[CANCER RESEARCH 49. 706-710, February 1, 1989]

Establishment of Ascitic Tumor of Human Pre-B Acute Lymphoblastic Leukemiain Nonconditioned Nude Mice1

Yi Luo, Hideki Hará,Yuro Haruta, and Ben K. Seon2

Department of Molecular Immunology, Roswell Park Memorial Institute,' Buffalo, New York 14263

ABSTRACT

In the present study, an ascitic tumor of NALM-6, a human pre-Bacute lymphoblastic leukemia cell line, was established in nude micewhich had not been subjected to any preconditioning such as X-irradiationand/or splenectomy. The ascitic tumor was also established in X-irradi-ated mice. Under various conditions, the tumor transplantability was100%. NALM-6 cells were initially inoculated i.p. into 8-day-old nudemice after being admixed with X-irradiated III -1080, a human fibrosarcoma cell line. Then, the in vivo grown (for 10 wk) tumor cells wereserially transplanted i.p. into X-irradiated adult (10 to 12 wk old) nudemice. After the serial passages, we succeeded in establishing a highlytransplantable NALM-6 ascitic tumor in nonpreconditioned as well asX-irradiated nude mice without the addition of I IT-1((SOcells. The ascitictumor cells do not lose transplantability after in vitro culture for 20 days.Cellular radioimmunoassay and fluorescence-activated cell sorter analysis indicated that the in vivo established NALM-6 tumor cells retainedthe antigenic phenotype of the parental NALM-6 cells. Titration experiments revealed a reciprocal relationship between survival time of themice and the number of tumor cells inoculated. When appropriate numbers of tumor cells (e.g., 4 x 10'' cells) were used for the inoculation,

survival times of individual mice fell within a relatively narrow range;this narrow range will facilitate the experiments where the present tumormodel is used for evaluating the in vivo efficacy of antitumor agents. Thepresent ascitic tumor models, particularly the one established in nonpreconditioned nude mice, will be useful for evaluating the in vivo efficacyof anti-human leukemia agents as well as for studying the in vivobiological behavior of the transplanted leukemia cells.

INTRODUCTION

Human tumors transplanted into athymic nude (nu/nu) miceprovide a very useful in vivo model for studying therapeuticefficacy of various antihuman cancer agents (1-11). In thisregard, various human tumors, particularly solid tumors, werepreviously transplanted successfully into nude mice by a numberof investigators (1, 4, 6, 8-10). However, it has been difficultto transplant human ALL4 cells into nude mice (12, 13). Thisis particularly so for non-T/non-B (including pre-B) ALL cells.ALL of non-T/non-B phenotype is the major subtype of ALL(14-16).

Recently, we succeeded in efficiently establishing solid formsof human non-T/non-B ALL in nude mice (11). However, therehas been no dependable ascitic tumor model of human non-T/non-B ALL in nude mice. An ascitic form of human ALL innude mice will represent the original ALL better than solidforms of the tumor. Therefore, we initiated a study to establishan ascitic form of human non-T/non-B ALL in nude mice.

Received 7/28/88; revised 10/14/88; accepted 10/24/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by USPHS Grants CA19304 and CA42683 awarded

by the National Cancer Institute.2To whom requests for reprints should be addressed.3A unit of New York State Department of Health. Roswell Park Memorial

Institute is a Comprehensive Cancer Center-NCI designated.4The abbreviations used are: ALL, acute lymphoblastic leukemia; McAb.

monoclonal antibody; CALLA, common acute lymphoblastic leukemia antigen;RIA, radioimmunoassay; FBS, fetal bovine serum; FACS, fluorescence-activatedcell sorter; HEPES. 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; FITC.fluorescein isothiocyanate; SaMIg, sheep anti-mouse immunoglobulin.

