Mediastinal epithelial neoplasms with recurrent molecular alterations

Introduction

The mediastinum is subject to a bewildering array of benign and malignant epithelial tumors, owing in large part to the wide variety of neoplasms that arise from the thymus and lung. Over the last several years, the expansion of molecular genetic testing has led to the discovery of recurrent genetic alterations in a subset of these tumors with these mutations frequently occurring within morphologically and immunohistochemically coherent groups. Diagnostic recognition of tumors with recurrent molecular changes within the larger context of traditional histopathologic classification is critical in today’s age of targeted treatments, allowing for the possibility of genetically informed personalization of therapy. The following discussion serves to help the surgical pathologist become aware of specific molecularly defined entities that one may encounter in practice and their genetic underpinnings.

The genetic landscape of thymic epithelial neoplasms is notable for a recurrent hotspot mutation, GTF2I p.L424H, occurring in the majority of type A and AB thymomas. Among more rare tumors, metaplastic thymoma is a biphasic tumor that harbors a YAP::MAML2 rearrangement. However, other MAML2 rearrangements can also be seen in a subset of otherwise unremarkable type B thymomas. Thymic carcinomas overlap molecularly with thymomas but show increased mutational burden and higher frequency of TP53, CDKN2A, and KIT mutations.

A variety of poorly differentiated epithelial tumors affect the mediastinum and a subset of these show recurrent genetic alterations. NUT carcinoma and SWI/SNF (switch/sucrose non-fermentable) complex deficient tumors, exemplified by thoracic SMARCA4-deficient undifferentiated tumor, represent aggressive, poorly differentiated tumor types that can show morphologic and immunohistochemical overlap with other malignancies; diagnosis is aided by immunohistochemical markers specific for their defined genetic changes.

Thymic epithelial neoplasms

Perhaps reflecting a tumor mutational burden among the lowest of all adult human malignancies, thymomas mirror normal thymus to an extent, showing an organoid growth pattern and a variable accompaniment of immature thymic T lymphocytes (1,2). The neoplastic epithelial cells of thymoma can be round, stellate, or spindle-shaped, and generally show open, evenly dispersed chromatin. Thymomas are divided into histopathologic World Health Organization (WHO) types A, AB, B1, B2, and B3 based on the predominating epithelial morphology and admixture of lymphocytes (2). Types A and AB show spindled thymic epithelial cells with AB containing a subpopulation of lymphocyte-rich (type B-like) areas, usually with abundant T cells. Types B1, B2, and B3 thymomas have variable amounts of thymic lymphocytes with B1 showing the most and B3 showing the least.

The Cancer Genome Atlas (TCGA) consortium described a number of recurrent genomic alterations in thymomas, lending support for the distinctiveness of the WHO histopathologic types (3,4). Specifically, GTF2I p.L424H is present in a large minority of all thymomas but is mostly restricted to spindled thymomas (types A and AB) (4-6). Mutations of GTF2I are rare in human cancers and L424H mutations have only been described in thymoma, making it potentially diagnostically useful to establish thymic origin (4).

Micronodular thymoma with lymphoid stroma and metaplastic thymoma represent rare tumors that do not neatly conform to WHO subtypes (7,8). Unlike the comingling of thymic epithelial cells and T lymphocytes seen in types A and B thymomas, micronodular thymoma is made up of multiple small, variably sized tumor nodules with sparse lymphocytes surrounded by a lymphoid stroma devoid of thymic epithelium (Figure 1) (9). Though data is limited, recent studies have shown recurrent GTF2I p.L424H in nearly all analyzed cases, suggesting a relationship between micronodular thymoma and type A and AB thymomas (10,11). Indeed, associations of micronodular thymomas with other thymoma types have been described with type A being seen in up to 30% of tumors (12,13). However, micronodular thymomas generally behave indolently, with only rare reports of recurrence or distant metastasis (13).

