Identification of amplified and highly expressed genes in amplicons of the T-cell line huT78 detected by cDNA microarray CGH

Background  

Conventional Comparative Genomic Hybridization (CGH) has been widely used for detecting copy number alterations in cancer and for identifying regions containing candidate tumor responsible genes. Recently, several studies have shown the utility of cDNA microarray CGH for studing gene copy changes in various types of tumors. However, no such studies on T-cell lymphomas have been performed. To date T-cell lymphomas analyzed by the use of chromosome CGH have revealed only slight copy number alterations and not gene amplifications.     

Combined use of expression and CGH arrays pinpoints novel candidate genes in Ewing sarcoma family of tumors

Background  

Ewing sarcoma family of tumors (ESFT), characterized by t(11;22)(q24;q12), is one of the most common tumors of bone in children and young adults. In addition to EWS/FLI1 gene fusion, copy number changes are known to be significant for the underlying neoplastic development of ESFT and for patient outcome. Our genome-wide high-resolution analysis aspired to pinpoint genomic regions of highest interest and possible target genes in these areas.     

Identification of differentially expressed genes in cutaneous squamous cell carcinoma by microarray expression profiling

Background  

Carcinogenesis is a multi-step process indicated by several genes up- or down-regulated during tumor progression. This study examined and identified differentially expressed genes in cutaneous squamous cell carcinoma (SCC).     

Identification of differentially expressed genes in hepatocellular carcinoma and metastatic liver tumors by oligonucleotide expression profiling

BACKGROUND: The characterization of differentially expressed genes between cancerous and normal tissues is an important step in the understanding of tumorigenesis. Global gene expression profiling with microarrays has now offered a powerful tool to measure the changes of thousands of genes in any carcinoma tissues in an effort to identify these key disease-related genes. To compare the gene expression of a primary liver carcinoma, metastatic carcinoma to the liver, and normal liver, the authors analyzed tissue from six primary hepatocellular carcinomas (HCCs), five colorectal adenocarcinoma metastases to the liver, and eight normal livers. METHODS: Samples were processed from total RNA to fragmented cRNA and hybridized onto Affymetrix GeneChip(R) expression arrays. Analyses were performed to determine the consensus pattern of gene expression for primary liver carcinoma, metastatic liver carcinoma, and normal liver tissue and their changes in expression level. RESULTS: In hepatocellular carcinoma, 842 genes were overexpressed, and 393 genes were underexpressed in comparison with genes of normal liver tissue. Of note, 7 of the 20 most increased identified known genes previously have been associated with liver carcinoma or other types of cancers. The 13 additional identified genes until now have not previously shown strong association with cancers. Furthermore, the authors identified 42 genes and 24 expressed sequence tags that are expressed at a significant level in both HCC and metastastic tumors, presenting a list of marker genes indicative of cancerous liver tissue. CONCLUSIONS: In this study, genes that can be involved in the production of and maintenance of hepatic carcinomas were identified. These data offer new insight into genes that are potentially important in the pathogenesis of liver carcinoma, as well as additional targets for new strategies for cancer therapy and treatment.     

Identification of target genes in laryngeal squamous cell carcinoma by high-resolution copy number and gene expression microarray analyses

Molecular mechanisms contributing to initiation and progression of head and neck squamous cell carcinoma are still poorly known. Numerous genetic alterations have been described, but molecular consequences of such alterations in most cases remain unclear. Here, we performed an integrated high-resolution microarray analysis of gene copy number and expression in 20 laryngeal cancer cell lines and primary tumors. Our aim was to identify genetic alterations that play a key role in disease pathogenesis and pinpoint genes whose expression is directly impacted by these events. Integration of DNA level data from array-based comparative genomic hybridization with RNA level information from oligonucleotide microarrays was achieved with custom-developed bioinformatic methods. High-level amplifications had a clear impact on gene expression. Across the genome, overexpression of 739 genes could be attributed to gene amplification events in cell lines, with 325 genes showing the same phenomenon in primary tumors including FADD and PPFIA1 at 11q13. The analysis of gene ontology and pathway distributions further pinpointed genes that may identify potential targets of therapeutic intervention. Our data highlight genes that may be critically important to laryngeal cancer progression and offer potential therapeutic targets.     

