Mucinous ovarian carcinoma (MOC) is an epithelial ovarian cancer subtype that is frequently misclassified as extraovarian mucinous metastasis (EOM) because of overlapping features. To address this diagnostic challenge, we perform genome-wide DNA methylation profiling of 58 MOCs, 38 EOMs, and 18 mucinous borderline ovarian tumors (mBOTs) collected from six institutions. Methylation analysis defines two mBOT groups, one epigenetically similar to normal ovary and one resembling MOC. Unsupervised clustering reveals two distinct MOC methylation subtypes with potential prognostic relevance in the internal cohort. Using these data together with 389 external profiles, we develop and validate a three-step machine-learning classifier that distinguishes MOC from EOM with 95.5% accuracy. External validation of this classifier on 21 MOCs and 24 EOMs yields an accuracy of 91.11% for differentiating MOC from EOM. These findings establish an epigenetic framework for mucinous ovarian tumors and provide a robust clinical classification tool.

PURPOSE:Biomarkers to predict relapse and guide adjuvant therapy selection are needed in endometrial cancer (EC), one of the few cancers increasing in incidence and mortality. Assessment of circulating tumour DNA (ctDNA) at multiple perioperative time points has not been explored in EC. This study aimed to evaluate the prognostic utility of ctDNA across four perioperative time points, explore ctDNA dynamics, and compare ctDNA-derived tumour burden with 18F-fluorodeoxyglucose (FDG)-PET/CT and MRI. PATIENTS AND METHODS:Participants with Type 2 EC planned for surgery and consenting to the collection of biospecimens were enrolled prior to surgery and followed prospectively. Participants had preoperative imaging, and tumour sequencing was performed using hybrid capture, which guided the design of droplet digital PCR (ddPCR) assays for ctDNA analysis. The primary study endpoint was relapse free survival. RESULTS:Fifty participants were enrolled in the CODEC study. Genomic findings were consistent with the established molecular landscape of EC. Detection of ctDNA postoperatively at 24 h (Time Point 2B) and at 2-6 weeks (Time Point 3) was associated with poorer relapse-free survival. No significant association with outcome was observed for preoperative (Time Point 1) or intraoperative (Time Point 2A) ctDNA. Preoperative ctDNA correlated with FDG-PET- and MRI-defined tumour volume. CONCLUSION:These findings are hypothesis-generating and support the prognostic relevance of postoperative ctDNA assessment in EC with the 2-6 week postoperative window the most clinically informative time point for detecting minimal residual disease. Further studies are required to validate these findings and determine whether ctDNA can guide adjuvant therapy selection.

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 poor outcomes. In a large HGSC cohort (n = 1389) including 282 individuals with pathogenic germline BRCA variants (gBRCApv), residual disease after primary surgery has limited prognostic effect in gBRCApv-carriers compared to non-carriers, and prognostic outcomes differ based on the mutation location within functional domains of the BRCA genes. Multi-omic profiling is performed on 154 tumors, enriched for patients with BRCA-deficient tumors that experienced short overall survival ( ≤ 3 years, n = 42). Patients with BRCA2-deficient HGSC and loss of NF1 survive twice as long as those without NF1 loss, whereas PIK3CA, RAD21 and MYC amplification define BRCA2-deficient HGSC with exceptionally short survival. Patients with BRCA1-deficient HGSC and a more elevated HRD score survive significantly longer. BRCA1-deficient tumors in short survivors have evidence of immunosuppressive c-kit signaling and EMT. Our findings confirm that outcome is not determined by BRCA status alone, but rather a combination of co-occurring genomic alterations, the extent of DNA repair deficiency, and the tumor-immune microenvironment.

