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.
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 wild-type 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.
Dieterle
Christoph P
CP
Unit of Toxicology and Chemotherapy, German Cancer Research Center, Heidelberg, Germany.
Conzelmann
Michael
M
Linnemann
Ulrich
U
Berger
Martin R
MR
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000483:Alleles; D001853:Bone Marrow / Q000378:metabolism; D002277:Carcinoma / Q000378:metabolism; D045744:Cell Line, Tumor; D003062:Codon; D003106:Colon / Q000473:pathology; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004996:Ethidium / Q000494:pharmacology; D005260:Female; D011905:Genes, ras / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D008099:Liver / Q000378:metabolism; D019656:Loss of Heterozygosity; D008207:Lymphatic Metastasis; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009362:Neoplasm Metastasis; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D011379:Prognosis; D011446:Prospective Studies; D012007:Rectum / Q000473:pathology; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D003062:Codon; D017931:DNA Primers; D004247:DNA; D004996:Ethidium