Gene Fusion EWS/FLI1 Differentiation Express the Ewing's Sarcoma ...

Biphenotypic Sarcomas with Myogenic and Neural Differentiation Express the Ewing's Sarcoma EWS/FLI1 Fusion Gene Poul H. B. Sorensen, Hiroyuki Shimada, Xian F. Liu, et al. Cancer Res 1995;55:1385-1392. Published online March 1, 1995.

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

Biphenotypic Sarcomas with Myogenic and Neural Differentiation Express the Ewing'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©tique des Tumeurs, Institut Curie. Paris. France Â¡G.7*./

ABSTRACT

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 Accurate diagnosis of primitive childhood sarcomas continues to be a fusion genes are capable of cell transformation and appear to act as formidable problem because these malignancies generally demonstrate aberrant transcription factors (3). Identification of EWS/FLIl and very little morphological evidence of their tissue of origin. One of these EWS/ERG fusion transcripts by the RT-PCR has formed the basis of tumor classes, the Ewing's sarcoma family of peripheral primitive neua sensitive and specific diagnostic test for pPNETs (7). Gene fusions roectodermal tumors (pPNETs), are thought to have a neural histogenesis have been identified and characterized in other pediatrie sarcomas. based on evidence of neuroectodermal differentiation. Greater than 95% The t(2;13)(q35;ql4) and t(l;13)(p36;ql4) translocations of alveolar of pPNETs carry t(ll;22) or K2I;22) chromosomal translocations which fuse the EWS gene from chromosome 22ql2 in-frame with either /â€¢'/.// RMS result in the expression ofPAX3/FKHR (8) and PAX7/FKHR (9) from chromosome Hq24 or ERG from chromosome 21q22. The pPNETs fusion transcripts, respectively, while the recently described intraab are considered to be histogenetically distinct from rhabdomyosarcomas, dominal DSRCT (10) has been shown to express a gene fusion myogenic tumors lacking these EWS gene fusions and hypothesized to involving EWS and the WTl tumor suppressor gene located at chro derive from immature skeletal muscle precursors. In the present study, we mosomal band Ilpl3 (11). Detection of these genetic abnormalities describe a unique set of childhood soft tissue sarcomas that show both thus provides a potentially powerful modality for the diagnosis of neural and myogenic differentiation. These biphenotypic tumors express primitive childhood sarcomas, and classification based on their iden myogenic regulatory factors and muscle-specific antigens and also show tification may ultimately provide a more accurate predictor of clinical neuroectodermal differentiation with ultrastructural evidence of neurosebehavior than current morphological classification systems. cretory granules and expression of neural-associated genes. Northern The pPNETs are a family of poorly differentiated tumors that analysis and reverse transcriptase PCR reveal expression of EWS/FLIl include Ewing's sarcoma, peripheral neuroepithelioma, and Askin gene fusions in all biphenotypic sarcomas analyzed. Chimeric EWS/FU1 transcripts and fusion proteins in these tumors are identical to those described for pPNETs. Our results provide evidence for a class of biphe notypic childhood sarcomas with myogenic and neural differentiation and suggest that these tumors may be related to the Ewing's sarcoma family of pPNETs.

INTRODUCTION Primitive sarcomas occurring in childhood have traditionally pre sented significant diagnostic challenges due to their relative lack of distinctive morphological features. One group, the SRCTs3 of child hood, are composed predominantly of poorly differentiated small round 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 histo lÃ³gica!cell of origin; therefore, they are often difficult to distinguish from each other based on morphological features. The identification of tumor-specific fusion genes resulting from chromosomal translocations in human malignancies is not only yield ing profound insights into oncogenesis but also holds great promise for tumor diagnosis. Two translocations, the t(ll;22)(q24;ql2) and t(21;22)(q22;ql2), characterize pPNETs and are considered specific for 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 with 18 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 Endow ment 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

