Development and clinical approbation of miR-THYROID test-system for differential diagnosis of thyroid follicular adenomas and carcinomas

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Introduction. Absence of objective morphological signs of malignancy complicates the problem of preoperative differential diagnosis of nodular thyroid lesions with follicular structure. In ambiguous cases, selection of surgical treatment is justified but results of histological examination of operative material often show benign nature of the nodules. A possible solution for this problem is development of molecular diagnostic methods for this pathology. For example, the miR-THYROID test system is based on the differences in expression patterns of regulatory microRNAs (miRNAs) in follicular adenoma cells and follicular thyroid cancer cells. Aim. To develop and validate a set of reagents (miR-THYROID) for differential diagnosis of follicular adenoma and follicular thyroid cancer using reverse transcription with subsequent polymerase chain reaction (RT-PCR). Materials and methods. Selection of potential marker molecules (n = 53) is based on the results of previous study of histologically verified samples of follicular adenoma and follicular thyroid cancer using high-throughput sequencing. In this study, a test system based on RT reaction with two-tailed primer and quantitative PCR with TaqMan probe was developed. To evaluate analytical characteristics of this technology, synthetic analogues of miRNA molecules were used; for analysis of diagnostic characteristics of the method, cytology samples of follicular adenoma (n = 20) and follicular thyroid cancer (n = 20) were used. Results. Studies of analytical characteristics of RT-PCR system and concentration of potential marker molecules in cytology samples were performed which allowed to shorten the list of marker miRNAs from 53 to 7 (hsa-miR-15a-5p, -20-5p, -24-3p, -106b-5p, -143-3p, -146b-5p, -192-5p). Algorithm of RT-PCR result analysis was developed based on calculation of molecule concentration ratios relative to oppositely directed follicular cancer-associated changes in expression, combination of these results, and calculation of the miR-T diagnostic parameter. miR-THYROID test system based in RT-PCR technology allowed to differentiate follicular adenoma from follicular cancer with sensitivity 89.47 % and specificity 90 %, positive prognostic significance 89.47 %, negative prognostic significance 90 %, and accuracy 89.74 %. Conclusion. Test system miR-THYROID based on 7 microRNA molecules was validated on 40 samples (20 samples of follicular adenoma and 20 samples of follicular cancer) and demonstrated high diagnostic significance.

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