Enrichment of putative PAX8 target genes at serous epithelial ovarian cancer susceptibility loci.

BACKGROUND: Genome-wide association studies (GWAS) have identified 18 loci associated with serous ovarian cancer (SOC) susceptibility but the biological mechanisms driving these findings remain poorly characterised. Germline cancer risk loci may be enriched for target genes of transcription factors (TFs) critical to somatic tumorigenesis. METHODS: All 615 TF-target sets from the Molecular Signatures Database were evaluated using gene set enrichment analysis (GSEA) and three GWAS for SOC risk: discovery (2196 cases/4396 controls), replication (7035 cases/21 693 controls; independent from discovery), and combined (9627 cases/30 845 controls; including additional individuals). RESULTS: The PAX8-target gene set was ranked 1/615 in the discovery (PGSEA<0.001; FDR=0.21), 7/615 in the replication (PGSEA=0.004; FDR=0.37), and 1/615 in the combined (PGSEA<0.001; FDR=0.21) studies. Adding other genes reported to interact with PAX8 in the literature to the PAX8-target set and applying an alternative to GSEA, interval enrichment, further confirmed this association (P=0.006). Fifteen of the 157 genes from this expanded PAX8 pathway were near eight loci associated with SOC risk at P<10-5 (including six with P<5 × 10-8). The pathway was also associated with differential gene expression after shRNA-mediated silencing of PAX8 in HeyA8 (PGSEA=0.025) and IGROV1 (PGSEA=0.004) SOC cells and several PAX8 targets near SOC risk loci demonstrated in vitro transcriptomic perturbation. CONCLUSIONS: Putative PAX8 target genes are enriched for common SOC risk variants. This finding from our agnostic evaluation is of particular interest given that PAX8 is well-established as a specific marker for the cell of origin of SOC.

Epithelial-Mesenchymal Transition (EMT) Gene Variants and Epithelial Ovarian Cancer (EOC) Risk.

Epithelial-mesenchymal transition (EMT) is a process whereby epithelial cells assume mesenchymal characteristics to facilitate cancer metastasis. However, EMT also contributes to the initiation and development of primary tumors. Prior studies that explored the hypothesis that EMT gene variants contribute to epithelial ovarian carcinoma (EOC) risk have been based on small sample sizes and none have sought replication in an independent population. We screened 15,816 single-nucleotide polymorphisms (SNPs) in 296 genes in a discovery phase using data from a genome-wide association study of EOC among women of European ancestry (1,947 cases and 2,009 controls) and identified 793 variants in 278 EMT-related genes that were nominally (P < 0.05) associated with invasive EOC. These SNPs were then genotyped in a larger study of 14,525 invasive-cancer patients and 23,447 controls. A P-value <0.05 and a false discovery rate (FDR) <0.2 were considered statistically significant. In the larger dataset, GPC6/GPC5 rs17702471 was associated with the endometrioid subtype among Caucasians (odds ratio (OR) = 1.16, 95% CI = 1.07-1.25, P = 0.0003, FDR = 0.19), whereas F8 rs7053448 (OR = 1.69, 95% CI = 1.27-2.24, P = 0.0003, FDR = 0.12), F8 rs7058826 (OR = 1.69, 95% CI = 1.27-2.24, P = 0.0003, FDR = 0.12), and CAPN13 rs1983383 (OR = 0.79, 95% CI = 0.69-0.90, P = 0.0005, FDR = 0.12) were associated with combined invasive EOC among Asians. In silico functional analyses revealed that GPC6/GPC5 rs17702471 coincided with DNA regulatory elements. These results suggest that EMT gene variants do not appear to play a significant role in the susceptibility to EOC.

Evaluating the ovarian cancer gonadotropin hypothesis: a candidate gene study.

