Small biopsy diagnosis of anterior mediastinal masses: histologic, immunohistochemical, and molecular considerations

Introduction

The mediastinum is the site of origin for a wide spectrum of neoplastic and non-neoplastic lesions that can pose significant diagnostic challenges in clinical practice (1,2). Although their incidence in the adult general population is only about 0.01%, mediastinal lesions can be associated with substantial morbidity through the compression of vital structures and the inherent risk of malignancy. Computerized tomography (CT), magnetic resonance imaging (MRI), fluorodeoxyglucose (FDG) positron emission tomography combined with CT (PET/CT) and other imaging modalities are useful for detecting mediastinal solid and cystic lesions (3-5). Serum markers for alpha-fetoprotein and human chorionic gonadotropin can help establish the diagnosis of mediastinal germ cell tumors, but the definitive diagnosis of practically all other mediastinal mass lesions requires examination of tissue biopsies or fine needle aspiration (FNA) biopsies obtained by various techniques (6-11). While many anterior mediastinal lesions can be diagnosed radiologically or managed with direct surgical resection (e.g., resectable thymoma, thymic cysts, mature teratoma), tissue diagnosis remains essential for locally advanced thymic tumors requiring neoadjuvant therapy, suspected lymphoma, and lesions with indeterminate imaging features.

This review focuses on the pathologic evaluation of anterior mediastinal masses, a diagnostically diverse group encompassing thymic epithelial tumors (TET), lymphomas, neuroendocrine neoplasms and germ cell tumors, in which accurate interpretation of small biopsies is critical for guiding clinical management.

Biopsy techniques for the diagnosis of anterior mediastinal lesions

The selection of a biopsy technique is guided by the safety profile of the particular procedure and whether cytologic or histologic materials are needed for accurate diagnosis. Mediastinoscopy and left mediastinal mediastinotomy (Chamberlain procedure) have traditionally been considered the gold standards for obtaining tissue samples from the mediastinum, but various minimally invasive techniques have gained prominence in recent years (12,13). They include image-guided percutaneous core needle biopsy (CNB) (Figure 1), transthoracic fine needle aspiration biopsies (TTFNA) (Figure 2), endobronchial ultrasound guided transbronchial biopsies (EBUS-TBNA) and EBUS mediastinal cryobiopsies (EBUS-TMC) (13-15).

Figure 1 Image-guided percutaneous core needle biopsy of thymoma showing lymphoepithelial areas and fibrous tissue septa (hematoxylin and eosin staining, 2× original).

Figure 2 Image-guided percutaneous core needle biopsy of anterior mediastinal mass showing a dual population of lymphocytes and larger, more cohesive epithelial cells (SurePath, Papanicolau stain, 60× original).

Percutaneous CNB and TTFNA use CT, CT-fluoroscopy, cone beam CT, or ultrasonography to obtain tissue or cytologic samples from mediastinal lesions. CNB yields sensitivities and specificities of 90–100% for the diagnosis of TET, although these lesions can exhibit considerable morphologic heterogeneity (16). Samples from TTFNA yield lower sensitivity of approximately 50–82% for detecting lymphomas and are often insufficient to accurately classify lymphomas due to limited tissue for histologic, immunophenotypic, and genetic evaluations (17,18). Although a meta-analysis of percutaneous biopsy across all mediastinal compartments reported overall complication rates of up to 13%, anterior mediastinum is relatively accessible via safe extrapleural access which avoids lung transgression and is associated with significantly lower complication rates (16,19). EBUS-TBNA and EBUS-FNA have become the preferred procedures for sampling mediastinal lymph nodes in lung cancer patients with 84–95% sensitivity and 100% specificity (20-22). EBUS-TMC allows for the collection of larger tissue samples than traditional EBUS-TBNA (15); this is particularly helpful for the diagnosis and subtyping of lymphomas and the performance of molecular tests in metastatic lung neoplasms and other mediastinal tumors.

EBUS-TBNA yields predominantly cytologic material via suction through a 21–22G needle, which is usually sufficient for diagnosing metastatic carcinoma and lung cancer staging. EBUS-TMC uses a 1.1 mm cryoprobe to obtain architecturally preserved histologic specimens, that require ancillary studies including molecular testing.

The multicenter Lancet Respiratory Medicine RCT confirmed that combining both techniques achieves 94% overall yield compared to 82% for TBNA alone (P=0.0026) (23).

EBUS guided sampling is primarily useful for mediastinal lesions involving or extending to the paratracheal or peribronchial regions. Its applicability to purely anterior (prevascular) mediastinal masses is limited, but it may provide diagnostic material when nodal or posterior extension is present, particularly in lymphoproliferative disorders (23).

Meticulous specimen triage is paramount, and the radiologist, pulmonologist or surgeon performing the biopsy procedure needs to communicate with the pathology laboratory to ensure that samples that are adequate for various tests are collected in the appropriate media. For example, for masses suspicious of lymphoproliferative disorder, obtaining fresh tissue in Roswell Park Memorial Institute (RPMI) medium for flow cytometry (FC) is highly recommended. The increased tissue volume from CNB and EBUS-TMC is especially valuable for modern molecular and immunohistochemistry (IHC) testing.

