Advances in Cancer Research Volume 40

ADVANCESINCANCERRESEARCH VOLUME 40 Contributors to This Volume Vladimir N. Anisimov Daniel Meruelo Richard Bach Ca...

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ADVANCESINCANCERRESEARCH VOLUME 40

Contributors to This Volume Vladimir N. Anisimov

Daniel Meruelo

Richard Bach

Cathleen T. Moore

Gosta Gahrton

Dan H. Moore

Peter A. Jones

Dan H. Moore II

H. Kirchner

Arthur D. Riggs Howard E. Skipper

ADVANCES IN CANCERRESEARCH Edited by

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

SIDNEY WEINHOUSE Fels Research Institute Temple University Medical School Philadelphia, Pennsylvania

Volume 40- 1983

ACADEMIC PRESS A Subsidiary of Harcourt Brace Jovanovich, Publishers

New York London Paris San Diego San Francisco Slo Paulo Sydney Tokyo Toronto

COPYRIGHT @ 1983, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECtlANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATlON STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

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United Kingdoit2 Edition published by ACADEMIC PRESS, I N C . (LONDON) LTD. 24/28 Oval Road, London N W l 7DX

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ISBN 0 - 1 2 - 0 0 6 6 4 0 - 8 PRlNTED IN THE UNITED STATES OF AMERICA 83 84 85 86

9 X 7 6 5 4 3 2 1

- 1 3360

CONTENTS CONTRIBUTORS TO VOLUME 40

........................

iX

5.Methylcytosine. Gene Regulation. and Cancer

.

ARTHURD RIGGSAND PETERA . JONES

I. I1. 111. IV. V. VI . VII . VIII. IX . X.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Basic Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence for Gene Regulation by DNA Modification . . . . . . . . . Cause or Effect: Necessary but Not Sufficient . . . . . . . . . . . . Induction of Gene Expression by 5-Azacytidine . . . . . . . . . . . Cancer-A Disease Resulting from Abnormal Differentiation . . . . . 5-Methylcytosine Levels in Tumorigenic Cells . . . . . . . . . . . . Carcinogens and Enzymatic DNA Methylation . . . . . . . . . . . . Oncogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 3 6 10 11 14 15 18 22 23 25

lmmunobiology of Infection with Human Cytomegalovirus H . KIRCHNER I. I1. I11. IV. V. VI VII . VIII . IX. X. XI * XI1 XI11. XIV

.

. .

xv.

XVI. XVII .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Herpesviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytomegaloviruses. HCMV . . . . . . . . . . . . . . . . . . . . . HCMV-Host Cell Interactions . . . . . . . . . . . . . . . . . . . . Epidemiology of the Infection with HCMV . . . . . . . . . . . . . . Diagnosis of HCMV Infection . . . . . . . . . . . . . . . . . . . . Clinical Significance of HCMV Infections . . . . . . . . . . . . . . Oncogenic Potential of HCMV. . . . . . . . . . . . . . . . . . . . Immunopathology . . . . . . . . . . . . . . . . . . . . . . . . . LatencyIReactivation . . . . . . . . . . . . . . . . . . . . . . . . Replication of HCMV in Leukocytes . . . . . . . . . . . . . . . . . Effects of HCMV on Leukocytes . . . . . . . . . . . . . . . . . . . Immunity against Infections with HCMV. General Aspects . . . . . . Humoral Immune Responses . . . . . . . . . . . . . . . . . . . . Cell-Mediated Immunity . . . . . . . . . . . . . . . . . . . . . . Nonspecific Defense Mechanisms against HCMV Infection . . . . . . Vaccine and Therapy Problems . . . . . . . . . . . . . . . . . . . V

32 35 37 39 41 45 47 55 61 63 64 67 69 72 76 83 91

vi

CONTENTS

XVIII . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

93 96

Genetics of Resistance lo Virus-Induced Leukemias D.ANIEL \fERC.ELO

ANU

RICHARVBACH

107 I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . The Initial Link between I‘iruses and Leukemias . . . . . . . . . . . 108 1x1 . Characteristics of the Retrovirus Family . . . . . . . . . . . . . . . 109 IV . Expression in Inbred Mouse Strains of Antigens Associated with MULV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 v . Genetics of Susceptibility to Viral Infection . . . . . . . . . . . . . 138 VI . Prospects for Control of Human Leukemia . . . . . . . . . . . . . . 173 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

Breast Carcinoma Etiological Factors DAN

I. I1 . 111. I\’.

v.

\‘I . \TI . VIII . IX . X. XI . XI1 . XI11 . XIV.

H . X~OORE.

DAN

H . ~ I O O R E11.

i\NU CAFHLEEN

Introduction . . . . . . . . . . . . . . . . . Heritage . . . . . . . . . . . . . . . . . . . Menses. Marital State. Parih . . . . . . . . . Breast-Feeding . . . . . . . . . . . . . . . . Contraceptives . . . . . . . . . . . . . . . . Benign Epithelial Diseases of the Breast . . . Hormonal Factors . . . . . . . . . . . . . . . Cancer . . . . . . . . . . . . . . . . . . . . Iatrogenic Factors . . . . . . . . . . . . . . Immunological Factors . . . . . . . . . . . . Viral Aspects of Human Breast Cancer . . . . Dietan. Factors . . . . . . . . . . . . . . . . Psychosomatic Factors . . . . . . . . . . . . Discussion and Concluding Remarks . . . . . References . . . . . . . . . . . . . . . . . .

T . MOORE

189 . . . . . . . . . . . 191 . . . . . . . . . . . 197 . . . . . . . . . . . 204 . . . . . . . . . . . 205 . . . . . . . . . . . . . . . . . . . . . . . 207 . . . . . . . . . . . 208 215 . . . . . . . . . . . 216 . . . . . . . . . . . 219 . . . . . . . . . . . . . . . . . . . . . . . 223 227 . . . . . . . . . . . 236 . . . . . . . . . . . . . . . . . . . . . . . 239 244 . . . . . . . . . . .

Treatment of Acute Leukemia-Advances in Chemotherapy. Immunotherapy. and Bone Marrow Transplantation COSTAGAHRTOH

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 I1. The Strategy for Treating Acute Leukemia . . . . . . . . . . . . . . 256 111. Classification and Prognostic Factors . . . . . . . . . . . . . . . . . 262 IV . Chemotherapy of Acute Leukemia . . . V . Imniunotherapy . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

271 297

vii

CONTENTS

VI . Bone Marrow Transplantation . . . . . . . . . . . . . . . . . . . . VII . Supportive Treatment . . . . . . . . . . . . . . . . . . . . . . . . VIII . Prospects for the Future . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

304 311 312 313

The Forty-Year-Old Mutation Theory of Luria and Delbruck and Its Pertinence to Cancer Chemotherapy HOWARDE . SKIPPER I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. The Somatic Mutation Theory (1943). . . . . . . . . . . . . . . . . 111. The Fluctuation Test of Law Pointing to the Origin of Methotrexate-Resistant Leukemia Cells . . . . . . . . . . . . . . . IV. Wide FIuctuations in the Degree and Duration of Response of Neoplasms to Chemotherapy in Similarly Staged and Treated Individuals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Effective but Noncurative Chemotherapy Consistently Increases the Survival Time Variance of Treatment Failures; Ineffective Chemotherapy Does Not . . . . . . . . . . . . . . . . . . . . . . VI . Idealized Surviving Fraction Curves That Are Compatible with Large Bodies of Experimental and Clinical Data . . . . . . . . . . . . . . VII . The Origin of Doubly and Multidrug-Resistant Neoplastic Cells . . . . VIII . Mathematical Relationships and Models . . . . . . . . . . . . . . . IX . Criteria for Optimum Delivery of Non-Cross-Resistant Combinations ofDrugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . Closing Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

331 333 334 335 338 342 346 348 356 362 362

Carcinogenesis and Aging VLADIMIR N . ANISIMOV I. I1. 111. IV. V. VI . VII . VIII . IX. X.

. . . . . . . 365 Introduction . . . . . . . . . . . . . . . . . . . . . Spontaneous Carcinogenesis and Aging . . . . . . . . . . . . . . . 367 Chemical Carcinogenesis and Aging . . . . . . . . . . . . . . . . . 370 Carcinogenesis Induced by Foreign Bodies and Aging . . . . . . . . 379 Radiation Carcinogenesis and Aging . . . . . . . . . . . . . . . . . 380 Hormonal Carcinogenesis and Aging . . . . . . . . . . . . . . . . . 385 Viral Carcinogenesis and Aging . . . . . . . . . . . . . . . . . . . 388 Mechanisms of Modification of Carcinogenesis by Aging . . . . . . . 390 Factors Modifying Rate of Aging and Carcinogenesis . . . . . . . . . 404 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415 . . . . . . . 415 References . . . . . . . . . . . . . . . . . . . . . .

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF PREVIOUS VOLUMES. . . . . . . . . . . . . . . . . . . . .

.. ..

425 431

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CONTRIBUTORS TO VOLUME 40 Numbers in parentheses indicate the pages on which the authors’ contributions begin.

VLADIMIRN. ANISIMOV,Laboratory of Experimental Tumors, N . N . Petrov Research Znstitute of Oncology, U S S R Ministry of Public Health, Leningrad 188646, U S S R (365) RICHARDBACH,Zroington House Institute, Department of Pathology, New York University Medical Center, New York, New York 10016 ( 107) G ~ S T GAHRTON, A Division of Clinical Hematology and Oncology, Department of Medicine, Huddinge Hospital and Karolinska Znstitute, S-141 86 Huddinge, Sweden (255) PETERA. JONES, Departments of Pediatrics and Biochemistry, Childrens Hospital of Los Angeles, USC School of Medicine, Los Angeles, Calqornia 90027 ( 1 ) H. KIRCHNER,Znstitute of Virus Research, German Cancer Research Center, 6900 Heidelberg, Federal Republic of Germany (31) DANIEL MERUELO,Zroington House Institute, Department of Pathology, New York University Medical Center, New York, New York 10016 (107) CATHLEEN T. MOORE,Department of Humanities and Social Sciences, Philadelphia College of Pharmacy and Science, Philadelphia, Pennsylvania 19102 (189) DAN H. MOORE, Department of Microbiology and Immunology, Hahnemann University Medical College, Philadelphia, Penns ylvania 19102 (189) DANH. MOORE11, Biomedical Sciences Division, University of California Lawrence Livermore Laboratory, Livermore, California 94550 (189) ARTHURD. RIGGS,Division of Biology, City of Hope Research Znstitute, Duarte, California 91010 (1) HOWARD E. SKIPPER,Southern Research Institute, Birmingham, Alabama 35255 (331) ix

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5-METHYLCYTOSINE, GENE REGULATION, AND CANCER Arthur D. Riggs Division of Biology, City of Hope Research Institute, Duarte. California

Peter A. Jones Departments of Pediatrics and Biochemistry. Childrens Hospital of Los Angeles. USC School of Medicine, Los Angeles, California

.................................................... . . . . . . . . . . . . ... . .. . . . . . . . . , . . . . . . ... . . . . . ... . . . . A. A New Information Coding System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Models for Cellular Differentiation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Evidence for Gene Regulation by DNA Modification.. . . . . . . . . . . . . . . . . A. Maintenance Methylases.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Clonal Inheritance of Methylation Patterns , , . , . . . , . . . . . . . . . . . . . , . C. I n Vitro Methylation and in Vivo Gene Activity . , . , . , . . . , . . . . . . . . . D. Hypomethylation and Gene Activity.. . . . . . . . . . . , . . . . . . . . . . . . . . . . IV. Cause or Effect: Necessary but Not Sufficient.. . . . . , . . . . . . , . . . . . . . . . . V. Induction of Gene Expression by 5-Azacytidine . . . . . . . . . . . . . . . . .. A. Fibroblast Cells to Muscle Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Other Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Conclusion: A Significant Gene Silencing Factor . . . . . . . . . . . . . . . . . . VI. Cancer-A Disease Resulting from Abnormal Differentiation . . . . . . . . . . . .MI. 5-Methylcytosine Levels in Tumorigenic Cells . . , . . , . . , . . . , . . . . . , . , . . VIII. Carcinogens and Enzymatic DNA Methylation . . . . . . . . . . . . . . . . . . . . . . . A. Inhibition of DNA Methylation in Vitro by Chemical Carcinogens . . . I. Introduction

11. BasicConcepts..

B. Inhibition of DNA Methylation in Living C IX. Oncogenes.. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . X. Conclusions.. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . A. A Cancer Model.. . . . . . . B. Main Points.. . . . . . . . , . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . XI. Addendum ...................................................... References, , . , . . . . . . . . . . . . . . . . . . . . . . . , . . , . . . . . . . . . . . . . . . . . , . . . . .

1 3 3 4 6 7

7 8 8 10 11 11 13

14 14 15 18 18

23

25 25 25

I. Introduction

The regulation of mammalian gene expression clearly is accomplished by multiple control systems operating at several levels. Some obvious levels of control include chromosome condensation, chromatin structure (e.g., DNase sensitivity), transcriptionaI control by re1 ADVANCES IN CANCER RESEARCH, VOL. 40

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-006640-8

2

ARTHUR D. RIGCS AND PETER A. JONES

pressors and activators, RNA processing, and translational control. Our purpose in this article is to present the argument for a newly recognized additional component of mammalian gene control, enzymatic DNA methylation, and to point out the relevance of this gene control system to cancer. Mammalian DNA is modified shortly after replication by the enzymatic conversion of about 3% of cytosines to 5-methylcytosine. A transmethylase(s) transfers a methyl group from S-adenosylmethionine (SAM) to the 5-carbon in the pyrimidine ring of cytosine (Fig. 1). 5-Methylcytosine is the only naturally occurring modified base yet found in mammalian DNA. Ninety percent or more of 5-methylcytosine is found in the sequence CG, and between 50 and 70% of CG sites are methylated, depending on the species and the tissue. Presently identified DNA methylases show little specificity for sequences flanking the CG site. Razin and Riggs (1980) published a short review presenting the case for DNA modification as an important component of mammalian gene control systems. Since the publication of that review, much additional experimental support has accumulated favoring the involvement of 5-methylcytosine in gene control. There have also been several other recent reviews of the methylation literature (Burdon and Adams, 1980; Drahovsky and Boehm, 1980; Razin and Friedman, 1981; Ehrlich and Wang, 1981; Doerfler, 1981; Felsenfeld and McGhee, 1982). In this article, we will A

I

SAM

SAH

B CHJ

-I -CG GC-

I

5- Methylcytosine

Cytosine

MAINTENANCE

DNA METHYLASE

-CG -GC-

FIG.1. The enzymatic formation of 5methylcytosine. (A) A DNA methylase acts on DNA and converts cytosine to 5methylcytosine. (B) By definition, a maintenance methylase acts on half-methylated sites and converts them to fully methylated sites, with methyl groups symmetrically located in both strands of the DNA duplex. From Riggs and Reilly (1981).

5-METHYLCYTOSINE, GENE

REGULATION, AND CANCER

3

focus primarily on the work of the last 2 years, considering first the general aspects of methylation and gene control, and then the cancerrelated aspects. I t . Basic Concepts

A. A NEW INFORMATION CODINGSYSTEM The notion that 5-methylcytosine may be a significant factor in mammalian gene control rests on an interesting theoretical framework built from established principles of protein-DNA interactions. Many of the recently established facts were predicted by Riggs (1975) and Holliday and Pugh (1975). Experimentally verified predictions now include: (1)methylation affects protein-DNA interactions; (2) methylation is symmetrical in both strands; (3)methylation patterns exist; (4)methylation patterns are tissue-specific; (5) methylation patterns are somatically heritable; and (6)maintenance methylases exist. Most of the literature supporting these statements will not be discussed here. Some of the references are listed in Table I, but the reader is encouraged to read the more comprehensive reviews, and also earlier seminal papers by Scarano (1971). The maintenance methylase concept is important and is illustrated in Fig. 2. A methylation site (CG) can exist in three states-unmethylated, methylated in both strands, and methylated in one strand (hemimethylated). Hemimethylated sites are generated from methylated sites by DNA replication, because only deoxycytidine is incorporated by DNA polymerase. By definition, a maintenance methylase acts on hemimethylated sites, but has little activity on unmethylated sites. It MAINTENANCE METHYLATION

F"3

-CG-CG.. -GC -GC-

-CG-CG-GC

-GC -

DNA REPLICATION

FIG. 2. Maintenance of methylation patterns in DNA. Given a maintenance type methylase, methylated sites stay methylated and unmethylated sites stay unmethylated through DNA replication cycles. From Riggs and Reilly (1981).

4

ARTHUR D. RIGGS AND PETER A. JONES

is apparent from Fig. 2 that two identical base sequences, even on the same DNA molecule, can be maintained in different methylation states by a maintenance methylase system. Thus, a methylation pattern can be maintained through DNA replication. Methyl groups in a CG site are, in effect, semiconservatively replicated, and become a heritable entity. In considering gene control during cellular differentiation, we would like to emphasize three points: (1) differentiation rests on a foundation of specific protein-DNA interactions; (2) DNA modification by the formation of 5-methylcytosine profoundly affects specific protein-DNA interactions; and (3) infomiation encoded in methylation patterns can be stably maintained through repeated DNA replication cycles by the maintenance methylase system. Since maintenance methylases exist (see below), the potential for a new coding system relevant to mammalian gene control during development certainly exists.

B. MODELSFOR CELLULAR DIFFERENTIATION How might the methylation patterns be established during development? What imparts specificity for establishment of patterns? One model for cellular differentiation relevant to the cancer model to be described later is illustrated in Fig. 3. It was proposed (Singer et al., 1979a; Razin and Riggs, 1980) that the ground state in the early embryo is full methylation, where most CG sites that ever will be methylated are methylated by this stage. Present data are most consistent with the ground state being reached in the inner cell mass (the embryo proper) of the late blastocyst at about the time of implantation. Specific demethylation events at critical control sites during development could be directed by sequence-specific proteins, which might just sterically inhibit the relatively nonspecific maintenance methylase(s). Alternatively, one could have demethylases that act in a sequenceand developmentally specific manner. Gjerset and Martin (1982) have recently published the first report indicating the existence of 5methylcytosine demethylase activity in mammalian cells. We think their enzyme is possibly a 5-methylcytosine aglycosidase rather than a true demethylase that removes only the methyl group, but at any rate, this type of specific demethylation during differentiation must be considered. A third possibility for changing methylation patterns is to have some

5-METHYLCYTOSINE, GENE REGULATION, AND CANCER

5

UNIFORMLY M ETHYL ATE D DE NOVO

UNDIFFERENTIATED EMBRYONIC CELLS

DIFFERENTIATED CELL TYPES

/G

/G

GE

Gf

EG

CG

'k

G$

GC

GE

--- ---GC GC

GC

1

''

/G

GE

+- - - - - - -METHYLATION - - - -- - - - - GERM LINE OR

SPECIFIC DEMETHYLATION CG

/G

GC

GE

G!' G$

CG EG ___---___ GC GC

CG GC

MAINTENANCE METHYLASE

1

/G

CG

EG

CG

/G

/G

GE

GC

GE

GC

GE

Gf

CELL TYPE 1

CELL TYPE 2

FIG.3. A demethylation model for the establishment and maintenance of a differentiated state. Inhibition of methylation by sequence-specific proteins during DNA replication leads to demethylation and the establishment of specific methylation patterns (closed circles represent methyl groups) in various cell types. The maintenance methylase system ensures heritability of the methylation pattern. From Razin and Riggs

(1980).

de no00 methylation (methylation of unmethylated sites) occurring at all developmental stages. Recent work suggests that de no00 methylation may be most active in the preimplantation embryo (Stewart et ul., 1982; Jahner et aE., 1982). In more fully differentiated cells, it has been established (see Section 111) that the predominant methylase activity is maintenance type, but at the same time, there is no doubt that some de novo methylation does occur. After gene transfer of viral or cellular genes, integrated copies occasionally become methylated and inactive (Doerfler, 1981). Therefore, it certainly is possible to have sequence-specific de novo methylation events occur during differentiation. DNA sequence-specific and developmental stage-specific maintenance methylase inhibitors, demethylases, and de novo methylases behave mechanistically differently from activator and repressor proteins found in bacteria. We propose that proteins that control development but which need not necessarily have immediate effects on transcriptional activity be called determinator proteins. An important point is that methylation models propose that specific determinator proteins directly or indirectly create a methylation pattern, which

6

ARTHUR D. RlGGS AND PETER A. JONES

then is somatically heritable even in the absence of the determinator proteins because of the maintenance methylase system. The stage can be prepared in advance; genes can be set, but not necessarily activated (or repressed). Central to the methylation models is that, for some genes (but not necessarily for all), methylation of critical sites in control regions prevents high level expression. Methylation locks the gene in an inactive state. The methyl groups must be absent for efficient transcription. In some systems, the removal of the locking methyl groups would be sufficient for expression, but in other systems the removal of methyl groups would be necessary but not sufficient, because of other regulatory factors. Weintraub et al. (1982)have recently discussed the possibility of gene control by multiple independent events.

111. Evidence for Gene Regulation by DNA Modification

Table I outlines the various types of experiments that have yielded important information with respect to 5-methylcytosine and gene control. Most of the earlier experiments have been discussed in earlier TABLE 1 EVIDENCE SUPP~RTING GENEC O ~ OBYL METHYLC CYTOSINE Type of experiment 1. 5-Methylcytosine affects proteinDNA interactions 2. 5-Methylcytosine stabilizes Z-form DNA 3. Methylation is symmetrical in CG 4. Maintenance methylases exist

5. Methylation patterns exist and are tissue-specific 6. Methylation patterns are somatically heritable 7. Gene activity correlates with hypomethylation of control regions 8. I n uitro methylation reduces in oitio gene activity 9. Inhibition of methylation activates

genes

Reference R a i n and Riggs (1980); Fisher and Caruthers (1979); Smith (1979) Behe and Felsenfeld (1981);Singleton et al. (1982) Bird (1978); Cedar et al. (1979) Sneider et al. (1975);Jones and Taylor (1981); Gruenbaum et al. (1982) Waalwijk and Flavell (1978b); see also Table I1 Pollack et al. (1980); Wigler et al. (1981); Stein et al. (1982a) See Table I1 Pollack et al. (1980); Wigler et al. (1981); Stein et aE. (1982b); Vardimon et al. (1982); Fradin et ol. (1982) See Table 111

5-METHYLCYTOSINE, GENE REGULATION, AND

CANCER

7

reviews and are not controversial. Some of the more recent experiments will be summarized here.

A. MAINTENANCEMETHYLASES Several studies have firmly established the existence of a maintenance methylase system in mammals. Sneider et al. (1975) first reported that a methylase from rat Novikoff hepatoma cells had increased activity when DNA from ethionine-treated cells was used as a substrate. The increased methyl accepting ability of DNA from ethionine-treated cells has been confirmed by Christman et a2. (1977) and Cox and Irving (1977). Ethionine inhibits most transmethylation reactions, and hemimethylated sites presumably are present in the DNA from ethionine-treated cells. Confirmation of this interpretation has been obtained recently. Jones and Taylor (1981) used 5-azacytidine to inhibit methylation and produce undermethylated DNA, which was then shown by direct measurement to be hemimethylated and to be a much better substrate for a partially purified mouse spleen DNA methylase. Gruenbaum et al. (1982) prepared a hemimethylated substrate by in vitro DNA replication with 5-methyldeoxycytidine triphosphate substituted for dCTP. This hemimethylated DNA was at least a 200-fold better substrate for Ehrlich ascites DNA methylase than the control DNA made with dCTP. There is now no doubt that the predominant methylase in mammalian cells has properties of a maintenance methylase, suggesting that methylated sites should be somatically heritable.

B. CLONAL INHERITANCE OF METHYLATION PATTERNS DNA-mediated gene transfer experiments have provided direct experimental support for somatic inheritance of methylation patterns (Pollack et al., 1980; Wigler et al., 1981). As an example, the recent experiments of Stein et al. (1982a) will be described. 4x174 doublestranded DNA is normally unmethylated and remains so after introduction into mouse cells by DNA-mediated gene transfer. However, when 4x174 DNA is methylated in vitro at its CCGG sites (by HpaII methylase), about 30% of the methylated CCGG sites are lost initially on transfer, but the residual methylation pattern is faithfully copied through at least 100 cell generations. It was concluded that “the inheritance of the cellular DNA methylation pattern is based on a CG-

8

ARTHUR D. RiCCS AND PETER A. JONES

specific methylase that operates on newly replicated hemimethylated DNA.”

C. In Vitro METHYLATIONAND in Vivo GENEACTIVITY Enzymatic methylation of CCGG sites in vitro (by HpaII methylase) reduces the efficiency of DNA-mediated gene transfer of selected markers such as thymidine kinase ( t k ) (Pollack et al., 1980; Wigler et aE., 1981) and adenosine phosphoribosyltransferase (aprt) (Stein et al., 1982b). These results probably reflect an inhibition of in vivo transcription from methylated control regions, because the recipient cells found to express the transferred marker were also found to have lost methylation at CCGG sites in the 5’-flanking regions. Introduction of a single pair of methyl groups (N-methyladenosine) per 5OOO base pairs in the promoter of Herpes thymidine kinase can abolish in uiuo expression after microinjection into mouse cells (Waechter and Baserga, 1982). In uitro methylation of CCGG sites by HpaII methylase reduces expression of genes after injection into frog oocytes. Vardimon et al. (1982) found that a methylated DNA fragment carrying the gene for the adenovirus DNA-binding protein was transcriptionally inactive after injection. There are three CCGG sites in the vicinity of this gene’s promoter, but it is not yet clear which sites are critical. A similar result has been reported by Fradin et al. (1982). There is a single HpaII site (CCGG) in the genome of SV40 and it is located in the control region of the late genes. Methylation of this site greatly reduces transcription from the late promoter after oocyte injection, but has no effect on transcription from the early promoter.

D. HYPOMETHYLATION AND GENEACTIVITY The correlation between undermethylation of the promoter or control regions of genes and their high-level expression has grown very strong, as evidenced by the list in Table 11. There are about 40 publications indicating that CCGG sites in nonexpressing tissues or cell cultures are more highly methylated than in expressing tissue. The correlation is excellent for the 5‘-flanking region and, in most cases, the coding region and even introns are also less methylated when the gene is being expressed.

5-METHYLCYTOSINE, GENE REGULATION, AND CANCER

9

TABLE I1 HYPOMETHYLATION AND GENEEXPRESSION Genes Nonviral Globin genes: chicken, rabbit, human

Ovalbumin, ovotransferin, and ovmucoid Metallothionein a-Fetoprotein ViteIIogenin, chicken J-chain gene Immunoglobulin H chains Growth hormone and chorionic somatomammotropin Ribosomal RNA Cytochrome P-450 Delta-crystalline Hepatic protein 22 Adenosine phosphoribosyltransferase Thymidine kinase, chicken Type I collagen Albumin Viral Thymidine kinase, HSV Herpes simplex virus Avian endogenous retroviruses Adenoviruses Epstein-Barr virus Herpesvirus saimiri Avian sarcoma virus Mouse mammary tumor virus Moloney murine sarcoma virus Moloney murine leukemia virus

AKR endogenous leukemia virus

References McGhee and Cinder (1979); Van der Ploeg and Flavell (1980); Shen and Maniatis (1980); Weintraub et al. (1981); Sanders-Haigh et al. (1982) Mandel and Chambon (1979) Compere and Palmiter (1981) Andrews et al. (1982) Wilks et at. (1982) Yagi and Koshland (1981) Rogers and Wall (1981); Dackowski and Morrison (1981) Hjelle et al. (1982) Tantravahi et al. (1981); Kunnath and Locker (1982); Reilly et al. (198213) Chen et al. (1982) Jones, R. E. et al. (1981) Nakhasi et al. (1981) Stein et al. (1983) Wigler et al. (1981) Parker et al. (1982); McKeon et al. (1982) Ott et al. (1982) Pollack et al. (1980); Wigler et al. (1981); Christy and Scangos (1982) Youssoufian et al. (1982) Groudine et al. (1981) Sutter and Doerfler (1980); Vardimon et al. (1982); Kruczek and Doerfler (1982) Kintner and Sugden (1981) Desrosiers et al. (1979) Guntaka et al. (1980) Cohen (1980); Breznik and Cohen (1982); Fanning et al. (1982) Gattoni et al. (1982) Stuhlman et al. (1981); Stewart et al. (1982); Jahner et al. (1982); Van der Putten et al. (1982); Hoffman e t al. (1982) Hofhan et al. (1982)

10

ARTHUR D. FUGGS AND PETER A. JONES

IV. Cause or Effect: Necessary but Not Sufficient

A question often raised is whether the undermethylation seen in actively transcribed genes is just a trivial consequence of the real controlling events. It is also pointed out that the correlation between undermethylation and activity is not perfect. For example, no change in methylation was seen in a type 1collagen gene between expressing and nonexpressing tissue (McKeon et al., 1982). In SV40-transformed cells, which do not express type 1 collagen, the gene does become methylated (Parker et al., 1982).Sheffery et al. (1982) have observed no change in globin gene methylation after induced differentiation of erythroleukemia cells. In response to these questions and experimental results, we wish to make three important points. First, most experiments correlating methylation and transcriptional activity have used the HpaIIIMspI restriction enzyme assay. This useful assay is based on the observation that MspI cleaves CCGG sites methylated at the internal C, whereas HpaII does not (Waalwijk and Flavell, 1978a; Singer et al., 1979b; Cedar et al., 1979). However, methylation of only CCGG sites is measured by this assay, and such sites represent only about one-sixteenth of the total methylated sites. Changes in critical sites could easily be missed. Sano and Sager (1982) have reported that tissue-specific differences in bovine satellite DNA are not very apparent at CCGG sites, but such differences are readily seen at TCGA sites. In this special case, it was possible to determine methylation patterns by direct DNA sequencing (Ohmori et al., 1978); the HpaIIIMspI assay would have missed the major changes. Second, the correlation between undermethylation and gene activity is strongest for the 5’-flanking regions, which contain the control sequences. In fact, even some of the apparent exceptions, where coding regions and introns remain methylated during transcription (e.g., collagen, vitellogenin, and aprt), support the correlation between transcription and hypomethylation of critical sites. Each of these systems has undermethylated 5’-flanking regions. The aprt system is particularly interesting, since aprt is expressed constitutively in all tissues, and the S’-flanking control region is unmethylated even though it contains a statistically significant high number of CG sites (Stein et al., 1983).In vitro methylation of the aprt gene greatly decreases its efficiency in gene co-transfer experiments (Stein et al., 1982b). In spite of the deficiencies in the assays, the correlation between undermethylation of control regions is very striking. Among the more than 30 systems analyzed in detail, all show a good correlation in the

5-METHYLCYTOSINE7 GENE

REGULATION, AND CANCER

11

5' or 5'-flanking regions. With present data and thinking, the surprising result is the strength of the correlation for the entire transcriptional domain. It couId be that the methylation of the coding regions and introns is relatively unimportant in comparison to methylation of the control regions, and there may be two levels of control operating independently. Methylation of the control region could be the primary switching mechanism; methylation of the coding region and introns could provide only fine tuning. Third, it has never been proposed that the absence of methylation in the coding region or even in the control region of a mammalian gene would necessarily result in transcriptional activity. In fact, some of the very first experiments on the globin genes (Van der Ploeg and Flavell, 1980) clearly indicated that the absence of methylation was not sufficient for expression. If methylation is primarily a locking mechanism as proposed by Razin and Riggs (1980), the necessary but not sufficient idea is easily understood. An unZocked door is not necessarily open. However, the 5-azacytidine experiments discussed next and listed in Table 111 indicate that in a surprisingly high percentage of systems, the inhibition of methylation seems sufficient to activate genes. V. induction of Gene Expression by 5Azacytidine

A. FIBROBLAST CELLSTO MUSCLECELLS Constantinides et al. (1977) and Taylor and Jones (1979) reported that the base analog, 5-azacytidine7 could change gene expression in mouse fibroblasts. Their basic observations are illustrated in Fig. 4. Strain 10T1/2 fibroblasts were treated for 24 hr with 5-azacytidine and then, after the analog was washed out, the cells were allowed to grow about 10 generations to confluency. Foci of differentiated cells (muscle cells, adipocytes, and chondrocytes) were found in the cultures. Striated muscle fibers were most common and muscle cells are never seen in untreated 10T112 cultures. It is apparent that 5-azacytidine switches on a new developmental pathway. Moreover, once the switch in programming occurs, it can be clonally propagated (Taylor and Jones, 1979). A somatically heritable change has been made, even though 5-azacytidine is not significantly mutagenic in this system (Landolph and Jones, 1982). 5-Azacytosine differs from cytosine only in that a nitrogen is substituted for the 5-carbon in the pyrimidine ring. This might inhibit 5-

12

ARTHUR D. RIGGS AND PETER A. JONES

TABLE I11 INDUCTION OF GENEEWRESSION BY ~AZACYTIDINE System

Species

References

Striated muscle cell formation

Mouse

Adipocyte and chondrocyte formation Muscle, adipocyte, and chondrocyte X chromosome reactivation

Mouse

Constantinides et al. (1977, 1978); Mondal and Heidelberger (1980) Taylor and Jones (1979)

Chinese hamster

Sager and Kovac (1982)

Human and mouse

Mohandas et al. (1981); Jones et al. (1982); Graves (1982); Lester et al. (1982) Compere and Palmiter (1981)

Metallothionein-1 inducibility Emetine resistance Herpes simpler thymidine kinase Cellular thymidine kinase Macrophage differentiation Friend cell differentiation Promyelocytic leukemia (HLW Fetal hemoglobin Melanocyte differentiation T lymphoma differentiation Prolactin Rous sarcoma virus Chicken endogenous virus expression Epstein-Barr virus Mouse endogenous type C Virus Mouse endogenous viral proteins Moloney murine leukemia Virus

Mouse Chinese hamster Mouse

Baboon Human Mouse Mouse Rat Hamster Chicken

Worton et ol. (1983) Clough et ol. (1982); Christy and Scangos (1982) Harris (1982) Boyd and Schrader (1982) Creusot et al. (1982) Bodner et ol. (1981); Christman et al. (1983) DeSimone et al. (1982) Ley et al. (1982) Silagi and Graf (1981) MacLeod et al. (1983) Ivarie and Morris (1982) Altanerova (1972) Groudine et al. (1981)

Human Mouse

Ben-Sasson and Klein (1981) Niwa and Sugahara (1981)

Mouse

Tennant et al. (1982)

Mouse

Hoffman et al. (1982); McGeady et al. (1982)

Chinese hamster Mouse Mouse Human

methylcytosine formation. DNA methylation is, indeed, inhibited after S-azacytidine incorporation into DNA (Jones and Taylor, 1980). Even low levels of incorporation of 5-azacytosine into DNA reduce DNA methylase activity in cell extracts for 1or 2 days after treatment (Tanaka et al. 1980; Creusot et al., 1982; Taylor and Jones, 1982). The current model consistent with present data is that DNA containing 5-

5-METHYLCYTOSINE, GENE REGULATION, AND CANCER

13

/ MUSCLE CELLS

lOT$ Mouse Fibroblosts

- --2PM AZO-C

Remove AZO-C

___)

24 hr

CHONDROCYTES

10 Doys

Many Cell Divisions

Foci of differentiated cells

FIG.4. Multiple new phenotypes induced by treatment with 5-azacytidine. See text and Taylor and Jones (1979).From Riggs and Reilly (1981).

azacytidine in place of C functions as an irreversible inhibitor of the maintenance methylase (Friedman, 1981).

B. OTHERSYSTEMS Gene switching by 5-azacytidine is not a rare phenomenon, and there are now at least 26 reports of 5-azacytidine treatment activating previously silent genes (Table 111). We will discuss a few of these systems. The first is the reactivation of genes for hprt, pgk, and g 6 p d on an inactive X chromosome, a system notable for its stability. In more than 20 years of numerous experiments, reversion of X-linked genes to activity was extremely rare, even where strong selection pressure was applied (in the case of h p r t ) (Kahan and DeMars, 1975). However, Mohandas et al. (1981), Jones et al. (1982), and Lester et al. (1982) have discovered that treatment of mouse-human hybrids with 5-azacytidine results in up to 1% of surviving cells expressing the hprt from the previously inactive human X chromosome. Moreover, about 10% of cells expressing human h p r t also expressed p g k or g 6 p d . After treatment with 5-azacytidine, the hprt gene on the human X chromosome functions much more efficiently in gene transfer experiments (Venolia et al., 1982; Lester et al., 1982), confirming that the reactivation is caused by a change at the level of the DNA, presumably undermethylation. The methylation state of the hprt gene has not yet been ascertained, but total 5-methylcytosine levels are significantly reduced by the 5-azacytidine treatment (Jones et al., 1982). Another remarkable observation has been published by Harris (1982). He studied a Chinese hamster cell line that had been selected (by BrdU) for a very stable thymidine kinase negative ( t k - ) phenotype, reverting to tk' with a frequency of only about However,

14

ARTHUR D. RIGGS AND PETER A. JONES

after treatment with 5-azacytidine, up to 10% of the cell population expressed the previously silent gene. Other methylation inhibitors, ethionine and 5-deaza-adenosine, also activated this t k gene. This result and those of Worton et aZ. (1983)show that heritable epigenetic changes (methylation) can masquerade as mutations. Ivarie and Morris (1982)found that 5-azacytidine efficiently reactivates a rat prolactin gene in a tumor cell line where the prolactin gene had been silenced by treatment with ethylmethanesulfonate. Also potentially relevant to cancer is that 5-azacytidine efficiently activates some silent endogenous retrovirus genes in chicken and rodent cells (Table 111).In the chicken system of Groudine et d . (1981), the endogenous virus ev-1 had been detected by hybridization experiments, but expression had never been observed. 5-Azacytidine treatment resulted in easily detectable transcription of the viral sequences. In this system, and also for the metallothionein gene (Compere and Palmiter, 1981), the activated genes were demonstrated to be hypomethylated.

C. CONCLUSION: A SIGNIFICANT GENESILENCING FACTOR When these 5-azacytidine experiments and the others listed in Ta-

ble I11 are considered along with the evidence coming from other lines as discussed above and outlined in Table I, there seems little remaining doubt that, in mammalian cells, 5-methylcytosine functions as part of a gene silencing system. In some cases, removal of the methyl groups is sufficient to give large increases in the transcriptional activity. We now turn to the question: is this gene silencing system relevant to cancer? VI. Cancer-A

Disease Resulting from Abnormal Differentiation

There is an emerging consensus that many human cancers are caused by the abnormal regulation of developmentally important genes that are always present in the genome (Comings, 1973).Cancer may result when these developmental stage-specific genes produce an active product in a cell type in which there would normally be little active product. The concept that cancer may be regarded as a disease of differentiation (Markert, 1968; Comings, 1973) is supported by the fact that many transformed cells retain the potential to respond to differentiation signals, and are capable of undergoing differentiation to end-stage nondividing cells (Pierce, 1974) and participating in normal development (Gootwine et al., 1982). An obvious example is pro-

5-METHYLCYTOSINE, GENE REGULATION, AND CANCER

15

vided by teratocarcinomas , which can be induced to differentiate and participate in normal development by being placed in an early embryo (Mintz and Illmensee, 1975; Papaioannou et al., 1975). Even after years in tissue culture, cancer cells can revert to normal. This dramatic result establishes without doubt that some cancers are the result of somatically heritable derangements in gene control. It should be emphasized that teratocarcinomas are just one example of tumor cells in which differentiation can be induced. The previous sections of this article have strongly suggested that DNA methylation plays a fundamental role in governing gene expression during normal cell differentiation. Therefore, it is certainly possible that derangements in DNA methylation patterns are responsible for the aberrant gene expression seen in cancer. This general idea is not new. Most investigators studying enzymatic DNA methylation have been aware of it and many have discussed it (Srinivasan and Borek, 1964; Gantt, 1974; Drahovsky and Wacker, 1975; Rorhanov and Vanyushin, 1980; Christman et al. 1977; Holliday, 1979; Lapeyre and Becker, 1979). In this article we will try to update the concepts and incorporate the recently published data on methylation with the rapidly progressing data and thoughts on gene regulation and cellular oncogenes. The following sections will review the evidence that methylation levels are altered in certain animal cancers, and that many carcinogenic agents can interfere with DNA methylation in uitro and in living cells. VII. 5-Methylcytosine Levels in Tumorigenic Cells

The evidence that DNA methylation controls cellular gene expression has prompted several investigators to determine overall levels of cytosine methylation in transformed cell systems (Table IV). Most of the studies reported in Table IV have measured the level of modified cytosine by chromatographic methods or by the use of restriction enzymes. Earlier studies (Silber et d., 1966), in which radioactive methionine was used as the methyl donor, have not been included because there are many potential artifacts that can influence the data obtained in such experiments. Thus, changes in intracellular S-adenosylmethionine pools or variations in amino acid transport, as responses to toxic damage, may markedly influence the determinations. Primary hepatocarcinomas and transplantable mouse liver tumors contain decreased levels of 5-methylcytosine relative to normal liver (Lapeyre and Becker, 1979; Lapeyre et al., 1981). The decreases in 5methylcytosine found in the hepatocarcinomas were not caused by

16

ARTHUR D. RIGGS AND PETER A. JONES

TABLE IV METHYLATIONLEVELS IN TUMORS AND TUMORIGENIC CELLLINES System Primary hepatocarcinomas and transplantable hepatocarcinomas Cattle leukemia Friend erythroleukemia Human leukemia HeLa cells Human fibrosarcoma cells Diverse range of human tumor lines HeLa cells and four lymphoblastoid lines Hamster fibrosarcoma transformed in oitro Chemically transformed mouse cells Mouse mammary tumor virusinduced neoplasms Polyoma-transformed B HK21 SV40-transformed human fibroblasts Adenovirus-transformed hamster cells Naturally occurring tumors

Methylation change

Reference

Decreased

Lapeyre and Becker (1979); Lapeyre et al. (1981)

Decreased Decreased Increased Decreased Decreased Decreased in 19 out of 20 Decreased

Romanov and Vanyushin (1981) Smith et al. (1982) Federov et al. (1977) Diala and Hoffman (1982b) Wilson and Jones (1983b) Diala et al. (1982)

Decreased

Wilson and Jones (1983b)

No change Variable Decreased Decreased

Diala and Hoffman (1982a) Wilson and Jones (1983a) Reilly et al. (1982a) Cohen (1980)

Increased No change

Rubery and Newton (1973) Diala et al. (1981)

Increased

Gunthert et al. (1976)

Decreased in 4 out of 5

Feinberg and Vogelstein (1983a)

Ehrlich et al. (1982)

deficiencies in DNA methylase activity within the tumors. Importantly, the results were duplicated with three different carcinogens, including acetylaminofluorene, chlordane, and 3-methyl-4-dimethylaminobenzene. This work (in which the 5-methylcytosine decreases were of the order of 15%) is particularly significant because it represents the only data on chemically induced tumors in uiuo. The 5methylcytosine content of cattle lympholeukemia has also been reported to be decreased relative to normal DNA (Romanov and Vanyushin, 1981); however, this is at variance with earlier reports suggesting that methylation levels in leukemia may in fact be increased (Silber et aZ., 1966; Federov et al., 1977). Several lines of cultured human tumor cells have been examined for

5-METHYLCYTOSINE7 GENE

REGULATION, AND CANCER

17

total DNA methylation and found to have decreased levels relative to human fibroblast DNA (Diala and Hoffman, 1982b; Ehrlich et at., 1982; Wilson and Jones, 1983b). Therefore, there is substantial evidence that the majority of human tumor celI lines contain lowered levels of DNA methylation. We also found decreased levels of DNA methylation in the highly tumorigenic hamster cell line A(TI)CL3, which was derived by the chemical transformation of diploid hamster cells in culture (Benedict et al., 1975). Recently, Feinberg and Vogelstein (1983a) found hypomethylation at specific sites in four out of five naturally occurring human tumors. The situation with chemically transformed mouse cell lines is not as clear as the earlier studies dealing with cells transformed in vivo or derived by transformation of primary cultures. No changes in overall cytosine methylation were observed in 3T3 cells transformed by chemicals (Diala and Hoffman, 1982a).Wilson and Jones (1983b) also found no changes in some 3T3 lines transformed by benzo(a)pyrene, but definite decreases in overall methylation occur in some tumorigenic lines. Tumorigenic viruses induce substantial changes in genomic 5methylcytosine levels, and tumors arising in mice infected with retroviruses have shown decreased DNA methylation (Cohen, 1980; Smith et at., 1982). In contrast, hamster cells transformed by polyoma or adenoviruses show substantial increases in 5-methylcytosine (Rubery and Newton, 1973; Gunthert et al., 1976). However, cell lines were used for transformation in the latter studies and the results may be open to various interpretations. Human fibroblasts transformed by SV40 virus have been examined and show no changes in 5-methylcytosine content (Diala et al., 1981). Table IV shows that decreases in 5-methylcytosine levels have been observed in most cases in which transformation was induced in uiuo, or in which low-passage diploid cells were used for the transformation studies. This is important because recent work (Wilson and Jones, 198313) has demonstrated that diploid cells explanted into culture rapidly lose 5-methylcytosine groups as they divide. The rate of loss of 5-methylcytosine is most rapid in mouse embryo cells that rapidly age in culture, and the achievement of a stable methylation level may be associated in some way with cell immortality (Wilson and Jones, 1983b). Thus, the use of cell lines as the starting point for transformation studies may be inappropriate in experiments to determine the relationship between DNA methylation and oncogenicity.

18

ARTHUR D. RIGGS AND PETER A. JONES

VIII. Carcinogens and Enzymatic DNA Methylation

A. INHIBITION OF DNA METHYLATIONin Vitro CHEMICAL CARCINOGENS

BY

Several alkylating agents have been shown to inhibit the methylation of a variety of DNA substrates in uitro (Table V). Drahovsky and Morris (1972) showed that E . coli DNA containing guanine residues modified by treatment with dimethyl sulfate was able to bind tightly to the DNA methylase. Later studies by Drahovsky and Wacker (1975) showed that MNNG was a powerful inhibitor of the rat liver methylase preparation. This inhibition was caused by an irreversible inactivation of the enzyme and was confirmed by later studies of Cox (1980) in which several alkylating carcinogens were tested for their abilities to inhibit in zjitro DNA methylation. MNNG was the only carcinogen studied that altered the methylase activity, and prevention of the MNNG effect by dithiothreitol suggested that the methylase was a sulkydryl-containing enzyme that was inactivated by MNNG binding to the active site. More recent work by Cox (1982) has shown that TABLE V INHIBITION OF DNA METHYLATION in Vitro BY CHEMICAL CARCINOGENS ~~

-~

~

Carcinogen

DNA substrate

Dimethyl sulfate

E. coli DNA

N-Methy1-N-nitro-N-

E . coli DNA

nitrosoguanidine (MNNG) N-Methyl-N-nitro-hinitrosoguanidine (MNNG) N-Methyl-N-nitrosourea (‘MNU) I-(Acetoxyacetylamino) fluorene (AAF) and Nmethyl-hi-nitrosourea (MNU) Benzo(a)pyrene diol epoxide and a diverse range of alkylating agents

Methylase

Reference

Mouse spleen Rat liver”

Drahovsky and Morn s (1972) Drahovsky and Wacker (1975)

Methyl-deficient rat liver DNA

Rat liver’

Cox (1980)

Methyl-deficient rat liver DNA Chicken erythrocyte (single- and double-stranded)

Rat liver

Cox (1982)

Rat brain

Salas et al. (1979); Pfohl-Leskowicz et al. (1982)

Hemimethylated DNA

Mouse spleen

Wilson and Jones (1983a)

* Direct effect on methylase.

5-METHYLCYTOSINE7 GENE

REGULATION, AND CANCER

19

methylnitrosourea (MNU) also can inhibit the methylation reaction if preincubated with the DNA substrate. Thus, alkylating agents may inhibit DNA methylation by reaction with either the enzyme or the substrate. Salas et al. (1979) showed that the aromatic hydrocarbon N-acetoxyN-acetylaminofluorine (AAF),which reacts covalently with DNA, inhibited the transfer of methyl groups to chicken erythrocyte DNA in the presence of a rat brain methyltransferase preparation. The mechanism of enzyme inhibition by AAF was investigated in more detail (Pfohl-Leskowicz et al., 1982), and a relationship was observed between the degree of DNA modification by AAF and its inability to accept methyl groups from the rat brain enzyme. The AAF substituted DNAs had a higher affinity for the enzyme than native DNA; however, this probably was not due to the known ability of AAF to induce a local destabilization of the helix. These authors suggested that the ability of AAF to inhibit methylation might have been caused by the presence of bulky AAF-guanine residues on the substrate, which would hinder the scanning action of the DNA methyltransferse. PfohlLeskowicz et al. (1982) also found that MNU adducts inhibited DNA methylation in the test tube and MNU was more active at inhibiting methylation on a molar basis than was AAF. AAF-substituted DNA had a higher affinity for the enzyme than did native DNA, and behaved as a methylation inhibitor of unmodified DNA in mixing experiments, whereas DNA alkylated with MNU did not inhibit the methylation of native DNA. Recently, hemimethylated DNA extracted from cells treated with low levels of 5-azacytidine has been used as a substrate for a maintenance methylation assay in vitro (Jones and Taylor, 1981). In this assay, methyl groups are transferred specifically to cytosine residues in the hypomethylated strand, thus satisfying the criteria of a maintenance methylation assay. The ability of hemimethylated DNA to accept methyl groups was inhibited markedly by a wide range of alkylating agents and benzo(a)pyrene diolepoxide (Wilson and Jones, 1983a). The induction of alkali-labile sites by depurination, or by UV light irradiation of bromouracil-containing DNA also lessened its ability to accept methyl groups in vitro, but the methylation reaction was much less sensitive to thymine dimers or to double strand breaks. Carcinogens induced the formation of alkali-labile DNA lesions, but the degree of subsequent methyltransferase inhibition we observed was greater than that expected for this damage alone. Therefore, the presence of carcinogen adducts on the DNA may have played a role in decreasing substrate efficiency.

20

ARTHUR D. RIGGS AND PETER A. JONES

The results summarized in Table V show that several alkylating agents and benzo(a)pyrene diolepoxide are inhibitors of DNA methylation in the test tube. Detailed mechanisms for this inhibitory activity are not known, although most investigators have suggested that it may be caused by carcinogen binding to guanine residues, which occur both opposite and adjacent to CG methylation sites. The occurrence of a carcinogen adduct in the vicinity of a modification site may alter its recognition by the enzyme and thereby inhibit methylation at that site. Alternatively, the carcinogen may prevent the scanning function of the enzyme so that modification sites downstream become undermethylated. Most of the work thus far has been done with impure enzyme preparations and uncharacterized DNAs. Since the details of the methylation reaction remain relatively obscure, it will be some time before the mechanisms of inhibition of DNA methylation by carcinogens are completely understood. Carcinogenic agents may cause heritable changes in 5-methylcytosine patterns by a variety of mechanisms, including adduct formation, induction of apurinic sites, single strand breaks, and direct inactivation of the DNA methyltransferase enzyme. B. INHIBITIONOF DNA METHYLATIONIN LIVING CELLSBY CARCINOGENS Several investigators have shown that the hepatocarcinogen ethionine substantially inhibits methylation in DNA made shortly after or during carcinogen exposure (Table VI). The ability of the ethionine to inhibit methylation was demonstrated first by Sneider et al. (1975). Cox and Irving (1977)found a similar result for regenerating rat liver at doses of ethionine only twice that used to induce hepatocarcinomas. Therefore, this inhibition may be linked to ethionine’s carcinogenic activity. Because ethionine is not usually considered to be mutagenic, the results were important in suggesting an alternative mechanism of action for the amino acid analogue. Ethionine also inhibits DNA methylation in Friend cells induced to differentiate with the analog (Christman et al., 1977), and DNA extracted from cells treated with ethionine is an efficient methyl acceptor in uitro. Boehm and Drahovsky (1979) studied the ability of ethionine to inhibit methylation in the DNA of P815 mastocytoma cells and showed that the methylation of the inverted repeat sequences in the DNA was more sensitive to inhibition than was the methylation of other classes of sequences. Ethionine is perhaps unique among other carcinogens that inhibit DNA methylation in that it is a competitive

5-METHYLCYTOSINE, GENE REGULATION, AND CANCER

21

TABLE VI INHIBITION OF DNA METHYLATION IN LIVING CELLSBY CARCINOGENS Carcinogen Ethionine

N-Methyl-N-nitrosourea MNNG 5-Azacytidine Benzo(a)pyrene Excision repair induced by ultraviolet light or N-(acetoxyacetylamino) fluorene

Cell type Novikoff hepatoma Regenerating rat liver Friend erythroleukemia cells P815 mastocytoma Raji cells Raji cells Mouse embryo cells (10T1/2) 3T3 but not 10T1/2 cells Human fibroblasts

Reference Sneider et al. (1975) Cox and Irving (1977) Christman et al. (1977) Boehm and Drahovsky (1979) Boehm and Drahovsky (1981a) Boehm and Drahovsky (1981b) Jones and Taylor (1980) Wilson and Jones (1983a) Kastan et al. (1982)

inhibitor of the enzyme after conversion to S-adenosylethionine, whereas the inhibition seen with other compounds tends to be noncompetitive. Boehm and Drahovsky (1981a,b) were able to show clearly, by restriction enzyme analysis, that the DNA of Raji cells exposed to MNU or MNNG was substantially undermethylated. In contrast, Craddock and Henderson (1979) failed to detect any effect of dimethylnitrosamine or methylmethanesulfonate on DNA methylation in regenerating rat liver. However, two precursors ( [3Hlmethionine and labeled nucleosides) were used to measure the level of methylation during carcinogenic treatment in these studies. Because of potential variations in amino acid transport, utilization, and sizes of the S-adenosylmethionine pool, it is difficult to determine the exact significance of these data. Benzo(a)pyrene induces a 12% decrease in DNA methylation in transformable 3T3 cells exposed to this important carcinogen (Wilson and Jones, 1983a).The maximum decrease was seen 24 hr after carcinogen treatment, but we were unable to detect similar changes in the DNA of treated 10T1/2 cells, which are used extensively for oncogenic transformation studies. The fact that gross alterations in the level of 5-methylcytosine did not occur does not mean that point changes (which might ultimately give rise to the transformation) were not induced at the level of specific genes. Also, it may be inappro-

22

ARTHUR D. RIGGS AND PETER A. JONES

priate to use cell lines for these studies for the reasons discussed earlier. 10T1/2 cells are also transformable by treatment with 5-azacytidine (Benedict et al., 1977). 5-Azacytidine may be carcinogenic in certain animal systems, but the data are not unequivocal (IARC Monograph, 1981).The nucleoside analog is a powerful inhibitor of DNA methylation within 10T1/2 cells (Jones and Taylor, 1980)but is, at best, a very weak mutagen in these cells (Landolph and Jones, 1982). The ability of 5-azacytidine to induce oncogenic transformation may be related to its ability to inhibit DNA methylation. Kastan et al., (1982) have recently studied the methylation of deoxycytidine incorporated into DNA during excision repair in human diploid fibroblasts. DNA damage was induced by ultraviolet radiation, MNU, or AAF, and it was found that methylation in repair patches induced by all three agents was slow and incomplete. The methylation of cytosine incorporated in cells damaged during logarithmic phase was much faster and reached an almost normal level in ten to twenty hours. Hypomethylated repair patches in confluent cells became more methylated when the cells were stimulated to divide, but the repair patch may not become fully methylated before cell division. Therefore, DNA damage and repair may lead to heritable loss of methylation in some sites. These results contrasted with the initial work of Drahovsky et al. (1375), Hilliard and Sneider (1975), and Lowe et al. (1976), but two precursors were used in the earlier work, so that accurate quantitation of changes in DNA methylation could not have been done. The studies of Kastan et al. (1982) suggest that carcinogens may induce hypomethylation of CG sites if DNA methylation is not completed before S-phase. This would agree with the earlier hypothesis by Holliday (1979) and might ultimately lead to inappropriate gene expression in treated cells. IX. Oncogenes

Considerable interest has been generated by the finding that normal cells contain sequences within their DNAs that are homologous to the transforming genes (u-onc)of retroviruses (for reviews, see Weinberg, 1982; and Cooper, 1982). Shih et al. (1979) and Cooper et al. (1980) first reported that DNA from chemically induced or spontaneous human tumors will transfer the transformed phenotype to mouse 3T3 cells. Some of the transformed mouse cells are also tumorigenic. These gene transfer experiments have resulted in the identification of several human cellular oncogenes (c-onc),and it is clear that the cellu-

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lar oncogenes from tumor cells are different from their normal counterparts, which do not function in these gene transfer experiments. In one case (c-Ha-ras),the difference has been found to be a single base mutation (Reddy et al., 1982; Tabin et al., 1982). However, even in these papers the authors discuss the multistage nature of human neoplasia and point out that abnormal expression of the normal c-Ha-ras gene can transform (Chang et al., 1982). Given the fact that viral and cellular gene expression can be suppressed by methylation (Section 111; Tables I1 and 111), it seems plausible that the expression of the cellular oncogenes may likewise be suppressed by methylation and induced by hypomethylation. To date, only one study has compared the state of methylation of such an oncogene in normal and chemically transformed cells (Gattoni et al., 1982). These studies showed that the cellular homologs of Moloney murine sarcoma sequences (c-mos) were hypermethylated and transcriptionally silent in a variety of normal and transformed rodent cells. This was in contrast to a Moloney sarcoma virus-transformed cell line in which the integrated viral sequence (v-mos) was hypomethylated and transcriptionally active. Thus, the c-mos sequences did not appear to play a role in the transformation of rodent ceIIs by chemical or physical agents, but the study did not exclude the possibility that other sequences might be activated during oncogenesis. X. Conclusions

A. A CANCER MODEL Figure 5 outlines our current working model for cancer. This model is similar to that of Holliday (1979), but some key points of emphasis are different. Holliday suggested that methylation (not demethylation) activates gene transcription. Demethylation events were thought to affect differentiation by inactivating genes. The experimental data are much more supportive of direct activation by demethylation. Although demethylation by a postreplication recombination event, for example, is possible, we think that a simpler mechanism is inhibition of the DNA methylase. Either the initial event or a later event could be a mutation, but we wish to stress the alternative possibility that an important event for some cancers is an inappropriate demethylation. There is no doubt that methylation changes can masquerade as mutations (see Section V). Carcinogens might inhibit methylases or otherwise interfere with

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Norrnol Division ond Mointenonce Methylotion

I

I

Corcinogenic ( I NI T I AT ION 1 Insult DNA Domoge Corcinogen Adducts Methylose lnhibition

Mutotion or Altered Methylotion

M

M

m

M

m M

?Ei=mmT REPLICATION PRESSURE

4

J

PROMOTION AND PROGRESSION 0

4

Mutation ond/or Methylotion Changes

DERANGED REGULATION, REPLICATION, HORMONES, AND SURFACE PROTEINS

FIG. 5. A demethylation and gene activation model for neoplastic initiation and/or progression. M represents methylated sites in DNA. Shading indicates genetic inactivity. Lack of shading indicates “unlocked” genes, with actual or potential transcription. The X in the left pathway represents a true mutation in a structural gene or control region. We wish to emphasize the pathway on the right with demethylation potentiating abnormal gene expression.

normal methylation. The initial event could result in inappropriate activity of a cellular oncogene, but another likely possibility would be the inappropriate expression of other normal genes which might not be recognized as oncogenes by transfection experiments. Given the initial event and resulting replication pressure, there is now strong selective pressure favoring cells with abnormal gene expression. Since the inheritance of methylation patterns is not totally rigid (Shmookler-Reis and Goldstein, 1982;Wilson and Jones, 1983b), it is likely that aberrant cell division would increase the chances of altered methylation.

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A critical point is that aberrant methylation patterns induced by carcinogenic exposure could be propogated in the absence of further carcinogenic treatment, and might potentiate a progressive change in gene expression within exposed cells. One of the singular advantages of the demethylation model proposed is that the transformed state could be truly reversible if the oncogene became remethylated under the action of de nouo methylases (in the early embryo, for example) or, alternatively, if there was a demethylation of a proper controlling gene (e.g., a repressor). Many of the individual steps in the model shown in Fig. 5 are known to occur; it remains to be seen if the demethylation pathway is significant in the progression from normal cells to full neoplasia.

B. MAIN POINTS In conclusion, we would like the reader to consider and remember three points. (1)Enzymatic DNA methylation is involved in mammalian gene control, apparently as part of a gene silencing mechanism. (2) Methylation changes are somatically heritable and can masquerade as mutations. (3)Carcinogens can interfere with enzymatic DNA methylation. XI. Addendum

Feinberg and Vogelstein (1983b) found hypomethylation of a c-Hain six out of eight colon and lung tumors examined. Also, clear evidence that 5-azacytidine causes multiple primary tumors in rats has been obtained (Carr, Reilly, Winberg, Smith, and Riggs, unpublished). Frost and Kerbel (1983) independently suggested a role for demethylation in the genesis of tumor heterogeneity, particularly with regard to the metastatic phenotype. TUS oncogene

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Tanaka, M., Hibasami, H., Nagai, J., and Ikeda, T. (1980).Aust. J . E x p . Biol. Med. Sci. 58,391-396. Tantravahi, U., Guntaka, R. V., Erlanger, B. F., and Miller, 0. J. (1981). Proc. Natl. Acad. Sci. U S A . 78,489-493. Taylor, S. M., and Jones, P. A. (1979). Cell 17,771-779. Taylor, S . M., and Jones, P. A. (1982).J . Mol. B i d . 162, 679-692. Tennant, R. W., Otten, J. A., Myer, F. E., and Rascati. R. J. (1982).Cancer Res. 42,30503055. Van der Ploeg, L. H. T., and Flavell, R. A. (1980).Cell 19, 947-958. van der Putten, H., Quint, W., Verma, I. M., and Berns, A. (1982).Nucleic Acids Res. 10, 577-582. Vardimon, L., Kressmann, A., Cedar, H., Maechler, M.,and Doerfler, W. (1982).Proc. Natl. Acad. Sci. U S A . 79, 1073-1077. Venolia, L., Gartler, S. M., Wassman, E. R., Yen, P., Mohandas, T., and Shapiro, L. J. (1982).Proc. Natl. Acad. Sci. U S A . 79, 2352-2354. Waalwijk, C.. and Flavell, R. A. (1978a).Nucleic Acids Res. 5, 3231-3236. Waalwijk, C., and Flavell, R. A. (1978b).Nucleic Acids Res. 5,4631-4641. Waechter, D. E., and Baserga, R. (1982).Proc. Natl. Acad. Sci. U S A . 79, 1106-1110. Walker, M. S., and Becker, F. F. (1981). Cancer Biochem. Biophys. 5, 169-173. Weinberg, R. A. (1982).Cell 30, 3-4. Weintraub, H., Larsen, A., and Groudine, M.(1981).Cell 24, 333-344. Weintraub, H., Beug, H., Groudine, M., and Graf, T. (1982).Cell 28,931-940. Wigler, M., Sweet, R., Sim, G. K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, s.,and Axel, R. (1979).Cell 16, 777-785. Wigler, M., Levy, D., and Perucho, M. (1981).Cell 24, 33-40. Wilks, A. F., Cozens, P. J., Mattaj, I. W., and Jost, J-P. (1982). Proc. Natl. Acad. Sci. U.S.A. 79,4251-4255, Wilson, V. L., and Jones, P. A. (1983a).Cell 32,239-246. Wilson, V. L., and Jones, P. A. (1983b).Science 220, 1055-1057. Woodcock, D. M.,Adams, J. K., and Cooper, I. A. (1982).Biochim. Biophys. Acta 696, 15-22. Worton, R. G., Grant, S. G., and Duff, C. (1983).I n “Gene Transfer and Cancer” (N. L. Sternberg and M. L. Pearson, eds.). Raven, New York (in press). Yagi, M., and Koshland, M. E. (1981).Proc. Natl. Acad. Sci. U S A . 78,4907-4911. Youssoufian, H., Hammer, S. .M., Hirsch, M.S., and Mulder, C. (1982).Proc. Natl. Acad. Sci. U.S.A. 79, 2207-2210.

IMMUNOBIOLOGY OF INFECTION WITH HUMAN CYTOMEGALOVIRUS H. Kirchner Institute of Virus Research. German Cancer Research Center. Heidelberg. Federal Republic of Germany

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I Intduction ................................................... I1 Herpesviruses.................................................. 111 Cytomegaloviruses. HCMV ...................................... Defective HCMV Virions ........................................ IV HCMV-Host Cell Interactions .................................... “Malignant” Cell Transformation in Vitro by HCMV ................. V Epidemiology of the Infection with HCMV ......................... VI . Diagnosis of HCMV Infection .................................... VII Clinical Significance of HCMV Infections .......................... A . HCMV as a Cause of Congenital Diseases and of Diseases Acquired around Birth ....................................... B Infectious Mononucleosis Caused by HCMV (HQMV-IM) ......... C . HCMV Infections in Transplant Recipients ...................... D . Patients Receiving Immunosuppressive Therapy ................. E . HCMV Infections in Patients with Neoplasms . . . . . . . . . . . . . . . . . . . VIII Oncogenic Potential of HCMV.................................... A Miscellaneous Data on the Role of HCMV in Cancer .............. B Kaposi’s Sarcoma ............................................ C . In Vitro Malignant Transformation of Cells by HCMV ............ IX Immunopathology .............................................. X. Latency/Reactivation ............................................ XI Replication of HCMV in Leukocytes ............................... XI1 Effects of HCMV on Leukocytes .................................. XITI Immunity against Infections with HCMV. General Aspects . . . . . . . . . . . . A Antigenic Heterogeneity of HCMV., ........................... B Cell Surface Phenomena Associated with HCMV Infection . . . . . . . . . . C . Soluble Antigens ............................................ D Respective Roles of Humoral and Cellular Immune Responses . . . . . XIV Humoral Immune Responses ..................................... XV Cell-Mediated Immunity ........................................ A Lymphocyte Cytotoxicity ..................................... B Lymphokines ............................................... C. Lymphocyte Proliferation ..................................... D Overview of the Investigations of HCMV-Induced Lymphoproliferation ......................................... XVI Nonspecific Defense Mechanisms against HCMV Infection ........... A NKCells ................................................... B. Potential Role of Macrophages in Defense against HCMV ......... C ADCC ..................................................... D. Role of Complement ......................................... 31

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E. Role of Interferon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVII. Vaccine and Therapy Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References

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I. Introduction

The immune response against viruses is considerably more complex than the immune response against inert antigens. In vitro studies are often performed using inactivated viruses. These studies are complex, since viruses, particularly herpesviruses, contain a large number of different proteins and glycoproteins. In uiuo, however, the defense system has to deal with infectious viruses which sometimes are able to infect the cells of the immune system. Thus, a situation ensues the complexity of which is often not realized by immunologists who mainly work with in vitro systems. The infection of immunocytes may be one of several types. Some viruses productively infect immunocytes, i.e., infectious progeny will be produced. This may cause cell death as is the case with DNA viruses, particularly with herpesviruses. Some RNA viruses, however, may be replicated without cell destruction. Viruses, furthermore, are able to transform lymphocytes, for example, Epstein-Barr Virus (EBV) which has a strong selectivity for human B lymphocytes which are polyclonally activated to proliferate and to produce antibodies of all classes and of many specificities. B lymphocytes transformed by EBV, besides other typical characteristics, acquire the capacity for indefinite in vitru growth. The activation of B lymphocytes by EBV is immunologically nonspecific. EBV acts on all B cells, even on immunologically virgin B cells of newborns. EBV, therefore, in a certain sense resembles a mitogen. However, mitogens, as far as one knows at present, appear to act on the cell membrane and thereby initiate a sequence of events culminating in DNA synthesis and mitosis. EBV, in contrast, has to be infectious in order to stimulate cellular DNA synthesis and the mitogenicity appears to be the consequence of the interaction of the viral genome with the cellular genome. There are various other types of infection including abortive infection and chronic infection. Finally, leukocytes may be the site of viral Eatency, as has been suggested for human cytomegalovirus (HCMV) and as will be discussed below. Obviously, infection of leukocytes or lymphocytes by viruses may

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have important implications for the pathogenesis of viral infections. First, leukocytes that are mobile cells represent ideal carriers for the distribution of viruses in the body. Second, it is tempting to speculate that viruses are able to multiply in (and destroy) those cells that carry the immunological memory and by this mechanism undermine the immune system. Viruses have been reported to have a variety of effects on the cells of the immune system both in vivo and in vitro, including both immunopotentiating effects and immunosuppressive effects. In many of these systems, however, it has not been ruled out that the immunoregulatory effects were caused by interferon. Interferons are known to have such effects and various subtypes of interferons are produced by white blood cells, some of them fairly rapidly. We believe that many of the reported effects of viruses on immunocompetent cells have, in fact, been interferon effects, and in further experiments care has to be taken to exclude this source of artifacts when studying virus effects in cell cultures. Experimental controls will require the use of antiinterferon sera. When reviewing the defense system against viruses, we believe it is important to distinguish between primary (natural) defense and immune reactions. Immune effector functions, by definition, are reactions that follow a secondary contact with the pathogen and are characterized by “immunological specificity.” The study of specific immune mechanisms is relevant for the development of vaccines. Immunity also has to be considered relevant in infections that tend to be recurrent as is typical for herpesviruses including HCMV. The problem of some of these recurrent infections is that they do occur despite the fact that specific immunity appears to be unimpaired. Maybe our current knowledge and technologies do not allow detection of subtle defects of the immune system that are connected with recurrent disease. It is an important biological issue to define the relationship between recurrent viral disease and the status of the immune system. Usually, one distinguishes between the humoral immune response as measured by antibody titers and the cellular immune response which represents a function of predominantly T cells and which can be measured by a variety of techniques. This distinction, although commonly used, may be misleading since there are many indications of collaborative functions between these two systems. Thus, the production of many antibodies, including certain antiviral antibodies, requires the participation of both B cells and T (helper) cells. There are quite a few other examples of the interplay between T and B cells

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and the distinction between cellular and humoral immunity may b e relatively artificial. Primary defense mechanisms, in contrast to specific immunity, are operative during the first encounter of the host with a pathogen. In certain experimental situations and with certain viruses, primary defense mechanisms appear to be very effective. They function poorly in other systems in which a primary infection leads to disabling disease or death of the host. From a number of experimental virus infections it appears that primary defense mechanisms are genetically controlled. It is not known if the primary antiviral defense in man is under genetic control. Primary antiviral defense includes preformed mechanisms such as the phagocytic system and rapidly induced mechanisms such as interferons. Again one distinguishes between cellular and humoral systems. It has to be emphasized that the so-called “primary” defenses are not restricted to the primary contact with a given pathogen. There is ample evidence to indicate that they are also activated during a specific immune response and serve as important amplifier mechanisms. The different candidate cells and molecules will be discussed below. It has to be kept in mind that there are additional mechanisms of defense, in a broader sense, that are usually not considered by immunologists. These include, for example, the absence of virus receptors on cells which may stop virus spread in the body. The presence or absence of virus receptors in certain systems appears to be genetically controlled. The replication of viruses may also be restricted within cells. Abortive infection, for example, of macrophages may effectively terminate a viral infection. Finally, the genetic make up of the virus itself decisively influences the fate of the virus infection, There is wide variability between different strains of certain herpesviruses in their pathogenicity for experimental animals ranging from total lack of pathogenicity to high virulence. It is not unreasonable to speculate that different strains of human herpesvirus differ in their pathogenicity for man, their natural host. An important additional aspect is, that not all virions that are produced are infectious and that defective interfering (DI) particles may be produced during the course of a viral infection which inhibit replication of the “normal” infectious virions. An additional interesting finding has been recently reported in studies of Herpes Simplex Virus (HSV) infection of C57BL/6 mice (Zawatzky et al., 1982).It was found that the mice survived high doses of virus (250 LDWS),whereas lower virus doses were lethal. Evidence

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was presented that this “survival of 250 L D ~ s ’was ’ due to interferon produced at the infection site. Lower virus doses that killed the mice did not induce measurable titers of interferon at the infection site. In this article we shall review antiviral defense and specific immunity in one viral system, which is HCMV. We will occasionally refer to observations made with other viruses, in particular with other human herpesviruses. In many aspects more extensive studies have been recently performed with HSV than with HCMV. Since we have recently reviewed the immunobiology of the HSV infection (Kirchner, 1982) we will frequently refer to studies with HSV, particularly when studies with HCMV are lacking. The reasons why we wish to review the immunobiology of the infection with HCMV are severalfold. First, HCMV is a virus of great clinical relevance. HCMV infections are prevalent in patients receiving organ grafts, and HCMV is one of the few viruses for which intrauterine infection has been established. Second, there are obvious interactions between the cellular components of the immune system and HCMV, and leukocytes are perhaps a site of replication and of latency of the virus. HCMV, in addition, has been found to be immunosuppressive and may cause secondary infections with other pathogens. The time has come (Rubin et al., 1979) to begin to unravel many of the unresolved issues regarding HCMV infection. Many aspects, as for example the question as to the sites of latency within the body, have not been clarified. Cellular immunology and molecular biology, however, have provided tools that will allow us to address questions that have not been studied with the proper methods before. This article does not intend to cover all virologic and clinical aspects of HCMV, since there have been a number of reviews which cover the older literature (Hanshaw, 1968, 1971; Krech et al., 1971a; Weller, 1972; Plummer, 1973; Michelson-Fiske, 1977).It is, therefore, not intended to completely review the extensive literature on HCMV. Rather, it is the aim of this article to pinpoint interactions between HCMV and the cells of the immune systems that urgently need further clarification and by doing this we hope to stimulate further research. II. Herpesviruses

Herpesviruses are large DNA viruses that occur in man and many different animal species. There are numerous herpesviruses that have the same morphological appearance and that cannot be identified microscopically, but can be by other criteria. Herpesviruses are often

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quite restricted in their host range and in their target cell range, at least as far as virus replication in uitro is concerned. Thus, one possibility for differentiation between different herpesviruses is on the basis of their cytopathic effect in different tissue culture systems. Other methods are based on antigenic differences between different herpesviruses (serology) or on the analysis of their DNA (restriction enzyme patterns). There are five human herpesviruses, including HCMV, EBV, Herpes Simplex Virus type 1 (HSV-l), Herpes Simplex Virus type 2 (HSV-2), and Varizella Zoster Virus (VZV). There is about 50% nucleic acid homology between HSV-1 and HSV-2 and thus, these two viruses are considered to be closely related or to represent subtypes of one virus. Nonetheless, HSV-1 and HSV-2 can be clearly distinguished by various biological criteria and unequivocally by restriction enzyme analysis. It is not known if there are subtypes of other human herpesviruses that allow one to establish groups of clinical isolates. Except for HSV-1 and HSV-8, there is no apparent nucleic acid homology between any other two human (or animal) herpesviruses. Herpesviruses appear to have coevolved with the species in which they are now found. In this species they are usually ubiquitous and in normal individuals they rarely cause disease. Thus, probably by selection, the individuals who have survived are in the possession of the appropriate defense systems against this virus and the virus lives as a harmless saprophyte with the host. There is evidence to indicate that HCMV is the most ancient of human herpesviruses. Pathogenicity due to HCMV is observed when by iatrogenic measures the defense systems are massively impaired or if the defense system is yet not fully developed, as in the case of the fetus or the newborn. There are other rare occasions when herpesviruses cause significant disease, the exact pathogenesis of which is poorly understood. Examples are HSV-encephalitis or community-acquired HCMV-infectious mononucleosis (IM). There is, however, evidence to indicate that a significant percentage of HCMV-IM cases are caused by blood transfusions. Most human herpesviruses are restricted to humans. An exception is HSV which infects (and kills) newborn mice and also adult mice of appropriately selected inbred strains after various schedules of infection. Certainly, there are differences between the virulence of different clinical isolates of HSV in mice. There are numerous data on mouse models of HSV infection which we have previously reviewed (Kirchner, 1982). However, there is a certain degree of artificiality in such models. Defense mechanisms may be activated in the non-

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adapted “foreign” host that are different from those in the host that has been living in harmony with the virus for millions of years. The host cell range of the human herpesviruses is also restricted, most remarkably in the case of EBV, which appears to have only two types of targets, the human B lymphocyte and tumor cells in nasopharyngeal carcinoma (Klein, 1975). HSV, generally, is not considered to be a lymphotropic virus, but, provided the conditions are carefully selected, it can be demonstrated that HSV is capable of in vitro replication in lymphocytes (Kirchner et al., 1977; Kirchner and Schroder, 1979). HCMV, in contrast, which is considered to be associated with lymphocytes in vivo, can only be replicated to significant titers in vitro in fibroblasts (and perhaps a few other cell types). There are in vitro systems in which a small percentage of lymphocytes can be productively infected with HCMV (see below). In vivo, based on virus isolation and even more on clinical symptoms and histopathology (Rosen and Hajdu, 1971), HCMV appears to replicate in many additional tissues, e.g., in salivary glands, lung, liver, kidneys, and endometrial tissue. Furthermore, there appear to be various forms of “atypical” interactions between HCMV and different cell types in vitro. Ill. Cytomegaloviruses, HCMV

As we have described above, herpesviruses are large DNA viruses that share an identical morphological appearance. A general definition has been given by Honess and Watson (1977). Herpesviruses are viruses of eukaryotes with linear double-stranded DNA genomes of more than 80 x lo6 mol wt which are replicated in the nucleus of infected cells, assembled into 100 nm diam. icosahedral capsids composed of 162 prismatic capsomeres which are enclosed in glycoprotein and lipid (ether sensitive) envelopes to give the normally infectious form of the virus.

Recently, subgroups of herpesviruses have been proposed (Herpesvirus Study Group, 1978), although these classifications have not as yet found wide acceptance. The group of betaherpesvirinae contains the viruses that are usually called cytomegaloviruses, the prototype of these is HCMV. Betaherpesvirinae have the following properties: (1) narrow host range; (2) relatively low reproductive cycle, slowly progressing lytic foci in cell culture; (3) enlargement of the infected cell in v i m and often in vitro (cytomegalia); (4) inclusion bodies may be present in the nucleus and in the cytoplasm; ( 5 ) latent virus infection

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frequently demonstrated in the salivary glands and/or other tissues; and (6) DNA has a molecular weight of 130-150 X lo6. The genome of HCMV consists of a linear double-stranded DNA with a molecular weight of approximately 150 X lo6 (Kilpatrick and Huang, 1977; Stinski et d . ,1979). HCMV DNA, similar to HSV DNA, contains long and short segments which can invert relative to each other, producing four different isomeric forms (Weststrate et al., 1980). The DNAs of different strains of HCMV demonstrate at least 80%homology by reassociation kinetics (Huang et al., 1976; Pritchett, 1980).However, HCMV DNA has less than 5%homology with HSV-1, HSV-2, MCMV, and simian cytomegaloviruses. Thus, there is no more nucleic acid homology between HCMV and “cytomegalovirus” of other species than between HCMV and HSV. It remains to be determined how useful the classification of the so-called cytomegaloviruses into one group is. Ho (1981) has pointed out that the biological relationship between HCMV and cytomegaloviruses of other species is probably not closer than that between other members of the family Herpetoviridae. In addition to a number of international standard strains, there have been numerous clinical isolates of wild-type strains of HCMV. Typical for all herpesviruses is the complexity of their DNA and it is, therefore, not surprising that there are differences between individual strains of HCMV. In contrast to the clear-cut differentiation between HSV-1 and HSV-2 that can be made b y restriction enzyme analysis, there are as yet no defined subgroups of HCMV. As with other herpesviruses, “molecular epidemiology” will probably have great impact on further approaches to this problem. The technique of restriction enzyme analysis, furthermore, will be important to determine whether several isolates recovered from one patient at different times are identical or if they represent different strains. The complexity in the DNA of HCMV is reflected in the complexity of the coded proteins. The practical aspect of this problem is the question as to the antigenic cross-reactivity of individual HCMV strains and the question whether humoral (or cellular) immunity against HCMV, as developed by infection with one strain, is protective against infection with all strains of HCMV. The situation with HSV serves as a good example: prior infection with HSV-1 is not protective against subsequent infection with HSV-2. However, the clinical course of a genital HSV-2 infection tends to be less severe in patients seropositive for HSV-1 than in seronegative individuals (Nahmias and Roizman, 1973a-c).

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HCMV VIRIONS DEFECTIVE Defective HCMV particles have been described by DeMarchi and Kaplan (1977). These were obtained after serial undiluted passages as with other herpesviruses. It was found that defective particles stimulated cellular DNA better than stocks of standard virus. Ramirez et al. (1979) have found that serially passaged HCMV populations contain defective genomes of lower buoyant density. Similarly, Stinski et al. (1979) have reported defectiveness resulting from serial undiluted passage of HCMV. The majority of defective DNA molecules had a molecular weight of approximately 100 x lo6 (as compared to the normal molecular weight of HCMV of 150 X lo6). IV. HCMV-Host Cell Interactions

Typical for HCMV is a very slow replicative cycle in permissive tissue cultures. This is particularly true for primary isolates. There is a considerable variability between primary isolates, how long it takes to detect a cytopathic effect in tissue culture and it may take several weeks for certain isolates. The reasons for the slow rate of replication are unknown. Standard strains that have been passaged in the laboratories may behave differently in that they cause a cytopathic effect faster. The principal cytopathic effects of HCMV infection are the formation of nuclear and cytoplasmatic inclusion bodies. The former may be areas ofvirus assembly (Iwasaki et al., 1973), whereas the significance of the latter is not completely understood. Typical is the accumulation of homogeneous electron-dense material in the cytoplasm, especially in the Golgi region. The dense material “buds” into cytoplasmic tubules. These structures exhibit spherical configuration and are referred to as dense bodies. They consist of viral structural proteins (Sarov and Abady, 1975).Additionally, there is an apparent increase in cell size after infection with HCMV (cytomeglia). Furukawa et al. (1973) have reported that in high-multiplicity infection of human fibroblasts HCMV produced early cell rounding 6 to 24 hr after inoculation. Kanich and Craighead (1972) have reported on differences between the cytopathologic effects induced by an adapted and a wild strain of HCMV. Albrecht and Weller (1980) have reported heterogeneous morphologic features of plaques induced by five strains of HCMV. It is not known what the reasons are for the slow replication of

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HCMV. A theory proposed by St. Jeor and Hutt (1977) implies that HCMV replication is dependent on cell DNA synthesis. Consequently, whenever a cell becomes infected, it will not synthesize new virus DNA until the cell goes through the S phase of the cell cycle. When the virus is released to the surrounding cells, each of these cells will have to enter the S phase of the cell cycle before new virus DNA is produced. It has been thought that only fibroblasts are permissive for the replication of HCMV. There are, however, reports of atypical virus-host cell relationships between HCMV and a number of different tissues. Also, heterologous infections have been discovered, many of which are transitory and/or produce no infectious particles. For example, HCMV was capable of adsorbing to and penetrating guinea pig cells, but was unable to replicate new virus (Fioretti et al., 1973). However, cultures infected with virus inoculum of high titer showed a CPE characterized by cell rounding. Bovine and Vero (simian) cells infected with HCMV exhibited cytopathic changes and produced specific antigens (Waner and Weller, 1974). Furthermore, human lung epithelial ceIls have been shown to be permissive to productive infection with HCMV in uitro, and to support viral replication without manifesting cytopathogenic effects or undergoing cell lysis (Michelson-Fiske et al., 1975). Knowles (1976) has grown several strains of HCMV, including recent isolates, in epithelial cells derived from thyroid tissue. The failure to isolate further strains in cultures from other specimens may indicate that a higher multiplicity of infection is required to infect epithelial cells than fibroblast cells. Human embryonic kidney cells are epithelioid cells which are normally nonpermissive for in vitro replication of HCMV. The cells were converted to a permissive state by prior treatment with 5-iodo-2'-deoxyuridine (St. jeor and Rapp, 1973). After infection of confluent human embryonic lung cells with HCMV, some of the cells in the culture become productively infected, whereas others are induced to synthesize cellular DNA (DeMarchi and Kaplan, 1977). Only a small percentage of those cells in the culture which are stimulated to synthesize cellular DNA also synthesizes detectable amounts of viral antigens during the first 48 hr after infection. In addition, under conditions of infection in which practically all of the cells in culture become productively infected by 48 hr no stimulation of cellular DNA is observed. Thus, stimulation of cellular DNA synthesis and productive infection appear to be mutually exclusive events.

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HCMV induces production of nuclear antigens resembling the EBV-induced early nuclear antigen (EBNA) as early as 3 hr after infection. These early antigens can be detected only by the anticomplement immunofluorescence technique (Geder, 1976). Evidence has been also reported for nuclear antigens in HCMV-transformed human cells (Geder and Rapp, 1977). HCMV induces membrane antigens which are detectable by immunofluorescence techniques as early as 24 hr after infection (Tanaka et al., 1981). ‘‘MALIGNANT” CELL TRANSFORMATION in Vitro BY HCMV St. Jeor and Rapp (1973) and Albrecht et al. (1976) have documented that cellular DNA synthesis is induced following HCMV infection. This may occur not only in permissive fibroblasts but also in nonpermissive cells. Boldogh et al. (1978) have shown that the ability of HCMV to stimulate host DNA synthesis is an early function of the viral genome and shows a high resistance to UV irradiation. Albrecht et al. (1976) and Lang et al. (197413) have reported that HCMV-infected cells show an increase in mitotic activity. The latter authors have also demonstrated growth in agar of HCMV-infected cells. All this evidence, i.e., increase in cellular DNA synthesis, increase in mitotic activity, and growth in agar, is typical of though not a definitive proof for cell transformation. Albrecht and Rapp (1973) have isolated clones of non-contact-inhibited hamster embryo fibroblasts after exposing them to UV-irradiated HCMV. A continuous cell line was established from one of these clones which proved oncogenic when inoculated into weanling golden Syrian hamsters. The studies of Lausch et al. (1974) and of Murasko and Lausch (1974) have presented data indicating that hamster cells transformed by HCMV express virus-related membrane antigen(s) and that such antigen(s) can induce a cell-mediated immune response in the tumor bearing host. V. Epidemiology of the Infection with HCMV

Like the other human herpesviruses, HCMV is an ubiquituous virus. Epidemiological analyses reveal that infections occur in endemic patterns. The percentage of seropositive individuals depends on socioepidemic factors and may be between 40 and 100%(Krech, 1973). The number of seropositives is higher among the poor than among the affluent. In underdeveloped countries the percentage of seropositive individuals may be very high. Krech and Tobin (1981) have reported

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that children in Ibadan and Entebbe have rates approaching 100%at an early age. This study, furthermore, has shown that in five areas of the world the number of children with HCMV antibodies increased with age, suggesting that there was some child-to-child transmission of HCMV infection in these regions. In other regions the absence of any significant age-related increase indicated that the main pathway of HCMV infection in early life was by transmission from mothers to their infants. The modes of spread of HCMV in human populations are not in all instances completely understood. For example, it is not known how community-acquired CMV infection is transmitted. It is of interest that community-acquired HCMV-IM is almost exclusively observed in patients older than 30 years (Stem, 1968). During a 12-month prospective study in a hemodialysis unit with frequently documented infections in the patients, none of 26 staff members developed active HCMV infection (Tolkoff-Rubin et al., 1978).Furthermore, in this study it was reported that 4 dialysis nurses who had been working in the unit for longer than 5 years remained seronegative. Similar data documenting the absence of infection in staff members were reported by Fiala et u1. (1975) and by Betts et ul. (1979). In contrast, Yeager (1975) showed an approximately 5% per year rate of HCMV seroconversion among pediatric nurses working with virus-excreting infants. Thus, the infectivity of HCMV for healthy individuals, if existent, appears to be very Iow. HCM is one of the few viruses that are unequivocally transmitted vertically and cause congenital disease. HCMV has been shown to infect, i n utero, 0.5 to 2.0% of all newborn infants (Hanshaw, 1971). Originally, it had been thought that only primary infections of the mothers lead to infection of the fetus. However, it is now known that congenitally infected infants can be born to mothers immune to CMV (Stagno et al., 1977) and also that a mother can give birth to infected infants in consecutive pregnancies (Embil et al., 1970). These observations are of relevance for vaccination problems and will be discussed below. It has to be questioned, why infection in utero occurs in less than 1% of all gestations when at least 50% of all women are seropositive. One wonders how high the frequency of congenital HCMV infection is in areas in which nearly 100%of the population is seropositive, as, for example, in parts of Africa. In a recent report by Schopfer et al. (1978)the rate of intrauterine infection was found to be about 1.4%in populations in which the prevalence of HCMV infection was almost 100%.The data clearly suggested that intrauterine infections in anti-

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body carriers is due to reinfection or reactivation of a previous infection. A certain number of women shed HCMV in the cervical excretion and in the urine. However, this number is quite low in the first trimester of pregnancy. There is a significant increase toward the end of pregnancy. Most interestingly, the shedding of HCMV from the cervix or in the urine is observed exclusively in women under the age of 30 (Knox et aZ., 1979). The reasons for this observation are unexplained and the clarification of this issue might provide some keys to the pathobiology of HCMV infection. One wonders if congenitally infected children are also observed exclusively from women younger than 30 years of age. Our search of the literature has revealed that this is indeed the case, and that the majority of connatal infections occur in young women considerably younger than 30 years, even than 20 years. Additionally, it has been found that the highest percentage of women shedding HCMV from the cervix is found between the ages of 14 and 18 years. Thus, the occurrence of connatally infected infants born to young primaparae does not at all imply that the infections are primary infections. Besides congenital infections, infections intrapartum by genital secretions and postpartum by breast milk (Stagno et al., 1980) and perhaps by other routes appear to be frequent (for a review see Panjvani and Hanshaw, 1981). Thus, a significant percentage of children are infected very early in life. The early infected children appear to shed HCMV for long periods of time and probably spread infection to other children. In most cases, then, the primary infection occurs in a clinically inapparent fashion. Numazaki et al. (1970) have reported that, in Japan, 60%of infants between 5 and 9 months of age excreted HCMV and that complement fixing (CF) antibodies were present in over 60% of infants between 6 and 12 months of age. Later on, when the infants reached 10 to 12 months of age, the incidence of excretion diminished. The results have suggested that primary infection with HCMV occurs in Japan in over 60%of healthy infants living at home, without clinical manifestations by 5 months of age. Levinsohn et aZ. (1969) observed a cohort of 100 apparently normal newborn infants in Seattle, Washington, five times during the first year of life. HCMV was isolated from oropharyngeal or urine specimens at least once from 15 of the infants. It was suggested that the mothers were the most likely source of the infants’ infections. There are quite a few indications that HCMV may be transmitted sexually. First, as discussed above, HCMV is excreted from the cervix

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uteri and in the urine by a significant number of women. HCMV isolation and antibody are significantly more prevalent among women with documented past or active gonococcal infection (Jordan et al., 1973). Furthermore, HCMV has been isolated from semen (Lang et al., 1974a). Lately it has been reported that the prevalence of anti-HCMV antibodies is significantly higher in homosexual than in heterosexual men (Drew et al., 1981). The reasons for these findings are not clear as yet. There have been clusters of Kaposi’s sarcoma (KS) described recently in male homosexuals that may have an association with HCMV (Ziegler, 1982). HCMV may also be transmitted by iatrogenic measures, for example, by blood transfusions, as will be discussed below. In transplantation medicine, HCMV may be transmitted with the grafted organ. However, as far as renal allografting is concerned, the study of Naraqi et al. (1978) has documented that allograft kidneys were infrequently infected with CMV (6%).The kidney parenchyma appears to be an uncommon site of latent CMV infection and may not be the usual source of virus in patients with viruria. Also in this study, the reported frequency of CMV inclusion bodies in renal allografts in the literature has been summarized. However, a different opinion was expressed in the paper of Ho et al. (1975), who reported that of 10 seronegative patients who received kidneys from seronegative donors, only three became infected. However, of 12 seronegative patients who received kidneys from seropositive donors, 10 became infected. The study of Betts et al. (1975)has reinforced that transmission of HCMV infection does occur with the renal allograft. Finally, it has to be stressed again, that latency is the typical feature for HCMV as for other herpesviruses. Reactivations do occur and thus, clinical disease may either be caused by exogenous infection or by endogenous reinfection. It has to be discussed what the clinical signs are that allow the diagnosis of a recurrence: usually a fourfold rise in the C F antibody titer is considered pathognomonic. In pregnancy, women do not present early enough for paired sera to be drawn during the first trimester. Griffiths et al. (1982) have shown that primary infection with HCMV in the first trimester of pregnancy can be diagnosed by testing a single serum sample by RIA for IgM antibodies. It is often difficult to decide if a clinical syndrome is caused by reactivation of latent virus or by de n o w infection. It is also not quite clear if the presence of antibodies (and latent virus) is totally excluding exogenous reinfection with another strain. There is marked heterogeneity between different HCMV strains and perhaps there is not

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a complete cross-protection between different strains. Newborns do get infected despite the presence of passively acquired antibodies. Perhaps even in the adult, the presence of antibodies is not protective against exogenous infection and perhaps all “recurrences” are de nooo infections. Finally, as we will discuss below, perhaps the number of seropositive individuals is somewhat higher than presently appreciated, because of the lack of sensitivity of the antibody tests used. Thus, a certain number of infections that are considered to be “primary” may, in fact, be reinfections. VI. Diagnosis of HCMV Infection

A variety of techniques are available for the diagnosis of infection with HCMV. For surveys of populations usually complement fixing (CF) antibodies are determined, although there have been some doubts about the sensitivity of this assay. For example, some subclasses of IgG do not fix complement and certain sera show anticomplementary activity. The failure of CF tests to detect antibodies in congenitally infected infants during the neonatal period was thought to be dependent on the lack of sensitivity of complement fixation for the IgM antibody (Dudgeon et al., 1969). Alternative methods for the determination of antibodies are discussed in Section XIV. An exhaustive study of CF, immunofluorescent, and neutralizing antibodies in HCMV infections has been presented by Krech et al. (1971~). It is believed that seropositivity by these assays indicates previous infection whereas seronegative individuals have not been infected as yet. However, data of Waner et al. (1973) have indicated that there are oscillations of antibody titers and perhaps at certain times antibody titers fall below the detection level and thus patients may be classified as seronegative, despite the fact that they have had a previous infection. However, in the study of Yeager (1975), no fourfold rises or falls in titer were seen over a 19 to 27 month period among 71 persons with initially positive HCMV C F titers. It is generally accepted that the documentation of IgM antibodies indicates recent infection. It has been found that about 1%of all newborns possess IgM antibodies against HCMV indicating that they are infected (IgM antibodies do not cross the placenta). Reynolds et al. (1974)found 18 patients with inapparent HCMV infections among 267 neonates with elevated umbilical cord IgM levels. All newborns born to seropositive mothers have passively acquired antibodies of the IgG class. Interestingly, a large number of babies get

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infected at birth or shortly afterwards despite the presence of maternal IgC antibodies and they continue to shed virus during the first year of life despite the fact that they have developed their own IgM (and IgG) antibodies. HCMV infection is proven by the detection of the virus in tissue culture. For that purpose the test fluid is inoculated into tissue cultures of fibroblasts that are permissive for HCMV. The diagnosis is made from the typical cytopathic effect which may take several weeks to develop. HCMV is most commonly found in the urine and also quite frequently in saliva. It has already been mentioned that HCMV can also be found in semen and cervical fluid. Isolation of the virus from urine has been shown to be the most sensitive means of detecting HCMV infection (Hanshaw, 1971). For fast detection of HCMV in the urine, electron microscopy has been proposed (Lee et al., 1978). This rapid detection method, however, should be complemented by tissue culture, since there is no way of differentiating between different herpesviruses by morphology. However, besides HCMV, no other herpesvirus appears to occur in the urine. There has been a report that laboratory strains of HCMV are relatively unstable at 4°C (Vonka and Benyesh-Melnick, 1966). However, Feldman (1968)has shown that there is clearly sufficient time for isolation of wild-type HCMV from urine even after storage of a sterile specimen of urine for several days after collection. During acute HCMV-IM, HCMV may be detected in the blood, where it is usually found to be associated with the cellular components and not with the serum. Armstrong et al. (1971) have isolated HCMV from the erythrocyte layer on repeated occasions. However, it could be that the erythrocytes were contaminated with leukocytes. It has been claimed (Diosi et al., 1969), but refuted by extensive investigations (Mircovic et al., 1971; Perham et aZ., 1971) that infectious HCMV may also be found in the blood of apparently healthy blood donors. The diagnosis of acute HCMV-IM can be clinically suspected. However, it closely resembles EBV-IM. For differential diagnosis it is necessary to perform the test for heterophile antibodies, which is usually negative in HCMV-IM. Honvitz et al. (1977), during a 50-month period, diagnosed heterophile antibody-negative IM in 43 patients. EBV-related serologic tests revealed that 7 patients had primary EBV infections, whereas 30 cases were due to HCMV infection. Of the remaining six cases, one was due to rubella, one to toxoplasmosis, and four were of undetermined etiology. Lemon et al. (1979) have presented data on active

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dual infection with HCMV and EBV and have suggested the possibility of multiple infections whenever determining the specific viral etiology of heterophile-negative IM. VII. Clinical Significance of HCMV Infections

Although HCMV is an ubiquituous virus and although infections with HCMV are often clinically inapparent, there have been a number of diseases associated with HCMV. The full range of clinical impIications has probably not been exhausted as yet. Within the scope of this article we shall discuss only the most important clinical entities and their implications for immunobiology, including the following: (1) HCMV as a cause of congenital diseases and of diseases acquired around birth; (2) HCMV-induced IM; (3) HCMV infections in transplant recipients; (4) HCMV infections in patients receiving immunosuppressive therapy; and (5) HCMV infections in cancer patients. In a separate section we shall discuss the suspected role of HCMV as a causative agent of neoplastic disease, particularly with respect to Kaposi's sarcoma. A. HCMV AS A CAUSEOF CONGENITAL DISEASES AND OF DISEASES ACQUIREDAROUND BIRTH HCMV is the most commonly recognized cause of viral-induced psychomotor retardations. Approximately 1%of newborn infants are infected and at least 10%of these will have some degrees of central nervous system dysfunction (Hanshaw, 1971). Intrauterine infections with HCMV may result in fetal growth retardation, embryopathy, and central nervous system involvement with subsequent perceptual and cognitive disabilities. Typical cytomegalic inclusion disease (CID) in the newborn or young infant, as a manifestation of transplacental infection with HCMV, may include hepatosplenomegaly, jaundice, thrombocytopenic purpura, microcephaly, and/or mental retardation (Weller and Hanshaw, 1962). Congenital HCMV infection may be clinically inapparent at birth (Stan et aE., 1970) and sequela of infection may only appear months or years later (Reynolds et al., 1974; Hanshaw et al., 1976). Children with symptomatic congenital infection are at very high risk for handicaps that will significantly impair development. Birnbaum et al. (1969)have studied 545 newborn children. Three of these were excreting HCMV in the urine during the first' 24 hr of life, but none of the three had classical CID. Larke et al. (1980) have

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studied 15,212 infants born in Hamilton, Ontario, Canada during a period of 44 consecutive months. Congenital HCMV infection was diagnosed in 64 infants, but only 4 had clinical or laboratory symptoms in the first week of life that suggested CID. Mothers of HCMVpositive infants were predominantly younger, primiparous women of lower educational and economic status, and the number who were unmarried was about threefold greater than among mothers of uninfected infants. The study of Larke et aZ. (1980)was continued by Saigal et al. (1982) who followed up on the infected infants. Of 64 infants, there were 3 deaths of 11 infants that could be located, 1 infant had quadriplegic cerebral palsy, and 7 had varying degrees of sensorineural hearing loss. Thus, this study confirms the high incidence of audiological problems in infants with HCMV reported by others (Pass et al., 1980b). In addition, it appeared as if behavioral problems were significantly greater in the patient group of Saigal et al. (1982) than in the matched controls. Besides intrauterine infection, infections during delivery or shortly after birth appear to be common (Reynolds et al., 1973). Excretion of HCMV via the breast milk has been documented (Stagno et aZ.,1980) but there may be other sources of infection for the newborn baby. There is some indication that infection at birth, although asymptomatic at first, may have significant later clinical sequels. Viruria in these children is almost uniformly detected at 3 to 6 months of age. Bray et aE. (1981)described two infants who presented with clinical and virological signs of HCMV infection. Progressive destructive changes in brain parenchyma were reported. The authors suggested that HCMV infection may act like other “slow” virus infections rather than as an isolated, nonprogressive teratogenic insult. In regard to intrauterine infection with HCMV there are several pressing problems. The first important question to ask is why less than 1% of all infants get infected in utero despite the wide-spread occurrence of the virus in the mothers. Depending on socioepidemic conditions and/or geographic patterns, 50 to almost 100% of all women are seropositive, indicating previous infection. One would tend to assume that many of these women also harbor a latent virus that may potentially be reactivated. The latter point, however, is not proven. Perhaps primary infection with HCMV leads to latency of the virus only in some women and not in others and only the former are the ones that activate the virus in pregnancy. Schopfer et al. (1978)have studied HCMV infections in infants in an

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African population in which all adults had experienced previous infection. Of 2032 newborns 28 had viruria, a rate of 1.4%congenital HCMV infection. Either reactivation of latent maternal HCMV infection or recurrence of infection during pregnancy despite the presence of antibodies may explain these findings. Excretion of the virus from the cervix or in the urine has been documented in a large number (about 10%)of all pregnant (and nonpregnant) women (Stagno et al., 1975). Perhaps, the women that suffer from primary infection during pregnancy infect the fetuses. If this were the case, of course, vaccination would be expected to be of great clinical significance to prevent primary infection. However, obviously connatal HCMV infections do occur in babies born to women seroimmune to HCMV prior to pregnancy (Stagno et al., 1977). One still may argue that the possession of antibodies does not sufficiently protect against de no00 infection from a different strain. In this case, however, one may expect even less from vaccination with an adapted laboratory strain of HCMV which in its properties may be very distinct from wild-type strains. Stagno et at. (1977)examined the offspring of 239 women. Intrauterine infection with HCMV occurred in 7 of 208 seroimmune women. Three neonates with congenital infection were born to 31 initially seronegative women. All the congenitally infected infants had subclinical involvement. This study makes it clear that large prospective studies involving thousands of births will be required to decide if primary infections lead to more serious sequels than reactivated endogenous infections. Also very sensitive antibody tests should be used in such prospective studies. Again, the question has to be stressed, why only 10%of all women reactivate HCMV. Interestingly, virus shedding from the cervix is found only in women under the age of 30 (Knox et al., 1979). This observation appears not to be related to hormonal status or similar problems, since in male homosexuals viral excretion also is no longer seen in individuals above the age of 30 (Drew et al., 1981). Thus age per se appears to be important in an as yet unexplained fashion; there may be a gradual build up of immune forces which finally takes care of virus shedding. Altern’atively,the virus loses virulence and eventually the virus infection “burns out.” All of these issues are totally unexplained. Finally, it appears from the work of Stagno et al. (1975) that a different possibility exists to explain the situation. These authors have reported that the percentage of women shedding HCMV is identical in late pregnancy and in nonpregnant women. They observed a suppres-

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sion of excretion (1.6 vs 9.5%) in early pregnancy. Thus there may be defense mechanisms that normally prevent virus reactivation in early pregnancy to protect the fetus. Only if these are impaired, the possibility of infection of the fetus may ensue. Interestingly, the percentage of women shedding HCMV in early pregnancy (1.6%) is quite close to the percentage of infected newborns observed (0.5-1.5%). Recently, very important data have been published by Huang et al. (1980).By means of restriction enzyme analysis of purified viral DNA, these authors have shown that, although HCMV strains from unrelated persons were always different, strains from five of six congenitally infected babies were identical to or very closely related to those from their mothers.

