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.

Supplementary Figure 1: Methylation Signature Approximation

OBJECTIVE:Most co-existent endometrial and ovarian carcinomas are clonally related and exhibit an indolent disease course. Pathologic assignment and clinical management of this entity vary greatly. The International Federation of Gynecology and Obstetrics (FIGO) 2023 endometrial carcinoma staging/risk stratification system introduced a new substage for co-existent endometrial and ovarian carcinomas that meet strict pathologic criteria (stage IA3, distinct from IIIA1). Our aim was to validate if FIGO IA3 identifies a subset of co-existent endometrial and ovarian carcinomas at very low risk of recurrence and determine whether further refinement, through molecular features and expanded ovarian pathologic criteria, could improve prognostic discernment and direct more patients for consideration of de-escalation. METHODS:Clinicopathologic, molecular, and outcome data were collected on patients with co-existent endometrial and ovarian carcinoma, extracted from pathology archives and molecularly classified endometrial carcinoma cohorts. RESULTS:Among the 154 co-existent endometrial and ovarian carcinoma patients, higher recurrence rates were observed with the p53abn (2/6, 33%), mismatch repair deficiency (MMRd) (7/34, 21%) or no specific molecular profile (NSMP) estrogen receptor (ER) negative-low (2/15, 13%) molecular sub-types, compared with patients with POLEmut or NSMP ER strong positive tumors. Thirty-two patients met FIGO IA3 criteria, with one recurrence and death event (MMRd). Eliminating patients with adverse molecular features (p53abn or MMRd endometrium or ovary, or NSMP ER negative-low endometrium) and expanding criteria to include any POLEmut or cases with bilateral ovarian involvement, intra- or pre-operative ovarian rupture, or ovarian surface involvement significantly improved risk stratification (p = .008) and added 48 co-existent endometrial and ovarian carcinoma patients (>2-fold increase) with no recurrence events (mean follow-up: 6 years). There was 91% concordance of molecular sub-type assignment between endometrial and ovarian tumors. CONCLUSIONS:FIGO IA3 criteria identify a subset of co-existent endometrial and ovarian carcinomas with excellent outcomes. However, incorporating molecular features into the definition enables greater prognostic discernment and supports the inclusion of patients with a broader range of pathologic features with indolent disease (increased from 20% to 49% of the cohort, 0 recurrences) who may be candidates for treatment de-escalation.

Supplementary Table 1: Characteristics of Study Participants with High Grade Serous Tubo-Ovarian Carcinoma by Contributing Study

Supplementary Table 3: Differential Methylation Analysis for Mayo Training and Testing Sets for all QC-passed CpG loci from 450k

Supplementary Figure 3: Immune Analysis

Mucinous ovarian carcinoma (MOC) is a rare histotype of epithelial ovarian cancer. Its origins are obscure: while many mucinous tumours in the ovary are metastases from the gastrointestinal tract, MOC can occur as an ovarian primary; however, the cell of origin is not well established. In this review we summarise the pathological, epidemiological, and molecular evidence for the cellular origins of MOC. We propose a model for the origins of the various tumours of the ovary with mucinous differentiation. We distinguish Müllerian from gastrointestinal-type mucinous differentiation. A small proportion of the latter arise from teratoma and a distinct terminology has been proposed. Other gastrointestinal mucinous tumours are associated with Brenner tumours and arise from their associated benign lesions, Walthard nests. The remaining mucinous tumours develop either through mucinous metaplasia in established Müllerian tumours or with even greater plasticity through gastrointestinal metaplasia of epithelial or mesothelial ovarian inclusions. This model remains to be validated and mechanistically understood and we discuss future research directions. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Endometriosis is a common gynecologic condition that causes chronic, life-altering symptoms including pain and infertility. There is an urgent need for new non-hormonal targeted therapeutics to treat endometriosis, but until very recently, the cellular and molecular signatures of endometriotic lesions are undefined, hindering the development of clinical advances. Integrating inherited risk data from analyses of >45 0000 individuals with ≈35 0000 single-cell transcriptomes from 21 patients, M2-macrophages as candidate drivers of disease susceptibility are uncovered, and nominating IL1 signaling as a central hub impacted by germline genetic variation associated with endometriosis risk. Extensive functional follow-up confirmed these associations and revealed a pleiotropic role for this pathway in endometriosis. Population-scale expression quantitative trait locus analysis demonstrates that genetic variation controlling IL1A expression is associated with endometriosis risk variants. Manipulation of IL1 signaling in state-of-the-art in vitro decidualized endometrial organoids impacts epithelial differentiation, and in an in vivo endometriosis model, treatment with anakinra (an interleukin-1 receptor antagonist) results in a significant, dose-dependent reduction in spontaneous and evoked pain and dampened pro-angiogenic signaling. Together, these studies highlight non-diagnostic cell types as central to endometriosis susceptibility and support IL1 signaling as an important actionable pathway for this disease.

