Approach to mediastinal cytopathology: a diagnostic overview

Introduction

The mediastinum is a complex anatomic compartment that harbors a diverse array of tissues, including thymic, lymphoid, germ cell, and mesenchymal elements. Because of this heterogeneity, mediastinal lesions present a broad diagnostic spectrum, ranging from benign cysts and inflammatory processes to primary and secondary malignancies. Establishing an accurate diagnosis is critical, as it directly influences clinical management strategies, which may range from surgical resection to systemic therapy.

Fine-needle aspiration (FNA) has become an essential, minimally invasive tool for evaluating mediastinal masses and lymphadenopathy. With the advent of endobronchial and endoscopic ultrasound-guided techniques, cytologic sampling of deep mediastinal structures can now be performed more safely and less invasively. These advances have markedly reduced the need for open surgical biopsies and have positioned cytology at the forefront of mediastinal diagnostics (1).

However, the cytopathologic evaluation of mediastinal specimens remains challenging due to overlapping morphologic features among various entities, limited material for ancillary testing, and the need for careful correlation with clinical and radiologic findings. Reactive lymphoid hyperplasia, granulomatous inflammation, thymic epithelial neoplasms, lymphomas, germ cell tumors (GCTs), and metastatic carcinomas can all mimic one another cytologically, requiring pathologists to integrate morphologic detail with immunocytochemical, flow cytometric, and molecular data for accurate classification.

This review provides a comprehensive overview of the cytopathology of mediastinal lesions. It highlights key cytomorphologic patterns, diagnostic pitfalls, and the expanding role of ancillary studies in establishing definitive diagnoses. Emphasis is placed on a practical, pattern-based approach that reflects current diagnostic workflows and incorporates recent developments in imaging-guided sampling, immunodiagnostics, and molecular testing. By synthesizing current evidence and expert practice, this review aims to serve as a resource for cytopathologists, residents, and clinicians involved in the diagnosis and management of mediastinal diseases.

Sampling techniques and specimen preparation

Accurate cytologic evaluation of mediastinal lesions depends on the adequacy and preservation of diagnostic material. Several minimally invasive procedures are available for sampling the mediastinum, each with distinct advantages depending on the anatomic location of the lesion, available expertise, and clinical context.

Sampling techniques

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has revolutionized mediastinal cytology. Using real-time ultrasound guidance, FNA can be performed through the bronchial wall to target paratracheal, subcarinal, and hilar lymph nodes with high precision. EBUS-TBNA provides excellent diagnostic yield for both benign and malignant lesions, and it is now considered the first-line approach for evaluating mediastinal lymphadenopathy, particularly in lung cancer staging and diagnosis of granulomatous diseases (2-4). Overall sensitivity and specificity of EBUS-TBNA in differentiating benign versus malignant lesions of the mediastinum or hilum are very high, at 95% and 100%, respectively (5). However, compared to traditional carcinomas, sensitivities for other lesion types are much lower: 93% for sarcoidosis, 75% for tuberculous mediastinal lymphadenitis, and 66% for lymphoproliferative lesions (5-9).

Endoscopic ultrasound-guided FNA (EUS-FNA) complements EBUS by providing access to posterior mediastinal and subaortic regions through the esophageal wall (10). Together, EBUS and EUS enable comprehensive sampling of virtually all mediastinal compartments while minimizing patient morbidity. The combined “medical mediastinoscopy” approach has largely replaced traditional surgical mediastinoscopy in many institutions (4,11).

Percutaneous computed tomography (CT)-guided FNA remains valuable for lesions located adjacent to the chest wall or for masses not accessible via endoscopy. This method allows for precise localization under imaging guidance and is particularly useful for large anterior mediastinal tumors such as thymic or germ cell neoplasms. However, CT-guided FNA carries a significantly higher risk of complications (8–30%), such as pneumothorax, pulmonary hemorrhage, or hemoptysis, compared to endoscopic techniques (1–2%) (11-13). Compared to surgical specimens, FNA cytology of the mediastinum has been found to have an overall sensitivity of 78% and a specificity of 98% for establishing a definitive diagnosis. Sensitivity varies markedly based on the type of lesion, with sensitivity of 33% for inflammatory lesions, 25% for benign cysts, 93% for metastatic carcinoma, 84% for lymphoma, 100% for GCTs, 100% for soft tissue tumors, and 87% for thymomas (1,14).

In touch imprint cytology, a minute fragment of tissue (i.e., core biopsy) is lightly “touched” to the slide, scattering cellular material over the slide without disrupting the integrity of the specimen. Advantages of touch preparations include their simplicity and reliability, rapid determination of specimen adequacy, and potential for diagnostic classification. However, artifacts such as thick three-dimensional clusters, necrosis, or abundant blood may obscure cellular morphology. Additionally, interpretation is complex and requires considerable training and experience on the part of the cytologist or cytopathologist. It is important to note that touch imprint cytology is primarily meant to complement frozen section analysis; it should not be used as the sole diagnostic technique (15).

Surgical sampling, including mediastinoscopy or video-assisted thoracoscopic surgery (VATS)-guided biopsy, is now reserved primarily for cases where cytologic methods fail to yield a definitive diagnosis. Even with surgical sampling, cytologic assessment remains an integral part of intraoperative consultations and frozen section correlation.

Specimen handling and preparation

The quality of cytologic interpretation is highly dependent on proper specimen processing. Aspirated material should be triaged appropriately for direct smears, liquid-based cytology, and cell block preparation. Air-dried smears stained with Diff-Quik or Giemsa allow rapid evaluation of cellular detail and background, while alcohol-fixed smears stained with Papanicolaou highlight nuclear morphology and chromatin features. Cell block preparations are essential for performing immunocytochemistry (ICC), special stains, and molecular assays (16).

