High-grade Serous Ovarian Cancer Tumors Research Articles

Trastuzumab deruxtecan (DS-8201a), a HER2-targeting antibody-drug conjugate, demonstrates in vitro and in vivo antitumor activity against primary and metastatic ovarian tumors overexpressing HER2.

High-grade serous ovarian cancer (HGSOC) and ovarian clear cell carcinoma (CC), are biologically aggressive tumors endowed with the ability to rapidly metastasize to the abdominal cavity and distant organs. About 10% of HGSOC and 30% of CC demonstrate HER2 IHC 3 + receptor over-expression. We evaluated the efficacy of trastuzumab deruxtecan (T-DXd; DS-8201a), a novel HER2-targeting antibody-drug conjugate (ADC) to an ADC isotype control (CTL ADC) against multiple HGSOC and CC tumor models. Eleven ovarian cancer cell lines including a matched primary and metastatic cell line established from the same patient, were evaluated for HER2 expression by immunohistochemistry and flow cytometry, and gene amplification by fluorescence in situ hybridization assays. In vitro experiments demonstrated T-DXd to be significantly more effective against HER2 3 + HGSOC and CC cell lines when compared to CTL ADC (p < 0.0001). T-DXd induced efficient bystander killing of HER2 non-expressing tumor cells when admixed with HER2 3 + cells. In vivo activity of T-DXd was studied in HER2 IHC 3 + HGSOC and CC mouse xenograft models. We found T-DXd to be significantly more effective than CTL ADC against HER2 3 + HGSOC (KR(CH)31) and CC (OVA10) xenografts with a significant difference in tumor growth starting at day 8 (p = 0.0003 for KR(CH)31, p < 0.0001 for OVA10). T-DXd also conferred a survival advantage in both xenograft models. T-DXd may represent an effective ADC against primary and metastatic HER2-overexpressing HGSOC and CC.

Abstract 1059: Biomarkers analysis on samples from patients in EVT801 clinical trial: Patient characterization and immunomonitoring

Abstract Introduction: EVT801 is a highly selective small molecule inhibitor of VEGFR3 that acts by inducing tumor (lymph)-angiogenesis normalization in and around the tumor. EVT801 has shown compelling activity in a wide range of cancer models showing a decrease of tumor hypoxia and an increase of CD8pos T-cells infiltration into the tumor micro-environment. EVT801 was well-tolerated in preclinical toxicology studies. A phase I study is underway focusing primarily on understanding the safety, tolerability, and pharmacokinetics of EVT801 across a range of doses (NCT05114668). Methods: The first stage of the phase I trial aims to determine the maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) for EVT801. Clinical samples from enrolled patients have been collected to explore preliminary signals of the biological activity of the drug and characterize tumor phenotypes. Patient characterization includes histology and mRNA signature in archival biopsies. Target engagement and pharmacodynamics effects are investigated by immunomonitoring as well as assessment of a defined set of proteins as markers of inflammation and angiogenesis as identified in preclinical in vivo models. Conclusion: Our first biomarkers analyses on a subpopulation of patients with High Grade Serous Ovarian Cancer (HGS-OC) enrolled in the first stage of the study showed that levels of VEGFR3 expression had a negative correlation with CD8 positive T-cells infiltration in the tumor microenvironment and a tendency to positive correlation with a PD1 mAb resistance signature. VEGFR3 expression also tends to be correlated with higher levels of hypoxia (CAIX labelling). Our working hypothesis is that patients with hypoxic HGS-OC tumors with high VEGFR3 expression may benefit from EVT801 treatment; this will need to be reinforced by inclusion of additional patients in dedicated PD biomarkers cohorts during stage 2 of the clinical trial. Citation Format: Lise Davenne, Michael Fitzgerald, Pierre-Benoit Ancey, Oona Delpuech, Céline Poussereau-Pomié, Michael Esquerre, Michael R. Paillasse, Marie Mandron, Philippe Rochaix, Maha Ayyoub, Clara Maria Scarlata, Christine Caux, Christophe Caux, Philippe Cassier, Carlos Gomez-Roca, Jean-Pierre Delord, John Friend, Pierre Fons. Biomarkers analysis on samples from patients in EVT801 clinical trial: Patient characterization and immunomonitoring [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 1059.

