Primary High-grade Serous Ovarian Cancer 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 A057: Inter-tumor heterogeneity revealed by multi-site whole genome sequencing of high grade serous ovarian cancer

Abstract A057: Inter-tumor heterogeneity revealed by multi-site whole genome sequencing of high grade serous ovarian cancer

Abstract A069: High-resolution volumetric imaging of primary and metastatic high-grade serous ovarian cancer explants

Abstract Introduction: As high-grade serous ovarian cancer (HGSOC) rarely shows symptoms, most patients present with metastases. Tumor heterogeneity between metastases may impede a patient’s response to therapy and thus impact survival. We aim to decipher the heterobiology of HGSOC lesions using high-resolution imaging of patient-derived explants (PDEs), to determine the differential treatment response between primary and metastatic tumors. Methods: PAX8, CK7, RAD51, γH2AX, Ki67, cPARP, and CD3 were chosen to enumerate tumor and T cells, and their activity. Antibodies were optimized in monolayer and spheroid cultures of HGSOC cell lines, COV318 and OVCAR4. CD3 was optimized in FFPE human tonsillitis tissue. Primary and metastatic HGSOC explants were cultured for 48h followed by 24h fixation. Several immunostaining and optical clearing protocols were tested on PDEs, which were imaged using a fully automated confocal high-content screening system. Results: Immunostaining and automated image analysis facilitated the identification of individual nuclei within PDEs without Refractive Index (RI) matching or optical clearing. However, high background impeded further image analysis, preventing the quantification of certain antibodies. Glycerol-based RI matching and ScaleS tissue clearing were implemented to try and overcome this. However, neither generated sufficiently high-quality images for analysis. A novel Clearing-enhanced 3D (Ce3D) immunostaining system recently developed for high-quality multiplex fluorescence microscopy of large tissue volumes was tested. While individual nuclei were recognized, as well as RAD51 foci and CK7 cytoskeleton staining, image analysis was still impeded due to high background. Conclusions: Protocols without RI matching and optical clearing can stain large, complex structures such as PDEs. However, high background impedes image analysis suggesting the need for optical clearing and RI matching. The inclusion of various RI matching and/or optical clearing steps failed to overcome high background staining. Further work is ongoing to refine immunostaining, RI matching, optical clearing, and mounting of PDEs to allow the quantification of all antibodies. Citation Format: Adele E. Connor, Joanne Lysaght, Donal J. Brennan, Jeremy C. Simpson, Antoinette S. Perry. High-resolution volumetric imaging of primary and metastatic high-grade serous ovarian cancer explants [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 A069.

Antitumor effects of redox-responsive nanoparticles containing platinum（Ⅳ）in ovarian cancer

Objectives: To explore the antitumor effects of redox-responsive nanoparticles containing platinum(Ⅳ)-NP@Pt(Ⅳ) in ovarian cancer. Methods: Redox-responsive polymer carriers were synthesized. Polymer carriers and platinum(Ⅳ)-Pt(Ⅳ) can self-assemble into NP@Pt(Ⅳ). Inductively coupled plasma mass spectrometry was performed to detect the platinum release from NP@Pt(Ⅳ) in reducing environment and the platinum content in ovarian cancer cells ES2 treated with cisplatin, Pt(Ⅳ) and NP@Pt(Ⅳ). The proliferation ability of the ovarian cancer cells were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular apoptosis was assessed by flow cytometry. Collection of primary ovarian cancer tissues from patients with primary high-grade serous ovarian cancer who were surgically treated at the Cancer Hospital of the Chinese Academy of Medical Sciences from October to December 2022. The high-grade serous ovarian cancer patient-derived xenograft (PDX) mice were intravenously injected with Cy7.5 labeled NP@Pt(Ⅳ) followed by in vivo imaging system. Mice were treated with PBS, cisplatin and NP@Pt(Ⅳ). Tumor volume and weight were measured in each group. Necrosis, apoptosis and cell proliferation of tumor tissues were detected by hematoxylin-eosin (HE) staining, TUNEL fluorescence staining and Ki-67 immunohistochemistry staining. Body weight and HE staining of heart, liver, spleen, lung and kidney of mice in each group were measured. Results: The platinum release of NP@Pt(Ⅳ) after 48 hours in reducing environment was 76.29%, which was significantly higher than that of 26.82% in non-reducing environment (P<0.001). The platinum content in ES2 cells after 4 hours and 7 hours of treatment with NP@Pt(Ⅳ) (308.59, 553.15 ng/million cells) were significantly higher than those of Pt(Ⅳ) (100.21, 180.31 ng/million cells) and cisplatin (43.36, 50.36 ng/million cells, P＜0.05). The half inhibitory concentrations of NP@Pt(Ⅳ) in ovarian cancer cells ES2, A2780, A2780DDP were 1.39, 1.42 and 4.62 μmol/L, respectively, which were lower than those of Pt(IV) (2.89, 7.27, and 16.74 μmol/L) and cisplatin (5.21, 11.85, and 71.98 μmol/L). The apoptosis rate of ES2 cells treated with NP@Pt(Ⅳ) was (33.91±3.80)%, which was significantly higher than that of Pt(Ⅳ) [(16.28±2.41)%] and cisplatin [(15.01±1.17)%, P＜0.05]. In high-grade serous ovarian cancer PDX model, targeted accumulation of Cy7.5 labeled NP@Pt(Ⅳ) at tumor tissue could be observed. After the treatment, the tumor volume of mice in NP@Pt(IV) group was (130±98) mm3, which was significantly lower than those in control group [(1 349±161) mm3, P<0.001] and cisplatin group [(715±293) mm3, P=0.026]. The tumor weight of mice in NP@Pt(IV) group was (0.17±0.09)g, which was significantly lower than those in control group [(1.55±0.11)g, P<0.001] and cisplatin group [(0.82±0.38)g, P=0.029]. The areas of tumor necrosis and apoptosis in mice treated with NP@Pt(Ⅳ) were higher than those in mice treated with cisplatin. Immunohistochemical staining revealed that there were low expressions of Ki-67 at tumor tissues of mice treated with NP@Pt(Ⅳ) compared with cisplatin. The change in body weight of mice in NP@Pt(Ⅳ) group was not significantly different from that of the control group [(18.56±2.04)g vs.(20.87±0.79)g, P=0.063]. Moreover, the major organs of the heart, liver, spleen, lung, and kidney were also normal by HE staining. Conclusion: Redox-responsive NP@Pt(Ⅳ), produced in this study can enhance the accumulation of cisplatin in ovarian cancer cells and improve the efficacy of ovarian cancer chemotherapy.