OBJECTIVE: Ovarian cancer is a hormone-related disease with a strong genetic basis. However, none of its high-penetrance susceptibility genes and GWAS-identified variants to date are known to be involved in hormonal pathways. Given the hypothesized etiologic role of gonadotropins, an assessment of how variability in genes involved in the gonadotropin signaling pathway impacts disease risk is warranted. METHODS: Genetic data from 41 ovarian cancer study sites were pooled and unconditional logistic regression was used to evaluate whether any of the 2185 SNPs from 11 gonadotropin signaling pathway genes was associated with ovarian cancer risk. A burden test using the admixture likelihood (AML) method was also used to evaluate gene-level associations. RESULTS: We did not find any genome-wide significant associations between individual SNPs and ovarian cancer risk. However, there was some suggestion of gene-level associations for four gonadotropin signaling pathway genes: INHBB (p=0.045, mucinous), LHCGR (p=0.046, high-grade serous), GNRH (p=0.041, high-grade serous), and FSHB (p=0.036, overall invasive). There was also suggestive evidence for INHA (p=0.060, overall invasive). CONCLUSIONS: Ovarian cancer studies have limited sample numbers, thus fewer genome-wide susceptibility alleles, with only modest associations, have been identified relative to breast and prostate cancers. We have evaluated the majority of ovarian cancer studies with biological samples, to our knowledge, leaving no opportunity for replication. Using both our understanding of biology and powerful gene-level tests, we have identified four putative ovarian cancer loci near INHBB, LHCGR, GNRH, and FSHB that warrant a second look if larger sample sizes and denser genotype chips become available.

Large-scale genome-wide association study of 398,238 women unveils seven novel loci associated with high-grade serous epithelial ovarian cancer risk.

Background: Nineteen genomic regions have been associated with high-grade serous ovarian cancer (HGSOC). We used data from the Ovarian Cancer Association Consortium (OCAC), Consortium of Investigators of Modifiers of BRCA1/BRCA2 (CIMBA), UK Biobank (UKBB), and FinnGen to identify novel HGSOC susceptibility loci and develop polygenic scores (PGS). Methods: We analyzed >22 million variants for 398,238 women. Associations were assessed separately by consortium and meta-analysed. OCAC and CIMBA data were used to develop PGS which were trained on FinnGen data and validated in UKBB and BioBank Japan. Results: Eight novel variants were associated with HGSOC risk. An interesting discovery biologically was finding that TP53 3'-UTR SNP rs78378222 was associated with HGSOC (per T allele relative risk (RR)=1.44, 95%CI:1.28-1.62, P=1.76×10-9). The optimal PGS included 64,518 variants and was associated with an odds ratio of 1.46 (95%CI:1.37-1.54) per standard deviation in the UKBB validation (AUROC curve=0.61, 95%CI:0.59-0.62). Conclusions: This study represents the largest GWAS for HGSOC to date. The results highlight that improvements in imputation reference panels and increased sample sizes can identify HGSOC associated variants that previously went undetected, resulting in improved PGS. The use of updated PGS in cancer risk prediction algorithms will then improve personalized risk prediction for HGSOC.

PROM1, CXCL8, RUNX1, NAV1 and TP73 genes as independent markers predictive of prognosis or response to treatment in two cohorts of high-grade serous ovarian cancer patients.

Considering the vast biological diversity and high mortality rate in high-grade ovarian cancers, identification of novel biomarkers, enabling precise diagnosis and effective, less aggravating treatment, is of paramount importance. Based on scientific literature data, we selected 80 cancer-related genes and evaluated their mRNA expression in 70 high-grade serous ovarian cancer (HGSOC) samples by Real-Time qPCR. The results were validated in an independent Northern American cohort of 85 HGSOC patients with publicly available NGS RNA-seq data. Detailed statistical analyses of our cohort with multivariate Cox and logistic regression models considering clinico-pathological data and different TP53 mutation statuses, revealed an altered expression of 49 genes to affect the prognosis and/or treatment response. Next, these genes were investigated in the validation cohort, to confirm the clinical significance of their expression alterations, and to identify genetic variants with an expected high or moderate impact on their products. The expression changes of five genes, PROM1, CXCL8, RUNX1, NAV1, TP73, were found to predict prognosis or response to treatment in both cohorts, depending on the TP53 mutation status. In addition, we revealed novel and confirmed known SNPs in these genes, and showed that SNPs in the PROM1 gene correlated with its elevated expression.

