High-grade Serous Carcinoma Patients Research Articles

Single-cell Transcriptomics Reveals the Aggressive Landscape of High-Grade Serous Carcinoma and Therapeutic Targets in Tumor Microenvironment

High-grade serous carcinoma (HGSC) is characterized by early abdominal metastasis, leading to a dismal prognosis. In this study, we conducted single-cell RNA sequencing on 109,573 cells from 34 tumor samples of 18 HGSC patients, including both primary tumors and their metastatic sites. Our analysis revealed a distinct S100A9+ tumor cell subtype present in both primary and metastatic sites, strongly associated with poor overall survival. This subtype exhibited high expression of S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1, and C15orf48. Individual knockdown of these ten marker genes, validated through in vitro and in vivo models, significantly inhibited ovarian cancer growth and invasion. Around S100A9+ tumor cells, a population of HK2+_CAF was identified, characterized by activated glycolysis metabolism, correlating with shorter overall survival in patients. Notably, similar to CAFs, immunosuppressive tumor-associated macrophage (TAM) subtypes underwent glycolipid metabolism reprogramming via PPARgamma regulation, promoting tumor metastasis. These findings shed light on the mechanisms driving the aggressiveness of HGSC, offering crucial insights for the development of novel therapeutic targets against this formidable cancer.

Podocalyxin promotes the formation of compact and chemoresistant cancer spheroids in high grade serous carcinoma

High grade serous carcinoma (HGSC) metastasises primarily intraperitoneally via cancer spheroids. Podocalyxin (PODXL), an anti-adhesive transmembrane protein, has been reported to promote cancer survival against chemotherapy, however its role in HGSC chemoresistance is unclear. This study investigated whether PODXL plays a role in promoting chemoresistance of HGSC spheroids. We first showed that PODXL was expressed variably in HGSC patient tissues (n = 17) as well as in ovarian cancer cell lines (n = 28) that are more likely categorised as HGSC. We next demonstrated that PODXL-knockout (KO) cells proliferated more slowly, formed less compact spheroids and were more fragile than control cells. Furthermore, when treated with carboplatin and examined for post-treatment recovery, PODXL-KO spheroids showed significantly poorer cell viability, lower number of live cells, and less Ki-67 staining than controls. A similar trend was also observed in ascites-derived primary HGSC cells (n = 6)—spheroids expressing lower PODXL formed looser spheroids, were more vulnerable to fragmentation and more sensitive to carboplatin than spheroids with higher PODXL. Our studies thus suggests that PODXL plays an important role in promoting the formation of compact/hardy HGSC spheroids which are more resilient to chemotherapy drugs; these characteristics may contribute to the chemoresistant nature of HGSC.

Abstract 1617: Tumor evolution during therapy in ovarian cancer

Abstract The evolution of tumors from diagnosis to relapse in ovarian high-grade serous carcinoma (HGSC) patients is poorly understood. Consequently, it is unknown whether diagnostic samples are representative enough to guide treatment directions at relapse, or whether acquired changes during therapy provide targets in patients with relapses. To answer these questions and characterize tumor evolution during treatment, we used data from >700 tissue and plasma samples from 200 HGSC patients belonging to the observational, longitudinal DECIDER trial (NCT04846933). Mutations from whole-genome sequencing data from tissue samples (2-12 per patient) were used to construct phylogenetic trees. The trees were corrected by the copy-number alterations and curated to integrate circulating tumor DNA (ctDNA) exome- and targeted-sequencing data from plasma samples. Evolutionary states presenting within and between sample heterogeneity were evaluated through clonal complexity and divergence. Additionally, genetic differences between persistent and non-persistent subclones were studied to identify features affecting treatment resistance. Our results indicate that the majority of subclones survive chemotherapy treatment and major subclonal replacement takes place in only a few patients. Within-sample heterogeneity was higher in plasma samples than in ascites, highlighting the importance of ctDNA samples to estimate persistent subclones. Additionally, our recently reported evolutionary states (Lahtinen et al. Cancer Cell 2023) were often altered by the treatment induced selection pressure. At late relapses, subclone compositions typical of maintaining evolutionary state tumors were less common, and a higher number of acquired subclones were detected, suggesting further evolution of the persisting clones. In summary, chemotherapy does not suppress heterogeneity in HGSC tumors. Interestingly, patients with the worst prognosis have a stable composition of multiple clones in their tumors, and tumors with low heterogeneity responded better to primary treatment. Relapse samples show lower within-sample heterogeneity and further evolution. Furthermore, we detected several evolutionary trajectories from stable, persistent cancer subclone populations to highly dynamic subclone structures. This renders diagnostic samples suitable to guide relapse treatment decisions in a subset of patients with HGSC but not for all. Our characterization of the dynamics of tumor clonal heterogeneity during treatment allows to define better therapeutic interventions for HGSC patients with relapsed disease. Citation Format: Sampsa Kalervo Hautaniemi, Jaana Oikkonen, Alexandra Lahtinen, Mai T.N. Nguyen, Giovanni Marchi, Kari Lavikka, Anna Rajavuori, Sakari Hietanen, Anni Virtanen, Johanna Hynninen. Tumor evolution during therapy in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1617.