Combined Interval Cytoreductive Surgery and Carboplatin-Based Hyperthermic Intraperitoneal Chemotherapy in Advanced Primary High-Grade Serous Ovarian Cancer.

Combining interval cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) improves survival in advanced epithelial ovarian carcinoma (EOC). Although limited, growing evidence regarding carboplatin-based HIPEC highlights its potential. This retrospective study included all patients with advanced primary high-grade serous ovarian cancer who underwent interval CRS combined with carboplatin-based HIPEC at our Canadian tertiary care center between 2014 and 2020. We identified 40 patients with a median age of 61 years. The median peritoneal cancer index was 13 and complete cytoreduction was achieved in 38 patients (95%). Median hospital stay was 13 days and there were four admissions to the intensive care unit (10%) and six readmissions (15%). Severe adverse events occurred in eight patients (20%) and there was no perioperative death. Recurrence was seen in 33 patients (82%) with a median DFS of 18.0 months and a median overall survival of 36.4 months. Multivariate analyses showed that age, peritoneal cancer index, completeness of cytoreduction, occurrence of severe complications, and bowel resection did not significantly impact DFS or OS in our cohort. Interval CRS combined with carboplatin-based HIPEC for advanced primary EOC is associated with acceptable morbidity and oncological outcomes. Larger studies are required to determine the long-term outcomes.

Increased expression of IDO1 is associated with improved survival and increased number of TILs in patients with high-grade serous ovarian cancer

BackgroundThe enzyme indoleamine 2,3-dioxygenase 1 (IDO1) plays a crucial role in regulating the immune system's response to tumors, but its exact role in cancer, especially in high-grade serous ovarian cancer (HGSOC), remains controversial. We aimed to investigate the prognostic impact of IDO1 expression and its correlation with tumor-infiltrating lymphocytes (TILs) in HGSOC. MethodsImmunohistochemical (IHC) staining and bioimage analysis using the QuPath software were employed to assess IDO1 protein expression in a well-characterized cohort of 507 patients with primary HGSOC. Statistical evaluation was performed using SPSS, and in silico validation considering IDO1 mRNA expression in bulk and single-cell gene expression datasets was conducted. Additionally, IDO1 expression in interferon-gamma (IFNG) stimulated HGSOC cell lines was analyzed. ResultsOur findings revealed that IDO1 protein and mRNA expression serve as positive prognostic markers for overall survival (OS) and progression-free survival (PFS) in HGSOC. High IDO1 expression was associated with a significant improvement in OS by 21 months (p < 0.001) and PFS by 6 months (p = 0.016). Notably, elevated IDO1 expression correlated with an increased number of CD3+ (p < 0.001), CD4+ (p < 0.001), and CD8+ TILs (p < 0.001). Furthermore, high IDO1 mRNA expression and protein level were found to be associated with enhanced responsiveness to pro-inflammatory cytokines, particularly IFNG. ConclusionsOur study provides evidence that IDO1 expression serves as a positive prognostic marker in HGSOC and is associated with an increased number of CD3+, CD4+ and CD8+ TILs. Understanding the intricate relationship between IDO1, TILs, and the tumor microenvironment may hold the key to improving outcomes in HGSOC.