Supplementary Table 5: Modeling of Time to Recurrence/Death Among Studies with Gene Expression Data, excluding data from Previously Published Participants

The molecular landscapes of epithelial ovarian cancers (EOCs) have largely been profiled using bulk sequencing techniques. Consequently, biologically relevant signals from the tumor and its microenvironment (hereafter stroma) are often conflated, dampening both tumor and stroma-specific observations. We profiled the carcinoma and stroma compartments separately across clear cell (CCOC), endometrioid (ENOC), and high-grade serous ovarian cancer (HGSC) using laser capture microdissection followed by sample-matched whole genome bisulfite sequencing and RNA-seq. Histotype designations drove sample segregation in the carcinoma compartment yet were not sufficient to explain top expression or methylation variations in the stroma. Unsupervised clustering of the top variable genes in the stroma identified four stromal subtypes (S1-S4) shared across the three histotypes. Of these, S1, S2, and S4 were the most distinguishable and could be recovered in external datasets. S1 was enriched for RNA splicing processes (SFSWAP, RBM25), S2 for metabolism (GATM, ALDH1A1) and S4 for remodeling of the extracellular matrix (FN1, POSTN, SFRP2). While the characteristics of S4 echo previous reports of the desmoplastic/highly fibrous stroma reported in HGSC, we show that S4 is also present in CCOC and ENOC tumors. Using HGSC samples from TCGA, we observed a strong correlation of S4 markers with the Mesenchymal subtype and an inverse correlation of S4 and S2 markers, suggesting mutual exclusivity of these stromal subtypes. Examining the TCGA-derived subtype markers in our microdissected data revealed that the canonical Immunoreactive and Mesenchymal subtypes were driven by signals from the stroma, rather than the carcinoma. Conversely, Differentiated and Proliferative markers were enriched in the carcinoma. Clustering of pure carcinomas revealed a Proliferative versus Differentiated dichotomy within the carcinomas, particularly within ENOC and HGSC tumors. CCOC tumors were systematically depleted of the Proliferative subtype, consistent with their indolent nature. We next investigated if genetic, epigenetic or transcriptional cues in the carcinoma compartment are associated with the characteristic of its TME but failed to detect differences at 5% FDR. This suggests that the stromal subtype may not be shaped solely by instructions from the cancer cells and that a more complex, reciprocal cross-talk exists between the carcinoma and its TME. Epigenetic and transcriptional differences in CCOC, ENOC and HGSC carcinomas can be largely attributed to histotype, yet this is not the case for their associated TMEs. With the growing importance of TME in influencing patient outcomes, and response to treatment, understanding the nature of a tumor’s TME is crucial. Thus, coupling the stromal subtypes (S1-S4) with the well-established histotype designations of EOCs may provide a more robust way to classify EOCs and inform optimal treatment strategies that account for the characteristic of the tumor stroma. Svetlana Djirackor, Karolin Heinze, Ian Beddows, David Sokol, Bianca Ribeiro de Souza, Martin Koebel, Marie Adams, Michael Anglesio, Hui Shen. Parallel epigenetic and transcriptomic profiling of carcinoma and stroma compartments across major ovarian cancer histotypes [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 B007.

