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·Sponsored by LOUISIANA DIVISION, AMERICAN CANCER SOCIFTY, INC Louisiana State Unnersity School of Medicine Tulane Umersity School of Medicme ·ETIOLOGY and TREATMENT of LEUKEMIA Proceedings of the First Louisiana Cancer Conference Edited by WALTER } BURDETTE, PH.D., MD, FACS Professor and Head of the Department of Surgery and Director of the Laboratory of Chnocal Biology, Umersity of Utah College of Medicine, Surgeon-in-Chref. Salt Lake County Hospital, Chref Surgical Consultus, Veterans Administranon Hospitals, Sul Lake City, Utah ST LOUIS 1958 THE CV. MIOSBY COMPANY ·COPYRIGHT © 1958 BY THE C V MOSBY COMPANY (All rights reseried) Prmted m the United States of America Library of Congress Catalog Carl Number 58-6574 ·IN MEMORIAM Arthur Kirschbaum

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·PARTICIPANTS William R. Arrowsmith, MD Associate Professor of Clinical Medicine, Tulane University School of Medicine, The Ochsner Clinic and Foundation Hospital, New Orleans, La Joseph H Burchenal, MD The Chemotherapy Service, Memortal and James Ewing Hospitals, The Division of Clinical Chemotherapy, Sloan-Kettering Institute, and Cornell University Medical College, New York, NY, WalcerBurdetre, PhD. MD Professor and Head of the Department of Surgery. University of Utah College of Medicine, and Surgeon-in-Chiel, Salt Lake County Hospital, Salt Lake City, Utah John P Fox, MD, PhD Professor of Epidemiology, Tulane University School of Medicine, New Orleans, La Jacob Furth, MD Associate Director of Research and Chief, Division of Experimental Pathology, The Children's Cancer Research Foundation, Boston, Mass Alfred Gellhorn, MD Director of the Institute of Cancer Research and Associate Professor of Medicine, Department of Medicine, Columbia University College of Physicians and Surgeons, and Chief of Medical Service, Francis Delafield Hospital, New York, N. Y 7 ·Ludwik Gross, MD Cancer Research Unit, Veterans Administration Hospital, Bronx, N. Y Arthur Kirschbaum, PhD. MD. Professor and Chairman, Department of Anatomy, Baylor University College of Medicine, and the University of Texas M. D. Anderson Hospital and Tumor Institute, Texas Medical Center, Houston, Texas Edward T, Krementz, MD Assistant Professor of Surgery, Tulane Unii crsity School of Medicine, New Orleans, La. Joseph V Schlosser, MD. Assistant Professor of Clinical Medicine, Tulane University School of Medicine, Charity Hospital of Louisiana, New Orleans, La. Albert Scgaloff, MD Assistant Professor of Clinical Medicine, Tulanc Unnersity School of Medicinc, The Ochsner Clinic and Foundation Hospital, New Orlcans, La Charles C Sprague, MD Associate Professor of Medicine, Department of Medicine, Tulane University School of Medicine, New Orleans, La Jerome T Syverton, MD Professor and Head, Department of Bacteriology and Immunology, University of Minnesota, Minneapolis, Minn Morwenna Till, MB, BCH (Camb) Chester Beatty Research Institute (Institute of Cancer Research Hospital), London, England Royal Cancer Arthur C Upton, MD Chief, Pathology-Physiology Section, Biology Division, Oh Ridge National Laboratory, O3k Ridge, Tenn George W Woolley. PhD Chief, Dission of Experimental Human Tumor Chemotherapy, Sloan-Kettering Institute for Cancer Research and Professor of Biology, Sloan-Keceri - sion, Cornell University Medical School. Vess York, NY 8 ·PREFACE The interest and color which sometimes illuminate recorded thought are often lost in the usual encyclopedic approach of scientific communication. The information included within these covers is set down with an effort to retain some of the spontaneity and flavor of its original presentation A short space of time was required for the presentation of the proceedings from which this volume arose, and the reader should be able to participate vicariously in an even more abbreviated interval. The ideas and exchanges between participants who are actuely obtaining and applying information about leukemia are recorded This volume represents an arrested instant during a memorable era when results justify hope that current methodology in the field of oncology may indeed lead to control of the disease The bibliography and summary passages may interest those who desire a slightly more comprehensive survey of ctiology and treatment. The proceedings from which this volume arose were first proposed as part of the professional education program of the American Cancer Society in Louisiana 2 "The Board of Directors made it possible through their active support and allocation of funds for the purpose Mr. Harry McEnerny, President, Dr E T. Krementz and his Committee on Local Arrangements, and Mrs. Marian Simmons, Xir Henry Houser, and Miss Brent Robertson of the Staff arranged for the meeting The editorial work was done for the most part under exceedingly pleasant circumstances and surroundings at St Louis University, and the secretarial work was performed by Miss Judy Felker, Mrs Helen Kane, and Mrs Esther Smith Acknowledgment is also due Dr M M. Wintrobe, who has been kind enough to review the final chapter. The competence, interest, and animated discussion of the participants are responsible for whatever profitable material is contained in the volume and for the hope that future conſerences will have equal value to them, the Board of Directors and the Staff of the Louisiana Division, American Cancer Society, the editor is deeply indebted WALTER J. BURDETTE Salt Lake Cits 9 ·Ludwik Gross, MD Cancer Research Unit, Veterans Administration Hospital, Bronx, NY Arthur Kirschbaum, PhD, MD Professor and Chairman, Department of Anatomy, Baylor University College of Medicine, and the University of Texas MD Anderson Hospital and Tumor Institute, Texas Medical Center, Houston, Texas Edward T Krementz, M.D Assistant Professor of Surgery, Tulane University School of Micdicine, New Orleans, La. Joscph V Schlosser, MD Assistant Professor of Clinical Medicine, Tulane University School of Medicine, Charity Hospital of Louisiana, New Orleans, La. Albert Segaloff, MD Assistant Professor of Clinical Medicine, Tulane University School of Medi cine, The Ochsner Clinic and Foundation Hospital, New Orleans, La Charles C Sprague, MD Associate Professor of Medicine, Department of Medicine, Tulane Unrversity School of Medicine, New Orleans, La Jerome T Syverton, MD Professor and Head, Department of Bacteriology and Immunology, Unuersity of Minnesota, Minneapolis, Mion Morwenna Tul, MB, BCh (Camb) Chester Beatty Research Institute (Institute of Cancer Research Hospital), London, England Royal Cancer Arthur C Upton, MD Chief, Pathology-Physiology Section, Biology Dassion, Oak Ridge National Laboratory, Oak Ridge, Tenn George Wl Woolley. PhD Chief. Division of Experimental Human Tumor Chemotherapy, Sloan Kettering Institute for Cances Kesearch and Professor of Biology, Slo.n-Kriterme Din slon ('ornel L'niversity Medical School. New York Y 8 ·CONTENTS Enology Jul Dugnosis of Leukemi VIRUSES AND CANCER (Jerome T. Syrerton, Al D.). 15 ETIOLOGY OF LEUKEMIA (Arthur Kirschbaum, Ph.D., AD.) 29 INFECTIOUS CONCEPT OF LEUKEMIA (Luduik Gross, M.D.). . 39 FILTERABLE AGENTS PREPARED FROM LEUKEMIC TISSUE CAUSING LEUKEMIA AND OTHER CANCERS IN MICE (George Vioolley, Ph.D.) 49 TACTORS INFLUENCING INDUCTION OF MYELOID LEUKE- MIA IN RF MICE BY IRRADIATION (Arthur C Uplon, MD, and Jacob Furth, AD.) 59 DIAGNOSIS OF LEUKEMIA (Charles C Sprague, AD) 71 Trulment of Leukem LABORATORY AND CLINICAL APPROACHES TO LEUKEMIA CHEMOTHERAPY (Alfred Gellhorn, AID). 80 IRRADIATION THERAPY OF LEUKEMIA (Joseph V. Schlosser, AD) 98

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·ETIOLOGY TREATMENT of LEUKEMIA ·THE USE OF CHLORAMBUCIL (CB 1348) AND BUSULFAN (MYLERAN) IN THE TREATMENT OF LFUKEMIA (Morn'enna Till, .1.B.), 102 TREATMENT OF LEUKEMIA ( Joseph II. Burchenal, M.D.). 109 CLINICAL MANAGEMENT OF LEUKEMIA (William R. Arron's smmh, M.D.). ETIOLOGY AND THERAPY OF LEUKIMIA /Walter 1. Burderte, PhD., N1.D.) 125 BIBLIOGRAPHY 146 12 ·ETIOLOGY TREATMENT of LEUKEMIA

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·Jerome T. Syrerton, MD VIRUSES and CANCER: When Gross' recently established the viral etiology of mouse leukemia, the role of viruses in the induction of neoplasms was re-emphasized. Consideration of now findings of this type is best based on a background knowledge of other types of ncoplasia now known associated with a virus as causative agent, and this latest characterization of a mammalian virus-induced tumor should not be used to advance again the general virus theory of cancer. That theory already has been responsible for its share of the hopeful enthusiasms and discouraging disappointments of cancer research. The secret of successful research involves as much the asking of proper questions as the secking of useful answers. Surely, the proper question is not whether all tumors are caused by viruses, but what tumors are so caused, and how. Rational consideration of our knowledge of viruses and tumors will avoid alike rejection by the urologist of the virus-induced growths as neoplasms and rejection by the oncologist of tumor agents as viruses. Between the two extremes, we can evaluate old accomplishments and plan new investigations. Both those familiar with the concepts of neoplasia and malignancy and those who feel they know what a virus is find it difficult to provide exclusive definitions As experimental biologists, we are not limited by the static conepts of pathology. The primary phenomenon of interest, neoplastic growth, is a growth of body cells capable of progression on transfer to other appropriate hosts The phenomenon of malignancy, recognized by cellular invasion or infiltration of contiguous fissue and dissemination by cellular metastasis, is a *Aided by grants from the American Cancer Society and the National Cancer Institute, National Institutes of Health 15 ·16 ETIOLOGY AND TREATMENT OF LEUKEMIA reflection of the degree of autonomy attained by the neoplastic cells. It is not necessary that neoplasia and malignancy be referable to the same ctiology. Virologists commonly conceive of a virus as an obligate intracellular parasite, that is, an agent capable of inducing its own replication by susceptible cells on which it is utterly dependent and yet to which it is essentially foreign. We recognize a virus by characteristic histo- or cytopathic cffect; wc know that the effect is associated with physical replicating particles, 8 to 300 mu in size, of nucleoprotein constitution, with antigenic capacity, usually insusceptible to chemotherapeutic and antibiotic agents. The concept of "virus" is fully as complex as that of “ncoplasm"; in cither case it is much casier to distinguish the normal and abnormal in practice than it is to isolate the essence of difference. The working definitions are designed mostly to differentiate types of abnormality. The oncologist is intent on the separation of hyperplasia from neoplasia; the virologist, on the distinctions among viruses, bacteria, fungi, and tickettsia The transmissible tuniors, like the latest instance which is the subject of this conference, demand more subtle decisions, such as whether the inducing agent is responsible for the neoplastic quality of cellular proliferation, and whether the behavior of the inducing agent is consistent with what we should expect of a virus With these questions in mind, it is profitable to examine the tumors for which decisions alrcady have been rendered AVIAN SARCOMAS The study of virus-induced avan tumors was initiated in 1910 with the discovery of the Rous sarcoma of chickens and the Fujinamı sarcoma of ducks The Rous sarcoma or Chicken Tumor 1, which now is a classic, experimental tumor, originated in a pedigreed stock of domestic fowl? Like mammalian transplanted tumors, it could at first be passed only in blood-related hosts, failing in other strains of the same stock By slow adaptation, the host range of the tumor gradually transcended the barriers of stock, breed, and cven species Rous's work showed that Chicken Tumor I possessed and propagated an acellular agent capable of inducing the de novo origin of other typical neoplasmı classifiable as malignant Distinct transmissible agents, viruses, now are known to be concerned in the etiology of many other fowl tumors, including fibrosarcomas, polymorphic sarcomas, round cell sarcomas, ordinary spindle cell sarcomas, osteochondrosarcomas, and endotheliomas Is yet, no avian carunoma has been attributed to tral infection Except for their rus etiology these autan connective tissue tumors are entirely analogous to the wide variety of mammalian sarcomas, and hence have been accepted as typical neoplasms The viruses originating these transmissible avian tumors not only are responsible for the neoplastic conversion, but also determine the structural and developmental properties of each particular tumor type. As yet, it has not heen ·VIRUSES AND CANCER possible to establish beyond doubt which cell types of the connective tissue series commonly give rise to Virus-induced tumors. The direction of tumor desclopment dictated by the infecting virus njay result from infection of different cells by the same or varant forms of visus, or by modulation of cells aſtes infection in response to the influence of host factors. Recent adı ances in tissuc culture, whereby homogeneous cell populations can be propagated from single cells with rcasonable regularity,' may hasten solutions to these problems. The influence of host factors on the primary cellular phenomenon has been demonstrated dramatically by the work of Duran-Reynals.' Virus introduced intravenously into young chicks produced a lethal hemorrhagic discase; injection of older birds or those previously protected with antibody resulted in typical sarcomas. The properties of Rous sirus are interesting but not extraordinary. It is about 48 mu in size"; it is extremely labile in the absence of protein, punſied preparations rapidly losing infectiuity at 37° C., although they can be stored indefinitely at -70° C. In the presence of protein, Rous vírus is remarkably resistant to desiccation, exposure to glycerol, or to irradiation This resistance to irradiation has been interestingly demonstrated by Peacock.' Primary sarcomas vanished upon irradiation with doses of 4,000 to 10,000 r; the virus, not inactivated, was resorbed through lymphatic channels, entered the general cos. culation, and was fixed at the periphery of the irradiated zone, where it induced formation of satellite tumors The biologic behavior of the Rous agent in no way gres reason for its rejection as a virus The painstaking efforts of Rubin" latchy have demonstrated that (a) a single particle can initiate tumor development, (b) virus of a single clone can inſect ectodermal or mesodermal cells, and (c) successive generations of multiplying sarcoma cells continually release a few virus particles from cach cell, so that virus production keeps pace with cell dission The problem of carlier sporadic failures to recoier virus from sarcomas, which occasioned some doubt of the causal relationship between urus and neoplastic induction, has been resolved. Rubin's results showed that the proportions of infectious and thermally inactivated ssrus present in sarcoma cell cultures were determined by the rate of cellular multiplication further, Bryan and his associates" ſound that the amount of virus recoverable from tumors was a function of initiating dose In spite of the weight of evidence accumulated from such careful studies, a considerable confusion of thought still exists with respect to the role of Rous virus in the production of neoplasms For example, Warburg," writing "On the Origin of Cancer Cells," had this to say If the Rous agent is inoculated into the chorion of chick embryos, tumors originate in the course of a few days as rapidly as in the transplantation of cancer cells The tumors formed are not chorion tumors, ·18 ETIOLOGY AND TREATMENT OF LEUKEMIA but Rous sarcomas. Thc Rous agent to which a particle weight of 150 million is ascribed at present is therefore capable of transmitting the morphological properties of the Rous sarcoma; and whatever we call the Rous agent hereditary unit," cell fragment, microcell or sporethe transmission of the Rous sarcoma by thc Rous agent is, in any case, nothing more than a transplantation, and is to be differentiated strictly from the production of a chicken sarcoma by methylcholanthrene, which is a neoformation of a tumor from normal body cells and as such takes a long time. Despite Professor Warburg's misconception of the common meaning of "transplantation," which is the transfer of intact cells from one host to another, the properties of Rous virus cited in his comnients are evidence of tumorigenic action on initially normal cells. AVIAN LEUKOSES Avian Icukosis represents a complex of malignant ncoplastic processes with viral etiology proved" by Ellermann and Bang in 1908. In retrospect, it is understandable why this discovery was not given adequate recognition: avian leukosis was considered a "spontaneous" ncoplastic disease because it occurred sporadically, was refractory to transmission even by grafting, and seldom yielded a filtrable agent. Since 1917, the small flocks of mixed breeds maintained in earlier practice have been replaced with inbred lines of birds selected by trap nesting for egg production. These superior stocks and enormous incubators resulted in vast numbers of chickens--deal populations for virus propagation. Veritable cpizootics of avian leukosis were the result. Lines of chickens have been evolved with incidences of leukosis limited only by dcath of the pullets before egg-bearing maturity; mortalities exceed 40 per cent. In these circumstances, the disease is readily transmissible. A sporadic descasc which closely simulated human leukemia in its epidemiologic features thus has been convened by selective host breeding mio an mfectious disease of epizootic manifestation Genetic and endocrine influences play a determining role in the etiology of avian Icukosis, with virus as the causative agent There are two types of asian leukosis. lymphomatosis and erythromyeloblastosis Lymphomatosis originates with cells of the lymphocytic series, and varies principally with respect to the location of the affected primitive cells. The disease commonly infiltrates the viscera to produce visceral lymphomatosis, it can enter nerie trunks to result in neurnlymphomatosis, but rarely inuades the circulation. Isolated lymphosarcomas represent another manifestation While lymphomatosis is common, erythromyeloblastotic leukosis is rare, 7 he latter condition, which essentially is a highly malignant leukemia of erythroblasts or myeloblasts, has been subjected to intense and rewarding laboratory study ·VIRUSES AND CANCER 19 Although many questions yet remain without answers, the systematic approach of Beard and associates' already has illuminated broad arcas of the problem of cry thromycloblastoxis, and it has provided a model for investigation of other ncoplasms of known or suspected virus origin. It appears that lymphomatosis and crythromycloblastosis are transmitted Specifically and independently by different viruses The virus of erythromycloblastosis (EMB) has received the bulk of study. It has been visualized by electron micrography and studied by physicochemical means. A single viral particle is about 140 mp in diameter. Plasma from infected chickens can contain from 60,000 to 2 trillion (2 x 10') such particles per milliliter; usually more than 60,000 particles are required to initiate infection. Similar to influenza virus, erythromyeloblastosis virus has an intimately associated enzymatic component, in this instance, adenosinc and inosine triphosphatasc. The virus now can be assayed quantitat cly by electron micrographic particle enumeration, infectivity bioassay, or enzyme-activity assay. Although infection is not always accompanied by production of neutralizing antibody, specific hyperimmunc serums can be produced in rabbits or chickens. Such serums do not affect enzyme activity. The intimacy of relationship between virus and host cell is emphasized (a) by the neutralization of inſectivity by antiserums induced in rabbits, guinca pigs, or micc against either concentrated virus or normal chick tissue and (b) by the presence of Forssman antigen in viral concentrates The cvidence found by Board and his associates" strongly indicates that normal chick and Forssman antigens cxist as integral parts of the virus. Since typical antiviral antibodies can be Produced experimentally with adequate doses of virus, it is evident that the irregular natural immune response derives from low dosage. Investigators of other neoplastic conditions should note that lack of an antibody response is not sufficient to rule out virus etiology. MAMMARY CARCINOMA OF MICE Since the turn of the century, the mouse mammary tumor probably has been the most popular tool for cancer research. Prior to 1936, many thousands of hours had been invested in the delineation of its biology, it was the prototypical example of an experimental, spontaneous tumor, a true mammalian malignant neoplasm, ideal for intestigation With the development of inbred or homozygous strains of mice," contnbuted by the outstanding efforts of Strong, Little, and others, the mammary tumor could be transplanted at will Work with inbred stocks proved the importance of genetic factors in the development of cancer of the breast In this early period, the investigations of Lathrop and Loeb" emphasized the role of hormonal factors The suggestion in 1933 by Little and Korteweg of the essential action of an extrachromosomal influence, and the culminating demonstration in 1936 by Bittner"of a causative, transmissible agent, finally characterized mouse mammary cancer as a virus-induced ·20 ETIOLOGY AND TREATMENT OF LEUKEMIA ncoplasm." 18 The virus has been hedgingly described by such noncommittal terms as milk influence," "milk factor," and "Bittner agent," but it evinces the properties by which a virus is known, and it can be so called. Mlouse mammary tumor virus is distributed throughout the bodies of mice of strains with a high incidence of mammary cancer. It is rcadıly transmissible to susceptible mice through mothers' milk by nursing, and by milk or extracts of assorted tissues by injection. The precise site of infection is not known, nor is it known whether the overt manifestation of mammary adenocarcinoma rcsults from an actuating effect of the virus or from a provocative influence similar to that cxerted by Shope virus in the rabbit papilloma-to-carcinoma sequence. Strains of mice exist with a fair incidence of breast cancer, from which no virus has been isolated. We are, howeves, reminded by the work with avian neoplasms that inability to recover virus by bioassay of crude extracts is no guarantec of its absencc. Mammary tumor virus has not been as well characterized biologically, physically, or immunologically as the other oncogenic viruses, for reasons of obvious difficulty. The latent period of infection alone is a formidable barrier to bioassay, ranging from six to twelve or more months. It is known that the virus is antigenic in heterologous species, but that, curiously, it is not antigenic in mice. This lack of immune response in the susceptible host cannot be attributed to lou dosage; detectable antibodies are not produced by hyperimmunization procedures. It is possible that host proteins are even more intimately associated with the virus than is the case with avian erythromyeloblastosis virus The common des elopment of breast cancer in mice, however incomplete our knowledge of it, reflects an essential role for a causative virus, and the inseparable influence and splay of genetic factors and hormones as well. PAPILLOMATOSIS OF RABBITS A cutaneous papilloma occurs enzootically among wild cottontail rabbits in states bordering the Mississippi River. In 1933, Shopes demonstrated that the benign papilloma was virus-induced. Reports by Rous and Bcard" that the horny growths could progress to frank epidermoid carcinoma raised hopes that a mammalian cancer of viral origin was available for study. During the twenty years from 1934, in our own laboratory more than 2,000 wild, cattontail, and domestic Dutch belt rabbits with papillomatosis were ob. served 20 Weekly examination of 600 of these, kept as undisturbed as possible, revealed the natural pattern of tumor growth Papillomatosis was found to progress predictably through three phases proliferature, stationary, and involutionary The proliferatre phase begins seven to forty-two days after exposure of injured epidermis to virus, when tiny vesiculord papules appear fected epithelial cells multiply into growths ranging in size from small nodules The in- ·VIRUSES AND CANCER 21 to enormous verrucous masıcs and cutancous horns In three to six months, a stationary phase supervenes in which lesions are quiescent, without evidence of degeneration or further proliferation. This phase commonly is of shorter duration in the experimental host, the domestic rabbit. The growth cycle terminates in an involutionary phase. Either the final failure of the germinal layer to con. tinue growth results in regression and disappearance of the lesion, or renewed proliferative sigor ensues, with replacement of benign by malignant cells and formation of epidermoid carcinoma The progression to carcinoma has been observed in 75 per cent of domestic and 25 per cent of wild rabbits naturally or experimentally infected, The causative agent of papillomatosis, Shope's virus, is about 44 to 66 m in size," and the virus is extremely resistant to deterioration. All of the antigenic and infective properties of the virus arc attributable to the clementary particles; no evidence of associated enzymes or host antigens exists Cytotrophism is limited strictly to certain skin cells If virus is inoculated along a long scarification passing across epidermis and mucous membranc, papilloma formation stops abruptly at the beginning of mucous membrane. Virus must reach the cpidermal basal cell layer to become effective. The number of par. ticles reaching the critical site determine the incubation period and the number, extent, and confluence of lesions Virus cannot be isolated from growths with low content of virus because virus concentrations sufficient to infect the critical site in the test host are not obtained Such difficulty of isolation with domestic rabbit papillomas led to the postulation of a "masked" state of virus, it seems clear now that insufficient virus was the case. Infectie virus is recoverable from proliferating, quiescent, or even regressing papillomas It has been recovered commonly from these papillomas in cottontails, but rarely from domestic rabbits; it has not been recovered from carcinomas. Apparently unquestionable evidence of a persistent association between virus and cancer was obtained, howeier, by Kidd and Rous"? with the V-2 carcinoma, with which successive transplantations over many years regularly induced virus-neutralızıng antibodies in recipient hosts The immune response ccased after 50 passages." We know that papilloma formation is a direct result of virus infection, we do not know the role of sirus in the neoplastic transformation The papilloma-to-carcinoma sequence suggests three possibilities (1) the ultimate cancer is produced by papilloma virus; (2) a second virus specifically inducing the carcinoma is activated by the antecedent papilloma, (3) papilloma virus may persist in the succeeding carcinoma as an inci- If either of the first and second alternatives is true, virus should be recoverable not only from the primary carcinoma but also from the secondary, pulmonary metastases It is not. The relation between virus and carcinoma remains unknown. dental passenger ·22 ETIOLOGY AND TREATMENT OF LEUKEMIA In summary, it can be said that five avian and mammalian tumors, at least threc primarily malignant, has been accepted as virus-induced. The complexity of the agents varies from that of rabbit papilloma virus to that of chicken sarcoma virus with its variability in mode of inſection, pathogenicity, and cytotrophism by spccics, race, tissue, and cell. The range of complexity and the properties involved are no stranger than those of other viruses. Oncogenic viruses certainly are responsible for the prolilerative origin of the tumors; whether they are always necessarily responsible for the ultimate neoplastic quality of the growths is not decided. The answer seems to vary from a definite "yes" with avian sarcoma and erythromyeloblastosis, to a "maybe" with mouse mammary carcinoma, to a "probably not" with rabbit epithelioma. We may hope that extension of the study of mouse leukemia virus, already so vigorously prosecuted by Gross,24 Law and associates," and Stewart, 26 will shed further light on the relationship of viruses to cancer. DISCUSSION DR. BURDETTE. Dr. Fox, will you open the discussion of Dr. Syverton's paper? Dr. Fox: Thank you, Dr. Burdette. I have been trying to relate the information presented about rodent disease to human application. Each of us, of course, does this in his own way, conditioned by his own peculiar experience. Like Dr Syverton, I am interested in viruses and sometimes labor under the handicap of being called a virologist Also, I am interested in epidemiology, and I wish to say at this point that the abundant information presented by Dr. Kirschbaum and Dr Syverton as to the multiplicity of factors which enter into the ultimate determination of whether or not, or when, leukemia occurs provides one with the basis for setting up a number of hypotheses with respect to human experience that are very stimulating to an epidemiologist. Obviously, the long, generation time of man enters into this picture and makes the direct execution of such experiments rather difficult, but I still think that an appropriate, welldevised study might yet be informative. I was very much interested in Dr Syverton's views concerning antibody response to some of these viral agents in their native host, implying that this was possibly because the viral agent itself contained the host protein. I am wondering whether it might not also be due in part to the fact that these infections may be quite commonly congenital I am thinking now of the virus of lymphocytic choriomeningitis in the mouse. A mouse infected after birth develops antibodies to the virus in the usual fashion, whereas the mouse infected in utero, that is, congenitally, harbors the virus throughout its life but never develops antibody In this case, apparently, the virus, having been present in the developmental stage, does not elicit the customary response to foreign protein. Would this apply to leukemia?

·VIRUSES AND CANCCR 23 Dr SYVERTON: It seems to me that four possibilities deserve consideration: 1. In agent and its specific antibody can coexist with any free agent or virus being neutralized by free antibody. This state of cocxistence of antibody and infectious agent, known as prcmunition, is cxemplified by psittacosis in its natural host, a prittacine bird. The bird commonly shows apparent recovery from clinical discase, but continues to carry psittacosis virus in the presence of specific antibody. This silent or inapparent infection continues for variable periods of time until some adıçre circumstance such as inclement weather, intercurrent infection such as salmonellosis, starvation, or other reason upset the resistance of the bird, resulting in recurrence of clinical disease. 2. A second category is cxcmplified by acquired tolerance in which exposure to an agent or antigen before or immediately after birth results in loss of the host's ability to react to that same agent upon recxposure later in life. 3. A third type is the liberation by certain agents of soluble antigens which persist widely distributed in the body and result in immunc paralysis (fixed antigen presents effective antibody formation by the host, the antibody-forming capacity of the host is blocked). 4. A fourth category is the case of agent, virus, or otherwise, so intimately associated with host tissues as to become essentially inseparable and to persist in what appears to be host nucleoprotein. DR. KIRSCHBAUN: Although antibodies have not been demonstrated, Dr Syverton, is it not true that as mice age they become resistant to the development of mammary cancer following introduction of the agent. Has anything been done to determine whether neutralizing antibodies are present in the serum of these older animals? DR. SYVERTON. To my knowledge, no. Dr KIRSCHBAUM Would you suspect there may be neutralizing antibodies? DR SYVERTON A reasonable, working hypothesis is the concept that animals acquire before or at birth a variety of agents that maintain and remain dormant unless activated by provocative measure De KIRSCHBAUM. Dr Fox, were you suggesting that, after the agent became available to the fetus, the fetus was desensitized, and then in later life the individual was able to form antibodies to the agent? DR Fox Yes, essentially what I was suggesting is that, because of contact with the agent during the fetal period, the agent in effect was not a foreign protein to that particular individual, although it might be to other members of the species DR KIRSCHBAUM That seems not to be applicable to the mamman tumor agent because it is not available to the embryo That is, the agent cannot ·24 ETIOLOGY AND TREATMENT OF LEUKEMIA be obtained from the embryo. The mammary tumor agent is obtained postnatally. DR. SYVFRTON: Is it not true that the same applies to other species within the first few days of life? Dr. WOOLLEY: That was the point I would like to stress, too. Apparently for the first few hours after birth, the animal is similar to the embryo in some respects. Dr. Syverton, would you discuss the time of fixation of viruses to tissue in relation to the appearance of disease at a later date. Dr. Syverton: It is not clear to me what you have in mind. If you refer to the incubation period of Shope rabbit papilloma, papilloma virus demands direct contact with the stratum germinativum of the epidermis to evoke infection For example, when papilloma virus is inoculated by a scratch that is made to extend over the juncture of skin and mucosa and onto the mucosal surlace, papillomatous proliferation is limited to where contact was made with basal cells of epidermis, the papillomatous growth stops abruptly at the mucosal juncture. As for the incubation period of Shope rabbit papilloma, virus once brought into contact with basal cells requires from seven to forty-two days before yıclding evidence of its presence by the development of small cpithelal excrescences which become papillomas or warts. Once that incubation period has been achieved, cells reduplicate quite rapidly and continue to do so for from six weeks to about three months, at which time multiplication ceases and the virus tumor remains in its stationary phase. Up to and during the stationary phase in the tumor's developmental cycle, virus in varying amounts can be demonstrated in the papilloma cells. It is not unlikely that virus recovered during the stationary phase represents preformed virus kept intracellularly for weeks or months Papilloma virus is a most resistant virus, as measured by heat inactivation, desiccation, or other adserse circunistance The final stage in the develormental cycle, the stage of regression, is manifested in one of two ways as the involutionary phase when the lesion becomes dried, atrophic, and sloughs. The skin site heals, leaving little or no evidence of pre-existing tumor Contrariwise, the regressive state may be manifested by renewal of proliferative capacity to result in an cpidermoid carcinoma which infiltrates underlying tissues, metastasszes, and kills the host Is it this well-established sequence that you had in mind, or was at the relationship of papilloma urus to Rous's V-2 carcinoma? Virus was demonstrated over several years in this transplantable tumor while maintained in Dr. Rous's laboratory Evidence for the presence of virus by antibody production was lost after many generations It is not clear whether the virus under consideration represented a passenger virus or a virus of signifiance in the pathogenesis of the V-2 carcinoma DR WOOLLEY, What happens to the virus in the end organ, during this long period?

·VIRUSES AND CANCER 25 DR. SYVERTON: I do not know. Mammary tumor virus obviously must be present, since it is recoverablc both from mammary tissues and a variety of other tissues during that time. DR. BURCHENAL: Are there any insect or fowl vectors for the virus of the Shopc papilloma in the wild rabbit? DR. SIVERTON: The natural mode or mechanism for infection of the cottontail rabbit by papilloma virus is not known It is known that lesions, under natural conditions, occur chiefly on head, face, buttocks, and legs This anatonic distribution suggests that the cottontail acquircs virus from its burrow. Dr. Shope has attempted, by ingenious approaches, to find the answer, even studying ground toads, ground owls, and other natural inhabitants of burrous It is my understanding that Icuko-is is a respiratory infection. Certainly, birds in the same enclosure contract the disease Dr BURCHEXAL. But no insect vectors are known? DR. KIRSCHBAUM: It occurs to me that virologists have the happy faculty of beleving that virus is present even though one cannot denonstrate it IC has been demonstrated that if the mammary tumor agent was not made available to mice of the high-tumor C3H strain by foster-nursing, as many as 30 per cent of the animals des eloped mammary cancer, and from these mammary cancers, the mammary tumor agent could not be demonstrated, using the methods that are ordinarily used to demonstrate it. In this case, the agent is said to be present because there is a method 10 demonstrate it. If the mammary tumor agent is excluded by foster-nursing and then mammary cancer is induced by the administration of methylcholanthrene and it does not appear when one extracts the mammary tumors and injects the most appropriate strain of test animals, one must come to the conclusion that in carcinogen-induced mammary tumors of mice the agent is not acquired if it is not present at the outset There may be other methods of demonstrating the agent, but at least tentatively I have to come to the conclusion that it is not demonstrable under these conditions DR WOOLLEY Are you referring to the same morphologic tumor when you speak of the carcinogen-induced tumor? DR KIRSCHBAUBT. I would say that in about 95 per cent of methylcholanthrene-induced mammary tumors there are areas of carcinoma. There are also areas of squamous metaplasia One might feel that this squamous metaplasia indicates that this is a different kind of cancer and that there is no agent found because this is not the kind of mammary cancer that occurs spontaneously, but I do not think one can brush aside the findings of Heston with that argument He finds mammary cancer with histology typical for the spontaneous neoplasm, although exhaustive experiments reseal no agent It seems to me that the agent ·26 ETIOLOGY AND TREATMENT OF LEUKEMIA accelerates the onset of the disease. There are other potent factors, and the agent is not infectious in the usual sense but is an accelerating factor, DR. SYVERTON: I follow your line of thought. Certainly, it is not known why some people harbor xanthelasma and xanthoma tumors; the presence of such tumors is of significance to the attending physician. DR. BURDETTE: One of the things you mentioned in your talk a moment ago was that masking of viruses may sometimes be explained on the basis of the small number of particles present. It occurs to me that this removes the masking of viruses from the occult into something which may be tested. Would you comment? Dr. SYVERTON: The term "masking" has been used in description of biological findings to indicate: (a) a latent, noninfectious form of virus which may or may not give rise to discernible antibodies, (b) inhibition or "mashing" of an infectious agent or virus by specific antibody, and (c) a quantitative relationship of viral particles to susceptible cells which demands a minimal number of particles for host transfer, as has been emphasized adequately by Beard. Bryan, at the National Cancer Institute, has provided convincing examples of why the term "masking" should not be used. He found that terminal dilutions of chicken tissue containing Rous sarcoma virus can result in the production of a tumor which eventually kills the host fowl. Yet, from such tumors, virus may not be recoverable. The more concentrated of successive twofold dilutions of the same chicken tissue induces tumors which contain approximately the same amount of virus as the Original source tumor. Failure to demonstrate virus in tumors provoked by terminal dilutions is subject to interpretation as absence of virus or masked virus when antibodies in recipient hosts are demonstrated Bryan's studies have shown that a critical number of ural particles are essential for induction of a tumor and that this number varies from onc virus tumor to the next DR BURDETTE Dr Syverton, would you hazard any comments about the manner in which tumor uruses act within the cell, whether actually this is only a matter of macromolecular structure, since an agent such as polyvinyi pyrrolidone has been shown by Hueper to be tumongenic, and cellophane, as well as other polymers, will produce tumors, as shown by Oppenheimer and others, or whether, as Darlington has suggested, the way these viruses act is by entering the cell and acting through competition with essential cellular systems DR. SYVERTON. I am glad to comment if you will permit me conyderable latitude. Let us assume that a mammalian cell consists of a bag of enzynies and genes subject to rearrangement, replacement, and variable functions Infection by a virus, which is nucleoprotein foreign to the cell, introduces a new battery or bag of enzymes to take over and convert the cell into a factory capable of pro- ·VIRUSES AND CANCCR 27 ducing, within predictable periods of time, an abundance of new nuclcoprotein or virus. The time required for this process for bacteria can be as short as twenty minutes, for poliomychtis, about cight hours; for many viruses, much langer. The result is production and release of a vast number of newly formed particles of nuclcoprotein, new virus In this process, virus, the product, acquires materials from the host cell and in turn, upon transfer to a new host cell, alters its cnzymic productive capacity. Dr. BURDFTTE: Do you think this is a sort of transduction? Is that what Jou are implying? Dr. SYVERTON: Yes, but transduction significs transfer between cells of a new property which may be biochemical, physical, or genic. Transfer by transduction is limited to a single characteristic. More nearly what I had in mind is the lysogenic relationship of bacterium and its bacterial virus, since by that relationship the bacterial cell maintains and transmits virus by its genic mechanism to its bacterial progeny. Dr. KIRSCHIBAUM: Dr. Syverton, would the results of experiments with the Shope papilloma not suggest that the virus is responsible for the initiation of the ncoplasm, but that continuing growth is not virus-dependent? Dr. Syverton: The findings can be so interpreted. An adequate explanation is not known for the disappearance of papilloma virus from tumor during its regressive stage inclusive of the carcinomatous lesion. Of examples acceptablc as virus tumors, certainly the epidermoid carcinoma that follows the Virus-induced papilloma has weal supportisc evidence for a virus causation, DR. GROSS Dr. Kirschbaum remarked about nonviral etiology of cer. tain types of mammary tumors You must keep in mind that our means at the present time to establish the viral etiology of a tumor consist mainly of bioassay; He inject cell-free extracts into susceptiblc animals and observe animals for the development of tumors We know that in many instances the extract docs contain virus and yet no tumor results Never can we state that the extract does not contain the virus just because the injection did not result in the deselopment of a tumor It is quite possible, in fact, perhaps probable, that there are several types of mammary tumor viruses The fact that some mice may still develop mammary tumors following foster-nursing suggests that such tumors may be transmitted just like the leukemic agent, directly through the embryo DR. BURDETTE Do you have any evidence that another virus is involved in the causation of mammary tumors, or is this only speculative? DR GROSS I have no evidence whatever, but I assume that not all mammary tumors are caused by the same agent, and that not all of them are transmitted through the milk ·26 ETIOLOGY AND TREATMENT OF LCUKEMIA accelerates the onset of the disease. There are other potent factors, and the agent is not infectious in the usual sense but is an accelerating factor. Dr. SYVERTON: I follow your line of thought. Certainly, it is not known why some people harbor xanthelasma and xanthoma tumors; the presence of such tumors is of significance to the attending physician. Dr. BURDETTE: One of the things you mentioned in your talk a moment ago was that masking of viruses may sometimes be explained on the basis of the small number of particles present. It occurs to me that this removes the masking of viruses from the occult into something which may be tested. Would you comment? DR. SYVERTON: The term "masking” has been used in description of biological findings to indicate: (a) a latent, noninfectious form of virus which may or may not give rise to discernible antibodies, (b) inhibition or "masking" of an infectious agent or virus by specific antibody, and (c) a quantitative relationship of viral particles to susceptible cells which demands a minimal number of particles for host transfer, as has been emphasized adequately by Beard. Bryan, at the National Cancer Institute, has provided convincing examples of why the term "masking" should not be used He found that terminal dilutions of chicken tissue containing Rous sarcoma virus can result in the production of a tumor which eventually kulds the host fowl. Yet, from such tumors, virus may not be recoverable The more concentrated of successive twofold dilutions of the same chicken tissue induces tumors which contain approxi. mately the same amount of virus as the original source tumor Failure to demonstrate virus in tumors provoked by terminal dilutions is subject to interpretation as absence of virus or masked virus when antibodies in recipient hosts are demonstrated. Bryan's studies have shown that a critical number of viral particles are essential for induction of a tumor and that this number varies from one virus tumor to the next DR BURDETTE Dr Syserton, would you hazard any comments about the manner in which tumor viruses act within the cell, whether actually this is only a matter of macromolecular structure, since an agent such as polyvinyl pyrrolidone has been shown by Hueper to be tumorigenic, and cellophane, as well as other polymers, will produce tumors, as shown by Oppenheimer and others, or whether, as Darlington has suggested, the way these viruses act is by entering the cell and acting through competition with essential cellular systems Dr SYVERTOX I am glad to comment if you will permit me considerable latitude. Let us assume that a mammalian cell consists of a bag of enzymes and genes subject to scarrangement, replacement, and variable functions Infection by a virus, which is nucleoprotein foreign to the cell, introduces a new battery or bag of enzymes to take over and convert the cell into a factory capable of pro- ·Arthur Kirschbaum, PhD, MID ETIOLOGY of LEUKEMIA Leukemia was recognized as a distinct clinical entity more than one hundred years ago, but its relationship to lymphosarcoma and other neoplasms remained quite obscure Most of the worh was descriptne untıl approximately 1908, when Ellermann," working on leukemia in the domestic lowl, ſound that this disease can be transmitted to susceptible animals by the inoculation not only of cells but also of cell-free extracts, at this time, the possibility of the Viral etiology of leukemia became apparent. It has taken more than forty years before the work of Gross” has made it seem likely that mouse leukemia may also have a viral etiology. The first experimental study in mammals appeared in 1927 when a group Working in Holland, Snijders and Tio Tjwan Gie,'' descnbed leukemia and lymphosarcoma in a homogeneous stock of guinea pigs These diseases could be transmitted to closely related animals by inoculating intact viable cells, the introduced cells grew at the site of inoculation to form a tumor, even though these cells were dernied from the blood stream Thus, these workers demonstrated that leukemia cells of the guinea pig may grow as a tumorous mass. When the cells of local tumors were inoculated intravenously into closely related animals, the recipients des eloped leukemia In 1929, Richter and MacDowell" described leukemia in the C58 strain of mice. This is a highly inbred strain in which 90 per cent of the animals develop lymphatic leukemia, and these workers began a systematic study on the genetics of this disease One of their most important contributions was the demonstration that nongenetic as well as genetic factors determine the genesis of leukemia. Although 90 per cent of the animals of this genetically homogene. 29 ·28 ETIOLOGY AND TREATMENT OF LEUKEMIA DR. BURDETTE: In closing, Dr. Syverton, would you carc to set up some rules for proving that a given virus is responsible for a given tumor Would you alter Koch's postulates in any way? DR. SYVERTOX: It has proved difficult to formulate rules to prove a given virus responsible for a given tumor. Evidence accumulated by Beard and his associates for crythromyeloblastosis virus as the causative agent of erythromyelo. blastosis of chickens is conclusive. Yet, if one examines the origin of tumors of mice, or the rabbit papilloma-to-carcinoma sequence initiated by Shope's rabbit papilloma virus, the problem is difficult. In each instance, the cudence or find. ings are subject to the interpretation that the causative agent consists of indistinguishablc host and virus constituents. Finally, as Rivers pointed out some years ago, Koch's dicta or postulates can be applied to virus infection. Dr BURDETTE: Later you will have an opportunity to make additional comments on the proofs offered for the viral etiology of leukemia.