tumor. These tumors are thought to have a neural cell of origin based on limited neuroectodermal development, and the different family members show varying degrees of neural differentiation (reviewed in Ref. l). They display ultrastructural evidence of primitive neurites and neurosecretory granules and show immunoreactivity for several neu ral-associated markers, including neurofilament proteins (12). In ad dition, neural differentiation can be induced in these tumors by treat ment with retinoic acid and nerve growth factors (13, 14). The pPNETs are considered to be clinically and histogenetically distinct from RMS, malignancies with skeletal muscle differentiation hypoth esized to derive from immature muscle precursor cells (reviewed in Ref. l). These tumors do not display neural features, containing instead tumor rhabdomyoblasts with immunoreactivity for skeletal muscle-associated antigens including desmin and muscle-specific actin. Ultrastructurally, they show muscle-specific cytoplasmic interme diate 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 diagnostic criterion for RMS (17, 18). These myogenic regulatory factors are thought to control the initiation and maintenance of myogenic differ entiation 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 morpholog ical evidence of both myogenic and neuroectodermal differentiation (1, 19). DSRCT demonstrates multilineage differentiation with epi thelial, neural, and skeletal muscle immunophenotypic features (10). Other entities remain poorly defined, but at least some of these cases may represent examples of malignant ectomesenchymoma (20-22).

Malignant ectomesenchymomas are predominantly pediatrie soft tis sue tumors characterized by the presence of neural sarcomatous primitive neuroectodermat tumor; RMS, rhabdomyosarcoma; DSRCT, desmoplastic elements in addition to one or more malignant mesenchymal compo SRCT; RT-PCR, reverse transcription-PCR; bp, base pair(s); kDa, kilodalton(s); NF, nents, including RMS. Molecular studies may be useful to clarify the neurofilament; CAT, choline acetyltransferase; CGA, chromogranin A; NSE, neuronrelationship of these tumors to other neural and myogenic soft tissue specific enolase. 1385 3V4, Canada. ' The abbreviations

used are: SRCT, small round cell tumor: pPNET. peripheral


EWS/FLII GENE FUSION IN BIPHENOTYPIC SARCOMAS Table 1 Pathological features of biphenot\pic

Age/sexSiteTC-13119/MSoft

mos./MSoft tissue, chest wallTC-1749/FSoft

Diagnosis

tumors and celi lines

Alveolar RMS

tissue, thighTC-2065

Alveolar RMS

tissue, upper armTC-25325/MRetroperitoneumTTC-54713/FRetroperitoneum Primitive RMS

Alveolar RMS

Alveolar RMS

IHC"Desmin â€¢H-Positive

Actin NeurofilamentEM*Myogenic

Positivet(ll;22Xq24;ql2)Â£Positive Positivet(ll;22Xq24;ql2)*Positive Positivet(ll;22Xq24;ql2)â€¢H- Positivet(ll;12;22Xq24;ql4;ql2)

NeuralCytogenetics of bands md22ql27PositivePositivet(ll;22Xq24;ql2)+;Positive 11424

" 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 tumors that were originally diagnosed as primitive forms of RMS based on morphological evidence of myogenic differentiation. These tumors expressed myogenic regulatory factors and demonstrated immunoreactivity for muscle-specific antigens. However, ultrastructural analy sis revealed both myogenic and neural features, and molecular studies demonstrated 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 biphenotypic tumors. Our results suggest that a subset of biphenotypic sarcomas with myogenic and neural features may be members of the pPNET family of childhood tumors, a finding which could have important implications in the diagnosis and treatment of this group of childhood malignancies. 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-32 and SAN-2 (28) have been described previously. Other cell lines were estab lished 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's Hospital, Memphis, TN (Birch)4 from primary tumor cells using standard

