CGH, cDNA and tissue microarray analyses implicate FGFR2 amplification in a small subset of breast tumors.

Multiple regions of the genome are often amplified during breast cancer development and progression, as evidenced in a number of published studies by comparative genomic hybridization (CGH). However, only relatively few target genes for such amplifications have been identified. Here, we indicate how small-scale commercially available cDNA and CGH microarray formats combined with the tissue microarray technology enable rapid identification of putative amplification target genes as well as analysis of their clinical significance. According to CGH, the SUM-52 breast cancer cell line harbors several high-level DNA amplification sites, including the 10q26 chromosomal region where the fibroblast growth factor receptor 2 (FGFR2) gene has been localized. High level amplification of FGFR2 in SUM-52 was identified using CGH analysis on a microarray of BAC clones. A cDNA microarray survey of 588 genes showed >40-fold overexpression of FGFR2. Finally, a tissue microarray based FISH analysis of 750 uncultured primary breast cancers demonstrated in vivo amplification of the FGFR2 gene in about 1% of the tumors. In conclusion, three consecutive microarray (CGH, cDNA and tissue) experiments revealed high-level amplification and overexpression of the FGFR2 in a breast cancer cell line, but only a low frequency of involvement in primary breast tumors. Applied to a genomic scale with larger arrays, this strategy should facilitate identification of the most important target genes for cytogenetic rearrangements, such as DNA amplification sites detected by conventional CGH. Figures on http://www.esacp.org/acp/2001/22-4/heiskanen.htm     

Lack of an Additive Effect between the Deletions of Klf5 and Nkx3-1 in Mouse Prostatic Tumorigenesis

Prostate cancer is one of the most common malignancies.The development and progression of prostate cancer are driven by a series of genetic and epigenetic events including gene amplification that activates oncogenes and chromosomal deletion that inactivates tumor suppressor genes.Whereas gene amplification occurs in human prostate cancer,gene deletion is more common,and a large number of chromosomal regions have been identified to have frequent deletion in prostate cancer,suggesting that tumor suppressor inactivation is more common than oncogene activation in prostatic carcinogenesis (Knuutila et al.,1998,1999;Dong,2001).Among the most frequently deleted chromosomal regions in prostate cancer,target genes such as NKX3-1 from 8p21,PTENfrom 10q23 andATBF1 from 16q22 have been identified by different approaches (He et al.,1997;Li et al.,1997;Sun et al.,2005),and deletion of these genes in mouse prostates has been demonstrated to induce and/or promote prostatic carcinogenesis.For example,knockout of Nkx3-1 in mice induces hyperplasia and dysplasia (Bhatia-Gaur et al.,1999;Abdulkadir et al.,2002) and promotes prostatic tumorigenesis (Abate-Shen et al.,2003),while knockout of Pten alone causes prostatic neoplasia (Wang et al.,2003).Therefore,gene deletion plays a causal role in prostatic carcinogenesis (Dong,2001).     

Adipose tissue gene expression analysis reveals changes in inflammatory, mitochondrial respiratory and lipid metabolic pathways in obese insulin-resistant subjects

Background

To elucidate gene expression associated with copy number changes, we performed a genome-wide copy number and expression microarray analysis of 25 pairs of gastric tissues.

Methods

We applied laser capture microdissection (LCM) to obtain samples for microarray experiments and profiled DNA copy number and gene expression using 244K CGH Microarray and Human Exon 1.0 ST Microarray.

