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Background: Genomic studies suggest that ∼50% of high-grade serous ovarian cancers (OC) have homologous recombination deficiency (HRD). Germline BRCA1/2 mutations (gBRCAmut) are expected to account for 1/3 of HRD in OC, and identification of non-gBRCAmut HRD tumors likely to respond to PARP inhibitors (PARPi) remains a challenge. Using comprehensive next generation sequencing (NGS)-based tumor genomic profiling, we developed a companion diagnostic HRD assay to predict sensitivity to the PARP inhibitor rucaparib by combining tumor BRCA1/2 status (germline and somatic) and genomic loss of heterozygosity (LOH). The HRD assay is being validated in a Phase 2 study (ARIEL2) and will be prospectively applied to the primary analysis of the ongoing Phase 3 study (ARIEL3) of rucaparib. Methods: The HRD assay uses 50-200ng of DNA from tumor FFPE specimens, which undergoes sequencing library construction and hybrid-capture of all coding exons from 100s of cancer-related genes. Libraries are sequenced to high, uniform depth (>500× unique coverage, Illumina® HiSeq) and data are processed by a customized pipeline that accurately detects all classes of genomic alterations, including BRCA1/2 base substitutions, indels, and homozygous deletions. Genomic LOH is assessed by a CGH-like analysis of sequencing coverage and >3,500 genome-wide SNPs and a tumor is classified as HRD with either BRCA1/2 alteration or high genomic LOH (LOH+). Somatic/germline status of discovered BRCA1/2 alterations is assessed by a previously-presented computational approach (“SGZ”, AACR 2014 abstract #1893), and verified against medical records where available. ARIEL2 is an ongoing single-arm (n = 180), open-label study of rucaparib in recurrent, platinum-sensitive OC patients. The primary objective is to evaluate clinical activity of rucaparib among 3 prospectively defined subgroups: tumor BRCAmut, BRCAwt/LOH+ (“BRCAness”) and BRCAwt/LOH-. Response is determined by RECIST and/or GCIG-CA125 criteria. Results: The HRD assay was performed on tumors from 121 patients, of whom 25% were found to be BRCA mutant (17 germline/12 somatic), 42% had the BRCAness signature (BRCAwt/LOH+), and 33% were biomarker negative (BRCAwt/LOH-). Efficacy data available for 61 patients revealed objective response rates (combined RECIST/CA125 criteria) at 70%, 40% and 8%, respectively. Responses were observed for all classes of genomic alterations, and in gBRCAmut and non-gBRCAmut tumors. Conclusions: Preliminary clinical data indicates that the HRD assay identifies OC patients likely to respond to rucaparib and highlights the potential for innovative companion diagnostics enabled by comprehensive genomic profiling based on NGS. Citation Format: James Sun, Iain McNeish, Robert L. Coleman, Amit Oza, Clare Scott, David M. O'Malley, Kevin K. Lin, Christine Burns, Christine Vietz, Philip J. Stephens, Murtaza Mehdi, Matthew Hawryluk, Heidi Giordano, Mitch Raponi, Lindsey Rolfe, Jeff Isaacson, Vincent A. Miller, Andrew Allen, Elizabeth Swisher, Roman Yelensky. A novel companion diagnostic predicts response to the PARP inhibitor rucaparib in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4670. doi:10.1158/1538-7445.AM2015-4670

5550 Background: Maintenance monotherapy with the PARP inhibitor olaparib prolongs progression-free survival in PSR SOC pts with no adverse impact on HRQoL. BRCAm pts derive greater benefit. Nausea...

5579 Background: Treatment options for women with high-grade serous ovarian cancer (HGSC) remain limited. Defective DNA repair capability may underlie response to standard chemotherapy and to PARP ...

5508 Background: At least 50% of high-grade serous ovarian cancers (OC) may have homologous recombination deficiency (HRD). Germline BRCA1 and BRCA2 mutations (gBRCAmut) account for ~1/3. Identific...

5566 Background: BRCA1/2 mutations (BRCAm) predict benefit from olaparib (vs placebo) in platinum sensitive high grade serous ovarian cancer (HGSOC). However not all patients (pts) with BRCAm respo...

