High-grade Serous Carcinoma Samples Research Articles

RAD51/geminin/γH2AX immunohistochemical expression predicts platinum-based chemotherapy response in ovarian high-grade serous carcinoma.

The RAD51 assay is a recently developed functional assay for homologous recombination deficiency (HRD) that reflects real-time HRD status. We aimed to identify the applicability and predictive value of RAD51 immunohistochemical expression in pre- and post-neoadjuvant chemotherapy (NAC) samples of ovarian high-grade serous carcinoma (HGSC). We evaluated the immunohistochemical expression of RAD51/geminin/γH2AX in ovarian HGSC before and after NAC. In pre-NAC tumors (n=51), 74.5% (39/51) showed at least 25% of γH2AX-positive tumor cells, suggesting endogenous DNA damage. The RAD51-high group (41.0%, 16/39) showed significantly worse progression-free survival (PFS) compared to the RAD51-low group (51.3%, 20/39) (p=0.032). In post-NAC tumors (n=50), the RAD51-high group (36.0%, 18/50) showed worse PFS (p=0.013) and tended to present worse overall survival (p=0.067) compared to the RAD51-low group (64.0%, 32/50). RAD51-high cases were more likely to progress than RAD51-low cases at both 6 months and 12 months (p=0.046 and p=0.019, respectively). Of 34 patients with matched pre- and post-NAC RAD51 results, 44% (15/34) of pre-NAC RAD51 results were changed in the post-NAC tissue, and the RAD51 high-to-high group showed the worst PFS, while the low-to-low group showed the best PFS (p=0.031). High RAD51 expression was significantly associated with worse PFS in HGSC, and post-NAC RAD51 status showed higher association than pre-NAC RAD51 status. Moreover, RAD51 status can be evaluated in a significant proportion of treatment-naïve HGSC samples. As RAD51 status dynamically changes, sequential follow-up of RAD51 status might reflect the biological behavior of HGSCs.

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 < 0.001) pathway activity and higher median PI3K (p < 0.001), Hedgehog (p < 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.

High-grade serous ovarian tumor cells modulate NK cell function to create an immune-tolerant microenvironment.

SUMMARYTubo-ovarian high-grade serous carcinoma (HGSC) is unresponsive to immune checkpoint blockade despite significant frequencies of exhausted T cells. Here we apply mass cytometry and uncover decidual-like natural killer (dl-NK) cell subpopulations (CD56+CD9+CXCR3+KIR+CD3−CD16−) in newly diagnosed HGSC samples that correlate with both tumor and transitioning epithelial-mesenchymal cell abundance. We show different combinatorial expression patterns of ligands for activating and inhibitory NK receptors within three HGSC tumor compartments: epithelial (E), transitioning epithelial-mesenchymal (EV), and mesenchymal (vimentin expressing [V]), with a more inhibitory ligand phenotype in V cells. In cocultures, NK-92 natural killer cells acquire CD9 from HGSC tumor cells by trogocytosis, resulting in reduced anti-tumor cytokine production and cytotoxicity. Cytotoxicity in these cocultures is restored with a CD9-blocking antibody or CD9 CRISPR knockout, thereby identifying mechanisms of immune suppression in HGSC. CD9 is widely expressed in HGSC tumors and so represents an important new therapeutic target with immediate relevance for NK immunotherapy.

Identification of signal transduction pathway activity with potential clinical target in high-grade serous ovarian and tubal carcinoma.