Results

Obviously, gain at 8q was detected at the highest frequency (70%) and 20q at the second (63%). We also identified molecular genetic divergences for different TNM-stages or histological subtypes of gastric cancers. Interestingly, the C20orf11 amplification and gain at 20q13.33 almost separated moderately differentiated (MD) gastric cancers from poorly differentiated (PD) type. A set of 163 genes showing the correlations between gene copy number and expression was selected and the identified genes were able to discriminate matched adjacent noncancerous samples from gastric cancer samples in an unsupervised two-way hierarchical clustering. Quantitative RT-PCR analysis for 4 genes (C20orf11, XPO5, PUF60, and PLOD3) of the 163 genes validated the microarray results. Notably, some candidate genes (MCM4 and YWHAZ) and its adjacent genes such as PRKDC, UBE2V2, ANKRD46, ZNF706, and GRHL2, were concordantly deregulated by genomic aberrations.

Conclusions

Taken together, our results reveal diverse chromosomal region alterations for different TNM-stages or histological subtypes of gastric cancers, which is helpful in researching clinicopathological classification, and highlight several interesting genes as potential biomarkers for gastric cancer.     

Coupled analysis of gene expression and chromosomal location

Microarray technology can be used to assess simultaneously global changes in expression of mRNA or genomic DNA copy number among thousands of genes in different biological states. In many cases, it is desirable to determine if altered patterns of gene expression correlate with chromosomal abnormalities or assess expression of genes that are contiguous in the genome. We describe a method, differential gene locus mapping (DIGMAP), which aligns the known chromosomal location of a gene to its expression value deduced by microarray analysis. The method partitions microarray data into subsets by chromosomal location for each gene interrogated by an array. Microarray data in an individual subset can then be clustered by physical location of genes at a subchromosomal level based upon ordered alignment in genome sequence. A graphical display is generated by representing each genomic locus with a colored cell that quantitatively reflects its differential expression value. The clustered patterns can be viewed and compared based on their expression signatures as defined by differential values between control and experimental samples. In this study, DIGMAP was tested using previously published studies of breast cancer analyzed by comparative genomic hybridization (CGH) and prostate cancer gene expression profiles assessed by cDNA microarray experiments. Analysis of the breast cancer CGH data demonstrated the ability of DIGMAP to deduce gene amplifications and deletions. Application of the DIGMAP method to the prostate data revealed several carcinoma-related loci, including one at 16q13 with marked differential expression encompassing 19 known genes including 9 encoding metallothionein proteins. We conclude that DIGMAP is a powerful computational tool enabling the coupled analysis of microarray data with genome location.     

Identification of frequent cytogenetic aberrations in hepatocellular carcinoma using gene-expression microarray data

Background  

Hepatocellular carcinoma (HCC) is a leading cause of death worldwide. Frequent cytogenetic abnormalities that occur in HCC suggest that tumor-modifying genes (oncogenes or tumor suppressors) may be driving selection for amplification or deletion of these particular genetic regions. In many cases, however, the gene(s) that drive the selection are unknown. Although techniques such as comparative genomic hybridization (CGH) have traditionally been used to identify cytogenetic aberrations, it might also be possible to identify them indirectly from gene-expression studies. A technique we have called comparative genomic microarray analysis (CGMA) predicts regions of cytogenetic change by searching for regional gene-expression biases. CGMA was applied to HCC gene-expression profiles to identify regions of frequent cytogenetic change and to identify genes whose expression is misregulated within these regions.     

