Ultrastructural and morphometric features of follicular thyroid adenomas

The new molecular markers DDIT3, STT3A, ARG2 and FAM129A are not useful in diagnosing thyroid follicular tumors

258P - Comparison of results of cytological and hystological research of thyroid gland nodes

Fine needle aspiration (FNA) is a valuable technique in the evaluation of thyroid nodules. A total of 182 FNAs of the thyroid performed at the Sultan Qaboos University Hospital were reevaluated and 153 were found to be adequate (84%). The cytological results of FNA were divided into neoplastic and non-neoplastic groups. Subsequent histology was obtained in 53 patients with an intraoperative frozen actions (FS) in 27 patients. Papillary carcinoma was found to be the most common, accounting for 70% of these cases with histological follow-up in the neoplastic group. The utility of performing FNA in all thyroid nodules and an intraoperative frozen section only in selected cases is discussed.

The potential value of DNA aneuploidy, in distinguishing benign from malignant follicular thyroid neoplasms, was studied. The nuclear DNA content of 65 follicular thyroid neoplasms (52 adenomas and 13 carcinomas) was determined by flow cytometric analysis of paraffin embedded material; in 58 cases preparations were technically satisfactory. In 22 follicular neoplasms DNA analysis was also performed on fresh material obtained by fine needle aspiration of surgical specimens. Cell cycle analysis was performed on both fresh and fixed specimens. An aneuploid DNA profile was found on analysis of fixed tissue in eight of 45 (18 per cent) follicular adenomas and four of 13 (31 per cent) follicular carcinomas. DNA aneuploidy was also found in six of the 22 (27 per cent) fresh preparations from follicular adenomas. The frequency of DNA aneuploidy in apparently benign and malignant follicular neoplasms was similar. Follicular thyroid neoplasia are best regarded as a single entity with a low incidence of local and distant spread. All follicular neoplasia are therefore best excised.

Background. The purpose was to study the cytomorphological structure of autonomously functioning thyroid nodules and the prevalence of cancer in this group based on a retrospective analysis of laboratory, instrumental and morphological data. Materials and methods. Patients with toxic adenoma (TA) were included in the study, who were treated in the V.P. Komisarenko Institute of Endocrinology and Metabolism of the National Academy of Medical Sciences of Ukraine (IEM) in 2010–2019. With the help of the medical information system TerDep from the clinical database of the IEM, patients with TA were selected who were operated at the Department of Endocrine Surgery in this period. There were 188 patients with thyroid TA, 156 (83 %) women and 32 (17 %) men. Their average age was 53.9 ± 1.2 years. To study the structural state of the thyroid, the results of ultrasound examination were analyzed, as well as cytological, immunocytochemical and morphological characteristics of TA. In patients with nodular/multinodular goiter, the size of formations was assessed, the data of cytological and immunocytochemical studies were analyzed. The prevalence of thyroid cancer among patients with TA was evaluated based on histological findings. Results. According to the ultrasound data, the sizes of TA ranged from 1.1 to 8.6 cm, with an average of 4.30 ± 0.09 cm. Echographic signs of nodular vascularization were recorded in 36 % of patients. Microcalcifications were observed in 9.4 % of cases. In all cases, the nodules had the correct shape, in 83.9 % — a clear contour, in 16.1 % — not clear contour. In 67.1 % of patients, the nodules were described as isoechogenic, in 32.9 %, a moderately reduced echogenicity of the nodules was noted. Fine needle aspiration biopsy with cytological examination was performed in 121 patients. In 88.5 % of cases, a cytological conclusion corresponded to Bethesda II (nodular goiter or adenomatous nodules), in 9.1 % — to Bethesda IV (follicular neoplasia), in 0.8 % — to Bethesda III (signs of epithelial atypia), in 0.8 % — to suspicion of malignancy (Bethesda V), and 0.8 % of patients had an uninformative cytological conclusion (Bethesda I). The frequency of detecting thyroid cancer among the total number of patients with TA was 3/188 (1.6 %). A detailed analysis of these cases showed that one patient had noninvasive encapsulated papillary carcinoma with oxyphil cell changes (cytological conclusion of follicular neoplasia with oxyphil cell metaplasia (Bethesda IV)), one had follicular carcinoma, and one had a tumor with uncertain malignant potential (cytological conclusion of adenomatous nodules (Bethesda II)). Conclusions. The sizes of the toxic adenoma ranged from 1.1 to 8.6 cm. The frequency of thyroid cancer among the total number of patients with TA was 1.6 %. The presence of hyperfunctioning thyroid TA cannot completely exclude the presence of malignancy. Complex ultrasounds studies with fine needle aspiration biopsy from different areas of nodules can be an adequate approach to rule out malignancy among toxic adenomas.

