Advances in Cancer Research, Volume 27

ADVANCES IN CANCER RESEARCH VOLUME 27

Contributors to This Volume J. I. Brewer

Martin Lipkin

C. Dean Buckner

Paul E. Neiman

Sushilkumar

G. Devare

Fred H. Reynolds, Jr.

Alexander Fefer

John Roboz

B. Halpern

C. R. Stanhope

Ronald B. Herberman

John R. Stephenson

Harold B. Hewitt

Rainer Storb

Howard T. Holden

E. Donna11 Thomas

B. D. Kahan

E. E. Torok Alice S. Whittemore

ADVANCES IN CANCER RESEARCH Edited by

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

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

Volume 27-1978 ACADEMIC PRESS

New York San Francisco London

A Subsidiary of Harcourt Brace Jovanovich. Publishers

0

COPYRIGHT 1978, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY. RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM T H E PUBLISHER.

ACADEMIC PRESS, INC.

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United Kingdom Edition published by ACADEMIC PRESS, MC. (LONDON) LTD. 24/28 Oval Road, London N W 1 7 D X

LIBRARY OF CONGRESS CATALOG CARD NUMBER:52- 13360 ISBN 0-12-006627-0 PRINTED IN THE UNITED STATES OF AMERICA

CONTENTS CONTRIBUTORS TO VOLUME27 ..............................................

ix

Translational Products of Type-C RNA Tumor Viruses JOHNR. STEPHENSON. SUSHILKUMAR G. DEVARE. AND FREDH . REYNOLDS. JR. I . Introduction ......................................................... I1. Type-C Viral Genome Structure and Complexity ........................ 111. Proteins of Type-C Tumor Viruses ..................................... IV. Genetic Mapping of the Type-C Tumor Virus Genome .................. V. Relatedness of Structural Proteins Coded for by Leukemia and Sarcoma Type-C Viral Genomes ......................... VI. Type-B and T4pe-D Oncomavirus Structural Proteins ................... VII . Summary and Conclusions ............................................ References ...........................................................

1 5 6 27

35 39 42 43

Quantitative Theories of Oncogenesis ALICE S . WHITTEMORE

I . Introduction .......................................................... I1. Expected Rates of Tumor Appearance .................................. 111. The Single Stage Theory of Iversion and Arley ........................ IV. The Multicell Theory of Fisher and Holloman .......................... V. The Multistage Theory with Negligible Cell Loss ....................... VI. The Multistage Theory with Non-Negligible Cell Loss .................. VII . The Multistage Theory with Proliferative Advantage of Intermediate Cells .................................................... VIII. Single Stage of Multistage Theory with Variation in Transformed Cell Types ............................................... IX . Implications for Dose-Response Relationships .......................... X . Conclusion ........................................................... References ........................................................... V

55 56 57 62 65 68 73 78 83 86 87

vi

CONTENTS

Gestational Trophoblastic Disease: Origin of Choriocarcinoma, lnvasive Mole and Choriocarcinoma Associated with Hydatidiform Mole, and Some Immunologic Aspects J . 1. BREWER,E. E. TOROK, 8. D. KAHAN, C. R. STANHOPE,AND B. HALPERN I. The Origin of Gestational Choriocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. lnvasive Mole and Choriocarcinoma Associated with Hydatidiform Mole ................................................... I I I. Immunobiology of Trophobl astic Disease ............................... References ...........................................................

89 125 138 145

The Choice of Animal Tumors for Experimental Studies of Cancer Therapy HAROLDB. HEWITT I. Introduction

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

11. Analysis of Species Used in Current Cancer Research . . . . . . . . . . . . . . . . . . . 111. Origin and ?ifaintenance of Animal Tumor Systems i n

149 153

Relation to Their Validity as Xlodels of Clinical Cancer. ................. 157 192 I\'. Reflections and Conclusions ........................................... 196 References ...........................................................

Mass Spectrometry in Cancer Research JOHN

ROBOZ

I. Scope of Applications and Analytical Techniques ....................... 11. Identification, Quantification, and Metabolism of Carcinogens . . . . . . . . . . . . 111. Metabolism and Monitoring of Antineoplastic Agents . . . . . . . . . . . . . . . . . . . IV. Biological Markers .................................................... References ...........................................................

202 2 15 233 253 260

Marrow Transplantation in the Treatment of Acute Leukemia E. DONNALL THOMAS,C. DEAVBUCKNER,ALEXANDERFEFER, PAUL E. NEIMAN,AND W N E R STORB I. introduction

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

............ 111. Analysis of Survival ................................................... 11. Patient Selection, Methods. and Surnniary of Clinical Results

269 270 271

CONTENTS

I V. Nature of Recurrent Leukemia ......................................... V. Efforts to Prevent Leukemia Relapse ................................... VI. Graft versus Leukemia ................................................ VII . Transplantation in Remission .......................................... VIII . Conclusions .......................................................... References ...........................................................

vi i 273 275 276 277 278 278

Susceptibility of Human Population Groups to Coion Cancer MARTINLIPKIN

I . Introduction ......................................................... I1 . Role of Environment in Increasing the Susceptibility of

Individuals to Colon Cancer ........................................... 111. Inherited Diseases that Increase Susceptibility to Colon Cancer ......... I V. Proliferative Abnormalities and Susceptibility to Colon Cancer .......... V. Newer Immunologic Studies .......................................... VI . Nuclear Protein and Enzyme Alterations ............................... VII . Studies of Cutaneous Cells ............................................ VIII . Examination of Fecal Contents ........................................ IX . Conclusion ........................................................... References ............................................................

281 282 287 293 296 296 299 300 300 301

Natural Cell-Mediated Immunity RONALD B . HERBERMAN AND HOWARD T. HOLDEN I . Introduction .......................................................... I1 . Characteristics of Natural Cytotoxicity ................................. 111. Specificity of Natural Cell-Mediated Cytotoxicity ....................... IV. Nature of Effector Cells ............................................... V. Relationship of Natural Cell-Mediated Cytotoxicity to Antibody-Dependent Cell-Mediated Cytotoxicity ....................... VI. Model for Placement of NK and K Cells in Pathway of Differentiation of T Cells .............................................. VII . Discrimination between Natural Cell-Mediated Cytotoxicity and Cytotoxicity by Other Effector Cells ............................... VIII . In Viuo Relevance of Natural Cytotoxicity .............................. References ...........................................................

305 307 324 333

SUBJECTINDEX ............................................................. CONTENTSOF PREVIOUSVOLUMES ..........................................

379 383

345 351 354 361 370

This Page Intentionally Left Blank

CONTRIBUTORS TO VOLUME 27 Numbers in parentheses indicate the pages on which the authors’ contributions begin.

J . I. BREWER,Department of Obstetrics and Gynecology and the Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611 (89) C. DEANBUCKNER,The Fred Hutchinson Cancer Research Center, Seattle, Washington 98104 and The Department of Medicine, Division of Oncology, University of Washington School of Medicine, Seattle, Washington 98195 (269) SUSHILKUMAR G. DEVARE, Laboratory of RNA Tumor Viruses, National Cancer Institute, Bethesda, Maryland 20014 (1) ALEXANDER FEFER,The Fred Hutchinson Cancer Research Center, Seattle, Washington 98104 and The Department of Medicine, Division of Oncology, University of Washington School of Medicine, Seattle, Washington 98195 (269) B. HALPERN, Department of Obstetrics and Gynecology and the Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611 (89) RONALDB. HERBERMAN,Laboratory of Immunodiagnosis, National Cancer Institute, Bethesda, Maryland 20014 (305) HAROLDB. HEWITT,Department of Morbid Anatomy, King’s College Hospital Medical School, London S.E.S., England (149) HOWARDT. HOLDEN,Laboratory of Immunodiagnosis, National Cancer Institute, Bethesda, Maryland 20014 (305) B. D. KAHAN,* Department of Surgery, Northwestern University Medical School, Chicago, Illinois 60611 (89) MARTIN LIPKIN,Memorial Sloan-Kettering Cancer Center, New York, New York 10021 (281) PAUL E. NEIMAN,The Fred Hutchinson Cancer Research Center, Seattle, Washington 98104 and The Department of Medicine, Division of Oncology, University of Washington School of Medicine, Seattle, Washington 98195 (269) * Present address: Divisions of Organ Transplantation and Immunology, Departments of Surgery and Biochemistry, University of Texas Medical School at Houston, Houston, Texas 77030. ix

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CONTRIBUTORS TO VOLUME

27

FREDH. REYNOLDS, JR., Viral Oncology Program, Frederick Cancer Research Center, Frederick, Maryland 21 701 ( 1) JOHN ROBOZ, Department of Neoplastic Diseases, Mount Sinai School of Medicine, The City University of New York, New York, New York 10029 (201) C . R. STANHOPE,Department of Obstetrics and Gynecology and the Cancer Center, Northwestern University Medical School, Chicago, lllirwis 60611 (89) JOHN R. STEPHENSON,Laboratory of RNA Tumor Viruses, National Cancer Institute, Bethesdu, Maryland 20014 ( 1 ) RAINER STORB,The Fred Hutchinson Cancer Research Center, Seattle, Wushington 98104 and The Department of Medicine, Division of Oncology, University of Washington, Seattle, Washington 98195 (269) E. DONNALL THOMAS, The Fred Hutchinson Cancer Research Center, Seattle, Washington 98104 and The Department of Medicine, Dioision of Oncology, University of Washington, Seattle, Washington 98195 (269) E . E. TOROK,Department of Obstetrics and Gynecology and the Cancer Center, Northwestern University Medical School, Chicago, lllinois 60611 (89) ALICE S. WHITTEMORE,Department of Environmental Medicine, New York University Medical Center, New York, New York (55)

ADVANCES IN CANCER RESEARCH, VOL. 27

TRANSLATIONAL PRODUCTS OF TYPE-C RNA TUMOR VIRUSES

John R. Stephenson, Sushilkurnar G. Devare, and Fred H. Reynolds, Jr. Laboratory of RNA Tumor Viruses, National Cancer Institute, Bethesda, Maryland and Viral Oncology Program, Frederick Cancer Research Center, Frederick, Maryland

I. Introduction .................................................... 11. Type-C Viral Genome Structure and Complexity.. ................ 111. Proteins of Type-C RNA Tumor Viruses ................................. A. RNA-Dependent DNA-Polymerase .................................. B. Structural Proteins .......................... .... ...... C. Src Gene-Coded Transforming Protein(s) ............................ IV. Genetic Mapping of the Type-C RNA Tumor Virus Genome . . . . . . . . . . . . . . A. Location of gag, pol, and env within the Viral Genome. ............... B. Intracistronic Mapping of the gag Gene of a Prototype Mammalian ............... Type-C Virus .................................. C. Identification of Functionally Analogous gag GeneMammalian Type-C Virus Isolates of Diverse Origin .................. V. Relatedness of Structural Proteins Coded for by Leukemia and Sarcoma ........... .......... Type-C Viral Genomes ....... VI. Type-B and Type-D Oncornavirus Structural Proteins ............. VII. Summary and Conclusions .... ........... References . . . . . . . . . . . . . . . . . . . ...........

6 7 8 24 27 27 30 31 35

I. Introduction

The existence of oncornavirus genetic sequences in a naturally integrated state within the cellular genome of a broad spectrum of vertebrate species is well established (Lieber and Todaro, 1975; Aaronson and Stephenson, 1976). Release of infectious virus particles, while generally repressed, can occur both spontaneously (Hartley et al., 1969; Aaronson et al., 1969; Stephenson and Aaronson, 1972b; Lieber et al., 1973) and following treatment with chemicals (Lowy et al., 1971; Aaronson et al., 1971b). Following activation, infectious virus may be transmitted horizontally both between individual animals of the same species (Hardy et al., 1973; Jarrett et al., 1973) as well a5 to other species (Benveniste and Todaro, 1976).The association of infectious oncornavirus particles with lymphoid tumors of many species has also been demonstrated (Gross, 1959; Lilly et al., 1975; Essex, 1975). Moreover, there is accumulating evidence that elevated endogenous virus expression may be an important determinant of host susceptibility to neoplastic transformation (Niman et al., 1977). 1 Copyright @ 1978 b y 4rademic €‘re\\,Inc

All light\ nf ieproduction in any toim re\ewed l5HN 0-12-006627-0

2

JOHN R. STEPHENSON ET AL.

Studies of the translational products of oncornaviruses have been initiated in efforts to gain insight into the role that these viruses may have both in normal cellular processes and in the etiology of tumors of their natural hosts. As early as 1958, Bernhard proposed a classification scheme for the diverse group of RNA viruses now included under the general term " oncornavirus". According to this system, RNA tumor viruses are designated as type-A, type-B, type-C (Bernhard, 1958, 1960), or type-D (Dalton et al., 1974) primarily on the basis of morphologic criteria. Intracellular virus-like particles occurring in a variety of mouse tumors have been designated type-A (Dalton et al., 1961). These are distinguished from other oncornaviruses mainly by virtue of their association with the endoplasmic reticulum rather than plasma membrane (Dalton, 1962). The second class of oncornaviruses, designated as type-B, have eccentrically located nucleoids and their envelope possesses characteristic projections or spikes (Sarkar et al., 1972). While mouse mammary tumor virus (MMTV), the prototype virus of this group, has been studied extensively, much less information is currently available regarding type-B viruses of other species of origin, such as the guinea pig (Opler, 1967; Nadel et al., 1967) and domestic cow (Miller et al., 1969; Van Der Maaten et al., 1974). The possibility that type-B particles may represent maturational products of intracytoplasmic type-A particles has been suggested on the basis of apparent similarities in the immunologic properties of their major structural proteins (Sarkar and Dion, 1975; Tanaka, 1977). The most extensively studied class of oncornaviruses are the type-C RNA tumor viruses. This group of viruses is characterized by their centrally located nucleoid and a pattern of virion assembly which occurs as a budding process at the plasma membrane (Sarkar et al., 1972). Type-C oncornaviruses can be distinguished from either type-B and type-D viruses on the basis of both morphologic criteria (Bernhard, 1958; Dalton et al., 1974) and the divalent cation preference of their RNA-dependent DNA-polymerase (Scolnick et al., 1970; Howk et al., 1973; Abrell and Gallo, 1973).In addition, many type-C virus structural proteins can be readily distinguished from those of type-B and type-D viruses. Moreover, a number of structural proteins of all type-C oncornavirus isolates examined to date have been found to share crossreactive interspecies antigenic determinants (Gilden, 1975; Stephenson et al., 1977b), and the major structural proteins of type-C isolates of several species have been shown to exhibit extensive regions of primary structure homoIogy (Oroszlan et al., 1975, 1976). Another characteristic property of type-C RNA tumor viruses is their unique

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

3

ability to provide helper functions for replication-defective sarcoma viruses (Hartley and Rowe, 1966; Huebner, 1967; Sarma et al., 1970; Aaronson and Rowe, 1970). In view of the fact that type-C viruses represent the major emphasis of the present review, the origins of many of the presently available isolates are summarized in Table I. It should be noted that in several instances endogenous type-C viruses of one species were transmitted to and became stably associated with the germ line of a second species (Benveniste and Todaro, 1974, 197513). In fact, the majority of type-C virus isolates can be traced back to two main lineages of ancestral viruses, one of rodent origin and the second, endogenous to primates. An understanding of the relatedness of different type-C virus isolates is important in the evaluation of much of the currently available information regarding properties of their structural proteins. Type-C viruses of a number of mammalian species, including endogenous viruses that have existed within the pig genome for millions of years (Benveniste and Todaro, 1975b), as well as a group of infectious horizontally transmitted isolates of gibbon apes (Kawakami et al., 1972) and a woolly monkey isolate (Theilen et al., 1971) are all related to known endogenous mouse type-C virus isolates and appear to be TABLE I MAMMALIAN TYPE-C ONCORNAVIRUSES Species of origin Rodent Mouse Mus musculus Mus caroli Mus cervicolw Rat Rattus norvegicus Hamster Cricetulus griseus Carnivores Cat Felis catus

Felis sylvestris ArtiodactyIs Pig Sus scrofa Deer Odocoileus hemionus Primates Baboon Papio cynocephalus Papio hamadyas Gelada Theropithecus gelada Woolly monkey Lugothrix spp. Gibbon ape Hylobates lar

Prototype virus isolate

Ancestral origin

R-MuLV, AKR-MuLV, etc. CERO CI CERV CI, CII RT 21C, SF-1, RMTDV CCL 14.1

Rodent Rodent Rodent Rodent Rodent

RD 114 FeLV FS-1, WCV-1

Primate Rodent Primate

CCL-33, PK(15) DKV

Rodent Unknown

M7, M28, BAB8-K BILN TG-1-K SSAV-1 GALV

Primate Primate Primate Rodent Rodent

4

JOHN R. STEPHENSON ET AL.

evolutionarily related to ancestral mouse viruses (Lieber et al., 197513). While other endogenous rodent viruses, such as those of hamster (Graffi et al., 1968; Kelloff et al., 1970) and rat (Bergs et al., 1970) origin, have not been as well studied, these also appear to constitute a highly related group (Benveniste and Todaro, 1975a). In addition, feline leukemia virus (FeLV), a horizontally transmitted type-C virus of cats, has been shown to possess significant nucleic acid sequence homology with, and was apparently derived from an endogenous rodent virus (Benveniste and Todaro, 1975a). Endogenous type-C viruses of Old World monkeys, apes, and possibly man constitute the second major lineage of mammalian type-C viruses. While isolation of infectious viruses of this group have been limited to baboon species of the genus Papio (Todaro et al., 1976; Stephenson and Aaronson, 1977), the presence (Benveniste and Todaro, 1976) and partial expression (Stephenson and Aaronson, 1977)of related nucleic acid sequences within the genomes of a much broader range of Old World primates has also been demonstrated. A class of endogenous feline viruses, the prototype of which is designated RD114 (McAIlister et al., 1972), are apparently of primate origin, having entered the germ line of an ancestral cat 20-30 million years ago (Benveniste and Todaro, 1974). In addition, there is suggestive evidence that a less well-characterized group of type-C viruses, endogenous to ungulates may be somewhat more closely related to primate than to rodent viruses (Aaronson et al., 1976; Tronick et al., 1977). The fourth major group of oncornaviruses, designated as type-D (Dalton et al., 1974)were described subsequent to the original oncornavirus classification proposed by Bernhard. These particles are somewhat larger in size than type-B or type-C viruses and have pleomorphic bullet-shaped nucleoids. The prototype isolates of this cIass include the Mason-Pfizer monkey virus (MPMV) (Chopra and Mason, 1970; Kramarsky et al., 1971) and a recently reported endogenous virus of the langur (Todaro et al., 1977a). A number of oncornavirus isolates of squirrel monkey origin have also been tentatively classified as type-D viruses (Heberling et al., 1977; Todaro et al., 1978). In addition to the four classes of oncornaviruses summarized above, there is a category of RNA tumor viruses generally known as RNA sarcoma viruses. These are replication-defective, transforming viruses which appear to have arisen as a result of genetic recombination between type-C viral and host cell genetic sequences (Scolnick et d., 1973, 1975; Frankel and Fischinger, 1977). Mammalian sarcoma isolates studied to date, while competent for transformation, have invariably been found to require type-C leukemia helper viruses for their

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

5

replication (Hartley and Rowe, 1966; Huebner, 1967; Aaronson and Rowe, 1970). Isolates of this group of viruses have been restricted to four mammalian species; these include two rodents, mouse (Moloney, 1966; Levy et al., 1973) and rat (Harvey, 1964; Kirsten and Mayer, 1967), one carnivore species, cat (Snyder and Theilen, 1969; Gardner et at., 1971),and one primate, woolly monkey (Wolfe et al., 1971). II. Type-C Viral Genome Structure and Complexity

The single-stranded type-C viral genomic RNA has a sedimentation coefficient of about 70 S, corresponding to an estimated molecular weight of approximately 1.2 x lo7 (Robinson et al., 1965; Duesberg, 1968; Montagnier et al., 1969). In addition, smaller RNA species with sedimentation values of 4 S and 7 S are found within the virion (Bishop et al., 1970a,b). Denaturation of the 70 S genomic RNA leads to production of two 35 S RNA subunits (Duesberg, 1968). On the basis of electrophoretic mobility (Duesberg and Vogt, 1973) and end-group analysis (Keith et al., 1974), a molecular weight of about 3 x lo6 was derived for each 35 S subunit. That the viral genome is polyploid and all 35 S subunits are similar in their sequence has been demonstrated by oligonucleotide fingerprinting analysis using ribonuclease TI (Billeter et al., 1974; Duesberg et al., 1974; Coffin and Billeter, 1976), size measurements of infectious DNA (Hill and Hillova, 1974) as well as by molecular hybridization (Baluda et al., 1974). Moreover, the recent application of heteroduplex mapping techniques to studies of type-C viruses have indicated the viral RNA to consist of two 35 S monomeric subunits, attached near their 5’ ends in a dimer linkage structure (Kung et al., 1975, 1976; Bender and Davidson, 1976). The 4 S virionassociated RNA species has been shown to represent tRNA (Erikson and Erikson, 1971; Bonar et al., 1967; Travnicek, 1968) and has been identified as tRNAnp for avian type-C viruses (Dahlberg et al., 1974b; Harada et al., 1975) and tRNAP” in the case of some but not all mammalian type-C virus isolates (Peters et al., 1977). Studies on Rous sarcoma virus (Furuichi et al., 1975; Keith and Fraenkel-Conrat, 1975) and the Moloney strain of murine leukemia virus (MuLV) (Stoltzhs and Dimock, 1976; Bondurant et al., 1976; Rose et al., 1976) have shown that the 5’ end of the viral RNA is capped by the structure m7G(5’)ppp(5‘)NmpNp.Such capping structures are common among eukaryotic mRNAs and may act to protect the RNA from attack by phosphatases and other nucleases and in addition may promote initiation of translation (Shatkin, 1976). The 3’ terminus of each 30-35 S RNA has a poly(A) sequence of about 200

6

JOHN R. STEPHENSON ET AL.

nucleotides (Lai and Duesberg, 1972; Ross et al., Ic172; Keith et al., 1974; Wang et aE., 1975). A tRNA molecule is associated with the viral 35 S RNA and functions as the primer for RNA-directed DNApolymerase (RDDP), initiating synthesis of complementary DNA at a unique site located within 150-200-nucleotide residues from the 5’ terminus of the viral genome (Faras et al., 1974; Taylor and Illmensee, 1975; Cashion et al., 1976; Haseltine et al., 1976). Recently, the sequence of the first 101 bases beginning at the 5’ end of the Prague RSV-C genome has been determined (Haseltine et al., 1977; Shine et al., 1977).These studies have resulted in the identification of a possible initiation triplet (AUG) for protein synthesis located 85 bases from the 5’ cap structure. Moreover, a sequence of 21 nucleotides immediately preceding the 3’ poly(A) of a prototype avian type-C virus, PrRSV-C, has been identified as: 5’GCCAUUUUACCAUUCACCA poly(A) 3’ (Schwartz et al., 1977). The fact that this sequence is identical to that of the first 21 nucleotides located at the 5’ end of the 35 S RNA indicates that the viral genome is terminally redundant. This possibility has recently been confirmed (Coffin and Haseltine, 1977). Independent evidence for terminal redundancy was derived !?om restriction endonuclease mapping of DNA sequences complementary to the Moloney sarcoma virus genome (Canaani et ul., 1977). This terminal redundancy provides for the possibility of circularization of the viral genome prior to integration into host cellular DNA. In fact, circular structures have been visualized by electron microscopy heteroduplex analysis and a replication mechanism involving a circular intermediate has been proposed (Junghans et at., 1977). 111. Proteins of Type-C RNA Tumor Viruses

In view of the above findings indicating the complexity of the type-C viral genome to be of the order of 2-3 x 106, the maximum size of the translational product for which it can code is about 300,000. Studies on type-C virus-coded proteins have now led to identification and characterization of a sufficient number of proteins to essentially account for this entire coding capacity. These consist of a protein with RNA-dependent DNA-polymerase enzymatic activity as well as structural components of the virion, including a 70,000 molecular weight envelope glycoprotein and several low molecular weight nonglycosylated proteins. In addition, a number of type-C virus isolates are known to have acquired transformation-specific sequences by recombination with host cell genes. Such recombinant viruses, which in general are replication-defective, apparently code for one or more pro-

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

7

teins associated with malignant transformation. In the following sections, currently available information regarding the properties of type-C viral translational products, with emphasis on possible functions, is reviewed. In addition, an attempt has been made to define and genetically map regions of the viral genome coding for individual translational products. A. RNA-DEPENDENTDNA-POLYMERASE

The RNA-dependent DNA-polymerase (RDDP), also known as “reverse transcriptase,” has the capacity to use both, polyribonucleotides and polydeoxyribonucleotides as template to synthesize complementary DNA (Baltimore, 1970; Temin and Mizutani, 1970; Baltimore and Smoler, 1971; Spiegelman et al., 1970a,b; Temin and Baltimore, 1972; Verma, 1977). The purified RDDP also exhibits ribonuclease activity “RNase H” which can selectively degrade the RNA moiety of RNADNA hybrids (Moelling et al., 1971; Baltimore and Smoler, 1972; Keller and Crouch, 1972; Leis et al., 1973). Analysis of mutants of avian and mammalian type-C viruses, characterized by temperaturesensitive lesions in their RNA-dependent DNA-polymerase, DNAdependent DNA-polymerase and RNase H activities convincingly demonstrated these activities to be virus-coded and essential for integration of the viral genome into the cellular DNA (Linial and Mason, 1973; Mason et al., 1974; Verma et al., 1974, 1976; Tronick et al., 1975). Most of the viral RDDP requires a primer such as transfer RNA and some metal ions for activity (Dahlberg et al., 197413; Hasteline and Baltimore, 1976; Grandgenett, 1976b). Thus, the type-C viral enzyme prefers Mn2+ ions while type-B and type-D viruses prefer Mg2+ions for their activity (Scolnick et al., 1970; Howk et al., 1973; Abrell and Gallo, 1973; Michalides et al., 1975). In addition to transcription of their natural template, all viral polymerases faithfully copy synthetic template-primers, such as poly(A) - oligo(dT), poly(C) oligo(dG), to various extents (Spiegelman et al., 1970a,b; Mizutani et al., 1970; Riman and Beaudreau, 1970). Recently, optimal conditions for reverse transcription of complete copy of the viral genome in vitro have been described (Rothenberg and Baltimore, 1977). Under conditions of limiting Mg2+ ion concentration, full length, apparently infectious (Rothenberg et al., 1977) complementary DNA copies of the viral RNA can be synthesized. The RDDP from the murine leukemia viruses has been shown to consist of a single polypoptide of about 70,000 molecular weight (Ross et al., 1971; Tronick et aZ., 1972; Gerwin and Milstein, 1972;

8

JOHN R. STEPHENSON ET AL.

Hurwitz and Leis, 1972). In contrast, the avian type-C viral reverse transcriptase contains two subunits, a (70,000) and /3 (110,000) (Temin and Baltimore, 1972; Verma et al., 1974; Gibson and Verma, 1974; Kacian et al., 1971; Grandgenett et al., 1973). The a subunit exhibits both polymerase and nuclease activities while the p subunit apparently enhances the binding of a to the template or substrate (Verma et al., 1974; Gibson and Verma, 1974; Grandgenett and Green, 1974; Moelling, 1974; Grandgenett, 1976a). Pulse-labeling of the Rauscher (R)-MuLV infected mouse cells has indicated that RDDP is initially synthesized in the form of a large precursor protein of about 200,000 molecular weight (Naso et al., 1975; Arlinghaus et al., 1976). Posttranslational cleavage of this high molecular weight precursor gives rise to an 80,000gag gene-coded precursor and the viral RDDP ofabout 75,000 molecular weight (Naso et al., 1975; Arlinghaus et al., 1976). The reverse transcriptase also provides a useful antigentic marker for the identification and characterization of type-C viruses of diverse origin (Aaronson et al., 1971a; Scolnick et al., 1972a). Antisera prepared against the enzyme of a given mammalian type-C virus most strongly inhibits the activity of the homologous enzyme and to a lesser degree enzymes of type-C virus isolates of other mammalian species (Aaronson et al., 1971a; Scolnick et al., 1972a; Parks et al., 1972). However, antisera to mammalian type-C viral enzymes do not inhibit the reverse transcriptases of avian type-C viruses or of mammalian oncornaviruses that are not type-C viral in origin (Aaronson et al., 1971a; Scolnick et a1., 1972a). Recently, radioimmunoassays for the RNA-dependent DNA-polymerases of avian (Panet et al., 1975; Reynolds and Stephenson, 1977) and mammalian (Krakower et al., 1977) type-C viruses have been described. By use of homologous competition assay for R-MuLV it was possible to distinguish R-MuLV enzyme from that of other murine viruses while in heterologous more broadly reactive assays, a number of mammalian type-C viruses showed immunologic cross-reactivity (Krakower et al., 1977). Application of competition immunoassays for the viral reverse transcriptase to studies of intracellular RDDP expression have led to the demonstration that translation of the type-C viral genome must involve more than one initiation site (Reynolds and Stephenson, 1977). B. STRUCTURALPROTEINS

Mammalian type-C viruses of diverse species of origin exhibit marked similarities in their structural components. Thus, it has been

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

9

possible to identify functionally analogous structural proteins of different type-C RNA viruses on the basis of their biochemical and immunologic properties. Type-C viral structural proteins can be separated into two groups on the basis of the map positions at which they are coded within the viral genome. One group, which includes the major envelope glycoprotein (gp70) and a nonglycosylated 15,000 molecular weight protein (p15E) are synthesized in the form of a common precursor coded for by a viral gene generally referred to as enu. The remaining viral proteins are characterized by molecular weights in the 10,000 to 30,000 range and are synthesized as a 65,000 molecular weight precursor protein coded for by a region of the viral genome designated gag. These latter proteins are nonglycosylated and are generally thought to be located in the nucleoid or core of the virion. The biochemical and immunologic properties and posttranslational processing of enu and gag coded structural proteins are considered below.

1. Env Gene-Coded Proteins There is accumulating evidence from a number of laboratories consistent with the possibility that the major envelope glycoprotein, gp70, and a lower molecular weight, nonglycosylated virion structural protein, p15E, are initially synthesized in the form of a common precursor. This is indicated b y the demonstration of an 85,000-90,000 molecular weight glycoprotein in R-MuLV infected cells which is precipitable by anti-gp70 and anti-p15(E) sera and which by pulse chase experiments gives rise to cleavage products of around 70,000 and 15,000 molecular weights, respectively. Methionine-labeled peptide sequences analogous to those of gp70 and p15(E) within this precursor have been identified b y tryptic digest analysis (Arcement et al., 1976; Shapiro et al., 1976; Van Zaane et al., 1976; Famulari et nl., 1976). Inhibition of glycosylation of the primary en0 gene product by use of 2-deoxy-D-glucose or cytocbalasin B leads to formation of a 70,000 molecular weight nonglycosylated protein (Shapiro et a1., 1976; W. J. M. van de Ven, personal communication) which presumably represents enu gene translational product prior to glycosylation. The product-precursor relationships between these various enu gent coded proteins have been confirmed by in uitro protein synthesis studies (Gielkens et al., 1974; Van Zaane et al., 1977). For instance, in a rabbit reticulocyte cell-free system, 22 S mRNA, isolated from R-MuLV infected cells, was shown to code for synthesis of a 70,000 nonglycosylated protein containing antigenic determinants in common with gp70 (Gielkens et al., 1974). Injection of the same mRNA

10

JOHN R. STEPHENSON ET AL.

into axenopus laevis oocyte translation system resulted in synthesis of a glycosylated 82,000 molecular weight protein as well as significant amounts of the two env gene cleavage products, gp70 and p15(E) (Van Zaane et al., 1977). In addition to the major 70,000 molecular weight envelope glycoproteins and the nonglycosylated envelope protein, p15(E), there have been reports of the presence of a 45,000 molecular weight glycoprotein constituent of type-C viruses (August et al., 1974; Fleissner et al., 1974; Ikeda et al., 1975; Moroni, 1972; Moennig et al., 1974). However, on the basis of amino acid composition, immunologic crossreactivity (Marquardt et al., 1977; Krantz et al., 1977; Charman et al., 1977) and peptide map (Elder et al., 1977), this latter glycoprotein appears to represent a breakdown product of gp70. a. Major 70,000 Molecular Weight Envelope Glycoprotein (gp70). The 70,000 molecular weight envelope glycoproteins of mammalian type-C virus isolates of mouse (Strand and August, 1974a; Hino et al., 1976), woolly monkey (Hino et al., 1975), baboon (Stephenson et al., 1976a), and feline (Stephenson et al., 1977a) origin, have been isolated and studied in detail. Immunologic characterization of gp7Os of these diverse viruses have indicated the presence of type, group, and interspecies-specific antigenic determinants (Strand and August, 1973, 1974a; Hino et al., 1975, 1976; Stephenson et al., 1976a). That these antigenic specificities reside in the protein moiety and not the carbohydrate residues was demonstrated by the use of glycosidase enzymes to remove selectively the carbohydrate portions of the molecule (Bolognesi et al., 1975). Competition immunoassays which measure type-specific antigenic determinants of these viral envelope glycoproteins have proven useful in discriminating closely related type-C virus isolates (Strand and August, 1974a; Hino et al., 1975, 1976; Stephenson et a1., 1976a), while interspecies immunoassays have been primarily utilized for detection and characterization of type-C virus isolates of diverse mammalian species (Strand and August, 1973; Stephenson et al., 1976a).These viral-coded glycoproteins have been shown to represent the major constituents of the viral envelope and to be associated with the spikes or surface projections on the virion surface (Nermut et al., 1972; Witte et al., 1973; Kennel et al., 1973; McLellan and August, 1976). As a consequence of their location within the virion envelope, viral glycoproteins represent the major target of neutralizing antibody (Ikeda et al., 1974; Steeves et al., 1974; Hunsmann et al., 1974). Studies of endogenous type-C viruses of mouse cells have led to the demonstration of multiple classes of biologically distinguishable vir-

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

11

uses (Stephenson et al., 1975a). Structural proteins, including gp70, coded for b y one such virus, designated Class 111, are expressed in the mouse throughout embryonic life (Stephenson et al., 1974; Hino et al., 1976). As a result, mice appear to develop immunologic tolerance to these proteins (Huebner et al., 1970, 1971; Stephenson et al., 1976b). In contrast, expression of translational products of other endogenous mouse type-C viruses, including a mouse cell tropic virus, designated Class I, is more tightly regulated (Stephenson et al., 1975a). Occasional spontaneous or chemical activation of virus of this class leads to its spread throughout the host (Stephenson and Aaronson, 1972b; Kawashima et al., 1976). Since proteins of Class I virus are not expressed during embryonic life, tolerance to antigenic determinants unique to this gp70 species fails to develop and virus replication is thus subject to regulation by host immune surveillance mechanisms (Stephenson et al., 1976b). The results of recent in vivo studies indicate that spontaneously activated Class I mouse cell-tropic virus can, in certain instances, recombine with and acquire genetic sequences coding for immunologic determinants of an endogenous viral envelope glycoprotein (Hartley et al., 1977; Elder et at., 1977a). These findings are consistent with early in vitro studies demonstrating genetic recombination between endogenous and exogenous mouse type-C viral genomes (Stephenson et al., 1974) and may provide one means by which exogenous virus can partially circumvent its recognition by host immune surveillance. There is evidence that a type-D oncornavirus of primates, MPMV, may have acquired glycoprotein antigenic determinants from its host, the rhesus monkey, by similar means to that described above for Class I ecotropic mouse type-C viruses (Stephenson et al., 1976a). This is suggested b y the fact that the envelope glycoproteins of MPMV and those of type-C viruses of the baboon-RD114 group exhibit immunologic cross-reactivity while other structural proteins of these viruses lack shared determinants. The phenomenon of interference between closely related oncornaviruses has been well established. Thus, infection of cells by a given oncornavirus isolate renders them resistant to superinfection b y the same or highly related viruses. Moreover, there has long been known to be a close correlation between host range, interference, and neutralization properties of related oncornavirus isolates. While these phenomena were initially demonstrated in the case of different subgroups of avian type-C viruses (Vogt and Ishizaki, 1965), such studies have been more recently extended to mammalian type-C viruses (Todaro et al., 1973; Levy, 1973). The correlaton between these various

12

JOHN R. STEPHENSON ET AL.

properties indicated the involvement of cellular receptor sites for virus infection. The availability of 1251-labeledgp70 has led to the identification and development of techniques for quantitation of such receptors (DeLarco and Todaro, 1976). Thus, 1Z51-labeledR-MuLV gp70 has been shown to bind and form a stable complex with receptor sites on murine but not other mammalian cells. Purified gp70-binding to mouse cells is prevented if the cells are actively producing related ecotropic virus, presumably because the receptors are occupied and are not available to bind exogenously applied gp70 (DeLarco and Todaro, 1976). Moreover, the presence or absence of appropriate receptors for different viruses may be an important host range determinant. For instance, mouse xenotropic viruses appear to use a different family of receptors from mouse ecotropic viruses, since they neither interfere with viral infectivity (Todaro et al., 1973; Levy, 1973; Fischinger et al., 1975) nor with gp7O-binding to mouse cells (DeLarco and Todaro, 1976). Endogenous type-C viral translational products, including gp70 (Hino et al., 1975), appear, in general, to be expressed throughout the life of their host. Thus, the resistance that cells of most species exhibit to infection b y such endogenous viruses may simply be due to the ability of endogenous gp70 to occupy available receptor sites. A major difficulty, however, is that expression of endogenous type-C viral glycoprotein, especially during embryonic life, leads to the development of immunologic tolerance (Stephenson et al., 1976b).Thus, strong evolutionary selective pressure may exist for an exogenous virus to recombine with genetic sequences coding for host glycoprotein determinants in order to circumvent the hosts immune surveillance, but retain the ability to utilize cell receptors other than those occupied by the host glycoprotein. Such a situation could account for much of the recent data obtained from studies of recombinant mouse (Stephenson et al., 1974; Fischinger et al., 1975; Hartley et al., 1977) and possibly primate (Stephenson et al., 1976a) oncornavirus isolates. There is accumulating evidence indicating that gp70 expression in the mouse may in some way be linked to differentiation and development. By use of immunofluorescence techniques, expression of type-C viral gp70 was demonstrated to be restricted to certain anatomical sites and to b e at much higher levels in lymphoid and epithelial cells than in other cells (Lerner et al., 1976). In fact, the major site of gp70 expression was reported to be the male genital tract (Lerner et al., 1976) and a protein immunologically and biochemically related to gp70 was found at high levels in secretions of the epididymis and ductus deferens (Del Villano and Lerner, 1976). In another study,

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

13

McClintock et al. (1977) reported constitutively high levels of AKRMuLV gp70 expression in the absence of overt virus in bone marrow cells of all strains of mice examined, including several which possess, at most, only a portion of the Class I type-C viral genome (Chattopadhyay et al., 1974). Moreover, the results of independent reports by several laboratories have established gp70 to be a constituent of the surface of normal thymocytes and to share immunologic and biochemical properties with the thymocyte differentiation marker Gix (Tung et al., 1975; Obata et al., 1975; Del Villano et al., 1975). b. Nongl ycosylated Envelope Protein ( p l 5 E ) . A nonglycosylated mouse type-C viral envelope protein of about 15,000 molecular weight (p15E) has also been described (Ikeda et al., 1975; Schafer et al., 1975). This protein has a marked tendency to aggregate in the absence of detergent and generally chromatographs in the void volume of agarose gel filtration columns even in the presence of 6 M guanidine hydrochloride (GuHC1) (Ikeda et al., 1975). Biochemical and immunologic characterization of p15E has shown it to be distinct from the 15,000 molecular weight gag gene-coded protein (p15) (Ikeda et al., 1975; Schafer et al., 1975), although both p15 (Strand et al., 1974; Barbacid et al., 1977) and p15E (Schafer et al., 1975) have been shown to possess group and interspecies antigenic determinants. Moreover, p15E has been shown to be a surface component of the virion and appears to represent the 15,000-17,000 molecular weight protein immunoprecipitable by normal mouse sera (Ihle et al., 1974, 1975; NOwinski and Koehler, 1974). There is evidence that in some instances p15E may be cleaved, giving rise to a low molecular weight polypeptide of about 12,000 (p12E) (Arcement et al., 1976).This possibility is supported by the results of pulse chase experiments as well as by peptide mapping data (Naso et al., 1976; Van Zaane et al., 1976). Recently, two groups of investigators have independently reported that in type-C viral particles, the envelope glycoprotein, gp70, is frequently found to be linked by disulfide bonds to a nonglycosylated protein of about 15,000 molecular weight. This was shown by a comparison of the electrophoretic mobilites of Moloney leukemia virus proteins under both reducing and nonreducing conditions (Leamnson et al., 1977; Witte et al., 1977). While the relationship of this 15,000 molecular weight protein to p 15E remains speculative, the possibility that they represent the same protein is supported by their similar electrophoretic mobilities and the fact that both have been shown to be constituents of the virion envelope (Leamnson et al., 1977; Witte et al., 1977). Moreover, there is evidence that both the protein disulfide bonded to gp70 and p15E have relatively acidic isoelectric points

14

JOHN R. STEPHENSON ET AL.

(Witte et al., 1977), while the 15,000 molecular weight gag gene coded proteins of mouse type-C viruses are more basic (Stephenson et al., 197%). 2. The gag Gene-Coded Virion Structural Proteins The nonglycosylated structural proteins coded by the avian type-C viral gag gene were shown in early studies to be synthesized in the form of a high molecular weight precursor polypeptide (Vogt et al., 1975). An analogous situation has more recently been described for a prototype mammalian type-C virus, R-MuLV (Naso et al., 1975; Stephenson et al., 197513; Van Zaane et al., 1976; Shapiro et al., 1976). Posttranslational cleavage of the 65,000-70,000 molecular weight R-MuLV precursor results in proteins of molecular weights of about 30,000 (p30), 15,000 (p15), 12,000 (p12), and 10,000 (p10). At present, the most widely adapted system for identification of the virus coded proteins is based on molecular weights as determined by gel filtration in the presence of 6 M GuHCl (Nowinski et al., 1972; August et al., 1974). Although this convention is useful in identification of proteins of murine type-C virus isolates, differences in the molecular weights of analogous proteins of virus isolates of evolutionarily diverse origin frequently lead to considerable confusion. This difficulty can be avoided, however, if gag gene-coded proteins are identified by a more generalized nomenclature system taking into account both immunologic and biochemical properties. For these reasons, mammalian type-C viral gag-coded proteins are designated in the following sections as: (a) major virion group-specific antigen, (b) hydrophobic structural protein, (c) type-specific RNA binding protein and (d) basic ribonucleoprotein. a. Major Virion Group-Specijc Antigen. The most extensively studied of the type-C viral gag gene proteins, to date, has been the major group-specific virion antigen (p30). This viral-coded structural protein is generally identified on the basis of its molecular weight of about 27,000-30,000 as determined by agarose gel filtration in the presence of 6 M GuHCl and its broadly reactive immunologic properties (Fig. 1).The initial identification of p30 was based on the results of complement-fixation tests indicating the expression of a virus-specific antigen in various mouse tissues (Hartley et al., 1965, 1969; Geering et al., 1966). Subsequent to these studies a number of groups successfully purified the protein associated with this immunologic reactivity by the application of gel filtration and preparative isoelectric focusing techniques (Oroszlan et al., 1970, 1971; Gilden and Oroszlan, 1972). An independent approach to purification of p30 involved agarose gel filtration in the presence of 6 M GuHCl (Nowinski et al., 1972).

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

30

50 70 FRACTION NUMBER

15

90

FIG. 1. Molecular weight analysis of type-C virus gag gene-coded structural proteins of a prototype RNA tumor virus, FeLV, by agarose gel filtration in the presence of 6 M guanidine hydrochloride. Individual column fractions were tested in competition immunoassays for FeLVp30 (A),p15 (B), p12 (C),andplO(D).[From Khan and Stephenson (1977) with permission of American Society for Microbiology.]

Immunologic analysis of type-C viral p30 by use of complementfixation (Huebner et al., 1964; Huebner, 1967; Hartley et al., 1969) and gel immunodiffusion analysis (Geering et al., 1966; Schafer et al., 1969; Gilden and Oroszlan, 1972) led to the demonstration of pronounced group-specific antigenic determinants. Immunologic assays recognizing such determinants provided one of the first means of discriminating type-C virus isolates of different species of origin. Subsequent independent studies b y several groups of investigators showed additional antigenic determinants shared by the major internal antigens of type-C RNA tumor viruses of diverse species of origin (Geering et al., 1970; Gilden et al., 1971; Schaferet al., 1972).With the development of sensitive and specific competition immunoassays, direct confirmation of these findings was obtained (Parks and Scolnick, 1972; Tronick et al., 1973). Moreover, in addition to these group- and interspecies-specific antigenic determinants, the major structural proteins of murine type-C viruses were shown to possess less pronounced type-specific determinants (Stephenson et al., 1974). The immunologic relatedness of the major structural proteins of type-C virus isolates of diverse species of origin appears to be reflected in their primary structure. Partial amino acid sequences have been determined for internal antigens of type-C virus isolates of a number of mammalian species including the cat, baboon, mouse, rat, gibbon ape, and woolly monkey (Oroszlan et al., 1975, 1977). The amino terminus of p30 proteins of each of these virus isolates have

TABLE I1 EXPRESSIONOF ANTIGEN RELATED TO THAT OF THE MAJOR STRUCTURAL OF THE P. cynocephalus BABOONVIRUSa

PROTEIN

(p30)

Viral p30 antigenic r e a c t i v i v

Family

Genus

Common name

Cercocebus

C . atys

Sooty white-crowned

Cynopithecus Papio

C . niger P. cynocephalus P. anubis P. hamadryas P. papio P. sphinx C . neglectus C . aethiops C . patas M. mulatta M . arctoides M. nernestrina M. irus P. pygmaeus P. troglodytes G. gorilla H. lar H . sapiens

mangabey Celebes (black) ape Yellow baboon Olive baboon Hamadryas baboon West African baboon Mandrill De Brazzas guenon Grivet monkey Patas monkey Rhesus macaque Stump-tailed macaque Pig-tailed macaque Crab-eating macaque Orangutan Chimpanzee Gorilla Gibbon ape Man

Cercopithecidae Cercopithecus Macaca

Pongo Pongidae < Z i , l a Homoinidae

Species

Hylobates Homo

Number tested

1 1 1 1 1 2 1

1 1 2 10 2 2 3 4

14 1 3 119

Number positive 0 1 1 1

0 0 0 1 0 0 10

Range (ng ~ 3 0 1 ~ cellular protein) <1 10 2 3 <1
2

40-60

2 3 0 0 0 0 0

25-70 30-80

<1 i l

From Stephenson and Aaronson (1977) with permission of Macmillan Journals Ltd. Partially purified liver extracts from primate tissues were tested in competition immunoassays for P. cynocephalus baboon virus p30 as previously described (Stephenson and Aaronson, 1977).

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

17

been shown to consist of Pro-Leu-Arg followed by a variable region (Oroszlan et al., 1975).A subsequent highly conserved region, eleven residues in length, beginning with residue number 11 was determined for each p30 examined with the exception of the major structural proteins of the gibbon ape and woolly monkey virus isolates in which this conserved sequence begins at residue 18 (Oroszlan et al., 1977). Of interest, the initial sequence of 15 residues of the major internal antigens of RD 114 and the baboon BAB8-K virus were found to be similar (Oroszlan et al., 1975). This observation is consistent with the results of immunologic (Sherr et al., 1975; Stephenson et al., 1976c; Stephenson and Aaronson, 1977; Barbacid et al., 1977) and hybridization (Benveniste et al., 1974) studies indicating a close relationship between RD114 virus of cats and endogenous viruses of Old World primates. The highly sensitive and broadly reactive nature of competition immunoassays for the major type-C viral structural protein, p30, have made such assays valuable for analysis of type-C viral antigen expression in virus-negative tissues. That such expression can occur in the absence of complete virus in tissues of a wide range of mammalian' species, including mouse (Huebner et al., 1970), cat (Stephenson and Aaronson, 1976, 1977), deer (Tronick et al., 1977), and Old World monkeys (Stephenson et a1 ., 1976c; Stephenson and Aaronson, 1977, Table 11) is well established. While the possibility of type-C viral p30 expression in human tissues has been raised (Sherr and Todaro, 1974, 1975; Strand and August, 1974b), the results of subsequent studies suggested that these reactivities may have been somewhat nonspecific (Stephenson and Aaronson, 1976,1977). In fact, in view of the extent of evolutionary divergence of endogenous primate type-C viral-coded antigens (Fig. 2), detection of p30 expression in higher ape and human tissues is likely to require the development of more sensitive and broadly reactive immunoassays than those presently available. While the significance of subviral p30 expression is not known, in recent studies Niman et al. (1977) have demonstrated elevated RD114 p30 expression to be common in lymphomatous tissues of cats. b . Hydrophobic Structural Protein. Molecular size analysis of proteins of mammalian type-C viruses has consistently revealed the presence of a low molecular weight gag gene-coded protein which chromatographs in the form of a 60,000-70,000 molecular weight complex in the presence of Triton X-100 (Barbacid et al., 1977).This phenomenon is consistent with reports indicating formation of specific complexes between hydrophobic membrane proteins and Triton X- 100 (Makino et al., 1973). The hydrophobic properties of these proteins

18

JOHN R. STEPHENSON ET AL.

100

4

3

2

1

0

RECIPROCAL OF ANTIGEN DILUTION (log ,J

FIG.2. Immunologic divergence of endbgenous type-C viral major structural proteins (p30) expressed in tissues of representative species of the primate subfamily, Cercopithecinae. Liver extracts partially purified b y ion exchange chromatography and gel filtration were tested in competition immunoassay using antiserum to P. cynocephalus virus to precipitate 1251-labeledP. cynocephalus viral p30. Purified extracts tested were from: P. cynocephalus (O), M . mulatta (a),C . niger ( A ) and C . neglectus (V). [From Stephenson and Aaronson (1977) with permission of Macmillan Journals Ltd.]

have been confirmed by affinity chromatography using phenyl sepharose, a hydrophobic resin consisting of phenyl groups covalently linked to Sepharose 4-B (Stephenson et al., 1977b). In the case of murine type-C viruses such as R-MuLV, the most hydrophobic protein has been found to be of about 15,000 molecular weight, while the analogous proteins of the RD114 cat virus as well as endogenous viruses of baboons exhibit molecular weights of about 12,000 (Barbacid et al., 1977). The hydrophobic nature of these proteins is consistent with earlier reports of their tendency to aggregate in the absence of nonionic detergents (Strand et alq,,, 1974; Schafer et al., 1975).Although these hydrophobic proteins form specific high molecular weight complexes under nondenaturing conditions, they nonetheless chromato-

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

19

graph at their expected molecular weights, when subjected to molecular size analysis under denaturing conditions (Fleissner, 1971; Barbacid et al., 1977). Interestingly, the isoelectric points of the hydrophobic protein of all t y p e 4 virus isolates examined to date have been found to approximate closely the values obtained for the major group-specific antigens of the same viruses (Stephenson et al., 197713; Fig. 3). Although the primary structure of R-MuLV p15 has not yet been determined, a partial sequence of a 19,000 molecular weight protein of a prototype avian type-C virus, Pr RSV-C, exhibiting similar properties and mapping at an analogous gag gene map position (Reynolds et al., 1977b) may be inferred from knowledge of the partial sequence of the viral RNA. The polynucleotide sequence of the 5' end of' the genomic RNA of this virus has been independently determined in two laboratories (Haseltine et al., 1977; Shine et al., 1977), and, on the basis

-

12

-

10

-8

- 6

- 4

- 2

FRACTION NUMBER

FIG.3. Analysis of type-C virus gag gene-coded structural proteins of aprototype RNA tumor virus, FeLV, by preparative isoelectric focusing. Individual column fractions were tested in competition immunoassays for FeLV p12 (A), p15 (B), p30 (C), and p10 (D). [From Khan and Stephenson (1977)with permission of American Society for Microbiology .I

20

JOHN R. STEPHENSON ET AL.

of the results obtained, the sequence of the amino terminal region of p19 has been predicted as NHzTMet-Lys-Gln-Lys-Ala-Ser-C0OH. Moreover, the amino-terminal residue of Pr RSV-C p19 and the p15 of several murine type-C viruses has been found to be blocked (Herman e t al., 1975;Oroszlan et at., 1978).In the case of Pr RSV-C, the blocked residue of p19 has been tentatively identified as a modified methionine. The blocked amino termious for R-MuLV is probably different from Pr RSV-C in that R-MqLV p15 apparently lacks methionine residues (Van Zaane et al., 1975, Arlinghaus et al., 1976). However, the blocked R-MuLV amino terminus could result from acetylation or other modifications of an amino terminal residue following cleavage. In this regard it should b e noted that in addition to the wellcharacterized 65,000 molecular weight R-MuLV gag gene-coded precursor (Stephenson et al., 197513; Van Zaane et al., 1975; Jamjoom et al., 1975), inhibition of precursor processing by use of the arginine analog canavanine results in accumulation of a somewhat larger gag gene-coded precursor (Van Zaane et al., 1976). Conceivably a methionine and perhaps a few additional residues are cleaved from the amino terminus of the primary gag gene translational product resulting in a 15,000 molecular weight protein that lacks methionine. The 15,000 molecular weight gag gene proteins of murine type-C viruses were shown by Strand et al., (197413) to possess broadly reactive interspecies antigenic determinants. Subsequent characterization of the hydrophobic 12,000 molecular weight viral proteins of RD 114, WCV- 1 and severaI endogenous baboon viruses revealed these proteins to share immunologic cross-reactivity with murine type-C viral p15 (Barbacid et al., 1977; Stephenson et al. 1977b). These findings are consistent with the possibility that the hydrophobic, methioninefree structural proteins of diverse mammalian type-C viruses represent functionally analogous proteins of their respective viruses. While little or no information is currently available concerning the function of these proteins, the fact that the mouse type-C viral p15 proteins are apparently found only at very low concentration in viral cores (Bolognesi et al., 1973) suggests a possible role within either the viral or celliilar membrane. Virus-specific cytotoxic antibodies against cell surface antigens have been detected in mouse sera (Old et al., 1964; Boyse, 1973; Lilly and Steeves, 1974). Moreover, a protein with the immunologic characteristics of R-MuLV p15 has been localized on the surface of leukemia cells of Rauscher virus-infected BALB/c mice and CC57BR mouse lymphosarcoma cells by immunofluorescence and im-

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

21

munoelectron microscopic analysis (Lejneva et al., 1976).In this same study, however, it was not possible to detect p15 on the surface of intact virions. These results suggest that p15 either is not on the virion membrane or that it is covered by the major envelope glycoprotein and therefore not accessible to react with antibody. A complete understanding of the architecture of the virion awaits further study. c . Type-Specific RNA-Binding Phosphoprotein. The most extensively characterized of the type-C viral gag gene-coded proteins include the highly acidic, immunologically type-speci fic phosphoproteins. In the case of viral isolates of mouse, pig, woolly monkey, and gibbon ape origin as well as the infectious feline virus, FeLV, proteins of this nature are of about 12,000 (p12) molecular weight. In contrast, analogous proteins of endogenous type-C viruses of the RD114baboon related group have molecular weights of about 15,000 (p 15) (Barbacid et al., 1977; Stephenson et al., 1977b).The isoelectric points of these proteins have been shown to be in the 4.2 to 5.4 range, establishing these to be the most highly acidic of type-C viral gag genecoded structural proteins. Moreover, structural proteins of this class are highly phosphorylated (Pal et al., 1975). The major phosphoamino acid of p12s of murine type-C viruses, as well as of FeLV, is phosphoserine. In contrast, the gibbon ape leukemia virus p12 was found to contain both phosphoserine and phosphothreonine, while the p15 proteins of RD114 and a prototype endogenous baboon type-C virus, BKD, contained phosphothreonine as their major phosphoamino acids (Pal and Roy-Burman, 1975). A unique property of the 12,000-15,000 molecular weight gag genecoded phosphoproteins is their ability to bind specifically RNA isolated from the same virus, but not to RNAs of heterologous type-C viruses (Sen et aE., 1976; Sen and Todaro, 1977). In such experiments, the virion phosphoproteins were found to bind both the 70 S genomic RNA as well as the component 35 S subunits in the form of complexes that are stabilized b y formaldehyde cross-linking. From such studies, it has been estimated that fewer than 15 molecules of p12 bind each RNA molecule. The specificity of this interaction is indicated by the ability of unlabeled p12, but not p30, to compete with 1251-labeledp12 for binding sites on the homologous viral RNA. The association of virion phosphoproteins with 70 S genomic RNA in intact virions has been independently demonstrated by ultraviolet irradiation of virion particles to cross-link the RNA-protein complex and subsequent electrophoretic analysis of the bound protein (Sen and Todaro, 1977).For type-C viruses of murine origin, the phosphoprotein found in associa-

22

JOHN R. STEPHENSON ET AL.

tion with the RNA genome by this means was p12 while a 15,00016,000 molecular weight phosphoprotein was found in association with the genomic RNA in the case of RD114. These findings are consistent with the contention that the murine type-C viral p12s and the p15s of RD 114 and endogenous primate viruses represent functionally analogous proteins. Further, analysis of a prototype mouse type-C virus isolate, R-MuLV, has indicated the extent of p12 phosphorylation to be highly variable (Pal et aZ., 1975)and to be a major factor influencing its RNA binding capacity (Sen et d., 1977). Only those R-MuLV molecules with minimal amounts of covalently linked phosphate were found to bind viral RNA raising the possibility that phosphorylation of RNA binding proteins may have important implications for viral regulatory hnctions. Immunologic analysis of g a g gene-coded phosphoproteins by competition immunoassay has demonstrated these proteins to b e highly type-specific (Stephenson et al., 1974; Tronick et aZ., 197413).Application of such assays to studies of diverse type-C virus isolates has been of value in discriminating and identifying closely related viruses. For instance, the existence of three immunologically distinct classes of endogenous type-C viruses of mouse cells were originally identified on the basis of the immunologic type-specificity of their p12 structural proteins (Stephenson et aZ., 1975a). The 12,000 molecular weight structural proteins of infectious type-C virus isolates of primates such as the woolly monkey and gibbon ape isolates are also highly typespecific and assays for such proteins provide a means of discriminating these closely related viruses (Tronick et al., 197413, 1975).While FeLV p 12 exhibits type-specific antigenic determinants, it also shares minor determinants in common with murine type-C viruses further indicating a high degree of relatedness between FeLV and endogenous type-C viruses of mouse cells (Khan and Stephenson, 1977). In contrast to the viruses described above, the most highly typespecific of the gag gene-coded structural proteins of endogenous type-C virus isolates of baboon origin are of about 15,000 molecular The RD114 virus (McAllister et al., weight (Stephenson et al., 1976~). 1972) as well as a related isolate of the European wild cat F. syluestris (Lieber et al., 1975a) also contain highly type-specific 15,000 molecular weight proteins (Stephenson et d., 1977b). Immunological assays for the p15 proteins isolated from P. cynocephalus baboon and RD114 viruses readily discriminate between viruses of cat or primate . addition these assays are suffiorigin (Stephenson et al., 1 9 7 6 ~ )In ciently type-specific to permit discrimination even between endogenous type-C viral isolates of different species of baboons such as P.

FWA TUMOR VIRUS TRANSLATIONAL PRODUCTS

23

cynocephalus, P. hamadryas, and P. anubis (Stephenson et al., 1976c; Stephenson and Aaronson, 1977). The ability to discriminate highly related type-C virus isolates of primates has had important application in the evaluation of type-C viruses of putative human origin. For instance, by this means, HL-23 a potential human type-C virus, isolated from a patient with acute myelogenous leukemia (Gallagher et al., 1975) was shown to consist of a mixture of two viruses, one indistinguishable from the woolly monkey isolate and the second indistinguishable from the endogenous P. cynocephalus baboon virus (Okabe et al., 1976). d . Basic Ribonucleoprotein. In addition to the major internal antigen and two lower molecular weight gag gene-coded proteins described above, a highly basic structural protein of about 10,000 molecular weight (p10) has been identified (Nowinski et al., 1972). This protein has been reported to be associated with the viral RNA in the form of a ribonucleoprotein complex within the mature virion (Fleissner and Tress, 1973; Bolognesi et al., 1973). On the basis of amino acid composition, murine type-C viral p10 has been shown to possess a high content of arginine residues (Fleissner and Tress, 1973) indicating it to be a relatively basic protein. The basic nature of p10 has been confirmed more recently b y preparative isoelectric focusing. By this means, the 10,000 molecular weight proteins of a broad range of mammalian type-C viruses were shown to exhibit isoelectric points in the 9.1 to 11.6 range (Fig. 4). As a result of its highly basic nature p10 has a high affinity for viral RNA as well as for single-stranded DNA (Davis et al., 1976). Though this interaction is relatively nonspecific with regard to source of RNA or DNA, it has been suggested that such binding may have a role in prevention of complementary strand formation by the viral reverse transcriptase thereby increasing the pool of single-stranded genomic RNA for viral maturation (Davis et al., 1976). The highly basic 10,000 molecular weight structural proteins of a number of diverse mammalian type-C viruses have been isolated and subjected to extensive immunologic. and biochemical analysis (Barbacid et al., 197613,1977; Stephenson et al., 197%). Early reports by Parks et al. (1974b, 1975) suggested the p10 and p12 proteins of mouse type-C viruses may be immunologically cross-reactive, thus raising the possibility of a precursor-product relationship between the two proteins. However, later studies using more extensive purification methods conclusively demonstrated that while both proteins were virus-coded they represented immunologically and biochemically distinct entities (Barbacid et al., 1976b). The plOs isolated from mouse RNA tumor viruses have been shown

24

JOHN R. STEPHENSON ET AL.

to possess strong group-specific antigenic determinants. Thus mouse type-C viruses are indistinguishable in competition immunoassays for p10 using any of a number of antisera to mouse type-C viruses for precipitation of either 1251-labeledRauscher or Moloney-MuLV p 10 (Barbacid et al., 1977). Interspecies antigenic determinants of the p10 polypeptide have also been demonstrated in studies using 12sI-labeled mouse and woolly monkey type-C viral antigens (Barbacid et al., 1976b) and more recently p10 isolated from the endogenous cat virus, RD114 (Barbacid et al., 1977).When compared with the properties of the major structural protein of the mammalian type-C viruses, p10 has been shown to possess equally broad immunologic cross-reactivity. Based on these findings, as well as its amenability to purification from tissue extracts, competition immunoassays for p10 provide a potentially valuable approach for analysis of viral gene expression in tissues of mammalian species from which infectious type-C virus has not been isolated to date. e . Glycosylated gag Gene-coded Polyprotein. Recently, evidence has been obtained indicating the expression of an uncleaved gag gene precursor in a glycosylated form at the surface of leukemia mouse cells (Snyder et al., 1977; Ledbetter et al., 1977). This glycosylated core polyprotein was shown to exist in at least two serologically distinguishable forms including a 95,000 molecular weight polyprotein which contains p30, p15, p12, and p10 and an 85,000 molecular weight precursor containing p30, p15, p12, but not pl0. These glycosylatedgag gene precursors apparently correspond to the Gross cell surface antigen (GCSA) associated with endogenous Gross-type murine leukemia virus expression in mouse cells (Snyder et al., 1977; Ledbetter et al., 1977). The surface localization of these proteins may be of considerable biological importance in that they may provide targets for host immune responses to leukemic cells. The possibility that the Moloney leukemia virus-determined cell surface antigen (MCSA) (Siegertet al., 1977)may alsocorrespond to a glycosylated gag gene precursor must also be considered.

C. SRC GENE-CODEDTRANSFORMING PROTEIN(S) Type-C viral genetic sequences associated with sarcomagenic transformation have been designated src (Baltimore, 1974). These sequences are presumably of cellular origin and are deleted or absent from the genomes of transformation-defective type-C viruses ( h i et al., 1973; Scolnick et al., 1974; Stehelin et al., 1976).In contrast to the extensive characterization of the gag, pol, and enu gene translational products, studies of src gene-coded proteins have been limited. In

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

25

fact, much of the evidence for a src gene translational product has been derived by indirect approaches. For instance, existence of such a protein was initially based on the isolation of temperature-sensitive mutants of avian (Biquard and Vigier, 1970; Martin, 1970; Friis et d., 1971; Kawai and Hanafusa, 1971; Bader and Brown, 1971) and mammalian (Scolnick et al., 1972b) type-C sarcoma viruses which reversibly lose expression of the transformed phenotype at their respective nonpermissive temperatures. The possibility that the src gene may code for more than one protein was suggested by results of early studies reporting complementation between individual temperature-sensitive avian sarcoma virus isolates (Wyke, 1973). However, this apparent complementation was later shown to be due to intracistronic recombination thus favoring the possibility of a single src gene product’(Wyke et al., 1974). Independent evidence arguing for the involvement of a single src gene product in transformation was derived from the inability to demonstrate complementation between a number of mammalian sarcoma virus isolates exhibiting nonconditional transformation-specific lesions (Green: berger et al., 1974a). Although these findings argue for a single complementation group, the possibility of two or more src gene-coded proteins sharing a single initiation site for translation cannot be ruled out. More direct evidence arguing for the existence of a src-coded protein has been derived by use of cell-free in vitro translation systems (Parks and Scolnick, 1977; Purchio et al., 1977). In studies by Parks and Scolnick, RNA of the Harvey strain of murine sarcoma virus, partially purified by density gradient centrifugation, was translated in vitro, and shown to code for a protein of about 21,000 molecular weight. This protein was distinct from translational products obtained in parallel experiments utilizing RNA of the helper type-C virus used to propagate Harvey sarcoma virus. Moreover, translation of the 21,000 molecular weight src-specific protein was initiated with [35S]formylmethionine from f~rmyl-[~~S]methionine-tRNA indicating it to represent a faithful translational pi-oduct of at least the N-terminal portion of a protein coded for by the sarcoma virus genome (Parks and Scolnick, 1977). However, the possibility that the sarcoma virusspecific protein detected in these studies may be coded for by acquired cellular sequences other than src cannot be ruled out. In other studies, Purchio et al. (1977) have demonstrated the synthesis of a 60,000 molecular weight polypeptide in a reticulocyte cell-free translation system using subgenomic regions of the avian sarcoma virus (ASV) genome as a mRNA. This protein appears analogous to a 60,000 molecular weight transformation-specific protein demonstrated

26

JOHN R. STEPHENSON ET AL.

in ASV-transformed chicken cells and ASV-induced hamster tumor cells b y immunoprecipitation of radiolabeled cell extracts with serum from tumor-bearing rabbits (Brugge and Erikson, 1977). A second approach to the characterization of the src gene product has involved efforts to identify antigens unique to sarcoma virus transformed cells b y in uiuo immunologic techniques. In initial studies along these lines, murine sarcoma virus-transformed nonproducer mouse cells were shown to exhibit weak transfonnationspecific antigenic determinants as measured b y immunoelectronmicroscopy (Aoki et al., 1973).However, it was not resolved as to whether these antigens represented src gene-coded proteins or whether they reflected expression of transformation-associated cellular proteins. Moreover, these antigens were not sufficiently immunogenic in the mouse to be recognized by transplantation rejection techniques (Stephenson and Aaronson, 1972a; Strouk et al., 1972; Greenberger et a / . , 1974b). Attempts to detect and study sarcoma virus-specific antigens have also been undertaken in the feline system. A major advantage of this system results from the early identification of a feline oncornavirusassociated cell membrane antigen designated FOCMA (Essex et al., 1977).Expression of FOCMA is a major factor in determing whether or not an animal successfully resists tumor development following infection with either feline sarcoma virus (Essex et al., 1971; Essex, 1971)or feline leukemia virus (Essex, 1976). FOCMA has been shown to be distinct from the major FeLV structural proteins (Stephenson et a1., 1977a; Essex et al., 1977).While nontransformed fibroblasts of diverse mammalian species become FOCMA positive upon infection with feline sarcoma virus, the same cells remain FOCMA negative when infected with feline leukemia virus alone (Essex et al., 1977). Moreover, mink cells nonproductively transformed by feline sarcoma virus express FOCMA (Sliski et al., 1977) in the absence of detectable levels of known FeLV translational products except for the two amino terminal gag gene proteins, p12 and p15, (Khan and Stephenson, 1977).The results of recent studies have shown that in both mink and rat cell lines nonproductively tranformed by FeSV, FOCMA, p15, and p12 are initially synthesized in the form of an 80,000-100,000 molecular weight precursor which upon posttranslational cleavage gives rise to a 65,000 molecular weight component which contains FOCMA and a 25,000 molecular weight component containing p 15 and p12 (Stephenson et al., 1977~). Feline lymphoma cells, including those from several tumors which lacked detectable levels of FeLV structural protein expression, were shown to be FOCMA-positive.

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

27

These findings strongly suggest FOCMA to represent an FeSV-coded transformation specific protein and provide preliminary information regarding the position within the FeSV genome coding for its synthesis (Stephenson et al., 1977~). An independent approach to identification of transformation-specific proteins has involved studies of low molecular weight polypeptide growth factors. Such factors have been a topic of recent interest because of their ability to bind cell surface receptors and modify cell growth (Cohen and Taylor, 1974; Carpenter et al., 1975).Recent data from Todaro’s laboratory suggests that one of these factors, designated epidermal growth factor (EGF), may be functionally related to the mammalian type-C viral STC gene product (Todaro et al., 1976). This conclusion has been arrived at based on the inability of cells transformed by murine and feline sarcoma viruses to bind EGF, whereas the cells transformed b y pol yoma, SV40, or chronically infected with nontransforming RNA tumor viruses exhibit normal levels of E G F binding capacity (Todaro et al., 1976). The impairment to E G F binding appears to b e due to production of excess levels of an EGF-like protein which occupies all available binding sites. While the binding of a second polypeptide growth factor, multiplication stimulating activity (MSA), to a distinct class of cell receptors is not reduced in murine and feline sarcoma virus transformed cells, two cell lines established from human fibrosarcomas were found to have lost the ability specifically to bind MSA (Todaro et al., 1977). Whether growth factors such as E G F and MSA represent src gene-coded proteins or alternatively whether their synthesis reflects secondary influences of src gene expression is not resolved.

IV. Genetic Mapping of the Type-C RNA Tumor Virus Genome

A. LOCATIONOF gag, pol, AND enti WITHIN THE VIRAL GENOME While the emphasis of the present review pertains to type-C RNA viruses of mammalian origin, much of our current knowledge concerning the organization of the type-C viral genome has been derived fiom studies of avian RNA tumor viruses. Thus, studies leading to a determination of the relative positions of the gag, pol, env, and src genes within the avian type-C viral genome are reviewed below. Avian type-C RNA tumor virus mutants utilized in mapping the viral genome have been described in several recent reviews (Hanafusa, 1975; Vogt, 1977).These are of several types, including deletion

28

JOHN R. STEPHENSON ET AL.

mutants as well as conditional lethal mutants exhibiting temperaturesensitive (ts) lesions in both replication- and transformation-specific functions. Examples of deletion mutants include the Bryan high titer strain (BH) of Rous sarcoma virus (RSV) and the NY 8 mutant of the Schmidt-Ruppin strain (SR) of RSV, both of which contain deletions in the env gene (Hanafusa et al., 1963; Kawai and Hanafusa, 1973). LA335 and LA337 represents examples of replication-defective mutants of the Prague strain of RSV exhibiting ts lesions in pol (Mason et al., 1974; Verma et al., 1974). Additional mutants will be discussed as they pertain to the biochemical mapping of the avian RNA sarcoma virus genome. In order to utilize the above mutants effectively for mapping, biochemical methods were necessary to locate positions of the mutations within the viral genome physically. For this purpose, T, ribonuclease-resistant oligonucleotide mapping techniques have been used (Wang and Duesberg, 1974; Joho et al., 1975). Mapping of ribonuclease TI resistant oligonucleotides involves partial hydrolysis of the viral RNA at high pH, oligo (dT)-cellulose chromatography to separate out 3’ poly(A) containing fragments, and sizing of the 3’ poly(A) containing fragments. The TI ribonuclease-resistant oligonucleotides are then fractionated by two dimensional thin layer chromatography and electrophoresis (Brownlee and Sanger, 1969; Barrell, 1971; Beemon et al., 1974; Duesberg et al., 1974). Analysis of genomic RNA of transformation-defective avian sarcoma viruses have indicated the presence of deletions, corresponding to src, representing approximately 15% of viral genome (Duesberg and Vogt, 1970, 1973; Martin and Duesberg, 1972; Neiman et al., 1974). These deleted regions were shown to be represented b y several large oligonucleotides restricted to TI ribonuclease digests of transforming viral RNAs (Lai, et al., 1973).Application of oligonucleotide mapping procedures as described above made possible the localization of these src-specific oligonucleotides within the avian sarcoma virus genome at a position adjacent to the 3’ poly(A) end (Wang et al., 1975, 1976a,b; Joho et al., 1975). Using similar techniques, a constant region(c), common to both transforming and transformation-defective viruses, has been located between the 3’ poly(A) end of the genome and the src gene (Wang et al., 1975; P6rez-Bercoff and Billeter, 1976). The identification of the src region of the avian sarcoma virus genome, near but not at, the 3‘ poly (A) has been recently confirmed by heteroduplex mapping procedures (Junghans et al., 1977).Application of TI ribonuclease-resistant oligonucleotide mapping techniques to analysis of recombinant RNA genomes from viruses of different sub-

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

29

groups has led to the location of the enw gene at a position adjacent to al., 1976a; Joho et al., 1975). This order is further indicated by the location of deleted oligonucleotide sequences in the genome of the glycoprotein-deficient mutant, NY 8, located at a position 2800 to 5000 nucleotides from the poly(A) end of the genome (Wang et al., 1976a). Analysis of a series of genetic recombinants between parental viruses differing both in host range and in the presence or absence of a ts reverse transcriptase lesion has led to the demonstration of a highly conserved cluster of oligonucleotides apparently corresponding to pol and mapping between 6000 and 8000 nucleotides from the 3' poly(A) end of the genome (Duesberg et al., 19761 Wang et al., 1976c; Joho et al., 1976). Finally, B77 avian sarcoma virus mutant, LA334, blocked in virus assembly at the nonpermissive temperature (Hunter et al., 1976; Friis et al., 1976) has been shown to possess a ts lesion located in p27, one of the gag gene-coded proteins (Rohrschneider et al., 1976).The availability of this mutant made possible identification of TI ribonuclease-resistant oligonucleotides corresponding to p27 near the 5' end of the avian type-C viral genome (Wang et al., 1976c) and thus completed the tentative gene order as: 5' gag-pol-enw-src(c) poly(A) 3'. In contrast to the extensive analysis of the avian type-C viral genome, available information concerning the overall organization of the mammalian type-C viral genome is quite limited. Approaches utilized in attempting to establish the relative positions of gag, enw, pol, and src within the mammalian type-C viral genome have been very different and much less direct than those applied to studies of avian RNA tumor viruses. This is primarily due to the lack of wellcharacterized mammalian type-C viral mutants such as those available in the avian RNA tumor virus system. The results of immunoprecipitation studies suggesting the possible existence of a common pol and gag gene product precursor (Arlinghaus et al., 1976; Jamjoom et al., 1977) support the possibility that the genes coding for these proteins occupy adjacent positions within the viral genome. Attempts to translate the mammalian type-C viral genomic RNA in uitro have generally resulted in synthesis of only gag gene-coded proteins (Gielkens et al., 1976; Kerr et al., 1976) consistent with gag being located at the 5' terminus of the genome, Moreover, addition of specific suppressor tRNAs to in vitro systems result in translation of pol, although at much lower efficiency than gag (Philipson et al., 1978).It has thus been proposed that synthesis of pol generally results from occasional read through of a termination codon located at the 3' terminus of the gag gene (Jamjoom et aZ., 1977). This hypothesis, if correct, would not only account for the src (Wang et

30

JOHN R. STEPHENSON ET AL.

fact that pol is generally expressed at much lower eficiency than gag, but would also place pol adjacent to the 3' terminus of gag within the type-C viral genome. The position of enu would thus be adjacent, or at least closer to, the poly(A) sequences at the 3' terminus of the viral genome. On the basis of these observations, and by analogy to avian RNA tumor viruses, the sequence of the mammalian type-C viral genome can tentatively be assigned as 5'-gag-pol-env- 3'. In view of the fact that all mammalian sarcoma virus isolates described to date are replication defective, mapping of the position of src by methods such as those described for avian sarcoma viruses has not been possible. However, data pertaining to the location of src within the genome of replication-defective mammalian sarcoma viruses have been recently obtained and will be discussed in later sections of this chapter. B. INTRACISTRONIC MAPPING OF THE gag GENE OF PROTOTYPEMAMMALIAN TYPE-CVIRUS

A

The four major nonglycosylated type-C viral gag-coded proteins are initially synthesized in the form of a common precursor which subsequently undergoes post-translational cleavage (Van Zaane et al., 1975; Jamjoom et al., 1975; Stephenson et al., 1975b). Moreover, several conditional lethal replication mutants of R-MuLV characterized by late maturation blocks (Stephenson and Aaronson, 1973, 1974) have been shown to possess temperature-sensitive defects in posttranslation cleavage of this gag gene-coded precursor (Stephenson et al., 1975b). The primary cleavage block exhibited by two such mutants is early and leads to accumulation of the primary 65,000 molecular weight gag gene translational product in an uncleaved form (Barbacid et al., 1976a; R. K. Reynolds and Stephenson, 1977). Additional mutants are characterized by the accumulation in infected cells of an intermediate cleavage product of about 40,000-50,000 molecular weight containing p30, p12, and p10, in the absence of detectable p15 (R. K. Reynolds and Stephenson, 1977). On the basis of these results, the 40,000-50,000 molecular weight peak would appear to consist either of a precursor Containing p30, p12, and p10, or two separate precursors, one containihg p30 and p10 and a second containing p30 and p12. Either of these possibilities would argue for p15 being located at a terminal position within the primary gag gene product. Molecular size analysis of viral antigen expression in each of several ts mutant-infected cultures has led to the demonstration of a promi-

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

31

nent peak at a molecular weight of about 25,000 containing reactivity in competition immunoassays both for p12 and p15 (Barbacid et al., 1976a; R. K. Reynolds and Stephenson, 1977).Characterization of this intermediate cleavage product by preparative isoelectric focusing has shown the two antigenic reactivities to cochromatograph at a position corresponding to an isoelectric point of 5.5, and thus intermediate between the values of 4.5 and 7.6 obtained for R-MuLV p12 and p15, respectively (R. K. Reynolds and Stephenson, 1977). These findings establish that p12 and p15 must occupy adjacent positions within the gag gene primary translational product. To determine the relative positions of p30 and p10, cell extracts prepared from ts 29 infected cultures were subjected to partial purification on an anti-R-MuLV p10 affinity column. The nonbound material was subsequently analyzed by agarose gel filtration under denaturing conditions. By means of this affinity chromatography step, it was possible to eliminate specifically cleavage intermediates containing p 10 and thus enrich for a 42,000 molecular weight p30-pl2 precursor at a molecular weight of around 40,000. Thus, on the basis of these findings, the sequence of the R-MuLV gag gene product was deduced as p15-p12-p30-p10 (R. K. Reynolds and Stephenson, 197713). The fact that of the four gag gene-coded structural proteins of R-MuLV only p15 exhibits a blocked amino terminus (Oroszlan et al., 1978) suggests its location at the amino end of the gag gene product and thus defines the polarity of the R-MuLV gag gene.

c. IDENTIFICATION

OF FUNCTIONALLY ANALOGOUS gag GENE-CODEDPROTEINS OF MAMMALIAN TYPE-C VIRUS ISOLATES O F DIVERSE ORIGIN

Evidence arguing for the possibility that all type-C RNA viruses share a common progenitor is rapidly accumulating. Initial indications supporting such a possibility were based on similarities in morphology and protein composition of type-C virus isolates of diverse mammalian species. More direct evidence for a common progenitor has been derived from studies demonstrating biochemically analogous structural proteins of type-C virus isolates of different species of origin to possess immunologically cross-reactive antigenic determinants. This was initially demonstrated in the case of the major structural protein, p30 (Gilden et al., 1971; Parks and Scolnick, 1972), and the envelope glycoprotein, gp70 (Strand and August, 1973, 1974a; Hino et aE., 1975). More recently, the 12,000-15,000 molecular weight hydrophobic pro-

32

JOHN R. STEPHENSON E T AL.

teins of widely diverse type-C virus isolates (Strand et al., 1974; Barbacid et al., 1977) as well as the lowest molecular weight gag gene product, p10 (Barbacid et al., 1976b), have also been shown to share cross-reactive determinants, The results of primary structural analysis of the 27,000-30,000 molecular weight structural proteins of representative type-C virus isolates provide direct molecular evidence that the genomic sequences coding for the major structural proteins of diverse mammalian type-C RNA viruses are evolutionarily related (Oroszlan

et al., 1975). In view of the above considerations and assuming that no major rearrangements of type-C viral genomes have occurred subsequent to their evolutionary divergence, it has been possible to identify analogous gag gene-coded proteins of different mammalian type-<=viruses, and thus, on the basis of immunologic as well as biochemical criteria, to predict the map positions of such proteins within the genomes of their respective viruses (Stephenson et al., 1977b). A prediction of the internal arrangement of the gag genes of a broadly representative group of mammalian type-C virus isolates derived on the basis of such considerations is summarized in Fig. 4. For instance, by analogy to the R-MuLV gag gene sequence, the amino-terminal gag gene protein appears to be a slightly basic, hydrophobic protein with broadly reactive interspecies antigenic determinants. In the case of the endogenous baboon type-C virus isolates, as well as closely related endogen-

Artiodactyls S. scrofo : CCL-33

I

6.1 IP151

Primates P cynocephalus : M7

-

L spp. : SSAV-1

I I

7.5 (PlZI

6.8 (PIS)

@4.!-Ta IPl21

B:49sg lP151

E4.6-24 IP121

6.7

I 10.1 1

(Pa)

IP101

7.0 1~271

8.8 IPW

1

9.9

1

IP10)

1

9.9 IP101

FIG. 4. Proposed arrangement of viral proteins within representative mammalian type-C viral gag gene-coded precursor polypeptides. The indicated values within tne bar graphs represent the isoelectric points of the individual proteins, while the values in the brackets represent the molecular weights as determined by agarose gel filtration in the presence of 6 M GuHCl. The lengths of bars for the gag gene proteins are drawn in proportion to their molecular weights.

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

33

ous viruses of the domestic cat and European wildcat, this protein has a molecular weight of 12,000, while the amino-terminal gag gene proteins of other mammalian type-C viruses examined to date have molecular weights of about 15,000. The protein adjacent to the amino-terminal gag gene-coded protein is acidic and possesses highly type-specific antigenic determinants, Because of the lack of broadly reactive interspecies antigenic determinants, the identification of proteins of different viruses located at this position within the gag gene has been somewhat more difficult. However, among closely related viruses of this group, proteins are immunologically cross-reactive. For instance, the p12s of each of several type-C viruses of mouse origin tested to date have been shown to share group-specific antigenic determinants (Stephenson et aZ., 1975a) and, in addition, to possess less pronounced interspecies antigenic determinants in common with feline leukemia virus (Khan and Stephenson, 1977). Moreover, primary structure analysis of the 12,000 molecular weight proteins of several mouse type-C viruses have indicated the presence of conserved regions of amino acid sequence (Oroszlan et a,?., 1978). The proteins of the baboon and related endogenous feline viruses located at this position within the gag genecoded precursor have molecular weights of about 15,000. Analogy between the p12 proteins of the mouse and woolly monkey viruses and the p15 protein of the baboon and RD114 virus are based upon their similar ionic and hydrophobic properties. Moreover these proteins exhibit highly specific RNA-binding properties (Sen et d.,1976; Sen and Todaro, 1977) and represent the major phosphoproteins of their respective viruses (Pal et al., 1975; Pal and Roy-Burman, 1975). In an attempt to account for the reciprocal relationship between the molecular weights of the 12,000 and 15,000 molecular weight aminoterminal proteins among various type-C virus isolates, a shift of about 30 amino acids in position of the posttranslational cleavage site separating the two proteins within the primary gag gene product has been proposed (Barbacid et al., 1977; Stephenson et al., 1977b). The protein occupying the third position from the amino-terminal end of the gag gene product is the 27,000-30,000 molecular weight, major virion structural protein. Finally, the carboxy-terminal gag gene product is highly basic and of about 10,000 molecular weight. The analogies between these two proteins among type-C virus isolates has been established based on their broadly reactive interspecies antigenic determinants. Moreover, as discussed above, in the case of the 27,000-30,000 molecular weight major structural protein, this has been confirmed b y direct amino acid sequence analysis. On the basis of the results summarized in Fig. 4, the isoelectric

34

JOHN R. STEPHENSON ET AL.

points of type-C virus structural proteins mapping at corresponding positions within their respective viral genomes appear highly conserved (Stephenson et al., 197713). In those instances in which the isoelectric point of any one of the gag gene proteins deviates somewhat from that of the corresponding proteins of other viruses, this invariably is accompanied by changes of similar magnitude in all four gag-coded proteins. For instance, the isoelectric points of the proteins occupying the third position from the amino terminus of the gag gene product in each case closely approximate the values determined for the amino-terminal proteins of the same virus, even though both may vary to some extent from one virus to the next. The conservation of the ionic properties of functionally analogous proteins of type-C viruses (Stephenson et al., 1977b), despite extensive variation in primary structure (Oroszlan et al., 1975), may reflect mutually compensating evolutionary changes. Such a situation has been postulated to explain conservation of charge of cellular proteins such as hemoglobin (Drysdale et al., 1971) and cytochrome c (Barlow and Margoliash, 1966).In the case of type-C viruses maintenance of a favorable balance of ionic charge may be necessary for functions of these proteins as structural components of the virion. As discussed in preceding sections, type-C viral structural proteins are presently designated according to their molecular weight as determined by agarose gel filtration under denaturing conditions (August et al., 1974). I n view of the considerations discussed above, a much preferable nomenclature system would appear to be one which also takes into account their immunologic and biochemical properties. For instance, as summarized in Table 111, intracistronic regions of the gag gene could be designated gag-a, gag-b, gag-c, and gag-d and individual viral proteins identified on the basis of the positions within gag coding for their synthesis. By adaptation of this nomenclature system, confusion such as that which frequently arises in relying on molecular weight for protein identification would be avoided. While the present review is primarily focused upon an analysis of translational products of mammalian type-C viruses, it is of interest to consider related studies of type-C viruses of avian origin. For example, the translational products of the avian type-C viral gag gene have been shown to include proteins of 27,000 (p27), 19,000 (p19), 15,000 (p15), and 12,000 (p12) molecular weight. The results of pactamycin mapping (Eisenman et al., 1974; Vogt et al., 1975) and intermediate cleavage product analysis (Reynolds et al., 1977a) have provided evidence indicating the arrangement of these proteins within the gag genecoded primary translational product as NH2-p19-p 12-p27-p15-COOH. Assuming this tentative sequence to be correct, and considering the

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

PROPERTIES OF

35

TABLE I11 KNOWN MAMMALIANTYPE-CVIRAL TRANSLATIONAL PRODUCTS

Viral gene

Protein product

Molecular weighta

Isoelectric point

gag

gag-a

15,000-[ 12,0001

6.1-8.3

gag-b

12,000-[ 15,OOOl

4.1-5.9

gag-c

26,000-30,000

6.2-8.0

gag-d

10,000

9.1-11.6

pol

RDDP

70,000

ND

enu

gp70

70,000

4.15.3

p15E

15,000

ND

Other properties (primary antigen determinants) Hydrophobic, aggregation in absence of detergent (type, group, interspecies) Hydrophylic, major virion phosphoprotein, specifically binds viral RNA (type, group) Major internal antigen (group, interspecies) Ribonucleoprotein (group, interspecies) RNA-dependent DNApolymerase activity (group, interspecies) Viral envelope glycoprotein (type, group, interspecies) Nonglycosylated envelope protein, aggregation even in presence of 6 M guanidine hydrochloride (group, interspecies)

Molecular weights were determined by agarose gel filtration in the presence of 6 M GuHCl (Fleissner, 1971). Values in brackets indicate molecular weights for structural proteins of the RD114-baboon virus group (Barbacid et al., 1977; Stephenson et al., 197713).

biochemical properties of avian type-C viral proteins, it would appear that avian and mammalian type-C viral proteins with similar properties occupy analogous positions within the genomes of their respective viruses (Reynolds et al., 1977a). These findings provide evidence supporting the possibility that even type-C viruses as diverse as those of avian and mammalian .origin may have been derived from a common progenitor. If so, it will be of interest to determine whether divergence of these viral genes occurred prior to or subsequent to the evolutionary divergence of their hosts. V. Relatedness of Structural Proteins Coded for by Leukemia and Sarcoma Type-C Viral Genomes

Currently available information regarding the biochemical and immunologic characterization of mammalian type-C viral proteins has

36

JOHN R. STEPHENSON ET AL.

primarily been derived from studies of leukemia viruses. In addition, however, in certain instances, cells nonproductively transformed by replication-defective sarcoma viruses also express type-C viral struetural antigens. This was originally indicated by the demonstration that a clonal cell line nonproductively transformed by a murine sarcoma virus designated Moloney-S+L- (Bassin et al., 1971)reacted positively by complement fixation for the murine leukemia virus “groupspecific” antigen (Fischinger et al., 1972; Aaronson et al., 1972). Further analysis of such cells b y much more sensitive and specific techniques has demonstrated the expression of three gag gene-coded proteins, p15, p12, and p30 in the absence of detectable levels of either the carboxy-terminal gag gene protein, p10, or the major enw gene product, gp70 (Tronick et al., 1973; Barbacid et al., 197613). The stable association of the region of the viral genome coding for structural proteins with genetic sequences coding for malignant transformation has been demonstrated b y rescue of the S+L- genome and subsequent isolation and analysis of new nonproductive transformants (Peebles et al., 1976; Barbacid et al., 197613). These findings, in combination with the results of nucleic acid hybridization studies (Scolnick et al., 1973, 1975; Frankel and Fischinger, 1977) indicate that mammalian sarcoma viruses contain genetic sequences for at least certain leukemia virus functions and thus are consistent with the possibility that replication-defective sarcoma viruses arise as a consequence of recombinational events between the leukemia virus genome and host genetic sequences (Scolnick et al., 1973). An extension of the above analysis to additional mammalian sarcoma viruses revealed considerable heterogeneity with regard to the extent of gag gene expression. Moreover, such expression invariably occurs in a progressive manner from the amino to the carboxy terminus of the gag gene product (Table IV). For instance, two sarcoma virus isolates, one of mouse origin, designated Moloney sarcoma virus, and the second of rat origin, Kirsten sarcoma virus, fail to code for the expression of any of the four gag gene proteins (Barbacid et al., 1976a). In contrast, cells transformed by the replication-defective Friend spleen focus forming virus have been shown to express only the aminoterminal gag gene protein, p15 (Bernstein et al., 1977). Cells nonproductively transformed by either a natural sarcoma virus isolate of the BALBlc mouse (MAI-3206) (Barbacid et al., 1976a), the Abelson lymphosarcoma virus (Barbacid et al., 1976a), or the Theilen strain of feline sarcoma virus (Khan and Stephenson, 1977) have been shown to express p15 as well as the adjacent protein, p12. All of these findings taken together with the fact that S+L- nonproducer cells express p15,

TYPE-C VIRAL

TABLE IV EXPRESSIONIN MAMMALIAN SARCOMA VIRUS TRANSFORMED NONPRODUCER CELL h N E S OF DIVERSEORIGIN

ANTICEN

Type-C viral gene product expressiona gag gene-coded

Sarcoma virus origin Mouse

Virus isolates NP-Moloney-M SV Friend SFFV BALB-MSV Abelson Leukemia Virus S+L- Moloney-MSV

Rat Cat Woolly monkey

KiMSV FeSV

wsv

Transformed nonproducer cell line M-NIH M-NRK NP9 MAI-3206 ANN-1 S+L- N R K HuACI, K-NRK FeSV-CCL 64 wsv c 1 2 wsv c 1 9 wsv c1 11

gag-a (P15)

gag-b (~12)

gag-c (~30)

gag-d (~10)

enu gene-coded (gp70)

-

+ + + + -

+ NT NT NT

+ + +

a Cell extracts were prepared and tested for type-C viral antigen expression by competition immunoassay according to previously described methods (Barbacid et al., 1976a).

