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HR pathway mutations

Supplementary Methods, Figures 1-5. Supplementary Figure 1. Outline of cohort selection. Supplementary Figure 2. Example of whole-genome single-nucleotide polymorphism (SNP)array profile and genome alteration print (GAP). Supplementary Figure 3. Pre-treatment serum CA125 levels. Supplementary Figure 4. Progression-free and overall survival in patients with SBT-EOC and EOC without adjacent SBT. Supplementary Figure 5. Progression-free and overall survival of patients with Grade 3 SBTEOC and Grade 3 EOC without adjacent SBT

Purpose: Women with epithelial ovarian cancer generally have a poor prognosis; however, a subset of patients has an unexpected dramatic and durable response to treatment. We sought to identify clinical, pathological, and molecular determinants of exceptional survival in women with high-grade serous cancer (HGSC), a disease associated with the majority of ovarian cancer deaths.Experimental Design: We evaluated the histories of 2,283 ovarian cancer patients and, after applying stringent clinical and pathological selection criteria, identified 96 with HGSC that represented significant outliers in terms of treatment response and overall survival. Patient samples were characterized immunohistochemically and by genome sequencing.Results: Different patterns of clinical response were seen: long progression-free survival (Long-PFS), multiple objective responses to chemotherapy (Multiple Responder), and/or greater than 10-year overall survival (Long-Term Survivors). Pathogenic germline and somatic mutations in genes involved in homologous recombination (HR) repair were enriched in all three groups relative to a population-based series. However, 29% of 10-year survivors lacked an identifiable HR pathway alteration, and tumors from these patients had increased Ki-67 staining. CD8+ tumor-infiltrating lymphocytes were more commonly present in Long-Term Survivors. RB1 loss was associated with long progression-free and overall survival. HR deficiency and RB1 loss were correlated, and co-occurrence was significantly associated with prolonged survival.Conclusions: There was diversity in the clinical trajectory of exceptional survivors associated with multiple molecular determinants of exceptional outcome in HGSC patients. Concurrent HR deficiency and RB1 loss were associated with favorable outcomes, suggesting that co-occurrence of specific mutations might mediate durable responses in such patients. Clin Cancer Res; 24(3); 569–80. ©2017 AACR.See related commentary by Peng and Mills, p. 508

Revised Supplementary Data, containing Supplementary Methods, Supplementary Figures, Supplementary Tables Supplementary Figure S1. Outline of cohort selection and analyses. Supplementary Figure S2. Clinical response and therapy course of 96 patients with exceptional responses to chemotherapy. Supplementary Figure S3. Distribution and type of TP53 mutations. Supplementary Figure S4. RB1 protein expression altered by genomic inactivation. Supplementary Figure S5. Characterization of CD8 and Ki-67 in tumors according to homologous recombination mutation status. Supplementary Table S2 Immunohistochemical analysis: primary antibodies and staining conditions Supplementary Table S3 Homologous recombination and DNA repair panel Supplementary Table S6 Comparison of molecular alteration prevalence between clinical subgroups Supplementary Table S7 Patient characteristics of tissue microarray cohort

Supplementary Highlighted Methods. This document contains the revised supplementary methods and results with changes highlighted

Supplementary Tables 1-7. Supplementary Table 1. SBT-EOC cases used for molecular analyses Supplementary Table 2. Mutations Screened on the Sequenom/OncoMap3 Platform Supplementary Table 3. Primer sequences for the indicated exons of TP53, ERBB2 and NRAS Supplementary Table 4. Clinico-pathologic characteristics of the subset of SBT-EOC used for molecular analysis compared to the entire SBT-EOC cohort Supplementary Table 5A. Somatic mutations found in borderline and invasive ovarian tumors Supplementary Table 5B. Genes in which somatic mutations were found according to grade and co-existing serous borderline regions Supplementary Table 6. Genes differentially expressed between paired borderline and invasive regions of SBTEOC (p

