Combined quantitation of HMGA2 mRNA, microRNAs, and mitochondrial-DNA content enables the identification and typing of thyroid tumors in fine-needle aspiration smears.

BACKGROUND: Analysis of molecular markers in addition to cytological analysis of fine-needle aspiration (FNA) samples is a promising way to improve the preoperative diagnosis of thyroid nodules. Nonetheless, in clinical practice, applications of existing diagnostic solutions based on the detection of somatic mutations or analysis of gene expression are limited by their high cost and difficulties with clinical interpretation. The aim of our work was to develop an algorithm for the differential diagnosis of thyroid nodules on the basis of a small set of molecular markers analyzed by real-time PCR. METHODS: A total of 494 preoperative FNA samples of thyroid goiters and tumors from 232 patients with known histological reports were analyzed: goiter, 105 samples (50 patients); follicular adenoma, 101 (48); follicular carcinoma, 43 (28); Hürthle cell carcinoma, 25 (11); papillary carcinoma, 121 (56); follicular variant of papillary carcinoma, 80 (32); and medullary carcinoma, 19 (12). Total nucleic acids extracted from dried FNA smears were analyzed for five somatic point mutations and two translocations typical of thyroid tumors as well as for relative concentrations of HMGA2 mRNA and 13 microRNAs and the ratio of mitochondrial to nuclear DNA by real-time PCR. A decision tree-based algorithm was built to discriminate benign and malignant tumors and to type the thyroid cancer. Leave-p-out cross-validation with five partitions was performed to estimate prediction quality. A comparison of two independent samples by quantitative traits was carried out via the Mann-Whitney U test. RESULTS: A minimum set of markers was selected (levels of HMGA2 mRNA and miR-375, - 221, and -146b in combination with the mitochondrial-to-nuclear DNA ratio) and yielded highly accurate discrimination (sensitivity = 0.97; positive predictive value = 0.98) between goiters with benign tumors and malignant tumors and accurate typing of papillary, medullary, and Hürthle cell carcinomas. The results support an alternative classification of follicular tumors, which differs from the histological one. CONCLUSIONS: The study shows the feasibility of the preoperative differential diagnosis of thyroid nodules using a panel of several molecular markers by a simple PCR-based method. Combining markers of different types increases the accuracy of classification.

miRNA profiling, detection of BRAF V600E mutation and RET-PTC1 translocation in patients from Novosibirsk oblast (Russia) with different types of thyroid tumors.

BACKGROUND: The postoperative typing of thyroid lesions, which is instrumental in adequate patient treatment, is currently based on histologic examination. However, it depends on pathologist's qualification and can be difficult in some cases. Numerous studies have shown that molecular markers such as microRNAs and somatic mutations may be useful to assist in these cases, but no consensus exists on the set of markers that is optimal for that purpose. The aim of the study was to discriminate between different thyroid neoplasms by RT-PCR, using a limited set of microRNAs selected from literature. METHODS: By RT-PCR we evaluated the relative levels of 15 microRNAs (miR-221, -222, -146b, -181b, -21, -187, -199b, -144, -192, -200a, -200b, -205, -141, -31, -375) and the presence of BRAF(V600E) mutation and RET-PTC1 translocation in surgically resected lesions from 208 patients from Novosibirsk oblast (Russia) with different types of thyroid neoplasms. Expression of each microRNA was normalized to adjacent non-tumor tissue. Three pieces of lesion tissue from each patient (39 goiters, 41 follicular adenomas, 16 follicular thyroid cancers, 108 papillary thyroid cancers, 4 medullary thyroid cancers) were analyzed independently to take into account method variation. RESULTS: The diagnostic classifier based on profiling of 13 microRNAs was proposed, with total estimated accuracy varying from 82.7 to 99% for different nodule types. Relative expression of six microRNAs (miR-146b, -21, -221, -222, 375, -199b) appeared significantly different in BRAF(V600E)-positive samples (all classified as papillary thyroid carcinomas) compared to BRAF(V600E)-negative papillary carcinoma samples. CONCLUSIONS: The results confirm practical feasibility of using molecular markers for typing of thyroid neoplasms and clarification of controversial cases.

