An ex vivo assay of XRT-induced Rad51 foci formation predicts response to PARP-inhibition in ovarian cancer


      • Homologous recombination (HR) defects are common in ovarian cancer, suggesting a role for PARP inhibitors.
      • No predictive assay for HR defects exists, but Rad51 is a reliable marker for HR.
      • An ex-vivo IR assay using Rad51 foci formation accurately predicts PARP-inhibitor response.



      BRCA-positive ovarian cancer patients derive benefit PARP inhibitors. Approximately 50% of ovarian cancer tumors have homologous recombination (HR) deficiencies and are therefore “BRCA-like,” possibly rendering them sensitive to PARP inhibition. However, no predictive assay exists to identify these patients. We sought to determine if irradiation-induced Rad51 foci formation, a known marker of HR, correlated to PARP inhibitor response in an ovarian cancer model.


      Ovarian cancer cell lines were exposed to PARP-inhibitor ABT-888 to determine effect on growth. Rad51 protein expression prior to irradiation was determined via Western blot. Cultured cells and patient-derived xenograft tumors (PDX) were irradiated and probed for Rad51 foci. In vivo PDX tumors were treated with ABT-888 and carboplatin; these results were correlated with the ex vivo ionizing radiation assay.


      Three of seven cell lines were sensitive to ABT-888. Sensitive lines had the lowest Rad51 foci formation rate after irradiation, indicating functional HR deficiency. Approximately 50% of the PDX samples had decreased Rad51 foci formation. Total Rad51 protein levels were consistently low, suggesting that DNA damage induction is required to characterize HR status. The ex vivo IR assay accurately predicted which PDX models were sensitive to PARP inhibition in vitro and in vivo. ABT-888 alone reduced orthotopic tumor growth by 51% in A2780ip2 cell line, predicted to respond by the ex vivo assay. Three PDX models' response also correlated with the assay.


      The ex vivo IR assay correlates with response to PARP inhibition. Analysis of total Rad51 protein is not a reliable substitute.


