A novel method for gene analysis of colorectal carcinomas using a crypt isolation technique

Abstract

Genetic studies of tumor masses require relatively pure populations of tumor cells. Substantial contamination by stromal cells, however, usually prevents detailed analysis of tumor cells. To improve the accuracy of gene analysis of tumor cells, a crypt isolation technique was introduced to separate neoplastic crypts from nonneoplastic stromal cells. Thirty-five specimens of colorectal carcinomas were examined for genetic alterations by using a PCR-based method combined with crypt isolation. K-ras gene mutations were detected by single-strand conformation polymorphism analysis. Allelic deletions of the p53 loci were estimated quantitatively based on loss of heterozygosity as determined by an automated DNA sequencer. Mutations of K-ras genes and allelic deletions at the p53 loci were detected in 18 (51%) and 15(68%) of 22 cases, respectively. The crypt isolation technique reduced ambiguity and provided quantitatively better results than conventional procedures. Evaluation of the allelic ratio for cases with loss of heterozygosity revealed that isolated neoplastic crypts were an almost pure population of tumor cells at the DNA level (>90%). Crypt isolation allowed sensitive and reliable analysis of the genetic alterations in colorectal carcinomas. This technique is applicable to biologic studies of colorectal tumorigenesis and genetic heterogeneities. To our knowledge, this is the first study in which genetic information has been derived directly from surgically resected primary colorectal carcinomas.