Figure 1 Micronodular thymoma with lymphoid stroma. (A) Micronodular thymoma with lymphoid stroma shows multiple coalescing nodules. Tumors characteristically show separation between the epithelial component and lymphocyte component in contrast with the comingling seen in other thymoma types. (B) The epithelial cells are short, spindled to ovoid cells with scant cytoplasm and contain few interspersed lymphocytes. HE staining; magnification, 20× (A), 200× (B). HE, hematoxylin and eosin.

Metaplastic thymoma is a histologically unique biphasic spindled and epithelial tumor harboring a recurrent YAP::MAML2 translocation (14,15). The epithelial component is comprised of plump to ovoid cells arranged in solid nests that merge with variable amounts of bland, elongated, fibroblastoid spindled cells (Figure 2) (14,15). Lymphoid stroma is not a feature of metaplastic thymoma. Immunohistochemistry targeting the Yap protein’s c-terminus is diagnostic when there is loss of expression (16,17). Other MAML2 translocations have been described in thymoma, however, including KMT2A::MAML2 and KMT2A::MAML3 in B2 and B3 thymomas, the clinical implications of which are not yet clear (18,19).

Figure 2 Metaplastic thymoma. (A) Metaplastic thymoma is a biphasic tumor made up of plump, ovoid epithelial nests and ribbons merging with variable amounts of fibroblast-like spindle cells (HE staining; magnification, 100×). (B) Immunohistochemistry for the c-terminus of YAP shows loss of expression, diagnostic for the YAP::MAML2 translocation seen in metaplastic thymoma (magnification, 100×). There is retained staining in the adjacent normal thymus. Notably, lymphocytes also show variable loss of expression. HE, hematoxylin and eosin.

Thymic carcinomas comprise a heterogeneous group of malignant epithelial tumors that can evade specific classification as thymic in origin, and consequently, are ideally excluded from originating elsewhere (20). Thymic squamous cell carcinomas show morphologic similarity to squamous cell carcinomas of other sites with a few distinctive features. Smooth contoured expansile nests separated by fibrous banding is more commonly seen in tumors of thymic origin, reminiscent of the growth of invasive thymoma. Keratinization is typically limited. Immunopositivity for CD117 and/or CD5, when present, helps distinguish from non-thymic squamous cell carcinomas. Thymic carcinomas lack an ultraviolet (UV) signature by next-generation sequencing, enabling their differentiation from cutaneous primaries.

Differentiation of type B3 thymoma from thymic carcinoma can occasionally be difficult. Carcinomas usually show overt features of malignancy including frank invasion, frequent foci of necrosis, and pleomorphism, but these features may not be well-developed. Thymic carcinoma-specific immunohistochemistry including c-kit and CD5 as well as evaluation for immature thymic lymphocytes [terminal deoxynucleotidyl transferase-positive (TdT+)] found in thymoma can be helpful in the differential diagnosis.

Thymic epithelial neoplasms: genetic landscape and molecular subtyping

Rapid expansion in molecular genetic testing has led to a greater understanding of the genetic underpinnings and predicted behavior of thymic epithelial tumors. Tumor mutational burden has gained traction as an important genetic measure, with studies showing possible trends in mutational burden based on WHO histotype. Type B thymomas have shown a trend of increasing mutational burden as lymphocyte infiltration decreases (4,21), however some have reported no trend (6). Multiple groups demonstrated that type A and type B3 thymomas frequently harbor more somatic mutations than other thymoma types, although data is somewhat mixed (4,22,23). Lastly, most data points towards thymic carcinomas as having significantly higher mutational burdens than thymomas (4,21,23-25). Tumor mutational burden has the potential to predict immune checkpoint inhibitor response, however, higher baseline immune-related phenomena i.e. paraneoplastic autoimmunity, contraindicate their use in the treatment of thymomas and even thymic carcinomas (26).

In addition to demonstrating WHO histopathologic type and molecular genetic congruence, TCGA data has been further used to propose molecularly informed subtypes of thymic epithelial neoplasms. Radovich et al. described four molecular subtypes through integrated clustering of thymomas and thymic carcinomas, resulting in A-like, AB-like, B-like, and thymic carcinoma-like clusters (4). These groups demonstrated rough concordance with WHO histopathologic subtypes and showed stratification with respect to survival time, suggesting a prognostic value. GTF2I mutations, as they are prevalent in WHO types A and B, were seen in A- and AB-like groups (Figure 3).

