[CANCER RESEARCH 30, 1883—1897,June 1970]

eradicate the 1 @8(or less) cells which survive remissioninduction—without unacceptable toxicity—is a problem westill face.

During this period of stepwise advancement in therapy ofleukemias, our knowledge of normal leukocyte kinetics andwhite blood cell cycle characteristics has also expanded.Leukemia is no longer considered simply as a disordercharacterized by rapid and wild proliferation of the myeloidor lymphoid series (i.e., a markedly shortened generationtime of the dividing cells), although the much used transplantable mouse leukemias and human acute leukemias dohave total population doub!ing times which clearly are muchshorter than the doubling times of most solid tumors whenthese are measurable.

Am overall dynamic difference between normal and leukemic leukocyte populations that is consistent, and whichprovides the base line for most of our present perspective,lies in the fact that the normal leukocytes are in a steadystate (cell production cell death), whereas untreated leukemic cell populations usually are not in a steady state (cellproduction is greater than cell death). If we start from thispremise and the evidence that the generation time or mediancycle time of dividing cells has not changed significantly, wemust assume that the biochemical lesion(s) responsible forleukemia results in: (a) a relative increase in leukemic cellproduction due to a higher fraction of the viable populationin a proliferative state; or (b) a relative decrease in leukemiccell death and resorption; or (c) both.

At the onset, it might be of value to review briefly thecurrent concepts of leukocyte kinetics. For more detailedreviews, the reader may consult several recent publications(11, 15, 36, 56).

Nonnal Myeloid Series

There is a great deal of experimental data concerning thebehavior of hematopoietic stem cells in animals, but in manthere is relatively little information in this important areaand stem cells have not been clearly identified.

In the normal individual, there is an orderly progression(differentiation) of the myeloid series in the bone marrowbeginning with the earliest recognizable precursor, the myeloblast, through the promyelocyte, myelocyte, metamyelocyte,band, and finally the mature polymorphonuclear leukocyte.Data on the cell cycle time of the parent stem cells arerelatively sparse and indirect, but their generation time is

SUMMARY

There have been several recent comprehensive reviews ofnormal and leukemic leukocyte kinetics in man (15., 36, 56),but this review purposely has been limited in scope. It iswritten not as much for investigators who are engaged instudy of leukemic cell or normal cell kinetics as for thosewho are primarily involved in design of therapeutic trials,experimental and clinical.

The purpose of the review is to bring together some“kinetic―information which appears to have special significance to therapy. Such information on human and animalleukemic cell populations and certain normal cell populationsis spread so widely throughout the scientific literature thatits consideration in an overall context is very difficult. Thusattempts at interdigitation (e.g., “kinetic―versus therapeuticresponse data), in order to work toward unifying conceptsand more effective applications, are both difficult and timeconsuming for anyone who wishes to try. We have attemptedto tabulate diverse data in such a manner that comparisonsare more feasible. Certain current interpretations or concepts, from numerous sources, have been narrated or ifiustrated. Several questions have been posed which seem to beespecially pertinent to therapy.

INTRODUCTION

Clearly, human leukemia remains a difficult disease totreat, but in the last 15 years remarkable progress has beenmade because of the discovery and development of newtherapeutic agents and more effective regimens and advancesin our knowledge of the disease(s). There are now more thana half-dozen agents which singly or in combination caninduce clinical and hematological remissions and can result inprolonged survival. In the context of this review, it isimportant to emphasize that such remissions may imply areduction in the patient's leukemic cells by about 99.99% orgreater (26). However, eradication of approximately 1 trillion leukemic cells (a 4-log reduction from i0' 2) leavingperhaps 100 mfflion (108), leaves us far short of our goalsince many of the remaining cells retain proliferative integrity. How best to choose and schedule agents which might

Received April 21, 1969; accepted February 23, 1970.

JUNE 1970 1883

Kinetics of Normal and Leukemic Leukocyte Populations andRelevance to Chemotherapy

Howard E. Skipperand SeymourPerryKettering-Meyer Laboratory, Southern Research Institute, Birmingham, Alabama 35205 [H. E. S.] , and the National Cancer Institute, NIH,Bethesda, Maryland 20014 [S. P.1

Howard E. Skipper and Seymour Perry

considered to be in the range of 1 to 3 days (15). The fullydeveloped cells remain in the bone marrow for 2 or 3 daysand constitute the mature granulocyte reserve which maintains the circulating granulocyte pool and upon which thebody may draw when there is need to mobilize largenumbers of granulocytes. There is evidence that only afraction of the cells in the normal bone marrow are in activeproliferation at a given moment in time. Cells are released bya poorly understood “bonemarrow release mechanism―which responds to a variety of stimuli including infection,bacterial endotoxic, etiocholanolone, etc. (14, 55, 82). Leukocytes less mature than the band are rarely released regardless of the stimulus. In the steady state, approximately 8x 10' granulocytesenterthe bloodeveryminute.

In the peripheral blood, granulocytes are distributed equally between the circulating and marginated pools which are ininstant equilibrium. The cells circulate in the blood for arelatively short period of time (t½ 6.6 hi) and then enterthe tissues (48). Once in the tissues, under normal circumstances, granulocytes do not return to the blood (59).

Normal Lymphocytes

The lymphocyte presents a different picture from theforegoing. Lymphocytes are produced in the lymph nodesand pass to the vascular tree by way of the minor and majorlymphatic vessels. The thoracic duct has been the moststudied of these in both man and animals. The rate of entry oflymphocytes and lymph into the blood via the thoracic ductvaries greatly with white blood counts ranging from 2,000 to20,000/cu mm and lymph flow up to 100 nil/hr. The majorityof the cells are small lymphocytes with 10 to 20% large

lymphocytes (many of which are in DNA synthesis) and smallnumbers of other mononuclear cells. The lymphocytescirculate in the blood and then may reenter the lymphaticsystem to begin the circuit again (57).

