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Background: Ovarian cancer is a leading cause of cancer-related death among women. In an effort to understand contributors to disease outcome, we evaluated single-nucleotide polymorphisms (SNP) previously associated with ovarian cancer recurrence or survival, specifically in angiogenesis, inflammation, mitosis, and drug disposition genes. Methods: Twenty-seven SNPs in VHL, HGF, IL18, PRKACB, ABCB1, CYP2C8, ERCC2, and ERCC1 previously associated with ovarian cancer outcome were genotyped in 10,084 invasive cases from 28 studies from the Ovarian Cancer Association Consortium with over 37,000-observed person-years and 4,478 deaths. Cox proportional hazards models were used to examine the association between candidate SNPs and ovarian cancer recurrence or survival with and without adjustment for key covariates. Results: We observed no association between genotype and ovarian cancer recurrence or survival for any of the SNPs examined. Conclusions: These results refute prior associations between these SNPs and ovarian cancer outcome and underscore the importance of maximally powered genetic association studies. Impact: These variants should not be used in prognostic models. Alternate approaches to uncovering inherited prognostic factors, if they exist, are needed. Cancer Epidemiol Biomarkers Prev; 22(5); 987–. ©2013 AACR.

Enzymatic factors driving cancer-associated chromatin remodelling are of increasing interest as the role of the cancer epigenome in gene expression and DNA repair processes becomes elucidated. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) is a central histone modification that functions in histone cross-talk, transcriptional elongation, DNA repair, maintaining centromeric chromatin and replication-dependent histone mRNA 3'-end processing, as well as being required for the differentiation of stem cells. The loss of global H2Bub1 is seen in a number of aggressive malignancies and has been linked to tumour progression and/or a poorer prognosis in some cancers. Here, we analyse a large cohort of high-grade serous ovarian cancers (HGSOC) and show loss of global H2Bub1 in 77% (313 of 407) of tumours. Loss of H2Bub1 was seen at all stages (I-IV) of HGSOC, indicating it is a relatively early epigenomic event in this aggressive malignancy. Manipulation of key H2Bub1 E3 ubiquitin ligases, RNF20, RNF40 and BRCA1, in ovarian cancer cell line models modulated H2Bub1 levels, indicative of the role of these RING finger ligases in monoubiquitination of H2Bub1 in vitro. However, in primary HGSOC, loss of RNF20 protein expression was identified in just 6% of tumours (26 of 424) and did not correlate with global H2Bub1 loss. Similarly, germline mutation of BRCA1 did not show a correlation with the global H2Bub1 loss. We conclude that the regulation of tumour-associated H2Bub1 levels is complex. Aberrant expression of alternative histone-associated 'writer' or 'eraser' enzymes are likely responsible for the global loss of H2Bub1 seen in HGSOC.

Ovarian Cancer Association Consortium, Breast Cancer Association Consortium, and Consortium of Modifiers of BRCA1 and BRCA2: et al., [Objective]: Clinical genetic testing is commercially available for rs61764370, an inherited variant residing in a KRAS 3' UTR microRNA binding site, based on suggested associations with increased ovarian and breast cancer risk as well as with survival time. However, prior studies, emphasizing particular subgroups, were relatively small. Therefore, we comprehensively evaluated ovarian and breast cancer risks as well as clinical outcome associated with rs61764370. [Methods]: Centralized genotyping and analysis were performed for 140,012 women enrolled in the Ovarian Cancer Association Consortium (15,357 ovarian cancer patients; 30,816 controls), the Breast Cancer Association Consortium (33,530 breast cancer patients; 37,640 controls), and the Consortium of Modifiers of BRCA1 and BRCA2 (14,765 BRCA1 and 7904 BRCA2 mutation carriers). [Results]: We found no association with risk of ovarian cancer (OR = 0.99, 95% CI 0.94-1.04, p = 0.74) or breast cancer (OR = 0.98, 95% CI 0.94-1.01, p = 0.19) and results were consistent among mutation carriers (BRCA1, ovarian cancer HR = 1.09, 95% CI 0.97-1.23, p = 0.14, breast cancer HR = 1.04, 95% CI 0.97-1.12, p = 0.27; BRCA2, ovarian cancer HR = 0.89, 95% CI 0.71-1.13, p = 0.34, breast cancer HR = 1.06, 95% CI 0.94-1.19, p = 0.35). Null results were also obtained for associations with overall survival following ovarian cancer (HR = 0.94, 95% CI 0.83-1.07, p = 0.38), breast cancer (HR = 0.96, 95% CI 0.87-1.06, p = 0.38), and all other previously-reported associations. [Conclusions]: rs61764370 is not associated with risk of ovarian or breast cancer nor with clinical outcome for patients with these cancers. Therefore, genotyping this variant has no clinical utility related to the prediction or management of these cancers., The COGS project is funded through a European Commission's Seventh Framework Programme grant (agreement number 223175 — HEALTH-F2-2009-223175). The scientific development and funding for this project were in part supported by the US National Cancer Institute GAME-ON Post-GWAS Initiative (U19-CA148112). Funding for the project was provided by the Wellcome Trust under award 076113. This work was partially supported by Spanish Association against Cancer (AECC08), RTICC 06/0020/1060, FISPI08/1120, Mutua Madrileña Foundation (FMMA) and SAF2010-20493. Was supported by a grant RD12/00369/0006 and 12/00539 from ISCIII (Spain), partially supported by European Regional Development FEDER funds. Contract grant sponsor: Asociación Española Contra el Cáncer, Spanish Health Research Fund; Carlos III Health Institute; Catalan Health Institute and Autonomous Government of Catalonia. Contract grant numbers: ISCIIIRETIC RD06/0020/1051, RD12/0036/008, PI10/01422, PI10/00748, PI13/00285 and 2009SGR290.

Background: Many epithelial ovarian cancer (EOC) risk factors relate to hormone exposure and elevated estrogen levels are associated with obesity in postmenopausal women. Therefore, we hypothesized that gene–environment interactions related to hormone-related risk factors could differ between obese and non-obese women. Methods: We considered interactions between 11,441 SNPs within 80 candidate genes related to hormone biosynthesis and metabolism and insulin-like growth factors with six hormone-related factors (oral contraceptive use, parity, endometriosis, tubal ligation, hormone replacement therapy, and estrogen use) and assessed whether these interactions differed between obese and non-obese women. Interactions were assessed using logistic regression models and data from 14 case–control studies (6,247 cases; 10,379 controls). Histotype-specific analyses were also completed. Results: SNPs in the following candidate genes showed notable interaction: IGF1R (rs41497346, estrogen plus progesterone hormone therapy, histology = all, P = 4.9 × 10−6) and ESR1 (rs12661437, endometriosis, histology = all, P = 1.5 × 10−5). The most notable obesity–gene–hormone risk factor interaction was within INSR (rs113759408, parity, histology = endometrioid, P = 8.8 × 10−6). Conclusions: We have demonstrated the feasibility of assessing multifactor interactions in large genetic epidemiology studies. Follow-up studies are necessary to assess the robustness of our findings for ESR1, CYP11A1, IGF1R, CYP11B1, INSR, and IGFBP2. Future work is needed to develop powerful statistical methods able to detect these complex interactions. Impact: Assessment of multifactor interaction is feasible, and, here, suggests that the relationship between genetic variants within candidate genes and hormone-related risk factors may vary EOC susceptibility. Cancer Epidemiol Biomarkers Prev; 25(5); 780–90. ©2016 AACR.

High-grade serous cancer (HGSC), the most common subtype of ovarian cancer, often becomes resistant to chemotherapy, leading to poor patient outcomes. Intratumoral heterogeneity occurs in nearly all solid cancers, including ovarian cancer, contributing to the development of resistance mechanisms. In this study, we examined the spatial and temporal genomic variation in HGSC using high-resolution single-nucleotide polymorphism arrays. Multiple metastatic lesions from individual patients were analyzed along with 22 paired pretreatment and posttreatment samples. We documented regions of differential DNA copy number between multiple tumor biopsies that correlated with altered expression of genes involved in cell polarity and adhesion. In the paired primary and relapse cohort, we observed a greater degree of genomic change in tumors from patients that were initially sensitive to chemotherapy and had longer progression-free interval compared with tumors from patients that were resistant to primary chemotherapy. Notably, deletion or downregulation of the lipid transporter LRP1B emerged as a significant correlate of acquired resistance in our analysis. Functional studies showed that reducing LRP1B expression was sufficient to reduce the sensitivity of HGSC cell lines to liposomal doxorubicin, but not to doxorubicin, whereas LRP1B overexpression was sufficient to increase sensitivity to liposomal doxorubicin. Together, our findings underscore the large degree of variation in DNA copy number in spatially and temporally separated tumors in HGSC patients, and they define LRP1B as a potential contributor to the emergence of chemotherapy resistance in these patients. Cancer Res; 72(16); 4060–73. ©2012 AACR.