Rapid on-site evaluation (ROSE) by a cytopathologist or trained cytologist plays a critical role in ensuring specimen adequacy and optimizing triage. ROSE enables immediate feedback to the proceduralist regarding cellularity, diagnostic material, and the need for additional passes, particularly for ancillary studies such as flow cytometry or molecular testing (3). In many cases, adequacy of a surgical specimen can quickly be determined using touch imprint cytology (15).

In cases where lymphoma or metastatic carcinoma is suspected, sample triage becomes especially important. A portion of the aspirate should be placed in Roswell Park Memorial Institute (RPMI) medium for flow cytometry, while the remainder can be processed for smears and cell blocks. Cytology material from EBUS-TBNA and EUS-FNA has been shown to be suitable for a broad range of molecular assays, including mutation analysis and fluorescence in situ hybridization (FISH) (16).

Normal and reactive findings

Accurate interpretation of mediastinal cytology requires familiarity with the spectrum of normal and reactive cellular elements that may be encountered in FNA specimens. Recognition of these patterns helps prevent overdiagnosis of benign processes as malignancy and ensures appropriate clinical management. The background of mediastinal aspirates may contain thymic tissue, lymphoid elements, or inflammatory debris, depending on the anatomic site and patient’s clinical condition.

Thymic elements

The normal thymus, particularly in younger patients, may be sampled inadvertently during aspirations of anterior mediastinal lesions. Cytologic smears typically reveal a heterogeneous lymphoid population consisting of small, mature lymphocytes admixed with scattered epithelial cells; Hassall’s corpuscles may rarely be seen. The epithelial component appears as cohesive clusters or single spindle to polygonal cells with oval nuclei, fine chromatin, and inconspicuous nucleoli. Recognition of this “thymic microenvironment” is crucial to avoid misinterpretation as thymoma or lymphoma.

In adults, thymic involution leads to predominance of adipose tissue and sparse epithelial elements, but residual thymic cells may still be observed. In some cases, thymic hyperplasia associated with autoimmune disorders (e.g., myasthenia gravis) can yield abundant lymphocytes and epithelial clusters that mimic neoplasia. Correlation with clinical history and radiologic imaging is essential.

Ectopic thyroid and parathyroid

Rarely, image-guided FNA of the mediastinum reveals ectopic thyroid or parathyroid tissue. The presence of these normal tissues in an abnormal location reflects a defect in early thyroid or parathyroid embryogenesis. These “lesions” appear benign due to their bland cytology and occasional fragments with a “honeycomb” architecture; see Figure 1. Immunocytochemical stains such as thyroglobulin, TTF-1, and parathyroid hormone (PTH) can be used to support thyroid or parathyroid origin. Parathyroid tissue is usually positive for neuroendocrine markers such as synaptophysin and chromogranin, and it exhibits nuclear GATA-3 positivity as well (17,18).

Figure 1 Ectopic parathyroid. (A) An FNA biopsy of ectopic parathyroid tissue shows cytologically bland cells with a moderate amount of cytoplasm, round to oval nuclei, and fine chromatin arranged in tight clusters (Diff-Quik, 20×). (B) The cell block has cells in a similar arrangement, with more apparent vacuolated cytoplasm (hematoxylin and eosin, 20×). FNA, fine-needle aspiration.

Reactive lymphoid hyperplasia

Reactive lymphoid hyperplasia represents one of the most common benign findings in mediastinal cytology. Smears are moderately to highly cellular, and they contain a polymorphous population of lymphoid cells including small mature lymphocytes, centroblasts, plasma cells, and tingible body macrophages. The presence of lymphoglandular bodies in the background supports a benign reactive process (19).

Importantly, cytologic polymorphism and the absence of monomorphic large atypical cells help differentiate reactive hyperplasia from lymphoma. However, certain low-grade lymphomas may closely resemble reactive proliferations; thus, cell block preparation and flow cytometric immunophenotyping are often critical for distinguishing between lymphoid hyperplasia and low-grade lymphomas (17).

Benign cysts

Benign mediastinal cysts are a heterogeneous group of lesions that are frequently encountered in cytopathology practice. Cystic masses make up 15% to 30% of all mediastinal masses. Overall, two-thirds of all mediastinal lesions are benign, and they are usually discovered incidentally during imaging. Mediastinal cysts can be congenital or acquired; most congenital cysts are unilocular, while neoplastic cysts are more likely to be multilocular.

FNA of these cysts commonly yields cystic contents composed of proteinaceous debris and macrophages, as shown in Figure 2. A definitive diagnosis generally requires the presence of cyst lining epithelium or associated tissue components. If an FNA completely drains a non-neoplastic simple cyst, it can be curative without recurrence. Recurrence of a cyst after complete aspiration, however, may suggest a neoplastic process.

Figure 2 Cystic contents commonly consist of proteinaceous debris and scattered macrophages. Hemosiderin may be present within the macrophages (Diff-Quik, 10×).

The location of the cyst is critical for narrowing the differential diagnosis, as different components are prone to the development of different types of cysts. The most common epithelial-lined cysts and their associated compartments are shown in Table 1. Additionally, distinguishing benign cysts from cystic thymomas or other cystic neoplastic lesions requires correlating FNA results with clinical history and imaging characteristics (17,20-22).

