High-grade Serous Ovarian Carcinoma Samples Research Articles

Single-cell RNA sequencing reveals the change in cytotoxic NK/T cells, epithelial cells and myeloid cells of the tumor microenvironment of high-grade serous ovarian carcinoma

BackgroundThe heterogeneity of high-grade serous ovarian carcinoma (HGSOC) has hindered the clinical treatment, and our current study aims to characterize the change in tumor microenvironment (TME) with the progression of HGSOC via single cell RNA sequencing (scRNA-seq).MethodsThe single-cell landscape in HGSOC was downloaded from the dataset GSE184880, which included 7 HGSOC and 5 normal samples and then applied for the filtering and annotation of cell clusters. The differentially expressed marker genes in these clusters were analyzed via “FindAllMarker” function in Seurat package and the functional enrichment analyses were implemented using clusterProflier package. Finally, the CellChat package was applied for the cell–cell communication analysis. Cellular experimental were determined Real-time Reverse Transcription Polymerase Chain Reaction (RT-qPCR).Results45,448 single cells were categorized into 10 cell clusters. The proportion of NK/T cells (49.5%), epithelial cells (15.3%) and myeloid cells (14%) was higher in the HGSOC samples. The heterogeneity and different enriched pathways of epithelial cells have been revealed with the progression of HGSOC from early to late stage, concurrent with the reduced activity of cytotoxic NK/T cells and the decreased capabilities of recruiting immune cells and presenting antigens in macrophages. Besides, the cell–cell communication analysis has revealed a strong communication of CXCL and CCL signal between M1 macrophages and cytotoxic NK/T cells in early stage of HGSOC. Moreover, RT-qPCR indicated that CCL4/5 and CCR1/5 levels were upregulated in tumor cell SK-OV-3.ConclusionThe investigation using scRNA-seq has depicted the change in cytotoxic NK/T cells, epithelial cells and myeloid cells of the TME of HGSOC, which may provide another insight into the specific mechanisms underlying the progression of HGSOC.

Chemotherapy drives tertiary lymphoid structures that correlate with ICI-responsive TCF1+CD8+ T cells in metastatic ovarian cancer.

Patients with high-grade serous ovarian carcinoma (HGSOC) are virtually insensitive to immune checkpoint inhibitors (ICIs) employed as standalone therapeutics, at least in part reflecting microenvironmental immunosuppression. Thus, conventional chemotherapeutics and targeted anticancer agents that not only mediate cytotoxic effects but also promote the recruitment of immune effector cells to the HGSOC microenvironment stand out as promising combinatorial partners for ICIs in this oncological indication. We harnessed a variety of transcriptomic, spatial and functional assays to characterize the differential impact of neo-adjuvant paclitaxel-carboplatin on the immunological configuration of paired primary and metastatic HGSOC biopsies as compared to NACT-naïve HGSOC samples from 5 independent patient cohorts. We found neo-adjuvant chemotherapy (NACT)-driven endoplasmic reticulum stress and calreticulin exposure in metastatic HGSOC lesions culminates with the establishment of a dense immune infiltrate including follicular T cells (TFH cells), a prerequisite for mature tertiary lymphoid structure (TLS) formation. In this context, TLS maturation was associated with an increased intratumoral density of ICI-sensitive TCF1+PD-1+ CD8+ T cells over their ICI-insensitive TIM-3+PD-1+ counterparts. Consistent with this notion, chemotherapy coupled with a PD-1-targeting ICI provided a significant survival benefit over either therapeutic approach in syngeneic models of HGSOC bearing high (but not low) tumor mutational burden. Altogether, our findings suggest that NACT promotes TLS formation and maturation in HGSOC lesions, de facto preserving an intratumoral ICI-sensitive T-cell phenotype. These observations emphasize the role of rational design, especially relative to the administration schedule, for clinical trials testing chemotherapy plus ICIs in patients with HGSOC.

Single-cell transcriptomics reveals tumor landscape in ovarian carcinosarcoma.

