Chromosomal aberrations reflect the selective retention of genomic fragments housing driver genes, whose abnormality contributes to tumorigenesis. The metaphase CGH assay has been used for the identification of novel driver genes, and its profiles correspond well to the chromosomal location of some known or suspected oncogenes and TSGs in lung tumors [7–10
]. However, for other frequently observed aberrations, no specific driver genes have yet been implicated because the method has relatively low resolution. We demonstrated in our study that the use of cDNA microarray CGH analysis may address this issue because cDNA micro-arrays represent high-resolution maps (in our study, one clone every 376 kb through the human genome), an approximately 20-fold higher mapping resolution than that attained by metaphase CGH. With the completion of the human genomic database, cDNA microarray CGH can map genomic gains and losses by their gene position rather than their chromosomal band, and therefore can immediately provide a list of candidate genes that occur within the region of interest.
The genomic copy number imbalances identified by our cDNA microarray CGH analysis appear comparable to those found in a recent study of lung cancer that used a BAC array CGH technique [11
]. Furthermore, because the cDNA microarray has a much higher mapping resolution (376 kb) than that achieved by BAC array CGH (1.4 Mb), our study restricted the larger fragment of genomic copy number changes to a small focal point of copy number aberrations of individual genes in primary lung tumors. For example, we determined two peaks of genomic gain at 5p13 and 5p35.3 in both the SQCAs and ADCAs that were not detected by the BAC array CGH, suggesting that the use of cDNA microarrays for analysis of DNA copy number variation has marked advantages over the use of large genomic DNA clone array-based CGH methods. Moreover, we determined the pattern of composite genomic losses of variable regions on several chromosomal loci in lung cancer, which was in keeping with the complex pattern of chromosomal rearrangements observed for deletions discovered by LOH [4,5
]; however, our results defined a narrower region and even identified the individual genes with genomic deletion. Thus, our cDNA microarray CGH analysis has a higher resolution than other methods and can be used to detect a small region or individual genes of amplification or deletion, and, finally, define the unique genomic signatures associated with lung tumorigenesis.
The results of our cDNA microarray CGH analysis also imply that primary SQCAs and ADCAs share common recurrent DNA copy number gains and losses of certain gene clusters, confirming previous findings that lung tumors involve a series of clonal molecular-genetic alterations [11,50
]. We also showed that substantial genomic differences exist between SQCAs and ADCAs. For example, when using metaphase CGH, we detected genomic gains in both ADCAs and SQCAs; the frequency of the genomic copy number of 3q was higher in SQCAs (100%, 6/6) than in ADCAs (50%, 4/8) (P
< .001). Correspondingly, when conducting cDNA array analysis of the same specimens, gene amplification of chromosome 3q was more frequent in SQCAs than in ADCAs. ADCAs tended to have a more heterogeneous gene transcript pattern and, in some cases, to exhibit a genomic profile in 3q more similar to that of nonneoplastic parenchymal lung tissues. The genomic copy number difference between primary SQCAs and ADCAs suggests that they may differ in the level of genomic instability or mechanisms by which they initiate and progress; the genomic aberrations of specific genes in the genomic phenotype of each of the histologic subtypes reflect their different genomic clonal evaluations and appear as different diseases at the molecular level [11
]. Most importantly, these unique genomic abnormalities may be developed as predictor sets of biomarkers for the early detection and classification of lung cancers.