·ETIOLOGY OF LEUKCMIA 31 The relationship of the chemical and physical leulemogenic agents (carcinogenic hydrocarbons, estrogenic hormone, and ionizing radiation) to the virus remains to be determined. Do these non iral agents merely act by way of a filterable acent? In other words, is a filterable agent the mediator for the carcinogenic hydrocarbons and x-irradiation? Thynionta (lynıphosarcoma) of the mouse has attracted considerable attention becausc cxtirpation of the thymus has reduced the incidence of leukemia in certain high-leukemia stocks. In strains of mice susceptible to the induction of leukemia by carcinogenic hydrocarbons or v-rays, thymectomy may also reduce the incidence of the induced disease. There is something definitely peculiar about the thymus as far as the gencsis of leuhemia is concerned. Perhaps the environment provided by the thymus may be responsible for the leulemic transformation of cells which invade the thymus. Injection of thymoma cells intravenously into closely related animals may result in the appearance of systemic leukemia. Systemic leukemia frequently is characterized by large lymph nodes, spleen, thymus, and liver. There is considerable variation in mouse leukemia from case to case, both histologically and etiologically Some inicstigators compare mouse leukemia and human lcuhemia, since the same forms of lymphomatous disease are seen in the mouse which are scen in the human species. I am inclined to think that the discase is so similar in the two species that perhaps we should not speak of mouse and human leukemia, but of the types, such as granulocytic and lymphocytic We have emphasized the thymus as a focus of origin of leukemia Howaver, the mesenteric lymph nodes may become lymphomatous, and metastasis to the Iner from such a lymphosarcoma may occur. It is to be emphasized that leukemia in the mouse is quite variable. Considerablc variation may be encountered in hybrids, but the discase tends to be more uniform in inbred strains of mice. On occasion, some question arises as to whether a lesion is hyperplastic or neoplastic. Some lesions of this type are not transplantable, and transplantation of mousc cells with progressise growth is a biologic test of neoplastic activity. Kaplan recently shoned that early in the genesis of thymomas they may not be transplantable, whereas subsequently they transplant readıly into genetically similar hosts Chlorotic leukemia may prove very valuable in testing such agents as busulfan In chronic, granulocytic leukemia, mice may have Jeukocyte counts as high as one million per cubic millimeter and become Intensely anemic, and this disease may become acute terminally as in the human subject In strain A, although highly susceptible to the induction of pulmonary tumors, no leukemia was induced when methylcholanthrene was fed Therefore, the pulmonary tissue is susceptible to the action of the carcinogen, whereas the lymphoid and myeloid tissues are resistant In the DB.1 strain, on the other ·30 CTIOLOGY AND TREATMENT OF LCUKEMIA ous stock developed Icukemia, approximately 10 per cent remained leukemiafree. The progeny of this 10 per cent, however, did develop leukemia, the incidence being 90 per cent, suggesting that the 10 per cent nonlcukemic were genetically similar to the 90 per cent which devcloped the disease. Therefore, it was assumed that nongenetic factors were responsible for the nonappearance of the disease in 10 per cent. The next most important step was the observation by Furth and Furth" that leukemia may be induced in low-leukemia strains of mice by exposure to x-rays. This was the first experimental demonstration that the disease may be induced in animals which otherwise remain leukemia-free. The unfortunate cvents in Hiroshima demonstrate that ionizing radiations may induce the same disease in the human species as well,92 so that leukemia of mouse and man may have a common etiology. Mider and Morton," in 1939, showed that the carcinogenic chemicals which induce various neoplasms quite readily in mice may, in certain genetically susceptible strains, cause the appearance of leukemia. This is further evidence that leukemia in mice is a neoplasm; chemicals which induce obvious cancers also induce leukemia Transplantation studies also indicate that leukemia of mice is a neoplasm because leukemia in mice transplants as do the other neoplasms of this species A third leukemogenic agent, estrogenic hormone, was discovered by Lacassagne," in France, and this work was expanded by Zondek, and by Gardner and Dougherty is in this country. This finding is rather difficult to reconcile with the possible etiology of the disease in man. However, related steroids from the adrenal cortex may modify the progress of leukemia in the human species, so that there may then be some fundamental link between the induction of leukemia in mice by estrogenic hormone and the appearance of the disease in man Recently, the development which has attracted the most attention is the discovery of a possible biologic leukemogen, a filterable agent which can be obtained not only from leukemic tissue of mice, but also from the embryonic tissue of high-leukemia strains When this material was inoculated into certain lowleukemia strains, then the incidence in recipients was higher than in controls. Drs Gross and Woolley will discuss this work at length, and also Dr Syverton has reported on the viral aspects of etiology We have mentioned that leukemia may appear spontaneously or be induced. I would like to emphasize that we cannot generalize about these agents which induced leukemia, they are operative depending on the genetic soil Not all mice are susceptible to the induction of leukemia by carcinogenic hydrocarbons, not all mice are susceptible to the induction of leukemia by estrogenic hormone, and not all mice des elop leukemia spontaneously. Genes determine the response of the host. Both genetic and nongenetic factors are involved ·I TIOLOGY OF LEUKEMIA 31 The relationship of the chemical and physical leukemogenic agents (carcinogenic hydrocarbons, estrogenic hormone, and ionizing radiation) to the virus remains to be determined. Da theve nonviral agents merely act by way of a filterable ageni? In other words, is a filterable agent the mediator for the carcinogenic hydrocarbons and x-irradiation? Thymoma (lymphosarcoma) of the mousc has attracted considerable at. tention because extirpation of the thymus has reduced the incidence of leukemia in certain high-leukemia sacks. In strains of mice susceptible to the induction of leukenug by carcinogenic hydrocarbons or X-rays, thymectomy may also ren duce the incidence of the induced disease There is something definitely peculiar about the thymus as far as the genesis of leukemia is concerned. Perhaps the environment provided by the thymus may be responsible for the leukemic transformation of cells which insade the thymus. injection of thymoma cells intracnously into closely related animals may result in the appearance of systemic leukemia. Systemic leukemia frequently is charactenzed by large lymph nodes, spleen, thymus, and Iner. Ihere is considerable variation in mouse leukemia from case to case, both histologically and etiologically Sunie iris estigators compare mouse leukemia and human leukemia, since the same forms of lymphomatous disease are seen in the mouse which are seen in the human species. I am inclined to think that the discase is so sinular in the two species that perhaps We should not speak of mouse and human Iculemia, but of the types, such as granulocytic and lymphocytic We have emphasized the thymus as a focus of origin of leukemia However, the mesenterx lymph nodes may become lymphomatous, and metastasis to the liver from such a lymphosarcoma may occur It is to be emphasized that leukemia in the mouse is quite variable. Considerable variation may be en. countered in hybrids, but the disease tends to be more uniform 11 inbred strains of mice. On occasion, some question arises as to whether a lesion as hyperplastic or neoplastic. Some lesions of this type are not transplantable, and transplantation of mouse cells with progressive growth is a biologic test of neoplastic activity. Kaplan recently showed that carly in the genesis of thymomas they may not be transplantable, whereas subsequently they transplant readily into genetically similar fonts Chlorotic leukemia may prove sery valuable in testing such agents as busulfan In chronic, granulocytic leukemia, mice may have leukocyte counts as high as one million per cubic millimeter and become intensely anemic, and this disease may become acute terminally as in the human subject In strain A, although highly susceptible to the induction of pulmonary tumors, no leukemia was induced when methylcholanthrene was fed. There fore, the pulmonary tissue is susceptible to the action of the carcinogen, whereas the lymphoid and myeloid tissues are resistant In the DBA strain, on the other ·32 CTIOLOGY AND TREATMENT OF LEUKEMIA hand, another highly inbred strain, feeding the same carcinogen results in the induction of leukemia, whereas pulmonary tumors are not induced. This sug. gests that the target tissue determines the response to the Icukemogen Genedetermined, systemic metabolism of the carcinogen does not determine leukemo. genesis, but something within the tissue itself docs. The site of gene action is thus the target tissue. Examination of multiple tumors in the mammary glands of strain A mice, carcinogen-induced, reveals hundreds of foci where the carcinogen acts, but the lymphoid tissue does not respond at all. Experiments to illustrate that the site of gene action is in the target tissue are of interest. The Bagg, albino strain is susceptible to the development of pulmonary tumors if urethan is gnen, urethan being the carcinogen. The DBA strain is urethan-resistant. Only an occasional tumor nodule appears. If the two strains are crossed, a susceptible F, hybrid results, this host being susceptible to the induction of pulmonary tumors. Prior to the administration of urethan, the pulmonary carcinogen, normal lung tissue from newborn micc of the susceptible parent stock, was grafted into the subcutaneous tissue of one car, and newborn lung tissue from the resistant parent strain into the other ear. Thus, in an F., hybrid host susceptible to the induction of pulmonary tumors were normal lung tissue grafts from susceptible and resistant strains. After the grafts had taken, the animals were given the carcinogen. Tumors appeared in situ in the host, and in the susceptible tissue grafts (12 of 17). One small nodule was seen in a single, resistant, tissue graft (1 to 17). This illustrates that the site of gene action is in the target tissue and that the host, in this case susceptible, did not alter the resistance of the resistant tissue. The same principle holds for leukemia. Law has crossed mice with a high incidence of leukemia to those in a strain having a low incidence of leukemia to obtain F, hybrids which were thymectomized Removal of the thymus inhibited the development of leukemia, although these F. hybrids were otherwise susceptible if thymus from the high-leukemia parent stock was grafted into F; hybrids early in life, then the F, hybrids developed leukemia On the other hand, if the thymectomized, F. hybrids recened grafts from the low-leukerta strain, then leukemia did not develop 69 He then transplanted the leukemic cells which appeared if the leukemia had arisen from the thymic graft of the parental stock, then this leukemia should have been transplantable not only into the parental stock from which the tissue came, but also into F, hybrids The latter was the case. In other words, on the basis of transplantation experiments, Law had excellent evidence that the leukemic cells originated not from the thymic graft, derned from the high-leukemia strain, but from the Fi, hybrid host. Histologic examination revealed that the grafted thymus was reconstituted by infiltration of cells from the host. Although it did not become leukemic, the grafted thymus induced leukemic transformation of host lym- ·LTIOLOGY OF LEUKEMIA 33 phocytes which had invaded the graſt. In other wordi, the grafted chymus ol the high-leukeria train represented a leukemia-promoting environment. Kaplan and a socialcy" found that thymectomized mice do not de clop leukemia following irradiation because the thymus has been extirpated. If the thymus is grafted into these animals after irradiation, then leukemia de elope, indicating that the thymus responds to some secondary factor following x-ir. radiation. This demonstrates that expause of the thymus itself to x-rays is not necessary for the induction of leukemia. Approximately 80 per cent of mice of the 258 strain des clop leukemia spontancourly. If the animals are led methylcholanthrene, leukemia occurs no carlier, and the incidence is ultimately no higher than spontaneously. In other words, although the animals are susceptible to sponlaneous leukemia, they do nor respond to the lcuiemogenic action of the carcinogen. Susceptibility to the spontaneous des clopment of leukemia does not imply susccptibility to the leulemogenic action of the carcinogen. Although a low-leukemia strain, «Omne leukemia docs appear in DBA mice, more in the female than in the male animals. When this strain receive methylcholanthrene cither bs skin-painting or feeding, leukemia de clops. Eighteen skin-painungs were given over a period of sat seeks to a group of DB.A mice. When the dove of carcinogen was low. then thymectomy inhibited the induction of leulemma II, honeer, the dose of methylcholanthrene was doubled, then thymectomy did not inhibit Scukemon genesis. It would appear that the thymus is preferentially susceptible to the leuiemogenic action of methylcholanthrene, but other lymphord tisuces quite «usceptible when large dove are given. If a strain of mice is susceptible to the independent Icukemogenic actiary of certain agents, and if these agents are given in combination, leukemia appears carlier and in higher incidence. When whole-body. T-irradiation, 200 r. was supplemented with 5 ug etradiol dipropionate estrogen, weekly after s-irradation, leukemia appeared carlier in Balb c mice, and the incident was higher than if the agents were administered independently. The tkimus was che primary site of response To demonstrate the pecificity of this sydcrgism. mure of this strain were gusen methylcholanthrene Out of 535 mice, only 6 de claped thymomas, whereas 18 of 48 mice which recensed the t-rans plus estrogen des eloped thymomas Age has a stnking influence on susceptibility to leukemogenic action vost trains are susceptible to leukemogenic agents only if they are treated early in life In the high-leukemia C38 strain, we see that leukemia appears carlier in female than in male mice, but the ulumare incidence is not much different Three different conces were made between the high-leukemia C58 strain and thrre low-leukemus trains, the CBS, C57, and SH, all of which are low-leulemia straits The leukemias appeared in female mice carlier than in male mile in all crosses. The age of occurrence of leukemia in hybrids is much later ·34 ETIOLOGY AND TREATMENT OF LEUKEMIA than in the pure C58 stock. The F, hybrids have a longer life expectancy and develop Icukemia late in life. In reciprocal crosses between the C58 and NH strains, leukemia appears later in life if NH female rather than male mice are used. in our laboratory, the work of Woolley on effects of cortisone on leukemogenesis has been confirmed. High-leukemia C58 nice werc given large doses of cortisone at monthly intervals for several months beginning at 2 months of age. Although the ultimate incidence of spontaneous leukemia was the same, leukemia appeared significantly later in life Cortisone inhibited induction of leukemia by methylcholanthrene MacDowell found that iſ C58 female mice were foster-nursed by animals of the StoLi strain, their life expectancy was greater Not only did these animals live longer, but ako the incidence of leukemia was lower. A factor in the breast milk reduced the incidence of leukemia We think of carcinogens as initiating leukemogenesis. We do not think usually of carcinogens as having any effect upon the host. If the Gardner lymphosarcoma of C3H origin is transplanted within this strain, growth is progressive in 100 per cent of the recipients. If, however, this lymphosarcoma of C3H origin is grafted into a foreign strain, DBA, there is temporary growth with regression, and then the animals resist transplantation. We have done this as many as eleven successive times. After each inoculation of lymphosarcoma tissue, there is absolutely no growth The animals appear to be permanently immune. If immune mice were skin-painted with methylcholanthrene, and then challenged by tumor inoculation, growth was progressive in one-third of the animals in methylcholanthrone-treated DBA mice, the progressively growing tumor transplants as it did originally, indicating that there is no change in the tumor itself, but more likely a change in the host as a result of treatment with methylcholanthrene. In summary, we can say that genetic factors influence the deselopment of spontaneous and induced mouse leukemia. Leukemogenic agents include ionizing radiation, chemical carcinogens, and estrogenic hormone Certain influences, for example, administered cortisone, may modify the growth of leukemic cells, although initiation of the disease is not inhibited Other factors actually may inhibit leukemogenesis (caloric restriction, administered androgen, thymectomy) After gro DISCUSSION DR BURDETTE: Dr. Sprague, would you comment on similarities between leukemias in mice and leukemias in man? DR. SPRAGUE I thought there was striking similarity between the morphology of leukemia in these animals and what we see in man This seemed to ·ETIOLOGY OF LEUKEMIA 35 be particularly truc of the acute lymphocytic and chronic granulocytic leukemia that Dr. Kirchbaum howed. DR. BURCHEXAL: That was strongly evident. Was the leukemia shown the type tending to respond to arcnic DR. KIRSCHRAUM: Yes, if we transplant chronic granulocytic leukemia and administer arenite, in some instances we may actually present the growth of the transplanted cells Dr. BURCHIENAL: In other words, one might say morphologically and biochemically it resembles the granulocytic strain in man. Arsenic would affect the chronic discasc in man as well DR KIRSCHBAUM: That is right. Dr. Tull: Did I understand you to say that transplanting this chronic granuloleukemic disease turned it into acute leukemia? Dr KIRSCHBAUM: That is the impression we had, judging from the morphology. In certain instances, there was a shift to the left just prior to death but not invariably. It is true, too, that if we take one of these chronic leukemias and transplant it many times so that it becomes more acute, then there is a progressive shift to the left with less and less differentiation on passage through multiple hosts DR SCHLOSSER Do androgens have a similar effect? DR KIRSCHBAUM In the case of methylcholanthrenc-induced leukemia in DBA mice, the administration of androgen results in a decreased incidence of induced leukemia, and castration has been demonstrated in AKR mice to increase the incidence of spontaneous leukemia We also have endence that the incidence is higher in females than in males I would like to mention the work of Kaplan and of Gardner on radiation-induced leukemia The administration of androgen presents induction At least, there is a markedly lower incidence in irradiated animals that allo received androgen, indicating that androgen is antileukemic. One thing that docs disturb me is that I am not sure we have been as careful as we should have been in controllir the weight of these animals In all this therapy one wonders about debilitating effects, whether caloric restriction and inanition resulting from the administration of these agents is at least contributing toward the effect that is obtained In cortisone-treated animals we have observed some decline in weight after the administration of these rather large doses, although the ultimate weight may not be altered very much Perhaps Dr Woolley would like to comment on this. DR WOOLLEY Using the strain AKR, we found that there was a depression in weight after administration of cortisone which lasted almost two weeks, followed by a gradual gain in weight until the time of the next monthly treatment, when the animals were up to full weight If one prepared a curve of weights immediately before therapy, there was no depression If one prepared a curve of weights soon after treatment, then there was a depression in hods weight.

·34 ETIOLOGY AND TREATMENT OF LEUKEMIA than in the pure C58 stock. The Fi hybrids have a longer life expectancy and develop leukemia late in life In reciprocal crosses between the C58 and NH strains, Icukemia appears later in life if NH female rather than malc mice are used, In our laboratory, the work of Woolley on cffects of cortisone on leu. kemogenesis has been confirmed. High-leukemia C58 mice were given large doses of cortisone at month); intervals for several months beginning at 2 months of age. Although the ultimate incidence of spontaneous leukemia was the same, leukemia appeared significantly later in life. Cortisone inhibited induction of leukemia by methylcholanthrene. MacDowell found that if C58 female mice were foster-nursed by animals of the StoLi strain, their life expectancy was greater. Not only did these animals live longer, but also the incidence of leukemia was lower. A factor in the breast milk reduced the incidence of Icukemia. We think of carcinogens as initiating leukemogenesis. We do not think usually of carcinogens as having any cffect upon the host If the Gardner lymphosarcoma af C3H origin is transplanted within this strain, growth is progressive in 100 per cent of the recipients. If, however, this lymphosarcoma of C3H origin is grafted into a foreign strain, DBA, there is temporary growth with regression, and then the animals resist transplantation We have done this as many as eleven successive times After each inoculation of lymphosarcoma tissue, there is absolutely no growth. The animals appear to be permanently immune If immune mice were skin-painted with methylcholarthrene, and then challenged by tumor inoculation, growth was progressive in one-third of the animals. After growth in methylcholanthrene-treated DBA mice, the progressively growing tumor transplants as it did originally, indicating that there is no change in the tumor itself, but more likely a change in the host as a result of treatment with methylcholanthrenc In summary, we can say that genetic factors influence the development of spontaneous and induced mouse leukemia Leukemogenic agents include 1onizing radiation, chemical carcinogens, and estrogenic hormone Certain infuences, for example, administered cortisone, may modify the growth of leukemic cells, although initiation of the disease is not inhibited Other factors actually may inhibit leukemogenesis (caloric restriction, administered androgen, thymectomy). DISCUSSION Dr. BURDETTE. Dr Sprague, would you comment on similarities between leukemias in mice and leukemias in inan? DR. SPRACUE: I thought there was striking similarity between the morphology of leukemia in these animals and what we see in man. This seemed to ·ETIOLOGY OF LEUKEMIA 37 agents. For example, the right combination of cortisone and x-irradiation will alter natural resistance of any species. Dr. Kirschbaum, do you consider the agents, steroid hormones and x-irradiation, provocative agents or causatis e agents of leukemia? If the various types of leukemia are distincth different diseases, one could suspect separable causative agents. DR. KIRSCHBAUBI: In a single inbred strain of mice, granulocytic and Ivmphocytic leukemia may appcar, One mas be seeing different manifestations of the same discase with the same provocative agent, or this may represent a strain susceptible to the induction of leukemia by different agents, perhaps different viruses. Granulocytic leukemia, chronic in FB mice, never appears before 400 days of age; by treatment with carcinogenic hydrocarbons, the discase will appear earlier, It so happens that this strain is susceptible to the spontancous disease and also susceptible to carcinogenic induction. do not know what effect estrogenic hormone or x-rays would have. Is that what you have in mind. DR. SYVERTOV: The experiences you citc are examples of extrinsic agents influencing innate resistance. Precisely what takes place when methucholanthrene, x-irradiation, or other extrinsic factor is operative is not known, but cach acts as a prolocative carcinogenic agent. Dr. KIRSCHBAUM: At this time I would like to make a distinction between transplanted and spontaneous leukema. Dr. Syverton has rnentioned resistance; he implied that this resistance is something directed both against the transplanted and spontaneous disease, and that x-rays and consone will facilitate or permit the growth of human neoplasms in animals Also, I know that Dr. Burchenal is transplanting leukemia of other species into hamsters. To me, the important biological characteristic of Icukemia is its progressive growth. However, the sporttaneous discase differs from the transplanted disease in that one can immunize against transplanted, but not against spontaneous disease. I suspect that spontaneous mouse leukemia is more the human leukemia than human leukemic cells growing in a hamster DR. BURCKENAL. In bamsters se have never succeeded in growing leukemic cells that we were sure were human, leukemic cells agree that spon. taneous leukemia in the mouse or in any other animal is much more like spontancous leukemia in man than transplanted leukemia, but, as Dr Kirschbaum knons, it is easier to work with transplanted leukemia for certain technical rea- Such studies are usually done with the hope that an analogy between the behavior of transplanted leukemia in the mouse or in any other mammal and human spontancous disease may be found. A great deal of my interest is in acute leukemia and transplanted Icukemjas As Dr. Kirschbaum has mentioned, no matter how chronic it may be in the mouse, the spontaneous disease usually tends to become acute after a few transplant generations, and there has been some similants between transplanted leukemias. sons ·36 ETIOLOGY AND TREATMENT OF LEUKCALIA DR. KIRSCHBAUM: Have you depressed the body weight by restricting caloric intake? I think this should be done, and we have not donc it. Dr. WOOLLEY: We have not done that cither. You will recall that Dr. Furth performed a caloric-restriction experiment, and caloric restriction prolonged the time of appearance of Icukemia Alst, the ultimate incidence of leuhemia was probably reduced by caloric restriction alone. DR KIRSCHBAUM: Of course, there is some difference in reducing the body weight of animals by one-third temporarily and keeping them at that level. Here we have a situation where there are fluctuations in body weight, depression after cortisone, and return to what is considered to be a normal weight for the strain. Dr. WOOLLEY: It seems to me the hormonal factor can be separated quite easily. Testosterone, a protein anabolic substance, has been used by us (unpublished data) to reduce the incidence of leukemia in the high-leukemia strain, AKR. DR. KIRSCHBAUM: Another argument against the caloric factor is that animals treated with estrogenic hormone may look debilitated, but still develop leu. kemia. Dr. WOOLLEY. I agree. Results must be compared experiment by experiment and strain by strain. In our experience, the incidence of leukemia in the susceptible, AKR strain has been reduced, although an increased incidence of leukemia in resistant animals has been reported. ACTH is a substance which presumably acts by means of cortisone production, causing lymphoid tissue destruction, Dr SPRACUE. In acute leukemia, I would like to ask what percentage of these animals show that change, and if a similar change is seen following treatment with either arsenic or urethan DR KIRSCHBAUM I cannot give you any figures, but the important thing is that there is a relationship between chronic and acute leukemia. Invariably, these chronic granulocytic leukemias become acute if thev are transplanted many times Dr. BURDETTE Dr. Syverton, will vou comment on the action of leukemogens in relation to host resistance? Dr. SYVERTON. Among factors which influence disease irrespective of one type are genetic make-up, age of the host, and hormonal balance These intrinsic factors determine natural, innate resistance as opposed to natural, innate susceptibility. For example, poliomyelitis and measles viruses are highly infectious for man, whereas an enormous amount of these viruses can be administered to other species, such as dog or rat, without effect Yet the clinical manifestations of both diseases in man are influenced by age, genetics, and hormones Why is an occasional person naturally resistant? Natural resistance for man and other species is subject to experimental manipulation by using a variety of provocative ·Ludwik Gross, AID) INFECTIOUS CONCEPT O LEUKEMIA (Based on Recent Experiments on Coll-Fre Transmission of Leukemian Afia* 1, 39-1 It has been suspected for many sears that leukemia developing "spontaneously" in sanous species, including human beings, may be caused by transmissible viruses. Thus far, however, this assumption can be confirmed experimentally in two species only, namely, in chickens and, recently, also in mice Ellermann and Bang (1908)?? were the first to determine that chicken leukemia could be transmitted by inoculation of filtered extracts The viral nature of mouse leukernia was revealed in our laboratory several years ago Filtered extracts prepared from leukemic mice of high-leukemic strains, such as AK or C58. nere inoculated into newborn mice of strains C3H, C3H (1), or C57 brown," which are, for all practical purposes, free from leukemia More than 30 per cent of inoculated mice de cloped typical generalized leukemia after a latent penod vaning from a few months to one and one-half years (Fig 1) These resulty now have been confirmed by Woolles and Small" at the Sloan-Kettenng Institute. In our experiments, the leukemic agent prosed to be highly specific, requinng particular strains of mice for inoculation Thus, the Bittner substrain of the C3H line proved to be susceptible, whereas the closely related National Cancer Institute substrain of the same inbred line prosed to be substantially more resistant *Aided in part by a grant from the Damon Runyon Memonal Fund 39 ·38 ETIOLOGY AND TREATMENT OF LEUKEMIA With regard to Dr. Syverton's question, Dr Furth tricd to obtain myeloid leukemias in the AK strain by performing thymectomy, followed by irradiation. His idea was that since most of the leukemias were lymphatic, usually thymomas in that strain, the incidence of these leukemias was reduced markedly, allowing the animals to live to an age when they might develop the granulocytic type of leukemia if one removed the thymus. DR. KIRSCHBAUA. I am not sure about the nature of the induction. I do know thymectomy has prevented the development of lymphocytic leukemia and the mice lived to develop granulocytic leukemia, which suggests the genesis of the granulocytic and lymphocytic drease is different, also the primary locus is in the thymius in only one of the two Dr SYVERTON: In their discussion, Drs Burchenal and Kirschbaum illustrated the influence of extrinsic and intrinsic factors upon the host As they point out, a given host under influence by one or several modifying factors can show widely variable manifestations of its disease Dr BURCHENAL The other point be brought out is that first or second generation transplants from spontaneous leukemia probably are much closer to the spontaneous form than those transplanted for several generations. Would you agree? Dr. KIRSCHBAUM I would indeed MacDowell, in classical experiments, demonstrated that one can immunize by injection of normal cells of certain genetic type This immunization is against transplanted leukemia, but not against spontaneous leukemia nor from the cells of a spontaneous case In other words, one cannot immunize against the first transplantation of leukemic cells so that the first transfer generation would apparently be more like spontaneous leukemia than is the usual transplanted disease DR BURDETTE Dr Gross, you speak frequently about the relation of viruses to the etiology of leukemia Would you comment on the maternal resistance factor in milk which Dr Kirschbaum mentioned? Do you regard it as a virus? DR LUDWIK GROSS I do not have enough information on this factor. I would like to comment on the difference between spontaneous and transplanted leukemia First, I would like to question the terminology ''spontaneous" because, of the work which we are doing is coriect, there is no such thing as spontaneous leukernia in mice Spontaneous smallpox or spontaneous measles could then be compared to spontaneous leukemia What we call spontaneous would be leukemia naturally induced DR. KIRSCHBAUM This is purels a question of terminology If there is a virus which is responsible for the development of leukemia in mice without man's intervention, then virus leukemia is spontaneous leukemia ·INTECTIOUS CONCEPT OF LEUKEMIA 41 *. Fig 1--Leukemic infiltration of liser This C57 brown/cd female, No 173, Exp 1 Irina ·40 ETIOLOGY AND TREATMENT OF LEUKEMIA TABLET INOCULATION OF FILTERED (SELAS OR BERKEFELD) AK LEUKEMIC EXTRACTS INTO NEWBORY C3H OR C3H (1) Mice of Two SUBSTRAINS FRESH EXTRACTS NUMBER NUMBER NUMBER DEVELOPED INOCU DEVELOPED PAROTID SUBSTRAIN LATED LEUKEMIA TUMORS* Bittner C3H 138 32 (23%) 24 Bittner C3H (F) 182 58 1327) 9 Bittner total 320 90 (286) 33 NCI C3H (1) 162 74%) *Average age, months leukemia. 11, parotid tumor, 5 HEATED EXTRACTS (65° TO 68° c 72 HR ) NUMBER NUMBER DEVELOPED NUMBER DEVELOPED PAROTID INOCULATED LEUKEMIA TUMORS 73 1 93 4 166 5 45 0 ONNO 9 Recent experiments carried out in our laboratory, utilizing electron microscopy, ultracentrifugation, and filtration through gradocol membranes, suggest that the mouse leukemic agent is a particle having a diameter less than 100 mu 45 TABLE II BASIC DIFFERENCES BETWEEN LEUKEMIA TRANSPLANTED (BY CELL GRAFT) AND INDUCED (BY INOCULATION OF CELL-FREE* CXTRACTS) TION RESULTS OF INOCULATION INTO MICE OF SUSCEPTIBLE LEUKEMO- FOREIGV STRAIN GENIC RESULTING THE SAME (c3H OR 057 INCUBA TUMOR AT LEUKEMIA STRAIN (AK) BROWN) SITE OF CAN BE NEW- NEW- TIME INOCULA TRANS- BORN ADULT BORN ADULT MOVTHS TION PLANTED TO Transplantation Positive Positive Positive Negative to 1 Usually Donor of leukemic present strain cells Inoculation of ?+ Negative Positive Negatise 3 to 24 Absent Recipient cell-free* AK strain leukemic agent *Centrifuged 7,000 g or filtered Posibly accelerated development of spontaneous leukemia Sufficient experimental information not yet available TABLE III TRANSPLANTATION OF LEUKEMIC CELL SUSPENSIONS (FROM SPONTANEOUS, TRANSPLANTED, OR INDUCED LEUKEMIA INTO ADULT MICE OF AK AND C3H STRAINS) AK RESULTS OF INOCULATION OF LEUKEMIC DONOR ADULT RECIPIENT MICE DONOR ORICIN OF DO VOR STRAIN AK STRAIN c3H Spontaneous Positive Negative C3H Inoculation of AK leukemic cells* Positive Negative CH Inoculation of cell-free AK agentt! Negative Positive *AK leukemic cells gralted into newborn C3F muce cause Jeukemja in two to three werks Following inoculation of cell-fier AK extracts into newborn C3H mie, leukemia develops after three to thenty four months Among the C3H or C3H (f) mice that have been inoculated in our laboratory with filtered leukemic extracts, some developed tumors of the parotid glands and/or subcutaneous fibromyxosarcomas instead of leukemia (Figs 2 and 3) ·INTICTIOUS CONCEPT OF LEUKEMIA Fıs 9 -- This C311 (1) female, No 156 Exp 261+-L was inorulated when less than 5 hours old with a filtered (Selas 03) AK leukemic extract As a result, this touse developed a subcutaneous spindle cell sarcoma in the right groin at 18 5 months of age (Hematorylın and eosin magnification *300 ·42 ETIOLOGY AND TREATMENT OF LEUKEMIA Fig 2-Parotid gland tumor This C3H male, No 363, Exp 2709-C was inoculated when less than 15 hours old with a centnfuged (7,000 * ) AK leukernic extract, diluted 1 100 As a result, this mouse developed bilateral parotid gland tumors at 45 months of age (Hematoxylın and eosin magnification, *315 ) ·INFECTIOUS CONCEPT OF LEUKEMIA 45 cxtracts prepared from mouse leukemic tissues were inoculated into newborn micc of susceptiblc strains, conceivably leukemic cells contain ses cral distinct oncogenic agents in certain forms of leukemia in mice. The fact that mixing the leukemic extract with ether" destroyed Icukemogenic potency, Icaving unchanged the extract's ability to inducc parotid tuniors or sarcomas, is consistent with the assumption that the leukemic extract contained scicral oncogenic agents and that the leukemiogenic agent in such an extract was more labile and TABLE VII INOCULATION OF ETHER-TREATED LEUKEMIC EXTRACTS Isto XEM BOR C311 OR C311 (1) MICE UMBER OF SUMBER OF BEICP EXPERIMENTA IYOCULATED 6 53 Chron c form of leukemia NUMBER DEVEL- OPED LEUXF3174 1. UBFR DEVEL- OD PAROTID TUMOR 6 NUSTAFR DEVEL- OPEP SARCOMA 2 thereforc destroyed by the action of cther in vitro. Should the latter assumption prove to be correct, the presence of ses cral oncogenic agents in the leu- Lemic extract may not be due to a mere accident alone. Whatever the theoretical interpretation, the fact remains unchanged that leukemia in mice of either the AK or C58 high-leukemic lines is caused by a filterable and thermolabile agent which is transmitted under natural conditions of life from one generation to another directly through the embryo Mouse Sculemia, like chicken lymphomatosis, appears, therefore, to be an egg-borne vrus discase DISCUSSION DR. KIRSCHBAUM Dr Gross, you cmphasize the fact that the wrus must be introduced soon after birth of Iculcmogenesis is to proceed How do you reconcile this with the fact that leukemia may be induced in mature animals of low-leukemia stocks by the administration of carcinogen X-rays, or estrogenic hormones? In those situations, do you believe there is a virus available which mediates the action of the carcinogen' Dr. GROSS It is very difficult to answer the question I suspect that the leukemic agent is widespread, perfectly adapted in the host, causing no symptoms unless activated by such factors as x-rays or carcinogens DR. BURCHENAL' Dr Gross, sou say that you think this virus is probably widespread In what other strains of mice have you found it besides the AK and C58? Has it becn demonstrated in other lines of leukemic or nonleukemic mice? DR GROSS Thus far I has been able to recover the agent only from AK and C58 mice DR. BURCHENAL, Hase you tried DBA mice? For instance, the DBA strain has a fairly low incidence of leukemia, but leukemia is readily induced with ·44 CTIOLOGY AND TREATMENT OF LEUKEMJA When cell-free, centrifuged or filtered extracts then were prepared from the induced parotid tumors or subcutaneous sarcomas and inoculated into newborn C3H micc, cither parotid tumors, subcutaneous sarcomas, or leukemia resulted. This immediately raised the question whether filtered extracts prepared from leukemic AK or C58 donors contain a single agent capable of causing different types of tumors, or whether they contain a mixture of oncogenic agents. TAHLE IV INOCULATION OF CELL-Free EXTRACTS FROM INDUCED C3H LEUKEMIAS INTO NEWBORN C3H AND C3H1 (f) MICE NUMBER OF NUMBER DEVELOPED NUMBER DEVELOPED NUMBER DEVELOPED NUMBER OF MICE Four developed spindle-cell sarcomas also TABLE V INOCULATION OF CELL-FREE EXTRACTS FROM INDUCED PAROTID TUMORS INTO NEWBORN C3H AND C3H (1) Mice NUMBER NUMBER NUMBER NUMBER OF NUMBER OF MICE DEVELOPED DEVELOPED DEVELOPED EXPERIMENTS INOCULATED LEUKEMIA PAROTID TUMOR SARCOMA 38 300 49 237 28 *Average are, months leukemia 14 parood tumor, 6, sarcoma 15 of these, 6 developed parotid tumors and spindle-cell sarcomas, 2 descloped parotad, submarillary, and adrenal tumors TABLE VI RESULTS OF INOCULATION OF CELL-FREE SARCOMA EXTRACTS INTO NEW BORN C3H1 OR C311 (1) MICE NUMBER OF EXTRACTS PREPARED 36 NUMBER OF MICE INOCU- LATED 316 NUMBER OF MICE DEVFI - OPED LEU KEMIA 8 NUMBER OF MICE DEVEL- OPFD PAROTID TUMORS 6 NUMBER OF MICE DEVEI - OPED SAR- COMAS 9 AVERAGE AGE LEUKEMIA OR TUMORS DE- VELOPED (soiris) 7 It is conceivable that a single oncogenic agent can induce different types of tumors as well as leukemia On the other hand, it is also possible to assume that leukemia or certain other neoplasms contain several distinct oncogenic agents Only one agent may be pathogenic at a given time, even though the others also may be present in the same tumor This would explain why parotid tumors or sarcomas could be induced with leukemic extracts, and why, when Graffi and his co-workers prepared filtered cell-free extracts from certain transplanted mouse tumors, such as the Ehrlich carcinoma, and inoculated them into newborn mice, leukemia resulted Since tumors other than leukemia, such as parotid carcinomas or subcutaneous fibromyxosarcomas, ,46 have been induced consistently when cell-free ·INTECTIOUS CONCEPT or LCUKOMA 45 cxtracts prepared from mouse Icukemic tissues were inoculated into newborn mice of susceptible strains, conceivably leukemic cells contain se cral distinct oncogenic agents in certain forms of Icukemia in mice. The fact that mixing the Iculcmic extract with ether" destroyed Icukcmogenic potency, leaving unchanged the extract's ability to induce parotid tumors or sarcomas, is consiste ent with the assumption that the Icukemic extract contained ses cral oncogenic agents and that the Icukcmogenic agent in such an extract was morc labile and Tagle VII IVOCL LATION OF CTHER-TREATER LELKEMIC EXTRACTS Into NEWBORN C311 OR C311 (i) VICE NUMAR DFIEL- OPED LFCREMIA SUMBER DEYEL- OPED PAROTID TUMOR VUBTR OF SUMBER OY MICE EXPERIMENTS IVOCL LATID 6 53 Chronic form of leukemia VUHRER DEVEL- OPZD SARCOMA 2 therefore destroyed by the action of cther in vitro. Should the latter assumption prove to be correct, the presence of several oncogenic agents in the Icu. kenic extract may not be due to a more accident alone Whatever the theoreti. cal interpretation, the fact remains unchanged that leukemia in mice of either the AK or C58 high-leukemic lines is caused by a filterable and thermolabile agent which is transmitted under natural conditions of life from one generation to another directly through the embryo Mousc leulcmia, like chicken lymphomatosis, appears, therefore, to be an egg-bornc virus disease DISCUSSION DR. KIRSCHBAUM: Dr. Gros, sou emphasize the fact that the virus must be introduced soon after birth of leukemogenesis is to proceed How do you reconcile this with the fact that leukemia mas be induced in mature animals of low-lcukemia stocks by the administration of carcinogens, X-rays, or cstrogenic hormoncs? In those situations, do you believe there is a virus available which mediates the action of the carcinogen? Dr Gross It is very difficult to answer this question I suspect that the leukemic agent is widespread, perfectly adapted in the host, causing no symptoms unless activated by such factors as x-rays or carcinogens DR. BURCHIENAL. Dr. Gross, sou say that you think this virus is probably widespread In what other strains of mice have you found it besides the AK and C58? Has it been demonstrated in other lines of leukemic or nonleukemic mice? DR GROSS Thus far I have been able to recover the agent only from AK and 658 mice. De BLRCHEVAL. Have you tried DBA mice? For instance, the DBA strain has a fairly low incidence of leukemia, but leukemia is readily induced with ·46 ETIOLOGY AND TREATMENT OF LEUKEMIA methyicholanthrene. Therefore, if the virus must be present for the induction of leukemia by a physical or chemical agent, it should appear in the strain, On the other hand, if the virus is only an additional carcinogen such as irradiation or methylcholanthrene, any one without the other would work Your hypothesis could thus be tested very well if you could demonstrate there is a vírus present in DBA mice Dr. Gross: I have not performed experiments with DBA mice. Dr. BURCHENAL: I wonder whether leukemogenesis is dependent always on the presence of the virus, or can onc agent cause it without the other: methylcholanthrene without the presence of virus, exposure to X-rays without the prescnce of virus, or the virus without the presence of, shall we say, cosmic rays or Konie other factor. Dr. Gross. Much additional work is required before this can be answered adequately. Dr SYVERTON: You referred several times to the use of temperature for inactuation Have you attempted to use graded temperatures for differential separation to learn whether you are dealing with one agent or three distinct agents? It seems reasonable that multiple agents should be separable by a temperature differential for inactivation. Dr Gross. Yes Such studies are in progress Dr. SYVERTON: Do I understand that a temperature of 80° is required to inactivate all three viruses or agents? DR Gross. Almost complete inactivation is obtained by heating to 650- 68° C for thirty minutes However, parotid rumors occasionally may be induced with heated extracts DR. KIRSCHBAUM Although you did not mention it today, Dr. Gross, you have taken adult C3H tissue, that is, tissue from a low-tumor strain and made extracts of that adult tissue, injected it into the newborn of the same strain, and obtained parotid tumors How do you explain this on the basis of viral ctiolog? A second question is whether parotid tumors can be induced by the administration of cortisone alone DR GROSS It is true that, in a small number of animals, tumors may be induced by injecting C3H mice with extracts from normal C3H or normal 057 brown mice I assume that the parotid tumor agent is present in many strains of However, it is probably so adapted in the host that it causes no disease in the carriers If cortisone is applied, one may occasionally induce parotid tumors in such hosts Following a blind transfer into newbom mice, one can occasionally also induce such tumors DR KIRSCHBAUME I am still curious If you can obtain parotid tumors by injecting adult C3H extracts, and if you feel these tumors appear only because you introduce the agent into the new born, how can the agent be there? The animal does not develop the tumor unless you provide the agent. These are animals mice ·INFECTIOUS CONCEPT OF LEUKEMIA 47 that have not been injected with the agent, and yet you are able to extract from their tissue something which induces the neoplasm. It would seem to me that the clement of nonspecificity cnters here. Do you really have reason to believe thus tissue has the agent? DR. Gross: Dr. Su crton, would you care to comment on this? DR. SYVERTOV: The problem as presented suggests that Dr. Gross is emploving an approach which has been used not uncommonly to demonstrate the presence of a latent agent. That approach is to use a provocative injection or implantation of foreign material, such as ticcuc, starch, or chemical, to lower host resistance to a point where overt evidence of cell injury by the infectious agent is shown by clinical disease. Actually, I am arguing both for and against Dr. Gross's point of view here. For example, if the agent responsible for any one or more of the thrce tumors vou describe were present as an indigenous, latent, virus infection, any inoculation may have served as provocative agent DR Gross: I assume that one can induce tumors in mice by blind passage Now, in the case of the parotid tumor agent in C3H mice, let us assume that many, perhap" all, C3H mice carri a small quantity of parotid tumor agent of low pathogenic potential Such low potential will not induce parotid tumors spontaneously. Houcier, if one tahes C3H normal tissue containing such an agent and injects it into a new born mouse, its potential may be sufficiently incrcased to induce the disease in the injected host. Dr. BURDETTE. Are you saying, Dr Gross, that this is a quantitative and not a specific phenomenon--that the agent exists in all your animals, and when jou inoculate them with extract, you are nierely increasing their quantıy? DR GROSS You increase the pathogenic potential of the agent. DR BURDETTE Would you define "pathogenic potential"? DR. GROSS: By passing surus from one host to another, potency to induce di case is very often increased Dr. BURCHEXAL. Does not such passage result in the enhancement of the virulence of the virus, presupposing recipient and donor are similar DR. SYVERTON There are several factors DR KIRSCHBAUM C3H mice, according to our hypothesis, develop neither leukemia nor parotid tumors because they do not have the agent Thes develop these diseases if sou introduce the agent from another strain at birth The thing I do not understand is how you can obtain this agent for inducing parotid tumors from adult C3H mice It would seem to me if such an agent for parotid tumors can be obtained similarly, vou might obtain an agent for leukemia DR WOOLLEY With the permission of Dr Gross I would like to discuss We have used cortisone for chronic treatment of strain C3H mice and obtained parotid gland tumors Both C3H mice from the National Cancer Institute and C3H mice from Dr Gross's laborators were treated with controne In the latter case, we secured a few parotid gland tumors, and we did not obtain this matter ·18 ETIOLOGY AND TREATMENT OF LEUKEMIA these tumors in the former. Apparently, the Bittner (Gross) subline is carrying a small amount of parotid tumor virus, and the other one, the NCI sublinc, is not. The amount of virus carried without concentration by inoculation into the newborn is probably not enough to lead to expression by tumorigenesis unless corti- Sone is used. Dr. BURDETTE: In any discussion of the interrelation between genetic susceptibility and the presence of viral particles within the cell, careful distinction must be made between the two. Attention so far has been focused on the existence and dosage of such particles within normal cells. Dr. WOOLLEY: Yes, some clarification is in order. Dr. Gross has just reported induction of leukemia in strains usually having a low incidence Perhaps the strain with low incidence will not casily support the leukemia virus gencration after generation. Otherwise, it would be a strain with high incidence. An example of this would come from studies on the milk factor utilizing a strain with a low incidence of breast cancer, such as the C57 black stock which cannot support the milk factor virus generation after generation, at least in a high enough concentration to lead to mammary cancer DR. KIRSCHBAUM. Dr. Woolley, !ou are implying that the virus is ubiquitous, present even in thc C3H strain without being introduced from a foreign strain, so some nonspecific factor, such as cortisone or something which is responsible for the secretion of adrenal cortical hormone, wall sinilarly induce the development of parotid rumors. DR. WOOLLEY: In the first place, one must further define strain C3H, Sublines may differ in character considerably. Skeletal variation within the Strain is one example De KIRSCHBAUM: Do C3H mice not develop parotid tumors unless injected with extracts at birth or unless they obtain corrisone? DR WOOLLEY I do not know whether this is due to a resistance factor or The fact is that in this particular experiment, where cortisone was gn en periodicalh throughout life, parotid gland tumors occurred in one subline and not in the other DR KIRSCHEAUX If the virus is there, how important is its introduction at birth? Is this not a rather redundant procedure? DR, GROSS It is very important DR BURDETTE If ny interpretation of the opinions of the participants is correct, most of the panel seems willing to accept the evidence that a virus is one of the factors which can be important in the etiology of leukemia in mice, but several members are still not quite convinced that this is always an essential facfor requisite for the development of leukemia not ·George Il' Woolley, PhD FILTERABLE AGENTS PREPARED from LEUKEMIC TISSUE CAUSING LEUKEMIA and OTHER CANCERS in MICE; During the past year, we have been investigating the relation of cell fractions prepared from leukemic tissues---centriſugates and filtrates to the occurrence of cancer in mice These and other studies have been undertaken to help resolve the leukemia problem into its component parts and thus make its control more precise. The methods of fractionation and injection have followed closely those des cloped by Gross." Extracts of 20 per cent concentration prepared with physiologic saline solution from leukemic ergans were centrifuged at 3,000 r.p m. for fifteen minutes, then at 9,500 rpm for ten minutes at 0° C. The supernatant fluid was used for inoculation or passed through a Selas 02 or 03 filter prior to use, All extracts were kept at 0o C and injected (005 ml ) subcu. taneously within forty-eight hours. Newborn mice were used for inoculations, and most animals were less than 12 hours old at the time of inoculation. Following weaning, the mire were aged without breeding and observed for tumor occurrence on The occurrence of tumors appeared to be modified by the type of prepara tion uscd and by the genetics of the hour receiving the preparation of 31 prep. *This investigation was supported par: by a research grant (CY-3781) from the National Cancer Institute of the National Institutes of Health, United States Public Health Service, and in part by & research grant from the Amentan Cancer Society (T-47) 49 ·50 ETIOLOGY AND TREATMENT OF LEUKEMIA arations (20 of AKn/Gs origin and 11 of C3H:/Gs origin), a total of 13 (6 AKn and 7 C3H) were active in inducing leukemias, tumors of the parotid gland, and sarcomas. The most active preparations were extracted from filtrate-induced tumors rather than from spontaneous tumors (Fig. 4). One such preparation, a filtrate, induced 4 leukemias out of 5 inoculated C3H/Bi mice (80 per cent) and 10 out of 17 C3H:/Gs mice (59 per cent). More tumors appeared in C3H/Gs and C3H/Bi hosts than in C3H,/Hu, C3H/Gs, and C3H/\ly hosts. Fig4 Section through liver of young (2 months) C3} [/B, mouse which recen cd 005 ml filtered leukemic tissu extract when newborn Note leukemic cell infiltration lymphocytic leukemia Use of a supernatant fluid which was diluted with an amount estimated to be 2 1 (2 parts of supernatant fluid to 1 part of salıne), after 7,000 g centrifugation, was followed by only parotid gland tumors and sarcomas-6 and 6. respectively-among 17 C3H, Gs injected and 1 sarcoma among C3H Gs mice injected. In some instances, parotid gland tumors and sarcomas occurred in the same individual, but neither appeared with leukema. Tumors arising in C3H mice were transplantable to other C3H mice but not to the strains of cxtract Origin-AKN, C58 and C57Br/ cd At the present stage of development, these studies confirm the new and basic findings by Gross that preparations such as those mentioned have cancer-inducing properties and that the induced tumors are of the recipient rather than of the donor type.