A molecular cytogenetic approach to studying platinum resistance

The technique of comparative genomic hybridisation (CGH) has until recently been used to screen for common genomic abnormalities in fresh tumour material; it has identified previously unrecognised regions of amplification associated with poor prognosis subtypes of breast cancer and lymphoma. Our group has applied this technique to resistant cell lines and their sensitive counterparts in order to define chromosomal abnormalities associated with acquired drug resistance. We have demonstrated the applicability of this technique to the study of drug resistance using cell lines with known mechanisms of resistance. The ability to detect novel genomic alterations in cell lines with novel mechanisms of resistance was also demonstrated. We subsequently examined the CGH profiles of seven different cell lines made resistant to three platinum analogues and showed the most consistent abnormalities to involve over-representation of regions 4q and 6q. More recently, we have applied the CGH technique to a series of testicular germ cell tumours (TGCTs) collected as formalin-fixed paraffin-embedded biopsy specimens from patients, both pre- and post-therapy using a platinum-based regimen (POMB/ACE). Previous reports have shown over-representation of X, 7q, 8q and 12p and loss of 13q to occur in 25% of primary TGCTs. Over-representation of 12p was confirmed in the majority of these biopsy samples; deletion of 13q was noted in the initial biopsies of several patients. We also demonstrated alterations of 4p, 4q, 5q and 6q in this series of patients. Newly acquired deletions of 2q and 18q and amplifications of 8q were frequently observed in post-chemotherapy samples from resistant tumours. The CGH studies on these patients with TGCT will not only enable us to correlate our observations on clinical material with those from long-term cell lines, but should also identify sites of key genes involved in clinical platinum resistance.     

Cutaneous melanoma: fishing with chips

DNA microarray technology is a versatile platform that allows rapid genetic analysis to take place on a genome-wide scale and has revolutionized the way cancers are studied. This platform has enabled researchers to characterize mechanisms central to tumorigenesis and understand important molecular events in the multi-step tumor progression model of cutaneous melanoma and other cancers. In melanoma, multiple global gene expression profiling studies using various DNA microarray platforms and various experimental designs have been performed. Each study has been able to capture and characterize either the involvement of a novel pathway or a novel cause-effect-relationship. The use of microarrays to define subclasses, to identify differentially regulated genes within a mutational context to analyze epigenetically regulated genes has resulted in an unprecedented understanding of the biology of cutaneous melanoma that may lead to more accurate diagnosis, more comprehensive prognosis, prediction and more effective therapeutic interventions. Related DNA microarray platforms like array-comparative genomic hybridization (CGH) have also been instrumental to identify many non-random chromosomal alterations; however, studies identifying validated targets as a result of CGH are limited. Thus, there exists significant opportunity to discover novel melanoma genes and translate such discoveries into meaningful clinical endpoints. In this review, we focus on various DNA microarray-based studies performed in cutaneous melanoma and summarize our current understanding of the genetics and biology of melanoma progression derived from accumulating genomic information.     

Origin and functional significance of large-scale chromosomal imbalances in neuroblastoma

Neuroblastoma is characterized by numerous recurrent large-scale chromosomal imbalances and gene amplifications which are associated with poor clinical outcome. The most common include MYCN amplification, loss of 1p, 3p and 11q, and gain of 17q genomic regions. Two of these abnormalities, MYCN amplification and loss of 11q, define different genetic subtypes of the disease with vastly different global gene expression profiles. The progress towards the identification of the genes and genetic pathways that have been affected by these abnormalities is reviewed and high resolution mapping of the chromosomal breakpoint regions using oligonucleotide array CGH (oaCGH) is discussed. oaCGH analysis is proving useful for both defining minimal regions of overlap of imbalances, as well as providing information on the molecular mechanisms that generate the chromosomal imbalances. These high resolution analyses have also permitted the detection of micro-deletions in the tumors that further assist in identifying genes important for neuroblastoma pathogenesis.     

Integrated Genomic Analysis Implicates Haploinsufficiency of Multiple Chromosome 5q31.2 Genes in De Novo Myelodysplastic Syndromes Pathogenesis

