Abstract
Introduction:
Nuclear protein in testis (NUT) carcinoma (NC) is an extremely rare and highly aggressive tumor. Non-canonical NC patients frequently have unique clinicopathological characteristics in terms of location, molecular alteration, and outcome, with morphological overlap with other tumors. Here, we report and summarize the non-canonical clinicopathological features of three unusual NC patients harboring BRD3::NUTM1 or WWTR1::NUTM1 gene rearrangement.
Methods:
The data of three patients with NC who were diagnosed between 2019 and 2022 at the Department of Pathology at West China Hospital or the Sichuan Kingmed Center for Clinical Laboratory were collected. Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and RNA sequencing were performed. A literature review was conducted for NC patients with non-canonical features.
Results:
All three NC patients were male and aged between 31 and 60 years. The primary sites were the gastric corpus, nasal cavity, and subcutaneous tissue of the calvaria vertex. Histologically, two patients harbored NC with glandular differentiation, and one harbored NC with small cell carcinoma-like morphology. IHC revealed positivity for Pan-CK (cytokeratin) (PCK) (3/3), Epithelial membrane antigen (EMA) (3/3), Cytokeratin 5/6 (CK5/6) (3/3), and P63 (2/3). The Ki-67 index ranged from 20% to 40%. The remaining IHC markers were negative. RNA sequencing revealed BRD3::NUTM1 fusion in two patients and WWTR1::NUTM1 fusion in one patient.
Conclusion:
In our study, the clinicopathological features of NC in patients with rare molecular alterations are reported and summarized. As increasing numbers of NUTM1-rearranged tumors with diverse histologic phenotypes have been reported, the traditional classification of NC as a purely epithelial malignancy may warrant reconsideration. Non-canonical NC needs to be further reported, integrated, and classified to precisely guide targeted therapy.
Introduction
Nuclear protein in testis (NUT) carcinoma (NC) is a rare, highly aggressive malignant tumor characterized by a fusion of the NUTM1 gene (referred to as the NUT gene) on chromosome 15 with other partner genes, with the most common partner gene being BRD4 (>70%). Although NC was initially reported in children and adolescents, it can affect individuals of any age with no gender predisposition. The predominant locations of NC are the head and neck, upper respiratory tract, and mediastinum. NC shows no clear histological specificity and mostly presents as poorly differentiated squamous cell carcinoma (SCC) (, ).
In this study, we evaluated three patients with non-BRD4 fusion-type NC that occurred in the stomach, nasal cavity, and top of the head. Histologically, two patients showed poorly differentiated carcinomas with glandular differentiation, and one patient showed small cell carcinoma-like morphology. Notably, BRD3::NUTM1 gene rearrangement was detected in one patient with glandular differentiation and in another with small cell carcinoma-like morphology, while WWTR1::NUTM1 gene rearrangement was detected in a patient with glandular differentiation.
As attention to NC has increased, increasing numbers of NCs with non-BRD4::NUTM1 fusion have been identified. These rare NC types often have non-canonical pathological manifestations and clinical processes (–), suggesting that non-BRD4 fusion-type NCs have the potential to be considered a special tumor subtype. By reviewing the literature and evaluating three unusual NC patients, we aimed to describe the clinicopathological features of these rare non-canonical NC and further expand the recognized molecular and histological spectrum of this entity.
Materials and methods
Case selection and literature search
Formalin-fixed paraffin-embedded (FFPE) samples diagnosed as NC with unusual morphological features were collected. These cases were diagnosed between 2019 and 2022 at West China Hospital of Sichuan University and Sichuan Kingmed Center for Clinical Laboratory. The Ethics Committee on Biomedical Research, West China Hospital of Sichuan University, provided institutional authorization and approval (approval number: 1016) for this retrospective observational research. Written informed consent was obtained from the patient’s next of kin for the publication of any potentially identifiable images or data included in this article.
In this study, “non-canonical NC” is defined as NUTM1-rearranged tumors with fusion partners other than BRD4. These cases often exhibit significant differences in clinicopathological features and biological behavior compared to canonical NC. For the literature review of NC patients, the keyword “NUT carcinoma” was searched in PubMed from 2010 to 2024. Table 1 further integrates non-canonical NC, excluding cases with canonical pathological features or incomplete clinical information (especially unclear fusion subtypes or incomplete prognosis).