B. INFECTIOUS MONONUCLEOSIS CAUSED BY HCMV (HCMV-IM) Infection with HCMV in adults may cause a clinical syndrome which closely resembles infectious mononucleosis caused by EBV (EBV-IM).In fact, in individual patients the two syndromes cannot be distinguished clinically. The symptoms observed in both diseases may be anemia, fever, hepatosplenomegaly, jaundice, malaise, etc. In the peripheral blood one finds the so-called “atypical” lymphocytes, as in other virus infections. Statistically, EBV-IM is more often associated with pharyngitis and lymphadenopathy. Differential diagnosis requires the documentation of heterophile antibodies which only occur in EBV-IM. Obviously, the production of heterophile antibodies is part of the polyclonal activation of B lymphocytes by EBV, which includes the clone that produces antibodies to foreign erythrocytes. It is not known why this clone is not activated in HCMV-IM. However, quite a number of other antibodies that are not produced in healthy individuals, particularly autoantibodies, are found in HCMV-IM (Kantor et al., 1970). However, it is totally unknown if HCMV, similarly to EBV, activates B cells. The widely quoted “civil war” of lymphocytes may apply only to EBV and not to HCMV. However, the similarity between the clinical pictures is striking and cytotoxic T cells that recognize virus-infected cells may play a role in both diseases. Generally one distinguishes between community-acquired disease, in which the mode of infection is unknown, and iatrogenic infection, which is observed in patients receiving massive blood transfusions, for example, during cardiac bypass surgery. Caul et al. (1972a) have reported that HCMV disease was observed only in patients receiving more than five units of blood. As reported by Tolkoff-Rubin et aZ.

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(1978) transfusion of frozen, deglycerolized erythrocytes that were free of leukocytes minimized the risk of HCMV-IM. Below, we will discuss some ideas about the role of blood transfusion in HCMV-IM. Besides these patients, HCMV-IM is sporadically observed in 0thenvise healthy adults (“community-acquired IM”). Presently, it is not known how infection is transmitted in these cases. Interestingly they occur predominantly in patients older than 30 years (Stern, 1968). We have stated above that virus shedding is not observed in women older than 30 years and that it appears as if at this age immune forces have been built up that are able to control the virus. It is tempting to speculate that primary infection with HCMV in individuals over 30 years of age resembles the situation observed in young adolescents upon primary infection with EBV, in that it represents an example of an effective immune response against primary virus infection which includes a transient immunopathological syndrome. Primary infections acquired in earlier postnatal life are inapparent. C. HCMV INFECTIONS IN TRANSPLANT RECIPIENTS The adverse effects of HCMV on the course of patients receiving transplants may be divided into three categories: (1) infectious disease syndromes produced by the virus itself; (2) a rather global suppressant effect on host defenses that predisposes that host to potentially lethal superinfection (see Rubin et al., 1977, for a discussion of this aspect); and (3) a possible effect in producing graft rejection. HCMV infection occurs in up to 90% of renal transplant recipients (e.g., Rifkind et al., 1967; Pass et al., 1978). The infection tends to occur early in the posttransplant course (6-15 weeks) and is associated with significant morbidity and mortality. Fiala et al. (1975) found active HCMV infection in 96% of patients after renal transplantation. HCMV viremia developed in 42% of patients an average of 2 months after renal transplantation, lasted about 2 months, and was followed by chronic viruria. Recently, a number of late posttransplant HCMV infections ‘have been observed, occasionally associated with serious complications (retinitis, liver failure, pneumonia, death) (Matas et al.,

1981). Pass et al. (1978) have suggested that the major factor in initiating and maintaining productive infection with HCMV was the host-vsgraft reaction. Naraqi et a,?. (1977) have concluded that immunosuppressive drugs, possibly augmented by a graft rejection response, account for the high incidence of recrudescent infections with HCMV. Rubin et al. (1981) reported that when antithymocyte globulin was

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given, in addition to conventional immunosuppression, there was an increased incidence of viremia and of clinical syndromes attributable to HCMV. Simmons et al. (1977) identified two patterns of HCMV infection following renal transplantation. In both patterns, fever and leukopenia occur within 6 months after the transplant. In addition, the benign form is characterized by renal biopsy evidence of rejection and brisk antibody responses to HCMV. The lethal syndrome runs a typical 4 week course, beginning with prostration, orthostatic hypotension, and progressing to severe pulmonary and hepatic dysfunction, central nervous system depression, and death. Antibody responses to HCMV are minimal. Again an important question (in regard to the usefulness of vaccination) is how many of the infections in renal transplant recipients are primary ones and how many of them represent reinfections. Suwansirikul et al. (1977) followed 47 patients who underwent renal transplantation. There were 18 cases of primary infection and 10 cases of secondary infection. These findings were based on whether the patient was seronegative or seropositive prior to transplantation. However, the antibody tests may not have been sensitive enough. Pien et al. (1973)have observed a fourfold or greater increase in C F antibodies against HCMV in eight of eight patients with a pretransplantation HCMV C F antibody titer 2 1:4; only two of nine patients with no detectable pretransplantation titer subsequently developed HCMV-CF antibodies. Lopez et al. (1974a) observed an association between renal allograft rejection and herpesviruses, especially CMV infection. However, such a relationship is difficult to establish or to exclude because both events occur so frequently after transplantation. May et al. (1978) performed an analysis of the effects of HCMV infection and HLA antigen matching on the outcome of renal transplantation. They concluded that HCMV infection had a more marked influence on allograft survival than did HLA antigen matching. Andrus et al. (1981)suggested that HCMV interferes with the beneficial effects of transfusion in renal transplant recipients. They have further shown that ABO type-0 individuals are relatively resistant to infection with HCMV. This observation may be very important since it is the first indication that susceptibility to HCMV in man may be genetically controlled. Glazer et al. (1979) vaccinated 12 seronegative renal transplant candidates with Towne 125 strains of live human cytomegalovirus. All vaccinees seroconverted and 10 of them underwent transplantation.

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Although HCMV was isolated from 6 patients after transplantation, the restriction endonuclease patterns of the viral DNA of these isolates differed significantly from those of the vaccine strain. Therefore, it appeared that the vaccine strain did not become latent in the host, at least not in a form that could be reactivated. One wonders, however, what the usefulness of a vaccine is, if the patients were nevertheless subsequently infected with wild-type strains of HCMV. HCMV infection is the most common and most important infection that occurs after allogeneic marrow transplantation. Although fever, arthralgias, arthritis, and hepatitis are associated, its most significant manifestation is disseminated infection with pneumonia. The incidence of HCMV pneumonia is about 20%and the mortality has been consistently >80% for histologically or virologically proven HCMV pneumonia (Neiman et al., 1977). The incidence of interstitial pneumonia in patients after syngeneic transplantation is much lower than that in allogeneic recipients and no cases have yet been associated with HCMV infection. The absence of any known pathogen from about 40% of the pneumonias in bone marrow transplant recipients is very noteworthy (Neiman et al., 1977). Meyers et al. (1980a) reported that the incidence of HCMV infection after allogeneic marrow transplant was high and approximately the same, regardless of the presence or absence of antibody to HCMV before transplant in either donor or recipient. The usual concepts of primary and recurrent infection may not be valid after marrow transplant because of the unusual immunologic situation in which the donor immune system is transferred to the marrow recipient. Specific cell-mediated immunity to herpesviruses after marrow transplant does not recover until the patient experiences an active virus infection. Thus, according to Meyers et al. (1980a), all HCMV infections &er marrow transplant may be considered primary infections.

D. PATIENTS RECEIVINGIMMUNOSUPPRESSIVE THERAPY We have discussed above the high prevalence of HCMV infections in transplant recipients. Based on the presence of serum antibodies against HCMV before transplantation it has been found that about half of the infections are primary, whereas the other half appear to represent reactivations of endogenous virus. However, these estimates may be wrong for a number of reasons (see Section XIV). In transplant patients a complex situation exists since there are a variety of factors that predispose them to virus infection including the following:

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1. The transplant itself may carry infectious (or latent) virus. We have discussed this possibility above for the kidney. Furthermore, transplant recipients often receive blood transfusions as well. 2. Allogeneic interactions of diverse types (rejection, graft-vs-host reaction, etc.) may cause activation of the latent virus. 3. Most transplant recipients receive immunosuppressive medication. One wonders if the latter condition alone is sufficient to cause reactivation of HCMV. Dowling et al. (1976), therefore, studied patients receiving cytotoxic immunosuppressive therapy for a rheumatologic condition. Eight of 14 patients followed after the initiation of therapy became infected with HCMV as demonstrated by a fourfold or greater rise in complement-fixing antibodies, viruria, or both. Seven of these 8 patients were seropositive before therapy suggesting that immunosuppression acts largely by reactivating latent infection. Perhaps the same explanation applies to the transplant recipients, i.e., most of the secondary infections are caused by immunosuppressive drugs. E. HCMV INFECTIONS IN PATIENTS WITH NEOPLASMS HCMV was isolated from 11 of 32 adult patients with leukemia, Hodgkin’s disease, and lymphomas (Duvall et al., 1966). Caul et al. (1972b) found 4 patients with HCMV infection among 22 children receiving therapy for leukemia. However, all 4 patients had received blood transfusions. Since, in addition, the therapy has to be considered as a cause of virus reactivation, this study provides little evidence that leukemia causes significant morbidity in children with HCMV. This conclusion is concordant with the data of BenyeshMelnick et al. (1964)demonstrating that there was no difference in the rate of urinary excretion of HCMV between leukemic children or other chronically ill children and “normal” children of comparable age in the same community. On the other hand, in a retrospective study, Henson et al. (1972) reported on 3 children with acute leukemia in whom CID appeared to be the cause of death. These data were derived from a series of 88 children among whom 24 had cultural evidence of HCMV infection at some point. Again, numerous variables of therapy may have contributed to virus activation besides the underlying leukemia. Thirteen cases of CID occurring in patients with acute leukemia were reported by Bodey et al. (1965). Eleven infections were diagnosed at autopsy within a 13-month period, suggesting the occurrence

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of an “epidemic,” which apparently originated in a 2-year-old child. Of the 13 infected patients 80% were under 10 years old. The clinical features of the 13individuals with CID were compared with a control group and no significant differences were found. Rosen and Hajdu (1971) have identified intranuclear inclusions of HCMV in 19 of 5788 consecutive autopsies of adult cancer patients. This low number again argues against the possibility that cancer per se may be frequently associated with secondary infections with HCMV. VIII. Oncogenic Potential of HCMV

Certain animal herpesviruses are proven tumorviruses, including herpesvirus saimiri, herpesvirus ateles, and the causative agents of Marek’s disease and of Luckk’s adenocarcinoma (Klein, 1972). There are very strong associations between EBV and two human tumors: Burkitt’s lymphoma and undifferentiated carcinoma of the nasopharynx (Klein, 1975). Associations have also been reported for cervical carcinoma with HSV (Melnick et al., 1974). The latter, however, are predominantly based on socioepidemiological data and are far from being conclusive. More recently, antigens induced by HSV-2 were found to be associated with squamous cell carcinoma in situ of the vulva (Kaufman et al., 1981). A. MISCELLANEOUS DATAON THE ROLE OF HCMV IN CANCER Wertheim and Voute (1976) have raised the possibility that neuroblastoma and Wilms tumors are induced by HCMV. They found that sera of 8 of 18 children with Wilms tumor and of 8 of 20 with neuroblastoma were positive for CF antibodies to HCMV. Since the two tumors appear to arise in embryonic life and since intrauterine infections with HCMV do occur, the hypothesis of Wertheim and Voute is interesting. A general theory of carcinogenesis may in fact imply initiation of tumor cells by a virus during a state of immunologic insufficiency as, for example, prenatally, at birth, or shortly after birth. It should be recalled that in the regions in which Burkitt’s lymphoma is frequently found, primary infections with EBV also occur very early in life (Klein, 1975). As it appears (Krech and Tobin, 1981) a high percentage of children in parts of Africa are infected with HCMV early in their life. In these regions, Kaposi’s sarcoma (KS) is a frequent tumor (see below). Hashiro et a2. (1979) isolated HCMV from cell cultures derived from

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3 of 16 surgical specimens of adenocarcinomas of the colon. Viral identification was accomplished through microscopic, cytochemical, and immunofluorescent procedures. Huang and Roche (1978) have investigated by membrane CRNA-DNA hybridization whether HCMV is present in adenocarcinoma of the colon. Four of seven tumors were definitively positive for CMV DNA (containing more than two genome-equivalents per cell) or repeatedly showed more than one genome-equivalent per cell. Melnick et al. (1978) isolated HCMV from cell cultures derived from 2 of 10 cervical cancer biopsies from patients in an advanced stage of the disease. Additional studies of the relation of HCMV infection with cervical neoplasia seem to be indicated. Rapp et al. (1975) reported that cells from prostatic tissue obtained from a 3-year-old donor exhibited scattered foci of cytopathology in primary culture. A virus was isolated and shown to be HCMV. After a number of cell culture passages a cell line was obtained in which virus could no longer be detected. Nucleic acid hybridization studies revealed that virus genetic material was carried by the cells and that the cells contained an average of 10 to 15 genome equivalents of HCMV-DNA. This finding may indicate that the cells have been transformed by HCMV, but it remains to be determined if this finding is largely due to laboratory manipulations or if it reflects an event that also can occur in oioo.

B. KAPOSI’S SARCOMA There has been a great deal written about Kaposi’s sarcoma (KS) during the past 50 years, which cannot be reviewed within the context of this article. A careful account of the older literature has been given in the proceedings of a conference held at Makerere College Uganda in 1961. The proceedings have been published in Acta Unio Znt. Cancrurn 18,1962. Reynolds et al. reviewed the topic of KS in 1965.A review of recent developments has been published by Safai and Good ( 1980). KS is a complex neoplasm, which might originate from endothelial cells and/or from the mononuclear phagocytic system (Anonymous, 1967).Multiple idiopathic pigmented hemangiosarcoma was the term given b y M. Kaposi to this tumor that now carries his name. KS usually presents as dark blue to reddish-purple macules, plaques, or nodules. The lesions are commonly located on the extremities, most often on the feet, but may appear anywhere on the skin or even in the mucous membranes. Lymph nodes and internal organs may be involved.

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The highest incidence of KS, including a cluster type of occurrence, has been reported in equatorial African blacks with a distribution strongly resembling that of African Burkitt’s lymphoma (Oettle, 1962). Maclean (1963) has reported on KS in Nigeria and has documented that KS is no rarity in this portion of the old Slave Coast. It was obvious that those territories from which the ancestors of today’s American blacks were derived contain some factors which provoke or encourage the development of KS (see below). In the United States, KS used to be a rare neoplasm seen predominantly in elderly men. In these men the disease is manifested by skin lesions and a chronic clinical course and it is rarely fatal. Recently, “epidemics” of KS have been reported in young male homosexuals in several American cities (Anonymous, 1982; Ziegler, 1982). In these cases KS appears to have a more malignant course. In these patients many infections are more prevalent than in heterosexuals, most notably Pneumocystis carinii and HCMV. Their respective roles, if they have any, in the genesis of KS will have to be determined. The similarities with Burkitt’s lymphoma are striking in that in both diseases a parasite (Plasmodium falciparum for Burkitt’s lymphoma, Pneumocystis carinii for KS) and a virus have been implicated (see below). The clinical findings in eight young homosexual men in New York with KS showed several unusual features. It affected younger men (fourth decade rather than seventh decade), the skin lesions were generalized rather than being predominantly in the lower limbs, and the disease was more aggressive (survival of less than 20 months rather than 8-13 years) (Hymes et al., 1981). There are a number of additional aspects of KS that are of great interest. First, whereas KS used to be a rare disease in Europe or in the United States, it is a fairIy common disease in Tanzania and Uganda, where it represents 5-10% of all cancers (Slavin et al., 1969; Taylor et al., 1972). In the United States it appears to be rare in nonwhites, whereas in Africa it is found almost exclusively in the black population and rarely ever in Caucasians and Asians. The majority of the patients, both in Uganda (35 of 37) and in Tanzania (108 of 117) have been male. KS occurs most commonly in adults and has a peak age incidence of 30-50 years. Only 4% of the cases occur in children, with a male/female ratio of 3/1. Slavin et al. (1969) have reported on 51 cases of KS in East African children. In African children the disease may have a very different presentation and course from that commonly seen in adults. The frequency of lymph node involvement in African children is particularly striking. KS in nonAfrican children is very rare (Dutz and Stout, 1960).

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Taylor et ul. (1971a) have reported two cases of KS during pregnancy. The low incidence of KS in females has been attributed by some to the influence of sex horniones. In view of its occurrence during pregnancy this assumption appears to be unlikely. There have been no clusters of KS observed among relatives, which argues against a simple Mendelian inheritance. However, the geographic and ethnic distributions referred to above suggest genetic influences plus environmental factors. In African blacks the disease is frequent whereas it is rare, for example, in whites in the Johannesburg area, even though the immigrant population has been present in the community for over 3 centuries (Oettle, 1962). On the other hand, KS has been found in black people in the regions from where the American black people came, but not in American blacks. KS has been recently subclassified into four groups. This, however, was done with African patients (Taylor et al., 1971) and because of the rarity of the disease in other parts of the world, it is not clear if this subclassification is generally applicable. The four subtypes have been termed nodular, florid, infiltrative, and 1ymphadenopathic (see Safai and Good, 1980, for a recent review). The lymphadenopathic form is disseminated and rapidly fatal. Giraldo et ul. (1972b) observed herpes-like virus particles in five of eight tissue culture lines derived from different cases of KS from the Congo and Uganda. In one line preliminary characterization suggested a virus resembling HCMV. These data from 1972 urgently need confirmation. Recent reports by Giraldo et u1. (1980) described CMV DNA sequences and CMV-determined nuclear antigens in KS biopsies and cell cultures of early passage history. Boldogh et ul. (1981) extended these findings by employing more sensitive 32P-labeled viral DNA for reassociation kinetics and in situ cytohybridization analysis. However, in all these studies it has to be considered that the “tumor cells” in KS neoplasms are of uncertain origin and that the tumors are usually composed of different tissues, including normal cells. The basic histologic lesion of KS is a proliferation of angiomatous tissue and plump spindle cells. The relation between the two is not understood. The nature and origin of the spindle cell is still in doubt. Niemi and Mustakallio (1965) investigated the fine structure of the spindle cell and suggested a multipotent perivascular mesenchymal cell (pericyte) for the cell of origin of KS. Dayan and Lewis (1967), from histological studies performed with silver inpregnation, suggested that the tumor originates from the reticuloendothelial tissue.

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Further aspects were discussed in Lancet many years ago (Anonymous, 1967). It appears that the relevant problems have not been resolved since then. It is also not known if KS is of multicentric or of monocentric origin, or in other words if the tumor is of monoclonal origin. Elegant experimental approaches are now available to address this question (Fialkow et al., 1970). For example, one benign virally induced “tumor”, i.e., the common wart, has been shown to be of monoclonal origin. Burkitt’s lymphoma, which shows a strong association with EBV, is also a monoclonal tumor, a finding which has considerable theoretical implications. There have also been extensive seroepidemiological studies in patients with KS. Giraldo et al. (1975, 1978) detected a specific serologic association of HCMV with European and American KS patients. In African patients the significance of the antibody titers was unclear due to the high background of HCMV infections in the control groups. There are also interesting connections between KS and the immune system. Although immune surveillance in the classical sense (as being a T cell-mediated mechanism) is probably nonexistent (Stutman, 1980), it is remarkable that KS, like other tumors of the lymphoreticular system, occurs in an increased frequency in transplant recipients (Penn, 1979). Any such patient who develops reddish-blue macules or plaques in the skin or oropharyngeal mucosa or has apparently infected granulomas that fail to heal should be suspected of having KS . The association of KS with other cancers, especially of the lymphoreticular system, has been frequently noted. Safai et al. (1980) reported a 20-fold increase in the incidence of lymphoreticular tumors after diagnosis of KS. It is also of interest that KS belongs to the class of tumors of which spontaneous regression has been reported (Anonymous, 1967). A case of KS in a patient, who has received a cadaveric renal transplant, was reported to show regression of extensive cutaneous and suspected pulmonary disease after therapy with bleomycine and vincristine (Hardy e t al., 1976). Thus, although many obstacles exist to a better understanding of the role of the defense system in the pathogenesis of tumors, studies of cellular immunity and/or other defense systems in patients with KS are of the greatest interest. Taylor and Ziegler (1974) sought defects in cellular immunocompetence in 25 patients with KS. Skin tests with recall antigens and phytohemagglutin (PHA) stimulation of lymphocytes in vitro were normal.

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However, attempted sensitization and subsequent challenge with dichloronitrobenzene (DCNB) demonstrated that the afferent limb of the response was impaired in some patients. Dobozy et al. (1973) reported similar data on six Hungarian patients with histologically confirmed KS. Taylor et al. ( 1 9 7 1 ~studied ) lymphocyte transformation in patients with KS. Lymphocytes from 7 of 21 patients showed increased transformation in cultures containing tumor cells and 6 of these 7 had nodular or infiltrative disease. An impaired response to both PHA and tumor cells was noted in patients with florid tumors, a form thought to be associated with a diminished host response. Recently an “epidemic” of KS was reported in homosexual men living in New York and California (Ziegler, 1982). Many of these patients are profoundly immunosuppressed and are afflicted with opportunistic infections such as Pneumocystis carinii pneumonia. So far at least a dozen microbial species have been shown to cause disease in these patients, including HCMV, HSV, bacteria, fungi, and protozoa (Durack, 1981). At this writing three reports have appeared, collectively indicating that this is a newly detected form of a severe acquired immunodeficiency, that predominantly occurs in previously healthy male homosexuals (Gottlieb et al., 1981; Masur et al., 1981; Siegal et al., 1981). Further reports will probably follow. Siegal et al. (1981) reported on four homosexual men presenting with gradually enlarging perianal ulcers from which HSV was cultured. Pneumocystis carinii pneumonia was the predominant clinical feature in the patients of Gottlieb et al. (1981) and of Masur et al. (1981).In each of the two latter series there was one case of KS. There is no doubt, that these patients are massively immunosuppressed, as for example, evidenced by a defect in natural killer (NK) cell activity (Siegal et al., 1981).An interesting finding is the virtual elimination of the helpedinducer subset of T lymphocytes, accompanied by an increase in the suppressor/cytotoxic subset (Gottlieb et al., 1981). It is, of course, of greatest concern to explore the etiology of this “new” disease entity. Obviously homosexuality is not a recent habit, and the pathogens, particularly HCMV, have been with humans for millions of years. Thus, the report that up to 88% of KS patients report regular use of nitrite inhalants is of greatest interest. Inhaled nitrites could combine with secondary amines to form highly carcinogenic nitrosamines (Ziegler, 1982).

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C. In Vitro MALIGNANTTRANSFORMATION OF CELLSBY HCMV There has been evidence for an oncogenic potential of HCMV from animal experiments. Albrecht and Rapp (1973) established a hamster embryonic fibroblast cell line which is transformed by UV-irradiated HCMV and is oncogenic in weanling Syrian hamsters. These data are important but it is obviously not possible to extrapolate from cell transformation studies in rodents to an oncogenic potential in man. Furthermore, there are serious problems with C-type viruses in such studies, as studies with HSV have documented (Hampar et al., 1976). Cellular transformation by HSV has been the subject of much controversy. Among other mechanisms, a “hit and run”-type phenomenon has been considered (Hampar, 1982). Reed and Rapp (1976) have reported that the Birch strain of HCMV enhanced the expression of murine leukemia virus P 30 expression in random bred Swiss/3T3A cells. Important data have been reported by Nelson et al. (1982). NIH 3T3 cells were transfected with cloned HCMV DNA fragments in order to identify the transforming region. This region was in the HindIII E fragment with the left boundary defined by the EcoRI-d-R junction and the right boundary by the HindIII E-T junction. IX. lmmunopathology

Viral immunopathology includes the kinds of diseases not caused by the virus itself but by the immune reaction of the body against the viral infection (Nathanson et al., 1975). A classical example is the infection of mice with lymphocytic choriomeningitis (LCM) virus. Encephalitis is caused by the immune reactions and therefore is prevented by immunosuppression, for example, when cyclophosphamide is injected. For the group of the herpesviruses, immunopathological syndromes in the usual sense, are not typical. For example, there is no indication that such symptoms play a role in the pathobiology of the infections with HSV or VZV. However, the situation appears to differ with respect to EBV and HCMV. It is thought that during acute primary infection in early adulthood EBV infects the B lymphocytes and that many of the symptoms in the patients are caused by the immune response against the virus-infected cells (Klein, 1975). For example, it has been found that the majority of the so-called atypical lymphocytes in the blood of EBV-IM patients represent T cells of the killer cell

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subtype that are directed against the infected B lymphocytes (Pattengale et al., 1974). Atypical lymphocytes are found in many other viral infections as well. Thus, the observation that they are found in HCMV-IM does not necessarily imply that HCMV infects B lymphocytes (Ho, 1981). It is widely held that infection with HCMV causes immunosuppression. Such immunosuppression may be detected by in uitro testing of specific cell-mediated immunity (see below). However, besides a defect in the tests of specific cell-mediated immunity a more general defect appears to occur which is, for example, manifested in a suppression of lymphocyte reactivity to general mitogens (e.g., Rinaldo et al., 1980). A most important cause of morbidity and mortality in transplant recipients in interstitial pneumonia, which may or may not be associated with detectable CMV infection. In a study of 80 bone marrow transplant recipients, there were 43 episodes of interstitial pneumonia, 28 of which were lethal. About 40%of the cases were idiopathic. CMV was the most common candidate pathogen, present in 47% of affected lungs (Nieman et al., 1977). Abdallah et aZ. (1976) presented evidence, which may indicate that in immunosuppressed patients HCMV can cause significant pulmonary disease alone, leading to respiratory failure and death. It is still a matter of dispute if HCMV, per se, is causing pneumonia or if immunosuppression by HCMV facilitates infection by other pathogens. In recipients of renal transplants it has been reported by Chatterjee et al. (1978)that death from pneumonia occurred in four of six patients who were seronegative for HCMV before transplantation and subsequently seroconverted. It is noteworthy, however, that these patients died from infections with Candida or Aspergillus. Thus, this report reinforces the possibility that HCMV infections cause immunosuppression which facilitates subsequent infection with opportunistic pathogens. Rubin et al. (1977) have also postulated that the major infectious disease importance of HCMV in renal transplant patients is its effect on host defense in predisposing the host to life-threatening infection with other microbial agents. Rand et al. (1978) have shown that cardiac transplant patients, who were seronegative for HCMV before transplantation, had both a significantly greater overall mortality and a higher bacterial pulmonary infection rate than those who were seropositive. Finally, Bale et al. (1980)provided additional evidence that, in children, HCMV infection may predispose the host to secondary infection. A variety of immunologic abnormalities in patients with HCMV-IM

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have been described by Kantor et al. (1970).In three anemic subjects erythrocyte autoantibodies were found. Additional immunologic aberrations included rheumatoid factor, antinuclear antibodies, cold agglutinins, and cryoglobulins which were detected in 9 of the 10 patients. X. LatencyIReactivation

The hallmark of infection with herpesviruses is the latency phenomenon and the possibility of periodic reactivation. A typical example for a latent herpesvirus infection is herpes labialis, the so-called fever blister, caused by HSV. It is widely held that the recurrences observed after various types of exogenous or endogenous stimuli are caused by reactivation of HSV in the trigeminal ganglion. The virus then travels down the nerve to cause a lesion in the skin close to the lip. These lesions are usually self-limited. There is not much evidence that links the development of the recurrence itself with immunologic factors; however, the local restriction of the lesion is probably a function of the immune system. Generalized infections do occur in immunosuppressed individuals (Kirchner, 1982). It is still a major challenge for molecular virologists to explore the molecular basis of latency of HSV in neural tissue (Roizman, 1974). It is well established, however, that infectious HSV cannot be recovered from ganglionic tissue, but prolonged in vitro cocultivation of the ganglionic tissue with cell cultures permissive for HSV is required to recover the virus. There is little doubt that HCMV is also capable of causing latent infection in the human organism and that a number of clinical syndromes associated with HCMV are caused by reactivation of the latent virus. Thus, in the recipients of kidney grafts, according to current estimates, about 50% of all HCMV infections represent reactivated endogenous infections. There has been a controversy over whether intrauterine infections of the fetus are due to primary or secondary infections of the mothers. We have discussed this issue above and have expressed the view that the evidence appears to support a predominant role of reactivated HCMV as a cause of intrauterine infection. Huang et al. (1980) have, by the techniques of “molecular epidemiology,” clearly shown that HCMV strains from five of six congenitally infected babies were identical or very closely related to those from their mothers. Strains from congenitally infected siblings were also concordant as were repeat isolates from three of four women.

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During acute infections, i.e., IM, HCMV can be recovered from the white blood cells that are acutely infected. Also, it appears that HCMV may be transmitted by blood transfusions although several recent careful investigations have failed to detect infectious virus in fresh blood donations (Perham et aZ., 1971; Mircovic et al., 1971). A possible explanation will be discussed below which implies that HCMV is latent in white blood cells of a yet unknown type and is reactivated by allogeneic stimulation. This hypothesis, however, is far from being proven. There are many gaps in our knowledge regarding the site of latency of HCMV. HCMV has been detected in a variety of different body excretions including cervical fluid, urine, semen, and saliva. Thus, HCMV appears to be harbored in the organs producing these fluids. Postmortem data have been presented by pathologists which document histological signs of HCMV infection in many additional organs that clinically have not appeared to be afflicted. Thus, HCMV infection is often more widespread than is clinically appreciated. XI. Replication of HCMV in Leukocytes

We have previously reviewed the relationship between HSV and leukocytes (Kirchner, 1982). HSV is generally considered to be a neurotropic virus but obviously it is lymphotropic as well, at least in vitro. HSV can be replicated both in T cells and in B cells, provided that the cells are preactivated by appropriate stimuli, for example, phytomitogens in the case of T lymphocytes (Nahmias et al., 1964; Kirchner et al., 1977).There is also some evidence to indicate that HSV replicates in cells of the monocyte/macrophage series (Daniels et al., 1978). EBV, in contrast to HSV, appears to have an exclusive target within the lymphoid system, the human B lymphocyte. EBV causes polyclonal activation and immortalization of B lymphocytes. Long-term lines of transformed B cells also produce low titers of EBV. From a number of reports it appears that HCMV can be isolated from human blood and that the virus in these instances is associated with “white blood cells.” However, the exact cell type that is infected with HCMV and the state of the virus in these cells need to be defined. It is of interest that HCMV also has been isolated from lymph nodes. Stulberg et aZ. (1966) reported on the isolation of HCMV from biopsied lymph nodes of two patients with hemolytic anemia. The approach, successfully used to replicate HSV in lymphocytes, i.e., preactivation with mitogens, has not supported significant repli-

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cation of HCMV (Rinaldo et al., 1978). Perhaps such experiments should be repeated using a variety of different strains of HCMV, including primary virus isolates. Furthermore, further sophistication of leukocyte culture techniques may also aid experimentation. In lymphoblastoid celI lines divergent results have been obtained. Rinaldo et al. (1978) failed to detect significant replication of HCMV in lymphoid cell lines of either T or B cell type or in hemic lines. In contrast, St. Jeor and Weisser (1977)and Furukawa (1979)successfully established “chronic” infections of lymphoid cells. These infections were different from the typical HCMV replication as observed in permissive human fibroblast cultures and rather resembled the type of infection observed in nonpermissive epithelial cells. Initially, Huang and Pagano (1974) suggested that HCMV replicates only in lymphoid cell lines that carry the EBV genome; however, the results of Tocci and St. Jeor (1979) have shown that chronic infection with HCMV occurs regardless of whether the lymphoid lines carry EBV genomes or not. The possibility of transmission of HCMV by blood transfusions has been investigated in a large number of laboratories. In 1975 a conference was held, the proceedings of which have been published in the YaleJournal of Biology and Medicine, 49,1976. The reader is referred to these papers for a review of the older literature. Transfusion of blood is sometimes followed by HCMV mononucleosis in the recipients, particularly if large amounts of blood have been transfused. There are basically three possibilities to explain this finding: 1. Stimulation of the recipient’s lymphocytes by the allogeneic leukocytes leads to activation of HCMV. This conclusion is less likely since HCMV mononucleosis has been reported to represent a primary infection (Lang and Hanshaw, 1969). Caul et al. (1971) have shown that patients with preoperative anti-HCMV C F antibody levels of more than 128 did not get infected. However, those with lower levels were infected as commonly as those without antibody. 2. The reverse situation may also be true; allogeneic interaction between these cells may lead to the activation of HCMV in the donor leukocytes. 3. Finally, there may be a simple transfer of cell-associated HCMV with the transfused blood without any need for activation of the white blood cells. The simple transfer of infectious particles with the transfused blood

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appears to be less likely. Although there has been an initial report of recovery of HCMV in donated blood from 2 of 35 individuals (Diosi et al., 1969), there have been several subsequent investigations that have completely failed to isolate HCMV from blood donations (in one study 223 blood donors were tested, in the other 290) (Mircovic et al., 1971; Kane et al., 1975). These negative results weigh heavily since they were obtained despite techniques that unequivocally yielded positive results when blood samples of patients with HCMV-IM were tested. There are many reports indicating that HCMV associated with WBC of these patients can be documented in cocultures of the leukocytes with permissive fibroblasts. It is also of interest that in whole blood under banking conditions experimentally inoculated HCMV was recovered after 28 days (Armstrong et al., 1971). In our opinion the second of the three possibilities mentioned above is the most likely one. Thus, we speculate that HCMV is transferred with the leukocytes but released only after these have been activated by confrontation with histoincompatible cells. This hypothesis can be tested in ljitro by cocultivation of mixtures of cells from different donors or by including agents in the assay that are known to activate lymphocytes, for example, mitogens. Option 2 is actually implied in the hypothesis published by Lang (1972). However, the reviewer failed to trace experiments in the literature that provide evidence that activation of lymphocytes by alloantigens or mitogens liberates infectious HCMV. There are a number ofadditional interesting aspects in regard to the transmission of HCMV by blood transfusions; it has been reported that transfusion of leukocyte-depleted blood was unable to cause infection in the recipient (Lang et al., 1977). This observation points to the role of leukocytes in transmission. Furthermore, in the study of Caul et al. (1971), only the recipients of five or more units of blood became infected. Also in the study of Prince et aZ. (1971)a positive correlation was observed between the volume of blood transfused and the risk of HCMV seroconversion. Although some authors believe that the chance of transmission of infection is greater if the units of blood are less than 24 hr old, others have shown no difference in the ability to transmit infection from units that are less than 3 days old as compared with those more than 3 days old (Prince et al., 1971). It appears that the transfusion of blood containing HCMV is particularly dangerous to newborns. Yeager et al. (1981)reported that newborns of seronegative mothers (and thus possessing no passively acquired antibodies) acquired lethal HCMV disease by blood

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transfusion. Thus, newborns should be transfused with blood from HCMV-seronegative donors, since they appear to be particularly vulnerable to systemic infection with HCMV. The data of St. Jeor and Weisser (1977), of Tocci and St. Jeor (1979), and of Furukawa (1979), referred to above, have documented infection of lymphoblastoid cells by HCMV. The infection observed was a chronic type, with low numbers of positive cells in infectious center assays. Overall viability of the cell cultures was not grossly altered, There are no reports of any other type of infection of human leukocytes by HCMV. There is no indication that HCMV is able to transform lymphocytes as EBV does with human B lymphocytes. Furthermore, there are no data on abortive replication of HCMV in macrophages such as has been reported for HSV and murine macrophages. It is totally unknown if HCMV may establish a latent infection in cellular components of the immune system. To our knowledge there has been only one report in which the HCMV genome has been found unexpressed in a lymphoblastoid cell line (Joncas et al., 1975). Finally, it needs to be determined which subpopulations of “leukocytes” harbor HCMV. Most investigators find that the titers of HCMV are associated with the mononuclear cell fraction of the blood cells of patients having viremia. Fiala et al. (1975) observed higher titers in the polymorphonuclear than in the mononuclear leukocyte fractions. Because of possible crosscontaminations more sophisticated separation techniques need to be used in the future. XII. Effects of HCMV on Leukocytes

Significant HCMV disease is most frequently seen in immunosuppressed patients. HCMV is probably immunosuppressive in uiuo, as discussed in Section IX. There are several reports that human herpesviruses, particularly HSV, affect the functions of lymphocytes and/or monocytes, which we have reviewed recently (Kirchner, 1982). Such experiments have, for example, shown that HSV in uitro inhibits lymphoproliferation induced by PHA (Plaeger-Marshall and Smith, 1978).We have failed to find similar in uitro experiments with HCMV in the Iiterature. Such experiments may in fact provide significant clues to the immunobiology of infection with HCMV. There are, however, two serious drawbacks associated with such experiments, as we have recently observed (unpublished data). First, many existing HCMV strains are contaminated with mycoplasmas and mycoplasmas have significant effects on lymphocytes. For example, they are mitogenic for lymphocytes (Gins-

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burg and Nicolet, 1973)and induce interferon production in lymphocyte cultures (Cole et al., 1976). Thus, it is an absolute requirement to have mycoplasma-free virus strains for studies of the effects of HCMV on lymphocytes. Second, it has to be considered that HCMV itself induces interferon of diverse subtypes in cultures of white blood cells (to be discussed in detail below). Interferon has profound effects on lymphocytes and/or macrophages as, for example, an inhibition of lymphoproliferation or an enhancement of the expression of certain membrane proteins (Gresser, 1977). Interesting data in regard to the T cell system in patients with HCMV-IM have been described by the group of M. Hirsch (Rinaldo et al., 1977, 1980; Levin et al., 1979; Carney and Hirsch, 1981). The patients, besides showing a delayed development of the specific lymphoproliferative response to HCMV (see below), have depressed responses in the lymphocyte proliferation (LP) test when HSV or VZV antigens were tested. Furthermore, the responses to pokeweed mitogen (PWM) or concanavalin A (Con A) were depressed, whereas PHAinduced lymphoproliferation appeared to be unimpaired. Later evidence was presented that the nonspecific depression was caused by monocytes, perhaps by HCMV-infected monocytes. Yet another possible explanation, which we suggest, is that HCMV caused interferon production by a yet unidentified cell population which caused monocyte activation; the monocytes then in turn suppressed lymphocyte function. Similar data have been reported by Ten Napel and The (1980a,b). These authors concluded that HCMV infection of adults resulted in a long-lasting cellular immunosuppression. In vitro responses to Con A and PWM appeared grossly disturbed as opposed to the PHA reactivity which was barely affected. Thus two groups have observed a dissociation between the responses to different mitogens. Another interesting set of data was obtained when analyzing T lymphocytes in patients with HCMV-EM (Carney et al., 1981).The acute illness is associated with a reversal in the normal ratio of helper to suppressor T lymphocytes with relative and absolute decreases in Thelper cells and corresponding increases in T-suppressor cells. It is of interest that in the newly discovered severe acquired immunodeficiency syndrome of male homosexuals a virtual elimination of the helperlinducer subset of T lymphocytes has been described (Gottlieb et al., 1981). Schauf et al. (1976) have studied the distribution of Ig-bearing, T, and null lymphocytes and the lymphocyte response to PHA and PWM in three infants with HCMV infection. The infants had significantly decreased percentages of T cells compared to age-matched controls. Compensatory increases in the percentages of Ig+ and null cells oc-

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curred. Decreased lymphocyte reactivity to PHA and PWM occurred in two patients. An increased number of “atypical” lymphocytes is frequently found in the circulation of patients with HCMV-IM. Elevated baseline levels of L3H]thymidine incorporation in the mononuclear cells of these patients (Rinaldo et al., 1977)may be related to this observation. Atypical lymphocytes may be identical with the so-called PTAlymphocytes, which are a subpopulation of lymphocytes identified with the electron microscope by the presence of cytoplasmic microtubule-like inclusions called “parallel-tubular arrays” (PTAs). The study of Payne and Tennican (1982) has shown that the percentage of these cells was increased during the acute phase of HCMV-IM. The authors further show that PTA cells contain Fc receptors and discuss their relationship to killer cells. The functions of polymorphonuclear leukocytes were studied in three patients with HCMV-IM by Rinaldo et al. (1979). These functioned within the expected normal range as measured by phagocytosis, reduction of nitroblue tetrazolium, and chemotaxis. XIII. Immunity against Infections with HCMV, General Aspects

A. ANTIGENICHETEROGENEITY OF HCMV Evidence that HCMV represents an antigenically heterologous group was reported in 1960 (Weller et d . ) Since . then, antigenic variation among HCMV strains has been demonstrated in neutralization tests (Krech and Jung, 1969; Andersen, 1972; Waner and Weller, 1978).Chiang et al. (1970) have used an IFA technique to investigate antigenic heterogeneity among HCMV strains. These appeared to fall into three antigenically distinct groups. Serological analysis of 15 naturally occurring HCMV strains with a panel of monoclonal antibodies to surface membrane proteins revealed that the antigenic determinants reactive with the antibodies tested were conserved in all strains, suggesting extensive cross-reactivity between strains (Pereira et aZ., 1982a). Probably these monoclonal antibodies will be very useful reagents for identification of virus isolates.

B. CELLSURFACE PHENOMENA ASSOCIATED WITH HCMV INFECTION Relevant to recognition phenomena by immunologically specific or nonspecific mechanisms are surface changes that are caused by virus

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infection of the cells. Particularly relevant are early changes since they make the infected cells vulnerable to cellular defense mechanisms well before virus replication is completed. As often referred to in this article there are comparably few data on HCMV in certain areas whereas there are many data on HSV. When cells are infected with HSV, changes in the plasma membrane occur, manifested by numerous phenomena which we have reviewed recently (Kirchner, 1982). HCMV induces membrane antigens which are detectable by immunofluorescent techniques as early as 24 hr after infection. These antigens appear on the surface of the infected cells in nonpermissive human epithelioid and animal cells in addition to permissive human cells (Tanaka et al., 1981). Stinski (1978) has investigated the sequence of protein synthesis in cells infected by HCMV. At least 10 distinct early virus-induced polypeptides were synthesized within 0-6 hr after infection of permissive cells. These polypeptides were synthesized before and independently of viral DNA replication. A majority of these early virus-induced polypeptides were also synthesized in nonpermissive cells which do not permit viral DNA replication. For the most recent information on HCMV-specified polypeptides the reader is referred to the papers by Pereira et al. (198213)and Siqueira-Linhares et al. (1981). Cultured human diploid fibroblasts after infection with HCMV could hemabsorb SRBC coated with rabbit anti-SRBC Ig (Rahman et al., 1976). Evidence was presented that suggested the existence of receptors on HCMV-infected cells that reacted specifically with the Fc region of human IgG. These receptors have independently been described by Furukawa et al. (1975). C. SOLUBLE ANTIGENS Soluble antigen preparations were prepared by Waner (1975) from cell cultures infected either with the Davis or AD 169 strains of HCMV. Fractionation of these preparations through Sephadex G-200 resulted in a molecular weight value ranging from 67,000 to 85,000. D. RESPECTIVEROLESOF HUMORAL AND CELLULAR RESPONSES IMMUNE Within the specific immune system one usually distinguishes between the humoral and the cellular immune response. The major components of the humoral immune response are the B cells and their products, the antibodies, whereas T cells and certain T cell-derived

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lymphokines are the main effectors within the cellular immune system. There are numerous interactions between T cells and B cells and the distinction between humoral and cellular immunity may turn out to be quite artificial. In the following we shall discuss the respective roles of humoral and cellular immunity in HCMV infection as presently understood. In the two subsequent sections we shall review the specific data that have been collected in regard to cellular and humoral immunity against HCMV infection. It is widely held that humoral immunity is of lesser relevance in herpesvirus infections. What are the reasons for this assumption? 1. Particularly with HSV, it has been shown that in vitro replication of the virus may take place even in the presence of a specific antibody. It is known that HSV can spread from cell to cell after cell fusion (Black and Melnick, 1955). Thus, the virus is passed without being exposed to antibodies. However, HSV-infected cells do express viruscoded antigens on their surface and thus may be recognized by cytotoxic effector cells. 2. For HSV, which may be studied in animal models, it has been shown that components of the cellular immune system, particularly T cells, are protective in adoptive transfer protocols whereas antibodies are not protective. However, more recently, several authors have stressed that passive immunization with antibodies against HSV may also be protective (Worthington et al., 1980). 3. Clinically, it is known for many herpesviruses that after primary infection they remain latent in the organism and periodically cause recurrent disease. An example is herpes labialis. In this situation it has been established clearly that recurrences do occur despite high and stable titers of antibodies and that antibodies only rarely fluctuate before or during recurrences. Similarly, HCMV is reactivated during pregnancy and again this occurs despite the presence of antibodies. Furthermore, the newborn may get infected during or after birth despite the possession of transplacentally acquired antibodies, and these children have been shown to shed HCMV for many months, again despite the presence of antibody (e.g., Numazaki et al., 1970). Thus, humoral immunity obviously does not interfere with chronic infection. It has been also shown in recipients of renal transplants in which the overall HCMV infection rate was 90% that despite immunosuppressant therapy, humoral immunity to HCMV was not impaired (Rytel and Balay, 1976). In fact, several studies (e.g., Lopez et al., 1974b) have documented that immunosuppressed renal transplant patients

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produced higher C F antibody responses to their HCMV infections than did normal persons.

A predominant role of cell-mediated immunity has therefore been assumed. Cell-mediated immunity is a novel field of research and great expectations have been placed on studies of cell-mediated antiviral immunity. In the case of HSV, we believe that the outcome of studies on cell-mediated immunity has thus far not significantly aided our understanding of the pathobiology of latency and recurrence. Collectively the data appear to suggest that in individuals with recurrences neither humoral nor cellular immunity is grossly impaired. Probably, because of the initial lack of sophistication in the assays, the role of cell-mediated immunity has been overestimated. It is also quite obvious that the assays of cell-mediated immunity are much more difficult to standardize than antibody tests and that in the future they will need much more refinement. Below we shall review the data in regard to cell-mediated immunity against HCMV in humans. Perhaps they ought to be viewed more optimistically than the data in the HSV system. The main drawback, however, remains that all herpesviruses are complex viruses, that their genomes code for many different proteins, and that it is not known which of them represent antigens relevant in cellular immunity.

XIV. Humoral Immune Responses

As mentioned above, herpesviruses of various types cannot be distinguished by morphology but (among other techniques) by serology based on the differences between the antigenic structure of the virons. Evidence has been accumulated suggesting that HCMV represents an antigenically heterogeneous group (Zablothney et a1., 1978). However, unlike the situation observed with HSV antigenic subtypes of the virus have not been unequivocally established. It is also not certain if prior infection with HCMV, which is evidenced by seropositivity, is protective against all wild-type strains. We shall discuss the following topics: (1)what is known about different HCMV proteins, both structural and nonstructural; (2) raising antisera against HCMV; (3)what is the significance of different antibody classes; (4)what is the respective usefulness of the different antibody tests; and ( 5 ) what is known about fluctuations of antibody titers and potential exogenous reinfections.

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1. There have been many investigations of virion and nonvirion antigens coded for by HSV. About 50 HSV-specific polypeptides have been defined, about half of which are structural (virion) and the other half nonstructural (nonvirion) (Honess and Watson, 1977). There are only a few studies about HCMV-specific polypeptides. However, Stinski (1978) has analyzed the sequence of protein synthesis in cells infected by HCMV and has identified early and late virus-induced polypeptides. Decisive for the recognition of virus-infected cells by effector cells or effector molecules in the immune system is the early expression of virus-coded membrane antigens; in the study of Stinski (1978) at least 10 distinct early virus-induced polypeptides were identified that were produced within 6 hr after infection. The new technology of raising monoclonal antibodies will probably lead to significant new clues about HCMV-specified polypeptides. At this writing there is only one report about monoclonal antibodies to surface membrane proteins on HCMV-infected cells (Pereira et al., 1982a). Additional reports will undoubtedly follow. 2. Neutralizing antibodies to HCMV, produced in animals, generally have been low in potency or cytotoxic. Krech and Jung (1969) reported on the development of neutralizing antibodies in guinea pigs following immunization with the Davis strain of HCMV. Waner and Weller (1978)have produced neutralizing antisera to HCMV immunogens obtained from infected cell cultures by an adaptation of the glycine buffer method of extraction. The antisera were used in neutralization kinetic tests to examine the antigenic relationships of several strains of HCMV. Huang et al. (1974) have prepared antisera with purified virions. Waner (1975) has prepared soluble antigen from cell cultures infected with HCMV. Antisera to this antigen lacked neutralizing activity but produced specific fluorescence confined to CMV intranuclear inclusion material. Specific high-titered antisera to two human and one simian CMV were produced by Huang et al. (1974) in guinea pigs. Both C F and immunofluorescence (IF) tests revealed that the two human strains are closely related and have little, if any, interspecific cross-reactivity with the simian strain. Again, it has to be stressed that the interpretation of HCMV antigens (like the antigens of many other viruses) will probably change drastically as soon as more monoclonal antibodies are available. 3. Following any primary virus infection, antibodies of different classes are produced in a predictable sequence. Early antibodies are the antibodies of the IgM and of the IgA class.

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IgA antibodies are considered to be important at mucosal sites. It is of interest that IgA antibodies against HCMV have been detected in saliva and cervical secretions (Tamura et al., 1980; Waner et al., 1977), but in neither the mouth nor the cervix does their appearance or concentration correlate with cessation of viral excretion. The presence, in the infant, of transplacentally acquired CMV antibody of the IgG class complicates the interpretation of serological results during the first 6 months of life. Detection of specific CMV IgM antibody in the serum of a newborn, however, usually indicates congenital infection since maternal IgM cannot cross the intact placenta. Because CMV IgM antibody may persist for months after primary infection its detection in a single serum sample from a postnatally infected child is of limited value in determining the timing of a primary infection (Waner et al., 1980). Hanshaw et al. (1976)found IgM antibodies directed against HCMV in the umbilical cord blood of 53 of 8644 newborns. Recently, an RIA for IgM antibodies has been reported by Griffiths et al. (1982),which has been useful in the diagnosis of primary HCMV infection during pregnancy. 4. Various tests have been developed to measure antibodies against HCMV. It is not within the scope of this article to describe the technical aspects of these tests. This has been done competently by Waner et al. (1980). The tests most commonly employed are the CF, the indirect fluorescent antibody (IFA), the indirect hemagglutination (IHA), and neutralization tests. In the future, ELISA will probably be used extensively because of its sensitivity and ease of performance. Castellano et al. (1977) tested 30 samples of serum for antibody to HCMV by ELISA and IHA and found the two tests to be in extremely close agreement. The C F test is commonly used for determining levels of HCMV antibody and is conveniently performed by microtiter technique. There have been doubts about the sensitivity of the test, particularly in HCMV-infected newborns (see above). The IFA test is a sensitive and broadly reactive method for determining antibody levels to HCMV. However, HCMV infection of human fibroblasts induces an Fc receptor in the cytoplasm of infected cells which may bind IgG nonspecifically, resulting in false-positive results in the IFA test. Swack et al. (1977) have shown that the substitution of a simian CMV strain eliminated this cytoplasmic reaction and allowed observation of virus-induced fluorescent intranuclear inclusions. Neutralizing antibody in human sera may show a degree of HCMV strain specificity (see below).

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Studies of HCMV-induced nuclear antigens have differentiated between early (HCMV-EA) and late (HCMV-LA) antigens (The et al., 1974). HCMV-EA are considered to be characteristic of a primary or acute HCMV infection. Griffiths et al. (1980) studied antibodies to HCMV-EA in pregnant women and concluded that these antibodies are not as transitory as has been suggested and that their presence, even at high titers, in a serum sample from a pregnant woman cannot be taken as presumptive evidence of recent primary infection. 5. The problems associated with studies of humoral immunity against HCMV in man are severalfold. a. Are the antibody tests used sensitive enough to detect all persons with previous HCMV infection? Furthermore, do all persons that have experienced a previous infection carry the latent virus in their body? Or to put it differently, does each primary infection lead to latency of HCMV, or are there infections that do not lead to latency? Perhaps the latter type of infection tends to cause only low and short-lived antibody titers. Perhaps endogenous reinfections that are clinically inapparent are required to sustain significant antibody titers. The dynamics of the antibody status have been stressed by Waner et al. (1973). Of 20 persons 11vacillated at least once between significant and undetectable levels in the test for C F antibody. These data were obtained from plasmapheresis donors and may not necessarily be applicable to all normal persons. Yeager (1975), in a longitudinal study of nurses, observed no fourfold rises or falls in titer over a 19 to 27-month period among 71 persons with initially positive CMV CF titer. b. Is there sufficient cross-reactivity between different laboratory and wild-type strains of HCMV? This question is relevant in regard to the possibility of repeated infections with different strains of HCMV and also in regard to laboratory techniques that require standard strains or their antigens in tests. Anderson (1972) tested 200 human sera for neutralizing antibodies against various HCMV strains. The individual sera in low dilutions were able to neutralize either all strains or none. On the basis of the kinetic studies, strain AD 169 was found broadly reactive and thus well suited for determination of neutralizing antibodies in human sera. c. Finally, it is important to stress again that perhaps humoral immunity is not of major importance in vivo, since it is known that there are instances in which infections with herpesviruses may

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proceed despite the presence of antibodies. Thus, exogenous reinfections may be possible even in seropositive individuals and not all reinfections in these are caused by endogenous reactivation of HCMV. From the questions posed above, it is obvious that there are many unresolved issues and that there is an urgent need for intensive studies utilizing very sensitive antibody assays and the tools of “molecular epidemiology” to determine if isolates obtained from one patient at various times represent an identical strain or not. In fact, Huang et al. (1980) reported that repeat isolates from three or four women were concordant by restriction enzyme analysis. XV. Cell-Mediated Immunity

We have described the reasons why it is believed that cell-mediated immunity plays an important role in the defense against herpesviruses and against HCMV in particular. We want to review the data which were obtained when testing cell-mediated immunity in healthy individuals and in patients with HCMV disease. Such testing has been done in u i t m using tests based on three different reaction principles of T lymphocytes: lymphocyte cytotoxicity, lymphokine production, and lymphoproliferation. A. LYMPHOCYTE CYTOTOXICITY

Predominantly from data in animal models, it is well established that sensitized T cells are cytotoxic against virus-infected cells, the cytotoxicity usually being restricted to histocompatible target cells. However, various subpopulations of white blood cells have been shown to be cytotoxic besides T lymphocytes. These include monocytes/macrophages, NK cells, and K cells, which will be discussed below. There has been a recent report of HLA-restricted cytotoxic T lymphocytes in bone marrow transplant recipients. Killer cells other than T lymphocytes were also reported in this paper (Quinnan et al., 1981). However, Kirmani et al. (1981)failed to detect a consistent pattern of HLA restriction when testing the killing of HCMV-infected human fibroblasts by human circulating mononuclear cells. They concluded that cytotoxic lymphocytes from nonimmune volunteers possessed characteristics of natural killer cells, whereas those from immune persons probably consisted of both natural killer cells and antibody-dependent cellular cytotoxicity (ADCC) effector cells.

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Quinnan et al. (1982) studied 58 recipients of bone marrow transplants to evaluate cellular cytotoxicity against HCMV-infected cells. All patients had absent or low HCMV-specific cytotoxic lymphocyte activity before the onset of infection. A specific cytotoxic response developed in all survivors, whereas only two patients with the fatal infection had even low cytotoxic responses. Natural and antibodydependent killer cell activities were depressed both before and during infection in patients with fatal infections, but not in those who survived. This report seems to suggest that both specific and nonspecific killer cells play a role in protection against HCMV in patients with bone marrow allografts.

B. LYMPHOKINES Specific immunity in animaI models is commonly estimated by the elaboration of specific lymphokines, such as macrophage migration inhibitory factor (MIF), macrophage activating factor, or gamma interferon. There have been a number of reports in which lymphokine production by immune lymphocytes, upon stimulation with HSV antigen, has been measured (e.g., Wilton et al., 1972). These studies, which we reviewed recently, have failed to unravel significant clues as to the pathobiology of infection with HSV. Fiorelli et al. (1982) used the direct leukocyte migration inhibition test to study the cell-mediated immune response in a group of children with HCMV infection. This test is assumed to measure a lymphokine which is specifically elaborated in the same reaction in which it is assayed. Thus, in a certain sense this test is a one-step assay for MIF. An interesting lymphokine is gamma interferon (see below). The measurement of gamma interferon production has been utilized to determine the specific cell-mediated immune response to HSV antigens. However, HSV also induces alpha interferon in human leukocytes regardless of whether they are derived from seropositive or seronegative donors (Green et al., 1981). When purified T cells supplemented with macrophages are cultured, the production of gamma interferon in response to HSV antigens can be measured. Thus, if one decides to test the production of gamma interferon as a parameter of the cell-mediated immune response, a11 parameters of the assay have to be monitored carefully and it has to be proven in each experiment that the interferon produced represents gamma interferon. Starr et al. (1980) studied interferon production in adult human leukocyte cultures stimulated with HCMV antigen. Alpha interferon

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was detected in supernatants of cultures stimulated with crude antigens regardless of the immune status of the donor. In contrast, when purified antigen was used as the stimulant, only cultures obtained from seropositive individuals produced detectable levels of interferon, which appeared to be predominantly gamma interferon.

C. LYMPHOCYTE PROLIFERATION The assay most commonly used for measuring the specific cellmediated immune response is the lymphocyte proliferation (LP) test. Various synonyma are used for this assay including lymphocyte blastogenesis, lymphocyte stimulation, lymphocyte transformation, etc. What one measures is the clonal proliferation of specifically sensitized lymphocytes upon in oitro reexposure to the specific antigen. This is usually done in a 4-6 day assay using populations of unseparated peripheral mononuclear white blood cells that contain T cells, B cells, and monocytes and by measuring the uptake of tritiated thymidine as an indication of DNA synthesis. The lymphoproliferation assay has been performed with many types of antigens including viral antigens. The first studies of this type were done in rabbits immunized with HSV (Rosenberg et aE., 1972). There have also been quite a few studies of HSV- and HCMV-induced lymphoproliferative responses in human leukocyte cultures. It should be realized that these studies are still much more difficult to standardize than antibody titrations, that they depend on tissue culture variables that are difficult to control, and that crude “antigens” (i.e., complete virions or even virus-infected cells) are usually used in these tests. Before reviewing individual studies that have been performed in groups of patients with HCMV disease, we shall discuss a few general aspects including the following: (1)the type of HCMV preparations used in the LP test; (2) the types of cell populations that are usually tested in the LP test and what is known of the nature of the cells participating in the reaction; and (3)how do the results of the LP test usually correlate with the testing of humoral immunity?

1. In the LP test, in most instances, crude virions are tested which are obtained from infected tissue cultures after removal of the cellular debris, Sometimes the virions are enriched by ultracentrifugation or by density gradients. In some studies antigens were prepared by glycine extraction (Waner and Budnick, 1977). In others the virus was inactivated by heating (Ten Nape1 et al., 1977) or by UV light (Wahren et al., 1981). We have failed to find investigations in which

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viruses inactivated by different methods were compared systematically. A number of investigators have used HCMV-infected cells instead of virions (Mgller-Larsen et al., 1975/76; Schirm et al., 1980). Generally, similar results were obtained when comparing the two. However, Schirm et al. (1980) presented data suggesting that HCMVinfected human fetal fibroblasts and cell-free HCMV are recognized by different populations of HCMV-specific memory lymphocytes. In our own (preliminary) studies a truly major concern has been that almost all strains of HCMV that we obtained contained mycoplasmas. Mycoplasmas have profound effects on the cells of the immune system, including lymphocyte stimulation and induction of interferon (e.g., Ginsburg and Nicolet, 1973; Beck et al., 1982). Thus, we realize the need to prepare virus stocks for LP tests by transfection and to carefully and repeatedly monitor all preparations for the presence of mycoplasmas. Yet another concern is the presence of antibodies in the serum used to support the LP test, traces of which may be left in the cell populations if they are not sufficiently washed. In an HSV system it has appeared as if antigen-antibody complexes stimulate as well as the virus antigens themselves (Fujibayashi et al., 1975). Wilhelm and Longthome (1980) found that the responses of lymphocytes from HCMV seropositive donors were decreased or abolished in the presence of HCMV seronegatiue plasma or serum. An important problem for studies of the LP test is the apparent heterogeneity of the different standard strains and probably of the wild-type strains of HCMV as well. In the study of Beutner et al. (1978), three strains of HCMV (AD-169, ADH-1-41, and Davis) and HSV-1 were compared in the LP test. No cross-reactivity was apparent between HSV-1 and HCMV, but the responses to the three HCMV strains suggested antigenic heterogeneity on the level of cellular immunity. 2. It is generally accepted that the cells reacting in the LP test are T lymphocytes. Depending on the mitogen or antigen used, the reaction of T lymphocytes is, to a greater or lesser extent, dependent on macrophages. In this context is appears worthwhile to explain the difference between mitogen- and antigen-induced lymphoproliferation, since these tests are used widely in cellular immunology laboratories. Antigens activate specific clones of lymphocytes. Since these clones are small, it takes 5-7 days until lymphoproliferation may be measured in the

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test. Mitogens, in contrast, cause a polyclonal activation, i.e., they (nonspecifically) activate a large number of clones at the same time. Thus, the optimal lymphoproliferation in this test is detected earlier, i.e., after 3-4 days. One generally distinguishes between T cell mitogens and B cell mitogens. Typical T cell mitogens are, for example, PHA and Con A. They are often used to test general immunocompetence in certain diseases as, for example, in HCMV-IM. A number of mitogens, such as endotoxin in mice or other bacterial products (for example, from Nocardia) or PWM, are B cell mitogens. The latter not only cause a lymphoproliferative response, but also cause the polyclonal production of antibodies, i.e., antibodies of many classes and specificities are produced. Mitogenic activation of T cells, in turn, causes the production of lymphokines. Generally, mitogens are considered useful models to study the cell biology and biochemistry of lymphocyte activation. However, it is our belief, that there may be an in r;i.t;orelevance of the recognition mechanisms induced by mitogens. Most mitogens are lectins and there is now increasing evidence to indicate that lectin-like structures play a role in cellular interactions of the immune system. Furthermore, both bacteria and viruses appear to have mitogenic structures on their surface and thus the immediate (nonspecific) responses elicited by these structures, such as lymphocyte activation and interferon induction, may have important consequences. Finally, infectious EBV itself phenotypically mimics the effects of mitogens, in that nonspecific lymphoproliferation and polyclonal production of antibodies by B lymphocytes is induced. This effect is clearly distinct from the T-lymphocyte proliferative response which is caused by antigenic structures of EBV and which is immunologically specific. There is no evidence of HCMV being capable of acting as a B cell mitogen. The specific lymphoproliferative response to HCMV will be discussed in detail below. Usually, in the LP test, leukocyte preparations are used which are prepared by the Ficoll-Hypaque gradient technique. For routine testing this method is fairly laborious, particularly when great numbers of individuals are to be tested. A considerably easier method is the whole-blood technique which has been used by Agatsuma et a2. (1979)to study HCMV-induced lymphoproliferation. This test is easy to perform and it has an additional advantage in that the cellular components are introduced into the test without disturbing the ratios of different cell populations relative to each other. Thus, one might expect, that the whole-blood technique represents a closer correlate to

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the clinical situation than the more commonly used test in which separated mononuclear cells are used. 3. The validity of the LP test depends on how closely it reflects the immune status of the tested individuals. Thus, it usually has to be determined if there is a correlation between the tests of humoral immunity, i.e., antibody testing, and those of cellular immunity. Certainly, there may be various problems in this regard. One has to be sure that the antibody test is sensitive enough to detect all individuals that have been previously infected. In studies of cell-mediated immunity against HSV, several investigators have noted that some individuals who appeared to be negative by conventional antibody assays, for example by the CF test, nevertheless reacted in the LP test. However, the study of Moser et al. (1981) has solved this problem, since they found that all individuals negative in ELISA for anti-HSV antibodies were also negative in the LT test. From these studies it appears that the LT test is a sensitive assay for previous exposure to the antigen, at least as far as HSV is concerned. However, in none of the studies which we have reviewed has a correlation been observed between the magnitude of lymphoproliferation and the actual titer of the antibody test. In the HSV system it has been shown that children, who were seronegative in the LT test at the onset of a primary HSV-induced gingivostomatitis infection, became positive within several weeks, indicating that primary infection with HSV causes a conversion of the LT test result (M@ller-Larsenet al., 1978).

D. OVERVIEW OF THE INVESTIGATIONS OF HCMV-INDUCED LYMPHOPROLIFERATION In systems of antigenic lymphocyte stimulation it is often useful to test cord-blood lymphocytes as a control, since they are usually negative. For example, cord-blood lymphocytes did not react in the LP test when HSV was tested as an antigen (Kirchner et al., 1978). Similarly, Beutner et al. (1978) has observed no HCMV-specific lymphocyte transformation activity in cultures of cord blood. A significant proportion of newborns get infected at birth or during the first weeks of life, but these infections are usually clinically inapparent. If one searches for these infections one may be able to detect them by the presence of antibodies of the IgM class (Reynolds et al., 1974) or by viruria. The latter, however, is seen only after an “incubation” period of 3 to 6 months. In our opinion, it would be very worthwhile to study a cohort of children in whom the LT test and the

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presence of IgM antibodies are sequentially studied and later supplemented by data on which of the infants developed viruria. Gehrz et al. (1977) found that neonates with CID were unreactive in the LP assay when HCMV antigens were tested. However, this defect was not a general defect of cell-mediated immunity since lymphoproliferation induced by mitogens was normal. It is also noteworthy that the children had antibodies to HCMV. Starr et al. (1979) found that six congenitally infected, viruric children responded poorly to HCMV in the LP test. Lymphocyte stimulation by HCMV in children who acquired infection within the first months of life was found to be weak or absent, as reported in children with congenital infection (Pass et al., 1981a). Again, this effect appeared to be unrelated to a generalized defect in cellular immunity. Reynolds et al. (1979) studied 35 mothers and 30 of their offspring with congenital or neonatal HCMV infection. Eleven offspring did not respond to the HCMV antigen and 15 of the 19 positive children displayed lower responses than those of normal adults. Productive infection in the younger children at the time of the assay and the presence of disease correlated strongly with the absence of responses. The mothers, as a group, also displayed diminished responses in the LP test. Mitogen stimulation was normal in all test subjects. Gehrz et al. (1981) studied the HCMV-specific cellular immune response to the LP test during human pregnancy. Seropositive pregnant women had lymphocyte proliferative responses that were markedly depressed at the end of the third trimester of pregnancy despite persistent levels of CF and IFA antibodies to HCMV. The reduced levels of reactivity in the LP test returned to levels detected in early pregnancy by 1 year after delivery. General parameters of cellular immunity, including T cell counts by sheep red blood cell (SRBC) rosetting and mitogen-induced lymphoproliferation were unaffected. Several authors studied patients receiving organ grafts. Pollard et al. (1978) studied cell-mediated immunity to HCMV in cardiac transplant recipients. Lymphocyte reactivity to the LP test in these patients was markedly depressed and returned to normal in 3 years. Rytel et al. (1978) found that six of nine HCMV-infected renal allograft recipients studied 6 or more months after transplantation were negative in the HCMV-specific LP test. Linnemann et a2. (1978) performed a study of 15 patients who received renal allografts. Of these, 14 developed HCMV infection and all infections were accompanied by a normal humoral immune response. Eleven patients had cellular immunity to HCMV before transplantation and all of these became negative in the first month after transplantation.

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Cell-mediated immunity was assessed in 38 seropositive recipients of renal transplants using the LP test with the HCMV antigen by Pass et al. (1981b). Before immunosuppression, responses of the patients were similar to those of the controls. After renal transplantation, lymphoproliferation was dramatically reduced for up to 18 months postoperatively, especially in patients treated with antithymocyte globulin. The adverse clinical effect of antithymocyte sera on HCMV infection in transplant recipients has been described before (Pass et al., 1980b). These authors indicated that renal transplant patients who receive both a poorly matched graft and antithymocyte globulin are at increased risk of morbidity due to HCMV. Finally, Meyers et aZ. (1980a) studied patients after marrow transplant to correlate HCMV infection with HCMV-specific lymphoproliferation. Of 158 patients 92 developed HCMV infection. The lymphocyte responses of patients who were seropositive before transplant was suppressed immediately after transplant; the lymphocyte responses of 73 long-term survivors were similar to those of normal persons. Lymphocyte responsiveness to HCMV was assessed in patients with HCMV-IM early in their illness by Levin et al. (1979). Levels of HCMV-specific antibody rose early in the illness but the proliferative response of mononuclear cells to HCMV antigen did not reach the levels characteristic of HCMV-immune donors until several months later. These patients also displayed nonspecific defects of T cell reactivity as discussed above. In the study of Ten Nape1 and The (1980a), the LP test was studied in 18 patients with documented acute HCMV infections. The development of a positive HCMV LP test lagged far behind the appearance of virus-specific antibodies. Again, the defect in cellular reactivity was nonspecific since mitogen reactivity was impaired also. In conclusion, in a variety of clinical states a defect of HCMVinduced lymphoproliferation was documented. This defect was observed to be specific in some of the studies (congenitally infected infants and their mothers), whereas, at least in the patients with HCMV-IM evidence exists for a more general defect of T cell immunity. XVI. Nonspecific Defense Mechanisms against HCMV Infection

We believe that it is important to clearly distinguish between immune effector mechanisms and nonspecific (primary) defense. Immunity is defined by “immunological specificity,” and the effector mech-

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anisms of the specific immune response are activated by a secondary contact of the organism with a pathogen. There is no doubt that, even in this situation, nonspecific amplifier mechanisms are activated. To give just one example: upon recognition of specific antigens, gamma interferon and other lymphokines are produced which then activate a variety of cells, for example, macrophages and NK cells. The nonspecific defense mechanisms are presently studied in many different animal models. There is no animal that can be infected with HCMV. However, infection of mice with MCMV may be studied in this regard. Below, we will refer to some data on the role of NK cells in the defense against MCMV. The problems associated with studying primary defense mechanisms against HCMV in the clinic are multifold. For example, primary infection with HCMV in most instances is clinically inapparent. A significant number of children get infected at birth or shortly thereafter. Three to 6 months later, one will be able to detect viruria persisting for a prolonged period. This viruria occurs despite the presence of high titers of specific antibodies. One wonders, what the decisive events are during the relatively long period between primary infection and the detection of viruria. A number of mechanisms ought to be discussed within the frame of the nonspecific antiviral defense. We will consider the following: natural killer (NK) cells, macrophages, antibody dependent cellular cytotoxicity (ADCC), the complement system, and the interferon system. A. NKCELLS

NK cells are a novel class of cells that morphologically resemble small lymphocytes and can be distinguished from mature monocytes. They are defined functionally by their capacity to lyse certain types of target cells in uitro without (in contrast to cytotoxic T lymphocytes) obvious preimmunization. Otherwise NK cells are characterized by the lack of some properties that are typical for T cells, B cells, and macrophages. However, certain markers are shared between NK cells and other members of the lymphoreticular community. For example, theta antigen or the antigens of the Qa series that typically occur on mouse T lymphocytes are present on mouse NK cells. Thus, NK cells cannot as yet be unequivocally defined. Besides definition of the place of NK cells within the hemopoietic system, there are numerous unresolved issues in the field of NK cell research. Most importantly, it is not known what structures on the

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target cells are recognized by NK cells. Obviously, NK cells could be prime candidates for cells functioning in antitumor surveillance mechanisms of some kind, if they are able to recognize the malignant cell surface phenotype. With respect to antiviral defense, it has recently been shown that virus-infected target cells, in contrast to their noninfected counterparts, may be lysed by NK cells. Yet another aspect of this interaction is that viruses in leukocyte suspensions induce interferon and interferon in turn may have a twofold effect. It appears to protect target cells against lysis by NK cells (Trinchieri et al., 1981) and it is wellestablished that interferon activates NK cells (see below). Presently it is not known if NK cells play an in uiuo role in antiviral defense. As it appears, only in the case of two herpesviruses, HSV and MCMV, in murine models has some evidence been obtained suggesting a role for NK cells in antivira1 defense. In animal models there are basically three approaches to test the in vivo role of NK cells including (1) adoptive transfer protocols, (2) use of immunosuppressive drugs in uiuo, and (3)the search for associations between resistance and the magnitude of the NK cell response. The former two approaches are notoriously troublesome in the NK cell field since there are no ways of preparing cell populations sufficiently enriched in NK cells and sufficiently depleted of other cell types that potentially play a role in antiviral defense. Furthermore, there are no immunosuppressants known that selectively affect NK cells in uiuo. However, associations have been observed between genetically controlled resistance of mice to HSV and the magnitude of the HSVinduced NK cell response (Engler et al., 1982). This was also observed when susceptible newborn mice of the C57BL/6 strain were compared with resistant adult mice (Zawatzky et aZ., 198213). However, there is the possibility that NK cell activation is simply a secondary parameter of a previous induction of interferon-which does occur in this model-and that NK cells are not of relevance in antiviral defense. That this may indeed be the case is suggested by data on SJL mice (Engler et al., 1982). SJL mice are genetically defective in the NK cell system but they produce interferon and they are resistant to HSV. A good case for an important role of NK cells in antiviral defense has been presented in the MCMV system. For example, bghg mice, that are genetically defective in their NK cell system, have been reported to be more susceptible to MCMV infection than controls (Shellam et al., 1981). Furthermore, a close correlation has been observed between the lethal effects of MCMV and the magnitude of the virus-

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induced NK cell activity in 10 of 11 mouse strains tested (Bancroft et al., 1981). Recently, natural killing of HCMV infected fibroblasts by human mononuclear leukocytes has been studied in two laboratories (Kirmani et al., 1981; Quinnan et aZ., 1981). In both reports, leukocytes from seropositive and from seronegative donors were able to lyse virus-infected target cells. Killing was mediated by non-T, non-B, Fc receptor-bearing cells. Similar data have been reported by Starr and Garrabrant (1981),who have shown that human peripheral mononuclear cells are capable of lysing HCMV-infected human fibroblasts. It has been reported by Diamond et al. (1977) that peripheral blood leukocytes from 14 healthy, nonimmune human donors were capable of destroying HCMV-infected human fibroblasts. Unlike adult leukocytes, leukocytes separated from cord blood were ineffective in destroying HCMV-infected target cells. We want to stress again the importance of the study by Quinnan et aZ. (1982), who showed that both HLA-restricted cytotoxic T cells and non-T killer cells were correlated with recovery from HCMV infection in bone marrow transplant recipients (see above).

B. POTENTIAL ROLE OF MACROPHAGES IN DEFENSE AGAINST HCMV It is generally assumed that macrophages play a major role in the defense against viruses and this topic has been reviewed repeatedly (Mogensen, 1979; Morahan and Morse, 1979). In animal models of virus infections, considerable evidence has been obtained to support the role of macrophages in antiviral defense. This is also the case for human viruses for which animal models exist, for example, HSV. There are considerable data on the role of macrophages in the defense of mice against HSV. It is not known if macrophages play a role in viral infections of man and very little is known about interactions between viruses and human macrophages in general. Murine macrophages are exquisite targets for a variety of viruses including, for example, MCMV (e.g., Brautigam et al., 1979). Several authors have reported replication of HSV in human monocytes whereas other laboratories have failed to observe replication of HSV in human monocytes. As it appears, certain technical aspects are of importance if one wants to document replication of HSV in monocytes. For example, Lopez and Dudas (1979) and Daniels et al. (1978), in a murine and a human system, respectively, have documented that monocytes have to be precultivated in vitro

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(“aged”) for several days before they become permissive for replication of HSV. The only indication that there are interactions between HCMV and macrophages is the study by Carney and Hirsch (1981). These authors investigated the depressed lymphoproliferative responses to T cell mitogens in patients with acute HCMV-IM. They obtained evidence that the reactivity in purified T-cell populations was restored, whereas it was suppressed in the presence of monocytes. Thus, it appeared that “suppressor monocytes” were activated by HCMV in uiuo which caused the suppression of T-cell mitogen responses. Carney and Hirsch (1981) isolated HCMV from blood monocytes of four patients with mononucleosis. Monocytes from uninfected control donors, when infected in uitro with HCMV, were found to be significantly more suppressive for autologous lymphocyte responses to Con A than were uninfected monocytes. Thus, there is a possibility that the “suppressor monocytes” isolated from patients with HCMV-IM are indeed HCMV-infected monocytes. C. ADCC ADCC is defined as a mechanism of cell-mediated target cell killing which occurs if the target cells are coated with anti-target cell antibodies. One may argue whether this is an immunologically specific mechanism. The antibody of course is specific, whereas the killer cell is not specific. It does not matter if the killer cells are derived from a nonimmune individual. The effector cells of ADCC have been termed K cells; they had been known before the description of the NK cells. The K cells share a number of properties with the NK cells and it is a matter of current controversy if the two cell types belong to a common group of cells. A remarkable feature of ADCC is that it can be demonstrated when using minute concentrations of antibody (i.e., high dilutions of serum). Nonetheless, the ADCC test is probably not useful as a routine test for anti-HCMV antibodies since one may envision various sources of artifacts. Furthermore, the in uiuo significance of ADCC is not at all understood. Perhaps it is merely a laboratory artifact. Numerous studies have been concerned with ADCC of human leukocytes against HSVinfected cells (e.g., Shore et d.,1979), whereas we failed to find, in the literature, a systematic analysis of ADCC against HCMV-infected cells. Kirmani et al. (1981) reported that ADCC was operative in their system.

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D. ROLE OF COMPLEMENT Cells infected with any of a large number of viruses, including HSV, are lysed following the interaction of complement with specific antiviral antibody (Oldstone and Lampert, 1979; Cooper and Welsh, 1979). Although HSV replicates in the nucleus, viral antigens appear at the cell surface rendering the cells susceptible to complement-dependent antibody lysis (Brier et al., 1971). In addition to lysing virus-infected targets, antiviral antibody and complement can lyse different virions, including HSV. This attack of complement appears to be directed against the lipid moiety of virions complexed with antiviral antibody. We failed to trace in the literature any report on the effects of complement on HCMV or HCMV-infected cells.

E. ROLE OF INTERFERON Interferon as an in vitro antiviral principle was discovered 26 years ago (Isaacs and Lindenmann, 1957),and very significant progress has been made during the past 3 years (for a review see DeMaeyer et al., 1981).Thus, it is now established that interferons are a group of many different proteins and that they are pleiotropic molecules that, besides the antiviral effect, have many additional effects in different biological systems (“nonantiviral” effects of interferons). Interferons are produced by a variety of different cells upon stimulation by different compounds. According to the new nomenclature, alpha interferon (formerly leukocyte interferon) is produced by cultures of white blood cells, when treated, for example, with viruses. As far as the human system is concerned, the amino acid sequence of alpha interferon has been identified and it has been established that there are at least 12 subtypes of alpha interferon that differ at defined areas within the molecule (Nagata et al., 1980). It is not understood which subtypes of leukocytes are the producers of alpha interferons and if, perhaps, different subtypes of white blood cells produce different subtypes of alpha interferons. Beta interferon (formerly fibroblast interferon) is produced by fibroblasts in oitro when treated, for example, by poly(1) . poly(C) or viruses. The amino acid sequence of human beta interferon is known and there is about 30% homology between human alpha and beta interferons. Gamma interferon used to be the least defined subtype of interferon. It was formerly called immune interferon or type I1 interferon and it is produced when T lymphocytes are confronted with “immu-

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nologic stimuli” such as specific antigens, alloantigens, or mitogens. Thus, gamma interferon, by definition, is a lymphokine. In fact it has been called a model lymphokine (Epstein, 1981) since much can be learned about lymphokines in general when studying interferon gamma. Interferons are not only produced in vitro but also in vivo. For example, interferons can be detected when mice are injected with virus or with different types of chemically defined interferon inducers. In this case, high titers of interferon are detected both at the injection site, for example, in the peritoneal cavity and in the serum. In most instances it is not known which are the producer cells of the interferons that are detected in the serum or in other body fluids. It is also not known if there are levels of interferon “naturally’’ occurring in the serum of healthy humans or if there are diseases that are associated with significant changes in serum interferon levels. This lack of knowledge is partially associated with problems of serum interferon measurement which precludes the measurement of low titers. It has been thought that herpesviruses, including cytomegaloviruses, are relatively insensitive to the action of interferon and that they are not very good inducers of interferon. However, these statements may not necessarily be true in the light of modem developments in interferon research. It has been recently shown that the replication of all conventional viruses appears to be sensitive to the action of interferons provided that appropriate conditions are selected. Furthermore we have shown that HSV, although a poor inducer of interferon in fibroblast cultures, is a good inducer of alpha interferon in human leukocyte cultures (Kirchner et al., 1979). The production of alpha interferon in human leukocytes, upon exposure to HCMV, has been reported by Emodi and Just (1974) and Starr et al. (1980). The effect of interferons on the replication of HCMV in tissue culture has been studied by Postic and Dowling (1977), who reported that human interferon inhibited HCMV replication in vitro. A prototype strain, Davis, and six clinical isolates of HCMV were tested. All six isolates showed uniform susceptibility exceeding that of the Davis strain by two- to fourfold. The effects of interferon in patients with HCMV-associated disease have been reported in two recent studies. A double-blind, placebocontrolled trial of interferon prophylaxis against viral infections was conducted using renal-transplant recipients. HCMV excretion began earlier and viruria was more frequent in placebo-treated than in interferon-treated patients (Cheeseman et al., 1979). In the study of

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Meyers et aZ. (1980), eight recipients of marrow transplants, with HCMV pneumonia diagnosed by open lung biopsy, were treated with doses of human alpha interferon of 2 x 104-6.4 x lo5 units/kg/day. All eight patients died from pneumonia and virus was still present in lung tissue from seven patients cultured after death. Since interferons better than those used in these studies will be available in the near future, and since HCMV infection is of serious concern one should pursue interferon treatment schedules with higher concentrations of improved purified interferons. Rhodes-Feuillette et al. (1981)discussed an interesting aspect in a preliminary note. They reported that after marrow transplant, patients had a circulating interferon which appeared to be gamma interferon. This interferon appeared not to be protective against HCMV, at least, in one of the patients. Similarly, serum interferon has been found in animal models of graft-vs-host disease. There are few data in the literature about interferon induction by HCMV in the disease state. There was an early report b y Emodi and Just (1974) that leukocytes from infants with CTD were unable to produce alpha interferon, then called type I interferon. This observation is very intriguing and seems to indicate a deficiency in the interferon system of these infants. Starr et al. (1979) also reported that six viruric children responded poorly to HCMV with interferon production. There has been controversy as to which type of interferon is synthesized in human leukocytes upon the addition of HSV. By a recent report this issue appears to be settled, in that predominant production of alpha interferon occurs in unpurified populations of leukocytes, which does not correlate with the immune status of the donor. However, in cultures of purified T lymphocytes, supplemented with macrophages, gamma interferon is synthesized corresponding to the immune status, i.e., only lymphocytes from seropositive donors responded (Green et al., 1981). Pollard et al. (1978) found that, upon addition of HCMV to leukocyte cultures of normal individuals, interferon was produced regardless of the serostatus of the donor, suggesting that alpha interferon was produced. A systematic comparative study, similar to the one described for HSV, has been performed by Starr et al. (1980). Again it is quite clear that when using HCMV as a source of antigen and the assay of gamma interferon as a parameter of cellular immunity, all parameters have to be controlled with great care. In fact, we do not advocate the gamma interferon assay as a routine assay for cellular immunity.

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XVII. Vaccine and Therapy Problems

The problems associated with active immunization against HCMV have evoked considerable controversy and to elaborate on all of these is beyond the scope of this article. The arguments for and against a live HCMV vaccine have been summarized recently by Plotkin et al. (1981), Plotkin (1981), and Osborne (1981). Plotkin and collaborators (1981) have presented the strongest push for a live HCMV vaccine. Several years before, Elek and Stem (1974) also reported on the development of a live HCMV vaccine. The reviewer has not found, in the literature, an attempt to prepare HCMV subunit vaccines, although in HSV research it is generally agreed that only subunit vaccines should be used clinically. A number of points cause concern.

1. First, all herpesviruses are candidate tumor viruses and thus the use of a vaccine other than a subunit vaccine is certainly something to worry about. Since the development of cancer may take decades, cohorts of patients who have received the vaccine for prolonged periods will have to be followed. 2. As outlined above, current antibody measurements probably underestimate the number of seropositive individuals and probably one wants to immunize seronegative individuals only. 3. It is not at all clear if intrauterine infections are caused by primary infections or by endogenous reactivation. Reactivations occur most frequently in very young women, and children of very young primaparae appear to be most commonly infected. Perhaps, all intrauterine infections are caused by endogenously reactivated HCMV (see also Huang et al., 1980). If this were so, one would certainly not want to infect seronegative young women with live HCMV vaccine. 4. The situation in transplant recipients is also not clear. As it appears, at least half of the infections with HCMV are endogenous reactivations. Perhaps many of the latter are induced by graft-vs-host disease or by immunosuppressive therapeutic schedules. Again, why would one want to increase the number of patients that carry endogenous virus which might be reactivated by immunosuppression. Furthermore, in transplant recipients, immunized with live HCMV, it has been found that infections with HCMV could not be prevented (Glazer et al., 1979), obviously because of insufficient cross-reactivity between the vaccine strain and wild-type strains. 5. The latter point seems to support another serious argument against the currently used vaccine. There is incomplete knowledge

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about HCMV antigens and it is doubtful if there are enough common antigenic sites on different HCMV strains that the presence of antibodies assures complete protection against de novo infection with a different wild-type strain. Further research has to reveal if, perhaps, multiple exogenous infections with different HCMV strains may occur during life. Particularly in immunosuppressed patients one will have to find out if all HCMV infections are endogenous reactivations rather than secondary exogenous reinfections. The problem in regard to vaccine strains appears to be very pressing since it may be doubtful if an “old’ laboratory strain will protect sufficiently against a variety of wild-type strains. 6. Yet a final problem is the consistency of properties in a vaccine strain. Antigenic modulation may be common and it is also known from animal models of MCMV infection that attenuation may not be stable and reversal of the phenotype to a pathogenic one may occur rapidly (Jordan, 1980). Thus, in conclusion, this reviewer shares the views presented by Osborne (1981)and fails to find the case for active immunization with live HCMV convincing. However, the reader is again referred to the paper by Plotkin et al. (1981). The vaccine problem, however, remains pressing since HCMV infections are of serious concern, particularly in recipients of bone marrow transplants among whom 20% die from interstitial pneumonia, a disease at least associated with if not caused by HCMV. No therapeutic approaches to control this disease have led to any significant improvement of the situation. Let us consider first if there are data on active immunization, i.e., on passive administration of anti-HCMV antibodies. Generally, it is held that antibodies are of less relevance in infections with herpesviruses than cell-mediated immunity. Evidence for this is represented by the finding that viruria in children is persistent despite the presence of antibodies, or that maternally transferred antibodies did not protect newborns from infections in the first place. A similar situation is seen in the case of HSV where over the years evidence from animal experiments argues against the usefulness of active immunization. Recently, the case has been reopened by a number of investigators who have shown that newborn mice could be protected from HSV-encephalitis by adoptive transfer of anti-HSV antibodies (e.g., Worthington et al., 1980).Thus, injection of anti-HSV antibodies into newborns suffering from generalized HSV disease has been suggested.

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In disease caused by VZV, the usefulness of passive antibody therapy has been documented. Results of current trials of passive antiHCMV antibody treatment as, for example, in transplant recipients have not been reported. However, Yeager et aE. (1981) reported a study suggesting that under some conditions antibody is protective. They studied newborns receiving HCMV positive blood and found that 4 of 10 infants born to seronegative mothers died of HCMV infection, whereas none of 31 infants, who were born to seropositive mothers and who, therefore, possessed passive antibodies, had serious or fatal disease. During the last few years a number of synthetic antivirals effective against HSV have been developed (for a review see Overall, 1981). Acyclovir, which is the latest addition to this list, appears to hold great promise against HSV. Acyclovir is a poor substrate for cellular enzymes and thus it accumulates only in HSV-infected cells. Although HCMV does not have a virus-specified thymidine kinase, in vitro activity of acyclovir against HCMV has been found (Plotkin et al.,

1981). It is not within the scope of this article to review the scattered data concerning the use of synthetic antivirals in HCMV infections in man. However, it appears that there is no series of patients that has been successfully treated by any antiviral. For example, Kraemer et al. (1978) attempted prophylactic treatment with adenine arabinoside in patients receiving bone marrow transplantation; 22 of 40 patients received prophylactic doses of 5 mg/kg/day administered intravenously on an intermittent schedule. No difference in overall survival between the two groups was detected. The drug did not reduce the frequency of interstitial pneumonia or viral isolation from routine cultures. Similarly, Rytel and Kauffman (1976) have reported the absence of response to therapy with adenine arabinoside in HCMV infections in renal allograft recipients. XVIII. Conclusions

In 1970 Weller contributed an illuminating article entitled Cytomegalovirus, the difficult years. This reviewer is a neophyte in HCMV research, but it appears to him that almost all the relevant problems addressed by Weller are still far from being solved and that the difficult years are still ahead of us. In this article we have attempted to focus attention on relevant problems of the immunobiology of infection with HCMV and on the

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interactions between HCMV and the cellular components of the immune system.

1. The prevalence of antibodies against HCMV varies in different parts of the world, depending on socioepidemiologic parameters. In certain regions up to 100% of the people may be seropositive. Among male homosexuals a higher percentage of seropositive individuals has been found than among male heterosexuals. 2. Although strains of HCMV do share common antigens, antigenic heterogeneity has been documented. It has not been possible to establish major subgroups of clinical isolates similar to the situation with HSV-1 and HSV-2. In the latter case it is known, for example, that previous infection with HSV-1 does not prevent infection with HSV-2. It is not known if previous infection with one strain of HCMV will induce protection against all strains of HCMV. It is also not known how frequently oscillations of antibody titers occur in healthy individuals and how often antibodies fall below detection levels. 3. One wonders if the presence of serum antibodies is indeed effective in preventing a secondary infection. Newborns get infected despite the possession of passively acquired maternal antibodies. Clearly, “endogenous reactivations” in pregnant women or in renal transplant recipients occur despite the presence of specific antibodies. One wonders, if reinfections are always endogenous reactivations or perhaps de no00 exogenous infections with strains against which there is not sufficient protection. 4. Latency is considered to be the hallmark of all herpesviruses and it is generally accepted that this applies to HCMV as well. However, it is not known in which tissues HCMV is latent. In fact it is not certain that latency does play a role. It is known that a certain number of persons are chronically infected and shed HCMV in various secretions. True latency is defined as the presence of the genetic information of the virus in an unexpressed state. Thus, in the case of HSV one is convinced that the virus is latent in the ganglion and by a variety of stimuli is activated and causes the typical fever blister. One wonders if all patients infected with HCMV will establish a lifelong latency and, also, if all of these will remain seropositive for long periods. Perhaps only some patients establish latency. 5. Leukocytes have been assumed to be a site of viral replication and of latency. Studies among recipients of blood transfusions have supported this view, yet there is little direct evidence of replication (and less so of latency) of HCMV in human leukocytes and there

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are no convincing data as to which type of white blood cells are involved. 6. HCMV belongs to the few viruses for which intrauterine transmission has been unequivocally proven and CID is a disease that deserves great concern. As it appears from data in areas in which 100% of the women are seropositive and from studies of molecular epidemiology, most cases of CID are caused by infection endogenously reactivated in the mother. Interestingly, the percentage of women shedding HCMV in late pregnancy and in the nonpregnant state is equal, whereas virus shedding is suppressed in early pregnancy. 7. About 1%of children are infected in utero, whereas a greater number of infants get infected around birth. The number of children with manifest CID is, however, much lower than 1%.Later consequences of intrauterine or perinatal infections, as represented by sensiauditory problems or behavioral abnormalities, appear to be important. 8. It has been observed that virus shedding is terminated both in women and men after the age of 30 years. It is unclear if this is caused by a gradual build up of immune forces that finally control the infection or if the virus infection burns out by changes that occur in the viral genome. Interestingly, de nouo primary infections do occur in persons older than 30 years. This so-called community-acquired IM resembles classical EBV-IM but the test for heterophile antibodies is usually negative. 9. Infections with HCMV are prevalent in recipients of transplants. Up to 90% of renal transplant recipients get infected with a variety of diseases. In recipients of allogeneic bone marrow, HCMV appears to be involved in the pathogenesis of interstitial pneumonia, which is often lethal. It is not clear how often HCMV directly causes pneumonia, because other pathogens such as Candida albicans or Pneumocystis carinii are frequently found. HCMV is known to be immunosuppressive and perhaps its major importance lies in suppression of the defense against lethal superinfection with other pathogens. 10. Various herpesviruses are oncogenic and there have been a number of approaches to find links between HCMV and human cancer. The best case may be made for KS, which is a frequent tumor in Africa (up to 9% of all tumors, for example, in Uganda) with a distribution similar to that of Burkitt’s lymphoma. In Africa, KS is observed exclusively in black persons with a male predominance of 13 to 1. In Europe and North America KS used to be a rare tumor, occurring predominantly in elderly whites. KS is relatively frequent in recipi-

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ents of renal transplants. Second primary tumors are observed in patients with KS and spontaneous regressions of KS have been reported. These observations do suggest a role of the immune system in oncogenesis. 11. There has been an alarming recent “epidemic” of fatal KS among male homosexuals in certain American cities. There appears to be a new type of an acquired immunodeficiency syndrome, associated with a depletion of helper T cells and other dysfunctions. At least 12 pathogens have been found to cause disease in these patients including HSV, HCMV, Candida,etc. Most prevalent is pneumonia caused by Pneumocystis carinii. In the same group of patients a drastically increased frequency of KS has been reported. It will have to be determined if in this immunodeficiency syndrome, which is associated with many types of infections, HCMV does indeed play a role as a carcinogenic agent. Certainly many cofactors may be involved, most notably nitrites that are in common use in these patients as inhalants.

ACKNOWLEDGMENT It is my pleasure to acknowledge the outstanding editorial assistance of Ms. Marion Kasamasch. Her help has greatly aided the progress of this article.

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GENETICS OF RESISTANCE TO VIRUS-INDUCED LEUKEMIAS Daniel Meruelo’ and Richard Bach lrvington House Institute. Department of Pathology. New York University Medical Center. New York New York

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I . Introduction ...................................................... I1. The Initial Link between Viruses and Leukemias ...................... 111. Characteristics of the Retrovirus Family .............................. A. Genome Organization .......................................... B. Similarities with Transposons .................................... C. Replication ................................................... D. Gene Expression ............................................... E . Transformation ................................................ F. Assembly ..................................................... G. Polymorphism ................................................. H . Proximity of Proviruses, Histocompatibility, and Lymphocyte Antigen Loci ....................................................... IV. Expression in Inbred Mouse Strains of Antigens Associated with MuLV . . . A . Glx and GCSA ................................................. B . X.l .......................................................... c. G(ruo~l1,G(ERLD), and G(AKSU) ..................................... D . PC.l ......................................................... E . TL........................................................... F. ML .......................................................... G . Other Antigens ................................................ V. Genetics of Susceptibility to Viral Infection ........................... A . Genes Affecting Virus Spread .................................... B. Adsorption and Penetration ...................................... C . Fu-I:Restriction of Integration ................................... D. Availability and Replication of Target Cells ........................ E . Transformation ................................................ F. Immune Surveillance against Viral Infection and Transformation . . . . . . VI. Prospects for Control of Human Leukemia ............................ References .......................................................

