antitumor activity of orally administered OK-432 on murine solid tumors. Noda et al"^^^ induced Interferon-gamma in human peripheral blood mononuclear cells by OK-432. Sekimoto et al^^'' showed the production of TNF by monocytes from cancer patients and healthy subjects induced by OK-432 in vitro, and its augmentation by human interferon gamma. OK-432 activated mononuclear cells were shown to be able to kill T98G glioblastoma cells by apoptotic mechanism through the Fas ligand/Fas system. Moreover, it has been demonstrated that the intrapleural administration of OK-432 in 70-80% of patients with malignant pleural efftision from metastatic lung cancer stimulated clinical improvement but more importandy, mediated a reduced suppressor macrophages and increased NK cell activity and cytokine production such as IL-1, MCF by macrophages and IL-2 and NK cytotoxicity factor by lymphocytes."^^^' ^^ These authors achieved similar results earlier in patients suffering from cancer of the stomach or the lung. ^^ Proteolytic Enzymes with Special Reference to Streptokinase Streptokinase is one of the active enzymes of the streptococcus strain used by William B. Coley and has a long history in cancer research ever since. Additionally, streptokinase has become an important tool in measuring plasminogen activation. However, there are scientists who think of streptokinase and other proteolytic enzymes to augment metastasis and others, who think of those enzymes as a promising adjuvant in cancer therapy. Both lines of thinking shall be briefly elucidated. While the external use of (proteolytic) enzymes in preclinical and clinical research has been long a domain of substituting genetically deficient enzymes (reviewed in ref 423), considerable evidence is mounting for their usefulness in a variety of immunologically mediated diseases including cancer and there has been increasing interest in the clinical use of proteolytic enzymes. ' ^ Clinical trials have examined the therapeutic efficacy of both single and

25^6^

Hyperthermia in Cancer Treatment: A Primer

combined application of these enzymes in individuals with a number of different conditions, including trauma, inflanrniatory and autoimmune diseases, mastopathy and cancer. ' The fact that tumors show enhanced fibrin deposits especially in the invasive periphery has been noted as early as 1958 and has subsequendy been used for targeting porphyrins as photosensitizers to tumor cells."^^^ Hence, the well known fibrinolytic effects of proteolytic enzymes have been suggested as a therapeutic rationale in tumor therapy. Additionally, numerous additional molecular mechanisms in support of this rationale have been elucidated lately. There is an increasing body of knowledge for the inmiunological mechanisms underlying the effects following exposure to proteolytic enzymes in vivo and in vitro. (For brief comments on the rationale of therapeutic use of proteolytic enzymes see refs. 428,429. As for the oral forms of proteolytic enzymes their enteric resorption as biologically intact macromolecules has beendescribed/^^-«''-^32 Already more than 20 years ago rejection of tumor allografts following treatment with enzymes from streptococcus pyogenes yf2& demonstrated by Mikolasek,^^^ activation of cellular immunity in cancer patients and enhanced activity of E rosette forming lymphocytes following proteolysis in vitro by Holland et al ^ and Thornes. These authors described anergic states in cancer patients, which were reversed by administration of proteolytic enzymes. Conversion back to an anergic state after stop of therapy however, resulted in recurrence of the disease. Later, Tomar et al ^ reported activation of NK cells in vitro by streptokinase. (See discussion of the observations by Mikolasek^^^ also in C.l of this review.) Additionally, successful therapy of accessible tumors like basal cell carcinomas, mycosis fungoides, and cutaneous metastasis of breast cancer have been reported by local appUcation of streptokinase-dornase. ^'^ Alteration of CAM expression has been described recendy ^^' ^^ and may be one underlying mechanism for clinical effects of application of proteolytic enzymes. Recently, enzyme-induced upregulation of lymphocyte beta2 integrins, and downregulation of L-selectin and CD44 has been observed in vitro, which may explain some of the immunologically mediated anti-tumor effects of proteolytic enzymes."^^^' NK cells utilize these beta2 integrins (CD 11/CD 18) for firm binding to tumor target cells,"^^"^^ and this class of cell adhesion molecules becomes rapidly increased on human NK cells upon activation. ^ Furthermore, cytotoxicity of lymphocytes against tumor cells has been shown to be gready inhibited when lymphocytes were treated with anti-CD 1 la and anti-CD 18, but not when treated with antiVLA-4 antibodies. Moreover, decreased CD 1 la/CD 18 cell surface expression has been shown to correlate with a decrease in NK cell activity, ^^ and an increase in CDl lb/CD 18 expression has been shown to enhance adherence of neutrophils to tumor cells. Additional clinical relevance for cancer therapy might be provided by these studies, which showed reduced expression of CD44 following enzyme treatment. High levels of CD44 expression on cancer cells facilitate malignant cell adherence to the extracellular matrix and thus are promoting metastatic tumor growth.^^^'"^^^ Compatible with this observation, an antimetastatic effect of in vivo application of bromelain ' and of streptokinase ' was observed in mice. It is well known that arresting tumor cell emboli in the microcirculation facilitates the development of blood borne metastases. Hence, additionally to the effects on cell adhesion molecule expression, it has been suggested, that enzyme-induced increased fibrinolysis caused a decrease in metastatic seeding. Uster et al showed bromelain to be effective in decreasing the attachment of human bladder and melanoma cells to extracellular matrix components. In this respect it is also of interest to note that the fibrinolytic system in aged rats, and its reactivity to endotoxin and cytokines shows significant decrease in activity which makes the individual more susceptible to endotoxin-induced effects, including microthrombosis and platelet aggregation."^^^ Consistent with these observations is also a study by Murthy et al who demonstrated a decreased tumor formation of TA3Ha mammary tumor cells in healing hepatic wounds of syngeneic strain A mice following treatment with human plasmin B-chain-streptokinase complex (B-SK) and recombinant tissue plasminogen aaivator (PA). Urokinase and heparin had no effect upon tumor formation in this model. PA was suggested to produce plasmin which, in turn, digests cell adhesion molecule protein structures and in due course inhibits tumor cell

Fever, Pyrogens and Cancer

297

attachment. Similar studies of the inhibitory effects of orally and systemically applied proteolytic enzymes on cancer growth and metastasis have been performed by Maruyama et al ^^ on sarcoma-180 ascites cells in vivo and by Thornes in clinical studies of postmenopausal patients with breast cancer and colorectal carcinoma. Thornes ^^' provided evidence that streptokinase treatment attenuated lymphocyte depletion following surgery and increased cellular immune functions. Szreder ' demonstrated remissions of different cancer types in humans and animals following artificially induced abacterial erysipelas and chronic aseptic abscesses. L-asparaginase has been proven to be a useful adjunct in the treatment of acute lymphoblastic leukemia, but additional experience also suggests a role in acute nonlymphoblastic leukemia. ^^ Higher levels of plasma fibronectin in patients with acute myeloid leukemias and blast crisis have been reported to decrease following streptokinase therapy. Earlier studies, which included streptokinase in an attempt to increase the response to chemotherapy with cyclophosphamide did not show a beneficial effect of the enzyme treatment in that model,"^^^' reported no increased effectiveness of electromagnetic radiation by the use of a single intravenous application of 350.000 lU of streptokinase. The other line of evidence is concerned with possible enhancement of tumor growth and metastasis induced by administration of proteolytic enzymes. Teuscher and Pester^ i.e., showed in an in vitro model that the application of antifibrinolytic drugs mediated the inhibition of vascularization of tumors. These authors hypothesized that inhibitors of serine proteinases and of plasminogen activators reduced the migratory behavior of tumor cells and that streptokinase, conversely stimulated cell migration. An earlier study reported increased spontaneous pulmonary metastasis in rabbits following treatment with human serum plus stteptokinase, an effect, which these authors attributed to fibrinolysis. McKinna and Rowbotham reported intravascular dissemination following streptokinase injection in an in vitro tumor colon cancer model. Staphylococcus Protein A The antitumor property of Staphylococcus protein A (PA) is well documented in the literature in various transplantable murine tumor models (reviewed in refs. 328,468,469). Protein A (PA) is an immunostimulating glycoprotein (mol. wt. 43,000 kDa) obtained from Staphylococcus aureus cowan I and attaches to the Fc fragment of IgG 1, 2 and 4, and preferentially binds to IgG included in immune complexes. Plasma absorption over PA has been shown to effectively reduce high levels of pathologic soluble circulating immune complexes (CIC), a method which has considerable less side effects and toxicity opposed to plasmapheresis. Animal and some human studies showed encouraging results in increasing cellular immunity, reduction in blocking activities and tumor regression with the use of plasma absorption over PA and direct administration of PA."^^^"^^^ Interestingly, some of these authors later reported that a leakage of bacterial products from staphylococcus species during plasmapheresis resulted in a general and unspecific immunostimulation and partially explained the beneficial effects of plasma absorption over PA.^^"^ Additionally, similar results have been observed when PA has been injected direcdy into tumor bearing animals. It is of interest to note that this approach did not elicit generalized toxicity. Also, PA injection has been shown to suppress the onset of tumorigenesis by inhibiting initiation and promotion of carcinogenesis. ' ^^ The indirect action by which PA may foster immunity and reduce CIC has been expressed by Zaidi et al. They suggest that the PA-induced depletion of B-lymphocytes leads to a decreased production of antibodies and subsequendy reduced levels of soluble immunosuppressive CIC. Furthermore, PA has been shown to exhibit potent cytokine stimulating properties and to enhance LAK cell induction and activity in lymphocytes from healthy volunteers and melanoma patients. These studies recendy led to the promising application of superantigen staphylococcal enterotoxin A (SEA) combined with the Fab fragment of a tumor-specific antibody^ as efficient immunotherapy for lung melanoma micrometastasis and lymphoma therapy^^^ in mice.

298

Hyperthermia in Cancer Treatment: A Primer

A new development for the administration of a staphylococcus derived enterotoxin has been investigated in China recendy. Researchers in China found that the Highly Agglutinative Staphylocoin (HAS), a super antigen biological product made by Shenyang-based Xiehe Group in China known as Gaojushengy can activate the patient s T-cells and repair damaged tissues by promoting or stabilizing the interaction between antigen-presenting cells and T-cells.^^Mt has been demonstrated that there is a clear relationship between the affinity of Superantigens for the T-Cell Receptor and their biological activity. Xiehe Group had put the super-antigen products into preclinical research in 1989 when the theory of super-antigen was proposed. In Western countries, super-antigen based research was first reported in 1997 for phase I clinical trials. Other Toxins Cholera toxin has been shown to effectively inhibit manmiary cancer growth in vivo and in vitro. This rejection has been associated with an increase in intracellular cyclic adenosine3":5''-monophosphate. Freunds adjuvant has a long history in active specific and nonspecific immunotherapy in cancer and is beyond the scope of this review to be discussed. NK-cell reactivity, i.e., in stage I and II nonsmall-cell lung carcinoma receiving adjuvant immunotherapy with Freunds adjuvant after surgery, was increased nonspecifically as demonstrated by Maroun et al. ^ Keyhole limpet hemocyanin (KLH) is derived from an inedible mollusk found on the pacific coast. Immunotherapy with ganglioside-KLH has been performed mainly in superficial bladder cancer and malignant melanoma next to BCG (for reviews see refs. 485,486,487). Gangliosides, containing glycosphingolipids that are anchored into the lipid bilayer of the plasma membrane, and which are overexpressed on tissues of neuroectodermal origin can be targets for the KLH-therapy of melanomas, sarcomas, neuroblastomas and astrocytomas. KLH acts as a potent vaccine targeted at these gangliosides to induce cytotoxic IgM antibodies, which are able to initiate complement mediated cytotoxicity. Intralesional KLH significandy reduced tumor incidence, growth rate, and mortality in the mouse bladder tumor model (MBT2).^^^ Moreover, instillation of KLH into the bladder has been found to have fewer side effects than BCG. It has been suggested that a local cytokine release of IL-2, IFNs, and TNF is involved in the effector pathway of KLH application. Sargent and Williams suggest that the lack of endogenous cytokine activity secondary to immunosuppressive events following cancer growth may be overcome by direct, local application of KLH. Interestingly, prevention of bladder recurrence correlated significandy with cutaneous delayed type hypersensitivity testing.^^i Viral Approaches There is an extensive literature on the use of viral approaches for the treatment of cancer (for reviews see refs. 492-499). First reports of leukemia treatments with viruses date back to mid sixties^^'^^^ of the 20th century. Wheelock and Dingle achieved febrile responses to repeated administration of six different viruses and observed clinical and hematological improvement in a patient suffering from AML. Importandy, in this patient, each treatment was followed by significant temperature surges. Recombinant viral protein derived vaccines for specific immunotherapy of cancer aim at the attempt to specifically target a T cell mediated immune response to cancer antigens. The vaccines are used to enhance the immunogenicity of cancer antigens. The lysis of carcinoma cells by T cells (CD8^ and or CD4^/CD8'^) was shown to be HLA restricted. Incorporating virus in autologous tumor vaccines has enhanced the antigenicity of tumor vaccines. Among viral treatments the Newcasde Disease virus (NDV) has most widely been used as a crude agent as well as using viral vectors.'^^^'^®^'^^^ The Newcasde Disease virus has been shown to be superior in preventing side effects over BCG admixed tumor vaccines.^ Antigenic targets include normal antigens that have a limited health-tissue distribution or expression (i.e., carcinoembryogenic antigen), viral proteins (i.e., E6 protein of human papillomavirus), and

Fevety Pyrogens and Cancer

299

mutated oncogens. Recent research has focused on peptide recognition by cytotoxic T-ceils, the expression of antigenic peptides bound to major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells, and the requirement for a second signal forT cell activation, such as the costimulatory molecule B7. It has been shown that viruses attached to autologous tumor vaccines deliver these costimulatory signals to tumor-reactive T cells following postoperative vaccination of tumor bearing hosts. ^^ Viral oncolysates induce two different types of inmiune response. NK cells induce target cell lysis primarily by the production of granzymes and poreforming proteins and do not need help from memory cells. In contrast, T cells lyse target cells primarily by the MHC-restricted release of lymphotoxin (TNF beta) causing programmed cell death (apoptosis) through endonuclease activation and target cell DNA fragmentation, a process which needs the assistance of memory cells. ^^ Shillitoe et al"^^^ used human papillomaviruses for gene therapy of cancer to target antisense or ribozyme molecules directed against these genes. The viruses are present in many cervical and oral cancers, and are likely to be etiological agents of the tumor. Recendy Hodge et al could show that the combination of a recombinant vaccinia virus containing the gene for the costimulatory molecule B7 and a recombinant vaccinia virus containing a tumor-associated antigen gene resulted in enhanced specific T-cell responses and antitumor immunity. Lately, defective presentation of MHC class I restricted antigens on a murine sarcoma have been identified by the failure of these tumor cells to present influenza virus antigen to virus-specific cytotoxic T cells.^^^ This approach in thefixturecould lead to ex vivo assessment of inmiunogenicity of tumors. Otherwise, it should be considered that MHC class I antigen defective cells are more likely to be detected by natural killer cells. ^^^'^^"^

Proposed Mechanism of Action Shear et al^ discovered in Coley s mixed bacterial vaccine (MBV) lipopolysaccharides (LPS) as the active substances. LPS, which are potent pyrogens, are the component of the outer membrane of gram-negative bacteria. Endotoxins do not kill tumor cells in vitro and therefore their antineoplastic effects have to be mediated by host-dependent mechanisms. These immunologic mechanisms include the activation of macrophages, natural killer cell (NK) (CD3-CD16/ 56^), cytotoxic T cells (CD3'^ CD16/56') and the release of cytokines.^^^ The prominent cytokines being secreted by activated macrophages and the RES are interleukin 1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF a), IL-12, GM-CSF and additionally TNF p (Lymphotoxin).^^^'^^'^

LPS Binding Sites At least five signal transducing binding sites expressed on the lymphocyte plasma membranes have been identified as LPS receptors including two proteins of 70-80 kDa and of 30-40 kDa, CD14, die GDI 1/18 family of adhesins and die 95 kDa scavenger receptor.^^^'^^^ The predominant discussed molecule in the literature is a circulating molecule named lipopolysaccharide binding protein (LBP) which forms a complex with endotoxins and was first thought to bind as a complex to the monocyte differentiation antigen, CD 14.^^^'^^'^'^'^^ Binding of the complex to monocytes and macrophages activates the cytokine secretion cascade of these cells. Lately it coidd be demonstrated that the picture is more complex. The LPS receptor CD 14 is a protein expressed on the surface of monocytes, macrophages, and polymorphonuclear leukocytes and a soluble, circulating protein in the blood. Both forms of CD 14 partake in the LPS response. Studies with recombinant LBP (rLBP) suggested that LBP functions catalytically, as a lipid transfer protein function basically to accelerate the binding of LPS to CD 14. On the other hand LPS and rsCDl4 complexes formed in the absence of LBP stimulate integrin function on PMN and expression of E-selectin on endothelial cells, indicating that LBP is not necessary for CDl4-dependent stimulation of cells. ^°

300

Hyperthermia in Cancer Treatment: A Primer

Recently a novel receptor for Escherichia coli heat-stable enterotoxin (ST) has been identified as a highly selective biomarker for metastatic colon cancer. ^^^'^^^ ST-receptor interaction was coupled to activation of guanylyl cyclase C (GCC) in all normal tissue samples of colon and rectum and all primary and metastatic colorectal tumors examined. However, neither ST binding nor ST activation of GCC was detected in any extraintestinal tissues examined. It may be hypothesized that these receptors may serve as a target for directing therapeutic administrations of bacterial vaccines to GCC expressing tumors in vivo. Furthermore, intracellular binding sites have been identified recently at microtubule proteins. This binding site is shared with the microtubule acting agent taxol. Microtubides are constructed from the heterodimers of alpha- and beta-tubulin along with microtubuleassociated protein-2. They are mediating mitosis and protein trafficking. ^^^'^^^

CellAdhesion The upregulation of cell adhesion molecules (CAM) following exposure to inflanmiatory cytokines and lipopolysaccharides has been established on a variety of cell types like endothelial cells, fibroblasts, synoviocytes, Langerhans cells, melanocytes, keratinocytes, mast cells, monocytes and eosinophils.^^^ CAM play a crucial role in endo-and enterotoxin-mediated lymphocyte distribution and target signaling. Alterations in the expression of CAM may have a profound impact on a wide range of immunologic processes (reviewed in refs. 566,563). Four major groups of cell adhesion molecules have been identified. First, members of the immunoglobidin superfamily (i.e., ICAM-1, ICAM-2, LFA-2), which facilitate cell adhesion byT and B lymphocytes. Second, members of the integrin family (i.e., LFA-1, MAC-1, p-150,95, VLA-1-6), whose fiinction is the dynamic regulation of adhesion and migration. Third, the selectin family (i.e., L-selectin, PADGEM, ELAM-1, LAM-1), which selectively targets leukocyte and neutrophil migration to tissues like lymph nodes or sites of acute inflanmiation. Last, CD44 which predominandy acts as the hyaluronate receptor, also fiinctions as a general cell adhesion molecule and, additionally, regulates activation thresholds for T cells. Thus, an endotoxin-mediated modulation of CAM can be expected to have crucial effects on leukocyte distribution and function. When T cells (CD3^ CD16/56-) and NK cells (CD3- CD16/56") encounter an antigen, mast cells secrete TNF a and IL-1 which in turn induces T- and NK cells to secrete IL-2 and Interferon-y (IFN-y).^^^'^^^ IFNy has been shown to affect the expression of MHC class II molecules by endothelial cells,^ ^ to increase the binding of T cells to endothelial layers, and to enhance the recirculation of lymphocytes through the lymph nodes.^^^ Furthermore it has been shown that IL-1, TNF and endotoxins enhance the binding of lymphocytes and neutrophils to endothelial cell monolayers.^^^'^^^ Pyrogenic substances are known to induce leukopenia followed by leukocytosis. This recruitment of lymphocytes from and to the blood stream occurs via the postcapillary venules. Cell adhesion molecules, expressed on lymphocytes and the endothelial cell layer, mediate rolling, adhesion and subsequent migration of lymphocytes.561'564-566 jj^^^ process is regulated by cytokines and is fundamental in understanding the early events following exposure to pyrogenic substances. Leukocyte cell adhesion is mediated by exposure to stimuli such as antigen for lymphocytes or complement factors and leukotriens for monocytes and granidocytes. The forming of cell a^regates or clusters to each other or to other cell t)yes such as vascular endothelial cells appears to be regulated by the activation state of the cell. When inflanmiatory mediators such as cytokines, thrombin and histamines are released following antigenic exposure (such as endotoxins) they cause the activation of blood vessel endotheUum. Qrtokines (IL-1, TNF) induce the expression of E-Selectin on endothelial cells after 3-6 \io\xTs, whereas thrombin and histamine lead to the release of P-Selectin on endotheUal cells within minutes. Additionally, in response to inflammatory cytokines such as IL-1, IL-4, TNF and IFN VCAM-1 and ICAM-1 are induced on vascular endothelium within 6-12 hours and 12-24 hours respectively. ^^^ Protein kinase C is a mediator of endothelial cell activation by LPS, TNF, and IL-1.^^^ The recruitment of lymphocytes into gut-associated tissues of

Fever, Pyrogens and Cancer

301

Peyer s patches and nonlymphoid villus regions of the small bowel is also mediated by cell adhesion molecules alpha 4-integrins and beta 2-integrins.^^^ IL-4, for example, a product of activated T cells, can interact with TNF to selectively elevate VCAM-1 expression and the induction of T cell-rich infiltrates.^"^^ It could be shown that the enterotoxin o^ Staphylococcal A (SEA) increased the cytotoxic T cell response to target cells by binding to major histocompatibility complex (MHC) class II molecules.^^ The cytotoxic activity clearly was mediated by HLA-DR2/ICAM-1 expressed on target cells binding to the integrin heterodimer CD 11 a/CD 18 expressed on effector cells as could be shown by anti-CD 11a or anti-CD 18 monoclonal antibodies (mAb), but not by anti-CD lib, anti-CD 11 c, or anti-CD2. Furthermore, it coidd be shown that resistance of choriocarcinoma cells^^ and melanoma cells to lysis by lymphocytes was partially due to a low expression of ICAM-1 and VCAM-1 respectively. Cell adhesion cell deficient mice exhibited impaired immune responses.^^^ Heat-inactivated gram-negative bacterium Brucella abortus not only has been shown to induce the secretion of IL-12 to differentiate Thl andTh2 cells but also to rapidly increase the expression of the costimulatory molecules B7.1 (CD80), B7.2, and ICAM-1.^^^'^^^ In these studies induction of IL-12 was confirmed by IL-12 p40 mRNA expression and protein secretion by isolated human monocytes. This initiation was blocked by an anti-CD 14 monoclonal antibody, su^esting that monocytes bound B. abortus visi their LPS receptor. Additionally, NK cell cytotoxicity against K562 target cells was enhanced.^^^ Interestingly, LPS induction of B7-1 on human monocytes was superior to IFN-gamma and no response was obtained with isolated IFN-alpha, grantdocyte-macrophage colony-stimidating factor (GM-CSF),TNF-alpha and GM-CSF^TNF-alpha.^^^ LPS, rhIL-I, and rhTNF-alpha act via common pathways in endothelial cell activation, a process that is being regulated by protein synthesis. Increased expression of cell adhesion molecules induced bv endotoxins is mediated via elevation of intracellular cAMP concentration activation in turn is sequentially mediated by proteinkinase C in the early phase of activation and by proteinkinase A in the later adhesion of lymphocytes. ^^^ The adhesion augmented by increased [cAMP]i is due to LFA-l/ICAM-l interactions between cells because it can be blocked by either anti-CD 11 a or anti-ICAM-1 mAb. A differential role of protein kinase C (PKC) in cytokine induced lymphocyte-endothelium interaction was established by Eissner et al^^ in vitro. TNF-alphaand LPS-induced ICAM-l expression on a human endothelium-derived cell line (EA.hy926) was imaffected by the PKC-inhibitor and thus appeared to be independent of PKC activation. In contrast, PKC-inhibitor significandy reduced ICAM-1 expression induced by IFN-gamma andIL-1. The functional implications of increased CAM expression on lymphocytes and endothelial cells following LPS challenge remain controversial. CAM clearly have been shown to costimulate cytotoxic T cells,5^5.586 jj^ ceUs^^^'^ss ^ ^ ^j^ cells.^^^'^^^'^^^ NK cells utilize die beta 2-integrins (CD18/CD1) for firm binding to target cells.^5-447.59i ^p^^^ activation, CDl lb and CDl Ic are rapidly increased on human NK cells."^^ Rat Kupffer cells mediated cytotoxicity against a syngeneic hepatoma cell line both by the production of nitric oxide and cell-to-cell adhesion via ICAM-1/CD 18.^^^ This cytolytic activity of lymphocytes against tumor cells is greatly attenuated when the lymphocytes are treated with anti- CDl la and anti- CD 18.^9 However, peritoneal PMNs derived from patients with bacterial peritonitis have been shown to have increased ICAM-1 levels but were functionally inactive to protect the host from microbial invaders.^^^ The authors speculate that an interaction between ICAM-1 and its' counter receptor CD18/CDlla may hinder effector functions. Furthermore, treatment of head and neck s e c cells with recombinant human interferon gamma (rHuIFN), a well known enhancer for the expression of CAM, did increase the ICAM-1/CD 11 a/CD 18 mediated binding of both LAK cells and PBM cells to tumor cells. But on the other hand, cytotoxicity of LAK cells against head and neck SCC cells was reduced after rHuIFN treatment.'^^^ Additionally, shedding of ICAM-1 from cultured tumors is able to inhibit the CDl la/CD 18/ICAM-l interaction between cytotoxic effector cells and ICAM-1^ target tumor cells.^^^ It is known that

302

Hyperthermia in Cancer Treatment: A Primer

shedding of CAM follows activation of resting leukoqaes with subsequent upregulation of CAM from intracellular storages.^^^'^^"^ Hershkoviz et al showed that heat-stressed CD4^ T lymphocytes exhibit differential modulations of adhesiveness to extracellular matrix glycoproteins, proliferative responses and TNF-a secretion. Heat-shock treatment of activated CD4^ T cells induced a decrease in the surface expression of beta 1 integrins, which in turn reduced T cell adhesion to fibronectin and laminin. On the other hand the potential of heat-stressed T cells to proliferate and to secrete TNF-alpha was increased. It should be noted that the expression of cell adhesion molecules not only mediates lymphocyte adherence, migration, extravasation and target cell recognition but also cancer cell metastasis.^ Analysis of soluble and target cell bound expression of cell adhesion molecules may be an important tool to measure the molecular effects of a given therapeutic intervention. In acute endotoxin overstimulation such as in patients with septic multiple organ failiue levels of soluble adhesion molecules (sICAM-1, sELAM-1, sVCAM) were significandy elevated. ^^^

Cytokines Exposure of the host to endo- and exotoxins initiates a complex imd multidirectional cytokine cascade. The physiological role of cytokines in our understanding of the cellular and humoral immune mechanisms involved in antitumor activities is a continuously growing body of knowledge and has been extensively reviewed.^^^'^^^"^^^ Cytokines include the interleukins, the interferons, tumor necrosis factor and the colony-stimulating factors. Clinically, the highest response rates to exogenous cytokine inmiunotherapy have been seen in malignant melanoma and renal cell cancer.""^ It has been shown that a variety of cytokines are needed for an effective CD8^ T cell mediated cytotoxicity by tumor cell-targeted gene transfer of interleukin 2, interleukin 4, interleukin 7, tumor necrosis factor, and interferon ganrnia.^^^ They have been widely used for immunotherapeutic approaches in cancer treatment (for reviews i.e., refs. 492,603-608). Recendy, different groups attempted to increase the therapeutic potential of these agents with genetic manipidation by introducing genes encoding cytokines into tumour-infdtrating lymphocytes and certain tumor cells. In this chapter some basic mechanisms of LPS induced cytokine secretion and their relevance to inmiunological responses to cancer shall be briefly summarized. Shieh et al^^^ for example, showed that LPS modulated CSF-1, granulocytemacrophage (GM)-CSF, G-CSF, IL-1, TNF, and Kit Ligand receptors on murine bone marrow cells (BMC) in vivo and in vitro. In vivo, LPS and LPS-induced cytokines (IL-1 and TNF) elicited the secretion of glucocorticoid and CSF activities, which revealed a mechanism for LPS up-modulation of IL-1 R on BMC in vivo. Interestingly, application of single cytokines like IFN-gamma could not activate macrophages for tumor cell killing, but required a second stimulus fromendotoxin.^^^'^^^ The cytokines shall be mentioned successively, although this mode of description is certainly not ideal since almost all immunological responses to LPS or other antigens involve a whole cascade of cytokines being induced and secreted. Not only LPS but also antigens from grampositive organisms evolve powerful immune responses. Two types of cytokine pattern and kinetics have been described after exposure of lymphocytes to (grampositive) Staphylococcus aureus enterotoxin A (SEA) and (gramnegative) lipopolysaccharide (LPS).^^^ First, LPS stimulation provoked strong production of IL-1 alpha, IL-1 beta, TNF-alpha, IL-6 and IL-8. After LPS exposure IL-1 alpha, IL-1 beta, TNF-alpha and IL-8 were peaking at or before 4 hours after cell stimidation. Also, IL-10 production was evident after 12 homrs of cell stimulation. TNF-beta, IL-2, IFN-ganuna and IL-4 were not detected in these cultures. All cytokine production, except IL-8, was downregulated at 96 hours. Second, SEA-stimulated cultures showed the highest point in production of IL-1 alpha, IL-1 beta and IL-8 later, after 12 hours. In addition, significant production of TNF-beta, TNF-alpha, IFN-gamma and IL-2 by T lymphocytes was found with peak production 12-48 hours after initiation of SEA IL-6 was only discovered in low amounts. Although in the

303

FeveTy Pyrogens and Cancer

Table 3. Mechanisms following stimulation of humoral and cellular defense

Effector Cell

Cytotoxic Effect

Macrophage NKcell LAKcell Granulocyte Mast cell Mast cell Mast cell Mast cell Mast cell Mast cell Mast cell

Direct cell-mediated Surface-TNF Perforines Cytokin-mediated TNF alpha LT(TNFbeta) IL-1 IFN- gamma LR NKCF

Secondary Cellular Activation

Cytokin-Activated Cell

IL-2

Macrophage (LAKcell) Macrophage (LAKcell) T- cell (LAKcell) LAKcell, NK-cell

IL-4 LR CSFs

LAK cell NK- cell bone marrow

TNF alpha IFN gamma IL-1

Abbreviations: CSFs: colony stimulating factor; IFN gamma: interferon gamma; IL: interleukin; LAK: lymphokine-activated killer cell; LR: leukoregulin; LT: lymphotoxin; NK: natural killer cell; NKCF: natural killer cell cytotoxic factor; TIL: tumor-infiltrating lymphocyte; TNF: tumor necrosis factor.

original formula of Coleys Toxin the ratio endotoxin/exotoxin was 7300:1 this observation may in part explain the higher success rate Coley observed after concomitantly administrating endotoxins and exotoxins to his cancer patients. As mentioned earlier exotoxins, also termed superantigens, are well known inducers of cell adhesion molecules in the inflammatory response^^"^ andTNF.^^^ Recently, superantigens could be shown to complex with the crystal structure of a T cell receptor beta chain. ^ Furthermore, staphylococcal enterotoxin B superantigen conjugated to tumor cells induced strong antitumor activity against Meth A-bearing mice, the antitumor effector cells having been V beta 7- 8- CD4" T ceUs.^^^ Moreover, even though endotoxin tolerance can be transferred between different bacterial species, it has been shown that the inflammatory response to grampositive and gramnegative infeaions differs profoundly. Riesenfeld-Orn et al i.e., showed that different cell wall components of grampositive organisms such as pneumococcus exhibited different cytokine induction profiles. Pneumococcal cell surface component did strongly induce IL-1 secretion, being up to 10.000-fold more potent than endotoxin, but did not induce TNF. Table 3 briefly summarizes the effects of the different effector cells and cytokines.

Tolerance The phenomenon of tolerance to repeated administrations of LPS in human and animals is an important chapter in the history of cancer treatment with bacterial products, since patients have been exposed to repeated vaccinations, often over prolonged periods of time. Beeson and Thomas give early reports on tolerance to bacterial antigens. Subsequent research concentrated on the role of the RES^^^'^^^ and demonstrated that splenectomized rabbits and humans show the same tolerance response as controls.^'^^ Since splenectomy impairs the production of circulating anti-endotoxin antibodies, it coidd be concluded that early tolerance up to 72 hours is not antibody mediated. For an early (review see refs. 234,621,624). More recent studies elucidated the molecular mechanisms of tolerance induction. Lindberg et al^^^ showed that tolerance could be induced with a nonpyrogenic, LPS-free O-antigenic polysaccharide hapten, when coupled to an immunogenic carrier protein. Johnson and Greisman^^^ established that endotoxin tolerance can be divided temporally in an early and a late phase response. Early tolerance occurs wdthin 24-96 hours following endotoxin exposure and is nonspecific and transient. Late-phase tolerance is mediated by the production of

304

Hyperthermia in Cancer Treatment: A Primer

anti-O-specific antibodies, occurs from one week to several weeks following initial endotoxin challenge and lasts for weeks to months. Williams,^^^ Madonna and Vogel,^ and Freudenberg and Galanos ^^ established that the early phase tolerance as well as lethality to LPS is a macrophage-mediated phenomenon. Haas et al^^^ demonstrated in vitro that monocytic cell lines can be prevented from a TNF response measured by decreased TNF mRNA by preincubating cells with low doses of LPS (10 ng/ml). Interestingly however, preincubation with the same dosage of LPS resulted in increased phagocytosis for the exotoxins oi Staphylococcus aureus^ indicating that some monocyte functions are still active whilst in a state of endotoxin tolerance. Furthermore, repeated administration of endotoxins in murine and human models showed downregulation ofTNF-alpha and IL-6, and upregulation of IL-1-beta and G-CSF.^^'''^^^'^^^ Decrease of TNF-alpha and IL-6 production resulted from an inhibition of gene transcription. Another study found different regulation pathways: Downregulation of TNF-alpha, IL-8, G-CSF, M-CSF and WBC count, and upregulation of IL-6.^^^0ther experiments showed that, preexposure of macrophages to very low doses of LPS (<1 ng/ml) inhibited the expression of TNF-alpha mRNA but not of IL-1 beta mRNA through a noncyclooxygenase-dependent mechanism.^^^ In vitro experiments, however, showed downr^ulation of the genes for TNF, IL-1 and IL-6 following preexposure of monocytes with low doses of LPS. Also, these authors noted again that the LPS tolerance coidd be transferred to resistance of a grampositive organism such as Staphylococcus aureus^ an observation later confirmed by Zhang and Morrison ^^ and in contrast to Haas et al and Mathison et al. Interestingly, the amount necessary for inducing a tolerant state is 1.000 time lower than required for the initial induction of TNF production, before tolerance is induced, (picograms vs. nanograms) as pointed out by Mathison et al. In contrast also, LaRue and McGall^^^ demonstrated that endotoxin tolerance is manifested by decreased LPS-induced IL-1-beta transcription. Protection against mucosal injury, nonelevated levels of ileal xanthine oxidase activity and no signs of bacterial translocation in response to repeated administration of LPS was shown by Deitch et al. ^^ The same principle could be demonstrated for the administration of TNF-alpha alone. By daily intravenous injections of recombinant human TNF-alpha (250 ug/kg per diem) healthy rat and mice became resistant to the hemorrhagic effect in the gastrointestinal system. While on the contrary, treatment at 5- or 10-d intervals produced similar results as the initial hemorrhage-causing injection. These results coidd not be confirmed by Vogel et al, who administered TNF and IL-1 and could not observe a tolerance-like reaction to single application of both cytokines. Combined administration however, induced synergistic toxicity in high doses but could reduce secondary CSF production and reduced the increase of macrophage progenitor cells in lower doses. These results were extended later by Gorgen et al,^"^^ who showed that pretreatment of mice with recombinant human granulocyte CSF (G-CSF) protected mice against septic shock, a mechanism mediated by reduced LPS-induced serum TNF activity. Interestingly, when tolerant macrophages were incubated with G-CSF in vitro, LPS induced high levels of TNF. These findings implicate that the protective effect of G-CSF is not direcdy acting on macrophages but acts as a negative feed back signal in vivo. Similar findings were published by Erroi et al, who coidd not achieve complete endotoxin tolerance by administering IL-6, TNF, or IL-1 alpha in an attempt to mimic LPS-induced tolerance. Some researchers found additional partial evidence for the molecular mechanisms involved in the phenomenon of endotoxin tolerance at the level of the LPS receptor CD 14. Mengozzi et al observed a 50% decrease in the CD 14 expression following repeated LPS exposure. Interestingly, cAMP, which is otherwise known to control TNF synthesis, was not affected by preexposure of monocvtes to LPS. Wakabayashi et al demonstrated that pyrogenic tolerance in the rabbit after a single LPS injection is associated with decreased circulating IL-1 beta and TNF levels as well as decreased production of these cytokines in vitro. However, after 7 days pyrogenic hyperresponsiveness to LPS was observed, which was associated with increased synthesis and secretion of IL-1 beta from PBMC in vitro.

