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Contains fulltext : 108021.pdf (Publisher’s version ) (Open Access) Recent Genome-Wide Association Studies (GWAS) have identified four low-penetrance ovarian cancer susceptibility loci. We hypothesized that further moderate- or low-penetrance variants exist among the subset of single-nucleotide polymorphisms (SNPs) not well tagged by the genotyping arrays used in the previous studies, which would account for some of the remaining risk. We therefore conducted a time- and cost-effective stage 1 GWAS on 342 invasive serous cases and 643 controls genotyped on pooled DNA using the high-density Illumina 1M-Duo array. We followed up 20 of the most significantly associated SNPs, which are not well tagged by the lower density arrays used by the published GWAS, and genotyping them on individual DNA. Most of the top 20 SNPs were clearly validated by individually genotyping the samples used in the pools. However, none of the 20 SNPs replicated when tested for association in a much larger stage 2 set of 4,651 cases and 6,966 controls from the Ovarian Cancer Association Consortium. Given that most of the top 20 SNPs from pooling were validated in the same samples by individual genotyping, the lack of replication is likely to be due to the relatively small sample size in our stage 1 GWAS rather than due to problems with the pooling approach. We conclude that there are unlikely to be any moderate or large effects on ovarian cancer risk untagged by less dense arrays. However, our study lacked power to make clear statements on the existence of hitherto untagged small-effect variants. 01 oktober 2012

Objectives: To assess the prevalence and predictors of depression and anxiety in women with ovarian cancer and their caregivers, to compare levels of depression and anxiety with community norms, and to explore the relationship between patients and their nominated caregivers. Design, setting and participants: Prospective cohort study of 798 women with invasive ovarian cancer recruited between 1 January 2002 and 30 June 2006 through the nationwide Australian Ovarian Cancer Study, and 373 of their caregivers. Main outcome measures: Depression and anxiety as assessed with the Hospital Anxiety and Depression Scale, and the role of demographic variables, disease and treatment variables, psychosocial variables, and use of mental health and support services as potential predictors. Results: Rates of anxiety and depression among patients were significantly lower than in previous reports, although clinical depression rates (5.9%) were significantly higher than community norms (3.0%; χ2=24.0; P

Molecular events leading to epithelial ovarian cancer are poorly understood but ovulatory hormones and a high number of life-time ovulations with concomitant proliferation, apoptosis, and inflammation, increases risk. We identified genes that are regulated during the estrous cycle in murine ovarian surface epithelium and analysed these profiles to identify genes dysregulated in human ovarian cancer, using publically available datasets. We identified 338 genes that are regulated in murine ovarian surface epithelium during the estrous cycle and dysregulated in ovarian cancer. Six of seven candidates selected for immunohistochemical validation were expressed in serous ovarian cancer, inclusion cysts, ovarian surface epithelium and in fallopian tube epithelium. Most were overexpressed in ovarian cancer compared with ovarian surface epithelium and/or inclusion cysts (EpCAM, EZH2, BIRC5) although BIRC5 and EZH2 were expressed as highly in fallopian tube epithelium as in ovarian cancer. We prioritised the 338 genes for those likely to be important for ovarian cancer development by in silico analyses of copy number aberration and mutation using publically available datasets and identified genes with established roles in ovarian cancer as well as novel genes for which we have evidence for involvement in ovarian cancer. Chromosome segregation emerged as an important process in which genes from our list of 338 were over-represented including two (BUB1, NCAPD2) for which there is evidence of amplification and mutation. NUAK2, upregulated in ovarian surface epithelium in proestrus and predicted to have a driver mutation in ovarian cancer, was examined in a larger cohort of serous ovarian cancer where patients with lower NUAK2 expression had shorter overall survival. In conclusion, defining genes that are activated in normal epithelium in the course of ovulation that are also dysregulated in cancer has identified a number of pathways and novel candidate genes that may contribute to the development of ovarian cancer.

Background The four-transmembrane MAL2 protein is frequently overexpressed in breast carcinoma, and MAL2 overexpression is associated with gain of the corresponding locus at chromosome 8q24.12. Independent expression microarray studies predict MAL2 overexpression in ovarian carcinoma, but these had remained unconfirmed. MAL2 binds tumor protein D52 (TPD52), which is frequently overexpressed in ovarian carcinoma, but the clinical significance of MAL2 and TPD52 overexpression was unknown. Methods Immunohistochemical analyses of MAL2 and TPD52 expression were performed using tissue microarray sections including benign, borderline and malignant epithelial ovarian tumours. Inmmunohistochemical staining intensity and distribution was assessed both visually and digitally. Results MAL2 and TPD52 were significantly overexpressed in high-grade serous carcinomas compared with serous borderline tumours. MAL2 expression was highest in serous carcinomas relative to other histological subtypes, whereas TPD52 expression was highest in clear cell carcinomas. MAL2 expression was not related to patient survival, however high-level TPD52 staining was significantly associated with improved overall survival in patients with stage III serous ovarian carcinoma (log-rank test, p < 0.001; n = 124) and was an independent predictor of survival in the overall carcinoma cohort (hazard ratio (HR), 0.498; 95% confidence interval (CI), 0.34-0.728; p < 0.001; n = 221), and in serous carcinomas (HR, 0.440; 95% CI, 0.294-0.658; p < 0.001; n = 182). Conclusions MAL2 is frequently overexpressed in ovarian carcinoma, and TPD52 overexpression is a favourable independent prognostic marker of potential value in the management of ovarian carcinoma patients.

Mouse mammary tumor virus (MMTV) is a hormonally regulated, oncogenic virus of mice. MMTV-like virus DNA has previously been detected in human breast cancers, liver disease, and liver cancers. It is hypothesized that local hormonal effects might be of primary importance in determining MMTV-like virus detection in human tumors. MMTV-like virus envelope (env) DNA was determined using nested PCR in 89 ovarian, 147 prostate, 50 endometrial, 141 skin, and 51 lung cancers. Viral-positive sequences were compared with published MMTV-like viral sequences from human breast cancer, liver cancer and MMTV. Immunohistochemistry for estrogen receptor (ER-α) and progesterone receptor (PgR) was performed on a subset of tumors. MMTV-like virus env DNA was detected in ovarian cancers (14/89; 16%), prostate cancers (53/147; 36%), endometrial cancers (5/50; 10%), skin cancers (13/141; 9%) but not in lung cancers (0/51). Phylogenetic analysis of the viral-positive sequences showed no clustering of the isolates according to tissue type. A significant association was observed between the presence of hormone receptors and detection of MMTV-like virus in the human cancers screened (P = 0.01). A significant association between MMTV-like virus and PgR was noted in skin cancers (P = 0.003). Therefore, unlike the mouse model, the detection of MMTV-like env sequences in human cancers in addition to breast indicates that MMTV-like viral expression is not breast cancer-specific and may relate to hormone-dependent viral expression. J. Med. Virol. 82:1044–1050, 2010. © 2010 Wiley-Liss, Inc.

Estrogen receptor (ER) binding has been shown to decrease in breast cancer cell lines exposed to sodium butyrate; however, the underlying mechanisms are unknown. In MCF-7 breast cancer cells, butyrate caused a rapid time- and concentration-dependent decrease in ER mRNA levels, apparent by 3 h at 3 mM butyrate. ER gene transcription rate was decreased and cycloheximide co-treatment did not relieve this inhibitory effect, suggesting that the butyrate effect was not dependent on ongoing protein synthesis. In both MCF-7 and T-47D cells the decrease in ER mRNA was mirrored by an increase in the level of epidermal growth factor receptor (EGF-R) mRNA. A marked inverse relationship exists between ER and EGF-R in human breast cancer biopsies and cell lines, and the reciprocal modulation of these genes by butyrate suggests that the expression of ER and EGF-R may be co-regulated. This relationship was further investigated in lines expressing only one or the other receptor. In the ER-positive EGF-R-negative line, MDA-MB-134-VI, butyrate exposure decreased ER mRNA levels, implying that the regulation of ER mRNA by butyrate is independent of EGF-R expression. However, butyrate decreased EGF-R mRNA in two ER-negative lines, MDA-MB-231 and HBL-100. As this effect differed from that in ER-positive lines, the regulation of EGF-R may depend on the expression of ER. The possibility that ER and EGF-R gene expression are closely linked has implications in the understanding of progression of human breast cancers to a hormone-independent phenotype and for the use of ER and EGF-R levels as independent prognostic indicators.

