High-grade Serous Ovarian Cancer Patients Research Articles

Harnessing Antitumor Immunity in Ovarian Cancer.

Despite progress in other tumor types, immunotherapy is not yet part of the standard of care treatment for high-grade serous ovarian cancer patients. Although tumor infiltration by T cells is frequently observed in patients with ovarian cancer, clinical responses to immunotherapy remain low. Mechanisms for immune resistance in ovarian cancer have been explored and may provide insight into future approaches to improve response to immunotherapy agents. In this review, we discuss what is known about the immune landscape in ovarian cancer, review the available data for immunotherapy-based strategies in these patients, and provide possible future directions.

Abstract 6553: Targeting macrophages and regulatory T cells improves response to chemotherapy in high-grade serous ovarian cancer

Abstract Despite the initial response to chemotherapy, most high-grade serous ovarian cancer (HGSOC) patients will suffer disease relapse within two years of diagnosis with no currently successful immunotherapy options.We performed single-cell RNA sequencing (scRNAseq) on omental metastasis from HGSOC patients. Analysis of 64,097 immune cells showed that neoadjuvant chemotherapy (NACT) induced immunosuppressive mechanisms that counteracted its potentially positive effects. We showed that while NACT enhanced phagocytosis and antigen presentation, this was counterbalanced by upregulation of Stabilin1 (STAB1) in eight out of eleven macrophage subpopulations we identified. The NACT associated increase in macrophage STAB1 expression was validated in a separate cohort of eighty HGSOC patients at the protein level. STAB1 is a macrophage scavenger receptor that transports phagocytosed targets to undergo significant lysosomal degradation reducing the efficiency of antigen presentation. In vitro anti-stabilin1 antibody (anti-stab1 ab) -treated macrophages had significantly reduced antigen degradation and higher CD11C expression than isotype-treated ones. Furthermore, T cells co-cultured with anti-stab1 ab -treated macrophages had increased T cell proliferation and killing.We identified thirteen CD4 subpopulations by scRNAseq, five of which were Tregs. We showed that NACT significantly upregulated and activated Tregs. Tregs displayed a shared developmental trajectory with naïve and effector T cells suggesting that those are induced rather than natural Tregs. This was supported by TCR clonal sharing between Tregs and other effector cells. In vitro T cells treated with FOXP3 anti-sense oligonucleotide (ASO), AZD8701, had an anti-tumor cytokine profile and enhanced tumor cell killing. Analysis of 69,781 scRNAseq cells from control and chemotherapy-treated HGSOC syngeneic mouse model showed similar responses. In two HGSOC syngeneic mouse models (HGS2 and 30200), combinations of chemotherapy with anti-stab1 ab and/or Foxp3-ASO significantly increased median survival of mice with established peritoneal disease compared to chemotherapy alone. Bulk RNAseq of tumors treated with anti-stab1 ab were enriched with CXCL9 positive macrophages, while Foxp3-ASO treatment showed significant increase in T helper cell infiltration and activation. Potentially cured long-term survivors (300 days+) were resistant to tumor rechallenge.In a third HGSOC mouse model (60577), in which tumors relapsed after a good initial response to chemotherapy, combinations of chemotherapy with anti-stab1 ab and/or Foxp3-ASO significantly increased the post relapse survival compared to chemotherapy alone.We believe that modulating Tregs and STAB1 could improve response to chemotherapy and can have translational potential as each AZD8701 and a humanized anti-stab1 ab are in phase I clinical trials. Citation Format: Samar Elorbany, Chiara Berlato, Larissa Carnevalli, Simon Barry, Eleni Maniati, Jun Wang, Ranjit Manchanda, Julia Kzhyshkowska, Frances Balkwill. Targeting macrophages and regulatory T cells improves response to chemotherapy in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6553.

Abstract 182: Cyclooxygenase-2 blockade is crucial to restore natural killer cell activity before anti-CTLA-4 therapy against high-grade serous ovarian cancer

