Introduction

Thyroid cancer is the most common endocrine malignancy. Fine-needle cytology (FNC) currently represents a reliable, accurate, and cost-effective diagnostic tool for the evaluation of thyroid nodules and for the management of nodular disease [1]. Testing FNC samples for a panel of molecular markers significantly increases the diagnostic accuracy of traditional cytology [2, 3]. We report a case of a 34-year-old woman who was diagnosed with a concurrent papillary thyroid carcinoma (PTC) and a mucoepidermoid carcinoma (MEC) based on FNC, whose diagnosis was supported by histology as well as immunocytochemistry. A panel of somatic mutations and rearrangements was also tested on FNC material in order to investigate on the molecular aspect of the PTC component of such tumor.

The current World Health Organization (WHO) classifies these neoplasms into two different categories: PTC belongs to the group of well-differentiated carcinomas of follicular cell origin, whereas MEC has been encompassed within the group of epithelial tumors of different or uncertain cell origin [4]. This may change in the future as new molecular studies will emerge.

Case Presentation

A 34-year-old woman was referred to our institute because of a firm and fixed nodule in the isthmic region of the thyroid. The patient underwent an ultrasound-guided FNC using a 23G needle without suction. The slides were stained with Diff QuikTM and Papanicolaou. On-site microscopic evaluation of the Diff QuikTM-stained slides disclosed a conventional PTC combined with a MEC. Two more FNC samples were collected, and the needle hubs were immediately washed in a 10-μL β-mercaptoethanol solution for subsequent genetic tests.

Immunocytochemistry was carried out for thyroid transcription factor-1 (TTF1) and cytokeratin 19 (CK19). Two months later, the patient underwent total thyroidectomy and immunohistochemical analyses were performed for TTF1; human thyroglobulin (hTg), and carcinoembryonic antigen (CEA). Dedicated FNC samples were tested for gene mutations (BRAF and RAS) and rearrangements (RET/PTC). Of RNA, 0.5 μg was reverse transcribed into cDNA and the applicability of the obtained RNA samples was verified analyzing the housekeeping gene GAPDH.

The primers employed for GAPDH, RET/PTC1, and RET/PTC3 gene rearrangements are reported in Table 1.

Table 1 Sequences of primers of GAPDH, RET/PTC1, and RET/PTC3 gene rearrangements
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Table 2 shows the primers for BRAF gene exon 15 and for point mutations in codons 12/13 and codon 61 (hot spots) of the H-RAS, K-RAS, and N-RAS genes.

Table 2 Sequences of primers of B-RAF, H-RAS, K-RAS, and N-RAS genes point mutations
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PCR amplifications were performed and gel-controlled in the same way in both rearrangements and point mutations. All PCR products were finally analyzed by capillary electrophoresis.

Cytologic Findings

The cytological examination revealed a peculiar epithelial malignancy displaying a mucinous background with necrosis, histiocytes, and lymphoid cells in different maturation stages. The tumor had both aspects of classic PTC and MEC.

The papillary component showed branching sheets and papillaroid clusters of cuboid cells with the typical nuclear features of PTC (ovoid nuclei with finely granular heterochromatin, occasional micronucleoli, nuclear grooves, and intranuclear cytoplasmic inclusions) (Fig. 1). MEC component was made up by strips, sheets, and loose groups of cubic-shaped cells with finely vacuolated cytoplasms in which the nuclei had basal position. These latter had vesicular chromatin structure with one or two small nucleoli; the cytoplasms tended to a triangular or polygonal shape, with their apical portions forming a continuous luminal border (Fig. 2). Sometimes, small nests of mucinous cells were found to have goblet cell differentiation (Fig. 3). Next to well-differentiated mucinous cells, several groups of neoplastic cells with squamoid differentiation (Fig. 4) or clusters of so-called intermediate cells (Fig. 5) could be observed.

Fig. 1
figure 1

FNC sample. Papillary carcinoma (PTC) component. A branching sheet of thyrocytes with evident nuclear grooves and micronucleoli can be observed in a necrotic background (DQ, ×400, original magnification)

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Fig. 2
figure 2

FNC sample. Mucoepidermoid (MEC) component. Some monolayered sheets of neoplastic cells of cubical shape can be seen. Notice the basal position of nuclei, with well-defined, finely vacuolated cytoplasms often aligned to define a luminal surface (left and right side of the picture). Papanicolaou, ×400, original magnification

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Fig. 3
figure 3

FNC sample. MEC component. A small nest of neoplastic cells with goblet cell formation can be seen. Papanicolaou, ×600, original magnification