Identification of a novel recurrent gain on 20q13 in chronic lymphocytic leukemia by array CGH and gene expression profiling

BackgroundThe presence of genetic changes is a hallmark of chronic lymphocytic leukemia (CLL). The most common cytogenetic abnormalities with independent prognostic significance in CLL are 13q14, ATM and TP53 deletions and trisomy 12. However, CLL displays a great genetic and biological heterogeneity. The aim of this study was to analyze the genomic imbalances in CLL cytogenetic subsets from both genomic and gene expression perspectives to identify new recurrent alterations.Patients and methodsThe genomic imbalances and expression levels of 67 patients were analyzed. The novel recurrent abnormalities detected with bacterial artificial chromosome array were confirmed by FISH and oligonucleotide microarrays. In all cases, gene expression profiling was assessed.ResultsCopy number alterations were identified in 75% of cases. Overall, the results confirmed FISH studies for the regions frequently involved in CLL and also defined a new recurrent gain on chromosome 20q13.12, in 19% (13/67) of the CLL patients. Oligonucleotide expression correlated with the regions of loss or gain of genomic material, suggesting that the changes in gene expression are related to alterations in copy number.ConclusionOur study demonstrates the presence of a recurrent gain in 20q13.12 associated with overexpression of the genes located in this region, in CLL cytogenetic subgroups.     

Identification of new candidate therapeutic target genes in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a subgroup of breast cancer that is negative for estrogen and progesterone receptor and ERBB2 protein expression. It is characterized by its aggressive behavior and by the lack of targeted therapies. To identify new therapeutic targets in TNBC, we used real-time quantitative RT-PCR to analyze 63 TNBC samples in terms of their mRNA expression of 26 genes coding for the major proteins currently targeted by drugs used to treat other cancers or undergoing clinical trials in breast cancer. Six of the 26 genes tested (VEGFA, SRC, PARP1, PTK2, RAF1, and FGFR3) were significantly upregulated in 13% to 46% of the TNBCs. None of the 6 genes was specifically upregulated in the TNBCs compared with 3 other classical breast tumor subtypes. No association was observed between overexpression of these 6 genes (except for FGFR3) and PIK3CA mutation status. These results confirm the interest of targeting VEGFA and PARP1 in ongoing clinical trials in TNBC patients and also identify new target genes (SRC, PTK2, RAF1, and FGFR3). Clinical trials could be initiated easily with existing drugs. Our results also suggest that these target genes might serve as predictive biomarkers of the TNBC treatment response.     

Characterization of the 12q amplicons by high-resolution, oligonucleotide array CGH and expression analyses of a novel liposarcoma cell line

The cytogenetic hallmark of well-differentiated liposarcoma (WDLS) is a giant marker chromosomes containing amplified genes from chromosome 12q13–q15. Here, we have employed SKY and high-resolution 244K oligonucleotide array CGH to characterize rearrangements and amplifications in a new WDLS cell line (GOT3) with a giant marker chromosome derived from chromosomes 12, 1, and X. The most prominent amplifications included 144 genes in 12q11–q21.2, 201 genes in 1q23.3–q44, and six genes in 13q32.1–q32.2. In the 12q amplicons, MDM2 showed the highest level of amplification followed by LYZ, HMGA2 (5′-part), TSPAN8, CNOT2, YEATS4, CDK4, GNS, HELB, and TSFM. Expression analysis of genes from the three major amplicons revealed that several highly amplified potential target genes, including HMGA2, MDM2, YEATS4, CDK4, PKP1, IPO9, and SOX21, were strongly overexpressed. Studies of cell cycle controlling proteins that interact with CDK4 and MDM2 revealed an abnormally strong expression of cyclins D1 and E. The selective high-level amplification of the 5′-part of HMGA2, including the DNA-binding domains, suggests that this gene is a major target of amplifications in WDLS. Our results also identify several novel candidate genes of potential pathogenetic and therapeutic importance for WDLS.     

Identification and validation of commonly overexpressed genes in solid tumors by comparison of microarray data

Cancers originating from epithelial cells are the most common malignancies. No common expression profile of solid tumors compared to normal tissues has been described so far. Therefore we were interested if genes differentially expressed in the majority of carcinomas could be identified using bioinformatic methods. Complete data sets were downloaded for carcinomas of the prostate, breast, lung, ovary, colon, pancreas, stomach, bladder, liver, and kidney, and were subjected to an expression analysis using SAM. In each experiment, a gene was scored as differentially expressed if the q value was below 25%. Probe identifiers were unified by comparing the respective probe sequences to the Unigene build 155 using BlastN. To obtain differentially expressed genes within the set of analyzed carcinomas, the number of experiments in which differential expression was observed was counted. Differential expression was assigned to genes if they were differentially expressed in at least eight experiments of tumors from different origin. The identified candidate genes ADRM1, EBNA1BP2, FDPS, FOXM1, H2AFX, HDAC3, IRAK1, and YY1 were subjected to further validation. Using this comparative approach, 100 genes were identified as upregulated and 21 genes as downregulated in the carcinomas.     

