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Tissue Microarray

      INTRODUCTION

      The tissue microarray (TMA) technique has been in use for 15 years. The technology was first described in 1987, but its use took off 11 years later, when Kononen and colleagues developed a device that could rapidly and reproducibly produce TMAs (
      • Kononen J.
      • Bubendorf L.
      • Kallioniemi A.
      • et al.
      Tissue microarrays for high-throughput molecular profiling of tumor specimens.
      ). This powerful, high-throughput technique can be used to assay hundreds of patient tissues arrayed on a single microscope slide.

      THE TECHNIQUE

      Following fixation, biopsies and excised tissue samples are usually embedded in paraffin blocks to facilitate their cutting on a microtome; this results in 5-μm tissue slides that can be stained with hematoxylin and eosin (H&E) and viewed under a microscope. The paraffin blocks can also be used as source of material from which to construct a TMA. In this procedure, areas of interest are marked on the H&E slides by a pathologist. Then, cylindrical tissue core biopsy specimens (Figure 1a) from the original formalin-fixed paraffin-embedded (FFPE) tissue donor blocks are punched out of the paraffin block using specialized TMA equipment and placed in a predrilled hole in a (new) recipient paraffin block at defined array coordinates (
      • Jawhar N.M.
      Tissue microarray: a rapidly evolving diagnostic and research tool.
      ;
      • Camp R.L.
      • Neumeister V.
      • Rimm D.L.
      A decade of tissue microarrays: progress in the discovery and validation of cancer biomarkers.
      ). Sectioning of this recipient paraffin block will reveal a slide with numerous small, round tissue sections through the cores punched out of the original blocks; hence, each original tissue sample is represented by one or more small “histospots” with a preset fixed diameter ranging from 0.6 to 2 mm (Figure 1b). The number of spots on a single slide depends on the core size, ranging from 40 to 800 spots (Camp et al., 2008). Cores are placed at specifically assigned coordinates, which typically are recorded in a spreadsheet. To facilitate “reading” of the TMA slide, different known tissue cores (e.g., liver, thyroid) are placed at the outer margins and empty holes are left at predefined places. After the TMA has been constructed, the recipient block is heated to 37 °C to fix the cores; then, using a microtome, sections are cut from the TMA blocks to generate TMA slides for analysis (
      • Jawhar N.M.
      Tissue microarray: a rapidly evolving diagnostic and research tool.
      ).
      Figure thumbnail gr1
      Figure 1Construction of a TMA. (a) Example of marked areas of interest on a hematoxylin and eosin slide indicating sites at which to punch out samples of normal skin and in situ and invasive squamous cell carcinoma (SCC). (b) Process of constructing a TMA. The area of interest is marked on a hematoxylin and eosin slide. The TMA core is taken out of the original donor block and arrayed in a recipient block using a TMA needle. Multiple sections can be cut from the recipient block for morphological and molecular studies. TMA, tissue microarray.
      Figure thumbnail fx1

      APPLICATIONS

      The increased use of new molecular biology techniques has revolutionized investigation of the pathogenesis and progression of diseases such as cancer. New markers, identified via molecular research in cellular and animal models, require clinical validation on histopathological human specimens. This translation from basic to clinical research is facilitated by the TMA technology, which enables investigators to screen for the expression of a specific protein on tissue samples from a large cohort of patients by immunohistochemistry or the presence of nucleotide sequences by in situ hybridization.
      All applications currently performed on standard histological sections from FFPE tissue are possible using TMA. In contrast to a series of whole FFPE-tissue sections, stained separately via immunohistochemistry, the use of TMA slides allows semiquantitative scoring of the intensity of staining because all tissue samples on a TMA slide, including the controls, have been exposed to the same amount of primary and secondary antibody and chromogen. For example,
      • Zhou H.
      • Ekmekcioglu S.
      • Marks J.W.
      • et al.
      The TWEAK receptor Fn14 is a therapeutic target in melanoma: immunotoxins targeting Fn14 receptor for malignant melanoma treatment.
      stained a TMA composed of melanoma samples from 169 patients to evaluate expression of fibroblast growth factor–inducible protein 14 (Fn14) and applied semiquantitative scoring (Figure 2) that enabled them to identify Fn14 as prognostic marker and therapeutic target in melanoma.
      Figure thumbnail gr2
      Figure 2Scoring of a TMA. Semiquantitative scoring of a TMA immunostained with an anti-Fn14 antibody containing melanoma tissue.
      Reprinted with permission from Zhou et al., 2013. TMA, tissue microarray.
      Detection of specific gene sequences using fluorescence in situ hybridization (FISH) of biopsy specimens from a large patient cohort (
      • Chen A.Y.
      • Chen A.
      Fluorescence in situ hybridization.
      ) is another application of TMA. FISH can be used to detect the presence or absence of specific gene sequences as well as their location; this technique has been found useful in the differential diagnosis of ambiguous melanocytic lesions (
      • Chen A.Y.
      • Chen A.
      Fluorescence in situ hybridization.
      ).