The present study used single-cell RNA sequencing (scRNA-seq) to characterize the cellular composition of ovarian carcinosarcoma (OCS) and identify its molecular characteristics. scRNA-seq was performed in resected primary OCS for an in-depth analysis of tumor cells and the tumor microenvironment. Immunohistochemistry staining was used for validation. The scRNA-seq data of OCS were compared with those of high-grade serous ovarian carcinoma (HGSOC) tumors and other OCS tumors. Both malignant epithelial and malignant mesenchymal cells were observed in the OCS patient of this study. We identified four epithelial cell subclusters with different biological roles. Among them, epithelial subcluster 4 presented high levels of breast cancer type 1 susceptibility protein homolog (BRCA1) and DNA topoisomerase 2-α (TOP2A) expression and was related to drug resistance and cell cycle. We analyzed the interaction between epithelial and mesenchymal cells and found that fibroblast growth factor (FGF) and pleiotrophin (PTN) signalings were the main pathways contributing to communication between these cells. Moreover, we compared the malignant epithelial and mesenchymal cells of this OCS tumor with our previous published HGSOC scRNA-seq data and OCS data. All the epithelial subclusters in the OCS tumor could be found in the HGSOC samples. Notably, the mesenchymal subcluster C14 exhibited specific expression patterns in the OCS tumor, characterized by elevated expression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1), collagen type XXIII α1 chain (COL23A1), cholecystokinin (CCK), bone morphogenetic protein 7 (BMP7), PTN, Wnt inhibitory factor 1 (WIF1), and insulin-like growth factor 2 (IGF2). Moreover, this subcluster showed distinct characteristics when compared with both another previously published OCS tumor and normal ovarian tissue. This study provides the single-cell transcriptomics signature of human OCS, which constitutes a new resource for elucidating OCS diversity.

Comparative Analysis of Gene Expression Analysis Methods for RNA in Situ Hybridization Images

Gene expression analysis is pivotal in cancer research and clinical practice. While traditional methods lack spatial context, RNA in situ hybridization (RNA-ISH) is a powerful technique that retains spatial tissue information. Here, RNAscope score, RT-droplet digital PCR (RT-ddPCR), and automated QuantISH and QuPath were employed for quantifying RNA-ISH expression values from formalin-fixed paraffin-embedded samples. The methods were compared using high-grade serous ovarian carcinoma samples, focusing on CCNE1, WFDC2, and PPIB genes. The findings demonstrate good concordance between automated methods and RNAscope, with RT-ddPCR showing less concordance. Additionally, QuantISH exhibits robust performance, even for low-expressed genes like CCNE1, showcasing its modular design and enhancing accessibility as a viable alternative for gene expression analysis.

EM-transcriptomic signature predicts drug response in advanced stages of high-grade serous ovarian carcinoma based on ascites-derived primary cultures.

Introduction: High-grade serous ovarian carcinoma (HGSOC) remains a medical challenge despite considerable improvements in the treatment. Unfortunately, over 75% of patients have already metastasized at the time of diagnosis. Advances in understanding the mechanisms underlying how ascites cause chemoresistance are urgently needed to derive novel therapeutic strategies. This study aimed to identify the molecular markers involved in drug sensitivity and highlight the use of ascites as a potential model to investigate HGSOC treatment options. Methods: After conducting an in silico analysis, eight epithelial-mesenchymal (EM)-associated genes related to chemoresistance were identified. To evaluate differences in EM-associated genes in HGSOC samples, we analyzed ascites-derived HGSOC primary cell culture (AS), tumor (T), and peritoneal nodule (NP) samples. Moreover, in vitro experiments were employed to measure tumor cell proliferation and cell migration in AS, following treatment with doxorubicin (DOX) and cisplatin (CIS) and expression of these markers. Results: Our results showed that AS exhibits a mesenchymal phenotype compared to tumor and peritoneal nodule samples. Moreover, DOX and CIS treatment leads to an invasive-intermediate epithelial-to-mesenchymal transition (EMT) state of the AS by different EM-associated marker expression. For instance, the treatment of AS showed that CDH1 and GATA6 decreased after CIS exposure and increased after DOX treatment. On the contrary, the expression of KRT18 has an opposite pattern. Conclusion: Taken together, our study reports a comprehensive investigation of the EM-associated genes after drug exposure of AS. Exploring ascites and their associated cellular and soluble components is promising for understanding the HGSOC progression and treatment response at a personalized level.

Targeted DNA sequencing of high-grade serous ovarian carcinoma reveals association of TP53 mutations with platinum resistance when combined with gene expression.

High-grade serous ovarian carcinoma (HGSC) is the most common subtype of ovarian cancer and is among the most fatal gynecological malignancies worldwide, due to late diagnosis at advanced stages and frequent therapy resistance. In 47 HGSC patients, we assessed somatic and germline genetic variability of a custom panel of 144 known or suspected HGSC-related genes by high-coverage targeted DNA sequencing to identify the genetic determinants associated with resistance to platinum-based therapy. In the germline, the most mutated genes were DNAH14 (17%), RAD51B (17%), CFTR (13%), BRCA1 (11%), and RAD51 (11%). Somatically, the most mutated gene was TP53 (98%), followed by CSMD1/2/3 (19/19/36%), and CFTR (23%). Results were compared with those from whole exome sequencing of a similar set of 35 HGSC patients. Somatic variants in TP53 were also validated using GENIE data of 1287 HGSC samples. Our approach showed increased prevalence of high impact somatic and germline mutations, especially those affecting splice sites of TP53, compared to validation datasets. Furthermore, nonsense TP53 somatic mutations were negatively associated with patient survival. Elevated TP53 transcript levels were associated with platinum resistance and presence of TP53 missense mutations, while decreased TP53 levels were found in tumors carrying mutations with predicted high impact, which was confirmed in The Cancer Genome Atlas data (n = 260). Targeted DNA sequencing of TP53 combined with transcript quantification may contribute to the concept of precision oncology of HGSC. Future studies should explore targeting the p53 pathway based on specific mutation types and co-analyze the expression and mutational profiles of other key cancer genes.