transcripts was performed as reported previously using total RNA as starting material (7). Oligonucleotide primers used included 11.3 (FLU) and 22.3 (EWS; Refs. 2 and 7). RT-PCR screening for neural-associated gene expression was performed on DNase I (Pharmacia)-treated total RNA using the manufac turer's protocols. Primers for CAT included 5'-GACCATCCTTTTCTGCATCT-3' (sense) and 5'-GGCTTGCTCTCAGTAGGC-3f (antisense); for CCA, they included S'-GACCGACTCTCGCCTTTCCG-S' (sense) and 5'GCTCCAAGACCTCGCTCTCC-3' (antisense); and for NSE, they included 5'-TCTGAACGTCTGGCTAAATACAAC-3' (sense) and 5'-GAAAAGGAAGAAGTGGAAAGTGCGG-3' (antisense). Amplified products were an alyzed by electrophoresis using 2% agarose gels and stained with ethidium bromide. Immunoprecipitation Analysis. Antisera were raised to a glutathione .V transferase-EWS/FLIl fusion polypeptide consisting of EWS amino acids 245-264 (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 line containing an 11;22 translocation (7) was subcloned into the expression plasmid pGEX-2T (Pharmacia). This construct was expressed in Escherichia coli, the fusion protein was purified using glutathione-agarose (32), and protein was injected s.c. into rabbits. This antisera recognizes epitopes from both EWS and FLU.5 Cells were metabolically labeled with ['15S]methionine, and whole cell lysates were subjected to two-cycle immunoprecipitation using EWS/FLII antisera. Immune complexes were precipitated with protein A-Sepharose (Pharmacia), fractionated by SDS-polyacrylamide gel electrophoresis, and analyzed by autoradiography.

methods. All cell lines were maintained in RPMI 1640 plus 20% fetal bovine serum, and all analyses were performed on early passage cell lines. Clinical and 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. Primary tumors stored frozen in liquid nitrogen were obtained from the Children's

RESULTS

Hospital of Los Angeles tumor bank. Electron microscopy and immunocytochemistry 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 according to 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, MYOGENIN, MYOD1, and for the neurofilament light and heavy genes (NF-L and NF-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 described previously (5). Probes for Northern and Southern analysis were radiolabeled by random primer extension. RT-PCR. Total RNA from cell lines or frozen primary tumor tissue was isolated as described previously (30). RT-PCR detection of EWS/FLII fusion 4 S. Piper, unpublished data.

Biphenotypic Tumors with Myogenic and Neuroectodermal Differentiation. Previous studies have documented the existence of childhood sarcomas with multilineage differentiation (1), and prelim inary studies in our laboratory indicated that a subset of childhood tumors with myogenic phenotype also express primitive neural fea tures. Therefore, we screened a series of tumors and corresponding cell lines of myogenic phenotypic features for evidence of neuroectodermal differentiation. Five cases were identified; pathological fea tures 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 primitive RMS. Morphological analysis revealed similar findings in all cases, with nests of poorly differentiated small round cells and clearly identifiable rhabdomyoblasts separated by fibrous septae, as shown in Fig. \A for the original tumor from case TTC-547. Each tumor 5S. L. Lessnick and C. T. Denny, unpublished data.

1386


WS/FUI

(il.NI-

HSION

IN lill'lll NOI Vl'IC SARCOMAS

Fig. 1. Pathology of biphenotypic tumors. A, histolÃ³gica! section of original tumor tissue from case TTC-547 showing primitive rhabdomyoblastic cells separated by fibrous septae. B, electron mi crograph of original tumor tissue from case TTC547 demonstrating muscle-specific cytoplasmic actin-like filaments (arrow). C. electron micrograph of the same cell as in (ÃŸ),showing dense-core neurosecretory granules (arrows).

