Ovarian Carcinoma Cohorts Research Articles

The human nucleoporin Tpr protects cells from RNA-mediated replication stress

Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.

Abstract 1427: Co-alteration of the nucleosome remodeler, ARID1A with the PI3K-pathway genes: A signaling opportunity in ovarian cancers

Abstract Objectives: We present mutations in the ARID1A gene and its co-occurrence with alterations of gene(s) of the PI3K pathway (GOP) in our clinical cohort of ovarian carcinomas (OC). We interrogated the signaling opportunity of the co-occurrence in tumorigenic transformation of OC. Methods: A comprehensive genomic-profiling (FoundationOne) was performed in 90 samples from patients admitted to the Avera Center Institute, USA. The mechanistic study included epithelial endometrioid adenoC cell line A2780 and A2780Cis. CRISPR-Cas9 edited OVCAR3 cell line was used for an ARID1A-incompetent model. Algorithmically chosen drugs, single or in combination were tested for their effect on cell cycle, proliferation, apoptosis, 3D growth, and cellular signals. Results: We recorded 87% metastatic and 11% adjuvant tumors with a predominant histological type of high grade serous (58%). A total of approximately 318 alterations in 100 genes was observed. Alterations (point mutation, frameshifts, and splice site) in the ARID1A was found in 8 patients whose tumors are predominantly clear cell carcinomas (6 out of 8). Interestingly none had alteration in germline BRCA1/2. All frameshift mutations were found in the adjuvant category while alterations in the metastatic category were more diverse. Alteration of GOP included PIK3CA, PIK3R1, AKT2, PTEN, AKT1, AKT3, MDM4, PIK3C2B, PIK3CB, TSC1, STK11, PPP2R1A, and FBXW7 with the most number of alterations in PIK3CA, PIK3R1, AKT2, PTEN genes. A heatmap showed that all patients with ARID1A alterations had alterations in at least one GOP. Treatment with PI3K inhibitor, copanlisib dose-dependently induced apoptosis both as a single agent and in combination with BMN673 in OC cells. Conclusion: OC expressing alteration of ARID1A frequently exhibit a co-alteration in at least one GOP, and the co-alteration provide a unique signaling opportunity to test rational combination of pathway-targeted drugs in OC. Our study provides an insight into the mechanism of how a loss of a tumor suppressor gene, ARID1A co-operates with gain of function mutation(s) of an oncogenic pathway, the PI3K pathway. Background: Nucleosome remodelers controls transcription of genes in human health and sickness by dynamic remodeling of human chromatin in a spatiotemporal manner. ARID1A, a core component of the mammalian SWI/SNF nucleosome remodeler complex, which interacts with DNA in a sequence-nonspecific way to regulate cellular processes, including proliferation and differentiation and hence reported to be involved in tumorigenesis (PMID: 25387058). The mutational (inactivating) status of ARID1A in a broad spectrum of gynecological neoplasms (PMID: 22009941) predominantly ovarian and endometrial cancers established its role as an epigenetic tumor suppressor (PMID: 23208470). The mechanistic study included OC cell line A2780 (epithelial), established from an untreated patient with ovarian endometroid adenocarcinoma and an isogenic cis-platin resistant cell line, A2780Cis. The results were being interrogated in the light of tumor cell signaling. Citation Format: Nandini Dey, Jennifer C. Aske, Ethan Thompson, Luis Rojas-Espaillat, David Starks, Pradip De. Co-alteration of the nucleosome remodeler, ARID1A with the PI3K-pathway genes: A signaling opportunity in ovarian cancers [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 1427.

Overexpression of SLC12A5 is associated with tumor progression and poor survival in ovarian carcinoma

IntroductionThe solute carrier family 12 member 5 (SLC12A5) gene is playing a putative oncogenic role in colorectal carcinoma. However, the status of SLC12A5 amplification and expression in ovarian carcinoma and...

Single-Arm Phases 1 and 2 Trial of Niraparib in Combination With Pembrolizumab in Patients With Recurrent Platinum-Resistant Ovarian Carcinoma

