SS18-SSX Fusion Gene Research Articles

Intriguing tumor, synovial sarcoma: hard to diagnose, harder to treat.

Synovial sarcoma (SS) is classified as an aggressive high-grade soft tissue sarcoma that predominantly affects the extremities. Despite its prevalence in the extremities (up to 80%), diagnostic and treatment challenges persist. This study aims to address these challenges by providing a comprehensive analysis of SS in extremities, focusing on diagnostic accuracy and treatment outcomes. The central questions of this study are: What are the diagnostic and treatment challenges associated with SS? How do tumor volume and histologic subtype influence prognosis? What role do immunohistochemistry and genetic markers play in SS diagnosis and management? We hypothesize that larger tumor volume and poor histologic differentiation are associated with worse survival outcomes in patients with SS. This retrospective study analyzed data from 63 patients diagnosed with SS between 2005 and 2020 at a single center. Patients with complete records of pathology, radiology, and surgery, and a minimum follow-up of 12 months were included. Tumor characteristics, treatment modalities, and follow-up data were reviewed. he study included 63 patients and 65 tumors. The mean age was 38 ± 17 years. There were 31 females (49%) and 32 males (51%). SS predominantly affected the extremities (n = 63, 97%), especially the lower limbs (n = 49, 75%). Tumor volume, with a mean of 110 ± 176 cm³, was a significant factor, with tumors >30 cm³ associated with higher rates of metastasis (p = 0,006) and reduced survival (p = 0,027). Histologically, 25 (38%) were monophasic, 24 (37%) were biphasic, and 16 (25%) were poorly differentiated, with poorer survival linked to poorly differentiated subtypes. Immunohistochemistry showed high positivity rates for Bcl-2 (89%, 17/19), EMA (88%, 52/59), and TLE1 (87%, 13/15). SS18-SSX fusion gene detected in 73% of cases (8/11). Metastasis occurred in 27 (42%) patients and recurrence in 24 (37%). 15 (23%) patients died from the disease. Accurate diagnosis of SS is crucial for effective management. Clinicians should be aware of negative predictive factors, including tumor volume >30 cm³ and poor histologic differentiation, when making treatment decisions. The study highlights the importance of extended follow-up due to the risk of late recurrence. IV.

Novel characteristics for immunophenotype, FISH pattern and molecular cytogenetics in synovial sarcoma

As a rare and highly aggressive soft tissue sarcoma, the new immunophenotype, atypical FISH pattern and relevant molecular cytogenetics of synovial sarcoma (SS) remain less known, although it is characteristically represented by a pathognomonic chromosomal translocation t (X; 18) (p11.2; q11.2). Methodologically, the morphology was retrospectively analysed by using H&E staining, and immunohistochemical features were investigated by using markers that have been recently applied in other soft tissue tumors. Moreover, FISH signals for SS18 and EWSR-1 break-apart probes were examined. Finally, cytogenetic characteristics were analysed via RT-PCR and Sanger sequencing. Consequently, nine out of thirteen cases that were histologically highly suspected as SS were finally identified as SS via molecular analysis. Histologically, nine SS cases were divided into monophasic fibrous SS (4/9), biphasic SS (4/9) and poorly differentiated SS (1/9). Immunohistochemically, SOX-2 immunostaining was positive in eight cases (8/9) and PAX-7 immunostaining was diffusely positive in the epithelial component of biphasic SS (4/4). Nine cases showed negative immunostaining for NKX3.1 and reduced or absent immunostaining for INI-1. Eight cases showed typically positive FISH signalling for the SS18 break-apart probe, whereas one case exhibited an atypical FISH pattern (complete loss of green signalling, case 2). Furthermore, the SS18-SSX1 and SS18-SSX2 fusion genes were identified in seven cases and two cases, respectively. The fusion site in 8 out of 9 cases was common in the literature, whereas the fusion site in case 2 was involved in exon 10 codon 404 in SS18 and exon 7 codon 119 in SSX1 (which has not been previously reported), which notably corresponded to the complete loss of green signalling in the FISH pattern. Additionally, FISH analysis of the EWSR-1 gene in nine SS cases demonstrated aberrant signalling in three cases that were recognized as a monoallelic loss of EWSR-1 (1/9), an amplification of EWSR-1 (1/9) and a translocation of EWSR-1 (1/9). In conclusion, SS18-SSX fusion gene sequencing is obligatory for a precise diagnosis of SS when dealing with a confusing immunophenotype and atypical or aberrant FISH signalling for SS18 and EWSR-1 detection.

