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.
Highlights
In recent years, the most important revolution in solid tumor management has been represented by the development of targeted therapies and the adoption of molecular criteria for precision diagnosis [1].The capability to perform molecular profiles of cancers has greatly contributed to the identification and the enlargement of approved anti-cancer drugs as well as to refining the prediction of response in subgroups of patients harboring specific alterations
In the context of somatic mutations detection, these systems are mainly represented by the array platforms, combined with the chemistry used in qPCR [2] and the analysis of multiple extension fragments by mass-spectrometry [3,4,5]
This study investigates the potentiality of a targeted Next Generation Sequencing (NGS) approach
Summary
The most important revolution in solid tumor management has been represented by the development of targeted therapies and the adoption of molecular criteria for precision diagnosis [1].The capability to perform molecular profiles of cancers has greatly contributed to the identification and the enlargement of approved anti-cancer drugs as well as to refining the prediction of response in subgroups of patients harboring specific alterations. The most important revolution in solid tumor management has been represented by the development of targeted therapies and the adoption of molecular criteria for precision diagnosis [1]. Diagnostics 2019, 9, 117 role in the era of personalized and precision medicine, the development of new molecular methods is a challenge in the research field and an important improvement for the diagnostic laboratories. Common methods applied to the molecular screening of tumor samples such as real-time polymerase chain reaction (qPCR) and Sanger sequencing are able to perform a single-mutation or single-exon test for each gene. In the context of somatic mutations detection, these systems are mainly represented by the array platforms, combined with the chemistry used in qPCR [2] and the analysis of multiple extension fragments by mass-spectrometry [3,4,5]
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