Analysis of Ki-ras gene mutations within the same tumor using a single tumor crypt in colorectal carcinomas

The point mutations occurring in codons 12 and 13 of the Ki- ras gene are useful genetic markers to identify intratumoral heterogeneity. A single tumor crypt, which consists of monoclonal cells, can be obtained using the crypt isolation method. Ki- ras gene mutations have been examined using the crypt isolation method to determine whether multiclonarity is present within the same tumor. Ki- ras gene mutations were analyzed using a crypt isolation technique coupled with polymerase chain reaction and direct sequencing in 21 sporadic colorectal carcinomas. The specimens were divided into two groups: a representative sample, which was composed of more than 50 tumor crypts, and a single tumor crypt sample. The latter consisted of 10 single tumor crypts, which were obtained from the same tumor separately. Ki- ras gene mutations were found in 11 of 21 representative samples and in 12 of 21 single tumor crypt samples. In the 11 samples with Ki- ras mutation, Ki- ras mutations were also found in most single tumor crypts. Among the 12 base substitutions found, G:C to A:T transitions were the most commonly observed. There were no differences between the two samples in the types of Ki- ras mutations found. One Ki- ras mutation that was not detected in the representative sample was observed in only a single tumor crypt. Most carcinomas appear to have a homogeneous composition that may result from the successful progression of one of the clones having a Ki- ras mutation. Additional mutations in the Ki- ras gene were rarely observed in colorectal carcinomas.