BACKGROUND:Ovarian cancer is characterised by high mortality and lacks effective screening, making prevention critical. Polygenic risk scores (PRS), which aggregate the effects of multiple common alleles, may capture a proportion of currently unexplained genetic risk. While PRS have been evaluated for risk prediction, their association with treatment response and survival remains unclear. This study assessed the utility of a PRS for predicting high-grade serous ovarian cancer (HGSOC) risk in an Australian population and its association with chemotherapy response and outcomes. METHODS:PRS were calculated for 1,097 HGSOC, and 812 controls using data from Australian research programs. Associations between PRS, OC risk, chemotherapy response, and survival were analysed. RESULTS:Each standard-deviation increase in PRS was associated with a 40% increase in HGSOC risk (OR 1.40, p < 0.001). Women in the top 1% of the PRS distribution had a lifetime OC risk approaching 3%. Higher PRS values showed a trend toward poorer outcomes, however these associations were not consistent across analyses. CONCLUSIONS:PRS were not clearly associated with chemotherapy response or survival but represent a significant risk factor for the development of HGSOC. Incorporating PRS into clinical models may improve risk stratification and support targeted prevention. IMPACT:As the first study to evaluate how PRS relate to both HGSOC risk and chemotherapy response and outcomes, we show that PRS are unlikely to serve as therapeutic biomarkers but support their use for enhanced risk-stratified prevention.

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.

The 15th Biennial Ovarian Cancer Research Symposium presented by the Rivkin Center for Ovarian Cancer and the American Association for Cancer Research held on September 20-21, 2024, in Seattle, WA covered cutting-edge research on the etiology and pathobiology of ovarian cancer. Several sessions focused on novel therapies including targeting the immunosuppressive microenvironment and advances in early detection. In this article we provide an overview of the key findings presented in the biology of ovarian cancer session, which covered novel model systems that accurately represent the disease, features of the tumor microenvironment (TME), and advances in understanding the genomic landscape and biomarkers of response.

Poly (ADP-ribose) polymerase inhibitors (PARPi) are approved for homologous recombination-deficient (HRD) high-grade serous ovarian cancers (HGSOCs), but their long-term effectiveness is limited by the emergence of resistance and hematological toxicity. Approximately 50% of HGSOCs are homologous recombination-proficient (HRP), and within this group, about 20% harbor CCNE1 amplification (cyclin E1-high). PARPi are especially ineffective in HRP tumors and in CCNE1 -high HGSOCs, which represent a major unmet clinical need. Loss of a chromatin remodeling enzyme, Amplified in Liver Cancer 1, ALC1, has been shown to enhance PARPi sensitivity. However, the clinical contexts in which ALC1 targeting will be clinically meaningful remains unclear. Here we demonstrate that ALC1 loss can enhance PARPi sensitivity across HRD and cyclin E1-high serous ovarian cancer lines, xenografts and patient-derived cells. ALC1 depletion can overcome several clinically relevant mechanisms of PARPi resistance, though its impact is limited in tumors with complete restoration of HR. Lower ALC1 expression correlates with longer progression-free survival and extended PARPi response. Importantly, ALC1 loss has minimal effects in BRCA -wild-type or BRCA -heterozygous non-cancerous fallopian tube cells, suggesting therapeutic safety. Increased PARPi sensitivity following ALC1 loss can be accurately predicted by endogenous single-stranded DNA levels, identifying a functional biomarker. Together, our studies define the clinical contexts in which the therapeutic utility of PARPi can be expanded by targeting ALC1, whose inhibitors are currently under evaluation in Phase I clinical trials.

