RNA expression profiling of archival formalin-fixed, paraffin embedded tissues

Abstract

One challenge limiting development of clinically useful biomarkers for cancer research lies in the paucity of well-annotated frozen specimens with long-term follow-up on which current high-throughput technologies largely rely. Archival formalin fixed paraffin embedded (FFPE) materials represent the only form of clinical specimens in large cohorts with extensive annotation and long-term clinical follow-up. Unfortunately to date no genomic platform has been identified which can accurately interrogate these specimens due to the extensive fixation- and storage-related RNA degradation. First, we compared two commercially available platforms for RNA expression profiling of archival FFPE specimens from clinical studies of prostate and ovarian cancer. The first platform was the Affymetrix microarrays following whole-transcriptome amplification using NuGEN WT-Ovation FFPE System V2. The second platform was the Nanostring nCounter without amplification. For each assay, we profiled 7 prostate cancer and 11 ovarian cancer specimens, with block age range of 4 to 21 years. Both platforms produced gene expression profiles with high sensitivity and reproducibility through technical repeats from FFPE materials of prostate and ovarian cancers. RNA-Seq allows to simultaneously observe gene expression levels, mutations in the coding sequences, splice variants and gene fusions, which are especially important in cancer studies. Therefore, next we determined feasibility of RNA-Seq from archival samples, and identified challenges in the data analysis specific to FFPE by head-to-head comparisons to fresh frozen (FF) specimens using 16 pairs of prostate tumor and adjacent normal samples. We observed high average correlations between gene expression values for FFPE technical replicates, and between FF and FFPE pairs. Conclusions: we assessed two high-throughput approaches ideal for discovery studies (microarray and sequencing) and a more economical approach ideal for simultaneous validation of a developed signature consisting of multiple biomarkers. All platforms generated robust, reproducible data with high-level of intra- and inter-platform concordance, regardless of FFPE block age and RNA integrity. Through this study, we have demonstrated the feasibility of using FFPE materials for biomarker discovery and validation for further integration into clinical practice. For more information on Svitlana Tyekucheva please visit: http://www.hsph.harvard.edu/svitlana-tyekucheva/

Date
Location
519 Wartik Lab at University Park; with video to room CG628 at Hershey
Event
Seminar