miR‑494.3p expression in synovial sarcoma: Role of CXCR4 as a potential target gene
Laura PazzagliaSerena PollinoMattia VitaleElisa BientinesiStefania BeniniCristina FerrariEmanuela PalmeriniMarco GambarottiPiero PicciMaria Serena Benassi
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Abstract:
Synovial sarcoma (SS) is a rare tumour, with dismal survival when metastasis occurs. SS contains a characteristic translocation (X;18)(p11;q11) and the fusion genes appear to be mutually exclusive and concordant in primary and metastatic tumours. Novel prognostic and predictive factors are required. The C‑X‑C motif chemokine ligand 12 (CXCL12)/C‑X‑C chemokine receptor 4 (CXCR4) axis is involved in tumour development and metastatic spread in many types of cancer and previous data have demonstrated a pivotal role of CXCR4 in SS cell migration and invasion. Bioinformatics and biological data indicated CXCR4 is a possible candidate target of miR‑494.3p, known to be involved in tumour progression. In this study, we analysed the expression of miR‑494.3p and its potential target, CXCR4, in a series of SS specimens. A significantly lower miR‑494.3p expression was found in the tumour compared to normal tissue associated with higher levels of CXCR4 both at the gene and protein level. The role of CXCR4 as a potential target of miR‑494.3p was assessed in two SS cell lines (SW982 and SYO‑I). Transfection with miR‑494.3p expression plasmid led to a marked decrease in CXCR4 gene and protein expression, concomitant with a transitory decrease in cell proliferation and migration. The SYO‑I cells also responded with an increased apoptotic fraction. The data of this study also demonstrate that the downregulation of miR‑494.3p in SS surgical specimens, concomitant with an increased expression of its potential target, CXCR4, was more evident in the metastatic subset. In vitro experiments confirmed that miR‑494.3p functioned as a tumour suppressor through the involvement of CXCR4 and ongoing studies are directed to better clarify its role in SS therapeutic strategies.Keywords:
Synovial Sarcoma
To investigate the feasibility of detecting SYT-SSX fusion gene in paraffin-embedded tissues and its diagnostic significance for synovial sarcoma (SS).Formalin-fixed, paraffin-embedded samples of 38 cases of SS and 40 cases of control tumors (including spindle cell sarcoma and metastatic adenocarcinoma) were retrived from archival materials. SYT-SSX fusion transcripts were detected in all samples by RT-PCR. House-keeping gene Porphobilinogen Deaminase (PBGD) was used to detect the quality of mRNA.PBGD mRNA was detected in 64 of the 78 tumor cases (82.1%). SYT-SSX fusion transcripts was detected in 33 of the 38 synovial sarcoma specimens. No SYT-SSX mRNA expression was found in control tumors. SYT-SSX mRNA expression rate reached 89.2% (33/37) in synovial sarcomas after exclusion of 1 case which was negative for both SYT-SSX and PBGD. In 33 SYT-SSX positive synovial sarcomas, 22 had SYT-SSX1 and 6 had SYT-SSX2 fusion transcripts. In 5 cases, the fusion type could not be distinguished. There was an association between SYT-SSX fusion type and histologic subtype. All 10 biphasic synovial sarcoma cases had the SYT-SSX1 fusion, whereas 12 of 18 monophasic synovial sarcoma had the SYT-SSX1 and 6 had the SYT-SSX2 fusion gene (P < 0.05).(1) Detection of SYT-SSX fusion gene in paraffin-embedded tissues was found to be a sensitive and specific method for the diagnosis and differential diagnosis of synovial sarcoma. (2) There was an association between SYT-SSX fusion type and histologic subtype.SYT-SSX2 fusion transcript could only be found in monophasic synovial sarcomas.
Synovial Sarcoma
Porphobilinogen deaminase
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Introduction: Synovial Sarcoma is a rare cancer and account for 5-10% of adult soft tissue sarcomas. The tumour exhibits unspecified histogenesis composed primarily of spindle cells with variable epithelial components. Despite establishment of some immunohistochemistry staining, making a definitive diagnosis of synovial sarcoma remains a challenging task. This is due to the histo-morphology and immunophenotypes similarities of this tumour to other types of soft tissue sarcoma. Objective: The current study aims to apply a molecular method for detection of SYT-SSX fusion transcript, a common molecular defect (>90% of the cases) in Synovial Sarcoma irrespective of the histologic subtypes. Method: Paraffin-embedded fixed-tissue (PEFT) blocks of 3 confirmed and 15 possible cases of Synovial Sarcoma were retrieved from Department of Pathology, Tengku Ampuan-Afzan Hospital, Kuantan and subjected to RNA purification using the standard spin column protocol. A one step direct RT-PCR was performed using SYT-SSX and PBGD primer sets for detection of SYT-SSX fusion gene and the reference gene PBGD respectively. Results: Our preliminary molecular analyses showed positive SYT-SSX fusion transcript in all 3 confirmed cases and 5 possible cases of synovial sarcoma. Further analysis is still on going for the remaining samples. Conclusion: Molecular detection of SYT-SSX fusion-transcript is useful in establishing the diagnosis of Synovial Sarcoma.