Exploring the Control of PARP1 Levels in High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is a leading cause of mortality from gynecologic malignancies worldwide. Although a transformative improvement has been shown with the introduction of PARP (poly(ADP-ribose) polymerase) inhibitors, the emergence of resistance to these drugs represents a therapeutic challenge. Hence, expanding our understanding of mechanisms behind the control of PARP1 expression can provide strategic guidance for the translation of novel therapeutic strategies. The Signal Transducer and Activator of Transcription (STAT) family of proteins consists of transcription factors critically involved in the regulation of important cellular functions. Notably, we recently demonstrated that, in cervical cancer cells, STAT1 controls PARP1 levels through multiple mechanisms, possibly involving also STAT3. Here, we tested the hypothesis that a similar mechanism might be operative in HGSOC. To this end, the impact of STAT1/STAT3 modulation on PARP1 expression was assessed in established and primary HGSOC cells, and molecular biology studies proved that STAT1 might act at both transcriptional and post-transcriptional levels to modulate the PARP1 level. Notably, bioinformatics analysis of TCGA databases demonstrated that increased STAT1 mRNA expression levels are associated with a favorable prognosis and with response to chemotherapy in HGSOC patients. Our findings suggest an alternative strategy for targeting HGSOC cells based on their dependency on PARP1.

The interaction of β-arrestin1 with talin1 driven by endothelin A receptor as a feature of α5β1 integrin activation in high-grade serous ovarian cancer

Dissemination of high-grade serous ovarian cancer (HG-SOC) in the omentum and intercalation into a mesothelial cell (MC) monolayer depends on functional α5β1 integrin (Intα5β1) activity. Although the binding of Intα5β1 to fibronectin drives these processes, other molecular mechanisms linked to integrin inside-out signaling might support metastatic dissemination. Here, we report a novel interactive signaling that contributes to Intα5β1 activation and accelerates tumor cells toward invasive disease, involving the protein β-arrestin1 (β-arr1) and the activation of the endothelin A receptor (ETAR) by endothelin-1 (ET-1). As demonstrated in primary HG-SOC cells and SOC cell lines, ET-1 increased Intβ1 and downstream FAK/paxillin activation. Mechanistically, β-arr1 directly interacts with talin1 and Intβ1, promoting talin1 phosphorylation and its recruitment to Intβ1, thus fueling integrin inside-out activation. In 3D spheroids and organotypic models mimicking the omentum, ETAR/β-arr1-driven Intα5β1 signaling promotes the survival of cell clusters, with mesothelium-intercalation capacity and invasive behavior. The treatment with the antagonist of ETAR, Ambrisentan (AMB), and of Intα5β1, ATN161, inhibits ET-1-driven Intα5β1 activity in vitro, and tumor cell adhesion and spreading to intraperitoneal organs and Intβ1 activity in vivo. As a prognostic factor, high EDNRA/ITGB1 expression correlates with poor HG-SOC clinical outcomes. These findings highlight a new role of ETAR/β-arr1 operating an inside-out integrin activation to modulate the metastatic process and suggest that in the new integrin-targeting programs might be considered that ETAR/β-arr1 regulates Intα5β1 functional pathway.

Poređenje PD-L1 ekspresije u citološkom i histološkom materijalu seroznog high-grade karcinoma jajnika

Introduction: Neoplastic cells in peritoneal lavage express various proteins with significant prognostic and therapeutic potential. Such expression could differ from the expression in a primary tumor or in metastases. In this research, we compared PD-L1 (programmed cell death ligand-1) expression on ovarian cancer cells in cytological material with its expression on peritoneal metastases and a primary tumor. Materials and methods: The study included 30 patients who had been operated on for high-grade serous ovarian cancer (HGSC) in FIGO IIIC, over the period of one year. Cytoblocks, cytological and tissue microarrays were assembled and immunostained with PD-L1 antibody. For each tumor compartment we determined four PD-L1 expression categories: negative, low, moderate, and strong expression, according to the percentage of membrane positive tumor cells. Moderate and strong positivity together were considered as high PD-L1 expression. Results: Moderate PD-L1 expression was the most frequent pattern in primary HGSC (50%) and in peritoneal metastases (omentum) (60%). Cytological samples mostly showed low PD-L1 expression (57%). Statistical analysis did not show a significant difference in PD-L1 expression between the study groups. We found a positive correlation of PD-L1 expression between different, matched tumor samples in every patient, with statistical significance (p &lt; 0.05) between all the analyzed samples. Conclusion: PD-L1 expression was similar in all three tumor compartments. This could point to similar peritumor regulatory mechanisms of HGSC in primary tumor tissue and cytology tumor samples. Immunohistochemical analysis of the assembled cytoblocks is sufficiently reliable in the assessment of PD-L1 expression on cancer ovarian cells from cytological material.

