Thymoma, a neoplasm of the thymic epithelium, is the most common primary neoplasm of the anterior mediastinum in adults, representing approximately 50% of tumors occurring at the site. However, thymomas are rare neoplasms [7]. The tumor can present with several morphologic variations and histological types which are a challenge for classification and diagnosis. The accuracy of aspiration biopsy and small biopsy for diagnosis of thymoma ranges from 80 to 100%, in several series [1–6]. The hallmark for the diagnosis of thymoma in a small biopsy sample is the presence of two cell populations, one composed of bland epithelial cells and the other of lymphocytes [8]. However, this classical feature may not be present in a large number of cases due to the variation of histological presentation seen in these tumors. Each histological type of thymoma elicits a different cytological pattern (Table 4.2) and therefore a different set of differential diagnosis.

In this chapter, we will use the 2015 WHO classification of thymic epithelial tumors in an attempt to correlate the cytological features with the histological classification. Briefly, in the WHO classification [9, 10], thymomas are classified as type A, which is composed predominantly of spindle-shaped cells (spindle-shaped pattern) and type B, composed of a mixture of bland epithelial cells and lymphocytes. Type B thymomas are subdivided into B1, B2, and B3, depending on the ratio of epithelial cells to lymphocytes. B1 thymomas are rich in thymic lymphocytes (lymphocyte-rich pattern) and B3 thymomas are predominantly composed of epithelial cell (epithelioid pattern, with or without lymphocytes) (Table 4.3). In addition, type B thymomas, often present with a combinations of different sub types in a spectrum of B thymoma within the same tumor mass (e.g., predominant type B2 with areas of B1). Another common combination is Type AB thymoma which is composed of a mixture of types A and B. Therefore, it may be very difficult to accurately classify a type B thymoma in small biopsy and cytological specimen due to sampling bias. In addition, there have been no studies that correlate histological classification with predictive response to therapy. Histologic subtype is not currently relevant for treatment and patient management. Therefore an accurate subclassification of thymomas in cytology is not desirable, whereas an accurate diagnosis of thymoma in conjunction with clinical and radiographic staging of the disease is important for patient management [10–12].

One of the most important pitfalls for the diagnosis of thymoma occurs when the tumor is not considered in the differential diagnosis of a biopsy of the anterior mediastinum. Similarly, thymoma should enter the differential diagnosis of FNAB of any site, especially pleural-based lesion, if the presence or history of an anterior mediastinal mass is noted. In fact, FNAB and small biopsies are often used in the diagnosis of metastatic site, whereas the primary diagnosis of thymoma is often made by surgical resection.

Pattern recognition approach: Spindle-shaped cells

The differential diagnosis for this group includes: spindle cell (type A) thymoma, spindle cell carcinoid tumor, sarcoma (primary or metastatic), and a spindle cell component of germ cell tumor.

This type of thymoma can occur at any age group, with a peak around the sixth to seventh decade of life. This tumor is often seen in a low clinical stage, most are confined to the thymus, although pleural implants and distance metastasis can occur [13, 14]. An aspiration biopsy of a Type A thymoma shows cohesive clusters of spindle-shaped cells arranged in bundles and large sheets. The cells have elongated nuclei and some of the nuclei may be wavy. Cytoplasm is evident but the cell border is poorly defined (Fig. 4.1). Lymphocytes can also be seen in the background or permeating the clusters of spindle-shaped cells. In a majority of cases, the lymphocytes are not prominent but can be seen within the epithelial cell clusters [3, 15]. Cell block preparations are helpful in identifying these features. The nuclei of the spindle-shaped cells are bland with clear, vesicular or evenly dispersed chromatin, which can suggest a neuroendocrine tumor (Fig. 4.2) [16].

Type A thymomas have a variety of histological patterns, which can lead to misdiagnosis, including a cylindromatous pattern mimicking adenoid cystic carcinoma [17]. The elongated nuclei and tight cell clusters may resemble a low-grade soft tissue tumor, such as solitary fibrous tumor. The diagnosis can be made with the use of ancillary test such as immunocytochemical stains. In thymomas, the epithelial nature of the spindle-shaped cells is revealed by a positive staining pattern for pan-keratin, and p63/p40, however, when present, the characteristic immunophenotype of the thymic T-cells can help in asserting the correct diagnosis. The thymic T-cells are positive for CD3, CD5, CD99, CD1a, and TdT. Therefore, the presence of these markers on the lymphocytes is suggestive of a thymic origin.

