Molecular inversion probe analysis of gene copy alterations reveals distinct categories of colorectal carcinoma

Abstract

Genomic instability is a major feature of neoplastic development in colorectal carcinoma and other cancers. Specific genomic instability events, such as deletions in chromosomes and other alterations in gene copy number, have potential utility as biologically relevant prognostic biomarkers. For example, genomic deletions on chromosome arm 18q are an indicator of colorectal carcinoma behavior and potentially useful as a prognostic indicator. Adapting a novel genomic technology called molecular inversion probes which can determine gene copy alterations, such as genomic deletions, we designed a set of probes to interrogate several hundred individual exons of >200 cancer genes with an overall distribution covering all chromosome arms. In addition, >100 probes were designed in close proximity of microsatellite markers on chromosome arm 18q. We analyzed a set of colorectal carcinoma cell lines and primary colorectal tumor samples for gene copy alterations and deletion mutations in exons. Based on clustering analysis, we distinguished the different categories of genomic instability among the colorectal cancer cell lines. Our analysis of primary tumors uncovered several distinct categories of colorectal carcinoma, each with specific patterns of 18q deletions and deletion mutations in specific genes. This finding has potential clinical ramifications given the application of 18q loss of heterozygosity events as a potential indicator for adjuvant treatment in stage II colorectal carcinoma.

Figure 1

Standard curve for MIP quantitation using X chromosome variants. Genomic DNA samples were analyzed, which had different copy numbers of the X chromosome. Using the average intensity determined from normal female genomic DNA samples, the mean log 2 ratio was calculated for the four MIPs on the X chromosome. Y axis, measured log 2 ratio; X axis, theoretical log 2 ratio based on the known X chromosome variant.

Figure 2

Heat map of cluster from significant gene copy alterations in colorectal cancer cell lines and carcinoma samples. Unsupervised hierarchical clustering was done on the data set of significant gene copy regions as determined by CLAC. The dendrogram from the clustering algorithm was used to generate a heat map, graphically depicting the relationship among the colorectal cancer cell lines. The heat map of the data uses a green (losses) versus red (gains) color scale. Each column represents an individual MIP arranged by chromosome and physical location in increasing order from left to right. A, each row represents a separate colorectal cancer cell line. The two topmost branches are designated as clusters 1 and 2. A subcluster branch representing MSI cell lines is labeled as 1a. B, each row represents a separate colorectal carcinoma sample. The two topmost clusters are labeled as clusters 1 and 2.

Figure 3

Overall frequency of deletions and amplifications of clusters 1 and 2 classification of primary colorectal carcinomas. The gene copy frequency between cluster 1 and 2 is separately plotted. Y axis, overall frequency of gene copy changes for individual MIP; X axis, location of probe as arranged by chromosome and their nucleotide location.