Ultrasensitive Detection of KRAS2 Mutations in Bile and Serum from Patients with Biliary Tract Carcinoma Using LigAmp Technology

Abstract

Patients with biliary tract carcinoma have a poor prognosis. Early detection efforts are urgently needed to ameliorate the dismal prognosis for these patients. Mutations of the KRAS2 gene are one of the most common genetic aberrations in this cancer. In this study, we used LigAmp, an ultrasensitive technology for detecting point mutations, to analyze KRAS2 mutations in patients with a variety of neoplastic and non-neoplastic pancreatobiliary diseases. DNA was isolated from 64 samples, including 44 bile samples and 20 serum samples. Oligonucleotides specific for KRAS2 G35A (GAT, G12D), G35T (GTT, G12V), and G34A (AGT, G12S) mutations were used. KRAS2 mutations were detected in 14 of 16 (87.5%) neoplastic bile samples and in 9 of 28 (32.1%) non-neoplastic bile samples. However, the mutation levels were significantly lower in the non-neoplastic bile (median = 0.4%) compared with those in the neoplastic bile (median = 5.1%). KRAS2 mutations were also detected in 9 of 11 (81.8%) serum samples from patients with biliary tract carcinoma, which was further confirmed by cloning BstN1-refractory PCR products and DNA sequencing. However, KRAS2 mutations were not present in the sera from eight patients with benign pancreatobiliary diseases. These data demonstrate that KRAS2 mutations are detectable in both bile and serum using LigAmp. This technology has the potential for early biliary tract carcinoma detection and possibly for residual disease monitoring post-therapy.