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 molecularly targeted agents, in both homologous recombinationproficient 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 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.

Phase I trial of TRC102 (methoxyamine HCl) in combination with temozolomide in patients with relapsed solid tumors and lymphomas

“…Baseline biopsies with high MGMT protein levels were not assessed for MMR proteins. * Significant elevations of biomarkers on-treatment (defined as ≥ 5% of tumor cells exhibiting ≥5 Rad51 nuclear foci or ≥4% of tumor nuclear area positive for γH2AX or pNbs1 staining [ 14 ]) are marked with asterisks. Abbreviations: Bx, biopsy site; CP, clinical progression; Dx, diagnosis; NP, not per protocol (i.e., no measurement of disease response available because patient refused further treatment); PD, progressive disease; Pre, biopsy taken before initiation of treatment; On-Tx, biopsy collected on cycle 1, day 4 or 5, 3–4 hours after treatment unless otherwise specified; SD, stable disease; Tx, treatment; %NAP, percent tumor nuclear area positive for the given biomarker.…”

“…After 4–5 days of combination drug treatment, nuclear Rad51, a homologous recombination biomarker, was significantly elevated in biopsies from 6 of the 12 patients. Elevated Rad51 levels were defined by the observation that at least 5% of tumor cells assessed demonstrated ≥ 5 Rad51 nuclear foci ( Table 3 ) [ 14 ]. Among these 6 cases, 3 also exhibited elevated nuclear γH2AX or pNbs1 (defined as ≥ 4% of tumor nuclear area positive for γH2AX or pNbs1 staining) [ 14 ].…”

“…Elevated Rad51 levels were defined by the observation that at least 5% of tumor cells assessed demonstrated ≥ 5 Rad51 nuclear foci ( Table 3 ) [ 14 ]. Among these 6 cases, 3 also exhibited elevated nuclear γH2AX or pNbs1 (defined as ≥ 4% of tumor nuclear area positive for γH2AX or pNbs1 staining) [ 14 ]. Of note, 4 of 5 patients in the expansion cohort with colorectal cancer exhibited a nuclear DDR biomarker response of some type.…”

Biodistribution and Anticancer Characteristics of Les-3833, A Novel 4-thiazolidinone-Based Lead Compound

“…These parameters can be used to predict the exposure of the investigated drug in the human body. Biodistribution can also show differences among various treatment groups, days of treatment, and other factors as well as estimate the variability among animals and identify cases with abnormal levels of the drug [8,25].…”

“…Pharmacokinetics describes how the body affects the specific chemicals in a drug compound that have been administered by an oral, intravenous (IV) or other route and how these chemicals are affected by the mechanisms of absorption, distribution, metabolism, and elimination; these mechanisms can have a dramatic impact on the biosafety and efficacy of the investigated compounds [6]. Pharmacokinetic studies during the preclinical stage support the improvement of drug candidates and provide information for evaluating their possible clinical applications [7,8]. The information obtained is also useful for choosing doses in efficacy studies, providing preliminary identification for drug-specific toxicity in the target organs, and, in some cases, revealing delayed toxicity [3].…”