Abstracts from the 5th Annual Biospecimen Research Network (BRN) Symposium Advancing Cancer Research Through Biospecimen Science February 22–23,2012 Bethesda,Maryland

1 Yale University School of Medicine, Department of Pathology, New Haven, Connecticut 2 Rochester University School of Medicine, Department of Pathology, Rochester, New York 4. The Tissue Quality Index: A Molecular Test to Estimate Time to Fixation for Formalin Fixed Paraffin Embedded Tissue

Background: Prolonged time to fixation is thought to be among the most significant pre-analytical variables that can affect biomarker assessment and ultimately patient care. Toward the goal of accurate measurement of breast cancer biomarkers, the ASCO/CAP committee issued guidelines capping time to fixation at 1 hour. The goal of our program is to build a tissue quality index (TQI) that represents a small series of molecular tests that can determine whether the time to fixation of a breast cancer specimen is less than 1 hour.

Methods: We constructed a tissue microarray (TMA) from 93 breast cancer specimens with known time to fixation ranging from 25 minutes to 415 minutes. We then validated a series of biomarkers including 45 antibodies, 2 mRNAs, 2 microRNAs and 1 snRNA, all of which were quantitatively measured using the AQUA technology on the TMA.

Results: We performed feature selection based on cross-validated stability with summed variables used to generate an empirical log odds ratio model that was constructed using the entire sample as a training set. The top two sums found where X1=Lamin + Hif2a and X2=MAPK + miR221. Then if X2 > X1 the sample is predicted to be fresh (fixed within 60 minutes). We restricted our preliminary analysis to the interval between 30 minutes and 100 minutes, which contains 77% of the total number of observations, and we show that a smoothed plot of the log-odds ratio is positive for times below 60 minutes and it is negative for times above 60 minutes.

Conclusions: Measurement of a series of biomarkers can be optimized to generate a TQI model that can predict whether time to fixation is less than one hour for breast cancer tissue. The model still needs to be validated on an independent series of breast tissue specimens and also may be improved by addition of other variables that change with time to fixation.

1 Kelly Government Solutions 2 Preferred Staffing Group 3 National Cancer Institute, Office of Biorepositories and Biospecimen Research 5. The Biospecimen Research Database: 1,100 Articles and Growing

The Biospecimen Research Database (BRD; https://brd.nci.nih.gov) is a free and publicly accessible web-based database that currently contains 1,114 published review and research articles covering a broad range of topics spanning the biospecimen lifecycle. To develop a searchable, user-friendly literature database, articles pertinent to human biospecimen science are meticulously categorized and annotated by a team of Ph.D. level scientists according to the type of biospecimen and technology platform used, the experimental variables investigated, and many other parameters. The current version of the BRD permits users to view newly curated articles on a daily basis resulting in an ever-expanding collection of literature that is useful during both experimental design and publication phases of biospecimen-related science. The BRD is particularly well-suited for performing meta-analysis using searchable fields. As an example, meta-analysis of morphological preservation and protein expression in formalin-fixed paraffin-embedded (FFPE) tissue was performed by examining the effects reported for several preanalytical and analytical parameters. In accordance with OBBR's intent to serve the biospecimen science community, a new version of the BRD is currently under production and will include such user-centric features as the ability to leave comments on specific article entries via the NCI Wiki platform and the ability to directly submit suggestions for articles to include in the BRD. Feedback and article referrals are welcome and currently can be submitted to biospecimens@mail.nih.gov.

1 Department of Medicine, University of Utah, Salt Lake City, Utah 2 Department of Experimental Pathology, School of Medicine, University of Utah, Salt Lake City, Utah 3 Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 7. Identifying Sentinel RNAs to Measure mRNA Integrity in Clinical Biospecimens

Clinical biospecimens are increasingly being analyzed for diagnostic or predictive panels of gene expression biomarkers in cancer, inflammatory or premalignant disorders. The integrity of mRNA in biospecimens is vital to the accuracy of such gene expression analysis. However, current measures of mRNA integrity primarily use rRNA as a surrogate marker. mRNA decay can also interfere with the accuracy of gene expression testing. Our goal was to develop an approach for identifying sentinel RNAs that represent improved markers of the intactness of most mRNA in a biospecimen. Since mRNA decay generally proceeds from the 3′ end and would be missed by analyzing polyA + selection mRNA, we used an efficient 5′ cap dependent purification of Pol II RNA (PNAS PMID 12777618, PLoS ONE PMID 21359205). 5′ capped RNA recovered from human liver specimens thawed at room temperature for 0, 5, 10, and 15 minutes was analyzed by RNA-sequencing (RNA-seq). Total RNA from these samples had RIN (bioanalyzer) values of 9.5, 8.9, 7.9, and 6.7, respectively. Surprisingly, RNA-seq analysis showed that only half of protein coding mRNAs at 10 and 80% at 5 minutes were ≥70% intact. 304 candidate sentinel mRNAs were identified by mining the data for sequence reads in the last and first 200 bp of the 3′ and 5′ end of transcripts over time (3′/5′ end ratio). qPCR of the 3′ and 5′ ends was used to develop a more rapid test for the integrity of sentinel mRNAs. We identified several sentinel RNAs, for the conditions cited, that reflect general mRNA decay that could be measured by 3′/5′ qPCR ratios. We conclude that RNA-seq analysis of differentially selected RNA in biospecimens, under specific conditions, can identify sentinel RNAs that provide improved measures of general mRNA integrity. Moreover, 3′/5′ qPCR assays measure their intactness.

Supported by CA148068.

1 Integrated Biobank of Luxembourg, Luxembourg 2 Manchester Cancer Research Centre Biobank, Paterson Institute for Cancer Research, Manchester, United Kingdom 3 Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 4 Damar Research Scientists, Damar, Drum Road, Cuparmuir, Cupar, Fife, Scotland, United Kingdom 5 Cancer Diagnosis Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland 6 Australian Prostate Cancer BioResource, Hanson Institute, Rundle Mall, Adelaide SA, Australia 7 Moffitt Cancer Center, Tampa, Florida 8 University of California, San Francisco, UCSF AIDS Specimen Bank, San Francisco, California 9 UZA, Edegem, Belgium 10 Michigan Neonatal Biotrust, Detroit, Michigan 11 Quintiles Laboratories, Marietta, Georgia 12 Kaiser Permanente, Portland, Oregon 13 Interinstitutional Multidisciplinary Biobank, Department of Laboratory Medicine and Advanced Biotechnologies, IRCCS San Raffaele Pisana, Rome, Italy 14 PPD Vaccines and Biologics Lab, Wayne, Pennsylvania 15 SeraCare Life Sciences, Gaithersburg, Maryland 16 Masonic Cancer Center's Tissue Procurement Facility, University of Minnesota, Minneapolis, Minnesota 17 Windber Research Institute, Windber, Pennsylvania 18 Karolinska Institutet Biobank, Stockholm, Sweden 19 Specimen Solutions LLC, Tucker, Georgia 11. Activities of the ISBER Biospecimen Science Working Group in 2010–2011

The ISBER Biospecimen Science Working Group was created in 2007 to advance biospecimen science. In 2010-2011, we compiled a significant update of biospecimen science literature. We developed and published the Standard PREanalytical Code (SPREC) to standardize traceability and reporting of the preanalytical steps involved with collection, processing, and storage of human biospecimens, as well as a paradigm of SPREC for environmental specimens and an IT SPREC tool (OLOSPREC). Next, we performed a critical review of our literature compilation and identified potential evidence-based quality control tools, critical information about biospecimens often lacking in research papers. One experimental project was conducted on the room temperature stability of extracted RNA, a second on the impact of logistics on cell viability, and results will be published in 2012. Finally, we developed and launched in October 2011 the first international Proficiency Testing schemes for biobanks - DNA quantification and purity and RNA integrity - in collaboration with the Integrated Biobank of Luxembourg (IBBL).

1 Clinical Research Directorate, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 2 Center for Global Health, National Cancer Institute, Bethesda, Maryland 3 Applied Developmental Research Directorate, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 12. Building Biorepositories for the United States–Latin America Cancer Research Network

The United States-Latin America Cancer Research Network (US-LA CRN) was ratified through bilateral agreements between the National Cancer Institute and the governments of Argentina, Brazil, Chile, Mexico, and Uruguay. The first US-LA CRN collaborative cancer research project, “Molecular profiling of stage II and III breast cancer in Latin American women receiving standard of care treatment,” is currently underway at multiple clinical sites in each of the countries. Key to the success of this project was the establishment of at least one centralized biorepository in each country; however, some countries have multiple biobanks because of the geographical distance between each participating clinical site. Each biorepository within the network presents different levels of infrastructure, capabilities and experience.

Preanalytical variations introduced from the collection to the storage of biospecimens may compromise research findings. Therefore, harmonization and annotation of quality parameters are key components in multinational clinical trial design. The efforts of the US-LA CRN have focused in (1) harmonizing and standardizing procedures taking into consideration local practices, restrictions and capabilities, and (2) developing the bioinformatics platform in three languages capturing clinical data, biospecimen quality data, and biospecimen tracking and inventory. Consequently, Standard Operating Procedures (SOPs) for tissue and blood collection, handling, processing, labeling, transport and storage were developed in addition to biospecimen quality forms that capture all relevant preanalytical variables. To ensure long term applicability and to provide optimal standardization, assessments were conducted through biobank and site visits, surveys, conference calls, and face-to-face meetings in an effort to achieve general consensus. SOPs and technical recommendations are in alignment with the 2008 Best Practices for Repositories from the International Society for Biological and Experimental Repositories (ISBER) and National Cancer Institute Best Practices for Biospecimen Resources.

Funded by NCI Contract No. HHSN261200800001E.

1 Office of Physical Sciences-Oncology, Center for Strategic Scientific Initiatives, Office of the Director, National Cancer Institute, National Institutes of Health 2 Office of Biorepositories and Biospecimen Research, Center for Strategic Scientific Initiatives, Office of the Director, National Cancer Institute, National Institutes of Health 3 Center for Strategic Scientific Initiatives, Office of the Director, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 13. A Physical Sciences Approach to Biospecimen Research via High Content Screening

The Office of Physical Sciences-Oncology (OPSO) has teamed with the Office of Biorepositories and Biospecimen Research (OBBR) to explore new approaches in biospecimen science using high content screening (HCS). HCS platforms bridge the gap between depth and throughput of biological experiments allowing one to capture large quantities of data not typically detected with standard throughput assays. OPSO and OBBR are currently initiating new projects to systematically develop best practices for biospecimen collection and handling for HCS technologies. HCS platforms yielding rich data sets will contribute important information for understanding the impact of cancer biospecimen collection, handling, and processing (CHP) variables on analytical outcomes. By using thoroughly annotated biospecimens, HCS platforms can be used to characterize and evaluate the impact of select altered biospecimen CHP variables on analytical outcomes. To date, OPSO investigators have used high-throughput, high definition microscopy based phenotypic assays to (1) classify and count circulating tumor cells in whole blood without enrichment as a biomarker for cancer prognosis and (2) identify a phenotypic signature for pancreatic cell metastasis by rapid characterization of over 11,000 nuclear and cell associated properties. This newly formed joint effort is expected to provide new knowledge in biospecimen research that has the potential to establish best practices and define new standards for cancer biospecimen analysis by cutting edge HCS platforms.

1 SomaLogic, Inc. Boulder, Colorado 2 New York University School of Medicine, New York, New York 3 University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15. Exposing the Criminal Record of Every Blood Sample: Use of SOMAmer Technology and Sample Mapping Vectors to Mitigate False Biomarker Discoveries

Biomarker discovery studies may fail to translate to the clinic because the study population does not match the intended clinical use or because hidden preanalytic variability in the discovery samples contaminates the apparent disease specific information in the biomarkers. This can arise from differences in blood sample processing between study sites or in samples collected differently at the same study site. To better understand the effect of different blood sample processing procedures, we evaluated protein measurement bias in a large multi-center lung cancer study using the >1000 protein SOMAscan assay. These analyses revealed that perturbations in serum collection and processing result in changes to families of proteins from known biological pathways. We subsequently developed protein biomarker signatures of cell lysis, platelet activation and complement activation and assembled these preanalytic signatures into quantitative multi-dimensional Sample Mapping Vector (SMV) scores. The SMV score provides critical evaluation of the quality of every blood sample used in discovery and also enables the evaluation of candidate protein biomarkers for resistance to preanalytic variability. Despite uniform processing protocols for each clinic, the SMV analysis revealed unexpected case/control bias arising from collecting case and control serum from different clinics at the same academic centers, an effect which created false or bias-contaminated disease markers. We therefore used the SMV score to remove bias-susceptible analytes and to define a well-collected, unbiased training set. An improved classifier was developed, resistant to common artifacts in serum processing. The performance of this classifier to detect lung cancer in a high risk population is more likely to represent real-world diagnostic results. We believe this approach is generally applicable to clinical investigations in all fields of biomarker discovery and translational medicine.