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

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

Abstract PR-003: Half of CCNE1-amplified high-grade serous ovarian carcinomas are homologous recombination deficient and platinum-sensitive

Abstract In ovarian high-grade serous carcinoma (HGSC), homologous recombination deficiency (HRD) sensitizes tumors to DNA-damaging platinum-based chemotherapy, as well as to poly-ADP-ribose polymerase inhibitors (PARPi), resulting in longer survival compared to HR-proficient patients. Cyclin E1 (CCNE1) amplified tumors are found in about 20% of HGSC patients and associate with poorer clinical outcomes. CCNE1 amplification is mutually exclusive with inactivating BRCA1/2 mutations, the best-established cause for HRD. This has led to the notion that CCNE1-amplified tumors are dependent on the HR pathway for viability i.e., are HR-proficient, but empirical data to demonstrate this are scarce. We set out to investigate whether in CCNE1-amplified HGSC, functional HR is indeed essential. Whole-genome sequencing (WGS) data from >200 HGSOC patients has to date been produced as part of the DECIDER project (https://www.deciderproject.eu/); from this cohort, we identified 51 CCNE1-amplified cases (defined as at least 8 CCNE1 copies and 2x tumor ploidy). As expected, no BRCA1/2-mutated patients were found in this cohort. For each CCNE1-amplified patient, we aimed to assess CCNE1 protein levels by IHC from at least one tumor site, obtain genomics-based HRD estimates and quantify functional HR capacity based on positivity for RAD51, a key HR protein marker that predicts clinically relevant HRD (Pikkusaari et al. 2023, PMID 36805632). So far, we have analyzed 29 CCNE1-amplified tumor specimens from 24 HGSC patients, all FIGO stage IIIB or higher. A third of samples displayed genomic HRD, i.e., were positive for mutational signature SBS3+ and/or ovaHRDscar+ (Perez-Villatoro et al. 2022, PMID 36581696); 50% were functionally HR-deficient, as measured by the RAD51 assay. Our findings thus challenge the dogma that CCNE1 amplification is mutually exclusive with HRD. Moreover, clinical outcomes of HR-deficient vs. HR-proficient CCNE1-amplified patients were significantly better (11/12 vs. 1/12 with complete response to primary therapy, median progression-free survival of 511 days vs. 316 days, respectively), indicating that our assays are measuring clinically meaningful HRD, and that HRD is a major driver of favorable platinum response also in the CCNE1-amplified cohort. It follows that the HR-deficient CCNE1-amplified subgroup would be expected to benefit from PARPi therapy as well. Our results indicate that CCNE1 amplification alone is not a surrogate marker for HR-proficient HGSC, nor an optimal predictive biomarker of chemotherapy resistance in HGSC. Instead, HRD – which in CCNE1-amplified tumors appears to not be consistently detectable by genomics-based HRD assays and therefore necessitates functional HRD testing – should be incorporated into patient stratification within the CCNE1-amplified patient population, to identify platinum-sensitive (and presumably, PARPi-sensitive) cases. Citation Format: Sanna Pikkusaari, Essi Kahelin, Jaana Oikkonen, Yilin Li, Giulia Micoli, Heidi Koskela, Anna Rajavuori, Johanna Hynninen, Sampsa Hautaniemi, Anni Virtanen, Liisa Kauppi. Half of CCNE1-amplified high-grade serous ovarian carcinomas are homologous recombination deficient and platinum-sensitive [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-003.

Abstract A003: Characterizing DNA methylation patterns driving chemotherapy resistance in ovarian high-grade serous carcinoma

Abstract To characterize the role of DNA methylation (DNAm) in chemotherapy response, we analyzed whole-genome DNAm data integrated with transcriptome from 67 samples of 17 patients with ovarian high grade serous carcinoma (HGSC) belonging to the prospective, longitudinal, multi-region DECIDER trial (NCT04846933). We used a novel decomposition method, which allows separating the effects of tumor microenvironment and treatment, tissue, and patient specific changes, facilitating the integration of the heterogeneous collection of samples. We firstly characterized DNAm alterations between tissue sites. Our results show that the cancer spread from tubo-ovarian sites to metastatic deposits is accompanied by moderate changes that employ cell junctions and focal adhesions. The most drastic change in the tissue intrinsic DNAm profiles implicated upregulation of members of the ATP-binding-cassette (ABC) family of transporters, including such multidrug resistance-associated genes as ABCC2, ABCC3, and ABCC4 in omentum. We then proceeded to characterize the DNAm changes during treatment. Our results reveal that DNAm plays a distinct role in emergence, driving, and regulation of both preexisting and acquired resistance to chemotherapy. We observed that patient-specific DNAm background shows enormous intra- and intertumor heterogeneity and sustains multiple driver mechanisms to resist chemotherapy-induced cell death already prior to treatment. We also observed that neoadjuvant chemotherapy (NACT) results in emergence of resistance via acquired, tissue specific DNAm alterations in DNA damage repair, apoptosis, cellular senescence, and drug efflux. Pathway analyses show that treatment-naïve tumors from patients with poor treatment outcome have DNAm-induced upregulation of key resistance promoting signaling pathways, such as PI3K/Akt, mTOR, and Rap1 cascades. Hypoxic transcriptional program activated via HIF-1 signaling was significantly altered in poorly responding patients, accompanied by changes in extracellular matrix instigated by integrins ITGB1 and ITGA5. Tumors of patients with refractory disease demonstrated DNAm changes in genes involved in tumor-promoting chronic inflammation driven by elevation of tumor necrosis factor. NACT treatment resulted in very few changes to these DNA aberrations and no acquired mechanisms, suggesting that intrinsic DNAm patient profiles accommodate multiple drivers for tumor progression and chemoresistance. In conclusion, using a large whole-sequencing DNAm dataset we elucidated the major underlying mechanisms of acquired platinum resistance, which provide possible targets for improving platinum sensitivity. Validation of these results and the intrinsic ability of HGSC to resist platinum-based treatment is currently on-going in our recently sequenced DNAm batch consisting of 400 samples of 135 HGSC patients. Citation Format: Alexandra Lahtinen, Giovanni Marchi, Kari Lavikka, Giulia Micoli, Susanna Holmström, Elsi Pöllänen, Ilari Maarala, Daria Afenteva, Yilin Li, Taru A. Muranen, Jaana Oikkonen, Kaisa Huhtinen, Anni Virtanen, Johanna Hynninen, Antti Häkkinen, Sampsa Hautaniemi. Characterizing DNA methylation patterns driving chemotherapy resistance in ovarian high-grade serous carcinoma [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 A003.