Background: Treatment options for women with ovarian cancer remain very limited and acquired resistance to current therapies is very common. Altered DNA repair capability in epithelial ovarian cancer (EOC) may underlie response to both standard therapy and novel treatments, such as PARP inhibitors. Molecular sub-classification of high-grade serous ovarian cancer (HG-SOC) may uncover potential drug targets and possible mechanisms of drug resistance. Understanding the contribution of DNA repair and other driver mutations to drug response and resistance requires the development of molecularly annotated preclinical models reflective of the clinic. Methods: A patient derived xenograft (PDX) cohort has been generated from consecutive, chemotherapy-naïve human HG-SOC and stratified according to in vivo response to standard chemotherapy, DNA repair capability and molecular characteristics, including next generation sequencing by Foundation Medicine. Resistance to therapy is driven by re-treating relapsed PDX in vivo providing invaluable “paired samples” (pre and post drug treatment), which are difficult to obtain from patients, to allow clonal evolution analysis of mechanisms of drug response and resistance. Results: The xenograft success rate was 83%. Of ten HG-SOC PDX, all exhibited mutations in TP53, five in BRCA1/2 (two of which were germline) and two were methylated for BRCA1. In vivo cisplatin response, determined as platinum sensitive (progression-free interval (PFI) ≥100 d, n=4), platinum resistant (PFI Conclusion: PDX with histologic, molecular and therapeutic annotation, as well as clinical outcome data allow interrogation of molecular aberrations and drug resistance in vivo. This will inform targeting of novel therapies and the design of clinical trials for women. Citation Format: Monique D. Topp, Lynne Hartley, Michele Cook, Valerie Heong, Emma Boehm, Lauren McShane, Jan Pyman, Orla McNally, Sumi Ananda, Maria I. Harell, Dariush Etemadmoghadam, Laura Galletta, Kathryn Alsop, Gillian Mitchell, Stephen B. Fox, Jeff B. Kerr, Karla J. Hutt, Scott H. Kaufmann, Australian Ovarian Cancer Study (AOCS), Elizabeth M. Swisher, David D. Bowtell, Matthew M. Wakefield, Clare L. Scott. Using molecularly characterized patient-derived models to delineate underlying drivers and vulnerabilities of epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 38. doi:10.1158/1538-7445.CANSUSC14-38

Introduction The Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) is a multidisciplinary, collaborative framework for the investigation of familial breast cancer. Based in Australia, the primary aim of kConFab is to facilitate high-quality research by amassing a large and comprehensive resource of epidemiological and clinical data with biospecimens from individuals at high risk of breast and/or ovarian cancer, and from their close relatives. Methods Epidemiological, family history and lifestyle data, as well as biospecimens, are collected from multiple-case breast cancer families ascertained through family cancer clinics in Australia and New Zealand. We used the Tyrer-Cuzick algorithms to assess the prospective risk of breast cancer in women in the kConFab cohort who were unaffected with breast cancer at the time of enrolment in the study. Results Of kConFab's first 822 families, 518 families had multiple cases of female breast cancer alone, 239 had cases of female breast and ovarian cancer, 37 had cases of female and male breast cancer, and 14 had both ovarian cancer as well as male and female breast cancer. Data are currently held for 11,422 people and germline DNAs for 7,389. Among the 812 families with at least one germline sample collected, the mean number of germline DNA samples collected per family is nine. Of the 747 families that have undergone some form of mutation screening, 229 (31%) carry a pathogenic or splice-site mutation in BRCA1 or BRCA2. Germline DNAs and data are stored from 773 proven carriers of BRCA1 or BRCA1 mutations. kConFab's fresh tissue bank includes 253 specimens of breast or ovarian tissue – both normal and malignant – including 126 from carriers of BRCA1 or BRCA2 mutations. Conclusion These kConFab resources are available to researchers anywhere in the world, who may apply to kConFab for biospecimens and data for use in ethically approved, peer-reviewed projects. A high calculated risk from the Tyrer-Cuzick algorithms correlated closely with the subsequent occurrence of breast cancer in BRCA1 and BRCA2 mutation positive families, but this was less evident in families in which no pathogenic BRCA1 or BRCA2 mutation has been detected.

A retrospective analysis of the use of 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) positron emission tomography (PET) was performed in patients with histologically proven disseminated carcinoma of unknown primary tumor (CUP).The records of 31 patients with CUP, excluding patients with isolated neck metastases, were reviewed to identify the ability of PET to detect the putative primary site (PPS) and/or to change therapeutic management.In eight out of 31 cases (26%), a PPS was confirmed, either definitively (one pathologically, one radiologically) (true positive) or clinically (six cases). For three cases (10%), histological evidence of a primary tumor distant from the PPS was found (false positive). In a further seven cases (23%), the PPS remained unconfirmed, whereas for 13 cases (42%) no PPS was identified. In five out of seven patients in whom the PET suggested a high probability of having identified the primary site, the PPS was confirmed definitively or clinically. PET altered clinical management in at least 12 cases (38%).PET contributed to the management of previously extensively investigated patients with CUP. Identification of a PPS and/or change in management was documented in 38% of cases, the majority of which were lung or pancreatic cancer. These findings are worthy of evaluation in a prospective study.