Abstract A007: DNA methylation analysis comparing high-grade serous ovarian cancer to the normal ovary with adjustment for cell type variation identifies tumor-specific methylation alterations

Abstract A007: DNA methylation analysis comparing high-grade serous ovarian cancer to the normal ovary with adjustment for cell type variation identifies tumor-specific methylation alterations

AHRR and SFRP2 in primary versus recurrent high-grade serous ovarian carcinoma and their prognostic implication

BackgroundThe aim of this study was to analyse transcriptomic differences between primary and recurrent high-grade serous ovarian carcinoma (HGSOC) to identify prognostic biomarkers.MethodsWe analysed 19 paired primary and recurrent HGSOC samples using targeted RNA sequencing. We selected the best candidates using in silico survival and pathway analysis and validated the biomarkers using immunohistochemistry on a cohort of 44 paired samples, an additional cohort of 504 primary HGSOCs and explored their function.ResultsWe identified 233 differential expressed genes. Twenty-three showed a significant prognostic value for PFS and OS in silico. Seven markers (AHRR, COL5A2, FABP4, HMGCS2, ITGA5, SFRP2 and WNT9B) were chosen for validation at the protein level. AHRR expression was higher in primary tumours (p < 0.0001) and correlated with better patient survival (p < 0.05). Stromal SFRP2 expression was higher in recurrent samples (p = 0.009) and protein expression in primary tumours was associated with worse patient survival (p = 0.022). In multivariate analysis, tumour AHRR and SFRP2 remained independent prognostic markers. In vitro studies supported the anti-tumorigenic role of AHRR and the oncogenic function of SFRP2.ConclusionsOur results underline the relevance of AHRR and SFRP2 proteins in aryl-hydrocarbon receptor and Wnt-signalling, respectively, and might lead to establishing them as biomarkers in HGSOC.

Tumor cell cytoplasmic metallothionein expression associates with differential tumor immunogenicity and prognostic outcome in high-grade serous ovarian carcinoma.

The underlying mechanism of high T-cell presence as a favorable prognostic factor in high-grade serous ovarian carcinoma (HGSOC) is not yet understood. In addition to immune cells, various cofactors are essential for immune processes. One of those are metallothioneins (MTs), metal-binding proteins comprising various isoforms. MTs play a role in tumor development and drug resistance. Moreover, MTs influence inflammatory processes by regulating zinc homeostasis. In particular, T-cell function and polarization are particularly susceptible to changes in zinc status. The aim of the present study was to investigate a possible role of MT-mediated immune response and its association with prognostic outcome in ovarian cancer. A retrospective study was conducted on a clinically well-characterized cohort of 24 patients with HGSOC treated at the University Hospital of Essen. Gene expression patterns for anti-cancer immunogenicity-related targets were performed using the NanoString nCounter platform for digital gene expression analysis with the appurtenant PanCancer Immune Profiling panel, consisting of 770 targets and 30 reference genes. Tumor-associated immunohistochemical MT protein expression was evaluated using a semi-quantitative four-tier Immunohistochemistry (IHC) scoring. MT immunoexpression was detected in 43% (10/23) of all HGSOC samples. MT immunoexpression levels showed a significant association to survival, leading to prolonged progression-free and overall survival in positively stained tumors. Furthermore, T-cell receptor signaling gene signature showed a strong activation in MT-positive tumors. Activated downstream signaling cascades resulting in elevated interferon-gamma expression with a shift in the balance between T helper cells (TH1 and TH2) could be observed in the MT-positive subgroup. In addition, a higher expression pattern of perforin and several granzymes could be detected, overall suggestive of acute, targeted anti-cancer immune response in MT-positive samples. This is the first study combining broad, digital mRNA screening of anti-tumor immune response-associated genes and their relation to MT-I/II in ovarian cancer. MT overexpression is associated with molecular characteristics of an anti-cancer immune response and is a strong prognostic marker in ovarian HGSOC. The observed immune cell activation associated with tumor MT expression comprises but is not limited to T cells and natural killer cells.

