A Tumor and Immune-Related Micro-RNA Signature Predicts Relapse-Free Survival of Melanoma Patients Treated with Ipilimumab

Abstract

Despite the unprecedented advances in the treatment of melanoma with immunotherapy, there continues to be a major need for biomarkers of clinical benefits and immune resistance associated with immune checkpoint inhibitors; microRNA could play a vital role in these efforts. This study planned to identify differentially expressed miRNA molecules that may have prognostic value for clinical benefits. Patients with surgically operable regionally advanced melanoma were treated with neoadjuvant ipilimumab (10 mg/kg intravenously every 3 weeks × two doses) bracketing surgery. Tumor biospecimens were obtained at baseline and surgery, and microRNA (miRNA) expression profiling was performed on the tumor biopsies. We found that an expression profile consisting of a 4-miRNA signature was significantly associated with improved relapse-free survival (RFS). The signature consisted of biologically relevant molecules previously reported to have prognostic value in melanoma and other malignancies, including miR-34c, miR-711, miR-641, and miR-22. Functional annotation analysis of target genes for the 4-miRNA signature was significantly enriched for various cancer-related pathways, including cell proliferation regulation, apoptosis, the MAPK signaling pathway, and the positive regulation of T cell activation. Our results presented miRNAs as potential biomarkers that can guide the treatment of melanoma with immune checkpoint inhibitors. These findings warrant further investigation in relation to CTLA4 blockade and other immune checkpoint inhibitors. ClinicalTrials.gov NCT00972933.

Keywords: biomarkers; immunotherapy; melanoma; miRNA; microRNA.

Figure 1

Biclustering analysis of differentially expressed genes by relapse-free survival (RFS). In the top bar, yellow represents samples of patients with RFS > 1 year, and blue represents those of patients with RFS of ≤1 year. In the body of the heat map, red shows up-regulated genes, and green shows down-regulated genes.

Figure 2

Time-to-event analysis comparing overall survival (OS) rate based on a 4-miRNA. Signature expression at baseline (before the initiation of neoadjuvant ipilimumab). The yellow line demonstrates patients with low expression of the 4-miRNA signature; the blue line demonstrates patients with high expression of the 4-miRNA signature. Y-axis represents the overall survival rate, and the X-axis represents the time in days.

Figure 3

Canonical pathways as adapted from Ingenuity Pathway Analysis (IPA). IPA was used to determine the functional annotation of target genes for the 4-miRNA signature differentially expressed in patients treated with neoadjuvant ipilimumab based on the relapse-free survival outcomes. The threshold for significance is depicted by the horizontal orange line and the five pathways that achieve significance are indicated on the abscissa with the most significant values to the left. The ratio is the number of molecules in a given pathway that meet the cutoff criteria divided by the total number of molecules that make up the pathway in the reference data set. A ratio of 1 indicates that every molecule in the canonical pathway is a target of at least one of the miRNAs in the signature. The p-values in the −log(p-value) are from Fisher’s exact test that measures the significance of the overlap between analysis-ready genes in the dataset and genes within a pathway or function.

Figure 4

Cell death and survival, cell cycle, and cellular movement molecular network. This pathway represented the top canonical pathway. Adapted from Ingenuity Pathway Analysis (IPA).