Abstract 4249: Establishment and analysis of patient-derived high-grade serous ovarian cancer organoids for disease modeling and drug testing

Abstract Purpose: Ovarian cancer is the most lethal gynecologic cancer. Majority of ovarian cancers belong to the subgroup of high-grade serous ovarian cancer (HGSC). Its standard treatment is surgery and chemotherapy with platinum and taxane-based compounds, for which almost all patients become resistant. We have established a panel of patient derived 3-dimensional (3D) HGSC long-term tumor organoid cultures for personalized disease modeling, drug screening and evaluation of the efficiency of potential new treatments to be used in preclinical research. Methods: Tumor tissues or ascites from &amp;gt; 30 HGSC tumors were set up as long-term 3D organoid cultures (PDOs). RNA and whole genome sequencing was performed from the original tumors and from PDOs. Pluripotency and proliferation were assessed via high-throughput immunofluorescent detection of PAX8 and Ki67. Resistance towards standard treatment and response to additional selected drugs and drug combinations were evaluated in cell death and proliferation assays. Imaging and image-based quantifications were performed under 3D setup. PDOs were set up for in vivo drug testing in immunodeficient NOD/Shi-scid/IL-2Rgnull (NOG) mice. Summary: A set of HGSC PDOs of different longitudinal, prospective, and multiregional samples has been established representing the three evolutionary states of HGSC: evolving, maintaining and adaptive state. They are ready to be used as rational and effective models for studying drug interventions. Supportively, results of preclinical studies with PI3K inhibitor Alpelisib matched the published patient responses. Tumor evolutionary analysis based on data of longitudinal, prospective, and multiregional collected samples suggest further targetable pathways that can mediate chemotherapy resistance and pinpoint potential targets for drug intervention. Conclusion: Inhibition of these pathways with simultaneous targeting of other cellular functions, such as lysosomal-mediated cell death, may be highly effective alternative treatment option. 3D imaging and efficient image analysis are necessary tools to understand tumor organoid biology and drug responses to assist development and pre-clinical evaluation of new treatments. This study is a part of the DECIDER project (https://www.deciderproject.eu/, NCT04846933). Citation Format: Tuula Kallunki, Anna R. Lauridsen, Aikaterini Skorda, Alexandra Lahtinen, Marie L. Bay, Benita Rasmussen, Kaisa Huhtinen, Taru Muranen, Jaana Oikkonen, Johanna Hynninen, Sampsa Hautaniemi. Establishment and analysis of patient-derived high-grade serous ovarian cancer organoids for disease modeling and drug testing [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 4249.

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.

Abstract B076: Targeting cancer glycomes sensitizes ovarian tumors to immunotherapy

Abstract Aberrant glycans on tumor cell surface contribute to immune escape. The purpose of this study is to establish novel immunotherapeutic strategies for HGSOC tumors by targeting glycans-mediated immune escape in high-grade serous ovarian cancer (HGSOC). Here, we performed in vivo screens targeting 96 glycan structures among a series of mouse HGSOC tumors with different genotypes. We identified β1, 6-GlcNAc branched N-glycan as a major determinant of immune escape in a homologous recombination (HR)-proficiency dependent manner in HGSOCs. Mechanistically, BRCA1/2 transcriptionally suppresses the expression of MGAT5 that produces β1, 6-GlcNAc branched glycans. Using in vitro T cell killing assay, we observed that removal of β1, 6-GlcNAc branched glycans by a small molecule inhibitor or genetically knocking down MGAT5, sensitizes HR-proficient human HGSOC cells to T cells assault. Consistently, using orthotopic syngeneic mouse models, we observed that inhibition of β1, 6-GlcNAc branched glycans sensitizes and synergizes with anti-PD-L1 in HR-proficient, but not HR-deficient HGSOCs. In summary, we identified β1, 6-GlcNAc branched N-glycan as a key mediator of immune escape in HR-proficient HGSOCs. Our findings indicate that MGAT5 represents a therapeutic target to overcome immune escape in a HR-status dependent manner and inhibition of β1, 6-GlcNAc branched glycans sensitizes HR-proficient HGSOCs to immune checkpoint blockades. Citation Format: Hao Nie, Pratima Saini, Heng Liu, Wei Zhou, Chen Wang, Daniel Claiborne, Nan Zhang, Mohamed Abdel-Mohsen, Rugang Zhang. Targeting cancer glycomes sensitizes ovarian tumors to immunotherapy [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 B076.