·FILTERABLE AGENTS PREPARED FROM LCUKEMIC TISSUC 51 GRANULOCYTIC DISEASE INDUCED BY FILTERABLE AGENT A second area of study in ok cs a filterable agent causing a malignant discase of the hematopoictic system thought to be granulocytic leukemia" (Fig. 5). Normally, this virus-induced discase can be transmitted rcadıly to adult individ. uaks of the Swiss strain but not to adult individuals of certain other strains, Recently, we haic becn able to transfer this disease to the resistant C3H/B. strain by means of inoculation of Selas porosity 03 filtrates into newborn C3H mice. Following this transfer, the disease has been pas-cd to both adult C3H,/B, and adult Swasi mice by both cell and cell-free filtrate material. Fig 5-Peroxidase reaction granules in stem cells in blood of Swiss mouse treated as adult with fitrate from spleen with "granulocytic leukemia" TREATMENT OF LEUKEMIC AND NONLEUKEMIC STRAINS WITH CORTISONE Another study whtch relates to the problem mentioned concerns the occurrence of parotid gland tumor in cortisone-treated mice of both leukemic and nonicukemic strains. In our first studies, we used strains AKR, C58, and a new strain developed by us following a cross between C58 and AKR Both C58 and AKR are high-leukemic strains which hase been studied intensively by many investigators In our study, treatment consisted only of Cortone administered subcutaneously (1 mg, per day) for three successive days once each month throughout life, starting when the mice were 1 month of age Following this treatment, no parotid tumors originated in strain AKR, but they ·52 ETIOLOGY AND TREATMENT OF LEUKEMIA Fug 6 --Parotid gland tumor in cortisone-treated female mouse descended from original mating of C58 AKR after inbreeding a few generations AKR cortisone-treated femail nouse Fig 7 - Section through parotid tumor of C58 (See Fig 6 ) ·FILTERABLE AGENTS PREPARED FROM LEUKEMIC TISSUS. 53 did originate in malc as well as in female mice of the other two strains A total of 6 were observed-2 in strain C58 and 4 in strain C58 AKR, or in 3.5 per cent and 80 per cent of the experimental animals, respectively. (Figs. 6 and 7) Nonc appeared in the control animals The ascrage age at autopsy with this type of tumor was 11 months. The tumors were, in most instances, first observed at a somewhat younger age More recently, these rare parotid gland tumors have been obscrved in our Cortone-treated C3H/Gs mice, but not in C3H,/Hu The results show that periodic treatment with Cortone has brought out a rare type of tumor in the mice mentioned, a type of tumor similar to that obsen ed following treatment of new born mice with Icukemic tissuc extracts and certain other cell-free preparations DISCUSSION DR SYVERTON: The significant experimental findings summarized by Drs. Woolley and Gross do not eliminate for a Sirologist the possibility that Dr. Gross's three tumors originated from three distinct agents which I would like to believe are viruses. Use of tissue transfer or cortisone could evoke a small percentage of tumors for a carrier strain of mice. Certain strains undoubtedly are free of tumor viruses, others may maintain viruses in the carrier state When a mouse is made highly susceptible from genetic background and hormonal inſluence, tuniors appear This sort of host-range response apples to all virus infections for example, the occurrence in mice of mammary tumors in from 1 to 5 per cent of a homozygous stock indicates to a virologist a latent or carrier infection with mouse mammary tumor virus X-irradiation, cortisone, tissue implantation, or almost anything can serve as a provocative agent. A most effectne means for crasıng genetic resistance and inducing receptivity is the purposeful use of cortisone and x-177adjauon singly or in combination These comments are all basic questions for Dr Woolles and Dr. Gross to answer. Dr. WOOLLEY“ Answering your last question first, thc C3H/An (National Cancer Institute) subline, which seems to be strain resistant to leukemia, may be taken as an example Leukemia has occurred only a few times in this subline and only from extracts which we considered very pocent. Perhaps with the addition of cortisone or X-rradiation an increased number of tumors will be found. DR SYVERTOV: That experiment should prove extremely interesting, particularly if cortisone or irradiation is employed We have learned, for example, that these two agents in combination can elicit evidence for a variety of infectious agents in hosts otherwise resistant. DR BURDETTE: In such an experiment, how does one separate alteration of the mechanism of immunity, direct action on the virus, and possible carcinogenic properties of the agent itself, such as irradiation ·S2 ETIOLOGY AND TREATMENT OF LEUKEMIA Fug 6 - Parotid gland tumor in cortisone-treated female mouse descended from orginal mating of C58 AKR after inbreeding a few generations Fig 7 - Section through parotid tumor of C58 * AKR cortisone-treated female mouse (See Fug 6) ·FILTERABLE AGENTS PREPARED FROM LEUKEMIC TISSUE 55 DR. KIRSCHBAUM: Vay I ask one more question? Have you injected cxtracts of embņo from strains other than the C58 or AKR, and, if so, what has happened? Dr. Gross: Nothing, other than the occasional appearance of parotid tuniors, a DR. SPRAGUE: I would like to ask Dr. Woolley a question concerning his interpretation of results with animals treated with cortisone. If cortisone is an agent which facilitates viral multiplication and at times actis ates latent viruses, one could anticipate that leukemia may occur at an earlier time or perhaps in higher percentage of such animal. Cortisone also has a striking lytic effect on lymphoid ti wuc, however, and perhap, this is the explanation of his results. I would appreciate his comments concerning this Dr. WOOLLEY: Cortionc is destructive and harmful to lymphoid tissue, whether it be normal or tumorous I would interpret the result as due to retarding or regressing influences on hmphaid tissue by cortisone. This is in contrast to findings for the parotid gland, where we know of no real effect of cortisone. Is that what you had in mind? DR. SPRAGCE. Yes Dr. BURDETTE. Dr. Woolles, do you have any results to indicate that the maternal resistance factor is an agent similar to these vral agents under dr-cussion, but with the opposite effect? De WoolLEY. I have no results DR. BURDETTE: Dr Syverton, would you comment on superinfection of viruses and competition between two separate particles of this type? DR. SYVERTON. When invasion or infection of cells is limited to two viruses, both present in adequate numbers, competition must occur. Failure or prevention of infection by one of these viruses is known as interference. This capacity of one virus to act as an interfering agent for a second virus is a temporal relationship limited to days Dr BURDETTE: Dr Woolley, would you comment on the incidence of leukemia among mice with mammary tumors, and vice versa? Dr. WOOLLEY The results which I presented, showing that leukemia did not occur with mamman tumors, may be interpreted as competition between tumor-inciting agents, or on the basis that the sublines had been separated for a number of generations Among our inbred, tumor races, we do not have leukemia competing with breast cancer, or vice versa These diseases of mice seem to be well separated by nature DR SYVERTON Is the inhibitor to which you refer labile and inactivated by heat? DR. WOOLLEY Yes ·54 ETIOLOGY AND TREATMENT OF LEUKEMIA Dr. SYVERTON: These remarks are in continuation of the cornments I just made. I assume that irradiation served as a provocative agent in contrast to the tumor-inciting agent or causative agent, virus or otherwise, already in the host. Activation can be effected by altering the host. For example, to cite our recent experiences, treatment of monkeys by use of irradiation and cortisone in combination revealed poliomyelitis virus in the vaccine responsible for the Cutter incident, while failure resulted from all other procedures for demonstration of polio virus. Dr. BURDETTE: In an ordinary type of viral infection, though, the result of the viral, infectious disease is not produced by either the cortisone or the irradiation DR SYVERTON. I did not make myself clear. Treatment with cortisone and x-Irradiation falls into two categories: (a) treatment to alter or abrogate natural host resistant to an infectious agent and (b) treatment for relcase of virus already present in cells. It would seem that the demonstration of tumorprovoking agent(s) in the C3H Bittner subline could be in this last category. DR KIRSCHBAUM: Has leukemia appeared in C3H mice that have been injected with parotid tumor extracts from nonleukemic mice? Dr Gross: No. Dr. WOOLLEY I have not had this experience either. DR. KIRSCHBAUM: Has leukemia appeared in C3H mice that have been injected with normal, enbryonic tissue of high-leukemic strains? DR. Gross. These extracts were prepared from mice of leukemic strains DR. KIRSCHBAUM Leukemia has also appeared, according to the work of Graffi, in mice which have been injected at birth with extracts of the Ehrlich, ascites tumor Parotid tumors have appeared in C3H mice injected at 1 day of age with normal adult C3H tissue Now, the question I would like to raise is this Has leukemia appeared in C3H mice injected at 1 day of age with normal adult C3H tissue? Has that experiment been done? Furthermore, has leukemia appeared in C3H 1-day-old mice injected with normal nonleukemic tissue of other strains? That, I think, is an extremely critical control. Dr GROSS The only manifestation of neoplastic condition produced in C3H mice by injection of normal tissues was that of development of parotid tumor. Neither leukemia nor sarcoma was induced with normal tissues. However, we can induce a certain per cent of leukemia in C3H mice with x-irradiation, Dr. BURCHEXAL Were you able to produce leukemia in young AKR mice from normal AKR embryos? DR. Gross That is something else again If one injects C3H normal tissues into newbom C3H mice, no leukemia results ·FILTERABLE AGENTS PREPARID TRON LTUKI MIC TISSUE 55 Dr. Kirscubav: Mas I ask one more question? Have you injecteil extracts of embro from trains other than the (58 or AKR, and, if so, what lias happened? Dr. Gross; Nothing, other than the occasional appearance of parotidi tumors. Dr. SPRAGUE: I would kle to ask Dr. Woolley a question concerning his interpretation of results with animals treated with cortisonc. If cortisone is an agent which facilitates viral multiplication and at times activates tatent viruses, one could anticipate that leukemia may occur at an carlicr time or perhaps in a higher percentage of such animals. Cortisonc alo has a striking lytic cllect on lymphoid antic, however, and perhaps this is the explanation of his results. I would appreciate his comments concerning this. DR WOOLIEY: Cortisone is destructive and harmlul to lymphoid tissue, whether it be normal or tumorous. I would interpret the result as duc to retard. ing or regressing influences on fymphoid tisuc bi cortisone. This is in contrast to findings for the parotid gland, where we know of no real effect of cortisone is that what you had in nund? Dr SPRAGUE.. Yes. Dr BURDETTE: Dr Woolley, do you have any results to indicate that the maternal resetancc factor is an agent similar to these viral agents under discus. sion, but with the opposite effect? DR. WOOLLEY I have no results DR. BURDETTE: Dr Syverton, would you comment on superinfection of viruses and competition between two acparate particles of this type? Dr SYVPRTOV. When invasion or infection of cells is limited to two viruses, both present in adequate numbers, competition must occur Failure or preven. tion of infection by one of these viruses is known as interference This capacity of one virus to act as an interfering agent fur a second virus is a temporal relationship limited to days DR BURDETTE. Dr Woolley, would you cominent on the incidence of leu. kemia among mice with mammary tumors, and vice versa? DR. WOOLLEY The results which I presented, showing that Icukemia did not occur with mammary tumors, may be interpreted as competition between tumor-inciting agents, or on the basis that the sublinex had been separated for a number of generations Among our inbred, tumor races, we do not havc lcu. kemia competing with breast rancer, or vice versa. These discases of mice mcm to be well separated by nature DX SYVYKTON. Is the inhibutor to which you relor labılc and inactivated by hrat 1)* texty. Yes, ·S6 ETIOLOGY AND TREATMENT OF LEUKEMIA Dr. Woolley. We have no data on parotid tumors, but we, too, have used the C3H, cell-free, leukemia preparations strain AK mice and have observed leukemia occurring at an early age (between 3 or 4 months). Dr. SYVERTON: This effect may be explained as dose and not superinfection in the sense it was used this morning. Use of newborn animals is a means for increasing the dosage effect, since animals before birth and within hours or days of birth do not respond as older or adult animals. Dr. BURDETTE. The viral etiology of leukemia has been emphasized in the preceding discussions, although Dr. Kirschbaum did review other factors known to influence the appearance of this type of neoplasm Since some information about the etiology of leukemia in humans is available, would the clinicians on the panel care to discuss how these results in animals may be carried over into the clinic? Dr. Schlosser, you may wish to discuss irradiation in relation to leukemia Dr. SchlossER: This is a problem in which I am personally interested because it is recognized that the incidence of leukemia among radiologists and others exposed to low, intensive irradiation over long periods of time is higher than one would otherwise anticipate In addition, the same may be true for those exposed to radioactive isotopes Dr. ARROWSMITH: The appearance of leukemia a considerable time after irradiation of the thymus in childhood also suggests that this is a long-term, late effect of irradiation. DR SCHLOSSER: I seem to recall a high incidence of carcinoma of the thyroid in children who had received radiation, although the dosage used for irradiation of the thymus is exceedingly small This may not be comparable to the irradiation received over a lifetime of handling radium and giving X-ray therapy. DR BURCHENAL On the other hand, the thymus may be a particularly sensitive organ, especially at the age when the children are generally irradiated. DR BURCHENAL Leukemias have been reported also. DR BURDETTE. What is the relation of hormones to differences between the incidence and response of leukemia in childhood and later in life? Dr. BURCHENAL There is not much I can say about that. It is fairly well established that children under the age of 10 respond differently to agents, particularly folic acid antagonists, than children over that age, or adults Whether it has any definite bearing on hormonal changes or not is not known Adults over the age of 30 respond less well to standard agents than the group between 15 and 30. Two other things regarding etiology could be mentioned. Bernard and his group believe that inhalation of benzol increases the incidence of leukemia Last, but perhaps not least, in our modern civilization it is not good from the point of view of leukemia to be within a thousand meters of the hypocenter of an atomic bomb when it explodes ·TILTERABLE, AGLNTS PREPARED FROM LEUKLMIC TISSUE 57 Dr. BURDETTE: Dr. Till, do you havc any comment? DR. TILL: Micntion should be made here of the rare fantilial incidence of leukemia It is surprising to me that if a genetic factor is responsible, scicral siblings in a family are not more often affected with this discase Dr. WOOLLEY: I do not think that is too surprising. If one looks at the mouse population, for example, one finds that there are ven fes high-leukemia strains. DR. ALFRED GELLITORX: In man, certain lculemuas hase been caucalls related to a variety of environmental factors Among radiologists and among many individuals exposed to the ionizing radiation of the atomic explosions in Japan, there is a high frequency of chronic mieloid leukemia It may be argued that radiation unmasks the latent Icuhemogenic sirus, but it is odd, if this bc true, that a chronic rather than an acute dyscrasia appears. I should like to return to Dr. Till's remarks. It is truc that an occasional case of congenital leukemia has been described, but this is a reportable rarity. It would be of interest to have the comments of those panel members who believe that a virus is important in leukemogenesis on the infrequency of leukemia in infants whose mothers develop the disease during the gestation period. It may be argued that the virus cannot pass the placental barrier However, one would still expect to see the disease in mother and child more frequently. DR. KIRSCHBAUM. In a discussion of the relationship of the work on mice to the discase in humans, the report of Schwartz from Chicago should be in. cluded. He has obtained filtrates of brain from human cases of leukemia and inoculated them intracerebrally into mice of a high-leulemua strain and reported an accelerated onset of leukemia Similarly, brain extracts from mice hase accelerated the onset of the disease in a high-leukemic stoch He reports having passed the brain extracts through mice for three passages and then recovered the activity DR. BURDETTF: Dr Sprague, did you have a comment DR, SPRAGUE I was interested in Dr. Burchenal's remarks concerning possible genetic factors Dr. Videbach" conducted a large family study in Copenhagen, Denmark, and, as a result of this studi, stated that the chance of developing leukemia by a member of a family where leukemia was present was 1 in 250 In a control group, the chance of such occurnng was stated to be 1 in 4,000 Dr. BURCHENAL. We have told our patients in the past that heredity played little role in leukemia, but in the past two weeks we have seen two families with two children affected with the disease This had not been encountered in the ten years previous DR BURDETTE Final remarks concerning etiology are now in order Dr. WOOLLEY: The results of studies on viral etiology gne the promise of opening up new vistas and creating nen problems not previously anticipated ·58 ETIOLOGY AND TREATMENT OF LEUKEMIA DR. SYVERTON: Our understanding of leukemia today is not unlike what was known twenty years ago of the rabbit papilloma-to-carcinoma sequence. Obviously, Dr. Gross and Dr. Woolley have made outstanding contributions in demonstrating that a transmissible agent is related to mouse leukenria. Since these agents provide opportunity for studies utilizing tissue or cell culture, it should be possible to arrive at three agents or a single agent, and, perhaps, to obtain cach in sufficient quantity for characterization by the techniques employed for the virus of erythromycloblastosis These findings are stimulating, since they indicate that agents or viruses may be shown to have causal relationship to mouse leukemia. DR. KIRSCHBAUM: I think the information discussed is confusing as well as exciting.

·Arthur C Upton, AD, and icob Furth, MD FACTORS INFLUENCING INDUCTION OF MYELOID LEUKEMIA I RF MICE by IRRADIATION: We have been interested in the induction of myeloid leukemia in the RF Touse for some time Most reports of experimental work with induction of leukemia deal with lymphoid rumors or reticulum cell neoplass Tuenty years ago, however, an increased incidence of myeloid leukemia was noted in mice of the RF stras exposed to tonizing radiation " The present report suriin marizes the results of continued studies of some of the factors that influence the induction of this disease The myeloid leukemias in the mouse, and their differentiation from leu. kemad reactions and extramedullary myeloponeses have been described in detau by Furth and Furth," Upton and Furth," and Bames and Sisman * Host spontaneous myeloid leukemas in RF mice occur in animais 11 to 18 months of age, their onset hastened in radiased animals. There is extensie infiltration of the bone marrow, spleen, and later, whereas the lymph nodes, thymus, and other organs are less affected Involsement of the peripheral blood is anable, as is the degree of maturation of the granulocytic series The disease, it genteral, »rcadıly transplantable into isologous hosts, but some leukemas do not take in 100 per cent of the recipients, cien on serial subpassages *Work performed at Oak Ridge under Contract No W.7405-eng-26 for the United States Aromuc Energy Commission 19 ·60 ETIOLOGY AND TREATMENT OF LEUKEMIA The thymic lymphomas of RF mice, as those in mice of most other strains, appear to arise in the thymus as lymphosarcomas. They may remain localized to the mediastinum, or they may become generally disseminated and thus involve the blood stream. The peak mortality from this disease occurs at the end of the first year of life in Irradiated mice, and it is hastened in relation to the amount of radiation received. In addition to myeloid leukemias and thymic lymphomas, other proliferative diseases of hemopoietic tissues occur in aging RF mice, but their incidence is not affected greatly by irradiation. Hence, they will not be discussed herein. The results of two investigations are presented in Table Viil. In oric, experimental animals received approximately 6 r of x-rays per minute and, TABLE VIII INCIDENCE OF MYELOID LEUKEMIA AND THYMIC LYMPHOMA IN RE MICE EXPOSED TO X-RAYS LEUKEMIA INCIDEACE (PER CENT) TYPE AYELOID LYMPHOID Tiryasia SEX MALE FEMALE MALE FEMALE (R/MIN) 6+ 600 6 601 6* 60+ 60+ Total dose (r) 0 4 19 128 19 19 14 350 29 18 17 38 512 4 17 35 *Data from Upton and associates Data from Upton and Furth 54 con in the other, approximately 60 r per minute. We do not know whether this difference in intensity influenced the induction of leukenua, but, at the higher dose rate, x-rays have a somewhat greater killing effectiveness In the nonirradiated mice of both experiments, the incidence of myeloid leukemia was slightly higher in male animals, and a single dose of only 128 r increased the incidence several fold in both male and female animals A higher rate of induc. tion was observed after 350 r, but, paradoxically, after 512 r the incidence of myeloid leukemia was lower than after either 128 or 350 r In contrast, the incidence of thymic lymphomas was higher in female animals, was not greatly clevated by exposure to 128 r, and increased progessively with the dose With this background, the present series of experiments was initiated To explore the influence of sex hormones, we castrated the mice at 4 to 5 weeks of age, one week prior to irradiation (Table IX) The induction of mycloid leukemia, which was more common in male than in female mice, was inhibited by removal of the testes, but it was unaffected by removal of the ovaries Conversely, the development of thymic lymphomas was enhanced by orchiectomy and inhibited by ovariectomy These observations suggest that in RF mice, as in mice of niost other strains, estrogen is leukemogenic to the thymus The development of mye. 57 55 ·INDUCTION or MYTLOID LEUKEMIA IN RE MICC BY IRRADIATION 61 loid leukemia, on the other hand, apparently is not affected by estrogen but may be potentiated by androgenic hormone The possible role of the thymus in the development of niyeloid leukemia was investigated in a study of the eſſects of thyniectomy. It was observed that removal of the thymus at 4 to 5 sechs of age, one week prior to irradiation, essentially abolished the induction of mediastinal lymphomas, as found previously by Kaplan" in C57BL mice, but it increased slightly the incidence of THYMECTOMY + 4501 CONTROL 450 MYELOID 30 20 سر 10 IL LL THYMIC LYMPHOID CUMULATIVE PER CENT DYING WITH LEUKEMIA 20 10 OTHER 30 20 10 10 20 30 to 20 30 10 20 30 AGE AT DEATH (MO) Fıs 8-Effects of thymectomy before irradiation on the induction of leukemia by X-rays in male mice of the RF strain myeloid leukemia. In the absence of the thymus, an enhanced induction of lymphomas in extrathymic lymphoid tissues was noted also (F1g 8). These lymphomas tended to occur at the same age as the thymic lymphomas in intact mice and significantly eather in life than the spontaneous nonthymic lymphomas in controls Thus, it appears that, in the absence of the thymus, other lymphoid ·62 CTIOLOGY AND TREATMENT OF LEUKEMIA tissues manifest susceptibility to induction of leukemia. This has been observed also by Kirschbaum and Licbelts in thymectomized mice treated with methylcholanthrene. The slightly greater frequency of mycloid leukemias after thymectomy is presumed to result from reduction in the number of micc dying from thymic lymphosarcoma. It is noteworthy, however, that in thymectomized mice the incidence of myeloid leukemia and the combined incidence of myeloid and lymphoid leukemias were lower after exposure to 450 r than in intact mice exposed to 300 r (Table IX). TABLE IX THE INFLUENCE OF GONADAL FACTORS ON THE INDUCTION OF LEUKEMIA BY X-RAYS IN RF MICE LEUKEMIA INCIDE\CE (PER CET) BALE FEMALE DOSE OTHER LYMPHOID, COM LSPIOID, COM- (R) TREATMENT DIYELOD THYMIC BINED MYELOID THYMIC PIVED 0 1 2 3 17 20 0 Gonadectomy 2 9 II 12 14 300 14 19 63 11 50 61 300 Gonadectomy 28 26 54 12 31 13 *In this experiment and those summarized in Tablet X and XI mice nere exposed in lots of 80 to 100 per treatment group with these factors of x-radiation 230 kip, 30 ma TSD. 93 7 cm, 70-80 /mn in air with alter, 3 mm Al filtration (Be wadon), hv. 04 mm Cu Because it was observed that shielding the thigh, that is, hemopoictic tissue in the bone marrows or spleen,42 during irradiation of the animal inhibited the induction of lymphoid leukemia, the influence of partial shielding on the production of myeloid leukemia was investigated. With the pelvis and lower extremitics shielded by lead, mice were given a single dose of 450 r at 5 to 6 weeks of age, the shielded tissue receiving only about 2.5 per cent of the total dose The results indicate that the induction of myeloid as well as of lymphoid leukemia was inhibited out of all proportion to the amount of tissue shielded (Table X) If it is assumed that the pelvis and lower extremities constitute one-third of the entire body, the total dose of x-irradiation in gram-roentgens applied to only part of the body was much less leukemogenic than the same total dose administered to the whole body (Table X) TABLE X EXPECTS OF PARTIAL SHIELDIG Or X-IRRADIATION OU LEUKEMIA INDUCTION IN RF MALF MICE LEUKEVIA INCIDENCE (PER CEVT) LYMPHOID, METOD THE DEIC COMBINED 26 INTEGRAL DOSE (R) DOSE (G-R) AREA EXPOSED 0 0 one 150 3 000 11 hole body 300 6,000 1 hole body 150 9 000 Whole body 450 6 000 Upper two-thirds body Pels and lower sufremties shielded 9 19 28 7 35 63 51 15 23 8 ·INDUCTION OF MYCLOIP LEUKEMIA IN RE MICC BY IRRADIATION 63 'The intra chous injection of nonirradiated marrow cells into totally irradıated mice has been observed to have a similar inhibiton) cffect on induction of lymphoma." The mechanism whereby the presence of nonirradiated hemopoictic cells inhibits the induction of leukemia is not known. Transplantation studies suggest that the implanted, nonirradiated marrow cells survive, prolif. crate, and cventually replace thone of the host that are destroyed by irradiation," Kaplan" suggested that irradiation causes an imbalance between forces that promote the regeneratie proliferation of hemopoietic cells and the capacity of the injured cells to respond He postulated that it is primarily this imbalance and the resulting overtimulation by growth-promoting factors, rather than direct cellular effects of radiation alone, that initiate the neoplastic change. in support of this concept are the observations that neoplasia arises in nonirradi. ated thymic tissue implanted in previously irradiated thymectomized hosts.*. "' A mere imbalance, however, should not yield cells autonomous in normal hosts. A "mutagen" produced by irradiation must be postulated. Creation of regulatory imbalance and altered responsiveness of irradiated cells actually have been demonstrated in tuntor induction by 1"** It appears that some substance or substances present in normal marrow will minimize the likelihood of my cloid leulemia de clopment after irradiation, but the mechanisms by which this protective power is excrted remain to be discloned In another experiment, the influence of fractionation of the dose of radiation was investigated, since it had been reported that the lymphoma-inducing effccuveness of a given dose may be appreciabiy enhanced if the radiation is divided in appropriately timed fractions " RF mice 5 to 6 weeks of age were gruen a total dose of 450 r of x-rays in three exposures, 150 r per treatment, with an interval either of two or of five days between exposures. The incidence of thymic lymphomas was significantly higher in the group exposed at inten als of five days than in those exposed at two-day intervals or in a single treatment (Table XI), thus confirming the obsenations of Kaplan and Brown." In con- TABLE XI EFECTS OF FRACTIONATION OF X-IRRADIATION ON LEUKEMIA INDUCTION RF MALE Mice NUMBER OF EXPOSURES (R) 0 450 450 INTERVAL BE- TWEE EXPO- SURES (DAYS) 0 LEUKEMIA INCIDENCE (PER CEST) LIMPHOTO, MYELOID THYMIC COMBI VED I 2 23 28 51 19 24 43 35 49 450 5) given in three doses 150 rpes exposure trast, the rate of induction of myeloid leukemia declined as the interval between exposures as prolonged from two to five days The mechanism of the observed enhancement of lymphoma induction by fractionation of the dose is unknown, but it has been suggested to be related to enhanced regenerative activity of ·64 ETIOLOGY AND TREATMENT or LEUKEMIA lymphoid tissues at the time of reirradiation. Howeier, a morphologic and physiologic correlation between mitotic activity and leukemia induction is yet to be demonstrated. In general, the effectiveness of a given amount of irradiation decreases as the dose rate is reduced, as observed with the induction of myeloid leukemia under the conditions described. SUMMARY In the RF mouse, myeloid leukemia is induced by a dose of radiation lower than that required for lymphoma induction. Furthermore, the rate of induction of myeloid Icukemia is maximal at levels of dosage below the lethal range and declines as the lethal level is approached, whereas the induction of lymphomas rises progressively with the dose Myeloid leukemias predominate in male mice, and lymphomas predominate in female mice. Induction of myeloid leukemia is inhibited by orchiectomy before irradiation and is not affected by ovariectomy. The induction and spon. taneous development of thymic lymphomas are increased by removal of the testes and reduced by removal of the ovaries. Thymectomy before irradiation prevents the development of mediastinal lymphomas but enhances that of extrathymic lymphomas. Removal of the thymus also results in a slightly higher rate of induction of myeloid leukemia, probably by curtailing the mortality from thymic lymphoma earlier in life. Shielding of the pelvis and lower extremities during irradiation of the animat inhibits the induction of myeloid as well as of lymphoid leukemia out of proportion to the amount of tissue shielded. Fractionation of the irradiation may increase the rate of induction of thymic lymphoma but has not been observed to enhance the formation of myeloid leukemia. DISCUSSION DR Gross I am not familiar with the RF strain and wish to know more about it, particularly the incidence of spontaneous leukemia DR. UPTOV The RF strain was des cloped by Dr Furth in the 1920's at the Rockefeller Institute, and it has been inbred by him since then the spontaneous incidence of leukemia in the strain vanes with sex. In males, the incidence of myeloid leukemia is generally around 3 per cent or less from experiment to experiment. The incidence of thymic lymphosarcoma (lymphoma arising in the thymus and becoming generalized to form lymphoid leukemia) is about 5 In the female, the incidence of myeloid leukemia is likewise approximately 3 per cent, but the incidence of thymic lymphoid leukemia is between 10 and 15 per cent, or even slightly higher in RF mice, as in mice of many other strains, there are prohferative diseases of reticular and lymphoid tasper cent or less ·INDUCTION OF MYCLOID LLUKENA IN RF MICE BY IRRADIATION 65 sues occurring late in life. One is a granulomatous lesion arising predominantly in mesenteric lymph nodles, having some of the features of Hodgkin's disease. Ds Thelina Dunnhas called this discase "reticulum cell tumor, type B" In addition, there are other types of reticulum cell sarcoma®, some of which may be referred to as moroctic leukemias llc hare endeavored to 1) pe most of these hy histologic examination only. We feel this is a complex group of conditions, the incidence of which is not markedly affected by radiation. However, we have scarcely studied them and are not prepared to say en much about them. As far as the transmissibility of the leukemias is concerned, we have altempted transmission of the nijclord leukemia in a few instances only, but the few cascs tested kase been found transmissible through successive passages In our experience, howeser, this discase has not produced 100 per cent takes in adult hosts We are hoping to obtain a transmissibic myeloid leukemia in the RF mouve that may be useful in the study of factors influencing the growth of myeloid leukemia. Honeser, we are discouraged alter observing, with the scveral cases tested, that we cannot obtain more than 30 to 30 per cent takes in a single passage and that the use of newborn recipients and preirradiation of the recipients does not influence the outcome sery much. As for the method of irradiation, 300 r was given in a single exposure at a rate of about 80 r per mmute, and the factors of irradiation were: 30 ma.,3 mm. of alumunum, TSD of 93,7 cm, and ful approximately 04 mm. of copper in the last experiments In myeloid leukemia, che spleen is uruformly greatly enlarged and pale ord! nants This appearance of the spleen contrasts with that of the levdemosd state 1 terms of color, since the spleen in the leukemord reaction is usually a dark purple or sntenseh red, whereas the spleen in mycloid lçukerna is very palc, often even ellowsh gras In some instances, there is slight enlargement of the lymph modes, although usually not much Occasionally, when green infiltrations are seen, se refer to the disease as chloroleukemua The bone marrow is uniformly infiltrated, and very often there is massa necrosis of the femoral bone marrow." The infiltrations are confined primaril to marrow, spicen, and user without much involvement of lymph nodes Maturation of the myeloid series in myeloid leukemias is sarabic Usually, che doughinul-shaped nucleus of the metamy clocy te is conspicuous in leukemic snfiltrations and in the blood Also, elevation of the peripheral leukocyte count occurs ordinaris Honeer, extensive studies on hematologic manifestations remain to be done in our laboratory The thymuc, lymphoid leukemia referred to in the present report is, we behere, a {\pe widely studied in many strains of mice and similar to the radiationinduced leukemia in C57 Black mue in estigated by Kaplan" The ces are usually lymphoblasts, occasionally, small cell lymphomas are obsersed We believe this disease arises m the thysnus as a lymphosarcoma, widely infiltrates the ·66 ETIOLOGY AND TREATMENT OF LEUKEMIA thymus and mediastinal structures, and may cventually become generalized and involve the blood stream, in which case we refer to it as lymphoid leukemia. Death in many instances results with mediastinal infiltration only, without any change in the peripheral blood. Dr. Gross: How high was the mortality within two weeks after the 200 rand 300 r doses? Dr. Upton: The LD 10/30 days in these mice would appear to be around 500 r. The animals in the present experiment were exposed at 5 to 6 weeks of age. We have not determined the LDs/30 days accurately in the RF strain at this age, but at 10 weeks of age the LD,/30 days is about 550 r. The mortality at the end of thirty days after 300 r is negligible DR. KIRSCHBAUM: I am glad that Dr. Upton emphasized that there are varving mechanisms of induction of leukemia in mice. Kaplan has emphasized the role of the thymus, and we, too, havc scen mesenteric lymphomas developing in thymectomized mice, indicating that extrathymic lymphoid tissue is susceptible to induction by x-rays, and this is also true for methylcholanthrene. Il large enough doses of methyicholanthrene are used, the incidence of induced leukemia is as high in thymectomized as intact mice Concerning the shielding, did it make any difference which area of the body was shielded, and did shielding of lymphoid as well as myeloid tissue prevent the induction of granulocytic leukemia? Do you have any ideas concerning the effect of shielding on regeneration of the bone marrow and the effect of the rapidity of regeneration on the ultimate incrdence of leukemia? Kaplan found by marrow suelding he had a more rapid regeneration of the thymus, and with a more rapid regeneration of the thymus, then leukemia did not occur DR UPTON Dr Kirschbaum, these are questions we have asked ourselves and are at present attempting to answer As yet, we have no information about shielding structures other than the lower extremities and pelvis. We have attempted to set up a more extensive shielding group, which is in progress, but we have no data to date We have not tried shielding lymphoid tissues as such to see whether this would influence the induction of myeloid leukemias it would be very desirable to know the rate at which hemopoietic cells recover following Irradiation when hemopoietic parts are shielded Evidence in the literature suggests that when hemopoietic cells are shielded, they repopulate the irradiated hemopoietic tissues elsewhere in the body Observations by Lindsley and anociates with transplantation of homologous bone marrow indicate that, when nonirradiated marrow I put into an irradiated host (in this case marrow from donor rats having D-antigen on the red blood cells was put into recipient rats having c-antigen on the red cells), increasing numbers of D-marked red cells occur in the peripheral blood Similarly, Nowell and associates found that rat bone marrow, when grafted into irradiated mice, produced circulating white blood ·INDUCTION OR MYELOID LEUKOWIA IN RT MICE RY ERRADIATION 67 cell containing alkaline phosphatase in the recipient mice, whercas leshocytes in the mouse ordinarily scre negative when tested for alkaline phosphatase. This edence, added to that of the experiments of Lindsley and associates, supports the vicw that the nonirradiated marrow cells inplant in the irradiated host, proliterate, and luncuon hemopoietically. When part of the body is shielded, nonirradiated hemopoietic celk are probably enabled to survive and to secd irradiated tsoves. However, this is extremely difficult to demonstrate conclushely, DR. KIRSCHBAUV: What you say is very true concerning the seeding, Howeser, Kaplan shicided bone marrow and still it was the thymus which regen. erated sapilly, Dr. Uprox: Further evidence regarding lymphoid regeneration has been reported recenth from Britain by Ford and associates, who have studied this question uath marrow containing chromosomes labeled with translocation. They found that the tagged chromosomes specific to the implanted marrow occurred in lymphose tresues in the irradiated recipicnt. Frorn this they inferred, akhough their results are not fully reported, that the lymphoid elements in the irradiated host, as well as the hemaporetic elements of the marrow, were derived from the implanted marrow tissue This is a critical question and one that needs to have continuing evidence. I do not know whether this errence is convincing, but it is strongly suggestie at least DR KIRSCHBAUN. Since genadal secretion has such an intluence on feukemogenesis, was any of the effect of radiation of the gonads, which might thus affect the induction of leukemia? Da UPTON The gonads in the male were not exposed if they were lying in the scrotum during irradiation, because the entire pels as was shielded In the female, it was doubtful whether the ouanes were included beneath the shield D& WOOLLEY I am wondering if transplantation results with lymphoid leukemia were much different than with mieloid leukemia You mentioned results with the myeloid tunsors DR UPTON I regret to say we have studied the shielded tissues nerther histologically nor from the hormonal standpoint Hence, I am unable to say what effect radiation mas kare had on the gonadal secretion We have made no study thus far of the transmissibility of the kmphoid tumors and have never attempted to prove thest buologie malignanrı by transplantation DR WOOLLEY: Dr Sprague, how would these reticulum cell tumors behave with the Brachet test DR SPRACUE We have had no experience with the Brachet test with tis. sues from patients with reuculem cell sarcoma We have had a single patient with replacement of the marrow with Ewing's sarcoma, and in this instance the cells behaved as mononuclear cells That is to say, the nucles of these cells stained normalls following ribonuclease hydrolysis I would like to ask Dr. Upton one ·66 CTIOLOGY AND TREATMENT OF LEUKEMIA thymus and mediastinal structures, and may eventually become generalized and involve the blood stream, in which case we refer to it as lymphoid leukemia. Death in many instances results with mediastinal infiltration only, without any change in the peripheral blood, DR. Gross: How high was the mortality within two weeks after the 200 rand 300 r doses? DR. UPTON: The LD../30 days in these mice would appear to be around 500 r. The animals in the present experiment were exposed at 5 to 6 weeks of age. We have not determined the LD50/30 days accurately in the RF strain at this age, but at 10 weeks of age the LD3n/30 days is about 550 r. The mortality at the end of thirty days after 300 r is negligible. Dr. KIRSCHBAUM: I am glad that Dr. Upton emphasized that there are varying mechanisms of induction of leukemia in mice. Kaplan has emphasized the role of the thymus, and we, too, have seen mesenteric lymphomas developing in thymectomized mice, indicating that extrathymic lymphoid tissue is susceptible to induction by X-rays, and thus is also true for methylcholanthrene. If large enough doses of methylcholanthrene are used, the incidence of induced leukemia is as high in thymectomized as intact mice Concerning the shielding, did it make any difference which area of the body was shielded, and did shielding of lymphoid as well as myeloid tissue prevent the induction of granulocytic leu. kemia? Do you have any ideas concerning the effect of shielding on regeneration of the bone marrow and the effect of the rapidity of regeneration on the ultimate incidence of leukemia? Kaplan found by marrow shielding he had a more rapid regeneration of the thymus, and with a more rapid regeneration of the thymus. then leukemia did not occur. Dr Upton: Dr. Kirschbaum, these are questions we have asked ourselves and are at present attempting to answer As yet, we have no information about shielding structures other than the lower extremities and pelvis. We have attempted to set up a more extensive shielding group, which is in progress, but we have no data to date It'e have not tried shielding lymphoid tissucs as such to see whether this would influence the induction of myeloid leukemias. It would be very desirable to know the rate at which hemopoietic cells recover following stradiation when hemopoietic parts are shielded Evidence in the literature suggests that when hemopoietic cells are shielded, they repopulate the irradiated hemopoietic tissues elsewhere in the body Observations by Lidsley and associates with transplantation of homologous bone marrow indicate that, when nonirradiated marrow is put into an irradiated host in this case marrow from donor rats having D-antigen on the red blood cells was put into recipient rats having c-antigen on the red cells), increasing numbers of p-marked red cells occur in the peripheral blood Similarlı, Nowell and associates*e found that rat bone marrow, when grafted into irradiated mice, produced circulating white blood ·INDUCTION OF MIYELOID LEUKENSEA IN RF MICE BY IRRADIATION 69 demic cell no longer invaded the peripheral blood, and more and niore primitise mycloid cells were seen in infiltrations of tissue on successive subpassages. Ulti. mately, the doughnut-shaped sucleus of the metam;elocyte became selatisek rare, and that of the mzeloblast occurred predominantis. Dr. GELLNORY: Does this persist, or do they die off? DR. UPTON: I am not sure that I understand your question, DR. GELLHORX: ... if scrial transplants are continued. Dr. Uprox: When we have done this, we have not obtained 100 per cent lain. We hasc had the unfortunate experience of losing two of our lines because the number of takes as small. Ictually, because of our lack of success ir obtaining uniformh tales, we have almost abandoned attempts to study these in scrial transplantation, DA. KIRSCHBAUV: Before one concludes that Icukemia is not transplantable, one must use a rather large number of animals in the first transfer. We have had the experience of taking a granulocytic leukem as donor, and inaces. laung twent mice, and having only one or ino take. From the one or two trans. plants, lines may be established in which takes us occur in 100 per cent of the cases. I think some of these are leukemias of cry lou malignancs or sery dilferentiated leukemias. Since they are differentiated, probably very few immaturc cells are in the moculum, and the immature cells are the ones responssble for transplantation that the investigator mas be doing is giving a very small dae of cells capable of transitting the discase, and this dose is then increased by frequent transfer. Of course, one may be dealing usth dependent lculemjas, and the hose must be conditioned before the inoculared cells can be grafted success DR. UPTOV* In the fent cases in which ne made serial subpassages, we did not see an increase in the number of takes although the cells appeared to be bem coming progressneh dedifferentiated From this we concluded that the neoplasm was conditioned in some way or that other factors were muertering **th the uniform occurrence of takos Dr WOOLLEY: Has I ask sour method of transplantation how you prepared the tissue, how you moculated, and whether it was subcutaneous or mtraperitoneal Dr UPTOV. The transfer of my clod leukemia was made by doiding splesse (suc into fine pieces with dusccting "ION (The imprint of the spleen wuall showed almost all melod cells) The splensc tassue was suspended in saline solution and injected sstraperitoneally We tried intravenous injection as well, but from the results we did not feel that this was a more effectne route DR GELLHORY From the vantage point of one who knows nothing about the field and one who neser expects to do an experiments in it, I would like to have you tell me what evidence exists that those really are leukerslas Will you ·68 ETIOLOGY AND TRCATMENT OF LEUKEMIA question regarding shielding. Jacobson has shown that animals whose spleen was shielded could withstand higher doses of total-body irradiation. Is it necessary that actise hematopoietic tissuc be present in the shielded pleen for such protection to be afforded? Dr. UPTON: The spleen is a very mysterious organ to me. I believe, how. ever, it has been the experience of those who have tried to promote recovery from radiation injury by shielding of the spleen that the technique yields best results in those animals which normally have hemopoietic activity in the spleen. Although there are differences between strains, mice usually have active myelo- and erythropoiesis in the red pulp. Hence, the shielding of the spleen under these condi. tions is, in fact, comparable to shielding the marrow. Furthermore, the em. bryonal spleen is characterized by actre hemopoiesis, and much of Jacobson's protection work in olved administration of embryonal tissues. In animals such as rabbits in which intrasplenic hemopoiesis does not occur, spleen shielding is not of much benefit.* Therefore, in answer to your question, the effect of spleen shielding probably amounts to shielding of hemopoietic tissue and is biologically equivalent to shielding of the marrow. DR. SYVERTON. Have you tried transmission with cell-free extracts? DR. UPION We have attempted nothing of that nature. We are, of course, keenly interested in understanding what radiation is doing I, myself, hase wondered if irradiation is unmasking a virus, let us say, and that we may have a lymphoid leukemogenic virus and possibly also a myeloid leukemogenic virus in the RF line A disease was discovered some scars ago by Dr Furth in the RF strain which is characterized by the development of ancmia, leukopenia, and erythropoietic splenomegaly is This condition was found to be transmissible with liophilized material or cell-free material A virus or viral agents could exist in the RF mouse capable of producing a variety of disorders together or individually in our studies with filtrates we concentrated on newborn recipients, since the experiments of Dr Gross, along with the work of others, indicate that sensetivity is maximal in newborn animals DR. GELLHORX What is the natural history of these leukemias? You say they do not transplant well. Do they destroy the host? DR. UPTON I am embarrassed to admit that we know very little about the natural history of myeloid leukemia When animals die with this disease, they are extremely anem. We have not performed extensie blood studies on them, but they are extremely pale, emaciated, and often exhibit massive necrosis of the femoral bone marrow The liver and spleen are usually extensively infiltrated by In the few attempts we have made to transmit the myeloid leukemias, the leukemias have undergone progressive dedifferentiation on successive passages In one of two cases in which the primary host had an elevated white cell count with many mature and immature granulocytic elements, finally the loumyeloid cells ·Charks C Spragul, D DIAGNOSIS of LEUKEMIA INTRODUCTION It is generally conceded that our current, conventional methods for diagnosis and classification of the loukemias are inadequart. Unul recently, it was not necessary to make a precise, morphology dagnosis in every unstance. How fler, with the rapidly expanding use of chemotherapeutic agents, we have learned that the sufferent leukemias tary greatly in their response to these agents. This makes it necessary that the best possible description be made in Csery case of leukemia One chemotherapeutic agent as presently avatlable that is highly effective in a single type of leukemia, and yet totally ineffective in other types of leukemia Certainly, more examples of this will probably be seen as time goes on The statement is made frequently that morphologkis harc had their day, and that ittle more will be achiered in this particular area of investigation I disagree with this viewpoint, and think that, with the application of new physical, chemical, and histochemical techniques, we all sce additional valuable contributions made in this fickt Later in this discussion, I wish to summarize the results of our own experience with some of these techniques. Initsally, I would like to review briefly some of the methods that have been der eloped in the recent past, largely in research laboratories Granted that many of these techniques and methods are not suitable as routine procedures, some of them can be adapted with a minimus of difficulty. I shall try to make the discussion of these briel, confining my remarks to their usefulness and relatuse advantages and disadvantages 73 ·70 ETIOLOGY AND TREATMENT OF LEUKEMIA tell me why I am wrong in thinking if you destroy the bone marrow with x-ray or with drugs, you might not get the same sort of change that you have described here? Dr. UPTON. The evidence we haic is that the animals all died of this discase, and, in thc few attempts we have made to transmit it, we have been able to do so in some instances The diagnosis was made at autopsy, and careful microscopic search did not disclose any other fatal process. There was widespread infiltration of primitive myeloid cells in the liver, spleen, and marrow, and foci of orderly maturation of granulocytes and erythrocytes were absent. This evidence causes us to view the disease as myeloid leukemia. Dr. KREMENT2: Do RF mice develop other tumors after radiation? DR. UPTON. The RF mouse will develop other neoplasms We think there is a shghtly increased incidence of pulmonary tumors in irradiated RF mice, but the major, neoplastic diseases are leukemias, lymphoid leukemia arısıng in the thymus and myeloid leukemia. DR. KIRSCHBAUM Dr. Gellhorn has raised a very pertinent question. Those of us who work on mice take pnde in having a biological test, and this biological test is transplantation In Dr. Upton's experiments, he has transmitted enough of these leukemias so he could assume those which do not transplant probably arc leukemia. However, myeloid metaplasta may closely simulate leukemia, and if one found it sporadically in a strain, it might be very difficult to determine whether or not this was leukemia or hyperplastic reaction. We should emphasize transplantation as biologic evidence that this is truly leukenria If one can transplant it, it is leukemia ·DIAGNOSIS OF LEUKEMIA 73 KIBONUCLEASE TEST The ribonuclease test probably is unfamiliar to most investigators. Dr. Carrera, in our laboratory, is largely responsible for the work which I would like to report at this time. Brachet" first descnbed the ribonuclease test in 1940. He showed that, following incubation in an aqueous solution of ribon nuclease, the basophilic granules in the cell cytoplasm lost their ability to take up pyronine in tissue «ections, whereas the staining of the chromatin of the cell nucleus by the methyl green component was unaffected. He, therefore, con cluded that ribonucleic acid was located primarily in the basophilic, cytoplasmic granules. Using a modification of the niechod, Lases and Thoma" described the unique differential behavior which is shonn by the nuclear chromatin of the white blood cells This tcst can be performed in one of two ways. llethanol-fixed slides ether of peripheral blood or of bone marrow can be hydrolyzed with a solution containing cathes ribonucleate or desoxs ribonuclease activity. Ribonuclease, we find, is much more satisfactory for everal reasons First, the hydrolysis can be carried out at 60° C., and the time is not critical. Filteen minutes has proved satisfactory. Second, it has been found that freshly 10ded urine is an excel lent source of ribonuclease. It is probable that there are factors in the urine other than ribonuclease responssble for its actim on the cells Desoxyriboryclease, on the other hand, is an expense enzyme, about six or seven hundred dollars a gram, and fydrolysis has to be carried out at 0° C for a period of fifteen seconds to one minute, the cmc being critical for optimal results After hydrolyzing with one or the other of these enzymes and staining with Gremsa stain, the following resuks are observed sssth ribonuclease hidrolyses, the nuclear matenal of the neutrophilic granulocyte loses its ability to fix the Alain, and it appears blanched The nucles of eosinophile granulocytes, lymphocytes, and monocytes, on the contrary, retain their ability to fix the Gremsa With desoxyribonuclease hydrolysis, the reverse attuatson prevails The nuclei of the mononuclear cells, monocytes, lymphocytes, and cosinophiles, are blanched, whereas the nucler of the neutrophilic granulocites stain normall The findings are illustrated in the photomicrographs In Fig 9, a monoblast is seen which had been hydrolyzed with a solution containing desoxyribonuclease actrity and then counterstasned with Giemsa stain The nucleus of this cell is entirely blanched, the nucleolus and an Aiser body are seen easily A neutrophil in the same held shows the nucleus to stain normally Fig 10 reproduces a peripheral blood film from a patient with acute lymphocytic leukemia The smear was hydrolyzed with a solution containing desoxyribonuclease actitty The nucleus of the lymphoblast is completely blanched, at was the monoblast in the previous illustration, and a neutrophilic granulocyte is included again for comparaure purposes an ·72 ETIOLOGY AND TREATMENT OF LEUKEMIA TECHNIQUES AND METHODS ELECTRON MICROSCOPY In electron microscopy, examination of cell sections has proved much preferable to the casting technique. It has been found possible to section a granulacyte into some seven hundred slices, these slices being of the order of 0.002 micron thick. Such sections have proved very satisfactory for electron microsсор; The instrument, of course, provides tremendous magnification up to a range of 100,000 diameters; with the very thin sections, it was hoped that this would make possible better differentiation of the various blast cells and so-called stem cell, but, unfortunately, this has not proved to be the case. Mature cells can be distinguished without difficulty with this technique, but the immature blast forms still offer the same problem as previously. Despite the potential usefulness of this technique, it offers two scrious ob. stacles. First, the cells have to be examined in a desiccated state, and, second, it is necessary that the material be very thin, thinner than an intact cell. These two obstacles make it difficult, if not impossible, to examine intact cells. For these reasons, electron microscopy and micrography have been used chiefly in attempts to identify virus particles within cells, and in describing the submicroscopic structure of the cells There is at least one report where virus particles were supposedly identified in leukemic cells by this method. However, other investigators who have had extensive experience with this method have failed to confirm this observation. PHASE CONTRAST MICROSCOPY This method allows one to examine le, unstained preparations providing a distinct image of the nuclear chromarin, nuclcoli, nuclcar membrane, mitochondria, specific granules, and Auer bodies. Those of you who have had an opportunity to visualize a vital preparation, using phase contrast microscopy, have been impressed undoubtedly with this as a technique In addition to allowing visualization of these cells in a vital state, it has been possible to utilize a time-lapse mouc technique, whereby the locomotion of the cells can be studied. It has been suggested that the actual locomotion of the cells may be important in terms of diagnosis Such a movie utilizing this time-lapse photographic technique has demonstrated beautifully the development of the lupus erythematous cell The nucical and granules are easil identified, and both Jones and Moeschlin pointed out the aid this provides in differentiating the blast cell of lymphosarcoma from the blast cell of lymphocytic leukenua They stressed that the lymphosarcoma blast cell has a prominent single nucleolus, whereas the lymphocytic leukemia cell has two to four nucleol Brausil" stated that she has been able to differentiate a lymphoblast and a myeloblast, using phase contrast microscopy. Others have been unahle to confirm her obserutrons.

