Fusions of the SYT and SSX Genes in Synovial Sarcoma

Fusions of the SYT and SSX genes in synovial sarcoma

Marc Ladanyi*,1

1Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Synovial sarcomas are high grade spindle cell tumors tumors. Ultrastructurally, the epithelial cells lining the that are divided into two major histologic subtypes, glandlike spaces in biphasic tumors show luminal biphasic and monophasic, according to the respective microvilli and are interconnected by various types of presence or absence of a well-developed glandular cell junctions. epithelial component. They contain in essentially all cases a t(X;18) representing the fusion of SYT (at 18q11) with either SSX1 or SSX2 (both at Xp11). Fusions of SYT with SSX1 or SSX2 in synovial Neither SYT, nor the SSX proteins contain DNA- sarcoma: prevalence and structural aspects binding domains. Instead, they appear to be transcriptional regulators whose actions are mediated primarily A t(X;18) is detected cytogenetically in over 90% of through protein-protein interactions, with BRM in the synovial sarcomas, regardless of histologic subtype. In case of SYT, and with Polycomb group repressors in the relation to this recurrent X chromosome translocation, case of SSX. Ongoing work on the SYT ± SSX fusion it is of incidental interest to note that the male to and synovial sarcoma should yield a variety of data of female ratio for this tumor is approximately even. broader biological interest, in areas such as BRM and Cloning of the translocation breakpoints initially Polycomb group function and dysfunction, transcrip- showed that the t(X;18) results in the fusion of two tional targets of SYT ± SSX proteins and their native novel genes, designated SYT (at 18q11) and SSX (at counterparts, dierential gene regulation by SYT ± SSX1 Xp11) (Figure 1) (Clark et al., 1994). SYT encodes a and SYT ± SSX2, control of glandular morphogenesis, protein of 387 amino acids, of which all but the C- among others. Oncogene (2001) 20, 5755 ± 5762. terminal 8 are fused to SSX. The exon structure of SYT is not available but the intron rearranged in the Keywords: chromosomal translocation; transcription t(X;18) appears greater than 13 kb (Clark et al., 1994; factor; nuclear localization Geurts van Kessel et al., 1997). Subsequent work showed that the Xp11 breakpoint actually involves either of two closely related genes, SSX1 (Xp11.23) and Introduction SSX2 (Xp11.21) (Crew et al., 1995; De Leeuw et al., 1995). These are respectively located in the vicinity of Synovial sarcomas account for almost 10% of all soft ornithine aminotransferase-like pseudogenes 1 and 2 tissue sarcomas and typically arise in the para-articular (OATL1 and OATL2), previously de®ned by ¯uores- regions in adolescents and young adults, but actually cence in situ hybridization (FISH) as alternative bear no biologic or pathologic relationship to breakpoints in synovial sarcomas (Shipley et al., synovium. They are considered high grade sarcomas 1994). The ratio of SYT ± SSX1 to SYT ± SSX2 cases which kill at least 25% of patients in the ®rst 5 years is close to 2 : 1 (Crew et al., 1995; Kawai et al., 1998; after diagnosis, in spite of the best currently available reviewed in Dos Santos et al., 2001). The term SYT ± management (Lewis et al., 2000). Synovial sarcomas SSX fusion is still used to refer to these fusions as a are divided into two major histologic subtypes, group. biphasic and monophasic. Biphasic tumors contain The SSX1 and SSX2 genes encode 188 amino acid both epithelial cells arranged in glandular structures proteins which are highly similar. The exon structure of and spindle cells, whereas monophasic types are the SSX1 and SSX2 genes has recently been published entirely composed of spindle cells, lacking the (Gure et al., 1997). In both genes, the fourth intron glandular epithelial component. Expression of epithe- is rearranged by the t(X;18). Whether the occurrence lial antigens is detected by immunohistochemical of translocations is purely random and their speci®city methods in the epithelial component in all biphasic re¯ects only the selection of functional oncogenic tumors and in scattered epithelioid or spindle cells in proteins (Barr, 1998), or whether the introns involved all biphasic tumors and most but not all monophasic are somehow more prone to recombine (Bouer et al., 1993) is an persistent issue in the pathogenesis of tumors with gene fusions. Because SSX1 is involved in the SYT-SSX fusion nearly twice as *Correspondence: M