Diagnostic pitfall: cystic changes within thymic cysts, bronchogenic cysts, or cystic metastases can yield fluid containing degenerated epithelial cells (e.g., squamous, mucinous columnar, ciliated columnar, or mesothelial cells), macrophages, and inflammatory debris, potentially leading to misinterpretation as a necrotic neoplasm. Similarly, inflammatory pseudotumors and granulomatous lesions may be mistaken for malignancy if only atypical epithelioid histiocytes or spindle cells are sampled. Correlation with radiologic features (e.g., well-circumscribed cystic lesion without solid components) and repeat aspiration may be necessary in such indeterminate cases.

Granulomatous inflammation

Granulomatous inflammation of the mediastinum is frequently encountered in association with sarcoidosis, tuberculosis, fungal infections, or foreign-body reactions. Cytologic smears show tight clusters of epithelioid histiocytes, often with multinucleated giant cells (Langerhans or foreign-body type) and a background of small lymphocytes. In sarcoidosis, the granulomas tend to be well-formed, non-necrotizing, and accompanied by a clean background. In contrast, necrotizing granulomas with granular necrotic debris suggest an infectious etiology (19,20,23).

Special stains such as Ziehl-Neelsen, Fite, or Grocott methenamine silver may be performed on cell block sections to identify acid-fast bacilli or fungal organisms. While granulomatous inflammation is a benign finding, its cytologic appearance may occasionally mimic metastatic carcinoma or thymic epithelial neoplasia, especially when granulomas are poorly formed or necrotic (2,4). Additionally, while granulomas themselves are non-neoplastic, they may be found in association with malignant lesions, including Hodgkin lymphoma or seminoma (19,20,23).

Other reactive and inflammatory conditions

Additional benign processes include reactive mesothelial proliferations and post-treatment changes. Mesothelial cells, if aspirated from cystic or pleural-associated lesions, appear as cohesive clusters with prominent nucleoli and peripheral cytoplasmic projections (the classic “lacy skirt”). These should be distinguished from adenocarcinoma cells by their uniform nuclei and two-tone cytoplasm on Papanicolaou stain. Reactive mesothelial cells may exhibit atypical nuclear or cytoplasmic changes, such as binucleation, increased nuclear-to-cytoplasmic ratio, or cytoplasmic blebbing, mimicking a malignancy. ICC using calretinin, WT-1, or D2-40 can be used to confirm mesothelial origin.

Post-irradiation or chemotherapy-related alterations may lead to cytoplasmic vacuolization, nuclear enlargement, or atypia in background cells; these changes must be interpreted with caution in the appropriate clinical context (17).

Neoplastic lesions

Mediastinal neoplasms encompass a diverse group of primary and secondary tumors, each with distinct cytomorphologic and immunophenotypic features. Cytologic evaluation plays a critical role in their initial diagnosis and guiding further management. The major categories include thymic epithelial tumors, neuroendocrine tumors (NETs), lymphoid neoplasms, GCTs, mesenchymal tumors, and metastatic malignancies (23). Lesions are most commonly encountered within the anterior mediastinum (24). Thymic or GCTs are identified more frequently in the anterior mediastinum, while a location in the middle or posterior mediastinum renders lymphoid or neurogenic lesions more likely (20).

Thymic epithelial lesions

Thymoma

Thymomas are the most common primary anterior mediastinal neoplasm in adults and display a biphasic population of epithelial cells and immature lymphocytes. Cytologic smears are often moderately cellular and show small, uniform epithelial clusters admixed with abundant lymphocytes. The epithelial component typically exhibits bland oval nuclei, fine chromatin, and scant cytoplasm, while the lymphoid background may obscure epithelial cells, particularly in lymphocyte-rich subtypes.

Two types of epithelial cells can be present in thymomas: “type A” cells are spindle or ovoid in shape, while “type B” cells are round or polygonal. Table 2 lists the spectrum of thymomas, including their cytologic features and prognosis. Images of these entities are illustrated in Figure 3. A “two-cell pattern”—cohesive epithelial clusters surrounded by lymphocytes—is characteristic. However, in lymphocyte-poor thymomas, epithelial cells may predominate, occasionally mimicking carcinoma. The use of ICC (cytokeratin-positive epithelial cells, TdT-positive immature lymphocytes) aids in confirming thymic origin. Recognizing this biphasic pattern and correlating with radiologic findings is key to avoiding overdiagnosis of malignancy. Additionally, type A thymomas are associated with GTF2I mutations, while type B thymomas tend to have loss-of-function mutations in TP53 (19,20,25,26).

Figure 3 Different types of thymoma. (A) A type A thymoma has abundant spindle-shaped epithelial cells with rare lymphocytes (Diff-Quik, 20×). (B) Nuclear features of the spindle cells seen in a type A thymoma are better visualized with a Papanicolaou stain (20×). (C) Immunocytochemistry for cytokeratins highlights the epithelial cells in a type A thymoma (20×). (D,E) A predominance of lymphocytes and rare round or polygonal epithelial cells are present in this type B1 thymoma (Diff-Quik, 20× and Papanicolaou, 20×). (F) A type B2 thymoma has relatively even proportions of lymphocytes and round or polygonal epithelial cells (Papanicolaou, 20×). (G) A marked predominance of round or polygonal epithelial cells as well as rare lymphocytes are present in a type B3 thymoma (Diff-Quik, 10×). (H) A type AB thymoma contains features of both spindle and round or polygonal cells (Papanicolaou, 20×).

