ABSTRACT Purpose: Synovial sarcomas (SS) constitute ~5-10% of soft tissue tumors with >90% SS having characteristic translocations due to the fusion of SYT gene (18q11) with either SSX1 (Xp11.23) or SSX2 (Xp11.21), making them useful diagnostic markers. The purpose of this study was to standardize a real-time polymerase chain reaction (RT-PCR) for detection of these fusion transcripts from formalin fixed paraffin embedded (FFPE) tissues establishing its usefulness in our settings. Material and Methods: Twenty five FFPE specimens (15 SS and 10 non-SS) were obtained from our archives (June September 2007). The 15 SS specimens included: Monophasic spindle (11); biphasic (1); poorly differentiated (1); and spindle cell tumor suggestive of synovial sarcoma (2). The non-SS included: Ewing’s (3); dermatofibrosarcoma (1); rhabdomyosarcoma (1); leiomyosarcoma (1); malignant peripheral nerve sheath tumor (MPNST, 1) and unclassified sarcomas (3). All cases were diagnosed based on histology and immunohistochemistry. RNA was extracted from four 20μ sections of each FFPE block. Reverse transcription was done in a 20 μl volume. RT-PCR was performed using validated primers and probes for both SYT-SSX1 and SYT-SSX2 (AB, USA) in two separate reactions, each containing probes for β-actin as an endogenous control. The reactions were amplified for 50 cycles with universal conditions using the 7500 real-time PCR system (AB, USA). Results: Thirteen of the 15 specimens for SS were positive for fusion transcripts (SSX1-62% and SSX2-38%) while all 10 non-SS specimens were negative demonstrating good sensitivity (87%) and specificity (100%) for the testing system. One poorly differentiated sarcoma and one malignant spindle sarcoma both morphologically suggestive of SS, were negative for translocations. A subset of positives with either translocations were confirmed by gel electrophoresis (product size) and sequencing. Conclusion: The detection of fusion transcripts for SS by RT-PCR will be a valuable adjunct to histopathological diagnosis and a useful molecular marker  Synovial sarcoma is an aggressive soft tissue tumor, accounting for 5-14% of soft tissue tumors 1  Classically occurs in the extremities of young adults, preferentially affects the para-articular regions, but has been described in a wide variety of anatomical locations  Includes two major histological subtypes, biphasic and monophasic types, characterized by the presence or absence of glandular epithelial differentiation in a background of spindle tumor cells 2,3 The monophasic subtype is more common representing 60-75% of these tumours 3 However, poorly differentiated synovial sarcoma, a third subtype, consisting of small round cells is also recognized It is essential to differentiate this type since it is associated with a worse prognosis 2,3,4 A characteristic feature detectable in most synovial sarcoma is the presence of a reciprocal translocation between chromosomes X and 18 t(X;18) (p11.2;q11.2)  The t(X;18) translocation results in the fusion of the proximal portion of the SYT gene at 18q11 to the distal portion of one of the two genes SSX1 or SSX2 at Xp11  SYT-SSX1 fusion transcripts have been detected in 61-64% of cases while SYT-SSX2 has been detected in the remainder of cases 2,4  SYT-SSX4 fusion and other variant chimeric transcripts have been detected occasionally in isolated cases  Detection of chimeric transcripts not only aids diagnosis, but has been debated to be of prognostic value influencing treatment and overall patient care 4,5,6  While the presence of SYT-SSX1 fusion gene has been found to be associated with worse prognosis by Ladanyi et al, more recent studies have not found significant correlation 5,6  Several methods have been used to detect these fusions including cytogenetic analysis, reverse transcriptase-polymerase chain reaction (RT- PCR) followed by gel electrophoresis or restriction fragment length polymorphism (RFLP), real-time PCR and fluorescent in-situ hybridization (FISH) with the latter two methods also providing the advantage of determining the transcript type A small study was undertaken using a real-time PCR based approach  to detect fusion transcripts (SYT-SSX1 and SYT-SSX2) in synovial sarcoma  to determine the usefulness of archived formalin fixed paraffin embedded tissues (FFPE) in detection of fusion transcripts in the absence of frozen/ fresh tissues  to determine the sensitivity, specificity and turnaround time of the real-time PCR in order to establish its utility in diagnostic settings METHODS Patient samples  The FFPE blocks of 15 samples with a diagnosis of synovial sarcoma were retrieved (June September 2007) from the archives at the department of pathology, Christian Medical College, Vellore, India  All histological slides were reviewed and the diagnosis confirmed in each case  The 15 samples were categorized as follows: Monophasic (11), biphasic (1), poorly differentiated (1) and ‘highly suggestive of synovial sarcoma’(2) REAL-TIME POLYMERASE CHAIN REACTION FOR THE DETECTION OF SYT-SSX1 AND SYT-SSX2 TRANSLOCATIONS IN ARCHIVAL SYNOVIAL SARCOMA SPECIMENS REAL-TIME POLYMERASE CHAIN REACTION FOR THE DETECTION OF SYT-SSX1 AND SYT-SSX2 TRANSLOCATIONS IN ARCHIVAL SYNOVIAL SARCOMA SPECIMENS Rekha Pai, Marie Therese Manipadam, Susanna N Thomas, Noel Walter; Department of Pathology, Christian Medical College, Vellore , India  A representation (10) of other sarcomas which are differentials for synovial sarcoma were also included: Ewing’s (3); dermatofibrosarcoma (1); rhabdomyosarcoma (1); leiomyosarcoma (1); malignant peripheral nerve sheath tumor (MPNST, 1) and unclassified sarcomas (3) Immunohistochemical (IHC) markers  The IHC markers used to aid the diagnosis of synovial sarcoma included epithelial membrane antigen (EMA, 1:10), cytokeratin (cytoK, 1:100), CD99 (1:50), CD34 (1:25), bcl2 (1:50), smooth muscle actin (SMA, 1:25) and S-100 (1:200). All antibodies were obtained from