Evaluation of pharmacodynamic responses to cancer therapeutic agents using DNA damage markers

Abstract

Purpose: We sought to examine the pharmacodynamic activation of the DNA damage response (DDR) pathway in tumors following anticancer treatment for confirmation of target engagement. Experimental Design: We evaluated the time course and spatial activation of 3 protein biomarkers of DNA damage recognition and repair (gH2AX, pS343-Nbs1, and Rad51) simultaneously in a quantitative multiplex immunofluorescence assay (IFA) to assess DDR pathway activation in tumor tissues following exposure to DNA-damaging agents. Results: Because of inherent biological variability, baseline DDR biomarker levels were evaluated in a colorectal cancer microarray to establish clinically relevant thresholds for pharmacodynamic activation. Xenograft-bearing mice and clinical colorectal tumor biopsies obtained from subjects exposed to DNA-damaging therapeutic regimens demonstrated marked intratumor heterogeneity in the timing and extent of DDR biomarker activation due, in part, to the cell-cycle dependency of DNA damage biomarker expression. Conclusions: We have demonstrated the clinical utility of this DDR multiplex IFA in preclinical models and clinical specimens following exposure to multiple classes of cytotoxic agents, DNA repair protein inhibitors, and molec-ularly targeted agents, in both homologous recombination–proficient and -deficient contexts. Levels exceeding 4% nuclear area positive (NAP) gH2AX, 4% NAP pS343-Nbs1, and 5% cells with 5 Rad51 nuclear foci indicate a DDR activation response to treatment in human colorectal cancer tissue. Determination of effect-level cutoffs allows for robust interpretation of biomarkers with significant inter-patient and intratumor heterogeneity; simultaneous assessment of biomarkers induced at different phases of the DDR guards against the risk of false negatives due to an ill-timed biopsy.