The Elusive Goal: Presidential Address1beginning to cure women with metastatic choriocarcinoma with methotrexate, and a few years later children with metastatic Wilms' tumor would

[CANCER RESEARCH 41, 4865-4884, December 1981]0008-5472/81 /0041-OOOOS02.00

The Elusive Goal: Presidential Address1

Bayard D. ClarksonLaboratory of Hematopoietic Cell Kinetics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

fi:.

Abstract

The advances in treatment of cancer which have taken placeduring the last 25 years are quite remarkable considering thattherapists have had at their disposal only what has been calledhalf-way technology. About 45% of serious cancers are now

curable with surgery, radiotherapy, and chemotherapy or combined forms of these treatments, but prospects for developingcurative therapy for the other half of cancer patients are notbright unless more selective forms of treatment can be developed. Recent advances in basic biology have opened doors tomarvelous new research opportunities hardly imaginable a fewyears ago, and the time is opportune to make a concertedeffort to develop more selective treatment.

' Supported in part by Grants awarded from the National Cancer Institute

(NIH-CA-19117; CA-20194; CA-08748), The American Cancer Society (ACS-CH-6L), and the United Leukemia Fund. Presented on April 28, 1981, at theSeventy-Second Annual Meeting of the American Association for Cancer Research, Washington, D. C.

I would like to share with you some personal reflectionsabout progress in cancer therapy during the last quarter-cen

tury. It was just about 25 years ago that I entered the cancerfield and I have thus had a good opportunity to observe howthe field has developed.

I had had excellent training in Internal Medicine at two of ourcountry's great medical centers in a time before the postwar

revolution in biomedicai research had gained momentum andbefore ultraspecialization had become the rule. The great professors were broadly conversant with recent advances in medical knowledge and seemed omnipotent to their students. Itwas in this setting, 30 years ago as a medical student, that Ifirst saw a patient with acute leukemia. She was a youngwoman whose first symptom was persistent bleeding followingan otherwise normal delivery. As soon as the diagnosis wasmade, she was transferred to the medical service and placedin a single room to await her demise. When the great professorsvisited her bedside, there was little learned discourse about thepathophysiology or treatment of her disease, as had been trueof other patients on the ward, but just expressions of sympathy.Fowler's solution and the recently reported trials of folie acid

analogs were mentioned perfunctorily for the benefit of thestudents but, since no one present had ever seen a beneficialeffect from arsenicals in acute leukemia and since the antifolswere not yet generally available, the patient received no treatment except blood transfusions. She died of uncontrolledbleeding a few days later.

I relate this brief case report to illustrate the lamentableignorance about cancer prevalent 30 years ago and the utterhelplessness of physicians to alter the natural course of disseminated forms of cancer. The leading pharmacology textbook at the time, which was originally published in 1941 (52),contained no section on treatment of neoplastic diseases, andthere was only passing mention of the use of arsenic andradiation for polycythemia vera and the chronic leukemias.When the second edition appeared in 1955 (53), it containeda chapter on "Chemotherapy of Neoplastic Disease" which

included discussion of the nitrogen mustards, folie acid analogs, the newly developed purine analogs, steroid hormones,and radioactive isotopes. However, in the preface to this chapter it was noted "... It should be stated at the onset that no

chemical compound has yet been found which is capable ofcuring any form of cancer." Fortunately, this statement would

soon become obsolete. Li and his colleagues (70) were alreadybeginning to cure women with metastatic choriocarcinoma withmethotrexate, and a few years later children with metastaticWilms' tumor would be cured with actinomycin D and irradiation

(46).Whereas other areas of clinical investigation were progres

sing rapidly, the assault on cancer within the discipline whichwas later to become known as medical oncology was justbeginning. The initial efforts were not always endorsed enthusiastically by the academic establishment. I am not sure of allthe reasons for the lack of approbation, but there still existed

DECEMBER 1981 4865

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S. D. Clarkson

on aura of mystery about the nature of cancer, and I suspectthat they believed that efforts to treat the disease with drugswere premature. For example, the leading Textbook of Medicine, published in 1951 (6), reveals there was still some doubtabout the neoplastic nature of leukemia. "There are two prin

cipal views concerning the nature of the leukemic process: onethat the condition is an infection, the other that it is a malignantneoplasm. The latter is the more commonly accepted theory atpresent." Furthermore, the cancer field was dominated by

strong-willed surgeons who were not interested in evaluating

alternate approaches to treatment because they themselveshad been taught that there was only one way to do things.Surgical training programs were governed with iron discipline,and little deviation from traditional practice was permitted. Afew pioneers were beginning to introduce methods for objectively comparing results of alternate methods of treatment, butsuch efforts met with formidable opposition, and it would besome years before the surgical establishment emerged fromthe dark ages.

When I declared my intention of entering the cancer field, Iwas strongly advised not to do so by my department chairman.He urged me instead to go into a more reputable disciplinesuch as endocrinology, which he assured me would be morerewarding. Nevertheless, I began by first experiments in cancerresearch during my residency in 1956. The objective was anambitious one, to cure mice with Friend leukemia. After administration of a lethal dose of total-body irradiation, bone marrow

obtained from healthy donors which had previously been immunized with formalin-treated virus in Freund's adjuvant was

injected i.v. into the irradiated leukemic mice. The experimentaldesign stemmed from a coalition of recent developments thencurrently popular. The viral etiology of several types of murineleukemia had recently been demonstrated by Gross (54) andFriend (48); effective vaccines had been available for years forvarious viral diseases, including one recently developed forpoliovirus; and Joseph Ferrebee and Donnall Thomas hadstarted their bone marrow transplantation experiments in Coo-perstown. I had excellent advisors; Charlotte Friend providedme with the virus, and RenÃ©Dubos taught me how to make thevaccine. However, in no way do I hold them accountable forthe results. Suffice it to say that the experiments were anunmitigated failure, and nothing will be gained by dwelling onthem further.

After completing my residency, I joined David Karnofsky'sand Joe Burchenal's Division of Cancer Chemotherapy at Me

morial Sloan-Kettering Cancer Center as a Fellow, spending

part of the time in clinical activities and part of the time inDave's chick embryo laboratory. Dave had found that certain

transplantable mouse leukemias grew readily in the chick embryo, and he was anxious to see if human leukemia would alsogrow in order to test the effectiveness of cytotoxic drugs.Despite many attempts, we never succeeded in growing humanleukemia in the embryo, and many of the drugs caused severeteratogenic effects. At the time, we knew little about differencesin growth rates of murine and human leukemias, but in retrospect it seems probable that the principle reason for failurewas the slower growth rate of human leukemia. With propertiming and dosage of mouse leukemic cells, the chicks hatchedand harbored viable leukemic cells for many weeks, until eventually they developed sufficient immunocompetence to rejectthem (24). The brain was invariably the last sanctuary.

In the course of these experiments, I became adept atpuncturing tiny embryonic blood vessels and began practicingthis new skill on progressively younger embryos. Knowing littleabout embryonic hematopoiesis, I was astonished to find thatthe circulating primary erythroblasts in the 2-day embryo had

a morphological appearance very similar to some of the acuteleukemias I was seeing in the clinic (72). By the fourth day,most of the primary erythroblasts disappeared from the bloodand were replaced by polychromatic erythrocytes already wellalong their maturation pathway. By the sixth day, the primarygeneration of erythrocytes reached maturity and would shortlybe replaced by maturing cells of later generations.

The primitive erythroblasts of human acute erythremia resemble closely the primary erythroblasts of the early chickembryo (Ref. 3, p. 131, Figs. 72 and 73), but unlike those inthe embryo, the later erythrocyte precursors in erythremiashow defective maturation with polylobed nuclei and deficienthemoglobin. Also, of course, in contrast to the developingembryo, if one reexamines the marrow of a patient with acuteerythremia at a later time, the picture remains unchanged withpersistence of the primitive cells and retention of disorderlymaturation. In contrast to acute erythremia in which the neoplastic cells are all erythroid precursors and the leukocytes arematuring normally, in erythroleukemia the neoplastic stem cellis not committed to a single line of differentiation but producesabnormal myeloblasts and monocytes as well as erythroblasts(Ref. 3, p. 191, Fig. 112).

The difference between the perfectly programmed and beautifully ordered differentiation and maturation of normal bloodcells in the embryo and the defective and chaotic developmentof leukemic cells is of course the core of the cancer problem.The extent of the defect is variable. In acute undifferentiatedleukemia, all cells are blocked at a primitive stage of development, while at the other end of the spectrum are what might becalled minimal-deviation leukemias, including CML2 and some

of the myelodysplastic states, in which the cells retain theircapacity for differentiation and maturation but are defective intheir response to regulatory influences.

In the course of our experiments, we found that certaintransplantable mouse leukemias, such as P388 and P815,when injected i.v. into the embryo caused anemia, leukoery-

throblastosis, and gross enlargement of the spleen which wasnot attributable to leukemic infiltration (24, 29). Whereas leukocytes do not normally appear in the blood until after hatching,the mouse leukemic cells caused an outpouring of embryonicleukocytes, especially granulocytes, several days earlier (Chart1). The leukemic cells were doing something which profoundlydisturbed normal embryonic hematopoiesis. The observationwas not surprising since all types of leukemia affect hematopoiesis and certain solid tumors were known to evoke granu-

locytic hyperplasia or other alterations (39). Nevertheless, theproblem was a fascinating one and the system seemed sufficiently promising to warrant pursuing this line of inquiry. However, at this point, my fledgling research endeavors abruptlyveered in a different direction.

