Relationship between subtyping methodologies. A, Comparison of transcriptomic subgrouping approaches: composition of Tothill subtypes across each of the TCGA subtypes; labeled P value represents comparison of Tothill subtype frequency across all TCGA subtypes by the Chi-squared test. B, Distribution of homologous recombination repair (HRR)-centric subtypes across each of the TCGA transcriptomic subtypes; labeled P value represents comparison of BRCA1/2m frequency across all groups by Chi-squared test; Bonferroni-adjusted P = 0.009. C, Distribution of HRR-centric subtypes across each of the Tothill transcriptomic subtypes; labeled P value represents comparison of BRCA1/2m frequency across all groups by the Chi-squared test; Bonferroni-adjusted P = 0.003. BRCA2m, BRCA2 mutant; BRCA1m, BRCA1 mutant; EMSY-overxp; overexpression of EMSY; nBRCA-HRRm, non-BRCA1/2 HRR gene mutation; CCNE1g, gain of CCNE1; HRRwt, non-CCNE1g HRR wild-type.

Molecular landscape of HGSOC. 1Mutation in non-BRCA1/2 HRR genes: 3 BRIP1, 2 CHEK2, 1 RAD51C, 1 PALB2, 1 concurrent BAP1 and NBN. CCNE1 CN gain, ≥4 copies by TaqMan CN assay. EMSY CN amplification, ≥6 copies by TaqMan CN assay.

Clinicopathologic features of patients with HGSOC.

OBJECTIVE:Clear cell ovarian carcinoma (CCOC) is generally associated with a favourable prognosis, however up to 30 % of low-stage cases relapse within five years. The benefit of adjuvant chemotherapy in early-stage disease (FIGO IA-IC1) remains uncertain. This study aimed to identify molecular and immune markers associated with relapse in a well-characterized CCOC cohort. METHODS:We analyzed 85 CCOC cases identified through the Edinburgh Ovarian Cancer Database. Targeted DNA sequencing assessed genomic alterations, while CD3 and CD8 immunohistochemistry evaluated tumour immune infiltration. Tumours were stratified by stage (IA-IC1, IC2-II, III-IV), and progression-free survival (PFS) analysis included stage, age, genomic features, and immune markers. RESULTS:Common genomic alterations included ARID1A (49 %) and PIK3CA (42 %) mutations, PIK3-AKT pathway perturbations (60 %), and mismatch repair-related mutational signatures (25.9 %). Genomic features were not significantly associated with tumour stage; however, low-stage tumours (IA-IC1) were enriched in CD3+ (40 % cases) and CD8+ (29 % cases) tumour-infiltrating lymphocytes (TILs) compared to higher-stage tumours (IC2-II: 13 %/7 %; III-IV: 9 %/0 %). In univariate analysis, low CD3+ TIL levels were significantly associated with reduced PFS (HR = 4.4, P = 0.042), and ARID1A wild-type status was linked to poorer PFS in low-stage tumours (HR = 7.2, P = 0.088). Notably, the combination of ARID1A wild-type status and CD3+ TIL depletion identified a high-risk subgroup with increased relapse risk (HR = 11.7, P = 0.051). CONCLUSIONS:The combination of ARID1A wild-type status and low CD3+ TIL levels suggests higher relapse risk in low-stage CCOC. These findings warrant further investigation into targeted and immune-based therapies for high-risk early-stage patients.

Tumor-infiltrating immune cells across high-grade serous carcinoma subtypes. A, CD3+ infiltration across HRR-centric subtypes; labeled P value represents comparison of BRCA2m and CCNE1g groups using the Mann–Whitney U test. B, CD3+ infiltration across TCGA transcriptomic subtypes; labeled P value represents comparison of IMR and PRO groups using the Mann–Whitney U test. C, CD3+ infiltration across Tothill transcriptomic subtypes; labeled P value represents comparison of C2 and C5 groups using the Mann–Whitney U test. BRCA2m, BRCA2 mutant; BRCA1m, BRCA1 mutant; EMSY-overxp; overexpression of EMSY; CCNE1g, gain of CCNE1; HRRwt, non-CCNE1g HRR wild-type.

PTEN and RB loss in HGSOC. A, Frequency of loss of PTEN protein expression across HRR-centric subtypes. B, Frequency of loss of RB protein expression across HRR-centric subtypes. C, Impact of RB loss on survival in patients based on HRR status. Multivariable hazard ratio (mHR) for HRR-ab: RB loss vs. HRR-ab RB-intact = 0.50; 95% CI, 0.30–0.84; mHR for HRRwt/CCNE1g: RB loss vs. HRRwt/CCNE1g: RB-intact = 0.71; 95% CI, 0.48–1.06. BRCA2m, BRCA2 mutant; BRCA1m, BRCA1 mutant; EMSY-overxp; overexpression of EMSY; nBRCA-HRRm, non-BRCA1/2 HRR gene mutation; CCNE1g, gain of CCNE1; HRRwt, non-CCNE1g HRR wild-type. HRR-ab, HRR-aberrant: BRCA1m, BRCA2m, EMSY-overxp, or nBRCA-HRRm.

