Targeting Treatment of Soft Tissue Sarcomas (Cancer Treatment and Research)

TARGETING TREATMENT OF SOFT TISSUE SARCOMAS

Cancer Treatment and Research Steven T. Rosen, M.D., Series Editor Klastersky, J. (ed): Infectious Complications of Cancer. 1995. ISBN 0-7923-3598-8. Kurzrock, R., Talpaz, M. (eds): Cytokines: Interleukins and Their Receptors. 1995. ISBN 0-7923-3636-4. Sugarbaker, P. (ed): Peritoneal Carcinomatosis: Drugs and Diseases. 1995. ISBN 0-7923-3726-3. Sugarbaker, P. (ed): Peritoneal Carcinomatosis: Principles of Management. 1995. ISBN 0-7923-3727-1. Dickson, R.B., Lippman, M.E. (eds.): Mammary Tumor Cell Cycle, Differentiation and Metastasis. 1995. ISBN 0-7923-3905-3. Freireich, E.J, Kantarjian, H. (eds): Molecular Genetics and Therapy of Leukemia. 1995. ISBN 0-7923-3912-6. Cabanillas, F., Rodriguez, M.A. (eds): Advances in Lymphoma Research. 1996. ISBN 0-7923-3929-0. Miller, A.B. (ed.): Advances in Cancer Screening. 1996. ISBN 0-7923-4019-1. Hait, W.N. (ed.): Drug Resistance. 1996. ISBN 0-7923-4022-1. Pienta, K.J. (ed.): Diagnosis and Treatment of Genitourinary Malignancies. 1996. ISBN 0-7923-4164-3. Arnold, A.J. (ed.): Endocrine Neoplasms. 1997. ISBN 0-7923-4354-9. Pollock, R.E. (ed.): Surgical Oncology. 1997. ISBN 0-7923-9900-5. Verweij, J., Pinedo, H.M., Suit, H.D. (eds): Soft Tissue Sarcomas: Present Achievements and Future Prospects. 1997. ISBN 0-7923-9913-7. Walterhouse, D.O., Cohn, S. L. (eds.): Diagnostic and Therapeutic Advances in Pediatric Oncology. 1997. ISBN 0-7923-9978-1. Mittal, B.B., Purdy, J.A., Ang, K.K. (eds): Radiation Therapy. 1998. ISBN 0-7923-9981-1. Foon, K.A., Muss, H.B. (eds): Biological and Hormonal Therapies of Cancer. 1998. ISBN 0-7923-9997-8. Ozols, R.F. (ed.): Gynecologic Oncology. 1998. ISBN 0-7923-8070-3. Noskin, G. A. (ed.): Management of Infectious Complications in Cancer Patients. 1998. ISBN 0-7923-8150-5. Bennett, C. L. (ed.): Cancer Policy. 1998. ISBN 0-7923-8203-X. Benson, A. B. (ed.): Gastrointestinal Oncology. 1998. ISBN 0-7923-8205-6. Tallman, M.S., Gordon, L.I. (eds): Diagnostic and Therapeutic Advances in Hematologic Malignancies. 1998. ISBN 0-7923-8206-4. von Gunten, C.F. (ed): Palliative Care and Rehabilitation of Cancer Patients. 1999. ISBN 0-7923-8525-X Burt, R.K., Brush, M.M. (eds): Advances in Allogeneic Hematopoietic Stem Cell Transplantation. 1999. ISBN 0-7923-7714-1. Angelos, P. (ed.): Ethical Issues in Cancer Patient Care 2000. ISBN 0-7923-7726-5. Gradishar, W.J., Wood, W.C. (eds): Advances in Breast Cancer Management. 2000. ISBN 0-7923-7890-3. Sparano, Joseph A. (ed.): HIV & HTLV-I Associated Malignancies. 2001. ISBN 0-7923-7220-4. Ettinger, David S. (ed.): Thoracic Oncology. 2001. ISBN 0-7923-7248-4. Bergan, Raymond C. (ed.): Cancer Chemoprevention. 2001. ISBN 0-7923-7259-X. Raza, A., Mundle, S.D. (eds): Myelodysplastic Syndromes & Secondary Acute Myelogenous Leukemia 2001. ISBN: 0-7923-7396. Talamonti, Mark S. (ed.): Liver Directed Therapy for Primary and Metastatic Liver Tumors. 2001. ISBN 0-7923-7523-8. Stack, M.S., Fishman, D.A. (eds): Ovarian Cancer. 2001. ISBN 0-7923-7530-0. Bashey, A., Ball, E.D. (eds): Non-Myeloablative Allogeneic Transplantation. 2002. ISBN 0-7923-7646-3. Leong, Stanley P.L. (ed.): Atlas of Selective Sentinel Lymphadenectomy for Melanoma, Breast Cancer and Colon Cancer. 2002. ISBN 1-4020-7013-6. Andersson , B., Murray D. (eds): Clinically Relevant Resistance in Cancer Chemotherapy. 2002. ISBN 1-4020-7200-7. Beam, C. (ed.): Biostatistical Applications in Cancer Research. 2002. ISBN 1 -4020-7226-0. Brockstein, B., Masters, G. (eds): Head and Neck Cancer. 2003. ISBN 1-4020-7336-4. Frank, D.A. (ed.): Signal Transduction in Cancer. 2003. ISBN 1-4020-7340-2. Figlin, Robert A. (ed.): Kidney Cancer. 2003. ISBN 1-4020-7457-3. Kirsch, Matthias; Black, Peter McL. (ed.): Angiogenesis in Brain Tumors. 2003. ISBN 1-4020-7704-1. Keller, E.T., Chung, L.W.K. (eds): The Biology of Skeletal Metastases. 2004. ISBN 1-4020-7749-1. Kumar, Rakesh (ed.): Molecular Targeting and Signal Transduction. 2004. ISBN 1-4020-7822-6. Verweij, J., Pinedo, H.M. (eds): Targeting Treatment of Soft Tissue Sarcomas. 2004. ISBN 1-4020-7808-0. Finn, W.G., Peterson, L.C. (eds.): Hematopathology in Oncology. 2004. ISBN 1-4020-7919-2.

TARGETING TREATMENT OF SOFT TISSUE SARCOMAS

edited by

Jaap Verweij Department of Medical Oncology Erasmus University Medical Center Rotterdam, The Netherlands and

Herbert M. Pinedo Department of Medical Oncology Free University Medical Center Amsterdam, The Netherlands

KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

eBook ISBN: Print ISBN:

1-4020-7856-0 1-4020-7808-0

©2004 Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow Print ©2004 Kluwer Academic Publishers Boston All rights reserved No part of this eBook may be reproduced or transmitted in any form or by any means, electronic, mechanical, recording, or otherwise, without written consent from the Publisher Created in the United States of America Visit Kluwer Online at: and Kluwer's eBookstore at:

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Table of Contents 1.

2.

Targeted therapy: Ready for prime time? C. Seynaeve and J. Verweij, Erasmus University Medical Center, Rotterdam, The Netherlands Volume-based radiotherapy targeting in soft tissue sarcoma Iain Ward, Tara Haycocks, Michael Sharpe, Anthony Griffin, Charles Catton, David Jaffray, Brian O’Sullivan, Princess Margaret Hospital, University of Toronto, Toronto, Canada

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3.

Preoperative therapy for soft tissue sarcoma 43 Janice N. Cormier, Howard N. Langstein, Peter W. T. Pisters, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

4.

TNF-based isolated limb perfusion: A decade of experience with antivascular therapy in the management of locally advanced extremity soft tissue sarcomas Dirk J Grünhagen, Flavia Brunstein, Timo L.M. ten Hagen, Albertus N. van Geel, Johannes H.W. de Wilt, and Alexander M.M. Eggermont, Erasmus University Medical Center, Rotterdam, The Netherlands

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5.

Pitfalls in pathology of soft tissue sarcomas Judith V.M.G. Bovée and Pancras C.W. Hogendoorn, Leiden Universtiy Medical Center, Leiden, The Netherlands

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Molecular biology and cytogenetics of soft tissue sarcomas: Relevance for targeted therapies Jonathan Fletcher, Brighams’ and Womens Hospital, Boston, MA, USA KIT and PDGF as targets Jaap Verweij, Erasmus University Medical Center, Rotterdam, The Netherlands Targeting mutant kinases in gastrointestinal stromal tumors: A paradigm for molecular therapy of other sarcomas Mike C. Heinrich, Christopher L. Corless, Portland, OR, USA Targeting other abnormal signalling pathways in sarcoma: EGFR in synovial sarcomas, in liposarcoma Jean-Yves Blay, Isabelle Ray-Coquard, Laurent Alberti, Dominique Ranchere, Hospital Edouard Herriot, Lyon, France Angiogenesis: A potential target for therapy of soft tissue sarcomas K. Hoekman and H.M. Pinedo, Free University Medical Center, Amsterdam, The Netherlands

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Index

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Preface The last decade we have witnessed a major change in the development of new techniques and agents for the treatment of cancer in general, and for soft tissue sarcomas in particular. The important achievements of molecular biology research have changed the landscape markedly. Increasingly subtypes of soft tissue sarcomas are shown to be related to changes in cellular growth factors in the cell signaling pathways. This in theory enabled to development of agents with specific activity against these factors. The presence of the KIT receptor at the surface of the gastrointestinal stroma tumor cell, and the constitutive activation by mutations, has lead to the discovery of the specific KIT tyrosine kinase inhibitor Imatinib, an agent with impressive activity in this disease. Before the era of Imatinib, GIST was an untreatable disease once metastasized. Imatinib clearly is a breakthrough in the approach of soft tissue sarcomas, and will likely serve as a role model for the development of other agents acting towards other receptors. Likewise, soft tissue sarcomas with their specific molecular characteristics, will likely serve as role model diseases for targeted treatment approaches. Whether the future lies in drugs with selective inhibition of only one receptor and one pathway, or multiple receptors and multiple pathways is currently a matter of debate, and again soft tissue sarcomas serve as role model. Fully in line with the more targeted approach in drug use, the changes in the field of radiation therapy and surgery basically also focus on a better targeting of the disease, albeit not based on the molecular characteristics yet. The present volume of this series reflects all of the above mentioned changes. World wide renowned experts have been willing to contribute to this book, and we would like to thank all of them for their efforts. Hopefully this book will contribute to a better understanding of the changes in the field, and will serve our patients in helping getting a better future. Jaap Verweij Herbert M. Pinedo Editors

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Chapter 1 Targeted therapy: Ready for prime time?

Caroline Seynaeve and Jaap Verweij

Dept. of Medical Oncology, Erasmus University Medical Centre-Daniel den Hoed Cancer Centre, Rotterdam, the Netherlands

Correspondence to: Caroline Seynaeve, MD, PhD Dept. of Medical Oncology, Erasmus University Medical Centre-Daniel den Hoed Cancer Centre, Groene Hilledijk 301 3075 EA Rotterdam, the Netherlands

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INTRODUCTION

Soft tissue tumours are a heterogeneous group of rare neoplasm’s with several histiotypes that all share a putative common mesenchymal origin, and account for only 1% of all adult malignancies. Variation in pathological definition has made it difficult to obtain exact numbers of patients with sarcomas. Based on the Surveillance, Epidemiology, and End Results (SEER) database, approximately 8,000 new cases of soft tissue sarcoma (STS) are diagnosed each year in the United States, while 4000 (50%) are dying per annum due to advanced or metastatic disease. This is a 10 times greater mortality that that of testicular cancer or Hodgkin’s disease, diseases with a similar or lower incidence (Stojadinovic et al, 2002). Since metastatic disease is only amenable to curative therapy in very selected cases (Blay et al, 2003) and very few drugs are available with meaningful activity, the search for effective systemic agents remains extremely important in order to improve the outcome and decrease mortality. Progress has long been modest and slow because of the rarity of the disease, the lack of systematic referral of adult patients to specialised centres also being influenced by the advanced age the disease mostly occurs (> 50 years), and the insufficient tumour selectivity of therapies. Further, research in the field of soft tissue sarcoma (STS) has been hampered by the wide range of histological appearances, with overlapping architectural and cytological characteristics, within which at this moment more than 50-100 different entities have been described (Weiss/Goldblum, 2001). Still new entities are being defined by means of improvement in light and electron microscopical appearances, immunohistochemical and molecular biological tools. While it has longer been recognised that many of the subtypes are associated with distinctive clinical and prognostic features, until recently a “onesize-fits-all” approach has been pursued because of the lack of truly targeted therapies. Being aware that this will change in the near future, results of randomised clinical trials on the efficacy of systemic therapy incorporating different subtypes are already difficult to interpret to day, and unfortunately probably will become even less informative in the future. Nevertheless, progress in diagnosis and therapy of adult STS over the last decades of the past century has been achieved resulting from improvement in pathological definition, imaging techniques, staging, surgical operating procedures and advances in limb preservation, the use of radiotherapy as an adjunct to other treatment modalities, a better delineation of the activity of the available chemotherapeutic agents doxorubicin and ifosfamide, and the search for new active drugs. Not to forget, advance has been obtained by the concerted action of the different specialists involved in the care of sarcoma patients working together in a multidisciplinary setting, while the efforts of co-operative

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groups facilitating cross-talk with respect to the communication of trial results and the initiation of new ideas for further studies greatly contributed. The start of this new century is characterised by exciting developments in all of the above as well as in genetic profiling of tumour specimens linking some sarcoma subtypes with distinct histopathological differences to each other and resulting in clarification of the classification of different sarcoma subtypes, and the molecular identification of oncogenes and protein products that will enable the development of targeted therapies. While the administered chemotherapy in the last century seemed independent of the subtype of STS, we are entering an era shifting towards targeted therapy for a specific subtype which hopefully will yield more benefit for both the patient and the scientific research group. As said, clinical trial design will also undergo change to reflect the nature of these therapies. However, since this strategy is not yet to-days practice, attempts to refine the currently available therapeutic armamentarium to maximise the therapeutic index by means of dose intensification and the identification of new agents with certain activity also remain of paramount importance. On the way to a new approach and further advance in systemic therapy in STS, various issues may be of importance which we will address in the following paragraphs: optimal use of systemic therapy at the beginning of the century, new and pipeline agents, molecular targets and signal transduction pathways, and changing methodology on testing new agents for activity.

2 CENTURY

CHEMOTHERAPY AT THE BEGINNING OF THE

Although over the last decades several known and new compounds have been tested for activity in STS, only doxorubicin and ifosfamide have meaningful activity. For both drugs a dose-response curve in STS has been identified, with higher response rates for doxorubicin administered at a cycle dose of and ifosfamide at a cycle dose of or more. Reported single agent response rates vary between 16-36%. Dacarbazine, while yielding some activity, has only shown short lasting responses of limited value (Seynaeve/Verweij, 1999; O’Sullivan et al, 2002). The value of these drugs has been studied in neo-adjuvant, adjuvant and metastatic setting.

2.1

(Neo-)Adjuvant Chemotherapy

The value of neo-adjuvant or adjuvant chemotherapy continues to be a matter of debate. Although the only randomised study investigating the value of neo-adjuvant therapy, by means of standard doses of adriamycin/ifosfamide, conducted by the EORTC failed to show any benefit in disease free and overall

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survival (Gortzak et al, 2001), the question remains actual since other groups have suggested impressive response rates using more dose-intensive regimens (Patel et al, 1998; Patel, 2002). The discussion has recently been stirred up by the results of the study by Delaney et al. reporting on the activity of MAID (mesna, adriamycin, ifosfamide and dacarbazine) interdigitated with radiotherapy and followed by surgery and postoperative chemotherapy in a subset of STS at very high risk of distant metastasis (Delaney et al, 2003, O’Sullivan/Bell, 2003). In comparison with a cohort of historical controls, the MAID regimen resulted in a dramatic improvement of distant disease free, disease-free and overall survival being 75% and 44%, 70 and 42%, and 87% and 58% respectively, all being statistically significant. As appealing these data may seem however, they have to be interpreted with great caution since all results come from non-randomised studies, while this approach should be investigated in randomised studies to avoid for bias in the comparative groups before it can be incorporated as standard of care. A similar discussion is ongoing with respect to the role of adjuvant therapy in STS (Verweij/Seynaeve, 1999). The most powerful evaluation of the value of chemotherapy (doxorubicin-based, standard doses versus control) originated from the Sarcoma Meta-Analysis Collaboration (SMAC) and showed a statistically significant improvement in local relapse-free survival (6%), distant metastasis-free (10%) and disease-free survival (10%) for treated patients, but only a trend toward an increased overall survival (4%) after a median follow-up of 9.4 years (SMAC, 1997). In 2001, the Italian Sarcoma Group reported that an intensified chemotherapy regimen consisting of epirubicin/ifosfamide in comparison with a control group, resulted in a significant increase of disease-free (48 versus 16 months, p=.04) and overall survival (75 versus 46 months, p=.03) in high-risk STS after a follow up of 59 months. Although this study was prematurely closed because of the interim results in favour of the chemotherapy group, it has to be noticed that where fewer metastatic events were seen at 2 years in the chemotherapy group (28% vs. 45%), identical metastatic rates were observed at the 4-year time point (Frustaci et al, 2001). Long term follow-up data from this study are therefore crucial, especially, where two other small studies using intensive anthracycline/ifosfamide regimens failed to confirm a benefit (O’Sulllivan/Bell, 2003). Further, the data of the ongoing EORTC study investigating the value of doxorubicin versus controls will hopefully add relevant information on this issue

2.2

Metastatic disease

In contrast with above-mentioned settings, the aim of systemic therapy in metastatic disease is disease control, symptom palliation and prolonged survival. As there are only modest gains in survival with the use of known chemotherapeutic agents, studies in the last decades of the century have focused on schedule optimisation and/or dose intensification (Bramwell et al,

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2000; Seynaeve/Verweij, 2002). Conclusions from these studies are that combination chemotherapy regimens produced higher response rates and more toxicity, but did not improve the complete response rate, the time to failure or overall survival. Some recent studies investigating more dose-intensive anthracyclin/ifosfamide regimens show unexpectedly high response rates, and resulted in a higher complete response percentage that may be important aiming at improved survival in selected individuals (Patel et al, 1998; Blay et al, 2003). However, these regimens have not yet been tested in a randomised setting. Therefore, the recently activated international world-wide EORTC-lead study, realised through the global co-operation of different collaborative groups, in which patients are randomised between doxorubicin (control) and a dose-intensified regimen (doxorubicin plus ifosfamide with G-CSF support) is warmly welcomed, while results are eagerly awaited and hopefully will bring an answer to a long-lasting question.

3 SYSTEMIC THERAPY – OLD DRUGS IN A NEW JACKET / NEW DRUGS Over the last decades of clinical research it has been recognised that many of the STS subtypes are associated with distinctive clinical and prognostic features. Therefore, it has already longer been questioned whether the “one-sizefits-all-approach” with respect to chemotherapy as has been applied till now is still appropriate. An analysis of the Soft Tissue and Bone Sarcoma Group Study (STBSG) of the EORTC into prognostic factors for the outcome of chemotherapy in advanced STS reported in univariate analysis an increased overall survival (OS) in lipo- and synovial sarcoma (SS), a decreased survival time in malignant fibrous histiocytoma (MFH), a lower response rate in leiomyosarcoma (LMS), and a higher response rate in liposarcoma (p<0.05, for all log-rank and X2 tests). In multivariate analysis, the subtype dropped out of the logistic model as independent prognostic factor (van Glabbeke et al, 1999). Comments that should be considered hereby are: in the multivariate analysis liver involvement, irrespective of the subtype, was included as covariate, being the more important with respect to the subtypes as for example synovial sarcoma where this metastatic pattern is only infrequently observed; the EORTC database has been set up at a moment that the histologic entity of “gastrointestinal stromal tumours” (GIST) had not yet been identified, and therefore a number of so-called leiomyosarcomas probably were GIST’s which at this moment are known to be chemotherapy-insensitive. In a more recent publication of the EORTC, progression-free rates (PFR) at 6 months (as an alternative for response rate) in non-pre-treated patients differed between subtypes, showing a PFR > 50% in synovial and liposarcoma, and in LMS and MFH (see figure 1) (van Glabbeke et al, 2002). This is in accordance with the clinical observation that SS is more chemo-sensitive. Whether this chemosensitivity indeed especially

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concerns high-dose ifosfamide as has been reported in small series (Rosen et al, 1994, Spillane et al, 2000), remains to be proven in larger studies. However, the above mentioned may partly explain why some investigators report impressive results of a certain agent in a specific STS subtype, albeit that this was never observed in properly designed studies allowing inclusion of a variety of subtypes. Paxlitaxel has been studied in patients after or without prior exposure to first line chemotherapy showing response rates between 0% and 12,5% (Blacerzak et al, 1995; Patel et al, 1997; Casper et al, 1998). Despite the lack of meaningful activity of paclitaxel for the whole STS group, responses in two of these studies were seen in patients with an angiosarcoma (Blacerzak et al, 1995; Casper et al, 1998). Based on these observations, Fata et al. reviewed their institutional experience with paclitaxel (different schedules) in nine patients with an angiosarcoma of the scalp/face, and found an impressive response rate of 89% with a median duration of 5 months (Fata et al, 1999). Attempts are being undertaken to try to investigate this further in a global study. Fewer studies have investigated docetaxel. In an initial phase II study conducted by the EORTC activity was seen in 18% of the patients. In a subsequent randomised study by the same EORTC group with doxorubicin as the control agent, docetaxel did not show any activity in 34 patients including 3 patients with an angiosarcoma, while a response was seen in 1/3 angiosarcoma patients in the doxorubicin arm (Verweij et al, 2000). In the EORTC analyses, LMS was reported to be less chemo-sensitive. One has to be aware that these data certainly are influenced by the fact that some of the LMS nowadays would be classified as GIST, known to be chemo-resistant. Investigating the value of gemcitabine in STS in small phase II studies some activity has been observed, with a pharmacological advantage being suggested with a 150-minute infusion. Responses to gemcitabine were particularly seen in uterine leiomyosarcoma whereas none were noted in gastrointestinal LMS (Spath-Swalbe et al, 2000; Patel et al, 2001; Svancarova et al, 2002; Okuno et al, 2002 and 2003). Interestingly, the combination of gemcitabine and docetaxel showed an impressive response rate of 53% in a nonrandomised phase II study in chemotherapy-naive or pretreated LMS, being 55% in uterine and 40% in other LMS, respectively. The performed pharmacokinetic evaluation in this study demonstrated that the 90-minute infusion time resulted in approximately 50% longer period of time above the gemcitabine concentration threshold of which may be important for greater DNA incorporation of gemcitabine affecting cell kill (Hensley et al, 2002). Although it is difficult to assess from this small study whether the high response rate is caused by the chemo-sensitivity of uterine LMS, which also has been suggested by others (Leyvraz et al, 2001; Pautier et al, 2002), the longer infusion schedule of gemcitabine or the synergistic activity of the chemotherapy combination, or just chance, these results are interesting enough to warrant further investigation into this issue.

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ET-743 (Trabectedin), a cytotoxic tetrahydroisoquinolone alkaloid isolated from a murine organism that binds to DNA and causes single strand breaks resulting in cell death, has over the last years been studied as novel antitumor agent in STS, because it had shown potent antiproliferative activity in vitro (Delaloge et al, 2001). While objective response rates in second line therapy for the whole group were modest, ranging between 6% and 8%, better response rates were seen in LMS and liposarcoma (14% and 16%) and the response duration was months (le Cesne, 2002). Even more important, in all studies performed in Europe and the United States impressive long-lasting major responders as well as a high number of durable stable diseases (+/- 50%) were seen. Overall survival at 12 months was consistently between 45-55%, for a category of patients being refractory to first line therapy and starting on the drug at documented progression (Demetri, 2002; Brain, 2002). The observation of a long durable response and stable disease, associated with clinical benefit in symptomatic disease, resulted in reconsideration of the optimal way of response assessment of novel antitumor agents in STS, as we will comment on later.

4 MOLECULAR TARGETS AND SIGNAL TRANSDUCTION PATHWAYS IN SOFT TISSUE SARCOMA

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The success of imatinib mesylate in treatment of GIST has led to a better appreciation of how studies of STS can enhance the understanding of cancer biology and development of targeted therapies. It also has taught us that expression of a target may not be enough to build a targeted clinical trial upon, but that it may be relevant to conduct model studies on the functional relevance of the target for tumour growth. Once this is established there is a justification to perform clinical studies directed towards this target. If functional relevance can not be proven because of the lack of appropriate assays, one has to be aware that the pragmatism of nevertheless studying the target in a clinical setting may be useful and may yield relevant information. The identification of new and relevant targets being involved in STS is one of the crucial steps. Microarray analysis allowing for the determination of gene expression profiles in sarcoma specimens may prove to be most useful hereby. Nielsen et al. recently reported data from a set of 41 sarcomas and described characteristic expression profiles for GIST (with kit being the discriminator gene), monophasic synovial sarcomas involving the retinoic-acid pathway and the epidermal-growth-factor receptor (EGF), subgroups of LMS (calponin-positive and –negative), while MFH and liposarcoma exhibited considerable heterogeneity (Nielsen et al, 2003). Lee et al., studying gene expression profiles in SS, LMS and MFH (n=9 in each group), also found a distinct pattern in SS, and identified a subset of MFH, but did not distinguish two separate LMS groups (Lee et al, 2003). Unfortunately, there was very little overlap between the identified genes in the two SS clusters, on one hand because other gene sets have been studied and selected, on the other hand possibly influenced by the subtype of SS studied which is not specified in the study of Lee et al. Further, these findings reflect that it is necessary to study large enough sarcoma samples before coming to conclusions. However, the observation that EGF is expressed in monophasic SS, while erb-B2 expression has been demonstrated in the epithelial component of biphasic SS (Nielsen et al, 2003; Borden et al, 2003) opens avenues to test the value of EGF-inhibitors or herceptin in this disease, which is further addressed in chapter 9. In addition, in vitro studies have shown that epithelioid sarcoma cells overexpressing EGFR1 respond to EGFR1-antibody therapy, lending support to test these inhibitors in epithelioid sarcoma. In inflammatory myofibroblastic tumours (IMT’s) composed of spindled mesenchymal cells admixed with a striking inflammatory infiltrate predominantly consisting of plasma cells and lymphocytes, the neoplastic nature became apparent by the observation that translocations and other rearrangements in the short arm of chromosome 2 occurred in the IMT spindle cells. These aberrations create fusion oncogenes that encode activated forms of the ALK receptor tyrosine kinase. ALK fusion oncoproteins are also characteristic for many anaplastic large cell lymphomas (Tuveson/Fletcher, 2001). Specific antagonists of these proteins may be effective in this type of diseases, but are not yet available.

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Most dermatofibrosarcoma protuberans tumours contain a translocation of chromosomes 17 and 22, resulting in oncogenic juxtaposition of the COL1A1 and platelet-derived growth factor beta genes. Since imatinib mesylate

is also an inhibitor of and it is assumable that glivec may be effective in this disease. Activity of glivec in this entity, indeed, has been reported in two case reports, underscoring this assumption (Schuetze et al, 2002; Labropoulos et al, 2003). Likely, most desmoplastic round cell tumours (DRCT’s) express an EWS-WT1 fusion oncogene resulting from a translocation of chromosomes 11 and 22. The oncoprotein EWS-WT1 is a transcriptional regulator inducing expression of which binds and activates both PDGFreceptors and (Tuveson/Fletcher, 2001). Since DRCT’s are quite chemotherapy-resistant it certainly is worthwhile to study the activity of glivec in this entity as well. This topic is further highlighted in chapter 9. The use of farnesyl transferase inhibitors (FTI) to target the oncogenic ras protein may be applicable in sarcomas overexpressing the ras protein, as is the case in the neurofibromatosis syndrome 1, which can predispose to malignant peripheral nerve sheath tumours (MPNST). FTIs can inhibit the trafficking of ras protein to the cell membrane by inhibition of the farnesylation of this protein (Scappaticci/Marina, 2001). Liposarcomas tend to be of low grade and generally have a better survival than many other STS subtypes. There is some laboratory evidence that the heterodimeric complex of peroxisome proliferator-activated receptor-gamma and the retinoid acid receptor (RAR) alpha functions as a central regulator or adipocyte differentiation, while it has been demonstrated that human liposarcoma cells can be induced to undergo terminal differentiation by treatment

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by PPAR ligand pioglitazone. Demetri et al. have used troglitazone, an analogue drug mediating differentiation, to attempt differentiation in poorly differentiated subtypes of liposarcoma (Scappaticci/Marina, 2001). This is further addressed in chapter 9. Finally, angiogenesis inhibitors are of great interest in STS, on one hand because of the important involvement of vessels in vascular sarcomas e.g. angiosarcomas and hemangioendotheliomas, and on the other hand because it may give us yet another mechanism to attack STS. In chapter 10 an overview is provided.

5 CHANGING METHODOLOGY ON TESTING NEW AGENTS FOR ACTIVITY Although phase II studies performed in a small group of patients suffer from biases with respect to random and prognostic variables (age, performance status, disease free interval, histotype....) or other unknown variables, they remain useful as screening studies to evaluate whether a new agent has biological activity (rather than therapeutic benefit) in the intended cohort of patients. If the results of the phase II trial are consistent with activity as expected from an active drug, the new agent deserves further testing. If results are consistent with the level of activity from an inactive drug, then the experimental agent is rejected from further investigation. Sample size is computed to ensure that these 2 decision rules are mutually exclusive. The statistical design to use by preference remains the two steps optimal and minimax design as proposed by Simon (van Glabbeke et al, 2002) Response to therapy, based on a measured decrease in the size of objective lesions, is considered the most effective end-point to document biological activity of cytotoxic agents. However, for non-cytoreductive anticancer drugs, as for example for signal transduction inhibitors and angiogenesis inhibitors, biological activity is frequently not expected to translate into a diminution of lesions, but rather in slowing down or arrest the growth acceleration. This still may result in clinical benefit (decrease of symptoms, improved quality of life, increase of progression free survival). The benefit of static disease (long term SD) during a certain therapy has since long been recognised for breast cancer, showing that survival in patients with durable SD (> 6 months) was similar to the survival in patients achieving an objective response for both first- and second-line endocrine therapies (Robertson et al, 1997 and 1999). However, these observations have been obtained in randomised studies, while data on stable disease rate in most phase II studies are lacking. Nevertheless, the data on ET-743 in STS suggest that stable disease induced by a cytotoxic agent may be worthwhile and actually underestimated in this entity. This is further substantiated by a recent retrospective literature review on a large number of cytotoxic agents tested in STS (Verweij/van Glabbeke, 2003).

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Since in phase II screening studies of biological agents response rate is not always an appropriate endpoint, an alternative endpoint may be “progression free survival/rate” (PFR) or “time to progression”. This endpoint has properly been defined by the RECIST Working Group and was considered a valuable alternative to estimate the biological activity of this type of agents in phase II studies by the EORTC and also by others (Therasse et al, 2000; Korn et al, 2001). However, the appropriate time point of assessing PFR has not been clearly defined, partly because this may be disease- or agent-specific and therefore impossible to define in general. The purpose of the earlier mentioned analysis of the EORTC studying the PFR in STS obtained by an active or an inactive agent, in the assumption that doxorubicin, ifosfamide and dacarbazine (as generally accepted) are the only active agents, has exactly been set up to clarify this for STS (van Glabbeke et al, 2002). In pre-treated patients (n=380), which is the relevant group for the purpose of phase II screening studies, the 3- and 6- months progression free rates were respectively 39% and 14% for an active drug (n=234), and 21% and 8% for an inactive agent (n=146). For the whole cohort, the rates were 28% and 10%. The Kaplan-Meier estimate of the PFR in patients treated with an active of an inactive drug is shown in figure 2 (standard error approximately 5%). The selection of an appropriate time point for PFR is known to be a compromise between the need to avoid false-positive trials, and practical burdens coincided with a long period of observation. If the disease is slowly progressing, absence of objective progression at the first evaluation (generally 6-8 weeks after the start of therapy) may not reflect any substantial drug activity. In the EORTC study there was no major discrimination between active and inactive agents at this time point. On the other hand, a study requiring a long treatment and follow-up period in this setting, which possibly can extend over years (as in GIST), may logistically be difficult to conduct and is unattractive in view of the purpose of a phase II study, which is to screen new agents for activity. Therefore, the EORTC suggested evaluating the progression free status at 3 and 6 months after the start of therapy. Further, the EORTC proposed to consider a drug as active in first line therapy of STS if the PFR at 6 months is 30-55% (depending on histology). For second line therapy, a PFR at 3 months of 40% would suggest drug activity, and 20% would suggest inactivity (van Glabbeke et al, 2002; Verweij/van Glabbeke, 2003). Another way of looking at the data could be to take together objective response and stable disease rate, resulting in the determination of the rate of no progression. Although van Oosterom already in 1986 proposed to use “progression arrest” as an endpoint in phase II studies (van Oosterom, 1986), the idea was only recently picked up by the NCIC that developed a multinomial phase II stopping rule using response and early progression. They showed that this was more efficient as compared to the usually used stopping rules (Dent et al., JCO, 2001). These data certainly would gain strength if PFR or progression arrest rates could be assessed by means of the use of the EORTC database in

12

combination with those of other co-operative groups, possibly even resulting in the determination of subtype specific progression arrest rates. Further, this would provide strong suitable tools that could serve as reference for future phase II studies aiming at more efficient screening of new and old drugs for activity in STS.

6

CONCLUSION

The fast development and application of microscopic and new immunohistochemical tools, molecular and cytogenetic analytical methods results in a better identification of specific or clusters of subtypes in STS, and provides specific molecular targets to which selected agents are being developed. This allows for a further evaluation of the characteristics and chemo-sensitivity of these different subtypes to known agents, and necessitates the prospective screening of selected (old and new) drugs in subtype specific cohorts of patients. Further, the drugs aiming at targeting the identified molecular target also have to be tested in subtype specific studies. In order to make this process as efficient as possible the endpoints of phase II screening studies should be clearly selected. We propose to use the 3and 6- month PFR as a reference value in phase II screening studies, while progression arrest also may be of relevance. Moreover, performing these screening studies of molecular targeting agents in a cohort of patients expressing the specific target in a disease already rarely occurring will not be possible

13

without a joined effort and a global co-operation of the several co-operative groups. We hope that the topics in this book may contribute to a better understanding hereby, and stimulate the co-operation and participation into initiated screening studies.

7

REFERENCES

1. Balcerzak SB, Benedetti J, Weiss GR et al. (1995) A phase II trial of paclitaxel in patients with advanced soft tissue sarcomas. Cancer 76, 2248-2252 2. Blay J-Y, van Glabbeke M, Verweij J, et al. (2003) Advanced soft tissue sarcoma: a disease that is potentially curable for a subset of patients treated with chemotherapy. Eur J Cancer 39: 64-69 3. Borden EC, Baker LH, Bell RS et al. (2003) Soft tissue sarcomas of adults: state of the translational science. Clin Cancer Research 9: 1941-56 4. Brain EGC (2002) Safety and efficacy of ET-743: the French experience. Anti-cancer Drugs 13 (suppl 1): 11-14 5. Bramwell V, Anderson HC, Charette ML et al. (2000) Doxorubicin-based chemotherapy for the palliative treatment of adult patients with locally advanced or metastatic soft tissue sarcoma: a meta-analysis and clinical practice guidelines. Sarcoma, 4: 103-112 6. Casper ES, Waltzman RJ, Schwartz GK et al. (1998) Phase II trial f paclitaxel i patients with soft tissue sarcoma. Cancer Invest 16: 442-446 7. Delaloge S, Yovine A, Taamma A et al. (2001) Ecteinasidin-743: a marine derived compound in advanced, pretreated sarcoma patients – preliminary evidence of activity. J Clin Oncol 19: 12481255 8. Delaney TF, Spiro IJ, Suit HD et al. (2003) Neoadjuvant chemotherapy and radiotherapy for large extremity soft tissue sarcoma. Int J Radiat Oncol Biol Phys, 56: 1117-1127 9. Demetri GD (2002) ET-743: the US experience in sarcomas of soft tissues. Anti-cancer Drugs 13 (Suppl 1): 7-9 10. Dent S, Zee B, Dancey J et al. (2001) Application of a new multinomial phase II stopping rule using response and early progression. J Clin Oncol 19: 785-791 11. Fata F, O’Reilly E, Ilson D et al. (1999) Paclitaxel in the treatment of patients with angiosarcoma of the scalp or face. Cancer 86: 2034-2037 12. Frustaci S, Gherlinzoni F, de Paoli A, et al. (2001) Adjuvant chemotherapy for adult soft tissue sarcomas of the extremities and girdles : results of the Italian randomized cooperative trial. J Clin Oncol, 19: 1238-1247 13. Gortzak E, Azarelli A, Buesa J et al. (2001) A randomised phase II study on neo-adjuvant chemotherapy for “high-risk”adult soft tissue sarcoma. Eur J Cancer 37: 1096-1103 14. Hensley ML, Maki R, Venkatraman E et al. (2002) Gemcitabine and docetaxel with unresectable leiomyosarcoma: results of a phase II trial. J Clin Oncol 20: 2824-2831 15. Korn EL, Arbuck SG, Pluda JM et al. (2001) Clinical trial designs for cytostatic agents: are new approaches needed? J Clin Oncol 19: 265-272 16. Labropoulos SV, Papadopoulos S, Hadjiyiassemi L et al. (2003) Response of metastatic dermatofibrosarcoma protuberans to imatinib mesylate. Proceedings ASCO 2003, J Clin Oncol 22: 830 (#3334) 17. Le Cesne A. (2002) Improving efficacy in soft tissue sarcoma. Satellite symposium ESMO 2002, 18 October, Nice, France. 18. Lee Y-F, John M, Edwards S et al. (2003) Molecular classification of synovial sarcomas, leiomyosarcomas and malignant fibrous histiocytomas by gene expression profiling. Brit J Cancer 88: 510-515

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19. Leyvraz S, Jundt G, Lissoni A et al. (2001) High-dose Ifosfamide and doxorubicin for the treatment of gynaecological sarcomas. Proceedings ASCO 2001, J Clin Oncol 20 (part 1): 362a (#1443) 20. Nielsen TO, West RE, Linn SC et al. (2002) Molecular expression of soft tissue tumours: a gene expression study. Lancet 359: 1301-1307 21. Okuno S, Edmonson J, Mahoney M et al. (2002) Phase II trial of gemcitabine in advanced sarcoma. Cancer 94: 3225-3229 22. Okuno S, Ryan LM, Edmonson J et al. (2003) Phase II trial of gemcitabine in patients with advanced sarcomas. Cancer 97: 1969-1973 23. O’Sullivan B, Bell RS, Bramwell V (2002) Sarcoma of the soft tissue. In: Souhami R, Tannock I, Hohenberger P, and Horiot JC (eds): Oxford Textbook of Oncology. Oxford University Press, Oxford, UK, 2002, 2495-2523 24.O’Sullivan B, Bell RS (2003) Has “MAID” made it in the management of high-risk soft tissue sarcoma? Int J Radiat Oncol Biol Phys, 56: 915-916 25. Pautier P, Genestie C, Fizazi K et al. (2002) Cisplatin-based chemotherapy regimen (DECAV) for uterine sarcomas. Int J Gynaecol Cancer 12: 749-754 26. Patel SR, Linke KA, Burgess MA et al. (1997) Phase II study of paclitaxel in patients with soft tissue sarcomas. Sarcoma 1, 95-97 27. Patel SR, Vadhan-Rai S, Burgess MA, et al. (1998) Results of two consecutive trials of doseintensives chemotherapy with doxorubicin and ifosfamide in patients with sarcoma. Am J Clin Oncol 21: 317-321 28. Patel SR, Gandhi V, Jenkins J et al. (2001) Phase II clinical investigation of gencitaine in advanced soft tissue sarcomas and window evaluation of dose rate on gemcitabine triphosphate accumulation. J Clin Oncol 19: 3483-4389 29. Patel SR (2002) Systemic therapy for advanced soft tissue sarcoma. Curr Oncol Rep 4: 299304 30. Robertson JFR, Wilsher PC, Cheung KL et al. (1997) The clinical relevance of static disease category for 6 months on endocrine therapy in patients with breast cancer. Eur J Cancer 33: 17741779 31. Robertson JFR, Howell A, Buzdar A et al. (1999) Static disease on anastrozole provides similar benefit as objective response in patients with advanced breast cancer. Breast Ca Res & Treatm 58: 157-162 32. Rosen G, Forscher C, Lowenbraun S et al. (1994) Synovial sarcoma: uniform responsee of metastases to high-dose ifosfamide. Cancer 73: 2506-2511 33. Sarcoma Meta-Analysis Collaboration (1997) Adjuvant chemotherapy for localised respectable soft tissue sarcoma of adults: meta-analysis of individual data. Lancet 350: 1647-1654 34. Scappaticci FA, Marina N (2001) New molecular targets and biological therapies in sarcomas. Ca Treatm Reviews 27: 317-326 35. Schuetze SM, Rubin BP, Eary JF et al. (2002) Molecular targeting of PDGF beta by imatinib mesylate in dermatofibrosarcoma protuberans. Proceedings CTOS 2002, Sarcoma 6 (suppl 2): 71 (#25) 36. Seynaeve C, Verweij J (1999) High-dose chemotherapy in adult sarcomas: no standard yet. Semin Oncol, 26: 119-133 37. Seynaeve C, Verweij J. (2002) High dose chemotherapy in sarcomas: science, fiction or science fiction? In: Lorigan P, Vandenberghe E (eds): High dose chemotherapy, Principles and Practice. Dunitz Publishers, London, UK, 2002, 167-179, 38. Spath-Schwalbe E, Genvresse I, Koschuth A et al. (2000) Phase II trial of gemcitabine in patients with pretreated advanced soft tissue sarcoma. Anti-cancer drugs 11: 325-329 39. Spillane AJ, A’Hern R, Judson I et al. (2000) Synovial sarcoma: a clinicopathologic, staging, and prognostic assessment. J Clin Oncol, 16: 1794-3803 40. Stojadinovic A, Leung DHY, Allen P et al. (2002) Primary adult soft tissue sarcoma: Timedependent influence of prognostic variables. J Clin Oncol 20: 4344-4352

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41. Svancarova L, Blay J-Y, Judson I et al. (2002) Gemcitabine in advanced adult soft tissue sarcomas. A phase II study of the EORTC soft tissue and bone sarcoma group. Eur J Cancer 38: 556-559 42. Therasse P, Arbuck GA, Eisenhauer EA et al. (2000) New guidelines to evaluate the response to treatment in solid tumors. J Natl Cancer Inst 92: 205-216 43. Tuveson DA, Fletcher JA (2001) Signal transduction pathways in sarcoma as targets for therapeutic intervention. Curr Opin Oncol 13: 249-255 44. Van Glabbeke M, van Oosterom AT, Oosterhuis JW et al. (1999) Prognostic factors for the outcome of chemotherapy in advanced soft tissue sarcoma: an analysis of 2,185 patients treated with anthracycline-containing first line regimens – an EORTC soft tissue and bone sarcoma group study. J Clin Oncol 17: 150-157 45. Van Glabbeke M, Verweij J, Judson I et al. (2002) Progression-free rate as the principal endpoint for phase II trials in soft-tissue sarcomas. Eur J Cancer 38: 543-549 46. Van Oosterom AT (1986) Phase II new drug trials in soft tissue sarcomas. In: Pinedo H and Verweij J (eds): Clinical management of soft tissue sarcomas. Boston, MA, Martinus Nijhoff Publishers, 131-138 47. Verweij J, Seynaeve C. (1999) The reason for confining the use of adjuvant chemotherapy in soft tissue sarcoma to the investigational setting. Semin in Radiation Oncology, 9: 352-359 48. Verweij J, Lee SM, Ruka W et al. (2000) Randomized phase II study of docetaxel versus doxorubicin in first- and second-line chemotherapy fr locally advanced or metastatic soft tissue sarcoma in adults: a study of the EORTC soft tissue and bone sarcoma group. J Clin Oncol 18: 2081-2086 49. Verweij J, van Glabbeke M (2003) Translating targets into treatment: changes in trial methodology and treatment approaches for soft tissue sarcomas. In: Educational book, ASCO 2003, 522-530 50. Weiss SW, Goldblum JR (2001) In: Weiss SW, Goldblum JR (eds): Enzinger and Weiss’s Soft Tissue Tumors. Mosby Inc, St Louis, Missouri, 2001: 1-19

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Chapter 2 Volume-based radiotherapy targeting in soft tissue sarcoma Iain Ward1 ,Tara Haycocks2, Michael Sharpe3, Anthony Griffin4, Charles Catton1, David Jaffray3, Brian O’Sullivan1

Departments of Radiation Oncology1, Radiation Therapy2, Medical Physics3, and Surgical Oncology4, Princess Margaret Hospital, University of Toronto, Toronto, Canada.