Previous reports using the serial analysis of gene expression, oligonucleotides, or cDNA array analysis have described sets of genes overexpressed or downregulated in primary SQCAs and ADCAs [21,22,30,31,36,51
]. In contrast to SQCAs, which always showed clusters as a distinct tumor group, ADCAs tended to have a more heterogeneous gene transcript pattern and, in some cases, to exhibit a profile more similar to that of nonneoplastic parenchymal lung tissues. Both of these facts make it difficult to molecularly classify ADCA using transcript signatures [51
]. In our study, cDNA microarray CGH analysis provided a clear genomic profile of genes in primary ADCAs that is distinctly different from that in normal lung tissues. That genes in primary ADCAs had a distinct genomic pattern in our study but no clear transcriptional profile in other study is not surprising for several reason: 1) genomic DNA is a different mixture from the mRNA representation of cells; 2) transcription of genes has different biologic changes and behaviors from their genomic ancestors in lung cancer; and 3) the level of mRNA expression does not completely reflect genomic copy number changes. In addition, the inclusion of some ADCAs with normal lung from the previous reports may due to the profiling of BAC bronchioloalveolar carcinomas. The comparison of ADCAs with normal lung at the genomic DNA level by cDNA microarray CGH analysis should reveal the differences. Future assessment of transcript level and gene copy number changes of the same set of lung tumors in parallel using the same array may define whether genomic structural abnormalities directly affect imbalances of expression in lung tumorigenesis. However, our findings showing that primary ADCAs have a set of genes with a unique genomic profile may be of interests because these minimal gene sets can be used for developing biomarkers for ADCAs. This finding is particularly important because ADCAs have become more prevalent than SQCAs—a trend that is occurring worldwide—and are more difficult to diagnose than SQCAs because they always arise from the smaller airways [1
There was no statistically significant relationship between smoking pack year and certain genomic aberrations; one possible reason may be the small sample size of the current study. Currently, we are analyzing a large cohort of clinical specimens in an ongoing study, assessing the concordance of the genomic findings detected by cDNA microarray CGH and correlating these data with smoking history, prognosis, tumor progression, and treatment of the patients.
Although we used only four genes for confirmation in this study, all four showed a strong correlation between FISH analysis and cDNA microarray CGH data for genomic copy number changes, indicating that the genomic signatures discovered by cDNA microarray CGH might be developed as biomarkers for early interventional strategies for lung cancer. Furthermore, our results may suggest that the genomic changes we observed are likely relevant to lung tumorigenesis. In fact, alterations of some of the genes have been previously reported in lung cancer (). For example, SFTPA1
is a phospholipid-protein complex that lowers the surface tension at the air-liquid interface in the alveoli of the lung and plays a key role in the innate host defenses there. The transcription-level and protein-level aberration of SFTPA1
has previously been observed in lung tumorigenesis [52–54
]. The product of GC20/Sui1
is a general monitor of the translational accuracy of proteins through recognition of the protein synthesis initiation codon, and the expression of GC20/Sui1
induced is related to cellular stress and may represent an important adaptive response to genotoxic agents [55
has been detected in normal liver cells but not in hepatocellular carcinoma cells [56
]. We found that the GC20/Sui1
transcript was diminished in 80% of lung cancer cell lines tested by using reverse transcription polymerase chain reaction (RT-PCR) (data not shown). Skp2
displays an S-phase-promoting function in the cell cycle and is implicated in the ubiquitin-mediated degradation of several key regulators of mammalian G1 progression, including p27, a dosage-dependent tumor-suppressor protein. Skp2 protein is overexpressed in oral epithelial carcinomas, and its expression levels correlate positively with prognosis [57
]. A positive correlation of an increased relative copy number of Skp2
with a transcriptional level was found in small cell lung cancers cell lines [58
is one of the components of the Skp1-Cullin1-F-box-Roc1 complex [59
]. Inui et al. [60
] recently found a high expression of Cks1
in ADCAs and suggested that such high expression may be involved in the pathogenesis of the diseases. In agreement with that report, our study's detection of genomic gain of Cks1
by FISH and cDNA microarray CGH analyses was common in ADCAs. However, further characterization of the genes with a genomic aberration identified in our study is needed to evaluate the effects of the genomic aberrations on transcriptional and protein levels in lung cancer.
In summary, we have generated a profile of genomic copy number aberrations in the two major histologic subtypes of primary NSCLC tumors. Our findings may be a step toward defining a new genomic taxonomy of such tumors and demonstrate the potential power of genomic copy number profiling in lung cancer diagnosis. The development and implication of a relevant panel of probes for detecting genomic signatures might be of great value in lung cancer diagnosis and surveillance strategies in a clinical laboratory setting. Nevertheless, double-blind, prospective, confirmatory studies by independent groups are necessary to further validate these findings.