demonstrated cytoplasmic immunoreactivity for muscle-specific desmin TC-131, TC-174, TC-206, TC-253, and TTC-547 as well as RMS and actin, which was maintained in cell lines (data not shown). Control control cell lines expressed MYF5. Expression varied among the cell lines, with TC-131, TC-253, and TTC-547 consistently showing pPNETs were negative. Ultrastructurally, original tumor tissue from each case showed cytoplasmic actin-like filaments characteristic of primitive lower levels. Myogenin expression was not observed in TC-131 RMS (1), as shown for case TTC-547 in Fig. IB. Moreover, neuroectoand TC-253 but was clearly evident in TC-174, TC-206, and dermal differentiation in these tumors could be demonstrated ultrastrucTTC-547. Interestingly, MYOD1 expression was virtually undeturally by the presence of primitive neurites and dense core neurosecre tectable in any of the biphenotypic tumor cell lines, while expres tory granules (33), shown in Fig. 1C for case TTC-547. Fig. l, B and C, sion was observed in all control RMS cell lines except Rhl8. As represent electron micrographs taken from the same cell, indicating the shown in Fig. 2B, MYOD1 expression could not be induced by divergent differentiation of these tumor cells. Each tumor also showed transfer to differentiation medium (2% horse serum), known to immunoreactivity for the neural antigen, neurofilament protein (Ref. 34; promote myogenic differentiation (15). Therefore, all five biphe notypic cell lines expressed MYF5, three of five expressed MYO Table 1), corroborating the biphenotypic nature of these tumors. GENIN, and none expressed MYODI. These findings were not due To confirm myogenic differentiation in these biphenotypic tumors, to the maintenance of primitive muscle cells in culture, as identical we examined cell lines for expression of the myogenic regulatory results were obtained using both early and late passage cell lines. genes MYF5, myogenin, and MYOD1 by Northern analysis (Fig. 2A). 1387


EWS/FUI

GENE FUSION

IN BIPHENOTYPIC

abcdefghijklm

D

abcdefghijkl

Fig. 2. Expression of myogenic regulatory factors in hiphenotypic tumors. Northern blot 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% horse serum containing 10 mg/ml insulin and transferrin (differentiation medium). Lanes a-j, as in (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 alterations of band 22ql2 in each case, and each had a t(ll;22) or variant (Table 1). This, along with evidence for neuroectodermal differentiation, prompted us to investigate whether the EWS/FLIl gene fusion might be present in these tumors. We first screened TC-131, TC-174, TC206, TC-253, and TTC-547 for EWS gene rearrangements by South ern blot analysis using genomic probes for the EWS locus. All five cell lines were rearranged at EWS using the indicated restriction enzymes as well as at least two others, while control RMS cell lines showed only germline bands (Fig. 4 and data not shown). Therefore, EWS is rearranged in these biphenotypic tumors. In addition, the Rhl8 cell line was also rearranged at EWS in these studies (Fig. 4). Subsequent cytogenetic analysis of Rhl8 did not reveal a t(ll;22) but did dem onstrate a t(10;22) involving band 22ql2 where EWS is located (data not shown). Interestingly, this cell line also lacked expression of MYODI (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 sarco mas, we screened cell lines for expression of EWS/FLII fusion tran scripts by RT-PCR using EWS and FLU oligonucleotide primers. This assay, used as a diagnostic test for pPNETs (7), detects several different amplification species (2, 7). These include 328-bp type 1 products, which represent in-frame fusion of exon 7 of EWS to exon 6 of FLU, 395-bp type 2 products 1 (fusion of EWS exon 7 to FLU exon 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 observed in TC-131, TC-174, TC-206, and TTC-547, while TC-253 showed a

All pPNET cell lines tested were uniformly negative for expression of these genes (Fig. 2 and data not shown). To demonstrate molecular evidence of neural differentiation, we analyzed the five biphenotypic cell lines for human neurofilament gene expression by Northern analysis. We used a cDNA probe cock tail that detects the 44.5-kb neurofilament heavy (NF-H) gene tran script as well as the 2.5- and 4.0-kb major transcripts of neurofilament light (A/F-L)'1 (35, 36). As shown in Fig. 3A, all five biphenotypic

abcdefghij

â€”NF-L -â€¢-NF-L

of dense core neurosecretory granules (41, 42), and NSE (43). As shown in Fig. 3ÃŸ,the five biphenotypic tumors, a neuroblastoma cell line, and two pPNETs all expressed the 135-bp CAT product, while RMS controls were negative. For CCA expression, we used primers that amplify both 485- and 583-bp fragments, as demonstrated for the SAN-2 neuroblastoma cell line (Fig. 3ÃŸ,Lane k). All five bipheno typic tumors and the two pPNET cell lines were positive for 485- or 583-bp fragments (or both), while RMS controls were negative. All cell lines were positive for a 333-bp fragment of NSE, known to be widely expressed (1), which was useful to confirm the presence of intact RNA in all samples tested. The above RT-PCR results were

s J-P. Julien, personal communication.