Cytological features

Spindle cell carcinoid is a rare neoplasm at this site, but nevertheless can be present in the mediastinum. The cells have the characteristic neuroendocrine morphology of “salt and pepper” chromatin distribution of tumor nuclei. The cell clusters may have similar arrangement to the one described for a spindle cell thymoma, but in carcinoid tumors, the cell clusters tend to be loosely cohesive whereas in thymomas the tumor cells form tight clusters. The presence of apoptotic bodies in carcinoid tumors may be confused with infiltrating lymphocytes thus mimicking a thymoma. However, immunocytochemical stains can help differentiate the two entities. In carcinoid tumor, the cells are positive for chromogranin, synaptophysin or other neuroendocrine markers with no predominant immature T-cell population. A positive labeling for neuroendocrine markers in the absence of immature T-cells points towards a diagnosis of a spindle cell carcinoid tumor.

Cytological features:

Schwannoma and malignant peripheral nerve sheath tumor are more commonly seen in the posterior mediastinum. In these cases, the presence of palisading spindle cells with wavy nuclei is suggestive of neural differentiation. The appropriate immunohistochemical stains guide the pathologist into the correct diagnosis. Tumor cells in these cases are positive for S-100 protein and negative for keratin. Solitary Fibrous Tumor (SFT) can be confused with Thymoma WHO type A. The tumors cells are bland and present as tight clusters. However, SFT are positive for CD34, BCL-2, and STAT-6, whereas thymomas are positive for keratins.

If the spindle-shaped cells mark for smooth muscle or skeletal muscle, a sarcoma with muscle differentiation or a component of a germ cell tumor enters the differential diagnosis. In these cases, careful observation of the cell type and characteristics of the cell are helpful features in pointing towards the correct diagnosis. Cells with malignant characteristics guide the pathologist toward the diagnosis of a sarcoma. Metastatic sarcomas are more commonly seen in the prevascular and visceral mediastinum, primary sarcomas of these sites are extremely rare.

Cells with a benign smooth muscle differentiation occurring in a cystic lesion in a young patient point towards a benign component of a mature teratoma. In addition, correlation with radiographic images is helpful in reaching the accurate diagnosis [18].

The differential diagnosis for this group includes: thymoma WHO type B, thymic carcinoma, carcinoid tumors, germ cell tumor, including seminoma, and metastatic carcinoma. Epithelioid lesions of the mediastinum can be further subdivided in two categories: those that contain lymphocytes and those without lymphocytes. The presence of lymphocytes in an epithelioid lesion of the mediastinum raises the possibility of a thymoma WHO type B, metastatic carcinoma to a lymph node or thymus and seminoma. Epithelioid pattern without lymphocytes, include thymic carcinoma, other germ cell tumor including yolk sac tumor and embryonal carcinoma, and metastatic carcinoma.

As discussed earlier, it is very difficult to accurately subclassify type B thymomas on a small biopsy or in an FNAB. Thymomas types B2 and B3 fall in the group of “predominantly epithelioid cells with lymphocytes”. The aspiration biopsy of a B2 or B3 thymoma is usually moderately to highly cellular, with a characteristic dual population of neoplastic epithelial cells and small lymphocytes (Fig. 4.3). The epithelial cells are polygonal shaped, mostly arranged in cohesive tridimensional clusters or in flat sheets (Fig. 4.4). The cells have regular nuclear contours with open or vesicular chromatin and delicate nucleolus. In other cases, the nucleoli can be prominent [8, 19]. The cytoplasm is delicate and the epithelial clusters may have interspersed lymphocytes (Fig. 4.5). The number of lymphocytes may vary, depending on the preparation and sampling of the tumor. Immunohistochemical stains are helpful to identify the origin of infiltrating lymphocytes. The epithelial cells are positive for pan-keratin and p63/p40. The lymphocytes have the characteristic immunophenotype of immature thymic lymphocytes.

There have been reports that thymic epithelial tumor can be positive for PAX-8 [20], however, the positivity for this marker in thymomas is highly dependent on the clone of antibody, whether it is a polyclonal or monoclonal antibody [21, 22].

