2.1. Demographic Features

HCC is more often observed in females, and the reported female to male ratio of HCC is 1.6–4.8:1 [4,16,17,18]. The affected age range is 54–62 years old [4,17,19,20,21,22]. In a retrospective study, tumor size, frequencies of extrathyroidal extension, lymph node metastasis, and distant metastasis, were similar between HCC and non-oncocytic follicular thyroid carcinomas [16]. To date, no correlation with radiation exposure has been reported [23].

2.2. Laboratory Tests

Thyroglobulin production varies by case. Both high thyroglobulin production (>500 ng/dL) [24,25,26], and limited production [5], which reflects less active follicular cells, have been reported. An increased thyroglobulin level is a biomarker for recurrence after thyroidectomy; however, some recurrent HCC cases have undetectable thyroglobulin levels. Clinical follow-up by thyroglobulin levels should be decided depending on the case.

2.3. Ultrasounds

Preoperative diagnosis by ultrasound is challenging. In ultrasounds, HCC shows a range of sonographic appearances from predominantly hypoechoic to hyperechoic lesions, and no preoperative features can differentiate HCC from adenoma [18,27]. Several attempts have been made to predict HCC before the operation. Ito et al. classified thyroid nodules into classes 1 to 5 with intermediate steps of 0.5 for classes 2 to 5, called ultrasound classes (USC) [28]. USC2 is defined as having a round and cystic nodule/isoechoic solid nodule—an adenomatous nodule or follicular adenoma. USC3 is defined as a round, hypoechoic solid nodule—a follicular adenoma, adenomatous nodule, or possibly carcinoma. USC4 is a solid nodule with an irregular border or the presence of psammoma calcifications—a carcinoma. Although USC is not related to the incidence of HCC, among patients diagnosed with oncocytic cell tumors by fine-needle aspiration cytology (FNAC), such patients are more likely to show malignancy when USC is 3 or greater.

2.4. Clinical Indicators

Indicators of malignancy among Hürthle cell tumors include male [29], tumor size >4 cm [18,23,26,29], US class ≥ 3 [26], older age (HCC 51.8 years old vs. Hürthle cell adenoma 43.1) [23]. Subsequently, surgical indications of Hürthle cell tumors are reported as USC ≥ 3, tumor size >4 cm, and thyroglobulin >500 ng/dL (with negative anti-thyroglobulin-antibody) [26].

4.1. Cytological Features and Differential Diagnosis

Using fine needle aspiration cytology (FNAC), clusters of the monotonous follicular cells with large eosinophilic, granular cytoplasm are observed, and usually, hypercellularity is noted (Figure 1). The cells often show irregular nuclear outlines, prominent nucleoli, and bland chromatin. The tumor cells appear scarcely accompanied by colloid, and/or inflammation.

Oncocytic cells are also observed in nodular goiter, chronic thyroiditis, and oncocytic variants of papillary thyroid carcinoma and oncocytic variant of medullary thyroid carcinoma (OV-MTC). Therefore, the presence of these cells should not engender a diagnosis of Hürthle cell lesion [5]. In chronic thyroiditis and nodular goiter, isolated or small cohesive clusters of oncocytic cells are common, representing follicular cells with oncocytic metaplasia. In addition, in nodular goiter, follicular cells with abundant colloids are observed, and the follicles are not uniform. In chronic thyroiditis, lymphoplasmacytic infiltrations are frequently observed. However, when a large irregular sheet of oncocytic cells dominantly appear, differentiating between chronic thyroiditis and Hürthle cell tumor may be difficult. Indicators of true Hürthle cell tumors are (1) microfollicular architecture, (2) absence of colloid, (3) absence of inflammation, and (4) presence of transgressing blood vessels [36,37].