Purpose: Serous ovarian carcinomas are the predominant epithelial ovarian cancer subtype and it has been widely believed that some or all of these may arise from precursors derived from the ovarian surface epithelium or fimbriae, although direct molecular evidence for this is limited. This study aimed to conduct copy number (CN) analysis using a series of benign and borderline serous ovarian tumors to identify underlying genomic changes that may be indicative of early events in tumorigenesis. Experimental Design: High resolution CN analysis was conducted on DNA from the epithelial and fibroblast components of a cohort of benign (N = 39) and borderline (N = 24) serous tumors using the Affymetrix OncoScan assay and SNP6.0 arrays. Results: CN aberrations were detected in the epithelium of only 2.9% (1 of 35) of serous cystadenomas and cystadenofibromas. In contrast, CN aberrations were detected in the epithelium of 67% (16 of 24) of the serous borderline tumors (SBT). Unexpectedly, CN aberrations were detected in the fibroblasts of 33% (13 of 39) of the benign serous tumors and in 15% (3 of 20) of the SBTs. Of the 16 cases with CN aberrations in the fibroblasts, 12 of these carried a gain of chromosome 12. Conclusions: Chromosome 12 trisomy has been previously identified in pure fibromas, supporting the concept that a significant proportion of benign serous tumors are in fact primary fibromas with an associated cystic mass. This is the first high resolution genomic analysis of benign serous ovarian tumors and has shown not only that the majority of benign serous tumors have no genetic evidence of epithelial neoplasia but that a significant proportion may be more accurately classified as primary fibromas. Clin Cancer Res; 17(23); 7273–82. ©2011 AACR.

Purpose: Cell-based approaches were used to identify genetic markers predictive of patients' risk for poor response prior to chemotherapy. Experimental Design: We conducted genome-wide association studies (GWAS) to identify single-nucleotide polymorphisms (SNP) associated with cellular sensitivity to carboplatin through their effects on mRNA expression using International HapMap lymphoblastoid cell lines (LCL) and replicated them in additional LCLs. SNPs passing both stages of the cell-based study were tested for association with progression-free survival (PFS) in patients. Phase 1 validation was based on 377 ovarian cancer patients receiving at least four cycles of carboplatin and paclitaxel from the Australian Ovarian Cancer Study (AOCS). Positive associations were then assessed in phase 2 validation analysis of 1,326 patients from the Ovarian Cancer Association Consortium and The Cancer Genome Atlas. Results: In the initial GWAS, 342 SNPs were associated with carboplatin-induced cytotoxicity, of which 18 unique SNPs were retained after assessing their association with gene expression. One SNP (rs1649942) was replicated in an independent LCL set (Bonferroni adjusted P < 0.05). It was found to be significantly associated with decreased PFS in phase 1 AOCS patients (Pper-allele = 2 × 10−2), with a stronger effect in the subset of women with optimally debulked tumors (Pper-allele = 4 × 10−3). rs1649942 was also associated with poorer overall survival in women with optimally debulked tumors (Pper-allele = 9 × 10−3). However, this SNP was not significant in phase 2 validation analysis with patients from numerous cohorts. Conclusion: This study shows the potential of cell-based, genome-wide approaches to identify germline predictors of treatment outcome and highlights the need for extensive validation in patients to assess their clinical effect. Clin Cancer Res; 17(16); 5490–500. ©2011 AACR.

A genome-wide association study (GWAS) identified single-nucleotide polymorphisms (SNPs) at 1p11.2 and 14q24.1 (RAD51L1) as breast cancer susceptibility loci. The initial GWAS suggested stronger effects for both loci for estrogen receptor (ER)-positive tumors. Using data from the Breast Cancer Association Consortium (BCAC), we sought to determine whether risks differ by ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), grade, node status, tumor size, and ductal or lobular morphology. We genotyped rs11249433 at 1p.11.2, and two highly correlated SNPs rs999737 and rs10483813 (r(2) = 0.98) at 14q24.1 (RAD51L1), for up to 46 036 invasive breast cancer cases and 46 930 controls from 39 studies. Analyses by tumor characteristics focused on subjects reporting to be white women of European ancestry and were based on 25 458 cases, of which 87% had ER data. The SNP at 1p11.2 showed significantly stronger associations with ER-positive tumors [per-allele odds ratio (OR) for ER-positive tumors was 1.13, 95% CI = 1.10-1.16 and, for ER-negative tumors, OR was 1.03, 95% CI = 0.98-1.07, case-only P-heterogeneity = 7.6 x 10 25]. The association with ER-positive tumors was stronger for tumors of lower grade (case-only P = 6.7 x 10 23) and lobular histology (case-only P = 0.01). SNPs at 14q24.1 were associated with risk for most tumor subtypes evaluated, including triple-negative breast cancers, which has not been described previously. Our results underscore the need for large pooling efforts with tumor pathology data to help refine risk estimates for SNP associations with susceptibility to different subtypes of breast cancer.

In the original publication of this article on page 6, paragraph “Discussion”, line 4, ‘In a U.S. population-based case–control study (n = 493 cases) Peres et al., reported a non-significant association between DII score and risk of developing ovarian cancer of similar magnitude (OR DII score 1.35, 95% CI 0.93–1.97) [20]’. It should read as ‘In a U.S. population-based case–control study (n = 493 cases) Peres et al., reported a significant association between DII score and risk of developing ovarian cancer (OR DII score 1.72, 95% CI 1.18–2.51) [20]’.

Tubo-ovarian high-grade serous cancer (HGSC) is the most common histotype of epithelial ovarian cancer (70%-80%) and the leading cause of death from gynecological malignancy. Novel monoclonal antibody and adoptive cell transfer immunotherapies have found success in cancer in the past five years; however success in HGSC treatment has been elusive thus far. We have recently shown that CD8+ T-lymphocytes infiltrating the tumor proper (TILs) associate in a dose response manner with longer patient survival time regardless of age, stage or residual disease. As understanding host factors associated with this clinically relevant tumor phenotype will contribute to the development of targeted therapies, we sought to identify germline genetic factors associated with CD8+ TIL levels in HGSC. We performed a germline genome-wide association study (GWAS) of CD8+ TILs using 1204 HGSC patients of European ancestry from twelve studies with centralized genotyping, immunohistochemistry, and harmonization of clinical data. Germline genotypes were assayed using the Illumina Infinium OncoArray Beadchip and were imputed to over 11.4 million variants based on the 1000 Genomes Project reference panel. CD8 immunohistochemistry was performed on tissue microarrays using the Leica Bond Rx stainer and scored into four levels of CD8+ TILs per x400 magnification of a 0.55-mm diameter field for each tumor hotspot: none (no CD8+ TILs), low (1-2 CD8+ TILs), moderate (3-19 CD8+ TILs), high (20 or more CD8+ TILs). GWAS used multinomial regression modelling of these four CD8+ TIL levels adjusted for study site, age, and European ancestry principal components. On the average, CD8+ TIL levels were 18%, 19%, 44% and 18% for none, low, moderate and high levels, respectively. As expected, CD8+ TIL levels associated with overall survival time (p < 2.6x10-8), and CD8+ TIL levels and age at diagnosis were similarly distributed across study sites. We identified a region on chromosome 22 with multiple, correlated variants (MAF > 20%) associated with CD8+ TIL levels at p-value < 2.0x10-5. The most statistically significant variant was an indel (rs557925408) located in the an intron of the MYO18B gene (Myosin XVIIIB, p-value < 8.7x10-6). Each copy of this variant associated with a reduced extent of CD8+ TIL infiltration (OR low v none = 0.92; OR moderate v none = 0.61). An independent set of 1200 OncoArray genotyped HGSC cases are undergoing CD8+ TIL scoring in December 2017 and will be included in final genome-wide analysis. This will provide improved statistical power and enable consideration of additional genetic models (e.g., linear trend tests). Comprehensive integrative analysis of germline, tumor, and clinical features provides a model for clinical molecular epidemiology studies and will be key to increasing our understanding of this important HGSC immunophenotype. Citation Format: Yanina Natanzon, Martin Köbel, Stacey J. Winham, Sebastian M. Armasu, Bryan M. McCauley, Robert A. Vierkant, Julie M. Cunningham, David Bowtell, Ian G. Campbell, Jenny Chang-Claude, Anna deFazio, Peter A. Fasching, Mark T. Goodman, Beth Y. Karlan, Francesmary Modugno, Kirsten B. Moysich, Roberta B. Ness, Weiva Sieh, Paul D. Pharoah, Susan J. Ramus, Ellen L. Goode. Tumor-infiltrating CD8-positive T-lymphocytes in tubo-ovarian high-grade serous cancer are associated with multiple germline variants in 22q12.1 in a genome-wide association analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 225.

