Abstract 2723: How BRCA1/2 mutations in TNBC affect TME and subsequently immune cell functions
Authors: Pueschl Dana , Oldrige Derek A. , Belman Jonathan , Shilan Jake S. , Nayak Anupma , Wubbenhorst Bradley , Pluta John , Vonderheide Robert H. , Feldman Michael , Maxwell Kara N. , Wherry E. John , Domchek Susan M. , Nathanson Katherine L. 
Published: July 2021

Abstract
Breast cancer type 1 and 2 susceptibility proteins (BRCA1/BRCA2) are well known breast cancer genes, mutations in which lead to defective homologous recombination repair (HRR). HR-based DNA repair deficiency (HRD) scores can be used to indicate DNA damage, genomic instability and may predict response to DNA damaging agents in BRCA1/2 mutated tumors. Tumors with a high HRD score caused by complete loss of BRCA1 or BRCA2 function locus-specific LOH are sensitive to DNA damage agents including platinum-based chemotherapy and poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). To understand the relationship between BRCA1/2 mutations and DNA damage in the tumor microenvironment (TME), we have characterized 107 BRCA1/2 tumors to determine HRD score using whole exome sequencing (WES) and simultaneously measured markers of DNA damage, PARP expression, tumor-infiltrating lymphocytes (TILs) and immune checkpoints to identify potential treatment targets on matching samples (n=47) using highly multiplexed fluorescence microscopy CO-Detection by indEXing (CODEX). We have established and validated a 40-plex breast cancer specific antibody panel consisting of markers to detect DNA damage, TILs and immune checkpoints to deeply profile how the TME is affected by BRCA1/2 mutations using CODEX. Computational image processing of CODEX data was performed to interrogate changes in number, size, morphology, and marker expression in tumor and immune cells. We have characterized BRCA1/2 tumors (n=47) on Tissue Microarrays (TMAs), and we have detected cytotoxic CD8+T and CD107a+NK cells in HRD low (&lt;42) and HRD high groups. Interestingly, their frequency and cytolytic function (measured by Granzyme A and perforin transcript level) appear to be associated with HRD scores. For instance, HRD low groups showed increased cytotoxic CD8+T and CD107a+NK cells whereas HRD high groups revealed decreased cell numbers as well as cytolytic function. We observed that HRD high, LOH positive was associated with increased DNA damage marker expression in tumor cells (H3pSer28, pATM, yH2AX) as well as NK cells (pATM) whereas immune checkpoint protein levels were decreased. We have planned quantitative analysis which allow us to determine the percentage of cell subtypes as well as the spatial compartmentalization of cells to interrogate the tumor microenvironment associated with BRCA1/2 mutations. In conclusion, BRCA1/2 mutated tumors with high HRD score revealed upregulated DNA damage expression in immune cells suggesting that BRCA1/2 mutations can impact HRR in CD8+T cells and CD107a+ NK cells and subsequently affect their ability to produce Granzyme A and Perforin. Our findings will decipher the role of DNA damage in BRCA1/2 mutated tumor cells and immune cell types. Outcomes can potentially predict treatment responses such as DNA damage, PARPi and checkpoint inhibitor therapies in TNBC BRCA1/2 breast cancer.
Citation Format: Dana Pueschl, Derek A. Oldrige, Jonathan Belman, Jake S. Shilan, Anupma Nayak, Bradley Wubbenhorst, John Pluta, Robert H. Vonderheide, Michael Feldman, Kara N. Maxwell, E. John Wherry, Susan M. Domchek, Katherine L. Nathanson. How BRCA1/2 mutations in TNBC affect TME and subsequently immune cell functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2723.