[CANCER RESEARCH 54, 4999-5004, September 15, 19941

have been treated with radiation or alkylating agents, and the medianinterval from diagnosis of the primary tumor to development ofleukemia was 45 months. Recently, the antineoplastic agent etoposidehas been associated with the development of acute myeloid leukemia,usually with specific chromosomal translocations involving 11q23(27—32). Thus, patients with a mediastinal GO', who are now fre

quently treated with etoposide-based regimens as well as radiation andalkylating agents, are at risk to develop a hematopoietic malignancyinduced by one of several possible mechanisms.

We report on a patient who developed an overt hematologicalmalignancy with features characteristic of acute lymphoblastic leukemia 5 months after the diagnosis and treatment of a mediastinal yolk

sac tumor. Cytogenetic analysis showed an i(12p) abnormality in thepatient's leukemia cells and in a cell line (UoC-B10) established fromthe leukemia cells. The i(12p) was retrospectively identified in themediastinal tumor cells by FISH analysis. The UoC-B10 cells possessed receptors for O-CSF, a cytokine which the patient received aspart of his treatment protocol.

MATERIALS AND METHODS

Case Report. A previously healthy 26-year-old male presented in September, 1991, with acute onset of back pain. Radiographic studies revealed anepidural mass with bony erosion and an anterior mediastinal mass. Thecomplete blood count was normal, AFP was 3210 ng/ml (normal, <5 ng/ml)and the f3HCGwas 18 milli-international units/mi (normal, <5 milli-international units/mi). The patient underwent a decompression laminectomy, andpathological review of the specimen revealed a yolk sac tumor (Fig. IA). In theBM presentwithin this specimen, the myeloid and erythroidlines appearednormal, micromegakaryocytes were observed, and focal involvement withtumor cells was noted. The patient was treated with cisplatin, etoposide, andbleomycin chemotherapy, followed by daily G-CSF on Intergroup protocol3887. He received 21-day cycles ofcisplatin 20 mg/m2/day X 5, etoposide 100mg/m2/day X 5, bleomycin 30 units i.v. on days 2, 9, and 16, and G-@SF (5

I.Lg/kgs.c.) from day 7 to day 18 with G-CSF doses omitted on days 9 and 16.During chemotherapy, the patient's hemoglobin gradually decreased from

14.3 to 8.2 g/dl, but this was felt to be related to cisplatin. After 4 cycles of

chemotherapy, the mediastinal mass had decreased markedly in size, and theAFP and f3HCGlevels had returned to normal. A bone marrow examinationwas done to assess cellularity and document the absence of bone marrowinvolvement by tumor prior to a bone marrow harvest for cryopreservation.The peripheral blood smear was notable for extreme red cell anisopoikilocytosis and marked polychromatophiia, as well as a large number of nucleatedRBC. The platelets were moderately decreased, and the WBC appeared normal. The bone marrow biopsy was hypercellular, approaching 100%.Megakaryocytes were plentiful, although many were small and immature.Erythroid hyperplasia was present, and the myeloid line showed a shift towardsimmaturity. There was no evidence of metastatic tumor or reticulum fibrosis.The overall impression of this bone marrow specimen was exuberant regencrating marrow in a patient receiving a hematopoietic growth factor followingintensive chemotherapy.

A bone marrowharvestandresectionof the residualmediastinalmass wasperformed. On histological review, the mediastinal mass showed extensivenecrosis and elements of mature teratoma which contained hematopoietictissue (Fig. 1, B and C). Prior to the resection and bone marrow harvest, mildthrombocytopenia was noted (70,000/pi). Postoperatively, the patient received

4999

Establishment of a Leukemia Cell Line with i(12p) from a Patient with a

Mediastinal Germ Cell Tumor and Acute Lymphoblastic Leukemia'

Peter A. Downie, Nicholas J. Vogelzang,2 Richard L. Moidwin, Michelle M. Le Beau, John Anastasi, Rudy J Allen,Susan E. Myers, Richard A. Larson, and Stephen D. SmithDepartments of Pediatrics [P. A. D., R. L M., R. J. A., S. D. S.], Medicine [N. J. V., M. M. L, R. A. L, S. E. MI, and Pathology [J. A. I. and the Cancer Research Center,University ofChicago, Chicago, Illinois 60637-1470