Cervicovaginal-Microbiome Analysis by 16S Sequencing and Real-Time PCR in Patients from Novosibirsk (Russia) with Cervical Lesions and Several Years after Cancer Treatment.

Disturbed cervicovaginal-microbiome (CVM) structure promotes human papillomavirus (HPV) persistence and reflects risks of cervical lesions and cancer onset and recurrence. Therefore, microbiomic biomarkers may be useful for cervical disease screening and patient management. Here, by 16S rRNA gene sequencing and commercial PCR-based diagnostic kits, we profiled CVM in cytological preparations from 140 HPV-tested women (from Novosibirsk, Russia) with normal cytological findings, cervical lesions, or cancer and from 101 women who had recently received different cancer therapies. An increase in lesion severity was accompanied by higher HPV prevalence and elevated CVM biodiversity. Post-treatment CVM was found to be enriched with well-known microbial biomarkers of dysbiosis, just as in cervical disease. Nonetheless, concentrations of some skin-borne and environmental species (which gradually increased with increasing lesion severity)-especially Cutibacterium spp., Achromobacter spp., and Ralstonia pickettii-was low in post-treatment patients and depended on treatment types. Frequency of Lactobacillus iners dominance was high in all groups and depended on treatment types in post-treatment patients. Microbiome analysis via PCR-based kits revealed statistically significant differences among all groups of patients. Thus, microbiome profiling may help to find diagnostic and prognostic markers for management of cervical lesions; quantitative PCR-based kits may be suitable for these purposes.

Selection of reference genes for quantitative analysis of microRNA expression in three different types of cancer.

MicroRNAs (miRNAs) are promising biomarkers in cancer research. Quantitative PCR (qPCR), also known as real-time PCR, is the most frequently used technique for measuring miRNA expression levels. The use of this technique, however, requires that expression data be normalized against reference genes. The problem is that a universal internal control for quantitative analysis of miRNA expression by qPCR has yet to be known. The aim of this work was to find the miRNAs with stable expression in the thyroid gland, brain and bone marrow according to NanoString nCounter miRNA quantification data. As a results, the most stably expressed miRNAs were as follows: miR-361-3p, -151a-3p and -29b-3p in the thyroid gland; miR-15a-5p, -194-5p and -532-5p in the brain; miR-140-5p, -148b-3p and -362-5p in bone marrow; and miR-423-5p, -28-5p and -532-5p, no matter what tissue type. These miRNAs represent promising reference genes for miRNA quantification by qPCR.

Preoperative Typing of Thyroid and Parathyroid Tumors with a Combined Molecular Classifier.

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 BRAFV600E 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 BRAFV600E 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%).

Detection of Cervical Lesions and Cancer in Air-Dried Cytologic Smears by Combined Analysis of mRNA and miRNA Expression Levels.

Cervical cancer screening is based on cytologic analysis and high-risk human papillomavirus (HR-HPV) testing, each having their drawbacks. Implementation of new biomarker-based methods may improve screening accuracy. Here, the levels of 25 microRNAs (miRNAs, miRs) and 12 mRNAs involved in cervical carcinogenesis in 327 air-dried Papanicolaou-stained cervical smears from patients with cervical precancerous lesions, cancer, or without the disease were estimated by real-time PCR. Using logistic regression analysis, small-scale miRNA-based, mRNA-based, and combined molecular classifiers were built based on paired ratios of miRNA or mRNA concentrations; their ability to detect high-grade cervical lesions and cancer was then compared. Combined mRNA-miRNA classifiers manifested a better combination of sensitivity and specificity than miRNA- and mRNA-based classifiers. The best classifier, combining miR-375, miR-20, miR-96, CDKN2A, TSP4, and ECM1, predicted high-grade lesions with diagnostic sensitivity of 89.0%, specificity of 84.2%, and a receiver-operating characteristic area under the curve of 0.913. Additionally, in a subsample of the same specimens, the levels of MIR124-2 and MAL promoter methylation, HR-HPV genotypes, and viral loads were analyzed. The relative high-grade lesion risk estimated by the classifier correlated with the frequency of MAL and MIR124-2 methylation but not with the HR-HPV genotype or viral load. The results support the feasibility of cellular biomarker-based methods for cervical screening and patient management.