kl -â€¢-NF-H

-Â»-ÃŸ-Actin

tumors expressed the NF-H transcript and at least one of the major transcripts of NF-L. Similar expression was observed in two neuro blastoma and two pPNET cell lines, while three RMS cell lines lacking EWS/FLIl fusion transcripts did not express these genes. As a further test for neural phenotype, we (37) and others have previously used cDNA sequence data to develop RT-PCR-based assays to screen for the expression of neural-associated gene expression, including the cholinergic cell-specific gene, CAT (38-40), CGA, the major protein

confirmed by probing blotted amplification products with cloned PCR fragments obtained from the SAN-2 neuroblastoma cell line for each assay (data not shown).

SARCOMAS

B -CAT

603 h 310 br/^

-NSE

Fig. 3. Neural differentiation in biphenotypic tumors. A, Northern blot hybridization of total 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 cell lines TC-131, TC-174, TC-253, TC-206, and TTC-547, respectively; Lunes f-h. RMS cell lines CTR, A204, and TC-280, respectively; Lanes i-j. pPNET cell lines TC-32 and TC-71; Lanes k-l, neuroblastoma cell lines IMR-32 and SAN-2. B, RT-PCR analysis of expression of the neural-associated genes CAT, CGA, and NSE. Total RNA extracted from cell 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, and TTC-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 at left.

1388


EWS/FUl

GENE FUSION

32 Bi a Fig. 4. EWS gene rearrangement in biphenotypic tumors. Genomic DNA from cell lines di gested with EcoRl, Pstl, or Hindlll was subjected to Southern analysis and hybridized with EWS probes PR.8 and RR2. Lanes tt and c-f. biphenotypic sarcoma cell lines TC-131, TTC-547, TC174, TC-253, and TC-206, respectively; Lane b. alveolar RMS line RhlS; Lanes 32 and 466. pPNET lines TC-32 and TTC-466, respectively; Lanes Bi and A. rhabdomyosarcoma lines Birch and A204, respectively. *, the positions of germline bands.

IN BIPHENOTYP1C

b c d A

SARCOMAS

32

Bi

e

466

Bi

f

A

â€¢ EcoRI/PR.8

type 3 amplified product. The two pPNET cell lines, TC-32 and TC-71, also expressed type 1 fusion transcripts, while the remaining RMS were negative (the presence of intact cDNA for the negative cases was confirmed by amplification using control primer sets). These results were confirmed by hybridizing the PCR products with an EWS oligonucleotide probe (Ref. 7; data not shown) and could be reproduced when several independently derived cryopreserved pas sages of each cell line were tested. Notably, the RhlS cell line was negative for EWS/FLI1 gene expression by RT-PCR, consistent with cytogenetic evidence of a t(10;22) rather than a t(ll;22). We are currently studying this cell line to determine if EWS is fused to a different gene (on 10q22) in RhlS. Because the five biphenotypic sarcomas described here were orig inally diagnosed as alveolar or primitive RMS, we tested these cell

