MicroRNA-30a-5pme: a novel diagnostic and prognostic biomarker for clear cell renal cell carcinoma in tissue and urine samples

Abstract

Background: The rising incidence of renal cell carcinomas (RCC) constitutes a significant challenge owing to risk of overtreatment. Because aberrant microRNA (miR) promoter methylation contributes to cancer development, we investigated whether altered miR-30a-5p expression associates with DNA promoter methylation and evaluated the usefulness as clear cell RCC (ccRCC) diagnostic and prognostic markers. Methods: Genome-wide methylome and RNA sequencing data from a set of ccRCC and normal tissue samples from The Cancer Genome Atlas (TCGA) database were integrated to identify candidate CpG loci involved in cancer onset. MiR-30a-5p expression and promoter methylation were quantitatively assessed by PCR in a tissue set (Cohort #1) and urine sets (Cohorts #2 and 3) from IPOPorto and Homburg University Hospital. Non-parametric tests were used for comparing continuous variables. MiR-30a-5p promoter methylation (miR-30a-5p me) performance as diagnostic (receiver operator characteristics [ROC]-validity estimates) and prognostic [metastasis-free (MFS) and disease-specific survival (DSS)] biomarker was further validated in urine samples from ccRCC patients by Kaplan Meier curves (with log rank) and both univariable and multivariable analysis. Results: Two significant hypermethylated CpG loci in TCGA ccRCC samples, correlating with miR-30a-5p transcriptional downregulation, were disclosed. MiR-30a-5p me in ccRCC tissues was confirmed in an independent patient's cohort of IPOPorto and associated with shorter time to relapse. In urine samples, miR-30a-5p me levels identified cancer both in testing and validation cohorts, with 83% sensitivity/53% specificity and 63% sensitivity/67% specificity, respectively. Moreover, higher miR-30a-5p me levels independently predicted metastatic dissemination and survival.

Epigenetic Regulation of microRNAs in Cancer: Shortening the Distance from Bench to Bedside

“…Comparing the methylation levels of miR-30a-5p in urine from patients with renal clear carcinoma (RCC) and asymptomatic controls, Outeiro-Pinho and colleagues have also shown the potential utility of this biomarker in the diagnosis of RCC. The overall accuracy of this assay was not very high (67%), but the results were validated in the second cohort of 171 RCCs [ 125 ]. Moreover, a panel of two microRNA methylated promoters composed of miR-663a and miR-129-2 was shown to accurately detect urothelial carcinomas in urine (85.85% accuracy) [ 126 ].…”

Epigenetic Biomarkers of Renal Cell Carcinoma for Liquid Biopsy Tests

“…Despite the high potential of DNA methylation as the disease biomarker, only 12 studies shed light on efforts to analyze DNA methylation in liquid biopsy specimens as noninvasive biomarkers for RCC [24][25][26][27][28][29][30][31][32][33][34][35] (Table 1), encompassing 25 individual genes. Four (33%) of 12 studies discussed used urine as the source of methylated DNA, six studies (50%) reported on serum or plasma, and two (17%) described both urine and serum/plasma.…”

“…For the validation, the ruling methods were the bisulfite conversion-based MSP or qMSP with rare exceptions (Table 1). Only two studies by Outeiro-Pinho et al [34] and Nuzzo et al [35] performed internal validation with training and test sample sets, while others missed this step. In addition, 58% of the studies included ≤50 RCC patients and only two studies [32,34] used a homogenous study cohort composed of patients with ccRCC, while others involved heterogeneous groups of patients with various types of renal tumors [24,[27][28][29]31,33,35] and, in some cases, the particular subtype was not specified [25,26,30].…”

“…Most studies compared circulating methylated DNA data with matched (or not) tissue samples [24][25][26][27]30,33,34], while no identical DNA methylation patterns between primary tumor and liquid biopsies were identified, and, as a rule, lower methylation frequencies or intensities in the body fluids were detected. The diagnostic sensitivity of various individual biomarkers varied from 6% to 83%, while the specificity was generally high and reached >90% for most of the biomarkers (Table 1).…”

The Role of Epigenetics in the Progression of Clear Cell Renal Cell Carcinoma and the Basis for Future Epigenetic Treatments

“…Regarding the prognostic role of miRNAs, overexpression of miR-221 and miR-32 are predictors of RCC mortality [ 146 , 147 ]. Similarly, miR-30a-5p downregulation, probably due to aberrant promoter methylation, is common in ccRCC and can be evaluated both in tumor tissue and urine samples to predict metastatic dissemination and worse survival [ 148 ]. Members of the miR-200 family and miR-205 promote EMT and reduced transcription and expression of E-cadherin [ 149 ].…”