e17541 Background: High-grade serous carcinoma (HGSC) is the most common subtype of epithelial ovarian and tubal 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 potential clinical target by comparing their activity assessment with STP activity in normal Fallopian tube epithelium (FTE), the tissue of origin of most HGSCs. Methods: We included 67 primary tumor samples taken prior to start of chemotherapy of postmenopausal patients diagnosed with advanced stage HGSC and 8 morphologically normal FTE samples of healthy postmenopausal women. Using OncoSignal pathway assays, we assessed functional pathway activity of the androgen receptor (AR), estrogen receptor (ER), PI3K, Hedgehog, TGF-𝛽 and Wnt 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 < 0.001) and Wnt (p = 0.046) pathway activity and higher median PI3K (p = 0.025) and TGF-𝛽 pathway (p = 0.030) activity as compared to the normal FTE group. In individual HGSC samples, aberrant activity was identified for the AR (n = 14), PI3K (n = 16), Hedgehog (n = 4), TGF-𝛽 (n = 36) and Wnt (n = 10) pathways. Frequently observed combinations of aberrant STP activity were AR/TGF-𝛽 (n = 8), TGF-𝛽/Wnt (n = 6) and PI3K/Hedgehog (n = 3). In total, we identified at least one STP with potential clinical target in 52 of the 67 HGSC samples. Conclusions: Our analysis enabled the identification of STP activity with potential clinical target in 78% 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.

The Claudin-Low Subtype of High-Grade Serous Ovarian Carcinoma Exhibits Stem Cell Features.

Simple SummaryHere, we identified and characterized a claudin-low subtype of high-grade serous ovarian cancer. This rare variant of undifferentiated neoplasm shares transcriptional features with the homonym subtype of breast cancer, including low epithelial differentiation, high mesenchymal signature, and enrichment for stem cell features. Since the claudin-low transcriptional signature is associated with poor prognosis, we believe that the identification of the claudin-low molecular profile may have important clinical implications, paving the way for personalized medicine in ovarian cancer patients.Claudin-low cancer (CL) represents a rare and biologically aggressive variant of epithelial tumor. Here, we identified a claudin-low molecular profile of ovarian high-grade serous carcinoma (HGSOC), which exhibits the main characteristics of the homonym breast cancer subtype, including low epithelial differentiation and high mesenchymal signature. Hierarchical clustering and a centroid based algorithm applied to cell line collection expression dataset labeled 6 HGSOC cell lines as CL. These have a high energy metabolism and are enriched in CD44+/CD24− mesenchymal stem-like cells expressing low levels of cell-cell adhesion molecules (claudins and E-Cadherin) and high levels of epithelial-to-mesenchymal transition (EMT) induction transcription factors (Zeb1, Snai2, Twist1 and Twist2). Accordingly, the centroid base algorithm applied to large retrospective collections of primary HGSOC samples reveals a tumor subgroup with transcriptional features consistent with the CL profile, and reaffirms EMT as the dominant biological pathway functioning in CL-HGSOC. HGSOC patients carrying CL profiles have a worse overall survival when compared to others, likely to be attributed to its undifferentiated/stem component. These observations highlight the lack of a molecular diagnostic in the management of HGSOC and suggest a potential prognostic utility of this molecular subtyping.

Genome-wide analysis of microRNA to evaluate prognostic markers in isolated cancer glands and surrounding stroma in high-grade serous ovarian carcinoma.

The molecular mechanisms responsible for the progression of ovarian cancer remain incompletely understood. By targeting multiple cancer-related genes, microRNAs (miRNAs) have been identified as key regulators of cancer development and progression. In addition, the microenvironment, which constitutes cancer glands and the surrounding stromal tissue at the invasive front, has an important role in cancer progression. Using array-based analysis of 14 cases (cohort 1), the aim of the present study was to evaluate global miRNA expression in cancerous glands and surrounding stromal tissues (isolated using a crypt isolation method), in order to identify potential prognostic markers of high-grade serous carcinoma (HGSC). Reverse transcription-quantitative PCR was also used to verify the results in cohort 1 (14 cases) and in 16 additional HGSC cases (cohort 2; verification cohort). Firstly, miRNA expression levels were compared between HGSC and normal samples among both the isolated cancer gland and stromal tissue samples. Secondly, miRNA expression was compared between HGSC cases with recurrence and those without recurrence among the isolated cancer gland and stromal tissue samples. The results revealed six and seven miRNAs identified in both of the aforementioned comparisons in isolated cancer glands and surrounding stromal tissue, respectively. Furthermore, downregulation of miRNA-214-3p in isolated cancer glands and downregulation of miRNA-320c in the corresponding stromal tissue were associated with a decrease in disease-free survival (without recurrence) in cohort 2. These findings indicated that specific miRNAs expressed in cancer cells and surrounding stromal cells of HGSC may be potential biomarkers predicting patient prognosis.

