Prediction of the Aggressive Clinical Course of Papillary Thyroid Carcinoma Based on Fine Needle Aspiration Biopsy Molecular Testing.

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.

MicroRNA Expression Profile in Bone Marrow and Lymph Nodes in B-Cell Lymphomas.

Hodgkin's lymphomas (HL) and the majority of non-Hodgkin's lymphomas (NHL) derive from different stages of B-cell differentiation. MicroRNA (miRNA) expression profiles change during lymphopoiesis. Thus, miRNA expression analysis can be used as a reliable diagnostic tool to differentiate tumors. In addition, the identification of miRNA's role in lymphopoiesis impairment is an important fundamental task. The aim of this study was to analyze unique miRNA expression profiles in different types of B-cell lymphomas. We analyzed the expression levels of miRNA-18a, -20a, -96, -182, -183, -26b, -34a, -148b, -9, -150, -451a, -23b, -141, and -128 in lymph nodes (LNs) in the following cancer samples: HL (n = 41), diffuse large B-cell lymphoma (DLBCL) (n = 51), mantle cell lymphoma (MCL) (n = 15), follicular lymphoma (FL) (n = 12), and lymphadenopathy (LA) (n = 37), as well as bone marrow (BM) samples: HL (n = 11), DLBCL (n = 42), MCL (n = 14), FL (n = 16), and non-cancerous blood diseases (NCBD) (n = 43). The real-time RT-PCR method was used for analysis. An increase in BM expression levels of miRNA-26b, -150, and -141 in MCL (p < 0.01) and a decrease in BM levels of the miR-183-96-182 cluster and miRNA-451a in DLBCL (p < 0.01) were observed in comparison to NCBD. We also obtained data on increased LN levels of the miR-183-96-182 cluster in MCL (p < 0.01) and miRNA-18a, miRNA-96, and miRNA-9 in FL (p < 0.01), as well as decreased LN expression of miRNA-150 in DLBCL (p < 0.01), and miRNA-182, miRNA-150, and miRNA-128 in HL (p < 0.01). We showed that miRNA expression profile differs between BM and LNs depending on the type of B-cell lymphoma. This can be due to the effect of the tumor microenvironment.

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.

New Opportunities for Preoperative Diagnosis of Medullary Thyroid Carcinoma.

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.

The Profile of MicroRNA Expression in Bone Marrow in Non-Hodgkin's Lymphomas.

Non-Hodgkin's lymphomas (NHLs) are a heterogeneous group of malignant lymphomas that can occur in both lymph nodes and extranodal sites. Bone marrow (BM) is the most common site of extranodal involvement in NHL. The objective of this study is to determine the unique profile of miRNA expression in BM affected by NHL, with the possibility of a differential diagnosis of NHL from reactive BM changes and acute leukemia (AL). A total of 180 cytological samples were obtained by sternal puncture and aspiration biopsy of BM from the posterior iliac spine. All the cases were patients before treatment initiation. The study groups were NHL cases (n = 59) and AL cases (acute lymphoblastic leukemia (n = 25) and acute myeloid leukemia (n = 49)); the control group consisted of patients with non-cancerous blood diseases (NCBDs) (n = 48). We demonstrated that expression levels of miRNA-124, miRNA-221, and miRNA-15a are statistically significantly downregulated, while the expression level of let-7a is statistically significantly upregulated more than 2-fold in BM in NHL compared to those in AL and NCBD. ROC analysis revealed that let-7a/miRNA-124 is a highly sensitive and specific biomarker for a differential diagnosis of BM changes in NHL from those in AL and NCBD. Therefore, we conclude that analysis of miRNA expression levels may be a promising tool for early diagnosis of NHL.

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.

Cytological and molecular diagnosis of Hürthle cell thyroid tumors: Analysis of three cases.

The cytological diagnosis of Hürthle cell (oncocytic) thyroid tumors by means of fine-needle aspiration biopsy represents a challenge, as Hürthle cell polymorphism and atypia alone are not indications of malignancy. In our recent work, an original diagnostic algorithm was proposed, which identified and typed malignant thyroid tumors by analyzing the molecular markers of cytological preparations. The aim of the present study was to assess the effectiveness of this algorithm at detecting Hürthle cell thyroid tumors in clinical samples used for cytological examination. Cytological and histological examinations of the biopsy material were performed for three patients with nodular neoplasms. Biopsy material of these patients was analyzed by quantitative PCR using preselected molecular markers [normalized concentrations of High-mobility group AT-hook 2 mRNA, three microRNAs (miRNAs or miRs; miR-146b, miR-221 and miR-375) and the mitochondrial (mtDNA)/nuclear DNA ratio]. The results revealed that the molecular test determined the malignancy of three cases of Hürthle cell tumor. This method may therefore be used to complement the cytological diagnosis of fine-needle aspiration biopsy. In all three cases, there was an increased content of mtDNA, indicating Hürthle cell malignancies. Furthermore, in the first case [Hürthle cell carcinoma (HCC)], increased miRNA-221 content was detected, which also indicated malignancy. In the second case (Hürthle cell papillary thyroid carcinoma), an increased level of miRNA-146b was present, which indicated papillary carcinoma. In the third case (Hürthle cell adenoma), no markers of malignancy were identified. The present study demonstrated that molecular testing together with cytological analysis can reduce the isk of error in the preoperative cytological diagnosis of unclear or ambivalent cases.

