Concurrent RB1 Loss and BRCA-Deficiency Predicts Enhanced Immunological Response and Long-Term Survival in Tubo-Ovarian High-Grade Serous Carcinoma.

PURPOSE: 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 7436 patients from the Ovarian Tumor Tissue Analysis consortium. We examined RB1 expression and germline BRCA status in a subset of 1134 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 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 to 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 interferon 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.

Mathematical modeling of the evolution of resistance and aggressiveness of high-grade serous ovarian cancer from patient CA-125 time series.

A time-series analysis of serum Cancer Antigen 125 (CA-125) levels was performed in 791 patients with high-grade serous ovarian cancer (HGSOC) from the Australian Ovarian Cancer Study to evaluate the development of chemoresistance and response to therapy. To investigate chemoresistance and better predict the treatment effectiveness, we examined two traits: resistance (defined as the rate of CA-125 change when patients were treated with therapy) and aggressiveness (defined as the rate of CA-125 change when patients were not treated). We found that as the number of treatment lines increases, the data-based resistance increases (a decreased rate of CA-125 decay). We use mathematical models of two distinct cancer cell types, treatment-sensitive cells and treatment-resistant cells, to estimate the values and evolution of the two traits in individual patients. By fitting to individual patient HGSOC data, our models successfully capture the dynamics of the CA-125 level. The parameters estimated from the mathematical models show that patients with inferred low growth rates of treatment-sensitive cells and treatment-resistant cells (low model-estimated aggressiveness) and a high death rate of treatment-resistant cells (low model-estimated resistance) have longer survival time after completing their second-line of therapy. These findings show that mathematical models can characterize the degree of resistance and aggressiveness in individual patients, which improves our understanding of chemoresistance development and could predict treatment effectiveness in HGSOC patients.

Small-scale mutations are infrequent as mechanisms of resistance in post-PARP inhibitor tumour samples in high grade serous ovarian cancer.

While the introduction of poly-(ADP)-ribose polymerase (PARP) inhibitors in homologous recombination DNA repair (HR) deficient high grade serous ovarian, fallopian tube and primary peritoneal cancers (HGSC) has improved patient survival, resistance to PARP inhibitors frequently occurs. Preclinical and translational studies have identified multiple mechanisms of resistance; here we examined tumour samples collected from 26 women following treatment with PARP inhibitors as part of standard of care or their enrolment in clinical trials. Twenty-one had a germline or somatic BRCA1/2 mutation. We performed targeted sequencing of 63 genes involved in DNA repair processes or implicated in ovarian cancer resistance. We found that just three individuals had a small-scale mutation as a definitive resistance mechanism detected, having reversion mutations, while six had potential mechanisms of resistance detected, with alterations related to BRCA1 function and mutations in SHLD2. This study indicates that mutations in genes related to DNA repair are detected in a minority of HGSC patients as genetic mechanisms of resistance. Future research into resistance in HGSC should focus on copy number, transcriptional and epigenetic aberrations, and the contribution of the tumour microenvironment.

Concurrent RB1 loss and BRCA-deficiency predicts enhanced immunological response and long-term survival in tubo-ovarian high-grade serous carcinoma.

