Citations

Abstract Background Breast cancer is etiologically heterogeneous, but which risk factors differ in their associations across tumor subtypes remains unclear. We conducted a large, pooled analysis to evaluate independent, dose-response associations between breast cancer risk factors and quantitative tumor features. Methods Analyses of 15,731 invasive breast cancers from 24 studies evaluated associations (p-trend) between reproductive and hormonal factors, body mass index (BMI), alcohol, smoking, and family history in relation to quantitative immunohistochemistry measures on tissue microarrays (ER, PR, HER2, KI67, TP53) and tumor grade. Analyses in a subset of 10 population-based studies estimated subtype-specific odds ratios (ORs) comparing cases to controls. A Bayesian False Discovery Probability (BFDP) <0.2 was used to identify associations with strong statistical evidence. Results Nulliparity and later age at menopause were associated with higher ER-positivity (p-trend=0.021 and 0.001, respectively), with corresponding OR[ER+] (95% CI) = 1.49 (1.16-1.90) for nulliparous vs. parous and 1.07 (1.03-1.11) per 5 years. Current combined menopausal hormone therapy (MHT) use was associated with lower grade (p-trend<0.001), with OR [grade1] = 3.37 (2.69-4.21) for current vs. never users. Higher BMI was associated with lower ER-positivity and higher grade in premenopausal women (p-trend<0.001 and <0.001), with OR[ER+] = 0.80 (0.74-0.87) and OR[grade1] = 0.75 (0.63-0.88) per 5 units, and with higher PR-positivity and higher grade in postmenopausal women (p-trend<0.001 and <0.001), with OR[PR+] = 1.08 (1.03-1.14) and OR[grade 3] = 1.10 (1.04-1.17) per 5 units. Conclusion This pooled analysis of 15,731 cases showed that nulliparity, age at menopause, MHT, and BMI have independent, dose-response associations with ER, PR, and grade, clarifying patterns of etiologic heterogeneity. Associations with HER2, KI67 and TP53, or other risk factors did not meet our threshold for strong evidence.

Abstract BRCA-associated homologous recombination deficiency (HRD) is present in ~ 50% of high-grade serous carcinomas (HGSC) and predicts sensitivity to platinum-based therapy. However, there is little understanding of why some patients with BRCA-deficient tumors experience unexpectedly poor outcomes. We profiled 154 tumors, enriched for patients with BRCA-deficient tumors that experienced short overall survival (≤ 3 years, n = 42), using whole-genome, transcriptome, and methylation analyses. All but one BRCA-deficient tumor exceeded an accepted HRD genomic scarring threshold. However, patients with BRCA1-deficient HGSC with a more elevated HRD score survived significantly longer. Patients with BRCA2-deficient HGSC and loss of NF1 survived twice as long as those without NF1 loss, whereas PIK3CA or RAD21 amplification defined BRCA2-deficient HGSC with exceptionally short survival. BRCA1-deficient tumors in short survivors had evidence of immunosuppressive c-kit signaling and EMT. In a large HGSC cohort (n = 1,389) including 282 individuals with pathogenic germline BRCA variants (gBRCApv), the location of the mutation within functional domains stratified clinical outcomes. Notably, residual disease after primary surgery had limited prognostic effect in gBRCApv-carriers compared to non-carriers. Our findings indicate that tumor HR proficiency in the context of therapy response and survival is not a binary property, and highlight genomic and immune modifiers of outcomes in BRCA-deficient HGSC.