Fever, Pyrogens and Cancer

305

As mentioned earlier, pretreatment of cancer patients with IFN-gamma receiving intravenous administration of purified LPS from Salmonella abortus f^«/prevented the downregulation of cytokine secretion, demonstrating that endotoxin tolerance is reversible.'^^ In this study, patients pretreated with 50 ug IFN-y 12 hours prior to endotoxin administration exhibited not only prevention of downregulation of endogenous cytokines (IL-6, IL-8, G-CSF, TNF alpha) normally observed after repeated endotoxin application, but also showed enhanced secretion of these cytokines to levels even higher than those achieved after the first LPS challenge. Therapeutic implications of this fact have been elucidated by Takahashi et al. ^ As already mentioned, the phenomenon of decreased TNF anti-tumor activity resulting from tolerance to repeated applications of exogenous TNF in vivo has been shown to be dependent on the histological tumor type, since this phenomenon has not been observed in all tumors. If tolerance induced decreased anti-tumor activity occurred (i.e., MCA sarcomas, Lewis lung carcinoma), it could be attenuated by addition of IFN-gamma.^"^^ The same authors ftirther showed that tolerance is a TNF-R55 mediated effect and selectively blocks the TNF-R75-mediated pathway, including IL-1 and glucocorticoid mediated pathways. As already mentioned, LPS exhibits selective and inverse priming effects on TNF alpha and nitric oxide (NO) production in mouse peritoneal macrophages. Low doses of LPS pretreatment of mouse macrophages increases LPS-dependent IL-6 and TNF alpha production in vitro, and decreases the synthesis of NO by macrophages.^^^'^^^'^"^^ Priming of macrophages with pertussis toxin has exacdy opposite effects: increased LPS-induced TNF-alpha production and inhibited LPS-dependent NO production.^^^ Furthermore, glucocorticoids have been shown to play an important role in the phenomenon of tolerance to bacterial products. Adrenalectomized mice do not develop endotoxin tolerance as demonstrated by Evans and Zuckerman^^^ and Zuckerman et al. They could show that LPS tolerance involved glucocorticoid-dependent and -independent mechanisms, since corticosterone levels in LPS-treated galactosamine-sensitized and not-adrenalectomized mice were similar to LPS-stimulated normal mice. The glucocorticoid antagonist RU-38486 abolished the development of tolerance induced by TNF and LPS.^^^ Furthermore, adrenalectomized mice exhibited an increased sensitivity to IL-1 and TNF.^^^ Glucocorticoids are known to suppress the synthesis of inflammatory cytokines,^^^'^^"^ and eicosanoids ^^ and downregulate inducible nitric oxide synthase.^^ Although most studies on endotoxin induced tolerance have concentrated on fever and lethality phenomenon, recent evidence suggests that tolerance in response to repeated endotoxin exposure also develops systemically as metabolic,^^'^ pulmonary,^^ and hemodynamic tolerance.

Translocation Systemic endo- and exotoxin exposure also leads to increased bacterial translocation from the gut which in turn may compromise the ability of the liver to clear translocated circulating LPS. Bacterial translocation from the gut first to the mesenteric lymph nodes and then to the systemic circulation mav play an important role in repeated administration of endo- and exotoxin based vaccines. ^ ^ ' ' The underlying mechanism for this phenomenon is mucosal injury, widening of the intercellular spaces due to tight junction failure below the brush border and capillary leakage.^^^'^^^"^^ Disruption of the normal gut flora results in overgrowth with gramnegative, enteric bacilli or aerobe species leading to bacterial translocation. In this respect it is interesting to note, that intraepithelial lymphocytes in the human gut have been shown to possess lytic potential and Thl and cytotoxic T cell functions, as measured by their cytokine profile. Translocation often has been seen after thermal or mechanical injury, and has been associated with altered host defense capability^ ^^ and multiple organ failure.^^'^^^ Tolerance to endotoxin-induced bacterial translocation in response to repeated administration of LPS has been shown by Deitch.^^ This protection against mucosal injury was mediated by nonelevated levels of ileal xanthine oxidase activity, since mucosal injury is known to be mediated by xanthine oxidase-generated production of free oxygen radicals. ^' ^

306

Hyperthermia in Cancer Treatment: A Primer

Most interesting, translocation has been associated with prolonged survival in patients with acute myelogenous leukemia (AML).^^^ These authors observed a "paradoxically prolonged survival'' in AML patients suffering from a common complication: post-transfusional hepatitis. They alleged the impaired hepatic endotoxin clearance in patients with acute viral hepatitis as the reason for endotoxemia and elevated TNF-a release and also observed virally induced IFN-y secretion, which in turn acts in synergy with TNF-a anti-proliferative and differentiation inducing.

Hyperthermia Exogenous induced hyperthermia as an attempt to mimick the physiological response to fever shall be briefly mentioned (for a review see ref 670). During the last decades a substantial body of laboratory and animal tumor data has been generated to evaluate the effects of heat on cell survival and growth. Temperature elevation in febrile response has been associated with effects on the recognition, recruitment, and effector phases of the inmiune response. Temperature elevation appears to affect primarily the phase of recognition and sensitization or activation of mononuclear leukocytes. Hyperthermia is direcdy cytotoxic to tumor cells and inhibits repair of radiation damage. These effects are increased by physiological conditions in the tumor bed including acidosis and hypoxia. Tumor blood flow often is reduced in relation to normal tissues, and hyperthermia leads to a further decrease in blood flow depending on temperature and thus augments heat sensitivity by reducing thermal outwash. A pioneer of hyperacidification in combination with hyperthermia has been von Ardenne in Germany.^^ He proposed the concept of hyperacidification plus hyperthermia, since hyperthermia is known to induce an acidic microenvironment, a concept later to become known as the cancer multiple-step therapy. '^ ' '^ Roberts and Steigbigel ^ demonstrated enhanced mitogen response and bactericidal capacity of polymorphonuclear leukocytes following in vitro exposure to febrile like temperatures. A number of in vitro and in vivo studies revealed specific effects of hyperthermia mimicking physiological temperature elevations seen in the febrile response (reviewed in ref 678). Yonezawa et al observed hvperthermia-induced apoptosis in malignant fibrous histiocytoma cells in vitro. Ensor et al^^ demonstrated a differential secretion of TNF-alpha and IL-6 in vitro in LPS-stimulated human macrophages (HuMoM phi) during 18-h incubation at 40 degrees C. While hyperthermia nearly completely inhibited TNF alpha release, IL-6 secretion remained unchanged. Also a 75-fold increase in the levels of the inducible heat-shock protein 72 (HSP-72) mRNA was observed. Another in vitro study showed increased NK cell cytotoxicity at febrile range (< or = 40 degrees C), but decreased cytotoxicity after exposure of cells to 1 h at 42 degrees C. ^^ Niitsu et al'^ reported the synergy of hyperthermia and rTNF on cytotoxicity and artificial metastasis in vitro and in vivo. Whole body hyperthermia increased natural killer cell activity, and cellular inmiunity in cancer patients, and demonstrated antitumor effects in synergy with exogenous TNF. ^ ' Synergistic anti-tumor effects of combined hyperthermia and inmiunotherapies have been documented by a variety of authors. Synergy of local water-bath hyperthermia and TNF alpha in cytotoxicity but also systemic toxicity were found by van der Zee et al. ^ The combination of local but not whole body hyperthermia and inmiunotherapy with LAK cells and IL-2 in the treatment of multiple pulmonary metastases in mice provided a significant reduction of pulmonary metastasis from MCA-105 sarcoma cells compared to the control group in a study by Strauch et al.^^^ Ex vivo experiments of Kappel et al showed no influence of whole body hyperthermia in subsequent in vitro stimulation of LPS-stimulated mononuclear (BMNC) cells on cytokine production. However, the study suggested that hyperthermia may have altered the sensitivity of BMNC to prostaglandins and in vivo significant cytokine induction was observed for G-CSF, IL-1 beta, IL-6, IL-8, IL-10, and TNF-alpha by Robins et al.^^^ Conversely, an earlier study found elevation of IL-1 alpha but not TNF-alpha following whole body hyperthermia.

Fever, Pyrogens and Cancer

307

Heat Shock Proteins The increased expression of heat shock proteins (HSPs) after hyperthermia treatment or LPS challenge has been shown to correlate with increased inmiunogenicity of cancer cells through their lysis by alpha/beta T cells. HSPs belong to a group of "stress proteins" secreted after a wide range of stimuli such as oxidative injury, heavy metals, exogenous heat and bacterial toxins. They are classified on the basis of their molecular weight and are divided in five families: low molecular weight family, hsp65, hsp70, hsp90 and hsplOO (for reviews see refs. 586,692,693). HSP have been su^ested to act as molecular chaperones in presenting protein structures to the lymphatic system. In this respect they may serve as carriers for antigenic tumor peptides and thereby increase the natural immunity to cancer.^^"^ Cancer cells have been reported to have increased expression of HSPs^^^ and it is well established that LPS challenge leads to increased expression of HSPs in macrophages,^^^'^^^ blood vessel endothelium^^^ and enterocytes.^^^ Most interestingly for this review, hsp70 induction has been shown following fever therapy with endotoxins in melanoma patients in vivo and in vitro.^^^ However, this study revealed hsp70 induction in vivo only in 50% of the cases exposed to a Coley Toxin like preparation (Vaccineurin®), while in vitro 100% of peripheral blood mononuclear cells could be shown to express hsp70 following endo- and exotoxin challenge, indicating additional mechanisms for control of expression in vivo. Increasing evidence su^ests that HSPs could confer protection against oxidative injury, noxious molecules, and bacterial toxins.^^^'^^^ In stressed cells HSPs 72 appears to be essential for survival during and after exposure to cellular injury. HSPs furthermore have been suggested to present cancer antigens to the human inmiune system, especially CD8^ T lymphocytes^ and have been suggested as potent cancer vaccines.^^ Moreover, it has been shown that exogenous cell components which normally are only presented in association with MHC class I proteins, can be directed into the endogenous pathway, conferred by MHC class I molecules, and also recognized by CTL.'^^^ Furthermore, it was shown that LPS induced a HSP 60 mediated increase in expression of ICAM-1 on blood vessel endothelial cells. This finding bears important implications for the attraction of leukocytes following the use of bacterial vaccines. HSP gene transcription increases during or direct after heating; a correlation between the synthesis of HSP and thermotolerance has been found in normal and malignant cells.^ HSPs appear after the activation of a so-called heat-shock transcription factor. This protein has been isolated and purified by Zimarino and Wu,^^^ and Wiederrecht et al.^^^ Nuclease digestion studies have clearly demonstrated that, until the cell is stressed, the protein does not bind to the appropriate promoter region of the gene. Upon activation, the factor binds to the heat-shock element and gene activation results. It is assumed that ubitiquin is involved in the activation process. It has fiirther been suggested that the signal for the induction of the heat shock response relates to the cell's reaction to the presence of abnormal proteins. Yet, a conmion way of gene activation is not known. Most organisms use transcription as the primary control, and translation control for „ fine tuning" for individual HSP synthesis.^^^ Signals involved in HSP synthesis use the second messenger cascades which possibly is triggered by an intra-membrane protein aggregation. However, it is not known which steps lead to the activation of the transcription faaor. Structure of genes and promoter rc^ons and the transcription factor are known. Interestingly, it has been shown that prior induction of HSPs protect the organism from subsequent LPS induced hypotension by inhibition of the overproduction of nitric oxide via reduced iNOS mRNA induction'^^^'^^^ and endothelial cells from apoptosis in vitro via hsp70 and inhibition of LPS-mediated O2 - generation.^^"^'^^^ However, while prior induction of HSPs exerted a posttranslational control of TNF alpha release in LPS-stimulated alveolar macrophages,^^ "^ '^ concomitant application ofTNF alpha enhanced LPS-induced heat shock protein production in vivo.^^^ Moreover, the myocardium has been shown to be protected from endotoxin induced ischemia by prior induction of HSPs.^^^ Additionally, macrophages exposed repeatedly to LPS and IFN-gamma have been shown to become resistant to the deleterious effects of nitric oxide by expressing hsp70.^^^ It also should be noted that HSPs contribute

308

Hyperthermia in Cancer Treatment: A Primer

to the development of drug resistance against chemotherapeutic against in cancer therapy and therefore extreme hyperthermia should not be applied inmiediately before chemotherapy/^^ From the preceding remarks on hyperthermia and HSPs it is tempting to speculate, that in the treatment of cancer patients with hyperthermia and bacterial vaccines it may be meaningful to expose patients first to hyperthermia and secondly to an endotoxin/exotoxin challenge. As it has been shown by Hotchkiss et A™ the protective effects of hyperthermia beginning 1 to 2 hours after heat exposure and reach a maximum at 12 hours. In this way patients would raise their core temperature first by induction of whole body hyperthermia and be prevented from experiencing unpleasant shivering and muscle cramps often following administration of fever induction. Additionally, it may be speculated that the protective effects of hyperthermia, i.e., induction of HSPs may aid in preventing the often intense side effects like hypotension and endothelial cell damage.

Alpha2 Macroglobidin Endotoxins and exotoxins have been shown to affect alpha2-macroglobidin (alpha2M) with their proteolytic enzymes.^^^ Although it has been shown that streptokinase suppresses some immune functions such as chemotactic activity,'^^^ or trypsin the IL-2 mediated proliferation of T cells,^^^ these enzymes may have interesting qualities on regulating the cytokine metabolism by activating alpha2M as shall be discussed further. The hypothesis is postulated that enzyme-activated alpha2M downregulates overexpressed cytokines and lymphocyte reactions in vivo, but may stimulate normal and desired immune responses. In this respect it has to be noted that most inmiunosuppressive actions of activated alpha2M and the respective enzymes have been reported in in vitro systems forT lymphocyte proliferation without'^ '^^^ and with^^ IL-2. Streptokinase has been shown to have inhibitory effects on in vitro tumoricidal activity of human serum, ^^ and in vivo it has been demonstrated to be a powerful inhibitor of tumor growth.^^^ Alpha2M is the regulator of distribution and activity of many cytokines including TNF alpha,^^^ TGF-beta l/^^-'^^^ TGF-beta 2,^2^-^26 pj^^^j^^ ^^^-^^^^ ^ ^ ^ ^ ^ ^^^^^ (PDGF),^^^'^^^ IL-1 beta^^^'^^^ and IL-6^^^ (reviewed in refs. 757 J?>^). Importandy, the alpha2M-bound cytokines and alpha2M-bound proteolytic enzymes both keep their biological activity.^^^ Alpha2M is a high molecular weight (Mrhuman =718,000) major plasma proteinase inhibitor and reacts with a broad diversity of endopeptidases. The enzymes are getting "trapped" in a well defined region of the alpha2M molecule, which then undergoes dramatic conformational changes, while the enzymes keep their proteolytic function.^ An enzyme carrying form of alpha2M is called the activated or "fast" form of alpha2M.^'^^''^'^^ The fast form of alpha2M preferentially binds TNF alpha, TGF-beta 1 and -beta 2, and IL-1 beta while PDGF, NGF and IL-6 bind to the native or "slow" form. Importandy, the fast form of alpha2M becomes activated for increased receptor-mediated endocytosis by exposing a latent alpha2M receptor-recognition domain to hepatocytes,^"^^ macrophages, and fibroblasts.^ ^' The cytokine carrying alpha2M then undergoes rapid clearance by binding to hepatic-, macrophagic-, and fibroblastic-alpha2M-receptors.^^^'^^ It is further su^ested that alpha2M plays an important role in cytokine testing bioassays, which may has been underestimated.'^^'^ However, in vitro exposure of macrophages to LPS and IFN gamma, but not to TNF, TGF beta-lor IL-6 induced a significant downregulation of the alpha2M-receptor/low density lipoprotein receptor.^^^ These studies need to be confirmed in vivo, but allow the hypothesis that the downregulation of the alpha2M-receptor/low density lipoprotein receptor may act as an inhibitory feedback mechanism for the binding of proteolytic enzymes.

Toxicity Toxicity of endo- and exotoxin-based cancer therapies can be considerable. The administration of such therapies therefore only should be performed by qualified medical providers and should include immediate access to conventional emergency support. Self-administration of salmonella endotoxin has been reported resulting in shock and multiple-organ dysfunction.

Fever, Pyrogens and Cancer

309

In the experienced hand however, die administration of this therapeutic approach does not impose a greater risk to the patient than conventional procedures such as chemotherapy. Informed consent may help to increase compUance and reframe the understanding of the patient in respect to beneficial effects of "fever-therapy". Additionally, psychoneuroimmunology research has shown that compliance with and possibly efficacy of therapy can be increased if the patient is fully informed. Even more, numerous authors report induction of fever and immunopotentiation with endotoxin without any toxic reactions (reviewed in refs. 106,757,758). Endo- and exotoxin mediated toxicities can include hypotension, hepatotoxicity, induction of herpes labialis, muscle spasms and cramps, and in severe cases shock and circulatory failure (cardiovascular effects reviewed in re£ 759). These side effects always should be noted and classified according to W H O criteria.^ High doses of endotoxin result in systemic effects such as circidatory failure and death.'^^^ A number of treatments have been suggested to diminish the dose limiting toxicities. In attempts to limit exogenous TNF toxicities Brouckaert et al^ ^ suggested "methylene blue, an inhibitor of the nitric oxide (NO)-induced activation of the cytosolic guanylate cyclase, without the indispensable protective properties of NO being affected" to prevent hypotension. Furthermore, they suggested anti GDI la to prevent IL-12 mediated sensitization to TNF and alpha 1-acid-glycoprotein and alpha 1-antitrypsin to protect against TNF-induced hepatotoxicity by reducing the release of platelet-activating factor.^ ^ LPS mediated toxicity and what is more lethality however, can not be explained by TNF induction alone. ^ Further toxicity is mediated by IL-1 as has been shown by the successful blocking of lethal LPS challenge with a recombinant receptor antagonist protein to IL-1.^ The same authors demonstrated manganous superoxide dismutase induced protection against lethal LPS challenge but not against LPS-mediated toxicities with a 24-hour pretreatment of a single dose of TNR^^5 Recombinant IL-1 receptor antagonist also protected against TNF-induced lethality.'^ Moreover, IL-1 alpha has been demonstrated to mediate the microcirculatory changes in the intestinal mucosa observed after systemic endotoxin exposure, including increased adhesiveness of leukocytes and mucosal damage.'^^'^ Recendy, the vascular pathophysiology of endo- and exotoxin induced hypotension and extravasation has been described as being the result of activation of the bradykininHageman-factor-kallikrein cascade. Inhibitors of kinin and kallikrein have been suggested subsequendy to block the shock induced by bacterial proteases by blocking the kallikrein-kinin cascade. Another study explained bacterial-associated vasculitis by IL-1 alpha mediated secretion of IL-6 and IL-8.^^^ Possible effects of endotoxin induced lung injury have been shown at the level of alveolar macrophages for enhanced secretion of IL-1, TNF-alpha, and prostaglandin £2.^^^ Finally, it is of interest to note that nontoxic polysaccharide derivatives free of lipid A have been shown equally effective in enhancing humoral immunity in FLV inmiunocompromised animals, both in vivo and in vitro.^^'^^^'^^ The nontoxic polysaccharide derivative however, did not induce TNF, and cells needed to be stimulated with IL-2 prior to LPS exposure to produce IFN.^^^ On the other hand the same authors reported effective inhibition of Meth A sarcoma with the use nontoxic LPS derivatives low in endotoxin.^^^ There are numerous substances, which efficiendy counteract the toxicities of endo-, and exotoxin based immunotherapies but it is important to keep the possible interactions with inmiunological effects in mind, which these preparations might elicit additionally and thus interfere with the efficacy of the treatment approach. The classical antipyretics include prostaglandin synthesis inhibitors indomethacin, ibuprofen and glucocorticosteroids next to antipyretics such as aspirin and paracetamol. Endogenous glucocorticoids play an important role in protecting the host against TNF mediated LPS sensitization.^"^ Additionally, chlorpromazine has been shown to protect against endotoxic shock by inhibiting peripheral and brain TNF, and upregulating IL-10 production.^^ Furthermore, the cytokine inhibitor pentoxifylline has been suggested as an antipyretic by lowering TNF and IL-6 levels.'^^

310

Hyperthermia in Cancer Treatment: A Primer

Acknowledgements The authors gratefully acknowledge the work of Dr. Helen Coley Nauts, Founder of the Cancer Research Institute in New York, who researched for over half a century the historical and clinical evidence of the use of mixed bacterial vaccines in medicine and who made so many of her records freely available to us.

References 1. Busch W. Einflufi von Erysipel. Berliner Klin Wschr 1866; 3:245-246. 2. Fehleisen V. VI. Cber die Zuchtung der Erysipelkokken auf kiinstlichem Nahrboden und Ihre Obertragbarkeit auf den Menschen. Dtsch Med Wochenschr 1882; 8:533-554. 3. Richter PF. Leukemia and Erysipel. Charite-annalens 1896; 21:299-309. 4. Laurence JZ. The diagnosis of surgical cancer (Lister Prize say for 1854). London: Churchill, 1854:56. 5. Coley WB. The therapeutic value of the mixed toxins of the streptococcus of erysipelas and bacillus prodigiosus in the teratment of inoperable malignant tumors. With a report of 160 cases. Am J Med Sci 1896; 112:251-281. 6. Coley WB. Treatment of inoperable malignant tumors with toxins of erysipelas and the bacillus prodrigiosus. Trans Am Surg Assoc 1894a; 12:183-212. 7. Coley WB. The treatment of inoperable malignant tumors with the toxines of erysipelas and the bacillus prodrigiosus. Am J Med Sci 1894b; 108:50-66. 8. Coley WB. Further observations upon the treatment of malignant tumors with the Toxins of erysipelas and Bacillus prodigious with a report of 160 cases. Bull John Hopkins Hospital 1896a; 7:175. 9. Coley WB. The therapeutic value of the mixed toxins of the streptococcus of erysipelas and bacillus prodigiosus in the teratment of inoperable malignant tumors. With a report of 160 cases. Am J Med Sci 1896; 112:251-281. 10. Coley WB. The treatment of malignant tumors by repeated inoculations of erysipelas: With a report often original cases. Am J Med Sci 1899; 105:487-511. 11. Coley WB. Inoperable sarcoma: A further report of cases successfully treated with the mixed bacterial toxins of erysipelas and bacillus prodrigiosus. Med Rec 1907; 72:129-137. 12. Coley WB. The treatment of inoperable malignant tumors with the toxines of erysipelas and the bacillus prodrigiosus. Am J Med Sci 1894b; 108:50-66. 13. Coley WB. A report of recent cases of inoperable sarcomas successfidly treated with mixed toxines of erysipelas and bacillus prodigiosus. Surg Gynec Obstetr 1911; 13:174-190. 14. Coley WB, Coley BL. Primary malignant tumors of the long bones. Arch Surg 1926; 13:780-836; 14:63-141. 15. Coley WB. EndotheUoma myeloma of tibia: Long-standing cure by Toxin treatment. Ann Surg 1931; 94:447-452. 16. Issels J. Immunotherapy in progressive metastatic cancer. Clin Trials J 1970; 3:357-366. 17. In: Hagcr ED, Abel U, eds. Biomodulation und Biotherapie des Krebses II. Endogene Fiebertherapie und exogene Hyperthermie in der Onkologie. Heidelberg: Verlag fiir Medizin Dr. E. Fischer, 1987. 18. Heckel M. Ganzkorper-Hyperthermie und Fiebertherapie. Hippokrates Verlag Stuttgart 1990. 19. Wiemann B, Starnes CHO. Coley's Toxins, Tumor necrosis factor and cancer research: A historical perspective. Pharmac Ther 1994; 64:529-564. 20. Kleef R, Jonas WB, Knogler W et al. Fever, cancer incidence and spontaneous remissions. Neuroimmunomodulation 2001; 9(2):55-64. 21. Wiltschke C, Krainer M, Budinsky AC et al. Reduced mitogenic stimulation of peripheral blood mononuclear cells as a prognostic parameter for the course of breast cancer: A prospective longitudinal study. Br J Cancer 1995; 71(6):1292-6. 22. Wiltschke C, Tyl E, Speiser P et al. Increased natural killer cell activity correlates with low or negative expression of the HER-2/neu oncogene in patients with breast cancer. Cancer 1994; 73(l):135-9. 23. Brunson KW, Goldfarb RH. In: Kaiser HE, ed. "Cancer growth and Progression". Dordrecht, the Netherlands: Kluwer Academic Publishers, 1989:133-138. 24. Cianciolo GJ. In: Gallin JI, Goldstein IM, Snyderman R, eds. "Inflammation: Basic Principles and CHnical Correlates". New York: Raven Press, 1988:861-876. 25. Nelson M, Bremner JA, Nelson DS. Tumour cell products inhibit both functional and immunoreactive interleukin 2 production by human blood lymphocytes. Br J Cancer 1989; 60(2):l6l-3. 26. Nelson M, Nelson DS. Inhibition of cell-mediated immunity by tumour cell products: Depression of interleukin-2 production and responses to interleukin-2 by mouse spleen cells. Immunol Cell Biol 1988; 66(Pt 2):97-104.

Fever, Pyrogens and Cancer

311

27. Schulof RS, Goldstein AL, Sxtein MB. In: Oldman RX, ed. "Principles of Cancer Biotherapy". New York: Raven Press, 1987:93-162. 28. Nelson DS, Nelson M. Evasion of host defences by tumours. Immunol Cell Biol 1987; 65(Pt 4):287-304. 29. Aune TM. Role and function of antigen nonspecific suppressor factors. Crit Rev Immunol 1987; 7(2):93-130. 30. Kamo I, Friedman H. Immunosuppression and the role of suppressive factors in cancer. Adv Cancer Res 1977; 25:271-321. 31. Stutman O. Immunodepression and malignancy. Adv Cancer Res 1975; 22:261-422. 32. Sulitzeanu D. Immunosuppressive factors in human cancer. Adv Cancer Res 1993; 60:247-67. 33. Friedman H, Blanchard DK, Newton C et al. Distinctive immunomodidatory effects of endotoxin and nontoxic Upopolysaccharide derivatives in lymphoid cell cultures. J Biol Response Mod 1987; 6(6):664-77. 34. Bokemeyer C, Kuczyk MA, Kohne CH et al. Risk of secondary neoplasia after treatment of malignant germ cell tumors of the testis. Med Klin 1996; 91(11):703-10. 35. Malpas JS. Long-term effects of treatment of childhood malignancy. Clin Radiol 1996; 51(7):466-74. 36. Shapiro CL, Recht A. Late effects of adjuvant therapy for breast cancer. J Natl Cancer Inst Monogr 1994; (16):101-12. 37. Krainer M, Wolf H, Michl I et al. Natural killer cell activity in long-term survivors of testicular cancer. Influence of cytostatic therapy and initial stage of disease. Cancer 1995; 75(2):539-44. 38. Rutqvist LE. Long-term toxicity of tamoxifen. Recent Results Cancer Res 1993; 127:257-66. 39. Albanell J, Gallego OS, Bellmunt J et al. Bladder neoplasm in a patient with panarteritis nodosa treated with cyclophosphamide. Rev Chn Esp 1992; 190(9):463-5. 40. Forbes JF. Long-term effects of adjuvant chemotherapy in breast cancer. Acta Oncol 1992; 31(2):243-50. 41. Zielinski CC, Muller C, Kubista E et al. Effects of adjuvant chemotherapy on specific and nonspecific immune mechanisms. Acta Med Austriaca 1990; 17(1): 11-4. 42. Zielinski CC, Muller C, Tichatschek E et al. Decreased production of soluble interleukin 2 receptor by phytohaemagglutinin-stimulated peripheral blood mononuclear cells in patients with breast cancer after adjuvant therapy. Br J Cancer 1989; 60(5):712-4. 43. Zielinski CC, StuUer I, Dorner F et al. Impaired primary, but not secondary, immune response in breast cancer patients under adjuvant chemotherapy. Cancer 1986; 58(8): 1648-52. 44. Tichatschek E, Zielinski CC, Muller C et al. Long-term influence of adjuvant therapy on natural killer cell activity in breast cancer. Cancer Immunol Immunother 1988; 27(3):278-82. 45. Kempf RA, Mitchell MS. Effects of chemotherapeutic agents on the immune response. II. Cancer Invest 1985; 3:23-33. 46. Cole WH, Humphrey L. Need for immunologic stimulators during immunosuppression produced by major cancer surgery. Ann Surg 1985; 202:9-20. 47. Cole WH. The increase in immunosuppression and its role in the development of malignant lesions. J Surg Oncol 1985; 30:139-144. 48. Miller Jr WH. Differentiation therapy of acute promyelocytic leukemia: Clinical and molecular features (see comments). Cancer Invest 1996; l4(2):l42-50. 49. Wotherspoon AC, DogUoni C, Diss TC et al. Regression of primary low-grade B-cell gastric lymphoma of mucosa-associated lymphoid tissue type after eradication of Helicobacter pylori (see comments). Lancet 1993; 342(8871):575-7. 50. Bailar Ilird JC, Smith EM. Have we reduced the risk of getting cancer or of dying from cancer? An update. Med Oncol Tumor Pharmacother 1987; 4(3-4):193-8. 51. Abel U. Die zytostatische Chemotherapie fortgeschrittener epithelialer Tumoren: Eine kritische Bestandsaufnahme. Stuttgart: Hippokrates-Verl, 1990. 52. Abel U. Chemotherapy of advanced epitheUal cancer-a critical review. Biomed Pharmacoth 1992; 46(10):439-452. 53. Schipper H, Goh CR, Wang TL. Shifting the cancer paradigm: Must we kill to cure? (editorial). J Clin Oncol 1995; 13(4):801-7. 54. Schipper H, Turley EA, Baum M. A new biologicalframeworkfor cancer research. Lancet 1996; 348(9035):1149-51. 55. Sporn MB. The war on cancer (see comments). Lancet 1996; 347(9012): 1377-81. 56. Office of Alternative Medicine: Expanding Medical Horizons: Report to the National Institutes of Health on Alternative Medical Systems and Practices in the United States. P. 170, 1992. ISBN 0-16-045479-4. 57. Teichert J, Schulze-Pillot T, Matthiessen PF. Zehn Jahre Forschungsforderung - „Unkonventionelle Methoden zur Krebsbekampfting". Deutsches Arzteblatt 1994; 91:A-3332-3336, (Heft 48).

312

Hyperthermia in Cancer Treatment: A Primer

58. Schmidt KL. Krebs und Infektionskrankheiten. Med Klinik 1910; 43:1690-1693. 59. Engel P. Ober den Infektionsindex der Krebskranken. Wien Klin Wschr 1934; 47:1118-1119. 60. Engel P. Cber den Einflufi des Alters auf den Infektionsindex der Krebskranken. Wien Klin Wschr 1935; 48:112. 61. Sinek F. Versuch einer statistischen Erfassung endogener Faktoren bei Carcinomerkrankungen. Z Krebsforsch 1936; 44:492-527. 62. Witzel L. Anamnese und Zweiterkrankungen bei Patienten mit bosartigen Neubildungen. Med Klin 1970; 65:876-879. 63. Newhouse ML, Pearson RM, Fullerton JM et al. A case control study of carcinoma of the ovary. Brit J Preventive Social Med 1977; 31:148-153. 64. Remy W, Hammerschmidt K, Zanker KS et al. Tumortrager haben selten Infekte in der Anamnese. Med Klinik 1983; 78:95-98. 65. Grufferman S, Wang HH, DeLong ER et al. Environmental factors in the etiology of rhabdomyosarcoma in childhood. J Nad Cancer Inst 1982; 68:107-113. GG. R0nne T. Measles virus infection without rash in children is related to disease in adult life. The Lancet 1985; 84l9i:l-5. G7. van Steensel-MoU HA, Valkenburg HA, van Zanen GE. Childhood leukemia and infectious diseases in thefirstyear of life: A register based case-control study. Am J Epidemiol 1986; 124:590-594. 68. Chilvers O, Johnson B, Leach S et al. The common cold, allergy, and cancer. Br J Cancer 1986; 54:123-126. 69. Abel U. Incidence of infection and cancer risk. Dtsch Med Wschr 1986a; 111:1987-81. 70. Abel U, Becker N, Angerer R et al. Common infections in the history of cancer patients and controls. J Cancer Res Clin Oncol 1991; 117(4):339-344. 71. Abel U. Cancer occurrence from the biometric viewpoint. Fortschr Med 1986b; 104(8): 158-62. 72. Schlehofer B, Blettner M, Becker N et al. Medical risk factors and development of brain tumors. Cancer 1992; 69:2541-2547. 73. Grossarth-Maticek R, Frentzel-Beyme R, Kanazir D et al. Reported Herpes-virus infection, fever and cancer incidence in a prospective study. J Chronic Dis 1987; 40:967-976. 74. Kolmel KF, Compagnone D. Melanom und Atopic. Dtsch Med Wschr 1988; 113:169-71. 75. Kolmel K, Gefeller O, Haverkamp B. Febrile infections and malignant melanoma: Results of a case-control study. Melanoma Res 1992; 2:207-211. 76. Cooper GS, Kamel F, Sandler DP et al. Risk of adult acute leukemia in relation to prior immune-related conditions. Cancer Epidemiol Biomarkers Prev 1996; 5:867-872. 77. Goldstein DJ, Austin JH, Zuech N et al. Carcinoma of the lung after heart transplantation. Transplantation 1996; 62(6):772-5. 78. Swinnen LJ. Transplant immunosuppression-related malignant lymphomas. Cancer Treat Res 1993; 66:95-110. 79. Hoover RN. Lymphoma risks in populations with altered immunity—a search for mechanism. Cancer Res 1992; 52(19 Suppl):5477s-5478s. 80. Thomas JA, Crawford DH. B-cell lymphoma in organ transplant recipients. Semin Thorac Cardiovasc Surg 1990; 2(3):221-32. 81. Shiba T, Noguchi S, Yao M et al. Carcinoma of the urinary bladder in a patient receiving cyclophosphamide for Wegener's granuloma: A case report. Hinyokika Kiyo 1991; 37(4):393-6. 82. Forbes A, Reading NG. Review article: The risks of malignancy from either immunosuppression or diagnostic radiation in inflammatory bowel disease. Aliment Pharmacol Ther 1995; 9(5):465-70. 83. Penn I. Posttransplant malignancies in pediatric organ transplant recipients. Transplant Proc 1994a; 26(5):2763-5. 84. Penn I. Malignancy. Surg CUn North Am 1994b; 74(5):1247-57. 85. Penn I, Draper GJ. General overview of studies of multigeneration carcinogenesis in man, particularly in relation to exposure to chemicals. lARC Sci Publ 1989; (96):275-88. (Cancers after cyclosporine therapy. Transplant Proc 1988; 20(1 Suppl l):276-9). 86. Tan-Shalaby J, Tempero M. Malignancies after liver transplantation: A comparative review. Semin Liver Dis 1995; 15(2):156-64. 87. O'Regan B, Hirshberg C. Spontaneous remission: An annotated bibliography. Institute of Noetic Sciences, Sausalito, ISBN 0-943951-17-8 1993. 88. Nauts HC. Bacterial vaccine therapy of cancer. Dev Biol Stand 1977; 38:487-494, (S. Karger, Basel 1978). 89. Nauts HC. Bacterial products in the treatment of cancer: Past, Present and Future (Meeting paper). International Colloquium on Bacteriology and Cancer 1982a. 90. Nauts HC. Bacterial pyrogens: Beneficial effects on cancer patients. Prog CHn Biol Res 1982b; 107:687-696.