Abstract A076: T-cell exhaustion is an independent predictive biomarker of clinical outcome in high grade serous ovarian cancer regardless of homologous recombination deficiency status

Abstract Epithelial ovarian cancer is the most lethal gynecologic malignancy, with 5-year survival rates of 46%. High grade serous carcinoma (HGSC) is the most frequent subtype of epithelial ovarian cancer. Approximately 50% of cases are homologous recombination deficient (HRD). Whereas maintenance treatment by PARP inhibitors has significantly improved the prognosis of patients with HRD tumors, clinical outcome in this malignancy is still poor. Despite the immunogenic nature of HGSC, in which T-cell infiltration correlates to improved clinical outcome following surgery and chemotherapy, the disease exhibits low response rates to immune checkpoint blockade (ICB) monotherapy in advanced line settings. We have previously identified epithelial malignancies including HGSC, CD4 and CD8 populations of tumor-infiltrating and antigen-specific T cells that display features of terminal exhaustion. These populations were key players in the response to anti-PD-1 in patients with epithelial tumors. Here, we set out to characterize tumor-infiltrating T cells in a cohort of 80 HGSC patients treated at our institution from whom we collected tumor samples prior to therapy in which HRD status was assessed. We used multiplex immunofluorescence of FFPE tumor samples to assess infiltration by CD4 and CD8 T cells and multiparametric flow cytometry of frozen viable cell suspensions to assess CD4 and CD8 T-cell exhaustion. The link between biological parameters and clinical outcome was assessed by univariable and multivariable analyses. Assessment of the T-cell infiltrate by immunofluorescence showed no difference in the absolute numbers, per tumor or stroma mm2, of CD8 or CD4 T cells between HRD and homologous recombination proficient (HRP) tumors. Instead, analysis of exhausted CD8 and CD4 T cells showed an increased frequency of terminally exhausted PD-1HighTIM-3+ CD8 T cells in HRD tumors. This population, which expresses the ectonucleotidase CD39, was significantly correlated to the presence of T regulatory cells (Treg), which agreed with previous studies showing comigration of effector and regulatory T cells in ovarian cancer. In addition, terminally exhausted CD4 T cells, expressing the immune checkpoints PD-1 and TIM-3 as well as CD39, were enriched in HRD tumors. Among HRD tumors, no difference was observed between the exhaustion profile in tumors presenting deleterious BRCA1/2 mutations compared to those with other mutations in the homologous recombination pathway. Univariable analyses showed exhaustion parameters to be significant predictors of progression-free survival (PFS). The frequencies of exhausted CD4 and CD8 T cells stood out as predictors of PFS in multivariable analyses. Altogether, these results provide the first evidence of a link between T-cell exhaustion and HRD status and reveal T-cell exhaustion as an independent predictor of disease outcome. They also underline the major role played by the antitumor immune response in HGSC and warrant the development of combination immunotherapies despite the lack of response to ICB monotherapy. Citation Format: Anna Salvioni, Mathilde Del, Marie Michelas, Pierre Vuattoux, Clara-Maria Scarlata, Carlos Martinez-Gomez, Nathalie Van Acker, François-Xavier Frenois, Guillaume Bataillon, Jean-Pierre Delord, Alejandra Martinez, Maha Ayyoub. T-cell exhaustion is an independent predictive biomarker of clinical outcome in high grade serous ovarian cancer regardless of homologous recombination deficiency status [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 A076.

