Feasibility of developing reliable gene expression modules from FFPE derived RNA profiled on Affymetrix arrays

The reliability of differential gene expression analysis on formalin-fixed, paraffin-embedded (FFPE) expression profiles generated using Affymetrix arrays is questionable, due to the high range of percent-present values reported in studies which profiled FFPE samples using this technology. Moreover, the validity of gene-modules derived from external datasets in FFPE microarray expression profiles is unknown. By generating matched gene expression profiles using RNAs derived from fresh-frozen (FF) and FFPE preserved breast tumors with Affymetrix arrays and FF/FFPE RNA specific amplification-and-labeling kits, the reliability of differential expression analysis and the validity of gene modules derived from external datasets were investigated. Specifically, the reliability of differential expression analysis was investigated by developing de-novo ER/HER2 pathway gene-modules from the matched datasets and validating them on external FF/FFPE gene expression datasets using ROC analysis. Spearman's rank correlation coefficient of module scores between matched FFPE/frozen datasets was used to measure the reliability of gene-modules derived from external datasets in FFPE expression profiles. Independent of the array/amplification-kit/sample preservation method used, de-novo ER/HER2 gene-modules derived from all matched datasets showed similar prediction performance in the independent validation (AUC range in FFPE dataset; ER: 0.93–0.95, HER2: 0.85–0.91), except for the de-novo ER/HER2 gene-module derived from the FFPE dataset using the 3'IVT kit (AUC range in FFPE dataset; ER: 0.79–0.81, HER2: 0.78). Among the external gene modules considered, roughly ~50% gene modules showed high concordance between expression profiles derived from matching FF and FFPE RNA. The remaining discordant gene modules between FF and FFPE expression profiles showed high concordance within matching FF datasets and within matching FFPE datasets independently, implying that microarrays still require improved amplification-and-sample-preparation protocols for deriving 100% concordant expression profiles from matching FF and FFPE RNA.