HRR pathway (HRR)–centric subtyping of high-grade serous carcinoma. A, HRR-centric classification taxonomy. B, OS profile of HRR-centric subtypes. C, Chemosensitivity of HRR-centric subtypes at first-line treatment (left) and treatment for disease relapse (right) as determined by CA125 tumor marker (top) and radiology (bottom). BRCA2m, BRCA2 mutant; BRCA1m, BRCA1 mutant; EMSY-overxp; overexpression of EMSY; CCNE1g, gain of CCNE1; HRRwt, non-CCNE1g HRR wild-type.

Supplementary tables 3a and 3b. Germline and somatic SNVs/indels at BRCA1/2.

Supplementary methods and supplementary figures

OBJECTIVES:Low-grade serous ovarian carcinoma (LGSOC) is a distinct, rare, ovarian cancer type characterised by younger patient age and intrinsic chemoresistance. Understanding the molecular landscape is crucial for optimising targeted therapy. METHODS:Genomic data from whole exome sequencing of tumour tissue was analysed in a LGSOC cohort with detailed clinical annotation. RESULTS:63 cases were analysed and three subgroups identified based on single nucleotide variants: canonical MAPK mutant (cMAPKm: 52%, KRAS/BRAF/NRAS), MAPK-associated gene mutation (MAPK-assoc: 27%) and MAPK wild-type (MAPKwt: 21%). NOTCH pathway disruption occurred across all subgroups. Tumour mutational burden (TMB), mutational signatures and recurrent copy number (CN) changes varied across the cohort with co-occurrence of chromosome 1p loss and 1q gain (CN Chr1pq) a recurrent feature. Low TMB and CN Chr1pq were associated with inferior disease-specific survival (HR 6.43; p < 0.001 and HR 3.29, p = 0.011 respectively). Stepwise genomic classification in relation to outcome resulted in four groups (TMB low; CN Chr1pq; MAPKwt/MAPKassoc; cMAPKm). 5 year disease-specific survival was 46%, 55%, 79% and 100% respectively for these groups. The two most favourable genomic subgroups were enriched for the SBS10b mutational signature, particularly the cMAPKm subgroup. CONCLUSIONS:LGSOC comprises multiple genomic subgroups with distinct clinical and molecular features. Chr1pq CN arm disruption and TMB represent promising methods to identify individuals with poorer prognosis. Further investigation of the molecular basis for these observations is required. MAPKwt cases represent around a fifth of patients. NOTCH inhibitors represent a candidate therapeutic strategy worthy of exploration across these cases.

<p>SNVs in non-BRCA1/2 HR genes</p>

<p>Genes in KEGG HR pathway</p>

<p>Supplementary methods and supplementary figures</p>

<p>Supplementary tables 3a and 3b. Germline and somatic SNVs/indels at BRCA1/2.</p>

Low grade serous ovarian carcinoma (LGSOC) demonstrates unique clinical and molecular features compared to other ovarian cancer types. The relationship between common histological features of LGSOC and molecular events, such as hormone receptor expression patterns and MAPK gene mutation status, remains poorly understood. Recent data suggest some of these molecular features may be biomarkers of response to recently introduced biologically-targeted therapies, namely endocrine therapy and MEK inhibitors. We utilize a cohort of 63 pathologically-confirmed LGSOC cases with whole exome sequencing and hormone receptor expression data to investigate these relationships. LGSOC cases demonstrated uniformly high oestrogen receptor (ER) expression, but variable progesterone receptor (PR) expression intensity. 60% and 37% of cases demonstrated micropapillary and macropapillary patterns of stromal invasion, respectively. 63% of cases demonstrated desmoplasia, which was significantly associated with advanced disease stage and visible residual disease after cytoreductive surgery. MAPK-mutant cases (KRAS, BRAF, NRAS) more frequently demonstrated macropapillary stromal invasion, while Chr1p loss was associated with desmoplasia and low PR expression. Presence of micropapillary stromal invasion and low PR expression were associated with significantly poorer survival after accounting for stage and residual disease status. Together, these data identify novel relationships between histopathological features and molecularly-defined subgroups in LGSOC.

Background Ovarian carcinosarcoma (OCS) is an uncommon, biphasic and highly aggressive ovarian cancer type, which has received relatively little research attention. Methods We curated the largest pathologically confirmed OCS cohort to date, performing detailed histopathological characterisation, analysis of features associated with survival and comparison against high-grade serous ovarian carcinoma (HGSOC). Results Eighty-two OCS patients were identified; overall survival was poor (median 12.7 months). In all, 79% demonstrated epithelial components of high-grade serous (HGS) type, while 21% were endometrioid. Heterologous elements were common (chondrosarcoma in 32%, rhabdomyosarcoma in 21%, liposarcoma in 2%); chondrosarcoma was more frequent in OCS with endometrioid carcinomatous components. Earlier stage, complete resection and platinum-containing adjuvant chemotherapy were associated with prolonged survival; however, risk of relapse and mortality was high across all patient groups. Histological subclassification did not identify subgroups with distinct survival. Compared to HGSOC, OCS patients were older ( P < 0.0001), more likely to be FIGO stage I ( P = 0.025), demonstrated lower chemotherapy response rate ( P = 0.001) and had significantly poorer survival ( P < 0.0001). Conclusion OCS represents a distinct, highly lethal form of ovarian cancer for which new treatment strategies are urgently needed. Histological subclassification does not identify patient subgroups with distinct survival. Aggressive adjuvant chemotherapy should be considered for all cases, including those with early-stage disease.

