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Cyclophosphamide treatment expands the

circulating hematopoietic stem cell pool in

dogs.

R A Abrams, … , C Bowles, A B Deisseroth

J Clin Invest. 1981;67(5):1392-1399. https://doi.org/10.1172/JCI110167.

Increased numbers of circulating granulocyte-monocyte precursor cells (CFUc) have been observed in the peripheral blood of man after antineoplastic chemotherapy. We have developed a canine model to study the biologic significance of this phenomenon for hematopoietic reconstitution following hematopoietically lethal exposure to total body irradiation (TBI). After cyclophosphamide administration, a 16-fold expansion of circulating CFUc numbers was observed during the period of rapid leukocyte recovery that occurred after the chemotherapy-induced leukocyte nadir. We had previously noted this association between leukocyte recovery and CFUc expansion in our human studies. After 900 rad TBI hematopoietic reconstitution was attempted with autologous, cryopreserved collections of peripheral blood mononuclear cells obtained either at times of post-cyclophosphamide CFUc expansion (group A, 14 dogs) or without CFUc expansion (group B, 12 dogs). Asd compared to group B collections, group A collections contained 11-fold more CFUc and were 12.5-fold more potent in fostering hematopoietic recovery after TBI. These results suggest that the expansion of CFUc numbers we observed was accompanied by a similar expansion of more primitive hematopoietic stem cell numbers. We conclude that

chemotherapy-induced expansion of circulating CFUc numbers appears to be of substantial import in effecting hematopoietic reconstitution--an observation that may be of significance for further studies of autologous hematopoietic reconstitution in man.

Research Article

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Cyclophosphamide

Treatment

Expands

the

Circulating

Hematopoietic

Stem Cell Pool

in

Dogs

R.A. ABRAMS, K. MCCORMACK, C. BOWLES, and A. B. DEISSEROTH,

Experimental HematologySection, Pediatric Oncology Branch, Division of CancerTherapy, National CancerInstitute, National Institutes of Health, Bethesda, Maryland20205

ABS T R A C T Increased numbers ofcirculating gran-ulocyte-monocyte precursor cells (CFUc) have been observed in the peripheral blood of man after antineoplastic chemotherapy. We have developed a canine modeltostudythebiologic significance ofthis phenomenon for hematopoietic reconstitution follow-ing hematopoietically lethal exposure to total body irradiation (TBI). After cyclophosphamide administra-tion,a16-fold expansion ofcirculatingCFUcnumbers was observed during the period of rapid leukocyte recoverythat occurred after the chemotherapy-induced leukocyte nadir. We had previously noted this asso-ciationbetween leukocyte recovery andCFUc expan-sionin ourhuman studies. After 900rad TBI hemato-poietic reconstitutionwas attempted with autologous,

cryopreserved collections ofperipheral blood mono-nuclear cells obtained either at times of post-cyclo-phosphamide CFUc expansion (group A, 14 dogs) or without CFUc expansion (group B, 12dogs). As

com-pared to group B collections, group A collections contained11-foldmoreCFUcandwere 12.5-foldmore potentin fostering hematopoietic recovery afterTBI. These results suggest that the expansion of CFUc numbers we observed was accompanied by a similar expansion ofmore primitive hematopoietic stem cell numbers. We conclude that chemotherapy-induced

expansion ofcirculating CFUcnumbers appearsto be of substantial import in effecting hematopoietic re-constitution-an observation that may be of signifi-cance for further studies ofautologous hematopoietic reconstitution in man.

Presented in abstract form at the Advisory Council on

Federal Reports, American Society of Clinical Investiga-tion and Association of American Physicians meetings in May 1980.

Dr. Abrams' current address is the Medical College of Wisconsin inMilwaukee.

Received forpublication 30 June 1980 and in revised form 16 January 1981.

INTRODUCTION

Hematopoietic stem cells have been observed to cir-culate in the blood of several mammalian species in-cludingman(1-5). Moreover, studiesin animalmodels have demonstrated that autologous mononuclearcells collectedsolely from theperipheralblood arecapable of effecting hematopoietic reconstitution after an otherwise hematopoietically lethal insult from cyto-reductive drugs orirradiation (6, 7).

Studies are currently being undertaken in man to define the importance of autologous hematopoietic reconstitution when usedinassociation withintensive antineoplastic therapy (8). In the allogeneic setting, donors of hematopoietic stem cells will by selection tend to be free of major illness. For these healthy donors theneed to collect hematopoietic stem cells bymeans of bulkbonemarrowcollectionisunlikely to beamajor problem even though such collection is an operative procedure and requires general or regional anesthesia. Even so,theabilitytocollecthematopoieticstemcells byaless morbid procedure might be of useinallogeneic settings for donors who are unwillingto submitto an operative procedure. Thispointwas recently demon-strated by Rich et al. (9) in a case report involving immune reconstitution with allogeneic peripheral blood leukocytes in the setting of adenosine deaminase-de-ficient severe combined immuno-deficiency. In this report the donor (the patient's father) refused to be a bone marrow donor, butconsented to leukopheresis.