Leukemic Cells (in Man)

In contrast to the orderly and well-disciplined production,development, and movement (and steady state) of leukocytesin the normal individual, the picture in leukemia is largelyone of disarray and confusion with marked deviation fromthe steady state. In acute leukemia, the normal cells arealmost completely replaced by large numbers of immaturecells, predominantly blasts, both in the marrow and theperipheral blood. The spleen, liver, and lymph nodes may besoinfiltratedwith thesecellsasto becomeenlarged.Information on cell cycle characteristics of leukemic leukocytes issparse and in certain instances conflicting, but most of thedata suggest that (a) cell generation times are in the normalrange or somewhat longer (12, 29, 61), (b) most of theblasts are nondividing or are in a prolonged G1 phase (36,50), and (c) the intravascular stay of leukemic cells isprolonged (1 , 10, 75), probably at least in part due to thepresence of immature leukocytes.

In chronic myelocytic leukemia, the bone marrow becomesvery cellular but the myeloid differential count is not greatlyaltered, at least “early―in the disease. The spleen and liver

and sometimes the lymph nodes are enlarged due to thepresence of cells in the myeloid series, both immature andmature. This excessive proliferation of myeloid cells isreflected in a rising peripheral white blood count with adifferential count quite similar to that found in marrow.There is good evidence that the normal “bonemarrowrelease mechanism― is partially disrupted (or overcome)allowing immature cells to appear in the blood. In addition,leukocytes may recycle from spleen to blood and fromblood to bone marrow (58). In the acute phase of chronicmyelocytic leukemia, the spectrum of myeloid cells is replaced by blast forms and the leukocyte kinetic patternresembles that in acute leukemia (31).

In chronic lymphocytic leukemia, large numbers of morphologically mature lymphocytes appear and result inenlargement of liver, spleen, and lymph nodes along withreplacement of the normal myeloid elements in the bonemarrow and blood. The leukocyte kinetic pattern as studied

with TdR-3 H' in vivo is quite different from chronicmyelocytic leukemia and suggests, as one would expect, thatthe marrow release mechanism does not play a significantrole in the high peripheral blood white count (88). Thekinetic patterns of lymphocytes once in the blood resemblethose observed in chronic myelocytic leukemia (22).

Much of the information concerning the dynamic behaviorof humanleukemiccellsof necessityhasbeenobtainedfromrather advanced disease, and the degree of advancement ofneoplastic disease often influences population doubling time,growth fraction, and “cellloss.―

If generalization is permissible at this point in time, itwould appear that human leukemia is a disease in which, asDameshek (18) has suggested, large numbers of cells haveaccumulated , many of which are not engaged in activeproliferation. Cells in the nonproliferating compartment maybe “sterile―or in prolonged G, or “G0―;in the latter state,they may retain proliferative integrity for some time. Thequantitative distribution between these compartments is acritical question from the standpoint of therapy, becauseavailable evidence suggests that temporarily and provisionallynondividing leukemic cells that retain capacity to revert to aproliferative state are partially to completely insensitive toantileukemic drugs (depending on the class of drug). Therelative proportion of cells in dividing versus nondividingcompartments (in relation to cell loss) presumably determines the rate of growth or doubling time of the totalleukemic cell population.

Such subdivision of proliferative state in relation to celldeath—which on alteration from the normal may lead toaccumulation of neoplastic cells and later slowing of tumorgrowth—has long been recognized in solid tumors. Forexample, what has been said about leukemia cell populationsis not at all incompatible with the “growthfraction― con

1The abbreviations used are: TdR-3H, tritiated thymidine; ALL,acute lymphocytic leukemia; nra-C, arabinosylcytosine; AML, acutemyelocytic leukemia.

1884 CANCER RESEARCH VOL.30

Norma! and Leukemic Leukocyte Kinetics

cept set forth by Mendelsohn (5 1) and the views of Steel(76) with respect to “cellloss―(in relation to cell birth) asneoplasms increase in cell number and mass.

A Framework from which to Consider Kinetic and Therapeutic Response Data

Before turning to leukemias in animals and examiningsimilarities and differences from what has been said abouthuman leukemias, it would be useful to consider the conceptdiagramed in Chart 1. The implications in this chart are notparticularly novel; however, a somewhat similar conceptualdiagram has proved useful in communication (73). By frequent reference to the compartments “defined―in Chart 1,we hope to avoid some semantic problems and reduceverbage. [If one does not like compartments, which sufferfrom the connotation of all-or-none, then Compartments A +B might be visualized as a continuous range of generationtimes (or G, ‘s)with A representing the shorter and Brepresenting the longer. In this context, it is assumed that acell with a long T@ (or a long G, episode) can give rise toprogeny with shorter generation times.]

If we wish to try to use the same sort of conceptualdiagram (Chart 1) to describe the behavior, proliferativeintegrity, and “sensitivity―of normal leukocytes (so thatdifferences from leukemic cell behavior may be discussed ina similar framework), we might assume that normal hematopoietic stem cells reside in Compartments A and B (with thehigher percentage probably in Compartment B), and thattheir sufficiently differentiated (and “sterile―)progeny mightbe represented by Compartment C.

In the context of the implications in Chart 1:Only cells in Compartment A are adding to the total

number of leukemia cells (or normal leukocytes) in the host.Progression of leukemia occurs when proliferation of cells

in A adds more to the intact population than is lost to D.

This is another way of saying that progression of diseaseoccurs when leukemic cell production is greater than celldeath; also, we might assume that normal leukocyte production is not lethal because of the existence of a steady state(proliferation in A loss to D).

Induction of remissions in leukemia results when loss to Dexceeds proliferation in A. Again, available results indicatethat in ALL this therapeutically induced “imbalance―in favorof cell loss to D must continue until about 99 .9% or greater of

the patient's intact leukemic cells (regardless of compartment)have been lost to D, and the normal order of events appears tobe restored, before we recognize a “complete―clinicalremission. In a somewhat similar vein, leukopenia induced bytherapy might be considered a situation in which loss to Dcontinues and proliferation in A fails to keep pace.

Advanced human leukemia in which “mostof the blasts arenondividing― might be considered a disease in which “mostof the blasts―arein CompartmentsB andC. If treatmentwith a truly cell-cycle-stage-specific agent induces completehematological remission in some human leukemias, it mightbe suggested that the fraction of cells which remained in Bthroughout treatment (effective blood levels of drug) wasrelatively small, but nevertheless significant, since “cure―isnot achieved.