Abstract A054: Interleukin-16 is a novel target to prevent age-associated epigenetic changes leading to malignant transformation associated with ovarian high-grade serous carcinoma (HGSOC)

Abstract Low-grade chronic inflammation and persistent oxidative stress are hallmarks of malignant transformation. High-grade serous ovarian carcinoma (HGSOC) is a fatal malignancy of women, and the median age of its incidence is 60-65 years. Our earlier studies showed enhanced expression of interleukin 16 (IL-16, an inflammatory cytokine), its receptor CD9, and glucose-regulated protein (GRP78, a marker of cellular stress and inhibitor of DNA damage repair mechanism) in postmenopausal ovaries and patients with HGSOC as compared with premenopausal subjects. It is possible that these age-associated increases in IL-16, CD9, and GRP78 expression may contribute to HGSOC-related epigenetic modifications of gH2A histone family member X (gH2AX, a marker for double-stranded DNA breaks), and histone deacetylase 1 (HDAC1), involved in DNA repair. The goal of this study was to examine if age-associated such changes lead to HGSOC-associated malignant transformation and if so, to determine mechanisms involved in such transformation. Materials and Methods: Exploratory study: Archived tissue specimens were selected for immunohistochemical, immunoblotting, genomic studies, and immunoassays based on the review of their hematoxylin and eosin staining and final pathological reports. Specimens were grouped as normal premenopausal women (30-50 years old), normal postmenopausal women (55-85 years old), and ovarian high-grade serous carcinoma (HGSC). In vitro study: human ovarian surface epithelium (HOSE) cells were treated with or without recombinant human IL-16 (rhIL-16) and recombinant human GRP78 (rhGRP78), and small interfering RNA-GRP78 (siGRP78) for 24 hours or 48 hours. Nuclear fractionations from treated- or untreated HOSE cells or OVCAR3 (HGSOC cell line) cells were extracted. Results: Compared to premenopausal women, a subset of postmenopausal women exhibited significantly elevated expression (P<0.01) of inflammatory markers IL-16, and its receptor CD9, as well as a marker of oxidative stress (GRP78). Additionally, markers of epigenetic changes, including gH2AX and HDAC1, presented similar patterns of increase in their expression. These observations were also consistent in HGSOC patients. Enhanced expression of GRP78, gH2AX, HDAC1, and CD9 in rhIL-16 treated HOSE cells like OVCAR3 cells and their reduction in siGRP78 treated HOSE cells suggests that age-associated increase in IL-16 in postmenopausal women may be a risk factor for malignant transformation and GRP78 may be a mechanism for IL-16-induced HGSOC development. Thus, IL-16 and GRP78 represent novel targets that may be used to develop novel targeted therapies to prevent HGSOC. Expression of gH2AX and HDAC1 increased in rhGRP78-treated HOSE cells. Conclusion: The results of this study suggest that age-associated increases in IL-16 (a marker of chronic inflammation) and GRP78 (a marker of oxidative stress) may contribute to the predisposition for HGSOC-associated malignant transformation. IL-16 and GRP78 offer potential targets for the prevention of HGSOC in women. Support: The Portes Foundation Citation Format: Jessica Ramirez, Animesh Barua. Interleukin-16 is a novel target to prevent age-associated epigenetic changes leading to malignant transformation associated with ovarian high-grade serous carcinoma (HGSOC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A054.

The prognostic significance of tumor-immune microenvironment in ascites of patients with high-grade serous carcinoma.

High-grade serous carcinoma (HGSC) is often associated with ascites at presentation. Our objective was to quantify immune cells (ICs) in ascites prior to any treatment was given and evaluate their impact on progression-free survival (PFS) and overall survival (OS). Forty-seven patients with primary HGSC and ascites were included. Flow-cytometric analysis was performed to detect percentages of CD3+ T cells (CD4+, CD8+, Tregs, and NKT cells), B cells, NK cells (CD56brightCD16- and CD56dimCD16+ subsets), macrophages and dendritic cells (DCs). Furthermore, CD103 expression was analyzed on T cells and their subsets, while PD-1 and PD-L1 expression on all ICs. Cut-off of low and high percentages of ICs was determined by the median of variables, and correlation with PFS and OS was calculated. CD3+ cells were the predominant ICs (median 51%), while the presence of other ICs was much lower (median ≤10%). CD103+ expression was mostly present on CD8+, and not CD4+ cells. PD-1 was mainly expressed on CD3+ T cells (median 20%), lower expression was observed on other ICs (median ≤10%). PD-L1 expression was not detected. High percentages of CD103+CD3+ T cells, PD-1+ Tregs, CD56brightCD16- NK cells, and DCs correlated with prolonged PFS and OS, while high percentages of CD8+ cells, macrophages, and PD-1+CD56brightCD16- NK cells, along with low percentages of CD4+ cells, correlated with better OS only. DCs were the only independent prognostic marker among all ICs. Our results highlight the potential of ascites tumor-immune microenvironment to provide additional prognostic information for HGSC patients. However, a larger patient cohort and longer follow-up are needed to confirm our findings.

TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion

A crucial requirement for metastasis formation in ovarian high-grade serous carcinoma (HGSC) is the disruption of the protective peritoneal mesothelium. Using co-culture systems of primary human cells, we discovered that tumor-associated NK cells induce TRAIL-dependent apoptosis in mesothelial cells via death receptors DR4 and DR5 upon encounter with activated Tcells. Upregulation of TRAIL expression in NK cells concomitant with enhanced cytotoxicity toward mesothelial cells was driven predominantly by T-cell-derived TNFα, as shown by affinity proteomics-based analysis of the Tcell secretome in conjunction with functional studies. Consistent with these findings, we detected apoptotic mesothelial cells in the peritoneal fluid of HGSC patients. In contrast to mesothelial cells, HGSC cells express negligible levels of both DR4 and DR5 and are TRAIL resistant, indicating cell-type-selective killing by NK cells. Our data point to a cooperative action of T and NK in breaching the mesothelial barrier in HGSC patients.

Survey of NF1 inactivation by surrogate immunohistochemistry in ovarian carcinomas

ObjectiveInhibition of the MAPK pathway by MEK inhibitors (MEKi) is currently a therapeutic standard in several cancer types, including ovarian low-grade serous carcinoma (LGSC). A common MAPK pathway alteration in tubo-ovarian high-grade serous carcinoma (HGSC) is the genomic inactivation of neurofibromin 1 (NF1). The primary objectives of our study were to survey the prevalence of NF1 inactivation in the principal ovarian carcinoma histotype as well as to evaluate its associations with clinico-pathological parameters and key biomarkers including BRCA1/2 status in HGSC. MethodsA recently commercialized NF1 antibody (clone NFC) was orthogonally validated on an automated immunohistochemistry (IHC) platform and IHC was performed on tissue microarrays containing 2140 ovarian carcinoma cases. Expression was interpreted as loss/inactivated (complete or subclonal) versus normal/retained. ResultsLoss of NF1 expression was detected in 250/1429 (17.4%) HGSC including 11% with subclonal loss. Survival of NF1-inactivated HGSC patients was intermediate between favorable BRCA1/2 mutated HGSC and unfavorable CCNE1 high-level amplified HGSC. NF1 inactivation was mutually exclusive with CCNE1 high-level amplifications, co-occurred with RB1 loss and occurred at similar frequencies in BRCA1/2 mutated versus wild-type HGSC. NF1 loss was found in 21/286 (7.3%) endometrioid carcinomas with a favorable prognostic association (p = 0.048), and in 4/64 (5.9%) LGSC, mutually exclusive with other driver events. ConclusionsNF1 inactivation occurs in a significant subset of BRCA1/2 wild-type HGSC and a subset of LGSC. While the functional effects of NF1 inactivation need to be further characterized, this signifies a potential therapeutic opportunity to explore targeting NF1 inactivation in these tumors.

Strategies for data normalization and missing data imputation and consequences for potential diagnostic microRNA biomarkers in epithelial ovarian cancer.

MicroRNAs (miRNAs) are small non-coding RNA molecules regulating gene expression with diagnostic potential in different diseases, including epithelial ovarian carcinomas (EOC). As only a few studies have been published on the identification of stable endogenous miRNA in EOC, there is no consensus which miRNAs should be used aiming standardization. Currently, U6-snRNA is widely adopted as a normalization control in RT-qPCR when investigating miRNAs in EOC; despite its variable expression across cancers being reported. Therefore, our goal was to compare different missing data and normalization approaches to investigate their impact on the choice of stable endogenous controls and subsequent survival analysis while performing expression analysis of miRNAs by RT-qPCR in most frequent subtype of EOC: high-grade serous carcinoma (HGSC). 40 miRNAs were included based on their potential as stable endogenous controls or as biomarkers in EOC. Following RNA extraction from formalin-fixed paraffin embedded tissues from 63 HGSC patients, RT-qPCR was performed with a custom panel covering 40 target miRNAs and 8 controls. The raw data was analyzed by applying various strategies regarding choosing stable endogenous controls (geNorm, BestKeeper, NormFinder, the comparative ΔCt method and RefFinder), missing data (single/multiple imputation), and normalization (endogenous miRNA controls, U6-snRNA or global mean). Based on our study, we propose hsa-miR-23a-3p and hsa-miR-193a-5p, but not U6-snRNA as endogenous controls in HGSC patients. Our findings are validated in two external cohorts retrieved from the NCBI Gene Expression Omnibus database. We present that the outcome of stability analysis depends on the histological composition of the cohort, and it might suggest unique pattern of miRNA stability profiles for each subtype of EOC. Moreover, our data demonstrates the challenge of miRNA data analysis by presenting various outcomes from normalization and missing data imputation strategies on survival analysis.

Stratification of ovarian cancer borderline from high-grade serous carcinoma patients by quantitative serum NMR spectroscopy of metabolites, lipoproteins, and inflammatory markers.

