High-resolution genomic and expression profiling reveals 105 putative amplification target genes in pancreatic cancer

Comparative genomic hybridization (CGH) studies have provided a wealth of information on common copy number aberrations in pancreatic cancer, but the genes affected by these aberrations are largely unknown. To identify putative amplification target genes in pancreatic cancer, we performed a parallel copy number and expression survey in 13 pancreatic cancer cell lines using a 12,232-clone cDNA microarray, providing an average resolution of 300 kb throughout the human genome. CGH on cDNA microarray allowed highly accurate mapping of copy number increases and resulted in identification of 24 independent amplicons, ranging in size from 130 kb to 11 Mb. Statistical evaluation of gene copy number and expression data across all 13 cell lines revealed a set of 105 genes whose elevated expression levels were directly attributable to increased copy number. These included genes previously reported to be amplified in cancer as well as several novel targets for copy number alterations, such as p21-activated kinase 4 (PAK4), which was previously shown to be involved in cell migration, cell adhesion, and anchorage-independent growth. In conclusion, our results implicate a set of 105 genes that is likely to be actively involved in the development and progression of pancreatic cancer.     

A comparative study of genome-wide SNP,CGH microarray and protein expression analysis to explore genotypic and phenotypic mechanisms of acquired antiestrogen resistance in breast cancer

Allelic imbalance is a common feature of many malignancies. We have measured allelic imbalance in genomic DNA from the breast cancer cell lines T47D, MDA-MB-231, two antiestrogen sensitive (MCF7N and MCF7L) and two resistant MCF7 cell lines (MMU2 and LCC9) using single nucleotide polymorphism (SNP) oligonucleotide microarrays. DNA from MCF7(L) and MMU2 cells was also analysed by comparative genome hybridisation (CGH) to compare with SNP microarray data. Proteins previously determined to be involved in disease progression were quantified by Western blot and compared to array data. The SNP and CGH array both detected cytogenetic abnormalities commonly found in breast cancer: amplification of chromosomes 11q13-14.1, 17q and 20q containing cyclin D1, BCAS1 and 3 (Breast Cancer Amplified Sequence) and AIB1 (Amplified in Breast cancer) genes; losses at 6q, 9p and X chromosomes, which included ERalpha (Estrogen Receptor alpha) and p16 ( INK4A ) genes. However the SNP chip array data additionally identified regions of loss of heterozygosity (LOH) followed by duplication of the remaining allele-uniparental disomy (UPD). Good concordance between SNP arrays and CGH analyses was observed, however there was poor correlation between gene copy number and protein levels between the cell lines. There were reductions in ERalpha, cyclin D1 and p27 protein levels whilst p21 protein levels were elevated in antiestrogen resistant MCF7 cell lines. Although protein levels varied there was no difference in gene copy number. This study shows SNP and CGH array analysis are powerful tools for analysis of allelic imbalance in breast cancer. However, the antiestrogen resistant phenotype was likely to be due to changes in gene expression and protein degradation rather than in altered gene copy number.     

Impact of DNA amplification on gene expression patterns in breast cancer

Genetic changes underlie tumor progression and may lead to cancer-specific expression of critical genes. Over 1100 publications have described the use of comparative genomic hybridization (CGH) to analyze the pattern of copy number alterations in cancer, but very few of the genes affected are known. Here, we performed high-resolution CGH analysis on cDNA microarrays in breast cancer and directly compared copy number and mRNA expression levels of 13,824 genes to quantitate the impact of genomic changes on gene expression. We identified and mapped the boundaries of 24 independent amplicons, ranging in size from 0.2 to 12 Mb. Throughout the genome, both high- and low-level copy number changes had a substantial impact on gene expression, with 44% of the highly amplified genes showing overexpression and 10.5% of the highly overexpressed genes being amplified. Statistical analysis with random permutation tests identified 270 genes whose expression levels across 14 samples were systematically attributable to gene amplification. These included most previously described amplified genes in breast cancer and many novel targets for genomic alterations, including the HOXB7 gene, the presence of which in a novel amplicon at 17q21.3 was validated in 10.2% of primary breast cancers and associated with poor patient prognosis. In conclusion, CGH on cDNA microarrays revealed hundreds of novel genes whose overexpression is attributable to gene amplification. These genes may provide insights to the clonal evolution and progression of breast cancer and highlight promising therapeutic targets.     