Abstract Objective: Here our goal was to evaluate the impact of the immunosuppressive enzyme cyclooxygenase-2 (COX-2) expression on the immune effector capacity against high-grade serous ovarian cancer (HGSOC) tumor cells through bioinformatic tools and in vitro validation. Methods: We performed an integrated bioinformatics analysis from transcriptomic data based on RNAseq (TCGA-OV, n=368; Australian cohort AOCS, n=80) and RNA microarrays (GSE26193, n=62; GSE30161, n=45). We estimated the proportion and characteristics of immune cells employing Gene Set Variation Analysis (GSVA), MIXTURE and Ecotyper cell deconvolution algorithms. Survival curves and Cox-regression models were built up according to known risk factors and immune scores predicted by in-silico analysis. Afterward, we validated these results using flow cytometry and cytotoxicity assays on circulating NK cells collected from advanced HGSOC patients treated at our institution (n=5). After three days of incubation, isolated NK cell cultures were stimulated with three pulses of a selective COX-2 inhibitor (celecoxib) or MOCK, daily. An anti-CTLA-4 monoclonal antibody (ipilimumab) or MOCK was added at the third pulse. Two ovarian cancer cell lines (SKOV3 and HeyA8) were treated similarly. Next, NK cells were co-cultured with SKOV3 or HeyA8 and cytotoxicity on these cells was examined using lactate dehydrogenase (LDH) cell viability assay. Results: Transcriptomic data evidenced a non-linear relationship between COX-2 gene, PTGS2, and CTLA4 in advanced stages of HGSOC. Hence, PTGS2/CTLA4 ratio allows us to investigate how COX-2 influences changes in the immune profile of these patients. Based on bioinformatics, a high PTGS2/CTLA4 ratio determines particularly dysfunctional NK cell states, along with the reduction of NK gene score associated with infiltration, cytotoxicity, and survival in HGSOC. Additionally, selective COX-2 blockade improves NK cell cytotoxicity against HGSOC cell lines independently of the CTLA-4 signaling. In fact, ipilimumab effect was almost absent in those cases with high COX2 activity. Interestingly, previous treatment with celecoxib on NK cells mainly explains the difference in cytotoxicity (p= 0.0253, two-way ANOVA test with Tukey’s multiple comparisons). Conclusions: This work represents the first evidence of the influence of COX-2 on NK cell function as a crucial factor for the efficacy of anti-CTLA-4 therapy in HGSOC patients who exhibited high tumoral levels of CTLA-4. HGSOC patients present dysfunctional circulating NK cells preferentially due to the strong CO-2 expression. Last, we emphasize the modulation of chronic inflammation to direct an immune pattern with greater antitumor capacity in HGSOC. (Funding: Fondecyt 1201083 and FONDAP 152220002 [MAC], CONICYT/Doctorado Nacional 2019-Folio 21190421 [FGV], CONICYT FONDAP 15130011 [MAC, IW]). Citation Format: Fernan Gomez-Valenzuela, Ignacio Wichmann, Felipe Suárez, Sumie Kato, Enrique Ossandón, Marcela Hermoso, Elmer Fernández, Mauricio A. Cuello. Cyclooxygenase-2 blockade is crucial to restore natural killer cell activity before anti-CTLA-4 therapy against high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 182.

Abstract 7662: Convolutional neural network algorithms and hematoxylin-and-eosin-stained images predict clinical outcomes in high-grade serous ovarian cancer patients with advanced age and stage

Abstract Ovarian cancer is the deadliest among gynecologic cancers. It is partly due to the mild symptoms in early stages and relatively high diagnostic age (about 63-year-old median age). These factors contribute to the short chemotherapy response time and survival time with about 18- and 36-months median progression-free survival and overall survival duration, respectively. The advancements of convolutional neural network (CNN) machine learning algorithms provide opportunities for the development of cost- and time-effective models which is urgently needed for ovarian cancer patients, especially for those with elevated risk. A CNN model was trained with hematoxylin and eosin (H&E) stained tumor whole slide images (WSIs) from ovarian cancer patients with advanced stages. 773 WSIs of 335 patients from TCGA-OV dataset were separated randomly for training and testing cohort. The model was then applied on testing cohort with advanced stage and age patients for H&E survival score. The c-index of the predicted risk on overall survival (OS) and progression-free survival (PFS) were 0.66 and 0.63, respectively. The log-rank p-values of patients between low and high risk were 0.00137 and 0.00138, respectively. The model was then further evaluated with an external dataset. The c-index of the predicted risk were 0.64 and 0.72 for OS and PFS, respectively. And the log-rank test between patients with low and high risk achieved p-values 0.017 and 0.0047 for OS and PFS, respectively. In conclusion, a novel prognostic CNN model was developed for ovarian cancer patients with advanced age and stage for better disease management. Citation Format: Chun Wai Ng, Kwong-kwok Wong, Berrett Lawson, Sammy Ferri-Borgogno, Samuel Mok. Convolutional neural network algorithms and hematoxylin-and-eosin-stained images predict clinical outcomes in high-grade serous ovarian cancer patients with advanced age and stage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7662.

Abstract 3682: Pre-surgical blood metabolites associated with ovarian cancer prognosis

Abstract Background: Ovarian cancer has a poor prognosis with a 5-year survival less than 50%, resulting > 14,000 deaths in the US annually and > 150,000 globally. Identifying prognostic biomarkers at time of diagnosis and elucidating the biological dysregulation among those who are more likely to progress after first-line treatment could inform personalized treatment strategies. Thus, we evaluated metabolites and metabolomic profiles in pretreatment blood associated with survival in women with high-grade serous ovarian cancer, the most common and most deadly histologic subtype. Method: We examined plasma metabolites in blood samples collected prior to ovarian cancer treatment in 80 high-grade serous ovarian cancer patients who participated in the PreOperative Pelvic Mass Study, a clinic-based study enrolling women undergoing surgery for a pelvic mass. Liquid chromatography tandem mass spectrometry was used to measure 524 known metabolites. We excluded metabolites with coefficient of variation ≥ 25% (n = 104), then removed metabolites with missing in > 20% of the samples (n = 3). Metabolites missing in < 20% of the samples were imputed to be half the minimum value for that metabolite, resulting in 417 unduplicated metabolites. All metabolite values were transformed to probit scores to achieve normality. Cox proportional hazard models were used to calculate the hazard ratios (HRs) and 95% confidence intervals (CIs) adjusting for age at diagnosis, body mass index (BMI), and stage. False discovery rate (FDR) was used for multiple testing correction. Metabolite set enrichment analysis (MSEA) was used to identify metabolite classes associated with prognosis. Results: Overall, none of the individual metabolites were significantly associated with survival after multiple testing correction (FDR > 0.05). However, 32 metabolites were associated with survival with p< 0.05, with 12 metabolites being associated with worse survival (HR range: 1.38 - 1.98) and 20 metabolites being associated with better survival (HR range: 0.54 - 0.73). MSEA revealed triglycerides (normalized enrichment score (NES) = 3.14), cholesteryl esters (NES = 2.43), and phosphatidylcholines (NES = 2.30) were positively associated with risk of death (FDR < 0.001). Steroids and steroid derivatives (NES = -1.93), sphingomyelins (NES = -2.45), and carnitines (NES = -2.27) were negatively associated with risk of death (FDR < 0.001). Diglycerides (NES = 1.96), lysophosphatidylethanolamines (NES = 1.72), and phosphatidylcholine plasmalogens (NES = 1.58) were positively associated and phosphatidylethanolamines (NES = -1.93) was negatively associated with risk of death with FDR < 0.05. Discussion: In this study, we explored plasma metabolites associated with mortality in high-grade serous ovarian cancer patients and observed multiple classes of lipid-related metabolites being associated with worse prognosis. Citation Format: Nan Lin, Oana A. Zeleznik, Julian Avila-Pacheco, Clary B. Clish, Allison F. Vitonis, Daniel W. Cramer, Kathryn L. Terry, Naoko Sasamoto. Pre-surgical blood metabolites associated with ovarian cancer prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3682.

