Proteogenomic analysis of chemo-refractory high-grade serous ovarian cancer.

To improve the understanding of chemo-refractory high-grade serous ovarian cancers (HGSOCs), we characterized the proteogenomic landscape of 242 (refractory and sensitive) HGSOCs, representing one discovery and two validation cohorts across two biospecimen types (formalin-fixed paraffin-embedded and frozen). We identified a 64-protein signature that predicts with high specificity a subset of HGSOCs refractory to initial platinum-based therapy and is validated in two independent patient cohorts. We detected significant association between lack of Ch17 loss of heterozygosity (LOH) and chemo-refractoriness. Based on pathway protein expression, we identified 5 clusters of HGSOC, which validated across two independent patient cohorts and patient-derived xenograft (PDX) models. These clusters may represent different mechanisms of refractoriness and implicate putative therapeutic vulnerabilities.

Heterogeneous Tumor-Immune Microenvironments among Differentially Growing Metastases in an Ovarian Cancer Patient.

We present an exceptional case of a patient with high-grade serous ovarian cancer, treated with multiple chemotherapy regimens, who exhibited regression of some metastatic lesions with concomitant progression of other lesions during a treatment-free period. Using immunogenomic approaches, we found that progressing metastases were characterized by immune cell exclusion, whereas regressing and stable metastases were infiltrated by CD8+ and CD4+ T cells and exhibited oligoclonal expansion of specific T cell subsets. We also detected CD8+ T cell reactivity against predicted neoepitopes after isolation of cells from a blood sample taken almost 3 years after the tumors were resected. These findings suggest that multiple distinct tumor immune microenvironments co-exist within a single individual and may explain in part the heterogeneous fates of metastatic lesions often observed in the clinic post-therapy. VIDEO ABSTRACT.

Ovarian cancer mutational processes drive site-specific immune evasion.

High-grade serous ovarian cancer (HGSOC) is an archetypal cancer of genomic instability1-4 patterned by distinct mutational processes5,6, tumour heterogeneity7-9 and intraperitoneal spread7,8,10. Immunotherapies have had limited efficacy in HGSOC11-13, highlighting an unmet need to assess how mutational processes and the anatomical sites of tumour foci determine the immunological states of the tumour microenvironment. Here we carried out an integrative analysis of whole-genome sequencing, single-cell RNA sequencing, digital histopathology and multiplexed immunofluorescence of 160 tumour sites from 42 treatment-naive patients with HGSOC. Homologous recombination-deficient HRD-Dup (BRCA1 mutant-like) and HRD-Del (BRCA2 mutant-like) tumours harboured inflammatory signalling and ongoing immunoediting, reflected in loss of HLA diversity and tumour infiltration with highly differentiated dysfunctional CD8+ T cells. By contrast, foldback-inversion-bearing tumours exhibited elevated immunosuppressive TGFß signalling and immune exclusion, with predominantly naive/stem-like and memory T cells. Phenotypic state associations were specific to anatomical sites, highlighting compositional, topological and functional differences between adnexal tumours and distal peritoneal foci. Our findings implicate anatomical sites and mutational processes as determinants of evolutionary phenotypic divergence and immune resistance mechanisms in HGSOC. Our study provides a multi-omic cellular phenotype data substrate from which to develop and interpret future personalized immunotherapeutic approaches and early detection research.

Functional analysis of the 1p34.3 risk locus implicates GNL2 in high-grade serous ovarian cancer.

The high-grade serous ovarian cancer (HGSOC) risk locus at chromosome 1p34.3 resides within a frequently amplified genomic region signifying the presence of an oncogene. Here, we integrate in silico variant-to-function analysis with functional studies to characterize the oncogenic potential of candidate genes in the 1p34.3 locus. Fine mapping of genome-wide association statistics identified candidate causal SNPs local to H3K27ac-demarcated enhancer regions that exhibit allele-specific binding for CTCF in HGSOC and normal fallopian tube secretory epithelium cells (FTSECs). SNP risk associations colocalized with eQTL for six genes (DNALI1, GNL2, RSPO1, SNIP1, MEAF6, and LINC01137) that are more highly expressed in carriers of the risk allele, and three (DNALI1, GNL2, and RSPO1) were upregulated in HGSOC compared to normal ovarian surface epithelium cells and/or FTSECs. Increased expression of GNL2 and MEAF6 was associated with shorter survival in HGSOC with 1p34.3 amplifications. Despite its activation of ß-catenin signaling, RSPO1 overexpression exerted no effects on proliferation or colony formation in our study of ovarian cancer and FTSECs. Instead, GNL2, MEAF6, and SNIP1 silencing impaired in vitro ovarian cancer cell growth. Additionally, GNL2 silencing diminished xenograft tumor formation, whereas overexpression stimulated proliferation and colony formation in FTSECs. GNL2 influences 60S ribosomal subunit maturation and global protein synthesis in ovarian cancer and FTSECs, providing a potential mechanism of how GNL2 upregulation might promote ovarian cancer development and mediate genetic susceptibility of HGSOC.