Comparative genomic hybridization analysis of 38 breast cancer cell lines: a basis for interpreting complementary DNA microarray data

Breast cancer cell lines provide a useful starting point for the discovery and functional analysis of genes involved in breast cancer. Here, we studied 38 established breast cancer cell lines by comparative genomic hybridization (CGH) to determine recurrent genetic alterations and the extent to which these cell lines resemble uncultured tumors. The following chromosomal gains were observed: 8q (75%), 1q (61%), 20q (55%), 7p (44%), 3q (39%), 5p (39%), 7q (39%), 17q (33%), 1p (30%), and 20p (30%), and the most common losses were: 8p (58%), 18q (58%), 1p (42%), Xp (42%), Xq (42%), 4p (36%), 11q (36%), 18p (33%), 10q (30%), and 19p (28%). Furthermore, 35 recurrent high-level amplification sites were identified, most often involving 8q23 (37%), 20q13 (29%), 3q25-q26 (24%), 17q22-q23 (16%), 17q23-q24 (16%), 1p13 (11%), 1q32 (11%), 5p13 (11%), 5p14 (11%), 11q13 (11%), 17q12-q21 (11%), and 7q21-q22 (11%). A comparison of DNA copy number changes found in the cell lines with those reported in 17 published studies (698 tumors) of uncultured tumors revealed a substantial degree of overlap. CGH copy number profiles may facilitate identification of important new genes located at the hotspots of such chromosomal alterations. This was illustrated by analyzing expression levels of 1236 genes using cDNA microarrays in four of the cell lines. Several highly overexpressed genes (such as RCH1 at 17q23, TOPO II at 17q21-q22, as well as CAS and MYBL2 at 20q13) were involved in these recurrent DNA amplifications. In conclusion, DNA copy number profiles were generated by CGH for most of the publicly available breast cancer cell lines and were made available on a web site (http://www.nhgri.nih.gov/DIR/CGB/++ +CR2000). This should facilitate the correlative analysis of gene expression and copy number as illustrated here by the finding by cDNA microarrays of several overexpressed genes that were amplified.     

Combined cDNA array comparative genomic hybridization and serial analysis of gene expression analysis of breast tumor progression

To identify genetic changes involved in the progression of breast carcinoma, we did cDNA array comparative genomic hybridization (CGH) on a panel of breast tumors, including 10 ductal carcinoma in situ (DCIS), 18 invasive breast carcinomas, and two lymph node metastases. We identified 49 minimal commonly amplified regions (MCRs) that included known (1q, 8q24, 11q13, 17q21-q23, and 20q13) and several uncharacterized (12p13 and 16p13) regional copy number gains. With the exception of the 17q21 (ERBB2) amplicon, the overall frequency of copy number alterations was higher in invasive tumors than that in DCIS, with several of them present only in invasive cancer. Amplification of candidate loci was confirmed by quantitative PCR in breast carcinomas and cell lines. To identify putative targets of amplicons, we developed a method combining array CGH and serial analysis of gene expression (SAGE) data to correlate copy number and expression levels for each gene within MCRs. Using this approach, we were able to distinguish a few candidate targets from a set of coamplified genes. Analysis of the 12p13-p12 amplicon identified four putative targets: TEL/ETV6, H2AFJ, EPS8, and KRAS2. The amplification of all four candidates was confirmed by quantitative PCR and fluorescence in situ hybridization, but only H2AFJ and EPS8 were overexpressed in breast tumors with 12p13 amplification compared with a panel of normal mammary epithelial cells. These results show the power of combined array CGH and SAGE analysis for the identification of candidate amplicon targets and identify H2AFJ and EPS8 as novel putative oncogenes in breast cancer.     

Genetic profiling of chromosome 1 in breast cancer: mapping of regions of gains and losses and identification of candidate genes on 1q