Pathway-level mutation analysis in primary high-grade serous ovarian cancer and matched brain metastases

Brain metastases (BMs) in ovarian cancer (OC) are a rare event. BMs occur most frequently in high-grade serous (HGS) OC. The molecular features of BMs in HGSOC are poorly understood. We performed a whole-exome sequencing analysis of ten matched pairs of formalin-fixed paraffin-embedded samples from primary HGSOC and corresponding BMs. Enrichment significance (p value; false discovery rate) was computed using the Reactome, the Kyoto Encyclopedia of Genes and Genomes pathway collections, and the Gene Ontology Biological Processes. Germline DNA damage repair variants were found in seven cases (70%) and involved the BRCA1, BRCA2, ATM, RAD50, ERCC4, RPA1, MLHI, and ATR genes. Somatic mutations of TP53 were found in nine cases (90%) and were the only stable mutations between the primary tumor and BMs. Disturbed pathways in BMs versus primary HGSOC constituted a complex network and included the cell cycle, the degradation of the extracellular matrix, cell junction organization, nucleotide metabolism, lipid metabolism, the immune system, G-protein-coupled receptors, intracellular vesicular transport, and reaction to chemical stimuli (Golgi vesicle transport and olfactory signaling). Pathway analysis approaches allow for a more intuitive interpretation of the data as compared to considering single-gene aberrations and provide an opportunity to identify clinically informative alterations in HGSOC BM.

Hormone Receptor Expression in Primary and Recurrent High-Grade Serous Ovarian Cancer and Its Implications in Early Maintenance Treatment.

Endocrine therapy is an effective treatment for low-grade serous ovarian cancer. However, the role of estrogen and progesterone receptors as biomarkers for high-grade serous ovarian cancer (HGSOC) is yet to be elucidated because not all estrogen and progesterone receptor-positive tumors benefit from anti-estrogen therapy. The degree of expression is presumed to play a vital role; however, that role is not well-defined in ovarian cancer. We aimed to determine the role of estrogen and progesterone receptor expression in primary and paired relapsed HGSOC. In this study, primary and matched relapsed tumor samples were collected from 80 patients with International Federation of Gynecology and Obstetrics Stage II-IV HGSOC. Tissue microarray was conducted and immunohistochemistry for estrogen and progesterone receptor expression was performed. Two independent pathologists performed the tissue microarray analysis with the Immunoreactive Score and Allred Total score. In the paired analysis, no significant difference in estrogen receptor expression was observed. However, progesterone receptor expression was significantly lower in patients with recurrent platinum-sensitive HGSOC. We conclude that anti-estrogen therapy targeting estrogen receptor positive HGSOC could be administered in primary and relapsed settings. The use of endocrine maintenance with an aromatase inhibitor in patients with estrogen receptor positive HGSOC needs to be further evaluated and validated in a randomized controlled trial.

High expression of CD8 in the tumor microenvironment is associated with PD-1 expression and patient survival in high-grade serous ovarian cancer.

Objective:The current study assesses programmed death-1 (PD-1) receptor expression and CD3, CD4, and CD8 tumor-infiltrating lymphocytes (TILs) in high-grade serous ovarian cancer (HGSOC) and associates our results with neoadjuvant chemotherapy history and disease prognosis.Materials and Methods:We included cases diagnosed with primary HGSOC with biopsy or surgical resection materials in this study. The immunoreactivity of CD3, CD4, CD8, and PD1 was assessed immunohistochemically in tumor tissue. We analyzed TILs in two predetermined groups of high and low TIL. The relationships between clinical characteristics, PD-1, and TIL were assessed. by the χ(2) test or Fisher’s Exact test. We used Kaplan-Meier survival analysis and Cox proportional hazards regression model to the connection between survival and the amounts of TIL, and PD1.Results:Univariate analysis demonstrated that optimal debulking (p<0.001), early International Federation of Gynecology and Obstetrics stage (p=0.046), and higher scores of stromal CD8+ TIL expression (p=0.028) in tumor cells were all substantially correlated with longer disease-free survival (DFS), whereas the remaining variables analyzed, including PD-1 positivity, stromal CD3+, and CD4+ TILs, and intraepithelial CD3+, CD4+, and CD8+ TILs, were not correlated with DFS. Also, univariate analysis revealed that optimal debulking (p=0.010), and higher scores of stromal CD8+ TIL expression (p=0.021) in tumor cells were all substantially correlated with longer overall survival (OS).Conclusion:Higher scores of stromal CD8+ TILs are substantially correlated with DFS and OS in univariate analyses, whereas scores of stromal CD3+ and CD4+ TILs, and intraepithelial CD3+, CD4+, and CD8+ TILs are not correlated with DFS and OS in both univariate and multivariate analyses. Also, we found a significant association between PD-1 positivity and the scores of stromal CD3+ TILs and intraepithelial CD8+ TILs. However, no remarkable relationship was revealed between PD-1 positivity and the survival of HGSOC cases.

