Data are presented on pharmacodynamic (PD) method validation and preliminary clinical qualification of three PD biomarker assays. M65 Elisa, which quantitates different forms of circulating cytokeratin 18 (CK18) as putative surrogate markers of both apoptotic and nonapoptotic tumour cell death, was shown to be highly reproducible: calibration curve linearity r2 = 0.996, mean accuracy > 91% and mean precision < 3%, n = 27. Employing recombinant (r) CK18 and caspase cleaved CK18 (CK18 Asp396 neo-epitope) as external standards, kit to kit reproducibly was < 6% (n = 19). rCK18 was stable in plasma for 4 months at -20 degrees C and -80 degrees C, for 4 weeks at 4 degrees C and had a half-life of 2.3 days at 37 degrees C. Cytokeratin 18 Asp396 NE, the M30 Apoptosense Elisa assay antigen, was stable in plasma for 6 months at -20 degrees C and -80 degrees C, for 3 months at 4 degrees C, while its half-life at 37 degrees C was 3.8 days. Within-day variations in endogenous plasma concentrations of the M30 and M65 antigens were assessed in two predose blood samples collected from a cohort of 15 ovarian cancer patients receiving carboplatin chemotherapy and were shown to be no greater than the variability associated with methods themselves. Between-day fluctuations in circulating levels of the M30 and M65 antigens and in XIAP mRNA levels measured in peripheral blood mononuclear cells by quantitative (q) RT-PCR were evaluated in two predose blood samples collected with a 5- to 7-day gap from 23 patients with advanced cancer enrolled in a phase I trial. The mean variation between the two pretreatment values ranged from 13 to 14 to 25%, respectively, for M65, M30 and qRT-PCR. These data suggest that the M30 and M65 Elisa's and qRT-PCR as PD biomarker assays have favourable performance characteristics for further investigation in clinical trials of anticancer agents which induce tumour apoptosis/necrosis or knockdown of the anti-apoptotic protein XIAP.