Penny Nymark

CDK4 is a probable target gene in a novel amplicon at 12q13.3–q14.1 in lung cancer

Several chromosomal regions are recurrently amplified or deleted in lung tumors, but little is known about the underlying genes, which could be important mediators in tumor formation or progression. In lung cancer, the RB1–CCND1–CDKN2A pathway, involved in the G1–S transition, is damaged in nearly all tumors. In the present study, we localized a novel amplicon in lung tumors to a fragment of less than 0.5 Mb at 12q13.3–q14.1 by using comparative genomic hybridization (CGH) on cDNA microarrays. This approach enabled us to identify 10–15 genes with the most consistent amplifications. Semiquantitative RT‐PCR analyses of 13 genes in this region showed that four of them (CDK4, CYP27B1, METTL1, and TSFM) were also highly up‐regulated. Immunohistochemical (IHC) analysis of 141 tumor samples on a tissue microarray showed that CDK4 was expressed at a high level in 23% of lung tumors. Six (21.4%) of the tumors with high CDK4 expression (n = 28) were shown by fluorescence in situ hybridization (FISH) to contain the 12q13.3–q14.1 amplification. For CDK4, a positive correlation was found between gene copy number (FISH and CGH array), mRNA expression (RT‐PCR), and level of protein expression (IHC). CDK4 expression did not correlate with CDKN2A methylation status. Amplification of CDK4 has been described in other tumor types, but its role in lung cancer remains to be elucidated. Although CDK4 amplification seems to be a relatively rare event (4.3%) in lung tumors, it indicates the significance of the RB1–CCND1 pathway in lung tumorigenesis.

Integrative analysis of microRNA, mRNA and aCGH data reveals asbestos- and histology-related changes in lung cancer

Lung cancer has the highest mortality rate of all of the cancers in the world and asbestos‐related lung cancer is one of the leading occupational cancers. The identification of asbestos‐related molecular changes has long been a topic of increasing research interest. The aim of this study was to identify novel asbestos‐related molecular correlates by integrating miRNA expression profiling with previously obtained profiling data (aCGH and mRNA expression) from the same patient material. miRNA profiling was performed on 26 tumor and corresponding normal lung tissue samples from highly asbestos‐exposed and non‐exposed patients, and on eight control lung tissue samples. Data analyses on miRNA expression, and integration of miRNA and previously obtained mRNA data were performed using Chipster. A separate analysis was used to integrate miRNA and previously obtained aCGH data. Both known and new lung cancer‐associated miRNAs and target genes with inverse correlation were discovered. Furthermore, DNA copy number alterations (e.g., gain at 12p13.31) were correlated with the deregulated miRNAs. Specifically, thirteen novel asbestos‐related miRNAs (over‐expressed: miR‐148b, miR‐374a, miR‐24‐1*, Let‐7d, Let‐7e, miR‐199b‐5p, miR‐331‐3p, and miR‐96 and under‐expressed: miR‐939, miR‐671‐5p, miR‐605, miR‐1224‐5p and miR‐202) and inversely correlated target genes (e.g., GADD45A, LTBP1, FOSB, NCALD, CACNA2D2, MTSS1, EPB41L3) were identified. In addition, over‐expression of the well known squamous cell carcinoma‐associated miR‐205 was linked to down‐regulation of the DOK4 gene. The miRNAs/genes presented here may represent interesting targets for further investigation and could eventually have potential diagnostic implications.

Identification of specific gene copy number changes in asbestos-related lung cancer

Asbestos is a well-known lung cancer-causing mineral fiber. In vitro and in vivo experiments have shown that asbestos can cause chromosomal damage and aberrations. Lung tumors, in general, have several recurrently amplified and deleted chromosomal regions. To investigate whether a distinct chromosomal aberration profile could be detected in the lung tumors of heavily asbestos-exposed patients, we analyzed the copy number profiles of 14 lung tumors from highly asbestos-exposed patients and 14 matched tumors from nonexposed patients using classic comparative genomic hybridization (CGH). A specific profile could lead to identification of the underlying genes that may act as mediators of tumor formation and progression. In addition, array CGH analyses on cDNA microarrays (13,000 clones) were carried out on 20 of the same patients. Classic CGH showed, on average, more aberrations in asbestos-exposed than in nonexposed patients, and an altered region in chromosome 2 seemed to occur more frequently in the asbestos-exposed patients. Array CGH revealed aberrations in 18 regions that were significantly associated with either of the two groups. The most significant regions were 2p21-p16.3, 5q35.3, 9q33.3-q34.11, 9q34.13-q34.3, 11p15.5, 14q11.2, and 19p13.1-p13.3 (P < 0.005). Furthermore, 11 fragile sites coincided with the 18 asbestos-associated regions (P = 0.08), which may imply preferentially caused DNA damage at these sites. Our findings are the first evidence, indicating that asbestos exposure may produce a specific DNA damage profile.