1 Department of Medical Oncology 2 Center for Molecular Oncologic Pathology 3 Dana-Farber Cancer Institute, Boston, Massachusetts 4 Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 5 Hospitalier Pitie Salpetriere, Service de Neurologie Mazarin, Paris, France 6 Department of Neurosurgery, Brain Tumor Research Center, University of California, San Francisco, California 7 Division of Pediatric Neurosurgery, Acibadem University Medical Center, Istanbul, Turkey 8 Department of Pathology, Acibadem University Medical Center, Istanbul, Turkey 9 Department of Pathology, Children's National Medical Center, Washington, DC 10 Department of Pathology, The University of Texas Southwestern Medical Center, Children's Medical Center, Dallas, Texas 11 Departments of Pathology, Oncology and Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland 12 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 13 Department of Pediatrics, Children's Hospital Boston, Boston, Massachusetts 14 Department of Neurology, Brigham and Women's Hospital, Boston, Massachusetts 15 Center for Neuro-Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 16 Department of Pathology, Children's Hospital Boston, Boston, Massachusetts 17 Harvard Medical School, Boston, Massachusetts 17. DNA Fragmentation Simulation Method and Fragment Size Matching Improve aCGH Performance of FFPE Tissues

Whole-genome copy number analysis platforms, such as array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) arrays, are transformative research discovery tools. In cancer, the identification of genomic aberrations with these approaches has generated important diagnostic and prognostic markers, and critical therapeutic targets. While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations. Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues. Using self-hybridizations of a single DNA sample we observed that aCGH performance is significantly improved by accurate DNA size determination and the matching of test and reference DNA samples so that both possess similar fragment sizes. Based on this observation, we developed a novel DNA fragmentation simulation method (FSM) that allows customized tailoring of the fragment sizes of test and reference samples, thereby lowering array failure rates. To validate our methods, we combined FSM with Universal Linkage System (ULS) labeling to study a cohort of 200 tumor samples using Agilent 1M feature arrays. Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA). This study demonstrates that rigorous control of DNA fragment size improves aCGH performance. This methodological advance will permit the routine analysis of FFPE tumor samples for clinical trials and in daily clinical practice.

1 Caprion Proteomics US, LLC, Menlo Park, California 2 Caprion Proteomics, Inc., Montreal, Quebec, Canada 19. Controlled Analysis of Preanalytical Variables in Clinical Blood Sample Collection, Processing, and Storage

Blood sample collection, processing, handling and storage protocols are based mainly on accepted practices rather than careful comparative analysis and testing. We set out, therefore, to examine variables intrinsic to each step in the process of obtaining and storing clinical samples, beginning with collection of samples from healthy subjects and cancer patients in controlled studies. Various tube types were tested including EDTA, heparin, serum and protease inhibitors. Various times on bench and temperatures of incubation were compared, before and after centrifugation of the blood. The effects of freeze-thaw cycles and time in freezer were also examined. Sample analysis has been performed by high-resolution mass spectrometry, leading to the identification of specific proteins that are affected by the various parameters tested. While different blood collection tubes can be used with reproducible results, there is a marked difference in the protein content obtained from each type, with protease inhibitor tubes offering significant protection from changes to the proteome. Freeze thaw cycles affect only a few specific proteins and only after multiple cycles. A multiplexed assay is currently being assembled for the analysis of stored samples in order to determine sample integrity and utility for use in clinical research.

Supported by NCI contract HHSN261200800001E.

1 Office of Biorepositories and Biospecimen Research, National Cancer Institute, National Institutes of Health 2 Kelly Services, Inc. 3 SAIC-Frederick, Inc. 4 Sapient Government Services 5 Critical Path Institute 20. caHUB: A Unique Post Mortem Biospecimen and Data Management Infrastructure Supporting the GTEx Project

The success of the GTEx program is founded on the collection of high quality, fit for purpose biospecimens recovered from post mortem (PM) tissue and organ donors. To date the GTEx biospecimen recovery program, operated through NCI's Cancer Human Biobank (caHUB) has performed nearly 100 individual donor biospecimen recoveries, averaging 20-30 tissues per donor totaling greater than 2,000 unique biospecimens in the collection. After rigorous quality check of biospecimens and data, and digital pathological evaluation, tissue and blood aliquots are distributed for analysis to the GTEx Laboratory, Data Analysis, and Coordinating Center (LDACC). GTEx and the caHUB program have worked to establish rigorous best practices for PM biospecimen recovery through engagement of industry experts in PM biospecimen recovery, stabilization, processing and storage, and supply chain logistics.

Currently, the caHUB team has developed over 150 project specific standard operating procedures that have been developed, reviewed and approved within the caHUB Quality Management framework. This framework together with regular training and the expertise of those performing tissue collection and processing has resulted in a collection that meets and exceeds molecular quality expectations for this project. With an average donor PM interval of between 2 and 10 hours, a specialized logistics chain procuring and redistributing tissues with a 2-4 day turnaround, and a dedicated data management team collecting >150 donor specific data elements within a 72-hour window, caHUB has established a one of a kind operational infrastructure to support the molecular characterization efforts of the GTEx project.

1 Yale University School of Medicine, Department of Pathology, New Haven, Connecticut 2 Rochester University School of Medicine, Department of Pathology, Rochester, New York 28. Quantitative Measurement of Expression of Common Breast Cancer Markers as a Function of Time Before Formalin Fixation

Background: Breast cancer therapy is based on expression levels of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). The accuracy of these measurements is affected by tissue handling prior to analysis and lack of standardization. Here, we used a systematic, prospectively designed approach to objectively measure the expression levels of these proteins, and Ki67 and p53, as a function of time to formalin fixation.

Materials and Methods: Two different breast cancer cohorts were used for this study: A TMA consisting of 93 breast cancer specimens with recorded time to formalin fixation (ranging from 25 to 415 minutes, with 74% of the samples having a time to fixation of 120 minutes or below). The second cohort consists of 25 matched pairs of biopsies and resections. Time to formalin fixation for the biopsies is minimal, while time to fixation for the resections is not recorded, averaging between 1 and 4 hours. Protein expression was measured using the AQUA method of quantitative immunofluorescence and the Aperio technology system to objectively quantify IHC/DAB staining.

Results: ER, PR, HER2, Ki67, and p53 were each analyzed with antibodies commonly used in the clinical setting. No statistically significant loss of protein expression as a linear function of prolonged time to fixation was seen within the range of 25 to 120 minutes. Trends toward loss were seen with longer time points, but none were statistically significant. Measurement of loss is confounded by tumor heterogeneity.

Conclusions: There is no evidence for loss of expression of critical breast cancer proteins as a function of time to fixation within a timeframe of 120 minutes, twice the current maximum recommended in the ASCO/CAP guidelines. Further evaluation of these proteins on normal breast tissue with an extended delay to fixation up to 72 hours is planned.

1 New York University School of Medicine, New York, New York 2 Johns Hopkins School of Medicine, Baltimore, Maryland 29. Ischemia/Fixation Trial Tissue Microarray

Since its introduction over 10 years ago, tissue microarray (TMA) technology has become an indispensible tool in biomedical research. Although there are clear benefits to their use in biomarker discovery, TMAs have some significant weaknesses. TMAs often use large cohorts of archival material from vast time spans, increasing the chance that variations introduced not only by the age of the archival tissue but also by diverse tissue handling and specimen preparation protocols can confound results. Preanalytical variables, specifically fixation and ischemia, have been implicated as key variables in the measurement of proteins by immunohistochemistry (IHC). To better understand the influence of fixation length and ischemic time on the final IHC result, the Prostate Cancer Biorepository Network (PCBN) collected tissue samples from radical prostatectomy specimens (n=42) with known ischemic intervals. The tissue samples were divided into four 5-7mm punches for delayed intervals to fixation (0, 1, 2 and 4hrs) (n=27) or five 5-7mm punches for fixation in 10% neutral buffered formalin at varied time lengths (4, 8, 12, 24 and 48hrs) (n=15). Samples for delayed fixation were kept in a moisture chamber to prevent them from drying out. Fixed samples were placed in PBS to prevent further cross-linking, prior to processing using a uniform protocol. A TMA will be constructed and assessed with known markers of ischemia, fixation and tissue quality (p27 and phospho-antibodies). Further, as the magnitude of ischemic change and fixation will be known, this TMA will be useful for determining their effects on tissue quality and marker reactivity. It is planned to make this TMA available to researchers accessing PCBN for validating antibodies.

*R. Pe Benito, and H.Fedor contributed equally to this work.

1 Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 2 Department of Chemical Engineering, Tuskegee University, Tuskegee, Alabama 30. The Effects on Immunorecognition of the Transfer of Cells From 10% Neutral Buffered Formalin to 70% Ethanol

Cells fixed in 10% neutral buffered formalin (NBF) may be transferred to 70% ethanol (ethanol) until immunostaining to reduce effects on immunorecognition of prolonged NBF fixation. Optimal times before transfer to ethanol and effects of transfer to ethanol on immunorecognition are not documented adequately.

DU145 and SKOV-3 cells were fixed in NBF for 5 minutes and 12, 15, 18, 36, 108, and 180 hours. After 12 hours in NBF, aliquots of the same batch of cells were transferred to ethanol for 3, 6, 24, 96, and 168 hours so the effects of comparable fixation on immunorecognition of PCNA, Ki67 (MIB-1), cytokeratins (AE1-AE3), and EGFr could be determined. A separate experiment compared NBF fixation for 5 minutes followed by 12 hours of fixation in NBF and 672 hours either in NBF or ethanol with and without antigen retrieval (AR, pH8.0 Tris-EDTA, boiling at 121 psi, 10 minutes).

NBF fixation decreased immunorecognition of PCNA, Ki67 (MIB-1), and AE1/AE3 after 12 to 18 hours (p<0.05), but the extent of these decreases were not important until between 18 and 36 hours (p<0.05). At 108 to 180 hours, there was almost complete loss of immunorecognition of PNCA, Ki67 (MIB-1) and AE1/AE3. Ethanol preserved immunorecognition at all points up to 180 hours. The effects on EGFr were less, but transfer to ethanol maintained cytoplasmic and membranous staining; however, transfer to ethanol for about 17 weeks was much less successful and varied with the antigen-antibody pair and cell lines. AR was not successful in complete recovery of immunorecognition but was best for NBF plus ethanol.

For preservation of immunorecognition of many antigens, transfer from NBF to ethanol after 18 hours will preserve immunorecognition for at least 1 week. However, cells should not remain in ethanol for many weeks, even if AR is used.

Supported by the CHTN (5U01CA44968); Breast (5P50CA0189019), Pancreatic (5P50CA0189019) and Cervical (5P50CA098252) SPORES; DOD Grant (W81XWH-10-1-0543) and U54 Grant (2U54CA118948).

1 Beaumont BioBank, Beaumont Health System, Royal Oak, Michigan 2 Department of Otolaryngology, Beaumont Health System, Royal Oak, Michigan 3 Department of Radiation Oncology, Beaumont Health System, Royal Oak, Michigan 31. The Development of a Protein Integrity Metric for Biorepository Specimens

With biorepositories so prominently featured in the modern diagnostic age, there is a crucial need to assure that biospecimen quality is maintained at the highest level possible. Sample quality as well as the validity of the subsequent analysis obtained from a sample is highly dependent on the preanalytical conditions to which a sample is exposed. The myriad of low abundance proteins likely to exhibit the greatest sensitivity to these variable conditions are also the likely biomarkers of tomorrow. Although quality metrics exist for the assessment of nucleic acids such as the RNA Integrity Number (RIN), there is no accepted index for the integrity of proteins contained within the samples that can be used to assess the reliability of samples processed and stored in today's biorepositories.

Here we illustrate the use of SELDI-TOF technology to develop a sample-specific protein integrity number (SPIN) that describes the state and thus utility of the sample irrespective of the processing procedure. High-throughput discovery proteomics is employed to identify proteins of differing but reproducible sensitivity to preanalytical factors. Following exposure to conditions designed to introduce preanalytical variability commonly encountered in biorepository laboratories, samples were subjected to SELDI analysis. The resulting spectra were normalized, and peak clusters under each experimental condition were created. After statistical analysis differences in spectra patterns and peak intensities were studied. Peak clusters with significant P values (<0.05) were then considered candidates for future identification and protein integrity focus. Interim SPIN numbers (ratio of non-stable to stable SELDI peak intensities) were assigned. A multiplex bead array was then performed on selected analytes and the results compared to the SPIN index.

