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Chromosome aberrations in solid tumors
Author: Joe W. Gray
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"REVIEW NATURE GENETICS VOLUME 34 | NUMBER 4 | AUGUST 2003 369 Human solid tumor genesis and progression are enabled by the aber- rant function of genes that positively and negatively regulate aspects of cell proliferation, apoptosis, genome stability, angiogenesis, inva- sion and metastasis 1 . Discovery and functional assessment of these genes is essential for understanding the biology of cancer and for clin- ical applications, including identification of therapeutic targets, early cancer detection and improved prediction of cancer risk and disease course. Many different factors can result in variant gene function, including polymorphisms, changes in genome copy number and structure, point mutations and epigenetic modifications. Furthermore, the mechanisms by which gene functions are altered vary between tumors. For example, tumor suppressor genes may be inactivated in some tumors by methylation and in others by mutation or physical deletion. Likewise, oncogenes can be activated by muta- tion, structural rearrangement or amplification. Because chromo- some aberrations are distinctive features of tumors that can be detected using both cytogenetic and molecular methods, we review here current knowledge regarding these aberrations in solid tumors and discuss how they arise and how assessment of recurrent chromo- somal changes can be used to improve understanding of tumor devel- opment. In addition, we consider the utility of specific aberrations as markers for prediction of disease outcome or response to treatment and as identifiers of genes to target for therapy or prevention. Chromosomal aberrations in tumors Chromosome aberrations can be analyzed using an increasing num- ber of efficient, large-scale genomic and molecular genetic technolo- gies, such as analysis of chromosome banding (Mitelman Database of Chromosome Aberrations in Cancer), high-throughput analysis of loss of heterozygosity (LOH; ref. 2), comparative genomic hybridiza- tion (CGH; refs. 3?6), fluorescence in situ hybridization (FISH; refs. 7?10), restriction landmark genome scanning (RLGS; ref. 11) and representational difference analysis (RDA; ref. 12). Some of these techniques, including RLGS, analysis of LOH and RDA, detect allelic imbalance that occurs by somatic recombination 13?15 or copy num- ber change, whereas others such as FISH and CGH are only sensitive to physical changes in genome structure or copy number. These analyses identify a broad range of chromosomal abnormalities in solid tumors, including altered ploidy, gain or loss of individual chro- mosomes or portions thereof and structural rearrangements (Fig. 1). The structural changes may involve equal exchange of material between two chromosome regions (balanced) or may be non-recipro- cal, such that portions of the genome are lost or gained. Restricted regions of the genome may be amplified and the amplified sequences present in small acentric fragments (double minutes), incorporated into tumor chromosomes in nearly contiguous homogeneously stain- ing regions (HSRs) or interspersed in the genome. Notably, individ- ual HSRs or other sites of amplified DNA may include genomic DNA originating from multiple different regions 16 . Extensive catalogs of recurrent abnormalities in a wide range of solid tumors have been compiled from cytogenetic (Mitelman Database of Chromosome Aberrations in Cancer) and CGH 17 studies and are available online (see list of URLs at the end of the article). These analyses indicate that there is considerable variability in the degree to which tumor genomes are aberrant at the chromosomal level. Some tumors have few chromosomal aberrations whereas oth- ers may contain dozens. The aberration spectrum, which comprises the numbers and types of aberrations and the regions that are recur- rently altered, differs in tumors that arise in different anatomical sites and in histologically distinct tumors that arise in the same anatomic location 18,19 . Tumor histology and aberration spectrum also vary with the genetic makeup of the affected individual. For example, tumors in individuals with hereditary non-polyposis colorectal can- cer are more likely to occur in the right colon 20 and to have diploid genomes, whereas the genomes of sporadic colorectal cancers are most often aneuploid 21?25 . Similarly, hereditary BRCA1 and BRCA2 breast tumors develop by specific and distinct evolutionary paths, as their gene expression profiles 26 and genome aberration spectra differ from each other and from those in