Abstract:

Objective To study the influence of common clinical interference factors on the detection of epidermal growth factor receptor（EGFR） T790M mutation of circulating tumor DNA（ctDNA） in plasma. Methods T790M wild-type plasma from 7 healthy donors was collected. Different concentrations of T790M mutant plasmid and certain interference factor（triglyceride,bilirubin and hemoglobin） were added into the T790M wild-type plasma to simulate interfered plasma. T790M mutation was detected by super amplification refractory mutation system（Super-ARMS） after DNA extraction. Each sample was detected for 3 times,the concentration and purity of DNA,Ct values of internal control and ΔCt values of T790M mutation were recorded. SPSS 22.0 software was used for statistical analysis. Results While the concentration of DNA of critically positive mutant samples could be affected by bilirubin（≥85.5 μmol/L） and triglyceride（≥9.25 mmol/L）（P<0.05）,the purity of DNA of them could not be affected. As low concentration of bilirubin（85.5 μmol/L） and high concentration of hemoglobin （2.0 g/L） affected the concentration of DNA of strongly positive mutant samples（P<0.05）,triglyceride （≥9.25 mmol/L） and hemoglobin（0.5-1.5 g/L） also affected the purity of DNA（P<0.05）. Lipemia had influence on the detection of T790M mutation by Super-ARMS,and triglyceride（≥9.25 mmol/L） affected the Ct values of internal control and ΔCt values of T790M mutation of both strongly and critically positive mutant samples（P<0.05）. Otherwise,high concentrations of bilirubin（≥256.5 μmol/L） affected the Ct values of internal control of strongly positive mutant samples,and middle concentrations of hemoglobin（≥1.0 g/L） affected their ΔCt values of T790M mutation（P<0.05）. Conclusions Different concentrations of hemoglobin,triglyceride and bilirubin have different effects on several indicators of detecting ctDNA EGFR T790M mutation by Super-ARMS. Plasma containing interference factors should be avoided in practical clinical detection as far as possible.

Key words: Circulating tumor DNA, Epidermal growth factor receptor, T790M, Interference factor, Triglyceride, Bilirubin, Hemoglobin