Acquired treatment resistance in homologous recombination (HR) deficient high-grade serous ovarian cancer (HGSOC) commonly involves restoration of HR, often through secondary, somatic reversion mutations in HR genes. Our recent analysis of HR deficient, end-stage HGSOC found that most resistance mechanisms, including reversion mutations, are subclonal. The aim of this study is to investigate how the subclonal, reversion-negative cells persist during treatment. We are investigating single cell sequencing approaches that enable genotyping of individual cells, such that the phenotype of cells without reversions can be studied. In a pilot experiment we have used two HGSOC cell lines with known reversion mutations. To genotype individual cells and thus overcome the limitations of standard single-cell RNA sequencing (scRNA-seq), we combined short-read whole-transcriptome scRNA-seq with long-read, targeted single-cell cDNA sequencing on an Oxford Nanopore MinION using a 44-gene hybridization capture panel. A custom bioinformatics pipeline, incorporating Flexiplex for multiallelic codon resolution, annotated cell barcodes with their HR driver, reversion and TP53 mutations. Genotypes were then transferred to the short-read dataset following outlier and doublet filtering, and transcriptomic differences between cells with and without reversions were identified through differential gene expression analysis. We have repeated this protocol on a patient ascites sample. Prior short-read DNA sequencing from the cell lines and matched tumor samples defined the driver BRCA1 mutation, reversion events, pathogenic TP53 mutations, and whether there was loss of heterozygosity (LOH) at the BRCA1 loci. We successfully genotyped BRCA1 driver and reversion alleles in both cell lines in the long-read data. Rare cells heterozygous wildtype for the BRCA1 driver mutation were detected, suggesting either back-reversion events or subclones that escaped LOH - cells that were undetectable by bulk short-read methods. Cell genotypes were mapped to the short-read whole transcriptome data, and transcriptional programs that may support cell survival were identified in the non-reverted cells. Our approach demonstrates the utility of unbiased long-read sequencing for detecting BRCA1/2 reversions at single-cell resolution, including potential back-reversion subclones that are challenging to identify through bulk sequencing approaches. The differential gene expression results have identified potential resistance mechanisms. Ongoing work includes incorporation of fusion detection, structural-variant calling, and splice isoform analysis in our bioinformatics pipeline to further elucidate resistance pathways. We anticipate this work will result in an increased understanding of the resistance mechanisms that arise in HGSOC patients with HR deficiency, including identification of subclonal resistance mechanisms that cannot be detected through bulk sequencing approaches. Lauren Tjoeka, Stuart Bencraig, David Yoannidis, Madelynne Willis, Joy Hendley, AOCS Study Group, Timothy Semple, Alicia Oshlack, Elizabeth L. Christie. Characterizing resistance at single cell resolution in HR deficient HGSOC [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 PR019.

Treatments for high-grade serous ovarian cancer (HGSOC) are initially effective but most invariably fail. Although they can successfully suppress the bulk of the tumour cell population, residual cancer cells can enter alternative therapy-resistant cell fates highlighted by proliferative arrest. Understanding the nature of these fates and how cells may resume uncontrolled proliferation will lead to the development of new treatments for HGSOC. In this study, we examine the response of HGSOC cells to standard of care cisplatin chemotherapy and to the RNA Polymerase I transcription inhibitor CX-5461/Pidnarulex, two drugs that elicit a potent DNA damage response and growth arrest. Here, we identify that HGSOC cells exposed to these therapies show multiple hallmarks of therapy-induced senescence (TIS) and derive a core TIS gene expression signature irrespective of genetic background or senescence trigger. Given that TIS is a potentially escapable state, we have performed a focussed drug screen to identify drugs that eradicate senescent HGSOC cells. We identify that therapy-induced senescent HGSOC cells, including those with decreased sensitivity to senolytic drugs that inhibit the pro-survival protein BCL-XL, can be eliminated using drugs that induce ferroptosis, an iron-dependent form of cell death. Mechanistically, we demonstrate that senescent HGSOC cells have altered expression of regulators of iron metabolism leading to intracellular iron overload that underpins this targetable vulnerability. Together, we highlight elevated levels of iron as a TIS biomarker in HGSOC and the potential of inducing ferroptosis to eradicate residual HGSOC cells following initial therapy.

Increased infiltration of CD3 + and CD8 + T cells into ovarian cancer (OC) is linked to better prognosis, but the specific antigens involved are unclear. Recent reports suggest that HLA class I can present peptides from noncoding genomic regions, known as noncanonical or cryptic peptides, but their immunogenicity is underexplored. To address this, we used immunopeptidomic analysis and RNA sequencing on five metastatic OC samples, which identified 311 cryptic peptides (40 to 83 per patient). Despite comprising less than 1% of total peptides, cryptic peptides from noncoding transcripts emerged as the predominant antigen class when compared to the other major classes of known tumor-specific and tumor-associated antigens in OC samples. Notably, nearly 70% of the prioritized cryptic peptides elicited T cell activation, as evidenced by increased 4-1BB and IFN-γ expression in autologous CD8 + T cells. This study reveals noncoding cryptic peptides as an important class of immunogenic antigens in OC.