Diagnosis and classification of thymomas on cytology can be challenging due to overlapping features, lack of architecture, and artifacts such as crush artifact. Smears are generally moderately cellular to hypercellular; type A thymomas are the exception, as it is not uncommon for them to be hypocellular or nondiagnostic. It can be difficult or nearly impossible to distinguish epithelial cells from atypical lymphocytes morphologically. Additionally, thymomas share many cytologic features with NETs, and neuroendocrine markers (e.g., CD56, synaptophysin) can be focally positive in thymomas. In such cases, ICC for squamous markers (e.g., p40, p63, CK5/6) to determine the presence of neoplastic epithelial cells is required. Subtyping based exclusively on cytological smears is generally not recommended (27).

Thymic carcinoma

Thymic carcinoma demonstrates marked cytologic atypia with irregular nuclear membranes, coarse chromatin, prominent nucleoli, and a necrotic background, such as that shown in Figure 4. The lymphoid component is sparse or absent. Cytomorphologically, thymic squamous cell carcinoma can closely resemble metastatic squamous cell carcinoma from the lung or head and neck region. Immunocytochemical profiling assists in this distinction; thymic carcinoma often expresses CD5 and CD117 (c-KIT), whereas metastatic squamous cell carcinoma is negative for both markers. Awareness of this pattern is critical, as clinical management differs substantially (19,20,26).

Figure 4 This case of thymic carcinoma exhibits a pleomorphic population of cells with scant cytoplasm, coarse chromatin, and variably prominent nucleoli. Necrotic debris is present in the background (Diff-Quik, 20×).

NETs

NETs in the mediastinum are relatively uncommon, accounting for approximately 5% of all mediastinal tumors. They are usually found in the anterior compartment. Like NETs in the lung, they are classified based on morphology, mitotic counts, and necrosis. Conceptually, NETs can be divided into low-grade tumors, which include typical carcinoid and atypical carcinoid, and high-grade tumors, including large cell neuroendocrine carcinoma and small cell carcinoma. Unlike the lung, the predominant NET in the mediastinum is atypical carcinoid.

Morphologically, NETs are composed of a monotonous population of round, oval, or spindle-shaped cells with characteristic coarsely granular chromatin (described as “salt-and-pepper”) and small, frequently indistinct nucleoli (Figure 5). Large cell neuroendocrine carcinomas and small cell carcinomas contain cells with a higher nuclear-to-cytoplasmic ratio and nuclear pleomorphism. Smears of small cell carcinoma also show nuclear molding and crush artifact; nuclear smearing may preclude a diagnosis based on morphology alone. The Ki-67 proliferation index is high in these high-grade NETs. Immunocytochemically, all NETs are positive for neuroendocrine markers: synaptophysin, chromogranin, CD56, and INSM1. Well-differentiated NETs are occasionally positive for PTH; however, nuclear positivity for GATA-3 supports normal parathyroid tissue rather than an NET (19,20).

Figure 5 Carcinoid tumor. (A) A carcinoid tumor of the mediastinum has a monotonous population of cells with round to oval nuclei and indistinct nucleoli. The lack of atypia makes this a typical carcinoid tumor (Diff-Quik, 20×). (B) The classic “salt-and-pepper” chromatin is more apparent in this Papanicolaou-stained slide (20×).

Diagnostic pitfall: the mediastinum may harbor primary thymic NETs or metastatic small cell carcinoma. Both show small, fragile cells with high nuclear-to-cytoplasmic ratios and nuclear molding. Distinguishing between the thymus versus lung as the primary site is crucial for management. Tumors from both sites express cytokeratin, chromogranin, and synaptophysin; however, a pulmonary small cell carcinoma exhibits TFF-1 positivity, while a thymic NET is usually negative for TTF-1.

Lymphoid lesions

The mediastinum is a frequent site for Hodgkin and non-Hodgkin lymphomas (NHLs), which may present as isolated masses or part of systemic disease. Cytologic diagnosis requires integration of morphology with flow cytometry and ICC.

Hodgkin lymphoma

Smears show a polymorphous background of small lymphocytes, eosinophils, plasma cells, and histiocytes with scattered Reed-Sternberg (RS) cells or their variants. Classic RS cells, as shown in Figure 6, are large and may have bilobed nuclei and prominent eosinophilic nucleoli, often in an inflammatory milieu. Necrosis and granulomas may also be present. Cell block sections and immunostains (CD30 positive, CD15 positive, and weakly PAX5 positive) confirm the diagnosis and distinguish Hodgkin lymphoma from reactive or metastatic processes (19,20).

Figure 6 Lymphomas. (A) Hodgkin lymphoma is defined by the presence of Reed-Sternberg cells, which are large, atypical cells with abundant cytoplasm, occasional binucleation, and prominent nucleoli. The background is composed of a heterogeneous population of plasma cells, histiocytes, and lymphocytes (Diff-Quik, 20×). (B) Abundant lymphoblasts are easily identified by their large size, high nuclear-to-cytoplasmic ratio, and fine chromatin (Diff-Quik, 20×). (C,D) A case of primary mediastinal B-cell lymphoma exhibits nuclear pleomorphism and prominent nucleoli (Diff-Quik and Papanicolaou, 20×).

NHL

NHLs show variable cytomorphology depending on the subtype. Low-grade lymphomas such as small lymphocytic lymphoma and follicular lymphoma have monotonous populations of small, mature lymphocytes. In contrast, high-grade lymphomas like diffuse large B-cell lymphoma consist of large, atypical cells with prominent nucleoli and less condensed chromatin. Two of the more common types of NHL occurring in the mediastinum are lymphoblastic lymphoma and primary mediastinal B-cell lymphoma, both of which are demonstrated in Figure 6. Lymphoblastic lymphoma consists of immature lymphoblasts forming a mass lesion. Primary mediastinal large B-cell lymphoma is an aggressive neoplasm of mature B-cells occurring in the mediastinum. Definitive subclassification requires flow cytometric immunophenotyping and sometimes molecular studies for clonality (28). Cytologic specimens obtained via EBUS-TBNA or EUS-FNA can yield sufficient material for both, obviating the need for excisional biopsy in many cases (19,28).