2 The abbreviations used are: CML. chronic myelogenous leukemia; La.,intraarterial; i.h.. intrahepatic; ara-C. 1-/?-o-arabinofuranosylcytosine; LI, labelingindex; ALL, acute lyymphoblastic leukemia; ANLL, acute nonlymphoblastic leukemia; 6-TG, 6-thioguanine; DAT. daunorubicin-l-ÃŸ-D-arabinofuranosylcylosine-6-thioguanine; Ph1 chromosome, Philadelphia chromosome; Â¡.t., intrathecal;MOPP, nitrogen mustard-vincristine-procarbazine-prednisone.

4866 CANCER RESEARCH VOL. 41



The Elusive Goal: Presidential Address

ABSOLUTE DIFFERENTIAL COUNTS PERIPHERAL BLOOD

o Gey's IV 10 days

Basophils10 P 815 IVIO days

0

10

0

10

o L

Platelets Eosinophils

Early polychromatic ./*

-o-^-aâ€”Â» -^ oâ€”o

10 Monocytes

Mature heterophils

O L-

10 14tP815

18 2210 14

l P815

Daysof incubation

Chart 1. Leukoerythroblastic reaction in chick embryo following i.v. injectionof P815 murine leukemic cells into 10-day-old embryo (24, 29). eryth., Erythro-

cytes.

Dr. Rhoads called me to his office where he and Dr. Karnof-

sky offered me a position on the attending staff. The positionwas to work with the Department of Surgery in examining theusefulness of infusion and perfusion techniques in treatinglocalized cancers. Although favorable results using regionalchemotherapy had recently been reported from several institutions, I had not been very impressed by the efficacy of thethen available cytotoxic drugs in solid tumors, and the proposaldid not seem to me to be very promising. We were still largelyusing single agents, although Min Chu Li and Bob Golbey werealready beginning to treat refractory germ cell tumors successfully with drug combinations and curing a small fraction ofpatients with metastatic disease. I tried to convince Dr. Rhoadsof the greater potential rewards of continuing studies on theinfluence of leukemia on embryonic hematopoiesis, but he wasunimpressed. Being a wise and practical man and a hematol-

ogist himself, he undoubtedly appreciated much better than Ithe enormous complexities of regulation of normal and leukemic cell growth which are still far from being unraveled (15).

I was persuaded to accept the offer and, working closelywith Walter Lawrence, we began a series of experiments thatwere to last for the next several years. We learned to snakecatheters into various arteries for long-term infusions and toisolate segments of the body for short-term perfusion of drugs.We also developed an animal perfusion model using the rabbit's

hind limb and the transplantable VX-2 tumor (79, 80), which I

obtained from Dr. Peyton Rous. With the help of Dorris Hutch

ison, Alex Bloch, Fred Philips, and Jack Fox, we set up a drugassay laboratory. We found that infusion of antimetaboliteswhich are rapidly degraded in the body into small arteries suchas the external carotid resulted in up to 100-fold higher con

centrations in the infused region than in the systemic circulationand that toxicity was strictly limited to the distribution of theinfused artery (25, 26, 45, 100). Infusion of larger arteries, ofcourse, had less differential effect. The i.a. infusions sometimescaused striking tumor regression, but they were usually ofshort duration (25, 36). by means of pneumatic tourniquets,clamps or intravascular balloons to occlude the great vessels,and a system of pumps and an oxygenator, it was possible toisolate any portion of the body desired for limited periods andto restrict the distribution of drug (8, 25, 67, 68). Using isotopeand bioassay techniques, we measured the leakage of labeledRBC and drugs from the isolated circuits and found that onecould achieve manyfold higher drug concentrations in theperfused region and that, if the drug was adequately flushedfrom the perfused circuit at the end of the procedure, littlesystemic toxicity would result. High concentrations of rapidlyacting drugs often caused impressive shrinkage of localizedcancer, but they also caused severe damage to the normaltissues in the perfused regions. In short, we found that neitherin our rabbit hind limb VX-2 tumor system nor in patients was

it usually possible by isolated perfusion to eradicate mosttumors without unacceptable local toxicity.

The same was true of prolonged i.a. infusions of drugs. Afterthe infusion was stopped, the tumor almost invariably regrewin the infused area, and commonly mÃ©tastaseslater appearedat distant sites which we had not suspected were involved. Weinitially blamed our poor results on the fact that most of thepatients referred to us had had extensive prior surgery orirradiation. On one occasion, we spent several weeks in another country where we treated patients with advanced cervicaland other localized cancers who had not had any prior treatment. But the results were the same; it was not possible toeradicate these tumors, at least without causing prohibitivelocal toxicity.

During this period, Dave and I wrote a review article aboutthe anticancer drugs then available, and I have selected astatement from its Introduction (63). "A review of anticancer

drugs would be expected to discuss those drugs which destroyor inhibit the growth of neoplastic cells without undue injury tothe normal tissues of the host ... Unfortunately, neither acommon cause nor a unique metabolic disturbance in canceris known, and drugs with specific anticancer activity have notbeen found."

I recently attended a conference on autologous bone marrowtransplantation, hoping to hear more favorable results than wehad experienced in treating tumors which were refractory toour best current treatment protocols. Satisfactory methods nowexist for procurement and cryopreservation of hematopoieticstem cells, and one can effectively rescue patients with theirown stem cells after administering doses of drugs or irradiationwhich would otherwise result in lethal myelotoxicity. I had anoverwhelming feeling of dÃ©jÃ vu as I listened to the litany ofpoor results. When the drug doses were significantly increased,other types of intolerable nonhematopoietic toxicity were encountered (such as severe mucositis and venoocclusive disease of the liver and nephropathy) just as we had experiencedduring our earlier attempts to extend the usefulness of the

DECEMBER 1981 4867



ÃŸ.D. Clarkson

cytotoxic drugs by regional administration.There is currently renewed interest in regional chemotherapy

of localized cancers in combination with hyperthermia, sincetumors are often more susceptible to heat damage than arenormal tissues. Fortunately, these new trials are being pursuedby a new cohort of less jaded investigators, and I wish themthe best of success.

Dave and I attempted in our review to relate the action of thecytotoxic drugs to particular events in the cell cycle, in thehope that such information would lead to more selective anti-

tumor chemotherapy (63). We uncovered very little in theliterature at the time about the relation of specific drug actionsto cell cycle events other than what was already well known,such as the facts that the actions of antimetabolites whichblock nucleic acid synthesis are largely restricted to the Sphase or that mitotic inhibitors interfere with spindle formation.We found that even less was known about the proliferationkinetics of human cells, and indeed there were many widelyprevalent misconceptions at the time. Attempting to discoverexploitable differences between normal and neoplastic populations, we began measuring various kinetic parameters,largely using [3H]thymidine and autoradiographic methods. In

pursuing these studies, we were greatly aided by many youngJapanese and American Fellows, too numerous to mentionand, in particular, by Jerry Fried and the late Sol Rubinow inanalyzing and interpreting the data and in constructing kineticmodels. Relatively few people were working in the field, largelyI suspect because of the tedious nature of the autoradiographicmethods, and it was quite easy to made new observations anddispel erroneous concepts.

We began studying solid tumors, and whenever possible wealso made comparable measurements in normal tissues suchas adjacent epithelium. Some of the patients in whom we hadinserted indwelling arterial catheters consented to let us injector infuse [3H]thymidine and to take serial biopsies prior to

beginning chemotherapy. Since I have been warned manytimes by my friends of the soporific effect of presenting detailedkinetic results, I will tread lightly and give just a few abbreviated

E.Y. HEPATOMA

10 DAY INFUSION TdR-H3 IMC/24 HR.

INTO HEPATIC ARTERY

Exposure=ll wks. â€¢6wks.%

LabeledtumorcellsD*2)3)Â«4)*5)6)7)8)9)10)11)75.790.374.094.738.342.69.185.310.862.847.0Average

graincount &range25.7

(5->50)28.0(5->100)43.0(5->100)20.

3 (5->50)21.8(5-43)23.0(5->100)8.8(5-

13)11.7(5->50)8.4(5-

20)14.1(5-32)9.315-

33)

Chart 2. Section of liver obtained at autopsy and prepared for autoradiogra-phy from a patient with hepatoma who died 3 days after completing a 10-day i.h.infusion of tritiated thymidine (ToW-ff3) (specific activity, 6.6 Ci/mmol, 1mCi(MC)/24 hr) followed by a 3-day infusion of 5-fluoro-2'-deoxyuridine (1.5

mg/kg/24 hr).

examples to illustrate some key points. Chart 2 shows a sectionof liver obtained at autopsy from a patient with hepatoma whohad completed a 10-day i.h. infusion of [3H]thymidine 3 days

before she unexpectedly died suddenly of massive hemorrhagefrom ruptured esophageal varices. An i.h. infusion of 5-fluoro-2'-deoxyuridine had been started immediately upon completionof the [3H]thymidine infusion and was continuing at the time ofher death. The dose of 5-fluoro-2'-deoxyuridine was probably

sufficient to destroy all cells in S phase in the infused liver andto prevent cell division during the last 3 days of life, inasmuchas no labeled or unlabeled mitoses were found at autopsy.Eleven small tumor nodules were present in the liver sectionshown. In some nodules, the majority of the tumor cells werelabeled, whereas in adjacent nodules only a minority werelabeled. The experiment illustrates two important points. Thefirst is that the tumor cells exhibit considerable heterogeneityin their kinetic behavior, just as other studies have shownheterogeneity within any neoplastic population in their meta-

static potential clonogenic capacity, expression of surfacecomponents, drug sensitivity and other properties of the cells.The second, is the problem of the dormant tumor cells. Five to90% of the tumor cells in different nodules failed to enter Sphase during the preceding 13 days. The existence of long-

term resting tumor cells, which we estimated could sometimesremain dormant for many months, was a constant finding in allof our studies in both solid and hematopoietic tumors (10, 12-14, 17, 19-22, 28, 30-32, 35, 66). Since such cells are

relatively invulnerable to antimetabolites and other drugs withlethal effects that are restricted to proliferating cells, it is notpossible to completely destroy human tumors with such drugs.Intolerable toxicity will be encountered long before all of thetumor cells can be destroyed.