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autolo-gous marrow collection may be quite difficult. If hematopoieticreconstitutioncould bereliablyeffected usingthe hematopoietic stemcellscollected with one or two leukopheresis procedures, the morbidity and cost of collection of autologous hematopoietic stem cellswouldbe reduced and collection could be doneon an out-patientbasis.Thesefactors might well broaden the applicability and indications forintensive antineo-plastictherapyrequiringhematopoieticreconstitution. Unfortunately, to date, efforts in man at effecting autologous hematopoietic reconstitution using cells collected solely from the peripheral blood have been disappointing(11, 12). One important exception tothis statement, however, hasbeen inthe settingof chronic phase, chronic myelogenous leukemia. In this setting circulatinghematopoietic stemcellnumbers,as meas-ured by circulating granulocyte-monocyte precursor cells (CFUc),1 are greatly expanded, and autologous collections ofperipheral blood leukocytes have been effectiveinpromoting hematopoieticreconstitution(13). Interestingly, expanded levels of circulating CFUc numbers have been transiently observed in man fol-lowing the administration of antineoplastic therapy consisting of cyclophosphamide (Cytoxan) and doxo-rubicin (Adriamycin) either alone (14) or in combina-tionwithother agents (15) to patients with malignancies other than chronic myelogenous leukemia. However, whether this chemotherapy-associated expansion of circulating CFUcnumbers might have biologic

signifi-cance for hematopoietic reconstitution has been unclear. Consequently, we have developed a canine model for studying the impact of cyclophosphamide-induced expansion of circulating CFUc numbers on the reconstitutive potency ofcollections of peripheral blood mononuclear cells. The impetus for the study reported here stemmed directly from ourobservations in man that chemotherapy-induced expansion of circulating CFUcnumbers occurredto amedian level of sevenfold in 85% of patients studied during

induction chemotherapy for extensive small cell

car-cinomaof the lung (15). METHODS

This study utilized a canine model specifically designed to

study hematopoieticreconstitution.The details of thismodel

with respect to animal selection, care, irradiation,

postir-radiation supportive care, andcriteria for engraftment have

been described (16, 17). The model utilizes a

hemato-poietically lethal dose of total body irradiation (TBI) (900

rad midplane dose at 9 rad/min) followed by supportive

care with parenteral fluids, antibiotics,andplatelets. Gastro-intestinal and other nonhematopoietic toxicity is mild, and there are no deaths related togastrointestinal toxicity. Dogs

1

Abbreviations

used in this paper:

CFUc,

circulating granulocyte-monocyte precursor cells; TBI, total body irradiation.

failing to achieve hematopoietic reconstitution die from

neutropeniaand sepsis.

Collection of peripheral blood mononuclear leukocytes

was performed utilizing a continuous flow blood cell sepa-rator (American Instrument Co., Inc., Silver Spring, Md.).

Circulatory access was obtained via a left-sided,

jugulo-carotid,arteriovenous shunt, maintained for as long as 7 d with

daily heparin flushing. During cell collection, blood flow ratethrough these shunts to the cell separator wasmaintained at40-50 ml/min. The bowl speed of the cell separatorwas maintained at 1,000 rpm. Leukocyte collections containing 3.0-5.0g/100 ml hemoglobinwere obtained at flowrates of 3-4ml/min and harvested into 600-mlplastictransfer packs containing 13-15 ml of acid citrate dextrose-National In-stitutes of Health (NIH) solution A. Duringcollection

anti-coagulation was maintained withheparin.

After cell collection had been completed, shunts were ligated, allowed to clot, andremoved21 d later. The resulting

neck woundwasirrigated with antibioticsolutionandallowed to heal for an additional 1-2 wk.

14dogs(group A)weregivenintravenous cyclophosphamide (16mg/kg) on each of2d 1 wkapart(days 0 and 7)inorder to produce expansion of circulatingCFUc numbers. This dose and timingwere based on clinical observations in patients (15) and preliminary experiments in dogs. Dogs receiving

cyclophosphamide underwentshunt insertion onday10or 13

after the initial cyclophosphamide dose, and cellcollection was performed on days 14-16. Twelve dogs (group B) had

collections obtained without cyclophosphamide pretreat-ment. Alldogs studiedwerehealthy male foxhounds of

com-parable age (8-12 mo) and weight (group A, mean weight = 31.1+0.7 kg; group B, mean weight=29.1+0.7 kg,

P>0.10).