The utility of fluorescence in situ hybridization (FISH) in determining DNA damage-inducible transcript 3 (DDIT3) amplification in dedifferentiated liposarcomas - an important diagnostic pitfall.

BACKGROUND AND OBJECTIVE: Dedifferentiated liposarcoma (DDLPS) is characterized by non-lipogenic sarcoma fields coexisting with adipocyte-rich well-differentiated areas. Amplification of the 12q13-15 region includes the MDM2 and DDIT3 genes. MDM2 amplification is considered a genetic hallmark of DDLPS, while DDIT3 is typically rearranged in myxoid liposarcoma. Recent studies showed that DDIT3 amplification is associated with myxoid liposarcoma-like (LPS-like) morphology in DDLPS. Our study aimed to evaluate the status of MDM2 and DDIT3 by FISH in DDLPS and correlate it with MLPS-like features. MATERIAL AND METHODS: Six patients with MLPS-like morphology DDLPS were investigated pathologically, immunohistochemically, and genetically. The control groups of patients with classical DDLPS morphology and well-differentiated liposarcoma (WDLPS) were established and molecularly assessed as well. Fluorescence in situ hybridization (FISH) used in routine diagnostics was performed to determine the status of MDM2 and DDIT3 genes. RESULTS: The patient's mean age was 64 (range from 43 to 85 years) with a 5:4 male to female ratio. Tumors were localized retroperitoneally (15) and extra-retroperitoneally (3). All cases demonstrated amplification of the 12q15 region containing MDM2 gene and co-amplification of the 5' DDIT3 FISH Probe representing DDIT3 telomeric tag. However, we did not find the relation of myxoid LPS-like morphology with DDIT3 amplification as previously reported. CONCLUSIONS: The biopsy material from DDLPS with myxoid areas can be misclassified as myxoid liposarcoma. Indeed, according to the histological image, DDIT3 status may be evaluated first. In these cases, we show that the DDIT3 telomeric tag amplification assessed by FISH, is a common, nonspecific feature, which is also found in classical DDLPS and WDLPS. Therefore, we believe that co-amplification of DDIT3 and MDM2 may be considered a spectrum of the 12q13-15 region amplification due to the specification of FISH methodology.

A novel germline PALB2 deletion in Polish breast and ovarian cancer patients.

BACKGROUND: PALB2 protein was recently identified as a partner of BRCA1 and BRCA2 which determines their proper function in DNA repair. METHODS: Initially, the entire coding sequence of the PALB2 gene with exon/intron boundaries was evaluated by the PCR-SSCP and direct sequencing methods on 70 ovarian carcinomas. Sequence variants of interest were further studied on enlarged groups of ovarian carcinomas (total 339 non-consecutive ovarian carcinomas), blood samples from 334 consecutive sporadic and 648 consecutive familial breast cancer patients, and 1310 healthy controls from central Poland. RESULTS: Ten types of sequence variants were detected, and among them four novel polymorphisms: c.2996+58T>C in intron 9; c.505C>A (p.L169I), c.618T>G (p.L206L), both in exon 4; and c.2135C>T (A712V) in exon 5 of the PALB2 gene. Another two polymorphisms, c.212-58A>C and c.2014G>C (E672Q) were always detected together, both in cancer (7.5% of patients) and control samples (4.9% of controls, p = 0.2). A novel germline truncating mutation, c.509_510delGA (p.R170fs) was found in exon 4: in 2 of 339 (0.6%) unrelated ovarian cancer patients, in 4 of 648 (0.6%) unrelated familial breast cancer patients, and in 1 of 1310 controls (0.08%, p = 0.1, p = 0.044, respectively). One ovarian cancer patient with the PALB2 mutation had also a germline nonsense mutation of the BRCA2 gene. CONCLUSIONS: The c.509_510delGA is a novel PALB2 mutation that increases the risk of familial breast cancer. Occurrence of the same PALB2 alteration in seven unrelated women suggests that c.509_510delGA (p.R170fs) is a recurrent mutation for Polish population.