A Deep Learning-Based Assessment Pipeline for Intraepithelial and Stromal Tumor-Infiltrating Lymphocytes in High-Grade Serous Ovarian Carcinoma.

Tumor-infiltrating lymphocytes (TILs) are associated with improved survival in patients with epithelial ovarian cancer. However, TIL evaluation has not been used in routine clinical practice because of reproducibility issues. The current study developed two convolutional neural network models to detect TILs and to determine their spatial location in whole slide images, and established a spatial assessment pipeline to objectively quantify intraepithelial and stromal TILs in patients with high-grade serous ovarian carcinoma. The predictions of the established models showed a significant positive correlation with the number of CD8+ T cells and immune gene expressions. Patients with a higher density of intraepithelial TILs had a significantly prolonged overall survival and progression-free survival in multiple cohorts. On the basis of the density of intraepithelial and stromal TILs, patients were classified into three immunophenotypes: immune inflamed, excluded, and desert. The immune-desert subgroup showed the worst prognosis. Gene expression analysis showed that the immune-desert subgroup had lower immune cytolytic activity and T-cell-inflamed gene-expression profile scores, whereas the immune-excluded subgroup had higher expression of interferon-γ and programmed death 1 receptor signaling pathway. The established evaluation method provided detailed and comprehensive quantification of intraepithelial and stromal TILs throughout hematoxylin and eosin-stained slides. It has potential for clinical application for personalized treatment of patients with ovarian cancer.

Mathematical modeling of the evolution of resistance and aggressiveness of high-grade serous ovarian cancer from patient CA-125 time series.

A time-series analysis of serum Cancer Antigen 125 (CA-125) levels was performed in 791 patients with high-grade serous ovarian cancer (HGSOC) from the Australian Ovarian Cancer Study to evaluate the development of chemoresistance and response to therapy. To investigate chemoresistance and better predict the treatment effectiveness, we examined two traits: resistance (defined as the rate of CA-125 change when patients were treated with therapy) and aggressiveness (defined as the rate of CA-125 change when patients were not treated). We found that as the number of treatment lines increases, the data-based resistance increases (a decreased rate of CA-125 decay). We use mathematical models of two distinct cancer cell types, treatment-sensitive cells and treatment-resistant cells, to estimate the values and evolution of the two traits in individual patients. By fitting to individual patient HGSOC data, our models successfully capture the dynamics of the CA-125 level. The parameters estimated from the mathematical models show that patients with inferred low growth rates of treatment-sensitive cells and treatment-resistant cells (low model-estimated aggressiveness) and a high death rate of treatment-resistant cells (low model-estimated resistance) have longer survival time after completing their second-line of therapy. These findings show that mathematical models can characterize the degree of resistance and aggressiveness in individual patients, which improves our understanding of chemoresistance development and could predict treatment effectiveness in HGSOC patients.

Mesonephric-like adenocarcinoma harbours characteristic copy number variations and a distinct DNA methylation signature closely related to mesonephric adenocarcinoma of the cervix.

