Grade Serous Epithelial Ovarian Cancer Research Articles

Immune profile of primary and recurrent epithelial ovarian cancer cases indicates immune suppression, a major cause of progression and relapse of ovarian cancer

BackgroundOvarian cancer is the third most prevalent cancer in Indian women. Relative frequency of High grade serous epithelial ovarian cancer (HGSOC) and its associated deaths are highest in India which suggests the importance of understanding their immune profiles for better treatment modality. Hence, the present study investigated the NK cell receptor expression, their cognate ligands, serum cytokines, and soluble ligands in primary and recurrent HGSOC patients. We have used multicolor flow cytometry for immunophenotyping of tumor infiltrated and circulatory lymphocytes. Procartaplex, and ELISA were used to measure soluble ligands and cytokines of HGSOC patients.ResultsAmong the enrolled 51 EOC patients, 33 were primary high grade serous epithelial ovarian cancer (pEOC) and 18 were recurrent epithelial ovarian cancer (rEOC) patients. Blood samples from 46 age matched healthy controls (HC) were used for comparative analysis. Results revealed, frequency of circulatory CD56Bright NK, CD56Dim NK, NKT-like, and T cells was reduced with activating receptors while alterations in immune subsets with inhibitory receptors were observed in both groups. Study also highlights differential immune profile of primary and recurrent ovarian cancer patients. We have found increased soluble MICA which might have acted as “decoy” molecule and could be a reason of decrease in NKG2D positive subsets in both groups of patients. Furthermore, elevated level of serum cytokines IL-2, IL-5, IL-6, IL-10, and TNF-α in ovarian cancer patients, might be associated with ovarian cancer progression. Profiling of tumor infiltrated immune cells revealed the reduced level of DNAM-1 positive NK and T cells in both groups than their circulatory counterpart, which might have led to decrease in NK cell’s ability of synapse formation.ConclusionsThe study brings out differential receptor expression profile on CD56BrightNK, CD56DimNK, NKT-like, and T cells, cytokines levels and soluble ligands which may be exploited to develop alternate therapeutic approaches for HGSOC patients. Further, few differences in the circulatory immune profiles between pEOC and rEOC cases, indicates the immune signature of pEOC undergoes some changes in circulation that might facilitated the disease relapse. They also maintains some common immune signatures such as reduced expression of NKG2D, high level of MICA as well as IL-6, IL10 and TNF-α, which indicates irreversible immune suppression of ovarian cancer patients. It is also emphasized that a restoration of cytokines level, NKG2D and DNAM-1on tumor infiltrated immune cells may be targeted to develop specific therapeutic approaches for high-grade serous epithelial ovarian cancer.

BRCA 1 and 2 somatic mutation in high grade serous epithelial ovarian cancer in Indonesian women that was operated on Dr. Soetomo General Hospital, Surabaya in 2019-2021

Link of Video Abstract: https://youtu.be/9kem7M9SNWIBackground: Ovarian cancer remains as major problem worldwide. BRCA1 and BRCA2 play a unique role in deoxyribonucleic acid (DNA) damage repair, especially in homologous recombination repair. There are no data on the prevalence of BRCA1 and BRCA2 somatic mutations in the Indonesian population. This study aimed to determine the incidence of BRCA1 and BRCA2 somatic mutations among a cohort of patients with high-grade serous epithelial ovarian cancer, and assess its correlation with clinical characteristics of the cohort. Methods: This was an analytical observational study of 26 patients admitted to our hospital from 2019 to 2021. Pathological specimens were obtained from the patients and examined for BRCA1 and BRCA2 somatic mutations. Data on clinical characteristics were collected from medical records. Statistical analysis was performed to determine the relationship between BRCA mutation status and clinical characteristics, including age, stage, menopausal status, parity, family history of malignancy, and patient outcomes. Results: Twenty-six patients with high-grade serous epithelial ovarian cancer were included in the study. Six patients (27% of 22 patients) had BRCA1 and BRCA2 somatic mutations, four did not pass the quality control test, and the remaining tested negative for these mutations. Most patients were over 50 years of age (73%). Additionally, 84% of patients were diagnosed at stage III, and only one patient had a family history of malignancy. At the end of the study, most patients (65%) had died. Statistical analysis showed no significant differences between the clinical characteristics of the groups. Conclusion: The incidence of BRCA1 and BRCA2 somatic mutations among patients with high-grade serous epithelial ovarian cancer at our hospital during 2019–2021 was 27%. There were no significant differences in the clinical characteristics of those with and without mutations, hence NGS screening following diagnosis establishment may be beneficial in further management plans.

Targeting Mucin Protein Enables Rapid and Efficient Ovarian Cancer Cell Capture: Role of Nanoparticle Properties in Efficient Capture and Culture.