107 108 109 109 111 111 114 117 117 119 124 132 132 134 135 135 136 137 137 138 138 139 140 143 151 161 173 176

1. Introduction

This article shall be concerned primarily with the genetics of susceptibility to leukemia in mice. the relevance of viruses as etiological agents. and the relationship of this information to leukemias and lymphomas in man . The studies that formed the foundations of present 1

Leukemia Society of America Scholar. 107

ADVANCES IN CANCER RESEARCH. VOL. 40

Copyright Q1.1983by Academic Press. Inc . All rights of reproduction in any form reserved. ISBN 0-12-006640-8

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day knowledge were begun several decades ago, with the period prior to 1960 being characterized by the gradual acceptance of the fact that viruses could cause cancer. This period also gave rise to the idea that genetically controlled factors could affect the degree and overall susceptibility to and latency of the disease. The 1960s and 1970s saw feverish activity, first defining the genes involved, and then probing cellular and molecular questions regarding their mode of action. Simultaneously, viruses were studied intensively to the extent that much of their molecular structure has now been elucidated. However, despite this intense activity, progress to date has not yet solved two fundamental questions: (1)Are viruses involved in human leukemias and lymphomas? (2) How can the knowledge on hand be applied to control or arrest the malignant process? It is hoped that this article summarizes and focuses the current state of knowledge in the field. While neither of the above stated questions can yet be answered, there is much reason to be excited and optimistic. The article shall first cover the virology of leukemia-inducing and related viruses, second discuss issues of specificity in virus-host genomic interaction, and third describe current knowledge of host genes conferring resistance or susceptibility to virus-induced neoplasia.

11. The Initial Link between Viruses and Leukemias

The proposition that leukemias are virus induced was suggested in studies of the infectious transfer of leukemias in chickens by Ellerman and Bang (1908) and in mice by Gross (1951). At that point in time, filtration had become an important tool in the study of virus diseases. If an extract prepared from diseased tissues could be filtered without losing its pathogenic potential (i.e., when the filtrate reproduces symptoms of the same disease following inoculation into a susceptible host) it was generally assumed that such a disease was caused by a virus (Gross, 1970). It was only the advent of the electron microscope that made it possible to visualize these pathogenic agents. Their electron microscopic morphology led to the use of the term “type C particles” for the leukemia viruses. In addition, the general class of oncogenic RNA tumor viruses was given the name “oncomaviruses.” However, many type C virus isolates do not show oncogenic activity. For this reason, a more general term was sought. All of these viruses have reverse transcriptase activity, hence the more recent term “retrovirus.”

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GENETICS OF RESISTANCE TO LEUKEMIAS

Ill. Characteristics of the Retrovirus Family

The designation “retrovirus” includes a variety of agents related to murine leukemia viruses. These have been classified into categories according to oncogenic properties. Broadly speaking, these classes include the sarcoma viruses, which induce rapid connective tissue neoplasms in vivo and transform fibroblasts in tissue culture; the acute leukemia viruses which cause rapidly identifiable hematologic neoplasms and generally possess some form of cell transforming potential in vitro; and the lymphatic leukemia viruses, whose inoculation can result in lymphocytic leukemia or lymphoma only after a long latency period and classically lack the ability to transform cells in tissue culture. In general, there are avian and mammalian examples of each of these categories. Structurally, the common characteristics shared by members of this family are genomes of diploid single-stranded RNA and a virion nucleotide polymerase capable of RNA-directed DNA synthesis. The basic genetic anatomy of the nondefective lymphatic leukemia viruses consists of three genes which code for products associated with the viral replicative cycle, termed gag, pol, and env.‘ The sarcoma and acute leukemia virus classes retain this basic framework while either adding an additional gene (Rous sarcoma virus, Fig. l),or acquiring new genetic material at the expense of replicative gene sequences, as has been demonstrated for Abelson-MuLV (Goff et al., 1980). These acquired sequences, apparently derived from cellular genes (Bishop, 1981), are thought responsible for neoplastic transformation by these viruses and have therefore been called oncogenes (see Section V,E,3).

A. GENOMEORGANIZATION The diploidy of retroviruses is unique among the genomes of known animal viruses and this property may explain the high recomgcnom IC IrrmnoL

genomtc

rcdundoncy

PO‘

,

env

I

src

1

A

(An)

7, t RNA Viral

RNA

FIG.1. The genome of avian sarcoma virus with redundant nucleotide sequences shown at the 3‘ and 5’ termini.

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DANIEL MERUELO AND RICHARD BACH

bination frequency obtained with these viruses (Weiss et aZ., 1973; McCarter, 1977). Figure 1 illustrates the composition, structure, and topography of a haploid subunit of one well-characterized retrovirus genome, the avian sarcoma virus (ASV). The general features outlined in this figure are applicable to most, if not all, retroviruses. The 5' termini of both subunits of the dimer are capped by the structure 5'-m7 GpppGm (Furuichi et al., 1975; Rose et al., 1976)and the 3' ends are polyadenylated (- 200 residues) (Bender and Davidson, 1976; King and Wells, 1976). About 10 specific sites within the 3' half of the genome, containing adenosine residues, are methylated (Furuichi et al., 1975; Beemon and Keith, 1976; Dimock and Stoltzfus, 1977). These fea" tures, i.e., polyadenylation, capping," and a low level of internal methylation, are common to eukaryotic mRNAs. It is not surprising therefore that the retrovirus genome can serve as a messenger for the synthesis of virus-specific proteins. Host cell tRNATp is bound to the genome of ASV at a site 101 nucleotides distant from the 5' terminus of the genome (Taylor and Illmensee, 1975) and serves as primer for the initiation of DNA synthesis by reverse transcriptase in uitro (Dahlberg et al., 1974; Harada et al., 1975).The identity and location of the tRNA primer vary among retroviruses. Other host cell tRNAs are also bound to the haploid subunit of retroviruses although less firmly (Sawyer and Dahlberg, 1973).Their function(s) (if any) are not known, but their importance is unclear since many of the represented isoacceptor species are not abundant enough to be associated with all molecules of viral RNA (Sawyer and Dahlberg, 1973). The haploid subunits of the genomes of most retroviruses are terminally redundant (Fig. 2). For example, Temin's group (Shimotohno et al., 1980a; Shimotohno and Temin, 1980) has shown that spleen necrosis virus contains a 5 bp direct repeat of cellular DNA next to a 3 bp inverted repeat of viral DNA. The inverted repeats formed the ends of a 569 bp direct repeat of viral DNA. Comparable results have been obtained by investigators working with proviruses of Moloney murine 3'-LTR

5'-LTR

- -4!r 3'

i

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t

3'

5'

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1

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FIG.2. Diagram of integrated viral DNA containing two long terminal repeats (LTRs).

GENETICS OF RESISTANCE TO LEUKEMIAS

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sarcoma virus (Dhar et al., 1980), mouse mammary tumor virus (Majors and Varmus, 1980), and Rous-associated virus 0 (Ju and Skalka, 1980). The lengths of the repeat differ in different viruses. The direct repeat of viral DNA is called LTR for long terminal repeat. These retroviral LTRs appear to function critically in the integration of proviral DNA into cellular DNA, and detailed analyses have revealed the presence of putative transcriptional promotor and polyadenylation signals (Ju and Skalka, 1980). WITH TRANSPOSONS B. SIMILARITIES

Several features of retrovirus organization, the presence of direct repeats of host DNA, inverted and direct repeats in the viral DNA, and unique sites on the virus DNA for insertion, are rather similar to those found in certain bacterial moveable genetic elements, namely, transposons (Shimotohno et al., 1980a). Other recently described moveable genetic elements of eukaryotic cells (yeast TY1 and Drosophila copia-see below) have these same features. In addition, all of these transposon-like structures end with the dinucleotides TG . . . GA (Allet, 1979; Kahnman and Kamp, 1979). Z Y I , copia, and retrovirus genomes have a 5 bp direct repeat of element or viral DNA flanking the inside ends of the two LTRs (Farabaugh and Fink, 1980; Dunsmuir et al., 1980; Gaffner and Philippsen, 1980). The similarities found are believed to be too strong to result merely from a chance coincidence. It should be noted that the only cellular moveable genetic elements described to date for vertebrates are the endogenously found retroviruses. If the retroviruses are derived from cellular genetic elements as is now presumed (Temin, l970,1971b), these movable genetic pieces may play a role in cell differentiation by regulating genetic rearrangements. For example, control of yeast mating type (Cameron et al., 1979) and immunoglobulin structure (Brack et al., 1978; Seidman and Leder, 1978) has been shown to involve DNA rearrangements. Whatever their function, the origin of retrovirus from cellular moveable genetic elements may provide a clue for carcinogenesis for which a viral etiology is not apparent. That is, such cancers may result from processes similar to those involved in the evolution of retroviruses. We shall return to this issue later in this article.

C. REPLICATION The viral envelope glycoproteins are primarily responsible for adsorption and penetration of virus into cells as demonstrated by the fact

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DANIEL MERUELO AND RICHARD BACH

that deletions in enu give rise to fully assembled, noninfectious virus particles (Duesberg et al., 1975). Similarly, temperature-sensitive conditional mutants in enu render virus particles noninfectious at the restrictive temperature (Vogt and Hu, 1977).The interaction between viral envelope glycoprotein and host celI receptors has been reconstructed in uitro with purified gp70 of MuLV and is highly specific (DeLarco and Todaro, 1976). The events that follow adsorption of the virus to the cell surface and onset of viral DNA synthesis are not well understood. Electron microscopy studies have suggested that the virus genome moves quickly after infection (within 10-60 min) into the nucleus of the host cell (Dales and Hanafusa, 1972). By contrast, biochemical data indicate that initial viral DNA synthesis starts in the cytoplasm of the cell and continues therein for the first 12-24 hr following infection (Varmus et al., 1974),after which integration occurs. It is presently not clear whether protein synthesis is or is not required as an early event in the establishment of infection by retroviruses. It has been shown that parental RNA associates with polyribosomes (Salzberg et al., 1977) and may, in some cases, be translated prior to the onset of (or in the absence of) viral DNA synthesis (Gallis et al., 1976). However, their data were obtained with extremely high multiplicities of infection and may not reflect the in vivo situation. The clearest experiments have shown that viral RNA may be expressed directly after introduction into the host cell by either microinjection (Stacey et al., 1977) or application in the presence of polycations. Figure 3 outlines the principal molecular events in the replication of retroviruses. The scheme is partly hypothetical but reflects current experimental findings. Following infection, virus-coded reverse transcriptase copies the virus-single stranded RNA genome into double-stranded DNA (Verma et al., 1976). Studies of reverse transcription in uitro indicate that DNA synthesis proceeds in a 5' to 3' fashion beginning at the priming tRNA molecule, polymerizing deoxyribonucleotides complementary to the RNA genome until the 5' end of the template is reached. The nascent single-stranded DNA molecule migrates then to the 3' end of the RNA template; there it partially pairs by virtue of complementary nucleotides with the string of approximately 60 bases immediately 5' to the poly(A)tail on the end of the genome (Coffin, 1979). After migrating to the 3' end of the RNA template, the nascent chain is elongated, presumably continuously, to the 5' end of the template producing a complete minus-strand DNA copy of the virus genome.

GENETICS OF RESISTANCE TO LEUKEMIAS c y t oplos in! c

v ~ r o l RNA

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113

events

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FIG.3. The replicative cycle of retroviruses. Portions of the scheme that are still hypothetical are indicated by a question mark.

The major stable products of synthesis are linear duplexes and closed circular duplexes (Fig. 3) (Shank et al., 1978; Hsu et al., 1978; Shank and Varmus, 1978); both forms are approximately the length of the haploid subunit of the viral genome. The mechanism for plus-strand DNA synthesis and for producing terminal repeats to form the double-stranded DNA molecule has not yet been elucidated. Nonetheless, a sequence just into the body of the virus from the repeat junction (in MuLV positions 516-560) is of interest. The upper strand of this sequence contains 15 pyrimidines in a row, followed by a 15 nucleotide sequence containing 12 purines and an 11 nucleotide sequence containing 10 pyrimidines (Sutcliffe et al., 1980). What makes this sequence of particular interest is that it occurs at the repeat junction, where the origin of plus-strand replication is thought to be localized (Mitra et al., 1979). When this sequence is examined closely, it can be shown to form a stem-and-loop structure (hairpin) (Fig. 4) similar to that for the single-stranded bacteriophage origins of replication (Sims and Benz, 1980).

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DANIEL MERUELO AND RICHARD BACH

FIG. 4. Origin of second strand synthesis can be drawn as a hairpin structure. A schematic minus-strand loop containing an inverted repeat is shown as derived from data of Sutcliffe et ol. (1980) for Moloney leukemia virus. Base complementation is indicated by heavy lines.

The double-stranded DNA molecule with repeated ends is thus the structure that probably integrates into the host chromosome. There it is inherited in a Mendelian fashion and acts as a substrate for transcription and generation of new virus particles. Retroviruses integrate at multiple sites in the host DNA (we shall return to this issue in Sections III,G,l and III,H,l-4). It is not known if the circular or linear form of viral DNA is integrated. However, presence of inverted repeats at the termini of either 5’ LTR or 3’ LTR suggests an analogy with the bacterial transposons (Kleckner, 1977) and suggests that the circular form is a better candidate for integration. How the circular form of the unintegrated viral DNA can be integrated has been the subject of several hypothetical models (Shapiro, 1979; Shoemaker et al., 1980). Viral DNA containing one LTR copy can integrate after generating two copies of LTRs in the models described by Shapiro (1979) or Shoemaker et al. (1980). However, only those molecules that contain two copies of LTR are able to transform or infect cells in the studies with cloned M-MSV DNA (Verma et al., 1980) and in ljitro reconstructed clones M-MLV DNA. D. GENEEXPRESSION The mechanisms that generate the final gene products of retroviruses are illustrated in Fig. 5, using ASV as a prototype. Of the four ASV genes, three are translated from mRNAs that contain the expressed gene at the 5’ end; g a g from 38 S mRNA, enu from 28 S mRNA, and src from 21 S mRNA. On the other hand, pol is probably expressed by the continuous translation from gag and pol in 38 S

GENETICS OF RESISTANCE TO LEUKEMIAS

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mRNA. This scheme is consistent with the notion that translation in eukaryotic cells initiates only at the 5’ termini of mRNA (Jacobson and Baltimore, 1968), and is well suited for independent expression of viral genes according to the relative need for gene products as is the case for plant (Hunter et al., 1976) and animal (Cancedda et al., 1975) viruses. The primary product of translation from gag is a polyprotein from which the individual polypeptides of the viral core are generated by cleavage (Vogt et al., 1975; Shapiro et al., 1976). The linear order of the individual core proteins within gag for ASV is illustrated in Fig. 5. The fact that cleavage of the polyprotein does not occur in certain host cells suggests that this processing is carried out by cellular enzymes (Eisenman et al., 1974), although enzymes associated with ASV and MuLV have been shown to cleave the precursor protein into the correct products (Helm, 1977; Yoshinaka and Luftig, 1977b). The nature of these virion-associated processing enzymes is unclear; in ALV such activities have been shown to be associated with p15 whereas in MuLV the protease activity apparently resides on a previously unrecognized protein (Yoshinaka and Luftig, 1977b). Particles of the gag polyprotein are often expressed on the cell surface in a glycosylated form that is neither cleaved nor incorporated into virions (Snyder et al., 1977; Ledbetter and Nowinski, 1977) giving rise to important antigenic determinants on the surface of leukemic cells in mice (see Section IV) (Snyder et al., 1977). A virus-specific RNA with the size and composition expected for a pol messenger has not yet been detected (Weiss et al., 1977; Hayward, 1977). The evidence, however, is consistent with the notion that pol is expressed from the continuous translation of gag and pol in 38 S mRNA. The molecular weight of the readthrough product is 180,000 (Pr 18OPO’) and contains the antigenic determinants and tryptic peptides of both the gag and pol proteins (Oppermann et at., 1977). The evidence supporting the readthrough model is as follows: (1)Pr 18OPo1 has been found in virus-infected permissive (Oppermann et al., 1977; Jamjoon et al., 1977) and nonpermissive cells and can be synthesized in vitro with either the viral genome (Kerr et al., 1976; Purchio et al., 1977) or 38 S RNA isolated from infected permissive cells as messenger; (2) kinetic analysis studies indicate that Pr 180P”’is not a precursor for any of the mature gag gene products (Oppermann et al., 1977), and that its turnover is related to the appearance of mature reverse transcriptase in virus particles (Oppennann et al., 1977); and (3) Pr 180P”’is present in extracellular virions where it gradually decays in concert with the appearance of the mature polymerase (Oppermann et

116

DANIEL MERUELO AND RICHARD BACH gp37-s-s-gp85

1 Pr 90'""

t Pr 70*n"

Pr180p0'

tt

5' cop

-

gag

,

pol

,

cnv

TrI

1

Pr 76"'

POl(fl)

, I

(7)

crc

I-

,

3' c

( A )n

pp 60"'

/ I \ \ p12 p i 5

p27

FIG.5. Gene expression for ASV. Messenger RNAs, polyprotein precursors, and cleaved mature virion molecules are shown.

al., 1977). The above data are, therefore, in line with the notion that reverse transcriptase is generated from a polypeptide precursor, and that this processing occurs in extracellular virus. The discovery of Pr 180p"'raises the issue of how readthrough translation is regulated in eukaryotes. In prokaroytes it is known to occur by suppression of a termination signal (Weiner and Weber, 1971).The fact that synthesis of Pr 180p"' in uitro with the genome of MuLV as messenger can be substantially augmented by the use of an amber tRNA isolated from yeast (Philipson et al., 1978), identifies the signal that terminates translation from gag but does not establish the mechanism by which Pr 180P"' is synthesized in the infected cell. Readthrough of amber codons is usually a very rare event in viuo, yet amber codons are not usually bypassed by means other than specific suppressor tRNAs (Philipson et al., 1978). Messenger RNA for en6 of various retroviruses has been identified by translation in uitro (Pawson et al., 1977) and in Xenopus oocytes (Van Zaane et al., 1977) and by microinjection of cells infected with a deletion mutant in enu (Stacey et al., 1977). It appears to be generated by RNA splicing, containing sequences from the 5' end of genomic RNA contiguous with enu homologous sequences (Shinnick et al., 1981).The primary product of translation from ASV enu is a protein of 70K MW (Pr 70""") (Moelling and Hayami, 1977). It has been shown to accumulate in infected cells in the presence of an inhibitor of glycosylation (Shapiro et al., 1976; Moelling and Hayami, 1977). While Pr 70""" has been shown to contain some carbohydrate residues (Moelling and Hayami, 1977), further glycosylation generates a second form, Pr 90""" (Moelling and Hayami, 1977; Famulari et al., 1976; England et al., 1977). The changed mobility of the polypeptide SDS gels

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results from the effect of carbohydrate residues on its electrophoretic mobility. When Pr 90”””is cleaved, it produces the mature envelope glycoproteins: gp85 and gp37 ofASV, gp70 and p15E of MuLV. Cleavage may be coordinated with migration of the glycoprotein to the surface of the infected cell (see Section 111,F).

E . TRANSFORMATION It should be noted that neoplastic transformation is not a necessary consequence of viral replication. A case in point are ASV and avian leukemia viruses (ALV), both of which replicate in avian fibroblasts, yet only ASV transforms these cells. In general, transformation by any single strain of virus is restricted to particular target cells (see Section V,D), whereas the host range for replication is less specific. While transformation can occur in the absence of viral replication, the efficiency of transformation of nonpermissive cells (e.g., mammalian fibroblast hosts for ASV) is usually quite low to (Temin, 1971a).It appears that a 21 S virus-specific RNA in cells infected with ASV encoding only src and “c” is likely to be the messenger for the transforming protein. Deletions in STC abolish the ability of the virus to transform fibroblasts but have no effect on viral replication and are, therefore, denoted “transformation defective” (tdASV). At least some strains of tdASV can induce lymphoid leukosis in birds (Biggs et al., 1973) and are, therefore, analogous to the naturally occurring avian leukosis viruses. Such deletions in src appear spontaneously in high frequencies for some strains of ASV (Vogt, 1971) or can be induced by mutagenesis (Biggs et al., 1973).

F. ASSEMBLY A model for the assembly of retroviruses has been proposed (Bolognesi et d.,1978) on the basis of several parameters, including arrangement of virion structural components in the assembled particles themselves, the fine genetic structure determined through numerous experiments, and available biosynthetic data. The model proposes the following: 1. Virus envelope components (e.g., gp70-pl5E) migrate to and are inserted on the cell surface at the site of virus budding, already in molecular complexes (i.e., polypeptide complexes). 2. Internal virus precursor molecules are then transported to the budding site where one end of the molecule is joined to the virus envelope complex, while the other end associates with the virus RNA.

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DANIEL MERUELO AND RICHARD BACH

3. Virus assembly progresses with the proteolytic cleavage of such precursor molecular complexes. 4. The cleaved virion components associate with each other to yield the substructures normally found in the mature, budded particles (i.e., the envelope, inner coat, core shell, and ribonucleoprotein complex). The mechanism of virus assembly seems to be greatly dependent on the proper association or “bonding” interactions between structural components which occur during the budding process. Thus specific recognition and noncovalent association between certain envelope and internal virion molecules is required for virus maturation. Whether such interactions serve an “aligning” function is not established, however some viral molecules remain associated during precipitation despite their noncovalent association (e.g., p19 and gp35 are brought down together by antiserum to gp85, the major glycoprotein) (Schlesinger, 1976). In addition, Rohrschneider et al. (1976) have shown that avian p19 is critical for virus synthesis and assembly since mutants with defective p19 molecules fail to assembly correctly. The evidence that precursor cleavage is a late step in virus maturation is as follows: First, the sequence of the structural components from exterior to interior of the mature virus is in good correspondence with the order they are found on identified precursor molecules (Barbacid et al., 1976; Eisenman et al., 1974; Jamjoon et al., 1977; Arcement et al., 1977). Second, several groups (Naso et al., 1976; Van Zaane et al., 1977; Famulari et aZ., 1976; Shapiro et aE., 1976) have identified nonglycosylated gag precusor molecules which appears to have escaped proteolytic digestion and processing. In addition, gag precusor molecules and precursor-specific proteases have been found in Rauscher leukemia virus. After appropriate incubation of these particles, cleavages result to give rise to the appropriate structural components (Yoshinaka and Luftig, 1977a,b). Third, it is known that some virus structural components (e.g., avian p15) are probably involved in this type of proteolytic activity (Von der Helm, 1977). The above data are consistent with the cleavage of gag precursor molecules at the cell surface, at a late stage of virus maturation and budding. Consistent with this notion is the fact that gag processing is usually associated only with virus-producing cells (Eisenman et al., 1974). The gag-pol molecule readthrough products often seen may not result from transcriptiodtranslation errors, but may function as a means of incorporating a small number of polymerase molecules into the virion. Viral RNA does not appear to be required for virion assembly, since murine

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leukemia virus produced in the presence of actinomycin D lacks 60 S to 70 S RNA, but contains normal amounts of virion structural polypeptides, including polymerase (Witte and Baltimore, 1977). Finally, in considering assembly, it was stated that the first step in virus assembly is the insertion on the cell membrane of viral envelope glycoprotein complexes. This notion is somewhat controversial in light of studies by Hanafusa and co-investigators (Scheele and Hanafusa, 1971; Kawai and Hanafusa, 1973) which have shown that defective Rous sarcoma virus (RSV) particles can be synthesized in the absence of detectable gp85 or gp35. The studies by Hanafusa’s lab (1971, 1973) involved analysis by polyacrylamide gel electrophoresis and it could not be excluded that very small amounts of the glycoprotein or a fragment of the envelope products were present in cells budding the RSV particles. This is important because Witte and Baltimore (1977) have shown that very little of env glycoproteins are required for the viral assembly process to occur.

G. POLYMORPHISM 1. Host Range Progress in the early years following discovery of avian and murine type C viruses was hindered by the fact that the only available assay for these viruses was leukemogenesis or tumorigenesis. The subsequent development of substantially more potent virus variants for several retroviruses and the isolation of several exogenous viruses (Pincus, 1980) considerably shortened the time required to observe leukemogenic effects. Further impetus to the study of viral oncogenesis came as a wide range of techniques ensued for rapid analysis of the biological functions and molecular biology of type C viruses. Studies made possible by these advances quickly revealed several different categories of host range polymorphisms for these viruses. For example, it was discovered that certain murine type C viruses can infect mouse cells but cannot infect cells of other species (these are designated ecotropic), while others cannot infect mouse cells but can infect cells of heterologous species (xenotropic viruses) (Levy and Pincus, 1970; Levy, 1978; Aaronson and Stephenson, 1973). Those than can infect cells of both the mouse and heterologous species are called dualtropic or polytroic viruses. In addition, a distinct category of viruses with this (dualtropic) same property, called amphotropic viruses, has been isolated from wild mice (only) (Hartley and Rowe, 1976; Rasheed et aZ., 1976; Chattopadhyay et aZ., 1978). Further poly-

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DANIEL MERUELO AND RICHARD BACH

morphism among the murine type C viruses has been recognized in their varying ability to replicate in cells of mice bearing different alleles at the Fu-1 locus and to express themselves in cells of different mouse tissues (organotropism). We shall return to polymorphism with regard to Fu-1 and organotropism later on in this article, but we would like to discuss the species type polymorphisms first. Biochemical studies have shown that the viral enu gene products are responsible for the type of host range variation which has classified viruses as ecotropic, xenotropic, polytropic, or amphotropic (Elder et al., 1977, 1978; Troxler et aZ., 1977). For example, polytropic viruses isolated from AKR mice show p30 peptide (gag gene product) maps similar to ecotropic AKR virus, but gp70 peptides (enu gene products) which differ from classical AKR virus in containing xenotropic gp70 sequences (Elder et al., 1977). The recombinant polytropic HIX virus, isolated from Moloney-MuLV virus-infected cells (Fischinger et aZ., 1975), also contains a p30 identical to its ecotropic Moloney-MuLV parental type, but a gp70 containing xenotropic virus determinants (Fischinger et al., 1978). The polytropic B-MuX virus, induced from BALB/c cells by iododeoxyuridine, again shows a p30 core protein similar to that of its ecotropic virus progenitor and a gp70 envelope protein which contains endogenous xenotropic determinants (Ihle et al., 1978). Indeed, an interesting characteristic of retroviruses is their high recombination frequency. Recombination increases polymorphism and occurs usually, but not always, in the enu gene. For example, analysis of gp70 tryptic peptides has shown, with one exception, that no two gp70s isolated from different viruses are identical (Elder et al., 1978). Almost every gp70 has distinct tryptic peptides. There are, nonetheless, resemblances which allow classification and subdivision of the viruses according to relatedness (Elder et al., 1977). In contrast to gp70, p30, which by mass is the major core protein, is highly conserved. However, not all p30s are identical, and thus far several distinct types have been defined in the mouse (Gautsch et al., 1981). Less is known about the relative structure of the polymerases, p12, p10, and p15, but available data suggest that polymerases p10 and p15 are relatively conserved. In contrast, p12, in accord with its specific RNA-binding function, is more polymorphic (Stephenson et al., 1974; Aaronson and Stephenson, 1975). 2. Polymorphism with Respect to Fv-1 As mentioned previously, replication by most murine type C viruses is subject to Fu-1 host range restriction (Rowe and Sato, 1973).

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Viruses, therefore, are designated as N-tropic or B-tropic according to their preferential growth on either N-type (Fu-1" homozygous) or Btype ( Fv-lb homozygous) mouse cells (Pincus et al., 1971b). The viral determinant of susceptibility to this restriction system has been biochemically assigned to the gag gene product (Hopkins et al., 1977; Schindler et al., 1977; Gautsch et al., 1978). N-tropic virus strains replicate 100-1000 times more efficiently in cells derived from NIH Swiss mice than in BALB/c cells (Pincus et al., 1971b). Conversely, B-tropic viruses replicate 30- to 100-foldbetter in BALB/c cells than in NIH cells (Pincus et al., 1971b). Fu-l permissiveness is governed by a single genetic locus on chromosome 4,whose two alleles (Fu-1" and Fu-lb) exert dominant restriction upon infection (Rowe et al., 1973). Early studies on the mechanism of Fv-l restriction showed, by using vesicular stomatitus virus (VSV) pseudotypes, that Fv-l did not influence virus adsorption or penetration. For example, VSV genomes encapsidated into coats derived from either N- or B-tropic viruses could infect normal cells of either Fu-1 type equally well (Huang et al., 1973; Krontiris et al., 1973). It was, therefore, concluded that an intracellular process specific to the type C virus infectious process must be affected by the Fu-l gene product. To study the mechanism of Fu-l restriction, several experiments were conducted. These revealed that viral DNA synthesis in Fu-l restrictive cells proceeds at normal levels (Jolicoeur and Baltimore, 1976; Sveda et al., 1976), but viral RNA synthesis is curtailed by Fu-l restriction (Jolicoeur and Baltimore, 1976). Association of viral DNA with cellular DNA is inhibited in restrictive cells, indicating that the Fu-l gene product interferes with integration of viral DNA into cellular DNA (Jolicoeur and Baltimore, 1976). Recently, it was demonstrated that a restrictive Fu-l gene product does not affect the production of linear viral DNA, but does markedly interfere with the appearance of supercoiled, closed circular viral DNA, possibly a crucial intermediate in proviral integration (Jolicoeur and Rassart, 1981). Fv-l restriction is, however, not absolute, since it can be overcome by infection ofa single cell with two or more virus particles (Pincus et al., 1975; Declkve et al., 1975; O'Donnell et al., 1976). The abrogation of Fu-l restriction by high multiplicity of infection has shown that infection by a single particle of the wrong tropism, even if it does not replicate, is sufficient to allow replication of coinfecting virus of the same tropism (Duran-Troise et al., 1977). The viral gene product determinants for N- and B-tropic host range appear to reside in the p30 structural protein by peptide mapping analysis (Hopkins et al., 1977; Schindler et al., 1977; Gautsch et al., 1978),although certain N- and B-

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DANIEL MERUELO AND RICHARD BACII

tropic isolates were found to differ in other structural proteins by use of isoelectric focusing (Pfeffer e t al., 1976). The viral determinants of N-, B-, and NB-tropism appear to be allelic on the basis of oligonucleotide mapping (Faller and Hopkins, 1977) and RNA sequencing (Rommelare et al., 1979). The gene product of this viral locus is present in infecting virions, and in Fv-l-restricted cells it participates in an interaction between newly synthesized viral DNA and the Fu-l gene product in some way which prevents successful integration of viral DNA into cellular DNA. Studies to identify the cellular gene product involved in Fv-1 restriction have shown that soluble extracts from uninfected Fv-l -restrictive cells are able to transfer resistance to Fv-1-permissive cells, if added shortly prior to or after virus infection (Tennant et al., 1974). Very little of this gene product, a cytoplasmic RNA molecule (Yang et al., 1978), can be found in cells. This scarcity of the material probably accounts for the fact that only relatively weak levels of resistance have been obtained in transfer experiments and for difficulties hampering progress in achieving a molecular understanding of the mechanism of Fv-1 restriction. While no mutants have been generated either in vivo or in vitro which fail to show FG-1restriction of either N- or B-tropic viruses, the study of Fv-l function might be helped by availability of Fv-1 congenic mice and the existence of several mouse cell lines which show an Fv-1 nonrestrictive phenotype (Hartley and Rowe, 1975). 3. Organotropism Newborn mice infected with murine type C viruses such as Gross, AKR, and RadLV viruses, develop a specific thymus-derived leukemia after a latency of several months (Gross, 1970; Kaplan, 1967). The development of leukemia follows the appearance of high titers of infectious leukemia virus in blood (Lilly et al., 1975). Using mice infected with Moloney-MuLV, molecular hybridization experiments have shown that virus-specific DNA sequences can be found only in target lymphoid organs, i.e., the thymus and the spleen (Jaenisch et al., 1975; Jaenisch, 1976).Virus-specific sequences are not detected in nontarget organs, such as kidneys, liver, brain, testes, muscle, and lungs. It would appear that injection of MuLV into newborn animals leads to specific infection of a restricted set of target cells, such as thymus-derived lymphocytes or other lymphocytic cells. It is probably through replication in the latter cells that virus makes its way into the bloodstream or infects mammary epithelial cells which then secrete high titers of infectious virus into the milk (Jenson et al., 1976).

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Other tissues, such as the germ line cells, very rarely seem to be infected. Shoyab and Baluda (1975) have made similar observations in chickens infected with avian myeloblastosis virus. In mice, McCrath et al., (1978a) have observed target specificity with mammary tumor virus (MMTV). Organotropism of infection, as described above, applies to the in uivo interaction, i.e., the infection of animals with virus and not necessarily to the in oitro situation. Thus cells which cannot be infected in uivo are known to support the replication of virus in uitro. For example, fibroblast cultures infected with Moloney-MuLV (M-MuLV) in vitro replicate virus efficiently and can be used to prepare virus stocks. In the animal, however, fibroblasts are not usually susceptible to infection and replication of M-MuLV. A second caveat in defining organotropism is that this tropism for infection can be different from that for transformation by any one virus. For example, while both T and B cells are susceptible to productive infection by MoloneyMuLV, virus-induced transformation (leukemia) is restricted to thymus-dependent T cells (DeclBve et al., 1974; Waksal et al., 1976; Baird et al., 1977). This tissue tropism (organotropism) has he€ped identify an additional polymorphism (to those described in earlier sections of this article) among viruses. For example, the polytropic viruses (PTV) of HRS/J mice differ from the HRS/J prototype ecotropic virus (ETV-1) in their tissue tropism. At least four isolates of PTVs are highly thymotropic in both HRS/J and CBA/J mice (Green et al., 1980). By contrast, ETV-1, when tested in virus-free CBA/N mice, infects thymus, spleen, and bone marrow to about the same extent (Green et al., 1980). Another example is provided by study of isolates of radiation-induced leukemia virus. Several type C viruses with distinctively different cytotropisms have been recovered from mice of strain C57BLKa (Dec k v e et al., 1976, 1978; Lieberman et al., 1979). The radiation leukemia virus (RadLV) and its tissue culture version (RadLVNL3) induce thymic lymphomas after inoculation into C57BWKa hosts, whereas three other isolates, designated BL/Ka (B), BUKa (N), and BL/Ka (X), are devoid of leukemogenic activity (Declgve et al., 1976; Lieberman et al., 1977). RadLV and RadLVNL3 are B tropic (for Fo-1) and thymotropic because they replicate preferentially in thymic lymphocytes of adult mice. The other three viruses are designated fibrotropic since they productively infect fibroblasts but not thymocytes ( i n uitro) of appropriate genotype: Fu-lbbfor BWKa (B), Fu-1"" for BWKa (N), and nonmurine cells for BL/Ka (X). The mechanism by which tissue-specific restriction operates ap-

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DANIEL MERUELO AND RICHARD BACH

pears to be distinct from that of Fu-1. For example, viral DNA from Band N-tropic virus-infected cells can transfect fibroblasts of either Fu-lbb or Fu-I"" genotype, because the viral integration block of Fu-1 is skipped. Blocks in the virus infection process such as Fu-1 are by-passed by transfection. On the other hand, the capacity of thymocytes and fibroblasts of different genotype to undergo transfection with DNA isolated from cell lines previously infected with the Becotropic, B-fibrotropic, or B-thymotropic-RadLV isolates i s not equal (Kopecka et al., 1980). For example, transfection of fibroblasts is not achieved with DNA from a thymotropic virus-producing cell line (Kopecka et al., 1980). Thus, viral organotropism is determined by mechanisms not surmountable by transfection.

H. PROXIMITY OF PROVIRUSES, HISTOCOMPATIBILITY, AND LYMPHOCYTE ANTIGENLOCI 1. A Hypothesis Several hypotheses have been postulated to account for the mechanism(s) responsible for organotropism. One of these is that tissuespecific activation of endogenous viruses might be a consequence of the specific chromosomal integration site of the virus. Expression or repression of genetic elements of different viruses might be under control of different cellular loci involved in normal tissue differentiation, These loci might exert their effect in a cis-specific manner, controlling gene expression downstream (Jaenisch and Berns, 1977). Thus, leukemogenic endogenous viruses might be integrated at chromosomal sites of the mouse which are not expressed early in embryogenesis. As a result of normal tissue differentiation, virus-related loci might become activated in cells of the lymphatic-erythropoietic lineages. If this were the case, integrated viral genomes would remain silent in all other organs and cells of the developing and adult mouse in which the specific differentiation loci remain silent (i.e., all nonlymphoid organs). Such a cis-acting mechanism for the regulation of tumor virus expression in avian cells has been suggested by the data of Cooper and Silverman (1978).The different patterns of virus gene expression seen in various mouse strains could also be explained by the simple assumption that in each instance a virus gene is integrated at a different chromosomal region which is active at specific stages of development or in certain tissues but not at other stages of development or in other tissues. We would like to propose that the speci$c integration sites are spec$cally near histocompatibility loci (minor and major) and lymphocyte differentiation loci encoding Ly antigens. We shall develop this hypothesis further shortly.

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This hypothesis to account for organotropism is reasonable in view of recent experiments by Jaenisch et al. (1981), in which strains of BALB/Mo mice were derived which carry the exogenous Mo-MuLV genome as stable Mendelian genes. Mice carrying virtually identical proviral sequences integrated at different chromosomal loci showed different phenotypes of virus expression, placing emphasis on the regulatory function of adjacent regions of cellular DNA (see Section 111,H74).By extension, this work implies that although proviruses may integrate randomly into cellular DNA during infection, the site of integration is not inconsequential for virus expression. This hypothesis is also compatible with recent experiments by Dina and Penhoet (1978), which suggest that the various integrated copies of Mo-MuLV and MSV in infected fibroblasts are expressed at different rates. How could one account for differential rates of expression? The answer might lie-in the chromatin structure. There are some suggestions that actively transcribing chromosomal sites have a different chromatin structure. Consistent with this notion is the fact that the chromatin structure of the actively transcribed Moloney-MuLV genomes in target cells of BALBfMo mice shows altered sensitivity to digestion with DNase I compared to the repressed Moloney-MuLV genome in nontarget tissues (Breindl and Jaenisch, 1979).Groudine et al. (1978) have made similar observations for the integrated genomes of several endogenous and exogenous avian tumor viruses. In addition, it has been demonstrated previously (Hayward and Hanafusa, 1976) that endogenous and exogenous viral genomes, which are presumably integrated at different chromosomal sites, are subject to different control mechanisms.

2. Studies with RadLV The genetic mapping of endogenous viruses in different inbred mouse strains is in its infancy. Thus it would appear premature to draw firm conclusions about the relation of chromosomal integration of a virus and its expression on the differentiated cell. Nonetheless, our studies with radiation leukemia virus (RadLV) have revealed some interesting, relevant findings. Association between malignant disease and the murine major histocompatibility complex H-2 (Fig. 6) was first noted by Gorer (1956)and Gross (1970), who observed that all strains showing a high incidence of spontaneous or induced leukemia had the same H-2 haplotype. Since then, H-2 associations with susceptibility or resistance to virally induced leukemias have been extensively demonstrated (Meruelo and McDevitt, 1978).

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DANIEL MERUELO AND RICHARD BACH

la-3 Sip --

10-1

MARKER LOCI

-\ SUBREGIONS REGIONS

H-2K

I r - U Ir-I9 10-4 la-5 Ir-1C

Ss

H-20 H-2L

00-100-2 TLo

-1

-A

-K

-B -J -E -C I -

5 -

0 -

TL -

FIG.6. Partial genetic fine structure of mouse chromosome 17 (linkage group IX) encoding H-2-TLa complex. The complex is divided into five main regions: K, I, S, D, and TLa.These regions are divided by marker loci H-2K, Ir-1, Ss(S l p ) , H-2D, and TL. The boundaries of each region are defined by inba-H-2 recombinations. The I region has been subdivided into five subregions by recombination: A, B, J, E, and C defined by marker loci Is-1A (la-l), lr-lE, Ia-4, IQ-5and Is-1C (la-3), respectively. By convention, the K end of the complex is the segment to the left of Ss. The D end is the segment to the right of Ss. Alleles are alternate genes at defined loci, and haplotype designates the specific combination of all alleles at all loci within the complex characterizing a given mouse strain. Allele and haplotype designations are noted in lower case letters, whereas regions, subregions, and marker loci are in capital letters. The TL region is subdivided by marker loci QQ-1,Qa-2, and TLa.

We have been investigating one of these associations after (Meruelo

et al., 197%) demonstrating that resistance to RadLV-induced neoplasia is associated with gene(s) in the D-region of the H-2 complex. Pertinent to the question of organotropism (as explained below) and relevant to the mechanism of action of H-2D genes in conferring resistance to RadLV is the finding that dramatic changes in the quantitative expression of cell surface H-2 antigens (particularly H-2D) occur following intrathymic RadLV inoculation (Meruelo et d., 1978). Studies measuring incorporation of [35Slmethionine strongly suggest that changes in expression of H-2D molecules reflect increased synthesis of this determinant after virus infection rather than simple uncovering of additional “buried” H-2D molecules (Meruelo et al., 1978). Several hypotheses to account for the observed induction of H-2D antigen(s) expression by RadLV have been suggested and tested (Meruelo, 1980). Our findings support the notion that increased H-2 expression results from RadLV integration at or near H-2D or genes that affect H-2D transcription. Thus our studies have shown (1)that ultraviolet inactivation (5020 ergs/mm2) or X-irradiation (400,000 rads) is sufficient to prevent RadLV-induced increases in synthesis and expression of H-2D antigens (Meruelo and Kramer, 1981). It has been shown (Decleve et aZ., 1977%)that such treatments are sufficient to lower infectivity drastically without affecting any of the viral proteins or viral penetration of target cells. Therefore, the effect of these treatments must be on the viral RNA so that it remains untranscribed or

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defective DNA copies result. (2) Intrathymic inoculation of RadLV in resistant mice leads to increased H-2D expression as long as these mice are of the Fu-lbb genotype. Mice of the Fu-1"" genotype which restricts RadLV replication fail to show increased H-2D antigen expression. The Fu-1 locus has been shown to restrict viral replication by blocking integration (Jolicoeur and Baltimore, 1976). (3)Inoculation of hybrid mice (resistant by susceptible F1) with RadLV leads to increased cellular expression of resistant but not of susceptible haplotype antigens. Thus the inductive mechanism augmenting H-2D synthesis does not operate in a trans mode (Meruelo and Kramer, 1981). (4) Agents that prevent integration of closed circular double-stranded DNA into the host genome, such as ethidium bromide (Guntaka et al., 1975) and fluorodeoxyuridine (FUdR) (Sveda et al., 1976), inhibit RadLV-induced increases in H-2D antigen expression (Meruelo and Kramer, 1981). Thymidine, which blocks the effect of FUdR on virus integration (Sveda et al., 1976), also blocks the effects of FUdR on RadLV-induction of H-2D antigen expression (Meruelo and Kramer, 1981). The findings described above encompass several concepts related to organotropism. First, RadLV integration after infection may occur randomly, but the interaction with or integration at one site must occur consistently, since RadLV infection always results in deregulation of H-2D synthesis in resistant mice. Second, such an interaction must occur early on and remain stable for a long period, since deregulation of H-2D synthesis is routinely observed by 36 hr after virus infection and persists for at least 12 weeks (Meruelo et al., 1978). Third, insertion at such a site must be kinetically important since H-2D synthesis and expression is enhanced. Furthermore, the integration site must be different between susceptible and resistant mice, since enhanced synthesis of H-2, which we currently attribute to increased transcription, occurs in resistant but not susceptible mice. Since resistant and susceptible are congenic pairs differing only at H-2D, one can go further and speculate that the integration of RadLV must occur at H-2D, or that H-2D has an effect on distant integration sites. The former possibility, which is easier to conceive (mechanistically), implies that H-2D DNA sequences determine the viral integration site(s). Recognition of some H-2D sequences will lead to integration and promotion of transcription, whereas other H-2D sequences will not lead to recognition and integration. Not yet discussed is the fact that overt leukemia, which occurs approximately 100% in susceptible mice and to a much lesser degree in resistant mice (Meruelo et al., 1977b), decreases H-2 synthesis to un-

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DANIEL MERUELO AND RICHARD BACH

detectable levels (Meruelo et al., 1978). Our preliminary results suggest that H - 2 methylation (Meruelo et al., unpublished) and rearrangements (Meruelo et al., 1983c) induced by RadLV transformation are responsible for the shutdown of H-2K and H-2D synthesis. While the results do not yet directly demonstrate the specific integration of RadLV related information directly at H-2, they suggest this very strongly. More recently examining a cosmid library of H - 2 genes we have found viral sequences proximal to H - 2 genes (Meruelo et al., unpublished). Therefore, the total evidence clearly supports the theory that specific viral integration sites are located at histocompatibility loci and loci encoding lymphocyte differentiation antigens. We shall provide additional data in the next section.

3. Differentiation-SpeciJc Loci and Genes Related to Leukemia Viruses and Leukemogenesis If the above findings are relevant to organotropism, one would expect to find additional examples of similar interactions. Figure 7 provides information suggesting that viral integration may occur often at or near loci coding for differentiation-specific determinants. “Productive” viral loci are those recognized to be of importance in some aspect of leukemogenesis or viral infection or induction. For example, the locus coding for xenotropic murine leukemia virus inducibility ( B m - I ) has been mapped to the same location on chromosome 1 (Kozak and Rowe, 1980a) as the major lymphocyte activating determinant 1

2

7

4

a

9

17

0

H-22 H-24

-H-2 -Tla

- H-31

-~-32

H-15

Ly-15

F s - -H-7 H-29

Fu-1 H-20

ECO/BC

FIG.7 . Correlation between virus-related mendelian loci and chromosomal sites rich in differentiation antigens.

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locus (Mls) (Festenstein et al., 1977). The recombination frequency between Pep-3 and Mls is 0.18 0.04 (Festenstein et al., 1977), and that between Pep-3 and Bxu-l is 0.20 k 0.05 (Kozak and Rowe, 1980a). Both loci are on the noncentromeric side of the Pep-3 locus. A locus on chromosome 4 affecting expression of xenotropic virus structural components, XenCSA (Morse et al., 1979),is flanked by loci coding for minor histocompatibility antigens H-15, H-16, H-20, and H-21, and by lymphocyte determinant(s) Ly-22.2 (Meruelo et al., 1983a).Aku-l, a locus encoding ecotropic viral genetic information on chromosome 7, is flanked by loci coding for minor histocompatibility antigens H-22 and H-24 (Rowe and Hartley, 1972). Fu-2, a locus that confers total resistance to the erythroleukemic disease induced by Friend virus (FV) infection, is situated adjacent to the locus for minor histocompatibility -7, H-7, on chromosome 9 (Lilly and Pincus, 1973; Axelrad, 1966). TL, a locus coding for antigenic determinants on thymocytes and leukemia cells, has been mapped less than 2 recombination units to the left of H-2D on chromosome 17 (Boyse et al., 1964, 1966). Ever since thymus leukemia antigens (TL) were first described, suggestions have been made about their possible origin from a viral genome integrated in chromosome 17. This was generally supported by the fact that TL- to TL+ phenotypic conversion was always diagnostic of malignancy (Stockert et al., 1971). Recently, this notion has come into question (Old and Stockert, 1977) principally because the molecular weight of the antigens on SDS gels is 45,000rather than the molecular weight expected for any known viral glycoproteins. However, Elder et aE. (1978) have shown that the enu product of MuLV, although 70,000 in molecular weight, is derived by glycosylation from a 45,000-molecular weight protein. Further support for the notion that TL may be somehow related to MuLV has been provided by Gazdar et al. (1977) and Ruddle et al. (1978). Their work on somatic cell hybrids indicates that all hybrid clones capable of replicating ecotropic MuLV retain mouse chromosomes 5, 15, and 17. These chromosomes may contain genes important in virus replication or cell-virus interaction. A locus involved in susceptibility to murine irradiation leukemogenesis, El-I, is adjacent to minor histocompatibility locus H-30 on chromosome 2. This region of chromosome contains in addition loci coding for minor histocompatibility antigens H-3, H-13, and H-30, and differentiation antigens Ly-6, Ly-8, Ly-11, Lym-11, H9/25, ThB, DAG, and ALA-1 (Meruelo et al., 1981, 1982). In addition another locus affecting susceptibility to RadLV induced leukemia is found adjacent to H-3 (Meruelo et al., 198313).Furthermore, in parallel with observa-

*

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tions that histocompatibility and differentiation loci are closely associated with genes affecting irradiation and RadLV leukemogenesis, Haughton and collaborators have shown (G. Haughton, personal communication) that H-4-H-2 interactions are critical for the development of B cell lymphomas. Among unmapped loci, Ac-1, a locus controlling susceptibility to Abelson virus-induced lymphogenesis in mice, is closely linked to H 36 (Risser et al., 1978), a minor histocompatibility locus mapping on chromosome 2 (Meruelo et al., 1982). These observations receive further support from our recent observations. Several polymorphic DNA restriction fragments by bridging with xenotropic and ecotropic envelope viral probes map adjacent to minor histocompatibility and lymphocyte antigen coding loci. Viral restriction fragments are associated with Ly-17on chromosome 1, H-30, H-3, and H - 1 3 on chromosome 2, H-16 on chromosome 4, Ly-21 on chromosome 7, H - 2 8 on chromosome 3, and H - 3 8 (chromosome location undetermined) (Meruelo et d.,1983d). It is, therefore, worthwhile to explore the concept of organotropism further. 4. The Integration Sites of Endogenous und Erogenous Moloney

Murine Leukemia Viruses Perhaps the strongest data supportive of the mechanism(s)proposed to account for organotropism come from the studies of Jaenisch and collaborators (1981).The integration site of a virus can be characterized by digestion of cellular DNA with a restriction enzyme that does not cleave within the viral genome itself. When coupled with separation of digested DNA fragments on agarose gels and hybridization with specific viral probes (“blotting” technique; Southern, 1975), such studies can serve as a sensitive method to compare integration sites of viruses and to investigate the specificity of integration as first exemplified by characterization of the integration site of DNA tumor viruses by Botchan et ol. (1976) and Ketner and Kelly (1976). The integration site of a number of type C viruses has now been studied using these techniques. Such studies have generally established the principle of random integration of viruses in host DNA. While Battula and Temin (1977) concluded that reticuloendothelius virus (REV) integrates at a unique site upon infection of chicken cells by infectivity studies, molecular hybridization has shown that multiple integration sites exist for REV (Battula and Temin, 1978; Keshet and Temin, 1978). The discrepancy between infectivity and hybridization experiments can probably be explained by postulating the existence of integrated subgenomic fragments which are not infectious

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but are detectable by hybridization. Similar studies by Steffen and Weinberg (1978) using rat cells infected with Moloney-MuLV and by Hughes et al. (1978) with chicken cells infected with avian sarcoma virus (ASV)have shown that viral genomes (or genome fragments) are found integrated at multiple sites. At the genetic level, the basis for the difference in expression of these genes is poorly understood. This is partially due to the presence of multiple copies of viral sequences in almost all mouse strains. Progress in this area, however, has recently been made by Jaenisch and coworkers (1981). These workers have introduced the well-defined exogenous Moloney leukemia virus (M-MuLV) into the germ line of mice, and prepared probes for M-MuLV which do not cross-hybridize with endogenous viruses (specific cDNA, Berns and Jaenisch, 1976; Jaenisch, 1977). After introducing the M-MuLV into the germ line of mice and preparing appropriate probes, Jaenisch et aZ. developed several new substrains of mice, BALB/Mo, which carry the exogenous MMuLV as an endogenous gene (Jaenisch et al., 1981). These sublines differ only in the fact that the M-MuLV genome is associated with different chromosomal sites as determined by restriction analysis. Thus, when the M-MuLV-specific cDNA probe was used, no labeled band was detected in EcoRI digested DNA isolated from uninfected mice (Jaenisch et al., 1981). In contrast, in DNA extracted from the nontarget organs of BALB/Mo mice, a single DNA fragment of 16 x 106 daltons was detected (van der Putten et al., 1979; Jahner et al., 1980). One of the substrains generated (BALB/Mo) carries M-MuLV specific sequences at a single Mendelian locus, designated Mou-1, and located on chromosome 6 (Jaenisch et al., 1978; Breindl et al., 1979). The presence of this dominant gene is associated with early viremia and high incidence of leukemia. Other BALB/c substrains of mice carrying M-MuLV at other distinct genetic loci were derived experimentally by similar germ line integration procedures (Jahner and Jaenisch, 1980). These three new substrains were designated as Mou-2, Moo-3 and Mov-4 (Jahner and Jaenisch, 1980). Each of these new substrains carry a single M-MuLV genome (integrated on a different chromosome (Jahner and Jaenisch, 1980). Mice carrying the Moo3 gene develop early viremia and die rapidly of leukemia, whereas animals transmitting the Mow2 gene express virus only occasionally, and then late in life (Jahner and Jaenisch, 1980). The M-MuLV proviruses integrated at the M o w 2 and M o o 3 loci are identical as judged by restriction enzyme analysis. In addition, the viruses activated from Mou-2, Mou-3, and BALB/Mo ( M o u - I )

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are identical by biological and biochemical criteria. The differences in expression thus appear to be determined by the chromosomal location of the virus, and cannot be explained b y arguments that differences in expression are due to defective virus genomes being associated with some chromosomes and not others. (However, the M-MuLV genome in Mou-4 mice was shown to have a partial deletion and no virus expression was observed in these animals.) Expression of the same endogenous virus in specific tissues and not in others, as observed in several systems (Cooper and Silverman, 1978; O’Rear et al., 1980), might be explained by the existence of cisacting cellular control elements which activate adjacent cellular genes during differentiation in specific tissues. This notion is supported by preliminary experiments (Jahner and Jaenisch, 1980; Jaenisch et al., 1981) in the M-MuLV system described above. Thus, transfection experiments performed with liver DNA from Mou-1,Mou-2 and Mou-3 mice indicated that XC plaques are induced only with Mou-3 DNA. This observation is compatible with a cis-acting control element determining the expression of the adjacent provirus, since the regulation of virus expression observed in uivo is not disrupted, despite the fact that DNA is sheared into several fragments to enhance transfection.

IV. Expression in Inbred Mouse Strains of Antigens Associated with MuLV

A. GIx AND GCSA The first definition of an antigen associated with MuLV and expressed at least under certain circumstances in normal cells became possible with the recognition that rat antisera to syngeneic MuLVinduced leukemias detected a broader spectrum of MuLV antigens than had been seen with mouse antisera (Geering et al., 1966). The probable explanation for this observation is that rats, which appear to lack endogenous MuLV, are not only highly susceptible to leukemia induction by MuLV, but also able to make a strong humoral response against these antigens because they are not part of the “self” repertoire. However, precisely for this reason, MuLV induced leukemias in rats are strongly immunogenic and readily rejected on transplantation unless they are passaged in immunologically immature recipients. Alternatively, the strong immunological response can be overcome by transplantation of sufficiently large numbers of leukemia cells in adult animals. This results in progressively growing tumors in hosts whose

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sera contain high levels of cytotoxic neutralizing and precipitating MuLV antibodies. The GIx antigen system was first identified using one such rat serum (Stockert et al., 1971). More recently, the development of methods to concentrate and purify MuLV has permitted preparation of heteroimmune sera in rabbits and goats to intact virus and to isolated structural components. Another early reagent, a mouse anti-murine Gross MuLV-induced tumor cell line, led to detection of the Gross cell surface antigen (GCSA) (Slettenmark-Wahren and Klein, 1962). A cell line, designated ESG2, was one of many obtained from a large series of C57BL mice injected as newborns with Gross virus (Old and Stockert, 1977). Immunization against antigens reacting with E S G2 cells was done indirectly. C57BW6 mice were injected with a transplantable AKR spontaneous leukemia, K36. The C57BL antiserum showing highest titer against ESG2 was absorbed with a variety of Gross MuLV induced and spontaneous leukemias arising in mice of high leukemia incidence and shown to detect a common antigenic determinant shared by ESG2, K36 cells and all leukemias induced by MuLVGross and spontaneous leukemias (Old and Stockert, 1977). Furthermore, the reactivity of this serum against tissues of normal young mice from different inbred strains indicated a high correlation between occurrence of antigen in spleen and other lymphoid tissues and incidence of spontaneous leukemia. Thus, high incidence strains, AKR, C58, PL, and CSH/Figge, were antigen positive, whereas low incidence strains, e.g., C57BL, A, and BALB/c, lacked the crossreacting antigen. Because of this relationship of the antigen to the leukemia incidence associated with Gross virus among many inbred mouse strains, the antigen was named G (Gross) cell surface antigen (GCSA) (Old et al., 1965). It soon became clear, however, that GCSA was found not only in lymphoid tissues of high leukemia incidence strains, but was also found in normal and malignant tissues of low incidence strains. Therefore, it became apparent that expression of GCSA among low incidence strains indicated widespread infection of mouse populations with MuLV. Further evidence favoring the link of GCSA with MuLV was provided by electron microscopy analysis which revealed an excellent correlation between GCSA expression and occurrence of MuLV particles in both normal and tumor tissue (Old and Stockert, 1977). Although initially it appeared that G I and ~ GCSA were identical antigenic determinants, further study has shown that this is not so. For example, anti-GIx antibody [(W/Fu X BN)FI rats anti-W/Fu leukemia cells] is cytotoxic not only for normal thymocytes from the high leuke-

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mia incidence, GCSA' strains but also for thymocytes from some GCSA- strains (Old and Stockert, 1977). The original designation G(129) (Old and Stockert, 1977) was changed to GIxwhen it appeared that one of the two genes responsible for antigen expression resided in linkage group IX of the mouse (chromosome 17). However, this linkage has been questioned (Old and Stockert, 1977). Although the studies described above clearly suggested that GIX and GCSA were antigens coded for MuLVs proper it was only recently that this has been shown directly. Advances in the biochemical analysis of MuLV and new methods to define cell surface molecules has permitted demonstration that both GIx and GCSA are in fact viral structural components incorporated into the cell surface. GIX is a typespecific antigen of the major envelope glycoprotein of MuLV, gp70 (Obata et al., 1975; Tung et al., 1975). Even more recently, comparisons of GIx+ viral gp70 and GIX- gp70 (Rosner et al., 1980) and of RNase T1 oligonucleotides from the genomes of these viruses (DonisKeller et al., 1980) have suggested that GN phenotype relates to the glycosylation of gp70. These studies reveal that Glx- gp70 has one additional glycosylation site compared to GIx+ gp70; the GIX-phenotype may result from masking of the GIX antigen by the extra oligosaccharide chain. GCSA is related to the internal core proteins of MuLV, p30 and p15, which occur as glycosylated polyproteins on the surface of infected cells (Tung et al., 1976a; Snyder et al., 1977). B. X.l The X . l system was first defined by the rejection of certain X-rayinduced BALB/c leukemias (Sato et al., 1973) in BALB/c hybrids. While resistance to these X-ray-induced lymphomas was not demonstrable in BALB/c mice, it was easily seen in (BALB/c x C57BL/6)Fl hybrids. It was subsequently shown that this hybrid resistance to leukemia transplants was under control of an H-2 linked Zr gene (see Section V,F,2,a) derived from the C57BL/6 parent. Furthermore, humoral immunity (responsible for resistance) could be shown to recognize an antigen, designated X.1, in the inoculated BALB/c leukemia RL6 1cells. This antigen is present on BALB/c, A, and AKR leukemias and is unrelated to GCSA or GIX(Tung et al., 1976b). X.l is present in normal tissues of high leukemia strains, but unlike GCSA and GIx is present in very low levels. While the presence of X.l in the normal tissues of high leukemia incidence strains and its induction in leukemias of X.l- strains suggests an association with MuLV, more direct

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evidence is still needed to show that X . l is an MuLV-related antigen (i.e., induction in MuLV-infected cells).

c*G(RADA1)7 G(ERLD)>and G(AKSL.2) Since these early studies, several additional MuLV-related cell surface antigens systems have been detected with the aid of murine normal sera. It appears that sera from normal mice, particularly F1 hybrids and random-bred Swiss mice, provide a rich source of antibodies reactive with MuLV-related cell surface antigens. Three re(Old and cent examples are the discovery of G ( R A D A ~ ) and G(ERLD) Stockert, 1977), and G(AKSLP) (Stockert et al., 1979). The notation used indicates their relation to MuLV-Gross with the subscript designating the prototype leukemia cell lines used in their definition. Randombred Swiss mice are the source of G(RADAI) antibody, and (C57BL x 129) FI mice are the source of G(ERLD) antibody. Both G(mDA1) and G(ERLD)are found in normal and leukemic lymphoid tissues of strains with a high incidence of leukemia, and both can be induced in fibroblasts by infection with N-tropic MuLV (Old and Stockert, 1977). Sera from a normal AKR-Fv-lb and (C3H X AKR)F1 mice was used to define the G(NSL.2)antigen. This determinant is also found in lymphoid tissue from high leukemia-incidence strains, but appears to be related specifically to dualtropic virus; it could not be induced by most ecotropic or xenotropic MuLV, but was expressed by fibroblasts infected with several different dualtropic MuLV (Stockert et al., 1979). The strain distribution patterns of these antigens clearly distinguishes them from one another as well as from GIX,GCSA, and X.l. The relationship of G(ERLD) and G ( R A D A ~ to ) MuLV structural components has been investigated, and it is suggested that they are associated with envelope glycoproteins (Old and Stockert, 1977).

D. PC.l Antisera prepared against the BALB/c myeloma MOPC-70A in H-2 compatible DBA/2 were shown to react with BALB/c myeloma cells but not with normal thymocytes or thymic leukemias (Takahashi et al., 1970). However, despite its lack of reactivity with T normal or malignant cells, the serum could be shown by absorption to react with normal BALB/c cells. The distinctive tissue distribution of the antigen recognized when compared with other known surface antigens indicated that a novel antigen was detected. Since normal and malignant

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plasma cells (myelomas) expressed this antigen, it was called PC.l. After the PC.l system was defined, it was observed that the serum of BALBlc mice, the prototype PC.l+ strain, had cytotoxic antibody to BALB/c myelomas and that this BALB/c antibody appeared to be detecting an antigen with a strain and tissue distribution that was identical with PC.l (Herberman and Aoki, 1972). Similar antibodies were subsequently found in the sera of a number of other mouse strains, both PC.1+ and PC.l-. The widespread distribution of naturally occurring antibody to PC.1+ myelomas and the presence of MuLV in high frequency in myelomas suggested to some investigators that PC.l was coded for by MuLV. However, a PC. 1-inducing virus might also be expected to occur in myelomas arising in PC. 1- strains. To date, only one such instance has been reported (Herberman and Aoki, 1972), but the issue remains controversial and some authors believe that PC.l is the product of a conventional Mendelian gene (Old and Stocked, 1977). A new surface antigen (PC.2) expressed exclusively on plasma cells, and distinct from PC.l, has been recently defined (Tada et al., 1980). Anti-PC.2 antibodies are not directed at MuLV associated antigens.

E. TL The TL system of cell surface antigens was recognized during the course of a study of radiation-induced leukemias of C57BL mice (Old et al., 1963). Among several C57BL antisera, one, prepared against ASL1, a transplantable A strain spontaneous leukemia, had a high titer against a transplantable, radiation-induced leukemia, ERLD. This serum was used to define the TL system of antigens. While the antiserum appeared to be specific for leukemia cells in C57BL/6 mice, it reacts with normal as well as leukemic cells derived from A strain animals. Among normal cells, however, only thymocytes expressed the antigen. In normal mice, T L is inherited as a Mendelian dominant trait. Linkage studies have mapped the TL locus, designated TZa,on chromosome 17, approximately 2 units to the left of the D end of the H-2 complex (Boyse et al., 1964). The anomalous appearance of TL+ leukemias in mice of TL- strains is thought to indicate that all mice possess the structural gene for TL but that in TL- strains, the gene is not normally expressed (Boyse and Old, 1969; Old and Boyse, 1973). During leukemogenesis, however, the TZa locus must be derepressed or activated to account for the expression of TL antigens on the surface of leukemia cells (Stocked et al., 1971). The latter suggestion led to

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the postulate that the Tla locus might encode an integrated viral genome (Stockert et al., 1971). Although this has not been formally excluded, current understanding of the TL system makes it most unlikely (Old and Stockert, 1977).

F. ML Antisera to DBN2 leukemias prepared in histocompatible mice and absorbed in vivo to remove alloantibody led to definition of the ML system (Stuck et al., 1964). The absorbed antisera still retained cytotoxic activity for the immunizing leukemia and other DBN2 leukemias but not to spontaneous or induced leukemias of any other mouse strains. The identity of the antigen recognized was first suggested by the finding that the sera reacted with normal mammary tissue and spontaneous mammary tumors of mice infected with mammary tumor virus (MTV). This restriction of antigen to leukemias of DBN2 mice and MTV-infected cells prompted the designation ML for mammaryleukemia. There is currently little doubt that the ML antigen is coded directly or indirectly by MTV (Old and Stockert, 1977). However, the reason for ML appearance in DBN2 leukemias is not clear. Molecular hybridization experiments have shown that genetic information related to MTV is present in all mouse strains (Varmus et al., 1973). However, expression is restricted to certain strains (MTV strains) and in these strains to certain tissues (as discussed previously in Sections III,G,3 and 11I,H7l.-4). Therefore, the detection of ML antigen in leukemia cells might indicate derepression of MTV genetic information in malignant lymphoid cells. Host regulatory factors would have to account for the fact that such derepression occurs only in DBN2 mice and, as recently reported, in GR mice (Hilgers et al., 1975). Alternatively, DBN2 and GR mice might express a unique leukemogenic virus that arose through genetic interaction with MTV. In fact, unique restriction fragments for MTV have been detected in GR mice (Michalides et al., 1981). Further biochemical definition of the ML antigen, examination of its relation to MTV structural components, and analysis of viruses obtained from ML+ leukemias are needed to definitively explain the above observations.

G . OTHERANTIGENS A diverse array of MuLV-related cell surface antigens have now been identified in mouse leukemia and there is every indication that the list will continue to grow. In this article we have listed only a few

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of these because we wish to make the reader aware of the existence of the class of antigens rather than attempt to provide a total compendium. The complexity and diversity of such antigens parallel that seen for MuLV envelope antigens (as alluded to in Section II1,G) (Aoki et al., 1974) and reflects the extensive MuLV polymorphism that has been previously discussed. The MuLV-related surface antigens do not appear to be transformation-specific, because they are also found on MuLV-infected but still untransformed cells. By contrast, avian and feline oncomaviruses express transformation-specific surface antigens that appear to be unrelated to viral structural proteins (Stephenson et al., 1977; Rohrschneider et al., 1975). Some investigators are actively pursuing detection and characterization of such antigens. V. Genetics of Susceptibility to Viral Infection

A considerable number of genes with the capacity to regulate or modify the replication and/or oncogenic process induced by C viruses have been identified. Although the detailed mechanisms by which they act are not fully understood for any of them, genes regulating neoplasia by retroviruses do so by acting at many different points in the development of the disease. We shall review the salient genes described to date. A. GENESAFFECTING VIRUSSPREAD To achieve substantial expression of type C viruses, several steps are required. First, one needs activation of chromosomally integrated viral genomes. Second, the infectious spread of virus from one cell to another within the population must occur. Exemplifying these steps is expression of virus in AKR mice. The ecotropic viral genomes represented by the Ako-l and Aku-2 loci are expressed as high levels of infectious virus in the tissues of AKR mice (Rowe et aZ., 1972). However, quantitative studies have revealed that only about 10%of spleen and thymus Iymphocytes initially produce virus (Mayer et at., 19781, followed by infectious spread of the virus pioducer trait to many other cells in the organ (Mayer et aZ., 1978). Similar findings have been obtained in ljitro (Rowe, 1972). Therefore, viral expression often occurs by exogenous infectious rather than by simultaneous activation of preexisting integrated viral genomes in many cells. Genes affecting induction and spread are, therefore, critical in susceptibility to leukemia. Induction genes generally represent integrated viral genomes and many of these have been defined including Akv-1, Akv-2 (Rowe et

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al., 1972; Kozak and Rowe, 1980a), Bxu-l (Kozak and Rowe, 1980a), Aku-3 (Rowe and Kozak, 1980),Aku-4 (Rowe and Kozak, 1980), Dbv (Jenkins et al., 1981), Blv (Jenkins et al., 1981), Fgu-l (Kozak and Rowe, 1982), Cu (Kozak and Rowe, 1979),Nxu-l and Nzu-2 (Datta and Schwartz, 1976,1977), Fgv-2 (Kozak and Rowe, 1982), C58u-2 (Kozak and Rowe, 1982), and Seu-l (Kozak and Rowe, 1982). Genes affecting virus spread are described below. B. ADSORPTIONAND PENETRATION Infection of cells by virus first requires successful adsorption and penetration. It has been shown by somatic hybridization that murine chromosome 5 contains a gene required for ecotropic virus infection (Gazdar et al., 1977; Oie et al., 1978) and coding for a cell surface receptor for ecotropic virus adsorption (Ruddle et al., 1978). Successful infection by pseudotype virus [consisting of a vesicular stomatitis virus (VSV) genome packaged into an ecotropic type C virus coat] of hamster-mouse hybrid cells is possible only in the presence of mouse chromosome 5, indicating an interaction between the host gene product(s) and the type C virus coat in adsorption. Likewise, host range restriction of xenotropic viruses by mouse cells appears to result from a virus penetration block, as even concentrated preparations of various xenotropic viruses show no replication in murine cells (Levy, 1978). Several experiments investigating blocks to xenotropic replication have shown an absorption/penetration block. For example, pseudotype particles with vesicular stomatitis virus cores and a xenotropic virus envelope were shown not to replicate (Besmer and Baltimore, 1977). The precise block has been more fully defined in experiments measuring absorption of xenotropic virus to nonpermissive mouse cells and permissive human and mink cells (Levy, 1978). In these experiments, residual titration experiments showed that absorption was similar in permissive and nonpermissive cells; therefore, the block operates at the level of virus penetration. Further experiments along these lines have shown that hybrids of mouse and human cells can be infected by xenotropic viruses only when somatic hybrid cells contain a complete complement of human chromosomes and very few mouse chromosomes (Gazdar et al., 1974; Scolnick and Parks, 1974), suggesting that an additional type of intracellular restriction to xenotropic virus replication may exist, regulated by the presence of murine DNA. However, the restriction in cells with murine genes has been explained by interference to viral penetration by viral genome glycoproteins on the surface of the so-

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matic cell hybrids derived from murine DNA (Besmer and Baltimore, 1977; Levy, 1978). Consistent with the blockage of penetration concept is the fact that embryo cells from some wild mice are susceptible to xenotropic virus infection. Susceptibility is dominant since hybrid mice of these wild strains with the usual inbred mouse strains are susceptible (Stephenson and Aaronson, 1977). Inbred mouse strains generally available must, therefore, lack the wild-type allele. In addition to restrictions by cellular membrane proteins or receptors, other factors can block adsorption of penetration by inactivating virus infectivity. For example, the sera of most normal mouse strains have been found to inactivate xenotropic type C virus infectivity (Aaronson and Stephenson, 1975), the NIH strain being a notable exception. This inactivating activity is not mediated by immunoglobulins (Levy et al., 1975; Fischinger et al., 1976), but rather by a serum lipoprotein (Levy, 1978). This lipoprotein is a high-density, triglyceride-rich lipoprotein, and can be converted from high-density to very low-density lipoprotein in uitro.

C. Fu-1: RESTRICTIONOF INTEGRATION Mouse chromosome 4 carries a gene closely linked to the Gpd-l locus that can interfere with infection of mouse cells by type C virus at a postpenetration step (Rowe and Sato, 1973). The mechanisms by which Fu-1 mediates restriction of virus spread have been alluded to earlier (see Sectioin 1117G,2). Restriction does not involve a membrane-receptor phenomenon and occurs after virus absorption and penetration, as “pseudotype” viruses with cytopathic vesicular stomatitis cores and N- and B-tropic type C virus envelopes showed similar cytopathic effects (Huang et al., 1973; Krontiris et al., 1973). Fu-1 resistance is relative and can be overcome at high input viral multiplicity of infection (Pincus et al., 1975; Declbve et aZ., 1975; O’Donnell et al., 1976). Once productively infected, Fu-1 -resistant and -sensitive cells produce virus in comparable amounts with similar latent periods (Pincus et al., 1975; O’Donnell et al., 1976); however, the length of the viral latent period varies with the multiplicity of infection, independent of Fu-1-mediated effects (O’Donnell et al., 1976).Abrogation can be affected using viruses inactivated by heat or X-irradiation (Bassin et al., 1978), suggesting that the virus need not replicate to overcome Fu-1 resistance. Early studies suggested that Fu-l restriction operates at a point prior to proviral integration (Jolicoeur and Baltimore, 1976).Jolicoeur and Rassart (1981) have shown recently that while synthesis of linear

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proviral DNA is unaffected by Fu-l, accumulation of closed circular DNA is impaired in resistant cells. They hypothesized that the Fu-l gene product may either block circularization of linear viral DNA directly or promote synthesis of a defective linear DNA which cannot circularize (Jolicoeur and Rassart, 1981).It is presumed that the supercoiled closed circular intermediate may be a critical species in the process of proviral integration. Tennant et al. (1974) have indicated that cytoplasmic fractions derived from Fu-l resistant cells transfer relative resistance to sensitive cells with two-hit dose-response relations (Tennant et al., 1974). Treatment with ribonuclease but not with deoxyribonuclease or proteases is able to abolish transfer of resistance (Tennant et d., 1976), and RNA preparations have been shown to transfer specific Fu-l resistance (Yang et al., 1978), implying that some RNA species are involved in Fu-I resistance. At least two Fu-l alleles have been described. Probably more than two exist since analysis of leukemogenesis in AKR X R F hybrid mice (both Fu-I"") has shown significantly lower leukemia incidence in hybrids of R F types Gpd-lamice than in those of the Am-type Gpd-lb (Mayer et al., 1978). Since Fu-l is linked to Gpd-I, and since both R F and AKR mice are supposedly Fu-I"", the Fu-l alleles of RF and AKR mice must differ. There is also accumulating evidence that the Fu-l" alleles in DBN2 and NIH mice are different. Cells of DBN2 mice show greater sensitivity to B-tropic viruses and somewhat reduced sensitivity to N-tropic viruses than other Fu-1" strains (Pincus et al., 1971a). In addition, DBN2 cells do not behave as NIH, C3H, and C57BL cells in sharing reduced efficiency in infectious center plating of certain B-tropic viruses (Pincus et al., 1975). Furthermore, B-tropic viruses which have been passaged on NIH cells so that they can grow in Fu-In or Fu-Ib cells with almost equal ease (i-e., with titers in NIH and BALB/c cells within 0.5 loglo of one another and single-hit titration patterns in both cells), do not grow efficiently in DBN2 cells, again suggesting a different allele at Fu-l for DBN2 versus NIH cells. Despite the almost universal restriction of virus replication in uiuo and in uitro associated with Fv-1, two cell lines have been identified which do not show evidence of Fu-I resistance. One of these is a subline of NIH 3T3 cells termed 3T3FL (Gisselbrecht et al., 1974), and a second group consists of a series of cloned cell lines derived from fetal mouse embryos, termed SC-1, SC-2, etc. (Hartley and Rowe, 1975).The SC-1 line known as IIIGA shows optimal sensitivity to both N- and B-tropic viruses in tissue culture assays, and has been used extensively for both isolation and titration of ecotropic viruses. SC-1

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cells have shown a stable phenotype of increased sensitivity to Btropic virus through greater than 100 passages (Hartley and Rowe, 1975). Its initial phenotype, however (the uncloned cell line), was found to be Fu-I", and many of the clones generated were insensitive to B-tropic virus (Hartley and Rowe, 1975). The interactions of N- and B-tropic viruses with Fu-ln and Fu-lb cells clearly constitute a reciprocal system, but N- and B-tropic viruses do not appear biologically equivalent for several reasons. First, all high-leukemia strains recognized, including AKR, C58, and C3H/Fg, are Fu-I", and show spontaneous infection with N-tropic viruses only. By contrast, B-tropic viruses are usually found in older mice and Fu-l mouse strains (e.g., BALB/c and C57BL/6) and are not generally associated with high frequencies of spontaneous leukemia. Second, Ntropic viruses are readily inducible by iododeoxyuridine from cells of most murine strains, as is xenotropic virus, whereas B-tropic virus is induced with difficulty in only a few strains (Stephenson and Aaronson, 1976). Third, while the phenotypes of N- and B-tropic viruses are in general quite stable, it is possible to broaden the host range of some viruses from B-tropic to NB-tropic after passage at high multiplicities of infection in NIH fibroblasts; however, broadening of host range for N-tropic viruses has never been found despite similar high multiplicity passage in BALB/c cells (Hopkins et al., 1976). Along with this extensively studied effect on exogenous infection of murine cells by MuLV, Fu-1 may also act at other levels in viral replication and expression. This has been suggested by work of Fenyo et al. (1980), who studied production of N-tropic virus in segregating populations of somatic cell hybrids. In these experiments, L cells (FuI") constituitively producing N-tropic "L virus" were fused with C57BL ( Fu-Ib) cells. The presence of chromosome 4 derived from C57BL (and, therefore, the restrictive Fu-Ib allele) in hybrids completely suppressed virus production, whereas loss of that chromosome during segregation resulted in reappearance of N-tropic virus production. These data would indicate that Fo-I restriction may have an affect in cells already carrying integrated proviral DNA. The in uitro data concerning Fu-1 function are reflected in studies of experimental leukemogenesis in mice. Thus, the susceptibility of mouse strains to Friend virus-induced disease (Lilly, 1967), RadLV and BE-L virus-induced leukemias, and Gross virus leukemia (Decleve and Kaplan, 1977) correlates largely with the permissive Fu-I allele. Studies of spontaneous leukemia in genetic crosses of AKR mice have also implicated the Fo-l gene as the major determinant of leukemia susceptibility (Nowinski et al., 1979).

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D. AVAILABILITY AND REPLICATION OF TARGET CELLS As stated earlier, infection and integration are not enough to ensure transformation. For example, MuLV infect fibroblasts in vitro, but such cells are not transformed either in vivo or in vitro. Transformation seems to require the availability of certain “target” cells whose growth regulatory program can be decontrolled by viral infection. Identification of these cells and the factors or genetic influence that mark them for transformation would significantly aid in understanding malignancy. Therefore, the subpopulations that serve as target cells for malignant transformation have been the subject of intense investigation. For example, radiation-induced leukemia appears to require an abundance of immature lymphoblastic cells that are present in the periphery of the thymic center during the first 2 weeks of life and decrease sharply in number thereafter (Kaplan, 1961; Axelrad and Van der Gag, 1962). After irradiation, the thymus has been shown to undergo injury characterized by a profound depletion of lymphoid cells followed by a period of regeneration, during which the same type of immature lymphoblastic cells are once again present for a period of several days (Kaplan and Brown, 1957). This short period of lymphoblastic cell regeneration may be critical for the restoration of thymus susceptibility to leukemogenesis in irradiated animals (Kaplan and Brown, 1957). A few relevant studies reflecting on target cell availability for leukemogenesis are described below in an effort to understand the role of these cells in the disease.

1. The Hairless Gkne During maintenance of the inbred mouse HRS strain, which carries the recessive hairless gene, hr (chromosome 14), it was observed that hairless homozygotes of this strain have a higher incidence (72%) of spontaneous lymphoma than their normal-haired littermates (20%) (Meier et al., 1969). Further study indicated that normal and hairless littermates had high titers of ecotropic type C virus in their tissues. On the other hand, hdhr mice have much higher titers of xenotropic virus activity in their thymuses shortly before developing lymphoma (Hiai et al., 1977). (The situation is analogous to the discussion of AKR leukemia in Section V,E,l.) Higher leukemia incidence in hrlhr mice may be related to an abnormality in differentiation associated with differentiation of the T lineage. At three months of age, hrlhr mice show increased Ly-1,2,3 and Ly-2 positive T cells, and decreased Ly-1+,2-,3- T lymphocytes when compared to their normal ( + / h r )littermates (Reske-Kunz et al.,

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1979). Whether this finding explains an immunological defect related to the inability of the hairless homozygote to suppress xenotropic virus expression and lymphomagenesis or provides a different ratio of target cells for xenotropic virus replication has not been investigated.

2. Fv-2 and Recruitment of Target Cells The recessive allele of Fv-2, FG-2‘ confers almost total resistance to the erythroleukemic disease associated with Friend virus infection. The mechanism of FG-2resistance is not yet understood, but it has been suggested that it might result from a reduced capacity of spleen virus (SFFV) to replicate in Fv-2’ cells (Blank et al., 197613).For example, it has been shown that Fu-2‘ impairs recruitment of neighboring target cells into infectious centers (Steeves et al., 1978). Similar restriction on SFFV replication is seen by other genes which reduce the number of erythroid target cells available for virus replication, such as loci involved in hereditary anemia, W (dominant spotting), S1 (steel), and f (flexed) (Meruelo and McDevitt, 1978). These loci probably have their effect because they alter the quality or quantity of target cells available to the virus. 3. T Lymphoma Retroviral Receptors and Control of T-Lymphoma Cell Proliferation

McGrath et al. (1980) have proposed a hypothesis for lymphoma induction based on the assumption of “fit” between a particular TMuLV and a particular subset of target cells in the thymus of susceptible hosts. Exact fit is expected to occur only on rare subsets of thymic cells whose receptors have specificity for the envelope glycoprotein of transforming MuLVs. According to their view, rare subsets of cells expressing receptors coded for by cellular genes are infected, transformed, and give rise to clonal progeny all bearing the same type of viral receptors. Furthermore, such receptors might represent antigenspecific receptors normally expressed by T lymphocytes. McGrath et al. (1980) have provided experimental data which they interpret to support this hypothesis. The evidence entails four principal observations. First, all cells in a particular thymic lymphoma bind T-MuLVs, whereas only 0.2-2% of normal thymocytes bind T-MuLVs (McCrath and Weissman, 1979). Second, binding appears to be fairly specific for the particular T-MuLV produced by the T lymphoma and is highly specific in competitive binding assays with even closely related T-MuLVs (McGrath et al., 1978a; McGrath and Weissman, 1979). Third, T-MuLV recognizes T cell receptors via their env glyco-

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protein products (McGrath et al., 1978b). Fourth, within the thymus of a T-MuLV infected host, only incipient T-lymphoma ,cells (not preleukemia cells) express cell-surface binding sites for leukemogenic TMuLV (McGrath and Weissman, 1979; McGrath et al., 1978~). These findings are of great interest in understanding the nature and availability of specific target cells for viral-induced leukemogenesis. Clearly depletion of target cells for a particular oncogenic virus should lead to enhanced resistance to the disease by the host. 4. Abelson Murine Leukemia Virus Target Cells in Mouse Bone Marrow Shinefeld et al. (1980)have reported the characterization of a monoclonal antibody which detects a surface antigen expressed by the bone marrow target cell of A-MuLV. Treatment of bone marrow cells with this antibody and complement results in a diminution of greater than 95% of the A-MuLV-derived in vitro transformed loci. The surface antigen detected by this antibody is also expressed on A-MuLV-transformed lymphoid cell lines, thymocytes, and some peripheral lymphocytes. This antigen is not expressed, however, by the pluripotent hematopoietic stem cells defined by the spleen colony-forming assay. The antigen detected is not a virally encoded product (Shinefeld et al., 1980).

5. Target Cell of Rauscher-MuLV Transformation Is a Pre-B Lymphoid Cell The phenotypic characteristics of Rauscher-MuLV-induced lymphoma cells appear to be those of immature B (pre-B) cells, which have been reported to synthesize small amounts of intracellular IgM, but to lack detectable surface immunoglobulin (Cooper et al., 1976; Owen et al., 1976, 1978; Raff et al., 1976). Recent evidence indicates that such cells express intracellular heavy chains in the absence of light chains (Burrows et al., 1979; Siden et al., 1979). Among cell extracts of different Rauscher-MuLV lymphoma lines tested in the competition immunoassay for mouse p chain, the level of p chain ranged from 30 to 700 nglmg of soluble cell protein (Premkumar et al., 1980). In contrast, neither spontaneous nor Moloney-MuLV-induced tumor cells contained detectable p chain. Neither kappa nor lambda chain was detectable (less than 1 ng/mg cell protein) in any of the Rauscher-induced lymphoma cell lines analyzed (Premkumar et al., 1980). These results suggest that the target cell for transformation by Rauscher-MuLV is a pre-B lymphoid cell.

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6 . Target Cells for Virat-Induced Leukemogenesis Are InfEuenced by the Organ Microenvironment Datta and Schwartz (1978) have studied the expression of ecotropic and xenotropic viruses, as well as the incidence of leukemia in the F1 progeny of crosses between AKR and NZB mice. These (AKR x NZB) Fl hybrids (ANFJ inherit four autosomal dominant loci which control expression of ecotropic and xenotropic viruses. Aku-1 and Akv-2, which determine the expression of ecotropic virus (Rowe, 1972; Chattopadhyay et id.,1975),are inherited from the AKR parent; and Nzv-1" and N z G - ~ "which , determine the expression of xenotropic virus (Datta and Schwartz, 1976, 1977), from the NZB parent. Despite the presence of these four loci, the incidence of leukemia in the ANFl mouse is very low and markedly delayed (Holmes and Burnet, 1966; Datta and Schwartz, 1978). In addition, restriction of ecotropic and xenotropic virus expression in ANFl is limited to thymocytes and peripheral T-lymphocytes. Datta and Schwartz (1978) have postulated that the thymus-specific restriction of virus expression (reduction in available target cells) in the ANFl mouse accounts for its unexpected resistance to leukemia. These investigators have postulated that the mechanism of retrovirus expression restriction in T lymphocytes of the ANFI mouse resides in the radiation-resistant thymic stroma (thymic reticuloendothelial elements) or in radiation-sensitive prothymic or thymic lymphoid cells. Datta et al. (1980) have shown that there is a specific augmentation of the expression of MuLV antigens and of ecotropic and xenotropic viruses in ANFl thymic lymphocytes when they were allowed to differentiate in irradiated, leukemia-prone AKR mice. By contrast, these phenotypic changes did not occur when ANFl thymocytes differentiated in irradiated ANFl and C57BR hosts, which have very low incidence of spontaneous leukemia (Datta and Schwartz, 1978; Rowe, 1972; Chattopadhyay et al., 1975). At least three interpretations are possible for these results. The first, postulated by Datta et al. (1980), is that the thymic microenvironment exerts a major influence on the expression of retroviral genes by thymocytes. This is supported by their argument that the thymic reticuloendothelial environment is solely responsible for the differentiation and maturation of bone marrow precursor cells to thymocytes (target cells) in lethally irradiated, bone marrow-restored chimeras (Von Boehmer and Sprent, 1976; Zinkernagel et al., 1978; Fink and Bevan, 1978; Sprent, 1978). Two other interpretations of their results are possible, one consistent with the hypothesis of McGrath et al. (1980) and the other with

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the findings of Teich and Dexter (1978). Both hypotheses would conclude that infection of ANFl cells by AKR virus in the ANFl- AKR chimeras provides proliferative potential to specific cells within the population giving them neoplastic potential. Teich and Dexter (1978) have presented strong evidence favoring the notion that murine leukemia virus infection of bone marrow cells in vitro can alter the program of differentiation. In fact, specific viral gene functions can, according to these investigators (Teich and Dexter, 1978),be defined in terms of capacity to induce specific differentiation properties. Furthermore, it is said that these effects are under control mechanisms of virus infection and replication by the cell analogous to the in uivo situation. On the other hand, the hypotheses of McGrath and Weissman (1979) and Lee and Ihle (1979) would postulate that chronic blastogenesis of ANFl lymphoid cells results in response to AKR viruses in the new chimera environment and the eventual proliferation of a malignant clone. Zielinski et al. (1982) have argued vigorously against alteration of differentiation and/or selective proliferation by MuLV infection. They argue that, as contrasted to the ANFl + AKR situation, there is no change in MuLV antigen expression or production of xenotropic virus in AKD/2 + AKR or ANFl-+ C57BR and AKR + ANFl chimeras. That is because C57BR and ANFl are nonproducer or low virus expressing strains, and AKD/2 donor mice lack the NZB genes needed for xenotropic virus expression. Furthermore, AKR or AKDI2 cells that were allowed to differentiate in AKR hosts did not generate either polytropic or xenotropic virus, although they did produce ecotropic viruses (Zielinski et aZ., 1982). In those chimeras the donor cells possess and express ecotropic viral genes, and they are not known to restrict the replication and spread of polytropic recombinant viruses (Hartley et al., 1977); yet, augmented expression of xenotropic and ecotropic viruses and generation of polytropic viruses (at 2 months of age) occurred only in the ANFl+ AKR chimeras. However, in the latter respect, the results of Zielinski et al. (1982) are different from those of Kawashima et al. (1976a,b). The latter authors have observed the amplification of MuLV antigens by thymocytes of syngeneic AKR + AKR chimeras. However, the difference reported by the two groups is age related. Kawashima et al. (1976a,b) did not observe MuLV antigen amplification in 2-month-old recipients, but only in 6-month-old preleukemic recipients, whose thymuses expressed xenotropic virus in high titers, as well as polytropic (MCF) particles. Zielinski et al. (1982) found MuLV antigen amplification in ANFl+ AKR chimeras when AKR recipients were as young

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as 2 months. At this age, the AKR thymus expresses only ecotropic virus. Zielinski et al. (1982) propose that the amplifying “signals,” which are unlikely to be either ecotropic, xenotropic, or polytropic virus, increase with time so that by the age of 6 months, the phenomenon can be detected in AKR thymocytes as observed by Kawashima et al. (1976a,b). Zielinski et al. (1982) concluded from their experiments that the functional status of the thymic reticuloendothelium is of particular importance in determining the expression of integrated retroviral genes by thymocytes, and the ANFl thymus lacks all of these properties despite the presence of target cells susceptible to transformation. 7. Ly-11.2: A Cell Surface Antigen of Znterest for Target Cell Studies Related to Leukemogenesis The above described results in different leukemia models reveal two important concepts. First, specific target cells exist for oncogenic viruses (e.g., Abelson, Rauscher, etc.). Second, the proliferation of such target cells before and after viral infection is affected by the microenvironment in which such cells replicate (e.g., the thymus epithelium). In contrast to the high selectivity of each virus for a particular target cell subpopulation, one subpopulation of T cells, those bearing Ly11.2, seems to proliferate rapidly during the preleukemic period in response to most thymoma-inducing agents (Meruelo et al., 1980a). Thus C58/J and AKWCum mice develop spontaneous leukemia some time after the fifth month of age (Meruelo et aZ., 1980a). Four split doses of X-irradiation (175 rads weekly) of 4- to 6-week-old mice have been shown to cause leukemia with incidences ranging from 75 to 100% (Kaplan, 1967; Pazmiiio et al., 1978). Intrathymic inoculation of RadLV into 3- to 6-week-old mice has been shown to induce leukemia with an incidence and latency period that depend on the strain of mice used (Kaplan, 1967). Whether leukemia arises spontaneously (as in A W J and C58/J mice) or is induced by RadLV inoculation or Xirradiation, a marked elevation in the expression of Ly-11.2 in the bone marrow and thymus is observed as animals progress from the healthy normal state to the preleukemic stage (Meruelo et al., 1980a). As the animal progresses to the more severe phase of the disease, a sharp drop in Ly-11.2 expression is observed (Meruelo et al., 1980a). The observed changes in Ly-11.2 are associated with leukemogenesis because no such changes occur in normal mice as they age (Meruelo et al., 1980a).The observed changes in Ly-11.2 expression during the

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preleukemic stage is due to an increase in the percentage of Ly-11.2 bearing cells. For example, during the preleukemic stages, the number of Ly-11.2 positive cells in the thymus increases from less than 5% to greater than 45% (Meruelo et al., 1980a). Several possibilities may account for the observed changes in Ly11.2 expression during leukemogenesis. One is that Ly-11.2 represents a virus-coded or virus-associated determinant. A comparison of the strain distribution of Ly-11.2 with that of other virally coded antigens or related traits (Meruelo et al., 1980b) shows that Ly-11.2 has a unique strain distribution when compared with Abelson, FE, XenCSA, X.1, G(ERLD),GIx, TLa, G(AKSLO), and G ( u D A 1 ) . In addition, no correlation can be found between Ly-11.2, Fv-1, and PC.1 phenotypes. Furthermore, a variety of tumor cells have been tested for Ly-11.2 expression and found negative (Meruelo et al., 1980a). Of these cells, several are actively producing complete, infectious virus and express viral components (gp70, p30, p15, p12, p10) on their cell surface. These data would tend to argue against a viral origin for Ly-11.2. Another possibility that could account for the observed changes in Ly-11.2 expression would be that during leukemogenesis, a particular functional subclass of T lymphocytes proliferates more rapidly than the rest. For example, Lee and Ihle (1979) have shown that during leukemogenesis, AKR-derived lymphocytes show an increased capacity to make a blastogenic response to purified viral antigens such as gp71. The response starts out low and peaks during the preleukemic period much in the same manner as Ly-11.2. However, changes in Ly11.2 occur only if leukemogenesis is under way, whereas increased blastogenic capacity occurs in mice that will not develop the disease (Lee and Ihle, 1979). Ly-11.2 antigens are present on prothymocytes, but not on several other functional lymphocyte subpopulations, including helper, suppressor, or cytotoxic T cells. The only subpopulation of cells involved in immune surveillance that have been found to bear Ly-11.2 to date, and hence that may proliferate in an effort to defend the host from neoplastic cells, have been natural killer (NK) cells (Meruelo et al., 1980a). Interestingly, in contrast to the increased percentage of Ly11.2 cells found in mice following leukemogenic fractionated X-irradiation, NK activity decreases sharply (Meruelo et al., 1980a). Another possibility that would account for the observed changes in Ly-11.2 expression during leukemogenesis is that Ly-11.2 is present in transformation-sensitive lymphocytes (TSL). During the early stages when these lymphocytes are proliferating, Ly-11.2 expression

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increases, but as the host’s immune responses begin to operate, the proliferating lymphocytes escape by shutting down Ly-11.2 expression. We have found that Ly-11.2 cells are singularly susceptible to infection by RadLV. During the first week after intrathymic inoculation of this highly leukemogenic virus, greater than 90% of virusinfected cells are Ly-11.2 positive (Meruelo et al., 1980a). In contrast, very few (10-20%) RadLV-infected thymocytes display other cell surface T cell phenotypes (e.g., Ly-1, Ly-2, Ly-3, Thy-1, etc.) (Meruelo et al., 1980a). The significance of this observation is strengthened by the fact that for a transient period of time (first 20 days) and then again in the preleukemic stage, Ly-11.2 antigen expression increases sharply on thymic cells of mice genetically susceptible to RadLV, but not in mice resistant to the virus (Meruelo et al., 1980~). If we presume Ly-11.2 to represent target cells for leukemogenesis, we are faced with the dilemma of explaining the distinct target found by others (described previously) for various MuLVs. At the moment, we have no explanation that accounts for this paradox. However, the importance of Ly-11.2 bearing cells in leukemogenesis cannot be easily discounted. We have mapped a major locus, Ril-1, conferring resistance to radiation-induced and RadLV-induced leukemogenesis to a site on chromosome 2 not distant from the locus coding for Ly-11.2 (Fig. 8). Furthermore, this chromosome has now been shown to contain the Abelson c-onc (Goff et al., 1981; P. D’Eustachio, personal communication). (A tentative location for this cellular oncogene is shown in Fig. 8.) Thus, a variety of loci involved in distinct types of leukemias are shown to map to the same chromosome, and this chromosome contains the locus coding for Ly-11.2. It is, therefore, clear that for the moment the concept of target cells for ieukemogenic MuLVs has to be qualified not only in terms of the role of the microenvironment, but also with regard to the fact that a T cell subpopulation singularly susceptible to viral infection proliferates rapidly during the preleukemic period in response to distinct oncogenic agents. B2M

Rii-l

e-jJ

H-30

Ly-8 ,The

Lr 6 I

I

H:13 a 1

1

FIG.8. Map of the H-30-Agouti region of chromosome 2. Markers shown are primarily lymphocyte and erythrocyte differentiation antigens coding loci. Only selected unrelated loci have been included in the map.

