biphenotypic sarcomas with myogenic and neural differentiation

[CANCER RESEARCH 55, 1385-1392, March 15, 1995]

Biphenotypic Sarcomas with Myogenic and Neural Differentiation Express theEwing's Sarcoma EWS/FLI1 Fusion Gene1

Poul H. B. Sorensen,2 Hiroyuki Shimada, Xian F. Liu, Jerian F. Lim, Gilles Thomas, and Timothy J. Triche

Department of Pathology ami Laboratory Medicine. British Columbia's Children's Hospital/University of British Columbia, Vancouver, British Columbia. Canada V6H-ÃŒV4[P. H. B. S., J. F. L.1; DÃ©partaientof Pathology anil Laboratory Medicine, Children's Hospital of Los Angeles/University of Southern California, Los Angeles, California W)027[H. S., X. F. L., T. J. T.]: and Lttboratoires de GÃ©nÃ©tiquedes Tumeurs, Institut Curie. Paris. France Â¡G.7*./

ABSTRACT

Accurate diagnosis of primitive childhood sarcomas continues to be aformidable problem because these malignancies generally demonstratevery little morphological evidence of their tissue of origin. One of thesetumor classes, the Ewing's sarcoma family of peripheral primitive neu-

roectodermal tumors (pPNETs), are thought to have a neural histogenesisbased on evidence of neuroectodermal differentiation. Greater than 95%of pPNETs carry t(ll;22) or K2I;22) chromosomal translocations whichfuse the EWS gene from chromosome 22ql2 in-frame with either /â€¢'/.//

from chromosome Hq24 or ERG from chromosome 21q22. The pPNETsare considered to be histogenetically distinct from rhabdomyosarcomas,myogenic tumors lacking these EWS gene fusions and hypothesized toderive from immature skeletal muscle precursors. In the present study, wedescribe a unique set of childhood soft tissue sarcomas that show bothneural and myogenic differentiation. These biphenotypic tumors expressmyogenic regulatory factors and muscle-specific antigens and also showneuroectodermal differentiation with ultrastructural evidence of neurose-cretory granules and expression of neural-associated genes. Northern

analysis and reverse transcriptase PCR reveal expression of EWS/FLIlgene fusions in all biphenotypic sarcomas analyzed. Chimeric EWS/FU1transcripts and fusion proteins in these tumors are identical to thosedescribed for pPNETs. Our results provide evidence for a class of biphenotypic childhood sarcomas with myogenic and neural differentiation andsuggest that these tumors may be related to the Ewing's sarcoma family of

pPNETs.

INTRODUCTION

Primitive sarcomas occurring in childhood have traditionally presented significant diagnostic challenges due to their relative lack ofdistinctive morphological features. One group, the SRCTs3 of child

hood, are composed predominantly of poorly differentiated smallround cells and include the Ewing's sarcoma family of pPNETs,

embryonal and alveolar subtypes of RMS, neuroblastoma, and others(1). These tumors may show minimal or no evidence of their histolÃ³gica!cell of origin; therefore, they are often difficult to distinguishfrom each other based on morphological features.

The identification of tumor-specific fusion genes resulting from

chromosomal translocations in human malignancies is not only yielding profound insights into oncogenesis but also holds great promisefor tumor diagnosis. Two translocations, the t(ll;22)(q24;ql2) andt(21;22)(q22;ql2), characterize pPNETs and are considered specificfor this tumor class. The translocations fuse the 5' portion of the EWS

Received 10/20/94; accepted 1/12/95.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 in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 P. H. B. S. was supported in part by a Medical Research Council of Canada

Centennial Fellowship, and the study was supported in part by the Las Madrinas Endowment for Molecular Pathology at the Children's Hospital of Los Angeles.

2 To whom requests for reprints should be addressed, at Department of Pathology,British Columbia's Children's Hospital, 4480 Oak Street, Room 2J8, Vancouver, BC V6H

3V4, Canada.' The abbreviations used are: SRCT, small round cell tumor: pPNET. peripheral

primitive neuroectodermat tumor; RMS, rhabdomyosarcoma; DSRCT, desmoplasticSRCT; RT-PCR, reverse transcription-PCR; bp, base pair(s); kDa, kilodalton(s); NF,neurofilament; CAT, choline acetyltransferase; CGA, chromogranin A; NSE, neuron-

specific enolase.

gene on chromosome 22ql2 to either the FLU gene on Ilq24 (2, 3)or the ERG gene on 21q22 (4-6). Chimeric proteins expressed by the

fusion genes are capable of cell transformation and appear to act asaberrant transcription factors (3). Identification of EWS/FLIl andEWS/ERG fusion transcripts by the RT-PCR has formed the basis of

a sensitive and specific diagnostic test for pPNETs (7). Gene fusionshave been identified and characterized in other pediatrie sarcomas.The t(2;13)(q35;ql4) and t(l;13)(p36;ql4) translocations of alveolarRMS result in the expression ofPAX3/FKHR (8) and PAX7/FKHR (9)fusion transcripts, respectively, while the recently described intraabdominal DSRCT (10) has been shown to express a gene fusioninvolving EWS and the WTl tumor suppressor gene located at chromosomal band Ilpl3 (11). Detection of these genetic abnormalitiesthus provides a potentially powerful modality for the diagnosis ofprimitive childhood sarcomas, and classification based on their identification may ultimately provide a more accurate predictor of clinicalbehavior than current morphological classification systems.

