Simon A. Gayther’s research while affiliated with The University of Texas Health Science Center at San Antonio and other places

Germline BRCA1 and BRCA2 mutations (BRCA1/2mut) predispose women to all subtypes of breast cancer (BC) - ER positive, ER negative and triple negative disease. The cumulative risks in women by age 80 years for all breast cancer (BC) range from 45-75% in BRCA1mut carriers and 41-70% in BRCA2mut carriers compared to the general population (∼13% lifetime risk of BC for women in the USA). Many aspects of the underlying biology of these genes in subtype-specific BC development remain unknown. Specifically, although BRCA1 & BRCA2 interact in the same DNA double-strand break repair pathway and BRCA1mut & BRCA2mut both cause basal, triple negative BC, BRCA2mut are more likely than BRCA1mut to cause luminal like ER positive BC. We hypothesize that exposure of normal mammary tissues to estrogens causes ER positive BC in women with BRCA2mut compared to women with BRCA1mut. We differentiated induced pluripotent stem cells (iPSCs) from subjects with and without BRCA1/2mut into models of normal mammary tissues using a patient-specific iPSC-derived 3D mammary organoid system and studied the neoplastic phenotypes of organoids with and without estrogen (E2) exposure. The iPSC model system enables self-renewal and differentiation into multiple cell type lineages. Intrinsically, this allows cells to self-organize into different cell partners to study the differential effects of specific genetic and/or environmental risk factors on neoplastic growth. Thus, iPSC-derived, mammary models can reproduce the status of germline genetic mutations associated with the development of breast cancers. We first developed a protocol to generate iPSC derived mammary models from multiple female subjects with and without BRCA1mut or BRCA2mut. None of the BRCA1/2mut carriers had a history of BC and all had had mastectomy for BC prevention because of their BRCA1/2mut status. Immunofluorescent cytochemical staining (ICC) confirmed that all models stained for lineage specific markers (CK18, mammaglobin, CDH1), confirming their mammary tissue status. iPSC derived mammary organoid models from both BRCA1 and BRCA2 heterozygous mutation carriers conferred a neoplastic phenotype reminiscent of a ductal carcinoma in situ (DCIS), a proposed precursor of BC (i.e., organoid models retained the wildtype copy of BRCA1 or BRCA2) compared to BRCA wildtype (BRCA1/2WT) controls, which retained both copies of both genes (confirmed by whole genome sequencing). This suggests haploinsufficiency contributes to the phenotype in both BRCA1 and BRCA2 carriers. We will present the data after differentiation of BRCA1mut, BRCA2mut and BRCAWT iPSC derived mammary models and then exposed to 150 picomol E2 treatment. The goal was to evaluate if mammary organoids from BRCA2mut carriers and quantify if they are more sensitive to E2 exposure than BRCA1mut and BRCAWT models. This may suggest that they are more likely to develop into BRCA2mut driven ER positive BC, which would suggest a hormone-dependent neoplastic transformation in BRCA2mut carriers reflecting the ER positive subtype. These finding could present opportunities for reducing mortality due to ER positive BC by driving BRCA2mut cells to an ER positive BC phenotype: This tumor subtype is know to have improved survival and therapeutic response through hormonal dependent treatments such as Tomoxifen. Citation Format: Simon Gayther, Alyssa Okimoto, Subash Dhungana, Kate Lawrenson, Xiaojiang Cui, Nur Yucer. Modeling BRCA1 and BRCA2 Mammary Tissues and Estrogen Positive Breast Cancer Using iPSC-Derived Organoid Models [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-04-07.