Synovial Sarcoma Preclinical Modeling: Integrating Transgenic Mouse Models and Patient-Derived Models for Translational Research.

Synovial sarcomas (SyS) are rare malignant tumors predominantly affecting children, adolescents, and young adults. The genetic hallmark of SyS is the t(X;18) translocation encoding the SS18-SSX fusion gene. The fusion protein interacts with both the BAF enhancer and polycomb repressor complexes, and either activates or represses target gene transcription, resulting in genome-wide epigenetic perturbations and altered gene expression. Several experimental in in vivo models, including conditional transgenic mouse models expressing the SS18-SSX fusion protein and spontaneously developing SyS, are available. In addition, patient-derived xenografts have been estab-lished in immunodeficient mice, faithfully reproducing the complex clinical heterogeneity. This review focuses on the main molecular features of SyS and the related preclinical in vivo and in vitro models. We will analyze the different conditional SyS mouse models that, after combination with some of the few other recurrent alterations, such as gains in BCL2, Wnt-β-catenin signaling, FGFR family, or loss of PTEN and SMARCB1, have provided additional insight into the mechanisms of synovial sarcomagenesis. The recent advancements in the understanding of SyS biology and improvements in preclinical modeling pave the way to the development of new epigenetic drugs and immunotherapeutic approaches conducive to new treatment options.

Abstract ND09: The discovery and characterization of CFT8634: A potent and selective degrader of BRD9 for the treatment of SMARCB1-perturbed cancers

Abstract Introduction: The chromatin factor BRD9 is a genetic dependency in some cancers, often referred to as SMARCB1-perturbed cancers. Two types of genetic alterations result in SMARCB1 perturbation: SS18-SSX gene fusion and SMARCB1 loss-of-function mutations. In synovial sarcoma, a rare and aggressive soft tissue malignancy comprising approximately 10% of all soft tissue sarcomas, the presence of the SS18-SSX fusion gene drives the disruption of SMARCB1 function and leads to a synthetic lethal dependence on BRD9. In SMARCB1-null solid tumors, for example malignant rhabdoid tumors (MRT), poorly differentiated chordomas, and epithelioid sarcomas, the absence of SMARCB1 protein results in a similar BRD9 dependence. Thus, in SMARCB1-perturbed cancers, including synovial sarcoma and SMARCB1-null cancers, degradation of BRD9 is hypothesized to result in an anticancer effect. CFT8634 is an orally bioavailable heterobifunctional degrader that induces ternary complex formation with BRD9 and an E3 ligase, leading to the ubiquitination of BRD9 and its subsequent degradation by the proteasome. Results: Here we describe the chemical structure of CFT8634 and an overview of the medicinal chemistry path leading to its discovery. In vitro, CFT8634 promotes rapid, potent, deep, and selective degradation of BRD9 with a half-maximal degradation concentration (DC50) of 2 nM in a synovial sarcoma cell line. In long-term growth assays, CFT8634 is effective at impairing cell growth in a concentration-dependent manner specifically in SMARCB1-perturbed contexts. In vivo, oral dosing of CFT8634 in xenograft models of SMARCB1-perturbed cancers leads to robust and dose-dependent degradation of BRD9, which translates to significant and dose-dependent inhibition of tumor growth in preclinical xenograft models. Conclusion: The preclinical data presented herein support the clinical development of CFT8634 for the treatment of synovial sarcoma and SMARCB1-null tumors. Citation Format: Katrina L. Jackson, Roman V. Agafonov, Mark W. Carlson, Prasoon Chaturvedi, David Cocozziello, Kyle Cole, Richard Deibler, Scott J. Eron, Andrew Good, Ashley A. Hart, Minsheng He, Christina S. Henderson, Hongwei Huang, Marta Isasa, R. Jason Kirby, Linda Lee, Michelle Mahler, Moses Moustakim, Christopher G. Nasveschuk, Michael Palmer, Laura L. Poling, Roy M. Pollock, Matthew Schnaderbeck, Stan Spence, Gesine K. Veits, Jeremy L. Yap, Ning Yin, Rhamy Zeid, Adam S. Crystal, Andrew J. Phillips, Stewart L. Fisher. The discovery and characterization of CFT8634: A potent and selective degrader of BRD9 for the treatment of SMARCB1-perturbed cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND09.

