Detection of Isolated Tumor Cells by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism for K-ras Mutations in Tissue Samples of 199 Colorectal Cancer Patients

Abstract

The aim of this study was to identify K-ras mutations as marker for isolated tumor cells in liver, lymph node, and bone marrow specimens of colorectal cancer patients. To detect these, a PCR-RFLP assay was used with a sensitivity exceeding that of routine histopathology by at least 1 order of magnitude. In addition, the ratio of mutated versus wildtype alleles was determined by an internal standard. Of 199 patients, 74 (37.5%) were found to bear a K-ras-positive tumor. Of these, 60 (81%) were mutated in codon 12 and 14 (19%) in codon 13 (P < 0.001). In addition, 14 organs were found K-ras positive, 13 of which were from 12 patients with a K-ras-positive tumor (16%) and 1 from a patient with a K-ras-negative tumor (0.8%). Eight patients exhibited liver involvement and 6 showed lymph node involvement. Remarkably, no bone marrow specimen was found K-ras positive (P < 0.017 versus liver involvement). Sequence analysis of tumor DNA revealed that GGT (Gly) was replaced by GAT (Asp; 35%), GTT (Val; 32%), AGT (Ser; 13%), GCT (Ala; 10%), TGT (Cys; 8%), and CGT (Arg; 2%) for codon 12, and by GAC (Asp) as the only type of mutation for codon 13. In colorectal carcinomas the ratio of K-ras mutated versus wild-type alleles ranged over 4 orders of magnitude (10 0 -10 ؊4 , median: 10 ؊2 ) and was correlated with both, residual tumor load (R1/2; P ‫؍‬ 0.028) and distant metastasis (M1; P ‫؍‬ 0.057). These results show that detection of K-ras mutated alleles by PCR-RFLP in patients with colorectal carcinoma may aid in the identification of isolated tumor cells. High ratios of K-ras alleles were correlated with certain negative prognostic parameters (R,M). In accord with its function as a primary filter for colorectal carcinoma cells, the liver was more often contaminated with K-ras-positive cells than bone marrow.

The balanced induction of K-ras codon 12 and 13 mutations in mucosa differs from their ratio in neoplastic tissues

“…Furthermore, the spectrum of mutations found in mucosa tissue shows a clear predominance of G:C➝A:T transitions in both, codon 12 and 13 mutations in concordance with other studies (13,17). For codon 12, this clear predominance is lost in neoplastic tissues.…”

“…Multiple samples of apparently normal mucosa tissue (group I) and of polyp tissue (group II) were compared with a sample from concomitantly growing colorectal carcinoma. In variation with previous, less detailed investigations on mucosa tissue (12,13), a higher percentage of mucosa samples (14.6%; corresponding to 30.5% of all patients) were found to harbour a K-ras mutation in their inconspicuous mucosa. More remarkable, however, is the finding that there was an even distribution between K-ras codon 12 and 13 mutations.…”

The prognostic value of c‐Kit, K‐ras codon 12, and p53 codon 72 mutations in Egyptian patients with stage II colorectal cancer

“…Mutations in codons 12 and 13 of K-ras were detected by polymerase chain reaction (PCR)/restriction fragmentlength polymorphism analysis with MvaI and XcmI restriction endonucleases (Amersham Life Science, Buckinghamshire, England) as described previously 27,28 ( Table 2). DNA from the CRC cell line HT-29 was used as a standard to control for the efficacy of the restriction enzymes, and double deionized water was used as a negative control.…”

Analysis of the K-ras/B-raf/Erk signal cascade, p53 and CMAP as markers for tumor progression in colorectal cancer patients

“…The detection of K-Ras mutations in codons 12 and 13 was performed by PCR-RFLP as previously described (17). In brief, DNA was amplified in the first PCR (30 cycles), using primers Ras A and -B (Table I) which generated an amplicon of 166 bp.…”

“…DNA sequencing was performed as previously described (17). Briefly, bands with a length of 134 bp, indicating the mutated DNA, were excised from 3% agarose gels.…”