sporadic breast tumors 27,28 . Functional importance There is wide agreement that recurrent genomic aberrations contain genes that are important for tumor development. The importance of recurrent aberrations involving gene dosage is particularly clear. In 1 Cancer Research Institute, 2 Department of Laboratory Medicine and 3 Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California 94143-0808, USA. 4 Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. Correspondence should be addressed to D.G.A. (albertson@cc.ucsf.edu) or J.W.G. (jwgray@lbl.gov). Chromosome aberrations in solid tumors Donna G Albertson 1,2 , Colin Collins 1,2 , Frank McCormick 1,3 & Joe W Gray 2,4 Chromosome aberrations in human solid tumors are hallmarks of gene deregulation and genome instability. This review summarizes current knowledge regarding aberrations, discusses their functional importance, suggests mechanisms by which aberrations may form during cancer progression and provides examples of clinical advances that have come from studies of chromosome aberrations. � 2003 Nature Pub lishing Gr oup http://www .nature .com/natureg enetics many cases, these aberrations contain known oncogenes or tumor suppressor genes whose expression levels are altered by the genomic changes. Classic examples in solid tumors include amplification of established oncogenes, such as ERBB2 (ref. 29), MYC 30 and CCND1 (ref. 31). Amplification also has a key role in the development of drug resistance. Cultured cells selected for resistance to N-(phospho- nacetyl)-L-aspartate 32,33 frequently amplify CAD, and development of resistance to methotrexate in cultured cells and in individuals with cancer is associated with amplification of DHFR 34 . Likewise, amplifi- cation of BCR?ABL is found in individuals resistant to STI571 (ref. 35), and amplification of AR occurs in prostate tumors that become resistant to endocrine therapy 36 . Other aberrations involve loss of specific regions of the genome. For example, deletions are important in the inactivation of tumor suppressor genes, such as PTEN 37 and CDKN2A 38 , and in elimination of the remaining normal alleles in carriers of inherited mutations involving RB1, BRCA1, BRCA2, PTPRJ and TP53 (refs. 39?42). Thus, it is reasonable to expect that additional genes important in cancer progression will be identified by assessment of other recurrently abnormal regions. There are, of course, many more aberrations in solid tumors than the highly recurrent ones. The sheer number and variety has led to the assertion that many, if not most, aberrations are noise. But some evi- dence supports another view, that the seemingly random aberrations generated by failures in processes that normally maintain genome integrity are the result of selection during evolution of the tumor. This notion is consistent with the observation that relatively simple, stereotypical genomic changes arise in cells selected for drug resis- tance 43 and in tumors that arise in mouse models of cancer 44,45 . The stereotypical nature of the aberrations suggests that essentially all are the result of selection during tumor progression. Assessing the extent to which seemingly random elements of the human tumor aberration spectrum contribute or collaborate in the genesis or maintenance of the tumor will be an important area for future study. Cytogenetic analyses have identified many structural chromosome changes in solid tumors (see the Mitelman Database of Chromosome Aberrations in Cancer), but relatively few are recurrent and have been shown to contribute to solid tumor development. In contrast, recurrent structural aberrations are frequent transforming events in sarcomas, leukemias and lymphomas 46 and include characteristic structural aberrations fusing BCR to ABL through the t(9;22) translocation in chronic myelogeneous leukemia 47 and FOXO1A to PAX3 or to PAX7 in rhabdomyosarcoma 48,49 . The paucity of impor- tant structural aberrations in solid tumors could be due to the diffi- culty of identifying and mapping structural rearrangements in these karyotypically complex genomes. But it also could be due to tissue- specific differences in mechanisms of aberration formation. For example, recombination is an essential step in the development of the hematopoietic progenitor cells in which leukemias arise, so there may be more opportunities for genes, such as MYC, EVI1 and CCND1, to be activated by recombination-mediated translocations in leukemias, whereas they are activated more often by amplification and other means in solid tumors 30,50,51 . Identifying the important cancer-related genes in recurrent abnor- malities is not always straightforward because the