The development of specific and sensitive immunomagnetic cell separation nanotechnologies is central to enhancing the diagnostic relevance of circulating tumor cells (CTCs) and improving cancer patient outcomes. The limited number of specific biomarkers used to enrich a phenotypically diverse set of CTCs from liquid biopsies has limited CTC yields and purity. The ultra-high molecular weight mucin, mucin16(MUC16) is shown to physically shield key membrane proteins responsible for activating immune responses against ovarian cancer cells and may interfere with the binding of magnetic nanoparticles to popular immunomagnetic cell capture antigens. MUC16 is expressed in ≈90% of ovarian cancers and is almost universal in High Grade Serous Epithelial Ovarian Cancer. This work demonstrates that cell bound MUC16 is an effective target for rapid immunomagnetic extraction of expressor cells with near quantitative yield, high purity and viability from serum. The results provide a mechanistic insight into the effects of nanoparticle physical properties and immunomagnetic labeling on the efficiency of immunomagnetic cell isolation. The growth of these cells has also been studied after separation, demonstrating that nanoparticle size impacts cell-particle behavior and growth rate. These results present the successful isolation of "masked" CTCs enabling new strategies for the detection of cancer recurrence and select and monitor chemotherapy.

Immune Profile of Blood, Tissue and Peritoneal Fluid: A Comparative Study in High Grade Serous Epithelial Ovarian Cancer Patients at Interval Debulking Surgery.

High-grade serous epithelial ovarian carcinoma (HGSOC) is an immunogenic tumor with a unique tumor microenvironment (TME) that extends to the peritoneal cavity. The immunosuppressive nature of TME imposes the major challenge to develop effective treatment options for HGSOC. Interaction of immune cells in TME is an important factor. Hence, a better understanding of immune profile of TME may be required for exploring alternative treatment options. Immune profiling of peritoneal fluid (PF), tumor specimens, and blood were carried out using flowcytometry, ELISA, and Procartaplex immunoassay. The frequency of CD56BrightNK cells and expression of functional receptors were reduced in PF. Increased activating NKp46+CD56DimNK cells may indicate differential antitumor response in PF. Functional receptors on NK, NKT-like and T cells were reduced more drastically in tumor specimens. Soluble ligands MIC-B and PVR were reduced, whereas B7-H6 was increased in PF. Dissemination of tumor cells contributes to soluble ligands in PF. A differential cytokine profile was found in serum and PF as IL-2, IL-8, IL-15, IL-27, IFN-γ, and GM-CSF were elevated specifically in PF. In conclusion, the differential immune profile and correlation of soluble parameters and NK cell receptors with chemo response score may add knowledge to understand anti-tumor immune response to develop effective treatment modality.

Survival and Chemosensitivity in Advanced High Grade Serous Epithelial Ovarian Cancer Patients with and without a BRCA Germline Mutation: More Evidence for Shifting the Paradigm towards Complete Surgical Cytoreduction.

Background and Objectives: Approximately 10-15% of high-grade serous ovarian cancer (HGSOC) cases are related to BRCA germline mutations. Better survival rates and increased chemosensitivity are reported in patients with a BRCA 1/2 germline mutation. However, the FIGO stage and histopathological entity may have been confounding factors. This study aimed to compare chemotherapy response and survival between patients with and without a BRCA 1/2 germline mutation in advanced HGSOC receiving neoadjuvant chemotherapy (NACT). Materials and Methods: A cohort of BRCA-tested advanced HGSOC patients undergoing cytoreductive surgery following NACT was analyzed for chemotherapy response and survival. Neoadjuvant chemotherapy served as a vehicle to assess chemotherapy response on biochemical (CA125), histopathological (CRS), biological (dissemination), and surgical (residual disease) levels. Univariate and multivariate analyses for chemotherapy response and survival were utilized. Results: Thirty-nine out of 168 patients had a BRCA ½ germline mutation. No differences in histopathological chemotherapy response between the patients with and without a BRCA ½ germline mutation were observed. Survival in the groups of patients was comparable Irrespective of the BRCA status, CRS 2 and 3 (HR 7.496, 95% CI 2.523-22.27, p < 0.001 & HR 4.069, 95% CI 1.388-11.93, p = 0.011), and complete surgical cytoreduction (p = 0.017) were independent parameters for a favored overall survival. Conclusions: HGSOC patients with or without BRCA ½ germline mutations, who had cytoreductive surgery, showed comparable chemotherapy responses and subsequent survival. Irrespective of BRCA status, advanced-stage HGSOC patients have a superior prognosis with complete surgical cytoreduction and good histopathological response to chemotherapy.

Modification of intrinsic ovarian cancer tumor factors by Wnt-pathway agents influences macrophage recruitment and activity.