38

JOHN R. STEPHENSON ET AL.

p12, and p30 argue that expression of the leukemia gag gene sequences in sarcoma virus nonproducer cells occurs progressively from the 5’ end of the viral genome. This possibility is further indicated from analysis of a series of clonal cell lines nonproductively transformed by the woolly monkey sarcoma virus (Aaronson et al.,

1975). Studies of intermediate cleavage products of the precursor proteins coded for by the portion of the gag gene expressed in various mammalian sarcoma virus nonproducer cells provide further information regarding the internal arrangement of the viral gene. For instance, analysis of either human or mouse cells containing the S+LMoloney-MSV genome revealed the presence of a 58,000-62,OOO molecular weight precursor containing p15, p12, p30, as well as a second precursor containing p30 and p12 in the absence of p15 or p10. Moreover, several nonproducer clones were found to express a 25,000 molecular weight intermediate cleavage product containing p 15 and p 12 (Barbacid et al., 1976a; Khan and Stephenson, 1977). These findings establish the order of the gag gene sequence within mammalian transforming viruses to be p 15-p 12-p30-p10, and thus to be identical to that in the original leukemia helper virus. Thus, it appears that the initial sequence of the leukemia virus gag gene remains unaltered during recombinational events leading to sarcoma virus formation, with deletions or translational blocks of varying extent occurring at its 3‘ terminus. As discussed in preceding sections, current evidence favors the possibility that the gag genes of mammalian, as well as avian, RNA tumor viruses occupy positions in close proximity to the 5’ end of their respective viral genome. However, the results of molecular hybridization studies indicate that the extent of leukemia virus-specific nucleic acid sequence homology within prototype mammalian sarcoma virus genomes is much greater than that which can be accounted for by only a limited region of the gag gene (Scolnick et al., 1973,1975; Frankel et al., 1976; Frankel and Fischinger, 1977). In order to reconcile these observations, the possibility that sarcoma virus formation may involve double crossover events between the leukemia helper virus and host cell genetic sequences, with host sequences acquired by recombination substituting for a portion of the 3’ end of the gag gene, has been proposed (R. K. Reynolds and Stephenson, 1977). Analysis of the extent of translation of the gag gene in cells nonproductively transformed by such viruses may thus provide a means of mapping the exact position of the recombinational site closest to the. 5’ terminus of the viral genome. Thus, if the extent of sequence homology between

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

39

the sarcoma virus genome and its leukemia helper virus is as extensive as has been reported (Scolnick et al., 1973, 1975) the major portion of leukemia virus sequences must be located near the 3’ end of the sarcoma virus genome. Such a possibility could account for reports by August and his collaborators of cell clones. nonproductively transformed by a mammalian sarcoma virus in which gp70 expression was observed in the absence of detectable p30 (Bilello et al., 1974, 1977).The involvement of a double crossover mechanism for sarcoma virus formation is further supported by studies of avian sarcoma virruses demonstrating the presence of short regions of leukemia virusspecific genetic sequences located at a position between src and the poly(A) sequences at the 3’ terminus of the viral genome (Wang et al., 1975; Pirez-Bercoff and Billeter, 1976; Varmus et al., 1977). However, this model should only be considered tentative in view of the possibility that secondary deletions or translation blocks within the gag gene of replication-defective sarcoma viruses, independent of the recombinational event resulting in sarcoma virus formation could also be responsible for incomplete gag gene expression. Recently, Bister et al. (1977) have isolated and characterized quail cell lines nonproductively transformed by an avian transforming virus designated MC29. Moreover, they have demonstrated the expression in such cells of a 110,000 molecular weight polyprotein containing p27 and p 19 in the absence of p15. These findings, in combination with oligonucleotide fingerprint analysis (Duesberg et al., 1977) raise the possibility that a portion of this 110,000 molecular weight precursor may correspond to the MC29 transforming protein. In this respect, MC29 may be somewhat analogous to feline sarcoma virus. These findings further support the possibility that at least in some instances, the 5’ terminus of the recombinational event leading to sarcoma virus formation may occur within the region of the type-C viral genome corresponding to gag. VI. Type-B and Type-D Oncornavirus Structural Proteins

The classification of RNA tumor viruses into four major classes is based primarily on the morphologic criteria. Thus, type-B and type-D viruses have diagnostic morphological features which discriminate these two groups of viruses, both from each other as well as from type-C virus isolates. In addition, the RNA-dependent DNApolymerase of the type-B and type-D viruses prefer Mg2+ions for their activity, distinguishing them from type-C RNA tumor viruses which are characterized by a Mn2+preference (Abrell and Gallo, 1973; Howk et al., 1973; Scolnick et al., 1970).

40

JOHN R. STEPHENSON ET AL.

The prototype type-B oncornavirus, and the member of this group that has been studied in greatest detail, is the mouse mammary tumor virus (MMTV). While a major nonglycosylated MMTV structural protein of 24,000 molecular weight as well as four lower molecular weight nonglycosylated structural proteins have been reported (Dickson and Skehel, 1974; Sarkar and Dion, 1975; Schochetman and Schlom, 1976), detailed immunologic and biochemical characterization of these proteins has not been completed to date. The major glycoprotein of MMTV, gp52, has a molecular weight of about 52,000-55,000 (Dickson and Skehel, 1974; Parks et al., 1974a; Ritzi et al., 1976; Teramoto et al., 1974; Sarkar and Dion, 1975; Schochetman and Schlom, 1976). Competition immunoassays which measure typespecific antigenic determinants of MMTV gp52, analogous to type-C viral gp70 immunoassays described previously, provide a means of discriminating MMTV isolates derived from different mouse strains (Teramoto et al., 1977). In addition, a second glycoprotein of 34,00036,000 molecular weight, gp36, has been described (Teramoto et a1., 1974; Sarkar and Dion, 1975; Dickson and Skehel, 1974). These two viral-coded proteins are initially synthesized in the form of a 75,000 molecular weight glycosylated precursor polypeptide (Dickson et ul., 1975; Schochetman et al., 1977). Studies on differential inhibition of gp52 and gp36 synthesis by hypertonic shock techniques have indicated the arrangement of the two component gl ycoproteins within this precursor as NH2-gp52-gp36-COOH (Schochetman et a1., 1977). Moreover, Schochetman and Schlom (1976) have reported that synthesis of these precursor glycoproteins involves a separate initiation site from that utilized for translation of the major nonglycosylated MMTV structural proteins. Recently, a 51,000 molecular weight envelope glycoprotein and a major 24,000 molecular weight internal structural protein of bovine leukemia virus (BLV) have been isolated and partially characterized (Devare et al., 1976; Devare and Stephenson, 1977). By use of competition immunoassays developed for each, these proteins have been shown to lack immunologic cross-reactivity with structural proteins of any of a broad range of other oncornaviruses tested (Devare et al., 1976; Devare and Stephenson, 1977). Thus, on the basis of reverse transcriptase divalent cation preference (Gilden et al., 1975; Kettmann et a1., 1976) and molecular weight of its major structural proteins, BLV appears to resemble MMTV closely, and has thus been tentatively classified within the type-B oncornavirus group (Devare and Stephenson, 1977).Although, less well studied to date, oncornavirus isolates of guinea pig origin (Nayak and Murray, 1973) also resemble MMTV

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

41

morphologically (Dahlberg et al., 1974a) and should probably be included in this class. Type-D oncornaviruses such as MPMV much more closely resemble type-C viruses than do the type-B viruses. This was initially indicated by similarities in the molecular weight of their component structural proteins. Thus, MPMV was shown by SDS gel electrophoresis to contain an envelope glycoprotein of about 70,000 molecular weight as well as nonglycosylated structural proteins of about 26,000 20,000, 15,000, 12,000 and 10,000 molecular weights (Tronick et al., 1974a; Schochetman et al., 1975). However, characterization of these nonglycosylated proteins has been minimal and the possibility that one or more may be of cellular, rather than viral origin has not been ruled out. The major 70,000 molecular weight envelope glycoprotein of MPMV has been isolated and studied in considerable detail (Stephenson et al., 1976a). In these studies, 1Z51-labeledMPMV gp70 was shown to be immunoprecipitated at high titers by homologous sera raised against MPMV. Of interest, the same anti-MPMV sera also precipitated '251-labeled70,000 molecular weight glycoproteins isolated from baboon and RD114 viruses but showed no detectable reactivity against labeled gp7Os of FeLV, R-MuLV, or woolly monkey virus. fn competition radioimmunoassays utilizing antiserum to MPMV to precipitate '251-labeledbaboon gp70, MPMV, baboon, and RD 114 viruses (Stephenson et al., 1976a) as well as the recent type-D oncornavirus isolates of langur and squirrel monkey origin (Todaro et al., 1977a) competed with similar efficiency while none of a large number of other type-C viruses showed detectable reactivity. These findings demonstrate that MPMV glycoprotein shares antigenic determinanQ with the major envelope glycoproteins of baboon-RD 114 virus group. In contrast there has been no evidence for immunologic crossreactivity between the major 26,000-30,000 molecular weight structural proteins of type-C and type-D oncornaviruses despite the fact that such proteins are generally much more broadly reactive and immunogenic than the envelope glycoproteins (Stephenson et al., 1976a). Since MPMV was originally isolated from the rhesus monkey which contains information for an endogenous type-C virus highly related to the baboon type-C virus (Beneveniste and Todaro, 1974)and expresses antigens in normal tissues (Stephenson et al., 1976c; Stephenson and Aaronson, 1977), the possibility that a portion of the MPMV gp70 may have been acquired b y a mechanism involving recombination with endogenous primate type-C viral genetic sequences must be considered.

42

JOHN R. STEPHENSON ET AL.

Vli. Summary and Conclusions

The type-C virus genome consists of two identical 35 S RNA subunits joined near their 5’ terminus in a hydrogen-bonded dimer linkage structure. The genome is terminally redundant containing short identical segments at its 5’ and 3’ ends that provide a means of forming circular structures during replication. On the basis of presently available data, the mammalian type-C viral genome appears to be arranged in the order 5’ gag-pol-enu 3’. In the case of replication-defective mammalian sarcoma viruses, src sequences acquired from the host cell genome by genetic recombination are located at varying positions, frequently extending into the 3’ terminus of the gag gene. The type-C viral gag gene codes for four structural proteins of 10,000 to 30,000 molecular weight. These proteins are initially translated in the form of a 65,000 molecular weight precursor polypeptide. By analysis of the intermediate cleavage products of this precursor, it has been possible to deduce the arrangement of the regions within the gag gene of a prototype murine type-C virus coding for each of the four structural proteins. Extensive immunologic and biochemical characterization of the gag gene-coded proteins of type-C viral isolates of diverse mammalian species has permitted the generalization of this sequence to a broad range of mammalian type-C viruses, The amino-terminal gag gene protein of such viruses is hydrophobic, relatively basic, and possesses broadly shared interspecies antigenic determinants. The second protein from the amino terminus of the gag gene-coded precursor is a highly acidic phosphoprotein characterized by specific RNA binding properties and type-specific antigenic determinants. The third protein is the major virion structural antigen characterized by a molecular weight of about 27,000-30,000 and broadly reactive antigenic determinants. Finally, the protein located at the carboxy terminus of the gag gene product is a highly basic ribonucleoprotein of about 10,000 molecular weight. Independent evidence for this sequence was derived by analysis of the extent of gag gene expression in cells nonproductively transformed by diverse mammalian sarcoma virus isolates (Fig. 5). The pol gene, located adjacent to gag within the viral genome, codes for the type-C viral reverse transcriptase. Translation of pol appears to be accomplished by occassional read through of the gag gene termination codon, thus providing a mechanism for regulation of the amount of reverse transcriptase synthesized. The primary translational product of the env gene, located adjacent to pol, is a precursor which is cleaved yielding two proteins, the major envelope glycoprotein, gp70, and a nonglycosylated 15,000 molecular weight protein designated p 15(E).

43

RNA TUMOR VIRUS TRANSLATIONAL PRODUCTS

5 -

NU2-

p15

pl2

P a

- 3

P10 -COOH

FIG.5. Model for translation oftype-C viral mRNA and posttranslational processing of virus-coded precursor polypeptides.

While our understanding of the molecular biology of type-C RNA tumor viruses is rapidly progessing, little information is presently available concerning either the normal cellular functions of these viruses or their etiologic involvement in tumors of their natural hosts. The availability of sensitive immunologic techniques for detection of expression of individual type-C viral translational products should provide a means of resolving such questions.

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ADVANCES IN CANCER RESEARCH. VOL . 27

QUANTITATIVE THEORIES OF ONCOGENESIS Alice S . Whitternorel

.

Department of EnvironmentalMedicine. New York University Medical Center and Department of Statistics. Stanford University

I . Introduction .............................. ........................ I1. Expected Rates of Tumor Appearance .................................. I11. The Single Stage Theory of Iverson and Arley .......................... A . The Theory ....................................................... B. Examples and Application to Data .................................. IV. The Multicell Theory of Fisher and Hollomon .......................... A. Temporal Behavior of Observed Incidence Rates ..................... B. Concentration Dependence of Observed Incidence Rates ............. V. The Multistage Theory with Negligible Cell Loss ................ A. The Theory ....................................................... B. Application to Terminated Exposures ............................... VI. The Multistage Theory with Non-Negrigible Cell Loss .................. A. Radiation-Induced Tumors ......................................... B. Urethane-Induced Murine Lung Tumors ............................ VII . The Multistage Theory with Proliferative Advantage of Intermediate Cells .................................................... A . TheTheory . . . . . . . . ............................................ .......................... B . The Armitage and Doll Two-Stage ............................ C . The Fisher Theory .............. inated Exposures ........... D . Application of the Fisher Theory t ......................... E . Increased Numbers of Target VIII . Single Stage or Multistage Theory in Transformed Cell Types A. The Initiation-Promotion Phenomenon .............................. B. Growth of Clones of Transformed Cells ............................. C. Application to Initiation-Promotion Experiments ..................... IX. Implications for Dose-Response Relationships .......................... A . The Problem ...................................................... B. Radiation Carcinogenesis ........................................... X . Conclusion ......... ........... References ...........................................................

55 56 57 57 58 62 62 64 65 65 67 68 68 71 73 73 74 75 76 77 78 78 78 79 83 83 86 86 87

I . Introduction

The purpose of this chapter is twofold: to review the quantitative theories of the origin of neoplasms. and to discuss a new theory . This On leave from Hunter College. CUNY. This work was supported by a grant from the Alfred P. Sloan Foundation to the SIAM Institute for Mathematics and Society and by a Rockefeller Foundation Fellowship in Environmental Affairs. 55 Copyright 0 1978 hy Academic Press. Inc. All rights of reproduction in any form resewed .

ISBN 0-12-006627-0

56

ALICE S . WHITTEMORE

theory synthesizes some of the others and describes aspects of initiation, promotion, and regression that have not been included previously. Armitage and Doll (1961) and Whittemore and Keller (1978) reviewed many of these theories &om a mathematical point of view. Here we discuss them without the use of mathematics. We shall examine them critically both qualitatively and quantitatively, by comparing their predictions with epidemiological and experimental observations. The theories relate the frequency and time of occurrence of tumors to the temporal and spatial distribution of oncogenic exposure. There are two main reasons for formulating such theories. One is to provide a framework for evaluating the consequences of proposed mechanisms of carcinogenesis. The other is to help determine allowable concentrations of known carcinogens in the environment, and to estimate the consequences of exceeding them. This is necessary because animal experiments must be done at concentrations high enough to cause some of the animals to develop tumors, while environmental concentrations must be low enough to produce very few tumors in man. Thus, apart from the great difficulties due to interspecies differences, animal experiments cannot be used directly to study low concentrations. Therefore some theory is needed to extrapolate the doseresponse relationships downward &om the high doses used in animal experiments to the low doses to be allowed in the environment. II. Expected Rates of Tumor Appearance

Oncogenesis is believed to involve two processes, transformation and growth. Transformation is the occurrence of one or more heritable changes in a cell which render it capable of generating a tumor. Growth is the process of multiplication by cell division whereby the clone of a transformed cell becomes a detectable tumor. Here the term transformed cell refers only to those altered cells that generate neoplasms, and not to their descendents. Cells which can be transformed to a state capable of generating neoplastic growth will be called target cells. A given tissue is assumed to contain some fixed number N of target cells, each of which has a chance of transformation which is unaffected by the tumorigenic fate of its neighbors. Then the expected rate of tumor occurrence in a tissue at time t is proportional to the number N of target cells. Thus it can be written Nr(t), where r(t) is the expected fraction of target cells which yield clinically detectable tumors per unit time. Similarly, the expected number of tumors which have appeared in the tissue by time t can be written NR(t). Here R(t) denotes the expected proportion of target cells which give rise to detectable tumors by time t .

QUANTITATIVE THEORIES OF ONCOGENESIS

57

The incidence rate is defined as the expected rate of tumor occurrence in tumor-fiee tissue. Under the above assumptions the incidence rate at time t can be shown to equal the expected rate Nr(t) of tumor occurrence in the tissue. Similarly the cumulative incidence at time t , defined as the sum of the incidence rates over all times prior to t , can be shown to equal the expected number NR(t) of tumors appearing in the tissue by time t . The time from initial exposure of a target cell to a carcinogen until it is transformed is called the transformation time. The growth time is the time required for a transformed cell to generate a clinically detectable tumor. All of the theories assume that the growth time is independent of the transformation time. The rate r(t) at which target cells yield detectable tumors can be expressed in terms of these two times. Therefore theoretical predictions for the transformation and growth times yield predictions for the incidence rate Nr(t). This is also true for the expected number NR(t) of tumors up to time t . Accordingly, we will present each theory by stating its assumptions about transformation and its assumptions about growth. These assumptions will imply the way in which r(t) and R(t) vary with time and with oncogenic exposure. The theoretical predictions of these quantities will be compared with empirical estimates of them. Ill. The Single Stage Theory of lverson and Arley

A. THE THEORY The earliest quantitative theory of carcinogenesis is that of Iverson and Arley (1950).This theory postulates that transformation of a target cell occurs as a single event, such as a somatic mutation or activation of an oncogenic virus. The rate 1 at which cells are transformed is assumed to be the sum of a spontaneous rate s of transformation in the absence of carcinogenic exposure, plus an induced rate due to exposure. The spontaneous rate s is assumed independent of time. The induced rate is taken to be proportional to the concentration c(t) of carcinogen at time t. The proportionality constant p , representing the transformation rate per unit of carcinogen, depends on the potency of the carcinogen and its metabolic derivatives, and on the sensitivity of the target cells. Since the concentration may vary with time, so may the rate 1 of transformation per cell. Thus we write

l(t) = s

+ pc(t).

(1) The rate of transformation of target cells in the tissue is l(t) times the number of unaltered target cells remaining in the tissue at time t. We

58

ALICE S. WHITTEMORE

assume that the rate Z(t) at which unaltered target cells are depleted is small at all times, and that the initial numberN of such cells is so large that depletion of target cells is negligible. Thus their number at all times is essentially N , and the rate of transformation in the tissue is NZ(t).The assumption of negligible target cell depletion is made in all the theories we consider. Iverson and Arley assumed that the clone of a transformed cell grows by the proliferation of its members, and that it is detected as a tumor when it contains some critical number a of cells. The probability per unit time that a cell in the clone divides is assumed to be a constant p. Since p is taken independent of carcinogen concentrations, carcinogenic exposure has no effect on the growth time of tumors. From these assumptions one can calculate the expected rate g(t) at which a transformed cell yields a detectable tumor at time t after transformation, and G(t), the proportion of transformed cells whose growth times are less than t . This theory ignores cell loss in a tumor due to necrosis, ignores the existence of nonproliferating tumor cells, and ignores variation in proliferation rates with time and with location within the tumor. However, it does provide for variation in tumor growth times, unlike the less plausible assumption of many theories that all tumors require a fixed time w for growth. The above assumptions for tumor growth and the expression (1)for the transformation rate l(t) complete the formulation of Iverson and Arley’s theory. Together they permit the calculation of the rate Nr(t) of tumor appearance and the expected number NR(t) of tumors in the tissue by time t . The theory involves five constants: s and p in (l),the growth constant p, the detectable size n, and the number N of target cells. In addition the concentration c(t) of carcinogen at the target cells must be specified.

B. EXAMPLESAND

APPLICATION TO

DATA

As a first example, let us suppose that c(t) is a constant c. Then (1) shows that the transformation rate I is the constants + p c . The rate r(t) of tumor appearance per target cell is in this case 1 times the proportion G(t) of all transformed cells whose growth times are less than t:

r(t) = lG(t) = sG(t) + pcG(t).

(2)

Here sG(t) is the spontaneous rate of tumor occurrence in the absence of the carcinogen, and pcG(t) is the induced rate due to concentration c of the carcinogen. If the spontaneous cell transformation rate s is

QUANTITATIVE THEORIES OF ONCOGENESIS

59

negligible, then b y (2) the rate Nr(t) = NpcG(t) of tumor appearance in the tissue at a given time t is proportional to carcinogen concentration c. It also follows that for t fixed, the expected number N R ( t ) of tumors per tissue is proportional to c. These results agree with Doll’s (1971) finding that the incidence rates of bronchial carcinoma among British physicians are proportional to smoking rate. This linear relationship between agestandardized incidence rates and average daily cigarette consumption is shown in Fig. 1. However the plots of cumulative incidence versus applied concentration of various carcinogenic agents shown in Fig. 2 exhibit a dose-response relationship that is more quadratic than linear. This disagreement with the Iverson-Arley theory indicates that the theory may be inappropriate for mouse skin carcinogenesis induced by benzpyrene and ultraviolet light. However the disagreement may be due to a discrepancy between applied carcinogen and actual concentration of the agent or its metabolites at and within the relevant target cells. The possible consequences of such a discrepancy will be discussed in Section IX,A. As a second example, suppose that a single dose C of carcinogen is administered at time t = 0. Then r(t) is the sum of the spontaneous rate sG(t) and an induced rate resulting from cells transformed at time zero by the carcinogen. The induced rate is pCg(t), where g(t) is the rate of

300

-

Age standardized incidence rate of bronchial carcinoma: 200 no. of -/lo5

man-mas 40050 -

I/ I

0 :

20 30 Cigarettes smoked/day 10

I

40

FIG.1. Incidence rate of bronchial carcinoma versus daily rate of cigarette smoking. The data points are from Doll (1971)and represent age-standardized incidence rates among men who started to smoke at ages 15 to 24 years and were not known to have changed their smoking habits. The straight line is eye-fitted to the data points.

60

ALICE S. WHJTTEMORE I A 5-

0

4-

e Cumulative tumor

m

3-

incidence

0

2-

1-

e-Le 0

c-

I

L

1

I

c

I

8

'

L

1

3

2

'

a

' 1

4

Dose rate of ultraviolet light, ergs/cm2/sec 5-

B

0

C

4-

Cumulative 3tumor 2incidence

0

t -

appearance at t of tumors resulting from cells transformed at time zero. Thus we have

r(t) = sG(t) + p C g ( t ) .

(3)

More generally, suppose that exposure to a carcinogen is terminated at some time to.At times t greater than to,the rate r(t)of tumor appearance is again the sum of a spontaneous rate sG(t) plus a rate induced by the carcinogen. In this case the induced rate is due to the appearance at time t of tumors whose progenitors were transformed prior to to,and whose growth time exceeded t - to.Thus for large times t when growth periods exceeding t - to are unlikely, the tumor appearance rate should equal the spontaneous rate. This is in disagreement with the data of Doll and Hill (Doll, 1971; Doll and Peto, 1976) on inci-

QUANTITATIVE THEORIES OF ONCOGENESIS

61

dence rates of bronchial carcinoma among former cigarette smokers. These data suggest that the excess over nonsmokers’ rates remains constant for as long as 20 years after termination of smoking, although the growth period for human lung carcinomas has been estimated at only 5 to 7 years. The prediction that tumor rates revert to the spontaneous rate after exposure termination is also in disagreement with the experimental data of Lee (1975), discussed in Section V and shown in Fig. 7 and 11. In this experiment the backs of mice were painted with benzpyrene, and treatment was discontinued at various stopping times in different groups. Although the tumor growth period was estimated to be 10 weeks, the incidence rates do not return to the spontaneous rates within the animals’ lifetime. As a final example, let the concentration c(t) of carcinogen be arbitrary. We shall examine the total expected number of carcinogen induced tumors, and its dependence upon exposure pattern and intensity. It follows from (1)that the induced transformation rate for the tissue is N p c ( t ) . Thus the expected number of target cells ever transformed by the carcinogen is NpC, where C is the carcinogen concentration integrated over time. The ultimate expected number N R ( m ) of induced tumors is then the ultimate expected number NpC of carcinogen-transformed cells, multiplied by the fraction G ( m) of these transformed cells which ever grow to detectable tumors:

N R ( a ) = NpCG(m).

(4)

Note that the ultimate expected number of tumors per tissue given b y (4)depends only on the total integrated concentration C and not upon the pattern c ( t ) of exposure. Moreover it is proportional to C. If spontaneous incidence is negligible, these remarks apply to total tumor numbers. These results may be compared with plots of mean number of pulmonary adenomas per mouse versus total injected dose of urethane and versus pattern of exposure shown in Fig. 3 (White et al., 1967). It is evident from Fig. 3 that fractionation reduces the tumorigenicity of doses 0.5 mg/gm and greater, arid that the dose-response relationships have a quadratic component. Both of these results are inconsistent with the Iverson-Arley theory. As noted by Armitage and Doll (1961), fractionation forward in time can reduce tumor incidence at any observation time because cells transiormed later have less time to develop to detectability by the time of observation. However this cannot account for the results of Fig. 3, because White et al. found a single dose of urethane more effective evein when administered at the end of the fractionation period.

62

ALICE S. WHITTEMORE

Dose oubdividrd into n frocii

40

Mean number 30 of tumors per 20 mwse 10

' 0

04

08

12

16

20

Total applied dOJe (mglgrn body weight)

FIG. 3. Average number of pulmonary adenomas per mouse as a function of total injected dose of urethane (ethyl carbonate) measured in milligrams per gram weight of mouse for five patterns of administration: subdivision of dose into le), 2(0), 4(A), 8( x), and 1 q O ) portions. The injections were given at intervals of 2 days. Reprinted with permission from White eb al. (1967).

Other experiments of White, discussed in Section IX,A, indicate that concentrations of urethane (or a carcinogenic metabolite) at the target cells may vary nonlinearly with injected dose. Such variation could account for both of the inconsistencies noted between the IversonArley theory and the data of Fig. 3. IV. The Multicell Theory of Fisher and Holloman

A. TEMPORAL BEHAVIOROF OBSERVED INCIDENCE RATES

At about the time that Iverson and Arley published their theory, a number of investigators noted that the death rates for many forms of human cancer increased proportionately with the fifth or sixth power of age. Fisher and Holloman (1951)observed this for death from cancer of the stomach in women in the United States. Nordling (1953) found it for death from all forms of cancer in men in England, France, Norway, and the United States. Stocks (1953)observed it in data on death from stomach cancer in both men and women in England and Wales. Armitage and Doll (1954)found it for mortality rates due to cancer of different sites and for each sex in England and Wales. This relationship does not hold for all types of cancer; notable exceptions are childhood cancers and hormone-related cancers, such as cancer of the femaIe breast or male testes. However, it does hold for the majority of adult cancers. Examples of data exhibiting this proportionality of incidence rate to a power of age are shown in Fig. 4. To interpret this observation, it was assumed that the time from

63

QUANTITATIVE THEORIES OF ONCOGENESIS

I

1000

t ! f

l0O0

4i

1oot

I

lncidsnw rats of nonmslanoma skin cancer 110. of c ~ / 1 0 man-yn. 5 '00 (log ralal

i Gastric cancer mortality rate: no. of casa/l05 man-yn. lo -

(log scale)

10 1-

- - t

20 40 80 Age in years (log scale 1

i

1'

i r' c

/

.l

20 40 80 Age in years (log scale)

FIG.4. Relationship between age anti incidence rates of (A) nonmelanoma skin cancer in Dallas white males for 1971-1972; (B) stomach cancer in Danish males for 19631967. Data points are from (A) Scotto et al. (1974), and (B) Clemmesen (1974). The straight lines, eye-fitted to the data, have slopes 5.66 and 5.47, respectively.

cancer detection until death was negligible compared to the time required for transformation and growth. It was also assumed that cancer fatality rates were independent of age. Therefore the incidence rate was assumed to be proportional to the mortality rate. Subsequent studies in which the age-specific incidence rates of cancer are recorded have confirmed this assumption. Furthermore the investigators assumed that the cancers were due to carcinogenic exposure that remained fairly constant from birth to death. Thus age was the same as the duration of exposure. Then the data show that the incidence rate is proportional to the fifth or sixth power of duration of exposure to a carcinogen of coristant concentration c. Two explanations of this power law relationship have been proposed. One, due to Fisher and Holloman, is that a certain number k (equal in this case to six or seven) of different cells have to be altered in a single tissue in order to forni a tumor. The other, due to Muller and Nordling, is that a single cell has to undergo k changes before it can generate a tumor. We shall first describe the consequences of the multicell theory of Fisher and Holloman. In the next section we shall treat the multistage theory of Mullev and Nordling.

64

ALICE S. WHITTEMORE

Let us suppose that the occurrence of a tumor in a tissue requires the presence of at least k altered cells, and that the fraction E of unaltered target cells which are changed per unit time is proportional to the concentration c of carcinogen:

1 =pc. (5) When p c is small and when the number k of altered cells is small compared to the number N of target cells in the tissue, it can be shown that the rate at which the kth alteration occurs in the tissue is approximately proportional to

(Npc)ktk-l

(6)

If we assume that the time for growth to detectable tumor is negligible compared with the time required for the k alternations, then the expression (6) is proportional to the incidence rate Nr(t). With k = 6 or 7, the incidence rate would then be proportional to the fifth or sixth power of age and to the sixth or seventh power of concentration. This age dependence agrees with the observations noted at the beginning of this section. However, the concentration dependence is incompatible with experimental and epidemiological data.

B. CONCENTRATION DEPENDENCEOF OBSERVED INCIDENCE RATES There is evidence that incidence rates vary as the &st or second power of c, rather than as c6 or c’. For example, Doll (1971) analyzed the incidence of bronchial carcinoma in physicians in England and its relation to cigarette smoking. He found that the incidence rate was approximately proportional to the number of cigarettes smoked per day, and to the fourth power of the duration of smoking (see Fig. 1and 5). If we suppose that the number of cigarettes per day is a measure of the carcinogen concentration c , and let t denote duration of smoking, then his result suggests incidence rates are proportional to ct4. The result (6) requires k = 5 to give t4, but then the rates would be proportional to c5, which disagrees with the data. Furthermore Lee and O’Neill (1971) and Altshuler et al. (1971) determined the incidence rate of tumors on the skins of mice which had been painted repeatedly with various concentrations of the carcinogens benzpyrene and dibenzanthracene, respectively. In both cases the rate was found to be proportional to c2. In addition Lee and O’Neill found that it was approximately proportional to the cube of treatment time t. Now (6) requires k = 4 to give t 3 , but then it also yields that the incidence rate is proportional to c4, in disagreement with the data.

QUANTITATIVE THEORIES OF ONCOGENESIS

65

I

i

1000 -

1

500 -

i

Dose-standardized incidence rat0 of bronchial carcinoma no. of c a r e r ~ 1 ~ 5 100 man-yrs. (log scale) 50 -

7

-

: i

- [

10 10

I

20

1

1

1

,

40 60

FIG.5. Incidence rate of bronchial carcinoma, standardized for rate of cigarette sm6king, versus years of smoking. The data points are from Doll (1971) and represent incidence rates among men who started to smoke at ages 15 to 24 years and were not known to have changed their smoking habits. The straight line is eye-fitted to the data points.

The proportionality of the tumor incidence rate to such a high power of c can be avoided in the multicell theory b y assuming that there are a number of different types of alteration, and that at least one of each of k types of altered cells must be present in the tissue in order for a tumor to occur. If exactly n of the k alterations are induced by the carcinogen at rates proportional to its concentration c, then in (6) the term ( N ~ C ) ~ will be replaced b y a term p,-oportional to c". Here n can be any integer between 0 and k. Nevertheless there remain some substantial objections to the multicell theory. One is the lack of specific assumptions about the spatial configuration of the altered cells in the tissue. Wright and Pet0 (1969) and Jones and Grendon (1975) have devised multicell theories incorporating such assumptions. However the main objection is the biological evidence for the monoclonicity of tumors. Thus w e shall describe the single cell multistage theory of carcinogenesis. V. The Multistage Thcory with Negligible Cell Loss

A. THE THEORY

According to the multistage theory of Muller (1951) and of Nordling (1953), a cell can generate a tusnor only after it has suffered a certain

66

ALICE S. WHITTEMORE

number, say k, of cellular events or changes. The quantitative consequences of this theory were derived by Stocks (1953) and by Armitage and Doll (1954). We assume that the k changes have different transition rates lxt), i = 0, 1, . . . , k - 1, given by

h(t) = SY + P d t ) ,

(7)

and that they must occur in the order 0, 1, . . . ,k - 1.A cell in stage i or an i-cell is one that has undergone the first i changes. Thus Zkt) represents the fi-action per unit time of i-cells which undergo their ( i + 1)st change. We also include the possibility of cell “death,” where death may be loss of ability to divide, with ddt) denoting the death rate for cells in stage i, i = 1, . , . , k - 1. These assumptions, which are represented schematically in Fig. 6, are a straightforward extension of the Iverson-Arley theory for transformation. In this section we shall assume that the loss of i-cells through death and through transition to stage i + 1 is negligible. If in addition the carcinogen concentration c(t) is constant at level c, then the transition rates I t are independent of time. I n this case the rate at which cells are transformed in the tissue is approximately proportional to NZ,,

. . . ,l!k-]tk-l.

If the growth time of a tumor is assumed to be a constant w, then the incidence rate is zero at times t less than w , and at times greater then w , it is approximately proportional to

Nl,,

. . . , l&l(t - wp-1.

(8)

An immediate consequence of (8)is that the logarithm of the incidence rate, when plotted against the logarithm of exposure duration t less w, forms a straight line of slope k - 1. Thus with w set equal to zero, this theory agrees with the power law relationships observed i n . Fig. 4. As was noted in the previous section, when exactlyn ofthe k changes

FIG.6. Schematic representationof the k-stage theory of transformation. Cells in the i* stage can either die at the rate d,(t) or be converted into cells in the (i stage at the rate ZQt). All target cells start as normal cells, in stage zero. Cells which reach stage k are transformed cells.

+ lr

QUANTITATIVE T:SEOFUES O F ONCOGENESIS

67

are induced by the carcinogen, then lo . . .l,+, and thus the incidence rate of (8) is proportional to c”, With n = 1,k = 5, and w = 0, (8) fits the cigarette data of Doll and Hill, as shown in Fig. 2 and 5. With n = 2, k = 4, and w = 10 weeks, it fits the mouse skin painting data of Lee and O’Neill. The quadratic dose-rate dependence of Lee and O’Neill’s cumulative incidence data is shown in Fig. 2C. Figures 2A and 2B show a quadratic dependence of cumulative incidence on dose rate of ultraviolet light and benzpyrene, respectively. The deviations from the quadratic relationship that occur at high incidence and high dose rates may reflect killing of transformed cells by the carcinogen.

B. APPLICATION TO

TERMIN4TED EXPOSURES

The multistage theory depirted in Fig. 6, with negligible cell loss, has been applied to the case in which the carcinogenic concentration is constant until some time to,after which it is zero. This is the case in the experiment of Lee (1975) in which the shaved backs of mice were painted periodically with bempyrene and other carcinogenic components of cigarette smoke. The treatment was discontinued at different stopping times in different groups of animals. Lee fit the multistage theory to these data, using maximum likelihood estimates of the relevant parameters. For the benzpyrene exposures, the estimates n = 2, k = 4, and w = 10 weeks were obtained. These results agree with the corresponding estimates of these parameters for the lifelong repeated benzpyrene treatment of Lee imd O’Neill (1971). Lee also found that benzpyrene appears to affect an early and a late change, with either p , or p , and either p 2 or p 3 approvimately zero. Evidence that p , is nonzero, i.e., that benzpyrene strongly affects the first change, is given by the experiment of Pet0 et al. (1975). They administered the same lifelong repeated benzpyrene treatment to mice starting at 10, 25, 40, and 55 weeks of age and found that tumor incidence after a given duration of treatment was no higher among the older mice. This suggests that benzpyrene acts on the first change and that other changes cannot tak,e place until this benzpyrene-related change is complete. Othenvisr older mice with more background exposure would have more one-cells available for the action of benzpyrene and thus a higher incidence of tumors. Lee’s terminated benzpyrerae data, and curves based on his estimates, are shown in Fig. 7. The curves fit the data well, despite the fact that the theory does not include possible retention of the benzpyrene or its metabolites in the #-issueafter termination of exposure, or variation in tumor growth times.

o.:l

68

ALICE S. WHITTEMORE 0

TUMOR INCIDENCE RATE (log scale) 0.01

/i [/

*

c = 36 pg/wk

--c = 36 pg/wk = 25 wkr

to

10

50 200 10

50 200 10

c = 36 pglwk lo = 35 wks

200 10

50

C = 36 pg/wk treated for life

50

200

i

I

I TUMOR INCIDENCE RATE (log scale)

o,l I

I

I

I 0.01

10

50

ZOO 10

50 ZOO 10

50

200 10

50 200

TREATMENT TIME IN WEEKS -10 (log scale)

FIG. 7. Tumor incidence rate for benzpyrene painted mouse skin versus time (in weeks) since start of treatment, less 10 weeks. The concentration cft) of benzpyrene is given by c ( t )= c, 0 5 t 5 t oand c ( t ) = 0, t o< t, with different values of concentration c and stopping time to.The values are shown on each graph. The data points were obtained by the interval technique given by Eq. (3)of Hoe1 and Walburg (1972)from unpublished data of P. N. Lee. The solid lines are the incidence rates predicted by the multistage theory, with Lee’s maximum likelihood estimates of the parameters. His estimates of the parameters k and w of (8) are k = 4 and w = 10 weeks. With sland p1given by (7),i = 0, 1, 2, 3, he obtains p o = p 2 = 0. His values for bfc)= ( N / 6 )so (sl+pic) s2(s3 + p g ) are b(0)= 0.0125 X b(36)= 4.78 x and b(60)= 21.84 x His estimates of p&s, are 5.21 and 7.56 for the 36-pg and 60-pg exposure groups, respectively. The dashed lines indicate the predicted background rate.

VI. The Multistage Theory with Non-Negligible Cell Loss

A. RADIATION-INDUCED TUMORS

We shall now consider the multistage theory with “death” of intermediate cells. This theory has been applied to radiation carcinogenesis, in which there is evidence that radiation causes loss of cellular ability to divide as well as malignant transformation. [A detailed review of the difficulties inherent in quantitative analysis of radiation carcinogenesis is provided by Mayneord and Clarke ( 1975).] The theory has also been used by Neyman and Scott (1967), as discussed in Section VI,B. Cell death was first included by Burch (1960) in the two-stage

QUANTITATIVE TH1;ORIES OF ONCOGENESIS

69

theory to analyze radiation-indLced cancer. H e was able to get rough agreement of the theory with data on leukemia incidence in survivors of the atomic bomb explosion at Hiroshima. More recently, Marshall and Groer (1977) have included dosedependent cell death in the three-stage theory to analyze radiationinduced bone cancer. They assume that the death rates d i are equal and are proportional to carcinogen concentration:

Here c(t) is the radiation dose late to a bone surface cell in rads per unit of time, and q is not small. ‘This assumption for ddt) leads to such depletion of target cells on the hone surface at high doses that tumors would be very unlikely to occur. However, osteosarcomas are observed at high doses. To account for this, target cells are assumed to be replenished at a large constant late. Then for dose rates less than one rad per day, depletion of targ3t cells can be neglected and their number remains essentiallyN. The theory for dose rates in this range is shown graphically in Fig. 8, The data for human and caninc: 226Raexposure at low doses indicates that tumor incidence varies as the square of dose. This follows from the theory if the transition rates for two cellular changes are proportional to dose. Therefore Marshall and Groer assumed that

Further, although the average glowth of an osteosarcoma is estimated at seven years, tumors may appear 10 to 20 years after exposures as short as a month. This can be explained by the third change, which occurs independently of radiation at the constant rate 1,. Stage two cells are assumed to have lost their ability to stop dividing. However, they behave as normal cells uatil they receive a signal to initiate mitosis. The third change is such a signal to divide, so that 1, is related to the host’s rate of bone remodeling. We now assume that 1,t and pC(t) are small relative to unity, where

FIG.8. Schematic representation of the three-stage theory of transformation used by Marshall and Groer and by Neyman and Clcott. It is a special case of the k-stage theory of Fig. 6.

70

ALICE S. WHITTEMORE 2.5

20

'

Incidence rate of human osteosamoma 1 0 0.5

' 0

10 x) 30 40 50 60 70 Time since intake (years)

FIG.9. Incidence rate of human osteosarcoma versus time since intake for the highest 15intake levels of 228Raand 226Rashown in Table 11. Curves are based on Marshall and Groer's minimum chi-square fits to human osteosarcoma data of the three-stage theory with loss of cells in stages one and two. The parameter estimates are given in Table 11.

C ( t ) is the dose to the target cells integrated over time prior to t . We also assume that the growth time is a constant w . Resulting incidence rate curves are shown in Fig. 9. They agree well with human 226Radata in three respects: First, the incidence rate at time t depends only upon the total dose C(t - w ) up to time t - w , and not upon the pattern of administration. Second, at low doses the incidence rate is approxiTABLE I OBSERVED AND EXPECTED~ NUMBERS OF WITHIN

Injection level (pCi/kg body wt)

Number injected

0.00 0.01 0.02 0.06 0.17 0.31 0.95 2.80 8.00

44

Total:

DOGS WITH

OSTEOSARCOMA

17 YEARS AFTER INJECTION OF 22sRab

Observed

Expected 0.0

12 13 10 -

0 0 0 1 2 5 11 12 9

-

0.0 0.0 0.6 1.1 9.9 10.5 11.9 6.0

164

40

40.0

10 25 23 14 13

a Expected numbers were obtained by multiplying the incidence rate of the threestage theory by the number of animals at risk during 2-month intervals, and summing the product over all 2-month intervals in the 17-year period. The parameters p = 5.0 x lo-' per rad, q = 0.01 per rad, 2, = 0.1 per year, w = 2.5 yr and f (conversion factor relating intake level to dose rate to the bone in rads per year) = 350 radslyr per pCi/kg were determined to minimize the value of a x2 goodness of fit test, subject to the constraint that total observed and expected numbers of osteosarcomas be equal. From Marshall and Groer (1977).

QUANTITATIVE THEORIES OF ONCOGENESIS

71

TABLE I1 OBSERVED AND EXPECTED~ NUMBERSOF HUMANSWITH OSTEOSARCOMA WITHIN 70 YEARS AFTER INITIAL INTAKE OF 22ERaPLUS =%ab Intake level (pCikg bone wt)

Nuriber exposed

1 - 3.90 3.91- 5.48 5.49- 7.70 7.71- 10.81 10.82- 15.19 15.20- 21.35 21.36 30.00 30.01- 42.15 42.16 59.23 59.24- 83.23 83.24- 116.94 116.95-164.32 164.33-230.88 230.89-324.42 324.43-455.85 455.86-640.52 640.53-900.00

l!iO !!9

Total:

Observed

0 0 0 0 0

Expected

--

-

0.2 0.2 0.3 0.3 0.6 1.3 1.4 4.0 6.2 8.4 7.6 5.5 4.9 6.4 5.4 1.0 0.5 -

4; 4

54

54.0

:\O .7 ‘!O !!4 9 :!6 :I2 :I8 !7 15 7 18 16 3 3

1 3 4 11 4 5 9 5 8 3 0 1

Expected numbers and parameters were obtained as in Table I, using one year rather than two month subintervals. The parameters are p = 4.7 x per rad, q = 0.01 per rad, Zz = 0.1 per year, w = 7 years andf = 15 raddyr per pCi/kg. From Marshall and Groer (1977).

mately proportional to the square of the dose C ( t - w). Third, for large doses, the incidence rate is approximately a constant, independent of time and dose. Observed and expected numbers of individuals with osteosarcoma following intake of 226Rain dogs and 226Raplus 228Rain humans, are shown in Tables I, and 11.

B. URETHANE-INDUCED MURCNELUNG TUMORS Shimkin and Polissar (1955)and others studied changes in the cell populations of mice’s lungs after a single injection of urethane. Examination of the lung tissue after sacrifice revealed clones of modified cells which were called hyperplastic foci. The number of such foci increased with time between injection and sacrifice, reached a maximum at about four weeks, and then declined. When a mouse was sacrificed after four weeks, pulmonary adenomas were found. Their

72

ALICE S. WHITTEMORE

number increased with time between injection and sacrifice. Because the question whether the hyperplastic foci were precursors of the tumors could not be answered by direct observation, a theoretical analysis was devised. According to the observations of White et aZ. (1967) as indicated in Fig. 3, the total number of tumors per mouse vanes as a quadratic function of the injected dose of urethane. Furthermore, as shown in Fig. 3, this number is decreased when a single dose is fractionated into several small subdoses. These observations contradict the IversonArley single stage theory discussed in Section 111. Therefore Neyman and Scott (1967) proposed a two-stage and a three-stage theory of cell transformation. They rejected the two-stage theory on the grounds that the resulting temporal behavior of the expected number of hyperplastic foci was inconsistent with the observed peak in their numbers at four weeks. Their three-stage theory is depicted in Fig. 8. Cells in stage zero are normal, cells in stage two are members of hyperplastic foci, and those in stage three are transformed cells. In the transition from stage one to stage two, a cell is assumed to produce two daughter cells in stage two by division. A possible biological basis for this assumption is that the one-cells have experienced a mutational event which is not expressed before mitosis. The rate 2dt) at which target cells undergo their first change is assumed proportional to carcinogen concentration c(t):

lo(t) = POdt).

(11)

However, the mitotic rate 1, of one-cells is taken to be a constant independent of carcinogenic treatment. The transition rate Z2(t) at which hyperfocal cells become malignant is given b y

12(t) = $ 2 + P24t).

( 12)

The death rates dl and d2 are constant, with d2 representing the excess of death over division rates for members of hyperplastic foci. According to (1l),all of the one-cells are produced by the urethane at or shortly after treatment. The continuous formation of hyperplastic foci at rate 22, by one-cell division offsets their net loss through death at rate d2 and their depletion to form tumors at rate Z2(t).Consequently this model predicts that the expected number of hyperplastic foci reaches a single maximum, in agreement with Shimkin and Polissar’s observations. However, if urethane is indeed eliminated from an animal’s system within 24 hours, then this version does not account for the quadratic dependence of total tumor numbers upon injected dose observed by White et al. (1967). Although the total expected number of

QUANTITATIVE THEORIES OF ONCOGENESIS

73

transformed cells per mouse docs contain a term proportional to total dose squared, the proportionality factor for the former is very small and probably cannot account for the observations. It corresponds to the occurrence of all three trailsitions in the short time (24 hours) during which the urethane is in the animal’s system. Experiments subsequent to the publication of Neyman and Scott’s theories have suggested that the quadratic dependence of tumor numbers on injected dose may be due to a quadratic relationship between actual dose to the target cells and injected dose. These experiments, discussed in Section IX,A, may account for the discrepancy between Neyman and Scott’s three-stagt: theory and the data. However the failure of subsequent investigations (White et al., 1970) to reproduce the hypercellular foci noted by Shimkin and Polissar casts doubt on the need for a three-stage theory for this tumor system. Alternate explanations for the decrease in tumor numbers with dose fractionation are discussed in Section IX,A. [f these explanations are valid, then urethane may indeed induce pulmonary adenomas in mice by a one: stage mechanism, as postulated by Iverson and Arley.

VII. The Multistage Theory with Proliferative Advantage of Intermediate Cells

A. THE THEORY Although the multistage theory with k greater than 2 accounts for some of the data, there is no direct experimental evidence that cancer occurs in more than two stage:...This led both Armitage and Doll (1957) and Fisher (1958) to modify the multistage theory so that a twoor three-stage theory could explain the observed data. Their modifications give a selective ad1,antage to cells in intermediate stages which enable them to multiply more rapidly than normal cells, a possibility which had been mentioned by Platt (1955) and Muller (1951, p. 130). In the theory of the preceding sections only the cells in the final or kth stage were assumed to increase in expected number through proliferation. The assumption of a selective advantage for intermediate cells is supported by observations of hyperplasia, dysplasia, and other cellular abnormalities preceding malignant transformation. To describe these modifications, we shall now reconsider the multistage theory in a more general foim. To do so we suppose that a cell in stage i greater than zero produces a clone of similar cells, called an i-clone. Then we introduce the age x of an i-clone, which is just the

74

ALICE S. WHITTEMORE

time elapsed since its progenitor suffered its ith change. This theory assumes that whole clones can be eliminated by death, and that the occurrence of a transitional event in more than one cell of a clone is unlikely. The expected fraction per unit time of i-clones of age x in which further transition occurs is denoted by Zkx,t). These assumptions permit the calculation of the rate of transformation in the tissue, which is just the expected number of newly fornied k-clones in the tissue at time t . In the case when the transition and death rates are independent of age, the result agrees with that of the multistage theory described in Section V. Both Armitage and Doll (1957) and Fisher (1958) assumed that the death rate of i-clones of age x is negligible, and that the transition rate Zi(x,t) for intermediate clones is proportional to the size of the clone. They differed however in their assumptions about this size. B. THE ARMITAGE AND DOLL TWO-STAGETHEORY

The schematic diagram shown in Fig. 6 with k = 2 and with d, set equal to zero depicts this theory. Cells in stage zero are normal, those in stage one generate clones which are assumed to grow exponentially at some rate p, and clones in stage two are tumors. The assumption of exponential growth of one-clones means that the size of a one-clone of age x is ear. Here pllog 2 represents the expected number of one-cell doublings per unit time. The transition rate Zl(x,t) is taken to be proportional to one-clone size with proportionality factor Zl(t) depending on the carcinogen concentration at time t. Thus we may write

1 l(x,t) = Zl(t)ePf. (13) Armitage and Doll assumed that carcinogen concentration is a constant independent of time. Therefore the rate Zo of transition to stage one and the proportionality factor 1, on the right-hand side of (13)are constants. If tumor growth time is w , then the rate Nr(t) of tumor occurrence in the tissue is the rate at which two-clones are formed in the tissue at time t - w . Curves comparing human cancer mortality data with this predicted rate, for Armitage and Doll’s estimates of NZ, Z1, p and w = 2.5 years, are shown in Fig. 10. Unlike the incidence rate given according to the multistage theory by (8), the incidence rate predicted by this theory does not yield a straight line when plotted against exposure duration less w on a double logarithmic scale. Although the curves in Fig. 10 fit the data well, the assumption of exponential growth for one-clones leads to difficulties. Armitage and Doll’s estimates of /3 for cancer of the stomach, intestine, rectum, and

QUANTITATIVE THE:ORIES OF ONCOGENESIS 1000

75

A

Mortality rate: no. of deathdl06 man-yrs.

(log scale)

'k

4'0

;O

$5

u 25 30 40 50 65

Ace in years less 2 5 (log scale 1

FIG.10. Mortality rate in men ( X ) and women (0) for cancer of the stomach (A) and of the rectum (B) versus age in years less 2.5 years. The data were recorded in England in 1951-1955. The solid lines are theoretical curves predicted by the Armitage and Doll two-stage theory with proliferative advarctage of one-cells. The parameters of the theory were determined by eye-fitting the curves to the data points. Reprinted with permission from Armitage and Doll (1957).

pancreas are fairly uniform, with an average value of 0.13. This implies that the selective advantage for one-cells enables them to double only once every 5 years. Thus a one-clone would contain, on the average, only 16 cells after 20 years. Such clones, even if formed in an individual at birth, would not become detectable as hyperplasia or dysplasia in a human lifetime, since growth to detectable size (about 4 x lo6 cells) would take 110 years. C-msequently these clones cannot be interpreted as observable precancerous lesions. Fisher's variation of the theory, involving quadratic rather than exponential growth for intermediate clones, does not require such slow initial growth.

C. THE FISHERTHEORY We now consider the multistage theory with the size of intermediate clones proportional to the square of their age. This modification is motivated by the fact that the stem cells in the germinal layer of adult mammalian epithelium stay essentially constant in number, with half the daughter cells moving outward and dying without further division. Therefore the selective advantage of a nonmalignant clone of altered cells is likely to be small, and the clone might plausibly expand laterally like a circular disk at some slaw constant rate. The radius of such a clone of age x will be proportional to x, and its area proportional to x2. Thus Fisher's assumption that the transition rate ZXx,t) of intermediate clones of age x at time t is propcational to their size leads to Z,(x,t) = Zr(t)x2,

i = 1,

. . . , k - 1.

(14)

This theory is depicted schem,rtically in Fig. 6, with d i set equal to zero for all i, and with intermediate transition rates lAx,t) given by (14), i = 1, . . . ,k - 1. When the concentration of carcinogen is constant, 1,

76

ALICE S. WHITTEMORE

and the proportionality factors li(t) on the right-hand side of (14) are constant. I n this case the rate of transformation in the tissue is approximately proportional to the 3(k - 1)st power of exposure duration times the product lo, . . . , 1 k - l of all k transition rates. For constant growth time w and for duration times t greater than w , the incidence rate is then approximately proportional to

Comparison of (15) with (8) shows that for constant exposures the consequences of this theory differ from those of the multistage theory only in the value of the power of time in the expression for the incidence rate. Here fewer stages are required to yield the observed high power of time dependence. A two-stage process with quadratic proliferation of one-cells results in the incidence rate increasing as the third power of time, in agreement with the mouse skin tumor data of Lee and O’Neill. A three-stage process with quadratic proliferation of one-cells and two-cells results in the incidence rate increasing as the sixth power of time, in agreement with some human cancer mortality data. This theory can be extended to allow a quadratic proliferative advantage to only some intermediate cells, such as those in the penultimate stage. In general, if n of the k - 1 intermediate clones grow in proportion to the square of their age, then the -incidence rate varies as the (2n k - 1)st power of exposure duration less growth time w . Thus a three-stage theory ( k = 3) with quadratic growth for two-clones ( n = 1)results in the incidence rate increasing as the fourth power of time, in agreement with the lung cancer data of Doll.

+

D. APPLICATIONOF EXPOSURES

THE FISHER

THEORYTO TERMINATED

We have noted that for constant exposures and for suitable values of the parameters, the age dependence of the incidence rates predicted by the Fisher theory is the same as that of the multistage theory. For example, the multistage theory with w = 10 weeks, k = 4 and with two of the four stages affected by benzpyrene accounts for the chronic mouse skin painting data of Lee and O’Neill discussed in Section V,A. The incidence rates given b y this theory are also predicted by the Fisher two-stage theory with w = 10 and with both stages affected b y benzpyrene. However the two theories differ in their consequences for nonconstant exposures, and it is therefore of interest to examine the Fisher theory applied to the terminated exposure data of Lee, discussed in Section V,B. Figure 11shows the incidence rates observed in

77

QUANTITATIVE THblORIES O F ONCOGENESIS

TUMOR INCIDENCE RATE 0.01 (log scale)

- - - L

10

lo

50 200

10

50 200

10

50 200

10

r

50 200

I

/

TUMOR

I

(log scale) c = 60 pg/wk

''Oo1

10

50 200

c = 60 ug/wk

10

50 200

c = 60 ug/wk

10

50 200

c = 60 po/wk

10

50 200

TREATMENT TIME IN WEEKS -10 (log scale)

FIG 11. The data of Fig. 7 compared with incidence rates given by the Fisher twostage theory, with w = 10 weeks. Fsx b(c)= N / 3 (so +poc)(sl+pic), the values were obtained by b(36) = 4.46 x and b(60)= 21.56 x b(0)= 0.0215 x equating total expected and observed numbers of tumor bearing mice in the control, 36and 60-pg lifetime exposure groups, respectively. The values for pds0 of 1.35 and 1.76 were obtained by equating total expected and observed numbers of tumor bearing animals in the 36- and 6@pg tenninatcd exposure groups, respectively. The dashed lines indicate the predicted background rate.

Lee's experiment versus those of the Fisher two-stage theory with w = 10 weeks and with both stages affected b y benzpyrene. Comparison of Fig. 7 and 11 indicates two differences between the incidence rates of the two theories. First, termination of treatment is followed by a more abrupt decrease in the Fisher rates than by those of the fourstage theory. Second, although the rates of both theories approach the background rate asymptotically d t e r treatment stops, those predicted by the Fisher theory do so mole slowly. Despite these differences, both theories provide acceptablt: fits to the data.

E. INCREASED NUMBERSOF TARGET CELLS Iverson (1954) has proposed another explanation for the proportionality of human cancer incidence to such a high power of age. H e modified the single stage theory of Iverson and Arley by assuming that the expected number of normal target cells in the tissue increases

78

ALICE S. WHITTEMORE

with age according to a logistic equation. The resulting incidence rate fits the observed cancer mortality data for suitable values of the parameters involved. Nevertheless because of its ad hoc nature, this theory has not gained acceptance. There has also been evidence that young tissue is just as susceptible to neoplastic induction from a given treatment as older tissue. This evidence is clearly demonstrated by the recent experiment of Pet0 et al. (1975), discussed in Section V,B.

VIII. Single Stage or Multistage Theory with Variation in Transformed Cell Types

A. THE INITIATION-PROMOTION PHENOMENON In the early 1940s Berenblum, Mottram, and Rous discovered that exposures of rabbit and mouse skin to carcinogens at concentrations too low to produce any tumors will, when followed by repeated paintings of a noncarcinogenic substance called a promoter, yield large crops of tumors. It was found that the order of application is important: Few if any tumors result when promoter paintings are followed by application of carcinogen. Thus it is possible to divide the process of carcinogenesis in mouse skin into two stages: initiation, i.e., alteration of the cells in the tissue by a carcinogen, called the initiator, and promotion, i.e., development of the initiated cells to detectable tumors by the promoter. Because the quantitative theories described in the preceding sections do not postulate alteration of tumor growth rates by carcinogenic exposure, none of them can account for the regression of tumors that occurs after termination of promoter painting, or the lack of tumors that accompanies short exposures to high doses of promoter. The theory developed in this section generalizes many of the previously described theories, and, by including variable tumor growth which may be affected by exposure, it provides a bamework for examining initiation-promotion data.

B. GROWTHOF CLONES

OF

TRANSFORMED CELLS

We shall now assume that there are different kinds of transformed cells, indexed by a parameter a.This parameter indicates the “degree of malignancy” of the cell, which increases with a. Some theory of transformation is assumed to determine the rate at which a normal

QUANTITATIVE THEORIES OF ONCOGENESIS

79

target cell becomes a transfornied ceIl of type a. For example, the k-stage theory will do this if Zk-,(t)depends upon a. We next assume that each transformed cell of type a generates a clone of similar cells, which wc: shall call an a-clone. Let n(t,.r,a) be the number of cells at time t in an a-clone generated by a cell which was transformed at time 7 . Since a newly formed a-clone consists of one cell, n(t,t,a) equals one. We assume that each clone grows to a maximum size M. The clone’s rate of cellular increase or decrease per unit time is proportional to its size n reduced b y the factor (M - n)/M. This growth decelerating factor is the fractional part of its ultimate size to be contributed by remaining growth. The proportionality factor, denoted by u, and measured in cells gained or lost per clonal cell per unit time, is called the intrinsic growth rate of the clone. Both u and M increase with a and both depend upon the concentrations of the various agents to which the tissue is exposed. A clone is assumed to become a detectable tumor when it reaches some critical number of cells rn. Thus an a-clone transformed at 7 will become detectable at time t determined by

n(t;a-,a)= rn.

(16)

To complete the formulation of the foregoing theory of growth, we must specify the critical number of cells m, the intrinsic growth rate u [ a , &t)l and the ultimate size M [ a , c ( t ) ] . With these specifications, the rate Nr(t) and expected amount N R ( t ) of tumor occurrence in the tissue can be deduced from the preceding assumptions. This theory generalizes the single or multistage theory with constant growth time by postulating instead a distribution of growth times corresponding to different transformed cell types.

c. APPLICATION TO

INITIATION-PROMOTION

EXPERIMENTS

We shall now apply a special case of the theory to analyze the effects of a single dose of initiator at time t = 0 and a promoter of concentration c. Although the promoter may play a role in transformation, we shall assume that only the initiator affects transformation and that only the promoter affects growth. We take the initiator’s effect to be virtually instantaneous, so that shortly after its application the tissue contains an expected numberf(a) Iif transformed cells of type a. The distribution f(a) of transformed cell types, shown hypothetically in Fig. 12, depends on the dose and type of initiator, as well as on the susceptibility of the host. A transformed cell of type a is assumed to generate a clone which

80

ALICE S . WHITTEMORE

EXPECTED NUMBER f ( a ) OF TRANSFORMED CELLS OF TYPE a

at

GROWTH PARAMETER a

Ultmot.

EXPECTED NUMBER f ( a ) OF TRANSFORMED CELL! OF TYPE a

expc1.d number

of

..... .

. . .,. . . -dl)

-u(d 0 GROWTH PARAMETER a

FIG. 12. A hypothetical distribution of transformed cell types in mouse epithelium shortly after initiation. Application of promoter at dose rate c increases the growth rate of an a-clone by an additive factor u(c). (A) The total number of tumors per mouse that have appeared by time t is the number of transformed cells whose parameters exceed a,, where a,+ u(c) is the growth rate of a clone formed at initiation and appearing as a visible tumor at time t . (B) The total number of tumors that ever appear with promoter application at does rate c is the number of transformed cells whose parameters exceed -u(c), as shown by the dotted area under the curve. Higher dose rates c', which increase growth rates by a greater factor u(c'), produce more tumors, as indicated by the striped area under the curve. Clones generated by transformed cells with negative parameters regress upon termination of promotion.

grows to size M > m at intrinsic rate a in the absence of promotion, and at rate a + u(c) with promotion at concentration c. Hence in this example clonal size M is independent of a and c, and u(a,c) = a

+ u(c),

with u ( 0 )= 0.

(17)

Thus the effect of promotion is clonal growth acceleration either through increased proliferation, decreased cell loss, or both. To account for regression we assume that there are clones for which the growth rate a in the absence of promoter is negative. A clone with a deterministic growth rate a Iess than zero would decrease in size and tend to zero as time passes. Alternatively the growth of a clone might be a probabilistic birth-death process with a equal to the birth (proliferation) rate minus the death rate. In the latter case a clone might

QUANTITATIVE THII3ORIES OF ONCOGENESIS

81

grow, but its probability of becoming detectable would be very small. When promoter is applied, the growth rate is increased to a + u ( c ) with u(c) greater than 0. Consequently some clones can grow in the presence of the promoter while they would decrease in its absence. Let at be the parameter required for a clone formed at initiation to appear as a tumor at time t . Of course at depends on promoter concentration c. The expected number NR(t) of tumors that have appeared in the tissue by time t is precisely the total number of transformed cells in the tissue whose growth parameters exceed at. This is illustrated in Fig. 12A. The value of at can be determined from the fact that the number of cells in an at-clone lit time t is m. The value is

at = aft - u(c),

( 18)

where a is a constant depending on m and M . The ultimate expected number N R ( m ) of tumors that ever appear in the tissue is the total number of transformed cells in1 the tissue whose growth parameters exceed am,which by (18) is -u’c). This is because promotion at conr centration c increases all clonal growth rates b y the additive factor u(c). Thus all clones with parameters exceeding -u(c) have positive growth rates with such promotion, and will ultimately appear as tumors. This theory predicts that the total expected number N R ( m ) of tumors in a tissue increases with promoter concentration c, provided that u(c) increases with c. This is because higher promoter concentrations make positive the growth rates of clones with greater excess of death over proliferation rates. The situation is illustrated in Fig. 12B. Promoter concentration c’ is higher than c , and clones with parameters in the range from -u(c’) to -u(c) will appear as tumors when the concentration is c’, but not when it is c. We next examine the relationship between the expected numbers NR(t,c) and NR(t,c’) of tumors per tissue at two promoter concentrations c and c’, for which u(c’; is greater than u(c). The theory indicates that the number of tumors expected at time t with concentration c is simply the number expected with concentration c’ at the earlier time t/(1 b t ) , where b = [u(c’) - u(c)]/u, and a is the constant appearing in (18). This can be summai~-izedas

+

NR(t,c)= N R ( t / (1 + bt),c’). The relation (19)is compared in Fig. 13 with experimental data from the initiation-promotion experiments of Van Duuren et at., (1973). For a suitable value of b the lower two curves satisfy (19), and for another value of this quantity the uppei two curves satisfy it. Another test of

82

ALICE S. WHITTEMORE 18

15

AVERAGE NUMBER 10 OF TUMORS PER MOUSE 5

1

TIME SINCE START OF PROMOTION (WEEKS)

FIG. 13. The average number NR(t,c) of tumors per mouse as a function of time in weeks after the start of promotion at promoter dose rate c . The data points are from Van Duuren et al. (1973). A single dose of 5 pg of 7,12-dimethylbenz[a]anthracene(DMBA) was followed by thrice weekly applications of phorbol myristate acetate (PMA) at three different dose rates in micrograms per application: 0.5(0),2.5(.), %(A). The two upper curves, for which c’ = 25 and c = 2.5, satisfy (19) with b = 0.05/week for t < 1/ 0.05 weeks. The two lower curves, with c’ = 2.5 and c = 0.5, satisfy (19) with b = 0.06/ week for t < U0.06 weeks. The upper curve was obtained by a least squares fit of the hnctionf(x) = 17 [ l - exp{-B(x - 5) + C(x - 5)*}]to the data; the values B = 0.083/wk and C = 0.0021wk2 gave the best fit. The-arrows are at the levels NR(U0.06, 2.5) and NR( 110.05, 25), which would be the asymptotes of the curves NR(t, 0.5) and NR(t, 2.5), respectively, if the above relations held at these values o f t .

(19) is shown in Fig. 14 based on experimental data of Burns (1976). Again a suitable choise of the constant b makes the curves forc = 1 and c’ = 2.5 satisfy (19). These two comparisons of (19) with experimental data lend some support to this theory. We shall now show how the theory can account for regression, as shown in Fig. 14 b y the curve NR,(t) and the associated data points. When the concentration is suddenly decreased from c to 0, those clones with a in the range 0 > a > - d c ) have their growth rates reduced from the positive value a u(c) to the negative value a.Therefore, these clones start to decrease. Any of them which had already grown to be detectable tumors regress and ultimately become undetectable. This explains the gradual decrease of the curve N R d t ) in Fig. 14 after the promoter concentration is reduced to zero at t = 42 days. We have seen that the total expected number NR(w,c) of clones

+

QUANTITATIVE THEORIES OF ONCOGENESIS

AVERAGE NUMBER OF TUMORS PER MOUSE

83

lo

TIME SINCE START OF PROMOTION (DAYS)

FIG.14. The average number NR(t,c) 3f tumors per mouse in each of three groups, as a function of time in days after the start cif promotion. A single 25-pg dose of the initiator DMBA was followed by thrice weekly applications of the promoter PMA. The PMA was applied to each group at one of the following dose rates c(t) in micrograms per application: c(t) = l(A); c(t)= 2.5(.); c(t) = L5, 0< t < 42, c(t) = 0, 42 < t < 100, c(t) = 2.5, 100 < t < 172,c(t) = 10,172 < t < m, ( 0 )The thickness ofthe horizontal bar indicates the dose rate of promoter applied to the third group. The curves N R , and NR, satisfy (19) with b = 0.004Yday for t < l/0.0042 clays. The curve N R , was obtained by a least squares fit of the hnction f ( x ) = 12 I1 - exp{-B(x - 36)}] to the data; the value B = .OSl/day gave the best fit. The curve N R s represents a visual fit to the data for the third group. The data points are kom Ewns (1976).

which ever become detectable tumors increases with promoter concentration c, provided that u(c) increases with c. Thus higher concentrations of promoter produce more growing clones. This explains why the curve NR,(t) in Fig. 14 increases from one level to another when c is increased at t = 100 days and again at t = 172 days. It also explains another phenomenon observed ,experimentally but not shown in Fig. 14. The phenomenon is that telmination of promotion at higher concentrations produces a greater j+action of regressed tumors. This can be explained because higher coricentrations c increase the range -u(c) < a < 0 of clones which decrease without promotion. This and the above qualitative agreement between the theory and the data provide further support for the theory, and concludes our discussion of it. IX. Implications for Dose-Response Relationships

A. THE PROBLEM Estimates of human risk (lifetime probability of cancer) &om exposures to low doses of carcinogenic agents often involve the use of animal experiments conducted at doses sufficiently high to produce

84

ALICE S. WHITTEMORE

tumors in an appreciable fraction of the animals tested. There are two major sources of uncertainty in obtaining such estimates. The first concerns the mechanism relating animal risk at high doses to animal risk at low doses. The second is the role of interspecies differences in extrapolating from animal to human risk. Risk estimates at low doses based on experimental results at high doses are extremely sensitive to the shape of the curve relating risk to dose. Thus it is desirable to obtain such estimates using a doseresponse curve which is based on a biologically plausible theory. The multistage theory, which is supported b y some experimental and epidemiological evidence, has been proposed b y many investigators. For chronic exposures to chemical carcinogens, Guess and Crump (1977)use a procedure that includes the predictions of the multistage theory, with the number of dose-related stages determined by the data. Their method indicates that animal experiments are not likely to establish upper bounds for risk estimates that decrease with dose at a faster than linear rate. A serious difficulty with reliance on quantitative theories of cancer mechanisms to determine dose-response relationships is the possible discrepancy between applied dose and cellular concentrations. The relationship between tumor production and actual cellular exposure may be obscured b y nonlinearity between cellular .and applied concentrations, For example White (1972), using urethane labeled with radionuclides, estimated the internal dose to a mouse over the 24-hour period subsequent to injection of a single dose of urethane. Her estimates of internal exposure, measured in milligram-hours per gram of body weight, are shown plotted against injected dose in Fig. 15A. It is evident that these estimates of internal exposure are quadratic rather than linear functions of injected dose. Plots of average number of tumors per mouse versus injected dose and versus estimated internal exposure are shown in Fig. 15B and C, respectively. They indicate that while tumor numbers depend quadratically on injected dose in agreement with the observations shown in Fig. 3, they vary nearly in proportion to estimated internal exposure. Thus a theory tailored to fit the observed quadratic external-dose-response relationship is likely to be an inappropriate description of the tumor producing mechanism, and as such could seriously underestimate risk at low doses of urethane. The relationship between estimated dose and cellular concentration is even more tenuous in epidemiological studies. The difficulty suggests that future modeling efforts should emphasize incorporation of physiological, pharmacological, and biochemical information. The

QUANTITATIVE THECIRIES OF ONCOGENESIS

85

30 -

A

25 -

Internal exposure (mg-hrs/gm) 15 (cumulative to 24 hrs)

o.o '

40 -

i

10 -

5-

B

ii

20 -

7

7

I

I

Ic

Mean number of tumors per mouse

'(Id .I 0

5

10

Internal exposure (mg-hrslgrn)

FIG. 15. (A) Internal exposure versus injected dose in mice treated with ethyl carbonate (carbonyl-"C). Internal exposure, measured in milligram-hours per gram weight of mouse, was estimated by computing the areas under the curves giving amount of expired and eliminated I4Catom as a function of tinie after urethane injection, for times up to 24 hours. The calculations assume that at any instant the amount of unrecovered '*C atom is still in the animal. The quadratic relationship between estimated internal exposure and injected dose observed in (A) is supported by the plots of mean number of tumors per mouse versus injected dose (B) and versus c5stimated internal exposure (C). The straight lines in (B) and (C) connect extreme poinis. It is evident from (B) and (C) that tumor yield varies quadratically with injected dose and linearly with estimated internal exposure. Reprinted with permission from White (1972).

problem has received considerable attention for radiation carcinogenesis (e.g., Mayneord and Clarke, 19i5) and for chemotherapeutic agents (e.g., Bischoff, 1977). However there is need for study of the distribution, excretion, and metabolism of' chemical carcinogens for the purpose of establishing effective dose ;in experimental work. The model of Swartz and Spear (1975) for the metabolism of hydrocarbons in mouse skin carcinogenesis represents a slep in this direction.

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ALICE S. WHITTEMORE

Models that include pharmacological and biochemical information would also be useful in reducing uncertainty due to interspecies differences. Furthermore their predictions for changes in internal exposure with different dosage schedules may explain anomalous experimental findings concerning the reducing and enhancing effects of dose fractionation. Klonecki ( 1976) has proposed another explanation for changes in tumorigenicity with fractionation: Changes occur because initial applications of fractionated dose alter the number of target cells in susceptible phases of the cell cycle.

B.

RADIATION CARCINOGENESIS

A full discussion of the dose-response curve and the effects of dose fractionation and protraction for radiation carcinogenesis is beyond the scope of this paper. Reviews of the subject are provided by Brown (1976), Shellabarger (1976), and the National Council on Radiation Protection and Measurements (1975). The following summary points of these reports are of interest. For low energy transfer (LET) radiation (x-rays and gamma rays), the form of the dose-response relationship varies widely depending on species and tumor site. Moreover, dose fractionation and protraction generally reduce tumor risk. For high LET radiation (neutrons) the dose-response relationship appears to be linear, and lengthening the exposure time of a given dose does not appear to reduce tumor yield. The differences between high and low LET radiation have been studied in terms of relative biological effectiveness (RBE), which is the ratio of low L E T radiation dose to high LET radiation dose for doses yielding equal tumorigenic effect. Experimental results indicate that RBE increases with decreasing neutron dose. All of these findings suggest that the carcinogenic effects of low LET radiation may require more than one radiation related event, while high LET radiation may induce tumors b y a one-stage mechanism. However there are human data indicating a linear dose-response curve for low LET radiation. X. Conclusion

Some of the defects of the theories discussed in the preceding sections are quite evident. For example the crucial distinction between benign and malignant tumors is ignored by many of them. Furthermore, none of them has included the possibility of cell repair or the action of the host’s immune system. Many of the assumptions underlying the theories represent extremely simplified versions of reality. An example of this is the as-

QUANTITATIVE TI-iEOFUES OF ONCOGENESIS

87

sumption that the target cells of a tissue are fixed in number and independent of each other with respect to likelihood of transformation. In addition, the sensitiviiy of target cells to transformation, as measured by the constants s i and p i of (7), is assumed constant. However, it may well vary with cell cycle time, with location in the tissue, and with hormone balance, level of immune response, and other factors in the cellular environment. Although we have presented the theories as deterministic, all of them were formulated as descriptions of the probabilities of transformation and tumor appearance. The quantities described here are the expected values of tumor rates and numbers. Most of the theories have emphasized these expected values and have not stressed variances and other statistics of the tumor incidence rates. To extract additional information fiom these statistics it will be necessary to consider two kinds of variation. One results from the stochastic processes within each individual, which have been treated in the present theories. The other is due to variation from one individual to another because of differences in susceptibility. It would be desirable to separate these two kinds of variation, and to determine what part of the observed variation each contributes. There is no reason why one thieory should apply to all tumor sites, to all agents, and to all species. It :eems likely that many different mechanisms are operating, and the theories proposed to describe them should reflect this variety. Despite all of their limitations, the single and multistage theories provide a flexible, broad, and biologically plausible fiamework in which to examine the gross behavior of oncogenesis data. They also providle a base for the development of investigations concerning the complicoating factors mentioned above, so that underlying mechanisms can b e elucidated. ACKNOWLEDGMENTS I wish to thank Norton Nelson for sug,jesting this review, and Bernard Altshuler and Joseph B. Keller for many helpful discussions related to it. REFERENCES Altshuler, B., Klassen, W., Troll, W., and Orris, L. (1971).Proc. Am. Assoc. Cancer Res. 12, 49. Armitage, P., and Doll, R. (1954). Br. J . ICancer 8, 1-12. Armitage, P., and Doll, R. (1957). B r . J . 17ancer 11, 161-169. Armitage, P., and Doll, R. (1961). In “Pioceedings of the Fourth Berkeley Symposium on Mathematical Statistics and Prot8ability” (J. Neyman, ed.), Vol. IV, pp. 19-38. Univ. of California Press, Berkeley. Bischoff, K. B. (1977). In ‘‘Environments1 Health: Quantitative Methods” (A. Whittemore, ed.), pp. 3-12. SOC. Itid. Appl Muth., Philadelphia, Penn\ylvania.

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Blum, H. F. (1959). “Carcinogenesis by Ultraviolet Light.” Princeton Univ. Press, Princeton, New Jersey. Brown, J. M. (1976). Health Phys. 31, 231-245. Burch, P. R. J. (1960).Nature (London) 185, 135-142. Burns, F., Vanderlaan, M., Sivak, A., and Albert, R. E. (1976). Cancer Res. 36, 14221427. Clemmesen, J. (1974).Acta. Pathol. Microbiol. Scand. S u p p l . 247, 1-255. Doll, R. (1971).J. R . Statistic. SOC. 134, 133-166. Doll, R., and Peto, R. (1976). Br. Med. J. 2, 1525-1536. Fisher, J. C. (1958). Nature (London) 181,651-652. Fisher, J. C., and Holloman, J. H. (1951). Cancer 4 , 9 1 6 9 1 8 . Guess, H. A., and Crump, K. S. ( 1977).In “Environmental Health: Quantitative Methods (A. Whittemore, ed.), pp. 13-30. SOC.Itid.A p p l . Math., Philadelphia, Pennsylvania. Hoel, D. G., and Walburg, H. E., Jr. (1972).J.Natl. Cancer Inst. 49,361-372. Iverson, S. (1954). Br. /. Cancer 8,575-584. Iverson, S., and Arley, N. (1950).Acta Pathol. Microbiol. Scand. 27, 773-803. Jones, H. B., and Grendon, A. (1975). Food Cosmet. Toxicol. 13,251-268. Klonecki, W. (1976).Zastosow. Mat. A p p l . Math. 15, 163-186. Lee, P. N. (1975). Tob. Res. Counc. Rev. Act. 1970-1974 (London), pp. 28-32. Lee, P. N., and O’Neill, J. A. (1971). Br. J. Cancer 25, 759-770. Marshall, J. H., and Groer, P. G. (1977). Radiat. Res. 71, 149-192. Mayneord, W., and Clarke, R. H. (1975). Br. J. Radiol. S u p p l . 12, 1-112. Muller, H. J. (1951). Sci. Prog. 7, 93-493. National Council on Radiation Protection and Measurements (1975). “Review of the Current State of Radiation Protection Philosophy,” Rep. No. 43. NCRPM, Washington, D.C. Neyman, J., and Scott, E. (1967).I n “Proceedings of the Fifth Berkeley Symposium on Mathematical Statistics and Probability” (L. M. Le Cam and J. Neyman, eds.), Vol. IV, pp. 745-776. Univ. of California Press, Berkeley. Nordling, C. 0. (1953). Br. J. Cancer 7,68-72. Peto, R., Roe, F. J. C., Lee, P. N., Levy, L., and Clack, J. (1975). Br. J. Cancer 32, 41 1-426. Platt, R. (1955). Lancet 1,867. Scotto, J., Kopf, A. W., and Urbach, F. (1974). Cancer 34, 1333-1338. Shellabarger, C. J. (1976). Cancer 37, 1090-1096. Shimkin, M. B., and Polissar, h4. J. (1955).J. Natl. Cancer Inst. 16, 75-93. Stocks, P. (1953).Br. J . Cancer 7,407-417. Swartz, J., and Spear, C. (1975). Math. Biosci. 26, 19-39. Van Duuren, B. L., Sivak, A., Segal, A., Seidman, I., and Katz, C. (1973).Cancer Res. 33, 2 166-2172. White, M. (1972). I n “Proceedings of the Sixth Berkeley Symposium on Mathematical Statistics and Probability” (L. M. Le Cam, J. Neyman, and E. L. Scott, eds.), Vol. IV, pp. 287-307. Univ. of California Press, Berkeley. White, M., Grendon, A,, and Jones, H. B. (1967).In “Proceedings of the Fifth Berkeley Symposium on Mathematical Statistics and Probability” (L. M. Le Cam and J. Neyman, eds.), Vol. IV, pp. 721-743. Univ. of California Press, Berkeley. White, M., Grendon, A., and Jones, H. B. (1970).Cancer Res. 30, 1030-1036. Whittemore, A., and Keller, J. B. (1978).SOC. Ind. A p p l . Math., Reu. 20, 1-30. Wright, J. K., and Peto, R. (1969). Br. J. Cancer 23,547-553.

ADVANCES IN CAhCER RESEARCH, VOL. 27

GESTATIONAL TROPHOBLASTIC DISEASE: ORIGIN OF CHORIOCARCINOMA, INVASIVE MOLE AND CHORIOCARCINOMA ASSOCIATED WITH HYDXTIDIFORM MOLE, AND SOME IMMUNOLOGIC ASPECTS' J. I. Brewer, E. E. Torok, B. D. Kahan2, C. R. Stanhope, and B. Halpern Department of Obstetricsand Gynecology, and lhe Cancer Center, Northwestern University Medical School. Chicago. Illinois

I. The Origin of Gestational Chorioca.rcinoma .............................. 89 A. Some General Clinical and Pathologic Aspects ....................... 89 B. Choriocarcinoma Arising durinK Seemingly Normal Pregnancy 97 with Fetus ........................................................ C. Comment ......................................................... 121 11. Invasive Mole and Choriocarcinoma Associated with Hydatidiform 125 Mole ................................................................. A. Incidence of Invasive Mole and Choriocarcinoma Associated with 125 Hydatidiform Mole ................................................ B. Follow-up of Patients after Evai:uation of Hydatidiform Mole .......... 132 111. lmmunobiology of Trophoblastic Disease ................................ 138 A. HLA Antigens and Trophoblastic Disease ............................ 138 B. ABO Blood Group Antigens in Trophobfastic Disease ................. 140 142 C. Native Host Reactions toward Choriocarcinoma ....................... D. Immunotherapy Trials .............................................. 143 145 References ............................................................

I. The Origin of Gestational Choriocarcinoma

A. SOME GENERALCLINICALWD PATHOLOGIC ASPECTS Recent advances have provided reasonable answers to many of the questions dealing with the origin and the primary lesion of gestational choriocarcinoma. These answers are to be found by the study of choriocarcinoma arising during 21. seemingly normal pregnancy which is accompanied by a fetus and by bringing together the observation and information provided in isolated reports. Positive answers are difficult to obtain in choriocarcinoma associated with hydatidiform mole and abortion. Much more concrele evidence of the origin and the early development and characteristics of choriocarcinoma can be obtained Supported by Grant Number CA-121Q9awarded by the National Cancer Institute, DHEW. Department of Surgery, Northwesterr University Medical School, Chicago, Illinois. 89 Copyright 0 1978 by Academic Press. Inc. All rights of reproducbon in- -any - .form reserved.

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and there is less need for interpretation and postulation in the former than in the latter. It is for these reasons that this presentation is restricted to a review of choriocarcinoma arising during prenancy that is accompanied by a fetus. It has long been believed that gestational choriocarcinoma almost invariably originates as a sequel to hydatidiform mole, abortion, term gestation, or ectopic pregnancy rather than having its origin within the conceptus during the course of the pregnancy. The last mentioned, although observed, has been thought to occur quite infrequently. Hertig (1968, p. 290) concluded that most choriocarcinoma following normal pregnancy must arise from bits of residual adherent trophoblast, focal placenta accreta, placental giant cells, or normal intravascularly growing trophoblast of the placental site, and that the vast majority of these patients destined to develop choriocarcinoma show clinical evidence of retained secundines. He also gave consideration to the possibility that the tumor could arise within the conceptus, rather than from retained trophoblast that underwent subsequent malignant change, but commented this was not usually subject to proof. Park (1971, chapter VIII, p. 134) stated it is customary to hold that choriocarcinoma may be a sequel to any type of pregnancy and noted that, as the clinical events usually unfold, choriocarcinoma does appear to follow a pregnancy. He added that in reality choriocarcinoma is itself a form of pregnancy, at least the later installment of the gestational process. Park and Lees (1950)stated that choriocarcinoma may arise at any time during the life of the trophoblast. Park (1971)raised the theoretical possibility that the zygote itself might be the first malignant cell, and Acosta-Sison (1955) offered the possibility that the trophoblast of the very early ovum might be malignant. Park (1971, p. 177) accepts the possibility that choriocarcinoma can arise in the placenta of a seemingly normal pregnancy, and also that it may arise by malignant transformation in normal trophoblast originally deported in physiologic fashion from the placenta, the neoplastic transformation taking place after the castingoff of the still normal parent tissue. He further has postulated that, in some instances in which the lesion is thought or considered to be gestational in origin, the neoplasm actually arises in a teratoma. Proof of each and all of these possibilities has been difficult to obtain. Park (1971)has raised a point which, if the answers were known, would add greatly to our knowledge of choriocarcinoma, namely, what is the time at which and how much of the trophoblast becomes a malignant neoplasm. Solutions to these problems and answers to the numerous questions have been difficult to come by because of the many varied clinical and pathologic aspects of choriocarcinoma. It has not been proven that normal trophoblast retained within the

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uterus after termination of a pregnancy or in a distant site to which it has been transported can b e transformed within a few days or weeks into choriocarcinoma. The chief support for this happening is the clinical sequence of events leading to the diagnosis of choriocarcinoma, nafiely, the delivery of the products of conception, abortion, term gestation, or molar pregnancy, followed by abnormal uterine bleeding and/or symptoms produced by metastatic disease. The uterine bleeding in one-half of the patients in which it occurs is of a continuous type from the time of the abortion, term delivery, or evacuation of the hydatidiform mole as noted by Ober et al. (1971). I n the patients he reported on, the diagnosis of choriocarcinoma was made within 2-8 weeks by curettage, which were performed for clinically presumed retained placental fragments. It ir not tenable to hold to the contention that in these patients the neoplasm arose and developed to full-blown tumor in 2-8 weeks, It is more realistic to believe that the tumor was present at the time the pregnancy was terminated and that it took 2 to 8 weeks for the clinician to establish the diagnosis. Certainly all those working with and studying this iumor know that it takes time for the clinician to become suspicious of the presence of this infrequent neoplasm and to accomplish the procedures leading to its diagnosis. Even a time longer than 8 weeks may bce required, since approximately onehalf of the patients in whom the choriocarcinoma appears to have developed after termination of the pregnancy has short or prolonged periods of amenorrhea following the pregnancy event before abnormal bleeding occurs or a metastatic lesion makes itself evident. The existence of amenorrhea does not arouse the suspicion of the clinician and the interspersed episodes of bleeding, if they occur, may be thought to be a return of irregular menstruation, or now more commonly to be the side effects of the contraceptive hormonal medication that is so frequently administered. This leads to a delay in diagnosis. Delays in diagnosis are encountered quite often and this makes it difficult to prove in some instances that choriocarcinoma did exist prior to the termination of pregnancy and that it produced the postpartum symptoms. There may be a prolonged timi: after termination of a gestation before a diagnosis of choriocarcinoma is made in patients who had no symptoms that might indicate the possible presence of a neoplasm. In most such instances, especially in those cases in which the time element is several months or years, the question of an intervening unrecognized new pregnancy with abortion giving rise to the choriocarcinoma always must be considered, and this clouds the issue. However, upon occasion, there is a sequence of events in a patient with this lesion that permits a more clear interpretation.

