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

ConditionsUnresectable Metastatic MelanomaBRAF V600E Mutation
Trial statusRecruiting
Trial phaseNot applicable
Trial typeInterventional
Biological sexAll
Age18+
SponsorMasaryk Memorial Cancer Institute

About this trial

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.

Eligibility criteria

This trial does not accept healthy volunteers

Qualifiers

Age ≥ 18 years.

Subject willing and able to provide written informed consent for biological sample collection.

Ability to communicate effectively with the investigator in the local language, as well as the ability to understand and comply with study requirements.

Histologically confirmed malignant melanoma.

Disqualifiers

Operable disease relapse.

Age under 18 years.

Pregnant or breastfeeding women.

Patients receiving palliative care with an estimated life expectancy ≤ 3 months.

Trial design

Design model

Parallel

Treatments tested in this trial

  • Novel CRISPR/Cas12a Electrochemical Bioassay

    Diagnostic test

    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.

  • Reference Standard Molecular Diagnostics (NGS and ddPCR)

    Diagnostic test

    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.

  • Serial Peripheral Blood Sampling for Liquid Biopsy

    Procedure/Surgery

    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.

Treatment groups

17 Participants
are divided into 2 treatment groups
Group A: Arm I (Active Monitoring)Experimental treatment 3 interventions
Group B: Arm II (Control)Active comparator 3 interventions

Trial outcomes

Primary outcomes

1

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).

Secondary outcomes

1

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.
2

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.
3

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.

Other outcomes

Sponsors and contacts

Click on the lead sponsor to view all of their trials.

Masaryk Memorial Cancer Institute

Lead sponsor

Source ClinicalTrials.gov
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