Effects of Wee1 inhibitor adavosertib on patient-derived high-grade serous ovarian cancer cells are multiple and independent of homologous recombination status.

ObjectiveA major challenge in the treatment of platinum-resistant high-grade serous ovarian cancer (HGSOC) is lack of effective therapies. Much of ongoing research on drug candidates relies on HGSOC cell lines that are poorly documented. The goal of this study was to screen for effective, state-of-the-art drug candidates using primary HGSOC cells. In addition, our aim was to dissect the inhibitory activities of Wee1 inhibitor adavosertib on primary and conventional HGSOC cell lines.MethodsA comprehensive drug sensitivity and resistance testing (DSRT) on 306 drug compounds was performed on three patient-derived genetically unique HGSOC cell lines and two commonly used ovarian cancer cell lines. The effect of adavosertib on the cell lines was tested in several assays, including cell-cycle analysis, apoptosis induction, proliferation, wound healing, DNA damage, and effect on nuclear integrity.ResultsSeveral compounds exerted cytotoxic activity toward all cell lines, when tested in both adherent and spheroid conditions. In further cytotoxicity tests, adavosertib exerted the most consistent cytotoxic activity. Adavosertib affected cell-cycle control in patient-derived and conventional HGSOC cells, inducing G2/M accumulation and reducing cyclin B1 levels. It induced apoptosis and inhibited proliferation and migration in all cell lines. Furthermore, the DNA damage marker γH2AX and the number of abnormal cell nuclei were clearly increased following adavosertib treatment. Based on the homologous recombination (HR) signature and functional HR assays of the cell lines, the effects of adavosertib were independent of the cells' HR status.ConclusionOur study indicates that Wee1 inhibitor adavosertib affects several critical functions related to proliferation, cell cycle and division, apoptosis, and invasion. Importantly, the effects are consistent in all tested cell lines, including primary HGSOC cells, and independent of the HR status of the cells. Wee1 inhibition may thus provide treatment opportunities especially for patients, whose cancer has acquired resistance to platinum-based chemotherapy or PARP inhibitors.

Predictive biomarker for surgical outcome in patients with advanced primary high-grade serous ovarian cancer. Are we there yet? An analysis of the prospective biobank for ovarian cancer

BackgroundHigh-grade serous ovarian cancer (HGSOC) is the most common subtype of ovarian cancer and is associated with high mortality rates. Surgical outcome is one of the most important prognostic factors. There are no valid biomarkers to identify which patients may benefit from a primary debulking approach. ObjectiveOur study aimed to discover and validate a predictive panel for surgical outcome of residual tumor mass after first-line debulking surgery. Study designFirstly, “In silico” analysis of publicly available datasets identified 200 genes as predictors for surgical outcome. The top selected genes were then validated using the novel Nanostring method, which was applied for the first time for this particular research objective. 225 primary ovarian cancer patients with well annotated clinical data and a complete debulking rate of 60% were compiled for a clinical cohort. The 14 best rated genes were then validated through the cohort, using immunohistochemistry testing.Lastly, we used our biomarker expression data to predict the presence of miliary carcinomatosis patterns. ResultsThe Nanostring analysis identified 37 genes differentially expressed between optimal and suboptimal debulked patients (p < 0.05). The immunohistochemistry validated the top 14 genes, reaching an AUC Ø0.650. The analysis for the prediction of miliary carcinomatosis patterns reached an AUC of Ø0.797. ConclusionThe tissue-based biomarkers in our analysis could not reliably predict post-operative residual tumor. Patient and non-patient-associated co-factors, surgical skills, and center experience remain the main determining factors when considering the surgical outcome at primary debulking in high-grade serous ovarian cancer patients.