Patients with recurrent ovarian carcinoma frequently develop resistance to platinum-based chemotherapy, at which time treatment options become limited. To evaluate the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor niraparib combined with pembrolizumab in patients with recurrent ovarian carcinoma. The TOPACIO/KEYNOTE-162 (Niraparib in Combination With Pembrolizumab in Patients With Triple-Negative Breast Cancer or Ovarian Cancer) trial, an open-label, single-arm phases 1 and 2 study enrolled women with advanced or metastatic triple-negative breast cancer (TNBC) or recurrent ovarian carcinoma, irrespective of BRCA mutation status. Median follow-up was 12.4 months (range, 1.2 to ≥23.0 months). Data were collected from April 15, 2016, through September 4, 2018, with September 4, 2018, as a data cutoff, and analyzed from September 4, 2018, through January 30, 2019. The recommended phase 2 dose (RP2D) was 200 mg of oral niraparib once daily and 200 mg of intravenous pembrolizumab on day 1 of each 21-day cycle. The primary objectives of phase 1 were to evaluate dose-limiting toxic effects and establish the RP2D and dosing schedule. The primary objective of phase 2 was to assess objective response rate (ORR; complete plus partial responses). Results from the phase 1 ovarian carcinoma and TNBC cohorts and phase 2 ovarian carcinoma cohort are reported. Because of the similarity in the phase 1 and 2 ovarian carcinoma populations, the data were pooled to perform an integrated efficacy analysis. Fourteen patients (9 with ovarian carcinoma and 5 with TNBC) in phase 1 and 53 patients with ovarian carcinoma in phase 2 were enrolled, for a pooled ovarian carcinoma cohort of 62 patients (median age, 60 years [range, 46-83 years]). In the integrated efficacy phases 1 and 2 ovarian carcinoma population (60 of 62 evaluable patients), ORR was 18% (90% CI, 11%-29%), with a disease control rate of 65% (90% CI, 54%-75%), including 3 (5%) with confirmed complete responses, 8 (13%) with confirmed partial responses, 28 (47%) with stable disease, and 20 (33%) with progressive disease. The ORRs were consistent across subgroups based on platinum-based chemotherapy sensitivity, previous bevacizumab treatment, or tumor BRCA or homologous recombination deficiency (HRD) biomarker status. Median duration of response was not reached (range, 4.2 to ≥14.5 months). At data cutoff, 2 patients with a response and 1 patient with stable disease continued to receive treatment. Niraparib in combination with pembrolizumab is tolerable, with promising antitumor activity for patients with ovarian carcinoma who have limited treatment options regardless of platinum status, biomarker status, or prior treatment with bevacizumab. Responses in patients without tumor BRCA mutations or non-HRD cancers were higher than expected with either agent as monotherapy. ClinicalTrials.gov identifier: NCT02657889.

Clinical and molecular characterization of ovarian carcinoma displaying isolated lymph node relapse

BackgroundDisease relapse is the primary cause of death from ovarian carcinoma. Isolated lymph node relapse is a rare pattern of ovarian carcinoma recurrence, with a reported median postrelapse survival of 2.5 to 4 years. To date, investigations have not compared isolated lymph node relapse ovarian carcinoma directly to a matched extranodal relapse cohort or performed molecular characterization of cases that subsequently experience isolated lymph node relapse.ObjectiveHere we seek to compare the clinical outcome, tumor-infiltrating lymphocyte burden, and frequency of known prognostic genomic events in isolated lymph node relapse ovarian carcinoma vs extranodal relapse ovarian carcinoma.Study DesignForty-nine isolated lymph node relapse ovarian carcinoma patients were identified and matched to 49 extranodal relapse cases using the Edinburgh Ovarian Cancer Database, from which the clinical data for identified patients were retrieved. Matching criteria were disease stage, histologic subtype and grade, extent of residual disease following surgical debulking, and age at diagnosis. Clinicopathologic factors and survival data were compared between the isolated lymph node relapse and extranodal relapse cohorts. Genomic characterization of tumor material from diagnosis was performed using panel-based high-throughput sequencing and tumor-infiltrating T cell burden was assessed using immunohistochemistry for CD3+ and CD8+ cells.ResultsIsolated lymph node relapse cases demonstrated significantly prolonged postrelapse survival and overall survival vs extranodal relapse upon multivariable analysis (HRmulti = 0.52 [0.33–0.84] and 0.51 [0.31–0.84]). Diagnostic specimens from high-grade serous ovarian carcinomas that subsequently displayed isolated lymph node relapse harbored significantly greater CD3+ and CD8+ cell infiltration compared to extranodal relapse cases (P = .001 and P = .009, Bonferroni-adjusted P = .003 and P = .019). Isolated lymph node relapse high-grade serous ovarian carcinoma cases did not show marked enrichment or depletion of cases with BRCA1/2 mutation or CCNE1 copy number gain when compared to their extranodal relapse counterparts (24.4% vs 19.4% and 18.2% vs 22.6%, P = .865 and P = .900).ConclusionIsolated lymph node relapse ovarian carcinoma represents a distinct clinical entity with favorable outcome compared to extranodal relapse. There was no clear enrichment or depletion of BRCA1/2 mutation or CCNE1 gain in the isolated lymph node relapse ovarian carcinoma cohort compared with extranodal relapse cases, suggesting that these known prognostic genomically defined subtypes of disease do not display markedly altered propensity for isolated lymph node relapse. Diagnostic tumor material from isolated lymph node relapse patients demonstrated greater CD3+ and CD8+ cell infiltration, indicating stronger tumor engagement by T cell populations, which may contribute to the more indolent disease course of isolated lymph node relapse.

Comprehensive analysis of PD-L1 expression, HER2 amplification, ALK/EML4 fusion, and mismatch repair deficiency as putative predictive and prognostic factors in ovarian carcinoma.