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E . TRANSFORMATION 1. Recombinant Viruses as Etiological Agents: The Evidence in Favor The role of endogenous MuLV in leukemogenesis is as yet controversial. The observations to be dealt with are as follows. First, let us examine the AKR disease. High titers of endogenous N-ecotropic MuLV are found in a wide variety of tissues of AKR mice throughout life (Rowe and Pincus, 1972; Kawashima et al., 1976b; Nowinski and Doyle, 1977). Expression of ecotropic MuLV seems essential for leukomogenesis and virus expression is required early in life (Meier et al., 1973; Lilly et al., 1975; Huebner et al., 1976; Nowinski et al., 1976; Schwarz et al., 1979). Thus a high degree of correlation was seen between ecotropic virus titer at 6 weeks of age and incidence of spontaneous leukemia in (BALB/c X AKR) X AKR backcross mice (Lilly et al., 1975). Furthermore, an Fv-l restriction greatly reduced the final incidence of leukemia. Thus, AKR x Fu-lbhybrids expressed much less virus at 6 weeks of age and a greatly reduced incidence of leukemia when compared with AKR X Fv-In hybrids (Rowe and Hartley, 1972). Again, other studies have shown that mice can be protected against leukemia if antibodies to MuLV are administered in the early neonatal period (Huebner et al., 1976; Schwartz et al., 1979; Nobis and Jaenisch, 1980). In contrast with the above information, some studies have shown that ecotropic virus expression in neonatal life does not invariably lead to leukemia. In (AKR X C3H) and (AKR X RF) hybrids, 100% of mice show spontaneous ecotropic virus expression at 6 weeks of age, yet only 22% of these hybrids come down with the disease (DuranReynals et al., 1978).Thus, neonatal virus expression appears necessary, though hardly sufficient, for development of leukemia. However, recent evidence has begun to downplay the role of ecotropic virus as the etiological agent per se. At 5-6 months of age, preleukemic changes in the AKR thymus seem to manifest themselves as amplified expression of MuLV-related cell surface antigens. These changes correlate with appearance of xenotropic MuLV (Kawashima et al., 1976a,b; Nowinski and Doyle, 1977). Along with these changes there appears to arise a novel type of recombinant viruses, having both xenotropic and ecotropic host range. These recombinant viruses, known as mink cell focus-inducing (MCF) (Hartley et al., 1977), are thought to be representative of genetic envelope recombinants between N-ecotropic and xenotropic MuLV (Hartley et al.,

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1977; Elder et al., 1977; Chien et al., 1978; Devare et al., 1978; Rommelaere et al., 1978; Lung et al., 1980; O’Donnell et al., 1980). Some of these recombinant MCF MuLVs have been shown to accelerate leukemia development after injection of newborn or young AKR mice (Nowinski and Hays, 1978; Cloyd et al., 1980; O’Donnell et al., 1980), suggesting that age-dependent formation of dualtropic recombinant viruses in thymus can account for at least part of the disease’s latent period. This notion is strengthened by the fact that neither ecotropic nor xenotropic virus accelerates the disease. In this light, earlier experiments showing that only extracts of thymus of older AKR mice could accelerate leukemia development (Kaplan, 1967; Nishizuka and Nakakuki, 1968; Hays and Vredevoe, 1977) may now be understood. Comparative study of MCF isolates obtained by Hartley et al. (1977) from individual leukemia mice has shown that these viruses generally fall into two groups. Class I accelerates leukemia, and in general were isolated from lymphomas arising in the thymus of AKR or C58 mice. Class 11, on the other hand, do not accelerate leukemia and were isolated from leukemias arising primarily in spleen and lymph nodes. In addition, Class I1 MCF viruses generally do not replicate efficiently in the thymus. The genomes of some of the cloned MCF viruses belonging to the two classes have been analyzed by RNase T1 fingerprinting and T1 oligonucleotide mapping and by restriction analysis using cloned viral probes. These studies have shown regions of identity between MCF viruses and ecotropic and xenotropic viruses (Rommelaere et al., 1977, 1978). From the fingerprints obtained, each of the MCF genomes examined appeared unique although related. All of the MCF viruses are altered in the 3’ third of the genome relative to their putative ecotropic parent (AKV). In addition, many of the new, presumably xenotropic, sequences they have acquired are held in common between them. Restriction studies have confirmed this general feature. Class I viruses share some oligonucleotides with AKV, but differ in the 3‘ terminal oligonucleotides (Rommelaere et al., 1978). Instead they have new MCF-specific oligonucleotides in this region. By contrast, Class I1 viruses share terminal T1 oligonucleotides with AKV in the 3’ end of the genome. One MCF, whose origin is unclear, has biological properties intermediate between the two classes, that is, it shares some T1 oligonucleotides with MCF (Class I property), but it has the same 3‘ terminal oligonucleotides as AKV (Class I1 property). Restriction analysis has shown that the principal difference between Class I and I1 MCFs is that the former share an XbaI site with ecotro-

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pic viruses that the latter lack. This XbaI site of the pathogenic MCF viral DNAs is located at 7.7 kb, in the coding region for p15E. However, the results of Chattopadhyay et al. (1982) suggest that the gp70 (and p15E) of at least some MCF viruses might be derived entirely from nonecotropic sequences. Furthermore, the env sequences, in such cases, might be found entirely in high-molecular-weight DNA of normal cells. This observation has suggested that embryo DNA of AKR mice contains preexisting (prior to any recombination event) copies of intact MCF like env gene sequences with a proviral structure. Evidence that recombination within the p15(E) region of MuLV may be significant for leukemogenicity is also supplied by the studies of Pedersen et al. (1981), where isolates of MuLV from a spontaneous AKR tumor which accelerated leukemia in AKR mice were found to share common oligonucleotide changes in the p15(E) coding region of the viral RNA. Thus, the emphasis on a role for MuLVs in leukemogenesis has shifted focus from the ecotropic to the MCF-like or recombinant MuLV. Molecular and biochemical studies have addressed the issue that some MCF viruses do not appear to be pathogenic; however, we shall return shortly to further arguments against a role for these viruses in the disease. Additional evidence favoring a viral etiology for leukemia are studies showing that MuLV infection of target cells is required for transformation. Thus, viral DNA sequences are amplified stoichiometrically in AKR thymomas (Berns and Jaenisch, 1976) as well as in lymphomas of BALB/Mo mice (Jaenisch, 1979) and often appear first in an unintegrated form (Jahner et aE., 1980) characteristic of acute virus infections (recall that approximately 10% of AKR cells are initially found to replicate virus and de novo infection appears to be required for virus expression in 100% of cells). Restriction endonuclease studies have further shown that newly integrated viruses are only partially homologous to the ecotropic MuLV-specific cDNA probes used (consistent with involvement of recombinant viruses), and that the resulting leukemias are of clonal origin, selected in some unknown way from a pool of infected target cells (Canaani and Aaronson, 1979; Steffen et al., 1979; van der Putten et al., 1979). Studies on the role of viruses in AKR leukemia have, in summary, established the following facts. Viremia in AKR mice, which occurs by spontaneous activation of ecotropic MuLV, is usually a first step in leukemogenesis and may provide a source of parental virus for the yet unspecified events of genetic recombination which ultimately yields dualtropic MCF viruses. The preleukemic changes of antigen amplifi-

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cation probably results from infection of susceptible target cells in the thymus by recombinant viruses. Antigen amplification in such cells probably accompanies viral DNA amplification. Infection of thymocytes may represent only an initiating event and other somatic alterations such as trisomy 15 (Dofuku et at., 1975; Klein, 1979, 1981) may actually be involved and account for the latent period of disease development. The involvement of MCF viruses in virally induced leukemia in uico has also been well studied in the Friend virus disease system. Work of Troxler et al. (1978)and Ruscetti et al. (1981) indicates that the pathogenicity of an ecotropic, molecularly cloned F-MuLV depends on its ability to efficiently generate high levels of replicating MCF virus in target tissues. In fact, Ruscetti et al. (1981) further showed that resistance to F-MuLV disease in certain strains of mice correlated with endogenous expression of MCF-related envelope glycoproteins, suggesting a viral interference mechanism. A final observation relevant to this discussion pertains to the finding by Fischinger et al. (1981) that a virus-negative cell line, NIXT, derived from an X-irradiation-induced Swiss mouse thymoma, expressed a glycoprotein which resembled MCF envelope glycoprotein by peptide mapping and viral interference data. Hence, recombinant viruses or expression of recombinant type glycoproteins may be critical factors in a variety of pathways for induction of murine leukemia. 2. Recombinant Viruses as Etiological Agents: Some Problems There are two basic difficulties with the notion that recombinant viruses are the causative agents for neoplasia as described above. First, in some instances (see below) leukemia occurs without evidence of viral replication. Second, the degree of viral recombination is not well correlated with the degree of oncogenicity in all cases (see below). Fractionated irradiation can cause leukemia (Kaplan and Brown, 1952). It is sometimes possible to isolate from irradiation induced tumors a recombinant virus(es) at the time of leukemia appearance (Deckve et al., 1977a; Haas, 1978). However, in contrast to the AKR lymphomas, most C57BL thymic lymphomas do not express viral antigens as detected by immunofluorescence or radioimmunoassay competition. The second problem discussed above is that a perfect correlation has yet to be found between recombinant virus structure and oncogenicity. Thus a high degree of oncogenicity has been found in some isolates, i.e., MCF247 (Hartley et a[., 1977), HIX (Fischinger et al.,

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1978), and C57BL/G-derived RadLV (Decleve et al., 1977b; Haas, 1978). However, the degree of recombination is not well correlated with the degree of oncogenicity in recombinant viruses of C3H origin (Devare et al., 1978). 3. Other Transforming Genes It is important to remember at this point that a viral association with lymphoma development is not obligatory (e.g., radiation-induced lymphomas-see above). Thus, it may be speculated that the crucial genes and gene products involved in oncogenic transformation in vivo are yet to be recognized. In this light, it is proper now to consider models for transformation based on other RNA tumor viruses and information regarding "cellular transforming" genes (see below). A general characteristic of RNA tumor viruses with transforming capacity is that they encode virus genomic sequences recombined with nucleotide sequences originating from cellular DNA. The substituted sequences are often found in the env and the c regions of the genome, but can stretch out to encompass the major portion of the viral RNA. The analysis of transformation competent sarcoma viruses has shown that viral RNA encodes the transforming gene proper, which has been termed STC. The analysis of src function has been possible because of the availability of viral mutants conditionally transforming at various temperatures (ts) (Stehelin et aZ., 1977; Hanafusa, 1977; Vogt, 1977) and the isolation and identification of the in vitro translation product of the src region (Beemon and Hunter, 1977; Purchio et al., 1977; Kamine et al., 1978; Levinson et al., 1978; Sefton et al., 1978). It is quite clear that src sequences constitute a gene whose product is responsible for, and maintains cellular transformation (Hanafusa, 1977; Vogt, 1977). The following specific information has been obtained for the avian sarcoma virus (ASV).A viral phosphoprotein of 60,000 daltons designated as pp60"" is detected in ASV-transformed cells of many species by immunoprecipitation of labeled cell extracts with serum from rabbits bearing ASV-induced tumors (Brugge and Erikson, 1977; Levinson et al., 1978; Sefton et at., 1978). Pp60"" is a transformation-specific phosphoprotein and not a viral structural antigen. Pp60"" is probably the product of src since translation in vitro of the region known to code genetically for src specified function results in the synthesis of a polypeptide of the same size and having a similar peptide map. In addition, normal chicken embryo fibroblasts contain a gene, c-STC, which is homologous to the avian sarcoma virus transforming gene src (Spector et al., 1978). Both genes

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code for a 60,000-molecular weight protein which is phosphorylated at serine and tyrosine residues (Collett and Erikson, 1978; Levinson et ul., 1978) and has the unusual function of phosphorylating tyrosine residues. The Rous sarcoma virus STC may be conceived as the model for virally associated transformation genes. In RSV STC, a cellular gene is combined with a retrovirus skeleton to generate a transforming virus. Similar findings have been made with at least seven additional avian transforming viruses: Fujinami, PRCII, avian erythroblastosis, MC29, avian myeloblastosis, and reticuloendotheliosis transforming viruses. These seven avian transforming viruses all have, as far as is known, non-cross-reactive cellular genetic information as a crucial component of their structure (Fischinger, 1980). For mammalian viruses, non-cross-reactive genes have been found in murine sarcoma virus, Abelson murine leukemia virus, and feline sarcoma virus. Recently, it has been shown that in the Harvey and Kirsten strains of rat-derived sarcoma virus, there is a segment of normal rat cell genetic information as well as a long stretch of sequence of a virus-like structure endogenous to rat cells (Fischinger, 1980). While these findings would suggest that in general these cell-derived proteins are probably responsible for viral transformation, hard evidence for their transforming potential exists in only the Rous sarcoma virus (RSV) system as described above. Nonetheless, transformation-defective mutants of Abelson virus have been isolated and shown to lack protein kinase activity (although they can act as acceptors in a trans-kinase reaction) (Reynolds et ul., 1980). Furthermore, mapping studies with fragments of the viral genome have located the murine sarcoma virus transforming activity in the cellular insert and similar evidence for Kirsten sarcoma virus has been gathered. Thus it seems increasingly evident that the putative transforming proteins are in fact responsible for changing the growth properties of infected cells. We shall return below to the question of whether leukemia viruses such as the MCFs may transform by a similar joining of the retrovirus skeleton to cellular oncogenes. However, it is important to realize that this concept may further stretch to apply to what is until now regarded as “nonviral” induced transformation, i.e., chemical carcinogenesis. The demonstration that the transforming genes of retroviruses are derived from cellular genes, or protooncogenes (Temin, 1974; Hayman, 1981; Bishop, 1981), has considerable implications. If certain cellular genes are oncogenic when placed under the influence of viral

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control elements, perhaps other genes, or indeed the same ones, mediate chemical carcinogenesis by being expressed at high levels as the result of carcinogen-induced mutations. Recent work from Shilo and Weinberg (1981) and Cooper et al. (1980) has shown that genes involved in chemical carcinogenesis can be detected by DNAmediated gene transfer experiments. Shilo and Weinberg (1981) have been able to induce transformation by transfecting into nontransformed mouse fibroblasts DNA from lines of mouse fibroblasts transformed in vitro by the chemical carcinogen 3-methylcholanthrene (MC). DNA from 15 transformed lines were tested and successful transfer of the transformed phenotype was obtained with DNA from five of these lines. Restriction endonuclease digestion of MC-transformed cell DNA was used to assess the number of transforming genes. The principle of this analysis is that a gene will be inactivated in transfection if an enzyme cuts within it, whereas enzymes that cut outside the gene will have no effect. In four of the cases tested, EcoRI and Hind111 abolish transformation, whereas digestion with B a d , XhoI, and SaZI had no effect, suggesting that the same gene is involved in transformation in each of the four cell lines. On the other hand, the transforming genes of other cell types, a rat neuroblastoma and a line of cells transformed by transfection with normal mouse DNA, exhibited distinct patterns of restriction enzyme sensitivity. The lines showing the same pattern were all derived by in vitro transformation of mouse fibroblasts with MC. Nonetheless, the DNA of many MC-transformed cells were not active in the transfection assay, and it is quite possible that only a subset of MC-responsive sequences was examined. The general picture that arises from these transfection experiments may be applicable to the virally associated oncogenes. The findings suggest that genes which during normal expression are not deleterious, and may even be vital for normal growth and development, can be oncogenic when joined to regulatory element which increase their expression. In these transfection experiments, transformation is induced when the DNA is sonicated to a small size before transfection, probably because sonication separates the transforming gene from regulatory sequences that normally control its expression. In the primary transfection, which is very inefficient, transformation occurs if the gene integrates close to a strong promoter. In a secondary transfection, a high efficiency is obtained because the lesion is now cis-dominant (Cooper et al., 1980). Viruses may work by similarly capturing such genes and promoting their transcription. This model has received direct support from experiments of Blair et al. (1981). They

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have cloned a protooncogene, the murine cellular src gene from normal mouse cells and shown that it does not transform when transfected into cells. However, when the cellular src gene is joined to sequences from the 5’ end of murine leukemia virus which contain the strong viral promoter, the resulting recombinant DNA molecules transform efficiently.

4 . Mechanisms for Leukernogenesis Let us now return to the issue of how “leukemia” viruses may cause leukemia. In view of the above discussion, it is difficult to resist suggesting that one of several mechanisms is responsible for their neoplastic effect. Important genes might be hidden in the virus, possibly at the 3’ end. This possibility is still viable, but the recent availability of the nucleotide sequence of the Moloney leukemia virus has not yielded data supportive of this notion (Shinnick et al., 1981).A second possibility is that the promoter function inherent in the terminal repeat structure (see Section I1,B) might, following integration in appropriate regions of host DNA, enhance downstream (or upstream) transcription and thus activate to high production cellular genes (oncogenes) that are normally regulated to low levels. A model consistent with this notion has recently been proposed for the induction of bursa1 lymphomas in chickens by ALV, and the evidence which suggests this model is as follows: lymphomas induced by ALV usually contain proviruses integrated at one or more sites. Nonetheless, at least some proviral information in each tumor is found integrated at one of a limited number of common sites (Neel et al., 1981; Payne et al., 1981; Fung et al., 1981). As proviral integration is known to occur at a larger number of sites on the host chromosome, possibly at random (Hughes et d.,1978; Steffen and Weinberg, 1978; Ringold et d . , 1979; Quintrell et al., 1980; Shimotohno et al., 1980a,b),this apparent specificity of integration in lymphomas suggests that neoplastic transformation requires integration adjacent to specific genes. In addition, it would appear that integration leading to transformation does not occur frequently, since most primary and metastatic tumors are clonal (Neel et al., 1981; Payne et uZ., 1981; Fung et al., 1981; Neiman et al., 1980); that is, neoplastic cells isolated from various organs of the same neoplastic animal contain similar or identical patterns of proviral integration as reflected by similar restriction maps of the integrated viral information throughout all organs. Recent evidence suggests that these viruses cause neoplasia by activating a normal cellular gene(s). It has, for example, been found that many ALV-induced lymphomas do not contain viral 35 S and/or 21 S

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mRNAs (Neel et al., 1981; Payne et al., 1981) and that many of the integrated proviruses in these tumors are defective (Neel et al., 1981; Payne et al., 1981; Fung et aZ., 1981).Therefore, it is unlikely that the replicating virus is itself able to sustain the transformed state. Furthermore, in at Ieast some cases, such as ALV, the oncogenic potential seems to reside within the 3' portion of the viral genome-within approximately 500 nucleotides from the poly(A) tract (Robinson et al., 1980; Crittenden et al., 1980, Tsichlis and Coffin, 1980).This segment of the viral genome does not seem to code for a viral protein (Czernilovsky et al., 1980). Finally, ALV-induced lymphomas contain new tumor-specific RNAs consisting of viral 5' terminal sequences covalently linked to cellular sequences (Neel et al., 1981; Payne et al., 1981).These mRNAs fall into a limited number of size classes (Neel et al., 1981) and these tumor-specific mRNAs are expressed at moderately high levels [loo-300 copies per cell (Neel et aZ., 1981)l. Based on the above observations, several investigators (Neel et al., 1981; Hayward et al., 1981; Payne et al., 1981; Robinson et al., 1980; Tsichlis and Coffin, 1980; Quintrell et al., 1980) have suggested a model for oncogenesis termed oncogenesis by promoter insertion (Neel et al., 1981; Hayward et al., 1981). We described earlier (see Section II1,B) that the integrated provirus consists of the viral structural genes flanked by sequences of approximately 600-350 nucleotides termed long terminal repeats (LTRs) (Hsu et al., 1978; Shank et al., 1978; Sabran et al., 1979; Hughes et aZ., 1979). These LTRs contain a putative promoter sequence (Czernilovsky et al., 1980; Shimotohno et al., 1980a,b; Yamamoto et al., 1980). Initiation of viral RNA synthesis normally occurs within the left LTR. However, initiation could also occur within the right LTR because the promoter sequence is repeated at the right end. If the provirus integrated adjacent to a potentially oncogenic cellular gene, transcription initiated from the viral promoter could generate an RNA molecule such as those found in ALV-induced lymphomas, containing both viral and cellular sequences. The resultant enhanced expression of this cellular gene might lead to neoplastic transformation. The model has found support recently from the identification of such a mechanism in at least one leukemia system. Using cDNA probes specific for five o-onc genes of avian acute transforming viruses, Hayward et al. (1981) have managed to identify one such gene. These authors have shown that most of the ALV-induced lymphomas they studied resulted from activation of the c-myc gene, the cellular counterpart of the transforming gene of MC29 virus, by a promoter insertion mechanism. More recently, however, Payne et al. (1982) have shown that enhanced expression of c-myc can

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occur when flanking proviruses are found in any of three configurations: (1)on the 5‘ side (“upstream”) of the c-myc in the same transcriptional orientation, (2) on the 3‘ side (“downstream”) of c-myc in the same orientation, or ( 3 )upstream, in the transcriptional orientation opposite to that of c-myc. These authors have, therefore, postulated that activation of adjacent cellular genes by retroviral DNA can involve mechanisms other than provision of a transcriptional promoter. An hypothesis concerning murine leukemia which is consistent with this protooncogene activation schema has been proposed by Dec k v e et al. (1977b) as a mechanism by which fractionated irradiation induces leukemia. As stated previously, most radiation-induced C57BLJKa thymic lymphomas do not express viral antigens detectable by immunofluorescence or radioimmune competitive assay (Ihle et al., 1976a) despite the fact that RadLV, a virus with thymotropicleukemogenic properties (Kaplan, 1967), can usually be recovered from such tumors by passage of cell-free extracts in uiuo. Decleve et al. (197713) have suggested that this paradox can be resolved by postulating that RadLV is initially activated by X-irradiation in a replication-defective form (RadLV-0) in which only the oncogenic segment of the RadLV genome is expressed (this would go undetected by immunofluorescence or radioimmune competition assays which use antibodies directed at non-oncogene-derived antigens) and that RadLV acquires infectivity in uiuo secondarily, possibly by a recombination mechanism. Such an infectious recombinant virus would be expected to carry the oncogenic element encoded by RadLV-0. Kaplan and collaborators have shown that the leukemogenic recombinant virus (but not the nonleukemogenic viruses) encapsulates two RNA species of molecular weights 8 and 5.6 kb. Nonleukemogenic viruses lack the 5.6-kb RNA species. This 5.6 RNA codes for 100,000- and 36,000-molecular weight species which have certain similarities to the putative transforming “fusion” proteins of certain acute defective viruses (e.g., Abelson MuLV). That is, they contain viral gag-derived determinants linked to possibly cell-derived sequences, and may be important for oncogenicity (Manteuil-Brutlag et al., 1980). It is possible that these findings will apply to leukemogenic (as opposed to nonleukemogenic) viruses of the MCF or recombinant type, although presently no such similarities have been uncovered. We hope the above thoughts have served to illustrate our ignorance regarding the transformation phase of leukemogenesis while reviewing current thoughts, and raising awareness to the fact that genetic elements are required on the part of both host and virus for transfor-

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mation to occur. The latter point is of special importance to this article and is, therefore, worth rephrasing. Clearly in the absence of genes favorable to recombination (e.g., those coding for ecotropic and xenotropic virus sequences, or facilitating recombination), resistance to the disease may ensue. Furthermore, if cellular genes are involved, the presence of the “transforming alleles” for the potential locus is required for susceptibility to the disease, unless of course the virus carries in its genome the required information. Much work will still be required to elucidate the mechanism(s) of transformation and genetic resistance at this level.

F. IMMUNESURVEILLANCE AGAINST VIRAL INFECTION AND TRANSFORMATION 1. The Murine Major Histocompatibility Complex, H-2 Once transformation of normal cells has occurred, the only mechanisms known which can interfere with the maintenance of transformation (short, of course, of the hypothetical loss of the gene for transformation itself) are immunological. While the host immune response may be capable of eliminating the transformed cells, the capacity of the host to mount an effective response is markedly influenced by its genetic constitution at a variety of loci. Perhaps the most important of these loci are clustered together and known as the major histocompatibility complex, H-2. This complex mapping on the mouse chromosome 17 affects a considerable number of immunological phenomena (Meruelo and McDevitt, 1978). It is a complex of closely linked genes responsible for the rapid rejection of skin grafts, graft-to-host reactions, and other immunologic as well as nonimmunologic phenomena. The present knowledge of the genetic map of the H-2 region (Fig. 6) is based on analysis of an extensive series of congenic resistant strains and their recombinants produced by numerous workers (Amos et al., 1955; David and Shreffler, 1972; Gorer and Mikulska, 1959; Klein et al., 1970; Shreffler and David, 1975; Snell and Cherry, 1974; Stimpfling and Reichert, 1970). The gene products of the H-2K and H-2D regions are detected serologically and are found on most cell types including lymphocytes, although their concentration may vary in different cell types. The normal physiologic functions of histocompatibility genes in the K and D regions of the mouse are still poorly understood, although tests employing mice with recombinant H-2 chromosomal segments have shown that incompatibility at either the K or D end of the H-2 complex causes rapid rejection of tumor and skin

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grafts (Shreffler and David, 1975). Biochemical analysis has shown that the D region product(s) has an apparent molecular weight of 43,000, while that of the K region product is 47,000 (Nathenson and Cullen, 1974). The complete amino acid sequence and molecular structure of some K and D region gene products has been obtained (Coligan et al., 1978; Vitetta and Capra, 1978; Hood et al., 1983). H-2K and H-2D gene products are often obligatorily involved in the induction and effector phases of T cell “killer” function. For example, in several studies cytotoxic T cells specific for viruses and for minor transplantation antigens appeared to recognize not only the antigen to which they had been sensitized, but also the H-2K and/or D gene product on the immunizing cells. In addition, products of the major histocompatibility complex (MHC) play an important role in cellular interaction in the immune response (Benacerraf and Katz, 1975) as well as during embryogenesis (Snell, 1968). The Z region was recognized as an important segment of the MHC when genetic control of immune responses to several synthetic polypeptides and low doses of natural antigens were shown to map in the chromosomal region lying between the K and S regions of the H - 2 system (McDevitt et al., 1972). It was subsequently shown that this region also codes for determinants on cell surfaces eliciting a proliferative cellular response in the mixed lymphocyte culture reaction and the graft-versus-host reaction (Bach et aZ., 1972; Meo et al., 1973). Efforts by several investigators (David et al., 1973; Gotze et al., 1973; Hammerling et al., 1974; Hauptfeld et al., 1973; Sachs and Cone, 1973), designed to raise antisera against Z region gene products, resulted in the identification of a new class of cell surface alloantigens, the I immune response region-associated (Ia) antigens. Biochemical analysis of these alloantigens showed them to be cell surface glycoproteins of two classes with molecular weights of approximately 25,000 and 33,000 (Cullen et al., 1974; Vitetta et al., 1974). Recent studies have identified a third “invariant” chain of MW 31,000 (Jones et ul., 1978; &cloosicet al., 1980). These antigens may be found on the surface of lymphocytes, niacrophages, and epidermal cells (Hammerling et al., 19’75). Within the last few years, systematic study of the Z region in a number of distinct inbred mouse strains, and in recombinant H-2 strains derived by crossing over between the H-2K and H-2D loci of distinct H - 2 haplotypes, led to the subdivision of the Z region into the Z-A, Z-B, Z-C, Z-E, and Z-J subregions (Lieberman and Humphrey, 1972; Murphy et al., 1976; Shreffler et al., 1977; Delovitch et al., 1977).In addition, this genetic analysis resulted in mapping of genes

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controlling the immune response to particular foreign antigens in the I-A, I-B, or I-C regions. These were designated Zr-lA, Zr-lB, and Ir-1C, respectively. In several instances, it also became apparent that two distinct I region genes could complement each other to develop a high response to a particular antigen (Benacerraf and Dorf, 1976). This complementation phenomena raised questions regarding the role of Ia antigens in immune responses. Recent evidence suggests that the two polypeptide chains that make up the I-E antigen are separately encoded within the I region. Jones et al. (1978), using two dimensional gel electrophoresis, discovered that the location of the I-E p chains on two-dimensional gels differed between BIO.A (5R) (AbEk)and B1O.A (AkEk).This suggested that expression of the I-E molecule is under two gene control with one gene mapping in the I-E subregion and another gene mapping to the left of the I-J subregion. This was confirmed by the peptide mapping studies of Cook et al. (1979, 1980). These authors showed that among recombinant mice B1O.A (5R) (AbEk),BIO.A (AkEk), and BIO.S (9R) (AsEk)heterogeneity exists in the peptide maps of the p chain of the I-E molecule, while no heterogeneity was found in the fy chain. Together these studies suggest that the I-E subregion moIecule is formed by complementation of a gene in the I-A subregion coding for the p chain (Ae) and a gene in the I-E subregion coding for the a chain ( E a ) .Tryptic peptide mapping studies from several other laboratories have confirmed these results (McMillan et al., 1981; Silver and Russel, 1979; Wakeland and Klein, 1979). The regulation of expression of cell surface antigens coded for by the I-A subregion by a locus mapping between I-J and H-2D suggests one mechanism by which two complementary genes might control immune responsiveness (Jones et al., 1978; McNicholas et al., 1982; Matis et al., 1982).As is true for Ir (immune response) and Is (immune suppression) genes, complementation allowing I-A antigen expression can occur in either the cis or trans position. Combining the b and k or d haplotypes allows the expression of the Aeb :E complex on the cell surface; this molecular structure is not found on cells of either parental haplotype. Functional capabilities unique to this complex of I-A and I-E polypeptide chains also would not be shared by either parental heplotype. In this context, it is interesting to note that in a number of complementary l r and I s gene systems, there is a good correlation between expression of Ia antigens coded for by the I-E or I-C and the presence of a complementary Ir or Is gene on the right side of the I region (Jones et al., 1978; McNicholas et al., 1982; Matis et al., 1982). The Ira gene thus might regulate the expression of Irp

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gene product (the Ae protein) on the cell surface, perhaps by coding for the I-E antigen. Further implications regarding the importance of immunologic mechanisms in MHC-associated phenomena come from the recent finding that the S gene of the H - 2 system, which was known to affect serum levels of a p-globulin, is in fact the structural gene for one of the polypeptide chains of the fourth component of complement, C4 (Shreffler et al., 1976). 2. H - 2 Linked Genes and Resistance to Virus-Induced Leukemogenesis

It was apparent from the initial studies of Corer (1956) and Gross (1970) that some relation existed between susceptibility to leukemogenesis and the H-Zk haplotype. The high leukemia mouse strains (AKR, C58, C3H/Figge, and RF), as well as two strains utilized by Gross to obtain a filterable agent from AKR mice, C3H/Bi and C57BR, were all of the H-Zk haplotype. In a formal genetic study of mice of various H - 2 types and their hybrids, Lilly and co-workers (Lilly, 1966; Lilly and Pincus, 1973)inoculated neonatal mice with Gross virus and recorded the ensuing development of the disease. The most extensiveIy studied hybrids were those of the C3H/Bi ( H-2k) and C57BL/6 (H-Zh) cross; in Fz and backcross hybrids, mice of the homozygous H-2klk type showed greater than 90% incidence of leukemia, whereas H-Zhb and H-2hlkmice showed leukemia incidence of approximately 30-50% which was also delayed in onset. Confirmation of the importance of H - 2 in affecting susceptibility to Gross virus-induced leukemia came from studies with congenic strains of mice, differing from each other only in the H - 2 chromosomal segment. Thus mice of the congenic C3H ( H - 2 k ) and C3H.SW (H-Zb) strain pair differed radically in their response to Gross virus, being susceptible and resistant, respectively. Similarly, C57BWlOSn (B10) mice, which are of the H-2b haplotype, were resistant to Gross virusinduced leukemia, but mice of the congenic B1O.BR strain ( H - 2 k ) were susceptible, although with latent periods significantly longer than those of C3H mice (probably because of their differences at Fu-1) (Lilly et al., 1964). Other lines of evidence indicated the importance of H - 2 in susceptibility to viral leukemia in mice. For example, Tennant and Snell (1968),studying leukemogenesis by the BT/L virus, observed a considerably greater level of resistance in C57BL/10 (H-Zb) than in congenic B1O.BR ( H - 2 k )mice.

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a. Role of Zmmune Response Genes in Leukemia Resistance. Studies with H - 2 recombinant mice were performed to localize the gene coding for resistant to Gross virus, Rgu-1 ,within the known fine structure of the H - 2 complex. The data obtained indicated that Rgu-1 maps toward the K-end of the H - 2 complex, comprising the K and Z regions (see Fig. 6). Evidence suggestive of the nature and mechanism of the Rgu-1 influence was obtained in studies with Friend virus (FV). Crosses between susceptible (DBN2 and BALB/c) and resistant (C57BL) mice showed the expected responses according to their Fu-1 and F v - 2 types, but in addition, they showed an H-2 specific component in their response. Because C57BL mice are rendered completely resistant by their Fv-2' genotype (which blocks the spleen focus-forming component of the virus from infecting such mice), Lilly et al. (1964) bred a strain of BALB/c mice (termed BALB.B) that was congenic at the H-2 locus, carrying the C57BL-derived H-2b haplotype instead of H-2d, and the permissive allele at Fu-2. BALB/c (H-2d)mice showed a 10fold lower virus dose threshold for splenomegaly induction and were much less prone to recovery from splenomegaly than BALB.B (H-2b) mice, indicating that the H - 2 difference of the host appeared to significantly alter the cause rather than the onset of the disease. The close or identical mapping of Rgu-l with the Z region, to which the great majority of Zr genes have been mapped, and the indication from FV disease studies that the H - 2 effect may influence a late event in the disease, namely recovery from splenomegaly (Lilly, 1968), led to the suggestion (Lilly et al., 1964; McDevitt and Bodmer, 1972) that the strength of the immune response to virus-specific or tumor-specific transplantation antigens (TSTA) might be regulated by Zr genes. Thus H-2 linked resistance to virus-induced leukemogenesis might result from a stronger immunologic response to a given virus induced antigen. One indication that this hypothesis could be correct was the finding of Aoki et al. (1966) that among progeny of the cross AKR ( H - 2 k and positive for the G antigen induced by Gross virus) x C57BL ( H-2b and G antigen negative), a significant number of mice homozygous or heterozygous for the H-2b haplotype showed detectable levels of anti-G antibodies but no H-2khomozygotes showed these antibodies. In addition, subsequent experiments by Sat0 et aZ. (1973) found that certain leukemias derived from BALB/c mice were rejected by hybrids of BALB/c with other inbred strains, contrary to the usual rules of transplantation. Similar studies with a series of recombinant hybrids estab-

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lished that the responsiveness to these tumors was linked with the K end of H-2, the location of Rgc-l and H-2-linked Ir genes. Furthermore, animals resistant to the tumor had high titers of antibody to Gross-specific antigens, whereas susceptible animals did not have similar titers. More recent studies by Lonai and Haran-Ghera (1977) showed a gene Rrtj-I located in the H-2 region, that influences resistance to the A-RadLV strain of radiation leukemia virus. This strain of RadLV is distinct from the Kaplan strain of RadLV (Lieberman and Kaplan, 1959). Chesebro and Wehrly (1978) also suggested an Z region influence on Friend virus (FV)-induced leukemogenesis Rfu-2, located in the K or Z regions, appears to affect recovery from FV leukemia. All of these results imply, but do not prove, that H-2-linked immune response genes are directly involved in conferring resistance to the development of malignancy. Recent studies from our laboratories have provided more direct evidence for the existence of H-2 linked immune response gene(s) to a virus- or tumor-specific transplantation antigens (Meruelo et aE., 1977b, 1980b). In these studies, AKR mice were crossed with animals of various H-2 congenic strains on the C57BL/10 or C3H genetic background and the hybrid mice injected intraperitoneally with AKR thymoma cell BW5147. (AKR X C3H.Q)Fl (H-2") but not (AKR X C3H)FI (H-2kk)mice were shown to generate a strong humoral response against BW5147 cells. A direct correlation could be demonstrated between survival to the injected BW5147 cells and humoral responsiveness. Cellular immunity appears to play no role in resistance to the proliferation of tumor cells. Humoral immunity and survival to BW5147 cells can be shown to be due to genes in either the B , J , or E subregions of H-2 (Meruelo et al., 1980b). The development of effective humoral immunity depends on B cells and Ly-1+,2-,3- helper T cells bearing the Z-Jk phenotype. These studies appear directly applicable to the spontaneous disease, and results of studies using transformed cells from an overtly leukemic AKR mouse parallel those obtained using BW5147 cells (Meruelo et al., 1980b). Further analysis of the humoral response has shown it to be directed against a protein, which appears to be distinct from virally coded gp70, p30, p12, and p10 by a variety of criteria (Zalman and Meruelo, 1982). Current investigations revolve around several issues: (1) Is this protein a true TSTA molecule; (2) Is it involved in transformation and, if so, does it bear any resemblance to other transforming proteins (e.g., like STC); and (3)What is the nature of the Zr defect in low responder animals (i.e., H-2kk). b. H-2D Effects on Susceptibility to Virus-Induced Leukemogene*

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sis. Resistance or susceptibility to FV (Chesebro et al., 1974) and the Kaplan strain of RadLV (Meruelo et al., 1977a) is associated with the D region of the H - 2 complex. It is important to note that while previous findings with FV were used to help explain the mechanisms of Rgu-I, the mapping location of the major component of resistance to FV is at the opposite ( H - 2 D ) extreme of the H - 2 complex from Rgu-l, which maps to the H-2K end (see Fig. 6). Such mapping argues that a second gene, distinct from Rgu-1, is responsible for the observed resistance. There are some other data indicating a second location for Ir genes within the H - 2 complex. For example, Young et al. (1976)have indicated the existence of an Ir-type mechanism to ferritin which maps to the TL region. However, most Ir genes mapped are located distant from H - 2 D . Therefore, the implied functional similarity between Rgu-l and Ir genes based on near if not identical mapping cannot be as readily applied in associating H - 2 D linked resistance to FV or RadLV with Ir gene effects. Experiments designed to test the role of H - 2 linked genes in resistance to FV and RadLV-induced neoplasia have revealed two general observations. First, virus infection alters quantitative expression of H-2 molecules. Second, cell-mediated immunity plays an important role in recovery from the disease. We shall detail these observations below. H-2 genes appear to affect the expression of surface antigens on FVinfected spleen cells (Lilly et al., 1964). One such antigen whose expression is affected by H-2 gene(s) is Friend-Moloney-Rauscher (FMR), an antigen probably encoded in the FV genome. FMR is abundantly present with the FV although not detectable on the surface of intact viruses. It appears, within 3-5 days after virus inoculation, on the surface of spleen cells of both BALB/c (H-2d, susceptible) and BALB.B (H-2", resistant) attaining its maximum level of expression in both strains 7-14 days after virus injection. Thereafter the level of expression of this antigen (as determined both by direct cytotoxicity of anti-FMR on the cells and by the quantitative ability of cells to absorb cytotoxic antibodies) declines in both strains of mice. However, in BALB/c mice (susceptible) the decline is rapid and much more complete, such that FMR is often difficult to detect at all during the terminal stages of the disease (about 21-28 days after virus administration). Associated with the apparent loss of FMR antigen in BALBlc mice is a concurrent and equally severe decrease in the level of expression of H-2 antigens that is not seen in BALB.B (resistant) mice. These changes in antigenic expression may significantly affect host defenses as discussed below. In addition, Bubbers and Lilly (1977) have obtained evidence that H-2" antigenic determinants might be incorpo-

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rated into virus particles in the process of budding from the membranes of resistant H-2b hosts, whereas H-2k antigenic determinants are never incorporated into virus budding off from H-2k (susceptible) hosts. Different levels of expression of FMR on infected cells could elicit different levels of immune reactivities in the two kinds of hosts and be the basis of H-2 linked resistance to FV-induced leukemia. Similarly, the appearance of H-2 antigenic determinants on budding virus may allow its rapid elimination by the host's immunologic defenses, while the lack of such H-2 determinants on virus may preclude such immune reactivity. Alternatively, quantitative differences in expression of cell surface antigens may, in and of themselves, constitute a host of defense mechanism independent of the immune surveillance system. Such perturbations on the cell surface may have profound effects on virus penetration in and out of cells. Studies from our laboratories during the past several years have shown that H-2 has a marked effect on RadLV-induced neoplasia. H-2associated resistance or susceptibility to RadLV maps to the D end of the complex (Meruelo et al., 1977a). The H-2Dd allele confers resistance to the disease, whereas the H-2Dq and H-2Ds haplotypes are associated with susceptibility. Thus, for example, BIO.S (7R) (H-2Dd) mice are resistant, while B1O.S (H-2Ds)animals are susceptible. H-2linked resistance to RadLV appears to be expressed as a dominant trait in hybrid offspring of crosses between susceptible and resistant mice. Further studies on virus replication have indicated no effect on actual infection (Meruelo et al., 1978). This is in accord with early studies with Friend virus by Lilly (1968) that indicates that H-2 effects on virus-induced leukemogenesis are noted at a late stage in the disease, namely recovery from splenomegaly. If, after intrathymic inoculation the course of virus replication in the thymus is followed over a 12-week period with the aid of an immunofluorescent anti-MuLV serum, a major difference is discernible 5 weeks after virus inoculation between susceptible BIO.S and resistant B1O.S (7R) mice. The number of immunofluorescence-positive thymus cells increases markedly during the 3- to 9-week interval after virus inoculation in susceptible mice, while little increase is seen in virus-positive cells among thymocytes of resistant mice. A remarkable effect on quantitative expression of H-2 antigens occurs on the cell surface following intrathymic RadLV inoculation (Meruelo et al., 1978). We have described some aspects of these changes in expression earlier in Section XII,H,2. Here we shall concentrate on the role of these changes in H-2D-linked resistance. When thymocytes

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from RadLV-inoculated mice and uninfected mice are incubated with alloantisera to H-2D and H-2K and then reacted with a fluoresceinlabeled rabbit anti-mouse IgG reagent, their cell surfaces can readily be seen stained with the aid of a fluorescent microscope or the fluorescent-activated cell sorter (Loken and Herzenberg, 1975). After intrathymic inoculation of RadLV, there is an early increase in cell surface expression of H-2K and H-2D molecules on thymocytes of BIO.S (7R) and B1O.S mice. The subsequent patterns of expression of H-2K molecules on thymocytes appear similar when these two strains of mice are compared. However, the subsequent effect of virus infection on levels of H-2D molecules differs in the two strains. Expression of H-2D molecules appears to be more markedly increased, for a more prolonged period in thymocytes of RadLV-infected BIO.S (7R) (resistant) mice than in thymocytes of RadLV-infected BIO.S (susceptible) animals. Furthermore, the increased expression of H-2D on B1O.S (7R) cells is greater and persists longer than the changes observed in expression of H-2K molecules in these same cells. It is remarkable that the differences observed with regard to H-2D expression in comparing BIO.S (7R) and BIO.S thymus cells correlate reciprocally with the changes in virus-positive cells seen in these same animals (Meruelo et al., 1978).BIO.S (7R) mice, which are resistant to leukemogenesis, do not appear to show a marked increase in the number of virus-positive cells in the thymus during the first 9 weeks after infection and show dramatically increased cell surface expression of H-2D molecules. Exactly the opposite phenomenon is seen when thymus cells of B1O.S mice (susceptible to RadLV) are examined. A number of additional factors suggest that changes in H-2 antigen expression may be important in the host’s response to infection by RadLV: (1)changes in expression occur very rapidly; (2) genes in the D region confer resistance to RadLV-induced neoplasia, and antigens coded for by the D region show the most marked and prolonged changes in expression and differential regulation between susceptible and resistant animals; (3) there is an inverse correlation between expression of H-2D and viral antigens (uide infra); and (4)finally, our studies (Meruelo et al., 1978) have shown that H-2 antigens disappear from the surface of RadLV-transformed cells. Thus, while resistance to the disease is associated with increased H-2 antigen expression, the neoplastic state is associated with disappearance of these antigens. Although the effects of increased H-2 expression on other steps required for oncogenesis remain to be evaluated, several observations suggest that elevated H-2 antigen expression enhances the effective-

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ness of the host’s cellular immune response to virus infected cells (Meruelo, 1979). First, cell-mediated immunity (CMI) against RadLVtransformed or infected cells can be detected with ease when H-2positive target cells are used in assaying ceil-mediated lympholysis ( C M L ) . Second, resistant mice develop greater numbers of effectors when injected with RadLV than do susceptible mice. Third, injection of normal (uninfected) thymocytes into syngeneic recipients of resistant or susceptible H - 2 type does not stimulate a CML response. However, injection of RadLV infected thymocytes from resistant mice produces a vigorous CML response and such thymocytes elicit the strongest response at a time when both H-2 and viral antigen expression is elevated. By contrast, injection of infected thymocytes from susceptible mice, which express viral antigens but low levels of H-2 antigens, does not stimulate a CML reaction. Thus, H-2D resistance to RadLV induced leukemogenesis has two important characteristics associated with it: (1)it is accompanied with specific changes in H-2 antigen expression; and (2) it appears to be mediated by a celi-mediated response. The effects of H-2D on FVinduced splenomegaly discussed previously strongly suggested similar ef’tects of H - 2 and viral antigen expression. However, the role of CMI has not yet been described. In a series of experiments designed to test the role of Zr genes on FV disease, Chesebro and Wehriy (1976a,b) found that while the antibody response showed little correlation with recovery from splenomegaly, there was a clearcut correlation between recovery from the disease and cell-mediated immunity. It was not clear, however, that CMI played a crucial role in the recovery or ensued after recovery had begun. In addition, no correlation existed between H - 2 type and ability to mount either a cell-mediated or humoral response. Both susceptible (H-2df and resistant ( H-2b) animals were capable of vigorous humoral and cell-mediated cytotoxic responses, despite the clear association between H - 2 type and resistance to the disease. An important consideration, however, is that Chesebro and Wehrly (1976a,b) tested for cell-mediated Zr gene(s) after inoculating virus concentrations so low that normally susceptible animals showed a high percentage of recovery from the disease-i.e., H - 2 control was not detectable. Under such circumstances, equal cell-mediated responsiveness in resistant and susceptible animals does not argue for the lack of an Zr gene. In fact, more recent experiments by Chesebro and collaborators (B. Chesebro, personal communication) has revealed that the kinetics of cell-mediated responses may be different with effectors from resistant animals proliferating earlier than those of

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Susceptible mice. Timing of the host response would, of course, be critical in containing the rapid spread of FV and the replicating erythroleukemic cells. Further evidence for the role of CMI in H-2D control of FV-induced erythroleukemia has come from the studies of Blank et al. (1976a). Using an experimental system different from that of Chesebro et al. (1976a), these authors have shown that resistant mice (H-2b) can mount a CMI response to FV-induced tissue culture-adapted tumor cells, whereas susceptible animals (H-2k)cannot. In the case of mice of the H-2b genotype immunized with FV, where the major factor governing the relative resistance of the mice to the virus-induced disease is the H-2Db-associated Rfu-l locus, resistance to the disease is associated with the generation of CTL specific for an unidentified viral antigen and the H-2Db gene product but not for the H-2Kb gene product (Blank and Lilly, 1977; Bubbers and Lilly, 1977). Present evidence indicates that FV-encoded molecules become associated with H-2Db molecules on the surface of infected cells, and that this molecular complex possess an antigenicity which induces the CTL response and which is recognized by the effector CTL population. In FV-infected cells of other H-2 genotypes, such H-2hiral protein complexes may involve H-2K or H-2D molecules, or both, or neither, and these complexes may or may not possess the capacity to induce a strong CTL response. By this mechanism, the H-2 genotype of the host plays a major role in determining whether or not virus-infected cells bear an antigen appropriate for eliciting a CTL response capable of destroying tumor cells.

3. Other Immune Reactivities Many indications are available that natural immunity against endogenous type C viruses in many mouse strains may be important in regulation of virus protein expression and virus replication. As discussed in Section IV, seroIogists had noted reactivity with type C associated antigens in a variety of reagents and normal mouse sera. More direct evidence for a role of humoral immunity in regulating virus protein expression came from studies of group specific antigen (gs) expression in R F mice. Gs expression was found to increase in these mice until approximately 50 days of age, at which time a sharp decrease in expression could be correlated with germinal center and gs antigen localization by immunofluorescence (Hanna et al., 1972). While R F autogenous mice seem to be protected by such type of immunity against lymphoid neoplasia, this response seemed detri-

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mental in the sense that it promoted glomerular disease (Hanna et al., 1972).By contrast, AKR mice also generate a natural humoral antiviral response, and while the antibodies can be eluted from kidneys (Oldstone et al., 1976), glomerular disease does not become fatal because death from leukemia occurs more rapidly. Alternatively, in AKR mice, production of immunoglobulin and immune complexes does not lead to glomerular damage. It should be noted that AKR mice have a complement deficiency (C5) which might explain why humoral immunity is not effective in protection against viremia and leukemia. With the introduction of potent radioimmunoassay techniques (Ihle et al., 1973), it was soon determined that occurrence of this type of immunological reactivity was found in many strains, including I, 129, NZB, C3H, A, and DBM2 (Nowinski et al., 1974). In most cases, the antiviral response was directed at gp70 and p15E (Ihle et al., 1976b; Oldstone et al., 1976). H-2-linked genes may affect the generation of these immune reactivities (Nowinski, 1975). However, the in vivo significance of these reactions remains unclear. Although selected normal mouse sera react with isolated viral gp70 (Ihle et al., 197613; Stephenson et al., 1976), many normal sera reactive with intact virus show no reactivity with isolated viral proteins. This result may be explained on the basis of weak-affinity interactions with determinants on gp70 and/or p15E which may be altered. However, the biological role of such antibodies is unclear, e.g., there exists high reactivity in AKR mice and low reactivity in C58 mice (Nowinski et al., 1974) although these strains have similar virology and leukemia incidence. It is of further interest that antibodies reactive with ecotropic viruses are seen only in certain strains, and antibodies to xenotropic viruses are seen only in the C57BLJ6 strain, but all strains appear to have antibodies to the recombinant MCF virus (Stockert et al., 1979). The relationship of these natural antibodies to prevention or enhancement of disease is under active investigation. Chesebro et al. (1979) and Doig and Chesebro (1979) have shown that H-2 and non-Hi-&linkedgenes are required for recovery from FVinduced leukemia. The non-H-2-linked gene, Rfu-3,appears to influence the production of anti-FV antibody independent of the H-2 genotype. Although mice of the Rfu-3‘l” genotype produce high levels of anti-FV antibody, they fail to recover from FV-induced leukemia unless the appropriate H-2 associated genes are present (Chesebro et al., 1979; Doig and Chesebro, 1979). Thus, FV leukemia cells continue to proliferate even in the presence of anti-FV antibody, possibly because of their resistance to antibody-complement-mediated lysis (Doig and Chesebro, 1979). Furthermore, FV leukemic spleen cells late in the

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disease express decreased amounts of FV cell surface antigens and release less infectious virus than leukemia spleen cells early in the course of the disease (Doig and Chesebro, 1979).The low levels of FV cell surface antigens and infectious virus observed in mice with antiFV antibody were found to be reversible after transfer of leukemic spleen cells into nonimmune animals. Therefore, anti-FV antibody appeared to play a role in altering the expression of viral antigens and infectious virus release in these cells. The presence of anti-FV antibody late in the disease is associated with decreased expression of Friend helper virus (F-MuLV)-encoded intracellular and cell surface proteins, whereas the expression of the replication-defective spleen focus forming virus (SFFV)-encoded proteins appeared to be minimally altered. Other investigators have suggested that cell surface antigen loss induced by antiviral antibody might protect infected cells from virus-specific cell-mediated lysis. This did not appear to be the case in the FV system. Anti-FV CMI effectors could recognize late leukemic spleen cells even though these cells expressed greatly decreased amounts of F-MuLV cell surface antigen. It appears likely that these CTL are able to recognize residual low amounts of F-MuLV gp70 on late leukemic cells. VI. Prospects for Control of Human Leukemia

We began this articIe with two questions: (1)Are viruses involved in human neoplasias? and (2) How can the knowledge on hand be applied to arrest or control the malignant process? In the face of the voluminous literature described, it is clear that biologists have done much to understand viral-induced leukemogenesis. The point is rapidly approaching when we can answer the first of these two questions and begin to provide an answer to the second question. Let us recapitulate the central core of knowledge that should permit such answers. First, with the demonstration that transforming genes of retroviruses are derived from cellular genes, designated protooncogenes, a door has been opened in search of the desired answers. Thus if certain cellular genes are oncogenic when placed under the influence of viral control elements, perhaps other genes, or indeed the same ones, mediate carcinogenesis by various agents when they are expressed at high levels as a result of exposure to harmful environmental agents. Second, identification and cloning of endogenous retroviral sequences present in human DNA appear to suggest an affirmative answer to the first of our two questions. For example, Martin et al. (1981) have shown that under nonstringent annealing conditions, a

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2.75 kb segment of cloned African green monkey DNA that specifically hybridizes to the provirus of AKR ecotropic murine leukemia virus (MuLV) and baboon endogenous virus (BaEV), detects related sequences in three different preparations of human brain DNA fragments. Furthermore, the evidence for human viral-like DNA is strengthened by the fact that restriction analysis of the annealing human sequences yields similar results to those obtained for mouse DNA annealing with the MuLV cDNA probe. In both cases, multiple bands suggested the presence of numerous copies of retrovirus-related sequences integrated in the genomes (mouse and human). Even more recent and convincing data come from the laboratory of R. C . Gallo (Poiesz et al., 1981a,b), which has directly isolated a newly discovered retrovirus in association with certain types of adult T cell lymphonidleukemia. The novel retrovirus has been isolated from a Tlymphoma cell line established in culture from a patient with mycosis fungoides. Subsequent results by Poiesz et al. (1981b) demonstrated the isolation of a similar virus from a patient with Sezary syndrome, and Kalyanaraman et al. (1981)showed that sera from two patients and one of the patient’s spouses contained antibodies which specifically reacted with the major core protein of the virus. Mycosis fungoides and Sezary syndrome are clinical variants of cutaneous T cell lymphoma and leukemia, respectively, a rare disease in human adults (Lutzner et al., 1975). Probably several technical difficulties account for the failure to detect such viruses earlier. One is the fact that culturing of leukemia cells is often required to detect virus expression, and second, culturing of human T cells was impossible until recently. With the advent of T cell grow factor (TCGF), in witro T cell culturing and detection of human retroviruses became possible. The virus called HTLV (for human T-lymphoma virus) seems to be quite distinct from the numerous types of animal retroviruses previously described. HTLV is morphologically and biochemically more closely related to bovine leukemia virus, which causes lymphoma in cattle, although the human and bovine viruses seem unrelated antigenically or by nucleic acid homology. The epidemiological most convincing data come from Japanese workers. Miyoshi et al. (1982a-c), by cocultivated T leukemic cells with umbilical cord leukocytes, have made the cord leukocytes TCGF-independent cells. These cultures have subsequently been shown to be producing virus similar to HTLV particles. These workers have thereby demonstrated for the first time with human cells experimental transmission of and transformation by a human virus.

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Even more striking, however, is the association between the virus and leukemia in uiuo. In major Japanese cities, there is a relatively high incidence of an aggressive form of adult T cell leukemia. An HTLV-like virus seems to be associated with such an aggressive, particularly malignant, fast growing type of T cell leukemia. Association of this type of leukemia with HTLV is strongest in Kyushu, Japan’s extreme southwest island. For example, a recent study (Uchiyamo et al., 1981) of 272 T cell leukemia cases shows a remarkable clustering of places of birth, in the Kagoshima and Nagasaki prefectures of Kyushu, and indicates that sera from all the leukemia patients tested and from 25% of healthy adults sampled react positively with the Miyoshi’s virus isolate (Miyoshi et al., 1982a) (similar or identical with HTLV). While not all T cell leukemia patients tested were positive (Kalyanaraman et al., 1981; Miyoshi et al., 1982a), the evidence for the involvement of viruses in human neoplasms becomes strengthened by such findings. Furthermore, the above evidence is, of course, not the only one supporting a role for viruses in human neoplasia. Previously, other viruses had been implicated as etiological agents for several human neoplasms. For example, Burkitt’s lymphoma and nasopharyngeal carcinoma have been associated with Epstein-Barr virus. Hepatitis B virus infection has been associated with liver cancer. Herpes simplex and papilloma virus have been suspect in the etiology of cancer of the uterine cervix. What is perhaps most clear now is that our ability to identify transforming genes, whether cellular or virus encoded, and our ability to isolate, clone, sequence, and study the transforming function of these genes and their regulatory mechanisms are increasing. At the same time, our understanding of the immune system, our ability to produce monoclonal antibodies of high specificity, and to identify other host mechanisms of defense against neoplasia are improving. Thus it is not unreasonable to foresee the day when this knowledge will be brought to bear successfully in the control of human cancer.

ACKNOWLEDGMENT Dr. Hugh 0. McDevitt introduced one of us (Daniel Meruelo) to the fundamental notions embodied in this article. His contributions in many of the areas covered, particularly those dealing with involvement of immune response and other H-2 and H L A genes in disease susceptibility, have been helpful in formulating our approach to the subject. His careful reading of this manuscript and constructive comments and advice are much appreciated.

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BREAST CARCINOMA ETIOLOGICAL FACTORS Dan H. Moore Department of Microbiology and Immunology. Hahnemann University Medical College, Philadelphia, Pennsylvania

Dan H. Moore II Biomedical Sciences Division, University of California Lawrence Livermore Laboratory, Livermore. California

Cathleen T. Moore Department of Humanities and Social Sciences, Philadelphia College of Pharmacy and Science, Philadelphia, Pennsylvania

I. Introduction ...................................... ......... Heritage ................................................. Menses, Marital State, Parity ...................................... Breast-Feeding .................................................. Contraceptives ...................... ........................ Benign Epithelial Diseases of the Breast ........................ Hormonal Factors.. .............................................. Cancer ........................ .............................. IX. Iatrogenic Factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Immunological Factors . . . XI. Viral Aspects of Human Breast Cancer .............................. XII. Dietary Factors .................................................. XIII. Psychosomatic Factors. ........................................... XIV. Discussion and Concluding Remarks. ............................... References. ...................................

11. 111. IV. V. VI. VII. VIII.

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I. Introduction

Breast carcinoma is the most prevalent type of cancer in American women. Approximately 8% of American women can expect to be stricken with this disease some time during their lifetimes. The rate in men is 1%that in women. Although many etiological factors are implicated, the causes or reasons for a woman being afflicted with this disease remain equivocal. 189 ADVANCES IN CANCER RESEARCH, VOL. 40

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There is an abundance of evidence that the incidence of breast carcinoma varies greatly from one population to another throughout the world and that in most populations it is increasing. Due to earlier detection? improving medical care, and possibly other factors, the death rate is not increasing as rapidly as the incidence rate. In general, the incidence is greatest in populations with the highest standards of living, such as those of Northwestern Europe and North America. Therefore? a woman’s heritage is usually a large factor in determining her risk of developing mammary carcinoma. But as the standard of living or “life style” of a population changes, so does the incidence of breast cancer change. For example, the life style in Japan has changed since World War 11, and during the last few years, mortality from breast carcinoma has dramatically increased as shown in Fig. 1 (Hirayama, 1978). 3500 3262

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It is not known how long it takes for a breast carcinoma to become large enough to be detected because cancers grow at different rates. It has been found, however, that the doubling time for breast cancer ceIls is anywhere from 81 to 900 days (Gershon-Cohen and Ingleby, 1959),which means that it would take 10 to 100 years for the tumor to reach 1 cm in diameter. It is concluded, therefore, that most breast cancers start relatively early in life, or they never reach a detectable size during a lifetime. The rate of growth may change for any given tumor, but even so, many years are required for it to become large enough to be detected by standard methods. Its growth is exponential, however, and once it reaches a diameter of 1 cm, with a constant doubling time (about 100 days is average), it would take less than a year for it to reach a diameter of 2 cm. The very long period of undetectability and the uncertainty of growth rate add to the difficulty of determining the initiating factors. That the period is long between initiation and presentation is supported by studies of atomic bomb victims of Hiroshima and Nagasaki. The data suggest that those irradiated near menarchal age were at highest risk and that the increased risk extended throughout their lifetimes (Tokunaga et al., 1979). There are many influences implicated in the etiology of human breast carcinoma, and it is the purpose of this article to report some of the recent findings. II. Heritage

Heritage, as we shall use the concept here, includes family, race, country of origin, religion, and any component of lifestyle that is firmly passed on from one generation to the next, such as abstention from certain foods on religious grounds. These factors seem to have a great influence on the incidence of breast cancer, but there is little agreement on which components of heritage are most important and how they operate. Since having a twin or other first-degree relative with the disease substantially increases the risk, it has been argued that there probably can be an inherited constitutional proclivity. Anderson (1978) has found that the familial risk is greater for premenopausal than postmenopausal breast cancer, that it is greater for bilateral than for unilateral, and that if a first-degree relative has both premenopausal and bilateral, a young woman’s risk is increased more than eightfold over another who has no breast cancer in her family. Adami et al. (1981), however, in their study of 1330 Swedish women with breast cancer, found that bilateral breast cancer in a first-degree relative increased the standardized relative risk (SRR) only 2.2 times,

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and that the age of presentation was not a very important hereditary factor. One or more first-degree relatives with breast cancer were reported by 11.2% of the 1330 breast cancer patients, but by only 6.8% of the controls (standardized relative risk = 1.7). For second-degree relatives, the SRR was 1.5. One or more sisters with breast cancer were reported by 10.1% of the patients and 5.1%of the controls (SRR = 2.0). A familial influence on breast cancer risk has been reported by many others (Kelsey, 1979; King et al., 1978,1980; King and Petrakis, 1977; Lynch and Guirgis, 1979; Maisin et aZ., 1978; Moolgavkar and Knudson, 1981; Speizer et al., 1978).Speizer et al. (1978) found that having a mother with breast cancer increased risk by 1.8times, but having a sister under 45 at presentation increased the relative risk up to 5.8 times; if, however, the sister was over 45 when the cancer appeared, the relative risk was increased only 2.0 times. In a study of twins, Holm et al. (1980) concluded that the increased risk was six times in monozygotic and two times in dizygotic siblings of breast cancer patients. Ottman et aZ. (1978)found that estrogen receptor levels were significantly lower in postmenopausal women who had a family history of breast cancer than in those who did not, but there was no difference in estrogen receptor levels among pre-, peri-, and postmenopausal women who had no family history of breast cancer, nor was there a difference in estrogen receptor levels between familial and nonfamilial premenopausal patients. It was concluded that with respect to this variable, familial breast cancer appears to be a homogeneous disease which is more similar to premenopausal than to postmenopausal nonfamilial breast cancer. From studies of many families at high risk for breast cancer, King et al. (1978) have postulated an autosomal dominant model for transmission of breast cancer susceptibility. They found that such a postulate fitted the data, particularly that of the largest families, signficantly better than an environmental model which assumes increased risk to be age but was not genotype dependent. On the other hand, King (1980) concludes that the ultimate development of breast cancer may be determined by physiological stress resulting from cultural practices. In a comparison of risk factors associated with familial and nonfamilial breast cancer, Macera et al. (1982) found that the two familial history groups showed consistent different risk profiles on various reproductive, hormonal, and environmental factors known to be associated with the disease. Menstrual activity in excess of 37 years resulted in an odds ratio of 12.3 in the family history group vs 1.1in the

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group without a family history. Other factors at significantly elevated risk for the family history group were more than 12 yeaSs from menarche to first birth, use of thyroid medication, and, for postmenopausal women, a Quetelet index score greater than 3.35. For the group without a family history, the only significant risk factors were large breast size and childhood spent in an urban area. No risk factor was significant in both family history groups, indicating that breast neoplasms in the two groups may have different etiologies and pooling the family history groups for analysis dilutes the magnitude of the associations. In a comparison of plasma hormone profiles of young women of high and of low risk for familial breast cancer, Fishman et al. (1978) found no statistically significant differences in the plasma concentration levels of prolactin, gonadotropin, estrone, estradiol, or estriol, although the high-risk group had consistently lower values for all the hormones except estriol. Fishman et al. (1979) also reported that familial high-risk women had lower urinary estrone and estradiol levels than carefully matched women with no family history of breast cancer and that the difference was highly significant. These data do not support the hypothesis suggested by Lemon and colleagues (1966) that the ratio estriol: estrone + estradiol was lower in high-risk women, although this hypothesis was supported by a study of Asian and Western populations. The urinary estriol ratio or quotient, as it is called, was found to be higher in Asian than in American women both in the follicular and luteal phases of the menstrual cycle (MacMahon et al.,

1971). A comparison of serum levels of prolactin, progesterone, estradiol, androstenedione, and dehydroepiandrosterone sulfate was made by Boffard et al. (1981) in 52 adolescent girls with a family history of breast cancer and 90 girls without such a history, but no significant differences were found in the endocrine status of the two groups. Consistently lower levels of plasma prolactin, gonadotropin, estrone, and estradiol for a familial high-risk population were recently reported by Lynch et al. (19811,but none of the differences was statistically significant. However, several characteristics of hereditary breast cancer were reported by the same authors: (1) significantly earlier age of presentation, (2) excess of bilaterality and/or specific other cancer associations, (3) vertical transmission, and (4)improved survival over nonhereditary forms. These authors also suggested that there is a genetic linkage between breast cancer susceptibility and the glutamate-pyruvate transaminase locus in breast cancer-prone families. In Tunisia, a form of breast cancer characterized by rapid progres-

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sion, inflammation, and edema has been reported by Mourali et aE. (1980).It occurred in more than 50% of the 581 breast cancer patients presenting at the Institute Salah Azaiz in Tunis during the period 1969-1974. This fulminating form was found more frequently in rural residents and women with blood type A and in those who had a recent pregnancy. In postmenopausal cases, late menarche was associated with increased risk. The risk factors for this disease progression were found to be quite different from those generally reported to influence the incidence of breast cancer. Breast cancer rates are usually found to be 40-70% higher in urban than in rural areas (Cumberbatch et al., 1979; Kelsey, 1979; Melton et al., 1980; Papolczy and Nagy, 1980; Pawlega, 1980; Thurmon and Robertson, 1979). Although diet and other factors have been implicated, the reasons for the difference are not understood. If it is fat in the diet, as some authors suggest, the incidence should probably be the other way around, because in general rural people have a higher fat diet than urban residents. The death rate from breast cancer in the Old Order Amish of Lancaster County, PA (rural) is roughly 1.7 times higher than the United States national average (Hamman, 1979), and diet is thought to be a principal factor because other elements of lifestyle-earl y marriage, early birth, low education, low professionalism except homemaking, and a relatively simple and primitive way of life-are associated with a low rate of breast cancer. The inbreeding of the Amish could be a factor. The high urban rate is also not likely to be population congestion or pollution, because Osaka, Bombay, or Mexico City, and most population concentration areas of the world have lower breast cancer rates than rural populations of North America or northern Europe or New Zealand or South Africa (white). Detection and reporting may influence to some degree the statistics of various areas but do not account entirely for the large differences. Cairns (1981)discusses genetic transpositions and genetic repair as possible mechanisms in genetic susceptibility to malignant processes, but at present there is no satisfactory explanation of how or why there is a familial influence on breast cancer risk. It is not a very stable characteristic, or is only one of many influences that operate synergistically and therefore is not generally a decisive factor in determining whether or not a breast cancer appears. The incidence of breast cancer does not remain constant in any population, but changes with environment. For example, the Japanese, who have been known to have one of the lowest breast cancer rates in the world, undergo an increase when they migrate to the United States (Buell, 1973). The incidence in Orientals living in Hawaii rises to that of the Hawaiian whites by

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the third generation (Dickinson et aZ., 1974). Similarly, black Americans have experienced an increased incidence as they have migrated from a rural to an urban environment and as their standard of living has risen (Austin et al., 1979; Devesa and Silverman, 1978). Since 1965, the incidence in Japan has increased dramatically (Hirayama, 1978). In most populations, the rate has shown an increasing trend, although some have shown periods of decrease (see Haagensen et al., 1981, p. 3). For example, breast cancer incidence in The Netherlands and in the United Kingdom decreased between 1935 and 1950, and it decreased in Australia and Norway between 1950 and 1975. There now exist many reports on the incidences of breast cancer in rather small ethnic or religious groups living within a larger population. One of these groups is the Parsis of Bombay, whose progenitors came to India from Persia soon after the Moslem invasions caused the fall of the Sassanian empire in A.D. 641. The Parsis, who are members of the Zoroastrian religion based on fire worship, are believed to have originated in the first millenium B.C. They were united and possibly isolated within Persia even longer than they have been in India. Due to their small number and their strict religious rules against marrying outside their sect, they have been inbred for at least 13 centuries. Although a few live in other places, including Iran, most of them (80,000 or 1.7% of Bombay’s population) live in Bombay. The Parsis stand out in that their breast cancer rate is three times that of the rest of the population of Bombay, and breast cancer accounts for one-half of all cancers in Parsi women (Paymaster and Gangadharan, 1970). [In the United States, the breast is the site of only one-fourth of all cancer in women.] Whether the unusual distribution has a genetic basis is unknown, but their lifestyle is more “Westernized” than that of the Hindus or Moslems. The Parsis marry later and have smaller families. The Parsi community has no poverty or illiteracy, and the diet contains more meat, particularly pork, but it is not known how much these factors contribute to the incidence of breast cancer. Another religious group of interest is the Old Order Amish (about 12,000) of Lancaster County, Pennsylvania, a sect of Christians who immigrated from Switzerland and Germany more than 200 years ago and maintain a lifestyle of that period. The values that govern their lifestyle are based on a literal translation of the Bible and a rigid adherence to Biblical teaching. They are mostly thrifty farmers who do not wish to be a part of modem industrialization, and therefore do not make use of electricity, automobiles, or tractors. Education is carried no further than the eighth grade. They exercise vigorously due to their occupation, produce most of their food, and abstain from the use

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of alcohol and cigarettes. Their diet is omnivorous, with no particular type of food excluded. They are reproductively isolated and rather highly inbred. They marry early (average age 21) and have large families (average 6 or 7 children). However, the Amish have a breast cancer death rate about 1.7 times that of non-Amish women of the same county (Hamman, 1979). Another somewhat similar religious sect is the Seventh-Day Adventists, who number about 2.5 million worldwide, with about 500,000 scattered throughout North America. About half follow a lacto-ovo-vegetarian diet; virtually all abstain from using pork products; they also abstain from the use of alcohol, tobacco, coffee, tea, and other hot condiments. Breast cancer risk, particularly of postmenopausal cancer, is lower than that of the general population (Enstrom, 1979),but evidence linking their low breast cancer ratio to diet has not been established (Phillips, 1975; Phillips et al., 1980). The Jews of New York City have a breast cancer incidence about 20% higher than the rest of the metropolitan population (Seidman, 1971). This increased rate prevails (S. Blumenthal, Director of Biostatistics, New York City Health Dept., personal communication) in the most orthodox, the Hassidim, who have a different heritage and lifestyle from the major Jewish population. They come from Russia and other eastern European countries, but all follow very rigorous dietary rules. They abstain completely from all pork products and shellfish, and consumption of any meat and dairy products is separated by a period of several hours. Marriage in the Hassidim is earlier than in other branches of Judaism, and no form of contraception is used, except in rare cases that are approved by a rabbi. In extensive studies of Jewish populations of Israel with eastern and western heritage, Schachter et al. (1980) concluded that way of life is more important than genetic factors in determining susceptibility to breast cancer, but King et al. (1980) have reported that an allele increasing susceptibility to breast cancer may be linked to the glutamate-pyruvate transaminase locus. The patterns of occurrence of breast cancer in 11 high-risk families were evaluated by segregation and linkage analysis. These patterns were consistent with the hypothesis that increased susceptibility to the disease was inherited as an autosomal dominant allele with high penetrance in women. However, until more work is done in this area, a true genetic propensity to breast cancer must be regarded with a great deal of caution. The authors state that there is no association in the general population between a woman’s glutaminate-pyruvate transaminase genotype and her cancer risk and that the linkage cannot be used as a screening test for breast cancer.

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Another curious factor associated with a woman’s heritage is the finding that if she is born of a young mother, she has a 25% lower risk of developing breast cancer (Rothman et al., 1980). Breast cancer in men occurs at about 1% the rate in women in North America and northern Europe, and also in the Parsi community of Bombay. However, in some populations where the incidence of breast cancer is low, it seems that the ratio of the disease in men to women is higher. In a survey of cancer at a mission hospital serving 56,000 Blacks in Northeastern Transvaal, South Africa, 13 cases of breast cancer were seen over a 9-year period and of these, three were in men (Sutherland, 1968). In a study of the relationship between male breast cancer and prostate cancer, Sobin and Sherif (1980)found that in most of the populations of the world, the ratio of male breast cancer to prostate cancer is low, but available data for Iran, Afghanistan, and Cairo, Egypt gave the ratio (based on relative frequency rather than incidence) of male breast to prostate cancer as high, i.e., there is a greater proportion of male breast cancers than prostate cancers in these populations. In Kampala (Uganda), 10% of all breast cancers were in males (Davis, 1957), but this high ratio is probably due to the low rate in females. According to some reports, the male carcinomas are highly endocrine sensitive. Estrogen treatment for prostate cancer increases risk of breast cancer (Sobin and Sherif, 1980). Practically all the male carcinomas are estrogen receptor positive, compared with only 60% of female, and three-fourths of the male cancers contain progesterone receptors (Ruff et al., 1981). Lin et al. (1980) claim that in America male breast cancer risk is higher in Jews, college graduates, professionals and managers, and men with testicular problems (mumps, orchiditis, injuries, undescended), men who marry late, and those who have no children. Most of these risk factors have also been implicated in the female diseases. We conclude that heritage has a strong influence, possibly the strongest of the several factors discussed here, on breast cancer risk, and true genetic propensity or weakness may be a reality in some people’s heritage; there may even be as yet undiscovered breast cancer genes. However, the present evidence seems to favor inherited environment or lifestyle over inherited genes. Ill. Menses, Marital State, Parity

In a study of breast cancer patients and other patients in hospitals in the cities of Athens and Piraeus, Greece, Valaoras, MacMahon, and others (1969) found that tbere was little difference in the menarchal age in the two groups unless the menarchal age was 16+ years. How-

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ever, there was a significant deficiency of women whose menarchal age was 16 or over in the breast cancer group compared to the nonbreast cancer patients. Many other surveys since then (Mirra et al., 1971; Craig et al., 1974; Lin et al., 1971) have supported these findings, although some other surveys have not. It is important to note also that in countries or populations where breast cancer rates are low, the age of menarche is older than in countries or populations where the breast cancer incidence is high. In the same population, the difference in menarchal age between breast cancer and non-breast cancer women is usually much less than the difference in menarchal age between North American or northern European women and women of many other parts of the world where the incidence of breast cancer is low. Usually, where the standard of living is high, menarche comes earlier than where the standing of living is lower. It has been proposed that menarchal age is a function of weight gain (Frisch and Revelle, 1970, 1971; Frisch and MacArthur, 1974), which may also be a function of diet. The hypothesis is that girls who grow up faster reach menarchal age earlier. Genetics may also be a factor. MacMahon (1979)has suggested that specific dietary factors associated with age at menarche may influence risk of breast cancer in later years. In an analysis of the many factors implicated in breast cancer risk, King (1980) has found that the number of years between menarche and the first full-term pregnancy is a significant variable, with the risk increasing with the interval. It has also been noted that early menarchal age is associated with premenopausal as opposed to postmenopausal cancer (Vakil and Morgan, 1978; Craig et al., 1974). Many publications report insignificant or no effect of menarchal age on breast cancer risk. In a study of 179 breast cancer patients and 179 matched controls in Sweden (Adami et al., 1978), no significant difference was found with respect to age at menarche, age at first birth, age at menopause, or number of children. In another study of 1155 breast cancer patients (27.7% unmarried) and 1000 controls (19.7% unmarried) in Hungary, there was no difference in menarchal age between the two groups. However, members of the breast cancer group reported a later menopause (Papolczy et al., 1978). Wynder et al. (1978) found age at menarche to have no effect in United States Caucasian women, but long menstrual periods, late age at first birth, and late menopause were found to increase risk, and Tulinius et al. (1978) found menarchal age to have only a slight influence. No significant effect of menarchal age was found by Hoff et al. (1980) in France. In a study of 487 breast cancer patients and 433 controls in Japan, Takatani and Wakabayashi (1979) found no difference in menarchal age or

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height and weight, but menstrual irregularity and pain at menses were found more frequently in the breast cancer women than in the controls. Juret et al. (1980), in France, found that the earlier the menarche, the poorer was the prognosis and also, the earlier the first delivery, the earlier the cancer onset. The prognosis was found to be worse when the first delivery occurred before age 23 or after age 30. Many menstrual disorders such as irregular periods, pain at periods, prolonged flow, premenstrual tension of breasts, and occasional amenorrhea have been associated with increased risk of breast carcinoma. In a prolonged study of 5000 women, Wallace et al. (1978) found that late menopause, longer and more variable cycles during the later 10 years, and a single or a small number of episodes of amenorrhea were associated with increased risk. Regular deviations from the standard 28 days was not as indicative as a single or small number of amenorrhea episodes. Premenstrual tension of the breasts was found to be a risk factor by Gluszek (1980). In a study of 164 breast cancer and 164 control women in Oklahoma City hospitals, Chen (1978) found that early menarche, low parity (<4), and late menopause were strongly associated with breast cancer. Surgical menopause decreased risk, but uterectomy increased risk. Sartwell et al. (1978) made an extensive study of benign and malignant breast tumors with the conclusion that artificial menopause decreased risk of breast cancer, but bilateral ovariectomy probably should take place before age 40 in order to be effective (Salber et al., 1969; Vorherr, 1981; Hirayama and Wynder, 1962; Feinleib, 1968; see MacMahon, 1973). Of the many factors now implicated in the etiology of breast cancer, marital status was the first to be recognized. It dates back almost 300 years to Ramazzini’s observation (1700) that cancer of the breast occurred more frequently in nuns than in other women. He attributed the increased risk to celibacy (Ramazzini, 1713). Later, Rigoni-Stern (1842) found the incidence of breast cancer in nuns in Verona to be about five times greater than in the rest of the population. Many studies have confirmed the association of breast cancer with the unmarried state, late marriages, or short marriages, as well as nulliparity. Zippin and Petrakis (1979) compared 45 pairs of sisters, one with breast cancer and one without. Twice as many patients (6) as their siblings (3) were unmarried. The average marriage age of patients was older (26.3 vs 22.6 years). In a study of 1155 breast cancer patients and 1000 controls chosen at random, Papolczy et al. (1978) found that 27.7% of the breast cancer patients and 19.7%of the controls were unmarried. Lilienfeld (1963)

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reported that, from New York State records exclusive of New York City, the incidence of breast cancer before age 40 was the same for never-married and married women (including those widowed and divorced), but after age 40, the age-specific incidence was higher for single women. Similar patterns have been observed in The Netherlands and in England and Wales. Lilienfeld (1963) suggested that one reason for the higher rate in older unmarried women may be that the frequency of artificial menopause is less in unmarried than in married women; thus, menopause is delayed in the never-married. Later data from the New York State registry (Janerich, 1979) show an actual lower rate in the never-married during the earlier part of life, up to age 40, with a crossover at about that age (see Fig. 2). It is suggested by Janerich that early breast cancer may be pregnancy related. The recent literature contains suggestions that pregnancy-lowered immune response may favor cancer growth. There are many conflicting reports on the effect of parity on breast cancer risk. Some conclude that an early first full-term delivery decreases risk; others find that early delivery increases risk or has no effect. Moreover, the effect of parity seems to depend on when and

AGE A T DEATH

FIG.2. Average annual age-specific death rates from cancer of the female breast for single, mamed infertile, and married fertile women, England and Wales, 1948-49. 0-0, Single; x -.- X , married infertile; 0---0,married fertile. Reproduced with permission from Lilienfeld (1963).

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how many births take place, and even on the sex of the child. Miscarriages and abortions are reported to have a different effect from fullterm deliveries, and all of these variables seem to influence the stage in life that a woman’s risk is increased or decreased. In a case-control study of 1868 breast cancer patients and 3391 control patients, Paffenbarger et al. (1980) found that late marriage and delayed first childbirth increased risk. In collecting data on more than 30,000 women (over 4000 were nuns) being screened for cancer at the Cancer Institute of the University of Louvain, Maisin et at. (1978) calculated the following approximate relative risk factors for breast cancer: 3.22 for menopausal vs nonmenopausal, 1.74 for breast cancer heredity vs no breast cancer heredity, 1.74 for without children vs with children, 1.71 for 4 or less vs more than 4 children, 1.45 for early (12 years) vs late (15 years) menarche, 1.22 for high education vs low education, 1.17 for breastfeeding vs no breast-feeding, and 1.16 for first delivery after 25 years vs first delivery before 20 years. These investigators found that both pre- and postmenopausal women without children were at higher risk and that after age 65, the risk increased rapidly while for women with more than four children, the relatively lower rate actually declined appreciably after age 65. It should be noted that the above data indicate that breast-feeding increased risk. Choi et al. (1978), in a study of 400 breast cancer patients and 400 matched controls in Canada, concluded that early menarche increased risk of postmenopausal breast cancer, late menopause increased risk after age 70, late age at first pregnancy had no influence, pregnancy of only 4 months or less increased risk, irregular periods increased risk, and weight and height were not factors except for women age 70 or over, where weight increase at menopause seemed to increase risk. They also concluded that the breast cancer group was less fertile and speculated that estrogenic stimulation without sufficient cyclic progesterone secretion may provide a favorable setting for the disease. Miller et al. (1980) have collected parity data on 11,127 ever-married women from the provincial cancer registers of Alberta, Manitoba, and Saskatchawan, Canada, and conclude that there is a consistent reduction of risk associated with four or more births except for those aged 20-34, where there was a suggestion of an increased risk. However, controlling for age at first pregnancy confirmed the hypothesis that age at first pregnancy rather than parity is the relevant factor for breast cancer. They also point out that parity is associated with socioeconomic status, but doubt that this is a factor in these prairie provinces.

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Juret et al. (1974) found that parous women present breast cancer at a significantly earlier age, that early menarche was associated with a more unfavorable prognosis (1980), and that a male first child was associated with a more favorable prognosis (1978). Janerich et al. (1978)suggest that an early first child of either sex has a protective effect against breast cancer occurring in the age range 3545 years, but only a male child gives protection against presentation at age under 35 years. In a survey of 216 breast cancer patients and 31,453 controls in Iceland, Tulinius et al. (1978)reported a linear increase in risk during the time span between menarche and first pregnancy and suggested that the breast tissue is particularly susceptible to causing and preventing influences during this period. Burns et ul. (1979), in a study of reproductive factors in Northern Alberta, Canada, found that although 15 years between menarche and first delivery was associated with increased risk over nulliparity, only in the postmenopausal group was less than 5 years protective. Menarchal age of itself, number of deliveries, early natural menopause, or early oophorectomy had little effect on breast cancer risk. Craig et al. (1974), in their Washington County, Maryland, survey of 134 breast cancer women and 260 controls, found no protective effect of childbearing prior to age 20. In an analysis of 236 records of breast cancer patients and 936 controls from the Utah State Cancer Registry, Hunt et a2. (1980)found that a late first delivery was associated with increased risk of breast cancer and that there was a strong interaction between age at first delivery and age at last delivery. The association of late age at first delivery with breast cancer was strongest for women whose age at last delivery was younger than 35 years, even after adjustment for parity. However, if age at last delivery was older than 35, age at first delivery had no effect. The total number of births was not significantly associated with incidence, but a late last delivery was associated with decreased risk. Wynder et al. (1978),however, found no effect of age at last delivery on breast cancer risk, but did find that late age at first delivery increased risk of presentation during the pre- and perimenopausal periods. Postmenopausal risk was not affected. In a case-control study in Finland of 122 breast cancer patients and 534 controls between ages 41 and 60, Soini (1977)reported that a first marriage between age 20 and 24 gave a relative risk of 1.5 and a first marriage at age 25 or after approximately doubled the risk over that for a first marriage under age 20. Soini also found a similar increase in risk with age at first delivery between ages 25 and 35 and a decrease in risk

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with the number of births, with the greatest decrease occurring between the second and third births. In another study at the University of Pennsylvania, Rosenwaike (1980) concluded that marriage before 20 decreased risk. Members of the University Department of Obstetrics and Gynaecology, Bristol Maternity Hospital, Bristol, England (University Dept., 1981) report that after a single pregnancy there are distinct histological and physiological changes in the breast. There is a decrease in the number of “clear” cells and an altered response to hormones. Menstrual cyclic variations in DNA and IgA synthesis are found only in parous women. This was shown in vitro with breast tissue taken at different days during the cycle. For parous women, the synthesis of DNA and IgA was found to be maximal during the luteal phase. Plasma concentrations of estrogen and progesterone were similar in both parous and nulliparous women, but pregnancy permanently altered the hormonal responsiveness of breast epithelium, and a carcinogenic effect of estrogen unopposed by progesterone on the breast is hypothesized. Prior to first pregnancy, a woman has an inadequate number of endogenous progesterone receptors, so that the breast tissue may, in effect, experience unopposed estrogen and thus be more susceptible to factors that may initiate breast cancer. From data on 164 breast cancer patients and 164 matched controls, Chen (1978)concluded that early menarche, low parity, and late menopause strongly increased risk. Surgical menopause decreased risk, while premenopausal noncastrating hysterectomy increased risk. Age at presentation was positively correlated with age at menopause for postmenopausal women. Some investigators have suggested that some risk factors are more influential on early (premenopausal) and others on later (postmenopausal) breast cancer. Those factors sometimes associated with premenopausal breast cancer are family history, high socioeconomic status, early age of menarche, high parity, and early parity. Those associated with the postmenopausal disease are late age at first marriage, nulliparity, more than 15 years between menarche and first delivery, late natural menopause, abortions, breast-feeding, and weight-height index. De Waard (1979) suggested that premenopausal breast cancer might be influenced by ovarian estrogens and postmenopausal by adrenal estrogens. Juret et al. (1980) state that early menarche and nulliparity or a first delivery at 23-29 provide a more favorable prognosis and that there is a marked decline in prognosis with multiparity, particularly after five deliveries. Although there is an abundance of literature on the influence of age

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of menarche, marriage, first delivery, menopause, and number and sex of children on breast cancer etiology, it is difficult to determine the magnitude of these influences. The final risk factor probably depends on interactions among all of these as well as many other variables, including genetics, diet, and psychosomatic influences. Without knowing all of the variables and the direction and magnitude of each one’s influence in an individual person, it is at present impossible to conclude the significance of any factor. IV. Breast-Feeding

Since the natural function of the mammary gland is to supply nourishment to offspring, it is logical to suspect that suppression of this normal function might be a factor in the etiology of mammary gland cancer, Over the years, in spite of no real confirmatory evidence, the impression developed that women with breast cancer had nursed less than controls. In 1960, Wynder, Bross, and Hirayama reported on a study of 632 breast cancer patients and 1253 control patients at the Memorial Hospital in New York City. They found no appreciable difference among those who never nursed (22% of the breast cancer patients and 23% of the controls). There was also no significant difference in the total time that members of the two groups nursed. The most extensive studies of the effect of breast-feeding were carried out during the 1960s by MacMahon and associates, who collected data on more than 4000 breast cancer patients and three times as many controls in seven areas of the world with widely differing incidences of breast cancer. To avoid inadvertent selection, a large percentage of the total breast cancer population was included in the survey in each area. The results of these surveys as reported by MacMahon et al. (1970a,b) showed that “even in areas where some women had lactated for a total of five years or more, such women occurred proportionally no less frequently among breast cancer patients than among unaffected women,” and that “it is unlikely that breastfeeding has any protective effect against breast cancer in women.” Haagensen et al. (1981) report that in their records of 981 women with breast cancer and 1371 without, 31.8% of the former nursed for an average of 4.4 months, and 28.0% of the latter nursed for an average of 3.1 months. More of the breast cancer women breast-fed for longer periods than the controls. Soini (1977), who studied 122 breast cancer patients and 534 controls in Finland, and Kalache et al. (1980), with 707 breast cancer

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patients and 707 controls in England, found no difference in breastfeeding among the different groups. Ratner (1980) found that breastfeeding for more than 6 months decreased risk and attributed the effect to the prolonged amenorrhea. Fernandez-Cid et al. (1980), with 343 breast cancer patients and 343 controls in Spain, concluded that short or artificial lactation was a risk factor. Some investigators have concluded that breast-feeding increases risk. Chapliuk (1978), in a survey of 189 breast cancer patients and 137 healthy control women in Russia, found that 28.9% of the former, but only 17.5% of the latter, had lactated for more than 1year. De Nigris et al. (1981), in a statistical analysis of “some elements in a mammography archive” in Ancona, Italy, found that when compared with healthy women or those with other diseases, the breast cancer patients distinguished themselves by, among other things, an increase in breast-feeding time. Salber et al. (1969), in their Boston, MA study, also found an increase in risk associated with breast-feeding. Many reports mention breast-feeding along with other data in breast cancer etiological studies, but its influence on the disease seems to be insignificant. Possibly there is some synergistic influence, but from available data it is not strong enough to establish the direction. V. Contraceptives

Almost all of the reports in the recent literature on contraceptives and breast cancer etiology deal with oral contraceptives (OC).In a prospective study of 1545 30- to 49-year-old OC users in northern Alberta, Canada, Lees et al. (1978) found some support for the theory (see Fasal and Paffenbarger, 1975) that preexisting subclinical breast cancer is promoted by OC after several months of use; the malignancy is identified only in ensuing years. However, Lees et al. concluded that OC apparently offer protection against the development of benign breast lesions. The women studied consisted of three groups: 301 with malignant breast cancer, 696 with benign breast disease confirmed by biopsy, and 548 who had no apparent breast disease. Total time of OC use was divided into 112 months, 13-59 months, and 260 months. Relative risk of breast cancer for the three groups was calculated to be as follows: 5 1 2 months = 0.6; 13-59 months = 1.4; and 259 months = 1.0. Members of the groups were also classified as recent users (within the last 12 months) and former users (not within the last 12 months). For all recent users, the relative risk was 21, while among former users, the relative risk was 0.3 for the 112month group, 1.3 for the 13- to 59-month group, and 0.5 for the 260-

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month group. Thus, in women without prior biopsy, only those in the middle group (13-59 month OC use) were at increased risk. The risk for the other two groups was reduced. In women with prior biopsy, the relative risk was significantly increased in recent users (who were likely to be also long-term users) and in all those taking OC for more than 5 years. Fasal and Paffenbarger (1975) reported that the relative risk for breast cancer was 1.9 for 2-4 year users but was only 1.1 for long-term “ever users.” Bock (1978), from a study in Denmark, suggested that steroid contraception may increase risk of breast cancer in young, nulliparous women and women with benign breast tumors and those with a familial history. Brinton et al. (1978) found a synergistic interaction between OC and several other risk factors. Black and Kwon (1980) concluded from a New York study that OC favor the transition from precursor to invasive cancer in women whose grandmothers and/or aunts had breast cancer but impaired this step in women who had no family history of the disease. LiVolsi et aE. (1978) suggest that the long-term use of oral contraceptives protects against the forms of fibrocystic disease that are not firmly associated with an increased risk of breast cancer, but not against the premalignant forms. From a study of 163 young women (age 532) in Los Angeles County, Pike e t al. (1981) found that the use of OC before a first delivery increased risk (RR = 2.2 at 6 years of use, p < 0.01), but after a first delivery, there was no increase in risk. An abortion before a first delivery increased risk ( 2 . 4 ~p; < 0.005). Trapido (1981), in a survey by questionnaire of 97,300 married women age 25-50 years, 622 with breast cancer, in eastern Massachusetts, found that the breast cancer rate was lower (RR = 0.84) in pill users except for nulliparous women, where the RR was 2.1. Many reports seem to suggest that OC increases risk in nulliparous women and may promote the growth of malignant neoplasia, but in parous women, if there is no incipient cancer present, the pill does not appear to increase risk and may even decrease risk. No significant effect on breast cancer risk was found in association with OC use in the following reports: Wynder et al. (1978), Spencer et al. (1978), Kelsey et al. (1978), Gross et al. (1979), Zippin and Petrakis (1979), Greg1 et ul. (1979), Huggins and Giuntoli (1979), McEwan (1979), Chowdhury (1980), Bokhman, et aZ. (1981), Royal College of General Practitioners (1981), and Vessey et a2. (1981, 1982). Barrier contraception (use of condom) has been reported to increase breast cancer risk (Gjorgov, 1980).

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VI. Benign Epithelial Diseases of the Breast

In the preceding section on contraceptives, we saw that some investigators found a greater frequency of breast cancer in those women using oral contraceptives who had previously had breast biopsies, but it was concluded that only those lesions which were already neoplastic (lobular neoplasias) were influenced by the contraceptive hormones to progress to breast carcinoma. The etiology of benign breast lesions is not understood. A familial history of breast disease, exposure to X rays, mastitis, and breast injury have been suggested (there is discrepancy in reports dealing with breast-feeding). There is more than one kind of benign breast lesion (see Haagensen et al., 1981; or Ernster, 1981), but the reports in the literature do not usually adequately differentiate them. According to Haagensen et al. (1978),it may take up to 25 years after diagnosis of gross cystic disease or lobular neoplasias for a carcinoma to develop, but the increased risk is always there. Other investigators also report a long latency between fibrocystic disease and cancer, if indeed a carcinoma eventually occurs. De Sanso et al. (1980)state that the highest percentage of carcinomas occurs 10 to 14 years after initiation of fibrocystic mastopathy, and Ismailov and Bakhtina (1980) found that dishormonal hyperplasia and carcinoma develop in the majority of women 20 to 25 years after the recording of mastitis or injury. It is of interest to note that both Ismailov and Bakhtina (1980) and Haagensen et al. (1981) report that breast carcinoma associated with gross cystic disease occurs more frequently in the right breast, while extensive international data show that overall breast cancer is about 10%more frequent in the left breast. According to Coombs (1978), benign breast disease may increase breast cancer risk by as much as threefold and may represent an earlier manifestation of an abnormal hormonal milieu (Coombs e t al., 1979). Grattarola (1978) has suggested that an abnormal hormonal status is manifested by anovulary menstrual cycles, increased androgenic activity, and endometrial hyperplasia, and that these conditions are the same in fibrocystic disease as in breast carcinoma. In a study of 1441 women with benign breast disease over a 12.9year period, Hutchinson et al. (1980)concluded that their breast cancer risk was 2.1 times higher than that of the general population and was greater if both breasts were involved. Buehring (1979) compared risk factors for benign breast disease in 283 women with those risk factors known for breast cancer and found

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several to be the same, but also some to be different. There was a positive association with familial history of benign breast disease and exposure to X rays, as with breast cancer, but there was a lack of association with parity, age at menarche, and first pregnancy, and an inverse relationship with use of oral contraceptives. Intrauterine device users had a lower than expected incidence of benign breast disease. There was no association of benign breast disease with history of breast-feeding. Tokuhata (1969) reported a marked increase in breast cancer and benign breast tumors in the offspring of breast cancer mothers and speculated on a causal relationship between benign and malignant breast tumors. There was no difference between those who were breast-fed and those who were not. Ismailov et al. (1981) concluded that breast-feeding was a factor in dishormonal hyperplasia, its incidence being higher in women who did not breast-feed or fed only for short periods. In a prospective study at Vanderbilt University, Dupont et al. (1980) reckoned that postmenopausal women with benign breast disease had a two to three times increased risk of developing breast cancer over the general population in the same age group. From the above, we conclude that benign epithelial lesions can be harbingers of breast carcinoma, but from the literature, it is not easy to conclude the nature of the most risky lesions nor their relative risks. Haagensen et al. (1981) found that 7.1% of his patients with proven gross cystic disease eventually developed breast cancer and that the risk factor increase was two to three times. VII. Hormonal Factors

The various hormones taken together probably constitute one of the most significant influences in the development of breast carcinoma because it is through hormones that many of the other known influences operate. However, in this section, we will deal with studies in which specific hormones or hormonal imbalances are directly associated with breast cancer. Prolactin, a polypeptide hormone secreted by the anterior hypophysis, and a promoter of milk synthesis, is at present being implicated by several investigators as being an important factor in the etiology of this disease. Plasma prolactin was measured in over 3500 women volunteers in a normal population by Kwa et al. (1981). In premenopausal women, there was a significant decrease in prolactin levels with increasing par-

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ity. The effect was transitory, however, since the plasma prolactin concentration rose with increasing time after the birth of the last child. These results may help explain the data of Hunt et al. (1980), which showed an association of age at first delivery with age at last delivery insofar as they influence breast cancer risk. In women whose age at last delivery was over 35 years, age at first delivery had little significance in determining risk. However, if the childbearing period was short, late age at first delivery increased risk. If childbearing decreases the circulating level of prolactin, and prolactin is a promoter of breast cancer, Hunt’s data would be explained, at least in part, by the lowered prolactin level resulting from late full-term pregnancy. Rose and Pruitt (1981) found that the plasma prolactin levels of 98 healthy women aged 22 to 65 years showed a strong inverse correlation with age, but there was no correlation with age in a wide age range of 110 breast cancer patients. When the patients and controls were classified according to their menopausal status, the premastectomy and postmastectomy early (recently diagnosed) breast cancer groups had prolactin levels that were significantly higher than those of the corresponding control groups ( p < 0.001 in all cases, pre- and postmenopausal). In advanced breast cancer, however, elevated plasma prolactin concentrations were found in only the postmenopausal patients. In a study of prolactin levels in Catholic nuns and their parous and nulliparous sisters, Henderson and Pike (1981) found little difference in the plasma prolactin levels of the nuns and their nulliparous sisters, but the parous sisters had lower levels that were highly significant. The mean prolactin levels in the nulliparous women were 24 to 35% higher than those in their parous sisters. Adjusting for possible effects of age and weight did not significantly change the differences. Furthermore, the prolactin levels were not significantly different in the parous women, irrespective of the number of deliveries. These investigators concluded that the protective effect against breast cancer of an early first delivery may be mediated, at least in part, by permanently lowering the level of circulating prolactin and that the lack of further decrease with subsequent deliveries is consistent with the claim by some that additional deliveries do not further lower breast cancer risk (MacMahon et al., 1973). It should be noted that Kwa et al. (1981) found that the decrease in prolactin level was transitory and rose with increasing time after delivery. In a 24-hr luteal phase study of prolactin secretion in 13 women whose mothers had breast cancer and 13 age and weight-matched

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controls, Levin and Malarkey (1981)found an increased mean proiactin level in the subjects over the controls. In addition, dopamine infusion experiments showed that the higher risk women had a relative resistance to prolactin suppression by dopamine. However, Costa et uZ. (1981) found no association between incidence of breast cancer deaths and neuroleptic therapy, which stimulates prolactin release. A few authors have suggested that the association of breast cancer with diet might be mediated partially by way of the diet’s influence on the prolactin level (Enig et al., 1978; Henderson, 1979; Wynder, 1979; Einhorn and Gustafsson, 1981). Upon measuring the prolactin level in over 4000 normal women, Bulbrook et al. (1981) found that the relationship between hormone levels and age fits a cubic equation and suggested that a curve of that nature would generate the observed age-incidence curve for breast cancer, if prolactin were a “carcinogen.” Prolactin receptors in breast tissue specimens from 110 women with various stages of breast cancer were studied by Ben-David et al. (1981). Specific binding sites for human prolactin were tested in uitro. Although the ages of the women, their hormonal status, and stage of breast cancer varied, binding sites for prolactin were found in a vast majority of the tissues, which was in contrast to the presence of steroid receptors. The results were interpreted to indicate that there may be a higher dependency of breast cancer on prolactin than on steroids. A human breast cancer cell line, T47D, was used b y Shiu (1982) in an in oitro study of the effect of human prolactin and growth hormones. It was found that the cells did not respond to either of the hormones alone, but there was a strong synergistic action in increasing the proliferation rate when 1 pglml each of hydrocortisone (H), insulin (I), and triiodothyronine (TG) was also added to the medium, although the (HITG) alone was not effective. Ovine prolactin produced a similar effect, but nonlactogenic hormones such as ovine growth hormone or human luteinizing hormone had no effect on the T47D cells. It has been demonstrated that prolactin stimulates DNA synthesis in rats and as a result increases the effectiveness of cyclic hydrocarbons in mammary carcinoma production (Nagasawa et ul., 1976; Yanai and Nagasawa, 1976). In a later publication, Nagasawa (1979) hypothesized that one of the roles of prolactin is to create conditions in the mammary gland favorable to the action of carcinogens via its stimulation of the rate of mammary gland DNA synthesis. Nagasawa’s hypothesis is depicted in Fig. 3. It is not presumed by Nagasawa that prolactin is a carcinogen but that it can prepare the ground for a carcinogen

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Normal mammary

I

CARCINOGEN

Mammary tumors

Mammary tumors

FIG.3. Scheme showing the role of prolactin in the development of mammary tumors. Prolactin increases the frequency of the cell division of normal mammary glands so that carcinogenic agents can act on the gland effectively. Reproduced with permission from Nagasawa (1979).

and act as a growth promoter. In a recent review, Nagasawa (1981, pp. 132-133) states: At present, the importance of human prolactin in the development of human breast tissues and in neoplastic changes is unknown. The suggestion that women developing breast cancer have an exaggerated prolactin secretion has not been supported by many studies (Brennan, 1977). Northrup et al. (1945) postulated that substances from breast tissue may sometimes cause the elevated pituitary prolactin secretion instead of being a consequence of it; co-culture of normal or malignant breast tissue with pituitary adenoma tissue resulted in an increased release of prolactin into the medium. The responsiveness of breast tissue to prolactin, or possible abnormal metabolism of the hormone in non-endocrine tissue such as breast tumors, may play a key role in the development and progression of breast cancer. Furthermore, experimentally, mammary gland DNA synthesis is a limiting factor in mammary tumorigenesis (Nagasawa, 1977), and prolactin plays a primary role in this process (Nagasawa et aE., 1976). Thus, it may be plausible to protect the genesis of breast cancer by decreasing pituitary prolactin secretion during the limited period of lifetime, especially when mammary gland DNA synthesis is so high (Nagasawa, 1977, 1979).