Fever, Pyrogens and Cancer

313

91. Nauts HC. Breast Cancer: Immunological, factors affecting incidence, prognosis and survival. New York: Cancer Research Institute Inc., 1984:261, (Monograph #18). 92. Nauts HC. Immuntherapie des Krebses. International Symposium on Endotoxin: Structural aspects and immunbiology of host responses. Giovinazzo, Bari, Italien: Riva del Sole, 1986, (29.05.-01.06.86). 93. Nauts HC. Bacteria and cancer-antagonisms and benefits. Cancer Surv 1989; 8:713-723. 94. Nauts HC. Beneficial effects of immunotherapy (Bacterial Toxins) on sarcoma of the soft tissues, other than lymphosarcoma. New York: Cancer Research Institute Inc., 1975, (Monograph #16). 95. Nauts HC. BiMiography of reports concerning the clinical or experimental use of Coley Toxins (streptococcus pyogenes and serratia marcescens), 1893-1984. New York: Cancer Research Institute Inc, 1997. 96. Nauts HC. New York: Cancer Research Institute Inc, 1975, (Monograph # 3 = Monograph #16). 97. Nauts HC. Ewing's Sarcoma of Bone: End results following immunotherapy (Bacterial Toxins) combined with surgery and/or radiation. New York: Cancer Research Institute Inc., 1974, (Monograph #14). 98. Nauts HC. Giant cell tumor of bone: End results following immunotherapy (coley toxins) alone or combined with surgery and/or radiation - 66 cases and concurrent infection - 4 cases. New York: Cancer Research Institute Inc., 1976, (Monograph #4). 99. Nauts HC. Historical perspective on the development of inbred mice. Introductory remarks. In: Morse Illrd HC, ed. Origins of inbred mice. New York: Academic Press, 1978:23-4. (QY 60.R6 069 1978). 100. Nauts HC. Host resistance and cancer. New York: Cancer Research Institute, Inc., Monograph #5 (unpublished). 101. Nauts HC. Osteogenic Sarcoma: End Reults following immunotherapy with bacterial vaccines, 165 cases or following bacterial infections inflammation or fever, 41 cases. New York: Cancer Research Institute Inc., 1974, (Monograph # 15). 102. Nauts HC. The beneficial effects of bacterial infections on host resistance to cancer. End results in 449 cases. A study and abstracts of reports in the world medical Uterature (1775-1980) and personal communications. New York: Cancer Research Institute Inc., 1980; ((1,032 references). Monograph No. 8, 2nd ed). 103. Nauts HC, Fowler GA. End results in lymphosarcoma treated by toxin therapy alone or combined with surgery and/or radiation or with concurrent bacterial infection. New York: Cancer Research Institute Inc., 1969, (Monograph #6). 104. Nauts HC, McLaren JR. Coley Toxins - the first century. Adv Exp Med Biol 1990; 267:483-500. 105. Nauts HC, Swift WE, Coley LB. Treatment of malignant tumors by bacterial toxins as developed by the late William B. Coley, M.D., reviewed in the light of modern research. Cancer Res 1946; 6:205-216. 106. Nauts HC, Fowler GA, Bogatko FH. A review of the influence of bacterial infection and of bacterial products (Coley's Toxins) on malignant tumors in man. Acta Med Scand 1953; l46(suppl 276): 1-103. (New York: Cancer Research Institute Inc., 1953, Monograph #1). 107. Nauts HC. Beneficial effects of acute concurrent infection, inflammation, fever or immunotherapy (bacterial toxins) on ovarian and uterine cancer. New York: Cancer Research Institute Inc., 1977, (Monograph #17). 108. Nauts HC. Enhancement of natural resistance to renal cancer: Beneficial effects of concurrent infections and immunotherapy with bacterial vaccines. New York: Cancer Research Institute Inc., 1970, (Monograph #12). 109. Nauts HC. Multiple myeloma: Beneficial effects of acute infections or immunotherapy (bacterial vaccines). New York: Cancer Research Institute Inc., 1975, (Monograph #13). 110. Vautier AH. Vue g^ndrale sur la maladie canc^reuse. Th^se de Paris 1813; 43:11. 111. Nowotny A. Antitumor effects of Endotoxins. In: Berry LJ (Hrsg.), ed. Handbook of Endotoxin. Vol. 3 Cellular Biology of Endotoxin. New York: Elsevier Science Inc., 1985:389-448. 112. Bruns P. Die Heilwirkung des Erysipels auf Geschwiilste. Beitr Z Klin Chirug 1887; 3:443-466. 113. Eschweiler R. Die Erysipel-, Erysipeltoxin- und Serumtherapie der bosartigen Geschwiilste. C.G. Naumann, Leipzig 1897. 114. Rohdenburg GL. Fluctuations in the growth energy of malignant tumors in man, with especial reference to spontaneous regression. J Cancer Res 1918; 3:193-225. 115. Wolfenheim W. Cber den heilenden Einfluf5 des Erysipels auf Gewebsneubildungen, insbesondere bosartige Tumoren. Z Klin Med 1921; 92:507-526. 116. Everson TC, Cole WH. Spontaneous regression of cancer: Preliminary report. Ann Surgery 1956; 144:366-383. 117. Everson TC. Spontaneous regression of cancer. Ann New York Acad Sci 1964; 114:721-735.

314

Hyperthermia in Cancer Treatment: A Primer

118. Everson TC. Spontaneous regression of cancer. Prog Clin Cancer 1967; 3:79-95. 119. Everson TC, Cole WH. Spontaneous regression of cancer. A study and abstract of reports in the world medical literature and personal communications concerning spontaneous regressions of malignant disease. Philadelphia, London: W.B. Saunders Co, 1966. 120. Stephenson HE, Delmez JA, Renden DI et al. Host immunity and spontaneous regression of cancer evaluated by computerized data reduction study. Surg Gynecol Obstetr 1971; 133:649-655. 121. Cole WH. Spontaneous regression of cancer and the importance of finding its cause. NCI Monogr 1976; 44:5-9. 122. Cole WH. Efforts to explain spontaneous regression of cancer. J Surg Oncol 1981; 17:201-209. 123. Dock G. The influence of compHcating diseases upon leukemia. Am J Med Sci 1904; 127:563-592. 124. Pelner, Fowler GA, Nauts HC. Effects of concurrent infections and their toxins on the course of leukemia. Acta Medica Scand 1958; I62(suppl 338):4-47. (New York: Cancer Research Institute, Inc., Monograph #2). 125. Kizaki M, Ogawa T, Watanabe Y et al. Spontaneous remission in hypoplastic acute leukemia. Keio J Med 1988; 37(3):299-307. 126. Huth EF. Die RoUe der bakteriellen Infektionen bei der Spontanremission maligner Tumoren und Leukosen. In: Lampert H, Selawry O, eds. Korpereigene Abwehr und bosartige Geschwiilste. Ulm: Haug-Verlag, 1957:23-37. 127. Takita H. Effect of postoperative empyema on survival of patients with bronchogenic carcinoma. J Thorac Cardiovasc Surg 1970; 59(5):642-4. 128. Ruckdeschel JC, Codish SD, Stranahan A et al. Postoperative empyema improves survival in lung cancer. Documentation and analysis of a natural experiment. N Engl J Med 1972; 287(20): 1013-7. 129. Matzker J, Steinberg A. Tonsillectomy and leukemia in adults (author's transl). Laryngol Rhinol Otol (Stuttg) 1976; 55(9):721-5. 130. Kapp JP. Microorganisms as antineoplastic agents in CNS tumors. Arch Neurol 1983; 40:637-642. 131. Nowacki MP, Szymendera JJ. The strongest prognostic factors in colorectal carcinoma. Surgicopathologic stage of disease and postoperative fever. Dis Colon Rectum 1983; 26(4):263-8. 132. Fucini C, Bandettini L, D'Elia M et al. Are postoperative fever and/or septic complications prognostic factors in colorectal cancer resected for cure? Dis Colon Rectum 1985; 28(2):94-5. 133. Treon SP, Broitman SA. Beneficial effects of post-transfusional hepatitis in acute myelogenous leukemia may be mediated by lipopolysaccharides, Tumor necrosis factor a and Interferon y. Leukemia 1992; 6:1036-1042. 134. Maurer S, Kolmel K. Spontaneous regression of melanoma. New York: Cancer Research Institute Inc., 1997, (Monograph #19). 135. Levin EJ. Spontaneous regression (cure?) of a malignant tumor of bone. Cancer 1957; 10:377-381. 136. Cole WH. Spontaneous regression of reticulum-cell sarcoma of bone. J Bone Joint Surg 1959; 4l-A:960-965. 137. Callan JE, Wood VE, Linda L. Spontaneous resolution of an osteochondroma. J Bone Joint Surg (Am) 1975; 57(5):723. 138. Copeland RL, Meehan PL, Morrissy RT. Spontaneous regression of osteochondromas. Two case reports. J Bone Joint Surg (Am) 1985; 67(6):971-3. 139. Eisenbud L, Kahn LB, Friedman E. Benign osteoblastoma of the mandible: Fifteen year follow-up showing spontaneous regression after biopsy. J Oral Maxillofac Surg 1987; 45(l):53-7. 140. CoUignon JC, Kalangu K, Flandroy P. Benign osteoblastoma of the spine. Apropos of 4 cases with a case of spontaneous recovery. Neurochirurgie 1988; 34(4):262-70. (Regression). 141. Margolis J, West D. Spontaneous regression of malignant disease: Report of three cases. J Am Geriatr Soc 1967; 15(3):251-3. 142. Rao S, Constantini S, Gomori JM et al. Spontaneous involution of an intra-axial brain stem lesion: A case report. Pediatr Neurosurg 1995; 23(5):279-81, (discussion 282). 143. Bluming AZ, Ziegler JL. Regression of Burkitt's lymphoma in association with measles infection. Lancet 1971; 2(715):105-6. 144. ReboUo J, Llorente I, Yoldi A. Spontaneous tumor regression in a patient with metastatic gastric cancer. Communication of an additional case. Rev Med Univ Navarra 1990; 34(3): 141-2. 145. Zambrana Garcia JL, Torres Serrano F, Lopez Rubio F et al. Spontaneous tumor regression and gastric cancer (letter). An Med Interna 1996; 13(l):47-8. 146. Friedrich Jr EG. Reversible vulvar atypia. A case report. Obstet Gynecol 1972; 39(2):173-81. 147. Temesrekasi D. Complete regression of 2 nonoperated hypopharyngeal carcinomas. Arch Klin Exp Ohren Nasen Kehlkopftieilkd 1969; 194(2):323-8. 148. Woods JE. The influence of immunologic responsiveness on head and neck cancer. Therapeutic implications. Plast Reconstr Surg 1975; 56(l):77-80. 149. Chien RN, Chen TJ, Liaw YF. Spontaneous regression of hepatocellular carcinoma. Am J Gastroenterol 1992; 87(7):903-5.

Fever, Pyrogens and Cancer

315

150. Grossmann M, Hoermann R, Weiss M et al. Spontaneous regression of hepatocellular carcinoma (see comments). Am J Gastroenterol 1995; 90(9): 1500-3. 151. Markovic S, Ferlan-Marolt V, Hlebanja Z. Spontaneous regression of hepatocellular carcinoma (see comments). Am J Gastroenterol 1996; 91(2):392-3. 152. Tarazov PG. Spontaneous necrosis of liver cancer: One more possible cause (letter; comment). Am J Gastroenterol 1996; 91(9):1872-3. 153. Hart S. Chronic lymphatic leukemia complicated by pneumonia. New York State J Med 1903; 78:220-227. 154. Dreyfus B. Les Remissions de la leucemie aigue. Sangre 1948; 1:35-40. 155. Bassen FA, Kohn JL. Multiple spontaneous remissions in a child with acute leukemia. The occurrence of agranulocytosis and aplastic anemia in acute leukemia and their relationship to remissions. Blood 1952; 7:37-46. 156. Paolino W, Sartoris S. Due casi di leucemia migliorati a seguito di complicazioni infettive. Minerva Med (Torino) 1960; 51:34554-3456. 157. Vladimirskaia EB. A case of prolonged spontaneous remission in a patient with chronic lymphatic leukemia. Problemy Gematologii I Perlevaniya Krovi 1962; 7:51-54. 158. Hardisty RM. Splenic aspiration in acute leukaemia. Lancet 1968; l(540):472-3. 159. Burgess MA, Gruchy GC de. Septicemia in acute leukemia. Med J Aust 1969; 1(22):1113-7. 160. Wyszkowski J, Armata J, Cyklis R et al. Remissions in acute leukemia resistant to treatment compUcated with steroid diabetes and severe infection. Ploski Tygodnik Lekaxski 1969; 24:1974-1975. 161. Wiernik PH. Spontaneous regression of hematologic cancers. Natl Cancer Inst Monogr 1976; 44:35-38. 162. Barton JC, Conrad ME. Beneficial effect of hepatitis in patients with acute myelogenous leukemia. Ann Int Med 1979; 90:188-190. 163. Conrad ME, Barton JC. Hepatitis and leukemia (letter). Ann Int Med 1979; 90:988. 164. Foon KA, Yale C, Clodfelter K et al. Posttransfusion hepatitis in acute myelogenous leukemia: Effect on survival. JAMA 1980; 244:1806-1807. 165. Vinogradova luE, Ivanina EK. Indicators of cellular immunity and the incidence of infectiousinflammatory diseases during clinicohematological remission in patients with acute leukemia. Ter Arkh 1984; 56(6):46-50. 166. Sanz GF, Sanz MA. Complete spontaneous remission in acute myeloblastic leukemia. Revista Clin Espanola 1986; 178:229-230. 167. Zhu XQ, Qian JW. Remission of acute lymphoblastic leukemia of childhood following acute infectious disease. A case report. Chin Med J (Engl) 1986; 99(5):433-4. 168. Maekawa T, Fujii H, Horiike S et al. Spontaneous remission of four months duration in hypoplastic leukemia with tetraploid chromosome after blood transfusion and infection. Acta haematologica Japonica 1989; 52:849-857. 169. Frick S, Frick P. Spontaneous remission in chronic lymphatic leukemia. Schweiz Med Wochenschr 1993; 123(8):328-34. 170. Delmer A, Heron E, Marie JP et al. Spontaneous remission in acute myeloid leukaemia (letter; comment. Br J Haematol 1994; 87(4):880-2. 171. Musto P, D'Arena G, Melillo L et al. Spontaneous remission in acute myeloid leukaemia: A role for endogenous production of tumour necrosis factor and interleukin-2? (letter; comment). Br J Haematol 1994; 87(4):879-80. 172. Jono K, Ikebe Y, Inada K et al. A case of spontaneous remission in chronic B-cell leukemia with virus infection. Nippon Naika Gakkai Zasshi 1994; 83(12):2159-60. 173. Lefrere F, Hermine O, Radford-Weiss I et al. A spontaneous remission of lymphoid blast crisis in chronic myelogenous leukaemia following blood transfusion and infection. Br J Haematol 1994; 88(3):621-2. 174. Greentree LB. Anaplastic lung cancer with metastases. Case report of a 15-year survival. Ohio State Med J 1973; 69(ll):84l-3. 175. Marcos Sanchez F, Juarez Ucelay F, Bru Espino IM et al. A new case of spontaneous tumor regression (letter). An Med Interna 1991; 8(9):468. 176. Sanchez-Cantu L, Rode HN, Yun TJ et al. Tumor necrosis factor alone does not explain the lethal effect of lipopolysaccharide. Arch Surg 1991; 126(2):231-5. 177. Mentzer SJ. Immunoreactivity in lung cancer. Chest Surg CUn N Am 1995; 5(1):57-71. (spontaneous remission REVIEW ARTICLE: 59 REFS). 178. Zygiert Z. Hodgkin's disease; remissions after measles. Lancet 1971; 1:593. 179. Ziegler JL. Spontaneous remission in Burkitt's lymphoma. Natl Cancer Inst Monogr 1976; 44:61-5. 180. Gattiker HH, Wiltshaw E, Galton DA. Spontaneous regression in nonHodgkin's lymphoma. Cancer 1980; 45(10):2627-32.

316

Hyperthermia in Cancer Treatment: A Primer

181. McClain K, Warkentin P, Kay N. Spontaneous remission of Burkitt's lymphoma associated with herpes zoster infection. Am J Pediatr Hematol Oncol Spring 1995; 7(1):9-14. 182. Kempin S, Cirrincione C, Straus DS et al. Improved remission rate and duration in nodular nonHodgkin lymphoma (NNHL) with the use of mixed bacterial vaccine (MBV). Proc Am Soc Clin Oncol 1981; 22:514. 183. Kempin S, Cirrincione C, Myers J et al. Combined modality therapy of advanced nodular lymphoma (NL) - The role of nonspecific immunotherapy (MBV) as an important determinant of response and survival. Proc Am Soc Clin Oncol 1983; 24:56. 184. Grem JL, Hafez GR, Brandenburg JH et al. Spontaneous remission in diffuse large cell lymphoma. Cancer 1986; 57(10):2042-4. 185. Drobyski WR, Qazi R. Spontaneous regression in nonHodgkin's lymphoma: CHnical and pathogenetic considerations. Am J Hematol 1989; 31 (2): 138-41. 186. Wolf JW. Prolonged spontaneous remission of case of malignant lymphoma. Mo Med 1989; 86(5):275-7. 187. Sureda M, Subira ML, Martin Algarra S et al. Spontaneous tumor regression. Report of 2 cases. Med Clin (Bare) 1990; 95(8):306-8. 188. De Berker D, Windebank K, Sviland L et al. Spontaneous regression in angiocentric T-cell lymphoma. Br J Dermatol 1996; 134(3):554-8. 189. Sawada M, Ohdama S, Umino T et al. Metastasis of an adenocarcinoma of unknown origin to mediastinal lymph nodes, and transient regression. Nippon Kyobu Shikkan Gakkai Zasshi 1994; 32(9):867-72. 190. Heinzlef O, Poisson M, Delattre JY. Spontaneous regression of primary cerebral lymphoma. Rev Neurol (Paris) 1996; 152(2):135-8. 191. Tsubura E, Hirao F, Fujisawa T et al. Tumor and infection. Saishin Igaku 1967; 22(10):2281-90. 192. Bagshawe KD. Tumor growth and anti-mitotic action. The role of spontaneous cell losses. Br J Cancer 1968; 22:698-713. 193. Muckle DS, Dickson JA, Johnston ID. High fever and cancer. Lancet 1971; 8:972, (regression). 194. Schwartz DB, Zbar B, Gibson WT et al. Inhibition of murine sarcoma virus oncogenesis with living BCG. Int J Cancer 1971; 8:320-325. 195. Cho-Chung YS, GuUino PM. Mammary tumor regression. V. Role of acid ribonuclease and cathepsin. J Biol Chem 1973a; 248(13):4743-9. 196. Cho-Chung YS, Gullino PM. Mammary tumor regression. VI. Synthesis and degradation of acid ribonuclease. J Biol Chem 1973b; 248(13):4750-5. 197. Cho-Chung YS, Gullino PM. Mammary tumor regression. VI. Synthesis and degradation of acid ribonuclease. J Biol Chem 1973c; 248(13):4750-5. 198. Remy W, Stuttgen G, Bockendahl H et al. Remission of skin melanoma metastases following BCG injection (letter). Dtsch Med Wochenschr 1976; 101 (39): 1435-6. 199. Berendt MJ. The immunological basis of endotoxin-induced tumor regression. Requirement for T-cell mediated immunity. J Exp Med 1978a; 148:1550-1559. 200. Berendt MJ. The immunological basis of endotoxin-induced tumor regression. Requirement for a preexisting state of concomitant anti-tumor immunity. J Exp Med 1978b; 148:1560-1569. 201. Pedersen NC, Johnson L, Theilen GH. Biological behavior of tumors and associated retroviremia in cats inoculated with Snyder-Theilenfibrosarcomavirus and the phenomenon of tumor recurrence after primary regression. Infect Immun 1984; 43(2):631-6. 202. Bolande RP. Spontaneous regression and cytodifferentiation of cancer in early life: The oncogenic grace period. Surv Synth Pathol Res 1985; 4(4):296-311. 203. Baker HW. Biologic control of cancer. The James Ewing lecture. Arch Surg 1986; 121(11): 1237-41. 204. Stone OJ. Acute local inflammation causing generalized increased ground substance viscosity: Guttate psoriasis, Reiter's syndrome, adjuvant disease, cancer regression. Med Hypotheses 1988; 25(3):l4l-5. 205. Jarpe MA, Hayes MP, Russell JK et al. Causal association of interferon-gamma with tumor regression. J Interferon Res 1989; 9:239-244. 206. Seachrist L. Spontaneous cancer remissions spark questions (news). J Natl Cancer Inst 1993; 85(23):1892-5. 207. Halliday GM, Patel A, Hunt MJ et al. Spontaneous regression of human melanoma/nonmelanoma skin cancer: Association with infiltrating CD4+ T cells. World J Surg 1995; 19(3):352-8. 208. Gunale S, Tucker WG. Regression of metastatic melanoma. Mich Med 1975; 74:697-698. 209. Wormald RP, Harper JI. Bilateral black hypopyon in a patient with self-healing cutaneous malignant melanoma. Br J Ophthalmol 1983; 67(4):231-5. 210. Wagner Jr RF, Nathanson L. Paraneoplastic syndromes, tumor markers, and other unusual features of malignant melanoma. J Am Acad Dermatol 1986; 14(2 Pt l):249-56, (Spontaneous regression).

Fever, Pyrogens and Cancer

317

211. Cook MG. The significance of inflammation and regression in melanoma (editorial). Virchows Arch A Pathol Anat Histopathol 1992; 420(2):113-5. 212. Grafton WD. Regressing malignant melanoma. J La State Med Soc 1994; l46(12):535-9. 213. Motofei IG. Herpetic viruses and spontaneous recovery in melanoma. Med Hypotheses 1996; 47(2):85-8. 214. London RE. Multiple Myeloma: Report of a case showing unusual remission lasting two years following severe hepatitis. Ann Int Med 1955; 43:191-201, (Regression). 215. Schurmans JR, Blijenberg BG, Mickisch GH et al. Spontaneous remission of a bony metastasis in prostatic adenocarcinoma. J Urol 1996; 155(2):653. 216. Katz SE, Schapira HE. Spontaneous regression of genitourinary cancer - an update. J Urol 1982; 128:1-4. 217. Mangiapan G, Guigay J, Milleron B. A new case of spontaneous regression of metastasis of kidney cancer (letter). Rev Pneumol Clin 1994; 50(3):139-40. 218. Edwards MJ, Anderson JA, Angel JR et al. Spontaneous regression of primary and metastatic renal cell carcinoma. J Urol 1996; 155(4):1385. 219. Hunter WS. Unexpected regressed retinoblastoma. Can J Ophthalmol 1968; 3(4):376-80. 220. Verhoeff FH. Retinoblastoma undergoing spontaneous regression. Calcifying agent suggested in treatment of retinoblastoma. Am J Ophthalmol 1966; 62(3):573-4. 221. Jain IS, Singh K. Retinoblastoma in phthisis bulbi. J All India Ophthalmol Soc 1968; 16(2):76-8. 222. Dobson L, Dickey LB. Spontaneous regression of malignant tumors. Am J Surg 1956; 92:162-173. 223. Sindelar WF. Regression of cancer following surgery. Natl Cancer Inst Monogr 1976; 44:81-84. 224. Challis GB, Stam HJ. The spontaneous regression of cancer. A review of cases from 1900 to 1987. Acta Oncol 1990; 29(5):545-50. 225. Kaiser HE. Biological viewpoints of neoplastic regression. In Vivo 1994; 8(l):155-65. 226. Watson AL. A case of recurrent sarcoma with apparendy spontaneous cure and gradual shrinking of the tumour. Lancet 1902; 1.300-301. 227. Shore BR. Spontaneous cure of congenital recurring connective tissue tumor. Am J Cancer 1936; 27:736-739. 228. Penner DW. Spontaneous regression of a case of myosarcoma. Cancer 1953; G:77G-77^. 229. Berner RE, Laub DL. The spontaneous cure of massivefibrosarcoma.Plastic Reconstructive Surg 1965; 36:257-262. 230. Weintraub LR. Lymphosarcoma. Remission associated with viral hepatitis. JAMA 1969; 210:1590-1591. 231. Mizuno S, Fujinaga T, Hagio M. Role of lymphocytes in spontaneous regression of experimentally transplanted canine transmissible venereal sarcoma. J Vet Med Sci 1994; 56(l):15-20. 232. Lei KI, Gwi E, Ma L et al. Spontaneous' regression of advanced leiomyosarcoma of the urinary bladder. Oncology 1997; 54(l):19-22. 233. Atkins E. Fever: Its history, cause and fimction. Yale J Biol Med 1982; 55:283-289. 234. Kluger MJ. Fever: Role of pyrogens and cryogens. Physiol Rev 1991; 71(1):93-127. 235. Roberts Jr NJ. The immunological consequences of fever. In: Mackowiak PA, ed. In Fever: Basic mechanisms and management. New York: Raven, 1991:125. 236. Roberts Jr NJ. Impact of temperature elevation on immunologic defenses. Rev Infect Dis 1991; 13(3):462-72. 237. Dinarello CA. Endogenous pyrogens. In: Mackowiak PA, ed. Fever: Basic mechanisms and management. New York: Raven, 1991:23. 238. Dinarello CA. Thermoregulation and the pathogenesis of fever. Infect Dis Clin North Am 1996; 10(2):433-49. 239. Burnet FM. The concept of immunological surveillance. Progr Exp Tumor Res 1970; 13:1-27, (Karger, Basel/Munch en/New York). 240. Burnet FM. Implications immunological surveillance for cancer therapy. Israel J Medical Sci 1971; 7:9-16. 241. Hanson DF, Murphy PA. Demonstration of interleukin 1 activity in apparendy homogeneous specimens of the pi 5 form of rabbit endogenous pyrogen. Infect Immun 1984; 45(2):483-90. 242. Rodbard D, Wachslicht-Rodbard H, Rodbard S. Temperature: A critical factor determining localization and natural history of infectious, metabolic, and immunological diseases. Perspect Biol Med Spring 1980; 23(3):439-74. 243. Hanson DF, Murphy PA. Temperature sensitivity of interleukin-dependent murine T cell proliferation: Q2 mapping of the responses of peanut agglutinin-negative thymocytes. J Immunol 1985; 135(5):3011-20. 244. Niitsu Y, Watanabe N, Umeno H et al. Synergistic effects of recombinant human tumor necrosis factor and hyperthermia on in-vitro cytotoxicity and artificial metastasis. Cancer Res 1988; 48:654-657.

318

Hyperthermia in Cancer Treatment: A Primer

245. Yamauchi N, Watanabe N, Maeda M et al. Mechanism of synergistic cytotoxic effect between tumor necrosis factor and hyperthermia. Jpn J Cancer Res 1992; 83:540-545. 246. Hanson DF. Fever and the immune response. The effects of physiological temperatures on primary murine splenic T-cell responses in vitro. J Immunol 1993; 151:436-448. 247. Roberts Jr NJ, Steigbigel RT. Hyperthermia and human leukocyte functions: Effects on response of lymphocytes to mitogen and antigen and bactericidal capacity of monocytes and neutrophils. Infect Immun 1977; 18(3):673-9. 248. Dinarello CA, Conti P, Mier JW. Effects of human interleukin-1 on natural killer cell activity: Is fever a host defense mechanism for tumor kiUing? Yale J Biol Med 1986a; 59(2):97-106. 249. Dinarello CA, Dempsey RA, Allegretta M et al. Inhibitory effects of elevated temperature on human cytokine production and natural killer activity. Cancer Res 1986b. 250. Boeye A, Delaet I, Brioen P. Antibody neutralization of picornaviruses: Can fever help? Trends Microbiol 1994; 2(7):255-7. 251. Coelho MM, Luheshi G, Hopkins SJ et al. Multiple mechanisms mediate antipyretic action of glucocorticoids. Am J Physiol 1995; 269(3 Pt 2):R527-35. 252. Shwartzman G. Phenomenon of local tissue reactivity. New York: PB Hoeber, 1937. 253. Heremans H, Van Damme J, Dillen C et al. Interferon gamma, a mediator of lethal lipopolysaccharide-induced Shwartzman-like shock reactions in mice. J Exp Med 1990; 171(6):1853-69. 254. Centanni E, Rezzesi F. Etude Experimentale sur I'antagonisme entre la tuberculose et le cancer. Neoplasmes 1926; 5:211-225. 255. Daels F. Beitrag zum Studium des Antagonismus zwischen den Karzinom-, Spirillen- und Trypanosomeninfektionen. Arch Hyg 1910; 72:257-306. 256. Gratia A, Linz R. Le ph^nom^ne de Shwartzman dans le sarcome du Cobaye. Compt rend Seanc Soc Biol Ses Fil-1931; 108:427-428. 257. Shwartzman G, Michailovsky N. Proc Soc Exp Biol Med 1936; 34:323. 258. Berendt MJ, Saluk P. Tumor inhibition in mice by lipopolysaccharide-induced peritoneal cells and an induced soluble factor. Infection Immunity 1976; 14:965-969. 259. Shear MJ. Studies on the chemical treatment of tumors. II. The effect of disturbances of fluid exchange on the transplanted mouse tumors. Am J Cancer 1935; 25:66-88. 260. Aoki N, Mori W. Effects of endotoxin administration on tumor and host: An experimental observation on tumor-bearing rabbits. In: Homma Y, Kanegasaki S, Luederitz O et al, eds. Bacterial Endotoxin. Weinheim, S: Verlag Chemie, 1987:205-221. 261. Shear MJ, Turner FC, Perrault A. Chemical treatment of tumors. Isolation of haemorrhage-producing fraction from Serratia marcescens (Bacillus prodrigiosus) culture filtrate. J Natl Cancer Inst 1943; 4:81-97. 262. Shear MJ. Chemical treatment of tumors. DC. Reactions of mice with primary subcutaneous tumors to injection of a hemorrhage-producing bacterial polysaccharide. J Natl Cancer Inst 1944; 4:461-476. 263. Westphal O. Bacterial endotoxins. The second carl prausnitz memorial lecture. Int Arch Allergy Appl Immunol 1975; 49(l-2):l-43. 264. Andervont HB. The reaction of mice and various mouse tumours to the injection of bacterial products. Am J Cancer 1936; 27:77-83. 265. Alexander P, Evans R. Endotoxin and Double stranded RNA render macrophages cytotoxic. Nature New Biol 1971; 232:76-78. 266. Hofstad T, Skaug N, Sveen K. Stimulation of B lymphocytes by Hpopolysaccharides from anaerobic bacteria. Clin Infect Dis 1993; 16(Suppl 4):S200-2. 267. DeFranco AL, Gold MR, Jakway JP. B-lymphocyte signal transduction in response to anti-immunoglobulin and bacterial Upopolysaccharide. Immunol Rev 1987; 95:161-76. 268. Jacobs DM. Immunomodulatory effects of bacterial Upopolysaccharide. J Immunopharmacol 1981; 3(2):119-32. 269. McGhee JR, Kiyono H, Alley CD. Gut bacterial endotoxin: Influence on gut-associated lymphoreticular tissue and host immune function. Surv Immunol Res 1984; 3(4):24l-52. 270. Nowotny A. Review of the molecular requirements of endotoxic actions. Rev Infect Dis 1987; 9(Suppl 5):S503-11. 271. Nowotny A, Moore ME, Nejman G et al. Time dependency of endotoxin-induced resistance to transplantable tumors in mice. Cancer Invest 1987a; 5(3): 195-203. 272. Nowotny A, Blanchard DK, Newton C et al. Interferon induction by endotoxin-derived nontoxic polysaccharides. J Interferon Res 1987b; 7(4):371-8. 273. Rietschel ET, Brade H, Brade L et al. Lipid A, the endotoxic center of bacterial lipopolysaccharides: Relation of chemical structure to biological activity. Detection of bacterial endotoxins with the limulus amebocyte lysate test. Prog Clin Biol Res 1987; 231:25-53.

Fever, Pyrogens and Cancer

319

TJ\. Rietschel ET, Brade H, Hoist O et al. Bacterial endotoxin: Chemical constitution, biological recognition, host response, and immunological detoxification. Curr Topics Microbiol Immunol 1996; 216:39-81. 275. Rietschel ET, Brade H. Bacterial endotoxins. Sci Am 1992; 267(2):54-6l. 276. Dabbert CB, Lochmiller RL, Zhang JR et al. High in vitro endotoxin responsiveness of macrophages from an endotoxin-resistant wild rodent species, Sigmodon hispidus. Dev Comp Immunol 1994; 18(2):l47-53. 277. Galanos C, Freudenberg MA, Luederitz O et al. Chemical, physicochemical and biological properties of bacterial lipopolysaccharides. Prog CHn Biol Res 1979a; 29:321-332. 278. Abel U. Die antineoplastische Wirkung pyrogener Bakterientoxine. In: Hager ED, Abel U, eds. Biomodulation und Biotherapie des Krebses. II. Endogene Fiebertherapie und exogene Hyperthermie in der Onkologie. Heidelberg: Verlag fiir Medizin Dr. E. Fischer, 1987:21-85. 279. Chun M, Hoffmann MK. Combination immunotherapy of cancer in a mouse model: Synergism between tumor necrosis factor and other defence systems. Cancer Res 1987; 47:115-118. 280. Giese M, Kirchner H. Interferons and their effects. Onkologie 1988; 11:151-154. 281. Engelhardt R, Mackensen A, Galanos C et al. Biological response to intravenously administered endotoxin in patients with advanced cancer. J Biol Response Mod 1990; 9:480-491. 282. Engelhardt R, Mackensen A, Galanos C. Phase I trial of intravenously administered endotoxin (Salmonella abortus equi) in Cancer patients. Cancer Res 1991; 51:2524-2530. 283. Engelhardt R, Otto F, Mackensen A et al. Endotoxin (Salmonella abortus equi) in cancer patients. CHnical and immunological findings. Prog CHn Biol Res 1995; 392:253-61. 284. Mackensen A, Galanos C, Engelhardt R. Modulating activity of Interferon-y on endotoxin-induced cytokine production in cancer patients. Blood 1991a; 78:3254-3258. 285. Mackensen A, Galanos C, Engelhardt R. Treatment of cancer patients with endotoxin induces release of endogenous cytokines. Pathobiology 1991b; 59:264-267. 286. Mackensen A, Galanos C, Wehr U et al. Endotoxin tolerance: Regulation of cytokine production and cellular changes in response to endotoxin application in cancer patients. Eur Cytokine Netw 1992; 3:571-579. 287. Knopf HP, Otto F, Engelhardt R et al. Discordant adaptation of human peritoneal macrophages to stimulation by lipopolysaccharide and the synthetic lipid A analogue SDZ MRL 953. Down-regulation of TNF-alpha and IL-6 is paralleled by an up-regulation of IL-1 beta and granulocyte colony-stimulating factor expression. J Immunol 1994; 153:287-299. 288. Conti P, Reale M, Nicolai M et al. Bacillus Calmette-Guerin potentiates monocyte responses to lipopolysaccharide-induced tumor necrosis factor and interleukin-1, but not interleukin-6 in bladder cancer patients. Cancer Immunol Immunother 1994; 38(6):365-71. 289. Otto F, Schmid P, Mackensen A et al. Phase II trial of intravenous endotoxin in patients with colorectal and nonsmall cell lung cancer. Eur J Cancer 1996; 32A: 1712-1718. 290. Moore MA, Gabrilove J, Sheridan AP. Therapeutic implications of serum factors inhibiting proliferation and inducing differentiation of myeloid leukemic cells. Blood Cells 1983; 9(1): 125-44. 291. Enterline PE, Sykora JL, Keleti G et al. Endotoxins, cotton dust, and cancer. Lancet 1985; 2(846l):934-5. 292. Kearney R, Harrop P. Potentiation of tumour growth by endotoxin in serum from syngeneic tumour-bearing mice. Br J Cancer 1980; 42(4):559-67. 293. Kearney R, Harrop P. Modulation of anti-tumour immunity and the effect of bacterial endotoxin on the growth of different syngeneic tumours from small inocula in mice. Br J Exp Pathol 1986; 67(3):371-81. 294. DerHagopian RP, Sugarbaker EV, Ketcham A. Inflammatory oncotaxis. JAMA 1978; 240(4):374-5. 295. DerH^opian RP. Inflammatory oncotaxis (letter). JAMA 1979; 24l(21):2264. 296. Sataline L, PeUiccia O. Inflammatory oncotaxis (letter). JAMA 1978; 240(22):243. 297. Shine T, Wallack MK. Inflammatory oncotaxis after testing the skin of the cancer patient. Cancer 1981; 47(6):1325-8. 298. Ben-Baruch A. Host microenvironment in breast cancer development: Inflammatory cells, cytokines and chemokines in breast cancer progression: Reciprocal tumor-microenvironment interactions. Breast Cancer Res 2003; 5(l):31-6, (Epub 2002 Oct 28). 299. Pollard JW. Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer 2004; 4(l):71-8, (Review). 300. Morita S, Yamamoto M, Kamigaki T et al. Synthetic lipid A produces antitumor effect in a hamster pancreatic carcinoma model through production of tumor necrosis factor from activated macrophages. Kobe J Med Sci 1996; 42(4):219-31. 301. Vosika GJ. Phase-I study of intravenous modified lipid A. Cancer Immunol Immunother 1984; 18:107-112.

