Clinicopathological Significance of NUT Rearrangements in Poorly Differentiated Malignant Tumors of the Upper Respiratory Tract

Abstract

Nuclear protein in testis (NUT) midline carcinoma (NMC) is a highly malignant carcinoma originating from the midline of the body. This study investigated the clinicopathological significance of NUT rearrangements in poorly differentiated malignant tumors (PDMTs) of the upper-respiratory tract (URT) in China. The clinical and pathological features of 155 PDMTs of the URT were reviewed. Epstein-Barr virus (EBV)-encoded RNA and NUT were investigated by in situ hybridization and immunohistochemistry (IHC), respectively. NUT-positive cases were examined by fluorescence in situ hybridization (FISH) and immunohistochemical staining with a set of cytokeratins (CKs) and neuroendocrine markers. One case was observed by transmission electron microscopy. Four cases of poorly differentiated squamous cell carcinomas and sinonasal undifferentiated carcinomas were diffuse positive for NUT by IHC and also stained for antibodies to CKs and P63 but were negative for neuroendocrine markers. Only 2 of these 4 cases showed rearrangements of the NUT and BRD4 genes by FISH; both these patients died within 12 months. The remaining 2 patients showed no NUT rearrangement by FISH and did not have an aggressive clinical course. NMC is a rare, poorly differentiated carcinoma, which occurs most often in midline organs, and in this first series from China, affected the sinonasal tract of older adults and was not associated with EBV infection. Determination of NUT protein expression and gene rearrangement can allow the differentiation of NMC from other URT PDMTs. The authors suggest that molecular determination of NUT gene rearrangements should therefore represent the gold standard for NMC diagnosis.

Keywords

NUT midline carcinoma BRD4-NUT poorly differentiated malignant tumor upper-respiratory tract

Introduction

Nuclear protein in testis (NUT) midline carcinoma (NMC) represents a rare, clinically aggressive carcinoma, molecularly defined by rearrangement of the NUT gene. These malignant tumors require distinct therapies. The NUT gene is located on chromosome 15q14 and is expressed exclusively in the testes under normal conditions. In BRD4-NUT NMCs, almost the entire gene is translocated and fused in-frame to the BRD4 gene on chromosome 19p13. Carcinomas with the specific t(15;19)(q14;p13) translocation comprise the majority of NMCs.¹ In about one third of NMCs termed NUT-variant tumors, however, NUT is fused to BRD3 or an unknown partner gene.² In 1991, Kees et al³ and Kubonishi et al⁴ found 2 cases of carcinoma in the thorax demonstrating t(15;19). Since then, more than 30 cases of NMC have been reported,^5-16 with similar incidences in men and women. Most have involved the midline organs (25/31), including the mediastinum (thymus; n = 2); 12 cases have involved the head and neck, and 10 have involved the upper-respiratory tract (URT). Although about 30 cases have been reported worldwide, only 1 case has been reported in China.⁵ We therefore sought to identify and characterize NMCs in a cohort of patients with poorly differentiated malignant tumors (PDMTs) of the URT in China using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In this study, we describe the clinical and morphological features of NMCs and compare them with those of other PDMTs of the URT.

Materials and Methods

Case Selection and Grouping

We reviewed 155 cases of PDMTs involving the URT, which were identified in the surgical pathology files of Beijing Tongren Hospital of Capital Medical University from 2002 to 2010. Cases were divided into 4 groups: group 1 was a collection of poorly differentiated squamous cell carcinomas (PDSCCs) and sinonasal undifferentiated carcinomas (SNUCs; n = 32); group 2 comprised nonkeratinizing and undifferentiated nasopharyngeal carcinomas (NPCs; n = 58); group 3 comprised small-cell carcinoma, neuroendocrine type (SCCNET; n = 10); and group 4 comprised a collection of other nonepithelial PDMTs (n = 55), including high-grade olfactory neuroblastoma (ONB; n = 40), Ewing sarcoma (EWS)/primitive neuroectodermal tumors (PNET; n = 5); and rhabdomyosarcoma (RMS; n = 10). Hematoxylin and eosin–stained or unstained 4-µm-thick, formalin-fixed, paraffin-embedded sections were collected from all the above cases and reviewed by 2 pathologists.

