Figure 5.

Detection of circulating BRAF^V600E gene copies from plasma of wild type (WT) metastatic melanoma (MM) patients. (A) BRAF^V600E/WT gene copies were isolated with PA or CF protocols from the plasma of WT cohort and quantified by digital PCR (dPCR). BRAF gene copies were expressed as gene copies per ml of plasma on a logarithmic scale. (B) Confirmatory study of the true positive status of plasma samples. Plasma samples from circulating BRAF^WT-positive patients, previously tested as mutation positive in the PA-processed plasma, were collected 6 months after the initial time point and processed with PA protocol. BRAF gene status was determined by digital PCR and expressed as gene copies per ml of plasma on a logarithmic scale. (C) Western blot analysis on exosomal markers from plasma of metastatic melanoma patients. Total proteins were extracted from EV pellets from PA and UC and loaded on SDS-page, blotted onto a nitrocellulose membrane and stained with antibodies against exosomal markers Alix, TSg101, CD9 and non exosomal marker APOA1. Fifteen micrograms of purified exosomes ExoRef™ were used as control. Results are representative of three independent experiments.