320

Hyperthermia in Cancer Treatment: A Primer

302. Goto S, Sakai S, Kera J et al. Intradermal administration of lipopolysaccharide in treatment of human cancer. Cancer Immunol Immunother 1996; 42(4):255-61. 303. Jimbo T, Akimoto T, Tohgo A. Systemic administration of a synthetic lipid A derivative, DT-546la, reduces tumor blood flow through endogenous TNF production in hepatic cancer model of VX2 carcinoma in rabbits. Anticancer Res 1996; l6(l):359-64. 304. Nowicki A, Ostrowska G, Aukerman SL et al. Effect of macrophage-modulating agents on in vivo growth of transplantable Lewis lung cancer in mice. Arch Immunol Ther Exp (Warsz) 1994; 42(4):313-7. 305. Freudenberg N, Joh K, Westphal O et al. Haemorrhagic tumour necrosis following endotoxin administration. I. Communication: Morphological investigation on endotoxin-induced necrosis of the methylcholanthrene (Meth A) tumour in the mouse. Virchows Arch A Pathol Anat Histopathol 1984; 403:377-389. 306. Boon T, Coulie P, Marchand M et al. Genes coding for tumor rejection antigens: Perspectives for specific immunotherapy. Important Adv Oncol 1994; 53-69. 307. Boon T, van der Bruggen P. Human tumor antigens recognized by T-lymphocytes. J Exp Med 1996; 183:725-729. 308. Gidlof C, Dohlsten M, Lando P et al. A superantigen-antibody fusion protein for T-cell immunotherapy of human B-lineage malignancies. Blood 1997; 89(6):2089-97. 309. Hansson J, Ohlsson L, Persson R et al. Genetically engineered superantigens as tolerable antitumor ^ents. Proc Nad Acad Sci USA 1997; 94(6):2489-94. 310. Litton MJ, Dohlsten M, Hansson J et al. Tumor therapy with an antibody-targeted superantigen generates a dichotomy between local and systemic immune responses. Am J Pathol 1997; 150(5):1607-18. 311. Jackson AM, Alexandroff AB, Mclntyre M et al. Induction of ICAM 1 expression on bladder tumours by BCG immunotherapy. J Clin Path 1994; 47:309-312. 312. Jackson AM, Alexandroff AB, Kelly RW et al. Changes in urinary cytokines and soluble intercellular adhesion molecule-1 (ICAM-1) in bladder cancer patients after bacillus Calmette-Guerin (BCG) immunotherapy. CUn Exp Immunol 1995a; 99(3):369-75. 313. Sung PK-L, Saldivar E, PhiUips L. Interleukin -Ip induces differential adhesiveness on human endothelial cell surfaces. Biochem Biophys Res Com 1994; 202:866-872. 314. Schumann RR, Pfeil D, Lamping N et al. Lipopolysaccharides induces the rapid tyrosine phosphorylation of the mitogen-activated protein kinases erk-1 and p38 in cultured human vascular endothehal cells requiring the presence of soluble CD14. Blood 1996; 87:2805-2824. 315. Puri RK, Rosenberg SA. Combined effects of interferon alpha and interleukin 2 on the induction of a vascular leak syndrome in mice. Cancer Immunol Immunother 1989; 28(4):267-74. 316. Economou JS, Hoban M, Lee JD et al. Production of tumor necrosis factor alpha and interferon gamma in interleukin-2-treated melanoma patients: Correlation with clinical toxicity. Cancer Immunol Immunother 1991; 34(l):49-52. 317. Edwards MJ, Abney DL, Heniford BT et al. Passive immunization against tumor necrosis factor inhibits interleukin-2-induced microvascular alterations and reduces toxicity. Surgery 1992; 112(2):480-6. 318. Tutor JD, Mason CM, Dobard E et al. Loss of compartmentalization of alveolar tumor necrosis factor after lung injury. Am J Respir Crit Care Med 1994; 149(5): 1107-11. 319. Carswell EA, Old LJ, Kassel RJ et al. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Nad Acad Sci 1975; 72:3666-3670. 320. Green S, Dobrjansky A, Chiasson M et al. Corynebacterium parvum as the priming agent in the production of tumor necrosis factor in the mouse. J Nad Cancer Inst 1977; 59:1519. 321. In: Homma JY, Kanegasaki S, Luederitz O et al, eds. Bacterial endotoxin: Chemical, biological and clinical aspects. Weinheim, Basel: Verlag Chemie, 1984. 322. Nowotny A, Behling UH, Chang HL. Relation of structure to function in bacterial endotoxins. III. Biological activities in a polysaccharide-rich fraction. J Immunol 1975; 115(l):199-203. 323. Mikolasek J, Direct evidence for rejection of tumour allografts in Str. pyogenes toxins-treated mice correlated with antistreptolysine O level in serum. Neoplasma 1972; 19(5):507-18. 324. Nativ O, Medalia O, Mor Y et al. Treatment of experimental mouse bladder tumour by LPS-induced epithelial cell shedding. Br J Cancer 1996; 74(4):603-5. 325. Behling UH, Nowotny A. Immune adjuvancy of lipopolysaccharide and a nontoxic hydrolytic product demonstrating oscillating effects with time. J Immunol 1977; 118(5): 1905-7. 326. Grohsman J, Nowotny A. The immune recognition of TA3 tumors, its facihtation by endotoxin, and abrogation by ascites fluid. J Immunol 1972; 109(5):1090-5. 327. Fidler IJ, Gersten DM, Riggs CW. Relationship of host immune status to tumor cell arrest, distribution, and survival in experimental metastasis. Cancer (Phila) 1977; 40:46-55.

Fever, Pyrogens and Cancer

321

328. Ray PK. Immunosuppressor control as a modality of cancer treatment: Effect of plasma adsorption with Staphylococcus aureus protein A. Contemp Top Immunobiol 1985; 15:147-211. 329. Chasseing NA, Eugui EM, Borda ES et al. Effects of sarcoma 180 growth on interleukin-1 and circulating immune complexes. Cancer Invest 1994; 12(4):390-4. 330. Das TK, Aziz M, Rattan A et al. Prognostic significance of circulating immune complexes in malignant tumours of head and neck. J Indian Med Assoc 1995; 93(l):3-7. 331. von Mensdorff-Pouilly S, Gourevitch MM, Kenemans P et al. Humoral immune response to polymorphic epithehal mucin (MUC-1) in patients with benign and malignant breast tumours. Eur J Cancer 1996; 32A(8): 1325-31. 332. Zhang K, Sikut R, Hansson GC. A MUCl mucin secreted from a colon carcinoma cell line inhibits target cell lysis by natural killer cells. Cell Immunol 1997; 176(2):158-65. 333. Jager E, Ringhoffer M, Karbach J et al. Inverse relationship of melanocyte differentiation antigen expression in melanoma tissues and CD8+ cytotoxic-T-cell responses: Evidence for immunoselection of antigen-loss variants in vivo. Int J Cancer 1996; 66(4):470-6. 334. Gonzalez FM, Vargas JA, Gea-Banacloche JC et al. Study of spontaneous cytotoxic activity in laryngeal carcinoma: Prognostic value. Acta Otorrinolaringol Esp 1995; 46(6):431-6. 335. Gupta SC, Agarwal J, Singh PA et al. A sequential study of humoral factors in ovarian neoplasms. Indian J Pathol Microbiol 1994; 37(3) :319-26. 336. Kuhl JS, Krajewski S, Duran GE et al. Spontaneous overexpression of the long form of the Bcl-X protein in a highly resistant P388 leukaemia. Br J Cancer 1997; 75(2):268-74. 337. Yang X, Page M. P-glycoprotein expression in ovarian cancer cell line following treatment with cisplatin. Oncol Res 1995; 7(12):6l9-24. 338. Hamre MR, Clark SH, Mirkin BL. Resistance to inhibitors of S-adenosyl-L-homocysteine hydrolase in CI300 murine neuroblastoma tumor cells is associated with increased methionine adenosyltransferase activity. Oncol Res 1995; 7(10-11):487-92. 339. Binaschi M, Supino R, Gambetta RA et al. MRP gene overexpression in a human doxorubicinresistant SCLC cell line: Alterations in cellular pharmacokinetics and in pattern of cross-resistance. Int J Cancer 1995; 62(l):84-9. 340. Graham CH, Kobayashi H, Stankiewicz KS et al. Rapid acquisition of multicellular drug resistance after a single exposure of mammary tumor cells to antitumor alkylating agents (see comments). J Nad Cancer Inst 1994; 86(13):975-82. 341. Shoulders HS. Observations on the results of combined fever and x-ray therapy on the treatment of malignancy. Southern Med J 1942; 35:966-970. 342. Zweifach BW, Kivy-Rosenberg E, Nagler AL. Resistance to whole body x-irradiation in rats made tolerant to bacterial endotoxins. Am J Physiol 1959; 197:1364-1370. 343. Donaldson SS, Cooper Jr RA, Fletcher WS. Effect of Coley's Toxins and irradiation on the A. melanoma # 3 tumor in the golden hamster. Cancer 1968; 21:805-11. 344. Chandler JJ, Stark DB, Allen CV et al. Treatment of cancer by bacterial toxins. Am Surg 1965; 31:443-449. 345. Nowotny A, Behling UH. Studies of host defenses enhanced by Endotoxins: A brief review. Klin Wochenschr 1987; 14:735-739. 346. Tang ZY, Zhou HY, Zhao G et al. Preliminary results of mixed bacterial vaccine as adjuvant treatment of hepatocellular carcinoma. Med Oncol Tumour Pharmacoth 1991; 8:23-29. 347. Morales A. From the 19th to the 21st centuries: BCG in the treatment of superficial bladder cancer. Eur Urol 1992a; 21(Suppl 2):2-6. 348. Hellstrom I, Hellstrom KE, Siegall CB et al. Immunoconjugates and immunotoxins for therapy of carcinomas. Adv Pharmacol 1995; 33:349-88. 349. Clark JI, Weiner LM. Biologic treatment of human cancer. Curr Probl Cancer 1995; 19(4): 185-262. 350. Vitetta ES, Thorpe PE, Uhr JW. Immunotoxins: Magic bullets or misguided missiles? Trends Pharmacol Sci 1993; l4(5):l48-54. 351. Cobb PW, LeMaistre CF. Therapeutic use of immunotoxins. Semin Hematol 1995; 29(3 Suppl 2):6-13. 352. Subira ML, Brugarolas A. Biotherapy of cancer. Rev Clin Esp 1992; 191(2):102-8. 353. Grossbard ML, Fidias P. Prospects for immunotoxin therapy of nonHodgkin's lymphoma. Clin Immunol Immunopathol 1995; 76(2): 107-14. 354. Ozaki S, Okazaki T, Nakao K. Biological response modifiers (BRM) as antigens. III. T cell lines specific for BRM kill tumor cells in a BRM-specific manner. Cancer Immunol Immunother 1995; 40(4):219-27. 355. Old LJ, Clarke DA, Benacerraf B. Effect of Bacillus Calmette-Giierin on transplanted tumors in the mouse. Nature 1959; 184:191-191.

522

Hyperthermia in Cancer Treatment: A Primer

356. Howard JG, Biozzi G, Halpern BN et al. The effect of mycobacterium tuberculosis (BCG) infection on the resistance of mice to bacterial endotoxin and salmonella enteritidis infection. Br J Exp Pathol 1959; 40:281-290. 357. Ruddle NH, Waksman BH. Cytotoxicity mediated by soluble antigen and lymphocytes in delayed hypersensitivity. J Exp Med 1968; 128:1267-1279. 358. Mastrangelo MJ, Kim YH, Bornstein RS et al. Clinical and histologic correlation of melanoma regression after intralesional BCG therapy: A case report. J Natl Cancer Inst 1974; 52(1): 19-24. 359. Hakim AA. Cyclic adenosine-3',5'-monophosphate in cellular immunity. Naturwissenschaften 1974; 6l(5):222-3. 360. Hakim AA, Grand NG. Mechanism of action of BCG vaccine on neoplastic proliferation and host immune responses. J Pharm Sci 1976; 65(3):339-43. 361. Vosika GJ. Clinical Immunotherapy trials of bacterial components derived from Mycobacteria and Nocardia. Review Article. J Biol Response Mod 1983; 2:321-342. 362. Morales A, Nickel JC. Immunotherapy for superficial bladder cancer. A developmental and clinical overview. Urol Clin North Am 1992b; 19:549-556. 363. Jurincic-Winkler C, Metz KA, Beuth J et al. Effect of keyhole Umpet hemocyanin (KLH) and bacillus Calmette-Guerin (BCG) instillation on carcinoma in situ of the urinary bladder. Anticancer Res 1995; 15(6B):2771-6. 364. Comeri GC, Belvisi P, Conti G et al. Role of BCG in T1G3 bladder transitional cell carcinoma (TCC): Our experience. Arch Ital Urol Androl 1996; 68(l):55-9. 365. Jackson AM, Alexandrov AB, Prescott S et al. Production of urinary tumour necrosis factors and soluble tumour necrosis fiictor receptors in bladder cancer patients after bacillus Calmette-Guerin immunotherapy. Cancer Immunol Immunother 1995b; 40(2):119-24. 366. Zhang Y, Broser M, Cohen H et al. Enhanced interleukin-8 release and gene expression in macrophages after exposure to Mycobacterium tuberculosis and its components. J Clin Invest 1995; 95(2):586-92. 367. Roszkowski W, Roszkowski K, Ko HL et al. Immunomodulation by propionibacteria. Zentralbl Bakteriol 1990; 274(3):289-98. 368. Turler A, Walter M, Schmitz-Rixen T. Current treatment strategy in malignant pleural effusion. Wien Klin Wochenschr 1996; 108(9):255-61. 369. Isenberg J, Stoffel B, Wolters U et al. Immunostimulation by propionibacteria-effects on immune status and antineoplastic treatment. Anticancer Res 1995; 15(5B):2363-8. 370. Chen MF, Suzuki H, Yano S. Induction of murine lymphokine-activated killer-like cells by Corynebaaerium parvum (C. parvum) in vitro: Lysis of tumor cells and macrophages by C. parvum-induced killer cells. Anticancer Res 1992; 12(2):451-6. 371. Bursuker I, Petty BA, Neddermann KM et al. Immunomodulation in an apparendy nonimmunogenic murine tumor. Int J Cancer 1991; 49(3):414-420. 372. Keller R, Keist R, Leist TP et al. Resistance to a nonimmunogenic tumor, induced by Corynebacterium parvum or Listeria monocytogenes, is abrogated by anti-interferon gamma. Int J Cancer 1990a; 46(4):687-90. 373. Karashima A, Taniguchi K, Yoshikai Y et al. Alteration in natural defense activity against NK-susceptible B16 melanoma cells after treatment with Corynebacterium parvum. Immunobiology 1991; 182(5):4l4-24. 374. Ko HL, Winkler C, Beuth J et al. Influence of propionibacterium avidum KP-40 on the proliferation, maturation, emigration and activity of thymocytes and monocytes. J Med Microbiol Virol Parasitol Infect Dis 1995; 282(1):86-91. 375. Pulverer G, Buss G, Ko HL et al. Propionibacterium acnes-metabolites inhibit experimental lung metastasis of murine sarcoma L-1 in BALB/c-mice. Int J Med Microbiol Virol Parasitol Infect Dis 1992; 277(3):364-70. 376. Pulverer G, Ko HL, Tunggal L et al. Combined immunomodulation (Propionibacterium avidum KP-40) and lectin blocking (D-galactose) prevents liver tumor colonization in BALB/c-mice. Int J Med Microbiol Virol Parasitol Infect Dis 1994; 281(4):491-4. 377. Lipton A, Harvey HA, Balch CM et al. Corynebacterium parvum versus bacille Calmette-Guerin adjuvant immunotherapy of stage II malignant melanoma (see comments). J Clin Oncol 1991; 9(7):1151-6. 378. Foresti V. Intrapleural Corynebacterium parvum for recurrent malignant pleural effusions. Respiration 1995; 62(l):21-6. 379. Isenberg J, Ko H, Pulverer G et al. Preoperative immunostimulation by Propionibacterium granulosum KP-45 in colorectal cancer. Anticancer Res 1994; 14(3B): 1399-404. 380. Raica M. Effects of intravesical Corynebacterium parvum on recurrences of superficial tumors of the urinary bladder. Anticancer Drugs 1992; 3(l):39-42.

Fever, Pyrogens and Cancer

323

381. Oettgen HF, Old LJ, Hoffmann MK et al. Antitumor effects of Endotoxin: Possible mechanism of action. In: Homma Y, Kanegasaki S, Luederitz O et al, eds. Bacterial Endotoxin. Weinheim, S: Verlag Chemie, 1984:205-221. 382. Galanos C, Luederitz O, Westphal O. Preparation and properties of a standardized lipopolysaccharide from Salmonella abortus equi (Novo-Pyrexal). Zbl Bakt Hyg, 1. Abt Org A 1979b; 243:226-244. 383. Galanos C, Lehmann V, Luderitz O et al. Endotoxic properties of chemically synthesized lipid A part structures. Comparison of synthetic lipid A precursor and synthetic analogues with biosynthetic Hpid A precursor and free Hpid A. Eur J Biochem 1984; 140:221-227. 384. Yamamoto A, Nagamuta M, Usami H et al. Release of tumor necrosis factor (TNF) into mouse peritoneal fluids by OK-432, a streptococcal preparation. Immunparmacol 1986; 11:79-86. 385. Furukawa H, Hiratsuka M, Iwanaga T et al. Adjuvant chemotherapy for advanced gastric cancer. Nippon Geka Gakkai Zasshi 1996; 97(4) :312-6. 386. Tsukuda M. Immunotherapy of patients with head and neck carcinomas. Gan To Kagaku Ryoho 1996; 23(3):283-90. 387. Kim JP, Kim YW, Yang HK et al. Significant prognostic factors by multivariate analysis of 3926 gastric cancer patients. World J Surg 1994; 18(6):872-7, (discussion 877-8). 388. Kim JP, Kwon OJ, Oh ST et al. Results of surgery on 6589 gastric cancer patients and immunochemosurgery as the best treatment of advanced gastric cancer. Ann Surg 1992; 2l6(3):269-78, (discussion 278-9). 389. Abel U. Gutachten zum Stand des Nachweises der Wirksamkeit der aktiven Fiebertherapie bei malignen Erkrankungen. In: Buehring M, Kemper FH, Matthiessen PF, eds. Naturheilverfahren und unkonventionelle medizinische Richtungen. Springer: LoseblattSysteme, 1996:1-17. 390. Torisu M, Uchiyama A, Goya T et al. Eighteen-year experience of cancer immunotherapies— evaluation of their therapeutic benefits and future. Nippon Geka Gakkai Zasshi 1991; 92(9):1212-6. 391. Clark JW. Biological response modifiers. Cancer Chemother Biol Response Modif 1991; 12:193-212. 392. Juranic Z, Tomin R, Spuzic I et al. The cytotoxic action of OK-432 from Streptococcus pyogenes. Med Hypotheses 1990; 33(2):73-4. 393. Cervical Cancer Immunotherapy Study Group. Immunotherapy using the streptococcal preparation OK-432 for the treatment of uterine cervical cancer. Cancer 1987; 60(10):2394-402. 394. Kikkawa F, Kawai M, Oguchi H et al. Randomised study of immunotherapy with OK-432 in uterine cervical carcinoma. Eur J Cancer 1993; 29A(11): 1542-6. 395. Okamura K, Hamazaki Y, Yajima A et al. Adjuvant immunotherapy: Two randomized controlled studies of patients with cervical cancer. Biomed Pharmacother 1989; 43(3):177-81. 396. Fujita K. The role of adjunctive immunotherapy in superficial bladder cancer. Cancer 1987; 59(12):2027-30. 397. Marumo K. Immunotherapy and urological malignancy. Nippon Hinyokika Gakkai Zasshi 1991; 82(3):361-71. 398. Hanaue H, Kim DY, Machimura T et al. Hemolytic streptococcus preparation OK-432; beneficial adjuvant therapy in recurrent gastric carcinoma. Tokai J Exp Clin Med 1987a; 12(4):209-14. 399. Hanaue H, Kim DY, Kubota M et al. Effects of biological response modifier on thoracic duct lymphocytes in recurrent gastric cancer. Evaluation of OK-432, a hemolytic streptococcus preparation. Tokai J Exp Clin Med 1987b; 12(2):97-102. 400. Nakazawa S, Yoshino J, Okamura S et al. Clinical efficacy of endoscopic injections of OK-432 in the treatment of gastric cancer. Scand J Gastroenterol 1988; 23(5):539-45. 401. Hattori T, Nakajima T, Nakazato H et al. Postoperative adjuvant immunochemotherapy with mitomycin C, tegafur, PSK and/or OK-432 for gastric cancer, with special reference to the change in stimulation index after gastrectomy. Jpn J Surg 1990; 20(2): 127-36. 402. Kyoto Research Group for Digestive Organ Surgery. A comprehensive multi-institutional study on postoperative adjuvant immunotherapy with oral streptococcal preparation OK-432 for patients after gastric cancer surgery. Ann Surg 1992; 2l6(l):44-54. 403. Maehara Y, Okuyama T, Kakeji Y et al. Postoperative immunochemotherapy including streptococcal lysate OK-432 is effective for patients with gastric cancer and serosal invasion. Am J Surg 1994; l68(l):36-40. 404. Sugimachi K, Maehara Y, Akazawa K et al. Postoperative chemotherapy including intraperitoneal and intradermal administration of the streptococcal preparation OK-432 for patients with gastric cancer and peritoneal dissemination: A prospective randomized study. Cancer Chemother Pharmacol 1994; 33(5):366-70. 405. Fukushima M. Adjuvant therapy of gastric cancer: The Japanese experience. Semin Oncol 1996; 23(3):369-78. 406. Watanabe Y, Iwa T. Clinical value of immunotherapy for lung cancer by the streptococcal preparation OK-432. Cancer 1984; 53(2):248-53.

524

Hyperthermia in Cancer Treatment: A Primer

407. Watanabe Y, Iwa T. Clinical value of immunotherapy with the streptococcal preparation OK-432 in nonsmall cell lung cancer. J Biol Response Mod 1987; 6(2): 169-80. 408. Watanabe Y, Shimizu J, Oda M et al. Clinical significance of immunotherapy for lung cancer— present and future. Nippon Geka Gakkai Zasshi 1991; 92(9):1217-20. 409. Luh KT, Yang PC, Kuo SH et al. Comparison of OK-432 and mitomycin C pleurodesis for malignant pleural eflfusion caused by lung cancer. A randomized trial. Cancer 1992; 69(3):674-9. 410. Lee YC, Luh SP, Wu RM et al. Adjuvant immunotherapy with intrapleural Streptococcus pyogenes (OK-432) in lung cancer patients after resection. Cancer Immunol Immunother 1994; 39(4):269-74. 411. Sakata Y, Komatsu Y, Takagi S et al. Randomized controlled study of mitomycin C/carboquone/ 5-fluorouracil/OK-432 (MQ-F-OK) therapy and mitomycin C/5-fluorouracil/doxorubicin (FAM) therapy against advanced liver cancer. Cancer Chemother Pharmacol 1989; 23(Suppl):S9-12. 412. Suto T, Fukuda S, Moriya N et al. Clinical study of biological response modifiers as maintenance therapy for hepatocellular carcinoma. Cancer Chemother Pharmacol 1994; 33(Suppl):Sl45-8. 413. Shibata S, Mori K, Moriyama T et al. Randomized controlled study of the effect of adjuvant immunotherapy with Picibanil on 51 malignant gliomas. Surg Neurol 1987; 27(3):259-63. 414. Nakano A, Kato M, Watanabe T et al. OK-432 chemical pleurodesis for the treatment of persistent chylothorax. Hepatogastroenterology 1994; 4l(6):568-70. 415. Nio Y, Shiraishi T, Tsuchitani T et al. Antitumor activity of orally administered streptococcal preparation, OK-432 on murine solid tumors and its absorption from the gut. In Vivo 1989; 3(5):307-13. 416. Noda T, Asano M, Yoshie O et al. Interferon-gamma induction in human peripheral blood mononuclear cells by OK-432, a killed preparation of Streptococcus pyogenes. Microbiol Immunol 1986; 30(l):81-8. 417. Sekimoto M, Kokunai I, Shimano T et al. Production of tumor necrosis factor (TNF) by monocytes from cancer patients and healthy subjects induced by OK-432 in vitro, and its augmentation by human interferon gamma. J Clin Lab Immunol 1988; 27(3):115-20. 418. Tabuchi K, Shiraishi T, Toda K et al. Expression of apoptosis-related gene products in human brain tumors and apoptosis-inducing therapy. Nippon Rinsho 1996; 54(7): 1922-8. 419. Hoshino T, Uchida A. Effective mechanisms of BRM, with special reference to induction of autologous tumor cell-kiUing (ATK) activity by OK-432. Can To Kagaku Ryoho 1986; 13(4 Pt 2):1277-84. 420. Uchida A, Micksche M, Hoshino T. Intrapleural administration of OK432 in cancer patients: Augmentation of autologous tumor killing activity of tumor-associated large granular lymphocytes. Cancer Immunol Immunother 1984; 18(1):5-12. 421. Uchida A, Hoshino T. Reduction of suppressor cells in cancer patients treated with OK-432 immunotherapy. Int J Cancer 1980; 26(4):401-4. 422. Lewis JG, Pizzo SV, Adams DO. Simple and sensitive assay employing stable reagents for quantification of plasminogen activator. Am J CHn Pathol 1981; 76(4):403-9. 423. Goldberg DM. Enzymes as agents for the treatment of disease. Clin Chim Acta 1992; 206(l-2):45-76. 424. Taussig SJ, Szekerezes J, Batkin S. Bromelain, the enzyme complex of pineapple (Ananas Comosus) and its clinical application. An update J Ethnopharmacol 1988; 22:191-203. 425. In: Gardner MLG, Steffen C-J, eds. Absorption of orally administered enzymes. Berlin, Heidelberg, New York: Springer, 1995:ISBN 3-540-58747-0. 426. O'Meara RA, Jackson RD. Cytological observations on carcinoma. Ir J Med Sci 1958; 6:327-328. 427. Musser DA, Wagner JM, Weber FJ et al. The binding of tumor localizing porphyrins to a fibrin matrix and their effects following photoirradiation. Res Commun Chem Pathol Pharmacol 1980; 28(3):505-25. 428. Smith CE. Microbial enzymes in clinical investigation, diagnosis and therapy. J Gen Microbiol 1971; 65(3):x. 429. Lehmann PV. Immunomodulation by proteolytic enzymes (editorial). Nephrol Dial Transplant 1996; ll(6):952-5. 430. Caspary WF. Physiology and pathophysiology of intestinal absorption. Am J CHn Nutr 1992; 55:299S-308S. 431. Lake-Bakaar G, Rubio CE, McKavanagh S et al. Metabolism of 1251-labelled trypsin in man: Evidence for recirculation. Gut 1980; 21:580. 432. Steffen C, Menzel J, Smolen J. Intestinal resorption with 3H labeled enzyme mixture (wobenzyme). Acta Med Austriaca 1979a; 6(1): 13-8. 433. Mikolasek J. Inhibitory effect of varidase on in vitro tumoricidal activity of human serum. Neoplasma 1974; 21(4):483-5.

Fever, Pyrogens and Cancer

325

434. Holland PD, Browne O, Thornes RD. The enhancing influence of proteolysis on E rosette forming lymphocytes (T cells) in vivo and in vitro. Br J Cancer 1975; 31 (2): 164-9. 435. Thornes RD. Unblocking or activation of the cellular immune mechanism by induced proteolysis in patients with cancer. Lancet 1974; 2(877):382-4. 436. Tomar RH, John PA, Lapham C. Activation of natural killer cells in vitro by a product of beta-hemolytic streptococci. Cell Immunol 1982; 69(2):388-94. 437. Klein E, Di Renzo L, Yefenof E. Contribution of C R 3 , C D l l b / C D 1 8 to cytolysis by human N K cells. Mol Immunol 1990; 27(12):1343-7. 438. Hale LP, Haynes BF. Bromelain treatment of human T cells removes C D 4 4 , CD45RA, E2/MIC2, C D 6 , C D 7 , C D 8 , and Leu 8/LAMl surface molecules and markedly enhances CD2-mediated T cell activation. J Immunol 1992; 149:3809-3816. 439. Munzig E, Eckert K, Harrach T et al. Bromelain protease F9 reduces the C D 4 4 mediated adhesion of human peripheral blood lymphocytes to human umbiUcal vein endothelial cells. FEBS Letters 1994; 351:215-218. 440. Kleef R, Delohery T M , Bovberg D H . Selective modulation of cell adhesion molecules on lymphocytes by bromelain protease 5. Pathobiology 1996; 63(6):339-46. 441. Batkin S, Taussig SJ, Szekerezes J. Antimetastatic effect of bromelain with or without its proteolytic and anticoagulant activity. J Cancer Res Clin Oncol 1988a; 114:507-508. 442. Batkin S, Taussig SJ, Szekerezes J. Modulation of Pulmonary metastasis (Lewis Lung Carcinoma) by bromelain, an extract of the pineapple stem (Ananas Comosus). Cancer Invest 1988b; 6:241-242. 443. Rokitansky OV, Stauder G, Streichhan P. Enzymtherapie als prae- und postoperatives Adjuvans bei der Brustkrebsbehandlung. Deutsch Zschr Onkol / J Oncol 1993; 25:130-136. 444. Uster S, Rimpler M. Influence of proteolytic treatment on the lectin-binding capacity of tumor cells. Forsch Komplementarmed 1995; 2:190-195. 445. Timonen T, Gahmberg CG, Patarrayo M. Participation of C D l l a - c / C D 1 8 , C D 2 and RGD-binding receptors in endogenous and interleukin-2-stimulated N K activity of CD3-negative large granular lymphocytes. Int J Cancer 1990; 46:1035-1040. 446. Timonen T, Jaaskelainen J, Maenpaa A et al. Growth requirements, binding and migration of human natural killer cells. Immunol Series 1994; 61:63-72. 447. Robertson MJ, Caligiuri MA, Manley TJ et al. Human natural killer cell adhesion molecules Differential expression after activation and participation in cytolysis. J I m m u n o l 1990; 145:3194-3201. 448. Werfel T, Witter W, Gotze O . G D I l b and C D l l c antigens are rapidly increased on human natural killer cells upon activation. J Immunol 1991; l47(7):2423-7. 449. Ferrini S, Sforzini S, Cambiaggi A et al. The LFA-1/ICAM cell adhesion pathway is involved in tumor-cell lysis mediated by bispecific monoclonal-antibody-targeted T lymphocytes. Int J Cancer 1994; 56(6):846-52. 450. Liu RH, Hotchkiss J H . Potential genotoxicity of chronically elevated nitric oxide: A review. Mutat Res 1995; 339(2):73-89. 451. Morisaki T, Torisu M. Enhanced adherence activity of OK-432-induced peritoneal neutrophils to tumor cells correlates to their increased expression of G D I l b / C D 18. CUn Immunol Immunopathol 1991; 59:474-486. 452. Stamenkovic I, Amiot M, Pesando JM et al. A lymphocyte molecule impUcated in lymph node homing is a member of the cartilage link protein family. Cell 1989; 46:1057-1062. 453. Gunthert U, Hofmann M, Rudy W et al. A new variant of glycoprotein C D 4 4 confers metastatic potential to rat carcinoma cells. Cell 1991; 65:13-24. 454. Brown D C , Purushotham AD, George W D . Inhibition of pulmonary tumor seeding by antiplatelet and fibrinolytic therapy in an animal experimental model. J Surg Oncol 1994; 55(3): 154-9. 455. Purushotham AD, Brown D C , McCulloch P et al. Streptokinase inhibits pulmonary tumor seeding in an animal experimental model. J Surg Oncol 1994; 57(l):3-7. 456. Emeis JJ, Brouwer A, Barelds RJ et al. O n the fibrinolytic system in aged rats, and its reactivity to endotoxin and cytokines. Thromb Haemost 1992; 67(6):697-701. 457. Murthy MS, Summaria LJ, Miller RJ et al. Inhibition of tumor implantation at sites of trauma by plasminogen activators. Cancer 1991; 68(8): 1724-30. 458. Maruyama H, Nakajima J, Yamamoto I. A study on the anticoagulant and fibrinolytic activities of a crude fucoidan from the edible brown seaweed Laminaria reUgiosa, with special reference to its inhibitory effect on the growth of sarcoma-180 ascites cells subcutaneously implanted into mice. Kitasato Arch Exp Med 1987; 60(3):105-21. 459. Thornes RD. Adjuvant therapy of cancer via the cellular immune mechanism or fibrin by induced fibrinolysis and oral anticoagulants. Cancer 1975; 35(l):91-7.

