DeFazio, Anna

Abstract A majority of patients with high-grade serous ovarian cancer (HGSC) develop progressive disease following primary treatment, with a five-year survival rate of approximately 40%, and less than 10% survive more than 10 years. Profound genomic instability, treatment with DNA-damaging drugs, and a large tumor burden promote the development of acquired resistance in a large proportion of patients, including multiple convergent resistant events within individuals. Despite the propensity of HGSC to develop drug resistance, a small proportion of patients with advanced disease at diagnosis become long-term survivors. These exceptional patients include those who had suboptimal surgical clearance, and therefore were not cured surgically, and others who have had no evidence of disease recurrence following initial surgery and adjuvant chemotherapy. Our study, which is part of the international collaborative Multidisciplinary Ovarian Cancer Outcomes Group (MOCOG), seeks to identify molecular, immunologic, and epidemiologic factors that influence long-term survival in HGSC. This presentation describes our genomic and immunologic analysis of exceptional survival of this deadly disease. Patient characteristics and clinical histories were evaluated to identify patients diagnosed with advanced stage (Stage IIIC/IV) and histopathologically confirmed HGSC with greater than 10-year overall survival. Whole-genome sequencing (WGS) was performed on primary tumors (median 78x coverage) and germline samples (median 39x coverage) of 55 long-term survivors. Primary tumor samples were also characterized by RNA sequencing, DNA methylation profiling, and immunohistochemistry. Thirty-eight (69%) of long-term surviving patients had residual disease following surgery, suggestive of highly chemosensitive disease. Most patients (41, 75%) were alive at last follow-up and 26 (47%) were progression free. Somatic mutation burden was higher in primary tumors of long-term survivors relative to controls. Genome-wide mutational signatures were predominantly Signature 3 (associated with homologous recombination deficiency), Signature 1 (age related), and Signatures 5, 8, and 16 (unknown etiology). Inactivation of the tumor suppressor RB1 by structural rearrangements or homozygous deletion was frequent in long-term survivors, with 33% of tumors showing associated loss of RB1 protein expression by immunohistochemistry compared to 13% of unselected HGSC controls (n=207; P = 0.001). In an independent HGSC cohort (n=847), RB1 protein loss was associated with prolonged survival (HR: 0.75, P < 0.001) compared to patients with RB1-positive tumors. Furthermore, co-occurrence of germline mutations in BRCA1 or BRCA2 and RB1 loss was associated with a significantly longer overall survival compared to patients with retained RB1 protein expression and no germline BRCA mutation (HR: 0.44, P < 0.001). Multiplexed immunohistochemical analysis with immune cell panels demonstrated distinct associations with long-term survivors. This study delineates the full landscape of genomic alterations in HGSC of long-term survivors. Our findings indicate that specific mutations are associated with enhanced host immune responses and long-term survival. Citation Format: Dale Garsed, Ahwan Pandey, Sian Fereday, Kathryn Alsop, Maartje Wouters, Flurina Saner, Catherine Kennedy, Celeste Pearce, Malcolm Pike, Susan Ramus, Martin Kobel, Anna deFazio, Ellen Goode, Brad Nelson, David Bowtell. Molecular analysis of exceptional response in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA05.