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The failure to find a primary lesion in the uterus in a patient with disseminated choriocarcinoma creates problems in the interpretation of the sequence of events since it would usually be expected that a primary lesion could be identified. However, in gestational choriocarcinoma with metastasis, it is not unusual for there to be no lesion found in the myometrium or endometrium. In a review of 80 patients with gestational choriocarcinoma registered in the Albert Mathieu Chorionepithelioma Registry, in whom the initial treatment was hysterectomy, J. I. Brewer (unpublished, 1962) found no residual tumor within the uterus in 12%of patients who had metastatic disease. Hertig (1968, p. 308) reported that, in postmolar patients who developed metastatic choriocarcinoma, the uterus when removed surgically or at autopsy was apparently free of choriocarcinoma in 50% of these patients. Hunter and Dockerty (1955)noted there was no primary lesion found in the uterus in 10-33% of patients who died of metastatic choriocarcinoma. Similar observations as these were reported by Hou and Pang (1956) and Mhrquez-Monter et al. (1968). Teacher (1903) recorded this occurrence and Schmorl ( 1904) proposed that “ectopic” choriocarcinoma might have as its primary source a chorionepitheliomatous lesion of a placenta completely expelled during labor, before the uterus is directly attacked but not until metastases had commenced. Later this was proved to occur (Brewer and Gerbie, 1966). A clear-cut explanation for this happenstance, which will further clarify and solve the problem, is provided later in the review presented here. In the detection, diagnosis, and follow-up and in the monitoring of therapy of patients with choriocarcinoma, the determination of the presence of and the quantity of human chorionic gonadotropin (hCG) is of utmost importance. In the past, as brought out in this review of case material going back over many years, the methods used were not adequately sensitive to measure low quantities of the hormone. This obviously compounded the clinical problems. Brewer and DeCosta (1967) analyzed the results obtained with the rather insensitive tests available in the past in 26 patients who developed histologic proven choriocarcinoma in association with hydatidiform mole. The tests were performed serially from the time the molar pregnancy was diagnosed and terminated to the time the diagnosis of choriocarcinoma was made. In the 26 patients, the test was positive at all times tested in only 4,was negative at all times tested in 6, and negative one or more times in 16. Inaccuracies of this magnitude make it mandatory to use more highly sensitive test methods, especially when low quantities of the hormone are present. From the standpoint of pathology there are many features that may

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create difficulties in making the histopathologic diagnosis of choriocarcinoma, in identifying the time at which the trophoblast becomes a malignant neoplasm, and in determining how much of the trophoblast is transformed to malignancy. The last two are often made difficult owing to a delay on the part of the clinician in becoming suspicious of the possible presence of choriocarcinoma and in performing diagnostic procedures, especially when the neoplasm is associated with term pregnancies and abortions. His index of suspicion is much greater and his action is more prompt in cases of choriocarcinoma associated with hydatidiform inole, but in this situation pathologic determinations are more difficult to make. Since uterine bleeding is the most frequent symptom produced b y the neoplasm, a curettage specimen is usually the first tissue submitted to the pathologist. The probllems encountered in making the diagnosis from a curettage specimen are well recognized. If the curettage was performed for bleeding after a term pregnancy or a pregnancy terminated in the later stages of the gestational period prior to term, and if the specimen contains masses of malignant-appearing trophoblast but no chorionic villi, the diagnosis is quite clear. If the curettage was performed for bleeding following an abortion, the trophoblast often is bizarre and poses a proldem of interpretation, especially if no villi are present. Usually this is a benign process as borne out b y the clinical progress of the patient. Villi, if present with such bizarre trophoblast, induce the pathologist to conclude with more assurance that the trophoblast even though abnormal is benign. However, this assurance cannot be complete a i exemplified b y a patient reported by Schumann and Voegelin (1937). The patient, 23 years of age, had had two term pregnancies and 5 years prior to the current pregnancy had had a hydatidiform mole, which was terminated without evidence of immediate or remote complications. The current new, apparently normal pregnancy ended in abortion with bleeding for which a curettage was performed. Microscopically, the tissue consisted of degenerated chorionic villi and “nests of abnormal syncytium.” Two months later, because of continued uterine bleeding another curettage was performed. The pathologist reported microscopic findings as being the same as those of the first curettage specimen. Six months later, uterine hemorrhage occurred and a nec I-otic mass projecting through the cervical 0s was found and removed. The pathologic diagnosis was choriocarcinoma. Chorionic villi were not identified. The patient subsequently died of choriocarcinoina, metastatic to the brain. This case not only demonstrates the probllems that arise but also lends insight into the time in pregnancy at which the neoplasm has its origin. The sequence of events starting with the nonmolar pregnancy, the pre-

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sumptive diagnosis of abortion with subsequent curettage which revealed abnormal trophoblast, and the persistence of symptoms and abnormal trophoblast until the diagnosis of choriocarcinoma was finally made strongly suggests that the neoplasm arose during the course of the pregnancy but was unrecognized pathologically. It demonstrates the difficulty in evaluating the future potential of the trophoblast. Without doubt, the presence of chorionic villi, even though degenerated, in the curettage specimen negated the diagnosis at the time of the abortion. While the presence of villi do detract from making the diagnosis of choriocarcinoma, it has been shown that villi are present in some stages of the disease, as will be documented later in this presentation. A personal appraisal of the illustration in the published article of Schumann and Voegelin (1937) leads to the conclusion that the trophoblast was malignant. It might be postulated that the choriocarcinoma in this case, which was diagnosed 8 months after the abortion, might have arisen in a new but undetected pregnancy, or that it might have arisen in persistent trophoblastic remnants of the benign hydatidiform mole which had occurred 5 years earlier. Neither of these seem good possibilities in view of the clinical course and the pathologic findings. The tissue obtained by curettage in some instances may reveal, upon histologic examination, individual cells and masses of trophoblast that have invaded the myometrium to an extent exceeding that usually observed in syncytial endomyometritis. Distinguishing this from choriocarcinoma is difficult, particularly if there are no villi in the specimen. In the clinical management of such a case, it is essential that serial testing for chorionic gonadotropin be conducted and that extremely sensitive test methods be utilized. A curettage specimen may consist of a large amount of blood and clots with a small cluster of abnormal trophoblastic cells, so meager that the pathologic diagnosis cannot be made with certainty. Both the clinician and the pathologist desire, in such cases, to have more tissue to examine, and, if a vaginal lesion is present, there is an urge to take a biopsy of it. This is an ill-advised procedure, since postbiopsy hemorrhage is frequently severe and is extremely difficult or even impossible to control, and death may result. The following account presents an example of this risk. The patient, age 33 years, had had a normal term delivery of a normal infant prior to becoming pregnant a second time. This second pregnancy ended in hemorrhage and abortion at 6 weeks gestation. A curettage was performed. Only two small clusters of abnormal appearing trophoblastic cells were in the curettage specimen (Fig. 1A). No chorionic villi were present. A diagnosis of choriocarcinoma was not made on these few cells. Four months later the patient

FIG.1. (A) One of only two clusters of zlbnormal hophoblast obtained by curettage for presumed incomplete abortion at 6 weeks. A diagnosis of choriocarcinorna was not made. (B) Four months later biopsy of :I vaginal lesion showed metastatic choriocarcinoma. The patient died of hemorrhage from the biopsy site.

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returned complaining of amenorrhea since the curettage, hematuria and vaginal bleeding. Examination revealed several purple vaginal lesions, multiple pulmonary metastatic lesions, tumor infiltrating the urinary bladder wall and a positive pregnancy test. A curettage and biopsy of a vaginal lesion (Fig. 1B) were performed, both revealing choriocarcinoma. Severe, uncontrollable bleeding ensued from the vaginal tumor biopsy site, and two days later, death from hemorrhage and shock occurred. A curettage may in other ways fail to provide a diagnosis. The curet may miss a small lesion or a critical portion of the lesion. On the other hand, at the time of curettage, the lesion may no longer be within the cavity of the uterus but rather within the myometrium beyond the reach of the curet or in some distant metastatic site. One thing that has made it possible to gain essential information and insight into the time of and the manner in which choriocarcinoma develops is the extensive and detailed pathologic examination of the placenta of term or near-term gestations in pateints who harbor the disease. The clinician and the pathologist are usually hindered in such examinations because the disease is not suspected at the time of delivery, and, when symptoms suggestive of disease occur, the placenta has long since been discarded. Furthermore, there is often no alteration in the gross characteristics of a placenta in which there is a choriocarcinorna. Extensive examination of all placentas is totally impractical, however desirable it might be. Occasionally, microscopic examination of the placenta may reveal an unsuspected early choriocarcinoma, such as reported by Driscoll (1963) and shown in Fig. 2. This early lesion had not extended into the maternal tissues and resolution of the disease was obtained by the spontaneous delivery of the placenta. In specimens other than a curettage specimen, it is equally true that the presence of chorionic villi with malignant-appearing trophoblast also leads the pathologist away &om the diagnosis of choriocarcinoma because it has been generally accepted that the absence of villi is one of the criteria needed for the diagnosis of this neoplasm. The usually accepted histopathologic criteria for the diagnosis of choriocarcinoma, namely, the presence of malignant trophoblastic cells, absence of chorionic villi, and hemorrhage and necrosis in maternal tissues, were developed and established by study of advanced disease, basically from specimens obtained at postmortem examinations. Inadequate consideration has been given to the possibility and probability that early stages of the neoplasm may have different histopathologic characteristics. One difference as shown b y Driscoll (1963) and b y Brewer and Gerbie (1966) is that villi are present. As Hertig (1968, p.

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FIG.2. A small primary lesion of choriocarcinomaarising in the trophoblast of villi (C) in an otherwise normal placenta (P) and completely contained within the placenta. It was revealed incidentally by microscopic examination of the expelled placenta. Specimen supplied by S . G. Driscoll, M.D.

294) correctly stated, it is doubtful that this malignancy follows a different pathway in its development from that of other cancers, and it is doubtful that it arises and develops suddenly as a full-fledged cancer. It is realistic to believe that an early stage exists. The best means of gaining factual information is b y the study of patients who have choriocarcinoma in seemingly normal pregnancies with fetuses present and b y detailed examination of the placentas.

B.

CHORIOCARCINOMA ARISINGDURING SEEMINGLY PREGNANCY WITH FETUS

NORMAL

A review of the literature and our own material reveals 29 cases of choriocarcinoma occurring during a pregnancy with a fetus suitable for study and evaluation (Bagshawe, 1969; Brewer and Gerbie, 5 cases, 2 published, 1966, 3 unpublished; Buckell and Owen, 1954; Cordua, 1949; Daamen et al., 1961; Dafoe, 1939; Davis and Brunschwig, 1936; Douglas and Otts, 1949; Driscoll, 1963; Emery, 1952; Felton and Smith, 1966; Fikentscher, 1941; Gusenleitner, 1954; Hutchinson et

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al., 1968; Lepow, 1959; MacRae, 1951; Maun and Green, 1943; Mercer et al., 1958; Resnick, 1945; Ringertz, 1970; Stahmann, 19S7; Steigrad et al., 1968; Walthard, 1907; M. Wheelock, personal communication; Witzleben and Bruninga, 1968). It should be obvious that all cases which occur are not published; we have personal knowledge of two additional cases managed at other institutions that have not been published. J. L. McKelvey (personal communication, 1965) described one case to us but did not publish it. In these cases, the disease was diagnosed during the gestation or immediately or very soon after delivery. Many cases diagnosed more remotely after termination of the pregnancy are not included in this report, since the time of origin of the neoplasm is more difficult to demonstrate and the needed interpretation of the sequence of events is subject to more wide variations. While this selection reduces the number of cases for consideration, it makes the data more meaningful and the interpretations more firm. For presentation, these 29 cases are divided into 3 groups: (1)those in which choriocarcinoma was shown to arise in the trophoblast of formed villi in an otherwise normal placenta, 15 cases; (2) those in which choriocarcinoma was present in the myometrium but was not identified in the placenta, 8 cases; and (3) those in which choriocarcinoma was not identified in either the uterus or placenta, but metastatic gestational choriocarcinoma was present, 6 cases.

1. Choriocarcinoma Arising in the Trophoblast of Chorionic Villi in an Otherwise Normal Placenta: 15 Cases It is in these 15 cases that the primary lesion can be identified, the site of origin of choriocarcinoma can be demonstrated, the association with chorionic villi in earlier stages of the neoplasm can be documented, and the explanation for the absence of villi in later stages of the disease can be provided. Five of the 15 cases were personally studied at our Trophoblastic Disease Center. Two of these 5 cases, which have been published (Brewer and Gerbie, 1966), will be reviewed briefly here, since they demonstrate the essential features; the other 3 cases had the same characteristics. In the first patient, during the fifth month of pregnancy, a bleeding, friable, hemorrhagic lesion appeared in the vaginal wall adjacent to the urethra. Biopsy revealed malignant-appearing trophoblast without chorionic villi. Eight days later, multiple bilateral pulmonary lesions were identified on roentgen examination. Abdominal complete hysterectomy and bilateral salpingo-oophorectomy were performed and the live fetus was removed from the extirpated uterus.

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The placenta and uterus appeared normal upon gross examination. The pathologist, at the hospital involved, fixed the entire uterus with the contained placenta, and then cut blocks and prepared microscopic slides from multiple blocks. This initial examination revealed normal villi and normal trophoblast consistent with the stage of gestation and no tumor in the myometrium. 'The entire gross specimen and blocks were sent to our Trophoblastic Disease Center where we methodically prepared microscopic sections of the entire specimen. In this secondary examination, a small primary choriocarcinoma lesion involving several villi was found in the otherwise normal placenta (Fig. 3); no lesion was found in the uterine wall. The lung and vaginal metastatic lesions increased rapidly in size; a cerebral lesion developed; death occurred; and an autopsy was performed. At autopsy, the findings were metastatic choriocarcinoma in the occipital'lobe of the right cerebral hemisphere with hemorrhage, parietal lobe of the left cerebral hemisphere, lungs, and vagina. No villi were found in these metastatic lesions. I n the second patient, microscopic examination of the grossly normal placenta, after its delivery and the delivery of a normal-term living

FIG.3. This shows a portion of a prim,irychoriocarcinomalesion arising from a villus and surrounded by normal chorionic villi which had produced distant metastases and death from disease without invading the endometrium or myometrium.

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infant, revealed choriocarcinoma within an otherwise normal placenta. The histopathologic findings were malignant-appearing trophoblast covering some stromal villi (Fig. 4) in a localized region of the placenta. The remainder of the placenta was composed of cbmpletely normal term chorionic villi. Shortly thereafter, and in the absence of symptoms, roentgen examination of the lungs was performed and revealed multiple pulmonary metastatic lesions. Curettage performed 2 weeks after the term delivery revealed typical choriocarcinoma; no villi were present. Death due to metastatic disease followed shortly. No lesions in the uterine wall were identified. In this case, thorough microscopic examination of the placenta at the time of an apparently normal-term delivery fortuitously provided scientific information of value. Had this not been done, it might be contended by some that the choriocarcinoma arose as a sequel to the termination of pregnancy in retained normal trophoblast which was transformed in 2 weeks to full-blown metastatic choriocarcinoma. These 2 cases as well as the other 3 cases we have studied show that the site of origin of the choriocarcinoma is in the trophoblast that covers formed villi of the placenta, and that, in one stage of develop-

FIG.4. High-power photomicrograph of a primary lesion of choriocaryinoma.arisingin the trophoblast of a villous (V) in an otherwise normal placenta which produced distant metastatic disease and death without involvement of the myometrium.

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ment of the neoplasm, chorioni~cvilli are present. They demonstrate that the lesion in the placenta may not interfere in some cases with the growth and development of the fetus and that it may not produce symptoms for a period of time. They show that the primary lesion in the placenta can involve only a few villi and be small and localized. They show that the neoplasm can escape detection grossly, and considerable effort may be required for the pathologist to find the lesion microscopically. They show that the primary lesion can be small and the metastases extensive. Had there not been metastases in these 2 cases, it is evident that the small primary lesion in each case could have been expelled with delivery of the placenta and gone undetected. The absence of villous structures in the metastatic lesions can be explained by the observations made in these patients. Within the mass of malignant trophoblast projectirng from the villus, there are regions of spontaneous degeneration (Fig. 5A). This causes fragmentation of the mass and sequestration of the outermost portion of it (Fig. 5A). This sequestered portion is then dislodged into the intervillous space, enters the maternal blood stream via a myometrial vein as shown by Brewer and Gerbie (1966), and is disseminated to distant sites without extending into or producing metastatic lesions in the myometrium. Such “skipping” of the uterus occurred in both cases, and this phenomenon may well account E x the failure to find a uterine lesion in some of the cases reported in the literature. The absence of villi in later stages of the disease is the result of spontaneous degeneration of the formed stromal villi, which was noted to occur in all 5 specimens. All stages of degeneration were observed, from that involving only the peripheral portion of the villous stroma to the stroma of the entire villus (Fig. 5B), to villous “ghost” formations, to a complete degeneration state in which villous structures are no longer recognizable (Brewer and Gerbie, 1966). With an individual stromal villus undergoing degeneration, the trophoblast may be involved in the same process or it may remain viable and luxuriant in its malignant-appearing state. Quite characteristically in this spontaneous process of necrosis within the placenta with choriocarcinoma was the re1ath.e absence of maternal lymphocytes and leukocytes, which is in contrast to the abundant infiltration of such cells in the masses of malignant tuophoblast located within myometrial veins and also in most metastatic lesions. Tumor invasion of the stroma of the villi was a rare occurrence. In only one villus in one of the cases were malignant trophoblastic cells identified within the stromal core and accompanied at this point b y loss of the basement membrane.

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FIG.5. These specimens were obtained from portions of the primary choriocarcinoma lesion in the placenta shown in Fig. 4. (A) Spontaneous degeneration (D) within the malignant trophoblast projecting from the villous into the intervillous space. Fragmentation and sequestration have resulted (F). (B) Spontaneous degeneration (D) of the stromal portion of villi (V) and the surrounding trophoblast.

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The malignant trophoblast in these specimens did not involve or destroy the endothelial wall of fetal capillaries in the villi even though the cells were in direct contact with each other (Brewer and Gerbie, 1966).A special search was made to identify such invasion, but none could be found. Steigrad et al. (1968) reported a patient with strikingly similar features as the 2 cases just described and as observed in other 3 cases we have studied. She was a 16-year-old primigravida, who first attended a prenatal clinic when 19 weeks pregnant. She had no symptoms. On physical examination all findings were normal and the pregnancy had progressed normally. A routine chest x-ray examination was reported as normal, but a subsequent revitew of the films revealed a lesion in the right lung field. One week later she developed a cough, pleuritic pain, dyspnea, and vaginal bleeding, which worsened during the following 7 days with the added symptoms of hemoptysis, vomiting, and passing blood per rectum. Examination revealed two purple, friable nodules in the vaginal wall beneath the urethral meatus, and one higher in the vagina, a uterus of 20 weeks gestational size, absence of fetal heart tones, and innumerable meta$)tatic lesions in both lungs. Further hemoptysis occurred and death ensued at 21 weeks of gestation. Au: topsy was performed. All the metastatic lesions were choriocarcinoma; none contained villi. In the placenta there were no grossly identifiable lesions but there were a few suspicious regions. Microscopic examination of the placenta demonstrated normal villi for the most part; however, in a local region of the placenta, some of the villi were covered with malignant trophoblast. Sections of the myometrium underlying this localized placental region revealed trophoblast in the myometrial venous spaces but none in the rnyometrium anywhere. Complete autopsy performed on the fetus failed to reveal any choriocarcinoma lesions. This case again demonstrates that choriocarcinoma originating as a small lesion in the placenta can skip the uterus and produce multiple metastases and death. Douglas and Otts (1949) reported a patient, who 10 months prior to the present pregnancy, had had a normal pregnancy and delivery of a normal infant. During the 10 months, menstruation was normal. In the third month of the present pregnancy, the first sign of any abnormality was the appearance of metastatic: choriocarcinoma vaginal and pulmonary lesions. The fetal heart tonr1:s were normal and the fetal skeleton was normal on x-ray examination. Radiation therapy was administered, and in the fifth month of gestation a hysterectomy was performed with delivery of a normal viable infant. The mother subsequently died of disease. No autopsy was perfoi~*med.Pathologic examination of the

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hysterectomy specimen demonstrated no gross abnormalities of the placenta or uterus. Upon microscopic examination of the placenta, a small lesion of malignant-appearing trophoblast arising in one villus was identified. No lesions were found in the myometrium or endometrium, but choriocarcinoma cells without villi were present in a myometrial vessel beneath the placental site. This is another example of a small lesion of choriocarcinoma arising in an otherwise normal placenta, producing multiple metastases while completely skipping the uterus, and resulting in death of the patient. In 1936, Davis and Brunschwig reported a patient in whom choriocarcinoma arose in the placenta during the course of an apparently normal pregnancy. Her only other pregnancy had terminated in an abortion at 8 weeks without complications. In the present pregnancy, her second, slight uterine bleeding occurred in the second, third, and fourth months, and in the sixth month severe bleeding developed. Examination revealed a mass in the cervical canal which was thought to be a placenta previa. The fetus was normal. Delivery of the infant was accomplished after burrowing through the presumed placenta previa and rupturing the membranes. The infant lived 1 hour. For 10 days postpartum, the bleeding continued. A supracervical hysterectomy was performed. During the operation, a tumor mass was observed to have extended through the uterus into the broad ligament and all of it could not be removed. Pathologic examination of the placenta revealed normal chorionic villi with normal trophoblast in some regions, but in other regions the villi were covered with large masses of abnormal trophoblast. Within some of these masses, necrosis of a portion of the trophoblast was evident. In the uterus, choriocarcinoma lesions were found in the myometrium, cervix, and a myometrial vein, none of which was accompanied by villi. Postoperatively, no distant metastatic lesions could be found. Radiation therapy was administered. The patient was alive and without demonstrable disease 5 months later. In this case, choriocarcinoma was shown to have arisen in the trophoblast covering the villi and within these masses of trophoblast there were localized regions of necrosis of the cells similar to that we described in our personally studied cases. The neoplasm described by Davis and Brunschwig directly invaded the uterine wall and the broad ligament but had not produced distant metastases even though tumor was found within myometrial veins. MacRae (1951) described a patient who, after 3 normal term pregnancies with normal infants, began to bleed vaginally in the 33rd week of an apparently normal fourth pregnancy. Examination revealed multiple purple vaginal lesions, which, on biopsy, were diagnosed as

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choriocarcinoma. There were 110 villi in these lesions. Hemoptysis developed shortly thereafter arid multiple pulmonary metastatic lesions were demonstrated by x-ray examination.. The infant’s heart tones were normal and its size was consistent with the weeks of pregnancy. A Cesarean hysterectomy was performed. The infant was normal and was living and well 1 year after delivery. The mother died. Autopsy revealed multiple, distant metastatic choriocarcinoma lesions, invasion into the myometrium, and small masses of choriocarcinoma in myometrial vascular sinuses; none was associated with villi. In the placenta, there was a grossly identified 2.0 x 0.75 cm yellow, crenated excrescence. Histologically, this lesion consisted of chorionic villi covered with heaped up mxses of trophoblast with marked anaplasia and complete absence of pattern. In the description it was noted that the abnormal trophoblast seemed to be growing from or invading the normal-looking villi. A review of the published illustrations show these lesions to be the same as those found in the placentas in the group of patients being reported here. In this patient reported by MacRae, the choriocarcinoma arising in the otherwise normal placenta was a grossly identifiable lesion, was localized to a small portion of the placenta, had invaded the myonietrium directly, was within myometrial vessels, and had produced distant metastases and eventual death. Ringertz (1970) reported the finding of a walnut-size focus of choriocarcinoma in an otherwise normal placenta which accompanied the delivery of a female stillborn weighing 1750 grams. Immediately postpartum, pulmonary metastases were identified and a curettage demonstrated choriocarcinoma invasion of the myometrium. The choriocarcinoma lesion in the myometrium contained no villi. Methotrexate was administered but the mother died of disease 4% months after delivery. This was the second pregnancy for this patient and both had ended in delivery of stillborn infants, one a male and one a female, both with multiple congenital anomalies. The choriocarcinoma arising from the trophoblast of the villi in a localized region of the placenta had extended into the myometrium and metastasized to distant sites. In 1954, Gusenleitner described a patient 38 years of age, who had had 5 normal pregnancies and 2 abortions prior to the present gestation. At 33 weeks in the present, apparently normal, pregnancy, vaginal bleeding occurred. It arose from 2 blue-black necrotic-appearing lesions in the introitus. A Papanicolaou smear test revealed groups of syncytial and cytotrophoblastic cells with large, irregular nuclei, and, upon the basis of this finding, a diagnosis was made of malignant cells, either ectopic or metastatic. No lung lesions were identified upon

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examination. Five days later another lesion appeared in the introitus. It was removed and a complete hysterectomy was performed. The infant was born alive, was of a size consistent with 33 weeks gestation, and appeared normal but died the next day. The postmortem examination of the infant revealed no tumor. During the first 3 weeks postpartum, new additional metastatic lesions appeared in the mother, and, despite radiation therapy, her condition worsened and she died. No gross abnormalities were noted in the placenta. In the prepared histologic sections of the placenta, there were chorionic villi with multiple abnormal trophoblastic cells invading their stroma and with trophoblastic proliferation. A diagnosis of choriocarcinoma was not made. There were no myometrial lesions. In this patient with a viable baby, metastatic choriocarcinoma developed during the course of the pregnancy and resulted in death of the mother. While a diagnosis of choriocarcinoma was not made from examination of the microscopic slides, the pathologist did describe abnormal trophoblastic cells. There was distant metastatic disease but no lesions in the myometrium. The 27-year-old patient reported by Resnick ( 1945) had previously had a normal-term pregnancy and delivery and an abortion. Following the abortion, her menstrual cycles were normal for 8 months. She then became pregnant for the third time. In the fifth month of pregnancy she developed a tumor mass in the breast but did not seek medical attention. In the seventh month, fetal movement ceased, and death of the fetus was confirmed by x-ray examination. Because of this and because the patient felt weak and listless and her general condition seemed poor, labor was induced, resulting in the delivery of a macerated fetus. The placenta looked unhealthy but no pathologic examination was made of it. Sepsis developed and transfusions were given. Shortly thereafter, the patient died. Autopsy demonstrated metastatic choriocarcinoma of the breast, lungs, liver, spleen, and kidneys. No choriocarcinoma was present in the uterus, despite the fact that the placental site could be recognized and a careful search was made. In view of the grossly abnormal placenta, it is most probable that it contained the primary lesion and that the disease had metastasized without involving the uterus. Two months before delivery of the fetus, metastases to the breast developed. A patient described by Fikentscher (1941) had an obstetric history of a normal pregnancy.with delivery of a normal child 7 years previously and an abortion without complications 2 years previously to the present pregnancy. During those 2 years, the menstrual cycles had been normal and regular until the third pregnancy ensued. In the 33rd week of the present pregnancy, vaginal bleeding began. One week later, a biopsy of a vaginal lesion revealed only necrotic mate-

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rial from which a diagnosis could not be made. At this time the fetal heart tones were normal. Shortly thereafter, the mother developed pulmonary symptoms, the fetal hleart tones were no longer heard, and vaginal bleeding continued, requiring blood transfusions. Abdominal complete hysterectomy and bilatc ral salpingo-oophorectomy were performed with delivery of a dead, partly macerated infant. In the myometrium beneath the placenta, 1 here were choriocarcinoma lesions; but no direct connection between the placenta and these myornetrial lesions could be demonstrated. A few microscopic sections of the placenta were prepared and they showed normal villi, some of which were accompanied by an excess of syncytium. The latter was interpreted as benign process. Adlowing the death of the patient, the autopsy revealed metastatic choriocarcinoma in the lungs. It is noteworthy that in the few microscopic sections studied, there was an excess of trophoblast on some of the villi which suggests that a more thorough study of the placenta might have provided more information. Driscoll ( 1963) reported the incidental finding of choriocarcinoma within a term placenta. The 23-ycar-old patient had previously had 3 normal term pregnancies and 3 abortions; one of the abortions was the last pregnancy preceding the present pregnancy. Curettage performed for this last abortion revealed normal villi, without trophoblastic proliferation or cellular abnormality and no hydatidiform swelling. In the present pregnancy, there was some scant bleeding in the first trimester, which subsided spontaneou,;ly. Otherwise, the prenatal course was uneventful. At 39 weeks, labor and delivery ensued. The infant was term size and normal. Upon gross pathologic examination, the placenta appeared normal except for a firm, yellow-white lesion 8-10 mm in diameter within the substance of the placenta. This resembled a small infarct. Microscopic examination of this lesion revealed choriocarcinoma, with the neoplasm arising in the trophoblast covering the villi, and with normal villi adjacent. In some instances, the trophoblast appeared to b e invaciing the stroma of the villus. There was a small aniount of necrosis of the trophoblast in some regions. No invasion of the decidua was demonstrated in serial sections. The postpartum course was normal, and both mother and child remained well, as evidenced by frequent examinations of the child and hCG testing and physical and radiologic examinations of the mother over a period of 9% months. This is the earliest stage in the development of choriocarcinoma in this group of patients and demonstrates the characteristics of the others described with the exception that no metastasis or extension of the lesion had occurred. Dr. Mark Wheelock recounted I:O us an unpublished case in which

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the patient had had a normal prenatal course and was delivered of a normal term infant. One week postpartum she had severe uterine bleeding and a metastatic tumor in the lung was demonstrated by x-ray examination. Curettage was performed. The pathologic diagnosis was normal chorionic villi and choriocarcinoma in the blood vessels and lymphatics of the myometrium. Death from choriocarcinoma occurred. It is reasonable to believe that the malignancy developed during the pregnancy, especially in view of the fact that a lung lesion was demonstrable 1week after the delivery. Experience has shown that it usually takes 10 to 12 days after the metastasis occurs in the lung before routine x-ray examination can detect it. In most instances it is hemorrhage associated with the metastasis that provides the size that makes it detectable by radiologic examination. These cases provide impeccable evidence on the origin and early development of choriocarcinoma in pregnancies not associated with hydatidiform mole. Choriocarcinoma arises in the trophoblast of the chorionic villi. In the early stages, choriocarcinoma in its original site is associated with and accompanied b y chorionic villi, which is quite contrary to the established pathologic criteria for the histologic diagnosis of this neoplasm. It may remain small and localized within the placenta and still produce widely disseminated metastases and death. The metastatic lesions are composed of malignant trophoblast with no formed villi. This is accounted for in the early stages by necrosis within the masses of trophoblast surrounding the villi, with a resulting sequestration of the distal portions of the trophoblast. The latter, since they lie in the intervillous spaces, are swept b y the maternal circulation into myometrial blood channels and then to distant sites. I n this manner, the neoplasm may completely skip the uterus, a not infrequent circumstance. This could account for the failure to find residual choriocarcinoma in the uterus in some patients with this disease. I n the latter stages of the disease, the necrosis also involves the stromal portion of the villi, and this degenerative change continuously progresses until the villi are no longer recognizable. In such circumstances only residual trophoblast may be found in the uterus. The neoplasm may extend from its primary site directly into the myometrium and cervix and from there be disseminated to other sites. The tumor may be present for some time before it produces symptoms. While the malignant trophoblast does invade the stromal portion of the villi, it is not a frequent occurrence. Choriocarcinoma invasion of the fetal vessels of the villi is extremely rare (Brewer and Gerbie, 1966), and seldom does the disease metastasize to the fetus. The fetus may continue its normal growth and development unless the neoplasm

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involves a large or an essential portion of the placenta. An early and small lesion of choriocarcinoma situated totally within the placenta may be expelled completely with the delivery of the plhcenta without evidence of further disease, as observed b y Driscoll (1963). 2. Choriocarcionoma Arising dwing Seeming1y Normal Pregnancy with Lesions in the Myometrium but None Identijied in the Placenta: 8 Cases Buckell and Owen (1954) reported a 22-year-old patient, nullipara, who had a normal pregnancy arid delivery of an apparently normalterm infant. The placenta was not examined microscopically. The baby appeared well for 5 weeks, then developed symptoms and died at age 7 weeks. Autopsy revealcd choriocarcinoma of the liver. The mother continued to bleed after the delivery and at 9 weeks postpartum a curettage revealed chorioc.ucinoma. At hysterectomy, the tumor involved the uterus and had extended into the right broad ligament. Radiation therapy was administered and 9 months later the patient had no evidence of disease. It is reasonable to conclude that the choriocarcinoma had developed in the placenta during pregnancy and had metastasized to the fetus. It s e e m quite clear that the neoplasm did not arise after the termination of pregnancy in residual placental elemenfs, as might be postulated. Stahmann (1957) described a patient who had had 3 previous term deliveries. Following the last pregnancy, the menstrual cycles were normal for 2 years, at which time she became pregnant. During the first 5 months she remained well and the fetus developed normally. In the sixth month of gestation, uterine bleeding began and metastatic lesions appeared in the vagina, lung, and pelvis. Biopsy of the vagina revealed choriocarcinoma. Fetal death occurred. The patient died 3 weeks after the bleeding began m d the metastatic disease was diagnosed. Autopsy examination dernonstrated a dead fetus and the presence of choriocarcinoma in the uterus, vagina, pelvis, and lung, but no tumor was found in the placenta; the chorionic villi were normal in the sections studied. An extensive search of the placenta for neoplasm was not made, and in light of present 1.rnowledgea small lesion could quite readily have been missed. The patient described by LepolN (1959)developed a metastatic lung lesion in the seventh month of her gestation and at 9 months was delivered of a normal living infant. The placenta appeared normal grossly and was not thoroughly examined microscopically. Six days after delivery, the lung lesion, which was removed surgically, was found to be choriocarcinoma. Hysterectomy was performed. The myo-

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metrium was invaded b y choriocarcinoma lesions without chorionic villi. The patient died. It must be concluded that the neoplasm arose in the placenta during pregnancy but was unrecognized. Felton and Smith (1966)reported a patient, who 3 years prior to her current pregnancy had a hydatidiform mole. For 2 years after termination of the molar pregnancy she had no symptoms, then she had an episode of amenorrhea for 4 months which was followed by 2 weeks of abnormal uterine bleeding. A diagnosis of dysfunctional bleeding was made. There were no further symptoms for the lY2 years, at which time she became pregnant. The pregnancy was uneventful until the 36th week when toxemia developed, followed by progressive anemia, melena, shock, and collapse, loss of fetal heart tones, and spontaneous onset of labor and delivery of a 5 pound 12 ounce stillborn infant without tumor. The placenta was normal grossly. Six days postpartum, chest pains occurred and x-ray examination of the lungs revealed multiple lesions. Methotrexate was administered, but 4 days later death occurred. Autopsy revealed choriocarcinoma in the body of the uterus, the intestines, lungs, brain, liver, and right kidney. The interpretation made by the author of this series of events was that the choriocarcinoma arose not from the current pregnancy but rather arose as a sequel to the hydatidiform mole which had been delivered 3 years previously. With the knowledge now gained concerning the development of this neoplasm during the course of an apparently normal pregnancy, it is preferable to interpret this series of events as follows: The lesion developed in the otherwise normal placenta during the current pregnancy, remained small in size, obscure in that it did not alter the gross appearance of the placenta, did not interfere with fetal development, metastasized during the course of the gestation, and caused the death of the mother after termination of the pregnancy. Maun and Green (1943) report a case of choriocarcinoma with regression of the primary uterine tumor in a 22-year-old7 nulliparous patient. The prenatal course, labor, and delivery at term were normal. The live born infant was normal and remained so. The placenta, which appeared normal grossly, was not examined microscopically. Two and one half months postpartum, the patient developed symptoms; no treatment was given, and at 4 months she died. Autopsy revealed a large choriocarcinoma of the liver with rupture of the liver and the peritoneal cavity filled with blood. In the fundus of the uterus was a soft hemorrhagic mass, 2.0 x 1.0 mm located in the deeper portion of the endometrium and adjacent myometrium. Histologically, the lesion was composed of degenerated material and shadows of decidual cells; no trophoblast was recognized. Thorough examination of the entire

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uterus failed to reveal any trophoblastic cells. The author considered this lesion to be the primary site of the choriocarcinoma which had undergone spontaneous regression. However, this might well be an instance in which the primary lesion in the placenta was expelled completely with delivery of the placenta. Mercer et al. (1958) reported a case in which the mother had choriocarcinoma that metastasized to the fetus. The prenatal course of the gestation and the labor and delivery were normal. The placenta was not examined. The baby at the age of 4 weeks had a tumor of the alveolar margin of the maxilla wlhich was choriocarcinoma. It was resected at 6 weeks of age but reciirred locally involving the head and face and the baby died of disease at 7 months. Following the delivery of the infant, the mother had continuous uterine bleeding for 8 weeks. Because of this and because the baby had been found to have choriocarcinoma, a curettage was performed, revealing choriocarcinoma in the cavity of the uterus Two weeks later (10 weeks postpaxtum), a hysterectomy was performed and choriocarcinoma was found in the myometrium and the interior portion of the uterus. Cobalt radiation therapy was administered. 4 lung lesion appeared 3% months postpartum. Six months after the delivery of the infant, the mother died of disease. An autopsy was performed. While the diagnosis of choriocarcinoma was not made until 8 weeks postpartum, the evidence is quite clear that the neoplasm arose in the placenta, involved only a portion of it as indicated by the unhampered continued growth and development of the fetus, and rnetastasized to the fetus, all prior to delivery in what appeared to be a normal pregnancy. Failure to examine the placenta histologically and completely negated the possibility of determining and demonstrating the site of origin of the primary lesion. Dafoe (1939)reported a patient 33 years of age who was operated on 2 months after her last menstrual period for a ruptured cornual pregnancy. Symptoms had begun 2 weeks prior to operation. At operation, the early fetus was found projectiing through a rent in the cornu of the uterus. These tissues were removed. The pathologic diagnosis was choriocarcinoma in an interstitial pregnancy with fetus. Six weeks postoperatively, a metastatic lung lesion was diagnosed. Three and one-half months after the hospital admission for operation, the patient died. The autopsy revealed metastatic choriocarcinoma in the cervix, pelvis, lumbar lymph nodes, lung, liver, spleen, kidney, and small bowel. The choriocarcinoma in the cornu of the uterus was at the site of the implantation and, while no normal placenta was identified, it must be assumed the neoplasm arose in the placental tissue. The onset

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of symptoms in the sixth week of pregnancy is much earlier than usually observed, and is undoubtedly the result of the implantation being in the cornual portion of the uterus. According to the report the patient had had no previous pregnancies. Cordua (1949) described a patient, who in the eighth month of pregnancy had hemoptysis and lesions in the lung demonstrated by x-ray examination. At the time of delivery the placenta was not studied thoroughly. Following term delivery she had a postpartum hemorrhage. A curettage revealed choriocarcinoma involving the uterus. In this group of patients, the findings indicate that the choriocarcinoma arose in the otherwise normal placenta during the course of an apparently normal pregnancy. The demonstration of choriocarcinoma in the uterus strongly supports this interpretation. The failure to identify the site of origin in the placenta was undoubtedly due to the fact that the placenta was not examined histologically or that the histologic search of the placenta was not sufficiently adequate. A complete search may be required, since it is well documented that the primary lesion may be extremely small and unrecognizable grossly and yet metastasize and kill the patient.

3. Choriocarcinoma Arising during Seemingly Normal Pregnancy with No Lesions Demonstrated in Either the Placenta or Uterus: 6 Cases In this group of 6 patients, metastatic choriocarcinoma occurred during the course of an apparently normal pregnancy with fetus but choriocarcinoma was not demonstrated in the placenta or the uterus. In 3 of the 6, the disease metastasized to the fetus. In the patient reported by Emery (1952),the prenatal course, labor, and term delivery were uneventful. Grossly, the placenta was normal. The infant appeared normal at birth but became ill at 2 weeks of age and died 6 weeks after birth. At autopsy, choriocarcinoma was found in the liver and lung. The mother had no symptoms or evidence of disease and 10 months after the delivery she was still well and no choriocarcinoma lesions were demonstrable. Emery stated it was possible that a primary choriocarcinoma lesion in the placenta had been completely expelled with the delivery of the placenta. This is entirely possible and is more plausible than assuming the malignancy arose as a primary lesion in the fetus or infant, metastasized, and killed within a period of 6 weeks. A primary lesion in the infant could not be demonstrated by postmortem examination of all tissues. In the absence of a thorough search of the placenta and since the uterus was not removed, proof cannot be established, but there is a good probability the author’s interpretation of the events is correct.

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Daamen et al. (1961) reported a patient who had a normal prenatal course and was delivered of an apparently normal infant at term. The infant developed anemia at 6 months of age, a liver lesion was detected, and the pregnancy test was positive. This test was performed, because one month earlier a duagnosis of choriocarcinoma had been established in the mother. A Iaparotomy performed upon the infant revealed tumor masses filling the abdominal cavity and involving the liver, the extent of which made surgical extirpation impossible. Methotrexate was administered, but failed to provide remission and the infant died of disease at 8 months of age. The mother for the first 4 days after delivery of the infant had continued, severe uterine bleeding requiring transfusion of 1600 ml of blood and a curettage. The tissue obtained at curettage was diagnosed as placental tissue and decidua. No choriocarcinoma was identified. During the next 5 months, the patient had irregular episodes of abnormal uterine bleeding, occurring at 2 to 5-week intervals. At 5 months postpartum, the presence of a pelvic mass was detected, which at operation was found to be a ruptured, cystic ovarian tumor and there was metastatic tumor in the omentuni. The ovary and a portion of the omentum were removed. The histopathologic diagnosis was “likely choriocarcinoma.” Six days postoperatively, the A schheim-Zondek pregnancy test was negative, One month later a second abdominal operation revealed choriocarcinoma masses filling the abdominal cavity and involving the uterus. The mother died 3 week:; later. An autopsy was not performed. The possibility of this being a case of primary ovarian choriocarcinoma is exceedingly remote, so remo:i:e that it should receive only fleeting consideration. The ovarian tumor was cystic in character, totally unlike a primary ovarian choriocar cinoma. A primary ovarian choriocarcinoma producing metastases in the fetus would be most unusual. The simultaneous occurrence of two primary choriocarcinomas, one in the maternal ovary, and one in the liver of the infant, is a most unacceptable theory. A better interpretation of the circumstances is that the choriocarcinoma in the mother and in the child represent one and the same tumor, that the primary lesion was gestational choriocarcinoma arising during pregnancy and metastasizing to the fetus. In the child, the disease was not diagnosed until the sixth month of age. In the mother, the diagnosis was not made until 5 months postpartum, despite the fact that repeated episodes of abnormal uterine bleeding occurred during that 5-month period. The severe, immediate postpartum bleeding indicated an abnormality, which upon pathologic examination revealed only placental tissue. From past and present experience, this placental tissue surgically obtained might represent a portion of the placenta not involved in a small primary choriocarcinoma. It

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is quite possible that the obtaining and studying of more tissue might have provided the diagnosis. It is also possible that the malignant trophoblast had already extended into the myometrium beyond reach of the curet at the time of curettage, or that it might have been completely expelled with delivery of the placenta. The subsequent course of the patient suggests the latter is true. Without pathologic examination of the uterus, either as a surgical or necropsy specimen, the sequence of events cannot be established with absolute certainty. It is most likely that the malignancy arose in an otherwise normal placenta and it seems improbable that it arose in fragments of normal placenta retained in the uterus, since the evidence strongly suggests the choriocarcinoma was already present and had metastasized to the fetus prior to the time the curettage was performed. A delay of 5 months in establishing the diagnosis of choriocarcinoma in a mother after a term delivery is not unusual and such a happenstance, although it can be avoided by proper testing, is not incompatible with the interpretation of events in this case as presented here. Witzleben and Bruninga (1968) reported a case of infintile choriocarcinoma and described a characteristic syndrome. The prenatal course of the pregnancy and the fetal growth and development were normal. The gestation terminated in the delivery of an apparently normal, term infant. The infant at 5 weeks of age developed melena and hemoptysis; at 7 weeks, pulmonary metastatic disease was demonstrated by x-ray examination; and, at 8 weeks of age, operation was performed for rupture of the liver and metastatic choriocarcinoma in the liver. Postoperative death of the infant occurred, and, at autopsy, choriocarcinoma of the lungs, liver, and brain was demonstrated. The mother had no evidence of disease and, at 8 weeks postpartum, when her baby died, an hCG test was reported as normal. Five and one-half months postpartum she had an elevated hCG titer and choriocarcinoma of the lung, liver, and small intestines. Evaluation of this case is hampered by the lack of definitive study and description of the placenta at the time of delivery. However, the presence of disease in both the baby and the mother points with a good degree of certainty that the malignancy arose in the placenta during pregnancy and metastasized to the fetus. Only in the postpartum period did the presence of choriocarcinoma become evident. The time element in the case of the infant, 5 weeks, is compatible with the contention, that the disease occurred as a result of metastasis during its intrauterine existence. The time element in the case of the mother, 5y2 months postpartum, is again not unusual and is in keeping with the findings in other cases. Walthard (1907) reported a patient in whom vaginal metastases of

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choriocarcinoma developed in the seventh month of pregnancy. The uterus was removed surgically and a search of the uterus and placenta failed to reveal any neoplasm. The infant was free of disease. Approximately 4 years later, the patient died of choriocarcinoma involving the lungs, liver, and kidneys. The circumstances indicate that the primary lesion was in the placenta, that it had metastasized during pregnancy, that the small primary had been expelled with the placenta, and that distal metastases had developed prior to the hysterectomy. Hutchinson et al. (1968) described a patient who progressed through the fist 28 weeks of her pregnancy without problems and with the fetal growth and heart tones normal. At that point in the pregnancy, she suddenly develciped pain and signs of intraperitoneal hemorrhage. At operation, one o {ary was removed for ruptured, cystic lobulated ovarian tumor with bleeding. The uterus was normal size for the gestation period and appealed to be a normally pregnant organ. The pathologic diagnosis was chioriocarcinoma. Postoperatively, x-ray revealed a pulmonary lesion. Immediately after operation, the hCG titer was 640,000 mouse units by the mouse uterine weight bioassay. Seventeen days later a second episode of intraperitoneal hemorrhage occurred. Operation revealed bleeding tumor masses involving the cecum, ileum, and omentum; all were removed including bowel resection. The pathologic report M'as choriocarcinoma. Several lung lesions were present at this time. Chemotherapy was administered, utilizing a triple therapy regimen consisting of vinblastine, methotrexate, and actinomycin D. Three courses were given starting at the thirtieth week of gestation. The hCG titers decreased and there was some resolution of the pulmonaiy metastases. At 37 weeks, labor was induced and a live infant was delivered. The placenta was normal grossly. Several portions of the placenta were examined, but no choriocarcinoma was found. In the'fu-st few days of life, the infant developed an encephalopathy with focal seizures, normochromic monocytic anemia, and urinary tract infection, probably the effect of chemotherapeutic agents administered to the mother during pregnancy. All were corrected with appropriate treatment. Follow-up of the infant and the mother was conducted for 4% months; the lung lesions disappeared, the hCG titers remained normal, and both patients were free of disease. In a patient mentioned b y Bag shawe (1969),respiratory symptoms, which were probably due to pulmonary metastases, developed during the last month of an otherwise normal fourth pregnancy. One month after normal-term delivery, she was found to have widespread carcinomatosis. N o choriocarcinomii or hydatidiform mole was noted in

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the placenta. It is reasonable tu conclude that this is a case of choriocarcinoma arising in the placenta and metastasizing during pregnancy with additional metastases appearing after delivery. The series of events in each of the patients in this group provide information for and lend support to the growing evidence that choriocarcinoma not associated with hydatidiform mole arises within a normal placenta of the current pregnancy. However, there is a matter of interpretation in the patients in this group, since the placenta and uterus contained no choriocarcinoma. Park (1971, Chapter VIII, p. 137), in his excellent works, has been foremost in providing interpretation of events, especially regarding the time at which gestational choriocarcinoma arises and its possible relationship to previous pregnancies. He has called attention to the possibility that the origin of choriocarcinoma may not be from the current gestation, as it seems, but rather may have had its origin in some pregnancy preceding the current one. H e has gathered published case reports in which the latter interpretation is a possibility and he has developed premises to support the possibility. In some instances, our interpretations of a specific case differs from his. An example is presented here. In the case report by Felton and Smith (1966), which is presented in this report, Park viewed the material and questioned whether the choriocarcinoma might not be a legacy from the distal hydatidiform mole rather than the proximal (current) pregnancy. The events in this case briefly were hydatidiform mole in 1961, amenorrhea, and episodes of abnormal uterine bleeding in 1963, a new pregnancy in 1964 which progressed normally until 28 weeks when toxemia, melena, shock, collapse, and death of the fetus occurred, labor ensued and the mother was delivered of a stillborn infant. Six days postpartum a pulmonary metastatic lesions was identified and 4 days later the patient died. On gross inspection, the placenta appeared normal. Autopsy revealed choriocarcinoma in the lung, intestine, liver, brain, and kidney. Our interpretation of this series of events is that the neoplasm arose during pregnancy in an otherwise normal placenta, remained small, and was delivered with the placenta but not before it had produced metastatic disease; its existence had escaped detection because the gross appearance of the placenta was not altered and because histologic examination of the placenta was not performed. The fact that the diagnosis was not made until 6 days postpartum does not detract from the interpretation, since the symptoms prior to delivery indicate the neoplasm was present during pregnancy. The specific, demonstrable findings in some of the other cases reviewed lend credence to this interpretation. More difficult to interpret are those cases in which choriocarcinoma

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is diagnosed more remotely to a known previous pregnancy. In such instances, a new but unrecognized pregnancy may have occurred which gave rise to the choriocarci~nomaand the possibility of neoplasm arising in a teratoma must be conlsidered. Park (1971, Chapter VIII, p. 135) called attention to some patients in whom choriocarcinoma makes its appearance in circumstance: in which, on grounds of “timing” alone, its relation to an earlier or even a current pregnancy is debatable. These are cases in which 1 he neoplasm is diagnosed after hysterectomy for choriocarcinoma, after the menopause, or after passage of a hydatidiform mole but separated from it by one or more intervening nonmolar pregnancies. In the pllisthysterectomy cases, the evidence indicates that choriocarcinoma may lie dormant for many years before it makes itself known. A patient with choriocarcinoma in whom the diagnosis was made for the first time many years after hysterectomy as well as after the menopause wa: encountered at our center. The patient was 59 years of age at the time the diagnosis was made. She had had 6 pregnancies, the sixth and last of which was a hydatidiform molar gestation occurring when 5 he was 48 years of age. It was terminated at her local hospital in the, fourth month of pregnancy by hysterectomy and was diagnosed h istologically as benign hydatidiform mole. There were no symptoms or clinical evidence of disease until 11 years later at which time she was operated on at her local hospital for a malignant abdominal tumor mass extensively involving the pelvis, bowel, and liver. Biopsy of the mass revealed choriocarcinoma. Postoperatively, chest films demonstrated pulmonary metastases. Chemotherapy was administered for 6 months but failed to provide remission. At autopsy the essentiiil findings were choriocarcinoma involving the abdomen, pelvis, liver, and lungs and no teratomatous tissues were found at any location in the body or the tumor. This case exemplifies well two of the elements of “timing” described by Park (1971, Chapter VIII, p. 135), nainely choriocarcinoma diagnosed 11 years after hysterectomy for hydatidiform mole and many years after the menopause. The series of events in this case might be interpreted as indicating the malignancy arose from residual benign molar tissue that had remained dormant in thl? benign state for years. There is no proof that such occurred. It is mole probable that choriocarcinoma had originated in a portion of the h>datidiform mole and had extended beyond the uterus prior to hysterectomy and that it had lain dormant for many years before clinical symptoms appeared and a diagnosis was made. An example that enhance: the latter probability is the patient who was managed at our Trophoblastic Disease Center, in whom all 3 trophoblastic diseases, hyditidifonn mole, invasive mole, and

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FIG. 6. Figures 6-8 show hydatidiform mole, invasive mole, and choriocarcinoma simultaneously present in a uterus. Figure 6 shows the hydatidiform mole in the uterine cavity.

choriocarcinoma were simultaneously present in the uterus (Brewer et aE., 1970) (Figs. 6, 7, and 8). The patient, when first seen by us, was 40 years of age. Her first 2 pregnancies were normal and the infants were normal; the second infant was born in 1952. The third pregnancy in 1955 was a twin gestation ending in term delivery of one normal, live infant and placenta and one stillbirth whose placenta was a hydatidiform mole. Because of postpartum bleeding, a curettage was performed; no trophoblast was found. Biologic pregnancy tests over a period of 4 months were negative. Menstrual cycles were normal for the next 2 years. In 1958, the patient was delivered of a normal infant at term; this fourth pregnancy had been normal throughout. For the next 4% years, the menstrual cycles were normal and the gynecologic and general medical examinations revealed normal findings. On March 3, 1963 the last menstrual period occurred. Slight uterine bleeding began on April 30 and continued until June 3, at which time a curettage was performed. This revealed hydatidiform mole. Four days later, radiologic examination demonstrated numerous metastatic

FIG. 7. (A) Invasive mole is shown i n the myometrium (M) with hemorrhage and degeneration (D). (B) Choriocarcinomai n the myometrium (M) is shown. These lesions are present in the uterus simultaneousl) with the lesions shown in Figs. 6 and 8.

FIG.8. (A) An invasive mole lesion in the cervix (C) and (B) a separate and distinct lesion of choriocarcinomain the cervix (C) accompanying, in the same uterus, the lesions shown in Figs. 6 and 7.

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lung lesions. At this point the patient was referred to our center, where the chorionic gonadotropin level was found to be 50,000 IU/24 hours. On June 12, 1963 a complete Zibdominal hysterectomy and bilateral salpingo-oophorectomy were ptbrformed. The uterus was the size of a 12-week gestational organ and the cavity was filled with molar tissue. Each ovary was 10 cm in diameter and contained multiple cysts. Microscopically, the curettage specimen revealed molar villi with some proliferation of the abnormal trol3hoblast. Within the extirpated uterus, the molar villi were covered witii a thin layer of trophoblast (Fig, 6). In the myometrium invasive mole (Fig. 7A) and choriocarcinoma (Fig. 7B) were identified. In separate and distinct lesions in the cervis, invasive mole (Fig. 8A) and ckioriocarcinoma (Fig. 8B) were found. The uterine vein adjacent to tlie uterus contained a small mass of malignant trophoblast. Chemotherapy administered over an 18-month period provided remission, whii:h has now endured for 12 years. The evidence in this case indicates that the choriocarcinoma arose in the current hydatidiform mole rathe -than in trophoblast from the previous molar gestation, which might have lain dormant in the host for 8 years. It reduces somewhat the probatiility of the theory that the trophoblast of a previous hydatidiform mole may reside in a dormant stage through the course of a subsequent norinal term gestation and then for some unexplained reason suddenly bcb transformed into a choriocarcinoma. C. COMMENT The cases presented here, in which choriocarcinoma occurred during a seemingly normal pregnarcy and in which a fetus was present, permit an excellent opportunity to study and describe the primary lesion of gestational choriocarcirtoma and its characteristics and to observe the earlier stages in its development. Under other circumstances, these things are rarely possible because the neoplasm has progressed beyond the earlier siages of development, the primary lesion has been expelled in part or in its entirety with the placenta or the fragments of it, or the neoplasm has grown to the extent that it involves the entire residual placental structure. In patients in Group 1, it is quite apparent that gestational choriocarcinoma had its origin in an otherwise normal placenta. I n the remainder of the 29 cases in which the placenta was either not available for histologic examination. not examined histologically, or not adequately examined, the sequence of events and the associated pathologic findings strongly indicate the primary lesion was in the placenta, although this was not proved. The exact time during the

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pregnancy at which the choriocarcinoma had its inception could not

be established in these 29 cases but it appears that it can arise at any time in the gestation. The earliest it was identified and diagnosed was the eighth week of gestation (Davis and Brunschwig, 1936; Dafoe, 1939), and, in these two cases, symptoms were present prior to the time of diagnosis, suggesting an earlier date of origin. In 29 cases the clinical and the pathologic findings indicate that the neoplasm was directly related to the current pregnancy and that it was not a sequel of a previous pregnancy. Two of the 29 patients had not had a previous pregnancy (Steigrad et al., 1968; Dafoe, 1939). The occurrence of choriocarcinoma in both the infant and the mother supports the premise that the neoplasm is directly related to the current pregnancy. In 5 of the 29 cases, the infants had choriocarcinoma; in 3 of the mothers, it was identified (Buckell and Owen, 1954; Daamen et al., 1961; Witzleben and Bruninga, 1968), but, in the other 2, it was not (Maun and Green, 1943; Emery, 1952). The reason for failure to find a lesion in the two mothers is explained satisfactorily in the citation of cases in this review. With due consideration of the premise presented b y Park (1971, Chapter VIII, p. 137), that a choriocarcinoma may possibly be a sequel to a previous, remote pregnancy rather than to the current one to which it might seem to be related, in none of the 29 cases did this seem probable. In none of the 29 cases or the 5 infants with choriocarcinoma was there any evidence of a teratomatous origin. The primary lesion in the patients in Group 1 had its origin in the trophoblast covering the chorionic villi. In no instance was choriocarcinoma observed to arise prior to the formation of chorionic villi, although it is believed possible that this neoplasm may arise at any stage in the growth and development of the fertilized ovum. The observations reported in Group 1 patients do show that during the early development of choriocarcinoma chorionic villi are present, contrary to the generally accepted diagnosis of this neoplasm. The explanation for the absence of villi in more advanced disease and in metastatic lesions is adequately given in this review. It was shown that the primary lesion may be of microscopic size, may involve the trophoblast of only a few villi in a localized region, and may not distort the normal gross appearance of the placenta. Even as the tumor grows, enlarges, and becomes visible to the naked eye, it remained localized and involved the villi in only a portion of the placenta in these patients with coexisting tumor and fetus. The primary lesion may behave in various manners. It may be contained completely within the placenta and be expelled in its entirety with delivery of the placenta. It may directly involve the uterus by

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extension from the primary sitc or indirectly b y vascular metastasis to the myometrium. In the latter process, a mass of malignant trophoblast within a myometrial vascular c:hannel becomes lodged, destroys the endothelium, and invades the myometrium. The case described by Fikentscher (1941)probably rel~resentsan instance of the latter type of uterine metastasis, since the re was no connection between the placenta and the endometrial-myometrial tissues but there were myometrial lesions, The primziry lesion may produce distant metastases without involvement of the uterus. It may infrequently metastasize to the fetus. From this primary site and Eiefore there is any local extension into the endometrium and myomet rium, the choriocarcinoma can metastasize via the intervillous space and myometrial venous channels to distant sites. That this does hallpen and that it happens frequently is demonstrated by the findings in the patients in Group 1. In 14 of the 15 patients with proved metastases, the primary lesion was identified in the otherwise normal placenta and the uterus was examined histologically either posthysterectomy or postmortem. One of the 15 cases is excluded because there were no metastases and because the entire lesion was contained within thci: placenta (Driscoll, 1963). In 6 of 14 patients, there were no lesions in the endometrium or myometrium despite the fact there were distant metastases. Thus, the neoplasm skipped the uterus in approximiitely one-half of the cases. The mechanism by which this happens wai demonstrated b y Brewer and Gerbie (1966). In the primary site the malignant trophoblast arising from the villi projects into the intervillou s space, Within this mass of malignant trophoblast there is spontaneou i degeneration of the centrally located cells that results in sequestraticin and fragmentation of the peripheral portion of the mass. This is a fi-equent finding. These fragments become dislodged and are Carrie13 by the intervillous circulation into my ometrial vessels unaccompariied by any portion of the stromal villi from which they arose. Once in ihe uterine circulation they are carried to various distant sites where they form metastatic lesions. The frequency with which this phenomenon occurred in the 14 patients and the demonstration of how it happens provide the information needed to explain why residual choriocarcinoma is often not found in the uterus in patients with advance13 metastatic disease, as observed and reported by others. Hertig (1968 p. 308) found the uterus free of tumor in 50% of patients with metastatic choriocarcinoma associated with hydatidiform mole. Hunter andl Dockerty (1955) reported it free of tumor in 10-33% of patients who died with metastatic choriocarcinoma irrespective of the typr of antecedent pregnancy. Hou and

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Pang (1956) found no residual tumor in 32% of patients with choriocarcinoma, and Mirquez-Monter et al. (1968) reported none in 29% of cases. Brewer (1962) observed the uterus removed surgically to be free of tumor in 12% of 80 patients with metastatic choriocarcinoma irrespective of the type of antecedent pregnancy. It is not possible to compare directly these various rates of frequency with which the phenomenon was observed since the pregnancy situations are different; but the combined data do show that rather frequently there will be no residual tumor in the uterus in patients with metastatic disease and that the pathologist and clinician can accept this as a characteristic of gestational choriocarcinoma. In the cases cited in this review it is demonstrated how infrequently the diagnosis is made b y examination of the primary, early lesion. There are many reasons for this, some of which are mentioned here. Quite commonly the disease is not clinically suspected at the time of the obstetric event, be it term or premature delivery, and the placenta containing the lesion is discarded without being examined histologically. The primary lesion is frequently small and does not distort from normal the gross appearance of the placenta. Later, when symptoms continue and/or when metastases appear, the placenta is no longer available for study and the opportunity to identify a primary lesion is lost. The placenta1 lesion may be so tiny, even though the metastases are large and extensive, that a diligent histologic search of the placentas may be required to locate the lesion, and this is not often done. The primary lesion may be completely expelled with the delivery of the placenta, leaving only the metastatic lesions in the myometrium or distant organs or tissues. At times, an abnormality in the placenta is observed but the histopathologic diagnosis is not made. An example of this is the case of abortion described by Schumann and Voegelin (1937) and mentioned previously in this review. The initial curettage specimen was diagnosed as degenerated chorionic villi and nests of abnormal syncytium. Although these tissue changes were identical to those in cases cited in this review, they were not recognized as malignant b y Schumann and Voegelin. That they were is evidenced by the death of the patient from metastatic choriocarcinoma within one year. In some instances the primary lesion is identified and is found to contain no villi. The evidence indicates this can be accounted for by the fact that, because of the delay in performing diagnostic procedures, the primary lesion had progressed beyond the early stage when villi were present, or they were absent because a portion of the primary lesion containing villi was spontaneously expelled, the more adherent invasive trophoblast remaining in place in the utenis.

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The 29 cases cited give insight into the primary lesion of choriocarcinoma. They form a basis for elaboration of some of its characteristics and lead to the concept that the gestational choriocarcinoma the origin of the neoplasm is in the trophoblast of an otherwise normal placenta. II. lnvasive Mole and Choriocarcinorna Associated with Hydatidiforrn Mole

In presenting data on invasiv 9 mole and choriocarcinoma associated with hydatidiform mole, the relationship of these trophoblastic diseases should be mentioned. The concept has long been accepted that hydatidiform mole develops as a basic or primary lesion and then may through cellular changes advance to invasive mole or to choriocarcinoma, or that it might progress to invasive mole and from invasive mole progress further to choriolzarcinoma. Park (1971, Chapter VI, p. 90) has proposed a concept, anc! has offered some evidence to support it that invasive mole is an entit:, unto itself with its invasive qualities present at the very beginning ol’the molar changes in the placenta. In addition, there is some possibility that choriocarcinoma is an early integral part of a hydatidiform mole, present at its inception. The case managed at our center and presented on page 118 may be an example of such a phenomenon. Since these concepts remain to be proved, the assumption to be used in this report is that hydatidiform mole is the primary lesion and that it may Ibrogress to invasive mole or choriocarcinoma.

A. INCIDENCE O F INVASIVE MOLE AND CHORIOCARCINOMA WITH HYDATIDIFORM MOLE ASSOCIATED In evaluating the data reported on the frequency with which invasive mole and choriocarcinoma are encountered in association with hydatidiform mole two groups of patients were established for study and comparision (Table I). The first group is comprised OF those patients in whom less sensitive hCG test methods were emplo:ied, hysterectomy was the major element in the treatment, and the diagnosis was established by histopathologic examination of the surgically removed tissues or of postmortem specimens. The data obtained in this group provide firm pathologic diagnosis upon which to base an incidence rate, but they are deficient in that they do not take into account those cases of invasive moles that undergo spontarl eous regression and escape detection and identification. The hCG test methods used were not adequately sensitive to detect persistent but degenerating disease or progressive

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TABLE I FREQUENCY OF INVASIVE MOLE AND CHOFiIOCARCJNOMA ASSOCIATED WITH HYDATIDIFORMMOLE, GROUPEDACCORDING TO DEGREEO F SENSITIVITY O F TEST METHODSUTILIZED BY INVESTIGATORS Number

of Authors Group I" Hertig and Sheldon Hertig Acosta-Sison and Baja Panlilio Acos ta-Sison Acosta-Sison Douglas Brewer and Gerbie Beischer Ringertz

patients

200 205 177 210 164 206 66 328 631 -

Total: Diagnosis known: Group I I b (a) Hamburger Delfs (b) Brewer Bagshawe Curry Goldstein

2187 1805

Total: Diagnosis known:

1446 911

129 7 2 ) 201 502 280 347 116 -

Percentage with I.M.

16

Percentage with CCA

2.5

7.6 6.1 7.3 7.6 2.7 2.1

8.6 10.9 2.4 3.0 0.9 1.6

5.5

3.4

23.2 42.0 48.2

4.7 3.0 0.7

Total percentage with I.M. and CCA

18.5 14.6 17.5 16.2 17.0 9.7 10.6 3.6 3.7 10.0

1

24.4

34.6,

40.6

'1 27.9

20

38.4 41.3

2.5

Group I = test method with low degree of sensitivity. Group I1 = test method with high degree of sensitivity; (a) degree of sensitivity is higher than in Group I, but lower than Group IIb; (b) degree of sensitivity is the highest.

disease with low hCG titer levels. The inadequacy of the test methods generally used was shown by Brewer and DeCosta (1967).In 26 of 137 patients who had hydatidiform mole and who were serially tested until invasive mole was diagnosed the test results were negative each time tested in 10 (38%),the results were negative one or more times they were performed in 14 (54%),and in only 2 patients (8%)were the results positive each time tested. The second group is made up of patients in whom extremely sensitive hCG test methods were utilized (mouse uterine weight bioassay, radioimmunoassay LH/hCG, and a modified procedure measuring

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specifically the native hCG ancl its p subunit), chemotherapy was the principal treatment with hysterectomy infrequently performed, and the diagnosis was established histopathologically in those with choriocarcinoma but much less often in those with invasive mole. The data obtained in this group provide, through the use of highly sensitive test methods, the firm evidence of surviving trophoblast and permit detection of the presence of continued disease long after the less sensitive methods become negative, which in some cases erroneously leads to the assumption that al; disease has disappeared. If the hCG titer is elevated beyond 60 days postevacuation and if the uterine cavity contains no residual hyd atidiform mole, the surviving trophoblast, thus detected, must be residing in the wall of the uterus and/or elsewhere in the body and must be either invasive mole or choriocarcinoma. Differentiation between these two lesions by histopathologic examination of tissue would be helpful, but, if this has not been done, the presumed and provisional diagnosis is invasive mole. Choriocarcinoma requires a pathologic diilgnosis. Only in a few instances, as tlie clinical course of the patient unfolds, will this provisional diagnosis be incorrect. Almost invariably, choriocarcinoma will make itself known before or after the sixtieth day and will be properly identified. The obvious deficiency in this method of arriving at an incidence rate is the lack of specific pathologic diagnosis in all cases. This is due to the fact chemotherapy has provided such excellent remission rates without surgery that hysterectomy or scrgical removal of other lesions is not justified for the establishment oj’ a pathologic diagnosis. The accuracy and the ability of the sensitive hCG test methods to detect disease, even though it may not be demonstrated by other procedures, provides a clinically acceptable base for determining the frequency with which invasive mole and choriocarcinoma are associated with hydatidiform mole after its evacuation.

1. sroup 1 Hertig and Sheldon (1947) rcported a study of 200 patients with hydatidiform mole, in whom 32 had an associated invasive mole (16%) and 5 had choriocarcinoma (2.5%),a combined incidence of 18.5%.In a subsequent series of 205 patierIts with hydatidiform mole studied by Hertig (1968) and analyzed by Dr. A. Brian Little, the overall incidence of invasive mole and choriocarcinoma was 14.6%. I n 1951, Acosta-Sison and Baja Panlilio reported an overall incidence of 17.5% of invasive mole and choriocarcinoma in 177 patients with hydatidiform mole. Acosta-Siso 1 (1961) found the incidence to be 16.2% in an additional 210 patiients; 16 patients had invasive mole

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(7.6%)and 18 patients had choriocarcinoma (8.6%).In another group of 164 patients with hydatidiform mole, Acosta-Sison (1964) reported the overall incidence to be 17% with 10 patients having invasive mole (6.1%)and 18 patients having choriocarcinoma (10.9%).Douglas (1962) in a study of 206 patients with hydatidiform mole found the following frequency of associated lesions: invasive mole, 15 patients (7.3%);choriocarcinoma, 5 patients (2.4%),an overall incidence of 9.7%. Brewer and Gerbie (1967) gathered data from 66 patients at Cook County Hospital which showed invasive mole in 5 (7.6%), choriocarcinoma in 2 (3%)with an overall incidence of 10.6%.In 1970, Beischer et al. studied 328 patients with hydatidiform mole and found that 9 (2.7%)had developed invasive mole and 3 (0.9%)had choriocarcinoma. The overall incidence was 3.6%.Two had disease they could not classify. Ringertz (1970) recorded 13 (2.1%)invasive.moles and 10 (1.6%)choriocarcinoma in 631 molar pregnancies, making an overall incidence of 3.7%. In Group 1 patients, the combined data of the reports show that in 2187 patients with hydatidiform mole, invasive mole and choriocarcinoma occurred in 222, an incidence rate of lo%, with a range of 3.6% to 18.5%. In determining the frequency with which invasive mole occurred and with which choriocarcinoma occurred, only 1805 of the 2187 patients are available for evaluation, since two of the reports reviewed did not give the data separately for each of these two lesions. Invasive mole occurred in 100 of the 1805 patients, a rate of 5.5%,with a range of 2.1% to 16%. Choriocarcinoma occurred in 61 of the 1805 patients, an incidence rate of 3.4%, with a range of 0.9%to 10.9%.

2. Group 2 Before presenting the data obtained from this group of patients it is essential that some premises be stated that will form the basis of the' evaluation to be made. The fiequency with which invasive mole and choriocarcinoma occurs in patients with hydatidiform mole is a totally different value fiom and is not to be confused with the frequency with which patients require treatment for these two tumors. This is mentioned because a distinction is not made between these two different incidence rates in some published reports. In our experience, the presence of these two tumors can be identified by sensitive hCG test methods where tests with less sensitivity fail. An example of this is an invasive mole undergoing spontaneous degeneration, as it fiequently does, with falling levels of hormone. The sensitive methods will detect the low level of hCG while the others will not. In relying upon these tests to identify the presence of

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the two tumors it is necessiry to know there is no residual hydatidiform molar tissue within the cavity of the uterus, since, if present, it could be the source ol'the hormone production. Curettage is adequate to ascertain this. Following complete terminat ion of a hydatidiform mole, the hCG titer as determined b y sensitive test methods will be in normal range within 60 days. This has been generally accepted. Thus, in those patients with elevated titers at and after 60 days postevacuation of a molar gestation and with no res dual hydatidiform mole in the cavity of the uterus, it can be concluded that either invasive mole or choriocarcinoma is present sornewhere in the body, possibly the myometrium, cervix, or more dijtant sites. Where it might be located and whether it is in a phase of regression, latency, or continued growth remains to be determined by fiirther gonadotropin testing, by other special tests and examinations, and by surgical procedures when indicated. In most instances of invasive mole, the determination of the quantity of hCG in the blood or urine will suffice to indicate its presence, to evaluate its course, and to permit its diagnosis clinically with reasonable assurance. I n choriocarcinoma, however, pathologic examination of tissue is required for its diagnosis. Hamburger (1944)studied 72 patients with hydatidiform mole using more sensitive hCG test methods than those employed in Group 1 patients, and found that 13 (18%)developed or had invasive mole or choriocarcinoma. Delfs (1959) r-ported a study of 129 patients with hydatidiform mole. She used hei sensitive bioassay in the follow-up of 119 patients after evacuation of hydatidiform mole and found that 26 (21.8%)had persistently elevated gonadotropin titers at 60 days postevacuation indicative of surviving trophoblast. The other 10 patients had hysterectomies as the primary treatment for invasive mole or choriocarcinoma and thus were excluded from the study of 119 patients in which the objective was to determine by hCG testing the natural history of the growths and the period of regression, latency, or transformation to malignancy. In the 129 patients, progressive trophoblastic disease, invasive Inole and choriocarcinoma, were present in 36 patients, an overall incidence of 27.9%.Invasive mole was present in 30 patients and choriov:arcinoma was present in 6, incidence rates of 23.2%and 4.7%, respectively. At the Northwestern University Trophoblastic Disease Center, the incidence has been determined in 502 patients with hydatidiform mole who were serially examin sd and had gonadotropin assays performed at frequent and regular intervals after evacuation of the molar gestation (Brewer et al., 1977). The test methods used were (1) the

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modified RIA-LHlhCG (Halpern et al., 1971) (2) the specific hCG procedure (Vaitukaitis, 1973) which measures native hCG and its p ~ u b u n i tmodified ,~ by us to measure as low as 1.6 mIU hCG/ml serum, and (3) the mouse uterine weight bioassay. The presence of invasive mole and choriocarcinoma in association with the molar gestation was determined by gonadotropin testing, clinical and radiologic examination, special procedures, and pathologic examination in some. All choriocarcinomas were proven by histopathologic study. Invasive mole and choriocarcinoma were present in 226 of the 502 patients, an overall incidence of 45%.Choriocarcinoma was present in 15 patients (3%)and invasive mole in 211 patients (42%). Bagshawe et al. (1973) reported the experience in the follow-up of 280 patients after evacuation of hydatidiform mole. Evaluation of his data based upon the premises established indicates that 137 of the 280 patients had invasive mole and choriocarcinoma, an incidence rate of 49%. Choriocarcinoma was present in 2 patients, 'a frequency rate of 0.7%.Invasive mole was present in 135 patients, an incidence rate of 48.2%.These are the frequencies at which the diseases occurred; they do not indicate the number of patients treated for these diseases. Curry et al. (1975) reported the findings in the follow-up of 347 patients with hydatidiform mole but did not provide individual data for invasive mole or choriocarcinoma. Of the 347 patients, 85 had elevated hCG titers at 60 days after evacuation of the hydatidiform mole, indicating rather clearly the presence of invasive mole or choriocarcinoma. In addition, 35 other patients were identified as having invasive mole or choriocarcinoma prior to 60 days postmolar evacuation. Thus, of the 347 patients, 120 had invasive mole and choriocarcinoma, an incidence of 34.6%. Goldstein (1972)reported data on the follow-up of 116 patients after evacuation of hydatidiform mole. Of these 116 patients, 23 had invasive mole or choriocarcinoma, an incidence rate of 20%. Categories of invasive mole and choriocarcinoma were not established, but the classification of metastatic and nonmetastatic trophoblastic disease was used. The combined data of Group 2 patients reveal that invasive mole and choriocarcinoma occurred in 555 of 1446 patients with hydatidiform mole, an incidence of 38.4%, with a range of 18% to 49%. In determining the frequency with which these tumors individually occurred, the total number of patients available for evaluation is 911 rather than 1446. Three of the reports reviewed gave data for the two combined tumor types and did not provide information for the tumor Immunochemical grade hCG is kindly provided by NIH-NIAMDD.

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types individually. Invasive mole occurred in 376 of the 911 patients, an incidence of 41.3% with a range of 23.2% to 48.2%. Choriocarcinoma was identified in 23 of ;he 911 patients, an incidence of 2.5%, with a range of 0.7% to 4.7%.

3. Sornment A comparison is made of the frequency with which invasive mole and choriocarcinoma, taken togcther, are associated with hydatidiform mole in patients in the two groups. In Group 1, in which less sensitive tests were used, invasive mole and choriocarcinoma occurred in 222 of 2187 patients with hydatidiform mole, an incidence of 10% and a range of 3.6%to 18.5%.I n Group 2, in which new and highly sensitive test methods were utilized, the ;e two lesions occurred in 555 of 1446 patients, an incidence of 38.4%,with a range of 18%to 49%.The latter higher rate of 38.4% is believed to be nearer the true incidence. It is evident from the analysis of th. data that the higher rate is a direct reflection of the greater ability ( I f the improved test methods to detect the presence of disease as compxed to the tests with lesser sensitivity. The results obtained by Hamburger (1944) and Delfs (1959) are of interest in this regard. They utilized test methods that were more sensitive than the older methods but less sensitive than the newer methods. We have included their data as a part of Group 2, despite the fact the sensitivity of their tests is intermediary between that of tests used in Group 1 and 2 patients. Their combined data show that invasive mole and choriocarcinoma occurred in 49 of 201 patients with hydatidiform mole, an incidence of 24.4%. The separate incidence rates for Group 1, the Hambuiger and Delfs' studies and Group 2 (excluding Hamburger and Delfs' patients) are respectively as follows: lo%,24.4%,and 40.6%.The incidence recorded by Hamburger and b y Delfs is intermediary to those recorded in Group 1 and in Group 2, which is consisent witk the intermediary position of sensitivity of their test methods. The fi-equency of choriocarciiioma in each of the groups is as follows: in Group 1, 61 of 1805 ratients, an incidence of 3.4% with a range of 0.9%to 10.9%;in Group 2,23 of 91 1 patients, an incidence of 2.5% with a range of 0.7% and 4.7%.The greater frequency noted in Group 1 is undoubtedly owing to) the inclusion of the data reported b y Acosta-Sison (1961, 1964), whick revealed the frequency to be 9.6%in 374 patients with hydatidifonrl mole, an incidence considerably greater than in the other patients in this group. Overall, it is quite apparent that the incidence of cnhoriocarcinoma remains at the same level as generally reported and accepted for the past 50 years.

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Such is not the case in the frequency of invasive mole. The incidence in patients of Group 1 is 5.5%as compared to 41% in those of Group 2. The range is 2.1%to 16%and 23.2%to 48.2%,respectively in the two groups. This higher incidence reflects the ability of the more sensitive test methods to identify persisting surviving trophoblast in degenerating invasive moles where less sensitive methods fail. While it can be expected that 38.4% of patients with hydatidifomi mole have or will have either invasive mole or choriocarcinoma, this in no way means that such a percentage of patients will require treatment. It remains for the physician to identify those who do and those who do not need treatment by appropriate testing and examination procedures. OF PATIENTS AFTER EVACUATION OF HYDATIDIFORM B. FOLLOW-UP

MOLE Delfs (1959) demonstrated that gonadotropin testing, using a method superior to those in general clinical use at that time, could detect the presence of invasive mole and choriocarcinoma after molar evacuation with more accuracy than previously and could bring about improved results with hysterectomy. Brewer et al. (1968) established and conducted a prospective protocol study using a more highly sensitive gonadotropin assay. It was shown that invasive mole was present in even greater frequency than reported by Delfs and that chemotherapy could provide 100% remission in both invasive mole and choriocarcinoma. Since then the effectiveness of such a plan of management has been repeatedly documented. At present the main issue in the follow-up management of patients after evacuation of hydatidiform mole is the more precise identification of the patients with invasive mole who must be treated. In this way it may be possible to reduce the number of patients subjected to the risks of chemotherapy. The number of patients diagnosed as having invasive mole has increased markedly with the utilization of sensitive test methods while the number with choriocarcinoma has remained approximately the same. This has resulted in more patients with invasive mole being treated. Since invasive mole often undergoes spontaneous degeneration and complete resolution without any treatment, it is desirable to review and evaluate the current published data regarding the numbers of patients treated and the indications for treatment. The purpose of this brief review is tn Dresent the data as reported

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from the United Kingdom (B:.gshawe et al., 1973) and the United States, to add the results of the latest study at Northwestern University Trophoblastic Disease Center reported here, and to evaluate and compare the data. Bagshawe et al. (1973) studied 280 patients following evacuation of hydatidiform mole and found that 143 (51%)had hCG titers in normal range by 60 days postevacuatic'n and remained normal thereafter. Of the remaining 137 patients, 3 were treated prior to the sixtieth day, 2 for invasive mole and 1 for choriocarcinoma. The other 134 patients had elevated hCG titers at 60 days postevacuation. Thirteen of these were treated subsequently, 5 at 4-5 months and 8 at 5-6 months postevacuation. One had choriocarci noma and 12 had invasive mole. In the 121, who were not treated, tho: hCG titers gradually fell to normal levels by day 280 post evacuation. In the total 280 patients in the series, he reported that spontar,eous complete regression occurred in 264 (94.3%)and that 16 (5.7%)were selected for treatment, 14 for invasive mole and 2 for chorio1:arcinoma. Bagshawe considers those lesions, which regressed spontaneously, to be invasive mole. He concludes from his study that continued gonadotropin testing after day 60 is preferable to treating patienis at or shortly after day 60 solely because of elevated hCG values. This is based upon the observation that practically all patients have inviisive mole and that most will undergo spontaneous regression. At the Northwestern University Trophoblastic Disease Center, our group has followed 502 patients after evacuation of hydatidiform mole (Brewer et al., 1977). The data are presented in Table 11. They show that in 276 (55%)of the 502 patients the hCG titers reached normal levels b y the sixtieth day, which is approximately the same as the 51% reported by Bagshawe et al. (1973). Of the remaining 226 patients, 18 were treated prior to day 60 and 208 still had elevated titers at 60 days. Of the 18 patients treated prioi to the sixtieth day, 3 had choriocarcinoma proved by curettage and 15 had invasive mole. Eight of the 15 had rising titers in the region of 100,000 IU/liter, 1had pulmonary and niesenteric metastases with a rising titer level of 50,000 IU/liter, 1 had pulmonary metastasis with plateauing titer at 40,000 IU/liter, and 5 had plateauing titers in the range of 4,000-20,000 IU/liter. Of the 208 patients, 121 had declining hC(; titers, which reached normal levels by day 170, evidence of spontaneous and complete regression of their invasive moles, The other 87 hzrd continued evidence of disease and were treated. Seventy-five had invasive mole and 12 had choriocarcinoma. The days postevacuation they were treated are shown in Table 11. Not all patients with elevated titers at day 60 are treated; in

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J. I. BREWER ET AL. TABLE I1

NORTHWESTERNUNIVERSITY TROPHOBLASTIC DISEASECENTERFOLLOW-UP POSTEVACUATION OF 502 PATIENTS WITH HYDATIDIFORM MOLE" Days after evacuation of hydatidiform mole ~

hCG Titers

30 days

60 days

Normal range Elevated

95 (19%) 407 (81%)

276 (55%)* 208 (41%Pd

Total patients treated: 105 (20.9% of the total 502 patients); 90 for invasive mole, 11 metastatic, 79 nonmetastatic (17.9% of the total 502 patients); 15 for choriocarcinoma, 3 metastatic, 12 nonmetastatic (3%of the total 502 patients). * 276 patients: none treated; all have remained free of disease 1-14 years. 226 patients remaining: 208 patients had continued diagnostic testing up to 60 days and afterward; 18 patients were treated prior to day 60 for invasive mole (15) or choriocarcinoma (3) and are thus excluded from the 60-day data. d208 patients with elevated hCG titer at day 60; 121 patients: not treated; all have remained free of disease 1-14 years; 87 patients treated: 62 at 60-90 days, 18 at 90-120 days, 4 at 120-150 days, 3 at 180-290 days.

the majority of patients testing is continued and decisions to treat may not be made for as long as 180 days, or in 3 patients even up to 290 days. These data indicate that the overall plan of hCG testing used by us and by Bagshawe et al. (1973) is comparable, but that some variations in the indications for treatment exist. The striking difference between the two reports appears in the percentage of patients treated, 20.9% versus 5.7%, respectively. While the difference can be accounted for in part b y the greater number of patients with choriocarcinoma in our group as compared to his, 15 (3%)versus 2 (0.7%),the fundamental reason for the difference is the greater number of patients treated for invasive mole in the former group. In none of 502 patients followed with hCG monitoring was there a recrudescence of trophoblastic growth after the hCG had fallen spontaneously below the concentration detectable by radioimmunoassay. All of the 502 patients, those treated and not treated, have remained well and free of disease for 1to 14 years. From the Southeastern Trophoblastic Disease Center at Duke University, Curry et al. (1975)reported the follow-up study of 347 patients after the evacuation of hydatidiform mole. By the sixtieth day postevacuation, 227 (65.4%)patients had hCG titers in normal range. Of the remaining 120 patients, 35 were treated prior to day 60, 12 with metastatic trophoblastic disease and 23 with nonmetastatic trophoblas-

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tic disease. Patients were not classified according to those who had choriocarcinoma and those who had invasive mole, so these data are not available. The other 85 patients had elevated titers at day 60 postevacuation; 51 of whom had gradually declining titers that reached normal range without therapy, and 34 of whom were treated. Thus, of the 347 patients with hydatidiform mole, 278 (80%)regressed without any therapy and 69 (20%)were treated. Again, for the entire group, disease categories of choriocarcinoma and invasive mole were not reported. All 347 patients are in remission. The 35 patients, who were treated prior to day 60, received therapy because of metastatic disease in 12 and rising hCG titers in 23 patients. The 34 patients treated after 60 days were treated because of plateauing titers or an elevated titer after day 60. These data indicate that a greater number of patients were treated (20%)than reported by Bagshawe et al. (5.7%)in 1973. Goldstein (1972) reported a study from the Trophoblastic Disease Center at Harvard of the follow-up of 116 patients after evacuation of hydatidiform mole, none of whom had prophylactic therapy. By the sixtieth day postevacuation, 93 (80%) of the 116 patients had hCG titers in the normal range. The other 23 patients (20%)were treated,, 5 for metastatic and 18 for nonmetastatic disease. Data were not reported according to disease categories of choriocarcinoma and invasive mole. All of the 116 patients, treated or untreated, are free of disease. The percentage of patients treated is greater than that cited by Bagshawe et al. (1973), 20% versus 5.7%,respectively. Delfs (1959) reported a follow-up study of 129 patients who had hydatidiform mole. She used a gonadotropin test method more sensitive than those used in Group 1 patients but less sensitive than those used in Group 2 patients, except the one reported b y Hamburger. The tests were performed every 1 to 3 weeks until negative or until treatment was indicated, The study was conducted during the time that hysterectomy was the standard treatment for invasive mole and choriocarcinoma and prior to the advent of chemotherapy. Ten of the 129 patients had hysterectomies early in the follow-up period, thus eliminating the local site of trophoblast. While the pathologic diagnoses are not recorded, the notation was made that one patient subsequently died of metastatic choriocarcinoma. Of the remaining 119 patients, 93 (78.2%)had normal gonadotropin titers and 26 had elevated titers at day 60 postevacuation. In 15 of the 26 patients the assays became negative later and in the other 11, hysterectomies were performed for choriocarcinoma in 5 and invasive mole in 6. Thus, 9.2% of the 119 patients required treatment. This

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does not represent the complete picture of the 129 patients, since it is noted that 1 of the other 10 not tested had choriocarcinoma and died, and it is not stated how many of the remaining 9 had invasive mole that might or might not have needed treatment. A review of these data reported from the United Kingdom (Bagshawe et al., 1973) and from the United States (Brewer et al., 1977; Curry et al., 1975; Goldstein, 1972) reveals that the percentage of patients with normal hCG titers at 60 days postevacuation of molar pregnancy is approximately the same, 51%, 55%, 65.4%, and 80%, respectively. Delfs’ data are excluded since the gonadotropin test used was of less sensitivity and accordingly the data obtained are not entirely suitable for comparison. The percentage of patients treated is markedly different: Bagshawe et al. (1973), 16 of 280 patients (5.7%);Brewer et al. (1977), 105 of 502 (20.9%);Curry et al. (1975),69 of 347 (20%);and Goldstein (1972), 23 of 116 (20%).These figures include all patients treated. Some were treated for definite indications prior to 60 days and thus did not complete the full 60-day testing schedule. It is apparent from the reports that the difference in the number of patients treated is largely due to some differences in the indications for therapy. A major variation is the indication for the treatment for certain metastatic lesions. K. D. Bagshawe (personal communcation, 1976) does not treat patients with a vaginal metastasis unless it is associated with hemorrhage or a pulmonary lesion unless the titer is rising. H e has shown that most of these are metastatic invasive mole and that they will regress spontaneously. The American workers treat all patients with metastases including these two exceptions made by Bagshawe. It is believed that treatment should b e administered to patients with vaginal lesions since a hemorrhage can occur at any time, which in some instances requires heroic measures to control and in others is completely uncontrollable. Another variation is in the management of histologically identified invasive mole. Curry et al. (1975), Goldstein (1972), and Jones and Lewis (1974) treat all such patients, while Brewer et al. and Bagshawe do not. The latter two require that other indications be present, such as severe uterine hemorrhage, uterine perforation with internal bleeding, or rising titers. Goldstein (1972) stated all patients with elevated titers at day 60 postevacuation were treated. Curry et al. (1975), Brewer et al. (1977), and K. D. Bagshawe (personal communication, 1976) have not used this as a definite indication, but rather have continued to follow the patient with hCG testing until other indications for treatment appear or until the disease regresses spontaneously. In this particular situation it is

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quite apparent that Bagshawe et al. (1973) carried the patients further without therapy than the others have been willing to do, and thus fewer patients are treated. He believes this is justified since most are invasive mole that will regress if given more time. His data (Bagshawe et al., 1973) indicate this to be true in the majority of instances. However, at the Northwestern University Center, we hesitate to continue testing the patient very long after day 60, unless the titers are progressively declining each time tested. Our experience has differed from that of Bagshawe et al. (1973; K. D. Bagshawe, personal communication, 1976). Of the 87 patients who had continued testing after day 60, choriocarcinoma was present in 13 in contrast to Bagshawe’s experience in which only 1 of 13 had choriocarcinoma. The groups in the United States believe a plateauing titer is an indication for treatment. K. D. Bagshawe’s (personal communication, 1976) list of indications do not include this, but is is presumed that titers plateauing at a high level would be sufficient reason to start treatment. All agree on certain indications : histologically proven choriocarcinoma, rising titers at any time, particularly if they are progressively increasing or rise sharply, a detectable level of hCG persisting and not falling by 6 months postevacuation, and hemorrhage or persistent bleeding &om the uterus or metastatic site. In view of the dissimilarities between the specific indications for therapy used by Bagshawe et al. (1973)and the reporting groups in the United States, the opportunity was taken to analyze the data further. A comparison from a different standpoint was made of the data reported by Bagshawe et al. (1973) in which 16 (5.7%)of 280 patients were treated and the data from Northwestern University reported here in which 105 (20.9%)of 502 patients were treated. We have used the indications for treatment specified by Bagshawe et al. (1973), from which his data were derived, to determine how many of the 105 patients at Northwestern University would have been treated if these indications were followed rather than our own. The result was as follows: 68 (13.5%)of the 502 patients would have been treated. It is apparent that a fewer number of patients would have been treated, if his indications had been used. It is also equally true that in our group of patients more required treatment than did those in the group reported by Bagshawe, namely, 13.5%versus 5.7%.The reason for this difference is not readily explained. It may be that more patients with actively progressing disease were by happenstance encountered in our group. In any case, these data indicate that in the United States a realistic figure for the number of patients with hydatidiform mole who

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will require subsequent treatment for invasive mole or choriocarcinoma is approximately 13.5%. Ill. lmmunobiology of Trophoblastic Disease

Gestational choriocarcinoma is derived from the trophoblast and may be considered a tumor graft having the potential to express paternal histocompatibility antigens absent on maternal cells. This being the case, choriocarcinoma could be susceptible to immunologic rejection; however, spontaneous rejection of choriocarcinoma seldom occurs. This suggests that choriocarcinoma cells do not express the same strong histocompatibility antigens found on the surface of erythrocytes or the pregnancy in some way masks the host immune system inhibiting rejection. There are at least three factors which may account for the failure of the host to reject allogenic trophoblastic tumors. (1) The absence of strong transplantation antigens would constitute major incompatibility and thus stimulate an isograft (Dowling, 1957). (2) In the presence of a state of immune unresponsiveness in the mother (Robinson et al., 1967) the tumor could induce maternal tolerance to the paternal antigens it carries. For example, Breyere and Barrett (1960) showed that postpartum murine females exhibited specific tolerance to the transplantation antigens of the mating male. (3) The deficincy of alloantigens, either due to poor expression on trophoblast (Beer and Billingham, 1971) or to shielding b y other surface moieties as suggested by Currie and Bagshawe (1967), could avert the immunologic rejection of the mammalian fetus and of trophoblast.