This study demonstrated preliminary findings which correlated sensitive, labile signature peaks of specific proteins to results in analyte quanititation. These proteins will form the basis of a future universally accepted simple assay.

1 PreAnalytiX GmbH, Hombrechtikon, Switzerland 2 Office of Biorepositories and Biospecimen Research, National Cancer Institute 3 Applied Biomedical Science and Informatics Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 33. Paxgene vs. Formalin-Fixed Tissue: A Comparison of Tissue Morphology and RNA Quality

Background: Preanalytical treatment of tissue samples can influence bioanalyte quality and, ultimately, outcome of analytical results. In this study, we compared RNA quality and tissue morphology from formalin fixed tissue to that of tissue treated with a two-step, formalin-free fixative, the PAXgene^® Tissue System (PAXgene). Snap-frozen tissue served as control for RNA quality. Effects of reversal of the PAXgene fixation/stabilization reagents were also examined.

Methods: Rat liver samples were fixed for 24 h in formalin, 4h in PAXgene Tissue Fix followed by 72 h storage in PAXgene Tissue Stabilizer, or snap-frozen in liquid nitrogen. One sample was incubated in PAXgene Tissue stabilizer first for 10 min then treated according to protocol. Sections of formalin fixed or PAXgene fixed, paraffin-embedded samples (FFPE or PFPE respectively) were stained with H&E. Total RNA was purified from FFPE tissue with miRNeasy™ and from PFPE tissue with the PAXgene Tissue miRNA kit. Real-time RT PCR was carried out using eight different SYBR-Green assays with amplicon lengths ranging from 109 to 610 bp.

Results: Histology of PFPE tissue was comparable to FFPE tissue; however, while RIN numbers for RNA from both fixed tissues were comparable, (FFPE=7.4-8.6; PFPE=7.2–7.6), RT PCR assays of FFPE tissue RNA were greatly inhibited relative to snap frozen tissue (Ct=3-11). In contrast, RNA from PFPE performed as well as RNA from snap-frozen tissue in these assays (Ct=0 to 1.0). Accidental immersion of tissue into PAXgene Tissue Stabilizer before fixation could be easily detected by histological examination.

Conclusion: Histology of PAXgene fixed tissue is comparable to formalin fixed tissue. While RNA with acceptable RIN values can be isolated from both FFPE and PFPE tissue, only RNA from PFPE tissue was comparable to RNA from snap-frozen tissue in RT PCR assays.

For research use only. Not for use in diagnostic procedures.

Funded by NCI Contract No. HHSN261200800001E.

1 National Human Genome Research Institute 2 National Institute of Mental Health 3 National Cancer Institute, National Institutes of Health, Bethesda, Maryland 35. The Genotype-Tissue Expression (GTEx) Project

Understanding the role of variation in the human genome is crucial to elucidating genetic contributions to human health and disease. Despite the results of genome-wide association studies (GWAS) documenting strong statistical associations between genetic variation and human traits, the functional role for most of these variants is largely unexplained. Nearly 90% of these GWAS-implicated sites lie outside of protein-coding sequences, suggesting that these variants might regulate gene expression. The Genotype-Tissue Expression (GTEx) project was launched in 2010 as a 2.5-year project sponsored by the NIH Common Fund with the goal of assessing the feasibility of collecting high-quality RNA from multiple tissues from healthy donors. The project will collect and analyze RNA levels in 30+ human tissues from 160 postmortem donors and 4-6 tissues from 100 surgical controls that have been characterized for germline genetic variation through dense genotyping. The resulting data will be made available to the scientific community through an NIH-supported GTEx database. By treating RNA expression levels as quantitative traits, expression quantitative trait loci (eQTLs) will be identified as sites containing genetic variation that correlate with changes in RNA expression. Such eQTLs have been associated with 4%-12% of expressed human genes, and with common complex human diseases, including obesity, atherosclerosis, type 2 diabetes, Crohn's disease, and asthma. Additionally, few studies have examined the tissue specificity of eQTLs. The GTEx project will thus serve as a resource database and tissue bank for many future studies, especially for understanding the functional basis of inherited susceptibility to disease.

1 Institute of Pathology, Medical University of Graz, Austria 2 PreAnalytiX GmbH, Hombrechtikon, Switzerland, c/o QIAGEN GmbH, Hilden, Germany 3 Institute of Pathology, Technical University Munich, Germany 4 Department of Pathology, Josephine Nefkens Institute, Rotterdam, The Netherlands 5 Dako Denmark A/S, Glostrup, Denmark 6 QIAGEN GmbH, Hilden, Germany 7 AROS Applied Biotechnology A/S, Aarhus, Denmark. 36. The Impact of Tissue Preanalytics and a New Stabilization Technology on the Quality of Tissue-Based Molecular Studies

Background: Molecular characterization of human disease requires analysis of multiple parameters ranging from classical histopathological features to a broad spectrum of molecular biomarkers. The morphological characterization is routinely based on the analysis of formaldehyde-fixed and paraffin-embedded (FFPE) tissues but it is known that formalin fixation impairs molecular analyses which typically require frozen tissue samples. Within the EU FP7 project SPIDIA we evaluated a new technology for combined tissue diagnostics and the impact of several pre-analytical variables on tissue sample quality and subsequent molecular analyses.

Methods: FFPE samples were compared to alternative fixatives, including a novel technology for simultaneous preservation of morphology and biomolecules (PAXgene Tissue System), and corresponding snap-frozen tissue samples served as reference. Morphology, antigenicity and different biomolecules were investigated with a focus on nucleic acids preservation.

Results: Established methods for quality control of RNA (e.g., 28s:18s ratio, RIN value) were well-suited for frozen tissue, but a more detailed analysis, like a qPCR assay based on different amplicon length, was needed to estimate the suitability of RNA from paraffin-embedded tissues for downstream applications. Results of qPCR were sensitive to RNA degradation introduced by preanalytical procedures such as time or type of fixation and storage. In contrast to FFPE, PAXgene-fixed and paraffin-embedded (PFPE) samples showed outstanding RNA preservation and strong correlation of multiple mRNA and microRNA profiles with snap-frozen samples as revealed by qPCR and microarray analysis. DNA isolated from PFPE tissues was of high molecular mass and well-suited for long-range and multiplex PCR, and different sequencing techniques. Protein extractions from PFPE samples showed comparable yield and preservation of phosphorylation levels to cryopreserved samples.

Conclusion: The quality of tissue-based molecular studies can only be defined in the context of the preanalytical procedures. The excellent preservation of biomolecules and morphology in PFPE samples provides new opportunities for comprehensive tissue diagnostics, biomarker discovery and reliable molecular analyses whenever a collection of snap-frozen material is impossible.

1 Institute of Pathology, Technische Universität München, Munich, Germany 2 Evotec AG, Martinsried, Germany 3 PreAnalytiX GmbH, Hombrechtikon, Switzerland 4 The SPIDIA Consortium, www.spidia.eu 5 Munich Biotech Cluster m4, www.m4.de 37. Delay to Preservation Does Not Induce a Systematic Phosphoprotein Response During Tissue Processing

Aims: The quality of tissue samples can have a significant impact on analytical data sets for biomarker research. In particular, posttranslational modifications such as phosphorylation need to be systematically investigated in that phosphorylated protein levels indicate the activation status of signal transduction pathways controlled by kinases. However, little is known about the impact of preanalytical factors on phosphoprotein stability. The aim of this study was to characterize the potential effects of delayed preservation and different preservation methods on the stability of phosphoproteins using targeted and non-targeted proteomic approaches.

Methods: Murine and rat liver samples were exposed to different ischemic conditions before preservation and either cryopreserved, formalin-fixed or fixed with the PAXgene Tissue System, a new non-crosslinking formalin-free fixative. The phosphoproteome was analyzed using quantitative tandem mass spectrometry (LC-MS/MS) and reverse phase protein array (RPPA) technology.

Results: The phosphoproteomic analysis of ischemic mouse liver tissue samples by LC-MS/MS indicated no significant global alterations of more than 5000 phosphosite-ratios analyzed during 60 minutes of delayed cryopreservation. The analysis of ischemic rat liver tissue samples by RPPA revealed similar results as investigated phosphoproteins, including phospho-Akt, phospho-p38 MAPK or phospho-p44/42 MAPK, showed very stable profiles during the time-course experiment, independent of the preservation method applied.

Conclusion: Since we could not detect significant global changes of the phosphoprotein profiles, neither with a targeted nor with a non-targeted approach, we conclude that the phosphoproteome seems to be more stable than expected with regard to delayed preservation. This allows accurate quantitative measurements of the activation state of signalling pathways of tissue samples which had not been immediately preserved. This result is essential for the development of new targeted therapies involving kinase inhibitors which have recently been a focus in the field of personalized medicine. Studies are ongoing to validate our results in human tissue samples as inter-patient variability may occur which is absent in our well controlled model systems.

This work has received funding from the Munich Biotech Cluster m4 (www.m4.de) and the European project SPIDIA (www.spidia.eu).

1 Women's Health Integrated Research Center at Inova Health System 2 Gynecologic Cancer Center of Excellence 3 The Henry M. Jackson Foundation for the Advancement of Military Medicine 4 Walter Reed National Military Medical Center 38. Improving Quality and Efficiency for Genomics and Proteomics: Advances for a Successful Biorepository

Purpose: Improving quality and efficiency are important facets for a successful biorepository and tissue processing facility.

Methods: At the Women's Health Integrated Research Center, molecular analyses are performed in frozen as well as formalin-fixed and paraffin-embedded tissues that undergo pre-analytic processing and laser capture microdissection (LCM).

Results: Standard operating procedures (SOPs) have been upgraded and streamlined to enhance quality and/or efficiency using new workflows for proteomics and genomics. A new storage configuration has been designed for OCT-embedded tissue to enhance storage capacity. A single-tube workflow from collection to injection was developed for proteomic profile to minimize loss of material due to pooling and facilitate the normalization process following mass spectrometry. Lysate volumes were modified to enhance peptide recovery of processed and digested tissue from formalin-fixed and paraffin-embedded tissue specimens following LCM. Recovery increased proportionately with lysate volume from ∼20 pg of peptides per cancer cell up to a maximum of 1,000 ng of total peptides. The single-tube workflow represents a scalable, straightforward, and reproducible sample preparation procedure. Inclusion of RNA inhibitors in fixatives and all aqueous solutions during preparation of membrane sides for LCM and gene expression array analysis of coding and non-coding RNAs resulted in a 25% increase in RIN (RNA Integrity Number) and a time-dependent stabilization of RIN. Tissue fixation prior to LCM and RNA extraction resulted in a 10% increase in RNA yield and a 5% increase in RIN. Transition to direct ultra-cold freezing of blood in PAXgene tubes did not compromise RNA yield or RIN. A layered storage box doubled storage for OCT-embedded and frozen tissue specimens.

Conclusions: Improvements in quality and efficiency during specimen procurement and pre-analytical processing have effectively enhanced genomic and proteomic profiling of gynecologic cancer and contributed to a culture of excellence in an integrated research center focused on women's health.