Diagnosis of anterior mediastinal lesions by FNA biopsies and core needle biopsies

As shown in Table 1, a large variety of primary neoplastic and non-neoplastic mass-like lesions and metastatic lesions can be seen in the anterior mediastinum (1,9). We will briefly review diagnostic considerations for the diagnosis of only the most common anterior mediastinal lesions: TET, lymphomas and neuroendocrine tumors (NET) with various diagnostic methods. Patients with selected mediastinal lesions, such as well circumscribed anterior mediastinal masses that are likely to be a thymoma, a thymic cyst or a teratoma with calcifications are sometimes managed with direct surgical resection but in our medical center, the vast majority of patients with anterior mediastinal lesions are biopsied with core biopsy needle, with diagnostic results comparable to those described below for various tumors.

Diagnosis of thymic epithelial cell neoplasms by core needle biopsies

TET include thymomas and thymic carcinomas (24). Thymomas can be diagnosed by percutaneous CNB with 50–90% sensitivity, but this procedure is not reliable for their subclassification, as these tumors exhibit overlap in World Health Organization (WHO) types in over 25% of cases (25,26). Thymomas are composed of cytologically bland neoplastic epithelial cells admixed with benign immature T-lymphocytes. They have been subclassified by the WHO into types A, AB, B1, B2, and B3 based on the morphology of the neoplastic epithelial cells and the epithelial-to-lymphocyte ratio (24). Histologically, WHO type A thymomas are composed of spindle-shaped epithelial cells with oval nuclei and scanty amphophilic cytoplasm exhibiting minimal anisocytosis and mixed with scant lymphocytes (Figure 3A). These thymomas can be mistaken for low grade spindle cell neoplasms. Immunostains for cytokeratins such as AE1/AE3, CAM 5.2, and Oscar highlight the epithelial nature of the neoplastic cells (Figure 3B).

Figure 3 Image-guided core needle biopsy of WHO type A thymoma with histologic and features. (A) Image-guided percutaneous core needle biopsy of WHO type A thymoma showing a lymphoid-poor neoplasm composed of oval and spindle cells showing minimal anisocytosis and scanty amphophilic cytoplasm (hematoxylin and eosin staining, 400× original). (B) The spindle cells of the WHO type A thymoma show cytoplasmic immunoreactivity for pancytokeratin AE1/AE3 (Polymer-based immunostain, 400× original). WHO, World Health Organization.

WHO type B1 thymomas resemble the normal thymus cortex and are composed of “polygonal” epithelial cells growing in a dendritic network scattered amongst numerous small lymphocytes (Figure 4A). On small samples, routine H&E alone is insufficient to reliably distinguish thymic hyperplasia from B1 thymoma, and ancillary immunohistochemical studies (Figure 4B) and/or surgical resection specimens are typically needed for definitive classification.

Figure 4 Image-guided core needle biopsy of WHO type B1 thymoma with histologic and immunohistochemical features. (A) Image-guided percutaneous core needle biopsy of WHO type B1 thymoma showing a lymphocyte rich neoplasm with inconspicuous epithelial cells. These lesions can be difficult to distinguish from lymphomas without the aid of immunohistochemistry for keratin and/or other epithelial markers (hematoxylin and eosin staining, 200× original). (B) The “polygonal” cells of the WHO type B1 thymoma show cytoplasmic immunoreactivity for pancytokeratin AE1/AE3. Note the meshwork of tumor epithelial cells exhibiting a growth pattern that resembles the architecture of normal thymus (Polymer-based immunostain, 200× original). WHO, World Health Organization.

WHO type B2 thymomas are composed of polygonal epithelial cells growing in small clusters and mixed with numerous lymphocytes (Figure 5A). WHO type B1 and WHO type B2 thymomas can be difficult to distinguish from lymphomas on core needle biopsies since the epithelial cells are often inconspicuous on hematoxylin and eosin (H&E) stained sections. Immunostains for various cytokeratins are very helpful in demonstrating the epithelial differentiation of the neoplastic cells (Figure 5B). Neoplastic thymic epithelial cells also stain with p40, p63, and desmoglein-3 (Figure 5C,5D).

Figure 5 Image-guided core needle biopsy of WHO type B2 thymoma with histologic and immunohistochemical features. (A) Image-guided percutaneous core needle biopsy of WHO type B2 thymoma showing a lymphocyte rich neoplasm with small clusters of “polygonal” epithelial cells (hematoxylin and eosin staining, 100× original). (B) The tumors cells of the WHO type B2 thymoma show cytoplasmic immunoreactivity for pancytokeratin AE1/AE3 (Polymer-based immunostain, 100× original). (C) The tumor cells of the WHO type B2 thymoma show nuclear immunoreactivity for p40. The presence of this immunoreactivity should not be interpreted as evidence for squamous cell carcinoma (Polymer-based immunostain, 100× original). (D) The tumor cells of the WHO B2 thymoma show cytoplasmic immunoreactivity for desmoglein-3. Although this marker also stains squamous cell carcinomas, it should not be used to distinguish this neoplasm from thymomas (Polymer-based immunostain, 100× original). WHO, World Health Organization.

