Nuclear localization of SYT, SSX and the synovial sarcoma-associated SYT-SSX fusion proteins

Synovial sarcoma is characterized by a prevalent chromosomal translocation, t(X;18)(p11;q11). As a result of this translocation the SYT gene on chromosome 18 fuses to either the SSX1 or the SSX2 gene on the X chromosome. In this study, we generated polyclonal antibodies against the SYT and SSX2 proteins. These antibodies specifically detected both these proteins and the SYT-SSX fusion proteins in transfected COS-1 cell extracts. Indirect immunofluorescence analysis of COS-1 cells expressing tagged or untagged SYT, SSX2, SYT-SSX1 or SYT- SSX2 indicated that all these proteins are localized in the nucleus, excluding the nucleoli. The SSX2 protein exhibited a diffuse staining pattern whereas both the SYT and SYT-SSX proteins appeared in several nuclear dots. Similar nuclear dots were also detected in primary synovial sarcoma cells growing in a short-term in vitro culture. Double immunofluorescence in conjunction with confocal laser-scanning microscopy revealed that the SYT and SYT-SSX nuclear dots do not co- localize with known nuclear structures as e.g. coiled bodies, SC35 interchromatin granules or PML bodies. The similar nuclear localization patterns of SYT and SYT-SSX suggest that the SYT-SSX fusion proteins are directed to SYT-associated nuclear domains where an abnormal function may be exerted.      

Dual colour fluorescence in situ hybridization to paraffin-embedded samples to deduce the presence of the der(X)t(X;18)(p11.2;q11.2) and involvement of either the SSX1 or SSX2 gene: a diagnostic and prognostic aid for synovial sarcoma

Identification of the t(X;18)(p11.2;q11.2) and the fusion gene products, SYT–SSX1 and SYT–SSX2, associated with a high proportion of synovial sarcomas, has been shown to be a useful diagnostic aid. This study demonstrates the application of dual colour fluorescence in situ hybridization to paraffin-embedded samples to deduce the presence of the derivative X chromosome and also the position of the breakpoint on chromosome X at either the SSX1 or the SSX2 gene. This used region specific markers from chromosomes X and 18 and an optimized protocol involving microwave exposure. Novel and rapid scoring criteria were validated which circumvented potential problems of nuclear truncation and defining cell boundaries. This involved blind analysis of two negative sarcoma samples and three synovial sarcomas in which corresponding frozen material had been previously shown to have the translocation involving different SSX genes. Six new cases diagnosed as synovial sarcoma were also analysed; two monophasic and two biphasic case were deduced to have a breakpoint in the SSX1 gene, one monophasic case an SSX2 breakpoint, and one case did not show rearrangement of the region. The ability to analyse formalin-fixed, paraffin-embedded samples in this way has practical implications for aiding the diagnosis of difficult cases, recently ascribed prognostic relevance, and allows further retrospective studies to be carried out. The methodology is also applicable to the identification of other tumour specific translocations in paraffin-embedded material. Copyright © 1999 John Wiley & Sons, Ltd.     

A synovial sarcoma with a complex t(X; 18;5;4) and a break in the ornithine aminotransferase (OAT)LI cluster on Xp11.2

The initial cytogenetic analysis of a biphasic synovial sarcoma revealed complex anomalies involving six different chromosomes: 46,Y,t(X185;4)(p11;q11;p13;q12),t(2;5)(q35;q11). After fluorescence in situ hybridization (FISH) analysis, using chromosome X-specific plasmid library and YAC probes, the situation appeared to be even more complex, with an insertion of part of the X chromosome short arm into the der(5)t(5;18). In spite of these complex chromosomal rearrangements, the Xp11 breakpoint could be mapped to within the ornithine aminotransferase (OAT)LI cluster, very similar to that reported previously for the standard t(X 18)(p11;q11) in synovial sarcomas. These findings suggest common pathogenetic pathways in these cytogenetically different but morphologically similar tumors. Genes Chrom Cancer 9:288-291 (1994). © 1994 Wiley-Liss, Inc.     