1 Van Andel Research Institute, Grand Rapids, Michigan 2 Translational Genomics Research Institute, Phoenix, Arizona 3 Integrated Cancer Genomics, Translational Genomics Research Institute, Phoenix, Arizona 4 Neurogenomics, Translational Genomics Research Institute, Phoenix, Arizona 5 Laboratory for Translational Medicine, Van Andel Research Institute, Grand Rapids, Michigan 6 Program For Biospecimen Science, Van Andel Research Institute, Grand Rapids, Michigan 39. Comparison of RNA Integrity From Morphologic Annotated Tissues by Varying Preservations

The current pace of assay development and accompanying large datasets increases the necessity for optimal quality tissue samples and methods to assess macroanalyte integrity. Herein we show the integrity of RNA from four independent sample sets gathered from different laboratories at our respective institutions to highlight aspects of sample collection, transport, and extraction with RNA integrity assessed by Agilent 2100 Bioanalyzer. Experimental data were gathered through an iterative process with fresh frozen aliquot stored at −86 °C as the comparative gold standard. Murine liver tissue stored for 4 yrs had (RIN=7.5) by whole tissue cryosections compared to laser capture microdissected (LCM) Histogene stained tissue with (RIN=6.8) and without (RIN=2.9) RNAse inhibitor. The second tissue preparation set used RNALater^® and H&E stained tissue for morphology evaluation with a specialized processing step to reduce associated high salt concentration. Similar to LCM, this approach provided a higher degree of confidence in the isolation of cellular components from heterogeneous tissue samples. The results gave better RIN scores (9.5) from specialized processed samples compared to the fresh frozen counterparts (RIN=9.2). A third collection media assessed was the PaxGene assessment of RNA integrity in samples subject to automated tissue processing and paraffin embedding. The fourth independent dataset demonstrated RNA stabilization from a neuronal subpopulation from the hippocampus utilizing LCM and downstream assay of RNA-seq. A direct comparison of split samples treated with and without RNAse inhibitors showed consistent improved RIN scores in time dependent manner from deltas 0.5 (10 min) to 2.0 (1 hr) to 4.7 (2 hr) and RIN >8 at 5 hr and demonstrates need for an RNAse inhibitor in any aqueous containing step. In conclusion, we show collaborative efforts in RNA analyte preparation from varied collection media and cellular harvests to ensure success in downstream transcriptomics to include excellent RNA-seq results.

1 University of California, San Francisco, California 2 University of California, San Diego, California 40. Method to Determine Adequacy of Frozen Brain Tumor Tissue

Glioblastoma (GBM) is the most common primary malignant brain tumor of adults, and banking of frozen tumor tissue samples is critical for both research and potential future diagnostic purposes. Due to tremendous tumor heterogeneity, including variation in the density of tumor cells, microvasculature, and necrosis, it can be difficult to obtain high-quality samples. We have devised a method to assess sample quality without compromising tissue integrity that relies on the histopathologic analysis of two ends of a flash-frozen tumor sample (∼200 mg). To demonstrate the usefulness of this technique we examined two sets of tumor tissue that represent two potential methods of collection. In the first set, we simulate retrospective tissue analysis and analyze the physical ends of a flash-frozen tissue sample (∼200 mg, n=57). Analysis of tumor density at both ends identified 19/57 (34%) samples as inadequate. Analysis of a single end identified 12/57 (21%) or 14/57 (25%) of the samples as inadequate. In the second set, we simulate prospective tissue banking and divide fresh tumor tissue (∼200 mg) in the operating room into three portions: the two ends are paraffin-embedded and the mid-portion is flash-frozen and banked (n=54). The results of these analyses will be reported. Due to GBM heterogeneity, we propose histopathologic analysis of banked frozen tissue is essential to confirm sample adequacy. Furthermore, for samples >200 mg we suggest analysis of a single sample end is inadequate to assess overall sample quality.

1 Moscow City Oncology Clinical Dispensary #1 and Central IHC Laboratory of the Moscow Health Department 2 Cureline, Inc. 3 I.M. Sechenov First Moscow State Medical University 41. Correlation Between Morphological Integrity of Brain Structures Inside and the Length of a Donor's Terminal Period

Histological evaluation of brain areas is a significant part of biomedical studies. Proper donor selection and brain autopsy sequence are important factors for procurement of high quality brain specimens. Hydration and ischemia are major contributing mechanisms of brain tissue damage. It has been previously reported that distribution of interstitial fluid and associated hydration in brain specimens collected post-mortem varies in different brain areas. We report here that interstitial fluid distribution and hydration in certain brain structures correlates with the length of a brain donor's terminal period.

This study was conducted at the Moscow Forensic Laboratory (Russia), where clinical and criminal death cases are autopsied for determination of the cause of death. Three hundred eighty six (386) cases with the cause of death being an acute hemorrhage were selected for this study, and distributed into two groups: (1) short terminal period (few minutes) and (2) prolong terminal period (longer than 24 hours). Post-mortem interval prior to specimen collection was identical for both groups (4–5 hours). Brain tissue samples (200mg) were collected from various brain structures including brain cortex, hypothalamus, cerebellum, sustancia negra, brain pons, brain stem and cerebellum. Exact wet weight of each specimen, as a measurement of interstitial fluid and hydration, was determined prior to placement into the laboratory oven. The brain tissue samples were dried at 100 °C for 5 hours, and weighed again for determination of the dry weight. We found that hydration has been significantly increased in the group with prolonged terminal period in brain cortex (49%) and cerebellum (13.3%). Moderate increase of hydration was observed in hypothalamus, brain pons and brain stem (8 – 11%). No change was found between substantia nigra hydration in two groups. These findings indicate that a prolonged terminal period increases brain hydration and causes morphological damage regardless of postmortem interval, and that these changes are dependent on the brain area. To avoid an accumulated damage, brain cortex and cerebellum should be harvested as a priority tissue while performing a brain tissue acquisition.

1 Clinical Molecular Profiling Core, Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 2 Biomatrica, San Diego, California 42. Long-Term Stability of Total RNA in RNAstable^® as Evaluated by Expression Microarray

Storage of normally labile RNA in laboratories is accomplished through ultra-low freezing of the nucleic acids. This however requires expensive freezers, a lack of convenient storage, and increased shipping costs. Biomatrica (San Diego, CA) has created RNAstable^®, a stabilization reagent that is used to store RNA in a dehydrated state at room temperature (RT) which protects the RNA from degradation. Previous studies have demonstrated that the integrity of RNAstable-protected RNA is maintained for at least four weeks and is suitable for quantitative PCR and expression microarray analysis. Our objective was to investigate the functionality of RNA stored in RNAstable^® at extended time periods through use of Illumina and Agilent RNA expression microarrays. We observed in Bioanalyzer electropherograms that RNA extracted from 293 cells stored at RT in RNAstable^® for 4.5 mon and 11.5 mon is similar in quality to RNA stored at −80°C. Illumina expression array QC metrics and gene expression patterns from RNAstable^®-protected RNA correlated well with those of freezer controls. In contrast, when RNA was stored at RT but without RNAstable^®, there were increased levels of degradation and a decrease in relatedness to both −80°C control and RNAstable^®-protected RNA. Significantly, when RNA was stored in RNAstable^® at 45°C for 4.5 months, equivalent to 22 months RT storage, RNA quality and expression microarray performance was similar to RNAstable^®-protected RNA at RT and −80°C controls. In addition to evaluating mRNA, a comparison of the storage conditions at 10.5 months of the miRNA expression was performed on Agilent Human miRNA v3 arrays. Here too we only found strong concordance when miRNA was stored in RNAstable^® or in the −80°C controls. We conclude that use of RNAstable^® holds promise as an effective stabilization reagent for total RNA and might be useful in situations where shipping and storage options are resource limited.

1 BioCision, LLC 2 Integrated BioTherapeutics, Inc., Gaithersburg, Maryland 3 Roslin Cellab, Roslin Biocentre, Roslin, Midlothian, Scotland, United Kingdom 46. Temperature-Controlled Manipulation and Alcohol-Free Cryopreservation: A New Era in Sample Handling and Biobanking

Standardization of sample handling and viable long-term storage is emerging as one of the most important challenges for biobanks, diagnostic companies and research institutes. As error is inherent in laboratory processes and often the end user is unaware of this, BioCision with the use of select materials such as highly thermo-conductive alloys and high-density crossed-linked foam, has precision engineered solutions to aid the standardization of sample handling.

Our research shows that when a 96-well plate is placed directly on ice, the wells do not actually reach 4 °C and there is a large variation of temperature, up to 3 °C. Conversely when the plate is placed on BioCision's highly-conductive CoolSink platform, all the samples are within the expected 0.5–4 °C range with <1 °C of variation and the “edge effect” is eliminated.

For cryopreservation, BioCision has developed an alcohol-free snap freezing CoolRack platform. For virus-infected samples, our experiments show that viruses such as Venezuelan equine encephalitis virus or influenza virus can be snap-frozen using a CoolRack on dry ice resulting in no loss of viral titer. With sensitive viruses such as dengue virus-2, we show only a minimal loss of 0.5–1 log pfu/ml titer post freezing, which is equivalent to the ethanol-dry ice slurry method but without the hazards and mess.

BioCision has also designed an alcohol-free controlled rate CoolCell freezing container. The use of the CoolCell container ensures equivalent or improved post-thaw cell viability (depending on the cell line or primary cell studied) and greater standardization when compared to current methods. With stem cells we obtain a significant increase (30–36%) in post-thaw viable cell counts compared to other bench-top freezing methods P<0.005. BioCision presents here a new standard for handling and freezing of samples making sample handling more precise while improving yields, and greater standardization.

1 Integrated Biobank of Luxembourg, Luxembourg 2 Laboratoire NorLux de Neuro-Oncologie, Centre de Recherche Public de la Santé (CRP Santé), Luxembourg 47. Impact of Tissue Heat Stabilization and Protein Extraction Method on Phosphoprotein Analysis

Preanalytical variables during sample collection and processing can impact the molecular integrity of specimens and introduce bias in downstream analyses. Protein modifying enzymes (e.g., phosphatases, kinases) may act during tissue processing and impair detection or quantification of unstable phosphoprotein biomarkers.

In our study, we used mouse brain tissue to apply and evaluate the impact of a heat stabilization method on the level of specific phosphoproteins. Heat stabilization was applied immediately after sample excision. The effect of stabilization type and time on phosphoprotein levels was evaluated using a BioPlex phosphoprotein assay. This is a multiplex bead based assay (xMAP technology) that allows quantification of the phosphorylated and total proteins in lysates from tissue samples. We compared the heat-stabilized and paired samples.

Our results showed that for snap frozen samples, the protein extraction yields were comparable in both buffer systems, while for heat-stabilized samples, the results were significantly different, with total protein yields being lower in heat-stabilized samples. However, the level of specific phosphoproteins was significantly higher in heat-stabilized samples as compared to the corresponding snap frozen samples, indicating the feasibility and phosphoprotein preservation ability of this tissue processing method. Our results also demonstrated significant differences between the measured phosphoproteins in heat-stabilized and snap frozen tissue, suggesting alterations within the short timeframe of snap freezing and stabilization.

Our results highlight the possibility to use tissue heat stabilization as a processing method in view of downstream phosphoprotein analyses, but also suggest the Bioplex phosphoprotein assay as a possible quality control method to assess tissue sample integrity.

1 Integrated Biobank of Luxembourg, Luxembourg 2 Paterson Institute for Cancer Research, Manchester, United Kingdom 3 Moffit Cancer Center, Tampa, Florida 4 University of California, San Francisco, UCSF AIDS Specimen Bank, San Francisco, California 5 UZA Antwerp Hospital, Edegem, Belgium 6 ISBER, Bethesda, Maryland 7 Michigan Neonatal BioTrust, Detroit, Michigan 8 IRCCS San Raffaele, Rome, Italy 9 Specimen Solutions, Tucker, Georgia 10 SeraCare LifeSciences, Gaithersburg, Maryland 11 Karolinska Institute, Stockholm, Sweden 48. ISBER Proficiency Testing Program for Biorepositories: Pilot Results for DNA and RNA Schemes

ISBER has developed a proficiency testing (PT) program for biorepositories in partnership with IBBL. The PT program allows biorepositories performing quality control assays/ characterization of the biospecimens to assess the accuracy of their testing and to compare their results with those obtained in other laboratories around the world. For biorepositories that want to pursue accreditation, the ISBER PT program will provide a necessary external quality assessment tool.

All the standard operating procedures have been written according to the requirements of the ISO17043 norm, reviewed by the ISBER PT Advisory Group and approved by the ISBER PT Coordinating Body. Specific software was configured for the needs of the biorepository PT schemes. The first two schemes that were launched in 2011 are “DNA Quantification and Purity” assessment and “RNA Integrity” assessment. The PT pilots on those schemes were successfully run in April 2011 among ISBER members of the Biospecimen Science Working Group. For the DNA Quantification/Purity scheme, the assigned value was 53.03ug/ml and all participants obtained /z/ < 1 scores. For the DNA ratio, the assigned value was 1.86 and all participants obtained /z/ < 1 scores. For the RNA Integrity scheme, the assigned value was RIN 8.76 and 66.6% of participants obtained /z/ scores < 1, 22.2% of participants obtained 1 < /z/ < 2 scores and 11.1% obtained 2 < /z/ < 3 scores.

The DNA Quantification/Purity and RNA Integrity schemes are open to all interested laboratories globally. Two additional schemes (Cell Viability and Tissue Antigenicity) will be added in 2012.