·DIAGNOSIS OF LEUKEMIA 75 Fig. 11 is from a patient with acute granulocytic leukemia. In this in. stance, the slide was hydrolyzed with a solution containing "ribonuclease" activity (Ireshly voided urine). The nuclear matcrial is blanched in all of the cells, as would be anticipated The results of these studies suggest that in the case of the neutrophil and bacophıl, the nuclear protein was largely ribonucleoprotein, whereas in the mononuclear cells and in the cosinophil, it was desoryribonucleoprotein This, I think, might explain in part the observation frequently made that cosinophils bchase more like lymphocytes than basophils and neutrophils, as in the case of the lymphopenia and eosinopenia observed following the administration Fug 11 --Appearance of strear from a patient with acute granulocytic leukemia after hydrolysis with a solution containing nbonuclease actisity of ACTH, for example This technique does not allow differentiation of the blast fornis oí lymphocytic and monocytic leukemia in that a lymphoblast and a monoblast behave the same way Fortunately, the differentiation of these two does not pose too difficult a problem as a rule I think you will agree that most of our difficulty is in differentiating monohiastic from myeloblasti jeukemia, and it is here that the method seems to have its greatest usefulness, DIOCHEMISTRY AND METABOLISM OF LEUKOCYTES Recent advances in the field of biochemistry and metabolism of leukocytes in the various leukemic states are of interest The level of amino acids has been found strikingly different in leukemic cells than in normal cells, this is ·74 CTIOLOGY AND TREATMENT OF LEUKEMIA ... comportar Fig 9-Monoblast hydrolyzed with a solution containing desoxyribonuclease actrity and then counterstained with Gremsa starn OOO o OG 00 C Fig 10 --Film of penpheral blood taken from a patient with acute lymphatic leukemia and hydrolyzed with a solution containing desoxyribonuclease actity ·DIAGNOSIS OF LCUKEMIA 77 The leukocrythroblastic peripheral blood picture that is observed in patients with metastatic carcinoma to the bone marrow can be confused easily with leukemia. The correct diagnosis usually is made at the time of bone marrow aspiration in such patients, although sery commonly aspiration is impossible, and bone marross section is required to provide the answer. The types of carcinoma most likely to produce this peripheral blood picture are carcinoma of the prostate, lung, and stomach. Multiple myeloma, on rare occasions, may be associated with such findings in the peripheral blood Wycloid nictaplasia commonly is niisdiagnosed as leukemia, but, again, a carclul cu aluation of the peripheral blood, and bone marrow history and physical camination usually allow this to be differentiated from leukemia. Red blood cells in the peripheral blood of such patients show pronounced anisocytosis and poikilocytosis almost invariably. The marrow may be extremely variable, ranging from hypercellular to hypocellular. Biopsy of the liver or of the spleen is the most reliable for establishing the diagnosis. Time will not perm a discussion of the interrelationship and some of the theoretical aspects concerning chronic granulocytic leukemia, polycythemia scra, myeloid metaplasia, and myelosclerosis. Probably they are closely related or actually variants of the same basic disorder. DISCUSSION DR BORDETTE Thank you, Dr Sprague, for revitalizing this morphologic subject and directing attention to pitfalls in diagnosis Discussion of Dr Sprague's paper is now open DR WOOLLEY: I would like to know whether there is any point in the blast cell «eries when differentiation breaks down. I believe that certain blast cell leukemias do respond to cortisone, whether or not in the lymphoid or granulocytic scries, and I wonder whether there is a point of cmbryonic differentation or lack of differentiation where this difference is obliterated DR SPRAGUE It is commonly recognized that there are some patients with acute leukemia where the cells are sufficiently immaturc to defy a morphologic diagnosis We have had occasion to study the blood and bone marrow of scveral such patients with the Brachet test and were able to classify them according to the behavior of the cells with ribonuclease hydrolysis. The subsequent course of the patient in each instance allowed us to confirm this initial impression DR. GELLHORN: Dr Sprague, first will you please comment on the report of Storti, from Italy, who recently described a simple test for differentiating between the various types of leukemia It consists of determining the osmotic fragility of the leukocytes of the buffy coat, using different concentrations of saline The leukocytic osmotic fragility is increased in acute and chronic ·76 ETIOLOGY AND TREATMENT OF LEUKEMIA particularly true of the leukocytes in the acute leukemias. The ratio of ribonuclease to desoxyribonuclease is markedly elevated in acute leukemia, as is the ratio of uracil to thymine. Desoxyribonuclease inhibitor is found in large amounts in normal leukocytes, yet very little is in the leukocytes of chronic granulocytic leukemia; almost none is in chronic lymphocytic leukemic leukocytes and in the leukocytes of various acute leukemias. Apparently, there is a normal range of this desoxyribonuclease inhibitor in leukocytes found in patients with leukocytosis and leukemoid reactions Valentine" found the alkaline phosphatase of cells in chronic leukemias to be less than normal, whereas in leukocytosis and in leukemoid reactions the level usually is normal or elevated Low values in leukemic cells do not return to normal even though a remission is induced Ellison" found the citrovorum factor content of leukocytes in acute leukemia to be elevated, whercas the level in cells of chronic leukemia usually is normal. Folic acid antagonist therapy markedly reduces the citrovorum level to values usually below normal A great deal of work has been done on culturing bone marrow, and it is now possible to keep bone marrow as well as peripheral leukocytes viable long enough for short-term studies in vitro. With the use of radioactive isotopes, notably carbon 14-labeled formate or adeninc, it has been possible to determine the rate of nucleoprotein synthesis, as well as to determine the effect of various chemotherapeutic agents on the rate of incorporation of one of these labeled precursors on the biosynthesis of nucleic acid In view of our rapidly increasing knowledge of the chemistry and metabolism of these leukemic cells, it is not improbable that leukemias soon may be classified chemically as well as morphologically. Also, the technique of bone marrow culture may prove a very useful technique in screening leukemic patients for therapy It is possible that in time something comparable to current methods of determining the sensitivity of bacteria to various antibiotics can be offered DIFFERENTIAL DIAGNOSIS Certain disorders may be confused with leukemia leukemoid reactions secondary to infections, particularly tuberculosis; collagen disorders, lymphomas, notably Hodgkin's disease; and liver disease, whether it be decompensated cirrhosis, metastatic cancer, or lıser abscess, are at times extremely difficult to differentiate from leukemia As a rule, a careful examination of peripheral blood and bone marrous films, plus a thorough history and physical examination, will allow a differentiation to be made This can be extremely difficult at times, however; I can recall one instance where it as impossible to determine with certainty whether the patient had leukemia or leukemoid reaction, even following autopsy ·DIAGNOSIS OF LCUKEMIA 79 factors present in urine, for they referred to the active principle as a "ribonuclease complex." Our results to date concerning the nature of the substance or substances in urine which account for the observed changes in the nucleus of neutrophilic granulocytes and its precursors may be summarized as follows: 1. Activity is not lost by dialyzing unne against tap water, but it is lost by dialyzing against distilled water. 2. The activity is slightly diminished after prolonged incubation at 58° C. at pH 7.7, and it is greatly diminished after heating to 100° C. for thirty minutes 3 The activity cannot be duplicated by commercial, crystallized ribonuclease, trypsin, or chymotrypsin. 4 The action can be closely simulated by sodium chloride solutions in concentrations ranging from 0.18 M. to 0 30 M and by phosphate buffer solutions of pH 7.7 of molarities between 006 and 008 5 The reaction of leukemic leukocytes appears similar to that of nommal leukocytes These results cast serious doubt on the hypothesis that the effect of urine is due to ribonuclease activity In fact, the only evidence suggesting any enzyme activity is the heat lability of the principle. Extraction of nucleoprotein by urine acting as a salt solution with the possibility of enzyme activity is now thought to be the most reasonable explanation of the reaction, although it has not been proved conclusively ·78 ETIOLOGY AND TREATMENT OF LEUKEMIA lymphatic leukemia, decreased in acute myeloid leukemia, and similar to normal leukocytes in chronic myeloid leukemia. Second, I would like to ask a rather impertinent question about the Brachet test. I had not known about this technique until reading your abstract, Dr. Sprague, and I was fascinated by it and very impressed by your report. Now, would you confirm once again for me, does it really work? DR. SPRAGUE: In answer to Dr. Gellhorn's first question, I was unaware of Dr. Storti's fragility studies until the leukemia symposium in Detroit last month We have had no experience with this method, but it certainly seems a simple method that may provide valuable information. We have been using the Brachet test for approximately one year and believe that it is quite reliable There has not been a single instance in which it has misled us. The test undoubtedly requires some refinements, and I am sure there are pitfalls in the procedure At the moment, however, we feel it is a very satisfactory laboratory method. DR. KIRSCHBAUM Are these really reflections of enzymatic activity? With ribonuclease, one could expect ribonucleic acid to disappear. Here, one has a complete washing out of the nuclear chromatin. Is this really cnzymatic action? Dr. SPRACUE I also question whether it is, actually, because it is contrary to what one would anticipate from the known composition of nucleoprotein We are not at all certain that the changes we observe in these cells are duc to enzymatic activity of either ribonuclease or desoxyribonucleasc alone. We are presently studying the effects of a purified enzyme to see how this compares with buffered urine as a source of ribonuclease activity Although I am not prepared to say at the moment what accounts for the observed changes, this does not detract appreciably from the practical application of the method if further study confirms its worth. DR TILL Did you perform the test more than once during the course of the disease in the same patient? Were the majority of tests performed when the diagnosis was first made DR SPRAGUE We have repeated the study on only one patient with chronic granulocytic leukemia In this instance, there was no difference in the behavior of the cells before and following treatment with Myleran. ADDENDUM Since the above report, additional studies have been carried out in an attempt to elucidate the mechanism of the so-called "ribonuclcase" test. in the watial studies, buffered urine was used as a source of "enzyme" as this had been reported by Lases and Thoma. They did not proude evidence of ribonuclease activity, however, and apparently believed that there were additional ·LARORATORY, CLINICAL APPROACHES TO LEUKEMIA CHEMOTHERAPY 81 basis for the discovery of compounds to use in the treatment of these dyscrasias Having justified the need for empiric screening of compounds in the treatment of leukemia, how is this accomplished? In the past few ycars, there has been a great deal of dircussion on the optimal method or methods for antitumor screening. A cooperative experiment conducted by thirty-nine insestigators in cight institutions has been addressed to the issue of what constitutes a reasonable biologic system for screening compounds which ultimately might be tested in the clinic against neoplastic disease. The over-all plan of the experiment was to test a standard group of compounds in as many diverse biologic systems as possible, correlating the results obtained with one another and with the effect of the compounds in human neoplastic disease TABLF XII I\STITUTIONS Bronx Botanical Garden, Bronx, NY Carnegie Institution, Cold Spring larbor, X Y Columbia University, New York, X Y Institute for Cancer Research, Philadelphia, Pa PARTICIPATING INVESTIGATORS Asheshos, IS Flon, H Hall, E A Demerec, M Hemmerly, J Dandson, JD Freeman, BB Gellhorn, A. Golino, M Hirschberg, E Kells, A Merson, G Andre, ) Aronson, M Creech, HJ Hankwitz, RF, Jr Hauschka, TS Littleton, BJ Rothman X Schultz, ) Clarke, D A Karnofsky, D1 Philips, FS Reilly, JIC Scholler. J Stock, CC Sugiura, K Tarnowski, G. S Schabel, FM, Jr Skipper, HE Thorson, JR Bittner, JJ Greenlees JL Heidelberger, C Keller, RA LePage, GA Bieber. S Elon GB Hitchines CH Sloan-Kettering Institute, New York, NY Southern Research Institute, Birmingham Ala University of Minnesota, Minneapolis, Minn University of Wisconsin, Madison Wis Wellcome Research Laboratones Tuckahoe, X Y ·Alfred Gellhorn, MD LABORATORY and CLINICAL APPROACHES 10 LEUKEMIA CHEMOTHERAPY THE SELECTION OF COMPOUNDS FOR CLINICAL TRIAL Unlike the effective methods which have been developed for the screening of potential antibacterial chemotherapeutic agents, the selection of compounds which merit clinical trial for the treatment of the leukemias poses a serious problem This is due to the fact that there is no curative drug which can be used as a standard against which to measure other compounds, and also because there is no biologic assay system which has been shown to be exactly comparable to human acute or chronic leukemia How does onc proceed? Ideally, we would like to have our colleagues in brochemistry and other basic disciplines provide sufficient information about the intimate characteristics of the leukemic cell so that it would be possible to des clop chemical agents which would selectively destroy the abnormal cells I few weeks ago, it was my pruulege to attend a conference at which a number of distinguished biochemists from this country and abroad discussed biochemical differences between normal and neoplastic cells After two days of vigorous discussion and examination of the evdence, it was agreed that there are a number of quantitative differences between neoplastic and normal cells, but that the fundamental biochemical characterization of abnormal growth still cludes definition Since the day-to-day clinical problem of leukemia is so pressing, empire methods of selecting drugs are in use, pending the development of a rational SO ·LABORATORY, CLINICAL APPROACHES TO ICUKEMIA CHICMOTHERAPY 83 if they proved effective as antitumor screens. Two bacterial mutation systems were included because of the possible relationship between mutagenic and anti. tumor action. The differentiation and developmental systems in slime mold. frog embryo, chich embryo, and the fruit fly, Drosophila, were studied as representing rapidly growing, normal tissues. The twenty-one biochemical indices of synthetic processes were included because of the possibility of more precise and sensitiic indicators of antitumor action. TABLE XIV LIST OF COMPOU\DS COMPOUND VUMBIA VALE IT I Mistargan Methyl carbamate Chloramphenicol (Chloromycetin) Potassium arsenite 8-Azaguanine(5-amino-7-hydroxy-1!1-triazolopyrimidine, Guanazolo) 8-Azaxanthine Benzimidazolc Saraprim, Pyrimethamine) 19 20 21 23 24 26 27 28 D.1-Ethionine 4.2-Thuenylalanine Desoxypyridoxine Colchicine 5-Methoxytoluquinone Metropsin Azaserine (0-duazoaretyl-L-serine) Table XIV presents the twenty-seven compounds which the group of investigators selected for study As can be seen, these included compounds which had been found to have activity against one or more of the experimental tumors, or to have strong activity against other biologic systems, as, for example, chloramphenicol, or were thought to be inactive, thereby terving as negative controls The action of these twenty-seven compounds was studied in each of the systems previously described, and each investigator sent his result to a central office, where the information was collated Eighteen hundred separate answers were obtained, an example of the flow sheet for two of the systems is shown in Tables XV and XVI Table XV summarizes the effect of the compounds on the fifteen experimental tumors The compound number is the same as glien in Table XIV Table XVI summarizes the effect of these compounds against the various microbiologic systems.

