COMBINED CLINICAL AND BASIC SCIENCE SEMINAR Selected Department

of Medicine,

and edited

by Richard

T. Silver,

The New York Hospital-Cornell

M.D. and Alexander

Medical

Center,

G. Bearn,

New York,

M.D.

New York

Some New Aspects of Modern Cancer Chemotherapy

RICHARD T. SILVER, M.D. New York, New York ROBERT C. YOUNG, M.D. Bethesda, Maryland JAMES F. HOLLAND, M.D. New York, New York

From the Oncology Service, The New York Hospital-Cornell Medical Center, New York, New York; the Medicine Branch, National Cancer Institute, Bethesda, Maryland: and the Department of Neoplastic Disease, Mount Sinai School of Medicine, New York, New York. Requests for reprints should be addressed to Dr. Richard T. Silver, The New York Hospital-Cornell Medical Center, 525 East 68th Street, New York, New York 10021. Manuscript accepted April 25, 1977.

772

November

1977

Dr. Richard T. Silver: The treatment of acute leukemia has progressed in a thrilling fashion in the past 20 years. This success has resulted both from the introduction of new and more effective drugs, and from the development of therapeutic regimens in man derived from concepts elaborated in the mouse model. In just two decades this approach has resulted in impressive improvement in remission duration in adults with leukemia, and in changing the prognosis in children with acute lymphocytic leukemia from one of universal fatality to one of long-term remission and even cure. Although the hematologic malignancies are responsible for less than 10 per cent of all cancer deaths, they are of considerable added importance by providing the basis for the chemotherapeutic strategy applicable to other more common malignancies of men and women. The design of intermittent combination chemotherapy, developed for the treatment of acute leukemia and Hodgkin’s disease, applied to adjuvant chemotherapy of breast cancer is a meaningful expression of this development. Cure of leukemia or, for that matter, any neoplastic disease requires killing the last viable neoplastic cell, either literally or functionally. Whether this can be accomplished by chemotherapy alone or whether it can be abetted by host immune mechanisms is a question unanswered. For many years, there has been an interest in the potentiation of host defense mechanisms against many forms of cancer. Now there exists the suggestion that tumor cell antigens recognized by the host elicit subsequent immune responses. Immunotherapy with BCG has been studied extensively in acute leukemia in children. Other interesting developments in clinical immunotherapy include the intradermal injection of neuraminidase treated cells and the methanol extraction residue of phenol killed BCG. In this Combined Clinical and Basic Science Seminar, Dr. Robert Young will first review some of the general concepts of tumor cell kinetics that have been developed and employed in modern, clinical, cancer chemotherapy. Then, Dr. James F. Holland will discuss some new results in the treatment of acute leukemia emphasizing the potential role of immunotherapy. Dr. Robert C. Young: The general concepts of tumor cell kinetics have been developed primarily by physicists, mathematicians and biochemists. Cell kinetics never caught the attention of clinicians until relatively recently when they became interested in the impact that cancer chemotherapeutic agents make on tumor growth.

The American Journal of Medicine

Volume 63

NEW ASPECTS OF MODERN CANCER CHEMOTHERAPY

Certain observations intrigued clinicians because they suggested differences in the growth characteristics of normal host tissues and various tumors. For instance, some tumors and some normal tissues were obviously much more sensitive to chemotherapeutic agents than others. In addition, tumors and normal tissues were initially sensitive but later became resistant to chemotherapeutic agents. Out of these perplexing clinical observations, clinicians sought a better basic understanding of the growth of normal tissues and tumors. Figure 1 is a Venn diagram of the kinetic behavior of normal tissues. All normal tissues grow, but the characteristics of their growth differ, depending upon the tissue involved. There are three growth classes of normal tissues relative to their individual growth characteristics. These have been termed a static pattern, a renewing pattern and an expanding pattern [ 11. The static population is comprised of relatively well differentiated cells which, after initial proliferative activity in the embryonic and neonatal period, rarely undergo cell division and proliferate. Typical examples are striated muscle and neurons. The expanding population of normal tissues is characterized by normal tissues that retain the capacity to proliferate, but in their adult state they do not ordinarily undergo continuous proliferation. However, if some special stimulus occurs, tissue injury for example, a tremendous surge of proliferation occurs with a regrowth of normal tissue. A good example is liver parenchymal tissue. The renewing population of cells is constantly in a proliferative state. There is a constant cell division, a high degree of cell loss and constant cell turnover. Cells comprising the bone marrow, the epidermis and the gastrointestinal tract are good examples of this kind of renewing population. Clinicians with experience in cancer chemotherapy realize that generally little toxicity is produced in static populations, but a tremendous amount of toxicity is associated with injury to cells in the renewing population. Thus, kinetic factors partly explain the different toxicities experienced by various normal body tissues as a result of chemotherapy. Many chemotherapeutic agents depend on the proliferative capacity of the tissue for their therapeutic effect. A schematic comparison of normal and cancerous growth is shown in Figure 2. In embryonic life, there is proliferation in all three normal populations mentioned. At some point in growth, a cellular brake mechanism must operate such that new cells are born with the same rate that cells die. A steady state is reached. In a renewing system, there is a great deal of tissue turnover and constant cell proliferation. In static systems, there is very little cell division and correspondingly

STRIATED

Figure 1. proliferation Populations.

MUSCLE

Classification of normal tissue,s according to &e/r characteristics. Static, Expanding and Renewing

less cell loss. In a simplified sense, cellular brake mechanisms which exist in normal tissues are injured or disorganized in cancerous tissues so that continued proliferation occurs and continues until the death of the host. We now know that tumors in human subjects do not always grow exponentially as illustrated in Figure 2. Apparently some sort of cellular brake mechanism in fact exists, even in cancerous tissues. Early growth in cancer cell populations does seem to be exponential in character, but in the late stages of growth, it is definitely not exponential in character (dotted line, Figure 2). Investigators have characterized tumor growth in basically three ways as shown in Figure 3. Exponential growth, characterized by line A, is a reasonable esti-

1 LOGCELL NUMBER

STEADY CELL a. b. c.

STATE

BIRTH = CELL Renewing Expanding stalk

LOSS

--

-----A

TIME

Figure 2. ization

November 1977

Lethal number of cells. Schematic

of normal

and cancerous

i character-

growth.