Deletions spanning chromosome 5q31.2 are among the most common recurring cytogenetic abnormalities detectable in myelodysplastic syndromes (MDS). Prior genomic studies have suggested that haploinsufficiency of multiple 5q31.2 genes may contribute to MDS pathogenesis. However, this hypothesis has never been formally tested. Therefore, we designed this study to systematically and comprehensively evaluate all 28 chromosome 5q31.2 genes and directly test whether haploinsufficiency of a single 5q31.2 gene may result from a heterozygous nucleotide mutation or microdeletion. We selected paired tumor (bone marrow) and germline (skin) DNA samples from 46 de novo MDS patients (37 without a cytogenetic 5q31.2 deletion) and performed total exonic gene resequencing (479 amplicons) and array comparative genomic hybridization (CGH). We found no somatic nucleotide changes in the 46 MDS samples, and no cytogenetically silent 5q31.2 deletions in 20/20 samples analyzed by array CGH. Twelve novel single nucleotide polymorphisms were discovered. The mRNA levels of 7 genes in the commonly deleted interval were reduced by 50% in CD34+ cells from del(5q) MDS samples, and no gene showed complete loss of expression. Taken together, these data show that small deletions and/or point mutations in individual 5q31.2 genes are not common events in MDS, and implicate haploinsufficiency of multiple genes as the relevant genetic consequence of this common deletion.     

Comprehensive genetic analysis of cancer cells

Human cancer is viewed as a disorder of genes originating from the progeny of a single cell that has accumulated multiple genetic alterations. The genetic alterations include point mutation, chromosomal rearrangements and imbalances. Amplifications primarily involve oncogenes whose overexpression leads to growth deregulation, while deletions commonly target tumor suppressor genes that control cell cycle checkpoints and DNA repair mechanisms. With the advent of molecular cytogenetics procedures for global detection of genomic imbalances and for multicolor visualization of structural chromosome changes, as well as the completion of human genome mapping and the development of microarray technology for serial gene expression analysis of the entire genomes, a significant progress has been made in uncovering the molecular basis of cancer. The major challenge in cancer biology is to decipher the molecular anatomy of various cancers and to identify cancer-related genes that now comprise only a fraction of human genes. The complete genetic anatomy of specific cancers would allow a better understanding of the role of genetic alterations in carcinogenesis, provide diagnostic and prognostic markers and discriminate between cells at different stages of progression toward malignancy. This review highlights current technologies that are available to explore cancer cells and outlines their application to investigations in human hepatocellular carcinoma.     

Identification of Genomic Alterations in Pancreatic Cancer Using Array-Based Comparative Genomic Hybridization

Background

Genomic aberration is a common feature of human cancers and also is one of the basic mechanisms that lead to overexpression of oncogenes and underexpression of tumor suppressor genes. Our study aims to identify frequent genomic changes in pancreatic cancer.

Materials and Methods

We used array comparative genomic hybridization (array CGH) to identify recurrent genomic alterations and validated the protein expression of selected genes by immunohistochemistry.

Results

Sixteen gains and thirty-two losses occurred in more than 30% and 60% of the tumors, respectively. High-level amplifications at 7q21.3–q22.1 and 19q13.2 and homozygous deletions at 1p33–p32.3, 1p22.1, 1q22, 3q27.2, 6p22.3, 6p21.31, 12q13.2, 17p13.2, 17q21.31 and 22q13.1 were identified. Especially, amplification of AKT2 was detected in two carcinomas and homozygous deletion of CDKN2C in other two cases. In 15 independent validation samples, we found that AKT2 (19q13.2) and MCM7 (7q22.1) were amplified in 6 and 9 cases, and CAMTA2 (17p13.2) and PFN1 (17p13.2) were homozygously deleted in 3 and 1 cases. AKT2 and MCM7 were overexpressed, and CAMTA2 and PFN1 were underexpressed in pancreatic cancer tissues than in morphologically normal operative margin tissues. Both GISTIC and Genomic Workbench software identified 22q13.1 containing APOBEC3A and APOBEC3B as the only homozygous deletion region. And the expression levels of APOBEC3A and APOBEC3B were significantly lower in tumor tissues than in morphologically normal operative margin tissues. Further validation showed that overexpression of PSCA was significantly associated with lymph node metastasis, and overexpression of HMGA2 was significantly associated with invasive depth of pancreatic cancer.

Conclusion

These recurrent genomic changes may be useful for revealing the mechanism of pancreatic carcinogenesis and providing candidate biomarkers.     

