An interventional study of Novel CRISPR/Cas12a Electrochemical Bioassay and Reference Standard Molecular Diagnostics (NGS and ddPCR) in Unresectable Metastatic Melanoma, sponsored by Masaryk Memorial Cancer Institute. Recruiting at 1 site in Czechia. Open to participants aged 18 Years and older. Per ClinicalTrials.gov, last updated 2026-10-16.
Sponsored by Masaryk Memorial Cancer Institute · Not applicable, Interventional, and Diagnostic
The goal of this prospective, low-interventional study is to develop and optimize a highly sensitive and specific method for the detection of the BRAF V600E mutation in liquid biopsy samples from patients with unresectable metastatic melanoma after disease relapse. The method will be based on a CRISPR/Cas12a system combined with isothermal amplification and endpoint electrochemical detection using electrode chips and low-cost instrumentation.
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.
3,073 studies on the registry are indexed under Melanoma; 529 are open to participants now.
This study's planned enrollment of 17 is below the median of 38 across 2,400 interventional studies indexed under Melanoma.
Browse Melanoma studies →Masaryk Memorial Cancer Institute is the lead sponsor of 12 studies on the registry; 9 are open to participants now.
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Exclusion Criteria:
BRAF V600E-Positive Metastatic Melanoma: Patients aged ≥ 18 years with histologically confirmed inoperable metastatic melanoma presenting with a documented BRAF V600E mutation (at primary diagnosis or disease relapse). A target cohort of 10 participants will undergo serial blood sampling for longitudinal monitoring of BRAF V600E mutation dynamics in cell-free DNA (cfDNA)
Diagnostic Test: Novel CRISPR/Cas12a Electrochemical Bioassay · Diagnostic Test: Reference Standard Molecular Diagnostics (NGS and ddPCR) · Procedure: Serial Peripheral Blood Sampling for Liquid Biopsy
BRAF V600E-Negative Metastatic Melanoma: Control group consisting of approximately 5 to 7 patients aged ≥ 18 years with histologically confirmed inoperable metastatic melanoma who are negative for the BRAF V600E mutation (BRAF wild-type). Participants undergo an identical serial blood sampling protocol to evaluate background signal, analytical specificity, and diagnostic performance.
Diagnostic Test: Novel CRISPR/Cas12a Electrochemical Bioassay · Diagnostic Test: Reference Standard Molecular Diagnostics (NGS and ddPCR) · Procedure: Serial Peripheral Blood Sampling for Liquid Biopsy
Experimental diagnostic platform utilizing the CRISPR/Cas12a system for selective enzymatic degradation of wild-type (WT) DNA sequences, followed by isothermal nucleic acid amplification (LAMP) and end-point electrochemical signal detection on microelectrode chips. Designed for sensitive and specific detection of the BRAF V600E mutation in circulating cell-free DNA (cfDNA) isolated from plasma.
Baseline baseline characterization of tumor tissue mutation status using Next-Generation Sequencing (NGS), accompanied by droplet digital PCR (ddPCR) for orthogonal measurement and quantitation of BRAF V600E allele frequency in circulating tumor DNA (ctDNA). Serves as the reference clinical comparator for evaluating sensitivity and specificity.
Venipuncture collecting 10 mL of peripheral blood into dedicated blood collection tubes at two specified clinical timepoints: Day 1 (prior to treatment initiation) and Day 1 + 6 months during ongoing therapy. Collected blood samples undergo immediate plasma isolation and cfDNA extraction.
Diagnostic Sensitivity and Specificity of the CRISPR/Cas12a Electrochemical Bioassay
Evaluation of the diagnostic performance (sensitivity and specificity) of the novel CRISPR/Cas12a-assisted electrochemical platform for detecting the BRAF V600E mutation in circulating cell-free DNA (cfDNA). Electrochemical current output measured on microelectrode chips will be validated against reference molecular diagnostics, specifically droplet digital PCR (ddPCR mutant allele count) and Next-Generation Sequencing (NGS).
Time frame: Up to 6 months post-treatment initiation (assessed at Baseline/Day 1 and Month 6).
Longitudinal Dynamics of *BRAF* V600E ctDNA Levels During Therapy
Quantitative monitoring of longitudinal changes in BRAF V600E circulating tumor DNA (ctDNA) levels in plasma across treatment timepoints. Statistical comparison of mutant DNA level variations over time will be conducted to characterize ctDNA kinetics during therapy.
Time frame: Baseline (Day 1 prior to treatment initiation) and Month 6 during treatment.
Correlation Between Electrochemical Current Signal and Reference Standard Quantitative Values
Statistical correlation between the raw electrochemical current signal (generated following Cas12a wild-type cleavage and isothermal amplification) and reference quantitative metrics, including ddPCR positive droplet count and NGS sequence variant frequency.
Time frame: Baseline (Day 1) and Month 6.
Molecular Signal Evaluation for Early Disease Progression and Resistance
Qualitative and quantitative assessment of serial liquid biopsy samples to evaluate the bioassay's utility for early identification of molecular disease progression or emerging therapeutic resistance during ongoing therapy.
Time frame: Up to 6 months of patient monitoring.
Plan to share: Undecided — A decision regarding the sharing of individual participant data has not yet been finalized. Formal data sharing policies for raw bioassay datasets will be determined prior to publication in accordance with institutional guidelines.
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Masaryk Memorial Cancer Institute