Abstract Improving risk prediction and prevention strategies through identifying new susceptibility genes for non-high-grade serous ovarian cancer (non-HGS) would represent an important advance in reducing incidence and mortality. Epithelial ovarian cancer (EOC) has five main histotypes that have distinct pathologies, molecular changes, clinical characteristics, and tissues of origin. They are classified as high-grade serous (HGS), low-grade serous (LGS), clear-cell (CCC), endometrioid (END), and mucinous (MUC). A family history of breast or ovarian cancer is the strongest single risk factor for EOC. Germline mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2 confer EOC risks of 44% and 17% by age 80, respectively. Only 40% of the excess familial risk is known, suggesting there are many other susceptibility genes yet unidentified. The non-HGS histotypes are poorly studied due to limited sample size within individual studies. Thus, families of cases with non-HGS tumors have few risk prediction options. Whole-exome sequencing (WES) analysis was performed on 251 non-HGS cases (56 LGS, 55 CCC, 117 END, 23 MUC). Cases were screened negative for BRCA1/2 mutations and selected for a family history of ovarian or breast cancer, or young onset (<45 years) of ovarian cancer. WES identified 1,278 genes with rare predicted truncating mutation in at least one case. Predicted truncating mutations in cases were compared to 171,584 controls from the Exome Aggregation Consortium (ExAC) and the Genome Aggregation Database (gnomAD). Gene-set enrichment analysis showed enrichment for genes involved in the DNA repair pathway (p=3.08 × 10−7). Twenty-five candidate genes (including 4 DNA repair genes) were selected for validation by targeted sequencing. Five genes important in HGS (BRIP1, FANCM, PALB2, RAD51C, RAD51D) as well as two additional candidates (XRCC2 and XRCC3) were also sequenced. Targeted sequencing was performed on 1,779 cases (669 END, 356 CCC, 327 LGS, 427 MUC) and 1,863 controls from studies in the Ovarian Cancer Association Consortium (OCAC). Library preparation and target enrichment was performed using the Fluidigm Juno System and Illumina sequencing was performed on a NovaSeq 6000 Sequencing System. A case-control analysis of predicted truncating variants in the 32 genes was performed. Using a minimum alternate allele frequency of 30%, we identified a higher frequency of mutations in non-HGS cases than controls in ERCC6 (p=0.034) and IL31RA (p=0.034). ERCC6 is involved in DNA repair and IL31RA is a cytokine receptor. Sanger sequencing validation of variants to confirm these results is ongoing. Several other genes showed suggestive higher frequencies of mutations in cases than controls. A larger case control analysis will be performed to confirm those findings. Identifying novel susceptibility genes for non-HGS may have clinical impact by reducing disease-associated mortality through improving risk prediction, identifying prevention strategies, and developing new targeted treatments. Citation Format: Marina Pavanello, Ed Dicks, Honglin Song, Amir Ariff, Adelyn Bolithon, Maria P. Intermaggio, Mark Pinese, Kirsten Moysich, Kunle O. Odunsi, Ellen Goode, David D. Bowtell, Peter Fasching, Jennifer A. Doherty, Francesmary Modugno, Susanne K. Kjær, Penelope M. Webb, Anna Wu, Anna deFazio, Ovarian Cancer Association Consortium, Paul James, Deepak Subramanian, Ian Campbell, Simon A. Gayther, Paul D.P. Pharoah, Susan J. Ramus. Germline mutations in new susceptibility genes for non-high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B37.

Abstract Purpose: Gene expression–based molecular subtypes of high-grade serous tubo-ovarian cancer (HGSOC), demonstrated across multiple studies, may provide improved stratification for molecularly targeted trials. However, evaluation of clinical utility has been hindered by nonstandardized methods, which are not applicable in a clinical setting. We sought to generate a clinical grade minimal gene set assay for classification of individual tumor specimens into HGSOC subtypes and confirm previously reported subtype-associated features. Experimental Design: Adopting two independent approaches, we derived and internally validated algorithms for subtype prediction using published gene expression data from 1,650 tumors. We applied resulting models to NanoString data on 3,829 HGSOCs from the Ovarian Tumor Tissue Analysis consortium. We further developed, confirmed, and validated a reduced, minimal gene set predictor, with methods suitable for a single-patient setting. Results: Gene expression data were used to derive the predictor of high-grade serous ovarian carcinoma molecular subtype (PrOTYPE) assay. We established a de facto standard as a consensus of two parallel approaches. PrOTYPE subtypes are significantly associated with age, stage, residual disease, tumor-infiltrating lymphocytes, and outcome. The locked-down clinical grade PrOTYPE test includes a model with 55 genes that predicted gene expression subtype with >95% accuracy that was maintained in all analytic and biological validations. Conclusions: We validated the PrOTYPE assay following the Institute of Medicine guidelines for the development of omics-based tests. This fully defined and locked-down clinical grade assay will enable trial design with molecular subtype stratification and allow for objective assessment of the predictive value of HGSOC molecular subtypes in precision medicine applications. See related commentary by McMullen et al., p. 5271

