Helen Moore

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

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 physiologic condition of the patient from which they were procured, or if such profiles are confounded by biologic effects from formalin fixation and processing. To assess the physiologic accuracy of genomic and expression data generated with FFPE specimens, we surveyed the literature for articles investigating genomic and expression endpoints in case-matched FFPE and fresh or frozen human biospecimens using the National Cancer Institutes 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. On the basis of 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.

Critical Issues in International Biobanking

Biobanking for clinical or research purposes includes the collection, processing, storage, and analysis of biological specimens. It is now well recognized that biobanking involves a complex array of technical and ethical/regulatory considerations. Biobanking policies and procedures are often documented by best practices that are usually voluntary but may be supplemented and reinforced by strict rules and regulations that govern informed consent, privacy, QC, and other critical issues. As biobanking has emerged as a global endeavor, with national networks and international collaboration becoming the norm, it has become even more critical that practices are coordinated and that quality standards are developed. Biobanking is also often a business endeavor, in that formal strategic and business plans need to be developed to ensure the long-term survival of the associated research programs. As new technologies are developed for using biospecimens to diagnose and treat disease, as well as to evaluate genetic risks, patients are becoming more aware of the importance and benefits of biobanking as part of the medical infrastructure. As a result, patients who donate biospecimens are becoming more interested in learning more about their own samples use and in seeing the actual results of the research. One of the aspects of these evolving attitudes toward biobanking was addressed in a previous QA Clin Chem 57:540–4).nnFrom the broad array of issues that could be addressed, this Q&A focuses on a few critical issues that many biobanks are facing today: quality management, biobank network design, long-term sustainability, conveying the importance of biobanking to the public, and the return of research results to biospecimen donors. Five experts who are engaged in national and international biobanking programs discuss these complex issues here.nnWhat are some of the important issues related to …

Fundamental Considerations for Biobank Legacy Planning.

Biobanking in its various forms is an activity involving the collection of biospecimens and associated data and their storage for differing lengths of time before use. In some cases, biospecimens are immediately used, but in others, they are stored typically for the term of a specified project or in perpetuity until the materials are used up or declared to be of little scientific value. Legacy planning involves preparing for the phase that follows either biobank closure or a significant change at an operational level. In the case of a classical finite collection, this may be brought about by the completion of the initial scientific goals of a project, a loss of funding, or loss of or change in leadership. Ultimately, this may require making a decision about when and where to transfer materials or whether to destroy them. Because biobanking in its entirety is a complex endeavour, legacy planning touches on biobank operations as well as ethical, legal, financial, and governance parameters. Given the expense and time that goes into setting up and maintaining biobanks, coupled with the ethical imperative to appropriately utilize precious resources donated to research, legacy planning is an activity that every biobanking entity should think about. This article describes some of the fundamental considerations for preparing and executing a legacy plan, and we envisage that this article will facilitate dialogue to help inform best practices and policy development in the future.

Understanding preanalytical variables and their effects on clinical biomarkers of oncology and immunotherapy

Identifying a suitable course of immunotherapy treatment for a given patient as well as monitoring treatment response is heavily reliant on biomarkers detected and quantified in blood and tissue biospecimens. Suboptimal or variable biospecimen collection, processing, and storage practices have the potential to alter clinically relevant biomarkers, including those used in cancer immunotherapy. In the present review, we summarize effects reported for immunologically relevant biomarkers and highlight preanalytical factors associated with specific analytical platforms and assays used to predict and gauge immunotherapy response. Given that many of the effects introduced by preanalytical variability are gene-, transcript-, and protein-specific, biospecimen practices should be standardized and validated for each biomarker and assay to ensure accurate results and facilitate clinical implementation of newly identified immunotherapy approaches.

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.