Ladanyi, Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY often as SSX2, we recently studied the sizes and 10021, USA; E-mail: [email protected] sequences of these introns for clues to why SSX1 Fusions of the SYT and SSX genes M Ladanyi 5756 opment to cartilaginous tissue and certain neuronal and epithelial cells (de Bruijn et al., 1996). The SSX genes appear to belong to a family of human tumor antigens whose expression in adults is restricted to tumors and germ cells (`cancer-testis' antigens) (Tureci et al., 1996; Gure et al., 1997). Most cancer-testis antigens are encoded by genes on chromosome X (Tureci et al., 1998). SSX expression during development has not so far been studied. There are no reported mouse knockouts of the SYT and SSX genes which could further illuminate their normal role in development. The SYT ± SSX chimeric transcript represents a highly sensitive diagnostic marker for synovial sarco- Figure 1 Schematic diagram of domain structure of the SYT, ma. The prevalence of this fusion in synovial sarcoma SSX, and SYT ± SSX proteins. The amino acid residues approaches 100% when there is adequate tumor RNA representing the boundaries of selected domains are indicated. for RT ± PCR. Its speci®city for synovial sarcoma, not The scale is approximate. Abbreviations: SNH: SYT amino terminal domain; QPGY: SYT glutamine-, proline-, glycine- and unexpected from the cytogenetic literature, has been tyrosine-rich domain; KRAB: KruÈ ppel-associated box; DD: SSX con®rmed by molecular screening of other morpholo- divergent domain; SSXRD: SSX repressor domain gically similar sarcomas (Hiraga et al., 1998; van de Rijn et al., 1999; Guillou et al., 2001). The clinical usefulness of its molecular demonstration in cases of intron 4 is more often rearranged than SSX2 intron 4 synovial sarcoma of unusual histology or location has (M Ladanyi and MY Lui, GenBank AY034079 and been reviewed elsewhere (Ladanyi and Bridge, 2000). AY034080). These introns are essentially of the same Molecular variants of the SYT ± SSX1 and SYT ± SSX2 size (SSX1: 1985 bp, SSX2: 1983 bp), arguing against a fusion transcripts are rare and none have been simple stochastic model of breakpoint occurrence. The recurrent (reviewed in dos Santos et al., 2001). The sequence analysis shows 88% identity between the two presence of the SYT ± SSX fusion in both the spindle introns (SSX1 and SSX2 are 89.1% identical in their cells and epithelial components of biphasic tumors has cDNA sequences). The SSX2 genomic sequence is also been demonstrated by a variety of methods (Hiraga et available in chromosome X working draft sequence al., 1998; Birdsall et al., 1999; Kasai et al., 2000). As segment NT 25348. The high level of identity and its expected, the formation of SYT ± SSX1 and SYT ± similar level in both intronic and exonic sequences of SSX2 is mutually exclusive and the fusion type is SSX1 and SSX2 highlights the recency of the concordant in primaries and metastases and constant duplication event leading to the formation of this gene over the course of the disease. family. Furthermore, sequence analysis of the SSX1 and SSX2 introns revealed no signi®cant dierence in repetitive elements, which have been proposed to be SYT ± SSX fusion proteins: functional aspects recombinogenic in other translocations. Speci®cally, both introns contain one ALU-Sc repeat in their 5' SYT and the SSX proteins lack recognizable DNA- portion and 2 MIR repeats at the 5' end. Since there is binding domains and are presumably transcriptional no obvious structural basis for the dierential involve- regulators whose actions are mediated primarily ment of SSX1 compared to SSX2, it is tempting to through protein-protein interactions. The SYT ± SSX speculate that it may instead re¯ect small functional chimeric proteins are likely to be involved in the dierences in the oncogenicity of SYT ± SSX1 vs SYT ± transcriptional deregulation of speci®c target genes. SSX2 leading to in vivo selection for the former or that The genes normally regulated by SYT or SSX proteins it may be due to dierences in chromatin structure through putative direct or indirect protein-protein around SSX1 and SSX2. At least four other SSX interactions with speci®c transcription factors are family genes have been identi®ed in Xp11 (Chand et presently unknown. al., 1995; De Leeuw et al., 1996; dos Santos et al., 2001). Of these, only SSX4 has been found to be SYT domain structure rearranged with SYT, and this only in two otherwise unremarkable cases of synovial sarcoma (Skytting