(CANCER RESEARCH 51. 336-340. January 1. 1991)

Biochemical and Genetic Characterization of the HBA71 Ewing's Sarcoma CellSurface Antigen1

Erich J. Fellinger, Pilar Garin-Chesa, Sai L. Su, Paula DeAngelis, Joseph M. Lane, and Wolfgang J. Rettig2Departments of immunology [E. J. F.. S. L. S., M'. J. K.J. Pathology ¡P.G-C., P. DJ, and Surgery, Orthopedic Service [E. J. F., J. M. L.J, MémorialSloan-Keltering

Cancer Center, New York, New York 10021

ABSTRACT

Monoclonal antibody HBA71 detects a cell surface antigen of humanEwing's sarcomas and peripheral neuroepitheliomas that distinguishes

these tumors from other small round cell tumors of childhood andadolescence. In the present study, we show that monoclonal antibodyHBA71 reacts with polypeptides of M, 32,000 and 30,000 and that theHBA71-coding gene segregates with human chromosomes X and Y inrodent-human hybrids. Therefore, we compared HBA71 to the T-cellleukemia antigen 12E7, which is encoded by the pseudoautosomal regionof chromosomes X and V. \Ye show that monoclonal antibodies HBA71and 12E7 (a) detect polypeptides of identical size, (b) react with mousecells transfected with complementary DNA corresponding to the 12E7-coding gene, MH 2. and (c) give similar patterns of reactivity with humantumor cell lines and small round cell tumor tissues. Thus, HBA71 and12E7 are identical or closely related antigens and the available M It.'2

probes will facilitate analysis of the molecular mechanisms that determinedifferential HBA71 expression in small round cell tumors of childhoodand adolescence.

INTRODUCTION

SRCT' of childhood and adolescence represent a diverse

group of malignant tumors of lymphoid, neural, and mesenchy-mal derivation that share a similar histological appearance,characterized by the presence of "small blue round cells," i.e.,

poorly differentiated cells with uniform nuclei and scanty cytoplasm (1). Based on clinical, biochemical, histochemical, andpathological criteria, distinct entities of SRCT have been defined, including several types of lymphomas, neuroblastoma,ES, and ERMS. These tumors differ in their biological behaviorand response to specific forms of therapy (2). Since the introduction of multiagent chemotherapy almost 20 years ago, survival rates of patients with various types of SRCT, includingES, ERMS, and advanced stages of neuroblastoma, have notimproved significantly (3-5). To achieve better treatment responses, it may be necessary not only to test new antitumoragents, but also to reevaluate existing criteria for SRCT classification and to define new biologically and prognostically distinct tumor subsets. In one recent series of studies, atypicalneuroblastomas (variously described as Askin tumors, PNET,or PN) have been shown to share a common chromosomalabnormality with ES, a reciprocal translocation between chromosomes 11 and 22, t(l I;22)(q24;ql2) (6-9). This translocation is not found in typical neuroblastomas, which instead mayshow homogeneously staining regions or double-minute chro-

Received 7/24/90; accepted 10/4/90.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.

' This work was supported, in part, by the Greenwall Foundation, the Stern

Fund for Pediatrie Bone Tumor Research, and the Society of Memorial Hospital.¡To whom requests for reprints should be addressed.' The abbreviations used are: SRCT, small round cell tumor(s); ES, Ewing's

sarcoma(s): ERMS, embryonal rhabdomyosarcoma(s); PN, peripheral neuroepi-thelioma(s): PNET. primitive neuroectodermal tumor(s); mAb. monoclonal antibody: MHA. mixed hemadsorption: PBS. phosphate-buffered saline: cDNA,complementary DNA; SDS, sodium dodecyl sulfate: PAGE, polyacrylamide gelelectrophoresis.

mosomes that reflect amplification of the N-myc oncogene (10).The t(l I;22)(q24;ql2) chromosome abnormality also distinguishes ES and PN/PNET from lymphomas and ERMS. Basedon this cytogenetic evidence, it has been proposed that PN/PNET are biologically more closely related to ES, a tumorwhich arises typically in bone, than to classical neuroblastoma(11). Accordingly, clinical trials have been initiated to treatpatients with PN/PNET according to ES protocols rather thanneuroblastoma protocols (11).

