Lulu Sun

Morphologic Correlations With Homologous Recombination Deficiency in High-grade Serous Carcinomas

High-grade serous carcinomas (HGSCs) with homologous recombination deficiency (HRD) respond favorably to platinum therapy and poly ADP ribose polymerase (PARP) inhibitors. Mutations in BRCA1 and BRCA2 commonly cause HRD and have been associated with Solid, pseudoEndometrioid, and Transitional-like (SET-like) histology. Mutations in other homologous recombination repair (HRR) genes as well as epigenetic changes can also result in HRD; however, morphologic correlates have not been well-explored in these cases. We hypothesized that HGSCs with HRD, regardless of the etiology, are associated with specific morphologic features. Forty-three cases of HGSC with genomic profiling, which included HRR gene mutation analysis and HRD score, were evaluated. The morphologic patterns, degree of nuclear atypia, necrosis, mitotic index, and tumor-infiltrating lymphocytes (TILs) were determined. The results showed that HRD-high status was significantly associated with the presence of BRCA1/2 mutation, SET-like morphology, geographic necrosis, and severe nuclear atypia. Additional HRR pathway genes with oncogenic mutations identified included ATM, BRIP1, BLM, FANCC, CDK12, CHEK2, RAD51C, and RAD51D. Almost one-third of HRD-high tumors did not have mutations in any HRR pathway genes identified. In conclusion, HGSC with HRD, regardless of BRCA1/2-status, was associated with SET-like morphology and more severe nuclear atypia. Identifying and reporting these patterns of tumor morphology can prompt genomic profiling with prognostic, therapeutic, and genetic counseling implications.

A fully next-generation sequencing-based method of classifying molecular sub-types of endometrial cancer retains prognostic value and expands biomarker targets

The molecular classification of endometrial cancer into POLE-ultra-mutated, mismatch repair-deficient, p53-mutated, and no specific molecular profile sub-types has significant prognostic value and is recommended in the evaluation of all patients with endometrial cancer. Nonetheless, there has been inconsistent clinical implementation. One possible barrier is the current practice of using several different assays, each with its own result, that subsequently need to be integrated. We developed a single, fully next-generation sequencing (NGS)-based assay that sub-types endometrial samples and evaluated it against an existing algorithm. Molecular sub-typing was retrospectively performed on 142 formalin-fixed, paraffin-embedded endometrial cancer samples using a clinically validated NGS panel targeting 133 genes and additional loci for micro-satellite instability and tumor mutational burden. In parallel, these same samples were classified by the World Health Organization algorithm using mismatch repair and p53 immunohistochemistry, and POLE sequencing. Concordance between algorithms was assessed, and the prognostic value of each was evaluated. We further explored racial disparities in outcomes and assessed other prognostic and potentially therapeutic biomarkers. The sequencing-based method was highly concordant with the World Health Organization algorithm (136/142 patients, Cohen's κ = 0.94) and retained prognostic value, with a significant difference in overall survival and progression-free survival by sub-type, and similar stratification to that initially identified in The Cancer Genome Atlas analysis. In addition, 11 cases had sequence variants in the previously established prognostic biomarker CTNNB1, and 3 cases had potentially targetable sequence variants in ERBB2. Endometrial cancer outcomes in Black individuals were worse, in part owing to the increased incidence of sub-types with a poor prognosis. A fully sequencing-based assay streamlines molecular classification of endometrial cancer and retains the prognostic value of other validated methods, which may aid clinical implementation. The additional genomic information obtained with an NGS panel, beyond the classification markers, can broaden potentially applicable therapies.

APOBEC3A drives ovarian cancer metastasis by altering epithelial-mesenchymal transition

High-grade serous ovarian cancer (HGSOC) is the most prevalent and aggressive histological subtype of ovarian cancer and often presents with metastatic disease. The drivers of metastasis in HGSOC remain enigmatic. APOBEC3A (A3A), an enzyme that generates mutations across various cancers, has been proposed as a mediator of tumor heterogeneity and disease progression. However, the role of A3A in HGSOC has not been explored. We observed an association between high levels of APOBEC3-mediated mutagenesis and poor overall survival in primary HGSOC. We experimentally addressed this correlation by modeling A3A expression in HGSOC, and this resulted in increased metastatic behavior of HGSOC cells in culture and distant metastatic spread in vivo, which was dependent on catalytic activity of A3A. A3A activity in both primary and cultured HGSOC cells yielded consistent alterations in expression of epithelial-mesenchymal transition (EMT) genes resulting in hybrid EMT and mesenchymal signatures, providing a mechanism for their increased metastatic potential. Inhibition of key EMT factors TWIST1 and IL-6 resulted in mitigation of A3A-dependent metastatic phenotypes. Our findings define the prevalence of A3A mutagenesis in HGSOC and implicate A3A as a driver of HGSOC metastasis via EMT, underscoring its clinical relevance as a potential prognostic biomarker. Our study lays the groundwork for the development of targeted therapies aimed at mitigating the deleterious effect of A3A-driven EMT in HGSOC.

