Abstract 3341: Development of a multiplex quantitative immunofluorescence assay to evaluate DNA damage repair deficient models in vitro and in vivo and the response to cytotoxic agents.
Authors: Allison M. Marrero, Scott M. Lawrence, Deborah Wilsker, Priya Balasubramanian, Robert J. Kinders, Ralph E. Parchment, Joseph E. Tomaszewski, James H. Doroshow
Journal: Cancer Research (2013)

Abstract The use of DNA damaging agents remains a mainstay in cancer therapy. Deletions and mutations of key DNA repair proteins in tumor cells, can dictate which of the DNA damage repair (DDR) processes will be critical following exposure to cytotoxic agents. Quantitation of repair pathway activation after drug treatment builds upon our validated qIFA for γH2AX, which scores individual nuclei as positive or negative for that biomarker and calculates the percent of nuclear area positive for γH2AX per imaged field. We have previously reported a prototype multiplex quantitative immunoflourescence assay (qIFA) for biomarkers of active Homologous Recombination (HR) and Nucleotide Excision Repair (NER) pathways. This first qIFA multiplex panel to quantify DDR response has been expanded to include Rad51, a key component of HR in addition to Nbs1 phosphorylated at S343 and γH2AX, both early markers of DNA double strand breaks, and ERCC1, critical to NER response. The assay currently in development uses highly specific antibodies directly conjugated to fluorophores or haptens, thereby eliminating the requirement for cross-absorbed anti-species antibodies. This in turn decreases reagent-caused autofluorescence and greatly simplifies assay QC. Addition of the Definiens analysis software has enhanced analysis of the DNA damage markers, including the ability to count foci per nucleus over an entire image field incorporating thousands of data points at a high-content capacity. This approach is useful in quantifying markers that occupy only a small intranuclear area, such as Rad51 foci, where total nuclear area measurements are less informative. Initial experiments using the multiplex qIFA in vitro were performed on cell lines characterized for DNA damage repair pathway defects. Differences in DNA damage marker Rad51 were observed between BRCA1 expressing and deleted cell lines post-irradiation, demonstrating utility of the marker. Examination of in vivo models revealed a differential activation of DDR pathways in irinotecan-treated xenograft samples of a BRCA-deficient tumor model, demonstrating the importance of using a multiplex approach in monitoring DNA damage. Our results suggest that monitoring markers from multiple DNA repair pathways using a multiplex approach could lead to important insights into global DNA repair responses in tumors. This assay will be expanded to clinical trials that obtain biopsies to assist in the evaluation of combination therapy approaches targeting DDR deficiencies. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Allison M. Marrero, Scott M. Lawrence, Deborah Wilsker, Priya Balasubramanian, Robert J. Kinders, Ralph E. Parchment, Joseph E. Tomaszewski, James H. Doroshow. Development of a multiplex quantitative immunofluorescence assay to evaluate DNA damage repair deficient models in vitro and in vivo and the response to cytotoxic agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3341. doi:10.1158/1538-7445.AM2013-3341