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Novel Lung Cancer-Related Genes Identified by a Non-Gapped Array-Comparative Genomic Hybridization Microarray in Asian and Caucasian Populations

ABSTRACT: This study constructed a non-gapped bacterial artificial chromosomes (BAC) array containing 3604 BAC clones covering 18 lung cancer-related chromosome imbalance hotspot regions. Using this specialized array, DNA from tumor and normal tissues of 40 Asian and 20 Caucasian non-small cell lung cancer (NSLCL) patients was analyzed by array-comparative genomic hybridization (array-CGH). Block-wise normalization and a Bayes regression approach were used to refine the chromosomal imbalance regions identified by array-CGH. The array-CGH results then analyzed by MetaCore software to identify potential cancer-related genes. Finally, 273 genes showing significantly associated with molecular pathway, cancer biomarker, and gene ontology database, such as ZNF322A on 6p22.1, ARHGAP19 on 10q24.1, FRAT2 on 10q24.1, and PAFAH1B1 on 17p13.3 functioning in MAPK, Rho GTPase, and Wnt, and motility control pathways with frequent copy number gain were selected. This study mapped concisely the novel oncogenes or tumor suppressor genes in lung cancer and revealed insights of difference on chromosomal imbalance between lung cancer from Asian and Caucasian.

ORGANISM(S): Homo sapiens

PROVIDER: GSE21276 | GEO | 2012/07/25

SECONDARY ACCESSION(S): PRJNA126579

REPOSITORIES: GEO

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Project description:This study constructed a non-gapped bacterial artificial chromosomes (BAC) array containing 3604 BAC clones covering 18 lung cancer-related chromosome imbalance hotspot regions. Using this specialized array, DNA from tumor and normal tissues of 40 Asian and 20 Caucasian non-small cell lung cancer (NSLCL) patients was analyzed by array-comparative genomic hybridization (array-CGH). Block-wise normalization and a Bayes regression approach were used to refine the chromosomal imbalance regions identified by array-CGH. The array-CGH results then analyzed by MetaCore software to identify potential cancer-related genes. Finally, 273 genes showing significantly associated with molecular pathway, cancer biomarker, and gene ontology database, such as ZNF322A on 6p22.1, ARHGAP19 on 10q24.1, FRAT2 on 10q24.1, and PAFAH1B1 on 17p13.3 functioning in MAPK, Rho GTPase, and Wnt, and motility control pathways with frequent copy number gain were selected. This study mapped concisely the novel oncogenes or tumor suppressor genes in lung cancer and revealed insights of difference on chromosomal imbalance between lung cancer from Asian and Caucasian. Clinical samples preparation and DNA extraction: Tissues were collected after obtaining appropriate institutional review board permission and informed consent from the recruited patients. Surgically resected tumor tissue and corresponding normal tissue were collected from 40 patients diagnosed with primary NSCLC admitted to Taipei Veterans General Hospital, Taiwan and 20 Caucasian NSCLC tumor tissues from University of Chicago, USA. Histological classification was determined according to the WHO classification and the tumor-node-metastasis system. Information on the age, sex, tumor type, tumor stage and smoking history of the patients was obtained from hospital records. The genomic DNA was prepared using proteinase K digestion and phenol-chloroform extraction. Array-CGH: The genomic microarray used containing 3604 BAC clones representing the human genome at 18 non-gap chromosome regions. Clone mapping was analyzed using the BAC end pairs database of UCSC Genome Bioinformatics (http://genome.ucsc.edu/). The BAC library used was RP-11, which was provided from Genome Research Center, University of Yang-Ming. The extracted BAC DNA was amplified to 10 μg by Phi29 DNA polymerase reaction (TempliPhi DNA Sequencing Template Amplification Kit, Amersham, Princeton, NJ), and digested by DNaseI (New England Biolabs, Ipswich, MA), and purified by MultiScreen-PCR Plates (Millipore, Danvers, MA). After digestion, the DNA fragments were resolved by gel electrophoresis and the fragment size were optimized to within 0.5–3 kb. Finally, the purified BAC DNA fragments were dotted triplicate in 72 blocks on the glass slides (Corning, NY, USA) with spotting solution (50% DMSO, 50% D2H2O). In brief, approximately 500 ng of tumor genomic DNA and reference DNA (for Asian samples: 28 matched-normal genomic DNA pool; for Caucasian samples: commercial human genomic DNA, Invitrogen) were labeled by random priming using BioPrime DNA Labeling System (Invitrogen) with Cy3-dCTP (tumor) or Cy5-dCTP (reference). Labeled tumor and reference DNA were combined together with 100 μg human Cot-1 DNA (Invitrogen). Hybridization was performed using MAUI hybridization system (BioMcro Systems, Salt Lake City, UT), agitating the hybridization solution for 50 hours at 50℃. The arrays were scanned using scanner GenePix4000B (Axon Instruments, Foster City, CA) and the images were segmented and transformed to Cy3-Cy5 log ratio using GenePix software by Axon Instruments, Inc. (http://www.axon.com). Further data processing, a normalization method called “block-wise normalization”, which normalized the array-CGH data by the log ratio of the self-self hybridization of reference DNA according to the mean and the standard deviations of each array block, was established.

Project description:Bronchioloalveolar carcinoma (BAC), a subtype of lung adenocarcinoma (ADC) without stromal, vascular, or pleural invasion, is considered an in situ tumor with a 100% survival rate. However, the histological criteria for invasion remain controversial. BAC-like areas may accompany otherwise invasive adenocarcinoma, referred to as mixed type adenocarcinoma with BAC features (AWBF). AWBF are considered to evolve from BAC, representing a paradigm for malignant progression in ADC. However, the supporting molecular evidence remains forthcoming. Here, we have studied the genomic changes of BAC and AWBF by array comparative genomic hybridization (CGH). We used submegabase-resolution tiling set array CGH to compare the genomic profiles of 14 BAC or BAC with focal area suspicious for invasion with those of 15 AWBF. Threshold-filtering and frequency-scoring analysis found that genomic profiles of noninvasive and focally invasive BAC are indistinguishable and show fewer aberrations than tumor cells in BAC-like areas of AWBF. These aberrations occurred mainly at the subtelomeric chromosomal regions. Increased genomic alterations were noted between BAC-like and invasive areas of AWBF. We identified 113 genes that best differentiated BAC from AWBF and were considered candidate marker genes for tumor invasion and progression. Correlative gene expression analyses demonstrated a high percentage of them to be poor prognosis markers in early stage ADC. Quantitative PCR also validated the amplification and overexpression of PDCD6 and TERT on chromosome 5p and the prognostic significance of PDCD6 in early stage ADC patients. We identified candidate genes that may be responsible for and are potential markers for malignant progression in AWBF. Keywords: array comparitive genomic hybridization, bronchioloalveolar carcinoma, non-small-cell lung carcinoma, prognostic markers Array comparitive genomic hybridization analysis of 29 Lung Adenocarcinomas with Bronchioalveolar Features.

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Novel Lung Cancer-Related Genes Identified by a Non-Gapped Array-Comparative Genomic Hybridization Microarray in Asian and Caucasian Populations

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