We went on to study tumors growing in an ascitic form andhematopoietic tumors, largely because of the greater ease inobtaining multiple samples and the greater reliability of measuring grain counts in intact cells than in tissue sections. Chart3 shows a labeled mitosis curve of the leukemic blasts in apatient with acute leukemia (27). In this case, the duration ofS phase was 19 hr and the mean intermitotic time, as estimatedfrom the grain count-halving time, was about 3.5 days. In alarge number of such studies performed in our laboratory andother laboratories, no consistent differences were found in theduration of the cell cycle phases of tumor cells and corresponding normal cells. When appreciable differences were found,they were usually attributable to overcrowding. Just as culturedcells enter a plateau phase of growth when they becomecrowded or nutritionally deprived (103, 106, 107), humantumor cells in vivo also slow their growth when their populationdensity increases. One example of this phenomenon is summarized in Table 1. The data shown are from experimentsperformed by Frank Sheehy in our laboratory and show thechanges in the cell cycle parameters of tumor cells in a patientwith ovarian cancer growing in an ascitic form before and aftertreatment with 1-/8-o-arabinofuranosylcytosine i.p. (96). After

the tumor cell concentration in the ascitic fluid was reducedfrom 1900 cu mm to 32/cu mm by treatment, there was amarked shortening of all phases of the mitotic cycle except forG2, the labeling and mitotic indices tripled, and the growthfraction more than doubled. In most cases, the effects ofreducing the cell density by treatment were less striking and,not surprisingly, we never came close to recruiting all of the





dormant tumor cells to begin proliferating (10, 19, 30, 32, 35,84, 88, 95).

There is great variability in the growth rates of different typesof tumors as well as within any single diagnostic category.Chart 4 summarizes some data compiled by Herbert Hansen(56) on the pretreatment pulse [3H]thymidine LI of lymphoma

cells grouped according to the Rappaport classification. Previous investigations have shown good correlation between theLI and the mean cell cycle time, the growth fraction, and thetumor volume-doubling time. The diffuse well-differentiated and

nodular poorly differentiated lymphomas have uniformly low Listhe nodular mixed and histiocytic types and diffuse poorlydifferentiated and diffuse mixed lymphomas have intermediatevalues, and the diffuse histiocytic and undifferentiated typeshave the highest proliferative rates. In general, the medianvalues for the different categories correlate well with their ratesof clinical progression, but there is considerable variability in

^ -Thymidine 20 me

LABELED MITOSES

IMarrow)

â€¢ â€¢ â€¢5 grains or more

Oâ€”o â€¢10grains or more

50 grains or more

-* â€¢100grains or more

I OATSir"-,

GRAIN COUNT DISTRIBUTION

MMMl

19M 4/21 4/22 4/26 4/28

Chart 3. Time of appearance of labeled marrow leukemic blasts In mitosisfollowing i.v. injection of 20 mCi of tritiated thymidine (specific activity, 6.6 Ci/mmol) in a patient with acute myelomonoblastic leukemia (M4) (27).

the proliferative rates of lymphomas with intermediate or highmedian values. Chart 5 shows a similar relationship betweenthe modal tumor cell volume and the Rappaport histologicaltype. The well-differentiated and nodular lymphomas have low

modal cell volumes, which fall in a narrow range similar to thatof normal lymphocytes, while the diffuse lymphomas havehigher median values with a much wider range. When thesetwo parameters are considered as a coordinate pair, a Cartesian kinetic-volumetric map can be constructed (Chart 6). A log

transformation of the modal cell volume and a square roottransformation of the labeling index have been used to compress the axes. The nodular lymphomas have uniformly lowvalues for both parameters. Most, but not all, of the diffuselymphomas have higher values, but there is considerable overlap among different histological types. Further follow-up and

analysis will be required to determine if the behavior of thedisease in individual patients correlates better with the loci oftheir tumor cells on this map than with histological type (56).However, since both cell size and LI have clear biologicalsignificance and can be determined accurately, we anticipatethat such objective measurements should correlate well withthe clinical behavior of the disease.

Chart 7 shows some data obtained by Zalmen Arlin in ourlaboratory on pretreatment pulse [3H]thymidine Lis of the mar

row leukemic cells in acute leukemia. The median value isslightly higher in ALL than in ANLL, and there is large variability

*â€¢

"2*A,OWDL

NPDL_L

;-Â«-~?~'TNML

NHL DPDLDMLRAPPAPORT

TYPE1'DHLDU + BURKITTS

Chart 4. In vitro pulse-tritiated thymidine Us of cell suspensions of tissuesinvolved by non-Hodgkin's lymphomas. Bars, group medians; the observationsare grouped according to Rappaport's classification system. DWDL. diffuse well-

differentiated lymphoma; NPDL, nodular poorly differentiated lymphocytic lymphoma; WML, nodular mixed lymphoma; NHL. nodular histiocytic lymphoma;DPDL, diffuse poorly differentiated lymphoma; DML, diffuse mixed lymphoma;DHL, diffuse histiocytic lymphoma; DU, diffse undifferentiated lymphoma (56).

Table 1Average durations of various phases of cell cycle and other properties before and after treatment with ara-C

Data from paper of Sheehy ef al. (96).

BeforetreatmentAftertreatmentGenera

tiontime(hr)9945G,

(hr)283S(hr)6234G2(hr)6.67.0M(hr)2.41.0ur Ml(TO)0.22

0.40.531.5Cellcount/LI

(%) cumm14

19004032Mediangramcount-halvingtime(hr)18073

GF, growth fraction; Ml, mitotic index.

DECEMBER 1981 4869



S. D. Clarkson

ui

2o

T -5-NHL DPDL

RAPPAPORTTYPE

DHL DU + BURKITT'S

Chart 5. Modal cell volumes of single-cell suspensions of tissues involved bynon-Hodgkin's lymphomas grouped according to the Rappaport classification

(56). Bars, group medians. For abbreviations, see legend to Chart 4.

Chart 6. Cartesian kinetic-volumetric map of cell suspension of tissues involved by non-Hodgkin's lymphomas grouped according to Rappaport's classi

fication. A square root transformation of the LI and a logarithmic transformationof the modal cell volume (MCV) have been used to compress the axes (56). DHL,diffuse histiocytic lymphoma; DU, diffuse undifferentiated lymphoma; DPDL,diffuse poorly differentiated lymphoma; DML, diffuse mixed lymphoma.

in both types. If one groups the ANLL results according to theFrench-American-British (FAB) classification (Chart 8), there

are no consistent differences among the morphological types,except that the M3 or promyelocytic type is significantly lowerthan the other subtypes (p < 0.001), and again there is largevariability within any one type. T-cell ALL has only a slightlyhigher mean value than do null or non-B, non-T cell ALLs, butthe L3 or Burkitt's type is invariably rapidly proliferating (not

shown).In an attempt to formulate some general principles based on

our experimental data, we developed a series of models ofnormal and leukemic cell growth. Chart 9 shows a model ofnormal steady-state erythropoiesis (19). Just as in acute leu

kemia, most of the normal stem cells are in a quiescent or G0state but, once triggered to divide, one of the daughter cells

becomes committed to one or another differentiation pathway,in this case erythropoiesis. The committed cell and its progenythen go through a series of amplification divisions during whichtheir level of maturation progressively increases until theyreach a state where they can no longer divide. Meanwhile, theother daughter of the original stem cell persists in an undifferentiated quiescent state until it is again called upn to meet thedemand for production of additional RBC. In the lower portionof the diagram, both homomorphogenic and heteromorpho-genic divisions are shown to express uncertainty about whichtype of division prevails. In the former, two undifferentiatedstem cells result while the latter results in one stem cell andone committed cell. In either case, the end result after aninfinite number of divisions is that the number of stem cellsremains constant.

In contrast, in acute leukemia, instead of the number of stemcells remaining constant during repeated divisions, there is aprogressive increase in the number of cells retaining stem cellcapability as well as of those committed to partial maturation.Since maturation is defective, many of the committed cells diewithout reaching full maturation (Chart 10).

25r

20

15

IO

52â€”i_, 26-

U*

I

â€¢

ALL ANLL

Chart 7. Pretreatment in vitro pulse-tritiated thymidine Lis of marrow leukemiccells obtained by marrow aspirates in ALL and ANLL. Bars, group median values.

25 r

20 -

â€¢50

IO ff

T

M1-M2 M4 MS, RAEB

Chart 8. Pretreatment in vitro pulse-tritiated thymidine Lis of marrow leukemic

cells obtained by marrow aspirates from patients with ANLL grouped accordingto the French-American-British (FAB) classification system. Bars, group medianvalues.