A. HLA ANTIGENSAND TROPHOBLASTIC DISEASE Studies of the association of HLA antigens with gestational trophoblastic disease began with a search for a correlation between the presence of HLA factor and disease incidence. While Robinson et al. (1967) found no relation between choriocarcinoma and the presence of antigens 4a, 4b, or 12 other leukocyte specificities, Lawler et al. (1971) found a statistically significant increased frequency of HLA-A 1 and B8. There was no correlation between disease incidence and the presence of specific HLA antigens in the analysis performed by us (Mittal

et al., 1975). Another possibility relating to HLA antigens and choriocarcinoma may be that no single HLA factor is associated with the disease, but rather the role of the HLA system relates to histocompatibility be-

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tween husband and patient, permitting trophoblastic tissue to escape from an immune response. Histocompatibility would render the emergence of cellular allograft rejection less likely, and indeed antibodies which were produced might even enhance cellular growth rather than be cytotoxic. Because of the strong immunogenicity of HLA-2, it was of particular significance to search for disturbances in the distribution of this antigen. The hypothesis that trophoblastic neoplasia could not progress in the presence of strong incompatibility was consistent with the finding that HLA-2 had a low frequency among patients and an increased frequency among their husbands as reported by Iviskova et al. (1968) and Mogensen and KissmeyerNielsen (1969) but not confirmed by Amiel and Lebovici (1970) or Amiel (1971). Mogensen and Kissmeyer-Nielsen ( 1969) correlated the degree of HLA compatibility between patient and husban‘d with prognosis in a preliminary study of 6 families. The tissue typing of 5 of these families could have produced mating zygotes which were histocompatible with their mother. In a further analysis by Mogensen and KissmeyerNielsen (1972), 21 of 27 patients suffering from choriocarcinoma ran a clinical course which fit the assumption that HLA incompatibility between tumor and host was associated with resistance to the neoplasm. While Lawler (1976) and Lewis and Terasaki (1971) found that the degree of HLA compatibility in these matings was higher than would be expected with random mating, HLA typing did reveal histocompatible children born antecedent to the development of choriocarcinoma. Mogensen and Kissmeyer-Nielson (1972) further argued that the paucity of HLA antigens represented in “pure” Eskimos of Greenland, which would result in a greater probability of husband-wife compatibility, may explain the great frequency of trophoblastic tumors in Eskimos as compared to Caucasians. On the other hand, Lawler et al. (1971) found that 57 choriocarcinoma patients and their husbands were no more, or less, compatible than controls. HLA incompatibility had no effect on the duration of therapy required to achieve remission. Further, the HLA type of an infant product of a pregnancy simultaneously associated with a disseminated choriocarcinoma was incompatible for the HLA antigen B12 and an unidentified first locus specificity. Klouda et al. (1972) confirmed that the offspring mating frequencies actually observed in these patients and their husbands were random for 7 HLA-A and 5 HLA-B specificities. Therefore, HLA specificity does not appear to have major influence in the etiology of trophoblastic tumors or their response to treatment.

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B. ABO BLOODGROUP ANTIGENS

IN

TROPHOBLASTIC DISEASE

ABO histocompatibility antigens are strong transplantation antigens which have proven to be important blood group substances associated with tolerance or rejection of transplanted tissue. ABO blood group substances are determined by three allels at a single gene site and are inherited in a codominant fashion. They are present not onlv on erythrocytes but also on other tissues. Fuchs et al. (1956) demonstrated their presence on fetal epidermal cells and Gross (1966) found erythhrocyte blood group A substance on early trophoblast. Mogensen and Kissmeyer-Nielsen ( 1972) reported an unpublished observation of Van Rood involving one patient where HLA antigens were found to be present on the cells of choriocarcinoma. The relationship between trophoblastic disease and ABO blood groups has been reviewed by many investigators. Erythroblastosis fetalis was found to be caused by immunologic responses evoked in the mother by fetal Rh antigens. Looking for a similar relationship, Schmidt and Hertz (1961) compared maternal and paternal blood group factors in 28 patients with histologically proven choriocarcinoma or invasive mole. There were no differences from the expected distribution (Table 111).

TABLE I11 DISTRIBUTTON OF ABO GROUPSIN TROPHOBLASTIC DISEASE

Number of patients

United States Schmidt and Hertz (1961) Scott (1962) Lewis (1973) Mittal et a l . (1975) Malaya Llewellyn-Jones (1965) Singapore Dawood et al. (1971) Great Britain Bagshawe (1976) a

Choriocarcinoma. Invasive mole. Hydatidiform mole.

Percentage observedlexpected

0

A

B

AB

46143 35/45 58/45 48/40

46146 4614 1 2614 1 32/44

417 11110 6/10 1518

414 914 1014 317

32.8143.5

27.9126

17.9125

37.9142.5 4 1.9142.5

40.2125.4 27.1125.4

13.8126.3 25.9126.3

8.115.8 5.115.8

3 1 4 " ~ ~ 40.5145.5

44.9140.7

11.5110.3

3.213.5

28"~~ 46" 31a.b 95a.b.c

89" 35lC

21.45.5

GESTATIONAL TROPHOBLASTIC DISEASE

141

On the other hand, a considerable body of evidence supports such a relationship. Scott (1962) reviewed ABO blood group data available on 46 of 175 patients with histologically proven choriocarcinoma registered in the Albert Mathieu Chorionepithelioma Registry of the American Association of Obstetrics and Gynecologists at Northwestern University Medical School in Chicago. H e noted a shift in the expected distribution &om blood group 0 to blood groups A, B, and AB (Table 111). In a study of 140 Chinese patients from Malaya with gestational trophoblastic disease (87.2%hydatidiform mole, 6.4% invasive mole and 6.4%choriocarcinoma), Llewellyn-Jones (1965)noted a shift from blood group 0 to groups AB and A without a shift to group B (Table 111). In Singapore, Dawood et al. (1971) found a shift from blood group 0 to group A in 89 patients with choriocarcinoma but not in 351 patients with hydatidiform mole. Bagshawe et al. (1971) and Bagshawe (1976), in patients requiring chemotherapy for treatment of gestational trophoblastic disease, noted a shift in the predicted frequencies from group 0 to A. Bagshawe et ul. (1971)attributed this shift and the increased risk of choriocarcinoma to the presence of a compatible paternal blood group. For example, an excess of blood group A patients with group 0 husbands and a deficiency of blood group A women with blood group A husbands was noted, Bagshawe et al. (1971) found group A to 0 matings 10.4 times as likely to have choriocarcinoma as group A to A matings. This effect appeared to be greatest when choriocarcinoma followed term delivery. It was concluded that group A to 0 and group A to A matings produce conceptions which are ABO compatible with the mother, and, therefore, that mechanisms other than ABO compatibility must be operative in the etiology of choriocarcinoma. However, such a conclusion ignores possible significant differences at A1 and A2 subgroups which could produce incompatibility. These findings are consistent with a role of ABO compatibility in the development of choriocarcinoma since women of blood groups A, B, or AB conceivably would be unable to destroy not only their own but also type 0 malignant cells. The early work of Schmidt and Hertz (1961) has been confirmed b y Lewis (1973)and b y Mittal et al. (1975) in small groups of patients and b y Tomoda et al. (1976) in a large group of patients. They found no shift from group 0 to A among patients with choriocarcinoma and no increase in the incidence of group A to 0 matings. In the final analysis, there is no consensus on the role of ABO matings in the etiology and clinical course of trophoblastic neoplasia. Such an association, if it exists, might be only incidental, not etiologic.

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c. NATIVE HOST REACTIONS TOWARD

CHORIOCARCINOMA

The histologic finding of a mononuclear cellular response surrounding choriocarcinoma cells provides suggestive evidence for cellular rejection of trophoblastic cells. This mononuclear cellular response involves lymphocytes, large monocytes and plasma cells surrounding histologically preserved malignant trophoblastic cells. Elston (1969) noted this response frequently in gestational choriocarcinoma but infrequently in nongestational choriocarcinoma. He reported that patients with choriocarcinoma, which was associated with a mononuclear cellular infiltrate consistent with allograft rejection, were more likely to respond to chemotherapy than patients with choriocarcinoma when this histologic pattern was not seen. This finding was confirmed by Park (1971, Chapter VII, p. 114) and Mogensen and Kissmeyer-Nielsen (1972).The intensity of the cellular reaction correlated positively with the response to chemotherapy reinforcing the concept of a synergystic role between chemotherapy and the immune response (Elston and Bagshawe, 1973). Evidence in favor of a defect in specific cellular resistance against the progression of the tumor has been obtained by several workers. Ben-Hur et a2. (1965) found that three patients with choriocarcinoma accepted their husbands skin grafts but rejected those from other donors. Robinson et al. (1963) noted that 6 of 7 patients, who had leukocyte agglutinin incompatibilities with their husbands, showed prolonged survival of paternal skin grafts without production of leuko-agglutinins. They attributed this finding to the presence of maternal immune tolerance due to a large amount of circulating, paternal, histocompatibility antigens. On the other hand, an unusually high percentage of transformed cells was noted in mixed lymphocyte culture reactions between choriocarcinoma patients and their husbands by Lewis et al. (1966) and Halbrecht and Komlos (1968), suggesting that these women could initiate a proliferative response, although they apparently had defective cytotoxic responses. Antileukocyte antibodies were identified in choriocarcinoma patients by Math6 et al. (1964),and by Mogensen and Kissmeyer-Nielsen ( 1968). Math6 noted agglutinating antibodies active against husbands’ leukocytes and platelets in the sera of five patients. In two cases he also noted that paternal skin grafts were surviving for longer periods than expected. Mogensen and Kissmeyer-Nielsen ( 1968) found antileukocyte antibody in 4 of 46 women with choriocarcinoma. Three of these antibodies were monospecific. None displayed complementfixing activity with the husbands’ platelets. IvLskova et al. (1968)suggested that these antibodies may have enhanced or even induced the

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growth of the tumor. However, in these cases, it was difficult to interpret the significance of the presence of antibody due to the possibility that it was provoked by prior pregnancies, blood transfusions, or by the disease. Lawler et al. (1974) reported 4 cases in which patients were presumably immunized by their first pregnancies, which were hydatidiform moles, against HLA antigens as evidenced by the presence of lymphocytotoxins. This finding suggests that HLA antigens are present on trophoblastic cells and may have the potential to induce a cytotoxic humoral response.

TRIALS D. IMMUNOTHERAPY It has been postulated that the effectiveness of chemotherapy in gestational choriocarcinoma may be related to concomitant maternal immune responses. The occurrence of spontaneous degeneration of choriocarcinoma, which has been reported rarely, further reinforces the hypothesis of immune rejection. Thus, investigators have employed immunologic manipulation in tumor-bearing hosts in attempting immunotherapy. Prior to the widespread acceptance of chemotherapy as the moqt effective form of treatment for choriocarcinoma, Doniach et al. (1958) attempted immunotherapy by deliberately immunizing a patient 3 weeks following removal of her uterus perforated b y choriocarcinoma. Paternal leukocytes were injected subcutaneously into the patient in an attempt to induce an immune response against residual tumor cells. This approach was based upon the animal experiments of Medawar (1944) and Billingham et al. (1956) wherein hosts could b e rendered resistant to allogenic tissue not only by application of a skin graft but also by intradermal injection of donor leukocytes. Following immuni: zation with leukocytes, Doniach‘s patient experienced accelerated rejection of a challenge graft of her husband’s skin suggesting successful immunization. However, the trophoblastic disease progressed with development of pulmonary and vaginal metastases, which were then successfully treated with methotrexate chemotherapy. Although Doniach suggested that immunity contributed synergistically to the response following chemotherapy, the objective role of immunotherapy in the successful outcome of this case is uncertain. Three years later, Hackett and Beech (1961) immunized a patient, who had choriocarcinoma with pulmonary metastases, with paternal leukocytes emulsified in Freund’s adjuvant. Two and four weeks after the active, specific immunization with this vaccine, the pulmonary metastases progressively had decreased and the patient’s clinical situ-

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ation had stabilized. The success of the immunotherapeutic regimen was transient; b y the twelfth week, new pulmonary lesions appeared. Challenge paternal skin grafts applied at this time did display accelerated rejection of husband’s skin indicating successful immunization, at least against skin. Subsequent 6-mercaptopurine chemotherapy did not prevent the progression of disease and the patient died. Necropsy material failed to demonstrate a cellular response or histologic evidence of trophoblast rejection. I n this case, accelerated skin graft rejection without an evident cellular response suggests that either the malignant trophoblastic cells had been altered by the deletion or modulation of the histocompatible paternal antigens or that the patient had developed specific antitumor unresponsiveness. Cinader et al. (1961) treated a patient who had choriocarcinoma, with both active and passive immunotherapy after a single course of vinblastine, postulating that antigenic differences between host and choriocarcinoma tissues were insufficient to stimulate maximum antibody formation. H e felt that the administration of additional antigen might increase antibody production by reaching sessile antibody forming cells not exposed to the surface antigens at metastatic sites. After three vaccinations of paternal leukocytes, his patient was treated with injections of rabbit-antipaternal sperm antibody. Urinary chorionic gonadotropin levels fell steadily to normal, and pulmonary metastases disappeared. Although the immunotherapy was deemed effective in this case, the authors cautioned that spontaneous degeneration of trophoblastic tissue could not be excluded. Robinson and Ratzkowski (1965) attempted active immunotherapy for a patient with advanced metastatic choriocarcinoma following failure of methotrexate and nitrogen mustard chemotherapy. Her husband’s leukocytes and Freund’s adjuvant were injected into separate sites in the patient in order to stimulate both antitumor antibody production and a cellular response. Post immunization, paternal skin graft tests did not show accelerated rejection and the patient did not develop antileukocyte antibodies. In an attempt to enhance the response to paternal leukocyte antigens, the hosts immune system was further boosted with Freund’s adjuvant alone. Pulmonary and abdominal metastases decreased in size, concomitant with a fall in urinary chorionic gonadotropin levels. Despite this apparent clinical improvement, the patient suddenly died due to sigmoid colon perforation, secondary to progressive choriocarcinoma. Bagshawe and Golding (1970) have reported the largest series of treated patients with invasive mole or choriocarcinoma using an immunotherapeutic approach. They began using immunotherapy in drug

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resistant cases as early as 1958 and have treated 34 patients with multiple immunizations of husband’s skin grafts and/or leucocytes. In addition, BCG was used in many cases. The results were disappointing. Of all these patients there was only one in whom it seemed probable that immunization had achieved remission. However, after remaining in remission for 8 years, the patient relapsed. She is the only patient of those treated with immunotherapy to have relapsed more than two years following completion of treatment (K. D. Bagshawe, personal communication, 1976). Bagshawe’s experience is similar to that of other investigators. Still attempts to control drug resistant trophoblastic disease using other immunotherapeutic methods are being made. K. D. Bagshawe (personal communication, 1976) is using in vitro activated lymphocytes from HLA identical siblings and complement dependent anti-LCG sera which are known to have in vitro cytotoxicity to choriocarcinoma cells. No conclusive evidence fiom these attempts have been obtained.

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ADVANCES IN CANCER RESEARCH, VOL. 27

THE CHOICE OF ANIMAL TUMORS FOR EXPERIMENTAL STUDIES OF CANCER THERAPY

Harold 8.Hewitt Department of Morbid Anatomy, King's College Hospital Medical School, London, England

I. Introduction ........................................................... 11. Analysis of Species Used in Current Cancer Research .................... 111. Origin and Maintenance of Animal Tumor Sytems in Relation to their Validity as Models of Clinical Cancer .................. ..... A. Introduction ........................................................ B. Spontaneous Tumors ................................................ C. Chemically Induced Tumors ........................................ D. Virus-Induced Tumors .................... ....................... E. Tumors Transplanted under Conditions of Incomplete Isogenicity ...... F. Relative Usage of Different Categories of Animal Tumors Used in Recent Experimental Studies of Cancer Therapy ...................... IV. Reflections and Conclusions ............................................. References ............................................................

149 153 157

157 159 163 167 176 182 192 196

I. Introduction

The subject of this chapter is too broad in scope to lend itself to the comprehensive treatment that is approachable under the more restrictive titles usual in this series. Historically, we have an extension back to the earliest years of the century; contemporaneously, we are confronted by some hundreds of communications a month which ostensibly deserve consideration under the title. It has to be admitted, however, that experimental workers in this field are under increasing pressure &om research funding bodies to represent their studies as being inspired by clinical demands for more effective therapeutic resources, and it follows that the impress of clinical relevance is often given to experimental data with imprudent latitude; profession of clinical relevance or actual recommendation of clinical application of animal tumor data is now the rule rather than the exception, although many of the investigations reported cannot be regarded as more than exploratory. It is the purpose of this chapter to survey current practice in the selection of animal tumors for studies of therapeutic agencies for cancer, and to examine the status of these tumors as models of naturally occurring cancers in man by reference to criteria of eligibility (or 149 Copyright @ 1978 b y Academic Press, Inc All rights of reproduction in any form reserved ISBN C-12-w)6627-0

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disqualification) imposed by their mode of origin and/or conditions of transplantation. Inevitably, the survey of practice has had to be b y sampling from the recent literature in a way that strictly avoids bias in respect of features affecting the assessment to be made. It may be revealed here, in advance of the detail to be presented, that some reform is long overdue in the methods of selecting animal tumor systems for use in studies of cancer therapy, and that such reform involves not merely the reinstatement of principles of selection which have long been recognized, but the provision of facilities b y which they can be more widely regarded. Cancer is distinguishable from all other categories of human disease in that its general biological features are faithfully represented in naturally occurring cancers of small mammals suitable for laboratory investigations: No species specification is required for definition of the carcinogenic potential of a chemical or physical agent; the mode of growth and pattern of spread of the disease are closely similar in the different mammalian species; and, most important in our present context, restraint of tumor growth is effected by similar agencies and by similar mechanisms in the different species. Indeed, the principal considerations disturbing the analogy between cancer in man and cancer in small mammals relate to differences of scale: between man and mouse, for example, there is an approximate 3000-fold difference of body size, and an approximate 25-fold difference of mean tumor volume doubling time, in necessary accordance with the species difference of life span (Hewitt, 1976).Thus, the student of cancer is much more generously provided with adequate animal models of the human disease than, for example, the investigator of clinical virus diseases or of such degenerative diseases as atheroma. It is the very abundance of the availability of animal models of cancer which imposes a need for discrimination in their selection. Clearly, a great part of cancer research using animal tumors is directed to the elucidation of rather general biological problems, neoplastic tissue being a convenient rather than a necessary material for the investigation. This is true of many biochemical studies and studies of transplantation genetics (Snell, 1958); there is no normal tissue of which the graft size can be as precisely quantitated and the success of grafting determined as readily, as by the use of tumor tissue. Also, the widespread use of transplantable ascites tumors peculiar to small rodents permits a degree of cellular individuation of unique technical convenience for a wide variety of studies. Such usage of animal tumors is a proper exploitation of resources provided that the technical convenience remains a separate consideration from that of appropriate

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modeling of autochthonous cancer in man. We are concerned here with a more limited use of animal tumors, with experiments undertaken to elucidate problems intrinsic to naturally occurring cancer, which envisage clinical relevance, or the results of which are to provide an instigation for clinical application. The requirements here are very much more stringent; considerations of technical convenience may often have'to be set aside in order to meet the exacting criteria for correct modeling of the human disease. The predominant, and most neglected, features required of an appropriate animal model are those reflecting the host/tumor relationship. Carter (1973), writing of the clinical correlations of experimental models used in the Division of Cancer Treatment (DCT) of the National Cancer Institute, and referring to the testing of chemotherapeutic and combined modalities, entertains the concept of tumor response as the manifestation of interaction between the drug, the host, and the tumor. Following prevailing trends of thought, it can be surmised that this concept entails the assumption of some possible contribution to tumor depredation of a hostile expression of the constitution of the host against the tumor, not triggered by an action of the drug on that constitution but exerted independently as an additive contribution and possibly stimulated b y absorption of material from cells killed directly by the drug. While such contribution of host exertion to drug-induced tumor restraint cannot be excluded u priori in the case of clinical cancer, it is to be noted that two principal murine tumors used for drug testing by the DCT are Leukemia L 1210 and Melanoma B 16: Both tumors have histories which make it highly probable, as we shall see in a later section of this contribution, that the immunogenicity each exerts (Math&,1972; Bystryn et al., 1974) is artifuctual. If this is so, and it is difficult to exclude, then their validity as models of naturally occurring autochthonous cancer is questionable. In turning our attention to host-mediated contributions to the response of a tumor to applied therapy, whether these be simply additive or induced by the therapy, we are confronted b y a long-standing prejudice, influential to a greater or lesser degree on thinking at different periods of the history of oncology. The most evident influence of the host on a tumor is the ample provision made for its relentless growth, continued almost invariably to the extremity of host death; spontaneous arrest of growth, or regression, of a clinical tumor is extremely rare and mostly confined to relatively uncommon tumors (Everson, 1964). In spite of this evident and usual manifestation of the untoward balance of the host/tumor relationship, the concept of a defensive posture of the host against the tumor has been obsessively

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retained in oncological thinking throughout the history of the subject and dominates the cancer literature at the present time. It is very curious that the absence of active host defense against cancer should be abjured as heresy and attention be directed to study of the mechanism of its failure. The evidential support for this orientation is slight and rests, in the clinical situation, very largely on the in uitro demonstration of autologous lymphocyte cytotoxicity against tumor cells. There is no doubt that these psychological or emotional influences favoring host resistance have encouraged a biased laxity in the choice of animal tumor systems for experimental studies to be accorded clinical relevance: The assumption of an ingredient of active host defense against autochthonous clinical cancers is felt to justify inclusion of that ingredient in the experimental system, even when it has the status of an artifact. It is very questionable whether recruitment of such possible artifactual resistance to the overall response of an experimental tumor to a therapeutic agent permits any generally valid concept of interaction between the agent, the host, and the tumor, as implied by Carter (1973) or stated by Maruyama (1968). This device, of allowing an artifact to persist in an experimental model in order to comply with an assumption concerning attributes of the clinical condition modeled leads to unresolvable problems when we come to assess the validity of attaching clinical relevance to experimental results obtained using the model. In the case of experimental immunotherapy, recent review of the animal tumors which have been used to promote clinical trial of this modality has shown the selection to be so biased as to require retrenchments concerning the immediate prospects for useful immunotherapy. Weiss (1977) has commented on the mounting evidence of the nonimmunogenicity of many spontaneous tumors, stating that “The assumptions which little more than a decade ago set a highly optimistic foundation for the new discipline of tumor immunology must today be deemed as simplistic.” To another prominent tumor immunologist (R. Baldwin) is attributed a recent conclusion that “the original concept that human tumors are antigenic and that immunotherapy may aid their elimination by an immune reaction was no longer tenable” (Leading Article, 1977). Klein and Klein (1977) have recently stated that the regularly demonstrated inability of spontaneous tumors to immunize autochthonous or syngeneic hosts is a crucial point that has not received the attention it deserves, Hewitt et al. (1976) had previously reported from their large experience of 27 different syngeneically transplanted murine tumors of spontaneous origin that they had encountered no evidence of tumor immunogenicity in any of a wide variety of quantitative experiments

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which could have revealed it, While these belated reappraisals of the experimental foundations of clinical immunotherapy may in due course compel a more realistic, and less attractive, valuation of the prospects of this modality of treatment, the harm done by excessive promotion of the topic is likely to endure longer. Inevitably, the latitude encouraged by experimental immunotherapists in respect of the criteria used to select animal tumor models of clinical cancer has weakened generally the discipline required to exclude laboratory artifacts; the use of questionable systems has spread to other fields of experimental therapy. The effect of artifactual immunogenicity in a system is almost invariably to flatter the potential of any therapeutic agent subjected to testing and so to bring it prematurely to a level of clinical significance. Artifactual immunogenicity in an experimental system may also, theoretically at least, influence the ranking of effectiveness of a series of therapeutic agents tested; this is so because antineoplastic agents are known to vary in their potential for suppressing host immunity; the difficulty of separately assessing the immunosuppressive and tumor inhibitory effects of a drug has been discussed by Schwartz (1968). This contribution has the limited purpose of reviewing the status of animal tumor systems in current use as models of autochthonous cancers of spontaneous origin, of discussing the conditions of origin and maintenance of animal tumors as these features affect their validity as models, and finally, of encouraging a reform of current practice in the selection of tumor systems as well as the creation of facilities by which such reform may be undertaken. II. Analysis of Species Used in Current Cancer Research

A representative survey of the use of animal tumors is provided by the annual publication: Research Using Transplanted Tumors of Laboratory Animals. A Cross-Referenced Bibliography, compiled by D. C. Roberts and co-authors of the Research Data Unit of the Imperial Cancer Research Fund, England. For Volume XI1 of this unique bibliography (Roberts and Drobycz, 1975), the compilers perused 92 relevant journals published in the English language that year, and listed from them 1381 articles recording experiments in which animal tumors were used. A valuable feature of this presentation for my purpose here is the Tumor Index, which lists all the named (or coded) and unnamed tumors used, segregating them by species of animal and tumor type and referring each tumor to all the articles reporting its use. To indicate the relative employment of different species (Table I), the unit of representation is one tumor employed in one separately

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RELATIVE USAGE

OF SPECIES FOR ANIMAL

TUMOREXPERIMENTS"

1975

1970

Species

Times used

% of total

Times used

Mouse Rat Hamster Guinea pig Fowl Dog Rabbit Bovine

1626 834

6 3 ) 95 32

99 I 504

64 55 4 3 3 1

2.5) 4.6 2.1

53 23 1 1 8 1

% of total

32 3.4) 4.9 1.5 <1

" Data compiled from bibliographies of Roberts (1970) and Roberts and Drobycz (1975). reported study; that is, a sum of 10 could be accumulated by one tumor being used in 10 different studies or 10 different tumors used in a single study, etc. Table I shows that the number of units for fowl, dog, rabbit, and bovine together contribute only 0.4% of the total; tumors of the guinea pig and golden hamster contribute under 5%. In the case of bovine, dog, fowl, and rabbit, it is probable that'experimentation is limited b y the absence or relative unavailability of fully isogeneic animal colonies as required for reliable serial transplantation of all but very rapidly growing tumors. Hamsters do not appear to provide any special experimental facility for in uiuo studies other than those associated with their possession of pouches, which are privileged sites permitting the temporary growth of potentially immunogenic tumor grafts and which allow observation of tumors by transillumination. Guinea pigs have a very low incidence of spontaneous tumors, and the great majority ofthe tumors used in this species during the last 5 years have been chemically induced. I shall refer later to the deficiencies of chemically induced tumors as models; it is significant that, with very few exceptions, the experiments done with guinea pig tumors were studies of tumor immunology. Thus, over 95% of all animal tumor experiments were done using mice or rats, rats being used one half as frequently as mice. A similar analysis of Vol. VII of the same bibliography, for 1970, showed that, although the total units recorded were only 61% of those for 1975, the relative frequency of usage of the different species was almost identical; again, 95% of experiments employed rats or mice, and rats were used half as frequently as mice. It is of interest in the context of this review to consider what criteria

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of choice may be influencing the rather stable relative utilization of rat and mouse tumors. The 33% preference for rat tumors requires the assertion of some advantage of that species which outweighs its considerable economic and other disadvantages: The cost of inbred rats on purchase is currently 60% greater than that of mice of equivalent category (M.R.C. Laboratory Animals Center, England); the approximately 10-fold greater body weight of adult rats implies a considerably greater demand per animal for animal house space, food, and servicing time. To the logistic advantages of the mouse must be added the technical advantages accruing from a much wider range of available strains, including many having a high incidence of various naturally occurring malignancies (Murphy, 1966), and a very much more detailed definition of histocompatibility and other genetical factors (see 47th Annual Report of the Jackson Laboratory, 1975-1976). Rats have no overall species distinction from mice in respect of gestation period, average litter size, life span, or oxygen consumption rate (Altman and Dittmer, 1964). In accordance with the overall similarity of life spans, the ranges of tumor growth rates in the two species are similar. Indeed, the greater body size of the adult rat emerges as the one distinction which could provide for certain technical advantages : Procedures requiring small vessel manipulation are more easily undertaken in the rat (Gullino et al., 1967; Kreel and Tavill, 1973); for a given level of humane observance tumors can be grown to a considerably larger size, an advantage for chemical studies requiring large quantities of starting material; surgical insertion of sizable mechanical devices is better tolerated in rats (Reinhold and Buisman, 1973). However, the 33% preference for rats over mice cannot be accounted for b y occasional specialized requirements of this kind. Questioning of individual researchers reveals that a preference for rats over mice in cancer studies is seldom dictated by specific theoretical or practical requirements. More often, the choice proceeds from traditional influences-familiarity with the species, logistic arrangements in the animal production facility used, or the availability of a particular rat tumor for which a large background of biological information has been previously obtained by others, permitting comparison of data obtained over an extended period. Particular named rat tumors have become so closely associated with experimental studies done in a particular institution as to identify the affiliation of any author reporting its use, as instanced by the R-I Rhabdomysarcoma (BA 1112) of the WAG/Rij rat strain; this arose in 1962 at the Radiobiological Institute of the Organization of Health Research TNO in the Netherlands and has since featured very largely in tumor studies reported from there.

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An analysis of the current usage of rat tumors, made in the light of considerations of artifactual tumor immunogenicity which were discussed in the previous section, reveals that the conditions of origin and transplantation of rat tumors very commonly disqualify them as suitable models for therapy research. Of the 834 items of rat tumor usage indexed by Roberts and Drobycz (1975),no less than 50% referred to rat hepatomas (as compared with an equivalent value of 1.2% for the mouse). Since spontaneous hepatomas do not arise frequently in rats, at least in those of the most commonly used (Wistar) strain (Lemon, 1967), it must be concluded that the overwhelming majority of rat hepatomas in use were originally chemically induced; 56 ascites rat hepatomas were established by Odashima (1964)and associates at the Sasaki Institute in Japan in the period 1951-1962,many of which have been extensively used in other laboratories. All of these hepatomas were chemically induced by 3‘-methyl-4-aminoazobenzene,4-dimethylaminoazobenzene, or O-aminoazotoluene; it is to be noted that only 30%of tumors so induced were found to be transplantable at first passage from the animal in which they were induced (Odashima, 1964),a feature which may proclaim a high frequency and potency of immunogenicity for tumors induced by these carcinogens. Of the remaining 50% of the items of rat tumor usage indexed by Roberts and Drobycz (1975),those not employing hepatomas, 23% refer to the use of Walker carcinosarcoma 256 or the Yoshida sarcoma, which arose in 1928 and 1943, respectively, in rats of unspecified genetic constitution (Stewart et al., 1959);it follows from their origin and history that all contemporary transplants of these tumors display artifactual immunogenicity attributable to their nonisogeneic transplantation. A cursory survey of the tumor designations and article titles referring to the remaining rat tumor systems used provides specific circumstantial evidence that a high proportion can be assumed to exhibit artifactual immunogenicity attributable to their chemical induction, their long history and indiscriminate transfer from one laboratory to another, or their nonisogeneic transplantation. In respect to this last feature, it has to be acknowledged that the designation of rat strains given in reported studies rarely attains to the degree of genetical discrimination implied in the designation of inbred mouse strains; most commonly, designation of the rat strain used does not go beyond “Wistar” or “Wistar-derived.” In fact, the Wistar Institute distributed a number of different strains to other laboratories over 30 years ago, and the unqualified term “Wistar” does not now carry any information b y which the histocompatibility status of a transplanted tumor can be assessed in relation to its origin (see discussion of a paper b y Lemon, 1967).

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It is concluded from this analysis and discussion of the differential use of mammalian species for tumor studies, that the mouse presents technical and economic advantages which are very rarely competed with by any other species. Choice of the rat for experiments employing transplanted tumors in studies of therapeutic agents invites more circumspection than is generally required in the case of the mouse; this is so because strain and genetical specifications of the rat are far less discriminatory, allowing a greater risk of the intromission of artifactual tumor immunogenicity into a transplant system, with impairment of its validity as a model of spontaneous autochthonous cancer. This is not to say that assiduous attention to the avoidance of artifacts in a rat tumor system, instituted and monitored within a single laboratory, cannot achieve the same reliability as an impeccable murine system.

Ill. Origin and Maintenance of Animal Tumor Sytems in Relation to their Validity as Models of Clinical Cancer

A. INTRODUCTION Comparison of treatments for cancer by clinical trial, while it is the ultimate means of establishing the clinical value of a therapeutic innovation, is a lengthy and expensive undertaking capable of discriminating only relatively large differences of treatment effectiveness. Fisher (1973) has concluded from an analysis of completed clinical trials that none has given decisive results concerning the long debated issue of simple versus radical mastectomy for surgical treatment of cancer of the breast; and Bross and Blumenson (1971) have predicted by the employment of deep mathematical models for analysis that the issue would be unlikely to be resolved by commonly suggested protocols even if 10,000 cases were entered in a trial. Clinical trial was also unable to discriminate any value of preoperative (Lindgren et al., 1968) or postoperative (Easson, 1968) radiotherapy as an adjunct to surgical treatment of cancer of the breast. Some loss of discrimination in such trials can b e attributed to “dilution” of the comparison by inclusion of a proportion of patients who succumb to progression of disease in sites remote from the treated region; but there is no doubt that the resolving power of a comparative trial is reduced to a large extent by the spread of intrinsic tumor potential among the cases entered, as represented by the range of tumor volume doubling times (Spratt and Spratt, 1964).Tumor growth rate has been shown to have a dominant influence on prognosis and treatment evalu-

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ation in breast cancer (Pearlman, 1976) and on the survival of cases of bronchogenic cancer treated by surgical resection (Meyer, 1973). There is no near prospect of any improvement in the resolving power of clinical trials or of a significant reduction of the time required for their completion. These enduring limitations are in striking contrast to the ability of a suitable animal tumor system to measure the potential value of a therapeutic innovation within a very few weeks; using murine tumor systems, the maximum time of observation required to finalize the cure rate in a group of tumor-bearing mice subjected to experimental therapy is unlikely to exceed 5 months, provided that exceptionally slowly growing tumors are excluded. It is clear that the development and refinement of therapeutic agents in the laboratory will always progress very much more rapidly than their clinical relevance can be confirmed. It is not inconceivable that this lag between laboratory findings and their clinical confirmation by formal trial, combined with an acceptance of the poor discrimination possible by clinical comparisons, may encourage a loosening of policy respecting the application of experimental findings to clinical practice: That is, the clinical relevance of a therapeutic advantage established using animal tumors may come to be taken for granted, and incorporated suitably into clinical practice without any sense of urgent obligation to confirm the clinical advantage statistically. It is, indeed, by such direct application that many valuable therapeutic resources have been introduced: x-rays were employed for treatment of clinical cancer within one month of Roentgen’s preliminary communication of his discovery (Del Regato, 1972), although in this case even animal studies were omitted. However, any tendency to more direct clinical application of animal tumor data must be discouraged until a much more fastidious selection of animal tumor models has become mandatory for studies which are to be accorded clinical relevance. There can be no confidence at the present time that such a rule is widely regarded by experimenters or insisted upon by editors. The deficiency is nowhere more evident than in the field of immunotherapy: Number 4 (August 1976) of the Compendium of Tumor Immunotherapy Protocols, collated b y the International Registry of Tumor Immunotherapy, lists over 330 clinical trials of immunotherapy projected or in progress; yet it is becoming increasingly apparent that the very large volume of evidence for the antigenicity of animal tumors, which has largely instigated this massive clinical enterprise, has been obtained almost exclusively from the use of systems incorporating immunogenicity as an artifact (Hewitt et al., 1976). Evidence for the antigenicity of autochthonous human cancer is, in fact, sparse (Klein and Klein, 1977; Weiss, 1977).

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I n the following subsections, the categories of mouse tumors that are available to experimental oncologists will be reviewed, with the limited objective of assessing their relative merits as valid models of the generality of clinical tumors. This may serve as a guide to investigators embarking on relevant researches who are able to make a free choice of their animal tumor systems.

B. SPONTANEOUSTUMORS The description “spontaneous” will be confined to tumors that arise in mice which have not been deliberately exposed to any carcinogenic agent and which are of strains which are not known to harbor any vertically or horizontally transmitted oncogenic virus; that is, subsequent discovery of the implication of such an agent in a mouse strain would require recategorization of the tumors arising in it. In general, spontaneous tumors, so defined, are those arising in “low cancer strain” mice. However, it should be mentioned that some substrains of commonly used inbred mouse strains do have incidences of up to 30% (in older mice) of specified tumor types in the absence of any proven oncogenic agent: hepatomas and ovarian tumors in the C3H strain, reticulum cell sarcomas in C57 B1 and DBN2, and hemangioendotheliomas in HWDe, to mention only a few (Murphy, 1966). Although such relatively high incidence tumors are acceptable as “ spontaneous,” a technical inconvenience may arise from the use of transplanted tumors in these strains: There is some risk of the occurrence of a second tumor arising in a mouse under observation for possible “cure” of its transplanted tumor. This may cause confusion or may require sacrifice of the mouse before the end of the observation period; in practice, the risk is negligible if the observations can be completed before the mice reach an age at which the incidence of the spontaneous tumors becomes significant. There can be no doubt that avoidance of artifactual immunogenicity in a transplanted tumor system is most reliably achieved b y use of a tumor arising spontaneously in, and tramplanted within, a colony of inbred mice maintained within the laboratory in which the experiments are undertaken. That the use of such systems is the exception rather than the rule in reported studies is a matter deserving inquiry. The requirements are simple: A colony of ex-breeder mice is retained, of which the individuals are subjected to careful “clinical” examination twice a week; subcutaneous tumors are detected by palpation or inspection; leukemias, by evident weakness, or pallor of the snout or feet, or by the occurrence of evident dyspnea associated with thymic enlargement; and abdominal malignancies are manifested b y abdom-

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inal enlargement or ascites. Mice suspected of malignant disease are dissected under aseptic conditions, and primary isogeneic transplantation is done as a planned operation. Failure to provide for relatively early detection of spontaneous malignant disease inevitably results in loss of a tumor from death and early putrefaction of the host. I n our experience of over 50 primary spontaneous malignant tumors, all were readily transplantable at the first and any subsequent passage, solid tumors being transplanted subcutaneously and leukemias by intraperitoneal transplantation of minces of infiltrated spleen, liver, or lymph node. Failure of primary transplantation indicates either bacterial infection of the graft or incomplete isogeneity in the mouse strain; we have very rarely encountered tumors in the mouse which, by clinical histopathological criteria, would be described as “benign.” I conclude from this experience that the relatively small usage of spontaneous tumors arising within the experimenter’s own colony is due to failure to retain a colony of aging mice, to subject it to regular surveillance by trained staff, or to maintain facilities for immediate primary transplantation under aseptic conditions. That the deficiency of one or more of these requirements is widespread is evident from the frequent resort to a few long transplanted tumors of spontaneous origin which have been transferred from one laboratory to another over many years (Melanoma B16, Lewis lung carcinoma, etc.); it is remarkable that some centers of cancer research of long standing and high reputation, whose hygiene regulations may prohibit importation of animal material, appear to confine their internal resources to virus-associated mammary tumors of high cancer strains or to chemically induced tumors. What may be envisaged as a discouragement to reliance upon locally available spontaneous tumors for experimental studies is the need for patience in their collection and some limitation of the free choice of tumor type. Our own collection (for mouse strains CBA/Ht and WHT/Ht) has included one or more examples of well or poorly differentiated mammary adenocarcinoma, gastric or dermal squamous carcinoma, soft tissue sarcoma, osteosarcoma, chondrosarcoma, lymphosarcoma, reticulum cell sarcoma, hemangioendothelioma, fibroadenoma, acute leukemia, and ascites tumor. The infrequency and fortuitous typing of spontaneous tumors make it unrealistic to await the appearance of a tumor of specified type or site of origin for particular studies, as may be sought in an attempt to secure accurate modeling of a clinical tumor in respect of these features. Many of the most common clinical tumors are not represented in the mouse, such as adenocarcinoma of the colon or squamous carcinoma of the uterine

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cervix; models of this kind can only be obtained by resort to chemical induction. For example, Corbett et al. (1975) induced colonic carcinomas in mice using one or other of 4 different chemical carcinogens ( 1, 2-dimethylhydrazine dihydrochloride, N-methyl-N ‘-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, or N-nitroso-N-methylurethan); of the 1011mice treated, only 67% survived at the 120th day after treatment, and only 4 serially transplantable tumors were obtained from this large undertaking. It is questionable whether the exotic specification of colonic origin was an important consideration for the chemotherapy assays in which the tumors were to be used. A more significant influence, in the context of this review, would be the high probability of artifactual tumor immunogenicity being introduced by the resort to chemical induction; such an artifact would be expected to have a more substantial influence on the results of the assays than defects of the model that could relate to its site of origin. The term “spontaneous” is very commonly applied to the tumors arising in “high cancer strain” mice and known to be associated with vertically transmitted oncogenic viruses. Such neoplasms are represented by the mammary tumors arising in strains C3H, A, and DBA/2, and by the lymphocytic leukemias arising in the high leukemia strains AKR and C58. It will be appreciated that the implication of viruses would exclude these tumors from the category “spontaneous” under the definition given. While it is true that expression of the oncogenic potential of the mammary tumor virus (MTV) requires a host susceptibility determined b y certain genetic and hormonal characteristics, the implication of MTV in tumor induction does confer a certain liability to the exertion of host tumor influences associated with the virus, and these influences can affect the response to therapeutic agents; a fuller discussion of these considerations appears in Section III,D. There have been instances of investigators preferring autochthonous tumors for use in experimental therapy, on theoretical grounds that transplantation may itself introduce artifacts which disqualify the animal tumor as a valid model of a human cancer to be treated in situ. In respect of the hosthumor relationship, however, it is a rule of transplantation genetics that strictly isogeneic transplantation introduces no alteration of histocompatibility from that obtaining in the autochthonous case or for autotransplants. It may be added that confinement of observations to autochthonous tumors is a condition incapable of providing an experimental system permitting comparison of treatment schedules: Strictly spontaneous tumors are too infrequent. Although a number of tumor bearers can be assembled for one experiment using MTV-induced mammary tumors from a high cancer strain,

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their use is subject to a reservation additional to that discussed briefly in the previous paragraph; this is, that any assembly of such tumors is heterogeneous in several respects. Tumors are differently located over the wide area occupied by the mammary glands (Hummel et at., 1966), a variation that requires many to be excluded from experiments involving localized radiotherapy; moreover, tumors of this class vary in their hormone dependence, degree of histological differentiation, and growth rate; Hawkes et al. (1968) found that the volume doubling times of 44 autochthonous mammary tumors of a C3H strain ranged between 5 and 22 days. It is proposed here that isogeneically transplanted tumors of strictly spontaneous origin are the only valid models of the generality of human cancers that can provide the operational requirements for quantitative assessment and comparison of the potency of therapeutic agents. Justification of the proposal rests very simply on the fact that clinical tumors, with rare exceptions, fall into the category “spontaneous,’’ as defined here. That is, they are not induced by the application of powerful chemical carcinogens and none has been proved to be associated with the oncogenic action of a virus. It is our experience that such tumors in the mouse do not evoke an immunological reaction of the host which has any restraining effect on the growth of primary implants or of metastatic tumors disseminated from them (Hewitt et al., 1976); we have done a great variety of experiments on a wide range of such tumors without disclosing any evidence of effective tumor immunogenicity. The immunogenicity of 7 different tumors was sought by the most sensitive test available: Groups of mice were “immunized” by multiple injections of lethally irradiated tumor cells; they, together with a group of untreated mice, were later challenged with graded inocula of viable homologous cells. From the results of these assays, we calculated the number of viable cells required for 50% successful transplantations to the normal and putatively immunized mice; in the case of all of the tumors so tested, the number of viable cells for 50% takes was rather less in the immunized than in the untreated mice. The conclusion from our experience is that spontaneously arising tumors are rarely, if ever, immunogenic in mice, and that these are, by definition, the only appropriate models of clinical cancer. As shall be discussed in the following two subsections, chemically and virally induced mouse tumors are usually immunogenic, and, in studies of therapeutic agents, the results obtained may represent the combined effects of the direct action of the agent and the host resistance.

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C. CHEMICALLY INDUCED TUMORS Almost 50% of the transplanted tumors used in recent experimental studies of chemotherapy or immunotherapy were originally chemically induced (see Section III,F, Table 11).It should be mentioned that many authors refer to the tumors used in their studies by letter/ number codes, and the fact of their induced origin is not always stated in the descriptions given; since many of the most commonly used tumors are now removed from their origin by periods approaching or exceeding a quarter of a century, bibliographic research is often re2 quired to establish the mode and time of origin of a tumor. For the origin of older tumors, the surveys of Dunham and Stewart (1953) and Stewart et al. (1959) provide a reliable and comprehensive listing. It is of interest that one of the most commonly employed transplantable tumors for current immunotherapy and chemotherapy research, Leukemia L 1210, was induced by methylcholanthrene almost 30 years ago. The importance of distinguishing this category of tumor origin in our present context refers to the peculiar propensity of such tumors to display antigenicity or immunogenicity, not only as isogeneic transplants but in the host in which they are induced (Klein et al., 1960; Haddow and Alexander, 1964). In this property they are distinguished from the overwhelming majority of tumors of spontaneous origin and thus forfeit their eligibility as suitable models of spontaneously arising cancer. In chemotherapy studies, the contribution of artifactual host resistance to the results obtained must be expected generally to flatter the prospective potential of chemotherapeutic agents in their clinical application. In the case of immunotherapy studies, the influence of artifactual host resistance attributable to artificial induction is more seriously misleading: Immunological manipulations which enhance the exertion of host resistance may depend entirely on the presence of artifactual immunity and may have no relevance to other experimental tumor systems, or clinical tumors, not embodying the artifact. Gross (1943) induced a fibrosarcoma in a C3H mouse using methylcholanthrene (MCA) and found that strictly isogeneic intradermal transplants of this tumor all grew readily, yet -20% underwent spontaneous regression between 16 and 45 days after transplantation; mice in which regressions had occurred were resistant to retransplantation of the same tumor but not of other tumors. His finding has been described as the foundation of modern “tumor immunotherapy” (Rapp,

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1972) in as much as it reinforced a principle of specificity and rescued the topic from the ignominy it had earned (Editorial, 1942) during a period of several decades in which allograft immunity had been presented as clinically relevant. Gross did not, however, report data for similar studies of spontaneous tumors done in parallel. Foley (1953) examined the immunogenicity of 3 C3H mammary tumors and 6 MCA-induced sarcomas, all in their first few passages, by a method in which transplanted tumors were induced to undergo necrosis by the progressive tightening of ligatures applied to their bases, with subsequent retransplantation of the same tumors to the treated hosts. Immunogenicity was demonstrated in all of the MCA tumors and in none of the mammary tumors. Prehn and Main (1957) used tumor excision and retransplantation to investigate the immunogenicity of 14 MCA-induced sarcomas and 7 histologically indistinguishable sarcomas of spontaneous origin: All of the chemically induced and none of the spontaneous tumor systems displayed tumor immunogenicity sufficient to entail a tumor rejection response. R6vksz ( 1960), using more quantitative techniques than Prehn and Main (1957), could demonstrate no tumor immunogenicity in any of 14 mammary carcinomas arising in mice of strains C3H, DBA, A, or ASW and examined as first generation isogenic transplants, or in any of 4 lymphomas arising spontaneously in mice of strains A or ASW, or in (A x ASW)F1 hybrids, examined as second generation isogeneic transplants. From a similar examination of 14 MCA-induced sarcomas, evidence was obtained of definite immunogenicity in 6 and of a high probability of immunogenicity in 5; in the remaining 3 cases, the evidence for immunogenicity did not attain significance. Combining the data of Foley (1953), Prehn and Main (1957), and R6vksz (1960), we see that immunogenicity was demonstrated in 31/34 of the chemically induced tumors and in 0/28 of the tumors not induced by chemicals. Since these early studies, evidence of the striking immunogenic propensity of chemically induced tumors has been abundantly added to, and the nonimmunogenicity of spontaneous tumors continues to be regularly confirmed (Hewitt et al., 1976) and to be commented upon in discursive considerations of the status of “immunotherapy” as evaluated b y evidence from animal tumor studies (Weiss, 1977; Klein and Klein, 1977). Among the chemical carcinogens employed to induce animal tumors, MCA very largely predominates and is commonly used in doses (0.5-1.0 mg) equivalent to those used by Prehn and Main (1957) and by RCvCsz (1960). It is, perhaps, the carcinogen that has been most intensively studied in attempts to clarify the relationship between

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chemical carcinogenesis and the immunogenicity of the tumors induced. Prehn ( 1963) has concluded that the immunosuppression induced by MCA in the doses commonly used may be a necessary component of the mechanism of its carcinogenicity. It is clear that the mutagenic action of a carcinogen would be expected to induce a proportion of clones combining malignant transformation with antigenic modification and that such clones would be able to progress to tumors in their coincidently immunosuppressed hosts; on isogeneic transplantation to normal recipients, the antigenicity of the tumor would become more evident. These general considerations must be expected to apply to any carcinogen that is demonstrably immunosuppressive, although this does not imply that immunosuppressive agents are necessarily carcinogenic. Certainly, immunosuppressive ability has been demonstrated for chemical carcinogens of most classes : polycyclic hydrocarbons other than MCA, such as 1,2-5,6-dibenzanthracene (Malmgren et al., 1952); hepatocarcinogens, including P-dimethylaminoazobenzene and 2-acetylaminofluorene (Baldwin and Glaves, 1968); 4-nitroquinoline 1-oxide, which induces moderately to highly immunogenic tumors (Outzen and Prehn, 1973); and even the rather weakly carcinogenic agent, urethan (Parmiani, 1970). M6nard et al. (1973) showed that the incidence of lung adenomas induced in mice by urethan was positively correlated with the degree of immunological impairment sustained by the individual. With carcinogenic nitrosamines, however, carcinogenic and immunosuppressive potency appear to be independent (Waynforth and Magee, 1974). These authors demonstrated that N-nitroso-Nmethylurea strongly depressed both cell-mediated and humoral immunity, whereas the related potent carcinogen dimethylnitrosamine, depressed cell-mediated immunity not at all and humoral immunity only slightly; they concluded that immunosuppression was not a necessary component of the mechanism of carcinogenesis, as previously suggested by Prehn (1963).Rice (1972) induced 733 lymphomas in a variety of mouse strains using 1-ethyl-1-nitrosourea; over 11% of the induced malignancies underwent incipient, partial, or complete regression in the original host; thus, regardless of whether this nitrosamine was immunosuppressive, a significant proportion of the induced malignancies displayed evidence of immunogenicity.. Initial growth of the lymphomas followed by their later regression is most readily explained by postulating that the drug induced initial immunodepression and that immune reactivity was recovered some time after the appearance of the lymphomas. An excellent review of the association between immunodepression and chemical carcinogenesis

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has been recently presented b y Stutman (1975), from which it can be gleaned that an association between these two effects is usual, if not invariable. The above cursory survey is sufficient to show first that chemically induced tumors are usually immunogenic on isogeneic transplantation whereas spontaneous tumors similarly tested are rarely so; and second that growth of the induced tumor in the primary host is commonly facilitated by coincident immunodepression. It is concluded that the immunogenicity of a chemically induced tumor must generally be regarded as a peculiarity of its mode of origin; that is, it is an artifactual property disqualifying such tumors as models of naturally occurring cancers. It is often asserted that use of chemically induced animal tumors as models of clinical cancer is justified b y the known implication of chemical carcinogens in the genesis of such clinical cancers as the bronchogenic cancers of smokers, or bladder cancer associated with industrial exposure to aromatic amines. Speculation has added to these examples of inadvertant exposure to carcinogens, the ingestion of aflatoxin (Warwick, 1976) or the endogenous production of nitrosamines (Hill et al., 1973) as possible causative factors in human cancer. However, the implied analogy between animal tumors induced after short latent periods by high concentrations of powerful carcinogens and clinical cancers arising in relatively very low frequency and after long latent periods foIlowing prolonged exposure to low concentrations of weak carcinogens breaks down in the light of more refined studies of tumor induction by MCA. Prehn (1969) has shown that the probability of MCA-induced tumors’ being immunogenic is inversely correlated with the length of latent period of their induction. More recently, Prehn (1975) has shown that tumors induced by relatively low concentrations (0.05-0.1%) of MCA were significantly less immunogenic than those induced by the concentrations more commonly employed (0.5-5.0%);furthermore, the effect of concentration was demonstrated independently of the influence of latent period. Prehn suggested that use of animal tumors induced b y low concentrations of oncogen wouId provide better models of “spontaneous” tumors. However, even the lowest concentration of MCA used b y Prehn is probably higher b y several orders of magnitude than the levels of carcinogenic potency likely to be attained in proliferating epithelial tissues intermittently exposed to carcinogenic pollutants in the human environment. Certainly it is possible b y control of the carcinogenic application and prudent selection from the tumors induced to obtain a transplant sys-

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tem in which the exertion of artifactual host resistance is minimal. However, it has to be realized that low levels of host resistance may not be revealed by gross tests for resistance, becoming manifest and influential only when the effectiveness of a therapeutic agent under test approaches “cure”; the contribution of resistance may then become decisive in attainment of that end point. A pertinent example of the critical influence of possibly artifactual host resistance is given by the studies of Maruyama (1968) using the radiation-induced LSA ascites lymphoma in C57 B 1 mice; he recorded that a radiation cell survival curve for this tumor irradiated in vivo was not fully exponential but showed an increasingly steep slope at survival levels below which he attributed to an immune influence. No such departure from continued exponentiality was observed by Hewitt (1962) in a similarly determined survival curve for a nonimmunogenic ascites lymphoma, although the curve was carried down to a cell surviving fraction of

10-8. It is concluded that no case can be made for deliberate selection of,a chemically induced tumor when the intention is to provide a model of spontaneous cancer for experimental studies of therapeutic agents, and that misleading data may be obtained by the use of such a tumor. The predominant use of chemically induced tumors in current therapy research projects must be ascribed to traditional influences, to their wider availability, and to a failure to develop local facilities for the isolation and maintenance of tumors of spontaneous origin.

D. VIRUS-INDUCEDTUMORS Of the animal tumors employed in recent experimental studies of cancer therapy only 14% were virus-induced (see Section III,F, Table 11).Although this overall relative usage is considerably smaller than that of chemically induced tumors (48%),virus-induced tumors have predominated in some studies of immunotherapy which have been given unquestioned clinical relevance and have led to direct clinical applications (e.g., Math&, 1972). The importance of such local representations of the clinical application of animal tumor studies, when they are given prominence, is that they invite further, imitative, clinical trials without review of the animal data providing the original instigation. It is true that the clinical application may prove to be valuable, justifying itself empirically and independently of questions concerning the validity of the animal models originally employed. But in the case where such fortuitous justification fails or when the clinical application proves to be actually harmful, a sweeping and undeserved

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discredit may fall upon animal tumor studies; such indiscriminate criticism is damaging to the ordered progress of laboratory-based medical science. An example of this possible discredit, pertinent to our immediate context, is provided by a clinical trial of BCG for the treatment of Burkitt’s lymphoma (Magrath and Ziegler, 1976). A climate encouraging to institution of the trial had been created by previous evidence of a restraining effect of BCG on the growth of immunogenic animal tumors, including virus-induced tumors, and by a generous evaluation of the circumstantial evidence that Burkitt’s lymphoma might itself be a virus-induced neoplasm. The result of the clinical trial was disappointing: The BCG treatment benefited none of the patients and was considered to have been deleterious to some. The question whether virus-induced animal tumors are valid models of clinical cancer requires some evaluation of the evidence for a viral origin of human cancers. And we here encounter the modern presentation of an obsessional concept which has prevailed in the annals of oncology for at least a century-the concept that an exogenous parasitic agent may commonly play a part in the etiology of cancers. Bland Sutton (1890) remarked that “bacteriologists have not yet succeeded in isolating a special bacterium for sarcomata in general; that such agents will soon be discovered is in the highest degree probable.” This confidence has been retained in its essentials over many decades of investigation, during which there has been a dominant preoccupation with the concept. Modern equivalents of Bland Sutton’s expression, with the attention turned from bacteria to viruses, are commonplace; Kessler and Lilienfeld ( 1969), for example, write “The viral hypothesis of leukemogenesis is in need of further testing. It has not as yet been substantiated b y the studies on space-time clustering or by the efforts to isolate viruses in cases of human leukemia.” It is true that the research of this century has yielded a number of oncogenic viruses in animals and that there is no reason to exclude the eligibility of the human species as a potential host for oncogenic viruses. But a veteran of the topic (Rous, 1965) has issued a sobering evaluation of the achievements at that time and a discouragement from “ ovelzealous projection: Except in chickens, neoplastic viruses are very rare.” And “Even if viruses turn out to be a cause of leukemia in human beings, and perhaps of Burkitt’s disease, this limited finding cannot be regarded as an Open Sesame to the cause of the generality of human growths. It certainly has not been proved to be such in mouse, rat and rabbit.” Leukemia and Hodgkin’s disease, among clinical cancers, have always been distinguished as the most likely candidates in speculations about virus causation, although the foundation for this

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distinction remains obscure. In fact, the emphasis on leukemia, by stimulating the most intensive search for a causative virus, has led to the accumulation of a larger volume of evidence against the hypothesis than attaches to any other form of human malignant disease. Epidemiological studies have provided no support for it (Kessler and Lilienfeld, 1969); leukemia of the mother during pregnancy, of which there have been over 50 recorded instances, has not been reported as having been associated with evidence of leukemia in the children born. The disease has not been transmitted in any of the 200 or more instances in which fresh leukemic blood has been transfused to another individual (see Bierman et al., 1956). The only human neoplasm that has been proved to be virus-induced is the common wart, and in this case the evidence for implication of a virus was adduced in the earliest days of virology. The infectivity of warts was proved by Variot (1894); the filtrability of the causative agent was demonstrated by Ciuffo ( 1907);complement-fixing antibodies were demonstrated in wart bearers by Maderna (1935);and the spontaneous arrest of growth, or actual regression, of warts is a matter of every day observation. These classical features of a horizontally transmitted oncogenic virus, with effective immunogenicity of the tumors induced, have never been demonstrated in the case of any other form of human neoplasia despite the enormous increase of knowledge and technical facilities which has ensued since the infectivity of warts was proved in the last century. The much studied association of EB virus with both Burkitt’s lymphoma and nasopharyngeal cancer is exceedingly complex, the malignant cells of Burkitt’s lymphoma commonly displaying both viral genome and “marker” cytogenetic aberrations. Since EB virus infects the majority of all human populations (Henle and Henle, 1970; Niederman et al., 1970), a concept of direct viral oncogenesis for these two malignant conditions is too simplistic (G. Klein, 1975). Similar reservations must be entertained concerning the association between HSV-2 virus and squamous carcinoma of the uterine cervix (De Thk et al., 1976). Thus, the evidence for implication of viruses in the generation of human cancers is very meager, having in mind the prolonged and intensive investigations to which the general hypothesis has been exposed. It would therefore appear to be imprudent to make a choice of virus-induced tumors to serve as models of the generality of human cancers when such choice can be justified only by the optimism of past endeavors and the anticipation of future discoveries. The moderately fi-equent use of virus-induced animal tumors is certainly far in excess of the frequency with which such tumors arise de

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no00 in nature. Their disproportionate representation among labora-

tory tumor systems must be attributed to the multiplication of examples by perpetuation of the few viruses that have been isolated or of the tumors induced by them, associated with their wide distribution to different laboratories. It is understandable that cytogeneticists, molecular biologists, immunologists, and some other specialists should be attracted to study of the mechanisms of viral oncogenesis by way of elucidating rather fundamental biological problems. For such inquiries, the use of virus-induced tumors envisages no necessary clinical relevance and demands no consideration of appropriate modeling. However, it is very probable that virus-induced tumors which have been introduced into an institution for such fundamental studies are commonly appropriated by other workers for therapy studies, their close availability taking precedence over their validity as models of spontaneously arising clinical cancer. As in the case of chemically induced tumors, the pecularity of virusinduced tumors which usually compromises their validity as models of autochthonous spontaneous cancer is the immunogenicity they almost invariably display when propagated as isotransplants, which is attributable to virus antigen, virus-associated antigen, or virus-induced transplantation antigen in the tumor cells. The immunology of virus-induced tumors has been very intensively studied and repeatedly reviewed in all its aspects, and it has been asserted b y Klein ( 1968)that tumor-associated transplantation antigens have been detected in all neoplasms induced by DNA and RNA viruses. That this peculiarity distinguishes virus-induced tumors from the generality of tumors of spontaneous origin is explicitly stated by the title of a recent paper by Klein and Klein (1977): “Rejectability of Virus-Induced Tumors and Non-rejectability of Spontaneous Tumors-A Lesson in Contrasts.” In what follows I shall restrict myself to limited aspects of the immunology of virus-induced tumors which seem to me to have particular significance in the context of this contribution in that they refer to considerations relevant to the acceptance or rejection of particular virus-induced tumors as models of spontaneous cancer. It is important to observe that the immunogenicity conferred on a tumor by its original virus induction will be retained independently of the continuance of infective viruses in it. Kobayashi and Kodama ( 1966) obtained nonvirus-producer (NVP) tumor by incubation of cells of a Friend virus-induced tumor with rabbit antiserum against Friend virus; they failed to detect infective virus in the NVP tumor even after 19 serial transplantations. Nevertheless, mice bearing NVP tumors

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developed neutralizing antibody against the virus, and the success of tumor transplantation to mice of the strain of origin was only 69%, indicative of a rejection response. Fieldsteel et al. (1973) obtained a virus-free tumor simply by culturing Friend virus induced tumor cells in uitro; although the virus-free tumor gave 100% successful transplantations using 103-104 cells, resistance could be readily induced in mice of the strain of origin by their treatment with infectious or noninfectious tumor cells. Thus, demonstration that a virus-induced tumor has become free of infective virus does not imply that the tumor has lost the immunogenicity associated with its viral induction. An understanding of this dissociation between the oncogenic capacity of a virus and its liability to confer antigenicity on the cells it has transformed is given by a consideration of virus-induced “allogenization” of normal tissues. That is, the skin of animals systemically infected with some oncogenic or nononcogenic viruses may acquire antigenicity of sufficient potency to evoke its rejection when isotransplanted to animals with no history of exposure to the virus (Svet-Moldavsky et al., 1970; Breyere, 1972). Allogenization of the skin did not require the continued presence of infective virus for its maintenance, in the same way as the persistence of infectious oncogenic virus is not required to maintain the immunogenicity of tumors which were originally virus-induced. A more subtle demonstration of the imposition of antigenicity by virus is reported by Kobayashi et al. (1969).They showed that two sarcomas of spontaneous origin and one chemically induced carcinoma, all three of which had been readily transplantable to rats of the strain of origin, acquired dramatically altered transplantation characteristics after they had been artificially infected with Friend virus: Infected tumor cells either failed to take or gave rise to tumors which underwent regression after temporary growth. They showed that rejection was due not to a cytolethal effect of the virus but to allogenization of the tumors associated with virus-induced neoantigens at cell sites accessible to immune attack. These observations of Kobayashi et al. (1969) raise the possibility that accidental virus infections may “spoil” otherwise suitable animal tumor systems when these are maintained in laboratories to which importation of tumor viruses is permitted. I shall refer later in this subsection to some recorded examples of such accidental contamination of biological materials. The widespread use of mouse mammary tumors in a variety of experiments, including assays of the effectiveness of therapeutic agents, warrants special attention here to their immunology. I have referred in Section II1,B to their frequent description as “spontaneous” tumors,

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which, under my definition of “spontaneous,” would be inappropriate except in the case of mammary tumors arising with low frequency in strains not harboring vertically transmitted mouse mammary tumor virus (MMTV). Mammary tumors arising in relatively high frequency in breeding females of “high cancer strain” mice (C3H, A, DBNZ, DD) are so highly dependent for their generation on the presence of MMTV intrinsic to these strains that they are more appropriately considered as virus-induced tumors; they are spontaneous only in the sense that the virus is not introduced into the individual by injection. However, there can be no doubt that the development of these high cancer strains by selective inbreeding “must be regarded as very serious experimental interference” (Klein and Klein, 1977), and it can be inferred that the effect of genetic manipulation is to fix and propagate a phenomenon that may be of infrequent occurrence in nature. The artifactual nature of MMTV-induction of mammary cancer becomes more evident when a low cancer strain such as CBA is converted to a high cancer strain by foster nursing mice which are to provide a breeding nucleus on mothers of a high cancer strain such as C3H. The purpose of drawing attention to these conceptual considerations is to discourage an impression, promoted b y their description as spontaneous, that MMTV-induced tumors can be regarded unreservedly as modek of naturally occurring sporadic cancers in man. It is generally true that the transplantation of MMTV-induced tumors to mice of the same MMTV-infected strain is not associated with a rejection response and that artifactual immunity rarely becomes a complicating influence on the results of therapy studies using these systems, They have been used very extensively in experiments designed to measure the dose of ionizing radiation required to cure transplanted tumors of specified sizes (Suit et al., 1965), and the results obtained accorded rather precisely with the prediction from radiation cell survival curves for tumor cells irradiated in vitro under similar conditions of oxygenation; these authors concluded that an immune response of the tumor-bearing mice had made no contribution to cure of the tumors. The response of tumors to irradiation is a unique facility for obtaining evidence of this kind because the dose of cytolethal agent actually delivered to the relevant cells can be very precisely stated; chemotherapeutic agents, by contrast, are subject to metabolic modification and diffusion barriers which make for unknown inconsistencies between the dose of active agent administered and the concentration achieved in the cells whose response provides the therapeutic end point measured. While transplanted mammary tumors appear to provide systems fiee

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from immunological contributions to therapeutic end-points, this does not imply that the tumor cells have no potentially antigenic characteristics which have been conferred by the MMTV which induced the tumors. The potential antigenicity of these virus-associated elements is not normally expressed in isotransplants made within an MMTV strain because the recipients have been rendered tolerant to them by neonatal infection with the virus present in the milk. When MMTV tumors are transplanted to isogeneic MMTV-free recipients, the antigenicity of the vims-associated elements in manifested, and immunological resistance is exerted against the graft. Lavrin et al. (1966) found that hyperplastic nodules from C3H mice would not grow in C3Hf mice over 6 weeks old, and the failed inocula displayed necrosis and lymphocytic infiltration characteristic of a rejection response. Suit and Silobrcic (1967) showed that MMTV-free (C57B1 x C3H) F1 mice could be immunized against MMTV tumors whereas MMTV-infected (C3H x C57B1) F1 mice could not; the essentials of these findings have been confirmed more recently by Lavrin (1970). The above considerations provide a convenient practical understanding of the part played by virus-associated antigenic elements in the immunology of mammary tumors of high cancer strains and give confidence that the use of isotransplanted MMTV tumors in MMTV mice for therapy studies is not associated with a significant risk of influential expressions of artifactual immunity. However, a more detailed consideration of the implications of virus-associated antigens is required for the interpretation of some immunization studies using MMTV-induced tumors. Neutralizing and cytotoxic antibodies are demonstrable in mice which have been infected with MMTV during the phase of life at which tolerance is established, and it has been asserted that “no true tolerance of MMTV exists in the mouse” (Hageman and Muhlbock, 1976). They suggest that some antigenic virus-associated proteins do not emerge or are not expressed in infected mice until after the period for induction of tolerance has passed. There is evidence that such “late” proteins can effect immunization of MMTV-infected mice (Attia and Weiss, 1966; Vaage, 1968; Vaage et a1 ., 1969).Weiss ( 1967) obtained a significant reduction of the success of first generation isografts of a mammary carcinoma in R 111 mice by treatment of recipients with a methanol extraction residue (MER) of BCG given some months before grafting. Similar treatment of C3H mice early in life significantly delayed, and reduced the incidence of, autochthonous mammary tumors appearing during adult life. He concluded that MMTV played a major role in any antigenicity displayed by mammary tumors. His experiments were distinguished

+ +

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by the considerable care taken to avoid imperfect isogenicity between donors and recipients of tumor grafts. It is clear that in experiments which reveal immunogenicity in isogeneically transplanted mammary tumors, even where this is only manifested after nonspecific stimulation by modulators of immune responsiveness, the indispensability of virus in conferring the antigenicity cannot be excluded. Hence, it is not allowable to extrapolate such findings to truly spontaneous tumors in which no virus is known to be implicated: The analogy between spontaneous tumors and MMTV-induced tumors is considerable, but is imperfect in respect of the host/tumor relationships. Quite apart from the deliberate use of virus-induced transplanted tumors as models of clinical cancer, we have to consider the inadvertent and unrealized use of such tumors which may result from errors of procedure within a laboratory. It must be acknowledged that laboratories to which a variety of oncogenic viruses and virus-induced tumors have been imported for experimental study is exposed not only to procedural errors within its walls but to those made previously in other laboratories supplying the imported materials: An error of labeling during cage changing could cause confusion between a truly spontaneous and a virus-induced tumor; imperfections of aseptic technique may result in contamination of a spontaneous tumor b y the virus of another tumor, and could bring about its allogenization; where tissue culture studies are done in association with in vivo studies of tumor cells, biological material is exposed to sources of contamination from two environments. Numerous examples are recorded of virus contamination of whole colonies of mice. Parker et al. (1966) used the hemagglutination-inhibition test to show that unsuspected polyoma virus was prevalent in 8/34 mouse colonies examined, and found that the infection was most likely to be prevalent in colonies housed in proximity to mice experimentally inoculated with the virus. Gaugas et al. (1969) encountered an outbreak of tumors in the immunosuppressed CBA mice they were using for studies of leprosy. These tumors were identified as polyomavirus-induced and the infection of their mice was traced to a holding cabinet in which pol yoma-virus-infected mice had been previously placed; only 318 of the tumors which they transplanted grew progressively in normal CBA mice, and one of these regressed within a few weeks. Fieldsteel et al. (1975) observed a rise from 2%to >50% in the incidence of mammary tumors arising in their colony of BALBkCrgl mice between the time they imported a breeding nucleus from the Cancer Research Genetics Laboratory at Berkeley in 1963, and 1967; the high incidence persisted. All of 6 of the tumors they examined con-

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tained B-type particles. After confirming that their BALB/c mice had not become confused with their MMTV-infected strain A mice, they concluded that the BALB/c mice had become contaminated with milk-borne MMTV and that the infection had subsequently been disseminated through their colony. Collins and Parker (1972) found viral contamination of 69% of the 465 specimens of leukemia virus suspensions or transplanted tumors which they examined from various sources; half of the 23 specimens of leukemia L1210 were contaminated, 5 with polyoma virus; the one specimen of P 388 Leukemia and all specimen of Sarcoma 180 and Lewis Lung Tumor were contaminated. Although most contaminations were b y the nononcogenic lactic dehydrogenase and mouse hepatitis viruses, it is clear that the presence of these viruses, especially if they can bring about allogenization of the tissues they infect, could invalidate immunological and other findings obtained using infected materials. These findings of Collins and Parker (1972) are a shocking revelation of the poor quality control of biological materials widely used in cancer research. It is clear from these examples that the immunological status of a spontaneous tumor is particularly in question when it arises and is maintained in a laboratory in which artificial induction of tumors is commonly practiced or to which there is free importation of oncogenic viruses or tumors induced b y them or of tumors which may have been transplanted and maintained within several previous laboratories. It may be significant that in my own laboratory, in which no immunogenicity has ever been demonstrated in any of the many spontaneous tumors that have been exclusively used (Hewitt et aE., 1976), importation of tumors or viruses (with the recent exception of MMTV intrinsic in a small C3H mouse colony) has been rigidly prohibited during the 20-year history of the two low cancer strain colonies maintained. To summarize this section on virus-induced tumors: Such tumors almost invariably exhibit evident or potential immunogenicity attributable to the virus or to virus-associated antigens, and their use for experimental studies intended to have relevance for spontaneous tumors is imprudent, unnecessary, and commonly misleading. In the case of MMTV-induced mammary tumors isotransplanted to MMTVinfected mice, immunity is unlikely to be manifest in a way that affects the results of assays of therapeutic agencies, but their use for study of immunotherapeutic procedures is more questionable. Evidence for implication of oncogenic virus in the generation of clinical malignant tumors is insufficient to justify use of virus-induced animal tumors as

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models. Finally, artifactual tumor immunogenicity may be associated with a nominally spontaneous tumor by unrealized contamination with oncogenic virus or by errors in tumor signification.

E. TUMORSTRANSPLANTED UNDER CONDITIONS ISOGENICITY

OF INCOMPLETE

The basic genetic principles governing the success of transplantations of normal or tumor tissue from one individual to another were established as long as 60 years ago using mice (Little and Tyzzer, 1916) and have since been refined and extended by further study. It is now evident that a multiplicity of alleles may exist at each of at least 15 different histocompatibility loci (Snell and Stimpfling, 1966); using tail skin exchange grafts, Bailey and Mobraaten (1964) estimated that the number of loci may be as high as 30. Although these loci vary considerably in their “strength,” as indicated by the ease or difficulty with which their associated histocompatibility barriers can be transgressed, it is known that all such barriers can be fortified b y preimmunization; indeed, the weaker loci have only been identified with the aid of immunization. The multiplicity of loci is an important consideration in respect of the liability of a strictly inbred mouse colony to undergo “genetic drift,” whereby a degree of histoincompatibility may arise between a transplanted tumor derived from an ancestral mouse and a descendant recipient of the same colony. Whatever the overall frequency of spontaneous mutation at any one locus may be, the risk of incompatibility arising must increase in proportion to the number of loci able to contribute to it. It is probably only at the H-2 locus that allelic transformations can result in absolute resistance to the growth of a transplanted tumor, and it appears that the supervention of such solid resistance to a tumor transplanted within its own substrain is very rare. I n over 20,000 intrastrain tumor transplantations, we have only encountered two absolutely resistant mice (resistant, that is, to all tumors which had arisen in that strain) in a colony continuously inbred without deliberate selection of sublines for over 20 years. Since no further resistant mouse has been encountered in the several years since then, it is concluded that progeny of the resistant mice did not contribute significantly to the breeding colony. However, allelic transformations at minor histocompatibility loci can be expected to introduce subtle artifacts into a transplanted tumor system being used to measure the potential of a therapeutic agent: The primary implant may grow readily, but the resistance induced by its growth may attain to a level sufficient to restrain its regrowth (or the

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emergence of metastases) following regression of the primary induced b y a cytotoxic effect of the agent under test. That is, the artifact associated with minor histoincompatibility may contribute substantially to the end-point of “cure,” and so invalidate extrapolation of the data obtained to a system in which the tumorhost relationship is free from such artifact. It seems that the risk of artifactual immunity being introduced into a tumor transplant system was at one time overrated and led to idealistic recommendations that transplantation should be confined within a litter; such a severe restriction clearly prohibits all assay procedures and cannot provide for any progressive studies. In our experience, using multiple sublines of an inbred strain, some of which are likely to have been divergent over as many as 30 generations, we have encountered no evidence of tumor immunogenicity even using (spontaneous) tumors which have been serially transplanted for over 8 years; our tests for immunogenicity have included comparative challenge assays of viable tumor cells in normal and putatively preimmunized mice. A similar prolonged freedom from evidence of “genetic drift” within an inbred mouse strain has been reported by Godfrey and Searle (1963), who used exchange skin grafting to seek evidence of histoincompatibility. Dr. M. F. W. Festing (personal communication) has calculated that there should still be a 96%chance that a tumor would be accepted by recipients of the inbred strain of origin after about 13 years of propagation of the strain b y brotherkister mating, assuming 3 generations per year and that mutation was the exclusive source of genetic variation. H e used Silvers and Gasser’s (1973) formula for calculating the probability that two sublines will be histocompatible after specified numbers of generations of brothedsister breeding, but modified their data by employing a preferred lower estimate of the mutation rate-that of J. Klein (1975), of 9.3 x l W 4 per generation for all histocompatibility loci. Thus, although the risk of genetic drift as a source of transplantation immunity cannot be eliminated from any transplant system, it certainly appears not to be great using an inbred strain of mouse maintained in the laboratory in which it is used and a tumor arising in it. It is unfortunate that many investigators secure these confined conditions for minimizing the risk of genetic drift by chemically inducing tumors in mice of their own substrain; they thus invite what is almost a certainty of artifactual tumor immunogenicity, however genetically homogeneous their inbred strain may be (see Section 111,C). “ Considerable emphasis has been given to progressive antigenic simplification” in serially transplanted tumors, whereby they acquire increasing ability to transgress a range of histocompatibility barriers.

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The phenomenon is referred to in two contexts: Either it is welcomed as an advantage because it widens the utility of a tumor by increasing the range of strains which can be employed to provide graft recipients, or it is advanced to “explain” an inability to demonstrate immunogenicity in a tumor which has undergone prolonged serial transplantation, as is required to sustain the hypothesis that autochthonous tumors are usually immunogenic (M. Woodruff, personal communication). However, there is little doubt that increasing transgression of histocompatibility barriers is attributable more to increasing tumor growth rate and “escape” from host immune influences than to total loss of antigenic elements from the tumor. Certainly, there are a great many tumors whose early immunogenicity was imposed by chemical induction or by their origin in an animal of unspecified genetic constitution which have retained their immunogenicity after continual serial passage for over 50 years; and in many cases, such as the Ehrlich tumor of mice, the transplantation history includes passage through a very wide variety of strains, giving every opportunity for eliminative immunoselection. There is no doubt that transplantation immunogenicity is commonly imposed on a tumor system b y transference of a tumor from one laboratory to another. When a tumor is of origin in a specified animal strain and mice of the same nominal strain are used as recipients in the receiving laboratory, a difference between the substrains used in the two laboratories is often overlooked as a possible source of relative incompatability between tumor and hosts. Substrains may often have been separately propagated for over 20 years, during which period some genetic dissociation is very likely to have taken place. Moreover, the long history of both substrains may imply propagation of each in a sequence of laboratories and/or commercial breeding establishments in any of which breeding errors may have been made. Thus, in many instances the description of a tumor as “isogeneically transplanted” is purely nominal and has not been validated b y histocompatibility tests done between mice of the substrains held by the donating and receiving laboratories. Smith and Scott (1972) obtained evidence of immunostimulation by C. pamum vaccine using a murine ascites leukemia of CBA mice which was transferred from our laboratory to theirs some years previously, although this tumor had not displayed evidence of immunogenicity in our hands. It was disclosed that this tumor had been transplanted at the receiving institution to a substrain of CBA mouse different from that in which it had arisen and with which our own experiments had been done. Graff et al. (1975) have given a striking example of the way in which

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subline differences between mice to which a tumor is transplanted a potentially serious problem for experimental present chemotherapists.” The distinction of their study is that they were alerted to the hazard by an incidence of spontaneous regressions and investigated it by quantitative transplantation assays. They assayed a transplantable leukemia (which had arisen in an AKR/J mouse in 1962) in 5 sublines of AKR obtained from different sources. Using AKWJ mice obtained directly from the Jackson Laboratory or mice of their own AKWJ colony derived 4 years previously, they found that very few mice survived the injection of 10 leukemia cells; a proportion of AKR mice from 2 substrains commercially bred but obtained through the U.S. National Cancer Institute rejected as many as lO3-10’ cells; and 75% of AKR mice obtained directly from a commercial breeder rejected lo7 cells. They demonstrated by exchange skin grafts a considerable degree of histoincompatibility between substrains and within some of them and concluded that the most likely source of the genetic inconsistencies was errors of breeding. Leukemia L1210, perhaps the leukemia most commonly used in experimental chemotherapy studies, has been shown to be immunogenic under practically all conditions of transplantation. It is likely that this property was partly conferred at its inception, for it arose in an MCA-treated mouse of the DBAI2 strain almost 30 years ago. Mihich (1969) refers to its immunogenicity in DBN2, (BALB/c x DBAI2) F1, and (C57B1/6 x DBAI2) F1mice. Although mice of the three DBAI2 substrains he examined all gave 100% takes with 10 cells, and evidently shared the H-2d histocompatibility factor, differences between the substrains at minor histocompatibility loci were associated with widely divergent results from chemotherapy studies: The percentage of leukemic mice surviving at 50 days after a standard combined chemotherapy treatment with 4, 4’-diacetyl-diphenylurea-bis-guanylhydrazone and arabinosylcytosine was 57% using DBAI2 HaDD, 16% using DBAIBJ, and 5 % using DBN2 Cr. Since “cured” mice resisted challenge with between 103 and lo6 L1210 cells, survivors evidently owed their cure to a contribution, varying with the substrain of mouse used, of host resistance mustered during temporary arrest of the disease by the drugs. Mihich (1969) demonstrated also that the presence or absence of immune exertion by DBAI2 HaDD leukemia recipients was associated with quite different evaluations of the combined chemotherapy schedule employed. Using whole-body irradiated mice, synergism between the two drugs was observed; using unirradiated mice, only an additive effect was obtained. A further commonly used MCA-induced ascites leukemia of “

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DBAI2 mice, P-388, also displays a wide range of differential transplantability to different substrains of DBAI2. Berry and Andrews (1961) required only 2 leukemia cells for 50% successful intraperitoneal transplantation to their DBNBJN mice and were unable to raise this value by “pre-immunization” of their mice using lethally irradiated leukemia cells. A corresponding value of -400 cells was obtained b y Birch et al. (1975) using DBAIBJ mice; and in their transplant system mice could be readily immunized against the leukemia. There can be no doubt that studies similar to those made by Graff et al. (1975) or Mihich (1969) would be equally revealing of inconsistencies between substrains if they were made of other transplantable animal tumor systems which are commonly used to assess therapeutic agents. In respect to immunity studies, it is to be noted that an immune response against a transplanted tumor which is, in fact, attributable to a histocompatibility difference between the mouse substrain in which the tumor arose and that to which it is transplanted is ostensibly deserving of the description “tumor specific,” although the term is not intended to have this connotation. A tumor-specific immune reaction can be identified as such only in the autochthonous host or in transplantees which have been shown to be isogenic with the host of tumor origin with respect to the transplantation of normal tissues. It is clear that the widespread use of familiar veteran tumors having a specified animal strain of origin does not at all achieve the consistency and comparability of data from different laboratories which is claimed for the practice. Substrain differences between recipients may very well be associated with a wider range of response to therapeutic agents than would be encountered for comparisons made between the responses of different nonimmunogenic tumors. It should be added that the older named or coded tumors are subject also to divergence between different lines of the tumor itself, which can have had different transplantation histories over several decades; some of this divergence may involve antigenic modifications. Atassi and Taignon (1974) reported the results of treatment of L1210 leukemic mice with adriamycin or adriamycin-DNA complex. The prognosis of treated mice bearing their own line of L1210 leukemia was significantly different from that of mice bearing a line of L1210 leukemia obtained from another laboratory. For logistic convenience, an increasing number of researchers now use as tumor recipients F1mice of a cross between two inbred strains of which one is the strain of tumor origin. While the laws governing the genetics of transplantation permit the assumption of histocompati-

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bility for such transplantation to F, hybrids, it should be appreciated that, in the case in which some residual heterozygosity remains in the relevant inbred strain of the F1 cross, the risk of histoincompatibility is substantially increased by using F, mice as recipients instead of mice of the original strain (M. F. W. Festing, personal communication). A further hazard of the use of F, mice as tumor recipients was referred to in Section 111,D: Transplantation of an MMTV-induced tumor to F, hybrid mice of a cross in which the strain of tumor origin is represented by the male, the female being of a MMTV-fi-ee strain, is associated with transplantation resistance against the tumor evoked by MMTV-associated antigens in it. Before concluding this subsection, reference must be made to the continued use of frankly allografted tumors, those of origin in animals of unspecified and technically unreproducible genetic constitution. These are all tumors which arise in exotic species from which homozygous inbred strains have not yet been derived, or tumors which arose in familiar laboratory species at a time before inbred strains were developed. It is remarkable that Ehrlich’s tumor and Crocker tumor 180 of mice, and Walker Carcinosarcoma 256 of rats, are still among the commonest tumors used in cancer research. Since the biological features of these tumors are adequately represented in any number of more recently arisen tumors, allowing isografting to the inbred strains of origin, their continued usage can be ascribed only to sentimental regard for them as memorials to the enterprising men who pioneered our present research facilities; the veteran tumors referred to arose respectively in 1907, 1914, and 1928 (Stewart et al., 1959). Use of the Ehrlich tumor was reported in 11%of the papers describing experimental studies with mouse tumors which were published in 1975 (Roberts and Drobycz, 1975),a drop of only 3% from the usage in 1970. All that need be said of frankly allografted tumors in the context of this essay is that results of therapy studies done with them can have no quantitative relevance for nonimmunogenic tumors, and that demonstration of their invariable antigenicity has no significance for the immunology of naturally occurring cancer. The large extent to which they are still employed is a poignant revelation of the difficulty encountered b y many contemporary cancer researchers in obtaining appropriate experimental tumor systems. In conclusion, it must be stated that the conference of partial allogenicity upon a transplanted tumor system by unrealized genetic contamination of the inbred strain of animal used, by failure to take account of possible genetic differences between the substrains of recipients during interlaboratory transfer of tumors, by contamination of

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tumors with possibly allogenizing viruses, or by errors of labeling resulting in confusion between tumors are among the most subtle and frequent sources of artifactual tumor immunogenicity. Because these influences are unrealized, a description of the tumors as “isogeneically transplanted” is nominally justified, and the deficiencies of the animal model are thus concealed and perpetuated. When confronted by a transplanted tumor system having a history of serial transplantation in one or more previous laboratories, it is rarely feasible to determine whether any tumor immunogenicity revealed in it has been superimposed as an artifact or represents the continuance of tumor specific antigenicity active in the original autochthonous host. It is certainly impossible to assess the relative contributions from the two sources when both may be exerting an influence on the effectiveness of a therapeutic agent under trial. These uncertainties deserve paramount consideration in deciding the criteria to be met by a transplanted animal tumor system which is to be adopted for use in programmed studies of either tumor immunology or experimental cancer therapy when the results obtained are to be given the status of clinical relevance. It is difficult to escape a conclusion that systems should be rejected for such studies if tumor immunogenicity can be demonstrated in them b y any of .the accepted immunization techniques available for revealing it.

F. RELATIVE USAGE OF DIFFERENT CATEGORIES OF ANIMAL TUMORSUSED IN RECENT EXPERIMENTAL STUDIES OF CANCERTHERAPY To assess current practice in the selection of animal tumor systems for therapy research I have sampled the recent literature in a way that should have avoided bias in reaching the conclusions to be drawn. I have perused the titles of all articles published in the British Journal of Cancer over the period 1974 through 1975 (Volumes 29-32) and in Cancer Research during 1975 (Volume 35) and have selected out for analysis all articles which report examinations of therapeutic or potentially therapeutic agents using transplanted animal tumor systems. The sample comprised 45 articles referring to the use of 71 tumors. Of the tumors used, 38 were employed in chemotherapy studies and 33 in immunotherapy studies (in the case of three articles in which a combination of two modalities was tested, the tumors used were allocated to the modality which appeared to be of predominant interest). Table I1 shows separately, for the chemotherapy and immunotherapy series, the distribution of the tumors used among the

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TABLE I1 ANIMALTUMORSUSED IN CHEMOTHERAPY OR IMMUNOTHERAPYEXPERIMENTS CATEGORIZED BY NATUREOF ORIGIN" Origin

Chemotherapy

Immunotherapy

Total

Chemically induced Virus-induced Radiation-induced Allografted Spontaneous

16 (42)a 5 (13) 2 (5) 3 (8) 12 (32)

18 (55) 5 (15) 2 (6) 1(3) 7 (21)

34 (48) 10 (14) 4 (6) 4 (6) 19 (27)

" Compilation from

relevant articles published in Cancer Res., Vol. 35 (1975) and

Br. J . Cancer, Volumes 29-32 (1974-1975). Numbers in parentheses indicate percentage of total in each column.

different categories of tumor origin: chemically induced, virus induced, radiation induced, allografted, or spontaneous. Allografted tumors are those arising in animals of unspecified genetic constitution or transplanted to animals of a strain different from that of the animal in which the tumor arose. Of the 4 radiation-induced tumors used, 3 were carried in congenic resistant mice, so that these systems embodied artifactual immunity deliberately introduced. There is no significant difference in the distributions for chemotherapy and immunotherapy experiments. This indicates neglect or unawareness of the need for particular care to exclude artifactual immunity from tumor systems to be used in immunotherapy studies. In the case of chemotherapy studies, the intromission of artifactual immunity may be expected only to augment the effectiveness of an agent under test, whereas in the case of immunotherapy studies, artifactual immunity in a system may be entirely responsible for any effect of immunostimulation demonstrated. Table I1 indicates that almost half the tumors used had been chemically induced, a condition that is almost invariably associated with artifactual tumor immunogenicity (Section 111,C). Only 27% of the tumors were of spontaneous origin, which is the category of tumor least likely to b e associated with immunogenicity and the one to which all but a very few clinical cancers belong. However, as discussed in Section III,E, artifactual immunogenicity may become superimposed upon a transplant system derived from a spontaneous tumor, and the risk of such contamination increases with the length of time elapsing since its origin. I have therefore analyzed the histories of the 19 tumors of spontaneous origin which appear in the sample for the purpose of assessing their status as models of autochthonous

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tumors of spontaneous origin with reference to an ideal that is easily attainable within a laboratory bred colony of mice. That is, a tumor arising spontaneously, preferably within the colony providing graft recipients, and used within 15 years of its origin. Table I11 lists the 19 tumors of spontaneous origin which appear in the sample, from which it is seen that in just over one half of the investigations, tumors were used which arose over 20 years previously [Lewis Lung Tumor (LLT), B16 Melanoma, Ridgeway Osteogenic Sarcoma and Neuroblastoma C 13001 and which have been heavily exposed to the numerous conditions which can superimpose artifactual tumor immunogenicity on a system, as discussed in Section II1,E. The predominant usage of LLT deserves discussion because it provides an understanding of the influences conducing to selection of experimental tumor systems. The advantage apparently distinguishing LLT is its large potential for pulmonary metastasis; it thus provides a convenient system for therapy studies directed to control of secondary disease following ablation of the primary implant (e.g., Mayo et al., 1972). However, LLT is not unique in its metastasizing potential, which is possessed by several of our own spontaneous carcinomas. As might be expected of a tumor which has undergone a great many serial passages and has been very extensively transferred from one laboratory to another since its origin, evidence concerning its immunogenicity after transplantation is inconsistent. DeWys (1972) associated its Gompertzian growth curve with a tumor-related systemic growth retarding factor, whereby the presence of a larger tumor in a nominally TABLE 111

TUMORS OF SFONTANEOUS ORIGINUSED IN CHEMOTHERAPY AND IMMUNOTHERAPY

Tumor Lewis Lung Tumor B1G Melanoma Ridgeway Osteosarcoma Neuroblastoma C 1300

EMTG Line 1 Lung Carcinoma Epithelioma Sp. 1 Mammary Cancer D7T4S SBI Lymphoma WHT Squamous Cancer 'D' a

EXPERIMENTS (1974-1975)" Strain of origin

C57B 116 C57B 116 AKm A/ Jax BALBIc BALBlc Wistar (rat) BALBic BALBIc WHTiHt

From same sample as used for Table 11.