The ALL cells we used were NALM-6, a pre-B ALL cell line;recently we established a solid form of NALM-6 tumor in nudemice (11). In our several preliminary experiments to establishNALM-6 ascitic tumor, NALM-6 alone or NALM-6 admixedwith X-irradiated HT-1080 (a human fibrosarcoma cell line)was transplanted i.p. into newborn (5 to 8 day old) nude mice.Then, the in vivo grown tumors were serially transplanted, withor without HT-1080, into X-irradiated adult nude mice to formascitic and solid tumors. Finally, we succeeded in establishingan ascitic NALM-6 tumor, without using HT-1080, into non-irradiated as well as X-irradiated adult nude mice. Under various conditions, all of the NALM-6-inoculated mice formedascitic tumors. In the ascitic tumor system, the 100% transplantability is very important for the clear interpretation oftumor suppression test results using various antitumor agents.The present tumor model meets this important requirement.Furthermore, the present establishment of human tumors intononirradiated mice may be particularly important in evaluatingthe in vivo efficacy of antihuman cancer agents as well as instudying the biological behavior of the transplanted tumor cells(see "Discussion").

MATERIALS AND METHODS

Mice. Pregnant BALB/c nude (nu/nu) mice as well as 6- to 8-wk-oldmale and female BALB/c (nu/nu) mice were obtained from HaríanSprague Dawley (Indianapolis, IN) through the Frederick Cancer Research Facility of the National Cancer Institute. Newborn mice wereobtained from either the purchased pregnant mice or from the matingof the nude mice housed in our laboratory. The mice were kept understerile conditions in cages with filter bonnets in a laminar flow unit(Lab Products, Maywood, NJ). They were given sterilized pellet foodand tap water (7).

Cell Lines. NALM-6, a human pre-B ALL cell line, was cultured inRPMI 1640 medium supplemented with 8% heat-inactivated FBS,penicillin (100 units/ml), and streptomycin (lOO/ig/ml) (17). HT-1080,a human fibrosarcoma cell line (18), was grown in monolayer culturein Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY)

supplemented with 10% FBS and gentamycin (50 /¿g/ml)(7).Establishment of NALM-6 Ascitic Tumor in Nude Mice. In several

preliminary experiments, newborn (5 to 8 day old) nude mice wereinoculated i.p. with various doses of NALM-6 cells with or without X-irradiated HT-1080 cells; HT-1080 cells were harvested after treatmentwith 0.2% trypsin, washed extensively, and X-irradiated with 6000 rads(1 rad = 0.01 Gy) (Ref. 7). Tumors with varying degrees of metastasiswere found in the inoculated mice. Based on these preliminary testresults, the following experiment was carried out. NALM-6 cells weremixed with a 2-fold excess of X-irradiated HT-1080 cells. The mixturewas washed and resuspended in saline to obtain a final concentrationof 6 x 107 mixed cells per ml. One-tenth ml of the cell mixture wasinjected i.p. into individual 8-day-old mice. Both ascitic and solid formsof tumor were found in the abdominal cavity of a mouse 2 mo after theinoculation of NALM-6. The ascitic and solid tumors were collectedseparately and mixed with '/isth the number of X-irradiated HT-1080cells. Approximately 5 x IO7 cells each of the two cell mixtures were

separately injected i.p. into individual adult (10 to 12 wk old) nudemice which had been X-irradiated weekly with 200 rads for 3 wk (11).The tumor cells were subjected to serial passages, initially with X-irradiated HT-1080 but later without HT-1080, until we obtained stable

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HUMAN NON-T-LEUKEMIA ASCIT1C TUMOR IN NUDE MICE

NALM-6 ascitic tumors from both ascitic and solid tumor preparations.In Vitro Culture of the in Vivo Grown NALM-6. The in vivo estab

lished NALM-6 cells were cultured in vitro under the same conditionsas those described for the parental NALM-6 cell line (see above).

Cellular RIA and FACS Analysis. Cell surface markers of the in vivoestablished NALM-6 tumor cells and the parental NALM-6 cells wereexamined in parallel by cellular RIA and FACS analysis. The details ofthe cellular RIA were described previously (19, 20). In the RIA, variousMcAbs were used under conditions of antibody excess. It should benoted that Fc receptors on the target cells were effectively blocked withhuman IgG during the assay (19, 20). FACS analysis was carried outas follows. Cells (1.5 to 2 million) were suspended in 10 to 20 i¿\ofRPMI 1640 medium containing 25 mM HEPES, 0.1% human IgG,0.5% bovine serum albumin, 2 mM EDTA, Trasylol (50 kallikreinunits/ml), and 0.1% NaN3 (Buffer A) and allowed to stand for 30 minat 4°C.Then, the cells were incubated with appropriate dilutions ofMcAb in Buffer A for 90 min at 4°C.After three washes with cold