Abstract 5947: The NCI Best Practices for Biospecimen Resources: 2016 revised recommendations

Improved biospecimen handling practices are increasingly important for cancer research as advanced molecular analysis becomes routine in clinical trials and more frequently available in standard of care medicine. Biospecimens and associated clinical data collected in a consistent, established fashion can greatly facilitate cancer biomarker validation and development and validation of clinical diagnostic assays. In order to establish a set of guidelines to improve the quality of biospecimen-related research, the NCI’s Biorepositories and Biospecimen Research Branch developed the NCI Best Practices for Biospecimen Resources which includes technical recommendations on biospecimen handling as well as ethical and regulatory best practices. The 3rd, 2016 revised version of these Best Practices focused on updating technical and operational best practices with recommendations based on more recent research, guidance and standards for collecting, processing and storing biospecimens; revised informatics best practices; and updated ethical, legal and policy sections describing new developments on return of research results, informed consent for genomics research, data sharing, and community engagement. These Best Practices aim to help patients by improving the reproducibility of cancer research data. The NCI Best Practices are also foundational to the NIH Precision Medicine Initiative, part of which aims to establish the world’s largest research biobank that will support studies that utilize biospecimens from a cohort of one million individuals in the United States. Citation Format: Abhi Rao, Jim Vaught, Ping Guan, Carol Weil, Helen M. Moore. The NCI Best Practices for Biospecimen Resources: 2016 revised recommendations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5947. doi:10.1158/1538-7445.AM2017-5947

Abstract 5264: The National Cancer Institute's biospecimen research database: A community resource for improving specimen quality

Tissue and blood biospecimens used in cancer research are collected, processed and stored using standard operating procedures (SOPs) that can vary tremendously across institutions. This lack of standardization in biospecimen collection and processing practices across sample sets and institutions can be a contributing source of research variability and can compromise analytical results. In an effort to minimize preanalytical variability and improve the quality of biospecimens used for cancer research, the National Cancer Institute9s Biorepositories and Biospecimen Research Branch (BBRB) developed the Biospecimen Research Database (BRD; http://biospecimens.cancer.gov/brd). The BRD is a free and publically accessible online database that aims to (i) improve access to peer-reviewed articles that investigate the impacts that biospecimen collection and handling practices can have on clinical, molecular, and proteomic endpoints, (ii) facilitate transparency between institutions and researchers by promoting SOP sharing and distribution, and (iii) improve the quality of biospecimens through the development of evidence-based procedural guidelines. To date, the BRD houses more than 2,300 articles and 200 SOPs from 30 different participating biobanking institutions. Articles are meticulously categorized and annotated by a team of Ph.D.-level scientists according to the type of biospecimen and technology platform used and the preanalytical factors investigated. Literature contained within the BRD serves as the foundation for internally developed reviewed papers as well as evidence-based procedural guidelines, termed Biospecimen Evidence-Based Practices, which focus on biospecimen collection, preservation, and processing. In addition, more than 90% of the BRD9s SOP library was contributed by other government offices, non-profit, private, and international biobanks and institutions. BBRB has invested a substantial and continued level of effort in the establishment of this scientifically accurate and robust database. Ongoing participation by the scientific community in the form of public commenting, article suggestions and SOP submissions is encouraged and will increase both the diversity and value of the BRD. Citation Format: Kelly B. Engel, Sarah R. Greytak, Ping Guan, Helen Moore. The National Cancer Institute9s biospecimen research database: A community resource for improving specimen quality. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5264.

Abstract 4500: Normal tissue and data resources for cancer research from the GTEx program