Background: Traditional diagnosis is based on histology or clinical-stage classification which provides no information on tumor metabolism and inflammation, which, however, are both hallmarks of cancer and are directly associated with prognosis and severity. This project was an exploratory approach to profile metabolites, lipoproteins, and inflammation parameters (glycoprotein A and glycoprotein B) of borderline ovarian tumor (BOT) and high-grade serous ovarian cancer (HGSOC) for identifying additional useful serum markers and stratifying ovarian cancer patients in the future. Methods: This project included 201 serum samples of which 50 were received from BOT and 151 from high-grade serous ovarian cancer (HGSOC), respectively. All the serum samples were validated and phenotyped by 1H-NMR-based metabolomics with in vitro diagnostics research (IVDr) standard operating procedures generating quantitative data on 38 metabolites, 112 lipoprotein parameters, and 5 inflammation markers. Uni- and multivariate statistics were applied to identify NMR-based alterations. Moreover, biomarker analysis was carried out with all NMR parameters and CA-125. Results: Ketone bodies, glutamate, 2-hydroxybutyrate, glucose, glycerol, and phenylalanine levels were significantly higher in HGSOC, while the same tumors showed significantly lower levels of alanine and histidine. Furthermore, alanine and histidine and formic acid decreased and increased, respectively, over the clinical stages. Inflammatory markers glycoproteins A and B (GlycA and GlycB) increased significantly over the clinical stages and were higher in HGSOC, alongside significant changes in lipoproteins. Lipoprotein subfractions of VLDLs, IDLs, and LDLs increased significantly in HGSOC and over the clinical stages, while total plasma apolipoprotein A1 and A2 and a subfraction of HDLs decreased significantly over the clinical stages. Additionally, LDL triglycerides significantly increased in advanced ovarian cancer. In biomarker analysis, glycoprotein inflammation biomarkers behaved in the same way as the established clinical biomarker CA-125. Moreover, CA-125/GlycA, CA-125/GlycB, and CA-125/Glycs are potential biomarkers for diagnosis, prognosis, and treatment response of epithelial ovarian cancer (EOC). Last, the quantitative inflammatory parameters clearly displayed unique patterns of metabolites, lipoproteins, and CA-125 in BOT and HGSOC with clinical stages I-IV. Conclusion: 1H-NMR-based metabolomics with commercial IVDr assays could detect and identify altered metabolites and lipoproteins relevant to EOC development and progression and show that inflammation (based on glycoproteins) increased along with malignancy. As inflammation is a hallmark of cancer, glycoproteins, thereof, are promising future serum biomarkers for the diagnosis, prognosis, and treatment response of EOC. This was supported by the definition and stratification of three different inflammatory serum classes which characterize specific alternations in metabolites, lipoproteins, and CA-125, implicating that future diagnosis could be refined not only by diagnosed histology and/or clinical stages but also by glycoprotein classes.

Abstract 1513: A multi-sample study reveals the evolution and heterogeneity in high-grade serous ovarian cancer

Abstract Ovarian cancer is a heterogeneous disease and high-grade serous carcinoma (HGSC) accounts for 70% of them. The pathogenesis of HGSC had remained obscure until the identification of its tubal origin; however, the carcinogenesis process remains largely underexplored. Meanwhile, targeted therapy using poly(ADP-ribose) polymerase (PARP) inhibitors have revolutionized the treatment of HGSCs and emphasizes the importance of patient stratification. Nonetheless, current clinical trials use different assays and a consensus approach for patient stratification is still lacking. In this study, whole genome sequencing technique was used to profile tumor-normal sample pairs, including some related tumors collected from different anatomical sites. This yields a multi-sample cohort comprising 55 pre-treatment samples from 33 HGSC patients and is suitable for addressing questions about molecular stratification and tumor pathogenesis. We first recapitulated two reported HGSC subgroups (H-HRD and H-FBI) in our cohort. Comprehensive analyses were conducted on genomic scars ranging from mutations, indels, structural variants and copy numbers. Our data suggests that the genomic subgroups are characterized by different extent and onset timing of homologous recombination repair defect (HRD) as well as CCNE1 activation. Of note, H-HRD group showed higher levels of copy number-based HRD score, which often serves as a surrogate biomarker for PARP inhibitor treatment response. On the other hand, tumor evolutionary trajectory was reconstructed based on in silico methods capable of inferring relative timing of genetic alterations. This highlights an early bifurcation of carcinogenesis paths in this HGSC dichotomy, despite a common scenario of a very early TP53 mutation, an often early whole genome duplication and a chromosomal instability phenotype seen eventually. Overall, these findings corroborate the concept of tumor-intrinsic genomic phenotypes by providing mechanistic underpinnings from the aspect of tumor evolution. This provides the rationale for studying HGSC disease biology in different contexts and formulating questions about subtype-specific pathogenesis and vulnerabilities. Translationally, it also holds the promise for better identifying the patient subset that might benefit from PARP inhibitor treatment. Citation Format: Siao-Han Wong, Kai Doberstein, Frederik Marmé, Benedikt Brors. A multi-sample study reveals the evolution and heterogeneity 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 1513.

Locally Performed HRD Testing for Ovarian Cancer? Yes, We Can!

Assessment of HRD status is now essential for ovarian cancer patient management. A relevant percentage of high-grade serous carcinoma (HGSC) is characterized by HRD, which is caused by genetic alterations in the homologous recombination repair (HRR) pathway. Recent trials have shown that not only patients with pathogenic/likely pathogenic BRCA variants, but also BRCAwt/HRD patients, are sensitive to PARPis and platinum therapy. The most common HRD test is Myriad MyChoice CDx, but there is a pressing need to offer an alternative to outsourcing analysis, which typically requires high costs and lengthy turnaround times. In order to set up a complete in-house workflow for HRD testing, we analyzed a small cohort of HGSC patients using the CE-IVD AmoyDx HRD Focus Panel and compared our results with Myriad's. In addition, to further deepen the mechanisms behind HRD, we analyzed the study cohort by using both a custom NGS panel that analyzed 21 HRR-related genes and FISH analysis to determine the copy numbers of PTEN and EMSY. We found complete concordance in HRD status detected by the Amoy and the Myriad assays, supporting the feasibility of internal HRD testing.

