Genomic profiles in stage I primary non small cell lung cancer using comparative genomic hybridization analysis of cDNA microarrays

Abstract

To investigate the genomic aberrations that are involved in lung tumorigenesis and therefore may be developed as biomarkers for lung cancer diagnosis, we characterized the genomic copy number changes associated with individual genes in 14 tumors from patients with primary non small cell lung cancer (NSCLC). Six squamous cell carcinomas (SQCAs) and eight adenocarcinomas (ADCAs) were examined by high-resolution comparative genomic hybridization (CGH) analysis of cDNA microarray. The SQCAs and ADCAs shared common frequency distributions of recurrent genomic gains of 63 genes and losses of 72 genes. Cluster analysis using 57 genes defined the genomic differences between these two major histologic types of NSCLC. Genomic aberrations from a set of 18 genes showed distinct difference of primary ADCAs from their paired normal lung tissues. The genomic copy number of four genes was validated by fluorescence in situ hybridization of 32 primary NSCLC tumors, including those used for cDNA microarray CGH analysis; a strong correlation with cDNA microarray CGH data emerged. The identified genomic aberrations may be involved in the initiation and progression of lung tumorigenesis and, most importantly, may be developed as new biomarkers for the early detection and classification of lung cancer.

Figure 1

The sensitivity of the cDNA microarray CGH analysis in detecting genomic copy number. Evidence of genes that increased in copy number (log₂ hybridization ratio >0) appears above the x-axis, whereas evidence of those that decreased in copy number (log₂ hybridization ratio >0) appears below the x-axis. (a) Chromosome X DNA copy number profile for male DNA compared with female DNA. The average log₂ Cy5:Cy3 hybridization ratio for X chromosome genes was -1, suggesting a 2:1 female-to-male X chromosome ratio. (b) Chromosome X DNA copy number profile for male DNA cohybridized with male DNA. The average log₂ Cy5:Cy3 hybridization ratio for X chromosome genes was 0, suggesting no X chromosome copy number change. (c) Chromosome 17 DNA copy number profile for one third the amount of the BT474 DNA used compared with the normal reference DNA; the log₂ hybridization ratio for the ERBB2 gene was 3.2, suggesting that the ERBB2 copy number was approximately 3 (arrow). (d) Chromosome 17 DNA copy number profile for DNA from NCI-H358 lung cancer cells cohybridized with normal reference DNA. The average log₂ hybridization ratio for TP53 genes was -1.2 (arrow), suggesting the ability of the cDNA microarray CGH to detect the losses of a single gene.

Figure 2

Comparison of metaphase CGH (a and b) and cDNA microarray CGH (c and b) for determining genomic copy number changes in 14 primary NSCLC tumors consisting of eight ADCAs and six SQCAs. Panels (a) and (b) are summaries of relative DNA sequence copy number changes detected by metaphase CGH in ADCAs and SQCAs, respectively. The vertical lines to the right of the chromosomal ideograms indicate copy number gains; those to the left indicate losses. Panels (c) and (d) are summaries of the cDNA microarray CGH profile of genomic imbalances for the ADCA and SQCA specimens tested, respectively, in (a) and (b). Evidence of genes that increased in copy number (log₂ hybridization ratio >0) appears to the right of the vertical line next to each chromosomal ideogram, and evidence of genes that decreased in copy number (log₂ hybridization ratio <0) appears to the left of the vertical line next to each chromosomal ideogram. The profile for chromosome Y is not shown because of the low representation of genes from this chromosome in the microarray.

Figure 2

Comparison of metaphase CGH (a and b) and cDNA microarray CGH (c and b) for determining genomic copy number changes in 14 primary NSCLC tumors consisting of eight ADCAs and six SQCAs. Panels (a) and (b) are summaries of relative DNA sequence copy number changes detected by metaphase CGH in ADCAs and SQCAs, respectively. The vertical lines to the right of the chromosomal ideograms indicate copy number gains; those to the left indicate losses. Panels (c) and (d) are summaries of the cDNA microarray CGH profile of genomic imbalances for the ADCA and SQCA specimens tested, respectively, in (a) and (b). Evidence of genes that increased in copy number (log₂ hybridization ratio >0) appears to the right of the vertical line next to each chromosomal ideogram, and evidence of genes that decreased in copy number (log₂ hybridization ratio <0) appears to the left of the vertical line next to each chromosomal ideogram. The profile for chromosome Y is not shown because of the low representation of genes from this chromosome in the microarray.

Figure 3

The 57 identified most informative gene associated with a P value of less than .05 in a paired t-test comparing eight ADCAs and six SQCAs. The heat map was made using TreeView [18]. The histologic type was given on top of the heat map.

Figure 4

Summary of the relative DNA sequence copy number changes detected by cDNA microarray CGH analysis in eight primary ADCAs and their paired normal lung tissues. Primary ADCAs have a clear genomic aberration profile of genes (a), which shows distinct differences from that of normal lung tissues (b). The vertical red bars represent the separation of chromosomes. Panel (c) shows the results of a permutation t test using 18 genes that have genomic changes and shows a significant difference between ADCAs and their paired normal lung tissues.

Figure 5

Validation of Skp2 genomic copy number changes in a SQCA specimen by metaphase CGH, cDNA microarray CGH analyses, and FISH. Metaphase CGH (a) shows a large amplicon in chromosome 5p, which is confirmed and precisely defined by cDNA microarray CGH and includes the Skp2 oncogene (b). The increased Skp2 copy number (c) was validated in the specimens by FISH using a Skp2 probe (green) and a centromeric probe (red), which was used as a reference. SQCA cells contain a two- to four-fold increase in the number of Skp2 probe signals compared with the number of the reference, indicating Skp2 amplification.