et In the SYT protein, sequence analysis has identi®ed al., 1999; Mancuso et al., 2000). SSX1 and SSX2 seem two distinct domains. On the basis of phylogenetic to escape X-inactivation (Miller et al., 1995; and M conservation, Thaete et al. (1999) de®ned a novel 54 Ladanyi, unpublished results). amino acid N-terminal domain (designated the SNH Studies of the pattern of developmental and adult domain). On the basis of the similarity of its amino expression of SYT and SSX have so far provided few acid composition to corresponding domains in other clues to their physiological function. Murine SYT is transcriptional regulators (such as EWS and TLS), it widely expressed in early embryogenesis and in adult appeared that a C-terminal domain rich in glutamine, tissues, and somewhat more restricted in fetal devel- proline, glycine and tyrosine (designated the QPGY

Oncogene Fusions of the SYT and SSX genes M Ladanyi 5757 domain), might function as a transcriptional activation transcriptional activating and repressing domains, an domain (Brett et al., 1997). Indeed, in standard observation which complicates models of SYT ± SSX- transcriptional assays, this portion of SYT, mapped mediated transcriptional deregulation. In transactiva- to residues 158 ± 387, when fused to the GAL4 DNA- tion assays, SYT ± SSX has a several-fold lower activity binding domain functions as a transactivation domain than native SYT (Brett et al., 1997). Unlike chimeric (Brett et al., 1997). Deletion of the SNH domain transcription factors presumed to function mainly at enhances transcriptional activation by SYT mutants, appropriate enhancers recognized by the intact DNA- suggesting that this domain acts as an inhibitor of the binding domain derived from one of the translocation QPGY activation domain. Thaete et al. (1999) have partners, the functional compartment within the also demonstrated the interaction in vitro of the SYT nucleus to which SYT ± SSX is targeted is more dicult (and SYT ± SSX) proteins with the SNF protein BRM. to de®ne and yet will form a key element in under- Thus, SYT (and SYT ± SSX) may interact directly with standing its aberrant function. This adds another level BRM, a component of the SWI/SNF complex involved of complexity to the oncogenic scenario in synovial in chromatin remodeling. The latter is a multiprotein sarcoma. complex which counteracts repression by chromatin structural proteins such as histones and the polycomb- Nuclear distribution of SYT, SSX, and SYT ± SSX group of proteins. The SNH domain is dispensable for proteins the BRM interaction, but the rest of the protein, including the portion between the SNH and QPGY Nuclear localization studies with deletion mutants have domains, appears necessary for the full interaction and found that the nuclear targeting domain of SSX colocalization of SYT (or SYT ± SSX) and BRM coincides with its C-terminal repressor domain (Thaete et al., 1999). The SYT-binding domain within (SSX ± RD), whereas in SYT, a domain within amino BRM maps to its amino terminal half. acids 51 ± 90 distinct from canonical nuclear localization signals is required for nuclear entry. Thus, both nuclear targeting signals are included in the fusion SSX domain structure protein. Nuclear localization of SYT ± SSX has also Sequence analysis and functional studies have identi®ed been con®rmed in synovial sarcoma tissues, using two major domains in SSX. The N-terminal regions of speci®c antibodies (dos Santos et al., 1997; Hashimoto both SSX1 and SSX2 show similarity to a transcription et al., 2000). Both native proteins and the fusion repression domain, the Kruppel-associated box proteins show nucleolar exclusion. The nuclear dis- (KRAB) (Witzgall et al., 1994; Margolin et al., 1994). tribution of the SYT and SSX proteins appears largely However, the SSX KRAB-like domain is an inecient determined by their respective interactions with BRM or even inactive repressor domain and a stronger novel and polycomb-group proteins. The SYT and SSX repressor domain (designated SSX ± RD) has been proteins have speckled and diuse nuclear patterns of functionally mapped to the C-terminal of both SSX1 distribution, respectively (Brett et al., 1997). Speci®- and SSX2 (Thaete et al., 1999; Lim et al., 1998). The cally, SYT localizes to distinct nuclear bodies, separate SSX ± RD domain is the most highly conserved portion from PML oncogenic domains and several other of the protein among SSX family members, further previously described nuclear bodies (dos Santos et al., supporting