UBC Faculty Research and Publications

Dual loss of the SWI/SNF complex ATPases SMARCA4/BRG1 and SMARCA2/BRM is highly sensitive and specific… Karnezis, Anthony N.; Wang, Yemin; Ramos, Pilar; Hendricks, William P.D.; Oliva, Esther; D’Angelo, Emanuela; Prat, Jaime; Nucci, Marisa R.; Nielsen, Torsten O.; Chow, Christine; Leung, Samuel; Kommoss, Friedrich; Kommoss, Stefan; Silva, Annacarolina; Ronnett, Brigitte M.; Rabban, Joseph T.; Bowtell, David D.; Weissman, Bernard E.; Trent, Jeffrey M.; Gilks, C. Blake; Huntsman, David G. 2016

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Accepted ArticleDual loss of the SWI/SNF complex ATPases SMARCA4/BRG1 and SMARCA2/BRM is highly sensitive and specific for small cell carcinoma of the ovary, hypercalcemic type  Anthony N. Karnezis1*, Yemin Wang1*, Pilar Ramos2*, William P.D. Hendricks2, Esther Oliva3, Emanuela D’Angelo4, Jaime Prat4, Marisa R. Nucci5, Torsten O. Nielsen1, Christine Chow6, Samuel Leung6, Friedrich Kommoss7, Stefan Kommoss8, Annacarolina Silva9, Brigitte M. Ronnett10, Joseph T. Rabban11, David D. Bowtell12, Bernard E. Weissman13, Jeffrey M. Trent2, C. Blake Gilks1#, David G. Huntsman1, 6, 14#  1. Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.  2. Division of Integrated Cancer Genomics, Translational Genomics Research Institute (TGen), Phoenix, AZ, USA.  3. Department of Pathology, Massachusetts General Hospital, Boston, MA, USA. 4. Department of Pathology, Hospital de la Santa Creu i Sant Pau, Autonomous University of Barcelona, Barcelona, Spain.  5. Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA.  6. Genetic Pathology Evaluation Centre, Vancouver General Hospital, University of British Columbia, Vancouver, BC, Canada. 7. Synlab MVZ Pathologie, Mannheim, Germany. 8. Department of Obstetrics and Gynecology, University Hospital of Tuebingen, Tuebingen, Germany.      This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/path.4633  This article is protected by copyright. All rights reserved.Accepted Article 9. The James Homer Wright Pathology Laboratories, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. 10. Department of Pathology, The Johns Hopkins Hospital, Baltimore, MD, USA. 11. Department of Anatomic Pathology, University of California San Francisco, San Francisco, CA, USA. 12. Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia. 13. Department of Pathology and Laboratory Medicine, Lineberger Cancer Center, University of North Carolina, Chapel Hill, NC, USA. 14. Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, BC, Canada.  * These three authors contributed equally. # Corresponding authors  Correspondence to:  David Huntsman, MD, FRCPC, FCCMG, Dr. Chew Wei Memorial Professor of Gynaecologic Oncology, UBC Professor, Departments of Pathology and Lab Medicine and Obstetrics and Gynaecology, UBC Director of OvCaRe, Vancouver General Hospital, BC Cancer Agency and UBC Distinguished Scientist, Department of Molecular Oncology, BC Cancer Agency Research Centre  #3427-600 West 10th Avenue Vancouver, BC V5Z 4E6 Canada Phone: 604.877.6000 x2301 dhuntsma@bccancer.bc.ca  This article is protected by copyright. All rights reserved.Accepted Articleor C Blake Gilks, MD, Room 1250, 1st floor JPPN Department of Pathology and Laboratory Medicine Vancouver General Hospital 910 West 10th Ave Vancouver BC V5Z 1M9 Canada Phone 604-771-7510 Blake.Gilks@vch.ca  Conflict of interest: None of the authors report any conflicts of interest.    This article is protected by copyright. All rights reserved.Accepted ArticleAbstract   Small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) is a lethal and sometimes familial ovarian tumour of young women and children. We and others recently discovered that over 90% of SCCOHT harbour inactivating mutations in the chromatin remodelling gene SMARCA4 with concomitant loss of its encoded protein SMARCA4 (BRG1), one of two mutually-exclusive ATPases of the SWI/SNF chromatin remodelling complex. To determine the specificity of SMARCA4 loss for SCCOHT, we examined the expression of SMARCA4 by immunohistochemistry in more than 3000 primary gynaecological tumours. Among ovarian tumours, it was only absent in clear cell carcinoma (15 of 360, 4%). In the uterus, it was absent in endometrial stromal sarcomas (4 of 52, 8%) and high-grade endometrioid carcinomas (2 of 338, 1%). Recent studies have shown that SMARCA2 (BRM), the other mutually exclusive ATPase of the SWI/SNF complex, is necessary for survival of tumour cells lacking SMARCA4. Therefore, we examined SMARCA2 expression and discovered that all SMARCA4-negative SCCOHTs also lacked SMARCA2 protein by IHC, including the SCCOHT cell lines BIN67 and SCCOHT1. Among ovarian tumours, the SMARCA4/SMARCA2 dual loss phenotype appears completely specific for SCCOHT. SMARCA2 loss was not due to mutation but rather from an absence of mRNA expression, which was restored by treatment with the histone deacetylase inhibitor trichostatin A. Re-expression of SMARCA4 or SMARCA2 inhibited the growth of BIN67 and SCCOHT1 cell lines. Our results indicate that SMARCA4 loss, either alone or with SMARCA2, is highly sensitive and specific for SCCOHT, and that restoration of either SWI/SNF ATPase can inhibit the growth of SCCOHT cell lines. Keywords: small cell carcinoma, hypercalcaemic type, rhabdoid tumour, SMARCA4/BRG1, SMARCA2/BRM, SMARCB1/INI1, SWI/SNF, HDAC inhibitor, trichostatin A, epigenetic silencingThis article is protected by copyright. All rights reserved.Accepted ArticleIntroduction Background – SCCOHT  Small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) is a rare ovarian cancer that predominantly affects young women in their teens and 20s, with an average age of 24 years. Although half of tumours are diagnosed at an early stage, the prognosis is dismal. For patients with stage IA disease, more than half will still die of disease, usually within two years[1]. Histologically, SCCOHT is characterized by sheets and poorly formed nests of small cells with scant cytoplasm, hyperchromatic nuclei and small nucleoli[1]. Approximately half of tumours also contain variable numbers of large cells with abundant eosinophilic cytoplasm, often rhabdoid inclusions, large nuclei and prominent nucleoli. The histologic differential diagnosis is broad and includes many primary and metastatic tumours to the ovary. The lack of familiarity of pathologists with this rare tumour and the lack of specific immunohistochemical markers can make its diagnosis challenging.  