ABSTRACT

We report the establishment of a leukemia cell line (UoC-B1O) from apatient who developed leukemia several months after the diagnosis of amedlastinal yolk sac tumor. The patient's yolk sac tumor responded tocombination chemotherapy, and a mature teratoma with focal areas ofhematopolesis was subsequently resected. However, 5 months after theInitial diagnosis, the patient developed an acute lymphoblastic leukemiawith a precursor B-cell phenotype. Cytogenetic analysis showed an i(12p)abnormalIty In the patient's leukemia cells and In the UoC-B1O cell line.Thei(l2p) wasalsoidentifiedretrospectivelyInthemedlastinaltumorcells by fluorescent in situ hybridization analysis. The UoC-B1Ocell line,which has been growing continuously for >24 months In culture, wasEpstein-Barr virus negative and was generally concordant with the padent's leukemia cells by analysis of Immunophenotype, karyotype, andgenotype. The UoC-B1O cell line possesses receptors for granniocytecolony-stimulating factor, a cytokine which the patient received as part ofhis treatment protocoL This cell line may be useful in studying therelationship between i(12p) and hematological differentiation of humanmediastinal germ cell tumors.

INTRODUCTION

Approximately 700 mediastinal OCTs@ occur each year in theUnited States (1). These tumors typically have yolk sac histology, arelocally extensive or metastatic at the time of diagnosis, respond wellto chemotherapy, but are cured in less than 50% of the cases (2).Recently, an association between mediastinal OCT's and malignanthematological disorders has been recognized. Over 30 cases havebeen reported and, while most of the second malignancies are AML,there has been a disproportionate number of uncommon subtypesreported, such as erythroleukemia and acute megakaryoblastic leukemia (3—26).In some patients, the leukemia was present at the time of(or even prior to) the diagnosis of the mediastinal OCF and, in most

patients, the hematological dysfunction was detected within 12months of the initial diagnosis (3—26).Thus, this syndrome differsfrom therapy-related AML that follows prior exposure to alkylating

agents, topoisomerase II inhibitors, or radiation therapy.In a review of 722 patients treated with non-etoposide-based regi

mens for germ cell tumors at Memorial Sloan-Kettering Cancer Center over a 30-year interval, Redman et a!. (1 1) observed an increasedincidence of leukemia. Patients who developed leukemia tended to

Received 3/28/94; accepted 7/20/94.Thecostsof publicationof thisarticleweredefrayedin partby the paymentof page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I SupportedbyaresearchgrantfromB.MeltzerandNationalCancerInstituteOrantsP01 CA 40046 and CA 14599. J. A. is a Special Fellow of the Leukemia Society ofAmerica.

2 To whom requests for reprints should be addressed, at Department of Medicine,

University ofChicago Medical Center, MC 2115, 5841 South Maryland Avenue, Chicago,IL 60637-1470.

3 The abbreviations used are: OCI', germ cell tumor; AML, acute myeloid leukemia;

UoC-, cell line established at the University of Chicago; FISH, fluorescence in situhybridization; O-cSF, granulocyte-colony-stimulating factor; AFP, serum a fetoprotein;PHCO, serum (3-subunitof humanchorionic gonadotropin;BM, bone marrow;MDS,myelodysplastic syndrome; BCP-ALL, B-cell precursor acute lymphoblastic leukemia;TdT, terminal deoxynucleotidyl transferase; ADA, adenosine deaminase; cDNA, complementaryDNA;EBV,EpsteinBarrvirus;PCR,polymerasechainreaction.

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LEUKEMIA CELL LINE WITH i(12P)

Source of Malignant Cells. Leukemia cells were obtained from peripheralblood and BM samples at the time of diagnosis of leukemia, anticoagulatedwith preservative-free heparin, and separated into aliquots for cell cultureexperiments, irnmunophenotyping, genotyping, and cytogenetic analyses. The

protocol procedures were approved by the Institutional Review Board andinformed consent was obtained.