·82 CTIOLOGY AND TREATMENT OF LEUKEMIA This experiment has been reported in detail elsewhere. The participating investigators and their institutions are shown in Table XII. Table XIII lists the biologic systems which were used in the evaluation. The spectrum of fifteen tumors included two in the rat, one in the rabbit, ten solid and two ascites tumors in mice. Among the microbiologic systems studied, the five mouse-adapted viruses were included because of the oft-repeated postulation that compounds which would be effective against viruses would very likely be active against tumors, because of the central role of nucleic acids in these two systems A number of bacteria and fungi were included because of the ease and rapidity of operating these systems, which would greatly facilitate screening TABLE XIII BIOLOGIC Systems EXPERIMENTAL TUMORS Sarcoma 180 Flexner-Jobling Walker 256 Carcinoma E0771 Sarcoma T241 Carcinoma 1025 Mecca lymphosarcoma Carcinoma RC Recently isolated mammary carciroma Carcinoma 755 DIFFERENTIATION AND BIOCHEMICAL SYN- MICROBIOLOGIC DEVELOPMENT THESIS Feline pneumonitis Escherichia colt muta- Tumor 755 DNA tion SD4 Vaccinia Escherichia coli muta- Ehrlich protein tion WP14 Influenza A Dictyostelium aggrega. Ehrlich adenine tion Western equine en- Dictyostelium culmina- Ehrlich guanine cephalitis tion Poho Frog embryo differen- Gardner protein tiation Bacteriophages Frog embryo drug Gardner adenine sensitivity Escherichia coli Chick embryo abnor- Gardner guanine malities Bacillus subtilis Chick ernbryo growth Flexner-Jobling Qur inhibition Serratia marcescens Drosophila laria Flexner-Jobling CO. Staphylococcus aureus Drosophila puparium Flexner-Jobling protein in vitro Mycobacterium smeg. Drosophila imago Flexner-Jobling promatis tein in VIVO Mycobacterium phler Drosophila larva/imago Plexner-Jobling nucleic acids Lactobacillus casei Drosophila phenotype Flexner-Jobling adcabnormalities ninc Kloeckera brevis Flexner-Jobling guanine Torulopsis utilis Drosophila chromo Spleen Q. some breakage Saccharomyces cere Drosophila crossing Spleen Co visiae 358 ouer Saccharomyces cere Drosophila variegation Spleen protein in vitro vistae 376 I59 21 Penicillum notatum Drosophila vanegatton Spleen protein in uno Ysc8 Aspergillus fumigatus Streptomyces griseus Streptomyces antibs. oticus Brown-Pearce carcinoma Ghoma 26 Leukemia L1210 Ehrlich ascites EF Ehrlich ascites ELD ·CON POUND SINUXELL III IIII ELSE PIEVYO VITIS VACCINZA T\FLVEZA WESTER EQUISE EXCEPHALITIS Not POLIO DACTERIOPHACES III+ ESCHERICHIA COLI + BACILLUS SUBTILIS 1+ + + 11+ ++ SERRATIA MARCESCENS STAPILYLOCOCCUS II+ + AUREUS It 3 MICRONOLOGIC SYSTEMS TANL2 .XVI 1 it II ++ MYCOBACTERIUM SMECMATIS **COBACTERIUM I + III+PHLEN I i III 1+ XLOECKERA BREVTS 1+1+1 it ! ! ! IIIIIITOR ULOPS1$ UTILIS 358 376 CIRFVISTAF Miche SACCHARO- *+ + It IIIIIIII PENICILLIL H VOTATUM ASPERGILLUS FUXIGATUS STREPTONCES GRISEUS ++ STREPTOMYCES 1 1 1 IIIIITATIBIOTICUS I **LACTOBACILLUS CASEI 58 AV.H.LONI VIJYJN11 OL SHYOUDY TYINI TO'XOLYYOIY I ·TABLE XV EXPERIMENTAL NEOPLASMS COM- POUND NUM BFR MAMMARE SAR- CAR- COMA FLFXNER WALKER CINOMA 180 JOBLING 256 E0771 | "SPONTA MAMMARI FOUS" MAMMARY SAR- CAR MECCA CAR- MAMMARY ETIRLICH CAR- LEU. COMA CINOMALYMPHO-CINOMA CAR CINOMA HROWY-GLIOMA KEMIA ASCITES 1241 | 1025 SARCOMARC CINOMA 755 PEARCE26L1210 EF ELD + + - د حيا به يې ونه 11 ++ ETIOLOGY AND TREATMENT OF LEUKEMIA + + + + + ++ + + + IIIIIIIIIII III+ I + IIIIIII1 i 1+1+ I + I + I+I+I+IIIIII+It It IIIt It II it + + + III+IIIIIII I+It ·Suso SUA CONTUUD UKBER 11 FELIVE PNEUMONITIS I VACCINIA - - donc don donor Nat IYFILENZAA IMESTERS EQLINE E\CEPHALTIS dour one POLIO BACTERIOPHACES 1+ (+ f+ . I ESCHERICHIA COL BACILLUS SLATIU + + + 1+ : 1+ LARATIA MARCESCENS STAPHYLOCOCCUS I+ + ALRELS MICROMOTORIC SISTENS TARL IVI + 11+ MYCOBACTLRILN 15XZCMATIS WYCOBACTERIUM 1+1 1+1.PELEJ 1+ .I+ LOECKLEA NAL115 1+ +1+ +TORLLOPSIS UTILIS 1+ + 1+ II+ + + CLATUS *ACCHIANO- PENICILLIL X VOTATEN 1 ASTHAGILLLS FUMIGATLS + STRIPTONCES GRISELS STREPTONICES # 1.IIIII ANTIBIOTICUS III : LACTOS ACILLLS CASEI 58 IdYKIONJHD VIXIT OL SIHOYONDDY IVOINTI JYOLVYOVT ·86 ITIOLOGY AND TREATMENT OF LIUKENETA From the raw data obtained from all of the systems, a further condensation was made This is shown in Table XVII. The significance of this table can best be explained through an example. Compound No. 1 is nitrogen mustard TAFLE XV21 RESPONSE OF TUMOR AND YOUTUMOR SYSTEMS TO TJIE AGENTS COUPOL VUMBER ATOLOGIC ACTIVITI + HIOT OGIC ACTUM + 9 6 + 16 21 2 3 4 5 6 TUMOR SYS TEMS PER CENT POS- ITIVF RESPOSF 73 10 60 73 40 87 60 0 0 33 67 0 NO TUMOR SISTEMS PER CENT POS- ITIVF RFSPO$$ 72 6 23 17 20 19 38 13 10 12 29 8 15 15 10 10 12 13 144 15 16 17 18 19 20 21 22 23 2.4 26 27 28 to 10 out wiono-obudowa 15 14 15 10 9 9 5 12 12 10 15 15 48 47 30 46 47 28 18 36 43 26 5+ 33 12 28 28 28 23 37 54 31 41 23 15 33 5 26 13 39 31 31 36 22 5 15 19 36 37 39 0 33 40 10 67 20 20 33 0 0 27 13 27 53 13 53 70 39 26 56 8 444 24 51 53 53 61 37 8 25 32 63 63 66 13 11 40 21 22 20 8 This compound produced a positive effect against eleven of the experimental tumors and no effect on four of the experimental tuniors, or a percentage positive response of 73 The compound also produced a positive effect on fortyone of the nontumor systems and no effect on sixteen, or again a percentage posttas e response of 72 The results for each of the other compounds have been summarized in a similar manner It was decided quite arbitrarily that a posithe biologic activity rating would be given to those compounds which affected one-third or more of the Systems studied Thus, Compound No 1, with a rating of 73 per cent sn both the tumor and nontumor systems, has a positive biologic activity rating, Having classified the compounds in this manner, it was possible to compare and contrast the effect of compounds on the tumor systems and on the nontumor systems. When this was done, It was found that the correlation between the action of a compound in the tumor systems and in the nontumor systems was poor, the oser-all matching of activity between the tuo systems was no better than 50 per cent From this evidence, as well as a number of other analyses carried out, it was concluded that the action of conta ·LABORATORY, CLINICAL APPROACHES TO LEUKEMIA CHCNOTHERAPY 87 pounds against tumors was not merely an expression of general biologic activity, but rather that there was some specificity of action This general conclusion was further supported by the fact that no nontumor system was found to respond sufficiently like the tumor system to be used as a substitute Finally, an examination of the response of the individual tumors indicated that no single cxperimental tumor responded in such a way that it alone could be used as an antitumor screening system from the evidence available, it was concinded that ccicral tumors should be utilized in such a screen. A partial test of the validity of the conclusions reached in this cxperiment can be made by comparing the results obtained against the experimental tumors with the results obtained in the clinic. Table XVIII presents those compounds TARLE XVIII CORRELATION BETH FE EFFact or "Ace" CovPOUNDS AGAINST EXPERIMENTAL TUMOR SCREEN AND ACAINST HUA TUMORS .. which were found to be positive in the experimental tumor screen, together with thesr effect against huinan neoplasms Table XIX summarizes this comparison for the compounds adjudged negative in the experimental tumor screen. Although not all of the compounds have been tested in the clinic, nor have the clinical trials been exhaustite, the correlation, on the whole, is encouraging TABLE XIX CORRELATION BETWEEN EFFECT OX "IVA TIYF CostPovaas AGAINST EXPERIMENTAL TUMOR SCREEN AD) Acalyst IIUMAN TUMORS ·88 ITIOLOGY AND TRCATMENT OF LEUKEMIA Alany investigators think that drugs now used in the clinic are relatively ineffective and that, therefore, the screening system must be relatively faulty Before accepting this rather discouraging point of view, it should be noted that the effect of these drugs on the experimental tumors also leaves much to be desired. In fact, the action of these compounds on human neoplastic disease is, in the main, greater than on the experimental tumors. It, therefore, would appear to be reasonable to hope that when compounds are found which destroy experimental tumors effectively, their clinical activity will be comparably great. CHEMOTHERAPY IN THE CHRONIC AND ACUTE LEUKEMIAS The clinical chemotherapy of the blood dyscrasias will be discussed at greater length and more authoritatively by Dr. Norwenna Till and Dr. Joseph H Burchenal, but I would merely like to offer our experience in the treatment of the chronic leukemias with two new drugs and also to present the treatment of acute leukemia in adults with massive doses of adrenal cortical steroids The work of members of the Chester Beatty Institute in London, under the direction of Dr Alexander Haddow, in the des clopment of new alkylating agents, has added two new compounds of interest. With Dr. George Timmis, Haddow described the effect of 1,4-dimethanesulfonyloxybutane, Myleran, an experimental tuniors ? From these observations, Drs D A. G Galton and M. Till carried out clinical studies which led to the use of this compound in the treatment of chronic myeloid leukemia. I wish to mention our results only because they are comparable to those obtained by Galton and Till, but our dosage system is somew hat different from that used by the British investigators.** In our series, we found that the daily oral administration of 10 mg. of Wyleran can be used safely. It is our practice to continue this dosage until the peripheral white count has been reduced to 25 per cent of its initial level At this time, the drug is discontinued to determine the level to which it will fall without treatment The objective in all instances is to bring the leukocyte count to the normal range. Table XX summarizes our results, together with TAALL XX TREATMENT OF CHRONIC MELOID LEUKEMIA WITH MYLERAS I\VESTIGATOR Galton and Toll Petrakus and associates $3 Haus and associates 86 Gigante and associates Pribylla and Stollbergs AUMBER OF CASES 30 11 16 14 10 81 21 102 DAILY DO SE (MO) 4.10 2-25 4- 6 10-20 2.6 2.25 2-20 2-25 DURATIO REMISSIOS REMISSION VUMBER PER CET (MONTIS) 26 86 0-48 7 64 0-19 14 88 2.21 12 86 0-5 9 90 4.15 68 83 0-48 17 61 0-48 85 83 0-18 Delafield-Presbyterian Group (1955)*4 Grand total ·LABORATORY, CLINICAL APPROACHES TO LEUKEMIA CHEMOTHERAPY 89 those obtained at other clinics. In our estimation, which reflects the consensus, Myleran is a useful addition in the treatment of chronic myeloid leukemia Bis-8-chlorcthylaminophenylbutyric acid, also known as Chester Beatty 1348 and chlorambucil, recently was introduced into clinical therapy by Galton and his associates" at the Royal Marden Hospital. Our experience with this compound in the treatment of chronic lymphoid leukemia is presented in Table XXI. As can be secn, the results are les favorable than those obtained in the treatment of chronic mycloid leukemia with Myleran. This is not unexpected, for cien radiotherapy does less well in the treatment of the chronic lymphoid dyscrasias than in the treatment of the my cloid varicty. TABLE XXI RESULTS OF CB 1318 THERAPY VUMBER OBJECTIVE TE PROVEMENT OF SLIOUT PATIENTS FXCELLENT GOOD VOE SUBJECTIVE IMPROVEMENT DIARKED OR MODERATE DIAGNOSIS Chronic lymphatic leukernta .twenty courses 18• 3 8 9 8 The treatment of acute leulenria in adults has lagged behind the treatment of this discase in children Recently, Hill and Vincent reported that large doses of cortisone in combination with 6-mercaptopurine and other compounds could produce remissions in the adult acute leukenna. We have carried out an ini estigation over the past eighteen months at our clinic on the effect of massive doses of adrenal cortical steroid in adults with acute leukemia.*2 In eight of sixteen adults, hematologic remissions were achicked with doses of cortisone up to 6 Gm a day, or of prednisone in daily doses of 1 Gm In four of these eight, the remissions were complete with res ersion of the bone marrow as well as the peripheral blood to normal. These results, together with trials in subacute leukemia and the blastic crisis of chronic myeloid leukemia, are summarized in Table XXII Although the remissions achieved were complete in a significant proportion of the cases, the duration was only measurable in months, and the longest duration of life achieved thus far has been one year The toxcity of massive, steroid dosage is appreciable, and it includes overwhelming TABLE XXII EFFECT OF PREDNISONE (1 000 MG DAILY) ACUTE LEUKEMIA OF ADULTS UITBER OF CASES 17 HEMATOLOGIC REMISSIONS PARTIAL COMPLETE DIAGNOSIS Acute leukemia Subacute leukemia Blastic Crisis of chronic myeloid leukemia no EFFECT 6 2 DURATION OP EFFECT {AO THS) 1-12 8 3 1 2 ·90 ETIOLOGY AND TREATMENT OF LEUKEMIA infection, gastrointestinal ulceration and perforation, electrolyte disturbances, steroid diabetes, and psychic disturbances from the results of our study, we concluded that the demonstration that hematologic remissions could be produced in adult, acute leukemia by large doses of adrenal cortical steroids was of greater heuristic than practical value. THE EFFECT OF BLOOD DYSCRASIAS ON HOST METABOLISM It is increasingly recognized that the destruction of the host by an invading tumor is less often due to impairment of vital functions through pressure or rcplacement of the infiltrating tumor cells than by more subtle and yet more devastating effects on the normal body economy. Fever, cachexia, and anemia constitute some of the manifestations of disruption of physiologic functions in patients with the leukemias. In the past few years, interesting and valuable insight has been cast on the mechanism of anemia in the blood dyscrasias º Marks and Bishop," at our institution, demonstrated a fundamental disturbance in carbohydrate metabolism in patients with epithelial tumors and blood dyscrasias. This is characterized by a decrease in the rate of glucose removal from the circulation as determined by an intravenous glucose tolerance test. Of particular pertinence to the present discussion has been the demonstration that individuals with chronic leukemia who have been treated effectively with available chemotherapeutic agents show an improvement in the abnormal carbohydrate metabolism as well. The precise mechanisms involved in this change in carbohydrate metabolism are under study In this investigation, the chemotherapeutic agent is of great value as a research tool. SUMMARY Problems involved in the selection of chemotherapeutic agents for the treatnient of the leukocite discrasias have been presented, together with methods of procedure currently employed. Three compounds which emerged from empirical antitumor screening and were applied to human leukemua therapy were presented briefly Finally, the use of chemotherapeutic agents as tools to study the pathologic physiology of leukemia was indicated The vigorous ini estigative pursuit in these three areas demonstrates that research in the leukemias is proceeding actucly and productively. It would appear that reasonable, if not spectacular, progress is being made DISCUSSION DR. SECALOFF Those of us who have followed the course of the experiments which Dr. Gellhorn outlined in the beginning have great admiration for the amount of work in olied, and are disappointed at the conclusions that of necessity must be drawn from the data A continuation of screcning proba- ·LABORATORY, CLINICAL APPROACHES TO LEUKEMIA CHEMOTHERAPY 91 bly will disclose a greater number of similar compounds, and perhaps some of the similar compounds will be somewhat better. Dr. Gellhorn, would you speculate as to the direction screening should take in an effort to obtain better, similar compounds? How would you select compounds for futher screening, and how would you select the dose of any given compound? It has been our experience in animal tumors that the largest tolerated dose is not always the optimum. DR. Gelliiory: Dr. Segaloft has asked searching questions which could be the basis of extended discussion. With regard to desclopments in the area of experimental cancer chemotherapy screening, I am sure that Dr. Burchena) could comment on the usefulness of using mouse leukemias as test objects for the sclection of drugs which may be effective in human blood dyscrasias. In our laboratory, we have attempted to develop a screening system which will aid in the discovery of drugs to inhibit the growth of, or destroy, human brain tumors. This system consists of a chemically induced glioblastoma multiforme in the mouse which can be serially transplanted in the strain from which it originally arose The tumor also is grown in tissue culture. Fragments of human brain tumors obtained at exploratory craniotomy are alo grown in vitro. The effect of compounds is determined on the mouse glioblastoma in vrlo and also in vitro and also on the human tumors in vitro. Ultimately, drugs which have an effect on some or all of the components of this multistrand screen are evaluated on patients with brain tumors. It is hoped that when sufficient data have been accumulated, this screen will be found to have relevance to a particular human tumor Dr Arthur Kirschbaum has forcefully and persuasiicly presented the position that spontaneous tumors in experimental animals should be used for cancer chemotherapy screening since they niay be more comparable to human The pragmatic reasons for eschewing spontaneous animal neoplasms for routine screening are that they are difficult to obtain in the requisite number, their natural history is so sanied that reasonable untreated control groups would be difficult to establish, and, further, no evidence has really been offered to indicate any real advantage of these tumors over the transplantable variety The selection of the optimal dosage schedule is difficult Since it is not possible to translate laboratory results on this issue to the clinic, the procedure is necessarily empiric The initial objective is to administer a maximal tolerated dose If this is effective, it is gradually reduced until experience in many chnics leads to the definition of a dosage regimen with the optimal therapeutic Index DR KIRSCHBAUM After viewing this array of tests in a quest for something which will have antitumor action, it seems to me a little unlikely that such a thing will be found After all, the effect of estrogenic hormone on prostatic cancer is due to its physiologic action, and why would estrogenic hormone cancer ·22 LTIOLOGY AND TREATMENT OF LEUKEMIA have any cffect on adenocarcinoma of the colon? One can easily understand why, in the case of prostatic cancer, there may be an effect, but not in the second instance. As a matter of fact, anything which affects all tumors would not be specifically antineoplastic, but its effect could be explained on some other basis. As far as transplanted and spontancous tumors are concerned, what we would really like to do is test something which closely simulates the human disease. Because there are not enough spontancous leukemias available at the present time is no reason to say that the transplanted disease is better merely because transplanted leukemias are more easily obtained. There is a very good reason why the transplanted leukemia is not a valid test medium. Trcatment is instituted within one to four days after transplantation, and this interferes with histocompatibility. Once the leukemia is established, then there is no antiluekemic effect, which strongly suggests that the drug is active in interfering with the establishment of the tumor. A correlation betucen the effect on the transplanted tumor and the effect on clinical disease is necessary for a working basis If one uses spontaneous leukemias of mice, the results are as discouraging as they are in clinical leukemia. In other words, if the experiment is loaded so that results will be obtained, the intestigator may be happy, but the patient is not any more improsed. Finally, it is possible to obtain large numbers of spontaneous leukemias. We do not have a tremendously large colony, but wc do have as many as six hundred spontancous leukemias occurring in a year. I think that testing on spontaneous Jeukemia might very well precede testing on the human disease I am not disparaging the effort with transplanted leukemias It series to provide useful Icads, but I think that we have to be realistic. The results are disproportionately encouraging, and I would question the statement that the effect on human leukemia is actually better than the effect of the corrcsponding drugs on mouse leukemia I would like to know the measure of effectiveness that is being used DR. BURCHENAL The important thing in a screening test in the first place is to have something that is moderately sensitie. If you screen, as uc have, ay 16,000 compounds in the past ten years, the important thing is not to miss one which might be effective against man That is one of the rca ons we like sarcoma 180, because it cems to be a fairly sensitnic test, and it is truc me are not treating established sarcoma 180, but rather putting it in bs trocar and starting to give the drug twenty-four hours alter the tumor implant is moculated That is not treacrteat. That is a test for inhibition of growth. and it is certainly true, as Dr Kirchbaum says, if one allow the sarcoma to be established for ninety-six hours, it is harder to treat. For preliminary screening, it does not matter too much whether bacteria or sarcoma 180 cells are screened, either in the growth inhibiton or the treatment stage The important ·LABORATORY, CLINICAL APPROACHES TO LEUKEMIA CHEMOTHERAPY 93 thing is to find something that indicates an effect. Then, later, after an effect is found, one can go on to something like established sarcoma 180, spontaneous leukemia, or established transplanted Iculcmia, and soc what these much more rigorous tests reveal We believe Dr Gellhorn has several discrepancies in his figures An example: Is methylformamide positive in mice and negative in patients? We have tried only five patients with this compound, and it is extremely toxic to the laer. It may be that if we could give twice the dose which we do give, it might have some effect on tumors As it is, we are limited by liver toxicity which Is even greater in man than mice. Azaguanine causes marked dermatitis in man, which does not occur in the dog, and may be a specific sensitivity of human skın. Perhaps if we could give larger doses, we could affect leukemias or tumors. The colchicine dervatives, for instance, were down as questionable I remem. ber initial positive work on colchicine in mouse leukemia by Dr Kirschbaum There is some question that it is active against Hodgkin's and other diseases, but it is pretty toxic. Some of the newer derivatives are positive. One of these newer agents, which is by far the most potent against sarcoma 180, is desacetylthiocolchicine It has an effect on chronic granulocytic leukemia The diamı. nopurine is effective in a fairly limited number of mouse tumors, and it is equally limited in its effect on leukemias In chronic granulocytic leukemia, it is possible to show an effect with this compound, but one must have a patient who is an iron man with copper-lined insides to do so, because it causes considerable nausea and vomiting If enough can be taken, it lowers the count We have seen that many times in the same patient To come back to spontaneous leukemia, the only thing that would concern me about deciding on a trial in patients, on the basis of final tests against spontaneous leukemia, would be that there are probably different kinds of spontaneous leukemia biochemically, even in the same strain of mice pound effective against one might not be effective against another, in patients as well as in mice I do think, however, that spontaneous mouse leukemia is closer to human leukemia than any of our transplanted leukemias DR GELLHORN. Both Drs Kirschbaum and Burchenal have stated that spontaneous mouse leukemia is more comparable to human leukemia than transplantable leukemia This appears reasonable, but only when the parameters are defined In the case of screening for antıleukemic drugs, I know of no evidence which clearly demonstrates the superiority of the spontaneous form over the transplantable varieties Dr Kirschbaum says that he has six hundred animals with spontaneous leukemias per year--in good years This number of animals would make possible the screening of about six compounds, which is scarcely a sufficient annual output of work DR BURCHENAL There must be correlation with human leukemia, of course. The sistem is significant as long as it correlates with respect to response. A com- ·94 ETIOLOGY AND TREATMENT OF LEUKCMIA DR. GELLNORN: Dr. Burchenal makes a plea for the use of microbiologic systems in screening. I reiterate that, in the study discussed, fifteen separate species failed to indicate responses which correlated well with results for clinical or animal tumors. I lasten to add that microbiologic systems can, and have, given valuable information on mechanisms of drug action which, in turn, have led to the development of new antitumor drugs Dr. Kirschbaum has asked for evidence that antitumor compounds are more effective in man than in animals This could be illustrated by many examples, but perhaps one will suffice. Nitrogen mustard can, and does, cause tumor regression in Hodgkin's disease and lympho arcoma. Against experimental tumors, at best, it produces partial inhibition of the growth ratc. Dr. SEGALOFF. How many compounds that are negative whers screened have been effective against leukemia? The use of neither spontaneous nor transplanted mammary cancer in mice has been particularly helpful as an inder for the most effective compounds for treating human breast cancer. Therefore, I wish to direct attention to possible screening of compounds which are not effective against tumors in animals but may be cffective against human tumors DR. KIRSCHBAUM I think that the correlation that Dr. Burchenal has demonstrated between transplanted leukemia and clinical leukemias is quite good, and I would be the last one to say that transplanted leukemia should not be used. I realize the number of spontaneous Icukemias is too few for a routine program, and think spontaneous leukemias should be included The question arises as to why the transplanted tumor is not as comparable or not as good an index as the spontaneous tumor. One can immunize against the transplanted tumor, but not against the spontaneous tumor, and, following the regression of transplanted tumors, the anima) is immune to renoculation We know this host-immune response may contribute to regression, and this may not be part of the effect of the drug This is not true for the spontaneous ncoplasm, and we have no evidence that the immune response in the patient contributes toward the regression DR. GELLHORN That is impressie I could not question differences between the spontancous and the transplantable animal tumor, but this is not the issue in our discussion The question is whether or not there is a peculiar value of the spontaneous versus the transplantable tumor in the selection of drugs which will be clinically useful In answer to Dr Segaloff's comments, we do not have a great deal of esidence on the question of the potentia) hazards of fake negatives in experi. mental chemotherapy screening. In the experiment which I haic summarized for you, there were ele en compounds which had no effect on animal tumors Cross correlation with available clinical evdence indicates that the only com- ·LABORATORY, CLINICAL APPROACHES TO LEUKEMIA CHEMOTIICRAPY 95 pound which might have been mixed by the experimental tumor screen is colchicine. This drug did not produce an effect on the tumors, although it ma), and note I say only may, have some ellect on human tumors. DR. SEGALOFF: Should you not rest at least af equivalent number of compounds that are negatn e in your screening test against human leukemia? D& BURCHENAL: Another thing to keep in mind ishen talking of screening tests is that there are many screcning tests Most spreching tests do not shon the adrenal steroids to be especially effective against mouse leukemia There are certain mouse leukemias that will respond to them, but there are not too many. Thouc might have been missed if only a couple of leukemias had been used, as to your ansser, 1 do not see how we can tell, short of ten thousand compounds found negative for patients, and I do not know how we will do that OR GELLTORY We do not have time for ten thousand, but the elenen for the most part have been tested This is my point. De BURCHEVAL. Colchicine is negatic because of toxicity in man There is nothung Practical about colchicine Dr. Kirschbaum uses it in his test against mouse leukemias. D& SECALOFF We are not talking about toxicity DR BURCUIENAL I am talkug about practicality. DR SEGALOFF The question is whether it works or not, whether it is toxic or whether st not 10xc. DR BURCIIEVAL. If I too toxic, only a relatne effect is obtained. No matter what the toxicity, the compound can be useful less than a toxic dose is effective, but if it is toxic at a dose which is ineffective, it is not useful Di SECALOFF. That is right However, the Ixer can be irreparably damaged with a compound such as one already mentioned, thus really has no bearing on the point as to whether it is inirically an effectie compound against leukemia itsell Admsttedly, the limits whether or not one is going to use it in patients, but the fundamental question as to its effectneness against leukemia per se has nothing to do with its toxicity DR BUKCHENAL I do not agree with you The whole idea of the screening program is to get a compound to be used in patients I do not care how effective it us against leukemir cells il u cannot be used in patients because of torsctv Do you see my point) DA SECALOFF We seem to have two different concepts beheue that it may be possible to alter a compound so as to change its toxicity and retain #5 effectnenes, against leukemia Dr GROSS I think there 4 a fundamental difference between the transplanted leukemia and spontaneous leukemia, and, while it may be expedient ·96 CTIOLOGY AND TREATMENT OF LEUKEATA at the present time to use transplanted leukemia for the tests, it is possible that certain conclusions can be missed and certain effective compounds may be missed if only transplanted leukemias are used for the screening tests. Anyone who has worked with both transplanted and spontaneous leukemias knows there are basic differences In the case of spontaneous leukemia, after a suitable length of time, one has a variety of forms of leukemias, some chronic, some acute. These different forms also occur in human leukemia. Spontaneous leukemia can be compared to the virus-induced leukemia in the laboratory. At the present time, we have virus-induced leukemia in chickens which appears in two or three weeks in 100 per cent of the chickens inoculated with cell-free extracts. Why are chickens not used for the screening test? We may have, within a few years, a similar virus-induced leukemia as in mice. Perhaps not all, but selected compounds should be checked against spontancous leukemia. Dr. Till: We may not be wise to focus all our energies in looking for substances that are antimetabolites, substances that will destroy the leukemic cells. Perhaps whatever causes leukemia causes a deficiency of some essential substance within the cells, and, as a consequence of this, the cells are unable to mature. DR, SPRACUE. From the preceding discussion, it seems that there is no satisfactory screening method at the present time. It would seem desirable that the method of screening that would approach human leukemia as closely as possible would be the most desirable. Considerable work has been done in the field of bone marrow cultures, and, perhaps by improving such methods, tissue culture of human leukemic cells might prove the most satisfactory in terms of screening antileukemic agents Certainly, it would seem that a concerted effort along this line would be justified Dk BURCHENAL· Theoretically, that is an excellent idea I do not know the percentage of human leukemic cells that can be cultured, but my impres. sion is that it is mighty low. Osgood has three lines of cells he has cultured, and is studying the use of synthetic media For the purpose of discussion, Jet us say the strains can be grown on synthetic medium and compounds can be tested against them The very fact that those three out of hundreds can grow in trssue culture must mean they are biochemically different from ordinary cells Until a method is avaşlable whereby we can culture cvery leukemic cell, question the value of such a method, but it is something that should be pursued as an important technique If only special cells are cultured, they may be of limited value as far as the usual leukemic cell which is encountered is concerned. DR. SYVERTOS To date, we haie failed to culture mouse leukemic cells, but it is not difficult to maintain them in vitro for tseke or more days Another approach is to emplos, in sitro, actuely metabolizing cells in combination or I ·LABORATORY, CLINICAL APPROACHES TO LEUKIMA CHEMOTHI RAM 97 admixture with a test antímetabolite or chemical and to add a virus to this ad. nixture. Production of virus by the cell series as an indicator of the ability of the lot antimetabolite or chemical to influence the metabolism and/or kill the host ccll In other words, the inherent capacity of the cell to produce vinus is a more delicate measure of its metabolism than direct manonictric measurement. This approach demonstrated that mout 50-called antiiral agents are anticellular metabolic agents which operate by inhibiting cellular metabolism and in turn by inhibiting formation of seius, DR. BURCHENAL' Is it not true that nomal human cells, growing in tissuc culture, have changed and become neoplastic, so it is hard to sas what a leukemic cell or any other cell will do in Irskue culture? Dr. SYVERTOV: 1 question the statement that human cells growing in tissue culture become neoplastic Supportive cridence consists of changes in anatomc or morphologic configuration, which can be attributed to mfluence of suboptimal growth medium, and to demonstration on transfer by injection of setcral million cells to man or animal is the production of a small nodule. The nodule shows on section cells at different issue kerels which competent pathologiats interpreted as euidence of cancer This eudence is subject to other interpretation is so far as it applies to cells derssed from cell cultures and not to spontaneous tumors I should appreciate Dr Burchenal correcting me if I misunderstand the evidence to date DR. GELLHORN In closing, I would like to take issue with Dr Sprague's statement that our screening methods are inadequate At the present time, I believe we can only say that the compounds which have been screened are Hadequate Until we have compounds which can cure mouse leulemsa but fail to modify human leukemia significantly, we cannot indict the screening methock personally feel that when an effectue drug against mouse leukemia is uncovered, it will also be of great value against human leukemia hope that the ume ss!! sos come when the screening method can be put to critical test Let us ·Joseph V. Schlosser, MD IRRADIATION THERAPY of LEUKEMIA Irradiation therapy is extremely useful in the management of leukemias, and it probably is responsible for more bencficial results than the majority of therapeutic chemical agents at hand. This does not mean that steroid or other chemotherapy is without salue, or that they necessarily are competitive methods of treatment As a matter of fact, the two methods of treatment often are integrated. In certain situations, chemotherapy has allowed irradiation therapy which otherwise would not have been possible. Good examples for this management are found in cases of acute leukemia. In addition, chemotherapeutic agents offer additional treatment to curb a crisis, and then irradiation therapy may be given again For example, patients with leukopenia who have become resistant to further irradiation treatment have been gi en steroid therapy with a change in the white count, and then additional irradiation therapy has been administered In general, we prefer to start treatment with irradiation therapy Perhaps, in a few tears, se mai say that he prefer to start with busulfan m myelogenous leukemia Certain differences exist between our approach to the creatment of lym. phocytic and of myelogenous leukemia by irradiation Usually, an attempt is made to kecp the blood count within fairly normal limits Most therapists attempt to do this whether treatment consists of irradiation or a chemotherapcutic agent However, we find that this is more important in the myelocytic group of leukemias than in the lymphocytic group in my clogenous leukemia, the blood count should be maintained as near the normal level as possible by irradiation, but, in lymphocytic leukemias, alteration of symptomatology in the more important. If the patient with lymphocytic leukemia has a tremen- 98 ·JRRADIATION THLRAPY OF LEUKEMIA 99 dous spleen, bulky lymph nodes, or painful arcas, or if he is extremely weak or has anemia, irradiation is gisen until the findings and symptoms are corrected We have found that patients with lymphocytic leukemia can have elevated leukocyte counts for considerable periods of time, and sull remain in cxcellent general condition with no particular complaints. On the other hand, in my clogenous leukemia, trouble usually follows an increase in the number of leukocytes. If the high count is alloned to persist, the pacient usually exhibits more pronounced symptoms. There are four major methods of x-ray therapy. The first is that most generally used, irradiation over the spleen The others are: local irradiation over either lymph nodes or visceral areas; the radiation of large segments of the body, usually hall of the trunk, either the chest or the abdomen, for extensiic in oliement in these areas; and total-body irradiation. There are some variations of these forms of r-ray therapy, but the small differences are of no consequence Splenic irradiation probably is the one that is most frequently used, and usually we use it first, regardless of which type of case we are treating. Fields usually are 15 by 15 cm, with treatment alternating to the anterior and posterior spleen, starting at dosage levels of 25 r and increasing the dosage up to 100 to 150 r per day. In unusual cases with bizarre skin and hemic manifestations, initial doses as small as 10 r over the spleen may be gren, gradually increasing the dosage as before in the case of lymphocytic leukemia, irradiation over the spleen sometimes will also allow assessment of the sensitivity of inohed lymph nodes to irradiation, since systemic effects may follow local irradiation over the spleen Sufficient regression in the size of peripheral lymph nodes may occur, so that much smaller doses need be administered to them subsequently in myelogenous leukemia, local irradiation over the spleen 19 continued if the response is good Ist appears that excessze irradiation over the spleen is required for the desired response, then total body irradiation is used. Local virradiation, of course, is one of the most useful ways of administering this type of therapy. Even if the patient is having some type of chemotherapy, very frequently local zones of involvement will not regress successfully eren in a patient who is showing a good general response to the drug, and residual zones of involvement may be left These can be in the form either of an enlarged spleen, enlarged local or mediastinal lymph nodes, or visceral involve- In many of these instances, doses in the range of about 1,000 r are delivered to the organ itself Olten, complete disappearance of the masses occurs within one week Local lymph node inval ement with lymphatic leukemia will regress completely with a similar dose in the same period of ment time, ·100 ETIOLOGY AND TREATMENT OF LEUKEMIA Occasionally, patients present themselves with massive liver and splenic involvement, or with extensive pulmonary infiltration, and in this instance we employ a technique or modification of it that we have used for pulmonary metastases in other types of carcinoma. Very large fields, 25 by 25 cm, or 30 by 30 cm., are employed a distance of 150 cm. from the tube, and daily doscs in air in the range of 50 r to 75 r are administered each day, alternating anterior and posterior ports. In the case of leukemia, the dosage required is lower than when carcinoma is treated. The usual dosage is 1,000 r to 1,250 r m air to the chest within a period of approximately one month. Leukemic infiltrations may disappear even sooner. Although a dose of 1,000 r in a month's time may seem smaller than 1,000 r delivered in one week to the small local areas of involvement mentioned previously, actually it represents higher energy absorption because of the tremendous volume of the field. In like fashion, when total-body irradiation is given, dosage is reduced even more than the usual 75 r per day tolerated by half of the trunk. The last method of treatment is the one we are using more and more. As a matter of fact, some of our more recent patients have been treated by totalbody irradiation almost exclusively, after a test dose of irradiation to the spleen to check the likelihood of a crisis with precipitous fall in the white count. We obtain blood counts at intervals of one month to six weeks, use total-body irradiation to bring the count to normal, and, when the count shows a tendency to rise, then gre more whole-body irradiation. The method of Mayneord” is used in calculating the dosage of total-body irradiation. You will recall that when Dr. Upton wished to determine the energy absorption in his mice, he considered only the roentgens delivered and the weight of the mouse, because there is not appreciable absorption from point of entrance to exit. This distance in patients is sufficiently great to make a correction for the intensity of irradiation necessary Dosage is calculated on the basis of energy absorption per unit mass of tissue Our plan of treatment has been to administer daily doses of one megagram roentgen, alternating anterior and posterior exposures for four doses Alter a minimum interval of one day, this may be repeated for a maximum total of sixteen to twenty megagram roentgens. We have compared the results obtained with wholc-body irradiation therapy of twenty-seven patients with my elogenous leukemia to the survival of twenty-four patients treated with splenic irradiation When all factors such as sunival for a period sufficiently long to recen e total-body irradiation are taken into consideration, we found no significant difference betwecn the survival following the two types of treatment, respectively Unfortunately, cases of lymphatic disease suitable for such a companson were not great cnough for valid cialuation We have changed our views recently concerning irradiation for acute leu- (hemohemia, due to the altered course of the disease following steroids ·IRRADIATION THERAPY OF LEUKEMIA 101 therapy. Although we continue to be extremely cautious about the amounts of irradiation used, these patients frequently have enlarged lymphoid tissuc in thc pharnyx, tonsillar arca, nech, and spleen which is symptomatic and may not only be very burdensome but also a factor in survival In one instance, lymphod tissue in the pharynx was encroaching on the laryngeal and esophageal inlet. Small doses of radiation over these areas may produce remarkable regression, and the symptomatic improvement is worth while, even though temporary. The remarks which have been made are based on an analysis of fifty-threc cases of lymphatic Icukemia and sixty-five cases of myelogenous leukemia trcated by irradiation therapy. Thesc data, as well as results obtained by others, indicate that survival of leukemic patients properly treated compares favorably to that following the onset of many types of cancer. (For Discussion, see p. 118 ) ·100 CTIOLOGY AND TREATMENT Or LEUKCMIA Occasionally, patients present themselves with massive liver and splenic involvement, or with extensive pulmonary infiltration, and in this instance we employ a technique or modification of it that we have used for pulmonary metastases in other types of carcinoma Very large fields, 25 by 25 cm. or 30 by 30 cm, are employed a distance of 150 cm, from the tube, and daily doses in air in the range of 50 r to 75 r are administered each day, alternating anterior and posterior ports. In the case of leukemia, the dosage required is lower than when carcinoma is treated. The usual dosage is 1,000 r to 1,250r in air to the chest within a period of approximately one month. Leukemic infiltrations may disappear even sooner. Although a dose of 1,000 r in a month's time may seem smaller than 1,000 r delivered in one week to the small local areas of involvement mentioned previously, actually it represents higher energy absorption because of the tremendous volume of the field. In like fashion, when total-body irradiation is given, dosage is reduced even more than the usual 75 r per day tolerated by half of the trunk. The last method of treatment is the one we are using more and more. As a matter of fact, some of our more recent patients have been treated by totalbody irradiation almost exclusively, after a test dose of irradiation to the spleen to check the likelihood of a crisis with precipitous fall in the white count. We obtain blood counts at intervals of one month to six weeks, use total-body irradiation to bring the count to normal, and, when the count shows a tendency to rise, then give more whole-body irradiation. The method of Mayneord” is used in calculating the dosage of total-body irradiation You will recall that when Dr. Upton wished to determine the energy absorption in his mice, he considered only the roentgens delivered and the weight of the mouse, because there is not appreciable absorption from point of entrance to exit. This distance in patients is sufficiently great to make a correction for the intensity of irradiation necessary. Dosage is calculated on the basis of energy absorption per unit mass of tissue Our plan of treatment has been to administer daily doses of one megagram roentgen, alternating anterior and posterior exposures for four doses After a minimum interval of one day, this may be repeated for a maximum total of sixteen to twenty megagram roentgens We have compared the results obtained with whole-body irradiation therapy of twenty-seven patients with myelogenous leukemia to the survival of twenty-four patients treated with splenic irradiation When all factors such as survival for a period sufficiently long to receive total-body irradiation are taken into consideration, we found no significant difference between the survi al following the two types of treatment, respectively Unfortunately, cases of lymphatic disease suitable for such a comparison were not great enough for valid evaluation. We have changed our siews recently concerning irradiation for acute leukemia, due to the altered course of the disease following steroids and chemo- ·CILORANBUCIL AND BUSULFAN IN TREATMENT OF LEUKEMIA 103 chronic myclogenous leukemia, busullan is as elicctise as repeated splenic irradiation, and it is more comicnient to the patient. We have not compared its cfficiency with that of whole-body irradiation or radioactive phosphorus administered according to Osgond's recommendation, but we believe it to be superior to other chemotherapeutic agents such as triethylene melamine, urethan, benzenc, or arsenic. The response is less crratic with busulfan, remissions are longer, and there are no side effects iſ the drug is administered properly. Patients have heen maintained in good chnical and hematologic condition for periods of two to fic vcars on busullan alonc. Although various douc schedules were employed in the carly stages of clinical trial, we have not used daily docs greater than 0065 mg. per kilogram (4 0 mg daily for most patients) in the last four cars. In initial loading dose was used a few time, but was not found helpful Doses larger than 60 mg daily offer no advantages and may cause seserc bone marron damage These large domes cause the Icukocyte count to fall as quickly as it does following splenic irradiation, but the subscquent ric in hemoglobin level is not greater. and does not occur sooner, than in patients receiving 40 mg. daily in our experience, hemoglobin lescls usually start to risc after four wechs of treatment and reach normal valucs after approximately three months This pattern is similar in patients treated with either splenic irradiation or busulfan, and the rate of elevation in hemoglobin level depends upon the initial value, being inde. pendent of the rate of decrease in leukocytes Patients receiving daily doses of 4.0 mg, busullan norinalls show some fall of leukocite count during the first month of treatment, but occasionally there is no significant fall for six or even eight weeks This may lead to the abandonment of the treatment by some clini. cians on the ground that the patient is resistant to busulfan Patients showing this delayed response are often those who have recensed several previous courses of splenic irradiation with remissions of shorter and shorter duration between These patients may do well on busulfan if it is administered continuously 1 small number of other patients who have recensed no previous radiotherapy may also respond slowls to busulfan. There usually is great subjective improvement, esen though the leukocyte count falls slowly and the number of immature cells in the peripheral blood remains high The spleen in such cases, although decreasing in size considerably, tends to remain moderately enlarged Il'e find continuous treatment is more satisfactory than intermittent treatment in these cases also Fig. 12 shows the slow response to busulfan in a patient who had received two previous courses of radiotherapy The long interval before the leukocyte count reached normal lesels should be noted, also, the hemoglobin continued to fall slowly during the fint eight weeks of busulfan treatment, but it rose subsc. courses ·MormenTill, MB THE USE of CHLORAMBUCIL (CB 1348) and BUSULFAN (MYLERAN) in the TREATMENT OF LEUKEMIA On this my first visit to the United States, it has been a great pleasure to meet so many distinguished workers in the field of leukemia, and to learn so much about their ideas and opinions on the subject in return, I should like to tell you something of the views of Dr. Galton and myself on the treatment of leukemi. with two drug« first synthesized at the Chester Beatty Rescarch Insti. tutc 'The first of these is busulfan (Mylcran), and the second, y-(p-di-2-chlorocthylaminophenyl) butyric acid (chlorambucil), has the code name GB 1348 and is an aromatic nitrogen mustard suitable for oral admunistration TREATMENT WITH BUSULFAN It is now widely accepted that busullan can induce remissions associated with fall of leukocyte count, regression of the spleen, and a rise of hemoglobin in patients with chronic my clogenous leukemia The only patients we have seen who proved resistant to the drug Initially were those already in blastic relapse, and one patient in whom there was no clear distinction from nonleukemic myelosis. We have found that in the long-term treatment of cases of *The work has been supported by grants to the Chester Beatty Rescarch Institute (Institute of Cancer Research Royal Cancer Hospttall from the British Empire Cancer Cam. paign, the Jane Coffin Childs Memohal Fund for fedical Research, the Anna Fulfer Fund. and the National Cancer Institute of the National Institutes of Health, United States Public Health Service 102 ·CHLORAMBUCIL AND BUSULSAN IN TRIATMENT OF LEUKEMIA 105 MYLERAN mo dally Bring нь%. (100%.148 mg per 100 ml) 100 000 TOTAL COUNT 50000! 2000! I9000 WHITE - CELL COUNT (per c. mm) spool 2poo 1ood IMMATURE MYELOID CELLS looL 1931 1952 1993 1954 Tig 13—Blood counts of a patient with chronic myelogenous leukemia who recen ed two courses of busulfan followed by continuous therapy (From Galton, DAG, and Toll, M Lancet 1: 425, 1955) Hb% (100%-14 89 per 100 ml) HAEMOGLOBIN 200000) logpool 50000! TOTAL LEUCOCYTES WBG per cu, mm 290001 10,0001 5000! IMMATURE 200g MYELOID CELLS 1000) HYT 700 SPLEEN 2 DAYS MAR 1953 MYLERAN 4mg daily. 2.1 103 DEC JAN MAY AUG DECIJAN 1954 MAY AUG 1955 DEC JAN 11956 TR 14--Blood counts of a patient with chronic myelogenous leukemia who recensed continuous therapy with busulfan following a single course of radiotherapy.

·104 CTIOLOGY AND TREATMENT OF LEUKEMIA quently without the aid of transfusion. This patient remained well on continuous treatment with busulan for more than two years During this time, he was very fit and able to carry on his business, which entailed extensive global traveling In new cases, we do not start treatment iſ the patient has no complaints. When ymptoms occur, we give a course of busulfan, 4.0 mg. daily, and iſ, as in the majority of cases, the leukocytes fall satisfactorily, the drug is stopped when the leukocyte count reaches a level of approximately 10,000 per cubic millimeter. Such a course may last two to three months. After this, we do not treat again until symptoms recur, which may not be for another twelve months IRRADIATION TO SPLEEN 20 MYLERAN mg dolly 1600r 1800r 4 (100% -14 8mg per 100ml) H0 % 100! BOL 601 200,000 100,000 50,000 INNOS 7730-LHM (per cmm.) 20,000 10000 Spoon Mar ร์ Moy Ring 1933 1954 Lig 12 Blood counts of a patient with chronic my clogenous touhemia who was treated with continuous busullan therapy after two courses of radrotherapy (From Galton, D. A God till, MT Lancet 1: 425 1955 ! or more Most patients shon a slow but steady increase in total Icukocyte count after the drug is stopped, and inimature leukocytes reappear in the peripheral blood within six months Symptoms usually do not recur until the hemoglonun has fallen to 10 Gm, at which time the leukocyte count may be up to 200,000 per cubic millimeter. When busulfan is given again, the immediate response us as good as i was to the first course, but relapse starts carlier, and then continuous administration is required Thc chart of a woman 40 cars of age with chronic my clogenous leukemia The diagnosis was first made nine months before the period coiered by this chart, at which time she reccited treatment with 3.5 mc pu, appears in Fig. 13 ·(HLORAMPUCIL AND BUSULTAN IN TREATMENT OF LEUKIMIA 107 as continuous therapy for escral nionthis with twice that daily dose has been required in other c3*es. Sumlar sariability response to treatment in chronic lymphocytic leukemia is well known with other forms of treatment We have Ircated only thirteen patients svith chronic lymphocytic leušenia, and, of these, seven responded cll. Thie is a small number of patients, and none has been followed for longer than three years; but our results suggest that the drug may prove useful in the managment of patienta tsith this disease. We do not treat symptomless patients, but, when symptoms occur, we start by using a dose of 0 1 mg. per kilogram (approximately 40 to 60 mg. in most cases) darly by mouth This dose is increased to 2 mg per llogram daily after four ceks if there has been no response, bust snipro cment is likely to occur during the third or fourth week of treatment We discontinue the drug when the teulocyte count reaches normal leiek or the lymph nodes and spieen hasc regressed satisfattorily Treatment is started again only when symptoms recur. We feel that repeated short courses of chlorambucil are betier than an artcmpt at maintenance Continuous administration would risk bone marrow damage and possibly would give an illusion of maintentance in a remission that ould have proceeded without further trcatment Severely anemic patients may do well Four parents wh hemoglobin less than 6 Gm had good remissions, with elevation of the value to approximately 12 Gm within five to 1 months from the beginning of treatment, but neither o two patients with «escre anema and a posee direct Coombs' test derised benefit from the drug There are no gastrointestittał side effects talos much larger doses are given tocrat enously A most important toxic effect of chlorambecil, as with all cytotoxic agents used in the treatment of this disease, ss the occurrence of purpura due to thrombocytopenia Patients with clinical evra dence of purpura are not suitable for treatment with this drug Instial thrombocytopenia, however, is not of itself a contraindication to the ure of chlorambucil, provided the daily dose does not exceed 0.1 mg per kilograin Only one patent in our series developed purpura Her platelet count initially was 45,000 per cubic millimeter, and, after four weeks' treatment with a dose of chlorambucil of 0 92 mg per kilogram (twice the dose ne now use), she de cloped widespread petechsae There sas no hemorrhage from mucous membranes The drug was discosttinued, and the platelet count subsequently rose to 20,000 per cubic millimeter Remmission continued for eighteen months Palients with lymphocytic lymphoma but no peripheral lymphocy tous ako vary in their sensitat o chlorambucił Those with heavy lymphocytic infiltraton in the bone marron require smaller doses than those tsità apparently normal bone marrow Chlorambuca es easy to administer, and in our hands appears to be much saler than treth lene melamine We feel that this drug is well worth further inal in the treatment of all types of chronic lymphocytic lymphoma ·106 CTIOLOGY AND TREATMENT OF LEUKEMIA The chart shows a satisfactory response to the first course of busulfan and a good initial response to a second course given fourteen months later. The second time, however, relapse occurred almost as soon as the drug was withdrawn, but maintenance therapy with busulfan effected control for a further twelve months. Blastic relapse occurred four years after the diagnosis was first made. Patients vary greatly in the dose required during maintenance. Some are sensitive and require only 0.5 mg. daily to maintain the leukocyte count at normal levels. Others need 4.0 mg dails. Adjustments of 0.5 mg are made in the daily dose at intervals, according to the level of the leukocyte count. Usually, a patient requires increasing doses of busulfan as time passes, to keep pace with the progress of the disease Such increasing doses usually do not exceed the daily dose given at the start of treatment, and thus there is little risk of thrombocytopenia Fig 14 shows the chart of a woman 21 years of age who received continuous treatment with busulfan. The increasing requirement of the drug as time passes is well demonstrated. It is of interest that this patient became amenorrheic a few months after starting busulfan, and subsequently developed severe menopausal symptoms. When resistance to busulfan eventually develops after long periods of maintenance therapy, this may manifest itself in three different ways There may be a relativ cly sudden rise in the peripheral iculocyte count, which consists mainly of blasts, there may be a fall in the leukocyte count to leukopenic levels, with a relatne increase in blasts, or therc may be a cry slowly increasing anemia, to which the patient becomes well adapted, together with an enlarging spleen The peripheral leukocyte count may remain constantly between 20,000 and 30,000 per cubic millimeter in the latter group of patients, but the spleen may enlarge so as to fill the abdomen and give rise to symptoms due to pres. The bone marrow in such cases usually is hy pocellular Il busulfan is discontinued, the peripheral count si«cs very rapidly and contains a high proportion of primitic cells The patient experiences sciere malaise, with high ſcver and generalized pains If another form of therapy as begun at this time, it should, therefore, be ghen in addition to busulfan, which may be withdrawn later when control has been secured with alternative therapy Radiotherapy to the enlarging splan sometimes may relieve the descomfort for a short while, but it does not reduce the size of the pleen significantly All three clinical tvpes of busulfan-resistant cases may respond for a short time to 6-mercaptopurine surc TREATMENT WITH CHLORAMBUCIL In marked contrast to the uniform response of patients with chronic mye logenous Icukemia to busullan, thone with chronic lymphocytic leukemia show wide variation in their response to chlorambucil Remision lasting a year has followed treatment with only o 1 mg per llogram daily for three weeks, where- ·Joapb H Burcheul, MD TREATMENT of LEUKEMIA* SCREENING OF AGENTS We wish primanly to discuss the treatment of acute leukemias. Certainly, we need new agents, since none of the drugs that we have at the present time are too satisfactory in the treatment of leukemia. There are many ways of looking for these agents, and, although screening programs have already been discussed, I would like to mention the one used at the Sloan-Kettering Institute Compounds from various pharmaceutical houses, from chemical companies, and from departments of chemistry at various unnersities are assembled, their prop. erties are catalogued, and a preliminan screening test with sarcoma 180 is used in mice in our estimation, this is the simplest technique and the one most economical of time and of mice Since 1946, over 16,000 crystalline compounds of known chemical structure and 10,000 additional unknown microbiologic filtrates have been included in these studies If they have an inhibitory effect or are promising otherwise, they may be tried in secondary tests. Scveral strains of mouse leukemia (both those sensitive to and resistant to the contentional methods of therapy of human leukemia) and a spectrum of some twenty mouse and rat tumors are included in the screening Effects on the developing chick embryo also may be scrutinized carefully, since embryonic tissue often will respond to the same drugs that will affect cancer They also may be added to tissue culture in which normal and ncoplastic cells are grown * These studies were supported by research grant CH-3215 from the National Cancer Institute of the National Institutes of Health, United States Public Health Service, institutional grants from the Amencan Cancer Society, the Damon Runyon Memorial Fund for Cancer Re. search, Lasker Foundation, and the Black-Stevenson Foundation 109 ·108 ITIOLOGY AND TRCATMENT OF LEUKEMIA REPRODUCTIVE SYSTEM AND THE THERAPY OF LEUKEMIA Busulfan and chlorambucil, like other alkylating agents, cause extensive damage to the gonads of cxperimental animals. Similar damage in patients under treatment with either of these drugs is manifest by the occurrence of amenorrhea in women and gynecomastia in men. Mention was made carler of our patient who became amenorrheic while on continuous treatment with busulfan. We have seen gynecomastia in two patients receiving busulfan and in one treated with chlorambucil. In all these cases, this condition was unilateral and lasted a few weeks only, in spite of the continued use of the drugs. Pregnancy in leukemia is rarc, but more than one author has described patients with chronic myelogenous leukemia surviving more than one pregnancy during the course of their discase There are various aspects to consider in the management of such cases, The effect of the pregnancy may be very slight. Patients with acute leukemia tend to abort, but patients with chronic leukemia, in spite of their potential hemorrhagic hazards, tend to have normal pregnancies with no excessive bleeding at term, and the general course of the discase is unaffected. The effect of the mother's leukemia upon the child cannot easily be ascertained from the literature All reported infants were normal at birth. None was followed more than a few years In view of Dr. Gross's work, it would seem most important to know what happens to these children in later life. It is the effect of the treatment of the leukemia upon the fetus that con. cerns us most today Small doses of splenic irradiation or radioactive phosphorus have been given to leukemic patients carly in pregnancy without ill effects, and no patients so treated have required further therapy until after delivery. We, personally, have not treated any pregnant patients with busulſan, and we would hesitate to do so Bollag gave a single dose (10 mg per kilogram) of this drug to rats late in pregnancy, and he found the infant rats to be sterile, with atrophic gonads In view of this, it would seem unwise to give maintenance doses of busulfan during pregnancy Patients already receiving contuous treatment with busullan are, hosyever, unlikch to become pregnant, owing to the sterilizing effect of the drug (For Discussion, see p. 118 ) ·TREATMENT OF LEUKCMIA 111 ually enlarge, or, toward the end of the disease, may des elop bone pains These localized symptoms also may respond well to treatment by localized irradiation. Patients who are not responsive to Anicthopterin may respond to steroids. and the opposite is also true. Between the groups of the steroids, the folic acid antagonists, and 6-mercaptopurine, there is no cross resistance. Usually, howcver, if a patient is resistant to one steroid, he will be resistant to all steroids If he is resistant to one folic acid antagonist, all will be incffective As to 6- mercaptopurine and thioguanine, if one has failed, the other probably will not bc usclul Steroids are useful in causing remissions in children with acute leukemia, quite useful in young adults up to the age of 30, and less useful oscr the age of 30, unless very high doses in the order of a gram of cortisone or Mícticorten daily are used. Even with high doses, results in adults over the age of 40 are not nearly as satisfactory as they are in children. Folic acid antagonists ace by conversion of folic acid to a reduced form, tetrahydropteroylglutamic acid, an intermediate in the production of biologically active folinic acid in the body These are the most useful of compounds in the acute leukemias of child. hood Unfortunately, in our experience, over the age of 8 or 10 they are not as satisfactory as they are in younger children, and over the age of puberty they are effective infrequently. Using folic acid analogues alone, we have rarely secn remissions in adults I have seen a remission in one woman 28 years of age, and I have heard of one in a person 35 years of age, but they are extremely Adults who have been treated with purine antagonists and either have had a remission and then des eloped resistance to it, or who have had treatment for a long period of time, may respond to Amethopterin. A trial, with cautious administration of the drug, seems justified in these patients Mercaptopurinc, thioguanine, and 6-chloropurine will produce remission in children and in adults with acute leukemia They also will produce remis. sions in patients with chronic granulocytic leukemia The effective dosage of these first two compounds in man is about the same We have not as yet seen any advantage of thioguanine over mercaptopurine, and we are not using it for that reason Also, 6-chloropurinc in children scems to be about as effective as mercaptopurine, but no better Dr Ellison, in our group, is now studying 6- chloropurine in adults with acute leukemia, and there are some fragmentary preliminary data which seem to show that adults, particularly adults over the age of 40, treated with 6-chloropurine have a higher incidence of remissions Several cooperative groups are attempting to determine whether this is a real superiority, or whether it is due to a relatively larger dose of 6-chloropurine Mercaptopurine is gn en orally, the usual dose is 2.5 mg per kilogram of body weight daily This amounts to approximately 50 mg per day in a child, and from 100 to 200 mg per day in an adult; it must be guien for long periods of time Remissions under three to eight weeks should not be expected. This rare.