Abstract 1553: S1PR1 directs tumorsphere proliferation in ovarian cancer

Abstract Despite the clinical efforts to improve detection and treatment of high-grade serous ovarian cancer (HGSOC), half of diagnosed women will die every year because of this disease. Notably, nearly all of them present with ascites at diagnosis, which is directly associated with poor prognosis. Malignant ascites production occurs when ovarian cancer cells shed from the primary tumor, creating a selective pressure environment that threatens their survival. Therefore, cancer cells cluster into tumorspheres, the main metastatic units in HGSOC. In recent years, valuable insights into the mutational and immunological landscapes of HGSOC have been gained. However, there is scarce evidence on the precise molecular mechanisms that enable tumorspheres to surpass the ascites bottleneck and metastasize. In this work, our group addresses this knowledge gap by deciphering the role of S1PR1 in ovarian tumorsphere biology. In this study, ascites samples and Formalin-Fixed Paraffin-Embedded (FFPE) tumors and peritoneal metastases from a cohort of 32 treatment-naïve HGSOC patients have been used. Furthermore, 3D spheroid models from four different ovarian cancer cell lines have also been developed for in vitro and in vivo assays. Our results indicate that up to 60% of cells within HGSOC tumorspheres are proliferating due to an increased expression of pro-proliferative receptors. Thus, cancer cells within tumorspheres that overexpress these receptors might benefit from autocrine and paracrine signaling through release of their ligands. In this work, we have identified S1P-S1PR1 as one of these ligand-receptor axes, which promotes an autocrine positive loop that increases the intermediates of sphingolipid metabolism in ovarian cancer spheroids. Here, we demonstrate that S1PR1 is highly expressed in ascites tumorspheres and, in consequence, overexpressed in metastases in comparison with primary tumors. Furthermore, we also report that proliferation levels of ovarian cancer spheroids positively correlate with S1PR1 endogenous expression. Consistently, these levels are significantly reduced when S1PR1 is knocked out or pharmacologically inhibited. In addition, our results indicate that MEK1/2-ERK is the key downstream pro-mitogenic pathway of S1PR1 in HGSOC tumorspheres, and that its pharmacological inhibition is effective in reducing growth and number of disseminations in vivo. Conclusively, our group is the first one in reporting that the axis S1P-S1PR1-MEK1/2 is the main proliferative mechanism in HGSOC tumorspheres, providing them with a selective advantage and, consequently, a greater metastatic potential. The preclinical findings presented here may represent an opportunity to further explore targeted therapeutic strategies to hamper disease progression in ovarian cancer patients with the poorest outcome. Citation Format: Núria Gendrau-Sanclemente, Agnès Figueras, Kristina Gracova, Elisenda Alsina-Sanchís, Juan Antonio Marín-Jiménez, Josefina Casas, August Vidal, Xavier Matias-Guiu, Sergi Fernandez-Gonzalez, Marc Barahona, Lola Martí, Jordi Ponce, Francesc Viñals. S1PR1 directs tumorsphere proliferation in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1553.

Abstract 1254: Integration of human omics analysis and new syngeneic tumor organoid models reveals that aberrant RAS/PI3K crosstalk triggers refractoriness in high-grade serous carcinoma