aberrations often contain multiple genes and more than one may be important. For example, regions that are highly amplified and contain known onco- genes, such as ERBB2 and CCND1, typically span only a few megabases, and yet they may contain more than one gene that con- tributes to tumor progression. Examples include the growth factors FGF19, FGF4, FGF3 and the actin-binding oncogene EMS1 that are located in close proximity to CCND1 and thus usually are amplified with CCND1 (ref. 51). Likewise, the gene encoding growth factor receptor?bound protein, GRB7, is in close proximity to ERBB2. Altered expression of multiple genes included in an amplicon proba- bly contribute to the tumor phenotype. For example, in a recent study of 14 breast cancer cell lines, >40% of highly amplified genes were overexpressed 52 . Moreover, because modulation of the expression of several of these genes influences aspects of tumorigenesis in model systems 53,54 , it seems likely that some genomic rearrangements are selected because they alter the expression of multiple genes that coor- dinately promote tumor progression. The functional consequences of recurrent gains or losses of single copies of regions of the genome, such as the losses of chromosomes 1p and 16q and gains of 1q and 3q that occur frequently in solid tumors, are even harder to establish, as the aberrations often extend over tens to hundreds of megabases and may affect hundreds to thou- sands of genes. The importance in cancer of the changes in gene expression that may be caused by these aberrations is supported by REVIEW 370 VOLUME 34 | NUMBER 4 | AUGUST 2003 NATURE GENETICS Polyploid Aneuploid Reciprocal translocation Non-reciprocal translocation Amplification (double minutes) Amplification (HSR) Banding SKY CGH LOH + + + + + + + + + + + + + + + + + + + + Normal diploid genome LOH (somatic recombination) Normal diploid genome Banding SKY CGH LOH + ab Technique Detection Technique Detection LOH (duplication/loss) + + + Amplification (distributed insertions) Figure 1 Schematic illustration of mechanisms by which chromosomal aberrations arise plus a summary of the ability of commonly applied technologies to detect the aberrations. (a) Aberrations that lead to aneuploidy. (b) Aberrations that leave the chromosome apparently intact. � 2003 Nature Pub lishing Gr oup http://www .nature .com/natureg enetics several reports linking haploinsufficiency of CDKN1B, CDKN2A, MAD2L1, TCF3, BLM and TP53 to cancer 55?60 , and moderately higher copy number of PIK3CA at 3q26 has been associated with activation of the phos- phatidylinositol 3-kinase (PI3-kinase) path- way 61 . In addition, extra copies of individual chromosomes are associated with greater cancer risk 62 and poor clinical outcome in leukemias 63 . Thus, the modest changes in gene dosage caused by recurrent single copy- number aberrations probably do contribute to tumor development or progression. Te chnologies to scan genomes, transcrip- tomes and proteomes 6,64?68 , optimized to allow reliable analyses of subtle changes in copy number or expression, should facilitate assessment of how the involved genes in these large regions exert their effects. Additional support for the functional importance of chromosomal aberrations comes from the observation that many aber- rations influence pathways that regulate cell growth or suppress apoptosis and that are known to contribute to tumor formation when deregulated. For example, Figure 2 shows more than 20 genes involved in cellu- lar signaling that have been found in regions of recurrent genomic abnormality and that may be deregulated by dosage changes. The rules governing how these pathways are deregulated are not yet clear but seem to vary according to tumor/tissue type and pathway. The RB pathway controlling entry into S phase provides an example. Inactivation of single genes in the path- way seems to be sufficient to alter cell cycle regulation in some tumors. In glioblastoma, for example, deletion of RB1, amplification of CDK4 or inactivation of CDKN2A each seems to be sufficient to inactivate the pathway 69,70 , whereas in melanoma, inactivation of RB1, mutation of CDK4 or deletion of CDKN2A seem to be sufficient and perhaps mutually exclusive events 71 . In other cases, alterations in two RB pathway members may cooperate. For example, in head and neck cancers, deletion of CDKN2A and amplification of CCND1 together are associated with a greater relative risk for recurrence, metastasis and death than either genetic alteration alone 72 . These observations are consistent with the effects of these genes on prolifer- ation of cells in culture, where deregulation of both genes