Abstract B65: Defining the tumor immune landscape in a mouse model of high-grade serous carcinoma

Abstract Clinical efficacy of immune checkpoint therapy in high-grade serous carcinoma (HGSC) has been disappointing to date and highlights the need to better understand the relationship between HGSC and its associated immune microenvironment. Tractable model systems for interrogating the tumor microenvironment in HGSC could be used to address this unmet need. We have generated genetically engineered mouse models (GEMMs) of HGSC utilizing oviduct-restricted tamoxifen-regulated Cre recombinase to drive conditional inactivation of key tumor suppressor genes (Brca1, Trp53, Rb1, Nf1) observed to be recurrently altered in human HGSC samples. These autochthonous tumors arise from the murine fallopian tube (oviduct) in the context of an intact immune system. We employed RNA sequencing (RNA-seq) of bulk tumors from 19 mouse HGSCs to compare their gene expression profiles to those of normal oviduct and ovary tissues (4 each), and 6 mouse endometrioid-like oviductal carcinomas based on conditional inactivation of Apc and Pten. We employed immunogenomic analysis of the RNA-seq data to characterize the intratumoral infiltrate of 28 immune cell types in these samples in silico. The analysis revealed a prevalence of gene expression signatures associated with immunosuppressive cell types, including myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in the mouse HGSCs, not observed in the other sample groups. Human HGSCs with available RNA-seq data (from TCGA samples) showed similar enrichment of MDSC and Treg gene signatures in the subset of human HGSCs classified as mesenchymal or immunoreactive expression subtypes. To further validate the findings in the mouse HGSCs, we used immunohistochemistry to detect subsets of tumor-infiltrating lymphocytes (TILs) in the tumor tissues. Specifically, CD8+ TIL infiltrates recapitulated epithelial (E-TIL), stromal (S-TIL) and combined epithelial/stromal (ES-TIL) patterns described in human HGSCs. Moreover, FoxP3+ Tregs were frequently observed to be co-infiltrated with CD8+ TILs as expected in an immunosuppressive tumor microenvironment. Gene set enrichment analysis showed that NF-kappa B signaling is among the top five pathways upregulated in HGSCs versus normal oviducts, suggesting a plausible mechanism by which tumor cells can alter and respond to the microenvironment during tumor development. These studies provide an experimental paradigm for improving our understanding of the role of the tumor microenvironment in HGSC pathogenesis and may aid in efforts to enhance the efficacy of immunotherapies in this lethal disease. Citation Format: Kevin W. McCool, Rork Kuick, Yali Zhai, Zach Freeman, Rong Wu, Eric Fearon, Kathleen R. Cho. Defining the tumor immune landscape in a mouse model of high-grade serous carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B65.

Combined CCNE1 high-level amplification and overexpression is associated with unfavourable outcome in tubo-ovarian high-grade serous carcinoma.

CCNE1 amplification is a recurrent alteration associated with unfavourable outcome in tubo‐ovarian high‐grade serous carcinoma (HGSC). We aimed to investigate whether immunohistochemistry (IHC) can be used to identify CCNE1 amplification status and to validate whether CCNE1 high‐level amplification and overexpression are prognostic in HGSC. A testing set of 528 HGSC samples stained with two optimised IHC assays (clones EP126 and HE12) was subjected to digital image analysis and visual scoring. DNA and RNA chromogenic in situ hybridisation for CCNE1 were performed. IHC cut‐off was determined by receiver operating characteristics (ROC). Survival analyses (endpoint ovarian cancer specific survival) were performed and validated in an independent validation set of 764 HGSC. Finally, combined amplification/expression status was evaluated in cases with complete data (n = 1114). CCNE1 high‐level amplification was present in 11.2% of patients in the testing set and 10.2% in the combined cohort. The optimal cut‐off for IHC to predict CCNE1 high‐level amplification was 60% positive tumour cells with at least 5% strong staining cells (sensitivity 81.6%, specificity 77.4%). CCNE1 high‐level amplification and overexpression were associated with survival in the testing and validation set. Combined CCNE1 high‐level amplification and overexpression was present in 8.3% of patients, mutually exclusive to germline BRCA1/2 mutation and significantly associated with a higher risk of death in multivariate analysis adjusted for age, stage and cohort (hazard ratio = 1.78, 95 CI% 1.38–2.26, p < 0.0001). CCNE1 high‐level amplification combined with overexpression identifies patients with a sufficiently poor prognosis that treatment alternatives are urgently needed. Given that this combination is mutually exclusive to BRCA1/2 germline mutations, a predictive marker for PARP inhibition, CCNE1 high‐level amplification combined with overexpression may serve as a negative predictive test for sensitivity to PARP inhibitors.