Parenteral antibiotics were used prophylactically while

shunts were in place, during cyclophosphamide-associated myelosuppression, and during wound healing after shunt

removal. Ampicillinandgentamicin were giventwicedaily in

doses of50mg/kg and3.0mg/kg, respectively.

Samples of blood and bone marrow forCFUc assay were

obtained in sterile syringes containing 200-300U of pre-servative-free heparin. Aliquots of peripheral blood cell collectionswere alsoanalyzed forCFUcgrowth.

Following collection, cells were cryopreserved in 10%

dimethyl sulfoxideasdescribed(16, 17),and thefrozen

prep-arations were immersed in liquid nitrogen (-196°C) until

needed for reinfusion. Time fromcell collection to cell

in-fusion (following TBI) averaged7wk. After TBI,dogs were

maintained with parenteral fluids, antibiotics, and platelets (transfused for thrombocytopenic bleedingorprophylactically

at a count of20,000/glor less). Beforeuse all routine blood products were irradiated to 2,500 rad. Blood counts were

obtained daily until death or full hematopoietic recovery occurred.

CFUc wereplatedin 0.3% agarusingtechniques described

from this laboratory (12) with the following modifications: erythrocyteswereremovedfrom both blood andbonemarrow usingdextran sedimentation (0.15 ml of3%Dextran [223,000 molwt]/mlofsample)at37°Cfor15-20 minfollowedby lysis of residual erythrocytes by ammonium chloride-potassium

lysing reagent obtained through the National Institutes of Health media room. Cultures were set up in supplemented

McCoy's mediacontaining 15% fetal calf serum (Gibco

Lab-oratories, Grand Island Biological Company, Grand Island,

N.Y.) (18)and8% (vol/vol) plasmacollected from a lethally

irradiateddog 10 d after 900 rad TBI and 4%(vol/vol)human placental conditioned media (19). These lattertwo reagents were added just before finalplating.Ameasured inoculum of cell suspension containing 1 x 106 peripheral blood

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nuclear cellsand<2.5 x 106granulocytes (20)wereplatedina final volume of1 ml in 35-mm Petri plates. Cultures were

incubated for 7d at 370C in a fully humidified ambient atmosphere containing 7.5%C02, and coloniescontaining a minimum of15cells (20) countedin duplicateor

quadrupli-cateplates.Bone marrowcellswereplatedinthesamemanner

utilizing 1 x 106 nucleated bone marrow cells in 35-mm

Petriplates.

CFUc values reported are means+1 (SEM). CFUc are

reportedin termsof CFUc/106 cells andasCFUc/ml.As

pre-viously noted (7) the latter value gives a better index of

CFUcavailable forcollection.This isparticularly important

following cyclophosphamide administration, when bothCFUc per 106 cells and cell counts per microliter are rapidly changing.

Statistical methods incorporated two-sidedP valuesin all

cases. Intergroup variation wascomparedusingthe nonpara-metricWilcoxonRankSum Test. Associations wereanalyzed

usingSpearman's Rank Correlation.Linear regressionanalysis

wasperformedutilizing themasterlibrarystatistics program

of a T1-58 programmable calculator (Texas Instruments,

Dallas,Texas).

RESULTS

Amplification of peripheral blood CFUc levels

during post-cyclophosphamide leukocyte recovery.

As shown in Tables I and II, groups A and B were comparable with respect tobase-line cell counts and CFUc levels in peripheral blood and bone marrow.

Intravenous cyclophosphamide, 16mg/kg,was admin-isteredon twooccasions 1wkapart(day0and7)toeach

group A dog. This cyclophosphamide regimen was

similarintimingandinresulting myelosuppressionto

the cyclophosphamide regimen thatwe hadobserved

to be associated with CFUc expansion during

leuko-cyte recoveryinman(15). Mean leukocyte andplatelet

nadirs in group A dogs were, respectively, 870+170 and

37,000+7,000/,ul.

As shown in Fig. 1, leukocyte nadir typically occurredonday10after the

administra-tionof cyclophosphamide andwaspromptly followed byaperiod of rapid leukocyte recoverybetween days 13 and 17.

