National Cancer Institute Biospecimen Evidence-Based Practices: Harmonizing Procedures for Nucleic Acid Extraction from Formalin-Fixed, Paraffin-Embedded Tissue

Variable and suboptimal biospecimen handling practices have been identified as impediments to biomarker discovery,1,2 including predictive biomarkers for oncology3 indicating a clear and present need for evidence-based, standardized practices. The United States and international efforts have been launched to better understand and mitigate variability during the preanalytical phase and decrease associated effects by promoting harmonization of procedures both within and across institutions. The Biorepositories and Biospecimen Research Branch (BBRB) of the United States National Cancer Institute (NCI) publishes best practice documents aimed at improving the quality of data generated from human biospecimens (https://biospecimens.cancer.gov/bestpractices/overview.asp) and sponsors research initiatives (https://biospecimens.cancer.gov/programs/default.asp) and the Biospecimen Research Database (BRD; http://biospecimens.cancer.gov/brd) to better understand thresholds and effects of individual preanalytical factors in biospecimen handling. The BRD allows users to query both a curated literature repository and standard operating procedure (SOP) library for a specific preservative, diagnosis, analyte, or preanalytical factor. The BRD incorporates information from international efforts, including those by the International Society for Biological and Environmental Repositories (ISBER) and the European Union-sponsored SPIDIA program (standardization and improvement of generic preanalytical tools and procedures for in vitro diagnostics; www.spidia.eu), among others. To facilitate the implementation of evidence-based practices in biospecimen handling, BBRB has developed a document series termed Biospecimen Evidence-Based Practices (BEBP), which contains step-by-step procedural guidelines derived from peer-reviewed primary research articles and expert experience.4 The aim of the BEBP series is to promote a practical level of standardization and improve overall biospecimen quality and data reproducibility by specifying both optimal methods and suitable alternatives, while merging published evidence with practical knowledge of experts in the field. The intent of the BEBP is not to serve as a SOP, but to facilitate the development of evidence-based SOPs by individual laboratories. The present BEBP focuses on nucleic acid extraction from formalin-fixed, paraffin-embedded (FFPE) tissue biospecimens (see Supplementary Data; Supplementary Data are available online at www.liebertpub.com/bio). Such biospecimens are being increasingly utilized in genomic research, and it has become clear over the past decade that variable and suboptimal FFPE biospecimen collection and processing practices can alter the quantity and/or quality of extracted DNA and RNA.5 Concordance between the molecular data generated with FFPE and snap-frozen biospecimens varies widely among reports, with correlations ranging from weak to very strong for the same analytical method.6 While lack of concordance may be partially attributable to differences in biospecimen handling during FFPE processing, available evidence suggests that the extraction method can compound or mitigate effects introduced during biospecimen collection, processing, and storage, thus affecting the suitability of samples for downstream analysis.7–11 When deciding on a nucleic acid extraction procedure, it is paramount to consider artifacts that may have been introduced during formalin fixation and processing, such as nucleic acid fragmentation,12 nucleic acid–protein crosslinking,13,14 denaturation,15,16 and additions of methylol groups to nucleic acids.15,17 Importantly, many such formalin-induced modifications to DNA and RNA are reversible13,18–21; for example, the addition of a demodification step as well as optimization of multiple steps during the extraction procedure can attenuate effects introduced during fixation and processing. However, FFPE-specific optimization steps are interdependent and must be considered collectively when developing a strategy for extraction. While several commercial extraction kits are marketed as being tailored for FFPE biospecimens, experts contributing to the BEBP advised validating any kit for the intended tissue type, processing regime, and analytical platform before implementation in experimental studies. Validation includes experiments to confirm the feasibility and accuracy of the intended analytical use, the reproducible performance of the kit, and the robustness to processing regimes.