Purpose The known epithelial ovarian cancer (EOC) susceptibility genes account for less than 50% of the heritable risk of ovarian cancer suggesting that other susceptibility genes exist. The aim of this study was to evaluate the contribution to ovarian cancer susceptibility of rare deleterious germline variants in a set of candidate genes. Methods We sequenced the coding region of 54 candidate genes in 6385 invasive EOC cases and 6115 controls of broad European ancestry. Genes with an increased frequency of putative deleterious variants in cases versus controls were further examined in an independent set of 14 135 EOC cases and 28 655 controls from the Ovarian Cancer Association Consortium and the UK Biobank. For each gene, we estimated the EOC risks and evaluated associations between germline variant status and clinical characteristics. Results The ORs associated for high-grade serous ovarian cancer were 3.01 for PALB2 (95% CI 1.59 to 5.68; p=0.00068), 1.99 for POLK (95% CI 1.15 to 3.43; p=0.014) and 4.07 for SLX4 (95% CI 1.34 to 12.4; p=0.013). Deleterious mutations in FBXO10 were associated with a reduced risk of disease (OR 0.27, 95% CI 0.07 to 1.00, p=0.049). However, based on the Bayes false discovery probability, only the association for PALB2 in high-grade serous ovarian cancer is likely to represent a true positive. Conclusions We have found strong evidence that carriers of PALB2 deleterious mutations are at increased risk of high-grade serous ovarian cancer. Whether the magnitude of risk is sufficiently high to warrant the inclusion of PALB2 in cancer gene panels for ovarian cancer risk testing is unclear; much larger sample sizes will be needed to provide sufficiently precise estimates for clinical counselling.

Abstract Purpose: The majority of patients with high-grade serous ovarian cancer (HGSC) develop progressive disease following primary treatment, with a five-year survival rate of ~30%. However, a subset of patients have an extraordinary response to treatment and ~15% survive more than ten years (long-term survivors). The Multidisciplinary Ovarian Cancer Outcomes Group (MOCOG) aims to uncover factors that influence long-term survival of HGSC patients. Here, we investigated the genomic and immunologic determinants of exceptional survival of this deadly disease. Experimental Design: Patient characteristics and clinical histories were evaluated to identify patients diagnosed with advanced stage (Stage IIIC/IV) and histopathologically confirmed HGSC with greater than 10-year overall survival. Whole-genome sequencing (WGS) was performed on primary tumors (median 78x coverage) and germline samples (median 39x coverage) of 55 long-term survivors. Primary tumor samples were also characterised by RNA sequencing, DNA methylation profiling and immunohistochemistry. Results: A total 38 (69%) of long-term surviving patients had residual disease following surgery, suggestive of highly chemo-sensitive disease. Most patients (41, 75%) were alive at last follow-up and 26 (47%) were progression-free. Somatic mutation burden was higher in primary tumors of long-term survivors relative to controls (316 unselected HGSC patients in The Cancer Genome Atlas). Genome-wide mutational signatures were predominantly Signature 3 (associated with homologous recombination deficiency), Signature 1 (age related) and Signatures 5, 8 and 16 (unknown etiology). Inactivation of the tumor suppressor RB1 by structural rearrangements or homozygous deletion was frequent in long-term survivors, with 33% of tumors showing associated loss of RB1 protein expression by immunohistochemistry compared to 13% of unselected HGSC controls (n=207; P = 0.001). Staining of adjacent tumor tissue revealed that RB1 loss was associated with increased numbers of PD-1+ tumor-infiltrating lymphocytes (P = 0.015) and MHC class I on tumor cells (P = 0.002). In an independent HGSC cohort (n=847), RB1 protein loss was associated with prolonged survival (HR: 0.75, P < 0.001) compared to patients with RB1 positive tumors. Furthermore, co-occurrence of germline mutations in BRCA1 or BRCA2 and RB1 loss was associated with a significantly longer overall survival compared to patients with retained RB1 protein expression and no germline BRCA mutation (HR: 0.44, P < 0.001). Conclusions: This study delineates the full landscape of genomic alterations in HGSC of long-term survivors. Our findings indicate that specific mutations might be associated with enhanced host immune responses and long-term survival. Citation Format: Dale W. Garsed, Ahwan Pandey, Sian Fereday, Kathryn Alsop, Maartje C. Wouters, Flurina Saner, Jessica A. Beach, Katy Milne, Catherine J. Kennedy, Joy Hendley, Nadia Traficante, Celeste L. Pearce, Malcolm C. Pike, Multidisciplinary Ovarian Cancer Outcomes Group, Susan J. Ramus, Martin Köbel, Brad H. Nelson, Ellen L. Goode, Anna deFazio, David D. Bowtell. The genomic landscape of high-grade serous ovarian cancer in long-term survivors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2722.

