Diagnostic significance and cytological features of NUT carcinoma by EBUS‐FNA, a case report and literature review

Cytomorphological features of NUT carcinoma include sheets or discrete nests of primitive, monotonous, round to oval shaped tumour cells with high N/C ratio and brisk mitotic figures. Abrupt squamous differentiation might be a diagnostic hint. More than 50% positivity of NUT immunohistochemistry staining is diagnostic. NUT carcinoma represents a poorly differentiated malignancy by extremely aggressive clinical course and poor prognosis. It frequently manifests in midline organs, notably in the mediastinum and lung. The rising preferences for utilizing the EBUS-FNA procedure in diagnosing thoracic and lung lesions stems from its high diagnostic yield. Hence, recognizing the cytomorphological features of NUT carcinoma is crucial for timely treatment and improved patient survival. NUT carcinoma, initially described in the early 1990s, has recently gained recognition due to its rarity and underdiagnosis. This aggressive tumour commonly arises in midline organs, such as the lung, thyroid, and salivary gland. It presents as an overall survival (OS) of 6. 7 months, with thoracic cases showing an OS of 4. 4 months, and head and neck cases exhibiting an OS of 9. 7 months. 1 NUT carcinoma is defined by NUTM1 gene rearrangements, featuring a distinctive chromosomal (15;19) translocation that results in the formation of a new fusion gene: BRD4: : NUT. While affecting both genders equally, this tumour demonstrates a higher incidence among children and young adults, though it can occur across all age groups. Approximately 20 new cases of NUT carcinoma are reported to the International NUT Midline Carcinoma Registry each year. Its fully recognition came in 2003 when scientists identified the BRD4-NUTM1 fusion gene as its' defining molecular base. 2 Subsequently, BRD3: : NUTM1 fusion gene and NSD3-NUTM1 fusion gene were also reported in isolated cases. Despite its common occurrence in midline anatomical sites, NUT carcinoma has also been described in various organs such as the parotid gland, pancreas, adrenal gland, bladder, and iliac bone. 3 In this report, we present a case of NUT carcinoma involving multiple midline structures diagnosed by endobronchial ultrasound-guided fine needle aspiration (EBUS-FNA). We discuss its distinctive cytomorphological features, highlighting diagnostic challenges, and pitfalls. A 40-year-old female presented to the emergency room with abdominal fullness and weakness. A CT scan showed a 12 cm solid, cystic, left ovarian mass along with multiple pelvic nodules concerning for carcinomatosis (Figure 1). In addition, a 1. 5 cm left pericardial soft tissue nodule was noted with extension to the left upper lobe, along with a nearly complete occlusion of the left pulmonary artery and left upper lobe airway. After 1 week, a repeat CT scan showed increasing peri-bronchial vascular consolidative, ground glass opacities in the left upper lobe, and septal thickenings consistent with lymphangitic carcinomatosis. An EBUS-FNA was performed with a rapid on-site evaluation (ROSE) for ovarian cancer staging. At ROSE, Diff-Quik smears showed primitive tumour cells arranged in sheets and clusters, displaying nuclear crush artefact and nuclear moulding. Scant cytoplasm with intracytoplasmic vacuoles was frequently observed, while definite glandular configurations were absent. Pap-stained smears revealed sheets, discrete nests, and isolated cells of primitive, monotonous, round to oval-shaped tumour cells with a high N/C ratio and brisk mitotic figures. Hyperchromatic/basaloid appearing nuclei resembling small cell neuroendocrine carcinoma were present, albeit with powdery to finely granular chromatin and prominent nucleoli instead of granular stippled chromatin. Additionally, cell block sections identified a minute focus of abrupt keratinized squamous cells (Figure 2). Immunohistochemical stains (IHCs) (Figure 3) showed tumour cells positive for p63, p40, CK7, and weakly positive for EMA; while negative for chromogranin A, synaptophysin, NapsinA, TTF-1, WT1, and PAX8. Anti-NUT antibody stain (clone C52B1, 1: 45; Cell Signaling) showed speckled nuclear positivity. A diagnosis of NUT carcinoma was confirmed. Furthermore, whole transcriptome sequencing (WTS) detected a BRD3: : NUTM1 fusion, with the splice site of exon 3 of NUTM1 (NM₁75741. 2) joining in-frame exon 11 of BRD3 (NM₀07371. 