provides a greater growth advantage than either alone 73 . Analyses of data from large-scale measurements of genome copy number, gene expression and methylation that are now underway should refine our under- standing of how the multiple aberrations interact. Evolution of chromosomal aberrations in tumors Tumor development typically proceeds through deregulation of gene expression such that the cells no longer respond to environmental cues and gain the capability to survive and proliferate inappropriately. Studies modeling tumor development in cultured cells or in rodents show that deregulation of only a few optimally chosen genes can initi- ate the process 74 and that the karyotypes of the resultant tumors can be near normal 75,76 . For example, overexpression of MYC and BCL2L1 establish conditions that favor cell proliferation and decrease Figure 2 Schematic representation of receptor tyrosine kinase?mediated signaling. Factors found to be amplified in human tumors are indicated in green and those found to be frequently lost are indicated in red. Black lines indicate direct activation (arrowheads) or inhibition (bars), blue lines indicate transcriptional activation and dotted black lines indirect effects. CDK2 EGFR, ERBB2, FGFR1 HRAS KRAS2 NRAS BAD RAL CCND1 CDK4 S phase CDKN1A Apoptosis CDKN2A RAF/MAP kinase cascade cascade Ets family Transcription factors Ets family transcription factors FOS SRC MYC NMYC PIK3CA PTEN PDK1 AKT1 AKT2 AKT3 RB1 E2F Transcription factors E2F5 Transcription factors CDKN1B Forkhead Transcription factors Forkhead transcription factors CDKN2AMDM2 MDM2MDM4 RPS6KB1 CCNE1 TP53 TNFSF6 RalGEFPRKCE PIP3 cell death and ultimately result in lesions that have the properties of human malignancies, including invasion, angiogenesis and metasta- sis. The genesis of human tumors, on the other hand, probably differs significantly from the relatively simple few-gene models that have been developed in vitro and in mice, because it is highly unlikely that altered expression of a few perfectly compensating genes occurs simultaneously by random mutation. More likely, human tumors evolve by accumulation of multiple alterations in small steps to tip the balance in favor of proliferation and tumor development. Chromosomal change is one mechanism by which cells might tip- toe towards cancer. But the importance of chromosomal aberrations in tumor development varies substantially between tumors (Fig. 3). Some tumors undergo marked chromosome rearrangement 77 whereas others may evolve by mechanisms that result in little chro- mosomal change 25 . This variability may be due to differences in the mechanisms by which tumors are initiated, the manner in which genome stability is compromised 78 , individual genotype or the par- ticular epithelial cell type in which the tumor arises. The aberration spectrum also may be influenced by the stochastic nature of aberra- tion formation. In those tumor types that are associated with chro- mosomal changes, the number of aberrations typically is small in premalignant, hyperproliferative lesions and substantially greater in more advanced lesions, supporting a role for acquisition of chromo- somal aberrations in tumor progression. In breast cancer, for exam- ple, the aberration frequency is low in hyperplasia but often sharply higher in carcinoma in situ 79,80 , whereas in colon cancer, genomic aberrations are present in high-grade dysplasias and adenomas 81 but REVIEW NATURE GENETICS VOLUME 34 | NUMBER 4 | AUGUST 2003 371 � 2003 Nature Pub lishing Gr oup http://www .nature .com/natureg enetics Figure 3 Schematic illustration of chromosomal evolution in human solid tumor progression. The stages of progression are arranged with the earlier lesions at the top. Cells may begin to proliferate excessively owing to loss of tissue architecture, abrogation of checkpoints and other factors. In general, relatively few aberrations occur before the development of in situ cancer. As indicated, a sharp increase in genome complexity (the number of independent chromosomal aberrations) in many (but not all) tumors coincides with the development of in situ disease. The types and range in aberration number varies markedly between tumors, probably owing to the specific failures in checkpoints or damage surveillance that are present, as illustrated by the whole-genome array CGH profiles of HCT116, a mismatch repair?defective cell line, and T47D, a mismatch repair?proficient cell line 64 . The copy number profiles of HCT116 and T47D are labeled as ?simple? and ?complex?, respectively, to distinguish between tumor genomes with few or many copy number changes. The spectrum of aberrations in in situ lesions