Mesonephric-like adenocarcinoma (MLA) of the female genital tract is an uncommon histotype that can arise in both the endometrium and the ovary. The exact cell of origin and histogenesis currently remain unknown. Here, we investigated whole genome DNA methylation patterns and copy number variations (CNVs) in a series of MLAs in the context of a large cohort of various gynaecological carcinoma types. CNV analysis of 19 MLAs uncovered gains of chromosomes 1q (18/19, 95%), 10 (15/19, 79%), 12 (14/19, 74%), and 2 (10/19, 53%), as well as loss of chromosome 1p (7/19, 37%). Gains of chromosomes 1q, 10, and 12 were also identified in the majority of mesonephric adenocarcinomas of the uterine cervix (MAs) as well as subsets of endometrioid carcinomas (ECs) and low-grade serous carcinomas of the ovary (LGSCs) but only in a minority of serous carcinomas of the uterine corpus (USCs), clear cell carcinomas (CCCs), and tubo-ovarian high-grade serous carcinomas (HGSCs). While losses of chromosome 1p together with gains of chromosome 1q were also identified in both MA and LGSC, gains of chromosome 2 were almost exclusively identified in MLA and MA. Unsupervised hierarchical clustering and t-SNE analysis of DNA methylation data (Illumina EPIC array) identified a co-clustering for MLAs and MAs, which was distinct from clusters of ECs, USCs, CCCs, LGSCs, and HGSCs. Group-wise comparisons confirmed a close epigenetic relationship between MLA and MA. These findings, in conjunction with the established histological and immunophenotypical overlap, suggest bona fide mesonephric differentiation, and support a more precise terminology of mesonephric-type adenocarcinoma instead of MLA in these tumours. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Proteomic Discovery of Plasma Protein Biomarkers and Development of Models Predicting Prognosis of High-Grade Serous Ovarian Carcinoma.

Ovarian cancer is one of the most lethal female cancers. For accurate prognosis prediction, this study aimed to investigate novel, blood-based prognostic biomarkers for high-grade serous ovarian carcinoma (HGSOC) using mass spectrometry-based proteomics methods. We conducted label-free liquid chromatography-tandem mass spectrometry using frozen plasma samples obtained from patients with newly diagnosed HGSOC (n = 20). Based on progression-free survival (PFS), the samples were divided into two groups: good (PFS ≥18 months) and poor prognosis groups (PFS <18 months). Proteomic profiles were compared between the two groups. Referring to proteomics data that we previously obtained using frozen cancer tissues from chemotherapy-naïve patients with HGSOC, overlapping protein biomarkers were selected as candidate biomarkers. Biomarkers were validated using an independent set of HGSOC plasma samples (n = 202) via enzyme-linked immunosorbent assay (ELISA). To construct models predicting the 18-month PFS rate, we performed stepwise selection based on the area under the receiver operating characteristic curve (AUC) with 5-fold cross-validation. Analysis of differentially expressed proteins in plasma samples revealed that 35 and 61 proteins were upregulated in the good and poor prognosis groups, respectively. Through hierarchical clustering and bioinformatic analyses, GSN, VCAN, SND1, SIGLEC14, CD163, and PRMT1 were selected as candidate biomarkers and were subjected to ELISA. In multivariate analysis, plasma GSN was identified as an independent poor prognostic biomarker for PFS (adjusted hazard ratio, 1.556; 95% confidence interval, 1.073-2.256; p = 0.020). By combining clinical factors and ELISA results, we constructed several models to predict the 18-month PFS rate. A model consisting of four predictors (FIGO stage, residual tumor after surgery, and plasma levels of GSN and VCAN) showed the best predictive performance (mean validated AUC, 0.779). The newly developed model was converted to a nomogram for clinical use. Our study results provided insights into protein biomarkers, which might offer clues for developing therapeutic targets.

Targeted Mass Spectrometry of Longitudinal Patient Sera Reveals LTBP1 as a Potential Surveillance Biomarker for High-Grade Serous Ovarian Carcinoma.

