UNCORRECTED PRO O F 1 An ex vivo assay of XRT-induced Rad51 foci formation predicts response 2 to PARP-inhibition in ovarian cancer 3 Monjri M. Q1 Shah a , Zachary C. Dobbin a , Somaira Nowsheen b , Monica Wieglos b , Ashwini A. Katre a , 4 Ronald D. Alvarez a , Panagiotis A. Konstantinopoulos c , Eddy S. Yang b , Charles N. Landen a, ⁎ 5 a Department of Obstetrics and Gynecology, University of Alabama at Birmingham, Birmingham, AL, USA 6 b Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL, USA 7 c Department of Medicine, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA 8 9 H I G H L I G H T S 10 11 • Homologous recombination (HR) defects are common in ovarian cancer, suggesting a role for PARP inhibitors. 12 • No predictive assay for HR defects exists, but Rad51 is a reliable marker for HR. 13 • An ex-vivo IR assay using Rad51 foci formation accurately predicts PARP-inhibitor response. 14 15 1 6 a b s t r a c t 1 7 a r t i c l e i n f o 18 Article history: 19 Received 7 February 2014 20 Accepted 11 May 2014 21 Available online xxxx 22 Keywords: 23 Ovarian cancer 24 PARP inhibitor 25 Homologous recombination deficiency 26 Rad51 27 Objective. BRCA-positive ovarian cancer patients derive benefit PARP inhibitors Q2 . Approximately 50% of 28 ovarian cancer tumors have homologous recombination (HR) deficiencies and are therefore “BRCA-like,” po 29 rendering them sensitive to PARP inhibition. However, no predictive assay exists to identify these patients. 30 sought to determine if irradiation-induced Rad51 foci formation, a known marker of HR, correlated to PARP 31 inhibitor response in an ovarian cancer model. 32 Methods. Ovarian cancer cell lines were exposed to PARP-inhibitor ABT-888 to determine effect on growt 33 Rad51 protein expression prior to irradiation was determined via Western blot. Cultured cells and patient- 34 derived xenograft tumors (PDX) were irradiated and probed for Rad51 foci. In vivo PDX tumors were treated 35 with ABT-888 and carboplatin; these results were correlated with the ex vivo ionizing radiation assay. 36 Results. Three of seven cell lines were sensitive to ABT-888. Sensitive lines had the lowest Rad51 foci fo 37 tion rate after irradiation, indicating functional HR deficiency. Approximately 50% of the PDX samples had 38 decreased Rad51 foci formation. Total Rad51 protein levels were consistently low, suggesting that DNA dam 39 induction is required to characterize HR status. The ex vivo IR assay accurately predicted which PDX model 40 sensitive to PARP inhibition in vitro and in vivo. ABT-888 alone reduced orthotopic tumor growth by 51% in 41 A2780ip2 cell line, predicted to respond by the ex vivo assay. Three PDX models' response also correlated w 42 the assay. 43 Conclusions. The ex vivo IR assay correlates with response to PARP inhibition. Analysis of total Rad51 pr 44 not a reliable substitute. 45 © 2014 Published by Elsevier Inc. 46 47 48 49 50 Introduction 51 Epithelial ovarian cancer is the most lethal gynecologic malignancy 52 in developed nations, with an Q3 estimated 14,270 deaths in the United 53 States alone in 2014 [1]. Even though initial treatment with a standard 54 platinum/taxane regimen is effective in about 80% of women diagnosed 55 with advanced stage disease, median progression-free interval is only 56 approximately 18 months, and median 5-year survival is approximately 57 40% [2,3]. 58 Over the past decade, great strides have been made in understand- 59 ing the genetic and molecular basis of cancer. In particular, hereditary 60 cancer syndromes have been very important in informing how specific 61 mutations can give rise to cancer and how those mutations can be 62 selectively therapeutically targeted. Hereditary breast and ovarian 63 cancers caused by mutations in the BRCA1 and BRCA2 genes, which are 64 important for homologous recombination (HR)-mediated DNA repair 65 when double-stranded DNA breaks (DSB) are encountered [4,5], are Gynecologic Oncology xxx (2014) xxx–xxx ⁎ Corresponding author at: University of Alabama at Birmingham, Department of Obstetrics and Gynecology, 176F RM 10250, 619 19th Street S, Birmingham, AL 35294, USA. E-mail address: clanden@uabmc.edu (C.N. Landen). YGYNO-975510; No. of pages: 7; 4C: http://dx.doi.org/10.1016/j.ygyno.2014.05.009 0090-8258/© 2014 Published by Elsevier Inc. Contents lists available at ScienceDirect Gynecologic Oncology j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / y g y n o Please cite this article as: Shah MM, et al, An ex vivo assay of XRT-induced Rad51 foci formation predicts response to PARP-inhibition in ov cancer, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.05.009