Increased FOXJ1 protein expression is associated with improved overall survival in high-grade serous ovarian carcinoma: an Ovarian Tumor Tissue Analysis Consortium Study

BackgroundRecently, we showed a >60% difference in 5-year survival for patients with tubo-ovarian high-grade serous carcinoma (HGSC) when stratified by a 101-gene mRNA expression prognostic signature. Given the varied patient outcomes, this study aimed to translate prognostic mRNA markers into protein expression assays by immunohistochemistry and validate their survival association in HGSC.MethodsTwo prognostic genes, FOXJ1 and GMNN, were selected based on high-quality antibodies, correlation with protein expression and variation in immunohistochemical scores in a preliminary cohort (n = 134 and n = 80, respectively). Six thousand four hundred and thirty-four (FOXJ1) and 5470 (GMNN) formalin-fixed, paraffin-embedded ovarian neoplasms (4634 and 4185 HGSC, respectively) represented on tissue microarrays from the Ovarian Tumor Tissue Analysis consortium underwent immunohistochemical staining and scoring, then univariate and multivariate survival analysis.ResultsConsistent with mRNA, FOXJ1 protein expression exhibited a linear, increasing association with improved overall survival in HGSC patients. Women with >50% expression had the most favourable outcomes (HR = 0.78, 95% CI 0.67–0.91, p < 0.0001). GMNN protein expression was not significantly associated with overall HSGC patient survival. However, HGSCs with >35% GMNN expression showed a trend for better outcomes, though this was not significant.ConclusionWe provide foundational evidence for the prognostic value of FOXJ1 in HGSC, validating the prior mRNA-based prognostic association by immunohistochemistry.

118. Single-cell RNA sequencing and co-occurring cellular state analysis of high-grade serous ovarian cancer

High-grade serous carcinoma (HGSC) is the most lethal subtype of ovarian cancer, and a majority of patients are diagnosed at advanced stages. One standard-of-care is neoadjuvant chemotherapy followed by cytoreductive surgery, however up to 80% of HGSC patients develop recurrent disease. There exists a critical need to better understand the features of this tumor microenvironment that may highlight potential therapeutic targets, help stratify chemotherapy responders from non-responders, and uncover novel cell states that may serve as prognostic or predictive biomarkers. We obtained multiple biopsies from five patients with advanced-stage HGSC, both pre- and post-treatment, and analyzed these samples using single-cell RNAseq. We identified 20 distinct transcriptional clusters of cells, including a well-defined tumor subset enriched for EPCAM and KRT8. We annotated each cluster using known marker genes, and additionally validated these data through genome-wide copy number and developmental maturity analyses. We then performed transcriptome deconvolution to identify co-occurring transcriptional states using EcoTyper. We then performed bulk RNAseq on paired pre- and post-treatment tumor biopsies from 23 HGSC patients. Applying our fingerprints of ecotypes from the scRNA-seq data, we identified multiple distinct transcriptional states within the pre- and post-treatment HGSC tumor microenvironment, which were enriched for distinct co-occurring states comprised of multiple immunologic lineages, including CD4 T and NK cell states. We plan to validate these ecotypes using spatial transcriptomics and compare clinical outcomes across transcriptional states to determine the potential prognostic or predictive implications of our discoveries.

Differential epithelial and stromal LGR5 expression in ovarian carcinogenesis

Lgr5 has been identified as a marker of the stem/progenitor cells in the murine ovary and oviduct by lineage tracing. However, little is known regarding LGR5 expression or its functional significance in human ovary tissues. Here, using RNA in situ hybridization and/or immunohistochemistry, we thoroughly investigated LGR5 expression in normal human ovaries, fallopian tubes and various ovarian tumors. We discovered that LGR5 expression is negligible in the human ovary surface epithelium, whereas ovarian stromal cells normally express low levels of LGR5. Remarkably, fallopian tube epithelium, inclusion cysts and serous cystadenomas with a Müllerian phenotype expressed high levels of LGR5, and LGR5 expression was restricted to PAX8+/FOXJ1− secretory cells of the tubal epithelium. Strong stromal LGR5 expression without epithelial LGR5 expression was consistently observed in the path from serous cystadenoma to serous borderline tumor to low grade serous carcinoma (LGSC). Unlike LGSC, high grade serous carcinoma (HGSC), clear cell carcinoma, endometrioid carcinomas displayed various epithelial-stromal LGR5 expression. Notably, high levels of LGR5 expression were observed in serous tubal intraepithelial carcinoma, which slightly declined in invasive HGSC. LGR5 expression was significantly associated with improved progression-free survival in HGSC patients. Moreover, in vitro assays demonstrated that LGR5 expression suppressed tumor proliferation and migratory capabilities. Taken together, these findings indicate a tumor-suppressive role for LGR5 in the progression of HGSC.