Purpose: Low-grade serous ovarian carcinomas (LGSC) are Ras pathway-mutated, TP53 wild-type, and frequently associated with borderline tumors. Patients with LGSCs respond poorly to platinum-based chemotherapy and may benefit from pathway-targeted agents. High-grade serous carcinomas (HGSC) are TP53-mutated and are thought to be rarely associated with borderline tumors. We sought to determine whether borderline histology associated with grade 2 or 3 carcinoma was an indicator of Ras mutation, and we explored the molecular relationship between coexisting invasive and borderline histologies.Experimental Design: We reviewed >1,200 patients and identified 102 serous carcinomas with adjacent borderline regions for analyses, including candidate mutation screening, copy number, and gene expression profiling.Results: We found a similar frequency of low, moderate, and high-grade carcinomas with coexisting borderline histology. BRAF/KRAS alterations were common in LGSC; however, we also found recurrent NRAS mutations. Whereas borderline tumors harbored BRAF/KRAS mutations, NRAS mutations were restricted to carcinomas, representing the first example of a Ras oncogene with an obligatory association with invasive serous cancer. Coexisting borderline and invasive components showed nearly identical genomic profiles. Grade 2 cases with coexisting borderline included tumors with molecular features of LGSC, whereas others were typical of HGSC. However, all grade 3 carcinomas with coexisting borderline histology were molecularly indistinguishable from typical HGSC.Conclusion: Our findings suggest that NRAS is an oncogenic driver in serous ovarian tumors. We demonstrate that borderline histology is an unreliable predictor of Ras pathway aberration and underscore an important role for molecular classification in identifying patients that may benefit from targeted agents. Clin Cancer Res; 20(24); 6618–30. ©2014 AACR.

Purpose: Women with epithelial ovarian cancer generally have a poor prognosis; however, a subset of patients has an unexpected dramatic and durable response to treatment. We sought to identify clinical, pathological, and molecular determinants of exceptional survival in women with high-grade serous cancer (HGSC), a disease associated with the majority of ovarian cancer deaths. Experimental Design: We evaluated the histories of 2,283 ovarian cancer patients and, after applying stringent clinical and pathological selection criteria, identified 96 with HGSC that represented significant outliers in terms of treatment response and overall survival. Patient samples were characterized immunohistochemically and by genome sequencing. Results: Different patterns of clinical response were seen: long progression-free survival (Long-PFS), multiple objective responses to chemotherapy (Multiple Responder), and/or greater than 10-year overall survival (Long-Term Survivors). Pathogenic germline and somatic mutations in genes involved in homologous recombination (HR) repair were enriched in all three groups relative to a population-based series. However, 29% of 10-year survivors lacked an identifiable HR pathway alteration, and tumors from these patients had increased Ki-67 staining. CD8+ tumor-infiltrating lymphocytes were more commonly present in Long-Term Survivors. RB1 loss was associated with long progression-free and overall survival. HR deficiency and RB1 loss were correlated, and co-occurrence was significantly associated with prolonged survival. Conclusions: There was diversity in the clinical trajectory of exceptional survivors associated with multiple molecular determinants of exceptional outcome in HGSC patients. Concurrent HR deficiency and RB1 loss were associated with favorable outcomes, suggesting that co-occurrence of specific mutations might mediate durable responses in such patients. Clin Cancer Res; 24(3); 569–80. ©2017 AACR. See related commentary by Peng and Mills, p. 508

OBJECTIVE: The ROR1 and ROR2 receptor tyrosine kinases have both been implicated in ovarian cancer progression and have been shown to drive migration and invasion. There is an increasing importance of the role of stroma in ovarian cancer metastasis; however, neither ROR1 nor ROR2 expression in tumor or stromal cells has been analyzed in the same clinical cohort. AIM: To determine ROR1 and ROR2 expression in ovarian cancer and surrounding microenvironment and examine associations with clinicopathological characteristics. METHODS: Immunohistochemistry for ROR1 and ROR2 was used to assess receptor expression in a cohort of epithelial ovarian cancer patients ( n =178). Results were analyzed in relation to clinical and histopathological characteristics and survival. Matched patient sample case studies of normal, primary, and metastatic lesions were used to examine ROR expression in relation to ovarian cancer progression. RESULTS: ROR1 and ROR2 are abnormally expressed in malignant ovarian epithelium and stroma. Higher ROR2 tumor expression was found in early-stage, low-grade endometrioid carcinomas. ROR2 stromal expression was highest in the serous subtype. In matched patient case studies, metastatic samples had higher expression of ROR2 in the stroma, and a recurrent sample had the highest expression of ROR2 in both tumor and stroma. CONCLUSION: ROR1 and ROR2 are expressed in tumor-associated stroma in all histological subtypes of ovarian cancer and hold potential as therapeutic targets which may disrupt tumor and stroma interactions.

