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British Journal of Cancer (2010) 102, 1524 1532 & 2010 Cancer Research UK All rights reserved 0007 0920/10 $32.00
http://www.bjcancer.com
Web End =www.bjcancer.com
Issues on fit-for-purpose validation of a panel of ELISAs for application as biomarkers in clinical trials of anti-Angiogenic drugs
K Brookes1,2, J Cummings*,1, A Backen2, A Greystoke1, T Ward1, GC Jayson2 and C Dive1
1Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK; 2Translational Angiogenesis Group, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK
BACKGROUND: 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.
METHODS: 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.
RESULTS: Method validation focused on measuring precision and showed that 15 of the 17 assays were within acceptable limits.
Stability at 801C 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 490% platelets from plasma requiring centrifugation at 2000 g for 25 min.
CONCLUSIONS: These studies highlight the need for assay validation and crucial assessment of sample handling issues before commencement of biomarker analysis in clinical trials.
British Journal of Cancer (2010) 102, 15241532. doi:http://dx.doi.org/10.1038/sj.bjc.6605661
Web End =10.1038/sj.bjc.6605661 http://www.bjcancer.com
Web End =www.bjcancer.com Published online 20 April 2010& 2010 Cancer Research UK
Keywords: biomarkers; angiogenesis; ELISA; method validation; stability; platelets
MolecularDiagnostics
Angiogenesis, the formation of new blood vessels from existing vasculature, is required for tumour growth (Folkman, 1971, 1990) and is orchestrated by coordinated release of multiple signals from tumour, endothelial and stromal cells depending both on tumour type and microenvironment (Bergers and Benjamin, 2003). Following the hypothesis that inhibition of tumour angiogenesis...