Single-cell analysis reveals the extent of genome doubling in ovarian cancer, its variability and its role in enabling tumours to evade the immune system. Single-cell analysis reveals the extent of genome doubling in ovarian cancer, its variability and its role in enabling tumours to evade the immune system.

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

Supplementary Table S5. Summary of cell lines used in this study.

Abstract High-grade serous ovarian cancer (HGSC) is the most common and lethal subtype of ovarian cancer. Extensive genomic instability and heterogeneity are characteristics of HGSC, with near-ubiquitous TP53 loss-of-function mutations, defects in homologous recombination (HR) repair, and extensive copy number aberrations contributing to the vast genomic heterogeneity observed. Most patients relapse and acquire resistance to platinum or PARP inhibitor (PARPi)-based therapy. Diverse mechanisms leading to therapy resistance and lack of predictive biomarkers means that matching the best treatment options to patients is difficult. Testing tumors for HR-Deficiency (HRD) status is now a clinical test to predict PARPi sensitivity in HGSC. We aimed to determine the influence of spatial and temporal heterogeneity on HRD status in HGSC. Patients (n=59) at Hammersmith Hospital (HH) underwent maximal-effort upfront cytoreduction for advanced HGSC, had their tumor dissemination mapped and tumor biopsies collected (range 4-15). Paired relapse samples were collected for 10 patients. Tumor DNA was extracted (n=5 tumors per case, plus relapse), Illumina Human OmniExpress genotyping performed for 59 cases and whole genome sequencing (WGS; 30x depth) performed for 21 cases. Allele-specific copy number (CN) was quantified using ASCAT. Multi-site CN data was also accessed from two cohorts (GSE38787, GSE40546). Tumors that failed QC or had an aberrant cell fraction <0.3 were excluded. HR scores were estimated in each cohort using different genomic instability score (GIS) or mutational signature-based algorithms and relevant cut-offs were applied to determine HRD and HR-Proficient (HRP) tumors. Variations in HR scores were observed across the cohorts with a proportion of patients presenting with a mixed HR score, displaying both HRD and HRP scores in their tumors: HH cohort (20%, 11/54 patients); GSE38787 (17%, 4/24 patients); and GSE40546 (28%, 4/14 patients) using a scarHRD algorithm. In the HH cohort (n=45), we examined if mixed HR status was associated with survival and demonstrated that patients with HR-mixed or all HRP tumors had poorer progression-free (p=0.0052) and overall survival (p=0.00092) than patients with all HRD tumors. Applying different approaches to the HH WGS data (scarHRD, CHORD, >63 score cut-off), approximately 20% of patients demonstrated HR-mixed scores in their tumors in each approach. However, depending on the approach chosen to define HRD, different cases showed HR-mixed scores, 2/21 mixed cases overlapped between the different algorithms applied with tumors changing from HRD to HRP and vice versa depending on the approach applied. Differences in HR-deficiency/proficiency scores suggests that HR status may not be consistent across advanced disseminated HGSC, indicating that a single tumor biopsy may not accurately depict spatial HGSC tumor biology. HR score variations in different methods (GIS or mutational signatures) indicates that these tests are not analogous for defining HRD and thus PARPi suitability. Citation Format: Elizabeth L. Christie, Marc Lorentzen, Maiqi Liu, Nikki Burdett, Edward Curry, Chun Hei Kwok, Ahwan Pandey, Katherine Nixon, Jennifer Ploski, James J. Clark, Iain McNeish, David D.L. Bowtell, Jonathan Krell, Christina Fotopoulou, Paula Cunnea. Effect of spatial and temporal inter-tumoral heterogeneity on homologous recombination deficiency scores in high grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B114.

Supplementary Table S4. Differential gene expression analysis comparing transcriptomes of tumors based on BRCA and RB1 alteration status.

Supplementary Table S15. Clinical characteristics of patients with HGSC according to RB1 and BRCA status.

Multivariate analysis of molecular alterations and OS in patients with HGSC and ENOC.

Supplementary Table S13. Clinical and molecular characteristics of ENOC.