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 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 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 PR012: Evolutionary dynamics of whole-genome doubling in ovarian cancer

Abstract Chromosomal instability (CIN) is a major driver of tumor progression and treatment resistance in many cancers. CIN is characterized by ongoing chromosome missegregation, segmental aneuploidy and whole-genome doubling (WGD), generating copy number heterogeneity that provides a substrate for natural selection. Although well characterized in model systems, the evolutionary dynamics and genomic impact of WGD is underexplored in primary tumors and metastases. Newly developed single-cell genomics assays now allow for the measurement of the whole genomes of single cells obtained from patient tissues, thus providing a direct view into the evolution of tumor genomes undergoing WGD. We performed multi-region single-cell whole genome sequencing (scWGS) in a cohort of patients with high-grade serous ovarian cancer (HGSOC), an archetype of high CIN cancers. We profiled a total of 70,644 single cells from 63 pre-treatment samples obtained for 36 HGSOC patients. For each patient, we computed total and allele-specific copy number per cell, and inferred the evolutionary history of each patient’s tumor, including clonal and subclonal WGD events and cell-specific whole-chromosome or arm-level copy number changes indicative of missegregations. To determine the role of CIN-associated micronuclei (MN) formation, we used high-resolution cGAS immunofluorescence (IF) microscopy to profile FFPE tissues site-matched to scWGS, and quantify spatially-resolved rates of MN formation and rupture across 120 samples. A majority of tumors were characterized by early clonal WGD (61%, 22/36 cases). The remaining patients exhibited subclonal WGD subpopulations averaging 10% of cancer cells in total. To understand the interplay between recent WGD and CIN across the cohort, we subset all patients cells into non-WGD, subclonal WGD, and clonal WGD and computed mis-segregation rates for these subsets. Non-WGD cells showed the lowest levels of mis-segregation. Subclonal WGD exhibited the highest CIN rates, including mis-segregation and multipolar cells indicative of CIN following a recent WGD event. Clonal WGD exhibited intermediate levels of mis-segregation indicative of stabilization following a historical WGD event. CIN following WGD often resulted in a reduction in ploidy relative to the ancestral WGD cell. Micronucleus rupture was ubiquitously detected in IF across HGSOC tumors, and MN rates varied widely between patients and across primary and metastatic sites. Micronucleus rupture commonly resulted from the impact of WGD, which increased sequestration of missegregated chromosomes into MN. Through multimodal single cell measurements of HGSOC tumors, we were able to characterize genomic complexity from individual cell division errors in HGSOC, and shed light into how the transient processes governing ovarian cancer cell evolution impact the genomes of cell populations. Citation Format: Ignacio Vázquez-García, Matthew Myers, Marc J. Williams, Samuel S. Freeman, Jun Li, Hongyu Shi, Gryte Satas, Nicholas Ceglia, Florian Uhlitz, MSK SPECTRUM Consortium, Britta Weigelt, Dmitriy Zamarin, Andrew McPherson, Samuel F. Bakhoum, Sohrab P. Shah. Evolutionary dynamics of whole-genome doubling in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr PR012.

Prediction of Chemoresistance-How Preclinical Data Could Help to Modify Therapeutic Strategy in High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is one of the most lethal tumors generally and the most fatal cancer of the female genital tract. The approved standard therapy consists of surgical cytoreduction and platinum/taxane-based chemotherapy, and of targeted therapy in selected patients. The main therapeutic problem is chemoresistance of recurrent and metastatic HGSOC tumors which results in low survival in the group of FIGO III/IV. Therefore, the prediction and monitoring of chemoresistance seems to be of utmost importance for the improvement of HGSOC management. This type of cancer has genetic heterogeneity with several subtypes being characterized by diverse gene signatures and disturbed peculiar epigenetic regulation. HGSOC develops and metastasizes preferentially in the specific intraperitoneal environment composed mainly of fibroblasts, adipocytes, and immune cells. Different HGSOC subtypes could be sensitive to distinct sets of drugs. Moreover, primary, metastatic, and recurrent tumors are characterized by an individual biology, and thus diverse drug responsibility. Without a precise identification of the tumor and its microenvironment, effective treatment seems to be elusive. This paper reviews tumor-derived genomic, mutational, cellular, and epigenetic biomarkers of HGSOC drug resistance, as well as tumor microenvironment-derived biomarkers of chemoresistance, and discusses their possible use in the novel complex approach to ovarian cancer therapy and monitoring.