·110 ETIOLOGY AND TREATMENT OF LEUKEMIA side by side by the roller-tube technique. Compounds also may be tried for their cffect against molds and fungi and bacteria. If a compound is effective against sarcoma 180, some of the mouse leukemias, and one or two other tests, clinical screening then is undertaken. For clinicians, two things are of primary interest, (1) the maximum tol. crated dose; (2) the system affected most. Dogs are used to investigate these questions, because organs damaged in the dog are likely to be the same ones affected first in the patient when he is treated with the drug Once data are obtained from canine experiments, cautious explorations with patients are possible. First, patients with far-advanced carcinoma arc trcated by increasing the drug gradually until beneficial effect is seen or the limit of toxicity is reached Calcium and uric acid excretion may aid in evaluating the effect of the agent used. Finally, the value of any new compound must be assessed in relation to other mcthods of treatment. PRINCIPLES OF TREATMENT Certain basic principles that we are using in the treatment of acute leukemias may bear repetition. In the first place, we have two classes of agents that can be used in the treatment of acute leukemia. We have the ancimetabolites, consisting of folic acid antagonists, purine antagonists, and, questionable, the glutamine antagonists The steroids comprise the other group Definite indications exist for the use of both types of agents. The steroids act more rapidly than the antimetabolites Imprarement may occur within a weck's time or even less when steroid is administered, whereas the antimetabolites rarely cxert any beneficial effect on the bone marrow for three to eight wechs. The discasc eventually becomes resistant to all these agents. This occurs morc quiche with the steroids than with the antimetabolites as a rule, but steroids are useful at times when antimetabolites cannot be used On the other hand, when the patient's condition allows three to eight wecks for an attempt to obtain a remis. sion, then the antimetabolites should be used and the steroids saved for the emergency, when time k at a premium. Conceivablv, one might use the combination of steroids and antimetabolites, but we think it is better to rescric the stcroids for the time which will surels come, when they and thcı alone will be ellectuvc. If the patient is acutch ill, has high fever, petechiac, and various cui dences of blecding, we think it is essential to start with steroids. In addition to using these specific forms of therapy, one should use antibiotics at the first sign of infection Transfusions should be used when indicated, also Patients with meningeal infiltration mai des elop headache, papilledema, and other esidence of increased cerebrospinal fluid They usually can be benefited by irradiation over the skull, even though bone marrow and peripheral blood may not require treatment Al come patients in fairly good remission, as far as marrow and peripheral blood arc concemed, have kidneys which grad.

·TREATMENT OF LEUKEMIA ually enlarge, or, toward the end of the di-casc, may develop bone pains These localized symptoms ako may respond well to treatment by localized irradiation Patients who are not response to Imethopterin may respond to steroids, and the opposite is also true. Between the groups of the steroids, the folic acid antagonists, and 6-mercaptopurine, there is no cross resistance. Usually, how - ever, if a patient is resistant to one steroid, he will be resistant to all steroida, If he is resistant to one folic acid antagonist, all will be ineffective. Is to 6 mercaptopurine and thioguaninc, if one has failed, the other probably will not be u«clul. Steroids are useful in causing remissions in children with acute leukemia, quite useful in young adults up to the age of 30, and less useful over the age of 30, unless 1 cry high doses in the order of a gram of cortisone or Meticorten daily are used. Even with high doses, results in adults over the age of 40 are not nearly as satisfactory as they are in children Folic acid antagonists act by conversion of folic acid to a reduced form, tetrahydropteroylglutamic acid, an intermediate in the production of biologically active folinic acid in the body These are the most uscful of compounds in the acute leukemias of child. hood Unfortunately, in our experience, over the age of 8 or 10 they are not as satisfactory as they are in younger children, and over the age of puberty they are effective infrequently Using folic acid analogues alone, we have rarely seen remissions in adults I have seen a remission in one woman 28 ycars of age, and I have heard of one in a person 35 years of age, but they are extremely rare. Adults who hasc been treated with purine antagonists and either have had a remission and then developed resistance to it, or who have had treatment for a long period of time, may respond to Imethopterin. A trial, with cautious administration of the drug, seems justified in these patients ſercaptopurine, throguanine, and 6-chloropurine will produce remission in children and in adults with acute lculemia They also will produce remissions in patients with chronic granulocytic leukemia The effective dosage of these first two compounds in man is about the same We have not as yet seen any advantage of thioguarune over mercaptopurine, and we are not using it for that reason Alo, 6-chloropurine in children seems to be about as effective as mercaptopurine, but no better Dr Ellison, in our group, is now studying 6- chloropurine in adults with acute leukemia, and there are some fragmentary preliminary data which seem to show that adults, particularly adults over the age of 40, treated with 6-chloropurine have a higher incidence of remissions Several cooperatne groups are attempting to determine whether this is a real superiority, or whether it is due to a relatnely larger dose of 6-chloropurine Jercaptopurine is given orally, the usual dose is 2 5 mg. per kilogram of body weight daily This amounts to approximately 50 mg per day in a child, and from 100 to 200 mg per day in an adult, it must be guien for long periods of time Remissions under three to eight weeks should not be expected This ·112 ETIOLOGY AND TREATMENT OF LEUKEMIA information has been circulated widely, but requests for advice from physicians indicate to us that it is not widely known. Although treatment lasts from six to twelve weeks as a rule, patients may at times become worse in a few wechs, and delaying steroid therapy obviously is inadvisable. In the absence of untoward symptoms, however, we continue treatment at least eight weeks before deciding it is ineffective. In some of the most satisfactory responses obtained, we have noted some evidence of remission in cight seeks, but not rcally a complete remission for ten or twchic wechs. The toxicity of the drug in this dosage level is minimal, but bone marrow depression occurs after excessive dosage When the leukemic count is high, the rapid fall in leukocyte count probably is therapcutic rather than toxic. In patients with a very high white count, particularly with extensic involvement of lymph nodes, a rapid fall in leukocyte count may be associated with abnormally high excretion of uric acid and with renal shutdown. In this group of patients, cautious administration is mandatory. Gastrointestinal complaints, including nausea and vomiting, may occur in adults given mercaptopurine, but they are rare. In contrast to the folic acid antagonists, which prevent the de novo synthesis of purines and pyrimidines, this compound intcrleres with the incorporation of preformed purines, their nucleosides and nuclcotides, into nucleic acid In general, the remissions are slightly shorter in a child with acute leukemia after treatment with mercaptopurine than those expected after treatment with folic acid antagonists Our series show approximately four months' actual rcmission as the average with mercaptopurine, exclusne of the time of treatment necessary to bring the patient into remission. The possibilities of conibining it with other substances which might incrcase the length of renuission will be discussed later Mercaptopurine and other purine antagonists will produce remissions in a high percentage of the patients with chronic granulocytic leukemia. It is imposible to say at the present time how it compares with busulfan or irradiation becausc cnough time has not elapsed for evaluation. The patient must be obencd more closely to regulate dosage properly than when either busulſan or irradiation as administered In most cases of chronje granulocytic leukemia treated with mercaptopurine, one must treat the patient continuously, although occasionally a patient with this type of leukemia docs well on intermittent therapy This is the exception rather than the rule The compound o-chazoacetyl-L-serine has a definite effect against many animal tumors Unfortunately, results are not promising when it is given to patients It lias 3 slıght cllect in terminal Hodgkin s discasc, with responses lasting two to three weeks. In children sith arute leukemia, it causes partial res. sons, again lasting three or four weeks in an occasional patient In combination with quarter doses of mercaptopurine, however, quarter doses of diazoarenilo. serine in sarcoma 180 in mice have been shown by Clarke and associates ", " to ·TRCATMENT OF LEUKEMIA 113 be more effective than full doses of either compound alone For that reason, we have treated children with acute leukemia with this combination of azacenine and mercaptopurine. We begin with 25 mg. mercaptopurine per kilogram of body weight and an equal dose of azaserinc. When the two compounds are given together, one is likely to encounter sore mouth, red tongue, and ulcerations of the mucosa of the tongue from the combination when given in this dosage. If this rather frequent complication occurs, the azaserine should be discontinued for a few days and then continued at one-half the original dosc. Most patients will tolerate approximately 10 to 1.5 mg per kilogram of the azascrine along with full doses of mercaptopurine. Nucleic acids are built in part from small units such as formate, glycine, glutamine, and CO:, and, as the body gradually assembles various components of the purine ring, certain steps in this synthetic pathway may be blocked by various antitumor agents. The antifolic compounds, for instance, are known to block the addition of one-carbon fragment to glycinc amide ribotide and are also known to block the incorporation of a one-carbon fragment into the 2 posi. tion, to make hypoxanthine ribotide. Jercaptopurine presumably blocks the conversion of a compound containing hypoxanthine into the adenine and guanine of nucleic acid. Azaserine blocks the conversion of formyl glycine amide ribotide to formyl glycine anidine ribotide Mercaptopurine and azaserine, in blocking the pathway at two different levels, should diminish the synthesis of certain nucleic acids necessary for leukenuc cells, and thus have more effect than the one compound alone This pnnciple of sequential inhibition was enunciated by Dr. Potter many years ago, and seems to have real possibilities in present-day chemotherapy For some reason, the combination of folic acid antagonists with mercaptopurine does not seem to be selectively synergistic in most tumor systems Slight synergistic effects are offset by additive toxicity We studied a series of twenty-nine patients selected because they were not critically ill Of these twenty-nine patients, a surprisingly high number, twenty, had complete remissions as defined by criteria which have been promulgated by the Clinical Studies Panel of the Cancer Chemotherapy National Service Cen- Three patients had partial remissions, and six were failures More important, the average length of complete remission was 6 2 months Remission was tabulated independently from the total time of treatment Remission after combined treatment is two months longer on the average than with mercaptopurine alone Control studies are not satisfactory as set in the group treated with mercaptopurine alone, and final conclusions must await a more comprehensive series For some reason, this combination of drugs has not yielded better results in our hands than mercaptopurine alone in adults with acute leukemia Progress definitely is being made in the treatment of leukemia Times selected 208 cases of leukemia in children who had no specific therapy. They were treated at another time than those with which they may be compared, they ter ·114 ETIOLOGY AND TREATMENT OF LEUKEMIA did not have comparable antibiotic and transfusion therapy, and many of the doctors who treated them did not have quite the zeal of modern chemotherapists. The median survival time in the series was 3.9 months from onset to death Among 154 children admitted to Memorial Hospital and trcated with antifolic acids and/or the steroids, the median survival time was eight months In a series of 174 consecutive cases of acute Icukemia in children treated since June, 1952, when mercaptopurine first became available to us in addition to the other agents, 50 per cent were alive over tseke months from the onect of their disease. It is not possible to report on the effect of mercaptopurme alone on survival time, since other therapy is instituted when relapse occurs. Some of these patients obviously were very ill on admission, were given a few doses of ACTH or cortisone, and died within a few hours or days after treatment was started They are all included in the series, honever, since the other agents were available for their treatment if they had been indicated. These results and those of others indicate that progress, though slow, is being made in the treatment of acute leukemia compared to the results obtained ten years ago. (For Discussion, sec p 118.)

·Villa R Anonsmuth, NID CLINICAL MANAGEMENT OF LEUKEMIA Some time ago, a well-known hematologist gave a talk on the use of urethan in the treatment of chronic mycloid leukemua and was quite enthusiastic. After the presentation, I had an opportunity to talk with him privately and asked him the kind of treatment he would request of hc had chronic myelord leukemia He said, “I would find the best radiologist I know and ask him to treat mc." One of the things we have to keep in mind in a conference like this, or in reading about therapeutic agents, is the fact that, when we are concerned with scrcening and the effort to find out the fundamental processes, someone nust study an entire series of patients on controlled or restricted methods of therapy On the other hand, if one has an individual patient who comes to him requesting treatment, no restriction as to the type of treatments from which to choose exists My remarks apply more to the latter situation Even though comparisons of treated untrcatcd leukemia offer certain dilficulties, there is no question about the prolongation of survival time and decreased morbidits in acute leukemia by means of current, therapeutic methods This is probably truc ako for chronic Icukemia We must remember, however, that in addition to what we like to attribute to specific therapy, supportile therapy has improsed and also influences survival time The widespread application of the Rh system and introduction of blood-banking which has enabled us to transfuse these people safely are two examples Antimicrobial drugs and steroids are additional examples In the management of chronic leukemias, I have been influenced by an earlier interest in radioactive phosphorus and have depended on it more than any other single therapeutic measure This is, of course, a form of whole-body radiation The policy we have followed has been to try to obtain remission with phosphorus and then not to allow serious clinical relapse. This usually means more frequent treatment with smaller doses One of the impressions ·116 ETIOLOGY AND TREATMENT OF LEUKEMIA obtained about the use of phosphorus in myeloid leukemia is that it will not reduce marked splenomegaly. However, if it does not respond to pº, local irradiation is an effective supplemental treatment Likewise, radioactive phosphorus has been effective in the treatment of chronic leukocytic leukemia in those who do not have massive splenomegaly or adenopathy, and we have found it as satisfactory as anything else tried. Again, it can be supplemented at any time with local irradiation, nitrogen mustard, or tricthylene melamine. Triethylene melaminc requires cautious administration One advantage is that it may be given orally, although the possibility that it may be neutralized before absorption must be realized. Since the patient with chronic meyeloid leukemia usually is symptomatic when seen, the trend toward treating patients with the disease at once is justified, particularly since the patient may not recognize the symptoms, and only after therapy does he realize the improvement that has occurred. Chronic lymphocytic leukemia is predominantly a disease of older patients. At times, it is difficult to know whether symptoms are due to Icukemia or to some other condition. Also, the patient frequently will account for symptoms on the basis of age or something else familiar to him, and only after therapy does he realize symptoms were present. Therefore, the pronouncement that therapy should be given only when symptoms are present is not always easy to follow, and a therapeutic test may be required to decide whether symptoms are duc to loukemia. Of course, cven then the patient who is receiving therapy thinks he ought to feel better, and the effect of therapeutic trial may be difficult at times to evaluate. Onc encounters complicating factors of hypersplenism in lymphocytic leukemia, particularly The possibility that really satisfactory control of a few of these patients may require splenectomy at some time during their illness should not be overlooked Purpural and hemolytic anemia are not at all un. common, even when a patient appears to be well controlled. Splenectomy in leukemia was donc fairly commonly in the earlier part of this century. It fell into disrepure, but now it is coming back into fashion In order to decide the difficult question of when to advise it, one must be sure that other measures to control the discase will not suffice and also that the gencral outlook of the patient is good enough to warrant major surgery When the diagnosis of leukemia, particularly in its acute form, is made, a common reaction is that the patient should be admitted to the hospital and transfused. Dr. Burchenal has already indicated that neither is essential in the management of leukemia Certainly, one of the things we ought to do with these people, since all we have to offer them really is a modest prolongation of comfortable life, is to promote as normal and as happy a situation as we can for as long as possible. Youngsters are not at all happy about being remoıcd from their home and placed in a hospital cien when it is necessary. Likewise, ·CLINICAL MANAGEMENT OF LI'UKIMIA 117 they are quite resistant to transfusions, and, while they may be anemic, most of these patients often are not having enough symptoms from the anemia itself to warrant hospitalization and/or transfusion. If therapy is at all satisfactory, clinical improvement will be rather prompt and will include a return of hemoglobin values to normal without transfusion; these measures, although they probably will be necessary later, should be avoided as long as possible. Another problem which arises is the question of infections in Iculemic patients and the advisahility of antibiotics. I see many patients who have been started on antibiotic therapy the day the diagnosis was made and continued on some antibiotic throughout the entire course of the illness. This is particularly true in those patients with a low leukocyte count which may alarm the physician. Even if the white count is 100,000, almost none of these cells are of any help in defense against infection, and yet the patients usually come in without infections Prescribing antibiotics is adding to the expense and adds another hazard to therapy, since reactions may occur. If the patient or parent as an intelhgent and responsible person who will recognize the indıcations for the use of antibiotics, then a prophylactic regimen can be avoided. If one does not have an intelligent patient, or parent, I think any type of program is more likely to be disappointing I was happy to hear Dr. Burchenal point out that the outlook is not uniformly bad for adults with acute leukemia I have seen one man in the fifth decade of life who had remission for a year on folic acid antagonist. With the advent of 6-mercaptopurine, certainly enough adults have remissions to warrant a trial on therapy Earlier, I became discouraged with treatment of leukemia, particularly myeloid and monocytıc, with the folic acid antagonists. Failure to respond and uniform toxicity make therapy secm inadvisable With 6-mercaptopurine, the incidence of remissions has been rewarding enough to warrant a trial in every adult and child who has the disease In conclusion, I wish to comment on the attitude of the physician who cares for the patient. Too often, the patient is told or interprets the remarks of the doctor to mean that nothing really can be done This is deplorable enough in acute leukemia, but with the chronic leukemias it may lead to a long period, sometimes a year or two, of purposeless existence At a time when the patient can be free from symptoms on adequate therapy, he has them unnecessarily because of a defeatist attitude The withholding of therapy in patients with symptoms for use later in the course of the disease often is unwise Whatever the method of therapy chosen, and particularly in chronic leukemias, as much treatment as is necessary should be giảen at the beginning to produce as good a remission as possible or as long as possible by whatever amount of therapy is required, without worry about a future rainy day It will come eventually, but by that time the patient's ability to respond probably will be so diminished that the response will be disappointing, and he will have been deprived of a long ·118 ITIOLOGY AND TREATMENT OF LEUKEMIA period of remission. Available methods of therapy should be exploited fully in management of the individual case, as well as in the optimistic search for improved methods and drugs. Certainly, the patient with leukemia can be treated today much more successfully than even a few short years ago. DISCUSSION Dr. BURCHEXAL: I would like to continue Dr. Arrowsmith's discussion. I certainly agree that there is no point in keeping children alive if they are in the hospital and living an uncomfortable life. We find, on the average, 90 per cent of our children with acute leukemia are out-patients at any one time, and only 10 per cent are in-patients I would certainly also agrce that they should not be transfused except when they have symptoms or when the hemoglobin suddenly drops to below 7.0 or 8.0 grams. We try to keep transfusions at a minimum, and usually we give transfusions on an out-patient basis. Antibiotics are used only when there are signs of infection, but they are then used quickly and in massive doses. DR. TILL: Do you consider frequent bone marrow examinations necessary for satisíactory management of your patients, or are these part of an investigational program of the new agents under trial? Dr. Burch[EXAL: We examine marrows more than is absolutely essential from the point of view of following the patient The habit arose because we were using intermittent theraps with folic acid antagonists. If marrows are cxamined csery two weeks on such patients, signs of relapse in the marrow arc seen when the clinical condition is excellent and the peripheral blood shows no Ngn of relapse Therefore, one is able to resume therapy and obtain remission as far as the marrow is concerned before there has even been clinical relapse From the rescarch point of view, it is important for detection of remission from compounds tested, and it is part of our protocol to do them in the out-patient department every two wechs in all children and in most adults ciery neck when they are in the hospital under treatment On the other hand, they are not necer kan that often if a new drug is not being "udied Dk SPRACLE. There are three questions that I would like to ask Dr Till Fint, what is known regarding the pharmacologic action of busulſan? Second, have you treated an group of patients with maintenance therapy, and, if so, that were the results achieved? Thurd, do sou fcel that in the busulfan-treated group there has been an increase in the incidence of blastic crises, and, in such patients, what do you feel is the optimal form of treatment? DR. TILL: Practically nothing - known of the pharmacology of busulfan Chemicalls, it is an alhs dating agent as is nitrogen mutard, but the effects produced by busulfan upon the hemopoietic system of the rat are very different from thone seen following comparable doses of nitrogen mustards. Studies are being planned with C'-labeled busullan, but I cannot give any data on this at present ·CLINICAL MANAGEMENT OF LEUKEMIA 119 In an er to the second question on the use of maintenance therapy, I do not know of any series of patients given buvullan continuously from the start of thera apy. We hase eight patients so treated; four of these received continuous treatment because the scre x-ray reistant and two because their initial response to busullan was slow. The other tuo patients reccived continuous therapy as a trial. One of the has done well for more than two years after the start of therapy. The other lised two years aíter the diagnosis was first made, but after the fint car her hemoglobin began to fall, in spite of continuous reductions of the cre of busullan, she slowly became leukopenic. When the drug was eventualls scopped, the leukocyic count sase to normal levels but showed a subxcantial prome portion of blasts. Her anenna perruled, trcatment with 6-mercaptopurine was nur helplul, and she required frequent transfusions during the last six months of her life. This case hestory would suggest that it is unwre to administer busulan continuously froin the uime of diagnoris as a routine. In ansnet to the question regarding the incidence of blastic cnsis in buvullantrcaled patients, I do not think the incidence differs from that in patients treated by other methods. Patients in blastic relape do not respond favorably to increase in doc of bu-ullan, but a minorits will respond for a while to 6-merraptopurine. DR ARROWS SITIO Would vou comment on morphologic findings in blast cell crisis? DR. TILL! Our specimens were obtained by a-piration, not trephine biopsy, In een case in which marrow puncture was performed at this stage of the discase, marrow was obtained onds with difficulty and fragments were feu, dicroscopically, there was marked hypoplasia, the commonest cells being bizarre megakaryocytes consisting of a single small nuclear fragment surrounded by platelets Dr. KIRSCHBAC. Dr Arrowsmith mentioned the improved methods of transfusion adminstration and the use of antibiotics To what extent can increased sunisal be attributed to specific antileulemic therap: and to what extent to better medical management? DR BLRCHENAL' Unfortunately, as tou know, there is no really good series which parallel in time to these more recent series since 1953, and treated with none of the specific agents The only figures I can give you are those from a small series of nineteen cases treated between 1946 and 1948 with the mustards, which are faith non-pecific agents in aiute leukemua They had the transfusions and some of the antibiotics that are available todas, and their survival was about Only one of thein had a presumable spuntaneous remission The only May we can tell we are making some progress i to uke the group treated with steroids and antifolics as control Mme of our «ases admitted up until June of 1972 had the antibiotics that are available todas, and they certain had as many transfusions There has been a considerable increase in the 50 per cent survival time with antilolies and steroids at about eight months and with mercaptopurine the same ·120 ITIOLOGY AND TREATMENT OF LEUKLMIA and possibly azaserine additions which bring it to about twelve months, although antibiotics and transfusions and zcal may prolong life somewhat, DR KIRSCHBAUM: To what extent is the difference between 6-mercaptopurine on the one hand and steroids and antifolics on the other due to selection of patients? Is it true that the acute case is treated with cortisone rather than with 6-mercaptopurine? Dr. BURCHENALIn this series, this is just a question of time. Before 1952, June or January, depending on which series is considered, mercaptopurine was not available for treatment. Both of these series are consecutive cases, no matter how ill the patients were. In the second group, mercaptopurine was available if indicated. If the patient was very ill, he was still included in the series and treated, DR. UPTON: I would like to make a comment which may or may not be out of place. Speaking of the therapy of leukemia, we are very interested at the Oak Ridge National Laboratory in the reversal or prevention of marrow injury by administration of nonirradiated bone marrow cells postirradiation. There is cudence that an irradiated animal will tolerate heterografts or homografts of marrow. Dr. C. C Congdon, who worked with Dr. E. Lorenz at the National Institutes of Health, tells me that he thinks they may have successfully trcated some guinea pigs with lymphoid leukemia by whole body radiation Although one may not be ready yet to try this approach on a clinical level, I would be interested to know what you consider the potential outlook for this type of treatment in man De BURCHENAL: I think that sort of thing is very fascinating. I have not talked to Dr Congdon, but I did talk to Dr Lorenz about this Studies that we did at one time in attempting to sterilize mousc leukemia with radiation showed that total body radiation in a single dose of 2,500 r or above prevented transplantation of the strain of leukemia used On the other hand, at 2,000 r, some would transplant and some would not. At 1,500 r all would transplant Therefore, I question whether one can protect an animal against quite large enough doses to sterilize all leukemic cells it would be fascinating if you could, but it is my impression that Dr Lorenz was not able to do it, although survival time was prolonged DR SCHLOSSER: Dr Collins has treated some patients in Houston with doses as high as 250 r in air in a single treatnient This is about ten times the amount we usually give in a day I would like to ask Dr. Till how many patients they have now treated with busulfan, and how man of them have been treated a year or more Alo, are you impressed that the survivals are as good as total-body or any other form of irradiation? Dr. Till: We have only thirty-two patients in the whole series, including those in the early trials which started over five years ago. Four of these died ·CLINICAL MANAGEMENT OF LEUKEMIA 121 within the first year of treatment, two of these being in the early trials when exces- Te dases were used, and another died of pulmonary embolus. Our impression of survival of busulfan-treated cases so far is that this may be as good as for the other forms of treatment. We cannot say more with so small a series. DR. KREMENTZ: Hase there been any crified cases of leukemias which have spontancously regres.cd? Dr BURCHENAL: They will certainly appear occasionally and can be very complete We saw onc child recenth who had a 100,000 white count which was alleged to bc entirely monocytes The marrow was reviewed by an eminent pathologist, and then lost, unfortunately. At any rate, there seemed to be no question that it was leukemia at that time. Transfusions were given as a sort of a last resort, and the patient recovered and was perfectly well for three and a half cars. Then she again presented a picture of leukemia and was treated with Amethopterin for about a month before we saw her. At the time we saw her, she was in remission and we stopped the drug. She ined another four years and finalls des cloped fulminating lculemia, with no question concerning the diagnosis She died despite all available therapy Post-mortem examination confirmed the diagnosis. I am certain the first remission was spontaneous, the second one I suspect was spontancous. We saw another girl who lived thrce and a half years after therapy with 2,6-diamınopurine, which is naturally a very cffective agent. This may have been a spontaneous remission as well So they do occur, but they arc rare, and I do not know any that has not recurred Of course, if a patient with a diagnosis recovers, the usual opinion is that it was only a leukemoid reaction originally. Spontancous semission before steroids and antibiotics became available may have been due to infection with an accompanying stress reaction which may have resulted in a response in those very sensitive to steroid stimulus DR. KIRSCHBAUM: Do you think that was a cortisone effect? Dr. BURCHENAL: I do not know, but would guess it probably was DR SCHLOSSER How frequently should blood counts be done in patients receiving busulfan? Dr. Burchcnal mentioned regression of large masses following 6-mercaptopurine, the development of high uric acid levels, and renal damage I would also like to hear some discussion of the problem of gouty kidneys. DR BURCHENAL: Apparently, the fall in white count, regresston of masses, and the increase in uric acid excretion depend on the agent used If an agent such as Amethoptern is used to treat chronic granulocytic leukemia, that agent is acting previous to the formation of the punne ring. For that reason, it seems to prevent the formation of any sort of purines which would then lead to uric acd, and the uric acid excretion level and level in blood decrease when therapy with Amethopterin is begun On the other hand, mercaptopurine, which interferes at a different level, presents the conversion of hypoxanthine to the guanine of nucleic acid, and this is probably an accumulation of the hypoxanthine which ·122 ETIOLOGY AND TREATMENT OF LEUKCAIA may be conierted to uric acid and excreted as such. If x-ray or nitrogen mustard or possibly even the steroids are used, they seem to break down the nucleic acids at higher levels, and again an outpouring of uric acid may occur. As to therapy, I have no suggestions other than caution about giving too much of the agent and administenng as much fluid as possible, particularly beforehand. Dr. ARROWSMITH: Nephrostomy has been reported as a lifesaving measure in these patients. I have seen tho patients who apparently were anuric. One was a case of myeloid metaplasia thought to be iculemic and had one dose of x-irradiation over a massive spleen. His uric acid level was extremely high, and he was anuric for forty-eight hours but recovered spontancously. The other onc, a patient I saw with Dr Gould Andrews, had a massivc lymphoma which quickly regressed with triethylene melamine and was accompanied by extremely high uric acid excretion and renal colic. This, as well as the previous case, subsided spontaneously after forcing fluids and without surgery, so I question whether nephrostom is necessary. Dr. BURCHENAL. Most of the time it is not necessary. DR. TILL In answer to the question about the frequency of blood counts in paticnts taking busulfan, a count once in four weeks is probably sullicient, but for someone inexperienced in the use of the drug it is probably safer to have counts cvery two weeks. Dr. Schlosser That would certainly be of no greater frequency than il \-ray therapy were used On the subject of high uric acid levels, we have obssersed patients in the past who were recciving irradiation over the spleen to collapse and expire within a few hours. Because of this, we now routineh start the dosage over the spleen extremely cautiously DR KIRSCHBAUM Dr Schloser, how do you explain the induction of rcmussion in chronic leukemia bi pleme irradiation DR SCHLOSSER Of course, I do not really know However, some cvidence nou suggests that it is probabk a chemical mechanism Whether it is necessary to irradiate the pleen or whether one can irradiate an arca in the body and achieve the same results in another pertinent question It is also though! if blood supph is richer in a gason arra, that the response, mar be better known, of course, in a wide varicts of biologic materials that increanng the ori. gen content of the medium increases the radiosensitivity of the organ I hanc been interested in this and have treated me recurrent brcast tuniors br baung the patients breathe pure oxygen while half was irradiated, and irradiating the other hall with no ou gen In two instances, I thought the ougenated fralf regressed faster This is something that is very difficult to be positic about, however Since then, I have treated more cases in this manner and did not note an difierence DR. KIRSCIBALI I am aware of the work on increased oxigenation, but I do not think you can explain ans effects of plenic irradiation on the basis of ·CLINICAL MANAGEMENT OF LEUKEMIA 123 increased ouigen tension in the plasma. lle arc assuming that there is a sig. nificant effect. Do you think it is likely that, as a result of irradiation of the loukemic spleen, there may be something produced in the spleen which then is responsible for this systemic effect? I think it might be very worth while to sec whether something might be catracted, let us say, from lcucmic spleens of micc that have been irradiated DR SYVERTOY: Further comment may be helpful as it applies to the clfects of irradiation X-irradiation of human cpithelial cells in culture, normal or malignant, by use of between 700 r and 750 r, results in no vistble cellular changes. The cells continuc to metabolize and to produce virus. X-irradiation with from 1,000 r to 5,000 r stops multiplication, but cells continue to metabolizc, and about half of them becomc hydropic and increase enormously in size. These cells, cell for cell, produce as much virus as nonirradiated cells. Internal cellular metabolic activits continues. Fmalls, from 15,000 to 30,000 roentgen units result in cellular shattering and total destruction. DR. SCHLOSSFR. Responses can be obtained no matter what portton of the bods one irradiates. It is not necessary to irradiate the spleen DR. KIRSCHBAUM Why do you choose the spleen? DR SCHLOSSER, Conscntionally, one is dealing with an enlarged organ, and I have found the response to be better and faster if irradiation is directed toward the spleen. If there is not an enlarged spleen and there are cnlarged mediastinal nodes, we start there Dr KIRSCHRAUN Why do sou not irradiate normal tusucs? DR. SCHLOSSER I think it would probabl work, although I have never tricd it. DR KRFMEYTZ Did Dr Kaplan do some work along this line with mice DR. KIRSCHRAUM. Kaplan's work was on the induction of leukemia. He irradiated thymectomized mice and postirradiation he transplanted thymus; in that thymus, Icukemic transformation of the cells wiis introduced That was the secondary effect of radiation, perhaps peroxide formation or whatever the chemical mechanism is DR KREMETZ Dr Rurchenal mentioned previously that the transplantation of human Icukemic cells and lymph nodes to animals has not been achieved Would you cnlarge upon this topic, Dr Burchcnalº Dr BURCJIENAL. I\'chase transplanted its marrows from children with acute leukemia and from some adults as well, some of whom were certainly in the cortisone-resistant stage of their drcase, into cortisonized hamsters, and none of them have grown The only thing we have been able to grow is a culture of feukernic ceils which was originally rolated by O good from a case of monocytic leukemia These cells were grown in (lue culture and injected as a suspension into the check pouch A slight growth was noted in two weeks. By the end of twenty-eight days, there was a tumos approximately 5 mm in diameter growing ·124 ETIOLOGY AND TREATMENT OF LEUKEMIA in each check pouch. Sections of that tumor were diagnosed as eittier anaplastic epidermoid carcinoma or reticulum cell sarcoma There is a question in everyone's mind as to whether the cells are leukemic or not. Dr. Osgood feels that the picture is perfectly consistent with a reticulum cell tumor, representing a solid form of monocytic leukemia. Others feel it is an epidermoid carcinoma. We would like to see transplantation of leukemic cells from human marrow into animals successful in 100 per cent of the cases. Then it might be useful as a screening agent Until that time, I question how much value it will be. Dr. KREMENTZ: Leukemic marrow has never been successfully transplanted into the anterior chambers of guinea pigs' eyes in our laboratory. I do not recall that anyone has ever successfully grown lymphomas on heterologous transplantation, either. DR. UPTON: In relative ignorance of biochemistry, I would like 10 ask a question of the panel myself. Dr. Gellhom presented some data showing changes in glucose tolerance associated with leukemia; there was a reversal of the abnormal glucose tolerance picture with remission. This calls to mind a recent paper by Warburgo concerning the altered respiration of tumor cells. Do other members of the panel have any cvidence about this change in glucose metabolism in various types of leukemia? Can anyone say more about it at this stage? Is there a disturbance that is peculiar to patients with cancer and with diabetes, or docs it occur in seriously debılıcated patients under scvere stress from other causes? Dr. BURCHENAL: The only evidence I hnow on it was that Dr Jorgen Kieler, from the University of Copenhagen, reported that he had studied human narrow by the Cartesian-drver technique, and found that the leukemic cells did respire. As to abnormal glucose tolerance, Dr. Glicksman's group at Memorial Hospital has seen that same sort of thing in patients with other types of cancer Incidence of abnormal glucose tolerance tests at Memorial Hospital in patients with cancer is higher than one would expect in the general population, so that is supportive evidence for Dr Gellhorn's work; but I think the fact in Dr Gellhorn's finding that when the leukemic cells were 100,000 to 400,000 in number there was an abnormal tolerance, and when they returned to normal numbers It also became normal, is much more exciting DR. UPTOV. Is this a disturbance that onc sces which is peculiar to patients with cancer, or is this something occurring in seriously debilitated patients under stress from other causes? Dr. BURCIIENAL. If I remember his criteria, they selected patients without diabetes and without a famils histor of diabetes who were not dcbilitated and were in good condition. This would be particularly important of additional proof cannot be obtained that an abnormal glucose tolerance is present before operation and a normal one is present afterward ·Walter / Burdelle, PhD, MD ETIOLOGY and THERAPY O LEUKEMIA* 127 ETIOLOGY DIAGNOSIS AND CLASSIFICATION. 131 SCREENING TOR CUFVIOTHERAPEUTIC AGENTS 132 133 134 134 135 TREATMENT Irradratrart. Chemotherapy Berzenc Arsenic Urethan Alkylating Agents Vechlorethamine Hydrochloride. Triethylene Velamine N-N-N" Triethylene Thiophosphoramide Busullan Chlorambucil Antymetabolitos Folic Acid Antagonists Purine Antagonists Steroids. Desacetylmethylcolchicine Surgery Supportive Therapy Management of Leukemia Acute Leukemia. Chronic Leukenja 135 136 137 137 138 139 140 141 112 142 143 143 144 *Aided by grants from the National Cancer Institute, National Institutes of Health, and the American Cancer Society 125 ·126 ETIOLOGY AND TRCATMENT OF LEUKEMIA EARLY HISTORY OF LEUKEMIA 1845 Discase first described. Named "leukemia" (evado + Virchow$8-100 awa) by Virchow after the "farblosen Körperchen" Craigielva Bennett 201 1857 Acute leukemia described Friedreich 10: 1858 Leukemia first described in horse and pis 1865 Case of leukemia without characteristic blood picture Leisering203 reported Cohnheim101 1865 First chemotherapy for leukemia (arsenic) 1866 First splenectomy for leuhemia 1868 Leukemia first described in birds Bryant to Roloff 100 1870 Mlyelogenous leukemia with changes in the marrow recognized 1871 Leukenua first described in dog and cat Neumann07 1872 First attempt at transmission of leukemia experi- Siedamgrotzky109 mentally, blood from human leukemia failed to induce leukemia in dogs and rabbits 1874 Leukemiz first described in mouse Moster109 1879 Rational classification of various forms of the discase Eberth 110 outlined as a result of microscopic studies of stained preparatiorts, as well as macroscopic observations Ehrlich 112 1900 Myeloblast identified, providing means for distinguishing between acute and chronic myelogenous discase 1903 First successful treatment of leukemia with roentgen Nacgel therapy Sonn113 1908 Avian leukosis transmitted by cell-free agent Ellermann and Bang21, 114 ·MIOLOGY AND THCRAPY OT LEUKEMIA 127 ETIOLOGY relative and absolute increase in acute leukemia's and in leukemia in the group of patients more than 50 years of age has been reported. This increasing incidence of leukemia in recent years is greater only for one other malignant disease, cancer of the lung. In the United States, the mortality rate from leukemia, which was 10 per million in 1900, rose to 63 per million in 1953. The disease is relaticly conimon in the first decade of life, and it is then encountered less frequently until the fifth and sixth decades arc reached. Lymphocytic leukemia predominates in childhood and in the older age groups, but myelocytic leukemia cxcecd, lymphocytic leukemia betwecn the ages of 20 and 50 years. In most series of cases there is a preponderance of males, ". 11 and among racial differences the incidence among Negroids is considerably less than among Caucasoid, 117, 130 The death rate for the two groups is equal in the nonwhite population, but lymphocytic leukemia is responsiblc for 41 per cent of the total deaths and myelocytic leukemia for 33 per cent of the total deaths from leukemia among Caucasoids " Although the familial occurrence of leukemia is sporadic and has not been reported as frequently as for many other neoplastic diseases, a number of reports support the premise that genetic factors for susceptibility are operative in the human disease. 131 124 Leukemia has been observed in siblings," in twins,'*' and two varieties have appeared in a single family." The age of onset often is similar for different, affected members of a family group. Insufficient data are available for any unequivocal view on the number, location, or kinds of genes involved, 123, 128 however. Leukemia, more commonly chronic than acute, occurs infrequently in pregnancy .129-105 The prognosis is better if pregnancy occur early in the course of the disease. Apparently, the child neither contracts the disease from the mother, nor has the mother been affected in the rare cases of congenital leukemia,138-139 A century after the original observations of Craigie, Bennett,'' and Virchow, 06-100 who recognized the disease independently, the first encouragement that the disease can be controlled since the adient of radiotherapy") is forthcoming Diagnostic methods have progressed more slowly since observations by these proncers and the microscopic observation of Donné, 1** Ehrlich,"! Friedreich,lº and Naegeli " It is only within the last decade that the obser ations of Ellermann and Banga* "** a half century ago hare been extended to any degree in mammals, and esidence that ruses are associated with the origin of human leukemia remains equivocal However, steady progress has been made in expanding knowledge about the role of filterable agents in inducing other tumors in animals and plants 13, 14, 15, 18, 19, 21, 45, 12, 141, 1 No longer are articles encountered attributing the onset of leukemija to trauma."*3-148 ·128 STIOLOGY AND TREATMENT OF LEUKÇAIA Possibly the experiments which have stimulated the most work and speculation in recent years have been those of Gross,1, 74, 75, 39, 40-49, 41, 46, 48, 49, 141, 147, 149 He was able to preparc extracts from leukemic tissues in the AK strain of micc by centrifuging and passing the material through Selas filters, which then was effective in producing leukemia when inoculated into newborn animals. This has been confirmed by Stewart,24 Woolley and Small, 4s, 59 Woolley, 52 and others. Not only were extracts prepared in this manner effective in producing leukemias, but also parotid tumors and sarcomas of soft tissues appeared and apparently depended on the introduction of a virus or viruses. The induced tumors which appeared were of the type similar to those appcaring in the recipient rather than in the strain of mouse from which the agent was derived. Stewart and co-workers' were able to culture this agent on renal tissue from monkeys in vitro. This agent obtained from mouse parotid tumors and leukemias was enhanced in effectiveness by serial passage. Friends, 185, 33* noted the cytoplasm of Ehrlich ascites tumor cells which were carried serially in Swiss mice contained particles suggesting inclusions which are commonly seen in cells infected with viruses. Following this lead, she was able to extract a cell-free agent which can be transmitted in adult mice. The virus produces a disease having the characteristics of leukemia, although the origin of the mononuclear cells scen characteristically in the peripheral blood of affected animals has not been determined. Schwartz and Schwartz and associates25% reported the isolation of a filterable agent from the brain of patients dying with leukemia which will accelerate the development of leukemia in AKR micc and induce leukemia in white Swiss mice Beard" clarified the etiology of avian leukosis, finding distinct but related viruses associated with avian leukemia, myeloblastosis, and crythroblastosis The threc agents of Icukosis in turn are but part of 2 larger system of agents responsible for the numerous avian lymphomas. Interest in the etiology of human leukemia has focused on the relation of irradiation to the origin of the disease It is eight to ten times as common in radiologists as in other physicians *15-339,34 The incidence of the diseasc has increased alarmingly in the last twenty years concomitant with the use of x-radiation, but the increment became apparent before the adient and common usage of radioactive isotopes. Evidence obtained from victims of the bombing of Hiroshima indicates that a single large dose of irradiation may result in leukemia Moloney and Kastenbaum reported that leukemia is twche times as common in survisors near the hypocenter at Hiroshima and Nagasakı as in survivors at the periphery. Additional evidence that leukemia may occur as a result of x-ray treatment and diagnostic procedures is found in the studies on individuals with leukemia treated for spondylitis by x-ray in Holland and Great Britain. Also leukemia has been reported following irradiation of the mediastinum in childhood, Difficulties in assessing and governing the use of therapeutic and diagnostic procedures with radiation are the deli- ·ETIOLOGY AND THERAPY OF LEUKEMIA 129 14 ciencies in our knowledge concerning maximum, sale dosage over various intervals of time and the biologic effects of extremely low dosage of irradiation. Current inquiry into possible deleterious effects of the amounts of irradiation commonly acquired by the population at large is cxccedingly disturbing. ***-341 The evidence from animal experimentation supports the inference that leukemia may occur in human beings as a result of irradiation. Furth and Furth" were the first to demonstrate the induction of leukemia by x-irradiation in mice with a los incidence of leukemia The experiments which Upton and Furth“ reported in the RF strain are particularly cont incing, since the type of leukemia appearing after irradiation is myeloid, whereas the type of spontaneous leukemia encountered in the strain is lymphoid. The induced type occurs with lower dosages niost frequently in males, whereas the spontaneous leukemia occurs predominantly in females. Although lymphatic leukemia may originate extrathymically in old age,' spontancous and induced lymphomas usually originate in the thymus, since early thymectomy reduces the incidence in most strains of mice.", *13-14 In the RF strain, hones er, thymectomy did not alter the incidence of myeloid leukemia induced by irradiation, suggesting that the thymus is the source of lymphogenous leukemia only Partial shielding, 67 of bone marrowel and spicen,"? administration of untreated, isologous, marrow cells, and fractionation of dosage inhibit the induction of lymphoid leukemia by irradiation. Estrogen may offset thc protective effect of shielding bone marrow but does not crert a similar effect on thymic shielding in C57BL micc,345 Partial shielding and dividing the dosage of irradiation also inhibit the myeloid leukemogenic effects of x-ray treatment. Kaplan and coworkers, 3* demonstrated that leukemia will appear in thymectomized and irradiated mice, which ordinarily do not develop leukemia, when they are inoculated subsequently with thymic tissue which has not been irradiated Therefore, the effect is apparently not a direct one on the thymic cell Genetic susceptibility to pulmonary tumors*** 159 resides within the target (issue when it is transplanted, rather than being a systemic influence Similarly, thymic cells retain their property for promotion of leukemia even though the leukemic cells themselves may arise from recipient tissue For example, Law and Potters found that leukemia appeared in thymectomized (C57BL * A) F, hybrids when thymus was transplanted from the susceptible (C57BL) parent and not when grafts from the resistant (A) parent were used Progenitor cells, as determined by histocompatibility characteristics, orginated most frequently from the F, host and less frequently from the nonirradiated C57BL graft. Snijders*9 and Tho Tywan Gie" performed the first experimental studies on leukemia in mammals They transmitted the disease in closely related guinca Pigs, producing lymphosarcoma after local inoculation and leukemia after intravenous passage Transplantation between strains is possible early in development before the mechanism of immunity?', 345 is mature, and both ·130 ETIOLOGY AND TREATMENT OF LEUKEMIA irradiation 349, 350 and cortisone, acting alone or synergistically, asi may reduce the immunologic response to transplantation of leukemic cells into foreign strains and species. Natural resistance to mouse leukemia may be transferred from rats without treatment to irradiated rat parabionts352 which otherwise would have been susceptible, but not from a resistant mouse to a susceptible parabiont without treatment, 253 Serial passage of leukemia in mice" can result in alteration of cellular virulence without concomitant changes in mor. phology, and the serum of immune animals can inactivate leukemia cells in vitro.Previous ideas about specificity and immunity have been challenged sharply by the polyomas encountered when Stewart and associates" inoculated hamsters with extracts from mouse leukemia. Pronounced differences in susceptibility to leukemia between inbred strains of mice were noted during the early work of Richter and MacDowell:0, 150 with the C58 strain, although nongenetic factors were also known to influence the appearance of leukemia since all of the genetically susceptible C58 mice did not develop the disease They were also able to demonstrate a maternal factor producing resistance to the development of leukemia so that incidence was reduced and latent period was prolonged. Although C58 mice have a high incidence of spontaneous leukemia, they are refractory to treatment with methylcholanthrene**; whereas DBA/2 mice are susceptible to the leukemogenic action of methylcholanthrene but have a low incidence of spontaneous leukemua *? Usually, F. hybrids show susceptibility to spontaneous and induced leukemia intermediate to that of susceptible and resistant parental strains, the degree depending on the specific strains used, and multiple genes for susceptibility are operaine. 315, 34 When two strains susceptible to estrogen lculem. ogenesis are crossed, the susceptibility of F, mice may be greater than that of either parent, however." Also, the F, animals may be resistant to the effect of estrogen when susceptible and resistant parents are matcd."** Age) alters susceptibility to induction of leukemia by transplantation of leukemic cells, 2*, 54 transmission by virus,'%, 341 and induction by irradiations and carcinogens As a rule, leukemogens are most effective when administered at an carly age, and resistance in F: hybrids is intensified with increasing maternal age Numerous studies have substantiated the increased incidence of leukemia in susceptible strains following administration of carcinogenic hydroand estrogen Lytrathy mic lymphoid tumors appear after thymectomy in DB1 2 mice treated with methylcholanthrene," "4 and Kirschbaum reported that lymphosarcoma from the C3H strain can be grafted into DBA mice prouided they are painted with methylcholanthrone L'rethan will increase the ellectus eness of other leukemogens, although it is not lcu- Acmogenic alone." Estrogen augments the leukemogenic action of irradiation, but androgen may counteract this action of estrogen as well as diminishi that of methylcholanthrenen and irradiation. On the other hand, castration enhances suscepubility to spontancous leukemia" " and leukemogenic effects 161 14 18 carbons 3,1 ·CTIOLOGY AND THERAPY OF LEUKEMIA 131 of methylcholanthrche.284 Contrary to the preponderance of leukemia in human males, it is interesting that thymomas and lymphocytic leukemia occur most frequently in female mice.** 38 Idrenalectomy increases the susceptibility of mice and rats to spontancous and transplanted leukemia'1, 372 and to that induced by irradiation, but cortisone delays the onset of spontancous lymphocytic" *** lculemia, thymic lymphosarcoma induced by r-irradiation,",$15 and leukemia induced by methylcholanthrenes if dosage is sufficiently great and prolonged. Idministration of pituitary growth hormone is associated with the appearance of pulmonary lymphoid tumors in rats if the hypophysis is intact, ato but tuniors do not appear in hypophyscctomized animals." "The enhancement of suceptibility to leulemia in mice by thyroid deficiency may be mcdiated by effects on body weight, since there is a direct correlation between weight and the appearance of leukemia." Reduction of caloric intake alone diminishes the number of spontancous lymphomas and prolongs the mean time of survival in transplanted leukemias. 158, 1** Interest in leukemis in cxperimental animals is heightened by the similarity between the discase in the mouse and in man In summary, experimental studies suggest that susceptibility to leukemia in animals is due to multiple genes governing not only the spontancous incidence but also response to various agents. Incidence is increased by irradiation, the presence of virus, estrogen, carcinogenic hydrocarbons, castration, adrenalectomy, and perhaps pituitary growth hormone under certain conditions Combination of different agents may act synergistically, and urethan, although inactive alone, potentiates the action of leukemogens Leukemia is inhibited by increasing age, caloric restriction, cortisone, androgen, thymectomy, and shielding of bone marrow, thymus, and spleen following irradiation Growth of leukemic cells in foreign strains may be promoted by inoculation early in development, cortisone, irradiation, and carcinogenic hydrocarbon Both the leukemogenic effects of irradiation and the intrinsic property of tissue from genetically susceptible donors to initiate the disease are apparently exerted indirectly DIAGNOSIS AND CLASSIFICATION Classification of the leukemias is often controuersial of detailed and too superficial simple Most of the difficulares in regimentation stem from un. certainties due to the limitations of morphologic criteria Sprague has emphasized the possibilities for differentiating between mieloid and mononuclear senes by means of ribonuclease and desoxyribonuclease in the future, pertinent information extending knowledge about the varieties of human leukemia should be forthcoming by means of cytochemical preparations, tissue culture, and microcinematography, utilizing electron, phase, reflecting, and interference microscopy. The nomenclature used in this chapter (Table XXIII) includes myelogenous, lymphogenous, monocitic, and stem cell forms of leukemia, designated ac- ·132 ETIOLOGY AND TREATMENT OF LEUKEMIA TABLE XXIII NO EXCLATURE OF LEUKEMIAS TYPE Myelogenous CLINICAL COURSE IN RELATION TO PREDOMINANT CELL ACUTE GITROVIC Myeloblast Myelocyte Neutrophil Eosinophil Basophit Lymphoblast Lymphocyte Monoblast Monocyte Undifferentiated stem cell Lymphogenous Monocytic Stem cell cording to the predominant type of cell and based on studies both of bone marrow and penpheral blood. In addition, the disease is divided temporally into the acute form, with blasts predominating, and into the chronic form of the disease, with either lymphocytes or myelocytes chiefly. Plasma cell leukemia has not been included because of uncertainties concerning its identity with multiple myeloma 181 The leukemic manifestations of malignant lymphoma have also been omitted In unusual cases of the chronic myclogenous diseise, the cosinophil or basophil may be the most common type of cell. However, the manifestations of leukemia range from a rather pure form to erythroleuko-prastrenemia, and from cases with pronounced leukocytosis to subleukemic forms of the malady, SCREENING FOR CHEMOTHERAPEUTIC AGENTS Possibly because thc leukemias occurring in laboratory animals fairly closely correspond to those appearing in the human population, remarkable success has been achici ed in a relatncly short time in the treatment of leukemia Despite problems in cvaluation due to advances in supportive therapy, variatrons in scquence and time of treatments, and scarcity of valid data on untreated cases, it is clear that the survival time of patients with Icukemia has hecn prolonged by chemotherapeutic agents 18! The hope that the difference in response between leukemic and normal cells will lead to cure of the discase, thereforc, has some justification In general, initial screening has consisted of testing an exceedingly sensitive transplantable tumor such as sarcoma 180 or the Walker carcinoma 256 for susceptibility to the compound chosen However, no single tumor can be used as an effective screen, and a spectrum of spontaneous and transplantable tumors should be used for an adequate suncy, as well as biologic systems for testing certain synthetic steps ar cytogenetic effects Suitable, actise matcrial then is used for clinical investigation after it has been tested for possible toxicity So far, screening procedures have failed to yield a good correlation between activity of compounds in the tumor sy sten and in the montumor systems of testing, indicating that the antitumor cffccis detected are somewhat specific.