NORMAL ERYTHROPOIESIS-STEADY STATE

El

Reticulocytes

O Stemcell DC

^Committed cell >CRestingstateL

lili1

2345GenerationsX16

Chart 9. Model of steady-state normal erythropoiesis (19).

In acute leukemia, the lethal number of cells is usually severaltrillion. It is worth emphasizing that their growth advantage isnot due to a faster growth rate than that of normal precursors,but rather because they do not respond normally to controlsand continue to recycle in the primitive blast cell pool. Indeed,as in most spontaneous human tumors, leukemic cells usuallyproliferate less rapidly than the corresponding normal precursors.

Unlike certain solid tumors which may be multifocal in origin,most hematopoietic neoplasms begin with transformation of asingle cell. In the case of leukemia, the leukemic populationundergoes silent expansion until it reaches a critical density inthe marrow (Chart 11 ). Production of normal cells is theninhibited, and lethal complications ensue as a result of theirdepletion (31 ). The rate at which any leukemic populationexpands and the density at which it inhibits normal cell production varies greatly. For example, with the same degree ofmarrow infiltration, slowly growing B-cell tumors such as nod

ular lymphomas or chronic lymphocytic leukemia usually havemuch less inhibitory effect than do more rapidly growing acuteleukemic cells. In chronic lymphocytic leukemia and hairy cellleukemia, an average of about 5 and not uncommonly 10 ormore years may elapse between diagnosis and death (86). Onthe basis of serial examinations of atomic bomb survivors whodeveloped CML, it has been estimated that there may be alatent period of 8 years between the original mutagenic eventand the development of clinical manifestations, and then another 3 years before death occurs, usually after acute transformation of the chronic-phase cells (62).

In the acute leukemias, the latent period is much shorter,probably 1 year or so. Since there are no methods to detect

small numbers of leukemic cells, such estimates are based oncircumstantial evidence such as the time to development ofconcordant leukemia in monozygotic twins (16). If the initialleukemic transformation occurs in a single cell in one twin inutero, the other twin will almost certainly be colonized by theprogeny of this clone because monozygotic twins share acommon placental circulation. Concordance of acute leukemiain identical twins can be as high as 20% and is frequentlydiagnosed nearly simultaneously. Identical chromosomal ab-

ACUTE LEUKEMIA

1118 14

O Leukemic uncommittedstemcell

â€¢Leukemiccell committedto maturationanddeath

Restingstate+Celldeath

~2!

o~â€”o

i 4 5Generations

Chart 10. Model of proliferation and maturation of acute leukemic cells (19).

OQ

14

1210

86420

N(NORMAL

6RANULOCYTES)

N1(LEUKEMIC CELLS)

I I

200 400

T DAYS

600

Chart 11. Schematic representation of the normal granulocyte population (W)and the leukemic population number (A/') on a logarithmic scale, as a function of

time, based on a mathematical model of the acute myeloblastic leukemic state.At time zero, one leukemic cell begins to proliferate; and when the leukemicpopulation approaches the normal population number, the normal granulocytesbegin to disappear (31 ).

DECEMBER 1981 4871



ÃŸ.D. Clarkson

normalities have been demonstrated in the leukemic cells ofboth twins (7), providing strong evidence that such leukemiashave a common monoclonal origin. Since the incidence ofconcordant leukemia is highest during the first year of life anddecreases with advancing age, the latent period must ordinarilybe about 1 year.

Dormer (44) has constructed a series of models to illustratethe variability in rates of expansion of different leukemic populations. The first model shown in Chart 12 is compatible withthe slow progression of some cases of acute leukemia whichare preceded by a preleukemic phase. In this population, mostof the leukemic blasts are capable of dividing only a few timesbefore becoming end cells and dying, without full maturation.Only Cell 13 is able to mature, and only one blast (X) persistsin a primitive state and recycles in the primitive blast cell pool.If no cells recycled, the cell loss factor would be unity and thepopulation would not expand. Since Blast X does not leave theproliferative fraction, the cell loss factor is slightly less thanunity and the population will expand, albeit very slowly. Basedon this model, in which the cell loss factor is 0.93, the growthfraction is 15%, and the cell cycle time is 40 hr, it takes morethan 10 years for the leukemic cells to outgrow the normalhematopoietic population (Chart 13). For reasons which arenot well understood, patients with a long preleukemic phaseare very refractory to treatment.

In the next model, which is compatible with overt leukemiawithout a preleukemic stage, again only one blast, Blast 14, iscapable of maturing, but 4 blasts instead of only one arerecycling (Chart 14). In this model, the cell cycle time remainsunchanged but, because the cell loss factor is lower (0.7) andthe growth fraction slightly higher (20%), only about 2 yearsare required for the population to expand from one cell to aclinically relevant fraction (Chart 15).

Like other workers in the field, we tried to apply cytokineticconcepts to devising improved treatment regimens, but suchefforts have been largely unsuccessful. Attempts to recruitdormant leukemic cells to begin proliferating by physicallyremoving large numbers by leukapheresis or by pretreatingpatients with cytotoxic drugs resulted in only a modest increasein the growth fraction or none at all. We also tried to takeadvantage of the fact that, since the normal stem cells aresuppressed at the time of diagnosis, it should therefore be

Â£2

1000

800

600

400

200

CELL LOSS FACTOR = 0.93GROWTH FRACTION = 0.15CELL CYCLE TIME = 40h

1 1 1 II8

1 !10

11424 6 8 10 12

YEARS AFTER TRANSFORMATION

Chart 13. Exponential growth of the leukemic population illustrated in Chart12 from the one-cell stage to a clinically relevant size. The time course of this

growth is compatible with the slow rate of progression in some cases of acuteleukemia when preceded by a preleukemic state [from Dormer (44)].

CELL LOSS FACTOR -11/15 -0.7 INFLUX FROMEARLY STEMCELL POOL?

Chart 14. Scheme of divisions in leukemic blast cell population in which onlyone of 15 cells differentiate, but 4 cells recycle in the primitive blast cell poolinstead of only one as in Chart 12 [from Dormer (44)].

DYING (?) ORRECYCLING*?)CELL

Chart 12. Scheme of divisions in a leukemic blast cell population in whichonly one of 14 cells differentiates and only one cell recycles in the primitive blastcell pool [from Dormer (44)].

possible to selectively kill a large fraction of the leukemicpopulation with drugs with actions that are restricted to proliferating cells. We used largely single drugs for treatment untilabout 1965; we found that the remission rate in ANLL was lessthan 25% and that the remissions were short; and we observedno significant change in the natural course of the disease (11 ).The median survival was only about 4 months, and no patientssurvived longer than 3 years. When we began treating patientswith a combination of active antimetabolites, ara-C and 6-TG,

the complete remission rate increased to about 50%, and therewas a modest increase in median survival from 4 to 10 monthsfor the whole group and to about 2 years for the responders,but again no patients were cured (49). We estimated that eachcourse of ara-C and 6-TG caused only a 1- or 2-logi0 cell kill

even in the most responsive patients. Because of toxicity toregenerating normal cells, the duration of treatment could notbe extended sufficiently to kill more of the dormant leukemic





1000

Â»180Â°600

O Z 400_JO

200

Otru_

CELL LOSS FACTOR = 0.7GROWTH FRACTION =0.2CELL CYCLE TIME = 40h

l S illlli

9 12 15 18 21 24 27 30036

MONTHS AFTER TRANSFORMATION

Chart 15. Exponential growth of the leukemic population illustrated in Chart14 from the one-cell stage to a clinically relevant size. The time course of thisgrowth is compatible with the rate of progression in acute leukemia without apreleukemic phase [from Dormer (44)].

cells as they gradually entered the proliferative cycle.Meanwhile, Skipper (97, 98) and his colleagues had shown

that certain drugs such as alkylating agents and anthracyclineswere quite effective in killing dormant tumor cells and that, withappropriate dosage schedules, it was possible to cure animalswith relatively advanced disease. It was only when we beganusing non-cell cycle-specific drugs after remission inductionthat we began to see long survivors. In the L-6 protocol, againabout one-half of the patients had complete remissions usingara-C and 6-TG for induction (18). After achieving remission,

they were treated for 3 more years with a regimen consistingof rotating sequences of combinations of antimetabolites aimedat destroying cells which had resumed dividing and of 1,3-bis(2-chloroethyl)-1-nitrosourea, cyclophosphamide, and dau-

norubicin to kill dormant cells. Using this strategy, about 25%of patients achieving remission or about 15% of the totalremained in long-term remission (Chart 16). With longer follow-

up, a few late relapses occurred, but the majority have remained in continuous remission (85).

Unfortunately, our further attempts to increase the numberof long survivors in ANLL have been unsuccessful. Using ananthracycline in combination with ara-C or with ara-C plus 6-TG for induction, remissions can now be rapidly achieved inabout 70% of patients, but most are still of short duration. TheL-12 and L-14 protocols included increasingly aggressive

treatment with multiple drugs during consolidation to circumvent development of drug resistance and to try to destroy asmany leukemic cells as possible (74, 75). However, as theintensity of treatment increased, we began to encounter deathsfrom myelotoxicity during remission. Moreover, most of thepatients who survived still relapsed, so there was no increasein the proportion of long survivors (Chart 17).

