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2 Determinants of the Research Value of Biospecimens Pathology is the study of the structural and functional changes brought about by disease or injury. Pathologists analyze biospecimens for such changes and thereby attempt to discern their causes. The present com mittee's statement of task poses a number of questions regarding the future use of the Joint Pathology Center (JPC) repository's biospecimens collection in clinical care, education, and research activities. This chapter lays the groundwork for addressing those questions by providing information on the means of preserving biospecimens, on methods for analyzing and as- sessing their research value, and on how the details of preservation, storage, documentation, and the applications for which they are intended may affect prospects for their use. It focuses on scientific and technical considerations; legal and ethical issues are addressed in Chapter 3. COLLECTION AND PRESERVATION OF BIOSPECIMENS Three types of biologic material may be collected during pathologic investigations: tissues and cells removed during surgery or obtained spe- cifically for diagnosis via biopsy; cytologic material, including that from fine-needle aspiration biopsy, brushings, or swabs; and whole blood. The main objective in diagnostic pathology and pathology laboratories is to provide accurate diagnosis of a disease and additional pathologic informa- tion needed to define a prognosis and determine appropriate therapeutic strategies. Clinical data--including information about the patient and her or his medical history, physical examination, and diagnostic imaging, such as X-rays, CT scans, and the like--are also collected to inform evaluations. 37

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38 FUTURE USES OF THE DOD JPC BIOREPOSITORY This section briefly addresses how specimens are handled after collec- tion, focusing on the types of samples found in the JPC repository. Fig- ure 2-1 shows the relationship between the various materials collected and their forms of preservation for pathologic analysis. The pathology workflow for tissues comprises collection or excision from the patient; visual examination of the macroscopic specimen (called a gross specimen); initial stabilization; transfer to a laboratory; selection of material from the gross specimen for further analysis; fixation; further visual examination; histopathologic, biochemical, or molecular analyses; and storage. After collection or excision and any initial diagnostic evaluation, speci- mens are typically either frozen or chemically stabilized for transport. The essential processing steps for laboratory preparation of samples that are not maintained in a frozen state are summarized below. Fixation. Fixative solutions stabilize tissue structure and biochemical constituents by coagulating (cross-linking, denaturing, and precipitating) proteins and thereby prevent cellular hydrolytic enzymes, which are re- leased when cells die, from degrading tissue components and rendering tissues inadequate for microscopy. Fixation also immobilizes fats and car- bohydrates, reduces or eliminates enzymatic and immunologic reactivity, Frozen Tissue Tissues/Cells Tissue - Tumor (Biopsy or - Non-malignant Resection) Gross Histology Gross Specimen: Slides Specimen: Fixed Fresh FFPE Tissue Tissue Microarrays Digital Blocks Images Cells Body Fluids FFPE Cell (Cytology) Blocks Patient Cytology Slides Cell Personal Smears Information Clinical Data Diagnosis History and Physical Exam Rx-CT Images FIGURE 2-1Relationship between pathologic materials collected and form in which they are preserved for analysis. Figure 2-1

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DETERMINANTS OF THE RESEARCH VALUE OF BIOSPECIMENS 39 and kills microorganisms that are present in tissues. The fixative routinely used in pathology is 10 percent neutral buffered formalin, a buffered aque- ous solution of formaldehyde. Fixation yields "wet tissue" that is either stored in an air-tight container or processed further as delineated below. Embedding. Specimen water (about 70 percent of tissue mass) is re- placed with paraffin wax, and the specimen is surrounded by paraffin in a mold to provide support during sectioning and to aid in preservation. Formalin-fixed paraffin-embedded (FFPE) tissue is one of the predominant forms in which pathologic specimens are stored. Sectioning. Sections are cut on a microtome, which has a blade similar to a single-edge razor blade, that is advanced through a block of paraffin- embedded tissue. The shavings--about 5 m thick, about twice the thick- ness of a human hair--are placed in water, and the floating shavings are picked up on 1 3-in. glass slides. A given tissue block can be recut many times, although specific slices may differ in the amounts and types of tissues (for example, primary tumor vs. normal tissue) present. Staining. Tissue components can be distinguished with selective absorp- tion of dyes to facilitate viewing under a microscope. The stain routinely used in histology is hematoxylin and eosin. There are special methods for highlighting components (such as microorganisms) that do not stain well with the customary preparations. Pathologists commonly use tissue sections prepared in this manner in their analyses. Researchers use tissue microarrays (TMAs) constructed from diag nostic blocks of FFPE tissue to permit simultaneous evaluation of expression of specific pathologic features--proteins by immunohistochemistry, for example--in hundreds of individual tissue samples from different patients on a single slide (Rimm et al., 2011; Voduc et al., 2008). TMAs are as- sembled by using a needle to core an FFPE tissue block and extract a 0.5- to 2.0-mm piece that is placed into a predrilled master paraffin block that may contain up to 400 cores. Sections from the resulting block may be cut with a microtome, placed on a slide, stained, and analyzed. In cancer research, TMAs are used to analyze the frequency of a molecular alteration in dif- ferent tumor subtypes, as detected by immunohistochemical and molecular techniques, to enable evaluation of potential diagnostic and prognostic markers by correlating staining patterns with light microscopy and clini- cal information, which may also contain outcome measures (Camp et al., 2008; Kapur, 2011). Advantages of using TMAs include minimal tissue use, lower reagent costs, faster results, and the ability to define a set of cases that