326

Hyperthermia in Cancer Treatment: A Primer

460. Szreder W. Effect of artificial abacterial erysipelas and prolonged sterile abscess on neoplastic diseases in man and animals. Przegl Lek 1968a; 24(4):425-8. 461. Szreder W. Effect of artificially induced abacterial erysipelas and of chronic aseptic abscess on human and experimental neoplasms. Pol Med J 1968b; 7(5):1122-9. 462. Bykowska K, Janczarski M, Wegrzynowicz Z et al. Plasma fibronectin in acute leukaemias and during streptokinase therapy. Mater Med Pol 1988; 20(2):ll4-8. 463. DeWys WD, Kwaan HC, Bathina S. Effect of defibrination on tumor growth and response to chemodierapy. Cancer Res 1976; 36(10):3584-7. 464. Holt JA. Alternative therapy for recurrent Hodgkin's disease. Radiotherapy combined with hyperthermia by electromagnetic radiation to create complete remission in 11 patients without morbidity. Br J Radiol 1980; 53(635):106l-7. 465. Teuscher E, Pester E. A possible explanation of mechanisms inducing inhibition of vascularization of tumours by antifibrinolytic drugs—the influence of migratory behaviour of endothelial cells. Biomed Biochim Acta 1984; 43(4):447-56. AGG. Sugimura M, Tsubakimoto, Kashibayashi Y et al. Effect of human serum plus streptokinase on spontaneous pulmonary metastases of Vx2 carcinomas transplanted in the maxillary sinus of the rabbit. Int J Oral Surg 1975; 4(3):112-20. 467. McKinna JA, Rowbotham HD. Experimental studies of factors causing blood-borne dissemination in cancer of the colon and rectum. Proc R Soc Med 1971; 64(5):569-70. 468. Ciavarella D. The use of protein A columns in the treatment of cancer and allied diseases. Int J Clin Lab Res 1992; 21(3):210-3. 469. Nand S, Molokie R. Therapeutic plasmapheresis and protein A immunoabsorption in malignancy: a brief review. J CHn Apheresis 1990; 5(4):206-12. 470. Dwivedi PD, Verma AS, Ray PK. Induction of immune rejection of tumors by protein A in mice bearing transplantable solid tissue Dalton's lymphoma tumors (published erratum appears in Immunopharmacol Immunotoxicol 1992; 14(4):981). Immunopharmacol Immunotoxicol 1992; l4(l-2):105-28. 471. Prasad AK, Singh KP, Saxena AK et al. Increased macrophage activity in protein A treated tumor regressed animals. Immunopharmacol Immunotoxicol 1987; 9(4):541-61. 472. Ray PK, Mohammed J, Allen P et al. Effect of frequency of plasma adsorption over protein A-containing Staphylococcus aureus on regression of rat mammary adenocarcinomas: Modification of antitumor immune response and tumor histopathology. J Biol Response Mod 1984a; 3(l):39-59. 473. Ray PK, Idiculla A, Mark R et al. Extracorporeal immunoadsorption of plasma from a metastatic colon carcinoma patient by protein A-containing nonviable Staphylococcus aureus: clinical, biochemical, serologic, and histologic evaluation of the patient's response. Cancer 1982; 49(9):1800-9. 474. Bandyopadhyay SK, Ray PK. Introduction of bacterial components in postadsorbed plasma during adsorption with Staphylococcus aureus. Cancer 1985; 56(2):266-72. 475. Ray PK, Bandyopadhyay S, Dohadwala M et al. Antitumor activity with nontoxic doses of protein A. Cancer Immunol Immunother 1984b; 18(l):29-34. 476. Ray PK, Bandyopadhyay SK. Inhibition of rat mammary tumor growth by purified protein A—a potential anti-tumor agent. Immunol Commun 1983; 12(5):453-64. All. Shukla Y, Verma AS, Mehrotra NK et al. Antitumour activity of protein A in a mouse skin model of two-stage carcinogenesis. Cancer Lett 1996; 103(l):4l-7. 478. Kumar S, Shukla Y, Prasad AK et al. Protection against 7,12-dimethylbenzanthracene-induced tumour initiation by protein A in mouse skin (published erratum appears in Cancer Lett 1992 Oct 21;66(3):255). Cancer Lett 1992; 6l(2):105-10. 479. Zaidi SI, Singh KP, Raisuddin S et al. Modulation of primary antibody response by protein A in tumor bearing mice. Immunopharmacol Immunotoxicol 1995; 17(4):759-73. 480. Singh KP, Shau H, Gupta RK et al. Protein A potentiates lymphokine-activated killer cell induction in normal and melanoma patient lymphocytes. Immunopharmacol Immunotoxicol 1992a; l4(l-2):73-103. 481. Guojun Wu et al. Intravesical instillation of highly agglutinative staphylococcin for preventing postoperative recurrence of bladder transitional cell carcinoma. Chinese Journal of Clinical Oncology 2003; P61-63 482. Leder L, Llera A, Lavoie PM et al. A mutational analysis of the binding of staphylococcal enterotoxins B and C3 to the T cell receptor beta chain and major histocompatibility complex class II. J Exp Med 1998; 187(6):823-33. 483. Cho-Chung YS, Clair T, Shepeard C et al. Arrest of hormone-dependent mammary cancer growth in vivo and in vitro by cholera toxin. Cancer Res 1983; 43:1473-1476.

Fever, Pyrogens and Cancer

327

484. Maroun JA, Pross HF, Stewart TH et al. The effect of specific and nonspecific immunotherapy on natural killer cell activity in patients with nonsmall-cell lung cancer. J Clin Oncol 1984; 2(11):1209-14. 485. Livingston PO. Approaches to augmenting the immunogenicity of melanoma gangliosides: From whole melanoma cells to ganglioside-KLH conjugate vaccines. Immunol Rev 1995; 145:147-66. 486. Kalble T, Otto T. Unconventional therapeutic methods in superficial bladder cancer. Urologe A 1994; 33(6):553-6. 487. Shapiro A, Kadmon D, Catalona WJ et al. Immunotherapy of superficial bladder cancer. J Urol 1982; 128(5):891-4. 488. Lamm DL, DeHaven JI, Riggs DR et al. Immunotherapy of murine bladder cancer with keyhole limpet hemocyanin (KLH). J Urol 1993; l49(3):648-52. 489. Schmitz-Drager BJ, Schattka SO, Ebert T. Immunotherapy of superficial bladder cancer. Urologe A 1993; 32(5):374-81. 490. Sargent ER, WiUiams RD. Immunotherapeutic alternatives in superficial bladder cancer. Interferon, interleukin-2, and keyhole-Hmpet hemocyanin. Urol Clin North Am 1992; 19(3):581-9. 491. Olsson CA, Chute R, Rao CN. Immunologic reduction of bladder cancer recurrence rate. Trans Am Assoc Genitourin Surg 1973; 65:66-72. 492. Slingluff Jr CL. Tumor antigens and tumor vaccines: Peptides as immunogens. Semin Surg Oncol 1996; 12:446-453. 493. Haas C, Schirrmacher V. Immunogenicity increase of autologous tumor cell vaccines by virus infection and attachment of bispecific antibodies. Cancer Immunol Immunother 1996; 43:190-194. 494. Schlom J, Kantor J, Abrams S et al. Strategies for the development of recombinant vaccines for the immunotherapy of breast cancer. Breast Cancer Res Treat 1996; 38:27-39. 495. Baltz JK. Vaccines in the treatment of cancer. Am J Health Syst Pharm 1995; 52:2574-2585. 496. Shillitoe EJ, Kamath P, Chen Z. Papillomaviruses as targets for cancer gene therapy. Cancer Gene Ther 1994; 1(3): 193-204. 497. Hu SL, Hellstrom I, Hellstrom KE. Recent advances in antitumor vaccines. Biotechnology 1992; 20:327-343. 498. Sinkovics JG. Viral oncolysates as human tumor vaccines. Int Rev Immunol 1991; 7(4):259-87. 499. loannides CG, Platsoucas CD, O'Brian CA et al. Viral oncolysates in cancer treatment; immunological mechanisms of action (review). Anticancer Res 1989; 9:535-544. 500. Wheelock EF, Dingle JH. Observations on the repeated administration of viruses to a patient with acute leukemia. New Eng J Med 1964; 27:645-651. 501. Webb HE, Whetherley-Mein G, Gordon Smith CE et al. Leukemia and neoplastic processes treated with Langat and Kyasanur forest disease viruses: A clinical and labortory study of 28 patients 1966. 502. Csatary LK. Viruses in the treatment of cancer. The Lancet 1971; 7728:825. 503. Csatary LK, Eckhard S, Bukosza I et al. Attenuated veterinary viruses vaccine for the treatment of cancer. Cancer Detection Prevention 1993; 17:619-627. 504. HagmuUer E, Beck N, Ockert D et al. Adjuvant therapy of liver metastases: Active specific immunotherapy. Zentralbl Chir 1995; 120:780-785. 505. Schirrmacher V. Immunity and metastasis: in situ activation of protective T cells by virus modified cancer vaccines. Cancer Surv 1992; 13:129-154. 506. Hodge JW, McLaughlin JP, Abrams SI et al. Admixture of a recombinant vaccinia virus containing the gene for the costimulatory molecule B7 and a recombinant vaccinia virus containing a tumor-associated antigen gene results in enhanced specific T-cell responses and antitumor immunity. Cancer Res 1995; 55(16):3598-603. 507. Restifo NP, Esquivel F, Asher AL et al. Defective presentation of endogenous antigens by a murine sarcoma. J Immunol 1991; 147:1453-1459. 508. Piontek GE, Taniguchi K, Ljunggren HG et al. YAC-1 MHC class I variants reveal an association between decreased NK sensitivity and increased H-2 expression after interferon treatment or in vivo passage. J Immunol 1985; 135(6):4281-8. 509. Karre K, Ljunggren HG, Piontek G et al. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature 1986; 319(6055):675-8. 510. Ohlen C, Bejarano MT, Gronberg A et al. Studies of subHnes selected for loss of HLA expression from an EBV-transformed lymphoblastoid cell line. Changes in sensitivity to cytotoxic T cells activated by allostimulation and natural killer cells activated by IFN or IL-2. J Immunol 1989; 142(9):3336-41. 511. Ljunggren HG, Sturmhofel K, Wolpert E et al. Transfection of beta 2-microglobulin restores IFN-mediated protection from natural killer cell lysis in YAC-1 lymphoma variants. J Immunol 1990; l45(l):380-6.

328

Hyperthermia in Cancer Treatment: A Primer

512. Ljunggren HG, Karre K. In search of the 'missing self: MHC molecules and NK cell recognition (see comments). Immunol Today 1990; ll(7):237-44. 513. Yamasaki T, Akiyama Y, Fukuda M et al. Natural resistance against tumors grafted into the brain in association with histocompatibiHty-class-I-antigen expression. Int J Cancer 1996; 67(3):365-71. 514. Salcedo M, Diehl AD, Olsson-Alheim MY et al. Altered expression of Ly49 inhibitory receptors on natural killer cells from MHC class I-deficient mice. J Immunol 1997; 158(7):3174-80. 515. Thomasson DL, Stewart CC. Macrophage tumoricidal activity: Activation and killing kinetics. In: Chirigos MA, Mitchell M, Mastrangelo MJ et al, eds. Mediation of cellular immunity in cancer by Immune modifiers. New York: Raven Press, 1981:1-7. 516. Henkart P, Millard P, Reynolds C et al. Cytolytic activity of purified cytoplasmic granule from cytolytic rat LGL tumors. J Exp Med 1974; 160:75. 517. GifFord GE, FHck DA. Natural production and release of tumour necrosis factor. Ciba Found Symp 1987b; 131:3-20. 518. Morrison DC, Lei MG, Kirikae T et al. Endotoxin receptors on mammalian cells. Immunobiology 1993; 187(3-5):212-26. 519. Morrison DC, Ryan JL. Bacterial endotoxins and host immune responses. Adv Immunol 1979; 28:293-450. 520. Takayama K, Qureshi N, Beuder B et al. Diphosphoryl lipid A from Rhodopseudomonas sphaeroides ATCC 17023 blocks induction of cachectin in macrophages by lipopolysaccharide. Infect Immun 1989; 57(4):1336-8. 521. Qureshi N, Honovich JP, Hara H et al. Location of fatty acids in Upid A obtained from lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023. J Biol Chem 1988; 263(12):5502-4. 522. Nathan CF. Secretory products of macrophages. J Clin Invest 1987; 79(2):319-26. 523. Wright SD, Jong MT. Adhesion-promoting receptors on human macrophages recognize Escherichia coU by binding to lipopolysaccharide. J Exp Med 1986; 164(6): 1876-88. 524. Wright SD, Ramos RA, Tobias PS et al. CD 14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein (see comments). Science 1990; 249(4975): 1431-3. 525. Ulevitch RJ, Mathison JC, Schumann RR et al. A new model of macrophage stimulation by bacterial lipopolysaccharide. J Trauma 1990; 30(12 Suppl):S 189-92. 526. Schumann RR, Leong SR, Flaggs GW et al. Structure and function of lipopolysaccharide binding protein. Science 1990; 249(4975): 1429-31. 527. Schumann RR, Rietschel ET, Loppnow H. The role of CD14 and Upopolysaccharide-binding protein (LPB) in the activation of different cell types by endotoxin. Med Microbiol Immunol 1994; 183:279-297. 528. Schuett C. Role of CD14 in cellular activation by endotoxins. Chemoth J 1991; 4:169-179. 529. Morrison DC, Kirikae T, Kirikae F et al. The receptor(s) for endotoxin on mammalian cells. Prog Clin Biol Res 1994; 388:3-15. 530. Hailman E, Lichenstein HS, Wurfel MM et al. Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14. J Exp Med 1994; 179(l):269-77. 531. Carrithers SL, Parkinson SJ, Goldstein SD et al. Escherichia coli heat-stable enterotoxin receptors. A novel marker for colorectal tumors. Dis Colon Rectum 1996a; 39(2): 171-81. 532. Carrithers SL, Barber MT, Biswas S et al. Guanylyl cyclase C is a selective marker for metastatic colorectal tumors in human extraintestinal tissues. Proc Nad Acad Sci USA 1996b; 93(25): 14827-32. 533. Carrithers SL, Parkinson SJ, Goldstein S et al. Escherichia coli heat-stable toxin receptors in human colonic tumors. Gastroenterology 1994; 107(6): 1653-61. 534. Ding A, Sanchez E, Tancinco M et al. Interactions of bacterial Hpopolysaccharide with microtubule proteins. J Immunol 1992; l48(9):2853-8. 535. Ding A, Sanchez E, Nathan CF. Taxol shares the ability of bacterial lipopolysaccharide to induce tyrosine phosphorylation of microtubule-associated protein kinase. J Immunol 1993; 151(10):5596-602. 536. Vale RD. Intracellular transport using microtubule-based motors. Annu Rev Cell Biol 1987; 3:347-78. 537. Mitchison TJ, Kirschner MW. Some thoughts on the partitioning of tubulin between monomer and polymer under conditions of dynamic instability. Cell Biophys 1987; 11:35-55. 538. Harlan JM. Leukocyte-endothelial interactions. Blood 1985; 65:513-525. 539. Dustin ML, Rothlein R, Bhan AK et al. Induction by IL-1 and interferon-gamma: Tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-l). J Immunol 1986; 137(l):245-54. 540. Detmar M, Tenorio S, Hettmannsperger U et al. Cytokine regulation of proliferation and ICAM-l expression of human dermal microvascular endothelial cells in vitro. J Invest Dermatol 1992; 98(2):l47-53.

Fever, Pyrogens and Cancer

329

541. Ishii T, Walsh LJ, Seymour GJ et al. Modulation of Langerhans cell surface antigen expression by recombinant cytokines. J Oral Pathol Med 1990; 19(8):355-9. 542. Lindsley HB, Smith DD, Cohick CB et al. Proinflammatory cytokines enhance human synoviocyte expression of functional intercellular adhesion molecule-1 (ICAM-1). Clin Immunol Immunopathol 1993; 68(3):311-20. 543. Wedi B, Eisner J, Czech W et al. Modulation of intercellular adhesion molecule 1 (ICAM-1) expression on the human mast-cell line (HMC)-l by inflammatory mediators. Allergy 1996; 51(10):676-84. 544. Czech W, Krutmann J, Budnik A et al. Induction of intercellular adhesion molecule 1 (ICAM-1) expression in normal human eosinophils by inflammatory cytokines. J Invest Dermatol 1993; 100(4):4l7-23. 545. Piela-Smith TH, Broketa G, Hand A et al. Regulation of ICAM-1 expression and function in human dermalfibroblastsby IL-4. J Immunol 1992; 148(5):1375-81. 546. Hogg N, Berlin C. Structure and function of adhesion receptors in leukocyte trafficking. Immunol Today 1995; 16(7):327-30. 547. Chong AFS, Aleksijevic A, Scuderi P et al. Phenotypical and functional analysis of lymphokineactivated killer (LAK) cell clones. Ability of CD3*, LAK cell clones to produce interferon y and tumor necrosis factor upon stimulation with tumor targets. Cancer Immunol Immunother 1989a; 29:270-278. 548. Chong AFS, Scuderi P, Grimes WJ et al. Tumor targets stimulate IL-2 activated killer cells to produce interferon-y and tumor necrosis factor. J Immunol 1989b; 142:2133-2139. 549. Pober JS, Gimbrone MA, Cotran RS et al. la expression by vascular endothelium is inducible by activated T cells and by human gamma-interferon. J Exp Med 1983; 157:1339-1353. 550. Yu CL, Haskard DO, Cavender D et al. Human gamma-interferon increases the binding of T lymphocytes to endothehal cells. Clin Exp Immunol 1985; 62:554-560. 551. Kimber I, Sparshott SM, Bell EB et al. The effects of interferon on the recirculation of lymphocytes in the rat. Immunology 1987; 60:585-591. 552. Schleimer RP, Rutledge BK. Cultured human vascular endothelial cells acquire adhesiveness for neutrophils after stimulation with interleukin-1, endotoxin and tumor-promoting phorbol diesters. J Immunol 1986; 136:649-654. 553. Yu CL, Haskard DO, Cavender D et al. Effects of bacterial lipopolysaccharide on the binding of lymphocytes to endothehal cell monolayers. J Immunol 1986; 136:569-573. 554. Schlievert PM, Watson DW. Group A streptococcal pyrogenic exotoxin: Pyrogenicity alteration of blood-brain barrier, and separation of sites for pyrogenicity and enhancement of lethal endotoxin shock. Infect Immun 1978; 21:753-763. 555. Schlievert PM, Bettkin KM, Watson DW. Production of pyrogenic exotoxin by groups of Streptococci association with group A. J Infect Dis 1979; 140:676-681. 556. Jones M, Hoover R, Meyrick B. Endotoxin enhancement of lymphocyte adherence to cultured sheep lung microvascular endothelial cells. Am J Respir Cell Mol Biol 1992; 7(l):81-9. 557. Carratelli CR, Nuzzo I, Bentivoglio C et al. CDlla/CD18 and CDllb/18 modulation by lipoteichoic acid, N-acetyl-muramyl-alpha-alanyl-D-isoglutamine, muramic acid and protein A from Staphylococcus aureus. FEMS Immunol Med Microbiol 1996; l6(3-4):309-15. 558. Henriques GM, Miotla JM, Cordeiro SB et al. Selectins mediate eosinophil recruitment in vivo: A comparison with their role in neutrophil influx. Blood 1996; 87(12):5297-304. 559. Heinzelmann M, Mercer-Jones MA, Gardner SA et al. Bacterial cell wall products increase monocyte HLA-DR and ICAM-1 without affecting lymphocyte CD 18 expression. Cell Immunol 1997; 176(2):127-34. 560. Picker L, Butcher E. Annu Rev Immunol 1992; 10:561-591. 561. Ottaway CA. In: Husband AJ, ed. Migration and Homing of Lymphoid Cells (Vol. II). CRC Press, 1988:167-194. 562. Springer TA. Adhesion receptors of the immune system. Nature 1990; 34:425-434. 563. Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell 1994; 76(2):301-14. 564. Pohlman TH, Stannes KA, Beatty PG et al. An endothelial cell surface factor(s) induced in vitro by lipopolysaccharide, interleukin-1, and tumor necrosis factor alpha increases neutrophil adherence by a CDwl8-dependednt mechanism. J Immunol 1986; 136:4548-4553. 565. Hynes RO, Lander AD. Contact and adhesive specificities in the associations, migrations, and targeting of cells and axons. Cell 1992; 68(2):303-22. 566. Mackay CR, Imhof BA. Cell adhesion in the immune system. Immunol Today 1993; 14:99-102. 567. Dinarello CA, Mier JW. Lymphokines. N Engl J Med 1987; 317:940-945.

330

Hyperthermia in Cancer Treatment: A Primer

568. Patarroyo M, Prieto J, Rincon J et al. Leukoq^e-cell adhesion: A molecular process fundamental in leukocyte physiology. Immunol Rev 1990; 114:67-108. 569. Patarroyo M. Adhesion molecules mediating recruitment of monocytes to inflamed tissue. Immunobiology 1994; 191(4-5):474-7. 570. Munro JM. Endothelial-leukocyte adhesive interactions in inflammatory diseases. Eur Heart J 1993; 14(Suppl K):72-7. 571. Magnuson DK, Maier RV, Pohlman TH. Protein kinase C: A potential pathway of endothelial cell activation by endotoxin, tumor necrosis factor, and interleukin-1. Surgery 1989; 106(2) :216-22, (discussion 222-3). 572. Miura S, Tsuzuki Y, Kurose I et al. Endotoxin stimulates lymphocyte-endothelial interactions in rat intestinal Peyer's patches and villus mucosa. Am J Physiol 1996; 271(2 Pt l):G282-92. 573. Briscoe DM, Cotran RS, Pober JS. Effects of tumor necrosis factor, lipopolysaccharide, and IL-4 on the expression of vascular cell adhesion molecule-1 in vivo. Correlation with CD3+ T cell infiltration. J Immunol 1992; l49(9):2954-60. 574. Dohlsten M, Hedlund G, Lando PA et al. Role of the adhesion molecule ICAM-1 (CD54) in staphylococcal enterotoxin-mediated cytotoxicity. Eur J Immunol 1991; 21(l):131-5. 575. Jarousseau AC, Thibault G, Reverdiau P et al. Adhesive properties of choriocarcinoma cells toward lymphocytes activated or not by interleukin-2. Cell Immunol 1994; 157(l):38-47. 576. Piali L, Fichtel A, Terpe HJ et al. EndotheHal vascular cell adhesion molecule 1 expression is suppressed by melanoma and carcinoma. J Exp Med 1995; 181:811-816. 577. SUgh Jr JE, Ballantyne CM, Rich SS et al. Inflammatory and are in intercellular adhesion molecule 1. Proc Nad Acad Sci USA 1993; 90(18):8529-33. 578. Schmittel A, Scheibenbogen C, Keilholz U. Lipopolysaccharide effectively up-regulates B7-1 (CD80) expression and costimulatory function of human monocytes. Scand J Immunol 1995; 42(6) :701-4. 579. Zaitseva M, Golding H, Manischewitz J et al. Brucella abortus as a potential vaccine candidate: Induction of interleukin-12 secretion and enhanced B7.1 and B7.2 and intercellular adhesion molecule 1 surface expression in elutriated human monocytes stimulated by heat-inactivated B. abortus. Infect Immun 1996; 64(8):3109-17. 580. Pohlman TH, Harlan JM. Human endotheUal cell response to lipopolysaccharide, interleukin-1, and tumor necrosis factor is regulated by protein synthesis. Cell Immunol 1989; 119:41-52. 581. Haverstick DM, Gray LS. Lymphocyte adhesion mediated by lymphocyte function-associated antigen-1. I. Long term augmentation by transient increases in intracellular cAMP. J Immunol 1992a; l49(2):389-96. 582. Galea P, Thibault G, Lacord M et al. IL-4, but not tumor necrosis factor-a, increases endothelial cell adhesiveness for lymphocytes by activating a cAMP-dependent pathway. J Immunol 1993; 151:588-596. 583. Haverstick DM, Gray LS. Lymphocyte adhesion mediated by lymphocyte function-associated antigen-1. II. Interaction between phorbol ester- and cAMP-sensitive pathways. J Immunol 1992b; l49(2):397-402. 584. Eissner G, Kolch W, Mischak H et al. Differential role of protein kinase C in cytokine induced lymphocyte-endothehum interaction in vitro. Scand J Immunol 1994; 40(4):395-402. 585. Lanier LL. Distribution and function of lymphocyte surface antigens. Molecules costimulating T lymphocyte activation and effector function. Ann NY Acad Sci 1993; 677:86-93. 586. Li GC, Mivechi NF, Weitzel G. Heat shock proteins, thermotolerance, and their relevance to clinical hyperthermia. Int J Hyperthermia 1995; ll(4):459-88. 587. Kobayashi T, Shiozaki H, Shimano T et al. Analysis of cytotoxic activity of the CD4+ T lymphocytes generated by local immunotherapy. Br J Cancer 1996; 73(1): 110-6. 588. Itoh Y, Koshita Y, Takahashi M et al. Characterization of tumor-necrosis-factor-gene-transduced tumor-infiltrating lymphocytes from ascitic fluid of cancer patients: Analysis of cytolytic activity, growth rate, adhesion molecule expression and cytokine production. Cancer Immunol Immunother 1995; 40(2):95-102. 589. Li R, Nortamo P, Kantor C et al. A leukocyte integrin binding peptide from intercellular adhesion molecule-2 stimulates T cell adhesion and natural killer cell activity. J Biol Chem 1993; 268(29):2l474-7. 590. Somersalo K, Carpen O, Saksela E et al. Activation of natural killer cell migration by leukocyte integrin-binding peptide from intracellular adhesion molecule-2 (ICAM-2). J Biol Chem 1995; 270(15):8629-36. 591. Scott CF, Bolender S, Mclntyre GD et al. Activation of human cytolytic cells through CD2/T11. Comparison of the requirements for the induction and direction of lysis of tumor targets by T cells and NK cells. J Immunol 1989; 142:4105-4112.

Fever, Pyrogens and Cancer

331

592. Saito H, Kurose I, Ebinuma H et al. Kupffer cell-mediated q^otoxicity against hepatoma cells occurs through production of nitric oxide and adhesion via ICAM-1/CD18. Int Immunol 1996; 8(7):1165-72. 593. Todd Illrd RF, Arnaout MA, Rosin RE et al. Subcellular localization of the large subunit of M o l (Mol alpha; formerly gp 110), a surface glycoprotein associated with neutrophil adhesion. J Clin Invest 1984; 74(4): 1280-90. 594. Diamond MS, Springer TA. The dynamic regulation of integrin adhesiveness. Curr Biol 1994; 4(6):506-17. 595. Hershkoviz R, Alon R, Mekori YA et al. Heat-stressed CD4+ T lymphocytes: Differential modulations of adhesiveness to extracellular matrix glycoproteins, proliferative responses and tumour necrosis factor-a secretion. Immunol 1993; 79:241-247. 596. Schadendorf D, Diehl S, Zuberbier T et al. Quantitative detection of soluble adhesion molecules in sera of melanoma patients correlates with clinical stage. Dermatology 1996; 192(2):89-93. 597. Endo S, Inada K, Kasai T et al. Levels of soluble adhesion molecules and cytokines in patients with septic multiple organ failure. J Inflamm 1995-96; 46(4) :212-9. 598. Musiani P, Modesti A, Giovarelli M et al. Cytokines, tumour-cell death and immunogenicity: A question of choice. Immunol Today 1997; 18(l):32-6. 599. Duff G W . Oppenheim JJ. Comparative aspects of host-parasite and host-tumor relationships. Cytokine 1992; 4:331-339. 600. Blankenstein T, Rowley DA, Schreiber H. Cytokines and cancer: Experimental systems. Curr Opinion Immunol 1991; 3:694-698. 601. Haas GP, Redman BG, Rao VK et al. Immunotherapy for metastatic renal cell cancer: Effect on the primary tumor. J Immunother 1993; 13:130-135. 602. Hock H, Dorsch M, Kunzendorf U et al. Mechanisms of rejection induced by tumor cell-targeted gene transfer of Interleukin 2, Interleukin 4, Interleukin 7, tumor necrosis factor, or interferon gamma. Proc N a d Acad Sci USA 1993; 90:2774-2778. 603. van der Schelling GP, Ijzermans JN, Marquet RL et al. Cytokines as immunotherapy in cancer. Ned Tijdschr Geneeskd 1992; 136(14):681-5. 604. Hill AD, Redmond HP, Croke D T et al. Cytokines in tumor dierapy. Br J Surg 1992; 79(10):990-7. 605. Holmlund JT. Cytokines. Cancer Chemother Biol Response Modif 1993; 14:150-206. 606. Kershaw M H , Trapani JA, Smyth MJ. Cytotoxic lymphocytes: Redirecting the cell-mediated immune response for the therapy of cancer. Ther Immunol 1995; 2(3):173-81. 607. Goey SH, Verweij J, Stoter G. Immunotherapy of metastatic renal cell cancer. Ann Oncol 1996; 7(9):887-900. 608. Kruit W H , Stoter G. The role of adoptive immunotherapy in solid cancers. Neth J Med 1997; 50(2):47-68. 609. Bubenik J. Cytokine gene-modified vaccines in the therapy of cancer. Pharmacol Ther 1996; 69(1):1-14. 610. Shieh J H , Peterson RH, Moore MA. Bacterial endotoxin regulation of cytokine receptors on murine bone marrow cells: In vivo and in vitro study. J Immunol 1994; 152(2):859-66. 611. Pace JL, Taffet SM, Russell SW. The effect of endotoxin in eUciting agents on the activation of mouse macrophages for tumor cell killing. J Reticuloendothel Soc 1981; 30(1): 15-21. 612. Pace JL, Russell SW, LeBlanc PA et al. Comparative effects of various classes of mouse interferons on macrophage activation for tumor cell kiUing. J Immunol 1985; 134(2):977-81. 613. Bjork L, Andersson J, Ceska M et al. Endotoxin and Staphylococcus aureus enterotoxin A induce different patterns of cytokines. Cytokine 1992; 4(6):513-9. 614. SchUevert PM, Bohach GA, Ohlendorf D H et al. Molecular structure of staphylococcus and streptococcus superantigens. J Clin Immunol 1995; 15(6 Suppl):4S-10S. 615. Fast DJ, Schlievert PM, Nelson RD. Toxic shock syndrome-associated staphylococcal and streptococcal pyrogenic toxins are potent inducers of tumor necrosis factor production. Infection Immunity 1989; 57:291-294. 616. Fields BA, Malchiodi EL, Li H et al. Crystal structure of a T-cell receptor beta-chain complexed with a superantigen. Nature 1996; 384:188-192, (See also comment: Nature 1996; 384:109-110). 617. Shimizu M, Yamamoto A, Nakano H et al. Augmentation of antitumor immunity with bacterial superantigen, staphylococcal enterotoxin B-bound tumor cells. Cancer Res 1996; 56(l6):3731-6. 618. Riesenfeld-Orn I, Wolpe S, Garcia-Bustos JF et al. Production of interleukin-1 but not tumor necrosis factor by human monocytes stimulated with pneumococcal cell surface components. Infect Immun 1989; 57(7):1890-3. 619. Beeson PB. Tolerance to bacterial pyrogens. I. Factors influencing its development. J Exp Med 1947; 86:29-38.

332

Hyperthermia in Cancer Treatment: A Primer

620. Thomas L. The physiological disturbances produced by endotoxins. Ann Rev Physiol 1954; 16:467-490. 621. Greisman SE, DuBuy B. Mechanisms of endotoxin tolerance. DC. Effect of exchange transfusion. Proc Soc Exp Biol Med 1975; l48(3):675-8. 622. Greisman SE, Hornick RB. The nature of endotoxin tolerance. Trans Am Clin Climatol Assoc 1975; 86:43-50. 623. Greisman SE, Hornick RB. Endotoxin tolerance. In: Beers Jr RF, Basset E, eds. The role of immunological factors in infectious, allergic, and autoimmune processes. New York: Raven Press, 1976:43-50, (W3 MI543 no.8). 624. Mengozzi M, Ghezzi P. Cytokine down-regulation in endotoxin tolerance. Eur Cytokine Netw 1993; 4(2):89-98. 625. Lindberg AA, Greisman SE, Svenson SB. Induction of endotoxin tolerance with nonpyrogenic O-antigenic oligosaccharide-protein conjugates. Infect Immun 1983; 4l(3):888-95. 626. Johnson CA, Greisman SE. Mechanisms of endotoxin tolerance. In: Hinshaw LB, ed. Handbook of Endotoxin. Vol 2: Pathophysiology of Endotoxin. Amsterdam: Elsevier Science PubUshers BV, 1985:359-401. 627. Williams JF. Induction of tolerance in mice and rats to the effect of endotoxin to decrease the hepatic microsomal mixed-function oxidase systtm. Evidence for a possible macrophage-derived factor in the endotoxin effect. Int J Immunopharmacol 1985; 7(4):501-9. 628. Madonna GS, Vogel SN. Induction of early-phase endotoxin tolerance in athymic (nude) mice, B-cell-deficient (xid) mice, and splenectomizcd mice. Infect Immun 1986; 53(3):707-10. 629. Freudenberg MA, Galanos C. Induction of tolerance to lipopolysaccharide (LPS)-D-galactosamine lethality by pretreatment with LPS is mediated by macrophages. Infect Immun 1988; 56(5): 1352-7. 630. Haas JG, Thiel C, Blomer K et al. Downregulation of tumor necrosis factor expression in the human Mono-Mac-6 cell Hne by Hpopolysaccharide. J Leukoc Biol 1989; 46(l):ll-4. 631. Zuckerman SH, Evans GF, Buder LD. Endotoxin tolerance: Independent regulation of interleukin-l and tumor necrosis factor expression. Infect Immun 1991; 59(8):2774-80. 632. Roth J, McClellan JL, Kluger MJ et al. Attenuation of fever and release of cytokines after repeated injections of lipopolysaccharide in guinea-pigs. J Physiol (Lond) 1994; 477(Pt 1): 177-85. 633. Takasuka N, Tokunaga T, Akagawa KS. Preexposure of macrophages to low doses of lipopolysaccharide inhibits the expression of tumor necrosis factor-alpha mRNA but not of IL-1 beta mRNA. J Immunol 1991; 146(11):3824-30. 634. Ziegler-Heitbrock HW, Blumenstein M, Kafferlein E et al. In vitro desensitization to lipopolysaccharide suppresses tumour necrosis factor, interleukin-l and interleukin-6 gene expression in a similar fashion. Immunology 1992; 75(2):264-8. 635. Zhang X, Morrison DC. Lipopolysaccharide-induced selective priming effects on tumor necrosis factor alpha and nitric oxide production in mouse peritoneal macrophages. J Exp Med 1993a; 177(2):511-6. 636. Mathison JC, Virca GD, Wolfson E et al. Adaptation to bacterial lipopolysaccharide controls lipopolysaccharide-induced tumor necrosis factor production in rabbit macrophages. J Clin Invest 1990; 85(4):1108-18. 637. LaRue KEA, McCall CE. A labile transcriptional repressor modulates endotoxin tolerance. J Exp Med 1994; 180:2269-2275. 638. Deitch EA, Specian RD, Berg RD. Induction of early-phase tolerance to endotoxin-induced mucosal injury, xanthine oxidase activation and bacterial translocation by pretreatment with endotoxin. Circulatory Shock 1992; 36:208-216. 639. Patton JS, Peters PM, McCabe J et al. Development of partial tolerance to the gastrointestinal effects of high doses of recombinant tumor necrosis factor-a in rodents. J Clin Invest 1987; 80:1587-1596. 640. Vogel SN, Kaufman EN, Tate MD et al. Recombinant interleukin-l alpha and recombinant tumor necrosis factor alpha synergize in vivo to induce early endotoxin tolerance and associated hematopoietic changes. Infect Immun 1988; 56(10):2650-7. 641. Gorgen I, Hartung T, Leist M et al. Granulocyte colony-stimulating factor treatment protects rodents against lipopolysaccharide-induced toxicity via suppression of systemic tumor necrosis fictor-alpha. J Immunol 1992; l49(3):918-24. 642. Erroi A, Fantuzzi G, Mengozzi M et al. Differential regulation of cytokine production in lipopolysaccharide tolerance in mice. Infect Immun 1993; 61(10):4356-9. 643. Mengozzi M, Fantuzzi G, Sironi M et al. Early down-regulation of TNF production by LPS tolerance in human monocytes: Comparison with IL-1 beta, IL-6, and IL-8. Lymphokine Cytokine Res 1993; 12(4):231-6.

