This study demonstrates that NMC represents a small but appreciable percentage of poorly or undifferentiated mediastinal malignancies, including examples of both epithelioid and round cell types. Using IHC, we detected overexpression of the NUT protein in 4 of 114 (3.5%) mediastinal tumors including thymic carcinomas and difficult-to-classify malignancies. The frequency of NMC was enriched among undifferentiated mediastinal tumors (10%). To confirm the mechanism of NUT overexpression, we performed FISH for NUT
; two cases contained a NUT
translocation and one contained a NUT
-variant translocation. One case lacked a demonstrable rearrangement using probes against NUT
despite high levels of NUT protein expression; this case likely harbored a cryptic translocation, described in detail below.10
The incidence of mediastinal NMC is within the same order of magnitude as that found at other anatomic sites. Previous reports that relied on FISH for screening demonstrated that 7% of midline carcinomas in children and young adults and 18% of undifferentiated carcinomas of the upper aerodigestive tract harbor a NUT
In contrast, only two new cases of NMC were detected amongst a series of >1000 neoplasms that were screened by NUT IHC without being selected for histologic type or anatomic location.10
The variable rate of detection is most likely influenced by case selection methodologies; in particular, the incidence reported in this and some other studies may be biased by the substantial number of cases drawn from personal consult files containing difficult-to-classify tumors. One additional previously unsuspected case of mediastinal NMC was identified in 2010 in consultation at BWH, following the introduction of routine IHC testing for NUT; this case is not included in this series. Ultimately, this study demonstrates that although NMC can be reproducibly detected by IHC among mediastinal tumors from a wide age range of patients, the incidence does not indicate a strong predilection for the thymus or the mediastinum in comparison to other sites.
The large number of consult cases in this study precluded our ability to systematically examine the clinical features of this patient cohort. However, all three NMC cases for which some clinical details were available presented with locally and/or systemically advanced disease, consistent with prior reports of aggressive behavior. While it is interesting to note that NMC was identified more frequently among cases with undifferentiated histology, our study was not powered to detect a statistically significant difference in incidence between different tumor subgroups. Histologically, all four mediastinal NMCs in this series were predominantly comprised of undifferentiated round-to-epithelioid cells growing in nests and sheets. With the exception of rare CK7 and CD5 immunoreactivity in one case, all NMCs in this study were negative for a broad panel of immunohistochemical markers. These results are consistent with previous reports, which have emphasized that NMCs typically exhibit the cytologic features of a poorly differentiated or undifferentiated carcinoma.1,10,19
Some cases of NMC show abrupt squamous differentiation, a feature that has been associated with NUT
-variant translocations in prior reports, although this was not true of the single case with squamous differentiation in this series.7
The frequency of squamous differentiation in this study (1 of 4) is similar to that described in a series of NMC from the upper aerodigestive tract (2 of 5).18
In contrast, squamous differentiation was common (9 of 11 cases) in one study examining a cohort of poorly differentiated epithelial tumors from patients <40 years old.7
The presence of a NUT
-translocation was confirmed in three of the four NUT-overexpressing tumors identified in this study. The single FISH-negative case that exhibited strong nuclear NUT protein expression is included in the series based on previous data demonstrating 100% specificity of the NUT C52 monoclonal antibody for tumors which harbor translocations involving NUT
As in our series, Haack et al.
identified two cases of NMC by NUT IHC that were negative using a similar FISH assay. Upon further study, these cases did in fact contain a NUT-BRD4
rearrangement, secondary to a small interstitial deletion involving the NUT
gene on chr15 that cannot be detected using the flanking probes employed in this study (a so-called cryptic NUT
rearrangement). In fact, these authors concluded that they could only attain 100% sensitivity for diagnosis of NMC by employing a combined NUT IHC and “gold standard” FISH (i.e. flanking probes) approach.10
Because we did not employ a combined screening approach in our series, our cumulative data may slightly underestimate the true incidence of mediastinal NMC seen at a tertiary care referral center.
A strength of our study is that it has examined a large number of primary mediastinal malignancies for the presence of NUT protein expression using whole tissue slides. While most of the NUT-positive cases were diffusely positive, there was some variable expression that may have led to false negatives had this study been performed using a tissue microarray. This study was limited by access to corresponding clinical data, therefore it is difficult to comment on patient features associated with mediastinal NMC such as smoking status or other potential risk factors.
Studies of the molecular pathogenesis of NMC have also implicated the BRD-NUT fusion oncoprotein in squamous cell carcinogenesis. While the role of NUT in normal tissues remains unknown, inhibition of BRD-NUT fusion proteins in NMC cell lines arrests cell growth and promotes striking and irreversible squamous cell differentiation in vitro.6,9
Consistent with the known role of BRD family members in chromatin remodeling, the fusion oncoproteins appear to promote epigenetic reprogramming via globally decreased histone acetylation and transcriptional repression, thus leading to undifferentiated cell growth.16,20
In combination, the histopathologic features described above and the laboratory data on BRD-NUT fusion protein-mediated oncogenesis support the notion that NMC is a distinctive genetic variant of squamous cell carcinoma, or perhaps a relatively undifferentiated tumor arising from a common squamous cell precursor.
Rapid advances in the targeted treatment of other translocation-driven tumors, such as anaplastic lymphoma kinase (ALK)-rearranged lung carcinoma and BCR-ABL in chronic myelogenous leukemia, serve as exciting precedent for diagnosis and treatment of this under recognized tumor.3,13
In fact, insights into the oncogenic role of BRD-NUT fusion proteins have triggered research into novel, directed therapies with histone deacetylase inhibitors and bromodomain-ET protein family (BET) members, including small molecule inhibitors of BRD4.2,4,15,16
As with other models of targeted therapy, advancing therapeutic research beyond the preclinical stage will by necessity require accurate recognition of NMC. Until the clinical features of this tumor type are better clarified, the diagnosis is almost entirely dependent upon the pathologist. Our findings indicate that the diagnosis of NMC should not be overlooked in the differential of any difficult-to-classify epithelioid mediastinal malignancy. In this series examining poorly differentiated malignancies of the mediastinum, NMC is relatively frequent, occurring in approximately 10% of undifferentiated tumors. Therefore, use of NUT IHC to evaluate for NMC should be considered in any poorly differentiated or undifferentiated epithelioid malignancy (with or without focal keratinization), particularly in the setting of otherwise negative immunohistochemical studies. Further advances in the treatment of NMC will require more widespread recognition of this aggressive malignancy.