In keeping with what already has been stated, it wouldappear that the “biochemical lesion(s)― associated withleukemia is reflected in deviation from the steady statebecause of the loss of some (but not all) response to“normalgrowth control― (15); i.e., the failure of the “normal fraction― of the cells to differentiate to a sterile pool ormovein a normalmannerfrom A to D.

Experimental Murine Leukemia Systems

We are not at all concerned (in the end) with just “curing―mice of leukemia. Our target is so obviously the control or

D.‘CellLoss―@ S Q.ILysisandResorption

Temporarily and ProvisionallyNondividing Leukemic Cells

CanRevert to A at Any Time(Long to Very Long ‘G,or

Compartment A

@nt.rru@$.dJ@[

Leukernic Cills

Engaged inDivision __________

Comportment B ComportmentC

Nondividlng LeukemicCslls Which Hove LostProliferative Integrity

(Forevermore)

Very Sensitive toEffective AgentsWhen Optimally Employed

Partially to CompletelyInsensitive to Drugs(Dep.nding on Class of Drug)

Of Little or Less Concern(By Definition)

Chart 1. On the proliferative behavior of leukemic cells and the “sensitivity―to therapy of those cells with proliferative integrity.

1885JUNE 1970

Howard E. Skipper and Seymour Perry

cure of human leukemia that it seems strange to feel calledon to say so. The only justification for working with animalleukemias resides in belief or faith (or past evidence) thatsome therapeutic agents uncovered in mouse leukemia“screens―will be useful in treatment of leukemias (or othertypes of human cancer), and that some important part ofwhat is learned about animal leukemia will add to ourunderstanding of human leukemia and how to control it.The point need not be belabored that basic understandingregarding animal leukemia is often much more easily andquickly gained—not because laboratory experimentalists arewiser, but because of ethical considerations involved instudies in man, and uniformity of the disease in animals andthe very large number of comparable leukemic hosts whichcan be studied. What is established beyond reasonable doubtregarding animal leukemias (transplanted or spontaneous)must be considered of basic scientific interest, but unreserved acceptance of carryover should await evidence gainedat the clinical level. Speculation is fun but it is better toobtain and look at the data.

Normal Leukocyte Production in the Mouse

Knowledge similar to that reported for man concerningdevelopment and movement of normal leukocytes in themouse is not extensive, but there is no available evidence ofqualitative difference (20). Quite clearly , the steady statesituation with respect to normal leukocyte behavior in themouse parallels that in man. However, much basic knowledgehas been gained in animals concerning hematopoietic colony-forming cells (78), their response to therapy, and theirrate of “recovery―following marrow damage (7 , 8 1). Theliterature in this area is extensive enough to warrant aseparate review . Herein we will simply mention that only asmall fraction of normal hematopoietic colony-forming cellsappears to be in a proliferative state (Compartment A) at agiven time unless or until bone marrow damage has occurred(3, 6, 80 ). Destruction by ionizing radiation of greater thanabout 99% of the normal hematopoietic colony-forming cellsin the mouse is usually lethal (47). Also , important andextensive observations show that these normal hematopoieticcolony-forming cells are much less sensitive to certain antileukemic drugs or to cytotoxic levels of TdR-3 H (whenoptimally used) than are long-transplanted AK leukemia(lymphoma) cells where about 99.99% of the cells withproliferative integrity pass through S phase during a periodof 24 hours (5, 7). Quite similar results have been obtained

in the Ll2 10 leukemia system (85 , 86).

Long-Transplanted Murine Leukemia

One or another of the available transplanted murine leukemias responds significantly to every agent which has beenshown to produce remissions in human leukemia, but anysingle line may fail to respond to one or more agents whichhave clinical activity. By the same token, all human leukemias do not respond to all the agents which have beenshown to be effective against a given type of humanleukemia. This evidence alone is perhaps justification for the

statement that “thesemouse leukemias often behave muchlike human leukemias at the biochemical level.―However,long-transplanted mouse leukemias, at the stage they areusually studied, appear to differ from advanced humanleukemias in at least one very important respect; namely, thegrowth fraction of the leukemic cells which retain proliferative integrity. Or more specifically, a very high fraction of,say, Ll 2 10 leukemia cells is in Compartment A until shortlybefore death of the host. The evidence for this conclusionmay be found in the following observations:

A single Ll 2 10 leukemia cell isolated with the aid of amicromanipulator often wifi (if successfully transplanted)give rise to l0@ leukemic cells (the lethal number) about 15days later (68, 71).

The doubling time of the total population and the mediancycle time of dividing Ll 2 10 leukemia cells are approximately the same until the number in the host approaches orexceeds 108 (68, 7 1). Also, the increase in cell number isessentially exponential over the range of I to approximately100,000,000 cells; then population growth becomes asymptotic (68).

ara-C, which is S phase-specific (27), will render about99.99 to 99.999% of Ll 2 10 leukemia cells permanentlysterile when administered to the host in effective doses every3 hr for 21 hr, thus providing effective blood concentrationsfor about twice the median cell cycle time (72). Repeatedcourses of such treatment regularly provide “cellcures―ifthe disease is not too far advanced (62, 72). This relationship between leukemic cell number and “curability―with acycle-stage-specific drug implies movement of L1210 leukemic cells from Compartment A to B with increase in thetotal number of leukemic cells.

The preferential cytotoxicity of very high levels of TdR-3 Hfor transplanted AK leukemia over hematopoietic colonyforming cells has been mentioned (5).

Spontaneous AK Leukemia (Lymphoma) in the AKR Mouse

Recent studies on the kinetic behavior and response totherapy of spontaneous (virus-associated) AK leukemia havehelped to bridge the gap between transplanted and spontaneous murine leukemias (53, 63, 70). Briefly, spontaneous AKleukemia which is very advanced when diagnosed has amedian cell cycle time that is not significantly different fromnormal dividing thymus lymphocytes (53). The doublingtime of such advanced leukemic cell populations is muchlonger than the median cell cycle time of the dividing cells,thus the growth fraction may be very significantly less than1.0 and the “cellloss―factor may be high relative to earlytransplanted murine leukemias. Some of the blasts are nondividing (53), and a relatively high fraction seem to lackproliferative integrity, although cells with capacity to giverise to a lethal population are to be found in most tissuesincluding the peripheral blood (63, 70). In therapeutic trials,the survival time of animals bearing advanced spontaneousAK leukemia has not been prolonged significantly by cellcycle-stage-specific agents, but host survival time is markedlyprolonged by cyclophosphamide alone or cyclophosphamidefollowed by ara-C in repeated courses (63, 70).