Serial measurements of blood and bone marrow

CFUc levels were made on days 7 and 13-16 in four dogs andondays 13-17in fourdogs. Day 7 measure-ments were made prior to administering the second (day 7)dose ofcyclophosphamide. These preliminary experiments were undertaken to document whether the association between chemotherapy-induced

ex-TABLE I

Peripheral Blood CellCountsand CellularContentof Cell Collections

ObtainedbyContinuousFlowCentrifugation*

Group At GroupB§ P

Base-line bloodcounts

Hemoglobin, g/lO0 ml 17.2±0.3 16.5±0.7

Leukocytes,per ,u 15,200±800 14,200+600

Platelets,perpi 306,000±28,000 315,000±17,000

Bloodcounts:immediatelybefore cellcollection

Hemoglobin,gllOOml 12.2±0.4 15.6±0.4 <0.01

Leukocytes,per ,u 8,600+1,500 14,500+1,000 <0.01

Mononuclear cells,% 36±6 30±2

Cell collections

Volume, ml 373±37 462±39 11

Hemoglobin,g/lOOml 3.2±0.4 3.7±0.4

Leukocytes,per ,u 40,300+8,200 35,700+4,600

Mononuclearcells, % 59±6 53±3

Mononuclearcells,perml 20.6±2.6 x 106 17.9± 1.9 x 106 II

Platelets,per ,u 440,000+70,000 804,000+116,000 <0.05

Blood counts: immediately after cellcollections

Hemoglobin,g/lO0ml 11.7±0.4 14.4±0.7 <0.01

Leukocytes,per/4 5,700±1,100 10,000+700 <0.01

Mononuclearcells,% 30±4 21+4 <0.05

Platelets,per /4 48,000+7,000 93,000±11,000 <0.01

*Resultsexpressedas mean+ 1 SEM.

$ 14dogs, cyclophosphamideadministereddays0and 7. Cell collection betweendays 14-16.

§12dogs,cyclophosphamidenotadministered.

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TABLE II

CFUcMeasurements:Marrow,Blood, and Cell Collections Obtained byContinuousFlow Centrifugation*

Group At GroupB§ P

Base-linebone marrow CFUc levels

per106nucleated cells 249±60 136±32

per mlof aspirate 14,000+3,500 7,000±2,200

Base-lineperipheral blood CFUC levels

per106mononuclear cells 12.9±3.8 9.8±3.0

per mlof blood 55±16 67±33

Peripheralblood CFUc levels immediately before cell collection

per106mononuclearcells 286±111¶ 8.8±3.6 <0.01

per mlof blood 860±421** 34±15 <0.01

CFUccontent of cell collections

per 106 mononuclear cells 185±47 17±6tt <0.01

per mlof collection 3,300±800 300±100tt <0.01

* Results expressed as mean±1SEM. t As in Table I.

§ As in Table I. "P<0.10.

¶ GroupAresult 32-fold greater than group B result and 22-fold greater than groupA pretreat-ment level.

** Group A result 25-fold greater than group B result and 16-fold greater than group A pre-treatmentlevel.

t tGroupAresult 11-foldgreater than groupB result.

pansion of circulatingCFUclevels andrapid postnadir leukocyterecoverypreviouslyobservedinmanwould also occur in ourstudy animals. Before cyclophospha-mide we observed CFUcvalues of 55±16/ml, and on days 7, 13, 14, 15, 16,and 17 after the initiation of cyclo-phosphamidemeanvaluesof2+1,210+ 115,825±420,

1162±651, 445±108, and 285±132 CFUc/ml, respec-tively, were observed. Thus, on days 14, 15, and 16

when leukocyte recovery was, in fact, occurring most rapidly, CFUc levelswereexpanded 15,21,and8-fold, respectively. In two dogs CFUc measurements on day 22following theinitial dose ofcyclophosphamide revealed CFUc per milliliter values of 76 and 22 (mean 49±38), suggesting return to base-line levels. These observations of CFUc expansion were, thus, consistent with our observations in man associating postnadir leukocyte recoveryandpeak periodsof post-chemotherapy expansionofcirculatingCFUc numbers. Basedontheseresultsdays14-16 were selected for cell collection in all dogs receiving cyclophosphamide. A

totalof14dogswere studied for CFUcexpansion and

peripheralbloodleukocytecollection following

cyclo-phosphamide andamedianCFUcexpansionof 18-fold (range 3-105)wasobserved.Asshownin Table II the mean expansion ofCFUc numbers per milliliter was

16-fold.