is similar to those found in more advanced malignancies. Thus, an early increase in chromosome aberration composition is followed by more modest chromosomal evolution. significantly greater in number in carcinomas 81,82 . Notably, the chromosome aberration spectrum seems to stabilize in advanced cancers, as judged by the similarities between tumor genomes in within-individual comparisons of in situ and invasive lesions 83 , pri- mary and recurrent tumors 84 and primary and metastatic tumors 85 . These observations may indicate that the rate of chromosomal evo- lution has decreased in carcinomas, which could occur if some of the causes of chromosomal instability, to be discussed below, were removed. It is also possible, however, that tumors seem stable because advanced tumors have evolved a genotype that is optimized for growth and dissemination, making it less probable that addi- tional lesions will confer further growth advantage. The remarkable karyotypic stability of established tumor cell lines in culture over many generations in many different laboratories supports this idea. These cells do show substantial cell-to-cell variability but the average genotype is stable 86 . Thus, selection is a strong force that eventually leads to an optimal, slowly evolving genotype. Deregulation of processes responsible for maintaining genome integrity is probably important in aberration formation. Eukaryotic cells are subject to continual DNA damage from both extrinsic (e.g., radiation, chemicals) and intrinsic (e.g., reactive oxygen, stalling of DNA replication forks) sources. In fact, it has been estimated that as many as ten double-strand breaks occur per cell cycle 87 , offering numerous opportunities for chromosomal rearrangements to occur. For this reason, elaborate systems have evolved to monitor genome integrity and coordinate cell cycle pro- gression with DNA repair. More than 70 genes have been identified that have roles in DNA damage surveillance and repair 88 . These include genes involved in mismatch repair (e.g., MSH2, MLH1), non-homologous end joining (e.g., XRCC5, LIG4, XRCC4, PRKDC), homologous recombination (e.g., RAD51, BRCA1, BRCA2) and signaling cascades responding to DNA damage (e.g., ATM, ATR, CHEK1, CHEK2, TP53, BRCA1, BRCA2, BLM and NBS1; refs. 89?94). Different aberration spectra are associated with failures in the various systems. For example, abrogation of the function of MSH2 or MLH1, genes involved in mismatch repair, result in tumors with few chromosomal aberrations but consider- able microsatellite alterations 21,22,24,25,95 . Defects in genes involved in the repair of DNA double-strand breaks may lead to chromo- some aberrations, such as translocations or amplifications (Fig. 1), and may be associated with high levels of chromosomal abnormal- ity as observed in tumors arising in individuals or mouse models carrying mutations in BRCA1 (refs. 27,96). Chromosome gains or losses also may be expected when genes involved in chromosome segregation or cytokinesis are deregu- lated 78,97?102 . Aberrant centrosome behavior, termed centrosome amplification, is associated with mutation or loss of function of such genes as TP53, STK15, RB1 and BRCA1. Centrosome amplification is characterized by the presence of abnormally large centrosomes, which may have >4 centrioles and altered functional properties, including hyperphosphorylation, increased microtubule nucleating capacity, abnormal centriole orientation and basal rather than apical position relative to the nucleus. It has been proposed as a primary source of genome instability in tumors, because it is associated with multipolar mitotic spindles, aneuploidy and unstable karyotypes. Thus, centrosome amplification may be a cause of both chromoso- mal instability and anaplasty in tumors. Another form of chromosome instability may occur if tumors originate in somatic cells with inactive telomerase 103,104 . Continued proliferation of these cells will result in progressive telomere short- ening. If surveillance mechanisms are sufficiently intact, cells will cease proliferating when telomeres reach a crucial short length 105?108 . Thus, telomere function provides a barrier to cancer development and may explain why benign lesions arrest 109,110 . If checkpoints are sufficiently compromised, however, then the chro- mosomes of cells with dysfunctional telomeres become susceptible to end-to-end fusions and subsequent breakage during cell division. The cells may undergo repeated rounds of aberrant cell division and genome reorganization. Most of the cells probably do not survive owing to abnormal and lethal chromosome rearrangements 108,111 . REVIEW 372 VOLUME 