The use of immunostains to characterize the lymphoid cells present in thymomas requires careful interpretation and may result in misdiagnosis if not evaluated in conjunction with epithelial markers and morphologic features. The thymocytes in these lesions are immature T cells that exhibit CD3, CD5 and TdT immunoreactivity (Figure 6). While these findings can be helpful in suggesting the thymic origin of a neoplasm, they can also result in a misdiagnosis of T-lymphoblastic lymphomas (T-LBLs) in cases where the presence of epithelial cells in a B1 or B2 thymoma is overlooked.

Figure 6 The immature lymphoid cells of the WHO type B1 thymoma show nuclear immunoreactivity for TdT. This finding confirms the presence of a thymic neoplasm and should not be interpreted as evidence for a lymphoblastic lymphoma. The latter is composed of larger, more atypical lymphoid cells that are not associated with keratin positive epithelial cells (Polymer-based immunostain, 200× original). WHO, World Health Organization.

WHO type B3 thymomas are lymphoid-poor lesions composed of oval or polygonal cells showing mild nuclear atypia and cytoplasmic immunoreactivity for keratins (Figure 7A,7B).

Figure 7 Image-guided core needle biopsy of WHO type B3 thymoma with histologic and immunohistochemical features. (A) Image-guided percutaneous core needle biopsy of WHO type B3 thymoma showing a lymphocyte poor neoplasm composed of solid sheets of slightly atypical epithelial cells (hematoxylin and eosin staining, 200× original). (B) The tumors cells of the WHO type B3 thymoma show cytoplasmic immunoreactivity for pancytokeratin AE1/AE3 (Polymer-based immunostain, 200× original). WHO, World Health Organization.

Thymic carcinomas comprise approximately 10% of thymic epithelial neoplasms and are composed of epithelial cells that exhibit overt cytologic features of malignancy, such as large nuclei with considerable anisocytosis, irregular nuclear membranes, clumped chromatin, and/or macronucleoli, high nuclear-cytoplasmic ratios, necrosis, and/or increased mitotic activity (1,27). The most common thymic carcinoma subtype is squamous cell carcinoma (Figure 8), composed of sheets of epithelial cells showing intercellular bridges, parakeratosis, keratin pearls and/or basaloid features similar to those seen in squamous cell carcinomas arising from the lung, head and neck and other areas. It is beyond the scope of this brief review to describe the histopathologic features of the other histopathologic types of thymic carcinomas listed on Table 2. Thymic carcinomas can often be diagnosed as epithelial malignancies without difficulty on percutaneous CNB, but it is often difficult to determine their site of origin as they show identical histopathological features to carcinomas arising in the lung and other organs (28). The diagnosis of primary thymic origin is often rendered by exclusion in a patient that shows on imaging studies a mediastinal mass and lacks clinical and/or imaging findings of a malignancy arising in another location. It has been proposed that the presence of immunoreactivity for CD5, polyclonal PAX8, GLUT-1 and/or CD117 (c-KIT) in a carcinoma favors thymic origin of the neoplasm, but these immunostains are not 100% specific for thymic origin (29,30). EZH2 and POU2F3 are two recently described markers that support the diagnosis of thymic carcinoma (31).

Figure 8 Image-guided percutaneous core needle of a squamous cell carcinoma of the thymus. The tumor shows identical histopathologic features to squamous cell carcinomas arising in the lung or other organs and the diagnosis of thymic primary is established by exclusion of extrathymic lesions (hematoxylin and eosin staining, 400× original).

Immunostain for NUT protein expression is used for the diagnosis of mediastinal midline carcinomas, rare poorly differentiated lesions resulting from a translocation of the NUT gene often resulting in fusion with the BRD4 gene (32,33).

Molecular studies are currently of limited clinical value for the management of patients with TET as thymomas generally lack actionable molecular variants (34). The GTF2I point mutation is seen in A/AB and B thymomas but is absent in thymic carcinomas. B2 and B3 thymomas rarely exhibit KMT2A-MAML2 translocations, and metaplastic thymomas show YAP1-MAML2 translocations. Thymic carcinomas exhibit complex karyotypes and frequently harbor KIT gene mutations (exon 11, 17), TP53 mutations, CDKN2A (p16) deletions and other molecular variants (35).

Diagnosis of mediastinal lymphomas by FNA biopsies and core needle biopsies

Mediastinal lymphomas are an important diagnostic consideration in patients presenting with an anterior mediastinal mass as lymphoproliferative disorders comprise approximately 50–60% of all mediastinal malignancies in both children and adults (36,37).

Mediastinal lymphomas may involve the thymus, mediastinal lymph nodes, or mediastinal organs, such as lungs, pleura, heart, and pericardium. Patients typically present with a bulky anterior mediastinal mass, symptoms related to mass effect such as superior vena cava syndrome, dyspnea, cough, and/or chest pain, and occasionally systemic B symptoms.

Primary lymphomas arising in the mediastinum include classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma (PMBL), mediastinal grey zone lymphoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT), and T-LBL. Non-neoplastic entities that can be encountered in the mediastinum include unicentric Castleman disease, IgG4-related disease, and Rosai-Dorfman disease (22).