Identification of stably expressed microRNAs in plasma from high-grade serous ovarian carcinoma and benign tumor patients

BackgroundOvarian cancer is a lethal gynecological cancer and no reliable minimally invasive early diagnosis tools exist. High grade serous ovarian carcinoma (HGSOC) is often diagnosed at advanced stages, resulting in poorer outcome than those diagnosed in early stage. Circulating microRNAs have been investigated for their biomarker potential. However, due to lack of standardization methods for microRNA detection, there is no consensus, which microRNAs should be used as stable endogenous controls. We aimed to identify microRNAs that are stably expressed in plasma of HGSOC and benign ovarian tumor patients.Methods and resultsWe isolated RNA from plasma samples of 60 HGSOC and 48 benign patients. RT-qPCR was accomplished with a custom panel covering 40 microRNAs and 8 controls. Stability analysis was performed using five algorithms: Normfinder, geNorm, Delta-Ct, BestKeeper and RefFinder using an R-package; RefSeeker developed by our study group [1]. Among 41 analyzed RNAs, 13 were present in all samples and eligible for stability analysis. Differences between stability rankings were observed across algorithms. In HGSOC samples, hsa-miR-126-3p and hsa-miR-23a-3p were identified as the two most stable miRNAs. In benign samples, hsa-miR-191-5p and hsa-miR-27a-3p were most stable. In the combined HGSOC and benign group, hsa-miR-23a-3p and hsa-miR-27a-3p were identified by both the RefFinder and Normfinder analysis as the most stable miRNAs.ConclusionsConsensus regarding normalization approaches in microRNA studies is needed. The choice of endogenous microRNAs used for normalization depends on the histological content of the cohort. Furthermore, normalization also depends on the algorithms used for stability analysis.

Integrative analysis reveals early epigenetic alterations in high-grade serous ovarian carcinomas.

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecological malignancy. To date, the profiles of gene mutations and copy number alterations in HGSOC have been well characterized. However, the patterns of epigenetic alterations and transcription factor dysregulation in HGSOC have not yet been fully elucidated. In this study, we performed integrative omics analyses of a series of stepwise HGSOC model cells originating from human fallopian tube secretory epithelial cells (HFTSECs) to investigate early epigenetic alterations in HGSOC tumorigenesis. Assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation sequencing (ChIP-seq), and RNA sequencing (RNA-seq) methods were used to analyze HGSOC samples. Additionally, protein expression changes in target genes were confirmed using normal HFTSECs, serous tubal intraepithelial carcinomas (STICs), and HGSOC tissues. Transcription factor motif analysis revealed that the DNA-binding activity of the AP-1 complex and GATA family proteins was dysregulated during early tumorigenesis. The protein expression levels of JUN and FOSL2 were increased, and those of GATA6 and DAB2 were decreased in STIC lesions, which were associated with epithelial-mesenchymal transition (EMT) and proteasome downregulation. The genomic region around the FRA16D site, containing a cadherin cluster region, was epigenetically suppressed by oncogenic signaling. Proteasome inhibition caused the upregulation of chemokine genes, which may facilitate immune evasion during HGSOC tumorigenesis. Importantly, MEK inhibitor treatment reversed these oncogenic alterations, indicating its clinical effectiveness in a subgroup of patients with HGSOC. This result suggests that MEK inhibitor therapy may be an effective treatment option for chemotherapy-resistant HGSOC.

Exploring the cellular and molecular differences between ovarian clear cell carcinoma and high-grade serous carcinoma using single-cell RNA sequencing and GEO gene expression signatures

The two most prevalent subtypes of epithelial ovarian carcinoma (EOC) are ovarian clear cell carcinoma (OCCC) and high-grade serous ovarian carcinoma (HGSC). Patients with OCCC have a poor prognosis than those with HGSC due to chemoresistance, implying the need for novel treatment target. In this study, we applied single-cell RNA sequencing (scRNA-seq) together with bulk RNA-seq data from the GEO (Gene Expression Omnibus) database (the GSE189553 dataset) to characterize and compare tumor heterogeneity and cell-level evolution between OCCC and HGSC samples. To begin, we found that the smaller proportion of an epithelial OCCC cell subset in the G2/M phase might explain OCCC chemoresistance. Second, we identified a possible pathogenic OCCC epithelial cell subcluster that overexpresses LEFTY1. Third, novel biomarkers separating OCCC from HGSC were discovered and subsequently validated on a wide scale using immunohistochemistry. Amine oxidase copper containing 1 (AOC1) was preferentially expressed in OCCC over HGSC, while S100 calcium-binding protein A2 (S100A2) was detected less frequently in OCCC than in HGSC. In addition, we discovered that metabolic pathways were enriched in the epithelial compartment of the OCCC samples. In vitro experiments verified that inhibition of oxidative phosphorylation or glycolysis pathways exerted direct antitumor effects on both OCCC and HGSC cells, while targeting glutamine metabolism or ferroptosis greatly attenuated chemosensitivity only in OCCC cells. Finally, to determine whether there were any variations in immune cell subsets between OCCC and HGSC, data from scRNA-seq and mass cytometry were pooled for analysis. In summary, our work provides the first holistic insights into the cellular and molecular distinctions between OCCC and HGSC and is a valuable source for discovering new targets to leverage in clinical treatments to improve the poor prognosis of patients with OCCC.