e14514 Background: The Wnt/B-catenin pathway is associated with a “cold” phenotype in the gynecologic malignancy tumor microenvironment (TME), resulting in immunosuppression. Research is limited regarding this pathway’s effects on TME myeloid cell populations. We aimed to characterize the impact of Wnt pathway modulation on macrophage activity in ovarian cancer. Methods: Syngeneic murine models harboring ID8 parental (ID8par) tumors were treated with DKN-01 and CGX-1321 as single agents or in sequence (DKN-01 followed by CGX-1321.) Flow cytometry analyses were performed on harvested omenta and blood. Ascites from 10 high grade serous epithelial ovarian cancer patients were treated with DKN-01 (Wnt activator) or CGX-1321 (Wnt inhibitor) for 48 hours; multiplex cytokine array was performed to determine changes in cytokine/chemokine expression. Lastly, we performed co-culture analyses on the effects of ID8par and ID8p53-/- isolated cell media on murine macrophage (RAW264.7) activity. Results: In the ID8par model, sequential DKN-01/CGX-1321 resulted in the greatest macrophage influx into the TME. CGX-1321 monotherapy increased the M2:M1 macrophage ratio (pro-tumor) while DKN-01 monotherapy resulted in decreased M2:M1 ratio (anti-tumor.) In human ascites, DKN-01 increased macrophage colony stimulating factor (M-CSF) expression. Of note, ascites from a patient harboring a p53 mutation demonstrated an increase in M1 polarization cytokines IFNy and TNFa in response to DKN-01; CGX-1321 treatment resulted in increased M2 polarization cytokines IL-4 and IL-13. In co-culture analysis, ID8par isolated media increased RAW264.7 migration and polarization towards an M2 phenotype; ID8p53-/- isolated media polarized towards an M1 phenotype. Conclusions: Macrophage activity influences immune responses and tumor behavior. For example, M2 macrophages are pro-tumor and can enhance tumor growth and immune evasion. In our studies, we demonstrated that macrophage recruitment and polarization (M1 vs M2) are influenced by genetic alterations that can be modified by treatment. The ID8par model positively influenced M2 macrophage activity; ID8p53-/- cells positively influenced M1 macrophage activity. Importantly, DKN-01 treatment reverses the M2-dominant phenotype in ID8par model towards M1. These results suggest that p53 mutation and/or DKN-01 treatment in ovarian cancer influences M1 anti-tumor macrophage activity, and posits a novel targetable pathway. Further studies should investigate Wnt/B-catenin modulation on macrophage activity for immunotherapy in gynecologic malignancies.

Neoadjuvant treatment for newly diagnosed advanced ovarian cancer: where do we stand and where are we going?

Newly diagnosed high grade serous epithelial ovarian cancer (EOC) patients are treated with radical surgery followed by adjuvant platinum and taxane combination chemotherapy. In EOC patients where upfront surgery is contraindicated for medical reasons (e.g., comorbidities or poor performance status), or where complete cytoreduction cannot be achieved, neoadjuvant chemotherapy (NACT) prior to interval debulking surgery (IDS), and adjuvant chemotherapy is an alternative therapeutic option. There is currently a lack of consensus about who are the best candidates to receive NACT, and some authors have even suggested that this approach could be harmful in a subset of patients via promotion of early chemoresistance. Standard and novel imaging techniques together with a better molecular characterization of the disease have the potential to improve selection of patients, but ultimately well designed randomised clinical trials are needed to guide treatment decisions in this setting. The advent of new and effective treatment options (antiangiogenics and PARP inhibitors), now approved for use in the first line and relapse settings has opened the way to clinical trials aiming to investigate these agents as substitute or in addition to chemotherapy in the neoadjuvant setting in molecularly selected EOC patients. Here, we will review the evidence supporting the use of NACT in newly diagnosed EOCs, data highlighting the importance of its use in selected patients, new imaging methodologies and biomarkers that can guide patient selection.

Abstract LB-268: Detection of TP53 clonal mutations in PAP test collected up to six years prior to high-grade serous epithelial ovarian cancer diagnosis

Abstract Background: The low five-years survival rate of High Grade Serous Epithelial Ovarian Cancer (HGS-EOC) is mainly related to late diagnosis. The anticipation of diagnosis constitutes a crucial step to increase the curability of this disease. Aim: This study explores an innovative potential HGS-EOC screening approach exploiting PAP tests routinely executed for cervical cancer surveillance and the presence of TP53 clonal alterations in almost all HGS-EOCs. Indeed, it investigates the possibility to detect, in DNA purified from PAP tests collected by patients years before the diagnosis, the clonal pathogenic TP53 variant identified in the matched primary tumor biopsy. Study Design: This retrospective and longitudinal study was conducted on 17 advanced HGS-EOC patients. Next Generation Sequencing (NGS) was used to identify TP53 clonal mutations in tumor tissue. The presence of these TP53 variants was then assessed by the droplet digital PCR (ddPCR) in DNA purified from all available patients' PAP tests executed up to almost six years before diagnosis. Results: In each patient, one TP53 clonal somatic variant was identified by NGS in the primary tumor samples (Table 1). The presence of the TP53 variant was then investigated by ddPCR in matched Pap tests. In twelve out of 17 patients the TP53 variant was detectable in PAP tests collected within six months before diagnosis (T1) or earlier (T2, T3 and T4) (Table 1). For two patients (21561 and 21521) more PAP tests collected at different time before diagnosis (two/four and three/six years before diagnosis, respectively) were analyzed and the TP53 clonal variant was detected at all time points (Table 1). Table 1:NGS and ddPCR resultsPatient IDTP53 mutationNGS tumor mutated %PAP test (months before surgery)T1(0-6)T2(7-24)T3(25-48)T4(≥49)Time intervalddPCR mutated %Time intervalddPCR mutated %Time intervalddPCR mutated %Time intervalddPCR mutated %21561c.818G>A65.010.20.24--250.21490.2621585c.817C>T40.94--11.30.21----21567c.281C>A71.9830.07------21587c.469G>T15.122ND------21586c.818G>A79.73--19.30.15----21569c.574C>T62.365.21.18------21624c.820G>T86.72----37.50.0465.3ND21570c.844C>T89.190.32.62------21627c.425_427del76.040.72.4------21640c.993+2T>G70.68--8ND----21507c.1025G>C91.23--9.20.06----21635c.844C>T66.241.3ND------21549c. 393_395del54.06----31.2ND65.3ND21521c. 722 C>G61.15----26.70.0567.30.0721654c.586 C>T49.354.70.09------21665c. 393_395del45.34----37.6ND--21683c.602 T>A33.42--18.50.06----ND Not Detected Conclusion: This study demonstrates that non-invasive early molecular diagnosis of HGS-EOC is feasible through detection of TP53 clonal mutations in the DNA purified from PAP tests performed during cervical cancer screening. Further developments in highly sensitive molecular approaches could dramatically improve early diagnosis of HGS-EOC. Citation Format: Chiara Pesenti, Lara Paracchini, Martina Delle Marchette, Luca Beltrame, Tommaso Bianchi, Tommaso Grassi, Alessandro Buda, Fabio Landoni, Lorenzo Ceppi, Cristina Bosetti, Maria Chiara Paderno, Marco Adorni, Debora Vicini, Patrizia Perego, Biagio Eugenio Leone, Sergio Marchini, Robert Fruscio, Maurizio D'Incalci. Detection of TP53 clonal mutations in PAP test collected up to six years prior to high-grade serous epithelial ovarian cancer diagnosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-268.