Thyroid carcinoma is a common endocrine cancer with a favorable prognosis if subjected to timely treatment. However, the clinical identification of follicular thyroid carcinoma (FTC) among patients with benign thyroid nodules is still a challenge. Preoperative fine needle aspiration-based cytology cannot always differentiate follicular carcinomas from benign follicular neoplasias. Because current methods fail to improve preoperative diagnosis of thyroid nodules, new molecular-based diagnoses should be explored. We conducted a microarray-based study to reveal the genetic profiles unique to FTC and follicular adenomas (FAs), to identify the most parsimonious number of genes that could accurately differentiate between benign and malignant follicular thyroid neoplasia. We confirmed our data by quantitative RT-PCR and immunohistochemistry in two independent validation sets with a total of 114 samples. We were able to identify three genes, cyclin D2 (CCND2), protein convertase 2 (PCSK2), and prostate differentiation factor (PLAB), that allow the accurate molecular classification of FTC and FA. Two independent validation sets revealed that the combination of these three genes could differentiate FTC from FA with a sensitivity of 100%, specificity of 94.7%, and accuracy of 96.7%. In addition, our model allowed the identification of follicular variants of papillary thyroid carcinoma with an accuracy of 85.7%. Three-gene profiling of thyroid nodules can accurately predict the diagnosis of FTC and FA with high sensitivity and specificity, thus identifying promising targets for further investigation to ultimately improve preoperative diagnosis.

This study describes two cases of signet ring cell thyroid adenoma. Signet ring cells contain immunoreactive thyroglobulin and stain positively for mucin by histochemical methods: previous interpretations of these peculiar findings suggested that signet ring celled thyrocytes may show dual endocrine and non-endocrine differentiation. In the present material the histochemical profile of mucinous collections was consistent with a glycoprotein containing 1:2-glycol groups and sialic acid residues. These biochemical features are the same as those of the thyroglobulin molecule although in normal thyroid tissue they can be only partially demonstrated by histochemical methods. It is suggested that the signet ring configuration may be solely due to the intracytoplasmic accumulation of thyroglobulin and that histochemically reactive protein-polysaccharide complexes derived from partial degradation of this glycoprotein may account for the positive mucin staining.

Follicular thyroid neoplasms are solitary nodules that present as a mass. Follicular adenomas are benign, encapsulated tumors with follicular differentiation that are more common in women, usually occur in adults, and are more common in areas of iodine deficiency. Follicular adenomas measure 1–3 cm and usually are smaller than follicular carcinomas, although size may overlap. Follicular neoplasms lack cytologic features of papillary thyroid carcinoma (PTC). Malignancy in follicular neoplasms requires capsular or vascular invasion. The capsule in follicular carcinomas often is thicker and more irregular than that of adenomas. Various histologic patterns, including microfollicular, macrofollicular, normofollicular, and trabecular growth, may be seen in follicular neoplasms. Areas of papillary architecture may be seen. Follicular neoplasms may have different cytomorphologies, including mucinous, signet ring cell, and clear cell, and may have intratumoral fat. Follicular neoplasms show immunopositivity for thyroid transcription factor 1 (TTF1), thyroglobulin, and keratin and are negative for chromogranin, synaptophysin, and calcitonin. Follicular neoplasms often show RAS mutations, and a few have t(2;3)(q13;p25) involving PAX8 and peroxisome proliferator-activated receptor-γ encoding a fusion protein. Follicular adenomas show increased expression of p27 cell cycle inhibitory protein and decreased Ki67 proliferative indices (LI) compared with follicular carcinomas, and carcinomas with metastases have higher Ki67 LI levels than those without. However, in an individual lesion, these and other immunohistochemical or molecular markers cannot separate benign from malignant follicular neoplasms definitively. Cytologic specimens cannot distinguish benign from malignant follicular neoplasms, as diagnosis of malignancy requires invasive growth. Follicular adenomas are treated with lobectomy and have an excellent prognosis. Follicular carcinomas show capsular and/or vascular invasion. Vascular invasion must occur within or beyond the capsule. Tumors with capsular invasion only (no vascular invasion) have more indolent behavior than those with vascular invasion. Follicular carcinomas with focal capsular and/or vascular invasion have been designated as minimally invasive, whereas those with more extensive invasion have been characterized as widely invasive, although this classification is being used less often. Tumors with extensive vascular invasion often are referred to as angioinvasive follicular carcinomas. Unlike PTC, which has frequent lymph node metastases, follicular carcinomas usually metastasize directly to viscera. Minimally invasive follicular carcinomas have a low mortality rate (3%–5% in some studies), and those with only capsular invasion (no vascular invasion) have a particularly good prognosis. Widely invasive carcinomas are aggressive, with a long-term mortality rate of 50 %.