Diagnostic pitfall: both lymphocyte-rich thymomas and lymphoblastic lymphoma may present with abundant small lymphocytes, leading to difficulty in distinguishing these entities. Thymoma typically shows a dual population of lymphocytes and epithelial cells, which are highlighted by cytokeratin on ICC. Acute lymphoblastic leukemia consists of a monomorphic population of lymphoid cells expressing TdT and CD3; an epithelial component is absent (29).

GCTs

The anterior mediastinum is the most common site for extragonadal GCTs. They are usually diagnosed via core biopsy or transthoracic FNA; GCTs are very rarely diagnosed via EBUS-FNA (30). Although typically more aggressive, extragonadal GCTs tend to share the morphological, immunocytochemical, and cytogenetic characteristics of their gonadal counterparts. A gain of chromosome 12p, considered pathognomonic for GCTs, is present in 80% of cases (31). Figures 7,8 show images of the more common GCTs, such as seminoma and embryonal carcinoma. Also in the differential diagnosis are yolk sac tumor and teratoma.

Figure 7 Mediastinal seminoma. (A,B) There are large dyscohesive cells with round nuclei and variably cleared chromatin in this case of mediastinal seminoma. Intervening lymphocytes are a common finding (Diff-Quik and Papanicolaou, 20×). (C) The cell block highlights the size difference between the neoplastic cells and reactive lymphocytes in this seminoma (hematoxylin and eosin, 20×). (D) Seminoma cells exhibit OCT3/4 positivity by immunocytochemistry (20×).

Figure 8 Embryonal carcinoma is composed of cells with nuclear pleomorphism, coarse chromatin, and prominent nucleoli. A necrotic background is a common finding (Papanicolaou, 10×).

Distinguishing GCTs from thymic or metastatic carcinomas relies heavily on clinical context, patient demographics, and ancillary testing (19,20,25,30,32). In about 10% of cases, extragonadal GCTs have been associated with concurrent somatic malignancies including sarcomas, leukemias, and carcinomas (31).

Diagnostic pitfall: mediastinal GCTs can display a wide morphologic spectrum and overlap with lymphoma or poorly differentiated carcinoma. Seminoma may mimic lymphoma due to its dyscohesive cells and lymphoid background. However, seminoma cells exhibit clear cytoplasm, prominent nucleoli, and a “tigroid” background on air-dried smears. Embryonal carcinoma and yolk sac tumor can mimic poorly differentiated carcinoma, but immunostains for OCT3/4, CD30, or AFP aid in differentiation. Clinical context (e.g., young male, anterior mediastinal mass) should always be integrated into the diagnostic approach (29).

Mesenchymal tumors

Mesenchymal tumors of the mediastinum are a rare and heterogeneous group of neoplasms, accounting for approximately 7% to 9% of all mediastinal tumors (33). They pose diagnostic challenges due to their diversity, overlapping morphologies, and the limited nature of tissue specimens obtained via FNA. Mesenchymal tumors are generally grouped based on their cell lineage (e.g., adipocytic, vascular, neurogenic, and tumors of uncertain differentiation). The differential diagnosis includes liposarcoma, schwannoma, ganglioneuroma, neuroblastoma, malignant peripheral nerve sheath tumor, solitary fibrous tumor, synovial sarcoma, and rhabdomyosarcoma. Neurogenic tumors, which are the most common mesenchymal tumors in the mediastinum overall, typically occur in the posterior compartment; other tumor types are usually found in the anterior region (20,33). Some of the most common mesenchymal mediastinal tumors are demonstrated in Figure 9.

Figure 9 Neurogenic tumors of the mediastinum. (A) An FNA biopsy of a schwannoma reveals a tight cluster of bland spindle-shaped cells with fibrillary cytoplasm (Papanicolaou, 10×). (B) Paragangliomas normally show moderately to highly cellular smears composed of round to oval cells with abundant finely granular cytoplasm. The classic Zellballen pattern is rarely identified in FNA specimens (Papanicolaou, 20×). (C,D) In a ganglioneuroma, occasional ganglion cells appear alongside a bland spindle cell component (Diff-Quik and Papanicolaou, 10×). (E) A solitary fibrous tumor consists of spindle cells tightly packed into a “patternless pattern” (hematoxylin and eosin, 10×). (F) Nuclear STAT6 positivity is nearly 100% sensitive for solitary fibrous tumors (10×). (G) This case of synovial sarcoma is composed of elongated, uniform cells with scant cytoplasm and fine chromatin arranged in fascicles (Papanicolaou, 20×). FNA, fine-needle aspiration.

Morphologically, these neoplasms are characterized by spindle, epithelioid, round, or pleomorphic cell morphology. Differentiating these lesions from each other can be extremely challenging, given the overlapping cytologic features in addition to the difficulty in obtaining diagnostic material. The correct diagnosis relies heavily on combining morphology with immunocytochemical and molecular testing. Techniques such as FISH are useful for confirming specific translocations characteristic of many sarcomas (19,20,25,31-35).

