Detection of SYT-SSX Rearrangements in Synovial Sarcomas by Real-Time One-Step RT-PCR

Abstract

Synovial sarcomas are aggressive tumors of adolescent and young adults that account for up to 10% of soft tissue sarcomas. Cytogenetically, they are characterized by translocation t(X;18), which is found in more than 95% of tumors. In most cases, it results in fusion of the SYT gene with the SSX1 or SSX2 gene, thus creating SYT-SSX1 or SYT-SSX2 rearrangement. The 2 types of gene fusion have been correlated with histologic variants and prognosis of synovial sarcomas. In this study, we developed a simple and rapid method for the simultaneous detection of SYT-SSX1 and SYT-SSX2 rearrangements by using a LightCycler real-time one-step reverse transcriptase polymerase chain reaction (RT-PCR) technology (Roche). Oligonucleotide probes were designed so that the donor probe would span a fusion point and the acceptor probe would be complementary to the SSX1 sequence but have 2 nucleotide mismatches with SSX2 sequence. Such a design allows simultaneous amplification of 2 types of rearrangement in the same reaction but distinguishes them based on differences in melting temperature detected by melting curve analysis after PCR. With this method, 27 tumors (9 synovial sarcomas and 18 nonsynovial sarcomas) were studied and showed SYT-SSX1 rearrangement in 6 cases and SYT-SSX2 in 3 cases. These results had complete correlation with the finding of conventional RT-PCR and direct sequencing. In conclusion, we have developed a fast, accurate, and simple method for the detection of 2 major types of SYT-SSX rearrangement by using LightCycler RT-PCR and melting curve analysis.