Method validation and preliminary qualification of pharmacodynamic biomarkers employed to evaluate the clinical efficacy of an antisense compound (AEG35156) targeted to the X-linked inhibitor of apoptosis protein XIAP

Abstract

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 r 2 ¼ 0.996, mean accuracy 491% and mean precision o3%, n ¼ 27. Employing recombinant (r) CK18 and caspase cleaved CK18 (CK18 Asp 396 neo-epitope) as external standards, kit to kit reproducibly was o6% (n ¼ 19). rCK18 was stable in plasma for 4 months at À201C and À801C, for 4 weeks at 41C and had a half-life of 2.3 days at 371C. Cytokeratin 18 Asp 396 NE, the M30 Apoptosense Elisa assay antigen, was stable in plasma for 6 months at À201C and À801C, for 3 months at 41C, while its half-life at 371C 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.

Evaluation of Circulating Tumor Cells and Serological Cell Death Biomarkers in Small Cell Lung Cancer Patients Undergoing Chemotherapy

“…While biomarker research is now an adjunct to most oncology drug trials, the data generated are usually exploratory, limited by the use of unvalidated assays and lacking sufficient robustness to inform on future clinical drug development. In our previous work these assays were validated and optimized to good clinical laboratory practice standard for clinical trials 8,9 and the CellSearch technology for CTCs currently dominates the technology platforms for CTC enumeration with respect to reproducibility. 15 Our current results will inform the application and interpretation of these bioassays when incorporated into upcoming trials of novel therapies for SCLC, not least those agents targeted to apoptosis regulatory components such as Bcl-2 family antagonists and inhibitors of the IAP family of proteins.…”

“…8,9 Serum samples were analyzed for nDNA [Cell Death Detection ELISA (Roche, Basel, Switzerland)] as previously described. 14 …”

“…7 nDNA release is also detected when levels of apoptosis overwhelm macrophage capacity for phagocytosis and a more necrotic cell fate ensues. 7 We have previously validated these cell death biomarkers 8,9 and optimized them for application to a busy, clinical setting. 6 Here we report on the behavior and clinical utility of these assays in patients with SCLC.…”

Preclinical evaluation of M30 and M65 ELISAs as biomarkers of drug induced tumor cell death and antitumor activity

“…The M30 and M65 ELISA kits were used as described previously in detail (17,18). Before analysis, plasma and tumor lysates, which were stored at -80jC, were allowed to thaw at room temperature.…”

“…M65 detects a common epitope present in the full-length protein as well as the 21-kDa caspase cleaved fragment (14) and is thus believed to measure, in addition to apoptosis, intact CK18 that is released from cells undergoing necrosis (16). Our laboratory has now validated both assays as fit for purpose in the analysis of plasma/serum collected from subjects entered into clinical trials (17,18). However, although both ELISAs have now been applied in several clinical trials of anticancer drugs as pharmacodynamic assays (19 -21), there have been few formal publications on the qualification of these assays as cell death markers in an in vivo model (22).…”