Supplementary Figures S1-S13

BACKGROUND:Ovarian high-grade serous carcinomas (HGSC) comprise four distinct molecular subtypes based on mRNA expression patterns, with differential survival. Understanding risk factor associations is important to elucidate the etiology of HGSC. We investigated associations between different epidemiologic risk factors and HGSC molecular subtypes. METHODS:We pooled data from 11 case-control studies with epidemiologic and tumor gene expression data from custom NanoString CodeSets developed through a collaboration within the Ovarian Tumor Tissue Analysis consortium. The PrOTYPE-validated NanoString-based 55-gene classifier was used to assign HGSC gene expression subtypes. We examined associations between epidemiologic factors and HGSC subtypes in 2,070 cases and 16,633 controls using multivariable-adjusted polytomous regression models. RESULTS:Among the 2,070 HGSC cases, 556 (27%) were classified as C1.MES, 340 (16%) as C5.PRO, 538 (26%) as C2.IMM, and 636 (31%) as C4.DIF. The key factors, including oral contraceptive use, parity, breastfeeding, and family history of ovarian cancer, were similarly associated with all subtypes. Heterogeneity was observed for several factors. Former smoking [OR = 1.25; 95% confidence interval (CI) = 1.03, 1.51] and genital powder use (OR = 1.42; 95% CI = 1.08, 1.86) were uniquely associated with C2.IMM. History of endometriosis was associated with C5.PRO (OR = 1.46; 95% CI = 0.98, 2.16) and C4.DIF (OR = 1.27; 95% CI = 0.94, 1.71) only. Family history of breast cancer (OR = 1.44; 95% CI = 1.16, 1.78) and current smoking (OR = 1.40; 95% CI = 1.11, 1.76) were associated with C4.DIF only. CONCLUSIONS:This study observed heterogeneous associations of epidemiologic and modifiable factors with HGSC molecular subtypes. IMPACT:The different patterns of associations may provide key information about the etiology of the four subtypes.

Supplementary Table 4: Multivariate Modeling of Methylation Signature Among Studies with Multiple Datatypes

Supplementary Table 2: CpGs That Defined Previously Reported Methylation Subtypes Using a Semi-Supervised Clustering Approach

Supplementary Figure 4: Correlations between Methylation at CpG Sites and TAP1 mRNA Expression

Supplementary Methods 1: The original 60 CpG loci defining methylation-based subtypes, and the approximation method for methylation signature

Supplementary Tables S1-S11

PURPOSE:Increasing genomics-based evidence suggests that synchronous endometrial and ovarian cancer (SEOC) represents clonally related primary and metastatic tumors. A systematic analysis of the global protein landscape of SEOCs, heretofore lacking, could reveal functional and disease-specific consequences of known genetic alterations, the directionality of metastasis, and accurate histologic markers to distinguish SEOCs from single-site tumors. EXPERIMENTAL DESIGN:We performed a systematic proteogenomic analysis of 29 patients diagnosed with SEOC at three international gynecologic oncology treatment centers (Chicago, Vancouver, and Tübingen). For direct comparison with single-site tumors, we included 9 patients with single-site endometrioid ovarian and 26 patients with single-site endometrioid endometrial cancer (EEC). For all 64 patients, we performed sequencing of a 275-gene cancer panel combined with compartment-resolved mass spectrometry-based proteomics of consecutive tissue sections to compare global (6,000+ proteins), tumor, and stromal proteomes. RESULTS:DNA-based panel sequencing confirmed that most SEOCs are clonally related. Global proteome profiling uncovered pronounced differences between SEOCs and single tumors and underscored the importance of the stromal proteome in defining and identifying SEOCs. We identified molecularly unique SEOC stromal proteomes, which were globally more related to single endometrial cancers. We finally derived a proteomic predictor distinguishing SEOCs from single-site ovarian and uterine tumors. CONCLUSIONS:The integrated proteogenomic data show that SEOCs are distinguishable from endometrioid endometrial or endometrioid ovarian cancer. Based on their proteogenomic similarity to EECs, we conclude that most SEOCs represent primary EECs that have metastasized to the ovary.

Supplementary Table S10. Number of patients by study and histotype.