Canine peripheral blood mononuclear cell col-lections obtained by continuous flow centrifugation. Immediately before cell collection blood counts in group A dogs (hemoglobin, leukocyte, and platelets) weresignificantly lower than those observed in group B

E E C.,

11,000 _

10,000

90000

80000

7000_

60000

5000

-4000

-3000

-2000

-I l I I A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 DAYSFROM START OF CYCLOPHOSPHAMIDE

FIGURE 1 Meanleukocyte responsetocyclophosphamide16

mg/kg given intravenopsly ondays 0and 7.Afterleukocyte

nadir onday10,postnadirrecovery occurredrapidlybetween days 13and 17. CirculatingCFUc levels were mostgreatly expanded on days 14, 15, and 16, andcellcollections were

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dogs (Table I). This differencewas adirect result of the myelosuppression that followed cyclophosphamide administration of group A dogs. Nevertheless, these differences did not hamper cell collection from the group A dogs and, as shown in Table I, comparable numbers of leukocytes and mononuclear leukocytes were collected from both groups. Collection was well tolerated, without mortality, in both groups. Imme-diately after cell collection, hemoglobin, leukocyte, andplateletcounts weresubstantiallylower ingroupA dogs (Table I), frequently to "clinically" significant levels, andplatelet and erythrocyte transfusions were givenprophylactically or because ofthrombocytopenic bleeding from puncture wounds to anumber of group A dogs.

These results show thatperipheral blood stem cell collectionwas possible andreasonably well tolerated in all animals studied, even those with

cyclophos-phamide-induced suppression of peripheral blood counts. Inaddition, they also suggestthat the myelo-suppression observed in this setting did not impair collection ofmononuclearleukocytes.Thisobservation is important because hematopoietic stem cells are, morphologically, mononuclear leukocytes (21).

CFUc content of cell collections was strikingly

in-creasedinthe groupAdogs.AsshowninTable II, col-lections from group A dogs contained 11-fold more CFUcper milliliterthancollectionsfrom groupBdogs.

For purposes of comparison we measured the CFUc content of20 normal canine marrows and observed thaton acell for cellbasis,CFUc contentofboth group A peripheral blood leukocyte collections and normal canine marrow werestrikingly similar (cell collection,

185±47; marrow,204±39/106cells; P> 0.10).

Survival afterTBI andinfusion of peripheral blood cell collections. After 900 rad TBI, group Adogs re-ceived thefollowingdoses ofcryopreservedautologous

peripheral blood mononuclear cells: 1 x 108/kg, four dogs; 0.5 x 108/kg, four dogs; 0.25 x 108/kg, one dog;

0.1 x 108/kg, five dogs. Group B (no cytoxan) dogs re-ceived doses of4 x 108/kg, fourdogs; 1 x 108/kg, four dogs; 0.5 x 108/kg, four dogs. 4 x 108 mononuclear

cells/kgwasthemaximum wecould consistently collect intwo,4-hcollections and theacutethrombocytopenia andanemiaproducedindogssomanageddiscouraged us from attempting a third collection during the 7-d life span of our arteriovenous shunts. In contrast, 1 x 108 mononuclear cells/kgcouldbe easilycollected in a single run lasting2-3 h.

InFig. 2we present the fraction of dogs at each cell dosewho recoveredleukocyte counts to aminimum of 1,000/mm3 and survived at least 40 d following TBI. Although the single groupA dog that was given 0.25 x 108 mononuclear cells/kg did recover counts and survive, he is notincluded in this figure, because re-sults in a single animal do not permit evaluation of

100

75F

.I

nh 50

25

a

,0

4/4 4/4

2/5

(1/4) (1

0.1

(3/4)

1/4)

4.0 10.0

FIGURE 2 Relationshipbetweensurvival and dose of periph-eral blood mononuclear cells (P. B. MNC) infused after900

rad TBI. GroupAdogsweregiven cells collectedattimesof cyclophosphamide-induced expansion ofcirculating CFUc levels (0). Group B dogswere given peripheral blood cells collected without CFUc expansion (a). Fractionsateach dose point indicate dogs surviving (at least 40d) over dogs tested.

fractional survival. In this model, as previously

de-scribed (16, 17), dogs achieving a leukocyte count of

1,000/,ul achieve full hematopoietic recovery with

granulocyte recovery paralleling leukocyte recovery

and platelet recovery occurringwith some delay, but

usually within 3-5 wk of leukocyte recovery. Our

experiencewasconsistentinthisregard; however,two

of our animals did experience "late" hematopoietic

deaths at day 36: one in group B (mononuclear cell

dose, 4 x 108/kg)thatengraftedonday22butwenton

to die ofthrombocytopenic hemorrhage, and one in

group A (mononuclear cell dose, 0.1 x 108/kg) that

engrafted on day 22 but in which hematopoietic

re-covery wasnotsustained andsepticdeathoccurredon

day 36with aleukocyte countof50/,l. Consequently,

we have chosen survivaltoday40as ourcriterion for

sustained and reliable hematopoietic survival. There

were no hematopoietic deaths (leukopenia, sepsis, or

thrombocytopenichemorrhage)seenafter day40. One

long-termsurvivorin groupAdiedonday76. At

post-mortem examination this dog was found to have a

ruptured urinary bladder. Because the two dogs

ex-periencing late hematopoieticdeath at day36did not

demonstrate sustained hematopoietic recovery, they

have notbeen included in Figs. 2and 3 (vide infra).

