Four sequential validation studies will be used to confirm the usefulness of the candidate biomarkers:
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Technical validation. An alternative technique will be used to validate the biomarker candidates in the same samples to ensure that they behave according to what was observed in the arrays. qPCR will be used to replicate the data of expression and CNVs. For genotyping or validation of methylation markers, TaqMan probes will be used.
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Biological validation. The candidates will be validated with an alternative technique in an independent series with similar characteristics, but more heterogeneous and with larger sample size. For this we will analyze at least 300 cases of colorectal cancer in stages I-IV, without excluding the MSI cases and 200 controls. Markers will be analyzed in tumor tissue and normal mucosa.
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Proof of concept. Tumor tissue or colonic mucosa is not useful for a screening test. We will attempt to identify those markers that pass the biological validation in blood or stool and test their predictive ability.
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Usefulness in standard practice. The markers that pass the proof of concept phase will follow to a validation study in similar conditions to practical use. We will use the population screening series (n > 700 with colonoscopy result, we expect lesions in 35% of them). The sensitivity and specificity of the marker will be compared with the FOB test.
