Objective: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and aggressive subtype of ovarian cancer with limited treatment options. Inactivating SMARCA4 germline and somatic mutations are present in nearly all SCCOHT cases, representing a signature molecular feature of this disease. Concomitant loss of SMARCA2, a mutually exclusive catalytic subunit of the SWI/SNF chromatic remodeling complex, has been attributed to worse overall survival in non-small cell lung carcinoma (NSCLC) when compared to cases with loss of SMARCA4 only. The objective of this study was to evaluate the mutational status and expression of SMARCA2 in SCCOHT.
Methods: We evaluated 10 archival cases of SCCOHT and 1 newly established case from a PDX model of recurrent disease. Specialty gynecologic pathologists reviewed all cases to confirm the diagnosis. DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tumors with at least 50% tumor cell nuclei. Using the MSK-IMPACT assay, samples were deep-sequenced for a panel of 341 known oncogenes and tumor suppressor genes frequently altered in cancer. Identified SMARCA4 mutations were validated using Sanger sequencing. Sequencing of the SMARCA2 coding regions and splice sites was done using AmpliSeq. We performed immunoblotting for SMARCA4 and SMARCA2 on available frozen tumor samples using standard protocol. To confirm loss of protein expression, immunohistochemistry (IHC) for the SMARCA4 and SMARCA2 was performed on FFPE slides of all cases; the absence of tumor cell nuclear staining in the presence of internal positive controls scored as loss-of-expression. For cell-line over-expression studies, SCCOHT BIN67 and NSCLC H1299 cells were transiently transfected with an expression plasmid containing cDNA for SMARCA4 using FuGene reagent (Invitrogen) according to manufacturer's instructions. Immunoblotting for SMARCA2 and SMARCA4 was performed on protein lysates extracted from cell pellets.
Results: IHC demonstrated loss of SMARCA2 expression in 9 (90%) of 10 archival SCCOHT cases and the PDX model. Sequencing of the SMARCA2 coding region and splice sites revealed no mutations, suggesting a non-mutational etiology of SMARCA2 protein loss. Previous work revealed biallelic SMARCA4 mutations in the 9 archival cases with SMARCA2 loss. SMARCA4 mutations were confirmed in both the PDX model and the one additional archival case, resulting in universal SMARCA4 mutations. The only case with normal SMARCA2 expression was also the only case with retained SMARCA4 expression. To explore the relationship between SMARCA2 and SMARCA4, we re-introduced SMARCA4 into BIN67 SCCOHT and H1299 NSCLC cell lines that lack expression. Wild-type BIN67 cells have very low levels of SMARCA2 expression, while wild-type H1299 cells express SMARCA2 normally. After transfection with plasmid cDNA and confirmation of SMARCA4 expression by immunoblotting, there was no increase in SMARCA2 expression in either BIN67 or H1299 cells.
Conclusions: Concomitant loss of SMARCA2 and SMARCA4 expression was seen in nearly all SCCOHT cases and our PDX model. While loss of SMARCA4 expression is due to biallelic mutations, the absence of SMARCA2 expression is non-mutational and potentially the result of epigenetic or post-translational silencing. Our data suggest that concomitant loss of SMARCA2 and SMARCA4 is another hallmark of SCCOHT. The reversal of SMARCA2 loss may serve as a potential novel therapeutic approach for SCCOHT.
Citation Format: Jill H. Tseng, Petar Jelinic, Brooke A. Schlappe, Niamh Conlon, Narciso Olvera, Fanny Dao, Jennifer J. Mueller, Yaser Hussein, Robert A. Soslow, Douglas A. Levine. Concomitant loss of SMARCA2 and SMARCA4 expression in small cell carcinoma of the ovary, hypercalcemic type. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B15.