Detection of the BRAF V600E Mutation in Liquid Biopsy Using the CRISPR/Cas12a System

This prospective, low-interventional study aims to develop and analytically and clinically validate a diagnostic tool for the detection of the BRAF V600E mutation in patients with unresectable metastatic melanoma after disease relapse. The developed method is intended for potential use in personalized medicine, particularly for optimization of treatment strategies and longitudinal monitoring of treatment response and disease progression. The diagnostic approach will be based on a CRISPR/Cas12a system combined with isothermal amplification and endpoint electrochemical detection. The CRISPR/Cas12a system will be used for selective degradation of the non-mutated (wild-type) DNA sequence, while isothermal amplification will provide a rapid and simple alternative to conventional PCR-based amplification. The amplified target will subsequently be detected using an electrochemical method based on electrode chips and relatively low-cost instrumentation. The primary objective of the study is to develop and validate the proposed electrochemical method for the detection of the BRAF V600E mutation in circulating cell-free DNA (cfDNA) isolated from plasma. The analytical and clinical performance of the new method will be assessed by comparison with established reference methods, including next-generation sequencing (NGS) and/or digital droplet PCR (ddPCR). Secondary objectives include evaluating changes in the presence and levels of BRAF V600E mutation in circulating DNA during treatment and assessing the potential of longitudinal changes in circulating tumor DNA (ctDNA) for early detection of disease progression or treatment resistance. The electrochemical signal obtained with the new method will be correlated with the corresponding results obtained using the reference methods. For NGS, the results will be compared with the corresponding sequencing findings, while for ddPCR, the electrochemical signal will be compared with the number of BRAF V600E-positive droplets. The sensitivity and specificity of the new electrochemical method will be evaluated in comparison with the reference methods. Approximately 10 patients with BRAF V600E-positive unresectable metastatic melanoma will be enrolled in the study. Patients may have melanoma diagnosed at relapse or may have a newly diagnosed melanoma presenting in an unresectable metastatic stage. Approximately 5-7 patients with BRAF V600E-negative unresectable metastatic melanoma will be included as negative controls. Peripheral blood samples will be collected at predefined time points, including before initiation of systemic treatment and after 6 months of treatment. Plasma will be isolated from the collected blood samples and used for cfDNA isolation and subsequent molecular analyses. The presence of the BRAF V600E mutation will be assessed using the newly developed electrochemical method and the established reference method(s). Longitudinal assessment of BRAF V600E in circulating DNA will be used to evaluate changes in mutation status during treatment and to explore the potential association between changes in ctDNA levels and treatment response, disease progression, or development of treatment resistance.