phosphate-buffered saline, the cells were incubated with FITC-conju-gated F(ab')2 SaMIg (Sigma, St. Louis, MO) for 90 min at 4°C.The

incubated cells were washed 3 times with cold phosphate-buffered salineand suspended in 25 mM HEPES buffer, pH 7.2, in RPMI 1640medium containing 2.5 mM EDTA and 5% fetal bovine serum. To fixthe cells, 1 ml of the cell suspension was mixed with an equal volumeof a 16% formaldehyde solution in RPMI 1640 medium. The fixedcells were kept in the cold room protected from the light until analyzedby FACS using a Becton Dickinson FACS 440. Each sample containingat least 10,000 cells was analyzed using the log amplification mode.Negative controls were target cells labeled with control mouse IgG andFITC-labeled second antibody, i.e., F(ab'>2-SaMIg.

Histology. Sections from the tumor, peritoneum, lymph nodes, mes-enterium, skin, livers, spleens, kidneys, lungs, and brains were fixed in10% formalin, stained with hematoxylin-eosin, and examined by lightmicroscopy.

RESULTS

Transplantation of NALM-6 into Nude Mice. In several preliminary experiments, 5- to 8-day-old newborn nude mice wereinoculated i.p. with various doses of NALM-6 cells with orwithout X-irradiated HT-1080 cells (see "Materials and Methods"). Both NALM-6 alone and a mixture of NALM-6 and

HT-1080 were able to induce tumors with varying degrees ofmetastasis in the majority of the inoculated mice which survivedsubsequent killing by their mother. The tissues involved withthe tumors were liver, lymph node, kidney, lung, skin, mesen-tcrium, peritoneum, and/or diaphragm. Based on these preliminary results, we undertook the task of establishing a stable,transplantable ascitic tumor of NALM-6 to use as a model ofhuman non-T/non-B ALL. After serial passages of NALM-6tumor derived from an 8-day-old mouse (see "Materials andMethods"), we obtained a stable, highly transplantable NALM-

6 ascitic tumor. Photographs of a nude mouse carrying such atumor and a control normal nude mouse are shown in Fig. 1.The results of the transplantation of the ascitic tumor aresummarized in Table 1. Various doses of tumor cells wereinoculated into either nonpreconditioned or X-irradiated adult(10 to 12 wk old) nude mice. The in vivo established tumorcells were either uncultured or cultured (for 10 or 20 days) invitro before transplantation. In each of the 7 experiments (Nos.1, 2, 3, 8, 9, 10, and 11) where 2 x 10" to 1.6 x IO7tumor cellswere inoculated i.p. into X-irradiated mice, the transplantabilitywas 100% (38 of 38 total mice). In the 9 experiments (Nos. 4,5, 6, 7, 12, 13, 14, 15, and 16) where 2 x IO6 to 2.5 x IO7

tumor cells were inoculated i.p. into nonpreconditioned mice,the transplantability was 100% (37 of 37 mice) except forExperiment 12 where relatively smaller numbers of the tumorcells were used and the transplantability was 80% (4 of 5 mice);

B

Fig. 1. Nude mouse bearing an ascitic tumor of NALM-6. The picture of thetumor-bearing mouse (.1 and It} was taken 3 wk after inoculation with 2.5 x IIIcells of the in vivoadapted NALM-6. For a comparison, a tumor-free nude mouseis shown (C and £>).