Granulosa-cell tumors (GCTs) are the most common type of malignant ovarian sex cord-stromal tumor (SCST). The pathogenesis of these tumors is unknown. Moreover, their histopathological diagnosis can be challenging, and there is no curative treatment beyond surgery.We analyzed four adult-type GCTs using whole-transcriptome paired-end RNA sequencing. We identified putative GCT-specific mutations that were present in at least three of these samples but were absent from the transcriptomes of 11 epithelial ovarian tumors, published human genomes, and databases of single-nucleotide polymorphisms. We confirmed these variants by direct sequencing of complementary DNA and genomic DNA. We then analyzed additional tumors and matched normal genomic DNA, using a combination of direct sequencing, analyses of restriction-fragment-length polymorphisms, and TaqMan assays.All four index GCTs had a missense point mutation, 402C--G (C134W), in FOXL2, a gene encoding a transcription factor known to be critical for granulosa-cell development. The FOXL2 mutation was present in 86 of 89 additional adult-type GCTs (97%), in 3 of 14 thecomas (21%), and in 1 of 10 juvenile-type GCTs (10%). The mutation was absent in 49 SCSTs of other types and in 329 unrelated ovarian or breast tumors.Whole-transcriptome sequencing of four GCTs identified a single, recurrent somatic mutation (402C--G) in FOXL2 that was present in almost all morphologically identified adult-type GCTs. Mutant FOXL2 is a potential driver in the pathogenesis of adult-type GCTs.

Fibroblast growth factor (FGF)-2 (basic) is a potent angiogenic molecule involved in tumor progression, and is one of several growth factors with a central role in ovarian carcinogenesis. We hypothesized that common single nucleotide polymorphisms (SNPs) in the FGF2 gene may alter angiogenic potential and thereby susceptibility to ovarian cancer. We analyzed 25 FGF2 tgSNPs using five independent study populations from the United States and Australia. Analysis was restricted to non-Hispanic White women with serous ovarian carcinoma (1269 cases and 2829 controls). There were no statistically significant associations between any FGF2 SNPs and ovarian cancer risk. There were two nominally statistically significant associations between heterozygosity for two FGF2 SNPs (rs308379 and rs308447; p < .05) and serous ovarian cancer risk in the combined dataset, but rare homozygous estimates did not achieve statistical significance, nor were they consistent with the log additive model of inheritance. Overall genetic variation in FGF2 does not appear to play a role in susceptibility to ovarian cancer.

Udgivelsesdato: 2009-Jun-18 The estimated prevalence of insomnia in cancer patients varies between 20% and 50%, which is substantially higher than the general population. To date, little is known about the risk factors for insomnia in patients with cancer. This study examines the prevalence and predictors of insomnia in a population-based sample of women with ovarian cancer. Participants were 772 women participating in the Australian Ovarian Cancer Study - Quality of Life Study. Insomnia was assessed using the Insomnia Severity Index (ISI). Demographic, disease and treatment variables, and psychosocial variables, including anxiety and depression, support care needs and social support and coping, were investigated as potential predictors of insomnia. Twenty-seven percent of women reported sub-clinical symptoms of insomnia (ISI score 8-14) and 17% reported clinically significant insomnia (ISI score 15-28). Three variables were significant predictors of clinically significant insomnia: young age (

Purpose: A significant number of women with serous ovarian cancer are intrinsically refractory to platinum-based treatment. We analyzed somatic DNA copy number variation and gene expression data to identify key mechanisms associated with primary resistance in advanced-stage serous cancers. Experimental Design: Genome-wide copy number variation was measured in 118 ovarian tumors using high-resolution oligonucleotide microarrays. A well-defined subset of 85 advanced-stage serous tumors was then used to relate copy number variation to primary resistance to treatment. The discovery-based approach was complemented by quantitative-PCR copy number analysis of 12 candidate genes as independent validation of previously reported associations with clinical outcome. Likely copy number variation targets and tumor molecular subtypes were further characterized by gene expression profiling. Results: Amplification of 19q12, containing cyclin E (CCNE1), and 20q11.22-q13.12, mapping immediately adjacent to the steroid receptor coactivator NCOA3, was significantly associated with poor response to primary treatment. Other genes previously associated with copy number variation and clinical outcome in ovarian cancer were not associated with primary treatment resistance. Chemoresistant tumors with high CCNE1 copy number and protein expression were associated with increased cellular proliferation but so too was a subset of treatment-responsive patients, suggesting a cell-cycle independent role for CCNE1 in modulating chemoresponse. Patients with a poor clinical outcome without CCNE1 amplification overexpressed genes involved in extracellular matrix deposition. Conclusions: We have identified two distinct mechanisms of primary treatment failure in serous ovarian cancer, involving CCNE1 amplification and enhanced extracellular matrix deposition. CCNE1 copy number is validated as a dominant marker of patient outcome in ovarian cancer.

Background: X chromosome inactivation, which silences gene expression from one of the two X chromosomes in females, is usually random. Skewed X inactivation has been implicated in both the expression and the suppression of X-linked disease phenotypes and has been reported to occur more frequently in breast and ovarian cancer patients, including BRCA1 or BRCA2 mutation carriers, than in control subjects. Methods: We assessed the pattern of X chromosome inactivation using methylation-specific polymerase chain reaction amplification of the exon 1 microsatellite region of the X-linked androgen receptor (AR) gene in DNA from blood samples obtained from control subjects without a personal history of breast or ovarian cancer (n = 735), ovarian cancer patients (n = 313), familial breast cancer patients who did not carry mutations in BRCA1 or BRCA2 (n = 235), and affected and unaffected carriers of mutations in BRCA1 (n = 260) or BRCA2 (n = 63). We defined the pattern of X chromosome inactivation as skewed when the same X chromosome was active in at least 90% of cells. The association between skewed X inactivation and disease and/or BRCA mutation status was assessed by logistic regression analysis. The association between skewed X inactivation and age at cancer diagnosis was assessed by Cox proportional hazards regression analysis. All statistical tests were two-sided. Results: The age-adjusted frequency of skewed X inactivation was not statistically significantly higher in ovarian cancer or familial breast cancer case subjects compared with control subjects. Skewed X inactivation was higher in BRCA1 mutation carriers than in control subjects (odds ratio [OR] = 2.7, 95% confidence interval [CI] = 1.1 to 6.2; P = .02), particularly among unaffected women (OR = 6.1, 95% CI = 1.5 to 31.8; P = .005). Among BRCA1 mutation carriers, those with skewed X inactivation were older at diagnosis of breast or ovarian cancer than those without skewed X inactivation (hazard ratio [HR] of breast or ovarian cancer = 0.37, 95% CI = 0.14 to 0.95; P = .04). Among BRCA2 mutation carriers, skewed X inactivation also occurred more frequently in unaffected carriers than in those diagnosed with breast or ovarian cancer (OR = 5.2, 95% CI = 0.5 to 28.9; P = .08) and was associated with delayed age at onset (HR = 0.59, 95% CI = 0.37 to 0.94; P = .03). Conclusions: Skewed X inactivation occurs at an increased frequency in BRCA1 (and possibly BRCA2) mutation carriers compared with control subjects and is associated with a statistically significant increase in age at diagnosis of breast and ovarian cancer. © The Author 2008

Although some high-risk ovarian cancer genes have been identified, it is likely that common low penetrance alleles exist that confer some increase in ovarian cancer risk. We have genotyped nine putative functional single-nucleotide polymorphisms (SNP) in genes involved in steroid hormone synthesis (SRD5A2, CYP19A1, HSB17B1, and HSD17B4) and DNA repair (XRCC2, XRCC3, BRCA2, and RAD52) using two Australian ovarian cancer case-control studies, comprising a total of 1,466 cases and 1,821 controls of Caucasian origin. Genotype frequencies in cases and controls were compared using logistic regression. The only SNP we found to be associated with ovarian cancer risk in both of these two studies was SRD5A2 V89L (rs523349), which showed a significant trend of increasing risk per rare allele (P = 0.00002). We then genotyped another SNP in this gene (rs632148; r2 = 0.945 with V89L) in an attempt to validate this finding in an independent set of 1,479 cases and 2,452 controls from United Kingdom, United States, and Denmark. There was no association between rs632148 and ovarian cancer risk in the validation samples, and overall, there was no significant heterogeneity between the results of the five studies. Further analyses of SNPs in this gene are therefore warranted to determine whether SRD5A2 plays a role in ovarian cancer predisposition. (Cancer Epidemiol Biomarkers Prev 2007;16(12):2557–9)