The pPNETs are a family of poorly differentiated tumors thatinclude Ewing's sarcoma, peripheral neuroepithelioma, and Askin

tumor. These tumors are thought to have a neural cell of origin basedon limited neuroectodermal development, and the different familymembers show varying degrees of neural differentiation (reviewed inRef. l). They display ultrastructural evidence of primitive neurites andneurosecretory granules and show immunoreactivity for several neural-associated markers, including neurofilament proteins (12). In ad

dition, neural differentiation can be induced in these tumors by treatment with retinoic acid and nerve growth factors (13, 14). ThepPNETs are considered to be clinically and histogenetically distinctfrom RMS, malignancies with skeletal muscle differentiation hypothesized to derive from immature muscle precursor cells (reviewed inRef. l). These tumors do not display neural features, containinginstead tumor rhabdomyoblasts with immunoreactivity for skeletalmuscle-associated antigens including desmin and muscle-specific ac-tin. Ultrastructurally, they show muscle-specific cytoplasmic intermediate filaments and primitive Z-discs (in more differentiated tumors).These tumors express the MyoD family of basic helix-loop-helix

regulatory factors, including MYOD1, myogenin, MRF4, and MYF5(reviewed in Refs. 15 and 16), and this has been used as a diagnosticcriterion for RMS (17, 18). These myogenic regulatory factors arethought to control the initiation and maintenance of myogenic differentiation by binding to muscle-specific enhancers and by activating

the transcription of genes involved in skeletal muscle development(15, 16).

Soft tissue sarcomas have been described which show morphological evidence of both myogenic and neuroectodermal differentiation(1, 19). DSRCT demonstrates multilineage differentiation with epithelial, neural, and skeletal muscle immunophenotypic features (10).Other entities remain poorly defined, but at least some of these casesmay represent examples of malignant ectomesenchymoma (20-22).

Malignant ectomesenchymomas are predominantly pediatrie soft tissue tumors characterized by the presence of neural sarcomatouselements in addition to one or more malignant mesenchymal components, including RMS. Molecular studies may be useful to clarify therelationship of these tumors to other neural and myogenic soft tissue

1385

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EWS/FLII GENE FUSION IN BIPHENOTYPIC SARCOMAS

Table 1 Pathological features of biphenot\pic tumors and celi lines

Age/sexSiteTC-13119/MSoft

tissue, chestwallTC-1749/FSoft

tissue, thighTC-2065

mos./MSoft

tissue, upperarmTC-25325/MRetroperitoneumTTC-54713/FRetroperitoneum

Diagnosis Alveolar RMS Alveolar RMS Primitive RMS Alveolar RMS Alveolar RMS

IHC"Desmin

ActinNeurofilamentEM*Myogenic

NeuralCytogenetics

of bands11424md22ql27PositivePositivet(ll;22Xq24;ql2)+;Positive

Positivet(ll;22Xq24;ql2)Â£PositivePositivet(ll;22Xq24;ql2)*PositivePositivet(ll;22Xq24;ql2)â€¢H-

â€¢H-Positive

Positivet(ll;12;22Xq24;ql4;ql2)

" IHC, immunohistochcmical studies performed on primary tumors; findings maintained in cell lines (data not shown). ++, strong staining in > 50% of cells; +, strong staining in

10-50% of cells.* EM, electron microscopy performed on primary tumors.

sarcomas. In the present study, we describe a set of childhood tumorsthat were originally diagnosed as primitive forms of RMS based onmorphological evidence of myogenic differentiation. These tumorsexpressed myogenic regulatory factors and demonstrated immunore-activity for muscle-specific antigens. However, ultrastructural analy

sis revealed both myogenic and neural features, and molecular studiesdemonstrated expression of neural-associated genes. We show here

that EWS/FLII fusion transcripts and chimeric EWS/FLII proteins,identical to those of pPNETs, are expressed in these biphenotypictumors. Our results suggest that a subset of biphenotypic sarcomaswith myogenic and neural features may be members of the pPNETfamily of childhood tumors, a finding which could have importantimplications in the diagnosis and treatment of this group of childhoodmalignancies.