In our previous serological analysis of SRCT, we have identified a highly restricted cell surface antigen, HBA71, which isshared by ES and PN/PNET but is absent from classicalneuroblastomas and lymphomas (12, 13). Thus, the HBA71tissue distribution parallels the distribution of thet(ll;22)(q24;ql2) marker chromosomes in SRCT. Little iscurrently known about the biochemical nature and function ofthe HBA71 antigen, the gene coding for HBA71, and its possible relationship to the t(l I;22)(q24;ql2) translocation. As afirst step in addressing these questions, we have used biochemical and genetic methods to characterize the HBA71 antigenand to identify and chromosomally map the HBA71-codinggene.

MATERIALS AND METHODS

Cell Lines. Human tumor cell lines were obtained from the cell bankat Sloan-Kettering Institute and maintained in culture as described (14).The derivation of the rodent-human somatic cell hybrids and characterization of their human chromosome content through karyotypeanalysis and testing for isoenzymes, cell surface antigens, and DNAsequences have been described (15-18). Several of the hybrid cloneshave been reanalyzed for their human chromosome content since theoriginal descriptions. The MIC2 transfectant CY2pp was derived bytransfection of mouse CY2 cells with a MIC2 cDNA clone, as described(19).

Monoclonal Antibodies. Mouse mAb HBA71 was raised against SIM-1 tumor cells (12, 13) and is an IgGl antibody, as determined by mAbcapture assay (Zymed Laboratories, San Francisco, CA). mAb 12E7has been described (20). Unrelated mouse IgGl immunoglobulins werepurchased from Becton-Dickinson (Mountain View, CA).

Serological Procedures. MHA rosetting assays for the detection ofsurface antigens on cultured cells were performed as described (15).Briefly, cells were seeded into 60-well MicroWell plates (Nunc, Ros-kilde, Denmark) (200 to 300 cells/well), grown for 18-48 h, washed,and incubated with serial dilutions of mAbs for 1 h. After repeatedwashes, target cells were incubated for 45 min with indicator cells,prepared by conjugating purified rabbit anti-mouse immunoglobulinheavy and light chain (DAKO Corp., Santa Barbara, CA) to humanRBC using 0.01% chromium chloride. Finally, plates were washed andreactivity was scored by determining the proportion of target cells withattached indicator cells (rosettes). The highest dilution of mAb givingrosette formation was defined as titration endpoint.

Flow Cytometry Analysis. Target cells were incubated with mAbs for1 h at 4"C, washed 3 times with PBS, incubated with fluorescein

isothiocyanate-conjugated rabbit anti-mouse immunoglobulin (Orga-non, Westchester, PA), and washed 3 times in PBS. Cells were analyzedon a FACSTAR PLUS laser flow cytometer system and histograms

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EWING'S SARCOMA ANTIGEN HBA71

Fig. 1. Immunoblot analysis of ES and PNcell lysates, separated by SDS-PAGE, withmAbs HBA71 and 12E7. A, lysate of TC-215PN cells tested with mAb HBA71 (lanes I and2) or unrelated negative control mAb (lanes 3and 4), following SDS-PAGE under reducing(lanes 1, 3, and 5) or nonreducing conditions(lanes 2 and 4). B, lysates of TC-215 PN cells(lanes I. 3) and 6647 ES cells (lanes 2, 4, and6) tested with mAb HBA71 (lanes 1 and 2),mAb 12E7 (lanes 3 and 4), or unrelated negative control mAb (lanes 5 and 6). Molecularweight determinations were derived from thepositions of prestained molecular weightmarkers (Rainbow; Amersham) included in adjacent lanes (not shown): myosin (M, 200,000),phosphorylase * (Mr 92,500), bovine serumalbumin (M, 69,000), ovalbumin (M, 46,000),carbonic anhydrase (M, 30,000), trypsin inhibitor (M, 21,500), and lysozyme (M, 14,300).Molecular weights (x 10~3)of the major bands

of HBA71 are indicated to the right of eachpanel.

1234

B

30^32^30

3456

were generated with the C30 and LYSIS data-handling systems (Bec-ton-Dickinson).