Abstract 1025: OvaPrint - a novel cfDNA methylation liquid biopsy for cancer risk assessment in high grade serous ovarian carcinoma

Abstract Background: High grade serous ovarian carcinoma (HGSOC) is a highly lethal gynecologic malignancy with a typical 5-year survival rate of 40% or less when diagnosed with late-stage disease. There is currently no single effective screening tool for high risk or community-based risk assessment for HGSOC and multivariate tests for preselected patients are suboptimal. In women with a known pelvic mass, surgery followed pathological assessment is currently the only definitive way to diagnose HGSOC. About 15% of women with a suspicious mass will have HGSOC and less than 15% will present with stage 1 cancer. A liquid biopsy assay based on cell-free (cf)DNA enables non-invasive dynamic assessment of disease status in patients with cancer where cfDNA methylation is particularly promising for detection of early stage HGSOC. The goal of the current study is to develop a cfDNA methylation liquid biopsy that can be used for the screening and risk assessment for HGSOC. Methods: Tissue and plasma samples were acquired under informed consent from multiple commercial and academic sources. Reduced representation bisulfite sequencing (RRBS) was performed on 33 HGSOC tissues as well as 8 normal ovary and 13 normal fallopian tube controls. Differentially methylated regions (DMRs) were called between HGSOC and normal controls using metilene and DSS. DMR calls with p-values &amp;lt; 0.05 and |αβ| &amp;gt; 0.45 (n = 1677 DMRs) were used to generate a targeted tissue-based methylation assay using the myBaits® hybrid probe capture platform (Daicel Arbor Biosciences). Next, targeted enrichment was performed on bisulfite converted plasma cfDNA on an independent set of 84 HGSOC samples (27 stage 1, 20 stage 2, 37 stage 3) and 100 samples from patients with benign ovarian masses. Mean beta value across DMRs were used to train a novel cfDNA methylation classifier model called OvaPrintTM to discriminate HGSOC from benign masses using a repeated 3-fold cross validation approach to obtain an unbiased estimate of the performance of the classifier. The average cross validated score for each sample was used to generate a receiver operator characteristic (ROC) curve. Results. RRBS analysis on HGSOC tissue and normal tissue controls demonstrated excellent separation of HGSOC from normal tissue samples using the selected DMRs. OvaPrintTM testing in an independent cohort of 184 plasma samples discriminated HGSOC from benign tumors with a high sensitivity and specificity (area under curve (AUC) = 0.931) using cfDNA methylation. Conclusions: OvaPrintTM is a novel cfDNA methylation liquid biopsy that can be used for effective cancer risk assessment for HGSOC in women presenting with pelvic masses. Citation Format: David N. Buckley, Gerald Gooden, Lee Shulman, Monica Newman, Mona Guo, Ben Yi Tew, Lynda Roman, Juan Pablo Lewinger, Bodour Salhia. OvaPrint - a novel cfDNA methylation liquid biopsy for cancer risk assessment in high grade serous ovarian carcinoma [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 1025.

Deciphering the immune landscape dominated by cancer-associated fibroblasts to investigate their potential in indicating prognosis and guiding therapeutic regimens in high grade serous ovarian carcinoma.

Limited immunotherapeutic effect in high-grade serous ovarian carcinoma (HGSOC) propels exploration of the mechanics behind this resistance, which may be partly elucidated by investigating characters of cancer-associated fibroblasts (CAFs), a significant population in HGSOC involved in shaping tumor immune microenvironment. Herein, leveraging gene expression data of HGSOC samples from The Cancer Genome Atlas and Gene Expression Omnibus datasets, we suggested that CAFs detrimentally affected the outcomes of HGSOC patients. Subsequently, we performed weighted gene co-expression network analysis (WGCNA) to identify a CAFs-related module and screened out seven hub genes from this module, all of which were positively correlated with the infiltration of immunosuppressive macrophages. As one of the hub genes, the expression of fibrillin 1 (FBN1) and its relevance to CD206 were further verified by immunohistochemistry staining in HGSOC samples. Meanwhile, we extracted genes that correlated well with CAF signatures to construct a CAFscore. The capacity of the CAFscore as an independent prognostic factor was validated by Cox regression analyses, and its relevance to components as well as signals in the tumor immune microenvironment was also investigated. Under the evaluation by the CAFscore, HGSOC patients with relatively high CAFscore had worse outcomes, activated mesenchymal signaling pathways, and immune checkpoint blockade (ICB) resistance signatures, which was consistent with the fact that non-responders in anti-PD-1 treatment cohorts tended to have higher CAFscore. Besides, the possibility of CAFscore to guide the selection of sensitive chemotherapeutic agents was explored. In conclusion, individualized assessment of the CAFscore could uncover the extent of stroma activation and immunosuppression and inform therapeutic strategies to improve the benefit of therapies.

