review September 2007 ⅐ Vol. 9 ⅐ No. 9 Detection of DNA copy number alterations in cancer by array comparative genomic hybridization Evi Michels, MSc, Katleen De Preter, PhD, Nadine Van Roy, PhD, and Frank Speleman, PhD Over the past few years, various reliable platforms for high-resolution detection of DNA copy number changes have become widely available. Together with optimized protocols for labeling and hybridization and algorithms for data analysis and representation, this has lead to a rapid increase in the application of this technology in the study of copy number variation in the human genome in normal cells and copy number imbalances in genetic diseases, including cancer. In this review, we briefly discuss specific technical issues relevant for array comparative genomic hybridization analysis in cancer tissues. We specifically focus on recent successes of array comparative genomic hybridization technology in the progress of our understanding of oncogenesis in a variety of cancer types. A third section highlights the potential of sensitive genome-wide detection of patterns of DNA imbalances or molecular portraits for class discovery and therapeutic stratification. Genet Med 2007:9(9):574–584. Key Words: Array CGH, cancer, genomics, copy number alteration, microarray, prognosis, therapy UNDERSTANDING CANCER THROUGH THE STUDY OF mutations, deletions affecting one (haplo-insufficiency) or CANCER GENOMICS both alleles, or epigenetic modifications. A particular class of oncogenic events most often (but not exclusively) observed in Cancer results from a series of genetic and epigenetic alter- leukemias, lymphomas, and soft tissue tumors are chromo- ations that allow cells to become independent of growth sig- some rearrangements causing in frame fusion of parts of two nals, to escape growth inhibitory and apoptotic signals, to ac- genes leading to the formation of hybrid genes with particular quire unlimited growth potential, and ultimately to invade oncogenic properties.3 neighboring tissues and metastasize to other organs.1 In the Classical cytogenetics has been extremely important in iden- past decades, research has been successful in identifying some tifying such recurrent rearrangements, in particular recurrent of the genes controlling these cellular processes, thereby con- translocations, which helped to uncover the position and al- tributing to the unraveling of the genetic basis of cancer. A lowed the molecular cloning of the genes implicated in such number of these genes emerged as key players with a central proto-oncogene activation or gene fusion events.3,4 This inter- role in the tight control of cell cycle, apoptosis, and DNA re- play between cytogenetic and molecular genetics allowed the pair. This multitude of genes controls networks of a restricted generation of new tools for diagnosis and follow-up of the number of signaling pathways. Two such pathways, centering disease. Furthermore, the discovery of gene fusions repre- around the RB1 and TP53 genes, respectively, are assumed to sented the first step toward the unraveling of the pathogenesis be disrupted in virtually all cancer types. Other major signaling of these particular cancers and, more importantly, for the de- pathways perturbed in cancer include the RAS/MAP kinase velopment of molecular-targeted therapy.5,6 Treatment of (growth control), PI3/AKT (survival, apoptosis), TGF␤, and chronic myeloid leukemia with the tyrosine kinase inhibitor JAK/STAT pathway.2 Cytogenetic and molecular genetic in- imatinib illustrates how the identification of such cancer genes vestigations provided insight into the plethora of mutations and perturbed signaling pathways revealed targets for less toxic that disrupt the normal function of genes implicated in cancer. and more efficient molecular therapy.7,8 Proto-oncogenes are known to be activated through gain-of- For some tumor entities, however, classical cytogenetics has function mutations at the base pair level or are over-expressed been less successful because of the complexity of karyotypes or because of copy number gain, amplification, or translocation. difficulty of culturing tumor cells in vitro. Chromosomal com- Tumor suppressor genes can be inactivated by loss-of-function parative genomic hybridization (CGH) opened the way for the study of DNA copy number alterations in such cancers. A par- From the Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium. ticular advantage of this technique is that dividing cells are not Frank Speleman, Center for Medical Genetics, Ghent University Hospital, Medical Research required, allowing the study of archived material such as frozen Building, De Pintelaan 185, B-9000 Ghent, Belgium; E-mail: [email protected] biopsies or paraffin-embedded material. A