We found no clear relationship between the growth rate ofthe leukemic cells and response to treatment. The medianpretreatment LI was not significantly different in patients withANLL having remissions on the L-12 or on other protocols

compared to those who failed to respond or who died duringattempted induction (Chart 18). The same was true of adultswith ALL treated with the L-10 protocol (not shown). Similarly,

there was no significant relationship between the pretreatment

LI and remission duration in ANLL. In the example shown inChart 19, both early and late relapses occurred on the L-12

protocol with approximately equal frequency in patients withboth slow and fast-growing leukemic populations. In patientswith ALL treated with the L-10 or L-1 OM protocols, the remission duration was slightly longer but only marginally so (p =0.060) in patients whose Lis were above the median of 10 thanthose with Lis at or below 10 (not shown). With respect tosurvival, patients with ANLL having Lis at or below the median

a Continues in remissioni Died in remissionâ€¢Marrow relapseâ€¢CNS relapse â€”Â»rrtorrowrelopse

6 mos iole*

20-

4 8 12 16 20 24 28 32 36 40 44 48 52Months after M-l marrow

Chart 16. Remission duration of patients with ANLL treated with the L-6

protocol (18). CWS, central nervous system.

SURVIVAL DURATION

By Protocol ForAcute IMonlymphoblastic Leukemia

o L-6 (101 PTS., 13 ALIVE)â€¢L-12 (104 PTS., 16 ALIVE)Â¿L-14 (58 PTS. .7ALIVE)

cr>

24 48 72 96 120

Months from diagnosisChart 17. Survival duration of patients (PTS ) with ANLL treated with the L-6.

L-12. and L-14 protocols (74, 75).

DECEMBER 1981 4873



S. D. Clarkson

Relation of Pretreatment LI in ANLL toResponseto LI2 Protocol

32

25r

20

Â£;oJ- 10

_LCR Failure Died during

TreotmentChart 18. Pretreatment Â¡nvitro pulse-tritiated thymidine Lis of marrow leu-

kemic cells obtained by marrow aspirates in patients with ANLL treated with theL-12 protocol (74). The patients are grouped according to whether they achievedcomplete remission (CR), failed to achieve remission affer several courses oftreatment, or died early during attempted remission induction. Bars, groupmedians.

of 7 had a longer median survival (12 months) than did thosewith higher Lis (5 months) (p = 0.035), but so many factorscan influence survival that the meaning of this observation isnot clear.

In a single disease entity, such as ANLL, drug sensitivityappears to be a more important determinant of response thandoes proliferative rate. With modern induction regimens, it ispossible to kill a large fraction of the leukemic population veryrapidly. By means of serial cytofluorometric measurements, itcan be shown that the proliferating cells are destroyed first,usually within the first day or so, but a large fraction of thenonproliferating cells are also killed (Chart 20) (1, 2). Somedata of Wolfgang Hiddemann and Michael Andreeff (59) areshown in Chart 21 which relate the extent of kill in the marrowas determined by serial biopsies to achievement of remissionin a series of patients treated wth a 5-day course of DAT. These

quantitative measurements are consistent with previous estimates, namely, that a leukemic cell kill of 2 logs or greater isrequired for achievement of remission.

The problem is how to destroy the remaining 9 or 10 logs ofleukemic cells, and this has proven to be extraordinarily difficult. One would think that, if a trillion or so cells can be rapidlykilled, it should not be too hard to kill the several billion cellsremaining. However, this is not a negligible number if every lastone, or at least all those with stem cell capability, must bedestroyed, and, unfortunately, cell kill follows the principle offirst-order kinetics (105). To appreciate the order of magnitude

we are discussing, it may be worth noting that a century, whichseems like a long time, is made up of approximately 3 billionseconds.

All of the drugs which are effective in ANLL are also very

damaging to normal precursors, especially when they are rapidly regenerating after the marrow has been depleted of leukemic cells by the initial course of treatment. Presumably, mostof the surviving leukemic cells are deep in G0 and are relativelyinvulnerable to the drugs. Reducing their density by treatmentmay trigger a certain fraction to begin proliferating, but no wayhas yet been found to force all of them to leave the G0 state sothat they will become susceptible to the next round of treatment. Moreover, even if a way were found, unless it was

Relation of L12 Remission Duration to Pretreatment LI

30

25

#

Â« 20_i

Â£ 15â€¢5

EÃˆ IO

in5

10 12 14

Months to relapse

18 20 22 >22

Chart 19. Pretreatment in vitro pulse-tritiated thymidine Lis of marrow leukemia cells of patients with ANLL who had complete remissions on the L-12 protocolplotted according to the duration of their remissions.

K.R. 63 Femole. AML

M300[- \

WBCÂ«I03/mm'

100

so

ft t CELLULAR1TY

Blasts

I

Bone Morrow Differentials

4/28/78 4/29 4/JO 5/1 5/2 5/3 5/4 5/5 5/15 5/19 5/30

FMF ANALYSIS - BONE MARROW CELLS I Propidium Iodide)

8IOO630045OO27009OO-4/28-P-.-

trvolmont_.---

7G0+G,.i,

O"00s""

7000G,t M.2650003000^

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4/5Â°-48hri..-?

;G0

+G, >964S-02Gj

+ M'14Uâ€”

. ., -..-

20 40 60 80 100 120 20 40 60 80 100 120

CHANNEL NUMBER (Fluorescence Intensity)

Chart 20. Flow microfluorometric (FMF) analysis of marrow leukemia cellsstained with propidium iodide in a heavily pretreated patient with acute myelo-blastic leukemia (AML) during treatment with DAT (1, 2). The fraction of marrowcells in S phase fell from 12.5% to 0.2% within 48 hr, and there was a markedreduction in the marrow leukemic population by the end of the 5-day course oftreatment. The patient had a brief remission, but relapse occurred after a fewweeks.





10log

mI3umm

2-

1-

â€¢â€¢ p <0.01

oo o

oo o

â€¢ responderso non-responders

Chart 21. Relationship between extent of cell kill of bone marrow blast cells(BMBC) per cu mm of bone marrow (BM) determined by serial bone marrowbiopsies and induction of remission in patients with ANLL after 5 days of treatmentwith DAT (59).

selective, one would expect the effects on the normal stem cellpopulation to be equally devastating. In some experimentaltumors and in a few human tumors, it is possible to cure evenrelatively large tumors without killing the host simply by increasing the doses of drugs to which the tumors are particularlysensitive. However, our own attempts to eradicate the remaining leukemic cells by escalating drug doses after remissionwas achieved were disastrous in both ANLL and ALL. Anunacceptable number of patients died from toxicity while still inremission.

I will show you one more example to illustrate the dilemma(Chart 22). I have chosen CML because in this disease onecan distinguish between the leukemic and normal populationsby the presence in the former of a cytogenetic marker, the Ph1

chromosome. Before treatment, almost all of the dividing marrow cells were Ph1 positive, but after splenectomy and one

course of DAT, most of the dividing cells in the marrow werenormal, lacking the Ph1 marker.3 Chemotherapy was continued

with the intent of further reducing the leukemic population, butthis proved more damaging to the normal cells than to theleukemic cells and the latter soon predominated again. Retreatment in October with DAT in the same schedule which hadinitially been effective failed to diminish the proportion of leukemic cells. The leukemic cells were still sensitive to the drugssince similar reductions in leukocyte and platelet counts occurred, but the normal cells had apparently been too severelydamaged by the treatment to permit them to again repopulatethe marrow.

3 T. Goto, M. Nishikori, Z. Arlin, T. Gee, S. Kempin. J. H. Burchenal, A. Strife,

D. Wisniewski, C. Lambek, C. Little, S. Jhanevar, R. Chuganti, and B. Clarkson.Growth characteristics of leukemia and normal hematopoietic cells in Ph' +

chronic myelogenous leukemia in vivo and in vitro and effects of intensivetreatment with the L-15 protocol, submitted for publication.

About 50% of patients with chronic-phase CML had completeremissions on the L-1 5 protocol,3 which is a moderately inten

sive regimen, but most were of short duration and, as in thecase of the earlier L-5 protocol (38), only a few lasted over 1

year. Physicians are understandably reluctant to treat a chronicdisease such as CML very aggessively because fatalities willsurely occur from toxicity. Complete destruction of the Ph'-

positive leukemic population is probably possible, but thus farit appears that it has been accomplished only with supralethaldoses of total-body irradiation and chemotherapy followed by

rescue with transplanted marrow from an identical twin (47).Only subtle differences have been found between CML cells

and normal hematopoietic cells, and their proliferation kinetics,functional properties, and sensitivity to cytotoxic drugs are verysimilar (31 , 83). 3 This is undoubtedly one of the major reasons

for our failure to substantially improve the treatment of thisdisease.

The major therapeutic advances which have occurred duringthe last quarter-century have been in tumors which are moreselectively damaged by one or more treatment modalities thanare the critical normal tissues. Most of the advances in chemotherapy have been empirical, using combinations of drugs,each of which has been shown to have significant activityagainst the particular tumor when administered as a singleagent.

