Detection of K-ras mutation by PNA-PCR/K-ras method in diagnosis of colorectal cancer tissues / 第二军医大学学报

ABSTRACT

Objective To determine the positive judgement standard of K-ras mutation detection method peptide nucleic acid (PNA)-PCR/K-ras (previously established in our laboratory) and to assess its diagnostic value for colorectal cancer tissues. Methods Plasmidswith K-ras codon 12 mutation and plasmids with K-ras wild-type plasmids were mixed and serially diluted into standard samples (mutation/total: 0, 1/3 200, 1/1 600, 1/800, 1/400, 1/200, 1/100) for six independent tests. The mutation CT, total CT and ΔCT (mutation CT-total CT) values were obtained by PNA-PCR/K-ras method. After the cut-off values of the mutation CT and ΔCT for K-ras diagnosis were identified by ROC analysis, the diagnostic criteria for K-ras mutation was defined by combining both the cut-off values of the mutation CT and ΔCT. A comparison was made between K-ras diagnostic rate by PNA-PCR/K-ns method and direct sequencing for 35 colorectal cancer tissues and their corresponding adjacent noncancerous tissues. Results The mutation CT and ΔCT values for 1/800 and the above standard samples were significantly different from those of the negative samples(P<0. 05), with the optimumcut-off values of mutation CT and ΔCT being 41. 7 and 15. 4, respectively. The diagnostic criteria (mutation CT≤41. 7 or ΔCT≤15. 4) for K-ras mutation was set up asAUC-ROC 0. 955 (P=0. 001). According this diagnostic criteria, the K-ras mutation diagnostic rates in each concentration gradient of the standard samples (0, 1/3 200, 1/1 600, 1/800, 1/400, 1/200, and 1/100) were 0%,66.7%,83.3%,100%, 100%, 100%,and 100%, receptivity, with the upper diagnostic limit being 1/800. The diagnostic rates of K-ras mutation by our method and by direct sequence method for colorectal cancer tissues were 45. 7% (32/70) and 18. 6% (13/70), respectively, showing significant difference (P = 0. 000). Conclusion PNA-PCR/K- ras method has higher sensitivity and positive detection rate than direct sequencing method for colorectal cancer tissues.