Chromosome 1 is involved in quantitative anomalies in 50-60% of breast tumours. However, the structure of these anomalies and the identity of the affected genes remain to be determined. To characterise these anomalies and define their consequences on gene expression, we undertook a study combining array-CGH analysis and expression profiling using specialised arrays. Array-CGH data showed that 1p was predominantly involved in losses and 1q almost exclusively in gains. Noticeably, high magnitude amplification was infrequent. In an attempt to fine map regions of copy number changes, we defined 19 shortest regions of overlap (SROs) for gains (one at 1p and 18 at 1q) and of 20 SROs for losses (all at 1p). These SROs, whose sizes ranged from 170 kb to 3.2 Mb, represented the smallest genomic intervals possible based on the resolution of our array. The elevated incidence of gains at 1q, added to the well-established concordance between DNA copy increase and augmented RNA expression, made us focus on gene expression changes at this chromosomal arm. To identify candidate oncogenes, we studied the RNA expression profiles of 307 genes located at 1q using a home-made built cDNA array. We identified 30 candidate genes showing significant overexpression correlated to copy number increase. In order to substantiate their involvement, RNA expression levels of these candidate genes were measured by quantitative (Q)-RT-PCR in a panel of 25 breast cancer cell lines previously typed by array-CGH. Q-PCR showed that 11 genes were significantly overexpressed in the presence of a genomic gain in these cell lines, and 20 overexpressed when compared to normal breast.     

Chromosomal aberrations and gene expression profiles in non-small cell lung cancer

Alterations in genomic content and changes in gene expression levels are central characteristics of tumors and pivotal to the tumorigenic process. We analyzed 23 non-small cell lung cancer (NSCLC) tumors by array comparative genomic hybridization (array CGH). Aberrant regions identified included well-characterized chromosomal aberrations such as amplifications of 3q and 8q and deletions of 3p21.31. Less frequently identified aberrations such as amplifications of 7q22.3-31.31 and 12p11.23-13.2, and previously unidentified aberrations such as deletion of 11q12.3-13.3 were also detected. To enhance our ability to identify key acting genes residing in these regions, we combined array CGH results with gene expression profiling performed on the same tumor samples. We identified a set of genes with concordant changes in DNA copy number and expression levels, i.e. overexpressed genes located in amplified regions and underexpressed genes located in deleted regions. This set included members of the Wnt/beta-catenin pathway, genes involved in DNA replication, and matrix metalloproteases (MMPs). Functional enrichment analysis of the genes both overexpressed and amplified revealed a significant enrichment for DNA replication and repair, and extracellular matrix component gene ontology annotations. We verified the changes in expressions of MCM2, MCM6, RUVBL1, MMP1, MMP12 by real-time quantitative PCR. Our results provide a high resolution map of copy number changes in non-small cell lung cancer. The joint analysis of array CGH and gene expression analysis highlights genes with concordant changes in expression and copy number that may be critical to lung cancer development and progression.     

Array-based comparative genomic hybridization reveals recurrent chromosomal aberrations and Jab1 as a potential target for 8q gain in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide. We have previously characterized global gene expression patterns in HCC using microarrays. Here, we report the analysis of genomic DNA copy number among 49 HCC samples using BAC array-based comparative genomic hybridization (CGH). We observed recurrent and characteristic chromosomal aberrations, including frequent DNA copy number gains of 1q, 6p, 8q and 20q, and losses of 4q, 8p, 13q, 16q and 17p. We correlated gene expression with array CGH data, and identified a set of genes whose expression levels correlated with common chromosomal aberrations in HCC. Especially, we noticed that high expression of Jab1 in HCC significantly correlated with DNA copy number gain at 8q. Quantitative microsatellite analysis further confirmed DNA copy number gain at the Jab1 locus. Overexpression of Jab1 in HCC was also validated using real-time RT-PCR, and Jab1 protein levels were studied by immunohistochemistry on tissue microarrays. Functional analysis in HCC cell lines demonstrated that Jab1 may regulate HCC cell proliferation, thereby having a potential role in HCC development. In conclusion, this study shows that array-based CGH provides high resolution mapping of chromosomal aberrations in HCC, and demonstrates the feasibility of correlating array CGH data with gene expression data to identify novel oncogenes and tumor suppressor genes.     

Detection of gene amplification by genomic hybridization to cDNA microarrays

Gene amplification is one of the major mechanisms of oncogene activation in tumorigenesis. To facilitate the identification of genes mapping to amplified regions, we have used a technique based on the hybridization of total genomic DNA to cDNA microarrays. To aid detection of the weak signals generated in this complex hybridization, we have used a tyramide-based technique that allows amplification of a fluorescent signal up to 1000-fold. Dilution experiment suggests that amplifications of 5-fold and higher can be detected by this approach. The technique was validated using cancer cell lines with several known gene amplifications, such as those affecting MYC, MYCN, ERBB2, and CDK4. In addition to the detection of the known amplifications, we identified a novel amplified gene, ZNF133, in the neuroblastoma cell line NGP. Hybridization of NGP cDNA on an identical array also revealed over expression of ZNF133. Parallel analysis of genomic DNA for copy number and cDNA for expression now provides rapid approach to the identification of amplified genes and chromosomal regions in tumor cells.     