Molecular Analysis of Short- versus Long-Term Survivors of High-Grade Serous Ovarian Carcinoma.

Simple SummaryOvarian cancer is commonly associated with poor survival; patients with a diagnosis of high-grade serous ovarian carcinoma (HGSC) have an overall survival of 39% at 5 years. For reasons not well known, about 32% of ovarian cancer patients survive 10 years or more. In this study, our goal was to determine the molecular differences that drive long-term survival (LTS) in patients with HGSC. Indeed, this study shows that patients with LTS have a distinct pattern of gene expression, with TMEM62 being related to LTS. Increased TMEM62 expression led to decreased proliferation of ovarian cancer cells in vitro and decreased tumor burden in vivo.Despite having similar histologic features, patients with high-grade serous ovarian carcinoma (HGSC) often experience highly variable outcomes. The underlying determinants for long-term survival (LTS, ≥10 years) versus short-term survival (STS, <3 years) are largely unknown. The present study sought to identify molecular predictors of LTS for women with HGSC. A cohort of 24 frozen HGSC samples was collected (12 LTS and 12 STS) and analyzed at DNA, RNA, and protein levels. OVCAR5 and OVCAR8 cell lines were used for in vitro validation studies. For in vivo studies, we injected OVCAR8 cells into the peritoneal cavity of female athymic nude mice. From RNAseq analysis, 11 genes were found to be differentially expressed between the STS and LTS groups (fold change > 2; false discovery rate < 0.01). In the subsequent validation cohort, transmembrane protein 62 (TMEM62) was found to be related to LTS. CIBERSORT analysis showed that T cells (follicular helper) were found at higher levels in tumors from LTS than STS groups. In vitro data using OVCAR5 and OVCAR8 cells showed decreased proliferation with TMEM62 overexpression and positive correlation with a longevity-regulating pathway (KEGG HSA04213) at the RNA level. In vivo analysis using the OVCAR8-TMEM62-TetON model showed decreased tumor burden in mice with high- vs. low-expressing TMEM62 tumors. Our results demonstrate that restoring TMEM62 may be a novel approach for treatment of HGSC. These findings may have implications for biomarker and intervention strategies to help improve patient outcomes

Single-Cell RNA-Sequencing Atlas Reveals the Tumor Microenvironment of Metastatic High-Grade Serous Ovarian Carcinoma

Ovarian cancer is the most common and lethal gynecological tumor in women worldwide. High-grade serous ovarian carcinoma (HGSOC) is one of the histological subtypes of epithelial ovarian cancer, accounting for 70%. It often occurs at later stages associated with a more fatal prognosis than endometrioid carcinomas (EC), another subtype of epithelial ovarian cancer. However, the molecular mechanism and biology underlying the metastatic HGSOC (HG_M) immunophenotype remain poorly elusive. Here, we performed single-cell RNA sequencing analyses of primary HGSOC (HG_P) samples, metastatic HGSOC (HG_M) samples, and endometrioid carcinomas (EC) samples. We found that ERBB2 and HOXB-AS3 genes were more amplified in metastasis tumors than in primary tumors. Notably, high-grade serous ovarian cancer metastases are accompanied by dysregulation of multiple pathways. Malignant cells with features of epithelial-mesenchymal transition (EMT) affiliated with poor overall survival were identified. In addition, cancer-associated fibroblasts with EMT-program were enriched in HG_M, participating in angiogenesis and immune regulation, such as IL6/STAT3 pathway activity. Compared with ECs, HGSOCs exhibited higher T cell infiltration. PRDM1 regulators may be involved in T cell exhaustion in ovarian cancer. The CX3CR1_macro subpopulation may play a role in promoting tumor progression in ovarian cancer with high expression of BAG3, IL1B, and VEGFA. The new targets we discovered in this study will be useful in the future, providing guidance on the treatment of ovarian cancer.