1 CryoXtract Instruments, LLC 2 Harvard Medical School, Boston, Massachusetts 53. Robotic Frozen Sample Aliquotter

Biological samples are invaluable resources that advance translational research, molecular medicine and biomarker discovery. It is imperative to protect sample integrity long term lest a study's results be compromised.

Banked samples typically are cryopreserved to protect their fidelity during long term storage; yet, exposing samples to repeated freeze-thaw cycles to prepare aliquots may compromise their fidelity. Sample thawing and mixing during aliquotting are manual and time-consuming. Biobanks face critical cost/efficiency trade-offs between freezing and storing samples in larger volumes (exposing them to freeze-thaw cycles) or in a larger number of lower-volume cryotubes (increasing storage, labware, and maintenance costs).

The robotic Frozen Sample Aliquotter enables the hands-free extraction of multiple frozen aliquots from a single frozen sample—allowing biobanks to distribute aliquots of valuable samples (e.g., plasma, serum) without thawing the parents repeatedly, and to preserve sample molecular fidelity and maximize sample useful life. Eliminating a number of freeze-thaw cycles enables sample storage in larger volumes up front, which improves laboratory efficiencies (e.g., reduced costs, storage space requirements).

The fully-automated platform delivers hands-free operation (tube management, tube de-capping and capping, sample coring, etc.) after loading samples into the system. The system maintains thermal control of the samples at ultra-cold temperature (−70 ^oC and below) throughout processing. Any sample material remaining after processing may be returned to storage still frozen.

Independent evaluations have demonstrated the technology's ability to consistently extract homogenous, uniform frozen aliquots representative of parent samples, and to eliminate carryover between samples. This presentation will describe the current generation of the technology and the most recent validation data.

The Automated Frozen Sample Aliquotter supports the needs of modern biobanking, strengthening a biobank's ability to fulfill its core mission to protect sample integrity and deliver quality samples for analysis, while improving laboratory efficiencies through automation.

1 Clinical Research Directorate/CMRP, SAIC-Frederick, NCI-Frederick, Frederick, Maryland 2 National Cancer Institute, Office of the Director, Coordinating Center for Clinical Trials, National Institutes of Health, Bethesda, Maryland 56. NCI Funding of Biomarker, Imaging, Quality-of-Life, and Cost-Effectiveness Analysis Studies

NCI's purpose for the Biomarker, Imaging, & QOL Studies Funding Program (BIQSFP) is to ensure that the most important, scientifically meritorious biomarker, imaging, quality of life studies and/or cost-effectiveness analyses (CEA) can be initiated in a timely manner in association with NCI clinical trials. The aim of the program is to fund studies embedded in selected large, randomized Phase 2 therapeutic trial concepts with a control arm and an integral study(ies), along with integral and/or integrated studies associated with Phase 3 therapeutic trials, cancer prevention trials, and primary symptom management trials.

The overall goal of the program is to provide opportunities to enhance clinical outcomes by validating targets, reducing morbidity, predicting treatment effectiveness, facilitating better drug /trial design, identifying populations that may better benefit from treatment, improving clinical trial accrual and retention, and modifying standards of care. BIQSFP applications are accepted from NCI Cooperative Groups and Community Clinical Oncology Programs (CCOP).

Since 2009, 26 BIQSFP funding applications have been reviewed with most evaluations via NCI's Scientific Steering Committees (http://ccct.cancer.gov/). Eighteen studies have been approved for BIQSFP funding with several others in evaluation. The approved studies have included both integral and integrated components (assays/tests/QOL tools). Integral components must be performed in order for the trial to proceed. Integral studies have the highest funding priority. Integrated components are clearly identified as part of the clinical trial from the beginning, are intended to identify or validate (in the patient population of interest) assays/tests/QOL tools that are planned for use in future trials, are performed in real time, and include complete plans for specimen collection, laboratory measurements, and statistical analysis. Integrated studies in general should not be exploratory but rather should be designed to test a hypothesis, not simply to generate hypotheses.

BIQSFP information presented in the poster includes scope of activity, process for application, and funded projects (http://biqsfp.cancer.gov/).

Funded by NCI Contract No. HHSN261200800001E.

1 Basic Biobehavioral and Psychological Sciences Branch, Behavioral Research Program (BRP), Division of Cancer Control and Population Sciences (DCCPS), National Cancer Institute (NCI) 2 Host Susceptibility Factors Branch, Epidemiology and Genomics Research Program (EGRP), DCCPS, NCI 3 Office of the Associate Director, EGRP, DCCPS, NCI 4 Office of the Associate Director, BRP, DCCPS, NCI 5 Office of Administrative Operations, Office of Extramural Research, Office of the Director, National Institutes of Health, Bethesda, Maryland 57. The Use of Biospecimens in Behavioral Research: A Portfolio Analysis of Research Supported by the Division of Cancer Control and Population Sciences at the National Cancer Institute

The mission of the Division of Cancer Control and Population Sciences (DCCPS) at the National Cancer Institute is to reduce the risk, incidence, and deaths from cancer as well as enhance the quality of life for cancer survivors. To achieve this mission, the division conducts and supports an integrated program of the highest quality genetic, epidemiological, behavioral, social, applied, and surveillance cancer research. The Behavioral Research Program (BRP) in DCCPS initiates, supports, and evaluates a comprehensive program of research ranging from basic behavioral research to the development, testing, and dissemination of interventions in areas such as tobacco use, screening, dietary behavior, and sun protection. Over the past five years, BRP has contributed between 34% and 38% of the division's funded portfolio. However, the extent to which biospecimens (e.g., blood, urine, saliva, tissue) and biodata are collected, used to address specific aims, stored, and shared in behavioral research projects funded through BRP is unknown. This descriptive portfolio analysis sought to address these gaps, determine the use of best practices, and explore the need for data harmonization. Preliminary analysis of research program projects funded between FY 2006 and 2010 revealed that 14% of grants coded for human subjects included the collection of biospecimens. Blood (58%) and saliva (35%) were the most common biospecimens collected. Biospecimens were often collected in research projects classified as biobehavioral in scope or in tobacco control. Results from more comprehensive analyses of the behavioral research portfolio and implications for biospecimen resource support will be reported.

1 Pediatrics and Child Health, Washington, DC 2 Biospecimen Repository/Biomedical Informatics Core, University of Hawaii 3 RCMI Translational Research Network and the University of Hawaii 4 Proteomics Core, Meharry Medical College Micro & Immunology, Nashville, Tennessee 58. Sharing a Model Consent Form to Increase Enrollment of Ethnically Diverse Populations in Research and Biospecimen Repositories

Enrollment of ethnically diverse participants is vital to advancing disparities research. Further, clear understanding of research participation, including opportunities and risks, is critical for true population engagement. As a consortium of 18 Research Centers in Minority Institutions (RCMI) grantee institutions, RCMI Translational Research Network (RTRN) members are instrumental in the recruitment of ethnically diverse communities in disparities studies. However, the consent process is often lengthy, leaving potential participants overwhelmed and disinterested, thus reducing enrollment. Consent for use of biospecimens in future studies presents another layer of required comprehension that may be compromised if embedded amongst the verbose narrative of a long consent form. More importantly, lack of comprehension clearly undermines the participant's ability to voluntarily provide consent. Though consent documents to participate in biospecimen repositories vary among Consortium members, they agree that simple and concise language is critical to ensuring that participants' agreement to enroll in research is informed, understood, and voluntary. Therefore the National Human Genome Center (NHGC) at Howard University (HU) developed a concise yet informative two-page consent document that can serve as a template for consent for research studies and biorepositories. The two-page consent form increased the collection of biological specimens, as well as epidemiological, family history, and medical data among African Americans at HU. These specimens are stored for use in future studies and hold tremendous promise for elucidating factors related to human disease, particularly related to cancer and diabetes, which disproportionately occur in communities served by RCMI institutions. By sharing its two-page form with RTRN members, the NHGC provides a model for ensuring human subjects protection pursuant to federal regulations through a process that effectively increases enrollment of ethnically diverse populations. Ultimately, such an increase in enrollment will enhance the quantity and quality of biospecimens available for research dedicated to reducing health disparities.

1 Washington University in St. Louis School of Medicine, Division of Public Health Sciences 2 Alvin J. Siteman Cancer Center 3 Washington University in St. Louis, George Warren Brown School of Social Work, St. Louis, Missouri 60. Participation in Biospecimen Research – Investigating the African American Male Perspective

Background: Research consistently demonstrates that the participation of minorities in Biospecimen (BsP) research is considerably low, particularly among African American men. Biospecimen science holds great potential for addressing concerns around healthcare disparities. Increasing participation among diverse audiences, such as African American men, provides researchers with necessary clinical and molecular data that can advance knowledge of prevention and treatment methods. To increase understanding around the barriers that African American men face when contemplating participation in such studies, focus group data was used to analyze health behavior themes.

Methods: Using convenience sampling, 70 African American men, between the ages of 40 and 80, were identified to participate in focus groups. Demographic data was collected and to control for group differences, individuals were assigned to groups based on their history of prostate cancer and educational background. Groups were held at community locations and lasted approximately an hour and a half. Using a semi-structured questioning route participants were asked about their preexisting knowledge, past experience, and general attitude around BsP research. Moreover, they were also asked to identify key factors that they believe promote or hinder participation in such studies.

Results: The average age of participants was 56 and most (94%) indicated they had never participated in any study where they gave a biological sample. Barriers were related to a number of social and behavioral themes, such as mistrust, lack of knowledge, interpersonal concerns, and social influences. Men commonly reported that they lacked knowledge of BsP studies and were never approached to participate in them. Physician endorsement increased their willingness to participate. Moreover, many of the men reported that if they clearly understood that participating in BsP research would “help someone down the line” or result in advancement in cancer care, treatment or prevention, they would more likely participate.

1 Consultant in Bioethics and Research Strategy, Bethesda, Maryland 2 Consultant in Research and Clinical Bioethics, Bethesda, Maryland 61. Sharing Results With Research Participants: Overcoming Obstacles

Research subjects who consent to collection of specimens for research often ask about whether their specimen characteristics and analytic data will be accessible to them. They understand that, although useful information is not available when specimens are collected, future research may render the information from specimens useful for themselves or others. They want to be informed when there is a possibility of clinically useful information, e.g., information that may be used to identify new clinical trials or personalized therapies.

Recent studies have shown that a drug to inhibit Janus kinase (JAK) activity is undergoing testing because of the strong association of certain of the mutations of JAK with better outcome treatment of B-cell Lymphomas (ClinicalTrials.gov Identifier NCT01431209).¹ Another study of BRAF mutations indicated that “ Kinase inhibitors targeting BRAF may be effective therapeutic options in a subset of gastrointestinal cancers.²

The 2011 revision of NCI's Best Practices for Biospecimen Resources acknowledges data and specimen sharing, but only with other investigators. They are silent on providing feedback or on giving information to research subjects at a future time.

The 2010 NCI Workshop Report, Release of Research Results to Participants in Biospecimen Studies,³ lays out and analyzes pros and cons of making such information available along with conditions that warrant its disclosure.

Research participants, during the consent process, will need to be informed of possible uses of materials. It must be made clear that although no biomarkers are identifiable at the present, should the research find potentially useful biomarkers in the future, that information will be accessible through scientific publications, patient advocacy groups, and/or clinicaltrials.gov or a cancer information database.

We recommend that NCI explore using clinicaltrials.gov to provide key words and summary information on studies involving specimen-related data. This would permit communication about early results of genomic analyses and allow research subjects to pursue those results through publications and other media.

As is done currently, the structure of the database would include educational material and tutorials on how to get information. The database would be searchable and include information on molecular markers and genomics so users might identify early clinical trials as well as more established ones.

References

1. Dr. Louis M. Staudt, deputy chief of the Metabolism Branch in NCI's Center for Cancer Research.

2. Novel V600E BRAF Mutations in Imatinib-Naive and Imatinib-Resistant Gastrointestinal Stromal Tumors Narasimhan P. Agaram, Grace C.Wong, Tianhua Guo, Robert G. Maki, Samuel Singer, Ronald P. DeMatteo, Peter Besmer, and Cristina R. Antonescu1, GENES, CHROMOSOMES & CANCER 47:853–859 (2008,

3. http://biospecimens.cancer.gov/resources/publications/workshop/rrra.asp, accessed January 7, 2012.