It can be very difficult to differentiate a thymoma type B3 from a thymic carcinoma on FNAB or even core biopsy if necrosis and cytological atypia is encountered. Pleomorphic nuclei can be seen in thymoma, thus resembling a poorly differentiated carcinoma (Fig. 4.6) [23]. However, most thymomas will retain an open or vesicular chromatin pattern, which can be differentiated from the hyperchromatic nuclei with dense chromatin pattern commonly seen in thymic carcinomas. When a predominantly epithelial neoplasm with cellular atypia is encountered, it is advisable to suggest these two entities in the differential diagnosis. Immunohistochemical studies can also be helpful in this differential diagnosis. The epithelial cells of a thymic carcinoma are positive for CD5 and CD117 (KIT) in approximately 40–60% of the cases [24–26]. Therefore, if the stains are positive, a diagnosis of thymic carcinoma can be suggested, but the stains are not useful if negative, since thymic carcinoma cannot be fully excluded. When lymphocytes are present, thymic lymphocytes (CD1a, TdT, and CD99-positive) are suggestive of a thymoma and not thymic carcinoma.

The differentiation between thymic carcinoma and thymoma is very important and clinically relevant. Most thymomas type B3 and thymic carcinomas present in advanced stage, where induction chemotherapy is used before resection, if the tumor is surgically resectable [27]. Different regimens of chemotherapy may be used depending on the diagnosis [28].

Another differential diagnosis of a B2 or B3 thymoma is a metastatic carcinoma to a lymph node and germ cell tumor such as a seminoma. Age and clinical presentation are very helpful in the interpretation of these biopsies as well as ancillary studies.

Type AB thymoma and other rarer types of thymomas such as micronodular thymoma with lymphoid stroma or metaplastic thymoma may be very difficult to classify correctly in a small biopsy or aspiration biopsy material. Similar to the more common types, the diagnosis of a thymoma can be made by the presence of a dual population of cells, namely bland epithelial clusters and thymic T-cells.

Cytological features

Thymic carcinoma is characterized by clearly malignant features of the epithelial cell component. Contrary to thymomas, the characteristic T-lymphocytic infiltrate is often absent. Thymic carcinomas can be present with several different histological types. The WHO has classified primary thymic carcinoma, into several histologic subtypes including neuroendocrine carcinomas. Squamous cell carcinoma is the most common type of thymic carcinoma, but it is morphologically indistinguishable from squamous cell carcinomas from other sites, therefore, clinical, radiographic, and immune profile of the tumor must be evaluated before ascribing the carcinoma as being of thymic origin.

Thymic carcinomas are seen in the group of “predominant epithelioid pattern without lymphocytes”. Cytology preparations of these carcinomas are similar to their more common counterparts, which arise in the lung and elsewhere. In general, the malignant cells have enlarged nuclei, coarse chromatin, discrete macronucleoli, and a moderate amount of cytoplasm (Fig. 4.7). Necrosis is frequently seen.

Lymphoepithelioma-like carcinoma of the thymus has been described in the cytology literature, in which the smears show tight clusters of large cells, surrounded and focally infiltrated by small mature lymphocytes. These cells show large rounded often overlapping nuclei. Nucleoli are easily seen, and in some cells are markedly enlarged. Lymphoepithelioma-like carcinoma can be particular difficult to be diagnosed in cytology preparation due the amount of lymphocytes present, thus mimicking the cytomorphological features of a thymoma type B.

Other histological types of thymic carcinoma such as adenocarcinomas, mucoepidermoid carcinomas, basaloid carcinomas, clear cell carcinomas, sarcomatoid carcinomas, and even neuroendocrine carcinomas follow the same cytological features of these tumors elsewhere [29–34].

Thymic squamous cell carcinomas are positive for keratin, p63/p40, similar to thymomas, and can be positive for CD5 and KIT (CD117). However, immature thymic T-cells should be absent in thymic carcinomas. Lymphoepithelioma-like carcinoma can be positive for EBV antigen, similar to other organs.

Cytological features

Neuroendocrine tumors of the thymus are very rare and can be classified, as typical or atypical carcinoid tumors, similar to the classification used for pulmonary carcinoid tumors. Atypical carcinoid is the most frequently encountered neuroendocrine tumor of the thymus. Atypical carcinoids of the thymus are more often seen in males in the age group of the fourth and fifth decade of life. The cytological criteria for carcinoid tumors of the thymus are similar to the criteria established elsewhere for these tumors [16, 35, 36]. Typically, aspiration biopsy smears show isolated round to plasmacytoid cells with typical salt-and-pepper chromatin pattern (Fig. 4.8). Additionally, small cohesive clusters of neoplastic cells with fine granular chromatin can be identified. Numerous apoptotic cells can be present on the smears, mimicking lymphocytes; therefore the differential diagnosis includes thymoma. Carcinoid tumors of the thymus are often misclassified as thymoma, due to failure to consider this diagnosis or lack of familiarity with this entity occurring in the thymus [16]. The differentiation between typical carcinoid and atypical carcinoid is difficult to make in a small biopsy sample similar to other sites of origin. However, the presence of necrosis or mitotic figures on the smears may suggest the diagnosis of atypical carcinoid. Other authors have suggested the use of proliferation markers such as Ki-67 (MIB-1) as a diagnostic tool to differentiate between low-grade carcinoid tumors, including an atypical carcinoid from a high-grade neuroendocrine neoplasm [35–38]. However, Ki-67 is not a reliable marker to separate typical from atypical carcinoid tumors. The diagnosis of high-grade neuroendocrine carcinomas (large cell neuroendocrine carcinomas and small cell carcinomas) follows the criteria for the diagnosis of these entities in the lung. It might be difficult to distinguish a high-grade neuroendocrine tumor of the thymus from metastases from a primary lung neoplasm.