Clinical importance of FANCD2, BRIP1, BRCA1, BRCA2 and FANCF expression in ovarian carcinomas.

OBJECTIVE: DNA repair pathways are potential targets of molecular therapy in cancer patients. The FANCD2, BRIP1, BRCA1/2, and FANCF genes are involved in homologous recombination DNA repair, which implicates their possible role in cell response to DNA-damaging agents. We evaluated a clinical significance of pre-treatment expression of these genes at mRNA level in 99 primary, advanced-stage ovarian carcinomas from patients, who later received taxane-platinum (TP) or platinum-cyclophosphamide (PC) treatment. METHODS: Gene expression was determined with the use of Real-Time PCR. The BRCA2 and BRIP1 gene sequence was investigated with the use of SSCP, dHPLC, and PCR-sequencing. RESULTS: Increased FANCD2 expression occurred to be a negative prognostic factor for all patients (PC+TP:HR 3.85, p = 0.0003 for the risk of recurrence; HR 1.96, p = 0.02 for the risk of death), and this association was even stronger in the TP-treated group (HR 6.7, p = 0.0002 and HR 2.33, p = 0.01, respectively). Elevated BRIP1 expression was the only unfavorable molecular factor in the PC-treated patients (HR 8.37, p = 0.02 for the risk of recurrence). Additionally, an increased FANCD2 and BRCA1/2 expression levels were associated with poor ovarian cancer outcome in either TP53-positive or -negative subgroups of the TP-treated patients, however these groups were small. Sequence analysis identified one protein truncating variant (1/99) in BRCA2 and no mutations (0/56) in BRIP1. CONCLUSIONS: Our study shows for the first time that FANCD2 overexpression is a strong negative prognostic factor in ovarian cancer, particularly in patients treated with TP regimen. Moreover, increased mRNA level of the BRIP1 is a negative prognostic factor in the PC-treated patients. Next, changes in the BRCA2 and BRIP1 genes are rare and together with other analyzed FA genes considered as homologous recombination deficiency may not affect the expression level of analyzed genes.

Loss of heterozygosity, microsatellite instability and TP53 gene status in ovarian carcinomas.

BACKGROUND: Microsatellite instability (MSI) and loss of heterozygosity (LOH) are frequent events in ovarian carcinogenesis; however, little is known as to their clinical significance and association with other molecular lesions. MATERIALS AND METHODS: Twelve microsatellite markers for MSI and LOH analysis were used in 64 ovarian carcinomas with known TP53 mutational status. The clinical importance of molecular alterations was evaluated in a uniform subgroup of patients treated with platinum-based regimens. RESULTS: LOH was detected in order of frequency at 17p13.3 (D17S926, 79%), 17p13.1 (TP53 locus, 69%), 13q14 (RB, 60%), 3p21 (D3S1611, 32.5%), 8q21 (D8S1811, 22%), 11p14/13 (D11S904, 19%), 10qter (D10S197, 13%) and 2p16-21 (D2S123, 11%). LOH at the RB1 locus showed association with LOH at the TP53 locus (p = 0.01). Platinum sensitivity was associated with heterozygosity at the TP53 locus (p = 0.006). Only one tumor displayed microsatellite instability in one marker (RB) only. CONCLUSION: Our results suggest that LOH at the 17p D17S926 locus in ovarian cancer is an earlier molecular event than LOH at the TP53 locus. Inactivation of TP53 and RB1 genes may have a synergistic effect in ovarian tumorigenesis.

Clinical importance of the EMSY gene expression and polymorphisms in ovarian cancer.

