BRAF, NRAS and TERT mutations occur in more than 2/3 of melanomas.,Its detection in patient’s blood, as circulating tumor DNA (ctDNA), represents a possibility for identification and monitoring of metastatic disease.,We proposed to standardize a liquid biopsy platform to identify hotspot mutations in BRAF, NRAS and TERT in plasma samples from advanced melanoma patients and investigate whether it was associated to clinical outcome.,Firstly, we performed digital polymerase chain reaction using tumor cell lines for validation and determination of limit of detection (LOD) of each assay and screened plasma samples from healthy individuals to determine the limit of blank (LOB).,Then, we selected 19 stage III and IV patients and determined the somatic mutations status in tumor tissue and track them in patients’ plasma.,We established a specific and sensitive methodology with a LOD ranging from 0.13 to 0.37%, and LOB ranging from of 0 to 5.201 copies/reaction.,Somatic mutations occurred in 17/19 (89%) patients, of whom seven (41%) had ctDNA detectable their paired plasma. ctDNA detection was associated with shorter progression free survival (p = 0.01).,In conclusion, our data support the use of ctDNA as prognosis biomarker, suggesting that patients with detectable levels have an unfavorable outcome.
Detection of BRAFV600E within cell free tumor DNA (ctDNA) is emerging as a promising means to improve patients’ stratification or enable BRAF inhibitor (BRAFi) therapeutic monitoring in a minimally invasive manner.,Here, we investigated whether extracellular vesicle-(EV)-associated-DNA (EV-DNA) has value as an alternative source of circulating BRAFV600E.,To do so, we identified a clinical practice-compatible protocol for the isolation of EV-DNA and assessed BRAF gene status on plasma samples from metastatic melanoma patients at the beginning and during BRAFi therapy.,This protocol uses a peptide with high affinity for EVs and it has been found to recover more mutant DNA from plasma than standard ultracentrifugation.,Molecular analyses revealed that mutant DNA is largely unprotected from nuclease digestion, interacting with the outer side of the EV membrane or directly with the peptide.,When used on clinical samples, we found that the protocol improves the detection of BRAFV600E gene copies in comparison to the reference protocol for ctDNA isolation.,Taken together, these findings indicate that EVs are a promising source of mutant DNA and should be considered for the development of next-generation liquid biopsy approaches.