In Situ Hybridization and IHC

Cases were examined for Epstein-Barr early mRNAs (EBER) by in situ hybridization (A500K.9901, PanPath, the Netherlands). All cases were stained with anti-NUT rabbit monoclonal antibody¹⁷ (clone C52B1Rabbit mAb #3625, Cell Signaling Technology, Danvers, MA), diluted (1:200) with antibody diluent (#8112, Cell Signaling Technology). Negative controls for NUT staining included cases of inflammatory and neoplastic lesions, including cervicitis, nasal polyp, breast invasive ductal carcinoma, and well-differentiated squamous cell carcinoma of the lung (n = 8). Cases that were positive for NUT by IHC were stained for cytokeratin (CK), CK7, CK8, p63,vimentin, neuron-specific enolase (NSE), synaptophysin (Syn), chromogranin A (CgA), S-100 protein, CD56 and CD99 (working solution, Lab Vision Thermo Fisher Scientific, Kalamazoo, MI). All antibodies were detected using antirabbit secondary antibodies (Maxvision, Maixin Bio, FuZhou, China). CK, CK7, CK8, vimentin, NSE, Syn, and CgA were located in the cell membrane/cytoplasm; S-100 protein was expressed in the nucleus/cytoplasm; CD56 and CD99 was located in the cell membrane; NUT and p63 were located in the nucleus. Samples were scored according to the staining intensity in the appropriate locations as follows: 0 (no stain); 1+ (1%-25% stained); 2+ (26%-50% stained), 3+ (51%-75%) stained, or 4+ (>75% stained).

Fluorescence In Situ Hybridization

Cases that were positive for NUT by IHC were subjected to FISH, as described previously.¹⁷ Chromosomes 19p13.1 BRD4 and 15q13 NUT breakpoints were evaluated by dual-color FISH assays in formalin-fixed, paraffin-embedded, unstained, 4-µm sections, as described previously. The probes used to detect the 19p13.1 BRD4 breakpoint included telomeric tandem bacterial artificial chromosome (BAC) clones RP11-319o10 and RP11-681d10 (digoxigenin-labeled, fluorescein isothiocyanate [FITC] antidigoxigenin-detected, green) and centromeric BAC clones RP11-207i16 and CTD-3055m5 (biotin-labeled, rhodamine-streptavidin-detected, red). The probes used to detect the 15q13 NUT breakpoint, flanking a 181-kb region, included telomeric BAC clones 1H8 and 64o3B (digoxigenin-labeled, FITC antidigoxigenin-detected, green) and centromeric clones 1084a12 and 3d4 (biotin-labeled, rhodamine-streptavidin-detected, red). Patients with greater than 80% hybridization efficiency in 4 areas (200 cells/area) of the tissue section were included in the analysis.

Transmission Electron Microscopy (TEM)

Tissue from case 4 was fixed immediately in 4% glutaraldehyde, postfixed in osmium tetroxide, and embedded in Epon 812. Then, 1-µm-thick sections were stained with toluidine blue and examined microscopically to confirm the presence of tumor. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a JEOL 1200 EX2 electron microscope (JEOL, Tokyo, Japan).

Results

EBER Detection

All but 1 patient from group 2 (NPC) were positive for EBER, whereas all cases from the other groups were negative. We performed IHC staining for NUT in all cases (n = 155).

Clinical Findings

The characteristics of the selected PDMT cases are listed in Table 1. There were 47 women and 108 men, with an age range of 4 months to 77 years. We found that 65 cases were primarily located in the sinonasal tract (Figure 1), 66 cases in the nasopharynx, 20 in the larynx, 2 in the hypopharynx, and 2 in the mandible. Also, 3 cases in group 1 (PDSCC/SNUC) were metastatic.

Table 1.