Correspondence: Brian O’Sullivan Department of Radiation Oncology Princess Margaret Hospital University of Toronto 610 University Avenue Toronto, Ontario Canada, M5G 2M9 Tel: 416 946 2123 Fax: 416 946 6556 Email: [email protected]

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1

INTRODUCTION

Although a highly effective adjuvant therapy, traditional radiotherapy (RT) target volumes used for STS have largely been constrained by available technology and are not ideal in some situations; this includes inadequate dose administration because of normal tissue constraints and/or the necessity for excessive volume coverage of normal tissue to encompass the tumor region. The advent of very precise treatment planning and delivery systems, including three dimensional conformal radiotherapy (3D CRT) and intensity modulated radiotherapy (IMRT), means it is now possible to select target volumes that more closely approach the optimum. Consequently, these new approaches provide great opportunity for treatment enhancement in the future. In this chapter, the principles of RT will be discussed as they relate to current or potential uses in the management of soft-tissue sarcoma (STS). Specific examples of situations in STS that lend themselves to volumetric-based RT planning approaches will be depicted to illustrate theses concepts and detailed background to the use of such approaches will be provided.

2

DEFINING TARGETS FOR RADIOTHERAPY OF STS

2.1

Tissues at risk

The choice of RT volumes in STS is profoundly influenced by the appreciation of the existence of a zone that may contain sub-clinical disease in proximity to the presenting site of the primary tumour. The size and extent of the putative ‘risk zone’ depends on a number of factors, and appreciating this will effect the target volume chosen for radiotherapy. Also, perhaps more than most cancers, the pathway to appropriate treatment may already have been declared by events that have taken place prior to referral. For example, the type of biopsy that may have already been performed, or a prior inappropriate excision may jeopardize the form and outcome of local treatment thereafter. 1

2.2

Local patterns of spread

In broad terms, soft tissue sarcomas tend to spread in a longitudinal direction within the muscle groups of the extremity. They generally respect barriers to tumor spread in the axial plane of the extremity such as bone, interosseous membrane, major fascial planes, etc. Thus the margins of radiation therapy must be wide in the cephalo-caudal direction but in the cross section there may be much greater security in defining non-target structures. For nonextremity lesions (e.g. head and and neck and torso lesions), the direction of sarcoma growth is also along the involved musculature but care must be taken to

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ensure that the fascial planes are appropriately recognized and encompassed in the radiation target volume. 2

2.3

Regional lymphatic pathways of spread

Regional lymph node involvement in STS is unusual and for most histological sub-types the nodal areas are not ordinarily treated electively. Important exceptions to this generalization include epithelioid sarcoma, clear cell sarcoma, angiosarcoma, and embryonal rhabdomyosarcoma.2 However the presence of overt regional lymph node disease generally prompts their inclusion, if the patient is being considered for curative management, although we recognize that institutional preference may vary in this regard. In targeting the lymphatic drainage areas, it is usual to treat the chain along the vascular supply to eventually reach the terminal group of lymph nodes that are at risk. 2

2.4

Paucity of available evidence

Unfortunately, with one exception,3 no formal assessment of target volumes in STS has been undertaken using contemporary hypothesis solving techniques such as comparative clinical trials. Also, problems in defining the gross tumor volume (GTV) will persist until resolution of dilemmas surrounding imaging characteristics occurs, at least when considering pre-operative radiotherapy where the volumes are most selectiveFor example, one obvious problem concerns the significance of peritumoral edema evident on magnetic resonance imaging4 and whether such areas should be considered part of the GTV (see figure 1).

2.5

Extracompartmental sarcoma

Certain anatomic areas are potential spaces without good definition from the standpoint of tumour containment and in such situations it is usual to design the radiotherapy target volume to encompass the extreme limits of the structure or region in question (generally determined by where the fascial reflections eventually merge). Areas such as the axilla, popliteal fossa, femoral triangle, and the entire subcutaneous compartment present problems of target volume delineation that must be evaluated on an individual basis.2 Evaluation of the region in terms of potential tissues involved or tissues that have already been surgically violated is paramount in deciding the most appropriate volume to treat. Again the principles are determined by the proximity of the most reliable barrier to tumor invasion (hopefully an intact durable anatomical boundary). Alternatively, an effective distance (e.g. 2-5 cm, where anatomically feasible) is maintained from the highest risk area, most typically manifested by the existing GTV or the pre-operative GTV; this should bear in mind the surgical-pathologic findings at the time of the resection.

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2.6

Margins for geometric uncertainty

An additional feature in radiotherapy target delineation concerns allowance for uncertainties in set up and treatment delivery. A region of additional margin must be defined to account for geometric uncertainty and is especially important where there is respiratory movement (e.g. abdominal and thoracic areas). The expansion of volume should be defined around the Clinical target volume (CTV) as the Planning Target Volume (PTV) to insure the inclusion of the areas at risk in the treated area.5, 6 A similar margin should be used when protecting normal tissues vulnerable to radiotherapy (e.g. the spinal cord in paraspinal, retroperitoneal or head and neck sarcomas) because

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uncertainties in set up and treatment delivery may equally result in inadvertent treatment of these structures. The additional zonal expansion surrounding these structures is termed a planning risk volume (PRV).6

2.7

Influence of scheduling of modalities

Pre-operative and post-operative RT represent the two usual approaches to external beam delivery for STS but effectively comprise two disease ‘scenarios’ from the standpoint of delineating targets. Pre-operative RT approaches can focus on the extent of definable disease (generally using imaging characteristics) and the choice of target is based on the anatomic location, containment by barriers to spread, estimated distance from the GTV that may contain microscopic disease, and allowance for geometric uncertainty. In contrast, post-operative radiotherapy volumes must encompass all surgically manipulated tissues and are often less specific because anatomic planes have been disrupted and no longer provide barriers to contain tumor growth and are consequently significantly larger.3, 7 Another consideration that is relevant to cases undergoing induction chemotherapy is the determination of the pre-chemotherapy volume in chemoresponsive tumors. In these situations, vigorous tumor response will likely have manifested by the time radiotherapy is ordinarily scheduled to commence and little if any radiologically apparent disease may be present at the time of radiotherapy planning. The initial pre-chemotherapy volume must be the reference for treatment planning and imaging studies must be carefully performed and recorded to facilitate subsequent RT planning.2

3 DELIVERY

3.1

VOLUMETRIC TREATMENT PLANNING AND Evolution of volumetric-based planning

The initial introduction of adjuvant radiotherapy more than two decades ago took place in an era when limb conservation had only recently become established as an alternative to amputation.8-10 The paradigm involved the use of radiotherapy to sterilize a broad field of tissue that generally targeted an entire muscle compartment from which the tumour had been resected and almost exclusively involved post-operative radiotherapy. Subsequently a desire to minimize morbidity resulted in progressive reduction in radiation treatment volumes and evidence accumulated that smaller radiation field margins could be used without compromising the very high rates of local control which were being achieved. 10-12 Refinement of external beam radiotherapy (EBRT) fields was limited by imprecise cross-sectional imaging

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and treatment planning and delivery systems could not tailor radiation dose to irregularly shaped volumes. Consequently, for many years conventional radiotherapy technique for limb STS essentially comprised two parallel opposed rectangular fields.10 Over the last decade unprecedented improvements in treatment planning and delivery have become available. Two new approaches, three-dimensional conformal radiotherapy (3D CRT) and intensity-modulated radiation therapy (IMRT), have been successfully applied to the treatment of other cancers, but their application to the management of STS is very new. The introduction of 3D CRT and IMRT is an important opportunity to reevaluate many aspects of the current treatment paradigm, which had evolved under the technological constraints of the past.

3.2

Three-Dimensional Conformal Radiotherapy (3D CRT)

3.2.1

The elements of 3D CRT

Three-dimensional conformal irradiation has been described as “external-beam radiation therapy in which the prescribed dose volume (treatment volume) is made to conform closely to the target volume”. 13 The increased conformality in comparison to two-dimensional techniques is made possible by innovations in hardware associated with the treatment apparatus, as well as in software which allows improved calculations of the absorbed dose and improved presentation of the results.

3.2.2

The multi-leaf collimator (MLC)

Simple modulation of the treatment beam to improve conformality may be achieved by physical blocks, missing-tissue compensators, physical wedges or dynamic wedges generated by moving collimators, but the single most important hardware innovation in the development of 3D CRT has been the multi-leaf collimator (MLC). This device consists of multiple interlocking finger-like blocks mounted in the treatment head of a linear accelerator, which may be advanced from either side by individual motors under computer control. The sides of the leaves interlock and are ‘dove-tailed’ together to minimize radiation leakage in between the leaves. In the past, conformation required the manufacture and handling of individual physical blocks. The ability of an MLC to rapidly change configuration under computer control, and to produce individualised beam shaping represents a significant advance and has made the delivery of complex multi-beam treatment plans practical and achievable. Furthermore, the MLC facilitates modulation of beam intensity by superimposing a smaller radiation field within a larger field (beam segmentation). 3.2.3 3D CRT plan calculation, visualization and evaluation

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The hallmark of three-dimensional planning is the calculation of the absorbed radiation dose throughout the volume of interest, rather than in selected axial cross-sections only. This allows accurate assessment of the dose deposited by beams that are not aligned with the axial plane (non-coplanar beams) and provides many more beam configurations, which may be utilized to produce optimal conformality to the target volume. Software that permits clear presentation of treatment data is an integral part of such planning systems. The effects of a candidate treatment plan on multiple sub-volumes may be evaluated, representing tumour and other structures of interest including normal tissues. These include features such as beam’s eye views (BEV) (i.e. visualization of any beam along its central axis irrespective of its direction and that include the target volume and tissues to be avoided in the view) (figure 2) and room’s eye views (that allow rapid visual

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assessment of coverage of the target volume by a single beam and the potential for collision of couch and gantry). Presentation of dose distributions in different planes and in three dimensions allow assessment of RT dose perturbation within and adjacent to the target volume, including ‘hot spots’ and ‘cold spots’. 3D CRT procedures facilitate development of a plan which deposits dose to the target volume with minimal dose to surrounding tissues. The degree of conformality may be quantified by the conformity index (CI), which is the ratio of volume enclosed by the prescription isodose surface to the planning target volume (PTV). 14 This is somewhat helpful in comparing candidate plans but in practice it is more useful to compare dose-volume histograms (DVHs) for volumes of interest (figure 3), 15 as the CI does not fully reveal the effects on critical structures.

3.3

Intensity modulated radiotherapy (IMRT)

3.3.1

The elements of IMRT

The impact of computer technology enhancements has provided the opportunity for extraordinary improvement in the physical basis of radiation therapy. Leading these advances in the contemporary era is the development of intensity-modulated radiotherapy (IMRT). IMRT is an advanced form of three-

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dimensional conformal radiotherapy in which radiation beams are not only shaped at their perimeters, but also include variable intensity across the profiles of the beams. This permits the creation of exquisite conformation of dose to targets of irregular shape while generating high dose gradients between tumor and normal tissues. In addition to the use of intensity modulated beams, the other key enhancement of IMRT over conventional conformal treatments, is the use of computer assisted iterative treatment plan optimization. In its complete form, IMRT combines inverse treatment planning (where dose to normal tissues as well as target regions is specified in advance), with computer controlled dynamic beam shaping and filtration (usually with MLC). 3.3.2

The mechanism of IMRT

The IMRT concept relies on the fact that multiple beams crossing a target from different directions do so with great redundancy that can be harnessed to provide substantial flexibility in distributing dose. Instead of permitting the full intensity of a beam to traverse the target, the dose in part(s) of the field aperture is reduced in a variable way (by programming the configuration and timing of the MLC leaf positions), according to the requirements. This can permit sparing of a structure on the one hand or delivery of relatively enhanced dose to the target in another part of the field aperture thereby causing deliberate perturbation of the RT dose distribution of a beam. At the same time, the variable dose incurred in components of the aperture of a given beam by this process can be compensated by enhancing or reducing the dose in components of the apertures of other beams directed at the target from other directions. In this way a ‘beam’ directed from a given direction, can be fashioned to comprise numerous (e.g. dozens to hundreds) of mini-beams (termed beamlets) with variable shape and intensity of radiation exposure. The ultimate result from the combination of numerous beams of different direction, intensity patterns, and shape provides an unprecedented three dimensional configuration to the composite dose distribution from all beams. Ironically, it is now reasonably certain that the capability of placing dose and avoiding tissues is significantly more precise than our knowledge of what issues are indeed involved and the measures required to minimize geometric uncertainty related to normal physiologic movements in the patient (eg. breathing, adjustment related to hollow viscous filling and emptying, etc). 3.3.3

Dose ‘sculpting’ and ‘painting’

Using the IMRT approach, the delivered RT dose can be fitted to volumes tailored to complex shape specifications. These can be both shaped externally (i.e. convex shaping), as in traditional fields, but also the external ‘surface’ of the intended RT dose region can be excavated thereby providing concave or indented shaping (‘dose sculpting’)16 that permit previously

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inaccessible target areas to be treated while avoiding adjacent vulnerable anatomy that may be partially surrounded by the target (figure 4). Most radiotherapy treatment plans, including 3D CRT plans and IMRT plans that use ‘dose sculpting’ strategies, accept a certain amount of dose heterogeneity within the target volume as a by-product of efforts to conform a given isodose to the periphery. This apparently anomalous situation can be further enhanced with IMRT where the opportunity exists to create deliberate variation in absorbed dose within the target. This has been termed ‘dose painting’ 16 and provides further opportunity to relatively spare normal tissues while delivering even more intense dose accumulation into diseased tissues.

3.3.4

Inverse Planning

IMRT targeting comprises a powerful engineering accomplishment employing concepts of radiation physics, advanced computation, and a revolution in hardware design. To achieve this an intended dose distribution is displayed on anatomic details obtained from thin CT scan slices upon which the target and the anatomic areas of risk are outlined. The CT scan also provides the radiation attenuation properties of tissues, which are necessary for the computation of absorbed dose. This is accomplished using numerical models that approximate the radiation transport physics in tissues. The planning objective includes two important specifications: a description of the required dose to each part of the target and the normal tissues in the treated region; and the importance of achieving that dose in relative terms compared to the other structures (target or normal tissues) as a ‘weighting’ expressed in a mathematical equation. Altogether this process is characterized as ‘inverse planning’, in which the requirements for the dose configuration in the patient is specified precisely in advance in mathematical terms and computer technology is then used to calculate the intensity pattern specifications of each beam to achieve the intended result. Thus the clinical objectives are specified mathematically in the form of an objective function (also called the score function or cost function)17 (see table 1). In practice the number of beams and selection of their orientation are chosen by the treatment planner because the computerized optimization process outlined below that searches for the optimal plan would not be possible, in practical terms, with our current computation tools. This process can be facilitated by the use of libraries of plans (including number and direction of beams) that successfully met a need in the past and the development of ‘class solutions’ that generally fit the problem at hand. Following the specification of treatment goals, an iterative search algorithm is used to compute the intensity patterns that optimize the dose distribution throughout the normal tissues and the target. Many thousands of

27

28

iterative calculations are generally required to solve the mathematical task of calculating an objective function that takes account of the ‘desired dose’, the ‘calculated dose’ (at each iteration), and the weighting (or priority) to eventually achieve a distribution of radiation dose in the volume that comes as close as possible to what is intended.

A variety of computational methods are available for this process, all with their own advantages and disadvantages, which in itself compromises an active area of research. These include different degrees of accuracy in accounting for tissue heterogeneity corrections, geometric dose reproduction (e.g. adjustment and synchronization of leaf position to take account of leakage between the leaves of the MLC and passage of radiation through multileaves of partial depth, the method of objective function calculation, and, of great

29

practical importance, speed of performing the calculations).17, 18 Unfortunately, the optimization process must be repeated several times if the initial result is not satisfactory. This may arise if the planning objectives cannot be met and may include excessive dosing of areas including ‘hot spots’ in normal tissues that exceed tolerance and rendering the plan vulnerable from a safety perspective, or if parts of the target do not receive reliable dose coverage.

4

RATIONALE FOR VOLUMETRIC-BASED PLANNING

4.1

Reducing late tissue adverse events

One of the main potential advantages to conforming the treated volume to the target volume is to reduce toxicity to surrounding tissues. Nonrandomised studies of limb STS have demonstrated an association of radiation field size with limb oedema and tissue induration 19 and of radiation dose with increased oedema, decreased muscle strength, decreased range of motion, fibrosis and worse functional outcome in general 19, 20. More recently, these findings have also been borne out in a prospective fashion in the comparison of different outcomes following pre-operative compared to post-operative radiotherapy. The latter generally requires higher doses and larger fields and is associated with significantly higher rates of fibrosis and edema two years following completion of treatment compared to the pre-operative approach.21 Because of its ability to produce high dose volumes with concave surfaces, IMRT will usually be able to spare some subcutaneous tissue to reduce the risk of lymphoedema.22 In addition, late (i.e. many years following treatment) irradiation induced bone fracture is evident with doses of exceeding 60 Gy in weightbearing bone 23, and minimization of dose to, or even complete avoidance of, such structures would seem desirable in reducing the risk of this debilitating complication. Dose modeling studies have shown that the ability of IMRT to produce high dose volumes with concave surfaces can substantially lower mean dose to the femur and the volume of bone irradiated to 95% of the prescription dose.24 This may reduce the incidence of late fracture, particularly in the setting of postoperative radiotherapy where higher doses are prescribed.

4.2

Facilitate curative radiotherapy

Lesions in proximity to vital structures may need specific volume modifications in order to encompass disease while sparing normal tissue. This may be particularly attractive in disease sites where critical organ tolerance may be paramount yet undertreatment of the target poses additional risks. This may involve potentially achieving kidney protection, spinal cord or

30

lung avoidance in different situations. This differs from the discussion of late tissue toxicity (section 4.1), because the context here concerns whether radiotherapy can be delivered at all with curative intent, out of concern for life threatening complications to critical anatomy. This approach may be best exemplified by retroperitoneal sarcomas and paraspinal tumours. Retroperitoneal sarcoma frequently recurs locally, despite optimal surgery, and radiotherapy is often used pre-, intra- or postoperatively to improve control.25-28 Local recurrences are still the rule, however. The need to observe the tolerance of bowel, liver, kidneys and other organs generally requires that lower doses be prescribed than for sarcomas at other sites and target coverage is often compromised. 3D CRT and MRT provide the opportunity to treat large and complex retroperitoneal tumor volumes that were previously close to impossible to treat. These modalities are especially well suited to pre-operative radiotherapy because the target is in situ with less risk of intra-abdominal contamination so that the region to be treated can be readily defined. In addition, bowel is both mobile and displaced by the tumor and treatment is well tolerated 28 in contrast to the post-operative setting 29 where the bowel is frequently tethered in the surgical bed making safe delivery of substantive doses of radiotherapy problematic. In addition, a problem that is especially difficult is presented by the large right sided retroperitoneal sarcoma where tumor is in direct proximity to the liver and frequently ‘hooded’ by this stucture. Standard radiotherapy cannot achieve safe coverage of the target while permitting the liver to be spared. Even 3D CRT is problematic for these lesions and IMRT presents many advantages (figure 5). Paraspinal tumors also pose prodigious problems for the safe delivery of radiotherapy. The real possibility exists for the spine to be severely injured by the treatments themselves (radiotherapy and surgery are both severely constrained by normal tissue tolerance) or by tumor. Target volumes partially enveloping the spinal cord pose a challenge in dose delivery not previously achievable with standard planning but that can be overcome with IMRT (see figure 3 and 4, and table 2).

4.3

Retreatment

Reirradiation of limb soft tissue sarcoma after local recurrence has been shown to contribute to limb conservation, although there is a risk of inducing radionecrosis, chronic ulceration and fracture. 30, 31 Brachytherapy has been advocated as the optimal radiation modality for this situation 31-34 but IMRT has the potential advantages, through inverse planning, to tailor the dose to treat sites inaccessible to brachytherapy. This approach has been used successfully to retreat vertebral metastases, including sarcoma,35 and it is well suited to locally recurrent STS.

31

32

4.4

Reduction of acute radiation morbidity

Combined modality treatment of STS improves local control,36, 37 but some structures may suffer enhanced toxicity. It is plausible that in the future IMRT may ameliorate this problem. For instance, preoperative radiotherapy increases the incidence of major wound complications compared with postoperative radiotherapy 3, despite reducing late fibrosis and oedema.21 One potential strategy to enhance wound healing might be to investigate the use IMRT to avoid irradiating skin in the region of the planned wound.38 Highly conformal RT may also reduce adverse interactions with chemotherapy. Ifosfamide is a drug that is being actively investigated in combination with radiotherapy because of its high activity against sarcoma,39, 40 but the incidence of moist desquamation of skin may be increased.39, 41 One suggestion is that IMRT may allow the radiation dose to skin to be limited to avoid this problem.38 3D CRT has also been used to reduce radiation toxicity to bone marrow, so that the intensity of chemotherapy is not compromised. 42

33

4.5

Radiation dose escalation

An intriguing aspect in considering the radiotherapy of many STS sites is that the results of treatment are generally satisfactory in terms of local control. The therapeutic strategy must therefore focus on the opportunity to either reduce the intensity of treatment application or modify radiotherapy volumes compared to traditional approaches. The exception may be retroperitoneal sarcoma where the local control rates, even with adjuvant radiotherapy, have been disappointing although the reason for this are multifactorial and with strong potential that the tumor is being underdosed and the target area at risk is not covered due to organ tolerance (see section 4.2). It is plausible that retroperitoneal sarcoma results could improve if doses to tumour were escalated while conformally avoiding organs at risk. This is probably achievable with IMRT.43

5

APPLYING VOLUMETRIC-BASED PLANNING

5.1

Multidisciplinary interactions

For several reasons, several specialties should be involved when very selected treatment RT volumes are proposed. Predominantly this applies to interaction between the radiation medicine disciplines and surgical oncology, medical imaging, and pathology, but also extends to medical oncology collaboration. We have already noted that prior surgical interventions may influence the choice of target tissues at risk in a striking fashion. We also discussed the implications of absence of pretreatment appropriate imaging since initial chemotherapy may eliminate all evidence of overt disease before radiotherapy can be administered. Clearly both circumstances may equally result in a situation where the use of selected conformal volumes may prove impossible. In retroperitoneal sarcoma, following pre-operative radiotherapy that may have treated a generous portions of involved or adjacent liver, it is very important that the surgical team be aware of the area of liver that has been treated so that sufficient liver is left intact in the patient if partial liver resection is contemplated. This information can only be imparted by detailed review of the planning and dosimetry records by the surgeons with the radiation oncologists and treatment planners 44 As high intensity dose escalation chemo-radiotherapy techniques evolve, there may be opportunity to spare superficial tissues using IMRT and prevent acute and long term skin and subcutaneous morbidity that may be a hallmark of the concurrent use of chemotherapy and radiotherapy. In this way as treatment strategies evolve, it is likely that the evolution to more selective radiotherapy volumes will require more rather than less interaction among the disciplines. Hopefully, it may be possible to develop

34

protocols where anatomic areas treated by one discipline may be avoided deliberately by the other to reduce the overall morbidity.

5.2

Imaging and image fusion

Imaging is key to the application of 3D CRT and IMRT. Targets as well as structures to be avoided must be identified accurately with respect to anatomical site and local extension to surrounding structures. They must then be delineated on the images of the radiotherapy planning system. Usually the radiation oncologist will examine the other images beside the planning CT images, mentally ‘coregister’ them and then electronically contour the structures by drawing on the latter. This obviously has potential to introduce error. An alternative, which is often used when adjacent critical structures require narrow margins for uncertainty, is to fuse the two sets of images. This is routinely done in the planning of stereotactic IMRT for skull base sarcoma. It is then relatively simple for the oncologist to trace around the MRI image of the structure and the resulting contour is registered on the CT image. The use of other methods of simulation are being investigated, such as MR simulators and combination PET / CT simulators.

5.3 variability

Reduction in geometric uncertainty and treatment

‘Geographic miss’ is a serious error as it exposes the patient to toxicity without the prospect of tumour control. However, as radiation treatment becomes more conformal the risk of geographic miss increases because any error is more likely to move the CTV out of the high-dose volume. Uncertainty is an important component in radiotherapy. The quality assurance requirements for IMRT present new and unique problems and involve technical issues at the radiotherapy delivery level as well as the planning problems already discussed. 45, 46 Consistency in set up and the use of appropriate immobilisation (see section 6.1) of anatomic regions should be integral to the planning process to insure that correct anatomic treatment and avoidance takes place. While strict immobilisation is not necessarily an integral part of 3D CRT or IMRT, in practice unless attention is directed towards reducing movement, much of the benefit of highly conformal treatment will be lost and the risk of unsatisfactory treatment increased.47

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6

DOSE HETEROGENEITY AND DOSE DISPERSION

6.1

‘Dose dumping’

An increase in dose heterogeneity within the target volume is commonly encountered in IMRT plans, frequently beyond the 95% -107% range that is commonly considered acceptable.5 This phenomenon has been noted in the treatment of many sites, including the thigh. 24 It is enhanced by a tendency for users to ascribe high priorities to conformality and target coverage rather than dose homogeneity in the objective function. 48 Generally, provided the radiotherapy is prescribed such that no part of the PTV is underdosed and the ‘hot spots’ fall within the GTV away from sensitive normal structures, adverse consequences are unlikely. When computer-assisted dose optimisation takes place in the IMRT planning process, the algorithm will consider all plans that satisfy the specified geometric and dosimetric constraints. This may result in high doses being dispersed into unexpected sites, and represents the converse of delivering inadequate dose to the target because unintended dose may be delivered to normal structures. This phenomenon is called ‘dose dumping’. Examples are the allocation of high doses to skin and subcutaneous tissues in a plan to treat limb sarcoma or to brain in the treatment of skull base disease. Experience generally allows prediction of when this might occur, but all IMRT plans should be carefully examined for unanticipated ‘hot spots’. If identified, the optimisation may need to be repeated after adjustment of contours for the relevant tissue and allocating appropriate constraints, and the incidence of ‘dose-dumping’ is reduced with experience.

6.2

Increased integral dose

In order to achieve highly conformal irradiation of the target, IMRT plans tend to use more beams than traditional RT approaches. Therefore more normal tissue can fall within the irradiated volume. Furthermore, the complexity of IMRT treatments, with many beamlets being delivered at less than 100% intensity, results in wider dispersion of lower doses of radiation but effectively a higher patient whole-body (or integral) dose than is the case with conventional external beam radiotherapy. 17 Concerns have been expressed that this could substantially enhance the rate of radiation carcinogenesis in cancer survivors49, 50 . This may be acceptable for older adults but in younger patients, particularly children, very careful consideration should be applied to balancing as yet unproven improvements in local control and toxicity against possible second malignancies. In principle, the excess radiation leakage that may lead to excess secondary cancers can be reduced by careful consideration in the design of radiation treatment technologies of the future.

36

7

OTHER MODALITIES

External beam photon therapy is only one method of delivering volumetric-based radiotherapy. Postoperative brachytherapy has been shown in a prospective randomised trial to improve local control of high grade STS over resection alone.36 However, it requires skills and facilities that are not available in many centers but the advantages of a short overall treatment time and irradiation of less normal tissue than traditional EBRT are understandably attractive.34 Technological enhancements now also permit full 3D planning for brachytherapy with dose-volume histograms and other tools employed by 3D CRT.51 There is also no reason why brachytherapy treatment planning systems should not be extended to include inverse planning capability, which might allow further sparing of nearby critical structures. The low integral dose resulting from brachytherapy makes it a useful option for recurrent tumour in previously irradiated tissue.52 In proton beam irradiation, high-dose volume treatment volumes can be made to conform precisely to the target by varying the depth and breadth of the ‘Bragg peak’. 53 If the peak is positioned at the target, there is no dose deposited in deeper tissues but also less dose in superficial tissues than if photon beams were used. Therefore proton RT has the advantage of a lower integral dose to normal tissues. The major drawback to the application of this technology is the cost of constructing facilities for delivery and currently it is only available at a handful of sites. Nevertheless, work is underway to improve access by increasing the number of treatment units and to improve its capability by introduction of intensity modulated proton beam (IMPT) planning and delivery. 53

Modulated electron therapy (MERT) is an investigational modality, which shows promise in the treatment of superficial targets. Modulation of electron beam energy, as well as intensity, allows limitation of the depth of penetration with a reduction in the dose to tissues that lie beyond tumour.54 This may result in a lower integral dose than photon IMRT produces. Theoretical applications include breast tumours (with sparing of lung) and situations where large areas of scalp need to be treated, such as for angiosarcoma (with sparing of underlying brain).

8

FUTURE PERSPECTIVES

8.1

Biological targeting / ‘dose-painting’

In STS, it has long been appreciated that biological heterogeneity exists. If different subvolumes have different biology, they may require different radiation doses to eradicate malignant clones. Larger radiation doses may be needed if clonogen density is greater, if hypoxia is present or if there is intrinsic

37

radioresistance. A number of functional imaging modalities are showing potential to identify such regions and could permit a biological target volume (BTV) to be conceived within the GTV on the basis of such imaging characteristics.16 These could include the incorporation of single positron emission tomography (PET), single photon emission computed tomography (SPECT)55, or proton MR spectroscopy (MRS).56, 57 Other novel applications of MRI include the identification of regions of hypoxia using blood-oxygen level dependent (BOLD) imaging sequences.58 The ability of IMRT to design and deliver specific nonuniform dose distributions within a target (‘dose painting’) has provided the capability to target different parts of the CTV. The BTV might be targeted to receive a higher radiation dose than the surrounding gross tumour, resulting in “biological conformality” as well as physical conformality.16 In this way, a paradigm of “multidimensional conformal radiotherapy” potentially acknowledges biological tumour heterogeneity in addition to the goals of physical conformality.16

8.2

Image-guided radiotherapy using on-line imaging

A highly active area of research is the use of CT imaging at the time of radiotherapy delivery. This reduces uncertainty regarding the positions of the target and organs at risk, as they no longer need to be inferred from images acquired days or weeks earlier. If the CT scanner and the linear accelerator gantry are mounted about the same axis, then the coordinates of structures of interest can be related directly to the radiotherapy delivery system, rather than to surface markings on the patient that are in turn related to the delivery system. One approach, under development at our centre is flat-panel cone-beam computed tomography where a kilovoltage photon source is mounted at 90° to the treatment head.59 A cone-shaped beam passes through the patient and is collected by a flat detector mounted on the opposite side during a single rotation to construct high-resolution images that allow adjustments to the treatment plan to be made to improve target coverage (adaptive radiotherapy). An alternative strategy is to mount a linear accelerator onto the ring gantry of a helical CT scanner to produce a treatment and imaging system known as helical tomotherapy. As well as delivering rotational IMRT, the transmitted portion of the beam is collected by a detector array on the opposite side of the gantry to produce megavoltage CT images that can be compared to the planning images to verify the accuracy of the delivered treatment.60

8.3

Clinical assessments

A key recommendation from the recent U.S. National Cancer Institute State-of-the-Science meeting on adult sarcomas in June 2002 included the view that IMRT) is a promising technique that should be actively studied in wellcontrolled trials of patients with soft tissue sarcomas.61 To achieve this,

38

prospective assessment of outcome should include established and validated methods of measurement for normal tissue toxicity, functional assessment, quality of life and careful evaluation of acute and late complications and local control of disease. There is an urgent need to correlate toxicity with modern measures of delivered dose, such as DVHs that allow assessment of the effects of partial organ tolerance and of inhomogeneous irradiation of whole organs. Certain structures, not previously considered avoidance targets, may be spared toxicity provided appropriate dose limits can be defined. For example, individual lymphatic trunks can be identified by indirect lymphography.62 Avoidance of these might allow more reliable prevention of lymphoedema following limb irradiation. In the long term, is conceivable that every part of the irradiated volume, including bone, joints and muscle beyond the PTV, might be contoured and assigned dose limits to reduce late toxicity.

9

CONCLUSION

RT targeting of STS presents considerable challenges in realizing optimum outcome in tissue and function preservation while maintaining high local control for most anatomic sites. While a highly effective adjuvant, RT delivered improperly may cause substantial disability by excessive volume or dose delivery. The advent of very precise treatment planning and delivery systems, including 3D CRT and IMRT, means it is now possible to choose to treat ideal volumes rather than ones that are merely feasible. At the same time precise knowledge of appropriate targets continues to evolve for the different clinical scenarios and will likely be greatly influenced in the future by enhanced imaging capability. Clinical trials are needed that include relevant end-points to measure improvements in the therapeutic ratio resulting from more precise RT targeting and without loss of local control. In addition, advancement of 3D CRT and IMRT over the next decade will rely on the consistent reporting and sharing of results concerning outcome of normal tissue from volumetric treatment planning.47

10

REFERENCES

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5. ICRU Report 50. Prescribing, Recording, and Reporting Photon Beam Therapy. Bethesda: International Commission on Radiation Units and Measurement; 1993. 6. ICRU Repor t62. Prescribing, Recording, and Reporting Photon Beam Therapy (Supplement to ICRU Report 50). Bethesda: International Commission on Radiation Units and Measurement; 1999. 7. Nielsen OS, Cummings B, O’Sullivan B, Catton C, Bell RS, Fornasier VL. Preoperative and postoperative irradiation of soft tissue sarcomas: effect of radiation field size. Int J Radiat Oncol Biol Phys 1991;21(6):1595-9. 8. Enneking WF, Spanier SS, Goodman MA. Current concepts review. The surgical staging of musculoskeletal sarcoma. J Bone Joint Surg Am 1980;62(6):1027-30. 9. Simon MA, Enneking WF. The management of soft-tissue sarcomas of the extremities. J Bone Joint Surg Am 1976;58(3):317-27. 10. Tepper J, Rosenberg SA, Glatstein E. Radiation therapy technique in soft tissue sarcomas of the extremity--policies of treatment at the National Cancer Institute. Int J Radiat Oncol Biol Phys 1982;8(2):263-73. 11. Lindberg RD, Martin RG, Romsdahl MM, Barkley HT, Jr. Conservative surgery and postoperative radiotherapy in 300 adults with soft-tissue sarcomas. Cancer 1981;47(10):23917. 12. Suit HD, Spiro I. Role of radiation in the management of adult patients with sarcoma of soft tissue. Semin Surg Oncol 1994;10(5):347-56. 13. Emami B, Graham MV, Michalski JM, Perez CA. Three-Dimensional Conformal Radiation Therapy: Clinical Aspects. In: Perez CA, Brady LW, editors. Principles and Practice of Radiation Oncology. 3rd ed. Philadelphia: Lippincott-Raven; 1998. p. 371-386. 14. Knoos T, Kristensen I, Nilsson P. Volumetric and dosimetric evaluation of radiation treatment plans: radiation conformity index. Int J Radiat Oncol Biol Phys 1998;42(5):116976. 15. Mohan R, Brewster LJ, Barest GD. A technique for computing dose volume histograms for structure combinations. Med Phys 1987;14(6):1048-52. 16. Ling CC, Humm J, Larson S, Amols H, Fuks Z, Leibel S, Koutcher JA. Towards multidimensional radiotherapy (MD-CRT): biological imaging and biological conformality. Int J Radiat Oncol Biol Phys 2000;47(3):551-60. 17. IMRT. Intensity-modulated radiotherapy: current status and issues of interest. IMRT Collaborative Working Group. Int J Radiat Oncol Biol Phys 2001;51(4):880-914. 18. Webb S. Advances in three-dimensional conformal radiation therapy physics with intensity modulation. Lancet Oncol 2000;1(1):30-6. 19. Robinson MH, Spruce L, Eeles R, Fryatt I, Harmer CL, Thomas JM, Westbury G. Limb function following conservation treatment of adult soft tissue sarcoma. Eur J Cancer 1991;27(12):1567-74. 20. Stinson SF, DeLaney TF, Greenberg J, Yang JC, Lampert MH, Hicks JE, Venzon D, White DE, Rosenberg SA, Glatstein EJ. Acute and long-term effects on limb function of combined modality limb sparing therapy for extremity soft tissue sarcoma. Int J Radiat Oncol Biol Phys 1991;21(6):1493-9. 21. O’Sullivan B, Davis A. A Randomized phase III trial of pre-operative compared to postoperative radiotherapy in extremity soft tissue sarcoma.[Abstract].Proc 43rd Annual Meeting, American Society of Therapeutic Radiology and Oncology. Int J Radiation Oncology Biol Phys 2001;51(3, supplement 1):151. 22. Millar BM, Bragg CM, Conway J, Robinson MH. Investigation of the use of intensity modulated radiotherapy (IMRT) in comparison with conformal radiotherapy in the management of soft tissue sarcoma.[Abstract]. Proc 43rd Annual Meeting, American Society of Therapeutic Radiology and Oncology. International Journal of Radiation Oncology Biology Physics 2001;51(3 (Supplement 1)):412. 23. Holt GE, Wunder JS, Griffin AM, Bell RS. Fractures following radiation therapy and limb salvage surgery for soft tissue sarcomas: high versus low dose radiotherapy.[Abstract] Proc Muskuloskeletal Tumor Society 2002:41.

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24. Hong L, Alektiar K, Hunt M, Leibel S. Intensity Modulated Radiotherapy for Soft Tissue Sarcoma of Thigh [Abstract]. Proc 44th Annual Meeting, American Society of Therapeutic Radiology and Oncology. Int J Radiat Oncol Biol Phys 2002;54 (2S):140-1. 25. Gieschen HL, Spiro IJ, Suit HD, Ott MJ, Rattner DW, Ancukiewicz M, Willett CG. Long-term results of intraoperative electron beam radiotherapy for primary and recurrent retroperitoneal soft tissue sarcoma. Int J Radiat Oncol Biol Phys 2001;50(1):127-31. 26. Petersen IA, Haddock MG, Donohue JH, Nagorney DM, Grill JP, Sargent DJ, Gunderson LL. Use of intraoperative electron beam radiotherapy in the management of retroperitoneal soft tissue sarcomas. Int J Radiat Oncol Biol Phys 2002;52(2):469-75. 27. Stoeckle E, Coindre JM, Bonvalot S, Kantor G, Terrier P, Bonichon F, Nguyen Bui B. Prognostic factors in retroperitoneal sarcoma: a multivariate analysis of a series of 165 patients of the French Cancer Center Federation Sarcoma Group. Cancer 2001;92(2):359-68. 28. Jones JJ, Catton CN, O’Sullivan B, Couture J, Heisler RL, Kandel RA, Swallow CJ. Initial results of a trial of preoperative external-beam radiation therapy and postoperative brachytherapy for retroperitoneal sarcoma. Ann Surg Oncol 2002;9(4):346-54. 29. Gilbeau L, Kantor G, Stoeckle E, Lagarde P, Thomas L, Kind M, Richaud P, Coindre JM, Bonichon F, Bui BN. Surgical resection and radiotherapy for primary retroperitoneal soft tissue sarcoma. Radiother Oncol 2002;65(3):137-43. 30. Graham JD, Robinson MH, Harmer CL. Re-irradiation of soft-tissue sarcoma. Br J Radiol 1992;65(770):157-61. 31. Nori D, Schupak K, Shiu MH, Brennan MF, Shupak K. Role of brachytherapy in recurrent extremity sarcoma in patients treated with prior surgery and irradiation. Int J Radiat Oncol Biol Phys 1991;20(6):1229-33. 32. Catton C, Davis A, Bell R, O’Sullivan B, Fornasier V, Wunder J, McLean M. Soft tissue sarcoma of the extremity. Limb salvage after failure of combined conservative therapy. Radiother Oncol 1996;41(3):209-14. 33. Pearlstone D, Janjan NA, Feig B, Yasko A, Hunt K, Pollock R, Lawyer A, Horton J, Pisters P. Re-resection with brachytherapy for locally recurrent soft tissue sarcoma arising in a previously irradiated field. Cancer J Sc Am 1999;5(1):26-33. 34. Crownover RL, Marks KE. Adjuvant brachytherapy in the treatment of soft-tissue sarcomas. Hematol Oncol Clin North Am 1999;13(3):595-607. 35. Milker-Zabel S, Zabel A, Thilmann C, Schlegel W, Wannenmacher M, Debus J. Clinical results of retreatment of vertebral bone metastases by stereotactic conformal radiotherapy and intensity-modulated radiotherapy. Int J Radiat Oncol Biol Phys 2003;55(1):162-7. 36. Pisters PW, Harrison LB, Leung DH, Woodruff JM, Casper ES, Brennan MF. Long-term results of a prospective randomized trial of adjuvant brachytherapy in soft tissue sarcoma. J Clin Oncol 1996;14(3):859-68. 37. Yang JC, Chang AE, Baker AR, Sindelar WF, Danforth DN, Topalian SL, DeLaney T, Glatstein E, Steinberg SM, Merino MJ, Rosenberg SA. Randomized prospective study of the benefit of adjuvant radiation therapy in the treatment of soft tissue sarcomas of the extremity. J Clin Oncol 1998;16(1):197-203. 38. O’Sullivan B, Bell R. Has “MAID” made it in the management of high-risk soft-tissue sarcoma. International Journal of Radiation Oncology Biology Physics 2003;56(4):915-916. 39. Cormier JN, Patel SR, Herzog CE, Ballo MT, Burgess MA, Feig BW, Hunt KK, Raney RB, Zagars GK, Benjamin RS, Pisters PW. Concurrent ifosfamide-based chemotherapy and irradiation. Analysis of treatment-related toxicity in 43 patients with sarcoma. Cancer 2001;92(6):1550-5. 40. Sauer R, Schuchardt U, Hohenberger W, Wittekind C, Papadopoulos T, Grabenbauer GG, Fietkau R. [Neoadjuvant radiochemotherapy in soft tissue sarcomas. Optimization of local functional tumor control]. Strahlenther Onkol 1999;175(6):259-66. 41. DeLaney TF, Spiro IJ, Suit HD, Gebhardt MC, Hornicek FJ, Mankin HJ, Rosenberg AL, Rosenthal DI, Miryousefi F, Ancukiewicz M, Harmon DC. Neoadjuvant Chemotherapy and Radiotherapy for Large Extremity Soft Tissue Sarcomas. International Journal of Radiation Oncology Biology Physics 2003;56(4): 1117-1127.