SMARCA4 in SCCOHT and cancer Recently, four groups independently identified inactivating SMARCA4 mutations in the majority of SCCOHT, resulting in loss of SMARCA4 protein[2-5]. SMARCA4 and the related protein SMARCA2 (also called BRG1 and BRM, respectively) are the two mutually exclusive ATPases of the SWI/SNF chromatin remodelling complex [6-8]. SWI/SNF subunits have been frequently implicated as tumour suppressors, with approximately 20% of cancers bearing mutations in these genes[9, 10]. Our initial analysis of a small collection of ovarian tumours indicated that SMARCA4 loss was highly specific for SCCOHT[2].  Potential therapeutic strategies for SCCOHT This article is protected by copyright. All rights reserved.Accepted ArticleThe function of the SWI/SNF complex in chromatin remodelling suggests that the pathogenesis of SCCOHT involves epigenetic dysregulation. This paradigm may offer treatment possibilities with agents that regulate the epigenome such as inhibitors of histone deacetylase (HDAC) or modifiers of histone or DNA methylation. The mutually exclusive nature of the SMARCA4 and SMARCA2 ATPases in the SWI/SNF complex has suggested that SMARCA2 may be a synthetic lethal target in SMARCA4-mutant cancers. Indeed, loss of SMARCA4 has been shown to lead to dependence on SMARCA2-containing SWI/SNF complexes for survival in some cell lines[11-13] raising the possibility of targeting SMARCA2 in SCCOHT using inhibitors of its ATPase or bromodomain.  Aims of this study The establishment of SMARCA4 mutation with accompanying loss of protein as the pathognomonic mutation in SCCOHT raises the need to explore the spectrum of tumours that share SMARCA4 (and perhaps SMARCA2) loss to understand the diagnostic utility of SMARCA4 immunohistochemistry (IHC). Because some ovarian and uterine tumours arise from common cell types (e.g. endometrial epithelium, either in the eutopic endometrium or ectopically as endometriosis), we also need to determine the diagnostic utility of SMARCA4 IHC in uterine tumours. Therefore, the goals of this study were to: 1) determine the specificity of SMARCA4 protein loss as a diagnostic marker for SCCOHT by studying its expression in a large cohort of ovarian and uterine tumours with an emphasis on entities in the differential diagnosis; and 2) determine whether SMARCA2 is expressed in SCCOHT and could be used as a therapeutic target.   Materials and methods Sample collection and tissue microarray construction This article is protected by copyright. All rights reserved.Accepted ArticleDuplicate 0.6 or 1.0 mm cores of formalin fixed, paraffin embedded tumour tissue from each case were used for tissue microarray (TMA) construction, as described previously[14]. Additional cases were studied by whole slide IHC. All samples were collected in accordance with institutional guidelines and protocols. For Vancouver samples, informed patient consent was obtained under research ethics board (REB)-approved protocols for all prospectively collected patient samples (REB H05-60 199), archived samples (REB H02-61 375) and for IHC analysis (REB H02-61375). Immunohistochemistry and scoring  TMAs were cut at 4 μm thickness onto Superfrost+ glass slides, and were processed using the Ventana Discovery XT, and the Ventana Benchmark XT and Benchmark Ultra automated systems (Ventana Medical Systems, Tucson, AZ, USA). Immunohistochemical staining was performed with antibodies to SMARCA4 (1:25, clone EPNCIR111A, ab110641, Abcam, Toronto, ON), SMARCA2 (1:50, clone HPA029981, Sigma, St. Louis, MO) and SMARCB1/BAF47/INI1 (1:50, 25/BAF47, 612110, BD Biosciences, Mississauga, ON).  All TMAs were scored twice by a pathologist (A.N.K.). For SMARCA4, tumours were scored as positive if any tumour cell nuclei showed staining; tumours scored as positive usually showed diffuse, moderate to strong staining. Tumours were scored as negative if tumour cell nuclei showed no staining only if adequate staining was detected in internal positive control cells (endothelium, lymphocytes or fibroblasts); absence of staining has previously been shown to correlate with the presence of inactivating mutations in SMARCA4[2-5, 15]. No cytoplasmic SMARCA4 staining was observed. Since SMARCA2 absence was due to non-mutational silencing, tumours were scored as positive for SMARCA2 if >5% of tumour cells showed nuclear staining. Tumours were scored as negative if tumour cells showed no nuclear staining, nuclear staining in up to 5% of cells, or only cytoplasmic staining. For both SMARCA4 and SMARCA2, tumours that showed no staining in tumour and stroma were considered technical failures and This article is protected by copyright. All rights reserved.Accepted Articlenot scored. Each case on a tissue microarray was represented as duplicate cores; one positive core was sufficient to count the case as positive. Cell lines BIN67, SCCOHT1 and KGN cells were all grown in DMEM/F-12 supplemented with 10% FBS. SVOG3e cells were cultured in 199/105 medium with 10% FBS. ES-2 cells were cultured in RPMI supplemented with 10% FBS. All the cells were maintained in a humidified 5% CO2-containing atmosphere at 37°C and tested regularly for Mycoplasma. Plasmids and lentivirus/retrovirus packaging The pLDpuro-SMARCA4 and pLDpuro-SMARCA2 plasmids were constructed by introducing the SMARCA4 and SMARCA2 from entry vectors (Genecopeia) into the pLDpuro-EnVA destination vector (a gift of Dr. Jason Moffat at the University of Toronto) using Gateway reactions (Life Sciences). To produce lentiviruses expressing SMARCA4 or SMARCA2, pLDpuro-SMARCA4 or pLDpuro-SMARCA2 was co-transfected with packaging plasmids psPAX2 and pMD2.G into HEK293T cells. Supernatants were collected at 72 h for lentivirus preparation and infection.   