Abstract 4002: Identification of signal transduction pathway activity with potential clinical target in high-grade serous ovarian carcinoma

Abstract Background: High-grade serous carcinoma (HGSC) is the most common subtype of ovarian cancer. It has a high mortality rate, even after successful first-line treatment with debulking surgery and chemotherapy. Although therapeutic options for targeted therapy are rapidly expanding, identification of patients who respond to these therapies remains a challenge. In recognition of the importance of the functional phenotype of cancer cells, Verhaegh et al. (Cancer Res 2014) developed assays to measure functional activity of signal transduction pathways (STPs) based on mRNA expression levels of pathway-specific target genes. In this study, we aimed to identify HGSCs with STP activity with a potential clinical target by comparing their activity with STP activity in normal Fallopian tube epithelium (FTE), the tissue of origin of most HGSCs. Methods: We included 50 primary tumor samples taken prior to start of chemotherapy of postmenopausal patients diagnosed with advanced stage HGSC and 9 morphologically normal FTE samples of healthy postmenopausal women. Using pathway assays, we assessed functional pathway activity of the androgen receptor (AR), estrogen receptor (ER), PI3K, MAPK, Hedgehog, TGFβ and Notch pathways. Differences in STP activity between groups were compared with Mann-Whitney U tests. Cut-off value for aberrant STP activity was defined as two standard deviations above the mean value of STP activity measured in FTE samples. Results: In the HGSC group we observed lower median ER (p &amp;lt; 0.001) pathway activity and higher median PI3K (p &amp;lt; 0.001), Hedgehog (p &amp;lt; 0.001) and TGFβ pathway (p = 0.020) activity as compared to the FTE group. In individual HGSC samples, aberrant activity was identified for the MAPK (n = 10), PI3K (n = 22), Hedgehog (n = 28) and TGFβ (n = 21) pathways. Frequently observed combinations of aberrant STP activity were Hedgehog/TGFβ (n = 12) and Hedgehog/PI3K (n = 9). In total, we identified at least one STP with potential clinical target in 88% (44/50) of HGSC samples. Conclusions: Our analysis enabled the identification of STP activity with a potential clinical target in 88% of the analyzed HGSC samples. Differentiation between normal and aberrant STP activity could have clinical utility in the selection of HGSC patients for targeted therapy. A prospective study (STAPOVER) has been designed to demonstrate clinical utility. Citation Format: Phyllis Van der Ploeg, Yvonne J.W. Wesseling-Rozendaal, Eveline C. Biezen-Timmermans, Jurgen M.J. Piek. Identification of signal transduction pathway activity with potential clinical target in high-grade serous ovarian carcinoma [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 4002.

BRCA1/2 genes mutations frequency in patients with various histological types of ovarian cancer (data for South of Russia).

e17548 Background: About 10-15% of malignant ovarian tumors (OC) are associated with hereditary diseases, and about 65-85% of hereditary OC have a mutation in BRCA1/2 genes. The aim of the study was to analyze BRCA1/2 genes hereditary mutation spectrum in patients in the South of Russia diagnosed with OC. Methods: We used EDTA venous blood and FFPE-blocks of 324 patients with histologically verified epithelial OC (T1-3cNx-1Mx-1, stage I-IV). BRCA1 gene mutations (185delAG, 300T &gt; C, 2080delA, 4153delA, 5382insC, 3819delGTAAA, 3875delGTCT) and BRCA2 gene mutations (6174delT) were determined by real-time PCR. Results: The most common types of OC were poorly differentiated serous carcinoma (high degree of malignancy) - 75.6%, well-differentiated serous carcinoma (low degree of malignancy) - 13%, mucinous tumors - 2.8%, endometrioid tumors - 4.3%, clear cell adenocarcinomas - 4.3%. BRCA1/2 genes mutations were found in 52 (16%) patients with OC. The frequency of detected BRCA1/2 genes mutations was: 5382insC - 53.8%, 4153 delA - 9.6%, 300T &gt; G - 21.2%, 2080delA - 7.7%, 185delAG - 3.8%, 6174delT - 3.8%. 3819delGTAAA and 3875delGTCT mutations were not found in the BRCA1 gene. All carriers were confirmed to have the same mutations in the tumor. There were no cases of sporadic changes in BRCA1/2. BRCA1 mutation carriers prevailed in the group of high-grade serous carcinoma patients (11.1%), where all cases of BRCA2 gene mutations were identified. In the group of low-grade serous carcinoma patients, the mutation rate was 1.5%, in the endometrioid adenocarcinomas group - 0.6%, in the mixed epithelial tumors group - 2.8%. In the group of patients with mucinous tumors, mutations were not detected. Conclusions: Thus, frequency of BRCA1/2 gene mutations in OC patients living in the South of Russia was 16.0%. Distribution of mutation types in the BRCA1 gene, with a predominance of 5382insC (53.8%) and 300T &gt; G (21.2%), corresponded to their occurrence ratio in the European populations. BRCA1/2 gene mutations were registered more often in the group of high-grade serous carcinoma patients.