Targeted metabolomic profiling of low and high grade serous epithelial ovarian cancer tissues: a pilot study.

Epithelial ovarian cancer (EOC) remains the leading cause of death from gynecologic malignancies and has an alarming global fatality rate. Besides the differences in underlying pathogenesis, distinguishing between high grade (HG) and low grade (LG) EOC is imperative for the prediction of disease progression and responsiveness to chemotherapy. The aim of this study was to investigate, the tissue metabolome associated with HG and LG serous epithelial ovarian cancer. A combination of one dimensional proton nuclear magnetic resonance (1D H NMR) spectroscopy and targeted mass spectrometry (MS) was employed to profile the tissue metabolome of HG, LG serous EOCs, and controls. Using partial least squares-discriminant analysis, we observed significant separation between all groups (p < 0.05) following cross validation. We identified which metabolites were significantly perturbed in each EOC grade as compared with controls and report the biochemical pathways which were perturbed due to the disease. Among these metabolic pathways, ascorbate and aldarate metabolism was identified, for the first time, as being significantly altered in both LG and HG serous cancers. Further, we have identified potential biomarkers of EOC and generated predictive algorithms with AUC (CI) = 0.940 and 0.929 for HG and LG, respectively. These previously unreported biochemical changes provide a framework for future metabolomic studies for the development of EOC biomarkers. Finally, pharmacologic targeting of the key metabolic pathways identified herein could lead to novel and effective treatments of EOC.

Abstract 725: Feasibility and evaluation of exosomal RNAs as novel diagnostic biomarkers for high grade serous epithelial ovarian cancer

Abstract Introduction: High-grade serous ovarian cancer (HGSOC) survival has been essentially unchanged over the last ten years despite new therapeutic strategies. In order to make a large-scale impact on overall survival, early detection strategies are imperative. Exosomes are cell-derived vesicles derived from multivesicular bodies or the plasma membrane that carry a variety of biomolecules, including protein, DNA, and RNA. Exosomal contents represent a novel and underexplored source of tumor biomarkers, in particular for HGSOC. This study aimed to first evaluate approaches to exosomal RNA purification and to then compare the exosomal RNA profiles from serum of patients with benign ovarian disease and HGSOC to determine if unique profiles exist, and to identify biomarkers useful for early detection. Methods: Cell free serum was obtained from patients in each of the following categories: HGSOC with early (n=24) or late stage (n=24) disease, benign ovarian masses (n=24) and healthy age-matched controls (n=24). We compared ultracentrifugation and a commercial precipitation solution to enrich and isolate exosomes. Exosomal enrichment was confirmed by both transmission electron microscopy (TEM) and western blot using anti-CD63 antibodies. RNA was extracted for each and deep sequencing analysis was performed for 12 candidates within each group. A second cohort of 48 samples will be used for validation of the best performing candidate biomarkers from the discovery cohort via RT-qPCR. Results: Enrichment of exosomes was confirmed via qualitative assessment using TEM and Western blot analysis. RNA quality was superior when RNA isolations were performed using the QIAgen RNeasy kit compared to the AllPrep kit. Mean RNA yield for each group ranged from 10.3-28.6ng/µl and RNA yield and quality was independent of sample age. RNAseq analyses are ongoing to identify biomarkers specific to HGSOC that can be detected at the earliest stages. The top differentially RNAs for the ovarian cancer groups will be validated in an independent cohort of specimens. A panel will then be generated in combination with CA125 to determine the clinical utility of an integrated -omics approach in early stage detection of HGSOC. Conclusions: Efficient isolation of exosomes and RNA can be achieved from archived specimens &amp;gt;10 years old. Evaluation of exosomal RNA in HGSOC provides a novel method for biomarker discovery. Citation Format: Emily N. Prendergast, Xianzhi Lin, Rosario I. Corona, Dennis J. Hazelett, Beth Y. Karlan, Kate Lawrenson. Feasibility and evaluation of exosomal RNAs as novel diagnostic biomarkers for high grade serous epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 725. doi:10.1158/1538-7445.AM2017-725

Molecular Mechanism Linking BRCA1 Dysfunction to High Grade Serous Epithelial Ovarian Cancers with Peritoneal Permeability and Ascites.