its critical function. The SSX-RD is also 2000). Thaete et al. (1999) have demonstrated that this required for the nuclear co-localization with Polycomb nuclear distribution represents co-localization of SYT group proteins, which function to repress transcription with BRM. SSX shows a diuse nuclear distribution through modi®cation of higher order chromatin with some punctate staining (dos Santos et al., 2000). structure (dos Santos et al., 2000). Taken together, SSX co-localizes with Polycomb group repressor these data suggest that transcriptional repression by proteins (e.g. HPC2, RING1, HPH1, ENX, EED, SSX is eected at least in part through association with and BMI1) (Soulez et al., 1999; dos Santos et al., or recruitment of Polycomb group repressors by the 2000), but no direct interaction has so far been SSX ± RD domain. identi®ed in co-immunoprecipitation and yeast two- hybrid assays between SSX or SYT ± SSX and selected Polycomb group proteins such as RING1 and BMI1 Domain structure of SYT ± SSX (Soulez et al., 1999). It initially appeared that SYT ± These recent data on the domain structure of SYT and SSX proteins followed a speckled distribution similar SSX proteins do not lend themselves to any simple to native SYT (Brett et al., 1997; dos Santos et al., models for the oncogenicity of SYT ± SSX. The 1997). However, recent data suggest a more complex chimeric transcript in synovial sarcoma replaces the 5' picture: in at least some cell types, the nuclear portion of SSX1 and SSX2 with all but the eight distribution of SYT ± SSX seems to follow that of the c-terminal amino acids of SYT. The loss of these eight punctate or non-diuse component of the staining terminal amino acids of SYT appears to have a observed for native SSX proteins, which parallels that negligible eect on transactivation by the SYT QPGY of Polycomb group repressor proteins (Soulez et al., domain. The C-terminal SSX ± RD domain present in 1999). Indeed, it has been suggested that some of the SYT ± SSX contributes a transcriptional repressor discrepant ®ndings in these nuclear localization studies domain to the protein. Thus, the fusion protein has are due to marked dierences in the distribution of

Oncogene Fusions of the SYT and SSX genes M Ladanyi 5758 Polycomb group repressors (and by association, of SSX is associated with the acquisition of novel DNA SSX proteins) between dierent cell lines (Soulez et al., or protein target speci®cities, as described for other 1999). chimeric transcriptional regulators (Lee et al., 1997; Bertolotti et al., 1998; Petermann et al., 1998). There are so far no reported mouse models transgenic for Models of SYT ± SSX function SYT ± SSX1 or SYT ± SSX2. Thus, models of SYT ± SSX oncogenesis need to Regarding the signi®cance of the C-terminal SSX consider which interaction is `dominant', BRM vs domain in vivo, a notable cell line has recently been Polycomb group, in terms of subnuclear targeting and described, HS-SY-3 (Sonobe et al., 1999). The SYT ± which transcriptional eect is dominant, transactiva- SSX1 fusion product in this cell line (and the original tion by the SYT QPGY domain or repression by the tumor material) is truncated at its 3' end, and thereby SSX ± RD. Dos Santos et al. (2000) provide evidence the protein lacks the entire SSX ± RD. Unlike other that the targeting signal contained in the SSX RD is synovial sarcoma cell lines, HS-SY-3 grows slowly in dominant over that in the SYT amino terminal vitro and is non-tumorigenic in nude mice (Sonobe et portion, and suggest that Polycomb-repression is al., 1999). The primary tumor was a 10 cm popliteal `dominant' over BRM. Thus, the prinicipal subnuclear monophasic synovial sarcoma in an 86 year old compartment aected by SYT ± SSX may be that woman, an unusually advanced age for this tumor. normally targeted by native SSX proteins in germ Assuming that SSX ± RD was absent from the time of cells. Therefore, one hypothesis suggested by these formation of the gene fusion in this unusual tumor, it considerations is that the t(X;18) leads to aberrant and is tempting to hypothesize that the SSX ± RD is constitutive re-expression of the SSX ± RD within the dispensable for synovial sarcoma tumorigenesis, SYT ± SSX fusion protein, resulting in the Polycomb- although it may contribute to tumor aggressiveness. mediated repression by the SSX ± RD of genes This cell line brings into question, albeit with inherent recognized by as yet uncharacterized protein-protein caveats, the model of SYT ± SSX oncogenesis which