Detection of salivary gland and sinonasal fusions by a next-generation sequencing based, ligation-dependent, multiplex RT-PCR assay.

AIMS: The discovery of tumour type-specific gene fusion oncogenes in benign and malignant salivary gland and sinonasal (SGSN) tumours has significantly increased our knowledge about their molecular pathology and classification. METHODS AND RESULTS: We developed a new targeted multiplexed next-generation sequencing (NGS)-based method that utilizes ligation dependent reverse-transcriptase polymerase chain reaction (LD-RT-PCR) to detect oncogenic fusion transcripts involving 116 genes, leading to 96 gene fusions known to be recurrently rearranged in these tumours. In all, 180 SGSN tumours (formalin-fixed, paraffin-embedded samples, 141 specimens and 39 core needle biopsies) from the REFCORpath (French network for rare head and neck cancers) with previously identified fusion genes by fluorescent in situ hybridisation (FISH), RT-PCR, or molecular immunohistochemistry were selected to test its specificity and sensitivity and validate its diagnostic use. Tested tumours encompassed 14 major tumours types, including secretory carcinoma, mucoepidermoid carcinoma, adenoid cystic carcinoma, salivary gland intraductal carcinoma, clear cell carcinoma, pleomorphic adenoma, adamantinoma-like Ewing Sarcoma, EWSR1::COLCA2 sinonasal sarcoma, DEK::AFF2 sinonasal carcinoma, and biphenotypic sinonasal sarcoma. In-frame fusion transcripts were detected in 97.8% of cases (176/180). Gene fusion assay results correlated with conventional techniques (immunohistochemistry [IHC], FISH, and RT-PCR) in 176/180 tumours (97.8%). CONCLUSION: This targeted multiplexed NGS-based LD-RT-PCR method is a robust, highly sensitive method for the detection of recurrent gene fusions from routine clinical SGSN tumours. It can be easily customized to cover new fusions. These results are promising for implementing an integrated NGS system to rapidly detect genetic aberrations, facilitating accurate, genomics-based diagnoses, and accelerate time to precision therapies in SGSN tumours.

A novel SMARCA2-CREM fusion expending the molecular spectrum of salivary gland hyalinazing clear cell carcinoma beyond the FET genes.

Hyalinizing clear cell carcinoma (HCCC) is a rare salivary gland carcinoma with a generally indolent behavior, characterized by recurrent chromosomal translocation involving EWSR1 (22q12.2) leading to two fusion genes EWSR1::ATF1 or EWSR1::CREM. We report one case of HCCC with a novel SMARCA2::CREM fusion, identified by targeted RNA next generation sequencing by LD-RT-PCR, which has until now never been described in salivary glands. The exon 4 of SMARCA2 is fused to exon 5 of CREM. This fusion has been described previously in only one tumor, a central nervous system tumor (intracranial mesenchymal tumor) but not in other FET::CREB fused tumors. This fusion was confirmed by CREM break-apart FISH and reverse transcriptase polymerase chain reaction (RT-PCR). The tumor cells showed retained expression of INI1, SMARCA2, and SMARCA4 by immunohistochemistry. We compare its clinical, histopathological, immunophenotypic, genetic features with those previously described in HCCC, FET::CREB fusion-positive. Our results added data suggesting that different histomolecular tumor subtypes seem to be included within the terminology "HCCC, FET::CREB fusion-positive," and that further series of cases are needed to better characterize them.