Establishment and Maintenance of the Cell Line. The technique forculturing leukemia cells was a modification of our previously reported method(33). Briefly, Ficoll-Hypaque gradient-separated cells were washed twice,

plated (1 x i0@cells/mi; 0.3 ml) onto 24-well Petri dishes, and cultured in anincubator gassed with 5% 02/6% C02/89% N2. Each well contained a feederlayer consisting of a mixture of media, agar (0.5%), and human serum (10%).

Characterization of Cellular Antigens. Cell surface antigens were evaluated on BM cells and the UoC-B10 cell line by indirect immunofluorescence using fluorescein isothiocyanate-conjugated antibodies and analyzedby flow cytometry.

Tumor Markers and Enzyme Evaluation. AFP and fJHCG were measured using the IMx AFP and IM.x total I3HCG assays, respectively (both fromAbbots Laboratories, Abbots Park IL). TdT activity was assayed by an ianmunofluorescencekit (Supratechs, Bethesda, MD), while ADA and nucleosidephosphorylase activity were measured as described previously (34).

Gene Rearrangement Studies. DNA was extracted from BM and theUoC-B10 cell line and analyzed by Southern blot hybridization (35). DNA wasdigested with two restriction enzymes (EcoRI and HindIII), electrophoresed on

0.8% agarose gels, transferred to nylon membranes, and hybridized withcDNAs from the human immunoglobulin heavy chain gene (J,@@)and theEpstein-Barr virus.

Cytogenetic Analyses. Cytogenetic analyses using a trypsin-Giemsa banding technique were performed on the “MDS―BM aspirate, the “ALL―BMaspirate, and the UoC-B10 cell line. Metaphase cells were prepared directly or

following short-term (24 or 48 h) culture without mitogens. Chromosomalabnormalities were described according to the International System for HumanCytogenetic Nomenclature (1991).

FISH Analysis. FISH analysis was performed using a biotinylated probespecific for the a-satellite repeat sequences in the centromeric region ofchromosome 12 (Oncor, Gaithersburg, MD). FISH analysis was performed onthe MDS specimen, on fresh leukemia cells, and tissue sections of the mcdiastinal germ cell tumor using methods described previously (36—38). The cell

nuclei in the tissue sections were counterstained with propidium iodide.G-CSF Studies. The expressionof G-CSFreceptorswas evaluatedon the

UoC-B10, U937 (positive control), and UoC-M1 (negative control) cell linesby PCR and binding studies. Reverse transcription was performed on mRNA

isolated from the cell lines. The resulting cDNA was amplified (35 cycles) byPCR using primers specific for the G-CSF message: 5' primer, 5'-CACCTGCCTCTOTGGAACTG-3'; 3' primer, 5'-CAGGTCTCTGAGCTGTTATG-3' (positions 2086 to 2105 and 2322 to 2303, respectively; Ref. 39).

The presence of cell surface G-CSF receptors was evaluated using aFluorokine G-CSF flow cytometry kit (R&D Systems, Minneapolis, MN).Cells were processed according to kit specifications and incubated with: (a)

unlabeled G-CSF; (b) streptavidin-phycoerythrin; (c) phycoerythrin-conjugated G-CSF (G-CSF-PE); or (d) G-CSF-PE after the cells had been preincubated (60 mm) with 100-fold molar excess of rG-CSF (Amgen, ThousandOaks, CA). Cells were analyzed by flow cytometry using a 488-nm wavelengthlaser excitation.

The effect of rG-CSF on leukemic cell growth was determined. Test cellswith >95% viability were evaluated while in log phase growth and whilegrowing in McCoy 5A media supplemented with either 10% fetal calf serumor serum substitutes (40). Cells (1—10X 10@/well)were cultured with andwithout supplemental rG-CSF (1—200U/mI) for 48 h, pulsed with 1 pCi[3H]thymidine (2 Ci/mmol; Amersham, Arlington Heights, IL) for 4 h, and

thymidine incorporation was determined by a liquid scintillation analyzer(TRI-CARB; Packard Instrument Co., Downers Grove, IL).

RESULTS

Establishment of UoC-B1O Cell Line. In the cultures of theperipheral blood sample, leukemia cell viability gradually fell to lessthan 1% during the first 14 days of culture. However, during the third

,

@..