interactions between SSX and putative DNA-binding depends on a functional SSX ± RD for subnuclear transcription factors. In the somatic precursor cells of targeting and overall transcriptional eect. synovial sarcoma, presumably lacking expression of native SSX, these unknown target genes would not be Interaction between SYT and p300 normally repressed. This scenario is complicated by the observation that at least some synovial sarcomas show Recently, a cellular role independent of transcriptional expression of the remaining unrearranged SSX gene or activation has been described for SYT, and by of other SSX family members, detectable by non- extension, for SYT ± SSX (Eid et al., 2000). Eid et al. nested RT ± PCR. Tureci et al. (1998) found expression (2000) report a direct interaction of SYT with the of the normal SSX2 gene in two synovial sarcomas nuclear protein p300, implicating SYT in the control of with SYT ± SSX1 gene fusions. We studied the cell adhesion in a transcription activation-independent expression of SSX1 and SSX2 in 30 synovial sarcomas manner, at least in part by allowing appropriate beta1 (from 15 men and 15 women) and found expression of integrin/®bronectin receptor function. Interestingly, SSX1 or SSX2 in eight tumors, six from female SYT deletion mutants lacking the eight C-terminal patients and two from male patients (M Ladanyi, I amino acids bound p300 but inhibited cell adhesion, Fligman, unpublished data). In the tumors from male presumably through a dominant negative mechanism. patients, the other SSX family gene was expressed, The inability of these SYT mutants to facilitate whereas tumors from female patients expressed either adhesion may be due to the loss of the SH2 binding the other SSX family gene, or the remaining normal motif within the deleted C terminal end. In preliminary allele of the SSX gene involved in the translocation (as assays, SYT ± SSX was found to be comparable to this identi®ed by appropriate PCR primers). The expression SYT deletion mutant in terms of dominant negative of normal SSX genes in synovial sarcoma may re¯ect eects on cell adhesion, suggesting that this may neoplasia-associated de-repression, as expected for another oncogenic eect of SYT ± SSX, mediated cancer-testis antigens, or may represent residual through the disruption of p300/SYT-dependent main- expression due to low level expression in the precursor tenance of adhesion responses. Current data on cells of synovial sarcoma. Thus, in at least some cases domains of SYT and SSX proteins de®ned by sequence of synovial sarcoma, SYT ± SSX could compete directly similarities or functional studies are schematized in with native SSX, if the above model of SYT ± SSX Figure 1. pathogenesis is correct. Alternative models of SYT ± SSX function, which may not be mutually exclusive, have recently been Clinical and phenotypic correlates of SYT ± SSX fusion proposed by Dos Santos et al. (2001). In another type in synovial sarcoma model, SYT ± SSX may redirect the `dominant' SSX ± RD domain to repress genes recognized and normally Studies in Ewing's sarcoma and alveolar rhabdomyo- activated through putative protein ± protein interac- sarcoma have established the concept that alternative tions between SYT and DNA-binding transcription forms of the speci®c fusion products seen in sarcomas factors. It is also possible that the formation of SYT ± with chromosomal translocations may have an impact

Oncogene Fusions of the SYT and SSX genes M Ladanyi 5759 on one or more features of the associated cancer, such al., 2000; Mancuso et al., 2000). This is also supported as age at diagnosis, primary site, proliferative rate, or by the results from additional studies, recently clinical outcome (Zoubek et al., 1996; Kelly et al., tabulated by Dos Santos et al. (2001). In any given 1997; de Alava et al., 1998). These dierent associa- individual analysis, the strength of this association with tions further support the notion that the fusion fusion type is obviously aected by the statistical proteins encoded by these chromosomal translocations power of a given study group and by dierences in are central to the biology of these sarcomas such that diagnostic criteria for biphasic histology. Nonetheless, the heterogeneity in fusion protein function associated occasional biphasic synovial sarcomas with the SYT ± with even minor structural dierences can have a SSX2 fusion product do exist, but gland formation in detectable and direct impact on either the cellular, these tumors may be less