Abstract Refractory high-grade serous carcinoma (HGSC) is one of the most severe clinical problems in gynecology, but we have barely grasped the underlying mechanism of aggressiveness and chemo-resistance. Previous study has shown that homologous recombination genes, BRCA1/2-unmutated HGSC patients possess the potential to be in refractory condition, and their primary response to chemotherapy was heterogenous; thus, there can be subtype-specific determinant factors hatching refractoriness in BRCA1/2-unmutated HGSC patients. To explore the determinant factors, we performed gene set variation analysis and comprehensive log-rank tests on TCGA advanced serous ovarian cancer datasets. As a result, regulation of small GTPase signaling prominently correlated with poor prognosis uniquely in BRCA1/2-unmutated HGSC patients (p-value < 0.001), and eventually, pathway clustering analysis by variational Bayesian Gaussian mixture model discovered aberrant RAS/PI3K crosstalk as peculiar poor prognostic signature of BRCA1/2-unmutated HGSC patients. To elucidate the mechanism of poor progression triggered by RAS/PI3K, we utilized our syngeneic HGSC organoid models newly established from murine fallopian tube epithelium by making Myc overexpressing, and knocking out Trp53 and Rb1; as a supplement, the propriety of these genetic alterations to develop HGSC models was backed up by genetic profiles of high-grade serous ovarian cancer patients in AACR Project GENIE. After that, we knocked out Nf1 and Pten whose deletions were detected relatively with high frequency in HGSC to induce aberrant RAS/PI3K crosstalk in the primitive HGSC model. In-vivo evaluation and growth comparison indicated that aberrant RAS/PI3K crosstalk transformed Brca1/2-unmutated HGSC modeling cells into aggressive phenotypes such as poor survival (p-value < 0.01) and high growth rate (p-value < 0.001). Chemo-sensitivity assay also showed that the genetic manipulation made Brca1/2-unmutated HGSC modeling cells more tolerant to anti-tumor agents like carboplatin (p-value < 0.001), paclitaxel (p-value < 0.01), and olaparib (p-value < 0.01). To investigate biological processes that induce the aggressive and chemo-resistant phenotype, we performed gene set enrichment analysis among our HGSC models. The result showed that aberrant RAS/PI3K crosstalk induces cell cycle acceleration and downregulation of apoptosis in the HGSC cells, which backed up aggressive phenotype and chemo-resistance in our HGSC models. In conclusion, aberrant RAS/PI3K crosstalk transforms Brca1/2-unmutated HGSC modeling cells more aggressive and chemo-resistant, which was well recapitulated by our HGSC modeling organoids. The integration of our computational approach and experimental models will lead us to detect peculiar therapeutic targets against refractory HGSC. Citation Format: Tomohiro Tamura, Kenta Masuda, Shimpei Nagai, Keiyo Imaeda, Juntaro Yamasaki, Eiji Sugihara, Hiroyuki Nobusue, Yuji Otsuki, Rui Yamaguchi, Kazuhiro Sakurada, Wataru Yamagami, Hideyuki Saya, Osamu Nagano. Integration of human omics analysis and new syngeneic tumor organoid models reveals that aberrant RAS/PI3K crosstalk triggers refractoriness in high-grade serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1254.

Abstract 2491: The Oxford Classic can identify HGSOC patients who may benefit from EMT-targeting therapies

Abstract Introduction: Despite development of novel targeted therapies such as PARP inhibitors and anti-angiogenic drugs, there is a clear lack of treatment options for High Grade Serous Ovarian Cancer (HGSOC) patients who are Homologous Recombination Repair proficient (50% of cases) and those with intrinsic resistance to these drugs. In this study we demonstrate the ability of the Oxford Classic (OxC)1,2, a non-genetic classifier, to identify HGSOC patients who may benefit from EMT targeting drugs. Methods: 139 HGSOC diagnostic tumor tissue (Brescia cohort) underwent RNA sequencing. Scottish cohort3 was used for external validation and TCGA, AOCS & OVCAD datasets were used for meta-analysis. Deconvolution of tumor RNAseq data, survival analyses, differential gene expression (DGE) analysis, gene pathway analyses in R and tumor immune profiling using CIBERSORT were performed. Results: Risk stratification of HGSOC using the Oxford Classic Patients with a higher OxC-EMT score had 3.6 times increased risk of death (95%CI: 1.6-8.0; p=2e-03) compared to patients with a lower OxC-EMT score by a multivariable cox regression analysis of Brescia cohort. By Kaplan-Meier survival analyses, a significant difference in overall survival in Brescia cohort (p=9e-06), Scottish cohort (p=2e-03) and a combined set of 1023 cases (p=1e-04), was observed between EMT-low risk patients (OxC-EMT score-0) and EMT-high risk patients (OxC-EMT score>0). Notably, 5-year median survival of EMT-low risk and EMT-high risk group was 50% and 13%, resp. (95%CI: 36.1%-69.3% vs 7.1%-23.5%) in Brescia cohort. Therapeutic options for OxC-EMT-high risk group DGE analysis of EMT-low patients and EMT-very high patients (OxC-EMT score>0.5) identified 404 differentially expressed genes common to the datasets. These included genes related to extracellular matrix organisation (VCAN, TGFβI), epithelial cell proliferation (RUNX2, FABP4, SERPINF1), cell chemotaxis (CCL19, DUSP1), angiogenic factors (VEGFC, CXCL12) and transmembrane kinase signalling pathways (TGFβ1/3, PDGFRα/β, IGFBP4/5/6, Wnt11). Of note, key EMT transcription factors, TWIST1/2, SNAI1/2, ZEB1/2, and stemness marker, ALDH1A3, were 2-7 times overexpressed in EMT-very high group. Furthermore, immune modulators, IL6 and IL10 were significantly upregulated and M2 macrophages were significantly more abundant in EMT-very high patients. Multiplex IHC is currently underway to confirm the abundance of TAMs and CD8-positive TRM in the two risk groups. Conclusions: 1) The Oxford Classic-based EMT is a robust prognostic biomarker of overall survival in HGSOC, that faithfully represents the complex circuitry of pathways which are a hallmark of Epithelial to Mesenchymal Transition. 2) OxC-EMT risk stratification can identify HGSOC patients who may benefit from EMT-targeting therapies. Reference: 1) Hu Z. Cancer Cell 2020 2) Hu Z. Clin Cancer Res 2021 3) Hollis R. Clin Cancer Res 2022 Citation Format: Lena Rai, Antonella Ravaggi, Eliana Bignotti, Robb Hollis, Joel Nulsen, Leticia Campo, Alistair Easton, Mara Artibani, Mike Churchman, Federico Ferrari, Christopher Yau, Charlie Gourley, Franco Odicino, Ahmed Ahmed. The Oxford Classic can identify HGSOC patients who may benefit from EMT-targeting therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2491.