3) resulting in the pathogenic fusion. The patient was recommended to enrol in the international NUT Carcinoma Registry and commenced treatment with both chemotherapy and immunotherapy. A restaging imaging study conducted 13-month later revealed disease progression, characterized by new pleural effusion, exacerbated mediastinal disease, and the emergence of liver and brain metastasis. The patient underwent a supracervical hysterectomy, bilateral salpingo-oophorectomy, and left lobe liver resection, confirming the involvement of NUT carcinoma in both ovaries and liver. NUT carcinoma is a poorly differentiated, clinically aggressive, and highly malignant entity recognized in the fifth edition of the World Health Organization (WHO) classification of Thoracic Tumours, characterized by NUT gene rearrangement. According to a comprehensive study analysing 10, 100 solid tumours, the estimated incidence of NUT carcinoma is 0. 21%. 4 Diagnosing NUT carcinoma via tissue biopsy presents challenges due to its broad range of differential diagnosis and rarity, resulting in limited reporting of cytological features. In this study, we present a case of NUT carcinoma diagnosed through EBUS-FNA cytology, initially suspected, and clinically described as an ovarian mass with pelvic carcinomatosis and distant metastasis to the mediastinum. Cytomorphological findings of NUT carcinoma exhibit distinctive characteristics, yet they often much overlap with features of other entities, posing diagnostic challenges. Smears typically display high cellularity, comprising cohesive clusters and isolated primitive, round to oval, medium-sized tumour cells. Enlarged nuclei with fine to coarsely granular chromatin, prominent nucleoli, and a high mitotic rate are common features. Additionally, nuclear moulding and chromatinic smearing may mimic neuroendocrine tumours. Background elements frequently include necrosis, bare nuclei, karyorrhectic cellular debris, and neutrophilic infiltrates. Clusters of cells with a moderate cytoplasm and distinct borders, suggesting squamous differentiation, may occasionally appear. However, definitive foci of abrupt squamous differentiation with keratinization are better appreciated on the cell block. While the presence of squamous differentiation aids in identifying NUT carcinoma, it may be missed on small samples. 5 Prior studies summarizing the cytomorphological features of NUT carcinoma are outlined in Table 1. In our case, during ROSE, malignant cells with neuroendocrine features were communicated to the interventional pulmonologist. A panel of immunohistochemical stains encompassing Mullerian, lung, and neuroendocrine markers was conducted. Positive staining for P63, p40, CK5/6, and CK7 along with negative Mullerian and negative neuroendocrine markers, essentially ruled out Mullerian origin. Considering the tumour's cytomorphology, presence of abrupt squamous keratinization on cell block, expression of squamous markers, thoracic location, and occurrence in a young, non-smoking female with advanced tumour stage, NUT carcinoma was strongly considered. Additionally, small cell carcinoma should be considered in the differential diagnosis if nuclear features resembling those of neuroendocrine tumours, such as moulding and chromatinic smearing, are observed. 5 However, small cell carcinoma typically exhibits more frequent nuclear moulding and chromatinic smearing compared to NUT carcinoma. Unlike NUT carcinoma, small cell carcinomas lack prominent nucleoli and abrupt foci of squamous differentiation. Synaptophysin, chromogranin A, and INSM1 can aid in this distinction, although NUT carcinomas may exhibit focal positivity, which can further complicate the diagnostic process. 6 Furthermore, electron microscopy usually reveals no ultrastructural evidence of neuroendocrine differentiation in NUT carcinoma. Other potential diagnostic pitfalls that may include poorly differentiated lung adenocarcinoma, as NUT carcinoma can occasionally express TTF-1, as reported by Hung et al. 6 However, NUT carcinoma typically lacks pseudo-glandular formations, and poorly differentiated lung adenocarcinoma would test negative for p40 and p63. NUT carcinoma has also been reported to be positive for CD99, which can be mistaken for Ewing sarcoma, 7 Nevertheless, Ewing sarcoma does not typically show squamous differentiation. NUT positivity is crucial for diagnosing NUT carcinoma, with the gold standard test for NUT carcinoma being FISH for NUT rearrangement. In a recent large cohort study by Farooq et al, findings from 55 NUT carcinoma cases revealed that p40 was positive in 65% of cases, p63 in 87%, and INSM1 in 54%. 8 However, immunohistochemistry results can be misleading, and without appropriate awareness, can lead to misdiagnosis. Therefore, it is advisable to order NUT IHC whenever primitive cells lacking discrete glandular configuration are identified, especially in the appropriate clinical setting. NUT IHC, developed in 2009, typically displays characteristic speckled nuclear staining for the NUT antibody. A study indicated that NUT immunostaining alone provided 100% specificity, 87% sensitivity, 99% negative predictive value, and 100% positive predictive value for NUT carcinoma diagnosis. 