Synovial sarcoma (SS): new perspectives supported by modern technology

Based upon the experience of 256 cases of synovial sarcoma (SS), the present review analyzes structural, biological and molecular pathology of this poorly known sarcoma. The histology displays a multiphenotype with two major components: biphasic and monophasic SS. In addition, a number of variants have been described: undifferentiated Ewing's like, myoxid and predominantly epithelial (monophasic epithelial sarcoma). Microcalcifications and squamous metaplasia are often seen in the tumor. Immunohistochemistry with EMA and cytokeratin in the epithelial or epithelioid component is diagnostic for SS together with vimentin positivity in the spindle cells. Several other epitopes are also expressed (CD99, CD56, C-MET, HGF/SF, CD44). The ultrastructure confirms the variegated pattern of the neoplasm demonstrating the epithelial component and the epithelioid or spindle cell type closely associated with each other. Transition of epithelial cells to epithelioid and spindle-like mesenchymal component is seen. Nude-mice xenografts and cell lines after in vitro culture confirm heterogeneity of this sarcoma. Molecular histology of the SS has provided high utility not only for their differential diagnosis due to a specific chromosomal translocation: t(X;18)(p11.2;q11.2) but also after cloning these breakpoints resulting in the fusion of two genes: SYT at 18q11 and SSX at Xp11. Further observations have lead to distinguish the existence of two related genes: SSX1 and SSX2, that provide a highly specific and sensitive diagnostic marker for SS. Moreover, clinical correlations have demonstrated that SYT-SSX1 leads to a poor clinical outcome while the fusion SYT-SSX2 provides survival advantages to the patients.     

滑膜肉瘤的融合基因检测分析

目的：基于存在染色体易位所致特异的SYT－SSX融合基因,证实可在滑膜肉瘤组织石蜡切片上检测出并探讨其在诊断中的价值。方法：采用逆转录－聚合酶链反应,检测并分析20例滑膜肉瘤（组织学亚型15例单相,5例双相）的SYT－SSX转录物,并对照相应病理学所见,结果：所检20例滑膜肉瘤中,有19例（95％）出现特异SYT－SSX逆转录聚酶链反应产物,其中13例具有SYT－SSX2融合基因的肿瘤有10例呈组织学单相分化。结论：SYT－SSX融合基因转录物可在石蜡切片和组织块中获得满意结果。具有较好的灵敏性,是滑膜肉瘤所特有的诊断标志物,它的亚类分型（SYT－SSX1和SYT－SSX2）,可能成为预后推测指征。     

滑膜肉瘤t(X;18)易位断裂点基因组DNA序列特征分析

目的 分析滑膜肉瘤t(X;18)染色体易位断裂点基因组DNA序列特征,探讨其与滑膜肉瘤t(X;18)染色体易位发生的关系。方法 采用长距离聚合酶链反应（PCR）及DNA测序技术,对2例滑膜肉瘤新鲜标本t(X;18)染色体易位断裂点基因组DNA序列进行了扩增及序列分析。结果 2例滑膜肉瘤标本均存在t(X;18)染色体易位,分别导致SYT-SSX1和SYT-SSX2融合基因形成,DNA序列分析显示,SYT基因第10内含子分别与SSX1和SSX2基因的第4内含子发生融合。在3种基因的断裂点附近均存在与共有易位素（translin)识别序列高度同源的序列,还发现3种基因的断裂点靠近,甚至位于呈回文结构的寡核革酸序列中,SSX1和SSX2基因第4内含子断裂点位于Alu重复序列附近,而SYT基因第10内含子断裂点上游和下游各500碱基内均未发现Alu或其他重复序列。在SYT两断裂点之间的DNA序列中存在一处拓扑异构酶Ⅱ的识别序列,但与两断裂点距离较远。结论 滑膜肉瘤染色体易位所涉及的3种基因的基因组断裂点区域存在特征性的序列,可能与滑膜肉瘤染色体易位的发生有关。     

Masked t(X;18)(p11;q11) in a biphasic synovial sarcoma revealed by FISH and RT-PCR

The initial cytogenetic analysis of a biphasic synovial sarcoma showed an apparently normal karyotype. After FISH using chromosome X- and 18-specific probes and RT-PCR using SYT- and SSX-specific primer sets, a cryptic synovial sarcoma-associated t(X;18)(p11;q11) could be revealed. The “masked” nature of the translocation may best be explained by a two-step scenario in which a genuine t(X;18)(p11;q11) has occurred as a first step and a reverse reciprocal X;18 translocation as a second step, leaving the synovial sarcoma-associated SYT–SSX1 fusion intact. The findings further underline our previous suggestion that SYT–SSX1 fusions may correlate with a biphasic nature of the tumor. In addition, our findings indicate that, in analogy to, e.g., the Philadelphia translocation in chronic myeloid leukemia, “masked” translocations may occur in soft tissue tumors and that, as a standard, RT-PCR and/or FISH analyses should be carried out in order to provide karyotypic information that may be relevant to tumor diagnosis and/or prognosis. Genes Chromosomes Cancer 23:198–201, 1998. © 1998 Wiley-Liss, Inc.     

A synovial sarcoma with t(X;18)(p11;q11) in a patient with Turner's syndrome.

The first case of synovial sarcoma in a patient with Turner's syndrome (45,X) is reported. Cytogenetic analysis of the tumor cells showed that the only X chromosome was involved in the t(X;18)(p11;q11) characteristic of synovial sarcoma.