The American Journal of Medicine

Volume 63

773

COMBINED CLINICAL ANDBASICSCIENCE SEMINAR -

t

:

I

;

‘i

A

I

B_--__-___

C. . . . . . . . . . . . . . . . . . . .

Time Graph of the different functions characterizing Figure 3. tumor doubling times. The ordinate is the tumor volume and the abscissa is time. A, Exponential tumor growth; B, Cube root function growth; C, Gompertzian tumor growth.

mate of tumor growth when a very small number of cells is involved. It is clearly not a good estimate when there is a large number of cells. Another equation characterizing tumor growth is shown in line B; it relates growth to a cube root function. This assumes that the tumor grows as a sphere, that the growth in the tumor is on the surface of the sphere and that it is not from the depths of the sphere. The most generally accepted equation currently in use is illustrated by line C. It is most consistent with all the experimental data available on animals and on man and is known as the Gompertzian function. Simply stated, it suggests that as the tumor mass increases, the time it takes to double its volume increases. Figure 4 is a theoretic tumor growth curve which assumes exponential growth and compares the number of cells in the tumor mass to the number of doublings 121. Obviously exponential growth is not strictly accurate throughout the range of tumor growth, but it does illustrate some important kinetic concepts. A good clinician might be able to recognize a 0.5 cm mass on a chest roentgenogram; he could likely palpate a 1 cm tumor mass. If such a lesion was discovered, he would assume that the tumor had been discovered quite early. Unfortunately, the tumor has undergone 30 doublings before discovery, which is equivalent to three-fifths of its entire

774

November 1977

The American Journal of Medicine

life span. Kinetically, it is far from an early tumor. Thus, our clinical technics, skilled as they are, recognize tumors late in their growth, after they have undergone a large number of doublings. This fact has several important clinical implications. First, metastatic disease may well have occurred long before obvious evidence of the primary lesion. Second, at later stages of tumor growth a very few doublings in the tumor mass make a very dramatic impact on the size of the tumor. When we therefore say that a patient had a small tumor that remained relatively dormant for a long time and then suddenly started rapid proliferation, that is probably correct as a clinical observation but probably kinetically and conceptually incorrect. A summary of the available information on the doubling times of tumors in human subjects is shown in Table I [3]. Unfortunately, only certain types of tumors can be studied this way because they must manifest themselves in a way that allows measurement. They must be relatively spherical, measurable on chest roentgenograms or by caliphers, or by some other means, and this restricts the spectrum of tumors that can be studied. Nevertheless, the information is useful despite its limitations. The mean doubling time is the time it takes for a mass to double its size. There is considerable variation in these times, in general, embryonal tumors, lymphomas and some of the malignant mesenchymal tumors have relatively fast doubling times, whereas the adenocarcinomas and squamous cell carcinomas have relatively slow doubling times. In addition, in almost every instance, metastases have faster doubling times than primary lesions. These estimates of the doubling times of tumors in human subjects are quite crude, but they suggest that the doubling time of these tumors approximates 50 days, with considerable variation about that estimate for individual tumors. Information I have presented thus far relates to the growth of the tumor mass as a whole. Now I will consider the kinetic behavior of individual tumor cells. Figure 5 illustrates a typical schematic view of the cell cycle first described by Howard and Pelt [4]. The mitotic phase, or M phase, of the cell cycle is the point of cell division. Following mitosis, there is a period of variable duration known as G,, or the post-mitotic phase. During G,, cellular activities, protein and RNA synthesis continue. There is a small amount of DNA repair during this period. These G, cells can either differentiate, or they can continue in the proliferative cycle. A burst of RNA synthesis occurs right before the onset of the S phase in which DNA proliferation or replication occurs. After completion of this DNA synthetic period, the cell enters the G2 period in which it has a diploid number of chromosomes and twice the DNA content of the normal cell. The cell remains in this

Volume 63

NEW ASPECTS OF MODERN CANCER CHEMOTHERAPY

IO00 Kilogram Mass

I Kllogram

Mass

;;_ I cm

J

32 cm Mass 8 cm Mass 4 cm Mass 2 cm Mass

Mass

0 5 cm Mass/

Tumor First Paipable (30 doublings) Tumor First Visualized (27 doubllngs)

5

IO NUMBER

Figure

4.

Theoretical

15 20 OF DOIJBLINGS

tumor growth

period for a relatively short period and then enters mitosis again. Variations occur around mean cycle times and in all phases of the cell cycle. The variation is greatest in the duration of G,. The events determining the variation in the G, period are not well understood, but they profoundly affect the proliferative behavior of the population of cells. If the G1 period is very short, then the cells cycle rapidly and are very proliferative. On the other hand, if they enter a very long G, period, they act almost as a resting cell. This unusually long post-mitotic resting state has been termed the (Go) phase and is characterized by a resting cell that retains the capacity for cell proliferation; during the course of any particular observation, however, it is not actively cycling. These cell cycle events have important implications as shown in Figure 6. Differential sensitivities to chemotherapy are associated with different proliferative states. The dividing cancer cells (compartment A), those actively engaged in the cell cycle, are very sensitive to our best chemotherapeutic agents. Cells in compartment B are a population of temporarily nondividing cells either in a long G, period or in Go. In the resting state, these cells are not very sensitive to chemotherapeutic agents; however, they can revert into the rapidly proliferating population after appropriate drug therapy and produce tumor regrowth. Finally, there is a third group of cells which is permanently nondividing; these cells occupy space and contribute to the bulk of the tumor.

curve.

25

Modified

30

35

from Collins

40

on X-Ray

45

50

et al. 121

However, because they do not divide, they are really of no major concern to the oncologist. The estimation of cell cycle times is made by using the morphologic appearance of cells in mitosis and the labelling of cells in the S phase with tritiated thymidine. Figure 7 illustrates the morphologic characteristics of cells in the various phases of the cell cycle. There are relatively subtle morphologic differences between G2, S, G, and Go cells; in fact, they are beyond our discriminatory technics. In contrast, mitosis is very well characterized. Thus, the morphologic characteristics of cells in mitosis allow one window into the cell cycle. The other window into the cell cycle is the S phase, and

TABLE I

Doubling Times (DT) for Tumors in Human Subjects (538 Cases from Worlds Literature)* Patients, Mean DT (no.1 (days) ___--

Tumor Pathology Embryonal

tumors

(lung metastasis)

Lymphomas Malignant mesenchymal tumors Squamous cell carcinoma (lung metastasis) Squamous

cell carcinoma

Adenocarcinoma Adenocarcinoma

* Modified

November 1977

(primary)

(lung metastasis) (primary)

from Charbit

f2

SD

76 51

27.0 28.9

22.5-37.2

a7

41 4 58.0

34.5-49.7 47.8-70.3

97 134

81.8

69.2-95.5

34

166.3

51

82.7

22.4-32.6

7 1.9-95.5 122.5225.7

et al. [3].

The American Journal of Medicine

Volume 63

775

COMBINED

CLINICAL AND

BASIC SCIENCE SEMINAR

I. Cell death 2. Different&ion 3. "Go"

PaOPHASE

state

MfilAPHASC

CELL CYCLE MODEL

MITOSIS

Figure 7. The morphologic appearance of cells in different phases of the cell cycle and mitosis.