Years since origin

24 21 27 35 ? ? 10 >5 ? 10

-

Instances in sample

5 3 1 1 2 1 2 2 1 1

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185

isogeneic mouse was associated with loss of carcass weight and depression of the growth rate of smaller tumors in the same animal. Although these findings are suggestive of the exertion of concomitant immunity, DeWys showed that the number of tumor cells required to initiate tumors in mice which had had primary tumor implants excised 8 or 15 days previously was not greater than that required in mice with no previous experience of the tumor; thus, in his system, tumor immunogenicity was excluded. However, Carnaud et al. (1974) revealed “weak but consistent” immunity in their LLT system by successful immunization of recipients using repeated small inoculations of viable cells; they admit that their finding could represent an artifact associated with prolonged serial passage of the tumor. The results of some chemotherapy studies using LLT have been so remarkably good, relative to the expectations for chemotherapy of solid clinical tumors, that a contribution from host resistance factors in the experimental system appears very likely. Mayo et at., (1972) obtained -40% cures of mice bearing 400-mg tumors using cyclophosphamide or methyl CCNU alone, or the two in combination, and 50% cures of mice whose primary tumors had been previously excised at a “quite advanced” stage of tumor growth. Variation of the level of artifactual immunogenicity in different transplant systems of LLT is likely to be responsible for inconsistent results obtained for the effectiveness of the nontoxic immunostimulant levamisole. Neither Spreafico et al. (1975)nor Johnson et al. (1975)could demonstrate any effect of levamisole on the growth of LLT, although the former obtained 90% cures of mice bearing the chemically induced leukemia L1210 and the latter obtained cures of MSV-induced rhabdomyosarcoma. On the other hand, Renoux and Renoux (1972) cured 3/12 mice bearing LLT by early treatment with a single subcutaneous dose of 0.5 mg/kg of levamisole. The findings oE Renoux and Renoux (1972) may very well exemplify the consequences of a change of mouse substrain associated with interlaboratory transfer of LLT. They used as recipients mice designated as C57BL/Rho, and acknowledge Dr. I. Gesser for his provision of the tumor; however, reference to a previous paper in the same journal by Gesser and Bourali-Maury (1972) reveals that their studies of LLT were made using mice designated as C57BL/6. A suggestion that LLT may have exhibited some immunogenicity very early in its history of interlaboratory transfer is gleaned from the report of Sugiura and Stock (1955), who obtained 100% successful transplantation of LLT to their nominally isogeneic mice, yet 4 % of the tumors spontaneously regressed; these regressions are an indication, at least, of some genetic heterogeneity among the recipients. It may b e assumed that the above incon-

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sistencies and uncertainties associated with the use of LLT would be equally displayed by the 21-year-old B 16 Melanoma. Certainly, circumstantial evidence of its immunogenicity has been revealed in some of its transplanted systems. For example, Chalmer et al. (1975) found that B 16 Melanoma grew faster and produced more lung metastases in mice chronically exposed to cigarette smoke than in untreated mice; they attributed this finding to an immunosuppressive effect of the exposure to smoke, and this interpretation is supported by their finding that the effect was greater using a more strongly immunogenic tumor induced by murine sarcoma virus. Although B 16 Melanoma provides a very special facility for metabolic studies of melanin production, this feature does not explain its very wide usage in therapy studies, which generally have no reference to it. It is concluded that LLT and B 16 Melanoma have become prominent not on account of unique features intrinsic in either tumor (LLT is described as an anaplastic derivative of a squamous cell carcinoma) but to thetr adoption b y the Division of Cancer Treatment of the U.S. National Cancer Institute for screening of chemotherapeutic agents. It is probable that this employment constitutes a recommendation which discountenances reservations invited b y the long transplantation history of both tumors. The Ridgeway Osteogenic Sarcoma, now almost 30 years from its year of origin, although it gives 100% takes in AKR mice and is known to recur after failed therapy (Laster, 1975), has given various indications suggesting a distinctive hosthumor relationship. The tumor is described as having arisen spontaneously in mice of the A h strain (Laster, 1975), but is currently transplanted in AKR mice, a condition which would appear to imply some lack of nominal isogeneity; it is described by Dunham and Stewart (1953)as being subject to 3% spontaneous regressions in AKR mice. It is remarkably susceptible to cure by actinomycin D; Schwartz et al. (1966) obtained 90-100% cures following a single intravenous dose of 800 pg/kg, but these authors rejected a contribution of host resistance to cure b y demonstrating that tumors were able to grow from secondary implants into mice during drug-induced regression of primary implants. Certain observations of Biano et al. (1971) suggest that the hosthumor relationship may be complex; a considerable splenomegaly accompanies tumor growth, but the number of antibody-forming plaques in the spleen was progressively reduced as the tumor increased in size. It appears possible that some immunosuppresive influence accompanies tumor growth, and that the system deserves a more exhaustive examination for features which could explain the apparent absence of immunogenicity despite the large opportunities for intromission of artifactual immunity

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to which the tumor has been exposed during its very long history of transplantation. Table I11 shows 2 instances of use of the EMTG tumor in therapy studies. This tumor has a complex history (Medina and DeOme, 1970; Rockwell et al., 1972): During early transplantation of a hyperplastic mammary nodule arising in a hormonally stimulated female mouse of strain BALBlc Crgl, the nodule transformed spontaneously to a histologically dimorphic malignant tumor, at which time it was transferred from the laboratory of origin (Cancer Research Genetics Laboratory, Berkeley) to Dr. R. F. Kallman of the Department of Radiology, Stanford University School of Medicine. Kallman transplanted the tumor (KHJ J) serially in BALB/c Ka mice (originally propagated from BALB/c Crgl), and over a period of at least 109 passages it remained free from any evidence of immunogenicity. The TD50 was small (2-16 cells) and could not be raised by preimmunization of recipients with lethally irradiated homologous cells or by addition of such cells to the inocula of viable cells. This is in accordance with expectations for a tumor whose origin involved no implication of chemical carcinogen or oncogenic virus, At the 25th serial passage of KHJJ, the tumor was adapted to growth in tissue culture and was propagated in culture for 33 serial passages as EMT; at the 33rd passage it was cloned as EMT6, and this derivative was henceforth alternately cultivated in vitro and in vivo in BALB/c Ka mice. However, EMTG was found to evoke “a decided immunologic response;” the TD50 was substantially higher in preimmunized mice and was boosted by addition of lethally irradiated cells to the inocula. Subsequent use of EMTG in other laboratories has revealed that the immunogenicity in BALB/c mice has been sustained at a level which can seriously complicate chemotherapy studies (A. Begg, personal communication). Since the immunogenicity of EMTG has run parallel with the nonimmunogenicity of KHJJ, from which it was derived, it is clear that the immunity is an artifact superimposed upon KHJJ by its adaptation to tissue culture; it is conceivable that the antigenic modification has been isolated and perpetuated by removal of the cell line from an influence of immunoselection. The history of EMTG is of interest because it illustrates another possible source of superimposed artifactual tumor immunogenici ty . Line 1 Lung Carcinoma (Table 111) is a tissue culture adapted cell line derived from a spontaneous alveolar carcinoma of the lung of a BALB/c mouse at its 12th serial passage in viuo; the tissue culture line was the source of cells used as inocula for subsequent in vivo studies of tumor growth (Yuhas et al., 1974). Although the original tumor

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maintained by serial passage in vivo has exhibited no failures to take or spontaneous regressions, more detailed study of the Line 1 Carcinoma transplanted in vivo has revealed a complex hostftumor relationship which Yuhas and his colleagues have interpreted immunologically. Yuhas et al. (1974) showed that subcutaneous tumors grew more slowly in mice immunosuppressed by whole-body irradiation (WBR) or by treatment with cortisone and that the growth depression induced b y WBR could be cancelled by the administration of syngeneic normal lymphoid cells to the mice after their exposure, Yuhas et al. (1975a) demonstrated later that there was mutual growth inhibition between the primary tumor and pulmonary metastases naturally disseminated from it or superimposed on the tumor-bearing mice by intravenous injection of tumor cells; this they interpreted as a manifestation of concomitant immunity in the system. Yuhas et al. (1975b) subsequently showed that growth of primary implanted tumors was significantly depressed by treatment of the mice with a combination of C. parvum vaccine with intravenous tumor cells. Thus, the Line 1 Lung Carcinoma must be regarded as a weakly inimunogenic system. Unfortunately the information in the relevant papers is insufficient to determine whether the immunogenicity is a consequence of the adaptation to tissue culture or was exhibited by the original unadapted tumor. Without the required information, and in the light of the clearer evidence for the EMT6 tumor, it seems wise to conclude that the immunity in the Line 1system is an artifact imposed by exposure to in vitro conditions. Epithelioma Sp. 1 (rat) arose spontaneously in a highly inbred Wistar rat and was used within the laboratory of origin in both the instances of its use which appear in the sample (Table 111); it was probably under 10 years old when used. Although this tumor has full eligibility as a spontaneous tumor and has been serially transplanted under conditions which minimize the risk of superimposed histoincompatibility, it is described as “weakly immunogenic” ( Baldwin, 1966).Rats in which primary implants had been destroyed by induction of ischemia in situ resisted challenge with viable cells of the tumor; they nevertheless went on to develop pulmonary metastases; rats could not be immunized using radiation-killed homologous cells. Thus, the evidence for immunogenicity is indeed weak. In the two investigations contributing to our sample, no immunotherapeutic effect on the growth of this tumor was obtained using either orally administered BCG or levamisole (Baldwin et al., 1975; Hopper et al., 1975). WHT Squamous Carcinoma ‘D’ arose spontaneously in my own laboratory and has been shown to be nonimmunogenic by repeated im-

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munization studies and numerous incidental observations (Hewitt et al., 1973, 1976; Hewitt and Blake, 1975). The investigation contributing this tumor to the sample (Table 111) was carried out in another laboratory, but the tumor and a breeding nucleus of WHT mice were supplied together &om my laboratory, so that no change in the substrain of recipients was associated with transfer of the system. Mammary Carcinoma D7T4S (Table 111) arose from a hyperplastic alveolar nodule appearing in a hormonally hyperstimulated MMTVfree BALB/c female mouse and had been serially transplanted in the strain of origin for several years. Eligible as a tumor model in respect of its origin and conditions of maintenance, it is nevertheless described as being slightly immunogenic (Dao et al., 1972). The SBI Lymphoma (Table 111)is strictly of spontaneous origin and was transplanted within the BALB/c colony in which it arose; over 80% successful transplantation is achieved with 5 cells, but no formal tests for its immunogenicity have been reported. The investigation reporting its use, in our sample, did not put to test, or require the exertion of, its immunogenicity. Summarizing the content of Table 111, I conclude that, out of the 19 tumors of spontaneous origin used in the investigations contributing to the sample, 10 have been serially transplanted over such long periods that they have been heavily exposed to conditions which could have imposed upon them artifactual features affecting the hosthumor relationship (LLT, B 16 Melanoma, Ridgeway Osteosarcoma and Neuroblastoma C1300), and 3 (EMT6 and Line 1 Carcinoma) exhibit tumor immunogenicity which is likely to be associated with their maintenance under in vitro conditions. Only the remaining 6 tumors, represented by Epithelioma Sp. 1, Mammary Carcinoma D7T4S, SBI Lymphoma, and WHT Squamous Carcinoma “D” conform to the ideal specifications I have suggested: spontaneous origin, less than 15 years transplantation history, and transplanted within the substrain of origin. Referring these 6 eligible tumors to the total sample of 71 tumor usages (Table 11), including tumors which were artificially induced or allografted, w e find that over 90% of the tumors currently used in experimental therapy research are of very questionable status as models of spontaneous autochthonous cancer. This does not, of course, imply that data obtained using tumor systems of relatively low status as models do not have validity within a limited context. But in as much as results obtained using questionable systems are subject to significant influence by artifacts, it is not allowable to extrapolate findings quantitatively to systems free of such artifact. That this limitation is very little regarded is evident from the frequency and im-

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prudence with which clinical implications are attached to reports of experimental studies. A similar analysis to that recorded in Table I11 has been made of the animal tumor systems selected for experimental studies of the action of C . pamum vaccine on tumor growth. This was undertaken for two reasons: I t employs a rather different method of sampling from the recent literature, covering a longer period (1972 through 1975) and a relatively unrestricted range of journals, and it confines attention to a sphere of interest in which clinical application of laboratory studies is being intensively promoted. (The Compendium of Tumor Immunotherapy Trials, No. 4, August 1976, records 48 clinical trials of the immunotherapy potential of C . parvum vaccine, either in progress or projected.) It is clearly an area of research in which a specially high standard of care is to be expected in the selection of animal models of human cancer. The method of sampling was by reference to all the articles indexed under “Corynebacterium paruum” in Volumes IX through XI1 (1972-1975) of the annual publication of Roberts or Roberts et al. entitled “Research Using Transplanted Tumors of Laboratory Animals.” The sample comprised 28 articles referring to the use of a total of 46 tumors. Table IV, recording the results of the analysis from C. parvum experiments, shows that the majority (76%)of tumors were chemically induced or virus-induced. The one radiation-induced tumor was, in fact, a partial allograft in the experiment describing its use (Smith and Scott, 1972). The two uses of a tumor specified as “ex-culture” were of CBAT-3 fibrosarcoma, a tumor resulting from malignant transformation in uitro of a culture of embryonic CBA fibroblasts; it is immunogenic. Thus, only 5 (11%) of the tumors used were of spontaneous origin in viuo and had been transplanted under moninally TABLE IV

c.

CATEGORIZATION OF ANIMAL TUMORSU S E D IN STUDIES OF pU?WU?Tl VACCINE (RANDOM SAMPLE OF PUBLICATIONS 1972-1975)

Tumor origin

Number

% of total

Chemically induced Virus-induced Radiation-induced Ex-culture Allografted Spontaneous

27 8 1 2 3 5

59 17 2 4 7 11

CHOICE OF ANIMAL TUMORS FOR THERAPY STUDIES

191

isogeneic conditions. All of these 5 spontaneous tumors appeared in the previous sample, were included in the analysis recorded in Table 111, and have been discussed above. (Three were representatives of the 24-year-old Lewis Lung Tumor, 1was B 16 Melanoma, and 1 was the Line 1 Lung Carcinoma.) It is remarkable that, in this series of experiments devoted to a clinically oriented topic, there was not a single example of the use of an ideal model of spontaneous autochthonous cancer, that is, a tumor of fairly recent spontaneous origin in the laboratory in which the experiments were done and having no history of maintenance in vitro. The descriptions of the experiments contributing to the sample commonly included the results of tests for the immunogenicity of the tumors used. These were almost invariably positive, as reported in the relevant papers or by reference to previous papers. For example, Smith and Scott (1972) employed 5 different tumors in their studies of C . parvum: 3 induced tumors (Hepatoma 129, Plasmacytoma PCGA, Leukemia L5178), a radiation-induced leukemia (R-I1)transplanted to a relatively histoincompatible mouse substrain, and fibrosarcoma CBAT-3 transformed from normal fibroblasts after maintenance in tissue culture. All of these tumors were immunogenic as shown by a rise in TD50 (challenge) after immunization of recipients with lethally irradiated homologous cells. This finding for the 5 tumor systems used by Smith and Scott (1972) is in striking contrast to ours (Hewitt et al., 1976) using a range of 7 spontaneous murine tumors having unexceptionable status as models and the same immunization procedure. We found that the TD50 (challenge) was less in immunized than in normal mice in the case of all 7 tumors. Detailed study of the results of the experiments by Smith and Scott (1972) reveals that the effectiveness of C. pamum vaccine as an immunostimulant was directly proportional to the strength of artifactual immunogenicity exerted in a system; for example, the vaccine was considerably more effective against allografts of Hepatoma 129 in BALB/c mice than against grafts in mice of the strain of origin (CBA). A high proportion of the authors who have demonstrated a restraining effect of C. parvum vaccine on the growth of artifactually immunogenic tumors induced by chemicals or viruses suggest by implication, or by forthright recommendation of clinical trial, that their results have significance for clinical therapy. This is so even when the animal model used is highly immunogenic and when the mode or time of administration of the vaccine in the experiments could have no realistic representation in the clinical situation. In many cases the vaccine has been adminstered before the tumor is grafted or has been mixed with the grafted cells at the time of grafting. Rarely have the

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authors explicitly entertained the possibility that their positive results may merely reflect the artifactual peculiarities of the experimental system they have used, as did Pimm and Hopper (1975). IV. Reflections and Conclusions

The discipline of cancer research can be roughly divided into two areas which are distinguished by a difference of orientation in relation to clinical cancer. “Basic” or “fundamental” investigations are addressed to the general biology of growth and encompass a broad spectrum of inquiries and approaches; the question whether the products of such research will conduce to better management of clinical malignant disease is left open, as indeed it must be if the research is to progress by internal instigation. The other area of research, to which this contribution is relevant, has been called “applied,” although the teasing phrase “mission oriented” nicely takes account of the sociological influences through which “applied” research seeks approval and gains favor. It must be acknowledged that there is growing impatience with the pace of progress in clinical treatment of cancer in relation to the volume of research afforded, to which research funding bodies have responded b y pretending to clairvoyant recognition of “relevance” in the evaluation of projects for which support is sought. Whatever may be the foundation or merits of this emphasis on relevance, there is no doubt that it has influenced the general direction of cancer research in a way that underlines the need to review and revise the criteria to be regarded in the selection of animal tumor models. Pressure on researchers to claim clinical relevance for their projects has had untoward effects on the style of presentation of fundamental and apparently esoteric investigations, whereby tortuous and highly speculative projections have been made from intimate studies of cell structure or function to the prevention or treatment of human cancer. Yet this tendency is in defiance of a more sober understanding that the utility of knowledge is an eventuality not susceptible to reliable prediction (Flowers, 1972). With respect to research for which more brash claims to relevance are made, by their description as “applied,” association between those who conduct animal experiments and those who treat patients has been fostered by placing the two in ever closer proximity. Indeed, there are now several situations in which large animal laboratory facilities and clinical undertakings fall under a single director. The danger of such intimacy is that it encourages a rather direct translation of animal data to clinical prescription in circumstances where knowledge and experience in one

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sphere may be unmatched by expertise in the other. Spectacular cures of animal tumors by treatments of negligible toxicity are regularly reported, and it is understandable that a clinical oncologist confronted b y such findings, and not made aware of the limitations of the animal model with which they were obtained, may feel an ethical obligation to confirm them clinically. Since most clinical oncologists are not equipped by training or experience to assess the status of an animal tumor as a model of clinical cancer, the responsibility for evaluating the clinical relevance of experimental data should reside with the experimenter. Tables 11, 111, and IV reveal that this responsibility is very far from being met by those on whom it rests: Only a small proportion of the animal tumor systems used in current therapy research can be claimed to be free from manifest or latent artifacts of a kind that can be highly influential on the data obtained by their use. Yet it is the rule, rather than the exception, for reports of such experimental studies to include a discussion of clinical implications if not actual recommendation of clinical application. Unrestrained enthusiasm for the general theory that spontaneously arising tumors are (at least potentially) immunogenic, based on a conviction that the creation of neoantigens is an invariable accompaniment of malignant transformation, has encouraged permissive acceptance of tumor antigenicity in an animal tumor system. It may be argued that artifactual introduction of this property serves merely to exaggerate, for experimental convenience, a feature of the natural disease. This is not an argument which can be rationally sustained. A belief in the natural immunogenic potentiality of an autochthonous tumor of spontaneous origin is adequately catered to by use of an animal tumor system which, by a priori reference to its history, can be considered to be free from a high risk of imposed artifacts. Any expression in the transplanted animal tumors of a primordial immunogenicity conceived to have been imposed b y spontaneous malignant transformation will then be exerted at a realistic level of effectiveness. Exaggeration b y artifact serves only to distort the model either quantitatively or absolutely. Since tumor immunogenicity is not detectable in many experimental systems derived from spontaneous tumors (Hewitt et al., 1976), and since distinction of natural from artifactual immunogenicity is rarely feasible, a strong case can be made for accepting for therapy studies only animal tumor models which have been shown to be nonimmunogenic. In circumstances in which the immune resistance of tumor-bearers does make a contribution to cure of the tumors by a therapeutic agent, it is often not appreciated how large the contribution may be. Mea-

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surement of the single dose of radiation required to cure half of a group of uniform tumors of specified size (the TCD5O) presents a specially favorable opportunity for assessing this contribution. This is so because of three features peculiar to the action of radiation on tumors. First, the dose of radiation actually delivered to every cell of the tumor, exposed under appropriate conditions, can be specified with great accuracy. Second, the cells of different tumors have closely similar radiosensitivities. And, finally, the approach to cure with increasing dose is described over the greater part of the dose/effect curve by an exponential relating the dose to the proportion of clonogenic cells reproductively killed. Thus, the TCD50 can be predicted from a generally applicable dose/effect curve in relation to an estimate of the average size of the clonogenic cell population in the tumors. For nonimmunogenic tumors of -8 mm diameter, the .TCD5O is predicted to be approximately 5000 rads, and this value has been verified in numerous radiobiological studies of such tumors (e.g., Howes, 1969). For their study of the adjuvant affect of C. purvum vaccine on the TCD50, Milas et al. (1975) employed an MCA-induced fibrosarconia displaying the immunogenicity characteristically associated with tumors so induced. Their TCD5O (without administration of C. paruum) was only 3400 rads. Thus, the TCD50 was less, b y 1600 rads, than the value of 5000 rads expected for nonimmunogenic tumors (1600 rads would reduce the clonogenic cell population of a nonimmunogenic tumor to less than 1%). Milas et ul. (1975) found that the injection of C. purvum vaccine into tumor-bearing mice on the day of irradiation reduced the TCD50 by 1000 rads. In terms of cell killing, therefore, stimulation of resistance by C. purvum added a contribution to tumor cure which was rather less than that already contributed by the unstimulated resistance intrinsic in the system. It is of interest to consider what may be the influences which are responsible for the predominant usage for therapy studies of tumor systems whose histories imply a high risk of the inclusion of artifacts. The question is most difficult to answer in respect of large, long established centers of oncological research which maintain their own breeding colonies of mice and therefore have ideal conditions for the isolation and maintenance of tumors of spontaneous origin. It is evident from a review of the relevant papers from several such large centers that the animal tumors used in them are induced or are veteran tumors imported from other laboratories. It must be conlcuded that regular surveillance of colonies of ex-breeder animals is not undertaken and that the value of accumulating a panel of internally evolved tumors of different types is not appreciated. Yet it is only by maintenance of such a facility that a need to import tumors, with inevitable

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risk to the hygiene of resident colonies, can be avoided. That the internal resources of some large centers are not adequate to meet the demands of workers within them is evident from requests made to our own laboratory for the provision of nonimmunogenic systems. In the case of small research facilities, the lack of intrinsically evolved tumor systems is more easily understandable, especially where a breeding unit is not maintained, and mice for experiment are obtained from a commerical source as required. Perhaps the most common encouragement to hasty importation of tumors having questionable status as models arises out of the situation confronting researchers embarking on short term limited projects; in such cases, there will be too little time to await the appearance of a spontaneous tumor having the features required for the study contemplated. The risk of superimposing immunological artifact on a tumor system is specially high in the case of facilities which maintain breeding colonies of inbred animal strains but do not have tumors of origin in the resident substrains. In these circumstances, a tumor that has arisen in the same nominal strain elsewhere is imported from another laboratory and is liable to acquire artifactual immunogenicity by its transplantation across a possible minor histocompatibility barrier between the substrain of origin and that in the receiving laboratory. It has been our practice in meeting requests for transplanted tumors from our own laboratory to advise the breeding up of a colony of the relevant mouse substrain from a nucleus we supply in advance of dispatch of the tumor, and it is our experience that the advice is appreciated and acted on. We have had no reports of tumors which w e have supplied under these conditions having developed immunogenicity after transfer. In the one case in which our advice proved to have been overlooked and the CBA leukemia line we supplied was transplanted to a different CBA strain, the leukemia exhibited immunogenicity (Smith and Scott, 1972). The important question arises whether the selection of tumor systems for immunotherapy studies has been subject to bias in favor of systems which are more likely to entaiI artifactual immunogenicity. From the chemotherapy column of Table 11, it is seen that 12/38 (32%) of the tumors used were of spontaneous origin. Combining the data of the immunotherapy column of Table I1 with the data of Table IV for studies of C. pamum vaccine shows that only 12/79 (15%) of the tumors used were of spontaneous origin; the difference approaches significance (0.1> p > 0.05). It is my belief that this evident bias reflects the results of excessive promotion of the topic of immunotherapy and betrays a response to pressures on researchers to obtain results which favor the promotion; it could imply that negative results obtained with tumors of spontaneous origin have gone unreported. My

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analysis confirms the previous assertion by Klein and Klein (1977) that bias has perverted this area of research. These are serious considerations which raise ethical issues concerning the clinical application of experimental findings. It is clear that the quality of experimental studies undertaken within limited research facilities could be much improved, and the high incidence of laboratory artifacts reduced, if the larger institutes of oncological research undertook to collect, maintain, and monitor transplanted animal tumor systems and made these available to researchers in smaller laboratories. The cost of maintaining such resources could be largely offset by realistic charges for material supplied. There are several national bodies in existence which presume to “coordinate” cancer research and to arrange facilities for intercommunication. It would be a valuable aid to researchers if such bodies undertook to organize facilities which would encourage the use and availability of experimental tumor systems which can be authenticated as acceptable models of naturally occurring cancer. It appears that such a service might have a more telling effect on the output of relevant research than arbitrary specification of the lines of research to be followed. I suggest also that referees of scientific papers and the editors of journals assume a responsibility to disallow the drawing of clinical implications from findings made by the use of animal tumor systems which entail obvious artifacts. It is appropriate to end this contribution by a quotation from a previous article in this series: “We should abandon the common models in animals (i.e., highly antigenic chemically induced tumors or those produced by ‘laboratory’ viruses) in the same way as we abandoned the use of allogeneic models for the study of relevant immunologic events , . . and concentrate on spontaneous tumors appearing in experimental animals” (Stutman, 1975).

ACKNOWLEDGMENTS The work of the author referred to in this chapter was supported exclusively by the Cancer Research Campaign. I gratefully acknowledge the skilled technical assistance of Miss Eileen Blake, A.I.M.L.T., and Miss Angela Walder, A.I.A.T., whose work has included the isolation and maintenance of over thirty murine tumors of spontaneous origin. I am grateful also to Mrs. Karen Jepson for assistance in preparing the manuscript.

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ADVANCES IN CANCER RESEARCH VOL . 27

MASS SPECTROMETRY IN CANCER RESEARCH'

John Roboz Department of Neoplastic Diseases. Mount Sinai School of Medicine. New York. New York

I . Scope of Applications and Analytical Techniques ......................... A . Advantages and Limitations ......................................... B . Nature of Mass Spectra .............................................. C . Components of Mass Spectrometer Systems .......................... D . Analytical Techniques .............................................. ..... I1 Identification. Quantification. and Metabolism of Carcinogens . A . Survey Analyses .................................................... B . Polycyclic Aromatic Hydrocarbons ................................... C Polychlorinated Biphenyls ................................... D . Bis-Chloromethyl Ether ............................................. E . Vinyl Chloride .................. F. Nitrosamines ................ ........................... G . Miscellaneous ............... H . Trace Elements . . ............................................. 111 . Metabolism and Monit of Antineoplastic Agents ..................... A . Cyclophosphamide ........................................... B . Nitrosoureas ............................ ......................... C . Purines. Pyrimidines. and Their Nucleosides ......................... D . Daunorubicin and Adriamycin ....................................... E . Antitumor Hormones ................................................ F. Hexamethylmelamine ............................................... G . Platinum Coordination Complexes ....................... H . Miscellaneous ...................................................... I V. Biological Markers ......................................... A . General ................................................... B. Polyamines ......................................................... C . Steroids .................................. ....................... D . Miscellaneous ...................................................... E . Conclusions ........................................................ References ......................................... ........

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Coplright @ 1978 b y Acsdeniir Prrc. Inc . A l l rights of reproduction in ;my fanil reserved . ISBN (I-12-006627-(I

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I. Scope of Applications and Analytical Techniques

A. ADVANTAGESAND LIMITATIONS Computerized GC/MS2 is probably the most comprehensive and versatile of all instrumental methods of analysis. A mass spectrometric analysis provides both unequivocal identification and quantification of the constituent(s) of interest, often in the presence of a host of similarly behaving native biological or artificial chemical materials. Recent developments in MS and computer instrumentation and analytical techniques elevated mass spectrometry to a unique status: high sensitivity and high selectivity are provided simultaneously, and are combined with general applicability and small sample requirements. Virtually any material, organic or inorganic, can be analyzed by MS as long as the molecular weight of the compound, or a suitable derivative of it, is less than about 1200. (Compounds with molecular weight up to 3000 may be analyzed when conditions are favorable). This limitation is imposed b y the requirement that the samples must be volatilized without decomposition, so that a vapor pressure of at least lo-' torr is obtained in the ion source of the MS. Although compounds of low rnolecular weight account for no more than 25% of the normal constituents of a cell, they include the end products of the endogenous metabolic processes in the body, volatile effluents in breath, and most drugs and carcinogens and their metabolites; all elements in the periodic table are also included. An important feature of MS is the small sample requirement: 1-5 p g for identification of an unknown, 100 ng for confirmation of expected identity, and 0.05-5 ng for quantification of known contituents in biological materials. Another advantage of MS is speed. Confirmation of identity and/or quantification can often be accomplished in minutes or hours. Identification of an unknown may, of course, take days or weeks. A disadvantage is that materials cannot be recovered after analysis. When a sample is ionized, ions are formed simultaneously from all constituents present, and the mass spectrum does not readily reveal *Abbreuiations: MS, GC, HPLC, TLC refer to the technique or instrument, depending upon context, of mass spectrometry, gas chromatography, high pressure liquid chromatography, or thin layer chromatography, respectively; G U M S and HPLC/MS refer to the combined techniques or instruments; Ion sources or ionization techniques (Section 1,G); EI = electron (bombardment) ionization, CI = chemical ionization, FD = field desorption ionization; pg, ng, and pg refer to microgram, nanogram, and picogram, respectively.

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whether the original sample was a mixture or a pure compound. Thus, a pure sample must be provided when the complete mass spectrum of an unknown is needed for identification. Samples may be purified by chemical or chromatographic methods and presented for MS analysis in solution; removal of solvents presents no problem. In another approach, a GC or LC is combined with the MS, and the pure sample is provided when the chromatographically separated component of interest arrives on-line into the ion source of the MS. There are four areas in which MS can be applied to problems in cancer research and therapy: (1) confirmation of identity, (2)identification of unknowns, (3) quantification of selected trace constituents, and (4)“profile” surveying, in a single analysis, of a large number of endogenous or drug metabolites, closely related groups of carcinogenic chemicals, or all elements in the periodic table. Mass spectrometry is also an important tool in the elucidation of the structure of complex molecules; however, the technique alone is usually not adequate. For example, NMR, IR, optical rotary dispersion, as well as MS were needed to establish the structure of daunomycin (Arcamone et al., 1968). Montgomery and Struck (1976) synthetized some 80 cyclic and acyclic anlogs of cyclophosphamide, isophosphamide, triphosphamide, and phosphoramide mustard; 19 were exclusively and 40 primarily characterized by mass spectrometry. To confirm the presence of a compound, one needs enough material to obtain a reasonably complete mass spectrum which is compared to that of the authentic compound. Mass spectra of more than 30,000 compounds are available in data libraries, and computerized matching may be made by telephone in minutes. “Unknowns” may be biologically active constituents isolated b y some means, peaks in the gas or liquid chromatograms obtained in profile searching, metabolites of an antineoplastic drug or a carcinogen, or impurities present in the drugs or carcinogens studied. The structure of these compounds is usually known once their identity is established. They may be of significance or they may be artifacts. Unfortunately, it takes just as much work to identify an unimportant constituent as it takes for a significant one. Identification is simple when the mass spectrum obtained is similar to one available in a data library. When the mass spectrum of the compound encountered is not available, the best approach is to determine the exact molecular weight of the molecular ion (accurate mass determination using high-resolution mass spectra) and calculate possible molecular compositions. The use of MS to quantify trace amounts, down to picomole levels of

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selected known components in relatively crude mixtures is based upon a new technique called selected ion monitoring (Section 1,D). In profile analysis, constituents of similar chemical nature are sepa: rated and quantified in a single analysis. Separation is accomplished by GC or LC followed by characterization and quantification using MS. Areas of application include searching for qualitative or quantitative changes in endogenous metabolic constituents caused by the disease (biological markers), or b y an antineoplastic drug or carcinogen, characterization and quantification of sets of isomeric carcinogens, and exploratory surveying of all inorganic elements in a single analysis. After a brief discussion of the basic principle of MS, three areas of application are reviewed: ( 1) identification, quantification, and metabolism of carcinogens, (2) metabolism and monitoring of antineoplastic agents, and ( 3 )searching for biological markers. The literature is covered for the period 1970 to early 1978. No attempt was made for completeness. Instead, a variety of approaches and techniques are described, and applications are illustrated in as many different areas as possible. A list of recommended texts and sources of current information is given at the end of the chapter.

B. NATUREOF MASS SPECTRA The process whereby a neutral molecule or atom becomes electrically charged in the vapor phase is called ionization. There is a minimum amount of energy (6-15 eV) that must be provided to remove an electron from the lowest energy orbital from a molecule. The unbroken molecule which thus becomes a positive ion is called a molecular ion (M+'). When excess energy is provided, it is transformed into vibrational energy by radiationless transitions, followed b y distribution over all integral degrees of freedom. When the vibrational energy concentrated in a particular bond becomes equal to the dissociation energy of that bond (2-5 eV for single bonds), the molecular ion dissociates and a fragment ion is formed. When there is still more excitation energy available, additional cleavages may take place and still smaller fragment ions will form. The fragmentation processes of polyatomic molecules are considered to be a series of competing and consecutive unimolecular reactions, similar to the rate processes characterizing ordinary chemical reactions. Mass spectrometers are ion optical devices which produce a beam of gaseous ions &om an evaporated sample, separate the resulting mix-

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ture of ions according to their mass-to-charge ( d e ) ratios, and provide output signals from which the nominal or exact mass and abundance of each detected species may be determined. The mass spectrum of a compound reveals in a graphical, pictorial, or tabular form the masses and intensities of those ionic species for which output signals were obtained. When the intensities of the masses are normalized with respect to the highest intensity (base peak), the array obtained is called the mass spectral fragmentation pattern. The nature and degree of the fragmentation of an organic molecule greatly depends upon the total energy received during ionization. Depending upon the type of ion source used (Section 1,C) and also upon experimental conditions for any particular ion source, a mass spectrum may contain only one or a few masses, or possibly as many as 100 or more masses. Thus, mass spectra are not well-defined properties of molecules, and there is no such thing as the “right” or “correct” spectrum of a compound. For example, the EI mass spectrum of the methyl derivative of 5fluoro-2’-deoxyuridine (FUdR) is so vastly different from the CI mass spectrum (Fig. 1) that one would not even suspect that both represent the same original compound. The fact that several kinds of mass spectra may be obtained for most materials is not a disadvantage. On the contrary, judicious selection of the technique of ionization and/or experimental conditions greatly expands the art. s of application of mass spectrometry. Although the fragmentation pattern of a compound may change significantly when experimental conditions (e.g., ion source temperature) are changed, the patterns are remarkably constant for a set of given conditions. In addition, most organic compounds are characterized by individual fragmentation patterns (“fingerprints”). These two features form the bases of the use of MS to confirm identity. Mass spectra provide two basic kinds of information: the “masses” of all ionic species detected and their corresponding intensities. “Mass” (mle)refers to mass-to-charge ratio, where mass is measured in atomic mass units (W = 12.00000 by definition) and “charge” refers to the number of positive charges acquired in the course of ionization. Usually one deals only with singly charged positive ions, i.e., e = 1. Negative ion MS, in spite of current advances (Hunt et al., 1977),is of only moderate interest at this time. When masses are determined to an accuracy of 0.5-LOU (atomic mass unit) in a low-resolution MS, the terms nominal or unit mass are used. When the accuracy of mass measurement is 0 . 0 0 1 ~ in high-resolution instruments, the terms exact or precise mass are employed. 8

JOHN ROBOZ

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FIG. 1. (A) Electron impact ionization mass spectrum of trimethyl-5-fluoro-2’deoxyuridine. The molecular ion atmle = 288 is present but of very low abundance. The peak at mle = 113 originates from the fragment of mle = 145 after further loss of -CH,OH. The peak at mle = 87 originates from the breakup of the pyrimidine ring. (B) Chemical ionization (methane as reagent gas) of methylated FUdR. The peak of highest intensity is the quasimolecular ion (M + 1).The small peaks at masses higher than the molecular weight originate from typical association reactions with the reagent gas and can usually be ignored.

c. COMPONENTS OF MASS SPECTROMETER SYSTEMS Most mass spectrometers consist of five functional elements: sample inlet system, ion source, mass analyzer, ion detector and recorder, and vacuum system. Dedicated minicomputers are employed so

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frequently that they soon may be considered integral parts of MS systems.

1. Inlet Systems Solid samples or compounds dissolved in a volatile solvent are placed in a small, one-end-closed capillary which is introduced into the middle of the ion source via a vacuum lock. Here the sample is vaporized by heating the capillary tube. These “direct insertion probes” may be initially cooled to prevent evaporation in the warm ion source before desired operational conditions are attained. The sample may be a pure solid, an isolated peak from GC, HPLC, or TLC, or even a crude biological mixture. Although the direct probe will yield mixed mass spectra when several components with similar vapor pressure are present, selected components may still be detected and/or quantified with selected ion monitoring (Section 1,D). The technique is sensitive: 50 ng material is often adequate to take several mass spectra. GCs are now routinely interfaced with both low- and high-resolution MS of all types. Since GC utilizes a carrier gas (usually He) at atmospheric pressure while MS must operate under vacuum torr in the ion source, in the analyzer) an interface (separator, enricher) is needed to remove the carrier gas without removing the sample being analyzed. In the jet separators, the GC effluent passes a small orifice as an expanding supersonic jet stream. The light helium atoms diffuse to the area around the heated jet and are pumped away, while heavier organic molecules continue on a straight line and enter the ion source. In the effusion-type separators the GC effluent passes cm. Helium passes through a heated glass tube with pores of through the pores and is pumped away while the organic molecules continue on a straight line. In the semipermeable membrane separators the organic materials d i h s e through a thin (0.0025 cm) membrane made of dimethyl-silicone polymer in which the carrier gas has poor solubility. With these separator$ 40-99% of the carrier gas is removed, and usually no more than 10-20% of the sample is lost. When all GC effluent enters the C I source, the same gas may be used as GC carrier and CI reagent gas (see next section), and the interface system, with its inherent problems of plumbing, may be eliminated. Combined HPLCiMS is currently under development; this technique is likely to become a major tool in biomedical analysis (McFadden and Schwartz, 1976; McFadden et al., 1977). Pilot studies for the separation of benzo(a)pyrene and pyrene with direct on-line combination of thin layer chromatography and mass spectrometry were reported by Issaq et al. (1977).

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2. lon Sources Of the 13 distinct methods available for ionization (Milne and Lacey, 1974), four are important in biomedical applications: electron (electron impact, electron bombardment), chemical, field, and spark ionization. The first three are used for the analysis of organic compounds, the last one for the elements. In the EI source the vapors of the sample are bombarded by energetic (70 eV) electrons emanating from a heated rhenium wire. All ions formed are repelled from the area of ionization by a weak positive voltage and leave the ion source via a small exit slit to be accelerated on their way into the mass analyzer. Because the amount of energy available is much more than needed for ion formation, fragment ions form in great abundance. In fact, the M+ ions often cannot he detected because all ions formed undergo additional fragmentation. In the CI source, a large amount of a reagent gas (1torr pressure) is cointroduced with the sample. The reagent gas is ionized with an electron beam in a conventional manner. For example, CH4+,CH3+, etc., ions form when methane is used as the reagent gas. Next, these primary ions enter into secondary ion-molecule reaction with unreacted methane molecules and form secondary ions such as CH5+and CzHs+,and even tertiary products such as C3H5+and C3H,+. These reagent gas ions collide with the neutral molecules of the sample, present only in very small quantity, and ionize them by ion-molecule collisions. If the sample is a good proton acceptor, a proton is added and an (M 1)+ion (quasimolecular ion) forms. Because the ions of the reagent gas do not have much excess energy to transfer, CI yields simple spectra with a high abundance of the (M 1)+ ions and relatively little fragmentation (Fig. 1B). In an FD ion source, the sample is absorbed from a solution onto an activated ion emitter which is a thin wire on which millions of microneedles of carbon are “grown.” In the presence of a strong electrostatic field ( lo8V/cm) created around the wire or a sharp blade, there is a finite probability for electrons to tunnel from the outside orbitals of molecules into unoccupied orbitals of the metal. The field desorption takes place on the multiple fine edges of the whiskers which also serve as sample reservoir. Since little energy is transferred, the spectra contain an abundance of M+‘ and (M 1)+or (M - l)+(hydrogen abstraction) ions. In the radiofrequency spark source, pulses of an ac potential of 20-100 kV are generated for a few microseconds between two electrodes formed by the sample material. Sparks may be made one at a time, or at a repetition rate as high 10“ per second. In the electrical

+

+

+

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discharge the sample material evaporates and a great abundance of ions of all types is formed. Most elements in the periodic table can be analyzed with similar sensitivity in a single analysis. Response is linear with concentration, and samples as small as a few micrograms may be analyzed. Nonconducting samples, such as water, are deposited onto a conducting electrode material of ultrahigh purity.

3. Mass Analyzers The objectives of mass analyzers are: first, to resolve an ion beam of mass m &om another beam of nearly the same mass m + Am (dispersive or prism action) and second, to maximize the resolved ion intensities (focusing or lens action). Most mass analyzers are based on either magnetic deflection or quadrupole filtration of ions. In magnetic analyzers, ions traveling through a magnetic field experience a force that is perpendicular to both the magnetic field and their direction of travel. The radius of curvature of the path of an ion depends on its mle value, the voltage through which the ion was accelerated prior to entering the magnetic field, and the strength of the magnetic field. For example, in a 90” sector magnetic field lighter ions describe a shorter path than heavier ions (Fig. 2). Under given conditions of accelerating voltage and magnetic field strength, only ions of a particular mle value will hit the electrical detector placed in a preselected place; all other ions will hit the walls. For successive focusing of ions of smaller or larger mle on the collector, the accelerating voltage or the magnetic field strength must be “scanned.” Singlefocusing mass analyzers focus ions homogeneous as to their mass and velocity but of slightly different initial directions. These analyzers have a quadratic mass scale due to the square functions of both magnetic field strength and radii of curvature. Single-focusing magnetic mass analyzers are normally used, alone or in GUMS combinations, to determine nominal masses. In double-focusing instruments an electrostatic field is added to the magnetic field to provide velocity focusing, i.e., focusing ions homogeneous as to their mass and initial direction but of different initial velocity, in addition to direction focusing. Of course, the magnetic field will also provide mass dispersion. There are several ways to combine electrostatic and magnetic fields. In the Mattauch-Herzog design an electrostatic field with a rotation angle of 30” is combined with a 90” magnetic field. When both magnetic field strength and acceleration voltage are kept constant (no scanning), all ions describe a different path length and are detected simultaneously on an ionsensitive photoplate. In another popular double-focusing arrangement

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FIG. 2. Schematic drawing of 90" sector field magnetic mass spectrometer. (HVP = high vacuum pumping line; EID = electron impact ionization detector used for the recording of a total ion chromatogram which resembles gas chromatograms obtained with flame ionization detectors; SEM = secondary electron multiplier ion detector.) (Courtesy of Varian Associates.)

(Nier-Johnson geometry), a 90" electrostatic analyzer is followed by a 60" magnetic analyzer; only electrical detection with scanning is available. Double-focusing instruments are used for exact mass determinations either when an unknown must be identified or when interferences of nearly identical masses must be resolved. In the quadrupole mass filters (Fig. 3) the accelerated ions enter along the long axis of four precisely aligned parallel cylindrical rods. A programmed combination of radiofrequency (rf) and dc voltages is applied to diagonal pairs of the electrodes. At any given combination of the fields, only ions of one particular mle value can maintain a stable trajectory and reach the detector at the other end; all other ions have unstable trajectories and eventually hit one of the rods. Mass scanning is by varying the dc voltage and the rf voltage in unison to maintain a constant ratio. The mass scale of quadrupole analyzers is linear and they provide only nominal masses. Quadrupole instruments have become the choice for many applications because of their adaptability

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FIG.3. Schematics of the operation of a quadrupole type mass spectrometer.(Courtesy of Finnigan Corporation.)

for combinations with GC, and recently LC, and relative ease of computerization. The most important measure of performance of an MS is resolution. Resolution of 600 means, for practical purposes, that masses of 600 and 601 can be distinguished. Low resolution instruments, including combined G U M S systems of both magnetic and quadrupole type, provide resolution of 600-2500. Double-focusing high-resolution magnetic instruments offer resolution in the 10,000-100,000 range. The importance of high resolution lies in the ability to separate ions of the same nominal mass that have differing elemental compositions and therefore different exact masses resulting from the packing fraction of the monoisotopic atomic weights of the elements making u p the compositions of the ion. When masses are determined to an accuracy of 0.003 u or better, unequivocal empirical formulas may be calculated. 4. Ion Current Detectors and Recorders Most MS systems employ secondary electron multipliers to detect and amplify the separated ion currents. Separated ion beams emerging from the mass analyzer are accelerated to 2-5 kV and impinge on a

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“conversion dynode” from which secondary electrons are released (Fig. 2). These, in turn, are focused on a second dynode from which the number of electrons released is greater than the number impinging upon it. In a 20-stage multiplier structure the amplification is about 10’. The final collector is connected to a high-speed electrometer the output of which is fed into a high-speed oscillographic recorder, an oscilloscope, a magnetic tape recorder, or a dedicated minicomputer, Ion currents measured are normally in the 10-8-10-16 A range, but even the arrival of 1 iodsec (1.6 x A) may be measured. Ion-sensitive photoplates are composite collector-transducer detectors. Exact masses are determined from the position of the lines in comparison with coanalyzed mass standards (perfluorokerosene) with the aid of a microdensitometer. Ion intensities are determined from the blackness of the lines.

5. Computerization Dedicated minicomputers, with continually increasing perfonnancelcost ratios, are becoming the rule rather than the exception for the acquisition and handing of data. They maximize the productivity of both tfie instrument system and operator. In interactive data systems, computers control the operation of both the MS and GC, acquire all data regardless of their significance, display data during acquisition so that immediate decisions may be made to change operational conditions if needed, and permit interrogation of all data at the convenience of the operator so that unwanted data can be discarded but useful data will not be missed (Roboz, 1977). As data processors, computers calculate peak intensities, background corrections, nominal and exact masses, chromatographic retention times, peak areas, and concentrations. They normalize spectra in several ways so that highspeed comparisons with libraries becomes possible, calculate possible molecular compositions corresponding to a determined exact mass value, and have many other uses of convenience.

D. ANALYTICALTECHNIQUES 1. Nominal and Exact Mass Measurements For confirmation of identity, a reIatively complete mass spectrum determined to an accuracy of 0.5-1.0 mass unit is obtained and compared to spectra in a data library (Milne and Heller, 1976). For quan-

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tification at trace levels, nominal masses of only one or two selected ions are monitored (see below). Masses determined to an accuracy of 0.001 mass unit are used for the identification of unknowns and in cases in which interfering constituents of nearly identical mass cannot be resolved chromatographically. Nominal or exact mass measurement techniques may be used in straight mass Spectrometry with all types of ion sources and in GUMS.

2. Selection of Zonization Technique The mode of ion production determines if the ions will indeed be representative of the sample, and also if the mass spectra will provide the kind of information desired. Current tendency is for combined operation of sources, e.g., EI/CI or EI/FD (Gierlich et al., 1974).When structural information is desired, EI spectra is the first choice because of the considerable fragmentation, particularly involving the skeleton of the compound. Thousands of spectra are available in libraries and much is known about correlations between structure and spectra; one disadvantage is that the molecular ion is often not detectable. The main advantage of CI is the great abundance of the protonated ions of the intact molecule. C I provides high sensitivity and specificity for assaying known compounds. There are also additional advantages such as the possibility of using different reagent gases to induce various chemical reactions in the ion source and the possibility of eliminating interfacing in GUMS b y using the same gas for GC carrier and reagent gas. Both E I and C I require the samples to be vaporized, thus derivatization is frequently needed. FD spectra provide little formation on structure. The M+' or (M 1)+ ions are abundant, derivatization is not required, samples may be applied in aqueous solution, polar and thermally unstable materials may be studied, and the molecular weight limit may be extended to several thousands. The disadvantages are the experimental difficulties with biological materials; current methodology is not adequate for quantification. After technological developments this technique is expected to assume major importance (Schulten, 1977). For the survey analysis of elements in multicomponent systems the obvious choice is spark source mass spectrometry.

+

3. Selected Zon Monitoring When the mass spectrum is scanned in conventional MS, most of the scan time is spent recording the intensities of masses containing little or no useful information. For example, if the mass range 100-600 is

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scanned in 5 seconds, only 0.01 second is spent on each mass. In selected ion monitoring (mass fragmentography, multiple ion detection), intensities or one, two, or more preselected masses are monitored simultaneously or in rapid repetitive succession. For example, the (M 1)’ ions in the chemical ionization mass spectra of 5-fluorouracil and an internal standard (6-mercaptopurine) are monitored to assay the former in blood (Section 11,C). The use of this technique is obvious when “gentle” ionization techniques, such as chemical ionization, is used. I n this technique the MS is used as a specific detector. It may be made even more specific if not the nominal but the exact mass of the compound of interest is monitored (Section 11,D). The technique is eminently suited for analysis of partially purified biological samples. In addition to specificity, the technique is about 103 times more sensitive than conventional G U M S or GC with flame ionization detection. A mass fragmentogram is similar in appearance to a gas chromatogram, except that there are several traces, each corresponding to the monitoring of a preselected mass. Identity is confirmed by checking GC retention times and the known relative intensities of the preselected peaks. For quantification, peak areas of the compound and internal standard are integrated. Stable-isotope enriched internal standard may be used to eliminate inaccuracies due to losses in sample preparation or on the GC column. Selected ion monitoring is perhaps the single most useful analytical technique currently available for trace quantification. It is sensitive (pmole quantities), specific, and may be employed to virtually all materials. Many applications are reviewed in later sections.

+

4. Stable Isotope Dilution In stable isotope dilution the concentration of a component in a matrix is determined horn the change produced in its natural isotopic composition by the addition of a known quantity of the same component the isotopic composition of which has been changed b y incorporation of stable isotopes. A potentially important use of stable isotopes is the administration of labeled, but not radioactive, drugs to humans followed by a search for metabolites in wiwo. When the degree of labeling with 15N, 13C, or 2H is so high that the Mf’ and (M 1)+ions are of comparable abundance, both the molecular and several fragment ions of the metabolites will appear as “twin ions” with characteristic intensities. A search for such ion pairs will reveal metabolites. When selected ion monitoring is used in searching for doublets, the isotopic method actually augments specificity.

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It. Identification, Quantification, and Metabolism of Carcinogens

A. SURVEYANALYSES 1. General Of some 6000 compounds tested for carcinogenic activity, only 1000 showed positive response (Ember, 1975). The 9 published volumes of the continuing series “Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man” (IARC, 1972-1975) are an indispensable source of information on the carcinogenicity of chemicals. Of the 222 chemicals evaluated in detail, 111 are unquestionably carcinogenic to experimental animals and 19 to man. Occupational exposure is known for 95 compounds, medicinical exposure for 29, and general environmental exposure for 52, including 15 polycyclic aromatic hydrocarbons. While it is clear that these carcinogens are hazardous when one is exposed to them as an “occupational hazard,” there is virtually nothing known about the hazards of long-term exposure at trace or ultratrace levels as “environmental hazard.” In “survey analysis” the main objective i s to detect, identify, and perhaps quantify as many constituents as possible so that their potential health hazards may be evaluated. Often no particular class of compounds is surveyed. Instead, such physical properties as volatility or polarity determine what kind of compound will be analyzed. G U M S has become the method of choice for survey analysis since dozens or possibly hundreds of compounds may be detected and identified in a single collected sample. In what follows, a few representative techniques are reviewed for surveying water and air. In the rest of this section, advances are discussed on those carcinogens known to be hazardous to man, and examples are shown for the study of the metabolism and mode of action of some carcinogens. In two outstanding general reviews on the environmental applications of MS, Alford (1975b, 1977) lists 668 references. In some publications reviewed, hundreds of pollutants were identified, in others only one or a few. In another fine review, Oswald et al. (1974) d‘iscusses factors affecting methodology as well as several compound classes. These reviews provide an overview of the all-important sampling techniques, analytical methodology developments, and applications in virtually all areas of environmental research, but contain relatively few references specifically dealing with carcinogens.

2. Water In a review of the analysis of organic compounds in water to support health effects studies, Garrison ( 1976) quotes some interesting statis-

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tics. To date, 1296 compounds have been identified in water and more than 90% were identified by combined GC-MS. About one-half of these were found only once, the majority less than five times. All these compounds are volatile or were made volatile by derivatization. It is estimated that 95% of all organic contaminants in water are nonvolatile. Progress in combined LC/MS will soon permit the systematic identification of nonvolatiles so that their possible health hazards may be evaluated. A comprehensive review of techniques for the analysis of organic pollutants in water together with applications to waters of various origin was published in a book (Keith, 1976a). A detailed description of sampling, separating, and identifying hundreds of organic substances in potable water (Grob, 1973) and the application of the technique to waters in the vicinity of Zurich, Switzerland (Grob and Grob, 1974) is typical of the many publications available in this field. Average sensitivity is 1 part in lOI3 (w/w). Once pollutants are identified, the next step is to trace the orgin of the pollution or, when that is known, trace the efficiency of various cleanup treatments. An instructive example is the identification and quantification of organic compounds in unbleached, treated kraft paper mill wastewaters after various treatments (Keith, 197613). Most analytical techniques employ charcoal, macroreticular resins, polyurethane foams, or solvent extraction for the collection and concentration of organics (Webb, 1975). Direct injection of aqueous samples into GClMS has obvious limitations; however, it is a fast and convenient tool, particularly for highly volatile constituents which might be missed in conventional techniques (Harris et al., 1974).

3. Air The general survey analysis of pollutants of air has received much attention in recent years and dozens of publications appeared suggesting techniques to improve the efficiency of sample collection, to increase resolution of constituents, and to provide specificity. Grob and Grob (1971) used cigarette-filter charcoal to trap volatile organics in the air of Zurich, Switzerland. After extraction with carbon disulfide, components were separated by capillary column GC and identified by MS. Over 100 components were identified, most of them aliphatic and aromatic hydrocarbons. Ciccioli et al. (1976) compared Tenax GC and Carbopack B in terms of sample recovery in “personal” samplers and found their performance comparable in many respects. Versino et al. (1976) collected organic micropollutants on a porous polymer glass absorption column and thermally eluted them with the aid of helium

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into a GUMS. In still another approach to sampling, Pellizzari et al. (1976) collected pollutants on a bed of Tenax GC, thermally eluted the vapors into a capillary trap cooled to -196"C, metered the samples into a capillary column for separation followed by MS identification. Typically, 80 to 150 compounds were found in air samples and several carcinogenic compounds were detected. A problem in cryogenic preconcentration of air samples is the presence of water. Swofford et al. (1976) exploited the presence of water and used it as the reagent gas in the CI mode. Lao et al. (1976a) and Thomas and Lao (1976, 1977) developed MS methodology for the detection and quantification of at least 16 known carcinogens which may be present in both urban and industrial atmospheres. These are: 4,4'-dinitrobiphenyl, 6-9-dimethyl- and 5,7-dimethyl benzacridine, 3,4-benzacridine, 2-aminochrysene, 6-aminocry6,12-diazaanthrene, 2-azafluoranthrene, sene, 1,2,7,8-dibenzcarbazole, ISazafluoranthrene, 1-azapyrene, 2-aminofluorene, 2-aminoanthracene, 4-aminofluoranthrene, and Saminopyrene.

B. POLYCYCLIC AROMATIC HYDROCARBONS

1. Isolation, Fractionation, Analysis Since polycyclic aromatic hydrocarbons (PAH) are usually present in environmental samples only as traces, and also accompanied by a variety of other extractable materials, isolation and fractionation of varying sophistication are needed. Furthermore, since individual polycyclic hydrocarbons vary considerably in their toxicities and even more in their carcinogenic properties, identification and quantification of specific compounds must be made in the presence of several compounds of very similar composition. For example, chrysene and the 1-,2-,4-, and 6-methylchrysenes exhibit moderate carcinogenicity, whereas the 5-methyl (also 3-methyl) chrysenes are strong tumor initiators. Caustland et al. (1976) synthetized many derivatives of benzo[alanthracene, dibenz[a,h]anthacene, 3-methylcholanthrene, and 7,12dimethylbenz[a]anthracene. The E I mass spectra of these compounds exhibit features characteristic of aromatic compounds, such as relatively intense molecular ions, abundant doubly charged ions, and other similarities. The phenols show M - 1,M - 28, and M - 31 peaks in addition to the intense and characteristic M - 29fragment. The epoxides exhibit small but characteristic M - 16 ions in addition to those ions common in phenols. The ortho-quinones of K-region have their

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most intense peak (base peak) at M - CO with less intense molecular ion, while with the non-K-region quinones just the opposite is true. PAH can also be analyzed by CI (Janini et al., 1976b). As expected CI spectra are simple; however, because the molecular ions of PAH are very abundant even in the EI mode, CI does not offer significant increase in sensitivity. Still another approach is to utilize EI at low ionization voltage (Blumer, 1975). Here the E I ion source is operated at 12 eV, at which point there is enough energy to form the molecular ions only and there is virtually no fragmentation. This is an old technique in the petroleum industry; however, sensitivity is rather poor. Inspection of the mass spectra of PAH reveals that MS alone does not readily distinguish between isomeric PAH because very few fragment ions form and the molecular ions tends to dominate the spectra regardless of the method of ionization employed. Thus, to analyze mixtures of isomeric ring systems or to distinguish among alkyl isomers, components must first be separated b y GC or’HPLC. Lao e t al. (1973) developed a GC method, using packed Dexsil-300 columns, for the separation and subsequent MS identification of some 70 PAH (from 3 to 7 rings), present in nanogram quantities. In subsequent developments, Lao et aE. (1976b) utilized Dexsil-400 and Dexsil-410 packing materials and succeeded in separating benz [alanthracene, a carcinogen, from chrysene and triphenylene. These components usually appear unresolved in gas chromatograms. Because benz[a]anthracene is often used as an “indicator” of PAH in environmental samples, this separation is an important advance in methodology. The same columns also separate benzo[a]pyrene and benzo[e]pyrene. In another approach, Lee et al. (1976a,b) used prior LC separation, followed by capillary column G U M S (Novotny et al., 1974) to resolve toxicologically important alkyl derivatives. The initial separation step by HPLC is particularly important in the analysis of airborne particulates which have relatively low concentrations of alkylated PAH with respect to the parent compounds. This technique was utilized to identify some 150 PAH in smoke condensates of tobacco and marijuana, and some 100 PAH in airborne particulates. I n a novel approach, Janini et aE. (1975, 1976a,b) utilized nematic liquid crystals to separate PAH prior to MS identification and quantification. Nematic crystals are unique compounds which do not melt sharply into a disordered liquid upon heating. Instead, at well-defined temperatures they pass through one or more intermediate stages in which some structural order still exists. For example, N,”-bis(p-methoxybenzy1idene)-cup’-bi-p-toluidine (BMBT) has a nematic phase ( 181”-320“C range) where the molecules are oriented with their long

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axes parallel. These liquid crystal will attract more, and retain longer, those structural isomers which are elongated than isomers which are more compact in shape. Accordingly, the order of elution is not in order of boiling points. With the development of two improved liquid crystals (Janini et aE., 1976b), N,N’-bis(p-phenylbenzylidene)a,a’-bip-toluidine (BPhBT) and N,”-bis(p-hexyloxybenzylidene)a,a’-bip-toulidine (BHxBT), several previously impossible separations were accomplished (Figs. 4,5, and 6 ) . Separation on liquid crystals is followed by analysis with CIiMS operated in the selected ion monitoring mode. For example, the base peak (highest intensity) in the mass spectra of all components shown in Fig. 4 is the same (mle 252); without the GC separation these compounds could not be distinguished by MS. Using this technique, as little as 4 ng ofbenzo[a]pyrene could be detected and quantified. 2. Air

A11 techniques reviewed in the previous section have been applied to analyze PAH in air at various locations. The references given contain numerous examples of survey analysis of PAH, including the detection of carcinogenic PAH. 3. Tobacco Smoke The widely publicized health effects of cigarette smoking have prompted numerous investigations concerning carcinogenic materials in tobacco smoke. These studies can be divided into three major areas:

I

5

7 9 Time (minutes)

11

FIG.4. Total ion chromatogram of 20-carbonpentacyclic arenes on liquid crystal phase (BPhBT) column at 275°C. (Reprinted from Janini et al., 197613, with permission.)

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FIG. 5. Total ion chromatogram of five 22-carbon arenes on liquid crystal phase (BPhBT) column at 275°C. Compounds in order of elution: 1,2,3,4-dibenzanthracene, 1,lZ-benzoperylene, 1,2,5,6-dibenzanthracene, 1,2,7,8-dibenzophenanthrene (picene), and 2,3,6,7-dibenzanthracene (pentacene). Reprinted from Janini et d., 1976b with permission.

In

I

1

0

b

8

12

16

20

24

28

Time (minutes)

FIG.6. Chromatogram of five 24 = carbon arenes on BPhBT liquid crystal column at 290°C. Compounds in order of elution: 4,5,6,7-dibenzopyrene, 4,5,7,8dibenzopyrene, coronene, 2,3,6,7-dibenzopyrene, and 1,2,6,7-dibenzopyrene. Reprinted from Janini et d., 197613 with permission.

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(a) problems in sampling and fractionation, (b) identifying tumorpromoting subfractions, and (c) identifying constituents. Collection techniques differ according to the objectives: In cigarette smoking PAH are inhaled in undiluted form, whereas in indoor pollution PAH are inhaled in highly diluted form. Among the many subfractions isolated, the neutral and weakly acidic fractions appear to contain most carcinogens (Severson et at., 1976). Tumor promoting subfractions are identified by bioassay. When individual identifications are needed, combined GC-MS is used. Hechtet al. (1975)obtained various subfractions of the weakly acidic fraction of cigarette smoke particulate matter, tested them for carcinogenicity, and identified by GUMS a variety of compounds in the active subfractions ranging from fatty acids to PAH. Holzer et al. (1976) identified 133 components in cigarette smoke, including many PAH. Hecht et al. (1978b)studied the reaction of nicotine and sodium nitrate: several nitrosamines and fragmentation products of the pyrrolidine ring were identified by MS. The methodology for analyzing PAH in smoke is now available, and more than 500 PAH, ranging from indene to dimethylcoronene, have been identified (Severson et al., 1976). These techniques may now be applied to the various types of tobaccos and cigarette preparations. For example, air-cured tobacco contains about one-half as much PAH than flue-cured tobacco, and the former is significantly less carcinogenic (Brunnemann and Hoffmann, 1976).

4. Carbon Black Recent suggestions connecting excess stomach cancer among workers in the tire industry focused attention to identify and quantify carcinogenic PAH in carbon blacks. Gold (1975) separated CH,Cl,extracted oil furnace black into neutral and hydrocarbon fractions. The latter was further fractionated, yielding a concentrated PAH fraction in which at least a dozen compounds, including naphthalene, acenaphthalene, pyrene, and benzo[g,h,i]fluoranthene, were identified. One peak, present in considerable quantity, could not be identified by low resolution MS or UV analysis. Precise mass measurement yielded the formula C18Hlowhich agreed with that of a previously isolated but not yet characterized carcinogen. Additional work resulted in the identification of the carcinogen as 3,4-dihydrocyclopenta[c,d]pyrene. Lee and Hites (1976) and Hase et al. (1976) utilized capillary column GUMS to identify some 28 compounds in carbon black. Seven were sulfur-containing polycyclics, including dibenzothiophene, benzo[a]dibenzothiophene, benzo[d,efldibenzothiophene, and ben-

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zo[d,efJnaphtobenzothiophene.There is evidence that substitution of a sulfur for an ethylene group in a ring may increase or decrease the carcinogenic potency of the particular compound. Thus, the methodology developed should be applicable to future work concerning the carcinogenic components of carbon black, and also air particulate matter in which sulfur-containing carcinogens have also been detected (Lee et al., 197613). Soot. Some 40 polycyclic aromatic hydrocarbons (concs. > l%), including benzo[a]pyrene (0.2%), pyrene, fluoroanthene, benz[a]anthracene, and phenanthrene, were identified in soot of an A 1 electrolysis furnace (Tausch and Stehlik, 1977).

5. Metabolism of Benzo [alpyrene Polycyclic aromatic hydrocarbons are metabolized into organic solvent-soluble phenols, trans-dihydrodiols, quinones, and watersoluble glutathione conjugates. Both the detoxification and metabolic activation to toxic derivatives or to active carcinogens are catalyzed by aryl hydrocarbon hydroxylase which is a microsomal enzyme system containing several P450 cytochrome enzymes. Because of its ubiquitous appearance, benzo[a]pyrene (BP), has been used as the prototype for the PAH. Gelboin and co-workers (Selkirk et al., 1974a,b, 1975; Yang and Gelboin, 1976) studied the metabolism of BP and utilized MS, TLC, and UV to identify metabolites. Benzo[a]pyrene was incubated with rat liver microsomes prepared from rats treated with methylcholanthrene. After stopping the reaction with acetone, the mixture was extracted with ethyl acetate, the solvent-soluble metabolites concentrated by evaporation to dryness, dissolved in a few drops of methanol, and metabolites were separated 700

65

50

75

100

12s

FRACTION

FIG. 7 . High pressure liquid chromatographic profile of the metabolites of benzo[ulpyrene (BP) after incubation with liver microsomes prepared from Bmethylcholanthrene treated rats. Fractions of 0.2 ml were collected at 20-second intervals. Q = quinone, OH = hydroxy. Metabolites were identified by mass spectrometry. Reprinted from Selkirk et ul., 1974b with permission.

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using HPLC (Fig. 7). Next, the HPLC peaks were collected and analyzed individually in a MS using the solid probe introduction system. Among the eight metabolites detected and identified, there were three dihydrodiols(9,lO-, 7,8-, and 4,5-dihydrodihydroxybenzo[a]pyrene), three quinones (benzo[u]pyrene-1,6-dione,and -6,12-dione), and two phenols ( 8 ,and 3-hydroxybenzo[a]pyrene).The three dihydrodihydroxy metabolites have the same molecular weight so that MS determination of the molecular weight alone, even by high-resolution MS, would not be adequate for characterization. Although several fragmentation peaks are the same in all three compounds, e.g., loss of water, loss of water plus a CHO fragment, and the doubly charged ion, there are different fragmentations that permit differC2H2in the 9, 10-diol, loss of HOH CO entiation: loss of HOH in the 7,8-diol, and the absence of both these ions in the 4,Sdiol metabolite. This is a good illustration of how fragmentation by E I may be utilized for identification and characterization. The areas of the HPLC peaks were utilized to quantify the various metabolites, and also to study how enzyme inhibitors affect the formation of each metabolite. For example, the inhibitors 7,8-benzoflavone and 1,2-epoxy-3,3,3-trichloropropane selectively affect the hydrase rather than the oxidase activity of the enzyme system resulting in both qualitative and quantitative effects on several metabolites (Selkirk et al., 1974a,b). In a subsequent study (Selkirk et al., 1975), the pH of the eluting solvent in the HPLC analysis was changed resulting in the detection of still another metabolite in the presence of the epoxide hydrase The unknown was ideninhibitor, 1,2-epoxy-3,3,3-trichloropropane. tified by MS as benzo[a]pyrene-4,5-epoxide.This identification provides considerable support to the suggested role of epoxides in the intermediary metabolism of BP. In still further studies by the same group (Yang et al., 1976; Yang and Gelboin, 1976) on BP diol-epoxides by nonenzymatic reduction, trihydroxypentahydrobenzo[alpyrenes were detected and characterized by obtaining the mass spectra of the isolated HPLC fractions. The activity of epoxide hydratase toward epoxides of different PAH, including benzo[a]pyrene-4,5-oxide,benzo[a]anthracene-5,6-oxide, and 3-methylcholanthrene-11, E-oxide, can be determined with specificity and sensitivity (1 ngiml incubation mixture) by GCiMS quantification (in selected ion monitoring mode) of the TMS derivatives of the corresponding trans-diols formed during incubation (Bettencourt et al., 1977).

+

+

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C. POLYCHLORINATED BIPHENYLS There are 210 possible polychlorinated biphenyls (PCBs) and many are present in commercial preparations. Although PCBs are not known to be carcinogenic to humans, they induce pathological changes upon long-range exposure, are highly toxic, even lethal, when high concentrations are inhaled, and several of them cause cancer in experimental animals. Since PCBs usually occur together with common chlorinated insecticides and related compounds which have very similar chromatographic retention times, GUMS is essential for separation and positive identification in environmental samples. The chromatographic (and also biological) aspects of PCB have been reviewed by Fischbein

(1972). The EI fragmentation of PCB was discussed by Safe and Hutzinger (1972),while the use of isotopic abundance ratios in the identification of PCB compounds in the presence of related substances was developed by Rote and Morris (1973).These two papers, together with a text on the MS of pesticides and related pollutants (Safe and Hutzinger, 1973), provide basic information needed for the identification of individual PCBs even in complex mixtures. To increase sensitivity, so-called ion abundance chromatograms may be used (Eichelberger et al., 1974). Starting with an idealized set of mass spectra for PCB, these authors evaluated several sets of candidate masses for monitoring PCB in the presence of those pesticides likely to be in the solvent extract of environmental samples. Quantification is possible using area measurements on the ion abundance chromatograms obtained in subset mass scanning. An alternative approach is to employ capillary column chromatography for high-resolution separation followed by MS identification, either on the basis of complete mass spectra for characterization or selected ion monitoring quantification at highest sensitivity (Schulte and Acker, 1974). In recent years there has been an abundance of publications on the metabolism of PCB in humans and experimental animals. Jensen et al. (1974) studied PCB in human adipose tissues and concluded that two adjacent unsubstituted carbon atoms are required for rapid metabolism. Curley et al. (1975) identified tetrachlorodibenzofuran in the urine of rats following dietary exposure to Aroclor 1254. Burse et al. (1976) identified (or at least characterized) six hydroxylated biphenyl metabolites in rats following prolonged dietary exposure to Archlor 1242 and 1016. In both studies, the metabolites were ex-

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tracted from urine with organic solvents, purified by some form of chromatography, methylated with diazomethane, and analyzed by GC/M S . In a different approach, Safe et al. (1975) utilized high-resolution MS with ion-sensitive photoplate detection to detect and identify several oxygenated metabolites of PCB in crude goat urine extract (also in other biological materials, see references in Safe et al., 1975). This technique utilizes the fact that chlorine-containing xenobiotics exhibit a large negative mass deficiency, while natural compounds containing C,N,O, and H have positive mass deficiency. When molecular formulas are determined with a precision of a few millimass units, oxygenated pentachloro-, tetrachloro-, and monochloro-biphenyl metabolites can easily be identified in crude mixtures,

D. BiS-CHLOROMETHYL ETHER

A high-resolution MS method for the detection of bis-chloromethyl ether (BCME) in air with a detection limit of 0.1 ppb was developed by Collier (1972). A 15,000-fold increase in concentration was provided when 15 liters of air was forced through Poropak Q (bead form) adsorber at a rate of 1.5 liter/min. Next, the adsorber was attached to the inlet system of the MS, the system pumped to lo+ torr, and the adsorbed organic components eluted into a reservoir b y heating (18OOC for 5 minutes). The collected sample was leaked into the ion source and the narrow mass range of 78.9-79.1 scanned (EI)at a resolving power of 1/3500. After mass calibration, the most abundant ion, at mle = 78.9950 (C1CH2-O-CH2)was quantified. The high resolution used provided separation from most of the ions that might be present from other contaminants, e.g., CH,SiCl+, CH3S2+,C2HSN+,and C,H,+. Two possible contaminants were not resolved: CH,O,P+ (mfe= 78.9949) would require a resolving power of 1/79,000; however, this ion does not contain chlorine so evaluation of BCME may be made on the basis of the chlorine-containing isotope at mle = 80.9921. The other possible unresolved interference is the C,HClF+ ion (mle = 78.9751), which may be problematic in the absence of BCME. When this is suspected, a small amount of BCME is added to see if the two peaks are identical or not. An alternative technique (Shadoff et al., 1973) utilized lowresolution GC/MS, which is fast and convenient to separate all compounds present so there was no need for high resolution to surmount interferences. Simultaneous monitoring of the mle = 79 peak (base

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JOHN ROBOZ

peak) and also the mle = 81 peak (chlorine isotope peak) provided selectivity combined with high sensitivity (fractional ppb level, accuracy within 10%). The advantages of both of these techniques were combined by Evans et al. (1975) who first passed the desorbed gas mixture through a GC and then monitored the C,H,OCl+ peak at high resolution (resolving power 3800) so that single ion monitoring at high resolution was achieved and the highest degree of specificity maintained (Fig. 8). With this method BCME is detectable to 0.01 ppb (10-liter air sample). The concern over the carcinogenicity of BCME increased considerably when Frankel et al. (1974) reported that it could be formed spontaneously in air or solution from hydrochloric acid and formaldehyde. The gas-phase reactions were carried out either in glass flasks or Saran bags under controlled conditions and BCME was both identified and quantified by high-resolution MS or GUMS. Reactant concentrations strongly affected BCME yield, while temperature or humidity were less important variables. Although the amount of BCME formed was only about 0.01 mol%, the strong carcinogenicity of BCME requires alertness about this hazardous chemical. Since BCME is an impurity in chlormethyl methyl ether (CMME), a commonly employed chlormethylating agent, a technique was developed (Tou and Kallos, 1974) to study the kinetics of the decomposition of both compounds in humid air. Both the nature of the reactor surface (e.g., glass, Teflon, etc.) and experimental conditions (e.g., humidity, temperature) were varied and preselected fragment ions were monitored for each compound. The upper limit of the rate of hydrolysis was only 0.00047 per minute for BCME at 2.6% water concentration; thus routine monitoring presents no problem.

FIG.8. Appearance of the mle = 79 region of the mass spectrum at resolving power 3800 in the analysis of bis-chloromethyl ether. The ions at mle = 78.9950 correspond to (ClC2H40)+.Reprinted from Evans et al., 1975, with permission.

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Two homologs of BCME, bis-chloroethyl ether and bis-chloroisopropyl ether, are suspected carcinogens and have been found in the environment. These compounds may be analyzed in water, after extraction with petroleum ether and concentration by evaporation, b y utilizing mass fragments formed by the loss of the CH&1 group (M+ - 49). Selection of these ions assures specificity and a detection limit of 10 ppb (Bonelli et al., 1975).

E. VINYLCHLORIDE Recent concern about the carcinogenic effects of vinyl chloride prompted analytical activities in three areas: (a) detection in various environments, (b) routine monitoring of the level of vinyl chloride, and (c) understanding action by exploring the metabolism. For the positive identification of vinyl chloride in various environments, MS is the ideal tool because of both high specificity and sensitivity. For example, Pelizzari et al. (1976) obtained profiles of organic vapor pollutants in the atmosphere in the Houston and Los Angeles areas. Some 120 peaks were detected in the total ion current chromatogram of the thermally desorbed (from Tenax cartridges) vapors after separation in a capillary column in GUMS. Vinyl chloride was present in the atmosphere in the Houston area but not in the area sampled near Los Angeles. Incidentally, a total of 21 halogenated hydrocarbons were detected, including chloroform, carbon tetrachloride, and the strongly carcinogenic trichloroethylene. For routine monitoring, as required by the Occupational Safety and Health Administration, the problems of sample collection for analysis by GC (flame ionization) were investigated (Ahlstrom et al., 1975; Levine et al., 1975). Vinyl chloride can be analyzed with adequate sensitivity and reproducibility by monitoring the mle = 62 peak (molecular ion) by GUMS (Bonelli et al., 1975; Taylor and Springer,

1975). There are three GC/MS techniques for the analysis of vinyl chloride in water (Alford, 1975a). When 2 pl of water is injected directly, detection limit is 0.05 mg/liter. For lower levels, extraction with CCl, is needed and the limit of detection is 2 pg/liter (0.14 ng injected). The vinyl chloride content of wastewater from a plastics manufacturing plant was in the 0.2-9.3 mg/liter range. Using simultaneous recording of the mle = 62 and mle = 64 peaks, and direct injection of 1ml water, Fujii (1977) was able to detect 0.1 ppb of vinyl chloride. Because proposed limits ofvinyl chloride in both food and drinks are at the ppb level, VanLierop and Stek (1976) used selected ion monitoring and

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integrated the areas for quantification. As little as 0.3 ppb of vinyl chloride was determined in salad oil. Preliminary work on the metabolism of vinyl chloride suggested that, similar to aromatic hydrocarbons, epoxidation may be responsible for the formation of the carcinogenic metabolite. The suspected epoxide, chloroethyleneoxide, in the pure state slowly rearranges to chloroacetaldehyde (ClCH,CHO). Gothe et al. (1974) used prepared rat liver homogenate in a conventional manner but, in addition to adding the necessary cofactors for the mixed-function exygenase system, 3,4-dichlorobenzenethiol(a nucleophile) was also added to trap metabolites. A mixture of air and vinyl chloride was exposed to the fortified liver homogenate for 2 hours, the reaction mixture extracted with hexane, the extract divided into bicarbonate soluble (acid) and neutral fractions, and both fractions were further fractionated by TLC. The zones of the postulated metabolites (determined with authenic compounds) were collected and analyzed by GUMS. The expected 3,4dichlorophenylthioacetaldehydewas found in the neutral fraction. The bicarbonate-soluble fraction yielded, after methylation, a peak corresponding to 3,Cdichlorophenylthioaceticacid methyl ester. In the second set of experiments, the trapping agent was not added to the liver homogenate but instead the vapors leaving the area of incubation were passed over the reagent in a second tube. The same metabolic products were found as in the first type of experiment. Watanabe et al. (1976a,b) detected three metabolites in the urine of rats using 14C-labeled vinyl chloride. One metabolite, corresponding to one-third of the total urinary radioactivity, was identified b y MS as N-acetyl-S-(2-hydroxyethy1)cysteine. The second metabolite (onefourth of radioactivity) was shown by MS as thiodiglycolic acid; the third metabolite could not be identified. It was concluded that the major metabolic pathway of vinyl chloride in the rat is conjugation with glutathione. Mueller et al. (1976) used GUMS to identify thiodiacetic acid and S-(carboxymethy1)cysteine in the urine of rats after a 48-hour exposure to 1000 ppm vinyl chloride.

F. NITROSAMINES Pensabene et al. (1972) synthetized 25 nitrosamines and provided basic data on their IR, GC, and MS properties. Chemical ionization mass spectra of nitrosamines was obtained by Gaffield et al. (1976). Gough and Webb (1973) developed a GUMS technique for the analysis of trace nitrosamines (1mg/kg) in foodstuffs. This technique employs a carrier gas pressure programming and peak-cutting device

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which utilizes a silicone membrane separator between GC and MS to remove excess carrier gas and enrich nitrosamine concentration, permits analysis of dimethyl- and diethylnitrosamines which would elute on the tail of the solvent in conventional analysis. To eliminate possible interferences, high resolution (e.g., 12,000) was employed. Gough et aZ. (1977) applied the technique for the quantification of N-nitrosodimethylamine and N-nitrosopyrrolidine in a variety of food products and their vapors, urine, and vegetation. Hecht et al. (1978a) studied the metabolic a-hydroxylation of N-nitrosopyrrolidine and identified a-hydroxynitrosopyrrolidine and 3-formyl- l-propanediazonhydroxide as unstable intermediates which decompose to Zhydroxytetrahydrofuran. For routine analysis at high sensitivity Brooks et al. (1972) converted several volatile N-nitrosamines into electron capturing derivatives with heptafluorobutyric anhydride (HFBA) using pyridine as catalyst. The structure of these derivatives was determined by obtaining both nominal and exact mass measurements on the molecular ions and important fragmentation products such as (M - F - H,O)+, (M - F C,H,)+, and ( M - C,F, - H,O)+ (Gough et al., 1975). The molecular weights of the HFBA-derivatives of N-nitrosodiethyl-, dipropyl-, and dibutylamines, N-nitrosopiperidine, and N-nitrosopyrrolidine correspond to the expected molecular ions; also the difference in molecular weight of any two of the derivatives is the same as that ofthe corresponding nitrosamines, indicating that all of them reacted with HFBA in a similar mode. In the case of N-nitrosodimethylamine the M+' ion was observed at mle = 522 which could only be explained by assuming two molecules of NDMA reacting with HFBA to form a stable structure containing a saturated ring with two nitrogen atoms. Sen (1972) and Sen et al. (1972) analyzed 59 samples of various meat products for the presence of dimethynitrosamine (DMN). DMN was present in five samples of uncooked salami and dry sausage in quantities in the 0.01-0.08 ppm range and in three samples at the 0.005 ppm (detection limit) level. The presence of DMN was confirmed using the mle = 74 (M+') and the mle = 30 (NO+)peaks in G U M S at low resolution and also b y high-precision mass measurement of the molecular ion (M+ = 74.0480 k 0.0004) of underivatized DMN. Sen et al. (1973) also investigated if nitrosopyrrolidine (NPy) might form during the cooking of nitrate-treated cured bacon. NPy probably originates from naturally occurring nitrosoproline (noncarcinogenic) which loses the carboxyl group upon heating. The level of NPy found in commercial bacon products upon frying was in the 0.004-0.02 ppm level; no NPy was detected in uncooked bacon. Considering that 50

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JOHN ROBOZ

ppm of a nitrosamine in the diet of rats induces a large incidence of tumors (Lijinsky, 1976), 100 ppb of NPy in bacon may be hazardous. Since 3-hydroxyproline and 3-hydroxypyrrolidine are also known to be present in pork, Sen et al. (1976) developed methodology to identify S-hydroxy-l-nitrosopyrrolidine (HNPy) which might also be present in fi-ied bacon. Since HNPy is nonvolatile, the 3-methoxy derivative was formed by methylating with sodium hydride and methyl iodide in ethyl acetate at room temperature. To eliminate interferences and maintain specificity, two ions were monitored at a MS resolution of 5000: the M+' ion (mle = 130.0742) and NO+ (m/e = 29.9980). No data have yet been reported on HNPy in bacon. The question of the presence, and importance, of N-nitrosodimethylaniine (DMN) in air is a subject of current debate. Fine and co-workers (1975) used GC and HPLC for separation, and MS for identification to confirm the presence of DMN in the air over Baltimore, Maryland and in Belle, West Virginia, at various locations. In Baltimore, D M N levels were found in the range of 1000-36,000 ng/m3 in the vicinity of a chemical factory where DMN is used as an intermediate. In other areas, less DMN was found; however, levels appeared higher in areas where chemical factories were located. The authors concluded with a thought-provoking comparison of data on DMN uptake: 4 slices of fried bacon = 500 ng, 20 cigarettes = 1000 ng, breathing air containing 1000 ng/m3 for 1day = 14,000 ng. These numbers are to be considered in light of the facts that 50,000 ng/kg/day of DMN fed twice weekly is clearly carcinogenic in the mink, and 200,000 ng/m3 DMN in air is carcinogenic to mice and rats. A cause for further concern are recent reports that the N-nitrosodimethylamine content of some commercial weed killer products is as high as 640,000 mg/liter (Rawls, 1976), and that N-nitrosodiethanolamine was separated by HPLC and identified by MS in toiletry products (Editorial, 1977b). Preussmann et a l . ( 1976) utilized 3,4dichlorothiophenol (Section I1,E) to trap indirectly alkylating electrophiles formed in the presence of rat liver microsomes. Ten nitrosamines, l,%dimethylhydrazine, and 3,3dimethylphenyltriazene (all known carcinogens) were tested and all gave the appropriate 3,4-dichlorophenyl alkyl thioethers as confirmed by MS. The noncarcinogenic methyl-t-butylnitrosamine did not alkylate. Positive relationship was established between carcinogenicity and alkylating capacity of nitrosamines. This technique may be of value for rapid screening of suspected indirect alkylating agents. In studying the conditions for the formation of nitrosamines in the

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digestive system, Wishnok and Tannenbaum ( 1977) reacted morpholine with saliva. I n addition to the expected N-nitrosomorpholine, a morpholinocyanamide was identified by high-resolution mass spectrometry, suggesting a hitherto unknown metabolic pathway for secondary amines.

G. MISCELLANEOUS

1. Phorbol Myristate Acetate Mouse skin tumorigenesis has been conceptualized to proceed in two steps: first, normal cells are initiated to a potential cancer state in an irreversible process by a chemical or physical agent; next, a promoting agent, when applied repeatedly, produces a reversible process leading to the cancerous state. The active principles of croton oil, a known tumor promoter in mouse skin, are the diesters of the parent diterpene alcohol, phorbol. Phorbol is a nonpromoter; phorbol myristate acetate ( 12-0-tetredecanoyl-phorol- 13-acetate) is the most biologically potent ester isolated from croton oil, Crombie (1968)investigated the structure and EI mass spectra of the parent diterpene alcohol and some derivatives. Segal et al. (1975) identified phorborol myristate acetate as a metabolite in mouse skin. The metabolite was also synthetized by reducing the c-5 carbonyl group of phorbol myristate acetate to a secondary alcohol with NaBH,, and its structure confirmed by MS. The parent compound exhibited a weak M+. ion, and major peaks corresponding to the loss of water, acetic acid, and myristic acid. The metabolite had no M + ion but showed intense fragmentation peaks corresponding to the loss of water, water + acetic acid, myristic acid, and myristic acetic acids. The FD spectrum of intact phorbol shows intense ( M = l)+and M+’ ions, and little fragmentation (Schulten, 1973).

+

2. AfEatoxins Aflatoxins in food and feed are usually assayed by employing TLC or LC for separation, densitometry, UV, or fluorescence for quantification, and relative chromatographic retention time or a separate microbiological assay for confirmation. Haddon et al. (1977)developed a high-resolution selected ion monitoring technique for the positive confirmation of Aflatoxin B1 in complex mixtures. Surface bonding between aflatoxins and glass sample containers increases sensitivity to such an extent that 0.1 ng can be positively identified.

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3. Dieth ylstilbesterol This synthetic estrogen is no longer used to prevent miscarriage, but quantification is still needed in foodstuff. The EI spectrum of the dimethylated compound exhibits a strong M+’ ion which may be monitored so that 10 pg injected material can be detected b y G U M S (Abramson, 1972). The dihydroxy and monomethoxydiethyl metabolites were identified in rat liver (Engel et ul., 1976), and the monoglucuronide metabolite was identified in the urine of meat producing animals (Aschbacher, 1976) by mass spectrometric analysis of chromatographically separated products.

4. Stilbenes Microsomal N-hydroxylation is believed to be essential, though not necessarily sufficient, for the metabolic activation of carcinogenic aromatic amides Mass spectrometric identification of metabolites of truns-4’-halo-4-acetamidostilbenes was reported by Gammans et al. (1977); the analytical approach developed may be employed in other studies of this type.

H. TRACEELEMENTS Results of investigations on the role of trace metals and other elements in cancer are contradictory and conflicting (Schwartz, 1975; Editorial, 1977a). Selenium appears to have a protective effect which may be offset b y zinc, iron, and nickel which are suspected carcinogens, while manganese possibly reduces the effects of nickel, and copper might inactivate carcinogenic viruses. Traces of chromium, lead, and nickel found in fibrous silicates may be involved in asbestos carcinogenesis. The “elemental fingerprints” obtained by spark source MS (Section 1,C) may be used to study subtractive, additive, or synergistic interactions of metals in a comprehensive survey of all elements at ultratrace levels in a single analysis. Biological samples must first be ashed or digested in oxidizing acids to eliminate interferences from hydrocarbons formed in the spark. In wet ashing at low temperature with perchloric or nitric acid (in quartz dishes) losses are low and group separation and/or isotope dilution may be employed. Direct analysis of serum, urine, and homogenized tissues may be made after lyophilization (Fitchett et al., 1974). After pretreatment, the sample must be imbedded into a conductor so that a spark can be created: Ultrapure graphite is often used. Evans and Morrison (1968)and Morrison (1967, 1969) provided experimental details and data for some 30 metals in

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serum, bone, lung, and kidney tumor. Sensitivity is 10 ppb for most elements in the periodic table, reproducibility is ~fr25%.Applications of surveying cancer-related biological materials include the analysis of iron, copper, and zinc in the serum of cancer patients (Brown and Jacobs, 1972) and the determination of some 72 elements in coal dust and in the ashed lung tissue of coal miners (Brown et al., 1972). A thought-provoking finding of the latter work was the positive correlation in trends found when the relative concentrations of a variety of elements, ranging fi-om major constituents such as calcium to trace contaminants such as scandium, in coal dust and lung tissue were plotted together. Survey analyses were also made of human hair (Yurachek et al., 1969) and human fingernails (Harrison and Clemena,

1972). Numerous publications appeared on the use of spark source MS to survey elements in air, domestic tap water, industrial waste waters, sediments, and fossil fuels. For references consult the reviews by Alford (1975b, 1977). Because of the complexity of the technique, spark source MS is the method of choice only in survey analysis. Once the significance of a particular element is established, other techniques (e.g., atomic absorption) should be employed for routine monitoring. Ill. Metabolism and Monitoring of Antineoplastic Agents

A. CYCLOPHOSPHAMIDE

1. Metabolism It is now well established that the effectiveness of cyclophosphamide (CP) arises via activation b y hepatic microsomal enzymes; the active metabolite(s) reach the target sites through the systemic circulation. Of the 32 papers presented at a symposium on the metabolism and mechanism of action of CP [Cancer Treatment Reports, 60, 299-525 (1976)] six were devoted exclusively to mass spectral studies and nearly 50% reported results obtained, at least in part, b y MS. In addition, at least 15 publications appeared during the last 3 years on the identification of major and minor metabolites of CP by MS. Evaluation of the cytotoxicity, both in uivo and in uitro, of the metabolites resulted in conflicting opinions about the reasons for the comparative selectivity and effectiveness of CP. The most important steps in the metabolism of CP (Fig. 9, Struck et al., 1975) are the ring-nitrogen oxidation to 4-hydroxycyclophosphamide or its tautomer aldophosphamide, followed by p-elimi-

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JOHN ROBOZ

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nation of acrolein to yield phosphoramide mustard. Phosphoramide mustard[N,N-bis( 2-chloroethyl)phosphorodiamidic acid] was isolated both from liver microsomes incubated with CP and from patients treated with the drug. It is a potent alkylating agent with strong antitumor activity and is now thought of as the chemotherapeutically active metabolite of CP. Whether the intermediate aldophosphamide breaks down in the liver and releases phosphoramide mustard into the circulation, or is metabolized intracellularly after reaching tumor cells is the subject of current studies. Carboxyphosphamide and 4-ketocyclophosphamide are major metabolites found in urine, but have no cytotoxic or alkylating activity and are probably products of detoxification. Nornitrogen mustard [N,N-bis(chloroethyl)amine] and 4-hydroxycyclophosphamide are cytotoxic, both in vitro and in vivo; their importance is not known.