Abstract 2128: Single-cell replication dynamics in genomically unstable cancers

Abstract Background: Single-cell whole genome sequencing (scWGS) methods such as direct library preparation (DLP) provide amplification-free capture of cells in all cycle phases and have enabled rich interrogation into the cell to cell genomic diversity of cancer genomes. Previous DLP-driven clonal evolution studies removed S-phase cells as replicated loci confounded phylogenetic inference from somatic copy number aberrations (CNAs), leaving single-cell replication timing (scRT) as an unstudied property. We thus developed a method that assigned S-phase cells to phylogenetic clones and used such assignments to infer scRT profiles in aneuploid cell lines and tumors. We applied this method to determine the relative proliferation rate between clones and obtain a single-cell picture of DNA replication fork progression for genomically unstable cancers. Methods: S-phase cells were assigned to phylogenetic clones based on copy number profile similarity and subsequently scRT profiles were inferred by normalizing out the effects of clonal, subclonal, and cell-specific CNAs before binarizing each genomic bin as replicated or unreplicated. scRT heterogeneity was quantified via a summary statistic representing the aggregate difference between expected and observed replication times (T-width). The scRT profiles were grouped into clone- and sample-level pseudobulks (aggregations of single cells) to determine genomic regions with differential replication timing. We then assessed the ability of S-phase enrichment to predict clonal expansions on time-series patient-derived xenografts of HER2+ and triple negative breast cancers that were either treatment-naive or exposed to cisplatin. In addition, we studied genetically engineered cell lines with inactivated DNA damage response genes (TP53, BRCA1, BRCA2) and primary high-grade serous ovarian cancer samples to quantify scRT heterogeneity at different states of genomic instability. Results: We analyzed 15 datasets containing over 25,000 cells with ~1/3 of cells in S-phase. Time-series datasets revealed that S-phase enriched clones were highly proliferative in the treatment-naive setting and were most sensitive to cisplatin. T-width values calculated from inferred scRT profiles increased as a function of genomic instability, with the degree of heterogeneity varied between genomic regions. Clone-level pseudobulk replication timing profiles revealed that whole-chromosome aneuploidies were more likely to have unperturbed replication timing compared to complex structural variants such as translocations, inversions, and micronuclei. Conclusions: Our time-series results show that S-phase cells enable the prediction of clone-specific proliferation and chemosensitivity using scWGS data from a single time point. The quantification of scRT heterogeneity across our collection of data implicates dysfunctional DNA replication as both driver and consequence of genomic instability. Citation Format: Adam C. Weiner, Andrew McPherson, Sohrab P. Shah. Single-cell replication dynamics in genomically unstable cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2128.

Abstract 1551: miR-625-3p enhances chemosensitivity in ovarian cancer cells through exosomal export of cisplatin and paclitaxel

Abstract Although a majority of women with high-grade serous ovarian cancer (HGSC) achieve a complete response to first-line platinum- and paclitaxel-based chemotherapy, around thirty percent of patients are identified as incomplete responders. Their diseases typically either does not respond or progresses during treatment (refractory) or recurs within 6 months of completing treatment (resistant), suggesting the inherit or intrinsic characteristics of the tumor. Currently, there are no biomarkers that can predict intrinsic resistance, which can avoid unnecessary treatment and toxicity. MicroRNAs (miRNAs) provide a novel master layer of regulation of gene expression. However, the molecular mechanisms by which miRNAs confer a chemoresistant phenotype in ovarian cancer have not been elucidated. To identify miRNAs that are associated with intrinsic chemoresistance in HGSC, Ion Torrent next-generation sequencing analysis of miRNAs was performed on microdissected chemosensitive and chemorefractory primary HGSC cases. Amongst all the miRNAs that were significantly down-regulated in chemorefractory cases, lower miR-625-3p expression was found to be associated with poorer overall and progression-free survivals. Further functional studies showed that overexpression of miR-625-3p significantly decreased cisplatin and paclitaxel resistance in ovarian cancer cells in vitro and in vivo. Online target prediction algorithms together with transcriptome profiling of HGSC cells transfected with miR-625-3p mimics or control mimics identified SSX2IP as a direct target of miR-625-3p, suggesting that SSX2IP may play a role in conferring cisplatin/paclitaxel resistance in HGSC by mediating the effect of miR-625-3p. SSX2IP is a microtubule anchoring and centriolar satellite protein, which plays a critical role in controlling microtubule length and orientation, and centrosome maturation. We demonstrated that HGSC cells transfected with SSX2IP had marked increases in microtubule masses, intracellular multivesicular body trafficking and exosomal cisplatin level; and a decrease in intracellular cisplatin level. In conclusion, down-regulation of miR-625-3p confers cisplatin/paclitaxel resistance in ovarian cancer cells via its novel direct target SSX2IP. SSX2IP alters the microtubule dynamics and subsequently mediates inter-organelle crosstalks between microtubules and multivesicular bodies, which facilitate exosomal export of cisplatin and paclitaxel of HGSC cells. This study is crucial for developing new predictive biomarkers for intrinsic chemoresistance, and new treatment strategies for HGSC based on upregulating miR-625-3p or downregulating SSX2IP expression in ovarian cancer cells, which will enhance cisplatin and paclitaxel sensitivity, and improve patient survival rates. Citation Format: Chi Lam Au Yeung, Tetsushi Tsuruga, Kay-Pong Yip, Sammy Ferri-Borgogno, Samuel Mok. miR-625-3p enhances chemosensitivity in ovarian cancer cells through exosomal export of cisplatin and paclitaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1551.