It remains unclear whether physical activity is associated with epithelial ovarian cancer risk. We therefore examined the association between recreational physical activity and risk of ovarian cancer in a national population-based case-control study in Australia. We also systematically reviewed all the available evidence linking physical activity with ovarian cancer to provide the best summary estimate of the association. The case-control study included women ages 18 to 79 years with a new diagnosis of invasive (n = 1,269) or borderline (n = 311) epithelial ovarian cancer identified through a network of clinics, physicians, and state cancer registries throughout Australia. Controls (n = 1,509) were randomly selected from the national electoral roll and were frequency matched to cases by age and state. For the systematic review, we identified eligible studies using Medline, the ISI Science Citation Index, and manual review of retrieved references, and included all case-control or cohort studies that permitted assessment of an association between physical activity (recreational/occupational/sedentary behavior) and histologically confirmed ovarian cancer. Meta-analysis was restricted to the subset of these studies that reported on recreational physical activity. In our case-control study, we observed weakly inverse or null associations between recreational physical activity and risk of epithelial ovarian cancer overall. There was no evidence that the effects varied by tumor behavior or histologic subtype. Twelve studies were included in the meta-analysis, which gave summary estimates of 0.79 (95% confidence interval, 0.70-0.85) for case-control studies and 0.81 (95% confidence interval, 0.57-1.17) for cohort studies for the risk of ovarian cancer associated with highest versus lowest levels of recreational physical activity. Thus, pooled results from observational studies suggest that a modest inverse association exists between level of recreational physical activity and the risk of ovarian cancer. (Cancer Epidemiol Biomarkers Prev 2007;16(11):2321–30)

Background p53 is commonly inactivated by mutations in the DNA-binding domain in a wide range of cancers. As mutant p53 often influences response to therapy, effective and rapid methods to scan for mutations in TP53 are likely to be of clinical value. We therefore evaluated the use of high resolution melting (HRM) as a rapid mutation scanning tool for TP53 in tumour samples. Methods We designed PCR amplicons for HRM mutation scanning of TP53 exons 5 to 8 and tested them with DNA from cell lines hemizygous or homozygous for known mutations. We assessed the sensitivity of each PCR amplicon using dilutions of cell line DNA in normal wild-type DNA. We then performed a blinded assessment on ovarian tumour DNA samples that had been previously sequenced for mutations in TP53 to assess the sensitivity and positive predictive value of the HRM technique. We also performed HRM analysis on breast tumour DNA samples with unknown TP53 mutation status. Results One cell line mutation was not readily observed when exon 5 was amplified. As exon 5 contained multiple melting domains, we divided the exon into two amplicons for further screening. Sequence changes were also introduced into some of the primers to improve the melting characteristics of the amplicon. Aberrant HRM curves indicative of TP53 mutations were observed for each of the samples in the ovarian tumour DNA panel. Comparison of the HRM results with the sequencing results revealed that each mutation was detected by HRM in the correct exon. For the breast tumour panel, we detected seven aberrant melt profiles by HRM and subsequent sequencing confirmed the presence of these and no other mutations in the predicted exons. Conclusion HRM is an effective technique for simple and rapid scanning of TP53 mutations that can markedly reduce the amount of sequencing required in mutational studies of TP53.

Cancers arise owing to mutations in a subset of genes that confer growth advantage. The availability of the human genome sequence led us to propose that systematic resequencing of cancer genomes for mutations would lead to the discovery of many additional cancer genes. Here we report more than 1,000 somatic mutations found in 274 megabases (Mb) of DNA corresponding to the coding exons of 518 protein kinase genes in 210 diverse human cancers. There was substantial variation in the number and pattern of mutations in individual cancers reflecting different exposures, DNA repair defects and cellular origins. Most somatic mutations are likely to be 'passengers' that do not contribute to oncogenesis. However, there was evidence for 'driver' mutations contributing to the development of the cancers studied in approximately 120 genes. Systematic sequencing of cancer genomes therefore reveals the evolutionary diversity of cancers and implicates a larger repertoire of cancer genes than previously anticipated.

Epithelial ovarian cancer (EOC) is a devastating disease which accounts for a large proportion of gynaecological cancer-related deaths. Poor survival is largely due to late diagnosis of the disease which typically presents with peritoneal dissemination. In order to achieve reduced tumor burden and improved survival, it is imperative to identify new prognostic markers and potential therapeutic targets. Recent work by our group identified high ATP- Binding Cassette A1 (ABCA1) transporter expression as being associated with poor outcome in EOC. ABCA1 has been widely studied as a cholesterol transporter, however there is little research investigating its potential roles in cancer biology. The aim of this work was to ascertain whether ABCA1 may promote aggressive characteristics in EOC. siRNA-mediated suppression of ABCA1 in EOC cell lines led to reduced colony forming ability, growth and migration by comparison with controls. By culturing EOC cell lines (Skov3, 27/87 and HEY) in a 3-dimensional spheroid model, we have shown that ABCA1 suppression significantly reduced overall spheroid volume (p = ≤0.0003, ANOVA with Dunnett's multiple comparison test). In Skov3 and 27/87 spheroids, reduced volume was associated with cell death; however in HEY spheroids, ABCA1 suppression significantly reduced cell numbers in the absence of detectable cell death (p = ≤0.0002, ANOVA with Dunnett's multiple comparison test). Detailed characterisation of spheroid architecture revealed an increased volume-to-cell ratio in those depleted of ABCA1, indicating a lower degree of compaction compared to controls. It was further observed that whilst HEY spheroids derived from control siRNA-transfected cells develop a prominent necrotic core this feature was dramatically impaired by ABCA1 suppression. Compaction and core necrosis are both well-documented processes in spheroid biology that are indicative of cellular contractility and a hypoxic tumour environment, both key characteristics of more aggressive, chemo-resistant and recurrent tumors. Collectively these data suggest that ABCA1 may be involved in regulating multiple processes associated with aggressive and invasive characteristics of EOC, highlighting ABCA1 as a potential therapeutic target. Citation Format: Rebekka Williams, Amanda Russell, Angelika Bongers, Sharon Sagnella, Christopher Fife, Wendy Jessup, Anna DeFazio, Georgia Chenevix-Trench, Michelle Haber, Murray Norris, Michelle Henderson. Examining the role of ABCA1 cholesterol transporter in ovarian cancer spheroids. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 313. doi:10.1158/1538-7445.AM2015-313

Germline genetic variation can affect chemotherapeutic drug disposition, toxicity and efficacy. Identifying single nucleotide polymorphisms (SNPs) that influence chemotherapy disposition may help to personalize individual treatment. Compared to the traditional candidate gene approach, a genome-wide association study (GWAS) is unbiased but usually requires a large sample size. Since most cytotoxics have a short half-life and are given cyclically, multiple blood samples are therefore required to adequately define drug disposition and systemic exposure. This limits patient sample sizes in pharmacokinetic studies. The aim of this study was to identify SNPs that are associated with carboplatin and paclitaxel pharmacokinetic parameters using a GWAS approach in two small patient cohorts. Methods: Two independent pharmacokinetic cohorts were recruited in Australia (N = 61, both carboplatin and paclitaxel) and the Netherlands (N = 35, paclitaxel only). A total of 719,665 SNPs were genotyped using Illumina Human OmniExpress arrays. Linear regression was used to test the association between each SNP and the trait of interest, both unadjusted and adjusted for corresponding covariates using PLINK. We performed separate analysis within each population and then ran meta-analysis combining results from different populations weighted by sample sizes using METAL. Results: Carboplatin and paclitaxel disposition is a relative stable phenotype. The genomic control parameters in the test statistics for the adjusted carboplatin and paclitaxel analysis were 1.03 and 1.02 respectively, suggesting that there was little concern for population substructure or other artefacts. We identified highly significant SNPs in ABCC2 associated with carboplatin clearance (asymptotic p = 5.2 × 10−6, empirical p = 1.4 × 10−5 from 107 permutations) which are highly plausible pharmacogenomic markers given the known role of ABCC2 in carboplatin efflux. We also identified a novel SNP associated with paclitaxel disposition with genome wide significance in chromosome 1 (rs17130142, asymptotic p = 2.0 × 10−9, empirical p = 1.3× 10−7), in an intergenic region, neighboring PKN2. Conclusion: Although these findings requires a validation in a larger study, our work demonstrated that GWAS of chemotherapeutic drug disposition can be effective even when performed in a relative small sample number and can be adopted in drug development and treatment programs. Citation Format: Bo Gao, Yi Lu, Annemieke J.M. Nieuweboer, Hongmei Xu, Jonathan Beesley, Ingrid Boere, Anne-Joy M. de Graan, Peter de Bruijn, Howard Gurney, Catherine Kennedy, Yoke-Eng Chiew, Sharon E. Johnatty, Philip Beale, Michelle Harrison, Craig Luccarini, Alison M. Dunning, Ron H.J. Mathijssen, Paul Harnett, Rosemary L. Balleine, Georgia Chenevix-Trench, Stuart MacGregor, Anna deFazio. Genome-wide study of carboplatin and paclitaxel disposition in ovarian cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5493. doi:10.1158/1538-7445.AM2015-5493

In rodents, progesterone receptors (PRs) A and B have different and often nonoverlapping roles, and this study asked whether different activities of the PR proteins in mouse are related to differences in their expression in reproductive tissues. The individual expression of PRA and PRB was determined immunohistochemically in mammary gland and uterus during the estrous cycle or in response to endocrine manipulation. In the mammary gland, PRA and PRB were colocated in PR+ epithelial cells, with little change during the estrous cycle. In the uterus, PRA was not detected in luminal epithelium at any stage of the cycle, and PR+ luminal cells expressed only PRB. In the stroma and myometrium, PRA and PRB levels fluctuated with cyclical systemic hormone exposure. Observation of functional end points suggested that augmented stromal and/or myometrial PRA in proestrus inhibited estrogen receptor expression and epithelial proliferation. Colocation of PRA and PRB was hormonally regulated, and ovariectomy did not reproduce the expression of PRA and PRB in the uterus during the estrous cycle. Whereas PRB was the only PR in the luminal epithelium in cycling mice, ovariectomy restored PRA expression, resulting in PRA-PRB colocation. In stroma and myometrium, PRA and PRB colocated in PR+ cells, but ovariectomy reduced PRA levels more than PRB, resulting in PRB-only-expressing cells. This study has shown that nonoverlapping PRA and PRB expression in the uterus, in particular the lack of PRA, and expression of PRB only in the luminal epithelium throughout the estrous cycle, is likely to contribute to the distinct roles of PRA and PRB in the adult mouse.