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40 FUTURE USES OF THE DOD JPC BIOREPOSITORY have related diseases and clinical annotations and to use many such cases in direct parallel analysis on the same slide. Advances in technology also permit high-fidelity capture of the infor- mation contained on slides and in diagnostic images in digital form. Digiti- zation removes the opportunity for further biologic analysis but facilitates storage, sharing, and, if desired, deidentification of specimens. USES OF BIOSPECIMENS Educational, clinical consultation, and research uses impose different demands on the physical state of a specimen and the documentation that accompanies it. This section presents a brief summary of the considerations that influence the assessment of fitness for those uses. Educational Uses Educational uses have the lowest bar for molecular quality and physi- cal integrity of material and therefore can, in principle, permit the greatest variety of samples. However, it is uncommon to keep fresh, frozen, or even preserved samples in a manner that would allow ready distribution beyond the location at which they were generated. Indeed, unfixed tissue poses a risk of infection and should be handled only by persons who are trained in handling potentially infectious agents. The risk associated with fixed tissues is much lower, but such agents as prions are not inactivated by normal fixa- tion methods. Fixed specimens can be encased in plastic to facilitate han- dling, eliminate the risk of contagion, and enhance the educational value, but this is not commonly done. For those reasons, in most contexts, gross specimens are likely to have their most effective and widespread educational use in image form. Microscope slides are extremely useful for education, are easy to ship and return, and carry a very low risk of contagion. They have a long but finite shelf-life. However, there is little need for samples of extremely rare entities, except in advanced residency and fellowship training, and slides of common entities are abundantly available. The current trend is to- ward archiving of teaching slides digitally and viewing them with virtual microscopy. That avoids the problems of image or slide degradation and of slide distribution. Digital microscopy allows a teacher and a student to collaborate as though they are at the same microscope even though they may be separated by large distances. Accompanying clinical information often improves the pedagogic value of specimens, but it is not always required.

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DETERMINANTS OF THE RESEARCH VALUE OF BIOSPECIMENS 41 Clinical Consultation Uses Clinical consultation was a major function of the Armed Forces Insti- tute of Pathology, and the JPC continues to serve this function for the Mili- tary Health System, the Department of Defense, and other federal agencies. In the past, it was common to limit the materials for consultation to stained microscopic sections. Advances in diagnostic procedures now require the ability to extend the histopathologic description with immunohistochemical and molecular probes. That may require that unstained slides be included in the materials for consultation and, less often, that the tissue block or fresh-frozen tissue be available. Consultation also requires that detailed clinical information be provided to the consulting physician. Large medi- cal centers can often carry out the more advanced procedures on their own and need only send appropriately stained slides to the consultant, whereas smaller centers might require that the consultant carry out the procedures. In general, fresher, unfixed tissues give better results in assays than do samples that have had longer intervals at room temperature or long periods in fixatives. When consultation is limited to examination of slides, digital (or "virtual") microscopy via scanning of glass slides (Pantanowitz et al., 2011) allows rapid, interactive consultation without the need to transport and retrieve slides. Research Uses Research comprises a broad array of activities and a correspondingly broad array of requirements for pathologic specimens. Case reports and historical studies might need slides alone and be limited only by the condi- tion of the slides, but more extensive studies of the mechanism of disease require the freshest materials possible with cryopreservation, snap freezing in liquid nitrogen, or relatively brief times in fixatives to obtain the best results. That means not that older materials or materials that have not been obtained under those conditions are without value--DNA sequences have been obtained even from Neanderthal bones--but simply that they make studies technically more difficult and limited in scope and preclude some studies as the signal-to-noise ratio tilts toward the noise. In most cases, the more complete the accompanying clinical informa- tion is, the more valuable the sample. Nonetheless, many otherwise undocu- mented samples that have been well characterized histopathologically can be of value in genetic, microRNA (miRNA), and other functional studies. Studying diseases that have a high incidence, such as breast cancer, need not depend on suboptimal tissues or special collections inasmuch as specimens are readily available, whereas studying rare diseases may require greater