Western blotting Whole-cell extracts were obtained for SDS-PAGE electrophoresis using the SMARCA4 and SMARCA2 antibodies described above. Ponceau S staining or Western blotting for vinculin (clone hVIN-1, V9131, Sigma, St. Louis, MO) was used to confirm equal protein loading. Real-time RT-PCR Total RNA was extracted from cells using RNeasy kits (Qiagen) and quantitated by Nanodrop spectrophotometry. One microgram of total RNA was reverse-transcribed into cDNA using random primers and Superscript III reverse transcriptase (Invitrogen), followed by PCR amplification of SMARCA4 or SMARCA2 using the Power SYBR® Green Master Mix (Life This article is protected by copyright. All rights reserved.Accepted ArticleTechnologies) according to the manufacturer’s instruction. GAPDH was used for normalization. The relative levels were calculated using the ∆∆Ct method. RNA isolation and gene expression microarrays Frozen tumour tissue was disrupted by sonication using the Covaris S-2 system (AQ: Details of supplier please) and stabilized by addition of equal volumes of TRIzol (Life Technologies). BIN67 and SCCOHT1 cells were washed with PBS and lysed using TRIzol. Total RNA was purified using RNeasy micro kits (Qiagen). RNA from two premenopausal normal ovary donors (ages 32 and 37) was obtained from OriGene (Rockville), pooled (100 ng each) and used as a common reference. RNA integrity and purity was measured using a 2100 Bioanalyzer (Agilent) and a Nanodrop ND-1000 Spectrophotometer. Labelled cRNA probes were prepared using Agilent's Low Input Quick Amp Labeling Kit, and hybridized to Agilent Human Gene Expression 4x44K microarrays following manufacturer protocols. Slides were washed and scanned at 5 μm using an Agilent Microarray Scanner (model G2505B) in an ozone-controlled environment, and data extracted, processed and normalized using Agilent Feature Extraction (FE) software (v10.5). Raw and processed data files have been deposited in GEO at accessions GSE49887 and GSE66434. Cell growth assay Cells were infected with lentivirus containing pLDpuro-GFP, -SMARCA4 or -SMARCA2. One day later, cells were selected in 2 µg/ml of puromycin for 3 d. The remaining cells were then harvested, counted and re-seeded at a density of 4000 cells/well in 96-well plates. Cell growth was monitored using the Incucyte Zoom live cell imaging monitor (AQ: Details of supplier please). The percentage of confluence in each well was calculated and plotted over a 7-d period to determine the effect of SMARCA4 or SMARCA2 re-expression on cell growth.   This article is protected by copyright. All rights reserved.Accepted ArticleResults SMARCA4 expression in gynaecological and non-gynaecological tumours We previously studied SMARCA4 protein status in a small series of 485 primary ovarian tumours where besides SCCOHT, only two tumours (0.4%), both clear cell carcinomas, were negative for SMARCA4[2]. In order to perform a definitive analysis of the sensitivity and specificity of SMARCA4 loss for SCCOHT and, thus, its potential as a diagnostic immunohistochemical marker, we expanded SMARCA4 IHC analysis to additional cases of SCCOHT and a large series of 3048 primary ovarian and uterine tumours and non-gynaecological mimics of SCCOHT (Table 1). Examination of SMARCA4 expression in 50 SCCOHT-derived samples (20 reported previously[2, 15] and 30 new cases reported here) revealed SMARCA4 loss in 42/46 (91%) primary tumours, 2/2 patient-derived mouse xenografts and 2/2 SCCOHT-derived cell lines (Table 1). Of the four tumours that retained SMARCA4 expression, three lacked SMARCB1 (also called BAF47 or INI1), a different core member of the SWI/SNF complex. One of the three SMARCB1-negative tumours showed biallelic inactivation of SMARCB1. In the remaining SMARCA4-positive case, exome sequencing revealed no mutations in SWI/SNF genes. We examined SMARC4 expression in 3048 primary ovarian and uterine tumours and non-gynaecological mimics of SCCOHT. Among 2324 primary ovarian epithelial, sex cord-stromal, and germ cell tumours (including the previously reported 485 tumours[2]), SMARCA4 loss was only observed in clear cell carcinoma (15/360, 4%) (Table 1), a tumour that is not in the histologic differential diagnosis of SCCOHT. All ovarian tumours in the histologic differential diagnosis of SCCOHT (including small cell carcinoma of pulmonary type, adult and juvenile granulosa cell tumours, Sertoli-Leydig cell tumours, endometrial stromal sarcoma, and dysgerminoma) retained SMARCA4 expression.  This article is protected by copyright. All rights reserved.Accepted ArticleAmong 680 primary uterine tumours, SMARCA4 loss was observed in endometrioid carcinoma (2/338, 1%) and endometrial stromal sarcoma (ESS, 4/52, 4%, Table 1). Both endometrioid carcinomas were FIGO grade 3; one was a dedifferentiated carcinoma[16], a tumour composed of low-grade endometrioid carcinoma (FIGO grade 1 or 2) and undifferentiated carcinoma. Among the ESS cases, two were low-grade, and two were high-grade. One high-grade ESS showed rhabdoid features. For the SMARCA4-negative dedifferentiated carcinoma and the high-grade ESS cases, only the high-grade/undifferentiated portions were represented on the TMAs. We also examined SMARCA4 expression in 44 extra-ovarian, non-gynaecological tumours that resemble SCCOHT histologically and can present with intra-abdominal or ovarian disease mimicking ovarian carcinoma clinically, including alveolar and embryonal rhabdomyosarcoma, desmoplastic small round cell tumour, Ewing’s sarcoma, synovial sarcoma, follicular lymphoma