Most ovarian carcinomas (OC) are characterized by poor prognosis, particularly the most frequent type high-grade serous carcinoma. Besides PARP inhibitors, target-based therapeutic strategies are not well established. We asked the question which other therapeutic targets could be of potential value and, therefore, analyzed a large cohort of OC for several predictive factors. Two hundred eighty-eight (288) cases of OC including the major histological types were analyzed by immunohistochemistry for PD-L1HER2, ALK, and the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6. HER2 amplification and ALK/EML4 fusion were assessed by fluorescence in situ hybridization. The most frequent finding was PD-L1 expression ≥ 1% in 19.5% of the cases, which correlated with a significantly better overall survival in multivariate analysis (p < 0.001). HER2 amplification was detected in 11 cases (4%), all high-grade serous carcinomas. Amplification of HER2 did not correlate with patients' survival. ALK/EML4 fusion was found in two cases (0.74%): one high-grade serous and one endometrioid carcinoma. MMR deficiency was only present in one case of stage IV high-grade serous carcinoma. Subsets of high-grade serous carcinomas show PD-L1 expression and HER2 amplification, respectively, and, therefore, could qualify for immune checkpoint inhibitor therapy or anti HER2 therapy. PD-L1 is also of prognostic impact. ALK/EML4 fusion is very rare in OC and not a putative therapeutic target.

The clinical importance of BRCAness in a population-based cohort of Danish epithelial ovarian cancer.

Germline mutations in BRCA1/2 genes predict improved survival and sensitivity to treatment with poly(adenosine-diphosphate-ribose) polymerase inhibitors in epithelial ovarian carcinoma. The prognostic importance of other genetic alterations leading to homologous recombination deficiency, collectively BRCAness phenotype, is unresolved. The aim was to analyze the distribution of homologous recombination deficiency in epithelial ovarian carcinoma caused by mutations in a panel of homologous recombination genes (including BRCA1/2) or epigenetic alterations. A further aim was to investigate the prognostic importance of homologous recombination deficiency, the BRCAness phenotype. We assessed 380 patient specimens from a Danish population-based epithelial ovarian carcinoma cohort for germline and somatic mutations in 18 different homologous recombination genes, including BRCA1 and BRCA2, using next generation sequencing. Epigenetic alteration due to BRCA1 hypermethylation was assessed by pyrosequencing and BRCA1 protein expression was evaluated by immunohistochemistry. Seventeen percent of patients with epithelial ovarian carcinoma carried a germline (9.8%) and/or somatic (6.3%) mutation in 12 (BRCA1, BRCA2, CHEK2, ATM, RAD51D, EMSY, PALB2, BRIP1, ERCC1, RAD50, ATR, RAD51C) of 18 sequenced homologous recombination genes. The homologous recombination mutation rate was similar among the different histologic subtypes, however the type of mutation (BRCA1/2 and other homologous recombination mutations) differed, p=4×10-4. BRCA1 hypermethylation was present in 7.4% of patient specimens for a total BRCAness phenotype of 23.9%. The BRCAness phenotype was associated with improved overall survival in the high-grade serous carcinoma subgroup with a median overall survival of 4.4 years (95% CI 3.0 to 5.3) versus 2.2 years (95% CI 1.9 to 2.4) in BRCAness wildtype, p=0.0002. Multivariate analysis confirmed an independent prognostic value of the BRCAness phenotype among the high-grade serous carcinoma subgroup, hazard ratio 0.65 (95% CI 0.47 to 0.92), p=0.014. The BRCAness phenotype is present in almost one-fourth of epithelial ovarian carcinoma and holds important prognostic information. The implications of our findings in relation to poly(adenosine-diphosphate-ribose) polymerase inhibitor treatment call for further investigation.

Abstract NTOC-115: ASSOCIATION BETWEEN NUCLEAR SYK PHOSPHORYLATION AND EGFR/ERBB2 PATHWAY AND POOR SURVIVAL IN HIGH GRADE SEROUS OVARIAN CANCER

Abstract BACKGROUND: Spleen tyrosine kinase (SYK) has emerged as a cancer associated kinase of which aberrant activity contributes to tumor development and drug resistance in several types of human cancer including ovarian carcinoma. To better understand the mechanism how the SYK pathway participates in tumor progression, we studied the biochemistry and transcriptional regulation of SYK in high-grade ovarian serous carcinoma. METHODS: Immunohistochemical analyses were performed for phosphorylated SYK (Y525/526) and total SYK levels in 42 primary serous ovarian carcinomas and 112 tumor ascites formalin-fixed paraffin embedded tissues. The relative expression of SYK long and short isoforms in peritoneal ascites and cell lines were examined by reverse transcriptase-PCR. Transcriptome analysis of gene regulated by SYK inhibition was carried out using Illumina Human HT-12 v4 arrays. Results from array was analyzed for upstream regulators. IHC data was correlated with pEGFR (Y1197) and pERBB2 (Y877) nuclear expression. Association with survival was determined by Kaplan-Meier analysis and the log-rank test. RESULTS: We observed that active phosphorylated SYK (p-SYK, Y525/526) is mainly localized in the nucleus and cytoskeleton in cell lines and tissues. Investigation of SYK mRNA spliced variants in ascites tumors showed predominant SYK long isoform expression, consistent with its nuclear localization. Survival analyses revealed that intense nuclear p-SYK correlated with poor overall survival in two ovarian carcinoma cohorts. Patients with relatively high p-SYK in the nucleus had approximately half the median survival compared to those with undetectable or low nuclear p-SYK. SYK inhibition affected changes in gene expression that are downstream of ERBB2 and EGFR signaling. Correlation analyses in tissues suggested a relationship between p-SYK and pERBB2 or pEGFR signaling. CONCLUSIONS: Our results suggest that activation of the SYK pathway leads to its phosphorylation and localization in cell nucleus. SYK activity plays a role in regulating EGFR and ERBB2 phosphorylation and activation. Thus, SYK may regulate tumor-promoting gene transcription through cross-talks with the EGFR/ERBB2 pathways. Citation Format: Yu, Yu; Ardighieri, Laura; Suryo Rahmanto, Yohan; Chen, Lin Y.; Tessarollo, Nayara G.; Mizushima, Taichi; Ayhan, Ayse; Davidson, Ben; Wang, Tian L., and Shih Ie M. ASSOCIATION BETWEEN NUCLEAR SYK PHOSPHORYLATION AND EGFR/ERBB2 PATHWAY AND POOR SURVIVAL IN HIGH GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-115.

