Analytical Evaluation of an NGS Testing Method for Routine Molecular Diagnostics on Melanoma Formalin-Fixed, Paraffin-Embedded Tumor-Derived DNA.

ABSTRACT

Next Generation Sequencing (NGS) is a promising tool for the improvement of tumor molecular profiling in view of the identification of a personalized treatment in oncologic patients. To verify the potentiality of a targeted NGS (Ion AmpliSeq™ Cancer Hotspot Panel v2), selected melanoma samples (n = 21) were retrospectively analyzed on S5 platform in order to compare NGS performance with the conventional techniques adopted in our routine clinical setting (Sequenom MassARRAY system, Sanger sequencing, allele-specific real-time PCR). The capability in the identification of rare and low-frequency mutations in the main genes involved in melanoma (BRAF and NRAS genes) was verified and integrated with the results deriving from other oncogenes and tumor suppressor genes. The analytical evaluation was carried out by the analysis of DNA derived from control cell lines and FFPE (Formalin-Fixed, Paraffin-Embedded) samples to verify that the achieved resolution of uncommon mutations and low-frequency variants was suitable to meet the technical and clinical requests. Our results demonstrate that the amplicon-based NGS approach can reach the sensitivity proper of the allele-specific assays together with the high specificity of a sequencing method. An overall concordance among the tested methods was observed in the identification of classical and uncommon mutations. The assessment of the quality parameters and the comparison with the orthogonal methods suggest that the NGS method could be implemented in the clinical setting for melanoma molecular characterization.