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42. Coles CE, Twyman N, Earl HM, Burnet NG. Conformal radiotherapy facilitates the delivery of concurrent chemotherapy and radiotherapy: a case of primitive neuroectodermal tumour of the chest wall. Sarcoma 2000;4(3):129-133. 43. Haycocks T, Kelly V, Islam M, O’Sullivan B, Swallow CJ, Catton CN. High resolution, intensity modulated radiation therapy (IMRT) for retroperitoneal soft tissue sarcoma (RPS) [Abstract]. Proceedings of the 7th Annual Meeting of the Connective Tissue Oncology Society. Sarcoma 2001;5(Supplement 1,):S24-S25. 44. O’Sullivan B, Wylie J, Catton C, Gutierrez E, Swallow CJ, Wunder J, Gullane P, Neligan P, Bell R. The local management of soft tissue sarcoma. Semin Radiat Oncol 1999;9(4):32848. 45. Low DA. Quality assurance of intensity-modulated radiotherapy. Semin Radiat Oncol 2002;12(3):219-28. 46. Dixon P, O’Sullivan B. Radiotherapy quality assurance: time for everyone to take it seriously. Eur J Cancer 2003;39(4):423-9. 47. Purdy JA. Dose-volume specification: New challenges with intensity-modulated radiation therapy. Semin Radiat Oncol 2002;12(3):199-209. 48. Pirzkall A, Carol M, Lohr F, Hoss A, Wannenmacher M, Debus J. Comparison of intensity-modulated radiotherapy with conventional conformal radiotherapy for complexshaped tumors. Int J Radiat Oncol Biol Phys 2000;48(5):1371-80. 49. Verellen D, Vanhavere F. Risk assessment of radiation-induced malignancies based on whole-body equivalent dose estimates for IMRT treatment in the head and neck region. Radiother Oncol 1999;53(3):199-203. 50. Hall EJ, Wuu CS. Radiation-induced second cancers: the impact of 3D-CRT and IMRT. Int J Radiat Oncol Biol Phys 2003;56(1):83-8. 51. Kovacs G, Hebbinghaus D, Dennert P, Kohr P, Wilhelm R, Kimmig B. Conformal treatment planning for interstitial brachytherapy. Strahlenther Onkol 1996;172(9):469-74. 52. Catton CN, Swallow CJ, O’Sullivan B. Approaches to local salvage of soft tissue sarcoma after primary site failure. Semin Radiat Oncol 1999;9(4):378-88. 53. Suit H. The Gray Lecture 2001: coming technical advances in radiation oncology. Int J Radiat Oncol Biol Phys 2002;53(4):798-809. 54. Ma CM, Pawlicki T, Lee MC, Jiang SB, Li JS, Deng J, Yi B, Mok E, Boyer AL. Energyand intensity-modulated electron beams for radiotherapy. Phys Med Biol 2000;45(8):2293311. 55. Nishizawa K, Okunieff P, Elmaleh D, McKusick KA, Strauss HW, Suit HD. Blood flow of human soft tissue sarcomas measured by thallium-201 scanning: prediction of tumor response to radiation. Int J Radiat Oncol Biol Phys 1991;20(3):593-7. 56. Nelson SJ. Multivoxel magnetic resonance spectroscopy of brain tumors. Mol Cancer Ther 2003;2(5):497-507. 57. Sijens PE. Phosphorus MR spectroscopy in the treatment of human extremity sarcomas. NMR Biomed 1998;11(7):341-53. 58. Baudelet C, Gallez B. How does blood oxygen level-dependent (BOLD) contrast correlate with oxygen partial pressure (pO2) inside tumors? Magn Reson Med 2002;48(6):980-6. 59. Jaffray DA, Siewerdsen JH, Wong JW, Martinez AA. Flat-panel cone-beam computed tomography for image-guided radiation therapy. Int J Radiat Oncol Biol Phys 2002;53(5):1337-49. 60. Mackie TR, Kapatoes J, Ruchala K, Lu W, Wu C, Olivera G, Forrest L, Tome W, Welsh J, Jeraj R, Harari P, Reckwerdt P, Paliwal B, Ritter M, Keller H, Fowler J, Mehta M. Image guidance for precise conformal radiotherapy. Int J Radiat Oncol Biol Phys 2003;56(1):89-105.

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61. Borden EC, Baker LH, Bell RS, Bramwell V, Demetri GD, Eisenberg BL, Fletcher CD, Fletcher JA, Ladanyi M, Meltzer P, O’Sullivan B, Parkinson DR, Pisters PW, Saxman S, Singer S, Sundaram M, Van Oosterom AT, Verweij J, Waalen J, Weiss SW, Brennan MF. Soft tissue sarcomas of adults: state of the translational science. Clin Cancer Res 2003;9(6):1941-56. 62. Partsch H, Stoberl C, Wruhs M, Wenzel-Hora BI. Indirect lymphography with iotrolan. Fortschr Geb Rontgenstrahlen Nuklearmed Erganzungsbd 1989;128:178-81.

Chapter 3 Preoperative therapy for soft tissue sarcoma

Janice N. Cormier, MD, MPH, Howard N. Langstein, MD, Peter W. T. Pisters, MD

The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030-4009

Correspondence to: Peter Pisters MD The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard Box 444 Houston, TX 77030-4009 USA

44

1

INTRODUCTION

A number of theoretical advantages are associated with the use of preoperative therapy for solid tumors. For patients with soft tissue sarcomas, a survival advantage has been difficult to establish, but there is evidence that induction therapy (e.g., chemotherapy, radiation therapy, or multimodality regimens) may result in cytoreduction that facilitates less radical surgical resections with improved postoperative function. This is particularly important for patients presenting with large tumors that are initially resectable only by means of amputation. The focus of this review is to summarize the rationale, treatment options and ultimate impact of induction therapy for patients with soft tissue sarcoma.

2

RATIONALE FOR INDUCTION THERAPY

2.1

Solid Tumors

The primary goal of induction therapy for solid tumors is to decrease the tumor burden. The concept of induction therapy, consisting of either preoperative local therapies (e.g., radiation, chemoradiation, or limb perfusion) or systemic therapy (e.g., chemotherapy), arose because surgical resection alone was inadequate for a number of tumors (1). There are several theoretical reasons for using induction or preoperative therapies for localized tumors. First, reducing the size of the tumor may enable margin-negative (R0) resections, resection of tumors that were initially unresectable, or less radical resections that allow preservation of function. Second, there may be systemic benefits from the early delivery of cytotoxic agents, such as elimination of micrometastatic disease and improved vascular delivery of therapies to undisturbed tumors (1). Finally, induction therapy allows rapid assessment of tumor response in situ. This is not possible with postoperative therapy, which requires long-term follow-up for assessment of its effectiveness (2). There are also potential disadvantages to induction therapy, including the associated toxicity (morbidity), possible ineffectiveness of induction therapy and delayed local treatment of the primary tumor, cost of therapy, and obscuring of pathologic staging information (3). When induction therapy includes radiation, the risk of wound-healing complications after surgery is increased. The effectiveness of induction therapy must be demonstrated prior to its generalization because many patients may be cured by locoregional treatment alone (1,3). The sequence of treatments -- chemotherapy, radiation therapy, and surgery-- may be important in determining outcomes for patients with some tumor types. Three categories of tumors have been defined with regard to

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induction chemotherapy: (1) tumors for which chemotherapy has been demonstrated to be the primary therapeutic modality (e.g., embryonal rhabdomyosarcoma, small cell lung cancer, and lymphoma), (2) tumors for which there is evidence of downstaging with chemotherapy (e.g., osteogenic and Ewing’s sarcoma, locally advanced breast cancer, anal carcinoma, and laryngeal cancer), and (3) tumors for which the benefits of induction chemotherapy have not been scientifically validated (e.g., esophageal cancer, gastric and pancreas cancers, non-small cell lung cancer, prostate cancer, cervical carcinoma, nasopharyngeal cancer, and soft tissue sarcomas) (1).

2.2

Sarcomas

Sarcomas are among the group of solid tumors for which the benefits of induction therapy have been difficult to establish. Survival benefits have not been definitively demonstrated because of the paucity of adequately powered randomized clinical trials. However, single-institution reports suggest that preoperative therapy enables tumor downstaging and organ sparing in some patients. Several distinct groups of sarcomas are recognized: soft tissue sarcomas, bone sarcomas (osteosarcomas and chondrosarcomas), Ewing’s sarcomas, and peripheral primitive neuroectodermal tumors. Since the late 1980s, preoperative chemotherapy has been the standard treatment for patients with osteosarcoma based on data from randomized controlled trials demonstrating a significant survival advantage with systemic therapy (4,5). The histologic subtypes rhabdomyosarcoma and Ewing’s sarcoma have been demonstrated to have a higher propensity for systemic metastases, and for these histologies, the addition of chemotherapy may have survival advantages and is considered standard care (6,7). The use of chemotherapy (preoperative or postoperative) for other sarcomas remains controversial. Patients with large (> 5 cm), high-grade, deep, extremity soft tissue sarcomas (American Joint Committee on Cancer stage III) commonly develop distant recurrence and subsequently die of sarcoma. Consequently, pre- or postoperative anthracycline-based chemotherapy is often considered in these patients.

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3

INDUCTION THERAPY OPTIONS

3.1.

Preoperative Radiation Therapy

3.1.1. Extremity and Trunk Sarcomas The primary goal of pre- or postoperative radiation therapy is to maximize local tumor control. Surgical resection and radiation therapy (external beam or brachytherapy) are generally considered standard treatment modalities for most patients with large, high-grade extremity soft tissue sarcomas. This therapeutic approach is based on data from two phase III trials demonstrating improved local control with the addition of radiation therapy for patients with localized extremity and trunk sarcomas (8,9). In the randomized trial from the National Cancer Institute (NCI), 91 patients with high-grade extremity tumors were treated with limb-sparing surgery followed by chemotherapy alone or by radiation therapy plus adjuvant chemotherapy. A second group of 50 patients with low-grade tumors were treated with resection alone or resection plus radiation therapy. The 10-year rate of local control for all patients receiving radiation therapy was 98%, compared with 70% for those not receiving radiation therapy (p = 0.0001) (8). Similarly, in the randomized trial from Memorial Sloan-Kettering Cancer Center, 164 patients with extremity or trunk sarcomas underwent observation or brachytherapy after conservative surgery. The 5-year local control rate for patients with high-grade tumors was 66% in the observation group and 89% in the brachytherapy group (p = 0.003) (9). No consensus exists on the optimal sequence of radiation therapy and surgery. Proponents of preoperative radiation therapy cite several advantages. First, multidisciplinary planning with radiation oncologists, medical oncologists, and surgeons is facilitated early in the course of therapy while the tumor is in place. Second, lower doses of preoperative radiation can more easily be delivered to an undisturbed tumor bed, which may have improved tissue oxygenation (10-12). Third, the size of preoperative radiation fields is smaller and the number of joints included in those fields is fewer than in postoperative radiation fields, and this may result in improved functional outcome (13). And finally, preoperative radiation may induce tumor shrinkage and thus facilitate surgical resection (11). On the other hand, critics of preoperative radiation therapy cite as disadvantages the difficulty of pathologic assessment of margins in irradiated specimens and the increased rate of wound complications. The only randomized comparison of preoperative and postoperative radiation therapy for soft tissue sarcoma conducted to date was performed by the National Cancer Institute of Canada Clinical Trials Group (14). This trial was designed to compare complications and functional outcomes of sarcoma patients treated with preoperative or postoperative external-beam radiation

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therapy. From October 1994 to December 1997, 190 patients were randomized to receive preoperative radiation therapy (50 Gy) or postoperative radiation therapy (66 Gy). At a median follow-up of 3.3 years, wound complications had occurred in 35% of patients given preoperative radiation therapy but only 17% of patients given postoperative radiation therapy (p = 0.01). The majority of wound complications occurred in patients with lower extremity sarcomas. Both groups achieved similarly high rates of local control and progression-free survival at 3 years (14). Interestingly, overall survival appeared to be improved in patients treated with preoperative radiation. These findings suggest that preoperative external-beam radiation therapy is effective but that patients should be informed of the increased risk for major wound complications — particularly for patients with lower extremity soft tissue sarcoma.

3.1.2. Retroperitoneal Sarcomas Based on the high rates of local control obtained with surgery plus radiation therapy in patients with extremity and trunk sarcomas, there has been interest in attempting such strategies for patients with retroperitoneal sarcomas. Administering preoperative radiation therapy to retroperitoneal soft tissue sarcomas is complex. Large tumors in proximity to vital radiosensitive anatomic structures frequently hinder safe delivery of treatment. However, there are several advantages to administering radiation therapy preoperatively for retroperitoneal sarcomas: the gross tumor volume is definable, which allows accurate treatment planning; tumors often displace radiosensitive viscera outside of the radiation field; and biologically effective radiation doses may be lower in the preoperative setting (15). Several groups have prospectively examined the effects of preoperative and intraoperative radiation therapy administered to patients with retroperitoneal sarcomas (16-19). These studies demonstrate that preoperative radiation doses of 45 to 50.4 Gy can be delivered to the retroperitoneum with acceptable treatment-related toxicity.

3.2

Preoperative Chemotherapy

Chemotherapy given either preoperatively or postoperatively for soft tissue sarcomas remains controversial. Soft tissue sarcomas encompass a diverse group of cancers that vary greatly in natural history and response to treatment, and the results of conventional chemotherapy regimens have generally been poor. While some histologic subtypes of sarcoma are very

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responsive to cytotoxic chemotherapy, most subtypes are resistant to current agents. The most active chemotherapeutic agents for bone sarcomas are doxorubicin, methotrexate, cisplatin, and ifosfamide. For Ewing’s sarcoma, doxorubicin, vincristine, cyclophosphamide, and ifosfamide have demonstrated response rates of up to 90% (20,21). Dactinomycin, vincristine, and etoposide are active only against small-cell sarcomas, including Ewing’s sarcoma, rhabdomyosarcoma, primitive neuroectodermal tumor, and neuroblastoma. For other subtypes of sarcoma doxorubicin and ifosfamide are the two most active agents, with consistently reported response rates of 20% to 40% (20,22,23). Both agents have demonstrated positive doseresponse curves (24,25). Response rates to ifosfamide at higher doses or in combination with doxorubicin have been reported to range from 20% to 60% in single-institution series (24,26-29). There are several theoretical benefits associated with early systemic treatment. In addition to early treatment of micrometastatic disease, preoperative systemic chemotherapy may induce primary tumor shrinkage, resulting in increased rates of limb salvage. In addition, a major deterrent to the use of postoperative chemotherapy has been the risk of adverse toxic effects in patients who do not respond to therapy. The ability to assess the effects of treatment by assessing tumor response in situ argues in favor of preoperative chemotherapy. Such an approach spares those patients who fail to respond to therapy from the prolonged toxicities of an ineffective treatment. Patients who are deemed to be responsive to preoperative chemotherapy can be treated postoperatively as well with hope of improving their outcome.

3.3

Multimodality Therapy / Chemoradiation

3.3.1

Extremity and Trunk Sarcomas

The objectives of sequential multimodality therapy or concurrent chemoradiation therapy are similar to those of other induction regimens, to achieve tumor reduction and provide local control while allowing the timely administration of systemic therapy to eradicate potential micrometastatic disease. Treatment approaches that combine systemic chemotherapy with radiosensitizers and concurrent external-beam radiation may improve diseasefree survival by treating microscopic disease while enhancing the treatment of macroscopic disease. Concurrent chemoradiation with doxorubicin-based regimens reportedly produces favorable local control rates for patients with soft tissue sarcoma (30,31). The initial experience involved intra-arterial doxorubicin combined with high-dose-per-fraction radiation therapy in patients with extremity soft tissue sarcomas. Since those findings were

49

published, several groups have attempted to evaluate the optimal route of administration (30,32-35), alternative chemotherapeutic agents (36-38), and the toxicity of combined therapies (39). The route of administration of doxorubicin, intra-arterial versus intravenous, was evaluated in a small phase III trial, which demonstrated no difference between the two routes in limb salvage, local recurrence, complications, or pathologic response (40). Given these results and the complexity of intra-arterial administration, intravenous administration of chemotherapeutic agents during concurrent chemoradiation is the current standard (31). Doxorubicin is the most commonly studied radiosensitizer for soft tissue sarcoma (31). A number of other agents have been evaluated, including idoxuridine (38), razoxane (36), and ifosfamide (41). In addition, various chemotherapy approaches have been studied including short-duration chemotherapy with concurrent rapid-fractionation radiation therapy (31), lowdose continuous-infusion chemotherapy with concurrent radiation therapy (42), and sequential chemotherapy and radiation therapy (43). The toxicity and responses for each approach appear acceptable, but additional survival data are required to determine if one is superior. It is difficult to make comparisons between chemotherapeutic agents and regimens because of the diversity of the patient populations, but in general preoperative chemoradiation combined with surgery has been demonstrated to be feasible, to have acceptable toxicity, and to result in favorable local control rates for patient with localized and locally advanced soft tissue sarcomas. Theoretical advantages of concurrent treatment notwithstanding, the concurrent use of local and systemic therapies decreases the total treatment time for patients with high-risk sarcoma. This decrease represents a specific advantage over current sequential multimodality treatment approaches, for which the total time for radiation, chemotherapy, surgery, and rehabilitation frequently exceeds 6 to 9 months. The toxicity associated with chemoradiation depends on the particular chemotherapeutic agent and route of administration as well as the radiation dose/fractionation regimen. Tumor factors such as size and anatomic site may also be critical in treatment-related toxicity. As with preoperative radiation alone, the rate of postoperative wound complications for patients treated with preoperative chemoradiation is high, reported as 26% in some series (30).

3.3.2

Retroperitoneal Sarcomas

Anatomic considerations favor preoperative over postoperative radiation for patients with retroperitoneal sarcomas (31), and pilot and phase I studies using idoxuridine-based and doxorubicin-based (44,45)

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chemoradiation have demonstrated that these approaches are safe and feasible for patients with retroperitoneal sarcomas. The Radiation Therapy Oncology Group is conducting a multicenter phase II trial of combined multimodality treatment for patients with intermediate- or high-grade retroperitoneal sarcomas (RTOG S-0124, www.rtog.org). Patients are given preoperative systemic therapy with doxorubicin and ifosfamide (up to 4 cycles) followed by preoperative external-beam radiation therapy (45 to 50 Gy) and then surgical resection with an intraoperative or postoperative radiation boost. The objective of the trial is to assess the feasibility, toxicity, and complications of this multimodality treatment regimen.

4

IMPACT OF PREOPERATIVE THERAPY

4.1

Scope of Surgery

The goal of surgical therapy for soft tissue sarcoma is to achieve grossly and microscopically negative (R0) margins of resection with the best possible functional result. Experience has demonstrated that surgical resection alone is generally inadequate treatment except for patients with small, superficial, well-differentiated lesions (46-48). The translation of a response to induction therapy (e.g., radiation therapy or chemotherapy) into a clinically meaningful outcome — reduction in the scope of surgical resection — is not well defined. A recent retrospective analysis examined the impact of preoperative chemotherapy on the scope of surgery. The study included a blinded assessment of imaging studies obtained before and after induction chemotherapy in 65 patients with stage II or III soft tissue sarcomas (49). The impact of induction chemotherapy was classified into one of three categories based on the perceived impact of induction chemotherapy on the planned surgical procedure: no change in planned surgical resection, decreased scope of resection, or increased scope of resection. The radiographic responses to preoperative chemotherapy included partial responses in 34%, minimal responses in 9%, stable disease in 31%, and progressive disease in 26%. Only 8 patients (12%) were believed to have downstaging sufficient to decrease the scope of their operation, and 6 patients (9%) had disease progression sufficient to increase the scope of their operation (49). Of interest, none of the 9 patients who were determined to require amputation prior to chemotherapy were able to undergo limb salvage after chemotherapy. Thus, there was no clear evidence to support the perception that patients with locally advanced extremity soft tissue sarcoma (resectable only by amputation) could be “downstaged” to permit function-preserving, limb-sparing surgery.

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4.2

Functional Status and Quality of Life

Treatment for sarcomas may result in significant functional disability and reduced quality of life (50). An adequate margin-negative (R0) tumor resection for soft tissue sarcoma includes removal of the tumor with a margin of normal tissue confirmed to be tumor-free by microscopic evaluation. The proximity of some tumors to vital anatomic structures often requires resection of such structures, including major motor nerves, resulting in significant postoperative functional impairment. In a prospective study evaluating functional outcome in patients with soft tissue sarcomas, large tumor size, postoperative complications, and neural sacrifice were associated with poor functional outcome (51). Treatment sequencing may have an impact on long-term functional outcome. Preoperative external-beam radiation therapy allows the delivery of lower doses of radiation to smaller volumes of tissue with potentially less long-term morbidity from tissue damage. In a large retrospective study, the incidence of delayed radiation-associated complications including soft tissue necrosis, bone fractures, osteonecrosis, edema, and fibrosis was 9% for postoperative radiation therapy and 5% for preoperative radiation therapy (p = 0.03) (52). The impact of the timing of radiation therapy on functional outcome was also examined in a randomized clinical trial supported by the National Cancer Institute of Canada (50). Two years following surgery, there were no differences in function or quality of life, with all measures returning to pretreatment levels, between the preoperative and postoperative radiation therapy groups. However, long-term follow-up is needed to assess the late manifestations of preoperative and postoperative radiation therapy, particularly with respect to physical function, limb edema, and bone fractures (14).

4.3

Pathologic Response

An advantage of preoperative therapy is the ability to assess tumor response in situ using radiologic imaging as well as pathologically following surgical resection. This strategy allows patients with unresponsive tumors to be identified and spared the toxicity and cost of additional treatment with a regimen that is not effective. The pathologic responses identified following induction chemotherapy have ranged from no response to complete response with no residual viable cancer cells. In most cases, responses are partial or incomplete. Studies have demonstrated that cells located in the wellvascularized tumor periphery are most likely to be affected by induction

52

chemotherapy and that if left untreated, these peripheral tumor cells are likely to be responsible for tumor recurrence (53). Chemotherapy-induced pathologic necrosis is a predictor of survival in patient’s who receive preoperative chemotherapy for osteogenic and Ewing’s sarcoma (54-57). However, the incidence of treatment-induced pathologic necrosis and its correlation with clinical outcomes are not well defined in patients with soft tissue sarcomas (2). Small studies have reported rates of complete pathologic tumor necrosis following doxorubicin-based induction chemotherapy of only 5% to 9% (58-60). In a retrospective analysis of 496 patients with intermediate- and high-grade extremity soft tissue sarcomas who were treated with preoperative therapy (consisting primarily of doxorubicin-based chemotherapy and radiation), complete pathologic responses were noted in 69 patients (14%). With a mean follow-up of 10 years, the 10-year local recurrence rate for patients with complete pathologic necrosis was 11%, compared to 23% for patients with less than 95% pathologic necrosis. However, the 10-year survival rate for patients with complete pathologic responses was 71%, compared to 55% for other patients (p = 0.0001)(2). Based on these results, the authors concluded that pathologic assessment of necrosis can be considered a surrogate endpoint for survival outcomes in patients with soft tissue sarcomas and, as such, can be used as a valid and timely endpoint by which novel agents and treatment protocols are evaluated (2).

5

SURVIVAL

5.1

Radiation Therapy

The relationship between local control and distant metastasis/diseasespecific survival has long been debated in the treatment of solid tumors. It is biologically plausible that with high-risk disease, more complete eradication of aggressive residual sarcoma could reduce the risk of distant progression and sarcoma-specific death. Important issues in this debate include (1) whether a local treatment modality can impact distant metastasis and tumorrelated mortality and (2) whether patients who have increased rates of local failure have an increased risk of subsequent distant metastasis. However, the prevention of local recurrence by either amputation or radiation therapy has not translated into a survival benefit in sarcoma patients (61). Examination of the published phase III trials of postoperative radiation for sarcoma indicates that local tumor control does not have an impact on disease-specific survival (62,63). However, the existing phase III trials (8,9,62,63) of postoperative radiation included relatively small numbers of patients at risk for recurrence — 52 patients in the NCI trial (J. Yang, personal communication) and 77 patients in the Memorial Sloan-Kettering

53

trial (M.F. Brennan, personal communication). If one accepts that the association between local control and survival is best addressed through studies of high-risk patients, then it is conceivable that the existing phase III trials may not be adequate for the assessment of a potential association. At least two large retrospective studies have examined the outcomes associated with preoperative versus postoperative radiation therapy (52,64). Both studies found no difference in the rates of local control and diseasespecific survival between the two treatment sequence groups. The only randomized comparison of preoperative and postoperative radiation therapy is the multi-institutional study performed by the National Cancer Institute of Canada Clinical Trials Group (14). With a median followup of 3.3 years, there were no differences in local recurrence, distant recurrence, or progression-free survival but there was a slightly higher overall survival rate in patients who received preoperative radiation therapy than in those who were treated with postoperative radiation therapy (p = 0.0481). It has been noted that there was a relative imbalance in the number of patients who died of other causes between the pre- and postoperative radiation arms. It is not clear whether this imbalance or other confounding factors could account for the significant survival difference or whether this observation indicates a significant clinical advantage associated with preoperative radiation. Interestingly, an improvement in disease-specific survival associated with the use of radiation has been demonstrated in phase III trials in other malignancies, including node-positive breast cancer (65-68) and some squamous cell carcinomas of the head and neck (69). A common theme in those reports was an examination of the relationship between local control and survival in subgroups of patients at high risk for distant metastasis.

5.2

Chemotherapy

In a meta-analysis of patients with extremity soft tissue sarcomas, the estimated survival benefit attributable to postoperative chemotherapy was only 7% (70). More recently, one randomized prospective trial of 104 patients with heterogeneous tumors reported an overall survival rate of 75 months for patients treated with surgery and chemotherapy versus 46 months for those treated with surgery with or without radiation (p = 0.03) (71). Two additional smaller randomized trials of patients with primary or locally recurrent soft tissue sarcoma also showed higher 5-year overall survival rates associated with chemotherapy, although the differences were not statistically significant (72,73). The interpretation of this complex literature is difficult, and there is no consensus on the role of chemotherapy for patients with localized high-risk soft tissue sarcoma (74,75).

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Although radiographic response rates to chemotherapy have been 20 to 30%, there have been conflicting reports regarding whether tumor response is associated with improved rates of disease-specific and overall survival (76,49,58). A major goal of preoperative or induction systemic therapy is the early eradication of micrometastatic disease. A single randomized phase II/III study attempted to examine the survival benefits associated with preoperative chemotherapy for soft tissue sarcoma (77). However, the study was closed prior to initiation of the phase III arm because of slow accrual. In the 134 patients randomized for the phase II study, the 5-year disease-free survival rate was 52% for the no-chemotherapy arm and 56% for the chemotherapy arm (p = 0.35).

6

SURGICAL COMPLICATIONS

6.1

Radiation Therapy

There are several studies which have examined the rate of postoperative wound complication following preoperative radiation therapy in patients with extremity soft tissue sarcoma (Table 1). In two large retrospective studies of preoperative versus postoperative radiation therapy (64,52), rates of postoperative wound complications were reported as 31% for preoperative radiation compared to 8% for postoperative radiation. Similarly, there was an increased rate of 18% (95% confidence interval 5% to 30%, p<0.01) of wound complications associated with the use of preoperative radiation therapy in the only randomized comparative study (14). Tissue transfer techniques are being used more often in patients undergoing preoperative radiation therapy with better postoperative outcomes (Fig 1)(79,81). Centers with experience in pedicled and free tissue flaps have demonstrated high success rates (> 90%) in ensuring healed wounds in a single-stage operation (81). Unlike the results with extremity sarcomas, there have been no reports of increased rates of wound complications in patients with retroperitoneal sarcomas who received preoperative radiation therapy (16). Long-term toxic effects of preoperative radiation therapy have been reported in only a small fraction of patients. In a report from the Massachusetts General Hospital, 4 of 37 retroperitoneal sarcoma patients treated with preoperative radiation therapy suffered from neuropathy, hydronephrosis, fistula formation, vascular injury and/or bowel complications (18). Similary, 4 of 41 patients treated at the Mayo Clinic were reported to have significant late toxic effects associated with combined therapy; these effects included duodenitis/gastric outlet obstruction, chronic pain, bowel obstruction requiring surgery, and, in one patient, death (16).

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6.2

Chemotherapy

There are few clinical data examining the relationship between preoperative chemotherapy and surgical complications following resection of soft tissue sarcomas. A single-institution retrospective analysis compared the rate of postoperative complications in 105 patients who received preoperative chemotherapy to that in 204 patients who were treated with surgical resection alone (82). The characteristics of the two treatment groups are presented in Table 2. The median interval between the end of chemotherapy and surgical resection was 45 days. Postoperative complication rates were similar in the

57

two treatment groups in both those with extremity sarcomas (34% versus 41%) and those with retroperitoneal/visceral sarcomas (29% versus 34%). The most common complications in both groups were wound infections.

7

CONCLUSIONS

Soft tissue sarcomas continue to represent a therapeutic challenge because of their rarity and the heterogeneity of the many histologic subtypes. The efficacy of preoperative therapy has been difficult to establish. However, there is evidence that induction therapy may result in reduction of tumor size that facilitates less radical surgical resections with improved postoperative function. Clinical evidence supports continued investigation of preoperative treatment approaches for soft tissue sarcomas. The use of preoperative therapy reinforces the need for cooperation among oncologic disciplines. The timing and sequence of treatment modalities should be discussed at the time of tumor diagnosis. Individual treatment decisions must weigh the estimated likelihood and severity of potential short- and long-term complications associated with one treatment sequence versus another.

8

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38. Sondak VK, Robertson JM, Sussman JJ. Preopertive idoxuridine and radiation for large soft tissue sarcomas: clinical results with five-year follow-up. Ann Surg Oncol 1998;5:106-112. 39. Pisters PW. Chemoradiation treatment strategies for localized sarcoma: conventional and investigational approaches. Semin Surg Oncol 1999;17(1):66-71. 40. Eilber FR, Giuliano AE, Huth JF, Weisenburger TH, Eckardt J. Intravenous (IV) vs. intraarterial (IA) Adriamycin, 2800r radiation and surgical excision for extremity soft tissue sarcomas: a randomized prospective trial [Abstract 309]. Proc Am Soc Clin Oncol 1990. 41. Cormier JN, Patel SR, Herzog CE, et al. Concurrent ifosfamide-based chemotherapy and irradiation. Analysis of treatment-related toxicity in 43 patients with sarcoma. Cancer 2001;92(6):1550-5. 42. Toma S, Palumbo R, Vincente M. Concomitant doxorubicin (DOXO) by continuous infusion (CI) and radiotherapy (RT) at low doses in locally advanced and/or metastatic soft tissue sarcomas (STS): long-term results of a phase II study [Abstract 520]. Proc Am Soc Clin Oncol 1995. 43. Kraybill WG, Spiro IJ, Harris J. Radiation Therapy Oncology Group (RTOG) 9514: a phase II study of neoadjuvant chemotherapy (CT) and radiation therapy (RT) in high risk (HR), high grade, soft tissue sarcomas (STS) of the extremities and body wall: a preliminary report [Abstract 348a]. Proc Am Soc Clin Oncol 2001. 44. Eilber F, Eckardt J, Rosen G, Forscher C, Selch M, Fu YS. Preoperative therapy for soft tissue sarcoma. Hematol Oncol Clin North Am 1995;9(4):817-23. 45. Pisters PWT, Patel SR, Pollock RE. Phase I trial of preoperative doxorubicinbased concurrent chemoradiation and electron-beam intraoperative radiation therapy (IORT) for resectable retroperitoneal sarcomas [Abstract 103]. Cancer J Sci Am 1998. 46. Alektiar KM, Leung D, Zelefsky MJ, Brennan MF. Adjuvant radiation for stage II-B soft tissue sarcoma of the extremity. J Clin Oncol 2002;20(6):1643-1650. 47. Fleming JB, Berman RS, Cheng SC, et al. Long-term outcome of patients with American Joint Committee on Cancer stage IIB extremity soft tissue sarcomas. J Clin Oncol 1999;17(9):2772-80. 48. Baldini EH, Goldberg J, Jenner C, et al. Long-term outcomes after functionsparing surgery without radiotherapy for soft tissue sarcoma of the extremities and trunk. J Clin Oncol 1999;17(10):3252-9. 49. Meric F, Hess KR, Varma DG, et al. Radiographic response to neoadjuvant chemotherapy is a predictor of local control and survival in soft tissue sarcomas. Cancer 2002;95(5):1120-6. 50. Davis AM, O’Sullivan B, Bell RS, et al. Function and health status outcomes in a randomized trial comparing preoperative and postoperative radiotherapy in extremity soft tissue sarcoma. J Clin Oncol 2002;20(22):4472-7. 51. Bell RS, O’Sullivan B, Davis A, Langer F, Cummings B, Fornasier VL. Functional outcome in patients treated with surgery and irradiation for soft tissue tumours. J Surg Oncol 1991;48(4):224-31.

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52. Zagars GK, Ballo MT, Pisters PW, Pollock RE, Patel SR, Benjamin RS. Preoperative vs. postoperative radiation therapy for soft tissue sarcoma: a retrospective comparative evaluation of disease outcome. Int J Radiat Oncol Biol Phys 2003;56(2):482-8. 53. Stephens FO. Induction chemotherapy: to downgrade aggressive cancers to improve curability by surgery and/or radiotherapy. Eur J Surg Oncol 2001 ;27(7):67288. 54. Lindner NJ, Scarborough MT, Spanier SS, Enneking WF. Local host response in osteosarcoma after chemotherapy referred to radiographs, CT, tumour necrosis and patient survival. J Cancer Res Clin Oncol 1998;124(10):575-80. 55. Rosen G, Caparros B, Huvos AG, et al. Preoperative chemotherapy for osteogenic sarcoma: selection of postoperative adjuvant chemotherapy based on the response of the primary tumor to preoperative chemotherapy. Cancer 1982;49(6):1221-30. 56. Wunder JS, Paulian G, Huvos AG, Heller G, Meyers PA, Healey JH. The histological response to chemotherapy as a predictor of the oncological outcome of operative treatment of Ewing sarcoma. J Bone Joint Surg Am 1998;80(7):1020-33. 57. Picci P, Bohling T, Bacci G, et al. Chemotherapy-induced tumor necrosis as a prognostic factor in localized Ewing’s sarcoma of the extremities. J Clin Oncol 1997;15(4):1553-9. 58. Ottaiano A, De Chiara A, Fazioli F, et al. Neoadjuvant chemotherapy for intermediate/high-grade soft tissue sarcomas: five-year results with epirubicin and ifosfamide. Anticancer Res 2002;22(6B):3555-9. 59. Henshaw RM, Priebat DA, Perry DJ, Shmookler BM, Malawer MM. Survival after induction chemotherapy and surgical resection for high-grade soft tissue sarcoma. Is radiation necessary? Ann Surg Oncol 2001;8(6):484-95. 60. Rahoty P, Konya A. Results of preoperative neoadjuvant chemotherapy and surgery in the management of patients with soft tissue sarcoma. Eur J Surg Oncol 1993;19(6):641-5. 61. Brennan MF. More is less: systemic treatment for local control in soft tissue sarcoma. Ann Surg Oncol 2001;8(6):480-1. 62. Brennan MF, Alektiar KM, Maki RG. Sarcomas of the soft tissue and bone. In: DeVita V, Hellman S, Rosenberg SA, eds. Cancer: Principles & Practice of Oncology. Philadelphia: Lippincott Williams & Wilkins, 2001:1841. 63. Espat NJ, Lewis JJ. The biological significance of failure at the primary site on ultimate survival in soft tissue sarcoma. Semin Radiat Oncol 1999;9(4):369-77. 64. Cheng EY, Dusenbery KE, Winters MR, Thompson RC. Soft tissue sarcomas: preoperative versus postoperative radiotherapy. J Surg Oncol 1996;61:90-99. 65. Overgaard M, Jensen MB, Overgaard J, et al. Postoperative radiotherapy in highrisk postmenopausal breast-cancer patients given adjuvant tamoxifen: Danish Breast Cancer Cooperative Group DBCG 82c randomised trial. Lancet 1999;353(9165):1641-8.

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66. Overgaard M, Hansen PS, Overgaard J, et al. Postoperative radiotherapy in highrisk premenopausal women with breast cancer who receive adjuvant chemotherapy. Danish Breast Cancer Cooperative Group 82b Trial. N Engl J Med 1997;337(14):94955. 67. Ragaz J, Jackson SM, Le N, et al. Adjuvant radiotherapy and chemotherapy in node-positive premenopausal women with breast cancer. N Engl J Med 1997;337(14):956-62. 68. Whelan TJ, Julian J, Wright J, Jadad AR, Levine ML. Does locoregional radiation therapy improve survival in breast cancer? A meta-analysis. J Clin Oncol 2000;18(6):1220-1229. 69. Mishra RC, Singh DN, Mishra TK. Post-operative radiotherapy in carcinoma of buccal mucosa, a prospective randomized trial. Eur J Surg Oncol 1996;22(5):502-4. 70. Tierney JF. Adjuvant chemotherapy for localised resectable soft-tissue sarcoma of adults: meta-analysis of individual data. Sarcoma Meta-analysis Collaboration. Lancet 1997;350(9092):1647-54. 71. Frustaci S, Gherlinzoni F, De Paoli A, et al. Adjuvant chemotherapy for adult soft tissue sarcomas of the extremities and girdles: results of the Italian randomized cooperative trial. J Clin Oncol 2001;19(5):1238-47. 72. Brodowicz T, Schwameis E, Widder J. Intensified adjuvant IFADIC chemotherapy for adult soft tissue sarcoma: a prospective randomized feasibility trial. Sarcoma 2000;4:151-160. 73. Petrioli R, Coratti A, Correale P, et al. Adjuvant epirubicin with or without ifosfamide for adult soft-tissue sarcoma. Am J Clin Oncol 2002;25(5):468-73. 74. Bramwell VHC. Adjuvant chemotherapy for adult soft tissue sarcoma: is there a standard of care? J Clin Oncol 2001;19(5):1235-1237. 75. Figueredo A, Bramwell VHC, Bell R, Davis AM, Charette ML. Adjuvant chemotherapy following complete resection of soft tissue sarcoma in adults: a clinical practice guideline. Sarcoma 2002;6:5-18. 76. Pisters PW, Patel SR, Varma DG, et al. Preoperative chemotherapy for stage IIIB extremity soft tissue sarcoma: long-term results from a single institution. J Clin Oncol 1997;15(12):3481-7. 77. Gortzak E, Azzarelli A, Buesa J, et al. A randomised phase II study on neoadjuvant chemotherapy for ‘high-risk’ adult soft-tissue sarcoma. Eur J Cancer 2001;37(9):1096-103. 78. Bujko K, Suit HD, Springfield DS, Convery K. Wound healing after preoperative radiation for sarcoma of soft tissues. Surg Gynecol Obstet 1993;176(2):124-34. 79. Peat BG, Bell RS, Davis A, et al. Wound-healing complications after soft-tissue sarcoma surgery. Plast Reconstr Surg 1994;93(5):980-7.

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80. Prosnitz LR, Maguire P, Anderson JM, et al. The treatment of high-grade soft tissue sarcomas with preoperative thermoradiotherapy. Int J Radiat Oncol Biol Phys 1999;45(4):941-9. 81. Langstein HN, Robb GL. Reconstructive approaches in soft tissue sarcoma. Semin Surg Oncol 1999;17(1):52-65. 82. Meric F, Milas M, Hunt KK, et al. Impact of neoadjuvant chemotherapy on postoperative morbidity in soft tissue sarcomas. J Clin Oncol 2000;18(19):3378-383.

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Chapter 4 TNF-based isolated limb perfusion: A decade of experience with antivascular therapy in the management of locally advanced extremity soft tissue sarcomas Dirk J Grünhagen, Flavia Brunstein, Timo L.M. ten Hagen, Albertus N. van Geel, Johannes H.W. de Wilt, and Alexander M.M. Eggermont Dept. of Surgical Oncology, Erasmus University Medical Centre-Daniel den Hoed Cancer Centre, Rotterdam, the Netherlands

Correspondence to: Alexander M.M. Eggermont, MD, PhD Professor Surgical Oncology, Head of Department Department of Surgical Oncology Erasmus University Medical Center - Daniel den Hoed Cancer Center 301 Groene Hilledijk 3075 EA Rotterdam The Netherlands Tel: 31-10-439 19 11 Fax: 31-10–439 10 11 E-mail: [email protected]

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1

INTRODUCTION

In the management of locally advanced extremity soft tissue sarcomas limb salvage has become all the more important in the light of evidence that amputations do not improve survival rates in patients with large (>5 cm) deep seated high grade sarcomas. Several studies have shown that marginal excisions with a high risk for local recurrence do not influence survival significantly (1-4). Of the 7800 new cases of STS diagnosed in the USA each year about 4700 occur in the extremities and tumors are often large at the time of diagnosis. (5) Treatment options for locally advanced extremity STS may consist of an amputation or a limb sparing extensive surgical procedure followed by radiation therapy. This combination may mutilate and compromise limb function considerably. Preoperative therapies to improve limb salvage rates have been propagated. Preoperative radiotherapy alone or in combination with intraarterial or intravenous chemotherapy has been reported to improve resectability rates of extremity soft tissue sarcomas. (6-8) Amputation may also be avoided and local control improved by combining a marginal resection in combination with brachytherapy to the tumor bed (9). Isolated limb perfusion is another strategy to deal with locally advanced soft tissue sarcomas which can be applied also in case of multifocal primary or multiple sarcoma recurrences in limbs, thereby expanding the patient population that can be successfully treated.

2

ISOLATED LIMB PERFUSION

The technique of isolated limb perfusion was pioneered by Creech and Krementz at Tulane University in New Orleans (10). Regional drug concentrations 15-25 times higher than those reached after systemic administration can be achieved by ILP without systemic side effects (11). Isolation of the limb is achieved by clamping and canulation of the major artery and vein, connection to an oxygenated extracorporeal circuit, ligation of collateral vessels and application of a tourniquet. Once isolation is secured, drugs can be injected into the perfusion circuit. Because of its efficacy and low regional toxicity profile melphalan (L-phenyl-alaninemustard) is the standard drug, most commonly used at a dose of 10 mg/L (leg) - 13mg/L (arm) perfused tissue (12). Tissue temperatures are monitored and radiolabeled albumen or erythrocytes is injected into the perfusion circuit to detect leakage into the systemic circulation by precordial scintillation probe (13). Leakage monitoring is mandatory especially for high dose tumor necrosis factor-alpha (TNF) perfusions in the treatment of soft tissue sarcomas. After 1-1.5 hours of perfusion the limb is rinsed with an electrolyte solution, cannulas are removed, and the vessels are repaired. Classification of acute tissue reactions after perfusion is done according to Wieberdink et al (14): (I) No reaction; (II) Slight erythema and/or edema; (III) Considerable

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erythema and/or edema with some blistering, slightly disturbed motility permissible; (IV) Extensive epidermolysis and/or obvious damage to the deep tissues, causing definite functional disturbances; threatening or manifest compartmental syndrome; (V) Reaction which may necessitate amputation.

