Cancer Therapy: Preclinical Development of a Functional Assay for Homologous Recombination Status in Primary Cultures of Epithelial Ovarian Tumor and Correlation with Sensitivity to Poly(ADP-Ribose) Polymerase Inhibitors Asima Mukhopadhyay 1,2 , Ahmed Elattar 1,2 , Aiste Cerbinskaite 2 , Sarah J. Wilkinson 2 , Yvette Drew 2 , Suzanne Kyle 2 , Gerrit Los 3 , Zdenek Hostomsky 3 , Richard J. Edmondson 1,2 , and Nicola J. Curtin 2 Abstract Purpose: Poly(ADP-ribose) polymerase (PARP) inhibitors selectively target homologous recombina- tion (HR)–defective cells and show good clinical activity in hereditary breast and ovarian cancer associ- ated with BRCA1 or BRCA2 mutations. A high proportion (up to 50%) of sporadic epithelial ovarian cancers (EOC) could be deficient in HR due to genetic or epigenetic inactivation of BRCA1/BRCA2 or other HR genes. Therefore, there is a potential for extending the use of PARP inhibitors to these patients if HR status can be identified. We developed a functional assay of HR status in primary cultures of EOCs based on Rad51 focus formation that correlates well with sensitivity to the potent PARP inhibitor AG014699. Experimental Design: Primary cultures were derived from ascitic fluid from patients with EOCs. HR status was investigated by γH2AX and Rad51 focus formation by immunofluorescence. Cytotoxicity to PARP inhibitors was tested by sulforhodamine B and survival assay. Results: Twenty-five cultures were evaluated for HR status and cytotoxicity to PARP inhibitor. Following exposure to AG014699, there was an increase in Rad51 foci (HR competent) in 9 of 24 (36%) but no increase (HR deficient) in 16 of 24 (64%) cultures. Cytotoxicity was observed in 15 of 16 (93%) HR- deficient samples but not in 9 of 9 HR-competent samples following 24-hour exposure to 10 μmol/L AG014699. Conclusion: HR status can be determined in primary cancer samples by Rad51 focus formation, and this correlates with in vitro response to PARP inhibition. Use of this assay as a biomarker now needs testing in the setting of a clinical trial. Clin Cancer Res; 16(8); 2344–51. ©2010 AACR. Genomic DNA is continually exposed to damage from exogenous or endogenous sources. However, most cells have a plethora of DNA repair mechanisms, including nu- cleotide excision repair, mismatch repair, base excision re- pair (BER), direct repair, and double-strand break (DSB) repair, through which they respond to this challenge and thereby restore their genomic integrity. Homologous re- combination (HR) is an error-free pathway of DSB repair in dividing cells. A faulty HR pathway has variously been associated with carcinogenesis (1). In recent years, poly(ADP-ribose) polymerase (PARP) inhibitors have emerged as a novel class of chemothera- peutic agent effective in tumor cells deficient in HR repair. PARP-1, the founding and most abundant member of a family of highly conserved enzymes, along with PARP-2, has an important role in signaling single-strand breaks (SSB) as a part of BER pathway (2, 3). Inhibition of PARP activity thus leads to an accumulation of SSBs that convert to DSBs and stalled replication forks in S phase, leading to cell death unless repaired by HR (4). HR-defective cells are exquisitely sensitive to PARP inhibition even in the ab- sence of DNA-damaging agents most likely because of the high level of naturally occurring endogenous base damage in the form of SSBs that would normally be re- paired by BER (5). Within the context of ovarian cancers, it is estimated that up to 15% of epithelial ovarian cancers (EOC) could be deficient in HR repair due to mutations in BRCA1 or BRCA2 genes (6, 7). BRCA1/BRCA2 proteins Authors' Affiliations: 1 Northern Gynaecological Oncology Centre, Queen Elizabeth Hospital, Gateshead, United Kingdom; 2 Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom; and 3 Pfizer Oncology, La Jolla, California Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Richard J. Edmondson, Northern Institute for Cancer Research, Newcastle University, Paul O'Gorman Building, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom. Phone: 44-191-445-6148; Fax: 44-191-445-2129; E-mail: Richard. edmondson@ncl.ac.uk. doi: 10.1158/1078-0432.CCR-09-2758 ©2010 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 16(8) April 15, 2010 2344 Downloaded from http://aacrjournals.org/clincancerres/article-pdf/16/8/2344/1994799/2344.pdf by guest on 13 June 2022