Clinical Interest of Circulating Tumor DNA in Oncology.

Genetic alterations in tumors, as predictor of response to targeted-therapies or as prognostic markers, are clinically relevant to determine adequate therapeutic management. Tumor biopsy is currently the golden standard for somatic alterations assessment, but this approach is invasive and does not consider tumor heterogeneity. In various body fluids like plasma, somatic mutations have been identified. Circulating tumor DNA (ctDNA) holds promises in tumor burden monitoring or malignancies early detection. Since allele frequencies of circulating somatic mutations are low, highly sensitive novel assays have been developed to allow the investigation of the tumor genome, leading to the emergence of the "liquid biopsy" concept. Despite these technological advances, other assays for identifying intratumor and intermetastases heterogeneity need to be developed. Before being applied to clinic, ctDNA analyses need to be harmonized and validated with well-powered, well-designed studies. One of the primary prerequisite to incorporation of ctDNA analysis in the follow-up strategy of malignancies is the checking of the concordance with golden standard detection methods, imaging, circulating proteins and biopsy. This review focuses on the clinical interest of ctDNA in solid tumors and hematological malignancies.

Detection of BRAF Mutations Using a Fully Automated Platform and Comparison with High Resolution Melting, Real-Time Allele Specific Amplification, Immunohistochemistry and Next Generation Sequencing Assays, for Patients with Metastatic Melanoma.

BACKGROUND: Metastatic melanoma is a severe disease with one of the highest mortality rate in skin diseases. Overall survival has significantly improved with immunotherapy and targeted therapies. Kinase inhibitors targeting BRAF V600 showed promising results. BRAF genotyping is mandatory for the prescription of anti-BRAF therapies. METHODS: Fifty-nine formalin-fixed paraffin-embedded melanoma samples were assessed using High-Resolution-Melting (HRM) PCR, Real-time allele-specific amplification (RT-ASA) PCR, Next generation sequencing (NGS), immunohistochemistry (IHC) and the fully-automated molecular diagnostics platform IdyllaTM. Sensitivity, specificity, positive predictive value and negative predictive value were calculated using NGS as the reference standard to compare the different assays. RESULTS: BRAF mutations were found in 28(47.5%), 29(49.2%), 31(52.5%), 29(49.2%) and 27(45.8%) samples with HRM, RT-ASA, NGS, IdyllaTM and IHC respectively. Twenty-six (81.2%) samples were found bearing a c.1799T>A (p.Val600Glu) mutation, three (9.4%) with a c.1798_1799delinsAA (p.Val600Lys) mutation and one with c.1789_1790delinsTC (p.Leu597Ser) mutation. Two samples were found bearing complex mutations. CONCLUSIONS: HRM appears the less sensitive assay for the detection of BRAF V600 mutations. The RT-ASA, IdyllaTM and IHC assays are suitable for routine molecular diagnostics aiming at the prescription of anti-BRAF therapies. IdyllaTM assay is fully-automated and requires less than 2 minutes for samples preparation and is the fastest of the tested assays.