High-grade serous ovarian carcinoma (HGSC) is the most prevalent subtype of epithelial ovarian cancer. The combination of a high rate of recurrence and novel therapies in HGSC necessitates an accurate assessment of the disease. Currently, HGSC response to treatment and recurrence are monitored via immunoassay of serum levels of the glycoprotein CA125. CA125 levels predictably rise at HGSC recurrence; however, it is likely that the disease is progressing even before it is detectable through CA125. This may explain why treating solely based on CA125 increase has not been associated with improved outcomes. Thus, additional biomarkers that monitor HGSC progression and cancer recurrence are needed. For this purpose, we developed a scheduled parallel reaction monitoring mass spectrometry (PRM-MS) assay for the quantification of four previously identified HGSC-derived glycopeptides (from proteins FGL2, LGALS3BP, LTBP1, and TIMP1). We applied the assay to quantify their longitudinal expression profiles in 212 serum samples taken from 34 HGSC patients during disease progression. Analyses revealed that LTBP1 best-mirrored tumor load, dropping as a result of cancer treatment in 31 out of 34 patients and rising at HGSC recurrence in 28 patients. Additionally, LTBP1 rose earlier during remission than CA125 in 11 out of 25 platinum-sensitive patients with an average lead time of 116.4 days, making LTBP1 a promising candidate for monitoring of HGSC recurrence.

N6-Methyladenosine-Modified KREMEN2 Promotes Tumorigenesis and Malignant Progression of High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) remains the most lethal female cancer by far. Herein, clinical HGSOC samples had higher N6-methyladenosine (m6A) modification than normal ovarian tissue, and its dysregulation had been reported to drive aberrant transcription and translation programs. However, Kringle-containing transmembrane protein 2 (KREMEN2) and its m6A modification have not been fully elucidated in HGSOC. In this study, the data from the high-throughput messenger RNA (mRNA) sequencing of clinical samples were processed using the weighted correlation network analysis and functional enrichment analysis. Results revealed that KREMEN2 was a driver gene in the tumorigenesis of HGSOC and a potential target of m6A demethylase fat-mass and obesity-associated protein (FTO). KREMEN2 and FTO levels were upregulated and downregulated, respectively, and correlation analysis showed a significant negative correlation in HGSOC samples. Importantly, upregulated KREMEN2 was remarkably associated with lymph node metastasis, distant metastasis, peritoneal metastasis, and high International Federation of Gynecology and Obstetrics stage (Ⅲ/Ⅳ), independent of the age of patients. KREMEN2 promoted the growth of HGSOC in vitro and in vivo, which was dependent on FTO. The methylated RNA immunoprecipitation qPCR and RNA immunoprecipitation assays were performed to verify the m6A level and sites of KREMEN2. FTO overexpression significantly decreased m6A modification in the 3' and 5' untranslated regions of KREMEN2 mRNA and downregulated its expression. In addition, we found that FTO-mediated m6A modification of KREMEN2 mRNA was recognized and stabilized by the m6A reader IGF2BP1 rather than by IGF2BP2 or IGF2BP3. This study highlights the m6A modification of KREMEN2 and extends the importance of RNA epigenetics in HGSOC.

Trps1 predicts poor prognosis in advanced high grade serous ovarian carcinoma.

TRPS1 is aberrantly expressed in a variety of tumors, including breast, prostate, and gastric cancers, and is strongly associated with tumorigenesis or prognosis. However, the role of TRPS1 in high grade serous ovarian carcinoma (HGSC) is unknown. We investigated the relationship between TRPS1 expression and clinicopathology in HGSC patients. The tumor-related regulatory mechanisms of TRPS1 was explored through in vivo and vitro experiments. The results showed that TRPS1 was highly expressed in HGSC compared to normal tissues. It was also linked to the cell proliferation index Ki67 and poor prognosis. In vivo experiments showed that knockdown of TRPS1 could inhibit tumor growth. In vitro experiments, knockdown of TRPS1 inhibited the proliferation of ovarian cancer cells. TRPS1 exerted its regulatory role as a transcription factor, binding to the PSAT1 promoter and promoting the expression of PSAT1 gene. Meanwhile, PSAT1 was positively correlated with CCND1 expression. These results suggest that TRPS1 affects HGSC proliferation and cell cycle by regulating PSAT1 and thus CCND1 expression.

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.

Targeting Galectin 3 illuminates its contributions to the pathology of uterine serous carcinoma.