Background: Somatic loss of the tumour suppressor RB1 is a common event in tubo-ovarian high-grade serous carcinoma (HGSC), which frequently co-occurs with alterations in homologous recombination DNA repair genes including BRCA1 and BRCA2 (BRCA). We examined whether tumour expression of RB1 was associated with survival across ovarian cancer histotypes (HGSC, endometrioid (ENOC), clear cell (CCOC), mucinous (MOC), low-grade serous carcinoma (LGSC)), and how co-occurrence of germline BRCA pathogenic variants and RB1 loss influences long-term survival in a large series of HGSC. Patients and methods: RB1 protein expression patterns were classified by immunohistochemistry in epithelial ovarian carcinomas of 7436 patients from 20 studies participating in the Ovarian Tumor Tissue Analysis consortium and assessed for associations with overall survival (OS), accounting for patient age at diagnosis and FIGO stage. We examined RB1 expression and germline BRCA status in a subset of 1134 HGSC, and related genotype to survival, tumour infiltrating CD8+ lymphocyte counts and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cell lines with and without BRCA1 mutations to model co-loss with treatment response. We also performed genomic analyses on 126 primary HGSC to explore the molecular characteristics of concurrent homologous recombination deficiency and RB1 loss. Results: RB1 protein loss was most frequent in HGSC (16.4%) and was highly correlated with RB1 mRNA expression. RB1 loss was associated with longer OS in HGSC (hazard ratio [HR] 0.74, 95% confidence interval [CI] 0.66-0.83, P = 6.8 ×10-7), but with poorer prognosis in ENOC (HR 2.17, 95% CI 1.17-4.03, P = 0.0140). Germline BRCA mutations and RB1 loss co-occurred in HGSC (P < 0.0001). Patients with both RB1 loss and germline BRCA mutations had a superior OS (HR 0.38, 95% CI 0.25-0.58, P = 5.2 ×10-6) compared to patients with either alteration alone, and their median OS was three times longer than non-carriers whose tumours retained RB1 expression (9.3 years vs. 3.1 years). Enhanced sensitivity to cisplatin (P < 0.01) and paclitaxel (P < 0.05) was seen in BRCA1 mutated cell lines with RB1 knockout. Among 126 patients with whole-genome and transcriptome sequence data, combined RB1 loss and genomic evidence of homologous recombination deficiency was correlated with transcriptional markers of enhanced interferon response, cell cycle deregulation, and reduced epithelial-mesenchymal transition in primary HGSC. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1. Conclusions: Co-occurrence of RB1 loss and BRCA mutation was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation.

The Prognostic Effect of Immune Cell Infiltration Depends on Molecular Subtype in Endometrioid Ovarian Carcinomas.

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.

p53 and ovarian carcinoma survival: an Ovarian Tumor Tissue Analysis consortium study.

Our 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.

Multiomic analysis of homologous recombination-deficient end-stage high-grade serous ovarian cancer.

High-grade serous ovarian cancer (HGSC) is frequently characterized by homologous recombination (HR) DNA repair deficiency and, while most such tumors are sensitive to initial treatment, acquired resistance is common. We undertook a multiomics approach to interrogate molecular diversity in end-stage disease, using multiple autopsy samples collected from 15 women with HR-deficient HGSC. Patients had polyclonal disease, and several resistance mechanisms were identified within most patients, including reversion mutations and HR restoration by other means. We also observed frequent whole-genome duplication and global changes in immune composition with evidence of immune escape. This analysis highlights diverse evolutionary changes within HGSC that evade therapy and ultimately overwhelm individual patients.

Methylation Signature Implicated in Immuno-Suppressive Activities in Tubo-Ovarian High-Grade Serous Carcinoma.

BACKGROUND: Better understanding of prognostic factors in tubo-ovarian high-grade serous carcinoma (HGSC) is critical, as diagnosis confers an aggressive disease course. Variation in tumor DNA methylation shows promise predicting outcome, yet prior studies were largely platform-specific and unable to evaluate multiple molecular features. METHODS: We analyzed genome-wide DNA methylation in 1,040 frozen HGSC, including 325 previously reported upon, seeking a multi-platform quantitative methylation signature that we evaluated in relation to clinical features, tumor characteristics, time to recurrence/death, extent of CD8+ tumor-infiltrating lymphocytes (TIL), gene expression molecular subtypes, and gene expression of the ATP-binding cassette transporter TAP1. RESULTS: Methylation signature was associated with shorter time to recurrence, independent of clinical factors (N = 715 new set, hazard ratio (HR), 1.65; 95% confidence interval (CI), 1.10-2.46; P = 0.015; N = 325 published set HR, 2.87; 95% CI, 2.17-3.81; P = 2.2 × 10-13) and remained prognostic after adjustment for gene expression molecular subtype and TAP1 expression (N = 599; HR, 2.22; 95% CI, 1.66-2.95; P = 4.1 × 10-8). Methylation signature was inversely related to CD8+ TIL levels (P = 2.4 × 10-7) and TAP1 expression (P = 0.0011) and was associated with gene expression molecular subtype (P = 5.9 × 10-4) in covariate-adjusted analysis. CONCLUSIONS: Multi-center analysis identified a novel quantitative tumor methylation signature of HGSC applicable to numerous commercially available platforms indicative of shorter time to recurrence/death, adjusting for other factors. Along with immune cell composition analysis, these results suggest a role for DNA methylation in the immunosuppressive microenvironment. IMPACT: This work aids in identification of targetable epigenome processes and stratification of patients for whom tailored treatment may be most beneficial.