1 University of Cincinnati Cancer Institute Tumor Bank 2 University of Cincinnati Department of Internal Medicine, Cincinnati, Ohio 62. Standardization of Consent Practices at the University of Cincinnati Cancer Institute Tumor Bank: Challenge in Biobanking

While intense labor is focused on biospecimen processing and storage conditions to obtain high quality samples, standardization of consent practices may be overlooked. To unify our banking procedures for multiple PI-initiated projects, the University of Cincinnati Cancer Institute Tumor Bank (UCCITB) developed and implemented standard operating procedures (SOP), not only governing all aspects of sample handling but also addressing subject consenting. Due to internal staffing limitations, we encourage medical personnel to consent their subjects. Our challenge is to expand banking efforts by involving more people in consenting while maintaining high quality consenting practices. In order to implement internal consenting procedures among clinical staff, we utilized various resources to introduce and follow-up on consent practices. First, consent documentation for new and existing banking projects was standardized. Providing a uniform presentation of critical information and questions for the subject ensures collection of key subject preferences regarding usage of biospecimens and associated protected health information. The standardized consent form was approved by the University of Cincinnati institutional review board. Second, a user-friendly one-page “Consenting Quick Reference” was developed to reinforce guidelines for appropriate consenting practices. This form was disseminated by e-mail to all consenters and published in our monthly electronic newsletter. It emphasizes how to identify and approach a potential participant, what information must be presented during consenting and how to keep standardized record notes in the electronic medical record. These procedures allow us to efficiently monitor newly consented subjects and alert all clinical staff about patient enrollment in specimen banking. Third, we developed an internal audit checklist for assessing compliance with our consenting SOP. Ultimately, excellent communication between clinical staff and tumor bank personnel is the key to successfully implementing our consenting SOP. Strong working relationships are maintained by daily interactions and our annual UCCITB meeting.

1 University of Cincinnati Cancer Institute Tumor Bank 2 University of Cincinnati Department of Internal Medicine, Cincinnati, Ohio 63. Improving Biospecimen Collection Practices at the University of Cincinnati Cancer Institute Tumor Bank

The University of Cincinnati Cancer Institute Tumor Bank (UCCITB) was established to collect, store and distribute human tumor, paired normal tissue, blood and other biofluid to basic, translational and clinical research. Specimens are collected from individuals with malignant diseases seen at the UC medical campus. De-identified medical and pathology information for all participants are available to researchers in strict compliance with privacy rules. All tissues are collected and stored as flash frozen cryovials and/or freezing media molds at −80 °C and/or in formalin fixed paraffin embedded blocks. Blood is collected in acid citrate dextrose and sodium citrate vacutainer blood tubes and stored as whole blood, plasma and nucleated cell pellet at −80 °C. To insure high specimen quality, UCCITB implemented a sample processing sheet where six time points from initial excision time to storage time are being recorded for each specimen. Through compiled data analysis, we were able to assess and address different issues and improve in several areas. As a result, we developed a specimen banking form with integrated labels that helped us accomplish several goals: (1) facilitate participant identification for specimen banking by medical staff at the clinics, operation rooms and pathology services which resulted in less confusion and better communication, (2) include brief instructions in the form for staff who may be unfamiliar with CITB procedures. This limited specimen mishandling, sped up sample processing and improved specimen quality, (3) the form encloses important information related to the sample handling which improved the CITB quality control and database, and (4) detachable labels ensured proper labeling and limit sample lost. To further address the problem of delay of tissue processing, CITB developed a travel processing kit to process tissue on site. This kit has allowed us to shorten tissue processing by 22 minutes on average.

1 Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 2 BioFortis Inc., Columbia, Maryland 65. Labmatrix: A Collaborative Research Information Management System at the NCI

Translational research programs have been created to provide a better understanding of disease susceptibilities and pathway mechanisms. These programs require integration and analysis of multiple disparate data sets that come from different sources such as the hospital and pathology/molecular laboratories. At the NIH, a subset of the patients treated at the Clinical Center consent into various Institutes' research protocols. Here we describe how intramural NCI investigators utilize a secure, centralized database system that (1) stores, annotates, curates, and tracks information such as patient data, biospecimen data, and experimental research data, (2) exchanges clinical and research information with other NIH data systems, (3) enables collaborative sharing of information amongst designated research groups, and (4) provides an intuitive environment for investigators to query and review all collected data with minimal need for direct IT support.

1 Clinical Monitoring Research Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 2 Research Data and Communication Technologies Corp., Garrett Park, Maryland 3 Malaria Research and Training Centre, University of Bamako, Bamako, Mali 4 Medical Science and Computing, Inc., Rockville, Maryland 5 Rakai Health Sciences Program, Kalisizo, Rakai, Uganda 6 Rakai Health Sciences Program, Uganda Virus Research Institute, Kalisizo, Rakai, Uganda 7 Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 67. Establishing FDA-Compliant Electronic Biospecimen Management and Environmental Monitoring Systems to Support Biomedical Research in Uganda and Mali

Regions endemic for HIV, TB, malaria and neglected tropical diseases frequently overlap with resource-constrained, geographically remote and harsh climate settings. The National Institute of Allergy and Infectious Diseases (NIAID) has developed and sustained international biomedical research programs in resource-constrained countries through partnerships with local scientists to facilitate training, improve laboratory and clinical infrastructure, and enhance information technology capacities. In accordance with FDA guidelines and NIAID policies, the NIAID's Office of Cyber Infrastructure and Computational Biology (OCICB) and NIAID intramural scientists have implemented 21 CFR compliant electronic biospecimen management systems (eBMS) for inventory, tracking and custodianship of human biospecimens; and electronic environmental monitoring systems (eEMS) for monitoring biorepositories at research sites in Mali and Uganda. The design of these systems required special consideration for the environments where they are located as both Mali and Uganda frequently experience unreliable delivery of basic infrastructure, such as phone and electricity. These systems required extra consideration of redundancy, use of wired systems if possible to avoid loss of alerting due to wireless network interruption and alerts sent through both SMS mobile networks and SMTP on Internet connections. Investing in and implementing these electronic systems has provided high-quality monitoring of biospecimens and biorespository facilities which in turn have provided scientists with quality assured biospeicmens for research.

1 Alberta Innovates Centre for Machine Learning, Computing Science, University of Alberta, Edmonton, Alberta, Canada 2 Canadian BioSample Repository, University of Alberta, Edmonton, Alberta, Canada 68. Open-Source Software for Sample Inventory

Introduction: The Canadian BioSample Repository (CBSR) has processed and stored biological samples since 2000. In 2008, CBSR began developing a comprehensive client-server Java application, named Biobank, to be used by nurses, lab technicians, researchers, and lab administrators. Nurses can enter samples into the system, technicians can process and transfer samples, researchers can request samples and view inventory information, and administrators can create comprehensive queries and manage users. Biobank is open source and free software, modelled on caTissue from the National Cancer Institute.

Methods: A team at the Alberta Innovates Centre for Machine Learning has programmed Biobank using an iterative approach with feedback from lab technicians at CBSR. Samples are assigned standards compliant machine-readable barcodes to minimize human errors and improve efficiency. Lab technicians use a downloadable Microsoft Windows thick-client that works with various flatbed scanners to scan sample barcodes.

Results: The software maintains chain of custody information, tracking samples from collection, through shipping, receiving, processing, storing, and retrieving. It allows user rights access, role configuration, and security. Custom reports can be created easily by administrators. User actions are audited, logged, and easily queried for regulatory purposes. Work is currently underway to integrate BioBank with TECAN EVO Freedom robots to automate sample processing. A web client, under development, will allow researchers and lab administrators access to BioBank's inventory information. Grid data services will allow sharing of inventory data between installations.

Conclusion: Over the last three years Biobank has been used and validated by technicians at CBSR, thirteen collection sites and three processing centres in North America and Europe. CBSR has partnered with the Ontario Health Study, in Toronto, Canada, who have assembled an additional programming team to implement further functionality. The Livestock Gentec team at the University of Alberta Faculty of Agriculture is currently implementing Biobank to track bovine samples.

Funding by Baxter BioSciences, Novo Nordisk, CSL-Behring, and Pfizer Ltd.

1 Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Rockville, Maryland 2 Department of Genitourinary Pathology, The Joint Pathology Center, Silver Spring, Maryland 3 Urology Service, Walter Reed National Military Medical Center, Bethesda, Maryland 69. Multidisciplinary Resource Informatics and Their Interactions

The dynamic, massive and multidisciplinary data involved in translational cancer research demands sophisticated and innovated information management strategies, which facilitate the collection, process, storage, transmission, and annotation within research arena. More importantly, the data integration and interactions should be addressed systematically to enhance disciplinary study design and knowledge sharing. CPDR's Integrated Biomedical Informatics Platform (IBIP) has set up Resource Informatics (RI) for each research domain: clinical care, biospecimen/biorepository, molecular bioinformatics, which (1) streamlines data management in research program with robust and reliable information systems, tools and applications; (2) establishes program-specific policies, standards and SOPs on information management, regulatory adherence, information security, privacy protection, etc.; (3) establishes infrastructure and framework to enhance platform-wide data search, integration, and information sharing among RIs. Multidisciplinary RIs, implemented and regulated by IBIP policies and standards, offer systematic, dynamic and seamless multidisciplinary data service and quality control mechanism. Multi-level user and application interfaces can be easily deployed on RIs to enhance user accessibility and interactions.

1 Department of Biomedical Informatics, University of Pittsburgh 2 Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 70. National Mesothelioma Virtual Bank: A Standardized Data and Specimen Resource to Expedite Translational Research

Background: The National Mesothelioma Virtual Bank (NMVB) was developed in 2006, and has been continuously supported by a grant from the CDC/NIOSH. Original collaborators include New York University, University of Pennsylvania, and the coordinating center, the University of Pittsburgh. In late 2011, the NMVB expansion included Mount Sinai School of Medicine. Each academic health center gathers clinically annotated biospecimens relevant to human mesothelioma research. Over this period of time, this robust resource has greatly increased its collection of specimens and has provided hundreds of tissue samples along with the associated data to investigators.

Design: The NMVB architecture consists of three major components: (a) standardization of data and specimen collection (b) collection of multi model dataset and mesothelioma specimen (c) on line data query tools for research investigators. In addition, marketing efforts have included presentations at both national and international conferences in order to expand the awareness and availability of the tissue and data bank. Furthermore, three Tissue Microarrays (TMAs) have been created at three separate academic health centers to provide for robust research.

Result: The NMVB database provides researchers real-time, interactive access to richly annotated mesothelioma specimens. The data disclosure and specimen distribution protocols are tightly regulated to maintain compliance with participating institutions' IRB and regulatory committee reviews. The NMVB currently has over 984 annotated cases available for researchers, including paraffin embedded tissues, fresh frozen tissue, three distinct tissue microarrays, blood samples and genomic DNA.

Conclusion: The National Mesothelioma Virtual Bank (NMVB) is a virtual biospecimen registry with robust translational biomedical informatics support to facilitate basic science, clinical, and translational research. Furthermore, it protects patient privacy by disclosing only de-identified data, making biospecimens readily and efficiently accessible to researchers free of charge.

1 Central Operations and Data Coordinating Center, AIDS and Cancer Specimen Resource 2 Mid-Region ACSR, AIDS and Cancer Specimen Resource 3 East Coast ACSR, AIDS and Cancer Specimen Resource 4 West Coast ACSR and Central Operations and Data Coordinating Center, AIDS and Cancer Specimen Resource Background 71. Informatics Solutions for Management of Unique Specimen Collections at a Multisite Biobank: The AIDS and Cancer Specimen Resource Experience

The AIDS and Cancer Specimen Resource (ACSR) was established by the National Cancer Institute in 1994 as a multi-center repository for biospecimens from patients with HIV-associated malignancies. The Resource has evolved to include access to large HIV-related specimen and data collections including AIDS-related cancer cohort studies. Coordination and integration of these “special collections” into the Resource has posed a unique challenge for database development and management.

Methods: ACSR Central Operations and Data Coordinating Center (CODCC) Standard Operating Procedures and Manuals of Operation were reviewed along with Material/Data Transfer Agreements, and Human Subjects approvals that govern each “special collection.” Information was abstracted to manage database integration of “special collections” data.

Results: We identified (1) common and unique data elements needed to effectively and efficiently merge “special collections” into the Resource database and; (2) rules required to control access, sharing and disbursement of “special collection” samples and data. Thus, database modifications will include elements for collection name, custodian, and dates. Individual collection-specific flexible rules will be overlayed to lock, share, collaboratively share and grant/revoke access to specimens individually or in defined groups.