9 Additionally, Zhuang et al. 10 reported that all seven NUT carcinoma cases in their small cohort tested positive for NUT IHC. The C52 antibody was found to stain NUT carcinoma cell line C-797, containing the BRD4: NUT4 fusion gene. Haack et al. noted that tumours positive for NUT (C52) IHC exhibited strong, diffuse speckled nuclear staining (>90%), while negative tumours lacked staining. However, they identified four false-positive cases: one autopsy case showed weak staining, possibly due to postmortem antigen degradation; two cases had NUT-variant translocations with potential lower NUT levels; and the fourth case was attributed to poor tissue preservation. 9 Weak cytoplasmic staining, although not uncommon, was considered nonspecific. Notably, hepatocytes and renal tubular cells may exhibit cytoplasmic staining, while oocytes may display weak nuclear and cytoplasmic reactivity due to low endogenous NUT expression. NUT IHC combined with FISH offers100% sensitivity and specificity, whereas FISH alone shows 93% sensitivity and 100% specificity. NUT IHC positivity was also seen in germ cell tumours, particularly dysgerminomas, due to the normal NUT expression. However, these cases lack NUT rearrangements found in NUT carcinomas. To further aid in this differentiation, germ cell tumours typically exhibits focal NUT IHC positivity and smooth nuclear staining contrasting with the diffuse speckled pattern seen in NUT carcinomas. Additionally, NUT carcinomas lack germ cell markers, aiding in their distinction from germ cell tumours. 11-13 The NUTM1 gene exhibits expression solely in post-meiotic spermatids during spermatogenesis. Roughly two-thirds of NUT carcinomas feature a reciprocal chromosomal translocation involving NUT on chromosome 15q14 and BRD4 on chromosome 19p13. 1, resulting in the form of an in-frame BRD4-NUT fusion oncogene. Rarely, NUT carcinoma may harbour BRD3-NUT rearrangements, with only eight reported cases of BRD3-NUT rearrangements thus far. Remarkably, seven of these cases manifested in the lung (refer to Table 2). In this report, we present another case of lung NUT carcinoma withBRD3-NUT rearrangement; however, further evidence is necessary to establish the correlation between the BRD3-NUT fusion gene and its propensity in lung carcinomas. Additionally, other NUT fusion partners such as NSD3 (nuclear receptor binding SET domain protein 3, chromosome 8), ZNF532 (zinc finger protein 532, chromosome 18), ZNF592 (zinc finger protein 592, chromosome 15), and CIC (capicua transcriptional repressor, chromosome 19) have also been reported. 3, 14-17 These fusions play a critical role in the pathogenesis of NUT carcinoma by impeding squamous cell differentiation and instigating uncontrolled proliferation of primitive cells. NUT carcinoma presents a poor prognosis, typically with a median overall survival of 6. 7 months. 1, 18 Currently, two clinical trials are underway to investigate the efficacy of Bromodomain and extra-terminal domain (BET) protein inhibitors in treating NUT carcinoma. One clinical trial is a phase 1/2 study of the Bromodomain Inhibitor ZEN003694 in combination with etoposide/platinum among patients with NUT carcinoma, expected to conclude by December 2023. The second clinical trial, a phase 1 study, assesses the efficacy of Bromodomain inhibitor BMS-986158 and BMS-986378 in the paediatric population with an estimated completion date of June 2024. In this case, the patient has survived for 14 months post-diagnosis while receiving ZEN003694 in combination with pembrolizumab; however, the most recent evaluation revealed disease progression with the emergence of new liver and brain metastasis. In conclusion, EBUS-FNA has emerged as a safe and effective procedure increasingly utilized in the diagnostic assessment and staging of intrathoracic lesions. It is of significant importance for cytopathologists to recognize NUT carcinoma in cytology specimens; however, given its rarity and the requirement for specific molecular study, there is a risk of underdiagnosed or misinterpretation. NUT carcinoma should be approached with caution, particularly in cases exhibiting high-grade cytomorphology without a glandular component in the midline organs. While the presence of abrupt squamous differentiation can serve as a helpful diagnostic clue, it may not always be evident in small cytology samples. 13 Yaping Ju: Data collection, drafted and edited manuscript. Miriam Velazquez: Data collection, edited manuscript. Andy Sherrod: Designed the study, edited the manuscript and made intellectual contribution. Tiannan Wang: Designed the study, drafted and edited the manuscript. N/A. All authors declared no conflict of interest. All data included in this study are available upon request by contact with the corresponding authors.