Karnezis, Anthony N.

This cohort study evaluates the reduction in risk of high-grade serous carcinoma among women undergoing opportunistic bilateral salpingectomy in Canada.

Abstract Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, deadly form of ovarian cancer that uniformly harbors mutations in SMARCA4, a member of the SWI/SNF chromatin remodeling complex. SWI/SNF impacts RNA splicing, and dysregulation of splicing can generate immunogenic tumor antigens. Here, we explored the relationship between SMARCA4 loss and RNA splicing dysregulation. SCCOHT primary tumors harbored tumor-associated outlier splicing events compared to normal tissues. Many of the tumor events were retained introns encoding novel peptides predicted to bind to MHC-I complexes. Immune cells were observed in primary SCCOHT tumors, suggesting a potentially immune reactive tumor microenvironment. Mutations in several SWI/SNF subunits were associated with higher rates of outlier retained introns across tumor types in TCGA data. Interestingly, RNA sequencing of isogenic SCCOHT cell lines demonstrated a role for SMARCA4 in intron retention. Distinct protein-protein interactions between splicing factors identified in SCCOHT cell lines supported a role for SMARCA4 in splicing regulation. Further, SWI/SNF localized to genes which were differentially spliced. Mass spectrometry analyses confirmed expression of some of these novel peptides and a subset of these are predicted to bind to MHC-I complexes. A pool of these novel peptides derived from retained introns in SCCOHT triggered proliferation and expression of TNFa and INFb in primary human T cells. Together, these data suggest that SMARCA4 loss in SCCOHT leads to intron retention. Furthermore, T cell activation by novel peptides encoded by these tumor-specific splicing events suggests intron retention could be a source of tumor-associated antigens in SCCOHT. Citation Format: Elizabeth Raupach, Apurva Hegde, Krystine Garcia-Masnfield, Marice Alcantara, David Rose, Rebecca Halperin, Krystal Orlando, Jessica Lang, Ritin Sharma, Victoria David-Dirgo, Salvatore Facista, Rayvon Moore, Rochelle Kofman, Zoe Jensen, Victoria Zismann, Anthony Karnezis, Yemin Wang, Lynda Bennett, Timothy Whitsett, Marcin Kortylewski, William Hendricks, David Huntsman, Lorna Rodriguez-Rodriguez, Bernard Weissman, Jeffrey Trent, Patrick Pirrotte. Loss of SMARCA4 leads to intron retention and generation of tumor-associated antigens in small cell carcinoma of the ovary, hypercalcemic type [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr B065.

Abstract Introduction: The purpose of this study is to evaluate the effectiveness of opportunistic salpingectomy (OS), a procedure that aims to remove only the fallopian tubes of general population risk women, for the prevention of epithelial ovarian cancer. Currently, the method of prevention for individuals at high risk of ovarian cancer involves a bilateral salpingo-oophorectomy (BSO), a procedure that removes both the fallopian tubes and ovaries. However, this procedure is not recommended for individuals of the general population, who make up about 80% of ovarian cancers, due to negative health implications to the cardiovascular and skeletal systems because of surgically induced menopause. The effectiveness of the OS in preventing ovarian cancers and how the procedure impacts the histotype distribution of ovarian cancers remains unknown. Study Methods: This study uses a population-based approach and a case-based approach to determine the effectiveness of OS and its impact on histotype distribution, respectively. For the population-based analysis, we will search through clinical databases to acquire cases of individuals who have had an OS and a tumor. A control population who have their fallopian tubes in-tact will also be acquired. A cox-proportional hazard analysis will be performed to determine the effectiveness of the procedure. Using pathological records, the case-based ascertainment of tumors from individuals who have had an OS will be selected and reviewed for histotype confirmation. Fallopian tubes of these patients will also be reviewed for precursor lesions. This cohort will be compared to a historical histotype distribution of ovarian cancers. Results: For the population-based approach, we have to date, identified less than five serous ovarian cancers in individuals who have had an OS (n=40, 477). A comparator surgery was used as a control and showed 21 cancers. For the case-based approach, we have partnered with 23 collaborating institutions from around the world to identify tumors in individuals who have had an OS. We have identified 26 epithelial ovarian cancers, with the most lethal gynecological histotype, high grade serous carcinoma (HGSC), accounting for 23.1% of the total proportion of epithelial ovarian cancer cases (n=6/26). This is significantly less than the historical histotype distribution of HGSC, which is 69.3% (Fisher’s exact test, p<0.0001). One HGSC case had a precursor lesion called a serous tubal intraepithelial carcinoma (STIC) while reviewing fallopian tubes of this individual. TP53 mutation analysis on one HGSC tumor sample showed a frameshift mutation in TP53 (c229fs) with the tumor and the corresponding STIC staining negative for p53 protein expression by immunohistochemistry. Conclusion: This evidence demonstrates the effectiveness of opportunistic salpingectomy for the prevention of ovarian cancer and shows a histotype distribution shift, with the most lethal gynecological malignancy having a significant reduction in cases following the procedure. Citation Format: Ramlogan Sowamber, Alice J. Mei, Paramdeep Kaur, Julianne McLeod, Emily McKay, Alex Lukey, Jamie Bakkum-Gamez, Natalia Buza, Paul Cohen, Kyle Devins, Rhonda Farrell, Christine Garcia, Blake Gilks, Ellen Goode, Anjelica Hodgson, Brooke Howitt, Pei Hui, Jutta Huvila, Anthony Karnezis, Kianoosh Keyhanian, Mary Kinloch, Martin Köbel, Felix KF. Kommoss, Lawrence Kushi, Janice S. Kwon, Kara Long-Roche, Anais Malpica, Jessica N. McAlpine, Dianne Miller, Esther Oliva, Andrea Palicelli, Aleksandra Paliga, Carlos Parra-Herran, Celeste Leigh Pearce, Sharnel Perera, Jurgen M. Piek, Haiyan Qiu, Joseph Rabban, Robert Rome, Miranda Steenbeek, Rebecca Stone, Aline Talhouk, Kristin M. Tischer, Britton Trabert, Penelope M. Webb, John R. Zalcberg, Gillian E. Hanley, David G. Huntsman. Evaluating the effectiveness of opportunistic salpingectomy (OS) for the prevention of epithelial ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr B045.