1886 CANCER RESEARCH VOL.30

Normal and Leukemic Leukocyte Kinetics

In general, it appears that advanced spontaneous AK leukemia resembles advanced human leukemias (with respect todynamic behavior) more than it resembles the long-transplanted lines of AK leukemia or Ll2lO leukemia in theirearly stages. On the very first passage of spontaneous AKleukemia cells to young AKR mice (smaller numbers thanare present in the spontaneous disease when diagnosed),these leukemic cells become very responsive to cycle-stagespecific agents (63, 70). Thus the smaller populations offirst-passage leukemic cells take on characteristics commonto long-transplanted lines.

Presentation of Diverse Data (Doubling Times, Cell CycleCharacteristics, Thymidine Indices) Obtained in Animal andHuman Leukemic and Normal Cell Populations

One purpose in tabulating the data presented in Tables 1 to4 was to provide a reasonable sample of the informationwhich (in part) underlies concepts already mentioned . Certain results are sparse, particularly cell cycle information forhuman cell populations. Such information is difficult toobtain, but, in a sense, the T@ data on human leukemic cellpopulations is bolstered by doubling time data from logphase cultures of human leukemic cells (25, 54, 79).

In Tables 1 to 4, the cell population sizes or degree ofadvancement of diseases are given when available . This isindeed essential for meaningful comparison of the databecause if we have learned anything about rates of growth ofcancer it is that such growth is rarely exponential over itsentire clinical history. Deviation from exponential growth(see Chart 2) can be the result of (a) decreasing growthfraction, (b) increasing cell loss, (c) increasing T@ in veryadvanced disease, or (d) a combination of these phenomena.

In the above context, Chart 2 presents a Gompertziancurve showing the rate of growth of a composite of “normaltissues,― the human fetus and man after birth. The curveshowing rate of fetal growth was adapted from an interestingobservation published by McCredie et aL (46). This chartmay seem out of place in this review , but in an overall senseit illustrates the end result of much of what we have beendiscussing; e.g., the steady state problem, differentiation, thegrowth fraction and movement of cells from A-@B and C,changes in doubling time with increase in mass, and “normalgrowth control.― Such Gompertzian growth curves seemalmost the rule in animal neoplasms, over their measurablegrowth ranges (Refs. 9, 39, 40, 46, 77 ; H. H. Lloyd,unpublished data). They imply exponentially retarded exponential growth. A major difference between neoplastic cellpopulations (including leukemias) and the fetus is that theformer often cause host death before a steady state isreached and, of course, fetal cells are not prone to metastasize to sites which cannot tolerate massive infiltration.

Relationships between “KineticData― and Response toTherapy

No attempt will be made to discuss the individual datum inTables 1 to 4; however, we would like to examine thedifferent types of results obtained with different leukemic

and normal cell populations from the standpoint of possiblerelevance to therapy. To facilitate examination, some datafrom Tables 1 to 4 have been brought together in Table 5.

Perhaps the most direct way to examine the data is withinthe context of several specific questions:

Question 1. Do leukemic cells divide more rapidly thannormal leukocyte precursors once both cell types have“decided―to divide? Or, more specifically, how do themedian Ta'S (or Ta's) of dividing leukemic cells comparewith those of dividing normal leukocytes (i.e., normal leukocyte precursors)?

Comment: The median Tc or Ts of L12l0 and advancedspontaneous AK leukemia is not shorter than the valuereported for normal thymus lymphocytes in the mouse noris it shorter than the median T@ br T@for normal intestinalepithelium or hair follicle cells in the mouse. The Ta's ofacute human leukemias are not shorter than the valuereported for normal myeloblasts in human marrow. Fromthe standpoint of chemotherapy, it is probably the coefficient of variation and range of Ta's or, more to the point,the relative coefficient of variation and range of@TM + 2 in normal and neoplastic cells (at the beginningand during treatment) that underlie our ability or inabilityto achieve remissions or “cures.―Better knowledge of theseranges in remission and relapse would be of theoreticalvalue in design of therapeutic trials.

Question 2. How do the doubling times of leukemic cellpopulations compare with those of normal leukocyte populations or other normal cell populations?

Comment: This question was posed to make a point.Obviously, normal cell populations which are in a steady statedo not havea “doublingtime―in theusualsense,whereasleukemic cell populations which are not in a steady state dohave a measurable and meaningful doubling time that increaseswith advancement of disease. It is pertinent that normalhematopoietic colony-forming cells in the marrow of mice,when regenerating after damage, have a doubling time of about1.2 days and are much more sensitive to cytotoxic agents,until such cells have returned to a steady state.

Question 3. Is there any relationship between doublingtime of leukemic cells and their sensitivity to S phasespecific agents? The answer to this question appears to be inthe affirmative : (a) ara-C kills a higher fraction of L12 10cells per given dose or course when the disease is notadvanced than when the disease in advanced and the doubling time has increased. (b) Log phase Ll 21 0 cells in cultureare sensitive to ara-C, whereas nondividing cells are notsensitive. (c) Advanced spontaneous AK leukemia with arelatively long average doubling time is not very sensitive toara-C, whereas on the very first passage of relatively smallnumbers (doubling time, ca. 1.1 day) these leukemic cellsbecome about as sensitive to ara-C as long-passage AKleukemia or Ll 2 10 leukemia. If first-passage AK leukemia isallowed to become about as advanced as the spontaneousdisease (when diagnosed) the doubling time increases, and itthen responds very poorly to ara-C. (d) It appears that thedoubling time of ALL and AML in beginning relapse isshorter than when the disease is still further advanced. Inthis context, some of the antimetabolites which kill cells in