Preoperative detection of malignancy in fine-needle aspiration cytology (FNAC) smears with indeterminate cytology (Bethesda III, IV) by a combined molecular classifier.

AIMS: Analysis of molecular markers in addition to cytological analysis of fine-needle aspiration (FNA) samples is a promising way to improve the preoperative diagnosis of thyroid nodules. Previously, we have developed an algorithm for the differential diagnosis of thyroid nodules by means of a small set of molecular markers. Here, we aimed to validate this approach using FNA cytology samples of Bethesda categories III and IV, in which preoperative detection of malignancy by cytological analysis is impossible. METHODS: A total of 122 FNA smears from patients with indeterminate cytology (Bethesda III: 13 patients, Bethesda IV: 109 patients) were analysed by real-time PCR regarding the preselected set of molecular markers (the BRAF V600E mutation, normalised concentrations of HMGA2 mRNA, 3 microRNAs, and the mitochondrial/nuclear DNA ratio). The decision tree-based classifier was used to discriminate between benign and malignant tumours. RESULTS: The molecular testing detected malignancy in FNA smears of indeterminate cytology with 89.2% sensitivity, 84.6% positive predictive value, 92.9% specificity and 95.2% negative predictive value; these characteristics are comparable with those of more complicated commercial tests. Residual risk of malignancy for the thyroid nodules that were shown to be benign by this molecular method did not exceed the reported risk of malignancy for Bethesda II histological diagnosis. Analytical-accuracy assessment revealed required nucleic-acid input of ≥5 ng. CONCLUSIONS: The study shows feasibility of preoperative differential diagnosis of thyroid nodules of indeterminate cytology using a small panel of molecular markers of different types by a simple PCR-based method using stained FNA smears.

Detection of high-grade neoplasia in air-dried cervical PAP smears by a microRNA-based classifier.

Recent studies have shown that changes in the expression levels of certain microRNAs correlate with the degree of severity of cervical lesions. The aim of the present study was to develop a microRNA-based classifier for the detection of high-grade cervical intraepithelial neoplasia (CIN ≥2) in cytological samples from patients with different high-risk human papillomavirus (HR-HPV) viral loads. For this purpose, raw RT-qPCR data for 25 candidate microRNAs, U6 snRNA and human DNA in air-dried PAP smears from 174 women with different cervical cytological diagnoses, 144 of which were HR-HPV-positive [40 negative for intraepithelial lesion or malignancy (NILM), 34 low-grade squamous intraepithelial lesions (L-SIL), 57 high-grade squamous intraepithelial lesions (H-SIL), 43 invasive cancers], were statistically processed. The expression level changes of various individual microRNAs were found to be significantly correlated with the cytological diagnosis but the statistical significance of this correlation was critically dependent on the normalization strategy. We developed a linear classifier based on the paired ratios of 8 microRNA concentrations and cellular DNA content. The classifier determines the dimensionless coefficient (DF value), which increases with the severity of cervical lesion. The high- and low-grade CINs were better distinguished by the microRNA classifier than by the measurement of individual microRNA levels with the use of traditional normalization methods. The diagnostic sensitivity of detecting high-grade lesions (CIN ≥2) with the developed microRNA classifier was 83.4%, diagnostic specificity 81.2%, ROC AUC=0.913. The analysis can be performed with the same nucleic acid preparation as used for HPV testing. No statistically significant correlation of the DF value and HR-HPV DNA load was found. The DF value and the HR HPV presence and viral DNA load may be regarded as independent criteria that can complement each other in molecular screening for high-grade cervical intraepithelial neoplasia. Although it has several limitations, the present study showed that the small-scale analysis of microRNA signatures performed by simple PCR-based methods may be useful for improving the diagnostic/prognostic value of cervical screening.

Selection and validation of miRNAs as normalizers for profiling expression of microRNAs isolated from thyroid fine needle aspiration smears.