Abstract 278: Investigating the functional role of cancer-associated fibroblasts in the emergence of drug-tolerant persisters in ovarian cancer

Abstract Chemoresistance remains a major clinical challenge for the treatment of high-grade serous ovarian cancer (HGSC), the most lethal gynecologic malignancy. The majority of patients who initially respond to first-line platinum/taxane-based chemotherapy eventually relapse with chemoresistant disease, leading to poor survival outcomes. It is increasingly recognized that cancer cells can enter a reversible drug-tolerant persister (DTP) state through non-genetic mechanisms to evade chemotherapy-induced cytotoxicity prior to the development of chemoresistance. However, how environmental signals and extracellular factors from the tumor microenvironment drive the process remains poorly understood. Cancer-associated fibroblasts (CAFs) are a functionally heterogeneous population of activated fibroblasts. CAFs play a critical role in shaping the tumor microenvironment and tumor behaviors, including promoting tumor growth and mediating therapy resistance. Here, we demonstrated that HGSC cell lines co-cultured with patient-derived FAP-high CAFs, a subpopulation of CAFs previously identified in our lab to be tumor-promoting, significantly increase the formation of DTPs upon prolonged carboplatin treatment compared to the mono-cultured condition. Interestingly, the extracellular matrix (ECM) organization signature is enriched in FAP-high CAFs compared to their FAP-low counterparts, and we found that ECM deposited by FAP-high CAFs also facilitates the emergence of DTPs in HGSC cells. Future work will focus on elucidating the mechanisms by which FAP-high CAFs and the associated ECM promote DTP formation in HGSC cells through transcriptomic and proteomic profiling. This will aid the identification of potential therapeutic vulnerabilities to target cancer cells in the DTP state in FAP-high HGSC patients. Citation Format: Angel S. Ng, Ngoc Bui, Laurie Ailles. Investigating the functional role of cancer-associated fibroblasts in the emergence of drug-tolerant persisters in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 278.

Abstract 6407: Identification of novel chemotherapy-related CHIP variations in patients with high-grade serous ovarian cancer

Abstract Background: Hematopoietic lineage differentiation is subjected to genetic mutations that, due to fitness advantages, might give rise to clonally expanded populations. Clonal hematopoiesis of indeterminate potential (CHIP) is defined as the outgrowth of a single clone driven by acquired somatic mutations in hematopoietic stem cells (HSCs), in the absence of hematological abnormalities. Previous studies have shown the association of CH with aging and a higher risk of developing secondary hematologic malignancies in cancer patients treated with chemotherapy agents. It is therefore of great interest to study CH incidence prior to and post chemotherapy exposure and its association with the evolution of hematologic malignancies. We aimed to characterize CHIP variants of a highly selected group of patients with high-grade serous ovarian cancer (HGSC) who underwent neoadjuvant chemotherapy (NACT). Methods: Comprehensive ultra-high-depth whole exome sequencing, employing unique molecular barcode technologies, was performed using plasma-derived cell-free DNA (cfDNA) and matched white blood cells (WBC) DNA and tumor DNA from pre-NACT (n=10) and post-NACT (n=10) samples of patients with HGSC. CHIP variants were defined as heterozygous variants found in both the WBC and cfDNA samples from the same patients with variant allele frequency less than 5% in the WBC and higher than 0.5% in cfDNA samples. Results: Our analysis identified 93,088 variants, impacting 13,780 genes. Among them, 463 variants were already known and published, and 5,548 variants were common to the COSMIC genes signature. The number of CHIP variants significantly decreased after chemotherapy (p = 0.004). We then focused on CHIP variants enriched after chemotherapy and identified 77 that showed higher VAF in post-NACT cfDNA samples, which may be associated with the development of therapy-related acute myeloid leukemia/myelodysplastic syndrome (t-AML/MDS). Among them, interestingly, we found a missense mutation with protein change in CDK12 (chr17_39492765_G_T). We noticed that 38% of the CHIP variants found in cfDNA samples, were also found in matched tumor samples. Among these, another CDK12 missense mutation (chr17_39492768_A_T) showed higher VAF in post-NACT tumors. Conclusions: Our innovative approach detected a higher number of CHIP variants than currently characterized. Analysis of cfDNA from HGSC patients consistently demonstrated a reduction in CHIP mutations post-NACT. The subset of variants with increased VAF post-NACT may be associated with therapy-related t-AML/MDS. The detection of cfDNA CHIP variants in matched tumors highlights the potential predictive role of CHIP in t-AML/MDS development, emphasizing its consideration in therapeutic protocols. Citation Format: Sara Corvigno, Amma Asare, Jun Yao, Li Zhao, Nicole Fleming, Joseph Celestino, Richard A. Hajek, Ency A. Arboleda Goette, Ridge T. Rogers, Tri V. Nguyen, Raymond N. Montoya, Karen H. Lu, Amir A. Jazaeri, Shannon N. Westin, Anil K. Sood, Sanghoon Lee. Identification of novel chemotherapy-related CHIP variations in patients with high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6407.

