Notable Histologic Findings in a "Normal" Cohort: The National Institutes of Health Genotype-Tissue Expression (GTEx) Project

CONTEXT.­: The National Institutes of Health (NIH) Genotype-Tissue Expression (GTEx) project was designed to evaluate how genetic variation and epigenetic effects influence gene expression in normal tissue. OBJECTIVE.­: To ensure that the grossly normal-appearing tissues collected were free from disease, each specimen underwent histologic evaluation. DESIGN.­: In total, nearly 30 000 tissue aliquots collected from almost 1000 postmortem donors underwent histologic review by project pathologists, and detailed observations of any abnormalities or lesions present were recorded. RESULTS.­: Despite sampling of normal-appearing tissue, in-depth review revealed incidental findings among GTEx samples that included neoplastic, autoimmune, and genetic conditions; the incidence of some of these conditions among GTEx donors differed from those previously reported for other populations. A number of age-related abnormalities observed during histologic review of tissue specimens are also described. CONCLUSIONS.­: Histologic findings from the GTEx project may serve to improve populational awareness of several conditions and present a unique opportunity for others to explore age- and gender-influenced conditions. Resources from the study, including histologic image and sequencing data, are publicly available for research.

Histologic and Quality Assessment of Genotype-Tissue Expression (GTEx) Research Samples: A Large Postmortem Tissue Collection.

CONTEXT.­: The National Institutes of Health Genotype-Tissue Expression (GTEx) project was developed to elucidate how genetic variation influences gene expression in multiple normal tissues procured from postmortem donors. OBJECTIVE.­: To provide critical insight into a biospecimen's suitability for subsequent analysis, each biospecimen underwent quality assessment measures that included evaluation for underlying disease and potential effects introduced by preanalytic factors. DESIGN.­: Electronic images of each tissue collected from nearly 1000 postmortem donors were evaluated by board-certified pathologists for the extent of autolysis, tissue purity, and the type and abundance of any extraneous tissue. Tissue-specific differences in the severity of autolysis and RNA integrity were evaluated, as were potential relationships between these markers and the duration of postmortem interval and rapidity of death. RESULTS.­: Tissue-specific challenges in the procurement and preservation of the nearly 30 000 tissue specimens collected during the GTEx project are summarized. Differences in the degree of autolysis and RNA integrity number were observed among the 40 tissue types evaluated, and tissue-specific susceptibilities to the duration of postmortem interval and rapidity of death were observed. CONCLUSIONS.­: Ninety-five percent of tissues were of sufficient quality to support RNA sequencing analysis. Biospecimens, annotated whole slide images, de-identified clinical data, and genomic data generated for GTEx represent a high-quality and comprehensive resource for the scientific community that has contributed to its use in approximately 1695 articles. Biospecimens and data collected under the GTEx project are available via the GTEx portal and authorized access to the Database of Genotypes and Phenotypes; procedures and whole slide images are available from the National Cancer Institute.

Single-nucleus cross-tissue molecular reference maps toward understanding disease gene function.

Understanding gene function and regulation in homeostasis and disease requires knowledge of the cellular and tissue contexts in which genes are expressed. Here, we applied four single-nucleus RNA sequencing methods to eight diverse, archived, frozen tissue types from 16 donors and 25 samples, generating a cross-tissue atlas of 209,126 nuclei profiles, which we integrated across tissues, donors, and laboratory methods with a conditional variational autoencoder. Using the resulting cross-tissue atlas, we highlight shared and tissue-specific features of tissue-resident cell populations; identify cell types that might contribute to neuromuscular, metabolic, and immune components of monogenic diseases and the biological processes involved in their pathology; and determine cell types and gene modules that might underlie disease mechanisms for complex traits analyzed by genome-wide association studies.

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.

RNA sequence analysis reveals macroscopic somatic clonal expansion across normal tissues.

How somatic mutations accumulate in normal cells is poorly understood. A comprehensive analysis of RNA sequencing data from ~6700 samples across 29 normal tissues revealed multiple somatic variants, demonstrating that macroscopic clones can be found in many normal tissues. We found that sun-exposed skin, esophagus, and lung have a higher mutation burden than other tested tissues, which suggests that environmental factors can promote somatic mosaicism. Mutation burden was associated with both age and tissue-specific cell proliferation rate, highlighting that mutations accumulate over both time and number of cell divisions. Finally, normal tissues were found to harbor mutations in known cancer genes and hotspots. This study provides a broad view of macroscopic clonal expansion in human tissues, thus serving as a foundation for associating clonal expansion with environmental factors, aging, and risk of disease.

Preanalytics and Precision Pathology: Pathology Practices to Ensure Molecular Integrity of Cancer Patient Biospecimens for Precision Medicine.