extensive than in SYT ± histologic, or clinical level (Lin et al., 1999). SSX1-positive tumors. The reproducible and statistically signi®cant nature of the association between t(X;18) fusion type and biphasic histology points to a Correlation with clinical behavior genuine biological phenomenon whose further elucida- This paradigm is also reinforced by the analysis of tion may provide insights into the basic histogenetic clinical-molecular correlates in synovial sarcoma. A process of mesenchymal to epithelial conversion and pilot study of this question showed that, among 39 architectural epithelial dierentiation (gland forma- patients with localized synovial sarcoma, cases with the tion). Hypothetically, the SYT ± SSX1 transcriptional SYT ± SSX2 fusion had a signi®cantly better metas- regulator may be more `permissive' than SYT ± SSX2 tasis-free survival than cases with SYT ± SSX1 (Kawai for this dierentiation program, or conversely, may be et al., 1998). The ®nding of a survival advantage for more repressive of the normal mesenchymal dierentia- SYT ± SSX2 was also reported two subsequent studies tion program of synovial sarcoma precursor cells. In with patient groups of similar or smaller size (Nilsson this context, it is interesting to note that epithelial et al., 1999; Inagaki et al., 1999). To con®rm these dierentiation may arguably represent a `default' correlations, we recently revisited this question in a cellular program, re¯ecting the absence or insucient retrospective multi-institutional series of 243 patients activity of transcription factors speci®c for the with synovial sarcoma (Ladanyi et al., submitted). In hematopoietic or mesenchymal lineages (Frisch, 1997). this more de®nitive analysis, SYT ± SSX2 fusion type is con®rmed as a signi®cant positive prognostic factor for Correlation with proliferative rate overall survival. It appears to exert part of its impact on prognosis prior to presentation, through an Two studies including in aggregate 52 cases, found a association with a lower prevalence of metastatic statistically sign®cant relation between SYT ± SSX disease at diagnosis. In patients with localized disease fusion type and tumor cell proliferative activity, with at diagnosis, SYT ± SSX fusion type appears to be the the SYT ± SSX1 tumors showing evidence of increased single most signi®cant prognostic factor by multivariate proliferation, as determined by quantitation of im- analysis. munostaining for Ki-67 (Nilsson et al., 1999; Inagaki et al., 1999). However, in a similar study of 73 cases, we could not con®rm this ®nding (Antonescu et al., 2000). Correlation with glandular epithelial differentiation Instead, a comparison of proliferative rate incorporat- Whether the exact location of the Xp11 breakpoint is ing fusion type, histologic type (monophasic vs related to the morphology of synovial sarcoma was a biphasic), and sample source (primary vs metastatic) controversial issue prior to the cloning of the t(X;18) revealed that the main determinants of proliferation breakpoints. FISH results from three separate groups rate in this study were the latter two factors. suggested a relationship between OATL1 (SSX1) vs Dierences in study results may re¯ect limitations of OATL2 (SSX2) breakpoints and histological subtypes the technique used or dierences in statistical of synovial sarcoma (De Leeuw et al., 1994; Janz et al., approaches (i.e. proliferative rate analysed as a 1995; Renwick et al., 1995). However, other results continuous variable vs a categorical variable). Further failed to con®rm these ®ndings (Shipley et al., 1994, studies using more precise methods of assessing this 1996; Crew et al., 1995). The initial molecular analysis parameter (e.g. ¯ow cytometry) could clarify this issue. of this issue found a statistically signi®cant relationship between histological subtype (monophasic vs biphasic) Possible functional basis for correlates of fusion type and SSX1 or SSX2 involvement in the fusion transcript, that could be summarized as follows: A direct comparison of the transforming or transacti- tumors containing an SYT ± SSX1 fusion transcript vating capabilities of the two dierent SYT ± SSX were monophasic or biphasic, whereas all (or almost fusion proteins has not been performed. The C- all) tumors with the SYT ± SSX2 fusion were mono- terminal 78 amino acids of SSX proteins included in phasic (Kawai et al., 1998). This association of SYT ± SYT ± SSX dier at 13 residues between SSX1 and SSX fusion type and biphasic histology (as de®ned by SSX2. These dierences are non-conservative, but 12 of the presence of glandular epithelial dierentiation with the divergent residues lie outside of the C-terminal SSX lumen formation) has since been upheld in several repressor domain (SSX-RD), arguing against signi®- independent studies (Nilsson et al., 1999; Antonescu et cant dierences between the two SSX proteins in terms

