Droplet Digital PCR Mutant and Longitudinal Monitoring of BRAF Mutations in Urinary cfDNA of Patients with Metastatic Cancers or ECD



  • BRAF mutations confer a survival and growth advantage to cancer cells and can be used for selection of targeted therapies.
  • Cell-free (cf) DNA detected in the urine of individuals with cancer offers an easily obtainable, low-risk, and inexpensive source of biologic material for mutation analysis.
  • Longitudinal assessment of BRAF mutations in urinary cfDNA can be used for monitoring of molecular changes throughout cancer therapy.


  • Quantitative assessment of BRAF V600E in urinary cfDNA was accomplished by droplet digital PCR (RainDance, Billerica, MA) with enrichment of mutant DNA fragments by pre-amplification of BRAF mutant alleles.
  • Concordance was determined between mutation analysis results from urinary cfDNA and tumor tissue obtained during routine diagnostic or therapeutic procedures for testing in the CLIA laboratory.
  • Longitudinal assessment of mutation status in sequential urine samples was performed whenever possible.

Urinary cfDNA Mutant limits for BRAF V600E mutations

  • V600E Mutant: > 0.107% of mutant DNA.
  • V600E Low Mutant: 0.05% – 0.107% of mutant DNA.
  • Wild type: < 0.05% of mutant DNA.

Positive and negative controls for BRAF V600E mutation

  • A total of 33 patients with diverse advanced cancers or Erdheim-Chester disease (histiocytic disorder with high prevalence for BRAF mutations), who were previously tested for BRAF V600E mutations in tumor tissue by a CLIA-certified laboratory were prospectively enrolled.
  • Single or multiple sequential urine samples (90-110 ml or 24 hour urine collection) for cfDNA mutation analysis were obtained at baseline and during therapy.

Highlighted Tables and Graphs

Longitudinal Assessment of Urine cfDNA Mutations

Concordance of BRAF V600E Tissue (CLIA) to Baseline Urine cfDNA and Any Assessed Point of Urine cfDNA


  • Detection and monitoring of actionable BRAF V600E mutations with droplet digital PCR in urinary cfDNA from patients with advanced cancers and Erdheim-Chester disease display acceptable preliminary concordance with mutation testing of tumor tissue in the CLIA laboratory.
  • Urine-based cell-free DNA BRAF mutation testing may offer a non-invasive alternative to mutational analysis in tumor tissue.
  • Mutations in urine cfDNA should be further investigated for monitoring mutation status during anticancer therapies.


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