BACKGROUND: Uterine serous cancer (USC) comprises around 10% of all uterine cancers. However, USC accounts for approximately 40% of uterine cancer deaths, which is attributed to tumor aggressiveness and limited effective treatment. Galectin 3 (Gal3) has been implicated in promoting aggressive features in some malignancies. However, Gal3's role in promoting USC pathology is lacking. METHODS: We explored the relationship between LGALS3 levels and prognosis in USC patients using TCGA database, and examined the association between Gal3 levels in primary USC tumors and clinical-pathological features. CRISPR/Cas9-mediated Gal3-knockout (KO) and GB1107, inhibitor of Gal3, were employed to evaluate Gal3's impact on cell function. RESULTS: TCGA analysis revealed a worse prognosis for USC patients with high LGALS3. Patients with no-to-low Gal3 expression in primary tumors exhibited reduced clinical-pathological tumor progression. Gal3-KO and GB1107 reduced cell proliferation, stemness, adhesion, migration, and or invasion properties of USC lines. Furthermore, Gal3-positive conditioned media (CM) stimulated vascular tubal formation and branching and transition of fibroblast to cancer-associated fibroblast compared to Gal3-negative CM. Xenograft models emphasized the significance of Gal3 loss with fewer and smaller tumors compared to controls. Moreover, GB1107 impeded the growth of USC patient-derived organoids. CONCLUSION: These findings suggest inhibiting Gal3 may benefit USC patients.

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

BACKGROUND: The aim of this study was to analyse transcriptomic differences between primary and recurrent high-grade serous ovarian carcinoma (HGSOC) to identify prognostic biomarkers. METHODS: We 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. RESULTS: We 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. CONCLUSIONS: Our 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.

ANGPTL4 accelerates ovarian serous cystadenocarcinoma carcinogenesis and angiogenesis in the tumor microenvironment by activating the JAK2/STAT3 pathway and interacting with ESM1.

BACKGROUND: Ovarian cancer (OC) is a malignant neoplasm that displays increased vascularization. Angiopoietin-like 4 (ANGPTL4) is a secreted glycoprotein that functions as a regulator of cell metabolism and angiogenesis and plays a critical role in tumorigenesis. However, the precise role of ANGPTL4 in the OC microenvironment, particularly its involvement in angiogenesis, has not been fully elucidated. METHODS: The expression of ANGPTL4 was confirmed by bioinformatics and IHC in OC. The potential molecular mechanism of ANGPTL4 was measured by RNA-sequence. We used a series of molecular biological experiments to measure the ANGPTL4-JAK2-STAT3 and ANGPTL4-ESM1 axis in OC progression, including MTT, EdU, wound healing, transwell, xenograft model, oil red O staining, chick chorioallantoic membrane assay and zebrafish model. Moreover, the molecular mechanisms were confirmed by Western blot, Co-IP and molecular docking. RESULTS: Our study demonstrates a significant upregulation of ANGPTL4 in OC specimens and its strong association with unfavorable prognosis. RNA-seq analysis affirms that ANGPTL4 facilitates OC development by driving JAK2-STAT3 signaling pathway activation. The interaction between ANGPTL4 and ESM1 promotes ANGPTL4 binding to lipoprotein lipase (LPL), thereby resulting in reprogrammed lipid metabolism and the promotion of OC cell proliferation, migration, and invasion. In the OC microenvironment, ESM1 may interfere with the binding of ANGPTL4 to integrin and vascular-endothelial cadherin (VE-Cad), which leads to stabilization of vascular integrity and ultimately promotes angiogenesis. CONCLUSION: Our findings underscore that ANGPTL4 promotes OC development via JAK signaling and induces angiogenesis in the tumor microenvironment through its interaction with ESM1.

The comparison of pure uterine serous carcinoma and mixed tumor with serous component: a single-institution review of 91 cases.

BACKGROUND: Pure uterine serous carcinoma (p-USC) and mixed tumors with serous component (m-USC) are aggressive subtypes of endometrial cancer associated with high mortality rates. This retrospective study aimed to compare clinicopathologic features and outcomes of p-USC and m-USC in a single center. METHODS: This study retrospectively reviewed patients diagnosed with USC at Peking University People's Hospital between 2008 and 2022. T-tests and chi-square tests were used to compare clinicopathological characteristics between p-USC and m-USC. Kaplan-Meier survival curve and Cox regression analysis were used to analyze the impact of clinical and pathological variables on OS and PFS. RESULTS: Among the 91 patients who underwent surgery, 65.9% (n = 60) were p-USC, and 34.1% (n = 31) were m-USC. Patients with p-USC had earlier menopause (P = 0.0217), a lower rate of progesterone receptor(PR) expression (P < 0.001), and were more likely to have positive peritoneal cytology (P = 0.0464). After a median follow-up time of 40 months, 28 (46.7%) p-USC and 9 (29%) m-USC patients had progression disease, 18 (30%) and 8 (25.8%) patients died of their disease. 5-year PFSR were 51.2% and 75.3%, respectively, and 5-year OS rates were 66% and 67.4%. Kaplan-Meier survival analysis showed that p-USC was more likely to relapse than m-USC (P = 0.034), but there was no significant difference in OS. Cox regression analysis showed that lymph node metastasis and surgical approach were risk factors for OS, and myoinvasion depth ≥ 1/2 was an independent risk factor for PFS. CONCLUSIONS: p-USC was more likely to relapse than m-USC, but there was no significant difference in OS between the two subtypes.

