Tissue Microarray Profiling of Cancer Specimens and Cell Lines: Opportunities and Limitations

0023-6837/01/8110-1331$03.00/0 LABORATORY INVESTIGATION Copyright © 2001 by The United States and Canadian Academy of Pathology, Inc. Vol. 81, No. 10, p. 1331, 2001 Printed in U.S.A. MINIREVIEW Tissue Microarray Profiling of Cancer Specimens and Cell Lines: Opportunities and Limitations Axel Hoos and Carlos Cordon-Cardo Departments of Surgery (AH) and Pathology (CC-C), Memorial Sloan-Kettering Cancer Center, New York, and Antigenics, Inc. (AH), New York, New York SUMMARY: The implementations of high-throughput genetic technologies, such as oligonucleotide microarrays, generate myriad points of data. The identified potential candidate genes need to be further characterized and selected using a large number of well-characterized tumors and stringent criteria. Tissue microarrays allow for such high-throughput expression profiling of tumor samples, providing, in addition, information at the microanatomical level. Different techniques could be applied for identification of specific phenotypic (immunohistochemistry and in situ hybridization) or genotypic (fluorescence in situ hybridization) alterations, holding strong potential for translational research. Tissue microarrays consisting of 0.6-mm biopsies of paraffin-embedded tissues are well validated and have been used for various clinicopathological studies. This review discusses the technical considerations for construction of such arrays from paraffin-embedded tissues and cell lines and outlines their potential for clinical research applications. The use of paraffin-embedded tissues has some limitations with regard to analysis of RNA or certain proteins. To overcome such limitations, we have developed a cryoarray strategy allowing for the processing of multiple frozen tissue specimens and/or cell lines on a single tissue block. These approaches offer the opportunity to conduct pilot and validation studies of potential targets using clinical samples linked to clinicopathological databases. (Lab Invest 2001, 81:1331–1338). R ecent advances in genetics and biotechnology have brought up new classification schemes based on biological markers rather than anatomical definition regarding the clinical extent of the disease and morphological evaluation. Novel targets identified by analyses using the newly released human genome information (Lander et al, 2001; Venter et al, 2001), together with the development of microarray techniques that allow rapid large-scale screening (Kononen et al, 1998; Lockhart et al, 1996) and progress in bioinformatics with regard to processing and evaluation of complex data sets (Akutsu et al, 2000; Kruglyak and Lander, 1998; Lander et al, 1987), will still need to be further investigated. These studies will center in further defining their biological activities and clinicopathological relevance. Translational research will particularly benefit from these developments. Tissue banks linked to comprehensive clinical databases, procured through patient consent and protected by stringent ethical criteria, will be one of the most crucial resources for discovery and validation studies. Molecular profiling of cancer using cDNA microarrays (Lockhart et al, 1996; Schena et Received April 23, 2001. Address reprint requests to: Dr. Axel Hoos, Antigenics, Inc., 630 Fifth Ave., Suite 2100, New York, NY 10111. E-mail: al, 1995) further dissected by tissue microarraybased studies (Hoos et al, 2001b; Kononen et al, 1998) are expected to yield information of clinical significance. This may include the definition of new phenotypic profiles ascribed to certain disease entities, genes involved in critical cellular programs altered in particular tumors, and molecular targets of predictive or therapeutic value. For example, DNA microarrays can be used for the identification of subsets of expressed genes that would confirm or redefine a clinical entity, a pathological lesion, or a given disease stage (Emmert-Buck et al, 2000). These genes can subsequently be investigated for their expression in a large number of tumor and normal tissues using tissue microarrays linked to databases for rapid and reliable clinicopathological correlations (Moch et al, 1999). Technical considerations and the potential of DNA microarrays have been thoroughly discussed in various publications within the last few years (Lipshutz et al, 1999; Lockhart and Winzeler, 2000). Tissue microarrays are now becoming a relevant tool for further characterizing information from DNA microarray studies (Mucci et al, 2000; Richter et al, 2000) (A Hoos, A Stojadinovic, R Ghossein, ME Dudas, D Kuo, DHY Leung, AR Shaha, MF Brennan, C Cordon-Cardo, and B Singh, unpublished data). This review summarizes our current experience with this field, discusses technical issues for tissue miLaboratory Investigation • October 2001 • Volume 81 • Number 10 1331 Hoos and Cordon-Cardo croarray construction, and indicates opportunities and limitations for their application. Microarrays Using Paraffin-Embedded Tissues Conventional techniques for analysis of cancer specimens on the molecular level are labor intensive and time consuming. In the long run, they will not allow us to keep up with the rate at which new targets for tissue investigation are identified by DNA microarray analysis. The recently developed tissue microarray technology allows for high-throughput molecular profiling of tumor specimens by several techniques, including immunohistochemistry, fluorescence in situ hybridization and RNA in situ hybridization (Kononen et al, 1998). To construct a tissue microarray, small core biopsies are taken from viable, morphologically representative areas of paraffin-embedded tumor tissues and assembled on a recipient paraffin block. This is done with a precision instrument (Beecher Instruments, Silver Spring, Maryland) that uses two separate core needles for punching the donor and recipient blocks and a micrometer-precise coordinate system for tissue assembly on a multi-tissue block. During the recent evolution of the technique, initial large-core biopsies of over 3 mm in diameter were minimized to 0.6 mm in diameter. This size is sufficient for assessing morphological features of the analyzed tissues and allows the combination of up to 1000 cores on a single paraffin block. Microtome sections taken from such tissue microarray and placed on glass slides can be used for rapid and efficient molecular analyses. Besides tumor tissues, microarrays can contain corresponding normal tissues and internal controls, which can be analyzed in one experiment. Validation Strategies The main concern regarding the tissue microarray technique is that 0.6-mm biopsies of tumor specimens on an array may not be representative of the whole tumor specimen because of tissue heterogeneity. In addition, if there were discrepancies between arrayderived and full-section– derived data, this may also lead to different results for clinicopathological correlations based on that data. Therefore, recent studi (...truncated)