MiR-146a inhibits ovarian tumor growth in vivo via targeting immunosuppressive neutrophils and enhancing CD8+ T cell infiltration

Immunotherapies have emerged as promising strategies for cancer treatment. However, existing immunotherapies have poor activity in high-grade serous ovarian cancer (HGSC) due to the immunosuppressive tumor microenvironment and the associated low tumoral CD8+ Tcell (CTL) infiltration. Through multiple lines of evidence, including integrative analyses of human HGSC tumors, we have identified miR-146a as a master regulator of CTL infiltration in HGSC. Tumoral miR-146a expression is positively correlated with anti-cancer immune signatures in human HGSC tumors, and delivery of miR-146a to tumors resulted in significant reduction in tumor growth in both ID8-p53-/- and IG10 murine HGSC models. Increasing miR-146a expression in tumors improved anti-tumor immune responses by decreasing immune suppressive neutrophils and increasing CTL infiltration. Mechanistically, miR-146a targets IL-1 receptor-associated kinase 1 and tumor necrosis factor receptor-associated factor 6 adaptor molecules of the transcription factor nuclear factor κB signaling pathway in ID8-p53-/- cells and decreases production of the downstream neutrophil chemoattractant, C-X-C motif chemokine ligand 1. In addition to HGSC, tumoral miR-146a expression also correlates strongly with CTL infiltration in other cancer types including thyroid, prostate, breast, and adrenocortical cancers. Altogether, our findings highlight the ability of miR-146a to overcome immune suppression and improve CTL infiltration in tumors.

Genetic characterization of primary and metastatic high-grade serous ovarian cancer tumors reveals distinct features associated with survival

High-grade serous ovarian cancer (HGSC) is the most lethal histotype of ovarian cancer and the majority of cases present with metastasis and late-stage disease. Over the last few decades, the overall survival for patients has not significantly improved, and there are limited targeted treatment options. We aimed to better characterize the distinctions between primary and metastatic tumors based on short- or long-term survival. We characterized 39 matched primary and metastatic tumors by whole exome and RNA sequencing. Of these, 23 were short-term (ST) survivors (overall survival (OS) < 3.5 years) and 16 were long-term (LT) survivors (OS > 5 years). We compared somatic mutations, copy number alterations, mutational burden, differential gene expression, immune cell infiltration, and gene fusion predictions between the primary and metastatic tumors and between ST and LT survivor cohorts. There were few differences in RNA expression between paired primary and metastatic tumors, but significant differences between the transcriptomes of LT and ST survivors in both their primary and metastatic tumors. These findings will improve the understanding of the genetic variation in HGSC that exist between patients with different prognoses and better inform treatments by identifying new targets for drug development.

Tumor and local lymphoid tissue interaction determines prognosis in high-grade serous ovarian cancer

Tertiary lymphoid structure (TLS) is associated with prognosis in copy-number-driven tumors, including high-grade serous ovarian cancer (HGSOC), although the function of TLS and its interaction with copy-number alterations in HGSOC are not fully understood. In the current study, we confirm that TLS-high HGSOC patients show significantly better progression-free survival (PFS). We show that the presence of TLS in HGSOC tumors is associated with B cell maturation and cytotoxic tumor-specific Tcell activation and proliferation. In addition, the copy-number loss of IL15 and CXCL10 may limit TLS formation in HGSOC; a list of genes that may dysregulate TLS function is also proposed. Last, a radiomics-based signature is developed to predict the presence of TLS, which independently predicts PFS in both HGSOC patients and immune checkpoint inhibitor (ICI)-treated non-small cell lung cancer (NSCLC) patients. Overall, we reveal that TLS coordinates intratumoral B cell and Tcell response to HGSOC tumor, while the cancer genome evolves to counteract TLS formation and function.