Figure 3 Thymic epithelial neoplasm common recurrent molecular alterations. Depicted are general molecular genetic trends in thymoma and thymic carcinoma. GTF2I mutations are highly prognostically relevant, and generally, tumors harboring this mutation do not recur. Most commonly, GFT2I mutations are seen in a large minority of A and AB thymomas as well as nearly all described micronodular thymomas. GTF2I mutations are rarely seen in other tumor types including thymic carcinoma. YAP::MAML2 mutations are specific to metaplastic thymoma. CDKN2A, TP53, and KIT mutations are seen most commonly in thymic carcinomas and to a lesser extent, in type B3 thymomas, reflecting their histopathologic overlap.

In contrast, Lee et al. grouped all tumors with GTF2I mutations into a single subtype, with the remainder divided into T-cell signaling enriched, chromosome stability, and chromosome instability tumors (27). They describe a stepwise molecular approach to arriving at each subtype, perhaps offering a practical approach to prospective testing. Their classification system parallels WHO histopathologic types less well than Radovich et al. and distributes B subtypes among several groups. Despite this, groups showed tight associations with disease free survival and overall survival, and in certain circumstances, showed higher relevance to prognosis over traditional histopathologic classification. For example, among patients diagnosed with B2 and B3 thymomas and thymic carcinoma, tumors with GTF2I mutations did not recur. Likewise, patients with Masaoka stage III–IV disease did not recur in the setting of GTF2I mutations, paralleling the more indolent behavior of micronodular thymoma with lymphoid stroma which harbors frequent GTF2I mutations. Currently available TCGA data confirms an absence of recurrence for B2 and B3 thymomas and thymic carcinomas harboring GTF2I L424H. Recurrence of a type B thymoma with a GTF2I mutation has been described in at least one study; however, this cohort was not subjected to central pathology review (28). Nonetheless, the presence of GTF2I mutations represents a highly prognostically relevant factor.

The overlap of B3 thymoma and thymic carcinoma in the above molecular genetic classifications mirrors the difficulty encountered in histopathologically separating tumors that straddle the borders of thymoma and thymic carcinoma. Both tumors can be infiltrative and metastatic and show morphologic and immunohistochemical overlap (Figure 4). B3 thymomas and thymic carcinomas frequently show more somatic mutations and large-scale copy number alterations compared to other thymic epithelial neoplasms (21). Although more commonly identified in thymic carcinomas, TP53 and CDKN2A mutations are also observed in B3 thymomas and cannot in isolation be used to distinguish between these tumors (23,27,29). Genetic differences have been described, however; the presence of KIT mutations, for example, favor thymic carcinoma (18,29,30).

Figure 4 Thymic carcinoma showing histopathologic overlap with thymoma. B3 thymoma and thymic carcinoma show relatively higher tumor mutational burden and chromosome instability compared to other thymic epithelial neoplasms, paralleling the difficulty in histologically separating cases with borderline features. (A,B) This case showed multifocal pulmonary metastases, and despite the presence of TdT+ lymphocytes, was favored to represent thymic carcinoma due to necrosis, pleomorphism, prominent mitotic activity including atypical mitoses, and immunopositivity for CD117. HE staining; magnification, 20× (A), 200× (B). TdT+, terminal deoxynucleotidyl transferase-positive.

KIT mutations occur in about one in ten thymic carcinomas and response to targeted small molecule inhibitors is well-documented to occur in a significant subset of patients (26,31). Alterations in epigenetic regulation including histone modification, chromatin remodeling, and DNA methylation appear to be more common in thymic carcinomas than thymomas and represent a potential, though largely unexplored therapeutic target (32).

