title: 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 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.
authors.affiliation: Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, England, UK. jcummings@picr.man.ac.uk
authors.forename: J
authors.initials: J
authors.lastname: Cummings
authors.forename: M
authors.initials: M
authors.lastname: Ranson
authors.forename: E
authors.initials: E
authors.lastname: Lacasse
authors.forename: J R
authors.initials: JR
authors.lastname: Ganganagari
authors.forename: M
authors.initials: M
authors.lastname: St-Jean
authors.forename: G
authors.initials: G
authors.lastname: Jayson
authors.forename: J
authors.initials: J
authors.lastname: Durkin
authors.forename: C
authors.initials: C
authors.lastname: Dive
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D014408:Biomarkers, Tumor / Q000097:blood*; D002138:Calibration; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000097:blood / Q000276:immunology; D005260:Female; D006801:Humans; D053538:Keratin-18 / Q000097:blood* / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000378:metabolism; D009841:Oligonucleotides / Q000493:pharmacokinetics*; D010051:Ovarian Neoplasms / Q000097:blood* / Q000188:drug therapy*; D010446:Peptide Fragments / Q000097:blood* / Q000276:immunology; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011933:Reagent Kits, Diagnostic / Q000592:standards; D012016:Reference Values; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013997:Time Factors; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000037:antagonists & inhibitors* / Q000235:genetics
chemical_list: C498209:AEG 35156; D014408:Biomarkers, Tumor; D000939:Epitopes; D053538:Keratin-18; C577325:M65 antigen, human; D009841:Oligonucleotides; D010446:Peptide Fragments; D012333:RNA, Messenger; D011933:Reagent Kits, Diagnostic; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human