PstI/RR2

HindIII/PR.8

lines for PAX3/FHKR or PAX7/FHKR fusion gene expression by RT-PCR using methods described previously(8, 9). All five cell lines were negative for the expected 409-bp PAX3/FHKR product (8) or the 695-bp PAX7/FHKR product (9), in contrast to positive control cell lines or tumor samples (data not shown). The RhlS cell line was similarly negative for PAX3/FHKR or PAX7/FHKR products by RT-PCR. We also used established methods ( 11) to test these tumors

for expression of EWS/WTl fusion transcripts derived from the t(ll;22)(pl3;ql2) of DSRCT, since this tumor is known to show multilineage differentiation. While the expected PCR product (11) was present in a positive control DSRCT, all five cell lines were negative (data not shown). To examine whether EWS/FLII fusion transcripts produced in biphenotypic tumors differ from those observed in pPNETs, we cloned 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 between exon 7 of EWS and exon 6 of FU-1 as described for type 1 fusions of pPNETs (Ref. 2; data not shown). The TC-253 product demonstrated abode k l mn o p q r 603bf/~ an in-frame type 3 fusion between exon 10 of EWS and exon 6 of FLI-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 transcripts 310bPx_ and demonstrate that junctional sequences are identical to those observed in pPNETs. We next determined whether EWS/FLII fusion transcripts identi fied by RT-PCR were functional. Northern blots of biphenotypic cell lines hybridized with an EWS cDNA probe demonstrated aberrant size (kPa) transcripts in addition to the expression of germline EWS (data not shown). To detect EWS/FLII chimeric proteins, immunoprecipitation ^â€¢-105.1 experiments were performed using antisera raised to an EWS/FLII ^â€¢-69.8 polypeptide fragment (4). As shown in Fig. 5B, immunoprecipitation from metabolically labeled TC-131, TC-174, TC-206, and TTC-547 revealed a 68-kDa band corresponding to the EWS/FLI1 fusion pro â€¢â€”43.3 tein (3). The size of this protein was identical to that of in vitro Fig. 5. Expression of EWS/FLII in biphenotypic tumors. A. RT-PCR analysis of cell translated EWS/FLII (Fig. 5B, Lane b). TC-253 (Fig. 50, Lane d), in lines. Total RNA extracted from cell lines was analyzed by RT-PCR using EWS (22.3) and which exon 10 rather than exon 7 of EWS is fused to exon 6 of FLU, h'LII (11.3) primers, and products were electrophoresed using 2% agarose gels. Lanes a showed an 80-kDa band. This protein is predicted to contain an and r, TC-32 and TC-71 (pPNETs); Lanes h-f. TC-131, TC-174, TTC-547, TC-2Ãœ6,and TC-253, respectively (biphenotypic tumors); Lanes g-l, RhlS, TC-147, TC-212, TC-280, additional 84 amino acids compared to the type 1 protein (2). These TTC-487, and A204, respectively (alveolar RMS); Lanes m-n. CTR and Birch (embryonal bands were not detected in lysates from negative control RMS TTCRMS); Lane o, TTC-442 (embryonal sarcoma of liver with RMS differentiation); Liine p, TTC-516 (malignant fibrous histiocytoma); Lane q. IMR-32 (neuroblastoma). Upper 487 or CTR cells, which lack EWS/FLII fusion transcripts. A 90-kDa arrow, type III product; lower arrow, type I product. B. immunoprecipitation of EWS/ species present in all cell lines tested corresponds to germline EWS. FLU fusion protein. Antisera generated from an EWS/FLII fusion polypcptide was used to immunoprecipitate 35S-labeled cell extracts from cell lines. Lane a, TTC-487 (alveolar EWS/FLII Expression in Primary Tumors. To rule out that RMS); Lane b, in vitro translated EWS/FLII protein; Lane c. CTR (embryonal RMS); EWS/FLII fusion transcripts observed in the above cell lines represent Lane d. TC-253; Lane e. TTC-547; Lanef. TC-174; Lane g. TC-131; and Lane h. TC-206 an artifact of in vitro cell culture conditions, we performed RT-PCR (biphenotypic sarcomas). Arrows, positions of type I (arrow /) and type III (arrow 2) on original patient material from biphenotypic tumors. Frozen primary fusion proteins. The band at 90 kDa likely represents germline EWS. 1389

B

h


EWS/FLIÃŒGENE FUSION IN BIPHENOTYPIC

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 tu mor tissue from ease TC-206 (Lane b), case TTC547 (Lane c), and case TC-253 (Lane d). Lane a, TC-32 cell line (pPNET); Lane e, TTC-487 cell line (RMS). B, RT-PCR analysis of five additional biphenotypic primary tumors (Lanes c-g). Lane a, TC-32; Lane b, TTC 487. Arrows, positions of amplified products.