A range of modalities including percutaneous and bronchoscopic techniques as well as more invasive surgical approaches can be used to diagnose mediastinal lymphomas. The choice of modality is governed by lesion location, size, relationship to critical structures, institutional expertise, and the intended downstream diagnostic tests (e.g., need for FC or molecular assays) (1,16). A description of some of these techniques with a focus on the benefits and limitations of that modality in terms of diagnostic yield and tissue triaging follows. In general, specialists performing these biopsies should be aware that routine H&E staining is insufficient to reliably distinguish thymic hyperplasia from B1 thymoma, and ancillary immunohistochemical studies and/or surgical resection specimens are typically needed for definitive classification (24,25).

TTFNA is minimally invasive and useful when the differential favors metastatic carcinoma, smallcell lung cancer, germ cell tumor, or infectious/inflammatory processes. When a lymphoma is suspected, touch imprints are preferred to evaluate for the presence of lymphoid tissue. Imprints can be stained on-site with a Romanowsky-type method, such as Diff-Quik, and air-dried (17). Distinguishing lymphomas from lymphoid rich TETs by cytology alone is challenging since architectural features are lacking. While cytologic samples combined with ancillary studies such as FC and cell block IHC improve diagnostic yield for lymphomas, the lack of tissue architecture in these samples may limit the ability to subtype and diagnose certain lymphomas. FNA should be accompanied by rapid on-site evaluation (ROSE) when possible to triage materials appropriately.

Percutaneous CNB provides tissue cores that preserve the tumor architecture, allowing routine histology, IHC, and molecular testing. It is currently a firstline option for mediastinal masses suspicious for lymphoma, reporting high diagnostic yield and low complication rates when performed by experienced teams (25,26). If a CNB sample is sent for intraoperative review, only a small portion of the tissue should be frozen to determine whether lymphoid tissue is present. Additional tissue cores may be requested if only fibrotic or necrotic tissue is seen on imprint/frozen evaluation. Ample tissue sampling should be obtained for classification and prognostication. CNB facilitates subclassification of mediastinal lymphomas in many cases when adequate tissue is obtained. Six to eight cores are recommended to obtain ample tissue blocks for FC, IHC, FISH studies, and molecular studies (38,39).

Surgical approaches (mediastinoscopy, VATS, or open thoracotomy) provide the most tissue and remain the gold standard when less invasive methods are nondiagnostic or when immediate, extensive sampling is required for accurate subtyping. Excisional or incisional biopsies provide the best assessment of nodal architecture—important for distinguishing nodular sclerosis CHL from other entities—and are preferable when initial minimally invasive sampling is inconclusive (1,38).

Recommended algorithms for the diagnosis of mediastinal lymphomas include obtaining at least one core or aliquot submitted fresh or in RPMI for FC and material fixed in formalin for histology, IHC, and molecular/FISH testing. Touch preparation slides are also useful for cytologic evaluation and potential FISH studies. If multiple core biopsies are received, embedding the core biopsies in separate blocks is recommended, allowing for potentially exhaustive IHC work-up on one or more blocks while preserving tissue for FISH and molecular studies in other blocks (33). For FNA/EBUS samples, preparing a cell block and ensuring adequate cellularity for immunostains and clonality (FC and/or molecular testing) testing are crucial (40).

Ancillary studies for the diagnosis of lymphoproliferative disorders include: FC, IHC, molecular studies, and FISH. FC provides rapid immunophenotypic characterization and is particularly valuable for B cell clonality assessment and detection of aberrant antigen expression (39). IHC also allows for immunophenotyping, assessment of markers that may not be available by routine FC (i.e., nuclear antigens, CD30, PD-L1, etc.), and evaluation of tissue architecture in core biopsies/surgical specimens. Molecular studies may include: clonality assessment by polymerase chain reaction-capillary electrophoresis (PCR-CE) or next generation sequencing (NGS) (IGH and IGK-gene rearrangement studies for B cell clonality and TCR beta and gamma gene rearrangement studies for T cell clonality) and NGS panels for DNA and RNA variants (41). Molecular studies may be particularly useful to support challenging diagnoses and to identify potentially actionable alterations. Additionally, FISH studies may be needed to assess for common alterations in certain lymphomas, i.e., MYC, BCL6, and BCL2 rearrangements. Utilization of FC, IHC, cytogenetic and molecular assays significantly improves diagnostic accuracy compared with morphology alone (39,41).

It is beyond the scope of this review article to discuss in detail the pathologic and cytologic features of all hematolymphoid entities involving the mediastinum, and we will review the diagnostic features on TTFNA and core biopsies of only CHL, PMBL, T-cell lymphoblastic lymphoma, thymic extranodal marginal zone lymphoma of MALT and mediastinal grey zone lymphoma (41).

CHL is subclassified into four recognized morphologic patterns, including: nodular sclerosis, mixed-cellularity, lymphocyte-rich, and lymphocyte-depleted subtypes. Subtyping CHL shows some correlation with epidemiology and clinical behavior and remains a “desirable” diagnostic criteria in the WHO 5th Edition (41-43). Involvement of the mediastinum is common in the nodular sclerosis subtype, particularly the unusual syncytial variant of nodular sclerosis (44-46). CHL incidence rates vary with age, with peaks in incidences (age-standardized incidence rates per 100,000 of 3.83 for men and 2.92 for women) occurring at age 20–29 years in non-Hispanic Whites (43). Nodular sclerosis CHL (NSCHL) subtype is the most common subtype (22,37). Male sex, adolescence and young adult age, prior EBV infection, HIV/AIDS, autoimmune diseases, cigarette smoking, and family history of CHL represent risk factors associated with CHL (41,43).