Characteristics of Patients Selected for NUT IHC

Age
Range	4 months to ~77 years
Mean	45 years
Sex
Male	108
Female	47
Primary site
Nasal cavity	40
Paranasal sinus	25
Nasopharynx	66
Larynx	20
Hypopharynx	2
Mandible	2
Original diagnosis
Sinonasal undifferentiated carcinoma	1
Poorly differentiated squamous cell carcinoma	31
Nasopharyngeal carcinoma	58
Small-cell neuroendocrine carcinoma	10
Ewing sarcoma/Primitive neuroectodermal tumor	5
Olfactory neuroblastoma	40
Rhabdomyosarcoma	10

Abbreviations: NUT, nuclear protein in testis; IHC, immunohistochemistry.

The clinical courses of the 4 cases positive for NUT by IHC are summarized in Table 2. All 4 cases were treated by radiation, and 1 received high-dose preoperative radiation treatment (4600 cGy/23f/32d), because the tumor was large (6.8 × 4.3 × 4.3 cm³) and had invaded into the front basicranium. Case 3 was a small tumor (1 × 1 cm²), with no invasion to the bone, and had therefore only received 25 doses of radiation (3400 cGy/17f/23d). These 2 patients remained alive. However, the 2 other NUT IHC–positive patients received postoperative radiation therapy. Case 2 received chemotherapy (paclitaxel, intravenous drip), but tumor recurrence and metastasis, with invasion into the right frontal lobe and orbit cavity, were noted by computed tomography and magnetic resonance imaging after 6 months. The patient underwent a second operation and combination chemotherapy but died 12 months after the initial diagnosis. Case 4 showed a highly aggressively clinical course. The patient presented with a 20-day history of headache, which had intensified over 6 days, accompanied by diplopia. The initial clinical diagnosis was a mass in the frontal sinus or invasive fungal sinusitis. The patient underwent emergency bilateral ethmoid sinus and maxillary sinus surgery but was unwell and drowsy postoperatively. The patient received no radiotherapy or drug treatment and died 1 month after diagnosis as a result of tumor metastasis into the intracranial tissue.

Table 2.

Clinical and Pathological Features of IHC NUT+ Cases

																FISH
Patient	Age (years)	Sex	Primary Site	Original Diagnosis	Rx	Metastases	Survival (months)	Sq Diff	Nec Diff	CK	CK7	CK8	p63	Neuroendocrine Markers	NUT	NUT	BRD4
1	55	M	Sinonasal	PDSCC	R	Intracranial, orbit	40ALWD	+	−	4+	3+	3+	4+	−	4+	−	−
2	42	M	Sinonasal	PDSCC	R,CR	Intracranial, orbit	9 12	+	−	4+	2+	3+	4+	−	4+	+	+
3	59	F	Nasal cavity	PDSCC	R	−	12ALWD	+	−	4+	1+	3+	4+	−	4+	−	−
4	50	M	Sinonasal	SNUC	−	Intracranial	1	−	−	3+	−	2+	4+	−	4+	+	+

Abbreviations: NUT, nuclear protein in testis; IHC, immunohistochemistry; CK, cytokeratin; Sq, squamous; Diff, differentiated; Rx, treatment; R, radiotherapy; CR, chemotherapy and radiotherapy; ALWD, alive with disease; NSE, neuron-specific enolase; Syn, synaptophysin; CgA, chromogranin A; Nec, neuroendocrine