C

B

A

\

CELL CYCLE

Figure 5. The cell cycle. M = mitosis; G1 = the postmitotic phase; S = the DNA synthetic phase; G2 = a postsynthetic phase.

DIVIDING CANCER CELLS

VERYSENSITIVE TO OUR BEST

DRUGS WHEN OPTIMALLY EMPLOYED

PARTIALLY TO COMPLETELY INSENSITIVETO DRUGS (DEPENDINGON CLASS1

OFLITTLE CONCERN EXCEPTFOR PHYSICAL PRESENCE

Figure 6. The different cell types in a tumor mass characterized by their proliferative state.

labelled thymidine can be administered and the ceils studied with autoradiography. With two windows into the cell cycle, one can quantify the number of cells in each of the phases of the cycles, and the number of cells in active cell proliferation. A schematic illustration of a cell cycle and a so-called per cent labelled mitosis curve (PLM curve) is shown in Figure 8. The duration of the first wave of mitoses is an estimate of the size of the S phase. There are two waves of labelled mitoses; the duration of time between the first and the second wave of labelled mitoses is an estimate of the cell cycle time or the generation time.

776

November1977 The American Journal of Medicine

An example of what an actual labelled mitosis curve looks like is shown in Figure 9. In this instance, the cells are L1210 leukemia in a CDF, mouse. Although the waves of labelled cells are not as sharp as the schematic, a double wave of labelled mitoses does occur and one can estimate the durations of the phases of the cell cycle and the generation time (Tg). Generation times of tumors in human subjects are much more difficult to obtain, but some information is available and is of interest. Figure 10 illustrates some studies performed in our laboratory on metastatic carcinoma of the breast [5]. In contrast to the animal

Volume 63

NEW ASPECTS

OF MODERN CANCER CHEMOTHERAPY

PLM

Schematic outline of a per cent labeled mitosis (PLM) curve. Upper graph Figure 8. is an idealization of a plot in which the abscissa represents time and the ordinate represents percentage of mitoses which are labeled. Lower panel represents the cell cycle.

models, there is a first wave of labelled mitosis but a very damped second wave. This finding is characteristic of studies in human subjects with tumors and simply means that there is great variation around mean cell cycle times. For most of the tumors studied, the duration of S phase, i.e., the length of time it takes a cancer cell to replicate its DNA, is roughly 24 hours. Computerized technics are presently available which allow further analysis of such data [6,7]. Figure 11 illustrates the generation of additional data by such computerized technics. The S phase is roughly a day, with a considerable amount of variation; G,, is about 35 hours with a tremendous variation. G2 is about 9 hours. The median cell cycle time or Tc is 51 hours. We have performed similar studies in patients with malignant melanoma [5]. They also illustrate the relative constancy of the duration of DNA synthesis and the damped second wave of labelled mitosis. One can estimate cell cycle times in these malignant melanomas at roughly 47 hours. The information presented earlier suggested that the doubling time of tumors in human subjects was approximately 50 days, but the studies on persons with breast cancer and malignant melanoma suggest that

November

the generation time of a tumor cell in a human subject is about 50 hours. If these estimates are correct, obviously a great number of influences and constraints other than the duration of the cell cycle operate to regulate the behavior of the tumor mass in man. The two most important influences are the growth fraction of the mass and cell death. The growth fraction is the number of cells in a tumor mass that are actively participating in the cell cycle. In the past, we have thought conceptually of a mass of tumor containing billions of cells all growing slowly. In actuality, in a tumor mass only a fraction of cells are rapidly proliferating; the remainder of the cells are out of cell cycle. The published information in man on solid tumor kinetics in vivo is presented in Table II [ 5,8- 121. First, it is remarkable that the duration of the DNA synthesis phase (Ts) is relatively similar for a number of tumors in human subjects, ranging from a low of 10 hours to a high of 31 hours. These are small varuations relative to the tremendous variation in doubling times of tumor masses (Figure 5). The length of the cell cycle in tumors varies from a little over half a day to perhaps three and a half to five days. Nevertheless, it is apparent that

1977

The American

Journal

of Medicine

Volume 63

777

COMBINED CLINICAL

AND BASIC SCIENCE SEMINAR

100 -

In

80-

f

60-

s

20

v, ?

I Olll

Tq =

I

14.0 hrs



’ 4

1

’ 8





I2





16





20



24







28

HOURS Figure 9. A per cent labeled mitosis curve of leukemia L 12 10 cells in vivo after treatment with BCNU.

CASE 1

CASE 2

L I -21.5 M. I. - 0.94

100

CASE 3

L I.--210 M. I. - 0.86

L.I.M.l.-

r

80 c

250 I.0

PLM

0

IO

20

30

40

HOURS Figure 70. PLM curves performed in three patients with carcinoma of the breast. Tumor analysed was located in multiple metastatic skin nodules.

L I =21-25X G F= 4%57%

PLM .

0

5

IO

I

I

15

20

.

.

.

.

.

I

I

25

30

35

40

45

50

55

60

65

HOURS

Figure 11. Breast carcinoma-three obtained from studies in Figure 10.

778

November 1977

patients. A computer analysis of data from PLM curves

The American Journal of Medicine

Volume 63

NEW ASPECTS OF MODERN CANCER CHEMOTHERAPY

TABLE II

Solid Tumor Kinetics in Vivo Growth T, (hours)

Tumor Ovarian

carcinoma

T, (days)

31

Patients

Fraction

Cell Loss

(%)

(%)

5

Studied Source

70

Clarkson

(ascites) Stomach

1965 26

carcinoma

3.8

95

et. al.

1965 19

Basal cell carcinoma

3

30

95

Frindel

20

et. al

epithelioma

Weinstein,

Squamous

12

1.5

IO

carcinoma

40

.6

90

Frindel

50 25

3-4

-70

2

33-68

Young, 1970

Breast

carcinoma

22

NOTE: Tg = generation

time;

2.1

T, = cell cycle

48-57

et al 1121

DeVita 1131

Young, DeVita 1970 1131

time

doubling times ranging from 10 days to a thousand days cannot be explained by this degree of variation in phases of the cell cycle or generation time. The two factors which are the major determinants in the speed with which tumors grow are the growth fraction and cell death. There is a marked variation in the growth fraction of these tumors in man, ranging from 25 per cent to almost 95 per cent in some ascites tumors (Table II). There is a tremendous amount of cell loss which occurs in human subjects with tumors. One does not

usually think of constant cell death in cancer; in fact, one thinks of the reverse. However, it is now clear that small alterations in cell loss can make a dramatic impact on the over-all doubling time of a tumor in man. Many factors can alter the behavior

of tumor growth

[ 13-201. Some of the major ones are listed in Table III. For example, the particular

cytotoxic chemotherapy, depending upon tumor and the particulair drug, can alter

the generation time and the growth fraction of tumors. Hormones appear to alter the kinetics of tumor growth by altering the growth without changing the generation

Factors Effecting the Kinetics of Tumor Growth

TABLE III

Primary Perturbing Cytotoxic

1111

Shirakawa 1970

24

9

et al.