3

TNF- BASED ISOLATED LIMB PERFUSION FOR STS

3.1 Inadequate results in STS with ILP with chemotherapeutic drugs only In contrast to the efficacy of melphalan- based ILP in patients with multiple intransit melanoma metastases, results wih ILP with melphalan, doxorubicin and a variety of other drugs for large soft tissue sarcoms were diiappointing. After studies in the seventies and eighties with poor response rates ILP for advanced SIS was largely abandoned (15-19). The reported studies are summarized in Table 1.

3.2 Results with ILP with TNF + Melphalan trials leading to approval of TNF Thanks to the pioneering work of Lejeune and Lienard this situation changed dramatically with the application of high dose TNF in the ILP setting (20). TNF-based ILP has been established as a highly effective new method of induction biochemotherapy in extremity soft tissue sarcomas with a 20-30% complete remission (CR) rate and about a 50% Partial Remission (PR) rate (2127). On the basis of results in a multicenter program in Europe TNF was

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approved and registered in Europe for the sarcoma-indication in 1998 (27). The European TNF/ILP assessment group evaluated 246 patients with irresectable STS enrolled in 10 years in 4 studies. All cases were reviewed by an independent review committee and compared with conventionally treated patients (often by amputation) of a population based Scandinavian STS database. In short: there were 246 patients with locally very advanced disease: Primary sarcomas in 55%, local recurrent sarcomas in 45%, multifocal primary or multiple local recurrences in 22 %. Overt concurrent metastatic disease in 15%. Tumors >10 cm in 46%. Grade III tumors in 66%. Previous radiotherapy (13%), chemotherapy (15%). Patients underwent 1 ILP (222 pts) or 2 ILPs (24 pts) of 90 minutes at 39-40° C with 2-4 mg TNF + melphalan (1013 mg/L limb volume). The first 56 pts also received A delayed marginal resection of the tumor remnant was usually (76%) done 2-4 months after ILP. Major responses were seen in 56.5 to 82.6 % of the patients after which usually resection of the sarcoma became possible. Limb salvage was achieved in 74%-87% in these 4 studies and in 71 % of the 196 patients who had been classified by the independent review committees as cases that normally could only have been managed by amputation (87%) or by functionally debilitating resection + radiotherapy (13%). Comparison with the survival curves based on a matched control study with cases from the Scandinavian Soft Tissue Sarcoma Databank showed that TNF had no negative effect on survival (p=0.96). It was concluded that the application of TNF in combination with melphalan in the setting of isolated limb perfusion represents a new and successful option in the management of irresectable locally advanced extremity soft tissue sarcomas (27).

3.3

Confirmatory single center reports on TNF + melphalan

Smaller single center studies with TNF+Melphalan have been reported recently by Lejeune (29) reporting a 17% CR and 64% PR rate in 22 STS patients treated for limb threatening STS tumors, achieving limb salvage in 77% of the patients. A similar limb salvage rate of 84% and excellent functional results were reported from the Berlin team regarding their experience in a series of 55 patients (30). We reported on very good outcome of 16 perfusions in 10 patients with multiple lymphangiosarcomas (Stewart Treves Syndrome), achieving a CR rate of 56% and a limb salvage rate of 80% (32). The Amsterdam group reported somewhat less favorable results in their experience in 49 patients. The limb salvage rate of 58% was felt to reflect the selection patients with particularly unfavorable characteristics (32). The French group reported recently on their experience in 72 patients. In a randomized phase II trial, utilizing various doses of TNF ranging from 0.5 mg to 4 mg, they observed a 35% CR rate and an overall limb salvage rate of 84%. No significant differences between the various TNF dosage groups were observed. In a particularly unfavorable group of patients with recurrent sarcomas after surgery

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and radiotherapy, with the recurrent sarcoma in the irradiated field, we reported on the Rotterdam experience in 24 patients. A response rate of 74% and a limb salvage rate of 67% was reported in these patients otherwise destined for amputation.

3.4

Safety in Elderly Patients

A very important message is given by the report on the Rotterdam experience with 50 TNF-based ILPs in patients older than 75 years with limb threatening tumors. Results were very favorable in the 34 perfusions for limb threatening sarcomas, with a 38% CR and a 38% PR rate, achieving limb salvage in 76% of the patients as well as in 16 perfusions for bulky melanoma intransit metastases resulting in a 75%CR and 25%PR rate. The procedure was proven safe in the elderly with the high reward of limb salvage which is of overriding importance in this age group as amputations lead to loss of independency in lives in the elderly (35). Moreover we reported on the absence of toxicity in patients without leakage and the relatively easy management and relative lack of toxicity in patients with high leakage of TNF during ILP (36, 37)

3.5

Results with TNF + Doxorubicine

Very similar results have been obtained by Italian perfusion groups with the drug doxorubicin in combination with TNF. Interestingly similar response and limb salvage rates are achieved while using lower doses of only 1 mg TNF instead of the usual doses of 2-4 mg used in combination with melphalan (28). The perfusions were performed at much higher temperatures (40-41 degrees), which leads to higher locoregional toxicity. Grade IV locoregional toxicity was reported in 25% as opposed to only 5% in the large TNF+Melphalan series (21,23,27). We found that with melphalan ILPs grade IV toxicity was clearly related to tissue temperatures of above 39 degrees when melphalan was administered (38) Therefore we have only allowed for tissue temperatures to rise to 39 degrees after melphalan has been added to the perfusion circuit the last 8 years and have hardly seen any cases with grade IV toxicity since, without a drop in response rates (27,31,34,35). Most likely therefore the higher regional toxicity in the Italian experience with doxorubicin is primarily related to the hyperthermia although doxorubicine may be responsible in part.

4

TNF-BASED ILP ACTIVE IN MANY HISTOLOGIES

Since the tumor vasculature in the target of TNF and of the TNF+chemotherapy combination it can be expected that this treatment is effective against a wide variety of tumor types as long as there is a welldeveloped vascular stromal component to the tumor. This is indeed the case.

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Apart from activity in some 20 different histological types of soft tissue sarcoma and activity in melanoma (39-43), the efficacy of TNF + melphalan ILP has also been demonstrated in various skin tumors (44), bony sarcomas (45) and limb desmoid tumors (46).

5 VASCULOTOXIC MECHANISM OF TNF + CHEMOTHERAPY The target of TNF is the tumor-vasculature. This common denominator in all these tumors makes the use of TNF very attractive and explains its efficacy in combination with chemotherapy across all these different histologies. The selective destructive effects of TNF-ILP on tumor-associated vessels have been illustrated in previous publications by means of pre- and post perfusion angiographies (23). Moreover in sarcoma patients’ magnetic resonance spectrometry studies we have clearly shown that the metabolic shut down of the tumor is virtually complete within 16 hours after the perfusion, confirming the likelihood of mediating its most important effects on the vasculature of the tumor (48). At the histopathological level we have also studied these intravascular effects such as thrombocyte aggregation, erythrostasis, endothelial and vascular destruction already in the early and late stages after ILP (48-49).

6

NEW INSIGHTS THROUGH LABORATORY MODELS

To further insight in the mechanisms underlying the positive results obtained with ILP in humans we developed in rats extremity perfusion models using the BN175 non immunogenic fibrosarcoma in Brown Norways rats and the ROS-1 osteosarcoma in WAG rats. In both models we could demonstrate that the tumor cells were resistant to TNF in vitro and that ILP in vivo with TNF alone had no major impact on tumor growth. In both models a strong synergistic antitumor effect leading to CRs in some 60-70 % was observed after ILP with TNF+Melphalan (50-51). TNF alone only caused some central necrosis and no regression of the tumor was observed as has been reported for the clinical setting as well. Histopathologically haemorrhagic necrosis was most prominent after ILP with both drugs. Early endothelial damage and platelet aggregation in the tumor vessels are observed after ILP with TNF + Melphalan and this is believed to lead to ischemic (coagulative) necrosis, which is in line with observations in patients. Our observations confirm that has its major effect on larger tumors, with well-developed vasculature in contrast to small tumors (diameter < 3 mm) with lack of developed capillary bed. TNF may exert its effect mainly through the neovasculature of the tumor, which is more abundant in large tumors. Moreover there are distinct similarities between tumor stroma generation and wound healing and observations by us that sites other than the tumor (recent wounds or skin overlying tumors only when invaded by tumor), which undergo

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angiogenesis, also become necrotic after ILP with TNF+ melphalan, but not after ILP with melphalan alone. We have demonstrated a number of crucial elements in our rat tumor models identifying the mechanisms for the strong synergy between TNF and Cytostatic drugs in ILP and have identified the prerequisites for an effective ILP:

6.1

Tumor vessel destruction

The vasculo-toxic effects of the combination of TNF + melphalan leading to haemorrhagic and anoxic coagulative necrosis as described above.

6.2

Enhanced drug uptake by the tumor

We have recently demonstrated that the addition of high dose TNF to the perfusate results in a 4-6 fold increased uptake by the tumor of the cytostatic drug. For Melphalan and for Doxorubicin is was demonstrated that this uptake was tumor specific and that no increased uptake was noted in the normal tissues, thus emphasizing the relatively selective action of TNF on the tumor-associated vasculature (52) This increase in concentration was also observed with doxorubicin (53). Moreover we have demonstrated that the effect correlates with the vascularity of the tumor. The more vascular the tumor the better the synergistic effect between TNF and the chemotherapeutic agent (54). Whether a TNF-mediated drop in interstitial pressure (55) in the tumor plays a role in this mechanism remains speculative.

6.3

Role of Leukocytes

We have shown that leukocytes play also an important role in the TNF-mediated antitumor effects. In rats that underwent total body irradiation and underwent an ILP at the time of absolute leukopenia the antitumor effect of an ILP with TNF+melphalan was very similar to the effects of a perfusion with melphalan alone. In the leukopenic rat the TNF-effect was lost and the synergy between TNF and Melphalan was no longer observed (56)

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6.4

Dose range for TNF

We demonstrated that 10 micrograms of TNF (a fivefold reduction of the “standard dose of 50 microgram” was the threshold dose for activity of TNF in our rat tumor extremity perfusion model. At 2 microgram all TNF-effects were lost (57). This finding would suggest that also in the clinical setting dose reduction without loss of activity could be explored as been also suggested by the clinical results in the UK (22) and in Italy (29) as well as the recent report from France (33).

6.5

Duration of ILP

As the pharmacokinetics of melphalan demonstrates that almost all melphalan uptake occurs over 20-30 minutes the minimal duration for an effective ILP should be 30 minutes. Shorter perfusion times are associated with a drop in CR and PR rates whereas longer than 30 minutes ILPs do not seem to further improve the results (57)

6.6

Mild Hyperthermia

Temperatures of 38-39 degrees Centigrade were shown to be essential for obtaining a good antitumor response without damage to the normal tissues in the limb. True hyperthermia (42-43degrees) resulted in an increase of CRs but was associated with very sever damage to the normal tissues. All antitumor efficacy was lost when perfusions were performed at room temperature. (57)

6.7

Hypoxia

We demonstrated that hypoxia can enhance the antitumor effects of an ILP with either TNF alone or Melphalan alone. Hypoxia did not further enhance the antitumor efficacy of an ILP with TNF+Melphalan as the synergy between these two agents “overrided” any minor enhancement mediated by hypoxia (57).

6.8

Interferon-gamma

In spite of many reports of the synergy between IFN-gamma and TNF both in vitro as well as in vivo in murine tumor models the role of IFNgamma not very strong in our rat models. We demonstrated that about a 10% increase in CR rate and an increase of about 20% in overall response rate was observed in our animal models (58), which resembles the situation in the clinic (43).

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6.9

Idiosyncratic toxicity

Interestingly unexpected interactions may lead to idiosyncratic reactions between TNF and certain cytostatic agents. Actinomycin D is commonly used in combination with melphalan in the clinical setting. When investigating whether TNF would enhance the efficacy of Actinomycin D we discovered that it did in an idiosyncratic and nondiscriminative way. The combination was more effective against the tumor than TNF+Melphalan, but this advantage was annulled by the toxicity of TNF+Actinomycin D to the normal tissues, resulting in the amputation of all extremities in these animal models. We sent out a strong warning to the clinic not to use TNF in combination with Actinomycin D (59).

6.10

Vasoactive drugs

Various vasoactive drugs have been and are being studied in our laboratory models. Nitric Oxide (NO) is an important molecule in the maintenance of both vascular tone and the integrity of the vascular wall and is highly produced in experimental and human tumors. We postulated that its inhibition could lead to hypoxia and an enhancement of TNF early vascular effects in the tumor. In our ILP BN 175 rat model we performed a response study with TNF in combination with the Arginine analogues L-NAME and LNA, which inhibit NO synthase. In rats treated with TNF combined with LNAME/LNA important and immediate antitumor effects were observed in all rats and necrosis of the skin at the tumor site. These effects are normally only observed when hypoxia or melphalan are added to TNF as described above. Typical TNF tumor response was observed, when NO synthase was inhibited during ILP (60). Another vasoactive drug is histamine, which is currently being studied. Also in this case we see a clear synergy with melphalan in our tumor models (61) These findings show the importance of agents that can change the pathophysiology of tumor vasculature, rheologic conditions en thereby can improve drug uptake in tumors. These findings underline the importance of investigating how to modulate tumor physiology and the potential that this approach has to improve efficacy of various standard agents.

7

CONCLUSIONS

Isolated limb perfusion methodology provides us an excellent tool in the clinic to obtain local control and avoid amputations of limbs in patients with limb threatening tumors. This has been largely achieved by the success of the antivascular TNF-based biochemotherapy in this setting. TNF, for the first time, has brought us an effective treatment against large, bulky tumors.

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Moreover Isolated limb perfusion is a albeit somewhat exotic, but very interesting research model to develop and study agents that modify the pathophysiology of large tumors that blocks effective penetration of cytotoxic drugs into the tumor. We can now manipulate and study the tumor vascular bed in ways that will identify “new” drugs that can enhance the activity of “old” drugs. Moreover it has proven to be a model system that may also facilitate the development of vector-mediated gene therapy and other innovative approaches. Much of these developments have been initiated by the application of TNF in this setting. TNF-based isolated limb perfusion is a very successful treatment option to achieve limb salvage in the management of advanced, multiple or drug resistant extremity tumors. TNF-based ILPs are now performed in some 30 cancer centers in Europe with referral programs for limb salvage. TNF-based antivascular therapy of cancer is here to stay and its potential needs to be studied further (62). Other drugs will follow and we may well learn through this model how to use them systemically more effectively as well.

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REFERENCES

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13. Klaase JM, Kroon BBR, Van Geel AN, Eggermont AMM, Franklin HR. Systemic leakage during isolated limb perfusion for melanoma. Br J Surg 1993;80:1124-1126 14. Wieberdink K, Benckhuijsen C, Braat RP, Van Slooten EA, Olthuis GAA. Dosimetry in isolation perfusion of the limbs by assessment of perfused tissue volume and grading of toxic tissue reactions. Eur J Cancer Clin Oncol 1982; 18:905-910 15. Krementz ET, Carter RD, Sutherland CM, Hutton I. Chemotherapy of sarcomas of the limbs by regional perfusion. Ann Surg 1977;185(5):555-564 16. Muchmore JH, Carter RD, Krementz ET. Regional perfusion for malignant melanoma and soft tissue sarcoma: a review. Cancer Invest. 1985;3:129-143 17. Pommier RF, Moseley HS, Cohen J et al. Pharmacokinetics, Toxicity, and Short-term results of cisplatin hyperthermic isolated limb perfusion for soft tissue sarcoma and melanoma of the extremities. Am J Surg 155:667-671, 1988 18. Klaase JM, Kroon BBR, Benckhuysen C, Van Geel AN, Albus-Lutter ChE, Wieberdink J. Results of regional isolation perfusion with cytostatics in patients with soft tissue tumors of the extremities. Cancer 64:616-621, 1989 19. Rossi CR, Vecchiato A, Foletto M, et al. Phase II study on neoadjuvant hyperthermicantiblastic perfusion with doxorubicin in patients with intermediate or high grade limb sarcomas. Cancer 73:2140-2146, 1994 20. Lienard D, Ewalenko, Delmotte JJ, Renard N, Lejeune FJ. High-dose recombinant tumor necrosis factor alpha in combination with interferon gamma and melphalan in isolation perfusion of the limbs for melanoma and sarcoma. J Clin Oncol 1992; 10:50-62 21. Eggermont AMM, Liénard D, Schraffordt Koops H, Rosenkaimer F, Lejeune FJ. Treatment of irresectable soft tissue sarcomas of the limbs by isolation perfusion with high dose TNF-a in combination with gamma-Interferon and melphalan. Fiers W and Buurman WA (eds), In: Tumor Necrosis Factor: Molecular and Cellular Biology and Clinical Relevance, Basel, Karger Verlag, 1993, pp 239-243 22. Hill S, Fawcett WJ, Sheldon J, Soni N, Williams T, Thomas JM. Low dose tumor necrosis factor-alpha and melphalan in hyperthermic isolated limb perfusion. Br J Surg 1993; 80:995-997 23. Eggermont AMM, Schraffordt Koops H, Lienard D, et al: Isolated limb perfusion with highdose tumor necrosis factor-alpha in combination with interferon-gamma and melphalan for nonresectable extremity soft tissue sarcomas: a multicenter trial [see comments]. J Clin Oncol 14:2653-65, 1996a 24. Santinami M, Deraco M, Azzarelli A, Cascinelli F, Chiti A, Costagli V, Manzi R, Quagliolo V, Rebuffoni G, Santoro N, Vaglini M. Treatment of recurrent sarcoma of the extremities by isolated perfusion using tumor necrosis factor alpha and melphalan. Tumori 1996;82:579-84 25. Eggermont AMM, Schraffordt Koops H, Klausner JM, et al: Isolated limb perfusion with tumor necrosis factor and melphalan for limb salvage in 186 patients with locally advanced soft tissue extremity sarcomas. The cumulative multicenter European experience. Ann Surg 224:75664; discussion 764-5, 1996b 26. Gutman M, Inbar M, Lev-Shlush D, Mozes M, Chaitchik S, Meller I, Klausner JM. High dose tumor necrosis and melphalan administered via isolated limb perfusion for advanced limb soft tissue sarcoma results in a > 90% response rate and limb preservation. Cancer 1997;79:112937 27. Eggermont AMM, Schraffordt Koops H, Klausner JM, Schlag PM, Kroon BBR, Gustafson P, Steinmann G, Lejeune FJ. Limb Salvage by Isolation Limb Perfusion with Tumor Necrosis Factor Alpha and melphalan for locally advanced extremity soft tissue sarcomas: results of 270 perfusions in 246 patients. Proceed ASCO 1999;11:497(abstract) 28. Rossi CR, Foletto M, Di Filippo F, Vaglini M, Anza M, Azzarelli A, Pilati P, Mocellin S, Lise M. Soft tissue limb sarcomas: Italian clinical trials with hyperthermic antiblastic perfusion. Cancer, 1999;86:1742-9 29. Lejeune FJ, Pujol N, Lienard D, Mosimann F, Raffoul W, Genton A, Guillou L, Landry M, Chassot PG, Chiolero R, Bischof-Delaloye A, Leyvraz S, Mirimanoff RO, Bejkos D, Leyvraz PF. Limb salvage by neoadjuvant isolated perfusion with TNFalpha and melphalan for nonresectable soft tissue sarcoma of the extremities. Eur J Surg Oncol 2000, 26:669-78

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30. Hohenberger P, Kettelhack C, Hermann A, Schlag PM. Functional outcome after preoperative isolated limb perfusion with rhTNFalpha/Melphalan for high-grade extremity sarcoma. Eur J Cancer 2001;37(6):S34-35 31. Lans TE, deWilt JHW, van Geel AN, Eggermont AMM. Isolated limb perfusion with tumor necrosis factor and melphalan for nonresectable stewart-treves lymphangiosarcoma. Ann Surg Oncol. 2002;9:1004-9 32. Noorda EM, Vrouwenraets BC, Nieweg OE, Slooten GW, Kroon BBR. Isolated limb perfusion with and Melphalan for Irresectable Soft Tissue Sarcoma of the Extremities. Ann Surg Oncol 2003;10;1:S36 33. Bonvalot S, Lejeune F, Laplanche A, Stoeckle E, Le Pechoux C, Vanei D, Lumbroso J, Terrier P, Aubert B, LeCesne A. Limb slavage by isolated limb perfusion (iILP) in patients with locally advanced soft tissue sarcoma (ASTS): a randomized phase II study comparing 4 doses of Proc. Am Soc Clin Oncol, 2003;22:823 34. Grünhagen D, Lans TE, de Wilt JHW, van Geel AN, Eggermont AMM. Management of Local Recurrences of Soft Tissue Sarcomas in an Irradiated Field after Prior Surgery and Radiotherapy: the Role of TNF-based Isolated Limb Perfusions to achieve Limb Salvage. Eur J Cancer 2003;39:in press 35. Etten van B, van Geel AN, de Wilt JHW, Eggermont AMM. Fifty Tumor Necrosis Factorbased Isolated Limb Perfusions for limb salvage in patients older than 75 years with limbthreatening soft tissue sarcomas and other exremity tumors. Ann Surg Oncol, 2003;27:32-37 36. Vrouwenraets BC, Kroon BBR, Ogilvie AC, Van Geel AN, Nieweg OE, Swaak AJG, Eggermont AMM. Absence of severe systemic toxicity after laekage controlled isolated limb perfusion with Tumor Necrosis Factor alpha and melphalan. Ann Surg Oncol, 1999;6:405-412 37. Stam, T. C., Swaak, A. J., de Vries, M. R., ten Hagen, T. L., Eggermont, A. M. Systemic toxicity and cytokine/acute phase protein levels in patients after isolated limb perfusion with tumor necrosis factor-alpha complicated by high leakage [In Process Citation] Ann Surg Oncol, 2000;4:268-75 38. Vrouenraets BC, Eggermont AMM, Hart AA, Klaase JM, van Geel AN, Nieweg OE, Kroon BBR. Regional toxicity after isolated limb perfusion with melphalan and tumour necrosis factor- alpha versus toxicity after melphalan alone. Eur J Surg Oncol. 2001;27:390-5 39. Lejeune FJ, Lienard D, Leyvraz S, Mirimanoff RO. Regional therapy of melanoma. Eur J Cancer 1993; 29A:606-612 40. Eggermont AMM, Liénard D, Schraffordt Koops H, Kroon BBR, Rosenkaimer F, Klaase JM, Schmitz PIM, Lejeune FJ. High dose tumor necrosis factor-alpha in isolation perfusion of the limb: highly effective treatment for melanoma in transit metastases or unresectable sarcoma. Reg Cancer Treat, 7:32-36, 1995 41. Eggermont AMM. Treatment of melanoma intransit metastases confined to the limb. Cancer Surveys, 26:335-349, 1996 42. Fraker DL, Alexander HR, Andrich M, Rosenberg SA. Treatment of patients with melanoma of the extremity using hyperthermic isolated limb perfusion with melphalan, tumor necrosis factor, and interferon gamma: results of a tumor necrosis factor dose-escalation study. J Clin Oncol, 1996;14:479-89 43. Lienard D, Eggermont AMM, Schraffordt Koops H, Kroon BBR, Towse G, Hiemstra S, Schmitz P, Clarke J, Steinmann G, Rosenkaimer F, Lejeune FJ. Isolated limb perfusion with tumour necrosis factor-alpha and melphalan with or without interferon-gamma for the treatment of in-transit melanoma metastases: a multicentre randomized phase II study. Melanoma Res, 1999;9:491-502 44. Olieman, A.F., Lienard, D., Eggermont, A.M., Kroon, B.B., Lejeune, F.J., Hoekstra, H.J. & Koops, H.S. Hyperthermic isolated limb perfusion with tumor necrosis factor alpha, interferon gamma, and melphalan for locally advanced nonmelanoma skin tumors of the extremities: a multicenter study. Arch Surg, 1999;134, 303-7 45. Bickels, J., Manusama, E.R., Gutman, M., Eggermont, AMM, Kollender, Y., Abu-Abid, S., Van Geel, A.N., Lev-Shlush, D., Klausner, J.M. & Meller, I. Isolated limb perfusion with tumour

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necrosis factor-alpha and melphalan for unresectable bone sarcomas of the lower extremity [In Process Citation]. Eur J Surg Oncol, 1999;25:509-14 46. Lev-Chelouche D, Abu-Abeid S, Nakache R, Issakov J, Kollander Y, Merimsky O, Meller I, Klausner JM, Gutman M. Limb desmoid tumors: a possible role for isolated limb perfusion with tumor necrosis factor-alpha and melphalan. Surgery 1999;126:963-967 47. Sijens PE, Eggermont AMM, Van Dijk P, Oudkerk M. magnetic resonance spectroscopy as predictor for clinical response in human extremity sarcomas treated by single dose melphalan isolated limb perfusion. NMR in Biomedicine 1995;18:215-224 48. Renard N, Liénard D, Lespagnard L, Eggermont AMM, Heimann R, Lejeune FJ. Early endothelium activation and polymorphonuclear cell invasion precede specific necrosis of human melanoma and sarcoma treated by intravascular high-dose tumour necrosis factor alpha Int J Cancer 1994;57:656-663 49. Nooijen PTGA, Eggermont AMM, Schalkwijk L, Henzen-Logmans S, DeWaal RMW, Ruiter DJ. Complete response of melanoma in-transit metastasis after isolated limb perfusion with tumor necrosis factor-alpha and melphalan without massive tumor necrosis: clinical and histopathological study of the delayed-type reaction patterns. Cancer Res 1998;58:4880-4887 50. Manusama ER, Nooijen PTGA, Stavast J, Durante NMC, Marquet RL, Eggermont AMM. Synergistic antitumour effect of recombinant human tumour necrosis factor with melphalan in isolated limb perfusion in the rat. Br J Surg 1996;83:551-555 51. Manusama ER, Stavast J, Durante NMC, Marquet RL, Eggermont AMM. Isolated limb perfusion in a rat osteosarcoma model: a new anti-tumour approach. Eur J Surg Oncol 1996;22:152-157 52. De Wilt JHW, ten Hagen TLM, de Boeck G, van Tiel ST, de Bruijn EA, Eggermont AMM. Tumour Necrosis Factor alpha increases melphalan concentration in tumour tissue after isolated limb perfusion. Br J Cancer 2000;82:1000-1003 53. Veen vd AH, Wilt de JHW, Eggermont AMM, van Tiel ST, ten Hagen TLM. augments intratumoural concentration of doxorubicin in isolated limb perfusion in rat sarcoma models and enhances antitumour effects. Br J Cancer,2000;82:973-980 54. B van Etten, M de Vries, M van IJken, T Lans, G Guetens, G Ambagtsheer, S van Tiel, G de Boeck, E de Bruijn, AMM Eggermont AMM, TLM Ten Hagen. Degree of tumour vascularity correlates with drug accumulation and tumour response upon TNF-based isolated hepatic perfusion. Br J Cancer. 2003;87:314-9 55. Kristensen CA, Nozue M, Boucher Y and Jain RK. Reduction of interstitial fluid pressure after TNF-alpha treatment of three human melanoma xenografts. Br J Cancer 1996;74:533536. 56. Manusama ER, Nooijen PTGA, Stavast J, de Wilt JHW, Marquet RL and Eggermont AMM. Assessment of the role of neutrophils on the antitumor effect of in an in vivo isolated limb perfusion model in sarcoma - bearing Brown Norway rats. J Surg Res 1998;78:169-175 57. DeWilt JHW, Manusama ER, van Tiel ST, van IJken MGA, ten Hagen TLM, Eggermont AMM. Prerequisites for effective isolated limb perfusion using tumour necrosis factor-alpha and melphalan in rats. Br J Cancer 1999;80:161 -166 58. Manusama ER, de Wilt JHW, ten Hagen TLM, Marquet RL, Eggermont AMM. Toxicity and antitumor activity of interferon-gamma alone and in combinations with TNF and Melphalan in isolated limb perfusion in the BN175 sarcoma tumor model in rats. Oncol Rep 1999;6:173-177 59. Seynhaeve ALB, de Wilt JHW, vanTiel SA, Eggermont AMM, ten Hagen TLM. Combination of Actinomycin D with TNF-a in Isolated Limb Perfusion results in improved tumour response in soft tissue sarcoma-bearing rats but is accompanied by severe dose limiting local toxicity. . Br J Cancer 2002; 86:1174-1179. 60. DeWilt JHW, Manusama ER, van Etten B, van Tiel ST, Jorna AS, Seynhaeve ALB, ten Hagen TLM, Eggermont AMM: Inhibition of Nitric Oxide Synthesis by L-NAME results in synergistic antitumour activity with melpahlan and tumour necrosis factor-alpha- based isolated limb. Br J Cancer, 2000:83: 1176-11

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61. Brunstein F, Hoving S, van Tiel S, ten Hagen TLM, Eggermont AMM. Synergistic antitumor activity of histamine in combination with chemotherapy in the regional treatment of soft tissue sarcomas. Eur J Cancer 2003;39:in press 62. Ten Hagen TLM, FJ Lejeune, Eggermont AMM. TNF is here to stay – Revisited, Trends in Immunology (Formerly Immunology Today), 2001;22:127-129

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Chapter 5 Pitfalls in pathology of soft tissue sarcomas

Judith V.M.G. Bovée and Pancras C.W. Hogendoorn

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands

Correspondence to: Pancras C.W.Hogendoorn Dept. of Pathology, Leiden University Medical Center, PO Box 9600 2300 RC Leiden, The Netherlands

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1

INTRODUCTION

Soft tissue sarcomas are rare, constituting fewer than 1% of all cancers. Soft tissue tumors are generally regarded difficult by surgical pathologists, since they constitute a very heterogeneous, relatively uncommon group of tumors comprising more than 40 entities, with considerable morphological overlap between the diagnostic entities (1). However, distinction is essential since these entities differ widely in treatment and outcome. Classification of soft tissue tumors is assisted by immunohistochemistry, confirming the line of differentiation, and in the last decade also by molecular diagnostics, detecting tumor-specific translations. Histological grading schemes have been developed for soft tissue sarcomas as a group and seem to be a valuable predictor of patient survival for many, but not all, types of soft tissue sarcomas. Accurate histological subtyping is essential for accurate histological grading. Clinicians and pathologists should be aware of the limitations, prognostic significance, and relationship of histological subtyping and grading in the therapeutic management of soft tissue sarcomas.

2

HISTOPATHOLOGICAL TYPING OF SOFT TISSUE TUMORS

2.1

Method: Needle Biopsy Versus Open Biopsy

There has been a continuous debate over the past years whether coreneedle biopsies, or open biopsies should be used in the diagnostic process of soft tissue tumors. Though a diagnostic accuracy has been documented as high as 90% in bone tumors (2), this number is debated widely for soft tissue tumors. Unfortunately due to increasing economic issues in health care and patient expectations the work up of patients with soft tissue tumors focuses on speed and patient friendliness instead of accuracy and a scientific basis for treatment. As a result core needle biopsies become more and more popular complicating accurate diagnosis and making grading virtually impossible. This is especially worthwhile realizing the more and more popular use of preoperative chemotherapy and isolated limb perfusion, which if successful leave virtual no tissue left for definite diagnosis, meaning that a substantial number of patients will be treated with toxic therapies, while one honestly does not know what kind and grade of tumor has been treated. Core needle biopsies are however very useful for the differential diagnosis with metastatic carcinoma, melanoma and to rule out lymphoma. In a specialized hospital setting it is useful for the diagnosis of a number of tumors with consistent genetic abnormalities which can be very accurately assessed by molecular techniques

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on needle biopsies as well (3).

2.2

Classification of Soft Tissue Tumors

Classification of soft tissue tumors is based primarily on the line of differentiation displayed by the tumor (the type of tissue formed by the tumor) rather than the type of tissue from which the tumor arises. The classification of soft tissue tumors has been changing over the past decades. An increasing knowledge of immunohistochemical and molecular genetic characteristics has resulted in a more accurate classification and a better understanding of the biology of these tumors. For instance, malignant fibrous histiocytoma (MFH) was for long considered the most common adult soft tissue sarcoma. However, its line of differentiation could not be established and the group of tumors seemed very heterogeneous in terms of clinical behavior and ultimate outcome, questioning its existence as a real entity. Fletcher et al (4) showed in a retrospective study that MFH could be subclassified, and that this subclassification was prognostically relevant. For instance, pleomorphic MFH could be subclassified as dedifferentiated liposarcoma (metastatic risk <25%), high grade myxofibrosarcoma (metastatic risk 35-40%) and pleomorphic rhabdomyosarcoma (most patients have metastases within 2 years) (4). In general, pleomorphic sarcomas demonstrating myogenic differentiation (high grade leiomyosarcoma, pleomorphic rhabdomyosarcoma, high grade myogenic sarcoma NOS) were shown to have a worse prognosis (4), underlining the need to minimize the use of the term MFH and necessitating the development of more intense therapeutic strategies for this category of tumors.

2.3

WHO Classification

The most recent classification is the WHO classification 2002, which is based upon consensus of several experts in the field (1)(table 1). This Working Group chose to divide the tumors according to their biological potential into four categories; benign, intermediate (locally aggressive), intermediate (rarely metastasizing) and malignant. Most benign soft tissue tumors do not recur locally, and if they do the recurrence is non-destructive and almost always cured by complete local excision. Soft tissue tumors in the intermediate locally aggressive category often recur locally with an infiltrative, locally destructive growth pattern, thus requiring wide excision with a margin of normal tissue. They do not have an evident potential to metastasize, in contrast to soft tissue tumors in the intermediate, rarely metastasizing category. In addition to the locally destructive growth pattern, the risk of distant metastases in this category is estimated at <2%, which is

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Table 1. WHO classification of soft tissue tumors ( 1 ). ADIPOCYTIC TUMORS Benign Intermediate (locally aggressive) Lipoma, lipomatosis, Atypical lipomatous tumor / well differentiated lipomatosis of Nerve liposarcoma Lipoblastoma/lipoblastomatosis Angiolipoma Myolipoma Chondroid lipoma Extrarenal angiomyolipoma Extra-adrenal myelolipoma Spindle cell / pleomorphic lipoma Hibernoma FIBROBLASTIC / MYOFIBROBLASTIC TUMORS Benign Intermediate (locally aggressive) Nodular / proliferative Superficial fibromatosis (palmar/plantar) fasciitis Proliferative myositis, Desmoid-type fibromatosis myositis Ossificans Ischemic fasciitis Lipofibromatosis

Elastofibroma

Fibrous hamartoma of infancy Myofibroma / myofibromatosis Fibromatosis colli Juvenile hyaline fibromatosis Inclusion body fibromatosis

Intermediate (rarely metastasizing) Solitary fibrous tumor and haemangiopericytoma Inflammatory myofibroblastic tumor Low grade myofibroblastic sarcoma Myxoinflammatory fibroblastic sarcoma Infantile fibrosarcoma

Fibroma of tendon sheath Desmoplastic fibroblastoma Mammary-type myofibroblastoma

Malignant Myxoid / round cell liposarcoma Dedifferentiated liposarcoma Pleomorphic liposarcoma Mixed-type liposarcoma Liposarcoma, not otherwise specified

Malignant Adult fibrosarcoma Myxofibrosarcoma Low grade fibromyxoid sarcoma / hyalinizing spindle cell tumor Sclerosing epithelioid fibrosarcoma

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Calcifying aponeurotic fibroma Angiomyofibroblastoma Cellular angiofibroma Nuchal type fibroma Gardner fibroma Calcifying fibrous tumor Giant cell angiofibroma SO CALLED FIBROHISTIOCYTIC TUMORS Benign Intermediate (rarely metastasizing) Giant cell tumor of tendon Plexiform fibrohistiocytic sheath tumor

Diffuse type giant cell tumor

Deep benign fibrous histiocytoma

Giant cell tumor of soft tissue

Malignant Pleomorphic “MFH” / undifferentiated pleomorphic sarcoma Giant cell “MFH” / undifferentiated pleomorphic sarcoma with giant cells Inflammatory “MFH” / undifferentiated pleomorphic sarcoma with prominent inflammation

SMOOTH MUSCLE TUMORS Malignant Benign Leiomyosarcoma (excl skin) Angioleiomyoma Deep leiomyoma Genital leiomyoma PERICYTIC (PERIVASCULAR) TUMORS Glomus tumor (and variants), malignant glomus tumor Myopericytoma SKELETAL MUSCLE TUMORS Malignant Benign Embryonal rhabdomyosarcoma (incl. spindle cell, Rhabdomyoma (adult, fetal, botryoid, anaplastic) genital type) Pleomorphic rhabdomyosarcoma Alveolar rhabdomyosarcoma (incl. solid, anaplastic)

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VASCULAR TUMORS Benign Haemangioma (incl.capillary, cavernous, arteriovenous, venous, intramuscular, synovial, subcutis / soft tissue) Epithelioid haemangioma

Intermediate (locally aggressive) Kaposiform haemangioendothelioma

Malignant Epithelioid haemangioendothelioma

Angiosarcoma of Intermediate (rarely soft tissue metastasizing) Angiomatosis Retiform haemangioendothelioma Papillary intralymphatic Lymphangioma angioendothelioma Composite haemangioendothelioma Kaposi sarcoma CHONDRO-OSSEOUS TUMORS Benign Malignant Soft tissue chondroma Mesenchymal chondrosarcoma Extraskeletal osteosarcoma TUMORS OF UNCERTAIN DIFFERENTIATION Benign Intermediate (rarely Malignant metastasizing) Synovial sarcoma Intramuscular myxoma Angiomatoid fibrous (incl. cellular variant) histiocytoma Juxta-articular myxoma Ossifying fibromyxoid Epithelioid tumor (incl. atypical / sarcoma malignant) Deep (“aggressive”) Mixed tumor / myoepiAlveolar soft part sarcoma angiomyxoma thelioma / parachordoma Clear cell sarcoma Pleomorphic hyalinizing of soft tissue angiectatic tumor Ectopic hamartomatous Extraskeletal thymoma myxoid chondrosarcoma PNET/ extraskeletal Ewing tumor Desmoplastic small round cell tumor Extra-renal rhabdoid tumor Malignant mesenchymoma

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PEComa / neoplasms with perivascular epithelioid cell differentiation Intimal sarcoma TUMOURS OF PERIPHERAL NERVES (5) Benign Malignant Schwannoma (incl. cellular, plexiform, Malignant peripheral nerve sheath Melanotic) tumor (MPNST) (incl. epithelioid, with divergent mesenchymal and / or epithelial differentiation, melanotic, melanotic psammomatous) Neurofibroma (incl. plexiform) Perineurioma (incl. intraneural, soft tissue)

not predictable on the basis of histology. Malignant soft tissue tumors, called soft tissue sarcomas, have a significant risk of distant metastases, ranging from 20-100% depending on histological type and grade (1). Some low grade sarcomas have initially a lower metastatic risk but they may advance in grade upon local recurrence, increasing the risk of distant spread. It is important to note that the intermediate categories of biological potential as defined in the WHO classification do not correspond to the histologically defined intermediate grade of malignancy (see histological grading) (1).

3

HISTOPATHOLOGICAL GRADING

Malignant soft tissue tumors (sarcomas) represent a heterogeneous group of neoplasms differing widely in their clinico-biological behavior, ranging from lesions that only very rarely metastasize to lesions that behave in a highly aggressive manner with metastases in most of the cases which may happen very rapidly or may take many years. Part of this behavior is histotype specific (e.g. well differentiated liposarcoma), underscoring the importance of adequate histopathological classification. Some lesions show however a broad spectrum of behavior not predictable from histological typing alone (e.g. leiomyosarcoma, gastrointestinal stromal tumor). Therefore, histopathological grading systems were developed in an attempt to identify histotypeindependent histological parameters that may be of help to predict prognosis (6). Thus, in addition to the histological type of a soft tissue sarcoma, histological grading also gives information about the degree of malignancy and the probability of distant metastases and survival. Histological grading is therefore a feature of the major tumor staging systems and constitutes important information for therapeutic decisions. Grading is of poor value for predicting local recurrence, which is mainly related to the quality of surgical margins.