34 | NUMBER 4 | AUGUST 2003 NATURE GENETICS Escape from tissue organization Normal epithelium Hyperplasia Carcinoma in situ Invasive cancer Metastatic cancer Enhanced proliferation Loss of cell cycle regulation Extended life span e.g. Loss of APC (colon) RAS CCND1 CMYC CDKN1A RB1 e.g. Stabilization Telomere stabilization Genome and chromosome instability Nucleotide level defects (mutation, microsatellite instability) Epigenetic alterations (methylation) Double-strand breaks Telomere erosion Cell division defects (centrosome amplification) 3 2 1 0 1 2 0 500 1,000 1,500 2,000 2,500 log2ratio Genome order 3 2 1 0 1 2 0 500 1,000 1,500 2,000 2,500 log2ratio Genome order Simple Complex Genome complexity � 2003 Nature Pub lishing Gr oup http://www .nature .com/natureg enetics Rarely, however, a cell with proliferative capacity may re-establish telomere function by re-activating telomerase 110,112 or an alternative telomere maintenance mechanism (ALT; ref. 113). The extent of chromosome rearrangement that might occur during such ?telomere crisis? may be substantial but probably varies depending on the num- ber of cell divisions in the presence of eroded telomeres, the func- tionality of other telomere maintenance genes and the types of damage surveillance systems that have been compromised. A role for telomere dysfunction in the generation of chromosomal aberrations is supported by several observations. First, telomerase is active in most cancers but is less commonly observed to be active in hyperplastic lesions 114?116 , and second, tumor telomeres are short rel- ative to normal cells, consistent with a period of erosion 103,117 . Furthermore, the high level of genome rearrangement that occurs in some late-generation telomerase-knockout mice supports a role for telomere dysfunction in the generation of copy number aberra- tions 118 . On the other hand, a large number of tumors show few copy number changes. These tumors may have arisen in epithelial stem cells with active telomerase 110,119?121 , telomere function may have been re-established before extensive genomic rearrangement, or they may be evolving by non-chromosomal mechanisms. Clinical utility Markers for chromosome aberrations that are important in solid tumor formation or progression facilitate cancer detection, predic- tion of clinical outcomes and response to therapy. In addition, identi- fication of the genes involved in regions of recurrent aberration may be attractive targets for the development of new therapies. Cancer detection. One promising approach to cancer detection uses FISH to detect cancer cells carrying specific numerical or struc- tural aberrations. This approach has been used to assay exfoliated urothelial carcinoma cells in voided urine 122 , lung cancer cells obtained using bronchoscopy 123 and cancer cells collected using fine needle aspiration 124 . FISH also has been used to detect rare dissemi- nated cancer cells. For example, one study used CGH to detect spe- cific genomic aberrations in primary tumors of acral melanoma and then used FISH to detect tumor cells carrying these aberrations in distant histologically normal regions to determine whether surgical margins were clear of tumor cells 109 . Extension of this approach to other tumor types promises to increase the accuracy of tumor margin assessment. FISH also has been used to detect circulating carcinoma cells in bone marrow and peripheral blood 125 . This assay eventually may be developed to identify individuals with metastatic disease who can then be offered systemic therapy. It is currently limited, however, by the low frequency of disseminated cancer cells and by an inability to distinguish between true metastatic cells and disseminated tumor cells that are not capable of proliferating at distant sites. In another approach, FISH has been used to determine if histologically ambigu- ous lesions, such as Spitz nevi, are benign by ascertaining if the lesions contain cells with aberrations characteristic of those that do not progress to cancer 126 . Analyses of microsatellite marker abnormalities also are proving useful for sensitive cancer detection. These PCR-based analyses screen for common tumor-associated genomic abnormalities in DNA collected from body fluids including feces, sputum, urine and serum. These analyses may target either exfoliated cells or free DNA. These assays have already been shown to be useful for early detection of can- cers of the lung 127 , colon 128 , kidney 129 , head and neck 130 and blad- der 131 . Combining these assays with sensitive imaging technologies may prove especially valuable in detecting tumors sufficiently early that they can be curatively