Expanding the Use of an SS18-SSX Antibody for Molecular Assays in Synovial Sarcoma

Abstract

Synovial sarcoma is an aggressive malignancy that generally affects adolescents and young adults and is characterized by high rates of recurrence and metastasis, with a 10-year survival rate of about 50%. The fusion oncoprotein SS18-SSX, the product of a pathognomonic chromosomal translocation t(X;18), is the oncogenic driver of this sarcoma, disrupting differentiation through widespread epigenetic dysregulation. Experimental research into SS18-SSX biology has been limited by the lack of an antibody that specifically detects the endogenous fusion oncoprotein as opposed to its native SS18 or SSX components. Recently, a rabbit monoclonal antibody was developed and made commercially available, which specifically detects the fusion junction site epitope of SS18-SSX as found in at least 95% of synovial sarcomas. Here, we characterize a suite of molecular biology assays using this new antibody, both confirming existing and reporting on novel applications. We demonstrate its high sensitivity and specificity for synovial sarcoma diagnosis on patient samples through positive immunohistochemical staining on synovial sarcoma, tissue microarray, and full face sections. In addition, we demonstrate detection of the human SS18-SSX protein when expressed in a genetically engineered mouse model of synovial sarcoma. We also demonstrate nuclear staining of SS18-SSX in synovial sarcoma cells using immunofluorescence, and visualize the interaction between SS18-SSX and the BAF complex member BRG1 through a proximity ligation assay. Lastly, we confirm the interaction between SS18-SSX and promoter regions of target genes through chromatin immunoprecipitation. This antibody represents a breakthrough in sarcoma research and has value in multiple applications to expand the knowledge of synovial sarcoma biology.

SS18-SSX Antibody: A Useful Tool to Save Time and Reduce Costs in Synovial Sarcoma Diagnosis. Proposal of a Novel Diagnostic Algorithm

“…The first goal we aimed at in this study was to confirm the high sensitivity and specificity of the SS18-SSX antibody in the diagnosis of SS in a retrospective cohort of SSs and its histological mimickers. Our results report a sensitivity of 95% and a specificity of 100% and are thus in perfect agreement with recently published data 13,17,19,22 –27 . In the largest reported series (400 tumors including 100 SSs and 300 mimics), Baranov et al who suggested for the first time that the IHC approach could replace molecular investigations to support SS morphological diagnosis 19 have obtained values perfectly overlapping with those presently reported.…”

Cytomorphology of metastatic colonic MXD4::NUTM1‐rearranged sarcoma

“…Our patient on the most recent clinical follow-up is 1) is extremely broad and includes NUT carcinoma, melanoma, neuroendocrine neoplasm, myoepithelial carcinoma, SMARCA4-deficient neoplasms, synovial sarcoma, and plasma cell neoplasm. [19][20][21][22][23][24][25][26][27][28][29][30] Immunohistochemical stains and/or molecular testing will be essential. That said, the tumor immunoreactivity with NUT comes with a caveat.…”

“…The differential diagnosis based on the epithelioid and rhabdoid/plasmacytoid morphology (Table 1) is extremely broad and includes NUT carcinoma, melanoma, neuroendocrine neoplasm, myoepithelial carcinoma, SMARCA4‐deficient neoplasms, synovial sarcoma, and plasma cell neoplasm 19–30 . Immunohistochemical stains and/or molecular testing will be essential.…”

Cell-of-origin epigenome underlies SS18::SSX-mediated transformation

“…Paraffin embedding, sectioning, and H&E staining were performed according to standard protocols at the UBC ACS Diagnostic & Research Histology Laboratory. FFPE tissues were also utilized for SS18::SSX IHC, as previously described 57 . Briefly, 4 um sections were subjected to heat-induced antigen retrieval for 20 min, followed by SS18::SSX antibody (1:300, 72364, Cell Signalling Technology) incubation for 15 min at 37°C.…”