The Biospecimen Preanalytical Variables Program: A Multiassay Comparison of Effects of Delay to Fixation and Fixation Duration on Nucleic Acid Quality.

CONTEXT.­: Despite widespread use of formalin-fixed, paraffin-embedded (FFPE) tissue in clinical and research settings, potential effects of variable tissue processing remain largely unknown. OBJECTIVE.­: To elucidate molecular effects associated with clinically relevant preanalytical variability, the National Cancer Institute initiated the Biospecimen Preanalytical Variables (BPV) program. DESIGN.­: The BPV program, a well-controlled series of systematic, blind and randomized studies, investigated whether a delay to fixation (DTF) or time in fixative (TIF) affects the quantity and quality of DNA and RNA isolated from FFPE colon, kidney, and ovarian tumors in comparison to case-matched snap-frozen controls. RESULTS.­: DNA and RNA yields were comparable among FFPE biospecimens subjected to different DTF and TIF time points. DNA and RNA quality metrics revealed assay- and time point-specific effects of DTF and TIF. A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was superior when assessing RNA quality, consistently detecting differences between FFPE and snap-frozen biospecimens and among DTF and TIF time points. RNA Integrity Number and DV200 (representing the percentage of RNA fragments longer than 200 nucleotides) displayed more limited sensitivity. Differences in DNA quality (Q-ratio) between FFPE and snap-frozen biospecimens and among DTF and TIF time points were detected with a qPCR-based assay. CONCLUSIONS.­: DNA and RNA quality may be adversely affected in some tumor types by a 12-hour DTF or a TIF of 72 hours. Results presented here as well as those of additional BPV molecular analyses underway will aid in the identification of acceptable delays and optimal fixation times, and quality assays that are suitable predictors of an FFPE biospecimen's fit-for-purpose.

Cold Ischemia Score: An mRNA Assay for the Detection of Extended Cold Ischemia in Formalin-Fixed, Paraffin-Embedded Tissue.

Although there are thousands of formalin-fixed paraffin-embedded (FFPE) tissue blocks potentially available for scientific research, many are of questionable quality, partly due to unknown preanalytical variables. We analyzed FFPE tissue biospecimens as part of the National Cancer Institute (NCI) Biospecimen Preanalytical Variables program to identify mRNA markers denoting cold ischemic time. The mRNA was extracted from colon, kidney, and ovary cancer FFPE blocks (40 patients, 10-12 hr fixation time) with 1, 2, 3, and 12 hr cold ischemic times, then analyzed using qRT-PCR for 23 genes selected following a literature search. No genes tested could determine short ischemic times (1-3 hr). However, a combination of three unstable genes normalized to a more stable gene could generate a "Cold Ischemia Score" that could distinguish 1 to 3 hr cold ischemia from 12 hr cold ischemia with 62% sensitivity and 84% specificity.

Small Nucleolar RNA Score: An Assay to Detect Formalin-Overfixed Tissue.

Although there are millions of formalin-fixed paraffin-embedded (FFPE) tissue blocks potentially available for scientific research, many are of questionable quality, partly due to unknown fixation conditions. We analyzed FFPE tissue biospecimens as part of the NCI Biospecimen Preanalytical Variables (BPV) program to identify microRNA (miRNA) markers for fixation time. miRNA was extracted from kidney and ovary tumor FFPE blocks (19 patients, cold ischemia ≤2 hours) with 6, 12, 24, and 72 hours fixation times, then analyzed using the WaferGen SmartChip platform (miRNA chip with 1036 miRNA targets). For fixation time, principal component analysis of miRNA chip expression data separated 72 hours fixed samples from 6 to 24 hours fixed samples. A set of small nuclear RNA (snRNA) targets was identified that best determines fixation time and was validated using a second independent cohort of seven different tissue types. A customized assay was then developed, based on a set of 24 miRNA and snRNA targets, and a simple "snoRNA score" defined. This score detects FFPE tissue samples with fixation for 72 hours or more, with 79% sensitivity and 80% specificity. It can therefore be used to assess the fitness-for-purpose of FFPE samples for DNA or RNA-based research or clinical assays, which are known to be of limited robustness to formalin overfixation.

Development of a consensus approach for return of pathology incidental findings in the Genotype-Tissue Expression (GTEx) project.

The active debate about the return of incidental or secondary findings in research has primarily focused on return to research participants, or in some cases, family members. Particular attention has been paid to return of genomic findings. Yet, research may generate other types of findings that warrant consideration for return, including findings related to the pathology of donated biospecimens. In the case of deceased biospecimen donors who are also organ and/or tissue transplant donors, pathology incidental findings may be relevant not to family members, but to potential organ or tissue transplant recipients. This paper will describe the ethical implications of pathology incidental findings in the Genotype-Tissue Expression (GTEx) project, the process for developing a consensus approach as to if/when such findings should be returned, possible implications for other research projects collecting postmortem tissues and how the scenario encountered in GTEx fits into the larger return of results/incidental findings debate.

Broad Consent for Research on Biospecimens: The Views of Actual Donors at Four U.S. Medical Centers.

Commentators are concerned that broad consent may not provide biospecimen donors with sufficient information regarding possible future research uses of their tissue. We surveyed with interviews 302 cancer patients who had recently provided broad consent at four diverse academic medical centers. The majority of donors believed that the consent form provided them with sufficient information regarding future possible uses of their biospecimens. Donors expressed very positive views regarding tissue donation in general and endorsed the use of their biospecimens in future research across a wide range of contexts. Concerns regarding future uses were limited to for-profit research and research by investigators in other countries. These results support the use of broad consent to store and use biological samples in future research.

A Novel Approach to High-Quality Postmortem Tissue Procurement: The GTEx Project.

The Genotype-Tissue Expression (GTEx) project, sponsored by the NIH Common Fund, was established to study the correlation between human genetic variation and tissue-specific gene expression in non-diseased individuals. A significant challenge was the collection of high-quality biospecimens for extensive genomic analyses. Here we describe how a successful infrastructure for biospecimen procurement was developed and implemented by multiple research partners to support the prospective collection, annotation, and distribution of blood, tissues, and cell lines for the GTEx project. Other research projects can follow this model and form beneficial partnerships with rapid autopsy and organ procurement organizations to collect high quality biospecimens and associated clinical data for genomic studies. Biospecimens, clinical and genomic data, and Standard Operating Procedures guiding biospecimen collection for the GTEx project are available to the research community.