1969 25

Frost 1101

1968 191 Bennington

cervix Melanoma

2

191

1970 Epidermoid

2

181

1968

(skin)

2

181

Clarkson

(ascites)

(no.)

et al.

Influence

chemotherapy

Growth

Fraction

t

Kinetic

Effect Example

Cell Cycle Time

t

Reference Young.

BCNU-L1210

t

Hormones

0

DeVita

1970 1131: Frei et al. 1964 1141

vincristine

Simpson-Herren,

Estradiol-

1970

1151

Tubiana 1971

/ 161

progesterone; mammary

Griswold

adenoca

mouse Roentgenogram

Skin tumors

Vascular

Mouse

supply

in man

Tannock

mammary

1968 Oxygen

Tannock.

tension

1171 Steel

1970 Immunologic Contact

cytolysis

inhibition

end-stage

and

growth

November

1977

Ehrlich

ascites

tumor

Ehrlich

ascites

tumors

Ehrlich

solid tumors

The American

DeCosse. Gelfant 1968 / 181 Lala. Patt 1966 1191 Lala 1970

Journal of Medicine

1201

Volume 63

779

COMBINED CLINICAL AND BASIC SCIENCE SEMINAR

TABLE IV

Classification of Chemotherapeutic Agents by Kinetic Effects Examples

Classification Cell cycle specificproliferation dependent Cell cycle specificless proliferation dependent Cell cycle nonspecificproliferation dependent Cell cycle nonspecificless proliferation dependent

Hydroxyurea,

ARA-C

5-FU, methotrexate

Cytoxan, actinomycin BCNU Nitrogen mustard

D,

NOTE: ARA-C = cytosine arabinoside; 5FU = 5-fluorouracil.

time of the tumor. X-ray therapy alters both the generation time and the growth fraction. Alterations in oxygen tension and vascular supply change the growth fraction rather than the generation time of growing tumors. Immunologic mechanisms seem to alter both generation time and growth fraction. Out of the background of basic cellular kinetics, certain concepts of chemotherapy have developed which allow a more learned approach to its use in the treatment of cancer. In tumor systems in animals, the survival of an animal is inversely proportional to the number of tumor cells implanted or to the size of the tumor at the time the study is initiated. Chemotherapeutic agents are effective by virtue of first order kinetics, i.e., they kill a constant fraction of the cells exposed to the chemotherapeutic agent, rather than a constant number of cells. This fact has several important implications. A single chemotherapeutic agent used as drug treatment for a tumor weighing 1 g (log cells) would produce 90 per cent cell kill but would only decrease the population of cells one log to 1O8.Thereafter, the tumor regrows at a relatively constant rate after exposure to the drug and kills the host a little bit later, but not too much later. It is only when a very large cell kill (99.999 per cent) is produced that one finds a significant delay in the regrowth of the tumor mass and prolongation of survival. This is one of the reasons clinicians have resorted to multiple drugs or combination chemotherapy and to

TABLE V

Site of Maximal Cycle

Portion of Cell Cycle Gl Early S Late S ‘32

M

780

November 1977

Drug Action in Cell

Drugs Actinomycin D Hydroxyurea, ARA-C 5-FU, methotrexate Adriamycin. daunomycin Bleomycin radiation Vincristine. vinblastine

The American Journal of Medicine

intermittent courses of chemotherapy to accomplish the tremendous amount of cell kill required to produce tumor regression and cure. Obviously, there are diseases in which single agents can produce this magnitude of cell kill without requiring combinations. People with tumors, such as Burkitt’s lymphoma, and choriocarcinoma can be cured with single drug therapy, but most human subjects with tumors are intrinsically less sensitive, and more sophisticated therapeutic technics are required to achieve adequate tumor control. When one achieves 90 per cent cell kill, in a solid tumor, there is still little prolongation of survival; the slow clearing of lethally-injured cells in a solid tumor prevents one from seeing the magnitude of the cell kill achieved. Only when the cell population is reduced to relatively low numbers (10’ to lo4 cells) can one achieve prolonged control or cure. Obviously, cell masses of 10’ to lo4 cells are below the size of clinical detection. This is the basis for using adjuvant chemotherapy in early stages of disease, when subclinical amounts of cancer cells are suspected. Chemotherapeutic agents have complex mechanisms of action and alter cells in a great variety of ways. It is now clear that different agents have different sites of action in the cell cycle. Their effectiveness is also a function of the proliferative capacity of the tissue involved. These observations have led to the classification of chemotherapeutic agents on the basis of their cell cycle specificity (Table IV). The cell cycle nonspecific agents are chemotherapeutic agents that kill in all phases of the cell cycle and are not too dependent upon proliferative capacity of the cells. One of the best examples of this class is nitrogen mustard, an alkylating agent which is effective against a number of solid tumors including those with relatively low growth fractions. This class of agents have found their utility in solid tumors in which the proliferative capacity is relatively low. In contrast, there are cell cycle specific agents which depend for their action on the proliferative capacity and on a particular phase of the cell cycle. An example of this class would be cytosine arabinoside (ARA-C), a drug that inhibits DNA polymerase. This class of agents kills in only one portion of the cell cycle, and cells not in that phase of the cell cycle will not be injured by the drug. This class of drugs tend to be most effective against tumors with relatively long S phases, and those tumors in which there is a relatively high growth fraction and a relatively rapid proliferative rate. The use of the cell cycle specific agents in slowly growing solid tumors is usually not successful as these agents are generally more injurious to the rapidly proliferating normal renewing tissues than the tumor. In contrast, there are cell cycle specific agents which act in a particular phase of the cell cycle but seem to

