Detection of Ki-ras gene point mutations in bile specimens for the differential diagnosis of malignant and benign biliary strictures

Abstract

Background and aim-The present study was undertaken to determine if detection of Ki-ras gene point mutations in bile specimens could diVerentiate between benign and malignant biliary strictures. Patients-Bile specimens were obtained from 117 patients exhibiting a stricture of the main bile duct, the nature of which was assessed by cholangiography, histology, and follow up. Methods-DNA from frozen bile specimens was extracted, amplified, and tested for codon 12 point mutations of Ki-ras gene using sequence specific oligonucleotide hybridisation and mutant allele specific amplification. Results-DNA amplification was successful in 110/117 bile specimens (94%). Detection of Ki-ras gene mutations in bile specimens was positive in 24.4% (22/90) of patients with malignant strictures, in 31.4% (22/70) when only primary malignant tumours were considered, and in 4% (1/25) of patients with benign strictures. Of the 49 patients with histological specimens obtained before surgery, the sensitivity of histology, Ki-ras mutation analysis, and combined methods was 59.2%, 28.6%, and 73.5% respectively. Conclusions-Our study showed that Ki-ras mutations may be detected in about one third of bile specimens from patients with primary tumours invading the main bile duct. Detection of such mutations appears to be specific and may help to diVerentiate between benign and malignant biliary strictures.

Methylation status of p14ARF and p16INK4a as detected in pancreatic secretions

“…Like other groups, we observed the frequent failure of PCR analysis in pilot studies (Saurin et al, 2000). In our experience, it is essential to find out the least possible amount of DNA allowing PCR amplification.…”

Rapid Detection of K-ras Mutations in Bile by Peptide Nucleic Acid-mediated PCR Clamping and Melting Curve Analysis: Comparison with Restriction Fragment Length Polymorphism Analysis

“…In this study, we developed a one-step real-time PCR and detected K-ras mutations in approximately one-third of patients with a malignant etiology for the biliary stenosis, whereas mutations were not present in any bile from patients with benign obstructions. The mutation frequency in bile was comparable to the frequencies reported in previous studies (3,4,8,31 ). This low frequency may be explained by the scirrhous nature of cholangiocarcinoma; the extraductal growth of pancreatic cancer, which makes it difficult for the cancer cells to be exfoliated into the bile; and/or the PCR inhibitors present in bile.…”

“…Cholangiocarcinoma also carries K-ras mutations, but the reported frequencies, as assessed in tissue DNA, vary considerably, from 20 -100% in cases in Western countries (10 -12 ) to 5-58% in Japanese cases (13 ). In general, the frequencies of K-ras mutations in biliary duct carcinoma were reported to be 8 -100% in tissue DNA, 5-30% in bile DNA (3,4,10 ), and 42% in brushing fluids (14 ). The point mutations reside mainly in the first two nucleotides of codon 12.…”

“…The presence of K-ras gene mutations in bile, duodenal juice, and pancreatic juice has been used to distinguish benign from malignant biliary strictures (3)(4)(5)(6)(7)(8). Although K-ras mutations have been detected in some patients with benign biliary strictures, such as chronic pancreatitis and gallstones (3,6 ), the detection of such mutations appears to be specific and is considered to be of help in differentiating benign from malignant strictures (3)(4)(5).…”

“…Although K-ras mutations have been detected in some patients with benign biliary strictures, such as chronic pancreatitis and gallstones (3,6 ), the detection of such mutations appears to be specific and is considered to be of help in differentiating benign from malignant strictures (3)(4)(5). Caldas et al (9 ) noted that activating point mutations in codon 12 of the K-ras gene occur in approximately one-half of nonpapillary duct lesions but were present in the majority of papillary, more advanced pancreatic cancers.…”