Which is the best methodology to validate a biomarker?

For each identified biomarker that is found relevant, specific bioassays are developed, optimized and validated for a reliable, cost- and time-effective follow-up. Since mass spectrometry was used to identify the biomarker, LC-MS and LC-MS/MS methods are usually well suited, and we use our expertise in the field to this end, typically based on FDA standards.

The relevance of identified biomarker candidates is first evaluated in silico, to evaluate if the corresponding molecule has a known link to the disease or not yet. Bioassays are then developped and validated (see above). The last step, which often is the most challenging, consists in the validation of the biomarker on large sets of samples, in different biological matrices. The biomarker has to be specific to the disease when compared to healthy samples, but its specificity versus other pathologies (which can sometimes be closely related) and versus different types of medical treatments (a biomarker can be induced by another drug used to treat other problems) remains a major challenge.

Newly discovered biomarkers are then used to develop and validate clinical assays for prognosis, for diagnosis and for the follow-up of medical treatments.

First is through means of a Microarray (for genes and gene probes), LC-MS or MALDI-TOF (for Proteins or metabolites). The next steps would be to carry out RT-PCR and Western on select candidate biomarkers.

For validating multiple protein biomarkers, protein microarray technologies offer great opportunities. The advantages of protein microarrays include high sensitivity, good reproducibility, quantitative accuracy, low sample volumes and parallelization when used in large populations of samples. However, the acquisition of a sufficient variety and quantity of various proteins for microarrays is a key challenge for such a technology.

PCR Array

For validating multiple RNA biomarkers, a promising technology is the PCR array, a high-throughput quantitative real-time (RT)-PCR technique, which combines the quantitative performance of RT-PCR with the multiple gene-profiling capabilities of microarrays to detect the expression of panels of genes simultaneously. The strengths of such a high-performance array platform include good reproducibility, specificity, high sensitivity and wide dynamic ranges. In particular, its flexibility and simplicity make it accessible for routine use in every laboratory, which is beneficial to large-scale analysis of biomarker validation across different laboratories. Currently, this technique has been used in the gene expression profiling of bladder, gastric and liver cancers.