Increased FOXJ1 protein expression is associated with improved overall survival in high-grade serous ovarian carcinoma: an Ovarian Tumor Tissue Analysis Consortium Study.

BACKGROUND: Recently, we showed a >60% difference in 5-year survival for patients with tubo-ovarian high-grade serous carcinoma (HGSC) when stratified by a 101-gene mRNA expression prognostic signature. Given the varied patient outcomes, this study aimed to translate prognostic mRNA markers into protein expression assays by immunohistochemistry and validate their survival association in HGSC. METHODS: Two prognostic genes, FOXJ1 and GMNN, were selected based on high-quality antibodies, correlation with protein expression and variation in immunohistochemical scores in a preliminary cohort (n = 134 and n = 80, respectively). Six thousand four hundred and thirty-four (FOXJ1) and 5470 (GMNN) formalin-fixed, paraffin-embedded ovarian neoplasms (4634 and 4185 HGSC, respectively) represented on tissue microarrays from the Ovarian Tumor Tissue Analysis consortium underwent immunohistochemical staining and scoring, then univariate and multivariate survival analysis. RESULTS: Consistent with mRNA, FOXJ1 protein expression exhibited a linear, increasing association with improved overall survival in HGSC patients. Women with >50% expression had the most favourable outcomes (HR = 0.78, 95% CI 0.67-0.91, p < 0.0001). GMNN protein expression was not significantly associated with overall HSGC patient survival. However, HGSCs with >35% GMNN expression showed a trend for better outcomes, though this was not significant. CONCLUSION: We provide foundational evidence for the prognostic value of FOXJ1 in HGSC, validating the prior mRNA-based prognostic association by immunohistochemistry.

CCNE1 and survival of patients with tubo-ovarian high-grade serous carcinoma: An Ovarian Tumor Tissue Analysis consortium study.

BACKGROUND: Cyclin 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. METHODS: Within the Ovarian Tumor Tissue Analysis consortium, amplification status and protein level in 3029 HGSC cases and mRNA expression in 2419 samples were investigated. RESULTS: High-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. CONCLUSION: This study provides large-scale validation that CCNE1 high-level amplification is associated with shorter survival, supporting its utility as a prognostic biomarker in HGSC.

Lifestyle and personal factors associated with having macroscopic residual disease after ovarian cancer primary cytoreductive surgery.

OBJECTIVE: The presence of macroscopic residual disease after primary cytoreductive surgery (PCS) is an important factor influencing survival for patients with high-grade serous ovarian cancer (HGSC). More research is needed to identify factors associated with having macroscopic residual disease. We analyzed 12 lifestyle and personal exposures known to be related to ovarian cancer risk or inflammation to identify those associated with having residual disease after surgery. METHODS: This analysis used data on 2054 patients with advanced stage HGSC from the Ovarian Cancer Association Consortium. The exposures were body mass index, breastfeeding, oral contraceptive use, depot-medroxyprogesterone acetate use, endometriosis, first-degree family history of ovarian cancer, incomplete pregnancy, menopausal hormone therapy use, menopausal status, parity, smoking, and tubal ligation. Logistic regression models were fit to assess the association between these exposures and having residual disease following PCS. RESULTS: Menopausal estrogen-only therapy (ET) use was associated with 33% lower odds of having macroscopic residual disease compared to never use (OR = 0.67, 95%CI 0.46-0.97, p = 0.033). Compared to nulliparous women, parous women who did not breastfeed had 36% lower odds of having residual disease (OR = 0.64, 95%CI 0.43-0.94, p = 0.022), while there was no association among parous women who breastfed (OR = 0.90, 95%CI 0.65-1.25, p = 0.53). CONCLUSIONS: The association between ET and having no macroscopic residual disease is plausible given a strong underlying biologic hypothesis between this exposure and diagnosis with HGSC. If this or the parity finding is replicated, these factors could be included in risk stratification models to determine whether HGSC patients should receive PCS or neoadjuvant chemotherapy.

Contrasting clinical characteristics and treatment patterns in women with newly diagnosed advanced-stage epithelial ovarian cancer in Australia, South Korea and Taiwan.

