Molecular characterization of advanced non-small cell lung cancer patients by cfDNA analysis: experience from routine laboratory practice

Background: Analysis of circulating free DNA (cfDNA) by the real-time PCR cobas (R) EGFR Mutation Test v2 (cobas (R) EGFR Test) is a diagnostic approach used in clinical practice for the characterization of advanced non-small cell lung cancer (NSCLC) patients. The test additionally outputs a semiquantitative index (SQI) which reflects the proportion of mutated versus wild-type copies of the EGFR gene in cfDNA with potential use as a biomarker. CfDNA concentration and cfDNA fragmentation pattern have also shown potential utility as biomarkers for cancer patients. We evaluated the implementation of EGFR testing and cfDNA related parameters in NSCLC patients in routine clinical setting as biomarkers for disease stage and diagnosis. Methods: A prospective cohort of 173 locally advanced or metastatic NSCLC TKI-naive patients analyzed by the cobas (R) EGFR Test were included in the study. Reproducibility of the test was assessed in 56 patients. The concentration of cfDNA and fragment size pattern was measured using fluorometry and microchip electrophoresis respectively. Results: The test showed high diagnostic accuracy when compared to the gold standard of biopsy tumor tissue testing. The SQI value showed a moderate reproducibility (r(2)=0.70) and did not correlate with cfDNA concentration (r(2)=0.17, P=0.28) or disease stage (stage III patients SQI=9.1 +/- 3.1 and stage IV patients SQI =11.5 +/- 4.8, P=0.41). We found differences in SQI values according to the type of EGFR mutation (Ex19Del mutations, SQI = 13.6; p.L858R, SQI=8.88; P=0.001). Stage IV patients had higher concentrations of cfDNA (P<0.0001) and higher fractions of cfDNA 100-250 base pairs (bp) fragments (P=0.01) compared to stage III patients. From the ROC curve analysis, cfDNA concentration showed higher AUC compared to cfDNA 100-250 bp fragments (0.86 vs. 0.71). We obtained a cut-off value for cfDNA concentration of 20.3 ng/mL with 72.3% sensitivity and 95% specificity for predicting disease stage in TKI-naive advanced NSCLC patients. Conclusions: The study indicates that cfDNA analysis in plasma for EGFR testing by RT-PCR is an accurate and fast method to initially stratify NSCLC patients in a real-world clinical setting. However, the SQI has limited clinical value. The cfDNA concentration and fragmentation pattern have clear potential clinical utility for tumor staging in NSCLC patients.