PATIENTS

Patients were referred to Edouard-Herriot Hospital, a tertiary care unit for endoscopy and radiology, for treatment of malignant or benign extrahepatic biliary strictures. Prospectively, 117 unselected patients were included in the study, between March 1994 and January 1997.

FINAL DIAGNOSIS

BILE SAMPLES AND DNA EXTRACTION

Fresh bile (1 ml) was collected in tubes at the time of drainage and immediately frozen in liquid nitrogen. For DNA extraction, 0.5 ml of lysis buffer (0.5 M Tris HCl, pH 9.0, 16 mM EDTA, 10 mM NaCl containing 0.5% sodium dodecyl sulphate (SDS), and 0.1 mg/ml of proteinase K) was added to the bile sample and incubated overnight at 50°C. DNA was purified by two successive extractions, first by addition of phenol-chloroform-isoamyl alcohol (25:24:1) and second by addition of chloroform-isoamyl alcohol (24:1). DNA was then precipitated by adding three volumes of ethanol in the presence of 0.1 M CH₃COONa.

DNA AMPLIFICATION

PCR followed by PCR-mutant allele specific amplification (PCR-MASA) were performed with primers identical to those described previously.19 The first PCR allows amplification of the sequence of Ki-ras gene coding for the first exon. To avoid false positive reactions by amplification of previous PCR products, deoxyuridine 5′-triphosphate (dUTP) was used instead of deoxythymidine 5′-triphosphate (dTTP), leading to new PCR products containing dU. The first PCR was performed in 1× PCR buffer (Eurobio, Les Ulis, France) with 10–100 ng of target DNA, 2 mM MgCl₂, 180 μM of each dNTP, 0.8 μM of each primer, 1 U of Taq DNA polymerase (Eurobio), and 0.1 U of uracyl-N-glycosylase (HK-UNG, Epicentre Technologie, USA) in a final volume of 50 μl. Each sample was first incubated at 37°C for 20 minutes and then denatured at 95°C for five minutes. DNA was amplified by 40 successive cycles (20 seconds at 94°C, 40 seconds at 55°C, and one minute at 72°C). PCR products were analysed by electrophoresis through a 2% agarose gel in Tris acetate-EDTA buffer (Sigma, St Quentin Fallavier, France) containing 0.5 μg/ml of ethidium bromide.

PCR-MASA was performed on previously amplified DNA by 40 successive cycles of 30 seconds at 95°C, 50 seconds at 62°C, and 50 seconds at 72°C. The reaction mixture contained 1× PCR buffer (Eurobio), 2 mM MgCl₂, 100 μM of each deoxyribonucleotide triphosphate (dNTP), 1 U of polymerase enhancer (Perfect Match, Stratagene, Montigny-le-Bretonneux, France), and 1 U of Taq DNA polymerase (Eurobio) in a final volume of 25 μl. Codon 12 point mutations of the first or second nucleotide were screened with two different mixtures of primers. Each set contains one common antisense primer and three discriminating sense primers at 0.35 μM except for primers exploring the mutations G12C (GGT→TGT) and G12D (GGT→GAT) at 0.45 and 0.50 μM, respectively. PCR-MASA products were subjected to electrophoresis through a 2% agarose gel containing 0.5 μg/ml of ethidium bromide. When DNA was amplified, giving a band of the expected size, three additional PCR-MASA were performed using the first PCR product as the starting material. Each was carried out with a single set of primers for discriminating each possible point mutation. Final analysis of the PCR-MASA products was carried out by agarose gel electrophoresis, as above.

SEQUENCE SPECIFIC OLIGONUCLEOTIDE HYBRIDISATION (SSOH)

The first PCR product of each sample was dot blotted onto positive nylon membranes (Appligene, Illkirch, France) and hybridised with radiolabelled oligomer probes. The membranes were prehybridised in 0.05 M phosphate buffer, pH 7.0, 0.1 M NaCl, 0.1% SDS, and 300 μg/ml of denatured herring sperm DNA for one hour at 54°C, and then hybridised overnight with ³²P labelled oligonucleotide probes recognising either wild-type Ki-ras gene or different Ki-ras mutants. Each olignonucleotide was labelled with 3.7×10⁶ Bq of [³²P] labelled γ ATP (16.6×10¹³ Bq/mmol; ICN pharmaceuticals, Orsay, France) and T4 polynucleotide kinase. Membranes were rinsed at room temperature in 3× standard saline citrate (SSC) buffer, 0.1% SDS, and then washed three more times in 3× SSC for 30 minutes, successively at 58°C, 60°C, and 62°C. Autoradiography was performed overnight at −80°C with an intensifying screen. In these conditions, no cross hybridisation was observed between mutated probes.

POSITIVE CONTROL

Six different synthetic DNAs, each with a single point mutation of either the first or second nucleotide of Ki-ras codon 12, were used as positive controls. They were prepared by PCR site directed mutagenesis. Each was included as a positive control in the PCR-MASA and SSOH analyses.