Abstract IA02: Functional role of endogenous retroviruses in cancer immunity

Abstract Endogenous retroviruses (ERVs) are derived from past exogenous retroviral infections of germ cells, which integrated into the genome of mammals and are inherited in a Mendelian manner. ERVs belong to the class of retro-elements possessing three main viral genes: group-specific antigen (gag), polymerase (pol) and envelope (env) flanked by two long terminal repeats (LTR). More than 8% of the human genome is considered of retroviral origin. Most ERV genes are protein non-functional due to recombination, mutations and deletions; however some ERV gag, pol and env genes produce functional proteins. Although largely gene defective, the LTRs of ERVs retained their regulatory activities resulting in high transcriptional levels; and in addition represent most of the ~10,000 annotated long non-coding (lnc) RNAs. In somatic cells ERVs are generally silent; however we and others demonstrated ERVs become activated during tumorigenesis. Recently we showed that induction of immune responses in human cancer cells by DNA methyltransferase inhibitors (DNMTi), like 5-azacytidine (Aza) and 2'-deoxy-5-azacytidine (Dac) is primarily due to the activation of (hyper)-methylated ERVs (Chiappinelli et al. 2015, Cell 162: 974-86). Furthermore, the unique genomic architecture of ERVs with 5 and 3 LTRs induced sense and antisense RNAs leading to formation of cytosolic dsRNA, which activated the cellular viral RNA sensing and defense pathway (like TLR3 and MAVS) triggering the type I interferon (IFN) response. This kind of response following ERV activation is called viral mimicry or endogenous viremia. Using ovarian carcinoma (OVCA) cell lines as a model, we showed that Aza significantly induced multiple ERV families and IFN stimulated genes (ISG). The induction was due to the formation of dsRNA, which was abolished with RNAse III (hydrolyzing dsRNA) and TLR3 or MAVS specific inhibition. ISGs were also directly induced by transfected ERV genes. Subsequently, a cohort of primary human serous OVCA tissues was analyzed and a significant positive correlation between ERV and ISG expression could be demonstrated. We and others previously showed that treatment with the DNMTi Aza positively contributed to immune checkpoint blockade therapy in a mouse model and in human cancers. Like exogenous retroviruses, which upregulate immune checkpoint genes (ICG), we also evaluated for a possible connection between ERV induction and ICG expression in primary tumors. Presently, we demonstrate that human serous OVCA tissues express significantly higher ICGs than control ovarian tissues, with VTCN-1 (B7-H4) as the highest expressed ICG. Using serous OVCA cell lines transfected with different ERV genes we could show an early induction of ICGs, primarily LGALS9 and PD-L1. Further analysis of tumor infiltrating immune cells using the same serous OVCA cohort and multiplex immunohistochemistry (IHC), we detect in some tumors high levels of tumor-associated CD79a, CD3+/CD8- and CD3+/CD8+ cells. Moreover, we find a direct link between ISG, ICG and infiltrating immune cells in OVCA. Analyzing other human tumors, e.g. brain tumors, like glioblastoma multiforme (GBM) and giant cell GBM (gcGBM) also demonstrate a significant correlation between high ERV induction and ISG as well as ICG expression. In contrast to serous OVCA, PD1 and VTCN-1 were the highest expressed ICG in GBM and gcGBM, which could also be shown with ERV transfected GBM cell lines. Using IHC gcGBM shows a high level of immune cell infiltration, which associated with giant cells and ICG expression, like PD-L1. Interestingly, the highest ERV expression was found in gcGBM, which correlated with endomitosis and cell fusion leading to giant nuclei and multinucleated cells as also shown with a stable ERV transfected GBM cell line. In summary, DNMTi mediated loss of methylation induced ERVs forming dsRNA in tumor cells and stimulated the innate immune system via ISGs. ERV RNA per se induced ISGs, but also ICGs similar to exogenous retroviruses in order to escape the immune system. Different upregulated ERVs, ISGs and ICGs were found in different cancers, like serous OVCA, GBM and gcGBM indicating diverse tumor and tumor microenvironment signatures. Thus ERVs have an ambivalent role in tumorigenesis, where tumor microenvironmental factors tip the balance being detrimental by generating immune evasion or beneficial for cancer therapy by stimulating the innate immune system. Citation Format: Reiner Strick. Functional role of endogenous retroviruses in cancer immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr IA02.