Table 1 Transplantability of the in vivoadapted NAIM-6 cells into nude mice

No. ofexperiments12345678910111213141516X-irradiation"

No. of In vitroof mice NALM-6 cells cultureTransplantability'+

1 XIO7+1 x 10'++1 xIO71

xIO72.5xIO72.5x IO7+1.5xlO7++

2x10«++4x10«++8x10'++

1.6x10'+2x 10«+4x

10«+8x10«+1.6

x IO7+1.4x10'4/410/104/45/55/54/44/45/55/55/55/54/55/55/55/54/4

"Mice were X-irradiated weekly with 200 rads (1 rad = 0.01 Gy) for 3 wk

before transplantation.b Results are expressed as number of mice with tumors/total number of mice

inoculated with NALM-6.

the effect of the tumor cell number on the transplantability andsurvival time is described below (see Fig. 2). The in vitro culture(for 10 or 20 days) of the in vivo established tumor cellsimmediately prior to the transplantation did not significantlyaffect the transplantability of the tumor cells in either X-irradiated or nonirradiated mice (Table 1). In a more recentexperiment, 1.5 x IO7 tumor cells which had been cultured in

vitro for 45 days were able to induce tumor in 4 of 4 nonirradiated mice.

We examined the effect of tumor cell number on the transplantability and survival time of the tumor-bearing mice forboth the X-irradiated and the nonirradiated mice (Fig. 2). Inthe case of the X-irradiated mice, the transplantability was100% between 2 x IO6 and 1.6 x IO7 tumor cells, and there

was an inverse relationship between the survival time and thenumber of tumor cells inoculated in the range of 2 x IO6 to 8x IO6 cells (Fig. 2A). Average survival times were 24.2, 28.4,

and 33.2 days, respectively, for the mice inoculated with 8 xIO6, 4 x IO6, and 2 x IO6cells. No significant difference in thesurvival time was observed between 8 x IO6and 1.6 x IO7cells

(24.2 versus 23.8 days) (Fig. 2A).In the case of nonirradiated mice, the transplantability was

100% between 4 x IO6 and 1.6 x IO7 tumor cells, and average

survival times were 27.8, 29.4, and 31.8 days, respectively, for1.6 x IO7, 8 x IO6, and 4 x IO6cell-doses (Fig. 2B). For the 2x IO6 cell-dose, three of the five mice tested died on Days 34,

36, and 37 after tumor inoculation, and one of the remainingmice was found to have a tumor 11 wk after tumor inoculation.

When the X-irradiated and the nonirradiated mice were

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HUMAN NON-T-LEUKEMIA ASCITIC TUMOR IN NUDE MICE

60

40

20

O»

»s100

80

60

40

20

A. X Irradiated MiceX l 6-10*o e. io'A 4. io'

123456

B. Non Irradiated Micel 6 ' IOe. to'

4 .IO*2.10'

Ot 234 56789 IO

Weeks After Tumor Inoculation

Fig. 2. Dose-dependent survival time of nude mice which were inoculated withthe in vivo adapted NALM-6. The mice had been either X-irradiated (A) ornonirradiated (B) before the inoculation. In both Experiments A and B, 20 micewere divided into 4 groups. Individual mice of each group were inoculated with1.6 X 107,8 X 106,4 X 10', or 2 x 106cells of NALM-6. One of the two surviving

mice of Group 4 of Experiment B was found to have tumor 11 wk after tumorinoculation.

compared, the average survival times were slightly shorter forthe X-irradiated mice at each of the tumor doses except that,at the lowest dose (i.e., 2 x IO6 cells), the difference became

pronounced (Fig. 2).Except for the 2 x IO6 cell-dose in the nonirradiated mice,

survival time for each group of either X-irradiated or nonirradiated mice was within a relatively narrow range (Fig. 2, A andB). This narrow range of survival time will facilitate the experiments where the present tumor model is used to evaluate thein vivo efficacy of antitumor agents.

Membrane Phenotype of the in Vivo Established Tumor Cells.Cell surface phenotypes of the in vivo established NALM-6tumor cells and the parental NALM-6 cells were examined inparallel by a cellular RIA and FACS analysis. The parentalNALM-6 cells expressed antigens of CD9, CD 10, CD24, andHLA-DR as well as a novel leukemia-associated cell surfaceantigen GP160 (21). They did not express CD20 antigen.Immediately prior to the testing, the in vivo derived tumor cellswere cultured in vitro for 10, 20, and 30 days to remove host-derived contaminating mouse cells. Before the in vitro culture,the tumor cells had been maintained in vivo for about 1 yr. Itshould be noted that the in vitro culture for 10 and 20 days didnot significantly affect the transplantability of the in vivo established NALM-6 tumor cells (see above Table 1). In the cellularRIA, we used 5 McAbs, i.e., SN3, SN4, SN5, SN6, and G4-3A7, all of which were generated in our laboratory and reactwith non-T leukemia cells including NALM-6. SN3 (anti-CD24) defines sialic acid of a B-lineage-restricted glycoprotein(22), whereas SN4 (anti-CD9) defines a cell surface antigentermed p24.5 SN5 (anti-CDIO) and SN6 define the commonALL antigen, termed CALLA (17), and a novel leukemia-associated antigen, termed GP160 (21), respectively. G4-3A7is directed to a public determinant of HLA-DR antigen.6 No