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Colvin et al. (1973) incubated mouse liver microsomes with C P (labeled with I4C universally in the side chain), separated the metabolic products on Sephadex LH-20 columns, isolated all radioactive fractions, derivatized them with diazomethane, and identified unreacted CP, phosphoramide mustard, and nornitrogen mustard by comparing their mass spectra to those of authentic synthetic compounds and also by confirming the molecular composition of both molecular ions and important fragment ions using exact mass measurements. The same group (Fenselau et d.,1975) identified circulating phosphoramide mustard in the blood and urine of patients receiving CP. Metabolites were separated in an Amberlite XAD-2 resin column, the methylated derivatives separated on silica plates, eluted with methanol, and analyzed by GUMS. Methylation of phosphoramide mustard led to a mixture of the mono-, di-, and trirnethylated parent compound, the monomethyl derivative comprising about 80% of the total. The EI mass spectra exhibited ions formed by cleavage in the C-C bond once removed from nitrogen and contained one chlorine atom so that ion pairs of predictable relative intensities ( 3 : 1) were obtained. The CI spectra (isobutane) yielded the (M + 1)+ions as the most abundant species with two chlorines resulting in a cluster of three peaks with intensity ratios of 9 : 6 : 1. Specificity was increased by monitoring all three masses (Fig. 10). Connors et al. (1974b) reported the identification of several metabolites of C P both in vivo and in vitro and offered detailed discussion of their possible significance. They have also investigated the metabolic activation of isophosphamide which appears to follow a pathway similar to that of CP. The important metabolite 4hydroxyphosphamide, which may act as “transport” from parent drug to either toxic or nontoxic final metabolites, appeared too unstable for direct identification. It was “trapped” with ethanol and identified by MS (Connors et al., 1974a). In the same study, 5 more metabolites, phosphoramide mustard, 4-keto-cyclophosphamide,carboxyphosphamide, 2-(2-chloroethylamino)tetrahydro-2H- 1,3,2oxyazophosphorine-2-oxide, and 3hydroxypropyl-N,N-bis(2-chloroethyl)phosphorodiamide,were also identified. This paper provides a great deal of information on in vitro methodology with rat liver microsomes as well as the implications of ) the results. This work was continued by Connors et aZ. ( 1 9 7 4 ~using cyclophosphamide-4d2to study the hydroxylation of CP by rat liver microsomes. I n still further studies (Cox et aE., 1976a), three alkylated analogs of C P (&methyl-, 4-methyl-, and 5,5-dimethyl-) were synthesized and incubated with various fractions of liver microsomes. Several products were isolated, identified, and their possible

236

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significance elucidated. When deuterium-labeled analogs of most known metabolites of CP were synthetized (Griggs and Jarman, 1975), it became possible to investigate the influence of deuterium substitution on the rates of metabolic pathways involving oxidation at the C-4 position as well as on the rate of elimination of acrolein from aldophosphamine. Isotope effects involving the 4-d, and 4,6-d4 analogs caused no change in antitumor activity compared to CP; however, 5,5-dideuteration caused a marked drop in potency. This appears important with respect to the proposed mechanism of

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activation involving the elimination pathway leading to the formation of the cytotoxic phosphoramide mustard (Cox et al., 197613). In a search for aldophosphamide in blood, Struck (1974, 1976; Struck and Hill, 1972) has isolated an intermediate product in a model oxygenase system in the presence of semicarbazide (NH,NHCONH,) utilizing the technique developed by Sladek (1973) and characterized it in a semicarbazone form. Aldophosphamide was considered to be unstable, particularly after unsuccessful attempts to detect it in the blood of CP-treated mice (Struck et al., 1975). Using a different approach, Fenselau et al. (1977) succeeded in isolating and identifying aldophosphamide as a cyanohydrine derivative both in vitro as well as in vioo in humans. First aldophosphamide was synthesized and characterized by mass spectrometry utilizing the trimethylsilyl derivative. The cyanohydrine derivative was prepared by treating aldophosphamide with sodium bisulfite and sodium cyanide; the product was characterized by chemical ionization mass spectrometry. In the in vitro studies, CP was incubated with rat liver microsomes, the metabolite extracted, derivatized, and characterized by mass spectrometry. In the in vivo studies, the metabolite was isolated fi-oni the blood of patients receiving CP, the derivative formed, and aldophosphamide identified by mass spectrometry. Administration of synthetic 4-hydroxy-CP (Struck et al., 1975) followed b y extraction with chloroform permitted in vivo recovery of this metabolite. Administration of phosphoramide mustard led to the identification of nornitrogen mustard and a new metabolite, 3(2-chloroethyl)1,2-oxazolidine-2-one. Investigating the alkylating capability of phosphoramide mustard and nornitrogen mustard, in terms of reacting with nitrobenzylphyridine, it was established that at physiological pH, phosphoramide mustard retained significant alkylating activity whereas nornitrogen mustard did not. On the bases of these experimental findings, the authors postulated that phosphoramide mustard is the most important active metabolite of CP, and it is formed from intermediates outside the target cells. In subsequent study, Colvin et al. (1976) confirmed the differences in alkylating capability between phosphoramide mustard and nornitrogen mustard. Both compounds were incubated with ethanethiol (C,H,SH) and the products were identified as N,N-bis(2-(ethylthio)ethyl)phosphorodiamidic acid and 2,2’-bis(ethy1thio)diethylamine. It was concluded that phosphoramide mustard alkylates as an intact molecule. Further experiments with deuterated analogs of both metabolites confirmed that CP alkylation proceeds via an aziridinium intermediate.

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In most studies of the metabolism of CP, EI/MS was employed; EI spectra often lack molecular ions of appreciable intensity. Przybylski et al. (1976) used both E I and FD ionization to characterize the metabolites of CP. The F D spectrum of 4-hydroxycyclophosphamide exhibits a strong (M 1)+ ion, permitting full characterization, whereas the molecular ion in the electron impact spectrum is not detectable. The E I and F D spectra of phosphoramide mustard complemented each other in establishing the structure of the free acid. Attempts to characterize aldophosphamide b y FD/MS failed, supporting the thought that this compound is too unstable to play an active role in the cytotoxic action of CP. FD/MS analysis of “activated” sulfur-containing C P derivatives confirmed several postulates concerning these compounds (Draeger et al., 1976).

+

2. Quanti5cation

The observation that patients could “taste” C P after intravenous infusion prompted Duncan et al. (1973) to develop a method for the identification of the drug in saliva, synovial fluid, milk, sweat, and cerebrospinal fluid. C P was extracted with chloroform, the layers separated by centrifugation, the organic layer concentrated by evaporation, and introduced into the ion source via a direct probe. Here the crude extract was evaporated and the presence of C P confirmed either by obtaining the complete mass spectrum at low resolution, or by measuring exact masses of selected fragments at high resolution. The most abundant ion, in the E I spectrum of C P (mle = 211) corresponds to the loss of the CH&l radical from the molecular ion. This peak is accompanied by another two mass units higher in the proper abundance ratio (3: 1) for one chlorine atom. The molecular ion of C P is weak but detectable. When the concentration of CP was 1 mg/ml body fluid, high-precision mass measurements were made on the peaks at mass 211 and 213, respectively. This was selected ion monitoring at high resolution and resulted in the combination of high sensitivity and complete specificity even in the presence of endogenous lipids of the same nominal mass. C P was identified in all body fluids studied. The presence of C P in milk contraindicates administration of the drug to nursing mothers. Its identification in cerebrospinal fluid is of significance in terms of the drug crossing the blood-brain barrier; and its appearance in saliva and sweat helps to explain observed incomplete recovery of the radioactively labeled drug in urine, feces, and respiratory gases. Griggs and Jarman ( 1975) synthesized cyclophosphamide-4,4,6,6-d4 and added a known quantity of it to blood and urine samples from

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patients. Blood was diluted with citrate-saline, red cells removed b y centrifugation, the drug extracted with chloroform, and concentrated to a small final volume. Urine was also extracted with chloroform. MS analyses were made by evaporating the crude chlorofomi extract inside the ion source from a direct probe and determining the relative heights of the peaks corresponding to the ( M - CH,Cl)- ion which are the most abundant peaks in the EI mass spectra of C P and the deuterated analog, respectively. For quantification, normal blood was spiked with the drug and a calibration curve obtained down to I pg/ml level. The technique was extended to include quantification of 4-ketocyclophosphamide in blood after extraction with ethyl acetate and methylation with diazomethane (Cox et al., 1976a). Analogs tetradeuterated in the bis-(2-chloroethy1)amino function were used as internal standard. The limit of detection was 1pg/ml, which was poorer than that of those methods involving prior gas chromatographic separation and selective ion monitoring (order of 10 ng/ml). The technique reported by Pantarotto et a2. (1974a) used the stable trifluoroacetyl derivative of C P with isophosphamide as internal standard. As confirmed b y MS, only one trifluoroacetyl group is picked u p b y the molecule. When CP is to be quantified in tissues, intereferences from endogenous substances require either laborious separation processes or high specificity. In a still further improved technique (Pantarotto et aE., 1976), the drug was determined by monitoring 4 selected masses in the CI mass spectra of the N-trifluoroacetyl derivatives of CP and the internal standard. The (M 1)+ions of both the drug and the internal standard are of the same mass, (mle = 357); however, GC retention times are different, thus monitoring presents no problem (Fig. 11).The other masses monitored are the isotope ion at mle = 357, and the two isotope ions corresponding to the loss of HC1 (mle = 321 and m/e = 323). The technique is linear in the 150 ng/ml to 100pg/ml range (serum). Incidentally, the technique can be used in reverse, i.e., for quantification of isophosphamide with C P as internal standard. The method is particularly u s e h l for the quantification of C P in tissues. For example, tissue concentrations determined in lung, kidney, heart, liver, spleen, and brain of C57BL16J mice bearing Lewis lung tumor were used to determine pharmacokinetic parameters of the 85 mg/kg C P injected intravenously 25 days after tumor implantation. Turning to the quantification of metabolites, Jardine et al. (1976) used GUMS with CI and selected ion monitoring to assay phosphoramide mustard and nornitrogen mustard in human serum and urine. Deuterium labeled analogs served as internal standards, and

+

240

JOHN ROBOZ

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FIG. 11. Mass hagmentogram of brain extract, trifluoroacetate derivatives. The first set of peaks represents isophosphamide (internal standard), the second set represents cyclophosphamide. Reprinted from Pantarotto et al., 1976, with permission.

trifluoroacetylation used to form derivatives suitable for GC separation. An alternative derivatization technique, using ethereal diazomethane, yields mono-, di-, and trimethyl derivatives. The dimethyl derivatives are most suitable for selected ion monitoring; however, when interferences occur due to endogenous constituents, the trimethyl derivatives may be utilized. (Fenselau et aZ., 1975; Jardine et al., 1976). In a typical analysis, 22 pg/ml phosphoramide mustard was found in the serum of a patient 75 minutes after receiving a l-hour infusion of C P (50mgkg). In another experiment, 10,30, and 15pg/ml of CP, nornitrogen mustard, and phosphoramide mustard, respectively, were found in the O-4hour urine collection from a patient who received a dose of 15 mg/kg C P intravenously.

MASS SPECTROMETRY IN CANCER RESEARCH

24 1

Jardine et al. (1978) extended the technique for the quantification of CP, phosphoramide mustard, and nornitrogen mustard in the plasma and urine of patients receiving C P therapy (60 or 70 mg/kg i.v.). All 3 compounds were available in deuterated form as internal standards. Peak plasma levels of C P and phosphoramide mustard (50-100 nmole/ ml) occurred at 3 hours after C P administration. Plasma half-lives, the origin of nornitrogen mustard, and the mechanism of action of CP are discussed. Both low- and high-resolution FD spectra have been obtained and interpreted for C P and almost all known metabolites (Schulten, 1974, 1976a,b). Quantification in tissues has been in the presence of interfering endogenous constituents. Once developed, this technique is likely to offer significant advantages over techniques requiring evaporation and derivatization, particularly for the analysis of nonvolatile (phosphorodiamidic acid) and thermally unstable (4-hydroxycyclophosphoramide, carboxyphosphoramide) metabolites.

B. NITROSOUREAS BCNU [ 1,3-bis(2-chloroethyl)-nitrosourea] was quantified (to 0.4 p M ) in plasma (Weinkam et al., 1978) by monitoring the (M + 1)+ ions of BCNU and its octadeutero analog (internal standard) upon evaporating the residue of a hexane-ether extract in a direct probe. May et al. (1974) investigated the role of hepatic mixed function oxidase in the metabolism of CCNU [ 1-(2chloroethyl)-3-cyclohexyl- 1nitrosoureal. Liver microsomes were prepared from both normal and phenobarbital treated rats and incubated with 14C-labeled CCNU. After removing excess CCNU with hexane, a metabolite was extracted with ether and purified by HPLC. EI mass spectra revealed that all fragments containing the cyclohexyl ring were 15 mass units higher in the metabolite than in intact CCNU. In addition, fragment ions of the metabolite indicated an unsaturated ring while the corresponding fragments of intact CCNU suggested a saturated ring. At the same time, the chloroethyl and nitroso groups were present in both compounds. These observations led to the identification of the metabolite as cis-4hydroxy CCNU, i.e., the cyclohexyl ring of the intact CCNU was stereospecifically hydroxylated by the liver microsomes. Additional evidence was obtained using cochromatography (thin-layer and liquid) with cyclohexyl (14C)-and2-chloroethyl (14C)-labeled CCNU, and NMR data on cis- and trans-Chydroxy CCNU. It is noted that, as expected, the hydroxylating activity of the NADPH- and 0,-fortified microsomes

242

JOHN ROBOZ

was induced by pretreatment with phenobarbital and inhibited by heating. In further studies on the metabolism of CCNU, and also methylCCNU ( 1-(2-chloroethyl)-3(trans-4-methyl cyclohexy1)-1-nitrosourea), Reed et al. (1975) identified 2-chloroethanol as the major product of nonenzymatic degradation in buffers, under physiological conditions, resulting from the deprotonation of the 3-nitrogen. The 2-chloroethyl moiety was trapped with C1-, Br-, and I- ions and the dichloroethane that formed identified by GC/MS. Other degradation products identified were acetaldehyde, vinyl chloride, ethylene, and cyclohexylamine. For the in v i m study of the metabolism of CCNU, Reed and May (1975) injected I4C-labeled CCNU into rats and isolated a metabolite from urine by HPLC which was identified by GUMS as a thiodiacetic acid. In addition, rapid microsomal hydroxylation of the cyclohexyl ring yielded five metabolites (cis or trans-2-hydroxy, trans-3-hydroxy, cis-3-hydroxy, cis-4-hydroxy, and trans-4-hydroxy-CCNU), all identified by MS. Essentially the same results were obtained by Hilton and Walker (1975) using HPLC for separation and MS for identification of the metabolites of CCNU both in vivo and in vitro. They concluded that although the hydroxylation of the cyclohexyl moiety of CCNU probably has no effect on the formation of the cytoxic intermediate, it certainly does effect protein binding, tissue distribution, and rate of excretion. The decomposition of BCNU ( l-3-bis(2-chloroethy1)-1-nitrosourea) in aqueous solution was investigated b y Colvin et al. (1974). A solution of 14C-labeled(in the chloroethyl group) BCNU was incubated in a buffer under various experimental conditions and volatile materials formed during decomposition were swept away with nitrogen and collected in ether at -70°C. Vinyl chloride, acetaldehyde, and 1,2dichloroethane were identified as volatile metabolites of BCNU by MS. When BCNU was allowed to decompose in aqueous solution in a gastight vial, followed by extraction of the product with ether, all the listed decomposition products were found again, plus another decomposition product (63% of the total), identified as chloroethanol. When chloroethylamine was reacted with nitrous acid in aqueous solution, the reaction products and their relative quantities were the same as observed in the decomposition of BCNU. The authors concluded that chloroethylcarbonium ion (or chloroethyldiazonium precursor) is generated during the decomposition of BCNU at physiologic pH and ternperature, and the reaction of this moiety with nucleic acids may explain the alkylating-like activity of BCNU in vivo. Hill et al. (1975)

MASS SPECTROMETRY IN CANCER RESEARCH

243

employed MS to characterize the microsomal metabolism of the nitrosoureas. The metabolic product of BCNU was 1,3bis(2-chloroethyl) urea, the products of CCNU and methyl-CCNU were ringhydroxylated derivatives. Numerical values obtained on the rates of the microsomal metabolism led to the conclusion that microsomal metabolism may take place before chemical decomposition of the administered drugs occur. Ludlum et d.(1975) incubated BCNU with the synthetic polynucleotides poly(C) and poly(G). After chemical and enzymatic digestion and separation from unreacted nucleotides b y column chromatography, 3 major derivatized (TMSi) nucleotides were identified b y MS (also by UV): 3-P-hydroxyethyl) cytidine, 3,N4-ethano-cytidine, and 7-(P-hydroxyethyl)-8-oxoguanosine. The authors concluded that BCNU generates active two-carbon fragments, probably chloroethyl carbonium ions, which confer alkylating activity when attached to CMP or GMP with a nucleic acid strand.

c. PURINES, PYRIMIDINES, AND THEIRNUCLEOSIDES Pantarotto et aZ. (1974a) developed a GUMS-CI-selected ion monitoring technique for the quantification of 5-fluorouracil (5FU), 5-fluoso-2‘-deoxyuridine (5-FUdR), 6-mercaptopurine (6MP), 6-mercaptopurine ribonucleoside (GMPR), and cytosine arabinoside (AM-C) in fluids down to a level of 100 ng/ml; 0.1 ml serum was diluted with water and extracted with n-butanol. An aliquot of the butanol phase was evaporated to dryness and the derivatizing reagent trimethylbenzylammonium hydroxide added. Methylation occurs “on-column” in the sample injection port of the GC. The (M + 1)+ ions of the drugs and the internal standard (imipramine) were monitored in the CI mode. An application of this technique (with minor modifications) is shown in Figs. 12 and 13. Several peaks appeared when the masses mle = 181 (5FU) and mle = 159 (6MP, used here as internal standard) were monitored in the GC effluent, but these did not interfere with those representing 5FU and 6MP. Peak areas were measured in terms of counts, and quantification was made with the aid of a calibration curve obtained by plotting area ratios of 5FU/6MP for known quantities of 5FU added to pooled normal serum. The same technique can also be used to quantify 6MP using 5FU as internal standard. Finn and Sadee (1975) quantified 5FU utilizing isotope dilution combined with selected ion monitoring. Highly enriched 14C-labeled 5FU (
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MASS SPECTROMETRY IN CANCER RESEARCH

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of thymidine, and its major metabolite, thymine, in serum of patients receiving thymidine in combination with 5FU, as a rescue in methotrexate therapy, or alone. In the same sample, 5FU and FUdR can also be quantified. Limit of detection: 50 ng/ml for each component. Rosenfeld et al. (1977) developed a technique for the quantification of 6-mercaptopurine in plasma with a limit of detection of 20 ng/ml. Simultaneous extraction and derivatization was achieved when 6MP was extracted by ion pairing with a quarternary amine into methylene chloride containing an alkylating agent (tetrahexylammonium hydroxide). The technique of silylation of purine and pyrimidine bases and nucleosides has been investigated in detail since multiple peak formation considerably reduces the utility of this otherwise desirable technique. Single chromatographic peaks were obtained for 5FU, 2-azacytosine, 5-azauracil, 6-azauracil, thymidine, uracil arabinoside, 5-azacytidine7and 6-azauridine. Unsymmetrical or multiple peaks and poor response were obtained for cytidine, cytosine, ARA-C, 6-azacytidine7 5-fluorodeoxyuridine, and several other compounds (Sadee et al., 1976).A detailed study on the silylation of nucleosides (effect of reagents, solvents, conditions) was undertaken by Gerhke and Patel (1977) and Patel and Gerhke (1977). An MS technique for the identification and quantification of compounds involved in the metabolism of pyrazofurin (3-/3-D-ribofuranosyl4-hydroxypyrazol-5-carboxamide), an antineoplastic drug currently undergoing clinical trials, was develaped by Roboz (1977). Free pyrazofurin was determined in the serum and urine, after removing proteins by ultrafiltration and interferences by rapid ion exchange chromatography, by monitoring the (M + 1)+ion of the fully silylated molecule (chemical ionization). The 5’-phosphate of pyrazofurin was identified in human leukemic cells incubated with the drug b y utilizing mass chromatography: the methylated crude extract was evaporated from the tip of a solid probe insertion system and the series of mass spectra obtained was searched for characteristic ions of the phosphate. These findings, together with results of reversal experiments with uridine or cytidine, support the assumption that the mode of operation of pyrazofurin is the interruption of the conversion of orotidine 5’-monophosphate to uridine 5’-monophosphate in the course of the biosynthesis of pyrimidine nucleotides. F D mass spectra of nanogram quantities of pure ARA-C and azacytidine were obtained without derivatization by Maurer and Rapp ( 1976). However, analytical procedures for biological materials are not yet available.

246

JOHN ROB02

D. DAUNORUBICIN AND ADRIAMYCIN Daunorubicin (daunomycin, DRB) and adriamycin (ADR) are anthracyclic antibiotics consisting of a tetracyclic quinoid aglycone in a glycosidic linkage to the amino sugar daunosamine. Although their structures appear similar, their antitumor properties are significantly different. This fact, together with the current eminence of adriamycin as one of the most useful antineoplastic agents, adds considerable importance to all metabolic and pharmacologic studies concerning these compounds and their analogs. The intact molecules of DRB and ADR cannot be analyzed b y conventional MS. The mass spectra of underivatized hydrolysis products were first obtained in connection with the original structure determination of these compounds (Arcamone et aE., 1968, 1972) and more recently their synthetic analogs (Arcamone et al., 1975). For example, the EI mass spectrum of adriamycione, the water insoluble aglycone of ADR, exhibits a molecular ion together with a series of predictable fragments, such as those resulting from the successive elimination of two molecules of water, the loss of the COCH,OH group b y the cleavage of the allylic bond between the ketone carbonyl and the quaternary carbon atom, and the loss of the CH,OH group. These observations together with data from NMR helped considerably to establish the structure of ADR. Purely MS studies on the EI fragmentation of several N-acyldaunosamine (Vigevani et aE., 1974; Roller et al., 1976) and trimethylsilylated DRB, ADM, and carminomycin (Roboz, 1974) derivatives are of potential importance for further developments in analytical methodology but need not be reviewed here. The major metabolites of DRB and ADR are daunorubicinol (13hydrodaunorubicin) and adriamycinol, respectively. These compounds and also their hydolytic products, the respective aglycones, were isolated from the urine of patients with acute lymphocytic leukemia by Bachur and co-workers who used TLC, IR, and MS to identify the compounds and derivatives by comparison with authentic products obtained in enzymatic reactions and hydrolyses. The metabolism of DRB (Takanashi and Bachur, 1975) is qualitatively similar to that of ADR (Takanashi and Bachur, 1976) (Fig. 14). Most metabolites detected were characterized by MS (Fig. 15) either in earlier studies (Bachur, 1971; Bullock et al., 1972; Asbell et al., 1972; Cradock et al., 1973) or in the present ones. The human metabolism of DRB involves carbonyl reduction, reductive glycosidic cleavage, O-demethylation, O-sulfation, and O-glucuronidation. Whether these multiple metabolites have significant cytotoxic activity is not known.

MASS SPECTROMETRY IN CANCER RESEARCH

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FIG. 14. The metabolism of Adriamycin in man. Heavy arrows indicate preferred positions. The following fluorescent metabolites were identified: adriamycin (I), adriamycinol (II), adriamycin aglycone (111), adriamycinol aglycone (IV), deoxyadriamycin aglycone (V), deoxyadriamycinol aglycone (VI), demethyldeoxyadriamycinol aglycone (VII), demethyldeoxyadriamycinol aglycone 40-sulfate (VIII), and demethyldeoxyadriamycinol aglycone 4-0-glucuronide (IX). Reprinted from Takanashi and Bachur, 1976, with permission.

About 10%of the administered drug fluoresence appears in urine in 5 days, and about 50% in the bile (preliminary studies show the same metabolites in bile as in urine). There are significant quantitative differences between the metabolism of ADR and DRB, e.g., daunorubicinol is present in larger proportion than adriamycinol indicating a preference for DRB as substrate for the cytoplasmic aldoketo-reductase and microsomal glycosidases. Since metabolites account for 60%of the fluorescence from ADR in urine, and even more for DRB, the monitoring of metabolites during therapy is indicated. (HPLC with fluorimetric detection is probably the best approach). It is noted that only fluorescent metabolites have yet been identified; a possible approach to detect other metabolites would be the administration of stable-isotope labeled ADR or DRB, followed by mass spectrometric search for the known stable isotopes.

248

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249

MASS SPECTROMETRY IN CANCER RESEARCH

The mass spectra of the unbroken, undervatized molecules of both DRB and ADR may be obtained by FD. Because the M+ and (M 1)+ ions have high abundances, there is a possibility of analyzing nanogram quantities of either drug with one serving as internal standard for the other by single ion monitoring and ion current integration techniques (Maurer and Rapp, 1976).

+

E. ANTITUMORHORMONES Hormones can be quantified b y radioimmunoassay (best for routine assays), GC (electron capture), enzymic methods, and selected ion monitoring. The sensitivity of these techniques is in the 10-11-10-14A4 range. Almost any steroid or related compound can be assayed alone or within a steroid profile by utilizing one of the GUMS techniques developed for the study of steroids and hydroxysteroids in pregnancy and infants (Adlercreutz, 1974; Miyazaki et al., 1977). This technique has two advantages over other methods for trace quantification: Crude extracts may be employed and positive identification is provided. Thus, the technique is the method of choice in nonroutine applications. When the objective is identification of a newly detected metabolite, or confirmation of the presence of a suspected metabolite, GUMS with full mass scanning or exact mass measurements is the only technique available. Among the steroidal hormones utilized in cancer chemotherapy, testosterone (androgen), diethylstilbestrol (estrogen), megestrol acetate (progestin), and prednisone and dexamethasone (corticosteroids) have been determined by MS. The use of MS in steroid-related biochemical markers is reviewed in Section IV, C, where several references are given for methodology applicable to most antitumor hormones. Only one example is discussed here. Adlercreutz et at. (1974, 1975; Adlercreutz, 1977) developed an assay for megestrol acetate in body fluids, and also studied the metabolism of the drug. Plasma was extracted with ether-chloroform, washed with distilled water, evaporated to dryness, picked up with ethyl acetate-benzene, purified on a silica gel column to remove cholesterol, and finally the compound was determined as the monomethoxime derivative by monitoring the molecular ions of the derivative and the internal standard (medroxyprogesterone acetate). Limit of detection: 30 pg injected material. When samples from patients with carcinoma corporis uteri were analyzed, plasma concentration varied in the 10-600 nmole/liter range, although most values fell into the 150-400 nmole/liter range (50 mg drug, twice daily). The reason for

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these great variations in individual absorption or metabolism is not known. NO metabolites were found in plasma, but two were isolated in both the unconjugated steroid and the glucuronide fractions of urine. Interestingly, both were characterized by MS as monohydroxysubstituted metabolites; however, their exact structure is not yet known. In a novel application, the same authors have also utilized selected ion monitoring to investigate the specificity of a radioimmunoassay developed for megestrol acetate and unmasked interferences caused by other compounds, possibly metabolites. The drug O , ~ ’ - D D D (Mitotane, bis(chloro-4’-phenyl-chloro-1phenyl)-2,2’-dichloro- 1,l’ethane) is used clinically for treatment pf carcinoma of adrenal cortex. Johnson et al. (1977) developed a mass fragmentographic technique for the quantification of the drug and its major metabolites in plasma in the 0.1-10 pg/ml concentration range. The drug and one of the metabolites was present in trace levels u p to 17 months after treatment even after brief exposure, suggesting that multiple daily doses may be unnecessary.

F. HEXAMETHYLMELAMINE Hexamethylmelamine (HMM),now under clinical trials, has activity against lung, breast, ovarian, and cervical cancer. GUMS was used to identify urinary metabolites (Worzalla et al., 1973, 1974; Johnson et al., 1973)in both humans and rats. The gas chromatogram of a 24-hour urine sample from a patient showed at least nine peaks, eight of which were identified: pentamethylmelamine, NZ,”2,”4f16-tetramethylmelamine, N2,N2,N4-trimethylmelamine,N2,N4,iV6-trimethylmelamine, N2,N2-dimethylmelamine, N2,N4-dimethylmelamine, monomethylmelamine, and melamine. It is clear that N-demethylation is the most important pathway in the metabolism of HMM in man (and also in rats), All metabolites were synthetized and tested for alkylating and antifolate activity; the results were negative. Johnson et al. (1974) developed MS techniques, using N-trimethylsilylimidazoleto silylate hydroxyl groups in the presence of amino groups, to detect and identify in blood several possible methylol-methylmelamine intermediates which might originate from HMM by an alternative metabolic pathway involving oxidative N-demethylation. Such compounds have not yet been found in biological samples. G. PLATINUhi COORDINATION COMPLEXES Haake and Mastin (1971) found that in simple square ,planar platinum(I1) compounds the M+ ions are detectable in the E I spectra

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of all complexes studied in spite of their relative involatility. The characteristic isotopic patterns of the C1 and Pt nuclei permitted the determination of the number of C1 atoms present, but the cis and trans isomers of Pt(HN,),Cl, and Pt(py),Cl, could not be distinguished. The site of action of the coordination complexes of platinum is thought to b e the nucleoside bases of DNA (Rosenberg, 1975).Roos et al. ( 1974) investigated the interaction of platinum(amine),Cl, complexes with nucleoside analogs by allowing the platinum complexes and the nucleosides, each at 10-3M concentration, to react in aqueous solution in the dark for a period of two weeks, after which the total volume was reduced by a factor of 10. Aliquots were evaporated, without derivatization, in a direct probe at 250-300°C. No reaction of any kind was observed with the trans isomers and only the starting platinum complexes and the nucleoside analogs were found. Using the cis isomers, 9-methyladenine and 1-methylcytosine reacted as monodentate ligands in replacing the chlorines. 9-Methylguanine reacted as a bidentate ligand, and 1-methylthymine did not react at all. The structure of the linkages could not be determined from the mass spectra. When uracil, thymine, and other substituted pyrimidines, and pyrimidine nucleosides and nucleotides are reacted with cisdiaquadiammineplatinum( 11) a class of potent antitumor agents, called “platinum blues,” is formed (Davidson et al., 1975). The nature of these complexes is not known. Because they do not crystallize, it is not even known if the complexes are monomeric or oligomeric. In a novel technique of ionization (Macfarlane and Torgeson, 1976), energetic fission fragments from the decay of californium-252 are utilized to volatilize and ionize nonvolatile biomolecules in aqueous solution in a single process. When a platinum-thymine blue was analyzed by this method, cluster ions were detected at regular intervals u p to mass 3000. Each group of clusters appeared to correspond to the additional of one platinum atom. The distribution of masses within individual clusters probably represents different fragments of the thymine moiety. This technique, when developed, may provide an entirely new approach to study the structure of complex molecules of low volatility.

H . MISCELLANEOUS 1. Methotrexate ( M T X ) MTX and its analogs are not amenable to conventional MS analysis because of their low volatility. Silylation yields a mixture of tri-, tetra-, and pentatrimethylsilyl derivatives of little practical significance. The

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FD mass spectrum of MTX shows an intense quasimolecular ion. Kirk et al. (1976) employed F D to characterize some 14 analogs of MTX synthesized to evaluate potential anticancer activity. In all cases, the (M + 1)+ion was the most intense peak (base peak) in the spectra. Hignite et al. (1978) identified several impurities (up to 4.8% concentration) in parenteral MTX dosage forms. Since “high dose” therapy may involve the administration of up to 20 gm MTWday, the impurities may be of considerable therapeutic/toxic interest. For the routine quantification of MTX in body fluids convenient assays, based on competitive protein binding, are available (Myers et al., 1975). 2. Aromatic Nitrogen Mustards Connors et al. (1972) suggested that “chemical trapping” of highly reactive metabolites to produce unreactive derivatives may be a general technique to study drug metabolism. Chemical trapping was accomplished with 35S-labeled sodium sulfide- in the study the metabolism of 2’-carboxy-4di-(2-chloroethyl) amino-2-methylazobenzene in rat liver homogenate. This compound is active against Walker 256 carcinosarcoma in the rat, and is typical of the “latent” compounds believed to be converted into more active alkylating metabolites by azoreductases in the liver. The chemically trapped metabolite was reduced to 4-di-(2-chloroethyl)amino-2-methylalanine. The technique is applicable to compounds containing the potentially reactive di-(2-chloroethy1)amino function.

3. Dinitrobenz yl Aziridines Connors et al. (1975) investigated the metabolism of 1( l-aziridinyl)-2,4-dinitrobenzene,an agent of high specific activity against Walker tumor in rats. Incubation with 9000 gm-liver homogenate and cofactors converted the compound to a metabolite, identified as 2-amino- 1-( l-aziridinyl)-4-nitrobenzene. This compound is more active in in vitro tests than the parent compound. While only one major metabolite was identified after incubation with liver homogenate, ten metabolites both polar and nonpolar were detected, isolated by TLC, and identified b y MS in the urine of treated rats.

4. E 11ipticine Two hydroxylated metabolites of ellipticine (5,1l-dimethyl-6fipyrido[4,3-blcarbazole) were identified in rat bile by Reinhold and Bruni (1976). Since no evidence was found for the conservation of deuterium at the position of hydroxylation using deuterated drug, it

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was concluded that the first step in the metabolism of ellipticine is aromatic hydroxylation, followed by conjugation and excretion in the bile.

5. Antiviral Drugs Because of possible direct or indirect association of herpes-type viruses with various forms of cancer, there is considerable current interest in antiviral drugs. Phosphonoacetic acid was quantified in the blood of experimental animals b y selected ion monitoring (Roboz et al., 1977). Ribavirin ( 1-p-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, Virazole) was determined in blood and urine in Phase I studies (Roboz and Suzuki, 1978) and several of its metabolites identified (Miller et al., 1977). Ara-A(9-p-D-arabinofuranosyladenoside)may also be quantified b y selected ion monitoring.

IV. Biological Markers

A. GENERAL An ideal biochemical marker of tumor activity should be tumorspecific, present in all cases, sensitive, and capable of reflecting the actual tumor burden of the host under therapeutic conditions. In fact, products of tumors may not be unique to the neoplastic state nor absolutely specific for a particular tumor. They are, however, still of major interest not only because of their potential diagnostic value but also because of the possibility of assessing how treatment is working (or failing) below the threshold of detection b y ordinary clinical means. I t is emphasized that most biochemical tests are performed on biological fluids remote from the tumor; thus they provide an indirect assessment of tumor behavior. This is not necessarily a disadvantage, and does not preclude the possibility of being relatively organ specific, e.g., acid phosphatase in carcinoma of the prostate, alkaline phosphatase in primary bone and liver tumors (Schwartz, 1976). Multiphasic biochemical screening to uncover occult disease, which has been in vogue for the last few years (Roboz, 1975), is of little importance in cancer diagnosis at the present time. One problem is the definition of what is meant by “normal” values. Here, individual biochemical profiling may be a promising approach since changes in individual norms, after corrections for aging, may signal the development of disease, including cancer (Nadel, 1969). A technique likely to

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produce progress in this field is “high resolution” analysis, i.e., the monitoring of a large number of constituents (e.g., carbohydrates, lipids) in a single analysis to screen a large population to discover differences. Both “within population,” i.e., cancer patients vs. normal controls, and “without population,” i.e., low-risk vs. high-risk groups, analyses may be made. Such “biochemical epidemiology” studies have been outlined for hormone-dependent cancers by Zumoff et al. (1975). The role of MS here is obvious: All separated compounds must be identified before their possible significance can be evaluated.

B. POLYAMINES After Russell (1971) reported increased excretion of putrescine (1,4diaminobutane), spermidine (N-(3-aminopropyl)-1,4-butanediamine), and spermine (N,N’-bis(3-aminopropyl)- 1,4-butanediamine), in the urine of patients with cancer, at least 30 papers appeared reporting elevated polyamine levels in patients with cancer, and suggesting that changing polyamine levels in the course of treatment be used to evaluate efficacy. Reviews on the role of polyamines as markers of response and disease activity in cancer chemotherapy (Durie et al., 1977) and on their relevance as markers of tumor kinetics (Russell, 1977) suggest that further work is justified. Analytical techniques developed to assay polyamines in body fluids include TLC, HPLC, GC, GClMS, and radioimmunoassay (Bartos et al., 1977). For routine assays, perhaps it is best to use a modified amino acid analyzer (Russell and Russell, 1975) which is sensitive to the pmole level and capable of assaying 20 samples a day. MS techniques have been used to identify polyamines and metabolites positively and to provide trace quantification with concurrent specificity. Walle (1973) developed a G U M S technique for polyamines and metabolites in urine: After extraction with alkaline butanol and purification by ion-exchange chromatography, trifluoracetic anhydride was used for derivatization. For best chromatographic performance, full acylation of all amino groups must be accomplished, i.e., two trifluoroacetyl (TFA) groups for putrescine, 3 TFA groups for spermidine, and 4 TFA groups for spermine. The EI mass spectra of the three compounds show small but discernible molecular ions and intense fragment ions corresponding to the loss of CF, and CF,CO groups, etc. Cadaverine ( 1,5-diaminopentane) served as internal standard for quantification. As little as 50 ng of a compound was adequate to obtain a full mass spectrum; with selected ion monitoring, the detection limit was 50 pg. All three polyamines were identified in hy-

MASS SPECTROMETRY IN CANCER RESEARCH

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drolyzed urines of patients with leukemia; free putrescine and spermidine were identified in unhydrolyzed urine. A large unknown peak appearing in the nonhydrolyzed urine of a patient with acute myelocytic leukemia was identified as monoacetylsperminidine (Fig. 16), a metabolic product of spermidine. Another unknown was identified as 1,3-diaminopropane, also related to spermidine metabolism. Significant increase was obtained in the acetylspermidine content of urine of leukemic patients under therapy with vincristine and prednisone after treatment with vincristine; the ratio of acetylspermidine to spermidine excretion appeared constant (Denton et al., 1973). Smith and Daves (1977) added deuterated putrescine-d,, cadaverine-d,, spermidine-d,, spermine-d,, and acetylspermidine-A-d2 to the samples prior to separation and derivatization. Thus, all possible losses during manipulations were the same for the compounds and internal standards. All nondeuterated trifluoroacetylpolyamines have their base peak at mle = 126 ( F,CCONH = CH,+); deuterated, derivatives appear 2 mass units higher. To increase sensitivity, ions resulting from the loss of CF, or CF,CO were monitored. This permitted the use of a large excess (a100 pmole) of deuterated standard without changing the peak height for the mass of the unlabeled polyamine, resulting in a “carrier effect.” Linear response curves were obtained in the 1-1000 pmole range.

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C. STEROIDS Zumoff et nl. (1975) reviewed the relationship of urinary excretion of androgen metabolites and estrogens to the natural history of breast cancer and concluded that the reported relationships of low urinary excretion of androgen metabolites to increased risk of breast cancer, and the protective value of a high ratio of urinary estriol to estrone estradiol are dubious. According to their hypothesis, the lack of active estrogen prior to age 30 is of protective value. MS has not been used extensively for the monitoring of steroids in large scale profile studies, although analytical techniques are readily available. Adlercreutz et nl. (1975) developed a technique for the determination of at least l l estrogens in body fluids or tissues with a sensitivity as low as 10-14M. Luyten and Rutten (1974) utilized highresolution capillary columns in the GC to separate urinary steroids so that a rather complete urinary steroid profile may be obtained in a single analysis. In what follows, several pilot studies are reviewed on the use of MS to identify potential markers in neoplasis. Reimendal and Sjovall ( 1973) developed computerized procedures for the rapid routine identification of a large number of steroids in profiles. For example, it took only 5 minutes to obtain a complete profile of steroids in the disulfate fraction from the plasma of a patient with choriocarcinoma. The concentration of the pregnanediol isomers was considerably higher in this patient than in normals; the significance of this finding is not known. Millington et al. (1974) and Millington (1975) developed a technique for the identification and quantification of endogenous steroids in human breast tumors by high-resolution mass fragmentography . Monitoring exact masses resulted in specificity and elimination of all interferences so that crude tissue extracts could be analyzed. Estradiol-l7/3 was the major classical estrogen in the primary breast tumors studied, dehydroepiandosterone was found in all tumors, and testosterone could not be found in any tumor. Most other steroids found were present in the range of 0.5-10 ng/gm tissue. Similar techniques were employed to the analysis of CI9 steroids in human benign hyperplastic prostate tissue (Millington et al., 1975).Sensitivity limit was at the feintomole level and quantification improved with the use of epimers as internal standards. The most predominant steroid found was 5a-dihydrotestosterone (0.8-40 ng/gm tissue). Other steroids of interest included the 3a, 17p and 3p, 17p isomers ofthe 5a-androstanediols (0.01-2.0 ng/gm range for most samples), and testosterone (0.07-5.0 nglgm).

+

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High-resolution mass fragmentography was used by Snedden and Parker (1976) to identify and quantify oestrone, oestradiol, oestriol, and progesterone directly in ovarian tissues (follicle corpus and various cysts). The specificity of the technique permitted the use of crude extracts. Different levels of progesterone were found in normal and diseased tissues of the same ovary; also, steroid levels changed parallel to the mentrual cycle. Maynard ( 1977) and Maynard et al. (1977) used GUMS to quantify silylated steroid derivatives in malignant and benign breast tissues. Levels of dehydroepiandrosterone varied significantly (malignant: 20-200 ng/gm tissue, benign: 200-1000 ng/gm), while 5-androstane3/3,17/3-diol levels were constant ( 10-200 ng/gm). Some 5 mg/day of an unknown steroid was detected in the urine of a patient with inoperable adrenal carcinoma (Fantl, 1973). After isolation, glucoronidase hydrolysis, chloroform extraction and TLC purification, the compound was identified by MS as &,16a-dihydroxyandrost-5-en-17one. This metabolite has a hydroxyl group in the C-16 position and apparently inhibits the action of steroid A4-5isomerase leading to the formation of a 5-ene-3a-hydroxy group. The only other known urinary metabolite with the same effect is pregn-5en-3a716a,20triol, identified by MS as a trimethylsilyl derivative in the urine of normal adult men and women, patients with various disorders, and patients with adrenal carcinoma (Fantl et al., 1973). The latter group excreted the compound u p to 15 mg/24 hour compared to 50-220 pg/24 hours for all other subjects. No other steroid hormones were excreted in excess amounts in the urine of the patients with carcinoma. Possible explanations for the appearance of these metabolites, are given in the references cited. D. MISCELLANEOUS

1. Catecholamines Abnormal secretion and metabolism of dopamine, norepinephrine, and epinephrine have repeatedly been shown to b e related to pheochromocytoma and neuroblastoma. Fluorimetry, GC, and GUMS have been used to quantify catecholamines. In a technique developed by Wang et al. (1975a) blood is deproteinized with perchloric acid and free catecholamines are derivatized with trifluoroacetic anhydride after several steps of purification. For the determination of total catecholamine content, an additional step of hydrolysis is included. To avoid interferences, selected ions may be

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monitored with the MS. Both the free and total catecholamine content was found considerably higher in patients with pheochromocytoma and neuroblastoma than in normal subjects. The more specific detection resulted in values lower than previously reported indicating that interfering materials might lead to false positives. This work was extended (Wang et al., 1975b) to include 3-methoxytyramine, normetanephrine, and metanephrine. All three 3-0-methylated catecholamines were found in much larger quantities in the plasma of patients with neuroblastoma and, to a lesser extent, pheochromocytoma, than in that of normal subjects, e.g., 510 pmole/ml vs 4-10 pmole/ml for 3-methoxytyramine. 2. Bile Acids and Sterols Methodology for the profiling and quantification of bile acids, such as cholic, deaxycholic, and lithocholic acids, as well as sterols, such as cholesterol arid coprostanol, has been available (Sjovall et al., 1971)for the investigation of the suggested association between concentrations of fecal bile acids and cholesterol metabolites and colon cancer. The fecal excretion of cholesterol, coprostanol, and cholestane-3/3,5a76/3trio1 was higher in patients with ulcerative colitis than in other groups (Reddy et al., 1977). The main role of mass spectrometry in such investigations is the positive identification of all components included in the profiling.

3. Volatiles G U M S has been extensively used to analyze underivatized volatile components in breath, body fluids, and tissues, both to detect and quantify preselected individual constituents and to obtain profiles in searching for aberrations associated with disease states. The analytical methodology is well developed [Politzer et al. (1976) reviews all aspects] and is ready for applications. Lovett et al. (1976) conducted a pilot study to detect differences in the volatiles from urine and ascitic fluid of normal and tumor bearing (Ehrlich ascites) mice. Some 20 compounds were identified and about one-half of these are also known to exist in human urine. The relative amounts of several components in urine changed as the disease progressed. The profiles of ascitic fluids contained several unidentified peaks not present in the salinewashed abdominal cavities of normal mice.

4. Lymphocytes In linear programmed thermal degradation MS, a small sample is placed at the tip of a direct insertion probe, the entire sample is slowly evaporated by programmed temperature, and certain preselected mas-

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ses are monitored so that individual profiles are obtained. Some 10- 100 p g of lyophilized lymphocytes from various leukemia patients were placed on the solid probe with drops of methanol, and a number of degradation profiles were obtained upon heating. The profiles showed similarities between patients with the same disease, while considerable differences were found between cells of chronic lymphocytic and hairy cell leukemia, It was concluded that future work was warranted and the technique should also be applicable to isolates of other cellular fractions (Yergey et al., 1978).

5. Carbohydrates

The carbohydrate moiety of carcinoembryonic antigen samples from four different tumors were investigated by Coligan et a,?. (1976). Fucose, mannose, galactose, N-acetylneuraminic acid, and N-acetyglucosamine were released, permethylated, and analyzed as alditol acetates by gas chromatography-mass spectrometry. No qualitative differences were found when different antigen preparations were compared. On the bases of the quantitative data, certain constrains were proposed on the structure of the carbohydrate moiety of carcinoembryonic antigen. A mass spectrometric technique was developed for the quantification of neuraminidase-susceptible and total N-acetylneuraminic acid (NANA) in biological materials (Roboz et al., 1978b).NANA is determined as the fully silylated compound by monitoring the protonated molecular ion produced by isobutane chemical ionization. Unlike the commonly employed thiobarbituric method (based on colorimetric detection), this technique provides positive identification of NANA, eliminates all interferences b y 2-deoxyribose and other carbohydrates, and is capable of detecting as little as 400 pg (0.6 x mole) of NANA. The first application of the technique was the determination of neuraminidase-susceptible NANA in leukemic cells employed in chemoimmunotherapy of acute myelocytic leukemia. 6. Thymidine Zncorporation Heck et al. (1977) studied in wiwo synthesis of DNA in the rat b y incorporating multilabeled, nonradioactive thymidine into the DNA of cells undergoing replication. The isotopic composition of the thymine products were determined b y both field desoprion mass spectrometry and scintillation counting in various organs.

E. CONCLUSIONS Combined gas chromatography-mass spectrometry is the best tool available currently for the multicomponent analysis of environmental

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pollutants and exogenous and/or endogenous metabolites in body fluids. The technique has been used to search for (i) environmental carcinogens samples, (ii) metabolites of both carcinogens and antineoplastic agents both in vivo and in vitro, and (iii) biological markers of cancer. Once detected, suspected identities of individual compounds are confirmed by comparing complete mass spectra to those of authentic compounds. Unknowns are identified by determining exact masses to a precision of 1millimass unit, and calculating all possible mol.ecular compositions. The computerized technique of selected ion monitoring, particularly in combination with chemical ionization, provides a unique combination of high sensitivity (picomole range), specificity, and general applicability for the quantification of selected carcinogens, antineoplastic agents, metabolites, and endogenous contituents in crude environmental or biological samples .

SELECTEDGENERALBIBLIOGRAPHY Budzikiewicz, H., Djerassi, C., and Williams, D. (1967). “Mass Spectrometry of Organic Compounds.” Holden-Day, San Francisco, California. McFadden, W. ( 1973). “Techniques of Combined Gas Chromatography/Mass Spectrometry.” Wiley (Interscience), New York. McLaffetry, F. (1973). “Interpretation of Mass Spectra,” 2nd ed. Benjamin, New York. Roboz, J. ( 1968). “Introduction to Mass Spectrometry.” Wiley (Interscience), New York. Waller, G., ed. (1972). “Biochemical Applications of Mass Spectrometry.” Wiley, New York. Watson, J. (1976:).“Introduction to Mass Spectrometry: Biomedical, Environmental, and Forensic Applications.” Raven Press, New York.

REFERENCES Abramson, F. P. (1972).Anal. Chem. 44,28A-35A. Adlercreutz, H. ( 1974). In “Mass Spectrometry in Biochemistry and Medicine” (A. Frigerio and N. Castagnoli, eds.), pp. 165-181. Raven, New York. Adlercreutz, A. ( 1977). I n “Quantitative Mass Spectrometry in Life Sciences” (A. P. De Leenheer and R. R. Roncucci, eds., pp. 15-28. Elsevier, Amsterdam. Adlercreutz, H., Nieminen, U., and Ervast, H. S . (1974)./. Steroid Biochem. 5,619-626. Adlercreutz, H., Martin, F., Wahlroos, O., and Soini, E. (1975)./. Steroid Biochem. 6, 247-259. AEI (1976). Application Note No. MS30819. AEI Scientific Apparatus, Manchester, England. Ahlstrom, D. H., Kilour, R. J., and Liebman, S. A. (1975). Anal. C h m . 47, 114-1412. Alford, A. (1975a).In “Environmental Applications of Advanced Instrumental Analysis,” EPA-660/4-75-004 (Environ. Monit. Ser.). U.S. Environ. Protect. Agency, Corvalhs, Oregon. Alford, A. (1975b). Biomed. Mass Spectrom. 2 , 229-253. Alford, A. (1977). Biomed. Mass Spectrom. 4, (in press).

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Wishnok, J., and Tannenbaum, S. (1977).Anal. Chem. 49,715A-718A. Worzalla, J. F.,Johnson, B. M., Ramirez, G., and Bryan, G. T. (1973). Cancer Res. 34, 2669-2674. Worzalla, J. F., Kaiman, B. D., Johnson, B. M., Ramirez, G., and Br)ian, G. T. (1974). Cancer Res. 34, 2669-2674. Wu, A., Au, Jo, and SadBe, W. (1978).Cancer Res. 38, 210-214. Yang, S. K., and Gelboin, H. V. (1976). Cancer Res. 36, 4185-4189. Yang, S. R., McCourt, M. D., Roller, P. P., and Gelboin, H. V. (1976). Proc. Natl. Acad. S C ~ U.S.A. . 73, 2594-2598. Yergey, A., Risby, T., and Golomb, H. (1978). Biomed. Mass Spectrom. 5, 47-51. Yurachek, J. P., Clemena, G. G., and Harrison, W. W. (1969).Anal. Chem. 41,1666-1668. Zumoff, B., Fischman, J., Bradlow, H. L., and Hellman, L. (1975). Cancer Res. 35, 3365-3373.

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ADVANCES I N CANCER RESEARCH, VOL. 27

MARROW TRANSPLANTATION IN THE TREATMENT OF ACUTE LEUKEMIA1s2 E. Donnall Thornas: C.Dean Buckner, Alexander F e f e ~Paul . ~ E. Neirnat~,~ and Rainer Storb The Fred Hutchinson Cancer Research Center, Seattle, Washington and the Department of Medicine, Division of Oncology, University of Washington School of Medicine, Seattle, Washington

I. Introduction .......................................................... 11. Patient Selection, Methods, and Summary of Clinical Results ............ 111. Analysis of Survival ................................................... IV. Nature of Recurrent Leukemia.. ....................................... V. Efforts to Prevent Leukemic Relapse ................................... VI. Graft versus Leukemia ................................................ VII. Transplantation in Remission .......................................... VIII. Conclusions .............................. References ...............................

269 270 271 273 275 276 277 278 278

I. Introduction

Almost 30 years have elapsed since the experiments of Jacobsen, Lorenz, and their colleagues demonstrated that mice could be protected against otherwise lethal irradiation b y shielding of the spleen or by intravenous infusion of marrow. By the mid 1950s, convincing evidence had accumulated indicating that the protective effect was due to the colonization of the recipient marrow b y donor cells. The implications of these laboratory studies for clinical application in man were 1957). A review of the early immediately apparent (Thomas et a?., studies on human marrow transplantation for the treatment of hematological disorders showed little or no evidence of success (Bortin, 1970). Work in the past decade, however, has produced a number of long-term survivors of marrow transplantation. The basic principles

* This investigation was supported by Grants CA 18029 and CA 15704, awarded by the National Cancer Institute, DHEW. Abbreviations used in this chapter are as follows: ALL, acute lymphoblastic leukemia; AML, acute myelogenous leukemia; CML-BC, chronic myelogenous leukemia in blast crisis; CY, cyclophosphamide; GVHD, graft-versus-host disease; and TBI, total body irradiation. Dr. Thomas is a recipient of Research Career Award A1 02425 from the National Institute of Allergy and Infectious Diseases. Dr. Fefer is an American Cancer Society Professor of Clinical Oncology. Dr. Neiman is a Scholar of the Leukemia Society of America. 269 Copyright @ 1978 h y Academic Press, Inc. All rights of reproduction in any form rererved.

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of marrow transplantation biology and its clinical application have been summarized in recent reviews (Thomas et al., 1975, 1977b). The present review is concerned with the clinical results of marrow transplantation in the treatment of patients with acute leukemia and the observations that have been made concerning the pathophysiology of acute leukemia. Chemotherapy of human leukemia is usually based on the premise that the therapeutic effect is due to the killing of leukemic cells. Since normal marrow cells are also killed by these drugs, the physician and the patient must tread a narrow path between therapeutic benefit and preservation of marrow function (Holland et al., 1976). I n theory, marrow transplantation should make it possible to kill the greatest number of leukemic cells with unusually large amounts of irradiation or chemotherapeutic drugs without regard to damage to normal marrow cells since the marrow transfused after therapy will restore normal marrow function. In addition, the transplanted marrow, since it is immunologically competent, might help in killing any residual leukemic cells by an immune attack directed at transplantation antigens or leukemia associated antigens on the leukemic cells. II. Patient Selection, Methods, and Summary of Clinical Results

Table I summarizes the results of marrow transplantation for 153 cases of acute leukemia carried out in Seattle between February of 1969 and October of 1975. The patients had either acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), or chronic myelogenous leukemia in blast crisis (CML-BC). Donors were either syngeneic (identical twin) or allogeneic (genotypically HLA matched sibling confirmed by nonreactivity in mixed leukocyte culture). All patients were given 1000 rad total body irradiation (TBI) from opposing 6oCosources shortly before the marrow infusion. Fourteen patients were given TBI only. Almost all the rest received cyclophosphamide [(CY), 60 mg/kg on each of 2 days] 4 and 3 days before TBI. Many patients in addition received other forms of antileukemic therapy either in an unsuccessful attempt to induce remission or to destroy leukemic cells prior to transplantation. Details of the preparation for engraftment and the transplant procedure have been presented elsewhere (Thomas and Storb, 1970; Thomas et al., 1975). Following engraftment, recipients of allogeneic marrow were given methotrexate for u p to 100 days postgrafting in an effort to reduce the incidence and severity of graft-versus-host disease (GVHD) (Storb et at., 1970). Recipients of syngeneic marrow were not given methotrexate but many

27 1

MARROW TRANSPLANTATION

TABLE I SEATTLEMARROWTRANSPLANT SUMMARY IN PATIENTS WITH ACUTE LEUKEMIAAS OF JUNE, 1977 Survival

Disease ALL” AML” CML-BCe a

Number

Donor Twinb HLA matched Twind HLA matched Twin HLA matched

15 52 14 58 5

10

Number with Number recurrent alive at leukemia 1 year 8 21 8 15 3 0

8 16

4 10 2 2

Number now alive

Longest living survivor (years)

5 9“ 3 6 1 2

7 7 5 5 1 1

Patients transplanted through October, 1975. Includes 2 patients with lymphosarcoma-leukemia. Includes 3 patients with recurrent leukemia after transplantation now in remission. Includes 1 patient with erythroleukemia evolving into AML. Patients transplanted through April, 1976.

received “immunotherapy” in the form of autologous killed leukemic cells plus added donor lymphocytes (Fefer et al., 1974). It should be emphasized that all of these patients had had extensive prior chemotherapy for leukemia, many had had prophylactic or therapeutic central nervous system irradiation, and almost all were in relapse at the time of transplantation (Thomas et al., 1977a). Ill. Analysis of Survival

Figure 1 shows a (Kaplan and Meier, 1958) plot of the survival of patients with ALL or AML who received either syngeneic (29 patients) or allogeneic (110 patients) marrow grafts. In the first 4 months after grafting, there was a significant mortality rate due to the complications of advanced illness at the time of grafting to recurrent leukemia, and, in the case of the allogeneic recipients, to complications of GVHD. Thereafter, in the first 2 years, the slope of the survival curve represents primarily loss of patients due to recurrent leukemia. The one death shown at 4 years is that of a patient with a central nervous system relapse 27 months after grafting. Most important, after approximately 2 years, the survival curve becomes almost flat. The flat survival curve extending from 2 years to 7 years constitutes an opera-

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a

3

4

12.5

- 8

1 0

\ ALLOGENEIC

1

2

3

4 YEARS

5

6

7

FIG. 1. Kaplan-Meier product limit estimates of the percentage surviving for 110 patients as of June, 1977. Day 0 is the day of marrow transplantation. The circles indicate surviving patients.

tional definition of cure in these patients, particularly since no maintenance chemotherapy was given after marrow grafting (Thomas et al., 1977c; Fefer et al., 1977). An analysis of a wide range of factors that might predict whether or not a patient will survive for the first 100 days after marrow grafting is underway. As yet, in this group of endstage patients, critical factors determining survival have not been identified. The most significant factor is an overall assessment of the general clinical condition of the patient at the time of grafting. Patients in good clinical condition in early relapse have a much better chance of survival than do patients in advanced relapse with lack of platelets and granulocytes and associated problems of infection and bleeding (Thomas et al., 1977a). Figure 2 shows a plot on a semilogarithmic scale of the probability that a patient will be in remission on a given day provided that he has lived to reach that particular day. This method of presenting the data permits the elimination of deaths from competing causes (such as GVHD and infection), The slopes of the curves thus reflect the rate of leukemic relapse. The results show that the rate of leukemic relapse is quite constant during the first lY2 years postgrafting. However, the curves are flat after approximately 2 years indicating no further relapses, The curve for the syngeneic graft recipients is not statistically significantly different from the curve for recipients of allogeneic grafts. The hypothesis that syngeneic marrow might be just as susceptible to a leukemogenic factor as the patient's own original marrow and the

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MARROW TRANSPLANTATION

ALLOCENEIC

a

I/12.5 0

1

2

3 4 YEARS

5

6

1

FIG.2. Kaplan-Meier product limit estimates of probability of being in remission. The circles indicate living patients in remission. The longest time to relapse with a syngeneic graft was I4 months and with an allogeneic graft 27 months.

related hypothesis that an allogeneic marrow graft might have a lesser susceptibility are not supported by these data. Rather, it would appear that the apparent eradication of leukemia in some patients, and the failure of eradication in others, is a reflection of the sensitivity of the leukemic cell population to the chemotherapy and TBI used in preparation for engraftment. At the present time there is no information to indicate whether sensitivity or resistance is a characteristic of the original leukemic cell population or is acquired during the course of therapy.

IV. Nature of Recurrent Leukemia

With a syngeneic marrow graft, donor and recipient are identical with regard to all blood genetic markers. Accordingly it is impossible to tell whether the recurrent leukemia represents regrowth from an original clone or represents reinduction. With an allogeneic graft, especially with a donor of opposite sex, it is possible to identify the origin of both the normal and abnormal cells following marrow grafting. In patients given 1000 rad TBI, the normal marrow cells seen after grafting have invariably been entirely of the donor type. During our early experience with marrow transplantation we observed a mechanism of leukemic relapse which was completely unanticipated, and which has significant implications for the etiology and pathogenesis of this disease (Fialkow et al., 1971;Thomas et al., 1972). Among six patients with ALL who were prepared with 1000 rad TBI alone, 2 were females who redeveloped active leukemia 62 and 135

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days following transplantation. Both of these individuals received allogeneic marrow &om histocompatible male siblings within 24 hours of irradiation. The normal marrow cells present after grafting and subsequently the leukemic cells after relapse were shown to have metaphases containing a Y chromosome and, in the second case, fluorescent staining of the leukemic cells showed the presence of a Y body. Apparently the leukemic cell population represented a malignant transformation of lymphoid cells in the donor marrow. Alternatively, it is possible that such an event represented a transfer of the Y chromosome to a surviving remnant of the original leukemic cell population following a cell fusion event. The occurrence of such an event in two of six comparable individuals, however, requires one to propose a fairly specific and complex series of steps for which no experimental evidence presently exists. Experimental model systems do suggest other possible explanations involving transformation of donor stem cells, one of which would be the existence of a leukemogenic virus resident in the host, and perhaps induced by the TBI. Unfortunately, there is at present no unambiguous evidence for the existence of infectious leukemogenic viruses in man, inducible or otherwise. While these apparent marrow graft transformation events may provide encouragement to those pursuing an infectious viral theory of leukemogenesis, recent research in tumor virology suggests alternative mechanisms. Several investigators have demonstrated that DNA proviruses or oncogenic retroviruses can transform target cells when such cells are exposed to free DNA from virus-infected cells (Hill and Hillova, 1972; Cooper and Temin, 1974). Further, this transforming potential of viral “oncogene”-containing DNA may be preserved in the absence of overt viral replication in the cells bearing the infectious provirus. It is intriguing, in this connection, that marrow graft transformation has not occurred in the 11 episodes of leukemic relapse in patients with donor of opposite sex that we have had the opportunity to study since the two original episodes. It may be significant that the protocols for preparation of engraftment in the later patients all incorporate significant cytoreduction prior to irradiation. Thus, the level and duration of exposure of newly infused marrow stem cells to material released from the dying leukemic cell mass may have been substantially reduced. The transfer of genes by free DNA is called “transfection,” and has, so far, only been demonstrated for viral genes. It remains possible that somatic cellular genes responsible for malignant transformation might have been transfected as well. One attractive aspect of the transfection theory is that, unlike the competing ideas mentioned above, it may prove susceptible to direct experimental testing.

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V. Efforts to Prevent Leukemic Relapse

In the initial group of 10 patients in the Seattle series prepared with TBI only, 4 patients with AML died too early to be evaluated for leukemic relapse while 5 of 6 patients with ALL suffered a relapse of leukemia. Therefore, in an effort to destroy more leukemic cells, high dose CY was added to the regimen before irradiation. In a group of 43 patients given CY plus TBI only, 14 relapsed. Other antileukemic drugs have been given before CY and TBI in an effort to reduce the leukemic cell population further. Depending on past drug exposure, 31 patients were given a variety of cytoreductive regimens, usually including cytosine arabinoside and rubidomycin prior to CY and TBI, and 11 relapsed. Nineteen patients were given 8-14 mg/kg 1,3bis(2-chloroethy1)-l-nitrosourea prior to CY and TBI. Three have relapsed but only 1 is a long-term survivor. In a more recent unpublished study, 7 patients were given a 5-day continuous infusion of cytosine arabinoside, 600 mg/m2/day, before CY and TBI. One of 7 patients survived over 100 days and he relapsed 1 year after transplantation. In an effort to avoid the problems of GVHD, Math6 et al. (1970) treated 11leukemic patients with antilymphocytic serum before infusing marrow from related donors, only 4 of whom were HLA identical with the recipient. Although GVHD was not observed, the grafts were transient and 10 surviving patients showed recurrent leukemia. This study points out the need for some form of leukemic cell cytoreduction in the preparative regimen. Other investigators have utilized vigorous chemotherapy instead of TBI before transplantation. In studies of a series of acute leukemia patients, Santos et al. (1976) used 50 mg/kg of CY given on each of 4 days in 25 patients and Graw et al. (1972)used 45 mg/kg of CY x 4 in 9 patients. All patients who survived developed recurrent leukemia. Graw et al. (1974) developed a BACT (bis-chloroethyl-nitrosourea, cytosine arabinoside, CY, 6-thioguanine) regimen. Ten patients were treated with this regimen, which proved to be very toxic with 5 deaths before day 35 (Buja et al., 1976). Four patients died with recurrent leukemia at 30-437 days postgrafting. However, one patient who had endstage AML is alive and well on no maintenance chemotherapy now 5 years after transplantation (Bleyer et al., 1975). The UCLA marrow transplant group has developed a regimen known as SCARI (6-thioguanine, cytosine arabinoside, rubidomycin, CY, TBI) which employs high dose chemotherapy prior to CY and TBI. Fifteen patients were treated with SCARI (UCLA Bone Marrow Transplantation Group, 1977). The regimen proved to b e quite toxic

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E. DONNALL THOMAS ET AL.

especially for adult patients. However, the preliminary results show 4 patients living at approximately 1 year after grafting and only 2 patients have suffered a relapse of leukemia. From these observations it appears that none of the regimens designed to increase the killing of leukemic cells has resulted in any overall improvement in survival in comparison to CY and TBI. Efforts to kill more leukemic cells in these endstage patients in relapse carry the price of increased toxicity. Since most of these patients have had a variety of chemotherapeutic agents before coming to marrow transplantation, it is difficult to find an effective drug to include in the regimen that has not already been employed. Dimethyl myleran is a drug that may show some promise in this regard (Floersheim, 1969; Buckner et al., 1975; Einstein et al., 1976; Kolb et al., 1974). It is an effective antileukemic agent which was abandoned because of excessive marrow toxicity (Bierman et al., 1958; Clifford et al., 1964). With the marrow transplantation regimen, excessive marrow toxicity can be ignored and preliminary studies to evaluate dimethyl myleran given before CY plus TBI are underway. VI. Graft versus Leukemia

Table I1 shows the incidence and seventy of GVHD in the 100 patients with acute leukemia transplanted after CY and TBI. One-half of the patients had significant GVHD, a continuing major problem in marrow transplantation biology which has been reviewed elsewhere (Thomas et al., 1975). Of particular interest here is the possible antileukemic effect of GVHD. Barnes et al. (1956) pointed out that a small residual population of leukemic cells might be killed off by a reaction of the engrafted marrow against the leukemic cells. Subsequently Math6 et al. (1965) enlarged upon this concept and coined

GRADEOF GVHD

AND

TABLE I1 RELATION TO RELAPSE OF LEUKEMIA"

Grade of GVHDb

Number of patients without relapse

Number of patients with relapse

0 I 11-IV

7 11 44

12 13 6

Ninety-three of 100 patients with acute leukemia who lived long enough to be evaluated after CY, TBI, and allogeneic marrow grafting. Grade 0, no CVHD; I, skin rash only; 11-IV GVHD in 2 or more target organs.

MARROW TRANSPLANTATION

277

the term “adoptive immunotherapy.” More recently Bortin (1974) and Fefer et al. (1976) have presented evidence in support of a graftversus-leukemia effect in murine systems. Table I1 shows the incidence of leukemic relapse in relation to GVHD in our patients given allogeneic grafts. It is apparent that some patients who had no identifiable GVHD have not had a relapse of leukemia. Many patients with severe GVHD do not survive long enough to have an opportunity to suffer a leukemic relapse. However, as indicated in Table I1,6 patients have suffered a relapse of leukemia in the face of severe active GVHD. Certainly, in these recipients of marrow from a donor matched at the major histocompatibility complex, if a graft-verus-leukemia reaction occurred it was not sufficient to prevent leukemic relapse. Figure 2 demonstrates a comparison of the leukemic relapse rate in patients given syngeneic marrow as compared to patients given allogeneic marrow and does not show a significant difference between these two groups. These data do not suggest that an allogeneic marrow graft had an antileukemic effect. This conclusion is only tentative, however, in view of the increased death rate from other causes associated with GVHD in the recipients of allogeneic marrow and of the unknown role of immunotherapy in the recipients of syngeneic marrow. VII. Transplantation in Remission

Since marrow transplantation appears to be curative for some patients with endstage acute leukemia, it is now ethically acceptable to consider transplantation earlier in the course of the disease. Marrow transplantation for patients in remission who are known to have a poor prognosis would entail the following advantages : ( 1) treatment before the leukemic cell population becomes resistant to therapeutic modalities, (2)treatment at a time when the body burden of leukemic cells is minimal, and (3) treatment when the patient is in excellent clinical condition and, therefore, better able to tolerate the antileukemic and transplantation regimes. In 1976 the Seattle marrow transplant team began to perform allogeneic marrow grafts in patients in remission after preparation with CY plus TBI. Almost all of the patients with ALL have had more than 1 relapse either in the marrow or in extramedullary sites. The majority of the patients with AML have been in the first remission. As expected, these patients tolerated the transplant regimen very well. The support requirements for platelet and white blood cell transfusions and time in the hospital were strik-

278

E. DONNALL THOMAS ET AL

ingly reduced. The earliest death was on day 62 following interstitial pneumonia due to cytomegalovirus. It is clearly too early to evaluate the results of this continuing study since the initial patients are now just beyond 1year after grafting. The crucial question of whether or not the increased risks of marrow grafting and GVHD and its complications can be balanced b y long-term remission without continued chemotherapy, and even “cure” in some patients, will take several years to answer. VIII. Conclusions

Marrow transplantation has now become established as a therapeutic option of definite benefit to some patients with acute leukemia. For the patient with an identical twin donor, marrow transplantation appears to be the treatment of choice since the complete remission rate even in adults with acute leukemia is approximately 90%. Some progress is being made toward resolution of the problems of transplantation biology that occur even though the sibling donor-recipient pairs are matched at the major histocompatibility complex. Efforts to reduce the incidence of recurrent leukemia by more intensive antileukemic therapy or b y transplantation in remission are under way. Marrow transplantation makes it possible to ignore the complications of marrow toxicity which usually limit the chemotherapy of leukemia. This will permit the exploration of other drug regimens and of drugs considered to be too toxic for ordinary use. Marrow transplantation remains a complex undertaking that requires a skilled team of physicians of many disciplines, nurses, and technologists. Pending development of better methods of specific treatment or prevention of hematologic malignancies, marrow transplantation is likely to be undertaken for an ever increasing number of patients in the coming decade.

REFERENCES Barnes, D. W. H., Corp, M. J., Loutit, J. F., and Neal, F. E. (1956).Br. A4ed.J.2,626-627. Bierman, H. R., Kelly, K. H., Knudson, A. G., Jr., Maekawa, T., and Timmis, G. M. (1958).Ann.N . Y.Acad. Sci. 68, 1211-1222. Bleyer, W. A., Blaese, R. M., Bujak, J. S., Herzig, G. P., and Graw, R. G., Jr. (1975).Blood 45. 171-181. Bortin, M. M. (1970). Transplantation 9, 571-587. Bortin, M. M. (1974). Clin. Immunobiol. 2, 287-306. Buckner, C. D., Ilillingham, L. A,, Giddens, W. E., Jr., and Thomas, E. D. (1975).Erp. Hematol. (Copenhagen) 3, 275-288. Buja, L. M., Ferrans, V. J., and Graw, R. G., Jr. (1976). Hum. Pathol. 7, 17-45.

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Clifford, P., Clift, R. A., Kahn, A. C., and Temmis, G. M. (1964). Br. J. Cancer 13, 435-438. Cooper, G. M., andTemin, H. M. (1974).J.Virol. 14, 1132-1141. Einstein, A. B., Jr., Cheever, M. A., and Fefer, A. (1976).J. Natl. Cancer Inst. 56, 609-613. Fefer, A., Einstein, A. B., Thomas, E. D., Buckner, C. D., Clift, R. A., Glucksberg, H., Neiman, P. E., and Storb, R. (1974).N . Engl. J. Med. 290, 1389-1393. Fefer, A., Einstein, A. B., Jr., and Cheever, M. A. (1976).Ann. N.Y. Acad. Sci. 277, 492-504. Fefer, A., Buckner, C. D., Thomas, E. D., Cheever, M. A., Clift, R. A., Glucksberg, H., Neiman, P. E., and Storb, R. (1977). N. E n g l . J. Med. 297, 146-148. Fialkow, P. J., Thomas, E. D., Bryant, J. I., and Neiman, P. E. ( 197l).Lancet 1,251-255. Floersheim, G. L. (1969). Lancet 1, 228-233. Craw, R. G., Jr., Yankee, R. A., Rogentine, G. N., Leventhal, B. G., Herzig, G. P., Halterman, R. H., Merritt, C. B., McGinniss, M. H., Krueger, G. R. D., Whang-Peng, J., Carolla, R. L., Gullion, D. S., Lippman, M. E., Gralnick, H. R., Berard, C. W., Terasaki, P. I., and Henderson, E. S. (1972).Transplantation 14, 79-90. Craw, R. G., Jr., Lohrmann, H.-P., Bull, M . I., Decter, J., Herzig, G. P., Bull, J. M., Leventhal, B. G., Yankee, R. A., Herzig, R. H., Krueger, G. R. F., Bleyer, W. A,, Buja, hl. L., McGinniss, M. H., Alter, H. J., Whang-Peng, J., Gralnick, H . R., Kirkpatrick, C. H., and Henderson, E. S. (1974).Transplont. Proc. 6, ,349-354. Hill, M., and Hillova, J. (1972).Nature (London), New Biol. 237,35-39. Holland, J. F., Glidewell, O., Ellison, R. R., Corey, R. W., Schwartz, J., Wallace, H. J., Hoagland, H. C., Wiernik, P., Rai, K., Bekesi, J. G., and Cuttner, J. (1976). Arch. Intern. Med. 136, 1377-1381. Kaplan, E. L., and Meier, P. (1958).J.A m . Stat. Assoc. 53, 457-481. Kolb, H. J., Storb, R., Weiden, P. L., Ochs, H. D., Kolb, H., Graham, T. C., Floersheim, G. L., and Thomas, E. D. (1974). Biomedicine 20, 341-351. MathC, G., Amiel, J. L., Schwanenberg, L., Cattan, A., and Schneider, M. (1965). Cancer Res. 25, 1525-1531. MathC, G., Amiel, J. L., Schwarzenberg, L., Choay, J., Trolard, P., Schneider, M., Hayat, M.. Schlumberger, J. R., and Jasmin, C. (1970). Br. Med.1. 2, 131-136. Santos, G. W., Sensenbrenner, L. L., Anderson, P. N., Burke, P. J., Klein, D. L., Slavin, R. E., Schacter, B., and Borgaonkar, D. S . (1976). Transplant. Proc. 8, 607-610. Storb, R., Epstein, R. B., Graham, T. C., and Thomas, E. D. (1970). Transplantation 9, 240-246. Thomas, E. D., and Storb, R. (1970). Blood 36, 507-515. Thomas, E. D., Lochte, H. L., Jr., Lu, W. C., and Ferrebee, J. W. ( 1957).N. EngZ.J. Med. 257,491-496. Thomas, E. D., Bryant, J. I., Buckner, C. D., Clift, R. A., Fefer, A., Johnson, F. L., Neiman, P., Ramberg, R. E., and Storb, R. (1972). Lancet 1, 1310-1313. Thomas, E. D., Storb, R., Clift, R. A., Fefer, A., Johnson, F. L., Neiman, P. E., Lerner, K. G., Glucksberg, H., and Buckner, C. D. (1975). N. Engl. J . Med. 292, 832-843 and 895-902. Thomas, E. D., Buckner, C. D., Banaji, M., Clift, R. A., Fefer, A., Floumoy, N., Goodell, B. W., Hickman, R. O., Lerner, K. G., Neiman, P. E., Sale, G . E., Sanders, J. E., Singer, J., Stevens, M., Storb, R., and Weiden, P. L. (1977a).Blood 49,511-533. Thomas, E. D., Fefer, A., Buckner, C. D., and Storb, R. (1977b). Blood 49,671-681. Thomas, E. D., Flournoy, N., Buckner, C. D., Clift, R. A., Fefer, A,, Neiman, P. E., and Storb, R. ( 1 9 7 7 ~ )L. e d . Res. 1,67-70. UCLA Bone Marrow Transplantation Group. (1977).Ann. Intern. Med. 86, 155-161.

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ADVANCES IN CANCER RESEARCH, VOL. 27

SUSCEPTIBILITY OF HUMAN POPULATION GROUPS TO COLON CANCER Martin Lipkin Memorial Sloan-Kettering Cancer Center, New York. New York

I. Introduction .......................................................... 11. The Role of Environment in Increasing the Susceptibility of Individuals to Colon Cancer. .................................. 111. Inherited Diseases That Increase Susceptibility to Colon Cancer.. . . . . . . . Inherited Adenomatosis and Related Disorders ......... IV. Proliferative Abnormalities and Susceptibility to Colon Cancer .......... V. Newer Immunologic Studies .......................................... VI. Nuclear Protein and Enzyme Alterations ............................... VII. Studies of Cutaneous Cells ............................................ VIII. Examination of Fecal Contents ........................ IX. Conclusion ........................................................... References ..................................

281 287 293 296 296 299 301

I. Introduction

Colorectal cancer poses a major problem in the United States and other countries. It is currently responsible for a high proportion of the malignant neoplasms found in the United States. Recent figures have indicated 99,000 new cases in the United States in 1975 and 49,000 resultant deaths (Silverberg and Holleb, 1975).The relative frequency of successful treatment of individuals varies with the stage at which the disease is detected; a higher rate of success is related to detection of disease at an earlier stage (Gilbertsen, 1974). Earlier detection of disease in the population aggregates having increased risk (listed in Table I) could reduce the burden denoted by the above figures. Recent studies indicate that improvements have been made in the identification of factors that are associated with the predisposition and development of colorectal cancer in several of these population groups. The early identification of abnormalities in cell development and related parameters, in individuals having high susceptibility to colon cancer can be carried out with greater precision than was formerly possible. Recent approaches to the identification of these abnormalities are enumerated in Table 11. This chapter will discuss current findings in this area, their contribution to our under28 1 Copyright Q 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 012-006627-0

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MARTIN LIPKIN

TABLE I POPULATION GROUPS AT INCREASED RISK FOR COLON CANCER Familial Polyposis Syndromes Inherited adenomatosis of colon and rectum Gardner’s syndrome (Oldfield) Turcots syndrome Diffuse gastrointestinal polyposis Colonic adenomas Previous colon, breast, endometrial, bladder cancer Site-specific colon cancer Cancer family syndrome Inflammatory bowel disease Common variable immune deficiency Residence in geographic areas having high frequencies of colon cancer

standing of the development of neoplasia in man, and their potential role in early detection and prevention of the disease. I I . The Role of Environment in Increasing the Susceptibility of Individuals to Colon Cancer

It is now well established that environment plays a role in the development of colorectal cancer. Numerous studies have shown a strong correlation between geographic, economic, and dietary exposure and colon cancer, and programs of surveillance are desirable for high-risk groups. Epidemiological studies of populations at high and low risk for colon cancer have shown striking differences in the composition of the diet in these population groups. In industrialized countries including those of northwest Europe and North America a great deal of animal TABLE I1 RECENT L4PPROACHESTO IDENTIFICATION OF HIGH RISK GROUPS Prior to clinically detectable lesions Study of environmental factors Proliferative abnormalities Immunologic measurements Study of cutaneous cells Nuclear protein and enzyme alterations Examination of fecal contents After clinically detectable lesions Hemoccult, radiologic, and endoscopic studies

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fat, protein, and refined carbohydrates are consumed; in these geographic regions the incidence of colon cancer is much higher than in the developing countries of Africa, South America (except meat-eating Argentina and Uruguay), rural India, and Japan, where much less meat is consumed and the diet is higher in vegetable fiber (Berg, 1973; Doll, 1969; Haenszel and Correa, 1971; Haenszel et al., 1973;Wynder and Shigematsu, 1967). These variations in incidence do not appear to be related to genetic differences, because migrant groups tend to assume colon cancer incidence rates of their adopted countries (Haenszel and Kurihara, 1968). In studies of Japanese Issei (first generation) and Nisei (subsequent generation) migrants in Hawaii, a higher incidence of large bowel cancer was observed in individuals who no longer continued the practice of eating at least one Japanese style meal daily (Haenszel et al., 1973). Colon cancer patients appeared to consume more beef and legumes than did controls. A rise in the consumption of meat accounted for the major difference in dietary habit between residents of Japan and Hawaii, and the increase in beef consumption paralleled the higher risk of bowel cancer among Japanese migrants. A correlation between the daily consumption of meat and the incidence of colon cancer also has been noted in individuals from many countries, suggesting an etiological role (Armstrong and Doll, 1975; Wynder and Shigematsu 1967). Observations of this type have led investigators to postulate that high animal protein and fat, characteristic of high-risk populations, are responsible for colon cancer. In one study of 28 countries positive correlations were found between the incidence of colon cancer and the amount of meat the various populations consumed (Gregor et al., 1969). On the basis of findings of this type, it has been postulated that the high incidence of colon cancer is due to the nature of the intestinal flora, which might possibly synthesize carcinogenic agents from food, and intestinal secretions such as bile acids. Accordingly, diet not only would be expected to influence the composition of the intestinal flora, but also the quantity of substrates available for the production of carcinogens. Burkett and his colleagues have also postulated that fiber content of diet may be associated with the development of colon cancer (Burkitt, 1975). Studies correlating the selenium levels with incidence of colon cancer are of recent interest (Andrews et UI?., 1968; Jacobs et al., 1976; Jansson et al., 1976).Previous epidemiologic studies had shown a high correlation between geographic regions having deficiency of selenium and increased incidence of colon cancer (Andrews et al., 1968; Jacobs et al., 1976; Jansson et al., 1976). Experimental carcinogenesis also has

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been inhibited by selenium administration (Harr et al., 1976; Schrauzer and Ishmael, 1974). Recently, addition of 4 ppm of selenium in drinking water reduced the number of rats developing dimethylhydrazine-induced colon tumors, and the total number of methylazoxymethanol-induced rat tumors (Jacobs et al., 1976). It is also true that other factors in these environments, for example the prevalence of infectious disease as well as other chronic illnesses, vary greatly among these populations. In addition, it is known that other factors not related to food also change simultaneously with dietary habits, in relation to industrial and economic development. The epidemiological evidence available can therefore suggest further studies that should be carried out, but does not supply definitive proof that any given dietary factors are causally related to variations in the incidence of colon cancer. In attempting to explore these leads to elucidate mechanisms related to pathogenesis, studies have been carried out to examine the fecal flora obtained from individuals in different parts of the world. Fecal samples were obtained from individuals residing in England, Scotland, and the United States, regions having high incidence of colon cancer, and from Uganda, India, and Japan where low incidence of colon cancer occurs. Although the same broad classifications of bacteria were found in feces from all the populations studied, differences in relative numbers of several of the bacterial groups were observed. The British and American subjects had more gram-negative anaerobes than did the Ugandans, Indians, or Japanese; in contrast, the latter had larger amounts of aerobic bacteria. The ratio of anaerobes to aerobes was therefore higher in individuals consuming a Western style diet than in those consuming largely vegetarian diets (Hill et al., 1971). The concentrations of acid and neutral steroids in the feces of those subjects also differed when individuals on high and low meat diets were compared. Fecal specimens from British and Americans on high meat diets contained higher amounts of steroids than feces of Ugandans, Indians, and Japanese, whose diets contained little or no animal fat and protein. Neutral steroid amounts were low in the feces of Ugandans and Indians, intermediate in the feces of Japanese, and high in the feces of British and Americans. I n addition, microbial conversion products of cholesterol, coprostanol and coprostanone, contributed a smaller amount to the total neutral steroid content of the feces of the Ugandans, Indians, and Japanese than the feces of the Western group. Interesting differences also were noted in the fecal composition ofacid steroids. The acid steroid concentrations were greater in the feces of British and Americans than in the Ugandans, Indians, and Japanese. The extent of con-

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285

version of acid steroids also appeared to be higher in the British and Americans than in other groups. Further observations were made in which daily fecal excretion of cholesterol metabolites was believed to be grater in Americans who consume a diet containing meat, than in Americans on a meatless diet (Reddy and Wynder, 1973). Despite these observations, other studies have suggested no significant differences in bacterial counts or species isolated from the feces of volunteers on high meat and meatless diets. When individuals at high risk were compared with low-risk populations, no major differences in varieties of intestinal bacteria were found. Minor quantitative variations were present, but characteristic organisms in high- or lowrisk groups were not described, and organisms present in individuals on high-risk diets were also found in individuals on low-risk diets. It was suggested that taxonomic grouping of bacteria is not important in analyzing the effects of diet on intestinal flora, but that the effect of altered diet on bacterial metabolic activity might be of interest, and in fact more useful in trying to assess significant factors present (Feingold et al., 1974; Moore and Holdeman, 1975). High-risk populations had a more heterogenous and variable microflora. Alterations in the flora were also believed to be effected by situations which produced anger or stress in the host. Similarly, concentrations of acid or neutral steroids and their metabolites were minimally effected during high meat compared to meatless diets (Hentges et al., 1976). However, some individuals excreted significantly higher amounts of cholesterol than the others. It has thus been suggested that ingestion of high animal protein itself does not result in major change in bacterial and chemical composition of feces. On the other hand, fat content may be more important since acid and neutral steroid concentrations were observed to decline when fat megt in the diet was replaced by lean meat (Hill, 1971; Reddy and Wynder, 1973). Secondary bile acids in the feces were also higher during fat meat ingestion when the concentrations of total bile acids were high, leading to the possibility that secondary bile acids are products of the dehydroxylation of primary fecal bile acids by intestinal bacteria. In further support of the above, high fat, high meat mixed Western diet, and nonmeat diet were compared in human volunteers for steroid content of feces (Reddy et al., 1975a), while protein content of both diets was similar. Findings indicated that total anaerobic microflora count, and fecal excretion of secondary bile acids and cholesterol metabolites, were greater during consumption of the mixed Western diet than the nonmeat diet, supporting a role of dietary fat on composition of intestinal flora and level of steroid conversion products in feces. Thus, individuals with colon cancer have been reported to have

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MARTIN LIPKIN

higher amounts of steroids in their feces than controls without cancer, and steroid conversion products such as deoxycholic acid, lithocholic acid, and cholesterol metabolities. Of further interest is the observation that the activity of fecal 7 a-dehydroxylase is higher in patients with colon cancer compared to controls, in association with conversion of cholic and chenodeoxycholic acids to deoxycholic and lithocholic acids. In individuals with cancer of the large bowel a high frequency were reported to have concentrations of bile acids in their feces greater than individuals with other diseases Hill et al., 1975; Reddy et al., 1975a). The colon cancer patients also had greater amounts of acid steroids in the form of secondary bile acids than did the other patients. These findings continue to support the view that an association of fecal steroids and the production of colon cancer is significant. In addition, it has been reported (Wilkins and Hackman, 1974) that normal North Americans shows two patterns of neutral sterol conversion as measured by fecal analysis: “High converters” appear to have a stable pattern of extensive conversion of cholesterol, sitosterol, and campesterol by the intestinal flora to degradation products, while “low converters” have little or no such conversign. This finding further points to the possibility that genetic, as well as environmental factors influence neutral sterol conversion. Other recent work has indicated that bile salt excretion can be affected differently by various dietary sources, and feeding wheat bran decreased the cholesterol saturation of bile, apparently b y increasing hepatic synthesis of chenodeoxycholic acid (Pomare et al., 1974). Related animal studies have been of interest in that rats fed high fat diets have been reported to be more susceptible to colon tumor induction by 1,2-dimethylhydrazine (DMH) (and more recently by azoxymethane) than animals fed a diet containing a normal amount of fat (Reddy et al., 1975b).Fecal excretion of acid and neutral steroids was higher in animals fed high fat diets than in animals on low fat diets, similar to human populations on high and low fat diets. Bile acids have a potentiating effect in inducing colon carcinoma in laboratory animals, and are believed to be promoting agents. The development of colonic tumors in rats exposed to the carcinogen N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) was increased by instilling lithocholic or taurodeoxycholic acids intrarectally (Narisawaet al., 1974); the carcinogenic effect of azoxymethane in rats was increased by increasing the concentration of bile reaching the colon, by feeding cholestyramine and by diverting the bile flow to the lower section ofthe small intestine (Chomchai et al., 1974; Nigro et al., 1973).Cholestyramine has been reported to increase the frequency of intestinal neoplasms induced with 1,2dimethylhydrazine in germfree rats (Asanoet al., 1975). It also has been

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reported that vitamin A deficient rats were more susceptible to dimethylhydrazine induced colon cancer (Rodgers and Nernberne, 1973; Rodgers et al., 1973). The possibility that significant bacterial conversion of bile acids and cholesterol may take place has been referred to. It is believed that the fecal concentrations of total acid steroids, deoxycholic acid and fecal bacteria containing the enzymes 7-hydroxycholanoyldehydroxylase and 3-oxo-cholanoyl A 4 dehydrogenase are of importance. The enzymes convert primary bile acids to secondary, and produce double bond formation on the bile acid nucleus. Thus, the possibility has been considered that both unsaturated and saturated bile acids contribute to the etiology of colon cancer, fulfilling roles of cocarcinogen and carcinogen (Hill, 1975). On the other hand, it was postulated (Mastromarino et al., 1976)that cholesterol metabolites may be important in the pathogenesis of colon cancer since fecal microbial 7-hydroxycholanoyl dehydroxylase and cholesterol dehydrogenase activities were noted to be higher in cancer patients compared to controls. Anaerobic intestinal bacteria contain enzymes that might induce the production of secondary bile acids and cholesterol metabolites. Thus, it is believed by some investigators that diets having high fat content, and perhaps a corollary low fiber content typical of the Western diet, are key factors in increasing colon cancer risk. These diets lead to changes in the lumenal contents of the large bowel as noted above. In attempting to understand the pathogenesis of colon cancer, key questions now concern the degree to which high-risk subgroups within the general population respond to dietary changes, and the degree and mode of interaction that may occur between dietary elements and colonic cells genotypically predisposed to neoplasia. Ill. Inherited Diseases That increase Susceptibility to Colon Cancer

As noted above, current findings suggest that the development of colorectal cancer is largely influenced by environmental factors, although attempts to identify specific elements, and in particular their mode of action, have not been definitive. At present, inheritance is believed to have a minor role in the genesis of colorectal cancer. This view is based largely on the frequency of identification of the clinically definable dominant inherited syndromes leading to colon cancer. Recent findings, however, have indicated again that familial associations in colon cancer are higher than in control groups, suggesting that inherited factors could have a greater role in the genesis of colorectal cancer than has been generally believed. Thus, it was recently noted

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that there was a significant increase in the number of deaths due to bowel cancer among first degree relatives of index cases compared with the expected incidence. Early age of onset, the presence of adenomas or other carcinomas in the operation specimens, and a history of previous carcinoma were found to be associated with an increased risk that the index case would have a positive family history (Lovett, 1974). It had been recognized that a significant percentage of colorectal cancer patients have family members who also have colon cancer, and that relatives of colorectal cancer patients were at an increased risk compared to the general population (Moertel et al., 1958). Familial associations could be influenced by genetic as well as environmental factors, and inherited predispositions could influence the development of colon cancer on the basis of cellular and related physiological abnormalities. Patients under age 40 who develop colorectal cancer have been reported more likely to have a family history of colon cancer than those over age 40 (Wynder and Shigematsu, 1967). In relatives of patients with multiple colonic malignancies the age of onset of colonic cancer has been reported to be significantly earlier than in the general population (Moertel et a1., 1958). The probability of developing a new colon cancer is increased in individuals who have had prior colon cancer or polyps, or female genital cancers, breast cancer, or bladder cancer. The degree to which inherited or environmental factors influence this predisposition remains to be determined. In individuals with previous colon cancer, the probability of developing a second or metachronous lesion is about three times higher than in the general population (Morson and Bussey, 1970). When the initial cancer was located in the cecum, a second primary colon cancer occurred most frequently. Associations of colonic and other neoplasms have similarly suggested that inherited factors are important. Female genital cancers, primarily endometrial, are twice as frequent as expected in colon cancer patients. Associations of breast cancer, endometrial, and ovarian cancer have been noted in women with colon cancer (Fraumeni, 1973; Lynch, 1967). A disease entity of hereditary adenocarcinomatosis has also been noted, an autosomal dominant with 90% penetrance (Anderson, 1970), and cancer families showing a striking incidence of primary malignancies at multiple anatomic sites including the colon, with an early age of onset have been identified in families highly predisposed to cancer (Lynch and Krush, 1967). Cancers that show patterns of inheritance that appear to be dominant can show tumor specificity, oc-

SUSCEPTIBILITY TO COLON CANCER

289

curring in the same tissue and in the same organs in family members, earlier age of onset than in the general population, and a tendency to develop at multiple sites within an organ (Anderson, 1970). The concept of cancer families has been broadened recently to include neoplasms of different types. The neoplasms in these families, which appear to b e influenced by genetic predisposition, affect diverse organs especially the colon and endometrium. They tend to develop earlier in life than usual and may occur separately or as multiple cancers in family members. Since adenocarcinomas of the colon and reproductive sites are known to coexist excessively in studies of multiple primary cancers, the familial syndrome may represent a scattering over the family tree of tumors that share etiologic influences (Fraumeni, 1973). Efforts to indicate a single-gene difference in colonic carcinomas with a genetic basis have largely been unsuccessful, except for the inherited disease adenomatosis of the colon and rectum (familial polyposis) (Alm and Licznerski, 1973; Gardner, 1962; Morson and Bussey, 1970). Ulcerative colitis, solitary polyps, and regional enteritis can also occur in familial aggregates and can develop into carcinomas. Although these syndromes suggest a multifactorial basis for possible genetic determination, the involvement of genetic mechanisms is still not clear. INHERITED

ADENOMATOSIS AND RELATEDDISORDERS

Several varieties of precancerous colorectal disease of genetic origin involving polyp formation have been well described. Znherited adenomatosis of the colon and rectum (A.C.R.; familial polyposis) is associated with innumerable colonic adenomas; it has been possible to estimate population frequency, relative fitness, and mutation rate. This is one of the few genetic disorders associated with malignancy offering such a quantity of these types of data, which enabled a fairly accurate description of the disease when clinically apparent. Various studies have estimated the disease frequency in the population based on clinically observable manifestations. These estimated frequencies range from 1 in 8300 in Michigan and 1 in 7150 in Kentucky, to 1 in 23,750 in St. Mark’s Hospital in London (Pierce, 1968; Reed and Neel, 1955; Veale, 1965).A penetrance rate of approximately 80% with an autosomal dominant mode of inheritance occurs. Fewer cases than expected have resulted in some families due to this incomplete penetrance. In A.C.R., the presence of hundreds to thousands of adenomatous

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MARTIN LIPKIN

polyps characterizes the classical expression of this disease. These may carpet the entire colon and rectum, with denser polyposis shown in the rectum. Sessile and pedunculated adenomatous and villous tumors are usually seen. It is of note that, in A.C.R., carcinomas develop with highest frequency in the lower colon, as occurs in the general population (Alm and Licznerski, 1973). To date no chromosomal entity can be cited as a useful diagnostic or prognostic marker for colon carcinoma or its precursor states, comparable to the Philadelphia chromosome in chronic granulocytic leukemia. Linkage between Duffy blood groups and the gene for A.C.R. susceptibility has been suggested (Veale, 1965). Earlier studies of chromosome pattern in adenomas and adenocarcinomas of the colon had shown hyperploidy in adenomas, and a degree of epithelial dysplasia related to a higher incidence of structural and numerical chromosome abnormalities. In well-differentiated adenocarcinomas, the degree of chromosomal abnormalities was pronounced (Enterline and Arvan, 1967). Abnormalities have more recently been reported involving chromosome numbers 8 and 14 in human colonic adenomas (Market aE., 1973; Mitelman et al., 1974). The changes preceded histologic evidence of invasiveness and were found with or without a hereditary basis. In A.C.R., excessive heteroploidy has been found in the cells of adenomas of A.C.R; while in sporadic polyps, the chromosome numbers were more normal. Cells with abnormal karyotype also showed involvement of C or D group chromosomes, both in A.C.R. and sporadic polyps. Histological changes in colonic mucosa of A.C.R. also have been found in association with the above findings. Minute mammillations of surface epithelium and localized hyperplasia were earlier described together with chronic inflammatory reaction and enlarged lymphoid follicles. Differentiation was made between adenomatous polyps having malignant potential, when arising in A.C.R. and sporadically in the general population, and hyperplastic polyps found in the general population. The proliferative abnormalities described in the next section occur in adenomatous and not in hyperplastic polyps. Figure 1 shows the cumulative percentage of incident cases having A.C.R. at various ages compared to the incident cases of colon cancer in other population groups. These data are useful in defining population groups at increased risk for colon cancer on the basis of age of onset of disease. The distributions of incident cases of (1) cancer in A.C.R., (2) familial colon cancer, (3) multiple primary cancers including the colon with family history of colon cancer, and (4) colon cancer

SUSCEPTIBILITY TO COLON CANCER *--a

Onsetof polyposisinA C R

291

Onset of colon and rectolconcei in general population

{ o---o Onset of concer in A C R

Onset of concer in familial colon cancer &---a Multipleprimary cancers including colon,mthfamily history of colon cancer n--q

u)

8 100 0 L

C

rl E I

0 0 0' 0 c

5 50

Y a 0)

-0 c

;20

(3

10

0 Age in years

FIG.1. Cumulative percentage of incident cases having inherited adenomatosis of the colon and rectum at various ages, compared to incident cases of colon cancer in other population aggregates. Data of A.C.R. are from Bussey (1975). Data of familial sitespecific colon cancer are from Dr. H. Lynch, and multiple primary cancers are from Memorial Hospital registry. The range of data of colon and rectal cancer in general population is from the Third National Cancer Survey, National Cancer Institute, and includes white and black males and females.

in the general population were subjected to statistical analysis. Pairs of distributions were compared by the Kolmogorov-Smirnov 2 sample test. All pairs of distributions shown in Fig. 1 were found to be significantly different ( p < .05, two sided test) indicating that the first three population groups are at increased risk for colon cancer compared to the general population. In A.C.R., cancer follows the development of adenomas with great regularity decades before the appearance of colon carcinoma in the general population; no disease leads to cancer with greater certainty. A delay between the onset of adenomatosis and cancer can also be noted, which is most pronounced in the younger age groups. In the older age groups, the development of adenomatosis and cancer appear in closer temporal sequence, in this respect resembling the onset of colon cancer that can be observed in the general population, in patients having one or several adenomas. Adenomatosis can also be detected near birth in a small fraction of individuals. In the past, estimates have indicated that when first seen, two-thirds of patients with adenomatosis will show evidence of cancer; by 40 years of age, over 50% will develop adenocarcinoma.