However, both animals had achieved leucocyte

re-covery to 1,000/,ul prior to death and have been

in-cluded in Fig. 4.

Although all of eight group A dogs given 0.5x 108/

kg or more mononuclear cells survived, we were

un-abletodemonstrate 100%survival for40datany dose

point forgroup B dogs including the 4 x 108/kg dose

which represented the maximal number of

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10,000

5,000

10

0

to

e~1000

500

U-S

5100

00

0o00

00

0

0

40

35

F 30 E 25

0

: 20

10

I0

10

0

O 0

0~~~~

0

0

0 0

Cytoxon

No

Cytoxon

FIGURE 3 Relationship between CFUc dose and survival in group A (Cytoxan) and group B (no Cytoxan) animals. Open circles indicate dogsachieving hematopoieticrecovery and surviving foratleast40d.Closedcircles indicate death

without complete hematopoietic recovery.

nuclear cells consistently collectable in two full 4-h runs. Atthis dose the nonsurviving group B dog did achieve a leukocyte count of

l,060/,ud,

but, as noted, died on day 36 while still receiving platelet support. From Fig. 2 the estimated doses of peripheral blood mononuclear cells associated with 50% survival are 2.5 x 108/kg for group B and0.2 x 108/kg for groupA. Theratioof thesetwoestimated50%survivalvaluesis 12.5 and is numerically comparable to the 11-fold mean increaseinCFUc content observed with groupA

collections.

Correlations amongCFUcdose,survival, andrateof

hematopoietic recovery. Fig.3relatessurvival,as de-fined for Fig. 2 (above), to CFUc dose per kilogram. In group A survival was consistently observed above 2 x 103 CFUc/kg and in group B survival was con-sistently observed above6x 103CFUc/kg. Inthe dose range of0.3 to 5 x 103 CFUc/kg more group A dogs

survived (fouroffive)than did group B dogs (one of

A R.-ar7

A 0 A

0 A A AA

100 1,000 10,000 100,000

Dos of CFUc/Kg

FIGURE 4 Relationship between CFUc dose and time to 1,000leukocytes/,ulafter 900 rad TBI. Increasing CFUc doses provided more rapid hematopoietic recovery. Correlation significant at P<0.01 level. 0, no cytoxan; A, cytoxan pretreatment.

five), but this difference was not statistically signifi-cant(P = 0.20, two-sidedttest).

Figure 4 relates rate of hematopoietic recovery

(daysto 1,000leukocytes/mm3) to CFUc dose. Linear regression analysis yielded an R value of 0.77 and seemed to provide a reasonable fit of the observed data, suggesting correlation between rate of hemato-poietic recoveryandCFUc dose. Subsequently, anal-ysis with Spearman's Rank Correlation confirmed sig-nificant association between CFUc dose and rate of hematopoietic recovery(P <0.01).

As shown by the data in Fig. 2 and including the single group A dog who received 0.25 x 108

mono-cytes/kg, 16 animals were long-term survivors. In

theseanimals median timetoleukocyte counts of500 and 1,000/mm3 and platelet countsof50,000/mm3were 13, 14,and45d, respectively.

Thus,inthis animal model, we observed that cyclo-phosphamide administration was associated with pre-dictable and reliable expansion of circulating CFUc numbers and that this expansion permitted collection of large numbers of peripheral blood mononuclear cells that contained increased numbers of CFUc. More-over, collections made during postcyclophosphamide expansion ofcirculating CFUc numbers were more potentinpromotinghematopoieticreconstitutionafter an otherwise lethal dose ofTBI than collections that did not contain increased numbers of CFUc. This sug-gests thatinthis setting, expansion ofCFUcnumbers was associated with a similar expansion of the more primitive hematopoietic stem cells (pluripotent) that are believed to be responsible for effecting hemato-poietic reconstitution.

DISCUSSION

Usinga caninemodel,wehave shown that cyclophos-phamide-induced expansion of peripheral bloodCFUc numbers is associated with an increased capacity of Biologic Significance of Expanded Circulating StemCell Numbers

I

%~%o

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peripheral blood mononuclear leukocytes topromote hematopoietic reconstitution after a hematopoietically lethal exposure toTBI. Based on oursurvival data in thisstudy we estimated the50% survivaldose ofcells collected duringCFUcexpansiontobe 0.2 x 108 mono-nuclear cells/kg. The estimated 50% survival dose of cells collected withoutCFUcexpansionwas2.5 x 108/ kg, 12.5 timeslarger,avaluewhichisnumerically com-parable to the 11-fold increased content of CFUc observed in cell collections made at times of CFUc

expansion.

