In previous studies, we described a method for detecting and typing malignant tumors of the thyroid gland in fine-needle aspiration biopsy samples via analysis of a molecular marker panel (normalized HMGA2 mRNA level; normalized microRNA-146b, -221, and -375 levels; mitochondrial-to-nuclear DNA ratio; and BRAF^V600E mutation) in cytological preparations by quantitative PCR. In the present study, we aimed to estimate the specificity of the typing of different thyroid tumors by the proposed method. Fine-needle aspiration cytological preparations from 278 patients were used. The histological diagnosis was known for each sample. The positive and negative predictive values of the method assessed in this study were, respectively, 100% and 98% for papillary thyroid carcinoma (n = 63), 100% and 100% for medullary thyroid carcinoma (n = 19), 43.5% and 98% for follicular carcinoma (n = 15), and 86% and 100% for Hürthle cell carcinoma (n = 6). Thus, we demonstrate that the diagnostic panel, including the analysis of microRNA expression, mRNA expression, the BRAF^V600E mutation, and the mitochondrial-to-nuclear DNA ratio, allows the highly accurate identification of papillary thyroid carcinoma, medullary thyroid carcinoma, and Hürthle cell carcinoma but not malignant follicular tumors (positive predictive value was below 50%).

The preoperative diagnostics of medullary thyroid carcinoma (MTC), including the measuring of the blood calcitonin level, has a number of limitations. Particular focus has recently been placed on the role of miRNAs in the development of various malignant tumors; a comparative analysis of accuracy of the existing methods for MTC diagnosis with a novel diagnosis method, evaluation of the miRNA-375 expression level, was performed in this study. The expression level of miRNA-375 in cytology samples from 555 patients with the known histological diagnosis, including 41 patients with confirmed postoperative diagnosis of MTC, was assessed. The diagnostic parameters of the basal calcitonin level, calcitonin in wash-out fluid from the FNAB needle, and miRNA-375 were compared. An assessment of the miRNA-375 expression level made it possible to detect all the MTC samples with a 100% accuracy among all the 555 cytology specimens, as well as in non-informative FNAB specimens, and specimens from the ipsilateral thyroid lobe. Parameters such as sensitivity, specificity, PPV, and NPV were 100%. The miRNA-375 level, unlike calcitonin, does not correlate with tumor volume, so it does not have the so-called “gray zone”. An assessment of the miRNA-375 expression allows one to accurately distinguish MTC from other malignant and benign thyroid tumors.

Molecular genetic events are among the numerous factors affecting the clinical course of papillary thyroid carcinoma (PTC). Recent studies have demonstrated that aberrant expression of miRNA, as well as different thyroid-related genes, correlate with the aggressive clinical course of PTC and unfavorable treatment outcomes, which opens up new avenues for using them in the personalization of the treatment strategy for patients with PTC. In the present work, our goal was to assess the applicability of molecular markers in the preoperative diagnosis of aggressive variants of papillary thyroid cancer. The molecular genetic profile (expression levels of 34 different markers and BRAF mutations) was studied for 108 cytology specimens collected by fine-needle aspiration biopsy in patients with PTC having different clinical manifestations. Statistically significant differences with adjustment for multiple comparisons (p < 0.0015) for clinically aggressive variants of PTC were obtained for four markers: miRNA-146b, miRNA-221, fibronectin 1 (FN1), and cyclin-dependent kinase inhibitor 2A (CDKN2A) genes. A weak statistical correlation (0.0015 < p < 0.05) was observed for miRNA-31, -375, -551b, -148b, -125b, mtDNA, CITED1, TPO, HMGA2, CLU, NIS, SERPINA1, TFF3, and TMPRSS4. The recurrence risk of papillary thyroid carcinoma can be preoperatively predicted using miRNA-221, FN1, and CDKN2A genes.