Immunoblotting. Cell extracts were prepared by lysing 0.1-0.2 ml ofpacked cells in 3 ml of ice-cold extraction buffer (0.5% Nonidet P-40,0.01 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.01 M MgCl2, 2 miviphenyl-methylsulfonyl fluoride, 20 units/ml aprotinin). Aliquots of cell extracts(60 ß\)were mixed with 3 x sample buffer [30 //I; 0.15 M Tris-HCl, pH6.8, 3% SDS, 6 mivi EDTA, 30% glycerol, 0.5 mg/ml bromophenolblue, with or without 3% (v/v) mercaptoethanol and 36 mg/ml dithiothrei-tol], boiled for 3 min, and separated by SDS-PAGE. Prestained molecular weight markers (Amersham, Arlington Heights, IL) were includedin all experiments. Proteins were transferred to nitrocellulose membranes (BAS NC; Schleicher & Schuell, Keene, NH) using a TransphorTE50 apparatus (Hoefer Scientific Instruments, San Francisco, CA) at0.9 amp for 1 h. Subsequently, membranes were soaked for 30 min inblocking buffer (PBS, 1% bovine serum albumin, 0.1% Tween 20),washed 3 times for 5 min, and incubated overnight with mAbs dilutedin the same buffer (mAb HBA71 and negative control IgGl, 20 Mg/ml;I2E7, ascites fluid at 1:100 dilution). After three washes, membraneswere incubated for 2 h with alkaline phosphatase-labeled goat anti-mouse IgG (Calbiochem, San Diego, CA) in blocking buffer. Followingthree washes in blocking buffer and one wash in 50 miviTris-HCl, pH9.6, substrate solution (10% nitroblue tetrazolium, 1% 5-bromo-4-chloro-3-indolyl phosphate in 50 iriMTris-HCl, pH 9.6, 5 miviMgCl2)was added. The reaction was stopped by washing the membranes withH2O.

Immunohistochemistry. Paraffin-embedded tissues were obtainedfrom the Department of Pathology at Memorial Hospital. Diagnoseswere established by routine light microscopic evaluation and electronmicroscopy in selected cases. Sections of 5-^m thickness were cut andpretreated with 0.05% saponin (Sigma, St. Louis, MO) in H2O for 30min at room temperature, as previously described (21). Endogenousperoxidase was blocked with 0.3% H2O2 in PBS for 30 min. Subsequently, sections were incubated with normal horse serum for 30 minand then with mAbs (HBA71 and negative control IgG I, 4 Mg/ml; mAb12E7, 1:1000 ascites Huid) for 12 to 18 h at 4"C, followed by biotiny-lated horse anti-mouse IgG (15 Mg/ml) and avidin-biotin horseradishperoxidase complex (1:1 ratio, 1:100 dilution; Vector Labs, Burlin-game, CA) for 30 min at room temperature. The final reaction productwas visualized by the H2O2-diaminobenzidine reaction. The sectionswere counterstained with Harris' hematoxylin.

RESULTS

Biochemical Characterization of HBA71. Two approacheswere used in this study to determine the biochemical nature of

the antigen recognized by mAb HBA71 on PN/PNET and EScells. First, cultured PN (TC-215) and ES cells (6647) weremetabolically labeled with ['Hjglucosamine or ["SJmethionine,

extracted with Nonidet P-40 lysis buffer, and used for immu-noprecipitation experiments using standard procedures (22);however, no specific immunoprecipitates were obtained. Second, detergent extracts of cell lines TC-215 and 6647 wereseparated by SDS-PAGE, transferred to nitrocellulose membranes, and tested with mAbs by the indirect immunoperoxidasemethod. As shown in Fig. IA for TC-215, mAb HBA71 detectstwo major bands, of M, 30,000 (p30) and M, 32,000 (p32), andminor bands of smaller size. Identical results were obtained intriplicate experiments, using TC-215 extracts separated underreducing or nonreducing conditions. A different banding pattern was observed in immunoblotting experiments with 6647ES extracts, which showed a p30 band of similar intensity butonly a faint p32 band (Fig. \B).