1887JUNE 1970

AverageSiteand size ofTime afterApproximate rangSite or distripopulationCell

populationinoculum orinoculationin populationbution of cellsdoublingtime(invivo unless otherwise indicatedstage of disease(days)size studiedstudied(days)MethodReference

Howard E. Skipper and Seymour Perry

Table 1

Doubling times of nwrlne and human leukemic cells, effects ofsize and site oflnoculation or stage of disease

4X 108 and>

l—lO@

i08—t09

l—l0@

1_106

102_ 106

<60—350mg350—900900 and>

104—106/ml2X 106 and>

10@_106 or

ca. 108_109

WiderangeWide range

i@@—i@''?

lO@—l0'@?10''?

lois to lethalNo.

ce 105@106/mI

10@—10'/ml

105—10'/ml

105—l0'/ml

71,8771,87

68

68

a

68

85

68

a

a

21

4

8

63,70

63,70

80

263424Estimated byauthors

54

25

79

79

3—66—8

Inoculation to death

Inoculationto death

0—6

7—8

8—99—10

0—7

3—1010—35

PeritonealcavityPeritoneal cavity

Widespread

WidespreadWidespread

Widespread

WidespreadWidespread

WidespreadMarrow

Brain (at onset)

Widespread

Subcutaneous;massSubcutaneous;massSubcutaneous;mass

Cell cultureCellculture

SpleenMarrowThymus

BloodWidespread

Widespread

Widespread

MarrowMarrow

MarrowMarrowMarrowWidespread

Culture

Culture

Culture

Culture

0.55Plateau

0.48

0.42ce.1.3

0.44

0.4ce.0.7

0.40.3

0.43

0.53

ce.0.450.751.7and>

0.59Plateau

0.480.480.470.440.44

1.1

ca5

1.2Approaching

plateau

45.8 (2—16)4—5Possibly 20—30

0.8—2.1

1—1.3

1.5

1.1

DirectcountDirect count

Host life spanafter singlecell

Host life span,titrationHostlife span,titration

Host life spanafter singlecell

Host life span,titrationHostlife span,titration

Host life span,titrationCoiony-forming units

Host life span,titration

Host life span,titration

Caliper measurementsCuliper measurementsCaliper measurements

DirectcountDirect count

Colony-forming unitsColony@formingunitsColony-forming unitsColony-forming unitsHost life span,titration

Host life span,titration

Diagnosisto median host death

Coiony-formingunitsColony-forming units

Serial marrow assessmentSerial marrow assessmentSerial marrow assessmentce.90 days(untreatedsurvival)/3.32 doublingsperdecade

Direct counts

Direct counts

Direct counts

Direct counts

Mouse

Ll2lOleukernia i.p.;@i.p.; lO@—iO'

i.p.; singlecell

i.p.; i—10@i.p.; i0@

iv.; single cell

iv.; i—iO@iv.;i0@

iv.; l—l0@i.v.;i0@ or 10@

intracerebral1—10'

s.c.;102_106

s.c.; 10'

L12l0 leukemiain logphasecultureCulture

AKleukemia.long-transplanted iv.; 10@

i.v.; 10@—l0@

AKleukemia,1stpassagefrom iv.; l0@—i0@spontaneous

AKleukemia,advancedspontaneousVeryadvanced

Normal femoral colony-forming iv.; 5 X 10@cells transplanted to lethallyirradiated mice

Man

ALL Beginning relapseBeginningrelapse

AML UntreatedAcuteleukemias After diagnosis

Leukemia cells in log phaseculture Culture(6 lines)

Leukemia cells in log phaseculture Culture(2 lines)

Cells from myeloblastic leukemia Cultureinlogphaseculture(SK-L7)

Normal lymphoid cells in log phase Cultureculture(SK-LN1)

Note: Populationdoublingtimesmay be iongerbut not shorterthanthe cycletime (Tc) of the dividingcells.Populationdoublingtimesof logphaseculturesareapt to havea highgrowthfraction and little cell loss(before growth becomesasymptotic). If this is the case,then their doubling time may approach T@.a@ M. Schabel.Jr.,unpublisheddata.

1888 CANCER RESEARCH VOL.30

Cellpopulation(In vivo unless otherwise indicated)@pproximate

populatioisizestudied orstageofdlseaseSite

ofcells

studiedpha@@

times(Iv)

J(TWTc) XT@ T@ Tcj@ Ty,j@@ 100 (%) T( T@MethodReference

Normal and Leukemic Leukocyte Kinetics

Table2

Median cell cycle characteristics ofdlvidlng murine and human leukemic cells and certain normal cells

1.81.4

1.5

3

3

2.2

1.3

1.5

2.0

3

3

1—2

2.2

106

10@I x 108 and>

a. lO'/mI

@dvanced

tandy state

@teadystate

lot insteadystate

teadystate

telapse“di@

Prednisoneday before)JntreatedJntreated(3 patients)

teady stateitcady stateteadystate

Steadystate

10s_ 106/ml

@ —106/ml

@eritonea1cavity@ritonea1cavity@ritonealcavity@ritonealcavity

eritoneal cavityeritoneal cavity

@ulture

Peritonealcavity

ulture

@hymus

isymus

)uodenum

@ipof tail

lair follicles

4anow4arrow

Marrow4arrow4arrow

MarrowMarrowMarrow

oion

:ulture

ulture

12.815.811.82112—1411.8

15.6

11.5

11.5

Lb 7.6

Mb20.2

Lt@6.8Mb8.2

11.5

9.0

10.8

60

60498340

242454

>15

17

9.011.88.9

10.7

11.3

7.0

7.0

6.06.0

5.51.5

5

6.3

6.0

202022

1916

12—2413—2413—24

10—15

11

Labeled mitosesLabeledmitosesLabeledmitosesLabeled mitosesGrain countsGrain counts

Labeledmitoses

Labeledmitoses

Labeled mimics

Graincountandlabeledmltoses

Grain count and labeledmitoses

Labeled mitoses

Labeledmitoses

Labeled mitoses

Labeled mitosesLabeled mitosesLabeled mitosesLabeledmitosesLabeledmitoses

Grain count and labeledmitoses

Labeled mitoses

Labeled mitoses

Labeled mitoses

a

838787a87

83

19

19

53

53

42

84

32

6161121229

15, 16, 37

44

79

79

Mouse

L1210 leukemia

L1210inlogphaseculture

L5178Yleukemia

Ii 178Y leukemia in log phaseculture

AX leukemia, advancedspontaneous

Normal lymphocytes (2-mo.-old AKRmice)