Table 1
| Case | Author | Gene fusion | Age (years) | Sex | Location | Histological feature | IHC positive | Treatment | Outcome (months) |
|---|---|---|---|---|---|---|---|---|---|
| Case 1 | Current study | BRD3::NUT t(9;15)(q34;q14) | 31 | M | Stomach | Small cell-like | PCK, CK5/6, CK7, P63 EMA | None | Alive (5) |
| Case 2 | Current study | WWTR1::NUT t(3;15)(q25;q14) | 60 | M | Calvaria vertex | Glandular differentiation | PCK, CK5/6, CK7, P63, EMA | Surgery and RCT | Dead (40) |
| Case 3 | Current study | BRD3::NUT t(9;15)(q34;q14) | 42 | M | Nasal cavity | Glandular differentiation | PCK, CK5/6, CK7, EMA | RCT | Dead (5) |
| Case 4 | Nishimura et al. () | BRD3::NUT | 46 | F | Inguinal subcutaneous mass | Glandular differentiation | CK5/6, CK7, P40 | Surgery and CT | Alive (132) |
| Case 5 | Lee et al. (50) | BRD4::NUT | 5 | M | Anterior medium | Glandular differentiation | CK, EMA, CEA | CT | Dead (1.5) |
| Case 6 | Xu et al. () | BRD4::NUT | 36 | F | Kidney | Glandular differentiation | CK7, CK20, 34βE12 | NA | Dead (6) |
| Case 7 | Stevens et al. () | BRD4::NUT | 44 | M | Lung | Glandular differentiation | PCK, CK5/6, TTF-1, P63, P16 | NA | NA |
| Case 8 | Chien et al. () | ZNF532::NUT | 21 | F | Mandible bone | Rhabdoid | CD34 | Surgery and RT | Alive (43) |
| Case 9 | Dickson et al. () | MXD1::NUT | 39 | F | Stomach wall | Rhabdoid | CK, GFAP | Surgery and CT | Alive (108) |
| Case 10 | Dickson et al. () | BCORL1::NUT | 45 | M | Arm | Pleomorphism | NA | Surgery and RCT | Dead (48) |
| Case 11 | Schaefer et al. () | CIC::NUT | 60 | M | Masticator space | Myoepithelial-like | CK7, ETV4 | CT and immunotherapy | Alive (10) |
| Case 12 | Le Loarer et al. () | CIC::NUT | 3 | M | Temporal | Epithelioid | PCK, ETV4 | Surgery and RCT | Dead (18) |
| Case 13 | Le Loarer et al. () | CIC::NUT | 5 | M | Occipital | Epithelioid | KL1, EMA, P40, CD99, ETV4 | CT | Dead (14) |
| Case 14 | Le Loarer et al. () | CIC::NUT | 7 | F | Paravertebral | Epithelioid | ETV4, CD99, SOX-2 | Surgery and CT | Dead (37) |
| Case 15 | Le Loarer et al. () | CIC::NUT | 27 | M | Lung | Epithelioid | ETV4, WT-1, SOX-2 | Surgery and CT | Dead (7) |
| Case 16 | Le Loarer et al. () | CIC::NUT | 22 | F | Lateral ventricule | Epithelioid | PCK, CD99, ETV4 | Surgery and RCT | Dead (17) |
| Case 17 | Le Loarer et al. () | CIC::NUT | 18 | M | Epidural space | Epithelioid | WT-1 | Surgery and RCT | Alive (40) |
| Case 18 | Goto et al. () | MGA::NUT | 49 | M | Lung | Sarcoma-like | BCOR, bcl-2, CD99, Syn, neurofilament, MUC4 | Surgery and RCT | Dead (13) |
| Case 19 | Stevens et al. () | MGA::NUT | 63 | F | Lung | Sarcoma-like | NA | NA | NA |
| Case 20 | Stevens et al. () | MGA::NUT | 61 | M | Chest wall/pleural | Sarcoma-like | CD34 | NA | NA |
| Case 21 | Diolaiti et al. () | MGA::NUT | 10 | M | Thigh | Sarcoma-like | CD99, CD34, bcl-2, desmin | Surgery and RCT | Alive (132) |
| Case 22 | Diolaiti et al. () | MGA::NUT | 10 | F | Dura mater | Sarcoma-like | CD99, desmin | Surgery and RT | Alive (15) |
| Case 23 | Wangsiricharoen et al. () | MGA::NUT | 10 | M | Thigh | Sarcoma-like | NA | Surgery | Alive (180) |
| Case 24 | Wangsiricharoen et al. () | MGA::NUT | 14 | M | Chest well | Sarcoma-like | S-100, CD56, neurofilament, CD99, calponin, TLE1 | Surgery | Alive (18) |
| Case 25 | Wangsiricharoen et al. () | MGA::NUT | 28 | M | Pelvis | Sarcoma-like | CD56, CD99 | Surgery | Dead (84) |
| Case 26 | Underwood et al. () | MGA::NUT | 48 | M | Foot | Sarcoma-like | CD99 | Surgery | Alive (6) |
| Case 27 | Mantilla et al. () | MGA::NUT | 61 | M | Pleura | Sarcoma-like | CD34 | NA | Alive (NA) |
| Case 28 | Stevens et al. () | MXD4::NUT | 65 | F | Colon (cecum) | Sarcoma-like | Vimentin | NA | NA |
| Case 29 | Van Treeck et al. () | MXD4::NUT | 38 | F | Sigmoid colon | Sarcoma-like | CD34, SMA | CT | Alive (15) |
| Case 30 | Van Treeck et al. () | MXD4::NUT | 40 | M | Ileocecal valve | Sarcoma-like | SMA, ERG | NA | Alive (5) |
| Case 31 | Van Treeck et al. () | MXD4::NUT | 65 | F | Cecum | Sarcoma-like | Syn | NA | Dead (30) |
| Case 32 | Van Treeck et al. () | MXD4::NUT | 44 | F | Descending colon | Sarcoma-like | CD99 | NA | Alive (10) |
| Case 33 | Van Treeck et al. () | MXD4::NUT | 67 | F | Descending colon | Sarcoma-like | CD99 | NA | NA |
| Case 34 | Tamura et al. () | MXD4::NUT | 34 | F | Ovary | Sarcoma-like | Vimentin, CD99 | Surgery and CT | Dead (9) |
| Case 35 | Xu et al. () | MXD4::NUT | 30 | M | Penis | Sarcoma-like | NA | Surgery | Dead (48) |
Summary of current reported cases of NUT-rearranged neoplasm with non-canonical clinicopathological features in the literature.
M, male; F, female; RCT, radiochemotherapy; RT, radiotherapy; CT, chemotherapy; NA, not available.
Immunohistochemistry and EBER in situ hybridization
Four-micrometer sections underwent hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) using the following series of antibodies: NUT (clone C52B1, CST, Massachusetts, USA), PCK (clone AE1/AE3, BIO, Fujian, China), Cytokeratin 5/6 (CK5/6, clone D5/16B4, MXB, Fujian, China), Cytokeratin 7 (CK7, clone RN7, BIO), EMA (clone GP1.4, BIO), Leukocyte common antigen (LCA) (clone 2B11 and PD7/26, BIO), CD56 (clone UMAB83, BIO), synaptophysin (polyclonal, MXB), S-100 (clone 4C4.9, MXB), myogenin (clone EP162, BIO), desmin (clone MX046, MXB), P63 (clone UMAB4, BIO), CD34 (clone EP88, BIO), Ki-67 (clone MIB-1, DAKO, California, USA), and PD-L1 (clone SP263, BIO). Epstein–Barr virus (EBV) in situ hybridization (ISH) (EBER1/2 probes, BIO) was also performed according to the manufacturer’s instructions. Staining was performed on a Roche Ventana system. The evaluation of these staining procedures was performed as previously described ().
Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) was performed using a NUTM1 dual-color break-apart probe (Anbiping, Guangzhou, China) to assess NUT gene translocation according to the manufacturer’s instructions. The slides were observed under a ×100 objective magnification using a fluorescence microscope (Leica DM6000, Wetzlar, Germany) equipped with the Applied Imaging 4.0 analysis software (Genetix, England, UK). The case was considered translocation positive if more than 15% of the scored tumor cells (at least 100 cells) showed split signals as previously described (, ) (note: ALK FISH diagnostic criteria were followed). The slides were independently evaluated by two pathologists (W Wang and X Wu) with expertise in FISH analysis.
Gene expression data analysis
RNA was extracted from tumor specimens using the RNeasy FFPE Kit (Qiagen, 73504, Hilden, Germany). The RNA sequencing method has been described previously (, ). Briefly, total RNA was extracted from tumor specimens using the RNeasy FFPE Kit (Qiagen, 73504) according to the manufacturer’s protocols. The NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB, #Z1955E, Massachusetts, USA) was chosen to remove the targeted ribosomal RNA (rRNA). After rRNA depletion and fragmentation, cDNA synthesis and next-generation sequencing (NGS) library preparation were performed using NEBNext® Ultra™ II Directional RNA Library Prep Kit (NEB, E7760L). RNA-seq libraries were sequenced on the Gene + Seq-2000 with a paired-end 2 × 100 bp protocol, resulting in 20 Gb per sample. After removal of terminal adaptor sequences and low-quality data using fastp (version 1.3.1) and removal of rRNA reads by aligning clean reads to the rRNA database (download from NCBI) using bowtie2 (version 2.5.5), clean reads without known rRNA were aligned to the reference human genome (hg19) through STAR (version 2.7.11b). Fusions were detected using a customized version of Arriba (version v1.0.0) and annotated using in-house software annoFilterArriba (version v1.5.5) with the NCBI release 104 database using default parameters. The filtering criteria were as follows: 1) the supporting read threshold for considering a non-hotspot fusion positive was conservatively set at 10 or more supporting reads. (2) For all known fusions, two supporting reads were used as the positivity threshold (). All final candidate fusions were manually verified using the Integrative Genomics Viewer browser. A series of quality control metrics were computed using RNA-SeQC (version 2.4.2) assessment. A threshold of ≥80 million mapped reads and ≥10 million junction reads per sample was set.
Results
Case 1
A 31-year-old male patient presented with generalized pain mainly in the large joints of the extremities, without an obvious pain-inducing event or factor. The patient also reported occasional headaches and abdominal distension. An X-ray finding demonstrated multiple osteolytic lesions in the vertebrae, pelvis, and other locations. Moreover, gastroscopy showed multiple ulcerative lesions in the greater curvature and anterior wall of the gastric body, with the largest being 1.2 cm in diameter (Figure 1A). Biopsy revealed diffusely distributed tumor cells with a small cell-like morphology, and poorly differentiated cancer cells were observed in some areas without obvious extrusion artifacts (Figures 1B, C). IHC was positive for PCK, EMA, P63, CK5/6, CK7, and NUT (Figures 1D–F). The Ki-67 index was 30%. RNA sequencing demonstrated that a chromosomal translocation t(9;15)(q34;q14) occurred between chromosomes 9 and 15 and produced the fusion oncogene BRD3::NUTM1 (Figure 2).
Figure 1
Figure 2
Subsequently, the patient developed fatigue and had a poor appetite. Additionally, the patient lost weight and gradually lost the ability to stand, walk, or defecate. He received acupuncture treatment at the local hospital, but the symptoms and pain were not significantly relieved. Five months later, a computed tomography (CT) scan revealed thickening of the gastric antrum wall and multiple enlarged lymph nodes around the gastric antrum, hepatogastric ligament, and para-aortic region. Multiple bone destructions were observed throughout the body, accompanied by multiple soft tissue nodules appearing in the liver, pancreas, adrenal glands, greater omentum, parietal peritoneum, peritoneal cavity, and retroperitoneum. He received palliative care to manage the pain with limited effectiveness and was ultimately lost to follow-up in the fifth month.
Case 2
A 60-year-old male patient presented with a gradually enlarging subcutaneous mass on the top of his head. The mass had a firm and darkened texture on the surface. CT imaging showed a 2.7 cm × 2.5 cm mass under the scalp (Figure 3A). Pathological examination revealed SCC accompanied by necrosis. Squamous differentiation and glandular differentiation were observed in focal areas. The tumor cells were positive for PCK, EMA, P63, CK5/6, CK7, and NUT, with a Ki-67 index of approximately 40% (Figures 3B, D, E). FISH analysis revealed a split signal in the NUTM1 gene, and RNA sequencing demonstrated a chromosomal translocation t(3;15) (q25;q14) that led to the fusion of WWTR1 and NUTM1. Subsequently, he underwent radiochemotherapy with regular follow-up.
Figure 3
Six months later, a CT scan revealed left cervical lymph node enlargement, which was further confirmed to be a metastasis after lymphadenectomy. One year later, he developed left ear pain with discharge and hearing loss. CT imaging revealed a solid mass with bone erosion behind the left auricle, which was surgically removed and confirmed to be a poorly differentiated SCC. Fourteen months later, a painful mass developed on his left neck, with PET–CT showing increased glucose metabolism in the left upper neck and parotid gland. SCC metastasis was suspected and subsequently confirmed through surgical resection. Two months later, a space-occupying lesion was identified in the right lower lobe on CT, and metastatic SCC was confirmed via fine-needle aspiration biopsy, with FISH indicating the existence of NUTM1 rearrangement. Six months after lung biopsy, he died of tumor progression, 40 months after the initial diagnosis.
