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Clinical pharmacodynamic assays need to meet higher criteria for sensitivity, precision, robustness, and reproducibility than those expected for research-grade assays because of the long duration of clinical trials and the potentially unpredictable number of laboratories running the assays. This report describes the process of making an immunoassay based on commercially available reagents "clinically ready". The assay was developed to quantify poly(ADP-ribose) (PAR) levels as a marker of PAR polymerase inhibitor activity for a proof-of-concept phase 0 clinical trial at the National Cancer Institute (NCI) and subsequent clinical trials. In this publication, we retrospectively examine the measures taken to validate the published PAR immunoassay and outline key lessons learned during the development and implementation of these procedures at both internal and external clinical trial sites; these measures included optimizing PAR measurements in tumor biopsies and peripheral blood mononuclear cells (PBMCs), reagent qualification, analytical validation and assay quality control, instrument qualification and method quality control, and support for external laboratories.

A forward mutation assay was developed to study mutagenic specificity induced by temperature-sensitive alleles of bacteriophage T4 gene 42, which encodes a thermolabile deoxycytidylate hydroxymethylase. Thymidine kinase (tk) mutations induced by T4 ts B3 at a semi-permissive temperature (34°C) were selected under near-ultraviolet light on synthetic agar plates containing bromodeoxyuridine, and sequenced after PCR amplification of the tk gene. 21 of 23 tk− mutations identified were C → T transitions, while the ramainder were C → A transversions. Analyses of the DNA sequence around each mutant site suggest that the mispairing of thymine with guanine in the template is suppressed when the next nucleotide is dGTP. The 5′ neighbor nucleotide of the mismatch may influence mutation frequency as well; no mutations with dAMP residues on the upstream side were seen. Our observations with the forward mutation assay here at consistent with previous results from an rII reversion assay, supporting our model that the mutator phenotype displayed by tsLB3 is a consequence of perturbation of dNTP supplies to replication sites due to partial impairment of thermolabile deoxytidylate hydroxymethylase at a semi-permissive temperature. The forward mutation assay described here is readily adapted for other studies of mutagenesis in T4 phage.

Based upon analyses of purified enzyme preparations, T4 bacteriophage-coded ribonucleotide reductase is considered to be relatively insensitive to control by allosteric inhibition. However, two factors suggest that CDP reduction to dCDP is feedback-controlled by dTTP in infected cells. First, the pool of 5-hydroxymethyldeoxycytidine triphosphate, which expands manyfold upon infection by a dCMP deaminase-deficient T4 mutant, shrinks to near-normal levels as a consequence of dTTP accumulation, and ribonucleotide reductase is the only apparent control point. Second, analysis of mutagenesis by 5-bromodeoxyuridine suggests that most induced mutations result from localized pool depletion of 5-hydroxymethyl-dCTP at replication sites, as if 5-bromo-dUTP were behaving like dTTP in inhibiting the CDP reductase activity of the phage enzyme. We found that CDP reductase activity in crude extracts of T4 phage-infected bacteria is sensitive to inhibition by either dTTP or 5-bromo-dUTP, at concentrations as low as 0.01 mM. However, in partially purified enzyme preparations that sensitivity is lost. Although we don't know the basis for this loss of feedback sensitivity, the results suggest that kinetic properties of enzymes in intact cells are determined by the cellular milieu in ways not apparent from analysis of purified enzymes.