A rare case of metastatic synovial sarcoma initially masqueraded as bronchogenic carcinoma: A case report

Synovial Sarcoma (SS) is a rare type of highly aggressive sarcomatoid neoplasm, that commonly affects the extremities. Primary pulmonary and intra-abdominal SS are extremely rare. However, metastasis to lungs from other sites is far more common. Diagnosis usually depends on histopathological evaluation (HPE) and immunohistochemistry (IHC) analysis. SS18–SSX fusion gene Cytogenetics is the confirmatory test. Here we report a case of a 54-year-old male smoker who presented with left-sided chest pain for one month primarily raised the suspicion of bronchogenic carcinoma based on chest radiograph. The further radiological evaluation demonstrated heterogeneously enhancing multiple soft tissue densities in the left hemithorax and the abdominal cavity. A diagnosis of monophasic metastatic SS was established after HPE and IHC analysis which showed spindle cells that were positive for CD 99, Bcl2, TLE 1 and D240. HPE with IHC analysis could serve as a helpful diagnostic tool to overcome the diagnostic dilemma. Metastatic workup is mandatory in all cases of SS because of the aggressive nature of the tumour.

Prognostic implication of desmoplastic stroma in synovial sarcoma: A histological review

Synovial sarcoma (SS) is a malignant soft tissue neoplasm harboring SS18-SSX fusion gene and is histologically characterized by spindle cells and epithelial components. Some investigations have demonstrated that desmoplastic reaction (DR) is an independent prognostic factor of cancers. However, it remains unknown whether DR is of predictive value for the prognosis of synovial sarcoma patients. Here, we reviewed the clinical and histological findings of 88 patients with SS. We defined DR as hyalinized collagenous structures and classified the degree of DR as follows: none, mild, moderate, and severe. Overall, 23 SS cases (24%) showed moderate or severe DR histologically. Statistically, the cases with moderate or severe degree of DR showed poorer prognosis than those with no or mild DR (local recurrence: P = 0.0059, distant metastasis: P = 0.0002, tumor death: P = 0.0382). The findings of the study suggest that the DR of synovial sarcoma could be an important prognostic factor.

Unmasking BCL-2 Addiction in Synovial Sarcoma by Overcoming Low NOXA.

Simple SummarySynovial sarcoma is a soft-tissue sarcoma that lacks effective systemic therapy and carries poor prognosis due to frequent late local recurrence and metastases. The cancer is known to be driven in part by increased expression of the pro-survival protein BCL-2. Surprisingly, synovial sarcoma proved resistant to BCL-2 inhibitors in pre-clinical trials. We identified increased activity of a second pro-survival protein, MCL-1, as responsible for this resistance. We showed that co-targeting both BCL-2 and MCL-1 proves to be an effective therapeutic approach both in cell culture and animal models of synovial sarcoma, supporting translation into clinical trials.Synovial sarcoma (SS) is frequently diagnosed in teenagers and young adults and continues to be treated with polychemotherapy with variable success. The SS18-SSX gene fusion is pathognomonic for the disease, and high expression of the anti-apoptotic BCL-2 pathologically supports the diagnosis. As the oncogenic SS18-SSX fusion gene itself is not druggable, BCL-2 inhibitor-based therapies are an appealing therapeutic opportunity. Venetoclax, an FDA-approved BCL-2 inhibitor that is revolutionizing care in some BCL-2-expressing hematological cancers, affords an intriguing therapeutic possibility to treat SS. In addition, there are now dozens of venetoclax-based combination therapies in clinical trials in hematological cancers, attributing to the limited toxicity of venetoclax. However, preclinical studies of venetoclax in SS have demonstrated an unexpected ineffectiveness. In this study, we analyzed the response of SS to venetoclax and the underlying BCL-2 family biology in an effort to understand venetoclax treatment failure and find a therapeutic strategy to sensitize SS to venetoclax. We found remarkably depressed levels of the endogenous MCL-1 inhibitor, NOXA, in SS compared to other sarcomas. Expressing NOXA led to sensitization to venetoclax, as did the addition of the MCL-1 BH3 mimetic, S63845. Importantly, the venetoclax/S63845 combination induced tumor regressions in SS patient-derived xenograft (PDX) models. As a very close analog of S63845 (S64315) is now in clinical trials with venetoclax in AML (NCT03672695), the combination of MCL-1 BH3 mimetics and venetoclax should be considered for SS patients as a new therapy.