Volume 83

NEW ASPECTS OF MODERN CANCER CHEMOTHERAPY

be less proliferation dependent. Primary examples are 5-fluorouracil (5-FU) and methotrexate. This initially seems curious because they are dependent upon the cell cycle yet they are not too proliferation dependent. The likely explanation is that each of these drugs has other mechanisms of action, including inhibition of RNA and protein synthesis, and affect the cell in other ways in addition to impairing its ability to replicate DNA. Lastly, there are cell cycle nonspecific agents which seem to be fairly proliferation dependent. Although they act on all phases of the cell cycle, they seem to be most active in those populations of cells that are rapidly proliferating. Examples of this group of compounds are cyclophosphamide, actinomycin D and BCNU (bis(2 chloroethyl)-1-nitroso-urea). In addition to cycle and proliferation sensitivity, chemotherapeutic agents may exert a greater effect on a particular phase of the cell cycle. Examples are listed in Table V. Actinomycin D, for instance, seems to primarily effect cells in the early S phase. Adriamycin and daunomycin seem to exert their major effect in the late S phase. The G2 population of cells seems most sensitive to bleomycin and radiation therapy, and cells in mitosis seem most sensitive to agents which act in part as mitotic spindle poisons, namely, the vinca alkyloids-vincristine and vinblastine. From Tables IV and V it is apparent that one can characterize chemotherapeutic agents with regard to their site of specific action in the cell cycle and their dependence, or lack of dependence, on proliferative characteristics. This has led to an effort by chemotherapists to utilize this knowledge to design better sequences of chemotherapy. One can now consider using combinations of drugs with different characteristic sites of activity or mechanisms of action to induce a marked regression of tumor. If one administers a cell cycle nonspecific agent which produces a two log kill in a host bearing a tumor mass with log cells, and no further therapy is given, the tumor would simply regrow. Death would be delayed only temporarily. If a cell cycle nonspecific agent produces this amount of cell kill, theoretically, a cell cycle-sensitive agent would be effective against those new cells coming into the cell cycle. Simply by altering, or using in sequence, cell cycle-specific and nonspecific agents, one hopes to eventually produce consistent repetitive log-kill in tumors, and finally, after using combinations and proper sequences of such agents, one may produce the magnitude of log kill required to induce cure. Several recent advances have occurred in the field of tumor cell kinetics which promise to provide additional new and important information to clinicians. One of the major problems in the past has been that information on tumor kinetics in man has been very difficult

TABLE VI

Type Conventional High specific activity autoradiography High speed scintillation autoradio-

graphs

Autoradiography Technics Specific Activity 3” - TdR

Conditions

1.9 (Ci/ mmol) 6.0

4’ AR10 stripping film 37’ NTB-2 emulsion

40-60

-85’C PPO-POPOP,NTB3 nuclear traclr

emulsion

Time To Completion 8 wk 24-48 hr

4-5

hr

_.___~

NOTE: SH-TdR = Vitiated; PPO-POPOP, NTB3 = ARlO.

to generate and very time consuming. Conventional autoradiographic technics using tritiated thymidine require at least eight weeks before a study may be completed. Now there are much more rapid technics. High specific activity autoradiography or, more recently, the high speed scintillation autoradiography, using very large doses of tritiated thymidine, now enable an investigator to obtain kinetic information in a clinical situation in 4 to 5 hours [22] (Table VI)‘. This allows the generation of information rapidly enough to be clinically significant. Finally, a very new technic called flow microfluorometry (FMF) [23] promises to provide a whole new mass of kinetic information of clinical usefulness. The technic involves the rapid flow of a stream of tumor cells through a lazor beam. Using a number of different cell fluorescence technics, one can kinetically characterize a population as large as 50,000 to 100,000 cells. Using DNA content determinations, one can define the stage of the cell cycle for each of the cells analyzed (Figure 12). The first peak includes cells with a haploid DNA content and, therefore, cells in the G, phase or the resting phase. There are a number of cells that are between the G, and G2 phase in which there is a diploid DNA content. These are the S phase cells. One can perform such an analysis in very rapid sequence after chemotherapeutic exposure and, for the first time, the kineticist now has a technic which will allow the sequential study of alterations of kinetics induced by chemotherapy. One can also sort cells, so if laboratory investigations required 5,000 tumor cells, all of which were in the G2 phase of the cell cycle, one could accomplish this with FMF. Further for the first time the sequential impact of kinetic alterations induced by particular chemotherapeutic agents can be studied with a large number of cells. Hopefully, these new technics will provide more useful information that will aid clinicians in structuring chemotherapy to events of the cell cycle, and understanding the proliferative characteristics of normal and

November 1977

The American Journal of Medicine

‘Volume 63

761

COMBINED CLINICAL

AND BASIC SCIENCE SEMINAR

c

-

G, Cells

,I

0

I 6N

II

o-1

4N

2N DNA

J

Content

Figure 12.

Typical flow microfluorometry data; abscissa is DNA content and ordinate is number of cells. A DNA distribution pattern of an exponentially growing cell population. Cells in particular phases of the cell cycle are noted.

tumor tissues. This will surely result in more appropriate chemotherapeutic technics with less toxicity and more therapeutic efficacy. Dr. James F. Holland: Acute leukemia is a model tumor occupying the fulcrum between experimentally induced tumors in animals and the large numbers of other types of cancer in human subjects. This is true, at least in part, because one can recognize a therapeutic end point rapidly, and the tumor can easily be sampled and roughly quantified by a simple, finger-stick or marrow aspiration. The first patient who was effectively treated by chemotherapists was one with acute lymphocytic leukemia of childhood. The Acute Leukemia Group B” has undertaken a number of prospective trials in the past two decades. In the first study, no child lived more than 15 months. Successively as we have adopted improved treatments, discovered in prior programs, there has been increasing survival [24,25,26]. The strategy of treatment requires reduction of the immediate toll of death from bleeding, infection and

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1976.

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other problems that occur in the florid leukemic state by the induction of remission. Using prednisone in combination with vincristine, with vincristine plus daunorubicin, or with 6-mercaptopurine, there is essentially no difference in the frequency with which one can induce remission in children. In several studies involving more than 2,000 children, the remission induction rate is approximately 85 per cent. There are other ways to measure the impact of drugs on the leukemic process, however, one of which is how long the leukemic cells remain absent. We have not found induction regimens which themselves lead to such lethal effect on leukemic cells that a prolonged disease-free state or, indeed, cure results. It is necessary to undertake an intensification or maintenance phase of treatment using drugs that, because of the change of cellular kinetics resulting from uncrowding, now have a greater impact. In our study of 1968, five choices of treatment at random [25] led to 32 different regimens. It would have taken a very high degree of prescience to have picked which arm would have been best, ahead of time. The results of treatment with vincristine and prednisone in