The tumor cells of oncocytic variants of papillary thyroid carcinomas show fine granular cytoplasms and elongated nuclei, and a nuclear groove. Intracytoplasmic inclusion can be sparse in some cases. The cytological features of papillary fronds and monolayered sheets suggest an oncocytic variant of papillary thyroid carcinoma rather than Hürthle cell tumors [38,39]. In the FNAC sample of the OV-MTC, amyloid deposit supports the diagnosis, but is not always observed [40]. OV-MTC cells show polygonal, plasmacytoid characteristics with eccentrically placed round nuclei with “salt and pepper” chromatin [41]. Multi-vacuolization resembling histiocytes and loose granularity is observed in OV-MTC. In contrast, granularity in HCC is dense and firm [42]. For accurate diagnosis, immunohistochemistry using calcitonin and thyroglobulin in combination [43], as well as consideration of family history of the patient and laboratory data are needed.

Cytologic features such as small cell dysplasia (bland nuclei, cell diameter less than twice the nuclear diameter), large cell dysplasia (cell diameter at least twice the nuclear diameter, with prominent nucleoli and irregular nuclear outlines), nuclear crowding, and dyshesion have been proposed as a diagnostic clue for HCC [44]. These criteria were later shown by others to be significant features in favor of HCC over adenoma and other benign lesions [45]. However, distinction of HCC from Hürthle cell adenoma by FNAC still remains to be challenging due to the lack of other supporting data [46]. In the daily practice using Bethesda reporting system for thyroid cytology (TBSRTC) [47], a Hürthle cell neoplasm is typically classified as atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS, Bethesda III) or benign (Bethesda II). The appearance of Hürthle cell is encouraged to comment. The presence of Hürthle cells itself does not increase the risk of malignancy [48], but > 75% Hürthle cells in a benign/Bethesda II aspirate may pose an increased risk of malignancy [49].

4.2. Macroscopic Features

Macroscopically, HCC shows a mahogany brown appearance (Figure 2), which is due to air contact [25]. Necrosis and hemorrhaging may be observed due to its degenerative nature. Sometimes calcification is observed and is more often found in HCC than in Hürthle cell adenoma. Calcification is a result of a physiochemical reaction to altered thyroglobulin within the colloid of the tumor follicles.

Figure 2. Gross findings of the HCC. (A) The tumor is mahogany brown color. Several foci of hemorrhage are noted. (B) Widely invasive HCC. Macroscopically, capsular invasion is evident (arrows). Scale bar indicates 2 cm.

4.3. Histological Features and Differential Diagnosis at Histology

In cases where the Hürthle cell tumor showed capsular invasion and/or vascular invasion, HCC is diagnosed (Figure 2B and Figure 3A,B). Based on its histology, the tumor is subcategorized as either minimally invasive or widely invasive. Minimally invasive HCC refers to encapsulated tumors with microscopically identifiable foci with capsular or vascular invasion less than four foci. Widely invasive HCC shows extensive capsular invasion and/or vascular invasion of four foci or greater.

HCC often causes infarction after FNAC. This may make it difficult to observe the capsular invasions. Mitochondrial DNA common deletion is reported to be less frequent in infarcted Hürthle cell tumors than in non-infarcted Hürthle cell tumors. Still, the mechanism leading to infarction is unknown [50]. For clear identification of capsular invasion, clinical information such as where the tumor cells were aspirated by FNAC is considered important. Widely invasive HCC often shows capsular invasion in the form of compacted nodules, lacking stromal desmoplastic reaction. In some cases, determining whether there is a capsule is difficult.

HCC is composed of pleomorphic or polygonal large cells with abundant, granular, and acidophilic cytoplasm and large nuclei with prominent nucleoli (Figure 3C) [51]. It often shows follicular, trabecular, and/or solid architecture, and rarely shows a predominantly papillary pattern [25]. A pure follicular pattern is less common in HCC [1], but when such a follicular pattern is seen, the tumor accompanies fibrous bands between nests.