SS18-SSX Rearrangement in Synovial Sarcoma Using Fluorescent in Situ Hybridization: A Report from an Iranian Population and Literature Review

Background: Synovial sarcoma is an aggressive soft tissue sarcoma. It has a wide spectrum of histopathologic patterns and uncertain immunohistochemistry, rendering it a diagnostic challenge. The objective of this study was to investigate the diagnostic utility and impact of SS18-SSX rearrangement evaluation in an Iranian population previously diagnosed with synovial sarcoma without such molecular tests. Method: We conducted this cross-sectional study on 44 formalin-fixed paraffin-embedded tissue blocks obtained from 23 synovial sarcoma patients (males, 69%; mean age, 36.4 years) and 11 cases with other neoplasms as negative controls. We assessed these specimens for SSX-SS18 gene rearrangement by break-apart fluorescent in situ hybridization (FISH) probes. Results: FISH study showed SS18-SSX fusion gene in 17 (73.91%) cases while six (26.09%) cases and negative controls did not show SS18-SSX fusion. Histopathologic type of tumor was significantly related to the presence of rearrangement (P=0.002) (rearranged: 11 biphasic and six monophasic tumors; non-rearranged: three monophasic and three poorly-differentiated tumors). Conclusion: This study supports the idea that molecular studies contribute to the confirmation of synovial sarcoma diagnosis, particularly in monophasic and poorly-differentiated subtypes, which show vague immunohistochemical results.

Evaluation of combined BCL-2/MCL-1 inhibition as a therapeutic approach for synovial sarcoma.

e23561 Background: While Synovial Sarcoma (SS) only accounts for about 10% of soft tissue sarcomas (STS), or ~10,000 cases/year, ~4,000 of these will be fatal. Little benefit has been seen with newer combination chemotherapies and immune checkpoint inhibitors. SS is a transcription factor-driven cancer characterized by an t(X;18) chromosomal translocation resulting in the SS18-SSX fusion gene. SS is also known to overexpress the anti-apoptotic protein BCL-2, which has been used as a diagnostic marker for SS. Venetoclax, the FDA approved BH3 mimetic which specifically inhibits BCL-2, has been used to treat BCL-2-driven hematological cancers. Yet, preclinical studies of venetoclax, currently in clinical use for BCL-2 related hematologic malignancies, have failed in SS, raising the possibility of other BCL-2 family members playing a role in resistance. Methods: Publicly available gene expression databases of SS were queried for expression of BCL-2 family member expression, including the endogenous MCL-1 inhibitor, NOXA. Six SS cell lines, including ASKA, HS-SY-II, Yamato, SW982, SYO.1, and an ex vivo line from a SS patient-derived xenograft (PDX) were tested in vitro with combinations of venetoclax and the MCL-1 inhibitor S63845, using cell viability assays. In vivo testing employed both a patient-derived xenograft (PDX) model of SS and a cell line derived (SYO.1) xenograft model. Results: Queries of published gene expression data for SS demonstrated that SS expresses low levels of the endogenous MCL-1 inhibitor, NOXA, nominating a mechanism for intrinsic venetoclax resistance in these tumors, and suggesting rational combination of venetoclax and the MCL-1 inhibitor, S63845. In vitro, SS all cell lines possessing the SS18-SSX fusion gene demonstrated synergistic sensitivity to combined S63845 and venetoclax, despite generally insensitivity to either drug as monotherapy. Importantly, in an SS PDX, we demonstrated S63845 and venetoclax synergize to induce tumor regression. In the SYO.1 xenograft model, combination therapy significantly reduced tumor growth compared to no treatment or single agent groups. Conclusions: These findings provide the preclinical rationale for translation of the rational combination of BCL-2 and MCL-1 inhibitors into clinical trials for SS.