Fine needle aspiration cytology (FNAC) is currently the method of choice for malignancy prediction in thyroid nodules. Nevertheless, in some cases the interpretation of FNAC results may be problematic due to limitations of the method. The expression level of some microRNAs changes with the development of thyroid tumors, and its quantitation can be used to refine the FNAC results. For this quantitation to be reliable, the obtained data must be adequately normalized. Currently, no reference genes are universally recognized for quantitative assessments of microRNAs in thyroid nodules. The aim of the present study was the selection and validation of such reference genes. Expression of 800 microRNAs in 5 paired samples of thyroid surgical material corresponding to different histotypes of tumors was analyzed using Nanostring technology and four of these (hsa-miR-151a-3p, -197-3p, -99a-5p and -214-3p) with the relatively low variation coefficient were selected. The possibility of use of the selected microRNAs and their combination as references was estimated by RT-qPCR on a sampling of cytological smears: benign (n=226), atypia of undetermined significance (n=9), suspicious for follicular neoplasm (n=61), suspicious for malignancy (n=19), medullary thyroid carcinoma (MTC) (n=32), papillary thyroid carcinoma (PTC) (n=54) and non-diagnostic material (ND) (n=34). In order to assess the expression stability of the references, geNorm algorithm was used. The maximum stability was observed for the normalization factor obtained by the combination of all 4 microRNAs. Further validation of the complex normalizer and individual selected microRNAs was performed using 5 different classification methods on 3 groups of FNAC smears from the analyzed batch: benign neoplasms, MTC and PTC. In all cases, the use of the complex classifier resulted in the reduced number of errors. On using the complex microRNA normalizer, the decision-tree method C4.5 makes it possible to distinguish between malignant and benign thyroid neoplasms in cytological smears with high overall accuracy (>91%).

Filaggrin mutations in a Western siberian population and their association with atopic dermatitis in children.

We determined the frequencies of null mutations of the FLG gene--2282del4, R501X, R2447X, 3702delG, S3247X, and the 12-repeat allele (rs12730241)--among 460 Caucasians of the city of Novosibirsk, Russia. The frequency was 17.7% for rs12730241, 2.73% for 2282del4, 0.22% for R501X, 0.33% for R2447X, and 0% for 3702delG and S3247X in a western Siberian population. A case-control study showed that the deletion 2282del4 was associated with atopic dermatitis in children (odds ratio 7.01; p<0.001). The other mutations were not.

Genetic variability of Anaplasma phagocytophilum in ticks and voles from Ixodes persulcatus/Ixodes trianguliceps sympatric areas from Western Siberia, Russia.

Anaplasma phagocytophilum is a causative agent of granulocytic anaplasmosis in different mammals. The presence of A. phagocytophilum was assayed in Ixodes persulcatus, Ixodes trianguliceps ticks and Myodes spp. voles from two I. persulcatus/I. trianguliceps sympatric areas in the Omsk region (Western Siberia, Russia). In total, A. phagocytophilum was found in 42/108 (38.9%) of vole blood samples, 13/34 (38.2%) of I. trianguliceps ticks removed from voles, 1/12 (8.3%) of I. persulcatus removed from voles, and 18/279 (7.2%) of questing I. persulcatus. GroESL operon sequence analysis of positive samples revealed three distinct A. phagocytophilum genetic groups previously identified in ticks and mammals in Russia. Genetic group 1 was found in 6/36 (16.7%) of sequenced positive blood samples; this group was previously revealed in I. persulcatus and Myodes spp. voles in different regions of Russia. Genetic group 2 was found in 30/36 (83.3%) of sequenced positive blood samples and all positive I. trianguliceps; this group was previously revealed only in Myodes spp. voles and common shrews (Sorex araneus) in I. persulcatus/I. trianguliceps sympatric areas in the Northern Ural. Genetic group 3 was found in all positive questing I. persulcatus and one blood sample; this group was previously revealed in I. persulcatus and Siberian chipmunks (Tamias sibiricus). We suppose that I. trianguliceps is the most probable vector for A. phagocytophilum of group 2. Analysis of the msp4 gene, intergenic region DOV1, and some other genetic loci has shown that isolates from different genetic groups significantly differ in all studied loci and that A. phagocytophilum of group 2 is closely related to A. phagocytophilum isolates revealed in voles and I. trianguliceps in Europe. A. phagocytophilum of groups 1 and 2 are the most similar to each other, while A. phagocytophilum of group 3 clusters with European A. phagocytophilum isolates from I. ricinus and various mammalian species.