Conclusions: A key responsibility of the ACSR CODCC is to develop and maintain the Resource database to coordinate the receipt and disbursement of biospecimens and data. The “special collections” are an added-value unique to the ACSR that allows researchers an opportunity to access comprehensive established collections. Development of a pre-configured specimen collection file within the new ACSR biospecimen management system that incorporates collection-specific elements and flexible rules will ensure improved access to current “special collections” and the ability to add future collections. This will increase the ACSR's effectiveness in supporting AIDs-related cancer research over the long-term and in initiating collaborative projects that leverage invaluable established resources and the expertise of researchers who are custodians of these collections.

1 Susan G. Komen for the Cure, ^® Tissue Bank at the Indiana University Simon Cancer Center and Division of Breast Surgical Oncology, Indiana University School of Medicine 2 Susan G. Komen for the Cure, ^® Tissue Bank at the Indiana University Simon Cancer Center and Indiana University School of Medicine 3 Department of Computer and Information Technology, Purdue University 4 Department of Computer and Information Technology, Purdue University and Bindley Bioscience Center in Discovery Park, Purdue University 72. Toward a Virtual Tissue Bank

To exponentially increase its utility as the world's only repository of normal breast tissue and matched serum, plasma and DNA, the Susan G. Komen for the Cure^® Tissue Bank at the Indiana University Melvin and Bren Simon Cancer Center (Komen Tissue Bank, or KTB) through the generous support of Oracle Giving and partnering with the Department of Computer and Information Technology at Purdue University is creating a virtual tissue bank (VTB) that will enable researchers from around the globe as they seek the cures and prevention of breast cancer. The KTB's VTB will serve as an “Omics” (Genomics, Proteomics, Metabolomics, and others) experimental data repository linked to anonymized annotations regarding the donor including demographics, medical history and breast cancer risk data. Also available will be image data, for example, digital mammograms, and hematoxylin and eosin stained sections of the tissue viewed with the aid of a virtual microscope. With the goal of enabling in silico experimentation, the VTB uses a “best of breed” approach that leans heavily on de facto standards promulgated by repositories such as the Gene Expression Omnibus; furthermore, the VTB couples this approach with a strong commitment to reduce information redundancies by streamlining the storage of content where appropriate and linking to curated resources such as PubMed. Initial data will include RefSeq data generated from normal breast epithelium, gene expression data from cell lines derived from the breast tissue, and serum metabolomics. In short, the VTB serves to maximize the KTB's specimens by extending the specimens' value beyond their physical dimensions.

1 Office of Rare Diseases Research, National Institutes of Health, Bethesda, Maryland 2 Office of Rare Diseases Research, ICF International, Rockville, Maryland 73. Accelerating Research for Rare Diseases: Rare Diseases Human Biospecimens RD-HUB

Rare diseases affect about 19-25 million people in the United States. Despite the health and financial burden on society, rare diseases are not given sufficient attention or funding for the development of needed research resources, and development of new therapies, leaving patients desperate for help. In addition to the general challenges associated with biospecimens of common diseases, rare disease biospecimens encounter many unique and difficult challenges specifically because patients with rare disease are dispersed over large geographical areas and the lack of sufficient specimens due to the rarity of the diseases. Thus, identifying and locating sources for these specimens is crucial for advancing research in rare diseases. To alleviate some of these problems and accelerate research in rare diseases the Office of Rare Diseases Research (ORDR) established the Biospecimen/Biorepositories: Rare Disease-HUB (RD-HUB) database and Web site: http://biospecimens.ordr.info.nih.gov/.

The main goals of RD-HUB are:

• To serve as a portal for researchers to locate and inquire on the availability of rare diseases specimens from existing biorepositories around the globe.

1 Biospecimen Repository/Biomedical Informatics Core, University of Hawaii 2 RCMI Translational Research Network, University of Hawaii 3 Microarray/Bioinformatics and Proteomics Core, Meharry Medical College 4 Santi, National Human Genome Center, Howard University 5 Genomics Core, Clark Atlanta University 6 Obstetrics and Gynecology, Meharry Medical College 7 Biomedical Informatics Core, University of Hawaii 74. A Biospecimen Repository Community of Best Practices Across Ethnically Diverse Institutions to Facilitate Informatics Solutions in Biospecimen Science

Maintaining high-quality biospecimen repositories (BSR) at institutions serving ethnically diverse populations is vital to ensure that such populations have ample opportunities to participate in and to drive emerging scientific innovation, particularly for diseases they disproportionately experience. Geographically dispersed institutions, funded by the NIH's Research Centers in Minority Institutions (RCMI) Program and facilitated by the RCMI Translational Research Network (RTRN), have formed a Community of Best Practices (CoBP) focused on BSR to share lessons learned about the maintenance of and informatics solutions for BSR. RCMI grantee institutions have historically faced significant resource limitations that hindered the development of robust BSR and informatics technical infrastructure to support clinical and translational science. Additionally, BSR at RCMI schools were often developed to support a specific study and were not driven by a vision that biospecimens would be used beyond that particular study. Such evolution of BSR often resulted in ad hoc, non-informatics-informed databases for specimen management, with limited ability to share specimens with outside researchers. RTRN and its BSR CoBP have catalyzed the ability of RCMI grantees to share resources and solutions, creating synergy for researchers using BSR to support their research programs. Collaborative informatics resources, such as RTRN's Data and Technology Coordinating Center (DTCC), promote efficient, distributed informatics solutions that can standardize BSR informatics practice in participating RCMI institutions, thereby increasing access and enabling more efficient management, tracking and dissemination of specimens. With prioritizing conversion of ad hoc legacy systems, adopting shared ontologies for repository information development, expanding BSR informatics best practices, and creating efficient specimen inventory tracking and control, the RTRN BSR CoBP and the DTCC are able to pool experience and enhance capacity across institutions to increase ethnically diverse population participation in clinical and translational science of particular relevance to the communities they serve. [Grant support from U54RR026136 and U54RR022762.]

1 University of Minnesota, Minnesota 2 Rajiv Gandhi University of Health Sciences, Bangalore, India 78. Development of Biorepositories and Translational Science Facilities Under the Aegis of Rajiv Gandhi University of Health Sciences

There is considerable interest among researchers in developing early markers, both genetic and molecular, and using them for clinical studies. According to the National Cancer Institute (NCI) of the National Institutes of Health (NIH), USA, “one of the most widely recognized and significant roadblocks to progress in cancer research is the lack of standardized, high-quality biospecimens.” If a state-of the-art biorepository is established in Bangalore, India, with international protocols, and NCI best practices procedures in place, it would provide a wealth of standardized clinical samples for all kinds of studies. The genetic diversity as well as cancer diversity in India is huge. We see a lot of collaborative projects spawning from such a platform. Kidwai Memorial Cancer Institute of Oncology (a national platform for oncology), Bangalore, has already established such multicenter collaborations with the Indian Institute of Sciences, National Center for Biological Sciences, St John's Medical Academy, Bangalore and prestigious Johns Hopkins University, USA and generated useful information on genomics and proteomics of squamous cell carcinoma. We are also contemplating establishing a biomarker development facility, to complement this effort. Furthermore, efforts are underway from RGUHS, to help develop centers of excellence in allied sciences, encourage collaborative research between various research institutions, in the State of Karnataka and build a common translational science platform, to promote the development of science from bench to clinic. An expert committee has been formed to coordinate these efforts. In this overview, we will present strengths and weakness of the programs in India and present a strong case for international collaboration to develop needed bio-repositories and translational science centers in India.

1 Erasmus MC Rotterdam, The Netherlands 2 Fondazione IRCCS Istituto Nazionale Dei Tumori 3 Karolinska Institute, Sweden 4 European Organisation for Research and Treatment of Cancer, Belgium 5 International Agency For Research on Cancer, France 6 Dutch Cancer Institute, The Netherlands 7 Oslo Universitetssykehus HF, Norway 8 Fundacion Centro Nacional de Investigaciones, Spain 9 Fundacion Instituto Valenciano de Oncologia, Spain 10 The Christie NHS Foundation Trust, United Kingdom 79. The Next Chapter of Sharing Medical Translational Cancer Research Samples in Networks: The EurocanPlatform Biobanking Approach

Sharing samples between hospital integrated biobanks is actually needed to perform medical translational cancer research with statistical significant impact on innovation of patient care. However, sharing samples is seen as a “no-go” area for most investigators, afraid to lose their institutional and departmental investment of resources. Biobanks have tried to set up networks enabling scientists to find samples they need. However, the enthusiasm to upload sample data is not always shared happily without knowing the benefits in advance. Especially in case there are language barriers, annotation problems, regulatory difficulties and quality issues to overcome.

The aim of the European project EurocanPlatform is to set up a European translational cancer research platform. Since biobanking is one of the major elements for such an infrastructure, it has become one work package within the project.

This project will involve bundling of good existing initiatives with alternative approaches developed in areas where obstacles are suspected. Working on awareness, motivation and even influencing work environments of primary investigators, collectors and patients through patient representative groups have become key targets. For instance: writing a letter of intent to boards of cancer centers and academic hospitals in which the effects on willingness to exchange samples is described on quality issues, institutional access rules and the use of Impact Factors to evaluate scientific achievement. Furthermore, distillation and publication of possible win-win situations in favour of both PIs and collectors can make both parties aware of opportunities.

The existing OECI-TuBaFrost project is adapted to serve the EurocanPlatform infrastructure needs. A biobank locator enables searching and contacting biobanks having interesting samples. The role of PIs involved in local research on the samples is enhanced. Identified banks can join in a closed project sample exchange platform, where only data uploads of samples that might be used in the project is needed.

1 City of Hope 2 California State University, San Bernardino, California 80. COH BrTM Registry: A Biospecimen Resource for Translational Research

Background: The NCI has cited several challenges with biorepositories, including clinical and pathologic data annotation and tracking of preanalytic variables. The City of Hope (COH) Circulating Breast Tumor Marker (BrTM) Registry collects peripheral blood samples from women with breast disease at the time of diagnosis and, for patients with malignancy, longitudinally through treatment and survivorship, recording long-term outcomes. We report compliance with strict standard operating procedures (SOPs) for the collection and processing of specimens for this biorepository.

Methods: Specimen data recorded include date and time of blood collection, time of centrifugation, and time of storage in −80 °C freezers and liquid nitrogen tanks. Patient demographic, tumor and treatment data are extracted onto standardized data forms and entered into a clinical database after QA review. These data are linked to the specimen tracking database. The elapsed time between blood collection, centrifugation and placement into freezer/tank was calculated for all blood components and compared to SOPs. Adequate specimen processing quality was defined as >80% compliance with each SOP measure.

Results: As of July 12, 2011, the COH BrTM Registry includes 666 women who have consented onto the study. Of these, 548 have donated blood samples, which were separated into a total of 16,019 biospecimens. Fully linked preanalytic data were available for 1,229 serum, 1,227 plasma, and 1,199 lymphocyte specimens. Adherence to all SOP requirements occurred >80% of the time. The biggest challenge was the strict clotting time for serum processing. Adherence to all other quality measures was >90%.

Conclusions: The COH BrTM Registry is a high quality resource that links serially collected biospecimens with demographic data and clinical outcomes to enable basic and clinical researchers to discover and validate diagnostic, prognostic, and predictive biomarkers for breast cancer.

1 Women's Health Integrated Research Center at Inova Health System 2 Gynecologic Cancer Center of Excellence 3 The Henry M. Jackson Foundation for the Advancement of Military Medicine 4 Walter Reed National Military Medical Center 5 Inova Fairfax Hospital 81. Biobanking in a Culture of Excellence

Purpose: The mission of the Biorepository at the Women's Health Integrated Research Center (WHIRC) is to provide researchers with well-annotated specimens of the highest quality for molecular analysis of gynecologic conditions including cancer.

Methods: We customized Freezerworks Sample Management Software (FW) to create a multi-dimensional database (MDMS) that provides barcoded annotation for aliquots, sub-aliquots, kits, components, projects and various types of data including clinical, imaging, epidemiologic and laboratory. Our consortium participates in multi-institutional clinical trials, banking protocols and research studies. Our collaborating institutions submit various types of data and specimens to the Data Management Center and the Biorepository at the WHIRC. Specimen kits are designed in a patient centric manner for individual protocols, and are distributed to our collaborating institutions. Specimen kits and components are tracked using MDMS, barcoded, and labeled with a de-identified Case ID. Sites batch ship frozen tissues, blood, serum and urine to the WHIRC monthly. Frozen tissues are grossed and undergo central pathology review within 1 month of receipt. DNA, RNA and microRNA are extracted from blood monthly. Serum and urine are processed and banked prior to testing. Barcoded specimens and associated clinical, specimen, quality control, quality assurance and laboratory data are readily imported and exported into and out of MDMS in batches. FW software has been customized by our team to create MDMS, which facilitates the integration and traceability of specimens and various types of data from study design through collection, receipt, processing, testing, analysis and publication.