Abstract 102: Transient chromosomal instability as a driver of ovarian cancer genome evolution

Abstract Chromosomal instability (CIN) is a major driver of tumor progression and treatment resistance in many cancers. CIN is characterized by ongoing chromosome missegregation, generating copy number heterogeneity that provides a substrate for natural selection. Although CIN has been well studied in model systems, the evolutionary dynamics and genomic impact of CIN is underexplored in primary tumors and metastases. Newly developed single cell genomics assays allow for the measurement of the whole genomes of single cells obtained from patient tissues, thus providing a direct view into the evolution of tumor genomes undergoing CIN. To study the single-cell and spatial variation of chromosomally unstable tumors, we performed multi-region analysis of 128 tumor samples in a cohort of 45 patients with high-grade serous ovarian cancer (HGSOC), an archetype of high CIN cancers. We performed single-cell whole genome sequencing (scWGS) and profiled a total of 70,644 single cells from 63 pre-treatment samples obtained across 43 patients. For each patient, we computed total and allele specific copy number per cell, and inferred the evolutionary history of each tumor, including clonal and subclonal whole genome doubling (WGD) events and cell-specific whole chromosome or arm level copy number changes, indicative of missegregations. To determine whether missegregated chromosomes fail to become part of the primary nucleus (PN) upon mitotic exit and become enclosed in a micronucleus (MN), we used high-resolution cGAS immunofluorescence (IF) microscopy to profile site-matched FFPE tissues and quantify spatially-resolved rates of MN formation and rupture across 128 site-matched samples. A majority of tumors were characterized by early clonal WGD (61%, 22/36 cases). The remaining patients exhibited subclonal WGD subpopulations averaging 10% of the patient’s cells in total. To understand the interplay between recent WGD and CIN across the cohort, we subset all patients cells into non-WGD, subclonal WGD, and clonal WGD and computed missegregation rates for these subsets. Non-WGD cells showed the lowest levels of missegregation. Subclonal WGD exhibited the highest rates of missegregation indicative of CIN following a recent WGD event. Clonal WGD exhibited intermediate levels of missegregation indicative of stabilization following a historical WGD event. CIN following WGD often resulted in a reduction in ploidy relative to the ancestral WGD cell. Using IF, we quantified the frequent breakdown of the MN membrane, and estimated spatially-resolved CIN rates by tracking localization of cGAS to the MN, which revealed elevated rates of MN formation and rupture. Through multimodal single cell measurements of HGSOC tumors, we were able to characterize genomic complexity from individual cell division errors in HGSOC, and shed light into how the transient processes governing ovarian cancer cell evolution impact the genomes of cell populations. Citation Format: Ignacio Vazquez-Garcia, Florian Uhlitz, Marc J. Williams, Jun Li, Hongyu Shi, Samuel Freeman, Matthew Myers, MSK SPECTRUM Consortium, Britta Weigelt, Dmitriy Zamarin, Andrew McPherson, Samuel F. Bakhoum, Sohrab P. Shah. Transient chromosomal instability as a driver of ovarian cancer genome evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 102.

Abstract LB156: Identified the ISG15 mediated extracellular vesicles drives ovarian cancer progression and metastasis: extracellular vesicular ISG15 is a potential biomarker and therapeutic target