Ovarian cancer constitutes the second most common gynecological cancer with a five-year survival rate of 40%. Among the various histotypes associated with hereditary ovarian cancer, high-grade serous epithelial ovarian carcinoma (HGSEOC) is the most predominant and women with inherited mutations in BRCA1 have a lifetime risk of 40-60%. HGSEOC is a challenge for clinical oncologists, due to late presentation of patient, diagnosis and high rate of relapse. Ovarian tumors have a wide range of clinical presentations including development of ascites as a result of deregulated endothelial function thereby causing increased vascular permeability of peritoneal vessels. The molecular mechanisms remain elusive. Studies have shown that fallopian tube cancers develop in women with BRCA1 gene mutations more often than previously suspected. Recent studies suggest that many primary peritoneal cancers and some high-grade serous epithelial ovarian carcinomas actually start in the fallopian tubes. In this article we have addressed the molecular pathway of a recently identified potential biomarker Ubc9 whose deregulated expression due to BRCA1 dysfunction can result in HGSEOC with peritoneal permeability and formation of ascites. We also discuss the role of downstream targets Caveolin-1 and Vascular Endothelial Growth Factor (VEGF) in the pathogenesis of ascites in ovarian carcinomas. Finally we hypothesize a signaling axis between Ubc9 over expression, loss of Caveolin-1 and induction of VEGF in BRCA1 mutant HGSEOC cells. We suggest that Ubc9-mediated stimulation of VEGF as a novel mechanism underlying ovarian cancer aggressiveness and ascites formation. Agents that target Ubc9 and VEGF signaling may represent a novel therapeutic strategy to impede peritoneal growth and spread of HGSEOC.

Abstract 1380: Genome-wide fingerprinting of regulatory chromatin to evaluate the tissue specific origins of high-grade serous ovarian cancer

Abstract The normal precursor tissue origins of high grade serous epithelial ovarian cancers (HSOC) are likely to be the fallopian tube epithelium (FTE) and/or the ovarian surface epithelium (OSE). To address this hypothesis, we used FAIRE-seq and ChIP-seq to evaluate the genome wide regulatory profiles of both tissue types and compared these profiles to well characterized HSOC cell lines (CaOV3 and UWB1.289). This analysis provided, for the first time, global high-resolution annotation of open chromatin and histone H3K27 acetylation and H3K4 monomethylation marks for these tissues types, which are consistent with sites of transcriptional regulation. Using these data, we identified over 10,000 non-promoter associated FAIRE/ChIP signal sites that represent candidate functional enhancers. The regulatory features provided a cell type-specific fingerprint for each cell type. While there were many similarities between OSE and FTE compared to other normal cell types, we also were also able to use these profiles to functionally distinguish OSE from FTE. When we compared profiles of HSOC cells, we observed stronger similarities in the regulatory chromatin landscape between FTE and HSOC cell types than between OSE and FTE. Supervised pathway analysis of genes located closest to putative enhancer sites provided additional evidence of signaling pathways that may underlie the neoplastic initiation and progression from normal tissue to HSOC phenotypes. Our results demonstrate the utility of this approach for identifying precursor tissue types for HSOC and other cancers based on the cell type specific chromatin datasets. Citation Format: Howard C. Shen, Simon Coetzee, Dennis J. Hazelett, Gerhard A. Coetzee, Houtan Noushmehr, Simon A. Gayther. Genome-wide fingerprinting of regulatory chromatin to evaluate the tissue specific origins of high-grade serous ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1380. doi:10.1158/1538-7445.AM2014-1380

Histones and their modifications in ovarian cancer - drivers of disease and therapeutic targets.

Epithelial ovarian cancer has the highest mortality of the gynecological malignancies. High grade serous epithelial ovarian cancer (SEOC) is the most common subtype, with the majority of women presenting with advanced disease where 5-year survival is around 25%. Platinum-based chemotherapy in combination with paclitaxel remains the most effective treatment despite platinum therapies being introduced almost 40 years ago. Advances in molecular medicine are underpinning new strategies for the treatment of cancer. Major advances have been made by international initiatives to sequence cancer genomes. For SEOC, with the exception of TP53 that is mutated in virtually 100% of these tumors, there is no other gene mutated at high frequency. There is extensive copy number variation, as well as changes in methylation patterns that will influence gene expression. To date, the role of histones and their post-translational modifications in ovarian cancer is a relatively understudied field. Post-translational histone modifications play major roles in gene expression as they direct the configuration of chromatin and so access by transcription factors. Histone modifications include methylation, acetylation, and monoubiquitination, with involvement of enzymes including histone methyltransferases, histone acetyltransferases/deacetylases, and ubiquitin ligases/deubiquitinases, respectively. Complexes such as the Polycomb repressive complex also play roles in the control of histone modifications and more recently roles for long non-coding RNA and microRNAs are emerging. Epigenomic-based therapies targeting histone modifications are being developed and offer new approaches for the treatment of ovarian cancer. Here, we discuss histone modifications and their aberrant regulation in malignancy and specifically in ovarian cancer. We review current and upcoming histone-based therapies that have the potential to inform and improve treatment strategies for women with ovarian cancer.