Malignant and benign thyroid nodule differentiation through the analysis of blood plasma with terahertz spectroscopy.

The liquid and lyophilized blood plasma of patients with benign or malignant thyroid nodules and healthy individuals were studied by terahertz (THz) time-domain spectroscopy and machine learning. The blood plasma samples from malignant nodule patients were shown to have higher absorption. The glucose concentration and miRNA-146b level were correlated with the sample's absorption at 1 THz. A two-stage ensemble algorithm was proposed for the THz spectra analysis. The first stage was based on the Support Vector Machine with a linear kernel to separate healthy and thyroid nodule participants. The second stage included additional data preprocessing by Ornstein-Uhlenbeck kernel Principal Component Analysis to separate benign and malignant thyroid nodule participants. Thus, the distinction of malignant and benign thyroid nodule patients through their lyophilized blood plasma analysis by terahertz time-domain spectroscopy and machine learning was demonstrated.

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.

Intratumoral Heterogeneity of Expression of 16 miRNA in Luminal Cancer of the Mammary Gland.

The purpose of this work is to determine the intratumoral distribution of miRNA expression profiles in luminal breast cancer (BC). The study included 33 certain BC cases of the luminal A or luminal B (Her2-) subtypes. The relative expression levels of miRNA-20a; -21; -125b; -126; -200b; -181a; -205; -221; -222; -451a; -99a; -145; -200a; -214; -30a; -191; and small nuclear RNAs U6, U54, and U58 were measured by RT-qPCR in four intratumor areas in each of 33 luminal BC specimens and in surrounding normal mammary gland tissues. Comparative analysis of miRNA expression levels between normal mammary gland tissue and different intratumor areas revealed that only four miRNAs (miRNA-21, -200b, -200a, -191) appear as consistently differentiating markers. A comparative analysis of miRNA expression levels between normal mammary gland tissue and the tumor border revealed statistically significant differences for ten miRNAs; 10 miRNAs show differential expression between normal mammary gland tissue and central tumor specimens; 9 miRNAs show differential expression between normal mammary gland tissue and tumor periphery 1; 13 miRNAs show differential expression between normal mammary gland tissue and tumor periphery 2. After comparing the tumor periphery 1 and tumor center, we found statistically significant differences in expression between five miRNAs and after comparing the tumor periphery 2 and tumor center, differences were observed for 12 miRNAs. MiRNA expression levels are subject to considerable variation, depending on the intratumor area. This may explain the inconsistency in miRNA expression estimates in BC coming from different laboratories.

Profiling 25 Bone Marrow microRNAs in Acute Leukemias and Secondary Nonleukemic Hematopoietic Conditions.

INTRODUCTION: The standard treatment of acute leukemias (AL) is becoming more efficacious and more selective toward the mechanisms via which to suppress hematologic cancers. This tendency in hematology imposes additional requirements on the identification of molecular-genetic features of tumor clones. MicroRNA (miRNA, miR) expression levels correlate with cytogenetic and molecular subtypes of acute leukemias recognized by classification systems. The aim of this work is analyzing the miRNA expression profiles in acute myeloblastic leukemia (AML) and acute lymphoblastic leukemia (ALL) and hematopoietic conditions induced by non-tumor pathologies (NTP). METHODS: A total of 114 cytological samples obtained by sternal puncture and aspiration biopsy of bone marrow (22 ALLs, 44 AMLs, and 48 NTPs) were analyzed by real-time PCR regarding preselected 25 miRNAs. For the classification of the samples, logistic regression was used with balancing of comparison group weights. RESULTS: Our results indicated potential feasibility of (i) differentiating ALL+AML from a nontumor hematopoietic pathology with 93% sensitivity and 92% specificity using miR-150:miR-21, miR-20a:miR-221, and miR-24:nf3 (where nf3 is a normalization factor calculated from threshold cycle values of miR-103a, miR-191, and miR-378); (ii) diagnosing ALL with 81% sensitivity and 81% specificity using miR-181b:miR-100, miR-223:miR-124, and miR-24:nf3; and (iii) diagnosing AML with 81% sensitivity and 84% specificity using miR-150:miR-221, miR-100:miR-24, and miR-181a:miR-191. CONCLUSION: The results presented herein allow the miRNA expression profile to de used for differentiation between AL and NTP, no matter what AL subtype.

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%).