Abstract P6-07-31: Assessing germline Homologous Recombination pathway deficiency in BRCA1 mutation carriers using Single Cell Network Profiling.

Abstract

Abstract Background: Inherited alterations in BRCA1/2 genes increase genomic instability and cancer susceptibility. DNA sequencing detects BRCA1/2 mutations, but has the following limitations; 1) mutations may have unknown functional significance, 2) epigenetic alterations and mutations in other Homologous Recombination (HR) pathway components are not detected, and 3) the combined effects of pathway mutations are not understood. Thus a functional assessment of HR competence at the single cell level remains an unmet need as BRCA1/2 sequencing does not holistically inform on functionality of the HR pathway. Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry-based assay that simultaneously measures, at the single cell level, extracellular surface markers and functional changes in intracellular signaling in response to extracellular modulators (Kornblau et al. Clin Cancer Res 2010). In this study, we tested the ability of SCNP to detect and quantify functional changes in HR signaling using peripheral blood mononuclear cell (PBMC) samples from BRCA1 mutation carriers (MUT) and wild type (WT) subjects. Methods: HR pathway activity was examined in PBMCs from BRCA1 MUT (n = 21) or WT (n = 20) subjects. Cell lines carrying BRCA1 MUTor WT genes were used as controls. PBMCs were stimulated with anti-CD3 and anti-CD28 for 24 hours to induce T cell proliferation then treated with PARP inhibitor (PARPi) AZD2281 +/− Temozolomide (TMZ) for 48h or 72h to induce DNA damage. DNA damage response (DDR) readouts were measured in both CyclinA2- and CyclinA2+ T cell subsets. Measurements included induced levels of p21, p53 and phosphorylation (p−) of p-H2AX, p-DNA-PKcs, p-RPA2/32, and p-BRCA1. Results: As expected based on the mechanism of action of PARPi, higher levels of induced p-H2AX and p53 were observed in CyclinA2+ cells of BRCA1 MUT versus WT cell line controls. In PBMCs, T cell proliferation (%CyclinA2+) was positively associated with PARPi induced DDR readouts. After controlling for proliferation, statistically significant differences in PARPi induced DDR signaling were observed between BRCA1 MUT and WT samples in many simultaneously assessed readouts including p-H2AX, p53 and p21 (increased in MUT), particularly in CyclinA2+ cells. Additionally, BRCA1 MUT samples displayed lower basal p-BRCA1 but higher induced p-BRCA1 levels compared to BRCA1 WT samples. Conclusions: SCNP was able to detect and quantify functional differences between PBMC samples from BRCA1 MUT (haploinsufficient) and WT donors by quantitatively assessing DDR signaling in CyclinA2+ T cells. Once verified on a larger data set, the assay could form the basis for the development of screening tests to identify subjects at higher risk of developing cancer or stratification tests to inform on cancer patient selection for treatment with PARP inhibitors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-07-31.