AbstractAn association between genetic variants in the vitamin D receptor (VDR) gene and epithelial ovarian cancer (EOC) was previously reported in women of African ancestry (AA). We sought to examine associations between genetic variants in VDR and additional genes from vitamin D biosynthesis and pathway targets (EGFR, UGT1A, UGT2A1/2, UGT2B, CYP3A4/5, CYP2R1, CYP27B1, CYP24A1, CYP11A1, and GC). Genotyping was performed using the custom‐designed 533,631 SNP Illumina OncoArray with imputation to the 1,000 Genomes Phase 3 v5 reference set in 755 EOC cases, including 537 high‐grade serous (HGSOC), and 1,235 controls. All subjects are of African ancestry (AA). Logistic regression was performed to estimate odds ratios (OR) and 95% confidence intervals (CI). We further evaluated statistical significance of selected SNPs using the Bayesian False Discovery Probability (BFDP). A significant association with EOC was identified in the UGT2A1/2 region for the SNP rs10017134 (per allele OR = 1.4, 95% CI = 1.2‐1.7, P = 1.2 × 10−6, BFDP = 0.02); and an association with HGSOC was identified in the EGFR region for the SNP rs114972508 (per allele OR = 2.3, 95% CI = 1.6‐3.4, P = 1.6 × 10−5, BFDP = 0.29) and in the UGT2A1/2 region again for rs1017134 (per allele OR = 1.4, 95% CI = 1.2‐1.7, P = 2.3 × 10−5, BFDP = 0.23). Genetic variants in the EGFR and UGT2A1/2 may increase susceptibility of EOC in AA women. Future studies to validate these findings are warranted. Alterations in EGFR and UGT2A1/2 could perturb enzyme efficacy, proliferation in ovaries, impact and mark susceptibility to EOC.

Abstract DNA methylation is instrumental for gene regulation. Global changes in the epigenetic landscape have been recognized as a hallmark of cancer. However, the role of DNA methylation in epithelial ovarian cancer (EOC) remains unclear. In this study, high-density genetic and DNA methylation data in white blood cells from the Framingham Heart Study (N = 1,595) were used to build genetic models to predict DNA methylation levels. These prediction models were then applied to the summary statistics of a genome-wide association study (GWAS) of ovarian cancer including 22,406 EOC cases and 40,941 controls to investigate genetically predicted DNA methylation levels in association with EOC risk. Among 62,938 CpG sites investigated, genetically predicted methylation levels at 89 CpG were significantly associated with EOC risk at a Bonferroni-corrected threshold of P < 7.94 × 10−7. Of them, 87 were located at GWAS-identified EOC susceptibility regions and two resided in a genomic region not previously reported to be associated with EOC risk. Integrative analyses of genetic, methylation, and gene expression data identified consistent directions of associations across 12 CpG, five genes, and EOC risk, suggesting that methylation at these 12 CpG may influence EOC risk by regulating expression of these five genes, namely MAPT, HOXB3, ABHD8, ARHGAP27, and SKAP1. We identified novel DNA methylation markers associated with EOC risk and propose that methylation at multiple CpG may affect EOC risk via regulation of gene expression. Significance: Identification of novel DNA methylation markers associated with EOC risk suggests that methylation at multiple CpG may affect EOC risk through regulation of gene expression.