MATERIALS AND METHODS

Cell Lines. The cell lines TC-131 (23), TC-206, TC-212, and CTR (24);A204 (25), Rhl8 (26), TC-32, TC-71, A4573, and 6647 (23, 27); and IMR-32and SAN-2 (28) have been described previously. Other cell lines were established at Children's Hospital of Los Angeles (TTC-442, TTC-487, TTC-516,and TTC-547), NIH (TC-147, TC-174, TC-253, and TC-280), or St. Jude'sHospital, Memphis, TN (Birch)4 from primary tumor cells using standard

methods. All cell lines were maintained in RPMI 1640 plus 20% fetal bovineserum, and all analyses were performed on early passage cell lines. Clinicaland pathological features of the five cell lines examined in detail, TC-131,TC-174, TC-206, TC-253, and TTC-547, are summarized in Table 1. Primarytumors stored frozen in liquid nitrogen were obtained from the Children's

Hospital of Los Angeles tumor bank. Electron microscopy and immunocyto-

chemistry were performed according to standard procedures.RNA and DNA Analysis. Poly(A+) or total RNA was isolated from cell

lines as described previously (29). Northern analysis was performed accordingto standard methods (29) using 2 fig of poly(A+) or 20 jig of total RNA for

each sample. cDNA probes for the myogenic regulatory genes MYF5, MYO-GENIN, MYOD1, and for the neurofilament light and heavy genes (NF-L andNF-H, respectively) were obtained from the American Type Culture Collection

(Rockville, MD). Conventional Southern blot analysis was performed using 10/xg genomic DNA/sample isolated from cell lines (29). Single-copy genomic

probes for the EWS locus, named PR.8, HP.5, and RR2. have been describedpreviously (5). Probes for Northern and Southern analysis were radiolabeled byrandom primer extension.

RT-PCR. Total RNA from cell lines or frozen primary tumor tissue wasisolated as described previously (30). RT-PCR detection of EWS/FLII fusion

transcripts was performed as reported previously using total RNA as startingmaterial (7). Oligonucleotide primers used included 11.3 (FLU) and 22.3(EWS; Refs. 2 and 7). RT-PCR screening for neural-associated gene expressionwas performed on DNase I (Pharmacia)-treated total RNA using the manufacturer's protocols. Primers for CAT included 5'-GACCATCCTTTTCTG-CATCT-3' (sense) and 5'-GGCTTGCTCTCAGTAGGC-3f (antisense); forCCA, they included S'-GACCGACTCTCGCCTTTCCG-S' (sense) and 5'-

GCTCCAAGACCTCGCTCTCC-3' (antisense); and for NSE, they included5'-TCTGAACGTCTGGCTAAATACAAC-3' (sense) and 5'-GAAAAG-GAAGAAGTGGAAAGTGCGG-3' (antisense). Amplified products were an

alyzed by electrophoresis using 2% agarose gels and stained with ethidiumbromide.

Immunoprecipitation Analysis. Antisera were raised to a glutathione .Vtransferase-EWS/FLIl fusion polypeptide consisting of EWS amino acids245-264 (2) fused to FLU amino acids 240-369 (31). A 450-bp BamHl-Pstl

fragment from a EWS/FLII fusion cDNA isolated from a tumor cell linecontaining an 11;22 translocation (7) was subcloned into the expressionplasmid pGEX-2T (Pharmacia). This construct was expressed in Escherichiacoli, the fusion protein was purified using glutathione-agarose (32), and protein

was injected s.c. into rabbits. This antisera recognizes epitopes from both EWSand FLU.5 Cells were metabolically labeled with ['15S]methionine, and whole

cell lysates were subjected to two-cycle immunoprecipitation using EWS/FLIIantisera. Immune complexes were precipitated with protein A-Sepharose(Pharmacia), fractionated by SDS-polyacrylamide gel electrophoresis, and

analyzed by autoradiography.

RESULTS

Biphenotypic Tumors with Myogenic and NeuroectodermalDifferentiation. Previous studies have documented the existence ofchildhood sarcomas with multilineage differentiation (1), and preliminary studies in our laboratory indicated that a subset of childhoodtumors with myogenic phenotype also express primitive neural features. Therefore, we screened a series of tumors and correspondingcell lines of myogenic phenotypic features for evidence of neuroec-

todermal differentiation. Five cases were identified; pathological features of the original tumors and corresponding cell lines (TC-131,TC-174, TC-206, TC-253, and TTC-547) are summarized in Table 1.

Each case was originally diagnosed as alveolar RMS or primitiveRMS. Morphological analysis revealed similar findings in all cases,with nests of poorly differentiated small round cells and clearlyidentifiable rhabdomyoblasts separated by fibrous septae, as shown inFig. \A for the original tumor from case TTC-547. Each tumor

4 S. Piper, unpublished data. 5S. L. Lessnick and C. T. Denny, unpublished data.

1386



Fig. 1. Pathology of biphenotypic tumors. A,histolÃ³gica! section of original tumor tissue fromcase TTC-547 showing primitive rhabdomyoblasticcells separated by fibrous septae. B, electron micrograph of original tumor tissue from case TTC-547 demonstrating muscle-specific cytoplasmic ac-tin-like filaments (arrow). C. electron micrographof the same cell as in (ÃŸ),showing dense-core

neurosecretory granules (arrows).