OBJECTIVE: The real-world INFORM study analyzed sociodemographics, treatment patterns and clinical outcomes for patients with newly diagnosed advanced epithelial ovarian cancer (EOC) in Australia, South Korea (S.Korea) and Taiwan preceding incorporation of poly(ADP-ribose) polymerase inhibitors into clinical practice. METHODS: Retrospective data from patients diagnosed with EOC (high-grade serous EOC for Taiwan) between January 2014 and December 2018 with ≥12 months follow-up from diagnosis were analyzed descriptively. Survival was evaluated by Kaplan-Meier with two-sided 95% confidence interval (CI). RESULTS: Of the 987 patients (Australia, 223; S.Korea, 513; Taiwan, 251), 98% received platinum-based chemotherapy (CT). In S.Korea and Taiwan 76.0% and 78.9% respectively underwent primary cytoreductive surgery; in Australia, 56.5% had interval debulking surgery. Bevacizumab was included in primary/maintenance therapy for 22.4%, 14.6% and 6.8% of patients in Australia, S.Korea and Taiwan, respectively. Patients receiving bevacizumab were high-risk (reimbursement policy) and achieved similar real-world progression-free survival (PFS) compared with CT only. Overall, the median real-world PFS (months; 95% CI) was similar across Australia (16.0 [14.63-18.08]), S.Korea (17.7 [16.18-19.27]) and Taiwan (19.1 [17.56-22.29]). CONCLUSION: This study reveals poor prognosis despite differences in demographics and treatment patterns for patients with EOC across Asia-Pacific suggesting the need for biomarker-driven novel therapies to improve outcomes.

Profiling the immune landscape in mucinous ovarian carcinoma.

OBJECTIVE: Mucinous ovarian carcinoma (MOC) is a rare histotype of ovarian cancer, with low response rates to standard chemotherapy, and very poor survival for patients diagnosed at advanced stage. There is a limited understanding of the MOC immune landscape, and consequently whether immune checkpoint inhibitors could be considered for a subset of patients. METHODS: We performed multicolor immunohistochemistry (IHC) and immunofluorescence (IF) on tissue microarrays in a cohort of 126 MOC patients. Cell densities were calculated in the epithelial and stromal components for tumor-associated macrophages (CD68+/PD-L1+, CD68+/PD-L1-), T cells (CD3+/CD8-, CD3+/CD8+), putative T-regulatory cells (Tregs, FOXP3+), B cells (CD20+/CD79A+), plasma cells (CD20-/CD79a+), and PD-L1+ and PD-1+ cells, and compared these values with clinical factors. Univariate and multivariable Cox Proportional Hazards assessed overall survival. Unsupervised k-means clustering identified patient subsets with common patterns of immune cell infiltration. RESULTS: Mean densities of PD1+ cells, PD-L1- macrophages, CD4+ and CD8+ T cells, and FOXP3+ Tregs were higher in the stroma compared to the epithelium. Tumors from advanced (Stage III/IV) MOC had greater epithelial infiltration of PD-L1- macrophages, and fewer PD-L1+ macrophages compared with Stage I/II cancers (p = 0.004 and p = 0.014 respectively). Patients with high epithelial density of FOXP3+ cells, CD8+/FOXP3+ cells, or PD-L1- macrophages, had poorer survival, and high epithelial CD79a + plasma cells conferred better survival, all upon univariate analysis only. Clustering showed that most MOC (86%) had an immune depleted (cold) phenotype, with only a small proportion (11/76,14%) considered immune inflamed (hot) based on T cell and PD-L1 infiltrates. CONCLUSION: In summary, MOCs are mostly immunogenically 'cold', suggesting they may have limited response to current immunotherapies.

The genomic and immune landscape of long-term survivors of high-grade serous ovarian cancer.

Fewer than half of all patients with advanced-stage high-grade serous ovarian cancers (HGSCs) survive more than five years after diagnosis, but those who have an exceptionally long survival could provide insights into tumor biology and therapeutic approaches. We analyzed 60 patients with advanced-stage HGSC who survived more than 10 years after diagnosis using whole-genome sequencing, transcriptome and methylome profiling of their primary tumor samples, comparing this data to 66 short- or moderate-term survivors. Tumors of long-term survivors were more likely to have multiple alterations in genes associated with DNA repair and more frequent somatic variants resulting in an increased predicted neoantigen load. Patients clustered into survival groups based on genomic and immune cell signatures, including three subsets of patients with BRCA1 alterations with distinctly different outcomes. Specific combinations of germline and somatic gene alterations, tumor cell phenotypes and differential immune responses appear to contribute to long-term survival in HGSC.