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42 FUTURE USES OF THE DOD JPC BIOREPOSITORY compromises with respect to sample quality and rely critically on special collections and repositories. Limitations on the use of pathologic samples in research are addressed in greater detail later in this chapter. TECHNOLOGIES USED TO MANAGE SPECIMEN ACQUISITION AND MANAGEMENT Over the last two decades, technology development has created both challenges to and opportunities for human biospecimen resources. The sensitivity, specificity, multiplexing capability, and speed of operation of technologies for molecular analysis of all classes of biomolecules in h uman specimens have undergone transformative improvements, and further re- finements are developed at ever-increasing rates. However, this greatly augmented analytic power has raised the bar for the quality of the bio- specimens that serve as the source of analytes for the technology platforms, which are increasingly used in research and clinical care. Biospecimens acquired in the setting of standard clinical practice that formerly served as adequate sources of research material despite the varied, undocumented, and uncontrolled sources of preanalytic variation1 to which they were exposed are no longer adequate, let alone optimal, for new molecular- assessment platforms. Technology development specifically directed to the challenges related to biorepository operation in this environment has been essential in addressing the gap between the demand for and supply of high- quality human specimens for molecular research. Biorepositories, such as the JPC, that comprise clinically derived sam- ples collected in diverse settings and referred for pathologic consultation on disease, typically face greater complexities in ensuring that their collections meet quality standards for sensitive analytic platforms than do biobanks that were established for the express purpose of collecting specimens for research. Clinical consultation biorepositories have difficulty in control- ling, recording, or assessing the sources of preanalytic variation that may compromise the molecular quality of their collections. Thus, technologic solutions for controlling processing and environmental variation and for assessing the molecular quality of processed or stored specimens have been essential for the continued evolution and usefulness of clinical consultation biorepositories for biomedical research. 1Preanalytic variation refers to any of the many biospecimen acquisition, handling, or pro- cessing procedures and environmental characteristics (such as temperature and humidity) to which a specimen may be exposed before analysis takes place. Preanalytic variation may alter the molecular quality or composition of a biospecimen and render it unsuitable for a specific type of analysis.

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DETERMINANTS OF THE RESEARCH VALUE OF BIOSPECIMENS 43 Technologic solutions for the problems in specimen acquisition, pres- ervation, and management include the following: Shipping technologies that maintain specific environments dur- ing transport of samples and thus help to maintain the molecular quality of the samples for analysis at remote sites. Specimen fixation and other stabilization technologies that preserve the quality of labile biomolecules, allow optimal molecular pres- ervation and histopathologic quality, or allow in situ stabilization of the specimen to preclude preanalytic variation incurred during specimen acquisition (for example, through surgical resection and pathologic handling). Information technology solutions that allow annotation of speci- men collections with clinical data about the individual from whom the specimen is derived; consent to specimen collection, transfer, storage, and use; authorization of use of protected health informa- tion; pathologic data on the specimen (such as gross description and accompanying diagnosis); collection, processing, transporta- tion, and storage data; quality-control data; specimen analysis data; radiologic imaging data; inventory tracking; overall (system- wide) quality-management data; and molecular analysis data. Specimen or molecular storage technologies, such as ambient- temperature ("dry-state") storage. Molecular quality-assessment technologies for RNA, DNA, and proteins. Digital imaging and image analysis technologies for precise structure-based data associated with each specimen. Those categories of technologies are not all equally developed, but all continue to improve rapidly and decrease in cost. Nevertheless, the rate of obsolescence of many of the technologies and the increasing knowledge of the effects of specific preanalytic factors on molecular analysis data require continual reassessment of the adequacy and functionality of tech- nologies that are in place in light of the repository's mission. TECHNOLOGIES USED TO ANALYZE SPECIMENS Researchers have several tools at their disposal for deriving clinical and research information from specimens. This section--which is based on review articles by West (2010) and Beck and colleagues (2010)--identifies some of the technologies used in analysis and discusses how preservation technique influences the ability to perform various types of analysis. Table 2-1 summarizes the results of some recent research on the influ-