Low-grade serous ovarian cancer (LGSC) responds poorly to platinum based chemotherapy and is characterized by activating RAS-MAPK pathway mutations, including oncogenic BRAF. Drugs that target this pathway are effective in BRAF-mutant melanoma but other cancer types, such as colorectal cancers, are less sensitive. Early phase trials report a 15% response rate to MEK inhibitors in LGSC patients, however it is not known which features may predict response. We aimed to determine clinical characteristics and treatment response in women with LGSC and to determine whether response to targeted pathway inhibition is associated with specific mutation profiles in LGSC cell lines. Tumor samples from a cohort of grade 1 and 2 serous ovarian cancer patients were analyzed using targeted, exome or whole genome sequencing. Patient characteristics, treatment and clinical outcome were assessed. Cell lines derived from patients with LGSC with known mutation profiles, AOCS2 (KRAS/BRAF/NRAS wild-type), MPSC1 (BRAFV600L, NRASQ16R), VOA1056 (NRASQ16R) and HCC5075 (KRASG12V), were treated with BRAF (dabrafenib) or MEK inhibitors (trametinib, pimasertib and binimetinib) and response was compared to cell lines derived from high-grade serous cancer (HGSC) and BRAF-mutant melanoma. Women diagnosed with grade 1 or 2 serous carcinoma between 1994-2015 were identified from 1654 invasive ovarian serous cancer cases in the Australian Ovarian Cancer Study and the GynBiobank. HGSC cases were excluded following histopathology review and TP53 mutation screening. Amongst 65 confirmed LGSC patients, 18 (27.7%) had a KRAS mutation, 9 (13.8%) had a BRAF mutation and 7 (10.8%) had a NRAS mutation. Women with advanced stage LGSC and residual disease following debulking surgery had a similarly poor progression-free and overall-survival compared with HGSC patients. We saw a dramatic response to BRAF inhibition in a patient with BRAFV600E-positive LGSC, however, the LGSC cell lines did not respond to dabrafenib. This is not surprising as the cell lines did not harbour the hot-spot BRAFV600E mutation. MPSC1 has a BRAFV600L and a NRASQ16R mutation, but dabrafenib did not inhibit growth. HCC5075 (KRASG12V) was sensitive to all three MEK inhibitors, but response to MEK inhibition in the other LGSC cell lines was modest. In conclusion, LGSC are generally chemotherapy resistant and molecular analyses can identify targetable mutations. However, LGSC are heterogenous with respect to underlying mutations and response to pathway inhibitors is likely to depend on which mutations and pathways are activated. BRAF mutations are not uncommon in patients with LGSC and should be routinely tested as BRAF inhibitors can be an effective treatment for these patients. MEK inhibitors may also be effective in a subset of cases. The results highlight the need for novel clinical trial design, as traditional clinical trials are unlikely to be effective in rare ovarian cancer sub-types. Citation Format: Tania Moujaber, Dariush Etemadmoghadam, Cristina Mapagu, Catherine Kennedy, Yoke-Eng Chiew, Casina Kan, Nikilyn Nevins, Sivatharsny Srirangan, Sian Fereday, Nadia Traficante, Australian Ovarian Cancer Study group, David Bowtell, Rosemary Balleine, Paul Harnett, Anna deFazio. Mutations in low-grade serous ovarian cancer and response to BRAF and MEK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2584.

Objective:As treatment costs for gynecological cancer escalate, real-world data on use of resources and costs becomes increasingly important. This study investigated medical costs, quality of life, and survival end points for women with ovarian cancer in Australia.Methods:Women with primary epithelial ovarian cancer referred for chemotherapy (n = 85) were recruited through 7 hospitals in Australia. Overall survival, progression-free interval, and quality-adjusted life years were assessed by stage using the Cox proportional hazards models. Direct medical costs, including those for surgeries, hospitalizations, supportive care, chemotherapy, and adverse effects (while on chemotherapy), were calculated over 2.5 years and assessed by nonparametric bootstrapping.Results:Quality-adjusted life years decreased with increased disease stage at diagnosis and ranged from 2.3 for women with stage I or II disease to 1.3 for those with stage IV disease. A total of AU $4.1 million (2008) were spent on direct medical costs for 85 women over approximately 2.5 years. Medical costs were significantly higher for women with stage III or IV disease compared with that for women with stage I or II disease ($50,945 vs $31,958,P< 0.01) and/or women who experienced surgical complications and/or adverse effects requiring hospitalization while on chemotherapy ($57,821 vs $34,781,P< 0.01). Costs after first-line chemotherapy were significantly higher for women with advanced disease (mean, $20,744) compared with those for women with early disease (mean, $5525;P< 0.01).Conclusions:Whereas for women with early-stage ovarian cancer, costs are concentrated in the period of primary treatment, cumulated costs are especially high for women with recurrent disease rising rapidly after first-line therapy.

10062Background: Patients with OC are followed clinically every 3 months (m) after 1LT with the aim of managing symptoms/late AEs and diagnosing recurrence. Studies report that clinicians are unawa...

10081Background: Women with ovarian cancer (OC) report high rates of anxiety (A) and depression (D), but most studies have used a cross-sectional design at a single time-point, not considered prior...

155 Background: Women with ovarian cancer (OC) report high rates of anxiety (A) and depression (D), but most studies have used a cross-sectional design at a single time-point, not considered prior history and not included affected women who are symptom-free due to medication. Our goals were to quantify (i) the total burden of A and D among women with newly diagnosed OC; the proportions who (ii) experience symptoms only after their OC diagnosis and (iii) with persistent symptoms; and (iv) use of appropriate medication/services by those affected. Methods: The OPAL (Ovarian Cancer Prognosis & Lifestyle) Study is a prospective study of Australian women diagnosed with OC from 2012-15 who agreed to complete questionnaires at baseline, 3, 6, 9, 12, 24, 36 & 48 months after diagnosis. At baseline, they were asked if they had ever been diagnosed with A or D and if they took medication for this in the year before their OC diagnosis. At follow-up they completed the Hospital Anxiety and Depression Scale (HADS) and were asked about current medication use. Results: Of 893 women with data for ≥1 time point, 216 (24%) reported clinical levels (HADS > 10) of anxiety (18%) and/or depression (15%) on at least one occasion during the first 3 years after diagnosis, with another 157 (18%) reporting use of anxiolytic or antidepressant medications. A further 167 (19%) reported subclinical (HADS 8-10) A or D. Of those with clinical levels of A/D and/or taking medication, 225 (60%) reported this at ≥3 time-points, 216 (58%) reported no prior history of A or D, and 271 (73%) reported no use of anxiolytic/antidepressant medication in the year before diagnosis. When women reported clinical levels of A or D, only 45% reported taking medication (37%) and/or seeing a psychiatrist or psychologist (19%). Conclusions: More than 40% of women with OC experienced clinical levels of A or D during treatment or the first 3 years of follow-up, for 25% this persisted. For 24% this was their first experience of distress and > 50% of those affected did not receive appropriate medication or psychological support. The hidden burden of anxiety and depression in this population is much greater than previously reported but is amenable to effective intervention if recognized.

88 Background: Five-year survival after a diagnosis of ovarian cancer (OC) is < 45% and women often ask what they can do, beyond following the recommended treatment, to improve their chance of survival. The OPAL Study addresses the question of whether lifestyle choices during or after treatment could positively impact quality of life (QoL) and, ultimately, survival. Methods: OPAL is a prospective study of Australian women newly diagnosed with primary OC from 2012-15 who agreed to complete questionnaires at baseline, 3, 6, 9, 12, 24, 36 & 48 months after diagnosis. Baseline data include sociodemographic, reproductive & hormonal, medical & family history, and lifestyle (diet, alcohol, smoking, physical activity, sitting time) information. At follow-up, women are asked about their current lifestyle, medication and complementary therapy use, and a range of patient-reported outcomes including the Hospital Anxiety and Depression Scale, Insomnia Severity Index and FACT instruments for ovarian cancer, neurotoxicity and fatigue. 85% also provided a blood sample at recruitment and/or 12 months after diagnosis. Detailed treatment and outcome data are collected annually from medical records. Results: Of 1451 eligible women identified, 219 were excluded and 274 declined leaving a cohort of 958; 72% have high-grade serous and 72% advanced stage disease. Follow-up is now close to 36 months with questionnaire response rates of 85-95% among active participants. At October 2017, we had received 734, 751, 636, 434 and 225 questionnaires at 6, 12, 24, 36 and 48 months, respectively; women have completed a mean of 5 questionnaires (range 1-8). A total of 558 (58%) have experienced disease progression or recurrence, 332 (35%) have died and 300 are still active in the study. The proportion of women reporting clinical levels of anxiety ranges from 6-9% at each time-point, depression from 5-7%, insomnia from 9-14% and fatigue from 30-57%. Conclusions: This is the first prospective study to collect detailed lifestyle, QoL, clinical and outcome information from a large population-based cohort of women newly diagnosed with OC. It provides a valuable resource to identify relationships between potentially modifiable aspects of lifestyle and outcomes (QoL & survival) in women with OC.