Background. The usefulness of fluorine 18-labelled deoxyglucose positron emission tomography (PET) in the detection of unknown primary tumor in patients seen with malignant cervical lymphadenopathy thought to arise from a head and neck primary differs in the published reports to date. To assess the role of PET in this scenario in our institution, an audit was performed. Methods. The records of 21 patients who met the clinical indication were reviewed. End points were the ability of PET to detect an unknown primary tumor and/or distant metastatic disease. Results. In 8 of the 21 patients, PET detected a potential primary site, although none was unequivocally PET positive. One case was pathologically confirmed. In five patients the potential primary site identified on PET could not be confirmed: two had negative biopsies, and three had no clinical evolution in the PET-suspect area for at least 24 months after the initial study. In the remaining two patients, the potential primary site detected by PET was treated without biopsy. PET detected additional regional and/or distant disease that had not been previously documented in nine cases. Conclusions. PET did not add significantly to the detection of an occult primary tumor in patients who had already been comprehensively evaluated by clinical and radiologic investigations. It was of substantial benefit, however, in detecting unsuspected distant disease in patients with undifferentiated nodal histologic findings and helped in delineating regional disease in patients with N2 disease. © 2003 Wiley Periodicals, Inc. Head Neck 25: 138–145, 2003

Aim: Maintenance monotherapy with the PARP inhibitor olaparib significantly prolonged progression-free survival (PFS) versus placebo in patients with PSR SOC, and subsequent analysis has shown that patients with a BRCAm receive greater treatment benefit (Ledermann et al Lancet Oncol 2014). As preserved HRQoL may support chronic administration in the maintenance setting, the effect of olaparib on HRQoL was evaluated in this randomized, double-blind Phase II trial (NCT00753545). Methods: Patient-reported HRQoL and disease-related symptoms were evaluated using the Functional Assessment of Cancer Therapy Ovarian (FACT-O) questionnaire, FACT/NCCN Ovarian Symptom Index (FOSI) and Trial Outcome Index (TOI). Patients completed the FACT-O questionnaire at baseline and every 28 days until progression. Individual symptom severity over 7 days was measured using the five-item Likert scale; physical, social/family, emotional, functional wellbeing and specific concerns to ovarian cancer patients were assessed. The TOI of the FACT-O was the primary HRQoL endpoint. FOSI is the sum of a subset of eight symptom-related items. Results: Of patients randomized to olaparib (n = 136) or placebo (n = 129), BRCA mutation status data were available for 254/265 (96%), of whom 136/254 (53.5%) had a known deleterious/suspected deleterious germline or somatic BRCA mutation. Most patients reported a best response of ‘improved’ or ‘no change’ on TOI (Table); this trend was also seen in other HRQoL measures. There were no statistically significant differences in improvement rates or time to worsening of TOI, FOSI and Total FACT-O. Overall BRCAm BRCAwt Olaparib Placebo Olaparib Placebo Olaparib Placebo TOI N = 115 N = 111 N = 64 N = 53 N = 49 N = 54 Improved * 23 (20.0) 20 (18.0) 16 (25.0) 10 (18.9) 7 (14.3) 10 (18.5) No change† 72 (62.6) 67 (60.4) 38 (59.4) 30 (56.6) 32 (65.3) 36 (66.7) Worsened†† 16 (13.9) 20 (18.0) 7 (10.9) 10 (18.9) 9 (18.4) 8 (14.8) Non-evaluable 4 (3.5) 4 (3.6) 3 (4.7) 3 (5.7) 1 (2.0) 0 BRCAwt, BRCA wild type, includes patients with no known BRCAm or a variant of unknown significance. * Best response of improved defined as two visit responses of 'improved' a minimum of 21 days apart without an intervening visit response of 'worsened'. †Defined as two visit responses of 'no change' or a response of 'no change' and a response of 'improved' a minimum of 21 days apart without an intervening visit response of 'worsened'. No change is defined as a change from baseline of greater than -7 but less than +7.††Defined as a visit of 'worsened' without a response of 'improved' or 'no change' within 21 days. Worsened is defined as a change from baseline of less than or equal to -7. Conclusions: HRQoL was not negatively impacted during maintenance therapy with olaparib. Phase III trials are enrolling. Disclosure: J.A. Ledermann: Travel grants from AstraZeneca; P. Harter: Received a grant from AstraZeneca; C. Gourley: Served on advisory boards for, and received travel grants from, AstraZeneca; G.J.S. Rustin: Served on advisory boards for AstraZeneca, Oxigene, Roche, Amgen, Boehringer Ingelheim and Clovis; C. Scott: Received travel grants from AstraZeneca; A. Fielding, S. Spencer, B. Bennett and D. Parry: Employee of AstraZeneca and stock ownership. All other authors have declared no conflicts of interest.