EMSY, a BRCA2-associated protein, is amplified and overexpressed in various sporadic cancers. This is the first study assessing the clinical impact of its expression and polymorphisms on ovarian cancer (OvCa) outcome in the context of the chemotherapy regimen used. In 134 frozen OvCa samples, we assessed EMSY mRNA expression with Reverse Transcription-quantitative PCR, and also investigated the EMSY gene sequence using SSCP and/or PCR-sequencing. Clinical relevance of changes in EMSY mRNA expression and DNA sequence was evaluated in two subgroups treated with either taxane/platinum (TP, n=102) or platinum/cyclophosphamide (PC, n=32). High EMSY expression negatively affected overall survival (OS), disease-free survival (DFS) and sensitivity to treatment (PS) in the TP-treated subgroup (p-values: 0.001, 0.002 and 0.010, respectively). Accordingly, our OvCa cell line studies showed that the EMSY gene knockdown sensitized A2780 and IGROV1 cells to paclitaxel. Interestingly, EMSY mRNA expression in surviving cells was similar as in the control cells. Additionally, we identified 24 sequence alterations in the EMSY gene, including the previously undescribed: c.720G>C, p.(Lys240Asn); c.1860G>A, p.(Lys620Lys); c.246-76A>G; c.421+68A>C. In the PC-treated subgroup, a heterozygous genotype comprising five SNPs (rs4300410, rs3814711, rs4245443, rs2508740, rs2513523) negatively correlated with OS (p-value=0.009). The same SNPs exhibited adverse borderline associations with PS in the TP-treated subgroup. This is the first study providing evidence that high EMSY mRNA expression is a negative prognostic and predictive factor in OvCa patients treated with TP, and that the clinical outcome may hinge on certain SNPs in the EMSY gene as well.

Variants in genes encoding small GTPases and association with epithelial ovarian cancer susceptibility.

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer mortality in American women. Normal ovarian physiology is intricately connected to small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran) which govern processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. We hypothesized that common germline variation in genes encoding small GTPases is associated with EOC risk. We investigated 322 variants in 88 small GTPase genes in germline DNA of 18,736 EOC patients and 26,138 controls of European ancestry using a custom genotype array and logistic regression fitting log-additive models. Functional annotation was used to identify biofeatures and expression quantitative trait loci that intersect with risk variants. One variant, ARHGEF10L (Rho guanine nucleotide exchange factor 10 like) rs2256787, was associated with increased endometrioid EOC risk (OR = 1.33, p = 4.46 x 10-6). Other variants of interest included another in ARHGEF10L, rs10788679, which was associated with invasive serous EOC risk (OR = 1.07, p = 0.00026) and two variants in AKAP6 (A-kinase anchoring protein 6) which were associated with risk of invasive EOC (rs1955513, OR = 0.90, p = 0.00033; rs927062, OR = 0.94, p = 0.00059). Functional annotation revealed that the two ARHGEF10L variants were located in super-enhancer regions and that AKAP6 rs927062 was associated with expression of GTPase gene ARHGAP5 (Rho GTPase activating protein 5). Inherited variants in ARHGEF10L and AKAP6, with potential transcriptional regulatory function and association with EOC risk, warrant investigation in independent EOC study populations.

No Evidence That Genetic Variation in the Myeloid-Derived Suppressor Cell Pathway Influences Ovarian Cancer Survival.

Background: The precise mechanism by which the immune system is adversely affected in cancer patients remains poorly understood, but the accumulation of immunosuppressive/protumorigenic myeloid-derived suppressor cells (MDSCs) is thought to be a prominent mechanism contributing to immunologic tolerance of malignant cells in epithelial ovarian cancer (EOC). To this end, we hypothesized genetic variation in MDSC pathway genes would be associated with survival after EOC diagnoses.Methods: We measured the hazard of death due to EOC within 10 years of diagnosis, overall and by invasive subtype, attributable to SNPs in 24 genes relevant in the MDSC pathway in 10,751 women diagnosed with invasive EOC. Versatile Gene-based Association Study and the admixture likelihood method were used to test gene and pathway associations with survival.Results: We did not identify individual SNPs that were significantly associated with survival after correction for multiple testing (P < 3.5 × 10-5), nor did we identify significant associations between the MDSC pathway overall, or the 24 individual genes and EOC survival.Conclusions: In this well-powered analysis, we observed no evidence that inherited variations in MDSC-associated SNPs, individual genes, or the collective genetic pathway contributed to EOC survival outcomes.Impact: Common inherited variation in genes relevant to MDSCs was not associated with survival in women diagnosed with invasive EOC. Cancer Epidemiol Biomarkers Prev; 26(3); 420-4. ©2016 AACR.