SARCOMAS

B b

c

d

e

a

b

tumor tissue was available for TC-206, TC-253, and TTC-547, as well as for five additional pediatrie soft tissue sarcomas with morpholog ical, ultrastructural, and immunocytochemical evidence of both myogenic and neuroectodermal differentiation.7 As shown in Fig. 6A,

e d

e

f

(17, 48, 49). Neuroectodermal differentiation in biphenotypic tumors was demonstrated by immunoreactivity for neurofilament proteins, as well as by ultrastructural identification of dense core neurosecretory granules and primitive neurites, which have not been described in original tumor tissue used to derive TC-206, TC-253, and TTC-547 all RMS. These structures are characteristic of cells of neural crest origin demonstrated identical findings as the corresponding cell lines. Fur and are found in neuroblastomas and pPNETs (reviewed in Refs. 1 thermore, all five additional biphenotypic tumors demonstrated type 1 and 33). In addition, all five cell lines as well as pPNET and neuro EWS/FLU fusion transcripts (Fig. 6B). Therefore, the EWS/FUl blastoma controls, but not conventional RMS controls, expressed fusion gene is expressed in primary tumors. multiple human neurofilament genes which encode the major inter mediate filaments of neural cells (12). Biphenotypic tumors, along with pPNETs and neuroblastomas, but not RMS controls, also ex DISCUSSION pressed CAT, specific for cholinergic cells (38) and CCA, which We demonstrate here that a subset of childhood sarcomas with both encodes the major protein component of neurosecretory granules (41). myogenic and neuroectodermal differentiation contain a chromosomal Interestingly, MYODl expression was not detected in any of the alteration resulting in the formation of the EWS/FLll fusion gene. The biphenotypic tumor cell lines tested and could not be induced by EWS/FLU gene fusion was described as a specific feature of the growth in differentiation medium. MyoDl may be dispensable for the Swing's sarcoma family of pPNETs (2, 3), resulting from the t(ll;22) initial stages of myogenesis, possibly due to functional redundancy found in 85% of these tumors (44). EWS is a ubiquitously expressed among the myogenic regulatory factors (50, 51). Transgenic mice gene of unknown function, while FLU (31, 45, 46) is a member of the lacking MyoDl express other myogenic regulatory factors, and skel Â£75 gene family of transcription factors. The EWS/FLU chimeric etal muscle formation appears to be normal (51). The EWS/FLll protein consists of the glutamine-rich amino terminal portion of EWS oncoprotein itself may inhibit MYODl expression, as has been dem fused to the carboxy terminal DNA binding domain of FLI1 (2, 3). onstrated for other oncogene or proto-oncogene products including This molecule efficiently transforms NIH3T3 cells, and this transfor Ras and c-Fos (52) as well as c-Jun (53). Lack of expression of mation ability requires both EWS and FLU domains (3). Recent MYODl may be a unique feature of these biphenotypic tumors. studies indicate that a second translocation in pPNETs, the t(21; The findings presented here that the EWS/FLU gene fusion occurs 22)(q22;ql2), fuses EWS with another ETS family transcription factor, in both biphenotypic sarcomas and in pPNETs suggests that these two ERG, located on chromosome 21q22 (4-6). The chimeric EWS/FLU classes of tumors are related. It is not clear why one set of tumors product (and likely EWS/ERG) appears to function as an oncoprotein expressing the EWS/FLll gene fusion demonstrates only a neuroec by acting as an aberrant transcription factor (47). Chimeric EWS/FLU todermal phenotype, while another set also shows myogenic features. transcripts and fusion proteins in the tumors described in the present One possibility is that expression of this chimeric gene somehow study were indistinguishable from those found in pPNETs, suggesting influences neural differentiation, such that neuroectodermal pheno that the EWS/FLll oncoprotein plays a similar role in biphenotypic type is a general feature of tumors with this gene fusion. In this tumors. context, the muscle phenotype observed in biphenotypic sarcomas The findings presented here provide a unique example of identical may be either a function of additional genetic alterations or a reflec gene fusions occurring in two phenotypically distinct solid tumor tion of the differentiation capacity of the cell of origin of these types. While pPNETs display exclusively neuroectodermal differen malignancies. Soft tissue sarcomas (in addition to DSRCT) have been tiation with no evidence of skeletal muscle development (reviewed in described which show both skeletal muscle and neural differentiation Ref. l), the biphenotypic tumors described here also demonstrated (1, 19). Malignant ectomesenchymomas occur primarily in childhood clear morphological, immunocytochemical, and ultrastructural evi and are composed histologically of tumor cells with neural differen dence of myogenic differentiation. All biphenotypic cell lines ex tiation as well as one or more malignant mesenchymal elements, pressed MYF5 and three of five expressed myogenin. These factors, including RMS (20-22). They are thought to arise from pluripotential along with MYODl and MRF4, constitute the MyoD family of myo embryonic neural crest tissue, or ectomesenchyme, which is known to genic regulatory factor genes (15,16). Constitutive expression of each be widely dispersed in human tissues (54). Experimental studies have factor is sufficient to convert a variety of nonmyogenic cells to demonstrated that these cells are pluripotential, being capable of skeletal muscle cells (15). Expression of this gene family is restricted differentiating not only into neuroectodermal tissue but also along to skeletal muscle and myogenic cell lines, and among human tumors, mesenchymal lineages, including skeletal muscle (54, 55). The biphe has only been described in entities with skeletal muscle differentiation notypic tumors described here may thus represent examples of ma lignant ectomesenchymomas in which neural development is very 7 Y. Hachitanda, C. Aoyama, J. K. Sato, and H. Shimada. The most primitive form of primitive and previously unrecognized. In this context, the malignant ectomesenchymoma: an immunohistochemically and ultrastructurally defined entity, manuscript in preparation. pPNETs, which are also thought to have a neural crest histogenesis 1390