Results: The Biorepository at the WHIRC has banked specimens from more than 1,500 women from at least 10 institutions to support at least 30 protocols and studies thus far, and is now gearing up to expand our operations to bank specimens from up to 900 patients annually to support and facilitate gynecologic research.

Conclusions: The Biorepository at the WHIRC provides well-annotated and high quality specimens to the Gynecologic Cancer Center of Excellence (GYN-COE) Consortium and the WHIRC for integrated molecular analyses as well as discovery, validation and mechanistic studies in endometrial cancer, ovarian cancer and other gynecologic conditions.

1 Department of Radiation Oncology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 2 X-Ray Science Division, Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 83. Radiobiological Tissue and Data Archive

Radiobiology studies in USA between 1950s and 1990s created large data and specimen archives. Thousands of mice, rats and dogs were exposed to external beam radiation with gamma rays and neutrons or internal emitters introduced by inhalation, intravenously, or through other routes. In vast majority of cases, moreover, animals were allowed to live out their complete lifespan, so that long-term effects of radiation could be evaluated. This type of country-wide effort is no longer feasible today, yet a full understanding of radiation exposure effects is an area of research still under investigation.

At this moment, most of the tissue and data from these experiments is housed at Northwestern University and is available to researchers interested in radiobiological studies. The data is freely accessible at the website http://janus.northwestern.edu, while specimens can be searched for using the same website and obtained by request. This approach to data and materials resource sharing will hopefully open a period of the re-exploration of this resource.

In addition to data and material sharing, our group is investigating the archive as well. For example, we are applying new statistical approaches on old datasets. Over the years, we have tested many different newly discovered approaches for investigation of formalin fixed paraffin embedded materials. Some of the samples at this time are hybridized against micro-RNA microarrays, while others undergo new types of histopathological analysis. We have found particular interest in using X-ray fluorescence microscopy for evaluation of elemental makeup of these samples. Two dimensional elemental mapping studies allow detection and quantification of both native biological elements and radionuclides present in the archival tissue samples.

1 Center for Prostate Disease Research (CPDR), Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, Maryland; Urology Service 2 Walter Reed National Military Medical Center, Bethesda, Maryland 3 Department of Urology, University Hospital, Rheinisch-Westfälische Technische University, Aachen, Germany 4 Department of Genitourinary Pathology, The Joint Pathology Center, Silver Spring, Maryland 84. Whole Mounted Prostate Specimen Bank Offers Unique Translational Research Opportunities in Prostate Cancer

Bio- and molecular specimen banks were established at CPDR since its inception in 1993, representing now a large repository of high quality specimens linked to clinico-pathological features of thousands of patients (Brassell et al., 2009). A vigorous translational research program has been built around these resources. The collection of over 1,700 whole mounted prostate specimens offer unique translational research opportunities in prostate cancer (CaP).

The ETS-related gene (ERG) proto-oncogene is frequently overexpressed in CaP as a result of a genomic rearrangement. Recently, ethnic differences in the frequency of the ERG fusion/overexpression have been noted.

Here we evaluate the frequency and pattern of ERG oncoprotein expression in whole mounted prostate specimens from rigorously matched Caucasian-Americans (CA) and African-American (AA) patients to better understand the biological basis for differences in CaP between the two populations. ERG oncoprotein was immunohistochemically detected by a specific ERG monoclonal antibody (clone 9FY). A higher percentage of CA (61.8%) than AA (28.2%) CaP patients had ERG positive index tumors and had at least one focus of tumor positive for ERG. There was high concordance of ERG positive prostatic intra-epithelial neoplasia (PIN) and ERG positive CaP in both AA and CA patients.

In conclusion, ERG expression is more prevalent among CA than AA CaP patients in matched cohorts. Differences in the pattern of ERG expression in CaP and differing trends in biochemical recurrence between CA and AA patients with ERG-positive index tumors suggest a dominant clonal selection of ERG-positive tumors in CA patients. These differences in ERG expression have the potential to delineate biological distinctions of CaP in the two patient populations with known differences in CaP incidence and mortality.

Taken together, our experience demonstrates the value of well annotated whole mounted prostate specimen bank in translational research.

1 Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 2 Department of Pathology, New York University School of Medicine, New York, New York 3 Department of Urology, New York University School of Medicine, New York, New York 4 Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland 85. Biobanking as Part of the Prostate Cancer Biorepository Network: A Focus on DNA, RNA, and Protein Derivatives From Radical Prostatectomy Specimens

The Prostate Cancer Biorepository Network (PCBN) is a collaboration between the Johns Hopkins University School of Medicine (JHU), the New York University School of Medicine (NYU), and the Department of Defense (DOD). The goal of the PCBN is to develop a biorepository with high-quality, well-annotated specimens obtained in a systematic, reproducible fashion using optimized and standardized protocols, and an infrastructure to facilitate the growth of the resource and its wide usage by the prostate cancer research community. One specific focus of the PCBN is to characterize critical parameters in the biospecimen “life cycle” that influence the molecular integrity of research tissues. We will describe our efforts to develop standard operating procedures (SOPs) for the extraction and biobanking of DNA, RNA and protein derivatives from frozen tissues harvested from radical prostatectomy specimens. This involved side-by-side comparison of extraction methods and optimization for prostate tissues. We have also developed a series of quality control (QC) procedures which included establishing standardized methods for quantification and assessing sample quality. We have developed a routine series of real-time PCR assays for DNA and RNA samples based on both housekeeping genes (GAPDH, 18S, β-globin) and markers differentially expressed in prostate cancer (Racemase, hepsin) that are performed on all samples included in the biorepository. The aim of performing these assays is to assure that the samples included in the PCBN are of sufficient quality for use in downstream applications. We will also plan to assess real-time PCR data in relation to pre-clinical variables such as warm ischemia time, time to tissue harvest, and age of the specimen. Additional efforts currently underway include determining global changes in RNA expression and protein quality in frozen tissues collected from open radical prostatectomy versus laparoscopic prostatectomy, and the development of SOPs for DNA/RNA extraction from archival formalin-fixed paraffin-embedded prostate tissues.

1 Department of Internal Medicine, Yale School of Medicine, New Haven, Connecticut 2 Department of Pathology, Yale School of Medicine, New Haven, Connecticut 3 Department of Surgery, Yale School of Medicine, New Haven, Connecticut 4 Departments of Epidemiology and Public Health, Yale School of Medicine, New Haven, Connecticut 87. The Yale Lung Cancer Biorepository: Merging Best Practices in Biospecimen Science and Clinico-Epidemiologic Annotation Enables Analysis of Gene-Environment Interactions Affecting Lung Cancer Prognosis

Priorities in lung cancer research include prognostic studies to identify early-stage patients at highest risk of recurrence following curative intent therapy, predictive studies advancing personalized medicine paradigms and etiologic studies of familial lung cancer and in never-smokers. Because tumor characteristics, germline genetic variation, environmental exposures and lifestyle factors all contribute, we designed the Yale Lung Cancer Biorepository to partner biospecimen science best practices with state-of-the-art clinico-epidemiologic annotations to facilitate robust investigation in lung cancer-related tumor-germline-environment interactions. Adult patients presenting to the Yale Thoracic Oncology Program (TOP) with lung cancer are eligible to participate. Enrolled participants complete an Intake Interview capturing demographics, medical comorbidities, family history of cancer, employment, quality-of-life, lifestyle choices (tobacco, alcohol and physical activity) and a food-frequency questionnaire. Intake biospecimens include a buccal swab, buffy coat and 20 mL plasma. Pre-treatment biospecimens include endobronchial brushings, pleural fluid and tumor/adjacent non-tumor samples. Touch-preparations are required for all tissue specimens frozen in RNA-Later to confirm malignancy. Surgical resections also yield OCT- and formalin-preserved specimens. Time to fixation is instant for biopsies and ≤30 minutes for resections. All biospecimens are stored at −190 °C. TOP faculty complete dedicated clinical/radiologic and pathologic staging. Regular medical record abstraction documents recurrence/progression, treatment and vital status. Repeat blood draws and quality-of-life and lifestyle reassessments occur every six months. Launched in 05/2011, enrollment is ongoing with 129 participants consented and over 2600 biospecimens archived through 12/2011. Surgical specimens are available from 70 participants. Seventy-eight percent completed the Intake Interview. The current population spans all stage groupings and histology reflects population-level distributions with 52% adenocarcinomas and 19% squamous cell carcinomas (SCCs). While SCCs occurred equally across sexes, adenocarcinomas predominated in women. Individuals smoking ≤10 pack-years represent 27.9% of adenocarcinomas but 0% of SCCs. The potential for contribution to lung cancer translational science will increase as this cohort matures.

1 Department of Mechanical Engineering, University of Minnesota, Minneapolis, Minnesota 2 Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota 3 Department of Medicine, University of Minnesota, Minneapolis, Minnesota 88. Stabilizing and Isolating Cellular Biomarkers in Lung Biospecimens

Lung disease remains a prominent cause of mortality in the U.S. with COPD being the third leading cause of death and lung cancer, accounting for 27% of all cancer deaths. There are few biomarkers for any lung diseases but a recent study suggests that low frequency cells present in bronchoalveolar lavage may at last provide a clinically valuable biomarker for lung disease. Unfortunately, the cells are collected in normal saline and lose viability rapidly with time post collection. Most samples need to be processed within an hour making use of this biomarker in a clinical context difficult. In addition, the cells of interest (epithelial cells or fibroblasts) are present at low frequency (<1%) and robust methods of isolating cells will be critical to establishing reliably the frequency of these cells and their biological characteristics. The objectives of this investigation are to design a short-term storage solution for the cells that will extend the viability of the cells for 6-8 h and develop methods of isolating these low frequency cells. A simple short-term storage solution was developed containing components generally regarded as safe (GRAS) and the viability of the cells was monitored over a 24 h period and this solution improved viability of the cells at least by 10% over controls stored in normal saline over the same period of time. Immunomagnetic depletion was used to remove all the hematopoietic cells in the sample. The remaining cells were fibroblasts and epithelial cells present at roughly 0.056% and 0.067% of the cells. Fibroblasts stained positive for vimentin and epithelial cells stained positive for cytokeratin. There is much interest in low frequency cells present in brushings, sputum and other biospecimens. This study demonstrates that it is possible to develop solutions and methods that permit the sample to be collected and processed for subsequent analysis.

1 Repository Services, National Marrow Donor Program, Minneapolis, Minnesota 2 Center for International Blood and Marrow Transplant Research, Minneapolis, Minnesota 3 Scientific Services, National Marrow Donor Program, Minneapolis, Minnesota 90. NMDP Research Sample Repository: 25 Years as a Worldwide HUB for Hematopoietic Stem Cell Transplant Research

Now in its twenty-fifth year, the National Marrow Donor Program (NMDP) maintains a centralized repository for the collection, processing and distribution of research samples to advance hematopoietic stem cell transplantation (HSCT). Through its worldwide network of partners, the NMDP collects pre-transplant blood samples from stem cell donors, umbilical cord blood units, and transplant recipients. The worldwide distribution of sample collection sites presents many challenges including consideration of each country's consent issues, international shipment of fresh blood samples, and the ability to link samples from donors and recipients when the respective sample collections occur between widely separated dates and locations. Special challenges exist in obtaining both the donor and recipient sample, considering that a planned transplant is frequently delayed or canceled if the patient's condition worsens. Sample submission compliance rates exceeding 90% are achieved by an effective system of monitoring and reporting to every NMDP center each trimester. The collection of more than 1.4 million aliquots contains sample sets from more than 41,000 unique donors, 4900 cord blood units, 41,000 transplant recipients, and 18,000 cell lines. Most useful for HSCT research are the paired samples from more than 26,000 transplants, where the NMDP has been able to obtain samples from both the donor/cord and the corresponding recipient of the transplant. The utility of the collection is distinguished by the complete and detailed HLA typing characterization that is performed on donor/recipient paired samples and is further complemented by comprehensive data collection on patient clinical outcomes. Sample and data distribution are facilitated through the NMDP research affiliation, the Center for International Blood and Marrow Transplant Research (www.cibmtr.org). More than 12,000 samples are shipped annually to researchers, with data generated submitted to the NMDP/CIBMTR and added to the knowledge base for the collection.