@ A@a4Fig. 1. A. yolk sac tumor from laminectomy specimen showing a myxomatous pattem

with strands of epithelial-like cells. B. resection of mediastinal mass showing features ofa mature teratoma with cartilage, intestinal epithelium, and respiratory mucosa. C. highpower of hematopoiesis (erythroid and granulocytic activity) within teratoma.

platelets and RBC transfusions for a mediastinal hemorrhage and persistentthrombocytopenia.

A bone marrow examination was then performed 3 weeks postoperativelyand, in comparison to the preoperative BM biopsy, showed a hypercellular

marrow with decreased megakaryocytes, increased blasts (5%), and dysplasticfeatures in all cell lineages, findings consistent with MDS. Two weeks later,blasts were noted in the blood, the serum lactate dehydrogenase rose dramat

ically, and a second bone marrow sample showed a marked hypercellularmarrow with 65% blasts. The blasts were myeloperoxidase negative andgenerally had L-1 lymphoid features, but some resembled erythroblasts while

others had L-3 morphology (Fig. 2A). The immunophenotype was consistentwith BCP-ALL (Table 1). Remission induction chemotherapy was started, butthe patient suffered an intracranial hemorrhage and died 10 days after the start

of therapy. Permission for an autopsy was denied.

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LEUKEMIACELLUNE V.11THi(12P)

L.!@ç@:@$:.

Fig. 2. A, cytospin preparation of leukemic blastsat the time of diagnosis of ALL (Wright's stain). B,leukemic blasts studied with FISH analysis with acentromeric probe to chromosome 12. Two brightsignals represented the two copies of chromosome12.,the small signal likely represents the i(12p). C,occasional cells from the MDS BM aspirate showingthe same three-signal pattern. D, tissue sections ofthe teratoma showing a cell with two bright and oneweak signal after in situ hybridization with the centromere 12 probe. A-D, X 1000.

week, cell proliferation was observed which continued after the cellshad been passed to suspension culture. The UoC-B10 cell line hassustained growth for more than 200 passages and has proliferated formore than 24 months in suspension culture.

Inununophenotype. The leukemia cells from the patient and thecell line expressed the immunophenotype of a B-cell leukemia:CD45+,HLA-DR+,CD1O+,CD19+,CD38+(Table1).Incontrastto the patient's leukemic cells, the UoC-B10 cell line expressed

IgD/lambdaand CD4 but none of the 6 other T-lineage antigenstested. While the patient's marrow expressed 30% positive cells forglycophorin, it was not directly determined whether the patient'sblasts expressed glycophorin. It is of interest that the cell line had

weak, but defmite and reproducible, expression ofglycophorin (12%).Cytochemical Stains, Tumor Markers, and Enzymes. Both the

patient's leukemic blasts and the UoC-B10 cells were nonreactivewhen stained with the myeloperoxidase, a-naphthyl acetate esterase,and PAS stains, and both lacked TdT activity. The UoC-B10 cells had

low ADA activity (4.6 EU/mg) compared to typical BCP-ALL cellsand BCP-ALL cell lines (41, 42). Also, AFP and carcinoembryonicantigen were not detected in either concentrated (10-fold) or unconcentrated UoC-B10 cell line culture media (the sensitivity of theassays was 1 ng/ml and 5 milli-international units/ml, respectively).These results are consistent with the results of Paiva et aL (43) whodemonstrated that somatic components within GC1's lack AFP.

Karyotype Analysis. Cytogenetic analysis on the MDS BM aspirate revealed a male karyotype with four chromosomally abnormal

clones (Table 2). The primary clone contained a Robertsonian translocation involving the chromosome 13 homologues [45,XY,der(13;13) (qlO;qlO); 21%]. A gain of an isochromosome for the short arm

of chromosome 12, i(12p), was observed in one clone (clone 4; 6%)and in eight of the nine nonclonal abnormal cells. In addition, therewere two additional unrelated clones (2 and 3) observed.

Analysis of the leukemic BM aspirate revealed three of the fourabnormal clones observed initially, as well as two new clones, andeight nonclonal abnormal cells. The i(12p) was noted in 80% of cellsexamined. Karyotype analysis of the UoC-B10 cell line revealedmany of the cytogenetic changes present in the predominate clone ofthe leukemic specimen, and an i(12p) was present in all of the cells.The Robertsonian translocation involving chromosome 13 homologues (45,XY,t(13q;13qJ was also present in all of the UoC-B10cells.