TIM-3 Dictates Functional Orientation of the Immune Infiltrate in Ovarian Cancer.

In multiple oncological settings, expression of the coinhibitory ligand PD-L1 by malignant cells and tumor infiltration by immune cells expressing coinhibitory receptors such as PD-1, CTLA4, LAG-3, or TIM-3 conveys prognostic or predictive information. Conversely, the impact of these features of the tumor microenvironment on disease outcome among high-grade serous carcinoma (HGSC) patients remains controversial. We harnessed a retrospective cohort of 80 chemotherapy-naïve HGSC patients to investigate PD-L1 expression and tumor infiltration by CD8+ T cells, CD20+ B cells, DC-LAMP+ dendritic cells as well as by PD-1+, CTLA4+, LAG-3+, and TIM-3+ cells in relation with prognosis and function orientation of the tumor microenvironment. IHC data were complemented with transcriptomic and functional studies on a second prospective cohort of freshly resected HGSC samples. In silico analysis of publicly available RNA expression data from 308 HGSC samples was used as a confirmatory approach. High levels of PD-L1 and high densities of PD-1+ cells in the microenvironment of HGSCs were strongly associated with an immune contexture characterized by a robust TH1 polarization and cytotoxic orientation that enabled superior clinical benefits. Moreover, PD-1+TIM-3+CD8+ T cells presented all features of functional exhaustion and correlated with poor disease outcome. However, although PD-L1 levels and tumor infiltration by TIM-3+ cells improved patient stratification based on the intratumoral abundance of CD8+ T cells, the amount of PD-1+ cells failed to do so. Our data indicate that PD-L1 and TIM-3 constitute prognostically relevant biomarkers of active and suppressed immune responses against HGSC, respectively.

Targeted Sequencing of Tubo-ovarian and Peritoneal High-grade Serous Carcinoma With Wild-type p53 Immunostaining Pattern.

We aimed to demonstrate the use of next-generation sequencing (NGS) to confirm the presence of tumor protein 53 (TP53) mutations in tubo-ovarian and peritoneal high-grade serous carcinoma (HGSC) with a wild-type p53 immunostaining pattern and investigate whether the TP53 mutational status is altered by chemotherapy. A commercial NGS panel comprising 171 genes was used to analyze the genetic profiles of 15 HGSC samples. Paired specimens obtained before and after chemotherapy were available for four patients. All examined samples exhibited TP53 mutations. For all the patients who underwent neoadjuvant or postoperative adjuvant chemotherapy, TP53 mutations identified in samples obtained after chemotherapy were the same as those detected in pre-chemotherapeutic samples. HGSCs exhibit TP53 mutations even though a subset of HGSCs displayed a wild-type p53 immunostaining pattern. Chemotherapy does not affect the TP53 mutational status in HGSC.

Metabolic Markers and Statistical Prediction of Serous Ovarian Cancer Aggressiveness by Ambient Ionization Mass Spectrometry Imaging.