The hypothesis that decreased pituitary prolactin secretion might reduce the mammary tumor incidence was tested in rats by Nagasawa and Morii (1981), who found that daily sc injections of 0.5 mg 2bromo-a-ergocryptine mesylate, a potent suppressor of pituitary prolactin secretion, into virgin rats for 7 weeks, beginning at 4 weeks of age, resulted in almost complete prevention of mammary tumor ap-

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pearance by 20 months of age. Only 1 in 30 rats developed a mammary tumor, while 10 of 21 controls developed mammary tumors. However, treatment given between 11 and 18 weeks of age decreased the incidence to a lesser extent-6 out of 30 rats developed mammary cancers, compared with 7 of 17 controls. In addition to prolactin, many other hormones and hormonal imbalance have been implicated in breast cancer etiology. Bulbrook (1981)has suggested several endocrine abnormalities that seem to be correlated with enhanced risk. The first is a qualitative, intermittent corpus luteum dysfunction in which progesterone deficiency leads to an estrogenic imbalance, The duration and intensity of the estrogenic action when not modified by progesterone may increase risk. The occurrence of this imbalance in adolescence or as menopause is approached might account, according to Bulbrook, for the increased risk associated with early menarche and late menopause as well as the protective effect of early first delivery. Another endocrine abnormality cited by Bulbrook (1981)is a low level of adrenal androgen synthesis, which may precede the clinical appearance of breast cancer by as much as 10 years. This abnormality has been identified in high-risk women, including patients with benign breast disease and relatives of breast cancer patients. There is also a correlation between this endocrine status and the likelihood of response to endocrine ablation. Grattarola (1978)observed that breast cancer patients had increased androgenic activity and endometrial hyperplasia. Another endocrine abnormality, according to Bulbrook’s hypothesis, involves pituitary function where some women with an increased risk show an elevated nocturnal peak in plasma prolactin concentration. Miller and Bulbrook (1980)emphasize that estrogens are the prime agents in breast tumor expression and suggest that estrogen action unopposed by progesterone during critical periods such as menarche or menopause may be an etiological factor. They note that there is an increased risk of breast cancer after a 10-15 year latency in women who have used noncontraceptive estrogens at the time of menopause. In a study of 400 breast cancer patients and 400 matched controls, Choi et aE. (1978) concluded that the members of the breast cancer group were less fertile and that estrogenic stimulation without sufficient cyclic progesterone secretion may have provided a favorable setting for breast cancer. Tramier (1979)compared the etiology of breast cancer with endometrial cancer and concluded that obesity, endometrial hyperplasia, and the contribution of endogenous and exogenous estrogens and the lack of progesterone were the most marked ele-

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ments common to cancer development in both of these organs. Furthermore, in premenopausal women, the level of nonprotein-bound estradiol (E2) is significantly higher in breast cancer patients than in matched controls, although the total serum concentrations of E2 are normal (Moore et al., 1982). This was found not to be due to low sex hormone binding-globulin binding capacities, which were within the normal range. The authors conclude that breasts of women with breast cancer may be or may have been exposed to elevated levels of biologically active E2, although the reason for this is obscure. Plasma progesterone levels were found by Berta et al. (1981) to be significantly lower in cystic breast disease patients than in controls, but estradiol (Ez) and estrone (El) levels were not significantly different. Also, sex hormone binding-globulin binding capacity (SHBG-bc) was lower in the patients. The authors suggest that, since the cystic breast disease patients are at high risk for breast cancer, the progesterone and SHBG-bc deficiency, respectively, induce a relative increase in plasma estrogen concentration that leads to an increase in proliferation of mammary epithelium. In a prospective study of 1083 women who had been treated for infertility between 1945 and 1965 at the Johns Hopkins private obstetricslgynecology clinic, Cowan et at. (1981) found that those women with a history of progesterone deficiency (the hormonal group) had a risk of premenopausal breast cancer 5.4 times that of women whose infertility was due to nonhormonal factors (nonhormonal group). The groups were comparable in terms of menarchal and menopausal age, oral contraceptive use, age at marriage, education, and history of benign breast disease. The incidence of postmenopausal breast cancer and all other cancers did not differ in the two groups. Age at first birth was not associated with breast cancer risk in either of the groups. Some sort of hormonal imbalance as a major factor in breast cancer etiology has been postulated by a large number of investigators: Korenman (1980), Bulbrook (1981), Coombs (1978), Dept. Obstetrics (1981), and Ismailov et al. (1981). Ismailov et al. (1980) studied menstrual function in 1066 women with various dyshormonal breast diseases and in 81 patients with breast cancer (61%of the patients were 31-50 years old). Dysfunction of the ovaries was diagnosed in 22.8%of the patients with dyshormonal disease and in one-third of the patients with breast cancer. The ratio of androsterone to tetrahydrocortisol, which is also an index to the aging process, was found by Kodama et al. (1977) to be significantly lower in breast cancer patients at both pre- and postmenopausal stages. This ratio was found to be highest in rural controls, followed

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by urban controls and breast cancer patients. The authors suggest that the ratio might be used as a measure of risk for breast cancer. Kiricuta and Frenkel (1982) compared some endocrine levels in patients with inflammatory breast carcinoma (IBC) with those in patients with noninflammatory breast cancer (NBC). Mean levels of urinary ethiocholanolone (Et) were lower in IBC than in NBC, and the androsterone/Et ratio was higher (1.00), and the Et/l l-oxygenated-17 ketosteroids ratio was lower (0.72) in the IBC than in the NBC patients. These and other observations led the authors to conclude that there was a greater hormonal imbalance in IBC than in NBC. I n a review of the endocrinology of breast cancer, Pineda (1980) concluded that ovariectomy causes regression in about 30% of all breast cancer cases, that gestation in advanced age favors its development, that although evidence suggests that prolactin-releasing hormone is tumorigenic, it appears that cells must be previously sensitized by ovarian hormones. Pearson and Ray (1959) and Manni et al. (1979a,b) have shown that metastatic breast cancer can in many cases be treated b y antiestrogen and antiprolactin drugs and that further improvement can be had by hypophysectomy, which indicates that both steroid and polypeptide hormones influence the growth of breast cancers. The more pronounced remission following hypophysectomy was interpreted to mean that growth hormone also plays a role. It is difficult to know how to interpret and summarize the massive literature dealing with the role of hormones in the etiology of breast cancer. Despite the major role that they play, we do not believe it is necessary to assume that hormones act as carcinogens in initiating the carcinomas, but that they are influential in the growth and expression of malignant breast tumors. They may also render the breast more susceptible to the action of carcinogens. Elevated quantities of plasma prolactin seem to be highly correlated with the expression of the disease. The lowered risk sometimes associated with late menarche, early first delivery, late last delivery, and early menopause could be accounted for primarily through the effect of these on the prolactin level. The other most influential hormone seems to be progesterone, whose plasma level seems to act in opposition to prolactin. Breast cancer is associated with lower levels of plasma progesterone. The eflect of the adrenal cortex-produced androgenic hormones and their metabolites seems to be equivocal. Estradiol and the esterol quotient do not seem to hold the interest that investigators placed on them only a few years ago. However, the

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interactions of so many hormones and their influences on so complex a development as breast cancer are not very far on the way to being understood. Perhaps certain deviations from the normal range that create unfavorable imbalances can influence the susceptibility of breast epithelium to carcinogens as well as influence the rate of malignant growth and can thus be important factors in breast cancer etiology* VIII. Cancer

A familial or personal history of cancer in any location or tissue may increase the risk of breast cancer. As mentioned in the section on heritage, cancer in one breast increases the risk of cancer in the other breast. In a review of the epidemiology of human breast cancer, Kelsey (1979) reported that tumors occur more frequently in the left than the right breast and that there is a large increase in relative risk of later having a cancer in the contralateral breast, particularly if there is a history of bilateral breast cancer in a first-degree relative. Kelsey also reported that a previous cancer of the ovary or endometrium increases the risk of breast cancer. Popova (1981)in Leningrad reports that the risk of a cancer in the second breast is increased 3-21 times over the general population and that the period of time between detection of the first and second tumors ranges from 3 months to 3 years for synchronous neoplasms and up to 29 years for metachronous neoplasms. Ricci et al. (1981), in Padova, Italy, found that of 564 breast cancer patients, 37 (6.5%) were bilateral and that mastectomized patients were at higher risk of developing cancer in the second breast, but the mortality rate was not increased. Surveys by Haenszel(l979) have shown that high colon cancer populations are high breast cancer populations. In an extensive survey of the association of endometrial and breast cancer at the Mayo Clinic, Annegers and Malkasian (1981)found that the relative risk of breast cancer after endometrial cancer was 1.3 times the general population rate, but this increase was confined to those who shared risk factors common to breast cancer-that is, nulliparity and, to a lesser extent, obesity. A study by Senie et al. (1980) of 980 patients at Memorial SloanKettering with unilateral breast carcinoma revealed a leftiright ratio of 1.26 and that when asynchronous bilateral carcinoma was documented, the disease first occurred more often in the left breast. Patients with simultaneous bilateral disease usually had a larger tumor

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in the left breast. In 174 healthy control women, the left breast was also usually larger than the right. In a review of multiple primary cancers, Newell (1980) reported that a lack of association between cancer of the cervix and breast and colon cancer suggests that this cluster is not related to hormonal and/ or dietary influences. However, as noted above, Haenszel found an association between breast and colon cancers. Newell further states that available evidence suggests a higher occurrence of multiple primary breast cancers among Hispanics than among whites or blacks in the United States. In a series of nearly 22,000 breast cancer patients, Prior and Waterhouse (1981a) found a 2.6-fold increase in the risk for a second primary tumor in the contralateral breast. When broken into three age ranges (at the time of diagnosis of the first primary) the corresponding risks were 5.3 (ages 15-44), 3.3 (45-59), and 1.5 (60+).A maximal risk of 5.0-fold was observed in the series as a whole during the third year after the diagnosis of the first primary. In a complementary analysis of 17,756 breast cancer and 4817 cervical cancer patients in England, Prior and Waterhouse (198lb) concluded that cancers of the breast and cervix are not etiologically refated. IX. Iatrogenic Factors

Ionizing radiation, oophorectomy, hysterectomy, and hormone therapy are the principal iatrogenic factors that influence the incidence of breast cancer. Radiation is known to be a most powerful carcinogen and is probably responsible for more cancers than any other iatrogenic factor. The average latent period between radiation and detectable breast cancer is long, probably averaging more than 30 years (Scanlon et al., 1978). The breast seems to be most sensitive to radiation-induced carcinogenesis in the 10- to 35-year age range. According to Land et al. (1980), risk increases linearly with dose and is heavily dependent on age when exposed. The breasts are often at high risk because X-ray examination of other organs often includes breast exposure. Ewen et al. (1980)estimate that more than 40% of all X rays are taken of the chest. In a follow-up survey of 1047 women with tuberculosis who had had multiple fluoroscopic chest examinations, 57 1 women irradiated for postpartum mastitis and 63,263 Japanese female survivors of the atomic bomb, Boice et al. (1979) conclude that risk of breast cancer increases significantly with increasing dose of radiation. Among the

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atomic bomb survivors who received more than 10 rads, 0.89%developed cancer while of those who received less than 10 rad, 0.49% developed cancer, giving a relative risk factor of 1.7 (p < 0.0001). Among the tuberculosis patients (estimated cumulative dose, 150 rads), 41 developed breast cancer compared with 23.3 expected. Among the mastitis patients, 36 of 571 (6.3%)of those irradiated developed breast cancer compared with 32 of 993 (3.2%)nonirradiated mastitis patients, giving a relative risk factor of 2.0 (p < 0,001). The average cumulative dose for those irradiated was 377 raddbreast and 247 raddpatient. Risk increased significantly with increasing dose (p < 0.00002). Fractionation did not seem to diminish the radiation risk, nor did time since exposure (45 years of observation). According to these investigators, the best estimation of risk among American women exposed after age 20 is 6.6 excess cancers per lo6 womanyears-rad. In examining the questions regarding tumor induction through routine mammography, Barke et al. (1980) offer the following advice: “because of the high radiation sensitivity of soft tissue, mammographies should be performed on young women only when the indications for this procedure are very marked.” According to Land (1980),excess risk is approximately proportional to dose and independent of ionizing density and fractionization of dose. Synergism is important. Radiation prior to menarche is associated with higher risk than radiation Iater (Tokunaga et al., 1979). Scatter radiation used to treat leukemia or Hodgkin’s disease may also affect the breasts. Li et al. (1981) reported that breast cancer developed in two sisters 4 years and 11 years, respectively, after Hodgkin’s disease was diagnosed and treated. Since a third sister and five relatives in the paternal line also had breast cancer, it was suspected that there may have been a synergistic action of genetics and radiation, although cytogenetic and HLA studies showed no markers of susceptibility to neoplasia. A synergism between radiation and diethylstilbestrol (DES) was found by Segaloff and Pettigrew (1978). In experiments on rats, X-ray radiation and/or diethylstilbestrol (DES) werelwas used. The combination of radiation and DES always produced a response that was greater than the sum of the response of DES alone, plus response to radiation alone. Malignant mammary tumors were relatively rare in groups that did not receive DES, and the optimal amount of synergism was dependent on the amount of radiation given. In experiments where the DES was given at various time intervals after the radiation,

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the synergistic interaction between the two did not decline with the time interval, indicating that the effect of radiation was not subject to repair . The effect of hormone therapy has been reported by many investigators. In a 25-year follow-up study of 693 mothers who had taken DES during pregnancy (12 g or less for 15-20 weeks) and 668 who had not, Bibbo et al. (1978) found breast cancer in 4.6%of the DES group and 3.1%in the control group, but the difference was not significant. The onset of the neoplasm was at slightly earlier age, and there were more deaths from breast cancer in the DES group, but these differences were not significant. However, in a period longer than 25 years, the differences may have become significant, as was found by Ryan (1978). Ryan’s group was exposed to 12 g or less for 15-20 weeks. In an extensive review of the role of exogenous female hormones in altering the risk of benign and malignant neoplasms, Thomas (1978) found no conclusive evidence that they influenced breast cancer. Also, Hulka (1980) reported no association between breast carcinoma and use of estrogens in treatment of postmenopausal women. However, conjugated estrogens were found to increase risk of breast cancer by Hoover et al. (1981). In a study of 345 women with breast cancer and 611 controls, the relative risk was found to be 1.4; 1.3 for menopausal women with intact ovaries and 1.5 for those with ovaries removed. Dose-effect was significant. The relative risk rose to about 2 for women with 10 or more prescriptions, with 5 years or more of use, and for those with a daily dose of 1.25 mg or more. There was also a synergistic effect for those women who had a family history of breast cancer. Brinton et al. (1981), in a study of 881 breast cancer cases and 863 controls, found that use of estrogens was associated with a relative risk of 1.24, with higher risks associated with users of high-dose preparations. Following bilateral oophorectomy, the RR was 1.54. RR was also higher in nulliparous women and those with a family history of breast cancer or benign breast disease. Use of estrogens in treatment of prostate cancer increases risk of breast cancer (Sobin and Sherif, 1980). Hawkins (1980)theorized that estrogen is probably involved in breast cancer in two main ways: (1) in the etiology of the disease as a promoting substance rather than as a carcinogen, and (2) in stimulating the growth of at least some breast cancers, acting through the estrogen receptors. Successful modes of endocrine therapy would then be interpreted as interfering with (1) the suppIy of estrogen to the tumor, ( 2 )the nuclear events in estrogen action, or (3) receptor replenishment.

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In connection with the effect of other drugs on breast cancer, Kewitz et al. (1980) found no increased risk with the long-term use of reserpine in the treatment of hypertension in postmenopausal women. In a study of 179 breast cancer patients, Stenkvist et al. (1980) noted that those who had been treated with glucosides for heart conditions had smaller tumors with less metastases, smaller cells with more monotonous morphology, and that the nuclear DNA-RNA content was lower. The authors concluded that the heart glucosides or their metabolites were able to interfere with the receptors of cells from breast cancer to lower their biologic potential. In a study of the use of thyroid supplements in relation to risk of breast cancer, Shapiro et al. (1980) compared 659 breast cancer women with 1719 controls whose rates of use of thyroid supplements were 9.1 and 8.7%,respectively. The study gave no evidence for increased risk even when the thyroid supplements were taken for more than 15 years. Although not an iatrogenic factor, hair dye has been implicated as a causative factor in breast cancer (Shore et al., 1979). A survey of 129 breast cancer patients and 193 controls who had attended a multiphasic screening center in New York between 1964 and 1976 showed that hair dye use for 10 years or more before cancer diagnosis was significantly related to the disease, suggesting that hair dyes act at some early stage of carcinogenesis and that the latent period is long. Also, the strongest association was in women more than 50 years old. In another survey (Nasca et al., 1980), a statistically significant increased risk of breast cancer was found for women with a history of benign breast disease (BBD) and exposure to hair dye as compared to women with BBD and no exposure to hair dye. The relative risk was calculated to be 4.5. The highest association between hair dye and breast cancer was for women in the age range 40-49 years, and there was a highly significant ( p = 0.0008) dose-response relationship among women who used dyes for changing the natural color as opposed to coloring gray hair. The authors caution that the number of patients (118) and controls (233) was small and that further studies are needed before firm conclusions can be reached. X. Immunological Factors

Low immunologic capacity is believed by many investigators to play an important permissive role in the development of cancer in general and of breast cancer specifically. The increased susceptibility

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to cancer with age is often associated with the decreasing immunologic capacity. “The best-known landmark in the programmed involution of the immune system with age is the involution of the thymus, which begins to occur at the onset of adulthood and is under hormonal control, most likely regulated by the central nervous system” (Good, 1980). The predominant immunologic deficiencies seen in the aging process are concomitants of T-lymphocyte attenuation. According to Herberman (1978), altered immune functions such as depressed T cells and other lymphocytes, lymphoproliferative responses, and delayed hypersensitivity reactions lead to increased risk. Moreover, it is possible that the protective effect of early first fullterm pregnancy is augmented by a supply of fetal antigens, a sort of natural vaccination which produces a change of state that discourages the inception and growth of tumors (Medawar and Hunt, 1981). Several serum protein levels in women with early (preoperation) breast cancer when compared with healthy matched controls were found to be low by Thompson et al. (1981).The ratios of the percentage of the breast cancer women to controls with lowered serum protein levels for the following components were albumin, 68%/4.7% (p < O.OOOl), IgG, 56%/21% (p < 0.02), IgM, 59%/19% ( p < 0.001), and transferase, 50%/4.7% (p < 0.0001). Using these measurements of serum proteins, the authors were able to assign degree of risk of malignancy for women found to have a breast mass. In another study of serum immunoglobulins in 120 women with breast cancer, a strong association between age at tumor diagnosis and serum IgA levels was found (Papatestas et al. (1979). A lowered serum level of IgA was associated with the appearance of tumors early in life and with a poor prognosis. The association was even greater if the patient had a family history of breast cancer. Also, there was a strong association between levels of IgG and parity; parous women and/or those who had a good prognosis had a higher level of IgG, compared to nulliparous women and those with poor prognosis. The authors suggest that immunoglobulin levels can be used to help identify women with a high risk of breast cancer and a poor prognosis. In a recent report by Lamoureux et al. (1982), profiles of serum protein levels were used to monitor disease stage and prognosis of 207 patients with breast cancer. Six of the serum proteins were found to be significantly elevated. They were a1-antitrypsin (a1AT), az-ceruloplasmin (cp), pl-transfemn, IgA, and complement components 4 and 5 (C4) and (C5).Seventy-two percent of the patients had at least two of

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these proteins elevated, of which q-AT (55%), C5 (38%),and IgA (36%)were most commonly raised. The number of elevated proteins was parallel to the progression of the disease. There was also a positive correlation between the number of elevated proteins and the prognosis of the patient. As a group, patients with no or only one protein elevated had a better prognosis than patients with two or more elevated. The authors conclude that these findings indicate that the high level of these serum proteins, reflecting an abnormal biochemical profile, provide valuable information concerning the stage of the disease and the prognosis of the patient. To summarize the above three papers by Thompson et al., Papatestas et al., and Lamoureux et al., reporting measurements of serum proteins in patients with breast cancer or with poor prognosis or in women at higher risk, it appears that there is agreement in associating these conditions with decreased level of albumin, IgA, IgG, and IgM, and transferase and increased level of al-antitrypsin, crz-ceruloplasmin, PI-transferrin, and complement components C4 and C5. There seems to be some discrepancy, however, concerning the role of IgA, with Papatestas et at. concluding that elevated IgA is associated with decreased risk, while Lamoureux et a1. conclude that elevated IgA, in association with the elevation of other serum proteins, predicts a poorer prognosis. Probably the level of IgA as well as some of the other serum proteins is reflecting balance or imbalance in organ functions and general state of health rather than measuring the status of a specific disease. Eilber and Morton (1970) found that a simple skin test could be helpful in determining the immune status of a patient and in predicting survival time. They found that patients with a positive skin test reaction to 2,4-dinitrochlorobenzene (DNCB) at the time of surgery showed a significantly decreased short-term recurrence rate when compared with patients with a negative DNCB response. With long follow-up periods, &own et al. (1980) have studied DNCB reactivity and its relation to survival and other measures of the severity of the disease in 202 breast cancer patients. When the survival distributions for all DNCB-positive and negative patients were compared, the DNCB-positive patients showed a signifiantly longer survival. However, if only advanced patients were compared, there was no difference in response rate or survival in DNCB-positive and negative patients. Moreover, other tests of immune function, including intradermal skin tests with microbial antigens, absolute lymphocyte counts, and lymphocyte responses to mitogens in vitro were not use-

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ful in distinguishing prognostically favorable groups among patients with advanced disease. These authors concluded that “although DNCB reactivity is progressively impaired in patients with increasing tumor burden and correlates with survival in breast cancer patients in general, in our experience, such tests do not provide prognostically important information above that given by careful clinical and pathological staging.” A breast cancer-associated antigen that has received considerable interest is the Thomsen-Friedeurich (designated as T) antigen. It occurs in normal tissues, but is masked by a carbohydrate so that it is unavailable to react with antibody. By treatment of red cells with sialidase, it can be made available in uncontaminated form. However, in human breast cancer tissue, it occurs naturally in the unmasked form and can be readily detected in blood serum (Springer et al., 1974),and breast cancer patients mount a profound humoral and cellular immune response against this antigen (Luner et al., 1977; Springer et al., 1980; Vos and Brain, 1981). It has been found by the last that producers of high levels of agglutinins for sheep red blood cells, whether cancer patients or not, also more often produce high levels of antibodies to T-antigen of human red blood cells. The only significant difference is that cancer patients have persistently depressed levels of anti-T, which provides further evidence that malignant tissues may have exposed T-antigen structures on their membranes. In order to test the hypothesis that a failure in the host’s immune system is responsible for or is a factor in the rapidly progressing breast cancer (RPBC) found in Tunisia (see section on Heritage), Levine et al. (1981)performed in vivo and in vitro assays of cellular immunity in Tunisian patients. Tests of delayed hypersensitivity using microbial antigens and in vitro studies including lymphocyte transformation tests and measurements of B and T cells indicated that the RPBC patients had response comparable to non-RPBC patients. The authors conclude that although there was some elevation in the frequency of blood group A in the RPBC patients, there was no specific RPBCassociated antigen and that this rapidly progressing breast cancer was not a reflection of immunodeficiency. A similar conclusion had been reached by Mourali et al. (1978). In theory, the immune system should be an important element in breast cancer etiology, and much effort has gone into experiments in both humans and animals in attempts to show the effectiveness of either cellular or humoral mechanisms. To date, however, it is difficult to find convincing evidence of their effectiveness.

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XI. Viral Aspects of Human Breast Cancer

Earlier reports, which will not be reviewed here, have shown that there exists in human breast carcinoma tissues and in milk entities which have many of the biophysical, biochemical, or immunological features characteristic of RNA tumor viruses, or components of them, and that many of these features can be found in murine mammary tumor virus (MuMTV). By means of an indirect immunoperoxidase technique, MesaTejada et al. (1978) demonstrated that an antigen immunologically related to a glycoprotein (gp52)of the mouse MTV can be identified in sections of human breast cancer. Later, Ohno et al. (1979)carried out experiments to show that this cross-reactivity was due to the polypeptide rather than the polysaccharide component of gp52. More recently, Keydar et al. (1982)have reported the presence of an antigen in cells of 128 of 204 (62.7%)human breast carcinomas that is immunologically related to the MuMTV envelope protein, gp52, and that this antigen is an indicator of the severity of the disease. The antigen was found in Stage IV carcinomas much more frequently (80%)than in Stage I (15%).Also, a significantly higher percentage of antigen-containing tumors was found among Israeli women born in North Africa than among those of European origin. Moreover, women whose Stage I1 carcinomas were positive for the antigen at the time of mastectomy usually had an unfavorable prognosis. In a study of human male and female mammary carcinomas at Memorial Sloan-Kettering Cancer Center, Lloyd et al. found positive reactions with gp52 antiserum in almost all (32 of 36,89%)carcinomas from male patients, but in only 14 of 50 (28%)carcinomas from female patients, and not in any normal breast tissue. The gp52-positive tumors were larger (significantly so in females), and the presence of this antigen seemed to be related to major prognostic factors. Other studies by Witkin et al. (1981) concluded that many breast cyst fluids contain IgA and IgG that cross-react with MuMTV. Specificity of the cyst fluid IgA for MuMTV was determined by comparing its binding to MuMTV B-type particles and to Rauscher leukemia virus (R-MuLV)C-type particles. Twenty-three of 40 breast cyst fluids that contained IgA reacted with MuMTV, and 12 of the 23 were also reactive with R-MuLV, while the remainder (11 of 23) were specific for MuMTV. This was taken to mean that there are at least two classes of IgA in breast fluid, one reactive with both B- and C-type viruses and the second reactive with only type B murine virus. The reactivity was detected by an enzyme-linked immunoabsorbent assay that measured

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antibody binding to both the envelope glycoprotein and the core protein of the virus. [Hanson (1982) claims that IgA is the dominant antibody of breast milk and suggests that cells which synthesize IgA are sensitized in the Peyer’s patches of the gut and are transported via lymph and blood to the breast, where they produce IgA during lactation.] Fractions with the buoyant density of retroviruses (1.16-1.18 g/ml) or their cores (1.21-1.25 g/ml) were isolated by Witkin et al. (1981) from breast cyst fluids. These fractions contained a DNA polymerase capable of using the same reverse transcriptase transcription-specific template (dClSl8 poly rCm)that is best used by MuMTV polymerase. Furthermore, the human cyst fluid fractions reacted with antiserum to MuMTV core component p28 but not with antiserum to R-MuLV component p30. Using the enzyme-linked immunosorbent assay (ELISA), Day et al. (1982) have shown that African breast cancer patients from Kenya exhibit the highest titers and the highest frequency (66%) of antibodies reactive with MuMTV. About 20-25% of American and Parsi (see section on Heritage) women with breast cancer also have MuMTV reactive antibodies. In marked contrast, sera from only 4% (1of 25) of breast cancer patients from the People’s Republic of China possess this antibody. The incidence of MuMTV-reactive antibody in American patients with benign mammary disease or colorectal cancer or in healthy females is 5-10%. Studies of Parsi and American families with high incidences of breast cancer revealed that apparently healthy family members frequently possess antibody reactive with MuMTV. Furthermore, aspirated breast cyst fluids from women with benign breast disease contained a particulate component (BCF-PC) that reacted with antiserum to MuMTV, and serum IgA from breast cancer patients was significantly more reactive with BCF-PC than was IgA from other categories, indicating that production of a specific IgA antibody to the BCF-PC increases in sera of patients with breast cancer. Tomana et al. (1981)found antibodies to MuMTV-related antigens in sera of patients with breast cancer. Using an indirect immunofluorescence procedure, these investigators found positive reactivities in the sera of 56 of 137 (40.9%) breast cancer patients, 5 of 27 (18.5%) patients with benign breast disease, 7 of 60 (11.7%) patients with neoplasms other than breast cancer, and 2 of 56 (3.6%) female controls. Immunochemical analyses indicated that the antibody activity was due to IgC and IgM. The antibodies could be removed from the human sera by preabsorption with disrupted MuMTV but not with the MuMTV polypeptides gp52 or gp34 or with Rauscher murine leuke-

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mia virus. In the breast cancer group of patients, a positive correlation was noted between age and the probability of detection of these antibodies (running from 18% in the 30- to 40-year age group ,to 50% in the 70+ year age group), but there was no correlation between the reactivity of patients’ sera and the stage of the disease. There was, however, a significantly higher incidence of antibody in patients with infiltrating ductal carcinoma of the mucinous type than in other histopathological types. Kryukova et al. (1981), using a highly sensitive electroimmunodiffusion method, found in two of five breast cancer extracts a protein of about 50,000 molecular weight that exhibited an antigenicity related to the major core antigen of MuMTV (p27). This antigenicity was not detected in the patients’ sera. Lopez et al. (1981) presented data demonstrating that lymphocytes from patients with metastatic breast cancer have markedly enhanced responsiveness, in blastogenesis assays, to MuMTV antigens as compared to lymphocytes from normal donors and that the responder cells can be separated with techniques used to purify lymphocytes of T-cell lineage. The enhanced blastogenesis, although not restricted entirely to breast cancer patients, was assumed to mean that the responding lymphocytes had been previously sensitized within the breast cancer patient to MuMTV-like antigen or possibly cross-reacting oncofetal proteins. Studies during the early 1970s have shown that RNA-dependent DNA polymerase activity (reverse transcriptase) occurs in human milk and breast cancer tissue. More recently, Kantor et al. (1979) reported on the purification of a DNA polymerase from human milk that resembled the reverse transcriptase of RNA tumor viruses. Viral reverse transcriptase could be distinguished from the various cellular DNA polymerases by their synthetic primer-template specificities. The enzyme purified from human milk was devoid of terminal transferase activity and was not immunologically related to human DNA polymerase a,p, or y. However, these investigators conclude that the viral origin of this human milk enzyme could not be established. Vogel and Chandra (1981) purified and characterized two forms of reverse transcriptase from the placenta of a patient with breast cancer. Although reverse transcriptases have been purified from tumor tissues by numerous investigators (Chandra and Steel, 1980; Ebener et al., 1979; Ohno et al., 1977; Poiesz et al., 1980), Vogel and Chandra and other investigators (Lowenstein et al., 1980) were unable to find similar enzymes in normal placentas. The significance of reverse transcriptases in the placenta of a breast cancer patient and their rela-

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tionship to the disease of breast cancer is at present not understood, although it is an interesting finding. Biologically active cellular DNAs of various tumors can induce oncogenic transformation of various cells by a process called transfection. The infecting cellular DNA is known as cellular transforming genes. DNAs of various tumors, particularly those transformed by virus, can induce transformation with high efficiency. For a review of transforming genes, see Cooper (1982). Lane et al. (1981) demonstrated that transforming genes contained within high-molecular-weight DNA from both mouse and human mammary carcinoma cells can induce cellular (NIH 3T3 mouse cells) transformation. Although transforming genes have generally been associated with oncogenic viruses, these were not. The mouse transforming genes were from either MuMTV-induced C3H mammary carcinomas or chemically (dirnethy1benzanthracene)-inducedBALB/c CRGL transplantable mammary carcinomas and the human transforming genes were from the human mammary carcinoma cell line MCF-7. Since the BALBlc CRGL mice have no demonstrable virus and the malignancy was chemically induced, it was concluded that the transmissible transforming DNA was not virus related. Furthermore, the NIH mouse cells transformed by DNAs of the MuMTV-induced tumors did not contain exogenous MuMTV DNA sequences. This indicated that MuMTV-induced mammary carcinomas contained active cellular transforming genes that were not linked to viral DNA. Further studies by Becker et al. (1982)resulted in the identification of an 86,000-dalton glycoprotein antigen that was associated specifically with the transforming genes of mammary carcinomas-both human and mouse. Sera from tumor-bearing mice immunoprecipitated the glycoprotein from extracts of NIH cells transformed by human mammary carcinoma DNA and by mouse mammary carcinoma DNA, and by DNAs from mice bearing primary mammary carcinomas, but sera from mice bearing many kinds of tumors except mammary carcinomas did not precipitate the glycoprotein antigen. It was not immunoprecipitated by extracts of NIH 3T3 cells, spontaneously transformed cells, NIH cells transformed by normal human DNA, NIH cells transformed by human bladder carcinoma DNA, nor by NIH cells transformed by Rous sarcoma virus DNA. The authors conclude that this glycoprotein antigen is specifically associated with expression of the transmissible transforming genes of human acd mouse mammary carcinomas. Although there are numerous reports of associations and cross-reac-

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tivities of MuMTV-related components and human breast cancer entities, we believe there is as yet no real basis to conclude that there is a virus, as in the mouse, involved in the etiology of human breast cancer. [The extensive literature before 1979 on this subject was reviewed by Moore et al. (1979).] There is at present no evidence for contagion or of transmission of a breast cancer virus via breast milk, although possible involvement of a MuMTV-related component in the etiology of some human breast cancers cannot be ruled out. XII. Dietary Factors

At the close of a conference on nutrition in the causation of cancer held at Key Biscayne, Florida, in 1975, Wynder, in his concluding remarks, stated that very few laboratories were concerned with the area of nutrition and cancer. Since 1975, however, there has been a noticeable swing in scientific interest toward dietary investigations. Publications dealing with the effect of nutrition on the etiology of breast cancer are appearing probably faster than in any other area dealt with in this review. The effects of overnutrition, high fat intake, and a wide range of dietary supplements are now of great concern to many breast cancer investigators. It is argued that overnutrition in early life causes rapid growth that results in early menarche, which in turn increases breast cancer risk, and that overnutrition and high fat consumption in later life results in breast cancer-promoting hormonal imbalances. Dickerson (1979), in a review on nutrition and breast cancer, states that the plasma of postmenopausal patients with breast cancer contains higher concentrations of total lipids, phospholipids, cholesterol, and lipase activity than that of age-matched women with cancers of other sites. Hirayama (1978) found a high positive correlation between per capita fat intake and adjusted breast cancer death rates in different districts in Japan and noted a close correlation with consumption of pork. Miller (1977) compared total caloric intake, total fat, saturated fat, oleic fat, linoleic acid, and cholesterol consumed by 309 breast cancer patients and the same number of matched controls. The intake of all of these food components by the cancer group was higher-the first three (total calories, total fat, and saturated fat) being significantly higher ( p < 0.05). The trend of the data was consistent for both premenopausal and postmenopausal women, except for those over age 70. For this small group (only 9), the breast cancer women consumed less. In a later report, Miller (1978) states that for both pre- and post-

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menopausal women, the relative risk for elevated total fat intake is approximately of the same order-the risk ratio being 1.6 for premenopausal and 1.5 for postmenopausal women. In order to test the hypothesis that diet can affect hormonal balance, Hill et al. (1980) measured plasma hormone levels in black (Bantu of the Transkei) South African women who had always maintained a strict vegetarian diet and distinct lifestyle and compared these levels with those of white (Caucasian) South African women who maintained a Western diet and lifestyle. After a hormonal baseline was established, the diet of black volunteers was switched to the Westerntype diet, consisting of meat, butter, milk, eggs, bread, and sugar. Blood was drawn every second day during a menstrual cycle before and after the diet change. Plasma testosterone, prolactin, and folliclestimulating hormone levels increased, while estradiol decreased in the black women after the diet change. The authors conclude that the hormonal changes in these women are concordant with hormonal changes found in women with anovulatory cycles, and that anovulatory cycles are associated with increased breast cancer risk (Bulbrook et al., 1978). In other experiments where North American women were put on a vegetarian diet, plasma prolactin and the nocturnal release of prolactin were decreased (Hill et al., 1981). A relationship of breast cancer mortality to diet rich in meat, fat, and sugar was found by Ingram (1981),who studied the trends in England and Wales for the period 1928-1977. With the onset of World War 11, there was a marked reduction in both breast cancer mortality and the intake of sugar, meat, and fat, but when consumption of these foods returned to pre-war levels by 1954, the breast cancer mortality did not rise to previous levels for some 15 additional years. Schoental (1981) has suggested that dietary fats may influence the incidence of breast cancer through their adventitious contamination with fusarium mycotoxins since the production of these mycotoxins is favored by wet, cool climate, and high breast cancer incidences are often found in such climates. Lubin et al. (1981) compared the diet of 577 breast cancer women aged 30 to 80 with 826 disease-free women selected from the general population in northern Alberta, Canada. Dietary components associated with increased risk were beef and pork, and those associated with decreased risk were cream and eggs. Gaskill et al. (1979)also reported a strong negative correlation of egg consumption with breast cancer, and suggest that egg consumption modifies the fecal flora environment in a direction unfavorable to the production of carcinogens and note that cancers of the intestines, colon, rectum, and uterus are also

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negatively correlated with eggs. On the other hand, Gaskill et al. find a positive correlation of breast cancer mortality with milk and milk product consumption as well as with the genetic ability (lactase sufficiency in adulthood) to digest milk. It is not understood why eggs and milk should have opposite effects on breast cancer etiology. However, the protective effect of high egg intake seems to be stronger. Enig et al. (1978) have challenged the relationship of dietary animal fat to cancer causation. Their extensive review of fat consumption and the sources of fat indicate that the per capita use of animal fat in the United States had declined from 104 g/day in 1909 to 97 g/day in 1972, while vegetable fat intake has increased from 21 to 59 g/day. According to American Cancer Society data, breast cancer death rate in the United States has not changed appreciably between 1930 and 1980 (no data given back to 1909), but the incidence rate has risen from under 60 to about 90/100,000 per year. Enig et al. argue that if fat in the diet is an important factor in cancer causation, it is more likely to be vegetable rather than animal fat. Processed vegetable fats such as the margarines, oils, and shortenings, which contain significant quantities of chemically altered unsaturated fatty acids containing trans double bonds in place of the natural cis double bonds, were held most suspect. The authors calculate that the trans fatty acid isomers are present in amounts up to 17% in commercial vegetable oils, 47% in margarines, and 58% in vegetable shortenings. They show that both breast and colon cancer mortality are negatively correlated with animal fat consumption but highly correlated with vegetable fat consumption. A comparison of 7J2-dimethylbenzanthracene (DMBA) induced mammary carcinogenesis in rats fed diets containing either 20% corn oil or 20% beef tallow was made by Clinton et al. (1979). The diets, administered for 4 weeks prior to DMBA, had no significant effect on food intake or weight gain, but 24 weeks after DMBA administration, 21 of 30 (70%)of the rats fed corn oil had mammary tumors vs 7 of 28 (25%)of those fed beef tallow. In experiments to test the effects of diets containing various amounts of saturated fats, unsaturated fats, and selenium, Ip and Sinha (1981) concluded that in rats receiving an adequate supplement of selenium, an increase in fat intake was accompanied by a marked increase in mammary tumor incidence when corn oil was used in the diets, but when the saturated fat, hydrogenated coconut oil, was used the tumor incidence was much lower. Only in those rats that were maintained on high polyunsaturated fat (5% corn oil) did selenium depletion result in a further increase in tumor incidence and in tumor

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DAN H. MOORE ET AL.

yield. The antioxidant property of selenium was suggested as a possible mechanism by which selenium protects against carcinogenesis. Kinlen (1982) studied cancer mortality between 1911 and 1978 in two groups of enclosed religious orders for women in Britain: one of 1769 nuns who ate no meat, and one of 1044 nuns who ate little meat. Mortality from breast cancer was not found to be significantly lower in either group than in the general population. The author suggested that preadult dietary practices may be more important in influencing breast cancer risk. However, the rates in nuns would have been higher due to celibacy or nulliparity if diet or other factors had not reduced it to normal. Gray et al. (1979), upon comparing breast cancer incidence and mortality rates in different countries, conclude that the former are highly correlated with animal protein consumption and the latter with total fat consumption. The effect of diet remained after adjusting for height, weight, and age at menarche. One way in which dietary fats may influence mammary tumor growth is by way of modification of the prolactin-binding capacity of tumor cell membrane. Cave and Erickson-Lucas (1982) fed rats carrying chemically induced mammary tumors either high fat (20% corn oil) or low fat (0.5% corn oil) diets, then analyzed the microsomemembrane fractions of the tumors for specific prolactin binding. The fractions from the rats on the high-fat diet had a significantly greater prolactin-binding capacity. In women, Kwa et al. (1981) found the plasma prolactin level in those overweight to be about 15% higher than in those underweight, but there was no sign$cant difference in prolactin level with respect to weight and height. Mason et al. (1982) found no association between body weight and tumor estrogen receptors and conclude that any data linking breast cancer risk with increased body weight cannot be explained by variations in estrogen receptor levels. Gray et al. (1982a) found no difference in age at menarche or in plasma or urinary levels of hormones in vegetarian and nonvegetarian California Seventh-Day Adventist teenage girls. Also, in an international study (Chile, Japan, Papua, New Guinea, United States) of postmenarchal girls age 16.5-17.7 years, Gray et al. (1982b) found no consistent relationship between blood levels of sex hormones and other variables studied, such as diet and anthropometric characteristics. The only significant differences were in urinary esterol and the esterol ratio. The Papua, New Guinea, girls had significantly higher

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levels. It was concluded that an elevated percentage of calories from fat did not increase prolactin levels. Hiatt et al. (1982)found no significant relationship between breast cancer and serum cholesterol and other serum lipids in 95,000 women (1035 with breast cancer) and conclude that the postulated causal relationship between dietary fat and breast cancer does not act via circulating lipid levels. De Waard et al. (1981)discuss a way in which overnutrition and obesity may influence the occurrence of breast cancer, especially in postmenopausal women. The effect of overweight is probably mediated through a menopausal estrogen mechanism. Estrogen-dependent breast cancer can be promoted by extraovarian estrogens from adipose tissue. In lean postmenopausal women, clonal selection is believed to lead to a preponderance of estrogen receptor-negative cells in any incipient cancer. Although initiation of breast cancer is probably unrelated to obesity and induction of the cancer may be rare after menopause, existing tumors will grow if they receive stimuli to which they are sensitive. Therefore, leanness or weight reduction may be a way of controlling estrogen receptor-positive breast cancer in its preclinical stages. Its growth could then be so slow that it is never detected within the lifetime. Mechanisms whereby obesity appears to increase the effect of estrogens on target tissues were studied by Siiteri et al. (1981),who found that the availability of serum estradiol (E2) to breast tissue was increased when the level of sex hormones binding globulin (SHBG) was reduced and since SHBG levels inversely correlate with overweight, the breasts of obese women would be exposed to excessive levels of E2, which may increase the risk of breast cancer. It should be remembered, however, that other studies did not find a correlation of E2 levels with breast cancer risk, although earlier measurements were usually of urinary E2 levels. M. M. Ip and C. Ip (1981) found no effect of dietary fat on the growth and estrogen sensitivity of a transplantable rat mammary tumor. Tumor growth was also unaffected by high-fat or low-fat diets given to ovariectomized (OV,), sham OV, Wistar-Furth rats, or to OV, rats with estradiol supplementation, nor did fat intake significantly alter progesterone receptor levels. However, many recent publications on diet conclude that high-fat diets, particularly animal fats in the diet, most closely correlate with breast cancer (Brammer and DeFelice, 1980; McMichael, 1980; Chan and Dao, 1981; Alexander, 1978; Gompel, 1980; Carroll, 1981; Lubin et al., 1981).

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Experiments carried out by Ross and Bras (1971, 1973, 1974) and Ross et al. (1979)have shown that in rats the important age for dietary influence on eventual development of cancer is the early life. In recent experiments, Ross et al. (1982)showed that animals that develop tumors could be distinguished from those that did not solely on the basis of age- or weight-specific information prior to maturity. In selfselection feeding experiments, individually housed rats were allowed to select from three isocaloric (443kca1/100 g) diets. The diets differed only in their protein (vitamin-free casein) and carbohydrate (sucrose) contents, but these two components always accounted for 80.5%of the diet (calories). The level of dietary fat (corn oil) was kept constant, as was the complement of minerals, trace elements, and vitamins. The combination of variables that maximized the probability of a neoplasm were (1)a high protein intake shortly after weaning, (2) a high degree of efficiency in converting consumed food into body mass at the time the individual entered puberty, (3)a high level of protein intake relative to body weight during the early adult period, and concomitantly (4)a high level of food intake, and (5) a rapid growth rate during the early postnatal life so that comparatively less time is required to attain a specified body weight increment. The opposite set of conditions reduced the probability of neoplasm occurrence. These data did not include breast cancer, but the results are probably useful in predicting mammary gland risk because there was a predominance of tumors in endocrine organs. Dietary deficiencies of selenium, zinc, copper, and manganese are at present being implicated in increased cancer risk. According to Beach et al. (1982), experimental animal studies have demonstrated that even marginal trace element deprivation during critical periods of growth and development, or alternatively, during prolonged deficiency in adulthood, can significantly alter immunologic function. From experiments in rats where DMBA was administered at various times during selenium (as sodium selenite, 5 ppm) supplementation periods, Ip (1981) concluded that (1) selenium can inhibit both the initiation and promotion phases of carcinogenesis; (2) a continuous intake of selenium is necessary to achieve maximum inhibition of tumorigenesis; (3) the inhibitory effect of selenium in the early promotion phase is probably reversible; and (4)the efficiency of selenium is attenuated when it is given long after carcinogenic injury. Ip also conducted experiments showing that selenium slowed the reappearance of mammary tumors after they had been caused to regress by ovariectomy. In other studies with rats, Ip et al. (1981)found that selenium defi-

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ciency caused a drastic reduction in the activity of hepatic glutathione peroxidase, but excess selenium had little effect on this enzyme’s activity. The effects of selenium (SeO2 and NazSeOs) on DMBA mammary tumorigenesis in BALB/c mice was studied by Medina et al. (19821, who found that selenite at 7 and 10.5 ppm reduced the mammary tumor incidence from 55 to 5 and lo%,respectively. From these and other experiments, these investigators conclude that selenium, in the form of inorganic selenite, is a potent chemoprotective agent for both viral and chemical carcinogen-induced mouse mammary tumorigenesis. Vitamin A and the retinoids are believed to be helpful in the prevention of breast cancer, as well as other types of cancer. Experiments with several species and organ cultures were recently reviewed by Axelsson (1981), who concludes that vitamin A apparently affects factors that are decisive for the malignant transformation of cells, such as cell differentiation and the synthesis of glycoproteins and proteoglycans. Administration of a dietary supplement of retinyl acetate beginning 1 week after carcinogen administration is highly effective in inhibiting mammary carcinogenesis in Sprague-Dawley rats (McCormick and Moon, 1982). If the dose of carcinogen (N-methyl-N-nitrosourea) was increased, or if the dietary retinoid was delayed, there was a decrease in the effectiveness of the diet, and the amount of decrease was greater with increasing carcinogen and with longer delay in starting the dietary supplement. Moon and Mehta (1982) have characterized a retinoid binding protein that is found in rat mammary tissue during normal and neoplastic differentiation. Relatively high levels of this protein were found in the mammary glands of pregnant animals, as well as in ovarian hormoneindependent mammary tumors. A possible correlation between endocrine and retinoid function in both normal and neoplastic differentiation of mammary tissue was indicated. C. Ip and M. M. Ip (1981) compared DMBA-induced mammary tumorigenesis in rats fed diets supplemented with selenium or retinyl acetate (RA) and a combination of both, starting 2 weeks before DMBA. The mammary tumor incidence without any dietary supplement was 90.0%, 53.3% with selenium alone, and 46.7% with RA alone, and 16.6%with the two supplements combined, in rats treated for 28 weeks. A shorter period (18 weeks) of treatment with combined supplement gave a tumor incidence of 50%. Aminoglutethimide has been added to the diet of rats treated with DMBA and shown to decrease the mammary tumor incidence and the

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number of tumors per rat (Rao et al., 1982). This drug has also been used in the treatment of breast cancer patients (Asbury et al., 1981; Kaye et al., 1981). A crude protein fraction from human urine was shown by Sarkar et al. (1981) to cause retardation or regression in DMBA-induced mammary tumors in rats, and asparagus racemosus was found to have an inhibitory effect on DMBA-induced mammary tumorigenesis in rats (Rao, 1981). A group of chemical compounds known as blocking agents prevent carcinogenesis by blocking carcinogens from reaching or reacting with critical target cells. There are a wide variety of such agents. Some are synthetic, but many are naturally occurring constituents of plants. They include indoles, phenols, flavones, aromatic isothiocyanates, coumarins, disulfram, and related chemicals (Wattenberg, 1979). Butylated hydroxyanisole (BHA), a phenolic antioxidant widely used as a food additive to prevent oxidative spoilage, also inhibits a broad range of carcinogens (Wattenberg, 1982). One of the ways by which BHA acts in an animal is by increasing the amount of an enzyme, glutathione S-transferase (GST), which is active in the carcinogen-blocking process. Recently, Lam et aZ. (1982) reported isolating two chemicals, kahweol and cafestol, from green coffee beans that, when added to diets of mice and rats, induced increased GST activity in the mucosa of the small intestine and in the liver. Thus, green coffee beans contain substances that are able to interfere with carcinogenesis. Roasted coffee also contains these substances, but much of their effectiveness is lost in the roasting process. It must also be remembered that coffee contains mutagens that may increase the risk of cancer. In other experiments, Cohen et al. (1982) showed that butylated hydroxytoluene (BHT) effectively reduced mammary and other tumors in animals given several different carcinogens. The degree of tumor reduction depended on both the amount of carcinogen used and the concentration of BHT, which ranged from 6000 to 300 ppm, added to the diet. The effect of simulated alcoholism on the development of spontaneous mammary tumors in C3H/St (a high mammary tumor strain) mice was studied by Schranzer et al. (1979). Immediately after weaning, in addition to a standard diet, female mice were provided with either water (controls), 12% ethanol, or red wine (11.5% alcohol) as the sole fluid. Eighty-two percent of the controls developed mammary tumors, with a median latency time of 14.2 months, while those receiving 12% ethanol had the same final incidence, but the latency time was reduced to 8 months. All of the wine-exposed animals grew slowly, were

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underweight, lost hair, and were in poor condition. Only a few very slowly growing mammary tumors developed. The relationship between breast cancer and alcoholic beverage consumption was evaluated by Rosenberg et al. (1982) in a case-control study of 1152 women with breast cancer, 519 with endometrial or ovarian cancer, and 2702 with nonmalignant disorders. For women who had ever drunk alcoholic beverages compared to those who had never drunk, the estimated relative risk of breast cancer was 1.4 (95% confidence interval, 1.0-2.0) when compared to the endometrial and ovarian cancer group, and 1.9 (1.5-2.4) when compared to the controls who had nonmalignant disorders. The association was evident for beer, wine, and spirits. The association could not be explained by any of the known risk factors for breast cancer, although the survey did not include other dietary factors. The authors conclude that alcohol consumption or related dietary factors increase the risk of breast cancer. In a short review of diet and cancer, McMichael(l980) suggests that diet can affect cancer causation and prevention in several ways. (1) Vitamin C can alter endogenous production of carcinogens such as the nitrosamines. (2) Endoles from cruciferous vegetables such as cabbage, cauliflower, Brussels sprouts, turnips, and broccoli can stimulate the microsomal enzyme aryl hydrocarbon hydroxylase, which appears to inactivate various noxious organic chemicals entering the GI tract, lungs, and skin via these epithelial portals. (3) Alcohol may enhance the effective contact of carcinogens with critical cellular molecules. It may act by altering cell membrane permeability to carcinogens or as a solvent vehicle for carcinogens, or additionally, it may act via the associated vitamin deficiencies that render the epithelium hyperplastic and otherwise vulnerable to the effects of carcinogens. (4) Vitamin A may protect against epithelial neoplasia through control of cell differentiation or the inhibition of carcinogen binding to DNA. The synergism between foods, chemicals, and other factors in causing, promoting, preventing, or controlling mammary adenocarcinomas in several animal species has been reported so frequently in the recent literature that no attempt has been made to include them in this article. Nevertheless, they provide an impressive testimony to the responsiveness of the mammalian system to these many factors. Different species may show some differences in the way they respond to the different variables, but in general, the responses are similar. One of the species differences that add to the difficulty of information transfer from one species to another is the great difference in lifespan. Most experimental animals have short lifespans with rapid development and change from one lifestage to the next, whereas man’s pro-

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gression through life may be 25 to 50 times slower. The cancer process is relatively very slow in human beings, and this makes it difficult to determine which factor acting at which stage in life can eventually bring on the end product. The number of incipient breast cancers may be many times the number that are detected. There are vast differences in the growth rate of different tumors, and the growth rate of any individual tumor may be changed at any time. Probably most of the documented risk factors are factors that accelerate growth rate. Those that decelerate growth rate are not so well known, because decelerations can be observed only after the tumor is large enough to be detected, and after it is this large, it has become more difficult to decelerate, although this does sometimes occur. XIII. Psychosomatic Factors

Mental stress must be considered as a possibly important factor in the etiology of breast cancer. This subject has been reviewed by Bahnson (1981),who gives evidence for specific personality and egodefensive characteristics that are found to be associated with cancer patients. Long-term psychodynamic aspects of personality such as repression, denial, poor emotional outlet, and a characteristic lack of self-communication are some of the characteristics considered by Bahnson. In comparison with patients with other illnesses and with healthy persons, many cancer patients seem to have lost awareness of their own needs and wishes and seem to present a realistic and pleasant interpersonal attitude, even though they may be seen by an observer to live a constricted and bleak life. Bahnson reports that a study by Wrye (1979) indicates that breast cancer patients perceive their mothers as unable or unprepared to assume the mothering role for them, and often their relationship with their fathers is stressed or lacking. Bahnson believes that cancer patients, unlike control groups, remember their parents as noninvolved, cold, and nonparticipatory in their early emotional lives. In a study of 160 women hospitalized for breast tumor biopsy, Greer and Morris (1975)found a significant association between a poor prognosis for breast cancer and a behavioral pattern existing throughout adult life, i.e., an abnormal release of emotion. In most cases, this abnormality was extreme suppression of anger, although in some cases, it was extreme expression of other emotions. Bahnson believes that people who rely on strong repression of emotional feeling do not develop alternative coping styles, but instead become overwhelmed by emotion when their usual strategies fail in situations of intense

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stress, and they may regressively discharge unrestrained degrees of emotion. In a personality inventory of women who were to undergo breast biopsies, Schonfield (1975) found that those under age 42 who were later diagnosed as having cancer had significantly higher covert anxiety scores by the Minnesota Multiphasic Personality Inventory (MMPI) test than did the women whose biopsies later were reported to be benign. Schonfield also found that the MMPI “lie scale” scores were significantly higher for the women later diagnosed as having malignant breast tumors. Simonton and Matthews-Simonton (1981) consider psychological factors as important agents in the etiology of cancer. Among them are psychological depression, impaired emotional outlets, and perceived lack of closeness to parents. Many publications show association of higher education with breast cancer (Hlaing and Myint, 1978; Lin and Kessler, 1980; Paffenbarger et al., 1980; Devesa and Diamond, 1980). It is not known whether the increased risk is due to increased stress of disciplined learning or to other factors in the lifestyle associated with higher education. A strong, positive association of income and education with breast cancer in United States white women was found by Devesa and Diamond (1980), but the strong association was with only education and not with income in United States black women. Animal experimentation has been widely used in attempts to discover some of the mechanisms by which stress can influence cancer incidence. Solomon (1969)found that ablation of the dorsal hypothalamus decreased antibody reaction, allowing accumulation of antigen in the host. Such animals were more receptive to tumor transplants and developed more malignant tumors, and the tumors had a more fulminating course. Amkraut and Solomon (1972) stressed BALB/c mice by electric shock after mammary tumor virus inoculation and showed that there was an increase in tumor size compared to nonshocked controls. Sklar and Anisman (1979) used the growth of a transplantable syngeneic mastocytoma in DBN2J male mice to determine the effect of stress from electric shock. A single session of inescapable shock resulted in an earlier tumor appearance, exaggeration of tumor size, and decreased survival time of the mice. In other experiments where the animals were allowed to initiate escape (escapable shock), the tumor growth was essentially normal, although the mean duration of shock for the escapable and inescapable shocked animals was the same. Long-term inescapable shock treatment mitigated the effect; the ani-

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mals adjusted somewhat to the stress; those that were exposed to 10 shock sessions adjusted better than those that had only 5 sessions. Those that received only one shock session had significantly faster growing tumors. The results indicated that inability to cope with stress behaviorally rather than physical stress per se was responsible for the effects of shock on tumor growth. Psychoneuroendocrine influences on immunocompetence and neoplasia in mice was studied by Riley and his associates over a period of several years (Riley, 1981).From these extensive studies, it was concluded that the most conspicuous biochemical factor associated with stress in mice was the increased concentration of corticosterone in the plasma, which when in high concentration is damaging to lymphoMonths

Age of mice (days)

FIG.4.Incidence and latent periods of mammary tumors in C3H female mice under various experimental and environmental conditions. Group A consisted of parous mice housed under conditions of chronic environmental and manipulative stress; group B, nonparous mice housed under the same conditions of chronic environmental and manipulative stress; group C, nonparous mice housed under protective conditions and subjected only to low or moderate environmental and manipulative stress; and group D, a combination of parous, nonparous, and virgin female C3H mice delivered by cesarean section and foster-nursed to deplete the milk-passaged MTV. However, gamete-transmitted viral genome or NIV is not eliminated by this procedure. Thus, mammary tumor production can occur under proper circumstances. This group was provided the maximum protection From environmental and manipulative stress. Reproduced with permission from Riley (1975).

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cytes and thymus elements that are essential for optimal cell-mediated immune defenses and possibly for effective immune surveillance involving the natural killer cells that may attack cancer cells. Riley (1975) found that protecting mice from the usual noise and handling procedures could delay and decrease the development of mammary tumors in MTV-infected high tumor strains of mice and that by regulation of stress, parity, and virus infection, the occurrence of mammary tumors could be almost completely controlled, as is illustrated in Fig. 4. XIV. Discussion and Concluding Remarks

In preparing this article, we have attempted to present a representative report of the recent data and the conclusions reached by the various investigators. These data and conclusions necessarily depict the many discrepancies and contradictions that now exist in the literature. From them, we are unable to deduct the degree of importance or relevance of the many factors implicated in the etiology of carcinoma of the mammary gland, but we suspect that most of them are involved in synergistic actions which either condition the tissue or initiate, promote, prevent, or retard mammary gland carcinogenesis. Under some conditions, any one of the many implicated factors may influence the manifestation of the disease in some members of some populations. This leads to the conclusion that none of the factors so far studied is of great importance singly; but in a situation where several act together, any one may appear to be significant. Factors that seem important in discriminating breast cancer from non-breast cancer women in one population do not seem to be very important in another population. For example, in some developing nations, low breast cancer rates are correlated with early marriage and early first delivery, whereas in other populations such as the Amish community of Lancaster County, PA (Hamman, 1979), or the Hassidic community of New York City (S. Blumenthal, personal communication, 1982), reproductive life is also started early, but breast cancer rates are unusually high. There are also implicated factors such as breast-feeding that were at one time thought to be influentia1 in many populations but are not now thought to be. Breast-feeding has not been thought to be important since the extensive studies of MacMahon and associates in the early 1960s (MacMahon et al., 1970a,b). Recently, we have made some preliminary studies on three different populations in South Africa that have strikingly different breast cancer rates, which further emphasizes the differences in significant

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factors. The South African data, obtained by questionnaires and interviews, were compared with similar data obtained in the United States [Philadelphia area and Lancaster Co. (PA) Amish]. Factors selected, in order of importance, by the BMPD stepwise linear discriminant analysis (Dixon, 1979) as discriminating between breast cancer and non-breast cancer groups are recorded in Table I. Of the more than 50 implicated risk factors included in the questionnaire, dietary components ranked high in discriminating breast cancer from non-breast cancer groups in all of the South African populations; they may also be important in the Philadelphia area population, but diet questions were not included. Number of years from menarche to delivery of first child was of high rank in the Philadelphia and urban vs rural Zulu comparisons, but was not significant in the other two comparisons. The Philadelphia breast cancer women had a shorter delay (Table V), which is opposite to the conventional dogma. Much of the data obtained through our questionnaire was subjective and therefore may contain error, but age at menarche and particularly age at first delivery are less likely to be in error. Other details of some high-ranking factors in discriminant analysis of our data are given in Tables I1 to V. In further commenting on this review, we are impressed by the present attempts being made to sort out the various dietary (nutritional, chemical, pharmaceutical) components that increase or decrease risk. In human beings, however, this is going to be difficult because of the inability to control the multitude of other factors that operate over a long period of time and bear their influences on the final outcome. Diets probably provide the greatest potential for influencing cancer development because they may contain a large number of substances that initiate, promote, inhibit, or retard cancer growth. Carcinogen inhibitors include the indoles, retinoids, and selenium, among many others that are at present being investigated. Among those being accused of initiating or promoting breast cancer are fats, both animal and vegetable, saturated and unsaturated. It is possible that vegetable oils, particularly those that have been chemically altered, are greater risk factors than unaltered vegetable or animal fats. The consumption of altered fats has increased in recent decades. We believe that the food substances (natural plant and animal products) consumed during human evolutionary development are less likely to be handled wrongly by the human organism. It seems that the early years of life are more important for dietary as well as for other factors to exert their influences. Many areas of investigation support this thesis. The work of Ross et al. (1982) in rats indicates that the diet before maturity influences cancer rates,

TABLE I FACTORS SELECTED BY STEPWISE LINEARDISCRIMINANT ANALYSIS

Rural Zulu vs

Rank

Urban Zulu

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Menarche to first delivery Fat in diet Grains in diet Breast stimulation Lamb + goat in diet Period length Number of children Enough money Like home Home big enough

Urban Zulu breast cancer vs no cancer Lamb + goat in diet Breast stimulation Age first delivery Age at menopause Grains in diet Home big enough

South African white breast cancer vs no cancer Worry Age at marriage Express emotions Breast-feed kids Vegetables in diet

Philadelphia breast cancer vs no cancer Menarche to first delivery Breast stimulation Age at frequent sex

Correct classifications No breast cancer Breast cancer 0

86% (59/64) 91% (69/76)"

Numbers for urban Zulu without cancer.

82% (62/76) 86% (18/21)

97% (35/36) 85% (11113)

63% (83/126) 58% (30/52)

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TABLE I1 RURALVERSUS URBANZULU(CANCER-FREE) WOMEN" Factor Number of years from menarche to first delivery Fat in diet (0-15) (butter, eggs, beef, pork, mi1k)b Grains in diet (0-9) (cereal, rice, nuts) Breast stimulation (0-2) (0 = never, 1 = some, 2 = often) Lamb and goat in diet (0-6) Length of menstrual period (days) Number of children Enough money (% yes) Like home environment (% yes) Home big enough (% yes)

Cancer incidence Number interviewed High fat (score range 0-15)

Rural Zulu"

Urban Zulub

Near 0 272

24/105

5.5

391 8.4

Rural

Urban

4.5 5.5

8.4 8.4

6.0

5.1

1.3

0.8

3.3 3.7 6.9 37 90 80

2.5 4.4 4.1 23 96 66

South African Caucasianb

72/105 256

9.3

Lancaster Co. (PA) Amishb

-

100/105 97

11.4

Breast cancer incidence: rural, 0; urban, 24 per 100,000. There was also a significant correlation between this dietary factor and breast cancer incidence in the four populations we studied (excluding women with breast cancer).

whereas the diet after maturity does not. Also, from the Japanese atomic bomb data, the young age is the most sensitive age to irradiation; exposure at this time may affect risk throughout the whole life span, but the effect of irradiation decreases with exposure age. Furthermore, the effect, if any, of oral contraceptive steroids seems to be greatest if used in the early postmenarchal years, and MacMahon has suggested that an early first delivery is the only delivery that offers protection. However, the effect of child-bearing does not seem to be simple. In some cases, the age at menarche, the age at first full-term pregnancy, the number of deliveries, miscarriages, or abortions, or the age at menopause seem to have an influence, but the influence of any of these is not universally confirmed, nor are the mechanisms through which they exert their influence understood. Nevertheless, it seems that a variety of hormonal imbalances and abnormalities may be im-

243

BREAST CARCINOMA ETIOLOGICAL FACTORS

TABLE I11 URBANZULUWOMENO Factor

No cancer

Breast cancer

Lamb and goat in diet (0-6)b Breast stimulation (0-2) Age at first delivery (years) Age at menopause (years) Grains in diet (0-9) Home big enough (% yes)

2.5 0.82 23.1 45.9 5.1 66

3.9 0.23 24.3 46.1 6.3 90

_ _ _ _ ~ ~

Cancer incidence Number interviewed Lamb, goat (score range 0-6)

Rural Zulub

Urban Zulub

South African Caucasianb

near 0 272 3.0

24/105 391 2.6

72/105 256 1.9

Lancaster Co. (PA) Amishb 97/105 97 0

Breast cancer incidence: 24 per 100,000 per year. There was a negative correlation between this dietary factor and breast cancer incidence in the four populations we studied (excluding women with breast cancer). b

TABLE IV SOUTHAFRICAN WHITEWOMEN" Factor

No cancer

Breast cancer

Do something about worries (0-6) Age at marriage Express emotions (0-6) Breast-feed children (% yes) Vegetables in diet (0-6)

3.9 24.2 5.8 83 4.0

2.4 22.7 4.4 69 5.0

a

Breast cancer incidence: 72 per 100,000.

TABLE V PHILADELPHIA AREAWOMEN^ Factor

No breast cancer

Breast cancer

Years from menarche to first delivery Breast stimulation Age started frequent sex (years)

12.7 1.25 22.3

10.6 1.00 22.0

a

Breast cancer incidence: 85 per 100,000.

244

DAN H. MOORE ET AL.

portant in the breast cancer process. Elevated blood level of prolactin, and possibly some of the corpus luteum hormones, and lowered progesterone seem to increase risk. Consumption of alcohol, use of hair dyes, and contact with several other synthetic chemicals may be factors that increase risk. Probably not enough emphasis has been placed on mental stress and other psychosomatic factors. It seem that ability to cope with stress may be more important than stress per se. Here again, the early life, particularly the parent-child relationship, seems to be important in building a body that may be able to successfully deal with later assaults. In our studies of the South African Zulu, we noticed that the rural mother living in her tribal habitat seemed to dedicate her entire life to child-raising with affection, while the urban Zulu mother was concerned with jobs and education, thus having less time to devote to child nurture. This may be a factor in the large difference in the breast cancer rates in the two populations. In contradiction to this, however, is the fact that the Old Order Amish children seem to have the same devoted nurture as the rural Zulu and yet the Amish have one of the highest breast cancer rates in the world. To us, this simply means that no one factor is, in itself, very important, but must be considered in the context of the whole universe of factors, many of them still uninvestigated or unknown. ACKNOWLEDGMENTS The preparation of this article and some of the results reported therein have been supported by the American Cancer Society Research Professors grant RDP-34 and the Biomedical Science Division, University of California, Lawrence Livermore National Laboratory, under contract number W-7405 ENG-48. We are also greatly indebted to Ms.Alice M. Smith for her patience and competence in typing the manuscript.

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TREATMENT OF ACUTE LEUKEMIA-ADVANCES IN CHEMOTHERAPY. IMMUNOTHERAPY.AND BONE MARROW TRANSPLANTATION G6sta Gahrton Division of Clinical Hematology and Oncology. Department of Medicine. Huddinge Hospital and Karolinska Institute. Huddinge Sweden

.

I . Introduction

....................................................

I1. The Strategy for Treating Acute Leukemia ...........................

111. Classification and Prognostic Factors ................................

A . Classification................................................. B . Prognostic Factors ............................................ IV. Chemotherapy of Acute Leukemia .................................. A . Acute Lymphoblastic Leukemia (ALL) ........................... B . Acute Nonlymphoblastic Leukemia (ANLL)....................... C. Duration of Chemotherapy and Survival.......................... D . New Drugs and Treatment of Refiactory Patients . . . . . . . . . . . . . . . . . . E . Carriers of Cytotoxic Drugs .................................... V. Jmmunotherapy ................................................. A . Active Immunotherapy ........................................ B. Passive Immunotherapy-Monoclonal Antibodies and Interferon ..... VI. Bone Marrow Transplantation ..................................... A. Allogeneic and Syngeneic Transplantation ........................ B. Technique for Bone Marrow Transplantation ...................... C . Survival after Allogeneic Bone Marrow Transplantation ............. D . Autologous Bone Marrow Transplantation ......................... VII. SupportiveTreatment ............................................ VIII . Prospects for the Future .......................................... References......................................................

255 256 262 262 263 271 271 278 283 287 295 297 298 302 304 304 305 306 310 311 312 313

1. Introduction

Prior to 1948. when Farber et at . introduced folic acid antagonists for the chemotherapy of acute leukemia. there was little hope of surviving once the diagnosis had been established.There was no therapy except for blood transfusions. and a limited number of moderately effective antibacterial agents. The median survival time for patients with acute leukemia was 2.2 months for both children and adults (Tivey. 1954). Occasionally. spontaneous remissions were reported. sometimes in association with blood transfusions or infectious epi255 ADVANCES IN CANCER RESEARCH. VOL. 40

Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-0066408

256

COSTA GAHRTON

sodes (Southam et al., 1951; Freireich et al., 1961).However, attempts to produce remissions b y transfusions or by inducing infections were usually unsuccessful (Bierman et al., 1950; Wetherly-Mein and Cottom, 1956; Weelock and Dingle, 1964). Aminopterin, the folic acid antagonist originally used by Farber et al. (1948),produced complete remissions, which means that no clinical or hematological signs of disease were observed, and hopes were immediately raised that leukemia was curable. However, the first patients treated with aminopterin relapsed after some months, and it soon became clear that there was much to learn about how to use cytotoxic drugs. Enormous efforts were also made to find new and more active antileukemic cytostatics. Corticosteroids were first used by Pearson et al., in 1950, 6-mercaptopurine by Burchenal et aZ., in 1953, and later, in the 1960s; there followed most of the drugs used today, i.e., cyclophosphamide, vincristine, daunorubicin, cytosine arabinoside, thioguanine, and L-asparaginase. Some new substances appeared in the 1970s, e.g., doxorubicin (adriamycin), the epipodophyllotoxins, and the nitrosoureas. The proper place of some of the newcomers in the treatment of acute leukemia has yet to be established. The improvement in treatment results seen during the last 10 years is as much due to improved methods of drug administration as to the use of new drugs. The importance of combining drugs with different mechanisms of action was first expressed by Frei et al. (1961). Since then an intensive search has been carried out to find the best possible combination for each particular type of leukemia. Improvement in the results with chemotherapy would not have been possible without the development of improved methods of supportive treatment, e.g., treatment of infections with aminoglycosides, intensification of blood product support, and improvement in intravenous feeding. For a minority of patients, i.e., those with an HLA-identical sibling, dramatic changes in the prospects for cure have been achieved by bone marrow transplantation. Immunotherapy, so far, has had little importance for the improved outcome for the patients. New ways to use monoclonal antibodies may change this poor role to the better. II. The Strategy for Treating Acute Leukemia

There are a number of factors to be considered when designing a treatment program for patients with acute leukemia. First, acute leukemia is a heterogeneous disease. There is acute lymphoblastic leuke-

TREATMENT OF ACUTE LEUKEMIA

257

mia (ALL)' and acute nonlymphoblastic leukemia (ANLL). Both of these main types can be further subdivided into moq3hological variants (Tables I and 11) and immunological variants (Table 111). In addition to this, certain leukemias are characterized by chromosomal markers, which appear to be distributed nonrandomly but whose significance is not clear in most cases. Second, patients with acute leukemia may be children or adults. So far, the two main discriminators affecting the choice of therapeutic program are the type of leukemia (ALL or ANLL) and the age group (children or adults). Some of the morphological and immunological subgroups differ in prognosis, but this has not yet been of much importance for the choice of chemotherapeutic program. However, some, such as acute promyelocytic leukemia (M3 type), may require additional supportive treatment and others, like the L3 type of ALL, may differ as regards indications for bone marrow transplantation. The therapy of ALL is different from that of ANLL, mainly because leukemic lymphoblasts and leukemic myeloblasts have different sensitivities toward the cytotoxic drugs. Methotrexate, prednisolone, vincristine, and cyclophosphamide are much more effective in lymphoblastic types of leukemia than in myeloblastic types, and daunorubicin and cytosine arabinoside are particularly suitable for the treatment of ANLL. However, these drugs are also quite effective against the lymphoblastic type of leukemia (Table IV). Children and young adults appear to tolerate high-dose chemotherapy much better than older adults do. This also has to be taken into account when planning the therapeutic program. The basis for chemotherapy is that leukemia is a clonaI disease (Fialkow et al., 1967, 1981; Gahrton et al., 1974a), i.e., the leukemic 1 Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeioblastic leukemia; ANLL, acute nonlymphoblastic leukemia; S-Aza, 5-azacytidine; ARA-C, cytoC, sine arabinoside; ASP, L-asparaginase; BCNU, 1,3-bis(2-chloroethyl)-l-nitrosourea; CCR, contincyclophosphamide; CCNU, l-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea; uous complete remission; CFU-C, colony-forming units; Ch, chemotherapy; ChIm, chemoimmunotherapy; CIr, cranial irradiation; CNS, central nervous system; CRR, complete remission rate; CSA, colony-stimulating activity; CSF, colony-stimulating factor; D, dexamethasone; Dact, dactinomycin; DHAD, dihydroanthracenedione; DMSO, dimethyl sulfoxide; DNR, daunorubicin; Dox, doxorubicin; GVHD, graft-versus-host disease; Hydrea, Hydroxyurea; IDM, intermediate dose methotrexate; LDL, low-density lipoprotein; Mesna, 2-mercaptoethane suIfonate; Mx, methotrexate; Mxi-t, intrathecal methotrexate; MP, 6-mercaptopurine; Ommaya, Ommaya reservoir; P, prednisone, prednisolone; PYR, pyrimethamine; TG, 6-thioguanine; Tdt, terminal deoxynucleotidyl transferase; Vc, vincristine; VM 26, teniposide; VP 16, etoposide; WBC, white blood cell count.

TABLE I FHEQIJENCY o~ FAB TYPES AMONG P x m w r s wm ANLL" Classification (%) ~~~~~

~~

type

Bennett et (11. (1976) (n = 272)

Zittoun et (11. (1976) (n = 93)

Whittaker et (11. (1979) ( n = 200)

Foon et (11. (1979) ( n = 56)

Sultan et (11. (1981) ( n = 250)

Mertelsmann et al. (1980) ( n = 202)

Lindquist et (11. (1982) (n = 73)

M1 M2 M3 M4 M5 M6 Other

20 25 7 14 17 2 15

12 31 9 27 9 5 7

7 64 2 17 2 8

27 28 4 23 9 0 7

21 32 16 16 12 3 0

9 28 15 8 32 3 4

14 43 5 15 23 0 0

FAB

FAB, French-American-British

0

classification (Bennett et al., 1976).

259

TREATMENTOFACUTELEUKEMIA

TABLE I1 FREQUENCY OF FAB TYPEAMONG CHILDREN AND ADULTSWITH ALL

ALL children (n = 566) ALL adults (215 years) (n = 324)

L1

L2

L3

(%)

(%)

(%)

84

15

1

Miller et ol. (1979)

29

64

3

Bloomfield (1982, review)

Reference

cell population has developed from a single cell and has some characteristics which differ from those of the normal hematopoietic cell population and these can be utilized in designing the treatment program. The goal of the therapy is to kill the last leukemic cell without killing too many of the normal hematopoietic stem cells. A certain level of effective normal bone marrow cell production must be retained, otherwise the patients will be killed by infections or thrombocytopenia during the treatment. The work by Skipper et al. (1964) in experimental L1210 mouse leukemia showed that the fraction of leukemic cells killed is the same for a given amount of treatment, regardless of the actual numbers of cells originally present. Provided the recovery of normal cells is more TABLE I11 OF ALL AND MAIN SURFACE MARKERSO IMMUNOLOGICAL SUBCLASSES Non-T, Non B-ALL Characteristics Percentage of all ALL Surface markers Common ALL antigens Ia-like antigens E-rosettes T-cell antigens (OKT3, Leu 1) Surface Ig Cytochemistry Terminal deoxynucleotidyl transferase Acid phosphatase Modified from Aisenberg (1981).

Common (CALL)

Other (Null-ALL)

-65

-15

+

T-ALL -15-20

+ t

B-ALL -2

260

COSTA GAHRTON

TABLE IV FOR INDUCTION OF REMISSION IN ACUTE LEUKEMIA SINGLE-DRUG THERAPY ~~

Complete remission

(a)

Drug

ALL

ANLL

References

Methotrexate

22

3-16

Prednisone

63

5-15

26-37

10-14

37

<24

18-23

15

L- Asparaginase

55

<10

Vincristine

47

0-20

Cytosine arabinoside

26

25

Frei et al. (1961); Frei and Freireich (1965); Vogler et al. (1967) Medical Research Council (1966); Wolff et al. (1967) Burchenal et al. (1953); Frei et al. (1961); Frei and Freireich (1965); Brubaker et nl. (1964) Murphy et al. (1955); Krakoff et al. (1961);Brubaker et al. (1964) Shnider et al. (1960); Bergsagel and Levin (1960);Tan et al. (1961); Yessayan et al. (1962); Fernbach et al. (1962); Dick (1965) Clarkson et al. (1970); Capizzi et al. (1970); Tallal et al. (1970) Heyn et al. (1966); Karon et al. (1966) Ellison et al. (1968); Howard et al. (1968); Wang et al. (1970); Bodey et al. (1974); Carey et al. (1975) Bernard et al. (1969); Weil et al. (1973, 1976); Wiernik et al. (1976) Wang et al. (1971); Bonadonna el al. (1972); Math6 et al. (1972); Benjamin et al. (1973); Tan et al. (1973) Jacquillat et al. (1972, 1976)

6-Mercaptopurine 6Thioguanine (or 6-Thioguanosine) Cyclophosphamide

Daunorubicin

45-60

35-50

Doxorubicin

11-38

7-30

Rubidazone

50

50-53

rapid than that of leukemic cells after a course of treatment, it should be possible, by giving repeated courses, to reduce the number of leukemic cells, while still retaining the normal cell population. When only a single leukemic cell is left, this will be killed in the course of treatment. In the practical clinical situation it is obviously difficult to reach this final goal, as can be seen from Table IV. Only a relatively small fraction of patients can obtain a complete remission with a single drug. Furthermore, it has been estimated that in bone marrow remission, there are still 10s to loLoleukemic cells (0.1-10 g) left in the body. In the untreated patient, the average number of leukemic Thus, although a 100- to 10,000-fold decrease in cells is probably

26 1

TREATMENT OF ACUTE LEUKEMIA

the number of leukemic cells may be obtained with a single drug, the prospects for cure are poor. In 1961, Frei and co-workers pointed out that the chances of cure would be much larger if drugs with different mechanisms of action were combined. Assuming that each drug acts independently, i.e., the leukemia is not cross-sensitive to the drugs, the complete remission rate (CRR) with a combination of two different drugs, A and B, can be calculated from the following formula (Frei and Freireich, 1965):

Practical experience with some of the original drug combinations (Table V) shows that in ALL in children, the observed frequency of remission was, in fact, somewhat higher than the expected one. In ANLL and with other drugs, the formula cannot be applied as easily in the clinical situation. The dosages used in single-drug treatment are sometimes too high to be used in combination with other drugs if normal stem cells are to survive. Nevertheless, it is clearly documented that combination chemotherapy is much more effective than single-drug therapy. The first part of the treatment, the induction phase, limits its goal to reducing the number of leukemic cells to about lo8 to 1O'O cells. This goal is generally reached after a single or repeated induction courses with two or more cytotoxic drugs. At this stage, i.e., the remission, the bone marrow is repopulated with normal hematopoietic cells and the patient is clinically well. The patient is now in an optimal condition TABLE V OFUCINAL OBSERVATION ON INDUCTIONOF REMISSION WITH SINGLE DRUGVERSUS DRUG COMBINATION IN ACUTE LEUKEMIA IN CHILDREN~ ~~

Complete remission (%) Drug combination with 6-Mercaptopurine

Vincristine

Drug

Single drug

Expected

Observed

Expected

Observed

Prednisone Methotrexate 6-Mercaptopurine Vincristine

57 22 26 47

70 42

82 44

77

84

Data from Frei et al. (1961), Frei and Freireich (1965), Karon (1963), and Acute Leukemia Group B (1965).

262

COSTA GAHRTON

for further treatment with the cytotoxic agents against the residual lo8 to 1O’O leukemic cells. A period of intensive treatment with various drug combinations, the so-called intensification or consolidation, is followed by a period of more or less intensive maintenance treatment until cessation of therapy. With the present treatment programs for ALL in children, there appears to be no advantage in continuing the maintenance treatment for more than 2 or 3 years. For ANLL, the optimal duration of the maintenance treatment has not yet been established. In practically all programs for maintenance treatment, drugs are given in intermittent courses at various intervals, usually 3 to 4 weeks. This gives a chance for normal hematopoietic bone marrow recovery between courses, and probably reduces depression of the immune system. Severe bone marrow depression, which occurs during induction treatment, necessitates extensive support with blood products, mainly platelets because of thrombocytopenia, and also with antibiotic agents because of frequent infections. For some patients, alternative methods of treatment might be of value when remission is obtained. Bone marrow transplantation appears to be the most promising method for the treatment of acute leukemia today, but it is limited to patients with HLA-compatible sibling donors. Immunotherapy seems to be of little importance, in contrast to what was thought a few years ago; but the new techniques being used to produce monoclonal antibodies directed against leukemic cells may change this situation. All phases of the treatment of acute leukemia are important. However, it is a common finding that the more aggressive the treatment the patient can tolerate during the initial induction period and the more rapidly that remission is obtained, the greater are the chances for a long-lasting remission. This was claimed long ago by the doyen of leukemia treatment research, Professor Jean Bernard, who in his rare utterances in the English language used to say, “Early aplasia is better than late aplasia.” 111. Classification and Prognostic Factors

A. CLASSIFICATION The traditional classification of acute leukemia into acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) is still the main basis for the choice of therapy. However, the use of cytochemical and immunological methods for the characterization of leu-

TREATMENT OF ACUTE LEUIU3MIA

263

kemic cells has led to a further subgrouping of the leukemias. Furthermore, since the traditional group of AML also includes acute monocytic leukemia and erythroleukemia, this group is frequently referred to as acute nonlymphoblastic leukemia (ANLL), as opposed to ALL. Based on cytochemical and morphological features, a working party consisting of French, American, and British (FAB)morphologists proposed a classification of ANLL into six subtypes (Bennett et d.,1976) (Table I), i.e., acute myeloblastic leukemia without maturation (M l), acute myeloblastic leukemia with maturation beyond the promyelocytic stage (M2), acute promyelocytic leukemia (M3), acute myelomonocytic leukemia (M4), acute monocytic leukemia (MS),and erythroleukemia (M6). Morphologically, ALL was subdivided into three groups (Table 11), one with mainly small cells (Ll), one with larger cells (L2), and one with cells which resembled Burkitt lymphoma cells and was called Burkitt type (L3). Although the L types in ALL have some prognostic value, the immunological cell-surface markers that appear on lymphoblasts (Table 111) appear to be more important determinants for prediction of the outcome, particularly for childhood ALL. The main subgroups of ALL according to the immunological markers are B-cell ALL (presence of surface immunoglobulin), T-cell ALL (sheep erythrocyte-rosette-positive cells), and “non-B, non-T” ALL (lacks T- or B-cell markers). Most patients with “non-B, non-T” ALL have cells which have a surface marker called the common-ALL antigen (cALLa). This group accounts for about 65% of all ALLs (Greaves et aZ., 1978; Sen and Borella, 1975; Pesando et al., 1979). The non-T, non-B ALL, in which the cells lack the cALLa antigen, may also be called unclassified ALL because of the lack of specific markers. It has some features in common with CALL, for example, the presence of Ia-like antigen on the leukemic cells in most patients, and the presence of terminal deoxynucleotidyl transferase (TdT). However, TdT is also present on T-ALL cells and Ia antigen is also found in cells of B-cell ALL (Aisenberg, 1981). Thus, unclassified ALL is probably a heterogeneous group of ALLs. Although there is no strict correlation between the morphological and immunological subtypes of ALL, it appears that the L3 subtype is practically always of the B-cell type.

B. PROGNOSTIC FACTORS A number of factors that can be analyzed before the institution of treatment are of importance for predicting its outcome (Tables VI, VII,

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COSTA GAHRTON

TABLE VI PRETRWTMENT PROGNOSTIC FACTORS IN ANLL ~

Favorable Low age, <50-60 years Acute promyelocytic leukemia (M3) Auer rods present CFU-C, colonies or small clusters CSA present or high Chromosomal pattern All metaphases normal t(8;2 1) +8 in M5 Terminal deoxynucleotidyl transferase negative

Unfavorable High age, >50-60 years Acute monocytic leukemia (M5) Auer rods absent CFU-C, no growth or large clusters CSA absent or low Chromosomal patterns All metaphases abnormal t(15;17) in M 3 -7 Terminal deoxynucleotidyl transferase positive Fever >100”F or infection Treatment-induced or previous dysmyelopoietic syndrome

and VIII). The two main morphological subtypes of leukemia (ANLL and ALL), by themselves, carry prognostic information, i.e., ALL has a much better prognosis than ANLL. However, it has to be considered that one prognostic factor may only be an “innocent bystander” to other prognostic factors. For example, the great majority of acute leukemias in children are ALL and children have a much more favorable TABLE VII PRETREATMENT PROGNOSTIC FACTORS IN ALL Favorable Age, 2-10 years LOWWBC, <20--50 x 109/liter (children) CALL L1 Chromosomes normal

Unfavorable Age, (2 years and >10 years High WBC >50 x 10g/liter (children) B cell T cell L3 Chromosomes Haploid Phl+ t(4;ll) Glucocorticoid receptors low CNS leukemia Mediastinal mass

265

TREATMENT OF ACUTE LEUKEMIA

TABLE VIII INFLUENCE OF AGEON REMISSIONINDUCTION IN ANLL Percentage complete remission at age (years)

Reference

Number of patients

<50 (or 15-60)

<60 (or 15-60)

>50

>60

~~

Ud6n et al. (1975) Wiernik et al. (1979) Glucksberg et al. (1981) Rai et al. (1981) Foon et al. (1981) Arlin and Clarkson (1982)

77 460 139 352 81 108

55 75

50 51 59 76 72

35 42

14 33 27 76 44

prognosis than adults. Thus, it might be difficult to decide which is the most important factor, age or the type of leukemia. However, a comparison of ANLL and ALL in adults suggests that there is a somewhat better prognosis for ALL patients in the same age group. Similarly, comparing ALL and ANLL in children shows that at least one subgroup of ALL (CALL)has a much better prognosis in this age group than ANLL. However, in many studies it is not easy to ascertain which prognostic factor is the most important. A somewhat better prediction of the outcome of treatment can be made after its initiation. For example, an early response to treatment, i.e., after one or two courses of chemotherapy, appears to be a favorable prognostic sign, while difficulty in inducing remission is an unfavorable sign. The most unfavorable sign of all is, of course, that the patient does not enter remission at all. Although some patients may survive for several months, or even years, without entering complete remission, the overall survival time is much shorter in this group than in patients that enter remission. Although the most studied factors for prediction of remission are pretreatment factors, early postinduction prognostic factors may be as important in the future, because they may guide the choice of consolidation and maintenance therapy. In particular, the selection of patients for bone marrow transplantation takes into account the predicted time from remission to relapse. Thus, a group of patients with a high probability of long-term survival with maintenance chemotherapy will not be transplanted in the first remission, while patients with a low probability of being maintained in remission with chemotherapy alone will receive transplants earlier in the disease.

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COSTA GAHRTON

1. Acute Nonlymphoblastic Leukemia a. Age. Age appears to be the most important factor for predicting remission (Arlin and Clarkson, 1982; Glucksberg et al., 1981; Rai et al., 1981; Uden et al., 1975; Wiernik et al., 1979) (Table VIII). Although there are exceptions to this (Foon et al., 1981), the conclusion reached from most studies that have included a large number of unselected patients is that remissions are rarely induced in patients over 70 years of age, and the frequency of remissions is considerably lower in patients over 50 than in those under 50 years of age. Thus, several groups do not advocate treatment with chemotherapeutic agents of patients above 70 years of age, unless it is necessitated by either (1)a rising white blood cell count, (2) a falling platelet count, (3) repeated life-threatening infections, or (4) increasing transfusion requirements (Arlin and Clarkson, 1982; Yates et al., 1981). It is also likely that if treatment is necessary, less intensive treatment modalities should be used in this age group than in younger patients. b. Morphological Subtypes. The morphological subclassification appears to be of moderate importance in predicting the outcome of treatment. In one study the remission frequency was lower in the M 5 type and the duration of remission was also considerably shorter in this (Jacquillat et al., 1980)and the M6 type of leukemia than in other M types (Bennett, 1982). The M 3 type, on the other hand, appears to have the best prognosis. Although the remission frequency does not appear to be substantially better than for most other M types, the duration of remission and survival of responding M 3 patients appear to be considerably longer than for other responding patients (Bernard et al., 1973; Jacquillat et al., 1979; Keating, 1982). However, in other studies no such differences in prognosis between FAB subtypes could be found (Mertelsmann et al., 1980; Arlin and Clarkson, 1982). c. Auer Rods. Although there is some controversy regarding the prognostic implications of M types, the morphological finding of Auer rods in the cytoplasm appears to indicate a comparatively favorable prognosis (Mertelsmann et al., 1980; Keating, 1982). Auer rods were present in 53% of all cases in one study and the complete remission rate was 68% in Auer rod-positive patients, but only 40% in those that were Auer rod-negative (Mertelsmann et al., 1980). d . Chromosomal Aberrations. In most studies, chromosomal aberrations are found in about half of the patients with ANLL (Sakurai and Sandberg, 1973; Golomb et al., 1976, 1978; Nilsson et al., 1977; Alimena et al., 1977; Hossfeld et al., 1979; Lindquist et al., 1982). Those who have 100% abnormal metaphases in their bone marrow cells have

TREATMENT OF ACUTE LEUKEMIA

267

a poor prognosis, while those who have only normal metaphases have a better one. Patients with both abnormal and normal metaphases have an intermediate prognosis. It is possible that this correlation is more quantitative than qualitative. Recently, it has been claimed that all patients with ANLL may have chromosomal abnormalities (Yunis et al., 1981). Subtle chromosomaI aberrations or a very low frequency of abnormalities among the bone marrow cells may not be detected in 10 to 20 metaphases, the number often investigated in current studies. It has been more difficult to determine the prognostic implication of a specific chromosomal aberration than of the quantity of abnormalities. However, the translocation between chromosomes 8 and 21, first reported by Rowley (1973) and present in 8 to 22% of patients with ANLL, may imply a favorable prognosis. However, this is true only if the patients do not have a missing sex chromosome; about 23% of the patients with the t(8;21) abnormality have such a deficiency. In these cases, the prognosis appears to be especially poor (Golomb, 1982). The +8 chromosome has recently been found by us to be present in a much higher frequency in the M 5 type of ANLL than in other M types, and it appears that in such cases the prognosis is relatively good, even if the aberration is present in all metaphases (Lindquist et al., 1982). Another specific chromosomal aberration with prognostic implications is the translocation between chromosomes 15 and 17, t(15;17), which occurs in about 50% of patients with acute promyelocytic leukemia (Rowley et al., 1977). Although the prognosis for M 3 patients appears to be comparatively favorable, those who carry the chromosomal aberration t(15;17) had a worse prognosis than those with a normal karyotype (Golomb, 1982). Another fairly specific chromosomal abnormality, monosomy 7, has been associated with a low frequency of remission and short survival times in most studies (Borgstrom et al., 1980; Borgstrom, 1981). However, there are several case reports to the contrary (Zech et al., 1975). It appears that the poor prognosis in one group of patients with monosomy 7 is associated with a higher incidence offever and infection, which is probably due to a defective response by the neutrophil granulocytes to chemotherapeutic stimuli (Ruutu et d.,1981). e. Cloning Characteristics. The cloning characteristics of leukemic cells cultured in vitro have also been reported to have prognostic significance (Moore et al., 1974; Vincent et al., 1977; Goldberg, et al., 1979; Gustavsson et al., 1981a; Hornsten et al., 1982). There are some differences between the results of these investigations, probably mainly due to the use of slightly different techniques and definitions

268

GOSTA GAHRTON

of colonies and clusters. We believe that colony formation, per se, is generally a favorable prognostic sign, while no growth is unfavorable (Hornsten et al., 1982). This also appears to be true in some of the previous studies (Moore et al., 1974; Gustavsson et al., 1981a). However, others claim that no growth is a more favorable sign (Vincent et al., 1977) and, similarly, that the presence of small clusters signifies a relatively favorable prognosis (Moore et al., 1974; Vincent et al., 1977; Goldberg et al., 1979). Thus, it appears that cloning characteristics do have prognostic implications. However, it is not yet possible to compare the results from one laboratory with those from another. It is hoped that better standardization of methodology will improve this situation. f Colony-S timulating Activity (Factor)(CSA or CSF).The colonystimulation activity of leukemic cells can also be studied in an agarculture system. Early studies indicated that the absence of CSA or a low level of CSA was associated with a poor prognosis, while a high level of CSA was a favorable sign (Granstrom and Gahrton, 1974). Later, this was documented by us in 87 patients with ANLL (Hornsten et al., 1977, 1982) (Fig. 1).The absence of CSA is a sign of very poor prognosis, even in age-matched patients. g. Fewer and Znfection. Fever and infection are common companions of acute leukemia. At diagnosis, about 50% of the patients have either fever above 101°F or proven infection (Gehan et al., 1976; Freireich et al., 1978; Rai et al., 1981). Both factors adversely affect prognosis, mainly because they reduce the frequency of remission. h. Other Prognostic Factors. Patients who have iatrogenic leukemia, i.e., leukemia due to treatment of Hodgkin’s disease, myeloma, or other malignant diseases, have a particularly unfavorable prognosis (Keating, 1982; Clarkson et al., 1982). Recently, it has been claimed that those 20% of patients with ANLL, who have leukemic cells that are positive for terminal deoxynucleotidyl transferase (TdT), have a poorer prognosis than those whose cells lack TdT (Bradstock et al., 1981; Benedetto et al., 1982). 2. Acute Lymphoblastic Leukemia As is the case for ANLL, the most important parameter for predicting prognosis in ALL is age. Other important factors are white blood cell count (WBC), CNS leukemia, morphological and immunological types of leukemia, and chromosomal aberrations. There are some minor differences between the importance of each such prognostic parameter in children and in adults.

269

TREATMENT OF ACUTE LEUKEMIA

PROBABILITY

5

10

15

\CSA 20

negative (n.31)

MONTH

FIG. 1. Survival of CSA-positive and CSA-negative patients.

In children, the most common prognostic parameters have already been used to divide patients into treatment-directed prognostic subgroups, i.e., standard-risk, intermediate-risk, and increased-risk groups (Table IX). Several centers use different programs for these three categories, or for standard- and intermediate-risk groups on the one hand and increased-risk groups on the other. a. Age. Several groups have demonstrated that the best treatment results are obtained in ALL patients between 2 and 10 years of age. The response in both younger and older patients is poorer (Zippin et al., 1972; George et al., 1973; Simone et al., 1975). Above 15 years of age the prognosis appears to be comparable to that for adults. b. White Blood Cell Count. In children, the initial white blood cell count seems to be of greater importance than in adults (George et al., 1978). A very high WBC seems to be an indication of a very poor prognosis in childhood ALL. TABLE IX GUIDELINES FOR RISKGROUPS OF ALL IN CHILDREN' Characteristics

Standard risk

Intermediate risk

Increased risk

Age WBC Immunology Morphology CNS Mediastinal mass

2-10 years <20 x lOg/liter Non B, Non T L1 or L2 No sign No sign

<2 years, >10 years 20-50 x 10Vliter Non B, Non T L1 or L2 No sign No sign

<2 years, >10 years >50 x 10g/liter Non B, Non T, T, B L1, L2, or L3b Presentb or absent Presentb or absent

Swedish ChiId Leukemia Group (1981, and unpublished). Only one of the criteria WBC >50 x 10g/liter,T cell, B cell, L3, CNS, or mediastinal mass have to be present for increased risk. 0

b

270

COSTA GAHRTON

c. Morphological Subtypes. The importance of the L types for prognosis has been debated. The L1 type appears to have a better prognosis than the L2 type (Brearly et al., 1979). However, the L1 type is also the most common type in children, while the L2 type appears to be the most common type in adults. Thus, the differences in prognosis in these two types of leukemia may merely reflect differences in ages. The =-type (Burkitt-type) leukemia has a well-defined morphological appearance. These patients usually have B-cell leukemia and a markedly poorer prognosis than those with other types (Flandrin et al,, 1975; Lister et al., 1978). d . Immunological Surfuce Markers. The immunological surface markers on lymphoblasts have been found to be important prognostic indicators, both in childhood and adult ALL (Chessels et al., 1977; Sen and Borella, 1975; Sallan et al., 1980; Greaves and Lister, 1981). CALLhas, by far, the best prognosis of all groups and B-ALL appears to have the poorest prognosis. T-ALL also appears to have a comparatively poor prognosis, while the prognosis for unclassified, or null, ALL is more variable. e. Chromosomal Aberrations. Chromosomal aberrations were found in 66% of the 330 patients with ANLL (157 children and 173 adults) studied by the Third International Workshop on Chromosomes in Leukemia (Bloomfield, 1982). In adults, it appeared that patients with normal karyotypes had a significantly higher frequency of remission (95%) than patients with the most common chromosomal aberrations, the Ph' chromosome and the t(8; 14) abnormalities, in which only 45% remission was achieved. In children, the longest remissions were obtained in those who had a modal chromosomal number of more than 50, while the shortest remissions were obtained in patients with the t(4;ll) or the t(8;14) abnormalities. In later studies of the t(4;ll) abnormality (Arthur et ul., 1982), six of seven patients had a poor prognosis. One never entered remission and five had remissions of short duration. Three of the poor-prognosis patients were 15 years old or older. Thus, the presence of the t(4;ll) chromosomal abnormality should be regarded as a poor prognostic sign in both children and adults. f. GZucocorticoid Receptors. Several groups have found a correlation between the response to corticosteroids and the presence of glucocorticoid receptors in patients with lymphoproliferative disorders (Mastrangelo et aZ., 1980; Bloomfield et al., 1981; Bloomfield, 1982). In one study it was also found that there was a correlation between glucocorticoid receptor levels and prognosis in childhood ALL (Lippman et al., 1978). It appears that the most significant factor is a very

TREATMENT OF ACUTE LEUKEMIA

271

low glucocorticoid receptor level, which is associated with a poor prognosis. However, a high glucorticoid receptor level is not necessarily associated with a particularly favorable prognosis. g. Other Prognostic Factors in Acute Lymphoblastic Leukemias. There are several other factors that have been claimed to have some prognostic implication in ALL. The most important of these appears to be CNS leukemia, which is known to be associated with a poor prognosis. In addition, patients with a mediastinal mass appear to have a poorer prognosis. However, these cases are most frequently of the Tcell type.