Synovial Sarcoma
Histogenesis
Fusion transcript
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To establish a method of SYT-SSX fusion gene detection by FISH and to explore its diagnostic value for synovial sarcoma.The presence of SYT-SSX fusion gene was determined by FISH using a tissue microarray containing 62 known synovial sarcomas, 60 non-synovial sarcomas and 133 equivocal synovial sarcomas. FISH results were compared with those of RT-PCR published previously.Overall, 96.9% (247/255) of the cases were successfully analyzed by FISH. The sensitivity of FISH for known synovial sarcomas was 96.7% (58/60), and the specificity for the non-synovial sarcoma was 100% (59/59). Moreover, SYT-SSX gene fusion was detected in 78.1% (100/128) of the equivocal synovial sarcomas. The concordance rate between FISH and RT-PCR was 83.6% (102/122) in those equivocal synovial sarcomas, and overall 79.7% (106/133) of these cases were confirmed as synovial sarcomas either by RT-PCR or by FISH.The sensitivity and specificity of FISH detection of SYT-SSX fusion gene are high. FISH and RT-PCR are complementary to each other in the confirmation of synovial sarcomas, particularly those questionable cases.
Synovial Sarcoma
Concordance
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To assess the diagnostic values of immunohistochemistry and SYT-SSX fusion gene detection for synovial sarcoma.Based on clinical features, histological and immunohistochemical profiles, 195 cases of tumors were divided into three diagnostic categories: definitive synovial sarcoma, probable synovial sarcoma and possible synovial sarcoma. RT-PCR Detection of the SYT-SSX fusion gene was performed using paraffin embedded tissue samples. Comparison between RT-PCR and immunohistochemistry results was carried out and their diagnostic value was evaluated.There were 62 (31.8%) definite synovial sarcomas, 49 (25.1%) probable synovial sarcomas and 84 cases (43.1%) possible synovial sarcomas. SYT-SSX fusion gene was detected in 140 (78.2%) cases overall, including 94.7% (54/57) definite synovial sarcomas, 86.0% (37/43) probable synovial sarcomas and 62.0% (49/79) possible synovial sarcomas. In tumors in the certain and probable synovial sarcoma categories, the positive rates of epithelial membrane antigen (EMA) were significantly higher in the SYT-SSX positive cases than SYT-SSX-negative cases (P = 0.022, P = 0.010, respectively). EMA was positively correlated with the presence of SYT-SSX (r(s) = 0.431, P = 0.001, r(s) = 0.463, P = 0.002, respectively). However, such a correlation was not seen in cytokeratin (CK), vimentin or S-100 protein immunostains (P > 0.05). In tumors of possible synovial sarcoma category, there were no significant differences of CK, EMA, vimentin or S-100 protein between SYT-SSX-positive and SYT-SSX-negative tumors.SYT-SSX fusion gene detection is not needed when the conventional approaches are diagnostic. EMA positivity has a similar diagnostic value to that of SYT-SSX by RT-PCR for tumors in the probable synovial sarcoma category. However, detection of SYT-SSX is very important for diagnosis of the tumors in the category of possible synovial sarcoma.
Synovial Sarcoma
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Objective To detect SYT-SSX fusion gene transcripts in fresh-frozen synovial sarcoma specimens and analyze the correlation between histological phenotypes and SYT-SSX subtypes. Methods Fresh-frozen specimens from 19 synovial sarcoma patients were studies from July 2000 to September 2004. SYT-SSX fusion gene and its subtypes were determined by RT-PCR. Simultaneously, definite pathological diagnoses were made and histological phenotypes were confirmed. The correlation between histological phenotype and SYT-SSX subtypes were analyzed.Results SYT-SSX fusion gene was detected in 15 cases(79%) and SYT-SSX1 and SYT-SSX2 was detected in 11 and 4 cases respectively. A rare variant of SYT-SSX2 was found. All these 15 cases were definitely confirmed as synovial sarcoma histologically and SYT-SSX fusion gene was detected in all these cases. 10 monophasic cases and 4 biphasic cases and 1 poorly differentiated case were confirmed and SYT-SSX2 did not exist in biphasic cases.Conclusions RT-PCR is a highly sensitive and specific method in detecting SYT-SSX fusion gene and that is specific for synovial sarcoma. To preserve fresh-frozen specimen and detect fusion gene by RT-PCR with proper primers is meaningful to clinical diagnosis as well as to basic research.