Mesenchymal tumors of the mediastinum occurring concurrently with other mediastinal neoplasms are rare but well documented, and they raise important diagnostic and pathogenetic considerations. Reported combinations include mediastinal liposarcoma, leiomyosarcoma, solitary fibrous tumor, or synovial sarcoma occurring synchronously with epithelial tumors such as thymoma, thymic carcinoma, or GCTs, as well as lymphomas. These tumors may arise as independent (“collision”) neoplasms, reflect a shared embryologic or microenvironmental predisposition within the mediastinum, or, less commonly, represent sarcomatous transformation or divergent differentiation within a single lesion (e.g., sarcomatoid thymic carcinoma). Recognition of concurrent tumors is clinically significant because management and prognosis are driven by the most aggressive component and require careful radiologic-pathologic correlation, thorough sampling, and judicious use of ICC and molecular testing to avoid misclassification as a single heterogeneous tumor. Overall, the literature emphasizes the rarity of these associations, the predominance of incidental discovery at resection, and the need for awareness among pathologists and clinicians when evaluating complex mediastinal masses (36,37).

Metastatic lesions

The mediastinum frequently harbors metastatic disease, most commonly from the lung, breast, gastrointestinal tract, kidney, or melanoma. Cytologic recognition of metastatic carcinoma is vital for staging and therapeutic planning. Common metastases include lung carcinoma, breast carcinoma, and melanoma.

One of the most challenging differential diagnoses in mediastinal cytology is distinguishing thymic carcinoma from metastatic squamous cell carcinoma of pulmonary or head and neck origin. Both may exhibit keratinization, necrosis, and prominent nucleoli. Clues favoring thymic origin include the presence of perivascular spaces, a lymphocyte-rich background, and positivity for CD5 and CD117. Metastatic squamous cell carcinoma typically expresses p40 and p63; it is negative for CD5. Occasionally, the site of origin is ambiguous even with ICC. When findings are equivocal, descriptive reporting (e.g., “epithelial neoplasm, favor thymic origin”) should be used rather than premature classification.

In addition to morphology and immunocytochemical workup, molecular profiling from the cell block material can be essential in the accurate identification of metastatic origin (19,20). Radiologic correlation can also be extremely helpful.

Ancillary techniques

Ancillary studies complement the morphologic assessment, resolve challenging differentials, and enable precise classification and prognostication. Advances in specimen processing have expanded the range of diagnostic, prognostic, and predictive tests that can be performed on cytologic material, including ICC, flow cytometry, and molecular assays.

ICC

ICC remains the cornerstone of ancillary testing in mediastinal cytology. When applied to cell block sections or direct smears, ICC facilitates lineage confirmation, subclassification, and distinction between morphologically overlapping entities. Table 3 summarizes the most useful immunocytochemical markers in mediastinal cytopathology. Optimization of cell block preparation is essential to preserve antigenicity and maintain staining reliability. Whenever possible, a panel-based approach should be employed rather than reliance on single markers, to avoid interpretive errors caused by aberrant or nonspecific expression.

While formalin-fixed, paraffin-embedded cell blocks are the ideal diagnostic material for ICC in cytology, smears may also be used as an alternative source. Both air-dried and alcohol-fixed smears can in theory be used for ICC if a cell block is hypocellular or unavailable. However, because there remains much variability in the preparation and processing of cytologic smears, validating and standardizing ICC interpretation on smears remains challenging. It is generally accepted that smears maintain their antigenicity following fixation (excluding acetone) and cover-slipping. Additionally, it is recommended to scan smears prior to utilizing them for ICC. Although previously stained smears can be re-used if they had negative results, the number of ICC stains performed on a given specimen is limited to those containing adequate diagnostic material (38,39).

Flow cytometry

Flow cytometric immunophenotyping is required for the accurate diagnosis and classification of lymphoid neoplasms, including those in the mediastinum. Fresh aspirated material should be placed immediately into RPMI or another isotonic medium to preserve cell viability.

Flow cytometry allows determination of B-cell or T-cell clonality, evaluation of antigen expression patterns, and detection of aberrant immunophenotypes that define specific lymphoma subtypes. Even limited aspirates from EBUS-TBNA or EUS-FNA can yield sufficient diagnostic information when properly triaged.

Molecular and cytogenetic studies

The use of cytology specimens for molecular testing has expanded considerably. Cell block sections and needle rinse material are suitable for polymerase chain reaction (PCR), FISH, and next-generation sequencing (NGS).

Cytologic smears have increasingly been shown to be feasible substrates for molecular testing, especially in settings where traditional tissue biopsies or cell blocks are limited or unavailable. Direct smears and liquid-based cytology often yield high-quality nucleic acids that can meet the input requirements for PCR and automated real-time PCR platforms, with studies reporting 100% concordance for common mutations (e.g., EGFR, KRAS) when validated protocols for DNA/RNA extraction are applied to stained smear slides. This demonstrates that cytologic preparations can successfully support targeted PCR assays and broader mutational analyses, offering faster turnaround and effective use of scarce material (40).

For FISH, cytology specimens perform well for detecting chromosomal rearrangements and other genomic alterations, with respiratory tract and other solid tumor cytology showing acceptable clinical results comparable to traditional preparations when validated appropriately (41). NGS analyses on cytologic smears also show high success and quality metrics; multiple studies report comparable sequencing success rates and even superior DNA quality metrics (e.g., higher mean target coverage and lower duplication rates) relative to formalin-fixed paraffin-embedded tissues. When cytologic smears are included alongside or instead of cell blocks, adequacy rates for RNA-based fusion NGS assays can increase markedly (42). Collectively, these data suggest that with appropriate validation and pre-analytic handling, cytologic smears are a robust and practical option for a range of molecular tests in diagnostic workflows.