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Gynecologic Oncology
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Siegel R.
        • Ma J.
        • Zou Z.
        • Jemal A.
        Cancer statistics.
        CA Cancer J Clin. 2014; 64: 9-29
      1. Ovarian cancer statistics.
        American Cancer Society, 2012 ([Accessed August 5, 2012, at])
        • Agarwal R.
        • Kaye S.B.
        Ovarian cancer: strategies for overcoming resistance to chemotherapy.
        Nat Rev Cancer. 2003; 3: 502-516
        • Moynahan M.E.
        • Chiu J.W.
        • Koller B.H.
        • Jasin M.
        Brca1 controls homology-directed DNA repair.
        Mol Cell. 1999; 4: 511-518
        • Patel K.J.
        • Yu V.P.
        • Lee H.
        • et al.
        Involvement of Brca2 in DNA repair.
        Mol Cell. 1998; 1: 347-357
        • Haaf T.
        • Golub E.I.
        • Reddy G.
        • Radding C.M.
        • Ward D.C.
        Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes.
        Proc Natl Acad Sci U S A. 1995; 92: 2298-2302
        • Scully R.
        • Chen J.
        • Ochs R.L.
        • et al.
        Dynamic changes of BRCA1 subnuclear location and phosphorylation state are initiated by DNA damage.
        Cell. 1997; 90: 425-435
        • Yuan S.S.
        • Lee S.Y.
        • Chen G.
        • Song M.
        • Tomlinson G.E.
        • Lee E.Y.
        BRCA2 is required for ionizing radiation-induced assembly of Rad51 complex in vivo.
        Cancer Res. 1999; 59: 3547-3551
        • Zhou C.
        • Huang P.
        • Liu J.
        The carboxyl-terminal of BRCA1 is required for subnuclear assembly of RAD51 after treatment with cisplatin but not ionizing radiation in human breast and ovarian cancer cells.
        Biochem Biophys Res Commun. 2005; 336: 952-960
        • Tsuzuki T.
        • Fujii Y.
        • Sakumi K.
        • et al.
        Targeted disruption of the Rad51 gene leads to lethality in embryonic mice.
        Proc Natl Acad Sci U S A. 1996; 93: 6236-6240
        • Lim D.S.
        • Hasty P.
        A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53.
        Mol Cell Biol. 1996; 16: 7133-7143
      2. Integrated genomic analyses of ovarian carcinoma.
        Nature. 2011; 474: 609-615
        • Zhao L.
        • Au J.L.
        • Wientjes M.G.
        Comparison of methods for evaluating drug–drug interaction.
        Front Biosci (Elite Ed). 2010; 2: 241-249
        • Chou T.C.
        Drug combination studies and their synergy quantification using the Chou–Talalay method.
        Cancer Res. 2010; 70: 440-446
        • Nowsheen S.
        • Bonner J.A.
        • Lobuglio A.F.
        • et al.
        Cetuximab augments cytotoxicity with poly (adp-ribose) polymerase inhibition in head and neck cancer.
        PLoS One. 2011; 6: e24148
        • Nowsheen S.
        • Bonner J.A.
        • Yang E.S.
        The poly(ADP-Ribose) polymerase inhibitor ABT-888 reduces radiation-induced nuclear EGFR and augments head and neck tumor response to radiotherapy.
        Radiother Oncol. 2011; 99: 331-338
        • Landen Jr., C.N.
        • Goodman B.
        • Katre A.A.
        • et al.
        Targeting aldehyde dehydrogenase cancer stem cells in ovarian cancer.
        Mol Cancer Ther. 2010; 9: 3186-3199
      3. Sanger Institute Cancer Genome Project.
        (Accessed February 5, 2014, at)
        • Konstantinopoulos P.A.
        • Spentzos D.
        • Karlan B.Y.
        • et al.
        Gene expression profile of BRCAness that correlates with responsiveness to chemotherapy and with outcome in patients with epithelial ovarian cancer.
        J Clin Oncol. 2010; 28: 3555-3561
        • Graeser M.
        • McCarthy A.
        • Lord C.J.
        • et al.
        A marker of homologous recombination predicts pathologic complete response to neoadjuvant chemotherapy in primary breast cancer.
        Clin Cancer Res. 2010; 16: 6159-6168
        • Domcke S.
        • Sinha R.
        • Levine D.A.
        • Sander C.
        • Schultz N.
        Evaluating cell lines as tumour models by comparison of genomic profiles.
        Nat Commun. 2013; 4: 2126
        • Weroha S.J.
        • Becker M.A.
        • Enderica-Gonzalez S.
        • et al.
        Tumorgrafts as in vivo surrogates for women with ovarian cancer.
        Clin Cancer Res. 2014; 20: 1288-1297
        • Audeh M.W.
        • Carmichael J.
        • Penson R.T.
        • et al.
        Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and recurrent ovarian cancer: a proof-of-concept trial.
        Lancet. 2010; 376: 245-251
        • Fong P.C.
        • Yap T.A.
        • Boss D.S.
        • et al.
        Poly(ADP)-ribose polymerase inhibition: frequent durable responses in BRCA carrier ovarian cancer correlating with platinum-free interval.
        J Clin Oncol. 2010; 28: 2512-2519
        • Risch H.A.
        • McLaughlin J.R.
        • Cole D.E.
        • et al.
        Prevalence and penetrance of germline BRCA1 and BRCA2 mutations in a population series of 649 women with ovarian cancer.
        Am J Hum Genet. 2001; 68: 700-710
        • Ledermann J.
        • Harter P.
        • Gourley C.
        • et al.
        Olaparib maintenance therapy in platinum-sensitive relapsed ovarian cancer.
        N Engl J Med. 2012; 366: 1382-1392
        • Gelmon K.A.
        • Tischkowitz M.
        • Mackay H.
        • et al.
        Olaparib in patients with recurrent high-grade serous or poorly differentiated ovarian carcinoma or triple-negative breast cancer: a phase 2, multicentre, open-label, non-randomised study.
        Lancet Oncol. 2011; 12: 852-861
        • Mukhopadhyay A.
        • Elattar A.
        • Cerbinskaite A.
        • et al.
        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.
        Clin Cancer Res. 2010; 16: 2344-2351
        • Pennington K.P.
        • Walsh T.
        • Harrell M.I.
        • et al.
        Germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian, fallopian tube, and peritoneal carcinomas.
        Clin Cancer Res. 2014; 20: 764-775
        • Chan N.
        • Pires I.M.
        • Bencokova Z.
        • et al.
        Contextual synthetic lethality of cancer cell kill based on the tumor microenvironment.
        Cancer Res. 2010; 70: 8045-8054
        • Barretina J.
        • Caponigro G.
        • Stransky N.
        • et al.
        The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.
        Nature. 2012; 483: 603-607
        • Forbes S.A.
        • Bindal N.
        • Bamford S.
        • et al.
        COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer.
        Nucleic Acids Res. 2011; 39: D945-D950
        • Lewis K.A.
        • Mullany S.
        • Thomas B.
        • et al.
        Heterozygous ATR mutations in mismatch repair-deficient cancer cells have functional significance.
        Cancer Res. 2005; 65: 7091-7095