The molecular determinants of thyroid follicular nodules are incompletely understood and assessment of malignancy is a diagnostic challenge. Since microRNA (miRNA) analyses could provide new leads to malignant progression, we characterised the global miRNA expression in follicular adenoma (FA) and follicular carcinoma (FC). Comparison of carcinoma and adenoma with normal thyroid revealed 150 and 107 differentially expressed miRNAs respectively. Most miRNAs were down-regulated and especially miR-199b-5p and miR-144 which were essentially lost in the carcinomas. Integration of the changed miRNAs with differentially expressed mRNAs demonstrated an enrichment of seed sites among up-regulated transcripts encoding proteins implicated in thyroid tumourigenesis. This was substantiated by the demonstration that pre-miR-199b reduced proliferation when added to cultured follicular thyroid carcinoma cells. The down-regulated miRNAs in FC exhibited a substantial similarity with down-regulated miRNAs in anaplastic carcinoma (AC) and by gene set enrichment analysis, we observed a significant identity between target mRNAs in FC and transcripts up-regulated in AC. To examine the diagnostic potential of miRNA expression pattern in distinguishing malignant from benign nodules we employed a supervised learning algorithm and leave-one-out-cross-validation. By this procedure, FA and FC were identified with a negative predicted value of 83% (data generated by microarray platform) and of 92% (data generated by qRT-PCR platform). We conclude that follicular neoplasia is associated with major changes in miRNA expression that may promote malignant transformation by increasing the expression of transcripts encoding tumourigenic factors. Moreover, miRNA profiling may facilitate the diagnosis of carcinoma vs adenoma.

Twenty-five adenomas of the thyroid were studied by transmission and scanning electron microscopy: 14 embryonal, fetal, and simple adenomas, 1 colloid adenoma, 2 toxic adenomas, 2 oxyphil adenomas, 2 "atypical adenomas" and 4 adenomas with capsular invasion, formerly designated "malignant adenomas." The only adenomas that presented with special ultrastructural features were the oxyphil and the toxic varieties. The toxic adenomas had organelle-rich cells with numerous and long microvilli on their surfaces. The oxyphil adenomas showed Hürthle cell changes, including abundant mitochondria, and smooth cell surfaces. The subdivision of the other forms of adenomas was impossible at the ultrastructural level; and this study did not reveal ultrastructural features which could distinguish the so-called "atypical" and "malignant" adenomas from ordinary thyroid adenomas. "Malignant" adenomas should preferable be called what they are: adenomas with capsular invasion. Neither does this study support the view that electron microscopy is a useful method to separate an adenoma from a well-differentiated follicular carcinoma in the occasional patient who presents problems.

Signet ring cells (SRCs) can be seen in a variety of thyroid tumors and can pose a diagnostic pitfall on cytology. This study describes the cytologic, histomorphologic, and molecular aspects of a cohort of primary thyroid tumors with SRCs. A search was performed of the Massachusetts General Hospital and Brigham and Women's Hospital (Boston, MA) pathology archives for the keywords thyroid, signet, and signet ring features between 2000 and 2014. Seven thyroidectomy specimens with corresponding thyroid fine-needle aspiration (FNA) were obtained. Cytology and histopathology slides were evaluated. Molecular analysis was performed using anchored multiplex polymerase chain reaction (AMP). The cohort consisted of four follicular adenomas (FAs), two noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTPs), and one secretory carcinoma (SC). The FNA diagnoses were atypia of undetermined significance (n = 3), suspicious for follicular neoplasm (n = 3), and suspicious for malignancy (n = 1). Molecular analyses revealed PTEN and FGFR3 mutations in an FA and NIFTP, respectively, and an ETV6-NTRK3 fusion in a case of primary thyroid gland SC. Our study demonstrates the range of thyroid tumors with SRCs. While most thyroid tumors with SRCs are benign, primary thyroid SC should also be considered in the differential diagnosis.