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44 FUTURE USES OF THE DOD JPC BIOREPOSITORY TABLE 2-1 Recent Research on the Influence of Preservation Method on Specimen Analysis Outcomes Collection or Preservation Methods Tissue Types Attributes Fresh-frozen (Snap-frozen) Pancreatic cancer RNA integrity was determined specimens with microcapillary electrophoresis, using RNA integrity number (RIN) algorithm and results of laser-capture microdissection (LCM). Various ex-vivo procurement times (up to 10 min, 1130 min, 3160 min, over 1 h); banked over three periods (20012004, 20042006, 20062008) Fresh-frozen Invasive breast Manual method: subjective cancer tissues evaluation of electropherogram; ratio method: ratio between 28S and 18S peaks; RIN Room temperature, iced, saline Normal tonsil Structural RNA integrity via solution, RNA-stabilizing Normal colon microchip electrophoresis buffer, snap-frozen (after 0.5, 1, 3, 6, 16 h) Snap-frozen (unfixed and Tonsil Microchip gel electrophoresis and immersed in RNA-stabilizing gene expression level via PCR buffer), thawed for 0, 5, and 45 min, 1, 3, 6, and 16 h Snap-frozen, formalin-fixed RNA quality paraffin-embedded (FFPE) FFPE Diffuse large B-cell Gene expression lymphoma

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DETERMINANTS OF THE RESEARCH VALUE OF BIOSPECIMENS 45 Effects Source 42 percent of human pancreas cancer specimens banked under a Rudloff et al., dedicated protocol yielded RNA with a RIN of 7 2010 Brief warm ex-vivo ischemia times did not adversely affect RNA quality (percentage of tissue with total RNA with RIN of 7 for 10 min, 42 percent; 1130 min, 58 percent; 3160 min, 33 percent; >60 min, 42 percent) Long-term storage of banked pancreas cancer biospecimens did not adversely affect RNA quality (total RNA with RIN of 7 banked in 20012004, 44 percent; 20042006, 38 percent; 20062008, 50 percent); RNA retrieved from pancreatic cancer samples with RIN of C7 subject to LCM yielded RNA suitable for further downstream applications Fresh-frozen pancreas tissue banked according to a standardized research protocol yields high-quality RNA in about 50 percent of specimens and can be used for enrichment with LCM; quality of tissues in the biobank was not adversely affected by slight variations in warm-ischemia times or different storage periods Comparison between RNA quality (RIN) and gene expression analysis Strand et al., shows dense clustering of high-quality samples but weak clustering of 2007 low-quality samples Manual and RIN methods are superior to ratio method RNA stable in both tissues under all conditions for up to 616 h Micke et al., Expression levels essentially stable when samples kept on ice 2006 Marked regulation of single genes observed during room-temperature storage in normal saline and RNA-stabilizing buffer RNA from 54 of 47 samples had proper ribosomal peaks Nonfixed specimens may be transported on ice for hours with minimal influence Minimal RNA degradation after 30 min Botling et al., Relevant changes in some gene-expression levels at 45 min 2009 Repetitive thawing cycles had similar effects on RNA integrity Incubation in RNA-stabilizing buffer prevents RNA degradation Introduced heating into extraction protocol to improve quality; Li et al., 2007 incubation at 70C for 20 min was applied to disrupt cross-links in FFPE without compromising RNA integrity TaqMan detection influenced by master mix, amplicon size, and use of preamplification step Comparable results in frozen and FFPE tissue Provided PCR protocol for gene-expression analysis Votavov et 62 of 65 samples "successfully" analyzed al., 2009 continued

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46 FUTURE USES OF THE DOD JPC BIOREPOSITORY TABLE 2-1 Continued Collection or Preservation Methods Tissue Types Attributes FFPE Parathyroid Proteome quality Fresh-frozen, FFPE Colon adenoma Proteome quality Liquid chromatography Frozen, FFPE Frozen/optimal Proteome quality cutting temperature Liquid chromatography (OCT)-embedded livers (rats)

