The pre-analytical phase of the liquid biopsy.

The term 'liquid biopsy', introduced in 2013 in reference to the analysis of circulating tumour cells (CTCs) in cancer patients, was extended to cell-free nucleic acids (cfNAs) circulating in blood and other body fluids. CTCs and cfNAs are now considered diagnostic and prognostic markers, used as surrogate materials for the molecular characterisation of solid tumours, in particular for research on tumour-specific or actionable somatic mutations. Molecular characterisation of cfNAs and CTCs (especially at the single cell level) is technically challenging, requiring highly sensitive and specific methods and/or multi-step processes.

Detection and Characterization of Circulating Tumor Cells by Quantitative Real-Time PCR.

We propose two different approaches involving the use of quantitative real-time PCR for the detection or analysis of circulating tumor cells. In one case cells are indirectly identified through the expression of a marker mRNA, while in the other one cells are enriched by size prior to be submitted to mutational analysis for a specific target. Both methods have been successfully applied to the study of circulating melanoma cells.

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

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 ( = 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 ( and genes) was verified and integrated with the results deriving from other oncogenes and tumor suppressor genes.