Poorly differentiated tumors with recurrent molecular alterations affecting the mediastinum

NUT carcinoma

NUT carcinoma is among the most aggressive human malignancies and frequently presents with metastatic disease (33-35). Tumors show nests and sheets of primitive, monotonous round cells with prominent nucleoli and areas of necrosis (Figure 5) (36). Areas of spindling are frequently present. Abrupt keratinization, small foci of squamous differentiation with sharp demarcation manifesting as cytoplasmic eosinophilia or clearing and occasional keratin pearl formation, is a clue to the diagnosis but only present in a subset of cases (36). The majority of cases will be cytokeratin positive but occasional cases can show minimal evidence of epithelial origin. On small biopsies, abrupt keratinization may not be appreciated.

Figure 5 NUT carcinoma. (A) NUT carcinoma of the mediastinum. NUT carcinoma should be suspected when encountering sheets of primitive-appearing, monotonous cells with prominent necrosis in the setting of an aggressive lesion (HE staining; magnification, 100×). (B) NUT carcinoma of the mainstem bronchus showing sheets of primitive cells with a prominent neutrophilic infiltrate, which is commonly seen (HE staining; magnification, 200×). (C) NUT carcinoma expressing focal synaptophysin (IHC; magnification, 200×). Neuroendocrine marker immunoreactivity in NUT carcinoma is typically limited to focal positivity and represents a potential pitfall. (D) NUT carcinoma expressing focal INSM1 (IHC; magnification, 200×). (E) Speckled nuclear immunoreactivity for NUT protein is highly sensitive and nearly entirely specific for the diagnosis (IHC; magnification, 200×). HE, hematoxylin and eosin; IHC, immunohistochemistry.

NUT carcinoma arises as a result of a complex catastrophic genomic rearrangement (“chromoplexy”) involving multiple simultaneous translocations, resulting in NUTM1 gene fusions with partners BRD4, BRD3, NSD3, and others (37,38). Tumor mutational burden is accordingly low, and genomic changes important to routine treatment paradigms including microsatellite instability and PD-L1 expression are absent (39-42).

Immunohistochemistry for NUT showing diffuse speckled nuclear labeling is highly specific and quite sensitive for the diagnosis (43). Germ cell tumors occasionally show weak, focal staining with NUT (43). A subset of NUT carcinomas show variable, usually weak neuroendocrine marker staining which can lead to misdiagnosis as a neuroendocrine neoplasm (42,44,45). CD34 and CD99 immunoexpression have also been reported but these are largely negative (42).

SWI/SNF complex deficient tumors

SWI/SNF describes a large family of chromatin-remodeling proteins that normally act in a tumor suppressive function, facilitating DNA repair (46). Alterations in SWI/SNF are among the most common genetic events in human cancers; however, for certain tumors, biallelic inactivation is a tumorigenic driver event.

Thoracic SMARCA4-deficient undifferentiated tumor is one such group that arises in the mediastinum and lung. These aggressive tumors show sheets of large, round cells with typically mild pleomorphism that can be marked in certain tumors (Figure 6). Rhabdoid morphology is not consistently seen, and overt spindling is rare; however, sarcomatoid variants have been well-described (47,48). As its name suggests, SMARCA4-deficient undifferentiated tumors express only focal or negative cytokeratins, TTF1, and p40. Epithelial origin is however evidenced by occasional cases showing an otherwise conventional non-small cell carcinoma component as well as an overall genetic similarity to lung adenocarcinoma and squamous cell carcinoma including a smoking signature, high tumor mutational burden, and mutations in KRAS, STK11, and KEAP1 (47,49). Despite this, claudin-4 is negative or only focally positive. SMARCA4-deficient undifferentiated tumors show a strong association with smoking that is perhaps greater than that seen in non-small cell lung carcinomas (47,49).