BACKGROUND Despite an overall poor prognosis, about 15% of patients with advanced-stage tubo-ovarian high-grade serous carcinoma (HGSC) survive 10 or more years after standard treatment. METHODS We evaluated the tumor microenvironment of this exceptional, understudied group using a large international cohort enriched for long-term survivors (LTS; 10+ years; n = 374) compared with mid-term (MTS; 5–7.99 years; n = 433) and short-term survivors (STS; 2–4.99 years; n = 416). Primary tumor samples were immunostained and scored for intraepithelial and intrastromal densities of 10 immune-cell subsets (including T cells, B cells, plasma cells, myeloid cells, PD-1 + cells, and PD-L1 + cells) and epithelial content. RESULTS Positive associations with LTS compared with STS were seen for 9 of 10 immune-cell subsets. In particular, the combination of intraepithelial CD8 + T cells and intrastromal B cells showed near 5-fold increased odds of LTS compared with STS. All of these associations were stronger in tumors with high epithelial content and/or the C4/Differentiated molecular subtype, despite immune-cell densities generally being higher in tumors with low epithelial content and/or the C2/Immunoreactive molecular subtype. CONCLUSION The tumor microenvironment of HGSC LTS is distinguished by the intersection of T and B cell coinfiltration, high epithelial content, and C4/differentiated molecular subtype, features which may inspire new approaches to immunotherapy. FUNDING Ovarian Cancer Research Program (OCRP) of the Congressionally Directed Medical Research Program (CDMRP), U.S. Department of Defense (DOD); American Cancer Society; BC Cancer Foundation; Canada’s Networks of Centres of Excellence; Canadian Cancer Society; Canadian Institutes of Health Research; Cancer Councils of New South Wales, Victoria, Queensland, South Australia, and Tasmania, Cancer Foundation of Western Australia; Cancer Institute NSW; Cancer Research UK; Deutsche Forschungsgesellschaft; ELAN Funds of the University of Erlangen-Nuremberg; Fred C. and Katherine B. Andersen Foundation; Genome BC; German Cancer Research Center; German Federal Ministry of Education and Research, Programme of Clinical Biomedical Research; Instituto de Salud Carlos III; Mayo Foundation; Minnesota Ovarian Cancer Alliance; Ministerio de Economía y Competitividad; Medical Research Council (MRC); National Center for Advancing Translational Sciences; National Health and Medical Research Council of Australia (NHMRC); Ovarian Cancer Australia; Peter MacCallum Foundation; Sydney West Translational Cancer Research Centre; Terry Fox Research Institute; The Eve Appeal (The Oak Foundation); UK National Institute for Health Research Biomedical Research Centres at the University of Cambridge; University of Pittsburgh School of Medicine; U.S. National Cancer Institute of the National Institutes of Health; VGH & UBC Hospital Foundation; Victorian Cancer Agency.