PRSS3 expression is associated with tumor progression and poor prognosis in epithelial ovarian cancer

ObjectivePRSS3 is an atypical isoform of trypsin that has been associated with breast, lung, and pancreatic cancers. This study aimed to elucidate the role of PRSS3 in tumor tissues of patients with epithelial ovarian cancer (EOC) and to investigate the prognostic value of this marker. MethodsPRSS3 expression was evaluated by immunohistochemistry and real-time PCR (RT-PCR) in ovarian cancers, benign ovarian tumors and the ovaries of age-matched normal patients. Correlations between clinicopathologic variables and PRSS3 expression in EOC tissues and the prognostic value of PRSS3 for progression-free survival (PFS) and overall survival (OS) were evaluated. ResultsPRSS3 expression was significantly elevated in EOC tissues compared to benign ovarian tumors and normal ovarian controls at both the mRNA and protein levels. There was a good correlation between the PRSS3 expression levels measured by the two different techniques. High PRSS3 expression in EOC tissues was significantly associated with advanced FIGO stage and lymph node metastasis. In a univariate survival analysis of the ovarian carcinoma cohort, positive expression of PRSS3 was significantly associated with shortened patient survival. Importantly, PRSS3 expression was a significant independent prognostic parameter in the multivariate analysis. ConclusionsThese findings indicate that PRSS3 overexpression can be used as a predictor of clinical outcome in patients with ovarian cancer and may therefore represent a new prognostic marker.

Decreased expression of H3K27me3 in human ovarian carcinomas correlates with more aggressive tumor behavior and poor patient survival.

It has been confirmed that trimethylation of lysine 27 on histone H3 (H3K27me3) plays an important role in epigenetic process of tumorigenesis. However, the status of H3K27me3 in ovarian cancer and its impact on patients' clinicopathologic characteristics and prognosis are unclear. In the present study, the immunohistochemistry (IHC) was utilized to detect protein expression of H3K27me3 in 12 normal ovaries, 26 ovarian cystadenomas, 31 borderline ovarian tumors and 168 ovarian carcinomas by tissue microarray. The association between H3K27me3 expression with clinicopathologic features and patient prognosis were also evaluated using various statistical models. The expression of H3K27me3 was decreased in 2 of 12 (16.7%) cases of the normal ovaries, 8 of 26 (30.8%) cases of cystadenomas, 12 of 31 (38.7%) cases of borderline ovarian tumors, and 93 of 168 (55.4%) cases of primary ovarian carcinomas, respectively (P<0.05). Further correlation analysis suggested that decreased expression of H3K27me3 in ovarian carcinomas was significantly correlated with more advanced pM and FIGO stages (P<0.05). In addition, asignificant association between decreased expression of H3K27me3 and shortened patient survival (mean 66 months versus 101 months, p=0.019) was demonstrated by univariate survival analysis of the ovarian carcinoma cohorts. Importantly, H3K27me3 expression provided asignificant independent prognostic factor in multivariate analysis (p=0.028). These findings confirmed that decreased expression of H3K27me3 in primary ovarian cancer might be correlated with the acquisition of an invasive and/or aggressive phenotype of tumor, and might serve as an independent biomarker for poor prognosis in patients with ovarian carcinoma.

Visual and automated assessment of matrix metalloproteinase-14 tissue expression for the evaluation of ovarian cancer prognosis

The purpose of this study was to evaluate whether the membrane type 1 matrix metalloproteinase-14 (or MT1-MMP) tissue expression, as assessed visually on digital slides and by digital image analysis, could predict outcomes in women with ovarian carcinoma. Tissue microarrays from a cohort of 211 ovarian carcinoma women who underwent a debulking surgery between 1993 and 2006 at the CHU de Québec (Canada) were immunostained for matrix metalloproteinase-14. The percentage of MMP-14 staining was assessed visually and with the Calopix software. Progression was evaluated using the CA-125 and/or the RECIST criteria according to the GCIG criteria. Dates of death were obtained by record linkage with the Québec mortality files. Adjusted hazard ratios of death and progression with their 95% confidence intervals were estimated using the Cox model. Comparisons between the two modalities of MMP-14 assessment were done using the box plots and the Kruskal–Wallis test. The highest levels of MMP-14 immunostaining were associated with nonserous histology, early FIGO stage, and low preoperative CA-125 levels (P<0.05). In bivariate analyses, the higher level of MMP-14 expression (>40% of MMP-14-positive cells) was inversely associated with progression using visual assessment (hazard ratio=0.39; 95% confidence interval: 0.18–0.82). A similar association was observed with the highest quartile of MMP-14-positive area assessed by digital image analysis (hazard ratio=0.48; 95% confidence interval: 0.28–0.82). After adjustment for standard prognostic factors, these associations were no longer significant in the ovarian carcinoma cohort. However, in women with serous carcinoma, the highest quartile of MMP-14-positive area was associated with progression (adjusted hazard ratio=0.48; 95% confidence interval: 0.24–0.99). There was no association with overall survival. The digital image analysis of MMP-14-positive area matched the visual assessment using three categories (>40% vs 21–40 vs <20%). Higher levels of MMP-14 immunostaining were associated with standard factors of better ovarian carcinoma prognosis. In women with serous carcinoma, high expression of MMP-14 was associated with lower progression.