Cytomorphologic features

Germ cell tumor of the mediastinum with seminoma, embryonal carcinoma, yolk sac tumor, and choriocarcinoma are also part of the group “epithelioid-rich pattern”. These tumors are present as high-grade malignant epithelioid neoplasm. As often in the case of germ cell tumors, serum markers and the young age of the patient are helpful clinical clues in establishing the diagnosis.

Aspiration biopsy of a seminoma can be moderately to highly cellular. The neoplastic cells are large and can be dispersed singly or in small flat aggregates. The seminoma cells have delicate cytoplasm and the nuclei have reticular to open chromatin with large prominent nucleolus (Fig. 4.9). When intact cells are present, they are described as having a “fried egg” appearance (Fig. 4.10). Due to the fragility of the cytoplasm, a so-called “tigroid” background is seen on the cellular smears. Although not pathognomonic, the presence of tigroid background, naked nuclei with prominent nucleoli, and lymphocytes are good criteria for the diagnosis of seminoma. In paucicellular smears, a tigroid background is often absent [39–42]. Poorly formed granuloma can be seen on smears in association with neoplastic cells. The presence of granuloma with a lymphocytic background and paucity of neoplastic cells may lead to misdiagnosis of the entity.

Similar features are described in core biopsies when large atypical epithelioid cells interspersed with lymphocytes are present. Knowledge of age of the patient, radiographic appearance of the lesion, and serum markers is helpful in establishing the diagnosis of germ cell tumors. Pure mediastinal seminomas are often associated with elevated serum levels of lactate dehydrogenase (LDH) and low levels of β-human chorionic gonadotropin (β-HCG); these findings however are not specific and can be seen in association with other germ cell tumor components. The presence of high serum levels of α-fetoprotein (AFP) and β-HCG raises a possibility of a mixed germ cell tumor, where the seminoma being the only sampled component.

Immunohistochemical studies show that seminoma cells are positive for OCT-4 and SALL4, both nuclear stains. The tumor cells are also positive for KIT (CD117), D2-40 and may show reactivity for keratin, often in a dot-like pattern [43].

Cytological features

Pure embryonal carcinoma is extremely rare. It is more commonly seen as a component of mixed germ cell tumor. Similar to seminoma, this is a tumor that occurs mostly exclusively in young men centered in the third and fourth decade of life. There is no specific serum marker associated with this tumor, like seminoma; there might be elevated serum levels of LDH and β-HCG.

The cytological features of embryonal carcinoma are that of a high-grade, poorly differentiated malignant carcinoma. The malignant cells are large, pleomorphic, and show dense hyperchromatic chromatin pattern. Nucleoli are prominent. The tumor cells are arranged in small tridimensional clusters often with the formation of papillary or acini structures (Fig. 4.11) [41, 44]. The correct diagnosis can be reached with immunohistochemical studies. Embryonal carcinoma cells are reactive to pan-germ cell tumor markers such as SALL4, as well as OCT-4. In addition, the tumor cells are positive for CD30 and keratin. Focal positivity for AFP can be present.

Cytological features

Yolk sac tumor of the mediastinum occurs almost exclusively in young men. This tumor has a high association with elevated serum levels of AFP, an important diagnostic clue. Pure Yolk sac tumors are rare, and similar to other germ cell tumors, they are more commonly seen as a histological component of mixed germ cell tumor.

Similar to embryonal carcinoma, the cytological features of yolk sac tumor are that of a high grade, poorly differentiated carcinoma. The cells are large, pleomorphic, and have prominent nucleoli with coarse chromatin pattern. Necrosis is often seen in the background. A helpful feature for the diagnosis of this tumor is the presence of intracytoplasmic or extracellular dense matrix (Fig. 4.12) best seen in modified Giemsa stains, where the dense matrix becomes metachromatic and identified as bright pink globules [41–46].