FeveVy Pyrogens and Cancer

333

644. Wakabayashi G, Cannon JG, Gelfand JA et al. Altered interleukin-1 and tumor necrosis factor production and secretion during pyrogenic tolerance to LPS in rabbits. Am J Physiol 1994; 267(1 Pt 2):R329-36. 645. Takahashi N, Fiers W, Brouckaert P. Anti-tumor activity of tumor necrosis factor in combination with interferon-gamma is not affected by prior tolerization. Int J Cancer 1995a; 63(6):846-54. 6AG. Takahashi N, Brouckaert P, Fiers W. Mechanism of tolerance to tumor necrosis factor: Receptor-specific pathway and selectivity. Am J Physiol 1995b; 269(2 Pt 2):R398-405. 647. Zhang X, Morrison DC. Lipopolysaccharide structurefiinction relationship in activation versus reprogramming of mouse peritoneal macrophages. J Leukoc Biol 1993b; 54(5):444-50. 648. Hirohashi N, Morrison DC. Low-dose lipopolysaccharide (LPS) pretreatment of mouse macrophages modulates LPS-dependent interleukin-6 produaion in vitro. Infect Immun 1996; 64(3): 1011-5. 649. Zhang X, Morrison DC. Pertussis toxin-sensitive factor differentially regulates lipopolysaccharideinduced tumor necrosis factor-alpha and nitric oxide production in mouse peritoneal macrophages. J Immunol 1993c; 150(3):1011-8. 650. Evans GF, Zuckerman SH. Glucocorticoid-dependent and -independent mechanisms involved in lipopolysaccharide tolerance. Eur J Immunol 1991; 21(9): 1973-9. 651. Lazar G, Agarwal MK. The influence of a novel glucocorticoid antagonist on endotoxin lethality in mice strains. Biochem Med Metab Biol 1986; 36(l):70-4. 652. Bertini R, Bianchi M, Ghezzi P. Adrenalectomy sensitizes mice to the lethal effects of interleukin 1 and tumor necrosis factor. J Exp Med 1988; 167(5):1708-12. 653. Beuder B, Krochin N, Milsark IW et al. Control of cachectin (tumor necrosis factor) synthesis: Mechanisms of endotoxin resistance. Science 1986; 232(4753):977-80. 654. Waage A, Slupphaug G, Shalaby R. Glucocorticoids inhibit the production of IL6 from monocytes, endothelial cells andfibroblasts.Eur J Immunol 1990; 20(ll):2439-43. 655. Parente L, Di Rosea M, Flower RJ et al. Relationship between the anti-phospholipase and anti-inflammatory effects of glucocorticoid-induced proteins. Eur J Pharmacol 1984; 99(2-3):233-9. 656. Radomski MW, Palmer RM, Moncada S. Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells. Proc Natl Acad Sci USA 1990; 87(24): 10043-7. 657. Fish RE, Spitzer JA. Continuous infusion of endotoxin from an osmotic pump in the conscious, unrestrained rat: A unique model of chronic endotoxemia. Circ Shock 1984; 12(2):135-49. 658. Demling RH, Lalonde CC, Jin LJ et al. The pulmonary and systemic response to recurrent endotoxemia in the adult sheep. Surgery 1986; 100(5):876-83. 659. Schlievert PM, Bettin KM, Watson DW. Inhibition of ribonucleic acid synthesis by group A streptococcal pyrogenic exotoxin. Infect Immun 1980; 27(2):542-8. 660. Deitch EA, Berg R, Specian R. Endotoxin promotes the translocation of bacteria from the gut. Arch Surg 1987; 122(2): 185-90. 661. O'Dwyer ST, Michie HR, Ziegler TR et al. A single dose of endotoxin increases intestinal permeability in healthy humans. Arch Surg 1988; 123:1459-1464. 662. Deitch EA, Ma L, Ma WJ et al. Inhibition of endotoxin-induced bacterial translocation in mice. J CHn Invest 1989; 84(l):36-42. 663. Deitch EA. The role of intestinal barrier failure and bacterial translocation in the development of systemic infection and multiple organ failure. Arch Surg 1990; 125(3):403-4. GCiA. Walker RI. The contribution of intestinal endotoxin to mortality in hosts with compromised resistance: A review. Exp Hematol 1978; 6(2): 172-84. 665. Lundqvist C, Melgar S, Yeung MM et al. Intraepithelial lymphocytes in human gut have lytic potential and a cytokine profile that suggest T helper 1 and cytotoxic functions. J Immunol 1996; 157(5):1926-34. G66. Winchurch RA, Thupari JN, Munster AM. Endotoxemia in burn patients: Levels of circulating endotoxins are related to burn size. Surgery 1987; 102(5):808-12. G67. Woodruff PW, O'Carroll DI, Koizumi S et al. Role of the intestinal flora in major trauma. J Infect Dis 1973; 128(Suppl):290-4. 668. Bahrami S, Redl H, Yao YM et al. Involvement of bacteria/endotoxin translocation in the development of multiple organ failure. Curr Top Microbiol Immunol 1996; 216:239-58. 669. Carrico CJ, Meakins JL, Marshall JC et al. Multiple-organ-failure syndrome. Arch Surg 1986; 121(2):196-208. 670. In: Kosaka M, Suguhara T, Schmidt KL et al, eds. Thermotherapy for Neoplasia, Inflammation, and Pain. Tokyo: Springer, 2001. 671. Burd, Dziedzic, Yan Xu et al. Tumor cell apoptosis, lymphocyte recruitment and tumor vascular changes are induced by low temperature, long duration (fever-like) whole body hyperthermia. J Cell Pysiol 1998; 177:137-147.

334

Hyperthermia in Cancer Treatment: A Primer

672. Ardenne M von. Spontaneous remission of tumors following hyperthermia - a feedback process? Naturwissenschaften 1965; 52(23):645. 673. Ardenne M von, Chaplain RA, Reitnauer PG. In vivo studies on cancer multiple-step therapy using the attack combination of optimum tumor overacidification, hyperthermia and weak X-irradiation. Dtsch Gesundheitsw 1969; 24(20):924-35. 674. Ardenne M von, Reitnauer PG. Measurements on selective damage to cancer cells in vitro by attack-combination with hyperacidification plus 40 degree C hyperthermia and various bile acids with favorable pH. Arzneimittelforschung 1970; 20(3):323-9. 675. Ardenne M von, Rieger F. On the present state of extreme total-body hyperthermia as element in the cancer therapy. Z Krebsforsch Klin Onkol Cancer Res Clin Oncol 1967; 69(4) :341-4. (Ardenne MV. Synergic therapeutic effect of selective local hyperthermia and selective optimized hyperacidity against tumors. Theoretical and experimental bases. Ther Ggw 1977 Jul; 116(7): 1299-316). 676. Kirsch R, Schmidt D, Fichler J et al. Problems of multiple step-therapy of carcinoma. II. Effect of hyperthermia on cancer tissue. Dtsch Gesundheitsw 1967a; 22(l6):732-5. 677. Kirsch R, Schmidt D, Schmidt H. Problems of multiple step-therapy of carcinoma. I. On the history of hyperthermic treatment. Dtsch Gesundheitsw 1967b; 22(15):678-81, (contd). 678. Pritchard MT, Ostberg JR, Evans SS et al. Protocols for simulating the thermal component of fever: PrecHnical and clinical experience. Methods 2004; 32(l):54-62. 679. Yonezawa M, Otsuka T, Matsui N et al. Hyperthermia induces apoptosis in malignant fibrous histiocytoma cells in vitro. Int J Cancer 1996; 66(3):347-51. 680. Ensor JE, Wiener SM, McCrea KA et al. Differential effects of hyperthermia on macrophage interleukin-6 and tumor necrosis factor-alpha expression. Am J Physiol 1994; 266(4 Pt l):C967-74. 681. Shen RN, Lu L, Young P et al. Influence of elevated temperature on natural killer cell activity, lymphokine-activated killer cell activity and lectin-dependent cytotoxicity of human umbilical cord blood and adult blood cells. Int J Radiat Oncol Biol Phys 1994; 29(4):821-6. 682. Zanker KS, Lange J. Whole body hyperthermia and natural killer cell activity (letter). Lancet 1982; l(8280):1079-80. 683. Park MM, Hornback NB, Endres S et al. The effect of whole body hyperthermia on the immune cell activity of cancer patients. Lymphokine Res 1990(Summer); 9(2):213-23. 684. Haranaka K, Sakurai A, Satomi N. Antitumor activity of recombinant human tumor necrosis factor in combination with hyperthermia, chemotherapy, or immunotherapy. J Biol Response Mod 1987a; 6:379-391. 685. Haranaka K, Satomi N, Sakurai A et al. Antitumour effects of tumour necrosis factor: Cytotoxic or necrotizing activity and its mechanism. Ciba Found Symp 1987b; 131:140-53. 686. van der Zee J, van den Aardweg GJ, van Rhoon GC et al. Br J Cancer 1995; 71(6):1158-62. 687. Strauch ED, Fabian DF, Turner J et al. Combined hyperthermia and immunotherapy treatment of multiple pulmonary metastases in mice. Surg Oncol 1994; 3(l):45-52. 688. Kappel M, Tvede N, Hansen MB et al. Cytokine production ex vivo: Effect of raised body temperature. Int J Hyperthermia 1995; ll(3):329-35. 689. Robins HI, Kutz M, Wiedemann GJ et al. Cytokine induction by 41.8 degrees C whole body hyperthermia. Cancer Lett 1995b; 97(2): 195-201. 690. Blake D, Bessey P, Karl I et al. Hyperthermia induces IL-1 alpha but does not decrease release of IL-1 alpha or TNF-alpha after endotoxin. Lymphokine Cytokine Res 1994; 13(5):271-5. 691. Multhoff G, Botzler C, Meier T et al. Proceedings of the fourth International Meeting of Heat Shock Response. Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor, 1994:330. 692. Parsell DA, Lindquist S. Heat shock proteins and stress tolerance. The biology of heat shock proteins and molecular chaperones. Cold Spring Harbor Laboratory Press, 1994a. 693. Benndorf R, Bielka H. Cellular stress response: Stress proteins—physiology and implications for cancer. Recent Results Cancer Res 1997; 143:129-44. 694. Fuller KJ, Issels RD, Slosman DO et al. Cancer and the heat shock response. Eur J Cancer 1994; 30A(12):1884-91. 695. Ferrarini M, Heltai S, Zocchi MR et al. Unusual expression and localization of heat-shock proteins in human tumor cells. Int J Cancer 1992; 51(4):6l3-9. 696. Zhang YH, Takahashi K, Jiang GZ et al. In vivo production of heat shock protein in mouse peritoneal macrophages by administration of lipopolysaccharide. Infect Immun 1994b; 62(10):4140-4. 697. Hirvonen MR, Brune B, Lapetina EG. Heat shock proteins and macrophage resistance to the toxic effects of nitric oxide. Biochem J 1996; 315(Pt 3):845-9. 698. Seitz CS, Kleindienst R, Xu Qet al. Coexpression of heat-shock protein 60 and intercellular-adhesion molecule-1 is related to increased adhesion of monocytes and T cells to aortic endothelium of rats in response to endotoxin. Lab Invest 1996; 74(l):24l-52.

Fevery Pyrogens and Cancer

335

699. Deitch EA, Beck SC, Cruz NC et al. Induction of heat shock gene expression in colonic epithelial cells after incubation with Escherichia coU or endotoxin. Crit Care Med 1995; 23(8): 1371-6. 700. Dressel R, Heine L, Eisner L et al. Induction of heat shock protein 70 genes in human lymphocytes during fever therapy. Eur J Clin Invest 1996; 26(6):499-505. 701.Jindal S. Heat shock proteins: Applications in health and disease. Trends Biotechnol 1996; l4(l):17-20. 702. Fracella F, Rensing L. Stress proteins: their growing significance in medicine. Naturwissenschaften 1995; 82(7):303-9. 703. Parsell DA, Kowal AS, Singer MA et al. Protein disaggregation mediated by heat-shock protein Hspl04. Nature 1994b; 372(6505):475-8. 704. Srivastava PK. Protein tumor antigens. Curr Opin Immunol 1991; 3(5):654-8. 705. Blachere NE, Udono H, Janetzki S et al. Heat shock protein vaccines against cancer. J Immunother 1993; l4(4):352-6. 706. Suto R, Srivastava PK. A mechanism for the specific immunogenicity of heat shock proteinchaperoned peptides. Science 1995; 269(5230):1585-8. 707. Burdon RH. The heat shock proteins. Endeavour 1988; 12(3):133-8. 708. Zimarino V, Wu C. Induction of sequence-specific binding of Drosophila heat shock activator protein without protein synthesis. Nature 1987; 327(6124):727-30. 709. Wiederrecht G, Shuey DJ, Kibbe WA et al. The Saccharomyces and Drosophila heat shock transcription factors are identical in size and DNA binding properties. Cell 1987; 48(3):507-15. 710. Giaccia AJ, Auger EA, Koong A et al. Activation of the heat shock transcription factor by hypoxia in normal and tumor cell lines in vivo and in vitro. Int J Radiat Oncol Biol Phys 1992; 23(4):891-7. 711. Hauser GJ, Dayao EK, Wasserloos K et al. HSP induction inhibits iNOS mRNA expression and attenuates hypotension in endotoxin-challenged rats. Am J Physiol 1996; 271(6 Pt 2):H2529-35. 712. de Vera ME, Wong JM, Zhou JY et al. Cytokine-induced nitric oxide synthase gene transcription is blocked by the heat shock response in human liver cells. Surgery 1996; 120(2): 144-9. 713. Feinstein DL, Galea E, Aquino DA et al. Heat shock protein 70 suppresses astroglial-inducible nitric-oxide synthase expression by decreasing NFkappaB activation. J Biol Chem 1996; 271(30):17724-32. 714. Wong HR, Mannix RJ, Rusnak JM et al. The heat-shock response attenuates lipopolysaccharidemediated apoptosis in cultured sheep pulmonary artery endothelial cells. Am J Respir Cell Mol Biol 1996; 15(6):745-51. 715. Chi SH, Mestril R. Stable expression of a human HSP70 gene in a rat myogenic cell line confers protection against endotoxin. Am J Physiol 1996; 270(4 Pt 1):C1017-21. 716. Snyder YM, Guthrie L, Evans GF et al. Transcriptional inhibition of endotoxin-induced monokine synthesis following heat shock in murine peritoneal macrophages. J Leukoc Biol 1992; 51(2): 181-7. 717. Ribeiro SP, Villar J, Downey GP et al. Effects of the stress response in septic rats and LPS-stimulated alveolar macrophages: Evidence for TNF-alpha posttranslational regulation. Am J Respir Crit Care Med 1996; 154(6 Pt l):1843-50. 718. Yoshida K, Maaieh MM, Shipley JB et al. Monophosphoryl Hpid A induces pharmacologic 'preconditioning' in rabbit hearts without concomitant expression of 70-kDa heat shock protein. Mol Cell Biochem 1996; 159(l):73-80. 719. SHutz G, Karlseder J, Tempfer C et al. Drug resistance against gemcitabine and topotecan mediated by constitutive hsp70 overexpression in vitro: Implication of quercetin as sensitiser in chemotherapy. Br J Cancer 1996; 74(2): 172-7. 720. Hotchkiss R, Nunnally I, Lindquist S et al. Hyperthermia protects mice against the lethal effects of endotoxin. Am J Physiol 1993; 265(6 Pt 2):Rl447-57. 721. Maeda H, Molla A, Oda T et al. Internalization of serratial protease into cells as an enzyme-inhibitor complex with alpha 2-macroglobulin and regeneration of protease activity and cytotoxicity. J Biol Chem 1987; 262(23): 10946-50. 722. Oda T, Kojima Y, Akaike T et al. Inactivation of chemotactic activity of C5a by the serratial 56-kilodalton protease. Infect Immun 1990; 58(5): 1269-72. 723. Legres LG, Pochon F, Barray M et al. Evidence for the binding of a biologically active interleukin-2 to human alpha 2-macroglobunn. J Biol Chem 1995; 270(15):8381-4. 724. Heumann D, Vischer TL. Immunomodulation by alpha 2-macroglobulin and alpha 2-macroglobulin-proteinase complexes: The effect on the human T lymphocyte response. Eur J Immunol 1988; 18(5):755-60. 725. Petersen CM, Ejlersen E, Moestrup SK et al. Immunosuppressive properties of electrophoretically "slow" and "fast" form alpha 2-macroglobulin. Effects on cell-mediated cytotoxicity and (alio-) antigen-induced T cell proUferation. J Immunol 1989; l42(2):629-35.

336

Hyperthermia in Cancer Treatment: A Primer

llCt. Borth W, Teodorescu M. Inactivation of human interleukin-2 (IL-2) by alpha 2-macroglobuhn-trypsin complexes. Immunology 1986; 57(3):367-71. 727. Wollenberg GK, LaMarre J, Rosendal S et al. Binding of tumor necrosis factor alpha to activated forms of human plasma alpha 2 macroglobulin. Am J Pathol 1991a; 138(2):265-72. 728. O'Connor-McCourt MD, Wakefield LM. Latent transforming growth factor-beta in serum. A specific complex with alpha 2-macroglobulin. J Biol Chem 1987; 262(29): 14090-9. 729. Danielpour D, Sporn MB. Differential inhibition of transforming growth factor beta 1 and beta 2 activity by alpha 2-macroglobulin. J Biol Chem 1990; 265(12):6973-7. 730. LaMarre J, Wollenberg GK, Gauldie J et al. Alpha 2-macroglobulin and serum preferentially counteract the mitoinhibitory effect of transforming growth factor-beta 2 in rat hepatocytes. Lab Invest 1990; 62(5):545-51. 731. Hall SW, LaMarre J, Marshall LB et al. Binding of transforming growth factor-beta 1 to methylamine-modified alpha 2-macroglobulin and to binary and ternary alpha 2-macroglobuhnproteinase complexes. Biochem J 1992; 281(Pt 2):569-75. 732. Huang JS, Huang SS, Deuel TF. Human platelet-derived growth factor: Radioimmunoassay and discovery of a specific plasma-binding protein. J Cell Biol 1983; 97(2):383-8. 733. Huang JS, Huang SS, Deuel TF. Specific covalent binding of platelet-derived growth factor to human plasma alpha 2-macroglobulin. Proc Nad Acad Sci USA 1984; 81(2):342-6. 734. Borth W, Luger TA. Identification of alpha 2-macroglobulin as a cytokine binding plasma protein. Binding of interleukin-1 beta to "F" alpha 2-macroglobulin. J Biol Chem 1989; 264(10):5818-25. 735. Legres LG, Pochon F, Barray M et al. Human alpha 2-macroglobulin as a cytokine-binding plasma protein. A study with rh-interleukin-1 beta and rh-interleukin-6. Ann NY Acad Sci 1994; 737:439-43. 736. Matsuda T, Hirano T, Nagasawa S et al. Identification of alpha 2-macroglobulin as a carrier protein for IL-6. J Immunol 1989; 142(l):148-52. 737. James K. Interactions between cytokines and alpha 2-macroglobulin (see comments). Immimol Today 1990; ll(5):l63-6. 738. Borth W. Alpha 2-macroglobulin. A multifunctional binding and targeting protein with possible roles in immunity and autoimmunity. Ann NY Acad Sci 1994; 737:267-72. 739. James K, van den Haan J, Lens S et al. Preliminary studies on the interaction of TNF alpha and IFN gamma with alpha 2-macroglobulin. Immunol Lett 1992; 32(l):49-57. 740. Barrett AJ, Starkey PM. The interaction of alpha 2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. Biochem J 1973; 133(4):709-24. 741. Starkey PM, Barrett AJ. Inhibition by alpha-macroglobulin and other serum proteins. Biochem J 1973; 131(4):823-31. 742. Rinderknecht H, Carmack C, Geokas MC. Effect of specific antibodies and a2-macroglobuhn on emzymatic activity of trypsin and chymotrypsin. Immunochemistry 1975; 12(l):l-8. 743. Delain E, Barray M, Tapon-Bretaudiere J et al. The molecular organization of human alpha 2-macroglobulin. An immunoelectron microscopic study with monoclonal antibodies. J Biol Chem 1988; 263(6):2981-9. 744. LaMarre J, Wollenberg GK, Gonias SL et al. Cytokine binding and clearance properties of proteinase-activated alpha 2-macroglobulins. Lab Invest 1991a; 65(1):3-14. 745. Feldman SR, Rosenberg MR, Ney KA et al. Binding of alpha 2-macroglobuHn to hepatocytes: Mechanism of in vivo clearance. Biochem Biophys Res Commun 1985; 128(2):795-802. 746. Kaplan J, Nielsen ML. Analysis of macrophage surface receptors. II. Internalization of alpha-macroglobulin trypsin complexes by rabbit alveolar macrophages. J Biol Chem 1979a; 254(15):7329-35. 747. Kaplan J, Nielsen ML. Analysis of macrophage surfece receptors. I. Binding of alpha-macroglobulin protease complexes to rabbit alveolar macrophages. J Biol Chem 1979b; 254(15):7323-8. 748. Imber MJ, Pizzo SV. Clearance and binding of two electrophoretic "fast" forms of human alpha 2-macroglobulin. J Biol Chem 1981; 256(15):8134-9. 749. Van Leuven F, Cassiman JJ, Van Den Berghe H. Demonstration of an alpha2-macroglobulin receptor in humanfibroblasts,absent in tumor-derived cell lines. J Biol Chem 1979; 254(12):5155-60. 750. Dickson RB, Willingham MC, Pastan I. Binding and internalization of 1251-alpha 2-macroglobuHn by culturedfibroblasts.J Biol Chem 1981; 256(7):3454-9. 751. Niemuller CA, Randall KJ, Webb DJ et al. Alpha 2-macroglobulin conformation determines binding affinity for activin A and plasma clearance of activin A/alpha 2-macroglobulin complex. Endocrinology 1995; 136(12):5343-9. 752. Gonias SL, LaMarre J, Crookston KP et al. Alpha 2-macroglobulin and the alpha 2-macroglobulin receptor/LRP. A growth regulatory axis. Ann NY Acad Sci 1994; 737:273-90.

Fever, Pyrogens and Cancer

337

753. Van Leuven F, Marynen P, Sottrup-Jensen L et al. The receptor-binding domain of human alpha 2-macroglobuhn. Isolation after limited proteolysis with a bacterial proteinase. J Biol Chem 1986; 261 (24): 11369-73. 754. Cunningham AJ, Elliott SF, Black JR et al. A simple method for isolating alpha 2 macroglobulincytokine complexes. J Immunol Methods 1994; l69(2):287-92. 755. LaMarre J, Wolf BB, Kittler EL et al. Regulation of macrophage alpha 2-macroglobulin receptor/ low density lipoprotein receptor-related protein by lipopolysaccharide and interferon-gamma. J CUn Invest 1993; 91(3):1219-24. 756. Taveira Da Silva AM, Kaulbach H C , Chuidian FS et al. Brief report: Shock and multiple-organ dysfunction after self-administration of salmonella endotoxin. N Engl J Med 1993; 328:1457-1460. 757. Hager ED. Mikrobielle immunmodulatoren: Aktive fiebertherapie mit bakterientoxinen. In: Buehring M, Kemper F H , Matthiessen PF, eds. Naturheilverfahren und unkonventionelle medizinische Richtungen. Springer LoseblattSysteme, 1996. 758. Nowotny A, Behling U H . Studies on host defenses enhanced by endotoxins: A brief review. Klin Wochenschr 1982; 60(l4):735-9. 759. Cooper KE. Some responses of the cardiovascular system to heat and fever. Can J Cardiol 1994; 10(4):444-8. 760. Miller AB, Hoogstraten B, Staquet M et al. Reporting results of cancer treatment. Cancer 1981; 47:207-214. 761. Parr I, Wheeler EA, Alexander P. Similarities of the antitumor actions of endotoxin, Hpid A and double-stranded RNA. Br J Cancer 1973; 27:370-389. 762. Brouckaert P, Fiers W. Tumor necrosis factor and the systemic inflammatory response Syndrome. Curr Topics Microbiol Immunol 1996; 216:167-187. 763. Libert C, Van MoUe W, Brouckaert P et al. alpha 1-Antitrypsin inhibits the lethal response to T N F in mice. J Immunol 1996; 157(11):5126-9. 764. Alexander HR, Doherty C M , Buresh C M et al. A recombinant human receptor antagonist to interleukin 1 improves survival after lethal endotoxemia in mice. J Exp Med 1991a; 173(4):1029-32. 765. Alexander HR, Doherty C M , Block MI et al. Single-dose tumor necrosis factor protection against endotoxin-induced shock and tissue injury in rats. Infect Immun 1991b; 59(ll):3889-94. 7G6. Everaerdt B, Brouckaert P, Fiers W. Recombinant IL-1 receptor antagonist protects against TNF-induced lethality in mice. J Immunol 1994; 152:5041-5049. 767. Fukumura D, Miura S, Kurose I et al. IL-1 is an important mediator for microcirculatory changes in endotoxin-induced intestinal mucosal damage. Dig Dis Sci 1996; 4l(12):2482-92. 768. Maeda H, Akaike T, W u J et al. Bradykinin and nitric oxide in infectious disease and cancer. Immunopharmacology 1996; 33(l-3):222-30. 769. Kaplanski G, Teysseire N , Farnarier C et al. IL-6 and IL-8 production from cultured human endothelial cells stimulated by infection with Rickettsia conorii via a cell-associated IL-1 alpha-dependent pathway. J Clin Invest 1995; 96(6):2839-44. 770. Smith PD, Suffredini AF, Allen JB et al. Endotoxin administration to humans primes alveolar macrophages for increased production of inflammatory mediators. J Clin Immunol 1994; 14(2):l4l-8. 771. Friedman H, Butler RC, Specter S et al. Nontoxic endotoxin polysaccharide induces soluble mediators which potentiate antibody production by murine retrovirus-suppressed splenocytes. Int J Immunopharmacol 1988; 10(3):283-92. 772. Friedman H, Klein T, Specter S et al. Immunoadjuvanticity of endotoxins and nontoxic derivatives for normal and leukemic immunocytes. Adv Exp Med Biol 1990; 256:525-35. 773. Nowotny A, Keler T, Pham PH et al. Isolation of a nonendotoxic antitumor preparation from Serratia marcescens. J Biol Response Mod 1988; 7(3):296-308. 774. Parant M, Le Contel C, Parant F et al. Influence of endogenous glucocorticoid on endotoxin-induced production of circulating TNF-alpha. Lymphokine Cytokine Res 1991; 10(4):265-71. 775. Mengozzi M, Fantuzzi G, Faggioni R et al. Chlorpromazine specifically inhibits peripheral and brain T N F produaion, and up-regulates IL-10 production, in mice. Immunology 1994; 82:207-210. 776. LeMay LG, Vander AJ, Kluger MJ. The effects of pentoxifylline on lipopolysaccharide (LPS) fever, plasma interleukin 6 (IL-6), and tumor necrosis faaor (TNF) in the rat. Cytokine 1990c; 2(4):300-6. 777. Eisner J, Sach M, Knopf H P et al. Synthesis and surface expression of ICAM-1 in polymorphonuclear neutrophilic leukocytes in normal subjects and during inflammatory disease. Immunobiology 1995; 193(5):456-64. 778. Scher RL, Carras A, Schwab D et al. Interferon gamma enhances lymphokine-activated killer cell adhesion but not lysis of head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 1995; 121(11):1271-5. 779. Koyama S. Immunosuppressive effect of shedding intercellular adhesion molecule 1 antigen on cell-mediated cytotoxicity against tumor cells. Jpn J Cancer Res 1994; 85(2):131-4.

CHAPTER 22

Future Perspectives of Interstitial and Perfiisional Hyperthermia Gian Franco Baronzio,* Michele De Simone, Gianmaria Fiorentini, Salvatore D'Ai^lo, Giovanni Viscond and E. Dieter Hager Imagination is more important than knowledge —^A. Einstein

Abstract

R

ecent developments and perfusion hyperthermia have expanded the treatment optionsinofthermal patientsablation with certain cancers. Initially thermal ablation was applied to liver tumor; later its application has been extended to focal malignancies confined% in other organs such as: breast, kidney, adrenal glands, pancreas, bone, and lung. Metastases to localized organs, such as liver, lung, and pleura are a common event. The inoperable tumors (primary or metastatic) are generally treated by systemic chemotherapy; however toxicity is very high. Some clinicians have developed regional therapies to reduce this toxicity. Perfiisional therapy permits a higher concentration of antineoplastic agents in the tumor target. Furthermore the combination of hyperthermia with appropriate antineoplastic agents has demonstrated enhancement of the single therapy and reduction of toxicity. Lung, pleura and liver perfusion in combination with hyperthermia, will briefly be described here. This review is not exhaustive; its purpose is to illustrate the applications that we hope will become routine in cancer therapy in the near future.

Introduction Image-guided ablation therapy with radiofrequency (RFA/HiTT), microwave, high-intensity focused ultrasound (HIFU), laser (LiTT) etc. has gained increasing attention by researchers as a tool for treating focal malignancies.^'^ As outlined by Goldberg,^ the methods offer many advantages compared to surgical resection, such as: reduction of morbidity and mortality, low cost and ability to perform ablative procedure on an outpatient basis. Initially RFA was applied only to tumors confined to the liver, later its application has been extended to tumors at different sites in the body (see Table 1). Some inclusion criteria are common to all patients treated with thermal ablation and are listed in Table 2. Even complications following treatment may be common or specific and confined to the organ treated (see Table 3). Specific criteria of inclusion and complications for singular treated tumors will be discussed later.

•Corresponding Author: Gian Franco Baronzio—Family Medicine Area, ASL-01 Legnano; Radiotherapy Unit, Policlinico di Monza, Via Amati 11, 20052 Monza (Mi), Italy. Office address: P.O.B. 5, 20029 Turbigo (Mi), Italy. Email: [email protected]

Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.

Future Perspectives ofInterstitial and Perfusional Hyperthermia

339

Table 1. RFA Primitives and metastatic human solid tumors treated References Liver Kidney Breast Lung Bone Adrenals Spleen metastases Thyroid Pancreas Brain Uterus Prostate Lymph nodes

7,8 10-13 20-23 27-31,33,34 35-38,40 41 42 43 44 4,45 46 4,45 4,45

Table 2. Patient inclusion criteria • • • •

Patients with no resectable primary or metastatic cancer Patients who have failed to respond to other convectional therapies Possible Improvement in life Other co-morbid conditions: age, anaemia, cardiac or anaesthesia contraindications

Table 3. Common complications following RFA Complications: generally are < 5% • Post ablation syndrome • Thermal injury • Collateral visceral injuries

Technical Aspects Thermal tumor destruction can be obtained by using radiofrequency, microwave, laser or ultrasound under the continuous and direct visualization by US, CT or MRI.^' The aims of these therapies are similar: coagulation of the tumor by sparing adjacent healthy tissue. Clinically this last aim is not completely attainable; in fact, tumor margins of normal parenchyma, comprised between 0.5-1 cm are often included during the treatment together. ^'^ At first glance this can be thought negative, but practically it has an important consequence in reducing the risk of tumor regrowth. Cancer cells at the periphery of the tumor mass are generally more active and proliferating; this is due to metabolic favourable conditions. These cells, which are not completely killed by radiotherapy/and or chemotherapy and are infiltrating normal parenchyma, can restart tumor growth. The temperatures reached by the two techniques are generally > 45°C, in general 90 to lOO'C and the treatment lasts between 15-30 minutes. In this range of heat, coagulative necrosis is developed and tumor cells death begins to occur 4 to 5

340

Hyperthermia in Cancer Treatment: A Primer

Figure 1. Schematic drawing of thermal ablation with RP-needle technique. minutes at 60°C and more, rapidly at increasing temperature.^'^ Performing thermal ablation, the borders of the tumor are defined using non-invasive ultrasound or magnetic resonance imaging (MRI). Hence, a needle is inserted into the tumor mass for delivering heat (Fig. 1). The radiofrequency technique will be briefly described.

Radiofrequency Ablation (RFA) With radiofrequency ablation (RFA) a highfi-equencyalternating current (100 to 500kHz) is generally delivered to tissue via an electrode tip inserted percutaneously or with minimal surgery. The electromagnetic energy transmitted is converted to heat by generating ionic agitation. As ions try to change direction and follow the alternating current, localized Trictional energy' is created in the area surrounding the electrode tip. The heat generated is at its maximum near the electrode tip and dissipates rapidly with increasing distance. Five RFA devices, which work on the same principles, are available at the time worldwide. The five devices currently in use (RITA medical System; Radionics Inc. Tyco Healthcare, Radio therapeutics Inc., Celon AG (Olympus) and Elektrotom 106 HiTT Berchtold Medical Electronics) differ each other for the following aspects:

Future Perspectives ofInterstitial and Perfusional Hyperthermia

V

341

V

a) Cooled needle

VV-

b) Wet needle

c) Deployable tines

Figure 2. Schematic images of three different RF needle appUcators.

Table 4. Characteristics of various RFA devices Model

Radionics®

Rita®

Radiotherapeutics®

Power of generator (W)

200

50-150-250

90 - 200

Needle dimensions (G)

17.5

14-15

15

Form of the needle

Single with tip holes

Movable hub + 4-9 hooks

Movable hub +10 hooks

Diameter of tumor treated (cm)

3-5

7

4

Methods of temperature measurement

Present

Present

Absent

1. Power of the generator, 2. The gauge and geometry of the needle. 3. The electrical parameter monitored. 4. Cooling needle tip.^ The various characteristics of the three devices are illustrated in Figure 2 and Table 4. The needles are generally constituted by an insulated shaft and a distal conducting tip. RITA device consists of a 50 W alternating current generator and a 15-gauge electrode. The needle electrode has a movable hub and 8 retracting curved electrodes that are deployed from the tip of the needle after its positioning into the tumor. Each tips of the needle contain a thermocouple that can register the temperature in the heated area. Radionics needle consists of a straight needle with an internal channel. Inside this channel a saline solution circulates for cooling. Some holes are present for permitting the leakage of saline solution. ' ' In this case the saline solution is used to increases the tissue conductivity. ' The Radiotherapeutic

342

Hyperthermia in Cancer Treatment: A Primer

Table 5. Tumor diameter-tumor control Diameter

% Local Control Achievable

< 2cm 2.5-3.5 cm 3.5-5 cm > 5 cm

90% 70-90% 50-70% < 50%

device is similar to RITA device. The movable hub can deploy 10 needles creating a more spherical heat distribution. This device differendy than RITA device, does not have the temperature surveillance at the tips of needles.^'^The Electrotom 106 HiTT system consists of a single or dual insulated RF needle system with a non-insulated tip where the RF energy applied ranges from 20 to 50 W. At the proximal end, the needle has an electric connector for the RF cable and a luer lock for the rinsing solution. This rinsing solution (isotonic sodium chloride) cools the heated area at the tip to prevent the occurrence of vapour or charring tissue, which could lead to a bad conductivity. The Celon POWER system is the first bipolar and multipolar ablation system. With the bipolar technique the current is restricted to the area to be treated. The tumor masses treated are generally of the order of less than 3 cm of diameter. RITA and Radiotherapeutic systems, that have expandable multiple arrays, can reach tumor masses of < 5 cm.^' With local perfusion of saline solution also with single needle systems tumors larger than 3 cm can be treated. But, with increasing tumor diameter the rate of complete responses is decreasing rapidly. A relation between tumor diameter treated and the achievable local control has been studied when applied to liver tumors (see Table 5). Multiple needle applicators for radiofrequency or microwave ablation can increase the coagulation zone enabling a higher rate of complete necrosis. If the multiple needle array is connected as a bipolar module to each other, the densities of the electric fields will be increased resulting in larger coagtdation zones. Bipolar needle applicators significandy reduce treatment time and may lead to a reduction in local tumor recurrence by improved homogeneity of the necrotic area.

Liver Cancer The first experimental treatments with thermal ablation have been tested in patients with primary or metastasized liver tumors (this kind of treatment is elsewhere described in the book). Other tumors than liver tumors have been treated with RFA and MWA devices. Further indications for RFA applicators are tumors of the kidney, adrenal gland, pancreas, breast, thyroid, parathyroid, lymphoma, bone, and osteoma. A brief description of the different tumors treated with RFA follows.