Tumor heterogeneity in the recurrence of epithelial ovarian cancer demonstrated by polycomb group proteins.

PurposeTo investigate tumor heterogeneity in the recurrence of epithelial ovarian cancer demonstrated by polycomb group (PcG) proteins.MethodsTissue microarrays containing matched primary and recurrent ovarian tumors from the same patients were constructed for detection of PcG protein expression. Survival analyses of clinicopathological parameters and expression of PcG proteins were performed on progression-free survival (PFS) and overall survival (OS) of patients. Genetic and epigenetic heterogeneity was explored in aspects of gene copy number and microRNA (miRNA) profiling.ResultsPcG proteins were heterogeneously expressed in primary versus recurrent tumors (P<0.05). In univariate survival analysis of the ovarian carcinoma cohorts, a significant association of intensive expression of BMI1 and EZH2 in first-onset lymph node metastases with shortened PFS was demonstrated (P=0.010, P=0.019); and a significant association of intensive expression of BMI1 and EZH2 in recurrent tumors with shortened OS was demonstrated (P=0.042, P=0.047). Importantly, BMI1 and EZH2 expression provided significant independent prognostic parameters in multivariate analyses (P<0.05). Gene amplification did not always coincide with PcG protein expression. Eight miRNAs were found to be downregulated in recurrent tumors, among which miR-298 might indirectly regulate the expression of EZH2 through transcription factor ILF3.ConclusionTumor heterogeneity exists in the recurrence of epithelial ovarian cancer, manifested by PcG protein expression and underlying genetic and epigenetic alterations. Intensive expression of BMI1 and EZH2 are predictors of earlier relapse and shorter OS, independent of grade and chemotherapy sensitivity. EZH2 and miR-298 have great potential to be new targets for treatment of recurrent ovarian cancer.

CYR61 overexpression associated with the development and poor prognosis of ovarian carcinoma.

Cysteine-rich 61 (CYR61) has been proven to be an oncogene with potential predictive and prognostic implications in a variety of human cancers. However, the expression pattern of CYR61 and its role in ovarian carcinoma remains largely unknown. In this study, the mRNA and protein levels of CYR61 in normal ovaries and ovarian carcinoma tissues were evaluated using reverse transcription-polymerase chain reaction, immunohistochemistry, and Western blotting. Compared to normal ovarian tissues, the mRNA and protein levels of CYR61 were significantly higher in ovarian carcinoma tissues. Moreover, receiver operating characteristic (ROC) curve analysis, Spearman's rank correlation, Kaplan-Meier plots, and the Cox proportional hazards regression model were used to investigate the potential association of the CYR61 protein with the development of ovarian carcinoma in an ovarian carcinoma cohort. Based on ROC curve analysis, high expression of CYR61 was defined as a tumor in which more than 70 % of cells were positively stained. Based on this cutoff value, high expression of CYR61 was detected in 51.5 % of invasive carcinomas, 35.3 % of borderline tumors, 25.9 % of cystadenomas, and 20 % in the normal ovaries. In ovarian carcinomas, CYR61 overexpression was associated with advanced FIGO stage. Univariate survival analysis on the ovarian carcinoma cohorts showed that overexpression of CYR61 was associated with poor survival of ovarian cancer patients. Multivariate analysis suggested that the protein level of CYR61 was an independent and significant prognostic factor for ovarian carcinoma. Our results suggest that the CYR61 protein is an important and independent biomarker for prognostic implications of ovarian carcinoma.

An EMT spectrum defines an anoikis-resistant and spheroidogenic intermediate mesenchymal state that is sensitive to e-cadherin restoration by a src-kinase inhibitor, saracatinib (AZD0530)

The phenotypic transformation of well-differentiated epithelial carcinoma into a mesenchymal-like state provides cancer cells with the ability to disseminate locally and to metastasise. Different degrees of epithelial–mesenchymal transition (EMT) have been found to occur in carcinomas from breast, colon and ovarian carcinoma (OC), among others. Numerous studies have focused on bona fide epithelial and mesenchymal states but rarely on intermediate states. In this study, we describe a model system for appraising the spectrum of EMT using 43 well-characterised OC cell lines. Phenotypic EMT characterisation reveals four subgroups: Epithelial, Intermediate E, Intermediate M and Mesenchymal, which represent different epithelial–mesenchymal compositions along the EMT spectrum. In cell-based EMT-related functional studies, OC cells harbouring an Intermediate M phenotype are characterised by high N-cadherin and ZEB1 expression and low E-cadherin and ERBB3/HER3 expression and are more anoikis-resistant and spheroidogenic. A specific Src-kinase inhibitor, Saracatinib (AZD0530), restores E-cadherin expression in Intermediate M cells in in vitro and in vivo models and abrogates spheroidogenesis. We show how a 33-gene EMT Signature can sub-classify an OC cohort into four EMT States correlating with progression-free survival (PFS). We conclude that the characterisation of intermediate EMT states provides a new approach to better define EMT. The concept of the EMT Spectrum allows the utilisation of EMT genes as predictive markers and the design and application of therapeutic targets for reversing EMT in a selective subgroup of patients.