Multi-omics analysis reveals spatial heterogeneity of infiltrating T cells in high-grade serous ovarian cancer.

e17603 Background: The effect of spatial immunologic variation, especially in T cell infiltration, recognition and expansion, across various tumor foci in the ovary (primary) and distant metastatic foci in the peritoneal cavity and their contribution to the limited response to immune therapy in High-Grade Serous Ovarian Cancer(HGSOC) remains unexplored. We aim to provide a detailed analysis of the landscape of heterogeneity of infiltrating T cells in primary and metastatic lesions and their differential characteristics in HGSOC. Methods: We performed multi-site sampling and simultaneous RNA sequencing (RNA-seq), whole-genome sequencing (WGS) and bulk T cell receptor (TCR) sequencing as well as single cell RNA sequencing (scRNA-seq) and paired TCR sequencing (scTCR-seq) in 9 patients (48 sites, including 19 ovarian, 11 omental, 18 other metastasis lesions) with untreated primary HGSOC. Validation by IHC, flow cytometry, another scRNA-seq data from 7 patients (32 sites, including 9 ovarian, 6 omental, 6 ascites and 11 other metastasis lesions) and spatial transcriptomics. Results: CD8 exhausted clusters that are enriched in ovarian tumors exhibited high exhaustion, tumor-specific score and low bystander score, whereas CD8 transition and NK-like clusters that are enriched in omental tumors exhibited the opposite characteristics, which was confirmed by flow cytometry. Of note, the exhausted CD8+ T cells exhibited GZMB, PRF1, FASLG and IFNG effector genes despite the expression of exhaustion markers, developing from an early differentiation state with high proliferative capacity and the process preferentially occurs in primary ovarian sites. Besides, T cells corresponding to the selected TCRs that had the same distribution as neoantigens were enriched in ovarian lesions, while the virus-specific TCR preferentially enriched in omentum samples. The association of TMB, HRD score, and COSMIC mutational signatures with antigen-engaged T cell subsets was not observed at the site level within individual patients. STARTRAC-migration analysis showed that T cell clusters in blood or other sites migrating to ovarian was extremely low or vice versa. The expression of MHC-I processing and presentation genes and MHC-I protein were higher in omental lesions, along with T cells were preferentially located in tumor region of ovarian tumors. Conclusions: We identify two different immune patterns in ovarian cancer: 1) ovarian lesions with low infiltration of mainly dysfunctional T cells and immunosuppressive Treg cells. These exhausted CD8 T cells with cytotoxic function are clonal expanded; 2) omental lesions infiltrated with non-tumor-specific bystander cells. Decreased major histocompatibility complex (MHC)-I antigen presentation ability and failure of T cell infiltration into omental tumors may contribute to lack of tumor-specific T cells in omental metastasis.

Copy number features as a novel biomarker of homologous recombination (HR) status in high-grade serous ovarian cancer (HGSOC).

5547 Background: Copy number alterations (CNA) arise as a result of somatic changes to chromosome structure, resulting in gain or loss of genomic regions. A likely source of copy number variation is an incorrect repair of DNA damage that causes chromosome instability (CI). Hence, many tumors with a high degree of CNAs suffer CI. This is the case of high grade serous ovarian cancer (HGSOC), where deficiency in homologous recombination (HRD) is highly prevalent, and so are CI and CNAs. Clinically, HR status in HGSOC is a biomarker of iPARP response and required for proper patient management. Available HRD tests measure genomic loss of heterozygosity (LOH) features, closely related to CI. Such features are complex to acquire in samples with low tumor content; overall, 15% of HGSOC specimens fail HR testing. CNA profiling is technically more amenable to lower tumor cellularity and may be captured by a wider range of techniques and applications (gene panels, low pass WGS...). We present here the analysis and identification of CNA features in HGSOC as a novel biomarker of HR status. Methods: A cohort of 123 primary HGSOC tumors were analysed with a custom hybrid capture-based NGS panel (VHIO-300) that provides, along mutations in 450 genes, genome-wide CNA profiles. B-allele fractions, obtained from single nucleotide polymorphisms also in the panel, allowed HRD-LOH score calculation (Marquard et al., 2015). 41 CNA segments correlated to HRD-LOH scores (proportions test, FDR &lt; 0.001) and selected as features to calculate a CNA-HGSOC score (range 0-100). Among them, 10 segments appeared altered almost exclusively in BRCA1/2 pathogenic/likely pathogenic mutant tumors. Results: The density plot of the CNA-HGSOC score showed a bimodal distribution with modes at 7 and 65. A cut-off value of 34 was selected based on the lowest density CNA-score value between the modes. Hence, high CNA-HGSOC was defined as tumors with scores ≥ 34. Our CNA-HGSOC score was strongly correlated as a continuous variable to HRD-LOH (Pearson correlation = 0.72; p &lt; 0.0001) and ROC analysis (AUC = 0.84; CI 95% 0.77-0.91; p &lt; 0.0001) demonstrated high predictability to classify tumors as if using an HRD-LOH test (HRD-LOH score; “very high”: 100-75, “high”: 74-50,”mid”: 49-25 and “low”: 24-0). BRCA mutation status was also accurately predicted using a subset of CNA features (AUC = 0.71; CI 95% 0.59-0.82; p &lt; 0.0001). Conclusions: CNAs may provide a new powerful genomic resource to HRD determination. We identified 41 CNA features in tumors to inform HR status and a subset of 10 revealed mutation status of BRCA1/2 in HGSOC. Upon further validation, a CNA-HCSOC score would be easily implemented in routine analysis pipelines in clinical labs, allowing HR testing or even broaden its application to emerging fields, such as liquid biopsy.