Abstract 3390: Preliminary results for a novel single extracellular vesicle assay for early stage ovarian cancer: The power of co-localized detection of surface biomarkers

Abstract Introduction: Ovarian cancer (OC) is one of the deadliest cancers, with 314,000 new cases and 207,000 deaths globally in 2020. Serum CA125 has been explored as an OC biomarker for the past 40 years, but lacks sensitivity for early stage OC and is not recommended for screening average-risk, asymptomatic women. We hypothesize that co-localization of biomarkers on the surface of individual extracellular vesicles (EVs), which are shed into the circulation by cancer cells, may lead to development of a blood test for early stage OC. We evaluated the potential of our approach in detecting early stage OC in clinical samples. Methods: We isolated EVs using size-exclusion chromatography and immunoaffinity capture, and detected biomarkers co-localized on the surface of individual EVs with proximity ligation qPCR. Using this approach, we evaluated 49 antibody combinations recognizing 2 or more biomarkers. Each combination consisted of 1 capture antibody and 2 oligonucleotide-tagged detection antibodies. We tested plasma samples from women with early stage I/II high-grade serous ovarian carcinoma (HGSOC)(n=18; 48-80 yr, med 57) and late stage HGSOC (n=24; 37-80 yr, med 54). HGSOC samples were sourced from 2 commercial vendors. Controls comprised samples from women with benign ovarian masses (n=26; 23-76 yr, med 39.5) sourced from a single vendor, and samples prospectively collected by Mercy from healthy women with no cancer history (n=24; 22-72 yr, med 52.5). PCR cycle threshold (Ct) values were measured for each of 49 combinations and data was evaluated using univariate analysis. Performance was compared to plasma CA125 measured at Mercy by commercial ELISA. Results: 8 of 49 combinations distinguished all stages of HGSOC relative to benign and healthy controls with AUCs ranging from 0.86 (95% CI 0.78-0.94) to 0.95 (95% CI 0.90-1.00), comparable to CA125 with an AUC of 0.87 (95% CI 0.79-0.95). One of the most effective combinations (STn, BST2, MUC1) had a sensitivity of 0.78 (95% CI 0.52-0.94) at a specificity of 0.96 (95% CI 0.87-0.99) in detecting early stage HGSOC. This combination also detected HGSOC in 6 of 11 women (3 early stage, 3 late stage) with normal CA125 (&amp;lt; 25 U/mL) and correctly classified 7 of 8 women with benign masses and high CA125 (&amp;gt; 25 U/mL). Conclusions: These preliminary data suggest that co-localization of surface biomarkers in single EVs may provide an effective means to identify women with early stage HGSOC, including those with normal CA125, while avoiding false positives in women with benign masses and high CA125. Despite the inherent challenges associated with commercial samples, our finding that several combinations detected early stage HGSOC is promising. Statistically powered studies with curated repository specimens are underway to refine combinations and independently validate our assay for early stage OC detection. Citation Format: Laura T. Bortolin, Daniel P. Salem, Sanchari Banerjee, Kelly M. Biette, Delaney M. Byrne, Anthony D. Couvillon, Peter A. Duff, Jonian Grosha, MacKenzie Sadie King, Christopher R. Sedlak, Ibukunoluwapo O. Zabroski, Claire Alexander, Karen Copeland, Daniel Gusenleitner, Emily S. Winn-Deen, Eric K. Huang, Bo R. Rueda, Joseph Charles Sedlak. Preliminary results for a novel single extracellular vesicle assay for early stage ovarian cancer: The power of co-localized detection of surface biomarkers [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 3390.

Abstract 1175: Spatially resolved transcriptomics to understand mechanisms of response to neoadjuvant chemotherapy

Abstract Background: Clinical response to therapy of patients diagnosed with high-grade serous ovarian carcinoma (HGSC) is highly variable. Mechanisms underlying response to treatment are not well understood. We previously performed bulk multi-omics analysis aimed at identifying biological differences between poor (PR) and excellent responders (ER) to neoadjuvant chemotherapy (NACT). Although differences were found, these were not as extensive as we had anticipated, suggesting that important molecular differences between such tumors may not be fully evident by bulk analyses. Thus, we carried out in-depth spatial analysis of HGSC to explore heterogeneity of disease and determine how the dynamic behavior between the tumor and the tumor microenvironment varies between PR and ER groups. Methods: We selected a cohort of patients with highly annotated HGSC samples categorized by response to NACT (PR and ER) and analyzed pre-treatment tumor tissues using bulk RNA sequencing (bRNASeq) and Visium Spatial Gene Expression. The definition of PR was stable or progressive disease after 3-4 cycles and/or suboptimal interval cytoreduction after NACT. ER was complete response or only microscopic disease left at the time of interval surgery. Results: To generate expression profiles, we used an unsupervised approach and identified nine distinct clusters, with at least 20 cell types. A comparison of the cell populations identified more stroma-dominated cell groups in the PR group. ER tumors contained more immune-related areas, with a high proportion of T, B and natural killer cells. This finding supports the established role of cytotoxic immune cells in strengthening chemotherapy response and better overall survival. In comparison with bRNASeq, we observed a blending phenomenon that hides deeper heterogeneity of the tumor. Some pathways, such as epithelial-mesenchymal transition (EMT), were statistically enriched in specific clusters of PR compared with the ER group, proving that deep characterization of tumor heterogeneity can be achieved only when exploring specific clusters of cells. The spatial distribution of the clusters showed that in the PR group, the clusters tended to be physically larger and distributed throughout the whole tissue. The ligand-receptor analysis revealed that co-expression of specific ligand-receptor pairing exists depending on their geographical localization, with closer interactions leading to stronger co-expression patterns. Conclusion: Two important findings emerged: importance of the stromal component as a potential driver of poor response to NACT; and identification of differential clusters (distribution and composition) in ER compared with PR tissues. Comparison of the in situ technique with bRNASeq allowed characterization of specific cell subpopulations that might be important determinants of lack of response to therapy, representing candidate therapeutic targets. Citation Format: Elaine Stur, Sara Corvigno, Mingchu Xu, Ken Chen, Sanghoon Lee, Jinsong Liu, Emilly Ricco, Nicole Fleming, Emine Bayraktar, Daniel Kraushaar, Jianhua Zhang, Anil K. Sood. Spatially resolved transcriptomics to understand mechanisms of response to neoadjuvant chemotherapy [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 1175.