treated using surgery. Prognosis and prediction. Numerous associations between genomic abnormalities and clinical behavior have been established. The association of amplification of ERBB2 with reduced survival duration in individuals with breast cancer is a prototypical example 29 . Amplification of MYCN in metastatic neuroblastoma in infants is an even stronger negative prognostic indicator 132 . Large-scale genome profiling analyses have established other prognostic associations 133 . Typically, these result in stratification strategies for affected individu- als that involve multiple genes. Although strategies based on detec- tion of chromosome aberrations show promise, those based on gene expression currently seem more powerful. Microarray-based analyses of gene expression, for example, have shown the ability to identify individuals with breast cancer with short disease-free survival 68,134,135 or with metastatic disease 136 . These observations are interesting as they suggest that propensity to recur or metastasize is an intrinsic property of the primary tumor. To date, prognostic associations have not been widely used clini- cally owing to the lack of alternative therapeutic strategies that can be applied to the different subgroups. But this situation is changing as gene-targeted therapeutics become available. Again, ERBB2 serves as an example. ERBB2 testing was of modest clinical interest until the development of Herceptin as a treatment for tumors that overexpress ERBB2. Now, analysis of ERBB2 expression and amplifi- cation is an essential guide to treatment 137 . Regrettably, response to Herceptin and other targeted therapeutics can be predicted with only moderate accuracy, even when using the best assays for target expression. For example, only 30?40% of individuals who overex- press ERBB2 will respond to Herceptin 138 . One possibility is that tumors may not respond because signaling pathways are deregu- lated at multiple points. The presence of activating aberrations downstream from ERBB2 or in parallel to it supports this possibility (Fig. 2). Large-scale, therapy-linked molecular profiling efforts now underway may resolve such issues. Ta rget identification. Recurrent genomic aberrations are good indicators of genes that contribute causally to cancer genesis or pro- gression and thus help to identify gene targets for therapy. For exam- ple, Figure 2 illustrates several genes involved in receptor tyrosine kinase?mediated signaling that are recurrently abnormal, including ERBB2, FGFR1, and EGFR. The encoded receptors are targeted by monoclonal antibodies IMC-C225 and Herceptin and the small mol- ecule tyrosine kinase inhibitors Iressa (ZD1839), Gleevec (STI571) and OSI-774 (refs. 139?141). Herceptin, a monoclonal antibody?based antagonist of ERBB2, is already approved for breast cancer 138 , and Gleevec is approved for the treatment of chronic myeloid leukemia and shows activity against gastrointestinal stromal tumors 142 . Aberrations downstream in signaling pathways also sug- gest therapeutic opportunities. For example, amplification of AKT2, RPS6KB1 and PIK3CA 61,143,144 and deletion of the negative regulator PTEN 37 all have been reported to activate PI3-kinase mediated signal- ing. These observations suggest the utility of PI3-kinase inhibitors, such as the rapamycin analog CCI-779 (ref. 145). These examples illustrate the potential of genome-based approaches to therapeutic target identification. It is important to note, however, that the absence of a genomic aberration does not indicate lack of response. As noted above, pathway activation may occur by genomic and non-genomic means, so, in general, recurrent genomic aberrations should be used to identify genes that are important therapeutic targets but not to estimate the fraction of tumors that might respond. In most cases, gene expression is expected to be a better indicator of response, but ERBB2 may be an exception because, at least in breast cancer, overex- pression is almost always caused by gene amplification 3 . REVIEW NATURE GENETICS VOLUME 34 | NUMBER 4 | AUGUST 2003 373 � 2003 Nature Pub lishing Gr oup http://www .nature .com/natureg enetics REVIEW The recurrent aberration maps assembled by Knuutila 17 and Mitelman suggest that many additional therapeutic targets remain to be discovered. Particular types of genome instability present in a tumor also may be of importance in planning treatment regimens. For exam- ple, cells with defects in mismatch repair are resistant to cisplatin 146 . Thus, identification of failures in particular mechanisms for mainte- nance of genome stability that might be present in tumors may provide both