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3.1

Grading Systems

Histological grading schemes have been proposed for soft tissue sarcomas as a group, since the relative rarity of soft tissue sarcomas severely hampers optimal and significant histotype specific clinicopathological studies. Grading aims at identifying and separating tumors with a favorable prognosis from those with a poor prognosis (6). Grading is based on histological parameters only and is very subjective. There is no ideal grading system and several different grading systems have been published so far and have been shown to correlate with prognosis. The two most widely used grading systems are the NCI (United States National Cancer Institute) system (7) and the FNCLCC (French Fédération Nationale des Centres de Lutte Contre le Cancer) system originally described by Trojani et al in 1984 (8), later modified by Guillou et al (9). The NCI system uses a combination of histological typing and histological parameters (cellularity, pleomorphism, and mitotic rate) for identification of grade 1 tumors. All the other types of sarcomas are classified as either grade 2 or 3, with 15% necrosis as the threshold for separating grade 2 from grade 3 tumors (1;7). The FNCLCC system is based on the evaluation of three parameters: tumor differentiation, mitotic rate and amount of tumor necrosis (table 2). There is however considerable interobserver variation in the assessment of these histological parameters and evaluation of the reproducibility of the FNCLCC system revealed a 75% agreement for tumor grade, while the agreement for the Table 2. FNCLCC grading system for (adult) soft tissue sarcoma (8;9)

TUMOR DIFFERENTIATION (see table 3) Sarcomas resembling normal adult mesenchymal tissue Score 1 Sarcomas for which histological typing is certain Score 2 Embryonal and undifferentiated sarcomas, sarcomas of Score 3 doubtful type, synovial sarcomas, osteosarcomas, PNET MITOTIC COUNT Score 1 0-9 mitoses per 10 HPF* Score 2 10-19 mitoses per 10 HPF Score 3 20 mitoses per 10 HPF TUMOR NECROSIS Score 0 No necrosis Score 1 <50% tumor necrosis Score 2 50% tumor necrosis HISTOLOGICAL GRADE Grade 1 Total score 2,3 Grade 2 Total score 4,5 Grade 3 Total score 6,7,8 * HPF (high power field) defined as

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histological typing was only 61% (10). When both grading systems are compared using the same set of 410 tumors concordance rates are only around 65% (9). The FNCLCC system is favored because it correlates better with overall and metastasis free survival, and it allocates less patients in the intermediate grade category and is more reproducible than the NCI system (9). The establishment of the tumor differentiation score in the FNCLCC system can however be problematic. A listing of the differentiation scores for the most common tumors has been reported (table 3)(9), but the rationale for some of these scores is not clear (11). Table 3. tumor differentiation score according to histological type in the updated version of the FNCLCC system (modified from Guillou et al (9) and the WHO classification (1))

HISTOLOGIC TYPE Well differentiated liposarcoma Myxoid liposarcoma “Round cell liposarcoma” Pleomorphic liposarcoma Dedifferentiated liposarcoma Fibrosarcoma Well differentiated Conventional Poorly differentiated Malignant peripheral nerve sheath tumor (MPNST) Well differentiated Conventional Poorly differentiated Epithelioid Malignant Triton tumor Myxofibrosarcoma (“myxoid MFH”) Pleomorphic sarcoma (“pleomorphic MFH”) With storiform pattern Patternless pleomorphic sarcoma With giant cells (“giant cell MFH”) With prominent inflammation (“inflammatory MFH”)

TUMOR DIFFERENTIATION SCORE

1 2 3 3 3 1 2 3 1 2 3 3 3 2 2 3 3 3

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Leiomyosarcoma Well differentiated Conventional Poorly differentiated / pleomorphic / epithelioid Biphasic / monophasic synovial sarcoma Embryonal / alveolar / pleomorphic rhabdomyosarcoma Myxoid chondrosarcoma Mesenchymal chondrosarcoma Conventional angiosarcoma Poorly differentiated / epithelioid angiosarcoma Extraskeletal osteosarcoma Ewing sarcoma / PNET Alveolar soft part sarcoma Epithelioid sarcoma Malignant rhabdoid tumor Clear cell sarcoma Undifferentiated sarcoma

1 2 3 3 3 2 3 2 3 3 3 3 3 3 3 3

3.2 Limitations and drawbacks of histological grading Grading soft tissue sarcomas as a group, due to their rarity, has several major disadvantages. One should realize that grading is not a substitute for the histological diagnosis, which should alsways be assessed first and may even be considered the most important predictor of outcome, illustrated by the fact that both th eNCI as well as the FNCLCC grading schemes are based on adequate histotyping. Some tumor types appear to be characterized by a biological behavior that can be predicted by histopathological classification alone (Table 4). Therefore, not all soft tissue sarcomas have to be graded since for some histological subtypes grade is of no prognostic value. Studying 1240 cases revealed that the histological grade (FNCLCC) is an independent predictor of metastasis development for the main histological types of adult soft tissue sarcomas, with the exception of MPNST and rhabdomyosarcoma (13). The WHO classification does not recommend histological grading for MPNST, angiosarcoma, extraskeletal myxoid chondrosarcoma, alveolar soft part sarcoma, clear cell sarcoma and epithelioid sarcoma (1). The latter three tumors will often metastasize within 10-20 years of follow-up (14). Histological grading is however useful in

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Table 4. Soft tissue sarcomas for which the histotype determines the biological behavior / grade (adapted from (6) and (14)). HIGH GRADE BEHAVIOR Angiosarcoma Desmoplastic small round cell tumor Extrarenal rhabdoid tumor Extraskeletal Ewing sarcoma / PNET Extraskeletal osteosarcoma Myxoid / round cell liposarcoma with >5% round cell component Mesenchymal chondrosarcoma Pleomorphic liposarcoma Rhabdomyosarcoma (except spindle cell variant) LOW GRADE BEHAVIOR Atypical lipomatous tumor / well differentiated liposarcoma Congenital fibrosarcoma Dermatofibrosarcoma protuberans Myxoid / round cell liposarcoma with <5% round cell component

tumors with varying behavior (e.g. leiomyosarcoma, myxofibrosarcoma, fibrosarcoma). One should also realize that grading does not differentiate benign from malignant lesions (1). Ideally, the clinicopathological study of sarcomas, including histological grading, should be histotype specific since evaluation of histological parameters in 1116 tumors revealed that there is no single universal histological prognostic parameter that is valid for all types of soft tissue sarcomas (15). However, due to the rarity of soft tissue sarcomas comparison of larger groups of individual tumor types is problematic, often hampering the identification of histotype specific prognostic histological parameters. Eventually, especially with targeted treatment becoming more and more applied, grading systems will be devised for an increasing number of individual sarcoma types. For instance, the biological behavior of myxoid / round cell liposarcoma can be predicted by the significance of the round cell component; if this component constitutes >5% and there is necrosis, the tumor will behave as a high grade sarcoma (1), although the mitotic rate can be low. Another example concerns gastrointestinal stromal tumors (GISTs). Stromal/mesenchymal tumors of the gastrointestinal tract have long been a source of confusion and controversy with regard to classification, line of differentiation and prognostication (16). With the discovery of targeted treatment using STI571 / Glivec, GISTs have become the center of attention. Since an accurate and reproducible diagnosis is essential to ensure appropriate treatment for GIST patients, a consensus approach to diagnosis and morphological prognostication has led to a scheme for estimating metastatic risk in these lesions, based on tumor size and mitotic count (Table 5) (16). Finally, one should be aware that only untreated primary soft tissue sarcomas should be graded, since radiation treatment and chemotherapy prior to

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surgical excision make grading of the resected specimen unreliable, for instance due to treatment induced necrosis, fibrosis and pleomorphism. Histological grading should only be performed on representative and well processed material. The mitotic rate for instance is greatly influenced by sampling error, tissue fixation, the presence of necrosis and section thickness (17). Table 5. Risk assessment for GIST (gastrointestinal stromal tumor) (16). Size* Mitotic rate <2cm <5 / 50 HPF# 2-5 cm <5 / 50 HPF <5 cm 6-10 / 50 HPF <5 / 50 HPF 5-10 cm >5 cm >5 / 50 HPF High risk >10cm Any mitotic rate >10 / 50 HPF Any size *size represents the single largest dimension. # HPF (high power field) not defined

Very low risk Low risk Intermediate risk

The distinction of mitoses from apoptosis and karyorrhexis can be difficult. The number of high power fields counted need to be standardized, taking into account the exact microscopic field size which may show marked variation between various types of microscopes. It is as yet unknown whether necrosis is best assessed microscopically or macroscopically (9;17). Since necrotic areas will be avoided when selecting blocks, microscopic assessment of necrosis will be an underestimate. Gross assessment of necrosis can be made only on the resected specimen, precluding retrospective (re)evaluation. Furthermore, naturally occurring intralesional heterogeneity also automatically implies the risk of sampling error. Thus, enough blocks should be sampled. The association of directors of anatomic and surgical pathology (ADASP) recommends sampling with an overall block number of approximately 1 per cm of the tumor’s greatest dimension (14). The consequence of these limitations is that reliable grading can rarely be performed on a needle biopsy or small incisional biopsy, unless it shows an obvious high grade tumor.

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4

ADDITIONAL TECHNIQUES

4.1

Immunohistochemistry

Immunohistochemistry is especially helpful in the classification of soft tissue sarcomas, since classification can be difficult due to their rarity, and their broad and partly overlapping morphological spectrum. Immunohistochemistry is a valuable diagnostic tool in determining the line of differentiation. In addition, in the past decade targeted treatment of soft tissue tumors has rapidly made its introduction as an adjuvant therapy, making immunohistochemistry a tool to investigate whether this treatment can be useful.

4.1.1

Immunohistochemistry for Classification Purposes

Immunohistochemistry is widely used as an adjunct to light microscopy aiding the classification of tumors. After the establishment of the differential diagnosis on conventional haematoxylin and eosin stained slides, the line of differentiation can be determined or confirmed using immunohistochemistry. In soft tissue tumors, skeletal or smooth muscle differentiation, nerve sheath differentiation, melanocytic, fibrohistiocytic, or endothelial differentiation can be distinguished (Table 6). A growing list of antibodies is available, each with its own sensitivity and specificity. Since no antibody is 100% specific for a certain tumor type, a combination of antibodies should be used. For instance, in daily practice a standard panel of antibodies is used in case of monomorphic spindle cell tumors or undifferentiated round cell tumors, two of the most difficult groups of soft tissue tumors in terms of differential diagnosis in which the application of immunohistochemistry is essential to come to an accurate diagnosis (Tables 7 &8). Several studies have been performed to detect proliferation markers, overexpression of oncogenes and loss of expression of tumor suppressor genes by immunohistochemistry in soft tissue sarcomas to gain further insight into their pathogenesis, and to investigate whether clinical behavior could be predicted. However, results so far have been disappointing and immunohistochemistry is not yet used as a prognostic tool in routine daily practice.

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Table 6. Immunohistochemistry as a tool to determine the line of differentiation.

Muscle differentiation Desmin, smooth muscle actin, muscle specific actin (HHF35), Myogenic transcription factors (MyoD1, Myf4 (myogenin)), myoglobin, heavy caldesmon, calponin Nerve sheath differentiation S100, CD57 Melanocytic differentiation HMB-45, Melan-A (MART-1), tyrosinase, microphtalmia transcription factor Endothelial differentiation Von Willebrand Factor (Factor VIII-related antigen), CD34 (human haematopoetic progenitor cell antigen), CD31 (platelet endothelial cell adhesion molecule-1), Ulex Europaeus Lectin Fibro-histiocytic differentiation CD68, Factor 13A, vimentin Epithelial differentiation EMA (epithelial membrane antigen), cytokeratin

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4.1.2

Immunohistochemistry for Drug Targeting

A number of molecular events at the DNA level, leading to altered protein expression can be identified by immunohistochemistry. A number of them have been implied as potential targets for drugs such as Her-2Neu, PDGF and c-kit. The most exciting results are presented by studies in gastrointestinal stromal tumors (GISTs) targeting c-kit. Overexpression as a result of an activating mutation can be identified by clear immunohistochemical positivity using antibodies directed against c-kit (CD117), which is of paramount importance in its differential diagnosis as well (18). Mutations in c-kit leading to constitutive activation of the tyrosine kinase are believed to play a role in the majority of GISTs. STI571 has been shown to be a selective inhibitor of the tyrosine kinase activity of kit, and was shown to be clinically effective in patients with GISTs (19-21). Eligibility for clinical trials is dependent on the expression of c-kit, as determined by immunhistochemical staining.

4.2

Limitations and Pitfalls of Immunohistochemistry

Since the introduction of (adjuvant) treatment of GIST by STI571, different tumor types have been investigated for c-kit overexpression using immunohistochemical staining, to investigate whether other tumor types might also benefit from STI571 treatment. These investigations demonstrated some important limitations. Variations in immunohistochemical techniques

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used in the different laboratories can lead to major discrepancies in positive staining. The type of antibody used can vary since several antibodies may be (commercially) available for the same antigen. In addition, the staining protocols used in different laboratories vary somewhat with regard to antigen retrieval. High background staining, as can be seen in some polyclonal antibodies or can be due to insufficient dilution of the antibody, may lead to false positive results. In general, it is of crucial importance to evaluate, if possible, positive internal controls, such as vessels walls (CD34, CD31, factor VIII, smooth muscle actin, muscle specific actin / HHF35, desmin), mast cells (c-kit/CD117) and nerve fibers (S100) to avoid false negative results.

4.3

Molecular Diagnostics

In the past decade, molecular diagnostics have played an increasing role aiding the pathologist in the differential diagnosis. Especially the detection of translocations, specific for certain tumor types (see next chapter) by reverse transcription PCR or by fluorescence in situ hybridization has found its way in routine daily practice in sarcoma specialized centers. Moreover, studies are beginning to indicate that the type of translocation found may be of prognostic value, and future clinicopathological studies and clinical trials will reveal whether these histotype specific prognostic parameters are superior to histological grading in making therapeutic decisions.

5

REFERENCES

1. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Soft Tissue and Bone. Lyon: IARC Press, 2002. 2. Van der Bijl AE, Taminiau AHM, Beerman H, Hogendoorn PCW. Accuracy of the Jamhidi trocar biopsy in the diagnosis of bone tumors. Clin Orthop 1997; 334:233-243. 3. Graadt van Roggen JF, Bovee JVMG, Morreau J, Hogendoorn PCW. Diagnostic and prognostic implications of the unfolding molecular biology of bone and soft tissue tumours. J Clin Pathol 1999; 52:481-489. 4. Fletcher CD, Gustafson P, Rydholm A, Willen H, Akerman M. Clinicopathologic reevaluation of 100 malignant fibrous histiocytomas: prognostic relevance of subclassification. J Clin Oncol 2001; 19(12):3045-3050. 5. World Health Organization Classification of Tumours. Pathology ans Genetics of Tumours of the Nervous System. Lyon: IARC Press, 2003. 6. Graadt van Roggen JF. The histopathological grading of soft tissue tumours: current concepts. Curr Diagn Pathol 2001; 7(1):1-7.

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7. Costa J, Wesley RA, Glatstein E, Rosenberg SA. The grading of soft tissue sarcomas. Results of a clinicohistopathologic correlation in a series of 163 cases. Cancer 1984; 53:530541. 8. Trojani M, Contesso G, Coindre JM, Rouesse J, Bui NB, De Mascarel A et al. Soft-tissue sarcomas of adults; study of pathological prognostic variables and definition of a histopathological grading system. Int J Cancer 1984; 33(1):37-42. 9. Guillou L, Coindre JM, Bonichon F, Nguyen BB, Terrier P, Collin F et al. Comparative study of the National Cancer Institute and French Federation of Cancer Centers Sarcoma Group grading systems in a population of 410 adult patients with soft tissue sarcoma. J Clin Oncol 1997; 15(1):350-362. 10. Coindre JM, Trojani M, Contesso G, David M, Rouesse J, Binh Bui N et al. Reproducibility of a histopathologic grading system for adult soft tissue sarcoma. Cancer 1986; 58:306-309. 11. Weiss SJ, Goldblum JR. Soft Tissue Tumors. 4 ed. St.Louis: the C.V. Mosby Company, 2001. 12. Brown FM, Fletcher CD. Problems in grading soft tissue sarcomas. Am J Clin Pathol 2000; 114 Suppl:S82-S89. 13. Coindre JM, Terrier P, Guillou L, Le D, V, Collin F, Ranchere D et al. Predictive value of grade for metastasis development in the main histologic types of adult soft tissue sarcomas: a study of 1240 patients from the French Federation of Cancer Centers Sarcoma Group. Cancer 2001; 91(10):1914-1926. 14. Recommendations for the reporting of soft tissue sarcomas. Association of Directors of Anatomic and Surgical Pathology. Hum Pathol 1999; 30(1):3-7. 15. Hashimoto H, Daimaru Y, Takeshita S, Tsuneyoshi M, Enjoji M. Prognostic significance of histologic parameters of soft tissue sarcomas. Cancer 1992; 70:2816-2822. 16. Fletcher CD, Berman JJ, Corless C, Gorstein F, Lasota J, Longley BJ et al. Diagnosis of gastrointestinal stromal tumors: A consensus approach. Hum Pathol 2002; 33(5):459-465. 17. Oliveira AM, Nascimento AG. Grading in soft tissue tumors: principles and problems. Skeletal Radiol 2001; 30(10):543-559. 18. Graadt van Roggen JF, Van Velthuysen MLF, Hogendoorn PCW. The histopathological differential diagnosis of gastrointestinal stromal tumours. J Clin Pathol 2001;(54):96-103. 19. Demetri GD, von Mehren M, Blanke CD, Van den Abbeele AD, Eisenberg B, Roberts PJ et al. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med 2002; 347(7):472-480. 20. Joensuu H, Roberts PJ, Sarlomo-Rikala M, Andersson LC, Tervahartiala P, Tuveson D et al. Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N Engl J Med 2001; 344(14):1052-1056. 21. Van Oosterom AT, Judson 1, Verweij J, Stroobants S, Donato dP, Dimitrijevic S et al. Safety and efficacy of imatinib (STI571) in metastatic gastrointestinal stromal tumours: a phase I study. Lancet 2001; 358(9291):1421-1423.

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Chapter 6 Molecular biology and cytogenetics of soft tissue sarcomas: Relevance for targeted therapies Jonathan A. Fletcher, M.D.

Department of Pathology; Brigham and Women’s Hospital Boston, USA

Correspondence to : Jonathan A Fletcher, MD Department of Pathology; Brigham and Women’s Hospital; 75 Francis Street Boston, MA 02115 USA

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1

INTRODUCTION

Various genomic mechanisms can result in activation of therapeutic targets in soft tissue sarcomas (Table 1). Some mechanisms involve rearrangements of sizeable chromosomal regions, and given that they can be demonstrated by conventional cytogenetic methods, they are often referred to as “cytogenetic aberrations”. A well known example is the rearrangement of chromosomes 17 and 22 (often in a circular “ring” form) which activates the platelet derived growth factor receptor beta (PDGFRB) pathway in dermatofibrosarcoma protuberans (1,2). Other genomic mechanisms cannot be shown by conventional cytogenetic methods, because they involve small deletions or point mutations of DNA material which are below the resolution of karyotyping and fluorescence in situ hybridization (FISH) methods. Such mutations can be referred to as “molecular aberrations”. A well known example in this category is the gain-of-function KIT gene point mutation found in most gastrointestinal stromal tumors (3). Most soft tissue sarcomas contain clonal cytogenetic and molecular aberrations, some of which are diagnostic for particular tumor types (4-8). Demonstration of characteristic chromosome abnormalities has been useful diagnostically, especially in undifferentiated small round cell or spindle cell soft tissue tumors (5). However, only recently have these aberrations become useful as therapeutic targets. The advantage of such targets is that their relevance to the sarcoma is clear, given that they are activated by specific genomic mutations, and that they are selected for during the clinical progression of the sarcoma. For example, the fusion oncogenes that result from chromosomal translocations in sarcomas are invariably retained by the neoplastic cells even when the sarcomas undergo dedifferentiation, or progress to a more advanced histological grade. These observations suggest that the oncogenes are essential in maintaining the transformed state of the sarcoma cells, and the proteins encoded by these oncogenes therefore represent compelling therapeutic targets. In general, the cytogenetic and molecular mutations of greatest relevance to targeted therapies are those which activate an oncoprotein, and which can be countered directly with a therapeutic inhibitor of the oncoprotein. Examples include the various tyrosine kinase proteins that are activated by cytogenetic or molecular aberrations in different types of sarcomas. Transcription factor proteins, which regulate gene expression, are also activated by many of the cytogenetic aberrations in sarcomas. Therapeutic inactivation of the transcription factor oncoproteins appears to be more challenging compared to inhibition of kinase proteins, but nonetheless, this family of proteins will doubtless provide many targets for future treatment strategies. Tumor suppressor mechanisms are also relatively challenging as therapeutic targets, in that the conceptual approach involves

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restoring the lost tumor suppressor function to the neoplastic cells. It has been difficult to devise methods in which such restoration of function is accomplished with high efficiency – and at physiological levels – across a heterogeneous neoplastic cell population. On the other hand, tumor suppressor mutations result inevitably in activation of downstream proteins whose function would normally be inhibited by the tumor suppressor. These downstream proteins might be more tractable to targeted therapies than the tumor suppressor proteins themselves.

2

METHODOLOGICAL CONSIDERATIONS

The classic cytogenetic approach requires fresh, viable, tumor specimens which should be processed rapidly and transported to the cytogenetics laboratory in sterile tissue culture media or in a physiologic buffer such as Hank’s Buffered Salt Solution. It is important that the cytogenetic sample be removed from the overall tumor specimen with sterile scalpel blades or scissors. Otherwise, bacterial or fungal contamination may lead to microbial overgrowth in the subsequent tissue cultures. Because viable tumor cells are essential in establishing the tissue cultures, it is also important that the specimen be selected carefully so as to contain a minimum of necrotic tissue. Also, it is crucial to minimize nonneoplastic components, particularly fibroblasts, lest these cells overwhelm the tumor population after the cultures are established. The success of the cytogenetic analysis depends largely on the quality of the tumor specimen, whereas the amount of tumor is less important: thus, percutaneous needle biopsies of small round cell tumors, and other cellular sarcomas, can be karyotyped (5). At least 80% of all soft tissue sarcomas can be cultured successfully if the specimens are carefully selected so as to minimize necrotic and nonneoplastic components. Fluorescence in situ hybridization (FISH), PCR and sequencing methods, unlike conventional karyotyping, can be performed in archival sarcoma specimens. These methods are most successful in fresh or frozen tumor specimens, but can also be performed in paraffin materials. FISH and molecular methods will likely play an increasing role in identifying therapeutic targets in sarcomas, and it is likely that one or another form of these analyses will be used routinely – as an adjunct to histological appraisal – in the near future.

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Several molecular cytogenetic methods have expanded the capabilities of FISH by permitting genome-wide evaluation of chromosomal aberrations. Examples include comparative genomic hybridization and spectral karyotyping. However, these methods are labor-intensive, and better suited to research applications than in routine clinical evaluation of sarcoma patients.

3

CYTOGENETIC MECHANISMS

As mentioned above, recurring chromosome rearrangements, particularly those that serve as diagnostic and/or prognostic markers, are critical events in sarcoma tumorigenesis (Table 1). The specific nature of each chromosome rearrangement whether translocation, deletion, or amplification – can identify the mechanisms by which specific genes are altered during tumorigenesis. Certain sarcoma translocation breakpoints interrupt genes directly, resulting in novel “fusion” oncogenes. Other translocation breakpoints are adjacent to a particular gene, and result in deregulated expression (typically overexpression) of the gene. Deletions of whole chromosomes, or chromosomal regions, generally signify loss of one or more tumor suppressor genes. Amplification events, whether extrachromosomal (double minute chromosomes) or intrachromosomal (homogeneously staining regions) signify increased copies - and associated overexpression - of one or more oncogenes.

4 MOLECULAR AND CYTOGENETIC ABERRATIONS IN SARCOMAS

4.1

Adipose Tumors – Diverse Genetic Mechanisms

Adipose differentiation is regulated by (peroxisome proliferator-activated receptor gamma), which is a nuclear receptor and transcription factor (9). Notably, ligands have shown promise as therapeutic adjuncts in liposarcoma, being most effective in myxoid/round-cell liposarcoma (10). The selectivity of targeted therapy is in keeping with molecular and cytogenetic evidence showing that liposarcomas have distinctive genetic and biological profiles. Indeed, most adipose tumors, whether benign or malignant, contain distinctive chromosome aberrations (Table 1). Useful diagnostic markers include 12q rearrangement in lipoma, ring chromosomes in welldifferentiated and dedifferentiated liposarcoma, t(12;16) translocations in mxyoid/round cell liposarcoma, and cytogenetic complexity in pleomorphic liposarcomas.

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The most diagnostically useful aberration, in malignant adipose tumors, is a translocation of chromosomes 12 and 16, t(12;16)(q13;p11). This translocation is found in myxoid liposarcomas (11-13), and is retained in those cases that acquire round cell features (14,15). The t(12;16) translocation results in fusion of the CHOP gene on chromosome 12 with the TLS gene on chromosome 16 (16,17), and the resultant fusion oncoprotein is an activated transcription factor. The t(12;16) translocation appears to be diagnostic for myxoid liposarcoma and has not been found in other subtypes of liposarcoma or in other types of myxoid soft tissue tumors (18,19). Detection can be accomplished by cytogenetics or reverse transcriptase PCR. Well-differentiated liposarcomas (atypical lipomas) contain large “giant marker” chromosomes or ring chromosomes. These chromosomes are generally comprised of chromosome 12 material, often admixed with components of several other chromosomes (13,20). The ring and “giant marker” chromosomes contain various amplified genes, but the essential gene amplification targets have not been pinpointed. Notably, these amplifications are retained when well-differentiated liposarcomas progress to clinically more aggressive, dedifferentatiated, liposarcomas. Therefore one can hope that eventual identification of the gene amplification targets will reveal therapeutic pathways that might be particularly useful in dedifferentiated liposarcoma. Pleomorphic liposarcomas differ cytogenetically from other liposarcomas in that they have exceedingly complex karyotypes with multiple clonal chromosome aberrations (12). The genomic complexity in pleomorphic liposarcomas has hampered attempts to define consistent chromosomal aberrations that might be of diagnostic and therapeutic utility.

4.2

Clear Cell Sarcoma (malignant melanoma of soft parts)

Clear cell sarcomas of soft tissues, also referred to as “melanomas of the soft parts”, share many phenotypic features with cutaneous malignant melanomas. Hence, it can be difficult to distinguish between clear cell sarcoma and cutaneous melanoma histologically. Despite the histologic similarities between clear cell sarcoma and cutaneous melanoma, these two tumors are quite different clinically. Clear cell sarcomas usually present as isolated masses located in deep soft tissues without apparent origin from skin. It is notable, therefore,

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that more than 75% of clear cell sarcomas contain a chromosome translocation, t(12;22)(q13;q12), that has never been reported in cutaneous melanoma. The t(12;22) translocation fuses the ATF1 gene on chromosome 12 with the EWS gene on chromosome 22 (21,22). ATF1 encodes a transcription factor, and the biological implications of the translocation are probably similar to those - as discussed below - in Ewing’s sarcoma translocations. The clear cell sarcoma t(12;22) translocation can be detected by karyotyping or in situ hybridization, whereas the associated ATF1-EWS fusion can be detected conveniently by reverse transcriptase PCR.

4.3

Desmoplastic Small Round Cell Tumor – Is PDGFRA a Target?

Desmoplastic small round cell tumors are aggressive and chemotherapy-resistant neoplasms that arise predominantly, but not exclusively, from intraabdominal soft tissues (23). These tumors are composed of undifferentiated malignant small round cells within a florid desmoplastic reaction (23), and virtually all cases express an EWS-WT1 fusion oncogene (24,25). The EWS-WT1 fusion oncogene results from translocation between the chromosome 11 short arm and the chromosome 22 long arm, juxtaposing the WT1 (Wilms tumor) and EWS (Ewing’s) genes, respectively (26,27). Notably, the EWS-WT1 oncoprotein, expressed in the neoplastic small round cells, is a transcriptional regulator that induces expression of platelet derived growth factor alpha (PDGFA) (28). PDGFA binds and activates both PDGFRbeta and PDGFRalpha, serving to activate potent mitogenic signaling pathways in fibroblasts (29). Therefore, it is likely that oncogenically induced PDGFA expression contributes to the prominent desmoplastic reaction in these tumors. However, it is unclear whether PDGFA also serves an autocrine or paracrine function in directly stimulating growth of the neoplastic cells. Irrespective, these observations have fueled hopes that therapeutic inhibition of PDGFRA (e.g. with imatinib) might benefit patients with this highly lethal disease.

4.4

Ewing’s Sarcoma

Most Ewing’s sarcomas and peripheral primitive neuroectodermal tumors contain chromosome translocations involving the Ewing’s sarcoma gene, EWS. More than 80% of Ewing tumors contain a cytogenetic translocation in which material is exchanged between the long arms of chromosomes 11 and 22, resulting in oncogenic fusion of

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the chromosome 11 FLI1 gene with the chromosome 22 EWS gene (3033). FLI1 encodes a transcription factor belonging to the ETS family of transcription factors, and the oncogenic EWS-FLI1 fusion gene encodes an activated version of this transcription factor. A smaller subset of Ewing tumors, perhaps 5-15% of the total, have variant translocations in which the EWS gene is fused with other ETS family transcription factor genes (Table 1) (34-38). The Ewing’s gene translocations are considered to be essential genetic aberrations because they are found in virtually all cases and are assumed to be the critical genetic aberration in these tumors. However, targeted therapies for the EWS oncoproteins are still developmental, with no substantial clinical responses in early studies of vaccine therapies to EWS (39). Alternate targets might be found amongst the kinase protein family, but thus far have not shown great promise in preclinical studies (40-42).

4.5 PDGFRB

Dermatofibrosarcoma protuberans – Activated

The typical cytogenetic abnormality in dermatofibrosarcoma protuberans (DFSP) is a ring chromosome composed of sequences from chromosomes 17 and 22 (43,44). The DFSP ring chromosomes contain multiple copies of a fusion gene, COL1A1-PDGFB, in which COL1A1 (a collagen gene) is contributed by chromosome 17 and PDGFB (platelet derived growth factor beta gene) by chromosome 22 (2,45). The diagnostic COL1A1-PDGFB oncogene fusion can be demonstrated by fluorescence in situ hybridization or reverse transcriptase PCR. Occasional dermatofibrosarcoma protuberans have balanced t(17;22) translocations - associated with the COL1A1 -PDGFB fusion gene - rather than the usual ring chromosomes. Irrespective of the cytogenetic mechanism, the COL1A1-PDGFB oncogene results in overexpression of PDGFB, which is a growth factor that activates platelet derived growth factor receptor beta (PDGFRB) and platelet derived growth factor receptor alpha (PDGFRA). This observation suggested the possibility that patients with inoperable DFSP might benefit from treatment with PDGFR inhibitors, e.g. imatinib, and this hypothesis has been confirmed by striking clinical responses in several patients (46,47).

4.6

Desmoid tumors – APC and beta-catenin

Deep fibromatoses (desmoid tumors) can contain various cytogenetic or molecular aberrations, including APC (adenomatous polyposis coli) and beta-catenin mutations (48,49), and trisomies for chromosomes 8 or 20 (50). Cytogenetic deletions of the chromosome 5

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long arm are seen in occasional desmoids, resulting in loss of the APC tumor suppressor gene, and the remaining APC allele in these cases is typically inactivated by a point mutation (51,52). The most common known mutations in sporadic desmoid tumors are activating beta-catenin mutations, which were demonstrated in 22 of 42 desmoids by Alman et al. (48,49). These mutations result in stabilization, and resultant overexpression, of the beta-catenin protein. Therefore, it is likely that targeted therapies of the Wnt-APC-beta-catenin pathway, will be useful in treatment of patients with advanced desmoid tumors. Notably, PDGFRB activation plays a highly mitogenic role in myofibroblasts, and this knowledge has led to therapeutic evaluation of PDGFRB inhibition (by imatinib) with promising preliminary results (53). It is unkown presently whether PDGFRB activation in desmoids results from WntAPC-beta-catenin pathway perturbations, or whether this is an unrelated biological mechanism.

4.7

Fibrosarcoma (Infantile/Congenital) and Mesoblastic Nephroma – Activated NRTK3/TRKC

Trisomies of chromosomes 8, 11, 17 and 20 are characteristic aberrations in infantile fibrosarcomas (54). It is interesting that one or more of this same group of trisomies is also found in the cellular variant of mesoblastic nephroma, which is an infantile renal tumor having substantial histological overlap with infantile fibrosarcoma (55). In addition, most infantile fibrosarcomas and cellular mesoblastic nephromas contain the same diagnostic chromosome translocation, t(12;15)(p13;q26) (56-58). This translocation results in fusion of the chromosome 12 ETV6 (also known as TEL) gene with the chromosome 15 NTRK3 (also known as TRKC) gene. The t(12;15) translocation is difficult to detect by conventional cytogenetic banding approaches, but is demonstrated readily by FISH or reverse transcriptase PCR (56-58). From a clinical standpoint, infantile fibrosarcomas and cellular mesoblastic nephromas are undifferentiated and mitotically active tumors that nonetheless have an excellent prognosis after excisional biopsy (59,60). Therefore, these tumors appear to be closely related at the pathogenetic, morphologic, and clinical levels, perhaps representing a single neoplastic entity, arising in either renal or soft tissue locations. Most infantile fibrosarcomas and cellular mesoblastic nephromas are cured by surgical excision, and those that are inoperable respond well to cytotoxic therapies. A small minority of cases progress, and can be lethal, on conventional therapies, and development of NTRK3 kinase inhibitors would doubtless be highly useful for this subgroup.

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4.8

Inflammatory Myofibroblastic Tumor – Activated ALK

Inflammatory myofibroblastic tumor (also known as “inflammatory pseudotumor”) is composed of fascicles of bland myofibroblastic cells admixed with a prominent inflammatory infiltrate. The inflammatory component of these tumors is reactive (and therefore normal cytogenetically), whereas the myofibroblastic cells contain clonal chromosome aberrations (61-63). A subset of inflammatory myofibroblastic tumors have cytogenetic rearrangements that activate the ALK receptor tyrosine kinase gene on chromosome 2 (64). This is the same gene activated, typically by fusion with the NPM gene on chromosome 5, in many anaplastic large cell lymphomas. Inflammatory myofibroblastic tumors with ALK gene rearrangements express the ALK protein strongly, and can be identified by immunohistochemical staining for ALK in tumor paraffin sections, or by FISH to demonstrate the ALK gene rearrangement. Therapeutic ALK inhibition would be a useful therapeutic adjunct in patients with oncogenic ALK activation, particularly those with extensive disease, where local control is occasionally difficult.

4.9

Gastrointestinal Stromal Tumors – Activated KIT and PDGFRA

The oncogenic molecular mutations in KIT and PDGFRA are discussed extensively in other chapters, and will not be detailed here. In brief, most gastrointestinal stromal tumors display strong immunostaining for the KIT receptor tyrosine kinase protein, and contain activating mutations of the KIT or PDGFRA oncogenes (3,65,66). These mutations have been targeted with spectacular success using the KIT inhibitor, imatinib (Gleevec) (67,68). In addition, germline (inherited) KIT mutations are responsible for rare syndromes of familial, multifocal, gastrointestinal stromal tumors (69). Both germline and somatic KIT aberrations are point mutations that are inevident at the cytogenetic level of resolution. However, gastrointestinal stromal tumors have distinctive karyotypes that generally include deletion of chromosomes 14 and 22 (66,70). Less often, gastrointestinal stromal tumors contain deletions of the chromosome 1, 9 and 11 short arms (Table 1). KIT or PDGFRA activation result from early – and even initiating – mutational events in gastrointestinal stromal tumors, and these kinase activations likely stimulate proliferation of the neoplastic progenitor cell. Subsequently, the various cytogenetic aberrations are acquired, which presumably drive the progression of the tumor from benign to malignant GIST (71).

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4.10

Malignant Peripheral Nerve Sheath Tumors – Ras pathway

Benign and malignant peripheral nerve sheath tumors are seen with greatly increased frequency in patients with the hereditary neurofibromatosis syndromes. These are the most common tumor predisposition syndromes, affecting 1 in 3500 individuals worldwide. Neurofibromas and malignant peripheral nerve sheath tumors are common in individuals with neurofibromatosis type 1, whereas benign schwannomas are associated with neurofibromatosis type 2 (central neurofibromatosis). Characterization of the neurofibromatosis syndrome genes has shed substantial light on the pathogenesis of peripheral nerve sheath tumors. The neurofibromatosis type 1 (NF1) and type 2 (NF2) genes are located on chromosomes 17 and 22, respectively, and both of these genes encode tumor suppressor proteins that normally constrain cell proliferation (72-77). Malignant peripheral nerve sheath tumors (MPNST) often have deletions of the NF1 gene, which can be demonstrated by FISH assays. The NF1 gene aberrations are accompanied by a generally complex karyotype, suggesting that genetic instability plays a prominent role in the development of MPNST (Figure). Notably, NF1 gene deletions can also be shown in the Schwann cell component of neurofibromas (78). This observation supports the view that neurofibromas are clonal schwannian neoplasms, whereas the other admixed cell lineages - including fibroblasts, mast cells, and perineural cells - are reactive. The NF1 gene encodes a large protein which is related to the p120 Ras GTPase Activating Protein (Ras-GAP) (79,80). Ras-GAP proteins diminish signaling through the Ras pathway by stimulating the hydrolysis of GTP to GDP, and resulting in conversion of the active RasGTP complex to the inactive Ras-GDP complex. The tumorigenic role of this function is highlighted by the finding that a subset of familial NF1 mutations are point mutations resulting in loss of Ras binding or RasGAP activity. The roles of NF1 in suppressing Ras activation, and the loss of this normal role in MPNST, suggest that inhibition of the Ras pathway might be beneficial in patients with MPNST, and in other NF1associated cancers. One potential site of intervention is Ras itself, which can be targeted with farnesyl protein transferase inhibitors (FTIs). FTIs are peptidomimetics which inhibit the farnesylation of ras and thus prevent the proper trafficking of Ras to the plasma membrane (81). Signaling pathways downstream of ras offer alternative targets for intervention, including members of the RAF, MEK and MAPK kinase families (82).

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4.11

Rhabdoid Tumor

Most malignant rhabdoid tumors - whether arising in soft tissues, kidney, or the central nervous system - are characterized by deletions of the chromosome 22 long arm. The chromosome 22 deletions target a tumor suppressor gene, INI1 (also known as SNF5, or SMARCB1, which encodes a protein involved in chromatin remodeling (83,84). The rhabdoid tumor karyotypic profile is quite characteristic inasmuch as the chromosome 22 deletion is often the only detectable cytogenetic aberration, suggesting that INI1 inactivation is a relatively early event in rhabdoid tumorigenesis. Additional evidence of an essential tumorigenic role includes the finding of germline INI1 mutations in some individuals with rhabdoid tumors (84,85), and the predictable development of rhabdoid tumors in mice with inactivating INI1 mutations (86). These observations suggest that novel therapies targeting the INI1 pathway might be very effective in patients with rhabdoid tumors .

4.12

Rhabdomyosarcoma

Cytogenetic analyses have been useful in reaffirming the distinct nature of embryonal and alveolar forms of rhabdomyosarcomas. Alveolar rhabdomyosarcomas are characterized by reciprocal chromosome translocations involving the FKHR (Forkhead transcription factor) gene on chromosome 13. In most alveolar rhabdomyosarcomas, the FKHR gene is fused with the PAX3 gene on chromosome 2 (87-89), but a minority of cases contain fusions of FKHR with the PAX7 gene on chromosome 1 (90). FKHR, PAX3, and PAX7 encode transcription factors, and the PAX3-FKHR and PAX7-FKHR fusion oncogenes encode activated forms of those transcription factors (91,92). By contrast, embryonal rhabdomyosarcomas typically lack FKHR translocations, but – in experimental models – can result from activation of various receptor tyrosine kinase proteins, including MET and ERBB2 (93,94), and can respond to inhibition of other receptor tyrosine kinase proteins, particularly IGF1R (95,96). Therefore, it is possible that therapeutic inhibition of these tyrosine kinase proteins might be useful clinically in patients with rhabdomyosarcoma.

4.13

Synovial sarcoma

Most synovial sarcomas contain a reciprocal translocation of chromosomes X and 18, t(X;18)(p11;q11), resulting in oncogenic fusion between one of several SSX family genes on chromosome X, and the SYT (SS18) gene on chromosome 18 (97). The t(X; 18) translocation is found in more than 90% of synovial sarcomas, but is not found in potential histologic mimics such as hemangiopericytoma, mesothelioma, leiomyosarcoma, or malignant peripheral nerve sheath tumor. There are no therapies as of yet which reproducibly inhibit the t(X;18) oncogenic mechanism, or the associated SYT-SSX fusion oncoproteins. In a

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preliminary report, Matsuzaki et al. described temporary stabilization of synovial sarcoma pulmonary metastases following immunotherapy with autologous dendritic cells that had been exposed to the SYT-SSX2 junctional region (98). Microarray expression profiling has shown higher ERBB2 (her2/neu) expression in synovial sarcoma compared to other soft tissue sarcomas, and the ERBB2 expression is found predominantly in the glandular components of the synovial sarcomas (99). Based on these findings, Allander et al. have suggested that ERBB2 might provide a therapeutic target in synovial sarcoma (99).

5

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84. Biegel, J. A., Zhou, J. Y., Rorke, L. B., Stenstrom, C., Wainwright, L. M., and Fogelgren, B. Germ-line and acquired mutations of INI1 in atypical teratoid and rhabdoid tumors. Cancer Res., 59: 74-79, 1999. 85. Sevenet, N., Sheridan, E., Amram, D., Schneider, P., Handgretinger, R., and Delattre, O. Constitutional mutations of the hSNF5/INI1 gene predispose to a variety of cancers. Am. J. Hum. Genet., 65: 1342-1348, 1999. 86. Roberts, C. W., Galusha, S. A., McMenamin, M. E., Fletcher, C. D., and Orkin, S. H. Haploinsufficiency of Snf5 (integrase interactor 1) predisposes to malignant rhabdoid tumors in mice. Proc. Natl. Acad. Sci. U. S. A, 97: 13796-13800, 2000. 87. Galili, N., Davis, R. J., Fredericks, W. J., Mukhopadhyay, S., Rauscher, F. J. 3., Emanuel, B. S., Rovera, G., Barr, F. G., and Rauscher, F. J. Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nat. Genet., 5: 230-235, 1993. 88. Barr, F. G., Galili, N., Holick, J., Biegel, J. A., Rovera, G., and Emanuel, B. S. Rearrangement of the PAX3 paired box gene in the paediatric solid tumour alveolar rhabdomyosarcoma. Nat. Genet., 3: 113-117, 1993. 89. Shapiro, D. N., Sublett, J. E., Li, B., Downing, J. R., and Naeve, C. W. Fusion of PAX3 to a member of the forkhead family of transcription factors in human alveolar rhabdomyosarcoma. Cancer Res., 53: 5108-5112, 1993. 90. Davis, R. J., D’Cruz, C. M., Lovell, M. A., Biegel, J. A., and Barr, F. G. Fusion of PAX7 to FKHR by the variant t(1;13)(p36;q14) translocation in alveolar rhabdomyosarcoma. Cancer Res., 54: 2869-2872, 1994. 91. Fredericks, W. J., Galili, N., Mukhopadhyay, S., Rovera, G., Bennicelli, J., Barr, F. G., Rauscher, F. J. 3., and Rauscher, F. J. r. The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3. Mol. Cell Biol., 15: 1522-1535, 1995. 92. Bennicelli, J. L., Fredericks, W. J., Wilson, R. B., Rauscher, F. J. 3., and Barr, F. G. Wild type PAX3 protein and the PAX3-FKHR fusion protein of alveolar rhabdomyosarcoma contain potent, structurally distinct transcriptional activation domains. Oncogene, 11: 119-130, 1995. 93. Sharp, R., Recio, J. A., Jhappan, C., Otsuka, T., Liu, S., Yu, Y., Liu, W., Anver, M., Navid, F., Helman, L. J., DePinho, R. A., and Merlino, G. Synergism between INK4a/ARF inactivation and aberrant HGF/SF signaling in rhabdomyosarcomagenesis. Nat. Med., 8: 1276-1280, 2002. 94. Nanni, P., Nicoletti, G., De Giovanni, C., Croci, S., Astolfi, A., Landuzzi, L., Di Carlo, E., Iezzi, M., Musiani, P., and Lollini, P. L. Development of Rhabdomyosarcoma in HER-2/neu Transgenic p53 Mutant Mice. Cancer Res., 63: 2728-2732, 2003. 95. Kalebic, T., Tsokos, M., and Helman, L. J. In vivo treatment with antibody against IGF-1 receptor suppresses growth of human rhabdomyosarcoma and down-regulates p34cdc2. Cancer Res., 54: 5531-5534, 1994. 96. Kalebic, T., Blakesley, V., Slade, C., Plasschaert, S., Leroith, D., and Helman, L. J. Expression of a kinase-deficient IGF-I-R suppresses tumorigenicity of rhabdomyosarcoma cells constitutively expressing a wild type IGF-I-R. Int. J. Cancer, 76: 223-227, 1998. 97. Clark, J., Rocques, P. J., Crew, A. J., Gill, S., Shipley, J., Chan, A. M., Gusterson, B. A., and Cooper, C. S. Identification of novel genes, SYT and SSX, involved in the t(X;18)(p11.2;q11.2) translocation found in human synovial sarcoma. Nat. Genet., 7: 502508, 1994. 98. Matsuzaki, A., Suminoe, A., Hattori, H., Hoshina, T., and Hara, T. Immunotherapy with autologous dendritic cells and tumor-specific synthetic peptides for synovial sarcoma. J. Pediatr. Hematol. Oncol., 24: 220-223, 2002. 99. Allander, S. V., Illei, P. B., Chen, Y., Antonescu, C. R., Bittner, M., Ladanyi, M., and Meltzer, P. S. Expression profiling of synovial sarcoma by cDNA microarrays: association of ERBB2, IGFBP2, and ELF3 with epithelial differentiation. Am. J. Pathol., 161: 1587-1595, 2002.