Abstract Despite homologous recombination (HR) deficiency being an important predictor of survival in high-grade serous ovarian carcinoma (HGSC), its status is rarely considered in large-scale studies of prognostic signatures and biomarkers. We sought to develop a gene expression-based classifier of HR deficient (HRD) HGSCs, suitable for archival FFPE tumor samples, to determine whether established prognostic markers were dependent on HR status. Using 361 HGSCs from The Cancer Genome Atlas (TCGA) where HR status was defined by the genomic gold standard, a HRD score, we trained and tested a ridge penalized logistic regression model. This 218-gene mRNA expression signature was then applied in 6002 HGSCs from the Ovarian Tumor Tissue Analysis (OTTA) consortium to predict the probability of a sample being HRD. In this large cohort of FFPE samples, we sought to understand the relationship between HR and overall survival. Applying Kaplan-Meier analyses and multivariate Cox proportional hazard regression modelling, adjusting for age and stratified by stage and study, we examined the relationship between HR status and the PrOTYPE subtype, SPOT prognostic score and other known prognostic markers in HGSC. Our machine learning model was 84% accurate in a held-out subset of the TCGA (n=73). Of the 6002 OTTA HGSCs, 40.4% were predicted to be HRD. These cases were significantly younger, were enriched for early stage (FIGO I/II) and had improved outcomes when compared to HR proficient (HRP) cases (hazard ratio [HZR]=0.71, 95% confidence interval [CI]=0.66-0.76, p<2x10-16). A linear increase in survival time was observed as the probability of a sample being HRD increased. HGSCs with >75% likelihood of being HRD had a median 5-year survival of 49.3% (95%CI=46.2-52.6%) which was a 17.7% increase from those with <25% likelihood. Both PrOTYPE and SPOT were significantly associated with survival, independent of HR status (p<2x10-16 and p<2x10-16 respectively). Notably the probability of being HRD was negatively correlated with an unfavorable SPOT score (R=-0.32, p<2x10-16), suggesting that SPOT partially captures the survival advantage of HR deficiency. Several individual biomarkers, assessed in 1248 to 2858 cases, showed HR dependent survival associations. HRD HGSCs with high GMNN expression had significantly improved, long-term outcomes (4-10years, HZR=0.39, 95%CI=0.20-0.78, p=0.01), and may be a marker of exceptional survival, while AR negative HRD cases had poorer outcomes, earlier in disease progression (0-4years, HZR=1.43, 95%CI=1.03-1.98, p=0.03). Significantly poorer prognoses were observed in HRP cases expressing PTEN (HZR=1.39, 95%CI=1.04-1.87, p=0.03), and in HRD cases with high expression of MyD88 (HZR=1.21, 95%CI=1.01-1.45, p=0.04). RB1 and FOXJ1 protein expression, and CD8+ T cell abundance were prognostic irrespective of HR status. Our study contributes to understanding the association between HR and HGSC survival in a statistically powered cohort and demonstrates heterogeneity in outcomes through clinically relevant prognostic signatures and biomarkers. Citation Format: Ashley L. Weir, Derek Chiu, Aline Talhouk, Mike S. Anglesio, Dale W. Garsed, Anna DeFazio, Holly H. Harris, Joellen M. Schildkraut, Andrew Berchuck, Ellen L. Goode, Paul D.P. Pharoah, David Huntsman, David Bowtell, Jen A. Doherty, Nadia M. Davidson, Martin Köbel, Susan J. Ramus. Homologous recombination DNA repair dependent survival associations in high-grade serous ovarian carcinoma: an Ovarian Tumor Tissue Analysis consortium study [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 B046.

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: The purpose of this study was to evaluate RB1 expression and survival across ovarian carcinoma histotypes and how co-occurrence of BRCA1 or BRCA2 (BRCA) alterations and RB1 loss influences survival in tubo-ovarian high-grade serous carcinoma (HGSC). Experimental Design: RB1 protein expression was classified by immunohistochemistry in ovarian carcinomas of 7,436 patients from the Ovarian Tumor Tissue Analysis consortium. We examined RB1 expression and germline BRCA status in a subset of 1,134 HGSC, and related genotype to overall survival (OS), tumor-infiltrating CD8+ lymphocytes, and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cells with and without BRCA1 alterations to model co-loss with treatment response. We performed whole-genome and transcriptome data analyses on 126 patients with primary HGSC to characterize tumors with concurrent BRCA deficiency and RB1 loss. Results: RB1 loss was associated with longer OS in HGSC but with poorer prognosis in endometrioid ovarian carcinoma. Patients with HGSC harboring both RB1 loss and pathogenic germline BRCA variants had superior OS compared with patients with either alteration alone, and their median OS was three times longer than those without pathogenic BRCA variants and retained RB1 expression (9.3 vs. 3.1 years). Enhanced sensitivity to cisplatin and paclitaxel was seen in BRCA1-altered cells with RB1 knockout. Combined RB1 loss and BRCA deficiency correlated with transcriptional markers of enhanced IFN response, cell-cycle deregulation, and reduced epithelial–mesenchymal transition. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1. Conclusions: Co-occurrence of RB1 loss and BRCA deficiency was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation.

