DETECTION OF RAS GENE-MUTATIONS IN PERIPHERAL-BLOOD OF CARCINOMA PATIENTS USING CD45 IMMUNOMAGNETIC SEPARATION AND NESTED MUTANT ALLELE-SPECIFIC AMPLIFICATION

We developed a sensitive technique of detecting circulating tumor cell s in carcinoma patients, using CD45 immunomagnetic separation to isola te epithelial cells in blood samples and specific polymerase chain rea ction analysis to identify point mutations of the K-ras gene. The meth od is based on the fact that the peripheral blood mononuclear cells (P BMC) that express CD45 antigen are trapped with anti-CD45 conjugated s upramagnetic microbeads while the carcinoma cells that do not express CD45 antigen are not trapped and pass through the magnetic fields. Thi s method concentrated the number of carcinoma cells 3.3 times. After t his separation, the modified method of mutant allele specific amplific ation was applied and this method was able to ten control carcinoma ce lls in a background of 10(7) PBMC. A preliminary clinical study demons trated that six cases of endstage carcinoma with K-ras mutations in th e primary tumor showed the same mutations in the peripheral blood samp les, while two cases without K-ras mutation in the primary tumor and 1 0 healthy volunteers showed no mutation in the peripheral blood sample s. The results suggest that this method may be very useful to detect c irculating carcinoma cells in the patient whose primary tumor shows K- ras mutations.