172 Background: Patients often ask if/when they will feel normal again following treatment for ovarian cancer (OC). There is a paucity of data on the trajectories of quality of life (QoL), physical (PWB), social (SWB), emotional (EWB) and functional (FWB) wellbeing over time following chemotherapy and especially regarding those who have persistent problems. Our aim was to quantify the proportion of women with significantly lower QoL/wellbeing than the general population at the end of treatment and determine if/when they return to normal. Methods: The OPAL (Ovarian cancer Prognosis & Lifestyle) Study is a prospective study of Australian women diagnosed with invasive OC from 2012-15 who agreed to complete regular questionnaires after diagnosis. 580 participants who received ≥3 cycles of platinum-based chemotherapy as primary treatment and completed a questionnaire while on or < 6 weeks after completing chemotherapy (baseline) were included. FACT-G data came from questionnaires at baseline and ~3, 6, 9 & 18 months post-baseline. Group-based trajectory models were used to identify groups with distinct patterns of QoL/wellbeing over time. Results: Overall, 44% (254) of women had QoL scores significantly lower than the general population at baseline; 35% (88) returned to normal by 3 months after treatment, 73% by 6 months and 27% (69) had not returned to normal by 18 months. The Table shows the comparable figures for the wellbeing subscales. Conclusions: While > 50% of women with OC can expect similar QoL, FWB and EWB to the general population at the end of chemotherapy, PWB was compromised in 3 of 4 women. For most, wellbeing recovered within 6 months but a substantial proportion reported ongoing deficits. A particularly prolonged impact was seen for those with poor EWB at baseline, warranting early intervention in this subset. [Table: see text]

Numerous studies have tested the association between TP53 mutations in ovarian cancer and prognosis but these have been consistently confounded by limitations in study design, methodology, and/or heterogeneity in the sample cohort. High-grade serous (HGS) carcinoma is the most clinically important histological subtype of ovarian cancer. As these tumours may arise from the ovary, Fallopian tube or peritoneum, they are collectively referred to as high-grade pelvic serous carcinoma (HGPSC). To identify the true prevalence of TP53 mutations in HGPSC, we sequenced exons 2–11 and intron–exon boundaries in tumour DNA from 145 patients. HGPSC cases were defined as having histological grade 2 or 3 and FIGO stage III or IV. Surprisingly, pathogenic TP53 mutations were identified in 96.7% (n = 119/123) of HGPSC cases. Molecular and pathological review of mutation-negative cases showed evidence of p53 dysfunction associated with copy number gain of MDM2 or MDM4, or indicated the exclusion of samples as being low-grade serous tumours or carcinoma of uncertain primary site. Overall, p53 dysfunction rate approached 100% of confirmed HGPSCs. No association between TP53 mutation and progression-free or overall survival was found. From this first comprehensive mapping of TP53 mutation rate in a homogeneous group of HGPSC patients, we conclude that mutant TP53 is a driver mutation in the pathogenesis of HGPSC cancers. Because TP53 mutation is almost invariably present in HGPSC, it is not of substantial prognostic or predictive significance. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Epithelial cell adhesion molecule (EpCAM)-targeted capture remains the most common isolation strategy for circulating tumor cells (CTCs). However, epithelial-to-mesenchymal transition (EMT) leads to decreased epithelial EpCAM expression affecting the optimal CTC capture. In this study, we tested a cohort of ovarian cancer cell lines using flow cytometry to identify N-cadherin as the additional immunomagnetic cell surface target for ovarian cancer cell isolation. Combined immunomagnetic targeting of mesenchymal N-cadherin and epithelial EpCAM enriched CTCs from advanced ovarian cancer patient blood approximately three times more efficiently than targeting of EpCAM alone. We also show that more EMT-phenotype CTCs are captured by including N-cadherin targeting into CTC isolation protocols. However, after N-cadherin-based CTC isolation, in some blood samples of healthy individuals, we also observed the presence of cells expressing markers common to CTCs. Our data show that these “false positives” can be largely distinguished from CTCs as circulating endothelial cells (CECs) by vascular endothelial–cadherin co-staining. CEC counts are highly variable in patients and healthy controls. Our data demonstrate that a combination of EpCAM with N-cadherin-targeted isolation can improve CTC detection and widen the EMT-phenotype spectrum of captured CTCs.

Purpose: The standard of care for ovarian cancer includes platinum-based chemotherapy. It is not possible, however, to predict clinical platinum sensitivity or to design rational strategies to overcome resistance. We used a novel approach to identify altered gene expression associated with high sensitivity to cisplatin, to define novel targets to sensitize tumor cells to platins and ultimately improve the effectiveness of this widely used class of chemotherapeutics. Experimental Design: Using differential display PCR, we identified genes differentially expressed in a mutagenized cell line with unusual sensitivity to cisplatin. The most highly differentially expressed gene was selected, and its role in determining cisplatin sensitivity was validated by gene transfection and small interfering RNA (siRNA) approaches, by association of expression levels with cisplatin sensitivity in cell lines, and by association of tumor expression levels with survival in a retrospective cohort of 71 patients with serous ovarian adenocarcinoma. Results: The most highly differently expressed gene identified was ANKRD1, ankyrin repeat domain 1 (cardiac muscle). ANKRD1 mRNA levels were correlated with platinum sensitivity in cell lines, and most significantly, decreasing ANKRD1 using siRNA increased cisplatin sensitivity >2-fold. ANKRD1 was expressed in the majority of ovarian adenocarcinomas tested (62/71, 87%), and higher tumor levels of ANKRD1 were found in patients with worse outcome (overall survival, P = 0.013). Conclusions: These findings suggest that ANKRD1, a gene not previously associated with ovarian cancer or with response to chemotherapy, is associated with treatment outcome, and decreasing ANKRD1 expression, or function, is a potential strategy to sensitize tumors to platinum-based drugs.

Approximately, 10% to 15% of serous ovarian tumors fall into the category designated as tumors of low malignant potential (LMP). Like their invasive counterparts, LMP tumors may be associated with extraovarian disease, for example, in the peritoneal cavity and regional lymph nodes. However, unlike typical invasive carcinomas, patients generally have a favorable prognosis. The mutational profile also differs markedly from that seen in most serous carcinomas. Typically, LMP tumors are associated with KRAS and BRAF mutations. Interrogation of expression profiles in serous LMP tumors suggested overall redundancy of RAS-MAPK pathway mutations and a distinct mechanism of oncogenesis compared with high-grade ovarian carcinomas. Our findings indicate that activating mutation of the RAS-MAPK pathway in serous LMP may be present in >70% of cases compared with ∼12.5% in serous ovarian carcinomas. In addition to mutations of KRAS (18%) and BRAF (48%) mutations, ERBB2 mutations (6%), but not EGFR, are prevalent among serous LMP tumors. Based on the expression profile signature observed throughout our serous LMP cohort, we propose that RAS-MAPK pathway activation is a requirement of serous LMP tumor development and that other activators of this pathway are yet to be defined. Importantly, as few nonsurgical options exist for treatment of recurrent LMP tumors, therapeutic targeting of this pathway may prove beneficial, especially in younger patients where maintaining fertility is important. (Mol Cancer Res 2008;6(11):1678–90)

Purpose: The study aim to identify novel molecular subtypes of ovarian cancer by gene expression profiling with linkage to clinical and pathologic features. Experimental Design: Microarray gene expression profiling was done on 285 serous and endometrioid tumors of the ovary, peritoneum, and fallopian tube. K-means clustering was applied to identify robust molecular subtypes. Statistical analysis identified differentially expressed genes, pathways, and gene ontologies. Laser capture microdissection, pathology review, and immunohistochemistry validated the array-based findings. Patient survival within k-means groups was evaluated using Cox proportional hazards models. Class prediction validated k-means groups in an independent dataset. A semisupervised survival analysis of the array data was used to compare against unsupervised clustering results. Results: Optimal clustering of array data identified six molecular subtypes. Two subtypes represented predominantly serous low malignant potential and low-grade endometrioid subtypes, respectively. The remaining four subtypes represented higher grade and advanced stage cancers of serous and endometrioid morphology. A novel subtype of high-grade serous cancers reflected a mesenchymal cell type, characterized by overexpression of N-cadherin and P-cadherin and low expression of differentiation markers, including CA125 and MUC1. A poor prognosis subtype was defined by a reactive stroma gene expression signature, correlating with extensive desmoplasia in such samples. A similar poor prognosis signature could be found using a semisupervised analysis. Each subtype displayed distinct levels and patterns of immune cell infiltration. Class prediction identified similar subtypes in an independent ovarian dataset with similar prognostic trends. Conclusion: Gene expression profiling identified molecular subtypes of ovarian cancer of biological and clinical importance.

Different subtypes of ovarian cancer appear to have different causes; however, the association between body mass index (BMI) and the different subtypes is unclear. We examined the associations between body-mass index (BMI) and weight gain and risk of the different histological subtypes of epithelial ovarian cancer in a case-control study in Australia. Cases aged 18-79 with a new diagnosis of invasive epithelial ovarian cancer (n = 1,269) or borderline tumor (n = 311) were identified through a network of clinics and cancer registries throughout Australia. Controls (n = 1,509) were selected from the Electoral Roll. Height and weight (1 year previously, at age 20 and maximum weight) and other risk factor information were ascertained via a self-administered questionnaire. Obesity was positively associated with clear cell tumors (Odds Ratio 2.3; 95% confidence interval 1.2-4.2) but not invasive endometrioid or mucinous tumors. Although there was no association with invasive serous tumors overall (0.9; 0.7-1.2), we did see an increased risk of serous peritoneal tumors (2.9; 1.7-4.9), but not of serous tumors of the ovary and fallopian tube. Of the borderline subtypes, obesity was positively associated with serous (1.8; 1.1-2.8) but not mucinous tumors (1.1; 0.7-1.7). Overweight was not associated with any subtype overall. There was no association with BMI at age 20, or weight gain for any of the histological subtypes. These results add to the current evidence that obesity increases a woman's risk of developing distinct histological subtypes of ovarian cancer.