Genomic and expression profiling of chromosome 17 in breast cancer reveals complex patterns of alterations and novel candidate genes

Chromosome 17 is severely rearranged in breast cancer. Whereas the short arm undergoes frequent losses, the long arm harbors complex combinations of gains and losses. In this work we present a comprehensive study of quantitative anomalies at chromosome 17 by genomic array-comparative genomic hybridization and of associated RNA expression changes by cDNA arrays. We built a genomic array covering the entire chromosome at an average density of 1 clone per 0.5 Mb, and patterns of gains and losses were characterized in 30 breast cancer cell lines and 22 primary tumors. Genomic profiles indicated severe rearrangements. Compiling data from all samples, we subdivided chromosome 17 into 13 consensus segments: 4 regions showing mainly losses, 6 regions showing mainly gains, and 3 regions showing either gains or losses. Within these segments, smallest regions of overlap were defined (17 for gains and 16 for losses). Expression profiles were analyzed by means of cDNA arrays comprising 358 known genes at 17q. Comparison of expression changes with quantitative anomalies revealed that about half of the genes were consistently affected by copy number changes. We identified 85 genes overexpressed when gained (39 of which mapped within the smallest regions of overlap), 67 genes underexpressed when lost (32 of which mapped to minimal intervals of losses), and, interestingly, 32 genes showing reduced expression when gained. Candidate genes identified in this study belong to very diverse functional groups, and a number of them are novel candidates.     

Expression profiling of Wilms tumors reveals new candidate genes for different clinical parameters

Wilms tumor is the most frequent renal neoplasm in children, but our understanding of its genetic basis is still limited. We performed cDNA microarray experiments using 63 primary Wilms tumors with the aim of detecting new candidate genes associated with malignancy grade and tumor progression. All tumors had received preoperative chemotherapy as mandated by the SIOP protocol, which sets this study apart from related approaches in the Unites States that are based on untreated samples. The stratification of expression data according to clinical criteria allowed a rather clear distinction between different subsets of Wilms tumors. Clear-cut differences in expression patterns were discovered between relapse-free as opposed to relapsed tumors and tumors with intermediate risk as opposed to high risk histology. Several differentially expressed genes, e.g.TRIM22, CENPF, MYCN, CTGF, RARRES3 and EZH2, were associated with Wilms tumor progression. For a subset of differentially expressed genes, microarray data were confirmed by real-time RT-PCR on the original set of tumors. Interestingly, we found the retinoic acid pathway to be deregulated at different levels in advanced tumors suggesting that treatment of these tumors with retinoic acid may represent a promising novel therapeutic approach.     

应用表达谱芯片筛选卵巢癌紫杉醇耐药差异表达基因

目的 应用表达谱芯片研究人卵巢癌紫杉醇耐药细胞株SKOV3/TAX300及SKOV3/TAX30与敏感细胞SKOV3之间的基因表达谱差异,筛选耐药相关基因.方法 分别提取细胞的总RNA并纯化mRNA,mRNA逆转录合成cDNA后,用Cy5和Cy3-dUTP标记,与基因芯片进行杂交反应,扫描芯片荧光信号图像,用基因图像分析软件对扫描图像进行数字化处理和分析.采用荧光定量PCR、RT-PCR验证差异表达基因TNC mRNA和MDR1 mRNA水平,Western blot法验证TNC和MDR1蛋白表达.结果 SKOV3/TAX300细胞与敏感细胞SKOV3筛选出202个差异基因,其中上调基因88个,下调基因114个;SKOV3/TAX30细胞与SKOV3敏感细胞筛选出358个差异基因,其中上调基因144个,下调基因214个.这些基因主要包括细胞外基质、细胞信号传递、细胞周期、细胞凋亡、药物代谢等方面.其中细胞外基质成分TNC在两种耐药细胞中均为明显上调.荧光定量PCR、RT-PCR检测TNC mRNA和MDR1 mRNA水平,Western blot验证TNC和MDR1的表达,结果均与芯片结果一致.结论 本研究筛选出的基因可能为进一步探讨卵巢癌肿瘤细胞耐药机制提供新的途径,细胞外基质成分TNC可能是紫杉醇耐药的相关基因.     