Chromosomal Assignment of HBA71. Serological analysis ofa panel of rodent-human somatic cell hybrids containing distinct subsets of the human chromosome complement was usedto determine the chromosomal location of the HBA71-codinggene. As a first step in this analysis, we determined the patternof HBA71 expression on human and rodent cell lines, includingthe cell lines used for somatic cell hybridization. Table 1 summarizes the results of MHA rosetting assays and shows thatHBA71 is most strongly expressed on ES and PN/PNET celllines. However, low to moderate levels of HBA71 expressionwere detected on most of the other cell lines tested. Theseresults, which suggest a broader representation of HBA71 oncultured human cells than previously described (13), were confirmed by cytofluorometric analysis. For example, we foundthat ES and PN/PNET cell lines (TC-215, 6647, SIM-1, andIARC-EW1) are high expressors, MOLT-4 T-cell leukemia isan intermediate expressor, IMR-32 and CHP-126 neuroblastoma are low expressors, and normal peripheral bloodlymphocytes and DAUDI Burkitt's lymphoma cells areHBA71" (Fig. 2). Table 2 shows that the rodent cells used for

somatic cell hybridization (mouse A9, LTK, and N4TG1 cellsand Chinese hamster YH21 cells) are HBA71". In contrast, 6of 11 hybrid clones tested were HBA71*. Comparison of the

subsets of human chromosomes present in these hybrid clones337

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EWING'S SARCOMA ANTIGEN HBA7I

Table 1 Reactivity ofmAb HBA 71 with cultured human cells

Cell type DesignationHBA71 cell

surface reactivity"

Ewing'ssarcomaPeripheral

neuroepithe-liomaNeuroblastomaAstrocytomaLymphoma/leukemiaMelanomaRenal

cancersOther

carcinomas6647.

IARC-EW1TC-215,

SIM-1LA-N-l.SMS-MSN

IMR-32CHP-126BE(2)-C,SMS-SANSK-MG-15

SK-MG-2, -13. -21SK-MG-8,-12U251MGRAMOS,

DAUDIHUT-78. SK-LV-18HPB ALL,SK-LY-16SK-MEL-179

SK-MEL-13SK-RC-45

SK-RC-17, -39SK-RC-8SK-OV3.

SK-UT2. SK-UT-3ASPC-I.SW1116HCT15. T2431,25031,25050

2501,25010

SO250

1,25050

25050

25010

2501,2502

250

* Cell surface reactivity was determined by endpoint titration with mixed

hemadsorption rosetting assays. Numbers indicate reciprocal of highest mAbdilutions (starting concentration. 1:2 HBA71 hybridoma culture supernatant)giving rosette formation with target cells; -, no reactivity with highest concentration of mAb tested.

controlHBA71

controlHBA71

ffl

10' 10'

MOLT 4

IIP

PBL

Fig. 2. Cytofluorometric analysis of TC-215 PN (A), MOLT-4 leukemia cells(B), and peripheral blood lymphocytes (PBL) (C) with mAb HBA71 or unrelatednegative control mAb. Histograms display relative fluorescence intensity (abscissa) versus relative number of antigen-expressing cells (ordinate).

with their HBA71+ phenotypes (discordancy analysis) was used

to determine which chromosome cosegregates with HBA71 cellsurface expression. For all human autosomes, this discordancyanalysis identified at least 4 independently derived hybrids thatwere HBA71* but lacked the respective chromosome. There

fore, it appeared unlikely that an autosomal locus controlsHBA71 expression. Instead, a close correlation was foundbetween HBA71 phenotype and the presence of human chromosome X, with 5 hybrids being HBA71 "^/chromosome X+, 5hybrids being HBA71 "/chromosome X~, and 1 hybrid beingHBA71*/chromosome X". This last hybrid was the only hybrid

in our test panel that contained human chromosome Y, suggesting that HBA71 may be encoded by a gene in the pseu-doautosomal region of the short arm of chromosome X, whichis shared by chromosome Y. Consistent with this interpretation,we found that an additional rodent-human hybrid cell line,G35CC, which contains only the long arm of human chromosome X and no chromosome Y, lacks HBA71 expression.

Comparison of HBA71 and 12E7 Cell Surface Antigens. Thereceptor for granulocyte-macrophage colony-stimulating factor