Unsupervised analysis reveals two molecular subgroups of serous ovarian cancer with distinct gene expression profiles and survival.

PURPOSE: Ovarian cancer is typically diagnosed at late stages, and thus, patients' prognosis is poor. Improvement in treatment outcomes depends, at least partly, on better understanding of ovarian cancer biology and finding new molecular markers and therapeutic targets. METHODS: An unsupervised method of data analysis, singular value decomposition, was applied to analyze microarray data from 101 ovarian cancer samples; then, selected genes were validated by quantitative PCR. RESULTS: We found that the major factor influencing gene expression in ovarian cancer was tumor histological type. The next major source of variability was traced to a set of genes mainly associated with extracellular matrix, cell motility, adhesion, and immunological response. Hierarchical clustering based on the expression of these genes revealed two clusters of ovarian cancers with different molecular profiles and distinct overall survival (OS). Patients with higher expression of these genes had shorter OS than those with lower expression. The two clusters did not derive from high- versus low-grade serous carcinomas and were unrelated to histological (ovarian vs. fallopian) origin. Interestingly, there was considerable overlap between identified prognostic signature and a recently described invasion-associated signature related to stromal desmoplastic reaction. Several genes from this signature were validated by quantitative PCR; two of them-DSPG3 and LOX-were validated both in the initial and independent sets of samples and were significantly associated with OS and disease-free survival. CONCLUSIONS: We distinguished two molecular subgroups of serous ovarian cancers characterized by distinct OS. Among differentially expressed genes, some may potentially be used as prognostic markers. In our opinion, unsupervised methods of microarray data analysis are more effective than supervised methods in identifying intrinsic, biologically sound sources of variability. Moreover, as histological type of the tumor is the greatest source of variability in ovarian cancer and may interfere with analyses of other features, it seems reasonable to use histologically homogeneous groups of tumors in microarray experiments.

The significance of c.690G>T polymorphism (rs34529039) and expression of the CEBPA gene in ovarian cancer outcome.

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<0.001) and tumor response to the PC therapy (p=0.014). The rs34529039 SNP did not affect the risk of developing ovarian cancer. This is the first study providing evidence that the c.690G>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.

Genome-Wide Meta-Analyses of Breast, Ovarian, and Prostate Cancer Association Studies Identify Multiple New Susceptibility Loci Shared by at Least Two Cancer Types.

UNLABELLED: Breast, ovarian, and prostate cancers are hormone-related and may have a shared genetic basis, but this has not been investigated systematically by genome-wide association (GWA) studies. Meta-analyses combining the largest GWA meta-analysis data sets for these cancers totaling 112,349 cases and 116,421 controls of European ancestry, all together and in pairs, identified at P < 10(-8) seven new cross-cancer loci: three associated with susceptibility to all three cancers (rs17041869/2q13/BCL2L11; rs7937840/11q12/INCENP; rs1469713/19p13/GATAD2A), two breast and ovarian cancer risk loci (rs200182588/9q31/SMC2; rs8037137/15q26/RCCD1), and two breast and prostate cancer risk loci (rs5013329/1p34/NSUN4; rs9375701/6q23/L3MBTL3). Index variants in five additional regions previously associated with only one cancer also showed clear association with a second cancer type. Cell-type-specific expression quantitative trait locus and enhancer-gene interaction annotations suggested target genes with potential cross-cancer roles at the new loci. Pathway analysis revealed significant enrichment of death receptor signaling genes near loci with P < 10(-5) in the three-cancer meta-analysis. SIGNIFICANCE: We demonstrate that combining large-scale GWA meta-analysis findings across cancer types can identify completely new risk loci common to breast, ovarian, and prostate cancers. We show that the identification of such cross-cancer risk loci has the potential to shed new light on the shared biology underlying these hormone-related cancers. Cancer Discov; 6(9); 1052-67. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 932.

