GENETIC-HETEROGENEITY OF THE C-K-RAS LOCUS IN COLORECTAL ADENOMAS BUTNOT IN ADENOCARCINOMAS

Background: Previous molecular genetics studies of colorectal cancer h ave identified multiple mutations in the c-K-ras gene (also known as K RAS2) in all phases of its development. Because of technical difficult y, prior studies rarely focused attention on the detailed distribution of c-K-ras mutations in multiple regions of the same primary tumor sp ecimen. However, with recent development of the selective UV radiation fractionation method, characterization of c-K-ras mutations in multip le regions of the same primary tumor specimen can be performed. Purpos e: Our purpose was to describe how c-K-ras mutations were distributed among cells obtained from multiple regions of the same primary tumor i n an attempt to describe differences between early and late colorectal carcinogenesis. Methods: Formalin-fixed, paraffin-embedded tissue blo cks were obtained. Seven adenocarcinomas and seven adenomas were selec ted for the presence of mutant c-K-ras genes and histologic transition s between normal and neoplastic tissue. Tissue sections were prepared for analysis by the selective UV radiation fractionation method by pla cing thin, fixed tissue sections on a plastic slide with no coverslip. Under the microscope, small ink dots from a felt-tip pen were manuall y placed directly on relatively pure cell subpopulations. The slides w ere placed with the tissue side exposed to a UV transillumintor for 2- 4 hours to inactivate the DNA present in the unprotected (''undotted'' ) cells. Individual dots were cut out of the plastic slide into 2 x 2- mm squares and placed into microfuge tubes. The DNA was extracted and supernatant used for polymerase chain reaction (PCR) analysis. Mutatio ns at c-K-ras codons 12 and 13 were detected. Results: The selective U V radiation fractionation method and PCR analysis revealed that c-K-ra s mutations never extended into normal mucosa and were present in all neoplastic cells regardless of phenotypes in all seven adenocarcinomas and three of the seven adenomas. Further examination of two carcinoma s for p53 (also known as TP53) mutations or loss of heterozygosity dem onstrated that these additional mutations were also present in all tum or cells, suggesting that a single transformed clone was responsible f or the majority of growth. However, in four other adenomas, tumor hete rogeneity was demonstrated, since c-K-ras mutations were detected only in discrete portions. Conclusions: Adenoma formation may include a st age in which multiple and genetically distinct neoplastic clones are p resent, while most carcinomas appear to have a homogeneous composition that may result from the successful progression of one of these clone s.