Abstract Purpose: Endometrioid ovarian carcinoma (ENOC) is the second most-common type of ovarian carcinoma, comprising 10%–20% of cases. Recently, the study of ENOC has benefitted from comparisons to endometrial carcinomas including defining ENOC with four prognostic molecular subtypes. Each subtype suggests differential mechanisms of progression, although tumor-initiating events remain elusive. There is evidence that the ovarian microenvironment may be critical to early lesion establishment and progression. However, while immune infiltrates have been well studied in high-grade serous ovarian carcinoma, studies in ENOC are limited. Experimental Design: We report on 210 ENOC, with clinical follow-up and molecular subtype annotation. Using multiplex IHC and immunofluorescence, we examine the prevalence of T-cell lineage, B-cell lineage, macrophages, and populations with programmed cell death protein 1 or programmed death-ligand 1 across subtypes of ENOC. Results: Immune cell infiltrates in tumor epithelium and stroma showed higher densities in ENOC subtypes with known high mutation burden (POLEmut and MMRd). While molecular subtypes were prognostically significant, immune infiltrates were not (overall survival P > 0.2). Analysis by molecular subtype revealed that immune cell density was prognostically significant in only the no specific molecular profile (NSMP) subtype, where immune infiltrates lacking B cells (TILB minus) had inferior outcome (disease-specific survival: HR, 4.0; 95% confidence interval, 1.1–14.7; P < 0.05). Similar to endometrial carcinomas, molecular subtype stratification was generally superior to immune response in predicting outcomes. Conclusions: Subtype stratification is critical for better understanding of ENOC, in particular the distribution and prognostic significance of immune cell infiltrates. The role of B cells in the immune response within NSMP tumors warrants further study.

Abstract Background: Accumulating evidence suggests a relationship between endometrial cancer and ovarian cancer. Independent genome-wide association studies (GWAS) for endometrial cancer and ovarian cancer have identified 16 and 27 risk regions, respectively, four of which overlap between the two cancers. We aimed to identify joint endometrial and ovarian cancer risk loci by performing a meta-analysis of GWAS summary statistics from these two cancers. Methods: Using LDScore regression, we explored the genetic correlation between endometrial cancer and ovarian cancer. To identify loci associated with the risk of both cancers, we implemented a pipeline of statistical genetic analyses (i.e., inverse-variance meta-analysis, colocalization, and M-values) and performed analyses stratified by subtype. Candidate target genes were then prioritized using functional genomic data. Results: Genetic correlation analysis revealed significant genetic correlation between the two cancers (rG = 0.43, P = 2.66 × 10−5). We found seven loci associated with risk for both cancers (PBonferroni < 2.4 × 10−9). In addition, four novel subgenome-wide regions at 7p22.2, 7q22.1, 9p12, and 11q13.3 were identified (P < 5 × 10−7). Promoter-associated HiChIP chromatin loops from immortalized endometrium and ovarian cell lines and expression quantitative trait loci data highlighted candidate target genes for further investigation. Conclusions: Using cross-cancer GWAS meta-analysis, we have identified several joint endometrial and ovarian cancer risk loci and candidate target genes for future functional analysis. Impact: Our research highlights the shared genetic relationship between endometrial cancer and ovarian cancer. Further studies in larger sample sets are required to confirm our findings.

Abstract Purpose: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by loss of the SWI/SNF ATPase subunits SMARCA4 and SMARCA2. A great need exists for effective targeted therapies for SCCOHT. Experimental Design: To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two patient-derived xenograft (PDX) models and one cell-line xenograft model of SCCOHT. Results: The receptor tyrosine kinase (RTK) family was enriched in siRNA screen hits, with FGFRs and PDGFRs being overlapping hits between drug and siRNA screens. Of multiple potent drug classes in SCCOHT cell lines, RTK inhibitors were only one of two classes with selectivity in SCCOHT relative to three SWI/SNF wild-type ovarian cancer cell lines. We further identified ponatinib as the most effective clinically approved RTK inhibitor. Reexpression of SMARCA4 was shown to confer a 1.7-fold increase in resistance to ponatinib. Subsequent proteomic assessment of ponatinib target modulation in SCCOHT cell models confirmed inhibition of nine known ponatinib target kinases alongside 77 noncanonical ponatinib targets in SCCOHT. Finally, ponatinib delayed tumor doubling time 4-fold in SCCOHT-1 xenografts while reducing final tumor volumes in SCCOHT PDX models by 58.6% and 42.5%. Conclusions: Ponatinib is an effective agent for SMARCA4-mutant SCCOHT in both in vitro and in vivo preclinical models through its inhibition of multiple kinases. Clinical investigation of this FDA-approved oncology drug in SCCOHT is warranted. Clin Cancer Res; 24(8); 1932–43. ©2018 AACR.