Alteration of hTERT full-length variant expression level showed different gene expression profiles and genomic copy number changes in breast cancer

We analyzed hTERT splicing patterns with respect to telomerase activity in breast cancer. Using a cDNA microarray in 22 cell lines, we observed the difference in expression profiling based on the different levels of full-length variant expression with 71 selected genes. Using 33 known genes that act with the telomerase complex, we performed unsupervised clustering with all cell lines, and found a clustering tendency related to the full-length variant expression level. Using array-based CGH, highly altered genomic copy number changes were found more often in MCF-7 (159 genes) than in MDA-MB-231 (109 genes) and MDA-MB-435 (49 genes), suggesting more genomic changes in MCF-7 cells. On comparing MCF-7 with MDA-MB231 and MDA-MB-435 cell lines, we identified 8 genes with different copy numbers, including dystroglycan, which is located in the p12-21.2 area of chromosome 3. In conclusion, alterations in the level of the full-length variant of hTERT showed different gene expression profiles and genomic copy number changes in breast cancer, which require further study into their cause-and-effect relationship.     

Recurrent amplification in the 22q11 region in laryngeal squamous cell carcinoma results in overexpression of the CRKL but not the MAPK1 oncogene

Thirteen laryngeal squamous cell carcinoma cell lines were recently studied by array comparative genomic hybridization (array-CGH) in order to identify recurrent DNA copy number alterations in the tumor genome. A highly amplified region 22q11.2 was found in two of the thirteen cell lines. Two established oncogenes CRKL and MAPK1 are localized in this region, but only CRKL was amplified in both cell lines. Therefore, to check if amplification of either CRKL or MAPK1 genes may be important in the pathogenesis of laryngeal squamous cell carcinoma, the DNA copy number and mRNA expression were measured in a cohort of 17 LSCC cell lines by quantitative real-time PCR (qPCR). For the CRKL gene gains of the copy number were found in 3/17 cell lines, while overexpression was found in 6/17 cell lines. Gains in the copy number for the MAPK1 gene were found in 1/17 cell lines, but overexpression was not detected in any cell line. A highly significant correlation between DNA copy number and expression for CRKL gene, but not for MAPK1 gene was established using the Pearson test. Thereafter, 46 primary samples of laryngeal cancer were tested by qPCR to check for possible gains in copy number of the CRKL gene. Gains were found in 3/46 cases. These results suggest that CRKL, but not MAPK1 is the target oncogene of the rare but recurrent amplification at 22q11.2 in laryngeal squamous cell carcinoma.     

Comparative genomic hybridization on mouse cDNA microarrays and its application to a murine lymphoma model

Microarray-based formats offer a high-resolution alternative to conventional, chromosome-based comparative genomic hybridization (CGH) methods for assessing DNA copy number alteration (CNA) genome-wide in human cancer. For murine tumors, array CGH should provide even greater advantage, since murine chromosomes are more difficult to individually discern. We report here the adaptation and evaluation of a cDNA microarray-based CGH method for the routine characterization of CNAs in murine tumors, using mouse cDNA microarrays representing approximately 14,000 different genes, thereby providing an average mapping resolution of 109 kb. As a first application, we have characterized CNAs in a set of 10 primary and recurrent lymphomas derived from a Myc-induced murine lymphoma model. In primary lymphomas and more commonly in Myc-independent relapses, we identified a recurrent genomic DNA loss at chromosome 3G3-3H4, and recurrent amplifications at chromosome 3F2.1-3G3 and chromosome 15E1/E2-15F3, the boundaries of which we defined with high resolution. Further, by profiling gene expression using the same microarray platform, we identified within CNAs the relevant subset of candidate cancer genes displaying comparably altered expression, including Mcl1 (myeloid cell leukemia sequence 1), a highly expressed antiapoptotic gene residing within the chr 3 amplicon peak. CGH on mouse cDNA microarrays therefore represents a reliable method for the high-resolution characterization of CNAs in murine tumors, and a powerful approach for elucidating the molecular events in tumor development and progression in murine models.     