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.

AbstractOur objective was to test whether p53 expression status is associated with survival for women diagnosed with the most common ovarian carcinoma histotypes (high‐grade serous carcinoma [HGSC], endometrioid carcinoma [EC], and clear cell carcinoma [CCC]) using a large multi‐institutional cohort from the Ovarian Tumor Tissue Analysis (OTTA) consortium. p53 expression was assessed on 6,678 cases represented on tissue microarrays from 25 participating OTTA study sites using a previously validated immunohistochemical (IHC) assay as a surrogate for the presence and functional effect of TP53 mutations. Three abnormal expression patterns (overexpression, complete absence, and cytoplasmic) and the normal (wild type) pattern were recorded. Survival analyses were performed by histotype. The frequency of abnormal p53 expression was 93.4% (4,630/4,957) in HGSC compared to 11.9% (116/973) in EC and 11.5% (86/748) in CCC. In HGSC, there were no differences in overall survival across the abnormal p53 expression patterns. However, in EC and CCC, abnormal p53 expression was associated with an increased risk of death for women diagnosed with EC in multivariate analysis compared to normal p53 as the reference (hazard ratio [HR] = 2.18, 95% confidence interval [CI] 1.36–3.47, p = 0.0011) and with CCC (HR = 1.57, 95% CI 1.11–2.22, p = 0.012). Abnormal p53 was also associated with shorter overall survival in The International Federation of Gynecology and Obstetrics stage I/II EC and CCC. Our study provides further evidence that functional groups of TP53 mutations assessed by abnormal surrogate p53 IHC patterns are not associated with survival in HGSC. In contrast, we validate that abnormal p53 IHC is a strong independent prognostic marker for EC and demonstrate for the first time an independent prognostic association of abnormal p53 IHC with overall survival in patients with CCC.

Abstract Mucinous ovarian carcinoma (MOC) is a rare histotype of ovarian cancer. Women with advanced disease (FIGO stage III/IV) have very poor outcomes, with 5-year survival of <20%. Little is known about the anti-tumor immune response in MOC, limiting potential immunotherapeutic options for patients. Immune data from 3 platforms were analysed. A NanoString mRNA expression panel with 39 immune-related genes (n=60 MOC, n=4198 other ovarian histotypes), a NanoString mRNA plexset with PD-1, PD-L1 (n=241 MOC, n=115 upper and lower gastrointestinal (GI) cancers), and 2 multicolor immunofluorescence (mcIF) panels (n=121 MOC) for CD68, PD-L1, PD-1, CD8 and FOXP3. Data overlapped all platforms for 22 MOC. Immune marker expression by ovarian cancer histotype was measured. Uni- and multivariable Cox Proportional Hazards assessed overall survival (OS) within MOC. Hierarchical clustering looked for subgroups of patients based on co-expression of immune genes. Compared to more common high-grade serous ovarian cancer (HGSC) and GI cancers, MOC were less immunologically active. High IGHM expression was associated with poorer OS (HR 1.31 (95% CI 1.02–1.69), p=0.033). High tumor densities of FOXP3 and PD-1 positive cells conferred poorer OS in a model with age, stage and site; however, these and PD-L1 and CD68 were associated with grade. The 39 gene NanoString and mcIF datasets revealed 4 clusters, roughly translating to immune cold, moderate and hot (groups 3,4). mcIF analysis revealed a subset with PD-L1 expression in the absence of T-regulatory cell infiltrates. This study provides novel insights into the MOC immune landscape. Despite relatively low immune activity overall, immunotherapy could be explored for a subset of patients with MOC. Supported by NSW Ministry of Health and UNSW Sydney, through the NSW Health PhD Scholarship Program 2017-2022; Translational Cancer Research Network top-up scholarship 2021

