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Tissue Microarrays: Potential in the Indian Subcontinent Review Tissue microarrays: Potential in the Indian Article subcontinent Venkataraman Girish, Ananthanaranayanan Vijayalakshmi* Department of Pathology, Loyola University Medical Center, Maywood, IL60153 and *Department of Preventive Medicine, Robert H.Lurie Cancer Center, Northwestern University Feinberg School of Medicine, Chicago IL60611 Correspondence to: Dr. Girish Venkataraman, E-mail: [email protected] Abstract Tissue microarrays (TMAs) are a means of combining hundreds of specimens of tissue on to a single slide for analysis simultaneously. The evolution of this technology to validate the results of cDNA microarrays has impacted tremendously in accurately identifying prognostic indicators significant in determining survival demographics for patients. TMAs can be generated from archival paraffin blocks, combined with sophisticated image analysis software for reading TMA immunohistochemistry, and a staggering amount of useful information can be generated in terms of the biomarkers useful in predicting patient outcome. There is a wide range of uses for the TMA technology including profiling of specific proteins in cancerous tissues and non-cancerous tissues. Given the wide variety of tissue resources available in India, investment in a dedicated TMA facility will be of immense use in the research arena in India. This review article discusses the basics of TMA construction, design, the software available for the analysis of this technology and its relevance to Indian scientists. A potential workflow structure for setting up a TMA facility is also included. Key Words: Tissue microarrays, histopathology Introduction institutions across the world have taken advantage of this fact and incorporated TMA facilities exclusively for In the current world of proteomics, the use of high this purpose. A thorough understanding of the potential density Tissue Microarrays (TMAs)—also called “tissue advantages of this technology coupled with a unified chips”—has been progressively increasing in the last system for generating TMA blocks using paraffin blocks decade ever since its inception in 1998. Arguably, it from high-histopathology-volume institutions in India might be touted as the most noteworthy development will help in establishing a good tissue database for the in histopathology techniques in the last decade.[1,2] Indian pathologists. The basics of TMA construction, While DNA microarrays permit expression analysis of hardware involved and scoring options available are thousands of genes from one tissue specimen on a discussed here. single array, TMAs make it possible to analyze hundreds or thousands of tissue specimens in a single experiment Basics of TMA construction using a single gene or antibody probe. Given the tremendous pace at which novel genes implicated in TMAs are a method of relocating tissue from standard cancer are being discovered using gene chips and histological paraffin blocks such that tissue from expression microarrays, TMAs hold an immense multiple donor blocks can be placed on the same potential to validate this genomic data across multiple recipient block. Therefore, the first step in the process tumor types in a limited time frame. Many pathology of creating a good TMA block is to locate the most Indian Journal of Cancer | January - March 2005 | Volume 42 | Issue 1 5 Venkataraman et al: Potential of TMAs representative area on each H/E slide and careful sufficient clinical data, an important issue that merits identification of the same area on the corresponding consideration is the number of cores per case to put paraffin block of the slide. Once this is done, the block into the TMA block while evaluating any biomarker is positioned underneath the TMA puncher such that expression by immunohistochemistry. Although this the representative area is directly under the punching number is variable, for most tumor TMAs, three to pins. Thereafter, small “core needle biopsies” of these four cores/block adequately represent a biomarker’s representative tissues are punched out directly from ability to predict survival outcomes.[8] Often two cores donor paraffin blocks and re-embedded onto a new are taken from the donor block; one core is taken from recipient TMA paraffin block. Many such cores can be the center of the donor block and the other close to embedded in such a “master-arrayer” block employing the periphery of the block. It is important to include this technique.