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Oncol Rep. 2011 May;25(5):1421-9. doi: 10.3892/or.2011.1203. Epub 2011 Mar 2.

Disseminated colorectal tumor cells in organs prone to metastasis detected by new double enriched nested-PCR in comparison with recognized assays.

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Toxicology and Chemotherapy Unit, German Cancer Research Center, D-69120 Heidelberg, Germany.


Hypothetically, K-ras mutations can be used as a marker of disseminated tumor cells (DTCs) in patients with K-ras mutated primary carcinoma. This study focused on the development of a useful assay for detecting low numbers of DTCs in potential target tissues of metastatic K-ras codon 12 mutated colorectal cancer. Tumor, liver, lymph node and bone marrow tissues from 46 colorectal carcinoma patients were examined for K-ras codon 12 mutations with a new double enriched nested (DEN)-PCR and the incidence of mutations was compared to those obtained from three established assays. DEN-PCR followed by sequencing found one mutated cell within 107 K-ras codon 12 wild-type cells and was more sensitive than other methods (1:106-1:102). Colon carcinomas (26/46) and adenomas (1/3) harbored mutations in K-ras codon 12. Sixteen of these 27 mutated tumors were found with all assays, two with three methods and one with the two most sensitive assays. In 8 cases, only DEN-PCR identified the K-ras mutation, and thus prevailed over the other methods used (p<0.002). Eight of 26 patients with K-ras mutated colorectal carcinoma also harbored K-ras mutated DTCs in liver and lymph nodes, respectively, and 4 in bone marrow. For liver and lymph node samples, DTC-mutations were identical to those in the primary carcinoma but those in bone marrow differed from the respective mutation in the primary carcinoma. In conclusion, DEN-PCR is a highly sensitive method for detecting K-ras mutations as marker of early and late tumor cell dissemination in tissues potentially harboring colorectal carcinoma metastases.

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