Culture models to define key mediators of cancer matrix remodeling.

High grade serous epithelial ovarian cancer (HG-SOC) is one of the most devastating gynecological cancers affecting women worldwide, with a poor survival rate despite clinical treatment advances. HG-SOC commonly metastasizes within the peritoneal cavity, primarily to the mesothelial cells of the omentum, which regulate an extracellular matrix rich in collagens type I, III, and IV along with laminin, vitronectin, and fibronectin. Cancer cells depend on their ability to penetrate and invade secondary tissue sites to spread, however a detailed understanding of the molecular mechanisms underlying these processes remain largely unknown. Given the high metastatic potential of HG-SOC and the associated poor clinical outcome, it is extremely important to identify the pathways and the components of which that are responsible for the progression of this disease. In vitro methods of recapitulating human disease processes are the critical first step in such investigations. In this context, establishment of an in vitro “tumor-like” micro-environment, such as 3D culture, to study early disease and metastasis of human HG-SOC is an important and highly insightful method. In recent years, many such methods have been established to investigate the adhesion and invasion of human ovarian cancer cell lines. The aim of this review is to summarize recent developments in ovarian cancer culture systems and their use to investigate clinically relevant findings concerning the key players in driving human HG-SOC.

Abstract 4024: Ten degrees of separation: novel genes affecting outcome in ovarian cancer.

Abstract Objective: To identify genomic predictors of overall survival of women with high grade serous ovarian cancer based on best separation overall survival Kaplan-Meier curves Methods: Comprehensive data analysis of the TCGA dataset of high grade serous epithelial ovarian cancer was carried out with a “training” (n= 375) and “validation” (n= 188) data sets. The R-code was written specifically to identify all genes with high expression that gave the greatest predictive potential for patient survival. The top ten cases with lowest sums of the log rank test p-values obtained for training and validation sets were chosen to be included with Kaplan-Meier survival curves for analysis. Results: The ten genes with high expression that had the smallest p-values in their respective order were: SLC6A1, LIPK, EHBP1, SUSD5, PEX3, SLC22A3, RABGEF1, PPM2C, KIAA1219, and GALNT10. The range of p-value sum ranged from 1.77E-3 to 2.09E-3. The lowest p-value was for SLC6A1, which encodes for GABA Transporter-1 that removes GABA from extracellular to intracellular space. Interestingly, four of the 10 genes predictive for poor outcome are directly involved in cell metabolism: LIPK, PPM2C, PEX3, and GALNT10. The median OS of the high gene expression group ranged from 38 to 48 months. The median OS of the low gene expression group ranged from 55-70 months. The median OS difference of high gene vs. low gene expression groups ranged from 15-25 months. EHBP1 gene, involved in endocytic trafficking of transferrin into endosomes and GLUT4 into adipocytes, had the greatest difference in median OS between high and low expressions at 25 months (p= 2.16E-3). Finally, low expression of PPM2C compared to its high expression was significantly associated with &amp;gt; 5 year survival (10.1% vs. 1.1%, p=0.001) and no recurrence in both training and validation sets (12.5% vs. 2.2%, p=0.04). PPM2C encodes for pyruvate dehydrogenase-phosphatase 1 that catalyzes reactivation of the alpha subunit of the E1 component of the pyruvate dehydrogenase complex. Pyruvate dehydrogenase complex in the active form transforms pyruvate from glycolysis to acetyl-CoA in the mitochondria. To evaluate whether similar findings are noted in other cancers, we also examined the TCGA data for breast, lung, colon cancer, and glioblastoma. PPM2C is amplified by 7% in invasive breast cancer, and leads to an overall worse survival (68.9 months vs. 129.5 months, p = 0.037). Conclusion: Genes involved in cell metabolism, neurotransmitter functioning, and endocytic trafficking are highly predictive of outcome in high grade serous ovarian cancer. High PPM2C expression and gene amplification leads to worsening overall survival in ovarian and invasive breast cancer, respectively. These pathways likely reflect novel and important therapeutic targets. Citation Format: Behrouz Zand, Cristina Ivan, Chad V. Pecot, Rajesha Rupaimoole, Heather J. Dalton, Justin Bottsford-Miller, Wei Hu, Alpa M. Nick, Anil K. Sood. Ten degrees of separation: novel genes affecting outcome in ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4024. doi:10.1158/1538-7445.AM2013-4024

Abstract 1123: OncomiR-569 hit p53 pathway and deregulate proliferation of ovarian epithelial cells.