Tumor suppressor microRNAs: A novel non-coding alliance against cancer

Tumor initiation and progression are the outcomes of a stepwise accumulation of genetic alterations. Among these, gene amplification and aberrant expression of oncogenic proteins, as well as deletion or inactivation of tumor suppressor genes, represent hallmark steps. Mounting evidence collected over the last few years has identified different populations of non-coding RNAs as major players in tumor suppression in almost all cancer types. Elucidating the diverse molecular mechanisms underlying the roles of non-coding RNAs in tumor progression might provide illuminating insights, potentially able to assist improved diagnosis, better staging and effective treatments of human cancers. Here we focus on several groups of tumor suppressor microRNAs, whose downregulation exerts a profound oncologic impact and might be harnessed for the benefit of cancer patients.     

Genome-wide combination profiling of copy number and methylation offers an approach for deciphering misregulation and development in cancer cells

Copy number changes and DNA methylation alterations are crucial to gene regulation in mammals. Recently, a number of microarray studies have been based on copy number and DNA methylation alterations in order to find clinical biomarkers of carcinogenesis. In this study, we attempted to combine profiles of copy number and methylation patterns in four human cancer cell lines using BAC microarray-based approaches and we detected several clinically important genes which showed genetic and epigenetic relationships. Within the clones analyzed, many contained cancer-related genes involved in cell cycle regulation, cell division, signal transduction, tumor necrosis, cell differentiation, and cell proliferation. One clone included the FHIT gene, a well-known tumor suppressor gene involved in various human cancers. Our combined profiling techniques may provide a method by which to find new clinicopathologic cancer biomarkers, and support the idea that systematic characterization of the genetic and epigenetic events in cancers may rapidly become a reality.     

Differential gene alteration among hepatoma cell lines demonstrated by cDNA microarray-based comparative genomic hybridization

We assayed chromosomal abnormalities in hepatoma cell lines using the microarray-based comparative genomic hybridization (array-CGH) method and investigated the relationship between genomic copy number alterations and expression profiles in these hepatoma cell lines. We modified a cDNA array-CGH assay to compare genomic DNAs from seven hepatoma cell lines, as well as DNA from two non-hepatoma cell lines and from normal cells. The mRNA expression of each sample was assayed in parallel by cDNA microarray. We identified small amplified or deleted chromosomal regions, as well as alterations in DNA copy number not previously described. We predominantly found alterations of apoptosis-related genes in Hep3B and HepG2, cell adhesion and receptor molecules in HLE, and cytokine-related genes in PLC/PRF/5. About 40% of the genes showing amplification or loss showed altered levels of mRNA (p < 0.05). Hierarchical clustering analysis showed that the expression of these genes allows differentiation between alpha-fetoprotein (AFP)-producing and AFP-negative cell lines. cDNA array-CGH is a sensitive method that can be used to detect alterations in genomic copy number in tumor cells. Differences in DNA copy alterations between AFP-producing and AFP-negative cells may lead to differential gene expression and may be related to the phenotype of these cells.     

In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma

Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.     

Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples

Analysis of genomic DNA derived from cells and fresh or fixed tissues often requires whole genome amplification prior to microarray screening. Technical hurdles to this process are the introduction of amplification bias and/or the inhibitory effects of formalin fixation on DNA amplification. Here we demonstrate a balanced-PCR procedure that allows unbiased amplification of genomic DNA from fresh or modestly degraded paraffin-embedded DNA samples. Following digestion and ligation of a target and a control genome with distinct linkers, the two are mixed and amplified in a single PCR, thereby avoiding biases associated with PCR saturation and impurities. We demonstrate genome-wide retention of allelic differences following balanced-PCR amplification of DNA from breast cancer and normal human cells and genomic profiling by array-CGH (cDNA arrays, 100 kb resolution) and by real-time PCR (single gene resolution). Comparison of balanced-PCR with multiple displacement amplification (MDA) demonstrates equivalent performance between the two when intact genomic DNA is used. When DNA from paraffin-embedded samples is used, balanced PCR overcomes problems associated with modest DNA degradation and produces unbiased amplification whereas MDA does not. Balanced-PCR allows amplification and recovery of modestly degraded genomic DNA for subsequent retrospective analysis of human tumors with known outcomes.     