Gene Rearrangement. Southern blot analysis of the patient's leukemia cells and the UoC-B10 cell line was done using the immunoglobulin heavy chain (J@J and EBV probes (Fig. 3). The rearrangednongermline band present in the cell line co-migrated with the rearranged band present in the patient's leukemia cells. The UoC-B10 cellline (and the patient's leukemia cells) lacked the EBV genome because the cells did not hybridize to the EBV cDNA probe (data notshown).

FISH Analysis Using the chromosome 12 probe, the leukemiacells showed three hybridization signals (Fig. 2B); two were ofnormal size and one was small, likely due to the centromeric structure

of the i(12p), as previously reported (36). The MDS BM aspirate was

studied retrospectively and occasional cells (<5%) were found withthe same three-signal pattern (Fig. 2C). Deparaffinized sections of thegerm cell tumor were studied according to a protocol of Hopman (38)and, although tissue sectioning made the analysis difficult, some cells

with two bright signals and one weak signal were observed (Fig. 2D).

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Table 2 CytogeneticanalysisNo.

ofCells

analyzedcells analyzedaoneKaryotype [no. ofcells]BM-MDS331

23445,XY,t(13;l3XqlO;qlO)

[7]45,idem,der(6)t(6,13)(j22;q12) [5]45,idem,der(18)t(?4;18),(q31;pll) [10]45,idem,+(12)(j,10),add(12@q24),+13,dic(13;18)(j13;p11),dic (14;l5XqlO;qlO) [2]9 nonclonal abnormal cells: 5 cells, der(13;13); 8 cells,i(12p)BM-ALL221

345645,XY,t(13;l3XqlO;qlO)

[1]45,idem,der(18)t(?4;18),(q31;pll) [1]45,idem,+(12)(plO),add(12Xq24),+13,dic(13;18)(j,13;pll),dic(14;l5XqlO;qlO) [2]46,idem,dic(8,22Xq24;pll),+11,add(llXq2l),del(11)(j,12),+i(12)(j10) [9]46,XY,dic(8,22@q24;p11),+11,del(11)(j,12),del(11@q13),+i(12)(j10),tri(13;13;22)(13qter—*13p11::13p11---@13q34::22p13---.22qter),+22[2]8 non-clonal abnormal cells: 7 cells, der(13;13); 6 cells,i(12p)UoC-B10

aTheabnormalcloneo20bserved in the UoC-B10 ce5@ll line was derived46,XY,dic(8;22Xq24;pl

1)+1 1,del(1 1)(j12),del(1 1@q13),+i(12)(@p10),der(13;13) (qlO;q10),der(20)t(17;20@q11;q13.3) [13]7 nonclonal abnormal cells; all with der(13;13) and i(12p)

from clone 5 observed in the leukemic bone marrow sample; however, clonal evolution has occurred.

LEUKEMIA CELL UNE wITH i(12P)

CD1a(T6)CD2(T11)CD3(T3)CD4(T4)CD5(Ti)CD7

(Leu9)CD8(F8)

1463653

000

981

0

CD1O(J5)CD19(B4}CD2O(B!)CD24 (BA!)KappaLambdaSIg-IgO

-1gM-IgD

49501827

5ND@NDND

100100

2

085

00

68

CD34 (HPCA-!) 7 1

CD56 (NKH-!) 0

CD13 (My7)CD14 (MO2)CD15 (Leu Mi)CD33 (My9)

4585

010

CD45 (KC56)CD38 (Leu 17)HLA-DR (12)CD61 (OPHIa)Olycophorin

623953

330

1009999

12

Table 1 Immunophenotypeof the patient's leukemia cells and the UoC-BI0 cell line[3H]thymidine uptake in the control cells, had no effect ontheClusterBone UOC-B10UoC@B10 cells cultured in either serum-free media or media with10%designation

marrow cell linefetal calfserum.Lineage(antigen) cells (%)(%)DISCUSSIONT-lymphoidWe

report the establishment of an i(12p) positive leukemia cell linecultured from a patient with a metastatic mediastinal GCF (yolk sac)who had progressive bone marrow dysfunction and i(12p) positive

leukemia. Of note, at initial diagnosis, micromegakaryocyteswereB-lymphoidobservedin the bone marrow, suggesting that abnormal hematopoietic

differentiation was already occurring. After the completion of chemotherapy, the patient developed thrombocytopenia andmultiineage1ProgenitorA

