Mutations in the KRAS2 oncogene during progressive stages of human colon carcinoma.

Abstract

A series of colon carcinomas, adenomas, and adjacent tissues were analyzed for ploidy alterations and mutations in KRAS2. To increase the sensitivity for identifying mutations, we used histological enrichment, cell sorting, and DNA amplification by the polymerase-catalyzed chain reaction followed by direct DNA sequence analysis. Of the 40 carcinomas analyzed, 27 contained aneuploid cells and 26 contained mutations at the first position of codon 12 ofKRAS2. Of the 12 adenomas studied, 4 contained aneuploid cells and 9 contained the same mutation at codon 12. In both adenomas and carcinomas, mutations were identified in both diploid and aneuploid cells. In some cases, regions of histologically benign mucosa adjacent to the carcinoma contained mutations. These combined results suggest that mutations in KRAS2 occur early in the development of human colon carcinoma, before change in ploidy, and that these mutations exist in diploid cells from which an aneuploid subpopulation arises. Furthermore, mutations may exist in histologically normal mucosa in regions adjacent to carcinoma, suggesting that a field of genetically abnormal mucosa may surround these tumors.Neoplastic progression is a multistep process characterized by genetic instability, loss of proliferative control, and clonal evolution (1-5). Mutations in cellular ras genes have been strongly implicated in this process (6). In humans, mutations within three ras genes, HRASI, KRAS2, and NRAS, have been reported in diverse tumors including myeloid leukemias, colon carcinomas, and pancreatic, thyroid, and renal cell carcinomas (7)(8)(9)(10)(11)(12)(13)(14). With colon carcinomas, investigators have further observed that ras gene mutations occur in villous adenomas adjacent to regions of carcinoma. These results suggest that ras mutations may not only be an early event in the temporal development of colon cancer but also contribute directly to the malignant phenotype (7)(8)(9).Efforts to elucidate the relationship between the occurrence ofras mutations and alterations in the behavior ofcolon tumors have used techniques of DNA amplification and oligonucleotide hybridization or RNase A mismatch-cleavage analysis on fresh or frozen human tissues (7-9). With these techniques -40%o of colon carcinomas and adenomas have been shown to contain ras mutations, the most frequent being a G --A transition at the second position of codon 12 or 13, resulting in a change from glycine to aspartic acid.However, these methods have several limitations. Quantitation of the frequency and nucleotide position of the mutation is difficult because most tumors consist of a heterogeneous population including epithelial cells, inflammatory cells, fibroblasts, and smooth muscle cells. The presence of nonneoplastic cells in these tumors can produce ambiguities in both the qualitative and the quantitative interpretation of results. The specific assignment of mutations to tumor cells requires purification of the tumor cell population. Moreover, oligonucleotide hybridization techniques hav...

Long-peptide vaccination with driver gene mutations in p53 and Kras induces cancer mutation-specific effector as well as regulatory T cell responses

“…To test for T cell responses against driver gene mutations of GI tumors we assembled a panel of long peptides comprising the most frequent KRAS 49-51 and TP53 52 hotspot mutations found in CRC and pancreatic cancer. We also included the BRAF V600E mutation 53 , which is a frequent driver mutation in melanoma but is also found to a lower percentage (10.7%) in GI tumors 51 .…”

The detection of K-ras mutations in colorectal cancer using the amplification-refractory mutation system

“…Many studies have examined the association of K-ras mutations with CRC (Bos et al, 1987;Forrester et al, 1987;Vogelstein et al, 1988;Bos, 1989;Burmer and Loeb, 1989;Vogelstein et al, 1989;Capella et al, 1991;Oudejans et al, 1991;Sidransky et al, 1992;Bell et al, 1993;Finkelstein et al, 1993a and b;McLellan et al, 1993;Urosevic et al, 1993;Breivik et al, 1994;Moerkerk et al, 1994;Morrin et al, 1994;Giaretti et al, 1995;Hasegawa et al, 1995;Hayashi et al, 1995;Ranaldi et al, 1995;Carpenter et al, 1996;Span et al, 1996;Villa et al, 1996). However, there are significant differences in reported frequencies of K-ras mutations in CRC (McLellan et al, 1993).…”

“…A significant proportion of CRCs have mutations in the K-ras oncogene (Bos et al, 1987;Forrester et al, 1987;Vogelstein et al, 1988;Burmer and Loeb, 1989;Delattre et al, 1989;Fearon and Vogelstein, 1990;Capella et al, 1991;Oudejans et al, 1991;Offerhaus et al, 1992;Bell et al, 1993;Finkelstein et al, 1993a and b;McLellan et al, 1993;Peltomaki et al, 1993;Urosevic et al, 1993;Breivik et al, 1994;Moerkerk et al, 1994;Morrin et al, 1994;Tanaka et al, 1994;Giaretti et al, 1995;Span et al, 1996). We report a study using 301 DNA samples extracted from a colorectal tumour bank.…”

“…The American Cancer Society estimated that in 1995 more than 130 000 new cases of CRC would be diagnosed in the USA and that there would be 54 900 deaths from the disease (American Cancer Society, 1995). CRC onset and its progression has been studied extensively at the molecular (Bos et al, 1987;Forrester et al, 1987;Vogelstein et al, 1988;Burmer and Loeb, 1989;Delattre et al, 1989;Kern et al, 1989;Vogelstein et al, 1989;Fearon and Vogelstein, 1990;El-Deiry et al, 1991;Oudejans et al, 1991;Houlston et al, 1992;Laurent-Puig et al, 1992;Offerhaus et al, 1992;Sharrard et al, 1992;Bell et al, 1993;Finkelstein et al, 1993a and b;McLellan et al, 1993;Peltomaki et al, 1993;Urosevic et al, 1993;Breivik et al, 1994;Dix et al, 1994;Moerkerk et al, 1994;Morrin et al, 1994;Tanaka et al, 1994;Giaretti et al, 1995;Laird et al, 1995;Lewis et al, 1996;Span et al, 1996) and genetic (Woolf et al, 1958;Macklin et al, 1960;Houlston et al, 1992;Zhao and Le Marchand, 1992;Peltomaki et al, 1993;Goldgar et al, 1994;Lewis et al, 1996) levels and there is a commonly accepted model relating tumour grade according to Dukes' stage (Dukes, 1932) to specific DNA changes …”

“…Although there are many examples of nucleic acid changes having potential as tumour markers (Bos et al, 1987;Forrester et al, 1987;Vogelstein et al, 1988;Burmer and Loeb, 1989;Delattre et al, 1989;Kern et al, 1989;Vogelstein et al, 1989; Fearon and Vogelstein, 1990;Jones and Buckley, 1990;Capella et al, 1991;El-Deiry et al, 1991;Oudejans et al, 1991;Stork et al, 1991;Laurent-Puig et al, 1992;Offerhaus et al, 1992;Sharrard et al, 1992;Bell et al, 1993;Finkelstein et al, 1993a and b;McLellan et al, 1993;Urosevic et al, 1993;Breivik et al, 1994;Dix et al, 1994;Magewu and Jones, 1994;Moerkerk et al, 1994;Morrin et al, 1994;Tanaka et al, 1994;Giaretti et al, 1995;Laird et al, 1995;Span et al, 1996), their value as clinical tools in cancer diagnosis, staging or even screening needs to be demonstrated and two important criteria must be met. First, adequate supplies of nucleic acid must be extracted from the clinical material; second, robust and accurate methods of analysis are required.…”