HRD related signature 3 predicts clinical outcome in advanced tubo-ovarian high-grade serous carcinoma.

OBJECTIVES: We evaluated usability of single base substitution signature 3 (Sig3) as a biomarker for homologous recombination deficiency (HRD) in tubo-ovarian high-grade serous carcinoma (HGSC). MATERIALS AND METHODS: This prospective observational trial includes 165 patients with advanced HGSC. Fresh tissue samples (n = 456) from multiple intra-abdominal areas at diagnosis and after neoadjuvant chemotherapy (NACT) were collected for whole-genome sequencing. Sig3 was assessed by fitting samples independently with COSMIC v3.2 reference signatures. An HR scar assay was applied for comparison. Progression-free survival (PFS) and overall survival (OS) were studied using Kaplan-Meier and Cox regression analysis. RESULTS: Sig3 has a bimodal distribution, eliminating the need for an arbitrary cutoff typical in HR scar tests. Sig3 could be assessed from samples with low (10%) cancer cell proportion and was consistent between multiple samples and stable during NACT. At diagnosis, 74 (45%) patients were HRD (Sig3+), while 91 (55%) were HR proficient (HRP, Sig3-). Sig3+ patients had longer PFS and OS than Sig3- patients (22 vs. 13 months and 51 vs. 34 months respectively, both p < 0.001). Sig3 successfully distinguished the poor prognostic HRP group among BRCAwt patients (PFS 19 months for Sig3+ and 13 months for Sig3- patients, p < 0.001). However, Sig3 at diagnosis did not predict chemoresponse anymore in the first relapse. The patient-level concordance between Sig3 and HR scar assay was 87%, and patients with HRD according to both tests had the longest median PFS. CONCLUSIONS: Sig3 is a prognostic marker in advanced HGSC and useful tool in patient stratification for HRD.

CD137+ tumor infiltrating lymphocytes predicts ovarian cancer survival.

OBJECTIVE: Ovarian cancer (OC) is the leading cause of death from gynecologic malignancy in the United States, and biomarkers of patient outcomes are limited. Data using immunohistochemical (IHC) analysis are mixed regarding whether and which tumor infiltrating lymphocytes (TILs) impact survival, and IHC does not adequately quantify rare cell populations, including CD137+ (4-1BB) tumor-reactive TILs. Our study investigates if a higher percentage of CD3+ CD137+ TILs is associated with improved overall survival (OS) in OC. METHODS: Flow cytometry was performed on viably banked OC digests. Chart review and statistical analysis were performed. Forty-seven patients were included, 40 of whom were diagnosed with high-grade serous ovarian carcinoma (HGSOC), papillary serous carcinoma, or undifferentiated histology. RESULTS: A high percentage of CD3+ CD137+ TILs correlated with improved OS (n = 40, r = 0.48, P = 0.0016). Subjects were divided into CD3+ CD137+ TIL high and low groups by the median. Subjects with high CD3+CD137+ TIL frequencies (>9.6%) had longer OS (Wilcoxon rank-sum test; P = 0.0032) and improved OS (logrank test; P = 0.007). Differences in CD3+ or CD3+ CD8+ TILs did not impact survival. CD3+ CD137+ TILs were predictive of OS regardless of germline mutation or debulking status. Analysis of subgroups including late stage HGSOC and late stage HGSOC with primary optimal cytoreduction indicated CD3+ CD137+ TILs correlated with improved OS after adjusting for age and PARP inhibitor use (P = 0.034 and P = 0.016, respectively). CONCLUSIONS: Prevalence of CD3+ CD137+ TILs in digested OC specimens is associated with improved OS, while general TIL markers are not. CD137 has the potential to be a novel biomarker for survival in OC.