Germline BRCA variants, lifestyle and ovarian cancer survival.

OBJECTIVE: Women with ovarian cancer who have a pathogenic germline variant in BRCA1 or BRCA2 (BRCA) have been shown to have better 5-year survival after diagnosis than women who are BRCA-wildtype (non-carriers). Modifiable lifestyle factors, including smoking, physical activity and body mass index (BMI) have previously been associated with ovarian cancer survival; however, it is unknown whether these associations differ by germline BRCA status. METHODS: We investigated measures of lifestyle prior to diagnosis in two cohorts of Australian women with invasive epithelial ovarian cancer, using Cox proportional hazards regression to calculate adjusted hazard ratios (HRs) and 95% confidence intervals (CIs). RESULTS: In the combined studies (n = 1923), there was little association between physical activity, BMI or alcohol intake and survival, and no difference by BRCA status. However, the association between current smoking status before diagnosis and poorer survival was stronger for BRCA variant carriers (HR 1.98; 95% CI 1.20-3.27) than non-carriers (HR 1.18; 95% CI 0.96-1.46; p-interaction 0.02). We saw a similar differential association with smoking when we pooled results from two additional cohorts from the USA and UK (n = 2120). Combining the results from all four studies gave a pooled-HR of 1.94 (95% CI 1.28-2.94) for current smoking among BRCA variant carriers compared to 1.08 (0.90-1.29) for non-carriers. CONCLUSIONS: Our results suggest that the adverse effect of smoking on survival may be stronger for women with a BRCA variant than those without. Thus, while smoking cessation may improve outcomes for all women with ovarian cancer, it might provide a greater benefit for BRCA variant carriers.

TRACEBACK: Testing of Historical Tubo-Ovarian Cancer Patients for Hereditary Risk Genes as a Cancer Prevention Strategy in Family Members.

PURPOSE: Tubo-ovarian cancer (TOC) is a sentinel cancer for BRCA1 and BRCA2 pathogenic variants (PVs). Identification of a PV in the first member of a family at increased genetic risk (the proband) provides opportunities for cancer prevention in other at-risk family members. Although Australian testing rates are now high, PVs in patients with TOC whose diagnosis predated revised testing guidelines might have been missed. We assessed the feasibility of detecting PVs in this population to enable genetic risk reduction in relatives. PATIENTS AND METHODS: In this pilot study, deceased probands were ascertained from research cohort studies, identification by a relative, and gynecologic oncology clinics. DNA was extracted from archival tissue or stored blood for panel sequencing of 10 risk-associated genes. Testing of deceased probands ascertained through clinic records was performed with a consent waiver. RESULTS: We identified 85 PVs in 84 of 787 (11%) probands. Familial contacts of 39 of 60 (65%) deceased probands with an identified recipient (60 of 84; 71%) have received a written notification of results, with follow-up verbal contact made in 85% (33 of 39). A minority of families (n = 4) were already aware of the PV. For many (29 of 33; 88%), the genetic result provided new information and referral to a genetic service was accepted in most cases (66%; 19 of 29). Those who declined referral (4 of 29) were all male next of kin whose family member had died more than 10 years before. CONCLUSION: We overcame ethical and logistic challenges to demonstrate that retrospective genetic testing to identify PVs in previously untested deceased probands with TOC is feasible. Understanding reasons for a family member's decision to accept or decline a referral will be important for guiding future TRACEBACK projects.

High Prediagnosis Inflammation-Related Risk Score Associated with Decreased Ovarian Cancer Survival.

BACKGROUND: There is suggestive evidence that inflammation is related to ovarian cancer survival. However, more research is needed to identify inflammation-related factors that are associated with ovarian cancer survival and to determine their combined effects. METHODS: This analysis used pooled data on 8,147 women with invasive epithelial ovarian cancer from the Ovarian Cancer Association Consortium. The prediagnosis inflammation-related exposures of interest included alcohol use; aspirin use; other nonsteroidal anti-inflammatory drug use; body mass index; environmental tobacco smoke exposure; history of pelvic inflammatory disease, polycystic ovarian syndrome, and endometriosis; menopausal hormone therapy use; physical inactivity; smoking status; and talc use. Using Cox proportional hazards models, the relationship between each exposure and survival was assessed in 50% of the data. A weighted inflammation-related risk score (IRRS) was developed, and its association with survival was assessed using Cox proportional hazards models in the remaining 50% of the data. RESULTS: There was a statistically significant trend of increasing risk of death per quartile of the IRRS [HR = 1.09; 95% confidence interval (CI), 1.03-1.14]. Women in the upper quartile of the IRRS had a 31% higher death rate compared with the lowest quartile (95% CI, 1.11-1.54). CONCLUSIONS: A higher prediagnosis IRRS was associated with an increased mortality risk after an ovarian cancer diagnosis. Further investigation is warranted to evaluate whether postdiagnosis exposures are also associated with survival. IMPACT: Given that pre- and postdiagnosis exposures are often correlated and many are modifiable, our study results can ultimately motivate the development of behavioral recommendations to enhance survival among patients with ovarian cancer.

