A cytological examination of the FNAB material and intraoperative imprint cytological analysis of the thyroid lesions were performed for three patients with nodular neoplasms. The cytological smears and intraoperative imprints was air-dried and stained by a standard procedure [May-Grünwald-Giemsa (MGG) staining]. Histological analysis of surgically removed thyroid tissue was conducted by staining tissue sections (5-µm thick) with hematoxylin and eosin. Briefly, after resection of the thyroid lobe, the tissue is fixed in formalin for 24 h at room temperature then histological processing of the tissue was carried out using vacuum infiltration processor Tissue-Tek^® VIP™ 6 (Sakura Finetek Europe B.V., The Netherlands) with the routine overnight run program (processing time: 12.7 h, temp=40-58˚C). After sectioning of the paraffin-embedded tissue, the sections were manually stained with hematoxylin (NPF ABRIS+) and eosin (ErgoProduction), following a basic protocol: Dewaxing (with xylene), dehydration (ethanol then water), hematoxylin (10 min), differentiation (acid alcohol), bluing (aqueous ammonium hydroxide), eosin (5 min), dehydration (ethanol then water), clearing (xylene), cover-slipping. The preparations were examined under a light microscope with a magnification of 100 and 400. The preparations were examined under a light microscope with a magnification of 100 and 400. The histological and cytological material was obtained in accordance with Russian laws and regulations; all data were depersonalized.

Nucleic acid was isolated from the cytological preparations (reviewed in ref. 13). The dried cytological preparation was washed into a microcentrifuge tube with three 200 µl portions of guanidine lysis buffer, then the sample was vigorously mixed and incubated in a thermal shaker for 15 min at 65˚C. Next, an equal volume of isopropanol was added. The reaction solution was thoroughly mixed and kept at room temperature for 5 min. After centrifugation for 10 min at 14,00 x g, the supernatant was discarded, and the pellet was washed with 500 µl of 70% ethanol and 300 µl of acetone. Finally, the RNA was dissolved in 200 µl of deionized water. If not analyzed immediately, RNA samples were stored at -20˚C until further use.

Assessment of relative expression levels of the HMGA2 gene (normalized to housekeeping gene PGK1), miR-146b, -221, and -375 (normalized to the geometric mean of miR-29b, -23a, and -197 levels) and calculation of the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) were done as described previously (13). The details are presented below.

The detection of the 6 miRNAs was performed on three samples of oncocytic tumors. To quantify miRNA, we followed the protocol published by Chen et al in 2005, which includes reverse transcription of mature miRNA using a long stem-loop primer followed by the detection of cDNA via real-time PCR (15). Reverse-transcription reactions were set up individually for each miRNA to be quantified. The obtained cDNA was subjected to further PCR analysis immediately. The reverse-transcription reaction and real-time PCR were carried out (reviewed in ref. 16). The miRNA content was normalized to the geometric mean of the amounts of the three reference miRNAs by the 2^-ΔCq method (17).

The relative concentration of HMGA2 mRNA was estimated by real-time RT-PCR, where PGK1 (phosphoglycerate kinase 1) mRNA served for normalization. The following RT-PCR program was employed: Incubation for 30 min at 45˚C, preheating for 2 min at 95˚C, and then 50 cycles of denaturation for 10 sec at 94˚C with annealing and elongation for 20 sec at 60˚C (13). The relative expression level was calculated by the 2^-ΔCq method.

Detection of specific sites in mtDNA and nDNA was performed independently by real-time PCR. The thermal cycling conditions were as follows: Preheating for 2 min at 95˚C, then 50 cycles of denaturation for 10 sec at 94˚C with annealing and elongation for 20 sec at 60˚C (13). The ratio was determined with the 2^-ΔCq method.

The classification method (reviewed in ref. 13) was used (Fig. 1). Thus, the specimens are classified as either benign (goiters and follicular neoplasms without markers of malignancy) or malignant (papillary thyroid carcinoma, medullary thyroid carcinoma, HCC, and a follicular neoplasm with markers of malignancy). Additionally, the ratio of mtDNA to nDNA allows to determine whether a given case of papillary thyroid carcinoma can be categorized as the Hürthle cell type.

Aspirates of FNAB from a 34-year-old female patient with a 2 cm nodule in the left thyroid lobe were investigated. The cytological report stated that this was a Hürthle cell (oncocytic) neoplasm, and that in the studied samples, there were groups of Hürthle cells of various sizes, with subpopulations showing pronounced polymorphism, abundant fine-grained cytoplasm, an eccentrically or centrally located nucleus, and enlarged nucleolus (Figs. 2 and 3).

Three slides were examined, and the results are given in Table I; our final opinion was HCC. The histological report on the surgical material indicated that this was Hürthle cell (oncocytic) thyroid carcinoma, and that the trabecular structure prevailed in the tumor tissue, with minimal invasive growth into the capsule. The report also stated that the Hürthle cells that formed the tumor featured pronounced cellular and nuclear polymorphism, eosinophilic granular cytoplasm, and enlarged nucleoli.

FNAB was performed on a 56-year-old female patient with a thyroid nodule. The cytological report indicated a suspected Hürthle cell (oncocytic) neoplasm and that the cellular aspirate consisted of cells with oncocytic signs, forming sporadic papillary structures; some cells had features characteristic of papillary carcinoma: Uneven nuclear contours and nuclear grooves. According to the cytological report, papillary thyroid carcinoma could not be ruled out (Figs. 4 and 5).

One slide was examined; the results are listed in Table II; our conclusion is Hürthle cell papillary thyroid carcinoma.

Total thyroidectomy was performed. The histological report stated that this was papillary thyroid carcinoma, and that the tumor tissue was predominantly papillar. Tumor cells belonged to the oncocytic type, with abundant eosinophilic granular cytoplasm and a moderately polymorphic nucleus with typical signs of papillary cancer. According to the report, the tumor showed invasive capsular growth, and there were regional lymph node metastases.

A 29-year-old woman with a solitary thyroid nodule in the right lobe. The cytological report on the FNAB samples indicated that this was a suspected Hürthle cell (oncocytic) neoplasm, and that in cytological samples, there were syncytial-like sheets and clusters of Hürthle cells with moderate cellular and nuclear polymorphism (Figs. 6 and 7).

One slide was examined; the results are presented in Table III; we concluded that this is a benign Hürthle cell tumor.

Surgical lobectomy was performed. The histological report stated that this was oncocytic adenoma, i.e., an encapsulated tumor with a solid and trabecular structure, consisting of cells of the oncocytic type, with abundant eosinophilic granular cytoplasm, a large nucleus, and enlarged nucleolus. According to this report, no capsular or vascular invasion was found.

We verified the validity of all the above reports. In the cases presented, the ratio of mtDNA to nDNA, which is a marker of Hürthle cells, was within previously described ranges (13) and helped to make the diagnoses (Fig. 8).