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DETERMINANTS OF THE RESEARCH VALUE OF BIOSPECIMENS 47 Effects Source 163 unique proteins identified via mass spectrometry Donadio et al., Similar results via sodium dodecyl sulfate (SDS)-out in gel-free method 2011 Antigenicity not always preserved in Western blot Despite some limitations due to extensive formalin-induced covalent cross-linking, results suggest that FFPE extracts may be an alternative source for large-cohort samples when frozen samples are unavailable "The major difference between frozen and FFPE proteomes was a Sprung et al., decrease in the proportions of lysine C-terminal to arginine C-terminal 2009 peptides observed, but these differences had little effect on the proteins identified." "Analysis of archival colon adenoma FFPE specimens indicated equivalent numbers of MS/MS spectral counts and protein group identifications from specimens stored for 1, 3, 5, and 10 years." "Analysis of the combined frozen and FFPE data showed a 92 percent overlap in the protein groups identified. Comparison of gene ontology categories of identified proteins revealed no bias in protein identification based on subcellular localization." "Archival samples displayed a modest increase in methionine oxidation, from approximately 17 percent after one year of storage to approximately 25 percent after 10 years." "These data demonstrate the equivalence of proteome inventories obtained from FFPE and frozen tissue specimens and provide support for retrospective proteomic analysis of FFPE tissues for biomarker discovery." "Comparable molecular mass representation was found in extracts Scicchitano et from FFPE and OCT-frozen tissue sections, whereas protein yields al., 2009 were slightly less for the FFPE sample." "The numbers of shared proteins identified indicated that robust proteomic representation from FFPE tissue and LCM [laser capture microdissection] did not negatively affect the number of identified proteins from either OCT-frozen or FFPE samples." "Subcellular representation in FFPE samples was similar to OCT- frozen, with predominantly cytoplasmic proteins identified. Biologically relevant protein changes were detected in atorvastatin- treated FFPE liver samples, and selected atorvastatin-related proteins identified by MS were confirmed by Western blot analysis. These findings demonstrate that formalin fixation, paraffin processing, and LCM do not negatively impact protein quality and quantity as determined by MS and that FFPE samples are amenable to global proteomic analysis." continued

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54 FUTURE USES OF THE DOD JPC BIOREPOSITORY cancer-related genes is being applied to DNA extracted from FFPE tumor- tissue specimens with PCR-based approaches. Recent advances in high- throughput next-generation sequencing methods permit the analysis of mutations in 200400 genes from small amounts of FFPE-extracted DNA. DNA, in general, is much more stable that RNA. However, all nucleic acids are susceptible to depurination in highly acidic environments, and the preservation of DNA in archival tissues depends heavily on the quality of the fixative used. Depurination can lead to strand cleavage, which would create problems for many of the modern molecular techniques. Thus, if unbuffered formalin was used originally to fix the archival material--as might be the case for older specimens in the JPC repository--it could lead to substantial degradation of the DNA (Akalu and Reichardt, 1999; Bonin et al., 2010). There are few published data on the quality of reads generated from high-throughput sequencing of archival DNA. Elemental and Chemical Studies Tissues are routinely analyzed for trace minerals in clinical pathology laboratories for diagnostic purposes. Trace minerals are preserved and can easily be localized and measured in FFPE tissues. For example, hepatic iron concentrations are essentially the same whether assessed in fresh tissues or in paraffin-embedded tissues (Torbenson, 2011). However, the altera- tions in tissue quality, such as a change in weight due to the replacement of water with less-dense paraffin, can necessitate correction factors (Bischoff et al., 2008). The routine fixation process, with numerous alcohol washes to dehydrate the tissue, results in the removal of many lipophilic small molecules. Elemental concentrations were studied in the past largely with atomic absorption spectroscopy or energy-dispersive X-ray analysis, but new methods are available now (Becker and Jakubowski, 2009; Harrington et al., 2010; Mizuhira et al., 2000). LIMITATIONS IN THE USE OF PATHOLOGIC SAMPLES IN RESEARCH Several factors influence whether pathologic samples obtained for clini- cal consultation purposes will be fit for use in research. This section iden- tifies the major issues that limit the research use of such specimens and addresses how the details of collection, preservation, storage, and docu- mentation can affect fitness for research use. The specimen preservationstabilization process might be inappropriate for or incompatible with the technologic or scientific demands of the re- search analysis.

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DETERMINANTS OF THE RESEARCH VALUE OF BIOSPECIMENS 55 The suitability of any specific specimen for analysis depends on the analyte that is to be assessed and the technique to be used for the a nalysis-- that is, whether it is "fit for purpose." As previously noted, the most com- mon stabilization method used in pathologic practice is 10 percent neutral buffered-formalin fixation followed by dehydration with graded alcohols, xylene exchange, impregnation by liquid paraffin, and permanent pres- ervation in paraffin. While the paraffin is maintained in liquid state, the specimen is spatially oriented in a small, thin rectangular container and then rapidly cooled until the paraffin is solidified. Adequacy of fixation of a pathologic specimen is determined largely according to the dimensions of the tissue (when it is submerged in formalin in the gross state and when it is sampled for single aliquots to be individually processed into tissue blocks, formalin penetrates about 1 cm into tissue) and the total time in fixative. In general, inadequate preservation due to underfixation is far more deleterious to samples than is prolonged fixation. Both fixation in a molecular cross-linking fixative, such as formalin, and paraffin embedding with exposure to high temperatures may compromise the molecular quality of samples. Other related issues concern variations in concentration, pH, or buffer- ing of formalin or the use of fixatives other than 10 percent neutral buffered formalin. The optimal formaldehyde concentration in a fixative and the optimal pH of the solution may depend on the biomolecule of interest, espe- cially in the case of immunohistochemical analysis. Practice is not standard- ized, and these measures are not always recorded in pathology reports and thus represent important unknowns in research. In the past, the use of such fixatives as Bouins solution (which contained picric and glacial acetic acid with formaldehyde) or special hematopathologic fixatives that contained metals, such as B5 zinc or B5 mercuric chloride, was common, and the fixative type was sometimes not recorded as a part of the pathologic record. Many of those fixatives are incompatible with modern molecular-analysis platforms. When specimens for research are accrued from multiple institu- tions that use different stabilization and preservation practices, data from molecular analyses may be neither reliable nor comparable or institution- specific batch effects may be seen. Important variation in pathologic processing also occurs before sta- bilization. For example, the length of time that elapses between specimen removal from a patient and specimen fixation ("time to fixation" or "cold ischemia time") and the conditions to which the specimen is exposed during this period, such as desiccation or various room temperatures (as opposed to refrigeration), may seriously alter both the molecular quality and the molecular content of the specimen and thus create artifacts that may be misinterpreted as reflecting disease biology. In some cases, the variations may even compromise histologic quality. Highly labile biomolecules may