Ovarian high-grade serous carcinoma (HGSC) results in the highest mortality among gynecological cancers, developing rapidly and aggressively. Dissimilarly, serous borderline ovarian tumors (BOT) can progress into low-grade serous carcinomas and have relatively indolent clinical behavior. The underlying biological differences between HGSC and BOT call for accurate diagnostic methodologies and tailored treatment options, and identification of molecular markers of aggressiveness could provide valuable biochemical insights and improve disease management. Here, we used desorption electrospray ionization (DESI) mass spectrometry (MS) to image and chemically characterize the metabolic profiles of HGSC, BOT, and normal ovarian tissue samples. DESI-MS imaging enabled clear visualization of fine papillary branches in serous BOT and allowed for characterization of spatial features of tumor heterogeneity such as adjacent necrosis and stroma in HGSC. Predictive markers of cancer aggressiveness were identified, including various free fatty acids, metabolites, and complex lipids such as ceramides, glycerophosphoglycerols, cardiolipins, and glycerophosphocholines. Classification models built from a total of 89,826 individual pixels, acquired in positive and negative ion modes from 78 different tissue samples, enabled diagnosis and prediction of HGSC and all tumor samples in comparison with normal tissues, with overall agreements of 96.4% and 96.2%, respectively. HGSC and BOT discrimination was achieved with an overall accuracy of 93.0%. Interestingly, our classification model allowed identification of three BOT samples presenting unusual histologic features that could be associated with the development of low-grade carcinomas. Our results suggest DESI-MS as a powerful approach for rapid serous ovarian cancer diagnosis based on altered metabolic signatures. Cancer Res; 77(11); 2903-13. ©2017 AACR.

Abstract A80: NAC1 attenuates BCL6 negative autoregulation and functions as a BCL6 coactivator of FOXQ1 transcription in ovarian cancer (OVCA).

Abstract Nucleus accumbens-associated protein 1 (NAC1) is a transcriptional co-regulator that lacks the DNA binding domain, thus relying on other co-factors to form higher-order transcription complexes. The present study aimed to elucidate the mechanism by which NAC1 modulates gene expression in OVCA. Nacc1-/- mice were euthanized, and spleens were collected. Promoter sequences were retrieved using Biostrings and BSgenome packages of Bioconductor 2.12. The position frequency matrix of BCL6 motif was obtained from Cistrome. BCL6 binding motifs on targeted promoters were identified by matchPWM, and log-odd scores calculated by PWMscoreStartingAt. For chromatin immunoprecipitation (ChIP), cells were fixed with 1% (w/v) paraformaldehyde prior to cross-linking with DTBP. Anti-BCL6, anti-NAC1 or rabbit IgG isotype were used for IP, and the genes of interest were amplified by qPCR. HEK293T cells were used for co-immunoprecipitaion (co-IP), and proteins were analyzed by Western blot. Luciferase reporter assays were performed in HeLa cells transfected with siNAC1 and/or siBCL6. Immunohistochemistry was done on tissue microarray (TMA) constituted of 51 primary ovarian high-grade serous carcinoma samples (HGSOC), using anti-NAC1 or anti-BCL6 antibodies. For microarray experiments, OVCAR5 cells were transfected with siNAC1 or siBCL6. Two-tailed t-test was used analyze data (means ± SD; p&amp;lt;0.05). BCL6 consensus binding motifs were mapped to the promoters of the NAC1 targeted genes FOXQ1 (positions A -1000, B -800, and C -150). NAC1 binds to BCL6 motifs as proved by ChIP and qPCR assays conducted in HeLa and MCF7 cells (p&amp;lt; 0.01). Reciprocal co-IP experiments done in HEK293T cells co-transfected with BCL6-FLAG and full-length NAC1-V5 indicated that NAC1 interacts with BCL6 via its C-terminal domain (NAC1-C186). Functional NAC1 is required to form putative NAC1/BCL6 transcription complex, and to modulate FOXQ1 expression in HeLa N130 tTA cells (p&amp;lt; 0.001). Luciferase reporter assays run in HeLa cells transfected with siNAC1 and siBCL6 proved that the genes interact and collaborate to regulate FOXQ1 transcriptional activity (p&amp;lt; 0.01). NAC1 and BCL6 transcriptional and protein expressions correlate in OVCA cell lines (R2=0.7025; p=0.0185), and in HGSOC TMA (R2=0.31; p=0.027), respectively. Furthermore, NAC1 modulates the expression of BCL6 as shown in OSE-NAC1 transfected cells (p=0.041), and in OVCAR5- (p=0.016), OVCAR3- (p=0.0089), SKOV3- (p=0.0216), and taxol-resistant SKOV3TR-siRNA-NAC1 (p=0.0002) transfected cells. BCL6 transcript expression was reduced by 50% in the spleen of Nacc-/- mice (p=0.0068). Endogenous NAC1 and BCL6 ChIP on the consensus BCL6 binding sequence within the BCL6 promoter in OVCA cells showed that BCL6 binds to its own promoter, and that NAC1 was enriched in the ChIP. Competition assay performed in NAC1-C186-OVCAR-3 cells revealed that BCL6 expression is modulated by NAC1 (p&amp;lt;0.0001), thus pointing to a direct interaction between NAC1 and BCL6 as a key phenomenon that attenuates BCL6 auto-downregulation by NAC1. Modulation of gene expression was assessed by cDNA microarray in siNAC1 or siBCL6 transfected OVCAR5 cells: 238 and 188 genes were down- whereas 139 and 113 genes were up-regulated by NAC1 and BCL6, respectively, amongst which 54 genes were down- and 25 genes were up-regulated concomitantly. In conclusion, our study establishes a new mechanism for NAC1-driven OVCA, in which it interacts with BCL6 via its C-terminal domain forming a transcription complex that modulates the expression of target genes. We have also described a novel function of NAC1 in attenuating BCL6 autoregulation in OVCA. Finally, we have found a significant overlap among NAC1- and BCL6-regulated, suggesting a transcription collaboration of NAC1 and BCL6 in OVCA cells. CORRESPONDING AUTHORS: RANGEL, LBA; SHIH, IM. Citation Format: Alice Laschuk Herlinger, Min Gao, Ren-Chin Wu, Tian-Li Wang, Leticia B A Rangel, Ie-Ming Shih. NAC1 attenuates BCL6 negative autoregulation and functions as a BCL6 coactivator of FOXQ1 transcription in ovarian cancer (OVCA). [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A80.

