Molecular profiling of cell-free circulating tumor DNA (ctDNA) in patients with advanced NSCLC has become a powerful diagnostic approach for targeted therapy selection and monitoring response. We carried out clinical validation of ctDNA testing as liquid biopsy in patients with advanced NSCLC in ISO15189-accredited biopathology laboratory.
We performed (i) pre-analytic and analytic validation in comparison with tumor tissue from 270 NSCLC patients; (ii) real-time evaluation of ctDNA analysis for EGFR mutations before anti-EGFR therapy in 16 patients, and (iii) the study of resistance mutations in 34 refractory patients under treatment. For blood collection, EDTA and Cell-Free DNA BCT tubes (Streck) were used. ctDNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen). ctDNA mutation status was assessed using (i) the Therascreen EGFR RGQ PCR kit (Qiagen), and (ii) NGS assay interrogating >1800 mutation hotspots in 22 cancer-associated genes using the Oncomine Solid Tumor DNA panel and Ion PGM sequencer (ThermoFisher).
The analytic validation demonstrated 70% sensitivity and 98% specificity for the EGFR RGQ kit with limit-of-detection »5%. 43% of samples were found mutant by tumor tissue analysis while 57% were found mutant by ctDNA NGS analysis, with limit-of-detection »1%. ctDNA yield was not affected by storage in Streck BCT tubes for up to 3 days. Real-time longitudinal ctDNA analysis showed that among 18% of initially EGFR mutated patients, 33% acquire T790M resistance mutation during treatment. 74% of patients had at least one somatic alteration detected by NGS assay, with druggable alterations in various genes (e.g. MET, FGFR3). Mean turnaround time was 13 [4-22] days for tumor tissue and 6 [1-11] days for ctDNA analyses. Mean turnaround time was 12.5 [4-23] days for tumor tissue and 4.68 [1-15] days for EGFR ctDNA analysis.
When tissue biopsy is contra-indicated or of insufficient quantity, rigorously validated ctDNA assays is an alternative approach for tumor tissue molecular analysis and may enable longitudinal examination of molecular profiling in advanced NSCLC patients.
Clinical trial identification
Legal entity responsible for the study
CHU Nice, France
CHU Nice, France
All authors have declared no conflicts of interest.