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56 FUTURE USES OF THE DOD JPC BIOREPOSITORY be lost altogether as time to fixation increases. Those factors are neither controlled nor recorded in common clinical practice. Rapid stabilization of tissue with cryopreservation of various kinds (for example, immersion in liquid nitrogen, freezing in the vapor phase of liquid nitrogen before im- mersion, immersion in dry iceisopentane slush, embedding in a freezing medium based on polyethylene glycolsucrose,3 and placement in a -80C freezer or a cryostat) may be used in exceptional circumstances but are not the standard of care for all tissues. Thus, if frozen (unfixed) tissue is needed for research, this requirement often cannot be met pathologic tissue. If viable cells or tissues are required for research, pathologic samples cannot fulfill this requirement unless viable aliquots are preserved appropriately at the moment of acquisition of the specimen and not used as a part of a retrospective pathologic sample set. Sample aliquot content might be unknown to the investigator, and too little of the lesion of research interest might remain in the residual tissue after clinical workup. Each paraffin tissue block processed for a case is thinly sliced (at 5-m intervals) with a microtome to produce sections that are placed on glass microscope slides and stained for histopathologic analysis under a light microscope. As previously noted, the standard histopathologic stain is hematoxylin and eosin, but a wide variety of stains may be used on adjacent tissue sections to reveal special features as a part of the pathologic workup. Unstained sections also are often cut and set aside for or used for immuno- histochemical analyses of various types required for pathologic diagnosis and characterization. In many cases, small amounts of tissue may remain in a block after complete workup, and this can result in inadequate residual amounts for research. Inadequacy of residual tissue for research is particu- larly problematic if the original specimen from the patient is very small or the lesion of interest is small, focal, or both and thus not of sufficient qual- ity (or, indeed, at present all) in the residual tissue in a block. The lack of quality control of pathologic tissue blocks made available for research, to verify the nature and content of the remaining tissue, can detract from their usefulness for research. It can also skew data from investigational studies if the residual blocks are assumed to be representative of the overall diagnosis but are depleted of diagnostic tissue. The original pathologic diagnosis might be unconfirmed or incorrect. Unconfirmed or incorrect diagnosis of pathologic material is u ncommon (Lind et al., 1995; Ramsey and Gallagher, 1992; Renshaw et al., 2003a,b; Safrin and Bark, 1993; Wakely et al., 1998), but in cases of rare diseases or 3Also known as optimal cutting temperature (OCT) compound.