Cytoplasmic granularity in Hürthle cells is due to the presence of numerous mitochondria (Figure 3D). The amount and morphological characteristics of the mitochondria vary greatly from case to case [52]. However, according to the diagnostic criteria, more than 75% of oxyphilic cells must be observed for diagnosis. The high prevalence of Hürthle cell changing into thyroid lesions may reflect high oxidative stress and reactive oxygen species production in thyroid cells during normal iodine and thyroid hormone metabolism [53,54]. These high reactive oxygen species levels can result in mutagenic genetic events in mitochondrial DNA, leading to mitochondrial dysfunction [7,55]. Abnormality of mitochondria leads to defects in the energy production process, and a compensatory mechanism for these defects is considered to come into play [7]. This results in increased proliferation of numerous mitochondria, which is observed as oncocytic cytoplasm. Over time, as the number of the mitochondria increases, Hürthle cell appearance is expressed [51], and this is a continuous process rather than a “black-and white phenomenon” [7]. In cases of HCC, necrosis frequently appears either spontaneously or after fine-needle aspiration. This observation is reported to be as a consequence of mitochondrial abnormalities [56].

Thyroid lesions containing oncocytic cells are nodular goiter, oncocytic variants of papillary carcinoma and medullary carcinoma. Oncocytic variants of medullary thyroid carcinoma (MTC) are rare, showing extensive mitochondrial hyperplasia, prominent nucleoli, well-defined cytoplasm, and polygonal cells, which is in contrast with the spindle-shaped cells of conventional MTC. Although MTC is derived from C cells (parafollicular cells), not follicular cells, oncocytic variant MTC may be difficult to histologically differentiate. MTC is often accompanied by amyloid deposits, and this aids in diagnosis. However, amyloid deposits sometimes occur as a result of systemic or secondary amyloidosis [57]. HCC with neuroendocrine differentiation [58] or thyroid tumors in patients with basal high calcitonin levels should be diagnosed with caution. For the appropriate diagnosis, consideration of molecular testing, as well as family history and serum calcitonin levels, is essential [59]. Oncocytic parathyroid adenoma and adenocarcinoma also require differential diagnosis. Neoplastic lesions showing extensive invasion without hormone production are especially difficult to differentiate. Immunohistochemically, parathyroid tissue tests positive for chromogranin A, GATA3, while thyroid tissue tests positive for TTF-1, PAX8. However, in the hormone non-producing tumors, neither calcitonin nor PTH is clearly expressed. Tumor location with images and invasive patterns can be diagnostic clues in these cases.

Immunohistochemistry is not mandatory for the diagnosis of HCC, except in cases to distinguish MTC or parathyroid tumors, and a specific diagnostic marker for HCC has not yet been obtained. HCC cells test positive for thyroid transcription factor-1 (TTF-1) and thyroglobulin, and negative for calcitonin and parathyroid hormone (PTH). Thyroglobulin may show perinuclear positivity. According to the literature, genes associated with Retinoid-interferon-induced Mortality-19 (GRIM-19) [60], p53 [56], and Cyclin D [61,62] can be immunohistochemically detected.

4.4. Ultrastructure

Using transmission electron microscopy, HCC cells are observed to have large, irregular nuclei with a deep notch [63]. Nuclei contain one to several nucleoli, and the cytoplasm is packed with several thousand mitochondria (Figure 4). A full-brown oncocytic cell is estimated to contain 4000 to 5000 mitochondria [64]. These mitochondria display two types of morphology. One is larger mitochondria with stacking “shelf-like” cristae [63,65]. The other is smaller and oval, and short cristae are peripherally arranged [63]. Other structural abnormalities are bundles of dense filaments, filamentous inclusions, and round electron-dense bodies in the matrix [5,51,66]. The amount and morphological characteristics of the mitochondria vary greatly from case to case [51,52]. The surface of HCC cells is irregular.

Figure 4. Electron microscopic images of HCC. (A) An HCC cell contains an irregular and notched nucleus. Cytoplasm is filled with numerous mitochondria. The bar indicates 2.0 micrometers. (B) Mitochondria display packed stacking cristae, arranged in the center. The bar indicates 200.0 nanometers.