Мета. Визначення експресії імуногістохімічних маркерів апоптозу р53 та bcl-2 і маркера проліферації Ki-67 у фолікулярних неоплазіях щитоподібної залози (ЩЗ). Матеріали та методи. Як критерії диференціальної діагностики фолікулярних неоплазій ЩЗ пропонуються численні імуногістохімічні маркери, серед яких найбільш корисними є маркери проліферації Ki-67 та апоптозу р53, bcl-2. Проведено імуногістохімічне дослідження операційного матеріалу 50 пацієнтів, оперованих у клініці ДУ «Інститут проблем ендокринної патології імені В.Я. Данилевського НАМН України» з приводу доброякісних (25 аденом) та злоякісних (25 карцином) фолікулярних новоутворень. Результати. Отримані дані показують, що фолікулярні аденоми солідної і фетально-ембріональної будови мають високий рівень спорідненості з фолікулярним раком ЩЗ за показниками проліферативної активності та порушень механізмів апоптозу, що, ймовірно, передбачає найбільший потенціал до малігнізації і подальшого метастазування. Висновки. Високий рівень експресії р53 та стабільно високий рівень експресії bcl-2 в клітинах фолікулярного раку ЩЗ і фолікулярної аденоми солідної та фетально-ембріональної будови вказує на глибокі порушення апоптотичних процесів при розвитку фолікулярних неоплазій ЩЗ.

The existence of lymphatic vessels in the dental pulp has been a matter of continuing controversy. We have now used light microscopy to examine semithin transverse sections of perfusion-fixed incisors and canines in cats. Lymphatics were found in all the teeth studied. In most teeth they were present in the coronal, middle, and apical regions of the pulp; but in a few they were lacking coronally and in the middle. Within individual teeth, lymphatics were found in the subodontoblastic zone or more centrally in the pulp; but none were found in the odontoblast layer or in the pulp horns. Vessels located by light microscopy were subsequently examined by transmission electron microscopy. Their ultrastructural features were typical of lymphatics and included irregular, attenuated endothelium with adjacent cells joined in different ways. Occasional gaps connected the extracellular spaces with their lumens, and abluminal endothelial projections appeared to form open end bulbs. There was very little basement membrane, but anchoring filaments were found near the abluminal surface of the endothelium and near collagen fibrils. The total cross-sectional area of lymphatic vessels was measured in semithin sections and, with pulp area, increased from the coronal region to the middle. However, both areas decreased from the middle to the apical region suggesting either that lymph flows faster as it reaches the foramens of the apical delta or that some vessels leave the tooth through lateral root canals. Using the methods of light and transmission electron microscopy, therefore, we have shown that pulp lymphatic vessels exist. Questions remain, however, about their distribution within teeth, variations between teeth, and routes of exit from teeth.