Aim: PARP inhibitors (PARPi) such as oral rucaparib are thought to be effective in cancers with homologous recombination deficiency (HRD), best shown to date in patients (pts) with a germline BRCA1/2 mutation (gBRCAmut). The Cancer Genome Atlas (TCGA) estimated ∼50% of pts with high-grade serous ovarian carcinoma (OC) have HRD tumors. Multiple mechanisms can lead to HRD, which in turn can lead to genomic loss of heterozygosity (LOH). Currently, the best molecular predictors of PARPi response (other than BRCA mutation) are not known and platinum-sensitivity, a surrogate predictive indicator for PARPi response, is inadequate. Molecular analysis of tumor tissue to assess BRCA mutations as well as genomic LOH, a phenotypic endpoint of HRD, could be a more inclusive method for selection of pts for PARPi therapy. Methods: The primary objective of Phase 2 study ARIEL2 is to identify a molecular HRD signature associated with clinical benefit from rucaparib treatment. This signature will be prospectively applied to the final analysis of the “all-comer” Phase 3 randomized pivotal trial (ARIEL3). ARIEL2 is an ongoing single-arm, open-label biomarker study designed to refine the molecular HRD signature associated with rucaparib response. Eligible pts (n = 180) have relapsed, platinum-sensitive, high-grade OC and measurable disease. Tumor HRD status is assessed using Foundation Medicine's next generation sequencing platform with the current HRD algorithm, based on BRCA status and genomic LOH, developed using in vitro/in vivo and TCGA (and similar) bioinformatic data. PFS and response by RECIST will be correlated with tumor HRD status. Enrollment of gBRCAmut pts is limited to maximize non-gBRCAmut response predictors. Results: Preliminary efficacy data from ARIEL2 indicate RECIST responses in patients who are BRCA wild-type and have high tumor genomic LOH as well as in BRCAmut pts. Data for approximately 75 pts is anticipated to be available in late September. Conclusions: Preliminary data indicate tumor genomic LOH as well as BRCA mutation analysis may predict OC pts likely to respond to rucaparib. Disclosure: I. McNeish: Interactions with Clovis relevant to rucaparib. Advisory Boards for AZ and Roche within the past 2 years also; R. Coleman: Membership on Clovis advisory board and clinical trial funding from Sponsor (Clovis); A.M. Oza: Clinical Trial Funding from Sponsor (Clovis) to Princess Margaret Cancer Centre; D. O'Malley: Clinical trial support from Sponsor (Clovis) Participated in an advisory board for Sponsor (Clovis); C. Scott: Corporate-Sponsored Research: Provision of drug and in-kind molecular analyses by Clovis Oncology of laboratory research not associated with these clinical trials Other Substantive Relationships: Clinical trial support for ARIEL2/3 at my site; A. Oaknin and A. Floquet: Clinical Trial support from Sponsor (Clovis); J. Brenton: Travel funding from Clovis Oncology for clinical trials meetings; K. Lin, S. Shetty, H. Giordano, M. Raponi and L. Rolfe: Clovis Employment and Stock Ownership. All other authors have declared no conflicts of interest.

Background Epithelial ovarian cancer (EOC) mortality rates have remained unchanged in 30 years. The most common EOC subtype, high-grade serous ovarian carcinoma (HG-SOC), can be divided into four subgroups on the basis of molecular characteristics. The C5 subgroup is defined by MYCN pathway activation and carries a poor prognosis. The commonly used EOC cell lines are poor models of HG-SOC, whereas patient-derived xenografts (PDX) retain pathological and immunohistochemical features of primary tumour, providing a valuable preclinical resource for therapeutic exploration. Methods Fourteen “C5-like” HG-SOC PDX were generated from tumour tissue transplanted into NOD/SCIDIL2R γ null mice. Three were from a consecutive cohort of HG-SOC PDX identified by qRT-PCR of the MYCN pathway and the rest from a separate cohort by gene expression analysis. Molecular analysis done included qRT-PCR for MYCN, HMGA2 , LIN28B, and sequencing of DNA repair genes. In-vivo response to cisplatin (intraperitoneal on days 1, 8, and 18) is underway, because platinum response provides prognostic information. Tumour volume is measured twice weekly and mice with tumours larger than 0.7 cm 3 are euthanised. PDX are deemed sensitive if response is maintained for 100 or more days; resistant if they had stable disease or relapsed in less than 100 days; and refractory if they progressed on treatment. Investigation of in vivo response to compounds targeting the MYCN pathway is underway and each response is considered in light of molecular phenotype. Findings Preliminary data revealed six “C5-like” PDX overexpressed LIN28B and eight overexpressed MYCN . Three overexpressed CCNE1 (drug resistance marker) and three PDX harboured mutations in DNA repair genes (marker of platinum sensitivity). Two PDX were sensitive to cisplatin, two were resistant, four were refractory, and one showed mixed response. Interpretation This cohort of “C5-like” HG-SOC PDX depicts molecular complexity and treatment response heterogeneity. Use of these molecularly annotated PDX to demonstrate novel effective therapies targeting the MYCN pathway will inform clinical trial design.

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

Mice lacking suppressor of cytokine signaling-1 (SOCS1) develop a complex fatal neonatal disease. In this study, SOCS1-/- mice were shown to exhibit excessive responses typical of those induced by interferon gamma (IFNgamma), were hyperresponsive to viral infection, and yielded macrophages with an enhanced IFNgamma-dependent capacity to kill L. major parasites. The complex disease in SOCS1-/- mice was prevented by administration of anti-IFNgamma antibodies and did not occur in SOCS1-/- mice also lacking the IFNgamma gene. Although IFNgamma is essential for resistance to a variety of infections, the potential toxic action of IFNgamma, particularly in neonatal mice, appears to require regulation. Our data indicate that SOCS1 is a key modulator of IFNgamma action, allowing the protective effects of this cytokine to occur without the risk of associated pathological responses.