(23) and a thymus-leukemia antigen, 12E7 (20), recognized bymAbs 12E7, 013, and F21 have been mapped to the pseudoau-tosomal region of chromosomes X and Y (18, 24, 25). Becauseof the similarity in molecular size between HBA71 and 12E7(18, 20, 26), we carried out experiments to determine therelationship between these antigens. First, immunoblotting experiments with cell extracts of TC-215 and 6647 showed thatthe HBA71 and 12E7 antigens comigrate and have identicalbanding patterns (Fig. IB). Second, immunofluorescence staining and Cytofluorometric analysis showed that mAbs HBA71and 12E7 react with mouse cells transfected with a cDNA ofthe 12E7-coding gene, A//C2, but not with nontransfectedmouse cells (Fig. 3). Third, we carried out cotyping analysis ofa panel of human tumor cell lines and normal peripheral bloodlymphocytes with mAbs HBA71 and 12E7 and obtained identical results, similar to those shown in Table 1 and Fig. 2 forHBA71. Finally, we compared the immunohistochemical staining patterns of mAbs HBA71 and 12E7 in selected tumortissues and found that both react with the three ES tested butnot with three neuroblastomas (illustrated in Fig. 4).

DISCUSSION

The HBA71 cell surface antigen is one of a small number ofserological markers that is consistently expressed in ES andPN/PNET (13, 21, 27, 28). HBA71 has been detected in all ES

Table 2 Serological analysis of HBA 71 cell surface expression on rodent-humansomatic cell hybrids

TargetcellsHybrid

cloneANK3.1312ANK3.11CE25/1GM7300NSK-4LC2/4LNK1.6LNK1.8NSK-3NSK-5LM29Rodent

cellsA9LTKYH21N4TGIHBA71reactivity"2502501,2501

,250250————_250—_—-Human

chromosomespresent5,

20,X5,20, (21)*, 22.X1e,

12.X6,8,

11,XIe,4', 6, 8, 11, 12, 14, 15, 19, 21,X5,6,7,

17, 18.213,6,8,10, 11, 16,17,203.

4, 8, 10, 11, 16, 17, 20,22lf,6, T. 8, 12, 14, 15, 17f, 19, 20,21T,3, 6, 7, 10, 11, 12, 14, 15, 17f, 20,2116,

17,Y—__-

" HBA7I cell surface expression was determined by MHA endpoint titration

assays. Numbers refer to reciprocal of highest mAb dilutions (hybridoma culturesupernatant: starting dilution. 1:2) giving rosette formation with target cells; —,no reactivity at highest concentration of mAb tested.

* Human chromosomes present in less than 40% of cells in hybrid cell line are

listed in parentheses.' Presence of rearranged or deleted copy of the human chromosome.

controlHBA71

10« III- 10«

CV2pp CY2 CY2pp

Fig. 3. Cytofluorometric analysis of Ai/C2-transfected mouse cells (CY2pp)and untransfected mouse cells (CY2) with mAb HBA71 (A and B). mAb 12E7(C). or unrelated negative control mAb (A-C).

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EWING'S SARCOMA ANTIGEN HBA7I

Fig. 4. Immunohistochemical analysis ofEwing's sarcomas and neuroblastomas withmAbs HBA71 and 12E7, using the avidin-biotin complex immunoperoxidase procedurewith formalin-fixed paraffin-embedded tissues.A, Ewing's sarcoma stained with mAb HBA71.

antigen positive; B, neuroblastoma stainedwith mAb HBA71, antigen negative; C, Ewing's sarcoma stained with mAb 12E7, antigen

positive; l>. neuroblastoma stained with mAb12E7, antigen negative. Original magnification, x 100; hematoxylin counter-staining.

and PN/PNET tumors tested to date, including over 100 specimens from the files of Memorial Hospital and the Vienna Boneand Soft Tissue Tumor Registry (12, 13).4 This extensive analy

sis has been facilitated by the fact that HBA71 can be detectedin paraffin-embedded tissues. Because of its limited distributionin normal tissues (13), HBA71 may be a useful target for tumorimmunolocalization and immunotherapy with cytotoxic mAbsor mAb conjugates. The present study was designed to investigate the biochemical and genetic nature of HBA71 and itscoding gene.