[2] Using a 0.6 mm diameter puncher, appropriate positive and negative controls besides nearly 600 or more tissue cores can be arrayed on a orientation cores. It would be useful to chart out an standard glass slide in a precise manner defined by X-Y MS Excel worksheet identifying the numbers of the coordinates. Although TMA block generation is a labor- various cores in the same pattern in which the cores are intensive and time-consuming process, the ability of this embedded in the TMA block. In addition, inclusion of high throughput technology to generate data from a irrelevant tissue at defined X-Y coordinates can help in staggering number of cases vastly improves statistical accurate orientation of the cores. Often cores from precision and power. tumors of same T size, stage or grade or tissue of origin can be clustered together on the TMA block. A TMA types, design and data handling options typical low-density TMA block and slide is shown in Figure 1. Last but not the least, it is important to Having such a large number of spots on one single ensure that all blocks used for TMA construction have slide calls for a precise organization of the tissue spots been fixed and processed similarly because some at three levels—firstly, in the design and placement of immunohistochemical markers may not work when the the tissue cores in the TMA block; secondly, in the tissue is fixed in a different fixative. linking of clinical and pathological data to the correct core; and lastly, a validated means of scoring and The technology used to generate TMA can range from analyzing any biomarker data so that a meaningful manual to fully automated systems. One of the more statistical analysis can be done. prominent companies include Beecher Instruments (San Prairie, Wisconsin, USA), which features manual, semi- Types of TMAs automated as well as fully automated systems. The cost can vary between $10,000 -$42,000 (USD) depending The nature of tissues that can be used on TMAs is on the type of the equipment. Cost-effective alternative varied, ranging from totally normal tissues from various methods of generating TMA blocks can also be adapted organs to non-neoplastic (like diabetes) and neoplastic ones and sometimes even cell lines.[3,4] The Cooperative Human Tissue Network (CHTN), a division of the National Cancer Institute (NCI) can provide investigators with a wide range of normal tissues placed on a TMA slide so the expression profile of a single protein in many tissues can be assessed simultaneously. The information from such TMAs can provide valuable information regarding the biology of diseases in which these proteins are altered. TMAs based on neoplastic tissues also termed as tumor TMAs, are broadly classified into three types—multi-tumor arrays, progression arrays (based on stage of tumor) and prognostic arrays where tumors with known clinical end points are arrayed.[5-7] Figure 1: A typical low-density TMA block containing 30 cores Design of TMAs (0.6mm each). The corresponding H/E slide with appropriate ID is depicted. Investigators musts be aware that individual cores can get depleted and the hence the need for duplicate representation An integral part of constructing TMAs is the design. of each core on the TMA block. Orientation cores have not been After deciding on the optimum number of cases with included here because of the low-density of cores on the block 6 Indian Journal of Cancer | January - March 2005 | Volume 42 | Issue 1 Venkataraman et al: Potential of TMAs if 60 or fewer number of cores are to be imprinted into worksheet is created and grid locations of the cores are the TMA block. A standard microscope fitted with a noted in rows and columns in the same pattern as in holing needle or a blunted 16G bone marrow trephine the TMA slide. Thereafter, hyperlinks are inserted for needle can be adapted to design TMA blocks at a each grid such that clicking on the hyperlink fraction of the price of commercial instruments.[9] corresponding to a specific core in the Excel worksheet will open the corresponding image of the same core for Data acquisition, analysis and integration scoring. This method is especially useful if the number of cores on a slide is not going to exceed 150 cores. Digital scanning and analysis For managing high-density TMAs with more than 600 The next issue of importance is reading the TMA spots cores per slide, an excellent set of software tools for in an orderly, reproducible and reliable manner. Of high-throughput analysis has been developed at Stanford course, the intuitive option would be to read one core University.[11] These investigators used the BLISS after another manually under a bright-field microscope. System mentioned previously for generating a database Even so, keeping track of the precise position of each of images. Immunohistochemical staining results are spot can become a painstaking process if there are more recorded into an MS Excel Worksheet and this Excel than 300 tissue spots on any single slide. Consequently, data is re-formatted by a program called “TMA- digital options are popular for analyzing biomarker Deconvoluter” which converts the Excel data into a text expression on TMA. Systems like the BLISS Imaging file so that a Hierarchical Cluster Analysis can be done (Bacus Labs, Lombard, IL) and ACIS system of using the “Cluster” and “TreeView” software—this Chromavision (www.chromavision.com) can scan and analyzes the relatedness within tumor subsets depending acquire images of all spots on the slide in one go on the immunohistochemical biomarker profile.
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