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combination were not improved upon by the administration of five days of asparaginase or by administering daunorubicin during induction. lntrathecal methotrexate administration was indispensable in treating the subclinical leukemia of the central nervous system, however, reducing to 25 per cent the frequency of this manifestation of the disease which had appeared in 60 per cent of untreated children by six years [ 25,261. Of the 32 regimens, the best were those in which patients were given vincristine and prednisone (with or without daunorubicin) during induction of remission and received intrathecal methotrexate. Among the four regimens of maintenance, 60 per cent of the children who received 6-mercaptopurine and methotrexate plus vincristine and prednisone (the two drug reinforcement regimen) are still, by actuarial calculations, in complete remission at seven and a half years. Remission in 15 children of an initial 32 is past five years: half of these children have had their treatment discontinued at random. Surely, among these children, in a disease in which 20 years ago none of our patients lived 15 months, there are some who are cured of acute lymphocytic leukemia [27]. The addition of daunorubicin in reinforcement to the exact same regimen sharply compromises the good therapeutic effect [ 261. Whereas the single drug regimen of methotrexate alone is also effective (nearly half the children are alive at six years), again an addition of daunorubicin in maintenance compromises its effect. The lesson is that more drugs are not necessarily good. The drugs that are used, their doses and their schedules are all of major consequence. In 1971 we accepted the data on this apparently optimal combination chemotherapy in remission and worked on other parts of the program. We used the same maintenance drugs in short courses at high dose in an intensification attempt to kill the leukemic cells, allowing normal cells to recover. Furthermore, we were unsatisfied with our own data on asparaginase since we had other data in children with advanced leukemia demonstrating that the drug was active. We established a more critical design than in 1968. Asparaginase was given over a period of 10 days before, during or after the administration of vincristine and prednisone, or not at all. This seemingly pedestrian question of when in the time sequence of treatment should a particular drug be given, produced a highly significant difference. Asparaginase given after the vincristine and steroid led to a significantly longer remission duration than when it was given before or during the administration of the other drugs [28]. I believe this observation is the type of stepwise progression that will be made in finding cures for acute lymphocytic leukemia. I do not expect single magnificent breakthroughs, but rather these kinds of component additions.

November

Acute lymphocytic leukemia is a highly dramatic success story because salvage of a child’s life means potentially a whole lifespan ahead. Adults in whom acute leukemia develops have an extraordinarily high mortality. The CALGB has been working in acute myelocytic leukemia for 20 years, but it was only about 10 years ago that concepts evolved in acute lymphocytic leukemia became applicable leukemia. Important contributions

to acute myelocytic came from access

to two new drugs that became available at that time, cytosine arabinoside and daunorubicin. In a three-way comparison of nearly 500 patients, Wiernik et al. [29] found that daunorubicin therapy produced complete or partial remission in 49 per cent of adults, cytosine arabinoside plus thioguanine therapy in 52 per cent and the combination of all three in 57 per cent. Similar studies for the period from 1967 to 1972 are characterized by slowly increasing survival. The time frame is in months, however, not years and although a tenfold improvement in two year survival appeared, it still was only from 2 per cent to 20 per cent [ 24.1. Looking at the results obtained with cytosine arabinoside and daunorubicin, studied many times in a regimen of five days of Ara-C and two days of daunorubicin, it became obvious that in virtually every instance in which induction was successful, it took a second course of drugs to induce remission. It seemed logical that one should incorporate the additional required drug into the first course of treatment. This would attain the necessary marrow aplasia early and diminish the time at risk for death from hemorrhage and infection during delayed aplasia. The extraordinary mortality from the disease occurs in the first month from these causes in some 40 per cent of patients [ 241. We did initiate a study in ,which Ara-C was given for seven days and daunorubicin for three days instead of the five and two day regimen [ 301. After the pilot study of this program, it was extrapolated as a comparative study to the full CALGB. Because the prognosis in patients over 60 years of age is even worse than in younger leukemic subjects, stratification by age was made for those under and over 60 years. The same 50 odd per cent remission rates was seen for those who received the standard five and two day regimen, but 70 odd per cent remission rates were observed for those who received the seven and three day regimen [31]. For failure to achieve remission on the first seven and three day course, a repetition of a five and two day course is possible. We have subsequently studied patients over the age of 60 years and have found that, indeed, the more intensive treatment is also more advantageous for them. The death rate is lower and the remission rate higher (55 per cent) with more intensive chemotherapy ]311. Patients

1977

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the disease, per se, but from the disorder that it causes in normal body systems, specifically, failure of normal bone marrow function. Impaired megakaryocyte growth and platelet production, and death from thrombocytopenic hemorrhage are common. Platelet transfusion has become an absolute requirement in the resourceful management of patients with acute myelocytic leukemia. Without this capability, one should refer patients to hospitals in which it exists. Bacterial infection requires a good laboratory, and early and intelligent antibiotic use. The transfusion of granulocytes makes a significant difference. Djerassi and Farber developed an extremely simple nylon filtration system [32], which we proved in a randomized clinical trial to be of value in preventing death of patients with acute leukemia from sepsis [33]. This is an important asset for the blood bank of community hospitals, let alone medical centers. Transfusion of granulocytes provides the kind of temporary support during chemotherapy of hematologic neoplasia that is the equivalent of the intensive care unit with its electronic and mechanical assistance for other areas of crisis medicine. It would be highly advantageous if one could prevent exogenous infection. In barrier isolation rooms with laminar air flow coming from the head wall down over the patient, where the nurse is completely excluded from the room, entering via a space suit, it is possible to cut down appreciably on the acquisition of airborne infections and pneumonias [34]. Now that a majority of patients with acute myelocytic leukemia enter remission, technics of therapy for use during the remission period are more urgently needed. In 1969, Mathe and his colleagues [35] adapted the original observations of Halpern that BCG could stimulate animals to augment their resistance to tumors. They inoculated children who had acute lymphocytic leukemia by flooding a scratch pattern 1 m long with Pasteur Institute BCG. Some children also received irradiated allogeneic leukemic cells. Some 35 per cent of these children have an extraordinarily long duration of remission and survival without evidence of disease, despite the fact that the chemotherapy had been suboptimal. Unfortunately, the observation has not been confirmed by any other group working with children with acute lymphocytic leukemia, perhaps because they have not identically reproduced the experiment. It became apparent later that acute myelocytic leukemia might be a far more suitable disease in which to undertake immunotherapeutic investigation. In mixed leukocyte cultures it has been domonstrated that the normal lymphocytes of a patient with acute myelocytic leukemia can be stimulated by his own myeloblasts. Furthermore, autologous myeloblast extracts elicited delayed cutaneous hypersensitivity in patients with acute myelocytic leukemia.