Clinicopathological analysis of 12 cases of children synovial sarcoma

Objective: To investigate histopathological characteristics, and differential diagnoses of childhood synovial sarcoma. Methods: HE staining, immunohistochemical staining and fusion gene detection by FISH were performed in 12 cases of synovial sarcoma in childhood at Beijing Children's Hospital from 2016 to 2018. Results: There were 6 cases of biphasic type, 1 case of monophasic epithelial type, 3 cases of monophasic spindle cell type and 2 cases of poorly differentiated synovial sarcomas. EMA, CKpan, bcl-2, CD99, TLE1 and CD34 immunostain positivities were observed in 10/12, 9/12, 12/12, 10/12, 10/12 and 0/12 cases respectively. Unique INI1 immunohistochemical staining was observed in 9/12 cases. SS18-SSX gene fusion was detected in 8 of 11 cases by FISH. Conclusions: Synovial sarcoma is rare in children. Histological morphology combined with immunohistochemistry and FISH SS18-SSX fusion gene detection are important for the diagnosis and differential diagnosis of synovial sarcoma in children.

Release of circulating tumor cells and cell-free nucleic acids is an infrequent event in synovial sarcoma: liquid biopsy analysis of 15 patients diagnosed with synovial sarcoma

BackgroundSynovial sarcoma is a rare soft tissue tumor which contains the unique SS18-SSX1, SS18-SSX2 – or, rarely, SS18-SSX4 - fusion transcripts. It is well known that some soft tissue tumors, like Ewing sarcomas and myxoid liposarcomas, can spread via the blood with free circulating tumor cells (CTC); this can be detected by several sensitive molecular biology methods. Here we report a study of fifteen synovial sarcoma patients with varied clinical backgrounds.MethodAfter blood withdrawal and nucleic acid isolation, we attempted to detect the SS18-SSX fusion genes from circulating tumor cells or cell-free nucleic acids with nested PCR and droplet digital PCR.ResultsSS18-SSX2 fusion transcript was identified in a small copy number with droplet digital PCR in one case. Nested PCR could not detect any of the fusion transcripts in the examined 15 synovial sarcoma cases.ConclusionsHeretofore two case reports could detect CTCs in synovial sarcoma - in the first paper, the patient was diagnosed with poorly differentiated type while the other had a rare primary gastric synovial sarcoma. However, until now, no other studies have detected CTCs in the peripheral blood of synovial sarcoma patients. Based on our findings, we can conclude that detection of the chimeric SS18-SSX fusion gene after surgical excision and/or chemotherapy/radiotherapy is a rare circumstance and hence in itself is not sufficient for monitoring the tumor recurrence. Therefore, monitoring of other possible biomarkers - for example synovial sarcoma specific miRNAs - is recommended.

Immunoreactivity of a Monoclonal Antibody to SS18-SSX Fusion Gene Product in Formalin-fixed Paraffin-embedded Synovial Sarcoma Tissue Section.

Synovial sarcoma is an aggressive sarcoma with specific reciprocal chromosomal translocation of SS18 (also known as SYT) and SSX genes. In the present study, we aimed to detect the SS18-SSX fusion gene product in routinely processed pathologic synovial sarcoma tissue section. Monoclonal antibodies to peptide QRPYGYDQ-IMPKKPA, which covered the fusion region of SS18-SSX, were newly established and subsequently characterized by enzyme-linked immunosorbent assay, Western-immunoblotting, and immunohistochemical staining. A monoclonal antibody designated BG35, reacted with boundary region of SS18-SSX chimera protein in a specific manner, when C-terminal or neighboring region of QRPYGYDQ-IMPKKPA is structurally exposed. Immunoreactivity of BG35 was localized in nucleus of synovial sarcoma cells, but not in other sarcoma cells, examined. Interestingly, synovial sarcoma cells with epithelial differentiation exhibited much strong BG35 immunoreactivity than synovial sarcoma cells with sarcomatous differentiation. BG35 may be useful for evaluating molecular kinetics of SSX-SS18 gene product in situ.