additional therapeutic targets and contraindications for others. For the future Identification of chromosomal abnormalities in solid tumors is becoming easier as genome-wide analysis technologies improve and as the genome sequence nears completion. Precise measurements of genome copy number analyses with sub-megabase resolution are now possible using array CGH with BAC arrays 45,64,147 , and CGH with cDNA arrays allows analysis of individual genes 6,52 , albeit at some- what lower measurement precision. Improvements of these technolo- gies in the near future will probably enable analyses that precisely interrogate essentially the entire genome for copy number changes. At the same time, multicolor FISH and optical mapping strategies based on microscopic analyses of individual DNA fibers digested with restriction enzymes 148 may facilitate identification of recurrent struc- tural aberrations at high resolution and low cost. Application of these techniques will provide a detailed view of the spectrum of chromo- some abnormalities that occur in human solid tumors. But chromo- some aberrations are only one of several mechanisms by which genes are deregulated in solid tumors. Other technologies are now emerging to provide equally compre- hensive information about gene sequence, methylation, transcrip- tion, protein composition and protein phosphorylation status so that our knowledge of the changes that occur to enable tumor progression are more complete. High-throughput mutation detection strategies are now sufficiently advanced that it is possible to screen for gene mutations and polymorphisms in entire tumor genomes 149 . This approach has already led to identification of BRAF as a tumor sup- pressor gene that is important in malignant melanoma and other tumor types 149 . Transcription analyses of most known genes and a substantial number of splice variants are already possible using cDNA 150 or oligonucleotide 151 arrays, although high-throughput, quantitative PCR approaches 152 may challenge array-based methods when measurement precision is paramount. Serial analysis of gene expression (SAGE) provides unbiased, quantitative analyses of gene expression but at comparatively high cost 153 . Other nucleic acid analysis techniques are allowing efficient assessment of allele-specific expression 154 , methylation status 65,149,155,156 and loss of heterozygos- ity (including homozygous deletions; ref. 37). Analyses of the cancer proteome using antibody arrays 157 , immunostained cell lysate arrays 158 and mass spectrometry 159 are allowing efficient assessment of differences in protein composition of tumors. Furthermore, these analytical approaches can be combined, as for example, ChIP-on- chips in which the identity of genomic DNA recovered by chromatin immunoprecipitation is read using microarrays 160,161 . The amount of information becoming available on the biology and genetics of human tumors is staggering. The challenge now is to inte- grate these diverse data with information on clinical behavior, pathol- ogy, drug response, deregulated pathways and processes and with comparable information for the mouse. Several integration efforts are now underway, including databases that map diverse data onto the human genome and mouse sequences. The US National Cancer Institute Cancer Genome Anatomy Project is one example of effort aimed at integrating information on chromosome aberrations, mole- cular profiles, genome sequences, clinical behavior and clinical trials. Successful integration of information from these approaches should provide a more complete picture of the ways in which gene deregula- tion occurs in solid tumors, how multi-gene deregulation leads to specific tumor phenotypes and how this information can be used to improve cancer management 162 . URLs. The Mitelman Database of Chromosome Aberrations in Cancer is available at http://cgap.nci.nih.gov/Chromosomes/Mitelman. More informa- tion about the University of Helsinki?s Laboratory of Cytomolecular Genetics is available at http://www.helsinki.fi/?lgl_www/CMG.html. More information about the US National Cancer Institute Cancer Genome Anatomy Project is available at http://cgap.nci.nih.gov/. ACKNOWLEDGMENTS This work was supported by the United States Public Health Service and the Avon Foundation. Published online 30 July 2003; doi:10.1038/ng1215 1. Hanahan, D. & Weinberg, R.A. The hallmarks of cancer. Cell 100, 57?70 (2000). 2. Hampton, G.M. et al. 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Subcellular Compartments
Cell Communication
Cell Cycle and Cell Division
Scientific Communication
Career Planning
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