and melanoma. All of these tumours retained SMARCA4 expression (Table 1). In summary, 46/50 (92%) of all SCCOHT-derived samples in our series lacked SMARCA4, and 49/50 (98%) lacked a core member of the SWI/SNF complex (either SMARCA4 or SMARCB1). When combined with other published SCCOHT cases [3-5], SMARCA4 loss has now been observed in 83/91 cases (91% sensitivity). Among primary ovarian tumours and non-gynaecological tumours that can mimic SCCOHT, we observed SMARCA4 loss in only 15/2368 tumours (15/2324 ovarian and 0/44 non-gynaecological) for a specificity of 99% among the tumours examined, and the only ovarian tumour that lacked SMARCA4 in our study (clear cell carcinoma) is not in the differential diagnosis of SCCOHT. These data demonstrate that the lack of SMARCA4 in ovarian tumours is a highly sensitive and almost completely specific IHC marker of SCCOHT. Absence of SMARCA2 protein in SCCOHT tumours This article is protected by copyright. All rights reserved.Accepted ArticleIn order to determine SMARCA2 status in SCCOHT and the potential for its development as a therapeutic target, we examined its expression in 45 cases. All 45 tumours (43 lacking SMARCA4 and 2 lacking SMARCB1) were negative for SMARCA2. However, rare tumour cells (usually <1% but up to 10% focally) still expressed SMARCA2. In areas containing only small cells, SMARCA2 staining was exclusively nuclear (Figure 1A, arrows). In tumours with a large cell component, the protein was exclusively localized to the cytoplasm of large cells (Figure 1B, bottom panels, arrowheads), with no cytoplasmic expression in the small cell component (Figure 1B upper panels).  We did not identify mutations in the SMARCA2 gene in SCCOHT, implicating silencing during tumorigenesis or in the cell of origin as the underlying mechanism explaining the absence of SMARCA2 protein. We therefore performed IHC for SMARCA2 in ovaries of premenopausal women and found uniform and strong expression in all ovarian cell types, including surface epithelial cells, stromal cells, granulosa cells, theca cells and oocytes (Figure 1C). This result suggests that SCCOHT loses SMARCA2 expression during tumorigenesis or, alternatively, SCCOHT arises from a rare SMARCA2-negative cell type in the ovary.  To determine whether dual loss of both SMARCA4 and SMARCA2 was specific for SCCOHT, we examined SMARCA2 expression by IHC in ovarian clear cell carcinoma, the only other tumour lacking SMARCA4 expression (Table 1). Tumours were deficient either for SMARCA4 or SMARCA2, but not both (Figure 2), indicating that dual deficiency of the two SWI/SNF ATPases is specific for SCCOHT. In uterine tumours, combined SMARCA4 and SMARCA2 deficiency was only observed in dedifferentiated carcinoma (n=1) and high-grade ESS (n=2) (Figure 3). Epigenetic silencing of SMARCA2 in SCCOHT cells The lack of mutations or deletions involving SMARCA2 in SCCOHT and the expression of SMARCA2 protein in rare tumour cells suggest that the absence of SMARCA2 protein in most This article is protected by copyright. All rights reserved.Accepted Articletumour cells arises from epigenetic silencing or mRNA degradation, as reported in other SMARCA2-deficient cell lines[17-21]. Compared to normal premenopausal ovary, SMARCA2 mRNA levels were decreased in seven SCCOHT tumour samples (four primary tumours and two mouse xenografts generated directly from patient tumours, and one SCCOHT cell line), an effect that was specific for SMARCA2 and not observed with genes encoding most other SWI/SNF family members (Figure 4A). Similar to the tumour samples, no SMARCA2 mRNA (Figure 4B) or protein (Figure 4C) was detected in two SCCOHT cell lines, BIN67 and SCCOHT1, and no underlying mutation was identified. SMARCA2 mRNA levels were significantly lower in BIN67 and SCCOHT1 cells compared to that in NOY1 (a yolk sac tumour cell line), SVOG3e (an immortalized primary granulosa cell line) or KGN cells (an adult granulosa cell tumour cell line) (Figure 4B), suggesting epigenetically silencing of the SMARCA2 gene or degradation of SMARCA2 mRNA as the mechanism underlying the lack of SMARCA2 protein in SCCOHT cells.  To determine whether we could reactivate SMARCA2 expression in SCCOHT cell lines, we treated BIN67 cells with inhibitors that target epigenetic regulators including the HDAC inhibitor trichostatin A (TSA), DNA methyltransferase inhibitor 5-azacytidine (5-AZA) and the EZH2 histone methyltransferase inhibitor GSK343. Of these reagents, only the HDAC inhibitor TSA induced the expression of SMARCA2 protein (Figure 4D), correlating with increased mRNA levels in BIN67 cells (Figure 4E). Furthermore, TSA stimulated the expression of SMARCA2 protein in a dose-dependent manner in BIN67 and SCCOHT1 cells (Figure 4F). These data implicate HDAC-mediated epigenetic silencing of the SMARCA2 gene in SCCOHT or, alternatively, an indirect inhibitory effect on SMARCA2 mRNA degradation.  Re-expression of SMARCA4 or SMARCA2 inhibits cell growth To assess whether the absence of SMARCA4 and SMARCA2 is crucial for tumour cell growth, we infected BIN67 cells with lentiviruses driving expression of SMARCA4 or SMARCA2. Overexpression of either protein robustly suppressed the growth of BIN67 cells (Figure 5A). This article is protected by copyright. All rights reserved.Accepted ArticleHowever, SMARCA4 expression did not significantly inhibit the growth of cells lacking only SMARCA4 (JHOC5 cells, ovarian clear cell carcinoma cell line[22]) or with normal SMARCA4 and SMARCA2 expression (ES-2 ovarian endometrioid adenocarcinoma cell line[22]) (data not shown). Previous studies have shown that the ability of SMARCA4 to induce cell cycle arrest in SMARCA4/SMARCA2-deficient cell lines is dependent on a functional retinoblastoma growth control pathway[17, 19, 23-26]. Therefore, the rapid growth arrest that SMARCA4 or SMARCA2 expression induced in SCCOHT cells is consistent with the absence of detectable RB1 gene mutations in all tumour samples and the presence of intact, full length RB1 protein (both hypo- and hyper-phosphorylated forms) in the BIN67 cells (data not shown).   