Biospecimens acquired during routine medical practice are the primary sources of molecular information about patients and their diseases that underlies precision medicine and translational research. In cancer care, molecular analysis of biospecimens is especially common because it often determines treatment choices and may be used to monitor therapy in real time. However, patient specimens are collected, handled, and processed according to routine clinical procedures during which they are subjected to factors that may alter their molecular quality and composition. Such artefactual alteration may skew data from molecular analyses, render analysis data uninterpretable, or even preclude analysis altogether if the integrity of a specimen is severely compromised. As a result, patient care and safety may be affected, and medical research dependent on patient samples may be compromised. Despite these issues, there is currently no requirement to control or record preanalytical variables in clinical practice with the single exception of breast cancer tissue handled according to the guideline jointly developed by the American Society of Clinical Oncology and College of American Pathologists (CAP) and enforced through the CAP Laboratory Accreditation Program. Recognizing the importance of molecular data derived from patient specimens, the CAP Personalized Healthcare Committee established the Preanalytics for Precision Medicine Project Team to develop a basic set of evidence-based recommendations for key preanalytics for tissue and blood specimens. If used for biospecimens from patients, these preanalytical recommendations would ensure the fitness of those specimens for molecular analysis and help to assure the quality and reliability of the analysis data.

The College of American Pathologists Biorepository Accreditation Program: Results from the First 5 Years.

The College of American Pathologists (CAP) developed the Biorepository Accreditation Program (BAP) in 2012. This program integrates best practices from the International Society for Biological and Environmental Biorepositories, the National Cancer Institute, the Organisation for Economic Cooperation and Development, the Center for Medicare and Medicaid Services, and the CAP Laboratory Accreditation Program. The goal of this elective program is to provide requirements for standardization in biorepository processes that will result in high-quality specimens that can be used to support research, drug discovery, and personalized medicine. CAP uses a peer inspection model to ensure the inspectors have proper expertise and to promote educational efforts through information sharing. Lead inspectors are comprised of pathologists, PhDs, and managers of biorepositories and they are often supported by CAP staff inspectors. Accreditation is a 3-year continuous cycle of quality with a peer inspection occurring at the start of year 1 and a self-inspection and CAP desk assessment at the start of year 2 and 3. At this time 53 biorepositories are fully CAP BAP accredited and 13 are in the process of obtaining accreditation. There are currently 273 established standards with requirement lists customized based on the scope of activities performed by a biorepository. A total of 90 inspections were completed between May 2012 and December 2016. Sixty-one were initial inspections and 29 were reinspections. A total of 527 deficiencies were identified in the areas of Equipment/Instrumentation (22%), Information Technology (18%), Specimen Handling and QC (15%), Quality Management (16%), Personnel (11%), Safety (10%), Facilities (6%), and Regulatory (2%). Assessment of common deficiencies identifies areas of focus for continuous improvement and educational opportunities. Overall success of the program is high based on the current enrollment of 66 biorepositories, anecdotal participant feedback and increasing national recognition of the BAP in federal documents.

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.

Papillary endothelial hyperplasia of the breast: the great impostor for angiosarcoma: a clinicopathologic review of 17 cases.

Seventeen cases of papillary endothelial hyperplasia (PEH, Masson's vegetant intravascular hemangioendothelioma) involving breast or mammary subcutaneous tissues are described. The mean patient age was 59; 14 (82%) were female and 12 (71%) presented with a mass. Nine women had mammographic evaluation, 3 of whom had microcalcifications. Five neoplasms were discovered by routine mammography. Sixteen cases were 2.7 cm or less in greatest dimension, and 8 (47%) were associated with a thrombus and/or cavernous hemangioma. Follow-up in 10 cases (up to nearly 8 years) showed no recurrences. Fifty-nine percent of the cases were received at AFIP for consultation with a working diagnosis of angiosarcoma. Features that help distinguish PEH from angiosarcoma include circumscription of the lesion, location in a vessel or association with thrombus, and papillary architecture without significant cytologic atypia or areas of solid growth. The recognition of the morphologic features of this lesion and its inclusion in the differential diagnosis of vascular mammary tumors will reduce the likelihood of its misdiagnosis as an angiosarcoma and avoid unnecessary and aggressive therapy.

A call to standardize preanalytic data elements for biospecimens.

CONTEXT: Biospecimens must have appropriate clinical annotation (data) to ensure optimal quality for both patient care and research. Clinical preanalytic variables are the focus of this study. OBJECTIVE: To define the essential preanalytic variables (data fields) that should be attached to every collected biospecimen and to provide a complete list of such variables, along with their relative importance, which can vary, depending on downstream use, institutional needs, and information technology capabilities. DESIGN: The College of American Pathologists Diagnostic Intelligence and Health Information Technology Committee sponsored a Biorepository Working Group to develop a ranked list of the preanalytic variables for annotating biospecimens. Members of the working group were experts in anatomic, clinical, and molecular pathology; biobanking; medical informatics; and accreditation. Several members had experience with federal government programs, such as the National Cancer Institute's Biospecimens and Biorepository Branch and the National Cancer Institute's Community Cancer Center Program. Potential preanalytic variables were identified and ranked along with available supporting evidence, definitions, and potential negative effects if the variable was not attached to the biospecimen. Additional national and international stakeholders reviewed the draft manuscript. RESULTS: The ranked listing of 170 preanalytic variables produced can be used as a guide for site-specific implementation into patient care and/or research biorepository processes. Conclusions.-In our collective experience, it is often difficult to choose which of the many preanalytic variables to attach to any specific set of biospecimens used for patient care and/or research. The provided ranked list should aid in the selection of preanalytic variables for a given biospecimen collection.