Figure 6 SMARCA4-deficient undifferentiated thoracic tumor. (A) SMARCA4-deficient undifferentiated thoracic tumor of the left mainstem bronchus showing sheets of round, loosely cohesive cells and occasional pleomorphic cells (HE staining; magnification, 200×). Rhabdoid features, as seen here, may be encountered. (B) SMARCA4-deficient undifferentiated thoracic tumor with multifocal pulmonary disease and extension into the mediastinum showing sheets of epithelioid to spindled cells with prominent necrosis (HE staining; magnification, 200×). (C) Immunohistochemically, cases lack evidence of a specific line of differentiation, i.e., TTF1 and p63/p40 negative (magnification, 200×). BRG1 (SMARCA4) loss of expression is diagnostic. Complete loss of BRG1, shown here, is most commonly observed and correlates with truncating mutations of SMARCA4. (D) Most cases show concurrent loss of SMARCA2 (IHC; magnification, 200×). (E) INI1 is intact; however, tumors with similar morphology can show loss of INI1 with retained BRG1 (IHC; magnification, 200×). HE, hematoxylin and eosin; IHC, immunohistochemistry.

BRG1, the protein product of SMARCA4, shows immunohistochemical loss of expression in the majority of cases with a minority showing severe attenuation diffusely in comparison to internal controls (50). Genetic inactivation of SMARCA4 results in reliance on SMARCA2 for chromatin remodeling and selective pressure for its subsequent silencing in tumor cells. Consequently, co-deficiency of SMARCA2 is observed in most cases. There is increasing recognition of isolated SMARCA2-deficient cases with SMARCA4 proficiency arguing for the application of both immunostains in diagnostic workup (51). SMARCB1-deficient tumors have been reported in the literature with a similar morphology and immunophenotype to thoracic SMARCA4-deficient undifferentiated tumor.

Thymic carcinomas frequently exhibit mutations in genes related to chromatin remodeling and epigenetic regulation, and a subset of thymic squamous cell carcinomas show biallelic SMARCA4 deficiency (32,52). These tumors would be expected to be diffusely p40 positive and show squamous differentiation. SMARCA4-deficient undifferentiated thoracic tumors, like SWI/SNF-deficient tumors of other sites, show variable degrees of neuroendocrine marker expression, and may also show CD34 and SALL4 positivity (50,53,54). Thymic large cell neuroendocrine carcinoma can overlap as well but shows a nested architecture and typically more keratin expression.

Conclusions

Thymomas can range from tumors that do not recur following definitive surgery to aggressive malignancies that present with metastatic disease. While traditional histopathologic classification and staging provide good prognostication, molecular genetic information can be used to predict behavior. The presence of GTF2I p.L424H mutations, which are present in most described cases of micronodular thymoma and a large minority of A and AB thymomas, is an indicator of good prognosis and tumors generally do not recur. Metaplastic thymoma is a histologically unique biphasic tumor harboring a YAP::MAML2 translocation that can be confirmed immunohistochemically through Yap c-terminus antibodies. Thymic carcinomas, and to a lesser extent B3 thymomas, have higher tumor mutational burden and frequent CDKN2A and TP53 mutations.

A variety of poorly differentiated malignancies arise in the mediastinum, and tumors can show morphologic and immunohistochemical profiles that overlap with other tumor types. NUT carcinoma should be suspected in the setting of an aggressive tumor showing primitive round cells with morphologic and/or immunohistochemical evidence of squamous differentiation. Immunohistochemistry for NUT is diagnostic. SWI/SNF complex deficient tumors are a group of poorly differentiated aggressive malignancies or epithelial origin that frequently do not show extensive cytokeratin labeling. Thoracic SMARCA4-deficient undifferentiated tumor is defined by BRG1 loss of expression and negativity for claudin-4, and generally lacks evidence of a specific line of differentiation. Typically, BRG1 shows complete loss, reflecting truncating mutations in SMARCA4. In contrast, SMARCA4-deficient non-small cell lung carcinoma is claudin-4 positive and may show evidence of pulmonary differentiation. BRG1 frequently shows partial loss or severe attenuation.

Acknowledgments

We are grateful to Dr. Peter Illei for generously providing case material for Figure 2. We are also grateful for the contributions of Dr. Lisa Rooper for generously providing case material for Figure 1 and Figure 5. The authors would also like to acknowledge the TCGA Research Network’s thymic epithelial tumors dataset for data used in this study (https://www.cancer.gov/tcga).

Footnote

Peer Review File: Available at https://med.amegroups.com/article/view/10.21037/med-2025-1-76/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-2025-1-76/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.

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