Background. Fine needle aspiration biopsy (FNA) of thyroid nodules under ultrasound control followed by cytological examination is the main method to assess the risk of malignancy of these formations. At the same time, vague and suspicious cytological findings (Bethesda III, IV) are indications for referring patients for surgical treatment, and in some cases this will be excessive. The analysis of molecular markers in the FNA material in addition to cytological examination is a promising way to improve the preoperative diagnosis of thyroid nodules. Aim. The aim of the study was improving accuracy of preoperative diagnosis of thyroid tumors. Materials and methods. The validation of the diagnostic capabilities of the domestic molecular genetic test (MGT), developed for the differential diagnosis of thyroid tumors, was carried out on the basis of a retrospective multicenter blind study. The test was based on the assessment of the relative levels of expression of GCM2 and HMGA2 genes, microRNA levels -146b, -221, -375, -31 and -551b, the ratio of mitochondrial and nuclear DNA, as well as the determination of somatic mutation V600E in the BRAF gene. 329 cytological samples were analyzed, which included: 22 (6.7%) with the conclusion of Bethesda II, 226 (68.7%) of Bethesda IV, 6 (1.8%) of Bethesda V, and 75 (22.8%) of Bethesda VI. Results. The following diagnostic characteristics were obtained for detection of malignant tumors by means of a molecular test in the study: specificity ― 90.8%, sensitivity ― 86.9%, positive predictive value ― 82.3%, negative predictive value ― 93.4%. The overall accuracy was 96.6% for detecting of papillary cancer with the molecular test, 94.4% for follicular cancer, 100% for medullary cancer, and 96.9% for Hürthle cell carcinoma. Conclusion. The performed study demonstrates the possibility of detecting and typing thyroid tumors with the use of a panel of a small number of molecular markers detected in cytological preparations with real-time PCR. The accuracy of the domestic MGТ is comparable with foreign analogues. Введение. Тонкоигольная аспирационная биопсия (ТАБ) узлов щитовидной железы под УЗ-контролем с последующим цитологическим исследованием является основным способом оценки риска злокачественности этих образований. При этом неопределенные и подозрительные на опухоль цитологические заключения (Bethesda III, IV) являются показанием для направления пациентов на оперативное лечение, в части случаев избыточное. Анализ молекулярных маркеров в материале ТАБ в дополнение к цитологическому исследованию ― многообещающий способ улучшения предоперационной диагностики узлов щитовидной железы. Целью исследования стало повышение точности дооперационной диагностики опухолей щитовидной железы. Материалы и методы. В данной работе на основании ретроспективного многоцентрового слепого исследования проведена валидация диагностических возможностей отечественного молекулярно-генетического теста (МГТ), разработанного для дифференциальной диагностики опухолей щитовидной железы. Тест основан на оценке относительных уровней экспрессии генов GCM2 и HMGA2, уровней микроРНК-146b, -221, -375, -31 и -551b, соотношения митохондриальной и ядерной ДНК, а также определение соматической мутации V600E в гене BRAF. Анализировано 329 цитологических образцов, из них: 22 (6,7%) с заключением Bethesda II, 226 (68,7%) Bethesda IV, 6 (1,8%) Bethesda V и 75 (22,8%) Bethesda VI. Результаты. В нашем исследовании получились следующие диагностические характеристики для выявления злокачественных опухолей молекулярным тестом: специфичность ― 90,8%, чувствительность ― 86,9%, положительная предсказательная ценность ― 82,3%, отрицательная предсказательная ценность ― 93,4%. При выявлении молекулярным тестом папиллярного рака общая точность — 96,6%, фолликулярного рака ― 94,4%, медуллярного рака ― 100%, Гюртлеклеточного рака ― 96,9%. Заключение. Проведенное исследование демонстрирует возможность выявления и типирования опухолей щитовидной железы с помощью панели из небольшого числа молекулярных маркеров, выявляемых в цитологических препаратах с помощью ПЦР в реальном времени. Точность отечественного МГТ сопоставима с зарубежными аналогами.

Background. Anaplastic thyroid cancer (ATC) is one of the most aggressive human tumors. Since median survival of ATC patients is only 4 months, its early diagnosis is very important. Although ATC has specific clinical manifestations, the analysis of expression of different microRNAs can facilitate preoperative diagnostics and help to detect its potential precursors among differentiated cancers and other thyroid malignancies.The study objective is to identify microRNAs specific for ATC that are different from microRNAs in other thyroid cancers.Materials and methods. We analyzed the expression of 14 microRNAs in histological specimens of 67 patients with ATC. The control groups included 25 patients with benign nodules, 36 patients with follicular adenomas, 32 patients with follicular cancer, and 152 patients with papillary thyroid cancer. For 7 out of 67 ATC patients, we compared mi-croRNA levels in histological and cytological specimens.Results. Patients with ATC demonstrated a statistically significant decrease in the expression of miR-145, miR-125b and increase in the expression of miR-155 and miR-21 compared to all control groups. We found two reliable diagnostic markers of ATC: relative miR-21 expression (at a cutoff of 14.9, sensitivity was 0.955 and specificity was 0.837) and the miR-21/miR-145 ratio (at a cutoff of 122, sensitivity was 0.955 and specificity was 0.955). The level of miR-21 expression and miR-21/miR-145 ratio in cytological specimens were accurate in all 7 cases (100 %).Conclusion. the level of expression of specific microRNAs can be used as a reliable biomarker for ATC. The consistency between the results obtained in cytological and histological specimens enables the use of stained cytological samples for this analysis.

Background. Urothelial carcinoma (UTC) is an aggressive disease with a known propensity for frequent recurrence. It is difficult to predict the velocity of the development of UTC recur using modern means of clinical diagnostics. Therefore, the development of the capabilities of histo-morphological study of tumor tissues is of particular relevance.Materials and methods. The materials of publications (PubMed, CrossRef) for 1990-2021, devoted to the choice of biomarkers for the diagnosis of UTC, the analysis of molecular pathways, progression and metastasis, were studied. The search was carried out for the key phrases "urothelial carcinoma", "recurrent UTK", "stem cells", "biomarkers of bladder cancer", "genetic changes in urothelium", "circulating tumor DNA".Results. Cancer stem cells serve as a source of UTC recurrence after removal from the primary focus, localizing in any areas of the urothelium, as well as outside the main tumor focus and are characterized by a common genotype, but different phenotypic manifestations. To predict the recurrence of the tumour is advisable to use gene expression signatures, since the subtypes of UTC are characterized by clear gene expression profiles. A larger sample and independent dataset is needed to confirm the clinical significance of the findings. Combined biomarkers predict UTC behavior, and FGFR3 and TP53 mutations can be components for a panel for predicting UTC recurrence. The use of the liquid biopsy method with the determination of the level of circulating tumor DNA is a promising diagnostic method that needs to evaluate the results of an initiated randomized trial.Conclusion. The accumulation of knowledge base about the molecular patterns of UTC will help bridge the gap between the results of molecular genetic and clinical diagnostics. Molecular changes in the transitional cell UTC demonstrates a high potential for determining the timing of tumor recurrence, assessing disease-free survival of patients and for planning the resource base of the healthcare system.