Extracellular microvesicle microRNAs as predictive biomarkers for targeted therapy in Stage IV cutaneous malignant melanoma. Homo sapiens

We analyzed the value of microRNAs (miRNAs) in plasma-derived extracellular microvesicles (EV) as biomarkers for treatment outcome in Stage IV CMM patients receiving MAPK inhibitors (MAPKis). Overall design: EV miRNAs were extracted from plasma samples from 28 Stage IV CMM patients collected before and during therapy with MAPKis. EV miRNA levels were measured by a quantitative PCR-array and correlated to therapy response and progression-free survival (PFS). Matched plasma samples before and during treatment were obtained from 25 of the 28 patients. MiRNA expression profiling was performed using miRCURY LNA Universal RT miRNA PCR Human panel I (Exiqon, Denmark) covering 372 human miRNAs. All miRNAs were polyadenylated and reverse transcribed in cDNA in a single reaction step. RNA isolation cDNA synthesis efficiency was examined by the detection of spike-in UniSp2, UniSp4 and UniSp6 to ensure that the quality of the input RNA was sufficiently high for effective amplification. cDNA and Exilent SYBR Green master mix (Exiqon, Denmark) were transferred to qPCR panels preloaded with primers. Amplification was performed in a Lightcycler 480 (Roche). We normalized miRNA expression to a set of miRNAs found to be most stable across conditions defined by the normfinder algorithm