5505 Background: Previously, we reported that maintenance treatment with the oral PARP inhibitor olaparib (400 mg bid) led to a significant PFS improvement vs placebo in patients (pts) with platinum-sensitive relapsed SOC (Ledermann et al NEJM2012). A preplanned subgroup analysis from this randomized, double-blind Phase II trial (NCT00753545) suggested that olaparib may lead to a greater PFS, and an OS, benefit in pts with a known germline BRCAm (gBRCAm). Since gBRCA wild-type (gBRCAwt) pts may develop somatic tumor (t)BRCAm, efficacy analyses were performed for all pts with BRCAm. Methods: gBRCAm status was determined retrospectively for all consenting pts (n = 166) using blood samples taken before randomization. tBRCAm status was determined from archival tumor samples of 196 pts. We analyzed PFS/OS by gBRCAm and total BRCAm status. Preliminary data are reported. Results: gBRCA status was known for 218/265 pts (gBRCAm, 96; gBRCAwt, 122). Including tBRCAm, 136 pts had a BRCAm (BRCAwt, 116). gBRCAm pts had the greatest PFS benefit with olaparib maintenance vs placebo (median: 11.2 vs 4.1 months [m]; HR, 0.17; 95% CI 0.09-0.32; P3 years. Olaparib tolerability was similar in BRCAm pts and the overall population. Conclusions: Olaparib maintenance treatment led to the greatest clinical benefit in pts with a BRCAm. These compelling data warrant confirmation in phase III trials. Clinical trial information: NCT00753545.

5073 Background: Recent molecular exploration of high-grade epithelial ovarian cancer (OC) has revealed potential targets for novel therapy based on altered DNA repair function, deregulated pathways and recurrent amplifications (Cancer Genome Atlas Research Network. 2011. Nature 474). Improved pre-clinical models allowing analysis of specific molecular subsets of ovarian cancer are urgently required to test novel treatment strategies. Methods: We have generated a novel xenograft model of human high-grade serous OC (HG-SOC). Histologic, functional and molecular analysis of the novel xenograft cohort (at baseline and following xenotransplantation) allows stratification of individual HG-SOC for testing with appropriate targeted therapy. We perform functional analysis of in vitro Homologous Recombination (HR) DNA repair and drug response capabilities on fresh human HG-SOC immediately following surgical resection. Molecular classification (similar to Tothill [Clin Canc Res. 2008;14]); analysis of NHEJ pathway (Proc Natl Acad Sci. 2011;108) and other DNA repair genes (Proc Natl Acad Sci USA 2011;108) is performed. In vivo drug response is studied in murine xenografts. Results: Sixteen chemotherapy-naive potentially HG-SOC samples and associated clinical data have been collected. Functional evidence of DNA repair (HR) capability and response to DNA damaging agents will be presented, including IHC for markers of DNA damage (gH2AX), DNA repair (RAD51AP1) and apoptosis (capsase 3 cleavage). Molecular classification, DNA repair gene and DNA repair pathway analyses are underway. Twelve HG-SOC have been transplanted and 6 of the first 8 have successfully xenografted, with serial transplantation and phenotyping of xenograft derivatives underway. In vivo drug response will be presented. Conclusions: This xenograft model will enable us to address hypotheses generated by recent molecular analyses of human HG-SOC (Cancer Genome Atlas Research Network. 2011. Nature 474; Clin Canc Res. 2008;14). Clinical, functional and molecular annotation will allow pre-clinical drug testing based on the plausible hypothesis approach.

The pro-apoptotic BH3-only protein, BIK, is widely expressed and although many critical functions in developmental or stress-induced death have been ascribed to this protein, mice lacking Bik display no overt abnormalities. It has been postulated that Bik can serve as a tumour suppressor, on the basis that its deficiency and loss of apoptotic function have been reported in many human cancers, including lymphoid malignancies. Evasion of apoptosis is a major factor contributing to c-Myc-induced tumour development, but despite this, we found that Bik deficiency did not accelerate Eμ-Myc-induced lymphomagenesis. Co-operation between BIK and NOXA, another BH3-only protein, has been previously described, and was attributed to their complementary binding specificities to distinct subsets of pro-survival BCL-2 family proteins. Nevertheless, combined deficiency of Bik and Noxa did not alter the onset of Eμ-Myc transgene induced lymphoma development. Moreover, although p53-mediated induction of Bik has been reported, neither Eμ-Myc/Bik(-/-) nor Eμ-Myc/Bik(-/-)Noxa(-/-) lymphomas were more resistant than control Eμ-Myc lymphomas to killing by DNA damaging drugs, either in vitro or in vivo. These results suggest that Bik, even in combination with Noxa, is not a potent suppressor of c-Myc-driven tumourigenesis or critical for chemotherapeutic drug-induced killing of Myc-driven tumours.