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MARTIN LIPKIN

The usual age in A.C.R. for diagnosis has been 25 years, while cancer in polyposis patients is usually diagnosed at later periods. This is about 20 years earlier than average figures for cancer without adenomatosis. Patients usually manifest multiple cancer: Approximately 50% of colectomy specimens show two or more cancers (Morson and Bussey, 1970). As observed, cancers have been reported in patients with adenomatosis at early ages, and after puberty adenoma formation and cancers increase. Individuals who are examined because of a known familial association are often successfully identified at a much earlier age as having A.C.R. without cancer, compared to those who seek medical advice due to symptomatic presentation of disease. As noted, most patients will die of colon carcinoma by age 50 unless a colectomy is performed. Gardner’s syndrome, a variant of A.C.R., is an autosomal dominant disorder showing a high degree of penetrance. Adenomatous polyps of the colon, and occasionally of the small intestine, are formed and there is propensity for adenocarcinoma development within the polyps. The syndrome has a lower incidence than A.C.R., extimated at 1 in 14,000 births. The disease is characterized b y colonic polyposis and soft tissue abnormalities such as sebaceous cysts, desmoid tumors, epidermal cysts, lipomas, subcutaneous nodules, and fibromas. In addition, bone abnormalities including osteomas of the skull, exostoses of the skeleton, cortical thickening of the long bones, and abnormal dentition can be found. Similar to A.C.R., the polyps are adenomas. They are distributed almost completely in the colon and rectum, although they can occur in the small intestine, especially in the region of the ampulla of Vater. Gene expression is variable, with some individuals having polyposis alone and other individuals having the various manifestations of the major triad. The extracolonic manifestations also can develop prior to the colonic adenomas. The age distributions of onset of colonic polyps and progression to colon cancer are similar to A.C.R. A particular problem can arise following surgery. Postoperative fibromas and low grade fibroblast or desmoid malignancies may arise in healing surgical wounds, and can result in invasion of mesenteric root or bowel. Other associated tumors can also occur, including carcinoma of the duodenum and papilla of Vater. Variants of the syndrome can include Turcot syndrome (polyposis coli associated with tumors of the central nervous system) and the Oldfield syndrome (extensive familial sebaceous cysts, polyposis coli, and adenocarcinoma). In Gardner’s syndrome, the adenomatosis and other features of the disease may be due to a single gene although it remains possible that additional genes are involved.

SUSCEPTIBILITY TO COLON CANCER

293

A third autosomal dominant inherited disease with variable expression is the Peutz-Jeghers syndrome, characterized by melanin pigmentation of the buccal mucosa, lips, face, fingers, toes, vagina, and anus. Polyps of the gastrointestinal tract, specifically the small intestine are found; about one-third are in the colon and rectum. However, the polyps are hamartomas rather than adenomas (Jeghers et al., 1949). This disorder appears to have very little malignant potential when compared to A.C.R. or Gardner’s syndrome. Nevertheless, some associated stomach and duodenal associated carcinomas have been reported (Dodds et al., 1972). The presence of one or more adenomas occurs in 5 to 10%of individuals in the general population, and can be associated with the development of adenocarcinoma. Kindreds have also been reported showing an association of single and multiple adenomas with adenocarcinoma, a link that appears to be genetically influenced. One study, by Woolf et al., (1975) showed that 45% of the adult members of one generation had solitary adenomas as well as the occurrence of adenomas in multiple generations. This family also had a high incidence of colon carcinoma. An autosomal dominant mode of inheritance is reinforced by these observations. Another inherited disorder is juvenile polyposis of the colon. These polyps are hamartomas and are not viewed as potentially malignant. Relatives of these juveniles do, however, express an above normal occurrence of adenomas and colorectal adenocarcinomas. IV. Proliferative Abnormalities and Susceptibility to Colon Cancer

Studies of cell proliferation have aided our understanding of events that develop during neoplastic transformation of colonic cells in A.C.R., and in individuals in the general population who have developed colon cancer. In individuals having A.C.R., colonic epithelial cells predestined to develop neoplasia show characteristic proliferative changes. During progressive stages of abnormal development, cell phenotypes appear in which epithelial cells gain an increased ability to proliferate and to accumulate in the mucosa. In the normal colon of man, the major proliferative activity occurs in the lower and midregions of the crypts adjacent to the base, occupying about three-quarters of the crypt columns. Approximately 15 to 20% of the proliferating cells are in DNA synthesis. The number of cells in the proliferative cycle diminishes as they advance to the mouth region of the crypts; within hours cells undergo further differentiation and proliferative activity ceases as they approach the crypt surface (De-

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schner et al., 1963; Lipkin and Deschner, 1968; Lipkin and Quastler, 1962; Lipkin et al., 1963; Messier and Leblond, 1960). In subjects with A.C.R, patches of flat mucosa can be detected having colonic epithelial cells that fail to repress DNA synthesis during migration to the surface of the mucosa (Deschner and Lipkin, 1970; Deschner et al., 1966). This finding has been observed in normal appearing colonic epithelial cells of subjects with A.C.R. before they develop adenomatous changes and before the cells begin to accumulate as polyps. It has been noted in 80% of random biopsy specimens (Fig. 2). It also was recently observed with high frequency in flat mucosa of individuals in the general population who developed primary colon cancer (Maskens and Deschner, 1977). In inherited adenomatosis, colonic epithelial cells develop additional properties. Cells that fail to undergo normal maturation with repression of proliferative activity also acquire altered morphological characteristics identified pathologically as “adenomatous.” These cells accumulate in the colonic mucosa and form tubular or villous structures initiating the formation of adenomas. Carcinomas develop with increasing frequency as these adenomatous excrescences enlarge (Morson, 1976). Figure 3 shows a sequence of events believed to lead to malignancy in inherited A.C.R. Proliferating colonic cells that have inherited the germinal mutation fail to repress DNA synthesis. Additional events then occur giving rise to new clones from the original cell population. The first leads to the development of the well-known

FIG.2. Quantitation with modified Venn diagram of a set of cases having two simultaneous phenotypic attributes. The diagram shows in 35 individuals in A.C.R. familial aggregates, the observed relationship between the presence of adenomas (n’)and failure of colonic epithelial cells to repress DNA synthesis (s’) in biopsies of flat mucosa. (s’) is present in 80% of random biopsy specimens of flat mucosa and (n’) is present in 54%of A.C.R. population, in contrast to controls without known gastrointestinal disease or history compatible with A.C.R. where (s‘) is present in 18%of random biopsy specimens and (n’) is zero. Numerical values are continually modified as new cases are added. (p) indicates fraction of cases where neither (s’) nor (n’)have been detected in colonic mucosa.

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295

FIG. 3. A sequence of events believed to lead to malignancy in inherited A.C.R. Notations s' and n' as in Fig. 2 (n'denoting development of adenomatous cells that proliferate and accumulate, initiating neoplastic growth) m' denoting malignancy. Proliferating colonic cells that have inherited the germinal mutationfail to repress DNA synthesis. Additional events are then believed to occur giving rise to new clones from the original cell population and leading to the development of adenomatous and malignant cells.

adenomatous cells that proliferate and accumulate on the surface of the mucosa. I n A.C.R., according to this concept, an additional event then occurs in the cells giving rise to invasive malignancy. Numerical estimates of the events that could lead to colon cancer have recently been made (Knudsen, 1976). following the approach of Fisher (1958). I n that analysis as well as our own, there is high agreement between the age-specific cumulative incidence rate of cancer in A.C.R. and numerical estimates when based on the occurrence of a low number of sequential events after the germinal mutation. A failure of colonic epithelial cells to repress D N A synthesis also occurs in ulcerative colitis (Eastwood and Trier, 1973). In ways similar to diseases of the colon, in atrophic gastritis, a condition associated with the development of gastric malignancy, epithelial cells also fail to repress D N A synthesis and undergo abnormal muturation as they migrate through the gastric mucosa (Deschner et al., 1972; Winawer and Lipkin, 1969). In precancerous disease of the cervical epithelium in humans (Wilbanks et al., 1967) and in the cervix of rodents after a chemical carcinogen (Hasegawa et al., 1976), a similar event occurs. Thus, during the development of neoplasms in other organs as well as in colon, persistent D N A synthesis has been observed in cells that would be normally expected to be terminal or end cells prior to invasive carcinoma. The findings support the possibility that common defects develop in the regulatory control of cell proliferation during neoplastic transformation in the epithelial lining of these various organs.

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V. Newer Immunologic Studies

Immunologic factors that may be related to increased susceptibility of familial aggregates to colon cancer are under investigation. An abnormality has recently been detected in some individuals at increased risk of colon cancer. When cancer free individuals from families highly predisposed to colon cancer (without classic A.C.R.) were studied to determine the nature of their cell-mediated immune capacities, 44% demonstrated an apparent perturbation of adherent cell function, manifesting itself as an inappropriate suppression of a potentially normal lymphocyte ability to respond to an allogenic stimulus. The defect in recognitive immunity appeared to be the same defect that was demonstrated in individuals with established malignancies (Table 111). Patients with recognized Gardner's syndrome also showed the deficit of recognitive immunity (Berlinger et al., 1977). These studies, which are being extended to additional disorders leading to colon cancer, offer the possibility of new means for the early detection of susceptible population groups. VI. Nuclear Protein and Enzyme Alterations

The enzyme composition of colonic cells has been shown to change in association with the development of adenomas and carcinomas in the colon of man. Activity of thymidine kinase is higher in neoplastic TABLE I11 MIXED LEUKOCYTE CULTURE RESPONSE OF UNAFFECTED MEMBERS OF COLON CANCERFAMILIAL AGGREGATES" Relative response of family members as percentage of controls

Relative response after filtration of cells through G I0 beads

P

38 48

88 77

M

36 66 53

70 78 60

C

63

68

J

61

75

V

39

71

Family

From Berlinger et al. (1977).

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SUSCEPTIBILITY TO COLON CANCER

than in mature colonic cells (Salser and Balis, 1973; Troncale et al., 1971). More recent studies have indicated that after administration of the carcinogen 1,S-dimethylhydrazine (DMH), thymidine kinase was altered both quantitatively and qualitatively, with properties resembling fetal enzyme after long term DMH treatment. I n human tissue, the enzyme from malignant cells was antigenically more like surface cell enzyme than from crypts. The enzyme from placental extracts and from adenomatous polyps was similar to tumor enzyme (Salser and Balis, 1973) (Table IV). Other cellular enzymes including ornithine decarboxylase increased after DMH in the colon but not in the liver, while the liver carcinogen acetylaminofluorene induced a marked increase in liver enzyme but not colonic (Ball et al., 1976).These findings have indicated specificity in enzyme complement associated with carcinogen-induced neoplastic transformation of the cells, and the possibility of indices denoting tumorigenesis in human populations highly susceptible to cancer. Recent studies have also shown that nuclei isolated from DMHinduced tumors contained characteristic complements of nonhistone nuclear proteins. These were not prominent in normal colonic epithelial nuclei nor in epithelial cells surrounding the tumors. Characteristic abnormal nuclear proteins were also detected in human colonic carcinomas and in a human colon carcinoma cell line (Fig. 4)(Boffa and Allfrey, 1976). Although these were not found in nonmalignant adenomatous polyps in A.C.R., the identification and selective accumulation of such proteins in colonic tumor nuclei and the developTABLE I V OF TK IN VARIOUS TISSUEEXTRACTS AFTER PREINCUBATION WITH ANTIHUMAN COLONIC T K FROM RABBITS~

RESIDUAL ACTIVITY

Source of TK

Partially purijied by a single precipitation with ammonium sulfate Normal colon Adenocarcinoma colonic Placenta I Crude extract Ovarian carcinoma PHA-stimulated lymphocytes Myeloblastic leukemic leukocytes From Salser and Balis (1973).

Control TK activity (in pica moles per incubation sample)

Undiluted

402 2 662 3 68 T 2

54 60

80 2 4 762 2 85? 4

35 95 95

8

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MARTIN LIPKIN

HN

HTI

HTn

C

4 Molecular weight x

lo3

FIG.4. Electrophoretic analysis of nuclei isolated from normal human colonic epithelial cells, from two different adenocarcinomas of the colon, and from a cell line HT-29 derived from an adenocarcinoma. Electrophoretic profiles of all tumor cells show prominent peaks (indicated by vertical lines) while normal colonic epithelial nuclei do not. (From Boffa and Allfrey, 1976.)

ment of analytical procedures for their detection in single cells offers a new approach to the early detection in single cells of molecular events associated with malignancy in man. In mice, morphologic and proliferative changes have been induced by carcinogens similar to those observed in man. After DMH was ad-

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ministered to mice, the distal colon was the main site of tumor nodule formation, a distribution common to that found in man. Multifocal tumors ranging from adenomatous polyps to metaplasias and carcinomas grew from the mucosa and then protruded into the lumen of the rectosigmoid. Early focal atypias and hyperplasia, located mainly on the folds, and adenomatous polyps and carcinomas appeared to be part of the progressive pathologic changes in mice and rats, and developed following administration. These changes were accompanied by an increased proliferative activity in the cells. Both DMH and MNNG induced an extension of the proliferative region of the flat mucosa toward the surface of the colonic crypt, as observed in man, with development of cells that continue to incorporate thymidine into DNA throughout their life span (Deschner, 1974; Kikkawa, 1974; Thurnherr et al., 1973; Wiebecke et al., 1973) (Fig. 5). VII. Studies of Cutaneous Cells

Recent studies have suggested that phenotypic expressions of the inherited disease ACR may extend to cutaneous cells. Increased heteroploidy in cutaneous epithelial cells derived from individuals

FIG. 5. Increase in proliferative compartment in mice after repeated injections of DMH (A, upper diagram, from Thurnherr et al., 1973) and after rectal installation in MNNG in rat (B, lower diagram, modified from Kikkawa, 1974).

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with Gardner’s syndrome was observed (Danes, 1976). It has also been noted that cutaneous fibroblasts derived from normal subjects grow in well-organized monolayers, in contrast to those from individuals with A.C.R., which have shown larger regions of criss-cross arrays and random orientation forming multilayers. A susceptibility to neoplastic transformation when exposed to Kirsten murine sarcoma virus has been reported (Pfeffer and Kopelovich, 1977).Of particular interest is the recent report of differences in actin distribution within cultured cells from individuals with A.C.R. compared to normals (Kopelovich e t al., 1977).In order to determine the specificity of findings of this type, the observations are presently being extended to include analysis of cutaneous cells from larger control groups and from additional families with various patterns of inherited polyposis and colon cancer. VIII. Examination of Fecal Contents

Additional studies are in progress to identify abnormal constituents of fecal contents and to examine their potential carcinogenic activity in colon cells. Studies of potential bacterial markers were referred to previously. The bile acids and their bacterial conversion products are a group of compounds currently under examination. Several recent reports compared the fecal neutral steroids and bile acids in patients with A.C.R. and controls other than relatives (Drasar et d., 1975; Reddy et al., 1976; Hackman et al., 1976; Watne and Core, 1975). Individuals with A.C.R. excreted higher amounts of cholesterol and lower levels of coprostanol and coprostanone. Nondegradation of cholesterol also was found in about one-quarter of individuals in the general population (Hackman et al., 1976). Further studies are also in progress to assess the utility of these variations in cholesterol and its metabolites in screening A.C.R. family siblings for disease. A further difference in fecal neutral steroid concentration of North American white and South African black populations was recently found, with most of the neutral steroids free (nonesterified) in North Americans; and a high proportion of plant and animal steroids esterified to long-chain fatty acids in Africans (Salyers, 1977). Recently, an additional and potentially important lead to the identification of increased susceptibility to colon cancer, was provided with detection of mutagenic activity in the feces of humans. It was suspected that N-nitrosa compounds might be associated with this mutagenic activity (Vargheseet al., 1977). Further work has suggested that anitroso group exchange reaction can occur with transfer from nitrosamine to amide moiety, resulting in the generation ofhighly reactive nitrosamide

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compounds in feces (Mandel, 1977). Several laboratories are now actively pursuing the question of whether fecal mutagens may interact with cells of the colon to induce the sequence of proliferative and morphologic changes that lead to the evolution of colon cancer. IX. Conclusion

The findings described have identified abnormal stages of development of colonic epithelial cells and related physiological and environmental factors that may influence the development of neoplasia, as well as individuals and familial aggregates at increased risk. Individuals and population groups are now classified on the basis of abnormal cell phenotype and related physiological and fecal content abnormalities. These classifications are leading to new indices that attempt to identify heightened degrees of susceptibility of individuals at increased risk of colon cancer, and the stages of development of their disease. With precise quantitative information available, elements that modify or accelerate the progression of disease in man can be studied. Future programs designed to identify high-risk population groups will require systematic analyses of mode and time of appearance of abnormal phenotypic expressions in these individuals and their families. Analysis of the risk factors together with corresponding study of interactions that take place with relevant carcinogens can lead to new means to identify individuals and population groups at increased risk of colon cancer, and studies are under way for that purpose. New programs designed to prevent the evolution of the cellular changes leading to malignancy are being considered at the present time utilizing the measurements described herein. ACKNOWLEDGMENTS Studies reported here from the author’s laboratory were aided by Contract 1-CP-43366 and Grant CA98748, from the National Cancer Institute, Department of Health, Education and Welfare.

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ADVANCES IN CANCER RESEARCH. VOL. 27

NATURAL CE LL-MEDlATE D I M MUNlTY Ronald 6. Herberman and Howard T. Holden

.

Laboratory of Immunodiagnosis.National Cancer Institute: Bethesda Maryland

I . Introduction .......................................................... I1. Characteristics of Natural Cytotoxicity ................................. A . Influence of Age . . . . . . . . . . . . B. Influence of Genetic Backgrot C . Influence of Environmental Factors and Disease ..................... D . I n Viuo Augmentation of Reactivity .................... E . Effects of in Vitro Cultivation of Lymphoid Cells . . . . . . . . . . I11. Specificity of Natural Cell-Mediated Cytotoxicity ................ A . Mice .............................................................. B. Rats . . . . . . . . . . . . . . . ............................................ C . Human ........................................................... IV. Nature of Effector Cells . . . . . . . . . . . . . . . . . ........................... A . Organ Distribution ................................................ .... B. Cell Surface Markers and Other Characteristics of N K Cells . C . Effect of Thymosin in Vitro ........................................ D . Effects of in Viuo Manipulations of Thymus Function ................ E Effect of Immunosuppression on NK Activity . . . . . . . . . . . . . . . V. Relationship of Natural Cell-Mediated Cytotoxicity to AntihodyDependent Cell-Mediated Cytotoxicity ...... A . Correlation of NK Activity with ADCC .............................. B. Comparison of Effector Cells Mediating NK Activity and ADCC . . . . . . C . Possible Mechanisms of Action of NK Cells ......................... VI . Model for Placement of NK and I; Cells in Pathway of Differentiation of T Cells .............................................. VII . Discrimination Between Natural Cell-Mediated Cytotoxicity and Cytotoxicity by Other Effector Cells ................................... A . Mice .......................................... B. Human ....... ............................................ a1 Cytotoxicity .............................. VIII . I n Viuo Relevance A . Correlation of Decreased Tumor Growth with Natural Cytotoxicity . . . . B . I n Viuo Relevance of N K Activity Against Nonmalignant Cells . . . . . . . C . Implications of Natural Cytotoxicity for Immune Surveillance ... Addendum ........................................................... References ...................... ..... ............

.

305 307 310 312 316 319 321 324 324 329 331 333 333 334 340 341 343 345 345 ,347 349 351 354 355 359 361 362 363 365 366 370

I . Introduction

In vivo resistance of the host against progressive tumor growth has mainly been attributed to mature ~Tcells. specifically immune against 305 Copyright 0 1978 by Academir Press. Inc . .411 iights of reproduction 111 iiny fwm reserved.

ISBN 0-12-(X)6627-0

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tumor-associated antigens. Indeed, in adoptive transfer experiments in mice and rats, in which the recipients were successfully protected against tumor cell challenge, such specifically immune T cells have been shown to b e necessary (Collavo et al., 1974; Gorczynski and Norbury, 1974; Berenson et al., 1975; Glaser et al., 1976b). Because of this important role of immune T cells, it was anticipated that nude mice and other T cell-deficient individuals would completely lack resistance against spontaneous or induced tumors, and that the incidence of rapidly progressive malignancies would be much higher than in individuals with competent T cell immunity. The failure to find such explosive tumor growth in nude mice, or a complete spectrum of malignant diseases in patients with immunodeficiency diseases, has been taken as strong evidence against the immunosurveillance theory (Moller and Moller, 1975; Outzen et al., 1975; Schwartz, 1975; Rygaard and Povlsen, 1976). In in vitro studies of cell-mediated immunity against tumors, the central role of mature T cells has also been stressed. Cell-mediated cytotoxicity against tumor cells has generally been thought to be mediated by three types of effector cells (Cerottini and Brunner, 1974); (a) specifically immune, mature T cells, (b) antibody-dependent cytotoxic cells, which bear surface receptors for the Fc portion of IgG molecules and thereby interact with IgG antibodies bound to target cells, and ( c ) activated macrophages or macrophages armed with specific antibodies. The recent recognition of the existence of natural cell-mediated immunity, particularly natural cell-mediated cytotoxicity (Oldham et al., 1973; Herberman et al., 1973, 1974a; Rosenberg et al., 1974), has substantially altered our concepts concerning the potential mechanisms for in vivo resistance against tumor growth and for in vitro cell-mediated immune reactions. Natural cell-mediated cytotoxicity has been found to be a general phenomenon in several species, in rats (Holtermann et al., 1973; Nunn et al., 1976; Shellam and Hogg, 1977) and in man (Oldham et al., 1973; Takasugi et al., 1973; Rosenberg et al., 1974) as well as in mice (Herberman et al., 1973, 1974a, 1975a; Kiessling et id., 1975a). With mice, almost all studies have involved the use of a short-term 51Crrelease cytotoxicity assay (CRA). With rats and humans, the phenomenon has been studied with both the CFL4 and also with long-term visual microcytotoxicity and radioisotopic cytotoxicity assays. Some studies with lymphoproliferative assays (Kanner et al., 1970; Shoji and McKhann, 1971; Forbes et al., 1973; Burk et al., 1975; Boyer and Fahey, 1976; Lopez et al., 1976; Krueger et al., 1977; Lee and Ihle, 1977) or migration inhibition assays (Lopez

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et al., 1976) have also shown that normal individuals may have cellmediated reactivity against tumor-associated or oncogenic virusassociated antigens. However, the characteristics of natural immunity detected in these noncytotoxic assays, and the nature of the effector cells mediating these reactions, have not been studied in detail. The findings of ubiquitous natural cell-mediated immunity must be considered in studies of cell-mediated immunity in tumor-bearing individuals and in discussions of immune surveillance. The occurrence of natural cell-mediated cytotoxicity against human tumor-derived cell lines has created particular problems in evaluating the reactivity of cancer patients (Herberman and Oldham, 1975).It is now clear that one must carefully evaluate the role of natural killer (NK) cells, as well as other, more well-known mechanisms of cytotoxicity, when cytotoxic reactions are measured. In this review, w e will concentrate on natural cell-mediated cytotoxicity, since most of the information in natural cellular immunity has been gathered with this assay. We will summarize the known information on the expression of natural cytotoxicity in rodents and in man, its specificity, the nature of the effector cells and their relationship to other immune mechanisms, and the possible in vivo relevance of natural cytotoxicity, particularly in regard to resistance against tumor growth. Since a large amount of information in each of these areas has been obtained and there are a number of differences among the different species studied, Table I represents an attempt to summarize many of the important points, which can be referred to for orientation by the reader while going through the details below.

II. Characteristics of Natural Cytotoxicity

The basic observation which initiated studies of natural cellmediated cytotoxicity was that lymphoid cells from some normal mice, rats, and human donors, which were not inoculated with tumor cells or other sources of antigen, had significant levels of cytotoxic reactivity against certain syngeneic or allogeneic tumor cells (Herberman et al., 1973, 1974a; Oldham et al., 1973; Rosenberg et al., 1974; Nunn et al., 1976). At first, only low levels of reactivity were observed. However, upon further investigations it was found that a large number of factors influenced the expression of NK activity and that with certain donors and tumor target cells, very high levels consistently could be detected. Such high natural cytotoxicity may exceed the levels of reactivity by specifically immune, mature T cells against highly antigenic

TABLE I SUMMARY OF CHARACTERISTICS OF NATURAL CELL-MEDIATED CYTOTOXICITY Mice LRvels of NK activity Age

Genetic background

Environmental factors and disease

In viuo boosting

I n vitro

Specijici t y

Absent at birth in most strains; peak levels at 5-8 weeks, low after 12 weeks Low activity in A strain against several target cells; high levels in CBA and nude mice; high reactivity dominant in F, Activity present in gem-free; some evidence for inhibitory effects of environmental factors, tumors, and other diseases Specific boosting (peak at 3 days) by some tumor cells, normal thymocytes, and bone marrow cells; apparently nonspecific boosting by murine viruses, BCG, C . parvurn Decrease in activity at 37°C within 2-3 hours and almost complete loss b y 24 hours At least 3 specific antigens detected with possible association of antigens with endogenous type-C viruses; antigens present on lymphomas, other mouse tumors, some nontransformed culture lines, normal thymocytes, bone marrow cells, PHA blasts; most xenogeneic cell lines negative but some human lines positive

Rats

Human

Absent at birth; peak levels at 5-8 weeks; low after 10-12 weeks Low activity in BN rats

No clear age relationship; some activity i n cord blood

Activity present in gem-fiee

Lab workers = other normal donors; possible increase with some viral infections; low activity in some cancer patients Boosting by influenza vaccine, peak at 2-3 days

No information yet available

No lability at 37°C; increased activity after 3-24 hours Antigens with possible association with endogenous type-C viruses mainly limited to rat cells; reactivity against some mouse tumor cells and human cultured cells

Males > female; low activity in males with HLA-A3, B7 haplotype

No lability at 37°C; increased activity after 5 days in medium with fetal bovine serum or other stimulants Probability of multiple antigens; broad specificity on human cultured cell lines, some human tumor cells, and some rodent tumor lines; not due to fetal bovine serum antigens

Eflector cells Organ distribution

Cell surface markers

Other characteristics

Effect of human thymosin in oitro I n uioo manipulations of thymus function

Relation of NK activity to ADCC

In viuo relevance

Present in spleen, lymph nodes, peripheral blood, peritoneal cavity, bone marrow; absent in thymus Weak expression of 0 antigen on NK cells after boosting and from nudes; Fc receptors present but difficult to detect; no complement receptor Nonadherent, nonphagocytic; inhibited by trypsin but recovers in 18 hours at 37"; moderately resistant to irradiation; sensitive to cyclophosphamide; not inhibited by ammonium chloride solution Small decrease in activity High in nude, nude-asplenic mice and neonatally thymectomized mice; low in thymus-grafted nude mice Good correlation between levels of activity with age, strain; effector cells have similar characteristics

Some tumors sensitive to NK grow poorly in nude mice, greater tumor resistance in young mice; may mediate bone marrow resistance and possibly anti-microbial resistance

Present in spleen, lymph nodes, peripheral blood, peritoneal cavity, bone marrow

Present in spleen and peripheral blood; absent in lymph nodes, tonsil, thymus

Activity not inhibited by anti-T cell serum; no easily detectable F c receptor or complement receptor

Low affinity receptor for sheep erythrocytes; easily detectable Fc receptor; no complement receptor

Nonadherent, nonphagocytic; inhibited by papain; moderately resistant to irradiation; not inhibited by ammonium chloride solution

Nonadherent, nonphagocytic; inhibited by trypsin and chymotrypsin; moderately resistant to irradiation; markedly inhibited by ammonium chloride solution but recovers after 18 hours at 37" Small decrease in activity

No information yet High in neonatally thymectomized and adult thyrnectomized, irradiated, bone marrow reconstituted rats No information yet

No information yet

No information yet

2 k

2 ir2 0

M

r

c:

s

U

GM

U

Same organ distribution, correlation between levels of activity among individuals; effector cells have same characteristics but difference in effects of trypsin and protein A No information yet

d

2

' w 0 co

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syngeneic tumor cells (e.g., in the mouse sarcoma virus (MSV) system; cf. Lavrin et al., 1973; Herberman et al., 1975a). In this section, we will discuss the major factors which have been found to affect the expression of natural cell-mediated cytotoxicity.

A.

INFLUENCE OF

AGE 1. Mice

In nude mice, as well as in conventional thymus-bearing mice, age has been shown to have an important and consistent effect on expression of natural cytotoxic reactivity. Our studies (Herberman et al., 1975a) and those of Kiessling et al. (1975a) indicated that lymphoid cells from mice less than 3 weeks of age lacked detectable cytotoxic reactivity, and that cytotoxicity appeared at about 4 or 5 weeks of age and reached peak levels between 5 and 8 weeks of age. Thereafter, there was a decline in activity to low levels. In conventional mice, there was almost no detectable activity in mice greater than 12 weeks of age. However, in nude mice, the decline has appeared to be more gradual, with persistence of some reactivity for longer periods of time. In some other studies, age was also found to be an important factor, but the kinetics of appearance and persistence of NK activity were substantially different. Gomard et al. (1974), in studies of AKR mice, found no natural reactivity against Gross virus-induced lymphoma cells in mice less than 3 months old, and detected considerable reactivity in 3- to 5-month-old mice and even in some mice which were more than 5 months of age. In contrast, we have found considerable levels of natural reactivity in 2-month-old AKR mice. Greenberg and Playfair (1974) only found natural reactivity in older NZB mice, mainly at 9 months of age. A small proportion of mice had very high levels of cytotoxicity against a subline of P815 ascites tumor cells. However, they did not test their mice at 2 months of age, and it is possible that a higher incidence of reactivity would have been found at that time. Indeed, Glimcher et al. (1977) and we have found that young NZB mice are strongly reactive against a different lymphoma target cell. It seems possible that the kinetics of natural cell-mediated cytotoxicity might vary with the target cells used. However, we found that there was a good correlation between the levels of reactivity against different target cells (Herberman et at., 1975a). Development of strong cytotoxic reactivity against one tumor line was always associated with high reactivity against other susceptible tumor cells. There

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were no instances of high reactivity against one target and low or absent reactivity against another target cell which was sensitive to cytotoxicity by effector cells from that strain.

2. Rats Age was found to play a similar important role in the expression of natural cell-mediated cytotoxicity in rats. Nunn et al. (1976) found that reactivity in CRA was maximal in 5- to 8-week-old rats. Lymphoid cells from 1-week-old rats never gave positive results, but, b y 3 weeks of age, significant reactivity was seen in some rats. The lymphoid cells from rats older than 10 weeks of age were either unreactive or produced levels of cytotoxicity only slightly higher than the baseline control. With the rats it was possible to perform serial studies with peripheral blood lymphocytes from individual animals. Reactivity was found to change abruptly, with a shift from high levels to unreactivity within 1week. Shellam and Hogg (1977) obtained a similar pattern of results, but the time course for appreciable levels of reactivity was more prolonged. Cytotoxic activity could be detected by 2 weeks of age, plateau levels were first reached at about 10 weeks, and the levels first declined at about 36 weeks. In longer term cytotoxicity assays, somewhat different results have been obtained (Oldham et al., 1977). In a [3H]-proline release assay, the highest activity was seen at 3-4 weeks of age, but activity then remained at about the level up to 16 weeks of age. In an [1251]iododeo~~ridine release assay involving a 48-hour period of incubation, there was no detectable 3- to 4-week peak but rather a similar incidence of high reactivity from 3 weeks to greater than 16 weeks of age. As discussed below (Section II,E), this persistence of high levels of reactivity of older rats in the assays with longer periods of incubation may be related to in vitro activation of cytotoxic reactivity.

3. Human In contrast to the marked age dependence of NK reactivity in rodents, age has not been found to have a major effect on human NK reactivity. Although some differences in reactivity have been associated with age, the general observation has been that most normal donors, over a wide age range, have easily detectable NK reactivity. The exceptions to this have been the finding of Rosenberg et al. (1972) that donors under the age of 16 had a considerably lower incidence of cytotoxic reactivity against leukemic target cells and the observation of Campbell et al. (1974) that cord blood lymphocytes had much lower

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reactivity than that of adults. However, several investigators have found that cord blood lymphocytes had moderate levels of reactivity (Levin et al., 1975; W. H. West and R. B. Herberman, unpublished observations) or levels indistinguishable from adults (Jondal and Pross, 1975). Takasugi et al. (1973) divided their adult donors by age into three groups, less than 29 years, 30-49 years, and greater than 50, and found no differences in the incidence of reactivity against most target cells. Similarly, Oldham et al. (1975) found no significant relationship between cytotoxicity and the age of normal donors. In contrast to the transient NK reactivity in young rodents, high levels of NK reactivity have been found to persist in some adult human donors for long periods of time (Rosenberg et al., 1974). B. INFLUENCE OF GENETICBACKGROUND

1. Mice

The levels of natural cytotoxic reactivity have been shown to vary widely among different strains of mice. The findings with lymphoid cells from a particular strain seem to be strongly influenced by the target cell used for the studies. In experiments with YAC and RBL-5 lymphoma cells (Kiessling et al., 1975a; Herberman et al., 1975a), CBA mice were found to have strong reactivity and A mice had low levels or undetectable activity. Based on such findings with YAC target cells, Kiessling and his associates have performed a detailed series of experiments to determine the genetic factors influencing levels of natural reactivity. Certain F1 hybrids with A mice as one parent showed good reactivity, whereas others had low reactivity (Petrinyi et al., 1975). From such studies, these investigators concluded that high natural reactivity was dominant. In further experiments, the levels of NK activity appeared to be influenced b y multiple genes, and the H-2 genotype appeared to be one important factor ( P e t r h y i et al., 1976). It seemed from such experiments that one of the genes affecting the levels of reactivity was present on the 17th chromosome, linked to H-2. From recent studies of several congenic resistant strains, the non-H-2 background of the mice appeared to have a considerable influence on reactivity and it has been concluded that the gene(s) must be situated outside of the H-2 locus (R. Kiessling, personal communication). From some recent studies in our laboratory, it seems unlikely that the genetic factors described in the above paragraph determine the overall levels of NK activity. Using RLd 1 lymphoma cells as targets,

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we have observed appreciable levels of cytotoxic reactivity in A mice (Herberman et al., 1977a).The median cytotoxicity values of A strain mice and some other strains against RLd1 are shown in Table 11. A strain mice and some other strains found by Petrcinyi et al. (1975) to be low reactors had levels of reactivity similar to those of C57BL/6 and BALB/c mice. It thus appears that the genetic factors described by Petr6nyi et al. (1975, 1976) affect the recognition of some target cells by NK cells and other factors may affect expression of natural cytotoxic reactivity. As will be discussed in Section 111, NK cells appear to react with a variety of antigenic specificities, and it seems likely that genetic factors help to determine which antigens are recognized. This would also explain a number of other apparent discrepancies among investigators as to the levels of natural cytoxicity in various strains. Even sublines of the same tumor cells may show consistent differences in sensitivity to lysis by NK cells from different strains. Sendo et al. (1975) and Glimcher et a2. (1977) found that spleen cells from BALB/c mice had no appreciable cytotoxic reactivity against cultured RLd 1 TABLE I1 NATURAL CELL-MEDIATEDCYTOTOXIC REACTIVITYOF SPLEEN CELLS FROM VARlOUS CONVENTIONAL STRAlNS OF MICE AGAINST RL61 TISSUECULTURECELLS Strain

A A.TL A.TH A.CA A.SW A.BY A.AL ATFR-1 ATFR-2 BALBJc C57BW6 AKR 129 SJL CBNN CBNH CBNCAHN CBNTG N ZB a

Effector cell :target cell ratio of 200: 1.

Median percentage of cytotoxici ty"

10 8 8 6

7 5 2

5 7 10

11 18 21 5 30 29 15 19 15

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RONAL~D B. HERBERMAN AND HOWARD T. HOLDEN

cells. In contrast, we have found that BALB/c mice react against an established culture line of RLd 1to about the same extent as C57BL/6 mice (Herberman et al., 1975a, 1977a). WP did find that C57BIJ6 and not BALB/c were reactive against RLd 1 ascites tumor cells. To examine directly the differences in reactivity of BALB/c mice against RLd 1 cells, we tested the same effector cells against our established RLd 1 tissue culture line and also against RLd 1 cultured cells from Dr. Cantor (Glimcher et al., 1977) and a short-term RLd 1culture that we had initiated 1 month before. The BALB/c spleen cells reacted well only with our established line, whereas effector cells from some other strains reacted well against all three target cells. Similar to the above differences noted for BALB/c, we and Gomard et al. (1974) observed appreciable reactivity in AKR mice, whereas Zarling et al. (1975) and Sendo et al. (1975) found AKR mice to be unreactive. Zarling et al. (1975) also found C58 mice to be unreactive, whereas Sendo et al. (1975) observed considerable reactivity. In contrast to the considerable variation among strains of conventional thymus-bearing mice in their cytotoxic reactivity against a given target cell, nude mice with a variety of different genetic backgrounds have been found to be highly reactive against several different target cells (Herberman et al., 1975a; Kiessling et al., 197513). Table I11 summarizes the reactivity of several types of nude mice against RLd 1 tissue culture target cells. Young nude mice with random-bred genetic backgrounds (NIH and Swiss), the type most frequently used b y investigators, have had quite high levels of reactivity. Nude mice which were obtained from the Animal Production Section of NIH after several backcross generations with inbred mice also were TABLE 111 NATURALCELL-MEDIATED CYTOTOXIC REACTIVITY OF SPLEEN CELLS FROM VARIOUS STRAINS OF NUDE MICE AND OF LYMPHNODE CELLS FROM LASAT

MICE

Strain of nude mice

Median percentage of cytotoxicitya

NIH Swiss BALBIc CBNN C3H C57BIJ6 Lasat

30 35 25 50 25 20 57

Effector cell :target cell ratio of 50 : 1 against RL8 1 tissue culture.

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highly reactive. Nude mice on a CBA background were somewhat more reactive than other nude mice, but the differences were not as striking as that seen with conventional CBA mice versus some other strains. Lymph node cells from lasat mice (Lozzio, 1976; Lozzio et al., 1976; Machado et al., 1976), which have the gene for asplenia as well as nu/nu genotype and are therefore asplenic as well as nude and have a more profound immunologic deficit, involving B cell as well as T cell functions, have been shown to have very high reactivity. The findings of high reactivity in nude and lasat mice with profound immunologic deficiencies raised the question of whether the observed high reactivity of CBNN might also be related to their defective B cell responsiveness to some antigens (Scher et al., 1975). We therefore compared the levels of NK activity in CBA/N mice with CBA/H and other CBA lines which do not carry this genetically determined defect (Table 11).All of the CBA sublines were found to be highly reactive, and it therefore seems unlikely that the B cell competence of the mice has a major influence on this phenomenon. 2. Rats In contrast to the extensive studies in mice of the influence of genetic background on NK activity, only a small amount of information is available on the variation in reactivity among strains of rats. Most of the studies have been confined to W/Fu rats (Nunn et al., 1976; Shellam and Hogg, 1977; Oldham et al., 1977). Random-bred SpragueDawley rats were shown to have activity against the (C58NT)D tumor which was comparable to that of W/Fu rats (Nunn et al., 1976). Shellam and Hogg (1977) tested a variety of strains, with different Ag-B genotypes, against another target cell, WIFuG-1, and found that most strains had comparable levels of reactivity. However, BDIX and (BDIX x W/Fu)F1 rats had lower reactivity, and BN rats were completely unreactive. Dr. J. R. Oehler in our laboratory has also observed low or absent reactivity in BN rats. It is of interest to note that the (BDIX x WlFu) hybrid rats had reactivity similar to that of the low reacting parent rather than that of WlFu, since, in analogous studies in mice, high reactivity appeared to be a dominant trait (see Section II,B,l). In contrast to the findings with the BDIX x W/Fu)F, hybrids, Williams et al. (1977) found that F1 hybrids of low reacting BN rats and WIFu rats had high NK activity against several clones of SV40transformed BN fibroblasts. Hybrids of BN with two other strains also had higher activity than BN. It is not clear whether these disparate results are due to the different strains used or to the different target cells.

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RONALD B. HERBERMAN AND HOWARD T. HOLDEN

3. Human There have been several reports of an association between reactivity and HLA phenotype. Petrinyi et al. (1974) initially reported that individuals with the HLA-A3, B7 haplotype had decreased cytotoxic activity. This has been confirmed and extended by Trinchieri and his associates (Santoli et al., 1976; Trinchieri et al., 1977). Male donors with HLA-B7 had significantly depressed activity and those with the HLA-A3, B7 haplotype had quite low activity. Significantly elevated reactivity was associated with HLA-B12. For no apparent reason these correlations were only significant for male donors, and female donors had activity only Vz to 2/3 that of male donors. It was also of interest, in regard to the possible in vivo role of natural cytotoxicity (see Section VIII), that the HLA-AS, B7 haplotype has been associated with an increased incidence of multiple sclerosis. The only other suggestion for a role of genetic factors in expression of natural cytotoxicity has come from the studies of Hellstrom et al. (1973), which indicated that adult black donors had significantly more reactivity than white donors in a visual microcytotoxicity assay against cultured melanoma cells. However, this report was based on tests of a small number of donors, and other investigators have failed to find a significant race-related difference in the reactivity of normal donors (e.g., Oldham et al., 1975).

c. INFLUENCE

OF

ENVIRONMENTAL FACTORS AND DISEASE

1. Mice The time course of spontaneous appearance of cytotoxic reactivity in mice within 8 weeks of age, persistence for only 2 to 4 weeks and then decline to low levels, is similar to that seen after immunization with alloantigens (Canty and Wunderlich, 1970)and tumor antigens (Lavrin et al., 1973). This suggested that most mice are exposed to antigen(s) within a few weeks after birth, which induced cytotoxicity shortly thereafter. Initially, environmental factors did not appear to play a major role in the kinetics or levels of natural cytotoxicity (Herberman et al., 1975a). Conventional and nude mice, whether raised conventionally or pathogen-free, seemed to have comparable reactivity. Furthermore, mice coming fiom a wide variety of sources developed similar levels of activity at approximately the same age. Because of these observations, it was suggested that endogenous factors in the mice, probably activation of endogenous type-C viruses, were responsible for producing sensitization. Recently we have noted considerable fluctuations in levels of activity in nude mice, partially associated with the source of the mice, the place for housing prior to testing, and

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the timing of transport to the testing laboratory. In addition, the general health of the nude mice when tested has sometimes been found to influence reactivity, with sick mice usually being less reactive than healthy mice. When differences have been noted, they have generally been in the direction of low activity at a time when high, peak reactivity was expected. The exact nature of the environmental factors producing these variations in nude mice has not yet been determined. However, these recent observations indicate that environmental factors may play an important role in either depressing or augmenting the innate cytotoxic reactivity of nude mice, and perhaps also of conventional mice. Mice bearing tumors have been found to have depressed levels of NK activity. Herberman et al. (19754 found that mice bearing murine sarcoma virus (MSV)-induced primary tumors had lower reactivity than uninoculated mice, Becker and Klein (1976) have confirmed this observation, and they obtained similar findings with conventional mice bearing a syngeneic methylcholanthrene-induced sarcoma and with nude mice bearing tumors induced by human lymphoblastoid cell lines. The finding of some NK activity within MSV tumors (Becker and Klein, 1976) indicates that this depressed reactivity in the spleen or other peripheral lymphoid organs could be due, at least in part, to a shift in distribution of NK cells.

2. Rats There have only been limited studies of the effects of environment on N K activity in rats and no information is available on the levels of reactivity in rats with tumors or with other diseases. Thus far, environmental factors have not been shown to play an important role. W/Fu rats in the United States (Nunn et al., 1976) and in England (Shellam and Hogg, 1977)have been shown to have appreciable levels of cytotoxicity. It is not possible to compare the levels seen in these studies directly since different target cells and other conditions were used. Shellam and Hogg (1977) found similar levels of reactivity in rats tested on the day of arrival in their laboratory and in rats of the same age housed for weeks or months in their animal house. Nunn et al. (1976) compared young germ-free Sprague-Dawley rats with conventionally raised rats and found similar levels of reactivity. Shellam and Hogg (1977) compared conventional and germ-free rats of another strain and also detected no differences.

3. Human It has been difficult to identify environmental factors which influence human NK reactivity. However, some data suggest that environ-

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mental factors may affect the levels of cytotoxicity. In contrast to the good reproducibility in results when the same donor is tested repeatedly over a short time period, several investigators have noted that some normal donors have considerable changes in levels of NK activity when followed for a long period of time (e.g., Rosenberg et al., 1974; Heppner et al., 1975). Usually there has been no obvious cause for such decreases or increases in reactivity. We have noted that some donors who usually have low levels of reactivity became highly reactive for a period of time after developing viral respiratory infections. This may be related to the augmenting effects of viruses, as discussed below (Section 11,D). The issue of environmental factors has also come up in studies of laboratory workers and of relatives and associates of cancer patients. Takasugi et al. (1973) noted that laboratory workers have a higher incidence of reactivity against various target cells than did other normal donors. We initially thought there was such an association (Oldham et al., 1973) but upon further testing this relationship was not seen (Oldham et al., 1975). Pross and Jondal (1975) also found no difference between laboratory workers and other normal donors in cytotoxic reactivity against the mouse tumor, P815X2. Rosenberg et al. (1972) detected cytotoxic reactivity of relatives of leukemia patients against leukemic target cells. However, other, unrelated normal donors were also reactive and there was no clear difference among donors in the same age range. Byers et al. (1975) described a significantly higher incidence of cytotoxic reactivity in household contacts of patients with osteogenic sarcomas or breast cancer against tumor-derived cell lines, which they concluded was specific for the type of cancer of the contact patient. However, the ALAb cell line was the main breast cancerderived cell line to show this reactivity, and Levin et al. ( 1976a) have found this to be an excellent target cell for more generalized natural cytotoxic reactivity. Although the possibility of increased cytotoxic reactivity in workers exposed to cancer cells and related materials or relatives exposed to cancer patients is intriguing, the data obtained thus far are equivocal or conflicting. Many investigators have compared the incidence of cytotoxic reactivity against tumor cell lines of cancer patients with that of normal donors. In most cases, the studies have focused on cell lines derived from the same type of cancer as that of the patients studied, and the results among studies have been conflicting and difficult to evaluate (Herberman and Oldham, 1975; Stevenson and Laurence, 1975). This may be partly due to the likelihood that several mechanisms of cytotoxicity are measured in such studies, including NK reactivity as

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well as disease-specific reactivity. To circumvent this problem and attempt to examine NK reactivity of carcinoma patients and of normal donors directly, some investigators have utilized lymphoblastoid, myeloid, or similar target cells (Rosenberg et al., 1974; Pross and Jondal, 1975). McCoy et al. (19734 and Pross and Jondal(l975) have suggested that such assays might be a useful measure of a form of lymphocyte function. Analogous to the decreased NK reactivity in tumor-bearing mice, McCoy et al. (19734 found that many patients with lymphomas, colon cancer, lung cancer, and melanoma had depressed reactivity against F265, a lymphoblastoid cell line, and that the reactivity of breast cancer patients was in the normal range. Similarly, Takasugi et al. (1973) reported that many normal donors were more reactive than cancer patients against a variety of cancer-derived target cells. Takasugi et al. (19774 have further reported that reactivity of cancer patients declined with tumor progression. In a recent study, Cannon et al. (1977) tested lymphocytes from normal donors and breast cancer patients against the myeloid cell line K562, as an indicator of NK reactivity, and against breast cancer cell lines. The reactivity against K562 was similar for each population, whereas (as will be discussed in more detail in Section VII) the breast cancer patients had higher levels of reactivity against the breast cancerderived cell lines. Heppner et at?.(1975) also found that the incidence of reactivity of cancer patients versus normal donors varied with the target cell line. With breast cancer-derived lines, cancer patients were usually more active than normal donors, but with some melanoma lines, the normal donors were frequently more reactive than the cancer patients. Oldham et al. (1975) also studied cytotoxicity against breast cancer and melanoma target cells, and observed that the incidence of reactivity of cancer patients and normal donors against all of the targets was similar.

D. In Vivo AUGMENTATION OF REALITY

1. Mice

The possibility for environmental factors to influence the levels of natural cell-mediated reactivity led to a series of experiments in mice to determine the effects of in vivo challenge by a variety of materials (Herberman et d., 1977a). Using the analogy to the kinetics of cytotoxicity induced by deliberate immunization, we were particularly interested in whether a “secondary” response could b e elicited in mice after decline in their spontaneous levels of cytotoxicity. We

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RONALD B. HERBERMAN AND HOWARD T. HOLDEN

found that reactivity in nude BALB/c mice, as well as in normal BALB/c mice, could be substantially augmented by inoculation of a variety of tumor cells. In young mice, the levels of reactivity were increased and in older mice, cytotoxicity rapidly reappeared. This augmented cytotoxicity reached a peak at 3 days after inoculation, and then declined to baseline levels b y 6 to 7 days. Only cells which appeared to bear antigens recognized by the NK cells were found to augment reactivity. Initially, only mouse cells were found to be active. However, after detecting cytotoxic reactivity against the human Chang liver tissue culture cell line (see Section III,A), we tested the ability of these xenogeneic cells to boost and found them to be active. Similarly, after recent indications that allogeneic bone marrow might contain antigens recognized by NK cells, we found that allogeneic bone marrow cells but not allogeneic spleen cells or syngeneic bone marrow cells could significantly augment cytotoxic reactivity. In addition to this apparently specific boosting, a variety of murine viruses, including murine sarcoma virus and lymphocytic choriomeningitis virus (LCMV), were able to cause marked increases in the levels of cytotoxicity in either nude or conventional mice (Herberman et al., 1977a). This effect appears to b e dependent on the ability of the viruses to replicate in the recipients since inactivated virus or viruses unable to infect mouse cells have been inactive. The viruses appeared to induce nonspecific, polyclonal activation of the natural cytotoxic mechanism, and the bacterial adjuvants, BCG and Corynebacteriurn purvum, has similar effects (Herberman et ul., 1977a). Wolfe et al. (1976) have also recently noted the boosting of natural cell-mediated cytotoxicity by BCG. In all of these studies, the characteristics of the effector cells after boosting were the same as those seen with NK cells (see Section IV). Pfizenmaier et al. (1975) observed a similar phenomenon, the rapid and temporary appearance of cytotoxicity against some cultured syngeneic target cells after inoculation of LCMV. On the basis of the effects of pretreatment of the effector cells with anti-8 plus complement, these investigators concluded that T cells were responsible for this activity. Good sensitivity to anti-8 would seem to be at variance with the experience with NK cells (see Section IV), but, in fact, depression of reactivity by such treatment was difficult to achieve (H. Wagner, personal communication). The ability of microorganisms to strongly augment the levels of NK activity may be related to some of the observed environmental effects on cytotoxicity. If mice were tested shortly after environmental exposure to a virus capable of augmenting, high levels might be seen. However, if they were tested several days later, low levels, possibly even below their baseline spontaneous levels, might be observed.

NATURAL CELL-MEDIATED IMMUNITY

32 1

The augmentation of NK activity by tumor cells bearing the relevant antigens might appear to be at variance with the observation of depressed reactivity in mice bearing primary MSV tumors (discussed in Section I1,C). However, as noted above, the timing may be critical. When mice are tested at 14 days or more after tumor cell or MSV inoculation, this is considerably beyond the period of augmentation. This would probably also account for the failure of Greenberg and Playfair (1974) to detect augmentation of natural reactivity in young NZB mice after inoculation of tumor cells, since they only looked at 12 days post inoculation. 2. Human There is very little information as yet on the ability to boost NK activity in rats or in man. It will be important to evaluate carehlly the effects on NK reactivity of inoculation of tumor cells, BCG, C. parvurn, and other agents which are being used for immunotherapy of cancer patients. Some evaluation of NK reactivity in patients receiving immunotherapy has been performed (e.g., Oldham et al., 1976a), but no studies have been reported on sequential daily testing of cytotoxicity after inoculation. This type of detailed kinetic study would probably be needed to detect augmentation analogous to that observed in mice. Based on the ability of several viruses to augment NK reactivity and the anecdotal observations of increased cytotoxicity in some donors with upper respiratory viral infections, we have recently performed a study on the effects of inoculation with swine influenza vaccine (W. H. West and R. B. Herberman, unpublished observations). A series of normal adult volunteers were tested for cytotoxicity against K562 target cells prior to vaccination and at frequent intervals thereafter. In the majority of donors, a significant increase in reactivity was seen 1to 3 days after inoculation. Thus, it appears that human NK activity can be augmented in a fashion quite analogous to that seen in mice.

E. EFFECTSOF in Vitro CULTIVATION

OF

LYMPHOID CELLS

In many studies, it has been of interest to test for NK reactivity after lymphoid cells were placed in culture for several days. In addition, many of the cytotoxicity assays involve incubation periods of 1 to 2 days. When reactive cells are cultured in the presence of tumor antigens or other possible stimulants, one might anticipate that in vitro augmentation of NK activity would result, The results of such studies have been complex, with different effects being observed in each species studied.

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RONALD B. HERBERMAN AND HOWARD T. HOLDEN

1. Mice One of the distinguishing characteristics of NK activity in mice has been its lability at 37°C. Incubation of reactive cells for 2 hours or more at 37°C has resulted in a substantial fall in cytotoxic activity (Herberman et al., 1975b). In contrast, such incubation of cytotoxic immune T cells has had no inhibitory effect (Herbermanet al., 1975b). The augmented reactivity seen after inoculation of tumor cells or microorganisms has also exhibited this lability (Herberman et al., 1977a; Wolfe et al., 1976). The loss in NK reactivity has not been accompanied by a large decrease in viable cells, and addition of 2-mercaptoethanol to the culture medium, which enhances in vitro survival of mouse lymphoid cells, has had no effect. The mechanism for this loss of functional activity, which has not been seen with rat or human cells, remains unclear. However, this lability of the effector cells probably accounts for our failure thus far to observe any evidence for in vitro augmentation of NK reactivity when we have used any of the procedures which work well with rat or human cells. Because of the lability of mouse NK activity, it has been essential to control carefully all in vitro manipulations with reactive cells to distinguish the lability from the effect of the particular treatment. Shustik et nl. (1976) were successful in generating cytotoxicity in normal cells cultured for 5 days, and the levels achieved were comparable to those achieved by sensitization against allogeneic stimulating cells. Gorczynski (1976a,b) had similar results, with cultured male spleen cells having considerably more cytotoxicity than those of female donors.

2. Rats Incubation of normal rat lymphoid cells at 37°C prior to testing has not resulted in a loss of reactivity but rather has been associated with significant increases in cytotoxicity. Shellam and Hogg (1977) observed that preincubation of cells for 3 hours resulted in consistent augmentation of cytotoxicity. Glaser et al. (1976a) measured at daily intervals cytotoxic reactivity of normal lymphoid cells cultured for up to 10 days. After 1 day of culture, normal spleen cells were significantly more cytotoxic than before culture; this cytotoxic reactivity then decreased to negligible levels with further time in culture. Culture of normal cells with (C58NT)D lymphoma cells resulted in persistent reactivity against that tumor, with a further rise at days 6 and 7. However, the characteristics of this augmented reactivity, and its relationship to NK reactivity versus specifically immune antitumor

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cytotoxic reactivity were not evaluated. Also the similarity of the augmented reactivity after several hours to 1 day of culture in medium alone to the in vitro augmentation of immune rat spleen cell cytotoxicity (Ortiz de Landazuri and Herberman, 1972a) has not been thoroughly expolored. Glaser et al. (1976~)found that incubation of rat spleen cells for 1 day in the presence of phytohemagglutinin (PHA) or endotoxin (LPS) resulted in a considerable augmentation in cytotoxic activity against a syngeneic tumor, (C58NT)D, and also against other syngeneic and xenogeneic target cells. These results are similar to the augmentation of cytotoxic reactivity of human lymphocytes by in vitro incubation with mitogens or other stimuli (see Section II,E,4). However, there were some observations with the mitogen-treated rat cells which complicate interpretation of the mechanism. The stimulated spleen cells reacted against a broader array of target cells than did unstimulated normal spleen cells, and different lymphoid cells appeared to be involved in generation of augmented cytotoxicity b y PHA as compared to LPS (see Section IV,B for more detailed discussion of the nature of effector cells).

3. Guinea Pigs There have been no reports, to our knowledge, of natural cellmediated cytotoxicity in guinea pigs. However, some recent studies of Dr. A. Altman (unpublished observations) with cultured normal guinea pig lymph node cells seem quite similar to those described in this section for cultured lymphoid cells of other species. After culture in medium containing fetal bovine serum (FBS), high levels of cytotoxicity against a syngeneic hepatoma cell and some other target cells have been observed. As described below for human PBL, this activity was dependent on culturing of the cells in heterologous serum (either FBS or horse serum) and was not seen in medium containing only serum from the same species (i.e., guinea pig serum).

4. Human As with rat lymphoid cells and in contrast to mouse lymphocytes, incubation of human peripheral blood lymphocytes (PBL) at 37°C has not led to loss of cytotoxic reactivity. Considerable increases in cytotoxicity upon in vitro culture under a variety of conditions have been noted. The kinetics of these increased levels of cytotoxicity have differed from those seen with rat cells, in that activity has usually remained high for 7 days or more of culture. The augmented levels of cytotoxicity have many characteristics which are similar or identical to NK reactivity, but, until the phenomena are shown to be the same,

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Bonnard (1978) has proposed the term CCC (cytotoxicity from cultured cells). Several investigators have reported that incubation of human PBL at 37°C in medium containing FBS resulted in a rapid increase in cytotoxic reactivity against tumor target cells (Stejskal et al., 1973; Levin et al., 1976b; Zielske and Golub, 1976; Ortaldo et al., 1977a,b). This phenomenon was found to b e highly dependent on the presence of FBS in the culture medium and did not occur in medium containing human serum (Zielske and Golub, 1976; Ortaldo et al., 1977a,b). However, cultures of PBL in the presence of mitogens, antigens, allogeneic PBL, or autologous or allogeneic lymphoblastoid, myeloid or tumor cell lines have also been associated with the development of high levels of cytotoxicity (Stejskal et al., 1973; Svedmyr et al., 1974; Martin-Chandon et al., 1975; Stejskal and Perlmann, 1976; Ortaldo et al., 1977a,b; Morales et al., 1977). With the mitogens and soluble antigens, the PBL were washed free of the stimuli prior to assay and therefore the effector phase was not dependent on the presence of the stimulant. There has generally been a good correlation between the development of a lymphoproliferative response and augmentation of cytotoxicity, and, since FBS as well as mitogens and antigens can stimulate proliferation, the same mechanism may underlie all of these observations. 111. Specificity of Natural Cell-Mediated Cytotoxicity

A. MICE

In virtually all of the studies of natural cell-mediated cytotoxicity, the investigators detected some degree of specificity in the phenomenon. However, the conclusions reached regarding the nature of the antigens have varied considerably depending on the tumor cells tested. In several studies, the detected antigens appeared to b e associated with type-C viruses (Herberman et al., 1975a; Kiessling et al., 1975a; Sendo et al., 1975; Zarling et al., 1975; Lee and Ihle, 1977). Kiessling et al. (1975a) tested for NK activity against a limited number of target cells and concluded that susceptibility to lysis was restricted to lymphomas induced by Moloney leukemia virus. However, in more recent studies in this laboratory (Becker et al., 1977; Kiessling, personal communication), this restriction was not found to hold. Becker et al. (1977) found no correlation between susceptibility to NK activity and any serologically defined group of type-specific antigen associated

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with type-C viruses. Furthermore, we (Herberman et al., 1975a) and others (e.g., Becker et al., 1976) have not seen any restriction of susceptibility to homologous H-2 type. Zarling et al. (1975) suggested that Gross leukemia virus antigens or embryonic antigens were being dected. Sendo et al. (1975) suggested that NK activity was directed against X. 1’ antigen, associated with expression of an endogenous type-C virus. Similarly, we have suggested (Herberman et al., 1975a) that NK activity is directed against several antigens associated with murine endogenous type-C viruses. Blair and Lane (1975a,b) and Gillette and Lowery (1976)have described reactivity in microcytotoxicity or cytostasis assays against mouse mammary tumor virus-infected target cells. In contrast to these reports of reactivity against virus-associated antigens, Small and Trainin (1975) and Pfizenmaier et al. (1975) have observed reactivity against autoantigens which bears some resemblance to NK activity. To determine the specificity of NK activity, two approaches have been taken. The first has been to test directly a variety of target cells for susceptibility to lysis. The other, which has provided more detailed information on the heterogeneity of the detected antigens, has been to test a wide variety of cells for thier ability to inhibit release of 51Cr from labeled target cells.

1. Direct Testing In almost all of the studies referred to above, normal lymphoid cells were tested against lymphoma target cells, and it has been widely assumed that only lymphoid target cells are susceptible to rapid lysis b y NK cells. However, we have recently found that many nonlymphoid target cells, harvested directly from in vivo tumors or from cultured cell lines, are susceptible to NK activity in a 4-hour 51Crrelease cytotoxicity assay (Table IV). Some sublines of 3T3, including the MSV transformed nonproducer line KA31 and SV40 transformed lines, were good target cells for nude spleen cells. Although most of the studies have been performed with established tumor cell lines, we have found that primary spontaneous thymomas of AKR mice are quite susceptible to NK activity. The low but significant levels of reactivity against the untransformed 3T3 cell line indicated that sensitivity to NK activity was not limited to transformed cultured cells. Similarly, Shustik et al. ( 1976) found that cultured lymphocytes developed high reactivity against cultures of syngeneic and allogeneic embryo fibroblasts. As discussed earlier (Section II,D), Pfizenmaier et al. (1975) observed transient cytotoxicity, against short-term cultures of syngeneic normal

326

RONALD B. HERBERMAN AND HOWARD T. HOLDEN TABLE IV NATURALCYTOTOXICITY O F NUDE SPLEEN CELLS AGAINST NONLYMPHOID CELL LINES Target cell

3T12 (embryo fibroblasts, transformed) 3T3 (embryo fibrobIasts) Type I CI 6 (spontaneously transformed) KA31 (MSV transformed, nonproducer) SV40 transformed: SV3T3 SVA 31C 14 E4 T-AUN lines (fibroblasts, spontaneously transformed) T-AUN C1, T-S-1 (SV40+) T-S-5 (SV40-) B16 (melanoma) LM5F-22 3LL (Lewis lung tumor) Mammary cancer cell lines TA3 L8a a

Percentage of cytotoxicitya 45

5 3.6 10 24 14 9

31 22 40 10 8 10 2.7 0.5

Effector cell :target cell ratio of 100: 1.

macrophages, after inoculation of mice with LCMV. Since most of the characteristics of their observations were compatible with augmented NK activity, it was of interest to examine directly the susceptibility to NK cells of overnight cultures of peritoneal macrophages. We found that spleen cells from NIH nude mice, and from CBA mice, 4 days after inoculation with LCMV, had significant levels of activity against these target cells. We have recently also observed low levels of NK activity against mouse PHA blasts. As already discussed earlier (Section II,B), the susceptibility to NK activity of a given tumor cell has been found to vary considerably with the growth conditions. In uiuo transplanted tumors have been more resistant to NK activity of most strains than in uitro cultures of the same tumors (Kiessling et al., 1975a; Herberman et al., 1975a), and some cultured cells have been much more sensitive to lysis (Kiessling et al., 1975a). This is consistent with the recent observations of Aoki et al. (1977) that several virus-associated antigens varied widely in expression, depending on whether the cells were grown in uitro or in uiuo. It initially appeared that mouse NK activity was restricted to mouse

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target cells. However, recently we have detected considerable reactivity of nude spleen cells against some human target cells but not against others (Herbermanet al., 1977a).The human Chang liver tissue culture cell line has been particularly sensitive to lysis. Similarly, R. Kiessling and his associates (personal communication) have observed lysis of the MOLT-4 cell line derived from a patient with acute lymphocytic leukemia. We have also observed some reactivity of nude and other mouse spleen cells against the rat lymphoma, (C58NT)D,which is also a susceptible target cell for rat NK activity (Nunn et al., 1976). We have also observed recently that mouse NK activity is not completely restricted to tumor or cultured target cells. The finding that normal mouse thymocytes could boost cytotoxic reactivity led us to test thymocytes as target cells (Herberman et al., 1977a). Table V shows the results obtained in tests of BALB/c nude spleen cells against syngeneic and allogeneic thymocytes and other normal target cells. Significant reactivity was observed against thymocytes and bone marrow cells but not against spleen or lymph node cells.

2. Inhibition of Cytotoricity Assays

The above results of direct tests with a variety of target cells indicate that many tumor cells and some normal cells are susceptible to N K activity. The resistance of some target cells, including some known TABLE V NATURALCYTOTOXICITY OF BALBlc NUDE SPLEENCELLS AGAINST NORMALTARGETCELLS Target cells

Tissue Thymus Thymus Thymus Thymus Thymus

Bone marrow Bone marrow Spleen Spleen Spleen Lymph nodes Lymph nodes

Strain BALBlc BALBlc C57 B L16 AKR NZB BALBlc WIFu rat BALBlc C57BLl6 NZB BALBlc C57BU6

Effector cell:target cell ratio of 200: 1.

Age of donor (weeks) 0 8 8 12 6

8 6 8 8 6 8 8

Percentage of cytotoxicity"

9 12 13 20 9 6 4 -1 1 1 -1 0

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to be susceptible to immune cytotoxic T cells, to lysis by normal lymphoid cells suggests that the reactions are specific. However, these data do not allow one to determine whether all susceptible target cells share the same antigen or whether several antigens are involved. To analyze the specificity of NK activity in more detail, we (Herberman et al., 1975a) and others (Kiessling et al., 1975a; Sendo et al., 1975; Zarling et al., 1975) have utilized the assay of inhibition of W r release cytotoxicity (Ortiz d e Landazuri and Herberman, 197213; Herberman et al., 197613). With each labeled target cell used in the assays, different antigenic specificities were determined. Because most of the tumor cells studied had some expression of endogenous type C viruses, it was postulated that the antigens detected by NK cells were associated with murine endogenous type-C viruses and these were designated MEV-SA2, 3 and 4 (Herberman et al., 1975a). In the inhibition studies of Sendo et al. (1975), an association of the antigens with endogenous type-C virus was also suggested. Consistent with this hypothesis, Lee and Ihle (1977) have found that NK activity of (B6C3)F1 mice could be specifically inhibited by gp69/71, the major envelope glycoprotein, of AKR endogenous leukemia virus. It therefore seems likely that murine endogenous type-C virus-associated antigens account for at least some of the NK activity. However, with the recent findings of NK activity against some xenogeneic tumor cells and some normal cells, it seems likely that additional, nonviral specificities are also involved. More inhibition studies, using a range of different target cells, are needed to sort out the distribution and nature of each antigen recognized by NK cells.

3. Examination of Tumors in Nude Mice for Antigenicity We are currently studying tumors which arise in nude mice for their susceptibility to NK activity and for the presence of the relevant antigens of these tumors. We received from Dr. 0. Stutman, under code, three tumors induced by methylcholanthrene. Two tumors were induced in CBA/H nude mice and the third in a normal CBA/H mouse. By direct testing and by the inhibition assay, the two tumors from nude mice lacked NK-related antigens, whereas the other tumor was positive. We have also received from Dr. H. Outzen five lymphocytic leukemias which arose spontaneously in BALB/c nude mice and three sarcomas of BALB/c nude mice induced by methylcholanthrene. All of these tumors also lacked NK-related antigenicity. The significance of this observation is somewhat confounded by the lack of antigenicity on several of Outzen’s spontaneous and induced tumors of normal BALB/c mice. It remains of interest, however, that none of the lym-

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phomas or sarcomas of nude mice studied so far has been susceptible to NK activity. As will be discussed later (Section VIII), this has important implications regarding the possible role of NK cells in immune surveillance. B. RATS

The studies of natural cell-mediated cytotoxicity in rats have also supported the specificity of the phenomenon. The approaches to analysis of specificity, i.e., direct testing and inhibition assays, and the pattern of results have been very similar to those in mice. However, in contrast to the studies in mice, little direct testing for specificity has been done, but rather the inhibition assays have been mainly relied upon. 1. Direct Testing

In the studies of Nunn et al. (1976), only the syngeneic (C58NT)D ascites tumor cell was used. Oldham et al. (1977) tested (C58NT)D ascites and tissue culture cells as targets and also the ERTh/V-G tissue culture line. In the short-term CRA, good reactivity was seen against the ascites (C58NT)D line but not against the cultured (CS8NT)D cells. The resistance of the cultured cells to NK activity could not be attributed to a general resistance to lysis since immune spleen cells reacted well against this line (Ortaldo et al., 1976; Oldham et al., 1977) and natural cytotoxicity could be detected against it in assays with longer incubation periods (Oldham et al., 1977). It is of interest to note, however, that the increased susceptibility of an ascites target compared to cultured cells is the opposite of that usually observed with mouse target cells (as discussed above, Section III,A,l). With another target cell, W/FuG-1, Shellam and Hogg (1977) found that the cultured cells were considerably more susceptible to rapid lysis than were the ascites cells. These investigators also tested a variety of other rat tumor cells and mouse cell lines for susceptibility to NK activity. Only rat tumors induced b y Gross or Moloney leukemia viruses were susceptible, and other rat tumor cells, normal rat lymphoid cells, and mouse tumor cells, including some induced by leukemia viruses, were resistant. Glaser et al. (1976~)tested the direct specificity of spleen cells cultured for 1day in medium alone or in the presence of PHA or LPS. The cells cultured alone reacted mainly against (C58NT)D, had low reactivity against another syngeneic line, LW-6, and no significant reactivity against mouse tumor cells. In contrast, the mitogenstimulated cells reacted well against all of the target cells tested. The

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extent of reactivity of rat NK cells against mouse tumor target cells needs to be more thoroughly investigated. We have recently found that RL81 mouse cultured cells are very sensitive to cytotoxicity by normal rat spleen cells. The human Chang and K562 cell lines were also sensitive to lysis b y rat NK cells. 2. Inhibition of Cytotoxicity Assays Nunn et al. (1976) tested a large number of rat and mouse cell lines for their ability to inhibit cytotoxicity of 51Cr-labeled(C58NT)D target cells. These studies demonstrated that the NK reactivity had specificity, with some cells inhibiting well and others giving little or no inhibition. The specificities of the immune reactivity against (C58NT)D and NK reactivity were found to be similar, but differed from any known serologic antigens. A variety of rat cells shared the antigens, but virusinduced mouse leukemias, including RBL-5 which is susceptible to mouse NK activity, were negative. This pattern of species-restricted specificity was analogous to MEV-SA-1,2,3, and 4 detected in studies of mice (Herberman et al., 1975a). Similarly, since there was a correlation between ability to inhibit and evidence for expression of rat endogenous type-C virus, it was suggested that the antigens detected were associated with rat endogenous viruses. Shellam and Hogg ( 1977)confirmed and extended these observations. They found inhibition with rat lymphomas induced b y murine leukemia viruses, with rat fibroblasts deliberately infected with these viruses, and with rat cell lines known to express rat endogenous virus or virus-associated antigens. Other nonvirus-induced tumors, tumors induced by pol yoma virus, and normal adult or fetal cells were negative. The mouse tumor cells tested were also negative. In addition to the inhibition assay, Shellam and Hogg (1977) performed adsorptions of effector cells on different monolayers and then tested for residual cytotoxicity against W/FuG-1. The same pattern of specificity was seen in these tests. They also tested murine sarcoma virus, feline leukemia virus, and Rous sarcoma virus for their ability to inhibit NK reactivity and found that only the murine virus gave a dose-responsive inhibition. However, this inhibition did not fit the species restriction seen with intact cells, and Shellam and Hogg cautioned that disrupted virus preparations may cause nonspecific inhibition. It should be noted that the evidence for species restriction of antigens was obtained when rat tumor cells were used as labeled targets. The recent finding of direct cytotoxicity of rat cells against mouse RL81 target cells requires that this issue be examined further to determine if RL81 cells can inhibit cytotoxicity of rat target cells or

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whether a separate antigenic specificity is being recognized on the heterologous cells.

C. HUMAN 1. Direct Testing Many investigators have tested lymphocytes from normal donors against a variety of human cell lines derived from tumors. In early studies, histologic type specific cytotoxicity of cancer patients was described, and reactivity b y normal donors was not noted (e.g., Hellstrom et at., 1971; Fossati et al., 1971; Sinkovics et al., 1971; O’Toole et al., 1972; Levy et d., 1972; Heppner et a l . , 1973). When investigators began to take notice of natural cytotoxicity, most described it as nonspecific (e.g., DeVries et al., 1975; Bukowski et al., 1976; Jondal and Pross, 1975; Hersey et al., 1975; Pierce and DeVald, 1975; Peter et al., 1976a,b). However, several investigators have recognized that lack of histologic type specific cytotoxicity should not be equated with complete lack of specificity, but rather that specificity or lack thereof needs to be carefully evaluated b y large “checkerboard” experiments (Herberman and Oldham, 1975). Klein has proposed (Bean et al., 1975) a useful categorization of the results of such tests and we will use this nomenclature in our discussion. She defined diseaserelated cytotoxicity as cytotoxicity solely against specific tunior target cells b y cells from patients with that type of cancer, selective cytotoxicity as cytotoxicity for some target cells of other histologic types but not for all target cells tested, and nonselective (presumably nonspecific) cytotoxicity as killing of all target cells tested, regardless‘ of histologic type. Most recent studies have described a lack of disease-related specificity, mainly because of natural cytotoxicity (e.g., Oldham et al., 1973, 1975; Takasugi et al., 1974; DeVries et al., 1975; Berkelhammer et al., 1975; Pavie-Fischer et al., 1975; Heppner et al., 1975; Canevari et at., 1976; Bukowski et al., 1976). However, several investigators have found selectivity in their cytotoxicity results (Matthews and MacLaurin, 1974; Rosenberg et aZ., 1974; Oldham et at., 1975 Pross and Jondal, 1975; Heppner et al., 1975). Others, on the basis of direct testing against many cell lines, have characterized their results as nonselective (Kiuchi and Takasugi, 1976; Takasugi et al., 1977b; Bakacs et al., 1977). In a workshop in which a number of groups of investigators performed tests with the same sources of effector cells and target cells, virtually all found some evidence for selec-

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tive reactivity by some donors (Bean et al., 1975). Takasugi’s group, while not finding selective cytotoxicity when analyzing their data by conventional means, have described selectivity revealed by a complicated procedure, termed interaction analysis (Kiuchi and Takasugi, 1976; Takasugi et d.,1977b). From those studies, they concluded that reactions were directed against at least two, widely distributed common antigens, TA1 and TA2 (Takasugi et al., 197713). Using a different procedure, Cannon et al. (1977) have also qualified the distinction between selectivity and nonselectivity b y demonstrating quantitative selectivity or relatively high cytotoxicity of some donors against breast cancer-derived cell lines. From all of these studies, it can be concluded that human natural cytotoxic reactivity, like that of rodents, is not nonspecific but rather appears to be directed against antigens which are represented on many different cell lines of various histologic types. An important observation has been that human PHA blasts are resistant to NK activity (Ortaldo and Bonnard, 1977), which probably accounts for the ability to detect specific histocompatibility antigen associated cytotoxicity with these targets. In one study (Santoli et d., 1976), reactivity seemed to be restricted to human and monkey cell lines, with little or no reactivity against mouse, rabbit, or hamster cell lines. However, others (PetrLnyi et al., 1974; Pross and Jondal, 1975) have detected strong reactivity against some mouse target cells. The detected antigens have been shown not to be due to fetal bovine serum, since target cells grown for some time in medium with human serum were also susceptible to cytotoxicity (McCoy et al., 1973b; Rosenberg et al., 1974). 2. Inhibition of Cytotoxicity Assays To analyze further the specificity of the antigens recognized by human NK cells, a few studies have been done with inhibition of cytotoxicity assays. In initial studies with the F-265 lymphoid cell line as target cells (McCoy et a1., 1973b; Rosenberg et a1., 1974), inhibition was seen with F-265 and another lymphoid cell line, whereas with a third lymphoid cell line, human PBL, thymus cells, and erythrocytes were negative. Ortaldo et al. (1977~)performed a more extensive study with the K562 target cell. A broad but reproducible pattern of specificity was demonstrated, with most but not all human established tissue culture cell lines being inhibitory. Some fresh single cell suspensions from human malignant tissues also were positive. Some other human established cell lines, leukemic blast cells, and normal PBL were negative. Of interest, in contrast to the species restriction generally found in the inhibition assays with rodent NK and labeled target

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cells, positive results were not limited to human cells; some mouse and rat lymphoma cells inhibited the cytotoxicity against K562. Takasugi et al. (1977b) have performed more extensive inhibition studies using several different target cells. They have also concluded that specific antigens are being detected and that their results support the expression of either one or both of two antigens, TA1 and TA2, on many cell lines. IV. Nature of Effector Cells

Much attention has been directed toward an understanding of the nature of NK cells. The findings of high levels of cytotoxicity reactivity in nude mice, the resistance of rat NK activity to anti-T cell sera plus complement, and the presence of cytotoxic reactivity in human PBL not readily forming rosettes with SRBC indicated that natural cellmediated cytotoxicity was mediated by effector cells different from the well-studied cytotoxic T lymphocyte, produced b y deliberate immunization in uiuo or in uitro (Cerottini and Brunner, 1974). Extensive investigations have further indicated that NK cells are not macrophages or B cells, but may be a subpopulation of T cells or pre-T cells. In this section, w e will review the large body of information which has been obtained in this area.

A. ORGANDISTRIBUTION 1. Mice

NK cells have been detected in most of the lymphoid organs of mice, with particularly high activity in the spleen, lymph nodes, and peripheral blood and low activity in the bone marrow and peritoneal cavity (Herberman et al., 1975a; Kiessling et al., 1975a). Only thymocytes were found to lack activity consistently. In our experience, the levels of reactivity in spleen and lymph nodes of individual mice were similar. In contrast, Greenberg and Playfair (1974) found that activities in these two organs did not correlate, and that the levels in peritoneal exudate cells were similar to those of lymph node cells. The reason for the discrepancy is not clear, but it may be related to the older age of the NZB mice studied by Greenberg and Playfair (1974). After boosting with tumor cells or microorganisms, high levels of augmented reactivity have been detected in spleen, lymph nodes, and peritoneal exudates (Herberman et al., 1977a; Wolfe et al., 1976). In a recent study, we have found that inoculation with LCMV also produced very

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high levels of reactivity in the bone marrow. This has important implications regarding the possible relationship of N K activity to bone marrow transplantation resistance (Section VII1,B) and the possible origin of NK cells from bone marrow stem cells (Section VI). Because of the latter possibility, we have examined the time of appearance of augmented reactivity in the bone marrow relative to that in the spleen. The kinetics for both organs were the same, with only a small increase after 1 day, and high levels on days 2 and 3. Similarly, we have found that reactivity can be first detected in the bone marrow of young mice at the same time as in the spleen.

2. Rats NK cells have also been found to be widely distributed among the lymphoid organs of rats. Nunn et al. (1976) found the highest levels of reactivity in spleen and lymph nodes, but also detected significant reactivity with peripheral blood lymphocytes, thymus cells, and peritoneal cells. This reactivity of thymus cells was not observed with mouse or human thymus cells and also has not been confirmed by other studies of natural cytotoxicity in rats (Shellam and Hogg, 1977; Oldham et al., 1977). This discrepancy needs to be reexamined. I t is possible that the observed cytotoxicity with rat thymus was due to contamination by adjacent mediastinal lymph nodes. Shellam and Hogg (1977) also tested bone marrow cells and thoracic duct lymphocytes and found no significant reactivity, even after preincubation at 37°C.

3. Human In almost all studies of human natural cell-mediated cytotoxicity, only PBL were tested. In collaboration with Drs. W. H. West and J. L. Weese, we have recently examined the distribution of NK cells in various lymphoid organs. Reactivity was readily detected with spleen cells, but thymus, lymph nodes, and tonsil had little or no cytotoxic activity. B. CELL SURFACE MARKERS AND OTHER CHARACTERISTICS OF

NK CELLS 1. Mice

The mouse NK cell generally has been thought to b e a non-T cell, particularly because of the high levels of reactivity in nude mice. The most important experiments to examine this question directly have

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involved the pretreatment of lymphoid cells from conventional and/or nude mice with anti-8 serum plus complement (Herberman et al., 1973, 197513; Gomard et al., 1974; Kiessling et al., 197513; Sendo et al., 1975).In none of the experiments with conventional, thymus-bearing mice was reactivity specifically decreased b y such treatment. Under the same conditions, immune cytolytic T lymphocyte activity was completely eliminated (Herberman et al., 1973). With spleen cells from nude mice, treatment with anti-8 plus complement resulted in a partial reduction in activity (Herberman et al., 197513). This effect appeared to be dependent on complement and on the presence of anti-BC3H (or Thy 1.2) antibody, since absorption with Thy 1.2 positive brain removed the inhibitory effect. Inhibitory activity of such treatment has been dependent on the use of high concentrations of antiserum, optimal amounts of rabbit serum as complement source, and optimal length of incubation. Kiessling et al. (1975b) failed to affect nude reactivity by anti-8 treatment, but they used diluted guinea pig serum as the complement source, We have repeatedly confirmed the partial inhibitory effects of anti-8 serum plus complement on nude spleen cell reactivity, and have seen as much as a 90% reduction in activity. We have also observed a similar effect on the reactivity of spleen cells from both nude and conventional mice after boosting (Herberman et al., 1977a).The cytotoxicity of normal spleen cells after culturing in vitro for 5 days was also partially susceptible to anti-8 serum plus complement (Shustik et al., 1976). It thus appears that at least some of the NK cells in nude mice and in mice after boosting have low but detectable expression of 8 antigen. The only concern relative to this conclusion is that the effects may have been due to another antibody in the antisera. Our absorption studies with brain tissues (Herberman et al., 1973, 1975b) have ruled out the possible effect of autoantibodies which may be present in such antisera (Dennert and Lennox, 1972). However, these antisera may also contain antibodies to other lymphocyte antigens (Frelinger and Murphy, 1976) and to virus-associated antigens. We have therefore recently examined the effects of an anti-Thy 1.2 serum, prepared in congenic mice and kindly provided by Drs. Boyse and Shen. Even after absorption to remove antiviral reactivity, this antiserum produced a 50% decrease in the reactivity of BALB/c nude spleen cells. Treatment of NK cells with a variety of other antisera plus complement has not produced any decrease in reactivity. Greenberg and Playfair (1974) observed no effect by a rabbit anti-T cell serum. Similarly, we have noted no inhibition by two different heterologous antisera to mouse brain, which have considerable reactivity against mouse

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T cells. We have also obtained no evidence for the presence of T L antigen (Boyse and Old, 1969), Th-B antigen (Yutoku et al., 1976), kappa light chain surface immunoglobulin, or Ia antigen (Niederhuber et al., 1976) on the NK cells. Gorczynski (197613) has reported that a heterologous anti-mouse brain serum plus complement could inhibit the cytotoxicity of cultured normal spleen cells of male mice but had much less effect on cells from female donors. However, treatment of female spleen cells prior to culture led to a considerable increase in the level of cytotoxicity generated in vitro (Gorczynski,

1976~). Very recently, Glimcher et al. (1977) treated NK cells with anti-Ly antisera plus complement to determine whether the reactive cells bore these T cell antigens. The anti-Ly2 antiserum had no effect, but an anti-Ly 1.2 serum abolished NK activity. However, the antigen recognized on the NK cells by this antiserum was shown not to be Ly 1.2, but rather was a unique specificity, provisionally designated NK. It is of interest to note that one of the antisera with potent anti-NK activity was made b y immunization with thymocytes. This is consistent with the NK cell being in the T cell lineage, as was suggested above by the piesence of a low density of 6 antigen. Most studies have indicated that the NK cells are nonadherent and nonphagocytic (Herberman et al., 197513; Kiessling et al., 1975b; Sendo et al., 1975; Zarling et at., 1975). After passage of reactive spleen cells over an adherence column, relative activity has usually been increased. By calculating recovery of total lytic units, we have found almost complete recovery of NK activity from such columns (Herberman et al., 197713). In contrast, in the study of Gomard et al. (1974), activity was much decreased or eliminated by column passage or by treatment with carbonyl iron and magnet. Therefore, the reactivity observed by those investigators appeared to be due to macrophages and is much different &om the NK activity observed by others. Consistent with our observation of no inhibitory effect by pretreatment of NK cells with anti-kappa serum plus complement, Kiessling et al. (1975b) found that passage of reactive cells over an antiimmunoglobulin column resulted in an increase in relative activity. Depletion of cells bearing receptors for complement also had no inhibitory effect on NK activity (Herberman et al., 197513). The initial experiments performed by us (Herberman et al., 1975b) and by Kiessling et al. (1975b)to deplete Fc receptor-bearing lymphocytes were interpreted as having no effect on NK activity. However, after studies in this laboratory (West et al., 1977a) and in others (Peter et al., 1975a) indicated that human NK cells possess Fc receptors, we

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reinvestigated this issue (Herberman et al., 197%). By absorption of cytotoxic spleen cells from nude or conventional mice on monolayers of sheep erythrocytes plus IgG antibodies to sheep erythrocytes, 50-90% of the total cytotoxic reactivity could b e removed. Parallel adsorption of cells on monolayers of sheep erythrocytes alone or on erythrocyte-antibody monolayers coated with protein A, to block the Fc portion of IgG, resulted in little or no depletion of NK activity. The presence of Fc receptors on the NK cells was confirmed by forming rosettes with IgG antibody coated sheep erythrocytes and then showing that this caused the NK cells to sediment more rapidly in a velocity sedimentation separation procedure. NK cells appear to have trypsin-sensitive sites on their surface which are required for cytotoxic reactivity. Brief exposure of NK cells to low concentrations of trypsin had no inhibitory effect on reactivity (Herberman et al., 1975b). However, Kiessling et al. (1976a) originally observed and we have confirmed that more prolonged treatment (30 to 45 minutes) with higher concentrations of trypsin resulted in a substantial decrease in NK activity as measured in a 4 hour assay. In contrast, cells treated in an identical manner continued to have substantial levels of cytotoxicity in an 18hour assay (A. Santoni, H. T. Hojden, and R. B. Herberman, unpublished observations). Kiessling et al. (1975b) directly studied the morphologic appearance of a highly reactive subpopulation of cells after depletion of most of the phagocytic cells, T cells, and B cells. Almost all of the remaining, highly cytotoxic cells had the morphologic characteristics of small lymphocytes. We have essentially confirmed these observations. In addition, with the kind help of Dr. T. Aoki, highly reactive subpopulations of nude spleen cells and lymph nodes were examined by immunoelectron microscopy. Almost all of the cells were small or medium sized lymphocytes, and a small proportion had detectable patches of 6 antigen.