5T. Fukukawa, Y. I lanini, and B. K. Seon, unpublished data.6 H. Matsuzaki and B. K. Seon, manuscript in preparation.

significant difference for any of the five cell surface markerswas observed between the parental NALM-6 and the in vivoderived NALM-6 (Table 2). This observation was made repeatedly in three separate experiments in which each of the 10-, 20-,and 30-day cultured NALM-6 tumor cell preparations wascompared with the parental NALM-6 (Table 2).

In another test, FACS was used to compare the phenotypesof the in vivo established tumor cells and the parental tumorcells. The results are summarized in Table 3. In the FACSanalysis, McAbs SN5 and SN6f plus SN6h were used. BothSN6f and SN6h define the novel leukemia-associated antigenGP160 as does SN6 (23). However, they are directed to differentepitopes from that of SN6. The combined use of SN6f andSN6h shows an additive effect.7 Consistent with the cellular

RIA results, the FACS analysis results indicate that the expression of CALLA and GP160 on NALM-6 was maintained afterthe in vivo adaptation of NALM-6.

Thus, both the cellular RIA and the FACS results indicatethat the in vivo adaptation of NALM-6 did not result in any

significant change of the cell surface phenotype.Histology. During the early stages of the transplantation,

metastasis of the tumor into various tissues was observed (seeabove). However, the established ascitic tumor which was obtained after serial transplantation without using HT-1080 cellsappeared not to involve metastasis into other tissues. Therefore,we examined the histology of the tissue sections of variousorgans (see "Materials and Methods") from three tumor-bear

ing mice. All the examined organs were tumor free except thatmesenterium contained adhering tumor cells. The ascites ofthese mice contained over 20 times more tumor cells as compared to the number of the inoculated NALM-6 cells.

DISCUSSION

A number of human solid tumors are readily transplantablein athymic nude mice (1, 4, 6, 8-10). However, tumors ofhematopoietic malignancies are generally more difficult totransplant in nude mice (12, 13, 24, 25). This is particularly sofor ALL. Because of this difficulty, most attempts at transplanting ALL cells into nude mice have been carried out usingimmunosuppressed nude mice (5, 11, 26-28). Among differenttypes of ALL, non-T/non-B (including pre-B) ALL is the mostdifficult to transplant in nude mice (11). Previously, we succeeded in efficiently transplanting NALM-6, a pre-B ALL cellline, into whole body X-irradiated nude mice to form subcutaneous solid tumors (11). This tumor model was successfullyused for evaluating the in vivo efficacies of immunotoxins (11).However, an ascitic tumor model could represent ALL betterthan the solid tumor model. But, no dependable ascitic tumormodel of non-T/non-B (including pre-B) ALL has been available. Therefore, we undertook the present study. Furthermore,we wanted to avoid using the widely used immunosuppressivepreconditioning (i.e., whole body X-irradiation and/or splenec-tomy) of nude mice before transplantation of human tumors.This immunosuppression of the host limits the usefulness ofthe nude mouse models for evaluating the in vivo efficacy ofantihuman cancer agents as well as studying the in vivo behaviorof the transplanted human tumors. In this regard, it may beworthy to mention that recently we found that interferon, abiological response modifier, substantially enhanced the in vivotherapeutic efficacy of immunotoxins in a nude mouse modelwhich was not immunosuppressed (29). The immunocompe-

7 Y. Harina and B. K. Seon, manuscript in preparation.

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Table 2 Cellular RIA to compare the cell surface markers of the in vivo adapted NALM-6 cells with those of the parental NALM-6 cellsThe in vivo adapted NALM-6 cells were cultured in vitro for 10, 20, or 30 days before the assay. In each assay, the parental cells were tested in parallel.