On gross examination, involved lymph nodes are enlarged and firm. In NSCHL, the lymph nodes may show nodularity. It is important to note that routine FC will not detect CHL, but it is helpful in excluding other lymphoma subtypes. Microscopically, NSCHL shows prominent fibrosis, including capsular involvement, a feature appreciated on low-power. The cellular portion of tissue demonstrates an atypical mixed inflammatory cell infiltrate (small lymphocytes, plasma cells, histiocytes, eosinophils, and/or neutrophils) that contains variable numbers of Hodgkin and Reed-Sternberg (HRS) cells. Hodgkin cells are enlarged, mononuclear cells, and Reed-Sternberg cells are binucleated/multinucleated cells with prominent eosinophilic nucleoli (Figure 9). Lacunar cells, which show cytoplasmic retraction, are typically also present. HRS cells may be scattered or present in clusters and sheets (i.e., syncytia). Syncytial variant of NSCHL denotes that the HRS cells are present in sheets (42).

Figure 9 Many Hodgkin-Reed-Sternberg cells are seen in this section from a 50-year-old female with nodular sclerosis classic Hodgkin lymphoma (hematoxylin and eosin staining, 200× original). The red arrows highlight the mononuclear Hodgkin cells with a prominent nucleolus. The blue arrow highlights a binucleated Reed-Sternberg cell with its characteristic owl’s eyes consisting of two separate nuclei with a large eosinophilic nucleolus in each. The inflammatory background consists of small lymphocytes, histiocytes, and scattered eosinophils.

HRS cells show down-regulation of the B-cell program, a diagnostic feature that is reflected immunohistochemically. HRS cells are positive for CD30, MUM-1, CD15 (75–85% of cases), PAX5 (weak), and thymus- and activation-regulated chemokine (TARC) (42). CD20 (30–40% of cases) can be variably positive in a minority of HRS cells, while other B-cell markers including CD19, CD79a, BOB1, and OCT2 are usually, but not always, negative (41,42,47). T-cell antigens may rarely be expressed (48,49). HRS cells are usually negative for CD45. EBV (i.e., EBER ISH) is positive in a subset of cases.

Molecular studies in CHL cases may be limited by the relative paucity of neoplastic cells in comparison to other neoplasms. Genes involved in immune evasion (e.g., PDL1/2, MHC I/II, CIITA, and B2M) and that result in activation of the NFKB (e.g., REL, TNFAIP3, TNFRSF14, NFKBIE, NFKBIA, CARD11, and IKBKB) and JAK-STAT pathways (e.g., STAT6, SOCS1, SOCS6, XPO1, CSF2RB, PTPN1, and PTPN6) in addition to other alterations are reported in CHL (50).

PMBL is an aggressive B-cell lymphoma thought to originate from thymic B-cells. The incidence of PMBL is 2.38 per million (SEER 9 database) and has been increasing since 1974 (51,52). This disease primarily affects young adults with a mean age of 36 years and a slight female predilection (51).

Histologically, PMBL shows an abnormal lymphoid infiltrate comprised of intermediate-to-large sized lymphoid cells with clear cytoplasm in a diffuse pattern (Figure 10A) (53). Occasional pleomorphism and HRS-like cells may be present. Varying degrees of fibrosis can be seen in PMBL, and the fibrosis often separates the lymphoma cells into small groups, forming an “alveolar” pattern (53).

Figure 10 Image-guided core needle biopsy of a primary mediastinal B-cell lymphoma of thymus. (A) Diffuse clusters of large lymphoma cells seen in a 23-year-old female with mediastinal grey zone lymphoma. The lymphoma cells demonstrate convoluted to occasionally multi-lobulated nuclei with vesicular chromatin and variably prominent nucleoli (hematoxylin and eosin, 200× original). However, the lymphoma cells exhibit a classic Hodgkin lymphoma-like immunophenotype with (B) strong, uniform CD30, CD15 (not shown), (C) TARC, and (D) weak PAX5, while predominantly negative for (E) CD20, (F) OCT-2, and CD45 (not shown). B-F: Polymer-based immunostain, 100×original.

Though fibrosis in PMBL may lead to inadequate flow cytometric studies (due to low cellular yield), when an adequate sample is analyzed, an abnormal B-cell population (CD19+/CD20+/CD22+) with high forward scatter light properties that lack surface kappa and lambda-light chains may be detected (53). By IHC, PMBL is typically positive for CD45, CD20, PAX5, OCT2, BOB1, CD79a, CD30 (most cases, variable and weak staining), MUM1, BCL2, CD23, MAL, and PD-L1/2 (Figure 10B-10F). A subset of cases expresses BCL6, and rarely, CD15-positivity may be encountered (41,52). EBV is typically negative.