Abstract Background The 3q26.2 loci amplification is present in a large proportion of high grade serous epithelial ovarian cancer (HGSEOC) is less frequently curable disease in the clinic. The 3q26.2 loci, which is present in at least 35% of HGSEOC, is large and structurally complex suggesting that multiple coding genes of the amplicon contribute to tumor initiation and progression either alone or through cooperative activity. Our high resolution SNP array mapping of 3q26.2 amplicon identified a microRNA named miR-569 is highly amplified in addition to the coding genes in a large set of ovarian cancer samples. Potential role of miR-569 is not characterized previously in the context of ovarian cancer and our study provide mechanism underlying the oncogenic role of miR569 in ovarian cancer. Results We demonstrated a strong correlation between 3q26.2 amplification and overexpression of miR569 in patient's samples of ovarian cancer. Importantly overexpression and knockdown of miR569 in normal epithelial cells altered their growth, proliferation, and lumen filling in 3-dimensional culture models in Matrigel. Ectopic expression of miR569 in epithelial cancer cells promoted tumor growth and metastatic potential of tumor cells in mouse xenograft models. In silico and in vitro experiments showed that miR-569 directly target and inhibited the expression of p53 induced tumor suppressor gene TP53INP1. TP53INP1 is transcriptionally regulated by p53 or p73 proteins, however miR-569 is a key post-transcriptional regulator of TP53INP1 expression independent of p53 or p73 proteins. Immunohistochemical and protein array analysis confirmed that TP53INP1 protein levels were higher in normal tissues compared to cancer tissues of breast and ovary. Reduced expression of TP53INP1 mRNA and protein were associated with worsened outcomes and promoted distant metastasis in the clinic. In consistent with our in vitro results, in vivo targeting of miR-569 markedly reduced tumor growth and metastatic spreads. Discussion Our studies combined with previous literature are compatible with a model wherein amplification of 3q26.2 increases miR569 levels resulting in decreased TP53INP1 levels independently of p53 or p73 function. Such a decrease in TP53INP1 subsequently reduces p53 phosphorylation resulting in a positive feedback loop and collapse the p53-mediated cell cycle regulation. Based on our preclinical results of antimiR-569 on cell survival, tumor growth and drug sensitivity inhibiting miR-569 activity or increasing TP53INP1 may be a valid therapeutic target to treat ovarian cancer. Citation Format: Pradeep Raghavan, Fan Zhang, Sunila Pradeep, Yiling Lu, Anne-Lise Borresen-Dale, Elsa Flores, Anil Sood, Gordon Mills. OncomiR-569 hit p53 pathway and deregulate proliferation of ovarian epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1123. doi:10.1158/1538-7445.AM2013-1123

Elevated levels of circulating microRNA-200 family members correlate with serous epithelial ovarian cancer

BackgroundThere is a critical need for improved diagnostic markers for high grade serous epithelial ovarian cancer (SEOC). MicroRNAs are stable in the circulation and may have utility as biomarkers of malignancy. We investigated whether levels of serum microRNA could discriminate women with high-grade SEOC from age matched healthy volunteers.MethodsTo identify microRNA of interest, microRNA expression profiling was performed on 4 SEOC cell lines and normal human ovarian surface epithelial cells. Total RNA was extracted from 500 μL aliquots of serum collected from patients with SEOC (n = 28) and age-matched healthy donors (n = 28). Serum microRNA levels were assessed by quantitative RT-PCR following preamplification.ResultsmicroRNA (miR)-182, miR-200a, miR-200b and miR-200c were highly overexpressed in the SEOC cell lines relative to normal human ovarian surface epithelial cells and were assessed in RNA extracted from serum as candidate biomarkers. miR-103, miR-92a and miR -638 had relatively invariant expression across all ovarian cell lines, and with small-nucleolar C/D box 48 (RNU48) were assessed in RNA extracted from serum as candidate endogenous normalizers. No correlation between serum levels and age were observed (age range 30-79 years) for any of these microRNA or RNU48. Individually, miR-200a, miR-200b and miR-200c normalized to serum volume and miR-103 were significantly higher in serum of the SEOC cohort (P < 0.05; 0.05; 0.0005 respectively) and in combination, miR-200b + miR-200c normalized to serum volume and miR-103 was the best predictive classifier of SEOC (ROC-AUC = 0.784). This predictive model (miR-200b + miR-200c) was further confirmed by leave one out cross validation (AUC = 0.784).ConclusionsWe identified serum microRNAs able to discriminate patients with high grade SEOC from age-matched healthy controls. The addition of these microRNAs to current testing regimes may improve diagnosis for women with SEOC.

BTN3A2 Expression in Epithelial Ovarian Cancer Is Associated with Higher Tumor Infiltrating T Cells and a Better Prognosis

BTN3A2/BT3.2 butyrophilin mRNA expression by tumoral cells was previously identified as a prognostic factor in a small cohort of high grade serous epithelial ovarian cancer (HG-EOC). Here, we evaluated the prognostic value of BT3.2 at the protein level in specimen from 199 HG-EOC patients. As the only known role of butyrophilin proteins is in immune regulation, we evaluated the association between BT3.2 expression and intratumoral infiltration of immune cells by immunohistochemistry with specific antibodies against BT3.2, CD3, CD4, CD8, CD20, CD68 and CD206. Epithelial BT3.2 expression was significantly associated with longer overall survival and lower risk of disease progression (HR = 0.651, p = 0.006 and HR = 0.642, p = 0.002, respectively) and significantly associated with a higher density of infiltrating T cells, particularly CD4+ cells (0.272, p<0.001). We also observed a strong association between the relative density of CD206+ cells, as evaluated by the ratio of intratumoral CD206+/CD68+ expression, and risk of disease progression (HR = 1.355 p = 0.044, respectively). In conclusion, BT3.2 protein is a potential prognostic biomarker for the identification of HG-EOC patients with better outcome. In contrast, high CD206+/CD68+ expression is associated with high risk of disease progression. While the role of BT3.2 is still unknown, our result suggest that BT3.2 expression by epithelial cells may modulates the intratumoral infiltration of immune cells.

