Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, UK.
Br J Cancer. 2010 May 11;102(10):1524-32. doi: 10.1038/sj.bjc.6605661. Epub 2010 Apr 20.
Successful introduction of new anticancer agents into the clinic is often hampered by a lack of qualified biomarkers. Studies have been conducted of 17 ELISAs representing a potential panel of pharmacodynamic/predictive biomarkers for drugs targeted to tumour vasculature.
The fit-for-purpose approach to method validation was used. Stability studies were performed using recombinant proteins in surrogate matrices, endogenous analytes in healthy volunteer and cancer patient plasma. The impact of platelet depletion was investigated.
Method validation focused on measuring precision and showed that 15 of the 17 assays were within acceptable limits. Stability at -80 degrees C was shown for 3 months with all recombinant proteins in surrogate matrices, whereas under the same conditions instability was observed with KGF in platelet-rich and platelet-depleted plasma, and with PDGF-BB in platelet-depleted plasma from cancer patients. For measurement of extracellular circulating analytes, platelet depletion should be conducted before freezing of plasma to prevent release of PDGF-BB, FGFb and VEGF-A. A protocol was developed to remove >90% platelets from plasma requiring centrifugation at 2000 g for 25 min.
These studies highlight the need for assay validation and crucial assessment of sample handling issues before commencement of biomarker analysis in clinical trials.
新的抗癌药物在临床上的成功引入往往受到缺乏合格的生物标志物的阻碍。已经对 17 种代表针对肿瘤血管靶向药物的潜在药效/预测生物标志物的 ELISA 进行了研究。
采用适用于方法验证的方法。使用重组蛋白在替代基质中进行稳定性研究，使用健康志愿者和癌症患者血浆中的内源性分析物进行稳定性研究。还研究了血小板耗竭的影响。
方法验证重点是测量精密度，结果显示 17 种测定法中有 15 种在可接受范围内。在替代基质中，所有重组蛋白在-80°C 下稳定 3 个月，而在相同条件下，血小板丰富和血小板耗竭的血浆中的 KGF 和癌症患者血小板耗竭的血浆中的 PDGF-BB 不稳定。为了测量细胞外循环分析物，在冷冻血浆之前应进行血小板耗竭，以防止 PDGF-BB、FGFb 和 VEGF-A 的释放。开发了一种从血浆中去除>90%血小板的方案，需要在 2000g 下离心 25 分钟。
这些研究强调了在临床试验中开始进行生物标志物分析之前，需要进行测定法验证和对样品处理问题进行关键评估。