The aim of the study was to investigate the development and differentiation of the adrenal glands in the grass snake (Natrix natrix L.) during the early stages of ontogenesis, i.e., from egg-laying to hatching of the first specimens. The material used for the studies consisted of a collection of embryos of the grass snake. The Natrix eggs were incubated in the laboratory at a constant temperature of 30 degrees C and 100% relative humidity. Embryos were isolated in a regular sequence of time from egg-laying to hatching. The age of the embryos was qualified according to normal tables for this species. For histological and histochemical investigations, the smallest embryos were fixed in toto. From the oldest embryos, the medial region with the mesonephros and adrenal primordium were resected. Depending on the requirements of histochemical methods, the material was fixed in various fixatives, namely, 10% formalin solution, Bouin, Wood and Millonig fluid, embedded in paraffin and sectioned into serial transversal, sagittal and longitudinal sections. The sections for review were stained with H&E and azan. For detection of adrenaline and noradrenaline in chromaffin tissue, the Wood and Honoré methods were used. SGC cells were detected with the silver stain method after Bodian. For electron microscopic studies, the adrenal gland was fixed in 2.5% glutaraldehyde and 2.0% paraformaldehyde 1:1 in 0.1 M phosphate buffer at pH 7.4 and post-fixed in 1.5% osmic acid in the same buffer. The fixed sections of the adrenal glands were embedded in Epon 812. Semithin and ultrathin sections were cut on ultramicrotome ultratome IV. Semithin sections were stained with methylene blue and ultrathin sections were routinely contrasted with uranyl acetate and lead citrate, then examined and photographed with the JEM JEOL 1220 electron microscope. According to morphological and metrical observation in the course of the grass snake embryo development, one can distinguish 12 stages of development. The primordia of the adrenal cortex appear at the first trimester of egg incubation as two asymmetrical strands between the mesonephros and aorta dorsalis. They are made of dense mesenchymal cells. At the second trimester of development, primordia are penetrated by chromaffinoblasts and capillaries. The mesenchymal cells differentiate into interrenal cells, while chromaffinoblasts are arranged dorsally of the gland. The glands are enclosed by the capsule which separates them from the mesonephros. At the third trimester of the eggs incubation, only noradrenaline appears in a chromaffin tissue. At the moment of snake hatching, the adrenal glands are completely differentiated, both in their structure and their function. The primordia of the interrenal tissue differentiate from mesenchymal cells similarly to mammals. During the development of the snake interrenal tissue, several types of cells can be recognized, varying in the degree of differentiation and in ultrastructural features: 1. Undifferentiated cells with features of mesenchymal cells 2. Differentiating mesenchymal cells 3. Transitional cells with features of mesenchymal and steroidogenic cells 4. Differentiating interrenal cells with pleomorphic mitochondria and numerous lipid droplets 5. Embryonic interrenal cells containing circular lipid droplets and underdeveloped smooth endoplasmic reticulum 6. Transitional interrenal cells containing mitochondria with tubular and vesicular cristae, large lipid droplets, numerous myelin structures, and well-developed smooth endoplasmic reticulum 7. Degenerating cells of embryonic interrenal tissue 8. Differentiating mesenchymal cells with features of fibroblasts The above classification is very schematic and presumptive. In developing adrenal glands at the first trimester of egg incubation type 1 and 2 cells predominate. Type 3 and 4 cells were observed at the second trimester of the adrenal primordia development. At the third trimester of egg incubation, embryonic adrenal glands were composed of the type 5 cells. At the moment of snake hatching, interrenal tissue contained type 5 and 6 cells. In the next days of the adrenal gland development, at the border between the cortex and in medulla as under the capsule, numerous cells were degenerated. During the entire development period the adrenal capsule was built from type 7 cells. The chromaffin tissue of the adrenal glands is derived from the neural crest. These findings agree with the findings of all embryologists. The first chromaffinoblasts infiltrated the adrenal cortex primordium around stage IV of development. They were mixed with interrenal cells and just at hatching they were localized dorsally of the gland. The chromaffinoblasts differentiated gradually from neuron-like cells to typical chromaffinocytes. All the chromaffinoblasts contained the chromaffin granules. The size and numerical density of the chromaffin granules increased with development. Just before hatching, the cells of the chromaffin tissue contained only noradrenaline. Differentiation chromaffinoblasts into chromaffin cells are probably stimulated and controlled by the influence of hormones, which are produced by the cells of the interrenal tissue. According to morphological, histochemical and ultrastructural observation in the course of adrenal differentiation and development in the grass snake, six morphological phases can be distinguished.

To improve preoperative examination of patients with follicular thyroid tumors using ultrasound-guided core-needle biopsy. All patients with Bethesda IV (follicular tumor) underwent ultrasound-guided core-needle biopsy of thyroid neoplasm according to original technique (patent No. 2826474 RU). Preoperative histological specimen after core needle biopsy was compared with urgent and elective histological examination. Statistical analysis was carried out using Kolmogorov-Smirnov and Kruskal-Wallis tests. Differences were significant at p<0.05. Thyroid tumors were available for core needle biopsy in all 45 cases (100%). Repeated cytological analysis confirmed TI-RADS category 3 in 22 (48.89%), TI-RADS 4 in 15 (33.33%) and TI-RADS 5 in 8 (17.77%) patients. Histological examination revealed colloidal goiter in 13 patients (28.89%), adenomatous hyperplasia in 15 (33.33%), follicular adenoma in 9 (20%), and follicular neoplasia with undetermined malignant potential in 2 (4.44%) cases. The quality of histological material made it possible to assess morphological type of tumor, capsule vascularization and cell atypia, invasion into capsule or vessels. In case of colloidal goiter, we were able to assess dimensions of follicles, presence of colloid, cell polymorphism, mitosis and cytoplasm content. Ultrasound-guided core-needle biopsy can reduce the number of unjustified surgical interventions in patients with follicular tumors and optimize management of patients with nodular thyroid lesions.