AbstractBackgroundCyclin E1 (CCNE1) is a potential predictive marker and therapeutic target in tubo‐ovarian high‐grade serous carcinoma (HGSC). Smaller studies have revealed unfavorable associations for CCNE1 amplification and CCNE1 overexpression with survival, but to date no large‐scale, histotype‐specific validation has been performed. The hypothesis was that high‐level amplification of CCNE1 and CCNE1 overexpression, as well as a combination of the two, are linked to shorter overall survival in HGSC.MethodsWithin the Ovarian Tumor Tissue Analysis consortium, amplification status and protein level in 3029 HGSC cases and mRNA expression in 2419 samples were investigated.ResultsHigh‐level amplification (>8 copies by chromogenic in situ hybridization) was found in 8.6% of HGSC and overexpression (>60% with at least 5% demonstrating strong intensity by immunohistochemistry) was found in 22.4%. CCNE1 high‐level amplification and overexpression both were linked to shorter overall survival in multivariate survival analysis adjusted for age and stage, with hazard stratification by study (hazard ratio [HR], 1.26; 95% CI, 1.08‐1.47, p = .034, and HR, 1.18; 95% CI, 1.05‐1.32, p = .015, respectively). This was also true for cases with combined high‐level amplification/overexpression (HR, 1.26; 95% CI, 1.09‐1.47, p = .033). CCNE1 mRNA expression was not associated with overall survival (HR, 1.00 per 1‐SD increase; 95% CI, 0.94‐1.06; p = .58). CCNE1 high‐level amplification is mutually exclusive with the presence of germline BRCA1/2 pathogenic variants and shows an inverse association to RB1 loss.ConclusionThis study provides large‐scale validation that CCNE1 high‐level amplification is associated with shorter survival, supporting its utility as a prognostic biomarker in HGSC.

AbstractBackgroundKnown risk alleles for epithelial ovarian cancer (EOC) account for approximately 40% of the heritability for EOC. Copy number variants (CNVs) have not been investigated as EOC risk alleles in a large population cohort.MethodsSingle nucleotide polymorphism array data from 13 071 EOC cases and 17 306 controls of White European ancestry were used to identify CNVs associated with EOC risk using a rare admixture maximum likelihood test for gene burden and a by-probe ratio test. We performed enrichment analysis of CNVs at known EOC risk loci and functional biofeatures in ovarian cancer–related cell types.ResultsWe identified statistically significant risk associations with CNVs at known EOC risk genes; BRCA1 (PEOC = 1.60E-21; OREOC = 8.24), RAD51C (Phigh-grade serous ovarian cancer [HGSOC] = 5.5E-4; odds ratio [OR]HGSOC = 5.74 del), and BRCA2 (PHGSOC = 7.0E-4; ORHGSOC = 3.31 deletion). Four suggestive associations (P < .001) were identified for rare CNVs. Risk-associated CNVs were enriched (P < .05) at known EOC risk loci identified by genome-wide association study. Noncoding CNVs were enriched in active promoters and insulators in EOC-related cell types.ConclusionsCNVs in BRCA1 have been previously reported in smaller studies, but their observed frequency in this large population-based cohort, along with the CNVs observed at BRCA2 and RAD51C gene loci in EOC cases, suggests that these CNVs are potentially pathogenic and may contribute to the spectrum of disease-causing mutations in these genes. CNVs are likely to occur in a wider set of susceptibility regions, with potential implications for clinical genetic testing and disease prevention.