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DETERMINANTS OF THE RESEARCH VALUE OF BIOSPECIMENS 57 diseases that have a documented, notoriously high degree of interobserver variation in diagnostic interpretation, it can be an important issue for both patient management and research. In such cases, it is considered standard of care to seek a second, specialty pathologic opinion, such as the consulta- tive opinions produced for specimens submitted to the JPC repository and its predecessors. Verification of diagnosis by obtaining a new pathologic analysis in every case used for research is recommended. However, it may also be problematic for research if the diagnosis that accompanies the case is outdated and no longer appropriately classifies the disease. Outdated diagnostic terminology (correct diagnosis but arcane language) or outdated diagnostic criteria for identification (classification according to a schema that is no longer in use) may cause considerable difficulty in mapping a historical case to a current diagnostic category accurately. Over the span of decades that the JPC repository has existed, pathologic classification of disease has evolved substantially. In some dis- ease categories, such a hematopathologic malignancies, entire disease clas- sifications have changed repeatedly because knowledge of pathogenesis has grown. Modern diagnosis of lymphoma and leukemia may require delinea- tion of specific molecular features that were never tested for in older cases. Depending on the specific preanalytic variation associated with a historical case, it may not even be possible to test accurately for the molecular fea- tures required for diagnostic classification. The more common problem in all disease categories is the lack of standardization in diagnostic terminology that was widespread in pathol- ogy for many years. That has been exemplified both by the use of a given diagnostic term for different disease entities and by the use of multiple diag- nostic terms for a given disease entity (Cooper, 2006). A researcher using a historical case may not have the requisite expertise to interpret the existing diagnostic terminology accurately and map it to the current diagnostic ter- minology standard correctly. Failure to reclassify cases correctly according to current diagnostic standards and current diagnostic terminology for any of the above reasons may skew research data. Preanalytic variations related to preoperative or intraoperative factors may create molecular artifacts. Many drugs used in preoerative and intraoperative periods and such surgical events as devascularization or arterial ligation with cessation of blood supply during resection (called warm ischemia time) may cause changes in the molecular profiles of resected tumor and normal tissues and preclude use of specimens for research. Shifts in molecular profiles due to iatrogenic interventions may not be recognized as artifacts and may be mis- takenly interpreted as disease signatures. Some drugs used in perioperative and interoperative periods have powerful molecular effects and are, in fact,

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58 FUTURE USES OF THE DOD JPC BIOREPOSITORY used specifically for these effects (Juhl, 2010), although little research has been published on this topic. In addition, the type of surgical procedure, the individual surgeon performing the procedure, and the use of robotic instruments are variables that affect the duration of the resection and the resulting tissue ischemia in the resection specimen. Although some informa- tion related to the number and types of drugs given before and during an operation may be available in clinical records, such as the anesthesia report, it is uncommon to have these data available to a biorepository. The com- mittee does not know whether or how many specimens in the JPC reposi- tory are annotated with data related to perioperative variables that may be pertinent to molecular research on the molecular profiles of tissue samples. Perhaps the most common preanalytic variable with an important effect on results of analytic tests, such as immunohistochemical staining, is the time that elapses after surgical removal of tissue until it is stabilized with fixation or freezing (cold ischemia time). The process of structural and bio- chemical tissue degradation occurring during this time is termed autolysis. The amount of time can be substantial, ranging from minutes to hours, and is rarely recorded in the pathology report. It may cause gain or loss of signal on molecular analysis, depending on the molecular entity in question. An analysis done without knowledge of the duration of the time to fixation and without an intrinsic control within the same specimen that can serve as a reference (for example, surrounding normal tissue that is known to express or not to express the molecule constitutively) may be misleading or even completely incorrect (Spruessel et al., 2004). Storage conditions or duration may compromise specimen quality. Oxidation occurs in both blocks and cut sections, but it is much greater on cut surfaces exposed to room air. Thus, tissue blocks stored at tem- peratures below the melting point of paraffin yield cut sections that show little deterioration of immunohistochemical signal compared with freshly embedded controls for as little as 2 years or as long as 25 years (Engel and Moore, 2011). RNAases are active even in FFPE tissues. For many antigens, immunoreactivity has been shown to deteriorate more rapidly if specimens are stored as cut sections rather than whole blocks; both the time line and the magnitude of the effect are antigen-dependent. On a crude level, paraffin blocks that have been stored under condi- tions that do not include climate control may lead to melting of paraffin blocks in warm weather or destruction from other causes. Gross specimens, albeit formalin-fixed, may undergo dehydration, fungal contamination, and putrefying deterioration when stored under conditions that expose them to environmental extremes. All those issues have affected specimens held by the JPC at some point over the course of the repository's history (Asterand, 2008).

