Evaluation of Pharmacodynamic Responses to Cancer Therapeutic Agents Using DNA Damage Markers

Clin Cancer Res. 2019 May 15;25(10):3084-3095. doi: 10.1158/1078-0432.CCR-18-2523. Epub 2019 Feb 21.

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 (γH2AX, 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 molecularly targeted agents, in both homologous recombination-proficient and -deficient contexts. Levels exceeding 4% nuclear area positive (NAP) γH2AX, 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 interpatient 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.

Publication types

  • Evaluation Study
  • Research Support, American Recovery and Reinvestment Act
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antineoplastic Agents / pharmacology*
  • Biomarkers, Tumor / metabolism*
  • Cell Cycle Proteins / metabolism
  • Clofarabine / pharmacology
  • Colorectal Neoplasms / drug therapy*
  • Colorectal Neoplasms / genetics*
  • Colorectal Neoplasms / metabolism
  • Colorectal Neoplasms / pathology
  • DNA Damage*
  • DNA Repair
  • Deoxycytidine / analogs & derivatives
  • Deoxycytidine / pharmacology
  • HCT116 Cells
  • HT29 Cells
  • Histones / metabolism
  • Humans
  • Mice
  • Mice, Nude
  • Nuclear Proteins / metabolism
  • Rad51 Recombinase / metabolism
  • Topotecan / pharmacology
  • Tumor Cells, Cultured
  • Xenograft Model Antitumor Assays

Substances

  • Antineoplastic Agents
  • Biomarkers, Tumor
  • Cell Cycle Proteins
  • H2AX protein, human
  • Histones
  • NBN protein, human
  • Nuclear Proteins
  • Deoxycytidine
  • Clofarabine
  • Topotecan
  • gemcitabine
  • RAD51 protein, human
  • Rad51 Recombinase