QUANTITATIVE-DETERMINATION OF K-RAS MUTATIONS IN PANCREATIC-JUICE FORDIAGNOSIS OF PANCREATIC-CANCER USING HYBRIDIZATION PROTECTION ASSAY

K-ms mutations at codon 12 (KRM) have been detected in similar to 80% of samples of purr pancreatic juice (PPJ) from patients with pancreati c cancer (PCa) and are a promising potential tumor marker. However, th e frequent presence of KRM? was reported in PPJ from noncancerous pati ents as determined by a highly sensitive method, raising questions as to the cancer specificity of this marker. Therefore we evaluated wheth er the hybridization protection assay (HPA). which can quantitatively determine KRM in PPJ, is useful for the diagnosis of PCa, differentiat ing from chronic pancreatitis (CP). PPJ was collected endoscopically f rom 29 patients with PCa, 26 patients with CP, and the 11 cases as the control group. Polymerase chain reaction (PCR) and HPA using an acrid inium ester-labeled DNA probe for KRM were performed with DNA extracte d from these samples. The results were compared with those obtained by PCR-restriction fragment length polymorphism (RFLP). The mean + 2 SD of chemiluminescence in the control group was 11,020 RLUs. When 11,020 RLUs was taken as the cut-off value, KRM was detected by PCR-HPA in 1 9 (66%) of 29 of PCa and one (4%) of 26 of CP cases. Analysis of PPJ b y PCR-RFLP demonstrated KRM in 22 (79%) of 28 of PCa and five (19%) of 26 of CP cases. However, four of five patients with CP who were KRM-p ositive by PCR-RFLP were defined as negative by PCR-HPA, suggesting th at PCR-HPA is superior to PCR-RFLP for the discrimination between PCa and CP. These findings indicate that quantitative analysis of KRM in P PJ using the PCR-HPA method is a promising approach for the diagnosis of PCa, differentiating from CP with a suitable cut-off value, as in t he case with the use of conventional serum tumor marker.