Abstract POSTER-TECH-1105: Assessment of ovarian cancer spheroid attachment and invasion of mesothelial cells in real time to profile the molecular signature of the invasive interface

Abstract Background and Aims: Ovarian cancer is the most common cause of death from gynaecological cancer worldwide (1). Often called the “silent killer”, the high mortality rate is associated with the late stage in which the disease is diagnosed (2). Unlike other solid cancers, ovarian cancer cells rarely spread via the vasculature, instead shed from the primary tumor and disseminate within the peritoneal fluid or ascites (2). The formation of spheroids within the non-adherent peritoneal environment is a barrier to effective treatment, due to the enhanced ability of spheroids to survive chemotherapies and seed distal metastases (3-5). Invading ovarian cancer cells interact with mesothelial cells lining the peritoneal surface, attach to, and invade the underlying basement membrane to establish secondary lesions (6). The molecular events at the interface between invading ovarian cancer and peritoneal cells at the onset of invasion have been poorly studied, existing methods capture events after invasion is well-underway. Therefore, the regulatory signals which govern the initiation of a metastatic lesion are poorly understood. We aimed: i) to develop an innovative method to co-culture ascites-derived primary ovarian cancer cells to measure invasion through peritoneal mesothelial cell layers in real time (7); ii) identify the unique protein expression profile at the ovarian cancer spheroid-peritoneal interface during early invasion using proteomics. Methods: Real Time Cell Analysis (RTCA) technology (xCELLigence, ACEA) was adapted to establish a three-dimensional co-culture model of the tumor microenvironment of the peritoneum (7). RTCA wells were coated with Matrigel (basement membrane matrix) and a confluent monolayer of human mesothelial cells. Multicellular spheroids were generated from primary ovarian cancer lines (high-grade serous carcinoma, leiomysarcoma, adenocarcinoma, carcinosarcoma) or benign ascites derived cells cultured under non-adherent conditions. Spheroids were added to the RTCA plate and real time measurements were conducted to determine the precise period when invasion commenced. Parallel cultures were histologically-preserved at invasion initiation and processed for assessment by Matrix-Assisted Laser Desorption/Ionization (MALDI) imaging. Results: We assessed invasion in a panel of ovarian cancer samples, benign ascites cells and the mesothelial cell line. Continuous real time measurements revealed that individual ovarian cancer samples exhibited differences in the timing of the onset of invasion. High-grade serous carcinoma samples initiated invasion rapidly after addition, with some samples detected in under 30 min, a time period not captured by traditional Transwell assays. The benign and mesothelial cells themselves did not invade. MALDI imaging allowed for the identification of peptides expressed at the tumor-mesothelial interface during early invasion. These molecules represent novel molecules involved in the initiation of the metastatic process and are the subject of further characterisation. Conclusion: This method represents a high-throughput quantitative analysis of ovarian cancer spheroid invasion of mesothelial and ECM barriers. The study has provided an unprecedented insight into the molecular mechanisms and molecules involved in the initiation of ovarian cancer invasion of the mesothelium, identifying new therapeutic targets.

