We have analyzed colon carcinomas by a combination of histological enrichment, cell sorting, polymerase chain reaction, and direct sequencing of the c-Ki-ras-2 gene. DNA was chemically extracted from 50-µm sections of paraffin-embedded colon carcinomas and amplified in vitro, and mutations were documented directly by DNA sequencing. Enrichment for tumor cells was obtained histologically and by sorting nuclei on the basis of DNA content differences. Mutations in codon 12 were present in both aneuploid and diploid subpopulations of sorted carcinomas, suggesting that these mutations precede ploidy alterations in the progression of these neoplasms. We have demonstrated the feasibility of utilizing DNA from tissues treated with different fixatives, including methyl carnoys, formalin, and Hollande's solution. This procedure allows one to retrospectively reconstruct the temporal relationship between the occurrence of mutations and sequential morphological changes during tumorigenic progression.
This work was supported by the National Cancer Institute Outstanding Investigator Award CA-39903 to Dr. Lawrence Loeb and by the NIH NIADDK Grant AM32971 to Dr. Peter Rabinovitch. Dr. Glenna Burmer was supported in part by NIH Grant AG00057 and by National Institute on Aging Grant AG07359.