Detection of tumor cells in blood using CD45 magnetic cell separation followed by nested mutant allele-specific amplification of p53 and K-ras genes in patients with colorectal cancer

• Published in: International journal of cancer 2000-07, Vol.89 (4), p.337-344
• Main Authors: IINUMA, H, OKINAGA, K, ADACHI, M, SUDA, K, SEKINE, T, SAKAGAWA, K, BABA, Y, TAMURA, J, KUMAGAI, H, IDA, A
• Format: Article
• Language: English
• Subjects: Adenocarcinoma - blood; Adenocarcinoma - genetics; Adenocarcinoma - pathology; Adult; Alleles; Antibodies, Monoclonal - immunology; Antigens, CD34 - blood; Antigens, CD34 - immunology; Biological and medical sciences; Colorectal Neoplasms - blood; Colorectal Neoplasms - genetics; Colorectal Neoplasms - pathology; Digestive system; Female; Genes, p53 - genetics; Genes, ras - genetics; Humans; Immunomagnetic Separation - methods; Investigative techniques, diagnostic techniques (general aspects); Magnetics; Male; Medical sciences; Middle Aged; Mutation; Neoplasm Recurrence, Local - blood; Neoplasm Recurrence, Local - genetics; Neoplasm Staging; Neoplastic Cells, Circulating - pathology; Nucleic Acid Amplification Techniques; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Prognosis; Sensitivity and Specificity; Survival Analysis
• ISSN: 0020-7136; 1097-0215
• DOI: 10.1002/1097-0215(20000720)89:4<337::AID-IJC4>3.3.CO;2-I

A new method for detecting circulating tumor cells that is based on magnetic-activated cell separation (MACS) and nested mutant allele-specific amplification (nested MASA) was evaluated in patients with colorectal cancer using the p53 and K-ras genes as genetic markers. By negative selection with anti-CD45 monoclonal antibody-conjugated supermagnetic microbeads, the proportion of tumor cells was enriched 9-fold. By the combination of MACS and nested MASA, 10 tumor cells in 10(7) normal peripheral blood mononuclear cells could be detected without false-positives. Using this method, we examined blood taken from the tumor drainage veins of 23 patients with colorectal cancer. Eighty-seven percent (20/23) of primary tumor tissues showed p53 and/or K-ras gene mutations. Forty-five percent (9/20) of patients with p53 and/or K-ras mutations in the primary tumor showed the same mutated genes in the blood samples. There was a significant association between the presence of p53 and K-ras gene mutation in the blood and tumor size, depth of invasion, and venous invasion. Blood gene mutation was detected in 80% (4/5) of samples from patients with synchronous liver metastases. Sixty percent (3/5) of patients with mutant genes in the blood developed asynchronous liver metastases after surgery. The overall survival of patients with p53 and/or K-ras gene mutation-positive findings in blood was significantly shorter than that of patients testing negative on Kaplan-Meier analysis. Our results suggest that the method may be useful for reliable detection of tumor cells circulating in the blood and may help to identify patients at high risk for relapse.