A spatial proteomic study of platinum refractory HGSOC implicates dual AKT and WNT activity linked to an immunosuppressive tumor microenvironment.

OBJECTIVE: Advanced-stage high-grade serous ovarian cancer (HGSOC) remains a deadly gynecologic malignancy with high rates of disease recurrence and limited, effective therapeutic options for patients. There is a significant need to better stratify HGSOC patients into platinum refractory (PRF) vs. sensitive (PS) cohorts at baseline to improve therapeutic responses and survival outcomes for PRF HGSOC. METHODS: We performed NanoString for GeoMx Digital Spatial Profile (G-DSP) multiplex protein analysis on PRF and PS tissue microarrays (TMAs) to study the bidirectional communication of cancer cells with immune cells in the tumor microenvironment (TME) of HGSOC. We demonstrate robust stratification of PRF and PS tumors at baseline using multiplex spatial proteomic biomarkers with implications for tailoring subsequent therapy. RESULTS: PS patients had elevated apoptotic and anti-tumor immune profiles, while PRF patients had dual AKT1 and WNT signaling with immunosuppressive profiles. We found that dual activity of AKT1 and WNT signaling supported the exclusion of immune cells, specifically tumor infiltrating lymphocytes (TILs), from the TME in PRF tumors, and this was not observed in PS tumors. The exclusion of immune cells from the TME of PRF tumors corresponded to abnormal endothelial cell structure in tumors with dual AKT1 and WNT signaling activity. CONCLUSIONS: We believe our findings provide improved understanding of tumor-immune crosstalk in HGSOC TME highlighting the importance of the relationship between AKT and WNT pathways, immune cell function, and platinum response in HGSOC.

Preclinical efficacy of RAF/MEK clamp avutometinib in combination with FAK inhibition in low grade serous ovarian cancer.

OBJECTIVES: Low-grade-serous-ovarian-carcinoma (LGSOC) is characterized by a high recurrence rate and limited therapeutic options. About one-third of LGSOC contains mutations in MAPK pathway genes such as KRAS/NRAS/BRAF. Avutometinib is a dual RAF/MEK inhibitor while defactinib and VS-4718 are focal-adhesion-kinase-inhibitors (FAKi). We determined the preclinical efficacy of avutometinib±VS-4718 in LGSOC patient-derived-tumor-xenografts (PDX). METHODS: Whole-exome-sequencing (WES) was used to evaluate the genetic fingerprint of 3 patient-derived LGSOC (OVA(K)250, PERIT(M)17 and A(PE)148). OVA(K)250 tissue was successfully xenografted as PDX into female CB17/lcrHsd-Prkdc/SCID-mice. Animals were treated with either control, avutometinib, VS-4718, or avutometinib/ VS-4718 once daily five days on and two days off through oral gavage. Mechanistic studies were performed ex vivo using avutometinib±defactinib treated LGSOC tumor samples by western blot. RESULTS: WES results demonstrated wild-type KRAS in all 3 LGSOC. OVA(K)250 PDX showed gain-of-function mutations (GOF) in PTK2 and PTK2B genes, and loss-of-heterozygosity in ADRB2, potentially sensitizing to FAK and RAF/MEK inhibition. The combination of avutometinib/ VS-4718 demonstrated strong tumor-growth inhibition compared to controls starting at day 9 (p < 0.002) in OVA(K)250PDX. By 60 days, mice treated with avutometinib alone and avutometinib/VS-4718 were still alive; compared to median survival of 20 days in control-treated mice and of 35 days in VS-4718-treated mice (p < 0.0001). By western-blot assays exposure of OVA(K)250 to avutometinib, FAKi defactinib and their combination demonstrated decreased phosphorylated FAK (p-FAK) as well as decreased p-ERK. CONCLUSION: Avutometinib, and to a larger extent its combination with FAK inhibitor VS-4718, demonstrated promising in vivo activity against a KRAS wild-type LGSOC-PDX. These data support the ongoing registration-directed study (RAMP201/NCT04625270).