Translation of a circulating miRNA signature of melanoma into a solid tissue assay to improve diagnostic accuracy and precision.

Aim: Successful treatment of cutaneous melanoma depends on early and accurate diagnosis of clinically suspicious melanocytic skin lesions. Multiple international studies have described the challenge of providing accurate and reproducible histopathological assessments of melanocytic lesions, highlighting the need for new diagnostic tools including disease-specific biomarkers. Previously, a 38-miRNA signature (MEL38) was identified in melanoma patient plasma and validated as a novel biomarker. In this study, MEL38 expression in solid tissue biopsies representing the benign nevi to metastatic melanoma spectrum is examined. Patients & methods: Nanostring digital gene expression assessment of the MEL38 signature was performed on 308 formalin-fixed paraffin-embedded biopsies of nevi, melanoma in situ and invasive melanoma. Genomic data were interrogated using hierarchical clustering, univariate and multivariate statistical approaches. Classification scores computed from the MEL38 signature were analyzed for their association with demographic data and histopathology results, including MPATH-DX class, AJCC disease stage and tissue subtype. Results: The MEL38 score can stratify higher-risk melanomas (MPATH-Dx class V or more advanced) from lower-risk skin lesions (class I-IV) with an area under the curve of 0.97 (p < 0.001). The genomic score ranges from 0 to 10 and is positively correlated with melanoma progression, with an intraclass correlation coefficient of 0.85 with stage 0-IV disease. Using an optimized classification threshold of ≥2.7 accurately identifies higher-risk melanomas with 89% sensitivity and 94% specificity. Multivariate analysis showed the score to be a significant predictor of malignancy, independent of technical and clinical covariates. Application of the MEL38 signature to Spitz nevi reveals an intrasubtype profile, with elements in common to both nevi and melanoma. Conclusion: Melanoma-specific circulating miRNAs maintain their association with malignancy when measured in the hypothesized tissue of origin. The MEL38 signature is an accurate and reproducible metric of melanoma status, based on changes in miRNA expression that occur as the disease develops and spreads. Inclusion of the MEL38 score into routine practice would provide physicians with previously unavailable, personalized genomic information about their patient's skin lesions. Combining molecular biomarker data with conventional histopathology data may improve diagnostic accuracy, healthcare resource utilization and patient outcomes.

Evolution of core archetypal phenotypes in progressive high grade serous ovarian cancer.

The evolution of resistance in high-grade serous ovarian cancer (HGSOC) cells following chemotherapy is only partially understood. To understand the selection of factors driving heterogeneity before and through adaptation to treatment, we profile single-cell RNA-sequencing (scRNA-seq) transcriptomes of HGSOC tumors collected longitudinally during therapy. We analyze scRNA-seq data from two independent patient cohorts to reveal that HGSOC is driven by three archetypal phenotypes, defined as oncogenic states that describe the majority of the transcriptome variation. Using a multi-task learning approach to identify the biological tasks of each archetype, we identify metabolism and proliferation, cellular defense response, and DNA repair signaling as consistent cell states found across patients. Our analysis demonstrates a shift in favor of the metabolism and proliferation archetype versus cellular defense response archetype in cancer cells that received multiple lines of treatment. While archetypes are not consistently associated with specific whole-genome driver mutations, they are closely associated with subclonal populations at the single-cell level, indicating that subclones within a tumor often specialize in unique biological tasks. Our study reveals the core archetypes found in progressive HGSOC and shows consistent enrichment of subclones with the metabolism and proliferation archetype as resistance is acquired to multiple lines of therapy.

Development and Validation of the Gene Expression Predictor of High-grade Serous Ovarian Carcinoma Molecular SubTYPE (PrOTYPE).

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.