Acute lymphoblastic leukemic cells are more sensitive toseveral drugs, including corticosteroids, vincristine, and L-as-

paraginase, than are normal stem cells. Although not curativeby themselves, the availability of such relatively nontoxic drugsgives the therapist an enormous advantage. Complete or partialremissions can be induced quite regularly without severely

41 yr d CML L-15

1,300900

x 103/mm3 500

100

8050

x I03/mm3 20

PLATELETS

KARYOTYPE

100 I 54 t 0 ! 28 ! 100 I(6/8) (15/28) (0/15) (7/25) (13/13)

98 O O 60 92i- (58/59) (0/22) (0/3) (18/30) (23/25)

THERAPY

RT-Â»SPLEENi\SeLEHfCToHff]DAT

â€¢â€¢Ara-C+EJHu_lililÃC

FEB MARMAY71978â€¢a

DaMTXTGCÂ»t.VCRPrÂ«d.1

1 1 1 1 1 1 11JULY

SEPT NOV JANMAR1979

Chart 22. Cytogenetic changes occurring in marrow population in a patientwith CML during treatment with the L-15 protocol.3 The patient had a briefremission with disappearance of most of the leukemic cells containing the Ph'

chromosome following splenectomy and one course of DAT in April, but therewas rapid repopulation of the marrow with Ph'-positive cells despite continuing

chemotherapy, and subsequent treatment with DAT in the same dosage schedulein October failed to reduce the percentage of Ph'-positive cells. RT. radiotherapy;

HU, hydroxyurea; MTX. methotrexate; Cyf., Cytoxan; VCR, vincristine; Pred.,prednisone.

DECEMBER 1981 4875



S. D. Clarkson

damaging the normal stem cells, and such drugs can also beinterspersed between courses of other cytotoxic drugs to prevent significant regrowth of leukemic cells while allowing recovery of normal cells. About one-half of children with ALL can

now be cured with the best modern treatment regimens (55),but until recently adults with ALL had a much less favorableprognosis. The natural course of ANLL and ALL is similar, themedian survival being only a few months without effectivetreatment, but our results in adults with ALL have been consistently better than in those with ANLL during the last decade(Chart 23). Moreover, as shown in Chart 24, the results withthe recent more intensive L-10 protocol appear to be significantly better than with the earlier L-2 protocol.

In the L-10 protocol, 11 drugs active against ALL are rotated

in a complex schedule to try to avoid intolerable toxicity to anyone organ system and development of resistance of the leukemic cells to any one component (33). The drugs are given inmaximally tolerable doses, and the sequences are administeredas close together as possible to minimize regrowth of leukemiccells during1 treatment-free intervals. The treatment, which in

cludes repeated intraventricular or i.t. methotrexate to preventmeningeal leukemia (23), is continued for about 3 years.

About 85% of previously untreated patients achieve remission on the L-10 protocol, and it appears that approximatelyone-half of the patients will remain in remission indefinitely.

Most of the patients who relapse do so within the first year anddie within 2 years; only one patient has thus far relapsed afterstopping all treatment at 3 years. Although several factors,such as age and initial leukocyte or platelet count, have someprognostic significance, as in the case of ANLL, no clearclinical or laboratory features have been identified which distinguish the long survivors from the rest of the patients. Type ofleukemia (i.e., null versus T-cell) and initial proliferative rate

are not clearly correlated with response, and although firm dataare still lacking, I suspect that the major determinant, as inANLL, is sensitivity of the leukemic cells to the drugs. The L-10

protocol is an arduous regimen for both patient and physician,and considerable perseverance, experience, and judgment arerequired for its successful implementation. Even in this tumor,

SURVIVAL DUR'ATION

By Disease CategoryFor Acute Leukemia

o ALL (101PTS., 49 ALIVE)â€¢ANLL (263 PTS.,36 ALIVE)

pxo.ooo;

24 48 72 96

Months from diagnosis

Chart 23. Survival duration of previously untreated patients with ALL andANLL treated during the last 10 years at Memorial Hospital.

REMISSION DURATIONL-2 VS.L-10/L-10M

o L-2 (23pts., 6 in remission)o L-ÃŒO/LÃŒOM (61 pis., 3censored,

37 in remission)j. Tick mark - last follow-up

100

0

Months from response 8/1/80

Chart 24. Remission durations of previously untreated patients (pis. ) withALL treated with the L-2 protocol and with the L-10 or L-10 M protocols at

Memorial Hospital (33).

which is highly responsive to many of the drugs we now haveavailable, we are treading the razor's edge between cure and

patient tolerance, and about one-half of the patients are still

dying of the disease.Another disease in which significant progress has been made

is Hodgkin's disease. Using a combination of irradiation to

involved areas and 3 cycles of MOPP before and after irradiation, a high percentage of patients with localized disease (i.e.,Stages I and II) remain disease free (Chart 25) (65). Whereasthese results with combined modality treatment presently appear better than with either involved-field or extended-field

irradiation alone, it is possible that late relapses or complications of the treatment will occur, and a much longer follow-up

will be necessary to determine if survival is significantly improved. Some patients with early disease are cured by localirradiation alone, and those who do relapse can often betreated successfully with chemotherapy; thus, it is argued thatwe may be overtreating some patients and increasing their riskof developing late complications such as leukemia. Thus far,no instances of leukemia have been observed in this group,but the risk is clearly still present.

Advanced Hodgkin's disease is still a formidable therapeutic

challenge. Chart 26 shows our results in Stages IIIB and IVHodgkin's disease using 2 alternating non-cross-resistant drug

combinations (MOPP and ABVD), plus radiotherapy to bulkytumor masses (99). As usual, previously untreated patientsresponded best, heavily pretreated patients worst, and minimally pretreated patients had an intermediate response. San-





J'lfi .'...1-ll-'l,r)0

100

EÂ£ 60

STAGES I & II DISEASE FREE SURVIVAL

â€¢5.

40IÂ»,I

a Involved fieldo Extendedfielda MOPP+ IF

(38pis . 14censored)(36pis.. 25 censored)(64pts.. 62 censored)

18 36 54 72 90Months from diagnosis

Chart 25. Disease-free survival durations of previously untreated patients(pts. ) with Stage I and II Hodgkin's disease treated at Memorial Hospital with

involved-field irradiation and extended-field irradiation and with 3 courses ofMOPP chemotherapy before and after involved field irradiation (MOPP + l.F.)(65).

toro et al. have obtained a similar response rate with MOPP-

ABVD (93), and this alternating combination appears superiorto MOPP alone.

As I have already indicated, the first human tumor to becured by chemotherapy alone was choriocarcinoma, and thistumor is unique in that it originates in the fetal portion of theplacenta and metastasizes to the mother. There are now several other solid tumors which are curable even when in advanced stages. Chart 27 shows progressive improvement inthe treatment of advanced testicular cancer with successiveprotocols at our institution (50, 104). VAB 4 includes 6 drugs,each with significant activity alone. All of the patients shownhad disseminated (or Stage 3) nonseminomatous cancer which,if not treated or inadequately treated, has a median survival ofabout 9 months with almost no patients surviving longer than3 years. Almost all patients with seminomas are curable withradiotherapy, and nearly all patients with early (i.e.. Stage II)metastatic nonseminomatous cancer can now also be curedeither with adjuvant chemotherapy administered prophylacti-

cally or when initiated as soon as evidence of recurrence isdetected.

De Vita ef al. (40) recently listed 12 human tumors in whicha significant fraction of patients can be cured by chemotherapyalone even when they have advanced disease (Table 2). Unfortunately, the list does not include the more common types ofcancer, and these 12 tumors comprise less than 10% of seriouscancers. Excluding patients with localized tumors currentlycurable by local or combined modality therapy, about 39,300patients per year present with advanced tumors of these typeswhich were previously uniformly fatal. De Vita4 has estimated

that about one third of these patients (i.e., 14,400) are nowbeing cured, and most of the remaining 25,000 patients havesignificantly extended survival." Most of the therapeutic ad

vances have been in the more rapidly growing tumors; slowlygrowing tumors such as chronic lymphocytic leukemia, multiplemyeloma, and most of the nodular lymphomas, even thoughquite responsive to chemotherapy, are conspicuously absentfrom this list.

To put this in perspective, about 785,000 new cases ofserious cancer occur annually in this country (Table 3), excluding skin cancer and cervical carcinoma in situ which are highly

4 DeVita, personal communication.

curable. About 505,000 have localized cancers and, of these,about 321,850 (about 40% of the total new cases) can now becured by surgery, radiotherapy, or combined modality treatment; this figure includes about 12,000 patients who are curedbecause chemotherapy was added to their surgical or radiationtreatment.

Of the 280,000 patients presenting with inoperable or disseminated cancer, about 157,000 (~56%) have tumors which

are moderately to highly responsive to chemotherapy, includingthe 39,300 patients with potentially curable cancers mentionedearlier of whom about one-third are already being cured. Of

the remaining 117,700 patients with responsive tumors, it isestimated that 63,000 have sufficiently good responses to havetheir survival prolonged. This leaves about 123,000 patients

SURVIVAL DURATIONAdvanced Hodgkin's Disease

MOPP/ABDV and RT

o PREVIOUSLY UNTREATED (67 PTS., 56 ALIVE)â€¢MINIMALLY PRETREATED (23 PTS. ,15 ALIVE)Ã»HEAVILY PRETREATED (28 PTS., 12 ALIVE)

1.0

-Â§0.8

0.6

0.4

0.2

0 12 24 35 48 60 72

Months from start of therapyChart 26. Survival durations of patients with advanced Hodgkin's diseases

(Stages 1MBand IV) treated at Memorial Hospital with 2 alternating non-cross-resistant drug combinations (MOPP and ABVD) plus radiotherapy (RT) to bulkytumor masses (99).

