Accuracy of Molecular Data Generated with FFPE Biospecimens: Lessons from the Literature

Abstract

Formalin-fixed and paraffin-embedded (FFPE) tissue biospecimens are a valuable resource for molecular cancer research. Although much can be gained from their use, it remains unclear whether the genomic and expression profiles obtained from FFPE biospecimens accurately reflect the physiological condition of the patient from which they were procured, or if such profiles are confounded by biological effects from formalin fixation and processing. To assess the physiological accuracy of genomic and expression data generated with FFPE specimens we surveyed the literature for papers investigating genomic and expression endpoints in case-matched FFPE and fresh or frozen human specimens using the National Cancer Institute's Biospecimen Research Database (http://biospecimens.cancer.gov/brd). Results of the survey revealed that the level of concordance between differentially preserved biospecimens varied among analytical parameters and platforms, but also among reports, genes/transcripts of interest, and tumor status. The identified analytical techniques and parameters that resulted in strong correlations between FFPE and frozen biospecimens may provide guidance when optimizing molecular protocols for FFPE use; however, discrepancies reported for similar assays also illustrate the importance of validating protocols optimized for use with FFPE specimens with a case-matched fresh or frozen cohort for each platform, gene or transcript, and FFPE processing regime. Based upon evidence published to date, validation of analytical parameters with a properly handled frozen cohort is necessary to ensure a high degree of concordance and confidence in the results obtained with FFPE biospecimens.

Exome-capture RNA sequencing of decade-old breast cancers and matched decalcified bone metastases

“…Furthermore, formalin-fixed, paraffin embedding (FFPE) -often performed in concert with decalcification -causes severe degradation and hydrolysis of RNA (18). In light of this, new capture-based methods of nucleic acid sequencing on aged FFPE specimens have shown efficacy in identifying DNA variants and even guiding care in academic centers (19)(20)(21). Exome-capture RNA sequencing (ecRNA-seq) is less well characterized in aged tumor samples, although recent studies on FFPE specimens have shown promising expression correlations with flash-frozen tissues (22)(23)(24).…”

Evaluation of the quality of RNA extracted from archival FFPE glioblastoma and epilepsy surgical samples for gene expression assays

“…This means that with downstream nucleic acid purification and enrichment, histopathological abnormalities (e.g. the malignant transformation of a cellular population or subpopulation) can be interrogated using molecular tools 7. This approach has been widely used in the discovery phase of biomarker investigation for large-scale validation and for subsequent implementation into routine clinical practice 8…”

Suitability of melanoma FFPE samples for NGS libraries: time and quality thresholds for downstream molecular tests

“…Hence, steps must be taken to improve the use of FFPE in molecular testing, especially those factors affecting downstream techniques, because this will permit the reevaluation of stored FFPE samples independently of their origin [4]. …”