Unlike other large B-cell lymphomas (not discussed in this review), MYC, BCL2, and BCL6-rearrangements are usually negative in PMBL (41,54). Molecular features of PMBL show overlap with CHL. Alterations in genes that result in activation of the NFKB pathway (e.g., TNFAIP3, NFKBIE, NFKB2, and IKBKB) and JAK-STAT pathways (e.g., STAT6, IL4-R, JAK1, SOCS1, and PTPN1) as well as in genes involved in immune evasion (e.g., CIITA, CD58, and B2M) are described in PMBL (55,56). With appropriate therapy, PMBL typically has a favorable prognosis (57).

Mediastinal greyzone lymphoma (MZGL) is a B-cell lymphoma that shows features overlapping between CHL and PMBL and poses a significant diagnostic challenge on CNB samples (41). Morphologically, MGZL can look like CHL, PMBL, or both in the same specimen (53). MGZL that morphologically resembles CHL (CHL-like MGZL) will show preserved expression of B-cell markers (CD20, CD19, CD79a, BOB1, OCT2), which are typically down-regulated/negative in CHL. On the other hand, cases that morphologically resemble PMBL (PMBL-like MGZL) will show variable loss of B-cell markers (which are typically uniformly expressed in PMBL) with strong and uniform expression of CD30 and/or CD15 (Figure 10). EBV is usually negative in MGZL. Molecularly, MGZL, CHL, and PMBL show similar mutational patterns (58). When small biopsies show equivocal or overlapping features of CHL and PMBL, pathologists should explicitly communicate uncertainty. It is generally not recommended to diagnose MGZL in a CNB. Obtaining generous samples, preferably incisional biopsy samples, is recommended for histologic and extensive immunophenotypic characterization. Multidisciplinary review to confirm the diagnosis is recommended since the distinction between CHL, PMBL, and MGZL can be subtle, but the therapeutic strategy may be significantly different (59).

T-LBL frequently presents with an anterior mediastinal mass in adolescents and young adults, most frequent in adolescent boys (60,61). T-lymphoblastic leukemia (T-ALL) is defined as >25% blood or bone marrow blasts and may occur in the setting of T-LBL. Morphologically, the lymphoblasts of T-LBL range in size from small to large, show a high nuclear-to-cytoplasmic ratio, may have fine to condensed chromatin, and have inconspicuous to prominent nucleoli (Figure 11). The blasts may also look like mature lymphocytes, which can pose a challenge in histologic sections and on touch preparation slides.

Figure 11 A core biopsy of a mediastinal lymph node from a 50-year-old male with lymphadenopathy shows small to medium-sized blastoid cells with round nuclei, dispersed chromatin, and small nucleoli (hematoxylin and eosin, 400× original).. By immunohistochemistry, these blastoid cells are positive for CD3, CD7, CD34, and Tdt, consistent with T-lymphoblasts. A concurrent bone marrow biopsy showed diffuse infiltrates of T-lymphoblasts. The findings support T-lymphoblastic lymphoma/leukemia.

FC is helpful in characterizing T-LBL and differentiating it from other types of acute leukemias, lymphomas, thymoma, and normal thymic tissue but should be correlated with morphology, IHC (e.g., for thymic epithelial networks), and molecular studies (e.g., T-cell gene rearrangements and T-LBL/ALL associated alterations) as applicable (62). While differentiating T-LBL from thymoma/thymic tissue may be challenging by FC, thymoma/thymic tissue will show preserved thymocyte (T-cell) maturational patterns and knowledge of these patterns is helpful in this differential diagnosis as there is overlap in the phenotype of T-LBL/ALL and thymoma/thymic tissue (63). The characteristic immunophenotype of T-LBL shows expression of cytoplasmic CD3, bright CD7, CD2, and CD5. Surface CD3 is usually decreased to negative. Though, there is variability in the expression and intensity of these markers. The lymphoblasts may be double-negative for CD4/CD8, double positive for CD4/CD8, or show expression of one/the other (64). T-lymphoblasts also demonstrate markers associated with immaturity such as CD1a, CD34, CD117, CD99, and TdT (41).

Early T-precursor lymphoblastic leukemia/lymphoma (ETP-ALL) is a subtype of T-ALL/LBL and distinct diagnosis. It is characterized by T-lineage blasts (demonstrated by cytoplasmic CD3 expression) which show dim/negative CD5, CD1a and CD8-negativity, and expression of one or more myeloid/stem-cell associated antigens (CD11b, CD13, CD33, CD34, CD117, and HLA-DR) without myeloperoxidase (41).

Molecularly, T-ALL shows a diverse spectrum of findings, beyond the scope of this review (65). T-LBL prognostic factors include response to chemotherapy, hematopoietic stem cell transplantation, age, white cell count at presentation, LDH levels, disease stage, and cytogenetic/molecular abnormalities. Minimal residual disease (MRD) detection also informs prognosis (66,67). Relapsed disease has a poor outcome.

Primary thymic MALT lymphoma is a low-grade B-cell lymphoma that typically presents asymptomatically (68). A significant minority of patients have a history of autoimmune disease, especially myasthenia gravis or Sjogren syndrome, sometimes preceding or concurrent with the lymphoma diagnosis (69).