WS/FUI (il.NI- HSION IN lill'lll NOI Vl'IC SARCOMAS

demonstrated cytoplasmic immunoreactivity for muscle-specific desmin

and actin, which was maintained in cell lines (data not shown). ControlpPNETs were negative. Ultrastructurally, original tumor tissue from eachcase showed cytoplasmic actin-like filaments characteristic of primitiveRMS (1), as shown for case TTC-547 in Fig. IB. Moreover, neuroecto-dermal differentiation in these tumors could be demonstrated ultrastruc-

turally by the presence of primitive neurites and dense core neurosecretory granules (33), shown in Fig. 1C for case TTC-547. Fig. l, B and C,

represent electron micrographs taken from the same cell, indicating thedivergent differentiation of these tumor cells. Each tumor also showedimmunoreactivity for the neural antigen, neurofilament protein (Ref. 34;Table 1), corroborating the biphenotypic nature of these tumors.

To confirm myogenic differentiation in these biphenotypic tumors,we examined cell lines for expression of the myogenic regulatorygenes MYF5, myogenin, and MYOD1 by Northern analysis (Fig. 2A).

TC-131, TC-174, TC-206, TC-253, and TTC-547 as well as RMS

control cell lines expressed MYF5. Expression varied among the celllines, with TC-131, TC-253, and TTC-547 consistently showinglower levels. Myogenin expression was not observed in TC-131and TC-253 but was clearly evident in TC-174, TC-206, andTTC-547. Interestingly, MYOD1 expression was virtually unde-

tectable in any of the biphenotypic tumor cell lines, while expression was observed in all control RMS cell lines except Rhl8. Asshown in Fig. 2B, MYOD1 expression could not be induced bytransfer to differentiation medium (2% horse serum), known topromote myogenic differentiation (15). Therefore, all five biphenotypic cell lines expressed MYF5, three of five expressed MYOGENIN, and none expressed MYODI. These findings were not dueto the maintenance of primitive muscle cells in culture, as identicalresults were obtained using both early and late passage cell lines.

1387



EWS/FUI GENE FUSION IN BIPHENOTYPIC SARCOMAS

abcdefghijklm

D abcdefghijkl

Fig. 2. Expression of myogenic regulatory factors in hiphenotypic tumors. Northernblot hybridization of poly(A*) RNA from cell lines using cDNA probes for MYF5.

myogenin (Myog), MYOD1, and CHOB (to assess loading of samples). A. growth in 20%fetal bovine serum (growth medium). Lanes a-e. biphenotypic cell lines TC-131, TC-174,TC-253, TTC-547, and TC-2Ãœ6,respectively; Lanes f-h. alveolar RMS lines Rhl8,TC-280, and TTC-487; Lanes i-j, embryonal RMS lines CTR and Birch; Lanes k-l,pPNET lines TC-32 and TC-71; m, neuroblastoma line IMR-32. B. growth in 2% horseserum containing 10 mg/ml insulin and transferrin (differentiation medium). Lanes a-j, asin (A); Lanes k-l, pPNET lines A4573 and 6647.

EWS Gene Rearrangements in Biphenotypic Tumors. The t(l 1;22), present in 85% of pPNETs, fuses the EWS gene to FU] (2, 3).Cytogenetic analysis of the biphenotypic cell lines revealed alterationsof band 22ql2 in each case, and each had a t(ll;22) or variant (Table1). This, along with evidence for neuroectodermal differentiation,prompted us to investigate whether the EWS/FLIl gene fusion mightbe present in these tumors. We first screened TC-131, TC-174, TC-206, TC-253, and TTC-547 for EWS gene rearrangements by South

ern blot analysis using genomic probes for the EWS locus. All five celllines were rearranged at EWS using the indicated restriction enzymesas well as at least two others, while control RMS cell lines showedonly germline bands (Fig. 4 and data not shown). Therefore, EWS isrearranged in these biphenotypic tumors. In addition, the Rhl8 cellline was also rearranged at EWS in these studies (Fig. 4). Subsequentcytogenetic analysis of Rhl8 did not reveal a t(ll;22) but did demonstrate a t(10;22) involving band 22ql2 where EWS is located (datanot shown). Interestingly, this cell line also lacked expression ofMYODI (Fig. 2), while it did express MYF5 and myogenin.