Case 3
A 42-year-old male patient with no underlying disease presented with recurrent epistaxis for 3 months, with a bleeding volume of 50–100 mL each time. Nasopharyngoscopy revealed mucosal erosion in Little’s area of the nasal septum, which was covered with a small amount of blood clots. A poorly defined mass was observed in the left nasal cavity, accompanied by ipsilateral exophthalmos and cervical lymph node enlargement. CT imaging demonstrated a mass spanning the left nasal cavity, nasolacrimal duct, and inner canthus. No distant metastasis was found in organs such as the chest, liver, or brain. Histopathological examination of the biopsy revealed that the tumor was surrounded by abundant interstitial fibrosis, forming strips, cords, and nests. The malignant tumor cells were uniformly round- or oval-shaped with minimal cytoplasm. The nuclei of the tumor cells were irregular with thick nuclear membranes, vesicular chromatin, and prominent large acidophilic nucleoli. Glandular differentiation with intraluminal mucin was observed in the focal area (Figure 3C). No definitive squamous differentiation could be observed. Mitotic figures, neutrophil infiltration, and extensive necrosis were prominent within the tumor. IHC staining for PCK, CK5/6, CK7, and NUT (Figure 3F) was positive, with a Ki-67 proliferation index of 20%–40%. EMA IHC staining was positive at the luminal border. RNA sequencing revealed BRD3::NUTM1 gene rearrangement (exon 10:exon 2), namely, a t(9;15)(q34;q14). Although timely and aggressive chemotherapy and radiotherapy were performed, the tumor progressed rapidly, and he died 5 months after the initial diagnosis.
In addition, negative results were obtained in all three cases for LCA, CD56, S-100, myogenin, desmin, CD34, PD-L1, and EBER in situ hybridization. FISH results for Case 2 and RNA sequencing results for all three cases are provided in Supplementary Material.
Discussion
NC accounts for approximately 1.1% of poorly differentiated/undifferentiated carcinomas of the head and neck (). The clinical course of NC is usually highly aggressive, with more than 80% of patients dying within the first year after initial diagnosis (). NC is usually resistant to non-specific radiochemotherapy, but most patients who are diagnosed in a timely manner benefit significantly from various NUT-rearranged targeted therapies (–). Therefore, precise and rapid identification of NC is crucial. However, the diagnosis of NC poses a great challenge to pathologists. The histology of canonical NC appears to reveal poorly differentiated carcinoma with or without squamous differentiation, typically presenting small- to medium-sized basaloid cells with one to two prominent nucleoli. In some cases, separation artifacts and clear cytoplasm could appear between the cytoplasm and cell membrane, resulting in a fried egg-like appearance (, ). Occasionally, atypical morphologies such as rhabdomyoid (), sarcomatoid (), and chondroid () features could also be present. Given the substantial morphological heterogeneity of NC, some scholars have suggested that all poorly differentiated carcinomas occurring in the chest, head, and neck should be screened with NUT-IHC staining (, , ).
Interestingly, we noted that all three NC patients in our study harbored non-canonical NUT fusion types, with the lesions frequently occurring in rare locations and showing non-canonical morphological characteristics. IHC was used to exclude more common tumors at these sites, including neuroendocrine tumors, malignant melanoma, hematolymphoid tumors, mesenchymal tumors, and EBV-associated tumors. A literature review of NC patients with unusual clinicopathological features was therefore conducted (Table 1; some cases were not included due to canonical morphology or unavailable genetic testing results).
Consistent with our findings, NC with non-canonical fusion types occurred at rare sites and had atypical morphological features in many cases: consistent with Cases 2 and 3 in our cohort, glandular differentiated NC was observed in the inguinal subcutaneous tissue of patients with BRD3::NUTM1 rearrangement (). NCs with epithelioid or myoepithelioid morphology were observed in the masticator space (), central nervous system, paravertebral body, epidural space, and lung (), accompanied by a CIC::NUTM1 rearrangement. Patients harboring ZNF532::NUTM1 or MXD1::NUTM1 fusion exhibited rhabdomyoid morphology in the mandible () or gastric wall (), respectively. In addition, NCs with sarcomatoid morphology were also common in patients with MGA::NUTM1 or MXD4::NUTM1 fusion, occurring in the lung (, ), thigh (, ), dura mater (), foot (), pleura (), chest wall (), colon (, ), pelvis (), ovary (), and penile shaft (). Thyroid-derived NCs often exhibit follicular architecture, colloid-like secretions, or even plasmacytoid morphology, with approximately 50% to 75% of them harboring NSD3::NUTM1 rearrangement (, ). Dickson et al. reported that an arm tumor harboring BCORL1::NUTM1 fusion displayed polymorphic morphology, including polygonal cells and spindle cells, with regions containing chrysanthemum-like structures and chondromyxoid matrix ().