Abstract Tubo-ovarian high-grade serous cancer (HGSC) is the most common histotype of epithelial ovarian cancer (70%-80%) and the leading cause of death from gynecological malignancy. Novel monoclonal antibody and adoptive cell transfer immunotherapies have found success in cancer in the past five years; however success in HGSC treatment has been elusive thus far. We have recently shown that CD8+ T-lymphocytes infiltrating the tumor proper (TILs) associate in a dose response manner with longer patient survival time regardless of age, stage or residual disease. As understanding host factors associated with this clinically relevant tumor phenotype will contribute to the development of targeted therapies, we sought to identify germline genetic factors associated with CD8+ TIL levels in HGSC. We performed a germline genome-wide association study (GWAS) of CD8+ TILs using 1204 HGSC patients of European ancestry from twelve studies with centralized genotyping, immunohistochemistry, and harmonization of clinical data. Germline genotypes were assayed using the Illumina Infinium OncoArray Beadchip and were imputed to over 11.4 million variants based on the 1000 Genomes Project reference panel. CD8 immunohistochemistry was performed on tissue microarrays using the Leica Bond Rx stainer and scored into four levels of CD8+ TILs per x400 magnification of a 0.55-mm diameter field for each tumor hotspot: none (no CD8+ TILs), low (1-2 CD8+ TILs), moderate (3-19 CD8+ TILs), high (20 or more CD8+ TILs). GWAS used multinomial regression modelling of these four CD8+ TIL levels adjusted for study site, age, and European ancestry principal components. On the average, CD8+ TIL levels were 18%, 19%, 44% and 18% for none, low, moderate and high levels, respectively. As expected, CD8+ TIL levels associated with overall survival time (p < 2.6x10-8), and CD8+ TIL levels and age at diagnosis were similarly distributed across study sites. We identified a region on chromosome 22 with multiple, correlated variants (MAF > 20%) associated with CD8+ TIL levels at p-value < 2.0x10-5. The most statistically significant variant was an indel (rs557925408) located in the an intron of the MYO18B gene (Myosin XVIIIB, p-value < 8.7x10-6). Each copy of this variant associated with a reduced extent of CD8+ TIL infiltration (OR low v none = 0.92; OR moderate v none = 0.61). An independent set of 1200 OncoArray genotyped HGSC cases are undergoing CD8+ TIL scoring in December 2017 and will be included in final genome-wide analysis. This will provide improved statistical power and enable consideration of additional genetic models (e.g., linear trend tests). Comprehensive integrative analysis of germline, tumor, and clinical features provides a model for clinical molecular epidemiology studies and will be key to increasing our understanding of this important HGSC immunophenotype. Citation Format: Yanina Natanzon, Martin Köbel, Stacey J. Winham, Sebastian M. Armasu, Bryan M. McCauley, Robert A. Vierkant, Julie M. Cunningham, David Bowtell, Ian G. Campbell, Jenny Chang-Claude, Anna deFazio, Peter A. Fasching, Mark T. Goodman, Beth Y. Karlan, Francesmary Modugno, Kirsten B. Moysich, Roberta B. Ness, Weiva Sieh, Paul D. Pharoah, Susan J. Ramus, Ellen L. Goode. Tumor-infiltrating CD8-positive T-lymphocytes in tubo-ovarian high-grade serous cancer are associated with multiple germline variants in 22q12.1 in a genome-wide association analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 225.

AbstractWe aimed to validate the prognostic association of p16 expression in ovarian high‐grade serous carcinomas (HGSC) and to explore it in other ovarian carcinoma histotypes. p16 protein expression was assessed by clinical‐grade immunohistochemistry in 6525 ovarian carcinomas including 4334 HGSC using tissue microarrays from 24 studies participating in the Ovarian Tumor Tissue Analysis consortium. p16 expression patterns were interpreted as abnormal (either overexpression referred to as block expression or absence) or normal (heterogeneous). CDKN2A (which encodes p16) mRNA expression was also analyzed in a subset (n = 2280) mostly representing HGSC (n = 2010). Association of p16 expression with overall survival (OS) was determined within histotypes as was CDKN2A expression for HGSC only. p16 block expression was most frequent in HGSC (56%) but neither protein nor mRNA expression was associated with OS. However, relative to heterogeneous expression, block expression was associated with shorter OS in endometriosis‐associated carcinomas, clear cell [hazard ratio (HR): 2.02, 95% confidence (CI) 1.47–2.77, p < 0.001] and endometrioid (HR: 1.88, 95% CI 1.30–2.75, p = 0.004), while absence was associated with shorter OS in low‐grade serous carcinomas (HR: 2.95, 95% CI 1.61–5.38, p = 0.001). Absence was most frequent in mucinous carcinoma (50%), and was not associated with OS in this histotype. The prognostic value of p16 expression is histotype‐specific and pattern dependent. We provide definitive evidence against an association of p16 expression with survival in ovarian HGSC as previously suggested. Block expression of p16 in clear cell and endometrioid carcinoma should be further validated as a prognostic marker, and absence in low‐grade serous carcinoma justifies CDK4 inhibition.