Oncogene Fusions of the SYT and SSX genes M Ladanyi 5760 of their putative interactions with the Polycomb group histiocytoma (Allander et al., 2001). By immunohisto- repressors. The role of the so-called `divergent domain' chemical analysis, we ®nd it expressed in the epithelial of the SSX proteins remains presently unknown component of biphasic tumors and in solid epithelioid (Figure 1). areas of monophasic tumors. FISH analysis demon- strates that this over-expression is not due to gene ampli®cation, and no correlation between SYT-SSX Expression pro®ling of synovial sarcoma fusion type and Her2/neu expression can be identi®ed so far (Illei et al., 2001). The inter-relationship of the High throughput studies of gene expression patterns in HGF/MET and Erb-B2/Her2/neu signaling systems is synovial sarcoma could begin to address several key potentially interesting. In breast, MET required for issues in the pathobiology of synovial sarcoma, tubulogenesis, whereas Her2 required for lobuloalveo- including cell lineage, control of glandular morphogen- lar dierentiation (Yang et al., 1995). HGF and MET esis, possible transcriptional targets of SYT-SSX (HGF receptor) can induce the heregulin/Her2 path- proteins, dierential gene regulation by SYT ± SSX1 way (Castagnino et al., 2000). Aside from its and SYT ± SSX2, as well as the establishment of fundamental interest, this ®nding also provides a `diagnostic' expression pro®les. Expression pro®ling biologic basis for evaluating HerceptinTM as a potential studies of synovial sarcoma using cDNA or oligonu- therapeutic agent in biphasic synovial sarcoma. cleotide microarrays are in progress in several laboratories, and are already yielding interesting leads in some areas (Nielsen et al., 2001; Allander et al., 2001). For instance, synovial sarcoma, as a monoclonal process recapitulating mesenchymal to epithelial con- Note added in proof version, presents us with naturally occurring setting in Nagai et al. (2001) recently examined the transforming which to examine this process at the level of gene activity of SYT-SSX1, its binding to BRM, and its eect expression. The strong association of the type of on gene expression in a small scale microarray experiment. SYT ± SSX transcriptional regulator and morphologic They found the N-terminal 181 amino acids of SYT-SSX1 epithelial dierentiation (biphasic histology) described to be necessary for its transforming activity. The same above suggests that putative functional dierences portionofSYT-SSX1wasfoundtointeractdirectlywith between SYT ± SSX1 and SYT ± SSX2 may result in BRM in both transfected heterologous cells and in a synovial sarcoma cell line. Along with the data of Thaete et dierences in the expression of direct or indirect target al. (1999), this would narrow the core BRM interaction genes involved in this process. Yet, there are few data domain of SYT-SSX1 to residues 73 to 181. By co- on the biological basis for glandular epithelial transfection of competitive BRM deletion constructs with dierentiation in this tumor. The MET receptor SYT-SSX1, they showed that the interaction with BRM is tyrosine kinase, implicated in mesenchymal to epithe- a major factor in transformation by SYT-SSX1. Finally, lial conversion in several in vivo settings, is co- using a 1176 gene microarray to analyse gene expression in expressed with its ligand, HGF, in the epithelial cells a cell line stably transfected with an inducible SYT-SSX1 of synovial sarcoma (Kuhnen et al., 1998; Oda et al., plasmid, they identi®ed DCC as a putative target of 2000). This suggests a possible autocrine basis for this transcriptional repression by SYT-SSX1. process, analogous to that observed in some in vitro systems (Tsarfaty et al., 1994). Recent cDNA microarray analyses of synovial sarcomas have identi®ed additional genes apparently relevant to this process including Erb-B2/Her2/neu (Allander et al., 2001). Acknowledgments Her2/neu is highly expressed in synovial sarcoma ML is supported by grant PO1 CA47179 from the National compared to other sarcomas, such as malignant ®brous Institute of Health.

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