The pathology group will apply additional methods including, but not limited to, in-situ hybridization, real-time PCR, and immunohistochemistry (IHC) to validate the expression level of single genes or proteins derived from array experiments. It will also validate single genes or gene combinations (molecular signatures) derived from the array experiments at the RNA and protein level. Furthermore, sub-cellular localization of specific protein markers from important signal transduction pathways based on scientific evidence (e.g. ß-catenin, MAPK-P) will be evaluated. SNP variations or mutation profiles will be confirmed by conventional capillary or pyrophosphate sequencing.

Once an experimental signature has been identified it needs to be confirmed in other and larger patient cohorts. For example, a signature identified in a phase II trial needs to be reproduced in a phase III trial. Moreover, molecular signatures will be specific for a patient subpopulation representing a particular ethnic background or a mix of different ethnicities. Therefore, signatures will have to be established in different geographic regions with different mixes of ethnicities.