Histopathological Findings

The cases were characterized histopathologically as PDMTs. All cases in group 1 (PDSCC and SNUC) had obvious focal necrosis; 19 cases had focal squamous differentiation, and 8 demonstrated minimal keratinization; 3 cases exhibited pseudolumen formation, and 2 cases had neuroendocrine structures. However, one case of SNUC showed no squamous or glandular differentiation. Some tumor cells were arranged in sheets, islands, or papillary structures (Figure 2A), whereas some were arranged in a palisading pattern. Two cases had sclerotic stroma, 1 with apparent chondroid differentiation. Most tumor cells were noncohesive, round, and ovoid, with scanty cytoplasm (Figure 2B). Two cases presented with foci of abrupt keratinization (Figure 2C). Only 1 case in group 2 (NPC) had vesicular nuclei and a single prominent eosinophilic nucleolus, and numerous lymphocytes were found infiltrating the tumor stroma in this case. SCCNET cases showed round, oval, or spindle-shaped tumor cells arranged in sheets or ribbons, with inconspicuous cytoplasm, dense chromatin, and absent nucleoli. Mitoses and apoptosis were easily seen. All cases showed focal necrosis, and 5 cases had rosette structures. Crush artifacts of the tumor cells were frequently seen. Tumor cells in cases with EWS/PNET were uniform, small to medium sized, and round, with a high nucleus-to-cytoplasm ratio; 3 cases showed fine chromatin, and 2 showed clumped chromatin. Only 1 case presented with Homer-Wright rosettes. Hyams et al¹⁸ developed a 4-tier grading scheme for ONB based on the growth pattern, amount of neurofibrillary stroma, frequency of rosettes, mitotic activity, presence of necrosis, and degree of nuclear atypia, with high-grade ONB comprising grades 3 and 4. The selected tumors in the current study were pleomorphic, and most demonstrated tumor cells growing in sheets, lacking a nested architecture. Large nucleoli were often present, and a neuropil was absent. Necrosis was common, and there was increased mitotic activity. The histological features of these ONBs mimicked SNUCs. In all, 9 cases of RMS were of embryonal type, and 1 was of alveolar type. The embryonal type is the most common type of RMS in the head and neck, followed by the alveolar type.¹⁹ Some tumor cells were spindle shaped, others were round, and many contained abundant eosinophilic cytoplasm. Mitoses and necrosis were commonly seen.

Immunohistochemistry

Of 97 EBER-negative cases, 4 were positive for NUT by IHC, all of whom belonged to group 1 (Figure 3A). The tumor cells in all cases revealed diffuse positive staining for CK and p63 (Figure 3B) and variable reactivity for CK7 and CK8. All were negative for vimentin, NSE, Syn, CgA, S-100 protein, CD56, and CD99. The results are summarized in Table 2.

Transmission Electron Microscopy

The original diagnosis in case 4 (Table 2) was SNUC. The tumor included 2 types of cells. Most were light cells with a vesicular nucleus and fine chromatin, usually with 1 or 2 prominent red nucleoli. Other, dark cells had a hyperchromatic, irregularly shaped nucleus (Figure 4A). Light cells were bigger than dark cells. All tumor cells were polygonal with a high nucleus-to-cytoplasm ratio and cytoplasm with few mitochondria. Tumor cells were closely opposed to each other with no explicit junctional structure (Figure 4B).

Fluorescence In Situ Hybridization

Dual-color FISH was performed on tissues from the 4 NUT-IHC-positive cases, using probes flanking the BRD4 and NUT genomic loci. However, only 2 of these cases showed NUT gene rearrangements. The tumors with NUT rearrangements also demonstrated rearrangement of BRD4, consistent with the presence of a BRD4-NUT fusion gene (Figures 5A and 5B). The results are summarized in Table 2.

Discussion

NMC is a rare and aggressive cancer. French et al¹ first identified the NUT rearrangement as a characteristic of this disease in 2003, and there are currently more than 30 reported cases in the literature. However, as a newly recognized tumor, the actual incidence of NMC remains unknown. One report suggested that NMC accounted for 18% of poorly differentiated carcinomas of the upper-aerodigestive tract.¹¹ The current study detected 2 (2/32, 6.25%) confirmed cases of NMC among tumors diagnosed as PDSCC and SNUC of the URT, all located primarily in the sinonasal tract. The sinonasal region is a common midline site for NMC. A review of the published cases shows that almost 90% of patients were younger than 40 years old, though this was likely to be influenced by selection bias. Most cases were in children and young adults, with male and female patients being similarly affected. In contrast, the average age of patients with NMC in our series was 51.5 years, probably largely because the average age of selected PDSCC cases was 58 years. These findings are consistent with the recent observation that NMC is not restricted to young adults and children.¹¹ NMC is not known to affect smokers; however, 2 of the current cases were smokers and both were Epstein-Barr virus (EBV) negative.