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For a number of years Dr. George Bekesi and I, among many others, have been working on ways to augment a host’s response against his own tumor. The most effective way to do this, we have found, is to incubate a tumor in the neuraminidase of Vibrio Cholerae, as others had done before us [36], but without quite the same incubation conditions (and without quite the same success) [ 371. Neuraminidase removes the outer layer of n-acetyl neuraminic (often called sialic) acid from the cell surface, exposing new molecular aspects and changing electrophysiologic conditions. In some way, not as yet clear, this eliminates the leukemogenicity of otherwise virulent cells. In syngeneic animals in which leukemia develops after the transplantation of a single leukemic cell, 10 million cells can be transplanted which have been incubated in neuraminidase without any evidence of leukemia arising [38]. One month later, however, these inoculated animals have become immune to the leukemia, and then they are able to withstand a challenge of 100,000 virulent cells. These were protection experiments, but they can be translated to the therapeutic model. Animals with established transplanted leukemia L1210, the classic tumor that has been used for experimental chemotherapy, exhibit 20 per cent survival after treatment with methyl CCNU, an experimental drug. If on day 3 or 6 or 9, chemotherapeutically treated animals are also given one injection of neuraminidase-treated leukemia L 12 10 cells, which are nonvirulent but highly immunogenic, 90 per cent survival results [39]. This immunity is quite specific; survival after transplantation of other carcinomas or sarcomas is not altered by the inoculation of neuraminidase-treated L1210 leukemic cells. Nonspecific immunotherapy with BCG or MER (the methanol extraction residue of BCG), although not as active, per se, does add to the effects of neuraminidase-treated cells. But leukemia L1210 is a transplanted tumor, and the possibility of genetic drift of the serially transplanted tumor away from the repetitively bred host could have occurred, giving the host a specific advantage. So we began to work with an inbred strain of AKR mice. In these mice leukemia develops spontaneously starting at about the sixth month of age, and by the 14th month, 95 per cent of the colony is dead of leukemia. The thymus, lymph nodes and spleen enlarge, leukemic cells appear in the blood, and lymphocytic leukemia can be diagnosed, just as in a child with leukemia. Half of these animals are dead in about 17 days after clinical diagnosis, with characteristic lymphoid neoplasm due to the Gross virus. When AKR leukemia is clinically evident, neuraminidase-treated AKR leukemic cells alone have no impact on survival from the disease without chemotherapy. When vincristine and a steroid are used as che-

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‘igure 13. Remission duration in adult patients (less than 60 years of age) wit1 acute myelocytic leukemia who were treated with cytosine arabinoside and daunorubicin to induce remission. Cyclical therapy for remission maintenance included monthly pulsed doses of cytosine arabinoside and thioguanine, cyclophosphamide, CCNU and daunorubicin. In the group receiving neuraminidase-treated myeloblasts remission duration is significantly longer than in the group treated with chemothera.py alone. Solid line = cells; broken line = control.

motherapy, they cause modest extension no cures. When the drugs are followed

in survival but by neuramin-

idase-treated AKR leukemic cells, a combined moimmunotherapy, however, extended survival

cheand

long-term disease-free status resulted in 20 to 40 per cent of the animals [40]. We have studied another leukemia in animals, E2G, caused by the Gross virus, which arises in C57 black mice, genetically dissimilar from AKR animals. Because E2G cells will not transplant in AKR mice, they could be tested live and were not found to be immunotherapeutic. After neuraminidase treatment, however, these virally infected cells also augmented immunity and produced major therapeutic improvement, suggesting that characteristics of surface antigens related to viral infection might be important in eliciting the immunologic response. This made it possible for us to justify using allogeneic leukemic cells in man, since we were aware of the nearly universal presence of reverse transcriptase in leukemic cells in man. This enzymatic footprint of viral activity suggests the possibility of common viral neoantigens on the cell surface of leukemic cells in man

1411. Leukemic myeloblasts are taken from volunteer patients with high peripheral counts, are purified to exclude granulocytes, frozen in nitrogen vapor and thawed at appropriate times for immunotherapy; the

viable cells are separated by flotation on human serum albumin. They are then treated with neuraminidase, washed and injected intradermally [4’1]. We use lOlo viable neuraminidase-treated cells injected intradermally in approximately 50 loci. These cells elicit delayed cutaneous hypersensitivity responses which sometimes persist for weeks. The intensive cyclic maintenance chemotherapy that we have used was not exceptionally successful and produced a rather short remission duration. The chemotherapeutic regimen was markedly improved by the immunization, however. Remission duration for thse chemoimmunotherapy group is better than that for any other treatment in a concurrent controlled trial that has been reported to date (Figure 13). The neuraminidase-treated cell immunization program is expensive, complex and dependent on the collection and storage of leukemic myeloblasts. We have looked for alternative simpler means of immunization. We chose to study MER, the methanol extraction residue of phenol killed BCG, choosing to avoid infection with live BCG because one cannot control the exact dose of the multiplying organism in the recipient. MER was discovered by David Weiss [42]. He was trying to immunize guinea pigs against tuberculosis with it, when an epidemic of pasteurellosis spread among his colony, attributed to pigeon droppings on cabbage. The disease

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was highly lethal except to the guinea pigs which had been immunized with MER. He recognized immediately that MER provided nonspecific immunity and confirmed this for several other bacterial organisms. He then studied animals with transplanted tumors and showed that MER caused major augmentation of host immunity against tumors. In mice in which spontaneous breast cancers develop, MER therapy causes a delay in the appearance of tumors [42]. Thus MER was thought to be an ideal immunostimulant for study. It was nonviable, precisely measurable and broadly effective. We have confirmed its activity in chemoimmunotherapy of transplanted and of spontaneous leukemias in mice. Thereafter, we have treated patients with acute myelocytic leukemia with chemoimmunotherapy using the same chemotherapy as for the neuraminidase-treated cell program, but with MER as immunotherapy instead. In a small comparative series, remission duration and survival are both more than twice as long in the group given MER as in the control group receiving identical chemotherapy [43]. We are hopeful that this kind of immunotherapy may prove as effective and cheaper than neuraminidasetreated cells. A broad clinical trial is currently in progress in the Cancer and Leukemia Group B. If the trial is as successful as the pilot studies, my expectation is that we will have substantially advanced

the practice of hematology for this catastrophic disease through utilizing a technic of study. The technic is the comparative, prospective, randomized trial in which we discipline ourselves to seek a specific answer. This technic is not new, of course, but it is a powerful tool of clinical investigation, too often overlooked, that has been used for a long time. In his classic treatise, “Of the Scurvy,” Sir James Lind reported: “I took 12 patients with the scurvy on board the Salisbury at sea. The cases were as similar as I could have them. They lay together in one place and had one diet common to them all. To two of them were given a quart of cider a day. To two, an elixir of vitriol, to two, vinegar, to two, oranges and lemons, and to the remaining two, an electuary . . recommended by a hospital surgeon. The most sudden and visible good effects were perceived from, the use of the oranges and lemons. One of those who had taken them being at the end of six days fit for duty. The other was appointed nurse to the rest of the sick” [44]. I think the concept of proposing problems in medicine, and setting about to solve them, will lead to more progress than being crisis-oriented, deciding what to do for a patient with a particular disease only after he shows up. The investigative approach has, despite its limitations, been actively applied for some years in cancer medicine, and now we are harvesting some of the fruits.