      ADVANTAGES

      Simultaneous analysis of a large number of specimens: TMA provides high-throughput data acquisition. For example, if 100 consecutive sections from a TMA block containing 200 cores are used, 20,000 data points are obtained. In addition, the original blocks from which the cores were taken are conserved; a similar approach using the original block would require an enormous amount of labor and time—and, more importantly, consume the original paraffin block.
      Construction of ex vivo tumor progression model: studying morphological and molecular changes through the stages of tumor progression is easily performed by constructing a TMA because it is possible to include different tumor progression stages. Figure 1a represents a TMA of squamous cell carcinoma (SCC) progression for which cores were taken from normal skin, in situ SCC, and invasive SCC of the same patient.
      Experimental uniformity: in a TMA, each tissue core is treated in an identical manner (same antigen retrieval, same temperature, same incubation time, same washing procedure, and same reagent concentration); therefore, samples can be compared and the immunohistochemical results can be read out in a semiquantitative way. The alternative of assessing the level of staining in separate histological whole sections is difficult because of subtle differences in intensity. When the cores are taken from pathologist-identified regions of interest, immunohistochemical staining can often be scored reliably by individuals with only rudimentary training or the scoring can be done by an automated reader (
      • Camp R.L.
      • Neumeister V.
      • Rimm D.L.
      A decade of tissue microarrays: progress in the discovery and validation of cancer biomarkers.
      ). Decreased assay volume, time, and cost: only a small amount of each reagent is needed to assay all cores at the same time, compared with separately assaying standard histologic whole sections from each donor.

      DISADVANTAGES

      Tissue heterogeneity: one of the most common criticisms of TMA is that the small cores may not be representative of the entire tumor, owing to tumor heterogeneity. This heterogeneity differs from cancer to cancer; for example, it has been estimated at 71% for SCC compared with 50% for breast cancer (
      • Li J.
      • Wang K.
      • Jensen T.D.
      • et al.
      Tumor heterogeneity in neoplasms of breast, colon, and skin.
      ). The problem of tumor heterogeneity and consequent false-positive or false-negative results in the TMA slide can be overcome by including enough tissue cores per sample. Ideally, this number should be based on careful consideration before constructing the TMA, but in practice cores should be taken from all histologically divergent areas in the original sample, as divergent histology may result in divergent phenotype or genotype. In a previous study we took three or four cores from each vertical-growth-phase melanoma and obtained good concordance between immunohistochemical and reverse transcriptase–PCR data (
      • Winnepenninckx V.
      • Lazar V.
      • Michiels S.
      • et al.
      Gene expression profiling of primary cutaneous melanoma and clinical outcome.
      ). Moreover,
      • Jensen T.O.
      • Riber-Hansen R.
      • Schmidt H.
      • et al.
      Tumor and inflammation markers in melanoma using tissue microarrays: a validation study.
      obtained up to 96% agreement between TMA and whole-slide immunohistochemical data. Finally, the statistical power of analysis of hundreds of cases will largely eliminate the effect of variability of a single data point.
      High cost: commercial TMA builder machines such as automated and semiautomatic tissue arrayers are expensive. A relatively simple and inexpensive alternative is the use of lab-made recipient paraffin blocks and ordinary cannula-piercing needles, skin biopsy punches, and bone marrow biopsy needles (
      • Choi C.H.
      • Kim K.H.
      • Song J.Y.
      • et al.
      Construction of high-density tissue microarrays at low cost by using self-made manual microarray kits and recipient paraffin blocks.
      ). However this alternative is time-consuming, and for large sample investigations, automated or semiautomatic arrayers may ultimately be less expensive.
      Variations in antigenicity: given TMA's ability to array many samples and to perform retrospective studies, variations in the antigenicity of stored archival samples may create problems.

      COMPARISON WITH DNA MICROARRAYS

      Every cell in the body contains a complete set of identical DNA; however, as a result of genetic and epigenetic mechanisms, only certain genes are active in a given cell, differentiating that cell from others (
      • Villasenor-Park J.
      • Ortega-Loayza A.G.
      Microarray technique, analysis, and applications in dermatology.
      ). The active genes are transcribed into messenger RNA (mRNA), which is subsequently translated into proteins. These proteins are responsible for the behavior and function of the cell (
      • Villasenor-Park J.
      • Ortega-Loayza A.G.
      Microarray technique, analysis, and applications in dermatology.
      ). In contrast to cDNA microarrays, which focus on gene expression at the mRNA level but do not yield information on the final steps of translation to a protein, TMAs can provide information on the expression level and activity of the final product, the protein.
      Figure thumbnail fx2

      SUPPLEMENTARY MATERIAL

      A PowerPoint slide presentation appropriate for teaching purposes is available at http://dx.doi.org/10.1038/jid.2014.277.

      CME ACCREDITATION

      This activity has been planned and implemented in accordance with the Essential Areas and Policies of the Accreditation Council for Continuing Medical Education through the joint sponsorship of the Duke University School of Medicine and Society for Investigative Dermatology. The Duke University School of Medicine is accredited by the ACCME to provide continuing medical education for physicians. To participate in the CME activity, follow the link provided. Physicians should only claim credit commensurate with the extent of their participation in the activity.

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        • Chen A.
        Fluorescence in situ hybridization.
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        Construction of high-density tissue microarrays at low cost by using self-made manual microarray kits and recipient paraffin blocks.
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        Tissue microarray: a rapidly evolving diagnostic and research tool.
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        Tumor and inflammation markers in melanoma using tissue microarrays: a validation study.
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        Tissue microarrays for high-throughput molecular profiling of tumor specimens.
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        Microarray technique, analysis, and applications in dermatology.
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        Gene expression profiling of primary cutaneous melanoma and clinical outcome.
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