Expression screening of 17q12-21 amplicon reveals GRB7 as an ERBB2-dependent oncogene

Gene amplification is a major genetic alteration in human cancers. Amplicons, amplified genomic regions, are believed to contain "driver" genes responsible for tumorigenesis. However, the significance of co-amplified genes has not been extensively studied. We have established an integrated analysis system of amplicons using retrovirus-mediated gene transfer coupled with a human full-length cDNA set. Applying this system to 17q12-21 amplicon observed in breast cancer, we identified GRB7 as a context-dependent oncogene, which modulates the ERBB2 signaling pathway through enhanced phosphorylation of ERBB2 and Akt. Our work provides an insight into the biological significance of gene amplification in human cancers.     

Identification of DNA copy-number aberrations by array-comparative genomic hybridization in patients with schizophrenia

Chromosomal abnormalities are implicated as important markers for the pathogenesis in patients with schizophrenia. In this study, with using bacterial artificial chromosome (BAC) array-based comparative genomic hybridization (CGH), we analyzed DNA copy-number changes among 30 patients with schizophrenia. The most frequent changes were partial gain of Xq23 (52%) and loss of 3q13.12 (32%). Other frequent gains were found in: 1p, 6q, 10p, 11p, 11q, 14p, and 15q regions, and frequent losses were found in: 2p, 9q, 10q, 14q, 20q, and 22q regions. The set of abnormal regions was confirmed by real-time PCR (9q12, 9q34.2, 11p15.4, 14q32.33, 15q15.1, 22q11.21, and Xq23). All real-time PCR results were consistent with the array-CGH results. Therefore, it is suggested that array-CGH and real-time PCR analysis could be used as powerful tools in screening for schizophrenia-related genes. Our results might be useful for further exploration of candidate genomic regions in the pathogenesis of schizophrenia.     

Genetics of early miscarriage

A miscarriage is the most frequent complication of a pregnancy. Poor chromosome preparations, culture failure, or maternal cell contamination may hamper conventional karyotyping. Techniques such as chromosomal comparative genomic hybridization (chromosomal‐CGH), array-comparative genomic hybridization (array-CGH), fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA) and quantitative fluorescent polymerase chain reaction (QF-PCR) enable us to trace submicroscopic abnormalities. We found the prevalence of chromosome abnormalities in women facing a single sporadic miscarriage to be 45% (95% CI: 38–52; 13 studies, 7012 samples). The prevalence of chromosome abnormalities in women experiencing a subsequent miscarriage after preceding recurrent miscarriage proved to be comparable: 39% (95% CI: 29–50; 6 studies 1359 samples). More chromosome abnormalities are detected by conventional karyotyping compared to FISH or MLPA only (chromosome region specific techniques), and the same amount of abnormalities compared to QF-PCR (chromosome region specific techniques) and chromosomal‐CGH and array-CGH (whole genome techniques) only. Molecular techniques could play a role as an additional technique when culture failure or maternal contamination occurs: recent studies show that by using array-CGH, an additional 5% of submicroscopic chromosome variants can be detected. Because of the small sample size as well as the unknown clinical relevance of these molecular aberrations, more and larger studies should be performed of submicroscopic chromosome abnormalities among sporadic miscarriage samples. For recurrent miscarriage samples molecular technique studies are relatively new. It has often been suggested that miscarriages are due to chromosomal abnormalities in more than 50%, but the present review has determined that chromosomal and submicroscopic genetic abnormalities on average are prevalent in maximally half of the miscarriage samples. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.