Abstract Background &amp; Objective: High-grade serous ovarian cancer (HGSOC) accounts for over 80% of all epithelial ovarian cancer (OC) diagnoses, and the majority of patients with HGSOC are diagnosed with advanced metastatic disease. Aberrant expression of interferon stimulated gene 15 (ISG15) has been demonstrated in human malignancies, and we have identified that ISG15 is highly expressed in HGSOC tumors and peritoneal ascites. This study seeks to determine whether ISG15 contributes to HGSOC progression and metastasis through extracellular vesicle (EVs) secretion. Method: ISG15 expression was analyzed in ascites samples and primary ovarian cancer cells (POCC) from different patients by ELISA and WB. Cell surface biotinylation assay was done to show the modulation of endo and exocytosis by ISG15 and STAT3. Immunoprecipitation pull down assay was done to demonstrate the interaction of ISG15 with activated STAT3. Confocal microscopy showed the co-localization of STAT3 with the endosome marker TSG101. In-vivo studies were done using bio-luminescence imaging in orthotopic ovarian tumor mouse models to measure tumor progression and metastasis. Results: ISG15 was found to be significantly elevated in HGSOC metastases (pelvic, mesenteric and diaphragmatic samples) as compared to primary ovarian tumors or benign samples. We observed that ISGylation was increased in POCC cells derived from ascites with increased USP18 expression. Our results confirmed a significant decrease in EV’s secretion in ISG15 KD POCC cells. Mice injected with ISG15 OE -POCC cells showed increased tumor burden when compared to the ISG15Kd cells in mice. Furthermore, we observed a significant reduction of ovarian tumor growth and metastasis in an orthotopic mouse model treated with a small molecule inhibitor-DAP5 that targeted ISG15 or exosome blocker (Amiloride) compared to untreated mice. In addition, we found the expression of ISG15 within the EVs represents a promising development in elucidating prognostic markers for HGSOC patients. Conclusion: Based on our results, ISG15 expression is elevated in HGSOC patient ascites and metastatic disease sites. The aberrant expression of ISG15 in patient ascites plays a key role in the secretion of EVs carrying ISG15, which contributes to HGSOC progression and metastases. Our study provides the pre-clinical evidence regarding new molecular targets, novel prognostic markers, and innovative therapeutic strategies for HGSOC, aiming to ultimately improve the survival of patients suffering from this disease. Citation Format: Kalpana Deepa Priya Dorayappan, Vincent Wagner, Dongju Park, Meghan M. Newcomer, Michelle DS Lightfoot, Deepika Kalaiyarasan, Takahiko Sakaue, Wafa Khadraoui, Casey Cosgrove, Larry J. Maxwell, Qi-En Wang Wang, David O’Malley, Raphael E. Pollock, David E. Cohn, Selvendiran Karuppaiyah. Identified the ISG15 mediated extracellular vesicles drives ovarian cancer progression and metastasis: extracellular vesicular ISG15 is a potential biomarker and therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB156.

Abstract LB195: Immunotherapies that repolarize macrophages and CD4 T cells enhance the effect of chemotherapy in high-grade serous ovarian cancer

Abstract We asked if single-cell RNA sequencing (scRNA-seq) analysis of tumor immune cells in high-grade serous ovarian cancer (HGSOC) would reveal ways to enhance effects of neo-adjuvant chemotherapy (NACT). Analysis of 64,097 immune cells from seven HGSOC omental metastases showed that positive effects of NACT on immune responses were counterbalanced by induction of immunosuppressive mechanisms, especially related to antigen presentation and Treg activity. We identified stabilin-1 (Clever-1) on macrophages and FOXP3 in Tregs as targets and validated actions of their inhibitors in vitro. ScRNA-seq analysis of 69,781 cells from chemotherapy-treated syngeneic mouse HGSOC tumors showed significant agreement with the patient results. Combinations of chemotherapy with anti-stabilin1 antibody and/or FOXP3 inhibitor (AZD8701), FOXP3i, significantly increased median survival of mice with established peritoneal disease. Long-term survivors (300 days+) were resistant to tumor rechallenge. Analysis of bulk RNA sequencing data from treated murine tumors confirmed our hypothesis and suggested potential mechanisms to explain the anti-tumor response seen in the murine models. Tumors treated with chemotherapy plus anti-stabilin1 antibody showed significant enrichment of anti-tumor macrophage signature and antigen presentation pathway compared to chemotherapy alone. Tumors treated with chemotherapy plus FOXP3i in combination with anti-stabilin1 antibody showed significant upregulation of antigen presentation, T cell activation and cytotoxicity and T helper 1 differentiation pathways. There was significant reduction in FOXP3 positive cells and significant increase of Tbet positive cells in FOXP3i treated murine tumors suggesting repolarization of naive T cells in the tumor microenvironment towards a more anti-tumor T helper phenotype. As there are early phase clinical trials of FOXP3 inhibitor and anti-stabilin1 (Clever-1) antibody in patients with advanced cancer, these results suggest ways of improving response to chemotherapy not only in HGSOC but in other cancers that are treated by NACT. Citation Format: Samar Elorbany, Chiara Berlato, Larissa Carnevalli, Eleni Maniati, Jun Wang, Simon Barry, Ranjit Manchanda, Julia Kzhyshkowska, Frances Balkwill. Immunotherapies that repolarize macrophages and CD4 T cells enhance the effect of chemotherapy in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB195.

