Cummings J, Ranson M, Lacasse E, Ganganagari J R, St-Jean M, Jayson G, Durkin J, Dive C
Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, England, UK.
Br J Cancer. 2006 Jul 3;95(1):42-8. doi: 10.1038/sj.bjc.6603220.
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.
本文展示了三种药效学生物标志物检测方法的药效学（PD）方法验证及初步临床鉴定数据。M65酶联免疫吸附测定法（ELISA）可定量循环细胞角蛋白18（CK18）的不同形式，作为凋亡和非凋亡肿瘤细胞死亡的假定替代标志物，该方法具有高度可重复性：校准曲线线性r2 = 0.996，平均准确度> 91%，平均精密度< 3%，n = 27。以重组（r）CK18和半胱天冬酶切割的CK18（CK18 Asp396新表位）作为外部标准品，试剂盒间的可重复性< 6%（n = 19）。rCK18在-20℃和-80℃下于血浆中稳定4个月，在4℃下稳定4周，在37℃下半衰期为2.3天。细胞角蛋白18 Asp396 NE，即M30凋亡检测ELISA法的抗原，在-20℃和-80℃下于血浆中稳定6个月，在4℃下稳定3个月，其在37℃下的半衰期为3.8天。在15名接受卡铂化疗的卵巢癌患者的两个给药前血样中评估了M30和M65抗原内源性血浆浓度的日内变化，结果显示其变化不大于方法本身的变异性。在一项I期试验中，从23名晚期癌症患者中采集了间隔5至7天的两个给药前血样，评估了M30和M65抗原循环水平以及通过定量（q）RT-PCR测定的外周血单核细胞中XIAP mRNA水平的日间波动。M65、M30和qRT-PCR的两个预处理值之间的平均变化分别为13%至14%至25%。这些数据表明，M30和M65 ELISA法以及qRT-PCR作为药效学生物标志物检测方法，在诱导肿瘤凋亡/坏死或抗凋亡蛋白XIAP敲低的抗癌药物临床试验中具有良好的性能特征，可供进一步研究。