Supplementary Data from Multiomic Characterization of High-Grade Serous Ovarian Carcinoma Enables High-Resolution Patient Stratification

Background Low-grade serous ovarian carcinoma (LGSOC) is a distinct, under-investigated and relatively chemotherapy-resistant ovarian cancer type. Understanding the molecular landscape is crucial to maximise the impact of molecularly-targeted therapy. Methods Whole exome sequencing and copy number data were integrated with histopathological patterns, ER/PR expression, and detailed clinical annotation, including survival, in a carefully curated LGSOC cohort. Results 63 tumours were analysed in the largest comprehensive genomic LGSOC study to date. Three genomic subgroups were identified: canonical MAPK mutant (cMAPKm: 52%, KRAS / BRAF / NRAS ), MAPK-associated mutation (27%, 14 MAPK-associated genes) and MAPK wild-type (MAPKwt: 21%). MAPKwt patients were younger at diagnosis (median 47 versus 62 years in the cMAPKm subgroup) and demonstrated shorter survival [multivariable HR (mHR) 4.17]. The inferior survival in the MAPKwt subgroup was due to shorter post-relapse survival (mHR 5.22) rather than shorter time to first progression (mHR 1.15). Patients in the MAPK-associated mutation subgroup had similar survival to cMAPKm cases. The cMAPKm subgroup more frequently demonstrated macropapillary invasion. Desmoplasia and micropapillary invasion were independently associated with poor survival. NOTCH pathway activation occurred independently of MAPK subgroup. Conclusions LGSOC comprises multiple genomic subgroups with distinct clinical, molecular and histopathological features. True MAPKwt cases represent around a fifth of patients: they are younger but have poorer survival. New therapeutic strategies with activity in this subgroup are urgently required. NOTCH inhibitors represent a therapeutic strategy worthy of exploration.

Purpose: The abundance and effects of structural variation at BRCA1/2 in tumors are not well understood. In particular, the impact of these events on homologous recombination repair deficiency (HRD) has yet to be demonstrated. Experimental Design: Exploiting a large collection of whole-genome sequencing data from high-grade serous ovarian carcinoma (N = 205) together with matched RNA sequencing for the majority of tumors (N = 150), we have comprehensively characterized mutation and expression at BRCA1/2. Results: In addition to the known spectrum of short somatic mutations (SSM), we discovered that multi-megabase structural variants (SV) were a frequent, unappreciated source of BRCA1/2 disruption in these tumors, and we found a genome-wide enrichment for large deletions at the BRCA1/2 loci across the cohort. These SVs independently affected a substantial proportion of patients (16%) in addition to those affected by SSMs (24%), conferring HRD and impacting patient survival. We also detail compound deficiencies involving SSMs and SVs at both loci, demonstrating that the strongest risk of HRD emerges from combined SVs at both BRCA1 and BRCA2 in the absence of SSMs. Furthermore, these SVs are abundant and disruptive in other cancer types. Conclusions: These results extend our understanding of the mutational landscape underlying HRD, increase the number of patients predicted to benefit from therapies exploiting HRD, and suggest there is currently untapped potential in SV detection for patient stratification.

Mucinous ovarian carcinoma (MOC) is a unique form of ovarian cancer. MOC typically presents at early stage but demonstrates intrinsic chemoresistance; treatment of advanced-stage and relapsed disease is therefore challenging. We harness a large retrospective MOC cohort to identify factors associated with recurrence risk and survival. A total of 151 MOC patients were included. The 5 year disease-specific survival (DSS) was 84.5%. Risk of subsequent recurrence after a disease-free period of 2 and 5 years was low (8.3% and 5.6% over the next 10 years). The majority of cases were FIGO stage I (35.6% IA, 43.0% IC). Multivariable analysis identified stage and pathological grade as independently associated with DSS (p < 0.001 and p < 0.001). Grade 1 stage I patients represented the majority of cases (53.0%) and demonstrated exceptional survival (10 year DSS 95.3%); survival was comparable between grade I stage IA and stage IC patients, and between grade I stage IC patients who did and did not receive adjuvant chemotherapy. At 5 years following diagnosis, the proportion of grade 1, 2 and 3 patients remaining disease free was 89.5%, 74.9% and 41.7%; the corresponding proportions for FIGO stage I, II and III/IV patients were 91.1%, 76.7% and 19.8%. Median post-relapse survival was 5.0 months. Most MOC patients present with low-grade early-stage disease and are at low risk of recurrence. New treatment options are urgently needed to improve survival following relapse, which is associated with extremely poor prognosis.