Epithelial ovarian cancer, the leading cause of death from gynecological malignancy in Western countries, is thought to arise from the ovarian surface epithelium (OSE). It has been postulated that the constant rounds of proliferation and repair following ovulation contributes to neoplastic transformation. However, there is little information on the genes and pathways which are involved in the normal functions of the ovarian epithelium, in particular genes that are hormone responsive and those central to functions such as proliferation and apoptosis during ovulation. We used laser microdissection and cDNA microarrays to profile gene expression specifically in mouse ovarian epithelial cells, first compared with other ovarian cells, and secondly between ovarian epithelium collected at different physiological stages. We identified over 1000 transcripts that were consistently more highly expressed in the ovarian epithelium compared with remaining ovarian cell types, including genes involved in cell growth, transcription, and cell adhesion. At the various physiological stages examined, the highest number of regulated genes was found during the estrous cycle, specifically on the evening of proestrus, coincident with the ovulatory surge of hormones and just prior to ovulation. The expression of several selected genes, identified by the microarray analysis, including Villin 2, Keratin 8, Arginine-rich mutated in epithelial tumors, and Tumor-associated calcium signal transducer 1, was validated by independent methods. The identification of genes expressed and regulated in the OSE, and characterization of the pathways involved, will contribute to a more detailed understanding of the ovarian epithelium transcriptome and ultimately lead to a better understanding of the aberrations leading to malignant transformation in the ovarian epithelium.

Chronic inflammation has been proposed as the possible causal mechanism that explains the observed association between certain risk factors, such as the use of talcum powder (talc) in the pelvic region and epithelial ovarian cancer. To address this issue we evaluated the potential role of chronic local ovarian inflammation in the development of the major subtypes of epithelial ovarian cancer. Factors potentially linked to ovarian inflammation were examined in an Australia-wide case-control study comprising 1,576 women with invasive and low malignant potential (LMP) ovarian tumours and 1,509 population-based controls. We confirmed a statistically significant increase in ovarian cancer risk associated with use of talc in the pelvic region (adjusted odds ratio 1.17, 95% CI: 1.01-1.36) that was strongest for the serous and endometrioid subtypes although the latter was not statistically significant (adjusted odds ratios 1.21, 95% CI 1.03-1.44 and 1.18, 95% CI 0.81-1.70, respectively). Other factors potentially associated with ovarian inflammation (pelvic inflammatory disease, human papilloma virus infection and mumps) were not associated with risk but, like others, we found an increased risk of endometrioid and clear cell ovarian cancer only among women with a history of endometriosis. Regular use of aspirin and other nonsteroidal anti-inflammatory drugs was inversely associated with risk of LMP mucinous ovarian tumours only. We conclude that on balance chronic inflammation does not play a major role in the development of ovarian cancer.

Genome-wide association studies have reported 11 regions conferring risk of high-grade serous epithelial ovarian cancer (HGSOC). Expression quantitative trait locus (eQTL) analyses can identify candidate susceptibility genes at risk loci. Here we evaluate cis-eQTL associations at 47 regions associated with HGSOC risk (P≤10−5). For three cis-eQTL associations (P, Genome-wide association studies have identified regions which confer risk of high-grade serous epithelial ovarian cancer. Here the authors use expression quantitative train locus analysis to identify candidate genes and functionally characterise them, identifying a role for HOXD9 in ovarian cancer.

© 2015 Cancer Research UK. All rights reserved. Background: Observational studies have reported a modest association between obesity and risk of ovarian cancer; however, whether it is also associated with survival and whether this association varies for the different histologic subtypes are not clear. We undertook an international collaborative analysis to assess the association between body mass index (BMI), assessed shortly before diagnosis, progression-free survival (PFS), ovarian cancer-specific survival and overall survival (OS) among women with invasive ovarian cancer. Methods: We used original data from 21 studies, which included 12 390 women with ovarian carcinoma. We combined study-specific adjusted hazard ratios (HRs) using random-effects models to estimate pooled HRs (pHR). We further explored associations by histologic subtype. Results: Overall, 6715 (54%) deaths occurred during follow-up. A significant OS disadvantage was observed for women who were obese (BMI: 30-34.9, pHR: 1.10 (95% confidence intervals (CIs): 0.99-1.23); BMI: ≥35, pHR: 1.12 (95% CI: 1.01-1.25)). Results were similar for PFS and ovarian cancer-specific survival. In analyses stratified by histologic subtype, associations were strongest for women with low-grade serous (pHR: 1.12 per 5 kg m -2) and endometrioid subtypes (pHR: 1.08 per 5 kg m -2), and more modest for the high-grade serous (pHR: 1.04 per 5 kg m -2) subtype, but only the association with high-grade serous cancers was significant. Conclusions: Higher BMI is associated with adverse survival among the majority of women with ovarian cancer.

BARD1 is a breast cancer tumor suppressor with multiple domains and functions. BARD1 comprises a tandem BRCT domain at the C-terminus, and this sequence has been reported to target BARD1 to distinct subcellular locations such as nuclear DNA breakage sites and the centrosome through binding to regulatory proteins such as HP1 and OLA1, respectively. We now identify the BRCT domain as a binding site for p53. We first confirmed previous reports that endogenous BARD1 binds to p53 by immunoprecipitation assay, and further show that BARD1/p53 complexes locate at mitochondria suggesting a cellular location for p53 regulation of BARD1 apoptotic activity. We used a proximity ligation assay to map three distinct p53 binding sequences in human BARD1, ranging from weak (425-525) and modest (525-567) to strong (551-777 comprising BRCT domains). Deletion of the BRCT sequence caused major defects in the ability of BARD1 to (1) bind p53, (2) localize to the cytoplasm and mitochondria, and (3) induce Bax oligomerization and apoptosis. Our data suggest that BARD1 can move to mitochondria independent of p53, but subsequently associates with p53 to induce Bax clustering in part by decreasing mitochondrial Bcl-2 levels. We therefore identify a role for the BRCT domain in stimulating BARD1 nuclear export and mitochondrial localization, and in assembling mitochondrial BARD1/p53 complexes to regulate specific activities such as apoptotic function.

Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.

Objective. The ABC transporter MRP2 (ABCC2) can mediate cisplatin efflux, and over-expression of MRP2 has been associated with cisplatin resistance in cancer cell lines. The aim of this study was to determine the role of MRP2 in modulating cisplatin cytotoxicity in normal cells as well as the relationship between MRP2 expression and clinical response to platinum-based agents in ovarian cancer. Methods. The effect of absence of MRP2 expression on cisplatin sensitivity was investigated using primary hepatocyte cultures from the TR− rat strain, which is deficient in Mrp2. We also examined MRP2 expression immunohistochemically in human ovarian tumors exhibiting extremes of clinical response to platinum-based chemotherapy, either absolute platin resistance or patients with residual disease after surgery who experienced extremely long complete response to primary platinum-based chemotherapy. Results. Primary hepatocyte cultures from Mrp2-deficient TR− rats were over threefold more sensitive to cisplatin and accumulated a twofold greater amount of platinum on DNA that wild-type rat hepatocytes. In human ovarian carcinomas, MRP2 was detected by immunohistochemistry in 3/13 (23%) tumors from patients with absolute platin resistance compared with 5/9 (56%) tumors from patients with prolonged survival following treatment including a platinum-based agent. Conclusion. These studies indicate that MRP2 may play an important role in modulating normal tissue response to cisplatin. However, MRP2 expression occurred only in a subset of primary ovarian cancers, was frequently aberrant in location and was not correlated with clinical response to platinum-based chemotherapy.

Patients with high-grade serous ovarian cancer (HGSC) have experienced little improvement in overall survival, and standard treatment has not advanced beyond platinum-based combination chemotherapy, during the past 30 years. To understand the drivers of clinical phenotypes better, here we use whole-genome sequencing of tumour and germline DNA samples from 92 patients with primary refractory, resistant, sensitive and matched acquired resistant disease. We show that gene breakage commonly inactivates the tumour suppressors RB1, NF1, RAD51B and PTEN in HGSC, and contributes to acquired chemotherapy resistance. CCNE1 amplification was common in primary resistant and refractory disease. We observed several molecular events associated with acquired resistance, including multiple independent reversions of germline BRCA1 or BRCA2 mutations in individual patients, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and recurrent promoter fusion associated with overexpression of the drug efflux pump MDR1.