Primary Intraprostatic Synovial Sarcoma.

Primary intraprostatic synovial sarcoma is a rare presentation of an otherwise well-studied disease, and it is one of the few primary sarcomas to occur in the prostate. Ancillary diagnostic techniques including immunohistochemistry and molecular genetics are useful to establish a definitive diagnosis. Despite its unorthodox location, it shares histologic and molecular genetic characteristics with tumors found elsewhere in the body. Most notably, the chromosomal translocation t(X;18)(p11;q11) encodes a chimeric transcription-activating protein, SS18-SSX, which has been identified as the primary driver mutation. The SS18-SSX fusion gene provides a consistent and dependable means of establishing a definitive diagnosis via reverse transcription-polymerase chain reaction or fluorescence in situ hybridization. Recent studies have continued to provide insight into the oncogenesis of this disease. The goal of this review is to elaborate on the clinicopathologic characteristics and underline those techniques that best facilitate the diagnosis of primary intraprostatic synovial sarcoma.

Abstract 969: Circulating cell-free microRNA as a novel biomarker for synovial sarcoma patients

Abstract INTRODUCTION Synovial sarcoma (SS) is characterized by the SS18-SSX fusion gene, and the presence of this chromosomal translocation is clinically useful as a diagnostic marker. However, SS18-SSX does not reflect disease progression and can be detected only with tumor specimens surgically resected. Therefore, novel non-invasive biomarkers would help clinicians to diagnose or detect tumor progression, which could improve patients’ prognosis. We investigated the expression of the serum microRNA (miRNA) of SS patients, and evaluated its significance using in vitro and in vivo experimental models. METHODS In the screening phase, global analysis by miRNA microarray was performed using extracted RNA derived from patients’ serum and culture medium. The serum samples were collected from SS patients, age-matched non-SS patients, and healthy volunteers. The culture mediums were obtained from human SS cell lines and human mesenchymal stem cells (hMSCs) as controls. In the validation phase, the expression levels of miRNA derived from culture medium were evaluated by quantitative real-time PCR (qRT-PCR) to determine the secretory potential of miRNA candidates. SS cell lines and hMSCs were cultured and the culture medium was collected at 0, 24, and 48 hours. Furthermore, serum miRNA levels of SS-bearing mice were analyzed by qRT-PCR according to the tumor growth and tumor resection. RESULTS A miRNA microarray profiling revealed that four miRNAs were significantly highly expressed (p < 0.05) in SS patients’ serum and cell culture medium, compared to control samples, which were selected as candidates for further analysis. We then identified that one miRNA expression among four candidates in the culture medium from all SS cell lines increased with time and with increasing numbers of tumor cells. These data suggested that the detected miRNA is a secretory miRNA derived from SS cells. Next, we investigated whether the serum expression of the detected miRNA correlates with tumor progression using SS-bearing mice, which demonstrated that the detected miRNA expression in mice serum increased size-dependently (R = 0.78, p < 0.05). Moreover, it significantly reduced after tumor resection (p < 0.05), indicating that the detected serum miRNA level reflects tumor burden in vivo. Furthermore, the expression of the detected miRNA in serum collected from SS patients was significantly higher compared with that in serum collected from other soft tissue sarcoma patients. CONCLUSIONS Our results indicate that the serum expression of the detected miRNA could be useful for tumor monitoring as a non-invasive biomarker for SS. Moreover, the detected miRNA may contribute to SS progression. Citation Format: Koji Uotani, Tomohiro Fujiwara, Aki Yoshida, Takuya Morita, Yutaka Nezu, Tadashi Komatsubara, Kazuhisa Sugiu, Takenori Uehara, Toshinori Omari, Ken Takeda, Toshiyuki Kunisada, Junya Toguchida, Akira Kawai, Takahiro Ochiya, Toshifumi Ozaki. Circulating cell-free microRNA as a novel biomarker for synovial sarcoma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 969.