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DETERMINANTS OF THE RESEARCH VALUE OF BIOSPECIMENS 59 Case-matched normal control samples from a tumor patient might be unavailable. A normal specimen is needed as a source of a reference genome in tumor patients in for correct identification of tumor-specific mutations. Depending on the disease, the diagnostic or therapeutic procedure that produced the specimen, or the tissue remaining in a case after diagnostic analysis, it may not be possible to meet this requirement. The clinical data associated with specimens may be inadequate, inaccu- rate, or nonstandardized. Even if biospecimens are of sufficient quantity and quality for a particu- lar molecular analysis, their value for translational research may be severely limited by the amount, type, and quality of clinical data that are available for an individual case and by the consistency of the data on the many cases that may be required for a study. Clinical data provide the essential func- tional or biologic behavioral correlations that define the medical relevance of the molecular data. The lack of relevant clinical data elements limits the value of the molecular analysis data for prediction and the conclusions that can be drawn. The type and amount of clinical data provided by the physician request- ing consultation was not prescribed in a standardized fashion by the JPC repository. Furthermore, it did not require that any clinical data submitted adhere to a standardized format.4 Thus, clinical data associated with cases varies widely in quality, quantity, and consistency, which may create impor- tant limitations in the utility of the JPC biospecimens for research studies. In some cases, it may be possible to acquire missing clinical data from the medical record in the institution in which the referred case originated. For some institutions, such as those within the Department of Veterans Affairs health system or the Department of Defense and many private insti- tutions, the acquisition of additional data elements for a case may be techni- cally facilitated by using an electronic medical record (EMR). Nevertheless, additional technical hurdles--such as the difficulty in combining data from an EMR with the JPC's information technology and the unreliability or unavailability of identifiers that would allow data from disparate sources to be combined--may limit the ease with which the data can be transferred or may preclude electronic transfer altogether. Acquiring or transferring additional data by nonelectronic means is labor-intensive, is expensive, and adds a risk of introducing errors. However, given the number of hospital mergers and closings in recent years, it may not be easy to trace a patient 4The Contributor's Consultation Request Form used in recent years does require that some standardized information be submitted. The most recent version of the form is reproduced in Appendix B.

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60 FUTURE USES OF THE DOD JPC BIOREPOSITORY record through the original submitting institution. The ability to acquire additional data may also be constrained by ethical and legal restrictions, such as privacy laws, as noted briefly below. Consent from the source might be inadequate for use, permission to re- contact might be lacking, or recontact might be prohibitively expensive. Although the acquisition of additional clinical data for a case may be technically possible, there may be circumstances where it would be prohib- ited or restricted by ethical or legal issues related to recontact or privacy laws, including the federal Health Insurance Portability and Accountability Act of 1996 (PL 104-191, 110 Stat. 1936, and accompanying regulations). In other situations--especially where a number of years have passed since the data or specimen were collected--it may require extensive research to locate the source individual. Those issues may render specific cases unusable for some types of research. Chapter 3 addresses consent and other ethical, legal, and regulatory aspects of use of the JPC materials for consultative, educational, and research purposes. REFERENCES Aebersold R, Mann M. 2003. Mass spectrometry-based proteomics. Nature 422(6928):198-207. Akalu A, Reichardt JK. 1999. A reliable PCR amplification method for microdissected tumor cells obtained from paraffin-embedded tissue. Genetic Analysis 15:229-233. Asterand. 2008. Assessment of the Department of Defense's tissue repository located at the Armed Forces of Pathology in Washington DC. Detroit, MI: Asterand, Inc. Azimzadeh O, Barjaktarovic Z, Aubele M, Calzada-Wack J, Sarioglu H, Atkinson MJ, Tapio S. 2010. Formalin-fixed paraffin-embedded (FFPE) proteome analysis using gel-free and gel-based proteomics. Journal of Proteome Research 9(9):4710-4720. Bataille F, Troppmann S, Klebl F, Rogler G, Stoelcker B, Hofstadter F, Bosserhoff AK, R ummele P. 2006. Multiparameter immunofluorescence on paraffin-embedded tissue sections. Applied Immunohistochemistry & Molecular Morphology 14(2):225-228. Beck AH, Weng Z, Witten DM, Zhu S, Foley JW, Lacroute P, Smith CL, Tibshirani R, van de Rijn M, Sidow A, West RB. 2010. 3-end sequencing for expression quantification (3SEQ) from archival tumor samples. PLoS One 5(1):e8768. Becker JS, Jakubowski N. 2009. The synergy of elemental and biomolecular mass spectrometry: New analytical strategies in life sciences. Chemical Societies Review 38(7):1969-1983. Bischoff K, Lamm C, Erb HN, Hillebrandt JR. 2008. The effects of formalin fixation and tissue embedding of bovine liver on copper, iron, and zinc analysis. Journal of Veterinary Diagnostic Investigation 20(2):220-224. Bonin S, Hlubek F, Benhattar J, Denkert C, Dietel M, Fernandez PL, Hofler G, Kothmaier H, Kruslin B, Mazzanti CM, Perren A, Popper H, Scarpa A, Soares P, Stanta G, Groenen PJ. 2010. Multicentre validation study of nucleic acids extraction from FFPE tissues. Virchows Archiv 457:309-317. Botling J, Edlund K, Segersten U, Tahmasebpoor S, Engstrm M, Sundstrm M, Malmstrm PU, Micke P. 2009. Impact of thawing on RNA integrity and gene expression analysis in fresh frozen tissue. Diagnostic Molecular Pathology 18(1):44-52.

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