Squamous cell carcinoma is the most commonly encountered malignant neoplasm of the URT, and includes keratinizing and nonkeratinizing types. Based on the frequent presence of squamous differentiation, NMC needs to be distinguished from ordinary squamous cell carcinomas by the presence of NUT gene rearrangements and often by the lack of a smoking history. Microscopically, NMC is composed of sheets and islands of noncohesive, round, ovoid cells with scanty cytoplasm and vesicular or fine/granular chromatin. Tumor cells usually have 1 or 2 prominent nucleoli and easily identifiable mitotic figures, and necrosis is frequently seen. Foci of abrupt keratinization are typical of NMC, occurring in approximately half of all cases,¹ which can help distinguish it from other small round-cell tumors. There was no glandular or neuroendocrine differentiation in any of the 2 confirmed cases of NMC in our study.

TEM also showed that NMC is a poorly differentiated carcinoma and identified 2 types of cells (dark and light cells). This new ultrastructural characteristic of NMC may prove useful for identifying future cases of NMC.

IHC is a useful technique for the differential diagnosis of NMC in relation to other PDMTs. A total of 4 cases in the current study demonstrated diffuse nuclear (≥50%) positivity in a characteristic speckled pattern, using a monoclonal antibody against NUT. This staining pattern was previously demonstrated to be 100% specific in an evaluation of nearly 1000 carcinomas.¹⁷ However, only 2 of these cases demonstrated NUT rearrangement by FISH. On the basis of their more aggressive clinical course, as well as the molecular findings, we consider that only the 2 NUT-IHC-positive cases confirmed by FISH could be diagnosed as NMCs. The apparent lack of NUT rearrangements in some NUT-IHC-positive cases may be attributable to cryptic NUT rearrangements, as previously demonstrated in 2 of 30 cases evaluated using the NUT monoclonal antibody.¹⁷ These cases involved an interstitial deletion of NUT and insertion of that fragment of DNA into either the BRD4 or another gene locus. This could be clarified by performing FISH using a probe that spans the NUT gene, rather than dual-colored flanking probes. However, this assay requires a highly specific signal and low background fluorescence, and unfortunately, both NUT FISH-negative cases in the current study had high background fluorescence because of resistance of the tissue to digestion, thus precluding FISH evaluation using the NUT-spanning probe. It is possible that these cases represent the first reported NUT-expressing cancers with no detectable NUT rearrangement. The 100% specificity of the NUT antibody should be demonstrated in a larger series. However, the possibility that both cases lacking BRD4 rearrangements by FISH may have been NUT variants or possessed cryptic BRD4 rearrangements cannot be excluded. Some previous data¹ as well as unpublished observations suggest that patients with NUT-variant NMCs may live longer than those with BRD4-NUT rearrangements.

The sinonasal area is a relatively small but complex anatomical region, which may give rise to a variety of other aggressive high-grade and/or undifferentiated malignant epithelial and nonepithelial neoplasms, such as NPC, SCCNET, EWS/PNET, ONB, and RMS. These tumors show poorly differentiated histological features that overlap with each other and with NMC. However, their clinical courses, treatments, and prognoses differ from those of NMC, and differentiating among these tumors is therefore important. Histological differentiation of such tumors generally requires the use of a panel of immunohistochemical markers and specific chromosomal translocations (Table 3). IHC for CK can distinguish cases of epithelial differentiation from these malignant tumors. CK reactivity may include a punctuate paranuclear or globoid pattern in SCCNET, which is variably reactive for neuroendocrine markers (NSE, SyN, S-100, and CD56). Focal CK expression is occasionally seen in EWS/PNET, which also demonstrates strong, diffuse CD99 and vimentin immunoreactivity. Molecular analysis by polymerase chain reaction can help in the diagnosis of EWS/PNET. Most EWS/PNETs have a characteristic t (11; 22) with EWS/FLI1 juxtaposition or other translocations involving EWS. ONB is usually negative for CK, though some cases have some CK-positive cells; NSE, CgA, and Syn are the most consistently positive immunohistochemical markers for the diagnosis of ONB.^20-23 No cases of NMC have demonstrated CgA and Syn expression (C.A.F., 2011).²³ S-100 protein is an important marker localized at the periphery of the neoplastic lobules in ONB. NPC has more of a tendency to originate in the nasopharynx and has a strong association with EBV. RMS is a malignant mesenchymal tumor of skeletal muscle that is positive for myogenic markers (desmin, myoglobin, MyoD1, and muscle-specific actin) and negative for CK. The molecular genetic features of embryonal RMS and alveolar RMS differ.^24,25 Cytogenetic evaluation can play a critical role in their distinction from NMC.

