Identification of mutation-independent BRCA2 protein deficiency expands diagnostics and selection of pancreatic cancer patients for personalized therapy with PARP1 inhibitors

Abstract

Background: Recently, therapies involving poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP1) inhibitors have been approved for metastatic BRCA1/2-mutated pancreatic ductal adenocarcinoma (PDAC). The current scheme of identification of patients with BRCA deficiency relies on genetic screening. Here, we tested the hypothesis that pancreatic tumors have a broader spectrum of BRCAness than can be identified solely by gene mutations. We focused on BRCA2 deficiency, which is predominant in pancreatic ductal adenocarcinoma (PDAC). Methods: Pancreatic cancer cell lines (wt or BRCA2mutated) were used to set up a protocol to verify antibody specificity and detect BRCA2 protein levels by flow cytometry.Post-surgery pancreatic tumor samples were assessed by spectral cytometry with unsupervised analysis to identify BRCA2-deficient clusters together with the expression of stemness and metastasis markers. Personalized tumor signatures specified BRCAness phenotype and cancer state to increase the accuracy of selection for therapy with PARP1 inhibitors (PARPis). In parallel, BRCA2 mutations were identified by NGS analysis. Results: We developed a cytometric panel to assess BRCA2 levels associated with sensitivity to PARPis (olaparib and talazoparib). Analysis of BRCA2 protein levels in patients’ samples showed high diversity. Unsupervised cluster analysis identified BRCA2-deficient clusters, together with metastasis and stemness markers, which indicated advanced tumors with dismal prognoses. Cluster composition confirmed the high heterogeneity of pancreatic tumors. In parallel, NGS did not recognize the BRCA2/1 mutations in any of the analyzed tumors. Therefore, based on the current selection, these patients would be excluded from PARPis therapy. Finally, analysis of each tumor personalized signatures of tumor cell subsets potentially sensitive to PARPis were demonstrated. Conclusions: We found that BRCA2 protein deficiency (BRCAness) is detected with metastasis/stemness markers in pancreatic tumors also in individuals lacking BRCA2 mutations. Our findings show that integrating the flow cytometry-based BRCA2 protein assessment with genetic screening is important to improve the effective selection of PDAC patients for therapy with PARPis. This might also be relevant for other BRCA-deficient tumors.