Primary and acquired chemotherapy resistance remains a major challenge in the treatment of ovarian cancer. We hypothesize that heritable polymorphisms might be associated with clinical outcome. Through the Ovarian Cancer Association Consortium, we have collected detailed clinical data from a large number of ovarian cancer patients. We have conducted association analyses of progression-free survival (PFS) and overall survival (OS) for 2,901 European women with ovarian carcinoma for ∼8 million imputed SNPs with minor allele frequency ≥ 0.02 and good imputation quality (imputation r2 ≥ 0.3) from the iCOGS experiment (Pharoah et al Nat Genet. 2013). Cox regression models adjusted for known prognostic factors were used to assess the log-additive effects of SNPs on PFS and OS in 2,825 patients who underwent cytoreductive surgery and received any chemotherapy (‘all chemo’) and in the subset of 1,098 patients who underwent cytoreductive surgery followed by ≥ 4 cycles of carboplatin AUC 5 or 6 and paclitaxel 175 or 135 mg/m2 intravenously (‘standard chemo’). We identified five SNPs at four loci that were significantly associated with outcome (p ≤ 10-7) in at least one of four analyses (Table). Analysis of expression data for genes within a 1Mb region flanking each lead SNP indicates significant associations between nearby genes and ovarian cancer survival. Of particular interest is the heat shock protein crystalline αB (CRYAB) at 11q23.1. We have also identified significant associations with KIAA1598 and RAVER2. Further analysis of these loci in a larger genotyped sample is necessary to definitively identify SNP(s) associated with outcome. Our study adds to the growing evidence that genetic variants are associated with ovarian cancer prognosis. Table: SNPs associated with ovarian cancer outcome and gene expression Overall Survival (P)*Progression-Free Survival (P)*Lead SNPChromosome LocusImputation R²Expression AnalysisAll chemoStandard chemoAll chemoStandard chemors1429768671p31.30.742.14E-0512.69E-044.68E-055.11E-073.64E-09rs559375217q32.30.600.198.58E-082.32E-052.80E-031.85E-03rs7885346210q26.110.602.23E-0724.57E-036.57E-081.65E-016.85E-03rs1089121911q23.10.841.53E-0531.51E-059.50E-081.50E-042.32E-04rs3579103211q23.10.811.53E-0531.66E-055.88E-086.46E-051.87E-04*P-values are adjusted for known confounders of ovarian cancer outcomes; FIGO stage, grade, histology, residual disease, and additionally for age at diagnosis for OS.1 RAVER2; 2 KIAA1598; 3 CRYAB Citation Format: Sharon E. Johnatty, Jonathan Tyrer, Jonathan Beesley, Bo Gao, Yi Lu, Stuart MacGregor, Anna deFazio, Paul Pharoah, Ellen Goode, Georgia Chenevix-Trench. Identification of genetic loci associated with ovarian cancer prognosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3286. doi:10.1158/1538-7445.AM2014-3286

Recurrent chromosome 8q gain in ovarian carcinoma is likely to reflect the existence of multiple target loci, as the separate gain of chromosome bands 8q21 and 8q24 has been reported in independent studies. Since tumor protein D52 (TPD52) has been identified as a chromosome 8q21 amplification target in breast and prostate carcinoma, we compared TPD52 expression in normal ovarian epithelium (n = 9), benign serous adenomas (n = 11), serous borderline tumors (n = 6) and invasive carcinomas of the major histologic subtypes (n = 57) using immunohistochemistry. These analyses revealed that all normal ovarian epithelium samples and benign serous tumors were predominantly TPD52-negative, whereas TPD52 was overexpressed in most (44/57; 77%) ovarian carcinomas regardless of histologic subtype. TPD52 subcellular localization was predominantly cytoplasmic, although nuclear localization was also frequently observed in mucinous and clear cell carcinomas. In an independent cohort of stage III serous carcinomas (n = 18), we also directly compared in situ TPD52 expression using immunohistochemistry and TPD52 copy number using interphase FISH analyses. This revealed that TPD52 dosage and TPD52 expression were significantly positively correlated. TPD52 therefore represents a novel molecular marker in ovarian cancer, which is broadly expressed across the different histologic subtypes and whose upregulation frequently reflects increased TPD52 copy number.

The progesterone receptor (PR) exists as two isoforms, PRA and PRB. In vitro studies have shown that these proteins are functionally distinct, suggesting that their relative expression can influence progesterone response. Low-grade endometrial stromal sarcoma (LGESS) is an uncommon tumor that usually expresses PR. In normal endometrial stroma, both PR isoforms are present with PRA predominant throughout the menstrual cycle. The relative expression of PRA and PRB in LGESS has not been previously reported. All nine cases of primary LGESS (seven uterine, two extrauterine) expressed PRB. Eight tumors also contained PRA and it was the predominant isoform in seven cases. These tumors had similar histopathologic appearances, whereas a case with approximately equal PR isoform expression showed features of sex cord or smooth muscle differentiation. An extrauterine tumor expressing only PRB had myxoid stroma. Recurrent tumor in two cases, which expressed predominantly PRA in the primary, contained reduced levels of PR consisting predominantly or entirely of PRB after prolonged interval progestin therapy. Most primary LGESSs showed PR isoform expression similar to normal endometrial stroma, consistent with the highly differentiated phenotype of this tumor. Variant differentiation or disease recurrence was accompanied by an altered PR isoform profile that could impact on hormone response.

Progesterone plays a central role in the regulation of ovarian function. The progesterone receptor (PR) has been shown to be essential for ovulation because mice lacking PR fail to ovulate and are infertile. PR is expressed as two isoforms, PRA and PRB, which have been shown to have different functional activities. In this study, we investigated the cellular distribution of PRA and PRB in the ovaries and oviducts of cycling mice using immunohistochemistry with isoform-specific monoclonal antibodies. In the ovary, on the evening of proestrus before ovulation, both the granulosa and theca cells of the preovulatory follicles expressed both PR isoforms. PRA and PRB staining was also observed in the theca cells of preantral and antral follicles, whereas only PRB was observed in the granulosa cells of primary, preantral, and antral follicles and in the corpus luteum. In the oviduct, PRA was the predominant isoform observed, expressed in both the epithelial and stromal cells, whereas PRB was only detected in the epithelial cells. The differences in PRA and PRB localization in the ovary and oviduct may reflect diverse functions for PRA and PRB in reproductive tissues and may have important implications in understanding the mechanisms of progesterone action.

The nuclear progesterone receptor (PR), which is expressed as two proteins with different functions (PRA and PRB), is expressed in the normal endometrium and endometrial cancer. Our previous work has shown that there is a disruption to the relative PR isoform expression in progression to malignancy in endometrial cancer in which expression of a single isoform is common. A consistent feature in these studies was discrete punctate distribution of PRA and PRB in the nuclei of endometrial cancer cells. In this study PRA and PRB distribution within the nucleus was examined in vivo in the normal endometrium during the menstrual cycle, and in endometrial cancer, by dual immunofluorescence and confocal microscopy using cohorts in which PRA and PRB expression levels have previously been characterized. In the normal endometrium, PR was distributed evenly within the nucleus and also localized in discrete subnuclear foci. In the proliferative phase, even PR distribution was predominant and both PR isoforms were colocated and distributed evenly. In the secretory phase, there was a marked increase in the proportion of nuclei containing PR distributed into discrete foci, and PRB was the predominant isoform in nuclear foci. There was an inverse relationship between even and focal PR distribution in the menstrual cycle, suggesting that hormonal fluctuations were involved in movement of PR into focal nuclear locations. In endometrial cancers colocalization of PRA and PRB was infrequent, and there was no relationship between even and focal PR isoform distribution, unlike the normal endometrium. PRA was predominantly evenly distributed in endometrial cancers, whereas PRB was focal. Even PRB distribution in endometrial cancer was not often noted. Multivariate analysis showed that PRA expression was highly predictive of even nuclear distribution in endometrial cancers and PRB expression of distribution into foci, and these associations were independent of total PRA and PRB levels. Nuclear distribution of PR isoforms was associated with clinical grade, where tumors of high grade had significantly fewer nuclei containing even PRA distribution and focal PRB distribution, compared with tumors of low grade. In the normal endometrium, localization of PR into nuclear foci coincides with high progesterone levels, suggesting that altered intranuclear PR distribution is hormonally regulated. Nuclear distribution may be an important component of gene regulation in target tissues, and disruptions in PR distribution in endometrial cancer could affect the function of PR and contribute to aberrant hormonal responses.