Our results show that in this setting CFUc values provide anexcellent index of theability ofapopulation

of peripheral blood mononuclear cells to promote hematopoietic reconstitution. In contrast, peripheral blood mononuclear cell dose, per se, does not. This result is similar to observations recently reported re-garding CFUccontentand theabilityof humanmarrow collections toeffecthematopoietic reconstitution(22). None of our results suggests that the reconstitutive potency ofCFUcnumberswas inany wayacutely

im-pairedby the use ofcyclophosphamide (cf. Fig. 3). In thisregard itis of interest thatconsistentsurvival was noted withfewer total CFUc (-2 x 103/kg)inthe cyclo-phosphamide-treated dogs (group A)than inthecontrol animals (group B, -6 x 103/kg). Although this differ-ence wasnotstatisticallysignificant, itis possiblethat the ratio of CFUc to pluripotent stem cells available in the peripheral blood was altered during periods of CFUc expansion.Specifically, if thiscyclophosphamide

regimen resulted in a relatively greater expansion of pluripotent stem cell numbers than CFUc numbers on the days when cell collections were obtained, the number of CFUc required for consistent survival would be decreased. Whether the same expansion andbehavior of both circulatingCFUcand pluripotent stem cell numbers would have been observed under different experimental conditions is a matter for further study. However, in murine systems, as re-cently noted byQuesenberryand Levitt(23), manipu-lations that result in CFU proliferation or migration also resultin CFUcproliferation ormigration.

Aninteresting comparison maybemadebetween our survival results with peripheral blood mononuclear cells containing increasedCFUcnumbers and previous observationswithbone marrow cells made in the same canine model. Utilizing normal, autologous cryopre-served marrow cells, Gorin et al. (16), obcryopre-served 43% survival among dogs given 0.1 x 108 nucleated bone marrow cells/kg. In the current study we observed 40% survival among group A dogs given 0.1 x 108 periph-eral blood mononuclear cells/kg. Similarly, Gorin et al. (16)observed 100% survival among all dogs given a minimum of 0.5 x 108nucleated bone marrow cells and inthecurrent study we observed 100%survival among

all group A dogs given at least 0.5 x 108 peripheral blood mononuclear cells/kg.

Unfortunately, CFUc data are not available from Gorin's study (16)topermitdirectcomparisonof CFUc contentbetween their bonemarrowand ourperipheral blood cell collections.

Ifourresultsinthegroup B animals weretechnically unfavorable, our group A results might appear artificially superior. However, our group B data com-pare favorably with prior studies using peripheral blood mononuclear cells collected from dogsinthe ab-senceofCFUcexpansion.For example, Sarpeletal.(6) observed survival in four of four dogs given 1 x 109 peripheral blood mononuclear cells/kg and Nothdurft et al. (7) observed long-term survival in three offour dogs given 3-6 x 108 mononuclear cells/kg. Our re-sultsinourgroup Bdogsarecompletelyconsistentwith thosepreviously published experiences (three of four survivors at adose of4.0 x 108mononuclear cells/kg). Our CFUc values are also consistent with those pro-vided in these same two studies, both in terms of levels intheperipheral blood prior to treatment and in terms ofdoses associated with survival.

These results also suggest that with properattention to postcollection blood counts, especially hemoglobin and platelet levels, large numbers of peripheral blood mononuclear cells and circulating hematopoietic stem cells can be safely collected after cyclophosphamide administration in spite of associated, transient myelo-suppression. After cell collection, acute hematopoietic recovery continues in an uninterrupted manner.

In conclusion, these results suggest that observed expansion ofcirculating CFUc numbers in man fol-lowing nonintensive, systemic chemotherapy (14, 15) may permit collection of circulating hematopoietic stemcellsin numbers adequate to effectively promote hematopoietic reconstitution. Such a result would be consistent with currentstudiesinpatients with chronic myelogenous leukemia (13), and would be a major step forwardin developing further clinical applications for autologous hematopoietic reconstitution.

REFERENCES

1. Goodman,J. W., and G. S. Hodgson. 1962. Evidence for stem cells in the peripheral blood of mice. Blood. 10: 702-714.

2. Debelak-Fehir, K. M., R. Catchatourian, and R. B. Ep-stein. 1975. Hematopoietic colony forming units in fresh and cryopreserved peripheral blood cells of canines and

man.Exp. Hematol.(Oak Ridge). 3: 109-115.

3. Storb,R.T.,T. C.Graham, R. B. Epstein, G. E. Sale, and

E. D. Thomas. 1977. Demonstration of Hematopoietic stem cells in the peripheral blood of baboons by cross circulation. Blood. 50: 537-542.