Background: The five year survival rate for women with ovarian cancer (OC) is less than 40%. Crucial to improving the outcomes for ovarian cancer patients is the development of accurate pre-clinical models of human epithelial OC, which can be used to unravel the mechanisms underlying its evolution and behaviour. Methods: We have generated a novel xenograft model of human high-grade serous OC (HG-SOC) in order to enable preclinical molecular and drug response assessment of individual HG-SOC: this includes a comparison of standard OC xeno-transplantion approaches (intra-peritoneal, subcutaneous and sub renal capsular), with the novel use of the rodent ovarian bursa, the orthotopic site. Utilising the rodent bursa may better replicate the relevant microenvironment and increase the success of EOC xeno-transplantion. Other aspects designed to optimize the model include the use of NOD-SCID-IL-2rg recipient mice, systemic estrogen supplementation and transplantation of fresh human HG-SOC fragments which have had no prior in vitro culture. Histological, functional and molecular analysis of the novel xenograft cohort (at baseline and following xenotransplantation) includes histological review; documentation of in vitro Homologous Recombination (HR) DNA repair and drug response capabilities (using novel α-irradiation and explant drug assays); classification according to molecular subtype (Tothill classfier); documentation of NHEJ pathway status, BRCA1/2 status and other DNA repair gene status. In vivo drug treatment studies are being performed, with the choice of treatment targeted to the specific molecular characteristics of the HG-SOC in question. Results: Fourteen consecutive chemotherapy naive potentially HG-SOC samples have been collected. Corresponding clinical data has been collected. Data concerning DNA repair capability and response to DNA damaging agents will be presented, including IHC for markers of DNA damage (γH2AX), DNA repair (RAD51) and apoptosis (capsase 3 cleavage). HG-SOC in this cohort have been classified according to Tothilll (Tothill et al 2008). Eight appropriate HG-SOC have been transplanted and 5 of the first 6 have successfully xenografted, with phenotyping of xenografts underway. In vivo analysis of response to cisplatin treatment and other relevant therapeutics will be presented. Conclusions: A novel xenograft model has been developed of human HG-SOC, which includes comparative characterization of important prognostic features both in the baseline panel of fresh human HG-SOC and in subsequent xenografts. This clinically, functionally and molecularly annotated consecutive xenograft cohort of HG-SOC will provide outstanding utility for the development of improved therapeutic approaches. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3276. doi:1538-7445.AM2012-3276

Background: Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that provides a critical hub for signalling from at least four different classes of cellular sensing mechanisms including growth factor receptors, GPCRs, integrins and mechanical stress forces. By temporal and spatial integration of signals from these sources, FAK plays a central role in cell migration, invasion and proliferation; processes vital for tumorigenesis. The significance of FAK to the function of signal transduction pathways provides a strong rationale for the combination of FAK inhibitors with other targeted agents to achieve improved efficacy against a range of cancers. Others have demonstrated the importance of FAK in angiogenesis and therefore combining a FAKi with anti-VEGF agents is attractive as it employs two complementary mechanisms of suppressing the formation of tumor vasculature. Here we present results from the co-administration of CTx-0294945, a highly selective FAKi, and bevacizumab (bev) in an orthotopic model of human breast cancer. Methods and Results: CTx-0294945 is an orally bioavailable small molecule ATP competitive inhibitor of focal adhesion kinase (FAK KD=0.21 nM). It exhibits high selectivity against a diverse panel of 125 kinases including the closely related Pyk2. CTx-0294945 inhibits autophosphorylation of 397Y-FAK in MDA-MB-231 cells with an IC50 = 7 nM and exhibits low general cellular toxicity (IC50 = 2.7 µM, MDA-MB-231 cells). CTx-0294945 is suitable for oral administration (%F=58 and t1/2=5.1 h at 20 mg/Kg in rat) and does not inhibit (IC50 >20 µM) any of the cytochrome p450 isoforms tested to date. To assess the co-administration of CTx-0294945 with bev, mice were injected orthotopically with MDA-MB-231 cells (106). After 14 days, when tumors were palpable, mice were randomized into 4 groups and dosing commenced. The groups were treated with CTx-0294945 (80 mg/kg QD, PO), bev (12.5 mg/Kg IP, x2/week), CTx-0294945 (80 mg/Kg QD, PO) and bev (12.5 mg/Kg IP, x2/week) or vehicle. Tumor growth was monitored and on day 28 animals in the vehicle and CTx-0294945 arms were culled when the size of the tumors reached ethical end point (1000 mm3). Tumor growth in the bev only and the CTx-0294945 + bev arms was significantly inhibited (75% and 88% TGI respectively). At this time the treatment regimes for both cohorts were stopped and tumor growth allowed to progress. After an additional 14 days the experiment was terminated when the bev treatment group reached ethical end point; however the average size of the tumors in the CTx-0294945 + bev cohort was still was only 562 mm3. Conclusions: Our data suggest the potential utility of combining a selective FAK inhibitor with bevacizumab to prevent tumour progression and enhance the durability of response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-308. doi:1538-7445.AM2012-LB-308

5003 Background: Olaparib (AZD2281) is an oral PARP inhibitor that has shown antitumor activity in patients (pts) with high-grade serous ovarian cancer (SOC) with and without BRCA1 or BRCA2 mutations. This randomized, double-blind, multicenter, placebo-controlled Phase II study evaluated maintenance treatment with olaparib in pts with high-grade PSR SOC (clinicaltrials.gov; NCT00753545). Methods: Pts with PSR SOC who had received ≥2 previous platinum regimens and were in a maintained partial or complete response following their last platinum-containing regimen were randomized to oral olaparib 400 mg bid or placebo. The primary endpoint was progression-free survival (PFS) by RECIST. Secondary endpoints included time to progression (TTP) by CA-125 (GCIG criteria) or RECIST, overall survival (OS) and safety. Results: 265 pts were randomized (136 to olaparib and 129 to placebo). Demographics and baseline characteristics were generally well balanced. At data cut-off there were 153 (58%) progression events. PFS...

