Circulating miR-1246 and miR-485-3p as Promising Biomarkers of Clinical Response and Outcome in Melanoma Patients Treated with Targeted Therapy

Abstract

Despite the significant improvements in advanced melanoma therapy, there is still a pressing need for biomarkers that can predict patient response and prognosis, and therefore support rational treatment decisions. Here, we investigated whether circulating miRNAs could be biomarkers of clinical outcomes in patients treated with targeted therapy. Using next-generation sequencing, we profiled plasma miRNAs at baseline and at progression in patients treated with BRAF inhibitors (BRAFi) or BRAFi + MEKi. Selected miRNAs associated with response to therapy were subjected to validation by real-time quantitative RT-PCR. Receiver Operating Characteristics (ROC), Kaplan-Meier and univariate and multivariate Cox regression analyses were performed on the validated miR-1246 and miR-485-3p baseline levels. The median baseline levels of miR-1246 and miR-485-3p were significantly higher and lower, respectively, in the group of patients not responding to therapy (NRs) as compared with the group of responding patients (Rs). In Rs, a trend toward an increase in miR-1246 and a decrease in miR-485-3p was observed at progression. Baseline miR-1246 level and the miR-1246/miR-485-3p ratio showed a good ability to discriminate between Rs and NRs. Poorer PFS and OS were observed in patients with unfavorable levels of at least one miRNA. In multivariate analysis, a low level of miR-485-3p and a high miR-1246/miR-485-3p ratio remained independent negative prognostic factors for PFS, while a high miR-1246/miR-485-3p ratio was associated with an increased risk of mortality, although statistical significance was not reached. Evaluation of miR-1246 and miR-485-3p baseline plasma levels might help clinicians to identify melanoma patients most likely to be unresponsive to targeted therapy or at higher risk for short-term PFS and mortality, thus improving their management.

Keywords: BRAF inhibitors; MEK inhibitors; circulating miRNAs; melanoma; resistance.

Figure 1

Box-and-whisker diagrams of miR-1246, mir-92b-3p and miR-485-3p levels in melanoma patients treated with targeted therapy. (A). cf-miRNA levels were measured by qRT-PCR in plasma samples obtained from 55 patients (45 Rs and 10 NRs) before the start of therapy (T0). (B). cf-miRNA levels were measured by qRT-PCR in matched plasma samples obtained from 34 patients at T0 and at progression (TP). The edges of each box represent the 75th and 25th percentile, respectively, and whiskers are defined according to Tukey method. The horizontal bar within each box indicates the median. The outliers are reported as dots. Relative expression levels of miR-485-3p and miR-92b-3p are multiplied by 100. Data were analyzed by nonparametric Mann–Whitney U test to compare differences between groups, (A) and by the Wilcoxon matched-pairs signed-rank test for the before–after differences (B). ** p < 0.01 and * p < 0.05.

Figure 2

Kaplan–Meier curves for progression-free survival (PFS) of melanoma patients treated with targeted therapy. Patients were stratified by T0 levels of miR-1246 (A), miR-485-3p (B), both miR-1246 and miR-485-3p (C), miR-1246/miR485-3p ratio (D). Group (a): miR-1246 < 8.637 and miR-485-3p ≥ 0.013; group (b): miR-1246 ≥ 8.637 and miR-485-3p ≥ 0.013, or miR-1246 < 8.637 and miR-485-3p < 0.013; group (c): miR-1246 ≥ 8.637 and miR-485-3p < 0.013. p-values are from log-rank test (A,B,D) and log-rank test for trend (C). The estimated percentages of PFS at 12 months from the start of therapy is also reported for each comparison.

Figure 3

Kaplan–Meier curves for overall survival (OS) of melanoma patients treated with targeted therapy. Patients were stratified by T0 levels of miR-1246 (A), miR-485-3p (B), both miR-1246 and miR-485-3p (C), miR-1246/miR-485-3p ratio (D). Group (a): miR-1246 < 8.637 and miR-485-3p ≥ 0.013; group (b + c): miR-1246 ≥ 8.637 and miR-485-3p ≥ 0.013, or miR-1246 < 8.637 and miR-485-3p < 0.013, or miR-1246 ≥ 8.637 and miR-485-3p < 0.013. p-values are from log-rank test. The estimated percentages of OS at 12 months from the start of therapy are also reported for each comparison.