Gene expression and copy number profiling suggests the importance of allelic imbalance in 19p in asbestos-associated lung cancer

Asbestos is a pulmonary carcinogen known to give rise to DNA and chromosomal damage, but the exact carcinogenic mechanisms are still largely unknown. In this study, gene expression arrays were performed on lung tumor samples from 14 heavily asbestos-exposed and 14 non-exposed patients matched for other characteristics. Using a two-step statistical analysis, 47 genes were revealed that could differentiate the tumors of asbestos-exposed from those of non-exposed patients. To identify asbestos-associated regions with DNA copy number and expressional changes, the gene expression data were combined with comparative genomic hybridization microarray data. As a result, a combinatory profile of DNA copy number aberrations and expressional changes significantly associated with asbestos exposure was obtained. Asbestos-related areas were detected in 2p21–p16.3, 3p21.31, 5q35.2–q35.3, 16p13.3, 19p13.3–p13.1 and 22q12.3–q13.1. The most prominent of these, 19p13, was further characterized by microsatellite analysis in 62 patients for the differences in allelic imbalance (AI) between the two groups of lung tumors. 79% of the exposed and 45% of the non-exposed patients (P=0.008) were found to be carriers of AI in their lung tumors. In the exposed group, AI in 19p was prevalent regardless of the histological tumor type. In adenocarcinomas, AI in 19p appeared to occur independently of the asbestos exposure.

Aberrations of chromosome 19 in asbestos-associated lung cancer and in asbestos-induced micronuclei of bronchial epithelial cells in vitro

Exposure to asbestos is known to induce lung cancer, and our previous studies have suggested that specific chromosomal regions, such as 19p13, are preferentially aberrant in lung tumours of asbestos-exposed patients. Here, we further examined the association between the 19p region and exposure to asbestos using array comparative genomic hybridization and fluorescence in situ hybridization (FISH) in lung tumours and FISH characterization of asbestos-induced micronuclei (MN) in human bronchial epithelial BEAS 2B cells in vitro. We detected an increased number of 19p losses in the tumours of asbestos-exposed patients in comparison with tumours from non-exposed subjects with similar distribution of tumour histology in both groups (13/33; 39% versus 3/25; 12%, P = 0.04). In BEAS 2B cells, a 48 h exposure to crocidolite asbestos (2.0 microg/cm(2)) was found to induce centromere-negative MN-harbouring chromosomal fragments. Furthermore, an increased frequency of rare MN containing a 19p fragment was observed after the crocidolite treatment in comparison with untreated controls (6/6000 versus 1/10 000, P = 0.01). The results suggest that 19p has significance in asbestos-associated carcinogenesis and that asbestos may be capable of inducing specific chromosome aberrations.

Molecular Alterations at 9q33.1 and Polyploidy in Asbestos-Related Lung Cancer

Purpose: Asbestos causes DNA damage and the fibers, together with tobacco smoke, have a synergistic effect on lung cancer risk. We recently identified 18 chromosomal regions that showed differences in DNA copy number between the lung tumors of asbestos-exposed and nonexposed patients. One of the previously identified asbestos-associated chromosomal regions at 9q was further analyzed for allelic imbalance and DNA copy number alterations (CNA) in the lung tumors of asbestos-exposed and nonexposed patients. In addition, the ploidy level of the tumors was studied. Experimental Design: Allelic imbalance was analyzed at 9q31.3-34.3 with 15 microsatellite markers in 52 lung tumor samples from asbestos-exposed and nonexposed patients. CNA at 9q32-34.3 were characterized by fluorescent in situ hybridization (FISH) with six bacterial artificial chromosome probes in 95 lung tumors. The ploidy level was analyzed in 100 lung tumors with FISH using three to five centromere probes. Results: Allelic imbalance at 9q31.3-q34.3 was found in all asbestos-exposed patient tumors (100%, 17 of 17) compared with 64% (14 of 22) in the nonexposed cases (P = 0.005). The most significant difference was detected at 9q33.1 (P = 0.002). FISH results showed that also CNA were more frequent at 9q33.1 in the three major histologic types of non–small-cell lung tumors of exposed patients, and the association showed a dose-dependent trend (P = 0.03). Furthermore, we detected more frequent polyploidy among the exposed (48%, 28 of 58) than among the nonexposed (29%, 12 of 42) patient tumors (P < 0.05). Conclusions: These results provide a basis for the development of a method to identify asbestos-related lung cancer on a molecular level.