Chapter 7 KIT and PDGF as targets

Jaap Verweij

Dept. of Medical Oncology, Erasmus University Medical Centre-Daniel den Hoed Cancer Centre, Rotterdam, the Netherlands

Correspondence to: Jaap Verweij, MD, PhD Dept. of Medical Oncology, Erasmus University Medical Centre-Daniel den Hoed Cancer Centre, Groene Hilledijk 301 3075 EA Rotterdam, the Netherlands

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1

INTRODUCTION

The development of one particular drug (STI571, Glivec®, Gleevec™) paired with one specific discovery of a gain of function receptor in Gastrointestinal stromal tumors has paved the way for a completely new approach in the treatment of soft tissue sarcomas. Gastrointestinal stromal tumors (GISTs) are rare diseases but they constitute the most frequent sarcomas of the gastrointestinal tract. At presentation, metastatic disease is found in up to half of patients [1]. Metastases are predominantly found in the liver and in the peritoneal cavity. The annual incidence of this rare disease[2] is estimated at 10-20 per 1.000.000 [3, 4], yielding 5000-10000 new cases each year in both the US A and Europe [5]. In 1983, Mazur and Clark introduced the term GIST to describe non-epithelial neoplasms of the gastrointestinal tract that stained positive for S100 protein, and showed neither smooth muscle nor Schwann cell features [6]. Diagnostic criteria have recently been more clearly outlined by a pathology consensus [4]. In this consensus, GISTs have been defined as spindle-cell epitheloid, or occasionally pleiomorphic mesenchymal tumors of the gastrointestinal tract that express the KIT receptor kinase, except for rare cases [2, 4]. Of crucial importance for our insight in molecular etiology, cellular origin, classification and treatment approach of GIST was the discovery of the gain-of-function mutations of the mentioned KIT receptor by Hirota et al [7]. The KIT proto-oncogene contains 21 exons and is located on chromosome segment 4q12 [8]. The frequency of gain-of-function mutations in KIT is high [9, 10]. These mutations result in the permanent activation of KIT signaling in the absence of binding of the stem cell factor (SCF), which leads to uncontrolled cell proliferation and resistance to apoptosis. The expression of the CD117 antigen, an epitope of the transmembrane KIT protein, has emerged as the most important defining feature for diagnosing GIST [11]. Staining positive for CD117 by immunochemistry suggests a histiogenesis from the interstitial cells of Cajal, the gastrointestinal pacemaker cells which control gut motility [12, 13]. Surgery is the standard of care for non-metastatic GIST. Five-year survival after surgical resection ranges from 35-65% [14]. Once metastasized, prognosis becomes poor. Independent risk factors for aggressive behavior following resection are: localization other than stomach, tumor size > 5 cm), and a high mitotic count [15]. Metastatic tumors are considered insensitive to conventional chemotherapy [15, 16] and median survival for metastatic or inoperable cases ranges from 9-12 months [17, 18].

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2

IMATINIB

As said, the development of a single drug, has been the backbone for considerable changes and improvement in the therapeutic approach in GIST. Imatinib Mesylate (imatinib; STI571; Glivec® , Gleevec™) [19] is a 2-phenylaminopyrimidine compound (Figure 1). Its molecular weight is 589.7 g/mol. Imatinib is highly soluble in water (50 mg/l at pH 7.4) [20]. About 90% of patients with metastatic or unresectable GIST, treated with imatinib in a phase I study showed clinical benefit [21]. The impressive response results obtained with imatinib have drastically changed the management of patients with unresectable or advanced GIST. Imatinib is the first rationally designed competitive inhibitor of several specific protein tyrosine kinases involved in signal transduction pathways [22, 23]. Imatinib competes with ATP for its specific binding site in the intracellular kinase domain, which inhibits the ability of the kinase to transfer phosphate groups from ATP to tyrosine residues on substrate proteins. By inhibiting substrate protein phosphorylation, imatinib prevents transduction of signals. Thus, the downstream signaling from the KIT-kinase that is activated by SCF or is abnormally activated in GIST, is inactivated by imatinib, which switches the fragile cell balance into apoptosis [24].

Imatinib inhibits is relatively selective and potently inhibits the tyrosine kinase activity of the intracellular Abelson (ABL) protein, the BCR-ABL fusion protein, the KIT protein, and the platelet-derived growth factor receptor (PDGF-R) [21, 25-27]. BCR-ABL is a fusion product of the Philadelphia chromosome which results from the

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translocation t(9;22), and plays a critical role in the development of chronic myelogenous leukemia (CML) [28], the disease for which Imatinib was initially developed [29]. In a GIST-cell line (GIST882) with a KIT-gene gain-of-function mutation, yielding constitutive activation of KIT, imatinib was found to switch off KIT-activity at [30]. In SCF dependent cell lines, imatinib inhibits KIT at an (inhibitory concentration of 50% of the maximal effect) of depending on the assay [25, 31, 32]. In view of the clinical activity in CML [29, 33] confirming proof of concept in that disease, the information that imatinib inhibits KIT [25] as well as proliferation of GIST in vitro [30], and a proof of concept of the activity of imatinib in a single GIST patient [34], imatinib was also developed in GIST [35].

3

PHARMACOKINETICS AND METABOLISM

When given orally, imatinib has a very good mean absolute bioavailability of 98% [36]. Food does not affect uptake [37]. Pharmacokinetic profiles in GIST and CML patients are reported to be similar [36, 38]. After a single 400 mg dose, the mean area under the plasmaconcentration curve approzimates 24.75 mg.h/mL at steady state [38]. After one month, GIST patients receiving a 600 mg daily dose showed at steady state a 1.2 fold higher AUC compared to patients receiving 400 mg daily [39]. Mean imatinib AUC increases proportionally with increasing doses up to 1000 mg. The estimated elimination half-life of imatinib is 18-20 hours [36, 38, 39], indicating that from this perspective once-daily oral dosing would be sufficient. On repeated dosing clearance tended to be somewhat lower [40], which may be related to improvement in albumin levels and higher creatinine levels. After long-term treatment (3-12 months), imatinib clearance seems to increase above the first day level [40], although the available data are yet not conclusive. Liver impairment does not require dose adaptation [41] while renal impairment does [43]. Importantly patients homozygous for one of three determined single nucleotide polymorphisms had a higher plasma AUC at steady state concentration [44].

4

KIT-DRIVEN GIST

The development of Imatinib in KIT-driven GIST initially involved parallel phase I and II studies. The European Organization for Research and Treatment of Cancer (EORTC) Soft Tissue and Bone Sarcoma Group performed a formal phase I study in which efficacy was a secondary objective[21, 35]. Eligible patients had to show histological evidence of soft tissue sarcoma, and GIST patients had to be positive for KIT, as indicated by expression of CD117 on immunohistochemical staining. This dose-escalation study included a total of 40 sarcoma

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patients, 35 of whom had GIST. The other 5 sarcomas did not express KIT. By common toxicity criteria of the National Cancer Institute (NCICTC version 2.0) [45], at the 500 mg twice daily dose-level 5 of 8 patients showed a dose-limiting toxicity (DLT), including nausea, vomiting, edema, and skin rash. A maximum tolerated dose of 800 mg per day, 400 mg bid, was advised for future studies. At a follow-up of 912 months, 82% of the included GIST patients still benefited from imatinib therapy (Table 1). At the same time an initial phase II study was performed by investigators in the USA and Finland to formally assess activity and safety in metastatic or unresectable GIST patients [39]. In this study, 147 patients were included. In 135 patients KIT positive GIST was confirmed by expression of CD117. In 10 cases no material was available to confirm this diagnosis. Patients were randomized to receive either a daily dose

of 400 mg or 600 mg imatinib. Side effects were frequent at both dose levels and comparable to those observed in CML, with the exception of a lower frequency of myelotoxicity and a higher frequency of serious gastrointestinal bleeding, which is probably disease related. Both doses were well tolerated. Because of lack of statistical power, this study was not primarily intended to compare efficacy between both doses. On neither dose, patients reached complete remission. Taking both doses together, 82% of patients showed remarkable benefit from therapy. The 1-year survival was estimated to be 88%. No statistically significant differences in outcome between both groups were found. In both studies in 5-10% of patients imatinib was totally inactive. Three of nine patients in the USA-Finland study, achieved a partial response or stable disease after dose escalation to 600 mg daily after experiencing progressive disease at 400 mg, indicating that there might be a dose-response relationship [39]. Finally the activity data were further confirmed by an EORTC phase II study [46] (See Table 1). The latter included 27 GIST

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patients treated with 400 mg imatinib bid. GIST patients again showed a high response rate, even one complete remission [46]. In view of remaining questions on dose-response relationships, subsequently 2 randomized phase III studies have been performed in pretreated patients with CD117-positive GIST receiving an oral dose of either 400 mg or 800 mg per day. Primary study objectives differ slightly. The study coordinated by the EORTC, in cooperation with the Italian Sarcoma Group and the Australian Gastrointestinal Tumors Group, has included 946 patients and had time to progression of disease as primary endpoint [47, 48], whereas the study coordinated by the South West Oncology Group (SWOG) accruing 746 patients has overall survival as primary endpoint [49, 50]. With a median follow-up of 8.4 months, at the time of the latest interim analysis, the EORTC study does show a better progression free survival for the higher dose group [48] (progression free survival at 6 and 12 months 78% and 69% versus 73% and 64%). Objective response rates by RECIST criteria [51] are reported to be identical in both dose groups (43%), with 2% to 3% complete responses. The SWOG study does not show significant differences between both dose groups at their most recent interim analysis as well [50]. Objective response rates by RECIST criteria [51] are reported to be comparable as well (Table 1). Data from cross-over from 400 to 800 mg are currently not yet available. Little is yet known about the potential of imatinib as neoadjuvant and/or adjuvant treatment. One study presents data of only 5 patients, who received adjuvant imatinib therapy after radical surgery. With a follow up of 7 to 13 months, none of these patients has developed recurrent disease yet [52], but clearly publishing such data is strange based upon flawed methodology, and surely these data to not justify routine use of the approach. The data on neo-adjuvant treatment are similarly scantly and incoclusive. A small study involved only 18 GIST patients that were operated after a period of imatinib treatment [53]. Radical resection was achieved in seven out of eight patients, who underwent the surgery because of residual tumor mass and had a partial response, and in two out of ten patients, who were operated because of progressive disease. Currently, various phase II and phase III studies are being performed investigating the potential role of imatinib in the treatment of resectable GISTs. Clearly only phase III studies can yield undebatable results in this setting. Most gain-of-function KIT mutations in GISTs are within the juxtamembrane region, encoded by exon 11 [54]. Mutations are also found in exon 9, and to a much lesser extent in exons 13 and 17, encoding for the extracellular region, the first part of the split kinase domain, and for the catalytic activation loop in the second part of the kinase domain, respectively [54, 55]. From in vitro studies, it was suggested that GISTs with a mutation of KIT in its regulatory part would respond better on imatinib as those with a KIT mutation in its enzymatic part [56]. Recently, it was reported that GIST patients with a KIT mutation in exon 11 have a significant higher partial response rate than patients with a

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mutation in exon 9 (72% versus 32%; p=0.033) as well as a longer time to progression of (23 months versus 6 months) [55, 57]. If no mutations were found, only 12% showed a partial response and the median time to treatment failure was approximately 3 months. More recent and extended data are discussed in chapter 8. Presented studies show that response to imatinib appears to be long lasting [21, 39, 48, 50]. However, resistance to imatinib, like in CML [59], after initial response or stable disease does occur. In GIST heterogeneous mechanisms may be responsible. Reported elucidating factors for acquired resistance in GIST are the overexpression of KIT due to genomic amplification of KIT, the acquisition of new mutations (e.g. in the enzymatic part of KIT), the loss of KIT expression, accompanied by gain-of-function mutations in other receptor tyrosine kinases, and not otherwise characterized functional resistance [60-62]. At doses up to 800 mg imatinib daily, side effects are generally mild to moderate and relatively easily manageable [21, 39, 46, 47, 49], and particularly include granulocytopenia, anemia, edema, fatigue, rash, and nausea. Higher dosing is associated with a higher frequency of some side effects, i.e. edema, fatigue, rash, and nausea [63]. Although most patients (90% to 100%) experience some toxicity, grade 3 and 4 adverse events are seen in 20% [39, 49],. Side effects seem to decrease in severity with ongoing treatment [21, 40, 46].

5

PDGF DRIVEN SARCOMAS

In view of the success of the inhibition of KIT in GIST by treatment with imatinib, and the fact that the agent in models also actively inhibited Platelet derived growth factor (PDGF), the latter target has also been assessed. Almost all sarcomas are reported to show some expression of PDGF although it is not known if this is truly the reflection of a tumor growth factor. activating mutations have meanwhile been described in GIST as a factor of responsiveness beside KIT mutations [64]. As indicated the EORTC phase I study [21, 35] already included 5 patients with other sarcomas than GIST, but none of them responded. In their phase II study the EORTC also included a stratum of 24 non-GIST sarcomas, not expressing KIT but assumed to be expressing PDGF. Importantly none of these patients had an objective response and the stable diseases obtained were so few and so short that it is unlikely that further studies will show any activity in such sarcomas [46]. The results of a phase II study with KIT expressing other sarcomas have yet not been published. An exception to the inactivity of imatinib in PDGF expressing tumors may be the activity of the agent in dermatofibrosarcoma protruberans (DFSP) and desmoid tumors. DFSP is an extremely rare disease that tends to remain local, but has a high potential for infiltration. Most DFSPs possess either ring chromosomes or translocations that result in fusion of 17q22 and 22q13, the location of the gene. Since imatinib was known to inhibit the

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latter it was given to a patient with DFSP, yielding such a dramatic reduction in tumor size that resection became possible [65]. Other case reports suggest the same. Similarly, two cases of PDGF (and KIT) expressing extraabdominal desmoid tumors have been reported [66], that both showed long lasting benefit. Further studies to assess the activity in these diseases are clearly warranted. Meanwhile several other inhibitors of KIT and PDGF have entered development. Although it is too early to assess activity without a reasonable level of doubt, early data are encouraging.

6

CONCLUSION

The development of Imatinib has clearly created a new paradigm for the treatment of soft tissue sarcomas. Although long term follow-up data are yet lacking it is already quite evident the drug is nothing less than a break-through in the treatment of GIST. It will be of crucial importance in the next coming years to establish a functional relationship between presence of a target for treatment and its involvement in actual tumor growth. For KIT this was evident in GIST and this is likely one of the reason for the success. And other development of signal transduction inhibitors may have failed due to the lack of such information.

7

REFERENCES

1. DeMatteo RP, Lewis JJ, Leung D, Mudan SS, Woodruff JM, Brennan MF. Two hundred Gastrointestinal Stromal Tumors: Recurrence patterns and prognostic factors for survival. Ann Surg. 231(1), 51-58 (2000). 2. Miettinen M, Lasota J. Gastrointestinal stromal tumors--definition, clinical, histological, immunohistochemical, and molecular genetic features and differential diagnosis. Virchows Arch. 438(1), 1-12 (2001). 3. Emory TS, Sobin LH, Lukes L, Lee DH, O’Leary TJ. Prognosis of gastrointestinal smoothmuscle (stromal) tumors: Dependence on anatomic site. Am J Surg Pathol. 23(1), 82-87 (1999). 4. Fletcher CD, Berman JJ, Corless C et al. Diagnosis of Gastrointestinal Stromal Tumors: A consensus approach. Hum Pathol. 33(5), 459-465 (2002). 5. Blanke CD, Eisenberg BL, Heinrich MC. Gastrointestinal Stromal Tumors. Curr Treat Options Oncol. 2(6), 485-491 (2001). 6. Mazur MT, Clark HB. Gastric Stromal Tumors. Reappraisal of histogenesis. Am J Surg Pathol. 7(6), 507-519 (1983). 7. Hirota S, Isozaki K, Moriyama Y et al. Gain-of-function mutations of c-KIT in human Gastrointestinal Stromal Tumors. Science. 279(5350), 577-580 (1998). 8. Giebel LB, Strunk KM, Holmes SA, Spritz RA. Organization and nucleotide sequence of the human KIT (mast/stem cell growth factor receptor) proto-oncogene. Oncogene. 7(11), 2207-2217 (1992). 9. Lux ML, Rubin BP, Biase TL et al. KIT extracellular and kinase domain mutations in Gastrointestinal Stromal Tumors. Am J Pathol. 156(3), 791-795 (2000). 10. Rubin BP, Singer S, Tsao C et al. Kit activation is a ubiquitous feature of Gastrointestinal Stromal Tumors. Cancer Res. 61(22), 8118-8121 (2001).

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11. Sarlomo-Rikala M, El-Rifai W, Lahtinen T, Andersson LC, Miettinen M, Knuutila S. Different patterns of DNA copy number changes in Gastrointestinal Stromal Tumors, Leiomyomas, and Schwannomas. Hum Pathol. 29(5), 476-481 (1998). 12. Sircar K, Hewlett BR, Huizinga JD, Chorneyko K, Berezin I, Riddell RH. Interstitial cells of cajal as precursors of Gastrointestinal Stromal Tumors. Am J Surg Pathol. 23(4), 377-389 (1999). 13. Kindblom LG, Remotti HE, Aldenborg F, Meis-Kindblom JM. Gastrointestinal Pacemaker Cell Tumor (GIPACT): Gastrointestinal stromal tumors show phenotypic characteristics of the interstitial cells of Cajal. Am J Pathol. 152(5), 1259-1269 (1998). 14. Roberts PJ, Eisenberg B. Clinical presentation of Gastrointestinal Stromal Tumors and treatment of operable disease. Eur J Cancer. 38 Suppl 5, S37-38 (2002). 15. Joensuu H, Fletcher C, Dimitrijevic S, Silberman S, Roberts P, Demetri G. Management of malignant Gastrointestinal Stromal Tumours. Lancet Oncol. 3(11), 655-664 (2002). 16. Plaat BE, Hollema H, Molenaar WM et al. Soft Tissue Leiomyosarcomas and malignant Gastrointestinal Stromal Tumors: Differences in clinical outcome and expression of multidrug resistance proteins. J Clin Oncol. 18(18), 3211-3220 (2000). 17. Mudan SS, Conlon KC, Woodruff JM, Lewis JJ, Brennan MF. Salvage surgery for patients with recurrent Gastrointestinal Sarcoma: Prognostic factors to guide patient selection. Cancer. 88(1), 66-74 (2000). 18. Van Glabbeke M, van Oosterom AT, Oosterhuis JW et al. Prognostic factors for the outcome of chemotherapy in advanced soft tissue sarcoma: An analysis of 2,185 patients treated with anthracycline-containing first-line regimens - a European Organization for Research and Treatment of Cancer Soft Tissue and Bone Sarcoma Group Study. J Clin Oncol. 17(1), 150-157 (1999). 19. Dagher R, Cohen M, Williams G et al. Approval summary: Imatinib Mesylate in the treatment of metastatic and/or unresectable malignant Gastrointestinal Stromal Tumors. Clin Cancer Res. 8(10), 3034-3038 (2002). 20. Manley PW, Cowan-Jacob SW, Buchdunger E et al. Imatinib: A selective tyrosine kinase inhibitor. Eur J Cancer. 38 Suppl 5, S19-27 (2002). 21. van Oosterom AT, Judson IR, Verweij J et al. Update of phase I study of Imatinib (STI571) in advanced soft tissue sarcomas and Gastrointestinal Stromal Tumors: A report of the EORTC Soft Tissue and Bone Sarcoma Group. Eur J Cancer. 38 Suppl 5, S83-87 (2002). 22. Kolibaba KS, Druker BJ. Protein tyrosine kinases and cancer. Biochim Biophys Acta. 1333(3), F217-248 (1997). 23. Druker BJ, Lydon NB. Lessons learned from the development of an abl tyrosine kinase inhibitor for Chronic Myelogenous Leukemia. J Clin Invest. 105(1), 3-7 (2000). 24. Taylor ML, Metcalfe DD. KIT signal transduction. Hematol Oncol Clin North Am. 14(3), 517-535 (2000). 25. Buchdunger E, Cioffi CL, Law N et al. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-KIT and platelet-derived growth factor receptors. J Pharmacol Exp Ther. 295(1), 139-145 (2000). 26. Druker BJ, Tamura S, Buchdunger E et al. Effects of a selective inhibitor of the abl tyrosine kinase on the growth of bcr-abl positive cells. Nat Med. 2(5), 561-566 (1996). 27. Verweij J, Judson I, van Oosterom A. Sti571: A magic bullet? Eur J Cancer. 37(15), 18161819 (2001). 28. Faderl S, Talpaz M, Estrov Z, O’Brien S, Kurzrock R, Kantarjian HM. The biology of chronic myeloid leukemia. N Engl J Med. 341(3), 164-172 (1999). 29. Druker BJ, Talpaz M, Resta DJ et al. Efficacy and safety of a specific inhibitor of the bcr-abl tyrosine kinase in Chronic Myeloid Leukemia. N Engl J Med. 344(14), 1031-1037 (2001). 30. Tuveson DA, Willis NA, Jacks T et al. Sti571 inactivation of the gastrointestinal stromal tumor c-KIT oncoprotein: Biological and clinical implications. Oncogene. 20(36), 5054-5058 (2001). 31. Heinrich MC, Griffith DJ, Druker BJ, Wait CL, Ott KA, Zigler AJ. Inhibition of c-KIT receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor. Blood. 96(3), 925-932 (2000). 32. Krystal GW, Honsawek S, Litz J, Buchdunger E. The selective tyrosine kinase inhibitor STI571 inhibits Small Cell Lung Cancer growth. Clin Cancer Res. 6(8), 3319-3326 (2000). 33. Druker BJ, Sawyers CL, Kantarjian H et al. Activity of a specific inhibitor of the bcr-abl tyrosine kinase in the blast crisis of Chronic Myeloid Leukemia and Acute Lymphoblastic Leukemia with the Philadelphia chromosome. N Engl J Med. 344(14), 1038-1042 (2001).

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34. Joensuu H, Roberts PJ, Sarlomo-Rikala M et al. Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic Gastrointestinal Stromal Tumor. N Engl J Med. 344(14), 1052-1056 (2001). 35. van Oosterom AT, Judson I, Verweij J et al. Safety and efficacy of Imatinib (STI571) in metastatic Gastrointestinal Stromal Tumours: A phase I study. Lancet. 358(9291), 1421-1423 (2001). 36. (imatinib mesylate) tablets prescribing information. 2003, East Hanover, New Jersey, USA: Novartis Pharmaceuticals Corporation. 37. Reckmann AH, Fischer T, Peng B et al. Effect of food on STI571 Glivec pharmacokinetics and bioavailability. Proc. Am. Soc. Clin. Oncol. 20, abstract 1223 (2001).38. Glivec®. Gist clinical monograph. 2002, Basel, Switserland: Novartis Pharma AG. 39. Demetri GD, von Mehren M, Blanke CD et al. Efficacy and safety of Imatinib Mesylate in advanced Gastrointestinal Stromal Tumors. N Engl J Med. 347(7), 472-480 (2002). 40. Judson IR, Donato Di Paola E, Verweij J et al. Population pharmacokinetic (pk) analysis and pk-pharmacodynamic (pd) correlations in phase I / II trial of Imatinib in Gastrointestinal Stromal Tumours (GIST) conducted by the European Organisation for Research and Treatment of Cancer Soft Tissue and Bone Sarcoma Group. Proc. Am. Soc. Clin. Oncol. 22, abstract 3287 (2003). 41. Ramanathan RK, Remick SC, Mulkerin D et al. P-5331: A phase I pharmacokinetic (pk) study of STI571 in patients (pts) with advanced malignancies and varying degrees of liver dysfunction (ld). Proc. Am. Soc. Clin. Oncol. 22, abstract 502 (2003). 42. O’Brien SG, Peng B, Dutrix C et al. A pharmacokinetic interaction of Glivec and Simvastatin, a cytochrome 3a4 substrate, in a patients with Chronic Myeloid Leukemia. Proc. Am. Soc. Hematology 98, 141a, abstract 593 (2001). 43. Remick SC, Ramanathan RK, Mulkerin D et al. P-5340: A phase I pharmacokinetic study of STI-571 in patients (pts) with advanced malignancies and varying degrees of renal dysfunction. Proc. Am. Soc. Clin. Oncol. 22, abstract 503 (2003). 44. Gurney H, Wong M, Rivory L et al. Imatinib elimination: Characterisation by in vivo testing of phenotype and genotype. Proc. Am. Soc. Clin. Oncol. 22, abstract 775 (2003). 45. Cancer therapy evaluation program. Common toxicity criteria, version 2.0. 1998, Bethesda, USA: National Cancer Institute. 46. Verweij J, van Oosterom A, Blay JY et al. Imatinib Mesylate (STI-571 Glivec®, Gleevec) is an active agent for Gastrointestinal Stromal Tumours, but does not yield responses in other softtissue sarcomas that are unselected for a molecular target. Results from an EORTC Soft Tissue and Bone Sarcoma Group phase II study. Eur J Cancer. 39(14), 2006-2011 (2003). 47. Casali PG, Verweij J, Zalcberg J et al. Imatinib (Glivec) 400 vs 800 mg daily in patients with Gastrointestinal Stromal Tumors (GIST): A randomized phase III trial from the EORTC Soft Tissue and Bone Sarcoma Group, the Italian Sarcoma Group (ISG), and the Australasian GastroIntestinal Trials Group (AGITG). A toxicity report. Proc. Am. Soc. Clin. Oncol. 21, abstract 1650 (2002). 48. Verweij J, Casali PG, Zalcberg J et al. Early efficacy comparison of two doses of Imatinib for the treatment of advanced Gastro-Intestinal Stromal Tumors (GIST): Interim results of a randomized phase III trial from the EORTC-STBSG, ISG and AGITG. Proc. Am. Soc. Clin. Oncol. 22, abstract 3272 (2003). 49. Demetri GD, Rankin C, Fletcher C et al. Phase III dose-randomized study of Imatinib Mesylate (Gleevec, STI571) for GIST: Intergroup s0033 early results. Proc. Am. Soc. Clin. Oncol. 21, abstract 1651 (2002). 50. Benjamin RS, Rankin C, Fletcher C et al. Phase III dose-randomized study of Imatinib Mesylate (STI571) for GIST: Intergroup s0033 early results. Proc. Am. Soc. Clin. Oncol. 22, abstract 3271 (2003). 51. Therasse P, Arbuck SG, Eisenhauer EA et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst. 92(3), 205-216 (2000). 52. Bumming P, Andersson J, Meis-Kindblom JM et al. Neoadjuvant, adjuvant and palliative treatment of Gastrointestinal Stromal Tumours (GIST) with imatinib: A centre-based study of 17 patients. Br J Cancer. 89(3), 460-464 (2003). 53. Hohenberger P, Bauer S, Schneider U et al. Tumor resection following imatinib pretreatment in GI Stromal Tumors. Proc. Am. Soc. Clin. Oncol. 22, abstract 3288 (2003).

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54. Fletcher JA, Fletcher CD, Rubin BP, Ashman LK, Corless CL, Heinrich MC. KIT gene mutations in Gastrointestinal Stromal Tumors: More complex than previously recognized? Am J Pathol. 161(2), 737-738; author reply 738-739 (2002). 55. Heinrich MC, Corless CL, Blanke C et al. KIT mutational status predicts clinical response to STI571 in patients with metastatic Gastrointestinal Stromal Tumors (GISTs). Proc. Am. Soc. Clin. Oncol. 21, abstract 6 (2002). 56. Heinrich MC, Rubin BP, Longley BJ, Fletcher JA. Biology and genetic aspects of Gastrointestinal Stromal Tumors: KIT activation and cytogenetic alterations. Hum Pathol. 33(5), 484-495 (2002) 57. Heinrich MC, Corless CL, Von Mehren M et al. PDGFRA and KIT mutations correlate with the clinical responses to Imatinib Mesylate in patients with advanced Gastrointestinal Stromal Tumors (GIST). Proc. Am. Soc. Clin. Oncol. 22, abstract 3274 (2003). 58. Heinrich MC, Corless CL, Duensing A et al. Pdgfra activating mutations in Gastrointestinal Stromal Tumors. Science. 299(5607), 708-710 (2003). 59. Deininger MW, Druker BJ. Specific targeted therapy of Chronic Myelogenous Leukemia with imatinib. Pharmacol Rev. 55(3), 401-423 (2003). 60. Fletcher JA, Corless CL, Dimitrijevic S et al. Mechanisms of resistance to Imatinib Mesylate (IM) in advanced Gastrointestinal Stromal Tumor (GIST). 22, abstract 3275 (2003). 61. Andersson J, Sjogren H, Meis-Kindblom JM, Stenman G, Aman P, Kindblom LG. The complexity of KIT gene mutations and chromosome rearrangements and their clinical correlation in Gastrointestinal Stromal (pacemaker cell) Tumors. Am J Pathol. 160(1), 15-22 (2002). 62. Eisenberg BL, von Mehren M. Pharmacotherapy of Gastrointestinal Stromal Tumours. Expert Opin Pharmacother. 4(6), 869-874 (2003). 63. Van Glabbeke MM, Verweij J, Casali PG et al. Prognostic factors of toxicity and efficacy in patients with Gastro-Intestinal Stromal Tumors (GIST) treated with imatinib: A study of the EORTC-STBSG, ISG and AGITG. Proc. Am. Soc. Clin. Oncol. 22, abstract 3286 (2003). 64. Heinrich MC, Corless CL, Duensing A, et al. PDGFRA activating mutations in gastrointestinal Stromal tumors. Science 229, 708-710 (2003). 65. Rubin BP, Schuetze SM, Eary JF, et al. Molecular targeting of platelet-derived growth factor B by Imatinib mesylate in a patient with metastatic dermatofibrosarcoma protruberans. J.Clin.Oncol. 20:3586-3591 (2002) 66..Mace J, Biermann JS, Sondak V, et al. Response of extraabdominal desmoid tumors to therapy with imatinib mesylate. Cancer 95:2372-2370 (2002)

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Chapter 8 Targeting mutant kinases in Gastrointestinal Stromal Tumors: A paradigm for molecular therapy of other sarcomas Michael C. Heinrich and Christopher L. Corless

Departments of Medicine and Pathology, Oregon Health Science University Cancer Institute and Portland VA Medical Center, Portland, USA

Correspondence to: Michael C.Heinrich, MD Departments of Medicine and Pathology, Oregon Health Science University Cancer Institute and Portland VA Medical Center, 3710 SW US Veterans Hospital Road Portland, OR 97201 USA

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1

INTRODUCTION

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasm of the gastrointestinal tract. The classification of these tumors has long been a subject of controversy in the pathology literature. Likewise, the medical management of metastatic or unresectable tumors has been problematic as these tumors are uniformly resistant to standard cytotoxic chemotherapy agents. Fortunately, recent advances in the understanding of the molecular biology and pathogenesis of these tumors has lead to advances in both diagnosis and medical therapy. In particular, the use of the tyrosine kinase inhibitor imatinib (formerly STI571; Gleevec® in the US and Glivec® in Europe, Novartis Pharma, Basel, Switzerland), has lead to a revolution in the therapeutic approach to metastatic GIST. In this chapter, we will review the development of targeted therapy for GISTs and will further suggest how this paradigm might be applied to other soft tissue sarcomas.

2 ONCOGENIC MUTATIONS OF KIT ARE COMMON IN GISTS

KIT is a 145 kD transmembrane glycoprotein that serves as the receptor for stem cell factor (SCF) and has tyrosine kinase activity.1,2 A member of the subclass III family of receptor tyrosine kinases, KIT is closely related to the receptors for PDGF, M-CSF and FLT3 ligand.3,4 Based on studies of naturally occurring loss-of-function mutations in mice, signaling through KIT is critical to the development of the interstitial cells of Cajal, as well as hematopoietic progenitor cells, mast cells and germ cells.5-9 Binding of SCF to KIT results in receptor homodimerization and activation of the tyrosine kinase activity, which leads to the phosphorylation of a variety of signaling intermediates.10,11 In many cases, these substrates are themselves kinases and serve to amplify intracellular signal transduction.12

2.1

Mutations of the Juxtamembrane Domain (Exon 11)

The juxtamembrane region of KIT (exon 11) functions to inhibit receptor dimerization in the absence of SCF. From in vitro mutagenesis studies it has been established that small, in-frame deletions, insertions or single amino acid substitutions in this domain disrupt its function, allowing spontaneous, ligand-independent receptor dimerization.13-16 In 1998, Dr. Seichi Hirota and colleagues discovered that GISTs not only express KIT protein, but that mutations in the juxtamembrane domain of the KIT gene can be found in these tumors.17 As predicted, the mutant KIT isoforms had constitutive kinase activity when expressed in vitro, that is, their kinase domains were active even in the absence of SCF. The oncogenicity of the KIT mutants was confirmed in a nude mouse tumorigenesis assay with transfected BA/F3 cells.

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supporting the view that activation of KIT plays an important role in the growth and survival of GISTs. The reported frequency of exon 11 mutations in GISTs has varied over a wide range (20% to 92%), but the highest frequencies have come from studies based on cDNA prepared from frozen tumor samples. Hirota and colleagues observed exon 11 mutations in 5 of 6 tumors (83%), while Rubin et al. uncovered 34 exon 11 mutations in 48 tumors (71%).17,18 In most studies that have used genomic DNA extracted out of paraffin-embedded tumor tissue the frequency has been lower (20% to 57%).19-24 This likely reflects technical issues related to the lower quality of DNA obtainable from such samples. In addition, the PCR primers used to amplify exon 11 in some paraffin-based studies did not permit analysis of the entire exon. Screening methodologies are another factor. Many investigators have relied on single strand conformation polymorphism (SSCP) to look for the presence of a deletion or point mutation, but this technique is not as sensitive as capillary gel electrophoresis, which in a recent study by Emile et al. yielded exon 11 mutations in 67.5% of 40 GIST samples.25 Denaturing HPLC (D-HPLC) is another highly sensitive screening methodology that has yielded high frequencies of deletions/insertions and point mutations in KIT exon 11. In a series of 322 paraffin-embedded GISTs for KIT gene mutations screened by this approach in our laboratory the frequency of sequence-confirmed exon 11 mutations was 66.1%. This series includes 127 malignant GISTs and 13 very low-risk GISTs that have previously been published.26,27 The spectrum of mutations, was similar to that reported by other groups. Deletions and insertions tend to affect the first part of the exon, particularly codons 557-559. Point mutations are limited to just four of the codons within the exon: 557, 559, 560 and 576. Internal tandem duplications are observed near the end of the exon. A subset of the tumors (17.8%) were either hemizygous or homozygous for the observed mutation, suggesting that there is negative selective pressure on expression of the wild-type KIT allele in exon 11-mutant tumors. In vitro experiments demonstrating that a peptide corresponding to the wild-type juxtamembrane domain is inhibitory to activated isoforms of KIT support this view.16

2.2

Mutations in the Extracellular Domain (Exon 9)

Exon 9, which encodes the extracellular domain of KIT, is the second most common site of mutation in GISTs. An insertion-type mutation that results in duplication of Ala501 and Tyr502, first reported by Lux and colleagues, was confirmed by Hirota et al. to encode a KIT isoform that has a constitutively activated kinase.28,29 Other groups have also observed this mutation and noted its preferential association with small intestinal origin.30-32 Among the 322 GISTs that we have analyzed, 42 (13%) had exon 9 mutations, only 1 of which was hemi/homozygous. All were the AY501-502 duplication/insertion, with the exception of a

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single FAF505-508 duplication/insertion.26 Based on our data and other published examples, 95% of exon 9-mutant GISTs arise in the small bowel, suggesting that the biology of this class of mutations differs from the exon-11 mutant tumors that most commonly occur in the stomach.

2.3

Mutations in the Kinase I Domain (Exon 13)

Lux and colleagues were the first to identify the K642E point mutation in KIT exon 13.28 Though this mutation is rare, it has since been observed by several other investigators in frequencies ranging from 0.8 to 4.1%.28,30,33 Our series of 322 GISTs yielded just 4 of these exon 13 mutations (1.2%). There is evidence that this mutation results in ligandindependent activation of the receptor, although it is unclear whether spontaneous receptor homodimerization is the mechanism.34,35

2.4

Mutations in the Activation Loop (Exon 17)

Mutations involving the activation loop of KIT are uncommon in GISTs. Rubin et al. reported an N822K and an N822H mutation in one case each.18 Two additional N822K mutants were detected in our series of 322 GISTs (0.6%), but no such mutations were observed by Kinoshita and colleagues amongst 124 GISTs.33 As is discussed further below, a germline D820Y substitution has been related to familial GIST;36 however, this mutation has not been reported in sporadic tumors. The N822K and D820Y mutations cause constitutive activation of the kinase domain, although the mechanisms remain unclear.26,36 It is interesting that a nearby codon in exon 17 (Asp816) is commonly mutated in other human malignancies, including mast cell disease, seminoma/dysgerminoma, acute myelogenous leukemia, and sinonasal natural killer/T-cell lymphoma.37-42 While mutations of this codon are highly activating, they have not been observed in more than 700 GISTs published to date. Conversely, activating mutations of KIT exon 11 have been found in only 5 cases of human mastocytosis. The implication of these observations is that the stem cells that give rise to GISTs have different transforming requirements than those that give rise to mastocytosis, and these requirements may be reflected in alternative signaling initiated by the various KIT mutations.

3 KIT MUTATIONS OCCUR EARLY IN GIST DEVELOPMENT Several lines of evidence support the hypothesis that activating mutations of KIT are the initiating event in most adult GISTs: 1) KIT mutations are common in small, incidentally discovered GISTs; 2) KIT mutation status does not correlate pathologic grade; 3) germline activating KIT mutations are associated with a heritable susceptibility to

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GIST; 4) expression of mutant KIT in mice results in GISTs; 5) KIT mutations antedate cytogenetic abnormalities. Early reports on KIT mutations in GISTs emphasized an apparent association with aggressive clinical behavior.19-21,23,24 These studies, however, were based on SSCP and/or direct sequence analyses of exon 11 amplimers prepared from paraffin-extracted DNA. Using D-HPLC, we screened 13 incidentally discovered, morphologically benign GISTs that were 1 cm or less in size. Ten of these sub-clinical tumors harbored exon 11 mutations (77%), and one had an exon 9 insertion.27 We have since analyzed 275 GISTs for which information on grade and stage was available. The exon 11 mutation frequency among tumors of low malignant potential (<5 cm and <5 mitoses/50 hpf) was 87.1%.26 Reports from Rubin and colleagues and by Wardelmann et al. show similar high frequencies of exon 11 mutations in low grade GISTs.18,43 Clearly, these mutations occur early in GIST oncogenesis and are therefore unlikely to influence malignant potential. In contrast to exon 11 mutations, the frequency of exon 9 mutations in our series of 275 GISTs was higher among malignant tumors (17.3%) than among high-risk (3.0%) and low-risk tumors (2.5%). These mutations appear to support altered intracellular signaling compared with exon 11-mutant tumors.44 Thus, the biology of exon 9-mutant tumors is inherently different and perhaps more aggressive than that of other GISTs. In theory, the progression of GISTs might be related to the accumulation of secondary mutations in KIT. For example, if a tumor harboring an exon 11 point mutation subsequently acquired an exon 9 deletion, it might have an additional growth advantage. This hypothetical phenomenon has not been observed in our series of GISTs. Among 127 tumors studied in detail for mutations in KIT exons 9, 11, 13 and 17, none had more than a single mutation.45 Germline mutations in the KIT gene have been reported in a number of kindreds, as summarized in Table 1. Individuals who inherit an activating mutation in exon 11 (V559A or deletion 559) are predisposed to skin hyperpigmentation, skin mastocytosis (urticaria pigmentosa), and the development of multiple GI stromal tumors. Interestingly, there is diffuse ICC hyperplasia in the GI tract in affected patients, consistent with continuous growth signaling from the mutant KIT isoform. GISTs that arise from this background hyperplasia are not usually clinically evident until early adulthood, suggesting that other mutations are necessary to allow full neoplastic progression. Other germline KIT mutations have been reported in exons 13 and 17 (Table 1). These mutations also lead to ICC hyperplasia and a predisposition to multifocal GIST formation, but they are not associated with skin pigmentation or mast cell proliferations. Thus, signaling from these particular tyrosine kinase domain mutations does not appear to influence melanocytes and mast cells in the same manner as the exon 11 mutations.