Epithelial ovarian cancer is a complex disease and patients vary considerably in response to treatment. A subset of patients responds well to endocrine therapy, however hormone receptor positivity does not predict response and there are no biomarkers in clinical use to help select patients that would benefit from endocrine agents. We aimed to identify markers of endocrine response in high-grade serous ovarian cancer (HGSOC) by analysis of genes that are differentially expressed in tumors from patients that are endocrine responders compared with non-responders, combined with analysis of estrogen (E2) regulated genes that differ between E2 sensitive and E2 insensitive HGSOC cell lines. Methods: Among women recruited to the Australian Ovarian Cancer Study, we identified 10 HGSOC patients treated with endocrine therapy (5 responders and 5 non-responders, based on GCIG CA125 response criteria) and performed RNAseq on cryopreserved tumor tissue. We also utilized paired E2 receptor (ER) positive HGSOC cell lines, derived from the same patient, with differential sensitivity to the growth effect of E2, PEO1 (E2 insensitive) and PEO4 (E2 sensitive). RNA was extracted following treatment with 0.1 nM E2 (or vehicle) for 24 hrs and gene expression changes in response to E2 were determined using RNAseq. Expression of selected ER responsive genes was validated using PCR array. Differential gene expression was determined using EdgeR. Pathway and Gene Ontology enrichment analysis of differentially expressed genes were performed using Metacore. Results: Between endocrine responders and non-responders, 27 genes were significantly differentially expressed (False Discovery Rate (FDR) Conclusion: The use of RNAseq in both tumors and cell line models was able to identify plausible sets of genes with prior evidence of association with endocrine sensitivity. In breast cancer, ANO1 has been associated with good prognosis following tamoxifen treatment, while NOTCH signaling, implicated in endocrine resistance, is negatively regulated by NRARP. Further validation is required to test the utility of these genes as predictive biomarkers that may aid in identifying HGSOC patients likely to respond to endocrine treatment. Citation Format: Cristina Mapagu, Sian Fereday, Australian Ovarian Cancer Study Group, David D. Bowtell, Paul R. Harnett, Anna deFazio. Identifying predictive markers of endocrine response in high-grade serous ovarian cancer using RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2787. doi:10.1158/1538-7445.AM2017-2787

Background ATP-binding cassette (ABC) transporters play various roles in cancer biology and drug resistance, but their association with outcomes in serous epithelial ovarian cancer (EOC) is unknown. Methods The relationship between clinical outcomes and ABC transporter gene expression in two independent cohorts of high-grade serous EOC tumors was assessed with real-time quantitative polymerase chain reaction, analysis of expression microarray data, and immunohistochemistry. Associations between clinical outcomes and ABCA transporter gene single nucleotide polymorphisms were tested in a genome-wide association study. Impact of short interfering RNA-mediated gene suppression was determined by colony forming and migration assays. Association with survival was assessed with Kaplan-Meier analysis and log-rank tests. All statistical tests were two-sided. Results Associations with outcome were observed with ABC transporters of the A subfamily, but not with multidrug transporters. High-level expression of ABCA1, ABCA6, ABCA8, and ABCA9 in primary tumors was statistically significantly associated with reduced survival in serous ovarian cancer patients. Low levels of ABCA5 and the C-allele of rs536009 were associated with shorter overall survival (hazard ratio for death = 1.50; 95% confidence interval [CI] =1.26 to 1.79; P = 6.5e-6). The combined expression pattern of ABCA1, ABCA5, and either ABCA8 or ABCA9 was associated with particularly poor outcome (mean overall survival in group with adverse ABCA1, ABCA5 and ABCA9 gene expression = 33.2 months, 95% CI = 26.4 to 40.1; vs 55.3 months in the group with favorable ABCA gene expression, 95% CI = 49.8 to 60.8; P =. 001), independently of tumor stage or surgical debulking status. Suppression of cholesterol transporter ABCA1 inhibited ovarian cancer cell growth and migration in vitro, and statin treatment reduced ovarian cancer cell migration. Conclusions Expression of ABCA transporters was associated with poor outcome in serous ovarian cancer, implicating lipid trafficking as a potentially important process in EOC. © 2014 The Author 2014. Published by Oxford University Press. All rights reserved.

Caregiver burden, quality of life (QOL) and unmet needs are poorly understood, particularly at the end of life. We explored these issues in caregivers of women with ovarian cancer.The Australian Ovarian Cancer Study (AOCS) is a prospective population-based study of women newly diagnosed with primary epithelial ovarian cancer. Ninety-nine caregivers of women participating in the AOCS QOL sub-study (88% response rate) rated their QOL (SF-12), psychological distress (HADS), optimism (LOT), social support (Duke) and unmet needs (SCNS-carers), and patients rated their QOL (FACT-O), every three months for two years. This analysis included measurements in the patient's last year of life.Caregivers had significantly lower mental and physical QOL than population norms (p0.01). Mean distress (p=0.01) and unmet needs increased over time, however social support remained constant. In linear mixed models, (using scores for each psychosocial variable over time), optimism (p0.0001), social support (p0.0001), higher unmet needs (p=0.008), physical wellbeing (p0.0001), and time to death (p0.0001) but not patient QOL, predicted caregiver mental well-being and distress. Highest unmet needs in the last 6months related to managing emotions about prognosis, fear of cancer spread, balancing one's own and the patient's needs, impact of caring on work and making decisions in the context of uncertainty.Aspects of caregiver functioning, rather than patient quality of life, predict caregiver quality of life and distress. Caregivers need help with managing emotions about prognosis, balancing their own and the patient's needs, work, and decision-making when there is uncertainty.

Objective Folate is essential for DNA synthesis and methylation and is implicated in tumour progression. Few studies have examined its role in ovarian cancer survival. Our objective was to determine relationships between intake of folate, related one-carbon nutrients, single nucleotide polymorphisms (SNPs) in folate-metabolising genes and survival following ovarian cancer diagnosis. Methods This analysis included 1270 women with invasive epithelial ovarian cancer diagnosed in 2002–2006. Pre-diagnostic and some post-diagnostic lifestyle, dietary, and sociodemographic information was collected via self-administered questionnaires. DNA samples were genotyped for SNPs in methylenetetrahydrofolate reductase ( MTHFR ), methionine synthase ( MTR ) and methionine synthase reductase ( MTRR ) genes. Adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated using Cox regression. Results Multivariate analyses did not identify associations between higher pre-diagnostic intake of folate, folic acid, vitamins B2, B6, and B12, methionine, betaine or choline and survival overall. In stratified analyses, higher folic acid and folate intake was associated with significantly worse survival among women with mucinous tumours (HRs per 100μg 1.30 and 1.43, respectively) and smokers (HRs per 100μg 1.23 and 1.16 respectively). There was also a suggestion that higher supplemental folic acid use post-diagnosis was associated with worse survival (HR per 100μg 1.03, 95%CI 1.00–1.05). MTHFR SNP rs2066470 was significantly associated with survival (per allele HR 0.81, 95%CI 0.67–0.98). Conclusions Our data provide little evidence that folate intake affects ovarian cancer survival. However, combined effects with smoking, and findings within the mucinous subtype and for post-diagnosis folic acid, warrant further investigation.

Obesity is an increasing health problem that is reported to influence chemotherapy dosing. The extent to which this occurs and whether this affects outcomes in ovarian cancer was unclear. To describe chemotherapy dosing practices in normal, overweight and obese patients treated for FIGO Stage III/IV serous ovarian cancer in the Australian Ovarian Cancer Study (AOCS). To evaluate the relationship between body mass index (BMI), dose intensity of chemotherapy received, overall survival (OS) and progression free survival (PFS).Patient characteristics including age, height, weight, FIGO stage, serum creatinine, primary chemotherapy received and outcome data were extracted from medical records and entered into the AOCS database. Outcomes were analysed against BMI and relative dose intensity (RDI) received, based on calculations derived from a standard regimen (carboplatin AUC 5 and paclitaxel 175mg/m(2)).333 women were included in the analysis. 27% were overweight and 21% were obese. In cycle 1 66% of obese patients received carboplatin doses more than 5% below their optimal calculated dose, and 32% received sub-optimal paclitaxel doses, compared to 25% and 13% of normal weight patients respectively. Obese women were more likely to have received85% RDI for carboplatin compared to normal weight women (p0.001). BMI group and RDI of carboplatin and paclitaxel were not predictors of OS. Women who received less than 85% RDI for carboplatin had a worse PFS (univariate analysis, median PFS 11 versus 15 months; p=0.04). There was no significant association between RDI and OS or PFS in multivariate analysis.Obesity is common in ovarian cancer patients, and commonly results in lower chemotherapy dosing than recommended. Analysis of chemotherapy dosing from this study suggests that reduced dose intensity of carboplatin, which was more common in obese women, may impact on PFS in patients with advanced serous ovarian cancer.

Objective\ud ABCB1 encodes the multi-drug efflux pump P-glycoprotein (P-gp) and has been implicated in multi-drug resistance. We comprehensively evaluated this gene and flanking regions for an association with clinical outcome in epithelial ovarian cancer (EOC).\ud Methods\ud The best candidates from fine-mapping analysis of 21 ABCB1 SNPs tagging C1236T (rs1128503), G2677T/A (rs2032582), and C3435T (rs1045642) were analysed in 4616 European invasive EOC patients from thirteen Ovarian Cancer Association Consortium (OCAC) studies and The Cancer Genome Atlas (TCGA). Additionally we analysed 1,562 imputed SNPs around ABCB1 in patients receiving cytoreductive surgery and either ‘standard’ first-line paclitaxel–carboplatin chemotherapy (n = 1158) or any first-line chemotherapy regimen (n = 2867). We also evaluated ABCB1 expression in primary tumours from 143 EOC patients.\ud Result\ud Fine-mapping revealed that rs1128503, rs2032582, and rs1045642 were the best candidates in optimally debulked patients. However, we observed no significant association between any SNP and either progression-free survival or overall survival in analysis of data from 14 studies. There was a marginal association between rs1128503 and overall survival in patients with nil residual disease (HR 0.88, 95% CI 0.77–1.01; p = 0.07). In contrast, ABCB1 expression in the primary tumour may confer worse prognosis in patients with sub-optimally debulked tumours.\ud Conclusion\ud Our study represents the largest analysis of ABCB1 SNPs and EOC progression and survival to date, but has not identified additional signals, or validated reported associations with progression-free survival for rs1128503, rs2032582, and rs1045642. However, we cannot rule out the possibility of a subtle effect of rs1128503, or other SNPs linked to it, on overall survival.