Previous attempts to characterize the HBA71 antigen withChromatographie procedures have produced conflicting results(13, 29) and led to the suggestion (29) that HBA71 may berelated to Thy-1, a well characterized cell surface glycoprotein.Our findings clearly show that HBA71 is not related to Thy-1,since the two antigens differ in molecular size, chromosomallocation, and tissue distribution (23, 30, 31). Instead, we demonstrate a close relationship between HBA71 and 12E7, anantigen previously studied in detail on hematopoietic cells (20),based on several lines of reasoning. First, mAb HBA71 andmAbs against the 12E7 antigen recognize similar polypeptides,p32 and p30, that comigrate in SDS-polyacrylamide gels [inprevious reports these polypeptides have also been referred toas p28 (20), p25/p30 (18), and p29/32.5 (26)]. Second, 12E7 isknown to be encoded by the pseudoautosomal region of humanchromosomes X and Y (18, 23, 26). Third, mAbs HBA71 and12E7 react with mouse cells tranfected with a human MIC2cDNA clone that was identified using mAb 12E7 as a probe(19). Finally, mAbs HBA71 and 12E7 show similar patterns ofreactivity with cultured human cells and SRCT tissues. Anunresolved issue remains the relationship between p32, p30,and the lower molecular weight bands seen in immunoblotswith mAbs HBA71 and 12E7. Conceivably, p32, which is onlydetected in some cell types (18, 26), could result from alternativeRNA splicing or protein processing. Immunoprecipitation ex-

4 E. J. Fellinger el al., Immunohistochemical analysis of Ewing's sarcoma cell

surface antigen HBA71. manuscript in preparation.

periments using mAb O13 and [3H]glucosamine-labeled cell

extracts have shown that both p30 and p32 are glycosylated(18), and differences in the extent of glycosylation may alsocontribute to the size differences between p30 and p32. Someof the lower molecular weight bands seen in the present studymay represent partial degradation products that arise in spiteof the presence of proteinase inhibitors during cell lysis, orprecursor molecules of HBA71. Since the HBA71 protein isnot efficiently labeled by conventional metabolic radiolabelingprocedures,5 we have been unable to test the latter possibilitywith pulse-chase experiments. Previous studies have shown thatmAb 12E7 recognizes a protein epitope (32), and the relationship between the HBA71 and 12E7 epitopes remains to bedetermined.

In view of the close similarity or identity of the HBA71 and12E7 antigens, we have reevaluated some of the discordantresults reported in past studies with mAbs HBA71 (13, 29),O13 (18), and 12E7 (20). With regard to HBA71, we haveextended our tissue analysis to include normal thymocytes,"

which are known to express 12E7 at high levels (20). We foundthat thymocytes are strongly HBA71*, while peripheral bloodlymphocytes are generally HBA71", the same pattern that has

been described for 12E7 (20). Conversely, we have shown thatmAb 12E7 reacts strongly with cultured ES and PN/PNET celllines and ES tissues. Finally, both O13 and 12E7 are known toreact with cultured cells derived from a wide range of tumortypes (18, 20). We demonstrate that HBA71 has a similardistribution in vitro and that quantitative differences in HBA71expression seen with different cell lines parallel those seen for12E7 (13, 18, 20). High level expression of the HBA71/12E7antigen on ES and PN/PNET cells and on normal thymocytesdoes not reflect any known functional or embryological similarities between these cell types. Instead, it seems to representyet another example of a differentiation antigen that showsexpression in unrelated cell lineages (33, 34). Precedents for

5E. J. Fellinger and W. J. Rettig, unpublished observations.

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EWING'S SARCOMA ANTIGEN HBA71

this type of antigen distribution include the common acutelymphocytic leukemia antigen (35), the neural cell adhesionmolecule (36), and the CD 13 antigen (37).

At present it is not known why ES and PN/PNET showstrong HBA71 expression while most of the other SRCT ofchildhood and adolescence are HBA71" (12, 13).4 One possible

explanation is that specific genetic abnormalities in ES andPN/PNET activate MIC2. However, MIC2 maps to chromosomes X and Y and the only consistent cytogenetic abnormalityknown for ES and PN/PNET is a reciprocal translocation,t(l I;22)(q24;ql2). Alternatively, HBA71 expression in ES andPN/PNET may reflect continued expression of a fetal mesen-chymal or neural phenotype that corresponds to the targetcell(s) of transformation in these tumors. This hypothesis canbe tested by analysis of HBA71 expression during early stagesof human mesenchymal and neural development.

ACKNOWLEDGMENTS

We thank Peter N. Goodfellow and J. Mark Hexham for providingmonoclonal antibody 12E7 and data on the serological analysis ofMIC2 transfectants.

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1991;51:336-340. Cancer Res   Erich J. Fellinger, Pilar Garin-Chesa, Sai L. Su, et al.   Sarcoma Cell Surface AntigenBiochemical and Genetic Characterization of the HBA71 Ewing's

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