PRESENTATION OF RESULTS

The frequency of Ki-ras gene point mutations detected in bile specimens was evaluated in different subgroups: malignant strictures (patients from groups 1 and 2 combined) versus benign strictures; primary carcinomas (pancreas, gall bladder, bile duct, or ampulla) versus metastases of other tumours; and primary tumours that developed in the main bile duct (bile duct and ampulla carcinoma) versus tumours possibly invading the bile duct (gall bladder and pancreas carcinomas). Statistical analyses were performed using Fisher's exact test (Statview, Berkeley, California, USA). p<0.05 was considered significant.

FINAL DIAGNOSIS

AMPLIFICATION OF DNA FROM BILE SPECIMENS

Amplification of the 286 bp of Ki-rasDNA was successful in 110 of 117 bile samples (94%). The wild-type sequence was detected in all 110 cases by both SSOH and PCR-MASA. To determine the cause of the failure of DNA amplification in the remaining seven bile specimens (obtained from patients with one benign and six malignant strictures, respectively), control DNA extracted from HeLa cells was added to these samples before the first PCR. This control DNA could not be amplified, suggesting that inhibitors of PCR were present in these seven bile specimens, as already suggested for stools.19

POINT MUTATIONS OF Ki-ras IN DNA AMPLIFIED FROM BILE SPECIMENS

Table 1 shows the sensitivity, specificity, and positive and negative predictive values of Ki-ras point mutations detected in bile specimens for confirmation of malignant strictures. Figure 1 shows the 22 point mutations detected in bile specimens sampled from the 90 patients in groups 1 and 2 (24.4% sensitivity, 95% confidence interval 15.5–33.3%). Ki-ras point mutations were detected in 31.4% (22/70) of cases of primary tumours compared with 0/20 metastases of other tumours (p<0.05), and in 27.3% (9/33) of tumours developed in the main bile duct (bile duct and ampulla carcinomas) versus 35.1% (13/37) of cases of gall bladder or pancreas carcinomas (NS). One mutation was detected in the 25 bile specimens originating from patients with benign strictures (96.0% specificity, 95% confidence interval 88.3–100.0%). The patient had a jejuno-biliary anastomosis previously performed for complicated cholecystectomy. He was followed for 36 months after bile drainage and did not exhibit any change in his general well being. He was therefore considered as having a benign biliary stricture. The predictive positive and negative values of Ki-ras mutation analysis for differentiating between benign and malignant biliary strictures were 96% and 25%, respectively.

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Figure 1

Gel electrophoresis analysis of the PCR-MASA products stained by ethidium bromide. Wild-type (G12) as well as mutated amplified DNA fragments are specified to the left. The origin of the amplified sample is indicated above (C, control without DNA; HeLa, HeLa cell DNA; G12A, G12D, G12V, and G12S, positive control DNA in which GGT codon 12 was changed for GCT, GAT, GTT, and AGT respectively; wild-type and mutated DNA originating from bile specimens of patients exhibiting pancreas (P), ampulla (Am), bile duct (BD), and gall bladder (GB) cancer.

PATIENTS WITH HISTOLOGICAL SPECIMENS OBTAINED BEFORE SURGERY

The diagnosis of malignant biliary strictures by non-surgical methods rests on examination of biopsies or cells obtained by endoscopy or percutaneous fine needle aspiration.6 ,20 However, such analyses are not 100% reliable and give negative results even in cases of definite malignant strictures, as demonstrated later by surgical means.6 The prevalence of Ki-ras point mutations was thus assessed in bile specimens from 49 patients with malignant strictures (groups 1 and 2) and with histological specimens obtained before surgery (endoscopic brushing in 16, endoscopic biopsies in 14, percutaneous cytology in 19). These histological specimens showed criteria for malignancy in 29 (59.2%) of the 49 cases. Ki-ras mutations were detected in bile specimens from 14 (28.6%) of these 49 cases, including seven of the 20 (35.0%) cases for whom the histological specimen was negative. In this group of 49 patients, the sensitivity of histology, Ki-ras mutation analysis, and combined methods were 59.2%, 28.7%, and 73.5%, respectively (table 1). The predictive negative value of histology and combined methods is not stated because histological specimens were available in only two patients with benign strictures.

COMPARISON BETWEEN SSOH AND PCR-MASA

Two G12A, 12 G12D, five G12V, and two G12S mutations were identified after PCR-MASA in bile samples from patients with malignant strictures. Concordance with the results given by the two methods was complete in all 15 cases that harboured a Ki-ras point mutation simultaneously detected by both methods. In seven cases, SSOH was not sensitive enough for detecting the point mutation found by PCR-MASA, irrespective of the nature of the mutation. One mutation identified by SSOH could not be screened by PCR-MASA (the sample was lost).