Abstract 600: Tumour and local lymphoid tissue interaction determines prognosis in high grade serous ovarian cancer

Abstract Tertiary lymphoid structure (TLS) is an ectopic lymphoid organ found in some tumors. The presence of TLS is recently proposed as a predictive biomarker for immune checkpoint inhibitors (ICI) in mutation-driven tumors. It is also associated with prognosis in copy number-driven tumors, including high grade serous ovarian cancer (HGSOC), although the function of TLS and its interaction with copy-number alterations in HGSOC is not fully understood. In the current study, we comprehensively analyze immune profile, copy number profile and quantitative imaging profile from 242 HGSOC patients (HH cohort), and combine these with two public single cell RNA-sequencing datasets (Lambrechts cohorts; n=9, 29,999 cells). TLS-high HGSOC patients show significantly better progression free survival in both HH and TCGA cohorts. We show that presence of TLS in HGSOC tumors is associated with B-cell maturation, resulting in higher and more specific immunoglobulin production; TLS also leads to more cytotoxic tumor-specific T-cells activation and proliferation. Through integrated analysis of single cell RNA-sequencing and whole exome sequencing data, we reveal that the copy number loss of IL15 and CXCL10 may limit TLS formation in HGSOC; we also propose a list of genes that may dysregulate TLS function. A candidate gene, DCAF15, is found to confer resistance to NK-mediated cytotoxicity in HGSOC cell lines. Lastly, we develop a radiomics-based signature to predict presence of TLS, which independently predicts PFS in both HGSOC patients and ICI-treated NSCLC patients. Overall, we reveal that TLS coordinates intratumoral B-cell and T-cell response against HGSOC tumor, while cancer genome evolves to counteract TLS formation and function. Citation Format: Haonan Lu, Georg Wengert, Hantao Lou, Reema Paudel, Naina Patel, Saral Desai, Bill Crum, Kristofer Linton-Reid, Mitchell Chen, Dongyang Li, Jacey Ip, Francesco Mauri, David J. Pinato, Andrea Rockall, Susan J. Copley, Sadaf Ghaem-Maghami, Eric O. Aboagye. Tumour and local lymphoid tissue interaction determines prognosis in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 600.

Abstract 206: Organoid models for predicting drug response in high grade serous and triple negative breast cancer

Abstract While most patients with high grade serous ovarian cancer (HGSC) respond to platinum-based chemotherapy, the response is rarely durable and recurrence almost inevitable. Women with triple negative breast cancer (TNBC) urgently require effective therapeutic options. In women with HGSC and TNBC (with mutations in BRCA1/2) treatment with a Poly-ADP-ribose inhibitor (PARPi) has emerged as a standard of care. In models of HGSC and TNBC senescence may play a role in PARPi resistance with i Bcl-xL, a member of the Bcl-2 family of proteins, preventing apoptosis. Therefore, we have initiated a clinical trial whereby a PARPi and then, an inhibitor of Bcl-xL, Navitoclax, will be added to their course of treatment [NCT05358639]. Bcl-xL is only one of the five known inhibitors of apoptosis, at present we do not know in vivo how these will impact the efficacy of the PARPi/Navitoclax combination or if they will provide other targets for effective therapy. Our hypothesis is that patient derived organoids can be used as a pragmatic way to assess the efficacy of new drugs and to identify for individual patients which drug(s) and drug combinations are likely to be most efficacious. In particular, with reference to the clinical trial, identify for individual patients which Bcl-2 protein inhibitor will synergize with a PARPi to optimize treatment. Currently we are using organoids to test novel drugs and drug combinations and to develop biomarkers for HGSC and TNBC treatment response. A limitation to the use of organoids generated from biopsy samples is the lack of efficient and reliable experimental models that recapitulate in vitro patient tumors faithfully enough to facilitate translation to therapeutic decisions for patients. By adapting the relatively new technique of conditional reprogramming and combining it with novel hydrogel based synthetic ECM supports we can reproducibly generate HGSC and TNBC patient-specific tumor organoids models in two weeks with greater than 90% success. Organoids grown in 384 or 1536 well format are stained with novel non-toxic dyes and chemoresponses to drugs are inferred from automatically acquired 3D confocal image stacks using deep learning AI algorithms that enable automated analyses. Our data suggest that this approach captures the inherent heterogeneity of the disease, albeit local to the sampled site. We are now employing deep learning AI algorithms to enable automated analyses of 3D confocal image stacks of organoids and infer drug responses that will be compared to patient responses in the clinical trial. Citation Format: Helen J. MacKay, Alla Buzina, Betty Li, Lilian Gien, David Andrews. Organoid models for predicting drug response in high grade serous and triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 206.