Traditionally, NC is considered to have an extremely bad prognosis, with a median survival of only 6–10 months (, ). Although some large cohort studies have shown no statistically significant difference in survival between patients with and without BRD4::NUTM1 rearrangement (, ), several reports have documented significant individual variation in survival, with patients with non-canonical fusion types showing a better outcome (Table 1). French et al. reported that the survival time of patients with non-BRD4::NUTM1-rearranged NC was nearly four times longer than that of patients with BRD4::NUTM1-rearranged NC (). Chau et al. reported that chest NC patients with BRD3::NUTM1 or NSD3::NUTM1 rearrangement had better outcomes than those with BRD4::NUTM1 rearrangement (). In addition, thyroid NC, characterized by a high frequency of NSD3::NUTM1 rearrangement, had a significantly better prognosis than other anatomical locations [5-year overall survival (OS) up to 58%], in which patients with NC-like morphology showed significantly worse OS than those with non-NC-like morphology (). Scattered case reports further supported this tendency as follows. Dickson et al. reported a gastric NC patient with MXD1::NUTM1 fusion demonstrating a 9-year survival period (). Wangsiricharoen et al. () and Diolaiti et al. () reported that MGA::NUTM1 fusion tumor patients exhibited an OS of 11 and 15 years, respectively, in the thigh and 7 years in the pelvis. Nishimura et al. reported a case of inguinal subcutaneous BRD3::NUTM1 fusion with long-term survival (11 years) after the initial diagnosis (). The second NC patient (Case 2) in our study with WWTR1::NUTM1 rearrangement also survived for 40 months after the initial diagnosis. The survival time of patients with these unusual fusion types of NC varied significantly, as shown in Table 1, from death within 1 month after diagnosis to long-term survival. Furthermore, some reports have suggested that non-BRD4::NUTM1-rearranged NCs may not be sensitive to bromodomain inhibitors, a currently available NUT-targeted therapeutic agent (). Therefore, in addition to IHC and FISH, NGS or RT-PCR may help identify specific fusion partners, which could provide valuable insight into tumor behavior and potentially inform therapeutic selection.
Based on the clinicopathological features of our cohort and the literature review above, it seems that the histological morphology and prognosis of NC appear to be associated with the fusion partner of the NUTM1 gene to a certain extent. According to the literature, non-BRD4::NUTM1 fusions were associated with distinct histologic phenotypes depending on the specific fusion type (e.g., CIC-, MGA-, NSD3-, and MXD4-associated fusions), while rare fusions (WWTR1, ATXN1, MXD1, etc.) were often associated with an extremely atypical location, histological morphology, and clinical course. If the grouping of NC patients can be based on the fusion partners, we speculate that the differences in treatment efficacy and prognosis may become more apparent. Unfortunately, case reports of NC with rare fusion types to date are still limited, so more NC reports are needed to investigate its detailed clinicopathological spectrum.
In recent years, with the widespread use of NGS, NUTM1 rearrangement has also been detected in tumors other than traditional NUT “carcinoma”, especially in skin adnexal tumors (, , –44) (Table 2). In a study of 67 NUT-rearranged poroid neoplasms, 64 patients (95.5%) harbored YAP1::NUTM1 fusion, two harbored WWTR1::NUTM1 fusion (, 44), and one harbored EMC7::NUTM1 fusion (). Notably, among the 67 poroid neoplasms, 27 were confirmed to be benign poroma, 26 (96.3%) harbored YAP1::NUTM1 rearrangement, and one harbored WWTR1::NUTM1 rearrangement (WWTR1::NUTM1 fusion was also detected via RNA sequencing in Case 2 of our cohort). Furthermore, NUTM1 rearrangement has also been detected in non-epithelial tumors, including sarcoma, acute lymphoblastic leukemia, and glioma (, 45–48) (Figure 4). In these studies, the terms “NUTM1-rearranged neoplasm” (, 45, 49) and “NUT sarcoma” (, ) were used to redefine these cases, which led to the following new proposition: does NUTM1 rearrangement, like BRAF mutation, ALK rearrangement, or NTRK rearrangement, act as a driver gene and widely exist in human tumors of various origins, even benign tumors?
Table 2
| Author | Age (years) | Sex | Location | Fusion partner/diagnosis | Outcome (months) | ||
|---|---|---|---|---|---|---|---|
| YAP1 | WWTR1 | EMC7 | |||||
| Hartsough et al. () | 55 | M | Scrotum | 1 porocarcinoma | Alive (4) | ||
| Parra et al. () | 81 | F | Foot | 1 porocarcinoma | NA | ||
| Christensen et al. (40) | 51 | F | Scalp | 1 porocarcinoma | Alive (25) | ||
| Agaimy et al. (41) | 28 82 | 2 M | External auditory canal | 2 porocarcinoma | Alive (12) Alive (6) | ||
| Sekine et al. () | NA | NA | Head and neck, trunk, extremity | 21 poroma 6 porocarcinoma | 1 poroma | NA | |
| Russell-Goldman et al. (42) | NA | NA | NA | 5 porocarcinoma | NA | ||
| Prieto-Granada et al. (43) | 50–83 | 3 M 2 F | Forehead, thigh, foot, knee | 2 poroma 3 porocarcinoma | NA | ||
| Macagno et al. () | NA | NA | NA | 12 poroid neoplasm | 1 poroid hidradenocarcinoma | NA | |
| Snow et al. (44) | 13–90 | 6 M 5 F | Abdomen, back, elbow, foot, knee, nose, temple | 3 poroma 6 porocarcinoma 1 dermal duct tumor | 1 poroma with porocarcinoma | NA | |
Summary of current reported cases of NUT-rearranged cutaneous poroid neoplasms in the literature.
M, male; F, female; NA, not available.
Figure 4
Canonical NC is predominantly an aggressive, poorly differentiated carcinoma arising from midline structures such as the thorax, head, and neck. Our study indicates that NUTM1-rearranged tumors occurring at rare locations are distinct from canonical NC and may require a different diagnostic framework. Case 2 particularly highlights this uncertainty: a calvaria vertex lesion with WWTR1::NUTM1 fusion showing glandular differentiation and a 40-month survival period. Therefore, not all NUTM1-rearranged tumors should be equated with canonical NC.
For tumors arising outside canonical midline sites, we recommend integrating histomorphology (e.g., glandular, poroid, or myoepithelial features), immunophenotype, and clinical behavior with molecular testing results. Before more explicit diagnostic criteria are established, the use of descriptive terms such as “NUTM1-rearranged tumor” with site and morphological modifiers may be considered. More cases of non-canonical NC need to be accumulated in the future to refine diagnostic criteria and construct a more unified conceptual framework for NUTM1-rearranged tumors.