Proper triage and preservation are critical. Samples designated for molecular analysis should be processed with minimal formalin exposure and adequate cellularity. Collaboration between cytopathologists and molecular laboratories ensures optimal test selection and interpretation (43-45).

Emerging technologies

Recent advances include digital image analysis, telecytology, and artificial intelligence (AI)-assisted diagnostics, which are increasingly applied to mediastinal cytology. Computer-assisted algorithms can quantify immunostain expression and identify subtle morphologic patterns predictive of malignancy (46).

Cryobiopsy

EBUS-guided transbronchial mediastinal cryobiopsy (EBUS-TMC) is a newer adjunctive technique that uses a cryoprobe introduced under EBUS guidance to freeze and extract larger, intact tissue specimens from mediastinal or hilar lesions. Cryobiopsy is gaining in popularity, particularly in the diagnosis of lung tumors. In this technique, an extremely cold cryoprobe is attached to the tissue of interest, and the tissue is removed during the freeze-thaw cycle. The larger sample size and preserved histologic architecture obtained by cryobiopsy can improve diagnostic accuracy, particularly in cases where FNA alone is insufficient, such as lymphoma or sarcoidosis. Meta-analyses and recent randomized trials have reported higher overall diagnostic yields for EBUS-TMC compared with EBUS-TBNA, with low rates of complications such as bleeding or pneumothorax, and improved performance in molecular and histopathologic assessments (47-49).

Robotic bronchoscopy

Robotic bronchoscopy has emerged as a complementary tool in mediastinal cytology by enhancing bronchoscopic reach, stability, and precision during sampling, particularly when combined with EBUS. Some platforms allow improved scope control and navigation, which can facilitate more accurate targeting of mediastinal lymph nodes and paratracheal or hilar lesions when performing needle aspiration. While robotic bronchoscopy alone does not replace EBUS for mediastinal staging, its integration may improve sample adequacy and procedural confidence, especially in anatomically challenging cases or when simultaneous evaluation of peripheral pulmonary lesions and mediastinal nodes is desired. Early studies suggest that robotic-assisted approaches can achieve diagnostic yields comparable to conventional techniques with acceptable safety profiles, though data specific to mediastinal cytology remain limited. As such, current evidence supports robotic bronchoscopy as an adjunct rather than a standalone solution, with further prospective studies needed to define its incremental value in mediastinal cytologic diagnosis (50,51).

AI for ROSE

Recent advances in cytopathology are increasingly focused on incorporating AI into ROSE workflows to augment diagnostic speed, accuracy, and scalability. Traditional ROSE—used during FNA and other minimally invasive procedures—helps ensure specimen adequacy and preliminary assessment in real time, guiding clinicians and improving yield. However, the reliance on specialized cytopathologists limits availability and timeliness in many settings. Deep learning and convolutional neural network (CNN) models have been developed to classify cytologic images with performance comparable to human experts and high area-under-curve metrics, demonstrating the feasibility of automated ROSE assistance across varied procedures including bronchoscopy and EUS-FNA. These AI systems may be deployed on cloud or mobile platforms to provide near-instant evaluation and help democratize access to ROSE services where cytopathologists are scarce (52,53).

Point-of-care (POC) molecular testing

Parallel to AI integration, POC molecular testing is emerging as an extension of the ROSE paradigm by enabling rapid, on-site genetic and biomarker analysis that can inform personalized therapy decisions within hours of sample acquisition. Novel cartridge-based molecular assays have demonstrated the feasibility of same-day detection of actionable mutations (e.g., EGFR, KRAS, BRAF, and gene fusions) directly from cytology and small biopsy samples, significantly shortening turnaround times relative to conventional laboratory diagnostics. This POC molecular capability complements ROSE by not only confirming sample adequacy but also providing immediate insight into mutation status that can guide clinical management and targeted treatment strategies. Together, AI-assisted digital evaluation and rapid molecular diagnostics represent frontier innovations aimed at enhancing the utility and impact of ROSE in modern precision cytopathology (54).

In addition, liquid-based cytology platforms facilitate cell capture for downstream molecular or digital applications, further enhancing the diagnostic and research potential of FNA material (55).

Limitations

Morphology and architecture

Accurate interpretation of mediastinal cytology requires recognition of several diagnostic pitfalls that can arise from overlapping cytomorphologic features, suboptimal specimen quality, or limited clinical information. Misinterpretation can lead to inappropriate clinical management, especially given the diversity of benign, primary, and metastatic lesions encountered in this anatomic compartment. Awareness of common diagnostic traps and systematic correlation with ancillary studies and imaging findings is essential for the cytopathologist or cytologist.

Cytology is often the first-line diagnostic approach for mediastinal lesions because it is minimally invasive and has a high yield for many malignancies. However, cytology may be insufficient when architectural features are essential for diagnosis. This is particularly true for lymphoproliferative disorders (especially Hodgkin lymphoma and some NHLs), where evaluation of nodal architecture, fibrosis, and growth patterns is critical (56). Similarly, thymic epithelial tumors, GCTs, and some metastatic carcinomas can be difficult to classify accurately on cytology alone due to overlapping cytomorphologic features, tumor heterogeneity, and limited ability to assess invasion or stromal components (57,58).