Comprehensive copy number profiles of breast cancer cell model genomes

Introduction

Breast cancer is the most commonly diagnosed cancer in women worldwide and consequently has been extensively investigated in terms of histopathology, immunochemistry and familial history. Advances in genome-wide approaches have contributed to molecular classification with respect to genomic changes and their subsequent effects on gene expression. Cell lines have provided a renewable resource that is readily used as model systems for breast cancer cell biology. A thorough characterization of their genomes to identify regions of segmental DNA loss (potential tumor-suppressor-containing loci) and gain (potential oncogenic loci) would greatly facilitate the interpretation of biological data derived from such cells. In this study we characterized the genomes of seven of the most commonly used breast cancer model cell lines at unprecedented resolution using a newly developed whole-genome tiling path genomic DNA array.

Methods

Breast cancer model cell lines MCF-7, BT-474, MDA-MB-231, T47D, SK-BR-3, UACC-893 and ZR-75-30 were investigated for genomic alterations with the submegabase-resolution tiling array (SMRT) array comparative genomic hybridization (CGH) platform. SMRT array CGH provides tiling coverage of the human genome permitting break-point detection at about 80 kilobases resolution. Two novel discrete alterations identified by array CGH were verified by fluorescence in situ hybridization.

Results

Whole-genome tiling path array CGH analysis identified novel high-level alterations and fine-mapped previously reported regions yielding candidate genes. In brief, 75 high-level gains and 48 losses were observed and their respective boundaries were documented. Complex alterations involving multiple levels of change were observed on chromosome arms 1p, 8q, 9p, 11q, 15q, 17q and 20q. Furthermore, alignment of whole-genome profiles enabled simultaneous assessment of copy number status of multiple components of the same biological pathway. Investigation of about 60 loci containing genes associated with the epidermal growth factor family (epidermal growth factor receptor, HER2, HER3 and HER4) revealed that all seven cell lines harbor copy number changes to multiple genes in these pathways.

Conclusion

The intrinsic genetic differences between these cell lines will influence their biologic and pharmacologic response as an experimental model. Knowledge of segmental changes in these genomes deduced from our study will facilitate the interpretation of biological data derived from such cells.     

Genetic aberrations in prostate cancer by microarray analysis

The aim of this study was to screen genetic as well as expression alterations in prostate cancer. Array comparative genomic hybridization (aCGH) to a 16K cDNA microarray was performed to analyze DNA sequence copy number alterations in 5 prostate cancer cell lines and 13 xenografts. The aCGH confirmed the previously implicated common gains and losses, such as gains at 1q, 7, 8q, 16p and 17q and losses at 2q, 4p/q, 6q, 8p, 13q, 16q, 17p and 18q, which have previously been identified by chromosomal CGH (cCGH). Because of the higher resolution of aCGH, the minimal commonly altered regions were significantly narrowed-down. For example, the gain of 8q was mapped to three independent regions, 8q13.3-q21.11, 8q22.2 and 8q24.13-q24.3. In addition, a novel recurrent gain at 9p13-q21 was identified. The concomitant expression analysis indicated that genome-wide DNA sequence copy number (gene dosage) was significantly associated with the expression level (p < 0.0001). The analyses indicated several individual genes whose expression was associated with the gene copy number. For example, gains of PTK2 and FZD6, were associated with the increased expression, whereas losses of TNFRSF10B (alias DR5) and ITGA4 with decreased expression. In conclusion, the aCGH mapping data will aid in the identification of genes altered in prostate cancer. The combined expression and copy number analysis suggested that even a low-level copy number change may have significant effect on gene expression, and thus on the development of prostate cancer.     

Genome-wide profiling of papillary thyroid cancer identifies MUC1 as an independent prognostic marker

