LAUSR

Objectives: To evaluate the potential of DNA damage response protein expression and foci formation to serve as biomarkers of resistance to platinum chemotherpay or Poly-(ADP)-Ribose Polymerase Inhibitors (PARPi) using immunohistochemistry (IHC). Methods: A collection of four patient-derived xenografts (PDX) mouse models with BRCA1 mutations in exon 11, a BRCA1 exon 13 mutant, and a BRCA1 wild-type tumor were analyzed. BRCA1 exon 11 mutants included: PDX196, mutation: c1961delA; PDX124, mutation: c196delA; PDX017, mutation: c1105_1106insTC; PDX056, mutation: c895_896delGT; BRCA1 WT control: PDX036 and BRCA1 exon 13 mutant: PDX1126, mutation: c4327C>T. Mice harboring PDX tumors were subjected to vehicle, PARPi (rucaparib), or platinum (cisplatin) treatment. Passaging and re-treatment of tumors was performed until minimal therapy response. Formalin-fixed paraffin embedded tumor samples was prepared for IHC analyses. IHC protocols were optimized to evaluate expression and foci formation for the following proteins: phosphorylated-RPA32, Rad51, phosphorylated-53BP1, 53BP1, BRCA1, Ki67. Results: BRCA1 foci formation was observed by IHC in PARPi and cisplatin resistant tumors. Reversion mutations were not detected and BRCA1 protein expression was due to increased levels of BRCA1-D11q. Increased RPA32 and RAD51 foci were also observed in several therapy resistant PDX models, which indicate active HR repair. Interestingly, acquired resistance to cisplatin and PARPi was associated with a reduction in 53BP1 and phosphorylated-53BP1. Finally, PARPi and cisplatin resistant tumors exhibited similar levels of BRCA1 and RAD51 foci. Conclusions: BRCA1-D11q protein expression correlates with platinum and PARPi response in several BRCA1 exon 11 mutant PDX models. We predict that increased BRCA1-D11q in combination with decreased 53BP1 expression drives RPA and RAD51 foci, resulting in active homologous recombination and therapy resistance. Our results suggest that IHC can be utilized for detection of DNA damage repair protein expression as well as foci formation, and could lead to the development of biomarkers that are predictive of therapy response.