Phospho-RPA2 predicts response to platinum and PARP inhibitors in homologous recombination–proficient ovarian cancer

BACKGROUNDTreatment of tubo-ovarian high-grade serous carcinoma (HGSC) includes cytoreductive surgery, platinum-based chemotherapy, and often poly(ADP-ribose) polymerase (PARP) inhibitors. While homologous recombination (HR) deficiency is a well-established predictor of therapy sensitivity, over 50% of HR-proficient HGSCs also exhibit sensitivity. Currently, there are no biomarkers to identify which HR-proficient HGSCs will be sensitive to standard-of-care therapy. Replication stress may serve as a key determinant of response.METHODSWe evaluated phospho-RPA2-T21 (p-RPA2) foci via immunofluorescence as a biomarker of replication stress in formalin-fixed, paraffin-embedded HGSC samples collected at diagnosis from patients treated with platinum chemotherapy (discovery cohort, n = 31; validation cohort, n = 244) or PARP inhibitors (n = 63). Recurrent HGSCs (n = 38) were also analyzed. p-RPA2 score was calculated using automated imaging analysis.RESULTSSamples were defined as p-RPA2-high if more than 16% of cells had ≥2 p-RPA2 foci on automated analysis. In the discovery cohort, HR-proficient, p-RPA2-high HGSCs demonstrated significantly higher rates of a chemotherapy response score of 3 to platinum chemotherapy than HR-proficient, p-RPA2-low HGSCs. In the validation cohort, patients with HR-proficient, p-RPA2-high HGSCs had significantly longer survival after platinum treatment than those with HR-proficient, p-RPA2-low HGSCs. Additionally, the p-RPA2 assay effectively predicted survival outcomes in patients treated with PARP inhibitors and in recurrent HGSC samples.CONCLUSIONOur study underscores the importance of considering replication stress markers, such as p-RPA2, alongside HR status in therapeutic planning. This approach has the potential to increase the number of patients receiving effective therapy while reducing unnecessary toxicity.FUNDINGThe Reproductive Scientist Development Program, GOG Foundation, Pilot Translational and Clinical Studies function of the Washington University Institute of Clinical and Translational Sciences, the Foundation for Barnes-Jewish Hospital, Washington University School of Medicine Dean's Scholar Program, The Cancer Biology Pathway Training Grant (5T32CA113275-17), The Lucy, Anarcha, and Betsey (L.A.B.) Award from the Department of Obstetrics and Gynecology at Washington University School of Medicine, and Veterans Affairs Office of Research and Development (I01BX006020).

RAD51 Foci as a Biomarker Predictive of Platinum Chemotherapy Response in Ovarian Cancer

Abstract Purpose: To determine the ability of RAD51 foci to predict platinum chemotherapy response in high-grade serous ovarian cancer (HGSOC) patient-derived samples. Experimental Design: RAD51 and γH2AX nuclear foci were evaluated by immunofluorescence in HGSOC patient-derived cell lines (n = 5), organoids (n = 11), and formalin-fixed, paraffin-embedded tumor samples (discovery n = 31, validation n = 148). Samples were defined as RAD51-High if >10% of geminin-positive cells had ≥5 RAD51 foci. Associations between RAD51 scores, platinum chemotherapy response, and survival were evaluated. Results: RAD51 scores correlated with in vitro response to platinum chemotherapy in established and primary ovarian cancer cell lines (Pearson r = 0.96, P = 0.01). Organoids from platinum-nonresponsive tumors had significantly higher RAD51 scores than those from platinum-responsive tumors (P < 0.001). In a discovery cohort, RAD51-Low tumors were more likely to have a pathologic complete response (RR, 5.28; P < 0.001) and to be platinum-sensitive (RR, ∞; P = 0.05). The RAD51 score was predictive of chemotherapy response score [AUC, 0.90; 95% confidence interval (CI), 0.78–1.0; P < 0.001). A novel automatic quantification system accurately reflected the manual assay (92%). In a validation cohort, RAD51-Low tumors were more likely to be platinum-sensitive (RR, ∞; P < 0.001) than RAD51-High tumors. Moreover, RAD51-Low status predicted platinum sensitivity with 100% positive predictive value and was associated with better progression-free (HR, 0.53; 95% CI, 0.33–0.85; P < 0.001) and overall survival (HR, 0.43; 95% CI, 0.25–0.75; P = 0.003) than RAD51-High status. Conclusions: RAD51 foci are a robust marker of platinum chemotherapy response and survival in ovarian cancer. The utility of RAD51 foci as a predictive biomarker for HGSOC should be tested in clinical trials.