·ETIOLOGY AND THERAPY OF LEUKEMIA 133 However, clinical effectiveness has been encouraging when promising compounds have been tested, and occasionally compounds are more effective against human, neoplastic disease than experimental tumor in animals Past deficiencies in the method may be attributed as much to the paucity of agents completely effective against experimental tumors such as leukemia as to an inadequate approach to screening. TREATMENT The quest for clicient therapeutic agents has not only been empiric, but rational selection of compounds to be tested more frequently has ultimately led to successful treatment. Choice of compound has been combined with a fortunate range of methods for testing cffectiveness in biologic systems The therapeutic effects of alkylating agents and antimetabolites which were chosen for testing in this fashıon 02-1** indicate the importance of knowledge concerning pathogenesis of leukemia in sclecting the proper sistem for screening, as well as the selection of agent to be tested however IRRADIATION The oldest'13 consistently successful treatment for leukerna is irradiation Careful comparisons by a number of therapists in the past?, 181.1"9 have led to the conclusion that irradiation prolongs the useful and efficient portion of the patient's remaining life span, but the average total duration of life probably is unchanged in irradiated groups In general, irradiation has been confined to chronic forms of the disease, but Schlosser suggests irradiation may now be combined with chemotherapeutic agents cien in acute leukemia For example, steroid therapy may be used to curb a crisis and irradiation tried subsequently to prolong the remission This plan of combined treatment for acute leukemia is not universally acceptable to all therapists at the present time, The usual form of roentgen therapy for leukemia is irradiation of the spleen, irradiation of lymph nodes or visceral areas, irradiation of large segments of the body, and total-body or body-bath irradiation. P" therapy *7. 186, 180-18* has the advantage of not being associated with radiation sickness, and it requires few special facilities Roentgen therapy is suprrior, howeser, when reduction sa size of spleen and lymph glands is desirable 18 p2 may be administered either orally or intravenously, and the usual dosage is 0.5 to 2 mc, with intervals between successive doses selected according to response Therapy with radium, 149 radioactive gold,190 191 and radioactive sodium has been reported, but it is not widely employed for leukemia. The method of titrated, regularly spaced roentgen or P therapy described by Osgood" has resulted in survival which is superior to other programs of therapy, according to the statistical analysis of Tey The salient features of this plan are therapy immediately after the diagnosis is made, irradiation ·132 ETIOLOGY AND TREATMENT OF LEUKCMIA TABLE XXI11 NOMENCLATURE OF LEUKEMIAS TYPE Myelogenous CLINICAL COURSE IN RELATION TO PREDOMINANT CELL ACUTE CHRONIC Myeloblast Myelocyte Neutrophil Eosinophil Basophil Lymphoblast Lymphocyte Monoblast Monocyte Undifferentiated Lymphogenous Monocytic Stem cell stem cell cording to the predominant type of cell and based on studies both of bone marrow and peripheral blood In addition, the disease is divided temporally into the acute ſorm, with blasts predominating, and into the chr form of the discasc, with either lymphocytes or myelocytes chiefly. Plasma cell leukemia has not been included because of uncertainties concerning its identity with multiple myeloma." The leukemic manifestations of malignant lymphoma have also been omitted. In unusual cases of the chronic myelogenous disease, the eosinophil or basophil may be the most common type of cell. However, the manifestations of leukemia range from a rather pure form to erythroleuko-piastrenemia, and from cascs with pronounced leukocytosis to subleukemic forms of the malady. 160 161 SCREENING FOR CHEMOTHERAPEUTIC AGENTS Possibly because the leukemias occurring in laboratory animals fairly closely correspond to those appearing in the human population, remarkable success has been achieved in a relatively short time in the treatment of leukemia. Despite problems in evaluation due to advances in supportive therapy, ariations in sequence and time of treatments, and scarcity of valid data on untreated cases, it is clear that the survival time of patients with leukemia has been prolonged by chemotherapeutic agents The hope that the difference in response between leukemic and normal cells will lead to cure of the disease, therefore, has some justification In general, initial screening has consisted of testing an exceedingly sensitive transplantable tumor such as sarcoma 180 or the Walker carcinoma 256 for susceptibility to the compound chosen However, no single tumor can be used as an effective screen, and a spectrum of spontaneous and transplantable tumors should be used for an adequate survey, as well as biologic systems for testing certain synthetic steps or cytogenetic effects Suitable, active material then is used for clinical investigation after it has been tested for possible toxicity So far, screening procedures have failed to yield a good correlation between activity of compounds in the tumor systems and in the nontumor systems of testing, indicating that the antitumor effects detected are somewhat specific ·TIOLOGY AND THERAPY OF LEUKEMIA 133 However, clinical clectiveness has been cncouraging when promising compounds have been tested, and occasionally compounds are more effective against human, ncoplastic disease than experinental tumors in animals. Tast deficien. cies in the method may be attributed as much to the paucity of agents completely effective against experimental tuniors such as leukemia as to an inadequate approach to screening. TREATMENT The quest for eficient therapeutic agents has not only been empiric, but rational sclection of compounds to lc tested more frequently has ultimately led to successful treatricnt. Choice of compound has been combined with a fortunate range of methods for testing effectus cness in biologic systems The therapeutic effects of alkylating agents and antinyetabolites which were chosen for testing in this fashion "02-*** indicate the importance of knowledge concerning pathogenesis of lcukcinia in sclecting the proper system for screening, as well as the sclection of agent to be tested IRRADIATION The oldest consistently successful treatment for leukemia is usradiation Carclul comparisons by a number of therapists in the past!ıs 191 179 have led to the conclusion that irradiation prolongs the useful and efficient portion of the patient's remaining life span, but the ancrage total duration of life probably is unchanged in irradiated groups. In gencral, irradiation has been confined to chronic forms of the disease, but Schlosser suggests irradiation may now be combined with chemotherapeutic agents even in acute leukemia For example, steroid therapy may be used to curb a crisis and irradiation tried subscquently to prolong the remission This plan of combined treatment for acute leukemia is not universally acceptable to all therapists at the present ume, however. The usual form of roentgen therapy for leukemia is irradiation of the spleen, irradiation of lymph nodes or visceral areas, irradiation of large segments of the body, and total-body or body-bath irradiation. p" therapy of, 160, 160 1** has the advantage of not being associated with radiation sickness, and it requires few special facilities Roentgen therapy is superior, however, when reduction in size of spleen and lymph glands is desirable 1 piz may be administered either orally or intravenously, and the usual dosage is 05 to 2 mc, with intervals between successive doses selected according to Therapy with radium, 'g radioactise gold, 190, 191 and radioactive sodium" has been reported, but it is not widely employed for leukemia The method of titrated, regularly spaced roentgen or P2 therapy described by Osgood'93 has resulted in survis al which is superior to other programıs of therapy, according to the statistical analysis of Tivey The calient features of this plan are therapy imniediately after the diagnosis is made, irradiation response ·134 ETIOLOGY AND TREATMENT OF LEUKEMIA of the entire body either by roentgen therapy or p', titration of the dose depending on the response of the patient, and initial weekly interval between dosage altered until suitable maintenance dosage and interval are obtained, When leukemia is being managed with chemotherapy, roentgen irradiation is useful supplemental therapy for refractory, localized discase. Localized lesions in the bones which are painful, cnlarged spleen, localized lymphatic involvement, enlarging kidneys, and meningeal infiltration, giving signs of increased intracranial pressure, all respond to irradiation with sufficient frequency to justify trial Variations in the sequence, treatment factors, dosage, and field irradiated are numerous and depend on the inclinations and experience of the radiologist, as well as the response of the individual treated. For example, doses of total-body irradiation, although usually in the range of 10 to 20 r, sometimes are given in massive amounts;194 and irradiation directed to the spleen usually is 200 to 400 r, but it may be as great as 2,000 r in refractory cases. More irradiation may be required for a response in myelocytic than in lymphocytic leukemia. Edema may follow irradiation of cnlarged lymph glands, and this can prove disastrous when impending venous or tracheal obstruction is present. The possibilities for improvement of therapy inherent in investigation of the effects of irradiation on normal and neoplastic tissues are as great as continued chemotherapeutic studies Actually, some of the effects of irradiation and chemotherapeutic agents are strikingly similar. Regression of tumors at a distance from the field of irradiation and changes in nonirradiated tissue transplanted to an irradiated field suggest humoral mediating factors. The reversibility of radiation damage by transplantation of bone marrow and the protective effect of shielded tissues have been studied extensively. 61, 67, 193 The proposal that damaged cells are repaired by a process akin to transduction and transformation 96 has not been confirmed, but methods of labeling cells® and information about mutagenic peroxides formed in irradiated material, as well as other studies on altering radiation effects, show real promise.", 197 CHEMOTHERAPY Benzene.—Exposure to benzene (benzol) may result in changes in the entire hematopoietic system, the most characteristic of which are found in the bone marrow Hyperplastic reactions occur more commonly in males and hypoplastic changes in females Chronic exposure is associated not only with anaplasia but also, less commonly, with leukemia.'” In the past, experience showed it to be useful treatment for leukemia" in daily dosage of 2 to 5 Gm Now it is no longer used therapeutically because of potential toxicity and the adıent of more satisfactory compounds Arsenic - One of the toxic effects of arsenic is to depress the leukocyte count, and compounds releasing inorganic arsenic have been employed in the ·ETIOLOGY AND THERAPY OF LEUKEMIA 135 past lor the treatment of Iculemia.**), ** Best results were obtained in chronic myclocytic dícase during continuous therapy. Fowler's solution, X.F. (potassium archite), usually was gisen in doses incrcasing from 0.3 mg. three times daily until response or toxic effect, ensued in addition to oral and recial routes of adminstration, potassium arsenite alo was administered intra cnously in diluic solution. This treatment has been superseded largely by other forms of therapy. Urethan.--Ethyl carbamate, which has been known for many years as a hypnotic, was tested for possible inhibition of various types of tumors by Haddon and Sexton" as part of a general program of screening of mitotic inhibitors. Leukopenia was observed, and Paterson and associates302 subsequently reported response of Icukemia to administration of the drug. The exact mode of action has not been clarified by tracer studies with labeled but it is of interest that the compound is carcinogensi in animals.23 Urethan is not effective in acute Iculcmia, and chronic myclocytic leukemia responds to it C2 Hs-O-C-NH2 L rethan (Ethyl Carbamate) lag in leumore favorably than chronic lymphocytic leukemia There is a kocytic response, so that the discontinuation of therapy s prudent when the count reaches fiftcen to twenty thousand leukocytes per cubic millimeter. The usual dose of the drug is i Gm after meals three times daliv, but there is considerable variation in response from patient to patient Smaller dosage may be effective in maintaining remissions In favorable cases, the total leukocite count recedes to the normal range, the differential count approaches the normal pattern, thc hemoglobin level rises, and the spleen and enlarged nodes are reduced in size Toxic effects are most commonly nausea and vomnting, but may alo include hypoplasia of the bone marrow, hepatic injur, and damage to the central nerious system Alkylating igents Vlechlorethamine Hydrochlonde Therapeutic possibilities of the radiomimetic nitrogen mustards were first recognized by Goodman and associates, 295 Goodman and Gilman, *"* and Gilman and Philips, 2* who found that transplanted lymphosarcoma regressed after treatment of mice with crystalline hydrochlorides of the nitrogen mustards Later, Lindskog ob- Cained clinical remission in a patient with lymphosarcoma, and hematologic studies by Dougherty showed resersible depression of bone marrow function Lymphocytopenia usually precedes granulocytopenía 29: Mjethyl-bis (2-chloro- ·136 ETIOLOGY AND TREATMENT OF LEUKEMIA ethyl) aminc hydrochloride (HN:, Mechlorethamine hydrochloride, Mustargen) causes a response in both chronic lymphogenous and chronic myclogenous leuhemia203-213 which may be prolonged, but chronic lymphocytic leukemia accompanied by anemia seldom responds satisfactorily,210 and acute leukemia cannot be managed with Mustargen. The usual course of therapy consists of 0 4 mg. in fresh solution per kilogram of body weight. Mustargen was used in far-advanced lymphogenous leukemia refractory to irradiation and in disseminatcd discase with visceral involvement.213 Because both rocntgen therapy and other chemotherapeutic agents yield longer remissions, Mustargcn now is seldom used even in combination with other treatment. 211, 212, 214 CH2 CH2 CI CH3-N CH2 CH2 CI I Mechlorethamine (HX, Mustargen. Methyl-bus (-Chloroethyl) Amine) Trrethylene Mlelamine. Triethylene melamine (2,4,6-triethylene-s-triazinc; TENI)215, 22* is a congener of Mustargen which is effective in chronic lymphogenous and myelogenous leukemua Acute Icukemia docs not respond S Triethylene Melamine (TEM, 2,4,6-Trethylene-s-Triazine) N-N-X"-Triethylene Throphosphoramide (ThioTEPA, TSPA) 226 Bur- This compound as synthesized by textile chemists and tested independently in England and in the United States Inhibition of neoplastic growth in animals was reported by Rose, Hendry, and Walpole, 225 Lewis and Crossley,?. chenal and co-workers, *2 and Philips and Thiersch 229 Clinical trials229-2!! were immediately encouraging One of the chief advantages of the drug is the fact that it is a nitrogen mustard analogue which can be administered orally.217 It ·ETIOLOGY AND THERAPY OF LEUKEMIA 137 may also be given in one-hall to one-third the oral dose intravenously. The compound, in acıd solution, forms ethyleneimonium cations which are biologically active and cytotoxic. For that reason, it is administered orally approximately two hours before breakfast, and 2 Gm. sodium bicarbonate is given with each 5 mg. triethylene melamine in order to prevent their appearance in the stomach.224 Injections of triethylene melamine into the pleural cavity can be effective in preventing the reaccumulation of pleural fluid, Nausea and vomiting are less troublesome with tricthylene melamine given by moutha3than with other nitrogen mustard therapy. Considerable delay in action of the drug occurs following administration, and successive doses must be given cautiously, especially if it is being given on an outpatient basis. Ulceration of the intestinal mucosa, thrombocytopenia, and hemorrhages have been reported in patients receiving triethylene melamine Higher doses are required to obtain a comparable response in chronic myelocytic than in chronic lymphocytic leukemia. Dosages recommended vary somewhat, but an initial dose up to 5 mg, in chronic lymphocytic leukemia and up to 10 mg in chronic myelocytic leukemia is reasonably safe. Subsequent treatment can then be planned on the basis of response within the period of a week or more. Triethylene melamine is now rarely used for the treatment of chronic myelogenous leuhemia N-N'-N" Tnethylene Throphosphoramide: Shav and associates, *** Zarafonetis and co-workers,*** and Leonard and associates reported response of chronic lymphogenous and chronic myelogenous leukemia to admunistration of triethylene thophosphoramide (thioTEPA). Acute leukemia not only does not respond, but also the experience of Smith and associates295 led them to advise that throTEPA is contraindicated for children with acute leukemia The drug is useful because it can be given intramuscularly and orally when the intravenous route is not desirable or practical Local tumefaction, hepatomegaly, splenomegaly, and leukocytosis regress with treatment Anorexia, nausea, and vomiting may occur, and therapy should not be given when thrombocytopenia is encountered А course consists of three to four weekly treatments of 25 mg and should not be repeated for one month to six weeks 218 Busulfan. Busulfan (1,4-dimethanesulfonylorybutane, Myleran, G T-41) is a polyfunctional alkylating agent depressing the granulocytic series which was synthesized by Haddow and Timmis in 195382 and found by Galton and 0 0 11 11 H3 C-S-O-CH2 CH2 CH2 CH2-O-5-CH3 Busulfan (GT-41, Myleran, 1,4-Dimethanesulfony loxybutane) ·136 ETIOLOGY AND TREATMENT OF LEUKCALIA ethyl) amine hydrochloride (HN2, Mechlorethamine hydrochloride, Mustargen) causes a response in both chronic lymphogenous and chronic myelogenous Jevkema18-213 which may be prolonged, but chronic lymphocytic Icukemia accompanied by anemia seldom responds satisfactorily;2!and acute leukemia cannot be managed with Mustargen. The usual course of therapy consists of 0.4 mg. in fresh solution per kilogram of body weight. Mustargen was used in far-advanced lymphogenous leukemia rcfractory to irradiation and in disseminated disease with visceral involvement 213 Because both roentgen therapy and other chemotherapeutic agents yield longer remissions, Alustargen now is seldom used cven in combination with other treatment, 215, 212, 216 CH₂ CH₂ CI CH3-N CH2 CHE CI Mechlorethamine (HX. Mustatgen, Methyl-bus IB-Chloroethyll Amine) Triethylene Melamine. Triethylene melamine (2,4,6-triethylene-s-triazine; TEM)*), 22" is a congener of Nustargen which is eficctive in chronic lymphogenous and myclogenous leukemia Acute leukemia does not respond An Triethylene Melamine (TEM, 2,4,6-Triethylene-s-Triazine) N-V-V-Triethylene Thiophosphoramide (THOTEPA, TSPA) This compound was synthesized by textile chemists and tested independently in England and in the United States, Inhibition of neoplastic growth in animals was reported by Rose, Hendry, and Walpole,” Lewis and Crossley, Burchenal and co-workers, and Philips and Thuersch *** Clinical trial,229-231 immediately encouraging. One of the chief advantages the drug is the fact that it is a nitrogen mustard analogue which can be administered orally.217 It 1 crc ·ETIOLOGY AND THERAPY OF LEUKEMIA 139 Considerable variation in the amount necessary for a response has been cncountered, and whether irregularity of absorption or utilization is responsible is not known Dosage has varicd from 0.1 to 13 mg. per kilogram of body weight. Damage to bone marrow is more likely during continuous therapy than after intermittent administration, but suppression of hematopoiesis usually is reversible,360 Lower dosage is advisable when initial thrombocytopenia is found, and such instances constitute a group in which chlorambucil is particularly useful.6%, 258 Anorexia, nausca, and vomiting occur only with large doses of the drug. When properly administered, chlorambucil rarely causes undesirable side effects. Approximately two-thirds of the cases of chronic lymphocytic leu. kemia trcated respond favorably*** with decrease in leukocyte count, correction of abnormalities in differential count and hemoglobin level, and diminution of hepatosplenomegaly and lymphadenopathy. Response occurs usually by the Sourth week of therapy and may last as long as six months Although a shorter course of tricthylene melamine may be required for remission, triethylenc melaninc is associated with more untoward side effects than chlorambucıl. The superiority of chlorambucil to irradiation is questionable,?** and additional experience will be required to establish its most useful role in the therapy of leukemia. Antimetabolites, Folic Acid Antagonists. The deleterious effects of folic acid when administered to patients with leukemia?), 202 are well known After the structure and function of this vitamin were established, analogues became available, and one of the striking effects of thesc antimetabolites proved to be leukopenia *64 The 4-amino analogues, 4-aminopteroylglutamic acid (Aminopterin), 4-amino-Nomethylpteroylglutamic acid (Methotrexate, Amethopterin), and 4-aminopteroylaspartic acid, were found to be active in mammals. However, the administration of pteroyIglutamic acid (PGA) does not reverse the effect of folic acid analogues in mammals because pteroylglutamic acid is transformed into folinic acid (citro- Forum factor) in the process of nucleic acid metabolism When the citrovorum factor is used to combat toxicity, it nullifies the therapeutic effects of folic acid antagonists as well The successful use of folic acid analogues in acute lymphoblastic and myeloblastic leukemias was first reported by Farber and associates 201 Some response is obtained in approximately two-thirds of children treated, 284, 285 and best results are obtained in acute lymphoblastic disease Chronic leukemias rarely268 respond. Of the 4-amino analogues, Methotrexate is the most satisfactory. 288, 287 Toxic side effects of continued therapy are stomatitis, diarrhea, pancytopenia, portal cirrhosis, and alopecia The alopecia is resessible during therapy. Folic acid antagonists should not be given during pregnancy because of deleterious effects on embryogenesis When renal insufficiency is present, plasma les els may con- 208 ·138 ETIOLOGY AND TREATMENT OF LEUKEMIA Tillº3, 236 to be effective palliation for chronic myelogenous leukemia. The effectiveness of busullan has been confirmed by numerous investigators, 84, 80, 237-233 and it generally is regarded as superior to triethylene melamine, urethan, and other chemotherapeutic agents. More experience will be sequired to determine how this drug compares to irradiation, but it seems to be equally effectue Busulſan is without significant toxicity when given properly, but excessive single and loading doses are unwisc since thrombocytopenia and hypoplasia of the bone marrow may ensue. Splenic irradiation may be necessary in addition to busulfan. Till cautions against continued dosage during pregnancy. Although patients may not be alert to symptoms of the disease and experience relicí after therapy, Till advises against beginning therapy in the absence of symptoms. The leukocytic response niay be delayed as long as six to eight weeks, particularly if splenic irradiation has preceded therapy, but usually occurs before this time. Increase in hemoglobin level may not begin earlier than one month and may not reach normal levels until three months have passed since the beginning of therapy, depending on the initial level but independent of the leukocytic response. Responses may last as long as twelve months. Relapses occur earlier with successive courses of therapy, and eventu. ally continuous administration of the drug may become necessary The recommended oral dosage is not greater than 0.065 mg per kilogram of body weight, which is approximately 4 mg daily for most patients. Dosage greater than 6 mg may depress myelopoiesis and offers no advantage. The biologic specificity of busulfan, although not absolute, and its occasional effectiveness in the management of cases refractory to radiotherapy make it exceptionally useful in the management of chronic myelogenous leukemia Chlorambucil. In the course of ini estigating biologic effects of aromatic alkylating agents on normal and neoplastic tissues, Haddow164, 25% found that 2 paraphenylbutyric acid derivative of mechlorethamine, chlorambucil (CB- 1318, p-NN-[d1-2-chloroethyl) aminophenylbutyric acid), inhibited the Walker с сне сна N CH2 CH2 CH2 COOH CI CH2 CH2 Chlosarnbucil (CB-1348, p.NN.{dı-2-Chloroethy!! Iminophe uylbutyric Acid) carcinoma 256 ?,5 Further studies by Elsons and also by Bollag?sruvealed that the agent depressed the lymphocyte count in the rai without a comparable change in the granulocytes Experience by Galton and associates and others252-259 soon demonstrated the uscfulness of chlorambucil in chronic lymphocytic leukemia.