Ovarian hormones have a pivotal role in the control of proliferation in the mammary gland, and cumulative life-time exposure to ovarian hormones is known to be a determinant of breast cancer risk. We have shown previously that a p.o.-active, long-acting butyrate analogue, sodium phenylbutyrate (PB), reduced proliferation in normal and malignant human breast cells in culture and reduced expression of ovarian hormone receptors, suggesting that PB had cellular effects consistent with decreasing breast cancer risk. The aim of this study was to determine the in vivo effects of PB in the normal mammary gland on epithelial cell proliferation, estrogen receptor alpha (ER alpha) expression, and cyclin D1 expression. BALB/c mice were treated with PB, delivered by mini-osmotic pumps, for 7 days. Moderate (250 mg/kg/day) and high (500 mg/kg/day) PB treatment resulted in a decrease in proliferation in mammary epithelial cells (P0.001), determined by bromodeoxyuridine incorporation. Analysis of ER alpha immunostaining revealed a significant reduction in moderate- and high-treatment groups (P = 0.01 and P = 0.02), and expression of cyclin D1 was virtually ablated (P0.001). Histone deacetylase inhibition is a known mechanism of butyrate action, and consistent with this, PB increased levels of acetylated histone H3 in the mammary gland. In summary, PB decreased proliferation in the mammary gland in vivo at clinically achievable doses. Decreased proliferation was accompanied by changes in the levels of ER alpha and cyclin D1. These data show that PB modulates parameters thought to be involved in the carcinogenic process in the normal mammary gland, and compounds in this class may therefore be useful candidates for breast cancer chemoprevention.

The aim of this Program is to study the association between epidemiologic risk factors, low-risk genes, and histologic and novel molecular subtypes of ovarian cancer. In December 2002, we received final approval from the Human Subject Research Review Board (HSRRB) to start recruitment at 13 of the proposed study sites and have since received approval to recruit at a further 2 sites. We have established a network of research nurses across the country and recruitment is now progressing well at 15 different sites. Recruitment of cases for the study began in January 2003 and control recruitment began in May 2003 and we have since recruited a total of 934 women with ovarian cancer and 569 control women. The recruitment, sample and data collection and processing systems are working well and we are continuously monitoring our performance against our targets as outlined in the reports from Core Components A (Epidemiology) and B (Biospecimens). In relation to this we also have established systems to manage data and samples from all of the different sites.

Summary After more than two decades of extensive use, drugs based on platinum continue to have a major role in cancer treatment. Although systematic approaches to the development of new analogues have produced agents with less toxicity and novel mechanisms of action, to date such approaches have not achieved more cures than can be achieved with the parent compound, cisplatin. Greater gains might be expected from accumulating knowledge about what makes cancer cells sensitive or resistant to platinum-based chemotherapy. Recent information on drug-efflux pathways, including expression of multidrugresistance protein 2, and on how tumour cells behave when their DNA is distorted by a platinum adduct, suggests new avenues for translational research. The prospects include modulation of cellular handling of platinum compounds and individualised therapy based on expression of molecules that determine platinum sensitivity.

5563 Background: Epithelial ovarian cancer (EOC) is generally treated by cytoreductive surgery and carboplatin/paclitaxel chemotherapy. Five-year survival is ~20% for patients with advanced disease...

e16519 Background: Olaparib is in development for maintenance treatment in patients with BRCA mutation positive (BRCAm) platinum sensitive recurrent (PSR) ovarian cancer. A phase 2 trial, NCT007535...

Purpose: Clinical observation has shown that paclitaxel ameliorates the antiplatelet toxicity of carboplatin when the two drugs are combined, although antitumour activity and white cell toxicity are at least additive. We hypothesized that this is due to an interaction between the two drugs at the level of the platelet precursor. Methods: We measured inhibition of growth of the megakaryoblast cell line MEG-01 following exposure to paclitaxel and carboplatin singly or combined. Drug interaction was assessed by median effect analysis. Results: An antagonistic interaction was observed, and this was most marked at drug concentrations giving a low level of growth inhibition (P

Purpose: To determine the relationship between obesity, diabetes, other major comorbidities and survival following a diagnosis of ovarian cancer. Methods: We examined the association between obesity, as measured by body mass index (BMI), diabetes and other comorbidities and survival from ovarian cancer in a large (n=1404), population-based cohort of women diagnosed with epithelial ovarian cancer between 2002-2006. Information about pre-diagnostic BMI and medical history was obtained via a self-administered questionnaire. BMI at the commencement of primary chemotherapy and clinicopathologic data were abstracted from medical records and deaths were ascertained up to October 2011 via linkage with the Australian National Death Index. We also examined short- (≥5 years) and long-term (>5 years) survival, as well as survival differences by histological subtype. Adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) were obtained using Cox regression. Results: After adjustment, a survival disadvantage was observed for women who were obese prior to diagnosis compared to women in the normal BMI range (BMI 30-34.9:HR 1.21, 95%CI 0.99-1.48; BMI≥35:HR 1.19, 95%CI 0.92-1.55). The association was similar for BMI at the commencement of primary chemotherapy. There was a suggestion that the adverse association between obesity and survival time was restricted to women with serous or endometrioid histological subtypes. Obesity conferred a survival disadvantage for both short- and long-term survival, with a stronger association in long-term survivors. Diabetes and other comorbidities did not appear to influence survival overall; however appeared to be important in the sub-group of women surviving longer than 5 years (HR1.44 and HR1.64, respectively). Conclusion: This is the first study to show that obesity is important in both short- and long-term survival following a diagnosis of ovarian cancer. More research examining diabetes, comorbidities and ovarian cancer survival should be conducted. Citation Format: Melinda M. Protani, Christina M. Nagle, Anna deFazio, Penelope Blomfield, Thomas Jobling, Yee Leung, James L. Nicklin, Lewis C. Perrin, Gerard Wain, Penelope M. Webb. Obesity, diabetes, other comorbidities and survival in a cohort of women with ovarian cancer. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr B45.

Background: Mucinous adenocarcinomas (MC) comprise a distinct group of neoplasms that can arise in multiple organs, such as the ovary, pancreas and gastrointestinal tract, but are relatively understudied. Mucinous ovarian carcinomas (MOC) are a distinct histological subtype of epithelial ovarian cancer, with late stage and high-grade MOC often resistant to standard platinum-based ovarian chemotherapeutic regimens. Controversy exists over whether high-grade MOC arise in the ovary or represent metastases from distant sites with secondary ovarian involvement. This study aims to determine if differences exist at the molecular level that distinguish low- and high-grade MOC, mucinous tumours of extra-ovarian origin (EOM) and primary MC from other sites, as this has significant implications for treatment strategies. Methods: We are identifying cases of low- and high-grade MOC from CART-WHEEL.org, the Australian Ovarian Cancer Study and national and international tissue banks. High-resolution molecular characterization of these cases using whole-exome sequencing, copy number arrays and RNAseq will be compared to similar data generated for mucinous cystadenomas and borderline ovarian tumours (the putative precursors to MOC), and cases of EOM, pseudomyxoma peritonei and primary MC from other sites. Results: Copy number analysis and mutation screening (KRAS, BRAF, NRAS, TP53, CDKN2A) of 22 cystadenomas, 22 borderline tumours and 31 MOC has identified activating RAS/RAF pathway mutations concurrent with CDKN2A loss by homozygous deletion, inactivating mutations and loss of heterozygosity as common to all mucinous ovarian tumours, while TP53 mutations were largely restricted to MOC. Exome sequencing of selected cases has revealed additional genes recurrently targeted by deleterious mutations. Conclusions: Preliminary findings demonstrate that the majority of MOC share genomic events with their putative precursors, supporting a cystadenoma-borderline-carcinoma progression for both low- and high-grade MOC. Intriguingly, exome sequencing suggests a significant overlap of mutated genes with mucinous tumours arising from other organ sites, although it remains to be seen whether gene expression analysis can distinguish site of origin for tumours with a mucinous histology. Citation Format: Ian Campbell, Sally Hunter, Wakefield Mathew, Yoland Antill, Susie Bae, Sumitra Ananda, Monique Topp, Lara Lipton, Winston Liauw, Thomas Jobling, Alex Boussioutas, Michael Christie, Prue Allan, Michael Friedlander Michael Friedlander, Stephen Fox, Anna Defazio, David Bowtell, Jan Pyman, Study Australian Ovarian Cancer, Clare Scott, Kylie Gorringe. Mucinous ovarian tumors: Are they all the same? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A3.

