Abstract P4-07-07: Evaluating Rad51/geminin protein expression as an indicator of homologous recombination deficiency in breast cancer models

Abstract

Abstract Background: Homologous recombination deficiency (HRD) in cancer cells can occur due to mutations (germline or sporadic), methylation or other epigenetic causes. HRD leads to a defect in the conservative, error-free DNA repair mechanism and is associated with enhanced susceptibility to DNA targeting chemotherapy. Currently functional HRD assays are not broadly available for clinical use. Many of the HRD assays used in the experimental setting require fresh frozen tissue for optimal results, or require specialized expertise to interpret the results. We evaluated an immunohistochemical (IHC) assay using formalin fixed paraffin embedded (FFPE) tissue to measure protein expression of Rad51 and geminin, a cell proliferation marker, to assess HRD in breast cancer cell line models and clinical breast cancer samples. We hypothesize that Rad51, which is involved in the later stages of HR, can serve as a functional marker of HRD. Methods: The MCF-7 human breast cancer cell line was used as a model with intact HR. Western blotting of total cell lysates from cells grown in culture was performed to confirm HR response following treatment with DNA damaging chemotherapeutic agents, cisplatin and doxorubicin. Paclitaxel, a microtubule targeting agent, was used as a negative control. Mice with MCF-7 xenograft tumors were also treated with cisplatin, or doxorubicin at two dose levels (low and high) and various time points post treatment to assess the dose and time response to HR markers. Tumors from mice treated with paclitaxel were used as a negative control. Xenograft tumors were fixed and analyzed by IHC using an antibody specific for total Rad51 and geminin expression. DNA damage was also assessed in a portion of the tumor using a pulse gel electrophoresis assay. We also analyzed FFPE breast cancer clinical samples from patients with BRCA1 mutations for Rad51 and geminin expression. Results: In MCF-7 grown in vitro, total Rad51 was elevated as soon as 4 hours following exposure to doxorubicin and cisplatin, but not in response to paclitaxel treatment. In xenograft tumors, baseline Rad51 and geminin expression were relatively high illustrating proficient HR in an actively proliferating tumor model. Rad51 expression increased post treatment with cisplatin and doxorubicin as early as 6hrs and peaked at 16-24hrs. Geminin expression correlated well with expression of Rad51 at baseline and in time response to treatment. Pulse gel electrophoresis in paired tumor samples confirmed DNA damage was occurring compared to vehicle control treated tumors. However, this technique did not show a strong dose or time response. Five breast tumors from patients with known BRCA1 mutations were stained for Rad51 and geminin expression. High geminin expression and low Rad51 expression was noted in the majority of these tumors. Conclusions: An IHC assay using FFPE tissue to measure Rad51/geminin is a promising method to assess HRD in breast cancer. Further analytical and clinical validation of this approach is ongoing. Citation Format: Chalasani P, Nagy D, Livingston RB, Weterings E, Nagle R, Singh S, Barnes M, Grogan T, Ridder R, Baker AF, Kandavel S. Evaluating Rad51/geminin protein expression as an indicator of homologous recombination deficiency in breast cancer models. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-07-07.