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From the above studies, it is clear that germline inheritance of an activating KIT mutation results in a greatly increased risk of developing one or more GISTs, strongly supporting the notion that KIT mutation is an initiating event in GIST pathogenesis. As GISTs in affected patients usually develop after the second decade of life, it is inferred that additional molecular events are required for development of a clinically advanced lesion. This inference is supported by studies of lesional clonality in two different kindreds with familial GIST (one kindred with an exon 11 point mutation, the other with an exon 17 point mutation).52 In the studied patients, the diffuse ICC hyperplasia present within the muscularis propria of the GI tract was found to be polyclonal in nature. In contrast, discrete mass lesions diagnosed as GIST were monoclonal, and different GIST lesions from the same patient were derived from independent clones. Recently, Sommer et al. have generated a murine model of GIST using a knock-in strategy to introduce a mutation found in human familial GIST (del V559 human = del V558 mouse) into the mouse genome.53 Heterozygous mutant mice developed patchy hyperplasia of KIT-positive cells of the myenteric plexus throughout the GI tract. The morphological, immunohistochemical, and ultrastructural features of these hyperplastic cells were consistent with ICC. In addition, nearly all mice developed stromal tumors of the cecum, with morphologic and immunophenotypic features similar to those seen in human GIST specimens. Relative to control mice, the heterozygous mutant mice had shorted survival due to bowel obstruction. None of the mice developed metastatic disease. It is interesting to note that GISTs occur spontaneously in dogs and juxtamembrane mutations in KIT are present in these tumors.54 Thus, the relationship between KIT signaling and GIST development is constant across several mammalian species. The majority of GISTs have one or more cytogenetic abnormalities, most commonly chromosomal deletions, and have a cytogenetic profile that is distinct from histologic mimics such as leiomyoma and leiomyosarcoma. These cytogenetic abnormalities are typically found in only a subset of GIST cells and thus are likely to be secondary events that occur after the initiating oncogenic KIT mutation. Indeed, some GISTs have normal karyotypes but have an activating KIT mutation. To summarize, the molecular, clinical, animal models and cytogenetic studies suggest a consistent pathogenic process for GIST progression. A simplified version of this progession is as follows: KIT

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activating mutation 14 q deletion 22q deletion 1p deletion 8q gain 11p deletion 9p deletion 17q gain.55 This schema is a generalization and is not meant to suggest that all GISTs acquire each of these cytogenetic abnormalities or that the abnormalities are necessarily acquired in this order. This model is analogous to the biology of chronic myelogenous leukemia where expression of BCR-ABL oncoprotein is the initiating event and additional cytogenetic abnormalities are acquired during progression to accelerated phase and blast crisis.56

4

PDGFRA IS AN ALTERNATIVE ONCOGENE IN

GISTS Some GISTs are negative for mutations in the KIT gene, despite best efforts to find these mutations, including complete cDNA sequencing. Heinrich, Fletcher and colleagues recently applied a novel methodology to search for other activated kinases in tumors wild-type for KIT (KIT-WT).57 A cocktail of antibodies to epitopes shared by a wide range of receptor tyrosine kinases was used to immunoprecipitate kinases from extracts of these tumors, and western blotting with a phosphotyrosine specific antibody revealed a novel band that was identified as PDGF receptor alpha (PDGFRA). Phosphorylated PDGFRA was detectable in a subset of KIT-WT tumors, but was not present in extracts of tumors with known KIT mutations. Conversely, extracts of KIT-mutant tumors had phosphorylated KIT, but were negative for phosphorylated PDGFRA. These results suggested that PDGFRA is the active kinase in some KIT-WT tumors. Examination of genomic DNA from KIT-WT tumors yielded a variety of mutations in the juxtamembrane (exon 12) and activation loop (exon 18) domains of the PDGFRA gene.57 When cloned and transfected into CHO cells, the PDGFRA mutant isoforms were found to be constitutively phosphorylated in the absence of PDGF-AA ligand, consistent with oncogenic activation. The frequency of PDGFRA mutations among KIT-WT GISTs is 34% (23/67 cases), and among all GISTs is 7.1% (23/322). PDGFRA exon 12 mutations appear to be less common than exon 18 mutations (1.5% versus 5.6%, respectively). PDGFRA -mutant tumors tend to have an epithelioid morphology, but are not histologically distinguishable from KIT-mutant tumors. Likewise, the signal transduction profiles for the two types of tumors are similar.57 No PDGFRA mutations have been found in 146 KIT-mutant tumors, so the activation of the two genes appears to be mutually exclusive. Although not as extensively studied as KIT mutations in GISTs, it seems likely that PDGFRA mutations might be an initiating event in some GISTs lacking KIT mutations. The impact of PDGFRA mutations on the diagnosis and treatment of GISTs is considered in later sections.

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5

OTHER GIST VARIANTS

As discussed above, we favor a model in which activating mutations of KIT or PDGFRA serve as the initiating event in the vast majority of these tumors. There are, however, a number of “variant” clinical syndromes in which GISTs appear to arise independent of KIT and PDGFRA mutations. The association of paraganglioma, pulmonary chondromas and “gastric leiomyosarcoma,” known as Carney’s triad, is a rare, sporadic syndrome that presents almost exclusively in young women.58 While the genetic basis for the syndrome is not known, virtually all reported patients have had one or more gastric tumors that were morphologically and immunophenotypically consistent with GIST.59 In most instances, the tumors are diagnosed before the patient reaches age 30. Preliminary studies of Carney triad-associated GISTs suggest that they do not harbor KIT or PDGFRA mutations (authors’ unpublished results). Gastric GISTs are occasionally diagnosed in pediatric patients outside of Carney’s triad.60 A malignant GIST of the stomach from one patient was recently screened for mutations in KIT exons 9, 11 and 13 and found to be negative.61 Preliminary studies of additional nonsyndromic pediatric GISTs indicate that KIT and PDGFRA mutations are much less common than in adult GISTs (authors’ unpublished results). Patients with neurofibromatosis type I (NF1, von Recklinghausen’s neurofibromatosis) often develop neurofibromas in the GI tract, but in addition, a subset of these patients suffer from one or more gastric, intestinal or colonic GISTs. Described as “autonomic nerve tumors”, “stromal tumors with skeinoid fibers” or “leiomyomatosis” in the older literature,62-66 the presence of strong KIT positivity in these tumors supports their designation as GISTs.32,67 Based on a Swedish study of 70 NF1 patients, the incidence of GISTs in this population is approximately 7%.68 Why the tumors arise in only a minority of these patients and yet may be multifocal remains an interesting mystery.

6 TARGETING ONCOGENIC KINASES IN GISTS USING IMATINIB The accepted standard treatment for localized GISTs is complete surgical resection.69 Recurrence after complete resection is common, occurring in up to 90% of patients with larger tumors,70 and survival following recurrence is short. Patients with advanced GIST have historically done poorly, with a 30% 6-month progression-free survival following initial chemotherapy.71 The sensitivity of GISTs to standard chemotherapy agents has been difficult to define because most published series have not adequately distinguished GISTs from leiomyosarcomas and neurogenic tumors. However, the published data suggest that the

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response rate of GISTs to single- or multi-agent chemotherapy regimens is less than 5%.72 The concept that patients with GISTs might benefit from treatment with imatinib was based in part on two experimental observations: 1) treatment of GIST cell lines with imatinib inhibited proliferation and induced apoptosis; 2) several GIST-associated mutant KIT isoforms were potently inhibited by imatinib in vitro at concentrations similar to wild-type KIT.73,74 Based on the identification of mutated KIT as a therapeutic target in GIST and the lack of an effective conventional medical therapy, a patient with chemotherapyresistant gastric GIST metastatic to omentum and liver was started on imatinib at 400 mg po daily in March 2000.75 The tumor in this patient expressed an exon 11 mutant KIT isoform and the patient responded dramatically to imatinib therapy. The strong pre-clinical rationale, coupled with the clinical success in this solitary case, led to the development of two proofof-principle GIST trials utilizing imatinib. An EORTC dose-escalation study of imatinib included 40 patients, 36 of whom had advanced GIST. The imatinib dose ranged from 400 milligrams daily to 500 mg BID. At the highest dose, five of eight patients had grade 3 toxicity (nausea/vomiting in 3, edema in 1, and dyspnea in 1) and 400 mg BID was considered the maximally tolerated dose. Nineteen patients (53%) had objective partial responses, while thirteen (36%) had stable disease and only four (11%) experienced frank progression. Two patients who progressed on 400 mg subsequently responded to 800 mg. With a minimum follow-up of 11 months, 29/36 remained on treatment. None of the four patients in the trial with a non-GIST sarcoma responded to imatinib.76,77 The GIST Working Group (a consortium of the Oregon Health & Science University Cancer Institute, the Fox Chase Cancer Center, the University of Turku, the University of Helsinki, and the Dana-Farber Cancer Institute) performed a phase II randomized trial of 400 mg daily versus 600 mg.78 One-hundred forty-seven patients with incurable GIST (51% pre-treated with systemic therapy) were accrued, with a median age of 54 and median performance status of 1. Ninety-four percent of the patients had undergone previous surgery, often involving the stomach or other areas in the GI tract, and therefore drug absorption was a concern in this trial. Nevertheless, pharmacokinetic assessments showed that drug levels were actually higher than that seen in leukemia patients given imatinib. Imatinib was highly effective in this advanced disease population. Sixty-two percent of patients on 400 mg daily, and 65% on 600 mg achieved a partial response.78,79 Stable disease was seen in 19 and 20% respectively, and 16 and 8% had primary resistance, manifested as initial disease progression. Twenty-eight percent of patients failing the lower dose responded when crossed over to the higher dose. With a median follow-up of 15 months, 73 percent of patients remained on study drug, with an overall median time-to-treatment failure of 72 weeks.

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7 USE OF KINASE MUTATIONS IN THE DIAGNOSIS OF GIST The use of KIT as an immunohistochemical marker for GISTs has helped to solidify an otherwise untidy field by engendering greater uniformity in both the diagnosis and the comparative study of these tumors. No immunohistochemical marker is perfect, however, and the heavy reliance on KIT staining has created some problems. The first is that there are several commercially available KIT antibodies and these antibodies are applied using different protocols in different laboratories. The result is disagreement as to the specificity of this marker for GISTs relative to other mesenchymal tumors in the abdomen, including fibromatosis (desmoid tumor), synovial sarcoma and leiomyosarcoma.8087 From our experience in a referral center for GIST patients, it is apparent that over-staining with inappropriately titered KIT antibodies is a problem in some laboratories. Educational efforts are underway in the U.S. to help pathology laboratories validate their KIT staining protocols, and improved commercial packages for KIT staining may soon be available. Thus, some of the confusion generated by improper immunohistochemical staining for this marker may abate in coming years. Perhaps a larger challenge created by the emphasis on KIT staining in GISTs is that approximately 5% to 10% of these tumors are either weak or negative for KIT expression. In our experience, KITnegative GISTs often have activating mutations of PDGFRA.57 In addition, KIT-negative GISTs are sometimes associated with activating mutations of KIT.88 In the latter situation it appears that KIT expression is either too low for detection with routine immunohistochemistry or is artificially absent due to problems with tumor fixation. We propose that genotyping for KIT and PDGFRA kinase mutations may be useful as a confirmatory test in most GISTs, particularly the subset of GISTs that lack KIT expression as assessed using a routine immunohistochemical assay. As discussed below, genotyping studies are useful not only providing molecular confirmation of the diagnosis, but in determining the prognosis of patients treated with imatinib.

8 KINASE MUTATIONS ARE PREDICTIVE OF IMATINIB RESPONSE The weight of existing evidence favors a model in which mutations of KIT and PDGFRA are the initiating event in the vast majority of GISTs. An additional prediction from this model is that imatinib treatment would be most effective in GISTs with sensitive kinase mutations. This prediction has been confirmed in correlative studies of patient specimens from the GIST working group phase II trial.

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Specifically, pre-treatment tumor specimens from 127 of the 147 treated patients were genotyped for KIT and PDGFRA mutations and the patient’s clinical response to imatinib, event-free and overall survival were correlated with tumor genotype. Additionally, the imatinib sensitivity of representative KIT and PDGFRA oncoproteins was determined in vitro.26 Activating mutations of KIT or PDGFRA were found in 112 (88.2%) and 6 (4.7%) GISTs, respectively. Most KIT mutations involved exon 9 (N = 23) or exon 11 (N = 85). The in vitro sensitivity of representative KIT exon 9 (reduplication of AY502-503), 11 (V560G, deletion WK557-557, deletion L579), 13 (K642E) and 17 (N822H, N822K) oncoproteins to imatinib was similar to that of ligand-activated wild-type KIT approximately 100-200 nM). In contrast, only some of the PDGFRA oncoproteins were sensitive to imatinib in vitro approximately 100-200 nM). Specifically, the D842V PDGFRA mutation seen in 3 patients was 10-20 fold more resistant to inhibition by imatinib approximately In patients with GISTs harboring exon 11 KIT mutations, the partial response rate (PR) was 83.5 percent, while patients with tumors containing an exon 9 KIT mutation or no detectable mutation of KIT or PDGFRA had PR rates of 47.8 percent (P=0.0006) and 0.0 percent (P<0.0001), respectively. Patients whose tumors contained exon 11 KIT mutations had a longer event free and overall survival than those whose tumors expressed either exon 9 KIT mutations or had no detectable kinase mutation. The difference in partial response rate and event free survival between the groups of patients whose GISTs had KIT exon 9 versus exon 11 mutations is particularly noteworthy, as the KIT oncoproteins encoded by exon 9 and exon 11 mutations were equally sensitive to imatinib in vitro. Preliminary studies suggest differences in downstream signaling in exon 9 versus exon 11 KIT-mutant GISTs,44 and such biological differences might influence the susceptibility of the tumor cells to apoptosis in response to kinase suppression by imatinib. Alternatively, the activation mechanisms for KIT exon 9 mutants might vary between the in vitro and in vivo settings. As only six genotyped patients in the trial had a PDGFRA mutation, it is difficult to draw firm conclusions regarding the response of such patients to imatinib. Nevertheless, it is noteworthy that 2 of 3 patients with an imatinib-sensitive PDGFRA mutation had a clinical partial response whereas all 3 patients whose GISTs expressed the imatinib-resistant D842V PDGFRA oncoprotein had progressive disease after initiation of imatinib therapy. The group of patients whose GISTs lacked a detectable KIT or PDGFRA mutant oncoprotein responded poorly to imatinib therapy. Of the nine patients whose GIST expressed wild-type KIT and PDGFRA kinases, none of the patients obtained a partial response and only three patients had stable disease as the best clinical response to imatinib therapy. Additionally, the event free and overall patient survival were

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significantly shorter in these patients. These results suggest that GISTs lacking a KIT or PDGFRA mutation are biologically distinct and are apparently less dependent upon these kinases that GISTs expressing mutant kinases. These data strongly support the notion that the responsiveness of GIST to imatinib is mechanistically linked to the expression of imatinibsensitive mutant kinases. Notably, the partial response rate of patients whose GIST expressed an imatinib sensitive KIT or PDGFRA mutant oncoprotein was 76%, whereas the partial response of GISTs lacking a kinase mutation or PDGFRA D842V was 0%. Overall, 100% (87/87) of the genotyped patients that achieved a partial response during imatinib therapy had GISTs expressing an imatinib sensitive mutant KIT or PDGFRA oncoprotein. Currently we are genotyping GIST specimens from patients enrolled in the US-Canadian Phase III trial of imatinib for metastatic or unresectable GIST. To date, GISTs from approximately 300 of the total 745 patients have been genotyped. Correlation with response data is not yet available, but the overall frequency and spectrum of mutations is similar to that observed in the GIST working group phase II trial (author’s unpublished observations). The larger patient numbers in the phase III trial should be more than adequate for confirmation and extension of the phase II genotyping results. The larger sample size of this trial will be particularly important for analyzing the less common mutations, such as PDGFRA D842V, and the group of GISTs without KIT or PDGFRA mutations. Routine genotyping of GISTs may be clinically useful in determining the likelihood of response to imatinib therapy. Such knowledge could prove useful in determination of prognosis and individualizing care plans. For example, patients whose GISTs express KIT exon 9, PDGFRA D842V, or wild-type KIT and PDGFRA proteins have a much higher rate of treatment failure than patients whose GISTs express KIT exon 11 mutation. Conceivably, these “high-risk” patients should be monitored more frequently than the “good-risk” patients, particularly during the first six months of therapy. PET imaging has proven to be a more sensitive and earlier predictor of imatinib response than conventional imaging. The optimal role of PET imaging in the management of GIST has not been defined, but it might be particularly useful in early assessment (days 21-40) of patients at moderate to high risk of treatment failure.77,89 As additional treatment modalities (alternative kinase inhibitors and/or combination therapy) are defined, the choice of upfront treatment regimen could be optimized using tumor genotype. Another area where genotyping may help guide therapy is in the use of adjuvant or neoadjuvant imatinib. Logically, the addition of imatinib to surgical resection should be most efficacious in situations where there is a high risk of failure with surgery alone and the tumor has an imatinib-sensitive genotype. For example, the use of neoadjuvant imatinib to treat a GIST lacking a kinase mutation seems unlikely to result in tumor shrinkage; there would be an approximately 70% chance

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of tumor progression and this might compromise surgical resectability of the tumor(s). Conversely, the 84% chance of a partial response of a GIST expressing an exon 11 KIT mutation to imatinib suggests a greater likelihood of benefit to combination therapy. Ongoing neoadjuvant and adjuvant trials of imatinib will define the overall benefit of such approaches. These ongoing trials are incorporating correlative genotyping studies that may define subsets of patients who will derive benefit from neoadjuvant and/or adjuvant therapy.

9

MOLECULAR CLASSIFICATION OF GISTS

Based on the studies presented in this chapter, it is clear that GISTs should be regarded as a group of closely related tumors rather than as a single, uniform entity. While it has been only a few years since KIT was identified as a highly sensitive marker for GISTs and imatinib was introduced as a new therapy, it is now apparent that there are clinically important differences among the oncogenic mutations that occur in KIT and PDGFRA, and that wild-type GISTs constitute yet another distinct subset. For this reason, we propose that GISTs be classified according to the scheme outlined in Table 2, which emphasizes the molecular context of the tumor and provides a quick reference for other syndromes with which it may be associated.

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To the extent that this classification is useful in identifying patients who are likely to fail initial imatinib therapy, or in identifying kindreds with possible germline KIT mutations, it is obvious that there will be an increasing role for mutation screening in newly diagnosed GISTs. Further progress in defining the oncogenic pathway(s) in wildtype GISTs, and in the development of new therapeutics, will certainly bring revisions to this classification, but in the meantime it may be helpful in interpreting the results from ongoing and future clinical trials of new targeted therapies for GIST.

10 EXTRAPOLATING THE GIST MODEL TO OTHER SOFT TISSUE SARCOMAS To date, the most effective use of small molecule kinase inhibitors has been in the treatment of hematological malignancies (Chronic Myelogenous Leukemia [CML], Chronic Myelomonocytic Leukemia, Hypereosinophilic syndrome) and sarcomas (GIST, Dermatofibrosarcoma Protuberans).77,78,90-97 A logical question is what is the biological relationship between these entities? As reviewed elsewhere, a striking common feature of hematological malignancies and sarcomas is 1) the association of these cancers with recurrent chromosomal translocations targeting transcription factors (e.g. AML1ETO in Acute Myelogenous Luekemia (AML) and PAX3-FKHR in alveolar rhabdomyosarcoma); and 2) constitutive activation of tyrosine kinases by translocation or intragenic mutation (e.g. KIT mutations in systemic mastocytosis and GIST) (Table 3).98-102 Accordingly, molecular analysis has become central to confirming the diagnosis of many hematological and mesenchymal malignancies (e.g. BCR-ABL to distinguish CML from other myeloproliferative diseases, PDGFRA mutations in KIT-negative GISTs). Abundant clinical and molecular evidence supports the hypothesis that chromosomal translocations targeting transcription factors serve to block hematopoietic stem cell differentiation. In contrast, kinase mutations serve to increase cellular proliferation (e.g. BCR-ABL, FLT3). The order of acquisition of these two types of genomic abnormalities determines the clinical phenotype. For example, if a kinase mutation develops first, the clinical phenotype is that of a myeloproliferative syndrome with a massive increase in the number of well-differentiated cells. Subsequent development of a differentiation block abnormality results in accumulation of immature precursors and transition to an acute leukemia/lymphoma syndrome (e.g. CML blast crisis).103 Conversely, a differentiation block may develop first resulting in a bone marrow/myelodysplastic syndrome with subsequent evolution to an acute leukemia/lymphoma syndrome after acquisition of an activating kinase mutation (e.g. AML1-ETO translocation with subsequent activating FLT3 internal tandem duplication mutation).101,102,104

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Based on the above model and reported trials of kinase inhibitors, the existing evidence suggests that kinase inhibitors will be most effective as monotherapy for those diseases in which the kinase mutation is the initiating event, as exemplified by the clinical response of chronic phase CML and GIST. Targeting the activity of fusion/mutant transcription factors might serve to reverse the differentiation block in sarcomas such as synovial sarcoma and rhabdomyosarcoma. Such a possibility is suggested by activity of all-trans-retinoic acid in targeting the fusion oncoprotein in some variants of acute progranulocytic leukemia.104,105 Even more speculatively, combined therapy targeting differentation blocks and genomically activated kinases might be particularly effective for those sarcomas associated with both abnormalities.106 To test the above model, we urgently need to define additional genomic mechanisms of kinase activation and differentiation blocks in sarcomas. It is likely that ongoing genomic and proteomic efforts will identify such abnormalities and will set the stage for new targeted approaches to sarcomas.107 In addition, and as discussed above in relationship to GISTs, identification of additional molecular mechanisms that initiate or enforce clinical progression of sarcomas will likely lead to additional subclassification and/or reclassification of various soft tissue neoplasms. Hopefully, refinements in diagnosis and new treatment approaches will lead to better treatment outcomes for patients with sarcoma.

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11

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Chapter 9 Targeting other abnormal signaling pathways in sarcoma: EGFR in synovial sarcomas, in liposarcomas Jean-Yves Blay 1,2, Isabelle Ray-Coquard3, Laurent Alberti2, Dominique Ranchère4 1. Unité d’Oncologie Médicale Hôpital Edouard Herriot Place d’Arsonval, 69003 Lyon 2. Equipe Cytokine et Cancer, Unité INSERM 590 Centre Léon Bérard 28, rue Laënnec, 69008 Lyon 3. Département de Médecine Centre Léon Bérard, 28, rue Laënnec 69008 Lyon 4. Departement of Pathology Centre Léon Bérard, 28, rue Laënnec 69008 Lyon Correspondance to: JY Blay Equipe Cytokine et Cancer, Unité INSERM 590 Centre Léon Bérard 28, rue Laënnec 69008 Lyon

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1

INTRODUCTION

Soft tissue sarcomas (STS) are a family of rare malignant diseases originating from mesenchymal cells whose incidence is around 2-3/100000 per year and causing to 2% of the total cancer-related mortality. According to the EUROCARE data (1) the 5-year survival in Europe of adult STS (excluding visceral ones) averages 60%. Further improvements in the treatment of these rare tumours are therefore needed.

2 SOFT TISSUE SARCOMAS: UNIFORM TREATMENTS FOR HETEROGENOUS DISEASES? There are multiple histological subtypes of STS and histological classifications have actually evolved constantly in the past years (2) as a consequence of the refinements of immunohistochemical, and molecular investigations. New molecular techniques, such as conventional cytogenetics, molecular cytogenetics, comparative genomic hybridization, or sequence of an overexpressed gene have enabled to identify specific molecular alterations associated with discrete histological subtypes (2,3). These specific molecular alterations are now used for diagnosis purposes: a diagnosis of Ewing family of tumors is now based on the detection of one of the specific translocations involving the EWS gene on chromosome 22. Other examples are detailed in different chapters of this book. These tools have enabled to profoundly modify the classifications of these tumors, with the identification of previously unrecognized subtypes of sarcoma, eg. gastrointestinal stromal tumors (GIST), or the splitting of malignant fibrous histiocytoma (MFH), into various new of other histological subtypes. Despite of these major improvement in the understanding of the pathogenesis of these sarcomas, until recently, most soft tissue sarcomas subtypes were grouped for the purpose of treatment. This remains the case regarding local treatment : standard treatment in localized tumors is generally a wide surgical excision combined with radiotherapy, whenever feasible, or in some cases radical surgery, i.e. compartmental resection or amputation of the extremity. Pre or post-operative radiotherapy reduces the rate of local recurrence significantly (4). Although the effect adjuvant chemotherapy has been studied in several studies, a recent international meta-analysis indicated an effect on progression free survival but no significant effect on overall survival (5). Chemotherapy is in contrast widely used in the treatment of advanced disease, basically with a palliative intent. Until recently, similar chemotherapy regimens were given to all types of sarcomas: doxorubicin and ifosfamide, yielding response rates in the range of 10-25% in monotherapy, of 20-35% in combination therapy are the most frequently prescribed agents (6). The systemic treatment of locally advanced or metastatic soft tissue sarcoma (STS) remains however unsatisfactory, with few long lasting response and only a small proportion of patients achieving long term survival (7,8). However, it has become clear that

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some subtypes have a distinct behavior with regards to response to chemotherapy. For instance, and as described elsewhere, gastrointestinal stromal tumors are resistant to all known cytotoxic agents, but exhibit specific sensitivity to the c-kit tyrosine kinase inhibitor imatinib (9,10). Synovial sarcomas, but not leiomyosarcomas, are characterized by high response rates to ifosfamide (6). We are therefore now moving from an undifferenciated therapy for all sarcoma subtypes to specific chemotherapy regimens for distinct sarcoma subtypes. One of the key questions regarding clinical sarcoma research in the next years is whether the knowledge gained in the understanding of the neoplastic transformation of various sarcoma subtype will be useful in routine clinical practice to select appropriate targets for the treatment of these patients. This has already been the case for tumors of the Ewing family, in which molecular diagnosis leads to specific therapeutic protocol. This has also been the case for the targeting of KIT and PDGFR in GIST and few other sarcoma subtypes (9-11). The purpose of this chapter is to discuss whether the recent identification of molecular alterations in specific sarcoma subtypes could lead to specific targeted treatment in these subtypes. Two histological subtypes, synovial sarcomas and liposarcomas, will be presented here and analyzed for possible new therapeutic strategies based on recent knowledge on the specific bioloy to these subtypes.

3

SYNOVIAL SARCOMA

Synovial sarcomas represent 5 to 10% of all sarcoma subtypes, and is the fourth most common type of sarcoma. Synovial sarcoma occur at all ages but predominantly in adolescent and young adults, between 15 and 40 years mostly with a slight male predominance (12-14). Typically, this tumor occurs in the lower (60%) or upper limbs (20%), in paraarticular areas of the tendon sheaths and joints, but can be encountered in regions with no relation to synovial structure, such as the head and neck (8%), thoracic and abdominal wall (8%) or even lungs and heart. Despite its name, there is no evidence that tumor cells come from synovial tissues and actually, some authors have suggested to rename them carcinosarcomas of the soft part (12). Synovial sarcomas comprises actually different histological subtypes: the biphasic type (Figure 1A), with distinct spindle cell and epithelioid cell components, the monophasic fibrous type, with no detectable epithelial cell component, and the rare monophasic epithelial subtype. Calcifying and poorly differentiated synovial sarcomas are also distinguished. Synovial sarcomas are grade 2 or 3 tumors according to the FNCLCC grading (1214). Local treatment follows the general rules of STS treatment with wide excision macroscopically and microscopically complete excisions followed by adjuvant radiotherapy chemotherapy , with or without adjuvant chemotherapy. Reccurence occur in 30 to 70% of patients in

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most series, mostly in the lung, lymph nodes and bones. 5 year survival rates range between 50 and 80% are reported in most series (12-14).

3.1

Specific translocations of synovial sarcoma

Specific t(X, 18)(p11.2;q11.2) translocations have been reported to be associated with synovial sarcomas. These translocations represent a hallmark of the tumor and is probably now a gold standard for the establishment of a diagnosis of synovial sarcoma in 2003 (12,15,16). It has for instance been observed in 100% and 96% of biphasic and monophasic synovial sarcomas in a series of 221 cases using RT PCR (17). This translocation involves the SYT gene on chromosome 18p11, and 3 of the 6 members of the SSX gene family on chromosome Xq11, namely SSX1 or SSX2 genes, and less frequently the SSX4 gene (16,17). These translocations can be detected using RT PCR or FISH techniques, on fresh or paraffin embedded tissues or even on FNA product with a high level of specificity and sensitivity. The nature of the SSX partner in the t(X, 18) translocation has been found to be correlated with distinct clinical presentation (smaller size for SSX2, female gender for SSX2) The biphasic subtype may more frequent harbor the translocation with the SSX1 partner, although this has not consistently been found (20). Synovial sarcomas with translocations involving SSX2 have also been

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reported to be associated with an improved outcome in particular in patients with localized disease (18-21). Other genetic changes can be detected in synovial sarcomas, including complex losses and gains of chemosomes, some of which (gains of 8p) being correlated with poor risk factors. Important genes reported to be altered in structure or expression levels in synovial sarcomas include the RB, p53 and p21 genes, overexpression of the antiapoptotic bcl-2 protein, overexpression of HER2/neu and the MET tyrosine kinase in the epithelial cell component compartment, a phenomenon which may contribute to epithelial mesenchymal transition with is a hallmark of these tumors (16). It is interesting to note in this sarcoma with an epithelial component that E-cadherin expression is frequently lost while translocation of the catenin in the nucleus is also frequently observed (16). These alterations are not consistently found in all tumors and are felt to represent late genetic alterations correlated with tumor progression. It is also likely that some of these phenotypic or genetic characteristics observed in synovial sarcomas are controled by the protein product of the t(X, 18) translocation. Indeed, the product of the genes rearrangements, SYT-SSX1 and SYT-SSX2, are transcription factors which also activates a discrete set of genes (16).

3.2 expression

Protein products of fusion genes regulate gene

The identification of specific translocations associated with sarcoma subtypes has enabled to improve the understanding of the deregulation of proliferation and apoptotic pathways in specific sarcomas subtypes. Usually, the translocations observed in soft tissue sarcomas fuse a gene with a wide pattern of expression to a gene encoding for a protein with a more restricted pattern of expression, leading to the creation of a new transcriptional activator with abnormal transcription function. For instance, it has been shown that the EWS-Fli1 fusion gene product induces the transcription of specific set of genes in the Ewing family of tumors, including PDGF C, myc, and MFNG, a soluble factor of the Fringe family with transforming properties (22). Protein product of fusion genes are therefore capable to stimulate the transcription of genes, which may play an important role in tumor proliferation. Similarly, the t(X,18) translocations results in a gene encoding for a protein in which the addition of the C terminal domain of ssx to the syt protein redirects the syt activation motif to different new genes, presumably with important transforming properties. The SYT gene is expressed in all embryonic and adult tissue, while the SSX genes have a much more discrete pattern of expression, in the testis and thyroid, and actually are members of the Cancer Testis Antigen family. The SYT-SSX translocation products contain both activation (in the syt part of the fusion protein) and repression motifs (in the ssx part) for the transcription of target genes. The gene product is detectable in the nucleus and inteferes with the SNF2/Brahma (brm) protein, a phenomenon which may be

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essential for transformation in synovial sarcoma cells (16). Specific target genes for the syt-ssx fusion protein have however not been precisely identified. The SYT-SSX gene product appears to act as a transcriptional inhibitor for some genes, in particular DCC, a tumor suppressor gene involved in cell adhesion. There is no direct evidence however that it triggers EGFR transcription. The specific pattern of gene expressed in synovial sarcoma has actually been investigated recently using expression microarray strategy.

3.3 of sarcomas

Gene expression patterns across histological subtypes

Four recent studies have investigated gene expression patterns in synovial sarcomas subtypes using microarrays (23-25). A summary of genes found overexpressed in these four papers is presented in Table 1 Nielsen et al (23) used a 42K gene array and identified a specific set of genes associated with the histological subtype of synovial sarcoma. Reference RNA was isolated from a pool of 11 cell lines. 13 genes were found overexpressed in this specific subtypes including the relevant SSX3 and SSX4 genes, previously identified in translocation variants of synovial sarcomas, cytokines BMP 2 and BMP7), and molecules involved in signal transduction such as and EGFR. The mechanisms through which these genes are overexpressed specifically in synovial sarcomas remain unclear. Other members of the EGFR family of genes may be overexpressed in synovial sarcomas: the HER2/neu gene was found to be overexpressed in a series of 37 synovial sarcoma tumor samples tested with expression microarrays containing 6548 sequence-verified human cDNAs in the study reported by Allander et al (24). In this study reference RNA was obtained from an osteosarcoma cell line. In the paper by Nagayama et al (25), using a 23K micrarray, 26 genes were found specifically overexpressed in synovial sarcomas, not including EGFR. Of note in this report, gene expression pattern of synovial sarcomas was found to be close to that of MPNST, including a large number of neural crest specific genes such as ephrins, N-myc or neurofilaments. In a fourth recent paper by Lee et al (25), 48 genes, for most of them different to those described in the three previously mentionned works, enabled to distinguish synovial sarcomas from other sarcoma subtypes. In these 4 studies, overexpressed gene were found in the cytokine and cytokine receptor genes, gene involved in transduction signal and regultation of expression, adhesion molecules, matrix components and cytoskeleton. Actually, only few genes were found overexpressed in synovial sarcomas in more than one study: these were the SSX4 gene (possibly because of its homology with the overexpressed SYT-SSX), the CRABP-1, PRAME- a melanoma Ag, IGF2, and a neuronspecific protein (23-26) (Table 1). The differences in the gene expression pattern of synovial sarcoma in these different works (single cell line, a panel of different tumor cell lines…) may be related to various technical issues,

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including the nature of reference cDNA used, the microarrays used in these works. It is however likely that the gene described in these studies contribute to tumor progression in synovial sarcoma. Whether they are

3.4

EGFR protein expression in synovial sarcomas

EGFR is actually the first member of a large family of growth factor receptor tyrosine kinases that share a common structure composed of an extracellular ligand binding domain, a short transmembrane domain, and an intracellular domain that has tyrosine kinase activity. Binding of the cognate ligand, for example, EGF or transforming growth factor to the extracellular domain of EGFR initiates a signal transduction cascade that can influence many aspects of tumor cell biology including growth, survival, metastasis, and angiogenesis, as well as tumor cell sensitivity to chemo- and radio-therapeutic drugs. Tyrosine phosphorylation provides docking sites on the EGFR for recruitment of proteins that are either direct substrates for EGFR-mediated phosphorylation, or adaptor proteins that link the receptor to a cascade of “downstream” biochemical reactions, for example the ras-raf-MAP-fos pathway, which drives tumor cell proliferation (27,28). Whether EGFR is indeed present at the surface of synovial sarcoma cells needed anyway confirmation using immunohistochemistry To our knowledge, there is no reported study investigating the expression of EGFR in fresh tumor samples of synovial sarcomas. In our hands, all 15 molecularly confirmed synovial sarcomas tested were found to have detectable expression of EGFR at the cell surface. Expression levels however vary considerably between each tumors, but also between the primary tumor and their metastasis. Figure 1 presents the expression of EGF using the Dako Ab in a patients with translocation positive biphasic synovial sarcoma. There is no demonstration to our knowledge that EGFR is expressed in its phosphorylated form in synovial sarcomas. However, the presence of this protein suggest that it may be an interesting target for a specific tyrosine kinase inhibitor in this tumor. We selected the HER1 protein to evaluate microarray-oriented targeted therapy in synovial sarcomas because 1) this gene is a well known protoncogene for a wide range of tumors, and therefore may contribute to transformation process and progression of synovial sarcomas. 2) EGFR tyrosine kinase inhibitors were available in clinical practice.

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Table 1 : Examples of enes preferentially expressed in synovial sarcomas as compared to other sarcomas in four different studies

Function

Ref

Receptor tyrosine kinase Cytokine Cytokine Cytokine Cytokine Ephrin A4 Ephrin B1 Ephrin B3 Fibroblast growth factor Fibroblast growth factor FGF receptor Receptor GDNF receptor Receptor TK for EGF Receptor TK Cytokine IGF binding protein Cytokine Ligand inhibitor

24 23 26 23 25 25 26 25 26 25 24 25 26 23 24 24,26 24 23 25

Transport of retinoic acid Transcription facor Transcription factor Nuclear receptor co-activator Transcription regulator Nuclear receptor Nuclear receptor RNA binding protein RNA binding protein Transcription factor Nuclear protein

23,24,25 26 25 26 24 26 23 23 23,26 24 24

CDK10 CNL1 MAPK10 NFAT4 PDK2 PTK7 PTPN2

Cdc2-related kinase GTP binding protein MAP kinase Nuclear factor Kinase Tyrosine kinase Nuclear Phosphatase

24 24 26 24 24 24 24

Matrix proteins COLIXa3 COLXVIII A1 THSB3

Collagen IX Collagen XVIII Thrombospondin 3

25 24 24

Family Cytokine and receptors Axl BMP-2 BMP-4 BMP-7 Endothelin 3 EPHA4 EPHB1 EPHB3 FGF9 FGF18 FGFF3 Frizzled homolog 10 GFRA1 HER1/EGFR HER2 IGF2 IGFBP2 TGFB2 WNT inhibitory factor

Transcription factors and related CRABP-1 MYB N-MYC NCOA3 PGK1

SSX3 SSX4 SOX9 TLE2 Transduction signal machinery

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Table 1 : Genes preferentially expressed in synovial sarcomas as compared to other sarcomas in four different studies (cont’d)

Adhesion molecules CDH1 PCAD GYPB ICAM1/CD54 ITGAM SELL

E Cadherin P Cadherin Glycophorin B Ig gene superfamily Integrin L selectin

26 25 26 26 26 26

Cytosketon ACTA2 ARPC1B BAT8 CTNNA Ectodermal-neural cortex 1 MSN Neurofilament heavy polypeptide, Neuron specific proteins Hs 79404 Spectrin

Alpha actin Actin polym. regulator Actin associated protein Alpha catenin Actin associated protein Moesin Actin associated protein

24 24 24 24 23 24 25 23,25 26

Other neuroectodermic specific proteins CNTNAP l Contactin associated prot. CST3 Cystatin 3 Dachshund Neural development Neural developemet DRPLA DYT1 Dystonia Granin-like neuroendocrin peptide IRX5 homeobox protein 5 MSX2 homeobox cont’ gene OLFM1 Olfactomedin FRAME Melanoma Ag Serin protease inhibitor Kunitz type, 2 Serin protease inhibitor Serin protease inhibitor SERPIN B6

3.5

24 24 23 24 26 25 23 23 24 24,25 25 24

Different types of targets for targeted therapy of

cancer Until recently, targeted therapy of solid tumors could be classified according in three different categories, according the the type of target: 1) “Early targets” displaying activating mutation(s) which play a causal role in the oncogenic process, acting presumably at an early step on oncogenesis; 2) “Late Targets” whose mutations occurs occuring at a later step of tumor progression; 3) “Uncertain targets” which are expressed in tumor cells, but whose role in the oncogenic process is uncertain in the pathogenesis of the tumor.

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1) The first example of “early target” is the efficacy of imatinib for the treatment of GIST As mentioned in a different chapter, GIST cells almost consistently contain activating mutations of the KIT gene. These mutations play an early and essential role in the oncogenesis of this tumor. This observation led investigators to investigate a specific inhibitor of the kit gene for the treatment of this tumor. The high response rate to a KIT tyrosine kinase inhibitor in 3 consecutive studies (9,10), has provided the first demonstration of the relevance of specific molecular approaches in solid tumors. 2) Treatment of breast carcinoma with Herceptin is an example of a “late target”. Only 15-25% of breast cancer are susceptible to this treatment which gives a relatively low response rate as single agent, but prolongs progression free and overall survival in combination with chemotherapy. 3) Regarding “uncertain targets”, PDGFR, an other target of imatinib, is almost consistently expressed at the surface of sarcoma cells, but its exact role is unknown in most sarcomas. The treatment of non GIST sarcomas with imatinib, with the hope of targeting this receptor whose role in the oncogenic process is uncertain, gives an example of the third type of target. In this situation, only occasional signs of antitumor activity were reported (29).

3.6

Targeting EGFR pathway in synovial sarcomas?

In view of the different tumor models studied so far in clinical practice, targeted therapy is therefore much more efficient when the target plays an early role in the oncogenic process. The observation of a consistent overexpression of the EGFR gene in synovial sarcoma in the study by Nielsen et al suggest that this protein may play an important role in the process of progression, possibly through an overexpression process driven by the product of the fusion gene. In this situation , however, the target is presumably a secondary event, whose over expression is the consequence of the initial genetic alteration caused by the translocation. To test this hypothesis, the Soft Tissue and Bone Sarcoma Group of EORTC is currently investigating the antitumor activity of a HER1 tyrosine kinase inhibitor, ZD1839, in synovialosarcoma overexpressing the HER1 receptor.

3.6.1

ZD1839 in human cancer

ZD1839 is a signal transduction inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase, and has been developed as an oral anti-tumor agent (30). ZD1839 has been found capable to delay growth and, at higher doses, cause regression in a wide range of other tumor xenografts. NSCLC express detectable levels of EGFR in 60-80% of the cases using immunohistochemistry, and, although the exact oncological role of EGFR in NSCLC remains unclear, ZD1839 was investigated in this model (31,32). In NSCLC clinical trials, clinically significant objective responses have been seen for both the 250 mg dose

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and 500 mg dose, in patients pretreated with platinum compounds and/or taxanes. Objective responses were seen in 10% of patients with large or bulky tumor and generally occurred by Day 28, although responses continued to occur for up to 4 months. Clinically significant improvements in disease-related symptom rates were also seen at both doses. Rate of disease-related symptom improvement was substantial with slightly more than 40% of the patients experiencing improvement for at least one month. The median time to symptom improvement occurred within 8 to 10 days. Approximately 80% of patients who had symptom improvement were still showing an improvement at the time of data cutoff. In addition to the efficacy findings, quality of life (QoL) improvement was seen in a significant proportion of patients and was consistent with disease-related symptom improvement, which reflects the lack of significant therapy-related toxicity observed. Tumor response and stable disease were associated with disease-related symptom improvement in the majority of patients; while association of better survival with objective response, disease control and disease-related symptom control was seen.

3.6.2

ZD1839 in EGFR+ synovial sarcomas

EGFR is one of the most overexpressed gene in synovial sarcomas in the study by Nielsen et al, and its protein product is detectable at the surface of most synovial sarcomas tested. Although the exact role of EGFR in the transformation process of synovial sarcomas, and the link between its expression and the specific genetic alterations associated with SS are unclear, EGFR may be a secondary target playing a role in tumor progression. The EORTC STBSG has initiated a clinical trial investigating ZD1839 for the treatment of synovial sarcomas expressing detectable EGFR at their cell surface. Translational research with investigation of the phosphorylation status of EGFR, as weel as gene expression profiles in SS prior and after ZD1839 treatment in scheduled. The trial is ongoing. This is to our knowledge the first example of conception of a targeted therapy trial based on results of expression microarrays demonstrating the presence of the target.

4

LIPOSARCOMA

The second example of such strategy is liposarcoma. Liposarcoma is one of the most common histological subtype of malignant soft tissue tumor occuring during adult life. 10 to 18% of malignant soft tissue tumors are liposarcomas, and this tumor may occur at virtually all ages (33). Its includes a wide variety of different histological subtypes with considerable heterogeneity regarding histological grade, molecular abnormalities, and clinical behavior. Liposarcoma may be classified in 3 major histological subtypes:

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1) The first subgroup comprises lipoma-like, well differentiated (WDLPS) and dedifferentiated liposarcomas (DDLPS), which represent more than one half of all LPS (Figure 2). These occur preferentially in the limbs, and in the retroperitoneum, less frequently in other parts of the body. 6 to 30% of these tumors exhibit a dedifferenciated phenotype, which increases the risk of metastatic relapse. In contrast, WDLPS are associated maily with a risk of local relapse. The risk of relapse and strongly correlates to the topography of the tumor. While limb LPS are most frequently efficiently treated, retroperitoneal LPS tend to reccur almost constantly when followed-up is sufficiently long, with a resulting risk of death which is considerably increased as compared to limb locations. Of note the number of liposarcomas belonging to this category may be higher than previously thought.

2) Myxoid and round cell liposarcoma, although they were given separated designation in the WHO classification of soft tissue tumors, share a similar set of molecular alterations and are distinguished essentially by the percentage of small round cell within the tumor mass. M/RLPS have a much more agressive behavior, with an increased risk of metastasis as compared to the remaining subtypes. In a large series of patients treated in a single institution and including 95 patients, the risk of metastasis ranged from 23% to 58% according to the percentage of round cell population in the tumor. Metastasis sites are somewhat different that

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those reported in other liposarcomas and soft tissue sarcomas: M/RLPS tend to relapse with metastasis in the soft part, in the bone and the lung. 3) Pleomorphic liposarcoma represent the third group of liposarcoma, including 10 to 15% of all liposarcomas. This is a less frequent tumor, less well characterized. These tumors may occur both in the limbs and retroperitoneum, later in life, and are associated with a high risk of relapse and death.