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(13, 14), may be derived from a distinct but related cell of origin that retains neuroectodermal features but has lost the capacity for myogenic differentiation. Our data indicate that a subset of childhood sarcomas with myo genic differentiation may be histogenetically distinct from conven tional RMS, tumors which are thought to arise from immature muscle precursor cells (reviewed in Ref. l). RMS are the most commonly diagnosed soft tissue sarcomas in children. However, primitive forms of this tumor class may be more heterogeneous than appreciated previously, and the differential diagnosis of primitive RMS appears to overlap with other nosological entities. Myogenic immunophenotype is known to be a feature of DSRCT, a tumor of multilineage differ entiation that appears to be a distinct tumor class associated with a characteristic gene fusion (10, 11). Other rare primitive sarcomas thought to be distinct from RMS have been described that demonstrate evidence of muscle differentiation, but these remain poorly charac terized and difficult to classify (1, 19). The biphenotypic tumors described here were all initially diagnosed as alveolar or primitive RMS 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/WTÃŒfusion transcripts found in DSRCT (11), observing instead pPNET-associated EWS/FUI gene fusions. These tumors, possibly representing primitive malignant ectomesenchymomas related to the pPNET fam ily, therefore, provide a further example of a primitive childhood sarcoma with myogenic differentiation. Preliminary studies in our laboratory suggest that these sarcomas may comprise 10% or more of childhood tumors with a primitive RMS phenotype.8 As described in the present study, these tumors can be distinguished by expression of the EWS/FLI1 gene fusion, and so it will be important to determine the incidence and prognostic significance of this tumor entity. The find ings presented here provide a further argument that classification of many forms of childhood solid tumors should be considered from a molecular 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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