The microRNA expression changes associated with malignancy and SDHB mutation in pheochromocytoma

Abstract

Currently, the diagnosis of malignant pheochromocytoma can only be made when there is clinical evidence of metastasis or extensive local invasion. Thus, there is a need for new diagnostic marker(s) to identify tumors with malignant potential. The purpose of this study was to identify microRNAs (miRNAs) that are differentially expressed between benign and malignant pheochromocytomas and assess their diagnostic accuracy. Toward this aim, we analyzed miRNA expression in benign and malignant pheochromocytoma tumor samples using whole genome microarray profiling. Microarray analysis identified eight miRNAs that were significantly differentially expressed between benign and malignant pheochromocytomas. We measured a subset of these miRNAs directly by RT-PCR and found that miR-483-5p, miR-183, and miR-101 had significantly higher expression in malignant tumors as compared to their benign counterparts. Area under the receiver operating curve (AUC) analysis indicated that miR-483-5p, miR-101, and miR-183 could be useful diagnostic markers for distinguishing malignant from benign pheochromocytomas. In addition, these miRNAs could be detected in pheochromocytoma patient serum. Overall our data suggest that misexpression of miR-483-5p, miR-101, and miR-183 is associated with malignant pheochromocytoma.

Figure 1

Number of differentially expressed miRNAs in pheochromocytoma tumors (benign, B; malignant, M) as compared to normal adrenal medulla tissue (NL) identified by microarray profiling. Differentially expressed miRNAs were defined using an FDR-adjusted P value ≤0.05.

Figure 2

Heatmap of the 50 most variably expressed miRNAs. Unsupervised hierarchical clustering was performed using Euclidian distance as the similarity metric. Expression variability was defined by greatest median absolute deviation across all samples. Each row represents an miRNA and each column an individual tumor sample. ‘miR-Plus’ miRNAs are proprietary to Exiqon. Tumor type is indicated below the heatmap as either benign (B) or malignant (M). Demographic and clinical data are plotted below the heatmap (red indicates data not available).

Figure 3

miRNA expression in benign and malignant tumors and normal adrenal medulla tissue (pool of 21 samples). The box plots represent (A) miRNA-483-5p, (B) miR-101, and (C) miR-183 expression as defined as the ΔC_t, as described in the Materials and Methods. A lower ΔC_t indicates higher miRNA expression level. In these graphs, potential outliers are indicated by either open circles (outlier) or asterisks (extreme outlier). B, benign; M, malignant; NLP, normal.

Figure 4

miRNA expression differences in samples categorized by various clinical factors. Expression is defined as the ΔC_t, as described in the Materials and Methods. A lower ΔC_t indicates higher miRNA expression level. miRNA expression differences were found in the comparison of (A) hereditary vs sporadic, (B, C and D) SDHB vs non-SDHB, (E and F) and adrenal vs extra-adrenal tumors. In these plots, potential outliers are indicated by either open circles (outlier) or asterisks (extreme outlier).

Figure 5

Receiver operating characteristic (ROC) curves for the differentially expressed miRNAs. The curves were generated from the ΔC_t measurements for (A) all tumor samples or (B) only non-SDHB tumor samples.

Figure 6

Expression level of miRNAs in patient serum. The scatter plot shows the miRNA expression as the C_t measured by qRT-PCR for each sample. Serum samples from patients with benign tumors (blue), patients with malignant tumors (red), or the no RNA template control (green) were included in this analysis. A lower C_t indicates higher miRNA expression level.