Impact of Muscle Mass Depletion on Surgical Outcomes in Patients with Primary High-grade Serous Ovarian Cancer Undergoing CRS and HIPEC.

To investigate the effect of sarcopenia on surgical outcomes in patients with primary high-grade serous ovarian cancer undergoing cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS and HIPEC). Descriptive study. Department of Surgical Oncology, Faculty of Medicine, Ankara University Ankara, Turkey, from January 2013 to January 2020. Forty patients, who underwent CRS and HIPEC with the diagnosis of primary high-grade serous ovarian cancer and peritoneal carcinomatosis were included in the study. Preoperative staging CT images were used to determine total psoas ındex values by measuring psoas muscle area at the level of L3 vertebra. Patients with total psoas ındex values below the cut-off levels were considered sarcopenic. Demographic, clinicopathological and perioperative results were compared between sarcopenic and non-sarcopenic patients. Serious postoperative complications (36.4% vs. 6.9%, p=0.039) and infective (54.5% vs. 17.2%, p=0.042) and pulmonary (72.7% vs. 34.5%, p=0.040 complications were significantly higher in sarcopenic patients. Hospital stay [20(12-25) vs. 12(9-16.5) days, p=0.017] and ICU stay [4(2-6) vs. 2(1-2.5) days, p=0.013)] were found to be longer in sarcopenic patients. In univariate analysis, advanced age (OR: 1.40 95% CI: 1.07-1.84; p=0.021) and sarcopenia (OR: 7.71% 95 CI: 1.17-51.06; p=0.039) were significantly associated with serious postoperative complications. The presence of sarcopenia (OR: 0.050 95% CI: 0.004-0.675; p=0.024) was found to be independent predictor of serious postoperative complications. Sarcopenia can easily be diagnosed without additional cost or radiation exposure with routine preoperative staging CT images. Identification of sarcopenic ovarian cancer patients in preoperative period may affect patient selection, predictability of possible serious complications, elective operation preparation process with a combination of nutrition and exercise therapy, thus postoperative complication rates may be reduced and short-term results may be improved. Key Words: Cytoreductive surgery, Ovarian cancer, Sarcopenia.

Primary high-grade serous ovarian cancer cells are sensitive to senescence induced by carboplatin and paclitaxel in vitro

BackgroundVarious types of normal and cancer cells undergo senescence in response to carboplatin and paclitaxel, which are considered the gold standard treatments in ovarian cancer management. Surprisingly, the effect of these drugs on ovarian cancer cell senescence remained unknown.MethodsThe experiments were conducted on primary high-grade serous ovarian cancer cells. Molecular markers of senescence were evaluated using cytochemistry and immunofluorescence. Cell cycle distribution was analyzed using flow cytometry. Expression of cyclins and signaling pathways was tested using western blot. Telomere length and telomerase activity were measured using qPCR, and the colocalization of telomeres with DNA damage foci using immuno-FISH. Oxidative stress-related parameters were quantified using appropriate fluorescence probes. Production of cancerogenic agents was analyzed using qPCR and ELISA.ResultsCarboplatin applied with paclitaxel induces senescence of ovarian cancer cells in vitro. This activity was reflected by permanent G2/M growth arrest, a high fraction of cells expressing senescence biomarkers (SA-β-Gal and γ-H2A.X), upregulated expression of p16, p21, and p53 cell cycle inhibitors, and decreased expression of cyclin B1. Neither telomere length nor telomerase activity changed in the senescent cells, and the majority of DNA damage was localized outside telomeres. Moreover, drug-treated cancer cells exhibited increased production of STAT3 protein, overproduced superoxide and peroxides, and increased mitochondrial mass. They were also characterized by upregulated ANG1, CCL11, IL-6, PDGF-D, TIMP-3, TSP-1, and TGF-β1 at the mRNA and/or protein level.ConclusionsOur findings imply that conventional chemotherapy may elicit senescence in ovarian cancer cells, which may translate to the development of a cancer-promoting phenotype, despite the inability of these cells to divide.