SURVIVAL DURATIONTesticular Cancer

o VASI (68PTS.,13ALIVE)â€¢VAB2(45PTS.,13ALIVE)Ã»VAB3 (66 PTS.,29 ALIVE)* VAB4 (68 PTS.,48 ALIVE)

Months from start of therapy

Chart 27. Survival durations of patients (PTS) with advanced (Stage 3) nonseminomatous testicular cancer treated at Memorial Hospital with successiveVAB protocols (50. 104).

DECEMBER 1981 4877



e. D. Clarkson

who present with advanced tumors which are largely unresponsive to existing treatment.

Since many of the responsive tumors occur in youngerindividuals, the cancer death rate of the population under theage of 40 has begun to fall significantly during the last decade(41). Although there are formidable problems, we can anticipate further improvements in the curability rate of some ofthese cancers with development of new active agents and moreskillful use of existing therapeutic modalities.

Another group in which we should see significant advancesin treatment are the 183,150 patients with largely localizedcancers which recur following primary treatment (Table 3).Impressive advances in surgical and radiotherapeutic techniques have occurred during the last several decades, and withfurther refinements and wider application it should be possibleto further improve the cure rate. It is estimated that about20,000 of this group of patients, including those with breastcancer, colorectal cancer, melanoma, and soft tissue sarcomas, are potentially curable with existing forms of adjuvantchemotherapy. However, this number should increase withselective targeting of potentially curable candidates andbroader application. Probably, some patients with small residual foci of disease will prove on longer follow-up to have already

been cured, but it is not yet possible to estimate the numberreliably. Some of the tumors may show late recurrence becausethey are naturally slow growing or because the therapy hasmerely delayed recurrence rather than being curative.

It is thus estimated that 356,250 patients (about 45% ofserious cancers) are now curable with current therapy.4 Per

haps we can roughly estimate that another 50,000 patients willbe cured with optimal use of existing therapy, but this stillleaves about one-half of the total 785,000 new cancer patients

who will continue to die of their disease.We can also anticipate further progress in reducing the

Table 2

Twelve cancers in which a fraction of patients with advanced disease can becured with chemotherapy

Data from paper of DeVita er al. (40).

ChoriocarcinomaALUHodgkin s diseaseDiffuse histiocytic lymphomaNodular mixed lymphomaTesticular carcinomaOvarian carcinomaAcute myelogenous leukemiaWilms' tumorBurkitt's lymphoma

Embryonal rhabdomyosarcomaEwing's sarcoma

mortality of some cancers by broader application of earlydetection or preventive techniques. Indeed, some of thesemeasures are undoubtedly already having a significant impact,such as screening for early cervical cancer, reduced inhalationof asbestos fibers, abandonment of the use of Thorotrast, andavoidance of exogenous estrogens during the first trimester ofpregnancy and in postmenopausal women (37, 43, 58, 60,76). In certain areas of the world where single types of cancerare unusually prevalent, such as primary liver cancer in Africaand Asia, and where discrete causative factors have beendefined, we can soon look forward to the development ofeffective preventive measures, such as a vaccine for hepatitisB virus and dietary changes to reduce ingestion of aflatoxinand other carcinogens. However, as is already evident, evenwhen major exogenous causative factors have been identified,such as cigarette smoking, alcoholic consumption, chewing ofbetel quid, sexual promiscuity, or excessive exposure to sunlight, it is difficult to persuade enough people to modify theirlife styles to substantially reduce the disease incidence. Exceptfor stomach cancer, no significant decrease is expected in theincidence of most major cancers during the next decade, andsome, especially lung cancer, are expected to increase (87).Thus, the need for improved treatment will remain an importantpriority for some time to come.

The therapeutic advances made during the last quarter century are quite remarkable considering that we have only had atour disposal what Lewis Thomas (102) has called halfwaytechnology. A massive effort has been mounted to utilize ourhalfway technology, and indeed, instead of the cancer patientbeing largely neglected as was true 25 years ago, sometimeshe is now in greater danger of being overtreated with drugswhich rarely produce a useful response. Whereas there wereonly a handful of medical oncologists 25 years ago, the sub-

specialty has grown exponentially during the last decade. Whilethis has resulted in vast improvement in the overall quality ofcare, there have also been some less desirable consequencesin terms of future therapeutic advances. In large metropolitancenters, there is now severe competition for patients, especiallythose with responsive types of cancer. Since in most instancesthe initial treatment has by far the best chance of producing acure, one of our most frustrating experiences is to see patientswith potentially curable tumors which have recurred followinginadequate treatment. Even with optimal available treatment,we are still failing to cure the majority of patients with the mostresponsive tumors; thus, there is no justification for complacency. Rather, we must intensify our efforts to sort out thereasons for success and failure and to develop better and lesstoxic treatment. Much has been learned from experimental

Table 3/980 cancer statistics taken from American Cancer Society facts and figures (1980)"

785,000 new cases

505.000 localized

321.850 cured by surgery and/or radia- 183.150 recurrences after primarytion therapy Â±chemotherapy treatment

i? 20.000 curable with adjuvant

therapy

280,000 metastatic orinoperable

1157,000 benefit from

chemotherapy

i39.300 potentiallycurable

I14.400 cured





tumors, but there are important differences between animaltumors and spontaneous human cancers. We must developbetter methods to cultivate fresh human tumors to make themavailable for basic research. This requires deliberate and closeinteraction between clinical and basic scientists, but unfortunately the current research climate is not always conducive tosuch a joint effort. Among the disturbing trends are antiintellec-

tual or cynical attitudes, not only on the part of the public atlarge but also among recently trained physicians: shrinkageand instability of funding for research: an ever increasingproliferation of paperwork; and excessively rigid regulationsfor accountability.

Clinical investigation flourished for about 25 years afterWorld War II with the strong endorsement of the public and ourgovernmental leaders. The National Cancer Act, passed in1971, provided greatly increased support for cancer researchwithout significantly detracting from support of other biomÃ©dical research. Benno Schmidt, Vincent DeVita, and Lee Clarkhave recently reviewed the main achievements of the NationalCancer Program, now in its tenth year, and it would be superfluous to reiterate these accomplishments (9, 40, 94). While itis inevitable that some would have preferred placing differentemphasis on particular areas of research, on the whole I believethe program was well planned, well balanced, and effectivelyadministered. Unfortunately, the appropriations have leveledoff during the last few years, and because of inflation progressively fewer new research applications are being funded.

While the number of clinical oncologists has greatly expanded, the number of physician-scientists has dropped alarm

ingly. In 1968, 15,000 physicians reported that their primaryactivity was research, but in 1975 only about half as many didso (69). Not only are we losing research-oriented medial school

faculty, but the number of physicians entering research andteaching careers is declining and not matching the attritionrate. The percentage of medical students graduating fromHarvard who assigned a high priority to research fell from 49%in 1963 to 2% in 1976(73).

Our system is faltering and many of the reasons are clear.Partly as a consequence of extravagant predictions of earlysuccess and in some cases injudicious use of cytotoxic drugs,we have lost the confidence of some segments of our society,and we are now experiencing a backlash of public opinion. Ina few highly publicized instances, young investigators haveeven been guilty of falsifying data, ostensibly because of competitive pressures. As is often the case, the majority of scientists are not to blame, but the transgressions of a few can domuch damage to our credibility. Moreover, it is quite understandable that the public becomes confused when prominentscientists adopt evangelical postures and loudly advocateforms of treatment for which there is no sound scientific basis.

Young physicians see their preceptors bogged down in administrative trivia and committee work, petty territorial disputes,struggling with endless grant applications and site visits tosupport their salaries and keep their research programs afloat,or even succumbing to social and political pressures to undertake clinical trials of laetrile or other substances of dubiousmerit. Little time is left for research, and too many of us devoteit to studying epiphenomena about which we are reasonablysure we can provide quick answers. While such studies oftenprovide useful information, they seldom get to the heart of theproblem.

Because of the many recent advances in knowledge andincreasingly sophisticated technology, longer training periodsand greater specialization than before are required for newinvestigators to become competent in their fields. Young physicians are disinclined to make the necessary long-term com

mitment to a research career when funding is unpredictableand when academic salaries are relatively parsimonious incomparison to the rewards of clinical practice. Not only aremost of our brightest young physicians avoiding the challengeof difficult research, but many of our basic scientists arelikewise limiting their activities to well-defined systems with

which they are familiar and which they believe have the bestchance of assuring renewal of their grants. Somehow we mustchange our ways of doing things so that we will encouragethese physicians and scientists to accept the difficult challenges. We must provide secure support for their research andkeep them free from entanglement in the bureaucratic web thatwe have created for ourselves.

The latest threat is commercialism. Some of our most talented scientists now devote substantial amounts of their timeto developing marketable products. I have heard the argumentsabout how a harmonious blending of academia and businesswill lead to immense benefits for mankind, but I am skeptical.It is hard to believe that business leaders' primary motivation

for profits and their penchant for secrecy are compatible withour traditions of free inquiry simply for the sake of seeking newknowledge and open exchange of ideas and information.Whether this partnership will be more advantageous than destructive remains to be seen.

As I have emphasized, what is needed is development ofmore selective therapy for cancer in the same sense thatspecific antibiotics cause selective damage to particular microorganisms, but this goal has proved to be extremely elusive.Although consistent differences have not yet been found between cancer and normal cells in their biochemical pathwaysor structural components, it may be that we just have notlooked hard enough. There is presently a great surge of interestin monoclonal antibodies which it is hoped will selectivelydestroy human tumors without damaging normal cells, but it isuncertain whether the present approaches, which so far havebeen rather empirical, will accomplish this goal without firstidentifying and purifying tumor-specific target antigens.