Normal intestinal crypt celia

Embryo(12—13days)

Normal hair follicle

Human

ALLAML

NormalMyeloblastPromyelocyteMyelocyte

intestinal crypt

Cells from myelobiastic leukemia(SK-L7)in logphaseculture

Normal lymphold cells (SK-LN1)inlogphaseculture

18 12 1.8

ce.1.0 1.0 70 3.80.58 2.1 75 4.0

‘75 2.9St 10.3

0.6 2.2 72 4.3

1.5 63 4.5

1.5 63 4.5

79 1.612 30 14.2

81 1.31.4 67 2.7

43 6.5

1.2 69 2.8

2.8 56 4.8

33 40

33 400.47 24 45 270.62 61 23 64

40 24

0.6 3.2 65 6

0.5 3.7 67 6

Note: The Tc . 7'@valuesare givenbecausethey are possiblyusefulin the selectionof dosageintervalsfor truly S phase-specificagents.aL Simpson-Herren, unpublished data.b@@ medium cells; small cells appear to have lost proliferative integrity. The volume and mass of all cells increase as they traverse the cycle, but a doubling in volume represents

slightly less than 30% increasein diameter.

1889JUNE 1970

CellpopulationSite

andsize of

inoculumPmiodafter

inoculationApproximatepopulation

sizeDistribution ofcellsMedian

labeling Index(%)MethodReferencePulseLonger

exposuretoTdR-3HL1210

leukemiacellsIn BDF, micei.p.; lO@l.p.;105ip.; 3 X i0@i.p.;3X1O5@y

57Day6Day 6Day710

i0@

2.5X105Pseltoneal

cavityPeritonealcavity

Pealtoneal cavityPeritoncalcavity60

55625421

he;93%

24 lv; 99.9%In

vivaInvivoIn vivoInvls'oa

8387

87L1210

leukemIa in log phase cultureLog phasecell culture-105-10@/ml6618

hi;>99%Culture83Spontaneous

thymic lymphomaSpontaneousAt diagnosisce. i0 or>ThymusCells%DistributionlnAKRmlceLarge77

Medium 30Small 4.1@‘All―(ce.37)“All―ce.5(25)

(56)

(20)Invivo52

aNormal

thymuslymphocytes inThymusCdl.%DistributionAKRmIceLarge86

Medium 75Small 2.1“All―(ce.10)(

1.8)( 9.4)

(89.0)Invivo52Epidermis.

basedlayerPulseEpithelium,bronchus0.4Invivo2Epithelium,crypts,duodenum0.4—1.7Epithelium,crypts,jejunum30Epithelium,crypts,ileum60Eplthellum,crypts, largeintestine40Epithelium,crypta.smallIntestine4Epithellum,smallbronchi47—SOKidney,tubules0.5Uver,

Kupffer'scells0.2Liver,littoralcells1.0Liver.parenchymalcells1.4Lymph

node,follicularcells0.1—0.8Lung.alveolarcells22.3—1.2Spleen.

folilcularcells1.0Spleen.redpulp0.5-19.2Thymus.

cortex6-17.5Thyroid6.1Trachea,

mucosa0.50.3

Table 3

Thymidine liidkeson rmirbse leukemicand n@nwl c&pop@sktions

Note: In terms of Cisart 1, the pulse thymidine index might be described as the fraction of A cells in S phase/A + B + C during a relatively thort intesval(ce@30 raIn). This indexoftenbearsanindirectrelationtothegrowthfraction(A/A+B+C)butnotneceswilytothegrowthfractionofcellsretainingproliferativeintegrity(A/A+B).

aL Slmpaon-Herren.unpublisheddata.

1890

Howard E. Skipper and Seymour Perry

CANCER RESEARCH VOL.30

Cell population% (median)No.of

atientsStage of diseaseSourceof

cellsPeriodof exposureto TdR@HMethodLabeling

index(%)ReferenceMedianRange

Normal and Leukemic Leukocyte Kinetics

Table 4

Thymidine indices—human leukemias and normal cell populations

MarrowBlood

MarrowMarrow

MarrowBloodBloodMarrow

MarrowBloodMarrowBloodMarrowBloodBloodMarrow

MarrowMarrowMarrowBlood

Blood

BloodBloodBloodBloodBlood

Bonemarrow

BloodBloodBonemarrowBonemarrow

Bonemarrow

Blood

Blood

MarrowBlood

In vitroIn vitro

In vitroIn vitro

In vitroIn vitroIn vivoIn vivo

In vitroIn vitroIn vivoIn vivoIn vivoIn vivoIn vivoIn vivo

In vitroIn vitroIn vivoIn vivo

In vivo

In vitroIn vitroIn vitroIn vitroIn vitro

In vitro

In vivoIn vivo

In vitro

In vitro

In vivo

In vivo

In vitroIn vitro

8.63.9

4349

122.73.72.8

5.61.33.40.76.10.2

10 (maximum)