Discussion SMARCA4 in ovarian and endometrial tumours Almost all SCCOHT contain inactivating somatic and germline mutations in SMARCA4 that result in loss of the SMARCA4 (BRG1) protein. We investigated SMARCA4 expression by IHC in a large collection of gynaecological and non-gynaecological tumours to determine the specificity of SMARCA4 loss for SCCOHT. All tumours in the differential diagnosis that we examined retained SMARCA4 expression. The only primary ovarian tumour other than SCCOHT that showed loss of SMARCA4 – clear cell carcinoma – does not enter the histological differential diagnosis of SCCOHT. Therefore, the absence of SMARCA4 is highly sensitive and essentially completely specific for SCCOHT. Though we only studied small numbers of germ cell tumours in the differential diagnosis of SCCOHT, our results are consistent with those of Witkowski and colleagues, who found no SMARCA4 mutations in 106 germ cell tumours, including 25 dysgerminomas and seminomas[27]. A few ovarian tumours in the differential diagnosis of SCCOHT were not This article is protected by copyright. All rights reserved.Accepted Articlestudied, including metastatic small cell lung carcinoma and oxyphylic tumours that can resemble the large cell variant of SCCOHT. In addition, we did not examine small cell carcinoma of the endometrium or cervix; notably, the latter has been reported to be deficient in SMARCA4[28]. Though SCCOHT and cervical small cell carcinoma have distinct clinical presentations, other IHC markers (e.g. WT1 and TTF1)[29] or molecular studies (HPV18 in the cervical tumours)[28, 30] can also be used to distinguish between these entities. The near universal expression of SMARCA4 as determined by IHC contrasts with the presence of SMARCA4 point mutations or deletions reported in several tumours in our study such as high-grade serous carcinomas[9, 10, 31-33]. Different mutation types in these tumours compared to those in SCCOHT likely underlie this apparent discrepancy. Of almost 100 SMARCA4 mutations reported in SCCOHT, all but three are destructive to the protein (truncating, frameshift, spice site or deletions)[15] with common bi-allelic inactivation due to mutation or loss of heterozygosity of the second allele[3, 5]. In contrast, approximately 80% of SMARCA4 mutations found in more than 500 other tumour samples are missense and do not typically involve inactivation of the second allele[9, 10, 31, 32]. Therefore, several tumours in our study may harbour missense mutations in SMARCA4 that, by their nature, were not suggested by IHC. Interestingly, although ovarian high-grade serous carcinomas reportedly show mutually exclusive homozygous deletions in SMARCA4 and SMARCA2[31, 32], we did not observe SMARCA4 protein loss in any of our samples. This apparent discrepancy might be explained by the extreme intratumoural heterogeneity[34] that characterizes this tumour. Accordingly, it seems unlikely that random selection of two different areas of a tumour for inclusion in a TMA would result in all tumour cells in both regions having homozygous deletions in SMARCA4. Silencing of SMARCA2 in SCCOHT Several groups recently demonstrated that SMARCA4-deficient lung cancer cells depend on SMARCA2, the other mutually exclusive ATPase in SWI/SNF complexes, for proliferation and This article is protected by copyright. All rights reserved.Accepted Articlesurvival[11, 12, 35], suggesting that SMARCA2 could be a therapeutic target in SMARCA4-mutant tumours. However, this requirement is not absolute, as some cell lines[11-13, 17, 19, 23-26] and lung cancers [36, 37] lack both ATPases. Similar to the lung cancers with poor prognosis that lack both SMARCA4 and SMARCA2[37], all SMARCA4-deficient SCCOHT were also deficient for SMARCA2. In addition, as previously reported for other SMARCA4-mutant tumours with loss of SMARCA2 expression[18-21], we could restore SMARCA2 expression by treatment of SCCOHT cell lines with the HDAC inhibitor TSA. We have found that HDAC inhibitors are potent inducers of SMARCA2 and growth arrest in SCCOHT cell lines (data not shown). Based on the pleotropic effects of HDAC inhibitors on global gene expression, we think it is unlikely that SMARCA2 upregulation is the sole mechanism by which these agents cause growth arrest. We are currently performing shRNA studies against SMARCA2 and with specific HDACs (guided by the known HDAC specificity of the particular HDAC inhibitors) to determine the role SMARCA2 plays in HDAC inhibitor-induced growth arrest and which HDACs mediate the growth inhibitory effect. Therefore, our results strongly support silencing, either transcriptional or post-transcriptional, as the mechanism underlying the absence of SMARCA2 gene expression in these tumours and implicate HDAC inhibitors as potential treatment options.  Among ovarian tumours, dual loss of SMARCA4 and SMARCA2 was only observed in SCCOHT. Ovarian clear cell carcinomas lacked SMARCA4 or SMARCA2 but not both. Similarly, Huang and colleagues recently reported no loss of SMARCA4 in 68 ovarian clear cell carcinomas, with only one tumour showing SMARCA2 loss[38], and although almost all SCCOHT tumour cells lacked SMARCA2, rare SMARCA2-positive tumour cells were identified, and the subcellular localization correlated with cell morphology – exclusively nuclear in small cells and either nuclear or cytoplasmic in large cells. Cytoplasmic SMARCA2 in large cells of SCCOHT is reminiscent of the cytoplasmic SMARCB1 staining observed in some cases of This article is protected by copyright. All rights reserved.Accepted Articleepithelioid sarcoma[39]. These findings suggest that in addition to mutation and gene silencing, SWI/SNF defects may also be due to protein mis-localization. Potential cells of origin  SCCOHT may arise from resident ovarian cells or from a cell type within an immature teratoma[4]. The conspicuous lack of SMARCA2 mutations in three papers that identified the SMARCA4 mutation in SCCOHT by next generation sequencing[2, 3, 5], together with the upregulation of SMARCA2 mRNA and translated protein by the histone deacetylase inhibitor trichostatin A, indicate that the lack of SMARCA2 protein expression is due to either epigenetic or post-transcriptional silencing. The lack of SMARCA2 expression in tumours may reflect either its original absence in the cell of origin or silencing as a pathogenic event during tumorigenesis.  