Post-translational modifications of histones are involved in diverse functions, including gene transcription and the DNA damage response. Targeting elements of the DNA damage response pathway is of increasing interest for the treatment of epithelial ovarian cancer (EOC). Monoubiquitination of histone H2B at lysine 120 (H2Bub1) is present at the coding region of highly expressed genes such as the gene encoding p21, CDKN1A, under conditions of DNA damage. Addition of this histone mark relies on the E3 ubiquitin ligase ring finger protein complex RNF20-RNF40 that we, and others, have previously shown interacts with members of the PAF1 complex necessary for RNA polymerase II-based transcriptional elongation. At 8.5 kDa, ubiquitin constitutes a bulky addition likely sufficient to interfere with condensed chromatin fibres, leading to a more open fibre conformation with greater accessibility for transcription factors and DNA repair proteins. H2Bub1 has been described as a master switch for mammalian gene regulation, with roles in histone cross-talk. Furthermore, H2Bub1 has recently been linked to the cellular DNA damage response, with the protein kinase ATM phosphorylating RNF20-RNF40 to induce H2Bub1 at sites of double strand breaks, leading to a more open chromatin structure for optimal repair. Global H2Bub1 loss has been reported in breast, colon, lung and parathyroid tumors1, and in some of these tumor types has been associated with advancing stage. We have undertaken immunohistochemical analyses of global H2Bub1 and total histone H2B using tissue microarrays of EOC sourced from the Australian Ovarian Cancer Study and the Gynaecological Oncology Biobank at Westmead. Expression of total histone H2B was high and consistent across all tumors, whereas over 70% of tumors showed total loss of global H2Bub1. Contrary to reports that global H2Bub1 levels decreased with advanced stage in breast cancer, we did not observe this for ovarian cancer. This may be related to the fact that cohorts of EOC are skewed towards tumors of advanced stage, as most women present with Stage III-IV disease. The mechanism of H2Bub1 loss in ovarian cancer is currently unknown, but is likely to involve genetic or epigenetic regulation of genes encoding proteins that function as components of E3 ubiquitin ligase machinery. We have previously shown that loss-of-function mutations in the tumor suppressor CDC73, a PAF1 complex member, lead to loss of H2Bub1 in parathyroid cancer1; however, we observed consistent staining of CDC73 in ovarian tumors. We are currently analysing EOCs for expression of RNF20 given that the RNF20 promoter is hypermethylated in breast cancer and TCGA-ovarian reports RNF20 missense mutations with neutral to medium predicted impact scores. This data will be presented. Given the apparent fundamental role of H2Bub1 in determining the accessibility of chromatin for molecules such as DNA repair proteins, H2Bub1 and/or its associated ubiquitin ligase machinery, may be considered as potential therapeutic targets for the treatment of EOC. 1Hahn MA, Dickson K-A, Jackson S, Clarkson A, Gill AJ and Marsh DJ. The tumor suppressor CDC73 interacts with the ring finger proteins RNF20 and RNF40 and is required for the maintenance of histone 2B monoubiquitination Human Molecular Genetics 2012; 21(3):559-568. Citation Format: Deborah J. Marsh, Anna deFazio, Adele Clarkson, Catherine Kennedy, Gregory G. Gard, Anthony J. Gill. Loss of histone H2B monoubiquitination in ovarian cancer – new therapeutic targeting opportunities based on chromatin relaxation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A29.

In human breast cancer cell lines, an inverse relationship exists between the basal levels of oestrogen receptor (ER) and epidermal growth factor receptor (EGF-R) gene expression. In addition, the tumour-promoting phorbol ester 12- O -tetradecanoyl-phorbol-13-acetate (TPA) inhibits ER and stimulates EGF-R expression in MCF-7 breast cancer cells. This study aimed to define further the potential mechanisms involved in the modulation of ER and EGF-R gene expression by TPA. ER mRNA levels were reduced after 3 h and declined to 30% of control between 12 and 72 h after exposure to 10 nM TPA. This decrease in mRNA levels was preceded by an apparent fall in ER transcription rate. There was no effect on the stability of ER mRNA following pretreatment for 3–24 h with TPA, supporting the conclusion that the fall in ER mRNA levels was predominantly due to a decrease in ER transcription rate. Levels of EGF-R mRNA increased 10-fold by 12 h due predominantly to an increased transcription rate. The TPA-induced decrease in ER mRNA was unaffected by the simultaneous administration of the protein synthesis inhibitor cycloheximide, whereas the increase in EGF-R mRNA was inhibited by co-incubation with cycloheximide. These data indicate a requirement for continuing protein synthesis for the TPA effect on EGF-R but not on ER mRNA levels. Because the modulation of ER and EGF-R gene expression by TPA is likely to involve the protein kinase C (PKC) signal transduction pathway, the effects of other known activators of PKC were investigated. The non-phorboid tumour promoter mezerein modulated ER (an 80% decrease) and EGF-R (a 20-fold increase) mRNA levels in a similar manner to TPA. In contrast, neither 1,2-dioctanoyl- sn -glycerol (DiC 8 ) nor 1-oleoyl-2-acetyl- sn -glycerol (OAG), both permeant analogues of the endogenous physiological activators of PKC, affected ER and EGF-R mRNA levels. These latter results were not due to a lack of efficacy because a single administration of DiC 8 was as effective as TPA in inducing c- fos mRNA at 30 min. However DiC 8 was less active in the later induction of c- myc mRNA. These data demonstrate reciprocal regulation of ER and EGF-R gene expression by TPA, involving effects on transcriptional events, which appear to be mediated by sustained activation of PKC.

To define the mechanisms by which antiestrogens inhibit breast cancer cell proliferation, the effects of the antiestrogen ICI 182780 on G1 cyclins and their cyclin-dependent kinase (CDK) partners were investigated in MCF-7 cells. Inhibition of entry into S phase became evident 9 h after treatment, with the proportion of cells in S phase reaching a minimum by 24 h. ICI 182780 increased the proportion of the hypophosphorylated, growth inhibitory form of the retinoblastoma protein (pRB). This change began at 4-6 h, preceding effects on S phase. This suggests that there are early effects on the activities of CDKs that target pRB that are not merely a consequence of changes in cell cycle progression. The kinase activity of Cdk2 decreased to low levels at 18-24 h when changes in S phase and pRB phosphorylation were well advanced. An earlier effect was seen on kinase activity associated with immunoprecipitated cyclin D1, which was reduced approximately 40% by 12 h, with further decreases at 18-24 h. Cdk2 and Cdk4 protein levels remained constant over 24 h. Cyclin D1 messenger RNA and protein were down-regulated by ICI 182780 from 2 h, with levels halved at 8 h. ICI 182780 also increased the expression of the CDK inhibitors p27KIP1 and p21WAF1/CIP1 at later times. These observations are compatible with the hypothesis that antiestrogens block entry of cells into S phase and inhibit cell proliferation as the consequence of an early decline in pRB phosphorylation contributed to by reduced cyclin D1/Cdk4 activity. At later times, increased CDK inhibitor abundance may act to repress Cdk2 and Cdk4 activities, causing additional reductions in pRB phosphorylation, thus maintaining the antiestrogen blockade of cell cycle progression.