382.005.7

10.34.10.13

55541130

85

18149.0446.0

34.014

1113.314.242

6.211.5

0.4310.7

0.027.90.90.5314.0

4949

6161

35353149

1212121212123130

653538

a

35

662813

65

3138

28

65

88

88

6513

1hz1hz

1hz1hz

lhr1hzPulsePulse

ImmediateImmediatePulse2 hrPulse2 hrPulse 2 hr1hzPulsePulse 1 hr

1hz1hzPulse 1 hrPulse 1 hr

Pulse

1 hr1hz1hz1hz1hz

lhr

PulsePulse

1hz

1hz

Pulse

Pulse

1hz1hz

1.5—6.2

34—5041—50

8.3—150.7—4.02.3—5.02.6—3.0

24.0—52.01.0—7.0

5.0—6.58.3—17.3

50—5546—58

9—1227—455—15

@—1117.5—19.06.4—208.5—9.535—52

2.5—12.018.0—88.0

23.9—483.7—10.410.5—12.5

O.3_I.89b10.3—15.6b

O.02_0.lb3.1_12.Sb0.6—1.50—1.80.60

2 At diagnosisAt diagnosis

4 At diagnosisRelapse

4 At diagnosisAt diagnosis

2 At diagnosis2 At diagnosis

1 Prednisone day beforePrednisoneday beforePrednisonedaybeforePrednisone day before

1 UntreatedUntreated

7 Untreated4 Untreated

3 Relapse5 Untreated1 At diagnosis

At diagnosis

3 Untreated

2 Untreated,earlyadvanced

2 Untreated11 Untreated10 Untreated

1 Untreated

1 Untreated1 Untreated

1 Untreated

2

Untreated4

Untreated3

3 Untreated6 Untreated

ALL

All blasts

Large blasts only 1041

Allblasts

AllblastsAllblasts

AML

All blasts

Allblasts

Allblasts

Large 9Medium 51Small 40

Chronic myelocytic leukemia

Blasts 2Promyelocytes 4Myelocytes 26Metamyelocytes 9Bands 26Segments 33All leukocytesAll leukocytes

Blasts only Not givenAll leukocytesImmature cellsonly 12Blastsonly Not given

Blastic crisis

11>14

Blasts>1 Sp Not given<15M

Notgiven

Osronic lymphocytic leukemia

“Early―Smallcells 99Largecells 1

AdvancedSmall 951.arge 5

Total leukocytesImmature cells only

1891JUNE 1970

—Cell

population% (median)No.of

atientsStage of diseaseSourceof

cellsPeriodof exposureto TdR.3HMethOdLabeling

index(%)ReferenceMedianRangeNormal1MarrowPulseIn

vivo16Myeloblast

Promyelocyte“Normaldifferential'85

65Myelocyte23Metamyelocyte0Bands

Granulocytes“Normaldifferential'2BloodPulseIn vivo55 (maximum)

55(maximum)171BloodPulseInvivo88Granulocytes5330.9(maximum)Smalllymphocytes451.0Large

lymphocytesLymphocytes23Thoracic ductPulseInvivo26.4Small1714.4CLarge

All leukocytes8348Blood1 hrIn vitro29.8CI)0—0.260IntestinalMucosal

cells2ColonPulse 2 hrInvivo13.9—3233@ryptcells

@ryptcells3 3Colon ileumPulse PulseInvivo Invivo16 1016—1745,4345,43Cells

from myeloblastic leukemia(SK-L7)in

log phase cultureCulturePulseInvitro4079Normallymphoidcells(SK-LN1)inlog

phasecultureCulturePulseIn vitro5079

Howard E. Skipper and Seymour Perry

Table 4 Cont

Thyrnidineindices-human leukemia, and normalcellpopulations

as P@ry,unpubliarseddata.blncludes maximum labeling indices for each patient.cFwe hr post-TdR.3H (maximum labelingindex).

S phase are better remission maintainers than remissioninducers. When human leukemic cells are “uncrowded―andadapted to log phase culture (doubling time , Ca. 1 day) theybecome very sensitive to S phase-specific drugs (23).

Question 4. How do the pulse thymidine indices of leukemic cell populations compare with those of normal leukocyte populations?

Comment: This question is complicated by our lack ofunderstanding (in depth) concerning the relationship betweenlabeling indices of large and small leukemic cells. Thelabeling index of L 12 10 leukemic cells is higher than allnormal thymus lymphocytes or all leukocytes in the marrow.There is wide range in the reported thymidine index ofspontaneous AK leukemic cells. The labeling index of normalhuman myeloblasts is very high and of metamyelocytes,bands, and polymorphonuclear cells is reported as zero. Thepulse thymidine index is a rough measure of the fraction ofCompartment A cells in S phase/A + B + C , during a shortperiod. A critical question is, “Whatfraction of normalhematopoietic stem cells is in S phase at a given moment intime?―All data available to us suggest that this fraction islow (3, 7, 41 , 86) in undamaged marrow.

Question 5. Is there any relationship between T@, T5 , or1@ TM@ 2 of different leukemias and their response

to chemotherapy, particularly cell-cycle-stage-specific agents?Comment: There is no obvious relationship between medi

an Ts and response to chemotherapy; however, there doesseem to be a relationship between@ + TM + TG2 (whichis reflected in TC). As mentioned above, a long median andwide range of TG@ TM + TG2 possibly underlies poorresponse to chemotherapy. This would apply to agents whichreact with or complex with DNA, as well as S phase-specificagents, if the cells with alkylated or complexed DNA loseproliferative integrity as they attempt DNA replication and along@@ TM@ 2 period allows for significant DNArepair.

The conclusions drawn from study of kinetic parametersversus response to chemotherapy in a much wider variety ofexperimental neoplasms, including solid tumors (69), aregenerally consistent with the above interpretations, namely:

“1. The doubling times of all of these tumors increase withincrease in tumor mass; the rates of increase in doublingtime vary.

“2.There is a consistent and unmistakable inverse relation.ship between advancement of neoplastic disease and ‘curabilty' by chemotherapy.

“3.There is an inverse relationship between the doublingtime of these tumors at initiation of treatment and responseto chemotherapy.“4.Thereisa directrelationshipbetweenthymidineindex

and response to chemotherapy.“5.There is an inverse relationship between@@ TM +

1892 CANCER RESEARCH VOL.30

Normal and Leukemic Leukocyte Kinetics

II.@

S

I

susss'e@ as s ss888$8@@r 5@ 8 88 88xz*zz@ xx z xx

28@

.-..—.4 u@@@ .5.5@

@ a@@@ e@@

@I:;H @ii@ ii <& 8@ !2 L@ i

@ei0 f'1@'4

@.UJ@ g@

@o!.@@ !@—ei.-.@ @,..@

1893JUNE 1970

I.. .@ @2li

L;.@ !.@@@.. S@@

+x@ @8'@@ +

@.@@@ :@@@

@, .- @2 h';@

II I ii! tilli@ @H1@

III 11111@O!@J

C2

g g@ I

5.5E: .a I'

@ @8 2

.E@ i1@ UI

H Hi

!! !!! @!@!