Synovial sarcoma: recent discoveries as a roadmap to new avenues for therapy.

Oncogenesis in synovial sarcoma is driven by the chromosomal translocation t(X,18; p11,q11), which generates an in-frame fusion of the SWI/SNF subunit SS18 to the C-terminal repression domains of SSX1 or SSX2. Proteomic studies have identified an integral role of SS18-SSX in the SWI/SNF complex, and provide new evidence for mistargeting of polycomb repression in synovial sarcoma. Two recent in vivo studies are highlighted, providing additional support for the importance of WNT signaling in synovial sarcoma: One used a conditional mouse model in which knockout of β-catenin prevents tumor formation, and the other used a small-molecule inhibitor of β-catenin in xenograft models. Synovial sarcoma appears to arise from still poorly characterized immature mesenchymal progenitor cells through the action of its primary oncogenic driver, the SS18-SSX fusion gene, which encodes a multifaceted disruptor of epigenetic control. The effects of SS18-SSX on polycomb-mediated gene repression and SWI/SNF chromatin remodeling have recently come into focus and may offer new insights into the basic function of these processes. A central role for deregulation of WNT-β-catenin signaling in synovial sarcoma has also been strengthened by recent in vivo studies. These new insights into the the biology of synovial sarcoma are guiding novel preclinical and clinical studies in this aggressive cancer.

The clinical implication of SS18 -SSX fusion gene in synovial sarcoma

Background:The aim of this study is to evaluate distribution and clinical impact of the SS18-SSX fusion gene in patients with synovial sarcoma in China.Methods:We collected and analysed the clinical data of 88 patients using univariate and multivariate survival analysis. HEK 293T and NIH 3T3 cell lines were transfected with the SS18-SSX1 or SS18-SSX2 gene to determine the respective involvement of these fusion genes in cell proliferation and invasion.Results:Overall survival was significantly better among SS18-SSX2 cases (P=0.001), FNCLCC grade 2 cases (P<0.001), and UICC stage 1 or 2 (P<0.001) by univariate and multivariate survival analysis. SS18-SSX1-positive cells were more proliferative and invasive than SS18-SSX2-positive cells.Conclusion:SS18-SSX fusion type is a significant prognostic factor for patients with synovial sarcoma.

Synovial Sarcoma Is a Stem Cell Malignancy

Synovial sarcoma (SS) is a malignant soft tissue tumor characterized by its unique t(X;18)(p11;q11) chromosomal translocation leading to the formation of the SS18-SSX fusion gene. The resulting fusion protein product is considered to play as an aberrant transcription factor and transform target cells by perturbing their gene expression program. However, the cellular origin of SS is highly debated. We herein established two novel human SS cell lines, named Yamato-SS and Aska-SS, and investigated their biological properties. We found the self-renewal ability of these cells to generate sarcospheres, to form tumors in serial xenotransplantation and reconstitute the tumor phenotypes without fractionation by any surface markers. Both SS cells as well as clinical tissue specimens from 15 patients expressed the marker genes-associated stem cell identity, Oct3/4, Nanog, and Sox2. We also found that both SS cells displayed limited differentiation potentials for mesenchymal lineages into osteocytes and chondrocytes albeit with the expression of early mesenchymal and hematopoietic lineage genes. Upon SS18-SSX silencing with sequence-specific siRNAs, these SS cells exhibited morphological transition from spherical growth in suspension to adherent growth in monolayer, additional expression of later mesenchymal and hematopoietic lineage genes, and broader differentiation potentials into osteocytes, chondrocytes, adipocytes, and macrophages in appropriate differentiation cocktails. Collectively, these data suggest that a human multipotent mesenchymal stem cell can serve as a cell of origin for SS and SS is a stem cell malignancy resulting from dysregulation of self-renewal and differentiation capacities driven by SS18-SSX fusion protein.