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Fig. 4
figure 4

FNC sample. MEC component. A loose cluster of large neoplastic cells (image center) with evident nuclear atypias and heavily orangiophilic (keratinized) cytoplasms can be seen admixed to smaller mucinous neoplastic cells. Papanicolaou, ×400, original magnification

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Fig. 5
figure 5

FNC sample. MEC component. A loose sheet of medium-sized neoplastic epithelial cells show intermediate features between mucinous and squamous differentiation (so-called intermediate cells). Notice the presence of larger, ovoid nuclei with small nucleoli and occasionally folded nuclear membrane, with moderate to ample, well-defined glassy cytoplasms. Papanicolaou, ×600, original magnification

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Immunocytochemical staining showed a marked and diffuse nuclear positivity for TTF1 in both papillary and mucoepidermoid components (Fig. 6) and a membranous positivity for CK19 in the portion morphologically identifiable as classic PTC, while it was only focally expressed in the MEC component (Fig. 7).

Fig. 6
figure 6

FNC sample. PTC component. A branching group of thyrocytes shows diffuse nuclear positivity for TFF-1. Immunoperoxidase, ×600, original magnification

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Fig. 7
figure 7

FNC sample. MEC component. A linear sheet of neoplastic mucinous-type cells shows membranous positivity for CK 19. Notice that the apical part of the cytoplasm does not stain for CK 19. Immunoperoxidase, ×400, original magnification

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Histologic Findings

A peculiar composite malignancy was found, which consisted of a minor component of classic PTC with psammoma bodies and small papillae and a predominant moderately/well-differentiated MEC component, in a context of a severe chronic thyroiditis (Fig. 8). The low-grade MEC focally infiltrated the peri-thyroid fat tissue.

Fig. 8
figure 8

Surgical sample. A composite neoplasm is shown, made up of a classical papillary carcinoma component with a psammomatous calcification (right half of the image) and of a classical low-grade mucoepidermoid carcinoma (lower and left part of the image). H and E, ×600, original magnification

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Immunohistochemistry revealed positivity for TTF1 in both papillary and mucoepidermoid areas, CEA was negative in the papillary areas but positive in the mucoepidermoid ones, hTg stained only the papillary part, and Alcian-Pas staining documented vast lakes of mucin-positive neoplastic cells in the context of the neoplasm (Fig. 9).

Fig. 9
figure 9

Surgical sample. Alcian-Pas staining shows wide mucin pools within the neoplasm (×600, original magnification)

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Molecular Findings

The sequence analysis showed neither mutations in BRAF gene nor point mutations in codons 12, 13, and 61 of H-RAS, K-RAS, and N-RAS genes, but it revealed the simultaneous presence of RET/PTC1 and RET/PTC3 rearrangements (Fig. 10).

Fig. 10
figure 10

The sequences show RET/PTC1 (a) and RET/PTC3 (b) rearrangements

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Discussion

FNC represents the gold standard for the differential diagnosis of thyroid nodules [5]. Not only molecular testing of FNC samples for gene mutations and rearrangements helps to improve the diagnostic accuracy of this technique, but it also allows a better stratification of the high-risk patients for clinical management [6].

A number of combined PTC and MEC carcinomas have already been described in the past [7–13]; nevertheless, the increasing number of these entities has not been enough to grant a definite position for these lesions in the WHO classification. Basically, two sets of hypotheses have been put forward: one is in favor of the origin of these tumors from ultimo-branchial bodies and/or solid cell nests [14, 15] and the other one is more in favor of metaplastic changes in well-differentiated thyroid carcinoma [16–18]. In our case, the papillary component was clearly of follicular cell origin, both immunocytochemically (TTF1 and CK19 positive) and under its molecular biologic profile. PTC is associated with constitutive activation of the RET-RAS-RAF-MAPK pathway, which transduces potent mitogenic and cell survival signals [19, 20]. Pathway activation is usually caused by point mutations in the BRAF or RAS family genes or RET/PTC gene rearrangements. These molecular alterations are mutually exclusive since activation of any single protooncogene confers uncontrolled functioning of downstream effectors [21]. The mutual exclusion also indicates that each of these mutations is independently sufficient to initiate thyroid tumorigenesis [22–24]. Our results showed neither BRAF nor RAS point mutations of the specific genes but a simultaneous presence of RET/PTC1 and RET/PTC3 rearrangements.

Double rearrangements have been already reported in previous studies [25, 26]. This simultaneous expression suggests that the additional variant of RET/PTC can arise as a progression within a single tumor or that papillary thyroid carcinoma can be of oligoclonal origin [27–29]. Although the latter hypothesis is more likely, further studies will be necessary not only to determine the origin and significance of the simultaneous presence of two or more RET/PTC rearrangements but also to clarify the genesis and the subsequent classification of combined tumors.