BNKAssociated2Macrophage/MyeloidOther234-a

Notdone.This

signal pattern was not observed in normal control cells, and threesignals were found in only 0.5% of nuclei (37).4.G-CSF

Studies. Using a reverse transcription-PCRprotocol,mRNAfor the G-CSF receptor was detected as a single 237-basepairband

in the U937 cell line (jxsitive control) and the UoC-B10cellline.The UoC-M1 cell line served as a negative control for boththereverse

transcription-PCR and binding studies. Using aFluorokineG-CSFkit, cell surface binding of G-CSF to the UoC-B10 celllinewas

detected (Fig. 4). G-CSF binding to the UoC-B10 cellswas4,4partiallyblocked (approximately 60%) when the UoC-B10 cells were

preincubated with 100-fold molar excess of G-CSF (data not shown).Supplemental rG-CSF, while it was associated with an increase inFig.

3. Southern blot analysis (JHprobe) of the patient's leukemia cells (columnA) andtheUoC-B10cell line(columnB).Arrow. therearranged,nongermlineband;—,germline@ HindIlldigestedlambdaphageDNAmolecularweightstandardsareshownonthe

6.6

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LEUKEMIA CELL LINE WITH i(12P)

(which were CD34+) within the yolk sac tumor component of theGCT and hypothesized that the leukemias were derived from pluripotent stem cells found within the yolk sac tumor (26, 5 1). Leukemiaswith i(12p) may represent a malignant counterpart of embryonalhematopoiesis because there is a higher frequency of histiocytic andmegakaryoblastic differentiation observed in mediastinal GCF patients when compared to the more mature myeloid subtypes observedin patients without mediastinal GC'F. This parallels hematopoieticdevelopment in the embryo where the development of macrophagesoccurs at 4 weeks gestation and thrombopoiesis at 8 weeks gestation(52, 53). Many features of the case presented here support this

hypothesis, including the rapid evolution of the patient's marrowdisease from a MDS involving myeloid, erythroid, and megakaryocyte lineages to a BCP-ALL.

There was good general concordance between the UoC-B10 cellline and the patient's leukemia cells as determined by a comparison ofthe morphology, immunophenotype, genotype, and karyotype results.Both had features of mature B-lymphocytes with the expression ofB-cell antigens (CD19, CD1O, and CD2O), the rearrangement of theimmunoglobulin heavy chain gene (54, 55) and enzyme activities(TdT and ADA) commonly observed in mature B-cell malignancies(41). In the cell line, the discordant expression of IgD/lambda mayrepresent B-lymphoid differentiation in culture, whereas the aberrantexpression of CD4 (along with the expression of glycophorin) suggests that the cell line may have the capacity to differentiate alongmore than one lineage.

Previously, a leukemia cell line with megakaryocytic features(EST-IU) was established from a patient with a mediastinal OCT whodeveloped AML 4 months after completing chemotherapy (56). Sincethis cell line failed to proliferate beyond 6 months in culture, UoCBlO appears to be the first leukemia cell line available to investigatorswith an i(12p) karyotypic marker. UoC-B10 cells, and the 1 liter of thepatient's cryopreserved BM which contains i(12p) blasts, will beuseful in: (a) studies of molecular consequences induced by allele losson chromosome 12; (b) determining the relationship between i(12p)

and hematological differentiation of GC'F; and (c) studies of acquiredcisplatin-resistance in human OCT (57). Finally, the accumulatingevidence suggests that patients with mediastinal GCFs should becarefully evaluated for a hematological disorder. Allogeneic bonemarrow transplantation performed early in the course of the diseasemay alter its devastating natural history.

ACKNOWLEDGMENTS

We thank the medical, nursing, and housestaff for their care of this patient,Dr. Bertil Glader for ADA analysis, the technologists in the Hematology/Oncology Cytogenetics Laboratory for their expertise, and Dr. PatriciaKampmeir for assistance in the care and evaluation of this patient. Weespecially thank the patient's wife and family for their support and devotion.