Lentivirus-mediated restoration of wild type SMARCA4 does not result in re-expression of endogenous SMARCA2 protein, whereas HDAC inhibitors do result in SMARCA2 protein expression (data not shown). This differential SMARCA2 protein upregulation is not likely due to differences in the effects of SMARCA4 expression or HDAC inhibitor treatment on the cell cycle since both manipulations result in growth arrest. We speculate that the inability of exogenous SMARCA4 to restore endogenous SMARCA2 expression reflects the origin of SCCOHT from a rare SMARCA2-negative cell.  Molecular pathogenesis The function of the SWI/SNF complex in transcriptional regulation, the absence of other recurrent mutations, and the diploid cytogenetic profile of SCCOHT indicate that the principle mechanism of oncogenesis is epigenetic dysregulation. The pathological features – poorly differentiated and highly proliferative – suggest that the principal defect is the failure to activate genes that promote or maintain terminal differentiation.  This article is protected by copyright. All rights reserved.Accepted ArticleThe role of the SWI/SNF complex in regulating differentiation as a potential mechanism of SCCOHT pathogenesis is also suggested by the uterine tumours with combined loss of both SMARCA4 and SMARCA2 – dedifferentiated carcinoma and high-grade ESS. These tumours are conceptually similar in that they both have low- and high-grade areas separated by an abrupt transition indicating a dedifferentiation event and, like SCCOHT, they both can show small cell or rhabdoid features in the high-grade areas[16, 45].  We propose that dual deficiency in core members of the SWI/SNF complex (e.g. SMARCA4 and SMARCA2 or SMARCB1 and SMARCA2) induces dedifferentiation from a normal cell or a low-grade tumour into an aggressive high-grade tumour with small cell and/or rhabdoid features. Similar to our findings in SCCOHT, malignant rhabdoid tumours also show dual deficiency for core SWI/SNF members: SMARCB1 by mutation and SMARCA2 by non-mutational silencing[48-50]. The shared clinicopathological features of SCCOHT and rhabdoid tumours of the brain and kidney – lethal behaviour, diploid cytogenetics, small cell/rhabdoid histology, and dual loss of core SWI/SNF components (one by mutation, one by silencing) – support the proposal that SCCOHT is a malignant rhabdoid tumour of the ovary, as proposed by Foulkes and colleagues[51]. More importantly, the shared genetics of rhabdoid tumours will hopefully predict their response to drugs that target epigenetic modifications. Our preliminary studies on HDAC inhibitors support this notion. This article is protected by copyright. All rights reserved.Accepted Article Acknowledgements We thank the SCCOHT families and patients for their important contributions to TGen’s institutional review board-approved study. We thank Drs. Ralf Hass and Barbara Vanderhyden for kindly providing SCCOHT-1 and BIN67 cells, respectively. Viable frozen SCCOHT tumour cells used to establish patient-derived xenografts were obtained from Molecular Response (San Diego, CA). This study was supported by grants from the National Institutes of Health (R01 CA195670-01, B.E.W., J.M.T. and D.G.H.), the Terry Fox Research Initiative New Frontiers Program in Cancer (D.G.H.), Canadian Cancer Society Research Initiative (D.G.H. and Y.W.), Terry Fox Foundation Strategic Health Research Training Program in Cancer Research at Canadian Institutes of Health Research (A.N.K.), the Anne Rita Monahan Foundation (P.R.), the Marsha Rivkin Center for Ovarian Cancer Research, the Ovarian Cancer Alliance of Arizona, the Small Cell Ovarian Cancer Foundation, and philanthropic support to the TGen Foundation.  This article is protected by copyright. All rights reserved.Accepted Article Statement of Author contributions  Author study design data/case collection performed experiments data analysis data interpretation literature search generation of figures writing of manuscript editing of manuscript ANK x x x x x x x x x YW x x x x x x x x x PR x x x x x x x x x WPDH x x   x x x x   x EO   x   x x     x x ED   x     x       x JP   x     x       x MRN   x     x       x TON   x     x       x CC     x x         x SL       x         x FK   x               SK   x               AS   x               BMR   x               JTR   x               DDB   x               BEW x x   x x       x JMT x x   x x       x CBG x x   x x       x DGH x x   x x       x   This article is protected by copyright. All rights reserved.Accepted Article   References 1. Young RH, Oliva E, Scully RE. Small cell carcinoma of the ovary, hypercalcemic type: a clinicopathological analysis of 150 cases. Am J of Surg Pathol 1994; 18: 1102-1116. 2. 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Distinct evolutionary trajectories of primary high-grade serous ovarian cancers revealed through spatial mutational profiling. J Pathol 2013; 231: 21-34. 35. Wilson BG, Helming KC, Wang X, et al. Residual complexes containing SMARCA2 (BRM) underlie the oncogenic drive of SMARCA4 (BRG1) mutation. Mol Cell Biol 2014; 34: 1136-1144. 36. Reisman DN, Sciarrotta J, Wang W, et al. Loss of BRG1/BRM in human lung cancer cell lines and primary lung cancers: correlation with poor prognosis. Cancer Res 2003; 63: 560-566. 