The proteins that control the number of primordial follicles initially established within the ovary are largely unknown. Here we investigated the hypothesis that PUMA, a pro-apoptotic protein belonging to the Bcl-2 family, regulates germ cell death during ovarian development and thereby determines the number of primordial follicles that make up the ovarian reserve. Ovaries were obtained from embryonic day 17.5 (E17.5) and post-natal day 10 (PN10) wild-type (wt) and puma–/– mice and subjected to morphological, molecular and stereological characterisation (n = 3-6 mice/genotype/age). At E17.5, ovaries were densely populated with germ cells and early meiotic oocytes. Immunostaining for MVH and PCNA confirmed the identity of germ cells and proliferating germ cells, respectively. Pyknotic nuclei and TUNEL positive germ cells were rarely detected, suggesting that cell death was uncommon at this age. At PN10, primordial follicle assembly was complete for both genotypes, as confirmed morphologically and by immunostaining for oocyte markers GCNA and MSY2. The number of germ cells in E17.5 wt and puma–/– ovaries was comparable (p=0.81, See Table 1). However, PN10 puma–/– ovaries contained significantly more primordial follicles than wt ovaries (P < 0.001, See Table 1), revealing an over-endowment of primordial follicles in the absence of PUMA. These data show that PUMA regulates the developmentally programmed death of germ cells between E17.5 and PN10 in the mouse and thereby determines the number of primordial follicles that make up the initial ovarian reserve. This work was supported by the NHMRC (Program Grants #494802 and #257502, Fellowships JKF (#441101), KJH (#494836), CLS (#406675), AS (#461299)); the Leukemia and Lymphoma Society (New York; SCOR grant#7015), the National Cancer Institute (NIH, US; CA80188 and CA43540) and Victorian Government Infrastructure Funds.

5500 Background: Olaparib (AZD2281; KU-0059436) is a novel, orally active PARP inhibitor that induces synthetic lethality in homozygous BRCA-deficient cells. A phase I trial identified 400 mg bd as the maximum tolerated dose (MTD) with an initial signal of efficacy in BRCA-deficient cancers (ASCO 2008; abst 5510). The primary aim of this study was to test the efficacy of olaparib in confirmed BRCA1/BRCA2 carriers with advanced chemotherapy-refractory ovarian cancer. The secondary aim was to assess the safety and tolerability profile in ovarian cancer patients with BRCA1/2 deficiency. Methods: In an international, multicenter, proof-of-concept, single-arm, phase II study, two patient (pt) sequential cohorts received continuous oral olaparib in 28-day cycles, initially at the MTD, 400 mg bd (33 pts), and subsequently at 100 mg bd (24 pts), a previously shown clinically active and PARP inhibitory dose. Eligibility criteria included confirmed genetic BRCA1/2 mutation and recurrent, measurable, incurable disease (previous chemotherapy, median 3 lines). The primary efficacy endpoint was best objective response rate (ORR; RECIST) post baseline. Change in CA125 was a secondary efficacy endpoint. All adverse events were reported using CTCAE v3. Results: At this interim analysis dated October 31, 2008, of 57 enrolled pts (39 BRCA1 deficient and 18 BRCA2 deficient), 33 were evaluable at 400 mg bd and 24 at 100 mg bd. The confirmed RECIST ORR was 33% at 400 mg bd and 12.5% at 100 mg bd. Clinical benefit rate (ORR and/or confirmed >50% decline in CA125) was 57.6% at 400 mg bd and 16.7% at 100 mg bd. Toxicity was mainly mild in severity, reflecting grade 1/2 nausea (44%); fatigue (35%); and anemia (14%). Grade 3 toxicity occurred infrequently, and comprised primarily nausea (7%) and leukopenia (5%). Conclusions: Oral olaparib is well tolerated and highly active in advanced, chemotherapy-refractory BRCA-deficient ovarian cancer, with greater activity seen at the higher dose. Toxicity in BRCA1/2 carriers was similar to that seen in non-carriers. This study provides positive proof of the concept of the activity and tolerability of genetically defined targeted therapy with olaparib in BRCA-deficient ovarian cancers. [Table: see text]

1009 Background: Targeting BRCA1 and BRCA2 mutation analysis to those most likely to carry a mutation is desirable. We examined the performance of 6 different methods of selecting individuals for B...

