DETECTION OF K-RAS POINT MUTATIONS IN THE SUPERNATANTS OF PERITONEAL AND PLEURAL EFFUSIONS FOR DIAGNOSIS COMPLEMENTARY TO CYTOLOGIC EXAMINATION

To determine whether DNA analysis can be performed using the supernata nts of body fluids after centrifugation at 2,000 rpm for 10 minutes, p eritoneal or pleural effusions or bile were examined for K-ras mutatio ns in 34 cases of pancreatic, colorectal, gastric, esophageal, or hepa tocellular carcinoma and 15 noncancer cases. The polymerase chain reac tion products for K-ms gene codons 2 to 97 of exons 1 and 2 were gener ated with 41 (93%) of 44 body cavity fluid and 5 (100%) of 5 bile samp les. By the single strand conformation polymorphism method, point muta tions were detected in the ascites supernatants of 8 (89%) of 9 cases of pancreatic carcinoma. In the remaining case, no point mutation was demonstrated because few malignant cells were present in the ascites f luid. Furthermore, K-ms point mutations were observed in the ascites s upernatants of 2 cases of colorectal carcinoma and 1 case of gastric c arcinoma. The DNA analysis of the supernatant of ascites fluid showed a K-ms point mutation in 3 cases of false-negative cytologic diagnosis (2 cases of pancreatic carcinoma and 1 case of colorectal carcinoma). Direct sequencing confirmed identical point mutations in the supernat ants, whole cell pellets, malignant cells from the cytologic smears of ascites fluid, and cancer tissues. This novel method allows simultane ous testing for genetic abnormalities in supernatants of body fluid, a fter removing cells for cytologic diagnosis.