Howard E. Skipper and Seymour Perry

I ru@ rrv@

I012

I0@

a,K@10@

E

be

I0@@

io6II0@I

A

Time

10,000

1,000

00

IC

.c

.2' 1.0.

0.1

0.01

0.001

0.000I

A tVSt'SAl

TG2 and response to chemotherapy, but no obvious relationship between median T@ of the dividing cells and ‘sensitivity.'“6.Alkylatingagents(whenoptimallyemployed)generally

appear more effective against tumors with longer doublingtimes, lower thymidine indices and longer@ + TM + TG2than the optimally employed S-phase-specific drugs (e.g.,arabinosylcytosine). Combinations which include alkylatingagents are sometimes even more effective when optimallyemployed.

“7.The odds against such relationships occurring by chancealone must be very high indeed―(69).

The authors of this review are quite frankly impressed bythe possibility that the growth fraction of the leukemic cellswhich retain proliferative integrity at the onset of treatment(A/A + B) and the influence of early treatment on theequilibrium A@±Boften may dictate the feasibility of leukemic cell eradication. This certainly is not a novel concept.This view first became attractive and tenable when it becameapparent that (a) neoplastic cell populations may contain

1894 CANCER RESEARCH VOL.30

Birth Age of Moles (in years)I 05 I 2 3 4 5 6 7 8 9 10 II 12 13 4 15 16 Il 18 19‘4'to to to to to to to to to to to to to to to to to to to to0 1.0 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 Il 18 1920

.@ ,‘,,, P1atsau@ co 70 k@

(overall for first year)

. .——.@—†5̃1@ day.

@_.Z!_e.—@@ 30

. a-@@

4 day-p' Human Fstus (wit weight);

@ 5.' 59;! @: 9.1 c;r@:P;@:,:r;<P@s@irs

,*4.8 ,—Thsorsticol Cons.@iusncssofI Leukemia CIII Accumulation

I at DifferentRatesOver. I Different Population Size Rongss

,—2Day DOubIkig Time

. a -

. a@@ • I I 5 I I I 5 1

0 20 40 60 80 100 120 140 160 180 200 220 240Age of Human Fetus (days)

Chart 2. Human fetal and childhood growth. The rate of fetal growth is an adaption from McCredie Ct a!. (46) in which the fit of the

Gompertzian equation, W 6.950 X 105e@ 7.97(1 . e@@t3Bt),@ m good agreement with extensive data. The computer-calculated doubling timeswere added by us. McCredieet a!. point out that “thecause of retardation in growth of the human fetus, which asymptotes at 3.8 kg is mainlydifferentiation of cells rather than a decrease in blood supply.―Also that “thevalue of W0 (6.9 X 105g) which represents the initial fetal weight,was about 5 volume doublings of the zygote. “Our computer extrapolation is in agreement.

The human body weight data are from the data of Spector (74) and the time scaleis in years rather than days.The lethal number of leukemia cells in man is thought to be of the order of 2 (about 1000 g) or slightly greater (26). The theoretical

consequences of accumulation of leukemic cells at differing rates over varying population ranges (or weights) can be visualized on the same plot.For example, assume that the growth fraction of ALL cells was very high in its earliest stages and thus the population doubling time was aboutequal to a T@of 2 days, and that after the population reached about i09 the doubling time increased to 4 days (close to that observed in beginningrelapse). Also assume that after the disease advanced to a population size of 10' â€t̃he doublingtime increased to almost 30 days (101 1 to a lethalnumber). Before the availability of chemotherapy, the median survival time (after diagnosis) of children was about 3 months. If this periodrepresented only a 1 log increase in leukemic cells, then the average doubling time over this range might be about 27 days (90 days for 10-foldincrease/3.32 doublings per decade 27 days). Dashed line, hypothetical rates of accumulation of leukemic cells under such roughly assumedcircumstances.

Normal and Leukemic Leukocyte Kinetics

provisionally “resting―cells which are not engaged in biochemical events peculiar to the process of cell division andthat these “resting―cells may return to a proliferative stateat any time (5 1), and (b) provisionally “resting―cells arepartially to completely insensitive to drugs which kill cellsbecause of interference with biochemical events peculiar todividing cells; e.g., DNA synthesis or function (7, 64, 67).

If it be true that the more “rapidlygrowing― leukemias aregenerally more responsive to S phase-specific drugs, themedian T@ of the dividing cells is not directly related tosensitivity (or growth rate) in a causative sense (but variationin 1@ TM@ TG' @550 related), and the sensitivity of thedividing fractions (@ompartment A) of various leukemias tochemotherapy is about the same, then it is reasonable tosuspect that both growth rate and responsiveness are relatedto the growth fraction (A/A + B + C) and that the feasibilityof leukemic cell eradication is related to A/A + B and ourability to influence B-÷Aby remission induction (or othermeans).

The above is not meant to imply that other approaches tothe control of leukemias should not be studied, developed,and applied at the clinical level whenever they reach thepoint when this seems feasible. For example : (a) somemethod which might induce leukemic cell populations toreturn to a permanent steady state (proliferation in A lossto D); (b) some method for converting all Compartment A

cells to Compartment B cells and maintenance therein, or,more desirable, converting all Compartment A and B cells toCompartment C cells (e.g., total sterilization by some mechanism akin to induced differentiation); (c) immunotherapywhich might destroy some fraction (or all) of CompartmentA and B cells, or immunotherapy which might eradicate, say,101 to 106 cells left after reduction to this range bychemotherapy.

Finally, the pragmatist might ask, “Howcan informationsuch as has been considered in this review advance ourefforts toward control of human leukemias?― If we couldprovide a “recipe―at this time, there would be no need tobring together such data for further study in relation totherapeutic response data. However, we believe that theseresults provided by many experimentalists and research cmicians are adding to our understanding of the leukemias andonhowto interpret“screening―resultsandmoresophisticated experimental therapeutic trials in terms of the targetproblem. To the degree that we understand why we succeedand why we fail in the treatment of neoplastic disease today,we should be able to design more critical experiments and,hopefully, more effective therapeutic regimens in the future.

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