McAbs"

or mousecontrolIgGSN3SN4SN5SN6G4-3A7Control

IgGExperiment

110-day

Parentalcultured cellscells4557

±395* 4355 ±196812+ 316 6139±1787

193 ±350 7408±9032264±667 3464 ±4274687±243 6220 ±346377±18 385 ±21Experiment

220-day

Parentalcultured cellscells4102

±377 3044±3915121+ 266 4581 ±1584892±107 4725 ±3221714+ 534 1527±5323890+ 331 3860 +311316±117

285±111Experiment

330-day

Parentalcultured cellscells4699

±65 4536 +6144541±74 4595 +2275448+143

5820+1111522±506 1529±5144520+

110 5091 +171599±146 529 ±49

" McAbs and the control IgG are all IgG I.* Mean + SD (cpm) of triplicate tests.

Table 3 FACS analysis to compare the cell membrane markers of the in vivoadapted NALM-6 cells with those of the parental cells

Values given are the percentage of positive cells.

McAbs"

or mousecontrol

IgG

Experiment 1 Experiment 2 Experiment 3

10-day 20-day 30-daycultured Parental cultured Parental cultured Parental

cells cells cells cells cells cells

SN5SN6f + SN6hControl IgG

94.872.1

1.1

95.295.4

5.3

93.073.3

1.2

95.376.5

6.7

83.252.34.1

82.143.7

5.5' McAbs and the control IgG are all IgGl.

tency of the host (i.e., nude mice) may be important in such anactivity of biological response modifiers.

In an attempt to establish an ascitic tumor model of non-T/non-B ALL, we inoculated i.p. NALM-6, with or without X-irradiated HT-1080, into newborn (5 to 8 day old) nude mice;HT-1080 probably provides an angiogenesis factor(s) and/or atumor-promoting factor(s) (11,27, 30). We repeatedly observedformation of metastatic tumors with varying degrees of asciticfluids in many of the inoculated mice which survived killing bytheir mother. After serial passages of the tumors, we obtaineda stable, highly transplantable ascitic tumor. The followingproperties of the established tumor point to the usefulness ofthe present tumor model in assessing the in vivo efficacy ofantihuman leukemia therapeutic agents, (a) Under various conditions, the transplantability was 100% (see Table 1). (b) Thehigh transplantability of the tumor applies to the nonprecon-ditioned nude mice as well as the X-irradiated nude mice (Table1). (c) The in vivo established tumor cells can be cultured invitro without any significant loss of transplantability (Table 1).Therefore, the number of tumor cells can be readily expandedin vitro and the in vitro cultured tumor cells which are free ofthe host-derived murine cells can be used for efficient transplantation, (d) Titration experiments showed an inverse relationship between the survival time of the mice and the numberof tumor cells used for inoculation, (e) The survival times ofindividual mice fell within a relatively narrow range at a fixedtumor-dose (Fig. 2). This rather homogeneous survival patternwill facilitate the interpretation of the test results in assessingefficacy of antitumor agents. (/ ) The in vivo adaptation of thetumor cells apparently did not cause significant change of themembrane phenotype of the parental tumor cells (Tables 2and 3).

We are currently using the present tumor model in assessingthe in vivo efficacy of antihuman non-T leukemia immunotox-ins. After further characterization of the in vivo adaptedNALM-6 cells, we intend to deposit the cells in a publicdepository such as the American Type Culture Collection.

ACKNOWLEDGMENTS

The authors wish to thank J. Prendergast and H. Tsai for theirexcellent technical assistance and Dr. J. Krasner, D. Ovak, and C.Zuber for their help in the preparation of the manuscript.

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HUMAN NON-T-LEUKEMIA ASCITIC TUMOR IN NUDE MICE

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1989;49:706-710. Cancer Res   Yi Luo, Hideki Hara, Yuro Haruta, et al.   Lymphoblastic Leukemia in Nonconditioned Nude MiceEstablishment of Ascitic Tumor of Human Pre-B Acute

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