ABCB1 (adenosine triphosphate-binding cassette transporter B1) mediates cellular elimination of many chemotherapeutic agents including paclitaxel, which is commonly used to treat ovarian cancer. A significant association between common single nucleotide polymorphisms (SNPs) in ABCB1 and progression-free survival has been reported in patients with ovarian cancer. Variable paclitaxel clearance due to genotype specific differences in ABCB1 activity in cancer cells and/or normal tissues may underlie the association. Using cell-based models, we evaluated the correlations between ABCB1 expression, polymorphisms, transporter activity and paclitaxel sensitivity in ovarian cancer (n = 10) and lymphoblastoid (n = 19) cell lines. Close associations between ABCB1 expression, transporter function and paclitaxel sensitivity were found in lymphoblastoid cell lines, although we could not demonstrate an association with common SNPs. In ovarian cancer cell lines, ABCB1 expression was low and the association between expression and function was lost. These results suggest that ABCB1 related survival difference in ovarian cancer patients is more likely to be due to differential whole body paclitaxel clearance mediated by normal cells rather than a direct effect on cancer cells.

Purpose: High-grade serous carcinoma (HGSC) accounts for the majority of epithelial ovarian cancer deaths. Genomic and functional data suggest that approximately half of unselected HGSC have disruption of the BRCA pathway and defects in homologous recombination repair (HRR). Pathway disruption is regarded as imparting a BRCAness phenotype. We explored the molecular changes in HGSC arising in association with specific BRCA1/BRCA2 somatic or germline mutations and in those with BRCA1 DNA promoter methylation. Experimental Design: We describe gene expression and copy number analysis of two large cohorts of HGSC in which both germline and somatic inactivation of HRR has been measured. Results: BRCA1 disruptions were associated with the C2 (immunoreactive) molecular subtype of HGSC, characterized by intense intratumoral T-cell infiltration. We derived and validated a predictor of BRCA1 mutation or methylation status, but could not distinguish BRCA2 from wild-type tumors. DNA copy number analysis showed that cases with BRCA1 mutation were significantly associated with amplification both at 8q24 (frequencies: BRCA1 tumors 50%, BRCA2 tumors 32%, and wild-type tumors 9%) and regions of the X-chromosome specifically dysregulated in basal-like breast cancer (BLBC; BRCA1 62%, BRCA2 34%, and wild-type 35%). Tumors associated with BRCA1/BRCA2 mutations shared a negative association with amplification at 19p13 (BRCA1 0%, BRCA2 3%, and wild-type 20%) and 19q12 (BRCA1 6%, BRCA2 3%, and wild-type 29%). Conclusion: The molecular differences between tumors associated with BRCA1 compared with BRCA2 mutations are in accord with emerging clinical and pathologic data and support a growing appreciation of the relationship between HGSC and BLBC. Clin Cancer Res; 19(13); 3474–84. ©2013 AACR.

Contains fulltext : 118401.pdf (Publisher’s version ) (Open Access) Various common genetic susceptibility loci have been identified for breast cancer; however, it is unclear how they combine with lifestyle/environmental risk factors to influence risk. We undertook an international collaborative study to assess gene-environment interaction for risk of breast cancer. Data from 24 studies of the Breast Cancer Association Consortium were pooled. Using up to 34,793 invasive breast cancers and 41,099 controls, we examined whether the relative risks associated with 23 single nucleotide polymorphisms were modified by 10 established environmental risk factors (age at menarche, parity, breastfeeding, body mass index, height, oral contraceptive use, menopausal hormone therapy use, alcohol consumption, cigarette smoking, physical activity) in women of European ancestry. We used logistic regression models stratified by study and adjusted for age and performed likelihood ratio tests to assess gene-environment interactions. All statistical tests were two-sided. We replicated previously reported potential interactions between LSP1-rs3817198 and parity (Pinteraction = 2.4 x 10(-6)) and between CASP8-rs17468277 and alcohol consumption (Pinteraction = 3.1 x 10(-4)). Overall, the per-allele odds ratio (95% confidence interval) for LSP1-rs3817198 was 1.08 (1.01-1.16) in nulliparous women and ranged from 1.03 (0.96-1.10) in parous women with one birth to 1.26 (1.16-1.37) in women with at least four births. For CASP8-rs17468277, the per-allele OR was 0.91 (0.85-0.98) in those with an alcohol intake of /= 20 g/day. Additionally, interaction was found between 1p11.2-rs11249433 and ever being parous (Pinteraction = 5.3 x 10(-5)), with a per-allele OR of 1.14 (1.11-1.17) in parous women and 0.98 (0.92-1.05) in nulliparous women. These data provide first strong evidence that the risk of breast cancer associated with some common genetic variants may vary with environmental risk factors.

Genomic instability and copy number alterations in cancer are generally associated with poor prognosis; however, recent studies have suggested that extreme levels of genomic aberrations may be beneficial for the survival outcome for patients with specific tumour types. We investigated the extent of genomic instability in predominantly high-grade serous ovarian cancers (SOC) using two independent datasets, generated in Norway (n = 74) and Australia (n = 70), respectively. Genomic instability was quantified by the Total Aberration Index (TAI), a measure of the abundance and genomic size of copy number changes in a tumour. In the Norwegian cohort, patients with TAI above the median revealed significantly prolonged overall survival (p

The EMS1 gene encodes an 80/85 kDa c-src substrate and localises with the CCND1 gene to chromosome 11q13. This locus is amplified in approximately 13% of human breast cancers. EMS1 gene amplification and expression were characterised in a panel of human breast cancer cell lines to determine at what levels expression is regulated. The degree of tyrosine phosphorylation of EMS1 protein was also determined and compared with the activity of src-family kinases. The EMS1 gene was amplified in 6 of 20 cell lines investigated: MDA-MB-134, -157, -175, -453, ZR-75-1 and MCF-7. In the MDA-MB-157 and MCF-7 cell lines, EMS1 was amplified in the absence of CCND1 gene amplification. EMS1 protein levels were increased relative to normal breast epithelial cells in 6 cell lines (ZR-75-1, MDA-MB-134, -175, -453, MCF-7 and BT-474). Of these, BT-474 is the only cell line that does not exhibit EMS1 amplification or increased EMS1 mRNA levels. EMS1 tyrosine phosphorylation was 3-fold higher in BT-474 and T-47D cells, which exhibited relatively high total src activity coupled with expression of both c-fyn and c-yes, than in MDA-MB-453 cells, which expressed only c-yes. Our results therefore demonstrate gene amplification to be the predominant mechanism underlying EMS1 over-expression in human breast cancer cell lines and identify tyrosine phosphorylation as a further level at which regulation of this protein may be perturbed. © 1996 Wiley-Liss, Inc.

Primary peritoneal cancer is rare and considered equivalent to stage III/IV ovarian cancer, but questions remain concerning its underlying biology, prognosis and optimal management.Clinico-pathological and treatment details of primary peritoneal (n=120) and ovarian cancer (n=635) were obtained on women recruited to the Australian Ovarian Cancer Study. Log-rank test was used to compare survival and cox proportional hazards models were fitted to obtain hazard ratios and 95% confidence intervals, both unadjusted and adjusted for age, grade, FIGO stage, residual disease and treatment with neoadjuvant chemotherapy. Molecular subtype was determined by gene expression profiling using published data.Compared with advanced serous ovarian cancer, primary peritoneal cancer patients were older (mean age 65.5 vs. 60.2years, p0.001), more often treated with neoadjuvant chemotherapy (38.4% vs. 11.4%, p0.001). Gene expression profiling classified a substantially higher proportion of primary peritoneal carcinomas as C1 (mesenchymal, reactive stromal infiltration) subtype (70.6% vs. 32.1%, p=0.029), which was associated with lower complete surgical resection rate. Women with primary peritoneal cancer had significantly shorter progression-free (11.6 vs. 13.6months, p=0.007) and overall survival (31.7 vs. 39.8months, p=0.012). In multivariate analysis, residual disease and neoadjuvant chemotherapy were both independently associated with increased risk of progression and death.Primary peritoneal cancer patients were more frequently treated with neoadjuvant chemotherapy and had inferior survival. Different tumor biology characterized by activated stromal fibrosis in primary peritoneal cancer may underlie the differences in treatment and clinical outcome.