The aim of this study was to compare response rates and survival in women with "platinum resistant" epithelial ovarian cancer (EOC) who received further platinum-based or non‑platinum chemotherapy for treatment at first relapse.Patients with high-grade EOC (including fallopian tube and peritoneal cancer) of all histologies recruited to the Australian Ovarian Cancer Study (AOCS) and treated with platinum-based primary chemotherapy were included. Response to second-line chemotherapy, overall survival (OS) and survival after treatment for first progression (OS2) were determined in all histologies and separately in women with high-grade serous tumors.Of the 341 patients classified as platinum-resistant by the 6-month threshold, 243 (71%) were treated with chemotherapy at relapse. CA-125 response rates to platinum-based chemotherapy were significantly higher compared to non‑platinum chemotherapy (51% vs 21%, P 0.001). Among patients with a platinum-free interval (PFI) of 3-6 months, OS2 in patients treated with platinum was significantly longer compared to individuals receiving non‑platinum-based treatment (median 17.67 months, 95% CI: 14.79-20.75 vs. 10.62 months, 95% CI: 8.02-12.72, P = 0.022). The patterns were similar when restricted to patients with high-grade serous histology. In patients with PFI3 months, there was no significant difference in response or survival according to type of second-line treatment.Our findings further question the use of a 6-month PFI as an arbitrary threshold for subsequent treatment decision-making. Some patients considered "platinum resistant" still derive clinical benefit from platinum-based chemotherapy. Biomarkers of platinum sensitivity are needed in clinical practice to identify potential responders who should be offered re-treatment with platinum.

Purpose: Low-grade serous ovarian carcinomas (LGSC) are Ras pathway-mutated, TP53 wild-type, and frequently associated with borderline tumors. Patients with LGSCs respond poorly to platinum-based chemotherapy and may benefit from pathway-targeted agents. High-grade serous carcinomas (HGSC) are TP53-mutated and are thought to be rarely associated with borderline tumors. We sought to determine whether borderline histology associated with grade 2 or 3 carcinoma was an indicator of Ras mutation, and we explored the molecular relationship between coexisting invasive and borderline histologies. Experimental Design: We reviewed >1,200 patients and identified 102 serous carcinomas with adjacent borderline regions for analyses, including candidate mutation screening, copy number, and gene expression profiling. Results: We found a similar frequency of low, moderate, and high-grade carcinomas with coexisting borderline histology. BRAF/KRAS alterations were common in LGSC; however, we also found recurrent NRAS mutations. Whereas borderline tumors harbored BRAF/KRAS mutations, NRAS mutations were restricted to carcinomas, representing the first example of a Ras oncogene with an obligatory association with invasive serous cancer. Coexisting borderline and invasive components showed nearly identical genomic profiles. Grade 2 cases with coexisting borderline included tumors with molecular features of LGSC, whereas others were typical of HGSC. However, all grade 3 carcinomas with coexisting borderline histology were molecularly indistinguishable from typical HGSC. Conclusion: Our findings suggest that NRAS is an oncogenic driver in serous ovarian tumors. We demonstrate that borderline histology is an unreliable predictor of Ras pathway aberration and underscore an important role for molecular classification in identifying patients that may benefit from targeted agents. Clin Cancer Res; 20(24); 6618–30. ©2014 AACR.