3. Conclusions

A number of pretreatment prognostic factors have now been identified in ANLL and ALL. In both groups the most important one appears to be the age of the patient. However, a number of other factors may help to predict the outcome of treatment. In the future, multivariant computer analysis of already known and new predictive parameters will probably be of value in identifying patients with either an extremely poor or an extremely good prognosis. However, it should always be kept in mind that a prognostic factor may only be valid for a specific treatment program. If various treatments have been used, errors in the interpretation of the information may be made. Thus, the implication of a prognostic factor may change with increasing intensification of therapy and with new treatment modalities. A factor which has prognostic implications for patients treated by chemotherapy may have a different value for predicting the outcome in patients who undergo bone marrow transplantation. In treating a patient, the importance of prognostic factors should not be overestimated. Each patient is unique, and the outcome of treatment may not always be that predicted to be most likely from the prognostic factors. IV. Chemotherapy of Acute Leukemia

A. ACUTELYMPHOBLASTIC LEUKEMIA (ALL) 1. Induction of Remission

The practical application of the concept that a higher frequency of remission could be induced by two drugs with different mechanisms

272

GOSTA GAHRTON

of action than by a single drug was first tried in children with ALL (Frei et al., 1961; Frei and Freireich, 1965, see above). In fact, a somewhat higher frequency of remission was observed than expected from the formula (see Section 11). Vincristine plus prednisolone is now the main drug combination used to induce remission in ALL. This drug combination gives complete remission in about 90% of the treated children (Table X). A somewhat higher frequency of remission (up to 96-98%) can be obtained if prednisolone and vincristine are combined with L-asparaginase or an anthracycline. However, this improvement is probably mainly due to a higher frequency of remission in increased-risk patients achieved by adding the third drug, while vincristine together with prednisolone without the third drug appear to give a similar high frequency of remission in standard-risk patients (Gustavsson et al., 1981b). In the study by Gustavsson et al., the frequency of remission was 95% in intermediate-risk patients and 97% in standard-risk patients. The overall incidence of remission was 96%. Similar results were obtained by Freeman et a2. (1982), using vincristine, prednisoIone, and L-asparaginase. Increased-risk patients had a frequency of remission of 88%, and standard-risk patients 96%. The overall frequency of remission was 91%. In adults, results of induction chemotherapy are not as good as in children (Table XI). However, combinations of vincristine, prednisolone, and daunorubicin, with or without an additional drug such as cyclophosphamide, now produce remission frequencies above 80%, i.e., comparabIe to the resuIts in children with increased-risk ALL. TABLE X INDUCTION OF REMISSION I N CHILDREN WITH ALL Reference

Number of patients

Drugs

CR (%)

Heyn et al. (1975) Sallan et ol. (1978) Aur et al. (1978) Gustavsson et al. (1981b)

502 137 268 367

Haghbin et al. (1980) Freeman et al. (1983)

133 600

vc + P vc + P Vc + P + ASP vc + P Vc + P + Dox Vc + P + Dox + ASP VC + P + DOX+ ARA-C Vx + P + ASP Vc + P + ASP + Mxi-t

86 94 95 96 96 96 96 98 91 96 88

[Standard risk] [Increased risk]

TABLE XI INDUCTION OF REMISSION, CONSOLIDATION, MAINTENANCE, AND SURVIVAL IN ADULT ALL ~

Reference

~~

Number of patients

Treatment for induction

CR (%)

Whitecar et oZ. (1972)

21

Smyth and Wiernik (1976)

17

VC + P + ARA-C + C (COAP) Vc + D + PYR + TG

Gahrton et oZ. (1974) Armitage and Burns (1977)

12 13

P + ARA-C + C + ASP VC + P + ARA-C

Lister et oZ. (1978)

51

Vc

Willemze et al. (1975) Henderson et al. (1979) Jacquillat et aZ. (1973) Esterhay et aZ. (1982)

21 149 30 24

+ P + Dox + ASP

Vc+P+DNR Vc + P + DNR + ASP Vc+P+DNR Vc + P + Mx + ASP

Gee et al. (1976)

23

Vc+P+DNR

Rodriguez et oZ. (1973) Omura et al. (1980)

14 99

Vc + P + Mx Vc+P+Mx

Clarkson et d . (1982)

72

Mayer et al. (1982a)

44

Vc + P + C + DOX Mxi-t (L-10 and L-1OM protocol) Vc + P + Dox

+ MP +

43

Consolidation, maintenance, and CNS prophylaxis VC + P

+ ARA-C + C

Median duration of CR (months)

Median survival (months)

14.5

5.5

Vc + D + PYR + TG + 6.0 CCNU 58 MP 4.1 67 Mx + MP + C + Mxi-t 11.0 + CIr 71 Mx + MP + C + Mxi-t 18.5 + CIr 72 Mx + MP + Mxi-t + CIr 15.0 72 Mx + MP + Mxi-t + CIr 15.0 73 Mx + MP + Mxi-t 11.0 75 Mx + ASP + MP + Vc 11.1 + DNR + iMx 78 L2 protocol: ARA-C + 25.0 TG + ASP + Vc + BCNU + DNR + Mx + C + Mxi-t and Ommaya 79 Vc + P + Mx + MP 8.0 80 VC + P + ASP + ARA-C 16.0 + T G + Mx + MP + C + Mxi-t + CIr 85 Vc + P + ARA-C + TG Not reached + ASP + Dox + Dact. + MP + Mxi-t

53

87

Vc + P + ASP + Dox + Mx + MP + Mxi-t + CIr

36

13.5 6.6 26.0 21.0 16.0 17.0 15.0 17.0 33.0

13.0 24.2 62.0

Not reached

274

GOSTA GAHRTON

2. Consolidation and Central Nemous System Prophylaxis a . Consolidation. Consolidation treatment is a controversial issue. Several reports claim that more intensive treatment shortly after remission does not have any substantial effect (Sallan et al., 1978; Sackman-Muriel et al., 1978). In others, consolidation may have increased the duration of remission and survival time in children with poor prognosis or in adults (Haghbin et al., 1980; Clarkson et al., 1982).The L-10 and L-1OM protocols used by Clarkson et al. (1982) utilized ARAC, thioguanine, L-asparaginase, and cyclophosphamide to consolidate the remission. In patients with a pretreatment WBC greater than 20 x 1Og/liter,a so-called Ommaya reservoir is implanted subcutaneously, and methotrexate is given directly into the ventricular system (Clarkson et aZ., 1979). The L-1OM protocol was mainly an intensification of the consolidation phase, and uses local irradiation against tumor masses. Compared to the previous less intensive protocol (the L-2 protocol), the results in adult ALL were significantly better. On the L-2 protocol, 6 of 29 (21%) of the patients are in continuous complete remission; on the L-10 protocol the proportion is 14 of 34 (44%), and on the L-1OM protocol it is 22 of 38 (48%). The follow-up times for the programs are 9 years for the L-2, 5 years for the L-10, and 2 years for the L-1OM. Despite the differences in follow-up times, it appears that intensive consolidation (L-10 and L-1OM) may significantly improve the results in adults. b. Central Nervous S ysteni Prophylaxis. Central nervous system leukemia occurs during bone marrow remission in 50 to 70% of children with ALL, unless effective prophylactic treatment is given (Evans et al., 1970). Most chemotherapeutic agents do not penetrate the blood-brain barrier in sufficient amounts to kill the leukemic cells in the cerebrospinal fluid or in microfoci within the CNS. Prophylactic CNS treatment was first studied by Pinkel’s group at St. Jude’s Hospital (Aur et al., 1971) who tried a number of prophylactic regimens during the 1960s. In the first study, 2400 rads was given to the cranium and methotrexate (5 doses of 12 mg/m2 twice weekly) was given intrathecally. The frequency of CNS relapses was thereby reduced significantly. A number of regimens were then tried which demonstrated that cranial irradiation in combination with intrathecal methotrexate was as good as full craniospinal irradiation for providing significant protection against CNS leukemia (Aur and Pinkel, 1973). CNS prophylaxis is not without problems. Demyelinating leukoencephalopathy occurs in high frequency in those patients who received

TREATMENT OF ACUTE LEUKEMIA

275

either cranial irradiation plus intrathecal methotrexate or craniospinal irradiation (Price and Jamieson, 1975; Pizzo et al., 1979). This syndrome is characterized by somnolence, headache, and vomiting. In addition, follow-up of these patients has revealed that many have poorer school performance, mental retardation, logical disability, decreased IQ, and EEG changes (Ochs et al., 1982; Rowland et al., 1982). The sequelae of CNS prophylaxis has led to attempts to find other treatment modalities for CNS leukemia prevention. In general, most of the complications have been attributed to the cranial irradiation, or cranial irradiation in combination with intrathecal methotrexate. Attempts have therefore been made to use intrathecal methotrexate without cranial irradiation, intermediate dose (IDM = 500 mg/m2) intravenous methotrexate with subsequent leucovorin rescue, or a combination of these two treatment modalities (Pizzo et al., 1979; Haghbin et al., 1980; Nesbit et al., 1981; Freeman et al., 1983). The results of such studies have been difficult to interpret. In general, central nervous system relapses have been less frequent in patients treated with cranial irradiation (Wang et al., 1976; Nesbit et al., 1981; Freeman et al., 1983) (Table XII). However, even if combinations with cranial irradiation are more effective in preventing CNS leukemia, they do not appear to be superior to intrathecal methotrexate plus IDM for the prevention of hematological relapse. In fact, the duration of remission was significantly longer in standard-risk children who received IDM in combination with methotrexate than in those who received cranial irradiation (Freeman et al., 1983). Among children with increased risk, there was no difference between the rate of hematological relapse in patients treated with cranial irradiation and those receiving IDM plus methotrexate. Thus, it appears that although cranial irradiation plus intrathecal methotrexate probably offers the best protection against CNS leukemia both in children and in adults (Table XII), treatment with intrathecal methotrexate plus IDM offers good protection against this complication and has other advantages. It is well known that relapses sometimes occur in the testes. The number of such relapses after treatment with IDM plus intrathecal methotrexate was significantly lower than that in patients treated with cranial irradiation plus intrathecal methotrexate (Freeman et al., 1983). No testicular relapses were seen in 66 children treated with a similar regimen by Moe et al. (1981). Thus, although it is too early to state that treatment with IDM plus intrathecal methotrexate is in all respects superior to cranial irradiation plus intrathecal methotrexate, most data at present speak in favor of this regimen.

276

GOSTA GAHRTON

TABLE XI1 CENTRAL NERVOUS SYSTEM PROPHYLAXIS IN ADULT ALL

Reference

Number of patients

CNS prophylaxis ~

Srnyth and Wiernik (1976); Esterhay et al. (1982) Smyth and Wiemik (1976); Esterhay et 01. (1982) Gee et al. (1976) Shaw and Raab (1977) Omura et al. (1980) Esterhay et oZ. (1982) Clarkson e t 01. (1982) Henderson et al. (1979) Lister et al. (1978) Willemze et al. (1975) Mayer et aZ. (1982a)

CNS leukemia (%) ~

40

None

42.5

18

Pyrimethamine PO

33.3

23 25 99 24

Mxi-t or Ommaya None or Mxi-t + CIr None or Mxi-t + CIr Mx iv 100 mgkg + 25% escalation Mxi-t or Ommaya Mxi-t + CIr Mxi-t + CIr Mxi-t + CIr Mxi-t + CIr

17.4 16.0 14.1 8.3

61 149 51 21 44

8.2 8.0 5.9 4.8 0.0

3. Maintenance Therapy and Duration of Remission The most important drugs for maintaining remission in children with ALL are methotrexate and 6-mercaptopurine. In most regimens 6-mercaptopurine is used on a daily oral schedule of 5 mg/m2, and methotrexate is given weekly intravenously at a dose of 20 mg/m2 (Aur et al., 1971). Although there seems to be little advantage in adding further drugs (Maurer and Simone, 1976), intermittent reinforcement with a combination of agents in addition to the 6-mercaptopurine-methotrexate regimen may further prolong disease-free survival (Fernbach et al., 1975; Gustavsson et al., 1981b; Freeman et d.,1983). Reinduction periods with vincristine and prednisolone, with or without additional drugs at monthly intervals, may therefore be used. This is probably most important in patients with increasedrisk ALL. The program presently used in the Swedish Child Leukemia Croup for maintaining patients with ALL is seen in Table XIII. With these types of program, more than 50% of the patients with ALL stay in continuous complete remission. With current programs, it appears that more than 70% of the standard-risk patients will stay in continuous complete remission (Gustavsson et al., 1981b; Freeman et al., 1983),while among increased-risk patients the median duration of complete remission is 2 to 3 years (Freeman et aE., 1983). The increasing proportion of patients in continuous complete remission is illustrated by the results of the Swedish Child Leukemia Group (Fig. 2). In

277

TREATMENT OF ACUTE LEUKEMIA

TABLE XI11 TREATMENT OF ACUTE LYMPHOBLASTIC LEUKEMIA IN CHILDREN".~

Year

Number of patients in remission

Program

1973 1976

162 87 39

111

IV, IVI

V,

1978

v c x 4, P Vc x 6, P Vc x 6, P, Dox x 2 ARA-C Vc X 6, P, Dox X 3 Vc x 6, P, Dox x 3 ASP

92 48

VI

Induction

CNS prophylaxis

Maintenance

CIr, Mx X 4 CIr, Mx x 6 CIr, Mx x 6

MP, Mx MP, Mx MP, Mx Reind

CIr, Mx X 6 CIr, Mx x 6

MP, Mx MP, Mx Reind

Swedish Child Leukemia Group (1981 and unpublished). Vc, Vincristine 2 mg/m2 (max 2 mg) iv weekly 4 or 6 weeks; P, prednisolone 60 mg/ m2 PO daily, day 1-35 then taper; Dox, doxorubicin 40 mg/m2 iv days 1, 22, (and 36); ASP, L-asparaginase 6000 IE/m2iv daily days 37-46; CIr, cranial irradiation; Mx (CNS), methotrexate 12 mg/m2 it weekly 4 or 6 times; MP, 6-mercaptopurine 75 mg/m2 daily; Mx (maintenance), methotrexate 20 mg/m2 PO weekly; Reind, vincristine 2 mg/mz iv days 1 and 8, doxorubicin 40 mg/m2 iv day 8, and prednisolone 60 mg/m2PO daily, days 1-8.

1.00

-

0.90 . 0.80 . 0.70

.

0.60

-

.. ............, ..........

_---

'-----.

..........

.-.-._ . :................ I

------i l I

! :...."..............

b,n-92

0.50 .

0.40 . 0.30 .

Ill,n-162

0.20 .

I

0

12

24

36

48 60 72 Time in CCR cmonthsr

84

96

MB

120

FIG.2. Continuous complete remission (CRR) in children with ALL. Data from the Swedish Child Leukemia Group (1981) and unpublished. Treatment programs according to Table XIII.

278

COSTA GAHRTON

a program started in 1978, the median time of complete remission has not yet been reached for either standard-risk patients or increased-risk patients. In adults, more intensive maintenance programs appear to be superior to less intensive ones (Table XI). Impressive results have recently been obtained by Clarkson et al. (1982) and Mayer et al. (1982a). In the L-10 and L-IOM protocols developed by Clarkson et al., a total of eight drugs (vincristine, prednisolone, methotrexate, adriamycin, 6mercaptopurine, bleomycin, BCNU, and cyclophosphamide) are used in a sequential schedule, and the sequence is repeated approximately every 20 weeks. At a median follow-up time of 5 years on the L-10 protocol 44% of the patients are in continuous complete remission, and at a median follow-up time of 2 years on the L-1OM protocol 58% are in continuous complete remission. The Sidney Farber Cancer Center Group (Mayer et al., 1982a) uses intermittent treatment with vincristine, methotrexate, 6-mercaptopurine, and prednisolone every 3 weeks. In this study, the median age was relatively low (23.5years). Nevertheless, 20 of 44 patients were in continuous complete remission as of April 1982. At this time 13 had been followed for more than 30 months. Thus, it appears that it may be possible to achieve approximately the same results in adults with ALL as are obtained with increased-risk children with this disease.

B. ACUTE NONLYMPHOBLASTIC LEUKEMIA (ANLL)

1. Induction of Remission The cornerstones in the induction treatment of ANLL are the anthracyclines combined with cytosine arabinoside (ARA-C). Table XIV illustrates the improvement in the frequency of remission with the best drug combinations before and after the discovery of the anthracyclines. Remission frequencies were constantly under 50% without the anthracyclines, but now some centers obtain between 70 and 80% remissions in both young and elderly patients. Daunorubicin is the most frequently used anthracycline in these combinations, but doxorubicin and rubidazone, a benzoylhydrazone analog of daunorubicin (Jacquillat et at., 1979), as well as the new aclacinomycin A (see below), are probably as effective. However, there are differences in sideeffects; for example, current studies indicate that the frequency of severe enterocolitis is higher in doxorubicin-treated patients than in patients treated with daunorubicin.

TABLE XIV INDUCTION OF REMISSION IN ANLL Reference

Number of patients

CR Drug combination

39 94 52 77 45

C + V + AM-C + P (COAP) DR + ARA-C C + ARA-C + P 2 ASP DNR + ARA-C 2 ASP Dox + ARA-C or DNR + ARA-C

Mertelsmann et al. (1980) Peterson and Bloomfield (1980) Rai et al. (1981) Foon et al. (1981) Keating et al. (1981) Paul et al. (1981a)

263 22 352 107 325 60

DNR + ARA-C + TC (L6 + L12 DOX+ ARA-C + TG + P + V DNR + ARA-C DNR + ARA-C + TG (TAD) DOX+ ARA-C + V + P DNR (or DNR-DNA) + ARA-C

Arlin and Clarkson (1982) Mayer et al. (1982b)

108 107

DNR + TG + ARA-C (L14 + L14M) DOX+ ARA-C + VC + P

Whitecar et al. (1972) Crowther et al. (1973) Gahrton et al. (1974) Ud6n et al. (1975) Preisler et al. (1979)

(%)

44 49 31 32 66

+ L14 protocols)

55 82 29-55 76 60 70 66 70

Comment

Median age 57 years Median age 60 years Median age 43 years; all patients <70 years Four different regimens Median age 46.5 years; all patients <60 years Median age 15 years

280

COSTA CAHRTON

A M - C is probably as important in the drug combination as the anthracycline. In a number of studies a third, or even fourth, drug may be used in the combination, i.e, 6-thioguanine, vincristine, or L-asparaginase (Table XIV). The best results have been obtained with the TAD regimen, i.e., a combination of daunorubicin, ARA-C, and 6thioguanine (Gale and Cline, 1977; Foon et al., 1981) (Table XV). Although two groups have obtained remission frequencies of about 80% with this drug combination, it is not entirely clear whether other factors, apart from the addition of a third drug, can explain the higher remission frequencies in some centers as compared to others. Differences in the selection of patients may well account to some of the difference in remission frequencies between 60 and 80%. In some centers, most patients over 70 years of age are not treated with chemotherapy, and in some the median age of the treated patients is astonishingly low. In some investigations, a prerequisite for entering the study is that the patient does not succumb to an infection during the first few days, or that severe infections are successfully treated. Such factors may help to explain why large cooperative studies almost always yield poorer results (Rai et al., 1981; Keating et al., 1981) than studies on a smaller number of patients. Different selection for age is probably one of the most important explanations of the variations in results. Although some centers have claimed that age does not play a role in remission induction (Foon et aZ., 1981), most centers have shown that this is the most important prognostic factor (Table VIII). Few centers obtain more than 40% remission in patients over 60 years and many larger studies obtain less than 30% remissions in such patients (Rai et d., 1981; Lonnqvist et al., 1982).

2 . Consolidation, Maintenance, and Duration of Remission Very early studies have shown that maintenance therapy given during the remission period resulted in a longer duration of remission TABLE XV TAD FOR INDUCTIONOF REMISSION IN ANLL',b

Drug

Protocol

Days

AM-C 6-Thioguanine Daunorubicin

100 mg/m2/12hr iv (30 min) 100 mg/m2/12hr PO 60 mg/mYday iv (4 hr after ARA-C)

1-7 1-7

~

~~~~~~~

~~

5-7 ~

~~

Gale and Cline (1977) and Gale et al. (1981). The TAD course is repeated once or twice at 14-21 day interval in patients who do not achieve remission on one course.

TREATMENT OF ACUTE LEUKEMIA

28 1

than if no maintenance was given (Freireich et al., 1963). In fact, Lewis et al. (1981) showed that even if intensive consolidation treatment is given for several months after a remission induction, the duration of remission is shorter than if maintenance treatment is continued. In their study, l-year survival was achieved in only 10% without maintenance treatment, but in 39% with maintenance treatment (Table XVI). The relatively low daily dose of 6-mercaptopurine used as continuous maintenance chemotherapy in earlier studies (Gahrton et al., 197413) has been replaced by more intensive intermittent courses of two or more drugs at about monthly intervals. Some programs use a number of different drugs in each treatment course and have a shorter interval than 1month between courses (Weinstein et al., 1980; Mayer et al., 1982b). In such programs there is no clear difference between consolidation and maintenance, since treatment during the maintenance period is as aggressive throughout the treatment period as consolidation in many other programs. The VAPA program (Table XVII) (Weinstein et al., 1980; Mayer et al., 1982b) is, so far, the one which has been shown to produce the longest duration of remission and the best survival. The median remission time has not yet been reached and the fraction still in remission at 1 year was 68%. However, more than half of the patients in this study were children, and results in adults were not as good. However, 11of 30 adults who obtained remission were still in continuous complete remission more than 18months after remission was obtained. Impressive results have also been obtained by Gale et al. (1981), who treated considerably older patients. Using daunorubicin, ARA-C, and thioguanine with intensive intermittent courses for 24 months, the fraction of patients still in remission at 1 year was 55%. Rather subtle changes in a treatment program may change the results. For example, in the study by Rai et al. (1981), 125 patients were randomized for maintenance treatment with five drugs-6thioguanine, ARA-C, CCNU, cyclophosphamide, and daunorubicin. Randomization was made to ARA-C, either given subcutaneously or intravenously (rapid iv bolus). The treatment was identical in other respects. The difference in the results was astonishingly large. Patients who received ARA-C subcutaneously had a median duration of complete remission of 18 months, as opposed to 8 months for those who received ARA-C intravenously. The fraction of l-year survivors was 58% in the group that received ARA-C subcutaneously and 42% in the group that received ARA-C intravenously. This illustrates that results may well improve considerably when more knowledge is gained about how to use the presently available drugs more efficiently.

M AINTENANCE AND

CONSOLIUATION

TABLE XVI CHEMOTHERAPY,REMISSION AND

SURVIVAL 1N

Number of patients CR

followetl

Reference

(%)

inCR

Wiernick and Serpick (1972) Gahrton et a1. (1974) Ud6n et al. (1975) Clarkson et al. (1975)

40 31 32 56

17 16 25 49

None None None ARA-C, T C

Burke et al. (1977) Chard et a / . (1978) Vaughan et ul. (1980) Lewis et al. (1981)

46 59 56 41

12 94 19 24

Lewis et al. (1981)

41

24

Peterson and Bloomfield (1981) Rai et al. (1981) Rai et al. (1981)

58 43

26 65

None None None ARA-C, TC (3 months) ARA-C, TG (3months) None None

43

60

None

Gale et al. (1981)

82

56

DNR, ARA-C, TG

Mayer et al. (1982b)

70

75

None

Consolidation

Maintenance None MP DNR, ARA-C, TC monthly DNR, TG, C, Vc, BCNU, Hydrea, Mx (L6 protocol) None ARA-C, TG, C, VO None None

ANLL

Median duration of CR (months)

In CR at 1 year

2 6 10 10

12 15 29 45

9 12 10

25 48 47

(%)

L6 protocol ARA-C, TG ARA-C iv, TC, CCNU, C, DNR ARA-C SC,TC, CCNU, C, DNR DNR, ARA-C, TG (24 months) Dox, ARA-C, J-Aza, Mx, MP, P, Vc (VAPA protocol) (16 months)

17

52

13

55

32

68

Comment

Randomized for maintenance or no maintenance Randomized for ARA-C iv or sc

Median age 14.5 years

283

TREATMENT OF ACUTE LEUKEMIA

TABLE XVII VAPA PROGRAM FOR MAINTENANCE OF REMISSION IN ANLL' Sequence I

Sequence I1

Doxorubicin, 45 mgimzlday iv, day 1

Doxorubicin, 30 mgim2/day iv, day 1

Sequence I11

Sequence IV

Vincristine, 1.5 mg/ ARA-C, 200 mg/m2/ m2/day iv, day 1 day continuous infusion, days

1-5 ARA-C, 200 mg/m2/ 5-Azacytidine, 150 mg/m2/day conday continuous tinuous infusion, infusion, days days 1-5 1-5

Methylprednisolone, 800 mg/m2/day iv, days 1-5 6-Mercaptopurine, 500 mg/m2/day iv, days 1-5 Methotrexate, 7.5 mgimziday, days

1-5 Given four times at 3-4 week intervals

Given four times at 4 week intervals

Given four times at 3-week intervals

Given four times at 3-4 week intervals

~

Weinstein et al. (1980) and Mayer et al. (1982).

C. DURATION OF CHEMOTHERAPY AND SURVIVAL

A large number of studies have shown that maintenance therapy is of benefit for the patient. However, there is considerable controversy over how long the maintenance therapy should continue. Earlier, when the duration of remission was short, this was not a problem. However, now that the remission period is much longer the question arises whether prolongation of the treatment really prolongs the remission period. The motivation of the patient to continue treatment decreases with the time spent in remission. Certain drugs, like the anthracyclines, have specific side-effects, i.e., cardiotoxicity after a certain cumulative dose. Infections related to bone marrow suppression and immunosuppression appear in many patients during therapy and are clearly therapy related. In ALL in children, cessation of therapy was tried in uncontrolled studies already in the 1960s (Pinkel, 1976; George et al., 1979). Earlier reports claimed that very few patients relapsed after cessation of therapy at 24 to 36 months. However, with longer observation times, the percentage relapsing after cessation of therapy now amounts to 15-20% (Table XVIII). This has resulted in a recent randomized

284

GOSTA GAHRTON

RELAPSEAFTER

Reference

TABLE XVIII CESSATION OF THE~UPY IN CHILDREN WITH ALL

Number of patients with discontinued therapy

Time of treatment before discontinuation of therapy (months) ~

Custavsson et al. (1981a,b) Rivera et ol. (1981) Nesbit et al. (1982)

95

288

] randomized

(160”)

36 24-36 36 60

~

Relapses after discontinuation of therapy (%) ~~~

20 20 22 17

After 36 months of maintenance treatment, randomization was made to discontinue (n = 156) or continue treatment for 60 months (n = 160).Seventeen percent of the patients relapsed in the latter group after the time of randomization.

study where one group of patients received therapy beyond this time, and the relapse rate was somewhat lower in children who continued on maintenance therapy (Nesbit et uZ., 1982). However, most centers advocate cessation of therapy at 36 months. Unless the patients relapse during the first 2 years after cessation of therapy, most centers regard them as cured. However, it has to be pointed out that relapses do occur at later times, although very rarely. In one case reported by Miller (1980) relapse occurred 9 years after diagnosis and 6 years after therapy had been discontinued. There are no studies which give an answer to the question of whether cessation of therapy in ALL in adults is of benefit for the patient. However, several studies use similar programs for the treatment of adult ALL to those now used in children with poor-risk ALL. In such programs, therapy is discontinued after 3 or 3.5 years. With the very intensive protocol developed by Clarkson et al. (1982), the curve for the duration of remission shows a plateau after 48 months of treatment. However, on the L-2 protocol only 6 patients of 23 (26%) who went into remission are on this plateau, and on the L-10 and L-10M protocols 11 of 61 patients who obtained remission have reached the plateau. The projected fraction of survivors among those who entered remission on these two programs (L-10 and L-1OM) is about 60%.Despite the very impressive results with the L-10 plus L10M protocols, they do not really give an answer to the question of whether cessation of treatment at 3.5 years is better than continuation. Similarly, in a study by Mayer et ul. (1982a),treatment was discontinued after 30 months in both adults and children. Although a plateau in

TREATMENT OF ACUTE LEUKEMIA

28.5

both duration of remission and survival is projected after 3.5 years for about 40 and 60% of the patients respectively, only 7 adult patients have so far reached this time and have been in continuous complete remission for between 3.5 and 6.5 years. Thus, although treatment has been terminated in several studies on adult ALL with apparent success, there is no definite answers to the question of whether this is really better than continuing the treatment. In ANLL this issue is still more controversial. Most centers continue treatment for about 5 years, but in recent studies with a more intensive maintenance program several centers discontinue treatment after 18 months to 3 years (Weinstein et al., 1980; Mayer et al., 1982b; Arlin and Clarkson, 1982). On the VAPA protocol (Weinstein et al., 1980; Mayer et al., 1982b), in which therapy is discontinued after about 16 months of intensive maintenance therapy, 8 of 37 patients who have completed the therapy have relapsed after this time. Four of 25 patients were children and 4 of 12 adults with a median age of 34.5 years. Some of the patients who entered remission and in whom therapy was discontinued have been followed for up to 66 months. In children, all relapses occurred very early after cessation of therapy. However, in the adults relapses have occurred up to 15 months after discontinuing treatment. Thus, although these results are the best so far presented in ANLL, they do not show that cessation of therapy is superior to continuation. In a recent randomized study by Sauter et al. (1982) 65 patients still in remission after 2 years of maintenance treatment were randomized to cessation or continuation of the maintenance treatment. After a median follow-up time of 29 months no significant differences in remission duration or survival between the two groups were found. However, when the results were presented at the ASCO meeting there appeared to be a tendency for a better prognosis in those who were on continuous maintenance treatment than in those in which therapy was stopped. Until further patients have been studied and the follow-up time is longer, it cannot be concluded from this investigation that cessation of therapy after 2 years should be advocated. The future results of this study will, however, be very important and it should be the aim of all centers who stop therapy to do this on a randomized basis. With long-term follow-up, most centers do not obtain more than 1020% survivors in adult ANLL (Table XIX). The results obtained by Mayer et al. (198213)may be due to a considerable extent to the large fraction of younger patients. Nevertheless, further intensification of treatment during the maintenance period may substantially increase the number of long-term survivors.

TABLE XIX LONG-TERM SURVIVAL IN ANLL Projected or obtained survival (%) at

Reference

Number of Datients treated

Lister et a!. (1980) Keating et al. (1981) Foon et 01. (1981)

86 245 107

Peterson and Bloomfield (1980) Rai et al. (1981) Mayer et al. (1982b)

58

0

b

352 107

3 years

Age Treatment

Range

Ch, ChIm Ch, ChIm Ch (50%) ChIm (50%) Ch

15-59 15->80 15-82

Ch Ch

Percentage of all patients. Percentage of patients who had a complete remission.

17-72 <20-84 0-50

Median

Total"

4 years

CR"

Total"

CR"

5 years Total"

CR"

12

19

10 16 29

38

48

22

35

18

27

18

23

14.5

16 48

30 68

14 43

27 62

11 43

21 62

9 12

TREATMENT OF ACUTE LEUKEMIA

287

D. NEW DRUGSAND TREATMENT OF REFRACTORY PATIENTS New combinations of drugs and more intensive regimens are generally tried in patients who relapse, or who are refractory to the most common drug combinations. Very high doses of ARA-C have proved to be efficient in some refractory and relapsed patients, which illustrates that in the same patients previously used drugs may induce remission at drastically higher dose levels (Frei et al., 1969; Rudnick et al., 1979). In such studies, a 30-fold increase in the ARA-C dosage (3 g/m2, 3-hr infusion every 12 hr for 4 consecutive days) has been used and proved to be effective. Also, it appears that at this high dose level, the efficacy of ARA-C can be synergistically improved by the addition of L-asparaginase (Capizzi, 1982), although L-asparaginase does not seem to improve the efficacy of combinations which include ARA-C at conventional dose levels (Gahrton et al., 197413; Ud6n et al., 1975). Adding L-asparaginase to high-dose ARA-C resulted in complete remissions in four of eight patients with refractory ANLL and in three of nine patients with advanced ALL (Capizzi et al., 1982). A number of new drugs have been tried in refractory and relapsed patients. Drugs that have proved to be effective in such patients (Table XX) are 5-azacytidine, amsacrine (m-AMSA),vindesine, the epipodophyllotoxins etoposide (VP 16) and teniposide (VM 26), ifosfamide, aclacinomycin A, dihydroxyanthracenedione (mitoxantrone), and 2'deoxycoformycin. A second remission is obtained much more frequently in children with ALL than in adult leukemia. Furthermore, remissions obtained in childhood ALL are generally longer than remissions obtained in adults.

1. 5-Azacytidine 5-Azacytidine is a pyrimidine-nucleoside analog. It was first isolated by a Czeckoslovakian group (Piskala and Sorm, 1964) from a species of Streptoverticillium. It resembles cytidine in chemical structure, and also in its mechanism of action. It interferes with DNA, RNA, and protein synthesis by inhibiting orthodylic acid decarboxylase. 5-Azacytidine is preferentially active in the S phase of the cell cycle, but has also some effect in resting cells. It appears not to exhibit cross-resistance with several of the commonly used cytotoxic drugs effective in acute leukemia, such as cytosine arabinoside, 6-mercaptopurine, vincristine, and the anthracyclines. 5-Azacytidine appears to be most active in ANLL (Karon et al., 1973; McCredie et al., 1973; Vogler et al., 1976; von Hoff and Slavik, 1977; Saiki et al., 1981). In a larger study of refractory patients, remis-

288

COSTA GAHRTON

NEW DRUGSIN

TABLE XX ACUTE LEUKEMIA, REFRACTORY OR IN RELAPSE

THE TREATMENT OF

Percentage remission in refractory or relapsed Drug SAzacytidine

ALL 0- 10

Amsacrine (m-ALMSA)

10-20

Vindesine

20-35

Etoposide (VP 16)

Teniposide (VM 26) Ifosfamide Aclacinomycin A

Dihydroanthracenedione (Mitoxantrone) 2-Deoxycoformycin

ANLL

References

8-24

Hrodek and Vesely (1971); McCredie et al. (1973); Vogler et al. (1976); Levi and Wiemik (1976); von Hoff and Slavik (1977); Saiki et al. (1977, 1981) Legha et al. (1980); Rivera et al. (1980); Arlin et al. (1980); Slevin et al. (1981) Baysass et al. (1979); Vats et al. (1981) EORTC (1973); Math6 et al. (1974); Smith et al. (1976); Bleyer et al. (1978) Rivera et al. (1975); Bleyer et al. (1979); Rodriguez et al. (1978) Rodriguez et al. (1978) Takahashi e t al. (1980); Yamada et 01. (1980); Jager et al. (1981); Warrell et al. (1981); Machover et al., (1982) Estey et al. (1982); Van Echo et al. (1982) Grever et al. (1980); Poplack et al. (1981)

17-25

0

0

8-35

3-13

5

33 12

0 21

4 (1/23) 17-22 (U6, 2/11) 7

0

sions were induced in 8% of those with ANLL and in none with ALL (Saiki et al., 1981) (Table XX). 5-Azacytidine has also been used for maintenance of remission in the VAPA program (Table XVII) (Weinstein et al., 1980). However, its contribution to the efficacy of this program, one of the best available for maintaining remission, has not been established.

TREATMENT OF ACUTE LEUKEMIA

289

Like other cytotoxic drugs, 5-azacytidine induces severe bone marrow depression. Gastrointestinal symptoms are severe in about 20% of the patients, and mild or moderate in about 40%. About 10% have diarrhea. Other side-effects that have been noted in some patients are fever, hypotension, myalgias, CNS symptoms such as coma, which appear in about 5%even at rather low dose levels, and moderate renal toxicity (Table XXI) (Saiki et al., 1981; Peterson et al., 1981; Case, 1982).Practical problems with 5-azacytidine include instability in solution. Therefore, it has to be freshly prepared and solutions must be changed every 4 to 6 hr. In summary, 5-azacytidine appears to be an interesting drug with efficacy in ANLL. However, there are several problems connected with its use, and therefore its place in leukemia treatment has yet to be established. 2. Amsacrine (m-AMSA) [4 I - ( 9-Acridinylamino)-methanesuZfon-m-anisidide]

Amsacrine is an acridine derivative, which was synthesized by Cain and Atwell (1974). It inhibits DNA synthesis by intercalating into DNA base pairs. It was shown to have a cytotoxic effect in several animal tumors, including the L1210 leukemias (Cain and Atwell, 1974). It has been studies in both ALL and ANLL refractory to other therapy or in relapse. Remission has been induced in between 10 and 25% of such patients (Table XX) (Legha et al., 1980; Rivera et al., 1980; Arlin et al.,1980; Slevin et al., 1981; Estey et al.,1982a). It has also been tried in combinations with thioguanine and ARA-C (Arlin et al., 1981) and has given promising results in refractory patients. The toxicity of amsacrine appears to be moderate in comparison to most other drugs which are effective in acute leukemia (Goldsmith et al., 1980; Slevin et al., 1981). In addition to bone marrow depression, nausea, vomiting, mucositis, and alopecia, hepatic toxicity has also been noted (Table XXI). Unusual complications are cardiac arrhythmias and grand ma1 seizures. Slevin et al. (1981) reported one patient who died with cardiac failure. However, this patient had received doxorubicin earlier, and it could not be concluded that amsacrine was the cause. Amsacrine appears to be a most promising drug in the treatment of acute leukemia. However, its place in therapy has not yet been established, and it is not known whether cumulative cardiotoxic effects may develop. Since the mechanism of action appears to be similar to that of the anthracyclines, trials with amsacrine should include a close watch for possible cardiotoxic side-effects.

TABLE XXI SIDE-EFFECTS OF NEW DRUGS IN ACUTE LEUKEMIAO

Bone marrow depression

Gastrointestinal (nausea, vomiting, diarrhea)

Neiirologic

Nephrotoxic and urothelial

5-Azacytidine

+++

+++

+

+

Amsacrine (m-AMSA) Vindesine Etoposide (VP 16) Teniposide (VM 26) Ifosfamide

+++ +++ +++

++ ++ ++ ++

++ + +

+++ +++

++ ++

++

Drug

Aclacinomycin A Dihydroxyanthracenedione (Mitoxantrone) 2-Deoxycoform ycin

++

+++

-, Not reported; (+), very rare; +, rare or mild;

++

+

+++

+

+ +, moderate; +++, severe.

Cardiac

+

Other and comments Fever, coma, hypophosfatemia

+ ++ -

-

++

++

Muco- Hepato- Alositis toxic pecia

-

++ + +

+

(+I

+

+

Fever Severe cystitis preventable with Mesna (see text) Fever

i+) Conjuctivitis

TREATMENT OF ACUTE LEUKEMIA

29 1

3. Vindesine Vindesine is a chemically derived structural analog of vinblastine (Barnett et al., 1978). In animals, it appears that the neurologic toxicity of vindesine is less than that of vincristine (Todd et al., 1976), although its spectrum of activity in tumors appears to be more like that of vincristine than that of vinblastine (Baysass et al., 1979).It has been tried alone or in combination with prednisolone in patients with ALL resistent to vincristine plus prednisolone (Math6 et al., 1978; Vats et al., 1981; Hulhoven, 1982). Remission was induced in 25 to 35% of such patients. However, other studies have demonstrated a significant degree of cross-resistance between the two drugs (Krivit et al., 1980). Vindesine has no effect in ANLL. Its toxicity is of the same kind as that seen with vincristine and vinblastine, but there are indications that bone marrow suppression may be somewhat less than with vincristine. If so, vindesine may be a valuable contribution to the treatment of ALL.

4. Etoposide (VP 16) and Teniposide (VM 26) The epipodophyllotoxins, etoposide and teniposide, are derivatives of podophyllotoxin, a substance known to induce metaphase arrest. In a search for less toxic podophyllotoxin derivatives, Stiihelin synthesized these two epipodophyllotoxins in 1970 (teniposide) and 1973 (etoposide),These substances appear to arrest cells not during mitosis but in the late S or Gz phase of the cell cycle (Grieder et al., 1977). Etoposide appears to have preferential action in ANLL, and particularly in myelomonocytic and monocytic types (Math6 et al., 1974; Smith et al., 1976; Bleyer et al., 1978,1979; Hurd et al., 1981; Look et al., 1981). In a compilation of several studies, the response rate in acute myelomonocytic leukemia was found to be 39% (Arnold, 1979). Etoposide has also been combined with other drugs. In one study, 10 of 22 patients with refractory or relapsed ANLL entered complete remission on etoposide plus 5-azacytidine (Look et al., 1981), and in another 3 of 7 patients with refractory myelomonocytic leukemia entered remission on etoposide combined with cyclophosphamide (Hurd et al., 1981). While etoposide appears to be most effective in acute myelomonocytic leukemia, teniposide is probably most effective in ALL (Dombernowsky et al., 1972; EORTC, 1972; Muggia et al., 1975; Bleyer et al., 1978, 1979). Several studies now use combinations of teniposide and other drugs, such as ARA-C, for refractory patients with childhood lymphocytic leukemia (Rivera et al., 1980, 1982) or poor-prognosis patients with ALL (Dahl et al., 1982). Treatment with teniposide and

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ARA-C in poor-prognosis patients resulted in 83% complete remissions in 33 patients. Toxicity, except for bone marrow depression, appears to be moderate with both etoposide and teniposide (Table XXI) and further studies are warranted.

5. lfosfamide (lsophosphamide) Ifosfamide is an analog of cyclophosphamide, which has an effect on a large number of tumors. Like cyclophosphamide, it acts by alkylation, Its limitation is mainly due to dose-limiting genito-urinary system toxicity. However, it was recently shown that this complication can be prevented by the simultaneous use of 2-mercaptoethane sulfonate (Mesna) (Brock et al., 1979; Scheef et al., 1979). Interest in ifosfamide was aroused by the finding that a response was obtained in cyclophosphamide-resistant L1210 leukemia (Brock, 1972). Ifosfamide has been shown to induce remission in refractory patients with ALL (Rodriguez et al., 1978). Some of these patients were considered resistant to cyclophosphamide. Ifosfamide has also been used in combination with doxorubicin in 32 patients with resistant or relapsed ALL by the Southwest Oncology Study Group. Complete remissions were obtained in 89% of previously treated patients (Ryan et al., 1980). The results of other studies also suggest efficacy in combinations with L-asparaginase and methotrexate (Yap et al., 1980). Ifosfamide has to be used in combination with Mesna, but in this combination it may prove to be a major contribution to the treatment of ALL. 6 . Aclacinom ycin A and Dihydroanthracenedione (Mitoxantrone)(DHAD) The cumulative dose-dependent cardiotoxicity of the most widely used anthracyclines, doxorubicin (adriamycin) and daunorubicin, seriously limits the use of these two drugs. Efforts have therefore been made in several laboratories to find analogs with less cardiotoxic sideeffects. Two of these are aclacinomycin A and dihydroxyanthracenedione (mitoxantrone) (DHAD). Aclacinomycin A was isolated in Umesawa’s laboratory in Japan (for review, see Oki, 1980). In experimental systems, aclacinomycin A has been shown to have an effect on a number of tumors, among them the L1210 leukemia. Its cardiotoxicity was compared to that of doxorubicin in mice with this leukemia. A fourfold increase in the dose of aclacinomycin A had to be used in order to induce the frequency of ECG changes that was induced by doxorubicin. Furthermore, several times higher doses of aclacinomycin A could be used before chronic myocardial damage was seen after prolonged treatment.

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Aclacinomycin has also been used in phase I1 trials. In one study of 32 patients with advanced colorectal carcinoma, no cardiotoxicity was reported (Korinek et al., 1982). However, in another, where aclacinomycin A was used to treat 55 patients with refractory malignant disease, one patient with previous myocardial infarction died in congestive heart failure, and two patients demonstrated increased systolic time intervals after 335 and 472 mg/m2.The two latter patients did not develop congestive heart failure (Roach et al., 1982).In another study, one patient showed dilatation of tubular structures and mitochondria and occasional myofibrillar loss on endomyocardial biopsy after receiving a cumulative dose of 700 mg/m2.However, this patient did not develop congestive heart failure (Unverferth et al., 1982). Thus, it appears that aclacinomycin A is less cardiotoxic than doxorubicin or daunorubicin. In addition to decreased cardiotoxicity, aclacinomycin A also appears to have the advantage of not inducing alopecia (de Jager et al., 1981), a common side-effect with doxorubicin and daunorubicin. Aclacinomycin A has been shown to induce remission in refractory or relapsed patients (Table XX) (Math6 et al., 1978; Takahashi et al., 1980; Yamada et al., 1980; de Jager et al., 1981; Warrell et al., 1981; Machover et al., 1982). Of 80 such patients with ANLL reported in these studies, 17 entered complete remission, and of 51 patients with ALL 6 entered complete remission. Thus, aclacinomycin A appears to be one of the most promising drugs for the treatment of acute leukemia, and phase I11 and IV studies should now be started. Dihydroxyanthracenedione (DHAD) was also developed during a search for anthracycline analogs with less or no cardiotoxicity (Von Hoff et al., 1980). DHAD is an anthraquinone drug which lacks the sugar molecule which has been blamed for the cardiotoxic side-effects of the anthracyclines (Adamson, 1974). In experimental systems, DHAD has been shown to have significant antitumor effect against several tumors, among them the L1210 leukemia. In addition, it appears to lack the cardiotoxic effect of the anthracyclines. Although the mechanism of action seems to be similar to that of the anthracyclines, i.e., inhibition of the DNA synthesis by intercalation between the base pairs of the DNA double-helix, no histological changes consistent with cardiotoxicity were found in experimental animals (von Hoff et al., 1980). Phase I1 trials with DHAD have been carried out in a number of studies, 17 of which were reported at the AACR and ASCO Meeting in 1982 (Schell et al., 1982; Knight et al., 1982; Stroehlein et al., 1982; Bonnem et al., 1982; Taylor and Von Hoff, 1982; Drelichman et al., 1982; Raghavan et al., 1982; Aapro et al., 1982; Neidhart and Roach, 1982; de Jager et al., 1982; Cowan et al., 1982;Van Echo et

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al., 1982; Callahan et al., 1982; Anderson et al., 1982; Estey et al., 1982b; Valdivieso et al., 1982; Pratt et al., 1982). Of 746 patients, 11 developed significant cardiac toxicity, most of them congestive heart failure. However, all of these patients had received significant amounts of doxorubicin before treatment with DHAD. In some of the patients, the association between DHAD and the cardiac failure was also questionable because of previous symptoms of heart disease. Thus, although it cannot be excluded that DHAD is associated with cardiac toxicity, maybe at higher cumulative dose than those that have been used so far, it appears that it is significantly less cardiotoxic than the anthracyclines doxorubicin and daunorubicin. Other sideeffects, commonly seen in patients treated with other cytotoxic drugs, seem to be acceptable. The efficacy of DHAD in refractory patients with acute leukemia appears to be comparable with that of the anthracyclines (Van Echo et al., 1982; Estey et al., 1982b) (Table XX). In summary, DHAD appears to be a promising drug for the treatment of acute leukemia and further studies are warranted. 7. 2’-Deoxycoformycin 2’-Deoxycofonnycin is an antibiotic nucleoside analog, which has been isolated from cultures of Steptomyces antibioticus (Woo et al., 1974). It is a powerful inhibitor of adenosine deaminase, an enzyme which has been found at increased levels in ALL leukemic cells (Smyth and Harrap, 1975). Some studies indicate that deoxycoformycin induces lysis of leukemic cells, while having little effect on myelopoiesis (Smith et aZ., 1980; Grever et al., 1980). Since adenosine deaminase values are usually high in leukemic cells from patients with T ALL, it has been suggested that its effect will be best in such patients (Prentice et al., 1981).However, an effect has also been seen in B-cell malignancies (Grever et al., 1980). Other studies indicate that deoxycoformycin does not inhibit normal erythroid, granulocytic, or T-lymphocytic colony growth, and this may indicate that deoxycoformycin may preferentially inhibit leukemic cell growth (Aye and Dunne, 1981). Deoxycoformycin has been tested in only a limited number of patients (Smyth et al., 1979; Koller et al., 1979; Prentice et al., 1981; Grever et al., 1980; Poplack et al., 1981). So far, remissions have been induced only in patients with lymphoblastic leukemias. Deoxycoformycin does not appear to depress bone marrow. The pronounced toxic effects are nausea, vomiting, diarrhea, hepatocellular enzyme elevation, conjunctivitis, and central nervous system symptoms, i.e., lethargy and seizures (Grever et al., 1980; Poplack et al., 1981). Deoxycoformycin is an interesting drug, particularly because of its lack of toxic effects on the bone marrow. It merits further study.

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8. Other Drugs Used in Refractory Patients Several new drugs are being tested in phase I and phase I1 trials. Indicine N-oxide is a pyrrolizidine alkaloid which has induced remission in both ALL and AML. However, its hepatic toxicity may be an obstacle to the further use of this substance (Letendre et al., 1981; Poster et al., 1981; Miser et al., 1982). The nitrosoureas, BCNU and CCNU, have been used in both refractory patients and in maintenance programs for the treatment of both ALL and ANLL, mainly in attempts to prevent or treat CNS leukemia (Sullivan et al., 1971; Smyth and Wiernik, 1976; Clarkson et al., 1982). However, their efficacy has not yet been tested in randomized trials, and at present they appear to be of limited value in the treatment of acute leukemia.

E. CARRIERS OF CYTOTOXIC DRUGS The use of cytotoxic drugs is hampered by their lack of selectivity for the tumor. This also applies to cytotoxic drugs directed against leukemic cells. Major efforts have therefore been made to find differences between leukemic cells and normal cells, which could be exploited to increase the selectivity of drugs. In 1972, Trouet and coworkers created the concept of lyzosomotropic cancer chemotherapy (Trouet et al., 1972). By coupling the anthracyclines to DNA as a carrier, it was thought that the complex thus created could be taken up more readily by leukemic cells by pinocytosis than by many other cells, such as cardiac cells. The cardiotoxicity of the anthracycline drugs could thereby be decreased. Liposomes have also been tested as carrier in experimental systems. A number of drugs can be incorporated into liposomes, and carried to the target. They too are thought to be taken up by endocytotic activity. An approach which appears to be more promising is the conjugation of cytotoxic drugs to monoclonal antibodies. However, so far this has not been exploited clinically. 1. Anthrac ycl ine-DNA Complexes In a series of studies in the early 1970s, Trouet and co-workers showed that anthracyclines could be coupled to DNA and still be used effectively for the treatment of experimental leukemia (Trouet et al., 1972, 1974; Trouet and de Campeneere, 1979). In fact, in the experimental situation the doxorubicin-DNA complex had an increased efficacy and decreased cardiotoxicity compared to unbound doxorubicin. Later studies by other groups have shown that a decreased cardiotoxic effect was also obtained in patients treated with DNA-bound anthracyclines compared to treatment with the unbound drugs (Ferrant et

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al., 1979; Gahrton et al., 1979; Paul et ul., 1981a,b). However, extensive pharmacokinetic studies in both plasma (Hulhoven et al., 1977, 1979; Andersson et al., 1979; Paul et al., 1980, 1981a,b) and leukemic cells (Andersson et al., 1979; Paul et al., 1979,1980,1982) have shown that the reason for decreased cardiotoxicity is probably that the binding of DNA leads to the slow release of the anthracycline from the complex, which prolongs the exposure of leukemic cells to the drug, but decreases the volume of distribution, and thereby probably decreases the cardiotoxicity. The daunorubicin-DNA complex has been tested for induction of remission and maintenance treatment of ANLL in adults (Ferrant et al., 1979; Gahrton et a/., 1979; Paul et al., 1981b). In the Swedish study (Gahrton et al., 1979; Paul et ul., 1981b) treatment resulted in complete remission in 70% of the ANLL patients under 60 years of age. The doxorubicin-DNA complex appears to be more stable than the daunorubicin-DNA complex. This indicates that doxorubicin-DNA might be less cardiotoxic than daunorubicin-DNA. It also appears that doxorubicin-DNA increases the concentration of the drug in leukemic cells more readily than either the free drug or daunorubicinDNA. This implies that the doxorubcin-DNA complex might also be more efficient in the treatment of acute leukemia. Studies by Lie et ul. (1979) and preliminary studies by the Leukemia Group of Middle Sweden (unpublished) seem to support this view. 2. Liposomes and Low-Density Lipoproteins Several cytotoxic drugs can be entrapped in either the aqueous phase of the liposomal structure or in the lipid phase (Kaye and Richardson, 1979) Drugs that can be entrapped rather easily are vinblastine and doxorubicine, while cytosine arabinoside, for example, shows low entrappment and higher rate of leakage. Some enhancement of efficacy and decrease in toxicity has been claimed with cytosine arabinoside and methotrexate entrapped in liposomes. However, the increase in efficacy has been marginal or doubtful in many trials, perhaps due to the fact that there are no known receptors specific for liposomes on tumors. However, such receptors for low-density lipoprotein (LDL) have been found in relatively high levels in human myelogenous leukemic cells (Ho et al., 1978; Vitols et al., 1983a). Evidence has been presented that once LDL is bound to its receptor, it is internalized and its components undergo hydrolysis, presumably in the lyzosome (Goldstein and Brown, 1977). In a current study, we have managed to incorporate lipophilic anthracyclines, i.e., AD 32 and aclacinomycin, into LDL (Vitols et al., 1983b). In vitro, the LDL-

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AD 32 complex was more readily taken up in ANLL leukemic cells than in control cells. Thus, it appears possible to further exploit LDL as a carrier for these anthracyclines, and hopefully this will make it possible to use these drugs more efficiently in leukemia. 3. Monoclonal Antibodies Attempts have been made earlier to use antibodies directed against tumor-associated antigens to carry cytotoxic drugs (Rubens, 1974). However, the difficulty in obtaining pure antibodies with absorption techniques, and the difficulties in producing large amount of antibodies, proved to be serious obstacles in pursuing some initially positive results in experimental systems (Math6 et al., 1958; Isliker et al., 1964). Using the hybridoma technique described by Kohler and Milstein (1975), monoclonal antibodies can now be produced in practically unlimited amounts. Although monoclonal antibodies per se can be used to kill leukemic cells together with complement, it appears that several of the cells to which binding occurs still survive such treatment (Ritz et al., 1981; see below). However, the fact that binding occurs seems to be the ideal prerequisite for obtaining an effect of a cytotoxic drug carried by the monoclonal antibody. So far, treatment with cytotoxic drugs carried by monoclonal antibodies has only been carried out in experimental system (Ghose and Blair, 1978; Arnon et al., 1980; Leserman et al., 1980). The prospect for the future is that monoclonal antibodies against such antigens as cALLa, as well as other leukemia-associated antigens, will be used to carry drugs which are effective in leukemia. Hopefully, this will result in increased efficacy and decreased toxicity. V. lmmunotherapy

The immunological approach to the treatment of tumors is based on results indicating that the neoplastic cells carry tumor-specific or tumor-associated antigens (Prehn, 1963; Old and Boyse, 1964; Klein, 1966). Such antigens also appear on blast cells in acute leukemia (Greaves et al., 1978), and recently it has been possible to raise a number of monoclonal antibodies against them (for reviews, see Aisenberg, 1981; Ritz and Schlossman, 1982). However, so far these antibodies have mainly been used for diagnostic purposes, and only recently have they been used for treatment in a few cases. Immunotherapy can be used in three principally different ways: passive, adoptive, and active. Passive immunotherapy involves the

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transfer of antibodies directed against antigens or the tumor cells. It may be nonspecific if antibodies are raised which are active against both tumor-associated antigens and normal antigens. Specific passive immunotherapy should only be directed against tumor-associated antigens. Treatment with monoclonal antibodies is an example of this approach. Adoptive immunotherapy involves the transfer of the whole immunological machinery to the patient, for example by transfusion of immunocompetent lymphocytes, bone marrow cells, or stem cells which can mature to immunologically competent cells. An adoptive immunotherapeutic effect by transplanted bone marrow cells seems to operate in the allogeneic situation (see Section VI). The most tested, but also the most debated, form of immunotherapy is active immunotherapy. This therapy aims to stimulate the patient’s own immune defense. The term specific active immunotherapy is used when the stimulatory agent is supposed to induce a reaction directed against the tumor-associated antigens. Nonspecific active immunotherapy means the general stimulation of the host immune response, which then hopefully directs some of its activity against the tumor cells. Trials with both specific and nonspecific active immunotherapy have been carried out extensively in acute leukemia. A. ACTIVEIMMUNOTHERAPY

1. Acute Lymphoblastic Leukemia (ALL) In 1969, Math6 et al. reported on the first study of active immunotherapy in 30 children with ALL. After induction of remission, 10 children received no further treatment, while 20 others were given weekly either Pasteur BCG by scarification or irradiated allogeneic ALL cells, or both BCG and leukemic cells. The untreated patients relapsed within 130 days, while 10 of the 20 patients who received immunotherapy survived for more than 295 days. Some of these patients have now been in complete remission for more than 10 years. This study started an ever-increasing number of trials with active immunotherapy in man, both in cases with solid tumors and in leukemia. In a study, initiated by the British Medical Research Council in 1969 (Medical Research Council, 1971; Kay, 1978) 121 patients with ALL in remission were randomized to no further treatment, treatment with Glaxo BCG by the multiple-puncture method (Heaf gun), or methotrexate. The group treated with methotrexate had a significantly

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longer duration of first remission and of survival than the other two groups, while the group treated with BCG did not differ from the group who received no treatment. In a number of studies similar negative results of immunotherapy in patients with ALL have been found (Heyn et al., 1978; Poplack et al., 1978; Ekert et al., 1980; Andrien et al., 1978). BCG from various strains has been used. It has been administered by multiple puncture or scarification, alone or together with allogeneic leukemic cells, but still results have always been negative. At the same time as these mainly negative results in ALL have been reported, the results of chemotherapy have continued to improve. At the present time, from the ethical point of view, it is obviously impossible to use BCG alone, or for that matter any form of immunotherapy, as the sole treatment for maintenance of remission in ALL. Further trials will have to be combined with chemotherapy, and will therefore be increasingly difficult to interpret.

2. Acute Nonlymphoblastic Leukemia (ANLL) The results of active immunotherapy in ANLL have been somewhat more positive than in ALL. In 1970, Powles et al. (Powles et al., 1977; Alexander and Powles, 1978) started the first randomized trial with adjuvant active immunotherapy in this disease. The patients were allocated to the two groups after the induction of remission. One group received intermittent chemotherapy monthly plus weekly immunotherapy with lyophilized Glaxo BCG administered with a multiple-puncture method (Heaf gun) and simultaneously lo9 stored allogeneic leukemic cells killed by irradiation. The other group received an identical regimen of chemotherapy, but no immunotherapy. Twenty-two patients received chemotherapy and 28 chemoimmunotherapy. The median survival time from clinical remission was 270 days in the chemotherapy group, and 510 days in the immunotherapy group. The follow-up of these results shows that the difference in median survival time was retained. However, after about 2 years, the curves overlapped, and there was no difference between the groups as regards the number of long-term survivors. In fact, the shapes of the curves were similar in the two groups. From 1973 to 1979 the Leukemia Group of Central Sweden made an attempt to repeat these results, the only difference being that live allogeneic cells were given instead. Twenty-two patients were randomized to chemoimmunotherapy, and 20 to chemotherapy only (Gahrton et al., 1976a; Lindemalm et al., 1978). The median survival time of the chemotherapy group was 344 days and that of the chemoimmunotherapy group 734 days, a difference that was statistically significant. Patients continued to enter this

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study until 1979, when 36 patients had entered the chemotherapy group and 40 patients the chemoimmunotherapy group. Both the duration of the first remission and the survival time were then significantly different for the two groups and to the advantage of the chemoimmunotherapy group. However, astonishingly enough, there was no difference between the two groups in the patients admitted to the study during the period 1976 to 1979, the whole difference being found in patients who entered during 1973 to 1976 (Reizenstein et al., 1982). No major changes had been made in the chemotherapy program, and those minor changes which were made, i.e., the treatment of one group of patients with daunorubicin-DNA complex and a short period of more intensive treatment with daunorubicin, were similar in the two maintenance groups (chemoimmunotherapy and chemotherapy) because of the random allocation of patients to the groups. However, during the latter period (1976-1979) the frequency of remission increased from about 50 to about 70%. Furthermore, the change in remission duration and survival, which resulted in the elimination of the significant difference, affected both groups but in opposite directions. The survival in the chemoimmunotherapy group decreased from the first time period (1973-1976) to the second (1976-1979) (from 24.0 to 16.0 months), while the survival in the chemotherapy group increased from 12.0 to 16.5 months. It was then speculated that increasing the fraction of patients in remission resulted in the entrance into the remission group of patients who responded better to maintenance chemotherapy and worse to immunotherapy than other patients. However, other data do not support this view, since patients who enter remission rapidly have a longer remission duration than late responders (see Section 111,A). Whatever the reason may be, in our hands increasingly better results with chemotherapy seem to abrogate the positive effect of immunotherapy. Other studies (Gale et aZ., 1981) have also failed to demonstrate an effect of irnmunotherapy with BCG plus leukemic cells in ANLL patients who receive intensive induction therapy and intensive intermittent maintenance chemotherapy. Active immunotherapy in ANLL has also been tried after attempts to increase the immunogenicity of the leukemic cells in oitro. This can be done by pretreating the leukemic cells with neuraminidase {Bagshawe and Currie, 1968; Bekesi et al., 1971). In a preliminary study by Bekesi and Holland (1982), 5 of 9 patients treated with chemotherapy plus neuraminidase-treated allogeneic leukemic cells became long-term survivors, and are still living 5-7 years after the induction of remission. All the control patients treated with chemotherapy only died within 1 year. Unfortunately, when larger

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numbers of patients entered a randomized trial the results were substantially poorer. In this study, 11 patients were randomized to chemotherapy only, 22 to chemotherapy plus neuraminidase-treated allogeneic leukemic cells, and 21 to chemotherapy plus MER (the methanol-retraction residue of BCG). The median duration of remission was 13 months in the neuraminidase-treated patients, but only 6 months in the control group. In the group which received both M E R and neuraminidase-treated cells the duration of remission was 10 months. Thus, the positive effect of the neuraminidase-treated cells was to some extent abrogated by the MER. Six of 22 patients treated with neuraminidase-treated myeloblasts are still alive 4-6 years after remission, 4 of them still in their first remission period. All patients in the other two groups have died by 3.5 years after the onset of treatment. Immunotherapy, with BCG as the only immunostimulant during remission has been used in a number of investigations (Peto, 1978; Whittaker et al., 1980;Lister et al., 1980;Bodey et al., 1980;Omura et al., 1982a,b).In two, a marginally positive effect has been claimed (Bodey et al., 1980;Whittaker et al., 1980),but in the other no effect could be demonstrated. One of the most significant studies even showed that BCG treatment might reduce the effect of chemotherapy in maintaining remission. This study by the Southeastern Cancer Study Group (Vogler et al., 1982)is important, particularly because of its size. Of the 690 patients who entered the chemotherapeutic induction program, 64% went into remission. After a period of consolidation chemotherapy for 9 weeks, 425 patients still in remission were randomized for (1)continuous infusions with cytosine arabinoside for 5 days plus daunorubicin for 2 days in courses which were repeated every 13 weeks for 12 months; (2)BCG (Tice strain) twice weekly for 4 weeks, then once monthly by intradermal injection with a Heaf gun; and (3)a combination of (1)and (2).Seventy-five patients were treated with chemotherapy only, 69 with BCG only, and 77 with the combination. The proportion of 1-year survivors was 63% with chemotherapy only, 28% with BCG, and 23% with chemotherapy plus BCG. Thus, BCG adversely affected the results, not only when given alone, but also when given together with chemotherapy. Other modulators of the immune system have been tried in an attempt to prolong remission and survival in ANLL. Levamisole hydrochloride, S-(2,3,5,6-tetrahydro-6-phenylimidazo)-2,lb-thiazole, is an anthelmintic which also has immunostimulatory properties. In a randomized study, the Finnish Leukemia Group (Lehtinen et d.,1982) found a significant prolongation of the duration of the first remission in 26 patients treated with chemotherapy plus levamisole, as com-

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pared to 25 patients treated with chemotherapy only. The difference was, however, marginal (195 days median duration of remission with chemotherapy only, and 243 days with chemotherapy plus levamisole), and significant only when analyzed with the x’ test. In two other studies, one by a Danish group (Thorling et al., 1978) and one by the NCI Baltimore Cancer Research Center Group (Chang et al., 1978), no effect of levamisole could be demonstrated. To summarize, the results of active immunotherapy in ANLL show that the place of this treatment still has to be defined. Specific immunotherapy with allogeneic leukemia cells may have a positive effect in the treatment of some patients. However, in comparison to the improved results obtained with chemotherapy during the last 10 years, the possible effect of immunotherapy appears marginal. In fact, in one important study treatment with BCG in ANLL even had an adverse effect.

B. PASSIVE IMMUNOTHERAPY-MONOCLONAL ANTIBODIESAND INTERFERON 1. Heteroantisera and Monoclonal Antibodies

Early attempts to use heteroantisera to treat patients with ALL were mainly unsuccessful. However, some response was obtained with a rabbit antimouse antilymphocytic serum in experimental leukemia. A prerequisite for efficacy in the experimental system appeared to be that the number of leukemic cells in the animals was small (less than lo3leukemic cells) (Math6,1972). After a dormant period in the 1970s, development of the technique for producing monoclonal antibodies (Kohler and Milstein, 1975) has revived interest in passive immunotherapy of hematological malignancies (Nadler et al., 1980; Ritz et al., 1981; Miller et al., 1982; Ritz and Schlossman, 1982). An antibody, 55, specific for the common acute lymphoblastic leukemia antigen (cALLa) was used in an attempt to treat four patients with cALLapositive non-T ALL (Ritz et al., 1981; Ritz and Schlossman, 1982). A rapid and dramatic decrease in the number of peripheral blood lymphocytes occurred within hours after administration of the antibody. However, after withdrawal of the treatment, there was also a dramatic rise to pretreatment levels, and no response was seen in the bone marrow. Thus, although these results are only weakly encouraging, the positive result of treatment of another patient, who had a B-cell lymphoma, with monoclonal antiidiotypic antibody, indicates that

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further trials in acute leukemia may be worthwhile. The patient with B-cell lymphoma (Miller et al., 1982) was a 67-year-old woman with nodular, poorly differentiated, lymphocytic lymphoma in relapse after previous chemotherapy. The treatment with the antiidiotypic antibody resulted in a complete remission, which at the time of writing had lasted for 6 months. Thus, it is possible that certain patients might respond to treatment with monoclonal antibodies. There are many possible mechanisms by which monoclonal antibodies may act, e.g., by antibody-mediated cell killing, complement-dependent cytolysis, antibody-dependent cytotoxicity by killer cells, or phagocytosis by the reticuloendothelial system of antibody-coated leukemic cells. So far, the use of monoclonal antibodies in leukemia treatment has been more important for purging marrow for autologous bone marrow transplantation, and for elimination of T cells in vitro for prevention of graft-versus-host disease following allogeneic bone marrow transplantation (see Section VI).

2. Znterjkron Interferon is an antiviral substance which was discovered in 1957 by Isaacs and Lindenmann. Apart from its antiviral properties, it also inhibits tumor progression in experimental systems (Gresser, 1977). The mechanism by which interferon exerts its antitumor effect is unknown, although an increase in spontaneous cytotoxicity has been demonstrated in man both in vitro and in vivo (review by Einhorn et al., 1980). Recently the Swedish Myeloma Study Group (Mellstedt et al., 1982) has shown that about 20% of patients with multiple myeloma respond to interferon treatment. Encouraged by these results we have used interferon to treat a patient with ALL and Down’s syndrome (Bratt et al., 1983). A second reason for the use of interferon in this patients was that the gene regulating interferon receptor level is probably located in chromosome No. 21 (Tan, 1976). Thus, an increase in the number of receptors on the leukemic cells is to be expected. Interferon treatment in this patient resulted in the disappearance of peripheral blast cells, and a concomitant increase in the natural killer cell activity in uivo. However, there was no bone marrow remission. Other previous attempts to treat children with acute leukemia have given either negative (Ahstrom et al., 1974) or moderately positive results (Hill et al., 1979). In the latter study, there was some response in peripheral blast count in four of five patients, and in one a short temporary remission was obtained. In one study (Vuopio et al., 1981), interferon, administered as a daily injection between chemotherapy

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treatment courses, was used during both the induction and the maintenance phase in ANLL. So far, there is no statistically significant difference between the group treated with interferon plus cheniotherapy and the group treated with chemotherapy only. In these studies, interferon produced by leukocytes (Cantell et al., 1974; Cantell and Hirvonen, 1977) was used at widely different dose levels. However, due to the scarcity of leukocyte interferon, the dosages used in most of the patients were not very high. Interferon can now be procured in unlimited amounts by various methods. In vitro assays with highly purified cloned interferon a2 produced by E. coli has shown suppression of the clonogenic capacity of cells from patients with ANLL (Hoffman et d., 1981). Thus, although the results of clinical trials with interferon in acute leukemia have been mainly negative, it is too early to say whether interferon will have a place in the treatment of this disease.

VI. Bone Marrow Transplantation

A. ALLOGENEICAF;D SYNGENEIC TRANSPLANTATIOK Bone marrow transplantation is now an established method for treating acute leukemia. It was first tried in humans by Thomas et al. (1957) and Math6 et ul. (1959). Although antigenic markers on red cells established engraftment in these early patients, it was only temporary. The patients either died rather quickly or lost markers of engraftment. Attempts to treat patients with acute leukemia with bone marrow transplantation were generally unsuccessful until the 1970s. Before that time, bone marrow transplantation was mainly attempted in terminal leukemia. However, in 1977 Thomas et al. reported that 13% of patients with acute leukemia in relapse became long-term survivors after bone marrow transplantation. It was also shown that patients who were in a less advanced stage of relapse fared better than those with advanced disease. This opened the door for treatment of patients in remission with bone marrow transplantation, and the results were dramatically changed to the better. Bone marrow transplantation has been made possible by advances in immunology, chemotherapy, and the treatment of infectious diseases. The most important developments have been (1)the definition of the HLA system (Ceppelini and van Rood, 1974) and the elucidation of its importance for transplantation (van Bekkum, 1974), ( 2 ) the

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development of chemotherapeutic and radiotherapeutic techniques to suppress the immune response in the host and to kill a maximum number of leukemic cells (Thomas et al., 1961; Santos, 1974), (3) the development of methods for prophylaxis and intensive treatment of infections in severely cytopenic and immunosuppressed patients during the posttransplantation period (Buckner et al., 1978), and (4) the development of methods to prevent graft-versus-host disease (GVHD) (Thomas et al., 1975). B. TECHNIQUE FOR BONEMARROWTRANSPLANTATION Bone marrow transplantation for the treatment of acute leukemia uses mainly HLA-identical sibling donors. Some centers have tried to use haploidentical donors and donors mismatched at selective loci (Clift et al., 1979; Powles et al., 1982a,b). This approach is still experimental, and for a high success rate it is essential to use an HLAidentical sibling (Thomas et al., 1975, 1979a). The pretransplant treatment includes high-dose cyclophosphamide, intrathecal methotrexate, and total-body irradiation with 10 Gy on the day of transplantation (Thomas and Storb, 1970; Thomas et al., 1975). Since a dose of 10 Gy has been associated with a high incidence of interstitial pneumonia (Keane et al., 1981; van Dyk et al., 1981; Bortin et al., 1982), shielding of the lungs to 800 or 900 R is common. Some centers use fractionated irradiation over several days (Peters et al., 1979; Shank et al., 1981; Clift et al., 1982a,b; Buckner et al., 1982a). Two to 2.5 x los bone marrow cellskg body weight of the patient are aspirated from the donor under sterile conditions in an operating room. Most part of the marrow is obtained from the iliac crest, particularly around the superior-posterior iliac spine. The marrow is prepared in a cell-culture medium to obtain a single-cell suspension. Generally, this is achieved by passing the marrow through one of two different gauge stainless-steel screens. The marrow is infused intravenously within 4 hr. The postengraftment regimen to prevent graftversus-host disease varies, but most centers use methotrexate (Thomas et al., 1975) for 3 months. Indirect signs of engraftment are reticulocytosis (generally the first sign) and increasing granulocyte and platelet counts. In most patients the marrow is completely repopulated in between 3 and 5 weeks posttransplantation. Chromosome markers are the most important direct markers. When the sexes of the donor and recipient are different, the X and Y chromosomes are distinct markers. Heteromorphic re-

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FIG.3. Chromosomal markers used to establish engraftment following bone marrow transplantation (BMT).The donor has fluorescent markers in the centromere region of chromosome Xo. 3 and fluorescent satellites on chromosome No. 22 which are found in the recipient after transplantation. Q-banding technique.

gions, best visualized by the Q-banding technique (Caspersson et al., 1971), have been successfully used to indicate engraftment when the donor and recipient are of the same sex (Gahrton et al., 197613) (Fig. 3).

c. SURVIVAL AFTER ALLOGENEICBONE MARROW TRANSPLANTATION Several factors influence the survival of patients after bone marrow transplantation. The most important is whether the patients are given a transplant in relapse or remission. In the largest series of patients

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treated in relapse (Thomas et al., 1977; Badger et al., 1982), 27 of 138 patients with ANLL or ALL are alive 3-9.5 years after transplantation. At present, bone marrow transplantation is mainly carried out in patients in remission. In general, ANLL patients receive a transplant during the first remission, increased-risk ALL patients during the first or second remission, and standard-risk ALL patients during the second or later remissions. Up to now, most centers have obtained results indicating a 50-60% probability of disease-free survival in ANLL (Thomas et al., 1979a; Blume et al., 1980; Powles et al., 1982; Zwaan and Hermans, 1982; Storb, 1982; Ringden et al., 1982) and a 40-50% probability of disease-free survival in ALL (Thomas et al., 197913; Johnson et al., 1981; Thomas and Johnson, 1982; Clift et al., 1982b; Zwaan and Hermans, 1982) (Fig. 4). It has to be pointed out that these figures generally pertain only to patients under 40 years of age, since most centers do not give transplants to older patients. This must always be kept in mind when the results are compared with those of chemotherapy. The most serious obstacles to successful treatment with bone marrow transplantation are septic infections during the early granulocytopenic period, GVHD, interstitial pneumonia, and relapse (Thomas et al., 1975).

1

0 ’

I

I

la0

I

1

360

5Qo

1

720

Days after BMT FIG.4. Survival of patients with ANLL (n = 144) and ALL (n = 92) transplanted in first (ANLL) or later (ALL) remissions. Data from the European Cooperative Group for Bone Marrow Transplantation (Zwaan and Hermans, 1982). ANLL = A-A, ANLL; 0--0, ALL.

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The most serious infectious complications are seen in the early posttransplant period. These infections may be similar to those seen in leukemia patients during induction treatment, i.e., primarily Gramnegative bacteria and fungi, but disseminated infections due to Herpes simplex, Herpes zoster, and adenovirus may also appear. During the early engaftment period, fatal infections with cytomegalovirus or Pneumocystis carinii may be seen. Interstitial pneumonia of known or unknown origin is one of the most serious complications in this period. The association with CVHD may be coincidental, but is has been speculated that such infection may either be activated by GVHD or promoted by this complication (Santos et aZ., 1982). Late in the posttransplantation period, Herpes zoster infection and infections with pyogenic bacteria are the most common ones. We have seen generalized Herpes zoster in a patient 4 years after transplantation. The spectrum of bacterial infections is similar to that seen in patients with h ypogammaglobulinemia. GVHD may be mild or fatal. It has mainly three target organs, i.e., skin, gut, and liver. Engrafted immunocompetent donor T lymphocytes respond to the alloantigens expressed on the host cell and injure host tissue, either directly or indirectly by educating other cell-effector systems. GVHD develops in 40-70% of patients transplanted with HLA-identical marrow (Thomas et al., 1975; Sullivan et al., 1981; Ramsay et al., 1982; Zwaan and Hennans, 1982). However, the degree of GVHD varies, and it is a direct cause of death in only about 5% of patients having received HLA-identical sibling marrow. The incidence of fatalities is higher in patients transplanted with haploidentical or partially HLA-incompatible marrow (Powles et al., 1982). The most common method used to prevent GVHD is the prophylactic ad1975). Other means have ministration of methotrexate (Thomas et d., recently been tried, such as methotrexate plus prednisolone (Ringden et al., 1981),cyclosporin A (Powles et al., 1982), antithymocyte globulin (Ramsay et aE., 1982), and T-cell-specific monoclonal antibodies (Prentice et al., 1982). Limited data on the efficacy of these new CVHD-prevention regimens are available. However, cyclosporin A and monoclonal antibodies may drastically change the concept that HLA-identical sibling donors are required. The combination of methotrexate and cyclosporin A has been used with some success to prevent GVHD in mismatched marrow transplantation (Powles et al., 1982). Most patients with acute GVHD will be cured. However, 15-30% of these patients will develop chronic GVHD. This is a serious and protracted disorder, which results in scleroderma-like changes in the

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skin, xerophthalmia, xerostomia, chronic hepatitis, malabsorption, and an anorexia-like syndrome (Graze and Gale, 1979). Some success in treating chronic GVHD has been achieved with a combination of azathioprine and prednisolone (Sullivan et al., 1981). Early treatment of acute GVHD with these drugs may prevent chronic GVHD (Groth et al., 1979; Ringd6n et al., 1981). Leukemia relapse is a serious obstacle to success in bone marrow transplantation. Relapse is much more frequent if patients are transplanted in partial remission or during relapse than if they are transplanted in complete remission (Buckner et al., 1982a,b; Clift et al., 1982a). It is also more frequent in patients with ALL transplanted in the second remission than in patients with ANLL transplanted in the first remission (Buckner et al., 1982a). There are no substantial data comparing these two diseases transplanted in the first remission, since most patients with ALL have been transplanted in the second remission. The overall incidence of relapse in ALL is 7-12% in recipients of HLA-identical sibling marrow. However, the incidence appears to be considerably higher if patients are transplanted with twin-marrow grafts. Also, patients who have received cyclosporin A as GVHD prophylaxis appear to have a higher incidence of relapse than patients treated with methotrexate (Zwaan and Hermans, 1982). This difference may be due to an increased risk of relapse in patients who have a decreased risk of GVHD, either because of identical-twin marrow or because of treatment with cyclosporin A. In fact, it has been shown that patients with GVHD experience a lower incidence of leukemia relapse (Weiden et al., 1981). Whether the graft-versus-leukemia effect can be separated from Tcell-mediated GVHD in humans remains to be determined. Experiments in rodents suggest that the graft-versus-leukemia effect can be distinguished from GVHD (Santos et al., 1982). Efforts to find inhibitors of GVHD, which do not affect graft-versus-leukemia, will certainly be made in the future. A further expansion of bone marrow transplantation as a method to treat leukemia is limited by the fact that only 25-30% of patients have an HLA-identical sibling donor. Attempts have therefore been made to use HLA-mismatched donors. In most such attempts haploidentical donors, either parents or siblings or children, have been used. Recipients have been selected for further identity in HLA-A, -B, -C, or -D loci, but all have by definition had a positive MLR test. In one study (Clift et al., 1979), 4 of 12 patients transplanted with mismatched marrow are alive 339 days after transplantation, and in another (Powles and Morgenstern, 1982a,b; Morgenstern et al., 1982) 13 of 27

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are alive, 6 of them more than 1 year after transplantation. In the latter study, cyclosporin A or methotrexate plus cyclosporin A were used during several months for GVDH prophylaxis. It is too early to predict whether these results will be superior to those with chemotherapy in an aged-matched group of patients. However, a number of techniques have now been developed, whereby GVHD-inducing T lymphocytes can be removed in oitro. Treatment with monoclonal antibodies appears to be the most promising method (Granger et al., 1982). Such antibodies are also being used in attempts to eliminate T cells in oioo by prophylaxtic treatment of the patient during the posttransplant period (Prentice et ul., 1982). Such methods, presently at the experimental stage, may well prove to be effective, and, if so, the indications for allogeneic bone marrow transplantation will be greatly widened.

D. AUTOLOGOUSBONEMARROWTRANSPLANTATION Autologous bone marrow transplantation is a rescue procedure which permits preceding supralethal doses of chemotherapeutic agents or irradiation. Its use in the treatment of patients with leukemia has been hampered by the fact that the marrow, even when sampled in complete remission, is contaminated with leukemic cells which eventually cause relapse. Methods have therefore been developed to circumvent this problem. Earlier studies used physical separation techniques (Dicke et al., 1978) or treatment with rabbit heteroantisera (Wells et al., 1979; Netzel et uZ., 1980) to eliminate leukemic cells prior to autologous transplantation. Fractionation methods have mainly been unsuccessful, but some promising results have been obtained with heteroantisera. The use of specific monoclonal antibodies to purge the marrow in r;itro in order to eliminate leukemic cells is more promising (Bast et d.,1982; Ritz et al., 1982). Attempts have now been made to use autologous bone marrow treated with such antibodies in oitro in order to rescue the patients after intensive chemotherapy and supralethal irradiation. In such studies, remission is induced by conventional chemotherapeutic agents. In most cases the marrow is sampled after intensification, during either the first or second remission. It is immediately treated with the monoclonal antibody and complement and then frozen in DMSO and stored in liquid nitrogen. The patient then receives chemotherapy and total-body irradiation similar to that given before allogeneic bone marrow transplantation. Twelve or 24 hr after total-body irradiation, the thawed marrow is given to the patient by intravenous infusion. Various antibodies have been used to purge the marrow. Ritz et al.,

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(1982) used the J5 antibody (murine IgG 2A), which is specific for the common ALL antigen (cALLa) and has been found to react with the leukemic cells from 80%of patients with non-T-cell ALL. It is a complement-fixing antibody, and was shown in vitro to specifically lyse cALLa-positive leukemic cells, while sparing normal hematopoietic stem cells. Two of four patients with CALLwho were pretreated with VM 26, ARA-C, cyclophosphamide, and total-body irradiation are still in remission 14 and 12 months after having received the J5 antibody and complement-treated autologous bone marrow. Kaiser et al., (1982) have used the LEU1 antibody together with complement to purge the remission marrow from four patients with T-cell ALL before autologous bone marrow transplantation after supralethal chemotherapy. Three patients are alive in complete remission 2, 5, and 17 months after transplantation. Other monoclonal antibodies that are being explored for treatment are the BA-1 and BA-2, which were raised against cells from the NALM-6-MI cell line, which has a pre-B-cell phenotype (Jansen et aZ., 1982). Recently, it has also been shown that at certain dose levels certain cytotoxic drugs may kill leukemic cells but spare normal hematopoietic stem cells. Kaiser et aE. (1982) successfully used prior incubation with 4-hydroxy-peroxy-cyclophosphamide (4-HC) to eliminate leukemic cells from a mixture with normal cells. The value of autologous bone marrow transplantation has yet to be determined. The relapse incidence may well be higher than with allogeneic bone marrow transplantation, since it is well known that this incidence is higher in patients transplanted with identical-twin marrow. However, the serious problem of GVHD is circumvened by autologous transplantation. Furthermore, 75% of patients eligible for bone marrow transplantation do not have an HLA-identical sibling donor. Thus, in the future autologous bone marrow transplantation may well be an important part of the treatment of patients with acute leukemia.

VII. Supportive Treatment

The advances in the treatment of patients with acute leukemia with chemotherapy and bone marrow transplantation has only been made possible through a parallel development in methods for supportive treatment. The most important of these has been the development of new antibiotics, e.g., the various aminoglucosides for treating infections with Gram-negative bacteria. Other important developments are prophylaxis in the treatment of Pneumoncystis carinii, the recognition and early treatment of fungal infections, and the development of new

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drugs for the treatment of viral infections, such as acycloguanosin and interferon. The importance of granulocyte transfusions and the need for laminar-air flow units and life islands is still debated. Most centers cannot afford extensive isolation methods, and the advantages appear to be marginal. Prophylactic granulocyte transfusions are probably of limited value, perhaps with the exception of prophylaxis during bone marrow transplantation. Other important supportive measures are adequate platelet and red blood cell support, recognition of electrolyte disturbances, and treatment of hyperuricemia. Indeed, advances in the aggressive methods of chemotherapy and bone marrow transplantation are only valuable if they give the patient a decent quality of life. The total care emphasized already by Sidney Farber also includes a strong psychological back-up of the patient and his family. The increasingly aggressive chemotherapeutic treatment methods, with accompanying frustrating side-effects during protracted periods, has increased the need for such support. Further advances in the recognition of psychological problems and methods to cope with them are needed. VIII. Prospects for the Future

The development of new and more effective chemotherapeutic agents, as well as the use of combinations of several drugs, has been the main reason for improved results in acute leukemia. The search for new drugs and new drug combinations will continue. Some drugs now being tested in phase I1 trials, such as aclacinomycin A and dihydroanthracenedione, are promising and ready for randomized trials. More specific new drugs will probably be found. One of the most interesting new enzyme inhibitors, deoxycoformycin, belongs to this category, but its place in leukemia treatment remains to be determined. A reduction of the cardiotoxic side-effects of anthracyclines, without reducing their efficacy in leukemia treatment, has been achieved not only by synthesizing new analogs, such as aclacinomycin A and dihydroanthracenedione, but also by using DNA as a carrier. The development of carriers for more specific chemotherapy is under way using, for example, low-density lipoprotein (LDL) or monoclonal antibodies. Although interest in active immunotherapy has decreased somewhat, it is possible that immunotherapy with monoclonal antibodies will be an important step forward. The importance of interferon may have been overestimated, but new production methods and the manu-

TREATMENT OF ACUTE LEUKEMIA

3 13

facture of new types of interferon may well lead to its gaining a place in leukemia treatment. The most important step forward during recent years is probably the use of bone marrow transplantation. So far, this method is only available for those who have HLA-identical sibling donors. However, methods are being developed to prevent GVHD, e.g., treatment with cyclosporin A, in vitro purging of the marrow with monoclonal antibodies directed against T cells, and direct treatment of the patients with such antibodies. Using such methods, trials have already been carried out using HLA-mismatched donors, and some success has been obtained. Development of autologous bone marrow transplantation will circumvent the problem of GVHD. Methods are being developed to purge the marrow for leukemic cells by in vitro treatment with monoclonal antibodies or drugs. The development of better supportive methods is a prerequisite for progress in chemotherapy and bone marrow transplantation. In this connection the development of new drugs for the treatment of viral infections, a growing problem during the maintenance phase of remission and in posttransplant patients, is of great importance. New drugs are being developed and one of them, acycloguanosine, has already proved to be effective for several herpes virus infections. Acute leukemia is no longer equated with early death. More than 50% of children will be cured, and the proportion of cured adults is growing.

ACKNOWLEDGMENTS This work was supported by grants from the Swedish Cancer Society, the Swedish Medical Research Council, and the Karolinska Institutets Forskningsfonder. The constructive criticism by Docent Curt Peterson and the devoted work on leukemia research, dealt with in this article, by my collaborators at the Division of Clinical Hematology and Oncology and many other departments, as well as the never-failing patience of my secretary Asa Johansson is greatly acknowledged.

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Saiki, J. H., Bodey, G. P., Hewlett, J. S., Aniare, M.,Morrison, F. S., Wilson, H. E., and Linman, J. W.(1981).Cancer 47, 1739-1742. Sakurai, XI., and Sandberg, A. A. (1973).Blood 41,93-104. Sallan, S. E., Camitta, B. M.,Cassady, J. R., Nathan, D. G., and Frei, E., 111 (1978). Blood 51,425-433. Sallan, S. E., Ritz, J., Pesando, J., Gelber, R., O’Brien, C., Hitchcock, S., Coral, F., and Schlossman, S. F. (1980). Blood 55, 395-402. Santos, C . V. (1974). Semin. Hernatol. 11,341-351. Santos, G . V., Kaizer, H., O’Reilley, R. J., and Spitzer, G. (1982). Educ. Symp. Educ. Workshop Bookl. Annu. Meet. Am. Soc. Clin. Oncol., 18th pp. 15-20. Sauter, C., Barrelet, L., Berchtold, W., Fopp, M.,Maurice, P., Tschopp, L., and Cavalli, F. (1982). Proc. Am. Soc. Clin. Oncol. 1, 128. Scheef, W.,Klein, 0. H., Brock, N.,Burkert, H., Giinther, U., Hoefer-Janker, H., Mitrenga, D., Schnitzler, J., and Voigtmann, R. (1979). Cancer Treat. Rep. 63, 501505. Schell, F. C., Yap, H. Y., Bltinienshein, G. R., and Bodey, G. P. (1982).Proc. Ant. Soc. Clin.Oncol. 1, 21. Sen, L., and Borella, A. (1975).N . E n g l . ]. Aled. 292, 828-832. Shank, B., Hopfan, S., Kim, J. H., Chu, F., Grossbard, E., Kapoor, N., Kirk-Finegan, D., and O’Reilly, R. J. (1981). Znt.]. Radiat. Oncol. B i d . Phys. 131, 1109-1115. Shaw, M. T., and Raab, S. 0. (1977). ,&fed.Pediatr. Oncol. 3,261-266. Shnider, B. I., Gold, L. G., Hall, T., Dederick, IM., Nevinny, H. B., Potee, K. G., Lasagna, L., Owens, A. H., Hreschyshyn, M., Selawry, O., Holland, J. F., Jones, R., Jr., Colsky, J., Franzino, A., Zubrod, C. G., Frei, E., 111, and Brindley, C., Jr. (1960). Cancer Chemother. Rep. 8, 106-1 11. Simone, J. V., Verzosa, M. S., and Rudy, J. A. (1975).Cancer 36,2099-2108. Skipper, H. E., Schabel, F. M., and Wilcox, W. S. (1964).Cancer Chemother. Rep. 35,l111. Slevin, M.L., Shannon, hl. S., Prentice, H. G., Goldman, A. J., and Lister, T. A. (1981). Cancer Chemother. Phormacol. 6, 137-140. Smith, C. M.,Belch, A., and Henderson, J. F. (1980). Biochem. Pharmacol. 29, 12091210. Smith, I. E., Gerken, M. E., Clink, H. M.,and McElwain, T. J. (1976).Postgrud. Med.]. 52,66-70. Smyth, A. C., and Wiernik, P. H. (1976).Clin. Phormacol. Ther. 19, 240-245. Smyth, J. F., and Harrap, K. R. (1975).Br. ]. Cancer 31, 544-549. Smyth, J. F., Chassin, M. M., Harrap, K. R., Adanison, R. H., and Johns, D. (1979).Proc. Am. Assoc. Cancer Res. 20,47. Southam, C. M., Craver, L. F., Dargeon, H. W., and Burchenal, J. H. (1951).Cancer 4, 39-59. Stiihelin, €1. (1970).Eur.]. Cancer 6, 303-311. Stahelin, H. (1973).Eur. /. Cancer 9, 215-221. Storb, R. (1982). In press. Stroehlein, J. F., Bedikian, A. Y., Karlin, D. A., Korinek, J. K., and Bodey, G. P. (1982). Proc. Am. Soc. Clin. Oncol. 1,94. Sullivan, G. W.,Shulman, H. M.,Storh, R., Weiden, P. L., Witherspoon, R. P., McDonald, G. B., Schubert, M. M., Atkinson, K., and Thomas, E. D. (1981). Blood 57, 267-276. Sullivan, bl., Vietti, T., Haggard, ht., Donaldson, M.,h a l l , J., and Gehan, E. (1971). Blood 38,680-688.

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THE FORTY-YEAR-OLD MUTATION THEORY OF LURlA AND DELBRUCK AND ITS PERTINENCE TO CANCER CHEMOTHERAPY Howard E. Skipper Southern Research Institute, Birmingham. Alabama

I. Introduction .................................................... 11. The Somatic Mutation Theory (1943) ............................... 111. The Fluctuation Test of Law Pointing to the Origin of Methotrexate-Resistant Leukemia Cells ............................. IV. Wide Fluctuations in the Degree and Duration of Response of Neoplasms to Chemotherapy in Similarly Staged and Treated Individuals. .......................................................... V. Effective but Noncurative Chemotherapy Consistently Increases the Survival Time Variance of Treatment Failures; Ineffective Chemotherapy Does Not. ......................................... VI. Idealized Surviving Fraction Curves That Are Compatible with Large Bodies of Experimental and Clinical Data ........................... VII. The Origin of Doubly and Multidrug-Resistant Neoplastic Cells . . . . . . . . VIII. Mathematical Relationships and Models. ............................ A. The Drug-Sensitive Neoplastic Cell Kill during Repetitive Doses or Courses of Chemotherapy. ..................................... B. Variables Which Influence the Rate of Selection of Drug-Resistant Neoplastic Cell Populations .................................... C. The Mathematical Model of Goldie and Coldman (1979)............ IX. Criteria for Optimum Delivery of Non-Cross-Resistant Combinations of Drugs .......................................................... A. Alternating Delivery of an Alkylating Agent and an Antimetabolite . . . B. Alternating, Simultaneous, and Sequential Delivery of a DNA Binder and an Alkylating Agent ....................................... X. Closing Remarks.. ............................................... References. .....................................................

331 333 334

335 338 342 346 348 348 350 354 356 357 360 362 362

I. Introduction

In 1964 we published a long paper concerning the curability of an experimental leukemia, at different stages of advancement, with a variety of single drugs (Skipper et at., 1964). Briefly, the following was noted.

1. There is an invariable inverse relationship between the total leukemia cell burden and curability with chemotherapy. 33 1 ADVANCES IN CANCER RESEARCH, VOL. 40

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-006640-8

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2. Dose-response relationships are apparent for all classes of anticancer drugs and are reflected in both the survival time of treatment failures and in cure rates. 3. A given dose of a given drug will kill approximately the same percentage, not the same number, of widely different-sized leukemia cell populations-so long as their growth fraction and degree of phenotypic heterogeneity are similar. During the succeeding years it was observed that the same principles obtain for a wide variety of solid tumors with different growth kinetics. In 1964 we were aware of the experiments and deductions of Luria and Delbriick that led to the somatic mutation theory (Luria and Delbruck, 1943) and the important fluctuation test carried out by Law using the L1210 leukemia system (Law, 1952). Unfortunately we did not immediately conceive that what we had observed was compatible with the mutation theory. Over the past 20 years we devoted much effort to the study of this question: Why do single drugs and combinations of drugs (used alone or in an adjuvant setting) cure disseminated animal and human cancers when they do, and why does chemotherapy fail when it fails? Prospective research on various facets of this question and retrospective analyses of experimental and clinical results obtained by many investigators eventually led us to these conclusions:

1. Selection and overgrowth of specifically and permanently drugresistant neoplastic cells are major causes of chemotherapeutic failure. 2. Two phenomena that have puzzled cancer chemotherapists for many years rather precisely describe what should be expected if we accept basic tenets of the mutation theory. 3. New and more quantitative approaches to interpretation of aviilable chemotherapeutic results-and planning of new combination chemotherapy regimens-are essential if we are to accelerate progress in curing disseminated cancers. In this article we will point to consistent experimental and clinical observations, and to current theory, that led to these conclusions. Mathematical relationships derived by Lloyd (1977)and Skipper et al. (1978) have been of help in the study of (1) the behavior of drugsensitive tumor cell populations during repetitive dose treatment, and (2) the variables which affect the rate of selection and overgrowth of

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drug-resistant neoplastic cell populations. The mathematical model of Goldie and Coldman (1979) has been of help in application of the fundamental implications of the mutation theory in the area of cancer treatment. II. The Somatic Mutation Theory (1943)

At this point I might suggest that reading or rereading the classical paper of Luria and Delbriick (1943) would be time well spent by almost anyone involved in research on cancer treatment in 1983. The following quotations from their paper give some insight into (1) the complicated problem they faced, (2)the reasoning they employed in shedding light on the origin of phage-resistant E . coli cells, and (3) the reasons for widely fluctuating numbers of resistant cells in independently grown populations of the same size. In the attempt to determine accurately the proportion of resistant bacteria, great variations of the proportions were found, and results did not seem to be reproducible from day to day. Eventually, it was realized that these fluctuations were not due to any uncontrolled conditions of our experiments, but that, on the contrary, large fluctuations are a necessary consequence of the mutation hypothesis and that the quantitative study of the fluctuations may serve to test the hypothesis. [Two hypotheses were considered by the authors: (1)resistant variants were induced by direct action of the virus, or (2) mutations had occurred in the cell populations prior to addition of the virus with the virus merely bringing the variants into prominence by eliminating the sensitive bacteria.] The present paper will be concerned with the theoretical analysis of the probability distribution of the number of resistant bacteria to be expected on either hypothesis and with experiments from which this distribution may be inferred. While the theory is here applied to a very special case, it will be apparent that the problem is a general one, encountered in any case of mutation in uniparental populations. It is the belief of the authors that the quantitative study of bacterial variation, which until now made such little progress, has been hampered by the apparent lack of reproducibility of results, which, as we shall show, lies in the very nature of the problem and is an essential element for its analysis. It is our hope that this study may encourage the resumption of quantitative work on other problems of bacterial variation.

In simple terms a large fluctuation in the proportion of resistant variants in similar-sized cell populations is a basic tenet of the mutation theory because mutations to a resistant state may occur at any time during the growth process. If the first “random” mutation occurs early, then the proportion of resistant cells will be much higher than if it occurs much later. (This seeming “randomness” does not imply that mutation rates are not useful and reproducible values.)

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In the years after the original deductions of Luria and Delbruck it was demonstrated that (1) the proportions of bacterial cells that are specifically resistant to different antibacterial drugs fluctuate widely in independent populations of the same size, and (2) a basic tenet of the mutation theory applies in mammalian neoplastic cells growing in uivo. Two recent papers by Goldie and associates succinctly describe the pertinence of implications of the mutation theory to some of the problems faced in cancer chemotherapy (Goldie and Coldman, 1979; Goldie et al., 1982). 111. The Fluctuation Test of Law Pointing to the Origin of Methotrexate-Resistant Leukemia Cells

In 1952 Law published the results of a very important experiment (Law, 1952).Briefly, he initiated 15 independent sublines of leukemia cells in mice, each originally from 150 leukemia cells from the same population. (The probability of the presence of methotrexate-resistant cells in inocula of only 150 leukemia cells is very low.) The leukemia cells were inoculated subcutaneously. These sublines were carried separately in serial transfers in untreated animals and were allowed to grow to measurable lymphomatous masses in each passage. At the seventh serial transfer, 8 x 105 leukemia cells were inoculated into 10 mice for each subline and the animals were treated with methotrexate (2.5 mglkgidose given every other day for four doses beginning 24 hr after inoculation). Subline 13 served as a control test. In this control series, 10 groups of 10 mice each were inoculated with cells from a single subline (No. 13) and treated as above. At 9 days postinoculation (2 days after the last dose of methotrexate) the animals were sacrificed and the lymphomatous masses were excised and weighed. The variance ratio of the mean lymphomatous masses, between the independent and single subline series, was about 21: Independent sublineshingle subline

=

4125/196 = 21 with p < 0,001

These observations by Law are indeed consistent with the deductions of Luria and Delbriick. In many chemotherapeutic trials carried out since 1952 it has been shown that repetitive passage of independent neoplastic cell populations in untreated animals is not necessary to demonstrate a highly significant variance in the proportion of drug-resistant phenotypes in individual animals that originally received implants or inocula from a single tumor or the same neoplastic cell suspension. This holds for

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variants that are specifically resistant to essentially all classes of drugs. This apparent reflection of implications of the mutation theory is observed in all types of experimental cancers we have studied. IV. Wide Fluctuations in the Degree and Duration of Response of Neoplasms to Chemotherapy in Similarly Staged and Treated Individuals

For many years chemotherapists have been puzzled by the wide variations in the degree and duration of response of neoplasms of the same type to chemotherapy in similarly staged and treated individuals (patients or animals). Had we conceived that implications of the mutation theory apply in independent cancer cell populations, in much the same way they do in independent bacterial cell populations, much of this puzzlement and frustration might have been avoided. The earlier quotations from Luria and Delbruck tell us a little of the frustration of bacteriologists in the first half of the twentieth century. To anyone who has followed the progress of cancer chemotherapy in humans, it is apparent that wide variations in the degree and duration of response of tumors of the same type in similarly staged and treated patients are the rule, not the exception. The same may be said for experimental cancers if animals are bearing tumor cell burdens that are too high to be cured by the drug or drugs being employed. The results in Fig. 1 (Schabel, 1979) and Fig. 2 (T. M. Corbett et al., personal communication) will serve as an illustration. In Fig. 1 we see the growth of a transplantable colon tumor in untreated animals and the response of small tumors to a slow-release form of ara-C in animals treated at 7-day intervals over a period of about 6.7 months. From these results we may deduce that at least 73% of the small tumors contained no tumor stem cells that were resistant to the ara-C levels provided by the doses of palmO-ara-C used; these animals were cured. After complete remissions the tumors in two of the animals recurred during continuing undiminished treatment. The tumors from the two animals that suffered recurrence were harvested and passed to other animals and shown to be resistant to palmOara-C. In Fig. 2 we see the diversity of response of larger tumors in animals treated with palmO-ara-C over a period of about 9.5 months. Significant tumor response was observed in all animals but the variation in the degree and duration of response in individual animals is quite large, e.g., some minimum responses, some partial responses (PRs), some complete responses (CRs) and some cures. Except for three

a

0

DAYS POSTIMPLANT

FIG.1. Long-term treatment of animals bearing small colon 36 tumors with palmOara-C (Schabel, 1979). (A) Untreated controls. (B) Treated with palmOLara-C, 63 mg/kg/ dose, q7d ( ~ 2 7 starting ) on day 21 after subcutaneous implantation of small trocar fragments. Drug deaths, 2/15; CRs, 13/15;cures, 11/15; recurrences, 2/15. Tumors from the two animals (1,2) that suffered recurrence during continuing undiminished treatment were harvested before death, passed to other animals, and observed to b e resistant to ara-C.

MUTATION THEORY OF LURIA AND DELBRUCK

10’

25

45

65

a5

337

105 125 145 165 185 205 225 245 265 285 305 325 DAYS POSTIMPLANT

FIG.2. The diversity in both degree and duration of response of 2-3 g colon 36 tumors on long-term treatment of animals with palmO-ara-C (T. H. Corbett et al., personal communication, 1980). PalmO-ara-C was administered in qd ( ~ 9courses ) at 19 mg/kg/ dose, with 10-day intervals of rest between, until death or for 313 days. Drug deaths, 2/14; minimum responses, 1/14; PRs, 5/14; CRs, 3/14; cures, 3/14. There was tumor progression after regressions in all animals, except those cured. The increase in survival time over untreated controls was 179%.

cures and two drug deaths, all tumors, after varying degrees of regression, resumed growth during continuing treatment and killed the hosts. These results and many more Iike them in animals bearing different neoplastic diseases and treated with various drugs and combinations of drugs are indeed compatible with implications of the mutation theory. In fact, I can think of no creditable interpretation of results of this nature, in animals or patients, except fluctuating numbers of drug-resistant phenotypes in individuals. In this context a minimum response or a partial response (PR) implies that by the time the drug-sensitive tumor cell mass has been reduced to unmeasurable dimensions, the drug-resistant tumor cell mass already has grown to measurable dimensions. On the other hand, a complete response (CR) implies that at the time the drug-sensitive tumor cells have been reduced to unmeasurable dimensions, the drug-resistant cells (if any) have not yet increased to measurable dimensions.

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HOWARD E. SKIPPER

The above views are supported by these experimental observations: 1. When neoplasms are caused to regress and then regrow during continuing treatment with single drugs, the surviving cells are invariably resistant to the drug that selected them (but not to other quite different classes of drugs). 2. When neoplasms are caused to regress by combinations of drugs and then recur during continuing treatment with the same combination, the surviving cells show resistance to one or more but not necessarily to all of the drugs in the combination. V. Effective but Noncurative Chemotherapy Consistently Increases the Survival Time Variance of Treatment Failures; ineffective Chemotherapy Does Not

The points to be made in this section are somewhat similar to those already emphasized, except that the analyses were carried out on therapeutic results in which the survival time of the treatment failures was the principal endpoint. For over 20 years w e have noted that effective but noncurative chemotherapy consistently increases the survival time variance of leukemic animals and animals bearing other neoplastic diseases.’ Such drug-induced increases in survival time variance are reflected in both remission and survival curves that are significantly less steep than those for ineffectively treated groups or untreated controls-even when the cures (if any) are excluded. Similar observations are seen in clinical results, e.g., recall the differences in the slopes of remission and survival curves for “responders” versus “nonresponders” in many clinical publications. At first we suspected that such drug-induced increases in survival time variance might be the result of selection of one or a few tumor stem cells (in some animals) that gave rise to progeny with slower than average repopulation rates. Later it became apparent that this interpretation often would not account for highly significant increases in remission and survival time variance. Much data such as that in Fig. 2 convinced us that more often the factor responsible for drug-induced increases in remission or survival time variance is the fluctuation in proportions and absolute numbers of permanently drug-resistant tu1 The survival time variance is the standard deviation of the survival times of the individual animals or patients in a group, squared. This is a simple and useful statistical value.

0

10 I

Time (Days) 20 30 I

I

50

40 I

A

-L1210/0; PalmO.

[-i"..; ..

1

loo 80

I

-

-

60

40 20 -

I

I

I

I

I

I

I

1

bA

CPA, dayZ+ (100% PalmO. curer) day 7

0

Pal4 b

I% LIPlO/ara-C cells

0-

I

I

I

I

1

I

FIG.3. Effects of adding a constant number of ara-C-resistant leukemia cells to the inocula received by variousIy treated animals-on the survival time variance and slopes of surviva1 curves. Inocula

A

B

L1210/0

Ll2lOfara-C

106 0 106

0 106 103

105

102

105

102

105

102

Treatment

Relative survival time variance

PalmO-ara-C PalmO-ara-C PaimO-ara-C

High Low" Low"

PalmO-ara-C CPA CPA -+ PalmO

LOW" High (100% cures)

Essentiaily the same survival time slope as in similarly inoculated untreated controls. Conclusions: Drug-induced increases in survival time variance are a reflection of a basic tenet of the mutation theory. All that is necessary to cover up the "naked variance" is to add to the inocula received by all animals a constant number of neoplastic cells (e.g., 0.1%)that are specifically resistant to the drug employed.

340

HOWARD E. SKIPPER Time (Days)

0

1I0

30

20 I

50

40

I00

A -L1210/0; CPA, 2OQmg/kg

60 40

same A,

Y

0

I

0

-0.1%LIZIOKPA cells, same R, I

I

1I0

2 I0

I

I

I

40 I

50 I

Time (Days)

30

60 1

0 1

In L

-

80

PalmO, tX,tng/kg

60-

4020

-

L I2IO/C PA same R,

0Time (Days)

0

10

20

30

40

50

CPA, day 5

60

I

-

40

-

20

-

0

I

\-

0.I % LIPIO/CPA cells

FIG.4. Effects of adding a constant number of cyclophosphamide-resistantleukemia cells to the inocula received by variously treated animals-on the survival time variance and slopes of survival curves.

34 1

MUTATION THEORY OF LURIA AND DELBRUCK

mor stem cells initially present in similarly staged individuals or emerging during repetitive treatment. It now seems evident that if a large group of animals are bearing, say, lo8 tumor cells that are sensitive to Drug A (T/O cells), and the number of stem cells that are specifically resistant to Drug A (T/A cells) fluctuates widely in individual animals, then repetitive treatment with Drug A until all or almost all of the drug-sensitive tumor cells are eradicated will leave widely fluctuating numbers of T/A cells in individuals (the “naked variance”). This in turn will result in a significant increase in the survival time variance over untreated controls or groups in which ineffective treatment failed to eradicate the vast majority of the originally preponderant drug-sensitive cells. The above interpretation is consistent with much data in animals and humans and with repetitive studies showing that all that is required to cover up the “naked variance” in leukemic animals is to add to the inocula received by all animals a constant number of leukemia cells that are specificaIly resistant to the drug to be used (e.g., 0.1%). Then the slopes of the survival curves for the treated and concurrent untreated controls are almost identical and steep (see Figs. 3 and 4, F. M. Schabel et al., unpublished data, 1978).

FIG.4 (continued) ~~

~

Inocula L1210/0

L121O/CPA

106 0

0 106 103

CPA CPA CPA

High Low“ Low“

106 0 106

0 106 103

PalmO-ara-C PalmO-ara-C PalmO-ara-C

High High High

106

103 103 103

CPA PalmO-ara-C Palm0 + CPAb

Low“ Higher High

lo6

106 106

Treatment

Relative survival time variance

Essentially the same as similarly inoculated untreated controls. The value of following palmO-ara-C by CPA in this group is interpreted as follows: the palmO-ara-C eradicated 103 L1210/CPA cells in most animals leaving some L1210/0 cells which could be eradicated by CPA. Conclusions: Same as in Fig. 3.

342

HOWARD E. SKIPPER

VI. Idealized Surviving Fraction Curves That Are Compatible with Large Bodies of Experimental and Clinical Data

The internal consistency of experimental and clinical data, gained by many investigators, leads us to feel confident regarding the points to be made in this section.

/I

,*'

treatment

,,/

1

1

3

5 7 9 TIME INTERVALS

11

FIG.5. Idealized representations of the influence of the tumor cell burden and mix and the duration of chemotherapy on the surviving total tumor cell burden. Curve

Intended implications

ab

Treatment to remission, cessation of treatment and relapse. After relapse a second remission might or might not be possible depending on the resistant : sensitive ratio at relapse Response pattern in the absence of singly and doubly resistant cells. Almost never observed with single drugs if the burden is 10a or greater. A more reasonable representation of the reduction of T/O cells A classical illustration of the selection and overgrowth of drug-resistant tumor cells during single-drug treatment or with less than optimally delivered combinations of drugs. Eradication of 104 residual tumor cells (after local treatment) by chemotherapy. Feasible because of the low probability of the presence of phenotypes with specific resistance to the drug or drugs used

ad ac

e

~

~

~~~~~~~~

These curves were not intended to illustrate the influence of other critical variables, e.g., fluctuating proportions of drug-resistant cells in similarly staged individuals, the doses and K , of all non-cross-resistant drugs as delivered, and the intervals between administration of non-cross-resistant treatments in relation to growth rate of a particular neoplasm.