Proteogenomic analysis of enriched HGSOC tumor epithelium identifies prognostic signatures and therapeutic vulnerabilities

We performed a deep proteogenomic analysis of bulk tumor and laser microdissection enriched tumor cell populations from high-grade serous ovarian cancer (HGSOC) tissue specimens spanning a broad spectrum of purity. We identified patients with longer progression-free survival had increased immune-related signatures and validated proteins correlating with tumor-infiltrating lymphocytes in 65 tumors from an independent cohort of HGSOC patients, as well as with overall survival in an additional 126 HGSOC patient cohort. We identified that homologous recombination deficient (HRD) tumors are enriched in pathways associated with metabolism and oxidative phosphorylation that we validated in independent patient cohorts. We further identified that polycomb complex protein BMI-1 is elevated in HR proficient (HRP) tumors, that elevated BMI-1 correlates with poor overall survival in HRP but not HRD HGSOC patients, and that HRP HGSOC cells are uniquely sensitive to BMI-1 inhibition.

Screening by Q Exactive liquid chromatography/tandem mass spectrometry identified Choline, 25-hydroxyvitamin D2, and SM(d18:0/16:1(9Z) (OH)) as biomarkers for high-grade serous ovarian cancer

High-grade serous ovarian cancer (HGSOC) has a high death rate and poor prognosis. The main causes of poor prognosis are asymptomatic early disease, no effective screening method at present, and advanced disease. Changes in cellular metabolism are characteristic of cancer, and plasma metabolome analysis can be used to identify biomarkers. In this study, we used Q Exactive liquid chromatography tandem mass spectrometry (LC–MS/MS, QE) to compare the differentiation between plasma samples (22 HGSOC samples and 22 normal samples). In total, we detected 124 metabolites, and an orthogonal partial least-squares-discriminant analysis (OPLS-DA) model was useful to distinguish HGSOC patients from healthy controls. Choline, 25-hydroxyvitamin D2, and sphingomyelin (d18:0/16:1(9Z) (OH))/SM(d18:0/16:1(9Z) (OH)) showed significantly differential plasma levels in HGSOC patients under the conditions of variable importance in projection (VIP) > 1, p < 0.05 using Student's t-test, and fold change (FC) ≥ 1.5 or ≤ 0.667. Metabolic pathway analysis can provide valuable information to enhance the understanding of the underlying pathophysiology of HGSOC. In conclusion, the Q Exactive LC/MS/MS method validation-based plasma metabolomics approach may have potential as a convenient screening method for HGSOC and may be a method to monitor tumor recurrence in patients with HGSOC after surgery SignificanceHigh-grade serous ovarian cancer (HGSOC) has a high death rate and poor prognosis. The main causes of poor prognosis are asymptomatic early disease, no effective screening method at present, and advanced disease. Changes in cellular metabolism are characteristic of cancer, and plasma metabolome analysis can be used to identify biomarkers. In this study, we used Q Exactive liquid chromatography tandem mass spectrometry (LC–MS/MS, QE) to compare the differentiation between plasma samples (20 HGSOC samples and 20 normal samples). In total, we detected 124 metabolites, and an orthogonal partial least-squares-discriminant analysis (OPLS-DA) model was useful to distinguish HGSOC patients from healthy controls. Choline, 25-hydroxyvitamin D2, and sphingomyelin (d18:0/16:1(9Z) (OH))/SM(d18:0/16:1(9Z) (OH)) showed significantly differential plasma levels in HGSOC patients under the conditions of variable importance in projection (VIP) > 1, p < 0.05 using Student's t-test, and fold change (FC) ≥ 1.5 or ≤ 0.667. Metabolic pathway analysis can provide valuable information to enhance the understanding of the underlying pathophysiology of HGSOC. In conclusion, the Q Exactive LC/MS/MS method validation-based plasma metabolomics approach may have potential as a convenient screening method for HGSOC and may be a method to monitor tumor recurrence in patients with HGSOC after surgery.

Constitutional BRCA1 and MGMT Methylation Are Significant Risk Factors for Triple-Negative Breast Cancer and High-Grade Serous Ovarian Cancer in Saudi Women.

Breast cancer (BC) and ovarian cancer (OC) are rapidly increasing in Saudi Arabia. BRCA1 and MGMT epimutations have been linked to a higher risk of these malignancies. The present research investigated the impact of these epimutations on the prevalence of BC and OC among Saudi women. DNA methylation was evaluated using methylation-specific PCR, whereas mRNA expression levels were assessed using qRT-PCR. We evaluated white blood cell (WBC)-BRCA1 methylation in 1958 Saudi women (908 BC patients, 223 OC patients, and 827 controls). MGMT methylation was determined in 1534 of the 1958 women (700 BC patients, 223 OC patients, and 611 controls). BRCA1 methylation was detected in 8.6% of the controls and 11% of the BC patients. This epimutation was linked to 13.8% of the early-onset BC patients (p = 0.003) and 20% of the triple-negative breast cancer (TNBC) patients (p = 0.0001). BRCA1 methylation was also detected in 14% of the OC patients (p = 0.011), 19.4% of patients aged <55 years (p = 0.0007), and 23.4% of high-grade serous ovarian cancer (HGSOC) patients. In contrast, the BRCA1 mutation was detected in 24% of the OC patients, 27.4% of patients aged ≥55 years, and 26.7% of the HGSOC patients. However, MGMT methylation was detected in 10% of the controls and 17.4% of the BC patients (p = 0.0003). This epimutation was linked to 26.4% of the late-onset BC patients (p = 0.0001) and 11% of the TNBC patients. MGMT methylation was also found in 15.2% of the OC patients (p = 0.034) and 19.1% of HGSOC patients (p = 0.054). Furthermore, 36% of the BRCA1-methylated patients and 34.5% of the MGMT-methylated patients had a family history of cancer, including breast and ovarian cancer. Notably, BRCA1 and MGMT mRNA levels were greater in the WBC RNA of the BC patients and cancer-free methylation carriers than in that of the OC patients. Our data indicate that BRCA1 and MGMT epimutations significantly contribute to the development of breast cancer and ovarian cancer in Saudi cancer patients. These blood-based biomarkers could help identify female patients at high risk of developing TNBC and HGSOC at an early age.