High expression of Ankyrin Repeat Domain 1 (ANKRD1) in ovarian carcinoma is associated with poor survival, and in ovarian cancer cell lines is associated with platinum resistance. Importantly, decreasing ANKRD1 expression using siRNA increases cisplatin sensitivity. In this study, we investigated possible mechanisms underlying the association of ANKRD1 with cisplatin response. We first demonstrated that cisplatin-induced apoptosis in ovarian cancer cell lines was associated with endoplasmic reticulum (ER) stress, evidenced by induction of Glucose-Regulated Protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153) and increased intracellular Ca(2+) release. The level of sensitivity to cisplatin-induced apoptosis was associated with ANKRD1 protein levels and poly (ADP-ribose) polymerase (PARP) cleavage. COLO 316 ovarian cancer cells, which express high ANKRD1 levels, were relatively resistant to cisplatin, and ER stress-induced apoptosis, whereas OAW42 and PEO14 cells, which express lower ANKRD1 levels, are more sensitive to ER stress-induced apoptosis. Furthermore, we show that overexpression of ANKRD1 attenuated cisplatin-induced cytotoxicity, and conversely siRNA knockdown of ANKRD1 sensitized ovarian cancer cells to cisplatin and ER stress-induced apoptosis associated with induction of GADD153, and downregulation of BCL2 and BCL-XL. Taken together, these results suggest that ANKRD1 has a significant role in the regulation of apoptosis in human ovarian cancer cells, and is a potential molecular target to enhance sensitivity of ovarian cancer to chemotherapy.

Background: Apoptotic endothelium has been suggested to have microthrombotic and microembolic potential. While some describe pro-coagulant activities and platelet binding, others demonstrate maintained fibrinolytic protein and anti-platelet aggregatory activity. Canalicular fragmentation is unique to apoptotic endothelium and is suggested to facilitate size reduction of apoptotic cells to reduce microembolic potential. Despite the potential importance of apoptotic microemboli, there are no reports characterizing changes in cell size and shape during endothelial apoptosis. Methods: Here, we describe transmission and scanning electron microscopic studies of apoptotic endothelium and compare changes seen with apoptotic HL-60 cells incapable of canalicular fragmentation. Results: We demonstrate reduced endothelial size (p < 0.05) with progressive apoptosis relative to apoptotic HL-60 cells. Mechanical stress accelerated size reduction of apoptotic endothelium (p < 0.01) but did not affect the size of apoptotic HL-60 cells. Mechanical stress also increased circularity in apoptotic endothelium (p < 0.01), suggested to facilitate passage through small vessels. Earlier work indicated that canaliculi form through plasma membrane invagination, but we report fusion of small vesicles contributing to canalicular growth, while canaliculi fuse to form large vacuoles and also dilate at late stages of apoptosis. Conclusions: These observations are consistent with the suggestion that endothelium is adapted to minimize microembolic potential and that canalicular fragmentation contributes to this.

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.

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.

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.

We earlier reported synergy between tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) for apoptosis in human umbilical vein endothelium (HUVEC). Here, we study morphological change by circularity measurement of HUVEC surviving this cytokine induced synergistic apoptosis. Contrasting with reports by others studying bovine endothelium, HUVEC did not change morphology in response to TGF-β1. TNF-α markedly elongated cells (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.

Although tumour cells are believed to migrate between endothelial cells early in metastasis, the possibility remains that endothelial apoptosis may also contribute to the tumour's breach of the vascular barrier. Although seemingly inconsistent with tumour angiogenesis, one publication describes the induction of contact-dependent apoptosis in cultured endothelium by tumour cells. The cell culture data are, however, open to challenge on technical grounds while there are no confirmatory reports. The present paper describes experiments overcoming these limitations. SAOS-2 human osteosarcoma cells and two rat carcinoma cell lines were co-cultured with human umbilical vein endothelial cells (HUVECs) and cultures labelled by surface lectin histochemistry for endothelium. The HUVEC culture density was determined and SAOS-2 cells, but not rat carcinoma cells, were found significantly to reduce HUVEC survival despite the release of potent growth factors as determined in separate experiments with tumour cell conditioned medium. Lectin labelling combined with light microscopy, transmission electron microscopy, flow cytometry for both lectin binding and DNA content, and DNA gel electrophoresis of SAOS-2/HUVEC co-cultures revealed extensive HUVEC apoptosis. These findings indicate contact-dependent endothelial apoptosis by SAOS-2, while this activity appeared weaker and overwhelmed by HUVEC proliferation with rat carcinoma cells. Importantly, this study supports the suggestion that endothelial apoptosis may be important for metastasis and suggests a complex interplay between endothelial proliferation and apoptosis in tumours.