MUTATION THEORY OF LURIA AND DELBRUCK

343

Figure 5 is from a chapter written by Blum, Frei, and Holland in the second edition of Cancer Medicine (Blum et al., 1982). These curves provide excellent conceptual representations which, with a little imagination, depict some of the staging and treatment variables that affect both the degree and duration of response to chemotherapy of many human and animal cancers. Points made in Fig. 5

1. Influence of the total tumor stem cell burden 2. Influence of the absence or presence of specifically drug-resistant neoplastic cells at the beginning of

See curves

ab and ac versus e ad versus ac versus e

treatment and/or emerging during treatment

3. Influence of the duration of treatment with the same doses and intervals of the same drug or drugs

ab versus ac; ad versus e

Obviously these curves were not intended to illustrate other critical variables already mentioned and to be considered later.

1. Curve “ad” in Fig. 5 This curve illustrates repetitive treatment with a particular drug or drugs killing 2 logs of tumor stem cells per dose or course with about a 1-log regrowth in intervals between treatment until cure is achieved. Unfortunately this sort of response is rare indeed in humans bearing 10’1-1012neoplastic stem cells and treated with single drugs. Exceptions may be choriocarcinoma or Burkitt’s lymphoma if patients cured by methotrexate or cyclophosphamide, respectively, were in fact bearing 1011-1012neoplastic cells with stem cell capacity. Combination chemotherapy now is used in treating patients with these cancers because drug resistance frequently has proved to be a limitation to cure. Combinations of drugs have provided results that are compatible with curve ad-in acute leukemias, some lymphomas, and certain other disseminated cancers-but in some instances the total burdens of neopfastic stem cells probably were much lower than 1011-1012at the initiation of chemotherapy. For example (1) Hodgkin’s disease where the measurable masses (nodes and elsewhere) often contain relatively small fractions of malignant cells associated with larger numbers of normal lymphocytes and other normal cells, and (2) Wilms’ tumor, testicular cancer, breast cancer, and others where local treatment often may leave relatively small numbers of disseminated neoplastic cells for chemotherapeutic eradication as in curve e . In this context the inverse relationship between tumor cell burden and cur-

344

HOWARD E. SKIPPER

ability with chemotherapy and the direct relationship between burden and the probability of the presence of drug-resistant tumor stem cells seems relevant (see Fig. 6 and Tables I and 11). Curve ad, in fact, may be a more reasonable representation of the response of the originally preponderant drug-sensitive cohort of tumor cells (T/O cells) in advanced human cancers that are quite responsive to single-drug treatment.

2. Cume “ac” in Fig. 5 This curve indicating successively smaller reductions in the total neoplastic cell burden (per dose or course) during repetitive treatment, a nadir after about six doses or courses, then burden progression Population Size I

101

lo3

12 lo5

lo6 ;I

10’

10’

1.0

0.7 0

c

‘j,

0.6

+!

0.5 n

2 0.4 an

N

6 0.3 * .- 0.2 f n

::0.1 0

L

a

0.0

LoglO N (Populotion S i z e )

FIG.6. A graphic illustration of the model of Goldie and Coldman relating the probability of the existence of zero resistant phenotypes to the mutation rate and the tumor size. In this redrawn plot we have marked percentage cures achieved by an optimum regimen of ara-C in relation to known burdens of intraperitoneally inoculated L1210 leukemia cells. These are pooled results from repetitive trials. Top arrow, very high probability of zero drug-resistant cells; bottom arrow, very low probability of zeroresistant cells. Mutation rates: A, 1 x B, 1 x C, 1 X D, 1 x E, 1 x 10-7;F, 1 x 10-8.

345

MUTATION THEORY OF LURIA AND DELBRUCK

TABLE I APPROXIMATE AVERAGENUMBERS OF SINGLY DRUG-RESISTANT NEOPLASTIC CELLS

EXPECTED IN RELATIONTO THE MUTATIONRATEAND TUMOR SIZEO

Total tumor stem cell burden Mutation rate 10-4 10-6

lo-*

104

106

108

10'0

10' 0 0

103 10' 0

105 103 10'

107 105 103

a In keeping with the mutation theory, considerable fluctuation in these numbers must be expected in similarly staged individuals. A simple approach to estimating the average number of singly resistant cells ( R )in a tumor ofN cells and a constant mutation rate (a)is R = d ( l n N ) (see Goldie et al., 1982). Thus when (Y = and N = lO'O, R = (10-4) (1010) (23) = 2.3 x 107.

during continuing undiminished treatment seems to represent a more common observation when patients bearing many advanced cancers are treated with chemotherapy. It should be emphasized that tumor volume or mass measurements always underestimate the rate and degree of tumor cell kill because tumor cell lysis and resorption is a relatively slow process and tumor cell proliferation is taking place in intervals between treatment. Curve ac is a classical illustration of chemotherapeutic selection and overgrowth of singly drug-resistant TABLE I1 ESTIMATES OF THE TOTAL NEOPLASTIC CELLPOPULATIONS EXPECTED TO CONTAIN ONE DOUBLY DRUG-RESISTANT CELL(AVERAGE) IN RELATIONTO THE MUTATIONRATESTO A STATEOF RESISTANCETO EACHOF THE NON-CROSS-RESISTANT DRUGS"

Rate of mutation of TI0 + TIA or TIB -+ TIBIAb

Rate of mutation of TI0 TIB or TIA -+ TIAIBb

10-8

10-6

10-4

10-8 10-6 10-4

> 10'9 1.5 x 10" 2.2 x 109

1.5 x 10" 2.2 x 109 3.3 x 107

2.2 x 109 3.3 x 107 5.7 x 105

Even higher fluctuations in the numbers of doubly resistant cells must be expected in similarly staged individuals. A simple approach to estimating the average numbers of doubly resistant neoplastic cells in reIation to mutation rates and tumor size may be found in a recent publication by Goldie et al. (1982). T/O, Cells sensitive to both drugs; T/A, cells resistant to Drug A only; TIB, cells resistant to Drug B only; TINB or T/B/A, cells resistant to both drugs. These estimates are based on the assumption that doubly drug-resistant cells arise from singly resistant cells. Q

346

HOWARD E. SKIPPER

neoplastic cell populations during single-drug treatment or doubly drug-resistant tumor cell populations during treatment with effective but noncurative combinations of drugs. If failures of this type are to be minimized it is necessary to pay close attention to the dose levels (and K J of all drugs in a combination and to the intervals between delivery of non-cross-resistant drugs (see Section IX).

3. Curue “ab” in Fig. 5 This curve depicts the achievement of a remission after three doses or courses of chemotherapy, and relapse after cessation of treatment. Depending on the ratio of drug-resistant to drug-sensitive neoplastic cells at the cessation of treatment and at relapse, a second remission might or might not be achieved by the same doses of the same drug(s). The second remission, if achieved, would likely be of shorter duration than the first. As already implied, had treatment been continued for a much longer period and progression been observed during continuing treatment-as in curve uc-a second remission could not be expected with the same treatment. 4. Curue “e” in Fig. 5 This curve illustrates eradication of about lo4 tumor stem cells remaining after surgery or radiotherapy by three or four doses or courses of a single drug or a combination of drugs. The relationships in Fig. 6 and Tables I and I1 suggest the reason why this is feasible. Often the median residual neoplastic cell burden after local treatment may be 104-106, but with ranges of lo9 in individuals. In those patients bearing very low burdens of viable tumor cells, after surgery or radiotherapy, single-drug treatment may be curative, but in those bearing lo9 or greater, single-drug treatment probably will result in curves like ac. For the latter individuals combinations of non-crossresistant drugs have a better chance of eradicating the residual tumor cells-drug-sensitive and drug-resistant alike. V11. The Origin of Doubly and Multidrug-Resistant Neoplastic Cells

For many years I was puzzled by a concept related to me by a few bacteriologists; namely, if there is only about one in lo8 bacterial cells that is specifically resistant to Drug A and only about one in lo8 that is specifically resistant to Drug B, then it is likely that there will be only about one in loi6that is specifically resistant to both Drug A and Drug B. At first I was not aware that this was only a guess made by someone

MUTATION THEORY OF LURIA AND DELBRUCK

347

(I know not who) and repeated by others. I searched the literature and found no experimental data to support this concept. Furthermore, this view seemed totally inconsistent with large numbers of experimental and clinical trials in which combinations of noncross-resistant drugs had been used in treating animals and patients bearing advanced cancers. Often such trials provided end-results that were consistent with curve ac in Fig. 5 rather than curve ad suggesting that neoplastic cell populations of 10l2or less in humans and lo9or greater in animals contain doubly drug-resistant neoplastic cells at the initiation of treatment or that doubly resistant phenotypes emerge during treatment. Also, it seemed to me that combinations of two noncross-resistant drugs should be more effective in treating advanced cancers than they usually are if doubly drug-resistant tumor cells are not a serious problem. The important studies of Schmid et aE. (1976) showed that when Ieukemia cells were repeatedly passed in animals treated with a sixdrug combination and tested for resistance to the combination (and each individual drug in the combination), after every fourth treated passage the following was observed.2 1. By the time the leukemia cells showed significant resistance to the combination, they were markedly resistant to two of the drugs in the combination, 6-MP and 6-thioguanine (a cross-resistant pair), but not to the other drugs. 2. With continued treated passage and selection the surviving leukemia cells became increasingly resistant to the combination as tests showed them to be comprised of doubly, then triply, and then multidrug-resistant phenotypes. Schabel and associates (unpublished data, 1978) carried out simple and easily interpretable experiments such as these. Leukemia cells already selected for a high degree of resistance to cyclophosphamide (L121O/CPA cells) were shown not to be cross-resistant to ara-C. If the burden of L1210/CPA cells was lo5 or less, animals were cured by optimum regimens of ara-C or a slow release form of ara-C. However, if L121O/CPA cells were allowed to increase to about lo8 before the animals were treated with ara-C, no cures could be achieved and the The six-drug combination was comprised of 6-MP, 6-thioguanine, &methyl MPR, methotrexate, 5-FU, and ara-C delivered in different ways. Only two of these drugs show cross-resistance, 6-MP and 6-thioguanine.

348

HOWARD E. SKIPPER

surviving leukemia cells (harvested after remission followed by relapse) were shown to retain their original resistance to cyclophosphamide and now to be solidly resistant to ara-C as well (L121OICPAI ara-C cells). These results imply that L121OKPA cells mutate to L1210/CPA/ara-Ccells at about the same rate as L1210/0 cells mutate to Ll2lO/ara-C cells. Other quantitative studies of this type provided strong support for the view that doubly drug-resistant cells arise from singly resistant phenotypes, hence the probability of the presence or emergence of doubly resistant cells in populations of, say, lo9 or greater may be quite high (see Table 11).

VIII. Mathematical Relationships and Models

Some seem to distrust mathematical relationships and models when they are applied to biological problems, most particularly to problems involving intact mammals. I suppose that this is because they feel, with some justification, that there are too many variables and too much “biological variation” in individuals and too little precise information to allow quantitative interpretations or predictions. I like mathematical relationships and models and like to try to apply them to problems involving intact mammals-so long as I think I understand the critical variables and the associated probability distributions. Often I can see no possible way to test theories having to do with experimental therapeutics or to test the compatibility or lack of compatibility of diverse biological data without reasonably quantitative analyses. The mathematical relationships described below have been useful to me in many analyses of chemotherapeutic trial results.

A. THEDRUG-SENSITIVE NEOPLASTICCELL &LL

DURING

REPETITIVE

DOSESOR COURSES OF CHEMOTHERAPY This simple relationship derived by Lloyd has been useful to me for many years (Lloyd, 1977; Skipper et al., 1978). The net drug-sensitive tumor cell kill during treatment (logs) =

[K,

X

n]

-

[(log 2/DT) (n - 1) ( I ) ]

where K , is the sensitive tumor cell kill per dose or course (logs) read from an experimentally established dose-response curve or an approximate dose-response curve deduced from clinical observations, n

MUTATION THEORY OF LURIA AND DELBRUCK

349

is the number of doses (or courses), DT is the doubling time of the neoplastic cells in near exponential phase, log 2/DT is the growth rate in fractions of a log per day, and I is the interval between doses. As indicated, this relationship does not take into consideration drug-resistant cells already present at the beginning of treatment or emerging during treatment. Suppose we were in possession of experimental or clinical trial results that were consistent (in principle) with curve ac in Fig. 5 and wished to test the hypothesis that overgrowth of drug-resistant cells was the cause of tumor progression after a temporary remission. For simple illustration we will refer to curves ad and ac in Fig. 5 and assume the following:

K , = 2 logs n = 11 doses I = 30 days DT = 9.1 days log 2/DT = 0.30U9.1 = 0.033 log per day or approximately 1 log per month (growth rate)

[2 x 111

-

[(0.301/9.1) X 10 X 301 = [22] - E9.921 = 12 log reduction of drug-sensitive cells at the end of eleven doses (cure after twelfth dose)

This calculation is consistent with curve ad and completely incompatible with curve ac in Fig. 5. Thus in actual trials where remissions were followed by tumor progression during continuing undiminished treatment with the same drug or drugs, we are almost forced to conclude that (1) each successive dose did not kill the same fraction of the total tumor stem cell burden, (2) the nadir was reached when selection had proceeded to the point where about 50% of the surviving tumor cells were resistant to the drug or drugs being used, and (3) after unequivocal tumor progression was observed the preponderance of the surviving tumor cells were resistant to the drug(s) being employed. The above deductions have been shown to be valid in many experimental chemotherapy trials. The best therapeutic regimen we have designed for cure of very advanced murine leukemias was based on calculations showing that a remission-inducing combination (ara-C

350

HOWARD E. SKIPPER

plus 6-thioguanine) would be curative if it were not for surviving doubly resistant leukemia cells. After this was deduced it was apparent that remission induction treatment should not be continued past the nadir achievable. Instead, we should switch to or begin to alternate with another non-cross-resistant combination at or near the nadir achievable by the remission-inducing combination. Of course, this is the strategy that first led to achieving cures in acute lymphocytic leukemia in children.

B. VARIABLESWHICHINFLUENCE THE RATEOF SELECTION OF DRUG-RESISTANT NEOPLASTICCELLPOPULATIONS It is easy to demonstrate in experimental neoplasms that clinical resistance to chemotherapy will be observed when selection has altered the resistant to sensitive neoplastic cell ratio from some originally low value (e.g., to about 1.0 (50%resistant cells). This has been done by inoculating known mixes of drug-sensitive and drugresistant leukemia cells and then treating with a particular drug. When the known mix ofL1210/0 and L1210/A cells approaches 50% of L1210/A cells, then Drug A provides little or no measurable therapeutic response. Not long ago we carried out an idealized analysis of the relative influence of five variables on the rate of selection of drug-resistant neoplastic cell populations. These variables included 1. K , is the sensitive tumor cell kill per dose (logs) which is directly related to the dose level. 2. R , is the original resistant: sensitive tumor cell ratio (e.g., This value is influenced by the tumor stem cell burden and the mutation rate (see Fig. 6 and Table I). 3. The mutation rate. In our experience the mutation rates to a state of resistance to antimetabolites (e.g., to usually are higher than the mutation rates to a state of resistance to alkylating agents (e.g., or less). In addition, stepwise increases in the degree of resistance to specific alkylating agents are common on repetitive exposure to higher levels, whereas variants with high degrees of resistance to purine and pyrimidine antagonists seem to arise in a single step. 4. Doubling time. 5. Interval between doses (manipulatable in therapeutic design).

351

MUTATION THEORY OF LURIA AND DELBRUCK

Using a computer program developed by Lloyd, idealized computations were carried out to gain better insight into the relative impact of each of the above variables on the number of doses required to select to the point of clinical resistance. Table 111 will serve to indicate the nature of these analyses. In fact, five sets of computations similar to those in Table I11 were carried out using wide ranges of values for each of the five variables. These analyses implied that K , is a dominant variable with respect to the rate of selection of drug-resistant populations. The other variables usually have a marked effect only when K , is low (e.g., < 1log). This view is supported by retrospective calculations that have been made and compared with observed results. The following simple relationship which takes into consideration only two of the above-mentioned variables ( K , and the original resistant : sensitive neopIastic cell ratio) will predict the number of doses TABLE 111 IDEALIZEDCOMPUTATIONS INDICATING THE RELATIVEEFFECTS OF FIVEVARIABLES ON THE NUMBER OF DOSESREQUIREDTO SELECT TO THE POINT OF CLINICAL RESISTANCE TO A SINGLE DRUG^.^ ~

Interval (days)

KS

1

0.1

**** **** ****

0.2

0.5 1.0 2.0 3.0

4.0 5.0 6.0

4

2

5 3 2 2 1 1

Doubling time: 0.5 dayc

**** **** **** ****

2 2 1 1 1

**** **** **** **** ****

1 1 1 1

Doubling time: 1 dayc

0.1 0.2

**** ****

0.5

10

**** **** ****

1.0 2.0 3.0 4.0 5.0 6.0

5

5

**** **** **** ****

3 2 2 1 1

3 2 2 1 1

2 2 1 1 1

7

14

21

**** **** **** **** **** **** ****

**** **** **** **** **** **** **** **** ****

****

1 1

**** **** **** **** **** 1 1 1 1

**** **** **** **** **** **** **** 1 1

****

**** **** **** **** **** **** ****

**** **** **** **** **** **** ****

**** **** ~

(continued)

352

HOWARD E. SKIPPER

TABLE 111 (Continued) Interval (days)

K,

0.1 0.2 0.5 1.o 2.0 3.0 4.0 5.0 6.0

1

**** 22 11 6 3 2 2 2 1

0.1 0.2 0.5 1.0 2.0 3.0 4.0 5.0 6.0

****

0.1 0.2 0.5 1.0 2.0 3.0 4.0 5.0 6.0

50 27 12 6 3 2

0.1 0.2 0.5 1.0 2.0 3.0 4.0 5.0 6.0

54

25 11 6 3 2 2 2 1

2

4

Doubling time: 2 days'

**** ****

10 5 3 2 2 1 1

**** **** ****

5 3 2 2 1 1

Doubling time: 3 dapsc

**** ****

**** ****

10 6 3 2 2 2 1

9 5 3 2 2 1 1

Doubling time: 7 days"

2 2 1

40 25 11 6 3 2 2 2 1

****

20 10 6 3 2

49 27 12 6 3 2 2 2 1

14

21

**** **** **** ****

**** **** **** **** ****

**** **** **** **** **** ****

1 1 1

1

**** **** ****

**** ****

3

2 1 38 24 11 6 3 2 2 2 1

**** **** **** **** 2 2 1 1 1

I

Doubling time: 14 days"

29 13 7 4 3 2 2 2

7

**** 21 11 6 3 2 2 2 1

**** ****

**** **** ****

**** **** ****

**** **** ****

5 3 2 2 1 1

4 3

**** ****

**** ****

2 2 1 1

7 5 3

2 2 1 1

TABLE I11 (Continued) Interval (days)

K,

1

0.1 0.2 0.5 1.0 2.0 3.0 4.0 5.0 6.0

56 30 13 7 4 3 2 2 2

Doubling time: 21 daysc 51 45 28 26 12 12 7 6 4 3 3 2 2 2 2 2 2 1

0.1 0.2 0.5 1.0 2.0 3.0 4.0 5.0 6.0

58 31 13 7 4 3 2 2 2

Doubling time: 30 daysc 54 48 29 27 13 12 7 6 4 4 3 3 2 2 2 2 2 2

0.1 0.2 0.5 1.0 2.0 3.0 4.0 5.0 6.0

61 33 14 7

Doubling time: 60 daysc 57 52 31 29 13 13 7 7 4 4 3 3 2 2 2 2 2 2

4

3 2 2 2

2

4

7

14

21

****

**** ****

**** ****

10 6 3 2 2 2 1

9 5 3 2 2

41 25 11 6 3 2 2 2 1

****

**** ****

47 27 12 6 4 3 2 2 2

38 24 11 6 3 2 2 2 1

23 11 6 3 2 2 2 1

20 10 6 3 2 2 2 1

1 1

10 6 3 2 2 2 1

**** 21 11 6 3 2 2 2 1

In other computations the effects of varying the mutation rate and the resistant to sensitive ratio were examined. The values in this table are the number of doses required to select to the point of clinical resistance. Where no value is given (four asterisks) the particular combination of K,, interval, and doubling time is such that no net reduction in the drug-sensitive tumor cells would be achieved by repetitive doses. This does not mean that selection toward a higher resistanVsensitive ratio is not occurring (relatively slowly). From these and other computations it appears that K , is a dominant variable; the original resistant/ sensitive ratio, mutation rate, doubling time, and interval between doses have relatively less effect unless K, is low. When combinations of non-cross-resistant drugs are employed all of these variables become critical, e.g., the K , for each drug and the intervals between administration of each drug may make the difference between success and failure (see Section IX). Mutation rate is and resistant/sensitive is for all doubling times.

354

HOWARD E. SICIPPEH

of a single drug required to select to the point of clinical resistance in experimental neoplasms (Skipper et al., 1978). nRes

=

log (Rcr/Ro)/Ks

where nRes is the number of doses that will select a drug-resistant neoplastic cell population, R,, is the resistant : sensitive ratio that will reflect clinical resistance (a ratio of 1.0 or 50% resistant cells), R, is the original resistant: sensitive tumor stem cell ratio (e.g., to or less depending on the resistant phenotype), and K , is the sensitive tumor cell kill per dose or course (logs). For example, assume

R, = 10-6 K,

=

0.5 log or 1.0 or 2 logs

then nRes

=

log (1.0/10-6))/0.5= 12 doses

nRes = log (l.0/10-6)/l.0 = 6 doses nRes

=

log (1.0/10-6)/2.0 = 3 doses

Not long ago we brought together 82 sets of observed data in which leukemia cells or solid tumor cells had been passed in animals that received known dose levels and numbers of doses of various drugsand were tested for resistance to the drug employed after each treated passage. These were tumor systems for which we had reliable doseresponse curves for the sensitive cells from which approximate K , values could be read. Simple calculations of nRes (as above) provided retrospective predictions that were in remarkable agreement with the observed data.

MODELOF GOLDIEAXD COLDMAN (1979) C. THEMATHEMATICAL In past reports I have expressed admiration for the study and the deductions that led to the well-known model of Goldie and Coldnian. Their model describes the probability of occurrence of drug-resistant cells within tumors of various sizes based on the somatic mutation theory. “The model predicts that there will be variation in the size of the resistant fraction within tumor colonies of the same size for any given value of the mutation rate, and furthermore that there will be a

MUTATION THEORY OF LURIA AND DELBRUCK

355

sudden decline in the expectation of cure as the cell number increases.” A graphic illustration is provided in Fig. 6. As mentioned earlier, the model of Goldie and Coldman has been of much help to me in application of the fundamental implications of the mutation theory in the area of cancer treatment. After careful study of their model, I reexamined the results of hundreds of experimental chemotherapeutic trials that we had carried out in the past and was able to strengthen interpretations in many instances and gain additional information in others. For example, over the years we had gained much data regarding the cure rates observed in the L1210 leukemia system related to the leukemia cell burden at the initiation of treatment with different drugs. When these results were plotted on replicas of their probability curves (as in Fig. 6) they frequently seemed to coincide with the probability of zero resistant phenotypes associated with a given mutation rate. All points for all drugs did not fall on a particular curve as nicely as in the one in Fig. 6, but trends seemed to be consistent. As is implied by these probabiIity curves, one should almost never observe that a given treatment which provides zero cures would approach 100%cures if we reduced the neoplastic cell burden by only 10-fold. This expectation is indeed consistent with our total experience. With encouragement from Goldie and Coldman we (Lloyd) developed a computer program not unlike one they had d e ~ e l o p e d It .~ includes the basic premises of Lloyd’s relationship with respect to the net sensitive tumor cell kill during repetitive treatment and their equation which related the probability of the existence of zero resistant phenotypes to the mutation rate (a)and the tumor size ( N ) . This program allows retrospective or prospective simulations of the continually changing burden and mix of sensitive and drug-resistant neoplastic cells during treatment with a single drug or two non-crossresistant treatments. Because of the number of variables involved one usually cannot intuitively visualize the effects of changes in a chemotherapeutic regimen without such simulations. For over 2 years we have used this simulation program and it has been helpful in deducing why some chemotherapeutic regimens are curative and why many others are not when used in treating a particular experimental cancer at a particular stage of advancement. For many years I had carried out hand-calculated simulations to test interpretations and in prospective planning of new chemotherapeutic regimens. These often required hours to days to carry out. The computer program will do the same things in minutes and without my quota of mistakes. It has been a joy to use for one who believes that interpretations that cannot be mimicked by prudent simulations using reasonable parameters are apt to be erroneous.

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HOWARD E. SKIPPER

IX. Criteria for Optimum Delivery of Non-Cross-Resistant Combinations of Drugs

In the past much effort was placed on the selection of therapeutically potentiating combinations of anticancer drugs and the best methods for their delivery. At first this was a slow process for several reasons.

1. Early experimental designs were primitive and our basic understanding of the quantitative implications of much used endpoints (percentage increase in survival time and tumor inhibition) was meager. 2. One of the original theories used for guidance was erroneous (i.e., therapeutic potentiation by combinations is a reflection of sequential or concurrent blockade of biochemical events leading to pol ynucleotides) . By now it seems clear that combinations of non-cross-resistant drugs (when properly delivered) are superior to single-drug treatment because they delay or prevent treatment failure due to overgrowth of specifically drug-resistant tumor cell populations. The fact that many combinations are less, or somewhat less, than additive in toxicity is an advantage. When we recently reviewed the results of hundreds of combination chemotherapy trials already carried out in experimental cancers-in the context of the mutation theory, with better understanding of the critical variables and with much better methods for deducing the effects of changes in the dose levels and schedules of two or more drugs-the criteria for optimum delivery of non-cross-resistant drugs seemed to become less puzzling. Based on the experimental and clinical results I have studied, there probably is no single best way to deliver all of the different combinations of drugs now used (or conceived) for the treatment of different disseminated neoplastic diseases. Combinations often are superior to single-drug treatment when they are administered simultaneously, in an alternating manner, sequentially, or according to some mix of these descriptive terms. However, this generalization holds only if attention is paid to the dose levels and effectiveness ( K , ) of each drug, the intervals between the administration of each non-cross-resistant drug in relation to the growth rate of a particular neoplastic disease and, of course, the toxicity of a combination as delivered. Poor choices of drugs, doses, or schedules-for a combination-often will result in responses that are not as good as can be obtained by optimum delivery

MUTATION THEORY OF LWRIA AND DELBRUCK

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of the best single drug in a combination. This has been demonstrated many times at the experimental level and probably at the clinical level as well. Recently we carried out retrospective analyses and simulations of large numbers of combination chemotherapy trials carried out in the Ridgway Osteogenic Sarcoma (ROS) system. The designs of these trials were such that it was rather easy to deduce the criteria for optimum delivery of two-drug combinations delivered in different ways.

A. ALTERNATINGDELIVERY OF AN ALKYLATING AGENT AND AN ANTIMETABOLITE A combination of cyclophosphamide (CPA) and 6-mercaptopurine (6-MP) delivered in an alternating manner was employed in treating groups of animals bearing 2-3 g ROS tumors. In all instances CPA was delivered in single doses alternating with qd ( X 11)courses of 6-MP. A single dose of CPA followed 4 days later by a qd ( x 11)course of 6-MP comprised a course of the combination. Four courses of the combination were employed and the dose levels of both drugs were varied, i.e., high doses of both, high doses of one and low doses of the other, or low doses of both. From other available data it was easy to construct reliable dose-response curves for each drug and to read K , values from them. Table IV provides a summary of an analysis of 27 internally controlled trials. Interpretation of the criteria for optimum delivery of this alternating combination was straightforward when we compared K , values for the dose levels of each drug with observed CR rates and cure rates. 1. In order for this Combination, when delivered in an alternating manner, to be maximally effective it was necessary for both treatments to approach equal effectiveness as suggested by Goldie et al. (1982). By this we do not mean that the effectiveness per dose of both treatments must be equal. (Note the K , per dose of CPA and the net K , per qd ( X 11)course of 6-MP in relation to observed results in Table IV.) 2. In this set of data it was necessary that the doses of both drugs be high enough and the intervals between delivery of both be short enough to kill the tumor cells that were sensitive to both drugs and those tumor cells that were specifically resistant to 6-MP or cyclophosphamide in order to achieve high CR and cure rates.

Approx. K , (logs) For dose levels employed

w

%

c

Dose level Best

A Per dose of CPA

3 4.5 2.5 2.5 3.8

3.8 2.5 1.6 1.6

B Per dose

of 6-MP 0.34 0.24 0.36 0.31 0.22 0.26 0.22 0.25 0.25

Net per qd ( x 11) course

Observed (%)

of 6-MP

Ratio (AIC)

2.5 1.5 2.8 2.2 1.2 1.7 1.3

1.2 3 0.9 1.1 ,I2 2.2

1.3

1.2 1.1

1.5

1 .I-)

CRs

Cures

100 100 100 100

100 100 90 80 80 80

100 100 100 100 100

60

30 30

Comment It is necessary for the K , per dose of CPA to be 1.6 logs or greater to eradicate the ROS/G-MP cells, and the net K , per course of &?VIP to be 1.2 logs or greater to eradicate the ROS/CPA cells (if any) 111 order to acliiew cures

Doses

0

%

Next best

1.1 1.6 2.5 1.6 1.2 2.5 1.1 1.6 1.6

0.21 0.15 0.18 0.31 0.39 0.18 0.21 0.15 0.18

1.1 0.5 0.8 2.2 3.1 0.8 1.1 0.5 0.8

1 3.2 3.1 0.73 0.39 3.1 1 3.2 2

100 100 100 100 100 90 80 80 70

0 0 0 0 0 0 0 0 0

Both too low 6-MP too low 6-MP too low CPA too low CPA too low 6-MP too low Both too low 6-MP too low 6-MP too low

Worst

1.1 1.1 0.8 0.75 0.75 0.5

0.25 0.15 0.36 0.13 0.21 0.31 0.17 0.25 0.21

1.5 0.5 2.8 0.4 1.1 2.2 0.7 1.5 1.1

0.73 2.2 0.29 1.9 0.68 0.23 0.71 0.23 0.18

0 0 0 0 0

0 0 0 0 0 0 0 0 0

CPA too low Both too low CPA too low Both too low Both too low CPA too low Both too low CPA too low Both too low

0.5 0.35 0.2

0 0 0 0

a This analysis provides strong support for the view that in order for non-cross-resistant treatments to b e maximally effective, when delivered in an alternating manner, they must approach equal effectiveness (Goldie et al., 1982). By this we do not mean that the K , values per dose of each must be equal. In this set of data it was necessary that the doses of both drugs were high enough, and the intervals between delivery of both were short enough, to kill the drug-sensitive and the drug-resistant cells as well. In instances where cures were achieved it is apparent that the tumor contained no cells that were doubly resistant, i.e., ROSI6-MPICPA cells, and that none emerged during treatment.

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HOWARD E. SKIPPER

In these trials there was little doubt that overgrowth of drug-resistant ROS populations was a major cause of treatment failure. When four or five maximum tolerated courses of 6-MP alone were delivered, tumor mass behavior curves consistent with curve ac in Fig. 5 were observed. Similar curves also were observed with less than optimum doses of the combination.

B. ALTEFWATINC, SIMULTANEOUS, AND SEQUENTIAL DELIVERY OF A DNA BINDERAND AN ALKYLATINCAGENT After gaining what we thought was important information regarding the criteria for optimum alternating delivery of cyclophosphamide and 6-MP, we reexamined the results (obtained in 1975) of 49 trials in which actinomycin D and cyclophosphamide were delivered in different ways to animals bearing 2-3 g ROS tumors. The same approaches were used in analysis and interpretation of observed results. Retrospective computer simulations were carried out and proved to be very helpful. Again we found that deducing the criteria for optimum delivery of actinomycin D and cyclophosphamide, according to different schedules (simultaneous, alternating, or sequential), was rather easy on ranking available data in decreasing order of cure rate and CR rate and comparing (1) approximate K , values for the varying doses used, and (2) the expected influence of the schedules employed. When this combination was delivered in an alternating manner both drugs had to be employed at levels that would be expected to eradicate the ROY0 cells, and the ROS/act D and ROS/CPA cells as well, in order to achieve high CR rates and high cure rates. When the two drugs were delivered simultaneously, q14d ( ~ 4 )low , dose levels of either or both drugs resulted in relatively poor endresults. Low levels of one or both drugs were not used in a sequential manner, but from trials carried out with many combinations in other tumor systems it seems safe to say that for sequential delivery to be curative the first treatment must eradicate all tumor cells that are resistant to the second treatment before switching to the second treatment. The uncertainty regarding this point (because of a basic tenet of the mutation theory emphasized earlier) often may make simultaneous or alternating delivery of non-cross-resistant drugs more attractive.

36 1

MUTATION THEORY OF LURIA AND DELBRUCK

TABLE V SOME RELATED OBSERVATIONS AND DEDUCTIONS HAVING TO D O DISSEMINATED CANCERS~ Observations and deductions 1. The direct relationship between tumor cell burden and the probability of the presence of drugresistant tumor stem cells 2. The expected fluctuation in the resistant: sensitive tumor cell ratio in similarly staged individuals 3. The inverse relationship between the neoplastic cell burden and curability with chemotherapy 4. The superiority of surgergy plus chemotherapy, over surgery or chemotherapy alone, in curing certain disseminated animal and human neoplasms-so long as the residual tumor cell burden after surgery is not too large 5. The diversity in the degree and duration of tumor response in similarly staged and treated individuals (humans and animals) 6. Effective but noncurative chemotherapy increases the remission and survival time variance of treatment failures; ineffective chemotherapy does not 7 . Combination chemotherapy, when optimally delivered, often is superior to single-drug treatment because it delays or prevents failures due to the selection and overgrowth of specifically drug-resistant neoplastic cells 8. Five variables affect the rate of selection of clinically resistant neoplastic cell populations. K , and the original resistant: sensitive tumor cell ratio are dominant variables 9. The criteria for optimum delivery of non-crossresistant combinations of drugs may be as simple (or complicated) as this: It is necessary that the doses (and %) of all drugs be high enough, and the intervals between each non-cross-resistant drug be short enough, to eradicate the neoplastic cells that are sensitive to all of the drugs and the pertinent drug-resistant phenotypes as well. Obviously the combination must be tolerated

WITH

TREATMENT OF

Comments 1. Consistent with the mutation theory 2. Consistent with the mutation theory 3. Related to 1 4. Related to 1 and 3

5. Related to 2 6. Related to 2

7 . Consistent with the mutation theory and other points listed 8. Appears consistent with the other points listed

9. Appears consistent with all of the points listed

This degree of compatibility between the observations and deductions of many experimentalists and clinicians is impressive (to me). The points discussed in Section VI support some of these deductions.

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HOWARD E. SKIPPER

X. Closing Remarks

In this article it may appear that I have tried to consider too many observations and analyses, some of which may not appear to be related. I suppose my decision to include what I did was influenced by a number of things, not the least of which were the quotations from Luria and Delbruck, most particularly (1) their lament that, at first, their results did not seem to be reproducible from day to day, and (2) their eventual deduction that large fluctuations in the proportion of resistant cells are a necessaw consequence of the mutation hypothesis and that quantitative study of the fluctuations may serve to test the hypothesis. In studying the reasons why cancer chemotherapy succeeds when it succeeds and fails when it fails, we eventually came to the conclusion that the diverse information and deductions listed in Table V are indeed related. Furthermore, as we examined the number of critical variables involved in optimum delivery of non-cross-resistant combinations of drugs (in the light of the fact that each year many thousands of patients with disseminated cancers respond to chemotherapy as depicted in curve ac in Fig. 5) it seemed increasingly important to try to apply more quantitative approaches to interpretation of available results and in planning new combination chemotherapy regimens. In my opinion the prudent use of available simulation programs can be helpful even though at first this may sound too complicated. Reasonable simulations almost surely will make the designers of new chemotherapeutic protocols think about limitations and possibilities that would not otherwise come to mind. Finally, the 40-year-old somatic mutation theory (or law) appears to be pertinent to problems faced in increasing the cure rates of disseminated cancers today-even those cancers that are relatively refractory to most available single drugs.

ACKSOWLEDGMEKT The experimental therapeutic research from Southern Research Institute referred to herein was supported by the Division of Cancer Treatment of the National Cancer Institute.

REFERENCES Blum, H. H., Frei, E., III., and Holland, J. F. (1982). 111 “Cancer Medicine” (J. F. Holland and E. Frei, 111, eds.), 2nd Ed., pp. 732. Lea & Febiger, Philadelphia, Pennsylvania. Goldie, J . H., and Coldman, A. J. (1879). Cancer Treat. R e p . 63, 1727-1731.

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Goldie, J. H., Coldman, A. J., and Gudauskas, G. A. (1982). Cancer Treat. Rep. 66,439449. Law, L. W. (1952). Nature (London) 169,628-629. Lloyd, H. H. (1977). (Uniu. Tex. System Cancer Center M . D. Anderson Hosp. Tumor Inst. Annu. Symp. Fundament. Cancer Res., 29th, 1976 pp. 455-469. Luria, S. E., and Delbruck, M. (1943). Genetics 28, 491-511. Schabel, F. M., Jr. (1979). In “Nucleoside Analogues: Chemistry, Biology, and Medical Applications” (R. T. Walker, E. DeClercq, and F. Eckstein, eds.), Vol. 26, pp. 363394. Plenum, New York. Schmid, F. A., Hutchison, D. J., Otter, G. M., and Stock, C. C. (1976).Cancer Treat. Rep. 60,23-27. Skipper, H. E., Schabel, F. M., Jr., and Wilcox, W. S. (1964). Cancer Chemother. Rep. 35,l-111. Skipper, H. E., Schabel, F. M., Jr., and Lloyd, H. H. (1978). Semin. Oncol. 15,207-219.

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CARCINOGENESIS AND AGING Vladimir N. Anisimov Laboratory of Experimental Tumors, N. N. Petrov Research Institute of Oncology, USSR Ministry of Public Health, Leningrad, USSR

I. Introduction ................................. 11. Spontaneous Carcinogenesis and Aging . . , . . . . . . . . . . . . . . . . . . . . . . . . . . A. Age-Associated Rise in Tumor Incidence in Animals and Humans . . . . B. Life Span and Tumor Incidence. . . . . . . . . , . , . . . . . . . . . . . . . . , . . . . . . 111. Chemical Carcinogenesis and Aging. . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Carcinogenesis Induced by Foreign Bodies and A V. Radiation Carcinogenesis and Aging. . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . A. Ionizing Radiation . . . . . . . . . . . . . . . . . . . . . . . . B. Ultraviolet (UV) Light.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Hormonal Carcinogenesis and Aging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Viral Carcinogenesis and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Mechanisms of Modifications of Carcinogenesis by Aging . . . . . . . . . . . . . . A. Initiating Effect of Carcinogens and Aging. . . . .... . . . . . . . . . . . . . . . . . . B. Promotion of Carcinogenesis and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Factors Modifying Rate of Aging and Carcinogenesis , . . . . , . . . . . . . . . . . . A. Carcinogens as Promoters of Aging . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Promoters of Aging and Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . C. Effect of Geroprotectors on Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . ............ X. Summary.................................... References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............

365 367 367 369 370 379 380 380 384 385 388 390 391 399 404 405 408 409 415 415

I. Introduction

The life span and rate of spontaneous neoplasm development in a species are among the most important integral indices of its biological characteristics. Both parameters depend upon a number of closely interconnected phenomena occurring in the organism during ontogenesis at various levels (subcellular, cellular, tissue, and organism). The existence of a close link between gerontogenesis and tumorigenesis is suggested, in particular, by the similarity of the equations for the curves showing death rate versus age (from 10 to 80) due to accidental causes and cancer (Dix et al., 1980). However, the essence of this link and causes for age-associated rise in tumor incidence are still obscure, notwithstanding the amount of evidence of similarity between the mechanisms of these two processes and related theories (Burnet, 1970; Pet0 et al., 1975; Pitot, 1977,1978; Doll, 1978; Dilman, 365 ADVANCES IN CANCER RESEARCH, VOL. 40

Copyright 8 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-006640-8

366

VLADIMIR N. ANISIMOV

1978, 1981; Magee, 1978; Dix et ul., 1980; Anisiniov and Turusov, 1981; Anisimov, 1981b, 198%). Recently, Xlartin (1980) analyzed 15 major ideas or concepts of the mechanism of aging. As changes in genome are believed to be the primary substrates of aging, it is possible, according to Martin, to divide the existing theories of aging into two groups. The first group suggests that modifications in gene structure are responsible (stochastic theories) and thus aging would appear to be a result of random irrepairable damage to niacroniolecules (nucleic acids and proteins) by different endogenous and exogenous factors, such as free radicals, mutagens, etc. The second group implicates modifications in gene expression (including theories of “programmed aging,” regulatory, neurohunioral, and immunologic theories). Therefore, from the gerontologist’s point of view, age-associated increase in tumor incidence may be either the result of the summation of random genome damage leading to malignant transformation of a cell, or the result of some “program” for cancer development at different levels of the organism which is produced during the course of natural aging. From the oncologist’s point of view, it is possible to explain ageassociated rise in tumor incidence in two ways: (1)as age advances, the duration of exposure to endogenous or exogenous carcinogens is increased and/or the total dosage of carcinogenic agents is increased; and (2) aging involves changes in the organism which promote tumor growth. Modern researchers consider carcinogenesis a two-stage process and lead to a concept which combines the above two hypotheses and explains the controversial results obtained in many experiments in which various carcinogens (chemical, radiation, hormonal, viral) are administered to animals of different ages. It is believed that during the first stage of carcinogenesis (initiation) the transformation of cells from normal to malignant takes place as a result of the effect of either exogenous or endogenous carcinogens. During the second stage (promotion), the fate of transformed cells is decided, that is, either they will be eliminated by the immune system or will be permitted unrestrained growth and progression (Berenblum, 1974; Pitot, 1978). For example, the initiating effect of chemical carcinogens on animals of various ages may be determined by many factors, such as the activity of metabolizing enzymes and their ability to produce mutagenic adducts, the binding rate of these with DNA, the efficiency of DNA repair systems, and the proliferative activity of target tissues. On the other hand, there is much evidence that age-associated changes occur-

CARCINOGENESIS AND AGING

367

ring in the internal environment of the organism promote the carcinogenic process.

II. Spontaneous Carcinogenesis and Aging

A.

AGE-ASSOCIATED RISE IN TUMOR INCIDENCE IN ANIMALSAND HUMANS 1. Tumors in Humans

There is much evidence that cancer incidence increases with age (Doll, 1973; Ponten, 1977; Dix et al., 1980; Napalkov et al., 1980). However, the rate of age-related development of neoplasms varies according to their localizations. Doll (1973) distinguishes four types of tumor distribution as age advances. The first includes the nephroblastomas and retinoblastomas related to early childhood, when the peak of tumor incidence is observed. After this period, such tumors are found rather infrequently; for instance, retinoblastomas are very rare in children over 5 years old. The second peak of tumor incidence is observed in middle age (over 40 years), an example of which is lung cancer. The third type (e.g., stomach cancer in Europe) is characterized by a sharp rise in tumor incidence in old age (over 60). The fourth peak of cancer incidence (such as breast and cervix cancer) is observed between 40 and 50 years, after which tumor incidence may even decrease. The classification suggested by Moolgavkar and Knudson (1981) differs only slightly. A simpler classification of curves illustrating the relationship between tumor rate and age is put forward by Dix et al. (1980). These authors subdivided all tumors found in humans (except for chlorionepithelioma) into two classes. The first class includes all tumors appearing in man after 50 years of age with onIy one peak in their rate, i.e., the majority of tumors. The second class is composed of tumors having two peaks of incidence (under 35 and over 50 years of age), such as lymphoid leukemia, bone and testicular tumors, and Hodgkin’s disease. Data on tumors of the first class showed that their incidence is exponentially increased between 10 and 80 years of age according to the following equations (Dix et al., 1980): male: log percentage total incidence = 0.03l(age) - 1.15 (I) female: log percentage total incidence = 0.027(age) - 0.897 (2)

368

VLADIMIR N. ANISIMOV

A number of researchers point out that the evaluation of age-associated dynamics of some tumors depends on their detectability, which may influence results for a given age. For instance, tumors of the skin, lips, throat, bladder, and cervix are diagnosed at an earlier stage than those of lungs, stomach, or pancreas (Dix et al., 1980). Cancer of the prostate seems to be found in old men much more frequently than would appear from the number of cases that have been registered (Ponten, 1977). It should be noted that the age-related dynamics of benign tumor incidence in man has not yet been studied, although this type of neoplasm is observed more frequently than malignant tumors (Ponten, 1977). According to Lever (1967), basal cell papillomas (with the exception of some inherited cases) are not found until puberty and are rare under 40 years of age, after which time their incidence quickly increases. Although there are no quantitative data available, it is assumed that all old people have verrucae senilis.

2. Tumors in Experimental Animals Spontaneous tumor incidence, localization, and type in experimental animals are greatly varied and depend on both endogenous (genetic) and exogenous factors (geophysical, dietary and housing conditions, pollution of water and food with carcinogenic and other agents, etc.). However, experimental animals, like humans, also demonstrate age-related increases in tumor incidence (Anisimov, 1976; Pour et al., 1979; Priestler and McKay, 1980). Inbred strains of mice, characterized by selective development of one or two tumor localizations, were found to manifest a definite age-related rise in tumor incidence. For example, 7- to 10-month-old male AKR mice develop leukemias followed quickly by death in 80-90% of the cases. The incidence of spontaneous lung adenomas approaches 90% in one strain of mice only when they reach the age of 18 months (Storer, 1966; Staats, 1980). However, noninbred mice, with a wider spectrum of spontaneous tumors, demonstrated a 64% age-related rise in tumor incidence by the age of 25 months (Andervont and Dunn, 1962). Oncological characteristics of rats of different stocks and strains are fairly stable, though the range of tumor development is wider than that of mice; for instance, rats develop more benign tumors. It should be noted that both humans and rats develop more benign than malignant tumors, and 80-95% of all tumors appear in the endocrine or reproductive systems in rats, but not in man (Anisimov, 1976). In female rats from the “Rappolovo” breeding farm of the USSR Academy of Medical Sciences, the majority of tumors were detected between days 801 and 900 of life, corresponding to the peak of tumor

CARCINOGENESIS AND AGING

369

incidence in the endocrine glands and reproductive organs that is characteristic of rats at this age (Anisimov et al., 1978).

INCIDENCE B. LIFESPANAND TUMOR Some investigators consider an increase in the dose of or exposure time to carcinogens as the main cause of age-related increases in tumor incidence (Peto et al., 1975). This is in accordance with the wellknown relationship between the dose of carcinogen and the time required for tumor development: dt" = const., where d = daily carcinogenic dose, t = mean tumor latency, and n = constant value (Druckrey, 1967). It can be seen that all the damage that is induced by exogenous or endogenous factors and causes malignant transformation is amassed at the moment of carcinogenic action, independently of the age, i.e., the effective dose of a carcinogen per unit of time is a constant value. In accordance with this, the probability of the development of spontaneous tumors should be greater with increases in the life span of the organism and in the life span of the population, respectively. But this is not the case; our data showed a decrease in relative spontaneous tumor incidence in rats aged over 900 days (Anisimov et al., 1978). Similarly, some authors observed an age-associated decrease in cancer incidence in humans over 80 years old (Ponten, 1977; Dix et al., 1980), though these results require further elucidation. Spontaneous tumor incidence does not correlate with the life span of a species and is approximately 30%for humans who live to the age of 69-77 years (Stukonis, 1979) and for noninbred rats that live for 3 years (Anisimov, 1976). Direct quantitative comparison between data on spontaneous and induced carcinogenesis in different species cannot be made. There are data on the inverse correlation between the duration of the life span of a species and the ability of tissues to activate procarcinogens into mutagenic adducts, and there is evidence for a direct correlation between the life span of a species and the repair efficiency of DNA damaged by different carcinogens (Table I). Therefore, long-lived species seem to be less sensitive to the initiating effects of carcinogens. It should be noted that some authors do not report a correlation between life span and the efficiency of systems responsible for DNA repair (Kato et al., 1980).This disparity may be a result of differences in techniques used to evaluate DNA repair efficiency. No significant positive correlation between life span and tumor incidence was found in long-lived or short-lived strains of mice by Storer (1966). Pour et aZ. (1979) stressed that genetic factors were much more responsible for the variation in hamster spontaneous tu-

370

VLADIMIR N. ANISIMOV

TABLE I CORRELATIOS BETWEEN SPECIESLIFE SPANAND SOMECHARACTERISTICS OF CARCINOGEN-TREATED TISSUES

Parameter Cytochrome PA48 level Rate of conversion of procancerogen to mutagen Binding of mutagens with DNA DNA repair accuracy

Carcinogen

Type of correlation

Fibroblasts

Benzo(a)pyrene

Inverse

Fibroblasts

DMBA

Inverse

Fibroblasts

DMBA

Inverse

Fibroblasts

UV light

Direct

Fibroblasts

Alkylating agents Alkylating agents

Direct

Tissue

Lymphocytes Liver

KMU

References Pashko and Schwartz (1982) Schwartz (1975)

Direct

Schwartz and Moore (1977) Hart and Setlow (1974) Francis et al. (1981) Medcalf and Lawley (1981) Harris et al. (1981)

Direct

Pegg et QZ. (1982)

mor incidence than the duration of life span of this species. It is a well-known fact that in some populations of animals of the same strain or stock, kept under standard conditions, certain variations in mean life span and spontaneous tumor incidence are observed (Turusov et al., 1973; Anisimov, 1976; Anisimov et al., 1978). We compared the results of observations on tumor incidence versus mean life span in 12 groups of noninbred rats (total number of animals, 561), that served as controls in our experiments (Anisimov, 1971, 1980; Anisimov et al., 1978), and failed to find a positive correlation between these two parameters. Thus, the available data show no positive correlation between spontaneous tumor incidence and (1)the life span of a species, ( 2 )the life span of different strains and stocks of the same species, and (3)the life span of separate populations of the same strain. This conclusion is in conflict with the concept suggesting a summation effect of the events that cause malignant transformation of the age-related increase in tumor incidence. 111. Chemical Carcinogenesis and Aging

The data obtained from a number of experiments involving administration of chemical carcinogens to experimental animals of various

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ages are inconsistent and rather controversial (Anisimov and Turusov, 1981; Anisimov, 1982b). Differences in results might be caused by many factors, such as the use of different carcinogens (direct or indirect), methodological approaches (route of administration, dosage per kilogram of body weight or per animal, single or chronic treatment), and specific organism features (species, strain, sex, age group). It is clear that the effective dose of indirect carcinogen requiring metabolic activation would vary in accordance with the age of the animals, since the activity of the corresponding enzymes in the liver or target tissue may also change with age. When a carcinogen is administered at a specific dose per unit of body weight, older animals may receive a greater absolute carcinogen dose. When large doses of carcinogens that induce tumors in internal organs are administered to animals, it is difficult to evaluate the age-related sensitivity to the carcinogen unless the experimental animals are killed at definite time intervals. When the carcinogen is administered in food or water, the quantity of feed or liquid consumed should be taken into account, as it may differ in animals of varying age. In one of our experiments young adult and old female rats were injected intravenously (iv) with N-nitrosomethylurea (NMU) (Anisimov, 1981a).Kidney tumors and mammary adenocarcinomas occurred mostly in 3-month-old NMU-treated rats; old (14-month-old) NMUtreated rats developed no mammary tumors and the incidence of kidney tumors was much lower than in the younger animals. However, cervicovaginal malignant tumors were observed in old NMU-treated rats only and the incidence of leukemias was the same in animals of both age groups. Thus, in the same experiment we observed both an age-associated decrease (mammary, kidney) and increase (cervix, vagina) in the sensitivity of tissues to a carcinogen, or no effect of age at all (hematopoietic system). This article will deal with the effect of aging on the sensitivity of some tissues to chemical carcinogens.

1. Skin Some authors report delayed development of skin tumors induced by polycyclic aromatic hydrocarbons (PAH) with advancing age. Many more 3-methylcholanthrene (MC) or 7,12-dimethylbenz(a)anthracene (DMBA)-induced tumors developed at a faster rate in 4-monthold NZB mice than in 12-month-old animals (Forbes, 1965; Morton et al., 1977).A single application ofa 0.5% solution of DMBA in benzene on the skin of St/Eh mice of various ages did not alter papilloma incidence, although latency of these tumors was greater in older animals (Engelbreth-Holm and Jensen, 1954). Stenback et al. (1981~)

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reported enhanced skin tumorigenesis induced by administration of different single doses (10-300 pg) of DMBA to female Swiss mice aged 48 weeks, as compared to that in 8-week-old animals. It should be noted that these authors did not observe the development of DMBA-induced skin tumors in 8-week-old mice treated with small doses of carcinogen (10 or 30 pg) over a period of 23 weeks after administration, while tumors developed in 2/72 (10 pg) and 6/63 (30 pg) 48-week-old mice over the same period. Experiments involving reciprocal syngeneic transplantation of skin grafts between animals of varying age (Ebbesen, 1977) showed that the blastomogenic effect of DMBA on mouse skin is diminished between 4 and 14-20 months of age, but increases thereafter. In carefully planned experiments by Pet0 et d.(1975), benzo(a)pyrene (BP) was applied chronically to the skin of female mice from the age of 10, 25,40, or 55 weeks. It was found that the rate and frequency of development of benign and malignant skin tumors were determined solely by the duration of exposure, i.e., total dosage of carcinogen, but bore no relation to the age at which treatment with BP was started. However, these experiments involved the use of a dose that induced tumors in all animals of all age groups, which might have eliminated any differences; moreover, no really old animals were used. In order to analyze the age-related dynamics of skin sensitivity to chemical carcinogens, it would be interesting to consider the results of studies on cocarcinogenesis, in which polycyclic aromatic hydrocarbon (PAH) was used as an initiator and the active ingredients of croton oil were used as promoters. These agents were applied to the skin of animals of varying age at different time intervals, 6-, 44-, and 56-week-old mice were treated with single doses of 20 pg DMBA in acetone and then 2 weeks later 2.5 p g of phorbol myristate acetate (PMA) was applied to the skin three times weekly for life (Van Duuren et al., 1978). A decrease in the incidence of papillomas and cancers was observed with an increase in the age of the mice at the start of the experiment. When application of the carcinogen to the skin of young (6 weeks) mice was not immediately followed by the promoter application, the carcinogenic process was increasingly delayed with increasing time interval between DMBA and promoter applications. Another carefully planned experiment (Stenback et al., 1981a-c) involved application of a single dose of 10-300 pg DMBA to the skin of female Swiss mice aged 8,48, or 68 weeks; 3.2 p g 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied as a promoter twice a week for 15 weeks starting 3 weeks after DMBA application. The authors

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did not observe any changes in the initiation effect of DMBA on mice aged 8 or 48 weeks; however, in the older age group the incidence of papillomas or cancers was considerably decreased. Skin tumor incidence was also decreased with increasing time interval between DMBA and TPA applications. The authors believe that the number of DMBA-transformed cells is not reduced as age advances, but that it is the age-related decrease in sensitivity to the TPA that leads to such a result. Data in the literature point to either delayed or lack of variations in blastomogenesis in mouse skin between 3 and 16-20 months of age with enhancement of the carcinogenic effect occurring after the age of 20 months. Ebbesen (1977) attributes the decrease in the carcinogenic effect of the PAH at months 14-20 to the age-related decline in the mitotic activity of mouse skin epithelium. At the same time, he believes that the increase in sensitivity to PAH in old mice is due to the low activity of chalones, or, more likely, to age-related disturbances in DNA repair efficiency. Such concepts are in accordance with data that demonstrate a decline in cell proliferation and in the rate of cell renewal in mouse skin between 3 and 19 months of age (Cameron and Thrasher, 1976)and correspond to findings on the age-dependent lowering of the percentage of "dark" skin basal cells which are regarded as stem cells (Klein-Szanto and Slaga, 1981). There is also some evidence for an age-related decrease in epidermal chalone activity in mouse skin (Olsson and Ebbesen, 1977). 2. Soft Tissues Mice and rats, aged 1-180 days and older, were injected with 1,2,5,6-dibenzanthraceneor BP (Dunning et al., 1936). While the frequency of tumor development was the same in all age groups, older animals developed more tumor nodes. Tumor latency was significantly shorter in the older age groups of both mice and rats. Subcutaneous injection of MC in C57BL mice aged 21 days and 6 or 20 months was followed by formation of tumors in all animals of the first group by day 428, in all those of the second group by day 180, and in all those of the third group by day 120 (Franks and Carbonell, 1974). Majski et al. (1978) injected BP sc in rats aged 1-9 or more than 12 months and established that aging involves an increased incidence of soft-tissue sarcomas (63% in 3-month-old rats and 87% in 9-month-old rats). Moreover, the mean latency of tumors in the old age group was significantly lower than in younger rats. Saxen (1954) injected MC sc in C3H mice aged 1-2 weeks or 3 and 12 months, and found that sarcomas were less frequent and tumor

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latency was longer in the older age group. Similar results were obtained by Stutman (1979) in experiments involving sc injection of 0.1 mg MC in 120- 210-, and 360-day-old CBA/H mice. Tumor incidence in these animals was 92, 76, and 26%, respectively; whereas in those receiving 0.02 mg MC it was 36, 12, and 0%, respectively. Thus, it seems that as a rule aging has a stimulating effect on sarcomogenesis after local exposure to PAH. The causes are still obscure. Age-related changes in the immune system do not seem to play a leading role in the age-associated enhancement of chemically induced sarcomogenesis. As was shown by Stutman (1979), sc MC injection in nude athymic mice of varying ages caused tumor development at the same rate as in mice with normal immune function. The agerelated decline in DNA repair should be recalled. The experiments by Fort and Cerutti (1981) demonstrated a slowing of excision DNA repair caused by ethylnitrosourea (ENU) in fibroblasts of 2-year-old rats as compared to 3-week-old rats. 3. Mammary Gland The results of experiments by Huggins et al. (1961), confirmed by multiple reports, showed that treatment of female rats with DMBA or MC between 50 and 75 days of age induced mammary adenocarcinomas in 90-100% of cases, whereas in younger and older animals tumors were much less frequent. An age-related decrease in rat mammary epithelium sensitivity to the carcinogenic effects of N-4-(4'-fluorobiphenyl)acetamide(FBAA) and NMU was shown by Stromberg and Reuber (1975) and Anisimov (1981a). Studies of the morphofunctional condition of mammary epithelium at the stage of its maximal susceptibility to carcinogens revealed that the DNA-binding capacity of DMBA in the mammary epithelium of 50-day-old rats is several times that in 80-day-old animals, which was in direct correlation with tumor incidence (Janss and Ben, 1978), mitotic activity, and DNA synthesis in mammary epithelium (Nagasawa, 1981; Russo et aZ., 1981). It was also shown that strain differences in the effect of PAH on rat mammary gland are determined by differences in the time of onset of puberty (Sydnor et al., 1962), which points to the hormonal background of changes in the intensity of proliferative processes in the mammary gland. Recent results by Tay and Russo (1981) show that in aged rats the efficiency of excision DNA repair is enhanced in mammary gland epithelium. This, however, does not rule out the possible role of the age-related decline in some enzyme activity which metabolizes carcinogenic substances, including PAHs (see below).

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4. Liver In numerous experiments (Reuber, 1975a), rats were fed various hepatocarcinogens [FBAA, methyl-4-dimethylaminoazobenzene,1,2fluorenyldiacetamide, and N-nitrosodiethylamine (DENA)] for equal periods of time, beginning at age 4,12,24, or 52 weeks. All the experiments showed 4-week-old rats to be the most sensitive to carcinogenic agents. Hepatocellular tumors were most frequent and in many cases were multiple, less differentiated, and had metastases in younger animals. Similar results were obtained after feeding dimethylaminoazobenzene (DAB), aflatoxin B1 (AFBl), and N-nitrosodimethylamine (DMNA) to rats of varying ages (Decloitre et al., 1973; Goes et al., 1975; Savchenkov et al., 1980). Mixed-function oxidases of the liver, which metabolize nitroso compounds, aromatic amines, and some other substances to proximal carcinogens, registered the peak of their activity in rats in the fourth week of life span; they decreased by month 2 (Decloitre et al., 1973), and seemed to reduce the total effective dose of the carcinogen and to be responsible for a considerable (70%)decrease in the binding of proximal carcinogen to liver DNA and proteins (Davies et al., 1976). It should be noted that carbon tetrachloride, which does not require such metabolic activation, was more effective in causing liver neoplasms in old animals (Reuber and Glover, 1967). In the experiments by Khudoley (1981), in which young and mature (6- to 7-week-old and 12- to 18-month-old) Rana temporaria (L) frogs were kept in water containing 5 ppm DMNA or dimethylnitramine (DMNO), tumor incidence in the liver (hepatocellular cancer) was greater in the older age group.

5. Gastrointestinal Tract Rats fed DENA from age 12 months did not develop tumors of the esophagus. However, maximal incidence of such tumors was observed after the carcinogen was administered from the age of 4 weeks (Reuber, 1976). The administration of DENA in drinking water for 11 weeks to mice aged 10 weeks or 9.5 and 17 months induced squamous cell tumors of the forestomach in 82, 85, and 53% of cases, respectively. However, the mean latency in the old group was shorter (108 days) compared to young and middle age groups (192 and 164 days, respectively) (Clapp et al., 1977). Kimura et al. (1979) administered N-methyl-N ‘-nitro-N-nitrosoguanidine (MNNG) in drinking water to rats aged 6, 20, or 40 weeks. The animals were sacrificed 50-60 weeks after the beginning of the experiment and the rates of gastrointestinal tumor incidence were 95,

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74, and 49%, respectively. It should be noted that animals in the young age group often developed adenomatous hyperplasia together with adenocarcinomas of the stomach, while the oldest group had gastric tumors only. Male and female BD-IX rats received 20 weekly sc injections of 1,2dimethylhydrazine (DMH) from age 35, 120, or 210 days (Moon and Fricks, 1977). The animals were killed 35 weeks after the last injection. Colon tumors were found in all males and 73%of females receiving treatment from day 35.In the other age groups, tumors were found in 74 and 73%of males, respectively, and the yield of tumors was the same. In females treated with DMH from day 120 or 210, colon tumors appeared in 35 and 9% of animals, respectively, and the number of neoplasms per rat decreased accordingly. In the experiments of Pozharisski et al. (1980)rats were treated with DMH from the age of 4, 8-10, or 18 months. WhiJe the number of intestinal tumor-bearing rats was practically identical in all age groups, the multiplicity of colon cancers diminished with advancing age. Large malignant tumors with pronounced invasive growth were more frequent in older animals, whereas younger rats more often revealed the early stages of malignancy (ca. in situ and superficial cancers). Among the important factors of the age-related decrease in the susceptibility of rats to the carcinogenic effect of DMH may be the agerelated decline in cell renewal rates and the activity of some enzymes which are vital for nucleic base metabolism in intestinal epithelium (Cameron and Trasher, 1976; Salser et al., 1976; Pozharisski et al., 1980).However, the rates of age-related changes are likely to be determined by some genetic or sex-dependent factors. Thus, in BD-I1 rats aging has no effect on the incidence of colon tumors (Moon and Fricks, 1977). Castration prior to DMH treatment did not influence carcinogenesis in female rats, while in the older age groups of male rats it resulted in a substantial decrease in tumor incidence and yield; administration of androgens brought these parameters to levels observed in uncastrated animals (Moon and Fricks, 1977). In a carefully planned experiment by Turusov et al. (1981), DMH was injected into CBA mice aged 2, 8, and 12 months. The animals were killed in groups every 2 weeks beginning at week 26 from the start of the experiment. Total colon tumor incidence in the 2-monthold mice appeared to be lower than that in the 8- and 12-month-old animals and was 13, 61, and 69%, respectively. The older age group showed a greater incidence of tumors with pronounced invasive growth, whereas younger mice had a considerably longer latency of colon tumors.

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6. Kidneys In female rats treated with FBAA or NMU at various ages the incidence of kidney tumors was found to be inversely related to age (Reuber, 1975b; Anisimov, 1981a). Savchenkov et al. (1980) administered DMNA by inhalation to young (1.5-month-old) and old (18month-old) male rats 5 days a week for 8 months, after which time all animals were killed. Kidney tumors (Wilms type) had developed in 11/70 young and in 1/70 old animals. Single ip injection of DMNA (30 mgkg) in rats of varying ages induced a bimodal distribution of mesenchymal and epithelial kidney tumors (Hard, 1979). New-born and immature rats developed mesenchymal tumors more frequently. The incidence of such tumors decreased to zero in rats treated with DMNA at the age of 5 months. However, epithelial tumors of the kidney cortex were more frequent during puberty, though the incidence of these tumors was lower in 5-month-old animals, and an agerelated decrease in kidney tumor malignancy was observed. The leading factors in the age-related decrease in the sensitivity of rat kidney to carcinogens seem to be reduced mitosis in kidney, prolonged time of cell renewal, and age-related decline in DNA replication intensity (Cameron and Trasher, 1976; Levitsky, 1980). 7. Bladder According to Hoover and Cole (1973), the relative risk of bladder cancer development is higher in people exposed to industrial carcinogens when young, as compared to those exposed later in life. However, some experiments in vitro give evidence of the possible enhancement of bladder epithelium sensitivity to the transforming effect of carcinogens in aged subjects. Neoplastic transformation was induced in primary cultures of C57BWIcrf mouse bladder epithelium by a single 24-hr exposure to DMBA (Summerhayes and Franks, 1979). In explants from older (28-30 months) mice, the transformed sites appeared earlier (by day 40-60) and were more frequent (in 28% of cases), whereas in the cultures obtained from younger (5-7 months) mice, the transformation had occurred by day 100 in 0.9% of the cases. Spontaneous transformation of bladder epithelium was observed only in explants derived from old mice.

8. Uterus and Vagina DMH treatment of CBA mice, aged 2-3 and 12-13 months, induced uterine sarcomas at the same incidence rate in both groups, although they appeared much earlier in older than in younger animals (Turusov et al., 1979). After iv injection of NMU into 14-month-old female rats,

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malignant tumors of the cervix uteri and vagina were registered in one-third of animals, while similarly treated 3-month-old rats did not develop tumors at these sites (Anisimov, 1981a). It may be suggested that the elevated serum estrogen level in female rats of this age, as well as the relative weight of the uterus (Anisimov and OkuIov, 1980), promote carcinogenesis in estrogen-dependent tissues of the uterus and vagina of older rats. Studies on ["]thymidine uptake by uterine DNA of 3- and 14- to 16-month-old rats showed an age-related increase of 21% (Likhachev et al., 1983). Cervicovaginal sarcomas were detected in rats treated with alkylnitrosoureas during pregnancy (Alexandrov, 1969), while the estrogen level is elevated. It should be noted that administration of estrogens in mice promoted DMH-induced carcinogenesis in the uterus (Ird and Smirnova, 1979). Thus, it may be suggested that age-related hormonal shifts promote proliferative activity in the uterus and vagina of 14-month-old rats, which is a critical factor in their high sensitivity to the blastomogenic effect of

NMU. Local intravaginal applications of DMBA in young (3 months) and old (18 months) SHR mice induced squamous-cell carcinomas of the cervix uteri and vagina in all animals. However, in older mice the tumor growth rate was higher and caused death more quickly than in younger animals (Anisimov, 1982b). 9. Lungs According to Doll (1978), the frequency of lung cancer in tobacco smokers is convincing evidence of the role played by increased smoking of more cigarettes for a longer time in the age-related rise in lung cancer incidence. This author also believes that the sensitivity of the organism to the carcinogenic effect of tobacco smoke is not increased with advancing age. It should be noted, however, that the decrease (occurring after 30 years of age) in inducibility of aryl hydrocarbon hydroxylase (Paigen et aE., 1978) is not taken into account in this case, nor is the age-associated decline in the lung ventilation ability (Klocke, 1977). According to the above data, daily doses of the carcinogen received by older tobacco smokers may be lower. Therefore, Doll's results on the exact correlation between lung cancer incidence in and the number of cigarettes smoked by individuals of varying age would indicate an increase in sensitivity to carcinogenic tobacco smoke components with advancing age. When mice aged 10 weeks or 9.5 or 17 months were treated with DENA in drinking water for 11 weeks, the incidence of lung adenomas in all age groups was identical but the latent period was shorter in the old age group (Clapp et al., 1977). Mice, 11-50 weeks old, were

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injected ip with single doses of urethane; 8 weeks after the start of the experiment the older mice had a lower incidence of lung adenomas, which were smaller in size than those in younger animals (Dourson and Baxter, 1981).According to Simnett and Heppleston (1966),proliferative activity of alveolar epithelium in mice is lower between 3 and 12 months of age, but then remains unchanged up to 24 months. It should be noted that in old rats the rate of lung metabolism of certain carcinogens producing mutagenic metabolites was somewhat higher than in rats of the middle age groups (Robertson and Birnbaum, 1982). There are no data on this problem obtained in experiments with mice.

10. Vascular Wall Mice, 10 weeks or 9.5 or 17 months old, were treated with DENA and the incidence of vascular tumor development was identical in all age groups, although latency was shorter in the old age group (Clapp et al., 1977).Vinyl chloride-treated 6-, 18-, 32-, and 52-week-old male and female rats developed angiosarcomas in the liver. Male rats had tumor incidences of 0, 0, 6.7, and 24%, and females 5.3, 15, 47, and 20%, respectively (Groth et al., 1981). The authors suggested a promoting effect of aging on the sensitivity of vascular walls to the carcinogenic action of vinyl chloride. 11. Hematopoietic System Experiments involving iv injections of NMU into rats of varying age failed to establish any age-related variations in the incidence of induced leukemia (Anisimov, 1981a). However, in BALB/c mice treated with pristan (2,6,10,14-tetramethylpentadecane)at the age of 1-2, 812, or 17-19 months, plasmacytoma incidence was 42, 29, and 88%, respectively (Sat0 et al., 1982). Rana temporaria (L) frogs, 6-7 weeks old, kept in water containing DMNA or DMNO, developed hemocytoblastosis more frequently than 12- to 18-month-old frogs under the same conditions (Khudoley, 1981). 12. Nasal Sinuses According to Doll (1978) the risk of nasal sinus tumor development in nickel industry workers increases with the age of the individual at the moment of the first contact with nickel. IV. Carcinogenesis Induced by Foreign Bodies and Aging

Data in the literature on the effect of aging on foreign body-induced carinogenesis are scarce. When Kogan (1959) implanted cellophane under the skin of rats of varying ages the animals developed sarcomas,

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with latency of tumor development being somewhat shorter in older rats than in younger animals. However, the author does not report the statistical analysis of the results and does not indicate the age of the animals used. In the experiments of Paulini et al. (1975), 1- to 15.5month-old male rats (total number 118) received sc polyester-polyurethane sponge implants of varying shape and size; tumor latency was inversely correlated with the age of animals at the time of foreign body implantation. Rats, 10 months old, treated intrapleurally with asbestos, developed pleural mesotheliomas earlier and with higher incidence than those treated at the age of2 months (Berry and Wagner, 1976). Epidemiological studies among asbestos industry workers by J. Pet0 et al. (1982) failed to establish the influence of age at first contact with asbestos on death rate caused by mesotheliomas. Thus, despite the paucity of data on the subject, the implantation of foreign bodies or asbestos injection into animals appears to enhance carcinogenesis as the age of an animal at the time of administration increases. V. Radiation Carcinogenesis and Aging

A. IONIZING RADIATION

The reduction of the life span of humans and animals after exposure to ionizing radiation generally depends on the increase in neoplasm incidence (Sacher, 1977; Bunger et al., 1981). A number of works showed, however, that life-shortening effects of radiation are more clearly pronounced in younger animals (Sacher, 1977; Yuhas, 1971). Several hypotheses are put forward to explain this phenomenon. One of them suggests that weaker animals die young, whereas those that reach old age are, to some extent, unaffected by damaging environment effects (e.g., radiation) (Yuhas, 1971). However, the analysis of data on the age-related sensitivity to the influence of carcinogens (Anisimov and Turusov, 1981) does not allow one to accept this concept. Another hypothesis suggests differences in the inhibiting effect of radiation on spontaneous tumor development in young and old organisms (Sacher, 1977). Doll (1978) showed that radiation-induced neoplastic development in patients with ankylosing sporidylitis is increased with increasing age of patients at the time of radiation exposure. He notes that these data are consistent with the observations made on individuals who had survived atomic bombardment, and on radiotreated female pa-

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tients of varying age with hemorrhagic methropatia. Doll concludes that age has a stimulating effect (age-related deterioration of immunity, in particular) on radiation-induced carcinogenesis. On the other hand, experiments with animals sometimes show different results. Tumor incidence was higher in young (1-to 3-month-old) rats exposed to fast neutron radiation (213-230 rad) as compared to older animals (10-15 or 21 months old) exposed to the same dose of radiation (Jones et al., 1968; Castanera et al., 1971). Single total-body radiation by X rays (300 R) of 7- to 52-week-old mice induced the development of tumors in 94% of the cases, whereas in 78- to 105-week-old animals tumor yield was 73% (Gajewski et al., 1977). On the other hand, Shikhodyrov et al. (1978) reported an increased tumor rate, yield, and malignancy in 9- to 12-year-old dogs exposed to y, neutron, or 210Poradiation, as compared to 3- to 9-year-old irradiated animals. 1. Hematopoietic System A number of experiments with mice showed that the incidence of radiation-induced lymphomas and leukemias reaches its maximal level in 1-to 2-month-old animals and decreases considerably when exposure to radiation occurs in older mice (Kaplan, 1950; Lindop and Rotblat, 1962a; Gajewski et al., 1977). However, Kobayashi et al. (1980) reported maximal thymic lymphoma incidence in the middle age group among 4-,19-, and 34-week-old mice exposed to y rays of 137Cs. In rats, aged 8-10 months, treated with 95Nb which selectively accumulated in bone, leukemia incidence was higher than in 3month-old rats (Streltsova and Moskalev, 1964). In 4-and 12-monthold W/Fu rats single exposure to X rays at a dose of 450 R resulted in leukemia in 14.8 and 26.9% of the cases, respectively, whereas similar treatment of 4-to 12-month-old Wistar rats had no effect on the incidence of leukemia, which was observed in 3.2 and 2.4% of the cases, respectively (Moloney et al., 1971). Local irradiation of the spinal column caused an increase in leukemia incidence in older patients in comparison to younger (Doll, 1978). However, Davis (1978) showed that the risk of leukemia in irradiated individuals after 10 years of age is one-half that of those under 10. This controversy might result from the nature and spread of radiation used: whether it is aimed mainly at bone marrow cells (local exposure or radionuclide injection) or at thymic cells too (total-body exposure), because thymectomy prevents the leukemogenic effect of total-body irradiation in mice (Kaplan, 1950). Moreover, the difference in age-dependent sensitivity to ionizing radiation in different species might result from the possible pecularities in the mechanisms of blastomogenic effects of radiation in different species of animals. It

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is known that the frequency of chromosomal aberrations in hematopoietic cells is increased with age, which may stimulate their agerelated sensitivity to the carcinogenic effects of ionizing radiation, as shown for humans and rats (Bochkov and Pilosov, 1968). However, in mice with leukemic retrovirus present in their genome (Huebner and Todaro, 1969), the age-related enhancement of viral genome expression may increase viral susceptibility to radiation, i.e., in older mice the exposure to radiation may have a therapeutic effect (Sacher, 1977). These results are supported by evidence on the radiation-induced decrease in mitotic activity of leukemic cells in mice (Beer et al., 1974), and the decrease in nonthymic lymphoma and granulocytic leukemia incidence in mice exposed to y- and neutron radiation (Upton et al., 1960). It is interesting that X-rays stimulate the ability of mouse thymic cells to incorporate leukemic virus (Latarjet, 1978). On the other hand, the experiments on autologous transplantation of bone marrow cells into irradiated mice raise doubts on the viral nature of myeloleukemogenesis (Alexandrov, 1972).

2. Ovaries Experiments involving irradiation of mice aged from 2 weeks to 6 months showed that maximal ovarian tumor incidence was observed in l-month-old animals (Kaplan, 1950; RiviCre et al., 1965). In 10- and 52-week-old LAFl mice the total-body exposure to X rays induced ovary tumors in 65 and 29% of cases, respectively (Cosgrove et al., 1965). Hyperstimulation of the ovary by pituitary gonadotropins, caused by an initial drop in estrogen level in radiation-damaged ovary, is believed to be of primary importance in the mechanism of ionizing radiation influence on ovarian tumor induction (Ird, 1966). The assumption that local radiation causes neoplastic transformation in mouse ovarian tissue seems inconsistent, because irradiation of one ovary does not induce tumors. We believe that irradiation of mice or rats in the postreproductive period when proliferative processes in the ovaries take place and gonadotropin level is high (contrary to estrogen level which is low) (Riegle and Miller, 1978; Dilman and Anisimov, 1979) should induce ovarian tumors with increased frequency. It is noteworthy that testicular tumor incidence was maximal in irradiated 1- and 21-month-old male rats, and minimal in 3-monthold rats (Castanera et al., 1971). 3. Mammary Gland Shellabarger et aE. (1966)showed that irradiation with y rays of 6oCo of female rats aged 40 and 160 days induced mammary tumors with

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the same incidence. However, in older animals the majority of tumors consisted of mammary adenocarcinomas, whereas in younger animals fibroadenomas prevailed. Similarly treated 120-day-old female rats had a rise in tumor incidence as compared with 14- and 30-day-old animals (Reincke et al., 1964).According to Moskalev and Shelesneva (1976), female rats aged 1and 4-6 months, exposed to p rays of wSr90Ytat doses of 2-16 Gy, showed identical sensitivity of mammary epithelium to the carcinogenic effects of radiation. The authors reported the earlier development of tumors by younger rats. It should be noted here that the above-mentioned experiments do not provide any data on the exposure of animals at the period of maximal mammary epithelium sensitivity to carcinogens (see Section II1,3). Dedov (1982)injected 12.2 Bq/kg of [75Se]selenomethionineinto 3-, 12-to 14-, and 24- to 26-month-old female rats. In the old age group benign mammary tumors appeared after 2.5-3.5 months, while in the middle age group tumor latency was 5.5-7 months. No mammary tumors were found in the young age group. This study failed to reveal malignant mammary tumors in the animals. 4. Bone In a number of experiments young, mature, and 8- to 10-month-old rats were treated with different radionuclides, which resulted in higher incidences of osteogenic sarcomas in the first age group (Sundaram, 1963; Streltsova and Moskalev, 1964; Sinjakov, 1976). The latency of the sarcomas was inversely correlated with the age of the animals at the time of isotope administration (Sinjakov, 1976). Castanera et al. (1971) showed that the proliferating bone tissue had a maximal sensitivity to total-body irradiation by fast neutrons in young rats. Another experiment, involving single injection of z24Raor 227Thto female rats aged 1or 5-6 months, which induced osteosarcomas, demonstrated decreases in tumor incidence in mature animals, while latency was considerably longer in younger rats (Luz et d.,1979). Also, it was shown that death risk resulting from bone neoplasms in irradiated individuals aged under 19.9 years is twice as high as for radiotreated subjects over 20 (Davis, 1978).

5. Skin Age of rats (1-100 days) at the moment of exposure to low voltage X rays failed to influence the incidence of skin tumors (Vanderlaan et al., 1976). Moskalev and Shelesneva (1976) reported more frequent skin sarcoma induction in 1-month-old rats by /3 rays of wSr-wYt at the dose of 1600 rad; whereas in similarly treated 4- to 6-month-old rats

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basal cell and squamous cell skin cancer was observed. The incidence of skin cancer induced in 28-, 200-, or 400-day-old rats by a single exposure to electron radiation (1200 rad) inversely correlated with the age of the animals (Burns et al., 1981). Similar correlation between skin tumor incidence and age at the time of exposure was found by Castanera et aE. (1971)who used 1-, 3-, and 21-month-old rats exposed to fast neutrons.

6. Lungs Single exposure to radiation induced lung tumors in mice with higher incidence as the age of the animals at the time of exposure increased (Lindop and Rotblat, 1962a; Gajewski et al., 1977). According to Castanera et al. (1971), lung tumor incidence in irradiated 1-,3-, and 21month-old rats was 15, 22, and 9%, respectively. According to the present concepts, two radiobiological effects form the basis of neoplastic pathogenesis in irradiated organisms, transformation of somatic cell gene structure and development of irreversible shifts causing persistent secondary cell proliferation (Alexandrov, 1972). Radiation-induced malignant cell transformation might be direct (mutation)or indirect (latent virus activation) (Latarjet, 1978).The available data show that maximal tissue sensitivity to carcinogenic effects of radiation in the organism is observed during puberty, which in rats and mice is over by month 3 of their life. High proliferative activity in growing tissues seems to play a leading role in age-related radiation carcinogenesis. This concept is confirmed by experiments on the artificial stimulation of proliferation in tissues, involving partial hepatectomy, heminephrectomy, and breaking bones, followed by tumor development at those sites exposed to irradiation (Alexandrov, 1972). B. ULTRAVIOLET (UV) LIGHT The incidence of neoplasms, the origin of which can be attributed to UV light, is considerably increased with advancing age (Forbes et al., 1979; Vitaliano and Urbach, 1980). Experiments with nude mice aged 6 and 16 weeks showed that sensitivity to UV light was considerably higher in younger than in older animals (Forbes et al., 1979). However, Israili et aE. (1982) failed to reveal any age-related difference in skin tumor incidence in 10- to 14- and 43- to %-week old nude mice exposed to UV light, although induction of skin tumors by UV light in 50% of 10-month-old mice ';'as significantly longer than in 50% of 2- to

CARCINOGENESIS AND AGING

385

3-month-old animals (Blum et al., 1942). It should be noted that no really old animals were used in these experiments. In a recent study by Ebbesen and Kripke (1982) BALB/c mice aged 4 weeks or 18 months were exposed to UV light three times a week; younger mice developed skin tumors earlier than older animals, although 32 weeks after the start of the experiment tumor incidence was identical in both age groups. The rate of UV light-induced squamous cell cancers and fibrosarcomas was also the same. Female 22- to 24week-old mice of the same strain developed UV-induced skin tumors in 70% of the cases after 31 weeks. When animals aged 70 weeks were exposed to UV light, tumors appeared in 55% of the cases with a mean latency of 35 weeks. Reciprocal transplantation of skin grafts showed that sensitivity to the carcinogenic effect of UV light was determined by the age of the host, and not the graft. However, when authors used larger skin grafts (16 mm2)from older animals, they observed a significant increase in the incidence of tumors in the grafted skin of 2-yearold mice as compared with l-year-old animals. Experimental data and clinical observations by Forbes e t al. (1979) suggest two aspects in which aging influences skin photocarcinogenesis: (1)UV light-induced damage accumulates with age; and (2) some biological changes in the skin (e.g., thinning of epidermal protective layers) and the organism (e.g., decreased DNA repair) may increase the sensitivity of the organism to the blastomogenic effects of UV light. In this connection, it is interesting that Olsson and Ebbesen (1977) found an age-related decrease in epidermal chalone activity in mouse skin, from which the elevation of sensitivity to the carcinogenic effect of UV light in the skin of senescent subjects may also be suggested. In fact, it was shown by Vitaliano and Urbach (1980) that the same dose of UV light increases the risk of skin cancer in humans aged over 60 years, as compared to younger subjects. This suggestion is also supported by the observations of Selmanovitz et al. (1977) on increased sensitivity to UV light in patients with various progeria syndromes accompanied by enhanced skin neoplasm incidence rate. VI. Hormonal Carcinogenesis and Aging

Nowadays, the ability of exogenous estrogens to induce neoplasms in both humans and animals is well known (IARC, 1979). However, even short-term estrogen administration during the perinatal period was followed by tumor development, while in maturity tumor induction requires prolonged estrogen treatment (IARC, 1979). These data

386

VLADIMIR N. ANISIMOV

are in accord with the concept of increased organism sensitivity to carcinogenic agents during the perinatal period (Alexandrov and Napalkov, 1981). Estrogen treatment does not induce tumors in mature female hamsters, but does so in males (IARC, 1979); whereas transplacental estrogen administration leads to neoplasm development in animals of both sexes (Rustia and Shubik, 1979). Estrogen (or other steroid) injections in pregnant or newborn rodents cause disturbances in hypothalamic sex differentiation, followed by the development of persistent estrus in adult life. This syndrome is characterized by an increase in neoplasm incidence (Anisimov, 1971)and by a number of hormone-metabolic shifts. Thus, persistent estrus might be regarded as intensified aging (see Section VII1,B).Adult female rats exposed in utero to diethylstilbestrol (DES) demonstrate decreased tolerance to glucose, hyperreactive insulinemia, disturbed regulation of serum somatomedine activity (Anisimov et al., 1981), and elevation of the hypothalamic thresholds of sensitivity to estrogen suppression (Napalkov and Anisimov, 1979), i.e., disturbances peculiar to old rats (Dilman and Anisimov, 1979). KaIland and Forsberg (1981) reported significant inhibition of cell immunity in animals treated with DES during the perinatal period. In these studies hyperplastic processes were observed with neoplasms of cervix uteri, vagina, and ovaries (Dunn and Green, 1963; Rustia and Shubik, 1979; Vorherr et al., 1979). This was accompanied by an increase in the incidence of tumors peculiar to the oncological characteristics of the animal strain used (Nomura and Kanzaki, 1977; Napalkov and Anisimov, 1979). It is known that the risk of cervicovaginal cancer in women exposed in utero to DES is high (Herbst et al., 1979). These women develop anovulatory syndrome and sterility, (Pomerance, 1973) which indicates increased risk of mammary and endometrial cancer (Bokhman, 1980). Estrogen uptake for different purposes by women during the reproductive and, especially, postmenopausal periods increases the risk of breast and endometrial cancer (IARC, 1979). It should be noted that the age-dependent increase in body fat in women might promote breast and endometrial cancer growth because obesity is accompanied by enhanced estrone synthesis (Kirschner et al., 1981). Ishak (1979)observed 11female patients who used hormonal contraceptives for a long time. Among those patients who later developed cancer of the liver, 7 women began taking the contraceptives at under 25 years of age, 2 patients between 28 and 30 years, and 2 after they reached 42

CARCINOGENESIS AND AGING

387

years of age. The author believes that the risk of liver tumor is directly proportional to the age of the patient at the start of estrogen intake. Geshickter (1939) observed mammary tumor development in estrogen-related 1- and 20-month-old rats with a latency of 9.5 and 3 months, respectively. Taking into account the data by Welsch and Nagasawa (1977)who reported the important role of prolactin in mammary carcinogenesis, and the data by Riegle and Miller (1978) who showed the age-related rise in prolactin concentration and elevation of hypothalamic sensitivity to estrogens resulting in the enhancement of prolactin secretion, it is possible to suggest that such a mechanism is involved in promotion of mammary carcinogenesis in aging rats. The majority of authors believe the cell genome to be undamaged by estrogens, which are assumed to stimulate cell proliferation, thus promoting the realization of blas tomogenic effects of chemical, radiation, and viral carcinogens (IARC, 1979). The Ames test did not reveal a mutagenic effect of estrogens, although the results obtained with DES are regarded by some authors (Rinkus and Legator, 1979) to be false-negatives. It was shown recently that microsomal enzymes in the liver cause biotransformation of DES into highly reactive epoxides and catechol-oxy-derivatives, such as dibenz(a)anthracene, capable of binding covalently with DNA (Metzler, 1979). Inhibition, by estrogens, of carcinogen-induced unscheduled DNA synthesis and excision repair might prove additional factors that promote induced mutations (Trosko and Chang, 1976). The effect of estrogens on peripheral target tissues and central chains of the neuroendocrine system may differ as age advances, causing significant modifications in age-related dynamics of organism sensitivity to estrogens. At least two factors might be responsible for modification of the estrogen effect on proliferation in target tissues: a considerable age-related decrease in estrogen receptors (Chang and Roth, 1979), and variations in chalone concentration caused by estrogens (e.g., in vaginal epithelium) (Anisimov and Okulov, 1980). Agerelated decreases in estradiol receptor levels and chalone concentration seem to lead to an age-related decrease in the sensitivity of homeostatic mechanisms, responsible for cell proliferation in tissues, to regulatory factors. Menczer et al. (1977) tried to induce folliculomas in the ovaries of mice aged 3 weeks and 6 or 10 months. Prolonged injections of pregnant mare gonadotropin into these animals caused only one ovarian tumor in the young age group, though the proliferation of lutein cells was observed in 48.9, 64.5, and 65% of cases, respectively. It should

388

VLADIMIR N. ANISIMOV

be noted that generalized lymphosarcomas appeared in 70% of the younger mice, in 71% of the animals of the middle age group, and in only 13% of the older mice. Rather interesting data may be obtained in the experiments involving ovariectomy followed by the transplantation of one of the ovaries into the spleen. Tumorigenesis in grafted ovary is believed to be caused by hyperstimulation of the ovary by pituitary gonadotropins, as transplant-secreted estrogens pass via the blood into the liver where they are inactivated (Biskind and Biskind, 1944). Li and Gardner (1950) reported a higher tumor incidence in 54- to 105-day-old than in 204- to 307-day-old similarly treated mice. However, Klein (1953) did not find any age-related variations in tumor incidence and rate in mice operated according to the Biskind method. Boe et aE. (1954) observed tumor development in grafted ovaries of 10-week-old rats with a latency of 450-520 days in all cases, while in similarly treated rats aged 18 months tumors did not appear. It should be noted, however, that older animals were observed only for 123-164 days following the operation, a period insufficient for tumor detection. The studies by Ber (1970) showed increased tumor incidence in ovaries transplanted into spleen in 3-week-old rats as compared to 14-week-old animals, but no really old rats were used in this experiment. It should be noted that all of these studies used rather small groups of experimental animals and have certain errors both in techniques and statistical evaluation of the material. Further studies on the subject would be useful. Vli. Viral Carcinogenesis and Aging

In the light of the viral theory of tumorigenesis, the age-related rise in tumor incidence might be explained by the increase in organism sensitivity to the effect of oncogenic viruses with advancing age (Ponten, 1977). This concept is matched by the results on correlation of tumor incidence with virus dose and level of viral expression (Atrock and Cardiff, 1979). It is well known that the carcinogenic effect of the majority of oncogenic viruses is confined mainly to newborn animals and is not manifested in the adult (Ponten, 1977). However, Eckerdt et al. (1956) showed that susceptibility to avian erythroblastosis virus was not altered in chickens between days 3 and 54 of life. Fischer 344 rats were intracerebrally inoculated with avian sarcoma virus B-77 (Copeland and Bigner, 1977). Brain tumors developed in all virus-injected 1-,9-, or 97- to 99-day-old animals, but in only 67% of 528-day-old rats. Survival rate of rats was increased with age, while tumor incidence was inversely correlated with age. As the latency of

CARCINOGENESIS AND AGING

389

brain tumors induced in rats by avian sarcoma virus is rather long (291 days for 99-day-old rats), the authors believe that brain tumor incidence in the older rats would have been higher, had they not died from the diseases of aging, including spontaneous tumors. Pazmino and Yuhas (1973) observed an abrupt decrease in the development of Moloney virus-induced sarcomas in mice from birth to puberty. However, in 1-year-old mice, the susceptibility to the virus effect was restored and progressed with advancing age, so that in 2.5to 3-year-old animals sarcomas appeared in all cases. It should be noted that serum of resistant adult mice contained antibodies responsible for the neutralization of oncogenic viruses, while in newborn and old mice the production of antibodies was significantly lowered. Similar results were obtained in recent experiments with monkeys (Eichberg et al., 1981). The authors reported the production of antibodies to C-type endogenous virus only in either very young (under 1 year of age) or very old (over 12 years) monkeys, Thus, deterioration of immunity in old age promotes virus-induced tumor development and growth. Experiments in vitro with simian virus 40 (SV40) showed that “old” fibroblasts of human embryonal lung cultures are transformed more easily than those cultivated for shorter periods (Ponten, 1977). Similar results were shown by Snyder and Sreevalsan (1973) on chicken embryonal fibroblasts and HeLa cells infected with SB virus. However, there is evidence for the decrease in sensitivity to transformation by avian sarcoma virus Semliki Forest B-77 and (SF)virus of “old” cultures of human and mouse fibroblasts (Reinerova-Hladka and Altaner, 1976; Eylan and Gazit, 1979). This controversy might be explained by the data on heterogeneity of “old” cultures with regard to the concentrations of cells in phase I1 and I11 in them (Ponten, 1977). This author believes that at present there is no reliable experimental evidence for the higher sensitivity to neoplastic virus-induced transformation in the cells in phase I11 (“old” cells) than in “young” cells, Besides, there are very important data on the increased sensitivity of fibroblasts obtained from patients with Down’s syndrome to the transforming effect of SV40, accompanied by a high incidence of neoplasms in such patients, as compared to healthy subjects (Todaro and Martin, 1967). According to Huebner and Todaro (1969), the age-related rise in tumor incidence may depend on age-related expression of a viral genome which is stored in the somatic cells of mammals. This theory agrees with the data on the age-connected derepression of the genome of mouse leukemia C-type virus and murine mammary tumor virus

390

VLADIMIR N. ANISIMOV

(MuMTV) (Florine et d.,1980; Tsubura et al., 1981). The results of the experiments involving ip injection of lymphoid cells of 1-and 14month-old BALB/c mice, characterized by a high incidence of spontaneous reticulosarcomas and leukemias in syngeneic animals of varying age (Ebbesen, 1971), may provide indirect evidence for the above concept. It turned out that the injection of cells from 14-month-old donors into l-month-old mice increased reticulocyte tumor incidence, whereas the inoculation of cells from l-month-old mice into both old and young animals did not influence tumorigenesis. Using the method of molecular hybridization, Schlom et al. (1977) showed that nucleotides of MuMTV, obtained from spontaneous tumors in younger C3H mice, were 25% noncomplementary to those obtained from tumors developed by older mice of this strain. Therefore, two factors might be responsible for the age-related dynamics in organism sensitivity to exogenous viruses and for the changes in virus genome expression: (1)age-dependent variations in corticosteroid and sex hormones level, which as was shown in vivo and in Gitro, significantly modifies the transforming effect of oncogenic viruses (Burnet, 1970; Ringold, 1979; Kohn et al., 1978); and (2) age-related changes in serum lipid concentration and in the whole system of immunologic surveillance (Burnet, 1970; Kohn et aE., 1978). The concepts of Huebner and Todaro give sufficient explanation to the age-related rise in the development of only certain types of virusinduced tumors (leukemias in mice, cats, and birds), but do not solve the entire problem (Ponten, 1977). VIII. Mechanisms of Modifications of Carcinogenesis by Aging

The data discussed in Sections III-VII seem rather controversial, which is, to a considerable degree, due to the use of different experimental approaches, carcinogenic agents, and animal strains and species. However, we shall try to evaluate the modifying effect of aging on carcinogenesis from the viewpoint of a two-stage model of cancer development. The concept of the multistage nature of carcinogenesis was formed in the oncologic literature long ago. A number of recent reviews deal with the data available on the peculiarities of realization of the twostage effect of different carcinogenic agents in various tissues (Berenblum, 1974; Foulds, 1975; Pitot, 1978; Day and Brown, 1980; Farber and Cameron, 1980).The period during which transformation of a normal cell into a malignant one occurs is called the initiation stage. This stage may consist of a succession of several events causing inherited changes in the cell before it becomes malignant. At this

CARCINOGENESIS AND AGING

39 1

stage susceptibility to carcinogenic factors depends on (1)metabolic pathways of carcinogen in liver and/or target tissue, (2) proliferative activity in the target tissue, (3) interaction of carcinogen (or its active metabolite) with DNA and protein, and (4) DNA repair (Montesano and Bartsch, 1976). During the second (promotion) stage, a transformed cell, if it is not eliminated by immunologic surveillance, goes into unrestrained division and progression.

