Not only gene mutation matters: Development of flow cytometry panel to determine BRCA2 deficiency for personalised therapy by PARP inhibitors in pancreatic cancers

Abstract

Abstract Background BRCA1/2 mutations are associated with several cancers. Our previous study indicate that microenvironmental stress conditions, induce BRCA1 protein deficiency not related to gene mutations, but resulting from attenuated translation. We propose that highly mortal pancreatic tumors might also belong to the BRCAness ones, even if not carrying BRCA1/2 mutations. In this study we developed and validated flow cytometry panel to estimate BRCA2 protein level, together with tumor and stem markers, to correlate with sensitivity to PARP1 inhibitors. Methods Antibody validation was performed in WT and mutant cells to setup the panel. Tumor cells were detected using CK19, CA19-9, CEACAM5 markers, together with CD44, CD133 and EpCAM for stem cell population, combined with viability dyes. For proof-of-concept experiments we used pancreatic adenocarcinoma (PDAC) cell lines: PANC-1 and BRCA2-defective CAPAN-1. Cells were treated with FDA approved Olaparib or BMN673, followed by apoptosis and cell cycle analysis. Finally, the primary cells from resected pancreas samples of PDAC patients were analysed in context of BRCA2 protein, tumor and stem cell markers expression. Results We found that selected BRCA-deficient cells show increased, dose-dependent, sensitivity to PARP1 inhibitors, compared to WT cells. This was manifested by significant G1 decrease, cell cycle arrest in G2/M and increased cell death induced by both drugs. Analysis of patient samples showed correlation between stem cell markers and BRCA2-deficiency. The validation process of antibody specificity, as well as the established protocol of cell isolation from pancreatic tumor samples will be also presented. Conclusions The developed flow cytometry panel can be applied to select BRCAness PDAC cells which has increased sensitivity to PARP1 inhibitors. Moreover, the selection of BRCA-deficient cohort based on flow cytometry test should be further investigated and considered as the diagnostic method for personalized therapies caused by PARP1 inhibitors. Legal entity responsible for the study The authors. Funding Foundation for Polish Science TEAM TECH Core Facility Plus/2017-2/2 (POIR.04.04.00-00-23C2/17-00) (KP). Disclosure All authors have declared no conflicts of interest.