Background: The traditional anesthetic used for collection of the serum culture medium for whole rat embryo culture studies has been ether. However ethical concerns have been raised due to the irritant nature of the vapour and safety concerns due to the risk of fire. Methods: Growth and development of gestation day 9.5 rat embryos cultured for 48 h in serum collected from rats anesthetised with either ether, isoflurane or halothane were compared. Results: There were no differences in any of the parameters used to assess embryonic development when embryos were grown in serum collected using either ether or isoflurane anesthetics. However, when embryos grown in serum collected using ether or halothane were compared, embryonic development was similar in all respects, except for a reduced number of embryos turned to become fully dorsally convex in the halothane group (p

Serum deprivation stimulates endothelial apoptosis while albumin inhibits this and has been proposed as important in confining apoptotic remodelling to poorly perfused vessels. Tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta are also reported to induce endothelial apoptosis. To investigate the comparative roles of these stimuli, the effect of TNF-alpha and TGF-beta, alone or in combination, in the presence or absence of serum or albumin was studied. There was strong synergy between the cytokines in inducing human umbilical vein endothelial cell apoptosis, but only in the absence of serum. Synergy was destroyed by boiling cytokines and was not affected by polymyxin B. Dose response experiments revealed greater activity of TGF-beta(1) than TGF-beta(2). The synergy was protein synthesis dependent and apoptosis was confirmed by DNA gel electrophoresis, transmission electron microscopy and FACS analysis. Data suggests a role for synergistic activation of endothelial cell apoptosis by TNF-alpha and TGF-beta(1) but perhaps only in poorly perfused vessels deprived of serum factors.

Primary and acquired chemotherapy resistance remains a major challenge in the treatment of epithelial ovarian cancer and there are currently few targeted treatments that improve clinical outcome. We screened for genes that could potentially be targeted to sensitise, or re-sensitise, ovarian cancer cells to chemotherapy and identified a novel candidate, Ankyrin Repeat Domain 1 (ANKRD1), not previously described in ovarian cancer. ANKRD1 is a putative transcription factor with roles in muscle development, inflammation and response to cell stress. ANKRD1 is a target of TGF-β/Wnt signalling, and is also a binding partner of Titin (TTN) which been identified as a candidate with a high probability of carrying ‘driver’ mutations in ovarian cancer. ANKRD1 was expressed in serous ovarian cancer samples and higher tumor levels were found in patients with worse outcome (overall survival, p=0.013), (Scurr et al, Clin Cancer Res 2008; 14: 6924-32). In this study we showed that ANKRD1 levels decrease in response to cisplatin, in concert with induction of PARP cleavage and apoptosis in human ovarian cancer cell lines, COLO316, PEO14 and OAW42. We showed that decreasing ANKRD1 expression using siRNA (ON-TARGETplus Human ANKRD1 siRNA) increased cisplatin-induced apoptosis. Increased cisplatin sensitivity was associated with endoplasmic reticulum (ER) stress, evidenced by induction of GRP78, GADD153 and increased intracellular Ca2+ release. We have screened drug libraries (Prestwick library) for agents that sensitise ovarian cancer cell lines to platins, via an ANKRD1 related mechanism, and have identified several potential candidate molecules. Taken together, these results suggest that ANKRD1 plays a significant role in the regulation of apoptosis in human ovarian cancer cells, and is a potential molecular target to enhance sensitivity of ovarian cancer to chemotherapy. Citation Format: Ying Lei, Beric Ross Henderson, Catherine Emmanuel, Paul Harnett, Anna de Fazio. Chemo-sensitisation in epithelial ovarian cancer cell lines by targeting Ankyrin Repeat Domain 1 (ANKRD1). [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 4616. doi:10.1158/1538-7445.AM2014-4616

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...

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.