REFERENCES

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2. Vogelzang, N. J., Anderson, R. W., and Kennedy, B. J. Successful treatment ofmediastinal germ cell/endodermal sinus tumors. Chest, 88: 64—69,1985.

3. Sales, L. M., and Vontz, F. K. Teratoma and Di Ouglielmo syndrome. South. Med.J., 63: 448—450, 1970.

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5. Sandberg, A. A., Abe, S., Kowalczyk, J. R., Zcdgenidze, A., Takeuchi, J., and Kakati,S. Chromosomes and causation of human cancer and leukemia. L. Cytogenetics ofleukemias complicating other diseases. Cancer Oenet. Cytogenet., 7: 95—136,1982.

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.@Ez

C,

Fig. 4. fluorescence-activated cell sorter analysis of binding of unlabeled O-CSF(Control), streptavidin-phycoerythrin (SA-PE), and phycoerythrin-conjugated G-@SF(GCSF-PE)to the UoC-B!0cell line.

dysplasia in the marrow and blood that rapidly evolved to BCP-ALL.The relationship between the progressive hematological changes andthe mediastinal GCF is not completely clear. However, since an i(12p)chromosome abnormality, which is a specific marker of GCT (21, 36,

44, 45), was detected by FISH and/or cytogenetic analysis in the bonemarrow specimens, it seems likely that the protean nature of the bonemarrow changes was mediated by evolving OCI' metastasis whichwere resistant to cisplatin, etoposide, and bleomycin chemotherapy.Patients with similar hematological changes, which were consistentwith GO' metastasis undergoing hematological differentiation andclonal evolution, have been reported previously (14, 17, 24, 26, 32).For example, among four patients with GCT and AML reported byBajorin et a!. (32), one was not karyotyped, but two of the remainingthree leukemias had complex karyotypes which included i(12p) andabnormalities of chromosome 13. The i(12p) chromosome abnormal

ity found in the germ cell tumor may have been directly involved inthe genesis of this patient's leukemia.

This patient received 36 doses of G-CSF (5 p@g/k@dose)to ameliorate the expected myelosuppression of chemotherapy. TheB-lymphoid cell line established from the patient's leukemia cellspossessed G-CSF receptors. While G-CSF receptors are generallyrestricted to hematopoietic cells of the myeloid lineage (46, 47),Drach et a!. recently reported G-CSF receptors on the leukemia cellsfrom each of the 21 patients with BCP-ALL.4 Also, in four of theireight patients studied, BCP-ALL colony growth was enhanced whenleukemia cells were cultured with G-CSF (50 ng/ml).4 Others have

reported a rise in the number of leukemic blasts in the blood ofpatients treated with G-CSF (48, 49), and in 2 patients with severeaplastic anemia, treatment with G-CSF was followed by the development of leukemia (50). However, exogenous G-CSF did not change

the growth rate or induced differentiation of the UoC-B10 cell line.Recently, Orazi et a!. (26) reported data on six patients with

secondary leukemia who initially presented with mediastinal GCT.They observed morphologically identifiable hematological cells

4D.Drach,Z. Estrov,S.Zhao,3.Drach,A. Cork,D.Collins,H. Kantaijian,andM.Andreeff. Granulocyte-colony stimulating factor, granulocyte-macrophage colony stimulating factor, PIXY-321, stem cell factor, interleukin-3, and interleukin-7: receptor bindingandeffectsonclonogenicproliferationinacutelymphoblasticleukemia,submittedforpublication.

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Vermaelen, K., and Van Den Berghe, H. Sq—anomaly in a patient with disseminatedteratoma. Cancer Genet. Cytogenet., 9: 239—244,1983.

8. Hagberg, H., Gustavson, K. H., Sundstrom, C., and Gerdes, U. Blastic phaseof myeloproliferative syndrome coexisting with a malignant teratoma. Scand. I.Haematol., 30: 36—42,1983.

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1994;54:4999-5004. Cancer Res   Peter A. Downie, Nicholas J. Vogelzang, Richard L. Moldwin, et al.   Leukemiawith a Mediastinal Germ Cell Tumor and Acute Lymphoblastic Establishment of a Leukemia Cell Line with i(12p) from a Patient

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