Abstract 4802: Metabolic distinctions in high grade epithelial ovarian cancer

Abstract Objective: To identify differences in overall metabolite profile of human high grade serous epithelial ovarian cancers (EOC) compared to normal ovarian tissues. Methods: Forty-five patient tissues were obtained (n= 30 EOC, n= 15 normal). Samples were analyzed by Gas Chromatography (GC)/mass spectrometry (MS) and Liquid Chromatography (LC)/MS platforms. The difference between the groups was reported in fold change (FC). Welch's two sample t-tests were used to identify metabolites that differed significantly between cancer and normal ovarian tissues and p&amp;lt;0.05 was defined as significant. Results: Using an existing library containing &amp;gt;2000 metabolites, there were 315 metabolites that were identified between EOC and normal ovaries. Of these 315 metabolites, 204 were significantly altered with 180 up regulated metabolites, and 24 down regulated. EOC had altered metabolism in the glycolytic, amino acid, lipid, nucleotide and eicosanoid pathways. There was an increase dependence on glycolysis consistent with the “Warburg effect,” as noted by elevated glucose-6-phosphate (FC= 2.8, p&amp;lt;0.003), fructose-6-phosphate (FC=2.9, p&amp;lt;0.001) and lactate (FC= 1.7, p=0.013). Ovarian cancer cells showed significantly elevated levels of long-chain fatty acids (e.g, docosatrienoate (22:3n3) [FC= 9.06, p &amp;lt; 0.001), including those derived from the diet (linoleate [FC=2.25, p&amp;lt;0.001] and linolenate [FC=2.37, p&amp;lt;0.001]). Furthermore, significantly high levels of components needed for phospholipid biosynthetic pathway (e.g. cytidine 5′-diphosphocholine [16.86, p &amp;lt; 0.001], choline [FC = 1.5, p&amp;lt;0.001]) were seen which along with the altered lipid metabolism supports membrane biosynthesis in cancer cells. The eicosanoid metabolism pathway had significantly higher levels of pro-inflammatory compounds such as prostaglandins E2 (FC= 17.11, p&amp;lt;0.001), I2 (FC=16.35, p&amp;lt;0.001) and 6-keto prostaglandin F1-α (FC= 16.77, p&amp;lt;0.001). Interestingly, compounds best characterized only in the central nervous system, were also significantly elevated in ovarian cancer tissues. Conclusion: High grade EOC shows distinct metabolic alterations compared to normal ovarian tissues, forming the basis for targeting key metabolic pathways for biomarker and/or therapeutic approaches in ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4802. doi:1538-7445.AM2012-4802

Androgen receptor expression is a biological marker for androgen sensitivity in high grade serous epithelial ovarian cancer

ObjectivesIn the present study we explore the effects of androgens and anti-androgens on primary cultures of EOC cells. We also investigate the effects of chemotherapy on AR expression. Epithelial ovarian cancer (EOC) arises from ovarian surface epithelial cells (OSE), which express the androgen receptor (AR). Androgen stimulation of OSE cells results in increased proliferation and protection from apoptosis. Nevertheless, in clinical trials anti-androgens have had a low objective response rate in relapsed ovarian cancer. Methods1. Androgen receptor (AR) expression and response to androgenic stimulation were correlated in primary ovarian cancer cells derived from ascitic fluid from patients with advanced ovarian cancer,2. AR expression in primary epithelial ovarian cancer was investigated before and after chemotherapy using paired histological samples which had been incorporated into a tissue microarray. ResultsEleven primary ovarian cancer cultures were established from ascitic fluid. There was wide variation of expression of androgen receptor mRNA between cultures. Cell division increased after dihydro-testosterone (DHT) stimulation in 6 out of 11 primary cultures. The fraction of cells in S-phase increased from 4.4% in cells grown in serum-free medium to 8.3% in cells stimulated with 100nM of DHT (P<0.001). The increase in S-phase fraction was abrogated after treatment with the anti-androgen, bicalutamide in 4 out of 5 responsive cultures. There was a strong correlation (r2=0.7) between nuclear AR expression by immunohistochemistry and S-phase fraction changes in primary cultures.Paired pre- and post-chemotherapy histological samples from 29 patients were incorporated into a tissue microarray (TMA). Nuclear and cytoplasmic AR expression by immunohistochemistry (IHC) decreased significantly after chemotherapy (P<0.01). ConclusionAR expression correlates with increased S-phase fraction in response to androgenic stimulation. Immunohistochemical analysis of AR expression needs to be further tested in clinical trials to select AR positive EOC for anti-androgen therapy. Anti-androgen use early in the course of ovarian cancer is more likely to be effective as these data suggest that androgen receptor expression decreases with exposure to chemotherapy and this may explain the low response rates seen in clinical trials of patients heavily pre-treated with multiple courses of chemotherapy.