The NIH Common Fund9s Genotype-Tissue Expression (GTEx) project aims to study gene expression and regulation across multiple human tissues from approximately 1000 healthy normal postmortem donors. GTEx will provide valuable insights into gene regulation and its tissue specificity, to identify correlations between genetic variations and variations in gene expression levels as expression quantitative trait loci (eQTLs), and to help understand inherited susceptibility to disease. Initiated in 2010, the GTEx program has generated a large volume of data associated with each donor, including clinical and histopathological, as well as genotyping and gene expression data from whole genome sequencing, whole exome sequencing, expression array, and RNAseq data. The program has published research results in multiple scientific journals over the past year. The following public resources are available from the GTEx program: 1. GTEx Portal: an open access database of GTEx expression data and analysis results: http://www.gtexportal.org 2. Database of Genotypes and Phenotypes (dbGaP): controlled access of comprehensive GTEx clinical data and raw sequencing data: http://www.ncbi.nlm.nih.gov/gap 3. Access to residual GTEx biospecimens for research: http://www.gtexportal.org/home/samplesPage 4. SOPs and best practices from GTEx biospecimen collections: http://biospecimens.cancer.gov/resources/sops/library.asp 5. GTEx histological image viewer: http://biospecimens.cancer.gov/resources/tissue_image_library.asp 6. GTEx donors’ families website: a lay description of the GTEx project tailored to the GTEx donors’ families: http://www.genome.gov/gtex GTEx data can be used in combination with other data sets such as The Cancer Genome Atlas (TCGA) and genome-wide association studies (GWAS) to further explore the genetic causes and biology of cancer. The eQTL data from multiple tissues will help prioritize candidate genes within GWAS-associated loci; allow evaluation of tissue specificity of associated loci, and pinpoint target tissues for disease studies. Researchers are using GTEx resources to: study cancer heterogeneity by subtracting normal tissue expression; identify mutations in protein-truncating variants which may cause cancer; and explore gene activity from multiple donors across multiple tissue types to better understand the age/gender bias in cancer and other diseases. Citation Format: Ping Guan, Abhi Rao, Simona Volpi, Susan Koester, Helen M. Moore, GTEx consortium. Normal tissue and data resources for cancer research from the GTEx program. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4500.

Abstract 1377: Impact of biospecimen pre-analytical factors on molecular analysis

The Biospecimen Pre-analytical Variables (BPV) Program of the Biorepositories and Biospecimen Research Branch (BBRB) at the National Cancer Institute (NCI) is designed to systematically investigate the effects of preanalytical factors on the molecular integrity of biospecimens. Specific parameters related to formalin fixation and paraffin embedding (FFPE) of cancer tissues were examined, including the effects of cold ischemic time (delay to fixation (DTF)) and time in fixative (TIF). Additional preanalytical studies focus on snap freezing method for tissue specimens (dry Ice vs. LN2 vapor), storage temperature (-80°C vs. LN2 vapor), and duration of storage for frozen specimens. Biospecimens for the BPV program were collected at four medical centers, within a tightly regulated infrastructure and strict adherence to SOPs, to enable consistent collection and handling across all sites. Biospecimens were collected from research participants undergoing surgical treatment for renal cell carcinoma; ovarian, fallopian tube, and peritoneal carcinoma; lung adenocarcinoma and squamous cell carcinoma; and colorectal adenocarcinoma. Biospecimens were subjected to specific experimental protocols to systematically vary the preanalytical factors of interest. Extensive annotation was performed with 300+ data elements that include steps in the collection, handling, and processing of the biospecimens, pathological evaluation of the tumor, and clinical information collected from donors. Biospecimen collections concluded in April 2015 with a total of 364 tumor tissue cases collected. The BPV program has conducted multiple, simultaneous molecular analyses to understand the impact of FFPE preanalytical factors on the expression and detection of various molecular analytes. Initial efforts were focused on evaluating the impact of DTF and TIF on the quality of DNA and RNA from FFPE samples using multiple methods and approaches such as NanoDrop 8000 UV-Vis spectrophotometer, Qubit 2.0 Fluorometer, Agilent Bioanalyzer and KAPA Human Genomic DNA Quantification. The QC data from both RIN and Kappa assays showed that FFPE samples have a significant drop in RNA and DNA quality compared with matched frozen samples. 72 hr TIF samples showed a significant drop in both RNA and DNA quality compared to shorter time points (6, 12, or 23 hr TIF), as measured by DV200 and Kappa assays. No significant differences were observed in RNA/DNA quality between the shorter TIF time points or between the DTF time points (1, 2, 3, 12 hr DTF). Further studies are now underway to evaluate additional preanalytical factors. The data from BPV studies will be widely shared with the research community through publication and deposition at a public data repository. The results from these studies will be used to develop evidence-based protocols and best practices for fit-for-purpose collection, processing, and storage of biospecimens. This project is funded by NCI Contract No. HHSN261200800001E. Citation Format: Rachana Agarwal, Ping Guan, Mary Barcus, Jasmin Bavarva, Robin Burges, Philip Branton, Latarsha Carithers, Corinne Camalier, Biswajit Das, Jason Lih, Hana Odeh, Nancy Roche, Dan Rohrer, Michael Sachs, Leslie Sobin, Jewell Scott, Anna Smith, Conrado Soria, Kimberly Valentino, Dana Valley, Mickey Williams, Helen Moore. Impact of biospecimen pre-analytical factors on molecular analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1377.