A. INITIATING EFFECTOF CARCINOGENS AND AGING 1. Effect of Aging on Pharmacodynamics of Carcinogens The relationship between the pharmacodynamics of carcinogens and aging has not been studied. Meanwhile, numerous data on the pharmacodynamics of drugs allow us, to some extent, to evaluate the effect of carcinogens on the aged organism. It was shown that thinning of the epidermis and lowering of the skin barrier function in old age (Selmanovitz et al., 1977), other factors being equal, may prove to be the factors increasing the rate of lipic-soluble carcinogen absorption or UV light penetration into the organism, thus contributing to enhanced carcinogenesis in older animals and humans. The age-related decrease in lung ventilation (Klocke, 1977) might reduce the effective dose of inhaled carcinogen. Although the majority of substances are absorbed in the gastrointestinal tract at the same rate independently of age, the absorption of some compounds was found to decrease as age advanced (Greenblatt et al., 1982). Due to the reduced evacuatory function of the gut in older organisms as compared to younger ones (Manier, 1974), carcinogenic agents or their metabolites might remain in the intestines for a longer time. The agerelated decrease in the serum albumin concentration (Dybkayer et al., 1981) seems to have a modifying influence on the metabolism rate of some drugs and carcinogens. Age-related obesity (Bruce et al., 1980) may significantly alter the distribution of some lipid-soluble carcinogens by varying their concentration in target tissues. Liver or kidney clearance may be lowered in age, thus increasing considerably the half-life of carcinogens or their active metabolites in the organism (Greenblatt et al., 1982).

2. Effect of Aging on the Activity of Carcinogen-Metabolizing Enzymes Metabolic activation and/or inactivation of a carcinogenic substance is of vital importance for estimating its effective dose. There are many data on age-dependent changes in the sensitivity of humans and ani-

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mals to drugs and xenobiotics (Schmucker and Wang, 1981). Many of these changes are caused by the age-related decline in the activity of liver microsomal enzymes which metabolize some carcinogens. It can be seen from Table I1 that liver microsomal enzyme activity is, as a rule, decreased with age. On the other hand, the activity of liver epoxide hydratase is increased in mice and rats with age (Birnbaum and Baird, 1979). The activity of nitroreductase and P-glucuronidase, which contribute to carcinogen detoxication, is enhanced in old age too (Yazawaet al., 1981).Greiner et al. (1980)observed maximal activity of epoxide hydratase in the mammary epithelium of 40-day-old rats. Between days 40 and 60 this parameter was lowered and remained unchanged up to day 100. These authors also found that the level of basal activity of mammary aryl hydrocarbon hydroxylase (AHH) in rats did not change between day 40 and 100, while its inducibility by MC was maximal on days 50-60, i.e., in the period of maximal sensitivity of mammary epithelium to the carcinogenic effect of PAH (see above). In rats much younger or older than that, AHH activity was low. The data on age-related dynamics of inducibility of TABLE I1 EFFECT OF AGING ON ACTIVITYOF Enzyme Cytochrome P450

CARCINOCEN-?dETABOLIZING ENZYMES IN RAT

Age group (months)

Effect of' aging

3, 12, and 27 N o effect 4, 12, and 36 Decreases 16 and 26 Decreases

NADP-cytochrome-c reductase

4,12, and 36 Decreases 3, 12, and 27 N o effect

Decreases Decreases Increases Decreases Increases

Nitroreductase Aryl hydrocarbon hydroxylase Epoxide hydratase

3, 12, and 24 3, 12, and 24 2 and 9 4, 18, and 31 3, 12, and 24

Glutathione-S-transferase

3, 12, and 24 N o effect

P-Glucuronidase Amidopyrine demethylase

LIVER

2 and 9 Increases 6 and 25 No effect

References Birnbaum and Baird (1978) McMartin et al. (1980) Schmucker and Wang (1981) McMartin et al. (1980) Birnbaum and Baird (1978) Lemeshko (1980) Yazawa et al, (1981) Yazawa et al. (1981) Baird et al. (1976) Birnbaum and Baird (1979) Birnbaum and Baird (1979) Yazawa et al. (1981) Paramonova (1981)

CARCINOGENESIS AND AGING

393

AHH in various rat tissues (liver, kidney, and lung) also seem very intriguing (Van Cantfort and Gielen, 1981). The AHH activity was studied in 282 human subjects aged from 10 to 69 years. It turned out that lymphocyte AHH inducibility was lowered after 30, then remained unchanged from 30 to 60 years (Paigen et al., 1978).The activity of demethylating liver enzymes, activating certain nitroso compounds, was found to be maximal in 29-day-old rats, while later it decreased considerably (Davies et al., 1976). It is noteworthy that the effect of aging on the activity of liver microsomal oxygenases depends to a considerable degree on the species, strain, sex, and substrate used (Birnbaum and Baird, 1979). Recently a technique based on bacterial tests involving the study of carcinogen mutagenicity was used. This technique employs the addition of intact or previously activated microsomal enzymes (or crude tissue extract) of young and old animals. Baird and Birnbaum (1979) used male rats and mice previously treated with the inducer of liver enzymes, Aroclor 1254. The Ames test showed enhanced synthesis of mutagenic derivatives of BP and 2-aminofluorene (2-AF) by liver homogenates or refined liver microsomes in 700-old-day CFN rats, against 300-day-old animals, as well as in 800-day-old C57BU6j mice, against 100-day-old ones. Jayaraj and Richardson (1981) showed a decrease by 40-50% in the rate of AFBl conversion into mutagenic adducts by S9 fraction and liver microsomes in F344 rats aged 12-18 months. However, AFBl activation remained unchanged between month 18and 27 of rat life. The authors believe that their results differ from those of Baird and Birnbaum (1979) because they had previously induced the production of microsomal oxydases by Aroclor 1254. Meanwhile, according to Baird and Birnbaum (1979) rats and mice demonstrated age-related enhancement in 2-AF mutagenicity both with and without previous treatment with Aroclor 1254. In the study by Jayaraj and Diller (1981), F344 12- to 27-month-old male rats showed a decrease in AFBl metabolism and activation rate into mutagenic derivatives by liver enzymes, whereas female rats of the same strain did not develop such changes. The experiments with SwissWebster female mice failed to reveal any significant age-related changes in the activation of 2-AAF, BP, and DMNA into mutagenic derivatives by S9 liver fraction, when using Salmonella typhimurium TAlOO (Guttenplan and Bliznakov, 1981).According to Robertson and Birnbaum (1982), mature female Long-Evans rats manifested decreased activation of AFBl , 2-AF, and 2-AAF into mutagenic derivatives by microsomal liver enzymes, as compared with younger animals. Minimal activity was observed before months 9-13 of their life.

394

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Variations in the activity of enzymes in liver and kidneys of animals between months 9-13 and 20-25 of their lives were small, whereas in the old age group the rate of lung metabolism of carcinogens was somewhat higher than in the middle age group. Thus, the available data give evidence for the existence of certain strain- and sex-related differences in the age-related dynamics of enzyme activity responsible for metabolizing carcinogens into mutagenic derivatives. This might explain the results of experiments with the same carcinogen (e.g., DMH) in rats of different sex and strain, when the animals’ age had quite a different effect on tumor incidence (Moon and Fricks, 1977). Also, there are some data on the species differences in the age-related ability of microsomal enzymes to activate carcinogens (Birnbaum and Baird, 1979; Poiley et al., 1981). It seems that at present the related data are too scarce to postulate a leading role for the age-related dynamics of carcinogen-metabolizing enzyme activity in the age-associated rise in cancer incidence (Baird and Birnbaum, 1979), or to ignore completely their contribution to this process (Poiley et al., 1981).

3. Effect of A g i n g on lnteraction between Carcinogens and Macromolecules There are rather few data on the effects of aging on the relationship between carcinogenic agents and cell macromolecules (DNA, RNA, and proteins). Thus, DMBA and DAB binding capacity with DNA and proteins in the mammary glands and liver was shown to decrease as age advances (Janes and Hadaway, 1977; Decloitre et al., 1973). Experiments in citro by Raineri and Hillesund (1977) demonstrated decreased PAH binding capacity with macromolecules of human embryonal cells in “old” cell cultures. Likhachev et al. (1983)showed that ip injection of methyl(acetoxymethy1)nitrosamine (DMN-OAc) to young and old rats did not show age-related variations in the methylation level of purine bases of ileum DNA, but in DNA of the colon this parameter was lower in older rats. These results are in accordance with the data on both the variations in DNA acceptability for methylation of different organs, and tissue-specific age-related changes in methylation of DNA from different organs (Kudryashova and Vanyushin, 1976). According to the above study, the ability of DNA to accept methyl radicals in the liver is not essentially changed as age advances, whereas in the brain this parameter is increased with age. I n the lungs this process is less pronounced.

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395

It was shown that the incidence of radiation-induced single-strand breaks in the DNA of liver, intestines, and lymphocytes is unaffected by age at the time of exposure (Konoplyannikova et al., 1980; Malakhova and Fomenko, 1981). The age-associated decrease in the quantity of receptors in tissues, particularly of some hormonal receptors (Chang and Roth, 1979) or in the biogenic amine level in the central nervous system (Anisimov, 1979; Finch, 1979), might in some way modify the effect of carcinogens both on target tissues and on systems of organism regulation (see below).

4. DNA Repair DNA repair systems, responsible for elimination of “spontaneous” or carcinogen-induced damage in DNA structure, are believed by some authors to play a leading role in maintaining genome stability (Hart and Daniel, 1980). Inaccurate or slowed DNA repair may, as it seems, contribute to the realization of the mechanisms of aging and carcinogenesis (Hart and Daniel, 1980; Gaziev et al., 1981; Gensler and Bernstein, 1981; Montesano, 1981). According to the data showing that subjects with syndromes of progeria and defects in DNA repair are prone to cancer development (Hart and Daniel, 1980), it is natural to suggest that the age-related decline in the DNA repair rate might increase organism sensitivity to carcinogens. Experiments with human blood leukocytes and lymphocytes whose DNA was damaged by UV light show an inverse correlation between age and DNA ability for reparative synthesis (Lambert et al., 1979; Dilman and Revskoy, 1981). However, excision repair of DNA in rat cell culture of retinal ganglion or lens epithelium (Ishikawa et al., 1978; Treton and Courtois, 1981) and in hamster brain, kidney, liver, and lungs (Gensler, 1981a,b) exposed to UV light was not changed with age. A number of experiments show that the age of mice at the time of exposure to ionizing radiation did not influence the number of singlestrand breaks occurring in the DNA of liver, intestines, and lymphocytes, and was approximately the same in the young (2-3 months) and old (15-20 months) animals. However, older animals demonstrated a lower level of radiation-induced DNA repair synthesis and the rate of excision of y-ray-damaged DNA base, against younger mice (Konoplyannikova et al., 1980; Gazyev et al., 1981; Malakhova and Fomenko, 1981). The authors believe these changes to be caused by age-associated limitations on the effect of repair enzymes on the cell chromatin DNA or in the intermolecular linkage of cell nucleus macromolecules.

396

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This suggestion is supported by evidence showing the different relaxation of liver nuclear nucleoides in young and old mice, when radiation-induced damage in DNA are identical (Gazyev et al., 1981). In the experiments of Bums et al. (1981),the skin of 28-, 200-, and 400-day-old rats was exposed to electron radiation (1200 rad). The number of DNA breaks in the epidermis was identical in all age groups, while the half-time of DNA repair was 21, 69, and 107 min, respectively. Meanwhile, Ono and Okada (1978) failed to reveal any difference in the DNA repair rate in the cerebellum of 2- and 22month-old irradiated mice. The ability of dog cerebellum neurons to repair y-ray-induced single-strand breaks in DNA also remained unchanged with age (Wheeler and Lett, 1974). The data on the age-dependent repair of DNA damaged by chemical carcinogens are rather controversial. According to Kanagalingam and Balis (1975) DNA repair of colonic enterocytes in older rats treated with DMH and some other alkylating agents was less efficient than in younger animals. On the other hand, Ishikawa et al. (1978) failed to reveal any age-associated changes in DNA repair accuracy in cell culture of rat retinal ganglion of varying ages treated with 4-nitroquinoline-oxide (4-NQO), NEU, and methylmethanesulfonate. DNA repair was less efficient in fibroblasts of 2-month-old rats treated with NEU, than in 3-week-old animals (Fort and Cerutti, 1981). It was also shown that fibroblasts of patients with syndromes of progeria were much more sensitive to the damaging effects of MNNG than fibroblasts of healthy donors (Scudiero et al., 1981). The ageassociated increase in human lymphocyte sensitivity to mutagenic effects of N-acetoxy-2-AAF was observed by Per0 and Norden (1981). According to Likhachev et al. (1981), the repair of DENA-alkylated DNA bases in the liver of young rats is more efficient than in the liver of old animals. Tay and Russo (1981) observed an age-associated increase in the efficacy of DNA repair in mammary epithelium of DMBA-treated female rats. The controversy on the available data on DNA repair at varying ages (Table 111) may, to some extent, result from the difference in the techniques used for the evaluation of DNA repair rate and accuracy. The nature of the damaging factor is of no small importance, either. For instance, it was shown (Hart and Daniel, 1980) that fibroblasts of patients suffering from xeroderma pigmentosum are not capable of DNA repair after exposure to UV light, acetoxy-AAF, and PAH; but the damage to DNA caused by X rays or alkylating agents might be efficiently repaired. These data indicate the difference in DNA repair accuracy depending on the nature of the damaging agents and, conse-

TABLE I11 EFFECTOF AGINGON DNA REPAIRACCURACYIN CARCINOGEN-DAMAGED TISSUES Tissue

Species

Carcinogen"

Age group (years/months)

Effect of aging

References

Mouse

UV light UV light UV light X ray N-OH-AAF Y ray

13-94 22 and 54 17-90 17-90 10-80 2 and 15-17

Decreases Decreases No effect Decreases Decreases Decreases

Fibroblasts Skin epidermis Liver

Rat Rat Mouse

NEU Electrons Y ray

1 and 24 1,6 and 13

2-3 and 18-20

Decreases Decreases Decreases

Colon epithelium

Rat Rat Rat

DENA DMN-OAC DMH, DMN-OAC

3 and 14 3 and 14 3-4 and 13-15

Decreases Increases Decreases

Mammary gland epithelium Retinal ganglion

Mouse Rat Rat

Y ray DMBA NEW, 4-NQO, UV light UV light Y ray Y ray

3 and 18 1.5 and 5 1-6 and 23

Decreases Increases No effect

Lambert et al. (1979) Dilman and Revskoy (1981) Kutlaca et nl. (1982) Kutlaca et al. (1982) Per0 and Norden (1981) Malakhova and Fomenko (1981) Fort and Cerutti (1981) Burns et d.(1981) Gasiev et al. (1981); Konoplyannikova et al. (1980) Likhachev et al. (1981) Likhachev et al. (1983) Kanagalingham and Balis (1975); Likhachev et al. (1982) Konoplyannikova et al. (1980) Tay and Russo (1981) Ishikawa et al. (1978)

14 and 40 2and22 7 weeks and 13 years

No effect No effect No effect

Treton and Courtois (1981) Ono and Okada (1978) Wheeler and Lett (1974)

Leukocytes Lymphocytes

Lens epithelium Cerebellum

Human Human

Rat Mouse Dog

~

~

~~

N-OH-AAF, N-Hydroxyacetylaminofluorene; NEU, N-nitrosoethylurea; DENA, N-nitroso-diethylamine; DMN-OAc, methyl(acetoxymethy1)nitrosamine;DMH, 1,2-dimethylhydrazine; DMBA, 7,12-dimethylbenz(a)anthracene;4-NQO, 4-nitroquinoline-1-oxide. a

398

VLADIMIR K. ANISIMOV

quently, on the systems responsible for DNA repair. On the other hand, genetic peculiarities of an organism are also of some importance. Thus, the damage caused to DNA of cells taken from patients with ataxia telangiectasia, by UV light or acetoxy-AAF, is repaired normally; whereas defects in excision DNA repair are observed when the damage is due to X rays or alkylating agents (Hart and Daniel, 1980). Recently, a correlation was found between the efficacy of DNA excision repair of promutagenic base in the tissues of animals treated with alkylating agents and their organotropism (Montesano, 1981). According to Table 111, the treatment of target tissues with alkylating agents is, as a rule, followed by deterioration in DNA repair, which is inversely correlated with age. However, this effect is not universal. For example, though DMH-induced enterocyte DNA repair is lowered with age (Kanagalingam and Balis, 1975), the carcinogenic effect of DMH is also decreased in old rats (Moon and Fricks, 1977; Pozharisski et ul., 1980). Similarly, a positive correlation was observed between the low rate of Ofi-ethylguanine excision from rat liver DNA and the resistance of these animals to the carcinogenic effect of DENA (Likhachev et d., 1981; Reuber, 1975a). On the other hand, the age-associated decrease in rat mammary epithelium sensitivity to DMBA is accompanied by an increase in the efficacy of repair of DNA damaged by this carcinogen (Tay and Russo, 1981). Hence, we niay conclude that age-associated changes in DNA repair play a permissive rather than a leading role in the mechanism of ageassociated dynamics of tissue sensitivity to carcinogens. 5. Prolijeratiue Acticity of Tissues The available data indicate a decrease in the proliferative activity of the majority of both quickly and slowly renewing tissues as age advances, (Cameron and Thrasher, 1972) though there is evidence for proliferative development in some tissues of senescent subjects (Martin, 1979). In view of the data available on a critical role of the proliferative activity in target tissue at the time of exposure to carcinogen (e.g., in the experiments involving partial hepatectomy) (Craddock and Frei, 1974), it may be suggested that this factor plays an important role in the age-associated decrease in the initiating effect of chemical carcinogens as well as ionizing radiation or hormones. This is supported by the results discussed in Sections III-VI. However, some investigators observed a greater incidence of tumors in old animals treated with direct-effect carcinogens, as compared to young ones. In our experiments (Anisimov, 1981a) when 3- and 14-month-old female

CARCINOGENESIS AND AGING

399

rats were intravenously injected with NMU, cervico-vaginal tumors were observed only in old NMU-treated rats, whereas mammary adenocarcinomas were only observed in the young group. A study on the level of t3H]thymidine incorporation into DNA of different rat organs showed that the DNA synthesis rate in liver, kidney, mammary glands, colon, and intestines is lowered, while it is enhanced in the uterus (Likhachev et al., 1983). In view of our data on the rise in serum estradiol level and increased weight of the uterus in older rats (Anisimov and Okulov, 1980), it may be suggested that it was the age-associated changes in hormone (estrogens) concentration that provided the enhanced proliferative activity in the uterus of aging rats and elevated sensitivity to carcinogenic effects of NMU. Thus, it may be suggested that the age-associated dynamics of proliferative activity in target tissues plays a key role in the modifying effect of age on the initiation of carcnogenesis.

B. PROMOTION OF CARCINOGENESIS AND AGING According to Burnet (1970), the age-associated rise in tumor incidence might be due to the decline in the immunologic surveillance system, occurring with age. Kroes et al. (1975) showed that suppression of immunity, as a rule, promotes carcinogenesis, while immune stimulators produce an antiblastomogenic effect. However, the enhanced frequency of tumor development in humans, as a result of treatment with immunodepressants, is limited to certain types of neoplasms (adenocarcinomas of lung, primary tumors of liver and bile ducts, cancer of urinary bladder, thyroid, sarcomas of soft tissues, and, possibly, tumors of cervix uteri); there is no proof of a similar relationship with respect to cancers of the stomach, colon, mammary gland, and bronchi (Doll, 1978). Dawson et al. (1978) did not observe a significant variation in spontaneous tumor development caused by prolonged immunodepression in neonatally thymectomized rats of two strains. This, as the authors believe, does not agree with the concept of immunosurveillance. In the experiments by Morrison et al. (1982) BALB/cGnDu homozygous recessive mice, with a mutagenic gene causing spontaneous thymus involution, were used. The authors transplanted 18 different strains of tumors and 3 in uitro transformed cell lines, injected 2 oncogenic viruses and 3 chemical carcinogens into these and control (normal) mice. Apart from Harding-Passey melanoma, all other tumors and/or agents had less success in immunodepressed mice with the defective gene. The original concept concerning the development of cancrophilia

400

VLADIMIR N. ANISIMOV

syndrome in the course of normal aging was suggested by Dilman (1978, 1981). According to this concept, a number of neurohormonal, metabolic, and immune shifts are developed in the organism with advancing age. These shifts, on the one hand, promote a pool of proliferative cells, thus increasing the possibility of malignant transformation of somatic cells. On the other hand, these metabolic shifts inhibit the division of thymus-dependent lymphocytes and suppress macrophage activity, which leads to metabolic immunodepression development. The most important among these shifts are lowered glucose tolerance, use of free fatty acids as the main energy substrate, hyperinsulinemia, and hyperlipidemia (Dilman, 1978, 1981). Experiments involving transplantation of tumors to animals of varying age might favor the concepts suggested by Burnet (1970) and Dilman (1978,1981), if the results would have shown enhanced tumor growth in older animals as compared to younger ones (Peto et al., 1975).These experiments rule out the effect of age on carcinogenesis induction and show the promoting effect of aging per se on tumor growth. The criteria for the evaluation of the results of such experiments might be (1)transplantability, (2) tumor growth rate, and (3)survival time of tumor-bearing animals. Age-associated differences in transplantability mainly illustrate the role of age in the decline of immunological vigor. When experimental animals are inoculated too many tumor cells, this difference might not be evident. However, in carefully planned experiments when strictly limited numbers of tumor cells are injected into young and old animals, there is no similar pattern between transplantability and the number of injected tumor cells. The results of Thompson (1976) showed that the incidence of lung tumor colonies in old (71 weeks) C3H mice was directly proportional to their age and the number of intravenously injected cells of Ehrlich ascites carcinoma. However, this correlation was not found in young (15 weeks) animals. These data provide convincing evidence for the existence of a certain “threshold” in the system of immune surveillance, and its level is lowered as age advances. When age has no effect on transplantability (when 100% of the animals, both old and young, develop tumors) the difference in tumor growth rate and life span of tumor-bearing animals is determined a priori by the age-associated hormone-metabolic shifts and some biological characteristic of the inoculated tumor. Inoculation of tumors originally derived from mammary gland epithelium (Ehrlich ascites, EMT6, A-755) to animals of varying age showed enhanced inoculation efficacy and tumor growth rate in older animals (Table IV). However

TABLE IV EFFECTOF AGINGON TRANSPLANTABLE TUMOR GROWTH IN RODENTS Tumor strain Human epidermoid carcinoma H.Ep.#3 Human gastric carcinoma (Shiraishi line) Squamous cell cervical carcinoma (SCC) Methylcholanthreneinduced sarcoma

Species

Age group (months)

Effect of agine

. References

Mouse

4-8 and 20-23

Increases

Teller et al. (1964)

Rat

1 and 3 3 and 5

Decreases Increases

Maruo et al. (1982) Maruo et al. (1982)

Mouse

3 and 18

Increases

Anisimov and Zhukovskaya (1981)

Mouse Rat

2-3 and 10-20 1-10 and 12-15

Increases Decreases

Fibrosarcoma Fibrosarcoma 1023

Rat Mouse

4 and 12 2 and 4-5

Ascites fibrosarcoma Sarcoma 180

Rat Mouse

3-4 and 16-18 3 and 18

Osteogenic sarcoma Mouse

3-4 and 10-17

Uterine sarcoma Mastocytoma P815 Reticulocell tumor type A Leukemia 1210 Leukemia La

Mouse Mouse Mouse

3 and 12 3 and 25 8 and 11-17

Mouse Mouse

3 and 11 3 and 18

Myeloma LCP-1 Mammary adenocarcinoma Spontaneous

Mouse

2-3 and 19-20

Ehrlich ascites EMT6 MAT-21

Mouse, d Mouse, 9 Mouse Mouse Mouse

3.5 and 16.5 3.5 and 16.5 3 and 16.5-18 3-4 and 20-28 2 and 4-5

A-755

Mouse

3 and 18

Walker-256

Rat

2 and 24

Mouse

2 and 24

Lewis lung carcinoma

3 and 18

Stjernward (1966) Hollingsworth et al. (1981) Decreases Loefer (1952) Increases Tagliabue et al. (1981) Increases Increases

Keller (1978) Anisimov and Zhukovskaya (1981) No effect Rodriguez et al. (1 976) No effect Baslova (1978) Increases Perkins (1977) Decreases Rodriguez et al. (1976) No effect Goldin et al. (1955) No effect Anisimov and Zhukovskaya (1981) Decreases Teller et al. (1974) Thompson (1976) Thompson (1976) Aoki et al. (1965) Rockwell (1981) Tagiiabue et al. (1981) Increases Anisimov and Zhukovskaya (1981) Decreases Bellamy and Hinsull (1978) Increases Gozes and Trainin (1977) No effect Anisimov and Zhukovskaya (1981) Increases Decreases Increases Increases Decreases

(continued)

402

VLADIMIR N. ANISIiMOV

TABLE IV (Continued)

Tumor strain

Species

Lung adenocarciMouse noma 1 Guelstein hepatoma Mouse 22a Novikoff hepatoma Rat

Age group (months)

Effect of aging"

3-8 and 18-23

Increases

3 and 14-16

Increases

4.5 and 27.5

Decreases

References Yuhas and Ullrich (1976) Anisimov and Zhukovskaya (1981) Bellamy and Hinsull (1978)

a Effect of aging on transplantability, growth rate, or survival of tumor-bearing animals.

development of spontaneous mammary gland adenocarcinomas was enhanced in old male C3H mice, as compared to old females (Thompson, 1976). The author believes this to be a result of age-associated hormone disturbances in old female C3H mice. In our experiments (Anisimov and Zhukovskaya, 1981) 3- and 14- to 18-month-old mice were inoculated with different tumors [ squamous cell cancer of cervix uteri (SCC), Lewis lung carcinoma, 22a-hepatoma, mammary gland adenocarcinoma A-755, and sarcoma-1801. As tumor transplantability was 100% in all cases, the rate of neoplastic growth was evaluated as a critical factor. It turned out that growth rate of squamous cell cancer of cervix uteri, 22a-hepatoma, sarcoma-180, and A-755 was greater in older animals, while Lewis lung carcinoma developed in both old and young mice at the same rate. The survival rate of young and old animals, inoculated with hemacytoblastosis La, was identical (Anisimov and Zhukovskaya, 1981). Histogenesis might prove an important factor in tumor growth and progression in young and old animals. It is seen in Table IV that hematopoietic neoplasms developed in animals of varying ages either at the same rate or faster in younger animals. On the other hand, old age promotes the growth of squamous cell epidermal cancer and lung adenocarcinomas. Inoculation of sarcomas of various origin to 3- to 12and 16- to 20-month-old animals showed that in older animals susceptibility to sarcomas and their growth rate were increased. Stjernward (1966) inoculated first generation MC-induced sarcomas into mice of varying ages. Six-month-old mice demonstrated maximal resistance to tumor transplantation, whereas l-week- and 22-month-old animals showed minimal resistance. The minimal level of T-cell-mediated immunity was also observed in l-week- and 22-month-old mice.

CARCINOGENESIS AND AGING

403

It was also shown that allogene inoculation in old NZB mice of MCinduced sarcoma of C3H mice was more successful in older animals, while reticulocellular tumor of A-type mice, inoculated syngeneically in animals of varying age, developed faster in younger mice (Rodriguez et al., 1976). However, we failed to find a similar correlation in our experiments (Anisimov and Zhukovskaya, 1981). In the light of differences in the average life span of animals of different strains, it may be suggested that the rate of age-associated tumor development in such animals will also be different. This may influence the comparative evaluation of the results of experiments with different animal strains. However, in our experiments involving inoculation of tumors, with different histogenesis, into C57BL/6 mice, variations in tumor growth rate were observed within each age group (Anisimov and Zhukovskaya, 1981). Thus, it may be suggested, that histogenesis of the inoculated tumor might prove an important factor for pattern growth in animals of varying ages. The experiments of Shapot (1979) and some of our early experiments showed that transplantation of tumors of different histogenesis is followed by the development of different hormone-metabolic shifts in these animals. Thus, in rats inoculated with Walker-256 or thyroid tumor (TT strain), a rise in serum insulin level was observed, while in the rats inoculated with Pliss sarcoma, the level remained unchanged, as compared to control animals. Considerable variation in cholesterol and triglyceride concentrations was also revealed. It is possible, that such shifts, caused by tumor inoculation, might in some cases stimulate age-associated hormone-metabolic disturbances promoting tumor growth, but these shifts may sometimes prevent the formation of such disturbances, thus decreasing tumor growth rate. It would be interesting to analyze data on the survival rate of cancer patients of varying ages. According to the detailed work of Axtell et al. (1976), survival rate is decreased proportionally to the age of a patient for the majority of cancer localizations. However, among patients with bone tumors, the prognosis was best for those aged 34-54, while for younger and/or older age groups it was worse. Survival rates, in children suffering from sarcomas and some types of lymphomas, were lower than in adult patients. Portnoy et al. (1980) analyzed 3877 stomach cancer patients aged from 18 to over 60. According to his observations, patients at the age of 18-35 revealed the shortest anamnesis, higher tumor growth rate, larger size of tumors, and earlier development of metastases. On the other hand, Hakama and Penttinen (1981) showed that in women under 50, suffering from cancer of the cervix uteri, tumors

404

VLADIMIR N. ANISIMOV

develop slower, have a stage of preinvasive cancer, and, as a rule, have a favorable prognosis, contrary to women over 70. It is noteworthy that in our experiments the survival rate of 3-month-old mice, with induced squamous cell cancer of the cervix uteri, was higher and tumor growth rate was lower, as compared with 18-month-old animals (Anisimov, 1982b). In related clinical work evaluating the influence of age on the prognosis of tumor growth, the stage of the disease and histogenesis of tumors are not taken into account, which complicates the analysis of this material. According to Petrov (1980), patients under age 30, with ovarian cancer (early stages), showed a worse survival rate than those aged 30-41, whereas in patients aged over 41, the 5-year-survival rate was again lowered. However, histogenesis of the ovarian tumors was not considered in this work. In our experiments (Pozharisski et aZ., 1980), in DMH-treated rats of varying age, multiplicity of colon tumors inversely correlated with the age of animals. However, large-size tumors were more frequent in older rats, and their morphology showed enhanced tumor progression in older animals. Turusov et aZ. (1981) observed greater incidence of intestinal tumors with invasive growth in 12-month-old DMH-treated mice, as compared to 8-month-old animals. These data provide evidence on the promoting effect of age per se on carcinogenesis. IX. Factors Modifying Rate of Aging and Carcinogenesis

For discussion of this problem it seems reasonable to define the meaning of the term “aging rate” as we understand it. There are many definitions resulting from the analysis of processes which take place both in a separate organism at different integration levels and in the whole population. The aging rate of individual organisms will be determined by the rate of age-associated changes of a certain number of factors responsible for the organism life potential (or vulnerability). The choice among such factors is widely argued in the available gerontological literature (Dilman, 1981; Everitt and Webster, 1976). As a matter of fact, present concepts and theories are based on these factors. It is not our task to analyze this problem. We shall just note here that the choice of the above factors might be made with regard to different integration levels: (1)at the subcellular and cellular level such factors may be the amount of damage in DNA and collagen cross-linkages, lipofuscine accumulation, enzyme activity, possible number of divi-

CARCINOGENESIS AND AGING

405

sions, etc.; (2) at the tissue and organ level-weight, cellularity, proliferative activity, amount of hormone and mediator receptors, functional activity, or barrier function of tissue or organ, etc.; and (3)at the organism level-concentrations of hormones and mediators in blood and regulatory centers (e.g., in the hypothalamus), functional activity of some organism systems (adaptive, energetic or reproductive). The problem of defining aging rate on a population level is also far from being solved. The Hompertz equation and its modifications suggested by many authors (Strehler, 1977; Sacher, 1977; Comfort, 1980) are sometimes satisfactory for the description of the correlation between death rate and age in populations:

R = Roeut

(3)

or In R = In Ro

+ at

(4)

where R = death rate, t = time, e = natural logarithm base, Ro = R when t = 0, a = constant, determining the slope of the Hompertz curve. As for the aging rate of the population, when the living (maintenance) conditions of a population are identical and do not change with time, then it might be expressed by means of the death rate index as:

dRldt = ROaeut

(5)

It is evident, that the aging rate of a population is determined by the constants a and Ro. Population aging rate is essentially characterized by the average and maximal life duration. However, these indices have different meanings, which will be discussed below (Section IX,B).

A. CARCINOGENS AS PROMOTERS OF AGING The promoting effect of carcinogenic and mutagenic agents on the process of aging is well known (Larionov, 1938; Dunjic, 1964; Anisimov, 1971; Lindop and Rotblat, 196213; Dilman, 1978, 1980; Alexandrov, 1978; Dilman and Anisimov, 1979; Gender and Bernstein, 1981). Table V illustrates data on hormone-metabolic shifts in the organism, and disturbances occurring at tissue and subcellular levels in the course of natural aging and in some types of carcinogenesis.

SIMILARITY OF

Parameter Hypothalamus Threshold of sensitivity to steroid feedback Catecholamine level and/or turnover Estradiol uptake Incidence of persistent estrus Serum estradiol level before switching-off of reproductive function Excretion of nonclassic phenolsteroids Adrenal cortex function Tolerance to carbohydrates Sensitivity to insulin Serum insulin level Oxidation of fatty acids Serum lipids level T-cell-mediated immunity DNA repair accuracy “Errors” in DNA synthesis Incidence of chromosome aberration Enzyme regulation Cell bioenergetics Clonal proliferation of some cells Derepression or activation of endogenous retroviruses Tumor incidence

TABLE V CHANGES DEVELOPING IN ORCANl5WS

Aging

DURING

Chemical carcinogens

AGINGA N U

Ionizing radiation

c \HC.INOLENF.SIS” Exogenic estrogens

Persistent estrus syndrome

Increases

Increases

Increases

Decreases

Decreases

Decreases

Decreases

Decreases

Increases Increases

Increases

Increases

Decreases Increases Increases

Increases

Increases

Increases

Decreases Increases Noncyclic secretion Increases

Hypercorticism Decreases Decreases Increases

Disfunction Decreases Decreases Decreases

H ypercorticisrn Decreases

H ypercorticism Decreases Decreases

Increases Increases Decreases Decreases Increases Increases Changes

Disfunction Decreases Decreases Increases or no changes Increases Increases Decreases Decreases Increases Increases Changes

Increases Increases Decreases Decreases Increases Increases Changes

No changes Decreases

Changes Increases Increases

Changes Increases Increases

Changes Increases Increases

Decreases Decreases Decreases Increases Increases Enzyme induction Changes Increases Increases

Increases

Increases

Increases

Increases

Increases

Increases Increases

CARCINOGENESIS AND AGING

407

Though these data are incomplete, it can still be seen that disturbances caused by natural aging and carcinogenic processes have something in common. Chemical carcinogens, ionizing radiation, and, possibly, certain hormones, have, on the one hand, a direct or indirect (latent virus activation) malignant effect on target tissues (initiation effect), and, on the other, provide the conditions for tumor growth and progression (promotion effect). It may be suggested that the key mechanism of the promoting effect of these carcinogens is their influence on the central chains of the neuroendocrine system, in particular on the hypothalamus (Dilman, 1978, 1981; Dilman and Anisimov, 1979, 1980; Alexandrov, 1978). It is assumed by Dilman (1981) that aging-associated elevation of the threshold of hypothalamic sensitivity to homeostatic inhibition by peripheral hormones is a key factor in the realization of neuroendocrine programs of organism development, aging, and formation of agerelated pathology, cancer included. As was shown in our experiments, some carcinogens (MC, DMBA, DMNA, NMU, NEU, DMH, DDT, and X rays) elevate the hypothalamic threshold of sensitivity to inhibition by estrogens in rats, while benz(a)anthracene and anthracene (which are noncarcinogenic agents for rats) had no effect on this parameter (Dilman and Anisimov, 1979; Anisimov et d., 1980d, 1982a). It is noteworthy that this phenomenon depends, first of all, on the aging-associated decrease in catecholamine and estrogen receptor concentrations in the hypothalmus (Dilman and Anisimov, 1979). The injections of DMBA or DMH into rats decreased the dopamine level in the hypothalamus (Anisimov et al., 1977, 1980d). Some investigators also reported on the competition between steroids and carcinogens for receptors (Kensler e t al. (1976). Age-related elevation of the hypothalamic threshold of sensitivity to inhibition by glucocorticoids was also shown (Dilman, 1978, 1981). In a series of experiments we studied the influence of carcinogenic agents on some lipid and carbohydrate parameters in rats treated with NMU, DMBA, DMH, DES, and X rays. All the models used demonstrated Iowered glucose toIerance accompanied by more or less pronounced reactive hyperinsulinemia and disturbances in the regulation of serum somatomedin activity. Variations in serum cholesterol and triglyceride level were not constant (Anisimov and Lvovich, 1976; Anisimov et al., 1980a-c; 1981; 1982a; Alexandrov et al., 1980). Raj and Venkitasubramanian (1974) reported lowered glucose tolerance in AFBI-treated chickens. These authors also showed that AFBl suppressed the activity of some glycolytic enzymes and stimulated the

408

VLADIMIR N. ANISIMOV

activity of the key enzymes of gluconeogenesis. It is noteworthy that DMBA and BP may induce atherosclerosis (Penn et al., 1981). The administration of some mutagens, e.g., 5-bromodeoxyuridine, alkylating agents, chemical carcinogens, and irradiation, was followed by the shortening of the animals’ life span and was considered to be a manifestation of accelerated aging (Lindop and Rotblat; 1962b; Dunjic, 1964; Anisimov, 1971; Ohno and Nagai, 1978; Kodell et al., 1980; Craddock, 1981). According to Ohno and Nagai (1978), neonatal injection of DMBA into female mice reduced their life span from 608 to 297 days. Untimely switching-off of the estrous function, weight loss, and graying were also observed in these animals. Dunjic (1964)reported the shortening of the life span of mileran-treated rats depending on the administered dose. This was accompanied by phenomena pertinent to old age, such as lens cataract and testicular atrophy. In our experiments (Anisimov, 1971) female rats perorally treated with MC demonstrated an early switching-off of reproductive function and a number of hormonal shifts peculiar to intensified aging. Thus, in spite of some differences in the nature of the shifts induced in the organism by natural aging and carcinogens, it should be acknowledged that, to a certain degree, it is possible to introduce such terms as radiation and carcinogenic aging (Alexandrov, 1978; Dilman, 1981; Gensler and Bernstein, 1981). B. PROMOTERS OF AGING AND CARCINOGENESIS It is seen from Table V that ionizing radiation, exogenous estrogens, or some carcinogens may induce persistant estrus syndrome. This syndrome involves some hormone-metabolic shifts promoting carcinogenesis and special attention should be attached to it. It is noteworthy that persistent estrus might be induced by the influences exerted on different chains of the neuroendocrine system by many other factors (Ird, 1966; Vishnevski et al., 1980). This state normally completes the reproductive period in rodents. Spontaneous or induced persistent estrus usually accompanies an increase in neoplasia incidence (Anisimov, 1982b). In our experiments (Alexandrov and Anisimov, 1976) rats were subjected to transplacental treatment with NMU. Tumors (mainly in the nervous system and kidneys) appeared in 37.5%of the experimental animals. Then, pregnant rats were injected with the same dose of NMU, and their offspring at the age of 3 months were subjected to ovariectomy followed by autoimplantation of an ovary into the tail. As

CARCINOGENESIS AND AGING

409

a result, persistent estrus was induced in these animals, and tumors were observed in 84.4% of the cases. Constant lighting of DMBAtreated rats also induced in them persistent estrus and increased tumor yield considerably (Khaetski, 1966). Obesity is another important factor promoting both the aging process and carcinogenesis. It is well known that other typical diseases of aging, such as hypertension, atherosclerosis, diabetes mellitus, and cancer, are very common in obese subjects (Dilman, 1978, 1981). it should be noted that rats with excessive weight often develop persistent estrus (Harris and Levine, 1965), and a high fat diet promotes carcinogenesis induced by various agents (Carrol and Khor, 1975). C. EFFECTOF GEROPROTECTORS ON CARCINOGENESIS We believe that mechanisms characterizing a close link between aging and carcinogenesis might be assessed by the effect of some drugs or factors (geroprotectors), which increase animal life span, on the incidence of neoplasms in these animals. According to different theories of aging approximately 20 drugs with geroprotective properties were suggested (Obukhova, 1975). A comparison of the available data on geroprotection mechanisms and their effect on tumor development would have been of assistance in understanding the interrelation between these two basic biological processes. The data on the effect of different geroprotectors on survival rate and tumor incidence in experimental animals are inconclusive. Moreover, the incidence of spontaneous neoplasms might be decreased and increased depending on the type of geroprotector (Anisimov, 1981b, 1982b). Emanuel and Obukhova (1978) suggested the classification of geroprotectors according to their type of effect on the rate of aging. These authors divided geroprotectors into three groups: (1)Group I, increasing the life span of the whole population; (2) Group 11, Iowering the death rate of long-lived individuals which leads to an increase in the maximal life duration; and (3) Group 111, increasing the life span of a short-lived subpopulation, while maximal life duration remains unchanged. According to their influence on aging rate, geroprotectors have different effects on spontaneous carcinogenesis (Fig. 1). As indicated in Table VI in mice infected with MuMTV, geroprotectors of the first type do not influence tumor incidence, but increase the latency of tumor development. At the same time, geroprotectors of the second type, which decrease rate of aging, also decrease tumor incidence. Mammary adenocarcinoma development in the MuMTVinfected mice is a two-stage process. Viral infection causes the trans-

410

VLADIMIR N . ANISIMOV 100

50

0 100

50

0 100

50

0

Frc. 1. Types of changes in survival (a) and tumor yield (b) curves caused by agents which affect the process of aging (geroprotectors).1-111, types of aging delay according to Emanuel and Obukhova (1978).Ordinate, a, surviving (%); b, tumor yield (%); solid line, control; dashed line, geroprotector.

formation of normal into malignant cells (initiation stage) and the promotion stage is due to age-associated hormone-metabolic and immune shifts. It may be suggested that the anticarcinogenic influence of the majority of geroprotectors is realized at the stage of virus-induced promotion of carcinogenesis. Only selenium is believed to inhibit oncornavirus replication (Medina and Shepherd, 1980). It is important that other antioxidants, e.g., 2-mercaptoethylamine or 2-ethyl-6methyl-3-oxypyridine, had no effect on mammary cancer incidence, but increase the latency of tumors. The viral nature of carcinogenesis in rats is not certain, and spontaneous neoplastic processes may be a result of the realization of a genetic program of aging. This suggestion is supported by the considerable stability of oncological characteristics of certain strains and stocks (Anisimov, 1976). The influence of exogenous carcinogens (radiation, diet, pollution of water and air with chemical carcinogens or their precursors, etc.), which, in principle, might be controlled, is of

CARCINOGENESIS AND AGING

411

TABLE VI EFFECTSOF GEROPROTECTORS ON DEVELOPMENT OF SPONTANEOUS MAMMARY ADENOCARCINOMAS IN FEMALE MICE Effect on Type of aging delay0 I I1

Geroprotector

Tumor latency

Tumor incidence

2-Mercaptoethylamine 2-Ethyl-6-methyl-3oxip yrid in e Caloric restriction

Increases No effect Increases No effect

Phenformin

Increases Decreases

Phenytoin

No effect

Decreases

L-Dopa

No effect

Decreases

Pineal factor Thymic factor Levamisol

Increases Decreases Increases Decreases Increases Decreases

Succinic acid

No effect

Increases Decreases

Decreases

References Harman (1972) Emanuel and Obukhova (1978) Tannenbaum and Sylverston (1953) Dilman and Anisimov (1980) Dilman and Anisimov (1980) Dilman and Anisimov (1980) Anisimov et al. (198213) Anisimov et al. (1982b) Bruley-Rosset et al. (1981) Anisimov and Kondrashova (1979)

According to Emanuel and Obukhova (1978).

lesser importance. From this viewpoint, the difference in the geroprotective effect on spontaneous tumor development in rats (Table VII) seems to be determined by its mechanism. According to this principle, all of them may be divided into two groups (Anisimov, 1981b). The first group consists of preparations which prevent occasional damage to macromolecules, preventing, thereby, errors during repair. The second group consists of drugs that delay the realization of the genetic program of aging and age-associated pathology. Antioxidants are the most typical representatives of the first group. Their geroprotective and antitumor effect seems to depend on the age at which their uptake was started, and inversely correlates with the dose of the damaging agent (i.e., the quantity of mutations or other damages in DNA or proteins per unit of time). It is suggested that antioxidants do not delay aging, but inhibit some exogenous factors which decrease the survival rate (e.g., they influence the number of free radicals in the food) (Kohn, 1971). The inhibiting effect of antioxidants on neoplasia is more clearly pronounced in carcinogen-treated animals (Wattenberg, 1978).

4 12

VLADIMIR N. ANISIMOV

The second type of geroprotector may be exemplified by antidiabetic biguanides, pineal and thymic factors, and by a calorie-restricted diet; These have multiple effects on hormone-metabolic and immune shifts in the organism (Table V), and their antitumor effect might be realized through the normalization of these shifts. It is noteworthy that these preparations also inhibited carcinogenesis induced by NMU, DMH, DMBA, and X rays in rats (Dilman et al., 1978, 1979; Alexandrov et al., 1980; Anisimov et al., 1980a,c,d; 1982a,b). Subdivision of geroprotectors into the above two groups is rather conventional. It was recently shown that some antioxidants are responsible for the enhanced immune response in old treated mice (Makinodan and Albright, 1979). On the other hand, phenformin may exert an antioxidant effect by decreasing fatty acids oxidation in the organism (Muntoni, 1974). Comparison of the available data suggests that tumor incidence is a function of the rate of aging. Taking into account the exponential character of dependencies between mortality and age and tumor incidence and age (Dix et al., 1980), we found it possible to calculate the correlation between rate of aging and tumor incidence, on the basis of the data on survival rate, spontaneous tumor incidence, tumor type, TABLE VII IN EFFECTOF CEROPROTECTORS ON DEVELOPhlENT OF SPONTANEOUS TUMORS

RATS

Effect on Type of aging delay" I I1

111

a

Ceroprotector Procaine (Gerovital) Caloric restriction Phenformin Buformin Pineal factor Tryptophan-deficient diet Phenytoin Ethylenediamine acetate-Nat Tocopherol : Benign tumors Malignant tumors Selenite: Malignant tumors

Tumor latency

Tumor incidence

References

No effect Increases Increases Increases Increases No data

No effect Decreases Decreases Decreases N o effect Decreases

Aslan et al. (1965) Ross and Bras (1971) Anisimov (1982a) Anisimov (1980) Dilman et al. (1979) Segall and Timiras (1976)

N o effect No data

Decreases Increases

Anisimov (1980) Dubina and Berlov (1974) Porta et al. (1980)

According to Emanuel and Obukhova (1978).

Increases Decreases Increases

Schroeder and Mitchener (1971)

CARCINOGENESIS AND AGING

413

and localization obtained from the experiments with 22 groups of both intact and geroprotector-treated rats (total number about 1900) (Anisimov, 1971, 1980, 1981a; Ross and Bras, 1971; Takizawa and Miyamoto, 1976). Life-tables were composed and death rate (R), mean life span, and rate of aging (a)were determined according to Eq. (3). Cumulative incidence of total or only malignant tumors was calculated according to the life-table for every group of animals by the age of 1000 days, and the parameter characterizing the slope of the tumor yield curve was determined. The results of the calculations showed a high degree of correlation between rate of aging (a)and total tumor incidence ( r = 0.453,p < 0.05) or total tumor yield slope ( r = 0.718, p < 0.01),and between (a)and malignant tumor incidence ( r = 0.600, p < 0.05) or malignant tumor yield slope ( r = 0.820, p < 0.01). From these results, we may suggest possible causes for the rise in malignant tumor incidence in the human population of industrial countries during this century. It is well known that in our century the Hompertz curve for human populations in industrial countries acquires a more rectangular form. The decrease in the mortality rate at younger ages is due to progress in medicine. However, the maximal life time of humans has remained unchanged for many centuries (Cutler, 1980). In other words, the rate of aging for senescent subjects is enhanced, corresponding to the third type of curve, characterizing the effect of geroprotectors on the survival in animals (Fig. 1,111).Hence, one may suggest that the observed increase in the sharpness of the Hompertz curve may be an important factor for the rise in tumor incidence in industrial countries (and, possibly, in the incidence of some of the other diseases of civilization, such as atherosclerosis, ischemic diseases, and diabetes mellitus). We believe that further progress in present prophylaxis is impossible without major changes in public health. In the burst of industrialization, urbanization, and increasing environmental pollution, one may hope only for partial aIleviation of their unfavorable effects on the organism. The achievement of significant results in this field will require the solution of very complex scientific and technical problems as well as considerable economic expense. The regulation of our “life-style” (diet, the age at which sex life is started, physical exercise, smoking, alcohol uptake, etc.) starting now, could be effective in cancer prevention and, as a result, in the increase of life span (Doll and Peto, 1981; Higginson, 1980).Treatment with drugs normalizing age-associated hormone-metabolic and immunologic shifts, thus delaying the realization of the genetic program of aging (aging rate is slowed down), should have considerable geroprotective and antitumor effects. Such factors as antioxidants and

-CARCINOGEN

me tabo 1 ic activation

...,

metabolic inactivation

I

T+/

transport

I

gland

NORMAL CELL

normal hypothalamus

damaged

DNA D S I I I L L V l l b e lV d L

Ive-

replication of

DNA

expression of transformation

KII=l-i

I

A-

' regulators of proliferation (growth factors, c ha lone s )

-

c

innnunocompetent

-

growth hormone

somatomedin

-c

-

4

VLDL, LDL

(cholesterol)

-

FFA

TRANSFORMED CELL c

CELL

TISSUE

ORGANISM

MALIGNANT TUMOR

FIG.2. An integral scheme of carcinogenesis. VLDL, very low density lipoproteins; LDL, low density lipoproteins; FFA, free fatty acids.

CARCINOGENESIS AND AGING

415

antimutagens may prevent the cancer-initiating effect of damaging agents, and might prove to be an important additional means for prevention of neoplastic processes and untimely aging. X. Summary

Figure 2 illustrates a suggested mechanism of carcinogenesis. This scheme takes into account the effect of carcinogens at different integration levels: subcellular, tissue, and organism. Any of these levels may be age dependent. Age-associated changes in the activity of enzymes responsible for activation and inactivation of carcinogens, and variations in concentrations of lipids and proteins contributing to the transport of carcinogenic agents into cells, may play an important role in the modifying effect of age on carcinogenesis. The effects of ageassociated changes in DNA repair need clarification. However, they are thought to exert a permissive influence on the age-associated rise in tumor incidence. It seems that proliferative activity of target tissues is the important modifying factor of carcinogenesis. Age-related changes of regulation at tissue and organism levels are also powerful factors in carcinogenesis modification. Age-dependent changes in the neuroendocrine system provide conditions for metabolic immunodepression and promotion of carcinogenesis. On the other hand, carcinogens per se (especially chemical and radiological) may intensify aging processes in the organism. Normalization, by drugs, of age-associated shifts requiring synthetic and energetic changes of a transformed tumor cells, and of immunological shifts, may exert both antitumor and geroprotective effects.

ACKNOWLEDGMENTS The author considers it his pleasant duty to thank Prof. N. P. Napalkov for his unflagging interest and valuable advice, Dr. K. M. Pozharisski, Dr. A. Likhachev, and Dr. R. Montesano for discussions of certain aspects of the problem, and Mrs. T. A. Shamova for assistance in the preparation of the manuscript for publication. We thank VEB Gustav Fischer Verlag (Jena) for kind permission to use their material in this article.

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INDEX

A 5-Azacytidine induction of gene expression by, 11Aclacinomycin A, 288, 290, 292-293 14 Acquired immunodeficiency syndrome, in treatment of acute leukemia, 28760,96 289,290 Actinomycin D, 360 Acyclovir, 93 ADCC, see Antibody-dependent cellular B cytotoxicity Adenine arabinoside, 93 Benign epithelial disease of breast, 207Adsorption of virus, gene product affect208 ing, 139 Betaherpesvirinae, 37 Age Bladder, sensitivity to carcinogens, 377 acute leukemia prognosis and, 266, Blood transfusion, cytomegalovirus in269 fections and, 50-51, 65-67 mammary gland tumors and, 374 Bone, radiation-induced carcinogenesis, tumor incidence and, 367-369 383 Aging Bone marrow transplantation chemical carcinogenesis and, 370-379 allogeneic and syngeneic, 304-305 foreign body-induced carcinogenesis autologous, 310-311 and, 379-380 survival after, 306-310 hormonal carcinogenesis and, 385-388 technique, 305-306 promoters of, 405-409 for treatment of leukemia, 304-311 radiation carcinogenesis and, 380-385 Breast, see also Mammary gland spontaneous carcinogenesis and, 367benign epithelial diseases of, 207-208 370 Breast carcinoma, 189-244 viral carcinogenesis and, 388-390 breast-feeding and, 201, 204-205 ALL, see Leukemia, acute lymphoblastic contraceptives, 205-206 AML, see Leukemia, acute myeloblastic dietary factors, 227-236 Amsacrine, 288, 289,290 heritage and, 191-197 ANLL, see Leukemia, acute nonlymhormonal factors, 208-215 phoblastic iatrogenic factors, 216-219 Anthracycline-DNA complexes, 295immunological factors, 216-219 296 incidence, 189-190 Antibody-dependent cellular cytotoxicmarital state and, 199-200 ity, 87 menses and, 197-199 L-Asparaginase, 260, 272, 273 other cancers and, 215-216 ASV, see Avian sarcoma virus parity and, 200-204 Auer rods, 266 psychosomatic factors and, 236-239 Avian sarcoma virus viral aspects of, 223-227 gene expression, 114-117 Breast-feeding, breast carcinoma and, 201,204-205 genome of, 109-110 425

426

INDEX

C Cancer abnormal differentiation and, 14-15 cytomegaloviruses and, 55-56 disseminated, treatment, 361 model for, 23-25 Carcinogen aging and, 390-415 inhibitors of DNA methylation, 18-22 Carcinogenesis aging and, 365-415 chemical, 370-379 foreign body-induced, 379-380 hormonal, 385-388 mechanism of, 415 modifying factors, 390-415 radiation, 380-385 spontaneous, 367-370 viral, 388-390 Carriers of cytotoxic drugs, 295-297 Cell surface antigens of leukemia cells, 132-138,270 Cellular transforming genes, 155-161 Central nervous system prophylaxis, 274-276 Cervical cancer, 56 Chemotherapy idealized surviving fraction curves, 342-346 fluctuations in response to, 335-341 mathematical relationships and models, 048-355 somatic mutation theory and, 331-362 Chromosomal aberrations, 266-267, 270 CID, see Cytomegalic inclusion disease Colon, adenocarcinoma of, 55-56 Colony-stimulating activity, 268 Coniplement, cytomegalovirus infection and, 88 Congenital disease, cytomegaloviruses and, 47-50 Consolidation treatment, 274, 280-281 Contraceptives, breast carcinoma and, 205-206 Cyclophosphamide, 260,273,357-360 Cytomegalic inclusion disease, 47-48, 54-55,95 Cytomegalovirus, human antigenic heterogeneity, 69

Cytomegalovirus, human (contd.) cellular immune response to, 70-72, 76-83 characteristics of, 37-39 cytopathic effects, 39 defective virions, 39 effects on leukocytes, 67-69 humoral immune response to, 70-76, 94 immunopathological syndromes, 6163 interactions with host cell, 39-41 latency and reactivation, 63-64 oncogenic potential, 55-61 replication, 39-40, 64-67 vaccines, 91-92 Cytomegalovirus infection in humans blood transfusion and, 50-51, 65-67 cell surface phenomena and, 69-70 clinical significance of, 47-55 diagnosis of, 45-47 epidemiology of, 41-45 immunity against, 69-72 immunobiology of, 32-96 immunosuppressive therapy and, 5354 nonspecific defense mechanisms against, 83-90 therapy problems, 92-93 in transplant recipients, 51-53 Cytosine, methylation of, 2 Cytosine arabinoside, 260, 278, 283

D Daunorubicin, 260, 278 Demethylation model, 5, 24 2-Deoxycoformycin, 288, 290, 294 Deoxyribonucleic acid interactions with carcinogens, 394-395 modification, 2 Deoxyribonucleic acid methylase, 2 Deoxyribonucleic acid repair, 395-398 Diet, breast carcinoma and, 227-236 Differentiation abnormal, cancer and, 14-15 cellular, models for, 4-6 Differentiation-specific loci, 128-130 Dihydroanthracenedione, 288, 290, 293294

427

INDEX

2,4-Dinitrochlorobenzene skin test, 221222 Doxorubicin, 260, 272, 273, 278, 283 Drug, cytotoxic carriers, 295-297 in leukemia therapy, 287-295 Drug combination, criteria for optimum delivery, 356-361 Drug resistance origin of, 346-348 selection for, 350-354

E eno gene, 109,114-117 Enzymes, carcinogen-metabolizing, aging and, 391-394 Estrogen breast carcinoma and, 212-215 as carcinogenic agent, 385-387 Etoposide, 288,290,291-292

F Fever, leukemia prognosis and, 268 Fluctuation test of Law, 334-335 Fv-1 IOCUS function, 140-142 polymorphism at, 120-122 Fu-2 IOCUS,129, 144

G gag gene, 109, 114-117

G(AKSL.B) antigen, 135 G(ERLD)antigen, 135 G(RADAI) antigen, 135 GIx antigen system, 132-134 Gastrointestinal tract, sensitivity to carcinogens, 375-376 GCSA, see Gross cell surface antigen Gene expression hypomethylation and, 8-11 induction by 5-azacytidine, 11-14 Gene regulation, 5-methylcytosine and, 1-25 Gene silencing factor, 14, 25 Geroprotectors, 405-415

Glucocorticoid receptors, 270-271 Gross cell surface antigen, 133-134

H H-2, see Murine major histocompatibility complex Hair dye, breast carcinoma and, 219 Hairless gene, 143-144 HCMV, see Cytomegalovirus, human Hematopoietic system radiation-induced carcinogenesis, 381382 sensitivity to carcinogens, 379 Herpesviruses, 35-37, see also Cytomegalovirus, human Hormones carcinogenesis and, 385-388 endogenous, breast carcinoma and, 208-215 Hormone therapy, breast carcinoma and, 218 Host range of retroviruses, 119-120, 139-140 Hypomethylation, gene expression and, 8-11

1 Ifosfamide, 288, 290, 292 Immune response to human cytomegaloviruses, 32-96 cellular, 70-72 humoral 70-76 Immune response genes, 165-171 Immune surveillance, against viral infection and transformation, 161-173 Immunocyte, viral infection of, 32 Immunopathological syndromes, cytomegaloviruses and, 61-63 Immunosuppression, cytomegalovirus infection and, 61-63, 67-69 Immunosuppressive therapy, cytomegalovirus infection and, 53-54 Immunotherapy for leukemia, 297-304 Infection, bacterial, leukemia prognosis and, 268 Infectious mononucleosis, caused by cytomegaloviruses, 50-51 Integration, gene product affecting, 140142

428

INDEX

Interferon cytomegalovirus infection and, 33,8890 for treatment of leukemia, 303-304 Interstitial pneumonia, 53, 92, 95

K Kaposi’s sarcoma, 44, 55, 56-60, 95-96 Kidney, sensitivity to carcinogens, 377 KS, see Kaposi’s sarcoma

L Leukemia associated cytomegalovirus infection, 54-55 genetics of susceptibility, 138-173 prospects for control, 173-175 Leukemia, acute, see also Treatment of acute leukemia chemotherapy, 271-297 immunotherapy, 297-304 remission, 260-261 types of, 256-258,262-263 Leukemia, acute lymphoblastic active immunotherapy, 298-299 chemotherapy, 27 1-278,283-285 prognostic factors, 268-271 types and subclasses, 259, 262-263 Leukemia, acute myeloblastic, types, 262-263 Leukemia, acute nonlymphoblastic active immunotherapy, 299-302 chemotherapy, 278-283,285-286 prognostic factors, 266-268 types, 258 Leukemia, experimental, curability, 331333 Leukemia, virus-induced, 107-108 Leukemia virus, 109, see also Murine leukemia viruses; Retroviruses differentiation-specific loci and, 128130 Leukemic cells cloning characteristics, 267-268 colony-stimulating activity, 268 drug resistance in, 346-348 methotrexate-resistant, 334-335 Leukemogenesis, 128-130 H-2 linked genes and, 164-171

mechanisms for, 158-161 organ niicroenvironment and, 146-148 Leukocyte cytomegalovirus replication in, 64-67 effects of cytomegaloviruses on, 67-69 Liposomes, as drug carriers, 296-297 Liver, sensitivity to carcinogens, 375 Long terminal repeat, 110-11 1 Low-density lipoproteins, as drug carriers, 296-297 LTR, see Long terminal repeat Lungs radiation-induced carcinogenesis, 384 sensitivity to carcinogens, 378-379 Ly 11.2 antigen, 148-150 Lymphocyte, viral transformation of, 32 Lymphocyte cytotoxicity, cytomegalovirus infection and, 76-77 Lymphocyte proliferation, cytomegalovirus infection and, 81-83 Lymphocyte proliferation tests, 78-81 Lymphokines, cytomegalovirus infection and, 77-78

M Macrophage, cytomegalovirus infection and, 86-87 Maintenance methylase, 2, 3, 7 Mammary gland, see also Breast radiation-induced carcinogenesis, 382383 sensitivity to carcinogens, 374 Marital state, breast carcinoma and, 199200 Menses, breast carcinoma and, 197-199 &Mercaptopurine, 260, 261, 283, 357360 Methotrexate, 260, 261, 283 Methylation, see aEso Hypomethylation gene regulation and, 3,6-11,23-25 inhibition by carcinogens, 18-22 in oitro, 8 Methylation pattern, 3-4 changes in, 4-6 clonal inheritance of, 7-8 5Methylcytosine formation, 2 gene regulation and, 1-25

429

INDEX

as gene silencing factor, 14 levels in tumorigenic cells, 15-17 Methylprednisolone, 283 Mitoxantrone, see Dihydroanthracenedione ML antigen system, 137 Monoclonal antibodies as drug carriers, 297 for treatment of leukemia, 302-303 Murine leukemia virus antigens of, 132-138 transformation by, 151-161 Abelson, target cells of, 145 MoIoney, integration sites, 130-132 Rauscher, target cells of, 145 Murine leukemia virus antigens, expression of, in mice, 132-138 Murine major histocompatibility complex, 161-171

N Nasal sinuses, sensitivity to carcinogens, 379 Natural killer cells, cytomegalovirus infection and, 84-86 Neoplasm, associated cytomegalovirus infection, 54-55 Nitrite inhalant, 60 NK cells, see Natural killer cells

0 Oncogenes, 22-23, 109 Oncornavirus, 108 Organ microenvironment, leukemogenesis and, 146-148 Organotropism, 122-132 Ovary, radiation-induced carcinogenesis, 382

P Parity, breast carcinoma and, 200-204 PC.1 antigen, 135-136 Penetration, control of, 139-140 pol gene, 109, 114-117 Polymorphism, in retroviruses, 119-132 Pr 180po', 115-116 Prednisone, 260,261,272,273

Prolactin, 208-212 Proliferative activity of lymphocytes, 81-83 of tissues, 398-399 Psychosomatic factors, breast carcinoma and, 236-239

R Radiation breast carcinoma and, 216-218 carcinogenesis and, 380-385 Radiation leukemia virus, 125-128 RadLV, see Radiation leukemia virus Recombinant virus, oncogenesis and, 151-155 Reticuloendothelius virus, 130 Retrovirus, 109-132 amphotropic, 119-120 assembly, 117-119 dualtropic, 119-120, 123-124 ectropic, 119-120, 123-124 gene expression, 114-117 genome organization, 109-111 host range, 119-120, 139-140 polymorphisms, 119-124 replication, 111-1 14 similarities to transposons, 111 spread, host genes affecting, 138-139 transformation, 117 xenotropic, 119-120 Retrovirus infection genetics of susceptibility to, 138-173 immune surveillance against, 161173 REV, see Reticuloendothelius virus Rubidazone, 260,278

S Sarcoma virus, 109, see also Avian sarcoma virus SFFV, see Spleen virus Skin radiation-induced carcinogenesis, 383384 sensitivity to carcinogens, 371-373 Soft tissues, sensitivity to carcinogens, 373-374 Somatic mutation theory, 333-334 Spleen virus, 144

430

INDEX

T Target cells, availability and replication of, 143-150 Teniposide, 288, 290, 291-292 6-Thioguanine, 260 6-Thioguanosine, 260 TL antigen system, 136 T lymphoma cells, 144-145 Transformation by HCMV in citro, 41, 61 immune surveillance against, 161-173 by leukemia viruses, 151-161 target cells and, 143 transforming genes, 155-161 Trdnsmethvh~se,function, 2 Transplantable tumors, aging and growth of, 400-403 Transplantation, see also Bone marrow transplantation cytomegalovirus infections and, 51-53 l’ransposons, similarities with retroviruses, 111 Treatment of acute leukemia, 255-313 bone marrow transplantation, 304-311 future prospects in, 312-313 pretreatment prognostic factors, 263271 strategy, 256-262 supportive, 311-312 Tumorigenic cell, 5-methylcytosine levels in, 15-17 Tumor incidence age and, 367-369 life span and, 369-370

U Ultraviolet light, 384-385 Uterus, sensitivity to carcinogens, 377378

v Vaccine, against cytomegaloviruses, 9192 Vagina, sensitivity to carcinogens, 377-

378 Vascular wall, sensitivity to carcinogens, 379 Vincristine, 260, 261, 272, 273, 283 Vindesine, 288, 290, 291 Viral latency, 32 Virus breast carcinoma and, 223-227 carcinogenesis and, 388-390 leukemia and, 107-108 Virus, type C, see Retrovirus

W White blood cell count, 269 Wilms tumor, 55

XYZ X-1 antigen system, 134-135 Zulu women, breast cancer and, 243-244

CONTENTS OF PREVIOUS VOLUMES

Volume 1 Electronic Configuration and Carcinogenesis C. A. Coulson Epidermal Carcinogenesis E. V . Cowdry The Milk Agent in the Origin of Mammary Tumors in Mice L. Dmochowski Hormonal Aspects of Experimental Tumorigenesis T. U . Gardner Properties of the Agent of Rous No. 1 Sarcoma R. J . C . Harris Applications of Radioisotopes to Studies of Carcinogenesis and Tumor Metabolism Charles Heidelberger The Carcinogenic Aminoazo Dyes James A. Miller and Elizabeth C . Miller The Chemistry of Cytotoxic Alkylating Agents M . C. J . Ross Nutrition in Relation to Cancer Albert Tannenbaum and Herbert Siluerstone Plasma Proteins in Cancer Richard J . Winder AUTHOR INDEX-SUBJECT INDEX

Volume 2

Carcinogenesis and Tumor Pathogenesis I . Berenblum Ionizing Radiations and Cancer Austin M . Brues Survival and Preservation of Tumors in the Frozen State James Craigie Energy and Nitrogen Metabolism in Cancer Leonard D. Fenninger and G. Burroughs Mider Some Aspects o f the Clinical Use of Nitrogen Mustards Caluin T. Klopp andJeanne C . Bateman Genetic Studies in Experimental Cancer L. w. Law The Role o f Viruses in the Production of Cancer C . Oberling and M . Guerin Experimental Cancer Chemotherapy C . Chester Stock AUTHOR INDEX-SUBJECT INDEX

Volume 3 Etiology of Lung Cancer Richard Doll The Experimental Development and Metabolism of Thyroid Gland Tumors Harold P. Morris Electronic Structure and Carcinogenic Activity and Aromatic Molecules: New Developments A. Pullman and B. Pullman Some Aspects o f Carcinogenesis P. Rondoni Pulmonary Tumors in Experimental Animals Michael B. Shimkin

The Reactions of Carcinogens with Macromolecules Peter Alexander Chemical Constitution and Carcinogenic Activity G. M . Badger 43 1

432

CONTENTS OF PREVIOUS VOLUMES

Oxidative Metabolism of Neoplastic Tissues Sidney Weinhouse AUTHOR INDEX-SUBJECT INDEX

Volume 4 Advances in Chemotherapy of Cancer in Man Sidney Farber, Rudolf Toch, Edward Manning Sears, and Donald Pinkel The Use of Myleran and Similar Agents in Chronic Leukemias D. A. G. Galton The Employment of Methods of Inhibition Analysis in the Normal and Tumor-Bearing Mammalian Organism Abraham Godin Some Recent Work on Tumor Immunity P. A. Gorer Inductive Tissue Interaction in Development Clifford Crobstein Lipids in Cancer Frances L. Hat;en and W . R. Bloor The Relation between Carcinogenic Activity and the Physical and Chemical Properties of Angular Benzacridines A. Lacassagne, N. P. Buu Hoi, R. Daudel, and F. Zajdela The Hormonal Genesis of Mammary Cancer 0. Miihlbock AUTHOR INDEX-SUBJECT INDEX

Volume 5 Tumor-Host Relations R. W. Begg Primary Carcinoma of the Liver Charles Berman Protein Synthesis with Special Reference to Growth Processes both Normal and Abnormal P. N. Campbell The Newer Concept of Cancer Toxin War0 h'akahara and Fumiko Fukuoka Chemically Induced Tumors of Fowls P. R. Peacock

Anemia in Cancer Vincent E. Price and Robert E. Greenfield Specific Tumor Antigens L. A. Zilber Chemistry, Carcinogenicity, and Metabolism of 2-Fluorenamine and Related Compounds Elizabeth K . Weisburger and John H . Weisburger AUTHOR INDEX-SUBJECT INDEX

Volume 6 Blood Enzymes in Cancer and Other Diseases Oscar Bodansky The Plant Tumor Problem A m i n C . Braun and Henry N . Wood Cancer Chemotherapy by Perfusion Oscar Creech, J r . and Edward T. Krementz Viral Etiology of Mouse Leukemia Ludwick Gross Radiation Chimeras P. C. Koller, A . J . S . Daoies, and Sheila M . A. Doak Etiology and Pathogenesis of Mouse Leukemia J . F. A. P. Miller Antagonists of Purine and Pyrimidine Metabolites and Folic Acid G . M . Timmis Behavior of Liver Enzymes in Hepatocarcinogenesis George Weber AUTHOR INDEX-SUBJECT INDEX

Volume 7 Avian Virus Growths and Their Etiologic Agents J . W . Beard Mechanisms of Resistance to Anticancer Agents R. W . Brockman

CONTENTS OF PREVIOUS VOLUMES

Cross Resistance and Collateral Sensitivity Studies in Cancer Chemotherapy Dorris J . Hutchison Cytogenic Studies in Chronic Myeloid Leukemia W. M . Court Brown and lshbel M . Tough Ethionine Carcinogenesis Emmanuel Farber Atmospheric Factors in Pathogenesis of Lung Cancer Paul Kotin and Hans L. Falk Progress with Some Tumor Viruses of Chickens and Mammals: The Problem of Passenger Viruses G. Negroni AUTHOR INDEX-SUBJECT INDEX

Volume 8 The Structure of Tumor Viruses and Its Bearing on Their Relation to Viruses in General A. F. Howatson Nuclear Proteins of Neoplastic Cells Harris Busch and WilliamJ . Steele Nucleolar Chromosomes: Structures, Interactions, and Perspectives M.]. Kopac and Gladys M . Mazeyko Carcinogenesis Related to Foods Contaminated by Processing and Fungal Metabolites H. F. Kraybill and M. B. Shimkin Experimental Tobacco Carcinogenesis Ernest L. Wynder and Dietrich Hoffman AUTHOR INDEX-SUBJECT INDEX

Volume 9 Urinary Enzymes and Their Diagnostic Value in Human Cancer Richard Stambaugh and Sidney Weinhouse The Relation o f the Immune Reaction to Cancer Louis V. Caso Amino Acid Transport in Tumor Cells R. M . Johnstonehnd P . G. Scholejield

433

Studies on the Development, Biochemistry, and Biology of Experimental Hepatomas Harold P. Morris Biochemistry of Normal and Leukemic Leucocytes, Thrombocytes, and Bone Marrow Cells 1. F . Seitz AUTHOR INDEX-SUBJECT INDEX

Volume 10 Carcinogens, Enzyme Induction, and Gene Action H . V . Gelboin In Vitro Studies on Protein Synthesis by Malignant Cells A. Clark Grifjin The Enzymatic Pattern of Neoplastic Tissue W. Eugene Knor Carcinogenic Nitroso Compounds P. N. Magee and]. M . Barnes The Sulfhydryl Group and Carcinogenesis J . S. Harrington The Treatment of Plasma Cell Myeloma Daniel E . Bergsagel, K . M. Grijjith, A. Haut, and W .1.Stuckley, J r . AUTHOR INDEX-SUBJECT INDEX

Volume 11 The Carcinogenic Action and Metabolism of Urethran and N-Hydroxyurethran Sidney S . Mirvish Runting Syndromes, Autoimmunity, and Neoplasia 0. Keast Viral-Induced Enzymes and the Problem of Viral Oncogenesis Saul Kit The Growth-Regulating Activity of Polyanions: A Theoretical Discussion of Their Place in the Intercellular Environment and Their Role in Cell PhysioIoay William Regelson

434

CONTEKTS OF PREVIOUS VOLUMES

Llolecular Geometry and Carcinogenic Activity of .4ramatic Compounds. New Perspectives Joseph C . Arcos and Mary F. Argus CL'MVLATIYE I S D E S

Volume 12 Antigens Induced by the Mouse Leukemia Viruses G . Pasternak Immunological Aspects of Carcinogencsis by Deoxyribonucleic Acid Tumor \'iruses G . 1. Deichmun Replication of Oncogenic Viruses in Virus-Induced Tumor Cells-Their Persistence and Interaction with Other \'iruses

H . Hanafusa Celltilar Immunity against Tumor Antigens Karl Erik Hellstroni and Ingegerd Hellstrom Perspectives in the Epidemiology of Leukemia Iming L. Kessler and Abraham M. I& ienfeld ?.UTHOR ISDEX-SVBJEC-r ISUEX

Volume 13 The Role of Innnunoblasts in Host Resistance and Inmunotherapy of Priman Sarcomata P . Alexander and J. G. Hall Evidence for the Viral EtioIogy of Leukemia in the Domestic Mammals 0 sIL' a Id Jurre t t The Function of the Delayed Sensitivity Reaction as Revealed in the Graft Reaction Culture Huint Cinsburg Epigenetic Processes and Their Relevance to the Study of Neoplasia Cujanati V. Sherbet The Characteristics of Animal Cells Transformed in Vitro laii Xfucpkerson

Role of Cell Association in Virus Infection and Virus Rescue J . Scoboda and 1. Hloiataek Cancer of the Urinary Tract D . 8 . Clayson and E . If. Cooper Aspects of the EB Virus M . A . Epsteiri hVTHOR INDEX-SUBJECT INDEX

Volume 14 Active Immunotherapy Georges Mathk The Investigation of Oncogenic Viral Genomes in Transformed Cells by Nucleic Acid Hybridization Ernest Winocour Viral Genome and Oncogenic Transformation: Nuclear and Plasma Membrane Events George Meyer Passive Immunotherapy of Leukemia and Other Cancer Roland M o f f a Humoral Regulators in the Development and Progression of Leukemia Donald Metcay Complement and Tumor Immunology Kusuyu Nishioka Alpha-Fetoprotein in Ontogenesis and Its Association with Malignant Tumors G . 1. Abelec; Low Dose Radiation Cancers in Man Alice Stewart AUTHOR INDEX-SUBJECT INDEX

Volume 15 Oncogenicity and Cell Transformation by Papovavirus SV40: The Role of the Viral Genome J . S . B u f e l , S . S. Teoethia, and J. L. Melnick Nasopharyngeal Carcinoma (NPC) J. H . C . Ho Transcriptional Regulation in Eukaryotic Cells A. J. MacGillitiruy, J . Puul, and G . Threljiall

CONTENTS OF PREVIOUS VOLUMES

435

Atypical Transfer RNA's and Their Origin in Neoplastic Cells Ernest Borek and Sylvia J . Kern Use of Genetic Markers to Study Cellular Origin and Development of Tumors in Human Females Philip J . Fialkow Electron Spin Resonance Studies of Carcinogenesis Harold M . Swartz Some Biochemical Aspects of the Relationship between the Tumor and the Host V . S . Shapot Nuclear Proteins and the Cell Cycle Gary Stein and Renato Baserga

Some Aspects of the Epidemiology and Etiology of Esophageal Cancer with Particular Emphasis on the Transkei, South Africa Gerald P. Warwick and John S. Harington Genetic Control o f Murine Viral Leukemogenesis Frank Lilly and Theodore Pincus Marek's Disease: A Neoplastic Disease of Chickens Caused by a Herpesvirus K . Nazerian Mutation and Human Cancer Alfred G. Knudson, Jr. Mammary Neoplasia in Mice S . Nandi and Charles M . McGrath

AUTHOR INDEX-SUBJECT INDEX

AUTHOR INDEX-SUBJECT INDEX

Volume 16 Polysaccharides in Cancer Vijai N . Nigam and Antonio Cantero Antitumor Effects of Interferon ion Gresser Transformation by Polyoma Virus and Simian Virus 40 Joe Sambrook Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overhealing? Sir Alexander Haddow The Expression of Normal Histocompatibility Antigens in Tumor Cells Alena Lengerova 1,3-Bis(2-ChloroethyI)-l-Nitrosourea (BCNU) and Other Nitrosoureas in Cancer Treatment: A Review Stephen K. Carter, Frank M . Schabel, Jr., Lawrence E . Broder, and Thomas P. Johnston AUTHOR INDEX-SUBJECT INDEX

Volume 18 Immunological Aspects of Chemical Carcinogenesis R. W. Baldwin Isozymes and Cancer Fanny Schapira Physiological and Biochemical Reviews of Sex Differences and Carcinogenesis with Particular Reference to the Liver Yee Chu Toh Immunodeficiency and Cancer John H . Kersey, Beatrice D. Spector, and Robert A. Good Recent Observations Related to the Chemotherapy and Immunology of Gestational Choriocarcinoma K . D. Bagshave Glycolipids of Tumor Cell Membrane Sen-itiroh Hakomori Chemical Oncogenesis in Culture Charles Heidelberger AUTHOR INDEX-SUBJECT INDEX

Volume 17

Volume 19

Polysaccharides in Cancer: Glycoproteins and Glycolipids Vijai N . Nigam and Antonio Cantero

Comparative Aspects of Mammary Tumors J . M . Hamilton

436

CONTENTS OF PREVIOUS VOLUMES

The Cellular and Molecular Biology of RKA Tumor Viruses, Especially Avian Leukosis-Sarcoma Viruses, and Their Relatives Howard M. Teniin Cancer, Differentiation, and Embryonic Antigens: Some Central Problems J. H.Coggin,Jr. and N. G. Anderson Simian Herpesviruses and Neoplasia Fredrich W . Deinhardt, Lawrence A. Falk, and Lauren G. W o l f . Cell-Mediated Immunity to Tumor Cells Ronald B. Herberman Herpesviruses and Cancer Fred Rapp Cyclic AMP and the Transformation of Fibroblasts Ira Pastan and George S . Johnson Tumor Angiogenesis Judah Folkman SUBJECT INDEX

Volume 20 Tumor Cell Surfaces: General Alterations Detected by Agglutinins Annette M. C. Rapin and Max M . Burger Principles of Immunological Tolerance and Immunocyte Receptor Blockade G. I. v . Nossal The Role of Macrophages in Defense against Neoplastic Disease Michael H. Levy and E . Frederick Wheelock Epoxides in Polycyclic Aromatic Hydrocarbon Metabolism and Carcinogenesis P. Sims and P. L. Grouer Virion and Tumor Cell Antigens of CType RNA 'Tumor Viruses Heinz Bauer Addendum to "Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overhealing?" Sir Alexander Haddow SUBJECT INDEX

Volume 21 Lung Tumors in Mice: Application to Carcinogenesis Bioassay Michael B. Shimkin and Gary D. Stoner Cell Death in Normal and Malignant Tissues E. H . Cooper, A. J . Bedford, and T. E. Kenny The Histocompatibility-Linked Immune Response Genes Baruj Benacerraf and David H . Katz Horizontally and Vertically Transmitted Oncomaviruses of Cats M . Essex Epithelial Cells: Growth in Culture of Normal and Neoplastic Forms Keef A. Rafferty, Jr. Selection of Biochemically Variant, in Some Cases Mutant, Mammalian Cells in Culture G. B. Clements The Role of DNA Repair and Somatic Mutation in Carcinogenesis James E. Trosko and Ernest H . Y . Chu SUBJECT INDEX

Volume 22 Renal Carcinogenesis I. M . Hamilton Toxicity of Antineoplastic Agents in Man: Chromosomal Aberrations, Antifertility Effects, Congenital Malformations, and Carcinogenic Potential Susan M . Sieber and Richard H . Adamson Interrelationships among RNA Tumor Viruses and Host Cells Raymond V . Gilden Proteolytic Enzymes, Cell Surface Changes, and Viral Transformation Richard Roblin, Iih-Nan Chou, and Paul H . Black Immunodepression and Malignancy Osias Stutman SUBJECT INDEX

CONTENTS OF PREVIOUS VOLUMES

Volume 23 The Genetic Aspects of Human Cancer W. E . Heston The Structure and Function of Intercellular Junctions in Cancer Ronald S. Weinstein, Frederick B . Merk, and Joseph Alroy Genetics of Adenoviruses Harold S. Ginsberg and C. S . H . Young Molecular Biology o f the Carcinogen, 4Nitroquinoline 1-Oxide Minako Nagao and Takashi Sugimura Epstein-Barr Virus and Nonhuman Primates: Natural and Experimental Infection A. Frank, W . A. Andiman, and G. Miller Tumor Progression and Homeostasis Richmond T. Prehn Genetic Transformation of Animal Cells with Viral DNA or RNA Tumor Viruses Miroslav Hill and Jana Hillova SUBJECT INDEX

Volume 24 The Murine Sarcoma Virus-Induced Tumor: Exception or General Model in Tumor Immunology? 1.P. Levy and J . C. Leclerc Organization of the Genomes of Polyoma Virus and SV40 Mike Fried and Beverly E . Griffin Pz-Microglobulin and the Major Histocompatibility Complex Per A. Peterson, Lars Rusk, and Lars Ostberg Chromosomal Abnormalities and Their Specificity in Human Neoplasms: An Assessment of Recent Observations by Banding Techniques Joachim Mark Temperature-Sensitive Mutations in Animal Cells Claudio Basilico

437

Current Concepts of the Biology of Human Cutaneous Malignant Melanoma Wallace H . Clark, Jr., Michael J . Mastrangelo, Ann M . Ainsworth, David Berd, Robert E . Bellet, and Evelina A. Bernardino SUBJECT INDEX

Volume 25 Biological Activity of Tumor Virus DNA F. L. Graham Malignancy and Transformation: Expression in Somatic Cell Hybrids and Variants Harvey L. Ozer and Krishna K.Jha Tumor-Bound Immunoglobulins: Zn Situ Expressions of Humoral Immunity Zsaac P. Witz The Ah Locus and the Metabolism of Chemical Carcinogens and Other Foreign Compounds Snorri S. Thorgeirsson and Daniel W. Nebert Formation and Metabolism of Alkylated Nucleosides: Possible Role in Carcinogenesis by Nitroso Compounds and Alkylating Agents Anthony E . Pegg Immunosuppression and the Role of Suppressive Factors in Cancer Zsao Kamo and Herman Friedman Passive Immunotherapy of Cancer in Animals and Man Steven A. Rosenberg and William D. Terry SUBJECT INDEX

Volume 26 The Epidemiology of Large-Bowel Cancer Pelayo Correa and William Haenszel Interaction between Viral and Genetic Factors in Murine Mammary Cancer J . Hilgers and P. Bentvelzen Inhibitors of Chemical Carcinogenesis Lee W. Wattenberg

438

CONTENTS OF PHEVIOUS VOLUMES

Latent Characteristics of selected Herpesviruses Jack G. Stecens Antitumor Activity of Corynebacterium part;unt Luka Milas and Martin T . Scott SUBJECT IXVEX

Volume 27 Translational Products of Type-C RNA Tumor Viruses ]ohn R. Stephenson, Sushilkumar G. Decare, and Fred H. Reynolds, J r . Quantitative Theories of Oncogenesis Alice S . Whittemore Gestational Trophoblastic Disease: Origin of' Choriocarcinoma, Invasive Mole and Choriocarcinoma Associated with Hydatidiform Mole, and Some Immunologic Aspects J . 1. Brewer, E . E. Torok, B. D. Kahan, C. R. Stanhope, and B. Halpern The Choice of Animal Tumors for Experimental Studies of Cancer Therapy Harold B. Hewitt Mass Spectrometry in Cancer Research John Robot Marrow Transplantation in the Treatment of Acute Leukemia E. Donna11 Thomas, C . Dean Buckner, Alexander Fefer, Paul E . Neiman, and Ruiner Storb Susceptibility of Human Population Groups to Colon Cancer Martin Lipkita Natural Cell-Mediated Immunity Ronald B. H e r h e m a n and Howard T. Holden SUBJECT INDEX

Volume 28 Cancer: Somatic-Genetic Considerations F . M. Burnet Tumors Arising in Organ Transplant Recipients Israel Penn

Structure and Morphogenesis of Type-C Retroviruses Ronald C. Montelaro and Dani P. Bolognesi BCG in Tumor Iminunotherapy Robert W. Baldwin and Malcolm V . Pimm The Biology of Cancer Invasion and Metastasis Isaiah J . Fidler, Douglas M. Gersten, and Ian R. Hart Bovine Leukemia Virus Involvenient in Enzootic Bovine Leukosis A. Burny, F . Bex, H. Chantrenne, Y. Cleuter, D. Dekegel, J . Ghysdael, R. Kettmann, M. Leclercq, J . Leunen, M. Mammerickx, and D. Portetelle Molecular Mechanisms of Steroid Hormone Action Stephen J . Higgins and Ulrich Gehring WBJECT INDEX

Volume 29 Influence of the Major Histocompatibility Complex on T-cell Activation J . F. A . P. Miller Suppressor Cells: Permitters and Promoters of Malignancy? David Naor Retrodifferentiation and the Fetal Patterns of Gene Expression in Cancer JOSI.? Uriel The Role of Glutathione and Glutathione S-Transferases in the Metabolism of Chemical Carcinogens and Other Electrophilic Agents L. F . Chasseaud a-Fetoprotein in Cancer and Fetal Development Erkki Ruoslahti and Markku Seppiilii Mammary Tumor Viruses Dan H. Moore, Carole A. Long, Akhil B. Vaidya,Joel B. Shefjeld, Arnold S. Dion, and Etienne Y. Lasfargues Role of Selenium in the Chemoprevention of Cancer A. Clark Griffin SUBJECT INDEX

CONTENTS OF PREVIOUS VOLUMES

439

Volume 30

Development of Human Breast Cancer Sefton R. Wellings

Acute Phase Reactant Proteins in Cancer E. H. Cooper and Joan Stone Induction of Leukemia in Mice by Irradiation and Radiation Leukemia Virus Variants Nechama Haran-Chera and Alpha Peled On the Multiform Relationships between the Tumor and the Host V. S . Shapot Role of Hydrazine in Carcinogenesis Joseph Bal6 Experimental Intestinal Cancer Research with Special Reference to Human Pathology Kazymir M . Pozharisski, Alexei J . Likhavchev, Valeri F. Klimashevski, and Jacob D. Shaposhnikov The Molecular Biology of Lymphotropic Herpesviruses Bill Sugden, Christopher R. Kintner, and Willie Mark Viral Xenogenization of Intact Tumor Cells Hiroshi Kobayashi Virus Augmentation of the Antigenicity of Tumor Cell Extracts Faye C. Austin and Charles W. Boone

INDEX

INDEX

Volume 33

Volume 31 The Epidemiology of Leukemia Michael Alderson The Role of the Major Histocompatibility Gene Complex in Murine Cytotoxic T Cell Responses Hemzann Wagner, Klaus Pfzenmaier, and Martin Rollinghoff The Sequential Analysis of Cancer Development Emmanuel Farber and Ross Cameron Genetic Control of Natural Cytotoxicity and Hybrid Resistance Edward A. Clark and Richard C . Harmon

Volume 32 Tumor Promoters and the Mechanism of Tumor Promotion Leila Diamond, Thomas G. O’Brien, and William M . Baird Shedding from the Cell Surface of Normal and Cancer Cells Paul H . Black Tumor Antigens on Neoplasms Induced by Chemical Carcinogens and by DNA- and RNA-Containing Viruses: Properties of the Solubilized Antigens Lloyd W . Law, Michael J . Rogers, and Ettore Appella Nutrition and Its Relationship to Cancer Bandaru S. Reddy, Leonard A. Cohen, G. David McCoy, Peter Hill, John H . Weisburger, and Ernst L. Wynder INDEX

The Cultivation of Animal Cells in the Chemostat: Application to the Study of Tumor Cell Multiplication Michael G. Tovey Ectopic Hormone Production Viewed as an Abnormality in Regulation of Gene Expression Hiroo lmura The Role of Viruses in Human Tumors Harald zur Hausen The Oncogenic Function of Mammalian Sarcoma Viruses Poul Andersson Recent Progress in Research on Esophageal Cancer in China Li Mingxin (Li Min-Hsin), Li Ping, and Li Baorong (Li Pao-Jung)

440

CONTENTS OF PREVIOUS VOLUMES

Mass Transport in Tumors: Characterization and Applications to Chemotherapy Rakesh K . Join, Jonas bl. Weissbrod, andlames Wei INDEX

Volume 34 The Transformation of Cell Growth and Transmogrification of DNA Synthesis by Simian Virus 40 Robert G. Martin Immunologic Mechanisms in UV Radiation Carcinogenesis Margaret L. Kripke The Tumor Dormant State E . Federick Wheelock, Kent J . Wuinhold, und Judith Leuich Marker Chromosome 14q- in Human Cancer and Leukemia Felix Mitelman Structural Diversity among Retroviral Gene Products: A Molecular Approach to the Study of Biological Function through Structural Variability James U'. Cautsch, John H . Elder, Fred C . Jensen, and Richard A. Lerner Teratocarcinomas and Other Neoplasms as Developmental Defects in Gene Expression Beatrice Mintz and Roger A. Fleischman Immune Deficiency Predisposing to Epstein-Barr Virus-Induced Lymphoproliferative Diseases: T h e X-Linked Lymphoproliferative Syndrome as a Model Daljid T . Purtilo INDEX

Volume 35 Polyoma T Antigens Walter Eckhart

Transformation Induced by Herpes Simplex Virus: A Potentially Novel Type of Virus-Cell Interaction Berge Hampar Arachidonic Acid Transformation and Tumor Production Luwrence Leuine The Shope Papilloma-Carcinoma Complex of Rabbits: A Model System of Neoplastic Progression and Spontaneous Regression John W. Kreider and Gerald L. Burtlett Regulation of SV40 Gene Expression Adolf Graessman, Monika Craessnaann, and Christian Mueller Polyamines in Mammalian Tumors, Part I Giuseppe Scalabrino and Maria E . Feriolo Criteria for Analyzing Interactions between Biologically Active Agents .Morris C . Berenbaum ISDEX

Volume 36 Polyamines in Mammalian Tumors, Part I1 Giuseppe Scalabrino and Maria E . Ferioli Chromosome Abnormalities in Malignant Hematologic Diseases Janet D. Rowley and Joseph R. Testa Oncogenes of Spontaneous and Chemically Induced Tumors Robert A. Weinberg Relationship of DNA Tertiary and Quaternary Structure to Carcinogenic Processes Philip D. Lipetz, Alan G. Galsky, and Rulph E . Stephens Human B-Cell Neoplasms in Relation to Normal B-Cell Differentiation and Maturation Processes Tore Godal and Steinar Funderud Evolution in the Treatment Strategy of Hodgkin's Disease Gianni Bonadonna and Armando Santor0

CONTENTS OF PREVIOUS VOLUMES

Epstein-Barr Virus Antigens-A Challenge to Modern Biochemistry DavidA. Thorley-Lawson, Clark M. Edson, and Kathi Geilinger INDEX

Volume 37 Retroviruses and Cancer Genes J . Michael Bishop Cancer, Genes, and Development: The Drosophila Case Elisabeth Gateff Transformation-Associated Tumor Antigens Arnold J . Levine Pericellular Matrix in Malignant Transformation Kari Alitalo and Antti Vaheri Radiation Oncogenesis in Cell Culture Carmia Borek Mhc Restriction and Ir Genes Jan Klein and Zoltan A. Nagy Phenotypic and Cytogenetic Characteristics of Human B-Lymphoid Cell Lines and Their Relevance for the Etiology of Burkitt’s Lymphoma Kenneth Nilsson and George Klein Translocations Involving lg Locus-Carrying Chromosomes: A Model for Genetic Transposition in Carcinogenesis George Klein and Gilbert Len0i.r INDEX

Volume 38 The SJL/J Spontaneous Reticulum Cell Sarcoma: New Insights in the Fields of Neoantigens, Host-Tumor Interactions, and Regulation of Tumor Growth Benjamin Bonaoida The Initiation of DNA Excision-Repair George W. Teebor and Krystyna Frenkel

441

Steroid Hormone Receptors in Human Breast Cancer George W . Sledge, Jr. and William L. McGuire Relation between Steroid Metabolism of the Host and Genesis of Cancers of the Breast, Uterine Cervix, and Endometrium Mitsuo Kodama and Toshiko Kodama Fundamentals of Chemotherapy of Myeloid Leukemia by Induction of Leukemia CelI Differentiation Motoo Hozumi The in Vitro Generation o f Effector Lymphocytes and Their Employment in Tumor Immunotherapy Eli Kedar and David W . Weiss Cell Surface Glycolipids and Glycoproteins in Malignant Transformation G . Yogeeswaran INDEX

Volume 39 Neoplastic Development in Airway Epithelium P. Nettesheim and A . Marchok Concomitant Tumor Immunity and the Resistance to a Second Tumor Challenge E . Gorelik Antigenic Tumor Cell Variants Obtained with Mutagens Thierry Boon Chromosomes and Cancer in the ‘Mouse: Studies in Tumors, Established Cell Lines, and Cell Hybrids Dorothy A. Miller and Orlando], Miller Polyomavirus: An Overview of Its Unique Properties Beverly E. Gr@n and Stephen M . Dilworth The Pathogenesis o f Oncogenic Avian Retroviruses Paula J . Enrietto and John A. Wyke Adjuvant Chemotherapy for Common Solid Tumors David A . Berstock and Michael Baum INDEX

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