Abstract Introduction: Small cell carcinoma of the ovary of hypercalcemic type (SCCOHT) is arguably the most aggressive ovarian cancer. Most patients are diagnosed at an advanced stage, do not respond to chemotherapy, and die of disease within 1-2 years. It affects children and young women and is reported to occur in families. The cause of the disease is poorly understood. Therefore, we used next generation sequencing technology to identify the genetic basis of the disease. Method: We performed whole genome, whole exome sequencing or targeted sequencing on tumours and germline samples from 17 SCCOHT patients and on the SCCOHT cell line BIN-67 and SCCOHT-1. Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded tumors from 23 patients and on a tissue microarray of 485 primary ovarian tumours of other subtypes. BIN-67 cells harbouring biallelic inactivation of SMARCA4 were transduced with a lentivirus expressing wild type SMARCA4. Result: We identified inactivating germline and somatic mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 79% (11/14) of SCCOHT patients, 2 of whom bore germline mutations, and in both cell lines. SMARCA4 protein was lost in 87% (20/23) of SCCOHT tumours but in only 0.4% (2/485) of other primary ovarian tumours. Reintroduction of wild-type SMARCA4 into BIN-67 cells resulted in altered cell morphology and growth arrest. Conclusions: The mutation spectrum, IHC profile and cell culture phenotype implicate SMARCA4 as a critical tumour suppressor in SCCOHT pathogenesis. Citation Format: Pilar Ramos, Anthony N. Karnezis, David W. Craig, Aleksandar Sekulic, Megan L. Russell, William P.D. Hendricks, Jason J. Corneveaux, Michael T. Barrett, Karey Shumansky, Yidong Yang, Sohrab P. Shah, Leah M. Prentice, Marco A. Marra, Jeffrey Kiefer, Victoria L. Zismann, Troy A. McEachron, Bodour Salhia, Jaime Prat, Blaise A. Clarke, Joseph G. Pressey, John H. Farley, Stephen P. Anthony, Richard B.S. Roden, Heather E. Cunliffe, David G. Huntsman, Jeffrey M. Trent. Small cell carcinoma of the ovary, hypercalcemic type displays frequent inactivating germline and somatic mutations in SMARCA4 [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1327.

Abstract Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) is a rare and highly aggressive malignancy that affects children and young women at a mean age of 24 (range 14 months - 58 years). SCCOHT is refractory to standard of care therapy for ovarian cancer, with ∼75% mortality within 18 months of diagnosis. The early age of onset of SCCOHT and reports of familial occurrence in some cases, strongly suggest an underlying hereditary etiology. To understand the molecular pathogenesis of SCCOHT, we performed next-generation genomic sequencing on a series of tumor and germline samples from SCCOHT patients. This analysis revealed germline and somatic inactivating mutations in SMARCA4, a subunit of the SWI/SNF chromatin-remodeling complex, in 75% (9/12) of SCCOHT patients. Moreover, immunohistochemical (IHC) analysis of 15 tumors revealed that 87% (13/15) of tumors lacked SMARCA4 protein. The high prevalence of SMARCA4 mutations in SCCOHT has not been previously reported in other, more common ovarian carcinomas. We therefore examined the expression of SMARCA4 protein in 300 ovarian carcinomas of different histologies by IHC and found SMARCA4 protein loss in only 6 tumors. In addition, the BIN-67 SCCOHT cell line, which harbors 2 splice site mutations in SMARCA4, showed complete absence of SMARCA4 protein by Western blot while representative cell lines from 4 other ovarian carcinoma subtypes as well as immortalized granulosa cells (SVOG) and adult granulosa tumor cells (KGN) all maintained SMARCA4 expression. The prevalence of germline and sporadic SMARCA4 mutations as well as frequent SMARCA4 protein loss in SCCOHTs implicates this gene as a tumor suppressor in this cancer and more broadly suggests a role for the SWI/SNF complex in its pathogenesis. In addition to providing evidence to the pathogenesis of SCCOHT, this finding provides the opportunity to develop treatment approaches for SCCOHT based on targeting vulnerabilities of SMARCA4-deficient cells. Citation Format: Pilar Ramos, Anthony Karnezis, David Craig, Aleksandar Sekulic, Megan Russell, William Hendricks, Michael Barrett, Karey Shumansky, Yidong Yang, Sohrab Shah, Leah Prentice, Marco Marra, Jeffrey Kiefer, Victoria Zismann, Troy McEachron, Bodour Salhia, Joseph Pressey, John Farley, Stephen Anthony, Richard Roden, Heather Cunliffe, David Huntsman, Jeffrey Trent. The rare, highly malignant small cell carcinoma of the ovary displays common inactivating germline and somatic mutations in the tumor suppressor SMARCA4. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-202. doi:10.1158/1538-7445.AM2014-LB-202