2. Rats NK cells in rats have been shown to be nonadherent, nonphagocytic cells (Nunn et al., 1976; Shellam, 1977; Oldham et al., 1977). The studies performed thus far have also indicated that the NK cell lacks cell surface markers of mature T cells. Treatment with heterologous anti-T cell antisera plus complement, which eliminated immune cytotoxicity against tumor cells, had no significant effect on the cytotoxicity by normal lymphoid cells (Nunn et al., 1976; Shellam, 1977; Oldham et al., 1977). However, these data do not definitely rule out the T cell nature of NK cells. In the studies with mouse NK cells,

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only anti-theta serum and not heterologous anti-T cell sera affected cytotoxic reactivity, and then only under certain circumstances. Nude spleen cells and cells obtained after boosting were sensitive to antitheta treatment, whereas lymphoid cells from young normal mice were resistant. The ability to inhibit, with anti-T cell serum plus complement, cytotoxic reactivity of normal spleen cells after stimulation in vitro with PHA (Glaser et al., 1976c) may be an analogous finding. This result is also similar to the sensitivity of cytotoxicity of cultured mouse lymphocytes to anti-8 plus complement (Shustik et al., 1976). Examination of rat NK cells for complement and Fc receptors has also yielded negative results (Nunn et al., 1976; Oldham et al., 1977). However, we have not yet had the opportunity to reexamine the possible expression of Fc receptors on these cells, using the same approach as that described for mouse NK cells. Shellam (1977) has described some other characteristics for rat NK cells: They had no detectable surface immunoglobulin, had the size of typical small lymphocytes in separations by velocity sedimentation at unit gravity, and were moderately sensitive to irradiation. Treatment of NK cells with papain resulted in loss of reactivity, which recovered when the cells were incubated for 4 to 5 hours at 37°C.

3. Human The characteristics of human NK cells have been extensively studied by many investigators. However, the results obtained regarding most of the cell surface markers have been conflicting. Almost the only point of agreement has been that most of the human NK activity is due to nonadherent, nonphagocytic cells (e.g., Peter et al., 1975b; Levin et al., 1975; Hersey et al., 1975; West et al., 1977a). Even in this respect, there has been one report of activity in a visual microcytotoxicity assay by granulocytes (Takasugi et d., 1975). Most investigators have concluded that human NK cells are non-T cells (DeVries et al., 1974; Jondal and Pross, 1975; Pross and Jondal, 1975; Peter et al., 1975b; Levin et al., 1975; Kiuchi and Takasugi, 1976; Bakacs et al., 1977). In all of these studies, cells forming rosettes with sheep erythrocytes (E-RFC) were separated from nonrosette forming cells, and considerable NK activity was seen with the non-E-RFC fractions. However, some of these investigators (DeVries et al., 1974; Levin et al., 1975) and others (Dean et al., 1975) have observed a significant amount of NK reactivity b y E-RFC. West et al. (1977a) found that most of the cytotoxic reactivity against K-562, more than 80% of the total lytic units (Kay et al., 1977), was associated with E-RFC. There are several likely explanations for these divergent re-

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sults. It has been shown that treatment of E-RFC with ammonium chloride solutions to remove SRBC contamination can result in loss of most NK activity for several hours (Kay et al., 1977). For studies using this procedure, this could have caused a falsely low estimation of reactivity in the E-rosetting population (e.g., DeVries et al., 1974; Jondal and Pross, 1975; Peter et al., 1975b; Kiuchi and Takasugi, 1976). Another, more fundamental explanation for the differing results is that NK cells appear to have low affinity receptors for SRBC (West et al., 1977a). If rosetting is not performed under optimal conditions of time, temperature, erythrocyte to PBL ratio, then many of the NK cells failed to rosette. However, by optimizing the conditions for rosetting or by using neuraminidase-treated SRBC, most NK cells were shown to reside in the E-RFC fractions (West et al., 1977a; Kay et al., 1977). The finding that human NK cells have a characteristic T cell marker but in lower density or lower affinity than the majority of T cells is quite analogous to the demonstration that mouse NK cells have a low density of e antigen. Hersey et al. (1975) found most of their cytotoxic reactivity in non-E-RFC, but they observed that many cells in that fraction reacted with an anti-T cell serum. On the basis of this, and other circumstantial evidence, they concluded that NK cells were activated T cells. Svedmyr et al. (1974, 1975) found that cultured PBL, which became cytotoxic in the presence of autologous lymphoid cell lines (see earlier discussion, Section II,E), lacked detectable receptors for SRBC. However, such activity could be generated from purified E-RFC (Svedmyr et a1 ., 1974) and it was shown that at the peak of stimulation in culture, the blast cells lost E receptors as measured by their assay (Svedmyr et al., 1975). The authors concluded that the cultured cytotoxic cells were of T cell origin. This would fit our earlier suggestion that this form of cytotoxicity is the same as NK activity and that NK cells are in the T cell lineage. The change in ability to rosette could represent a shift in differentiation from mature T cells to pre-T cells with concomitant acquisition of NK activity (see further discussion in Section VI) . There have also been many conflicting reports as to whether human NK cells have either Fc receptors or complement receptors. Some groups reported that the complement receptor was a characteristic marker for these cells (Pross and Jondal, 1975; DeVries et al., 1974). However, others have failed to detect this receptor on a substantial proportion of NK cells (Hersey et al., 1975; Kiuchi and Takasugi, 1976; West et al., 1977a). West et al. (1977a) have shown that rosette formation solely through the complement receptor requires the use of IgM

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antibodies to SRBC, without contamination by IgG antibodies which can mediate rosette formation through the Fc receptor. It seems likely that the studies reporting the presence of complement receptors used such mixed reagents. The presence of Fc receptors on human N K cells has been readily detected by many investigators (Peter et al., 1975b, 1976a, 1976b; Hersey et al., 1975; Kiuchi and Takasugi, 1976; West et al., 1977a; Kay et al., 1977; Bakacs et al., 1977) using several different separation techniques. By analysis of total lytic units, Kay et al. (1977) found that more than 80% of NK activity was associated with Fc receptor-bearing cells. Ortaldo et al. (1977a,b) have found that cytotoxicity of cultured PBL is also mediated by cells with Fc receptors. Of particular interest was the observation that cytotoxicity associated with Fc receptor-bearing cells was generated even when cells with Fc receptors were removed from PBL before culturing. These data indicated that some cells acquired Fc receptors in culture at about the same time they became cytotoxic. Human NK activity could be inhibited substantially by gamma globulin or by EA antigen-antibody complexes (Peter et al., 1975b; West et al., 1977a). However, soluble antigen-antibody complexes have not been inhibitory (H. D. Kay, unpublished observations). It seems likely that large aggregates or complexes could bind to the Fc receptors on NK cells and sterically interfere with their cytotoxic activity. Alternatively, if natural cytotoxicity is actually a form of ADCC (discussed below, Section V), then blocking of Fc receptors by some types of complexes may even more directly inhibit activity. Some other characteristics of NK cells have been examined. Treatment with cyclic AMP was markedly inhibitory (Rosenberg et al., 1974). Pretreatment of NK cells with trypsin or chymotrypsin has resulted in a loss of most or all NK activity (Kay et al., 1977). in Vitro C. EFFECTOF THYMOSIN

1. Mice

The presence of high levels of NK activity in nude mice and the detection of some 8 antigen on the NK cells suggested that the cytotoxic reactivity might be mediated by prethymic T cells (Pritchard and Micklem, 1973; Scheid et al., 1973; Loor and Roelants, 1974). Incubation of such cells with thymopoietin and other thymic humoral factors has been found to induce detectable quantities of 8 and TL antigens (Scheid et al., 1973; Goldstein et al., 1975). Incubation of nude spleen cells at 37°C with thymopoietin or with calf thymosin did

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not have a detectable effect on NK activity. It is somewhat difficult to relate our negative results with the effects described by others, since, in the one reported study on the effects of thymopoietin on functional activity of lymphocytes (Basch and Goldstein, 1975), only marginal effects on lymphocyte stimulation by mitogens were observed. In addition to the other thymic humoral factors, we have performed experiments with a human thymic hormone preparation of Dr. A. White. Incubation of nude spleen cells with this material at 37°C for 2 hours has produced a small decrease in cytotoxic activity relative to that seen with spleen cells incubated in medium without the factor. Incubation with Thymic Humoral Factor (THF, Umiel and Trainin, 1975) gave similar results.

2. Human Incubation of human PBL whith the human thymic hormone preparation has also resulted in a small but quite consistent decrease in cytotoxic activity against the K562 cell line (W. H. West and R. B. Herberman, unpublished observations). In contrast, incubation of cells under the same conditions with heated factor or with a control protein fraction had no inhibitory effect.

D. EFFECTSOF in Vivo MANIPULATIONSOF THYMUSFUNCTION 1. Mice The high levels of NK activity in athymic nude and lasat mice, and the inhibitory effects of in vitro incubation of nude spleen cells with human thymosin, indicated that the expression of natural cellmediated cytotoxicity might be inversely related to the degree of thymic function. To explore this further, we have performed a series of investigations in vivo to determine the effect of increased or decreased thymic function. Grafting of fetal BALBlc thymuses into young BALB/c nude mice resulted in a considerable restoration of mature T cell function, as evidenced by good lymphoproliferative responses to the mitogens, phytohemagglutinin (PHA), and concanavalin A (Con A). The grafted nude mice, at 6 weeks of age, had similar levels of NK activity as thymus-bearing nu/+ BALB/c littermates, and much lower activity than sham operated nude littermates. In contrast to the strong effects of thymus grafts, daily administration of calf or human thymosin for 3 to 4 weeks, according to the protocol of Ikehara et al. (1975), had no detectable effect on NK activity. These mice also had very little detectable restoration of lymphoproliferative responses to PHA and

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Con A. This and the weak or undetectable in vitro effects of thymic hormone preparations indicate that these materials lack the potent effects of intact thymus on functional activity. We have also performed thymectomies on newborn BALB/c mice to determine whether early removal of the thymus would mimic the athymic state of nude mice. In mice with partial thymectomy, some increase in NK.activity above that of sham operated was seen, and with total thymectomy, very high levels resulted. Nude mice are usually the offspring of heterozygous, nu/+, mothers and therefore may be affected by humoral thymic factors via the placenta before birth and through the milk thereafter. Hale et al. (1976) showed that homozygous nude females could produce offspring, and that such nude mice had a more profound deficit in T cell helper activity for antibody production. It was therefore of considerable interest to examine NK activity in such mice. Virtually all of the nude mice from homozygous nude mothers, when tested at 6 to 8 weeks of age, have displayed high levels of NK activity, very similar to that of nude mice from heterozygous mothers. These results indicate that NK activity can develop in the absence of any maternal thymic influence. Both types of nude mice may be at maximal levels of reactivity and therefore the further thymus-related immunologic deficit in the offspring of nude mothers may not result in even higher levels of reactivity. Consistent with this is the observation of Loor et al. (1975) that levels of &positive pre-T cells were similar in nude mice from homozygous and heterozygous nude mothers.

2. Rats Shellam (1977) examined the effects on NK activity of administration of an immunosuppressive antilymphocyte serum and found that it had no effect. H e then tested rats after neonatal thymectomy or after adult thymectomy, irradiation, and bone marrow reconstitution. Similar to the results in mice, these manipulations resulted in higher levels of NK activity.

3. Human Studies of the effects of altered in uivo thymic function, comparable to those in mice and rats, have not yet been reported. It will be of interest to study NK activity in patients with various types of T cell deficiencies, and particularly patients with failure of thymus development. In addition, patients receiving thymic hormone preparations can be studied to see if these cause some in vivo decrease in NK activity.

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E. EFFECT OF IMMUNOSUPPRESSION ON NK ACTIVITY In contrast to the extensive information on the effects of athymia and thymectomy on NK activity, there is little information available regarding the effects of other forms of immunosuppression. Such information could be quite useful in dissociating NK from other types of cytotoxicity and in evaluating the in vivo role of NK cells. Since there is such a paucity of data and since NK cells and K cells share many characteristics (see Section V), we will discuss together information on the effects on NK and K cells by immunosuppressive treatments. We recognize the obvious objection that even if the cells mediating both functions are similar the mechanisms of killing may be different and the treatments may act at different phases in the development of these functions. NK and ADCC activity in mice has been shown to be moderately resistant to the effects of irradiation in vitro.,Low doses of irradiation (350-700 R) did not appreciably decrease NK or ADCC activity while higher doses (>1000 R) produced a substantial reduction in cytotoxicity (Dr. A. Santoni, unpublished observations). Whole body irradiation with 350 R had no appreciable effect on NK activity when measured after 0 to 3 days, whereas 800 R caused a decrease of greater than 70% in NK and ADCC activity at 1 to 3 days (J. Y. Djeu and A. Santoni, unpublished observations). Shellam (1977) examined the effects of irradiation in rats. NK cells were somewhat more resistant to in vitro radiation than were immune T cells, with 50% of activity remaining after 1000 R and 30% after 5000 R. In contrast, whole body radiation had a greater effect, with a substantial loss of activity at 3 days after 500 R and complete elimination of reactivity with 900 R. Treatment of mice with cyclophosphamide resulted in a considerable decrease in NK reactivity, with a reduction of greater than 70% at 1 to 3 days after in vivo administration of 110 mg/kg (Dr. J. Y. Djeu, unpublished observations). Some limited information has been obtained on the effects of immunosuppression on human NK and K cell cytotoxicity. Rosenberg et al. (1974) found that 500 R x-irradiation, given 24 hours prior to assay, did not affect reactivity, whereas 1000 R or more caused a marked reduction in cytotoxicity. If the x-irradiation was performed just prior to assay, much of the activity persisted even after 15,000 R. Campbell et al. (1976) examined the effects of irradiation on various lymphocyte functions and found that antibody dependent cell-mediated cytotoxicity as well as some T cell functions and the number of B cells in the peripheral blood were substantially reduced after 3000-3500 R irradiation administered over a period of 1 month. Performance of the

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cytotoxicity assay in the presence of some immunosuppressive drugs, actinomycin D or methotrexate, reduced the activity of normal lymphocytes (Rosenberg et al., 1974). A few patients with collagen diseases who were receiving corticosteroid therapy were also found to have very low levels of NK activity (Rosenberg et al., 1974). This was confirmed in a more systematic study by Parrillo and Fauci (1977a), which showed that intravenous administration of hydrocortisone caused a profound inhibition of NK activity after 24 hours. It can be seen from the above summary that only fragmentary information has been gathered thus far on the effects of immunosuppressive agents on natural cytotoxicity or on ADCC. It will be important to perform more systematic studies and examine the kinetics of depression after various in vivo treatments. Furthermore, in order to assess the relative in vivo importance of NK activity, information must also be obtained at the same time on the effects of the treatment regimen on other immune functions, including immune T cell cytotoxicity, ADCC, and macrophage mediated effects. In previous studies, usually only one function was examined. Thus far, very little information of this type is available. Shellam (1977)found that the profound depression of NK activity in rats at 3 days after irradiation was followed at 10 days b y levels of activity substantially higher than that of untreated controls. Similarly, studies in our laboratory have shown that administration of cyclophosphamide (110 or 330 mg/kg) caused a marked suppression of NK activity in mice at 1to 3 days but that this was followed by a rebound to levels above those of the untreated controls (J. Y. Djeu, unpublished observations). In contrast to the full recovery of NK activity by 7 to 10 days after cyclophosphamide treatment, induction of immune T cell cytotoxicity by MSV was inhibited b y administration of the drug 7 days earlier (D. H. Lavrin, unpublished observations). However, NK activity was not examined in this study and such direct comparisons are needed. After therapeutic radiotherapy of patients, Campbell et al. (1976) examined the kinetics of the return to normal of various lymphocyte subpopulations, including K cells. K cell activity and B cell levels became normal by 3 to 6 months after treatment, whereas T cell activity (lymphoproliferative response to PHA) remained abnormal for over 1 year. We have also observed divergent temporal effects of cyclic combination chemotherapy on NK activity and lymphoproliferative responses in mixed leukocyte cultures (Herberman et al., 1972). Parrillo and Fauci (1977a) performed a kinetic study of the effects of corticosteroids on NK activity and on lymphocyte subpopulations. At 4 hours, NK activity was unchanged, E-RFC were decreased, and cells

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with Fc receptors and K cell activity (Parrillo and Fauci, 197713) was increased. These limited results suggest that the effects of immunosuppressive agents on NK activity can vary considerably with the type of treatment, dose and frequency of administration, and time of assay in relation to treatment. Because of these complexities and the differential effects on various immune functions, caution must be exercised in attributing the in vivo effects of a particular regimen of immunosuppression to inhibition of NK activity. V. Relationship of Natural Cell-Mediated Cytotoxicity to Antibody-Dependent Cell-Mediated Cytotoxicity

It has recently become apparent that the expression of NK activity in mice and in human donors is correlated closely with the expression of antibody-dependent cell-mediated cytotoxicity (ADCC). The effector cells mediating both forms of cytotoxicity also have very similar characteristics, and these observations raise the possibility that natural cell-mediated cytotoxicity is actually a form of ADCC. A. CORRELATION OF NK ACTIVITY WITH ADCC

1. Mice

Natural antibodies against mouse tumor cells and type-C virusassociated antigens have been detected by many investigators (Aoki et al., 1966; Mellors et al., 1969; Herberman and Aoki, 1972; Ihle et al., 1973; Sato et al., 1973; Nowinski and Kaehler, 1974; Aaronson and Stephenson, 1974; Martin and Martin, 1975) and this had led to consideration of the possible relationship of NK activity to ADCC. ADCC has been found to account for a major portion of the natural reactivity observed in microcytotoxicity assays with mouse mammary tumor virus-infected target cells (Blair and Lane, 1975a,b; Lane et al., 1975; Blair et al., 1976). Greenberg and Playfair (1974) and Kiessling et al. (1976a) failed to find any correlation between NK activity and ADCC against chicken erythrocyte target cells. However, the ADCC in their systems was primarily mediated by adherent phagocytic cells, and other types of effector cells, which are nonadherent and nonphagocytic, have been shown to play a role in ADCC of mouse tumor target cells (Blair and Lane, 1975b; Lamon et al., 1975). Therefore, it would seem more appropriate to compare NK activity with the latter type of ADCC against mouse tumor target cells. In collaboration with Dr. A.

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Santoni (unpublished observations), we have directly compared the expression of NK reactivity with the levels of ADCC against mouse tumor target cells coated with alloantiserum. The effect of age on reactivity in ADCC was very similar to that described (Section I1,A) for NK activity. In C57BL16, BALB/c and nude mice, little ADCC reactivity was detected prior to 4 weeks of age, and activity reached peak levels at 5 to 9 weeks of age and was usually low or undetectable thereafter. In addition to the overall correlation among groups of mice of different ages, there was a fairly good correlation in levels of NK activity and ADCC among individual mice. As observed by 0. Stutman (personal communication), C3HfBALB/c mice had detectable levels of ADCC activity at 1 to 2 weeks of age. In this strain, NK activity was also detectable in such young mice. As with NK reactivity, young nude mice with random-bred, BALB/c and C57BL/6 genetic backgrounds had high levels of ADCC activity. Among different strains of conventional, thymus-bearing mice, a good correlation was seen in tests for NK activity and ADCC activity against the same target cell (RBL-5). C3H/HeN (both high and low mammary tumor incidence sublines) and NZB had high levels of reactivity; C57BL/6 and BALB/c had intermediate levels; and A, AKR, and SJL had low levels. As a further correlation, inoculation of older mice with LCMV or C. parvum, which had been found to boost NK activity, also led to a rapid increase in levels of ADCC activity. Therefore, in contrast to the previous reports of a lack of correlation between NK activity and ADCC (Greenberg and Playfair, 1974; Kiessling et a1 ., 1976a),we have found the two types of cell-mediated cytotoxicity, against mouse tumor target cells, correlated very well for a variety of different characteristics. 2. Rats There have been very few efforts thus far to relate NK reactivity to ADCC. However, in view of the correlation between augmentation of human CCC and ADCC (see Section V,A,3), it is of interest to note that ) that incubation of rat spleen cells at 37°C Glaser et al. ( 1 9 7 6 ~found with PHA or LPS led to increased ADCC activity as well as to increased cytotoxicity against tumor cells (as discussed in Section II,E,2). Another observation of possible relevance, in view of the persistence of NK activity in thymectomized rats (Shellam, 1977), is that ADCC activity was also detectable in such animals (Harding et al., 1971).

3. Human Several investigators have recently examined the correlation of levels of NK reactivity with levels of ADCC reactivity against tumor

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target cells. In each of these studies, of normal donors and/or cancer patients, good correlations between NK and ADCC activities have been seen (Peter et al., 1975a,b; Santoli et al., 1976; Trinchieri et al., 1977; W. H. West and R. B. Herberman, unpublished observations). In addition, the organ distribution of effector cells for both forms of cytotoxicity were found to be the same, with considerable reactivity with PBL and spleen cells and little reactivity with thymus, tonsil, or lymph node cells (West et al., 1977b, and unpublished observations). When human PBL have been cultured at 37" in medium containing FBS (see Section 11,E73),additional ADCC reactivity was generated along with CCC (Ortaldo et al., 1977a,b). Stimulation of PBL in vitro with mitogens, soluble antigens, or allogeneic cells has also produced augmented ADCC reactivity (Connolly et al., 1975; MacDonald and Bonnard, 1975; Ortaldo et al., 1977a,b) in addition to CCC.

B. COMPARISON OF EFFECTORCELLS MEDIATING NK ACTIVITY AND ADCC 1. Mice

Previous studies of the relationship between the NK cells and eEector cells mediating ADCC in mice have indicated several major differences (Greenberg and Playfair, 1974; Kiessling et al., 197513, 1976a). ADCC activity was attributed to a monocytic cell (Greenberg and Playfair, 1974; Kiessling et al., 1975b), an adherent, phagocytic cell with a receptor for complement (Kiessling et al., 1975b, 1976a). However, as pointed out previously (Section V,A,l), ADCC was tested against chicken erythrocyte target cells, and some studies have indicated that other types of effector cells may also be responsible &r ADCC against mouse tumor target cells (Blair and Lane, 1975b; Lamon et al., 1975). Therefore, in collaboration with Dr. A. Santoni (unpublished observations), we have compared the characteristics of the NK cell with the effector cells (which we will call K cells) mediating ADCC against alloantibody coated mouse tumor cells in an overnight 51Crrelease cytotoxicity assay. In this system, like the NK cells, the K cells were shown to be nonphagocytic and nonadherent. Adsorption of spleen cells on EA monolayers to remove Fc receptor-bearing cells resulted in a moderate decrease in relative K cell activity, but as has been described for NK cell activity, the depletion was only partial. These experiments have indicated a relative difficulty, in comparison to human cells (West et al., 1977a; Kay et al., 1977), in removing mouse Fc receptor-bearing cells by such procedures. Pretreatment of spleen cells with anti4 serum plus complement has resulted in a substantial loss in ADCC activity as well as a partial decrease in NK

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activity. However, such treatment with anti4 without complement had similar effects on K cell activity, and it remains unclear as to whether the inhibition is due to selective action on K cells bearing 6 antigen or to nonspecific inhibition by antigen-antibody complexes. The sensitivity of N K cells and K cells to irradiation in vitro was quite similar, with little effect of 500R and then a progressive decline in activity with increasing dose. Both types of activity were found to be sensitive to in vivo cyclophosphamide treatment. Taken together with the close correlation in levels of N K and K cell activity in different mice, these data point to a very close similarity between N K and K cells. However, there have been some consistent differences between the characteristics of N K and K cells. In contrast to the lability of N K activity at 37"C, ADCC activity was unaffected by incubation for up to 3 hours at 37°C. Kiessling et al. (1976a) and we have obsetved than N K activity in a short-term assay is trypsin sensitive, in contrast to the lack of inhibitory effect of this enzyme on ADCC activity. Also, addition of aggregated gammaglobulin (Herberman et al., 1975b; Kiessling et al., 1976a) or an anti-immunoglobulin reagent (Herberman et al., 1975b), which can effectively inhibit ADCC, had no inhibitory effect on N K activity. It should be noted, however, that the inhibitory effects on ADCC of gammaglobulin and anti-immunoglobulin have been seen in experiments with antibody coated target cells, in which these agents would be expected either to block the free Fc receptors of the effector cells or to mask the Fc portion of the immunoglobulins on the target cells. These experiments tend to rule out ADCC against antibody coated target cells as a mechanism for N K activity but do not bear on the possibility of ADCC by K cells already armed in vivo (see Section V,C).

2. Human A series of recent studies have indicated that human N K cells and K cells have very similar cell surface markers and other characteristics. Lymphocytes appear to be responsible for most of the observed reactivities, but normal granulocytes have shown to be capable of mediating both direct cytotoxicity (Takasugi et al., 1975) and ADCC (Zighelboim et al., 1974). Peter et al. (1975b, 1976a) performed a series of separation procedures on human PBL and showed that N K and K cells were present in the same fractions. They found that both types of effector cells were nonadherent and nonphagocytic and had Fc receptors for IgG. These investigators, as well as many others (Wisloff et al., 1974; Zighelboim et al., 1974; Brier et al., 1975; MacDermott et al., 1975; Cordier et al., 1976), reported that the K cell was a non-T cell,

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which did not form rosettes with SRBC. However, this did not represent a discrepancy from the characteristics of NK cells, since Peter et al. (1975b) also concluded that NK cells lacked receptors for SRBC. Perlmann et al. (1975) initially reported that K cells could form rosettes with SRBC if the latter were pretreated with neuraminidase. By using the procedures previously described (Section IV, B), West et al. (1977b) have demonstrated that K cells, as well as NK cells, have low affinityreceptors for SRBC. In a direct comparison of the characteristics of NK and K cells, Kay et al. (1977) found that up the 80% of the total lytic units of both NK and ADCC activities were associated with E-RFC. As described by Peter et al. (1975b),both NK and K cells had Fc receptors. In studies of lymphocytes in various lymphoid organs, there has been an excellent correlation between the presence of low affinity E-RFC with Fc receptors (West et al., 1977c) and the expression of NK and ADCC (West et al., 1977b) activities. All of these were present in PBL and spleen, and virtually undetectable in thymus, tonsil, and lymph nodes. In addition, both types of cytotoxic reactivity were inhibited by pretreatment of the PBL with an ammonium chloride solution (Kay et al., 1977). As with the studies of mouse lymphocytes, some clear differences between NK reactivity and ADCC have been observed. NK reactivity is markedly inhibited after treatment of PBL with trypsin or chymotrypsin, whereas such treatments have no effect on ADCC (Kay et al., 1977). Further, when protein A was added to the cytotoxicity assays, ADCC, but not NK, was significantly inhibited.

c. POSSIBLE

MECHANISMS OF ACTION

OF

NK CELLS

All of the data gathered to date indicate that NK cells are directly cytotoxic and that their action is dependent on contact with the target cells. Although we and others have assumed that NK activity was a form of cell-mediated cytotoxicity in which the information for reactivity and specificity was an integral part of the lymphocyte, the possible role of ADCC must now be considered. The close correlation between levels of NK activity and K cell activity may be more than coincidental. Some of the natural cell-mediated cytotoxicity observed in a microctyotoxicity assay against mouse mammary tumors has been shown to be mediated by ADCC, with coating of target cells by antibody during the incubation period (Blair and Lane, 1975a,b; Blair et al., 1976). As discussed (Section V,B), it seems unlikely that coating of target cells by antibodies is involved in NK cell activity. However, since NK cells appear to possess Fc receptors, it is possible that these cells are armed

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in vivo with natural antibodies or with antigen-antibody complexes (Perlmann et al., 1972; Greenberg and Shen, 1973; Sakselaet al., 1975). The reduction in NK activity by trypsinization or by incubation at 37.T would be consistent with this since such treatments might remove the immunoglobulins needed for reactivity. Based on this possibility, we have performed a series of experiments in which untreated and trypsinized lymphocytes were incubated with autologous serum and with culture fluids from explanted lymphocytes. Thus far the cells incubated with serum or culture fluids have not been consistently found to have increased cytotoxic reactivity against the tumor target cells. However, there has been very little experience with armed lymphocytes. It is possible that some unidentified variables are critical to the success of such experiments. Pollack and Nelson (1974) described arming of lymphocytes for reactivity in a microcytotoxicity assay using serum from mice 1 to 2 days after inoculation of tumor cells or oncogenic virus. The arming factor in their system was shown not to be assocaited with either IgG or IgM (Pollack and Nelson, 1975),and it could be produced in lethally irradiated mice (Pollack and Nelson, 1976). Similarly, Peter et aZ. (1976b) have obtained evidence for an augmenting soluble factor, which may be produced by B cells, but which could not be removed b y anti-IgG. It will be necessary to rule out the role of K cells with such unusual arming or augmenting factors as well as with more conventional antibodies before it can be concluded that NK activity is a mechanism entirely distinct from ADCC. The diversity of natural antibodies against mouse tumor cells, type-C viruses, and other antigens would provide a ready explanation for the heterogenous specificities detected by NK cells. Another possible mechanism has been suggested for natural cytotoxicity by effector cells with Fc receptors (Rager-Zisman et al., 1976). In their study, Herpes simplex virus-infected target cells were shown to express Fc receptors and nonspecific killing appeared to occur b y cross-linking of effector and target cells by aggregated immunoglobulins through their F c receptors. Although this mechanism needs to be considered in each new system, it does not appear to explain most of the observations of NK activity discussed above. In both our mouse and human systems, we have found that some target cells without detectable Fc receptors to be quite susceptible to cytotoxicity. Furthermore, performance of the cytotoxicity assays in medium without serum or with agamma FBS had no effect on results. Rager-Zisman et al. (1976) also found that the effector cells for their cross-linking phenomenon were adherent cells with the characteristics of macrophages, which are different from the characteristics of NK cells described earlier (Section IV,B).

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35 1

VI. Model for Placement of N K and K Cells in Pathway of Differentiation of T

Cells The considerable evidence presented herein for an inverse correlation between levels of NK activity and thymic function might be explained by two alternative mechanisms: (a) NK cells are prethymic T cells and further differentiation under the influence of the thymus results in thymic and post-thymic T cells in which NK activity is no longer expressed or expressed only at low levels; and (b) NK cells are non-T cells, whose activity is modulated b y T suppressor cells. Most of the data presented thus far are consistent with either mechanism. However, the results in the mouse of treatment with anti-8 serum plus complement tend to rule out the suppressor cell model. With that mechanism, one would predict that removal of T cells from a lymphoid cell preparation of conventional mice by such treatment would lead to higher levels of activity and that such treatment of nude cells would either have no effect or would cause some increase. However, the data indicate that anti-8 treatment of lymphoid cells of normal mice had no effect and such treatment of nude cells resulted in partial depression of NK activity. We have further tested the suppressor cell hypothesis by mixing BALB/c nude spleen cells with spleen cells from syngeneic conventional mice. N o reduction of the high level of NK activity of the nude cells was detected. Similarly, the association of human NK cells with E-RFC does not support the suppressor model and is consistent with the T cell nature of NK cells. There is considerable evidence for the existence of prethymic T cells, with high levels of such cells in nude mice and in neonatally thymectomized mice. Raff (1973) originally described a low percentage (1-2%) of 8 antigen positive cells in nude mice, detectable by immunofluorescence. Somewhat higher values ( 5 4 % )were detected b y cytotoxicity assays (Raff and Wortis, 1970) and nude lymph node cells were shown to be capable of absorbing out anti-8 antibodies (Raff, 1971). Loor and Roelants (1974) subsequently reported that u p to 20% of nude mouse spleen cells contained a low density of e antigen, detectable by a rabbit antiserum to brain associated e antigen. The expression of low density 8 antigen on lymphocytes seemed to be independent of humoral thymic factors, since cells from nude mice born from homozygous nude mothers had comparable characteristics (Loor et al., 1975). This group also reported that T L antigen could be detected by immunofluorescence on as many as 16% of nude mouse spleen cells (Roelants et al., 1976). However, T L antigens have not been detected by cytotoxicity on lymph node cells of healthy nude mice (Scheid et at., 1975). Further support for the presence of prethymic T cells in nude mice has come from studies in which in v i t r o

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incubation of cells with thymic factors or other agents (Scheid et al., 1973; Goldstein et al., 1975) or in uiuo inoculation of some thymic factors (Scheid et al., 1975) led to a considerable increase in the proportion of lymphocytes expressing the T cell associated markers, 8 and TL. Environmental infection of nude mice with hepatitis virus produced similar effects (Scheid et al., 1975). In most of these studies, only antigenic markers of T cells were analyzed and little direct evidence for induction by humoral factors of functionally active T cells has been presented (Ikehara et al., 1975; Basch and Goldstein, 1975). Nonetheless, Pritchard and Micklem (1973) suggested that nude mice have precursors of functionally active T cells, and since several studies have either indicated the presence of some functionally active T cells in nude mice (Kirov, 1974; Hale et al., 1976) or the ability to induce functional reactivity in lymphocytes of nude mice (Ramseier, 1975). Based on the above information and the results obtained in our studies of the nature of the NK cells, William West and I have developed a model for placement of N K cells in the pathway of differentiation of T cells (Fig. 1). This model is similar to that proposed by Loor et al. (1976) for changes in cell surface antigens during early T cell differentiation. Prethymic T cells contain a low density of 8 antigen. Within this population are included cells with Fc receptors and cells with NK activity. The cells with NK activity and those with Fc receptor seem to b e overlapping if not identical subpopulations.

Mphrr

?mbymic T Wh

I I

I

I I

?

I

I

?

I I

I

I

I

L------+

I I L

Thymoun

I

- -- - - - - - - - - - - - -- - - - - -- E mtfmilp?

FIG. 1. Model for placement of NK and K cells in the pathway of differentiation of T cells.

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Human NK cells also fit this formulation, having easily detectable Fc receptors and low affinity receptors for SRBC, a marker as characteristic for human T cells as 8 antigen is for mouse T cells (West et al., 1977a). West et al. (1977~)have recently obtained considerable evidence that there is a separable subpopulation of human T cells, characterized by the presence of Fc receptors for IgG and low affinity receptors for SRBC. This popularion bears considerable similarity to the mouse pre-T cell subpopulation, and these may be analogous. It also seems likely that human K cells mediating ADCC are also included in this same population of lymphocytes (West et al., 1977b). In the studies with human effector cells, NK and K cells had virtually identical cell surface characteristics (Kay et aZ., 1977). In the mouse, the placement of K cells in this scheme is somewhat less certain. As discussed previously (Section V,B), we are not yet certain about the presence of 8 antigen on K cells. However, the correlation between NK cell activity and K cell activity in relation to age, strain, and nude mice (as discussed in Section V,A) has been striking, and both NK and K cells in mice have Fc receptors. In analogy with the human, it seems likely that mouse K cells will reside in the same subpopulations as NK cells. Prethymic T cells appear to originate in the bone marrow (Scheid et al., 1973; Loor et al., 1976). Similarly, Haller, Kiessling, and their associates have obtained evidence that the bone marrow contains precursors of NK cells (R. Kiessling, personal communication). Bone marrow cells from high responder mice were able to transfer NK reactivity to low responder recipients. We have failed to detect NK activity earlier, with age or after boosting, in bone marrow than in the spleen, but this may simply indicate that transfer of NK cells between the bone marrow and peripheral lymphoid tissues occurs rapidly. According to our model, when pre-T cells come under thymic influence, by passing through the thymus in the normal state, some differentiation occurs, with loss of Fc receptors and NK and K reactivity, and expression in the mouse of increased amounts of 8 antigen and increase in, or appearance of, TL antigen, and in the human expression of higher affinity receptors for SRBC. In the absence of the thymus in nude mice or neonatally thymectomized mice, high levels of pre-T cells with NK and K reactivity persist. Exposure of such cells to humoral thymic factors or development of some diseases in nude mice (Scheid et al., 1975)could also lead to some differentiation of the cells, with loss of their associated NK functional activities. After T cells leave the thymus, they further differentiate into mature T cells which in the mouse, continue to express moderate amounts of 8 antigen, lose T L antigen, and in the human, express receptors with

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moderate affinity for SRBC. These cells remain inactive in NK and K cell reactivity and usually continue to lack Fc receptors. Human effector cells which are generated in mixed lymphocyte cultures and have specific cytotoxic reactivity against allogeneic target cells appear to reside in this subpopulation (Ortaldo and Bonnard, 1977), and in general, cytotoxic specifically immune mature T cells may be generated from this population. This leaves us to account for the expression of NK and K cell activities in peripheral lymphoid organs. In the absence of the thymus in nude mice, this would simply involve a movement from the bone marrow into other organs. This process, bypassing the thymus, may also occur to some extent in conventional mice. Alternatively, or in addition, some cells leaving the thymus and some mature T cells in the periphery may be activated or de-differentiate back into cells with pre-T cell characteristics. Ortaldo et al. (1977a,b) have obtained a considerable amount of evidence to support this possibility, When human PBL were depleted of Fc receptor-bearing cells and then placed in culture, within 4 to 5 days cells with detectable Fc receptors appeared and these had high levels of direct cytotoxic activity and ADCC. Treatment of peripheral lymphocytes with human thymosin has been shown to decrease NK and K cell activities and to change some human PBL from low affinity E-RFC to high affinity E-RFC (W. H. West, A. White, and R. B. Herberinan, unpublished observations). Taken together, these observations support the possibility that lymphocytes can reversibly move from one subpopulation to the other. VII. Discrimination Between Natural Cell-Mediated Cytotoxicity and

Cytotoxicity by Other Effector Cells

The contribution of NK activity to the cytotoxicity measured in any in vitro assay needs to be carefully considered. NK cells may be present in immune lymphoid populations, and in fact, the immunizing procedure may have augmented NK activity as well as produced more specific reactivity. Exactly what percentage of the total reactivity is represented by this effector function is dependent on variables such as choice of target, species, age (in rodents), the organ used as the source of effector cells, previous exposure to modulating agents (e.g., viruses, bacteria, chemotherapeutic drugs, irradiation, tumor cells), and the nature of the in vitro assay. Therefore, in order to determine accurately the role of other cytotoxic effector mechanisms (e.g., specifically cytotoxic T cells, and macrophages), it is necessary to define, eliminate, or control for natural cytotoxicity in all in vitro cytotoxicity as-

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says. At the moment there are no readily available methods for specifically eliminating NK activity. However, b y utilizing the information we have at the present concerning the nature of the NK cell, it is possible to approach the problem in a systematic manner, keeping in mind that as we enrich, deplete, or control for NK activity we might be influencing other effector cell activities as well.

A. MICE Until recently, many investigators used lymphoid cells from normal mice as baseline controls for studies of cytotoxicity produced b y immunization. However, the observations of NK activity and particularly the considerable variability in levels of NK activity among individual mice have raised some serious concerns regarding the proper baseline to be used in cytotoxicity assays. Some aspects of this have been previously discussed (Herberman et al., 1976a). In order to assess the levels of NK activity, several possible baseline controls have been used, and the baseline of choice depends on the type of assay. The most commonly used control is the medium control, but as previously discussed (Herberman et al., 1976a), this is often an artificial control which has little relationship to the results obtained in any groups with lymphoid cells present. For short-term assays, e.g., 4-hour W r release assay, an autologous control has been quite satisfactory. This consists of the use of unlabeled target cells in place of, and at the same concentration as, the lymphoid effector cells. This has provided a reproducible baseline (Herberman et al., 1976a) and has been especially useful for studying low levels of NK reactivity. Another baseline control that has been useful in long-term assays (incubation periods of 12 to 18 hours or more) as well as in short-term assays is a thymus cell control in which normal thymus cells or thymus cells from immune animals are used in place of effector cells (Herberman et al., 1976a; Oldham et d., 1977; H . T. Holden and A. Santoni, unpublished observations). This has been suitable because, as discussed herein (Section IV,A), thymus cells from either normal or immune animals lack NK activity and they also lack detectable immune cytotoxic T cell activity. However, when thymus cells are used as controls, it is essential to avoid contamination by adjacent lymph nodes. When one is interested in studying non-NK immune cytotoxic activity, a baseline control of normal lymphoid cells may be quite useful and, in some cases, may be the best available baseline (e.g., Tinget al., 1977). At first glance, this seems very logical since it should reflect the

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amount of immune cytotoxicity above the natural levels. However, there are a few problems which need to be considered before this baseline is adopted. The most important is the susceptibility to NK activity of the particular target cell and the amount of variation among mice being studied in their levels of NK activity. If the target cell is reIativeIy resistant to NK activity and older mice are studied, with low levels of NK activity, a pool of normal lymphoid cells from several mice would seem reasonable. However, if much NK activity was seen against the target cell, the normal baseline controls would b e high and it would be important to know how much of that was due to N K activity versus poor condition of the target cells. In addition to these considerations, it should be realized that the immunization procedure itself could cause a change in the level of NK activity, and it would be very helpful to have a means to assess the contribution of this to the apparent level of immune cytotoxicity achieved. Any of the above methods could provide a stable baseline control for a given test. Nevertheless, it is still difficult to make a comparison between data obtained in tests performed on separate days. However, by using the autologous control in conjunction with cryopreserved target cells and a cryopreserved lymphocyte internal standard, assay variation can be monitored and controlled (Holden et al., 1976, 1977). These techniques keep assay to assay fluctuations to a minimum so that results obtained on different days can be evaluated and compared. An entirely different approach to the study of non-NK cytotoxic effector cells would be to deplete the NK cells selectively. Unfortunately, selective removal of NK cells is difficult in the mouse. There are several methods that can be employed to decrease the NK activity in mouse spleen cells but in most cases they give variable results and only remove part of that cytotoxicity. As discussed earlier (Section IV,B), mouse NK cells have an Fc receptor; however, adsorption ofcells on EA monolayers does not completely deplete NK cells even though a large percentage of the activity is lost after this treatment (Herberman et al., 197713).This may be explained by the absence of Fc receptors on some NK cells or by the inability of this technique to remove all cells with low affinity Fc receptors. A further problem with this approach is that at least some mouse immune cytotoxic T cells have Fc receptors and therefore this would not be a reliable discriminant (Stout et al., 1976). Two different antisera have been employed to decrease NK cytotoxicity, anti-8 (Herberman et al., 1975b) and anti-NK (Glimcher et al., 1977). However, treatment with anti-8 plus complement presents several problems. Although this will partially deplete NK activ-

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ity in some cases, it also will eliminate mature T cells and hence cannot be used to study residual immune T cell cytotoxicity. Furthermore, such treatment cannot be used to deplete NK activity reliably because it only causes up to 90% decrease in cytotoxic reactivity in nude mice (Herberman et al., 1975b) and in boosted animals (Herberman et d., 1977a) but it does not affect natural cytotoxic reactivity in uninoculated, conventional mice. However, since anti-8 plus complement can substantially reduce the levels of boosted NK activity, caution should be exercised in attributing cytotoxic reactivity to mature T cells as opposed to NK cells if the activity can only be removed after vigorous treatment with anti-8 plus complement or if the activity is only partially inhibitied by such treatment. Another problem which is associated with this antiserum is that treatment of spleen cells with anti-8 alone, without the addition of complement, often increases the cytotoxicity against some tumor targets, especially in long-term cytotoxicity assays (A. Santoni, unpublished observations). The mechanism of this effect is unknown and is currently under investigation. Anti-NK antiserum appears to be much more specific in its action, with selective and fairly complete elimination of NK effector cells, and therefore could potentially be the most useful method of depleting NK function from effector cell populations. The main limitation at present is that this antiserum only affects NK activity of some mouse strains. NK activity in the mouse can also be altered by incubation at 37°C (Herberman et al., 197513) or b y extended trypsin treatment (Kiessling et al., 1976a).Our laboratory has shown that NK activity is sensitive to incubations at 37°C for 2 hours or longer (Herberman et al., 1975b), and, in fact, overnight incubation at 37°C reduces the level of activity to almost baseline levels. Assuming that other effector functions are not so unstable, this technique can be very useful for depleting NK activity. However, its value might be limited in long-term assays since culture of normal cells for several days can actually lead to the generation of cytotoxicity (Shustik et al., 1976; Gorczynski, 1976a). Treatment with 1.0% trypsin for 30 to 45 minutes can decrease the level of NK activity to low levels if the cytotoxicity assay is short (4-6 hours). However, the technique is not effective in reducing NK activity in long-term (18 hour) cytotoxicity assays since the levels of activity are siniilar with or without treatment (A. Santoni, unpublished observations) and this appears to represent a regeneration of the cytotoxicity during the prolonged incubation period. NK activity may also be distinguished from specific immune activity by the specificity of the reactions. As previously stated (Section 111) most of the studies on NK activity in the mouse have demonstrated

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some degree of specificity. After defining the specificity that is being recognized by the immune cytotoxic cells, it should be possible to identify target cells which do not carry the relevant antigens (and hence are not lysed by immune cytotoxic effector cells) but are still sensitive to cytotoxicity by normal lymphoid cells from that strain of mouse. However, in many cases the cells which carry the relevant antigens for immune cytotoxicity also are sensitive to NK activity; nevertheless, the antigens which are recognized by the different effector populations do not appear to be the same, at least in the system that we have examined (Herberman et al., 1975a). Therefore by examination of the specificity by the inhibition of 51Crrelease cytotoxicity assay (Herberman et al., 1976b), one might be able to distinguish NK from immune cytotoxicity. A further approach along this line, which has already been found to work with human PBL, is to remove NK cells b y adsorption to monolayers of cells bearing NK-associated antigens and then test for residual activity against specific target cells. NK activity in uninocuIated mice has a consistent relationship to age, with the peak of natural killer activity at about 6 to 8 weeks of age with low or undetectable levels after 12 weeks of age. Therefore, the role of NK activity can be minimized in vitro by employing older mice for study. However, this is complicated by the discovery that many different agents are capable of boosting NK activity in mice. Immunization with a particular antigen or a particular tumor cell does not mean that the cytotoxicity measured in vitro is specifically against that tumor cell, especially when tests are performed within a few days after inoculation. Several investigators have reported the detection of cell-mediated cytotoxicity 2 to 3 days after inoculation of syngeneic or allogeneic tumor cells (Lamon et al., 1972; Forman and Britton, 1973; Pollack and Nelson, 1974). The observed reactions were thought to b e specific but the nature of the specificity at these earIy times was not extensively evaluated. Forman and Britton (1973)found that the effector cells harvested within a few days after inoculation were resistant to treatment with anti-8 plus complement, whereas later on they were quite sensitive. It is quite possible that at least part of the reactions observed at 2 to 3 days represented a boost in natural reactivity rather than early primary immunization to the specific antigen used for immunization. Pfizenmaier et al. (1975) found that early after immunization with LCM virus, autoreactivity separate from specific anti-LCM reactivity was seen. Again, as discussed above (Section II,D), it seems quite possible that their observations were related to the boosting of natural reactivity. Finally, many investigators have shown cytotoxic reactivity in peritoneal exudate cells after the animals have been in-

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jected intraperitoneally with BCG or C. paruum. We (Herberman et al., 1977a) with BCG and C. pamum and Wolfe et al. (1976) with BCG have shown that NK cytotoxicity is greatly enhanced after these treatments. Hence, at least some of the cytotoxic reactivity detected after these injections could be related to the NK activity which has been stimulated. We have recently observed that macrophages from C. parvum injected animals, purified by adherence on petri dishes, are contaminated by considerable numbers of NK cells (P. Puccetti and H. T. Holden unpublished observations). Hence, considerable caution must be exercised in attributing observed cytotoxicity to either immune, mature T cells or to macrophages, and in many cases, NK cells may play a significant role. B. HUMAN Discriminating specific immune cytotoxic activity from NK activity is considerably more difficult in studies of human lymphoid cells than it is in mice. The history of exposure to possible immunogens is less certain, and there are considerably more variable, and less predictable, levels of NK activity in human subjects. This has made it very difficult to study cell-mediated cytotoxicity of cancer patients against their own tumor cells or against allogeneic tumor cells of the same histologic type (Herberman and Oldham, 1975). Since the problems of natural cytotoxicity have been recognized, an assortment of possible baseline controls, similar to those discussed for studies in mice, have been considered. In order to measure the variation in reactivity among normal donors, it has been suggested that several normal individuals be tested in each experiment and that the least active normal (Oldham et al., 1975) or the median reactive normal (Herberman et al., 1976a) be used as the baseline. Although these approaches provide a reasonably good reflection of normal reactivity on a population basis, they are not adequate for following the reactivity of a given individual over time, since the baseline is not completely stable. The use of a standard cryopreserved population of normal PBL should be helpful for this purpose (Oldham et al., 1976b; Holden et al., 1977).However, it is even more important to develop methods for clearly discriminating between NK activity and other forms of immune cytotoxic reactivity. One approach, developed by Cannon et a l . (1977), has been to test simultaneously the cytotoxic reactivity of PBL against a good indicator cell line for NKactivity and against a target cell with antigens relevant to the system under study. By using a cell line derived from breast cancer

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and the K562 cell line, it was possible to identify some patients with breast cancer who had relative hyperreactivity against the breast cancerderived line. These data suggested that these patients had another type of effector cell activity in addition to NK activity. Another approach, based on differences in specificity of the various types of effector cells, has demonstrated the presence of cytotoxic activity mediated by cells with characteristics of NK cells in mixed lymphocyte cultures (Ortaldo and Bonnard, 1977).PHA blasts, which are good targets for specifically immune T cells and are resistant to NK activity, reflected one type of activity, whereas K562 target cells mainly reflected NK activity. More definitive discrimination would be expected to result from selective removal of NK cells from a mixture of lymphoid cells. By removal of cells forming rosettes with EAC (SRBC; anti-SRBC; and complement complexes), it was possible to identify effector cells specific for Epstein-Barr virus-associated antigens in the blood in patients with infectious mononucleosis (Svedmyr and Jondal, 1975)and in a biopsy of Burkitt’s lymphoma (Jondal et al., 1975). As discussed previously (Section IV,B,3), this depletion was probably through the Fc receptors on human NK cells. By adsorption of NK cells on monolayers of SRBC coated with IgG antibody (West et al., 1977a) or of immobilized soluble antigen-antibody complexes (Kay et al., 1977), it has also been possible to deplete NK activity almost completely. Since immune cytotoxic T cells do not appear to have detectable Fc receptors (Ortaldo and Bonnard, 1977), this approach would appear to b e a very useful one for making the needed discrimination. It has already been shown to identify effector cells in the peripheral blood of some breast cancer patients, which lack Fc receptors and are cytotoxic against breast cancer-derived target cells (W. West, G. 13. Bonnard, and R. B. Herberman, unpublished observations). The other marker on human NK cells which provides the basis for separation is the receptor for SRBC. As discussed above (Section IV,B,3), more than 80% of the NK activity can be removed by rosetting with SRBC (Kay et al., 1977).This b y itself would not be expected to separate NK cells from immune, cytotoxic T cells. However, it is possible at least partially to separate mature T cells with high affinity receptors for SRBC from NK cells in the low affinity E-RFC population b y rosette formation at 29°C (West et al., 1977a,b,c). Adsorption of NK cells on monolayers of target cells which have the relevant antigens offers yet another method for selective depletion of natural cell-mediated cytotoxicity. Studies in progress in our laboratory (W. West and R. B. Herberman, unpublished observations) indicate that this procedure can deplete most or all of the activity against

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K562 cells and not affect the reactivity of some individuals against a breast cancer-derived cell line. In summary then, it seems likely that reliable and effective methods for separating NK activity from other forms of human cytotoxic effector cells are available. In the near future, it should be possible to assess the distribution and specificity of these other types of cytotoxic cells which react with tumor cell lines and/or other target cells. VIII. In Vivo Relevance of Natural Cytotoxicity

Understanding of the in vivo relevance of natural cell-mediated cytotoxicity is the ultimate objective in this area of research. With the multiplicity of other possibly relevant effector mechanisms, it is very difficult to define the role of NK cells in resistance to tumor growth and other in vivo phenomena. It is particularly difficult to answer these questions in human studies since the opportunities for in vivo manipulations are obviously quite limited. Most of the information that has been, and will probably continue to be, gathered in this important area has come from studies in mice, as discussed below. Several investigators have noted that nude mice develop spontaneous tu-mors or carcinogen-induced tumors with relatively low incidence (Rygaard and Povlsen, 1976; Stutman, 1974; Outzen et al., 1975). In addition, transplanted tumors of mice and of heterologous species have not produced progressive tumor growth in nude mice with the consistency that one might have anticipated from the ready growth of skin allografts and xenografts. Bonmassar et al. (1975) have noted the impaired growth of an allogeneic lymphoma in nude mice and in lethally irradiated mice. Rotter and Trainin (1975) found that the Lewis lung carcinoma, 3LL, grew poorly in lethally irradiated, bone marrow reconstituted mice. More than one million 3LL tumor cells and B16 melanoma cells failed to produce tumors in some nude mice (Giovanella et al., 1974). Similarly, Gillette and Fox (1975) showed that several tumors grew less well in thymectomized, lethally irradiated, bone marrow reconstituted mice than in normal mice. Intravenously inoculated syngeneic or allogeneic tumor cells were found to produce fewer tumor colonies in the lungs of nude mice than in normal, nu/+, littermates (Skov et al., 1976; Fidler et al., 1976). Shin et a l . (1975) failed to produce tumors in nude mice by several clonal isolates of virus transformed 3T3 cells. Stutman ( 1975) observed that 120-day-old nude mice had some resistance to tumor induction by polyoma virus, and spleen cells from these mice were able to transfer partial resistance to this virus. Nude mice have become widely used for growth of human tumor

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cells. In the course of these studies, some resistance to tumor growth has been noted. Although metastatic spread of some transplanted tumors has been observed (Giovanella et al., 1973, 1974) many investigators have noted the rarity of metastasis in nude mice, even when metastatic deposits of human tumors were transplanted (Rygaard and Povlsen, 1969; Castro, 1972; Ozzello et al., 1974; Schmidt and Good, 1975, 1976; Maguire et al., 1976). Some human tumors only produced tumors when inoculated intracranially (Epstein et al., 1976) and others, especially gastric carcinomas, produced no tumors in nude mice (Schmidt and Good, 1975).Maguire et al. (1976) have even noted occasional regression of a highly malignant hamster tumor in nude mice. These many observations of some resistance to growth of syngeneic, allogeneic, and xenogeneic tumors in nude mice might be attributed to their high levels of natural cell-mediated cytotoxicity. The findings that some human cell lines are sensitive to NK activity and that inoculation of these cells can boost reactivity are consistent with this possibility. As more direct evidence, a series of experiments have already been performed which show a correlation of resistance to tumor growth with sensitivity of a tumor to cytotoxicity by NK cells.

A.

CORRELATION OF CYTOTOXICITY

DECREASED TUMORGROWTH WITH NATURAL

Kiessling and his associates (Kiessling et al., 1975c; Petranyi et al., 1976) have performed an extensive series of experiments which demonstrate a correlation between the levels of NK activity in different strains of mice and the resistance of F1 hybrids between each strain and A mice to the A strain lymphoma, YAC, the cultured line of which is very sensitive to natural cytotoxicity. Mice which were thymectomized, irradiated, and fetal liver reconstituted also showed this resistance (Kiessling et al., 1976b).The main problem in relating the in vitro and in vivo observations in the above studies is that the ascitic form of YAC was used for the in vivo studies, and Kiessling et al. (1975a) found this to be rather resistant to NK activity. Another type of correlation has been observed when some tumor cells have been transplanted into mice of different ages. Sendo et al. (1975)observed that young (B6 x BALB/c) F1mice, at the time of peak levels of NK activity, were more resistant to growth of R L d l than were older mice. Similarly, we have found that 6- to 8-week-old nude mice were more resistant to growth of a low dose of MCDV-12 lymphoma cells than were 12- to 14-week-old nude mice. The R L 8 1 tumor also grew poorly in 8-week-old nude mice, even after 350 R irradiation of the recipients, but it produced a significantly higher

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tumor incidence in nude mice pretreated with cyclophosphamide, which has been found to depress NK activity. Because of our interest in NK activity and its possible relevance to in viuo resistance to tumor growth, we have been quite interested in examining the characteristics of tumor cell lines which grew poorly in nude or thymectomized mice, to determine whether the resistance to such tumors could be explained by sensitivity to NK activity. All of the tumors in this category, the Lewis lung carcinoma (Rotter and Trainin, 1975), the B16 melanoma (Skov et al., 1976; Fidler et al., 1976), E4, an SV40 transformed 3T3 line (Gillette and FOX,1975), and the L5MF-22 lymphoma (Bonmassar et d.,1975) have been shown to be sensitive to natural cell-mediated cytotoxicity by nude spleen cells (see Table IV in Section 11).In collaboration with C. Chang, we have performed more extensive studies on some clones of the spontaneously transformed T-AL/N cell line. It was initially noted that these cells grew less well in allogeneic nude mice than in syngeneic AWN mice. With the T-S-5 clone, the TDS0was 200 to 400 times higher in nude mice than in conventional mice. I n addition, some of the tumors produced in nude mice were found to regress. When these cells were tested as target cells, they were observed to be highly sensitive to cytotoxicity by spleen cells from nude mice (Table IV) but resistant to, lysis by spleen cells from AL/N mice. As a further correlation with the ability of antigenic tumors to boost the levels of NK activity in nude mice, the inoculation of T-S-5 into nude mice increased the resistance to subsequent challenge with the same tumor. Based on these observations, we have formulated the hypothesis that the failure to observe a very high incidence of spontaneous carcinogen-induced tumors in nude mice might be due to their high levels of NK activity. Only tumors with low or no sensitivity to natural cytotoxicity would then be likely to be detected in nude mice. Consistent with this hypothesis, all of the tumors which arose in nude mice that we have studied thus far have been resistant to NK activity and, by inhibition studies, have lacked detectable antigens (see Section III,A,3). A further prediction from our hypothesis would b e that chronic suppression of NK activity in nude mice would result in a higher spontaneous tumor incidence, and we are setting u p experiments to examine this possibility.

B. In Vivo RELEVANCEOF NK ACTIVITYAGAINST CELLS NONMALIGNANT

The findings that NK activity is not completely restricted to tumor cells provide a possible explanation for some in vivo phenomena, par-

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ticularly that of resistance to bone marrow transplantation. When Bonmassar et al. (1975) observed the impaired growth of an Hhincompatible tumor in nude mice, they suggested that the same mechanism might be responsible for resistance to tumor cells and to bone marrow. Gallagher et al. (1976) have also suggested that the two phenomena might be related. In a recent workshop (Trentin and Bennett, 1977), the information on correlations between NK activity and bone marrow resistance was discussed. Despite some apparent differences in characteristics, a number of similarities was noted. Both activities develop at about 3 weeks of age, are relatively radioresistant and sensitive to cyclophosphamide, and are present in nude mice. In collaboration with J. J. Trentin and G. Cudcowicz, R. Kiessling (personal communication) performed some comparative experiments. The bone-seeking isotope, which depresses bone marrow resistance (Bennett, 1973), also was found to suppress NK activity. One discrepant finding has been the slight effects of silica on NK activity, in contrast to the depressive effects of this agent on bone marrow resistance (Lotzovi et al., 1975). Our finding that NK cells have some cytotoxic activity against bone marrow cells (Section II1,A) provides more direct evidence for the possible relationship between these phenomena. The ability to boost NK activity with allogeneic bone marrow cells (Section II,D) and the presence of high levels of NK reactivity in the bone marrow after boosting (Section IV,A) further support this possibility. However, in contrast to the results described in the previous paragraph, we have found an appreciable amount of NK reactivity persisting in mice after treatment with 89Sr,and inoculation of LCMV into such mice caused some, albeit lower than normal, augmentation of NK activity. The reactivity of NK cells against normal thymocytes could also explain the difficulties in transplanting thymus grafts from donors older than 3 weeks into nude mice (Radov et al., 1975). Also the higher levels of autoreactivity in neonatally thymectomized mice and the inhibition of this reactivity by T H F (Small and Trainin, 1975) raise the possibility that natural cell-mediated cytotoxicity is also involved in this phenomenon. It is also possible that NK activity is involved in resistance against infections by viruses and other microorganisms. Several studies have indicated that nude mice are relatively resistant to infection by some agents, e.g., myxoviruses (Haller and Lindenman, 1974), Listeria rnonocytogenes (Emmerling et al., 1975), Brucella abortus (Cheers and Waller, 1975), and Candida albicans (Cutler, 1976; Rogers et al., 1976). The observation that many microbial agents can induce a rapid

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augmentation of NK activity suggests that the microorganisms themselves or infected host cells may be targets for this reactivity.

C. IMPLICATIONS OF NATURALCYTOTOXICITY FOR IMMUNE SURVEILLANCE

The original formulations of the theory of immune surveillance (Burnet, 1957; Thomas, 1959) focused on the central role of the immune response as a natural defense against neoplasia. Only more recently has the theory been modified to stress the relationship of thymus-dependent immunity to immune surveillance (Burnet, 1970). It has been this modification of the theory which has aroused a series of criticisms of the concept of immune surveillance (Moller and Moller, 1975; Outzen et al., 1975; Schwartz, 1975; Rygaard and Povlsen, 1976), and which even led to a counter theory of immune stimulation (Prehn and Lappe, 1971). Much attention has been directed toward two apparent contradictions to the theory of immune surveillance, the relatively low incidence of tumors in nude mice and the failure of some tumors to develop in thymectomized mice. Although these data do challenge the modified concept of immune surveillance, in which thymus-dependent immunological reactions are required for effective antitumor resistance, they do not really bear on the basic theory itself. The discovery that nude mice and neonatally thymectomized mice and rats have high levels of NK activity, a potentially very effective alternative mechanism for immune surveillance, provides a good explanation for most of the available in vim data. The available information on the incidence of tumors in immunodeficient or immunosuppressed humans has also engendered controversy regarding the role of immune surveillance. With some forms of depressed immunity, the incidence of some types of tumors, especially those of the reticuloendothelial system, have been clearly increased. However, in other diseases associated with immune depression, e.g., leprosy, an increased incidence of cancer has not been noted (see review b y Melief and Schwartz, 1975). As discussed earlier (Section IV,E), this variable association of immune depression with elevated tumor incidence might be related to different effects of disease or immunosuppressive regimen on NK cell activity and other possible defense mechanisms. It will b e very important to evaluate the levels of these effector functions in the various conditions carefully to determine whether any correlate with the incidence of tumors in these patients. The other principal challenge to the concept of immune surveil-

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lance has been that, in contrast to the antigenicity of virus-induced tumors, spontaneous tumors frequently lack detectable antigenicity and therefore might not be susceptible to control by the immune system (reviewed very recently by Klein and Klein, 1977). Much has been made of the findings that tumors arising in uitro are not more antigenic than those arising in uiuo where the immune system might have been expected to select for tumors with weak or absent tumor associated antigens (Prehn, 1971; Heidelberger, 1973). However, almost all of the negative evidence has been obtained by procedures designed to detect transplantation resistance and other immune responses which have generally been associated with immune T cell activity. If, as w e suggest, there is a role for NK activity in immune surveillance, then the question of antigenicity and resistance to tumor growth needs to be asked by protocols designed to detect this function as well as that of immune T cell-mediated cytotoxicity. For example, to detect increased resistance to challenge by tumor cells which might be induced by augmentation of NK activity, the time of challenge would probably have to be much sooner after immunization than the 1 to 2 week interval usually employed, and attempts at hyperimmunization by repeated inoculation of tumor cells might be counterproductive. In addition, the antigens associated with NK activity may be quite distinct from those detected by immune T cells. We have found that the antigens recognized on RBL-5 tumor cells by the T cell immune response induced by MSV (Herberman et al., 197413) are different from those recognized by NK cells ( H e r b e m a n et ul., 1975a). If this is true for a wide variety of tumors, then the entire question of antigenicity, or lack thereof, with regard to the role of immune surveillance will need to be reexamined. Addendurn

In the last year, since this review was originally prepared, there have been several recent developments on the nature of NK cells and on factors influencing the levels of natural cell-mediated cytotoxicity, which add substantially to the understanding of this phenomenon. Therefore, the following additions should be related to the appropriate sections of the review.

To SECTION I I , D , l Ojo et al. (1978) have confirmed that Corynebucterium parvum can boost when given intraperitoneally, but they and Savary and Lotzovi

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(1978) found that intravenous inoculation led to depressed NK activity. Welsh and Zinkernagel (1977) have confirmed that LCMV induces rapid augmentation of NK activity. MacFarlan et a l . (1977) showed that Semliki Forest virus could also rapidly boost NK activity. In all of these studies, the characteristics of the effector cells after boosting were the same as those seen with NK cells. After Oehler et a l , ( 1978b) found that poly I : C would strongly boost NK reactivity in rats, we performed extensive studies in mice to determine the role of interferon in augmenting NK activity. Poly I : C and a variety of other interferon-inducing agents were shown to be able to induce considerable increases in cytotoxic reactivity, and the peak responses occurred at around the time of previously described rises in interferon levels (Djeu et al., 1978). Administration of crude or purified virus-induced interferon also induced an increase in NK activity and this could be detected within 2 hours. Gidlund et a l . (1978) have also shown that interferon inducers and interferon could induce augmentation of NK activity and that simultaneous administration of anti-interferon could efficiently block the effects of the interferon inducers and could partially block the effects of C. parvum. It therefore appears likely that interferon plays a central role in the boosting of NK activity and it will be of interest to determine what role it has in inducing or maintaining the spontaneous levels of natural cytotoxicity. The mechanism of the effects of interferon on NK cells remains to be determined, but these may be related in some way to the ability of interferon to also augment cytotoxic reactivity of immune T cells (Lindahl et al., 1972) and of macrophages (Schultz et al., 1977).

To SECTION II,D,2 Oehler et al. (197813) recently studied the effects of a variety of agents on the cytotoxic reactivity of normal rats. The results were quite similar to those in mice. C. paruum, LCMV, and Kilham rat virus all strongly boosted reactivity, with a peak in most lymphoid organs at around 3 days. The specificity of the augmented cytotoxicity, and the cell surface characteristics of the effector cells were indistinguishable from the specificity and characteristics of rat NK cells. Inoculation of BN rats, which have low levels of natural cytotoxicity, with C. parvum induced cytotoxic reactivity in the peritoneal cavity as high as that obtained with WIFu rats. In contrast, the boosted activity in BN spleens was substantially below that observed in WIFu spleens after C. paruum. A new finding which was made in the rats was that poly I : C was able

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to strongly boost NK reactivity, and the peak effects occurred within 1 day. Other polynucleotides, including poly A : U which has strong adjuvant properties, had no such effect on NK. Since poly I : C, and not the other polynucleotides tested, is a potent inducer of interferon, this led to the hypothesis that boosting of NK is mediated by interferon. As discussed above, this hypothesis has been correct for boosting of NK activity in mice.

To SECTION II,E,l With the recent findings that in uiuo administration of poly I : C or interferon could rapidly boost NK reactivity, we have also examined their effects on cultured NK cells (Djeu et al., 1978). Exposure of normal spleen cells to poly I : C for as little as 2 hours resulted in a substantial increase in cytotoxic reactivity. Crude and purified interferon preparations had similar effects and these could be seen after preincubation for only 45 minutes. These in uitro boosting effects were completely inhibited by the addition of small amounts of a specific anti-mouse interferon. Thus, as described above in the section on in uiuo augmentation of NK activity, interferon may also play a central role in the augmentation, and perhaps even the maintenance, of NK activity in uitro. We have recently observed that macrophages may influence the levels of NK activity in uitro. Overnight cultures of normal spleen cells in the presence of adherent splenic or peritoneal macrophages resulted in higher cytotoxic reactivity. At least some of this effect may be attributable to the production of interferon by these macrophages. The in uitro boosting of NK activity by poly I : C was found to be dependent on the presence of macrophages, whereas interferon was able to boost cultures of spleen cells depleted of adherent cells.

To SECTION IV,B,2 Initial studies of rat NK cells for complement and Fc receptors yielded negative results (Nunn et al., 1976; Oldham et al., 1977). However, using the same more sensitive approach as that described above for mouse NK cells, rat NK cells were also shown to have detectable Fc receptors (Oehler et al., 1978a). T o SECTION IV,B,3 Recent studies by Ortaldo and Robbins (1978) have shown that the in uitro generation of cytotoxic cells from Fc negative precursors in-

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volves the cooperation of radioresistant E-rosetting cells with radiosensitive null cells which lack detectable surface immunoglobulins. It will be of interest to determine what role, if any, interferon plays in the generation of this reactivity. T o SECTION IV,E Some interesting differences in the effects of immunosuppressive treatments have been noted when their effects on boosting of rat NK activity b y poly I : C were studied (Oehler and Herberman, 1978). Hydrocortisone, x-irradiation, and cyclophosphamide, at doses which had suppressive effects on NK, had little or no effect on the ability of rats to be boosted by poly I : C . To explain these findings, it was postulated that the precursors of NK cells were resistant to these treatments whereas the NK cells themselves were sensitive. T o SECTION V,B,2 Studies on the possible role of immunoglobulins in NK have continued. When protein A or F(ab’), anti-human F(ab’), was added to the cytotdxicity says, ADCC but not NK was significantly inhibited (Kay, 1978). However, Troye et a l . (1977) found that Fab anti-human Ig could inhibit the cytotoxicity of normal lymphocytes against bladder carcinoma-derived cell lines. In recent experiments, we have also examined the effects of a F(ab’), anti-mouse F(ab’), reagent (generously provided by Dr. T. Chusid), which would be expected to interact with cytophilic antibody. This had no inhibitory effect on mouse NK activity. T o SECTION V,C Recently, Koide and Takasugi (1977) have suggested that human NK is mediated b y arming of K by natural antibodies. The reduction in NK activity by trypsinization or by incubation at 37°C would be consistent with this, since such treatments might remove the immunoglobulins needed for reactivity. Based on this possibility, Kay (1978) has performed a series of experiments in which untreated and trypsinized lymphocytes were incubated with autologous or allogeneic serum and with culture fluids from explanted lymphocytes. Thus far he has been unable to confirm the results of Koide and Takasugi. The cells incubated with serum or culture fluids have not been consistently found to have increased cytotoxic reactivity against the tumor target cells. It is possible that some unidentified variables are critical to the success of

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such experiments. However, the failure to inhibit NK with anti F(ab’), reagents tends to rule out the central role of arming antibodies.

To SECTIONVIII,A Several investigators have recently noted a good correlation between sensitivity of tumor cells to cytotoxicity by normal lymphocytes and inhibition of growth in uivo. Warner et aE. (1977) showed that tumors sensitive to NK grew poorly in nude mice. Harmon et a l . (1977) found that rat methylcholanthrene-induced sarcomas that were sensitive to cytotoxicity by normal spleen cells were also inhibited in their in vivo growth when admixed with spleen cells.

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SUBJECT INDEX A ABO blood group antigens, in trophoblastic disease, 140-141 Adenomatosis, inherited colonic cancer and, 289-293 Adriamycin, mass spectometric determination of, 245-249 Aflatoxins, mass spectrometric detection of, 231 Air, mass spectrometry of pollutants in, 216-217 polycyclic aromatic hydrocarbons, 219 Animal tumors for use in cancer studies, 149-200 analysis of species of, 153-157 chemically induced, 163-167 origin of, 157-192 spontaneous tumors, 159-162 tumor transplants, 176-182 virus-induced, 167-176 Antiviral drugs, mass spectrometry of, 253 Armitage and Doll theory of oncogenesis, 74-75 B B-type oncornaviruses, structural proteins of, 39-43 Benzo[u]pyrene, metabolism of, mass spectrometric studies of, 222-223 Bile acids and sterols, mass spectrometry of, 258 Biological markers, mass spectrometry of, 253-259

C Cancer research, mass spectrometry in, 201-267 Carbohydrates, mass spectrometry of, 259 Carbon black, polycyclic aromatic hydrocarbons in, 221-222 Carcinogens, mass spectrometric studies of, 215-233 Catecholamines, mass spectrometry of, 257-258

Cell-mediated immunity, 305-377 age effects on, 310-312 augmentation of, 319-321 characteristics of, 307-324 decreased tumor growth in, 362-363 effector cells in, 333-345 effects of in vitro cultivation of lymphoid cells, 321-324 environmental factors in, 316-319 genetic factors in, 312-316 in immune surveillance, 365-366 immunosuppression effects on, 343-345 i n uiuo relevance of, 361-366 natural, 354-361 specificity of, 324-333 N K cells and K cells in, 351-361 relationship to antibody-dependent cell-mediated cytotoxicity, 345-350 his-Chloromethyl ether, mass spectrometric studies of, 225-227 Choriocarcinoma (gestational) with hydatidiform mole, 125-138 immunotherapy trials of, 143-145 native host reactions toward, 142-143 origin of, 89-125 from seemingly normal pregnancy, 97-125 Colon cancer cutaneous cells in, 299-300 environmental factors in, 282-287 fecal contents in, 300 human susceptibility to, 281-303 immunologic studies on, 296 inherited diseases and, 287-293 nuclear protein and enzyme alterations in, 296-299 proliferative abnormalities and, 293296 Computers, use in mass spectrometry, 212 C-type RNA tumor viruses enu gene-coded proteins of, 9-14 gug gene-coded proteins of, 14-24 genome structure and complexity of, 5-6 genetic mapping, 27-30 proteins of, 6-27

379

380

SUBJECT INDEX

SRC gene-coded transforming proteins of, 24-27 structural proteins of, 8-24 coded by leukemia and sarcoma genomes, 35-39 translational products of, 1-53 properties, 35 Cutaneous cells, in colonic cancer, 299300 Cyclophosphamide mass spectrometric determination of, 238-241 metabolism of, 233-238 Cytotoxicity, natural, characteristics of, 307-324

Gardner’s syndrome, colonic cancer incidence and, 292 Genetics of cell-mediated immunity, 312-316 of colonic cancer, 287-293 Gestational trophoblastic disease, 89-147 ABO blood group antigens in, 140-141 choriocarcinoma, 89- 125 HLA antigens and, 138-139 immunology of, 138-145 invasive mole, 125-138 Guinea pigs, cell-mediated immunity in, 323

D

Hexamethylamine, mass spectrometric determination of, 250 HLA antigens, trophoblastic disease and, 138- 143 Hoimones, antitumor type, mass spectrometry of, 249-250 Human chorionic gonadotropin (hCG), in trophoblastic choriocarcinomn detection, 92 Humans cell-mediated immunity in, 31 1-312, 316, 317-319, 321, 32Z324, 348 effector cells, 334, 338-342 specificity, 331-333 Hydatidiform mole, invasive mole and choriocarcinoma associated with, 125-138 follow-up studies, 132-138

Daunorubicin, mass spectrometric determination of, 245-249 Diethylstilbestrol, mass spectrometric detecton of, 232 Dinitrobenzyl aziridines, mass spectrometry of, 252 D-type oncornaviruses, structural proteins of, 39-43 E Ellipticine, mass spectrometry of, 252253 Enu gene-coded proteins, of C-type RNA tumor viruses, 9-14 Environment egects on cell-mediated immunity, 316319 role in colonic cancer, 282-287 Enzymes, in colonic cancer, 296-299

H

I

Feces, abnormal chemicals in, in colonic cancer, 300 Fisher theory of oncogenesis, 75-76 application to terminated exposures, 76-77

Immune surveillance, cell-metliated immunity in, 365-366 Iminunobiology, of trophoblastic disease, 138-145 Initiation-promotion phenomenon, of oncogenesis, 78 Inlet systems, for mass spectrometry, 207 Invasive mole, incidence of, 125-138 Ion sources, for mass spectrometry, 208-209

G

J

F

Gag gene-coded proteins, of C-type RNA

tumor viruses, 14-24

Juvenile polyposis of colon, colonic cancer incidence and, 293

38 1

SUBJECT INDEX K

of vinyl chloride, 227-228 of water pollutants, 215-216

K cells, in cell-mediated immunity, 351-354 L

Leukemia, marrow transplantation in therapy of, 269-279 Leukemia virus, structural proteins coded by, 35-39 Lung tumors, urethane-induced, multistage theory of oncogenesis in, 68-71 Lymphocytes, mass spectrometry of, 258-259 Lymphoid cells, in vitro cultivation of, effects on cell-mediated immunity, 321-324

M Marrow transplantation in leukemia therapy, 269-279 during remission, 277-278 graft vs. leukemia, 276-277 patient selection and clinical results, 270-271 recurrence data, 273-274 relapse prevention, 275-276 survival data, 271-273 Mass analyzers, for mass spectrometry, 209-2 11 Mass spectrometry advantages and limitations of, 202204 analytical techniques for, 212-215 of antineoplastic agents, 233-253 of antitumor hormones, 249-250 of biological markers, 253-259 of his-chloromethyl ether, 225-227 in cancer research, 201-267 components of systems for, 206-212 computer use in, 212 of nitrosamines, 228-231 of polychlorinated biphenyls, 224-225 of polycyclic aromatic hydrocarbons, 217-223 stable isotope dilution for, 214 theory of, 204-205 of trace elements, 232-233

Methotrexate, mass spectrometric determination of, 251-252 Mice cell-mediated immunity in, 310-311, 312-315, 316-317, 319-321, 322, 340-341, 345-346,347-348 effector cells, 333-337 specificity, 3 2 4 3 2 9 Multicell theory, of oncogenesis, 62-65 Multistage theory of oncogenesis, 65-68 with proliferative advantage of intermediate cells, 73-74 of radiation-induced tumors, 68-71 single stage of, 78-79 of urethane-induced tumors, 71-73 N

Nitrogen mustards, mass spectrometry of, 252 Nitrosamines, mass spectrometric studies of, 228-231 Nitrosoureas, mass spectrometric determination of, 241-243 NK cells, in cell-mediated immunity, 351-354 Nuclear proteins, role in colonic cancer, 296-299

0 Oldfield syndrome, colonic cancer incidence and, 292 Oncogenesis Armitage and Doll two-stage theory of, 74-76 clone growth of transformed cells and, 78-79 Fisher theory of, 75-76 implications for dose-response relationships, 83-86 initiation-promotion phenomenon of, 78 experiments involving, 79-83 multicell theory of, 62-65 multistage theory of, 65-68 single stage of, 78-83 quantitative theories of, 55-88 single stage theory of, 57-62

382

SUBJECT INDEX P

Peutz-Jeghers syndrome, colonic cancer incidence and, 293 Phorbol myristate acetate, mass spectrometric detection of, 231 Platinum coordination complexes, mass spectrometric determination of,

250-251 Polyamines, mass spectrometry of, 254-

255 Polychlorinated biphenyls, mass spectrometric analysis of, 224-225 Polycyclic aromatic hydrocarbons (PAH), mass spectrometric analysis of, 217-

223 Purines, mass spectrometric determination of, 243-245 Putrescine, mass spectrometry of, 254-

255 Pyrimidines, mass spectrometric determination of, 243-245 R

Radiation tumors induced by multistage theory of, 68-71 theories, 86 Rats cell-mediated immunity in, 310, 315,

317, 322-323, 342, 346 effector cells, 334, 337-338 specificity, 329-331 RNA-dependent DNA-polymerase, properties of, 7-8 RNA tumor viruses, C-type, translational products of, 1-53

Spermidine, mass spectrometry of, 254 Spermine, mass spectrometry of, 254-255 SRC gene-coded transforming proteins, of C-type RNA tumor viruses, 24-27 Steroids, mass spectrometry of, 256-257 Stilbenes, mass spectrometric determination of, 232 Structural proteins of B- and D-type oncornaviruses, 39-43 of C-type RNA tumor viruses, 8-24 of RNA-dependent DNA-polymerase, 8 T Therapy of cancer, animal tumors for study of, 149-200 Thymidine, incorporation of, mass spectrometry, 259 Thymosin, effects on cell-mediated immunity, 340-341 Tobacco smoke, polycyclic aromatic hydrocarbons in, 219-221 Trace elements, mass spectrometric determination of, 232-233 Translational products, of C-type RNA tumor viruses, 1-53 Tumors (See also Oncogenesis) in animals for therapy studies, 149-200 expected rates of appearances of, 56-57

v Vinyl chloride, mass spectrometric studies of 227-228 Viruses, tumors induced by, cancer studies using, 167-176 Volatiles, mass spectrometry of, 258

S W Sarcoma virus, structural proteins coded by, 35-39 Single stage theory, of oncogenesis, 57-62

Water, mass spectrometric analysis of pollutants in, 215216

CONTENTS OF PREVIOUS VOLUMES

Volume 1 Electronic Configuration and Carcinogenesis C. A . Coulson Epidermal Carcinogenesis E . V. C o w d y 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 . Winzler AUTHOR INDEX-SUBJECT INDEX

Volume 2 The Reactions of Carcinogens with Macromolecules Peter Alexander Chemical Constitution and Carcinogenic Activity G. M . Badger 383

Carcinogenesis and Tumor Pathogenesis 1. 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 I). Fenninger and C. Burroughs Mider Some Aspects of the Clinical Use of Nitrogen Mustards Calvin T. K l o p p and Jeanne C . Bateman Genetic Studies in Experimental Cancer L. W. Law The Role o f Viruses in the Production of Cancer C . Oberling and M . Cuerin 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 Arom'atic Molecules: New Developments A . Pullman and B. Pullman Some Aspects o f Carcinogenesis P. Rondoni Pulmonary Tumors in Experimental Animals Michael B. Shimkin

384

CONTENTS O F 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 o f 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 Goldin Some Recent Work on Tumor Immunity P. A. Gorer Inductive Tissue Interaction in Development Clifford Grobstein Lipids in Cancer Frances L. Haven and W. R. Bloor The Relation between Carcinogenic Activity and the Physical and Chemical Properties of Angular Benzacridines A. Lacassagne,N. P. BuuHoi, R. Daudd, and F. Zajdela The Hormonal Genesis of Mammary Cancer 0. Muhlbock 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 Nakahara 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 BFluorenamine 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 Armin C . Braun and Henry N . Wood Cancer Chemotherapy by Perfusion Oscar Creech, Jr. and Edward T. Krementz Viral Etiology o f Mouse Leukemia Ludwick Gross Radiation Chimeras P. C. Koller, A. J . S . Daoies, and Sheila M . A. Doak Etiology and Pathogenesis of Mouse Leukemia 1.F. A. P. Miller Antagonists of Purine and Pyrimidine Metabolites and of 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 1. W. Beard

CONTENTS OF PREVIOUS VOLUMES Mechanisms of Resistance to Anticancer Agents R. W. Brockman Cross Resistance and Collateral Sensitivity Studies in Cancer and 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 o f Neoplastic Cells Harris Busch and William J. Steele Nucleolar Chromosomes: Structures, Interactions, and Perspectives M . J . Kopac and Gladys M . Mateyko 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 Stambuugh and Sidney Weinhou se

385

The Relation of the Immune Reaction to Cancer Louis V. Caso Amino Acid Transport in Tumor Cells R. M . Johnstone and P. G. Scholefield 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 G r i . n The Enzymatic Pattern of Neoplastic Tissue W. Eugene Knor Carcinogenic Nitroso Compounds P. N . Magee and J . M . Barnes The Sulfhydryl Group and Carcinogenesis 1.S . Harrington The Treatment o f Plasma Cell Myeloma Daniel E. Bergsagel, K . M . Grifith, A. Haut, and W. J . Stuckley, Jr. AUTHOR INDEX-SUBJECT INDEX

Volume 11 The Carcinogenic Action and Metabolism of Urethan and N-Hydroxyurethan Sidney S . Miruish Runting Syndromes, Autoimmunity, and Neoplasia D. Keast Viral-Induced Enzymes and the Problem of Viral Oncogenesis Saul Kit

386

CONTENTS OF PREVIOUS VOLUMES

The

Growth-Regulating Activity of Polyanions: A Theoretical Discussion of Their Place in the Intercellular Environment and Their Role in Cell Physiology William Regelson Molecular Geometry and Carcinogenic Activity of Aromatic Compounds. New Perspectives Joseph C . Arcos and Mary F. Argus

AUTHOR INDEX-SUBJECT INDEX CUMULATIVE INDEX

Volume 12 Antigens Induced by the Mouse Leukemia Viruses G. Pasternak Immunological Aspects of Carcinogenesis by Deoxyribonucleic Acid Tumor Viruses C. 1. Deichman Replication of Oncogenic Viruses in Virus-Induced Tumor Cells-Their Persistence and Interaction with Other Viruses H . Hanafusa Cellular Immunity against Tumor Antigens Karl Erik Hellstrom and lngegerd Hellstrom Perspectives in the Epidemiology of Leukemia Irving L. Kessler and Abraham M . Lilienfeld AUTHOR INDEX-SUBJECT INDEX

Volume 13 The Role of Immunoblasts in Host Resistance and Immunotherapy of Primary Sarcomata P. Alexander and J. G . Hall Evidence for the Viral Etiology of Leukemia in the Domestic Mammals Oswald Jarrett

The Function of the Delayed Sensitivity Reaction as Revealed in the Graft Reaction Culture Haim Ginsburg Epigenetic Processes and Their Relevance to the Study of Neoplasia Gajanan V. Sherbet The Characteristics of Animal Cells Transformed in Vitro lan Macpherson Role of Cell Association in Virus Infection and Virus Rescue J . Svoboda and 1. Hloidnek Cancer of the Urinary Tract D . B . Clayson and E . H . Cooper Aspects of the EB Virus M . A . Epstein AUTHOR INDEX-SUBJECT INDEX

Volume 14 Active Immunotherapy Georges Math6 The Investigation of Oncogenic Viral Genomes in Transformed Cells by Nucleic Acid Hybridization Ernest Winocour Viral Genome and Oncogenic Transfonnation: Nuclear and Plasma Membrane Events George Meyer Passive Immunotherapy of Leukemia and Other Cancer Roland Motta Humoral Regulators in the Development and Progression of Leukemia Donald Metcalf Complement and Tumor Immunology Kusuya Nishioka Alpha-Fetoprotein in Ontogenesis and Its Association with Malignant Tumors G . 1. Abeler Low Dose Radiation Cancers in Man Alice Stewart AUTHOR INDEX-SUBJECT INDEX

CONTENTS OF PREVIOUS VOLUMES

Volume 15 Oncogenicity and Cell Transformation by Papovavirus SV40: The Role of the Viral Genome J . S . Butel, S . S . Tevethia, and J . L. Melnick Nasopharyngeal Carcinoma (NPC) 1.H . C. H o Transcriptional Regulation in Eukaryotic Cells A. 1. MacGilZiuray, 1. Paul, and C. Threlfall Atypical Transfer RNA's and Their Origin in Neoplastic Cells Ernest Borek and Sylvia]. Kerr 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 AUTHOR INDEX-SUBJECT INDEX

387

l,%Bis(Bchloroethy1)- 1-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 17 Polysaccharides in Cancer: Glycoproteins and Glycolipids Vijai N . Nigam and Antonio Cantero 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 of 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 . McCrath 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 Lengerovd

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

388

CONTENTS OF PREVIOUS VOLUMES

Recent Observations Related to the Chemotherapy and Immunology of Gestational Choriocarcinoma K . D. Bagshave Glycolipids of Tumor Cell Membrane Sett-itiroh Hakomori Chemical Oncogenesis in Culture Charles Heidelberger AUTHOR INDEX-SUBJECT INDEX

Volume 19 Comparative Aspects of Mammary Tumors J . M. Hamilton The Cellular and Molecular Biology of RNA Tumor Viruses, Especially Avian Leukosis-Sarcoma Viruses, and Their Relatives Howard M. Temin 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 d f e 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. J . V. Nossal The Role of Macrophages in Defense against Neoplstic Disease Michael H . Levy and E . Frederick Wheelock Epoxides in Polycyclic Aromatic Hydrocarbon Metabolism and Carcinogenesis P. Sims and P. L. Grover Virion and Tumor Cell Antigens of C-Type 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 Bamj Benacerrafand Daoid H . Katz Horizontally and Vertically Transmitted Oncomaviruses of Cats M . Essex Epithelial Cells: Growth in Culture of Normal and Neoplastic Forms Keen 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

CONTENTS OF PREVIOUS VOLUMES

Volume 22

Volume 24

Renal Carcinogenesis J . 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, lih-Nan Chou, and Paul H . Black Immunodepression and Malignancy Osias Stutman

The

SUBJECT lNDEX

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 of the Carcinogen, 4-Nitroquinoline 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 Mirosluv Hill and Juna Hillooa SUBJECT INDEX

389

Murine Sarcoma Virus-Induced Tumor: Exception or General Model in Tumor Immunology? J . P. Levy and J. C. Leclerc Organization of the Genomes of Polyoma Virus and SV40 Mike Fried and Beverly E. Grifin &-Microglobulin and the Major Histocompatibility Complex Per A. Peterson, Lars Rask, 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 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 Evefinu 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 . ]ha Tumor-Bound Immunoglobulins: In Situ Expressions of Humoral Immunity lsaac 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 Car-

390

CONTENTS OF PREVIOUS VOLUMES

cinogenesis by Nitroso Compounds and Alkylating Agents Anthony E . Pegg Immunosuppression and the Role of Suppressive Factors in Cancer lsao Kamo and Herman Friedman Passive Immunotherapy of Cancer in Animals and Man Steuen A . Rosenberg and William D .

Terry SUBJECT INDEX

Volume 26 The

Epidemiology Cancer

of

Pelayo Correa and William Haensrel Interaction between Viral and Genetic Factors in Murine Mammary Cancer 1.Hilgers and P. Bentuelzen Inhibitors of Chemical Carcinogenesis Lee W. Wattenberg Latent Characteristics of Selected Herpesviruses lack (3. Stevens Antitumor Activity of Corynebacteriun parvum Luka Milas and Martin T. Scott

Large-Bowel SUBJECT INDEX

A

B C 8

D 9

E D F 1 6 2 H 3

1 4 J

S