37. Matsubara D, Kishaba Y, Ishikawa S, et al. Lung cancer with loss of BRG1/BRM, shows epithelial mesenchymal transition phenotype and distinct histologic and genetic features. Cancer Sci 2013; 104: 266-273. 38. Huang HN, Lin MC, Huang WC, et al. Loss of ARID1A expression and its relationship with PI3K-Akt pathway alterations and ZNF217 amplification in ovarian clear cell carcinoma. Mod Pathol 2014; 27: 983-990. 39. Agaimy A. The expanding family of SMARCB1(INI1)-deficient neoplasia: implications of phenotypic, biological, and molecular heterogeneity. Adv Anat Pathol 2014; 21: 394-410. 40. McCluggage WG, Oliva E, Connolly LE, et al. An immunohistochemical analysis of ovarian small cell carcinoma of hypercalcemic type. Int J Gynecol Pathol 2004; 23: 330-336. 41. Kommoss F, Oliva E, Bhan AK, et al. Inhibin expression in ovarian tumors and tumor-like lesions: an immunohistochemical study. Mod Pathol 1998; 11: 656-664. 42. Cannon PM, Smart CR, Wilson ML, et al. Hypercalcemia with ovarian granulosa cell carcinoma. Rocky Mt Med J 1975; 72: 72-74. 43. Ulbright TM, Roth LM, Stehman FB, et al. Poorly differentiated (small cell) carcinoma of the ovary in young women: evidence supporting a germ cell origin. Hum Pathol 1987; 18: 175-184. This article is protected by copyright. All rights reserved.Accepted Article44. Song S, Walter V, Karaca M, et al. Gene silencing associated with SWI/SNF complex loss during NSCLC development. Mol Cancer Res 2014; 12: 560-570. 45. Lee CH, Marino-Enriquez A, Ou W, et al. The clinicopathologic features of YWHAE-FAM22 endometrial stromal sarcomas: a histologically high-grade and clinically aggressive tumor. Am J Surg Pathol 2012; 36: 641-653. 46. Rao Q, Xia QY, Shen Q, et al. Coexistent loss of INI1 and BRG1 expression in a rhabdoid renal cell carcinoma (RCC): implications for a possible role of SWI/SNF complex in the pathogenesis of RCC. Int J Clin Exp Pathol 2014; 7: 1782-1787. 47. Chapman-Fredricks JR, Herrera L, Bracho J, et al. Adult renal cell carcinoma with rhabdoid morphology represents a neoplastic dedifferentiation analogous to sarcomatoid carcinoma. Ann Diagn Pathol 2011; 15: 333-337. 48. Kahali B, Yu J, Marquez SB, et al. The silencing of the SWI/SNF subunit and anticancer gene BRM in Rhabdoid tumors. Oncotarget 2014; 5: 3316-3332. 49. Li L, Fan XS, Xia QY, et al. Concurrent loss of INI1, PBRM1, and BRM expression in epithelioid sarcoma: implications for the cocontributions of multiple SWI/SNF complex members to pathogenesis. Hum Pathol 2014; 45: 2247-2254. 50. Yamamichi N, Yamamichi-Nishina M, Mizutani T, et al. The Brm gene suppressed at the post-transcriptional level in various human cell lines is inducible by transient HDAC inhibitor treatment, which exhibits antioncogenic potential. Oncogene 2005; 24: 5471-5481. 51. Foulkes WD, Clarke BA, Hasselblatt M, et al. No small surprise - small cell carcinoma of the ovary, hypercalcaemic type, is a malignant rhabdoid tumour. J Pathol 2014; 233: 209-214.   This article is protected by copyright. All rights reserved.Accepted Article Table 1. SMARCA4 immunohistochemical analysis in ovarian, uterine and selected non-gynaecological tumours Tumour type n SMARCA4 negative (percent) SCCOHTPrimary tumours 46 42 (91%) Patient-derived xenografts 2 2 (100%) Cell lines 2 2 (100%) Subtotal 50 46 (92%) Ovarian epithelial tumours High-grade serous carcinoma 1198 0 (0%) Clear cell carcinoma 360 15 (4%) Endometrioid adenocarcinoma 258 0 (0%) Endometrioid adenocarcinoma, mixed 9 0 (0%) Endometrioid borderline tumour 1 0 (0%) Low-grade serous carcinoma 53 0 (0%) Serous borderline tumour 23 0 (0%) Mucinous carcinoma 98 0 (0%) Mucinous borderline tumour 6 0 (0%) Mixed mucinous tumour 5 0 (0%) Anaplastic carcinoma in mucinous carcinoma 1 0 (0%) Mixed carcinoma 1 0 (0%) Undifferentiated carcinoma 18 0 (0%) Carcinosarcoma 27 0 (0%)  Benign Brenner tumour 3 0 (0%) Borderline Brenner tumour 1 0 (0%) Malignant Brenner tumour 2 0 (0%)  Small cell carcinoma, pulmonary type 3 0 (0%) Ovarian carcinoma, not otherwise specified 5 0 (0%) Subtotal 2072 15 (1%) Ovarian/adnexal sex cord-stromal tumours Adult granulosa cell tumour 113 0 (0%) Juvenile granulosa cell tumour 29 0 (0%) Fibroma/fibrosarcoma 8 0 (0%) Thecoma 7 0 (0%) Sertoli-Leydig cell tumour 41 0 (0%) Leydig cell tumour 2 0 (0%) Sclerosing stromal tumour 6 0 (0%) Sex cord tumour with annular tubules 2 0 (0%) This article is protected by copyright. All rights reserved.Accepted ArticleSteroid cell tumour 1 0 (0%) Sex cord tumour, not otherwise specified 4 0 (0%) Female adnexal tumour of Wolffian origin 4 0 (0%) Gynandroblastoma 3 0 (0%) Endometrioid stromal sarcoma 1 0 (0%) Subtotal 221 0 (0%) Ovarian germ cell tumoursDysgerminoma 5 0 (0%) Yolk sac tumour 6 0 (0%) Dysgerminoma and yolk sac tumour 1 0 (0%) Mature teratoma 3 0 (0%) Immature teratoma 6 0 (0%) Carcinoid tumour 4 0 (0%) Strumal carcinoid in a mucinous cystadenoma 2 0 (0%) Sebaceous carcinoma in a dermoid cyst 1 0 (0%) Squamous cell carcinoma 3 0 (0%) Subtotal 31 0 (0%) Uterine epithelial tumoursEndometrioid adenocarcinoma 336 1 (0%) Clear cell carcinoma 28 0 (0%) High-grade serous carcinoma 94 0 (0%) Mucinous carcinoma 3 0 (0%) Mixed carcinoma 11 0 (0%) Large cell carcinoma 1 0 (0%) Small cell carcinoma 3 0 (0%) Undifferentiated carcinoma 3 0 (0%) Dedifferentiated carcinoma 2 1 (50%) Carcinosarcoma 65 0 (0%) Subtotal 546 2 (0%) Uterine mesenchymal tumoursLeiomyoma 49 0 (0%) Leiomyosarcoma 21 0 (0%) Endometrial stromal sarcoma 52 4 (8%) Undifferentiated endometrial sarcoma 1 0 (0%) Rhabdomyosarcoma 2 0 (0%) Adenosarcoma 7 0 (0%) Uterine tumour resembling ovarian sex cord tumour 2 0 (0%) Subtotal 134 4 (3%) Non-gynaecological tumoursThis article is protected by copyright. All rights reserved.Accepted ArticleAlveolar rhabdomyosarcoma 10 0 (0%) Embryonal rhabdomyosarcoma 10 0 (0%) Desmoplastic small round cell tumour 2 0 (0%) Ewing’s sarcoma 9 0 (0%) Synovial sarcoma, monophasic 3 0 (0%) Synovial sarcoma, biphasic 2 0 (0%) Melanoma 7 0 (0%) Follicular lymphoma 1 0 (0%) Subtotal 44 0 (0%) Total 3048 21 (1%)  This article is protected by copyright. All rights reserved.Accepted ArticleFigure and Table Legends   Table 1. SMARCA4 immunohistochemical analysis in ovarian, uterine and selected non-gynaecological tumours.  