Progression-specific genes identified by expression profiling of matched ductal carcinomas in situ and invasive breast tumors, combining laser capture microdissection and oligonucleotide microarray analysis

Becoming invasive is a crucial step in breast cancer oncogenesis. At this point, a lesion carries the potential for spreading and metastasis--a process, whose molecular characteristics still remain poorly understood. In this article, we describe a matched-pair analysis of ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of nine breast ductal carcinomas to identify novel molecular markers characterizing the transition from DCIS to IDC. The purpose of this study was to better understand the molecular biology of this transition and to identify candidate genes whose products might serve as prognostic markers and/or as molecular targets for treatment. To obtain cellular-based gene expression profiles from epithelial tumor cells, we combined laser capture microdissection with a T7-based two-round RNA amplification and Affymetrix oligonucleotide microarray analysis. Altogether, a set of 24 tumor samples was analyzed, comprised of nine matched DCIS/IDC and replicate DCIS/IDC preparations from three of the nine tumors. Cluster analysis on expression data shows the robustness and reproducibility of the techniques we established. Using multiple statistical methods, 546 significantly differentially expressed probe sets were identified. Eighteen candidate genes were evaluated by RT-PCR. Examples of genes already known to be associated with breast cancer invasion are BPAG1, LRRC15, MMP11, and PLAU. The expression of BPAG1, DACT1, GREM1, MEF2C, SART2, and TNFAIP6 was localized to epithelial tumor cells by in situ hybridization and/or immunohistochemistry, confirming the accuracy of laser capture microdissection sampling and microarray analysis.     

Chromosomal aberrations detected by comparative genomic hybridization (CGH) in human astrocytic tumors

We investigated chromosomal aberrations in 16 patients with astrocytic tumors of various histologic malignancies by comparative genomic hybridization (CGH). The degree of chromosomal loss was shown to be negatively correlated with histologic malignancy. Losses of portions of chromosomes 1p, 19q and 22q were the three chromosomal aberrations observed most frequently. Alterations in multiple chromosomes were observed more frequently in glioblastomas than in astrocytomas or anaplastic astrocytomas (P < 0.001). Primary glioblastomas showed a high frequency of genomic DNA gains (5/7), whereas recurrent glioblastomas from anaplastic astrocytomas did not (0/3). We found CGH to be a powerful tool for surveying DNA alterations in tumors and characterizing the biology of tumors of astrocytic lineage.     

Molecular profiling of low grade serous ovarian tumours identifies novel candidate driver genes

Low grade serous ovarian tumours are a rare and under-characterised histological subtype of epithelial ovarian tumours, with little known of the molecular drivers and facilitators of tumorigenesis beyond classic oncogenic RAS/RAF mutations. With a move towards targeted therapies due to the chemoresistant nature of this subtype, it is pertinent to more fully characterise the genetic events driving this tumour type, some of which may influence response to therapy and/or development of drug resistance. We performed genome-wide high-resolution genomic copy number analysis (Affymetrix SNP6.0) and mutation hotspot screening (KRAS, BRAF, NRAS, HRAS, ERBB2 and TP53) to compare a large cohort of ovarian serous borderline tumours (SBTs, n = 57) with low grade serous carcinomas (LGSCs, n = 19). Whole exome sequencing was performed for 13 SBTs, nine LGSCs and one mixed low/high grade carcinoma. Copy number aberrations were detected in 61% (35/57) of SBTs, compared to 100% (19/19) of LGSCs. Oncogenic RAS/RAF/ERBB2 mutations were detected in 82.5% (47/57) of SBTs compared to 63% (12/19) of LGSCs, with NRAS mutations detected only in LGSC. Some copy number aberrations appeared to be enriched in LGSC, most significantly loss of 9p and homozygous deletions of the CDKN2A/2B locus. Exome sequencing identified BRAF, KRAS, NRAS, USP9X and EIF1AX as the most frequently mutated genes. We have identified markers of progression from borderline to LGSC and novel drivers of LGSC. USP9X and EIF1AX have both been linked to regulation of mTOR, suggesting that mTOR inhibitors may be a key companion treatment for targeted therapy trials of MEK and RAF inhibitors.