4.1 sarcomas

Heterogeneity of molecular alterations in synovial

The two largest subgroups of liposarcoma are characterized by very different genetic alterations. M/RLPS exhibit a typic reciprocal translocation t(12,16)(q13,p11) resulting in the fusion of the CHOP gene on chromosome 12, with the TLS gene on chromosome 16 (33,34). Less frequently, alternative translocations t(12, 22) with a different partner for CHOP are observed. CHOP is a DNA binding protein , while TLS/FUS is an RNA binding protein capable to interact with several nuclear receptors for thyroid , steroids and retinoid receptors, a combination reminding the t(11,22) translocation associated with Ewing sarcomas. These translocations yield different transcripts, in particular a type II transcript capable to inhibit the adipocytic maturation of preadipocytes when transfered into adipocytes and to promote a transformed pheotype in these cells (35). In contrast, well differentiated and dedifferentiated liposarcomas exhibit typical genetic alterations characterized by giant marker and ring chromosomes. These giant markers or ring chromosomes consistently contain amplified portion of the 12q13-q15 portion of chromosome 12. This region contains a number of gene playing a potential role in the oncogenesis of these tumors in particular the mdm2 gene, CDK4, GLI1, HMGI-C, SAS…).The amplification of this region may be useful to distinguish benign adipose tissue from lipoma like, well differentiated liposarcoma

4.2 liposarcoma

The PPAR gamma pathway in adipogenesis and

Interestingly, despite a distinct set of genetic abnormalities, all liposarcoma subtypes still express an important nuclear receptor involved in adipocyte differentiation. The nuclear receptor peroxysome proliferator activated receptor gamma is an essential signaling molecule for the process of adipocyte differentiation. This nuclear receptor binds several natural or synthetic ligands, such as prostaglandins, and the thiazolidindione family of antidiabetic drugs including troglitazone, rosiglitazone. It interacts with to form a DNA binding complex which is capable to promote

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adipocyte differentiation (36,37). Indeed, this complex triggers the transcription of specific adipocytic gene, and its transfection into fibroblasts is capable to stimulate the ectopic expression of adipocytic genes and to promote the acquisition of an adipocytic phenotype (37). The combination of agonists of the and pathways exert a synergistic activity for adipocytic differentiation.

4.3 Targeting PPAR-gamma pathway in liposarcoma to force differentiation has been reported to be expressed in each of the major histological subtypes of liposarcoma. It is a physiological component of the molecular apparatus for adipocytic differentiation. Synthetic ligands of this receptor are capable to promote the terminal differentiation of liposarcoma cell lines, with an intracellular accumulation of lipids, and cell cycle arrest (39). A synergy with agonist was observed in vitro. This observation strongly suggested that these ligands could enable to promote the differentiation of liposarcoma cell in vivo, in the clinical setting (36). This induction of differentiation associated with cell cycle arrest could be of therapeutic benefit, since 1) WDLPS have a much slower progression rate and indolent course than DDLPS, 2) tumor stabilization in soft tissue sarcoma has similar impact on overall survival than partial response (39). The objective is therefore not to block a kinase will possible growth promoting properties, but to force the tumor cell towards their physiological differentiation pathway, on which they are blocked because of neoplastic transformation. In vivo, Demetri and colleagues (39) fist investigated the capacity of a agonist, troglitazone, to modulate the differentiation liposarcoma cells in vivo in 3 patients. Troglitazone was found capable to induce LPS diffenciation in vivo, as evaluated by histological reassessment showing an important intracellular lipid accumulation,, NMR analysis of triglyceride content in the tumor, reduction of the Ki67 positivity, induction of the expression of fat specific genes, such as aP2 and adipsin (39). On MRI imaging of the tumor, troglitazone induced subtle changes in tumor signal, compatible with intratumoral lipid accumulation. Still no responses were observed, but tumors exhibited a less aggressive phenotype with a slow growth or stabilization, consistent with the effects observed in tumor cells in vitro. Another trials on 49 patients from the same group was reported in an abstract form and confirmed these biological observations (40). Median progression free survival for WDLPS, DDLPS, MRLPS and pleomorphic liposarcoma were 408, 81, 92 and 89 weeks respectively. There results are encouraging and further investigation of this family of compounds in liposarcoma is therefore mandatory.

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5 CONCLUSIONS: IDENTIFY CAUSAL MOLECULAR ALTERATION IN SARCOMAS The observation that imatinib can induce a dramatic response and growth arrest rate in GIST associated with KIT mutation provided the first demonstation of the efficacy of a targeted therapy directed towards the initial molecular event causing tumor transformation, soon followed by the demonstration of its efficacy in dermatofiborsarcoma protuberans with a t(17,22) translocation yielding high levels of autocrine PDGF production (41). However, only a limited number of solid tumors have such a characteristic simple and “targetable” activating molecular alteration,and many are sarcomas. Indeed, specific molecular alterations associated with specific sarcoma subtypes have been characterized in a growing number of histological subtypes of sarcomas in the last years: these include specific reciprocal translocation resulting in an oncogene with translation activating properties, or more complex massive losses and gains of chromosomes in other histological subtypes. It is therefore essential to identify the target genes of the abnormal transcriptional activators encoded by most protein products of sarcoma translocations. Among these, it is necessary to identify which genes play an essential role in the transformation process of these sarcomas. With the identification of these genes, specific treatment directed towards their protein products may be proposed In the present paper, an example of this strategy is presented, with the clinical trial of the EGFR1 inhibitor ZD1839 in synovial sarcoma. Although the direct link between the malignant transformation and EGFR overexpression has not been formally demonstrated, this trial will enable to establish whether this inhibitor exerts significant growth inhibitory properties in this tumor. Presumably, it will be necessary to combine different inhibitors in such strategy. Other potential targets may be the ras family of proteins, using farnesyl transferase inhibitors in particular when this pathway is constitutionally activated through NF1 mutation. The inhibition of cdk4 in liposarcomas harbouring the 12p1315 amplification is another potential target. Another stragy is to try to circumvent the differentiation blockade observed in most cancer cells by triggering pharmacologically the physiological pathways of differenciation when they remain intact in the tumor cell. The example of agonist in liposarcomas provided here is the first piece of evidence showing that this strategy is relevant in vivo in cancer patients. Although no responses were observed, growth arrest could be feasible using this strategy. The identification and investigation of new and more active activators in liposarcoma remains a relevant strategy for the control of these tumors.

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22. De Alava E., Gerald W. Molecular diagnosis of the Ewing family of tumors. J Clin Oncol 2000;18:204-213. 23. Nielsen TO, West RB, Linn SC, et al. Molecular characterisation of soft tissue tumours: a gene expression study. Lancet 002;359: 1301-7 24. Allander SV, Illei PB, Chen Y, et al. Expression profiling of synovial sarcoma by cDNA microarrays: association of ERBB2, IGFBP2, and ELF3 with epithelial differentiation. Am J Pathol. 2002;161:1587-95. 25. Nagayama S, Katagiri T, Tsunoda T, et al. Genome-wide analysis of gene expression in synovial sarcomas using a cDNA microarray. Cancer Res. 2002; 62: 5859-66. 26. Lee YF, John M, Edwards S, et al. Molecular classification of synovial sarcomas, leiomyosarcomas and malignant fibrous histiocytomas by gene expression profiling. Br J Cancer. 2003; 88: 510-5. 27. Schlessinger J. Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell 2002; 110: 669-72 28. Olayioye MA, Neve RM, Lane HA, Hynes NE The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J 2000; 19:3159-67. 29. Judson I, Verweij J, Van Oosterom A et al. Imatimib (Imatinib) an active agent for gastrointestinal stromal tumors but not for other soft tissue sarcoma subtypes not characterized for KIT and PDGR-R expression, results of EORTC phase II studies Proc Am Soc Clin Onc 2002, 21 (abstract 1609). 30. Culy CR, Faulds D. Gefitinib. Drugs 2002; 62:2237-50 31. Bunn PA Jr, Franklin W. Epidermal growth factor receptor expression, signal pathway, and inhibitors in non-small cell lung cancer. Semin Oncol 2002; 29(5 Suppl 14):38-44 32. Herbst RS, Kies MS. ZD1839 (Iressa) in non-small cell lung cancer. Oncologist 2002;7 Suppl 4:9-15 33. Weiss SW, Goldblum JR. “Liposarcoma” in Enzinger and Weiss’s Soft Tissue Tumors 4th. edition, Sharon Weiss, John R. Goldblum eds. St-Louis, MO: Mosby, 2001. 34. Knight JC, Renwick PJ, Cin PD, Van den Berghe H, Fletcher CD.Translocation t(12;16)(q13;p11) in myxoid liposarcoma and round cell liposarcoma: molecular and cytogenetic analysis. Cancer Res 1995; 55:24-7 35. Kuroda M, Ishida T, Takanashi M, et al. Oncogenic transformation and inhibition of adipocytic conversion of preadipocytes by TLS/FUS-CHOP type II chimeric protein. Am J Pathol. 1997; 151: 735-44 36. Tontonoz P, Singer S, Forman BM, et al. Terminal differentiation of human liposarcoma cells induced by ligands for peroxisome proliferator-activated receptor gamma and the retinoid X receptor. Proc Natl Acad Sci U S A. 1997 Jan 7;94(1):237-41. 37. Tontonoz, P., Hu, E., Graves, R. A., Budavari, A. I. & Spiegelman, B. M. mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer. Genes Dev. 1994; 8, 12241234 38. Van Glabbeke M, Verweij J, Judson I, Nielsen OS; EORTC Soft Tissue and Bone Sarcoma Group. Progression-free rate as the principal end-point for phase II trials in softtissue sarcomas. Eur J Cancer. 2002; 38:543-9. 39. Demetri GD, Fletcher CD, Mueller E, et al. Induction of solid tumor differentiation by the peroxisome proliferator-activated receptor-gamma ligand troglitazone in patients with liposarcoma. Proc Natl Acad Sci USA. 1999; 96:3951-6 40. Demetri GD, Speigelman B, Fletcher CD, et al. Differenciation of liposarcomas in patients treated with the PPAR-g ligand troglitazone: documentation of biological activity in myxoid round cell and pleomorphic subtypes. Proc Am Soc Clin Oncol. 1999; 18: 535 a (abstract 2064). 41. Heinrich MC, Corless CL, Duensing A, et al. PDGFRA activating mutations in gastrointestinal stromal tumors. Science. 2003; 299:708-10.

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Chapter 10 Angiogenesis: a potential target for therapy of soft tissue sarcomas K. Hoekman and H.M. Pinedo

Department of Medical Oncology, Free University Medical Center, De Boelelaan 1117, 1007 MB Amsterdam.

Correspondence to : K. Hoekman, MD, PhD Department of Medical Oncology, Vrije Universiteit Medical Centre, De Boelelaan 1117, 1007 MB Amsterdam, the Netherlands. Telephone : 020 4444319 Fax : 020 4444355 E-mail : [email protected]

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1

INTRODUCTION

Tumors depend on neo-vascularisation (angiogenesis) during development (Folkman, 2002). The use of curative therapy in the treatment of soft tissue sarcomas (STS) is generally only possible in the initial stage of the disease, where surgery plus or minus adjuvant radiotherapy is used, and in a minority of cases where metastasectomy is possible. In advanced metastatic disease, treatment with the chemotherapeutic agents, adriamycine and ifosfamide, is associated with low response rates (20-35% when given as single therapy and up to 45% when given in combination) and with short progression free intervals. This means that there is a high need for additional chemotherapeutic compounds or effective alternatives. Tumor vascularisation might be a new target for anti-tumor therapy. The questions to be asked are therefore : what evidence is there that STSs are dependent on angiogenesis, and which factors are involved in STS-associated angiogenesis? which agents are available for anti-angiogenesis therapy, and what is the optimal strategy for using these compounds in patients with STS?

2

ANGIOGENESIS

Angiogenesis, the proliferation, migration and tube formation of endothelial cells, is a process regulated by many stimulating and inhibiting factors. In the tumor situation these factors are produced or generated by tumor cells and by activated stromal cells, such as fibroblasts and immune cells. The balance between pro and contra angiogenic factors determines the presence and activity of the angiogenesis process. While these factors may be found in the circulation, it is the local presence in tumor tissue that is of major relevance. Different families of proteins are involved in angiogenesis which can be classified into three main groups. The first group comprises several stimulating growth factors (vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietins, e.g., and cytokines (interleukin-6, e.g.) and several inhibiting factors such as thrombospondin, IL-12 and fragments of collagen and circulating proteins. VEGF, for instance, is an outstanding angiogenesis factor that is produced by all sorts of cells, with a unique characteristic, namely, the targeting of cancer endothelial cells. This growth factor induces the proliferation and migration of endothelial cells, and, at the same time, it is a survival factor and a permeability inducing factor for those cells (Ferrara, 2002). The second group comprises proteolytic enzymes belonging to the family of metalloproteases (MMPs) or plasminogen activators (uPA and tPA). These proteases are responsible for the degradation of the basal membranes and proteins of the extracellular matrix (ECM) during the migration of endothelial cells. The third group comprises adhesion molecules, e.g. integrins and cadherins. These molecules are a critical factor in the communication between cells, and in the communication between cells and constituents of the ECM, and determine largely the outcome of endothelial

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cell migration and tubular assembly. All these different regulators of angiogenesis are possible targets for anti-angiogenesis therapy. There are various reasons why tumor cells produce these angiogenic factors or induce the receptors for these factors on endothelial cells. Genetic abnormalities found in oncogenes, such as ras and c-myc, and suppressor genes, such as p53, are associated with on the one hand an ongoing production of pro-angiogenic proteins and on the other with an ongoing inhibition of the production of inhibitory factors. A mutated p53, for example, is associated with overexpression of VEGF and bFGF-binding protein, activation of and downregulation of thrombospondin-1, all of which producing a pro-angiogenic effect (Broxterman et al., 2003). In addition, hypoxia and stress are responsible for a conditional, physiologic angiogenic response. The ongoing, genetically determined production of angiogenic factors is responsible for tumors being “wounds that never heal”, while stress conditions further stimulate the angiogenic impulse. In addition, activated stromal cells contribute substantially to this physiologic angiogenic response in tumor tissues. Hypoxia is a common finding in tumor tissues because the proliferation of tumor cells, in general, outgrows the vascular supply. In the final stage of angiogenesis, the nascent vessel needs support from smooth muscle cells (pericytes) for further stabilisation. The recruitment of these cells is regulated by other growth factors like PDGF, angiopoietin-1 and A final characteristic of tumor-associated neovascularisation is the chaotic, immature aspect. This is caused by the unbalanced and ubiquitous presence of multiple angiogenic factors, and the chaotic gradients of angiogenic factors laid down by the variety of cells involved in the process of tumor-associated angiogenesis. While tumor cells generally are present as groups or bundles, stromal cells are found to be more dispersed in tumor tissue. The above is more even more evident in the periphery of tumors and might explain the chaotic appearance of tumor neovasculature locally. Tumor-associated endothelial cells differ from quiescent endothelial cells in several aspects: they have a higher rate of proliferation, they have a different phenotype under the influence of the mixture of angiogenic factors and tumor stress conditions, and they are less mature, often missing the support of pericytes. This means that the turnover of such vessels is high and that the tumor endothelial cells are more vulnerable to therapeutic interventions. In summary, the target of angiogenic therapy may be not only the factors involved in the process of angiogenesis (anti-angiogenesis) but also the tumor endothelial cells themselves (anti-vascular therapy). The implication of the above is that any therapy which reduces the tumor cell burden will also reduce the tumoral production of angiogenic factors, and any therapy which is toxic for endothelial cells will be anti-angiogenic, as well.

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3 THE ROLE OF ANGIOGENESIS IN THE BIOLOGY OF SARCOMAS Microvessel density (MVD) is often found to be a prognostic factor in the response to therapy and survival of cancer patients, although numerous articles on this topic are ambiguous in their conclusions. This may be due to technical problems regarding the way in which the tumor fields of interest are selected and/or the manner in which vessels are counted. In four independent studies examining MVD in soft tissue sarcomas (Yudoh et al., 2001; Kawauchi, 1999;Tomlinson et al., 1999; Saenz et al., 1998), MVD was not associated with histological type, grading, metastatic behavior or survival, but MVD was correlated with survival in a fifth study (Comandone et al., 2003). Tomlinson et al, describe a different pattern of angiogenesis in STS versus breast carcinoma. In breast cancer the capillaries were clustered in bursts within the stroma of the tumor, while the sarcoma capillaries were homogeneously distributed in the tumor stroma. They credit this difference to the greater number of activated fibroblasts in carcinomas, with their own gradients of angiogenic factors in the tissues. This aspect has been studied in carcinosarcoma, which contains both tumor types. In accordance with the findings of Tomlinson a study by Yoshida et al. describes a significant higher MVD in the carcinoma areas of the tumor than in the sarcoma parts (Yoshida et al., 2000). However, the consequences of the above for antiangiogenesis therapy of STS are not clear. Further evidence pertaining to the relevance of angiogenesis in STS biology comes from different studies. An animal study (Mori et al., 1999), shows that transfection of fibrosarcoma cells with VEGF resulted in massive angiogenesis around the tumor and in increased metastatic behavior, which findings confirm the importance of VEGF to the initiation of angiogenesis, but also to the most detrimental aspect of cancer, namely, the metastatic potential. The in vitro production of VEGF by different STS cell lines is, in general, abundant (Hu et al., 2000; own observations), which is in line with the high VEGF production also found in the conditioned medium of many carcinoma cell lines. The involvement of bFGF in the biology of STS was indicated in a fibrosarcoma development model. The authors showed that the ability to release bFGF was critical for malignant characteristics and was associated with the induction of neovascularization (Kandel, 1991). There have also been studies of the levels of angiogenic factors in the blood of STS patients. Serum VEGF was shown to be elevated in patients with STS in multiple studies (Graeven et al., 1999; Linder, 1998; Rutkowski, 2002;Heits, 1997), and correlated significantly with tumor stage and grading. Also plasma MMP-9 activity was shown to be enhanced in STS patients (Pallotta et al., 2000). A remarkable finding in the serum of STS patients was the elevation of endostatin, the COOH-terminal proteolytic fragment of the basement membrane component collagen XVIII and a potent angiogenesis inhibitor (Feldman, 2001). The findings of the study associated an increased serum endostatin level with an increased risk of tumor recurrence after resection. Investigation of STS tissue showed that the concentration of VEGF in STS tissue was enhanced and positively correlated with grading (Yudoh et

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al., 2001; Chao, 2001). STS tissue levels of VEGF appeared to be a prognostic factor in the study of Yudoh. VEGF overexpression might be expected in STS due to the fact that it is inter alia regulated by the p53/MDM-2 pathway, which shows aberrations in the majority of STSs (Zietz et al., 1998). The introduction of wild type p53 in sarcoma cells containing mutant p53 significantly reduced the expression of VEGF (Zhang, 2000), and induced angiogenesis-restricted dormancy in a mouse fibrosarcoma model (Holmgren, 1998). Overexpression of VEGF might also be associated with hypoxia in STS and consequently with regulated stimulation of VEGF expression (Nordsmark et al., 2001). Overexpression of the protease uPA, an essential enzyme for the migration of cells, was also associated with increasing grade, local recurrence and metastases in STS (Choong et al., 1996). Thymidine phosphorylase, another angiogenesis factor which is often coexpressed in tumor tissue, was overexpressed in uterine leiomyosarcomas and correlated with increased MVD (Hata, 1997). Expression of the receptors for angiogenic factors has been described. Angiosarcoma cells and Kaposi cells are large producers of VEGF and have VEGF receptors. It has been demonstrated that in these tumor types VEGF is active as an autocrine growth factor (Fujimoto et al., 1998; Hashimoto et al., 1995; Masood, 1997). The same suggestion has been advanced concerning bFGF in some leiomyosarcomas (Tamiya, 1998). The AIDS-induced Kaposi sarcoma (KS) is a tumor of vascular origin. KS cells produce and respond to VEGF and bFGF. The trans-activator HIV protein Tat activates VEGF and bFGF and the VEGF receptor-2. In addition, Tat and bFGF synergistically activate MT-MMP-1 and TIMP-1 and consequently induce the secretion of MMP-2 (Toschi et al., 2001). High serum FGF-2 levels were associated with a higher risk of death in infected patients (Ascher et al., 2001). A more or less unique finding in STSs is the presence of large cysts in advanced tumors. These cysts may develop due to hypoxia and, consequently, necrosis in the center of the tumor. Indeed, STSs harbour areas with very low oxygen pressures (Brizel et al., 1996; Nordsmark et al., 2001). Another or additional explanation may be sought in the presence of extreme local concentrations of VEGF-induced partly by hypoxia- which is a powerful permeability factor for endothelial cells (Ferrara, 2002). The cystic fluid may be regarded as the in vivo conditioned medium of the tumor, and is therefore greatly informative for the investigation of tumor biology. The VEGF concentration in intratumoral fluid collections was found to be extremely high in our own STS studies, many times higher than in the plasma of the same patients even reaching levels 450 times higher than found in normal plasma (Verheul, 2000). A possible explanation for this finding may be the fact that platelets contain substantial amounts of VEGF (Verheul et al., 1997), whereas activated endothelial cells promote the adhesion and activation of platelets (Verheul, 2000), which may result in a strongly enhanced local release of VEGF. The concentration of basic FGF was not enhanced in these STS fluids, except in a patient with a gastro-intestinal stromal tumor (GIST). The activity of uPA and MMP-9 was also enhanced in STS cyst fluid, confirming the role of these proteases in tumor-induced angiogenesis. Finally, we found high concentrations of a number of key

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coagulation factors in the fluid. Together with evidence of intratumoral platelet activation, the above findings confirm the hypothesis of a putative role of platelets and coagulation in tumor angiogenesis (Pinedo, 1998). In the cystic fluid of a patient with GANT we found, in addition to high VEGF, enhanced levels of endostatin, tPA and plasmin (Hansma et al., 2003). The demonstration of the generation of endostatin inside a tumor was a unique finding; together with the activation of tPA and consequent generation of plasmin in the fluid it is a likely mechanism of action of endostatin (Reijerkerk et al., 2003).

4 ANTI-ANGIOGENIC THERAPEUTIC OPTIONS FOR SOFT TISSUE SARCOMAS Preclinical research has brought to light many clues about antiangiogenic therapy of patients with STS. Antibodies directed at VEGF, or VEGF-receptor tyrosine kinase inhibitors (SU5416) decreased the growth of human sarcoma explants in immunocompromised rodents (Angelov et al., 1999; Wang et al., 1998). This growth inhibition was attributed to having the effect on tumor angiogenesis of leading to a reduction of tumor cell proliferation and increased apoptosis. Complete inhibition and regression of a rhabomyosarcoma explant could only be induced by blocking both tumor (human) and host (animal) VEGF (Gerber, 2000). Histological analysis of residual tumor tissue revealed an almost complete absence of host-derived vasculature and massive tumor necrosis. Combination of an antibody to the VEGF type 2 receptor with continuous low dose doxorubicin resulted in an enhanced growth inhibition of 2 different sarcoma xenografts associated with an increased apoptosis of endothelial cells in the tumors (Zhang, 2002). Together, these studies confirm the importance of VEGF in the initiation of sarcoma-induced angiogenesis and further encourage anti-VEGF based trials in STS patients. An internal 4 kringle containing fragment of plasminogen (angiostatin) and the 5 kringle fragment of plasminogen, the latter induced via the uPA-plasmin pathway, are both potent inhibitors of angiogenesis. Both compounds inhibited the growth of a murine fibrosarcoma in mice (Cao, 1998). Stable gene transfer of angiostatin cDNA in Kaposi sarcoma cells delayed tumor growth in nude mice, which was associated with reduced vascularization (Indraccolo et al., 2001). Endostatin, the fragment of collagen XVIII, is generated during breakdown of the basal membrane and ECM, and is also a natural angiogenesis inhibitor. Gene therapy with endostatin, which ensures a sustained endostatin overproduction, significantly inhibited tumor formation in the lung after intravenous injection of fibrosarcoma cells (Nakashima et al., 2003). Intermittent treatment of fibrosarcoma explants in mice with endostatin showed regression followed by regrowth of the tumor xenographs, but also showed no recurrence of the tumor after 4 cycles of endostatin treatment, which would suggest that the therapy does not induce acquired drug resistance (Boehm, 1997). These studies warrant a clinical follow-up.

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Anti-vascular therapy is another strategy in downgrading the vascular support of tumors. The tumor-associated endothelium has been described as being the target of metronomic therapy with low dose cyclophosphamide (Man et al., 2002). It proved to be a succesful approach in rats challenged with a sarcoma (Rozados, 2003). Low-dose cyclophosphamide eradicated a high percentage of sarcomas without evident toxicity. In studies with other tumor types, cyclophosphamide was combined with anti-angiogenic agents, which resulted in long-term remissions. This strategy should be further investigated in future sarcoma studies. Another compound is ZD6126, a vascular targeting drug that disrupts the tubulin cytoskeleton of proliferating endothelial cells, resulting in selective destruction and congestion of tumor blood vessels. ZD6126 induced extensive hemorrhagic necrosis in a rat fibrosarcoma model (Robinson et al., 2003). Combination of ZD6126 with cisplatin or radiotherapy enhanced significantly the anti-tumor effect in a mouse model with human sarcoma xenographs (Siemann, 2002). Further investigation of ZD6126, in combination with chemotherapeutic agents with proven efficacy for sarcomas, is warranted. Clinical experience with anti-angiogenesis agents for advanced STS is scarce. The NCI list of ongoing trials in STS records a total of 35 trials; only one of which, however, is an anti-angiogenesis based study, combining thalidomide with in the adjuvant setting. There was also a phase I study of IL-12 on the list, which included a number of STS patients. An important study was performed by Kuenen et al, who treated 31 STS patients with SU5416, which is a tyrosine kinase inhibitor of the VEGF receptors type 1 and 2 (Kuenen et al., 2003). The results include one minor response and 5 stable diseases (less than 25% increase during 3 months of treatment) of limited duration. In one patient a remarkable decrease of fluid collection within the tumor could be observed together with progression of tumor volume, which at least confirmed activity of this agent against the permeability effects of VEGF. Different reasons were given for the rather disappointing outcome. There was uncertainty about the bioavailability of SU5416, the complexity and redundancy of cytokines/growth factors involved in angiogenesis and also the fact that the vascular support of established tumors is mostly performed by mature vessels, which are less dependent on VEGF (Eberhard, 2000). The above suggests that modulation of this very compound, combination with other anti-angiogenesis agents or the use of other drugs which have an effect on established tumor vessels might deliver better results in STS. Tumstatin, a fragment of collagen IV (Hamano et al., 2003), and VEGF-trap, a soluble VEGF decoy receptor (Huang et al., 2003), have recently been described as agents that have the potency to abolish mature tumor vessels, followed by marked tumor regression, including regression of lung metastases. New studies, using these agents, eventually in combination or combined with chemo-therapeutics and/or other biological agents are warranted. There are a number of ways of achieving selective treatment of tumors: by targeting either the tumor cells or tumor-associated endothelial cells, by administering drugs in the tumor itself, or by administering drugs in the feeding arterial system of the tumor. The first-mentioned approach has

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been carried out extensively in animal studies using doxorubicin – a drug used for STS - conjugated with different kinds of peptides, which results in higher uptake in the tumor, less toxicity to the host, and less acquired drug resistance (van Hensbergen, 2002; Mazel et al., 2001). It is still a promising strategy, also for therapy of patients with STS. The latter strategy has been used in isolated limb perfusion using (Lejeune et al., 2000) for patients with melanoma or STS located in the extremities. TNF (plus interferon) induced apoptosis of tumor endothelium by reduced activation of leading to detachment of endothelial cells (Ruegg, 1998), while enhancing the selective uptake of tumor-toxic melphalan in the sarcoma. This therapy induced about 30% complete responses and offered the majority of patients the possibility for limb-sparing resection of the tumor. This experimental therapy affirms the principle that the systematic combination of a chemotherapeutic agent- even one that is not very effective- with an angiogenic intervention can be successful for patients with cancer. The importance of the way a drug is presented to tumor tissue or the way it inhibits functionally a target was underlined by the negative results of a trial with vitaxin, a humanized monoclonal antibody against the integrin (Patel et al., 2001), given intravenously to patients with a leiomyosarcoma. Reduced penetration of cytotoxic drugs in tumor tissue is an important aspect of drug resistance (Jain 2002). In two recent papers, it was shown that tumor drug uptake could be stimulated by low-dose given systematically (ten Hagen et al.; 2000), or by administration of STI571 (Pietras et al., 2002). STI571 (Glivec) is a tyrosine kinase inhibitor of signaling via the BCR-ABL fusion protein, but also via c-Kit and the PDGF receptors and Inhibition of the latter receptors, present on endothelial cell supporting cells, is thought to be responsible for a reduction in tumor interstitial pressure and, consequently, for stimulation of drug uptake. These findings should also be further elaborated in clinical studies. A different type of tumor is the Kaposi sarcoma, being of vascular origin. It was shown that overexpression of multiple angiogenic factors, such as VEGF, bFGF, IL-8, MMP-2 and -9 was present in KS tissue. Thus, it is not surprising that agents such as SU5416 (Arasleh, 2000), the antiangiogenic agent TNP-470 (Arbiser et al., 1999; Dezube et al,. 1998) and the MMP-inhibitor COL-3 (Cianfrocc et al., 2002) were successful in treating this tumor type, with an overall response rate of 44% in respect of the latter drug. In addition, the effect of the commonly used HIV protease inhibitors on Kaposi sarcoma are partly due to the fact that a decrease is brought about in the activity of MMP-2 (Sgadari et al., 2003). In conclusion, STS-associated angiogenesis and STS-vessels as such offer multiple specific avenues for further research and treatment strategies of patients with STS (see also Heymach, 2001 and Scappaticci, 2001). It is questionable if attacking one pivotal factor or phenotypic address of STSassociated vessels will result in substantial anti-STS responses. An important problem in animal studies lies in the difficulty of predicting results of the activity of these types of agents in patients with cancer. This may be explained by the fact that animal models only represent the initiation of angiogenesis in recently installed tumor explants. It will be a challenge to

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improve the design of animal studies and to look for theoretically optimal combinations of biologicals and chemotherapeutic agents for the therapy of patients with STS. In some STS patients the presence of large tumors offers the opportunity to get tumor tissue samples before and after therapy. Investigation of these tissues with microarrays and proteomic techniques will deliver a better understanding of STS biology and the effects of therapeutic interventions on these tumors (Borden et al., 2003); all of which will ultimately lead to better therapies for patients with soft tissue sarcomas.

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INDEX Actinomycin D, 74 Activation loop mutations in, 132 Acute myelogenous luekemia (AML), 142 Acute radiation morbidity, 33 Acute tissue reactions, 66–67 Adipogenesis PPAR gamma pathway in, 163–164 Adipose tumors cytogenetic aberrations in, 102–104 molecular aberrations in, 102–104 ALK activated, 108 ALK receptor tyrosine kinase, 8 Angiogenesis anti, 174–177 defining, 170 Angiopoietins, 170 Angiosarcoma, 173 treatment of, 6 Angiostatin, 174 APC, 106–107 Apoptosis, 92 Basic fibroblast growth factor (bFGF), 170 Beam’s eye view (BEV) of retroperitoneal liposarcoma, 23f Beta-catenin, 106–107 Biological target volume, 37 Blood-oxygen level dependent (BOLD) image sequences, 37 Brachytherapy, 30 postoperative, 36 CD117 antigen, 118 Chemoradiation for extremity/trunk sarcomas, 48–49 for retroperitoneal sarcomas, 49–50 Chemotherapy isolated limb perfusion and, 67 neo-adjuvant, 3–4 postoperative, 47–48, 56t surgical complications in, 56–57 survival in, 53–54 vasculotoxic mechanisms of, 71 Chronic myelogenous leukemia (CML), 142–143 Cisplatin, 48 Clear cell sarcoma cytogenetic/molecular aberrations of, 104–105 Clinical target volume (CTV), 20

Collagen, 170 Computerized tomography, 31f Conformal fields parallel opposed pairs, 31 CT scans, 26 Cyclophosphamide, 48, 175 Cytogenetic aberrations, 100 in adipose tumors, 102–104 Clear cell sarcoma, 104–105 Cytogenetic approach, 101 Cytogenetic mechanisms, 102 Cytokines, 170 Cytotoxic agents, 10 Dacarbazine, 11 Dermatofibrosarcoma protuberans, 106 desmoid tumors and, 123–124 Desmoid tumors, 106–107 dermatofibrosarcoma protuberans and, 123–124 Desmoplastic small round cell tumors, 105 Docetaxel, 6 Dose dispersion, 35 Dose heterogeneity, 35 Dose painting, 25–26 biological targeting and, 36–37 Dose sculpting, 25–26 Dose-volume histograms (DVH), 24 of POP plans, 32f Doxorubicin, 2, 11, 48, 152 dose-response curve of, 3 TNF and, 70 Drug targeting immunohistochemistry for, 95 Drug uptake, 72 Early targets, 159 Edema, 20f EGF expression of, 8 EGFR1-antibody therapy, 8 EGFR pathway targeting, 160 EGFR protein expression of, in synovial sarcomas, 157 ZD1839 and, 161 Elderly patients safety in, 70 Endothelial differentiation, 94 Enes preferential expression of, 158t–159t Epithelial differentiation, 94

Index

182 ET-743 (Trabectedin), 7, 10 Ewing’s sarcoma, 105–106, 152 Exon 9 mutations, 133 Exon 11 mutations, 133 External beam radiotherapy (EBRT), 21–22, 36 Extracellular domain mutations in, 131–132 Extracompartmental sarcoma, 19 Extremity sarcomas chemoradiation for, 48–49 multimodal therapy for, 48–49 preoperative radiation therapy for, 46–47 False-positive trials, 11 Farnesyl transferase inhibitors (FTI), 109 ras protein targeting by, 9 Fibro-histiocytic differentiation, 94 Fibrosarcoma, 107–108 Fluorescence in situ hybridization (FISH), 101 Fusion genes protein products of, 155–156 Gastrointestinal stromal tumors (GIST), 5, 91, 95, 108 diagnosis of, 138 gastric, 136 genotyping of, 140 imatinib treatment of, 121 KIT-driven, 120–123 KIT mutations in, 130, 132–136 molecular classifications of, 141t, 142 neo-adjuvant treatment of, 122 other sarcomas and, 142–143 PDGFRA and, 135 risk assessment for, 92t variants of, 136 Gene expression histological subtypes of sarcomas and, 156–157 in synovial sarcoma, 155 Geometric uncertainty margins for, 20–21 reduction of, 34 Gross tumor volume (GTV), 19, 24f Hematological disorders kinases in, 143t HER1 protein, 157 Histamine, 74 Histologies TNF-based ILP in, 70–71 Histopathological grading, 87–92 FNCLCC system for, 88–90 limitations of, 90–92 NCI system for, 88–90 systems for, 88–90 Histopathological typing, 62–67 HPLC denaturing, 131 Hyperthemia mild, 73 Hypoxia, 73, 171

Idiosyncratic toxicity, 74 Ifosfamide, 2, 11, 48, 152 dose-response curve of, 3 Imatinib clinical efficacy of, 121 dose-escalation of, 137 mesylate, 119f metabolism, 120 oncogene kinase targeting with, 136–137 pharmocokinetics of, 120 phase II studies of, 121 response, 138–141 Immunohistochemistry, 93 for classification, 93–95 for drug targeting, 95 limitations of, 95–96 for monomorphic spindle cell tumors, 94t for undifferentiated round cell tumors, 95t Induction therapy postoperative chemotherapy, 47–48 preoperative radiation therapy, 46–47 rationale for, 44–45 for sarcomas, 45 for solid tumors, 44–45 Integral doses, 35 Intensity modulated radiotherapy (IMRT), 18, 30 dose constraints for, 28t elements of, 24–25 five field, 31f inverse planning, 26–29 mechanisms of, 25 Interferon-gamma, 73 Isodose lines distribution of, 27f Isolated limb perfusion, 66–67 chemotherapy and, 67 with cytostatic drugs, 67t duration of, 73 in histologies, 70–71 TNF-based, 67–69 Juxtamembrane domain mutations of, 130–131 Kaplan-Meier estimates, 11 of progression-free rates, 12f Kaposi sarcoma, 173, 176 Karyorrhexis, 92 Kinase 1 domain mutations in, 132 Kinase mutations imatinib response and, 138–141 use of, 138 Kinases in hematological/mesenchymal disorders, 143t inhibitors, 143 KIT activated, 108 activation loop of, 132

Index extracellular domain of, 131 gain-of-function mutations, 122 germline mutations in, 134t inhibition of, 123 juxtamembrane region of, 130–131 kinase 1 domain of, 132 mutations of, 132–136 oncogenic mutations of, 130 KIT receptor, 118 L-NAME, 74 Laboratory models, 71–74 Late targets, 159 Late tissue adverse events, 29 Leiomyosarcoma (LMS), 5 Leukocytes role of, in TNF-mediated antitumor effects, 72 Liposarcoma, 9–10, 104 classifying, 161–162 dedifferentiated, 162 myxoid, 162–163 occurence of, 161 overall survival in, 5 PPAR gamma pathway in, 163–164 retroperitoneal, 23f round cell, 162 well differentiated, 162 LNA, 74 Local patterns of spread, 19 MAID regimen, 4 Malignant fibrous histiocytoma (MFH), 5, 55f, 83, 152 Malignant peripheral nerve sheath tumor, 109 T2-weighted magnetic resonance image of, 20f Melanocyte differentiation, 94 Melphalan TNF and, 67–70 Mesenchymal disorders kinases in, 143t Mesoblastic nephroma, 107 Metalloproteases, 170 Metastatic disease systemic therapy in, 4–5 Methotrexate, 48 Mitoses, 92 Modalities scheduling, 21 Modulated electron therapy (MERT), 36 Molecular aberrations, 100 in adipose tumors, 102–104 Clear cell sarcoma, 104–105 Molecular diagnostics, 96 Molecular targets, 7–10 Monomorphic spindle cell tumors immunohistochemistry for, 94t Multi-leaf collimator (MLC), 22 Multimodality therapy for extremity/trunk sarcomas, 48–49

183 for retroperitoneal sarcomas, 49–50 Muscle differentiation, 94 Myofibroblastic tumors, 108 Myxofibrosarcoma, 83 Needle biopsy versus open biopsy, 82 Nerve sheath differentiation, 94 Neurofibromatosis type 1 (NF1), 136 NF1 gene, 109 Nitric oxide (NO), 74 NTRK3 activated, 107 Oncogenic kinases in GISTS, 136–137 Oncogenic mutations of KIT, 130 Open biopsy versus needle biopsy, 82 Paraganglioma, 136 Parallel opposed pairs (POP) of conformal fields, 31 Paraspinal tumors, 30 Pathologic responses, 51–52 Paxlitaxel, 6 expression of, 9 Pericytes, 171 Peroxisome proliferator-activated receptor-gamma 9 Phase II studies, 10 of imatinib, 121 Phase III trials, 52–53 Planning target volume (PTV), 20, 24f Plasminogen activators, 170 Plasminogen (angiostatin), 174 Platelet derived growth factor (PDGF) sarcomas driven by, 123–124 Platelet derived growth factor receptor alpha (PDGFRA), 105, 176 activated, 108 as alternative oncogene, 135 D842V, 139–140 Platelet derived growth factor receptor beta (PDGFRB), 100, 176 activated, 106 Pleomorphic rhabdomyosarcoma, 83 Pleomorphic sarcomas, 83 Positron emission tomography (PET), 37 Postoperative therapy functional status and, 51 impact of, 50–52 pathologic response and, 51–52 scope of surgery and, 50 PPAR gamma pathway in liposarcoma, 163–164 Preoperative radiation therapy for extremity/trunk sarcomas, 46–47 for retroperitoneal sarcomas, 47 Progression-free rates, 5, 7, 11

Index

184 Kaplan-Meier estimates of, 12f Proton beam irradiation, 36 Pulmonary chondromas, 136 Quality of life, 51 Radiation doses escalating, 33 Radiation target volume, 27f Radiation therapy surgical complications of, 54–56 survival in, 52–53 Radiotherapy (RT), 18 conformal, 32 curative, 29–30 external beam, 21–22 image fusion in, 34 image-guided, 37 intensity modulated, 18 retreatment, 30–31 spread patterns and, 18–19 targets for, 18–21 three-dimensional conformal, 18 tissues at risk in, 18 Ras pathway, 109 Ras protein FTI targeting, 9 Regional lymphatic pathways of spread, 19 Retinoid acid receptor (RAR), 9 Retroperitoneal sarcoma chemoradiation for, 49–50 multimodality therapy for, 49–50 preoperative radiation therapy for, 47 RT plans for, 31f Rhabdoid tumors, 110 Rhabdomyosarcoma, 110 Signal transduction pathways, 7–10 Single photon emission computed tomography (SPECT), 37 Soft tissue sarcoma (STS) angiogenesis in, 172–174 classification of, 83 diagnosis, 2–3 histological subtypes of, 152–153 identifying causal molecular alteration in, 165 progression-free rates for, 7f therapuetic targets in, 9t Solid tumors induction therapy for, 44 Static disease, 10 SU5416, 175 Surgical complications of radiation therapy, 54–56 Survival, 52–54 Suveillance, Epidemiology, and End Results (SEER), 2 Synovial sarcoma, 110–111, 154f EGFR protein expression in, 157

enes expression in, 158t–159t gene expression in, 155–156 heterogeneity of molecular alterations in, 163 local treatment of, 153–154 occurence of, 153 overall survival in, 5 translocations of, 154–155 Systemic therapy drugs and, 5–7 for metastatic disesase, 4–5 Three-dimensional conformal radiotherapy (3D CRT), 30 elements of, 22 multi-leaf collimator, 22 plan calculation, 22–23 Thrombospondin, 170 Toxicity, 44 Transcription factors targeting, 142 Transforming growth factor 157 TRKC activated, 107 Trunk sarcomas chemoradiation for, 48–49 multimodal therapy for, 48–49 preoperative radiation therapy for, 46–47 Tumor necrosis factor-alpha (TNF), 66 dose ranges for, 73 doxorubicine and, 70 in histologies, 70–71 melphalan and, 67–70 vasculotoxic mechanisms of, 71 Tumor vessels destruction of, 72 Tyrosine kinase inhibitors, 176 Uncertain targets, 159 Undifferentiated round cell tumors immunohistochemistry for, 95t Vascular endothelial growth factor (VEGF), 170 receptor tyrosine kinase inhibitors, 174 serum, 172 Vasculotoxic mechanisms of chemotherapy/TNF, 71 Vasoactive drugs, 74 Vincristine, 48 Volumetric-based planning applying, 33–34 multidisciplinary interactions of, 33 rationale for, 29–33 Volumetric treatment, 21–29 evolution of, 21–22 WHO classification, 83–87 ZD1839, 160–161 EGFR protein and, 161