Abstract 1821: FFPE preanalytical variables: Investigating the effect of delayed times to fixation on the proteome and phosphoproteome for FFPE kidney tumor samples and a comparison of tumor versus normal for matching FFPE and OCT frozen tissue

Variations in how human biospecimens are collected, processed and stored have been shown to significantly affect downstream molecular analyses. FFPE (formalin-fixed, paraffin-embedded) specimens are valuable resources for biospecimen based research, since many such samples are routinely collected and stored in biobanks. With an aim to evaluate the impact of delay to fixation on total protein and phosphoprotein profiles, we used a label free mass spectrometry based proteomic and phosphoproteomic analysis on FFPE renal cell carcinoma (RCC) tumor tissues from 20 patients at four “delay to fixation” time points (1h, 2h, 3h and 12h delay to fixation), collected under the National Cancer Institute9s (NCI) Biospecimen Pre-analytical Variables (BPV) program. In addition, we compared tumor and adjacent normal kidney tissue at the four delay to fixation time points. This comparison was performed using both FFPE and frozen (OCT) tissue from case-matched tumor specimens to assess the relative impact of the two biospecimen preservation methods. We employed a label-free intensity based quantitation for the proteome profiling using high resolution Orbitrap mass spectrometry. A total of 3475 proteins and 1690 phosphoproteins were quantitated in the FFPE specimens and 3728 proteins and 1817 phosphoproteins in the OCT (frozen) tumor specimens. Very few significant changes were observed at the proteome level with different delay to fixation times. However, at the phosphoprotein level, significant numbers of phosphopeptides were observed to change and these changes were observed across the majority of patients. Approximately 8% of the phosphopeptides were significantly changed after a 12 h delay in time to fixation (vs. 1 h) compared to just 0.5% of the nonphosphopeptides. The observed changes to the phosphoproteome do not appear to be completely random. The phosphopeptides that are differentially expressed are enriched in proteins associated with renal cell death. In the tumor versus normal tissue comparison, large numbers of changes were observed at both the proteome and phosphoproteome level. These changes were consistent between the FFPE and OCTtumor specimens. In general, however, greater magnitude fold changes were observed in OCT versus the FFPE tumor specimens. Many of the differentially expressed proteins observed in this study have previously been identified in urine. These findings could potentially lead to the development of a urine-based biomarker assays for renal cell carcinoma that could aid in the early detection of this cancer. This work is funded by NCI Contract No. HHSN261200800001E. Citation Format: Fiona E. McAllister, Rachana Agarwal, Bich Nguyen, Yiyong Zhou, Sushmita Roy, Daniel Chelsky, Ping Guan, Mary Barcus, Hana Odeh, Lararsha Carithers, Helen Moore. FFPE preanalytical variables: Investigating the effect of delayed times to fixation on the proteome and phosphoproteome for FFPE kidney tumor samples and a comparison of tumor versus normal for matching FFPE and OCT frozen tissue. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1821. doi:10.1158/1538-7445.AM2015-1821