EWS/FLIl Fusion Gene Expression in Biphenotypic Tumors.To determine whether EWS is fused to FLU in biphenotypic sarcomas, we screened cell lines for expression of EWS/FLII fusion transcripts by RT-PCR using EWS and FLU oligonucleotide primers. This

assay, used as a diagnostic test for pPNETs (7), detects severaldifferent amplification species (2, 7). These include 328-bp type 1products, which represent in-frame fusion of exon 7 of EWS to exon6 of FLU, 395-bp type 2 products 1 (fusion of EWS exon 7 to FLUexon 5), and 581-bp type 3 products (fusion of EWS exon 10 and FLU

exon 6; Ref. 5). As shown in Fig. 5A, type 1 products were observedin TC-131, TC-174, TC-206, and TTC-547, while TC-253 showed a

All pPNET cell lines tested were uniformly negative for expressionof these genes (Fig. 2 and data not shown).

To demonstrate molecular evidence of neural differentiation, weanalyzed the five biphenotypic cell lines for human neurofilamentgene expression by Northern analysis. We used a cDNA probe cocktail that detects the 44.5-kb neurofilament heavy (NF-H) gene transcript as well as the 2.5- and 4.0-kb major transcripts of neurofilamentlight (A/F-L)'1 (35, 36). As shown in Fig. 3A, all five biphenotypic

tumors expressed the NF-H transcript and at least one of the majortranscripts of NF-L. Similar expression was observed in two neuro

blastoma and two pPNET cell lines, while three RMS cell lineslacking EWS/FLIl fusion transcripts did not express these genes. Asa further test for neural phenotype, we (37) and others have previouslyused cDNA sequence data to develop RT-PCR-based assays to screenfor the expression of neural-associated gene expression, including thecholinergic cell-specific gene, CAT (38-40), CGA, the major protein

of dense core neurosecretory granules (41, 42), and NSE (43). Asshown in Fig. 3ÃŸ,the five biphenotypic tumors, a neuroblastoma cellline, and two pPNETs all expressed the 135-bp CAT product, while

RMS controls were negative. For CCA expression, we used primersthat amplify both 485- and 583-bp fragments, as demonstrated for theSAN-2 neuroblastoma cell line (Fig. 3ÃŸ,Lane k). All five biphenotypic tumors and the two pPNET cell lines were positive for 485- or583-bp fragments (or both), while RMS controls were negative. Allcell lines were positive for a 333-bp fragment of NSE, known to be

widely expressed (1), which was useful to confirm the presence ofintact RNA in all samples tested. The above RT-PCR results were

confirmed by probing blotted amplification products with cloned PCRfragments obtained from the SAN-2 neuroblastoma cell line for each

assay (data not shown).

s J-P. Julien, personal communication.

abcdefghij kl-â€¢-NF-H

â€”NF-L-â€¢-NF-L

-Â»-ÃŸ-Actin

B

-CAT

603 h

310 br/^ -NSE

Fig. 3. Neural differentiation in biphenotypic tumors. A, Northern blot hybridization oftotal RNA from cell lines using cDNA probes for neurofilament heavy (NF-H) and light(NF-L) genes and ÃŸ-actin (to assess loading of samples). Lanes a-e, biphenotypic celllines TC-131, TC-174, TC-253, TC-206, and TTC-547, respectively; Lunes f-h. RMS celllines CTR, A204, and TC-280, respectively; Lanes i-j. pPNET cell lines TC-32 andTC-71; Lanes k-l, neuroblastoma cell lines IMR-32 and SAN-2. B, RT-PCR analysis ofexpression of the neural-associated genes CAT, CGA, and NSE. Total RNA extracted fromcell lines was treated with DNase I and analyzed by RT-PCR. Electrophoresis of amplified

products (CAT, 135 bp; CG-4, 485 and 583 bp; NSE, 333 bp) was performed using 2%agarose gels. Lanes a-e. biphenotypic cell lines TC-131. TC-174, TC-253, TC-206, andTTC-547; Lanes f-g. pPNET cell lines TC-32 and TC-71; Lanes h-j. RMS cell lines CTR,Birch, and A204, respectively; k, neuroblastoma cell line SAN-2. Markers are shown atleft.

1388



EWS/FUl GENE FUSION IN BIPHENOTYP1C SARCOMAS

32 Bi a b c d A 32 Bi e 466 Bi f A

Fig. 4. EWS gene rearrangement in bipheno-

typic tumors. Genomic DNA from cell lines digested with EcoRl, Pstl, or Hindlll was subjectedto Southern analysis and hybridized with EWSprobes PR.8 and RR2. Lanes tt and c-f. bipheno-typic sarcoma cell lines TC-131, TTC-547, TC-174, TC-253, and TC-206, respectively; Lane b.alveolar RMS line RhlS; Lanes 32 and 466. pP-NET lines TC-32 and TTC-466, respectively;Lanes Bi and A. rhabdomyosarcoma lines Birch andA204, respectively. *, the positions of germlinebands.