large body of liter- Disclosure: The authors declare no conflicts of interest. ature describes the findings by chromosomal CGH on such Submitted for publication May 14, 2007. tumors, which contributed to the mapping of tumor suppres- Accepted for publication June 13, 2007. sor genes and amplified proto-oncogenes and the identifica- DOI: 10.1097/GIM.0b013e318145b25b tion of prognostically relevant genomic subclasses.9,10 The role 574 Genetics IN Medicine Array CGH in cancer of low copy number gain in cancer is still poorly studied, but material, but difference in quality can occur depending on the evidence is accumulating that gain of one single copy of a gene, type of DNA isolation procedure. Therefore, when using kits chromosomal region, or entire chromosome can effectively for fast DNA isolation, an extra DNA purification step can be contribute to the tumor phenotype. useful to further improve DNA quality. The issue of DNA qual- Because the original CGH procedure used chromosomes as ity is of particular importance for formalin-fixed paraffin-em- the target for assessing DNA copy number alterations, the res- bedded tissue (FFPE) material. FFPE samples are of impor- olution of such analyses remained limited to approximately 5 tance for cancer research, as they are often more readily to 10 Mb.11 The use of DNA clones spotted in array format on available than frozen samples and thus represent an important slides as targets for hybridization of normal and test DNA (ar- source of archival tissue with long clinical follow-up. The ma- ray CGH) opened the way for a dramatic increase in resolution jor disadvantage to using FFPE samples is DNA degradation up to 30 kb12 and more rapid and streamlined handling of resulting from the type and duration of fixation. Procedures assays, avoiding the tedious production of well-spread chro- have been developed to obtain DNA of sufficient quality, mosome slides and subsequent karyotyping. As a result of these but array CGH on such samples remains challenging and typ- advantages, particularly the broader accessibility for laborato- ically generates lower signal-to-noise ratios. Given the high ries to implement array CGH, this new technique has now cost of array CGH analysis, it is essential to reduce the number become popular and widely applied, among other technologies of failures to a minimum through assessing DNA quality be- for studying gains and losses in tumors.13–16 This widespread fore hybridization. Although a simple DNA gel electrophoresis application in tumor genetics, however, only followed several can be performed for this evaluation, this may require too years after the first technical reports describing the much valuable patient material and may also insufficiently technique.13–15 This can be explained by the limited availability predict samples compliancy for further analysis, indicating of good quality slides with a high density of probes. cDNA that parameters other than size are important for success. It has arrays were more readily available, but the rather low sensitiv- been demonstrated that DNA cross-linking because of the fix- ity makes averaging across multiple clones necessary for scor- ation procedure is also an important factor influencing DNA ing gains and losses.11 BAC clones were suitable alternatives as quality. Therefore, an optimized selection process was devel- probes,17 but quality of spotted slides could vary greatly be- oped. This includes an improved DNA isolation protocol pro- cause of changes in temperature, humidity, BAC DNA concen- moting DNA de–cross-linking, followed by multiplex PCR- tration, and damage to spotting pins. The quality of sample based quality control to assess residual DNA cross-linking, and control DNA, batch variation of Cot I DNA, labeling, and resulting in a more precise estimation of archival samples suit- hybridization conditions are also important factors for able for array CGH analysis.31 Recently, another study pub- success.18 The availability of a 1-Mb BAC set and exchange of lished guidelines for qualifying FFPE DNA samples for geno- experimental information during various meetings18 greatly typing, loss of heterozygosity, and copy number analysis, triggered the dissemination of the technology. At the same including random amplified polymorphic DNA-PCR as a crit- time, the possibility of the use of SNP chips for assessment of ical evaluation step.32 In addition, commercial kits to deter- DNA copy number alterations was discovered,19–22 and im- mine the quality of genomic DNA isolated from FFPE tissue proved high-density oligonucleotide slides were produced, are becoming available. For example the BioScore screening further broadening the accessibility of array CGH platforms and amplification kit (Enzo