(9)

5. Barr, R. D., J. Whang-Peng, and S. Perry. 1975. Hema-topoietic stemcells inhuman peripheral blood. Science (Wash. D. C.) 190: 284-285.

6. Sarpel,S. C., A. R.Zander,L.Horvath,and R. B. Epstein. 1979. The collection, preservation and finction of'

pe-ripheral blood hematopoietic cellsindogs.Ex1).Hematol. (Oak Ridge). 7: 113-120.

7. Nothdurft, W., C. Bruch, T. M. Fliedner, and E. Ruber. 1977.Studiesonthe regenerationof the CFUc population in blood and bone marrow of lethally irradiated dogs

after autologous transfusions of cryopreserved

mono-nuclear cells.Scand.J. Haematol. 19: 470-481.

8. Deisseroth, A., and R. A. Abrams. 1979. The role of autologous stemcell reconstitution in intensivetherapy forresistantneoplasms.Cancer Treat. Rep. 63: 461-471. 9. Rich,K.C., C. M.Richman,S.E. Mejia,andP.Daddona.

1980. Immunoreconstitution by peripheral blood

leuko-cytes inadenosine deaminase-deficient severecombined

immunodeficiency. J. Clin. Invest. 66: 389-395. 10. Abrams, R. A., D. Glaubiger, R. Simon, A. Lichter, and

A. B. Deisseroth. 1980.Haemopoieticrecovery in Ewing's sarcomaafterintensivecombinationtherapy and

autolo-gous marrowinfusion. Lancet. II385-389.

11. Hershko,D., R. P.Gale,W.G. Ho, and M. V. Cline. 1979.

Cure ofaplastic anemia in proxysmal nocturnal

hemo-globinuria by marrow transfusion from identical twin:

failure of peripheral leukocyte transfusion to correct

marrowaplasia. Lancet. 1: 945-947.

12. Abrams,R. A., D.Glaubiger, F. R.Appelbaum, andA. B. Deisseroth. 1980. Result of attempted hematopoietic re-construction using isologous peripheral blood mono-nuclear cells: a case report. Blood. 56: 516-520. 13. Goldman,J. M. 1978. Modernapproachestothe

manage-mentof chronic granulocytic leukemia. Semin. Hematol.

15: 420-430.

14. Richman, C. M., R. S. Weiner, and R. A. Yankee. 1976. Increase incirculatingstemcells following chemotherapy

in man.Blood. 47: 1031-1039.

15. Abrams,R. A., A.Johnston-Early,C.Kramer,J. D. Minna, M. H.Cohen, andA. B.Deisseroth.1981. Amplificationof circulating granulocyte-monocyte stemcell(CFUc)

num-bers following chemotherapy in patients with extensive

small cellcarcinomaof the lung.Cancer Res. 41: 35-41. 16. Gorin, N. C., G. Herzig, M. I. Bull, and R. G. Graw, Jr.

1978. Long-term preservation of bone marrow and stem

cell poolindogs.Blood. 51: 257-265.

17. Appelbaum, F. R., G. Herzig, R. G. Graw, M. I. Bull, C.

Bowler, N. C. Gorin, and A. B. Deisseroth. 1978. Study

of cell dose and storage time on engraftment of cryo-preserved autologous bone marrow in a canine model.

Transplantation(Baltimore).26: 245-248.

18. Pike, B. L., and W. A. Robinson. 1970. Human bone

marrow colony growth in agar gel.J. Cell. Physiol. 76: 77-84.

19. Burgess, A. W., E. M. A.Wilson, and D. Metcalf. 1977.

Stimulation by human placental conditioned medium of hemopoietic colony formation by humanmarrowcells. Blood.49: 573-583.

20. Kovacs, P., C. Bruch,andT. M. Fliedner. 1976. Colony

formation bycanine hematopoieticcells in vitro.

Inhibi-tion by polymorphonuclear leukocytes. Acta Haematol. (Basel). 56: 107-115.

21. Ross,W. M., M. Korbling, W. Nothdurft, W. Calvo, and T. M. Fliedner. 1977. Characterization of bone marrow and lymph node repopulating cells by transplanting mononuclear cells into radiated dogs.In Experimental Hematology Today. S.J. BaumandG. D.Ledney, editors.

Springer-Verlag, New York.29-37.

22. Spitzer,G., D. S. Verma, R. Fisher,A.Zander, L. Velle-koop,J. Litam, K. B. McCredie, andK. A. Dicke. 1980.

The myeloid progenitor cell: its value in predicting hematopoietic recovery after autologous bone marrow

transplantation. Blood.55: 317-323.

23. Quesenberry, P., and L. Levitt. 1979. Hematopoietic stemcells. II. N.Engl.J. Med.301: 819-823.

References

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