Dako Inc, USA  Standard immunohistochemistry techniques were followed for performance of these tests using the avidin-biotin method 7 RNA extraction and reverse transcription  RNA was extracted from all 25 samples included in the study using the RecoverAll TM Total nuclei acid isolation kit (Ambion Inc, USA)  Briefly four-five 20μ sections obtained from the archived FFPE blocks were deparaffinized with xylene and digested with proteinase for 3 hours at 60 0 C, followed by several buffer washes, DNase treatment and final elution. RNA elute was quantitated using the NanoDrop (NanoDrop technologies, USA) and the 260/280 ratio determined  Samples (5 μl) were reverse transcribed using the High Capacity cDNA reverse transcription kit (Applied Biosystems, USA) to give a final volume of 20μl of cDNA and stored (-20 0 C). Real-time PCR  Each sample was separately amplified with validated primers and probes (Applied Biosystems, USA) to detect the presence SYT-SSX1 (Hs_ _ft) or SYT-SSX2 (Hs_ _ft) translocations  Primers and probes for the amplification of β-actin (endogenous control) were also included with each sample  Each sample was amplified to a final volume of 25 μl containing the 1X Taqman master mix, primers and probes for either of the two translocations and for β-actin, 5 μl target cDNA and sterile DEPC treated water  Amplification was performed using the universal thermal cycling conditions (50 0 C for 2 min, 95 0 C for 10 min, 95 0 C for 15 sec, 60 0 C for 1 min ) on the 7500 real-time PCR system (Applied Biosystems, USA) for 50 cycles  Each run also included positive and non-template controls and results were interpreted only if these controls were satisfactory. Each sample was considered positive if it reached the cycle threshold (Ct) within 40 cycles  The turnaround time was determined in each run by noting the time from the start of extraction to the finish of the real-time PCR assay  The product size was determined for a subset of positive samples by gel electrophoresis (2% gel) to yield 83 and 77 bp products for SYT-SSX1 and SYT- SSX2 respectively  Select products were also taken up for sequencing using the primers of Ladanyi et al (unpublished data) using the ABI 3700 automated sequencer RESULTS Of the 25 FFPE specimens included in the study, 15 were obtained from patients with a histopathological diagnosis of synovial sarcoma and clinicopathological features of this group are listed below  The median age was 25 years (range from years) with a male to female ratio of ~1:1  67% (10/15) patients presented with a primary tumor, 27% (4/15) had recurrence (within 7 months to 10 years) and only one presented with lymph node metastases; Nearly half the tumors (n=7) sampled were larger than 5 cms in greatest dimension  The lower extremity was the most common site of tumor (80%) followed by the upper extremity (12%) and only one case presented as an intra abdominal mass  73% of the tumors characterized were classified as monophasic synovial sarcoma, with fewer cases of biphasic and poorly differentiated types (7% each) ; 13% were ‘highly suggestive of synovial sarcoma’  Of the immunohistochemical markers used, EMA and CD99 were positive in most cases Results of real-time PCR  RNA extracted from FFPE specimens ranged from 22 to 1137 ng/μl with the quantity of extract showing no relation to age of the specimen  Thirteen of the 15 FFPE specimens included for analysis yielded a positive result giving the real-time PCR assay a sensitivity of 87%  More number of tumors had the SYT-SSX1 translocations (53%) while 33% of the specimens analyzed had SSX2 chimeric transcript Of the 11 monophasic type of tumor included 6 (55%) were SSX1; the biphasic and poorly differentiated tumors characterized in this study (one each) had the SSX1 translocation Both spindle cell tumors with a suggestion of synovial sarcoma did not amplify with probes for either of the two translocations, though β-actin was positive in both these cases  The average Ct value was 33 (ranging from cycles)  None of the 10 non-synovial sarcoma samples amplified with both the SYT- SSX1 and SYT-SSX2 primers yielded a positive result though they had amplifiable DNA as shown by the amplification of β-actin, therefore demonstrating 100% specificity of the testing system  Four positive samples with either of the translocations were subjected to analysis by gel electrophoresis and yielded a 83 or 77 bp product based on the fusion type  The time required from the start of extraction to completion of the real-time assay was calculated and was ~ 10 hours (one and quarter working days) CONCLUSION  The real-time PCR assay for synovial sarcoma assessed in our setting using 15 histopathologically diagnosed FFPE specimens was found to have good sensitivity and specificity  The PCR worked well with RNA extracted from archived (over a year) FFPE specimens indicating that the system can be used reliably for both prospective and retrospective diagnostic evaluation  The testing system may be particularly useful considering that the test can be completed in 10 hours and can be a valuable diagnostic aid to rule out synovial sarcoma when it considered as a differential  The ability of the testing system to provide a diagnosis in cases of poorly differentiated synovial sarcoma can be of added value  Though the test has provided data on the distribution of translocation types, characterization of larger number of samples and correlation with disease outcome and progression may generate more valuable information REFERENCES: 1. 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Genes Chromosomes Cancer 31: Cerilli LA and Wick MR. Immunohistology of soft tissue and osseous neoplasms. In Diagnostic immunohistochemistry, Dabbs D [ed], Elsevier publications, 2006, pp The work presented here was funded by the Department of Pathology, Christian Medical College, Vellore, India SYT-SSX1 β-actin SSX1 SSX2 Poorly differentiated type Monophasic spindle