The tumor is composed of a polymorphic infiltrate of small lymphocytes, plasma cells, and characteristic medium-sized cells with irregular nuclei, inconspicuous nucleoli, and moderate cytoplasm (centrocyte-like cells) (Figure 12). Plasma cells are present, particularly at the periphery of the lesion or in the interfollicular areas. Benign residual or reactive lymphoid follicles may be “colonized” and partially effaced by the marginal zone B-cells, though this is less common than in nodal MALT (68,70). A characteristic feature of MALT lymphoma in the thymus is infiltration of Hassall’s corpuscles thymic epithelial cells by the neoplastic lymphocytes, forming lymphoepithelial lesions (LELs). By IHC, tumor cells are positive for CD20 and CD79a while negative for CD10, BCL-6, LEF1, Cyclin D1 and SOX11 (71). However, weak CD10 and/or BCL-6 expression may be seen inside germinal centers with follicular colonization. Further, FC may be helpful in demonstrating kappa or lambda-light chain restriction in B-cells.

Figure 12 Image-guided percutaneous core needle of a marginal cell lymphoma (MALT lymphoma) of the thymus (hematoxylin and eosin staining, 100× original). MALT, mucosa-associated lymphoid tissue.

Diagnosis of neuroendocrine neoplasms by core needle biopsies

The mediastinum can give rise to low-grade and high-grade NET that show identical pathologic features to those seen in the lung and other organs (1,72-76). Low-grade neoplasms include typical carcinoid/neuroendocrine neoplasms grade I and atypical carcinoid/neuroendocrine neoplasms Grade II (77). These tumors can be readily diagnosed on percutaneous core needle biopsies in the presence of epithelioid cells showing round or oval nuclei with “salt and pepper” chromatin pattern, arranged in trabeculae, small islands or pseudorosettes. These neoplasms exhibit growth patterns suggestive of neuroendocrine differentiation, such as cells growing in cords, islands and/or pseudorosettes (Figure 13A-13D). Tumor cells exhibit immunoreactivity for “neuroendocrine markers”, such as chromogranin, synaptophysin, CD56 and/or insulinoma associated protein 1 (INSM1) (Figure 14A-14C). Typical carcinoid/Neuroendocrine grade I usually exhibit no necrosis and rare mitoses. Atypical carcinoid/Neuroendocrine Grade II exhibit presence of punctate areas of necrosis and greater than 2 mitoses in 10 high power fields (Figure 15). It is difficult to subclassify mediastinal NET based on the evaluation of necrosis and mitotic activity on core needle biopsies, due to sampling problems. The proliferative activity of neuroendocrine neoplasms, as measured with Ki-67 immunoreactivity, generally correlates with prognosis but it is not used to subclassify pulmonary or mediastinal lesions.

Figure 13 Image-guided percutaneous core needle biopsy of thymic typical carcinoid/neuroendocrine neoplasm grade I. (A) Cords of uniform tumor cells with round nuclei and amphophilic cytoplasm. The lesion exhibited only rare mitoses and no necrosis; (B) an insular growth pattern composed of small island of tumor cells; (C) pseudorosettes. The histologic growth patterns shown in (A-C) are characteristic of neuroendocrine differentiation and can be seen in carcinoids/NET and high-grade carcinomas, although the latter tumors are composed of considerably more atypical cells than those shown in these figures; (D) the tumor cells of carcinoid tumors/neuroendocrine neoplasm show round or oval nuclei with characteristic “salt and pepper” chromatin pattern. (A,B) Hematoxylin and eosin staining, 200× original; (C,D) hematoxylin and eosin staining, 400× original.

Figure 14 The tumor cells of a typical carcinoid/neuroendocrine tumor grade I show cytoplasmic immunoreactivity for (A) chromogranin A; (B) synaptophysin and (C) nuclear immunoreactivity for INSM1 (Polymer-based immunostain, 200× original). INSM1, insulinoma associated protein 1.

Figure 15 Image-guided percutaneous core needle biopsy of thymic atypical carcinoid/neuroendocrine neoplasm grade II showing punctate necrosis. The lesion also exhibited 4 mitoses/10 high power fields (hematoxylin and eosin staining, 200× original).

High-grade neuroendocrine neoplasms such as large cell neuroendocrine carcinoma and small cell carcinoma can also present rarely as primary thymic neoplasms (74,76,78). Large cell neuroendocrine neoplasms are composed of pleomorphic cells showing round or oval nuclei with variable macronucleoli and scanty amphophilic cytoplasm, arranged in cords, islands and pseudorosettes (Figure 16). Small cell carcinomas can rarely develop as primary mediastinal tumors but most mediastinal small cell carcinomas are metastatic from pulmonary primary lesions (79) (Figure 17).

Figure 16 Image-guided percutaneous core needle biopsy of thymic large cell neuroendocrine carcinoma composed of large, pleomorphic cells forming cords and focal pseudorosettes. The tumor cells exhibited synaptophysin and INSM1 immunoreactivity (hematoxylin and eosin staining, 400× original). INSM1, insulinoma associated protein 1.