This article is protected by copyright. All rights reserved.Accepted Article Figure 1. Immunohistochemical analysis of core SWI/SNF proteins in SCCOHT. (A) Dual loss of SMARCA4/BRG1 and SMARCA2/BRM in SCCOHT. Endothelium and lymphocytes are internal positive controls for both proteins. Arrows denote rare tumour cells expressing SMARCA2. SMARCB1/INI1 protein expression serves as a positive control for tumour cell immunoreactivity. This article is protected by copyright. All rights reserved.Accepted Article(B) SMARCA2/BRM expression and subcellular localization in regions of small cell vs. large cell morphology. When present, SMARCA2 only showed nuclear localization (arrows, Figure 1A and 1B). In contrast, scattered tumour cells with large cell morphology also expressed SMARCA2 in the cytoplasm (arrowheads). (C) SMARCA4/BRG1 and SMARCA2/BRM expression in normal ovary from premenopausal women. SMARCA4 was expressed strongly in oocytes and granulosa cells but was either weak or absent in stromal cells. In contrast, SMARCA2 showed diffuse and strong expression in oocytes, granulosa cells and stromal cells. In addition, both proteins showed uniform expression in ovarian surface epithelium (data not shown). H&E and SMARCA4 immunohistochemistry images are reproduced from our previous publication, Ramos et al.[2] (Supplementary Figure 4, panels A-D). This article is protected by copyright. All rights reserved.Accepted Article Figure 2. Immunohistochemical analysis of SMARCA4/BRG1 and SMARCA2/BRM in ovarian clear cell carcinoma. Tumours were negative for either SMARCA4 (middle panels) or SMARCA2 (bottom panels) or showed intact expression of both proteins (upper panels). No dual deficient tumours were identified. Similar to SCCOHT, rare scattered SMARCA2-positive cells (arrows) were identified in some SMARCA2-negative tumours. This article is protected by copyright. All rights reserved.Accepted Article   Figure 3. Immunohistochemical analysis of SMARCA4/BRG1 and SMARCA2/BRM in endometrial stromal sarcoma (ESS) and dedifferentiated carcinoma (DDC) of the uterus. Three uterine tumour types showed SMARCA4/BRG1-deficiency: low grade ESS (LGESS, n=2, upper This article is protected by copyright. All rights reserved.Accepted Articlepanels), high grade ESS (HGESS, n=2, middle panels) and DDC (n=1, lower panels). Both cases of SMARCA4-negative LGESS maintained expression of SMARCA2. In contrast, both HGESS and the DDC that lacked SMARCA4 were also deficient in SMARCA2.   Figure 4. SMARCA2 silencing in SCCOHT tumours and cell lines and reactivation by the histone deacetylase inhibitor trichostatin A. (A) Heat map depicting the expression of genes encoding SWI/SNF family members in SCCOHT tumours (SCCO-002, -012, -014 and -015), patient-derived mouse xenografts (PDX-040 and -065) and SCCOHT cell lines (BIN67 and SCCOHT1). SMARCA2 mRNA is strongly downregulated in contrast to almost all other This article is protected by copyright. All rights reserved.Accepted ArticleSWI/SNF genes. Premenopausal ovaries were used as a reference. (B) Real time PCR of steady state SMARCA4 and SMARCA2 mRNA levels in SCCOHT cell lines (BIN67 and SCCOHT1) compared to other ovarian cell lines. SVOG3e, immortalized granulosa cells. KGN, adult granulosa cell line. NOY1, yolk sac tumour cell line. Levels of mRNA were normalized to GAPDH. (C) Western blotting for SMARCA4 and SMARCA2 in SCCOHT cell lines. Vinculin served as loading control. Both SCCOHT cell lines expressed SMARCB1/INI1 (data not shown), similar to primary tumours (Figure 1A). (D) Specific upregulation of SMARCA2/BRM protein by the histone deacetylase inhibitor trichostatin A (TSA). BIN67 cells were treated for 72 h with TSA, GSK343, trametinib or 5-aza-cytidine, and SMARCA2 expression was analyzed by Western blotting. Ctrl denotes vehicle treatment. SMARCA2 (lane 2) denotes cells transduced with lentivirus expressing SMARCA2. KGN, adult granulosa cell tumour cells. Ponceau S staining served as a loading control. (E) Real time PCR of SMARCA4 and SMARCA2 mRNA levels in BIN67 cells after 72 h of treatment with 0.6 μM TSA. Although absolute baseline SMARCA2 mRNA levels are significantly lower than SMARCA4 (Figure 4B), both are arbitrarily set to 1 in this experiment. SMARCA2 mRNA levels are strongly upregulated by TSA compared to SMARCA4. (F) SMARCA2 protein upregulation is TSA dose-dependent in SCCOHT cell lines. BIN67 and SCCOHT1 cells were treated with increasing doses of TSA for 72 h, and SMARCA2 expression was analyzed by Western blotting. Ctrl denotes vehicle treatment. SMARCA2 (lane 2 for each cell line) denotes cells transduced with lentivirus expressing SMARCA2. ES-2, ovarian endometrioid adenocarcinoma cells. Ponceau S staining served as a loading control.     This article is protected by copyright. All rights reserved.Accepted Article   Figure 5. Re-expression of SMARCA4 or SMARCA2 suppresses SCCOHT cell growth. (A) BIN67 cells were transduced with lentiviruses expressing GFP, SMARCA4 or SMARCA2 and This article is protected by copyright. All rights reserved.Accepted Articleseeded at a density of 4000 cells/well in 96-well plates for monitoring cell growth using Incucyte Zoom live cell monitor. The percentage of confluence in each well was calculated and plotted to determine the effect of SMARCA4 or SMARCA2 re-expression on cell growth. (B) Re-expression of SMARCA4 or SMARCA2 was confirmed by Western blotting. Ponceau S staining served as a loading control.   This article is protected by copyright. All rights reserved.

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