Changes in the methylation levels of 21 microRNA genes in 91 breast cancer samples in comparison with paired samples of histologically unchanged tissue were studied by quantitative methylation-specific PCR. For 19 microRNA genes, a significant increase in the methylation level in tumors in comparison with normal tissues was shown (Mann-Whitney test). When considering the data for breast cancer samples only from patients with clinical stages I and II (59samples), 17 genes with a significantly increased level of methylation were identified. Increased methylation level for 11 genes (MIR124-1, MIR124-3, MIR125B-1, MIR127, MIR129-2, MIR132, MIR137, MIR193a, MIR34B/C, MIR375, and MIR9-1) compared to the paired norm was highly significant (p<0.001, FDR=0.01). The ROC analysis was used to optimize a set of markers for diagnosing breast cancer at the early stages consisting of 4 microRNA genes: MIR125B1, MIR127, MIR1258, and MIR132; the system is characterized by 100% specificity, 85% sensitivity, and AUC=0.924. Importantly, 100% specificity eliminates false positive results. Detection of methylation of at least one of the 4 genes of this set is sufficient to classify the patient's sample as breast cancer.
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