Figure 17 Image-guided percutaneous core needle biopsy of mediastinal small cell carcinoma composed of small cells with hyperchromatic nuclei and scanty cytoplasm. It was difficult to determine the tumor origin. The absence of a pulmonary mass or other primary sites on imaging studies was consistent with the possibility of a thymic origin of the tumor. However, small cell carcinomas of the lung can present as small perihilar lesions that can be difficult to detect on imaging studies (hematoxylin and eosin staining, 400× original).

Diagnosis of thymic epithelial cell neoplasms and neuroendocrine neoplasms by TTFNA

The use of TTFNA is challenging for the diagnosis of TET and neuroendocrine neoplasms due to the difficulty in obtaining cellular samples from an area that is difficult and somewhat dangerous to biopsy. However, experienced radiologists can obtain cytological preparations with moderate to high cellularity and the sensitivity of FNA for the diagnosis of mediastinal lesions approaches that of core needle biopsies (26,80). FNA from thymomas shows a dual cell population with varying proportions of neoplastic epithelial cells and reactive T-lymphocytes. In type A thymoma, the epithelial cells are spindle-shaped with fine chromatin and inconspicuous nucleoli. If the lymphoid populations are not prominent, neuroendocrine neoplasm may be considered in the differential diagnosis. Type B thymoma features epithelioid epithelial cells, with the lymphocytic component being most prominent in type B1 and less so in type B2 (Figure 18). Occasionally, type B thymomas with rare epithelial cells can be misinterpreted as benign lymph nodes or lymphomas. FNA from thymic carcinomas show high cellularity and high-grade cytological features. The atypical epithelial cells often appear in cohesive clusters, with enlarged, pleomorphic nuclei, coarse chromatin, and prominent nucleoli. The background may contain necrotic debris and mitotic figures. When a cell block is available, immunohistochemical studies are invaluable for diagnosis. Markers such as CD117 (c-KIT) and CD5 are commonly used to identify thymic origin of carcinoma, while TdT and CD3 help identify immature T-cells and T lymphocytes, respectively. CD99 assists in diagnosing the thymic origin of neoplasm, and p63 is useful for identifying epithelial components (81). FNA from thymic neuroendocrine neoplasms share cytological features with NET found elsewhere in the body. These features include organoid, trabecular, and rosette patterns, along with monomorphic cells with stippled chromatin. Necrosis and mitoses are key factors in determining the histologic grade of the tumor. Tumor cells strongly express keratins and neuroendocrine markers.

Figure 18 Fine needle aspiration biopsy of a WHO type B2 thymoma showing a dual population of lymphocytes and epithelial cells (Papanicolaou stain, 400× original). WHO, World Health Organization.

Diagnosis of germ cell tumors by needle aspiration and core needle biopsies

Primary mediastinal germ cell tumors are unusual neoplasms that are more frequent in young adults (82). They include seminomas with over 90% 5-year survival rates and non-seminomatous germ cell tumors with very poor outcome (Table 1). A few examples of mediastinal seminomas and non-seminomatous germ cell tumors diagnosed by core biopsy, FNA or cryobiopsy have been described (83-85). It is beyond the scope of this brief review to describe the microscopic pathology of all mediastinal germ cell tumors. Fichtner et al. recently reviewed their experience with 32 mediastinal germ cell tumors and proposed an algorithm for their diagnosis (7). Immunostains for octamer-binding transcription factor (Oct3/4), placental-like alkaline phosphatase, Sal-like protein 4 (SALL4), beta-human chorionic gonadotropin (HCG) and others are needed to diagnose and subcategorize mediastinal germ cell tumors (7).

Conclusions

Minimally invasive biopsy techniques play a central role in the evaluation of anterior mediastinal masses when tissue diagnosis is indicated (Figure 19). Optimal diagnostic accuracy depends on appropriate biopsy selection guided by lesion location and clinical suspicion, adequate tissue sampling to preserve architectural features required for histologic subtyping, and systematic integration of ancillary studies, including IHC, FC, and molecular testing. Image-guided percutaneous CNB remains the preferred first-line approach owing to its high diagnostic yield and favorable safety profile, while EBUS-guided sampling serves a complementary role in cases with lymph node involvement or posterior extension, particularly for lymphoproliferative disorders where tissue architecture and immunophenotyping are indispensable for classification. Close collaboration between interventionalists and pathologists, ideally with on-site adequacy assessment, is essential to ensure representative tissue acquisition and accurate diagnosis.

Figure 19 Diagnostic algorithm for anterior mediastinal lesions. *, submit fresh tissues to the intraoperative consultation laboratory to perform frozen section and/or rapid on site evaluation so that pathologist an arrive at a tentative differential diagnosis and triage tissues for flow cytometry, immunohistochemistry and/or molecular studies. β-hCG, beta-human chorionic gonadotropin; AFP, alpha-fetoprotein; CT, computerized tomography; MRI, magnetic resonance imaging.

Acknowledgments

I would like to thank the Cedars-Sinai staff member who assist in retrieving and preparing cases for their invaluable support and dedication.

Footnote

Peer Review File: Available at https://med.amegroups.com/article/view/10.21037/med-2026-1-0009/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://med.amegroups.com/article/view/10.21037/med-2026-1-0009/coif). A.M.M. serves as an unpaid editorial board member of Mediastinum from January 2026 to December 2027. The other 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.

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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