Cell-based models have shown that response to chemotherapy has a heritable component. We hypothesized that in order to identify loci associated with treatment outcome we should focus on cases known to have had uniform chemotherapy for epithelial ovarian cancer. We therefore performed a two-stage genome-wide association study (GWAS) of progression free survival (PFS) following first line carboplatin/paclitaxel chemotherapy in ovarian cancer cases. In the first stage, we genotyped (Illumina Omni1) 183 Australian cases selected using an extreme phenotype design, and also included data on 134 cases from the TCGA and 68 from Mayo Clinic. In this stage, 260 cases had ‘uniform’ treatment (“primary” group: at least 4 cycles of carboplatin 5-6 AUC and paclitaxel 135-175 mg/m2 every three weeks) and 125 cases (“secondary”) had an unknown amount of carboplatin/paclitaxel chemotherapy. In the 2nd stage we genotyped 156 of the top ranking genotyped and imputed SNPs in 4660 cases (1080 ‘primary’ and 1433 ‘secondary’) from 11 sites in OCAC. The additive allelic association with the PFS was assessed in a Cox Proportional Hazards model, adjusting for study site, histological subtype, grade, stage and residual disease. For the SNPs with low minor allele frequencies, we ran permutations to correct the asymptotic p-values. One SNP clearly replicated in the 2nd stage. The associations in both stages were strongest in the “primary” group (1st stage results for this imputed SNP: hazard Ratio (HR) per minor allele=6.30, 95% CI=[4.26, 9.30], two-sided asymptotic p-value=1.88e-6, permuted p-value=4.46e-5; 2nd stage: HR per minor allele=3.37, 95% CI=[2.48, 4.62], one-sided asymptotic p-value=5e-5, permuted p-value=2.9e-4; meta-analysis p-value corrected by permutation=1.7e-7). Furthermore, an independent cell-based GWAS conducted using HapMap lymphoblastoid cell lines showed that a more common SNP (MAF~4.5%) in moderate LD with our reported SNP (r2~0.28), was associated with carboplatin sensitivity (p-value=9e-3). Both these SNPs are located in a gene on chromosome 9 that is known to be associated with circulating levels of high-density lipoprotein cholesterol. Low levels of expression of this gene are associated with poor outcome for both progression free (P=.017) and overall survival (P=.001) from serous ovarian cancer in The Cancer Genome Atlas. We are now genotyping a panel of rare and common SNPs in this gene in additional validation cohorts from OCAC and conducting additional experiments to evaluate its biological relevance with respect to treatment outcome, including the potential role of cholesterol metabolism as a determinant of response to chemotherapy. Our findings may provide some insight into the suggested role of statins in improving outcome for ovarian cancer.

5026 Background: It has been accepted that 5-10% of all invasive epithelial ovarian cancer cases can be attributed to hereditary pathogenic mutations in cancer predisposition genes. Consequently ge...

Molecular subtypes of serous ovarian cancer have been recently described. Using data from independent datasets including over 900 primary tumour samples, we show that deregulation of the Let-7 pathway is specifically associated with the C5 molecular subtype of serous ovarian cancer. DNA copy number and gene expression of HMGA2, alleles of Let-7, LIN28, LIN28B, MYC, MYCN, DICER1, and RNASEN were measured using microarray and quantitative reverse transcriptase PCR. Immunohistochemistry was performed on 127 samples using tissue microarrays and anti-HMGA2 antibodies. Fluorescence in situ hybridisation of bacterial artificial chromosomes hybridized to 239 ovarian tumours was used to measure translocation at the LIN28B locus. Short interfering RNA knockdown in ovarian cell lines was used to test the functionality of associations observed. Four molecular subtypes (C1, C2, C4, C5) of high-grade serous ovarian cancers were robustly represented in each dataset and showed similar pattern of patient survival. We found highly specific activation of a pathway involving MYCN, LIN28B, Let-7 and HMGA2 in the C5 molecular subtype defined by MYCN amplification and over-expression, over-expression of MYCN targets including the Let-7 repressor LIN28B, loss of Let-7 expression and HMGA2 amplification and over-expression. DICER1, a known Let-7 target, and RNASEN were over-expressed in C5 tumours. We saw no evidence of translocation at the LIN28B locus in C5 tumours. The reported interaction between LIN28B and Let-7 was recapitulated by siRNA knockdown in ovarian cancer cell lines. Our results associate deregulation of MYCN and downstream targets, including Let-7 and oncofetal genes, with serous ovarian cancer. We define for the first time how elements of an oncogenic pathway, involving multiple genes that contribute to stem cell renewal, is specifically altered in a molecular subtype of serous ovarian cancer. By defining the drivers of a molecular subtype of serous ovarian cancers we provide a novel strategy for targeted therapeutic intervention.

Molecular events leading to ovarian cancer are poorly understood but ovulatory hormones and a high number of lifetime ovulations increase risk. The most common histotype, serous ovarian cancer, is thought to arise from ovarian surface epithelium or inclusion cysts although recent evidence has implicated fallopian tube epithelium and other Müllerian tissues as potential sites of origin. The aim of this study was to identify genes that may play a critical role in the pathogenesis of ovarian cancer from candidates that we have previously identified as regulated during the murine estrus cycle in microdissected ovarian surface epithelium and matched oviduct. We examined estrus regulated gene profiles in normal mouse ovarian and oviduct epithelium for genes (i) differentially expressed in ovarian cancer compared to normal controls; (ii) with copy number aberration in ovarian cancer; and (iii) with reported mutation in solid tumors. We identified over 350 genes that are regulated in the normal mouse ovarian epithelium during the estrus cycle and dysregulated in cancer including four genes found to be mutated in various tumour types (Catalogue Of Somatic Mutations In Cancer) and around 50 genes that have copy number aberration in ovarian cancer (The Cancer Genome Atlas). A similar number of genes were regulated in the normal mouse oviduct epithelium during the estrus cycle and dysregulated in ovarian cancer including six that are mutated in various tumor types and around 35 with copy number aberration in ovarian cancer. Interestingly, there was very little similarity between the ovarian and oviduct epithelium gene lists with only 19 genes in common, however, pathway analysis revealed over-representation of genes involved in cell cycle, particularly the spindle assembly checkpoint, and extracellular matrix/cell adhesion in both lists. We identified NUAK family, SNF1-like kinase, 2 (NUAK2) as upregulated in ovarian epithelium during the proestrus phase of the murine estrus cycle, upregulated in ovarian cancer and with a candidate driver mutation for breast and ovarian cancer. Expression of NUAK2 was examined by immunohistochemistry in human ovary (n=10), fallopian tube (n=9), and ovarian cancer (n=119). This showed that NUAK2 expression was highest in fallopian tube, intermediate in OSE and lost in ∼20% of ovarian cancer cases. Kaplan-Meier curves with log-rank test revealed reduced expression of NUAK2 was significantly associated with reduced overall survival in ovarian cancer (p Future work involves functional analysis of key genes, including NUAK2, in human cell lines. Defining genes that are activated in normal ovarian and oviduct epithelial cells in the course of ovulation that are also dysregulated in cancer has identified a number of molecular pathways and novel candidate genes that may contribute to the development of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3875. doi:10.1158/1538-7445.AM2011-3875

Ovarian cancer is a devastating disease. More than half the women diagnosed with advanced disease will die within five years. Tumours displaying serous histology are the most common, and although initially responsive to current chemotherapy regimens, the disease is characterised by a frequent rate of relapse, often with disease that is resistant to further treatment. Tumours displaying serous histology are the most common and often have the worst prognosis. The ATP-binding cassette (ABC) transporter superfamily consists of 48 functional transmembrane proteins which are further classified into seven subfamilies according to sequence similarity, designated A through G. Various members of the B, C and G branches are well-known for their abilities to convey drug resistance to cancer cells in vitro, but their precise roles in determining clinical outcome are still unclear. In addition, ABC transporters have important and diverse physiological roles through active transport of a variety of endogenous substrates, which could also contribute to tumour phenotype and treatment response. This is the first study to explore the entire ABC transporter gene family in relation to clinical outcome of ovarian cancer. Using a Taqman low density array format for real-time PCR, we examined expression of all ABC transporter genes in a cohort of 150 serous ovarian cancers. Kaplan-Meier survival analysis revealed that expression of multiple ABCA family transporters was significantly associated with progression-free or overall survival and multivariate modelling identified ABCA1, ABCA5, ABCA6, ABCA8 and ABCA9 as new prognostic markers for serous ovarian cancer that are independent of established clinical indicators. The prognostic significance of ABCA family transporter expression was validated in an independent microarray dataset consisting of 350 serous tumours available through The Cancer Genome Atlas. These ABCA family transporters are not known to transport drugs but instead function in cellular lipid trafficking and cholesterol homeostasis, suggesting that treatment failure may involve mechanisms other than simple efflux of chemotherapeutic drugs. In light of accumulating evidence for the importance of cholesterol and bioactive lipids in several cancers, this study highlights an important area for investigation into the biology and treatment of serous ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1660. doi:10.1158/1538-7445.AM2011-1660

Background MAL2 protein is frequently overexpressed in breast carcinoma, and MAL2 overexpression is associated with gain of the corresponding locus at chromosome 8q24.12. Independent expression microarray studies predict MAL2 overexpression in ovarian carcinoma, but this has not been confirmed. MAL2 binds tumour protein D52 (TPD52), which is frequently overexpressed in ovarian carcinoma, but the clinical significance of TPD52 over-expression is unknown. We sought to assess immunohistochemical expression of MAL2 and TPD52 in a range of ovarian tumours, and to correlate their expression with survival. Methods Immunohistochemical analyses of MAL2 and TPD52 expression were performed on tissue microarray sections of benign, borderline and malignant epithelial ovarian tumours. Staining intensity and distribution was assessed both visually and digitally. Results MAL2 and TPD52 expression was significantly higher in high-grade serous carcinomas (SCs) than in serous borderline tumours. MAL2 expression was highest in SCs relative to other histological subtypes, whereas TPD52 expression was highest in clear cell carcinomas. High-level TPD52 expression was significantly associated with improved overall survival in stage III SC patients (log-rank test, p n = 124), and was an independent predictor of improved survival in the overall carcinoma cohort (hazard ratio 0.498, 95%CI 0.34–0.73; p n = 221) and in SCs (hazard ratio 0.44; 95%CI 0.29–0.66; p n = 182). Conclusions MAL2 overexpression is frequent in ovarian carcinoma, and TPD52 overexpression is a favourable prognostic marker.