Synovial Sarcoma
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Synovial Sarcoma
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Synovial sarcomas account for up to 10 percent of soft-tissue sarcomas and include two major histologic subtypes, biphasic and monophasic, defined respectively by the presence and absence of glandular epithelial differentiation in a background of spindle tumor cells. A characteristic SYT–SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas. The translocation fuses the SYT gene from chromosome 18 to either of two highly homologous genes at Xp11, SSX1 or SSX2. SYT–SSX1 and SYT–SSX2 are thought to function as aberrant transcriptional regulators. We attempted to determine the influence of the two alternative forms of the SYT–SSX fusion gene on tumor morphology and clinical outcome in patients with this sarcoma.
Synovial Sarcoma
Fusion transcript
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Recent molecular analyses have reported that the specific translocation t(X;18) (p11.2;q11.2) in synovial sarcoma induces the fusion of the SYT gene on chromosome 18 to either of two duplicated genes on chromosome X, SSX1 or SSX2. As a result, two types of fusion genes SYT-SSX1 and SYT-SSX2, whose products play major roles in tumorigenesis, are formed. However, the precise function of the fusion genes is still unclear. To determine clinical relevance of the SYT-SSX fusion genes, we analyzed the fusion transcripts in 13 synovial sarcomas by reverse transcription-polymerase chain reaction (RT-PCR) and also determined the subtype by restriction enzyme assay. These molecular results were compared with certain clinical features of the tumors. The SYT-SSX fusion transcript was detected in the majority of the cases (11 / 13) with synovial sarcoma, suggesting that it is a useful diagnostic marker. The subtype analysis showed that only the recurrent tumors expressed SYT-SSX2. This may indicate that there are some biological differences between the two subtypes, such as transforming activity. On this basis, SYT-SSX2 may correlate with the progressive character of this tumor and could be a predictor of prognosis.
Synovial Sarcoma
Fusion transcript
Clinical Significance
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To assess the feasibility of detecting SYT-SSX fusion transcripts in paraffin-embedded tissues of synovial sarcoma by reverse transcription-polymerase chain reaction (RT-PCR).RT-PCR was used to amplify the SYT-SSX fusion transcripts using archival formalin-fixed paraffin-embedded tumor specimens from a series of 37 synovial sarcoma cases. To investigate the specificity of the SYT-SSX fusion transcripts, a variety of non-synovial sarcoma tumors were included in the study as negative controls. The detected messages derived from fusion genes were confirmed by subsequent sequence analysis.SYT-SSX fusion transcripts were detected in 33 of 37 (89.2%) synovial sarcomas. None of the 34 cases of non-synovial sarcoma tumors showed amplified products of SYT-SSX fusion transcripts, although PBGD mRNA was detected in all specimens. Among 33 SYT-SSX-positive synovial sarcomas, 22 tumors had an SYT-SSX 1 fusion transcript, whereas 6 tumors had an SYT-SSX2 fusion transcript. Fusion types can not be distinguished in the remaining 5 cases. There was a significant relationship between SYT-SSX fusion type and histologic subtype. All 10 biphasic synovial sarcomas had the SYT-SSX1 fusion, whereas all tumors with SYT-SSX2 were of monophasic morphology (P < 0.05).RT-PCR can be applied to archival formalin-fixed paraffin-embedded tumor tissues as a sensitive and reliable technique for the diagnosis and differential diagnosis of synovial sarcoma. There is an association between SYT-SSX fusion type and histological subtype. SYT-SSX2 fusion transcripts can only be found in monophasic synovial sarcoma.
Synovial Sarcoma
Fusion transcript
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To demonstrate that a characteristic SYT-SSX fusion gene resulting from chromosomal translocation t (X; 18) (p11; q11) was detectable in archival paraffin-embedded synovial sarcoma tissues.SYT-SSX fusion transcripts in 20 synovial sarcomas (histologic subtypes, 15 monophasic and 5 biphasic) were detected and analyzed by the reverse-transcriptase polymerase chain reaction and compared with the results from relevant pathological data.A specific SYT-SSX RT-PCR product was found in 19 of 20 (95%) synovial sarcomas tested and of the 13 tumors containing SYT-SSX2, 10 were monophasic.SYT-SSX fusion transcripts are considered as a defining diagnostic marker of synovial sarcomas and the subtypes of SYT-SSX fusion transcripts (SYT-SSX1 and SYT-SSX2) may yield prognostic information.
Synovial Sarcoma
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