Advantages and limitations of microarray technology in human cancer

Oncogene (2003) 22, 6497–6507 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Advantages and limitations of microarray technology in human cancer Giuseppe Russo1,2, Charles Zegar1,2 and Antonio Giordano*,1 1 Sbarro Institute for Cancer Research and Molecular Medicine, College of Science and Technology, Temple University, Philadelphia, PA 19122, USA; 2Department of Human Pathology and Oncology, University of Siena, Italy Cancer is a highly variable disease with multiple heterogeneous genetic and epigenetic changes. Functional studies are essential to understanding the complexity and polymorphisms of cancer. The final deciphering of the complete human genome, together with the improvement of high throughput technologies, is causing a fundamental transformation in cancer research. Microarray is a new powerful tool for studying the molecular basis of interactions on a scale that is impossible using conventional analysis. This technique makes it possible to examine the expression of thousands of genes simultaneously. This technology promises to lead to improvements in developing rational approaches to therapy as well as to improvements in cancer diagnosis and prognosis, assuring its entry into clinical practice in specialist centers and hospitals within the next few years. Predicting who will develop cancer and how this disease will behave and respond to therapy after diagnosis will be one of the potential benefits of this technology within the next decade. In this review, we highlight some of the recent developments and results in microarray technology in cancer research, discuss potentially problematic areas associated with it, describe the eventual use of microarray technology for clinical applications and comment on future trends and issues. Oncogene (2003) 22, 6497–6507. doi:10.1038/sj.onc.1206865 Keywords: microarray; proteomics; human cancer Introduction In April of this year, we witnessed one of the most monumental achievements in biology: the complete sequencing of the human genome. The decoding and database deposition of billions of bases of sequence is the starting point of postsequence functional genomics. The discovery of the Periodic Table had an important impact on chemistry. So too, the complete deciphering of the human genome will have impressive effects on human health and quality of life. Currently, we understand the function of only a limited number of human genes. To study all human genes function is a technological challenge. To face this challenge, new *Correspondence: A Giordano, Department of Biotechnology, Temple University, 1900 N. 12th Street, Room 333, Philadelphia, PA 19122, USA; E-mail: high-throughput tools have been developed. The microarray assay is a powerful molecular technology that allows the simultaneous study of the expression of thousands of genes or their RNA products, giving an accurate picture of gene expression in the cell or the sample at the time of the study. For example, the expression of all the genes for drug resistance and metabolism or all the known oncogenes in a cell can be detected and measured in the same timeframe (Brown and Botstein, 1999; Collins, 1999; Lander, 1999). The microarray can be defined as an ordered collection of microspots (the probes), each spot containing a single species of a nucleic acid and representing the genes of interest. This technology is based on hybridization between labeled free targets derived from a biological sample and an array of many DNA probes that are immobilized on a matrix (Southern et al., 1999). The targets are produced by reverse transcription and the simultaneous labeling of RNA extracts from a biological sample hybridized with DNA fragment probes. The hybridization signal produced on each probe is the mRNA expression level of the corresponding gene in the sample at the time of the study. The signals are detected, quantified, integrated and normalized with dedicated software and reflect the ‘gene expression profile’ or ‘molecular portrait’ for each biological sample. Many thousands or tens of thousands of distinct spots can be printed on a silicon or glass slide or a nylon solidstate base. There are mainly two variants of microarrays: cDNA and oligonucleotide microarrays (Schena et al., 1995, 1996; Lockhart et al., 1996). Although both types of microarray are used to analyse gene expression patterns, these variants are fundamentally different (Lipshutz et al., 1999). In cDNA microarrays, relatively long DNA molecules are immobilized on a solid surface. This type of microarray is mostly used for large-scale screening and expression studies. The oligonucleotide microarray is fabricated by in situ light-directed chemical synthesis or by conventional synthesis followed by immobilization on a glass matrix. This microarray is used for detection of mutations, gene mapping and expression studies and allows for the differential detection of gene family members or alternative transcripts that are not distinguishable by cDNA microarrays. The chemistry of the microarray in itself is not new, since hybridization technology has been well established for decades. However, the simultaneous study of Advantages and limitations of microarray technology G Russo et al 6498 thousands of genes transforms the microarray technique into a powerful whole system analytical tool. Almost 10 years have passed since the first microarrays were created, and yet this technology is still improving and advancing. Since its initial introduction, the number of microarray applications has expanded (Figure 1). Starting from their use in gene screening and target identification, this technology is finding new applications such as developmental biology, disease classification, pathway studies, drug discovery and toxicology. The technology involved in the production and use of the microarray is beyond the scope of this review, but has been extensively reviewed elsewhere (Schena et al., 1995; Niemeyer and Blohm, 1999; Bowtell, 1999; Brown and Botstein, 1999; Celis et al., 2000; Cheung et al., 1999; Duggan et al., 1999; Graves, 1999; Khan et al., 1999; Hegde et al., 2000; Meldrum, 2000). We describe here some of the recent developments and results in microarray technology in cancer research, discuss potential problems, describe clinical applications and comment on the future of this technology. The importance of measuring global gene expression in human cancers Characterizing the population of transcribed genes has led to the creation of a new term, the transcriptome (Su et al., 2002). This concept defines the complete set of transcribed genes expressed as messenger RNAs for a particular species. The transcriptome, therefore, represents the universe of RNA messengers that may code for proteins. Only approximately 5% of genes are active in a particular cell at any given point in time. Most of the genes are repressed, and this control may occur at either the transcriptional or th (...truncated)