Renal Cell Carcinoma For this kind of tumor the mainstay treatment is surgery, however when renal conservation is desired RFA provides an opportunity. ^° Conservation refers in primis on patients with unresectable tumors, singular kidney, non functioning kidney or bilateral recurrent tumors (von Hippel Lindau syndrome).^^ Renal tumors with a component in the renal sinus cannot be completely treated as demonstrated by Gervais.^^ Complications are generally not severe and include hemorrhage, ureteral stricture, and thermal injury to psoas muscle. No deaths were reported. ^^ The results of thermal ablation depend at least from two factors: tumor dimension (< 3 cm),^^ tumor location^"^'^^ and the power of the generator.^^ Rohde et

Future Perspectives ofInterstitial and Perjusional Hyperthermia

343

al^^ treated with RFA and cryoablation metastatic renal cell carcinoma. They concluded that for patients with low performance status this technique may become an elective and effective method of treatment. For a review of various clinical studies conducted until 2004 see Hines-Peralta^^ and Ogan.^^

Breast Cancer The advances on imaging devices have contributed to discover breast tumor masses at its beginning, with the consequence of reducing radical surgical interventions. The standard treatment has been radical mastectomy although actually a less surgical radical approach is used (lumpectomy) followed by radiation therapy. Notwithstanding these earlier interventions a non surgical approach with a less cosmetic defect is desirable. RFA is an ideal treatment at least for masses less than 1.5 cm, with negative sentinel lymph node and it is a useful preoperative antitumoral treatment in advanced breast carcinoma with or without chemotherapy.^^'^^ In animal models, the approach with chemotherapy, specifically with intratumoral doxorubicin determines and increases response in tumor destruction, compared to RFA alone. Studies with other methods (cryotherapy, laser hyperthermia, microwave hyperthermia) have been conducted to eradicate breast tumor. The purpose of these studies is similar as the initial clinical results to the RFA one.^^'^^

Lung Tumors Many patients with primary or secondary lung neoplasia are not surgical candidates. Radiotherapy or conformational radiotherapy is a treatment option not devoid of side effects. RFA is gaining acceptance as treatment modality, however heat distribution in lung tissue is irregular and not uniform. The reasons responsible for this no homogeneous heat diffusion are the presence of air filling zones and the large nearby vessels acting as heat sinks. To target the lesion to be treated with RFA, computed tomography or ultrasound are used. Akebosshi et al^^ have reported up to 46% of complete response in the treatment of primary lung tumors with minimal toxicity. Patient s pulmonary functions are preserved and the application of other therapies such as radiation or chemotherapy can be avoided. The major complications consist in pleural effusions and pneumothorax. Herrera et al treated 18 patients with inoperable primary lung tumors. They reported similar results and complications.^^ Diameter of the lesions greater than 5 cm seems critical as assumed by studies on liver tumors. A recent trial undertaken on 27 patients with non small cell lung cancer (NSCLC) or metastases to lung has clearly demonstrated that the size of the tumor is the major discriminator regarding patient survival and the necrosis obtained by RFA application.^^ In another trial not only primary and metastatic tumors were treated but also bone metastases to the ribs or sternum, in order to obtain pain relief Authors report that this last type of treatment is feasible and avoid of serious complications. Pain palliation is obtainable with this technique and an association to RFA and radiation therapy for larger masses can be used.^° A recent study by Steinke^^ on 14 centers around the world, reports that, on 493 RFA procedures performed two deaths occurred, pneumothorax in 30% and 10% of pleural effusion. In this author opinion pulmonary hemorrage is however more frequent than assumed and reached the 5.9 percent.^'^ Studies in Italy by Gadaleta et al^^ {stt Addendum) confirm the utility of RFA for primary and metastatic lung neoplasm, and in agreement with other authors, they report the following complications after post RFA procedure: 46% patients had moderate fever, 37% pain, 29% pleural effusion and cough, 16% pneumothorx and 12.5% dyspnea. Some authors have also demonstrated the possible use of RFA in combination with surgery. From these preliminary studies it is possible to conclude that pulmonary RFA appears to be safe, minimally invasive, with negligible mortality, litde morbidity, short hospital stay and gain in quality of life.^^'^^'^^

544

Hyperthermia in Cancer Treatment: A Primer

Bone Tumors Different malignant tumors such as from the breast, prostate, kidney and lung metastasize to bone. Bone metastases are lytic or plastic and associated with intermittent or constant pain. Sometimes fraaures and sensimotoric disorders are associated. RFA has been used to treat spinal metastatic lesions, osteoid osteomas, and lytic vertebral metastases. ^^'^^ The results of these groups are at its b^inning. In every case the study of Callstrom et al^^ reported, beyond a decrease in the pain degree, an objective decrease in analgesics medication. More randomized trials are in every case necessary, as oudined by Wood.^^ The only conclusions, in agreement with animal experiments,^^ that can be retained are: RFA application on bone metastases is a safe and potentially successful therapy. Pain relief seems fast and persistent, as demonstrated by Po^i et al.

Miscellaneous Tumors Treated with RFA RFA application on different other tumors are in progress and can be only pointed out. Wood ^' treated with his group adrenal and splenic metastases. In all the two cases an effective treatment was reproted. Pacella"^^ et al in Italy applied the technique of RFA to thyroid tissue. The treatment caused a decrease in dysphonia and symptoms from tumor-associated compression. However, the authors oudined leave also that it is hazardous to attempt a complete necrosis of the lesions because of proximity of vital surrounding structures. Resection was considered the gold standard of treatment for pancreatic tumor, unlikely most patients are at far advanced stage and cannot be cured. Different chemotherapeutic regimens, not avoid of severe toxicity are used. Actually application of RFA to primitive and metastatic pancreatic tumors has been performed and found a useful tool. ' Ghezzi et al demonstrated that RFA ablation of uterine fibroids during laparoscopy is feasible and safe. Furthermore, RFA potentially provides a safer option for removing prostate tumors and for treating brain tumors.

Perfusional Treatment Introduction The metastatic process is a multistep event and represents the most dreadful aspect of cancer. At the moment of diagnosis, cancers are relatively far advanced in their natural history and the presence of metastases is a conmion event. In fact, approximately 30% of patients will have detectable metastases at the moment of clinical diagnosis and a further 30% of patients will have occult metastases. Metastases can be disseminated and they can interest different organs at the same time, or can be localized to a specific organ."^^ In the case of localized disease, surgery

Table 6. Physical and phannacokmetic barriers to drug delivery Physical and physiological barriers Increased interstitial fluid pressure Hypoxic areas Increased distance from nutritive vessels Interstitium composition Pharmacokinetic factors Sex Age Inadequate drug concentration into tumor area Hepatic drug metabolism Renal cleareance Normal tissue side effects Decreased drug exposure time

Future Perspectives ofInterstitial and Perfusional Hyperthermia

345

Venous end

Arterial end C

Figure 3A. Example of circuit scheme of isolated perfusion. is the treatment of choice, however recurrence and prognosis depend on many criteria such as: resectability, patients cUnical situation, and number of metastases. After resection, recurrence is conunon suggesting that micrometastatic foci are present already at the moment of diagnosis. Chemotherapy is an ideal setting but only a few patients will be cured by it. However in the majority of cases systemic chemotherapy fails. Many physiological barriers and pharmacokinetics parameters"^^'^^ contribute to decrease its efficacy (see Table 6). Systemic chemotherapy compared to regional chemotherapy has limited benefits. Regional chemotherapy consists in the isolation of an anatomical region and in treating this by using chemotherapy at high doses with absence or minimum systemic toxicity.^^ Typical indications of regional chemotherapy are limbs, lung, liver, pleura, and pancreas. The method is common to all organs and consists in different sequential steps. The first step is the surgical isolation of the organ; the second is to keep the organ perftised. Oversimplifying the perfusion is kept by a circuit that consists of an out and inflow placed catheters, tubing, a roller pump, a reservoir, a heat exchanger and an oxygenator.^ A typical device with its circuitry is described in Figure 3A,B. Hyperthermia is applied to increase the sensitivity of tumor cells to antineoplastic agents to kill even more tumor cells and lowering the recurrence. ^^'^ The biological and physiological reasons for the combination of chemotherapy with hyperthermia are described elsewhere in this book. Aside from limbs, for which isolated chemohyperthermia is now an accepted treatment, the organs actually treated with perfusion chemohyperthermia are: lung, pleura and liver.

Clinical Applications Isolated Lung Perfusion Chemohyperthermia (ILUPH) The lung is the most common site of metastatic involvement beside of lymph nodes for all cancer types. Lung metastases occur in 50% of patients with cancer diagnosis.^^ Retroprospective studies have demonstrated that surgical removal, with an a^essive approach in selected patients, is the treatment of choice. However some technical and clinical limitations exist. Patients with unresectable lung metastases are candidates for Isolated Lung Perfusion Chemotherapy. This

346

Hyperthermia in Cancer Treatment: A Primer

Figure 3B. Example of device (Perfomer® Rand, Modena, Italy) for regional dierapy in which venous reservoir, oxygenator, heat exchanger, roller pump and temperature deteaors are included. (Reprinted with permission from Rand, Modena, Italy.) implies, as described above, a more efficient drug delivery to lung tissue. Different animals studies have been conducted demonstrating a superiority of perfusion technique compared to systemic chemotherapy. These authors determined that this kind of chemotherapy delivery method is reproducible and safe.^^'^^ The perfusion technique of these authors included the isolation of the two lungs, Jacobs in the 1961 ameliorated the technique isolating

Future Perspectives ofInterstitial and Perjusional Hyperthermia

347

the single lung. In the dogs he demonstrated the feasibility and the safety of the surgical technique. Pierpont^^ and Jacobs^^ demonstrated also, by using radioisotopes that no leakage of the drug was present outside the pulmonary circulation. In animal studies the method of Jacobs is still used today. Clinical studies were initiated by Creech^^ and have been continued by different authors.^^ The Johnston and Ratto groups^^'^^ used cisplatinum and demonstrated an high concentration of the drug inside the diseased lung. Other authors used or are using other antineoplastic agents such as: melphalan,^^ Doxorubicin^^ and TNF-a. Technical Aspects Different post-mortem studies have demonstrated that primitive lung tumors and lung metastases are supplied by bronchial arterial circulation.^^'^^ Furthermore primary lung tumors show differences depending on the tumor zone supplied. The inner part is generallv supplied by bronchial artery whereas its peripheral part is suppUed by pulmonary arteries. Because pulmonary arterial circulation, in a normal person, drains exclusively through the pulmonary veins, this permits a complete isolation of the lungs and a selective perfusion. ' At the moment we can distinguish three methods of regional lung perfusion (RLP) with: (1) inflow Pulmonary artery occlusion;^^ (2) Bronchial Artery infusion;^^ (3) no atrial appendage cannulation.'^^The first method consists of an unilateral perfusion with occlusion of the right or left pulmonary artery, using a balloon-tipped cardiac catheter, inserted percutaneously and radiographically positioned. After the occlusion of the ptdmonary arteries chemotherapy is infused distal to the balloon. The second technique consists of the infusion of antineoplastic agents through the bronchial artery and is performed by isolating the aorta from which bronchial arteries arose using a double-balloon catheter. Compared to the 1st technique this method permits to treat both lungs simultaneously. The third method is a technique of single isolated lung perfusion that is performed by cannulating the pulmonary artery and vein, instead of the left atrial appendage. The bronchial arteries were clipped temporarily to decrease leakage and toxicity of chemotherapeutic drugs. Another procedure is the ^Isolated Thoracic Perfusion (ITP) which is the most used actually. ITP consists of the insertion of two balloon catheters under X-ray control, one in the aorta, just above the celiac axis, and the other in to the inferior vena cava below the right atrium. In order to reduce the perfusion volume, Esmarch bandages are placed around the roots of both arms.^^ Side EflFects

Ratto et al^^ also studied the pulmonary function after 90 days from the perfusion. They demonstrated that forced vital capacity (FVC) decreased approximately 25% as the forced expired volume (FEVl). Furthermore a decrease of 20% in CO2 diffusion capacity (DLCO) was shown with no significant change in arterial PO2 and PCO2. Johnston reported that the use of doxorubicin (DOX) at 40 mg/m^ was safe and not associated with haematological, gastrointestinal or cardiac toxicity. Data at 8 weeks post perfusion showed no statistical significant difference on cardiac ejection fraction, on FEVl and DLCO.^^ All the authors have reported that drug leakage was minimal as well as the toxicity to normal tissues. ' ' ' ' Pleura Perfusion Treatment Metastatic pleura effusion is frequendy associated with the terminal stage of gastric, lung and breast cancer. The pleura is also the site of insurgence of rare primitive tumors such as mesothelioma- Mesothelioma diagnosis is generally difficult to obtain and its incidence is increasing in Western world. About 5% of patients have bilateral disease and 50% have distant metastases. Their survival at 5 years is less than 5% and their quality of life is poor. Today a standard treatment for mesothelioma does not exist.^^ Many treatment programmes combining surgery, chemotherapy, radiotherapy and inununotherapy have been used with more or less success. Radiotherapy has more a role on palliation and the response to chemotherapy is very low (< 20%). The highest response rate regards doxorubicin, mitomycin and ifosfamide. Biological response modifiers such as IL-2 and interferon have been associated with an

348

Hyperthermia in Cancer Treatment: A Primer

objective response for patient at early stage. '^^ Tumor localized to the pleura offers a local treatment with anticancer drugs. This treatment method has advantages: the tumor is direcdy exposed to a high drug concentration and the toxic side effects are limited. However as learned by peritoneal perfusion treatment, prerequisites for effective intracavitary chemotherapy are the absence of tumor outside the cavity and the surgical remove of all macroscopic tumor, as drug penetration by diffusion is limited to a few millimetres. Furthermore, the synergistic effect of heat and certain drugs like mitomicyn and cisplatin, have induced many groups to treat mesothelioma and metastases confined to pleura with intrathoracic chemohyperthermia. '^^ Technical Aspects The treatment method consists in two phases: the surgical one (thoractomy) followed by intrapleural perfusion thermochemotherapy (PPTC). '^^ The PPTC is however also possible without thoractomy^^ or performed under video-assisted thorascopic surgery (VATS).^^ The group of Matsuzaki^^ has demonstrated that a significant difference exists between the group treated with surgery followed by intrapleural perfusion thermochemotherapy and the group treated by surgery alone. The group treated only by (PPTC) without previous surgery had not advantage compared to group treated by surgery alone. Surgical procedure is done in lateral position, under general and thoracic epidural anesthesia and with a double-lumen endotracheal tube position. A posto lateral thoractomy through the fifdi intercostals space is performed. Extrapleural dissection follows, subsequendy the thickened pleural sheets are removed and separated by the underlying healthy tissue (pleura or lung parenchyma). After decortication if lung is damaged, a pneumectomy is performed. Small tumor nodules on diaphragm and pericardium are eliminated by coagulation, whereas larger masses are partially or completely resected.'^ '^^'^^ After cytoreduction pleural perfusion thermochemotherapy (PPTC) is carried

Figure 4. Methods for treating pleural cavity.

Future Perspectives of Interstitial and Perjusional Hyperthermia out keeping the patient in posterolateral position. A typical example of circuit used to treat pleura with (PPTC) is illustrated in Figure 4. Generally the lung, underlying the treated pleura, is kept collapsed or partially inflated as the controlateral lung is ventilated. The lung is kept collapsed to allow sufficient space between parietal and visceral pleura to adequate perfusion and to limit toxicity to lung. As shown from the circuit an inflow and outflow catheters are placed associated to silicone tubes for temperature measurement in the pleura and in the proximal oesophagus. The inflow outflow catheters are then connected to the roller pump the heat exchanger and the reservoir. Sometimes outflow liquid is filtered permitting the escalation of the total regional drug dose. The drugs used are cisplatin [CDDP] (50-80 mg/m^), adriamycin (15-25 m g W ) or mitomycin [MMC] (0.7 mg/Kg; maximum dose 60 mg), sometimes MMC ands CDDP are associated. The drugs are diluted in a sterile perfusate (4L) that is propelled by the roller pump in a closed circuit at the rate of 200 cm min'^ The temperature is kept between 40-42°C for 60-90 min. Every 30 min during PPTC different blood samples are measured. The temperature is monitored every 10 min.^'^^'^^ The second method of PPTC, not associated to thoracotomy, the so called video-assisted thorascopic surgery (VATS) is performed similarly, with the difference that no surgical resection of primary or secondary tumors is performed. This last method is however more palliative than the thoractomy combined with hyperthermia.^^ Side Effects All the authors have reported no major complications to lungs beside of wound infection or diaphragmatic prosthesis displacement. The overall procedure was completed without any death or toxicity^^'^^-^^ Liver Isolated Perfiision Liver, lungs and lymph nodes are filter organs and therefore inclined to metastasization. The poor chemonsensitiviity of metastases, peculiarly those of colorectal origin has forced many researchers to use methods for increasing the time and the concentration of drugs. The need for decreasing or limiting the side effects for this important and delicate organ, carried Aigner et al in 1981 to perform liver isolation for perfusion of antineoplastic agents.^^ Since 1981, modifications and technical improvements have been continuously introduced by Aigner s group and by other investigators.^^^^ Liver metastases may be of different origin and their chemonsensitivity may vary according to the histological type and their response in presence of heat. Technical Aspects Different surgical methods of liver isolation have been tested in both animals and patients.^^'^ Actually with different variations the methods can be classified as: A. Surgical hepatic isolation. B. Percutaneous isolated hepatic perfusion. Procedure A: Surgical Hepatic Isolation As first step an external extracorporal veno-venous bypass is established by placing a catheter in the saphenous vein, advanced into the infrarenal inferior vena cava, and a second cannula inserted through the axillary vein until the superior vena cava. The second step is the shunting of portal venous blood flow by placing a venous cannula into the mesenteric vein and incorporating this into the veno-venous bypass. The third step is the position of inflow cannula in the gastruodenal artery followed by a clamping of the common hepatic artery and of suprahepatic artery. After this positioning, the liver is isolated and ready to be perfused. For a schematic vision of the perfusion circuit (see Fig. 5)^^'^^'^'^ The perfusate is kept for 1 h at temperatures between 39.5°C and 40°C. Generally temperature is measured in the liver parenchyma using a Swan-Ganz catheter inserted into the portal vein. Three kinds of chemotherapeutic drugs are used: (a) melphalan, (b) mitomycin-C and (c) Tumor necrosis factor

349

350

Hyperthermia in Cancer Treatment: A Primer

Axillary v.

Gastroduodenal art.

Figure 5. Isolated hepatic perfusion (IHP) circuit scheme. Reproduced with permission from: Grover A, Alexander HR. The past decade of experience with isolated Hepatic perfusion. The Oncologist 2004; 9:653-664.^5 (TNF-a). All in different combinations are used to treat colorectal metastases or metastases of other origin such as malignant melanoma.^^'^^'^^'^^ Procedure B: Percutaneous Isolated Hepatic Perfusion Surgical procedure is complex and cannot be repeated. This drawback limits the results of this technique, and has forced many researchers to develop a simpler and repetitive procedure. The developed method is a partial isolation of the liver (isolated hepatic perfusion (IHP)) by closing the inferior caval vein above and below the hepatic veins using a four lumen /two balloon catheter. This positioning permits to collect blood from hepatic veins diverting it to a charcoal filter before it returns to patients systemic circulation. Drugs are administred via a percutaneously placed catheter in the femoral artery (see Fig. 6).^ '

Future Perspectives ofInterstitial and Perfusional Hyperthermia

351

Filter

V Flow probe

Pump

Figure 6. Percutaneous hepatic perfusion circuit scheme reproduced and modified with permission firom: Grover A, Alexander HR. The past decade of experience with isolated hepatic perfiision. The Oncologist 2004; 9:653-664.^5 Since 1993 358 patients, with hepatic metastases, were treated with IHP. The results of these clinical trials have been recently reviewed by Groover and Alexander.^^ On 15 trials reported, the majority have been conduaed with melphalan alone or in association with cisplatin orTNF-a, 12 of them with metastases from colorectal cancer, 3 malignant melanoma and five Mixed histology.^^'^^'^^ Some authors used mitomicyn-C for the treatment of colorectal metastases for the known synergistic effect with hyperthermia.^^ A 41% of partial response has been obtained with this procedure and in 6-9% of complete response. Ocular melanoma has the greater percent of complete and partial response compared to liver metastases of other origin. The median survival time has been > 10 months. Side Effects With the surgical procedure only a transient elevation of hepatic enzymes and bilirubin has been noted. However some fatal deaths for total liver failure and grade 4 leukopenia have been described. The mortality in the majority of the studies was of the order of 5%. An elevation of hepatic enzymes beyond 7 days after the perfusion procedure itself can be considered as melphalan-related.^^^^

Conclusions Radio frequency thermoablation (RFA) applied under sonographic guidance has certain features which may make it a first choice in the treatment of certain hepatocellular carcinoma (HCC) and make it a promising method for the curative and palliative treatment of secondary

552

Hyperthermia in Cancer Treatment: A Primer

tumors of the liver. Randomized controlled trials have been performed for HCC but not yet for secondary liver tumors likefix)mcolorectal or breast cancer. Acmally, the various procedures of perfusion, herein described are at their beginning. To our opinion RFA procedures have a greater future compared to some isolated procedures. RFA techniques are easier to learn and to be performed. They can be performed by few qualified persons, whereas perfusion procedures need a skilled and coordinated team. For perfusion chemotherapy less invasive methods have to be developed like the percutaneous intraperitoneal hyperthermic chemoperfiision or the percutaneous isolated hepatic perfusion, which in turn has to be compared with local chemoembolisation. Further randomized controlled trials are warranted.

Addendum: Radiofrequency Thermal Ablation in the Treatment of Lung Malignancies Cosimo Gadaleta, Anna Catino and Vittorio Matdoli"^ Summary Radiofrequency ablation is an advanced, minimally invasive technique used to treat several types of neoplasm. In the last few years, its application in the treatment of lung tumors has received considerable interest. In our experience, 10 unresectable primary lung tumors and 70 lung metastases from various solid tumors were treated with percutaneous RFA. The procedure was performed under CT scan guidance and general anesthesia; the rate of complete necrosis of the treated neoplasms was 94% while the most frequent complication was pneumothorax, requiring pleural drainage in 14% of sessions. CT scan and MRI with gadolinium have shown to be accurate and useful to assess the therapeutic adequacy of lung RFA. Lung RFA is a very promising technique, minimally invasive and well tolerated in the majority of patients; further investigation is strongly su^ested in order to define the optimal role of lung RFA in the multidisciplinary therapy of lung malignancies.

Introduction Among the new image-guided percutaneous techniques which are based on hyperthermic energy obtained from various sources, radiofrequency thermal ablation (RFA) has recendy received much attention as minimally invasive approach for the local treatment of solid neoplasms.^ The predominant mechanism of action is thermal injury; a needle-electrode with a non-insulated portion generates medium-frequency electromagnetic waves of 480 kHz, so producing a ionic agitation and frictional heat within the tissue surrounding the tip of the needle. The heat induces irreparable cellular damage leading to coagulative necrosis. Since its widespread application for the ablation of unresectable liver tumors,^ RFA has been tested in the treatment of various solid tumors. Lung tumors seem to be good targets for RFA because the surrounding air in the adjacent normal lung parenchyma provides an insulating effect, so concentrating the RF energy within the tumor tissue.^ As a result, less radiofrequency energy deposition is required to achieve adequate tumor heating than with intrahepatic pathology. In addition, experimental studies have reported that RFA of lung tumors results in the greatest coagulation diameter, depending on a variety of tissue-specific characteristics. •Corresponding Author: Vittorio Mattioli—Interventional Radiology Operative Unit, Critical Area Department, Oncology Institute, Bari, Italy.

Future Perspectives of Interstitial and Perfusional Hyperthermia

353

The interest on this image-guided procedure in the treatment of lung neoplasms is witnessed by the increasing number of published reports;'^'^ despite the heterogeneity of the studies, including various approaches (i.e., percutaneous or thoracotomic), type of device, patients' characteristics and duration of follow-up, the technique can be considered safe, feasible and well tolerated by patients.

Patients and Methods From February 2002 to September 2005, 45 patients with 80 lung neoplasms (10 primary NSCLC and 70 metastases from other solid tumors) underwent lung RFA. Lung lesions were subclassified as follows: "Paramediastinal'' were those in contact with mediastinal structures (without infiltration) or less than 1 cm from them, including fibrous pericardium, major vessels, cardiac pedicle, trachea and bronchi; "Central-parenchymal" lesions were those fiilly surrounded by pulmonary aerated parenchyma, more than 1 cm. from the mediastinal structures and visceral pleura; "Subpleural" lesions were those in direct contact (without infiltration), or distant less than 1 cm from the pleura. Patients were considered unresectable due to technical or anatomical/functional contraindications, or in case of refiisal of surgery. Main inclusion criteria were: cytohistologically proven lung neoplasm, presence of 3 lesions or less with total maximum diameter less than 10 cm, normal coagulation tests and platelets count, disease not infiltrating bronchi, big vessels and/or interstitial mediastinal tissue, absence of neoplastic or massive pleural effiision. The technique was performed percutaneously with CT scan guidance (Fig. 7). A 17-gauge, monopolar cooled electrode-needle was used, with lenght ranging between 10 and 15 cm, depending on the depth of the lesion to be treated. The non-insulated part of the needle was between 1 and 3 cm, depending on the diameter of the lesion. Lesions with diameter less than or equal to 2.8 cm were treated with a single placement of the needle, whereas larger lesions were treated after subdivision into sectors (each sector was treated as if it were a 2 cm lesion), and by overlapping the sectors to ensure full ablation of the entire nodule. The wattage-current setting is selected automatically by the system and is based on the amount of water in any specific tissue (i.e., the amount of free ions present). The system adjusts itself according to the level of resistance and impedance. A maximum treatment time of 12 min. Has been found to ensure complete coagulative necrosis of the tumor volume, according to the corresponding diameter of the exposed part of the needleGrounding pads (22 x 19 cm) were placed in the lumbar or gluteal r^ion, according to the position of the nodtde to be treated. Nodules close to the apex of the lung required pads placed in the lumbar region, and nodules close to the base of the lung required pad placement in the gluteal region. All subjects were treated under general anesthesia with intubation by a double-lumen tube. A CT scan with contrast medium was scheduled before treatment to evaluate baseline enhancement of the tumor, while repeated CT scans were performed during the procedure, without contrast medium and with a collimation of 5 nmi, in order to select the optimal access path of the needle as well as to detect and manage possible acute complications, and finally to monitor the treatment-related structural changes of the lesion (Fig. 7). Furthermore, because it is often necessary to make small changes of the position of the needle, as a result of the morphology and volume of the lesion, CT scan allows to monitor even the smallest repositioning of the needle. An additional unenhanced CT scan was performed inunediately afi:er the procedure, while chest radiographs to detect pneumothorax and other acute complications were obtained 2 hours after and daily until discharge of the patient from the hospital. All patients received antibiotic prophylaxis immediately before the procedure and during the whole hospitalization time thereafter.

354

Hyperthermia in Cancer Treatment: A Primer

Figure 7. Patient afFected by primary lung cancer (NSCLC). A-B) CT scans obtained during RFA show the multiple insertions of the electrode-needle inside the lesion, with different angulations The assessment of treatment efficacy included a contrast-enhanced CT scan and NMR with gadolinium, 1 month and then every 3 months after the procedure; it was based on the absence of contrast-enhancement in the treated area and, in addition, on the possible presence of cavitation.

Results Eighty lung neoplasms in 45 patients underwent RFA, while 58 sessions of treatment were carried out. Impedance values ranged between 75 and 140 Ohm; the internal temperature, close to the electrode needle, ranged between 45*'C and 75°C. Temperatures lower than 58°C were sometimes recorded during successive placements of the electrode needle into large lesions, which are treated in partially overlapping sectors to allow complete ablation of the tumor. Nevertheless, in all cases the temperature was never less than 45"C. The power generated during the procedure was between 85 and 140 W; values between 85 and 90 W were observed while treating nodules smaller than 3 cm, with the electrode needle placed partially outside the lesion. In these instances the impedance seemed inversely correlated.

Future Perspectives ofInterstitial and Perjusional Hyperthermia

355

\ ^^ffi^^^^^Mm^Km

" ' '^^VHI

H Figure 8. Patient affeaed by primary lung cancer (NSCLC) CT scans obtained after RFA show the treated neoplasm with "cockade phenomenon" visible by parenchymal window (A-B) and by mediastinal window {C-D).

With a median follow-up of 14 months, complete ablation of the entire lesion was achieved in 75/80 cases (94%). Local recurrence only in the treated area was observed in 2 cases (2.5%), whereas globally 5 cases showed relapse both in treated area and/or in other distant sites. One notable case involved one patient, affected by a large primary lung tumor (NSCLC) with a diameter of 7 cm, who showed early relapse in the treated nodule, not well detected by CT scan but visible by NMR with gadolinium; a FNAB in the doubtful area revealed the presence of neoplastic viable cells. This patient was successfully re-treated and experienced a progression-free interval of 10 months, until metastatic disease in other extrathoracic sites was found. Among the 5 patients who relapsed in the treated area, 3 had nodules larger than 3.5 cm. The major complication was pneumothorax, requiring chest-tube placement in 8/58 (14%) sessions. One case of bronchopleural fistiJa was observed, resolved after a prolonged pleural drainage; other side effects, considered to be moderate and easily manageable, were cough with rust-coloured spitting (18/58:31% of sessions), minimal pleural effusion (18/58:31%), thoracic pain (14/58:24%), transient dyspnoea (8/58:14%) and moderate-grade fever lasting more than 3 days in 10/58 sessions (17%). No treatment-related death occurred. Immediately after treatment, CT images showed wrinkling of the edges of the lesion with unchanged diameter and partial emptying, likely due to vaporization of tissue; in addition multiple concentric rings with various densitometric characteristics were visible in the pulmonary parenchyma around the lesion. This appearance, so-called "cockade phenomenon", was most evident 48-72 hrs after treatment (Fig. 8).

356

Hyperthermia in Cancer Treatment: A Primer

At the same time, a sectorial hyperemia surrounding the lesion, with a conical shape with the apex at the hilum, as well as a minimal pleural effusion were present, resolving itself in a few days. CT scans obtained after 30 days and successive revealed progressively less definition of the lesion, and ultimately a core of hyperdense scar tissue surrounded by a thin hyperdense ring with a distance from the nucleus that was inversely correlated to the size of the nodule. In case of complete ablation, the treated nodules appeared enlarged, most likely due to central necrosis and cavitation surrounded by reparative fibrosis, without contrast-enhancement.

Discussion Lung RFA has demonstrated to be safe and feasible; the percutaneous approach and imaging guidance make this procedure suitable for patients considered unfit for standard surgical reseaion. The usually short hospital stay and the good tolerability contribute to RFA being well accepted by patients, resulting in a favourable impact on quality of life. Pneumothorax is the most serious reported complication;^'^ in our experience it occurred in 14% of sessions. We think that an acceptable rate of major complications could be achieved by an increasing knowledge and training of the groups. Furthermore, the optimal selection of patients to be treated and the choice to use general anesthesia could contribute to improve the tolerability and the results of lung RFA. The use of general anesthesia is supported by multiple bio-medical factors: (1) guaranteeing a complete control of the patient's airway passages, especially in the event of a massive pneumothorax or serious parenchymal hemorrage; (2) optimizing the ventilatio/perfusion rate in case of bronchopathy, which is often concurrent, as well as physiological changes related to the lateral or prone position of the patient during treatment; (3) assuring unconsciousness, suppression of reflexes and muscular relaxation in order to obtain total immobility of the patient and to eliminate memory of the event; (4) eliminating the risk of accidental injury to the pleura and parenchyma due to unvoluntary respiratory movements as in case of transparietal punctures. The technique is extremely promising also with respect to the efficacy; in our experience it was possible to obtain a high rate of complete ablation in the treated nodules (94%); it is also interesting to consider the data from experimental animal models,^ ^ reporting that CTscanguided lung RFA appear to be very effective to achieve a "safety margin", also in comparison with RFA during surgery. In agreement with other authors,^^ we think that in case of lesions larger than 3.5 cm it is difficult to achieve complete necrosis; nevertheless, when the subdivision of the lesion in overlapped sectors is required, the use of repeated CT scan during the procedure could improve the technical residts, due to the capability of monitoring even the slightest repositioning of the electrode-needle. Lung RFA produces a typical radiological appearance which resembles a "cockade", so-called due to the formation of concentric layers surrounding the ablated lesion, most likely corresponding to histopathological changes and related to thermal gradients between the tumoral nodule and the surrounding parenchyma.^ CT scan is the most widely used imaging technique to evaluate lung tumors treated with RFA, able to detea changes in contrast-enhancement as well as the presence of cavitation;^'^^'^"^'^^ nevertheless, a careftil follow-up of treated patients have to be conducted to detect any infection in cavitated lesions, especially in immunocompromised patients.^^'^ In addition, NMR with gadolinium could be potentially useftil to evaluate the therapeutic efficacy of lung RFA, by providing information about tumoral density and composition as well as to accurately determine the extent of tissue necrosis.^'^^'^^ Nothwithstanding the need for more standardized methods, we consider CT scan and NMR with gadolinium reliable techniques for radiologic assessment of treatment efficacy and follow-up of lung neoplasms submitted to RFA.

Future Perspectives ofInterstitial and Perfusional Hyperthermia

357

In conclusion, the experiences on lung RFA suggest that this procedure could represent an important tool in the multidisciplinary approach of unresectable lung tumors. Further well-designed and long-term clinical trials are warranted: (1) to identify which subgroups of patients are ideal candidates to this procedure, (2) to determine the optimal combination of lung RFA with other antineoplastic therapies, and finally (3) to establish the impact of lung RFA on patients' survival with respect to standard therapy.

Addendum References 1. Wood BJ, Ramkaransingh JR, Fojo T et al. Percutaneous tumor ablation with radiofrequenqr. Cancer 2002; 94:443-51. 2. Liu LX, Jiang HC, Piao DX. Radiofrequency ablation of liver cancer. World J Gastroenterol 2002; 8(3):393-399. 3. Rossi S, Buscarini E, Garbagnati F et al. Percutaneous treatment of small hepatic tumors by an expandable RF needle electrode. Am J Roentgenol 1998; 170:1015-1022. 4. Solbiati L, Livraghi T, Goldberg SN et al. Percutaneous radiofrequency ablation of hepatic metastases from colorectal cancer:long-term results in 117 patients. Radiology 2001; 221:159-166. 5. Goldberg SN, Gazelle GS, Compton CC et al. Radiofrequency tissue ablation in the rabbit lung:efficacy and complications. Acad Radiol 1995; 2:776-784. 6. Ahmed M, Liu Z, Afzal KS et al. Radiofrequency ablation:efFect of surrounding tissue composition on coagulation necrosis in a canine model. Radiology 2004; 230:761-767. 7. Dupuy DE, Zagoria RJ, Akerley W et al. Percutaneous radiofrequency ablation of malignancies in the lung. Am J Roentgenol 2000; 174:57-9. 8. Gadaleta C, Mattioli V, Colucci G et al. Radiofrequency ablation of 40 lung neoplasms:preliminary results. Am J Roentgenol 2004; 183:381-386. 9. Van Sonnenberg E, Shankar S, Morrison PR et al. Radiofrequency ablation of thoracic lesions:Part II, Initial clinical experience. Technical and multidiscipUnary considerations in 30 patients. Am J Roentgenol 2005; 184:381-390. 10. Gadaleta C, Catino A, Ranieri G et al. Radiofrequency thermal ablation of 69 lung neoplasms. J Chemother 2004; 16(5):86-89. 11. Nomori H, Imazu Y, Watanabe K et al. Radiofrequency ablation of pulmonary tumors and ormal lung tissue in swine and rabbits. Chest 2005; 127:973-977. 12. Belfiore G, Moggio G, Tedeschi E et al. CT-guided radiofrequency ablation: a potential complementary therapy for patients with unresectable primary lung cancer-A preHminary report of 33 patients. Am J Roentgenol 2004; 183:1003-11. 13. Lee JM, Jin GY, Goldberg SN et al. Percutaneous radiofrequency ablation for inoperable non-small cell lung cancer and metastases: preliminary report. Radiology 2004; 230:125-134. 14. Suh RD, Wallace AB, Sheehan RE et al. Unresectable pulmonary malignancies: CT-guided percutaneous radiofrequency ablation-Preliminary results. Radiology 2003; 229:821-829. 15. Jin GY, Lee JM, Lee YC et al. Primary and secondary lung malignancies treated with percutaneous radiofrequency ablation: evaluation with follow-up helical CT. AJR Am J Roentgenol 2004; 183:1013-1020. 16. Marchand B, perol M, Roche EDL et al. Percutaneous radiofrequency ablation of a lung metastasis: delayed cavitation with no infection. J Comput Assist Tomogr 2002; 26:1032-1034. 17. Bojarski JD, Dupuy DE, Mayo-Smith WW. CT imaging findings of pulmonary neoplasms after treatment with radiofrequency ablation:results in 32 tumors. AJR Am J Roentgenol 2005; 185(2):466-71. 18. Oyama Y, Nakamura K, Matsuoka T et al. Radiofrequency ablated lesions in the normal porcine lung°: long-term follow-up with MRI and pathology. Cardiovasc Intervent Radiol 2005; 28(3):346-53.

References 1. Sackenheim M MCD. Radiofrequency Ablation The Key to cancer treatment. JDMS 2003;19:88-92. 2. Hanson PS, Soulen MC. Tumor ablation: a review of technique and outcomes. AppHed Radiology 2001. 3. Goldberg SN, Gazelle GS, Mueller PR. Thermal Ablation Therapy for focal malignancy. A unified approach to underlying principles, techniques, and diagnostic imaging guidance. AJR 2000; 174:323-331. 4. Nazir B, Chaturvedi AK, Rao A. Image-guided radiofrequency ablation of tumors: current status. Ind J Imag 2003; 13:315-322.

