Profiling DNA damage response in ATM/BRCA2 carriers to inform hereditary cancer risk.

Abstract

10574 Background: Approximately 10% of cancers stem from inherited germline pathogenic variants (gPV), predominantly in DNA repair genes including ATM and BRCA1 or BRCA2 . Identification of a gPV in high-risk families can guide management, however the clinical implications of variants of uncertain significance (VUS) identified by genetic testing remain unclear. Our aim is to develop a DNA damage response (DDR) activity profile of high-risk populations with/without cancer with ATM or BRCA2 gPV to assist in determining the relevance of ATM or BRCA2 gPV/VUS. Methods: We profiled several key proteins that play a role in DDR using a Luminex-based Multianalyte immunoassay (hereafter referred to as DDR xMAP) in peripheral blood monocytes (PBMCs) derived from whole blood. The standardized DDR xMAP assay was first applied to PBMC specimens from sporadic colorectal cancer (CRC, n = 95) patients and cancer-free age-matched controls (n = 47) at baseline. The DDR xMAP was used to profile seven DDR proteins, phosphorylated Chk1 S345 , Chk2 T68 , γH2AX S139 , p53 S15 and total ATR, MDM2, p21. Univariate classification and regression tree analysis was used to identify statistically significant cut points in DDR analyte levels. We then measured the DDR analyte levels in individuals with BRCA2 gPVs with (n = 11) and without a diagnosis of cancer (n = 11) as well as in cancer-free non-carrier controls (n = 15) at baseline. We compared these values using the two-sided Mann-Whitney test and the Benjamini-Hochberg false discovery rate method to account for multiple markers. Results: Using the initial set of CRC cases and healthy controls, we identified statistically significant cut points in multiple DDR analyte levels including total ATR ( > 81.8), Chk1 S345 ( > 28.0) and γH2AX S139 ( > 51.3) that can individually distinguish between CRC cases and cancer-free controls (P < 0.001). Next, in preliminary analysis of patients with BRCA2 gPV with and without cancer had increased levels of all DDR analytes (P < 0.05) compared to non-carrier cancer-free controls, except for γH2AX S139 . Levels of proteins involved in replication stress response, ATR and downstream Chk1 S345 , were elevated. Total MDM2 levels, negative regulator of p53, were highly elevated in BRCA2 gPV carriers. There was not a significant difference between DDR analytes for those with BRCA2 gPV with and without cancer. Conclusions: Profiling key DDR markers can significantly distinguish between CRC patients and cancer-free controls. DDR analytes are significantly increased in PBMC specimens from individuals with BRCA2 gPV (with or without a diagnosis of cancer) compared to non-carrier cancer-free controls. Ongoing research in high-risk groups will establish cut points for this assay, improving our understanding of DDR activity in individuals with ATM/BRCA2 gPV and clarifying the clinical significance of gPV/VUS.