Table 3.

Expression of Immunohistochemical Markers in URT Tumors

Tumor Type	CK	CK7	CK8	p63	VIM	NSE	S-100	CD56	Desmin	NUT
NMC	+	V	+	+	−	−	−	R+	−	+
SNUC	+	+	+	F+	−	V	−	−	−	−
NPC	+	+	V	+	−	−	−	−	−	−
ONB	F+	−	−	F+	−	+	+^a	−	−	−
RMS	−	−	−	−	+	−	−	−	+	−
SCCNET	+^b	−	−	−	−	+	V	+	−	−
PNET	F+	−	−	−	+	V	V	−	−	−

Abbreviations: URT, upper-respiratory tract; CK, cytokeratin; VIM, vimentin; NUT, nuclear protein in testis; NMC, NUT midline carcinoma; SNUC, sinonasal undifferentiated carcinoma; ONB, olfactory neuroblastoma; RMS, rhabdomysosarcoma; SCCNET, small-cell carcinoma, neuroendocrine type; PNET, primitive neuroectodermal tumor; R, rarely positive; V, variably positive; F, focal positive (1%-10% positive).

Positive in the peripherally situated sustentacular cells.

Weakly positive.

The 2 FISH-positive cases of NMC in the current study were located in the sinonasal tract. One case was originally diagnosed as SNUC, and the other was diagnosed as PDSCC. According to the World Health Organization International Histological Classification of Tumours, SNUC is a highly aggressive and clinicopathologically distinctive carcinoma of uncertain histogenesis that typically presents with locally extensive disease. It is composed of pleomorphic tumor cells with frequent necrosis and should be differentiated from lymphoepithelial carcinomas or ONB.²⁶ It has been histologically defined as being devoid of squamous or glandular differentiation, and neurofibrillary material and neural-type rosettes are not identified. Ejaz and Wenig²⁷ suggested that the histological definition of SNUC should be expanded to include tumors with differentiated foci only in cases where clinical parameters (ie, rapidly enlarging and destructive sinonasal lesions) and the majority of the histological findings (ie, undifferentiated pleomorphic cell population) are present. The nuclei of tumor cells in SNUC are medium to large sized, and some cases are immunoreactive for NSE. It is therefore possible to differentiate NMC from SNUC, which is composed of smaller-sized cells that lack neuroendocrine differentiation, by NUT IHC or NUT rearrangement by FISH. All NMCs in this study demonstrated diffuse positivity for p63, in contrast to SNUC, which is reported to show only weak or no expression of p63.²⁸ These results suggests increased squamous epithelial differentiation in NMCs in contrast to SNUCs.

There is still no successful treatment for NMC. However, its unique molecular cytogenetic characteristics mean that it may benefit from targeted therapy. In a recent report, a child with NMC received differentiation therapy with the FDA-approved histone deacetylase inhibitor vorinostat (SAHA) for 5 weeks, and a marked decrease in tumor avidity was observed by positron emission tomography. However, the patient unfortunately died as a result of recurrent and metastatic disease 11 months after the initial diagnosis.²⁹

NMC is currently poorly understood in terms of its etiology, histogenesis, biological behavior, and even its diagnostic criteria. The results of the current study suggest that all poorly differentiated, midline, EBV-negative carcinomas in any age group should be screened for NUT expression and NUT rearrangements by IHC and/or other molecular means. The determination of NUT rearrangements by molecular techniques should represent the gold standard for the diagnosis of NMCs.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Natural Science Foundation of China (81070769).

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