Downregulation of SS18-SSX1 expression by small interfering RNA inhibits growth and induces apoptosis in human synovial sarcoma cell line HS-SY-II in vitro

Substantial evidence indicates that the characteristic SS18-SSX fusion gene may play an important role in synovial sarcoma development and progression. For obtaining better insights into the genetic alterations and molecular mechanisms involved in synovial sarcomas and for developing novel therapeutic strategies for this disease, we first examined the efficiency of small interfering RNAs (siRNAs) targeting the SS18-SSX1 fusion gene in knocking down its expression in the human synovial sarcoma cell line HS-SY-II, and then evaluated the effects of downregulation of this gene on apoptosis, apoptosis-related gene expression, growth regulatory proteins, and the growth of tumor cells in vitro. We observed a marked decrease (by more than 87.6%) in SS18-SSX1 expression levels in cells transfected with a plasmid expressing hairpin siRNA for this gene, which was accompanied by (i) reduction in protein levels of cyclin D1 and cyclin A, (ii) reduction in antiapoptotic protein Bcl-2 and activation of caspase 3/apoptosis, and (iii) growth inhibition of HS-SY-II cells in vitro. Our results demonstrate that siRNA targeting of SS18-SSX1 may have therapeutic potential in the treatment of synovial sarcomas.

Differential roles of SS18–SSX fusion gene and insulin-like growth factor-1 receptor in synovial sarcoma cell growth

Recently we demonstrated that the synovial sarcoma specific fusion gene SS18–SSX is crucial for cyclin D1 expression and is linked to cell proliferation. In this report we explore the role of SS18–SSX and IGF-1R for their potential functions in cellular proliferation and survival in cultured synovial sarcoma cells. We found that targeting of SS18–SSX mRNA by antisense oligonucleotide treatment drastically and rapidly decreased cell proliferation but caused only a slight increase of apoptosis. The synovial sarcoma cells were confirmed to express IGF-1R, and treatment with an IGF-1R inhibitor resulted in substantially reduced cell viability by inducing apoptosis in these cells. Conversely, inhibition of the IGF-1R resulted only in a slight to moderate decrease in DNA synthesis. In conclusion, SS18–SSX and IGF-1R seem to play important but different roles in maintaining malignant growth of synovial sarcoma cells. Whereas SS18–SSX maintains cyclin D1 and cell proliferation, IGF-1R protects from apoptosis.

Molecular Detection of SS18-SSX Fusion Gene Transcripts by cRNA In Situ Hybridization in Synovial Sarcoma Using Formalin-fixed, Paraffin-embedded Tumor Tissue Specimens

SS18-SSX fusion genes resulting from a chromosomal translocation t(X;18)(p11.2;q11.2) are a genetic hallmark of synovial sarcoma. Although such cytogenetic or molecular aberrations have mostly been detected by fluorescence in situ hybridization or reverse transcription-polymerase chain reaction, the expression of SS18-SSX has been poorly investigated at a cellular or tissue level. In this study, biotinylated tyramide (BT)-based in situ hybridization (ISH) was performed to detect SS18-SSX transcripts using formalin-fixed, paraffin-embedded tissues from 15 synovial sarcomas. Digoxigenin-labeled cRNA probes flanking the fusion points of SS18-SSX1 and SS18-SSX2 were generated by in vitro transcription, and hybridized signals were detected by a streptavidin-biotin complex method after chemical enhancement with BT. The localizations of signals were compared with the immunohistochemical expressions of epithelial or neuroectodermal markers and those of cell adhesion including cytokeratins (CAM5.2, AE1/AE3, CK7), epithelial membrane antigen, E-cadherin, beta-catenin, c-erbB-2 (HER2/neu), CD56, and claudin-1. The ISH signals of the SS18-SSX transcripts were identified in 13 synovial sarcomas, and their fusion types correlated with those determined by reverse transcription-polymerase chain reaction. In biphasic tumors, the ISH signals tended to localize to epithelial areas, whereas spindle-cell areas or monophasic fibrous tumors showed a less intense or focal expression pattern. Notably, the expression patterns of AE1/AE3, CK7, and c-erbB-2 often colocalized with the ISH signals (7 of 11 cases positive for each marker). Our results suggest that BT-based ISH can be used as a molecular technique for the detection of SS18-SSX using formalin-fixed, paraffin-embedded tissues.