Pyman, Jan

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.

Abstract 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 <100 d, n=3) or platinum refractory (n=3), was largely consistent with patient outcome. Three of four platinum sensitive HG-SOC PDX contained DNA repair gene mutations, and the fourth was methylated for BRCA1. In contrast, all three platinum refractory PDX overexpressed dominant oncogenes (CCNE1, LIN28B and/or BCL2). Molecular analysis of this cohort has revealed actionable targets for novel therapeutic strategies. In vivo studies, including with PARP inhibitors, are underway. 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

Abstract 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