Biomarkers expression among paired serous ovarian cancer primary lesions and their peritoneal cavity metastases in treatment-naïve patients: A single-center study.

BackgroundHigh‐grade serous ovarian carcinoma (HGSOC), the most common histologic subtype of ovarian epithelial cancer, is associated with treatment resistance, enhanced recurrence rates, and poor prognosis. HGSOCs often metastasize to the peritoneal cavity, while fluid cytology examination could identify such metastases. This retrospective study aimed to identify potential biomarker discrepancies between paired HGSOC primary tissues and metastatic peritoneal fluid cytology samples, processed as cell blocks (CBs).MethodsTwenty‐four pairs of formalin‐fixed, paraffin‐embedded primary tissues and metastatic CBs from an equal number of treatment‐naïve patients were used, and immunohistochemistry (IHC) for epidermal growth factor receptor (EGFR), human epidermal growth factor receptor, programmed cell death‐1 ligand 1 (PD‐L1), and CD147 was applied.Results13/24 pairs showed discordant EGFR IHC results; in all these 13 patients, EGFR was positive (≥1+ membranous staining intensity found in at least 10% of the cancer cells) in the peritoneal, yet negative in the primary tissue samples. Notably, EGFR IHC was positive in 15/24 of the metastatic, whereas in just 2/24 of the primary HGSOC samples (p < 0.001). Although most PD‐L1 results were concordant, 5/24 and 6/24 pairs exhibited discordant results when stained with the E1L3N and 22C3 clones, respectively. Lastly, CD147 overexpression was found more often in the metastatic rather than the matched primary HGSOCs stained with CD147, though the difference was not significant.ConclusionsCytology from effusions could be considered for biomarker testing when present, even when tissue from the primary cancer is also available and adequately cellular, as it could provide additional information of potential clinical significance.

ACSM3 suppresses the pathogenesis of high-grade serous ovarian carcinoma via promoting AMPK activity.

Ovarian carcinoma is the fifth commonest malignancy in females and exhibits a high recurrence rate. High-grade serous ovarian carcinoma (HGSOC) is the main histologic subtype. It displays extensive genetic heterogeneity. Here, we aimed to identify potential therapeutic targets for HGSOC. Both bioinformatic data from TCGA and 73 pairs of tumor and normal samples from patients were analyzed to reveal the expression level of ACSM3 in HGSOC. Next, cellular and animal experiments, including cell proliferation, colony formation andxenograftassays were performed to explore the suppressive function of ACSM3. Finally, biochemical methods, AMP/ATP ratiomeasurements and Western blotting were used to elucidate the mechanismunderlying the ACSM3-AMPK axis in HGSOC. After analyzing transcriptome data of TCGA HGSOC samples, we found that ACSM3 is down-regulated in patient samples compared with normal controls. This observation was validated using data from primary clinical samples. Proliferation, soft agar colony formation and xenograft assays revealed that ACSM3 is able to suppress HGSOC tumor growth both in vitro and in vivo. Moreover, we found that ACSM3 overexpression increased the AMP/ATP ratio and the phosphorylation level of AMPK at threonine 172. In addition, we found that AMPK silencing in EFO21 and SKOV3 cells completely abolished the anti-oncogenic effect of ACSM3. Our data indicate that the ACSM3-AMPK axis is involved in the pathogenesis of HGSOC and, as such, may act as a therapeutic target for this cancer.