It may be that some leads towards development of moreselective treatment have already been discovered but have notyet received sufficient attention. I will give just one example.Doubtless many of you have others in mind; at least I hope so.Using current banding techniques, about one-half of human

acute leukemias have been reported to have visible chromosomal abnormalities (78, 89, 92), and recently, with high resolution cytogenetic analysis, it appears the majority of patientswith ANLL will be found to have defects (108). The examplesshown in Table 4, taken from a recent review by Janet Rowley(90), list several types of human leukemia or lymphoma inwhich characteristic chromosomal rearragements or deletionsoccur fairly consistently. As already noted, the translocationbetween chromosomes 22 and 9 in CML is associated withchanges which deviate only slightly from normal, whereasthose occurring in acute leukemia are associated with moresevere behavioral defects. In general, acute leukemias in whichall of the marrow cells have visible chromosomal abnormalitiesdo not respond as well to treatment as do those without

DECEMBER 1981 4879



ÃŸ.D. Clarkson

Table 4

Examples of consistent chromosomal abnormalities in human leukemias andlymphomas

Data from the paper of Rowley ef al. (90).

TumortypeCMLM2

(myeloblastic) typeofANLLM3

(promyelocytic) typeofANLLSecondary

ANLLB-cell

ALL andBurkitt'slymphomaAbnormalityt(9;22)t(8;21)t(15;17)â€”

5. 5q, â€”7,or 7q(also+8

or+21)Â«8;14)Approximate

incidence(%)858

(of allANLL)4085>90

demonstrable aneuploidy (51), but specific abnormalities appear to have different prognostic significance (90).

The 8;21 translocation is restricted to the M2 type of ANLL,which accounts for about 8% of all cases, and such patientsare reported to have longer than average survival (61, 90). The15;17 translocation is seen in about 40% of patients with acutepromyelocytic leukemia (90). As noted earlier, acute promyelocytic leukemia usually has a lower proliferative rate than othertypes of ANLL, and some investigators have found that a highpercentage of patients with acute promyelocytic leukemia become long survivors with effective treatment (5). Secondaryacute leukemias developing in patients previously treated forother diseases or in those with a history of occupational exposure to mutagenic agents have a very high frequency ofnonrandom loss of chromosomes 5 and/or 7, or of parts ofthese chromosomes (90), and such leukemias are notoriouslyrefractory to treatment.

The chromosome changes occurring in ALL are differentthan those in ANLL. In the former, gains of chromosomes 13,14, and 21 and a deletion of the long arm of No. 6 are mostcommon (90). Thus, different genes appear to be responsiblefor the growth advantage of leukemic lymphoid cells comparedto myeloid cells. A specific reciprocal translocation t(4;11) hasbeen reported in about 8% of cases and carries a poor prognosis (108). B-cell ALL, which has an L3 type of morphology

and accounts for about 4% of cases of ALL, has a highincidence of translocation of genetic material to chromosome14 (4, 77). The donor chromosome is usually No 8, and thesame balanced reciprocal 8; 14 translocation is found in virtually all cases of Burkitt's lymphoma (57). Both tumors grow

rapidly and are very responsive to treatment but, whereas ahigh percentage of children with Burkitt's lymphoma can be

cured, the cure rate in the L3 type of ALL has remained low. Itis still unsettled whether the site of the critical "oncogenic"

lesion is located on chromosome 8 or chromosome 14 (57,64). The incidence of 14q+ translocation is also high in otherlymphoid cancers, especially B-cell, although chromosomes

other than chromosome 8 may serve as donors. As shown byHecht and Kaiser-McCaw (57), the key breakpoint usually

occurs near the distal end of the long arm of chromosome 14in band 14q32. In contrast, a more proximal breakpoint, located in band 14q12, which occurs in benign lymphocytichyperplasia associated with ataxia-telangiectasia and occa

sionally in normal subjects, does not seem to be associatedwith cancer (57).

Although it has not been proven that such chromosomalchanges are responsible for the growth advantage of the neo-

plastic cells, this seems a good possibility. On repeated occasions in my laboratory, we have observed that, when the SK-L7 human lymphoid cell line, originally diploid, spontaneouslyacquired an additional chromosome 15 after prolonged cultivation, this change was invariably associated with a markedlyincreased cloning efficiency in soft agar and a decrease in thedoubling time of the cells from about 30 hr to 14 hr as the cellswith trisomy 15 outgrew the original diploid population (17, 34,103). One might look on this as an example of malignantprogression occurring in vitro. Progression from low-grade tohigh-grade malignant tumors is frequently observed clinically,

as in blastic transformation of CML or malignant progression oflymphomas, and these transformations are commonly associated with additional chromosomal changes (92).

The problem is to define precisely how specific genetictranspositions alter cellular behavior to give the leukemic cellsa survival advantage over normal stem cells. Such geneticrearrangements are undoubtedly associated with changes inregulation of gene expression which, in turn, must be reflectedin crucial phenotypic changes such as differences in antigenicexpression or unresponsiveness to regulators of cell growth ordifferentiation. However, dissection of the biochemical chain ofevents responsible for these changes and attempts to identifyinappropriate gene products or the absence of normal productshas scarcely begun. Some of my colleagues who are skilled inmodern molecular biology have told me the problem is toodifficult. Perhaps understandably, they prefer to restrict theiractivities to viruses or plasmids where they can manipulate therelatively few genes with considerable precision rather thantrying to unravel changes in the expression of the 100,000 orso genes present in human cells.

Cellular differentiation and maturation are admittedly staggeringly complex processes (15). Genes are switched on andoff and a bewildering array of different proteins are producedas stem cells commit themselves to differentiate and thenprogress along the various maturation pathways. However,there has been steady progress in assembling the human genemap and in linkage analysis, and the O'Farrell 2-dimensional

gel electrophoresis technique has sufficient resolving power topermit separation and identification of scores of specific proteins (42, 81, 82, 101). With these methods, it should not beimpossible to define the consequences of specific genetictranspositions which result in neoplastic behavior and, in fact,studies are already under way in several laboratories. Forexample, Sach's group has been dissecting the protein

changes associated with differentiation of normal murine my-

eloblasts in comparison to those occurring in myeloid leukemiccells (71, 91). Some leukemic clones can be forced to differentiate with various inducers whereas others cannot. Proteinchanges induced during normal differentiation do not occur allin one sequence, but rather as multiple, parallel, and separatelyinduced sequences resulting from separately programmedpathways of gene expression. The mutant clones can be induced to follow some of these sequences but are blocked inthe induction of other pathways. Moreover, all the leukemicclones constitutively express a common subset of proteinchanges different from those found in normal myeloblasts, andthe size of these subsets is inversely proportional to the competence of the leukemic clones to be induced to differentiate.Sachs has proposed that the constitutive expression of somepathways of gene expression results in leukemia, whereas the





expression of other pathways results in decreased capacity fordifferentiation.

We have an excellent opportunity to follow these leads indissecting the abnormalities in gene expression in human neoplasms which are already known to exhibit genetic rearrangements. In addition to the changes occurring in the leukemias,specific chromosomal defects have been described in a number of other human cancers, including deletion of the long armof chromosome 13 in hereditary retinoblastoma and deletionof the short arm of chromosome 11 in Wilms' tumor (64, 108).

Doubtless other specific genetic lesions will be discovered byhigh-resolution cytogenetic analysis. Whether unique proteins

will be found against which highly specific monoclonal antibodies can be prepared still remains to be seen, but with better

understanding of differences in gene expression between normal and malignant cells, the next step of developing selectivetherapy should not be too distant. The task will not be easy,but it will be infinitely simpler than trying to solve the truly mind-

boggling wonder of how in the first instance a single cell canproduce a chicken in just 21 days with rarely a mishap.

The beautiful place shown in Fig. 1 is the ball court ofChichen Itza. The object of the game was to put the ball throughone of the stone rings which you can see high on the upperwalls. Since the ball was scarcely smaller than the orifice andsince the players were not allowed to use their hands and onlythe team captain was permitted to ascend to the upper level ofthe court, the game often went on for days before a goal was

Fig. 1. Ball court at Chichen Itza.

Fig. 2. Mural on wall of ball court at Chichen Itza showing priest at left holdingthe severed head of the losing team captain and the kneeling figure of the captainat right with blood spurting from his neck.

DECEMBER 1981 4881



S. D. Clarkson

scored. The penalty for losing was severe. Shown in Fig. 2 onthe wall of the ball court is a high priest holding the losingcaptain's head; the blood is spurting from his neck and from

the severed head.Our goal is even more difficult and certainly more elusive,

but the penalty for failure is just as deadly. Much progress hasbeen made during the last several decades in understandingthe biology of cancer, in defining the different types and theirnatural rates of progression, and in developing reliable methodsfor measuring the influence of therapy. With skillful use of therelatively nonselective therapy now available, it has provedpossible to cure several types of advanced cancer. Recenttechnological advances in molecular genetics have openeddoors to marvelous research opportunities hardly imaginable afew years ago. The stage is set and the time is opportune tomake a concerted effort to develop more specific treatment.The number of truly creative people in each generation islimited. We must make every effort to capture the imaginationof our brightest young people and encourage them to reachout for the difficult goal which has thus far eluded us.

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1981;41:4865-4884. Cancer Res Bayard D. Clarkson The Elusive Goal: Presidential Address

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