Clinicopathological variables used at present for prognostication and treatment selection for papillary thyroid carcinomas (PTCs) do not uniformly predict tumor behavior, necessitating identification of novel prognostic markers. Complicating the assessment is the long natural history of PTC and our rudimentary knowledge of its genetic composition. In this study we took advantage of differences in clinical behavior of two distinct variants of PTC, the aggressive tall-cell variant (TCV) and indolent conventional PTC (cPTC), to identify molecular prognosticators of outcome using complementary genome wide analyses. Comparative genome hybridization (CGH) and cDNA microarray (17,840 genes) analyses were used to detect changes in DNA copy number and gene expression in pathological cPTC and TCV. The findings from CGH and cDNA microarray analyses were correlated and validated by real-time PCR and immunohistochemical analyses on a series of 100 cases of cPTC and TCV. Genes identified by this approach were evaluated as prognostic markers in cPTC by immunohistochemistry on tissue arrays. CGH identified significant differences in the presence (76 versus 27%; P = 0.001) and type of DNA copy number aberrations in TCV compared with cPTC. Recurrent gains of 1p34-36, 1q21, 6p21-22, 9q34, 11q13, 17q25, 19, and 22 and losses of 2q21-31, 4, 5p14-q21, 6q11-22, 8q11-22, 9q11-32, and 13q21-31 were unique to TCV. Hierarchical clustering of gene expression profiles revealed significant overlap between TCV and cPTC, but further analysis identified 82 dysregulated genes differentially expressed among the PTC variants. Of these, MUC1 was of particular interest because amplification of 1q by CGH correlated with MUC1 amplification by real-time PCR analysis and protein overexpression by immunohistochemistry in TCV (P = 0.005). Multivariate analysis revealed a significant association between MUC1 overexpression and treatment outcome, independent of histopathological categorization (P = 0.03). Analysis of a validation series containing a matched group of aggressive and indolent cPTCs confirmed the association between MUC1 overexpression and survival (relative risk, 2.3; 95% confidence interval, 1.1-5.5; P = 0.03). Our data suggest that MUC1 dysregulation is associated with aggressive behavior of PTC and may serve as a prognostic marker and potential therapeutic target in this disease.     

Genome-wide differences between microsatellite stable and unstable colorectal tumors

Genomic copy number changes are frequently found in cancers and they have been demonstrated to contribute to carcinogenesis; and it is widely accepted that tumors with microsatellite instability (MSI) are genetically stable and mostly diploid. In the present study we compared the copy number alterations and the gene-expression profiles of microsatellite stable (MSS) and MSI colorectal tumors. A total number of 31 fresh-frozen primary tumors (16 MSS and 15 MSI) were used. Twenty-eight samples (15 MSS and 13 MSI) were analyzed with metaphase comparative genomic hybridization (CGH), nine of which plus one additional sample (4 MSS and 6 MSI) were further analyzed by cDNA-based array-CGH. Gene expression analysis was performed with six samples [3 MSS and 3 MSI, four of these used in metaphase CGH (mCGH) analysis] to identify differentially expressed genes possibly located in the lost or amplified regions found by CGH, stressing the biological significance of copy number changes. Metaphase and array-CGH analysis of two colon cancer cell lines (HTC116 and SW480, reported as MSI and MSS archetypes) gave comparable results. Alterations found by mCGH in MSS tumors were +20, +8q, -8p and -18q. Interestingly, 1p22, 4q26 and 15q21 were found deleted preferentially in MSS tumors, while 22q13 was found gained in MSI tumors. The regions of alterations identified by array-CGH were gains at 8q24, 16q24.3 and 20q13, and the loss of 5q21, appearing in the both types of tumors. Gene expression analysis revealed genes with specific associations with the copy number changes of the corresponding genomic regions. As a conclusion, colorectal cancer is a heterogeneous disease, demonstrated by the genomic profiles of individual samples. However, our data shows that copy number changes do not occur exclusively in the MSS phenotypes.     

Notch3 gene amplification in ovarian cancer

Gene amplification is one of the common mechanisms that activate oncogenes. In this study, we used single nucleotide polymorphism array to analyze genome-wide DNA copy number alterations in 31 high-grade ovarian serous carcinomas, the most lethal gynecologic neoplastic disease in women. We identified an amplicon at 19p13.12 in 6 of 31 (19.5%) ovarian high-grade serous carcinomas. This amplification was validated by digital karyotyping, quantitative real-time PCR, and dual-color fluorescence in situ hybridization (FISH) analysis. Comprehensive mRNA expression analysis of all 34 genes within the minimal amplicon identified Notch3 as the gene that showed most significant overexpression in amplified tumors compared with nonamplified tumors. Furthermore, Notch3 DNA copy number is positively correlated with Notch3 protein expression based on parallel immunohistochemistry and FISH studies in 111 high-grade tumors. Inactivation of Notch3 by both gamma-secretase inhibitor and Notch3-specific small interfering RNA suppressed cell proliferation and induced apoptosis in the cell lines that overexpressed Notch3 but not in those with minimal amount of Notch3 expression. These results indicate that Notch3 is required for proliferation and survival of Notch3-amplified tumors and inactivation of Notch3 can be a potential therapeutic approach for ovarian carcinomas.