Issues on fit-for-purpose validation of a panel of ELISAs for application as biomarkers in clinical trials of anti-Angiogenic drugs.
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 -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.


CONCLUSIONS
These studies highlight the need for assay validation and crucial assessment of sample handling issues before commencement of biomarker analysis in clinical trials.
Brookes
K
K
Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, UK.
Cummings
J
J
Backen
A
A
Greystoke
A
A
Ward
T
T
Jayson
G C
GC
Dive
C
C
D020533:Angiogenesis Inhibitors / Q000097:blood / Q000627:therapeutic use*; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000097:blood; D002986:Clinical Trials as Topic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards*; D006801:Humans; D009369:Neoplasms / Q000097:blood / Q000188:drug therapy*; D055550:Protein Stability; D011786:Quality Control; D011933:Reagent Kits, Diagnostic / Q000592:standards; D013048:Specimen Handling; D054928:Validation Studies as Topic
D020533:Angiogenesis Inhibitors; D014408:Biomarkers, Tumor; D011933:Reagent Kits, Diagnostic
Curr Pharm Biotechnol. 2009 Sep;10(6):609-25
19619121
Cancer Invest. 2008 Feb;26(1):53-9
18181046
Pharm Res. 2003 Nov;20(11):1885-900
14661937
Eur J Drug Metab Pharmacokinet. 1991 Oct-Dec;16(4):249-55
1823867
Pharm Res. 2000 Dec;17(12):1551-7
11303967
J Natl Cancer Inst. 1990 Jan 3;82(1):4-6
1688381
J Pharm Sci. 2009 Sep;98(9):2909-34
18823031
Clin Cancer Res. 2005 Sep 1;11(17):6103-8
16144908
Expert Rev Mol Diagn. 2007 Jan;7(1):87-98
17187487
J Immunol Methods. 2006 Oct 20;316(1-2):1-7
16996079
J Natl Cancer Inst. 2001 Jul 18;93(14):1054-61
11459866
Rheumatology (Oxford). 2009 Sep;48(9):1036-44
19549789
Pharm Res. 2005 Apr;22(4):499-511
15846456
J Immunol Methods. 2009 Mar 15;342(1-2):106-14
19174166
Clin Pharmacol Ther. 2007 Jan;81(1):104-7
17186007
Protein Eng Des Sel. 2006 Apr;19(4):147-53
16478751
Nat Clin Pract Oncol. 2008 Jul;5(7):378-91
18560389
AAPS J. 2007 Apr 20;9(2):E117-22
17614353
MLO Med Lab Obs. 1994 Feb;26(2):22-6
10171791
J Immunol Methods. 2009 Jan 1;340(1):55-64
18983846
J Biol Chem. 1993 Jun 25;268(18):13372-7
8514775
Br J Pharmacol. 2008 Feb;153(4):646-56
17876307
J Cancer Res Clin Oncol. 2009 Feb;135(2):271-81
18642029
J Biol Chem. 2009 Sep 11;284(37):25388-403
19574212
Exp Mol Pathol. 2006 Feb;80(1):72-6
15961076
Nat Rev Cancer. 2002 Oct;2(10):727-39
12360276
Cardiovasc Res. 2004 Aug 1;63(2):226-35
15249180
Nat Rev Clin Oncol. 2009 Jul;6(7):395-404
19424102
PLoS One. 2009;4(4):e5108
19352513
Clin Vaccine Immunol. 2008 Jan;15(1):42-8
18003817
Clin Cancer Res. 2009 Nov 1;15(21):6674-82
19861458
Cancer Chemother Pharmacol. 2007 Nov;60(6):921-4
17333190
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Aug 1;877(23):2191-7
19022709
N Engl J Med. 1971 Nov 18;285(21):1182-6
4938153
Br J Cancer. 2005 Feb 14;92(3):532-8
15685240
Cytokine. 2002 Aug 21;19(4):201-5
12297114
Blood. 2009 Mar 19;113(12):2835-42
19036702
AAPS J. 2007 Apr 27;9(2):E123-7
17614354
J Pharm Biomed Anal. 2000 Jan;21(6):1249-73
10708409
Br J Cancer. 2006 Jul 3;95(1):42-8
16804528
Pharm Res. 2006 Feb;23(2):312-28
16397743
J Clin Oncol. 2010 Jan 10;28(2):193-201
19949019
Clin Diagn Lab Immunol. 1999 Jan;6(1):89-95
9874670
Lancet Oncol. 2002 Jul;3(7):425-30
12142172
Nat Rev Clin Oncol. 2009 Jun;6(6):327-38
19483739
Nat Rev Cancer. 2003 Jun;3(6):401-10
12778130
Cancer Epidemiol Biomarkers Prev. 2006 Jun;15(6):1078-82
16775163