The fluorescence was read by DyNA Quant? 200 fluorimeter, using the cuve and capillary DyDNA Capillary Cuvette Adaptor Kit (Pharmacia Biotech, Orsay, France). The threshold of DNA detection established by the manufacturer is 2ng��l?1, which corresponds to 100ngml?1 serum in our extraction first protocol (Coulet et al, 2000). Detection of KRAS2 gene mutations Only G12D mutations in codon 12 of the KRAS2 gene were searched for using allele-specific amplification, with the following primers: 5��-CTTGTGGTAGTTGGAGCTAA-3��, 5��-AATGGTCCTGCACCAGTAATATG-3��. Amplifications were performed with 0.3��M of each primers, 200��M of each deoxynucleotide triphosphate (dNTP), 1.5mM of MgCl2, 0.025units per ��l of AmpliTaq Gold polymerase Cetus (Perkin Elmer), 2.

5��l or 5��l of 10�� buffer, 5��l of the concentrated DNA elution was used as template in a 50��l volume reaction. Polymerase chain reaction (PCR) with serum DNA was performed 10min at 94��C, followed by 60 cycles of 94��C for 30s, 61��C for 30s, 72��C for 1min and a final extension of 10min at 72��C. Controls without DNA and positive controls were performed for each set of PCR reactions. PCR products were separated by electrophoresis in a 6% acrylamide gel and stained with ethidium bromide. Serum Ca 19.9 dosage The serum value of Ca 19.9 was measured with a commercial solid-phase double-antibody sandwich immunoassay (Roche Laboratories, Basel, Switzerland). The upper limit of normal value was 37UIml?1. All patients and controls also underwent biochemical liver tests. Cholestasis was defined by alkaline phosphatase levels above twice the normal value.

Statistical analysis For each marker (serum KRAS2 mutations and Ca 19.9), sensitivity, specificity, positive and negative predictive values were calculated. Thereafter, the combination of both markers (i.e., one and/or the other positive) was studied. The chi-squared test was used to compare the occurrence of KRAS2 mutations. Differences were considered significant when P<0.05. RESULTS Circulating DNA quantification Adequate DNA was extracted from the serum in sufficient quantities for analysis in all patients and controls. All patients but one had a serum DNA concentration higher than the threshold of detection of 100ngml?1. The mean concentrations of DNA extracted from serum of patients with pancreatic cancer and chronic pancreatitis were 730��90ngml?1 and 560��93ngml?1, respectively (P=0.

19). KRAS2 mutations in circulating DNA KRAS2 mutations were identified in the serum of 22 patients (47%) with pancreatic adenocarcinoma and in four patients (13%) with chronic pancreatitis (P<0.002). The sensitivity, specificity, positive and negative predictive values of serum KRAS2 mutations for the diagnosis of pancreatic cancer were 47, 87, 85 and 52%, respectively. GSK-3 There were no statistically significant differences in age, gender, smoking, tumour stage and survival, according to presence or absence of plasma KRAS2 mutations.