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

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 (H2AX, 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: Due to 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 intra-tumor 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 molecularly-targeted agents, in both homologous recombination-proficient and -deficient contexts. Levels exceeding 4% nuclear area positive (NAP) H2AX, 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 intra-tumor heterogeneity; simultaneous assessment of biomarkers induced at different phases of the DNA damage response guards against the risk of false negatives due to an ill-timed biopsy.

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