Abstract PR-012: Proteogenomic analysis of enriched tumor epithelium identifies prognostic signatures and an increased dependency of homologous recombination proficient cells on bmi1 in high grade serous ovarian cancer

Abstract Background: Proteogenomic characterization efforts of high-grade serous ovarian cancer (HGSOC) have largely assessed tumors with high tumor cellularity (“purity”) to enhance detection of cancer-related biomarkers. Comprehensive analyses of cancers independent of purity are needed to reflect a “real-world” spectrum of patient phenotypes. To improve identification of clinically relevant molecular alterations associated with HGSOC independent of tumor purity, we applied cellular enrichment techniques coupled with comprehensive multi-omic analyses in HGSOC patient tumors spanning a broad spectrum of purity. Methods: Seventy HGSOC patient tumors were selected exhibiting low to high tumor purity (20-90%) balanced by clinical outcomes. Whole genome sequencing (WGS), mRNA-seq, quantitative global proteomics, methylation array and reverse phase protein array analyses of Bulk Tumor and laser microdissected (LMD) Enriched Tumor (ET) cells separately for each case. Prognostic and homologous recombination deficient (HRD) expression signatures were validated in two independent cohorts. Results: Analysis of WGS in ET compared to WT resulted in significant increases in sensitivity to identify somatic SNV, indel, structural variants, and neoepitopes (SNV p = 4.8E−3; indel p = 7.7E−5; SV, p = 1.1E−8, neoepitopes, p= 1.0E−10). Following LMD, 63% of cases characterized as mesenchymal subtype (C4) in WT samples were reclassified to other molecular subtypes (1.0E−3) suggesting that historical HGSOC expression subtypes strongly reflect tumor purity. Analysis of paired primary and metastatic tumors demonstrated that WT proteomic profiles largely cluster by anatomic location while ET proteomic profiles co-cluster in a patient centric manner. Hierarchical cluster analysis identified patients with longer progression free survival associated with increased immune signatures and validated proteins correlating with tumor infiltrating lymphocytes (TILs) in 65 tumors collected from an independent cohort of 12 HGSOC patients, as well as with overall survival in an additional cohort of 126 HGSOC patients. We identified that homologous recombination deficient (HRD) tumors express transcriptomic and proteomic pathways associated with metabolism and oxidative phosphorylation that we validated in independent patient cohorts, including 69 HRD-positive HGSOC tumors. We further identified that polycomb complex protein BMI-1 is elevated in HR proficient (HRP) tumors, that elevated BMI-1 correlates with poor overall survival in HRP but not HRD HGSOC patients, and that HRP HGSOC cells exhibit increased sensitivity to BMI-1 inhibition. Conclusion: Proteogenomic alterations in HGSOC tumors uncovered using enrichment techniques highlight the importance of specimen preparation in the identification of tumor alterations. Our efforts provide insights into low purity HGSOC correlating TILs with improved disease prognosis, expression alterations associated with HRD status, and increased sensitivity of HRP tumor cells to BMI-1 inhibitors. Citation Format: George Larry Maxwell, Nicholas Bateman, Tamara Abulez, Anthony Soltis, Andrew McPherson, Seongmin Choi, Dale Garsed, Chunqiao Tian, Brian Hood, Kelly Conrads, Pang-ning Teng, Julie Oliver, Glenn Gist, Dave Mitchell, Tracy Litzi, Christopher Tarney, Clifton Dalgard, Matthew Wilkerson, Mariaelena Pierobon, Emmanuel Petricoin, Chunhua Yan, Daoud Meerzaman, Clara Bodelon, Nicolas Wentzensen, Jerry S. H. Lee, David Huntsman, Sohrab Shah, Craig Shriver, Neil Phippen, Kathleen Darcy, David Bowtell, Thomas Conrads. Proteogenomic analysis of enriched tumor epithelium identifies prognostic signatures and an increased dependency of homologous recombination proficient cells on bmi1 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 PR-012.

Abstract IA001: Placing our bets to make a difference

Abstract The presentation will focus genomics of high grade serous ovarian cancer (HGSC). I will briefly describe TRACEBACK, a study aimed at identifying deceased high-risk mutation carriers, and how a novel approach to patient ascertainment and testing may serve as a model for other familial cancer diseases. I will discuss some of key questions and directions arising from our studies of end-stage BRCA-related HGSC using samples collected through a rapid autopsy program. The major focus of the presentation will be on analyses of patients with exceptionally poor or favorable overall survival, including a cohort of BRCA1/2 patients with an unusually poor response to treatment and survival, and HGSC patients who survive more than 10 years following their diagnosis. Citation Format: David D. Bowtell. Placing our bets to make a difference [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 IA001.