Microarray analysis of differentially expressed genes in ovarian and fallopian tube epithelium from risk-reducing salpingo-oophorectomies.

Mutations in the BRCA1 and BRCA2 genes confer an increased lifetime risk for breast and ovarian cancer. Ovarian cancer risk can be decreased by risk-reducing salpingo-oophorectomy (RRSO). Studies on RRSO material have altered the paradigm of serous ovarian cancer pathogenesis. The purpose of this study was to identify candidate genes possibly involved in the pathogenesis of serous ovarian cancer by carrying out a microarray analysis of differentially expressed genes in BRCA1/2- mutation positive ovarian and fallopian tube epithelium derived from RRSO surgery. Freshly frozen ovarian and fallopian tube samples from nine BRCA1/2 mutation carriers scheduled for RRSO were prospectively collected together with five mutation-negative control patients undergoing salpingo-oophorectomy for benign indications. Microarray analysis of genome-wide gene expression was performed on ovarian and fallopian tube samples from the BRCA1/2 and control patients. The validation of microarray data was performed by quantitative real-time polymerase chain reaction (qRT-PCR) in selected cases of RRSO samples and also in high grade serous carcinoma samples collected from patients with a BRCA phenotype. From 22,733 genes, 454 transcripts were identified that were differentially expressed in BRCA1/2 mutation carriers when compared with controls, pooling all ovarian and fallopian tube samples together. Of these, 299 genes were statistically significantly downregulated and 155 genes upregulated. Differentially expressed genes in BRCA1/2 samples reported here might be involved in serous ovarian carcinogenesis and provide interesting targets for further studies.

Decreased progesterone receptor isoform expression in luteal phase fallopian tube epithelium and high-grade serous carcinoma

We previously reported that BRCA1/2-mutated fallopian tube epithelium (FTE) collected during the luteal phase exhibits gene expression profiles more closely resembling that of high-grade serous carcinoma (HGSC) specimens than FTE collected during the follicular phase or from control patients. Since the luteal phase is characterised by high levels of progesterone, we determined whether the expression of progesterone receptor (PR) and PR-responsive genes was altered in FTE obtained from BRCA mutation carriers during the luteal phase of the menstrual cycle. RT-qPCR confirmed a decreased expression of PR mRNA in FTE during the luteal phase relative to follicular phase, in both BRCA1/2 mutation carriers and control patients. Immunohistochemistry using isoform-specific antibodies confirmed a low level of both PR-A and PR-B in HGSC and a lower level of staining in FTE samples obtained during the luteal phase compared with the follicular phase. No significant difference in PR-A or PR-B staining was found based on patient BRCA mutation status. Analysis of our previously reported gene expression profiles based upon known PR-A- and PR-B-specific target genes did not partition samples by BRCA mutation status, indicating that overall FTE PR response is not altered in BRCA mutation carriers. HGSC samples grouped separately from other samples, consistent with the observed loss of PR expression. These findings indicate no overall difference in PR signalling in FTE as a function of BRCA mutation status. Thus, the molecular similarity of BRCA1/2-mutated luteal phase FTE and HGSC likely results from an altered response to luteal phase factors other than progesterone.