Sample inadequacy

Diagnostic yield remains an important metric for assessing mediastinal cytopathology, particularly for benign and rare lesions, yet current studies highlight significant variability. While diagnostic yields for malignant mediastinal disease with EBUS-TBNA and related techniques are generally high, the sensitivity for conditions such as lymphoma, benign inflammatory diseases (e.g., sarcoidosis), and uncommon tumors is more variable and often lower. Newer procedures like mediastinal cryobiopsy have demonstrated markedly higher yields in small cohorts, especially for benign and rare pathologies that are challenging with cytologic sampling alone, but robust evidence from larger prospective studies is still needed to define the true performance across disease types and to refine practice guidelines (59).

Several factors can limit diagnostic yield, including small lesion size, necrotic or fibrotic tissue, and poor needle targeting. Hypocellularity or excessive blood contamination may obscure key cytologic details. Interpretation can also be compromised by crush artifact and air-drying artifact (14,16,20,33,60). Close communication among the cytopathologist, radiologist, and proceduralist is essential to optimize outcomes.

Inadequate or poorly preserved samples are a frequent source of diagnostic error. Necrotic, hemorrhagic, or paucicellular aspirates may obscure diagnostic features, while crush artifact or air-drying artifact can distort cellular morphology. For example, necrotic debris in cystic thymoma, GCTs, or metastatic carcinoma may mimic suppurative inflammation. Chromatin smearing is a common problem in small cell carcinoma, in particular. Air-dried smears from endobronchial ultrasound-guided FNAs can exaggerate nuclear detail, potentially simulating malignancy. To minimize these errors, ROSE should be used when available to confirm specimen adequacy and guide triage for ancillary testing.

Cytology can also be inadequate when extensive ICC, molecular testing, or assessment of tumor microenvironment is required. Poor cellularity, crush artifact, necrosis, or sampling error may further limit diagnostic confidence, particularly in fibrotic, cystic, or necrotic mediastinal lesions. In such scenarios, core needle biopsy or surgical biopsy (e.g., mediastinoscopy or VATS) may be necessary to obtain sufficient tissue for histologic evaluation, ancillary studies, and definitive classification. Current guidelines therefore emphasize a tailored approach, reserving cytology for lesions with high pretest probability of diagnosis by needle sampling, while escalating to tissue biopsy when cytology is nondiagnostic or discordant with clinical and radiologic findings (57,58).

Lack of technique standardization

FNA and EBUS-TBNA remain minimally invasive mainstays, but the standardization of these techniques is incomplete, especially for newer procedures. For example, the variability in needle size, number of passes, and procedural strategies among operators contributes to inconsistent sampling quality, and guidelines on optimal sampling protocols are still evolving. Emerging sampling approaches like mediastinal cryobiopsy promise larger and more diagnostic tissue samples and have shown improved yield versus standard EBUS-TBNA in preliminary series, but large multicenter trials and consensus recommendations on when and how to use these techniques are lacking (59,61).

In mediastinal disease, imaging strategies are critical for directing cytopathologic sampling, yet evidence gaps remain regarding optimal imaging workflows. CT and ultrasound are widely used to localize lesions and guide biopsies, and guidelines like the ACR Appropriateness Criteria provide expert advice on modality selection for mediastinal masses. However, the integration of advanced imaging adjuncts—such as Doppler ultrasound to assess vascularity, or newer technologies like confocal endomicroscopy and AI—has not been systematically evaluated in high-quality studies specific to mediastinal lesions. There is limited comparative evidence on how different imaging strategies impact cytologic adequacy, reduce nondiagnostic results, or improve differentiation between benign and malignant processes prior to biopsy (62,63).

Diagnostic algorithm

Given the heterogeneity of mediastinal lesions, a structured and algorithmic approach greatly enhances diagnostic efficiency and accuracy. The integration of cytomorphologic assessment, ICC, flow cytometry, and molecular testing—interpreted alongside radiologic and clinical data—forms the foundation of modern mediastinal cytopathology. A simplified workflow for the diagnosis of mediastinal lesions is provided in Figure 10.

Figure 10 The flow chart provides a diagnostic algorithm for the diagnosis of mediastinal lesions in cytopathology. AFP, alpha-fetoprotein; β-hCG, beta-human chorionic gonadotropin; LDH, lactate dehydrogenase.

Conclusions

The cytopathologic evaluation of mediastinal lesions has evolved from a primarily morphologic discipline into a comprehensive, multidisciplinary field that integrates cytomorphology, immunophenotyping, molecular testing, and radiologic correlation. With advances in minimally invasive sampling techniques such as endobronchial and endoscopic ultrasound-guided FNA, high-quality cytologic material can now be obtained from nearly all mediastinal compartments, often obviating the need for surgical biopsy.

Cytopathology plays a pivotal role in the initial diagnosis, staging, and molecular characterization of mediastinal disease. Through careful attention to sample adequacy, judicious use of ancillary techniques, and algorithmic diagnostic reasoning, cytopathologists can achieve diagnostic accuracy comparable to histopathology while preserving tissue for targeted therapy testing.

Emerging technologies—including digital cytology, AI, NGS, cryobiopsy, and robotics —are reshaping the discipline, expanding its utility from diagnosis to disease monitoring and translational research. Continued collaboration among cytopathologists, cytologists, molecular scientists, radiologists, and clinicians will be essential to fully realize the potential of these innovations.

Acknowledgments

None.

Footnote

Peer Review File: Available at https://med.amegroups.com/article/view/10.21037/med-2025-1-58/prf

Funding: None.

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://med.amegroups.com/article/view/10.21037/med-2025-1-58/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All clinical procedures described in this study were performed in accordance with the ethical standards of the Ohio State University Institutional Review Board and with the Declaration of Helsinki and its subsequent amendments. Written informed consent was obtained from the participants for the publication of this article and accompanying images.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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