Decreased Expression of Beclin 1 Correlates Closely with Bcl-xL Expression and Poor Prognosis of Ovarian Carcinoma

BackgroundIt has been suggested that autophagy-related Beclin 1 plays a critical role in the regulation of tumor development and/or progression, but its prognostic significance and relationship with Bcl-xL expression in ovarian carcinoma are unclear.Methodology/Principal FindingsIn the present study, the methods of Western blotting and immunohistochemistry (IHC) were utilized to investigate the expression status of Beclin 1 and Bcl-xL in fresh ovarian tissues and paraffin-embedded epithelial ovarian tumor tissues. Decreased expression of Beclin 1 was examined by IHC in 8.3% of normal ovaries, in 15.4% of cystadenomas, in 20.0% of borderline tumors, and in 55.6% of ovarian carcinomas, respectively. In ovarian carcinomas, decreased expression of Beclin 1 was correlated closely with ascending histological grade, later pT/pN/pM status and/or advanced clinical stage (P<0.05). In univariate survival analysis, a highly significant association between low-expressed Beclin 1 and shortened patient survival was evaluated in ovarian carcinoma patients (P<0.01), and Beclin 1 expression was an independent prognostic factor as evidenced by multivariate analysis (P = 0.013). In addition, decreased expression of Beclin 1 was inversely correlated with altered expression of Bcl-xL in ovarian carcinoma cohort, and combined analysis further showed that the low Beclin 1/high Bcl-xL group had the lowest survival rate.Conclusions/SignificanceOur findings suggest that Beclin 1 expression, as examined by IHC, could be served as an additional tool in identifying ovarian carcinoma patients at risk of tumor progression, and predicting patient survival in ovarian carcinomas with increased expression of Bcl-xL.

Clinicopathologic Analysis of Low-stage Sporadic Ovarian Carcinomas

Current histologic criteria endorsed by World Health Organization for surface epithelial ovarian tumors suffer from less than optimal reproducibility and correlation with clinical outcomes. A modified approach for histologic subclassification of ovarian carcinomas (OCs), proposed by the group of Gilks and colleagues, has been shown to be reproducible and clinically meaningful. We hypothesized that this approach could be validated using a well-annotated, external cohort of OCs with detailed immunophenotypic, genotypic, and clinical data. The cohort comprised 91 patients with low-stage (FIGO stage I/II) OC who underwent primary surgical management at our institution from 1980 to 2000. The clinical and histologic features of this cohort were reported in 2004. In this study we rereviewed the hematoxylin and eosin slides and reassigned histologic type using the criteria proposed by Gilks and colleagues. p53 and Wilms tumor 1 (WT1) expressions were evaluated using standard immunohistochemical techniques on a tissue microarray. Absence of (null) or strong and diffuse p53 staining in >75% of tumor cells (overexpression) was interpreted as aberrant p53 expression, in contrast to the 2004 study in which the "null" result had been interpreted as negative. Direct TP53 gene sequencing of the entire coding region had been performed on all cases with available tissue. Relationships between survival and the following parameters were studied: tumor cell type, histologic grade, stage, p53 and WT1 expression, TP53 mutation, and presence of associated endometriosis. Results were compared with those of the 2004 study, at which time only TP53 mutation was shown to correlate with adverse survival; notably, TP53 mutation did not correlate with p53 expression by immunohistochemical analysis. After review, presence of TP53 mutations, WT1 positivity, and aberrant p53 expression were strongly associated with high-grade serous histology (P=0.0001). Modification of p53 staining assessment revealed strong correlations with TP53 mutation status, which was superior to the positive versus negative approach used previously (P=0.0005). Redefined histologic criteria revealed a stronger association between the high-grade serous type and WT1 positivity (P=0.02). Prolonged 10-year disease-specific and progression-free survival were associated with endometrioid histology (P<0.02; 94%±4% vs. 53%±7% and 96%±4% vs. 50%±7%, respectively). Inferior 10-year disease-specific survival and progression-free survival were associated with high histologic grade (vs. low and intermediate; P<0.01; 54%±7% vs. 89%±6% and 56%±7% vs. 89%±6%, respectively), presence of TP53 mutation (P<0.04; 54%±12% vs. 64%±10% and 34%±12% vs. 70%±6%, respectively), aberrant p53 expression (P<0.02; 35%±12% vs. 75%±7% and 36%±12% vs. 73%±7%, respectively), and absence of associated endometriosis (P=0.01; 48%±9% vs. 80%±6% and 51%±9% vs. 80%±6%, respectively). In addition, inferior 10-year PFS was associated with WT1 positivity (P=0.01; 42%±7% vs. 72%±7%) and high substage (IA-IB vs. IC-IIC; P=0.04; 53%±7% vs. 81%±8%). These results validate updated approaches to scoring p53 expression as well as the revised criteria proposed by the group of Gilks and colleagues.

Transitional Cell Carcinoma of the Ovary is Related to High-grade Serous Carcinoma and is Distinct From Malignant Brenner Tumor

Transitional cell tumors of the ovary include benign, borderline (atypically proliferating), and malignant Brenner tumors (BT), as well as transitional cell carcinoma (TCC). Some TCCs could conceivably be examples of malignant BT where the benign component has been overgrown. Our objectives were: (A) compare the immunophenotypes of BT and TCC and (B) examine a large cohort of ovarian carcinomas for cases with the immunophenotype of BT and transitional features but lacking a benign BT component. Seven BTs (3 benign, 3 borderline/atypically proliferating, 1 malignant) and 7 TCCs were stained for WT1, ER, p53, and p16(INK4a). The BTs were negative for WT1, p53 overexpression, ER (except for weak positivity in 1), and negative or weakly positive for p16(INK4a). In contrast, the TCCs stained as follows: 4/6 positive for WT1, 5/7 positive for ER, 2/7 strongly positive for p16(INK4a), and 6/7 showed abnormal p53, an immunophenotype resembling that of high-grade serous carcinoma. A database of 500 cases of ovarian carcinoma was searched and 116 showed an immunoprofile characteristic of BT: WT1 negative, ER negative, p16(INK4a) or weak positive, p53 negative (77 clear cell carcinoma, 14 endometrioid carcinoma, 12 mucinous carcinoma, 8 high-grade serous carcinoma). None of these tumors showed transitional features on review, indicating that if examples of malignant BT where there has been overgrowth of benign BT components exist, they are rare. Our results suggest that BT and TCC are unrelated, and should not be combined for classification purposes.

CHD1L Protein is overexpressed in human ovarian carcinomas and is a novel predictive biomarker for patients survival

BackgroundOur recent studies suggested that the chromodomain helicase DNA binding protein 1-like (CHD1L) gene plays an oncogenic role in human hepatocellular carcinoma. However, the status of CHD1L protein expression in ovarian cancer and its clinical/prognostic significance are obscure.MethodsIn this study, immunohistochemistry (IHC) for CHD1L was performed on a tissue microarray (TMA) containing 102 primary ovarian carcinomas and 44 metastatic lesions (omental metastasis). Receiver-operator curve (ROC) analysis was used to evaluate patients’ survival status.ResultsThere is an augmented tendency of CHD1L expression in ovarian carcinoma metastasis than in primary lesions (P<0.05). A significant association was found between positive expression of CHD1L and tumors histological type (P <0.05). By univariate survival analysis of the ovarian carcinoma cohorts, positive expression of CHD1L was significantly correlated with shortened patient survival (mean 66.7 months versus 97.4 months, P<0.05). Moreover, CHD1L expression was evaluated to be a significant and independent prognostic factor in multivariate analysis (P<0.05).ConclusionsThese findings provide evidence that positive expression of CHD1L protein is significantly correlated with the metastasis proceeding of ovarian carcinoma, and CHD1L protein expression, as examined by IHC, may act as a novel prognostic biomarker for patients with ovarian carcinoma.

Overexpression of SGLT1 is correlated with tumor development and poor prognosis of ovarian carcinoma

Overexpression of glucose transporters has been identified in a variety of human cancers. However, the expression status of Sodium dependent Glucose Transporter 1 (SGLT1) in ovarian carcinoma has not been investigated. In our study, protein expression levels of SGLT1 were explored by semiquantitative immunohistochemical staining on archival formalin-fixed paraffin-embedded pathologic specimen consisting of 178 epithelial ovarian tumors. Receiver operating characteristic curve analysis, Spearman's rank correlation, Kaplan-Meier plots and Cox proportional hazards regression model were utilized to analyze the data. The threshold for high expression of SGLT1 was determined to be above 40% (areas under curve = 0.683, P = 0.003) based on the area under curves. Significantly overexpression of SGLT1 was observed in 39.7% invasive carcinomas, 11.5% borderline tumors, 10% cystadenomas but in none of the normal ovaries (0%). In ovarian carcinomas, SGLT1 overexpression was positively correlated with later pT status (P = 0.029) and advanced FIGO stage (P = 0.024). By univariate survival analysis on the ovarian carcinoma cohorts, overexpression of SGLT1 was associated with shortened patient survival (mean 70.5 months in tumors with overexpression of SGLT1 versus 89.3 months in tumors with normal levels of SGLT1; P = 0.019). By multivariate analysis, SGLT1 protein expression remained as a significant and independent prognostic factor for the prediction of patient survival (P = 0.033). SGLT1 overexpression, as examined by immunohistochemistry, is an independent biomarker for poor prognosis of patients with ovarian carcinoma.