In conclusion, after reviewing the canonical NC represented by BRD4::NUTM1, our study focused on reporting and summarizing NC cases with non-canonical fusion variants, characterized by uncommon anatomical sites and distinctive histopathological morphology. We aim to help pathologists quickly and accurately identify and diagnose these non-canonical NC cases. When diagnosing and classifying NUT-rearranged malignant tumors, it is crucial to preliminarily classify them into carcinoma or sarcoma based on histomorphology and immunophenotype and to further classify them into canonical or non-canonical fusion subtypes, which could potentially inform treatment decisions with available targeted agents. Therefore, additional case reports and larger studies are needed to better understand the impact of different NUTM1-rearranged tumors on downstream pathways and biological behavior, thereby guiding drug development and improving the prognosis of NC.
Statements
Data availability statement
The original contributions presented in the study are publicly available. This data can be found here: https://ddbj.nig.ac.jp/search/, accession numbers SAMD01928453, SAMD01928454, and SAMD01928455.
Ethics statement
The studies involving humans were approved by The Ethics Committee on Biomedical Research, West China Hospital of Sichuan University. The studies were conducted in accordance with the local legislation and institutional requirements. Inform consent can be exempted in this retrospective observational research. Written informed consent was obtained from the patient’s next of kin for the publication of any potentially identifiable images or data included in this article.
Author contributions
ZL: Writing – original draft. CY: Writing – original draft. XZ: Writing – review & editing. YW: Writing – review & editing. MC: Data curation, Writing – review & editing. MT: Methodology, Writing – review & editing. LH: Methodology, Writing – review & editing. ML: Supervision, Writing – review & editing. PZ: Supervision, Writing – review & editing. XW: Methodology, Writing – review & editing. JB: Methodology, Writing – review & editing. DL: Writing – review & editing, Data curation, Resources. LJ: Writing – review & editing. WW: Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This study was supported by the Sichuan Science and Technology Program (No. 2021YJ0117) and the 1•3•5 project for disciplines of excellence–Clinical Research Incubation Project, West China Hospital, Sichuan University (Nos. 2019HXFH034 and ZYJC21074).
Conflict of interest
Authors MT and LH were employed by company Sichuan Kingmed Center for Clinical Laboratory Co., Ltd.
The remaining author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2026.1828484/full#supplementary-material
References
1
FrenchCA. Nut carcinoma: clinicopathologic features, pathogenesis, and treatment. Pathol Int. (2018) 68:583–95. doi: 10.1111/pin.12727
2
SalatiMBaldessariCBonettiLRMessinaCMerzVCerbelliBet al. Nut midline carcinoma: current concepts and future perspectives of a novel tumour entity. Crit Rev Oncol Hematol. (2019) 144:102826. doi: 10.1016/j.critrevonc.2019.102826
3
StevensTMMorloteDXiuJSwensenJBrandwein-WeberMMiettinenMMet al. Nutm1-rearranged neoplasia: a multi-institution experience yields novel fusion partners and expands the histologic spectrum. Mod Pathol. (2019) 32:764–73. doi: 10.1038/s41379-019-0206-z
4
Le LoarerFPissalouxDWatsonSGodfraindCGalmiche-RollandLSilvaKet al. Clinicopathologic features of CIC-NUTM1 sarcomas, a new molecular variant of the family of CIC-fused sarcomas. Am J Surg Pathol. (2019) 43:268–76. doi: 10.1097/pas.0000000000001187
5
WangsiricharoenSWakelyPEPrietoVGYuW. Sarcoma with MGA::NUTM1 fusion: a report of three cases and literature review. Histopathology. (2023) 83:712–21. doi: 10.1111/his.15004
6
Van TreeckBJThangaiahJJTorres-MoraJStevensTMRothermundtCFassanMet al. Nutm1-rearranged colorectal sarcoma: a clinicopathologically and genetically distinctive Malignant neoplasm with a poor prognosis. Mod Pathol. (2021) 34:1547–57. doi: 10.1038/s41379-021-00792-z
7
SekineSKiyonoTRyoEOgawaRWakaiSIchikawaHet al. Recurrent YAP1-MAML2 and YAP1-NUTM1 fusions in poroma and porocarcinoma. J Clin Invest. (2019) 129:3827–32. doi: 10.1172/jci126185
8
MacagnoNKervarrecTSohierPPoirotBHaffnerACarlottiAet al. Nut is a specific immunohistochemical marker for the diagnosis of YAP1-NUTM1-rearranged cutaneous poroid neoplasms. Am J Surg Pathol. (2021) 45:1221–7. doi: 10.1097/pas.0000000000001693
9
LiangZTangYLiCXieGChenMZhouPet al. Oral and oropharyngeal nut carcinoma: a multicentre screening study of poorly differentiated oral cancer. Histopathology. (2024) 85(6):868–78. doi: 10.1111/his.15245
10
ChenMYangJLvLLiYTangYLiuWet al. Comprehensive genetic profiling of six pulmonary nuclear protein in testis carcinomas with a novel micropapillary histological subtype in two cases. Hum Pathol. (2021) 115:56–66. doi: 10.1016/j.humpath.2021.02.004
11