Low-grade serous ovarian cancer (LGSC) is a challenging disease to treat effectively. It often occurs in young women and it is well-recognized to be resistant to standard chemotherapy. The underlying molecular driver mutations are now beginning to be understood and they are distinctly different from the more common counterpart, high-grade serous ovarian cancer. LGSC are characterized by somatic mutations in RAS/RAF genes and a number of new agents have been developed that target these mutations, and related activated pathways. However, it is not yet known which pathway-targeted drugs, or combination of drugs, will be effective in the treatment of LGSC. BRAFV600E mutations have been reported in LGSC and BRAF inhibitors have demonstrated significant improvement in progression-free survival in patients with BRAF-mutant melanoma. However, limited response is seen in other cancer types, such as colorectal cancers harboring the same mutation, suggesting that clinical benefit is tumor-type specific. In this study we aimed to characterize BRAF mutations in LGSC and to determine whether BRAF inhibitors could demonstrate a clinical benefit in ovarian cancer patients. Patients with LGSC were identified through the Australian Ovarian Cancer Study and the Westmead GynBiobank, Sydney, Australia. Tumor mutations were assessed using targeted and exome sequencing, and gene copy number was measured by whole genome SNP arrays. Tumor expression of BRAFV600E protein was also assessed by immunohistochemistry. Dabrafenib monotherapy was trialed in a patient with a somatic BRAFV600E mutation and progress was monitored clinically, biochemically using CA125 tumor marker levels and radiologically with PET imaging. Amongst Grade 1 serous ovarian carcinoma cases, 5/40 (12.5%) were shown to have a BRAFV600E mutation. Tumors with a BRAFV600E mutation had a relatively low degree of gene copy number change and were TP53 wild-type. The BRAF-inhibitor, dabrafenib was trialed in a heavily pre-treated BRAFV600E mutation-positive LGSC patient with progressive chemotherapy-resistant disease (n=1). Whole exome sequencing confirmed the BRAFV600E mutation was the highest frequency variant allele present and also identified deleterious mutations in other cancer-associated genes including CSMD1, BMP1 and DNM1 at lower frequencies, suggestive of sub-clonal events. The patient received dabrafenib monotherapy for 11 months, and demonstrated a substantial clinical, radiological and biochemical response, with complete normalization of her CA125 levels for the first time in six years. These results demonstrate that molecular analysis of low-grade serous ovarian carcinoma can identify targetable mutations and provide effective treatment options. The substantial response to dabrafenib suggests that BRAF inhibition represents a potential therapeutic option for ovarian cancer patients with somatic BRAFV600E mutations and should be tested in future trials. However, the results also highlight the need for novel clinical trial design, as traditional clinical trials are unlikely to be effective in such rare ovarian cancer sub-groups. Citation Format: Anna DeFazio, Tania Moujaber, Dariush Etemadmoghadam, Catherine Kennedy, Yoke-Eng Chiew, Rosemary L. Balleine, Catherine Saunders, Gerard V. Wain, Alexander Dobrovic, Australian Ovarian Cancer Study Group (AOCS), David DL Bowtell, Paul R. Harnett. BRAFV600E mutations in serous ovarian cancer and response to the BRAF inhibitor, dabrafenib. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A25.

While the genomics of high-grade serous carcinoma are well-studied in large international consortia, the less common subtypes have been neglected. We have sought to rectify this gap by analyzing international collections of low-grade serous and mucinous ovarian carcinomas and their putative benign and borderline precursors. Exome sequencing and copy number analysis of low-grade serous carcinomas (n=9) and serous borderline tumours (n=13) and targeted sequencing and copy number analysis in additional carcinomas (n=10) and borderline tumours (n=44) identified recurrent mutations in novel drivers such as EIF1AX and USP9X, as well as the known drivers KRAS, BRAF and NRAS. Copy number changes including 9p and 1p losses were significantly associated with progression from borderline to carcinoma. Exome and targeted sequencing analysis of mucinous carcinomas (GAMuT study) found a surprisingly high proportion (~50%) with TP53 mutations, and mutations in new drivers like RNF43 and ELF3. Despite similarities in early RAS/RAF pathway oncogenic drivers and CDKN2A disruption, the genetics of these two subtypes are otherwise distinct, suggesting differing etiologies and selective pressures. We also present here the first whole-genome sequencing analysis of a high-grade mucinous ovarian carcinoma collected from multiple sites at autopsy (CASCADE study). The patient, aged just 41 when diagnosed with Stage I mucinous ovarian carcinoma, had a 26-month progression-free interval, including normal CA125 and CA19-9 measurements at 21 months. The primary tumor was mostly borderline in appearance, with only a small focus of carcinoma. At autopsy, the carcinoma was widespread in the body, and whole-genome sequencing data was obtained from deposits in the omentum, iliac lymph node, para-aortic lymph node and upper diaphragm. These data were compared to the primary ovarian tumor and nine other sites sampled at autopsy. Citation Format: Kylie L. Gorringe, Matthew Wakefield, Sally M. Hunter, Georgina L. Ryland, Dane Cheasley, Michael S. Anglesio, Michael Christie, Raghwa Sharma, Antill Yoland, Simone M. Rowley, Jason Li, Blake Gilks, Prue E. Allan, Andrew N. Stephens, Sumi Ananda, Jan Pyman, Martin Koebel, Jessica McAlpine, Charlie Gourley, David G. Huntsman, Anna deFazio, David DL Bowtell, Ian G. Campbell, Clare Scott. Genomics analyses of less common epithelial ovarian cancer subtypes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B08.

Purpose: High-grade serous cancer (HGSC) is the most common cancer of the ovary and is characterized by chromosomal instability. Defects in homologous recombination repair (HRR) are associated with genomic instability in HGSC, and are exploited by therapy targeting DNA repair. Defective HRR causes uniparental deletions and loss of heterozygosity (LOH). Our purpose is to profile LOH in HGSC and correlate our findings to clinical outcome, and compare HGSC and high-grade breast cancers. Experimental Design: We examined LOH and copy number changes using single nucleotide polymorphism array data from three HGSC cohorts and compared results to a cohort of high-grade breast cancers. The LOH profiles in HGSC were matched to chemotherapy resistance and progression-free survival (PFS). Results: LOH-based clustering divided HGSC into two clusters. The major group displayed extensive LOH and was further divided into two subgroups. The second group contained remarkably less LOH. BRCA1 promoter methylation was associated with the major group. LOH clusters were reproducible when validated in two independent HGSC datasets. LOH burden in the major cluster of HGSC was similar to triple-negative, and distinct from other high-grade breast cancers. Our analysis revealed an LOH cluster with lower treatment resistance and a significant correlation between LOH burden and PFS. Conclusions: Separating HGSC by LOH-based clustering produces remarkably stable subgroups in three different cohorts. Patients in the various LOH clusters differed with respect to chemotherapy resistance, and the extent of LOH correlated with PFS. LOH burden may indicate vulnerability to treatment targeting DNA repair, such as PARP1 inhibitors. Clin Cancer Res; 18(20); 5806–15. ©2012 AACR.

Because of the high risk of recurrence in high-grade serous ovarian carcinoma (HGS-OvCa), the development of outcome predictors could be valuable for patient stratification. Using the catalog of The Cancer Genome Atlas (TCGA), we developed subtype and survival gene expression signatures, which, when combined, provide a prognostic model of HGS-OvCa classification, named “Classification of Ovarian Cancer” (CLOVAR). We validated CLOVAR on an independent dataset consisting of 879 HGS-OvCa expression profiles. The worst outcome group, accounting for 23% of all cases, was associated with a median survival of 23 months and a platinum resistance rate of 63%, versus a median survival of 46 months and platinum resistance rate of 23% in other cases. Associating the outcome prediction model with BRCA1/BRCA2 mutation status, residual disease after surgery, and disease stage further optimized outcome classification. Ovarian cancer is a disease in urgent need of more effective therapies. The spectrum of outcomes observed here and their association with CLOVAR signatures suggests variations in underlying tumor biology. Prospective validation of the CLOVAR model in the context of additional prognostic variables may provide a rationale for optimal combination of patient and treatment regimens.

Purpose The frequency of BRCA1 and BRCA2 germ-line mutations in women with ovarian cancer is unclear; reports vary from 3% to 27%. The impact of germ-line mutation on response requires further investigation to understand its impact on treatment planning and clinical trial design. Patients and Methods Women with nonmucinous ovarian carcinoma (n = 1,001) enrolled onto a population-based, case-control study were screened for point mutations and large deletions in both genes. Survival outcomes and responses to multiple lines of chemotherapy were assessed. Results Germ-line mutations were found in 14.1% of patients overall, including 16.6% of serous cancer patients (high-grade serous, 22.6%); 44% had no reported family history of breast or ovarian cancer. Patients carrying germ-line mutations had improved rates of progression-free and overall survival. In the relapse setting, patients carrying mutations more frequently responded to both platin- and nonplatin-based regimens than mutation-negative patients, even in patients with early relapse after primary treatment. Mutation-negative patients who responded to multiple cycles of platin-based treatment were more likely to carry somatic BRCA1/2 mutations. Conclusion BRCA mutation status has a major influence on survival in ovarian cancer patients and should be an additional stratification factor in clinical trials. Treatment outcomes in BRCA1/2 carriers challenge conventional definitions of platin resistance, and mutation status may be able to contribute to decision making and systemic therapy selection in the relapse setting. Our data, together with the advent of poly(ADP-ribose) polymerase inhibitor trials, supports the recommendation that germ-line BRCA1/2 testing should be offered to all women diagnosed with nonmucinous, ovarian carcinoma, regardless of family history.