â€¢EcoRI/PR.8 PstI/RR2 HindIII/PR.8

type 3 amplified product. The two pPNET cell lines, TC-32 andTC-71, also expressed type 1 fusion transcripts, while the remaining

RMS were negative (the presence of intact cDNA for the negativecases was confirmed by amplification using control primer sets).These results were confirmed by hybridizing the PCR products withan EWS oligonucleotide probe (Ref. 7; data not shown) and could bereproduced when several independently derived cryopreserved passages of each cell line were tested. Notably, the RhlS cell line wasnegative for EWS/FLI1 gene expression by RT-PCR, consistent with

cytogenetic evidence of a t(10;22) rather than a t(ll;22). We arecurrently studying this cell line to determine if EWS is fused to adifferent gene (on 10q22) in RhlS.

Because the five biphenotypic sarcomas described here were originally diagnosed as alveolar or primitive RMS, we tested these cell

abode k l m n o p q r603bf/~

310bPx_

Bh size (kPa)

^â€¢-105.1

^â€¢-69.8

â€¢â€”43.3

Fig. 5. Expression of EWS/FLII in biphenotypic tumors. A. RT-PCR analysis of celllines. Total RNA extracted from cell lines was analyzed by RT-PCR using EWS (22.3) andh'LII (11.3) primers, and products were electrophoresed using 2% agarose gels. Lanes a

and r, TC-32 and TC-71 (pPNETs); Lanes h-f. TC-131, TC-174, TTC-547, TC-2Ãœ6,andTC-253, respectively (biphenotypic tumors); Lanes g-l, RhlS, TC-147, TC-212, TC-280,TTC-487, and A204, respectively (alveolar RMS); Lanes m-n. CTR and Birch (embryonalRMS); Lane o, TTC-442 (embryonal sarcoma of liver with RMS differentiation); Liine p,TTC-516 (malignant fibrous histiocytoma); Lane q. IMR-32 (neuroblastoma). Upper

arrow, type III product; lower arrow, type I product. B. immunoprecipitation of EWS/FLU fusion protein. Antisera generated from an EWS/FLII fusion polypcptide was usedto immunoprecipitate 35S-labeled cell extracts from cell lines. Lane a, TTC-487 (alveolar

RMS); Lane b, in vitro translated EWS/FLII protein; Lane c. CTR (embryonal RMS);Lane d. TC-253; Lane e. TTC-547; Lanef. TC-174; Lane g. TC-131; and Lane h. TC-206

(biphenotypic sarcomas). Arrows, positions of type I (arrow /) and type III (arrow 2)fusion proteins. The band at 90 kDa likely represents germline EWS.

lines for PAX3/FHKR or PAX7/FHKR fusion gene expression byRT-PCR using methods described previously(8, 9). All five cell lineswere negative for the expected 409-bp PAX3/FHKR product (8) or the695-bp PAX7/FHKR product (9), in contrast to positive control cell

lines or tumor samples (data not shown). The RhlS cell line wassimilarly negative for PAX3/FHKR or PAX7/FHKR products byRT-PCR. We also used established methods ( 11) to test these tumors

for expression of EWS/WTl fusion transcripts derived from thet(ll;22)(pl3;ql2) of DSRCT, since this tumor is known to showmultilineage differentiation. While the expected PCR product (11)was present in a positive control DSRCT, all five cell lines werenegative (data not shown).

To examine whether EWS/FLII fusion transcripts produced inbiphenotypic tumors differ from those observed in pPNETs, wecloned and sequenced PCR products from TC-131, TC-174, TC-206,TC-253, and TTC-547. Sequence analysis of products from TC-131,TC-174, TC-206, and TTC-547 revealed in-frame fusions betweenexon 7 of EWS and exon 6 of FU-1 as described for type 1 fusions ofpPNETs (Ref. 2; data not shown). The TC-253 product demonstratedan in-frame type 3 fusion between exon 10 of EWS and exon 6 ofFLI-i as previously reported for pPNETs (Ref. 5; data not shown).These data confirm that RT-PCR products detected in these bipheno

typic cell lines represent amplification of EWS/FLII fusion transcriptsand demonstrate that junctional sequences are identical to thoseobserved in pPNETs.

We next determined whether EWS/FLII fusion transcripts identified by RT-PCR were functional. Northern blots of biphenotypic cell

lines hybridized with an EWS cDNA probe demonstrated aberranttranscripts in addition to the expression of germline EWS (data notshown). To detect EWS/FLII chimeric proteins, immunoprecipitationexperiments were performed using antisera raised to an EWS/FLIIpolypeptide fragment (4). As shown in Fig. 5B, immunoprecipitationfrom metabolically labeled TC-131, TC-174, TC-206, and TTC-547revealed a 68-kDa band corresponding to the EWS/FLI1 fusion pro

tein (3). The size of this protein was identical to that of in vitrotranslated EWS/FLII (Fig. 5B, Lane b). TC-253 (Fig. 50, Lane d), in

which exon 10 rather than exon 7 of EWS is fused to exon 6 of FLU,showed an 80-kDa band. This protein is predicted to contain an

additional 84 amino acids compared to the type 1 protein (2). Thesebands were not detected in lysates from negative control RMS TTC-487 or CTR cells, which lack EWS/FLII fusion transcripts. A 90-kDa

species present in all cell lines tested corresponds to germline EWS.EWS/FLII Expression in Primary Tumors. To rule out that

EWS/FLII fusion transcripts observed in the above cell lines representan artifact of in vitro cell culture conditions, we performed RT-PCR

on original patient material from biphenotypic tumors. Frozen primary

1389



EWS/FLIÃŒGENE FUSION IN BIPHENOTYPIC SARCOMAS

Fig. 6. EWS/FUl expression in primary tumors.A. RT-PCR using primers 22.3 and 11.3 was per

formed on total RNA extracted using primary tumor tissue from ease TC-206 (Lane b), case TTC-547 (Lane c), and case TC-253 (Lane d). Lane a,TC-32 cell line (pPNET); Lane e, TTC-487 cellline (RMS). B, RT-PCR analysis of five additionalbiphenotypic primary tumors (Lanes c-g). Lane a,TC-32; Lane b, TTC 487. Arrows, positions ofamplified products.

Bb c d e a b e d e f

tumor tissue was available for TC-206, TC-253, and TTC-547, as well

as for five additional pediatrie soft tissue sarcomas with morphological, ultrastructural, and immunocytochemical evidence of both myo-genic and neuroectodermal differentiation.7 As shown in Fig. 6A,

original tumor tissue used to derive TC-206, TC-253, and TTC-547 all

demonstrated identical findings as the corresponding cell lines. Furthermore, all five additional biphenotypic tumors demonstrated type 1EWS/FLU fusion transcripts (Fig. 6B). Therefore, the EWS/FUlfusion gene is expressed in primary tumors.

DISCUSSION

We demonstrate here that a subset of childhood sarcomas with bothmyogenic and neuroectodermal differentiation contain a chromosomalalteration resulting in the formation of the EWS/FLll fusion gene. TheEWS/FLU gene fusion was described as a specific feature of theSwing's sarcoma family of pPNETs (2, 3), resulting from the t(ll;22)

found in 85% of these tumors (44). EWS is a ubiquitously expressedgene of unknown function, while FLU (31, 45, 46) is a member of theÂ£75gene family of transcription factors. The EWS/FLU chimericprotein consists of the glutamine-rich amino terminal portion of EWS

fused to the carboxy terminal DNA binding domain of FLI1 (2, 3).This molecule efficiently transforms NIH3T3 cells, and this transformation ability requires both EWS and FLU domains (3). Recentstudies indicate that a second translocation in pPNETs, the t(21;22)(q22;ql2), fuses EWS with another ETS family transcription factor,ERG, located on chromosome 21q22 (4-6). The chimeric EWS/FLU

product (and likely EWS/ERG) appears to function as an oncoproteinby acting as an aberrant transcription factor (47). Chimeric EWS/FLUtranscripts and fusion proteins in the tumors described in the presentstudy were indistinguishable from those found in pPNETs, suggestingthat the EWS/FLll oncoprotein plays a similar role in biphenotypictumors.

The findings presented here provide a unique example of identicalgene fusions occurring in two phenotypically distinct solid tumortypes. While pPNETs display exclusively neuroectodermal differentiation with no evidence of skeletal muscle development (reviewed inRef. l), the biphenotypic tumors described here also demonstratedclear morphological, immunocytochemical, and ultrastructural evidence of myogenic differentiation. All biphenotypic cell lines expressed MYF5 and three of five expressed myogenin. These factors,along with MYODl and MRF4, constitute the MyoD family of myogenic regulatory factor genes (15,16). Constitutive expression of eachfactor is sufficient to convert a variety of nonmyogenic cells toskeletal muscle cells (15). Expression of this gene family is restrictedto skeletal muscle and myogenic cell lines, and among human tumors,has only been described in entities with skeletal muscle differentiation

7 Y. Hachitanda, C. Aoyama, J. K. Sato, and H. Shimada. The most primitive form of

malignant ectomesenchymoma: an immunohistochemically and ultrastructurally definedentity, manuscript in preparation.