SummarySince apoptosis is impaired in malignant cells overexpressing prosurvival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Of seven putative BH3 mimetics tested, only ABT-737 triggered Bax/Bak-mediated apoptosis. Despite its high affinity for Bcl-2, Bcl-xL, and Bcl-w, many cell types proved refractory to ABT-737. We show that this resistance reflects ABT-737's inability to target another prosurvival relative, Mcl-1. Downregulation of Mcl-1 by several strategies conferred sensitivity to ABT-737. Furthermore, enforced Mcl-1 expression in a mouse lymphoma model conferred resistance. In contrast, cells overexpressing Bcl-2 remained highly sensitive to ABT-737. Hence, ABT-737 should prove efficacious in tumors with low Mcl-1 levels, or when combined with agents that inactivate Mcl-1, even to treat those tumors that overexpress Bcl-2.

e22202 Background: The most common epithelial ovarian cancer subtype, high-grade serous ovarian cancer (HGSC), can be divided into four subgroups based on molecular characteristics. The C5/Stem-A s...

Histone deacetylase inhibitors (HDACi) can elicit a range of biological responses that affect tumor growth and survival, including inhibition of cell cycle progression, induction of tumor cell-selective apoptosis, suppression of angiogenesis, and modulation of immune responses, and show promising activity against hematological malignancies in clinical trials. Using the Eμ-myc model of B cell lymphoma, we screened tumors with defined genetic alterations in apoptotic pathways for therapeutic responsiveness to the HDACi vorinostat. We demonstrated a direct correlation between induction of tumor cell apoptosis in vivo and therapeutic efficacy. Vorinostat did not require p53 activity or a functional death receptor pathway to kill Eμ-myc lymphomas and mediate a therapeutic response but depended on activation of the intrinsic apoptotic pathway with the proapoptotic BH3-only proteins Bid and Bim playing an important role. Our studies provide important information regarding the mechanisms of action of HDACi that have broad implications regarding stratification of patients receiving HDACi therapy alone or in combination with other anticancer agents.

5534 Background: BRCA1/2 mutations (BRCAm) appear to predict benefit from PARP inhibition. However, non-mutation carriers also benefit and not all patients (pts) with BRCAm respond to PARP inhibito...

Background: The ATM gene encoding a putative protein kinase is mutated in ataxia-telangiectasia (A-T), an autosomal recessive disorder with a predisposition for cancer. Studies of A-T families suggest that female heterozygotes have an increased risk of breast cancer compared with non-carriers. However, neither linkage analyses nor mutation studies have provided supporting evidence for a role of ATM in breast cancer predisposition. Nevertheless, two recurrent ATM mutations, T7271G and IVS10-6T→G, reportedly increase the risk of breast cancer. We examined these two ATM mutations in a population-based, case-control series of breast cancer families and multiple-case breast cancer families. Methods: Five hundred twenty-five or 262 case patients with breast cancer and 381 or 68 control subjects, respectively, were genotyped for the T7271G and IVS10-6T→G ATM mutations, as were index patients from 76 non-BRCA1/2 multiple-case breast cancer families. Linkage and penetrance were analyzed. ATM protein expression and kinase activity were analyzed in lymphoblastoid cell lines from mutation carriers. All statistical tests were two-sided. Results: In case and control subjects unselected for family history of breast cancer, one case patient had the T7271G mutation, and none had the IVS10-6T→G mutation. In three multiple-case families, one of these two mutations segregated with breast cancer. The estimated average penetrance of the mutations was 60% (95% confidence interval [CI] = 32% to 90%) to age 70 years, equivalent to a 15.7-fold (95% CI = 6.4-fold to 38.0-fold) increased relative risk compared with that of the general population. Expression and activity analyses of ATM in heterozygous cell lines indicated that both mutations are dominant negative. Conclusion: At least two ATM mutations are associated with a sufficiently high risk of breast cancer to be found in multiple-case breast cancer families. Full mutation analysis of the ATM gene in such families could help clarify the role of ATM in breast cancer susceptibility.

Evasion of apoptosis contributes importantly to c-Myc-induced tumorigenesis. The BH3-only Bcl-2 family members Puma and Noxa are critical pro-apoptotic transcriptional targets of p53, a major mediator of Myc-induced apoptosis and suppressor of Myc-induced tumorigenesis. Hence, we have explored the impact of their individual or combined loss on myc-driven lymphomagenesis. Notably, Puma deficiency both increased B-lineage cells and accelerated the development of B lymphoma, accompanied by leukaemia, but not of pre-B lymphoma. Noxa deficiency alone also increased B-lineage cells but did not accelerate lymphomagenesis. However, its deficiency combined with loss of one puma allele produced more rapid onset of both pre-B and B lymphomas than did loss of a single puma allele alone. Nevertheless, the acceleration evoked by loss of both genes was not as marked as that caused by p53 heterozygosity. These results show that Puma imposes a significant, and Noxa a minor barrier to c-Myc-driven lymphomagenesis. They also indicate that additional BH3-only proteins probably also drive Myc-induced apoptosis and that non-apoptotic functions of p53 may contribute substantially to its tumour suppressor role.