High-grade serous ovarian cancer (HGSOC) is a highly aggressive and lethal form of ovarian cancer. Challenges to diagnosis and treatment include a lack of effective screening methods for early detection, the absence of cancer-specific symptoms, and the development of chemoresistance. The genomic instability of HGSOC, further complicated by homologous recombination deficiency (HRD), leads to heterogeneity in HGSOC tumors and patient response to treatment. This makes it challenging to develop a single, effective diagnostic and treatment approach for this disease. Proteogenomic studies have provided some insight into HGSOC biology, but a deeper understanding of the tumor proteome through chemotherapy and disease recurrence is needed. Here we have profiled the proteome of tumors from 29 HGSOC patients before and after multiple rounds of chemotherapy. The proteome of HGSOC tumors remained unchanged for individual patients even after numerous rounds of chemotherapy. Differential expression analysis revealed known and novel proteins associated with chemoresistance and HRD status, further supported by similar changes at the genetic and epigenetic levels. We found that HRD affected proteins related to immune pathways. HRD was also associated with more shared T cell receptor (TCR) CDR3 repertoires of tumor-infiltrating T cells and increased neoantigen counts. ERAP1, a protein involved in peptide trimming before antigen presentation to immune cells via MHC- class I, was overexpressed in homologous recombination proficient (HRP) tumors and negatively correlated to neoantigen count. Its potential role in immune suppression in HRP tumors makes it an attractive therapeutic target that may be effective in combination with immunotherapy, particularly for HRP tumors. 26-plex immunostaining of HGSOC whole tissues further revealed significant differences in the spatial proximities of immune cells to each other and to tumor cells based on HRD status. Through proteomic and imaging analysis, this work has shown that the immune landscape of HGSOC tumors is influenced by homologous recombination status and identified candidate drivers of HGSOC biology.

Antagonistic activities of the 53BP1 axis and the tumor suppressor BRCA1-BARD1 determine whether DNA double-strand breaks (DSBs) are repaired by end joining or homologous recombination. We show that the CTC1-STN1-TEN1 (CST) complex, a central 53BP1 axis component, suppresses DNA end resection by EXO1 and the BLM-DNA2 helicase-nuclease complex but acts by distinct mechanisms in restricting these entities. Whereas BRCA1-BARD1 alleviates the CST-imposed EXO1 blockade, it has little effect on BLM-DNA2 restriction. CST mutants impaired for DNA binding or BLM–EXO1 interaction exhibit a hyper-resection phenotype and render BRCA1-deficient cells resistant to poly(ADP–ribose) polymerase (PARP) inhibitors. Our findings mechanistically define the crucial role of CST in DNA DSB repair pathway choice and have implications for understanding cancer therapy resistance stemming from dysfunction of the 53BP1 axis.

Epithelial ovarian cancer (EOC) genetics research has been focused on germline or somatic mutations independently. Emerging evidence suggests that the somatic mutational landscape can be shaped by the germline genetic background. In this study, we aim to unravel the role of somatic alterations within EOC germline susceptibility regions by incorporating functional annotations. We investigate somatic events, including mutational signatures, point mutations, copy number alterations, and transcription factor binding disruptions, within 33 EOC germline susceptibility regions. Our analysis identifies significant associations between candidate germline susceptibility genes and somatic mutational signatures known to be key risk factors for EOC, such as mismatch repair deficiency, age-related mutagenesis, and homologous recombination deficiency. In addition, we find somatic point mutations and copy number alterations are significantly enriched in histotype-specific active enhancers and promoters within EOC risk loci. Furthermore, we examine the impact of germline variants and somatic mutations on transcription factor binding sites, identifying cancer developmental transcription factor motifs frequently affected by both types of mutations. Overall, our study highlights the importance of integrating germline and somatic mutations with regulatory and epigenomic data to gain insights into the genetic basis of EOC.

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.

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.

High grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy, killing more than 9,000 women each year in the United States alone. Nearly 80% of patients with HGSOC tumors will experience recurrence within 5 years, but little is known about the mechanisms that drive this process. Intratumor heterogeneity is believed to be a key feature of recurrence and resistance in HGSOC tumors, with early studies reporting diverse and complex mechanisms of the seeding of metastatic sites, including metastasis reseeding the primary tumor. Few studies have investigated temporal changes to clonality and structural variants through disease recurrence and the development of chemoresistance as surgical debulking is not frequently performed at this later stage. We performed multi-omic profiling in paired chemo-naive and chemoresistant tumors from 32 HGSOC patients to investigate the mutational and genomic landscape of disease progression. Somatic mutation profiles were largely conserved through disease progression. Mutational burdens did not significantly differ across recurrence but were driven by homologous recombination repair deficiency status. Clonal composition and dynamics were measured through variant allele frequency alterations as tumors progressed from primary chemo-naïve to recurrent chemo-resistant tumors. A novel candidate driver gene, MDC1 , from the homologous recombination repair pathway, was significantly mutated and over-represented in patients with homologous recombination proficient tumors, with somatic mutations clustered in a single exon. Tumor evolution and phylogeny revealed that few changes in clonal abundance and complexity occur across the disease course in these patients. Taking structural variants into account, homologous recombination repair proficient (HRP) tumors tend to be polyclonal while homologous recombination repair deficient (HRD) tumors tend to be monoclonal, accompanied by a longer progression-free survival than the HRP patients. Three distinct classes of tumors were identified by structural variant signature analysis: tumors defined by DNA losses, tumors defined by DNA gains, and tumors defined by copy number neutral changes, which were largely defined by HRD status. Each class displayed distinct regions of the chromosome that were frequently affected by large scale SV events (>5Mb). Although no regions were frequently altered in recurrent tumors, GO analysis revealed that recurrent tumors have a significantly reduced immune response, which was not seen in the primary tumors. Ultra long read sequencing validated a majority of the SVs identified in short read sequencing and identified additional SVs undetected by short reads. These analyses identify that the phenotype of high grade serous ovarian tumors as defined by mutation and clonality profiles is established early in disease development and remain largely unchanged through chemotherapy and recurrence. This, when considered with the significant inter-patient heterogeneity identified in HGSOC, demonstrates the need for personalized therapies based on tumor profiling.

Rare, germline loss-of-function variants in a handful of DNA repair genes are associated with epithelial ovarian cancer. The aim of this study was to evaluate the role of rare, coding, loss-of-function variants across the genome in epithelial ovarian cancer. We carried out a gene-by-gene burden test with various histotypes using data from 2573 non-mucinous cases and 13,923 controls. Twelve genes were associated at a False Discovery Rate of less than 0.1 of which seven were the known ovarian cancer susceptibility genes BRCA1 , BRCA2 , BRIP1 , RAD51C , RAD51D, MSH6 and PALB2 . The other five genes were OR2T35, HELB, MYO1A and GABRP which were associated with non-high-grade serous ovarian cancer and MIGA1 which was associated with high-grade serous ovarian cancer. Further support for the association of HELB association comes from the observation that loss-of-function variants in HELB are associated with age at natural menopause and Mendelian randomisation analysis shows an association between genetically predicted age at natural menopause and endometrioid ovarian cancer, but not high-grade serous ovarian cancer.

While polygenic scores (PGSs) have shown promise in advancing precision medicine by capturing the additive effects of common germline variants on inherited disease risk, they are presently limited by reduced performance outside of European-origin populations. We extend our previously developed Bayesian polygenic model (PGM) method, select and shrink with summary statistics (S4), to improve prediction accuracy in ancestrally diverse populations. We benchmark this multi-ancestry extension (S4-Multi) against alternative methods on both simulated and biobank data predicting type 2 diabetes, breast cancer, colorectal cancer, asthma, and stroke. In simulation tests, we find that S4-Multi achieves 169% improvement on average over its single ancestry S4 counterpart at prediction in non-European target populations. S4-Multi matches or exceeds top performing methods across the ancestry continuum. In biobank tests, we find that the top-performing PGM method varies considerably by target ancestry and phenotype, with S4-Multi achieving comparable performance to top multi-ancestry methods overall. However, S4-Multi does so while including between 9% and 77% fewer genetic variants relative to competing models, suggesting potential for robust performance in clinical settings with limited available genomic data.

Background As precision medicine advances, polygenic scores (PGS) have become increasingly important for clinical risk assessment. Many methods have been developed to create polygenic models with increased accuracy for risk prediction. Our select and shrink with summary statistics (S4) PGS method has previously been shown to accurately predict the polygenic risk of epithelial ovarian cancer. Here, we applied S4 PGS to 12 phenotypes for UK Biobank participants, and compared it with the LDpred2 and a combined S4 + LDpred2 method. Results The S4 + LDpred2 method provided overall improved PGS accuracy across a variety of phenotypes for UK Biobank participants. Additionally, the S4 + LDpred2 method had the best estimated PGS accuracy in Finnish and Japanese populations. We also addressed the challenge of limited genotype level data by developing the PGS models using only GWAS summary statistics. Conclusions Taken together, the S4 + LDpred2 method represents an improvement in overall PGS accuracy across multiple phenotypes and populations.