358

Hyperthermia in Cancer Treatment: A Primer

5. Neeman Z, Wood BJ. Radiofrequency ablation beyond the liver: Techniques in vascular and Interventional radiology 2002; 5:156-163. 6. Goldberg SN, Gazelle GS, Solbiati L et al. Radiofrequency tissue ablation: increased lesion diameter with a perfusion electrode. Acad Radiol 1996; 3(8):636-644. 7. deBaere T, Denys A, Wood BJ et al. Radiofrequency liver ablation: experimental comparative study of water-cooled versus expandable systems. Am J Roentgenol 2001; 176:187-192 8. Solbiati L, Goldberg N, lerace T et al. Hepatic metastases: Percutaneous radiofrequency ablation with cooled-tip electrodes. Radiology 1997; 205:367-373. 9. Livraghi T, Goldberg SN, Monti F et al. Saline-enhanced radiofrequency tissue ablation in the treatment of liver metastases. Radiology 1997; 202:205-210. 10. Fiske H. Radiofrequency ablation takes aim at kidney tumors. Radiology Today 2002; 19-22. 11. Jacomides L, Ogan K, Watumill L et al. Laparoscopic application of radio frequency energy enables in situ renal tumor ablation and partial nephrectomy. J Urol 2003; 169:49-53. 12. Gervais DA, McGovern FJ, Arellano RS et al. Renal cell carcinoma: clinical experience and technical success with radio-frequency ablation of 42 tumors. Radiology 2003; 226:417-424. 13. Pavlovich CP, McClellan MW, Choyke PL et al. Percutaneous radio frequency ablation of small renal tumors: initial results. J Urol 2002; 167:10-15. 14. Michaels MJ, Rhee HK, Mourtzinos AP et al. Incomplete renal destruction using radio frequency interstitial ablation. J Urol 2002; 168:2406-2410. 15. Corwin TS, Lindberg G, Traxer O et al. Laparoscopic radiofrequency thermal ablation of renal tissue with and without hilar occlusion. J Urol 2001; 166:281-284. 16. Gettman MT, Lotan Y, Corwin TS et al. Radiofrequency coagulation of renal parenchyma: comparison of effects of energy generators on treatment efficacy. J Endourol 2002; 16:83-88. 17. Rohde I, Alberts C, Mahnken A et al. Regional thermoablation of local and metastatic renal cell carcinoma. Oncology reports 2003; 10:753-757. 18. Hines-Peralta A, Goldberg SN: Review of radiofrequency ablation for renal cell carcinoma. Clin Cancer Res 2004; 10:6328s-6334s. 19. Ogan K, Jacomides L, Dolmatch B et al. Percutaneous radiofrequency ablation of renal tumors: technique, limitations, and morbidity. Urology 2002; 60:954-958. 20. Fujimoto, Kobayashi K, Takahasashi M et al: Clinical pilot studies on preoperative hyperthermic tumour ablation for advanced breast carcinoma using an 8 MHz radiofrequency heating. Int J Hyperthermia 2003; 19:13-22. 21. BuraK WE, Agnese DM, Povoski SP et al. Radiofrequency ablation of invasive breast carcinoma followed by delayed surgical excision. Cancer 2003; 98:1369-1376. 22. Goldberg SN, Saldinger PF, Gazelle et al. Percutaneous tumor ablation: increased necrosis with combined Radiofrequency ablation and intratumoral doxorubicin injection in a rat breast tumor model. Radiology 2001; 220:420-427. 23. Robinson D, Parel JM, Denham DB et al. Interstitial Laser Hyperthermia model development for minimally invasive therapy of breast carcinoma. J Am Coll Surg 1998; 186: 284-292. 24. Morin J, Traord A, Guy D et al. Magnetic resonance- guided Percutaneous cryosurgery of breast carcinoma: technique and early cHnical results. Can J Surg 2004; 47:347-351. 25. Gardner RA, Vergas HI, Block JB et al. Focused microwave phased array thermotherapy for primary breast cancer. Ann Surg Oncol 2002; 9:326-332. 26. Akimov A, Seregin VE, Rusanov KV et al. Nd:YAG interstitial laser thermometherapy in the treatment of breast cancer. Lasers Surg Med 1998; 22: 257-267. 27. Akeboshi M, Yamakado K. Percutaneous radiofrequency ablation of lung neoplasms: initial therapeutic response: J Vase Interv Radiol 2004; 15:463-470. 28. Herrera L, Fernando HC, Perry Y. Radiofrequency ablation of pulmonary malignant tumors in non surgiucal candidates. J Thor Cardiovasc Sur 2003; 125:929-937. 29. Lee JM, Jin GY, Golderg SN et al. Percutaneously radiofrequency ablation for inoperable non-small cell lung cancer and metastases:preHminary report. Radiology 2004; 230:125-134. 30. Dupuy DE, Mayo-Smith WW, Abbot GF. Clinical applications of radiofrequency tumor ablation in the thorax. Radiographics 2002; 22:s59-s69. 31. Steinke K, Sewell PE, Dupuy D et al. Pulmonary radiofrequency ablation-an international study survey. Anticancer Research 2004; 24:339-344. 32. Steinke K, King J, Glenn Det al. Pulmonary hemorrage during Percutaneous radiofrequency ablation: a more frequent complications than assumed. Interactive Cardiovascular Thoracic Surgery 2003; 2:462-465. 33. Gadaleta C, MattioU V, Colucci G et al. Radiofrequency ablation of 40 Lung neoplasms:Preliminary results. Am J Roentgenol 2004; 183:361-368.

Future Perspectives ofInterstitial and Perjusional Hyperthermia

359

34. van Sonnenberg E, Shankar S, Morrison PR et al. Radiofrequency ablation of thoracic lesions: part2, Initial clinical experience. Technical and multidisciplinary considerations in 30 patients. Am J Roentgenol 2005; 184:381-390. 35. Groenemeyer DHW, Schirp S, Gevargez A. Image-guided Percutaneous thermal ablation of bone tumors. Acad Radiol 2002; 9:467-477. 36. Groenemeyer DHW, Gevargez A, Schirp S. Image-giuided radiofrequency ablation of spinal tumors: preliminary experience with an expandable array electrode. Cancer J 2002; 8:33-39. 37. Callstrom MR, Charboneau JW, Goetz MP et al. Painful metastases involving bone: feasibility of percutaneous CT- and US-guided radio-frequency ablation. Radiology 2002; 224:87-97. 38. Wood JB. Feasibility of thermal ablation of lytic vertebral metastases with radiofrequency current. A commentary. Cancer J 2002; 8:26'-28. 39. Letcher FS, Goldring S. The effect of radiofrequency current and heat on peripheral nerve action potential in the cat. J Neurosurg 1968; 29:42-47. 40. Poggi G, Melazzini M, Bernardo G et al. Percutaneous ultrasound- guided radiofrequency thermal ablation of malignant osteolysis. Anticancer Res 2003; 23:4977-4983. 41. Wood JB Abraham J, Hzizda J. Radiofrequency ablation of adrenal tumors and adrenocortical cxarcinoma metastases. Cancer 2003; 97:54-60. 42. Wood JB, Bates S. Radiofrequency thermal ablation of a splenic metastasis. J Vase Interv Radiol 2001; 12:261-263. 43. Pacella CM, Bizzari GC, Spiezia S et al. Thyroid tissue: Us- guided Percutaneous laser thermal ablation. Radiology 2004; 280:272-280. 44. Elias D, Baton O, Sideris L et al. Necrotizing pancreatitis after radiofrequency destruction of pancreatic tumors. Eur J Surg Oncol 2004; 30:85-87. 45. Radiofrequency tissue ablation: an early Indian experience. Indian J Gastroeril 2003; 22:91-93. 46. Ghezzi et al Am J Obstet Gynecol 2005; 192:768-773. 47. Liotta L, Kohn EC. Invasion and metastases. In Cancer Medecine 6 Edition. Kufe; Pollock; Weichselbaum; Bast: Gansler; Holland; Frei Eds. BC Decker 2003; 151-159. 48. Munn LL. Aberrant vascular architecture in tumors and its importance in drug based therapies. Drug Discovery Today 2003; 8:396-403. 49. Curti BD. Physical barriers to drug delivery in tumors: Critical Reviews in Oncology/Hematology 1993; 14:29-39. 50. Jain RK. Transport of molecules, particles, and cells in solid tumors. Ann Rev Biomed Eng 1999; 01:241-263. 51. Aigner KR. Regional chemotherapy-editorial review article. Reg Cancer Treat 1994; 2:55-66. 52. Creech OJ, Krementz ET, Ryan RF et al. Chemotherapy of cancer : Regional perfusion utilizying an extracorporeal circuit. Ann Surg 1958; 148:616-632. 53. Weksler B, Burt M. Isolated lung perftision with antineoplastic agents for pulmonary metastases. Chest Surgery Clinics of North America 1998; 8:157-183. 54. Stehhn I. Hyperthermia perfrision with chemotherapy for cancer of extremities. Surg Gynecol Obstet. 1969; 129:305-308. 55. Van Schil PE. Surgical treatment for pulmonary metastases. Acta Clinica Belgica 2002; 57:333-339. 56. Van Putte BP, Hendricks J MH, Romijin S et al. Isolated lung perftision for the treatment of pulmonary metastases current mini-review of work in progress. Surgical oncology 2003; 12:187-193. 57. Ellis JL, Ng B, Port J et al. Isolated lung perftision with carboplatin for metastatic sarcoma in the F344 rat. Surgical forum 1994; 294:-295. 58. Weksler B, Lenert J, Ng B et al. Isolated single lung perftision with doxorubicin is effective in eradicating soft tissue sarcoma lung metastases in rat model. J Thorac Cardiovasc Surg 1994; 107: 50-54. 59. Pierpont H, Blades B. Lung perfusion with chemotherapeutic agents. J Thorac Cardiovasc Surg 1960; 39:159-165. 60. Jacobs JK, Flexner JM, Scott HWJ. Selective isolated perfusion of right or left lung: J Thorac Cardiovasc Surg 1961; 42:546-552. 61. Johnston MR, Minchen RF, Dawson CA. Lung perfusion with chemotherapy in patients with unresectable metastatic sarcoma to the lung or diffuse bronchioalveolar carcinoma. J Thorac Cardiovasc Surg 1995; 110:368-373. 62. Ratto GB, Toma S, Civalleri D et al. Isolated lung perftision with platinum in the treatment of pulmonary metastases from soft tissue sarcomas. J Thorac Cardiovasc Surg 1996; 112:614-622. 63. Burt ME, Liu D, Abolhoda A et al. Isolated lung perftision for patients with unresectable metastases from sarcoma: a phase I trial. Ann Thorac Surg 2000; 69:1542-1549. GA. Pass HI, Mew DJY, Kranda KG et al. Isolated Lung perftision with tumor necrosis factor for pulmonary metastases. Ann Thorac Surg 1996; 61:1609-1617.

360

Hyperthermia in Cancer Treatment: A Primer

65. Miller BJ, Rosenbaum AS. The vascular supply to metastatic tumors of the lung. Surg Gynecol Obstet 1967; 125:1009-1115. GG. Milne ENC. Circulation of primary and metastatic pulmonary neoplasm: a post-mortem microarteriographic study. AJR 1967; 100:603-619. 67. Pass HI. Isolated lung perfusion for pulmonary metastases. In: Lotze MT, Rubin TJ, eds. Regional therapy of advanced cancer. New York: Lippincott-Raven Press, Philadelphia, 1997:63-73. 68. Smyth NP and Blades B. Selective chemotherapy of lung during unilateral pulmonary arterial occlusion with a balloon-tipped catheter. J Thorac Cardiovasc Surg 1960; 40:653-666. 69. Cliflfton EE, Mahajan DR. Technique for visualization and perfusion of bronchial arteries: suggested cHnical and diagnostic applications. Cancer 1963; 16:444-452. 70. Liu D, Burt M, Ginsberg RJ. Lung perfusion for treatment of metastatic sarcoma to the lungs. In: Markman M, ed. Current Clinical Oncology: Regional chemotherapy: clinical research and practice. New Jersey: Humana Press Inc., 2000:87-99. 71. Guad^ni S, Miiller H, Valenti M et al. Thoracic stop -flow perfusion in the treatment of refractory non small Cell lung cancer. J Chemotherapy 2004; I6s:40s-43s. 72. Miiller H, Guadagni S. Regional plus systemic chemotherapy: an effective treatment in recurrent non-small cell lung cancer. EJSO 2001; 27:190-195 73. American Thoracic Society. Management of malignant pleural effusions. Am J Resp Care Med 2000; 1987-2001. 74. Astoul PH, Viallat JR, Laurent JC et al. Intrapleural recombinant 11-2 in passive immunotherapy for malignant pleural effusion. Chest 1993; 103:209-213. 75. Rosso R, Rimoldi R, Salvatri F. Intrapleural beta interferon in the treatment of malignant pleural effusions. Oncology 1988; 45:253-256. 7G. Sugarbacher DJ, Jacklitsch MT, Soutter AD et al. Multimodality therapy of malignant mesothelioma. In: Roth JA, Ruckdeschel JC, Weisenburger TH, eds. Thoracic Oncology. 2nd ed. Philadelphia: WB Saunders Co., 1996:538-555. 77. Pass HI, Robinson BW, Testa J et al. Emerging translational therapies for mesotheUoma. Chest 1999; ll6s:455s-460s. 78. Monneuse O, Beaujard AC, Guibert B et al. Long- term result of intrathoracic chemohyperthermia (ITCH) for the treatment of pleural malignancies. Br J Cancer 2003; 88:1839-1843. 79. Matsuzaki Y, Shibata K, Yoshioka M et al. Intrapleural perfusion hyperthermo-chemotherapy for malignant pleural dissemination and effusion. Ann Thorac Surg 1995:127-131. 80. Shigemura N, Akashi A, Mitsumori O et al. Combined surgery of intrapleural perfusion hyperthermic chemotherapy and panpleuroonectomy for lung cancer with advanced pleural spread: a pilot study. Interactive Cardiovascular and Thoracic Surgery 2003; 2:671-675. 81.Aigner KR. Isolated liver perfusion. In: Morris DL, McArdle CS, Onik GM, eds. Hepatic Metastases. Oxford: Butterworth Heinemann, 1996:101-107. 82. Rothbarth J, ToUenar RAEM, Schellen JHM et al. Isolated hepatic perfusion for the tteatment of colorectal metastases confined to the liver: recent trends and perspectives. European J of cancer 2004; 40:1812-1824. 83. Oldhafer KJ, Lang H, Frerker M et al. First experience and technical aspects of isolated liver perfusion for extensive liver metastases. Surgery 1998; 123:622-631. 84. Carroll NM, Alexander HR. Isolation perfusion of the liver. Cancer J 2002; 8:181-193. 85. Grover A, Alexander HR. The past decade of experience with isolated hepatic perfusion. The Oncologist 2004; 9:653-664. 86. Vahrmeijer AL, vanDierendonck JK, Keizer HJ et al. Increased local cytostatic drug exposure by isolated hepatic perfusion: a phase I clinical and pharmacologic evaluation of treatment with high dose melphalan in patients with colorectal cancer confined to the liver. Br J Cancer. 2000; 82:1536-1546. 87. Linden^r P, Fjjilling M, Hafstrom L et al. Isolated hepatic perfusion with extracorporeal oxygenation using hyperthermia, tumor necrosis factor alpha and melphalan. European J of Surgical Oncology 1999; 25:179-185. 88. Marinelli A, dE Brau LM, Beerman H et al. Isolated liver perfusion with mitomycin-c in the treatment of colorectal cancer metastases to the liver. Jpn J CUn Oncol 1996; 26:341-350. 89. Alexander HR, Libutti SK, Bartlett DL et al. A phase I-II study of isolated hepating perfusion using melphalan with or without Ttumor necrosis factor for patients with ocular melanoma netastatic to liver. Clinical Cancer Research 2000; 6:3062-3070.

Index Ablation 36, 39, 61, 135, 167, 171, 172, 185, 188, 190-197, 271, 338-340, 342-344, 351-356 Adenocarcinoma 71, 100, 114, 121, 143, 156, 183, 194, 237, 242, 245, 259, 260, 287, 290 Adriamycin 136, 138, 158, 349 Aldehyde 81,85, 116,143 Alkylating agent 103, 157, 159, 160, 321 Amine oxidase 115, 116 Anaerobic glycolysis 73, 111 Angiogenesis 67-70, 72, 73, 81, 92-97, 124, 130,156,249 Antenna array 39, 46, 51, 167 Antiblastic peritoneal perfusion 199 Antitumor immunity 252, 294, 299 Antivascuiar drug 99, 106, 107 Antivascular effect 104,142 Apoptosis 36, 82, 86, 116, 132, 135, 142, 143, 154, 157, 161, 184, 218, 219, 227, 228, 250, 259, 262, 263, 278, 299, 306, 307, 324, 337 Arrhenius plot 30, 115, 128, 129

B Bacillus Calmette Guerin (BCG) 223, 251, 252, 276, 277, 289, 290, 293, 294, 298, 300, 301, 318, 325, 329-331 Betulinic acid 81, 85, 136, 143 Bio-heat equation 21,22,32-34,41 Bioenergetic status dl, 73, 81 Bladder cancer 159, 178, 223, 278, 293-295, 298 Bleomycin 120, 123, 135, 136, 138, 143, 158,159,219 Blood flow modification 81 Brain tumor ^%, 85, 128, 137, 143-146, 148-150, 174, 176, 279, 280, 344

CA4DP 8 1 , 8 3 , 1 3 6 , 1 4 1 , 1 4 2 Calcium blocking agents 81, 83, 141 Calculation of the produced power deposition 19 Cancer 3, 27-29, 33, 36, 60, 61, 63, GJ, G^-79, 81-86, 92, 94, 96, 99, 110, 111, 114-116, 124, 129, 131-133, 135, 137, 138, 142, 145, 146, 156-159, 162, 167, 168, 174-178, 183-188, 197, 206, 209, 218-224, 228, 237, 240-243, 245, 247, 249-251, 253, 259, 263, 264, 267, 269-280, 282, 286-290, 292-300, 302, 303, 305-308, 338, 339, 342-345, 347, 351,352,354,355 Cancer of the rectum 63 Cancer dierapy 28, 92, 94, 96, 99, 111, 115, 116, 142, 156, 158, 167, 168, 278, 295, 296, 308, 338 Capacitive coupled antenna 43 Capacitive coupled hyperthermia devices 1 G7y \7A, 175 Capacitive coupled RF-device 176 Capacitive heating 12, 14, 17,40, 168, 170, 174, 175 Carboplatin 136, 159, 218, 219, 223, 242 Carcinoma 36, 62, 63, 71, 73, 77. 80, 82, 104, 111, 115, 119, 120, 123, 134, 141, 142, 157, 160, 172, 175, 178, 183, 184, 187, 190, 191, 197, 206, 220, 223, 251, 263, 278, 280, 286-288, 292, 294-298, 305, 342, 343, 351 Carcinomatosis 199, 201, 205, 206, 218, 219, 221,222,224 CD91 248,250,262 CEM 134, 135 Chemo-radiotherapy 86, 128, 143 Chemoembolization 81-83, 140 Chemotherapy 35, 55, 61, 82-84, 92, 94, 99, 103, 104, 107, 119-123, 128, 129, 134, 135, 137,139, 144, 146, 150, 156, 157, 159, 162, 167, 173-178, 187, 199, 206, 208, 218-223, 227, 235, 237, 238, 240, 252, 258, 261, 264, 276, 280, 292, 297, 308, 309, 338, 339, 343, 345-348, 352 Choriocarcinoma 301

362 Clinical effect 227, 296 Clinical thermal dosimetxy 134 Clinical trial 52, 60, 61, 94, 97, 124, 137, 142, 145,167,170,174,175, 178, 188, 220, 221, 250, 252, 278, 295, 298, 351, 357 "Closed" technique 199 Coleys Toxin (CT) 251, 253, 264, 267, 276-278, 290, 292, 293, 303, 307 Colon cancer 206, 259, 297, 300 Colorectal cancer 141, 175, 191, 197, 206, 222, 223, 237, 242, 245, 280, 294, 297, 351 Combretastatin-A4 96, 136, 141, 142 Conformational radiotherapy (CRT) 128, 145-150,343 Corynehacterium parvum 251,264,276,288, 289, 293 COX2 inhibitor 129, 136, 143 Crank-Nicolson 21 Cryoablation 343 Cytokines 68, 70, 142, 168, 170, 219, 247-263, 265, 267, 277, 278, 282, 288-290, 293, 295-305, 306, 308, 309 Cytoreduction 199,206,222,348 Cytotoxic T lymphocyte (CTL) 248,249, 258, 261, 263, 282, 292, 299, 301, 305, 307

D 5,6-dimethylxanthenone-4-acetic acid 136, 141, 142 Danger model 261,262 Deep hyperthermia (DHT) 167-170, 172-178, 186 Degradable starch microspheres (DSM) 83, 138 Dendritic cell (DC) 115, 247-254, 260-263, 265, 267 Destructive phase 36 Diamine oxidase (DAO) 115,116 DMXXA 81, 83, 136, l 4 l , 142 DNA 93, 103, 110-112, 115, 130, 132, 136, 137, 156-159, 161,167, 209, 251-253, 259, 299 Doxorubicin (DOX) 135, 138, 159, 199, 206,219,343,347 Drug heat sequence 137

Hyperthermia in Cancer Treatment: A Primer

Electromagnetic heating 3, 40, 50 Endocavitary hyperthermia 167, 168, 172, 173 Endothelium 70, 72, 79, 80, 83, 129, 145, 249,254,300,301,307 Endotoxemia 230, 280, 306 Endotoxin 114, 277, 280, 286-291, 293-296, 299-309 Essential fatty acid (EFA) 81,136,143 Exotoxin 276, 278, 282, 286, 289, 302-305, 307-309 Extracellular pH 68, 7 5 , 1 1 . 78, 85, ^(i. 131, 139 Extreme whole body hyperthermia 31, 230, 236-238, 240-242, 245, 276

Fever 28, 39, 110, 121, 196, 245, 247, 248, 253-257, 263, 265, 267, 276-286, 288, 294, 305-309, 343, 355 Fever-range whole body hyperthermia (FR-WBH) 245,276,277 Finite-difference time-domain method (FDTD) 20,21 Flavone acetic acid (FAA) 81, 83, 107, 136, 141, 142 Formaldehyde 230, 231

Gastric cancer 206, 220, 223, 280 Gastric carcinoma 206, 220, 223, 295 Genedierapy 55, 114, 124, 158, 161, 251, 252, 259, 267, 299 Glioblastoma 61-63, 128, 143, 144, 148-150, 171,176,178,295 Glioma 104, 143-145, 175, 176, 259, 260, 295 Glucose 68, 73, 75, 78, 79, 81-85, H I , 139, 144,146,148,150,159,250 Glycolysis 7 3 , 7 8 , 1 1 1 , 1 3 9 Granulocyte macrophage colony stimulating factor (GM-CSF) 248,251,252,257, 259, 260, 278, 288-299, 301

Index

H Heat deposition 4, 81, 86, 138 Heat sensitivity 56, 77, 110, 112, 159, 258, 306 Heat shock protein (HSP) 31, 34, 37, 80, 85, 93, 110, 114, 124, 135, 143, 159, 161, 167, 168, 218, 247, 248, 250-254, 259, 260, 262, 263, 265, 267, 306-308 HIV 227, 228, 233-236 Homing 255, 259-262, 265, 282, 284, 285 Homing of immune competent cells (T-iymphocytes) 265 Hydralazine 81, 83, 141 Hyperglycaemia 78, 79, 81, 83, 85, 240, 242 Hyperproteolysis 227, 230 Hyperthermia 3, 4, 11, 15, 22, 23, 27-32, 34-40, 52, 55, 56, 61, G7, 7'b-77. 79-86, 92, 99-107, 110-116, 119-121, 123, 128, 129, 131-139, 141-146, 148, 150, 156, 159-162, 167-178, 183-188, 199, 206, 208, 209, 218-220, 222-224, 227, 228, 230-242, 245, 247, 252-254, 256-261, 263-267, 276, 277, 306-308, 338, 343, 345,349,351 Hyperthermic antiblastic peritoneal perfusion (HAPP) 199,200,205,206 Hyperthermic isolated limb perfusion 208, 209, 214 Hypoxia 35, 67-73, 75, 77, 81-83, ^^, 93, 100, 102, 103, 129-132, 136-138, 141-143, 156-159, 162, 249, 250, 306 Hypoxia inducible factor-1 (HIF-1) 67-69, 73, 131

I IL-1 83, 219, 255, 257, 258, 278, 282, 283, 289, 292, 295, 299-306, 308, 309 IL-la 142,257 IL-lp 258 Immune response 113, 114, 168,247-250, 252-254, 256, 259, 262-267, 277, 281, 287-289, 291, 293, 294, 298, 299, 301, 302, 306, 308 Immunology 27, 277 Implicit method 19, 21 In transit metastases 208, 209 Infrared-A 237-242, 245 Infrared-B 237, 238

363 Infrared-C 237, 238 Innate immunity 247, 261, 263, 264, 267 Interferon 248, 251, 254, 256, 260, 265-267, 278, 282, 286, 288, 289, 293, 295, 300-302, 347 Interstitial fluid 68, 70, 73, 7A, 137, 259, 344 Interstitial fluid pressure (IFP) 68, 70-72, 137,344 Interstitial hyperthermia 15, 103, 145, 168, 171, 184,188 Interstitial thermobrachytherapy 122 Intracavitary hyperthermia 119,123 Intracavitary hyperthermic perfusion 218 Intracellular pH 74, 78, 79, 85, 138, 139, 148 Intraperitoneal hyperthermic perfusion chemotherapy (IPHC) 219-224 Invasive method 22, 23, 352 Isolated limb perfusion (ILP) 208, 209, 214-216,257

Lactic acid 73, 74, 78, 83, 106, 144, 265 Leakage 199, 200, 211, 213-215, 297, 305, 341,347 Lidocaine 85, 143 Limb perfiision 208-210, 214-216, 257, 261, 265 Liposarcoma 287 Liposome 110, 112, 113, 115, 138,251,253, 259, 260 Liver tumor 82, 83, 172, 174, 190, 294, 338, 342, 343, 352 Local hyperdiermia 22, 96, 99, 100, 105, 120, 123, 136, 137, 141, 150, 168, 170, 184,239,258-260,264,267 Locoregional hyperthermia 167,168,177 Lonidamine 7 4 , 8 1 , 8 5 , 136 Lung cancer 36, 174, 175, 280, 288, 289, 295, 343, 354, 355 Lymphoangiogenesis 69 Lymphocyte homing 261, 262 Lymphokine-activated killer (LAK) cell 250, 251, 256, 258, 260, 265, 266, 284, 293,

297,301,302,306

364

Hyperthermia in Cancer Treatment: A Primer

M

N

Magnetic drug 138 Magnetic induction 12, 15, 17, 40, 144 Magnetic resonance (MR) 19, 23, 24, 35, 73, 104, 120, 124, 145, 148, 150, 242, 340 Medical technology 240 Melanoma 85, 104, 110-113, 115, 120, 121, 123, 124,142, 159, 171, 178, 208, 209, 215, 217, 233, 251, 260, 261, 264, 265, 279, 280, 292-294, 296-298, 301, 302, 307, 350, 351 Melphalan 136, 158-160, 214, 257, 261, 347, 349,351 Membrane 31, 36, 52-55, 70, 82, 92-94, 96, 100, 103, 110-113, 116, 129, 135, 138, 139, 142, 143, 162, 167, 176, 177, 209, 218, 219, 261, 292, 298, 299, 307 Mesothelioma 199, 206, 220, 222, 347, 348 Metabolic abnormalities 73 Metabolic microenvironment 67,99, 106, 139 Metabolism 54, 55, 68, 69, 75, 81-83, 131, 135, 136, 139,157-159, 167, 218, 219, 308, 344 Metastases 34, 37, 63, 71, 82, 96, 120, 123, 162, 172, 174, 175, 178, 188, 190-192, 194, 196, 197, 208, 209, 215, 219, 233, 245, 247, 260, 264, 265, 292-297, 302, 306, 338, 339, 343-345, 347-353 Microelectrode 73, 85 Microenvironment 67y 68, 80-83, 93, 99, 106, 138, 139, 156, 167, 247-249, 256, 261,265,306 Microenvironment modification 81-83 Micromilieu modification 139 Microvasculature 71, 92, 93, 95, 261 Mild hyperdiermia 81, 86, 113, 183, 184 Mitomycin C 83, 136, 137, 138, 141, 157-160, 199, 206, 219, 220, 222, 223, 242, 349 Mixed Bacterial Vaccine (MBV) 253, 267, 276, 277, 287, 292, 299 Modification of blood flow 93 Monoclonal antibodies (MAbs) 251, 259, 260, 267, 276, 286, 301 Monocyte 250, 292, 293, 295, 299-301, 304 MRI 4, 35, 62,104,145, 191, 195, 196, 339, 340, 352

Nano(bio)technology 60, 63 Nanoparticle 60-62, 138, 168, 172 Nanotechnology 60-63 Natural killer (NK) ceU 70, 184, 248, 249, 254, 256, 258, 262-267, 282, 284, 292-296, 299-302, 306 Neuroleptic analgesia 242 Neutrophil 248, 256, 259, 263, 265, 282, 296, 300 Node dissection 208, 209, 211,215 Non invasive method 22-24, 35, 73, 134 Non-randomized clinical trial 121, 172, 175

o OK-432 264, 276, 295 "Open" technique 199,200 Ovarian cancer 178, 206, 218, 220, 222, 223, 276, 290 Ovarian carcinoma 63, 115 Oxaliplatin 159,160,218,223 Oxaliplatinum 199,206 Oxygen enhancement ratio (OER) iGy 77y 130-132 Oxygen fi-ee radicals 110, 115 Oxygenation 67, 81, 86, 93, 104, 134, 139, 158

Pancreatic cancer 175, 220, 288 Perfiision 4, 5, 22, 32-34, 43, 67y 70, 71, 74, 75, 78, 79, 81, 86, 100, 102, 104, 106, 107, 114, 129, 138, 139, 168, 170, 173, 178, 190, 194, 199-201, 205, 206, 208-211, 213-216, 218-223, 257, 259, 261, 265, 338, 342, 345-352, 356 Perfusion procedure 351,352 Perfusion time 208,213 Perfusional hyperthermia 168, 258, 260, 338 Peritoneal carcinomatosis 199, 201, 206, 218, 219,221,222,224 Peritoneal carcinosis 218 Peritonectomy 199,201-204 pH G7y 71, 73-79, 81-86, 100, 102, 106, 123, 129,131, 133, 135, 136, 138-140, 143, 144,148,162,167,249,265

Index pH regulatory mechanisms 7Ay 75, 140 Pharmacokinetics parameters 135, 136, 345 Phase I study 142 pHe 68, 73-75, 77, 78, 85, 136, 138, 139 pHi 7 3 , 7 4 , 7 7 , 7 8 , 8 5 , 1 3 8 , 1 3 9 Photodynamic therapy 99, 103-107 Physics 3, 4, 7, 29, 30, 32, 37, 56 Pleural effusion 294, 295, 343, 353, 355, 356 Polyamines 136, 143 Power deposition 19, 21, 145, 146 Propioni bacteria (PB) 276, 293, 294 Prostate cancer 63, 183-188, 259 Protein A 297 Protein denaturation 128 Protein synthesis 111,112, 250, 301 Proteinase inhibitor 230, 308 Proteolysis 227, 230, 233, 235, 253, 296 Pseudomyxoma peritonei (PMP) 199, 201, 203, 206, 220-222 Pyrogens 170, 255, 276, 282-285, 299

Quercetin 7^, 81, 85, 136, 143, 148

R Radiofrequency (RF) 25, 36, 38, 4 1 , 43, 80, 86, 103, 119-122, 143, 145, 168-172, 174-177, 184, 190-197, 338-342, 352 Radiofrequency hyperthermia 143 Radiofrequency thermal ablation (RFA) 190, 192, 194, 196, 197, 338-344, 351-357 Radioimmunodierapy (RI) 259, 260 Radiotherapy 4, 27, 35, 38, 61, 63, 78, 82-86, 92-94, 97, 99, 102-104, 107, 119-123, 128, 130, 132, 134, 137, 139, 141-143, 145-148, 150, 156, 158, 162, 167, 170-175, 177, 183, 185, 186, 227, 256, 259, 264, 276, 292, 339, 343, 347 Randomised studies 119, 121, 122, 145, 190, 220, 224, 264, 295 Rapid heating 81, 86 Reconstruction time 215 Regional chemotherapy 345 Regional hyperthermia 184 Rhabdomyosarcoma 103, 279, 292 RNA 93, 110-112, 161, 233, 234, 236, 282 ROS 115

365

Sarcoma 63, 171, 173, 175, 206, 208, 215, 220, 222, 253, 260, 261, 264, 265, 276, 277, 280, 286, 287, 292-294, 297-299, 305, 306, 309 Semiclosed HAPP technique 199,200,205 Serotonin 81, 83, 141, 142 Spherex 83, 138 Spontaneous remission 277, 280, 281, 290 Staphylococcus protein A 276, 297 Starch microspheres 83,138 Streptokinase 276, 290, 295-297, 308 Stroma 140, 183 Superficial hyperthermia (SHT) 167,170, 171 Systemic cancer multistep therapy (SCMT) 237, 240-245

T cell receptor (TCR) 247-249, 298, 303 TAA 247,251,252 TBF deprivation 140 TBF reduction 81, 140 Therapeutic use 162,277,289,296 Thermal ablation 135, 171, 172, 185, 197, 338, 340, 342, 352 Thermal enhancement ratio (TER) 30, 122, 132-134, 160 Thermo-chemo-radiotherapy 86, 128, 143 Thermoablation 39, 6 1 , 167, 188, 351 Thermocouples 19, 23, 24, 184, 341 Thermodynamic approach 54 Thermometry 4, 5, 19, 24, 121, 184, 193 Thermoradiotherapy 78, 83, 85, 107, 264 Thermoresponse G7y 81, 82 Thermosensitization 75, 135 Thermotherapy 39, 60, 61, 78, 83, 85, 168, 172, 184 Thermotolerance 77, 80, 82, 84-86, 93, 114, 115, 129, 133, 135, 139, 143-145, 148, 250, 307 T N F - a 141, 142, 168, 219, 248, 250-254, 256-261, 263, 267, 278, 280, 282, 289, 295, 299-302, 304-309, 347, 350, 351 Trimodality treatment 137,144 Trypsin 229-232, 235, 308, 309 Trypsinemia 230-232

366 Tumor 19, 27-31, 33-36,42, 55, 56, 60-63, 67-86, 92-97, 99-107, 120, 121, 123, 124, 128-131, 133-135, 137-150, 156, 160, 161, 167, 168, 170-176, 178, 183-187, 190-192, 194-197, 199, 201, 206, 208, 209, 213, 214, 218-220, 222-224, 247-254, 256, 257, 259-265, 267, 276-281, 286-303, 305-308, 338-345, 347-349, 352-357 Tumor bioenergetic status 73 Tumor blood flow (TBF) 71, 72, 78-84, 93, 99, 102, 103, 107, 129, 130, 138-142, 306 Tumor blood flow reduction 78, 83 Tumor immunity 247, 261-263, 267 Tumor immunotherapy 249 Tumor infiltrating lymphocytes (TIL) 250, 251,262,301,302 Tumor membrane 112, 138 Tumor microenvironment G7y 80-82, 99, 139, 247-249, 261 Tumor neovascularization 70, 97 Tumor perflision G7, 75, 81, 100, 104, 106, 107, 129,139 Tumor specific surface antigens 247 Tumor vascidar morphology G7, 70

u Ultrasound (US) 4, 5, 11, 12, 18, 23-25, 39, 120, 124, 168, 170, 172, 184-186, 191, 195,338-340,343

Hyperthermia in Cancer Treatment: A Primer

Vascular eff^ect 99, 100, 102, 104, 106 Vascular endothelial growth factor (VEGF) 68-70, 72, 92, 94-97, 129, 131, 249, 252,261,287 Vascular targeting agent (VTA) 81, 83, 136, 139-141 Vasculature 22, G7, 70, 7A, 79, 80, 92-97, 100, 104,107,140,141,261 Vasculogenesis 70 Vector potential 3,15 Vessel cooption 70

w Water-filtered infrared-A radiation (WIRA) 237-242, 245 Whole body hypertiiermia (WBHT) 31, 136, 227, 228, 230, 232-238, 240-242, 245, 247, 256-261, 263-267, 276, 277, 306, 308