Evaluation of mitochondrial biogenesis and ROS generation in high-grade serous ovarian cancer.

Ovarian cancer is one of the leading causes of death for women with cancer worldwide. Energy requirements for tumor growth in epithelial high-grade serous ovarian cancer (HGSOC) are fulfilled by a combination of aerobic glycolysis and oxidative phosphorylation (OXPHOS). Although reduced OXPHOS activity has emerged as one of the significant contributors to tumor aggressiveness and chemoresistance, up-regulation of mitochondrial antioxidant capacity is required for matrix detachment and colonization into the peritoneal cavity to form malignant ascites in HGSOC patients. However, limited information is available about the mitochondrial biogenesis regulating OXPHOS capacity and generation of mitochondrial reactive oxygen species (mtROS) in HGSOC. To evaluate the modulation of OXPHOS in HGSOC tumor samples and ovarian cancer cell lines, we performed proteomic analyses of proteins involved in mitochondrial energy metabolism and biogenesis and formation of mtROS by immunoblotting and flow cytometry, respectively. We determined that the increased steady-state expression levels of mitochondrial- and nuclear-encoded OXPHOS subunits were associated with increased mitochondrial biogenesis in HGSOC tumors and ovarian cancer cell lines. The more prominent increase in MT-COII expression was in agreement with significant increase in mitochondrial translation factors, TUFM and DARS2. On the other hand, the ovarian cancer cell lines with reduced OXPHOS subunit expression and mitochondrial translation generated the highest levels of mtROS and significantly reduced SOD2 expression. Evaluation of mitochondrial biogenesis suggested that therapies directed against mitochondrial targets, such as those involved in transcription and translation machineries, should be considered in addition to the conventional chemotherapies in HGSOC treatment.

Synergistic combination therapy delivered via layer-by-layer nanoparticles induces solid tumor regression of ovarian cancer.

The majority of patients with high grade serous ovarian cancer (HGSOC) develop recurrent disease and chemotherapy resistance. To identify drug combinations that would be effective in treatment of chemotherapy resistant disease, we examined the efficacy of drug combinations that target the three antiapoptotic proteins most commonly expressed in HGSOC-BCL2, BCL-XL, and MCL1. Co-inhibition of BCL2 and BCL-XL (ABT-263) with inhibition of MCL1 (S63845) induces potent synergistic cytotoxicity in multiple HGSOC models. Since this drug combination is predicted to be toxic to patients due to the known clinical morbidities of each drug, we developed layer-by-layer nanoparticles (LbL NPs) that co-encapsulate these inhibitors in order to target HGSOC tumor cells and reduce systemic toxicities. We show that the LbL NPs can be designed to have high association with specific ovarian tumor cell types targeted in these studies, thus enabling a more selective uptake when delivered via intraperitoneal injection. Treatment with these LbL NPs displayed better potency than free drugs in vitro and resulted in near-complete elimination of solid tumor metastases of ovarian cancer xenografts. Thus, these results support the exploration of LbL NPs as a strategy to deliver potent drug combinations to recurrent HGSOC. While these findings are described for co-encapsulation of a BCL2/XL and a MCL1 inhibitor, the modular nature of LbL assembly provides flexibility in the range of therapies that can be incorporated, making LbL NPs an adaptable vehicle for delivery of additional combinations of pathway inhibitors and other oncology drugs.