Major phases of biomarker development. Five major steps are required to develop a clinical test for standard practice. First, the discovery or pilot phase compares 20 to 40 cases and controls using antibody-based arrays or MS-based approaches or flow/mass cytometry. It is recommended that the term “candidate biomarker” be used to refer to findings of early phase studies when additional validation is needed. A protein will be considered a candidate marker worth being pursued if the area under the curve (AUC) of the receiver operating characteristic (ROC) is >0.70. Next, the validation phase is usually performed with a high-throughput immunoassay, and the cohort is created from retrospective longitudinal case-control repositories from multiple institutions. The fourth step focuses on few biomarkers and requires a prospective multicenter validation, typically on a large multicenter cohort with the goal to define cutoff for high and low risk for a specific outcome. The hope is that this step will lead to a clinical test that will be approved by the FDA. Finally, the biomarker can be used in a clinical trial. If the use of biomarker changes the patients’ outcome, it is likely that it will be used in standard practice and its impact on the reduction of disease burden in the population of interest is quantified.