ChenMZhaoSLiangZWangWZhouPJiangL. Nut carcinoma of the parotid gland: report of two cases, one with a rare ZNF532-NUTM1 fusion. Virchows Arch. (2022) 480:887–97. doi: 10.1007/s00428-021-03253-9
12
WangJLiRLiJYiYLiuXChenJet al. Comprehensive analysis of oncogenic fusions in mismatch repair deficient colorectal carcinomas by sequential DNA and RNA next generation sequencing. J Transl Med. (2021) 19:433. doi: 10.21203/rs.3.rs-544833/v1
13
WuSShiXRenXLiKPangJLiangZ. Evaluation of NTRK gene fusion by five different platforms in triple-negative breast carcinoma. Front Mol Biosci. (2021) 8654387. doi: 10.3389/fmolb.2021.654387
14
ZhaoSDuXZhangYBaiJMengLLiXet al. Development and application of a whole transcriptome sequencing assay for the detection of gene fusions in clinical cancer specimens. BMC Cancer. (2025) 25:842. doi: 10.1186/s12885-025-14186-w
15
LeeTChoJBaekCHSonYIJeongHSChungMKet al. Prevalence of nut carcinoma in head and neck: analysis of 362 cases with literature review. Head Neck. (2020) 42:924–38. doi: 10.1002/hed.26067
16
BauerDEMitchellCMStraitKMLathanCSStelowEBLüerSCet al. Clinicopathologic features and long-term outcomes of nut midline carcinoma. Clin Cancer Res. (2012) 18:5773–9. doi: 10.1158/1078-0432.ccr-12-1153
17
StathisAZuccaEBekraddaMGomez-RocaCDelordJPde La Motte RougeTet al. Clinical response of carcinomas harboring the BRD4-NUT oncoprotein to the targeted bromodomain inhibitor OTX015/MK-8628. Cancer Discov. (2016) 6:492–500. doi: 10.1158/2159-8290.cd-15-1335
18
MaherOMChristensenAMYedururiSYedururiSBellDTarekN. Histone deacetylase inhibitor for nut midline carcinoma. Pediatr Blood Cancer. (2015) 62:715–7. doi: 10.1002/pbc.25350
19
JungMKimSLeeJKYoonSOParkHSHongSWet al. Clinicopathological and preclinical findings of nut carcinoma: a multicenter study. Oncologist. (2019) 24:e740–8. doi: 10.1634/theoncologist.2018-0477
20
ChienYWHsiehTHChuPYHsiehSMLiuMLLeeJCet al. Primary Malignant epithelioid and rhabdoid tumor of bone harboring ZNF532-NUTM1 fusion: the expanding nut cancer family. Genes Chromosomes Cancer. (2019) 58:809–14. doi: 10.1002/gcc.22785
21
GotoTAraiYShibataTOyamaTYoshidaA. Sarcoma with MGA-NUTM1 fusion in the lung: an emerging entity. Virchows Arch. (2020) 476:317–22. doi: 10.1007/s00428-019-02623-8
22
den BakkerMABeverlooBHvan den Heuvel-EibrinkMMMeeuwisCATanLMJohnsonLAet al. Nut midline carcinoma of the parotid gland with mesenchymal differentiation. Am J Surg Pathol. (2009) 33:1253–8. doi: 10.1097/pas.0b013e3181abe120
23
BishopJA. Newly described tumor entities in sinonasal tract pathology. Head Neck Pathol. (2016) 10:23–31. doi: 10.1007/s12105-016-0688-7
24
NishimuraYRyoEYamazakiNYatabeYMoriT. Cutaneous primary nut carcinoma with BRD3-NUTM1 fusion. Am J Surg Pathol. (2021) 45:1582–4. doi: 10.1097/pas.0000000000001801
25
SchaeferIMDal CinPLandryLMFletcherCDMHannaGJFrenchCA. CIC-NUTM1 fusion: a case which expands the spectrum of NUT-rearranged epithelioid Malignancies. Genes Chromosomes Cancer. (2018) 57:446–51. doi: 10.1002/gcc.3
26
DicksonBCSungYSRosenblumMKReuterVEHarbMWunderJSet al. Nutm1 gene fusions characterize a subset of undifferentiated soft tissue and visceral tumors. Am J Surg Pathol. (2018) 42:636–45. doi: 10.1097/pas.0000000000001021
27
DiolaitiDDela CruzFSGundemGBouvierNBouladMZhangYet al. A recurrent novel MGA-NUTM1 fusion identifies a new subtype of high-grade spindle cell sarcoma. Cold Spring Harb Mol Case Stud. (2018) 4(6):a003194. doi: 10.1101/mcs.a003194
28
UnderwoodCIMCardonaDMBentleyRCShenGFengXJourGet al. Epithelioid hyalinizing sarcoma with MGA-NUTM1 fusion. Am J Clin Pathol. (2020) 154:859–66. doi: 10.1093/ajcp/aqaa113
29
MantillaJGRicciottiRWChenEHochBLLiuYJ. Detecting disease-defining gene fusions in unclassified round cell sarcomas using anchored multiplex PCR/targeted RNA next-generation sequencing-molecular and clinicopathological characterization of 16 cases. Genes Chromosomes Cancer. (2019) 58:713–22. doi: 10.1002/gcc.22763
30
TamuraRNakaokaHYoshiharaKMoriYYachidaNNishikawaNet al. Novel MXD4-NUTM1 fusion transcript identified in primary ovarian undifferentiated small round cell sarcoma. Genes Chromosomes Cancer. (2018) 57:557–63. doi: 10.1002/gcc.22668
31
XuBChenJFSarungbamJTickooSDicksonBCReuterVEet al. Nutm1-fusion positive Malignant neoplasms of the genitourinary tract: a report of six cases highlighting involvement of unusual anatomic locations and histologic heterogeneity. Genes Chromosomes Cancer. (2022) 61:542–50. doi: 10.1002/gcc.23046
32
BarlettaJAGildaySDAfkhamiMBellDBocklageTBoisselierPet al. Nutm1 -rearranged carcinoma of the thyroid : a distinct subset of nut carcinoma characterized by frequent NSD3 - NUTM1 fusions. Am J Surg Pathol. (2022) 46:1706–15. doi: 10.1097/pas.0000000000001967
33
ViswanathanKHahnEDoganSWeinrebIDicksonBCMacMillanCet al. The histological spectrum and immunoprofile of head and neck nut carcinoma: a multicentre series of 30 cases. Histopathology. (2024) 85:317–26. doi: 10.1111/his.15204
34