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Goss RJ: The strategy of growth, Control of Cellular Growth in Adult Organisms (Teir H, Rytomaa T, eds), New York, Academic Press, 1967, p 3. Collins VP, Loeffler RK. Tivey H: Observations on growth rates of human tumors. Am J Roentgen01 76: 988, 1956. Charbit A, Malaise EP, Tubiana M: Relation between the pathological nature and the growth rate of human tumors. Eur J Cancer 7: 307, 1971. Howard A, Pelt SR: Nuclear incorporation of P-32 as demonstrated by autoradiographs. Exp Cell Res 2: 178, 1951. Young RC, DeVita Vt; cell cycle characteristics of human solid tumors in vivo. Cell Tissue Kinet 3: 285, 1970. Barrett JC: A mathematical model of the mitotic cycle and its application to the interpretation of percentage labeled mitoses data. J Nat Cancer lnst 37: 443, 1966. Steel GG, Hanes S: A technique of labeled mitoses analysis by automatic curve fitting. Cell Tissue Kinet 4: 93, 1970. Clarkson B, Ota K, Ohkita T, et al.: Kinetics of proliferation of cancer cells in neoplastic effusions in man. Cancer 18: 1189, 1965. Frindel E. Malaise E, Tubiana M: Cell proliferation kinetics in five human solid tumors. Cancer 22: 611, 1968. Weinstein CD, Frost P: Cell proliferation in human basal cell carcinoma. Cancer Res 30: 724, 1970. Bennington JL: Cellular kinetics of invasive squamous carcinoma of the human cervix. Cancer Res 29: 2187, 1968. Shirakawa S, Lute JK, Tannock IF, et al.: Cell proliferation in human melanoma. J Clin Invest 49: 1188, 1970. Young RC, DeVita VT: The effect of chemotherapy on the growth characteristics and cellular kinetics of leukemia

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L1210. Cancer Res 30: 1789, 1970. Frei E, Whang J, Scoggins RB, et al.: The stathmokinetic effect of vincristine. Cancer Res 24: 1918, 1964. Simpson-Herren L, Griswold DP: Personal communication (Quoted by Tubiana M). The kinetics of tumor cell proliferation and radiotherapy. Br J Radio1 44: 325, 197 1. Tubiana M: The kinetics of tumor cell proliferation and radiotherapy. Br J Radio1 44: 325, 1971. Tannock IF: The relation between cell proliferation and the vascular system in a transplanted mouse mammary tumor. Br J Cancer 22: 258, 1968. DeCosse JJ, Gelfant S: Noncycling tumor cells: mitogenic response to antilymphocyte serum. Science 162: 698, 1968. Lala PK, Patt HM: Cytokinetic analysis of tumor growth. Proc Nat1 Acad Sci USA 56: 1735. 1966. Lala PK: Personal communication (Quoted by Tubiana M in

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Skipper HE: Cancer chemotherapy is many things: G. H. A. Clowes’ memorial lecture. Cancer Res 31: 1173, 1971. Durie BGM. Salmon SE: High speed scintillation autoradiography. Science 190: 1093, 1975. Tobey RA. Crissman HA: Preparation of larae auantities of synchronized mammalian cells in late G& the pre-DNA replicative phase of the cell cycle. Exp Cell Res 75: 460. 1972. Holland JF: The acute leukemias. Textbook of Medicine, 14th ed (Beeson PB, McDermott W, eds), Philadelphia, W.B. Saunders Co.. 1975. D 1485. Holland JF. Glidewell 0J:‘Chemotherapy of acute lymphocytic leukemia of childhood. Cancer 30: 1480, 1972. Holland JF. Glidewell OJ: Oncologists’ reply: survival expec-

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tancy in acute lymphocytic leukemia. N Engl J Med 287: 769, 1972. CALGB Minutes, March, 1976, Dr. Marise Weil, Study Chairman Unpublished data. Jones B, Holland JF, Glidewell OJ: Lower incidence of CNS leukemia using dexamethasone instead of prednisone for induction in acute lymphocytic leukemia. Proc Am Assoc Cancer Res 16: 183, 1975. Wiernik PH. Glidewell OJ, Holland JF: Comparison of daunorubicin with cytosine arabinoside and thioguanine, and with a combination of all 3 drugs for induction therapy of previously untreated AML. Proc Am Assoc Cancer Res 16:82, 1975. Yates JW, Wallace HJ Jr, Ellison RR, et al.: Cytosine arabinoside and daunorubicin therapy in acute myelocytic leukemia. Cancer Chemother Rep 57: 485, 1973. CALGB Minutes, March 1976. Dr. Kanti Rai, Study Chairman. Unpublished data. Djerassi I, Farber S: Control and prevention of hemorrhage platelet transfusion. Cancer Res 25: 1499, 1965. Higby DJ, Yates JW, Henderson ES, et al.: Filtration leukophoresis for granulocyte transfusion therapy, clinical and laboratory studies. N Engl J Med 292: 761, 1975. Yates JW, Wallace HJ Jr, Ellison RR, et al.: Cytosine arabinoside (NSC #63878) and daunorubicin (NSC #83142) therapy in acute myelocytic leukemia. Cancer Chemother Rep 57: 485, 1973. Mathe G, Amiel JL, Schwarzenberg L, et al.: Active immunotherapy for acute lymphoblastic leukemia. Lancet 1: 697, 1969. Bagshawe KD, Currie G: lmmunogenicity of L1210 murine leukemia cells after treatment with neuraminidase. Nature

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218: 1254, 1968. Bekesi JG, St. Arneault G, Holland JF: Increase of leukemia L1210 immunogenicity of Vibrio cholerae neuraminidase treatment. Cancer Res 31: 2130, 1971. Bekesi JG, St. Arneault G, Walter L, et al.: lmmunogenicity of leukemia L1210 cell after neuraminidase treatment. J Natl Cancer lnst 49: 107, 1972. Bekesi JG, Holland JF: Combined chemotherapy and immunotherapy of transplantable and spontaneous murine leukemia in DBA/P and AKR mice. Investigation and stimulation of immunity in cancer patients. Recent Results in Cancer Research, Vol 47 (Mathe G, Weiner R, ed), Berlin, Heidelberg, New York, Springer-Verlag, 1974, p 357. Bekesi JG, Roboz JP, Holland JF: Immunotherapy with neuraminidase-treated murine leukemia cells after cytoreductive therapy in leukemic mice. Modulation of host immune resistance in the prevention and treatment of induced neoplasias. Fogarty International Center Proceedings 28: 219; 1977. Holland JF, Bekesi JG: Immunotherapy of human leukemia with neuraminidase modified cells. Med Clin North Am 60: 539, 1976. Weiss DW: Nonspecific stimulation and modulation of the immune response and of states of resistance by the MER fraction of tubercle bacilli. Natl Cancer lnst Monogr 35: 157, 1972. Cuttner J, Holland JF, Bekesi JG, et al.: Chemoimmunotherapy of acute myelocytic leukemia. Proc Am Sot Clin Oncology 16: 264, 1975. Lind J: A treatise “Of the Scurvy,” containing an inquiry into the nature, causes and cure of that disease, 1st ed, London, Edinburgh, 1753.

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