·LTIOLOGY AND THERAPY OF LEUKEMIA 141 alin respond occasinnally. Buxullan and irradiation gic superior results in the therapy of chronic myclocytic leukemia Clinical trials followed the demonstration by Clarke and associates as that the drug not only inhibited but alan cured sarcoma 180 and increased the sursival of mice with transplanted leu. kemias, both sensitive and resistant to Amethopterin Some children resistant 10 therapy with Amethopterin and steroid therapy responded to G-mercaptopurine also. The dosage of 2.5 mg. per kilogram of body weight per day use. ally amounts to 5 mg, in a child and from 100 to 200 mg, in an adult. The drug must be given for long periods to be cffectue Remissions are not expected in less than from three to eight weeks, and Burchenal advises treatment at least cight wechs before abandoning trial with 6-mercaptopurine. On occasion, complete remission may not occur for ten to twch wecks. Although toxicity at the recommended dosage is minimal, bone marrow hypoplasia docs occur, and nausca and vomiting are other undesirable side effects Renal damage as a result of high uric acid levels following rapid fall in leukocyte count should be anticipated by regulating the amount of the drug administered Remissions SH NHL OH CH CH CH N AC N M H 6-Mercaptopurine (Puninethot) Aderine 11ypoxanthin Purine Analogues usually are shorter in childhood acute leukemia than remissions following therapy with folic acid antagonists Combined therapy with the glutamine antagonist, azascrine, *** gres results suggestie that complete remissions are somewhat longer than those with mercaptopurine alone 142 Hall and Vincent reported that large doses of fluorohydrocortisone in addition to 6-mercaptopurine may gne remissions in adults with acute leukemia Steroids---Cortisone, hidrocortisone, ACTH, prednisone, and prednisolone may produce prompt remissions and are particularly valuable in acute leukemia occurring in children. Farber and co-workerszt, 29 and Pearson and assocatesise were the first to recognize the usefulness of steroids in leukemia In comparison to the other steroids, prednisone and prednisolone produce comparable therapeutic effects with less undesirable changes in electrolyte balance 10 Bes. bard and Deltour, 19 Hill and associates, 91, 29% and otbers*2, 173, 27 administered cortisone in doses up to 5 Gm and prednisone and prednisolone in doses up to I om daily in adults to produce responses which would otherwise not be achieved. On the basis of response of the leukemia, there may be some justifica- ·140 ETIOLOGY AND TREATMENT or LEUKEMIA tinue to be high, and care should be exercised in calculating dosage to prevent cumulative toxic effects.269 Methotrexate may be given intramuscularly or orally in daily doses of 1.25 to 5 mg. The drug can be continued in daily doses up to two months,162 or until remission occurs. If continuous therapy then is elected, treatment on alternate days with lower dosage may be advisable. Serial studies of bone marrow and peripheral blood are advisable for most satisfactory management. Although relapse inevitably occurs and remission may be of only a few weeks' duration, the response may extend over a period of many months in favorable cases.241, 284, 265, 270-273 In general, remissions are longer than after therapy with 6-mercaptopurine and steroids.16 -NH₂ GOOH но HC --N-C- 1 CH3 -N-CH2 GHZ NH2 GN2 COOH Amethopterin (Methotrexate, 4-Amino-N10 Methylpteroylglutamic Acid) COOH 1 HCNC ỹ -NH2 -N-CH₂ OH CH2 CHE COOH Folic Acid (PGA, Pteroylglutamic Acid) Folic Acid Analogues Purine Antagonists Utilizing the compounds and information obtained by Elion and co-workers4-2and by Hitchings and co-workers?"8-2#1 on purines and pyrimidines in relationship to the biosynthesis of nucleic acid, Burchenal and co-workers?»2-3** found that a purine antagonist, 6-mercaptopurine (Purinethol), caused complete or partial remission and clinical improvement in approximately half of the children with acute leukemia treated. Adults with acute leukemia28s or afflicted with early and late chronic myelocytic leukemia ·ETIOLOGY AND TIIERAPY OF LEUKEMIA 1.3 has been re-evaluated as a procedurc to be ined only in selected cases rather than indiscriminately. Results «cem to justify the operation when the criteria of Ferrata and Fieschi'' arc followed with certain modifications Asindications, they included thrombocytopenia on the basis of hypersplenism, splenomegaly, and hemolytic anemia. The bone marrow in Teulemia will not likely present as favorable a picture as desirable, but operation is contraindicated only if the numbers of nucleated red cells and megaharyocytes are reduced to a pronounced degree. In the case of hyper-plenic thrombocytopenia and pancytopenia, the calculated risk of operative intervention in patients with leukemia may well be rewarding,"ss, 165-119 with improvement lasting for a number of years How ever, splenomegaly alone is not an indication for splenectomy, responding as it frequently does to chemotherapy, irradiation, or a combination of the two. When the great size of the spleen and associated pain are otherwise unmanageable, removal becomes justified Dameshch and associates," 5,"1 Wintrobe," and others advisc splenectomy only after failure of steroid therapy when hemolytıc ancmia is the rcason for considering surgery Better results are obtained il the spleen is removed carly in the course of the disease Occasionally, splenectomy is performed for the cytopenia before leukemia is recognized, ans and the diagnosis should always bc hept in mind in this group of cases Thymectomy" has not been bencficial in clinical cases of lymphogenous leukemia 313 SUPPORTIVE THERAPY $13-315 A forthright, positive, and checrful attitude on the part of the physician assures the patient and his famils that something can be done, and therapy should be directed toward prolonging the useful and happy period remaining before the inevitablc outcome In children, particularls, the periods of hospitalization should be reduced to a minimum Leukopenia is not necessarily an indication for transfusion, and the hemoglobin value may return to normal in anemic pa. tients without transfusion after appropriate therapy For this reason, transfusions should be deferred unless the necessity is obuous If transfusions do become necessary, they can be given on an out-patient basis Also, prophylactic antibiotic therapy ordinarily is not advisable in the absence of a specific infectious process. The therapist should guard against the mistake of deferring definitive therapy too long with the idea that the patient may need therapy more urgently in the future Treatment should begin when there are indications, since the patient's disease may become refractory to treatment, forfeiting palliation earlier in the disease MANAGEMENT OF LEUKEMIA161 311 316-328 Acute Leukemia -- Both lymphoblastic and myeloblastic leukemia respond to antimetabolites and lymphoblastic leukemia responds to steroids. The meta- ·142 ETIOLOGY AND TREATMENT OF LEUKEMIA tion for this type of therapy, although the usual side effects appear with doses of this magnitude. Ranney and Gellhornº advise antacids and 3 Gm. potassium chloride daily as part of the prednisone and prednisolone therapy. Fessas and co-workers?s confirmed the excellent results of treatment for acutc lymphoblastic Icukemia, but they advised caution in using cortisone and corticotropin in other forms of leukemia because response was not only less likely to occur but also the process seemed to be accelerated in some cases. Desacetylmethylcolchicine.---Desacetylmethy/colchicine (Demecolcin, Colcemid), extracted from Colchicum autumnale by Santavy and Reichstein, ** is thirty to forty times less toxic than colchicine in doses producing the same antimitotic cffects 207 Following initial studies of Moeschlin and co-workers, a others have tested its therapeutic effects.249-399 Remission of chronic granulocytic leukemia occurs frequently when this drug is administered, although relapse ensues rather promptly if it is discontinued. Leonard and Wilkinson 302 Снзо -NH-CH3 CH3 01 Hz OCH3 Desacery imethylcolchicme (Colcemid, Democolcin) pointed out that the aplastic anemia, thrombocytopenia, and precipitation of acute myeloblastic leukemia, which are disadvantages of busulfan therapy, were not scen in a small group of cases they treated with Demecolcın. Initially, Demecolcin may be given in daily doses of 3 to 10 mg. by mouth. When the lcukocyte count diminishes, a daily maintenance dose of 3 to 5 mg. is usually adequate with minimal side effects 299 A similar compound, N-desacctylthiocolchicine, has been found to have similar therapeutic effects by Huguenin and associatesana and by Burchenai and Krakoff," but additional data are desirable in order to evaluate the relative usefulness of this agent and Demecoicin in comparison to other types of therapy SURGERY Other than biopsy of soft tissues and bone marrow to establish the diagnosis, surgery is useful in leukemia only when splenectomy is indicated After the first splenectomy for leukemia by Bryanti's in 1866, the procedure was tried extensively and abandoned as inadusable. In recent decades, however, the operation ·ITIOLOGY AND THERAPY OF LEUKEMIA 145 mine the exact uscfulness of the colchicine group of compounds in the treatment of chronic my clogenous leukemia. A tremendous amount of cxperience has been accumulated in the use of localized and lotal-body irradiation in chronic leukemia. A comparison of the results of irradiation treatment and chemotherapy is difficult, but there is no clear-cut supcriority of chemotherapy to irradiation in the therapy of chronic leukemia. One type of treatment may be a usclul supplement to the other. Irradiation of the spicen and localized lesions oſten is a useful adjunct to chemiotherapy. The methods of continuous, titrated dosage of total body and P2 irradiation first described by O-good" have produced results which arc quite impressive. In the treatment of thc indavidual case of chronic lymphocytic and granulocytic leukemia, the therapist has at his command a comforting array of methods for definitive therapy (Table XXIV), as well as transfusions and antibiotics as supportive therapy for leukemia, Clearly, the possibulty that additional chemotherapeutic agents may be more effectue encourages laboratory and clinical screening programs, such as current investigations on the effect of antibiotics and biologic filtrates alone and supplemented with irradiation.320, 331 addition, much is to be Icarned about the most effective combinations and sequences for using the agents at hand In ·144 CTIOLOGY AND TREATMENT OF LEUKEMIA bolic antagonists of folic acid, purines, and glutamine are useful chiefly in children and more rarely in adults. Fortunately, resistance to one does not imply resistance to the remainder, and one agent can be used as supplemental therapy to the other. Serial treatment, therefore, may offer more than the use of one of the antimetabolites alone. Response to the antimetabolites requires a longer period of time, but remissions are longer in comparison to the results of steroid therapy. In acute leukemia, therefore, the folic acid, purine, and glutamine antagonists should be reserved for those cases in which waiting for a response for therapy is justified On the other hand, steroids exert their effects rapidly but the response is not prolonged. Therefore, in any patient who is acutely ill, particularly with hemorrhagic diathesis, steroids should be the initial form of therapy. In children, Amethopterin and mercaptopurine are the preferable drugs to use, whereas mercaptopurine probably is more advantageous in adults. Chronic Leukemia.-A wider choice of agents is available for the treatment of chronic granulocytic and lymphocytic leukemia in comparison to effective measures for the acute disease. Benzene and arsenic are no longer commonly used, and urethan is used much less frequently than formerly. The alkylating agents, nitrogen mustard, triethylene melamine, and thiorepa, may cause regressions in both granulocytic and lymphocytic discase, but probably should be rescrved as supplemental therapy with triethylene melaminc being the preferred treatment. Superior to these agents for granulocytic leukemia is busulfan, and discontinuous therapy is practically free from undesirable sccondary effects Chronic lymphocytic leukemia responds to chlorambucil, although somewhat inconstantly This, however, represents the most nearly specific of all the therapeutic agents for treatment of lymphogenous disease. In chronic granulocytic leukemia, the purine antagonists also may be effective occasionally. 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Massive Predatsone and Prednisolone "Therapy in Leukemia and Lymphomas in the Adult, J Am Geriatrics Soc 4.627, 1956 293 Dubois-Fernière, H, Zumstein, P, Bodier, C, Kalası, J, Sorg, D, Valthier, J, and Hausser, A Cortisone in Massive Doses and 6-ſercaptopurine in Treatment of Acute Leukemias in Adult, Possibility of Producing Successive Remissions, Praxis 45 93, 1956 ·134 BIBLIOGRAPHY 240 Fowler, 1. M, and Jolly, W P: Modern Concepts in the Treatment of Leukemia, Lancet 76: 2:1, 1956 241 Frost, J. W, and Jackson, CB, Ir. Myleran in the Treatment of Chronic Granulocytic Leukemia, J AM A 161: 54, 1956 242 Gheg. P. B. Myleran in the Treatment of Chronic Myelord Leukemia, Acta haemat 16. 171, 1956 243 Hyman, GA, and Gellhorn, A Myleran Therapy in Malignant Neoplastic Diseases. Use of 1,4-Dimethane-Sulfony łoxybutane With Emphasis on Chronic Granulocytic Leukemia, ) AMA 161: 811, 1956 244 Josephen, J.O. Myleran in Chronic Afyelogenic Leukerna, Nord, med 56: 1530, 1956 245 Kenis. . 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EL R. and Jukes, Acid in Mice and Chicks by Cherucal Anta, 65: 368-370, 1917 264 Proceedings of the Second Conference on Folic Acid Antagonists in the Treatment of Leukemia, Blood 7. 97-190, 1952 265 Chiarenza, ) ) The Folic Acid Antagonists in the Treatment of Leukemia, B MQ 7. 76, 1956 266 Ilays, E F, Scanlan, TC, and Engle, RL, JS Multiple Remissions in an Adult With Acute Leukern:4 Treated Principally With Amethopterin, Ann Int Med 45, 298, 1956 267 Burchesal, 3 st Clinical Effects of Analogs of Folic Acid, Purincs, Pyrimidines, and Amino Acids, Fed Prot 13. 700, 1951 268 Thiersch, J. B, and Philips, FS Efect of 4-Aminopteroyl-Glutamic Acid (Aminorterin) on Early Pregnancy, Proc Soc Exper Biol & Med 74: 201-208, 1950 ·BRLIOGRAPHY 157 323 Jolly, W. P., and lowler, M. The Treatment of Leukemia in Adults, Iowa M. Soc. 46. 635, 1936 324 Halyer, N. and Stein). A The Modern Chemothrrapy of Chronic Myelogenous Leukemua, llareluan, Tel-Aviv 51: 43, 1956 325 Innes, 3. The Leukemia, Practitiones 178. 135, 1957 336 Keris V. Le traitement chemiotherapique dins lucrmies aieurs et chroniques, Scalpel 109- 769, 1956 327. O.cood, E E, and Scaman, a). Treatment of Chronic Leukemias, MA $50: 1372, 1952. 338 Paterson, E.; Treatment of Leukemia, Canad. Mis 75: 599, 1956, 400 Torni Roentgen Taon D.. 1.t c Therapy (Actinomycin C) in Lymphoxranu. 10. 913. 1955 y 513 Professional Education Lousss Illu he Lymphomas in the United States, 1957. j 8 Lippincott Cop 305 Leuke ulur ce Walusu » Transmissible Agent, Ann New York Acad Sc 63 $22-532. 1957 335 Ulrich, Incidence of Leukema in Radiologists, New England) Med 234: 45, 3946 316. March, 1 C Leukemia in Radiologists ia 20-Year Period, Am J MSc 220 282, 1950 337. Peber, S. and Puck ? Leukemia and Other Malignancies in Physicians, Am J M Se 224: 15+ 1952 338 Abbott, 3 D. Farras, II E and Greene, R Acute ałyeloid Leukemia After Radioactive Therapy. Lancet 270. 78). 1956 339 Serdien, SAL, Srieel E, Salow, HA and Melamed, S Acuts Myeloid Leukema Following Proloneed lodine.531 Therapy for Metastanze Thyroid Carcinoma. Sesence 123. 800 1956 30 2a , s? ? 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Mouse Leukemia XI A Maternal Influence That Lowers the faced are of Spontaneous Caset, Proc Soc. Exper Blol & Med 69 57), 1918 348 Shay, R, Cruenstein M. and Glazer. I Uniform Transfer to Random Bred Rats of Lymphasic Leukemua Induced by Gastric Instillation ul Methylcholarshrene, Proc 349 Kaalund forrensen Soc. Exper Brol & Med 75: 753 1950 Experiments m Transmission of Leukoses From Mice to Roentgen-Irradiated Rass Arta radiol 2! 483, 1940 350 Seibold, I R, Rathbone, R R, and Furth, 1 Studies on Transmissible Lymphadenosis of Nice Pro Soc Exper Bio & died 29 629, 1932 351 Werder * A Fredman Mar Dowe!! E c. and Syverton, T. The Combined Effects of Cortisone and Roentgen Frradason Upon Natural and Induced Resistance to Homotransplantation of Afouse Leukemia, Line IB, Cancer Res 13. 158, 3953 352 Bibel ), and Holm-Jensen, Parabsosis and Resistance to Transplantaron 1. The Influence of Parshosts on the Growth of a alouse Leulosis in Irraduated Ratt Acta baih et microbiol scandinas 24: 531, 1917 ·156 BIBLIOGRAPHY 294 Dubois-Ferrière, I{ : Treatment of Acute Leukemia in Adult, Myeloblastic Development of Chronic Leukemia, Erythro-Leukemia and Malignant Lymphgranuloma Caused by Massue Doses of Cortisone or Prednisone, Schweiz med Wchnschr 86. 1460, 1956 295 Fessas, P, Wintrobe, MM, Thompson, RB, and Cartwright, GE Treatment of Acute Leukemia With Cortisone and Corticotropin, MA Arch Int Med 91: 384, 1954 296 Santavy, F, and Reichstein, T. Isolierung neuer Staffe aus den Samen der flerstzeitlose Colchicum autumnale, L Substanzen der Flerkezeitlose und shre Derivate, Hehet Chi Acta 33: 1606, 1950 297. Schar, B, Loustalot, P, and Gross, F Demecolcin (Substanz F), ein neues, aus Colchicum autumnale isoliertes Alkaloid mut starker antimstotischer Wirkung, Klin Wohnschr 32: 49, 1954 298 Moechlin, S, Meyer, H, and Lichtman, A Ein neues Colchicum-Nebanalkaloid (Demecolcin Ciba) als Cytostaticum myloischer Leukamein, Schweiz med Wchrschr 83.990, 1953 299 Boch, HE, and Gross, R Leukame und Tumorbehandlung mit inem Nebenalkaloid aus Colchicum autumnale (demecolcin), Acta haemat 11: 280, 1954 300 Gaafar, M, and Al-Badry, A Effect of Demecolcnt (Colcemde) on Chronic Leukemsas, J Egyptian 11 A 38: 385, 1955 301 Kostkowskt, A Effect of a Colchicine Deru alve, Demccolcine, on Leukernas and Malignant Lymphogranulomatosis, Polskse Arch Med Wohnschr 26. 1329, 1956 302 Leonard, BJ, and Wilkinson, I F: Desacetylmethyl-colchicine in Treatment of Myeloid Leukemia, Brit M 1 1: 874, 1955 303 Huguenin, R, Truhaut, R, and Saracino, R. Instal Results of the Use of a Colchicine Derivative, N-Desaccryl-Thiocolchicine, in the Chemotherapy of Cancer, Bull Assoc fr Cancer 42. 308-325, 1955 304 Ferrata, A. and Fiesch. A Spunti di Clinica e terapia delle splenopatic, Haematologica 20 1-58, 1939 305 Fisher, J H, Welch, CS, and Dameshek, W Splenectomy in Leukemia and Leukosarcoma. New England Med 24G 477, 1952 306 Puorger, G, and Aymon, G A propos de l'influence de la splenectomie sur le décours d'une leucémic myeloide, J Suisse de méd 78: 762, 1948 307 Andre, R, Dreyfus, B, and Bessis, M Lymphoid Leukemia With Leukopenia The Presence of Anti-Leukocyte Auto-Anti-Bodies Favorable Effect of Splenectomy, Bull et mém Soc med hop Paris 70, 384-392, 1954 308 Ferrata, A, and Storti, E Le malattie del sangue manuale per Medici e Studenti, Milano. 1946, Societa edit libraria, p 751 309 Ilagan, PS, and Watson, CJ Ilypersplenism and Hemolytic Anemia in Leukemia Results of Splenectomy, Pror Third Internat Cong Internat Soc Ilematol, New York 1951. Grune & Stratton, Inc, pp 95-98 310 Hunter, O B, and Kiernan, PC Splenectomy in Leukemia, Postgrad Med B. 307. 213, 1950 311 Dameshek, W, Rosenthal, MC, and Schwartz, LI Treatment of Acquired Hemolytic Anemia With Adrenocorticotrophic Hormone (ACTH), New England J Med 244 117-127, 1951 312 Dean. GO, Earle. AM and Reilley WA Failure of Thymectomy in Lymphatic Leukemia, AMA Arch Surg 63 695, 1951 313 Bierman, HR, Cohen, P, McClelland, ) N, and Shimkin, MB The Effect of Transfusions and Antibiotics Upon the Duration of Life in Children With Lymphoc. enous Leukea, Pediat 37. 455, 1950 31% Schoget, N H Transfusion Management of Chronic Leukemia, Geneesk gids 3-4 477, 1956 315 Il'etherley-Mein G and Cotton D G Fresh Blood Transfusions in Leukemia, Brit J Hacmat 2 25, 1956 316 Bousser, ) and Christol, D New Treatments of Myeloid Leukema, Semaine hóp Paris 32. 2208, 1956 317 Brem, RJ Management of the Leukemias. ] Tennessee M A 49 261, 1956 318 Burchenal, Jl, and Ellison, RH Chemotherapy of Human Leukemia, Prog Pleaf, s ; 1; 265, 1956 319 Engstedt, L Management of Acute Leukemia in Adults Current Therapeutsc P'osssbilties, Sienska lak tid 53. 2698 1956 320 Esensen, OK Chemotherapy of Leukemia Vord med 56 1171, 1956 Cur- 321 Tall, BE, Willett FM Feichtmeter TV Recd, E B, and Dowling, WF rent Trends in Cancer Chemotherapy, California Med 8+ 1, 1956 322 Haut, A, Altman, SJ, Cartwright CE, and Wintrobe, MM The Influence of Chemotherapy on Sun is al in Acute Leukemia, Blood 10. 875, 1955 ·INDEX A Acceleration of progression of leukemia after cortisone and corticotropin therapy, 142 ACTII, 36, 141, 142 Acute leukemia blood smear, hydrolized with ribonuclease actity, 75 6-chloropurine therapy, 151 Ruorohydrocortisone therapy in large doses, IS adults, 137 lymphoblastic, $39-140, 142, 144 management, 1++ 6-ercaptopurinc therapy, 140-1+1 myeloblasexe, 139-140, 141 results of therapy, 313-114 steroid therapy, massive dose, 89 therapy, 309-35+ throguanine therapy, }}} Acute dytnptablases leukemia Cole acid antagonists therapy, 119-140 management, 14+ steroid therapy. 142 Acute myeloblast leukera folic acid antagonists therapy, 139-140 management, 144 Adresale tomy, 131 Ademir, 14 Age, effect of and relation to therapy, 56-57 on induced and transplanted leukemia, $30 trauction of vino zh erobryonic extracts, 51-55 on leukemia, 33-34 Alkaline phosphatase content of leukemic cells, 76 Alkylasing agents, 135-139, 144 Amethopeeris, 111, 139-140, 141, 144 rompica tons of therapy, 139-140 dosage, 140 Amino acid ariation in levels between leukemic and normal cells, 75-76 4-Amino-10-1730 hylpleroyIglutamic acid (see Amcthopterin) Aminoptets), 133 +-Aminopteroylasparkc acid, 139 4-Aminopte ro, Iglutamic acid (Ammoptrrin). 139 Andrews, G. 122 Androgen, 35 ststeraction with arradiation, 35 Antibody response, 22-23 Annabsote therapy of leukemia, 110, 117, 143, 145 Antimetabolices, 110 139-141, 544 acson miermediary metabolsm, 113 Arrowsmeh, W R, S6, 115-118, 119, 122 Arsenac, 35 dosage, 135, 144 Tespone ut lukemsa sn mice to arsenate, 35 therapy or leukemia in man, 134-135, 144 Attitude of physician treating leukemia, 117. Auer's bodies, 72 Aulonory of target assue, 129 4van leukosts, 18, 128 Avian garcon3s, 16 1 Aquanine, 93 A225rine, combined therapy with mercaptor porine, 113 complications of therapy, 113 159 ·158 BIBLIOGRAPHY 353 Furth, OB, Barnes, W. A, and Brower, A B. Studies on Resistance to Transmissible Leukemias in Mice by Means of Parabiosis, Arch Path 29. 163, 1910 354 Hauschka, T. S, and Kiedar, BJ Loss of Virulence and Increased Specificity of Lymphosarcoma 6 C311 ED After Serial Passage Through Resistant Mice, Proc Am Assoc. Cancer Res 1: 19, 1954 355 Schrek, R, and Preston, F. W Toxicity of Homologous Immune Serum to Transplantable Tumor, Studies Using Phase Microscopy and Cinemicrography, J Nat Cancer Inst. 16. 1021, 1956 356 Kirschbaum, A. Etiology of she Leukemias Chemical and Hormonal Factors in Mice, Proc Thund Nat Cancer Coní, Philadelphia, 1957, J B Lippincott Co, P 331 357 Kirschbaum, A, and Mixer, HW: Induction of Leukemia in Esght Inbred Stocks of Mice Varying in Susceptibility to the Spontaneous Disease, Lab & Clin Med 32: 720, 1947 358 MacDowell, E C, and Richter M eukemia IX The Role of Heredity 359 Cole es on the Genetics of Spontaneous 360 McK Lot305, Ageing, and Longevity, Tr Coll Physicians, Phil, kol 10, 1942 361 Waters, N F, and Bywaters T 1. 362 Kapli on ni 363 Mortc G 364 Law, unul in Experimental Leukemia of Mice, Ann New York Acad Sc 57. 575, 1954 365 MacDowell, EC Mouse Leukema XV Resistance to Spontaneous Cases in Hybrids Induced by Milk, Cancer Res 15:19, 1955 366 Gardner, WU, Kirschbaum, A, and Strong, LC Recesving Estrogens, Arch Path 29: 1, 1940 Lymphoid Tumors in Mice 367 Kirschbaum, A, Shapiro, R, and Mixer, i W Synergistre Action of Leukerrogenic Agents, Cancer Res 13. 262, 1953 368 Kirschbaum, A. Lrebelt, AC, and Falls, NG. Influence of Conadectomy and Androgenic Hormone on the induction of Leukemia by Methylcholanthrene in Dba/2 Mice, Cancer Res 15: 685, 1955 369 Kaplan 1 s, and Brown, MB Inhibition by 'Testosterone of Radiation-Induced Lymphord Tumor Development in Intact and Castrate Male Mice, Cancer Res 12. 262, 1951 370 Murphy, J B The Effect of Castration, Theelin, and Testosterone on the Incidence of Leukemia in a Rockefeller Institute Strain of Alice, Cancer Res 4. 622, 1914 371 Law L W Effect of Conadectomy and Adrenalectomy on the Appearance and In. cidence of Spontaneous Lymphoid Leukemua in C58 Mice, J Nat Cancer Inst 8 157, 1947 372 Sturen, E, and Murphy, J B The Effect of Adrenalectory on the Suscepability of Rats to a Transplantable Leukemia, Cancer Res 4: 384, 1914 373 Kaplan, HS Brown, MB, and Marder, SN Adrenal Cortical Function and Lymphord Tumor incidence in Irradiated Mice, Cancer Res 11. 262, 1951 374 Woolley, GW and Peters. BA Prolongation of Life in High Leukemsa AKR Mice by Cortisone, Proc Soc Exper Biol & Med 82 286, 1953 375 Kaplan S. Marder, S N, and Brown, MB Adrenal Cortical Tunction and Radiation-induced Lymphoid Tumors of Mice Cancer Res 11. 629. 1951 376 Moon, HID, Simpson, ME, LI, CH. and Evans, H AI Neoplasms in Rats Treated With Pituitary Growth Hormone 1 Pulmonary and Lymphatic Tissues, Cancer Res 10; 297, 1950 377 Moon, II D. Simpson, ME, L. C1, and Evans, 11 M Neoplasms in Rats Treated With Pituitary Growth Hormone V Absence of eoplasms in Hypophyseetomuzed Rats, Cancer Res 11. 535, 1951 378 Grad, B , Berenson, J, and Caplan, L The Influence of Hyper. and 11ypothyroidisin on the Incidence of Lymphogenous Leukemia in AKR Mice, Proc. Am Assoc Cancer Res 2: 20, 1955 379 Saxton, } A, Jr, Boon, MC, and Furth, J Observations on the Inhibition of De. velopment of Spontaneous Leukemia in Mice by Underfeeding, Cancer Res 4 401, 1914 380 Sitwast, S E, Eddy, B E, Stanton, MF, and Berkeley, WHI Influence of Age on Tumor Induction in Mice and flamsters Injected 11th a Tumor Agent Carried in Tissue Culture, Proc An Assoc Cancer Res 2: 318, 1958 ·Wo0 $15 , 18, 19, 28, 58 Cohnheim, 137 ortlakehamile, 141 inerhylene melamine, 137 OR NOCX 161 Chronic leukemma, Symphocytic Coni'd Diagnosis of leukemia, 71-77, 131-132 treatment with urradiation, 99, 13+ diferential, 76, 77 trarthylene melamine therapy, 136-137 1,4Diamchantsulionyloxybutant (see Buxul. tracement, 144-145 myclocyic Differenpation of blast cells by Brachet fest, busulfan therapy, 88-83, 137-138 77 6-chloropunine therapy, I!! Discussion desacetylthrocolchicinc therapy, 93, 142 diagnosis of leukemia, 77.79 Traduation therapy, 135 ftiology of leulema, 35-38 6-Nettaptopurint therapy, 152, 140-141 flirable agents prepared from leukemie 17. chuo uansne therapy, 119 Sue, 53-58 {rethylene melamine, 136-137 induction of myeloid leukema in mice, 64- urethan therapy, 135 Cstronorum factor, 76, 139 infectious concept of Icukemia, 45-48 Classification of Irukemas, 132-132 istening programs for chemotherapy, 90. Clarke, D A, 112, 141 97 Clinical management of leukemia, 115-338 therapy of leukra, 118-124 126 viruses and cancer, 22-28 Colchicine, '93, 35 Donné, 127 Coskins, 120 Dosage Combined therapy (Azasecas, mercaptopu. Ameshoprera, 140 nae), 113 Brseme. 135 Comparison of effecta eness of antitumor comtrenerne, 13+ pounds in man and animals, 93, 94, busullan, 138 95 chlorambucil, 139 Complications of therapy desacetylmethylcolchicine, 142 Amrthopeesin, 139-140 azaserine, 113 hydrochloride, 236 busullan, 112 tricthylene thophosphoramidr, chlorambucil, 139 Srath following irradiation of spleen, 122 prednisolone, 141-142 failure in treatment of lymphogenous levprednisone, 141-142 teseshylene melamine, 137 kemia, [43 renal damage, 141 urethan, 135 Dougherty, TF, and Dougherty, ), 30. 135 Congdon, CG Duro, T. 65 Cortisont, 37, 55, 120, 139, 141-142 Duran-Reynals, F, 17 alteration of tatural resistance by cottssone and irradiation, 130 E provocative 53, 51 cross resistance between chemotherapeutic Eberth, G3, 126 Ehrlich P, $26, 127 agents, effect on body weight, 35-36 ascites tumor, 128 cffect on leukemagervels, 3+ fluorohydrocortisone, 145 Electron microscopy, 72 carcinoma, 44 paroid tumors, 47, 48, 51-53 Elson, GB, 140 steroid therapy, massise doses 89 Ellermann, , 18, 29, 39, 126, 127 therapy, 89 8.35 AK, 26, 127 A, Criste, blast cell Ethyl carbamate (see Urethan) in Chronic myeloid leukrrua, 89 128 incidence in patients treated with busulan, 19 Crosg tesistance between chemotherapeutic effect on myclaud leukemia. 60-61 agents 555 Etsology. 127-131 Crossley, ul., [36 of C'ulture of bone marrow and leukemie cells, leukemia, 125.145 calonc intake 35-36. 131 96-97 carcinogen, 25, 30, 31, 34, 37, 66, 130 cause of human leukemia, 56-57, 128-129 0 differences in response of target tissues, 31- 32 Dameshek, 13 effect of foster surine, 3 Darlington, D, 26 effect of ganaderiomy on induction of teu- Deltaus G, 1+1 Demcoldin desacetylmethicolchicine), 142 kertua by stay. 62 effect of inoculation of marrow on leukemia Dermaus Stom azaqualine, 93 induction by uradjation, 63 Desacts Imethylcatchicine, 142 effect of partial skselding on leukema in. Desareti thoxolchicine, 93, 142, 147 dutrion by radiation, 62 as açent, Crasgre, ·160 INDEX B Bang, 0, 18, 39, 126, 127 Barns, WA, 59 Beard, J W., 19-20, 26, 28, 128 Bennett, J H, 126, 127 Benzene, 131 dosage, 131, 141 Bernard, 7, 141 Biologic filtrates in leukemia, 145 Biopsy, 142-143 Bishop, J. 90 Bittner, II, 19 Blast cell crisis in chronic myeloid leukemia, 89 differentiation by Brachet test, 77 incidence of crisis in patients treated with busulfan, 119 Blood smear, hydrolyzed with ribonuclease activity, 75 transfusion, 110, 115, 117, 143 Bollac, W, 108, 138 Bone marrow. effect of inoculation on results of irradiaton, 63 effect of shielding, 129 examination and diagnosis, 76-77 use in following course of discase during therapy, 118 Bone marrow cells, administration of, 129 Brachet, J, 72 Brachet test, 67-68, 77-78 Brausil, B, 72 Breast carcinoma in mice, 19, 20, 25, 27, 48, 55 Brown, MB, 63 Bryan, 1R, 26 Bryant, T, 126, 142 Burchenal, T H , 25, 35, 37, 38, 45, 46, 47, 54, 56, 57, 88, 92, 93, 95, 96, 97, 109-114, 118, 119 120, 121, 122, 123, 12+, 136, 140, 141, 142 Burdette, W I, 22, 26, 27, 28, 34, 36, 38, 47, 48, 53, 54, 55, 56, 57, 77, 125. 143 Busulfan, 102-106, 112, 118, 120, 121, 137, 138, 144 compared with 6-mercaptopurine therapy, 141 complications of therapy, 142 continuous administration, 104, 106 dosage, 88-89, 98, 103-104, 106 effect on fetus, 108 maintenance therapy, 119 pharmacology of, 118 reproductre system damage by, 108 resistance to, 106 response to therapy, 103-106 structural formula, 137 C Caloric tattake, 35, 36, 131 Carcinogen, 30, 31, 31, 37, 130 extrathymic origin of leukemia, 66 methylcholanthrone, 25, 130 Carcinoma mammary, 19, 20, 25, 27, 48, 55 V-2, 20, 21, 21 11'alker 256, 132-133, 138 Carrera, GM, 73 Castration, 62, 130 CB-1348 (see Chlorambucil) Chemical formula adenine, 141 Ameshopterin, 140 busulfan, 137 chlorambucıl, 138 desacetylmethylcochicine, 142 folic acid, 140 hypoxanthine, 141 mechlorethamine hydrochloride, 136 6-mercaptopurine, 141 N-N'-N triethylene thiophosphoramide, 536 triethylene melamine, 136 urethan, 135 Chemotherapy of leukemia, 88-90, 134-14+ Amethopterin, 111, 139, 140 antibiotic therapy, 110 arsenic, 35, 134-135, 144 azaguanine, 93 azaserine, 113 benzene, 134, 14+ busulfan, 88-89, 102-106, 112, 137 chlorambucil, 89, 106-107, 138, 139 6-chloropurine, 111 colchicine, 93, 95 combination with iradiation therapy, 98 combined therapy, azaserine and mercaptopurine, 113 cortisone therapy, 89, 141, 142 cross resistance between chemotherapeu. tic agents, 111 desacetylthrocolchteine, 93, 142 folic acid antagonists, 110, 111, 112, 120, 139-140, 144 mechlorethamine hydrochloride, 136, 144 6-mercaptopurine, 89, 106, 111-112, 113, 140, 141 nitrogen mustard, 116, 135 N-N'-N" triethylene theophosphoramide, 137, 144 o-diazoacetyl-L-scrine, 112-113 purine antagonists, 110, 140-141, 144 steroid therapy, 89, 110, 111, 141 thioguanine, 111 toxicity of compounds, 95 treatment of acute leukemia, 110-114 triethylene melamine, 116, 136, 137 urethan, 36, 130, 135, 144 Chlorambucil complications. 139 damage to reproductive system, 108 dosage, 106-107 therapy, 89, 106-107, 138-139, 144 6-Chloropurine, 111 Chromosomes, labrled, 67 Chronic leukemia lymphocytic, 134 chlorambucil therapy, 89, 106-107, 138- 139 isradiation of spleen, 99 mechlorethamine hydrochloride therapy, 136 6-mercaptopurine therapy, 89 135, ·3 37: tion. 54-55 chloris, 71, 77 INDEX 163 Ilosi-Cont' resistance, 36-37, 47 Jacobson, LO, 68 alteration with methylcholanthrene, 34 Jones, O P., 72 factors affecting, 36-37 ilueprr, WC, 26 K Ilupucain, R, 142 Ilydrocortisone, 141.142 Kaplan, II S, 31, 35, 61, 63, 65, 123, 129 !!ypophysectomy, effect on leukemia, 131 Kastenbaum, XA, 128 Hypoxanthine, 141 Kidd, J C, 21 Kieler, 1. 121 Kirschbaum, A, 23, 25, 27, 29-34, 35, 36, 38, 45, 46, 47, 48, 54, 55, 57, 118,63 58, 62, 66, 67, 69, 70, 78, 91, 92, Immunization against transplanted leukemia, 93, 91, 119, 120, 121, 122, 123 38 Koch's postulates, 28 Inactivation of leukoma virus, 46 Korteweg, R, 19 Incidence of leukemia, 127 Krakoff, I H, 142 Increased sunsa! following specific feukemic Krementz, E T, 70, 121, 123, 124 therapy, 119-120 L Infections en leukemic paticats, 117 Inoculation of marrow, effect on leukemia Lacassagne, A, 30 induction by irradiation, 63 Lathrop, AEC, 19 of normal tissue in relation to tumor induc- Lanes, W, 73, 78 Law, W, 32, 129 Irradiation, 37, 38, 131, 145 Leisering, 126 alteration of natural resistance by cortisone, Leonard, BJ, 137, 142 130 Leukemia (see Acute leukemia, Chronic leu- As cause of leukemia in human beings, 129 kemia, Etiology) 31 as provocative agent, 53, 54 compared with 6-mercaptopurine trest. differential diagnosis, 76, 77 ment, 141 death following irradiation of spleen, 122 dulterentration between myeloid metaplasia and, 77 effect of inoculation of marrow on leukemsa induced by irradiation, 63 effect on, of age, 33 effect of shielding on wradiation leukemoof body weight, 35-36 62, 66-67, 129 of estrogen, 61-62 effect of thyme implant on thymectomized of gonadectomy on induction by X-ray, 62 effect of x-zradiation on cellular metaboof snoculation of mattow on induction by irradiation, 63 extrathymuc origin of leukemia, 66 lysm, 123 of partial shielding on induction by icradiation, 63 fractionation of irradiation, 63, 64, 129 of thymus, 61-62 1131, 63 ctiology, 29-31, 127-131 induction of leukemia in mice, 30, 31, 59 alterable agrats prepared trom tissue, 49-53 61, 129 heredity, 29-30 Interaction of siradiation and androgen, 35 leukema in mice following radiation, 59. 6+, 129 In physicians, 128 local, 99-100 mechanism of irradiation effect on spleen, induced, 130 122-123 snfectious concept, 39-45 of liver, 41,50 of spleen, 99, 122 123, 145 Brethod fos preparing oncogenic virus from P. 104, 115 116 relaton to hurgan leukemia, 56-57, 129 tissue, 49-50 methylcholanthrene, effect in CS8 strain, 33 shielding of bone marrow, 129 myeloid, 59 of spleen, 67-68 natural history in RF mice, 68-69 spontaneous and induced leukemia in the RF osmotic fragility of leukocytes, 77, 78 peroxidase therapy of leukemia, 98-101, 106, 133-134 fraction granules in stem cells combination with chemotherapy, 98, in mouse, 31 110-111 possible competition between tumor incit steroid therapy, 98, 110 ing agents, 55 with busulian as compared to splenic relation of genetics to induction of fumors Irradiation, 102-103 by oncogenie virus, 49-50 relationship between total bods, 100, 120, 134, 145 spontaneous and treatment of acuic leukemia, 100-101 transplanted, 37, 38 of myelorytic leukemia, 135 sex difference, 35 whole-body, 100, 120, 134, 145 similanty between mouse and human, 31, 34-35 genesis history, 126 12 KF strain, 64-66 infiltration stran, 64-66 ·162 INDEX Etrology-Cont'd Gonads-Cont'd effect of shielding on irradiation leukemo effect of gonadectomy on induction of levgenests, 66-67 kemia by X-rays, 62 factors influencing induction of myeloid effect of irradiation on Icukemogenesis, 67 leukemia, 59-64 Goodman, LS, 135 familial occurrence of leukemia, 127 Graffi, A, 44, 51 fractionation of irradiation, 63, 64 Grafting of thymus, 32-33 granulocytic disease in mouse induced by Granulocytic disease in mouse induced by hiterable agent, 51 filterablc agent, 51 heredity, 29-30 Gross, L, 15, 29, 30, 38, 39-45, 46, 47, 48. hormonc, adrenal cortical, 30, 131 49, 50, 54, 55, 58, 64, 66, 95, 128 estrogen, 30, 36, 130 GT.-41 (see Busullan) human, 56-57 induction of parotid tumors by cortisone, H 47, 48, 51-53 interaction of multiple agents, 31 Haddow, A, 135, 137, 138 leukemia in pregnancy, 57, 108 Haut, A, 88 multiple factors, 34 Hendry, J A, 136 spontaneous and induced leukemia in RF Heredity. strain, 64-66 familial occurrence of leukemia, 127 Synergistic action of leukemogens, 33 human leukemia, 57, 127 testosterone, 36 in induced and spontaneous leukemia, 130 viral, 30, 39-45, 46, 49, 51, 52, 53, 55, in leukemia, 26-27, 29-30, 130 56, 57, 68, 69, 127, 128 pulmonary tumors, 129 Extrathyme origin of leukemia, 66 relation to oncogenic virus, 49-50 site of çene action in target tissue, 31-32 F susceptibility, 48 HIJIT M89, 141 Farber, S, 139, 141 Hiroshima, 30, 128 Ferrata, A, 143 Hastology. ressas, P, 142 blood smear from patient with acute granu. Fibrosarcomas, 40, 44, 128 locytic leukemia hydrolyzed with following inoculation of leukemic extracts, ribonuclease activity. 75 40 diagnosis of leukemia, 71-17 histology, 43 leukemic infiltration of liver, 41, 50 specificity of oncogenic virus, 54-56 monoblast hydrolyzed with desoxyribonu- Fieschi, A, 143 clease activity, 74 Filterable agents prepared from leukemic tisnatural history of leukemia in RF mice, 68-69 Fluorohydrocortisone, 141 parotid gland tumor in cortisone-treated Folic acid antagonists, 110, 111, 112, 120, 139- mouse, 52 140, 144 peripheral blood hydrolved with desoxy- Folinic acid (citro orum factor), 76, 139 ribonuclease activity, 74 Ford, CE, 67 peroxidase reaction granules in stem cells Foster nursing, ellect on leukemia, 34 In mouse leukemia, 51 Fowler's solution (see Arsenic) Hitchings, G H, 140 Fox, P, 22 Hormone Fractionation of irradiation, 63, 64 acceleration of progression of leukemia after Triedreich, N, 126, 127 cortisone and corticotropin therapy, Friend, C, 128 142 Furth, 3, 30, 36, 38, 59-64, 128 ACTH, 36 adrenal cortical hormone, 30 androgen, 35 G cortisone, 34, 89 cross resistance between chemotherapeutic Galton, D AG, 88. 102, 104, 105, 137, 138| agents, 111 Gardner, W U. 30, 35 effect of castration on myeloid leukemia, Gellhorn, A, 57, 68, 69, 77, 80-90, 91, 93 60 91, 95, 97, 12+ effect of gonadectomy on induction of Icu. Genetics (see Heredity) kemia by x-ray 62 Gigante, D, 88 effect on results of shielding, 129 Gilman, LS, 135 estrogen, 30, 31, 36 130 Glicksman, A, 124 fluorohydrocortisone, 111 Glucose tolerance alteration in chronic leurelation to response of human brukemia to antleukemic agents, 56-57 in leukemia, 124 steroid therapy in massive doses, 89 Glutamine antagonists, 110, 111 testosterone, 36 Gonads Host metabolism, eflect of blood dyscrasas sues, 49-53 — kemia, 90 on, 90 castration, 130 ·Dada fluoroh, decortisone, 141 g110b motherapeutic agents, 132-133 INDEX 165 P-NX-d1-2-chloroethyl) aminophenylbutync Sexton, WA, 135 acid (see Chlorambucil) Shay, ii, 137 Polyomas, 130 Shilding of spleen, 67-68 Polyvinyl pyrrolidone, 26 partial, effect of, on leukemia induction by Potter, A1, 113, 129 radiation, 62, 66-67, 129 Prednisolone, 141, 142 Shope, R E, 20, 28 Prednisont, 141-142 Sedamgrotzky, 0, 126 Susman, I E, 59 effect of busulfan on fetus, 108 effect of leukemia, 103 Sites of origin in leukemia. leukemia in, 57 systemie, 31 thymus, 31 Pribilla, 1,88 Protein metabolism in leukemic cells, 75-76 Small, MC, 39, 128 Pteroylglutamic acid, 139 Souders, E. P, 29, 129 Pulmonary tumors in RF mice, 70 Specificity of oncogenic urus, 51, 56 genetic susceptibility of, 129 Spleen death following irradiation, 122 Purine antagonists, 110, 140-141, 141 Purineihol (6-Mercaptopurine) effect of shielding, 67-68, 129 irradiation therapy, 99, 145 mechanism of irradiation effect, 122-123 R surgery, 116, 143 therapy with busulfan compared to irradiation, 102-103 Splenectomy, 116, 143 failure in treatment of lymphogenous leu. kemia, 143 Spontancous leukemia, 58 H-11+ myeloid, 59 RF mice, 59-60, 61-66 Spontaneous regression, 121 action in urine, 78-79 Sprague, CC, 34, 36, 55, 57, 67, 71-77, Richter, M N , 29, 130 78, 96, 118, 131 Rivers, J P, 28 Roloff, f. 126 Steroid hormones, 37, 120, 140, 141-142, 144 Rosc, 2, 136 acceleration of leukemia after cortisone and corticotropin therapy, 142 Rous, P, 16, 20, 21, 21, 26, 27 ACTH, 36, 141, 142 Rous virus, 17, 26 cross resistance between chemotherapeu. Rubin, H, 17 S irradiation therapy, '98 Santavy, F, 112 therapy, 110, 111, 142-143, 144 Sarcoma 180,92-93, 109, 112, 132 massa e doses, 89, 141, 142 Sarcoma, asian, 16 Stewart, SE, 128 Fujinamı, 16 Stollberg, G, 88 Rous, 16, 25 Storti, E, 77, 78 Schlosser, J V, 35, 56, 97, 98-101, 120, 121, Strong, LC, 19 '122, 123, 133 Structural formula (see Chemical formula) Schwartz, S 0.57, 128 Summary 80-90, 109-110 alkylating agents, 135-139 biologic systems used in, 82-83 antimetabolites, i39-141 calcium and uric acid excretion, 110 chemotherapy, 131-144 correlation between results of clinical trial etiology and treatment of leukemia, 125- and experimental, 86-87 for etiology of leukemia in animals, 131 multiforme, 91 factors influencing induction of myeloid list of compounds used in cooperative proleukemia in mice, 6+ eram, 83 management of leukemia, 144-145 microbiologic systems, 94 oncogenic viruses, 22 sarcoma 180, 92-93, 109, 112 purine antagonists, 140-141 selection of compounds for clinical trial, response of leukemia to therapy (tabular), 80 88 spontaneous and transplanted leukemias screening of compounds for chemotherapy, compared, 91-96 90 use of dogs to test toxicity, 110 steroid therapy, 142-143 Segaloff, A, 90, 91, 95 surgery, 142-143 Senn N, 126 treatment of leukemia, 133-145 Sex difference in incidence of leukemia, 130 Superinfection of viruses, 55 131 Supportive therapy, 143 tic Screening, 80. 145 143 ·164 INDEX Leukemia-Cont'd Mice, strains--Cont'd sites of origin, 31 DBA, 31, 32, 33, 34, 45, 46, 130 specificity of oncogenic virus, 51-56 FB, 37 spontaneous, 58 NII, 33 spontaneous regression, 121 RF, 59-60, 61, 63, 64-66, 68, 70, 129 synergistic action of leukemogens, 33 Stoli, 34 transmission by inoculation, 29 Microscopy, 72 transplantation, 30, 129-130 treatment, 109-114 (see also Treatment of Mider, GB, 30 leukemia) Milk agent, 19, 20, 25, 48 Leukemogens, interaction of, 130-131 Moeschlin, S. 72, 142 Lewis, MR, 136 Moloney, J. B, 128 Liebelt, A G, 62 Monoblast hydrolyzed with desoxyribonuclcasc Lindskoc, GE, 135 activity, 74 Lindsley, D L, 66 Morton, 1, 30 Little, CC, 19 Mosler, F., 126 Loeb, J, 19 Myeloblastic leukemia (see Acute myeloblastic leukemia) Local irradiation, 99-100 Myeloblastosis, 128 Lorenz, E, 120 Myelocytic leukemia (see Chronic leukemia, Lymphoblastic leukemia (see Acute lymphomyelocytic) blastic leukemia) Mycloid leukemia in mice, 59-6+ Lymphocytic leukemia (see Chronic leu Myleran (see Busulfan) kemia, lymphocytic) Lymphogenous leukemia (see Chronic leukemia, Acute leukemia) N Lymphomatosis, 18 Naegels, O , 126, 127 Lymphosarcoma, 18, 29, 129, 130 Gardner, 34 Nagasakı, 128 transplantation of, 129-130 Natural history of leukemia in RF mice, 68-69 Natural resistance, alteration of, by cortisone Lysogeny, 27 under radiation, 130 N-desacetylthiocolchicine, 142 MI Nitrogen mustard, 144 MacDowell, E C, 29, 31, 130 therapy, 116 Mammary carcinoma in mice, 19, 20, 25, 27, N-N-N" Triethylene thiophosphoramide, 137, 48, 50 144 Marks, P, A, 90 Nephrostomy, 122 Masking of virus, 26 Neumann, E, 126 Massic steroid therapy. 141-142 Nowell, PC, 66 Maternal resistance factor, 38, 55 Mayneord, W V, 100 o Mechlorethamine hydrochloride, 135, 144 dosage, 136 o-diazoacetyl-L-serinc, 112-113 6-Mercaptopurine, 89, 106, 111-112, 113, Oscood, EE, 123, 133-134, 145 119, 121-122, 140-141, 144 Osmotic fragility of leukocytes in leukemia, compared with busulfan, 141 77-78 compared with irradiation, 141 Oppenheimer, BS, 36 dosage, 111-112, 120 Methods for preparing oncogenic virus from P Icukemic tissue, 49-50 Ps 104, 115-116, 133, 145 hlo dosage, 133 drotitrated therapy, 133-134, 145 Papillomitosis, 20, 21 mechanism for infection, 25 extrathy mic origin of leukemia, 66 papilloma-to-carcinoma sequence, 58 induced leukemia, effect of cortisone on Shope, RE, 24, 28 action of, 3+ Parotid gland tumors, 40, 42, 44, 128 Mrce, strains cortisone induction, 47, 48, 51-53 A, 31, 32, 129 specificity of oncogenic virus, 51-56 AK, 38, 39, 44, 45, 50 | Paterson, E, 135 AKR, 35, 36, 51, 52, 54, 55 Pearson, ( 11, 111 Bags albino, 32, 33 Peripheral blood hydrolyzed with desoxyribo- C3H, 25, 34, 39, 42, 43, 44. 45, 46, 47, nuclease activity, 74 48, 50, 51, 53, 5+ Petrakis, N L. 88 C57, 33, 39, 41, 46, 48, 61, 65, 129 Phase contrast microtropy, 72 C58, 29, 33, 34, 39, 44, 45, 50, 51, 52, Phillips, FS, 135, 136 55, 130 Pituitary growth hormone, 131 - - 1 ·INDEX 167 Viruses and cancer-Cont'd Shope's virus of papillomatosis, 20, 21, 28 size, 16 transmission, 25 carcinoma, 20, 21, 24 Viral ctrology of leukemia--Cont'd granulocytic disease in mouse induced by filterable agent, 51 Inactivation, 46 infectious concept, 39-45, 127 infiltration of lives, 41, 50 method for preparing oncogenic virus from tissue, 49-50 mouse agent, characteristics, 40 natural history in RF mice, 68-69 parotid gland tumor alter inoculation of extract, 40 in cortisone-treated mouse, 52 tumors, 128 peroxidase reaction granules in stem cells in mouse, 51 relation of genclics to induction of tumors by oncogenic virus, 49-50 relauon of viral to other leukemogens, 53 single vs multiple oncogenic viruses in extracts from mice, 44-45 specificity of oncogenic virus, 54-56 superinfection, 55 Virchow, R, 126, 127 Viruses and cancer, 15 chology of tumors, 22, 27, 29 fixation, 24 masking, 21, 26 milk agent, 19, 20, 25 relation to genes and enzymes, 26, 27 to host cell, 19 Rous, 17 Walker carcinoma-256, 132-133, 138 Walpole, A L, 136 Warburg, 0, 17-18, 124 Weight, effect on incidence of leukemia, 35. 36, 131 Whole-body irradiation, 100, 120, 134, 145 Wilkinson, JF, 142 Wintrobe, MM 143 Woolley, GW, 24-25, 30, 31, 35, 36, 39, 47, 48, 49-53, 54, 55, 56, 57, 58, 67, 69, 77, 128 х Xanthelasma tumors, 26 Xanthoma tumors, 26 X-irradiation (see Irradsation) z Zarafonetis, C JD, 137 Zordek, B, 30 ·166 INDEX Surgery, 142-143 Treatment of leukemia Cont'd Indications, 143 Synergism, 33 busulfan, 88-89, 102-106, 112 Syverton, J, T, 15, 22, 23, 24, 25, 26, 27, chemotherapy, 88-90, 134-14+ chlorambucil, 89 28, 30, 36, 37, 38, 46, 47, 53, 54, 55, 56, 58, 68, 96, 97, 123 6-chloropurine, 111 chronic, 141-145 clinical management, 115-118 T colchicine, 93, 95 TEM (tricthylene melamine), 116, 122, 136. cortisone, 89 137, 144 combination arradiation chemotherapy, Testosterone, 36 98 Therapy of leukemia, 109-114 (see also Irdesacetylthiocolchicine, 93 radiation, Treatment of leukemia) Infection, 117 Thiersch, J B, 136 Irradiation, 100-101 Thioguanine, 111 management with blood transfusions, 110, ThioTEPA (N-N-N" triethylene thiophos- 115, 117, 143 phoramide), 137, 144 6-mercaptopurinc, 89, 106, 111-112, 113 Thoma, K, 73, 78 nitrogen mustard, 116 Thrombocytopenia, 106-107 o-diazoacetyl-L-serine, 112-113 consideration of, in chlorambucil therapy, outpatient, 118 207 purine antagonists, 140-141 dosage, 106-107 radioactic gold, 193 Thymoma, 31, 38 radioactie sodium, 133 radium, 133 Thymeczorny, 31, 33, 38, 129 Thymus reproductive system damage due to al. ellect on leukemia, 61-62 kylarıng agents, 108 methylcholanthrene, effect on thymectomy, supportive, 143 130 steroid, 98, 115 spontaneous and induced leukemia in RF surgical, 142-143 strain, 64-66, 129 Symptoms as indication for, 116 surgery, 31, 33, 38, 129 thioguanıne, 111 triethylene melamine. 116 thymoma, 31, 38 Thyroid deficiency, 131 Triethylene melamine, 116, 122, 136-137, effect on leukemia, 131 Till, M, 35, 57, 78, 88, 96, 103-108, 119, complications, 137 dosage, 137 120, 122, 138 Timmis, CM 137 2,4,6-Triethylene-s-triazine (see Triethylene melamine) Tio Tywan Gie, 29, 129 N-N-N" Triethylene thiophosphoramide, 137, Tissue culture, conversion of normal to neoplastic cells, 97 Titration of irradiation therapy, 133 TSPA (N-N-N" triethylene thtophosphoram- Tivey, H, 113-114, 133 ide), 137, 144 Total body irradiation, 100, 120, 131, 145 Transduction, 27. 134 U Upton, AC, 59-64, 64, 66, 68, 69, 70, 100, 120, 124, 129 Urethan, 36, 130, 135, 147 alteration of effectiveness of leukemogens 2015 pidid oli, 14-IJU by, 130 compared to spontaneous leukemia, 37-38 dosage, 135 coniersion of spontaneous chronic leukemia I'ne acid levels after x-ray and nitrogen musto acute leukemia, 37 card treatment, 122 effect on transformation of chronic to acute following 6-merta propurine therapy, disease, 35-36 121-122 heterolocous, of leukeme rells, problems of, 123, 124 in RF mice, 69, 70 Trauma and leukemia, 137 Treatment of leukemia, 109-114 (see also Videbark, A, 57 Chemotherapy of leukemia irradia. Vincent, L, B9, 141 tson) I Viral etiology of leukemia acute, 100-101 accelerated onset in mice following injec. adiantage of ting, 117-118 tion of extracts of tissue from hu- Amethoptetin, 11 man cases, 57 antibiotic 110, 115, 117, 141, 145 as cause, 128 arsenic, 35, 134-135 effret of x-irradiation un multiplication and cellular metabolism, 123 araguanine, 93 hite sable agents prepared from tissur, 45.18 azaserne, 113

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