Background: Tumor protein D52 (D52) overexpression is characteristic of both adult and childhood cancers and has been associated with reduced overall survival in breast cancer patients and an increase in proliferation and metastatic capacity of transfected cell lines. However, little is known about the mechanism(s) by which D52 overexpression contributes to cellular transformation or tumour progression. Aims: We aimed to identify genes which are differentially expressed in response to D52 expression in 3T3 fibroblasts, and to use these gene expression changes to predict cellular phenotypes which are altered by D52 expression. Methods: Gene expression profiling was performed using Affymetrix expression microarray analysis. Up- and down-regulated gene lists were analysed using GOSTAT, Metacore and literature searches. Differential expression of candidate proteins were validated using Western blot analysis of intracellular protein extracts or precipitates from conditioned media. Migration and invasion assays were performed using Boyden chambers. Results: D52 expression was associated with over 300 genes being differentially expressed at least two-fold relative to vector controls. A number of overexpressed genes were found to be normally involved in the inflammatory response. We focused on validating candidates such as COX-2, MMP-2 and Osteopontin as being overexpressed in D52-transfected cells relative to vector controls. We also confirmed an upregulation of phospho-AKT in D52 expressing cells, as reported by others. GOSTAT analysis revealed that gene ontology terms related to the extracellular matrix (ECM) were most over-represented amongst differentially expressed genes. The implied resultant ECM re-modelling was examined through migration and invasion assays, in which D52 expressing cells showed a significantly increased migratory and invasive capacity relative to vector control lines. Conclusions: These results implicate D52 as a pro-inflammatory intermediate, whose expression may act to drive cancer progression via an increase in phospho-AKT, followed by the production of inflammatory molecules such as Osteopontin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2338.

Identifying patients prior to treatment that are less likely to benefit from or most likely to experience adverse events from chemotherapeutic agents is essential. Even though humans are the most relevant system, pharmacogenomic discovery in the field of oncology is plagued by difficulties in executing large clinical trials and confounding factors such as co-morbidities, dosage, and concomitant medications. Therefore, we developed a genome-wide cell-based approach evaluating single nucleotide polymorphisms (SNPs) and gene expression to predict chemotherapy-induced response and toxicity and then validated our findings in clinically relevant patient cohorts. Our model utilizes the International HapMap lymphoblastoid cell lines (LCLs). Genome-wide association studies were performed to identify SNPs significantly associated with carboplatin sensitivity through their effects on mRNA expression. The significant findings were evaluated in an independent LCL replication set and in patient samples obtained from the Australian Ovarian Cancer Study (AOCS) and two Phase II head and neck clinical trials (UC12019 and UC13881) conducted at the University of Chicago. Four hundred and nine ovarian cancer patients receiving carboplatin and paclitaxel were analyzed in AOCS; while 60 and 32 head and neck cancer patients were evaluated in UC12019 and UC13881 trials. Carboplatin was used as induction and concomitant chemo-radiation therapy in UC12019 and UC13881 trials, respectively. Using LCLs, our genome-wide model identified 65 SNPs that are associated with at least one carboplatin sensitivity phenotype through the expression of 61 genes. Five of them were replicated in a separate set of LCLs. In AOCS, SNP (rs1649942) was significantly associated with progression-free survival (Plog-rank=0.009) and overall survival (Plog-rank=0.03). In the head and neck cancer trials, 2 other SNP-phenotype associations were identified and replicated in both trials, including the association between SNP (rs4946514) and overall response to carboplatin, and the association between SNP (rs7134205) and post treatment platelet changes. Given the obstacles to performing large, replicable pharmacogenomic studies in patients, the cell-based model is proven to be an effective alternative in novel pharmaco-SNP discovery. We demonstrate germline SNPs identified through the cell-based genome-wide approach are clinically important predictors of chemotherapy response and toxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2761.

Despite a high initial response rate to first-line platinum/paclitaxel chemotherapy, most women with epithelial ovarian cancer relapse with recurrent disease that becomes refractory to further cytotoxic treatment. We have previously shown that the E3 ubiquitin ligase, EDD, a regulator of DNA damage responses, is amplified and overexpressed in serous ovarian carcinoma. Given that DNA damage pathways are linked to platinum resistance, the aim of this study was to determine if EDD expression was associated with disease recurrence and platinum sensitivity in serous ovarian cancer. High nuclear EDD expression, as determined by immunohistochemistry in a cohort of 151 women with serous ovarian carcinoma, was associated with an approximately two-fold increased risk of disease recurrence and death in patients who initially responded to first-line chemotherapy, independently of disease stage and suboptimal debulking. Although EDD expression was not directly correlated with relative cisplatin sensitivity of ovarian cancer cell lines, sensitivity to cisplatin was partially restored in platinum-resistant A2780-cp70 ovarian cancer cells following siRNA-mediated knockdown of EDD expression. These results identify EDD as a new independent prognostic marker for outcome in serous ovarian cancer, and suggest that pathways involving EDD, including DNA damage responses, may represent new therapeutic targets for chemoresistant ovarian cancer.

A lymphocyte clonal assay developed to quantitate in vivo somatic cell mutations at the hypoxanthine-guanine phosphoribosyltransferase locus was modified in order to study resistance to methotrexate. Even though nucleoside-free culture conditions were used methotrexate was not lethal to lymphocytes plated into micro-wells at greater than 10(2) cells/well. HPLC analysis of supernatants from wells plated initially with 10(4) cells/well in 100 microM methotrexate revealed the presence of micro-molar levels of hypoxanthine and thymidine by the 5th and 8th day of culture respectively. When lymphocytes were plated at less than or equal to 10(2) cells/well in nucleoside free medium, methotrexate was cytotoxic and micro-molar levels of thymidine together with hypoxanthine protected lymphocytes cultured under these conditions from toxicity. Modulation of nucleic acid antimetabolite cytotoxicity by nucleosides and bases has been recognised for some years. Nucleoside free culture conditions have been advocated for studying cellular sensitivity to antifolates to avoid such interfering factors. However our results indicate that metabolites from dying or damaged cells can prevent methotrexate cytotoxicity, further complicating the development of a suitable clonogenic assay for investigating antifolate sensitivity.

A rapid estimation of mutation frequency in tumours would be invaluable in cancer management. Considerable evidence suggests that drug resistance in tumours frequently arises as a consequence of spontaneous somatic mutation (Goldie & Coldman, 1979) and therefore the mutation rate of a tumour will be an index of the potential to develop drug resistance. Previous studies have compared the mutation rates of cell lines using cloning assays. Cifone & Fidler (1981) reported a higher mutation rate in metastatic variant of UV-2237 fibrosarcoma cells than in a clone of the same cell line with lower metastatic potential. Warren et al. (1981) reported that fibroblasts from patients with Bloom syndrome, which predisposes individuals to various cancers, had a 10-15 fold higher mutation rate than did fibroblasts from normal individuals. These findings have been important in correlating malignant capacity with genetic instability, but unfortunately, the techniques used have limited general application since few human tumour cells will form colonies on plastic. An alternative assay, soft agar cloning, has also been used for the determination of mutation rates in a variety of mammalian cell lines, including Chinese hamster ovary cells (Li & Shimizu, 1983) and the L5178Y mouse lymphoma cell line (Irr & Snee, 1982). Again, the low cloning efficiency of human tumour cells in soft agar (Hamburger et al., 1978) and the fact that this method selects only mutant cells which clone in agar limits the use of this assay to determine mutation frequency in human tumours. This latter source of error might bias the estimation of the mutation rate in favour of more malignant cells, which generally have higher cloning efficiencies (Elmore et al., 1983). Morley et al. (1983) and Albertini et al. (1982) have reported a limiting dilution technique for the measurement of mutation frequency in human lymphocytes. Culture conditions have been optimised for peripheral blood lymphocytes and a cloning efficiency of 20-60% has been obtained. Problems have been encountered, however, in studies of cultured lymphoblast lines with variable

Background: Median overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is similar to 4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC. Patients and methods: Expression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies. Results: Expression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years. Conclusion: The OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches.