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Introduction/Background* The PI3K/AKT signaling pathway is activated in a wide spectrum of human cancers. INPP4B is a tumor suppressor gene encoding lipid phosphatase type II - a negative regulator of the PI3K signaling. We aimed to determine mechanisms of INPP4B inactivation in ovarian cancer. Methodology Among 194 ovarian cancers studied, there were 126 serous, 23 endometrioid, 18 clear cell, 10 mucinous and 17 other type carcinomas. INPP4B mutations were analyzed in 52 carcinomas using Sanger sequencing method. Analyses of copy number alteration (CNA), mRNA expression and promoter methylation were performed with the use of quantitative PCR (qPCR) method for 194, 144 and 125 ovarian carcinomas, respectively. Five specimens of noncancerous fallopian tube constituted a control group. Statistical analyses were done with Fisher’s exact test, χ2 and Mann-Whitney U tests. Result(s)* One INPP4B missense mutation, c.1659G>A, p.(Gly554Ser), was detected in two carcinomas (3.8%, 2/52) of clear cell and serous type. The INPP4B CNA was found in 82 out of 194 (42.3%) ovarian cancers. There were 25.3% (49/194) allelic losses and 17% (33/194) amplifications at the INPP4B locus. Allelic loss was associated with high-grade (P = 0.031) and advanced FIGO stage (P = 0.011) tumors. Reduced copy number was more common in carcinomas with PIK3CA amplification (P = 0.014) and PIK3R1 allelic loss (P = 0.001). The INPP4B copy loss was mutually exclusive with PTEN mutations (P = 0.035). The INPP4B mRNA expression was significantly decreased in ovarian cancers compared with control tissues (P = 0.004). The difference in mean expressions between carcinomas and normal tissues was 57% (0.099 ± 0.12 vs 0.231 ± 0.11). We did not observe an association between decreased mRNA level and copy number loss of the gene. Lower levels of INPP4B mRNA were more frequent in cancers with wt PTEN, PIK3R1 and KRAS genes (P = 0.038). INPP4B promoter wasn’t methylated in any of 125 ovarian carcinomas. Conclusion* A part of ovarian cancers have a reduced INPP4B copy number and mRNA expression. This may be an alternative pathway of PI3K activation in these tumors. Copy number loss is more common in cancers with aggressive tumor phenotype. This study was supported by the grant no. 2016/23/B/NZ5/00572 of the National Science Centre, Poland

Background: Accumulating evidence suggests a relationship between endometrial cancer and ovarian cancer. Independent genome-wide association studies (GWAS) for endometrial cancer and ovarian cancer have identified 16 and 27 risk regions, respectively, four of which overlap between the two cancers. We aimed to identify joint endometrial and ovarian cancer risk loci by performing a meta-analysis of GWAS summary statistics from these two cancers. Methods: Using LDScore regression, we explored the genetic correlation between endometrial cancer and ovarian cancer. To identify loci associated with the risk of both cancers, we implemented a pipeline of statistical genetic analyses (i.e., inverse-variance meta-analysis, colocalization, and M-values) and performed analyses stratified by subtype. Candidate target genes were then prioritized using functional genomic data. Results: Genetic correlation analysis revealed significant genetic correlation between the two cancers (rG = 0.43, P = 2.66 × 10−5). We found seven loci associated with risk for both cancers (PBonferroni < 2.4 × 10−9). In addition, four novel subgenome-wide regions at 7p22.2, 7q22.1, 9p12, and 11q13.3 were identified (P < 5 × 10−7). Promoter-associated HiChIP chromatin loops from immortalized endometrium and ovarian cell lines and expression quantitative trait loci data highlighted candidate target genes for further investigation. Conclusions: Using cross-cancer GWAS meta-analysis, we have identified several joint endometrial and ovarian cancer risk loci and candidate target genes for future functional analysis. Impact: Our research highlights the shared genetic relationship between endometrial cancer and ovarian cancer. Further studies in larger sample sets are required to confirm our findings.

Accumulating evidence suggests a relationship between endometrial cancer and epithelial ovarian cancer. For example, endometrial cancer and epithelial ovarian cancer share epidemiological risk factors and molecular features observed across histotypes are held in common (e.g. serous, endometrioid and clear cell). Independent genome-wide association studies (GWAS) for endometrial cancer and epithelial ovarian cancer have identified 16 and 27 risk regions, respectively, four of which overlap between the two cancers. Using GWAS summary statistics, we explored the shared genetic etiology between endometrial cancer and epithelial ovarian cancer. Genetic correlation analysis using LD Score regression revealed significant genetic correlation between the two cancers (rG = 0.43, P = 2.66 × 10−5). To identify loci associated with the risk of both cancers, we implemented a pipeline of statistical genetic analyses (i.e. inverse-variance meta-analysis, co-localization, and M-values), and performed analyses by stratified by subtype. We found seven loci associated with risk for both cancers (PBonferroni < 2.4 × 10−9). In addition, four novel regions at 7p22.2, 7q22.1, 9p12 and 11q13.3 were identified at a sub-genome wide threshold (P < 5 × 10−7). Integration with promoter-associated HiChIP chromatin loops from immortalized endometrium and epithelial ovarian cell lines, and expression quantitative trait loci (eQTL) data highlighted candidate target genes for further investigation.

BackgroundBRCA1 methylation has been associated with homologous recombination deficiency, a biomarker of platinum sensitivity. Studies evaluating BRCA1-methylated tubal and ovarian cancer (OC) do not consistently support improved survival following platinum chemotherapy. We examine the characteristics of BRCA1-methylated OC in a meta-analysis of individual participant data.MethodsData of 2636 participants across 15 studies were analyzed. BRCA1-methylated tumors were defined according to their original study. Associations between BRCA1 methylation and clinicopathological characteristics were evaluated. The effects of methylation on overall survival (OS) and progression-free survival (PFS) were examined using mixed-effects models. All statistical tests were 2-sided.Results430 (16.3%) tumors were BRCA1-methylated. BRCA1 methylation was associated with younger age and advanced-stage, high-grade serous OC. There were no survival differences between BRCA1-methylated and non–BRCA1-methylated OC (median PFS = 20.0 vs 18.5 months, hazard ratio [HR] = 1.01, 95% CI = 0.87 to 1.16; P = .98; median OS = 46.6 vs 48.0 months, HR = 1.02, 95% CI = 0.87 to 1.18; P = .96). Where BRCA1/2 mutations were evaluated (n = 1248), BRCA1 methylation displayed no survival advantage over BRCA1/2-intact (BRCA1/2 wild-type non–BRCA1-methylated) OC. Studies used different methods to define BRCA1 methylation. Where BRCA1 methylation was determined using methylation-specific polymerase chain reaction and gel electrophoresis (n = 834), it was associated with improved survival (PFS: HR = 0.80, 95% CI = 0.66 to 0.97; P = .02; OS: HR = 0.80, 95% CI = 0.63 to 1.00; P = .05) on mixed-effects modeling.ConclusionBRCA1-methylated OC displays similar clinicopathological features to BRCA1-mutated OC but is not associated with survival. Heterogeneity within BRCA1 methylation assays influences associations. Refining these assays may better identify cases with silenced BRCA1 function and improved patient outcomes.

Objective TP53 mutation is the most frequent molecular event in BRCA1 -associated ovarian carcinomas. TP53 status may be a confounding factor in the evaluation of clinical importance of other proteins. We aimed to evaluate the clinical significance of BRCA1 mutations with respect to the TP53 accumulation status in 159 high-grade ovarian carcinomas. Methods Statistical analyses were done with the Kaplan-Meier method, log-rank test, the Cox's and logistic regression models for all patients, and in subgroups with and without TP53 accumulation (TP53+ and TP53−, respectively). Results Forty of 159 ovarian carcinomas (25.2%) were diagnosed in patients with BRCA1 germline mutations; 102 tumors (64.2%) were TP53+ and 57 (37.8%) were TP53−. Among patients with TP53+ carcinomas, BRCA1 carriers had increased odds of recurrence compared with sporadic cases (HR 2.25, P =0.003; median disease-free survival time 7.7 vs. 18.4months, respectively). In the smaller TP53− subgroup, BRCA1 mutation reduced the risk of death by 46% (HR 0.54, P =0.099, median overall survival time 42.7 vs. 28.1months), but beyond the border of significance. When the TP53 status was not taken into account, BRCA1 mutations did not show any significance, however, there was a trend toward increased odds of complete remission for women with BRCA1 mutations compared to non-carriers (OR 2.47, P =0.064). Taxane-platinum therapy showed advantage over the platinum-cyclophosphamide one in the entire group of patients and in the TP53+ subgroup. Conclusions Our results suggest that the TP53 accumulation status determines the prognosis of BRCA1 mutation carriers with high-grade ovarian carcinomas.

Introduction/Background The phosphoinositide 3-kinase (PI3K) signaling cascade is one of the most frequently deregulated cascades in human cancers. The possibility of its inhibiting makes the PI3K pathway an attractive target for personalized therapy. We aimed to determine mRNA expression of PIK3R1, a gene encoding the p85α regulatory subunit of PI3K. Methodology PIK3R1 mRNA expression analysis was carried out for 144 ovarian cancers and 5 specimens of noncancerous fallopian tube as a control group. Expression was evaluated using the quantitative PCR (qPCR) method and TaqMan Gene Expression Assays. Statistical analyses were done with the Mann-Whitney U test, the Cox’s and logistic regression models. Results PIK3R1 mRNA expression was significantly decreased in ovarian cancers compared with control tissues (P=0.0028). The difference in mean expressions between cancers and normal tissues was 70% (0.1167±0.34 and 0.3981±0.13 in ovarian cancers and a control group, respectively). Reduced expression may be a consequence of allele loss of the PIK3R1 gene, because there is an association between mRNA level and copy number alteration of the gene (P=0.0001). Lower level of PIK3R1 mRNA was observed in non-serous (P=0.0437), poorly or mostly undifferentiated ovarian cancers (P=0.0114) diagnosed in patients under 54 years of age (P=0.077). Reduced expression of PIK3R1 was associated with the presence of TP53 mutation (P=0.0106) but not with PTEN loss or PIK3CA amplification. Patients overall survival, disease-free survival, complete remission and platinum sensitivity were not associated with PIK3R1 mRNA expression level. Conclusion Ovarian cancers have decreased PIK3R1 expression at mRNA level and it may be a consequence of the PIK3R1 gene copy loss. Grant Support This study was supported by the grant no. 2016/23/B/NZ5/00572 of the National Science Centre, Poland. Disclosure Nothing to disclose.

// Bozena Konopka 1, * , Lukasz Michal Szafron 1, * , Ewa Kwiatkowska 2 , Agnieszka Podgorska 1 , Aleksandra Zolocinska 3 , Barbara Pienkowska-Grela 1 , Agnieszka Dansonka-Mieszkowska 1 , Anna Balcerak 3 , Martyna Lukasik 1 , Anna Stachurska 1, 4 , Agnieszka Timorek 5 , Beata Spiewankiewicz 6 , Mona El-Bahrawy 7 , Jolanta Kupryjanczyk 1 1 Department of Pathology and Laboratory Diagnostics, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland 2 Department of Genetics, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland 3 Department of Molecular and Translational Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland 4 Department of Applied Pharmacy and Bioengineering, Medical University of Warsaw, Warsaw, Poland 5 Department of Obstetrics, Gynecology and Oncology, 2nd Faculty of Medicine, Medical University of Warsaw and Brodnowski Hospital, Warsaw, Poland 6 Department of Gynecologic Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland 7 Department of Histopathology, Imperial College London, UK * These authors have contributed equally to this study Correspondence to: Lukasz Michal Szafron, email: lukszafron@gmail.com Keywords: ovarian cancer, CEBPA, synonymous polymorphism, gene expression, DNA-damaging chemotherapy Received: May 18, 2016 Accepted: August 25, 2016 Published: September 02, 2016 ABSTRACT The CEBPA gene is known to be mutated or abnormally expressed in several cancers. This is the first study assessing the clinical impact of CEBPA gene status and expression on the ovarian cancer outcome. The CEBPA gene sequence was analyzed in 118 ovarian cancer patients (44 platinum/cyclophosphamide (PC)-treated and 74 taxane/platinum (TP)-treated), both in tumors and blood samples, and in blood from 236 healthy women, using PCR-Sanger sequencing and Real-Time quantitative PCR (qPCR)-based genotyping methods, respectively. The CEBPA mRNA level was examined with Reverse Transcription quantitative PCR (RT-qPCR). The results were correlated to different clinicopathological parameters. Thirty of 118 (25.4%) tumors harbored the CEBPA synonymous c.690G>T polymorphism (rs34529039), that we showed to be related to up-regulation of CEBPA mRNA levels (p=0.0059). The presence of the polymorphism was significantly associated with poor prognosis (p=0.005) and poor response to the PC chemotherapy regimen (p=0.024). In accordance, elevated CEBPA mRNA levels negatively affected patient survival (p T, p.(Thr230Thr) (rs34529039) polymorphism of the CEBPA gene, together with up-regulation of its mRNA expression, are negative factors worsening ovarian cancer outcome. Their adverse clinical effect depends on a therapeutic regimen used, which might make them potential prognostic and predictive biomarkers for response to DNA-damaging chemotherapy.

Thymidylate synthase (TYMS) is a crucial enzyme for DNA synthesis. TYMS expression is regulated by its antisense mRNA, ENOSF1. Disrupted regulation may promote uncontrolled DNA synthesis and tumor growth. We sought to replicate our previously reported association between rs495139 in the TYMS-ENOSF1 3′ gene region and increased risk of mucinous ovarian carcinoma (MOC) in an independent sample. Genotypes from 24,351 controls to 15,000 women with invasive OC, including 665 MOC, were available. We estimated per-allele odds ratios (OR) and 95% confidence intervals (CI) using unconditional logistic regression, and meta-analysis when combining these data with our previous report. The association between rs495139 and MOC was not significant in the independent sample (OR = 1.09, 95% CI = 0.97–1.22, p = 0.15, N = 665 cases). Meta-analysis suggested a weak association (OR = 1.13, 95% CI = 1.03–1.24, p = 0.01, N = 1019 cases). No significant association with risk of other OC histologic types was observed (p = 0.05 for tumor heterogeneity). In expression quantitative trait locus (eQTL) analysis, the rs495139 allele was positively associated with ENOSF1 mRNA expression in normal tissues of the gastrointestinal system, particularly esophageal mucosa (r = 0.51, p = 1.7 × 10−28), and nonsignificantly in five MOC tumors. The association results, along with inconclusive tumor eQTL findings, suggest that a true effect of rs495139 might be small.

// Lukasz Michal Szafron 1, * , Anna Balcerak 2, * , Ewa Anna Grzybowska 2 , Barbara Pienkowska-Grela 1 , Agnieszka Podgorska 1 , Renata Zub 2 , Magdalena Olbryt 3 , Jolanta Pamula-Pilat 3 , Katarzyna M. Lisowska 3 , Ewa Grzybowska 3 , Tymon Rubel 4 , Agnieszka Dansonka-Mieszkowska 1 , Bozena Konopka 1 , Magdalena Kulesza 1 , Martyna Lukasik 1 , Jolanta Kupryjanczyk 1, * 1 Department of Pathology and Laboratory Diagnostics, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland 2 Department of Molecular and Translational Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland 3 Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland 4 Institute of Radioelectronics and Multimedia Technology, Warsaw University of Technology, Warsaw, Poland * These authors have contributed equally to this work Correspondence to: Lukasz Michal Szafron, e-mail: lukszafron@gmail.com Keywords: ovarian cancer, prognostic factor, CRNDE, gene expression, TP53 Received: June 18, 2015 Accepted: October 06, 2015 Published: November 04, 2015 ABSTRACT The CRNDE gene seems to play an oncogenic role in cancers, though its exact function remains unknown. Here, we tried to assess its usefulness as a molecular prognostic marker in ovarian cancer. Based on results of our microarray studies, CRNDE transcripts were further analyzed by Real-Time qPCR-based profiling of their expression. The qPCR study was conducted with the use of personally designed TaqMan assays on 135 frozen tissue sections of ovarian carcinomas from patients treated with platinum compounds and either cyclophosphamide (PC, N = 32) or taxanes (TP, N = 103). Elevated levels of two different CRNDE transcripts were a negative prognostic factor; they increased the risk of death and recurrence in the group of patients treated with TP, but not PC (DNA-damaging agents only). Higher associations were found for overexpression of the short CRNDE splice variant (FJ466686): HR 6.072, 95% CI 1.814–20.32, p = 0.003 (the risk of death); HR 15.53, 95% CI 3.812–63.28, p < 0.001 (the risk of recurrence). Additionally, accumulation of the TP53 protein correlated with decreased expression of both CRNDE transcripts in tumor cells. Our results depict CRNDE as a potential marker of poor prognosis in women with ovarian carcinomas, and suggest that its significance depends on the therapeutic regimen used.

Ovarian tumors from two patients, compatible by histological and immunohistochemical criteria with small cell carcinoma of hypercalcemic type (SCCHT) (WT1+, EMA dispersed+, synaptophysin+ or dispersed+), were extensively sampled in order to find clues to their histogenesis. Subsequently, small foci of immature teratoma were found in both of them (in 1/122 and in 3/80 tumor sections). In one case, microfoci of yolk sac tumor were also present within the teratoma area as well as in the background of the small cell tumor population - in the primary tumor and in omental metastasis. We found a resemblance of the microscopic patterns of SCCHT and atypical teratoid/rhabdoid tumor (AT/RT) of the central nervous system, and this prompted us to evaluate INI-1 and SMARCA4 immunohistochemical expression, because their alternative loss is regarded as a molecular hallmark of AT/RT. INI-1 expression was retained, while that of SMARCA4 was lost. We therefore analyzed tumor DNA by PCR amplification and sequencing for mutations in the SMARCA4 gene (NG_011556.1), which were identified in both tumors (c.2184_2206del; nonsense c.3277C>T - both in one tumor; nonsense c.3760G>T in another tumor). These data suggest that SCCHT is most likely an embryonal tumor originating from immature teratoma and related to malignant rhabdoid tumor. Further analyses are necessary to determine whether the tumors diagnosed as SCCHT constitute a homogeneous group or represent more than one entity.

Background SMARCA4 mutations have recently been identified as driving lesions of the ovarian small cell carcinoma of hypercalcemic type (SCCHT). Familial occurrence of this neoplasm was described previously. Methods We looked for germline SMARCA4 alterations in eight patients with the SCCHT. DNA was extracted from probands’ and their relatives’ blood. The SMARCA4 coding sequence, previously found altered in all the tumors, was PCR amplified and sequenced in the germline DNA. Results Two patients carried a heterozygous germline SMARCA4 alteration: c.3760G > T and c.2352insG, respectively. The analysis of the probands’ next of kins revealed that the c.3760G > T mutation was inherited by the proband and her sister from their father, and the sisters’ four children also carried the mutation. The proband’s sister was diagnosed with a carcinoma of the parotid gland at age 2. A brother of the other proband was tested negative. Conclusions Our study suggests that some women develop the ovarian SCCHT due to the inherited or possibly de novo-occurring germline alterations in the SMARCA4 gene, however, its penetrance appears limited. Nevertheless, because of high aggressiveness of the SCCHT, a molecular diagnostics of the SMARCA4 gene and careful follow-up should be offered to patients with this cancer and their families.

p19(INK4d) (CDKN2D) is a negative regulator of the cell cycle. Little is known of its role in cancer development and prognosis. We aimed to evaluate the clinical significance of p19(INK4d) expression in ovarian carcinomas with respect to the TP53 accumulation status, as well as the frequency of CDKN2D mutations. p19(INK4d) and TP53 expression was evaluated immunohistochemically in 445 ovarian carcinomas: 246 patients were treated with platinum-cyclophosphamide (PC/PAC), while 199 were treated with taxane-platinum agents (TP). CDKN2D gene expression (mRNA) was examined in 106 carcinomas, while CDKN2D mutations in 68 tumors. Uni- and multivariate statistical analyses (logistic regression and the Cox proportional hazards model) were performed for patient groups divided according to the chemotherapeutic regimen administered, and in subgroups with and without TP53 accumulation. High p19(INK4d) expression increased the risk of death, but only in patients with the TP53-negative carcinomas (HR 1.61, P = 0.049 for PC/PAC-treated patients, HR 2.00, P = 0.015 for TP-treated patients). This result was confirmed by the mRNA analysis (HR 4.24, P = 0.001 for TP-treated group). High p19(INK4d) protein expression associated with adverse clinicopathological factors. We found no alterations in the CDKN2D gene; the c.90CG (p.R30R; rs1968445) polymorphism was detected in 10% of tumors. Our results suggest that p19(INK4d) expression is a poor prognostic factor in ovarian cancer patients. Analyses of tumor groups according to the TP53 accumulation status facilitate the identification of cancer biomarkers.

CRNDE, recently described as the lncRNA-coding gene, is overexpressed at RNA level in human malignancies. Its role in gametogenesis, cellular differentiation and pluripotency has been suggested as well. Herein, we aimed to verify our hypothesis that the CRNDE gene may encode a protein product, CRNDEP. By using bioinformatics methods, we identified the 84-amino acid ORF encoded by one of two CRNDE transcripts, previously described by our research team. This ORF was cloned into two expression vectors, subsequently utilized in localization studies in HeLa cells. We also developed a polyclonal antibody against CRNDEP. Its specificity was confirmed in immunohistochemical, cellular localization, Western blot and immunoprecipitation experiments, as well as by showing a statistically significant decrease of endogenous CRNDEP expression in the cells with transient shRNA-mediated knockdown of CRNDE. Endogenous CRNDEP localizes predominantly to the nucleus and its expression seems to be elevated in highly proliferating tissues, like the parabasal layer of the squamous epithelium, intestinal crypts or spermatocytes. After its artificial overexpression in HeLa cells, in a fusion with either the EGFP or DsRed Monomer fluorescent tag, CRNDEP seems to stimulate the formation of stress granules and localize to them. Although the exact role of CRNDEP is unknown, our preliminary results suggest that it may be involved in the regulation of the cell proliferation. Possibly, CRNDEP also participates in oxygen metabolism, considering our in silico results, and the correlation between its enforced overexpression and the formation of stress granules. This is the first report showing the existence of a peptide encoded by the CRNDE gene.