(17, 48, 49). Neuroectodermal differentiation in biphenotypic tumorswas demonstrated by immunoreactivity for neurofilament proteins, aswell as by ultrastructural identification of dense core neurosecretorygranules and primitive neurites, which have not been described inRMS. These structures are characteristic of cells of neural crest originand are found in neuroblastomas and pPNETs (reviewed in Refs. 1and 33). In addition, all five cell lines as well as pPNET and neuroblastoma controls, but not conventional RMS controls, expressedmultiple human neurofilament genes which encode the major intermediate filaments of neural cells (12). Biphenotypic tumors, alongwith pPNETs and neuroblastomas, but not RMS controls, also expressed CAT, specific for cholinergic cells (38) and CCA, whichencodes the major protein component of neurosecretory granules (41).Interestingly, MYODl expression was not detected in any of thebiphenotypic tumor cell lines tested and could not be induced bygrowth in differentiation medium. MyoDl may be dispensable for theinitial stages of myogenesis, possibly due to functional redundancyamong the myogenic regulatory factors (50, 51). Transgenic micelacking MyoDl express other myogenic regulatory factors, and skeletal muscle formation appears to be normal (51). The EWS/FLlloncoprotein itself may inhibit MYODl expression, as has been demonstrated for other oncogene or proto-oncogene products includingRas and c-Fos (52) as well as c-Jun (53). Lack of expression of

MYODl may be a unique feature of these biphenotypic tumors.The findings presented here that the EWS/FLU gene fusion occurs

in both biphenotypic sarcomas and in pPNETs suggests that these twoclasses of tumors are related. It is not clear why one set of tumorsexpressing the EWS/FLll gene fusion demonstrates only a neuroectodermal phenotype, while another set also shows myogenic features.One possibility is that expression of this chimeric gene somehowinfluences neural differentiation, such that neuroectodermal phenotype is a general feature of tumors with this gene fusion. In thiscontext, the muscle phenotype observed in biphenotypic sarcomasmay be either a function of additional genetic alterations or a reflection of the differentiation capacity of the cell of origin of thesemalignancies. Soft tissue sarcomas (in addition to DSRCT) have beendescribed which show both skeletal muscle and neural differentiation(1, 19). Malignant ectomesenchymomas occur primarily in childhoodand are composed histologically of tumor cells with neural differentiation as well as one or more malignant mesenchymal elements,including RMS (20-22). They are thought to arise from pluripotential

embryonic neural crest tissue, or ectomesenchyme, which is known tobe widely dispersed in human tissues (54). Experimental studies havedemonstrated that these cells are pluripotential, being capable ofdifferentiating not only into neuroectodermal tissue but also alongmesenchymal lineages, including skeletal muscle (54, 55). The biphenotypic tumors described here may thus represent examples of malignant ectomesenchymomas in which neural development is veryprimitive and previously unrecognized. In this context, thepPNETs, which are also thought to have a neural crest histogenesis

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EWS/FUI GENE FUSION IN B1PHENOTYPIC SARCOMAS

(13, 14), may be derived from a distinct but related cell of originthat retains neuroectodermal features but has lost the capacity formyogenic differentiation.

Our data indicate that a subset of childhood sarcomas with myogenic differentiation may be histogenetically distinct from conventional RMS, tumors which are thought to arise from immature muscleprecursor cells (reviewed in Ref. l). RMS are the most commonlydiagnosed soft tissue sarcomas in children. However, primitive formsof this tumor class may be more heterogeneous than appreciatedpreviously, and the differential diagnosis of primitive RMS appears tooverlap with other nosological entities. Myogenic immunophenotypeis known to be a feature of DSRCT, a tumor of multilineage differentiation that appears to be a distinct tumor class associated with acharacteristic gene fusion (10, 11). Other rare primitive sarcomasthought to be distinct from RMS have been described that demonstrateevidence of muscle differentiation, but these remain poorly characterized and difficult to classify (1, 19). The biphenotypic tumorsdescribed here were all initially diagnosed as alveolar or primitiveRMS on the basis of morphological and immunophenotypic features.However, we failed to detect PAX3/FKHR (8) or PAX7/FKHR (9)fusion transcripts described for alveolar RMS or EWS/WTÃŒfusiontranscripts found in DSRCT (11), observing instead pPNET-associ-

ated EWS/FUI gene fusions. These tumors, possibly representingprimitive malignant ectomesenchymomas related to the pPNET family, therefore, provide a further example of a primitive childhoodsarcoma with myogenic differentiation. Preliminary studies in ourlaboratory suggest that these sarcomas may comprise 10% or more ofchildhood tumors with a primitive RMS phenotype.8 As described in

the present study, these tumors can be distinguished by expression ofthe EWS/FLI1 gene fusion, and so it will be important to determine theincidence and prognostic significance of this tumor entity. The findings presented here provide a further argument that classification ofmany forms of childhood solid tumors should be considered from amolecular genetic as well as from a morphological perspective.

ACKNOWLEDGMENTS

We thank C. T. Denny and S. L. Lessnick of the University of California(Los Angeles, CA) and J. Peters at Children's Hospital of Los Angeles for their

contributions to this study.

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1995;55:1385-1392. Cancer Res Poul H. B. Sorensen, Hiroyuki Shimada, Xian F. Liu, et al. Gene

FusionEWS/FLI1Differentiation Express the Ewing's Sarcoma Biphenotypic Sarcomas with Myogenic and Neural

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