Assessment of variation in immunosuppressive pathway genes reveals TGFBR2 to be associated with risk of clear cell ovarian cancer.

BACKGROUND: Regulatory T (Treg) cells, a subset of CD4+ T lymphocytes, are mediators of immunosuppression in cancer, and, thus, variants in genes encoding Treg cell immune molecules could be associated with ovarian cancer. METHODS: In a population of 15,596 epithelial ovarian cancer (EOC) cases and 23,236 controls, we measured genetic associations of 1,351 SNPs in Treg cell pathway genes with odds of ovarian cancer and tested pathway and gene-level associations, overall and by histotype, for the 25 genes, using the admixture likelihood (AML) method. The most significant single SNP associations were tested for correlation with expression levels in 44 ovarian cancer patients. RESULTS: The most significant global associations for all genes in the pathway were seen in endometrioid ( p = 0.082) and clear cell ( p = 0.083), with the most significant gene level association seen with TGFBR2 ( p = 0.001) and clear cell EOC. Gene associations with histotypes at p < 0.05 included: IL12 ( p = 0.005 and p = 0.008, serous and high-grade serous, respectively), IL8RA ( p = 0.035, endometrioid and mucinous), LGALS1 ( p = 0.03, mucinous), STAT5B ( p = 0.022, clear cell), TGFBR1 ( p = 0.021 endometrioid) and TGFBR2 ( p = 0.017 and p = 0.025, endometrioid and mucinous, respectively). CONCLUSIONS: Common inherited gene variation in Treg cell pathways shows some evidence of germline genetic contribution to odds of EOC that varies by histologic subtype and may be associated with mRNA expression of immune-complex receptor in EOC patients.

miR-7 expression in serous ovarian carcinomas.

BACKGROUND/AIM: miR-7 has recently been linked to cancer. Some miR-7 targets, including B-cell lymphoma 2 (BCL2) and epidermal growth factor receptor (EGFR), are involved in ovarian cancer (OC) pathogenesis. The majority of OCs display TP53 mutations, which are critically important for OC development. We aimed to study the expression level of miR-7 and of two of its postulated target genes, BCL2 and EGFR, in serous ovarian carcinomas of different TP53 status and tumour grade. MATERIALS AND METHODS: Gene and miR expression was assessed by real-time reverse transcription polymerase chain reaction in 45 clinical samples of low- (G1+G2) and high- (G3) grade primary serous OC with wild-type (wt) or mutated TP53, as well as in three OC cell lines, each representing a different TP53 status. The results obtained in patients with OC were analysed against their disease-free survival (DFS). RESULTS: In high-grade OC with TP53 mutations, the level of miR-7 expression significantly exceeded (by several fold) that in wtTP53 cancer (p<0.01). Within the wtTP53 tumour series, the level of miR-7 expression was significantly higher (by over 10-fold) in high-grade than in low-grade OC (p<0.01). miR-7 expression was not found to influence DFS. The differences in miR-7 expression depending on TP53 status found in clinical OC samples were not observed in OC cell lines. miR-7 overexpression correlated with diminished BCL2 expression, but there was no relationship between miR-7 and EGFR expression, neither in tumour samples nor in the cell lines. CONCLUSION: There is a link between miR-7 expression and TP53 status and tumour grade in serous OC. Molecular mechanisms of these relationships need to be elucidated. Of the two postulated miR-7 target genes we examined, BCL2, but not EGFR, seems to be a possible miR-7 target in OC.

Germline SMARCA4 mutations in patients with ovarian small cell carcinoma of hypercalcemic type.

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.

The putative oncogene, CRNDE, is a negative prognostic factor in ovarian cancer patients.

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.

The Novel Gene CRNDE Encodes a Nuclear Peptide (CRNDEP) Which Is Overexpressed in Highly Proliferating Tissues.

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.