ChauNGHurwitzSMitchellCMAserlindAGrunfeldNKaplanLet al. Intensive treatment and survival outcomes in nut midline carcinoma of the head and neck. Cancer. (2016) 122:3632–40. doi: 10.1002/cncr.30242
35
GiridharPMallickSKashyapLRathGK. Patterns of care and impact of prognostic factors in the outcome of nut midline carcinoma: a systematic review and individual patient data analysis of 119 cases. Eur Arch Otorhinolaryngol. (2018) 275:815–21. doi: 10.1007/s00405-018-4882-y
36
FrenchCAKutokJLFaquinWCToretskyJAAntonescuCRGriffinCAet al. Midline carcinoma of children and young adults with nut rearrangement. J Clin Oncol. (2004) 22:4135–9. doi: 10.1038/nrc3659
37
ChauNGMaCDangaKAl-SayeghHNardiVBarretteRet al. An anatomical site and genetic-based prognostic model for patients with nuclear protein in testis (NUT) midline carcinoma: analysis of 124 patients. JNCI Cancer Spectr. (2020) 4:pkz094. doi: 10.1093/jncics/pkz094
38
HartsoughEMMoranJDevinsKWszolekMCornejoKM. An unusual case of a scrotal porocarcinoma and review of the literature. Am J Dermatopathol. (2023) 45:51–5. doi: 10.1097/dad.0000000000002327
39
ParraOKerrDABridgeJALoehrerAPLinosK. A case of YAP1 and NUTM1 rearranged porocarcinoma with corresponding immunohistochemical expression: review of recent advances in poroma and porocarcinoma pathogenesis with potential diagnostic utility. J Cutan Pathol. (2021) 48:95–101. doi: 10.1111/cup.13832
40
ChristensenDJTulucM. Porocarcinoma with YAP1-NUTM1 fusion presenting as a nut immunohistochemistry-positive lymph node metastasis. J Cutan Pathol. (2023) 50:410–4. doi: 10.1111/cup.14413
41
AgaimyATögelLHallerFZenkJHornungJMärklB. YAP1-NUTM1 gene fusion in porocarcinoma of the external auditory canal. Head Neck Pathol. (2020) 14:982–90. doi: 10.1007/s12105-020-01173-9
42
Russell-GoldmanEHornickJLHannaJ. Utility of YAP1 and NUT immunohistochemistry in the diagnosis of porocarcinoma. J Cutan Pathol. (2021) 48:403–10. doi: 10.1111/cup.13924
43
Prieto-GranadaCMorloteDPavlidakeyPRodriguez-WaitkusPRamirezCFlorentoEet al. Poroid adnexal skin tumors with YAP1 fusions exhibit similar histopathologic features: a series of six YAP1-rearranged adnexal skin tumors. J Cutan Pathol. (2021) 48:1139–49. doi: 10.1016/b978-0-443-06654-2.00011-1
44
SnowJTGeorgantzoglouNGreenDCParraOLeBlancREYanSet al. Molecular analysis of nut-positive poromas and porocarcinomas identifies novel break points of YAP1::NUTM1 fusions. J Cutan Pathol. (2022) 49:850–8. doi: 10.1111/cup.14265
45
McEvoyCRFoxSBPrallOWJ. Emerging entities in NUTM1-rearranged neoplasms. Genes Chromosomes Cancer. (2020) 59:375–85. doi: 10.1002/gcc.22838
46
LilljebjörnHHenningssonRHyrenius-WittstenAOlssonLOrsmark-PietrasCvon PalffySet al. Identification of ETV6-RUNX1-like and DUX4-rearranged subtypes in paediatric B-cell precursor acute lymphoblastic leukaemia. Nat Commun. (2016) 7:11790. doi: 10.1038/ncomms11790
47
GuZChurchmanMRobertsKLiYLiuYHarveyRCet al. Genomic analyses identify recurrent MEF2D fusions in acute lymphoblastic leukaemia. Nat Commun. (2016) 7:13331. doi: 10.1038/ncomms13331
48
SiegfriedAMasliah-PlanchonJRouxFELarrieu-CironDPierronGNicaiseYet al. Brain tumor with an ATXN1-NUTM1 fusion gene expands the histologic spectrum of NUTM1-rearranged neoplasia. Acta Neuropathol Commun. (2019) 7:220. doi: 10.1186/s40478-019-0870-8
49
LuoWStevensTMStaffordPMiettinenMGatalicaZVranicS. Nutm1-rearranged neoplasms-a heterogeneous group of primitive tumors with expanding spectrum of histology and molecular alterations-an updated review. Curr Oncol. (2021) 28:4485–503. doi: 10.3390/curroncol28060381
50
LeeACKwongYIFuKHChanGCMaLLauYL. Disseminated mediastinal carcinoma with chromosomal translocation (15;19). a distinctive clinicopathologic syndrome. Cancer. (1993) 72:2273–6. doi: 10.1002/1097-0142(19931001)72:7<2273::aid-cncr2820720735>3.0.co;2-u
Summary
Keywords
BRD3, gene fusion, nuclear protein in testis (NUT) carcinoma, NUTM1, WWTR1
Citation
Liang Z, Yang C, Zhai X, Wang Y, Chen M, Tang M, Huang L, Li M, Zhou P, Wu X, Bao J, Liang D, Jiang L and Wang W (2026) Expanding the spectrum of non-canonical NUT carcinoma: clinicopathological and molecular characterization of BRD3::NUTM1 and WWTR1::NUTM1 fusion variants. Front. Oncol. 16:1828484. doi: 10.3389/fonc.2026.1828484
Received
11 March 2026
Revised
22 May 2026
Accepted
29 May 2026
Published
01 July 2026
Volume
16 - 2026
Edited by
Sharon R. Pine, University of Colorado Anschutz Medical Campus, United States
Reviewed by
Gopinath Prakasam, University of Texas Southwestern Medical Center, United States
Chih Chieh Yen, National Cheng Kung University, Taiwan
Zhuomiao Ye, Chongqing University, China
Updates
Copyright
© 2026 Liang, Yang, Zhai, Wang, Chen, Tang, Huang, Li, Zhou, Wu, Bao, Liang, Jiang and Wang.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Xiaoqian Zhai, xiaoqzhai@163.com; Dongni Liang, dongni1108@163.com
†These authors have contributed equally to this work
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.