Abstract A092: Ovarian tumor organotypic slices cultures for functional drug screening (TOSCA)

Abstract A092: Ovarian tumor organotypic slices cultures for functional drug screening (TOSCA)

Abstract B017: Understanding the impact of transporter heterogeneity on the efficacy of PARP inhibitors

Abstract B017: Understanding the impact of transporter heterogeneity on the efficacy of PARP inhibitors

Abstract B031: The PLK1 inhibitor Volasertib as a treatment for HSF1 and MYC coamplified ovarian cancer

Abstract Ovarian cancer is a disease that afflicts 1 in 78 women. This disease only has a 5-year relative survival rate of 49% due to the fact that the first line chemotherapy for this disease, platinum-based chemotherapy with taxanes, has an 80% recurrence rate. To improve the treatment for this disease, it would be wise to find biomarkers of sensitivity to other drugs. Using publicly available data from The Cancer Genome Project, our lab has shown that 36% of High Grade Serous Ovarian Cancers (HGSOC) have gene amplifications for both Cellular Myelocytomatosis (MYC) and Heat Shock Factor 1 (HSF1) genes. These genes encode for transcription factors and are known to contribute to malignant disease in multiple cancer types, including ovarian cancer. Evidence indicates that MYC and HSF1 are both essential to HGSOC cells with co-amplification of these genes, that MYC and HSF1 transcriptional activities are highly correlated in patient tumors, and the two transcription factors have a protein-protein interaction, all suggesting these two transcription factors cooperate to drive this subset of HGSOC tumors. Many drugs have been developed to inhibit MYC or HSF1 though they have not shown success in clinical development. We aimed to decrease the activity of these proteins by targeting polo-like kinase 1 (PLK1), a kinase that phosphorylates and regulates both MYC and HSF1 proteins. Active PLK1 levels were highly correlated with MYC and HSF1 protein levels and inhibition of PLK1 with volasertib reduced MYC and HSF1 protein levels and transcriptional activity. Furthermore, volasertib was found to have 155-fold greater potency in HGSOC cells with MYC-HSF1 co-amplification compared to cells without these genes amplified. The increased potency of volasertib in MYC-HSF1 co-amplified cells were also observed in clonogenic growth and spheroid models. Together, these data indicate that MYC and HSF1 cooperate in HGSOC when these genes are amplified and targeting a common kinase between them in PLK1 disrupts this cooperation. Furthermore, MYC and HSF1 co-amplification may serve as a biomarker for volasertib through a personalized medicine approach to treat HGSOC patients. Citation Format: Matthew O'Malley, Imade W. Williams, Bobby Walker, Haimanti Ray, Kenneth P. Nephew, Richard Carpenter. The PLK1 inhibitor Volasertib as a treatment for HSF1 and MYC coamplified 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 B031.

Abstract B089: Intratumoral expression analysis of mast cells in high grade serous ovarian cancer

Abstract Objective: The goal of this study is to examine intratumoral expression and phenotypic changes in mast cells following neoadjuvant chemotherapy (NACT) exposure in high grade serous ovarian cancer (HGSOC). Methods: 37 HGSOC matched pre-and post-NACT patient tumors were stained with a commercially available mast cell hemotoxic-based kit and both intratumoral and stromal mast cells were quantified. Immunohistochemistry was employed to stain for histamine in 19 matched HGSOC patient tumors and mean intensity and integrated optical density was quantified using Image J. Kaplan Meier curve analysis was employed to determine the relationship between mast cells levels and progression-free survival (PFS). Immortalized human mast cells, LUVA were treated with carboplatin and paclitaxel and specific genomic changes were measured by quantitative PCR (qPCR). Results. Mast cells were more prominently expressed in intraepithelial regions compared to stromal cells, however both populations were significantly (p&amp;lt;0.05) increased following NACT. Total mast cells stratified by upper and lower quartile demonstrated that higher levels were significantly (p&amp;lt;0.05) associated with improved PFS. Furthermore, histamine, a marker of mast cell degranulation was significantly (p&amp;lt;0.05) increased in HGSOC tumors post-NACT and significantly correlated (r=0.553, p=0.013) with the change in total mast cells. Following chemotherapy treatment LUVA cells demonstrated an over 7-fold increase in amphiregulin (AREG) expression (p&amp;lt;0.01), a key marker of mast cell activation. Interestingly, angiogenic and pro-tumorigenic factors IL-10, IL-8, and VEGFA exhibited no significant differences in expression following chemotherapy exposure. Conclusions: Overall, our findings suggest a correlation in NACT exposure and intratumoral mast cell recruitment, degranulation, and activation. Further studies include investigating differences in specific tumorigenic genes and cell signaling pathways in mast cells pre- and post- NACT, as well as determining if mast cells can impact HGSOC chemotherapy response. Citation Format: Julia McAdams, Jasmine Ebott, Payton De La Cruz, Nicole E. James. Intratumoral expression analysis of mast cells 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 B089.

Abstract A111: Development and analytical validation of a multiplexed targeted mass spectrometry assay to detect ovarian cancer protein biomarkers in sera

Abstract A111: Development and analytical validation of a multiplexed targeted mass spectrometry assay to detect ovarian cancer protein biomarkers in sera