Since tissue is not always available, biomarkers testing, can be performed on circulating free DNA (cfDNA). Compared to real time PCR, next-generation sequencing (NGS), covers also less common and novel variants. To increase sensitivity NGS can be narrowed to target a limited number of actionable genes. This strategy, known as ultra-deep sequencing, requires a careful validation. Here we validated a narrowed gene panel to produce a DNA library covering 568 actionable mutations in six gene (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα) involved in non small cell lung cancer, gastrointestinal stromal tumor, metastatic colo rectal carcinoma and melanoma (SiRe).
This study had a retrospective and prospective design. After in – vitro studies on cell lines aimed to assess the assay analytical performance, cfDNA from a retrospective series of cases including, lung (n = 51) and colon (n = 3) neoplasms and melanoma (n = 9) previously well characterized on both tissue and matched cfDNA was employed to validate the SiRe panel; then, blood samples prospectically collected from NSCLC patients (n = 87) were tested to assess this panel performance in daily clinical practice.
On cell lines, the SiRe had high intra – and inter – run reproducibility with 0.2% lower limit of mutation detection. In the retrospective series of cfDNA, a total of 54 mutations were detected by SiRe showing 100% specificity, confirming 39 EGFR, 9 KRAS, 1 NRAS, 5 BRAF mutations previously detected on matched tissue. Noteworthy, in 4 cases SiRe detected mutations that had been missed on cfDNA by real time PCR. On prospectically collected cfDNA, SiRe detected in 4/46 patients, without baseline tissue availability, activating EGFR mutation; at the time of tumor progression following the treatment with gefitinib, erlotinib and afatinib SiRe detected T790M in 30% (9/30). On the overall, the SiRe panel showed a sensibility of 93.4% and specificity of 100%.
The SiRe panel is an effective tool enabling a cost - effective implementation of NGS for cfDNA mutational profiling in molecular pathology practice
Clinical trial identification
Legal entity responsible for the study
University of Naples Federico II, Department of Public Health
University of Naples Federico II, Department of Public Health.
All authors have declared no conflicts of interest.