ABSTRACT
Objective To determine the lower limit of detection (LLOD) and cut off values of K-ras mutation detection by peptide nucleic acid (PNA) clamping-PCR.Methods The genomic DNA of pancreatic cancer cell lines (PANC1 and SW1990) with codonl2,13 mutation and the genomic DNA of placenta with K-ras wild type were mixed and diluted serially into samples with different mutation rate (0,0.1％,0.2％,0.4％,0.8％,1.6％,3.1％,6.25％,12.5％,25％,50％),PANC1 cells with 1％ mutation rate and SW1990 cells with 30％ mutation rate and 4 samples with the quantity of DNA was 50,20,5,1 ng and 50,10,5,1 ng was prepared.Codon 12,13 mutation of K-ras was determined by PNA-PCR,and the mutation Ct values,overall Ct values were collected,and the △Ct values (mutation Ct values-overall Ct values) were calculated,and the tests were repeated for 10 times.ROC curve was used to analyze the △Ct values and determine the best cut off values for K-ras mutation,and the positive diagnostic rate,LLOD was evaluated.Results The mutation Ct,△Ct values of codon 12 mutation of PANC1 and codon 13 mutation of SW1990 of all the different mutation rates were statistically significantly different (P ＜ 0.05) when compared with negative control group,but the overall Ct values were not statistically significantly different from that of negative control group.For detection of K-ras codon 12 mutation by ROC curve,the relevant area of ROC curve (AUC) was 0.926,the optimum cut off value of △CT was 11,the sensitivity and specificity were 84％ and 100％,respectively,and the LLOD was 0.4 ng.For detection of K-ras codon 13 mutation by ROC curve,the relevant AUC was 0.906,the optimum cut off value of △CT was 9.5,the sensitivity and specificity were 71％ and 100％,respectively,and the LLOD was 1.5 ng.The mutation detection results of fixed rate further confirmed the LLOD.Conclusions This study successfully defines LLOD and cut off value of PNA clamping-PCR/K-ras method in detection of K-ras 12 and 13 codon mutations.This method meets the requirement of clinical application.
ABSTRACT
Objective To explore the clinical significance of the detection of K-ras12 and K-ras13 mutations in the peripheral blood of population with high risk group of pancreatic cancer.Methods From May 2010 to November 2011,a total of 160 patients with clear diagnosis and high risk of pancreatic cancer were collected.Among those patients,36 cases were finally diagnosed as pancreatic cancer,36 cases were chronic pancreatitis (CP),six cases were autoimmune pancreatitis (AIP),16 cases were other malignant tumor,13 cases were pancreatic benign tumor,13 cases were bile duct stones and 40 cases were other benign diseases.Fasting peripheral venous blood was collected in all selected candidates.The condition of K-ras12 and K-rasl3 mutation was detected by peptide nucleic acid-mediated real time fluorescen quantitative polymerase chain reaction.Mann-Whitney U test was performed to compare the mutation between the two groups.Chi-square test was used to compare the rates.According to the receiver operator characteristic (ROC) curve,the mutation positive decision value and the mutation positive rate of K-ras12 and K-ras13 were calculated.Results There were significant differences in K-ras12 mutation between pancreatic cancer group (1154(2207)) and other benign diseases (476(973)),CP group (446(808)),bile duet stones group (357(568)) (U=502,446 and 117,all P＜0.05).There were no significant differences in K-ras13 mutation between pancreatic cancer group and other benign diseases,CP group,bile duct stones group (all P＞0.05).When the mutation positive decision value of K-ras12 was set as mutation copy over 1000,the positive mutation rate of pancreatic cancer and non-pancreatic cancer was 55.6％ (20/36) and 25.0％ (31/124),respectively.When the area under the ROC curve of K-ras12 was 0.664,there was differential diagnostic value in identifying pancreatic cancer and non-pancreatic cancer (P=0.003).When the area under the ROC curve of K-ras13 was 0.522,there was no differential diagnostic value in identifying pancreatic cancer and non-pancreatic cancer (P=0.695).There was significant difference in K-ras13mutation between smokers and non-smokers (943 (1510) and 571 (964)).Conclusion K-ras12mutation in peripheral venous blood may be used for pancreatic cancer screening.
ABSTRACT
ObjectiveTo investigate the clinical significance of quantitative detection of K-ras codon 12 and 13 mutations in the tissues of pancreatic cancer and related pancreatic diseaaes. Methods One hundred and thirty samples from surgically removed pancreatic tissue with a conclusive pathological diagnosis (105 cases of pancreatic ductal adenocarcinoma,8 cases of pancreatic adenosquamous carcinoma of the pancreas,2 cases of pancreatic mucinous adenocarcinoma,3 cases of pancreatic endocrine carcinoma,6 cases of duodenal and papillary adenocarcinoma and 6 cases of benign pancreatic diseases ) were collected.Quantitative detection of K-ras codon 12 and 13 mutations was performed by the method of peptide nucleic acidmediated PCR clamping with two different fluorescence labeled probes.Mutation number ＞ 100 copies was used as the criteria to calculate the positive mutation rate.ResultsThe median and quartile of K-ras codon 12mutations of pancreatic ductal adenocarcinoma,adenosquamous carcinoma of the pancreas,pancreatic mucinous adenocarcinoma,pancreatic endocrine carcinoma,duodenal and papillary adenocarcinoma and benign pancreatic diseases were 4062 (495,10800),238 (45,8420),15 (9,21),3 (3,16),2283 (73,5037)and 21(8,56),and the positive mutation rates were 84.8％ (89/105),50.0％ (4/8),0,0,66.7％ (4/6)and 16.7％ (1/6).The quantity of K-ras codon 12 mutation in pancreatic ductal adenocarcinoma was not statistically different from those of adenosquamous carcinoma,duodenal and papillary adenocarcinoma,but it was significantly higher than those in pancreatic mucinous adenocarcinoma,pancreatic endocrine carcinoma,and benign pancreatic diseases (P ＜0.05).The area under ROC of K-ras codon 12 mutation in pancreatic ductal adenocarcinoma was 0.727.The sensitivity and specificity of the K-ras codon 12 mutation for the diagnosis of pancreatic ductal adenocarcinoma were 84.8％,64.0％,respectively.The quantity of K-ras codon 12 was associated with survival of patients with pancreatic ductal adenocarcinoma.The quantity of K-ras codon 13 mutations and the positive mutation rates in pancreatic ductal adenocarcinoma was not statistically different from other pancreatic diseases.ConclusionsThe quantity of K-ras codon 12 mutation has good differential diagnostic and prognostic prediction value for pancreatic ductal adenocarcinoma.