Abstract and Introduction
Malignant melanoma is an aggressive form of skin cancer. Recently, drug therapy of advanced disease has been revolutionized by new agents. More therapeutic options, coupled with the desire to extend treatment to the adjuvant setting mean that prognostic biomarkers that can be assayed from formalin-fixed paraffin-embedded clinical would be valuable. microRNAs have potential to fill this need. We analyzed 377 microRNAs in 79 primary melanomas and 32 metastases using a split sample discovery strategy. From a discovery analysis using 40 thick primary melanomas (20 cases with metastasis and 20 controls without metastasis at 5 years), microRNA expression was measured by quantitative RT-PCR (QRT-PCR). MiR-10b emerged as a candidate prognostic microRNA. This was confirmed in an independent validation set of thick primary melanomas (20 cases with metastasis and 19 controls without metastasis at 5 years). In the combined discovery and validation cohorts (n=79), miR-10b expression showed a 3.7-fold increase in expression between cases and controls (P=0.005) and showed a trend of increasing expression between primary melanomas and their matched metastases (P<0.001). In situ hybridization showed expression was in melanoma cells and correlated with expression measured by QRT-PCR (P=0.0005). We used the combined discovery and validation samples to verify the prognostic value of additional candidate microRNAs identified from other studies, and proceeded to analyze miR-200b. We demonstrated that miR-10b and miR-200b showed independent prognostic value (P=0.002 and 0.047, respectively) in multivariable analysis alongside known clinico-pathological prognostic features (eg, Breslow thickness) using a Cox proportional hazards regression model. Furthermore, the addition of these microRNAs to the clinico-pathological features led to an improved regression model with better identification of aggressive thick melanomas. Taken together, these data suggest that miR-10b is a new prognostic microRNA for melanoma and that there could be a place for microRNA analysis in stratifying melanoma for therapy.
Melanoma is an aggressive skin cancer. Most tumors can be cured by local surgical excision but once spread to distant sites occurs the prognosis is poor. However, recent clinical trials have provided renewed hope for patients with advanced melanoma. These new therapeutic avenues mean that a search for tissue biomarkers is very timely, especially prognostic biomarkers that can stratify patients into subgroups with varying therapeutic need.
One of the major problems in finding relevant biomarkers is that macromolecules are poorly preserved in clinical melanoma samples because all of the tumor tissue is typically fixed in formalin and then paraffin embedded. However, microRNAs (miRNAs) have attracted attention because they are reasonably well preserved. These are small, non-coding single-stranded RNAs that are about 22 nucleotides in length and that function post transcriptionally as negative regulators of gene expression. Altered miRNA expression in cancer was first reported in 2002, with downregulation of miR-15 and miR-16 expression, which target B-cell lymphoma 2 (BCL2) in chronic lymphocytic leukemia. Many subsequent reports of miRNAs in cancer have been reviewed.
In melanoma, miRNAs can be readily analyzed from formalin-fixed paraffin-embedded tissue, show high frequency genetic alterations, and exhibit a melanoma-specific signature. In addition, miRNAs have shown prognostic value in melanoma.[7–11] A panel of six miRNAs was identified as a post-recurrence survival signature, and miRNA and mRNA networks were associated with the outcome, while a 4-miRNA signature was associated with brain metastasis. Several anti-miRNA agents are under development and therefore tissue miRNA analysis has potential predictive value in the future.
The evidence thus far shows that miRNAs are well preserved in formalin-fixed paraffin-embedded melanoma tissue and that they have a potential to be prognostic biomarkers. Therefore we sought to identify miRNAs with prognostic value and to determine whether they could provide independent prognostic significance beyond routine clinical and pathological factors used in clinical staging, focusing on at risk patients with thicker melanomas. To test the hypothesis that miRNAs have prognostic value we compared differential expression of 377 miRNAs in non-metastasizing and metastasizing primary melanomas using a four-step approach based on split-sample methodology. In addition, we also sought to validate a small number of prognostic miRNAs already identified by others in previous studies.
Mod Pathol. 2016;29(2):112-121. © 2016 Nature Publishing Group