Primary renal NUT carcinoma identified by next-generation sequencing: A case report and literature review

Background NUT carcinoma is a rare aggressive squamous cell carcinoma subtype genetically defined by NUTM1 rearrangements. NUT carcinoma usually has a primitive differentiation state and can be easily misdiagnosed as an undifferentiated carcinoma or Ewing sarcoma. Case presentation We report a case of NUT carcinoma of renal origin initially diagnosed as a malignant small round-cell tumor, likely to be Ewing sarcoma/primitive neuroectodermal tumor. Based on next-generation sequencing (NGS), the diagnosis was revised to NUT carcinoma with a characteristic NUTM1 rearrangement. The patient relapsed after surgery and received a standard NUT carcinoma treatment. However, due to advanced neoplasm progression, first-line chemotherapy failed and the patient died. Conclusion Routine NUT immunohistochemistry staining, NGS, and/or fluorescent in situ hybridization for poorly differentiated carcinoma and sarcoma tumors can help avoid misdiagnosis of NUT carcinoma-related tumors, allowing patients to benefit from bromodomain and extra-terminal motif inhibitor therapy.

NUT carcinoma generally exhibits primitive differentiation with an abrupt squamous epithelial component. However, nearly 50% of the cases lack a squamous epithelial component and are often misdiagnosed as undifferentiated carcinoma or Ewing's carcinoma. With the rapid development of molecular pathology, next-generation sequencing (NGS) has emerged as a means to provide unique advantages for the diagnosis of poorly differentiated cancers that lack characteristic histological features. We report a case of primary renal NUT carcinoma that was identified by NGS to have a 3 NUTM1 rearrangement and present a review of the relevant literature.

Case Report
A 41-year-old male visited a local hospital in September 2018 due to lumbago. His past medical history and family history were unremarkable. An enhanced computed tomography (CT) scan revealed a soft tissue density shadow 7.1 cm × 2.6 cm in the left kidney. The density was uneven with a local protrusion from the renal silhouette. Upon contrast enhancement of the CT scan, the density intensified. Laparoscopic radical excision surgery was performed. Based on gross examination, the resected kidney specimen was 14.0 cm × 10.0 cm × 6.5 cm with a mass at the inferior pole being 7.0 cm × 5.7 cm. The surface of the specimen was gray-white and gray-red of medium hardness with an unclear boundary. The mucosae of the renal pelvis and ureter were smooth. Microscopic examination revealed a diffuse, patchy infiltration of tumor cells. The cells were round or oval shape and ranged in size from small to medium. The cytoplasm was relatively rich and eosinophilic with plasmacytoid and epithelioid morphologies. Nuclear atypia was significant and some nuclei were vesicula-like. Nucleoli and karyokinesis were obvious. Focal patchy necrosis was present with perineural invasion in some foci. There was extensive vascularization of the tumor stroma (Fig. 1A).

Discussion
NUT carcinoma is a rare, highly malignant tumor of unclear origin that is often considered a poorly differentiated squamous cell carcinoma; however, its clinicopathological features are different from those of classical squamous cell carcinoma. NUT carcinomas can occur at any age (0-81.7 years), but primarily affects children, adolescents, and young adults. It typically develops at the midline with 50% of cases occurring in the aerodigestive tract and 41% occurring in the mediastinum [3]. However, it can also occur in parenchymal organs, including the urinary bladder, mammary gland, endometrium, kidney, and orbit, and is able to invade soft tissues and bone.
During the initial hospital visit by the patient described in the current case report, the tumor was limited to the kidney and imaging did not show significant abnormalities at any other sites. Accordingly, the kidney was considered to be the primary site. Primary renal NUT carcinoma is extremely rare. Our literature search identified reports of only five other cases of primary renal NUT carcinoma [4][5][6][7]. The clinicopathological features of the current patient and those of the previously reported five cases are summarized in Table 1.
Typical NUT carcinoma morphology is a patchy infiltration of small-to-medium-sized undifferentiated tumor components, low-to-moderate tumor cell cytoplasm volume, and active karyokinesis. Sudden triggering of squamous epithelial cell differentiation may occur in approximately half of the cases. In contrast to typical squamous cell carcinoma, which is characterized by rich polymorphism, the morphology of NUT carcinoma cells is 5 relatively consistent; however, NUT carcinoma is not typically found in situ [8]. The current case of NUT carcinoma was similar to the five other reported cases in that cell differentiation was relatively primitive, all cells lacked squamous epithelial characteristics, and tumors demonstrated diffuse but strongly positive expression of CD99. FISH was negative in our patient for an EWSR1 fusion. EWSR1 was reportedly fused with FLI1 in only 85% of cases [9];. Although the FISH results indicated that EWSR1 escaped probe detection of gene breakage and translocation, this negative result could not completely rule out a diagnosis of Ewing sarcoma. Therefore, a diagnosis of NUT carcinoma was made only after NGS analysis.
A review of the literature revealed that NUT carcinoma is often misdiagnosed as Ewing sarcoma/PNET. A characteristic feature of NUT carcinoma based on immunohistochemistry is that > 50% of tumor nuclei are positive for NUTM1, with a sensitivity and specificity of 87% and 100%, respectively. By combining immunohistochemistry with FISH, the diagnostic sensitivity can reach 100% [10]. While NUT immunohistochemistry can be used as a primary screen, a definitive diagnosis of NUT carcinoma requires the molecular detection of NUTM1 rearrangement/fusion. NUTM1 has multiple fusion partners. Two-thirds of the patients have fusions between NUTM1 and BRD4, while the remaining exhibit fusions between NUTM1 and BRD3 or NSD3 [11]. Although the partner gene of NUTM1 of both the five primary renal NUT carcinomas from the literature and our current case all shows BRD4, whether the partner gene is related to the primary site needs further investigation.
The rapidly increasing use of NGS has led to the discovery of additional partner genes for NUTM1, including CIC, BCORL1, MYXD1, and MGA [6 12 13]. This has also widened the range of tumor histology targets. A report by den which presented a myxoid chondrosarcoma morphology [13]. Therefore, tumors with NUTM1 rearrangements are not limited to NUT carcinomas, but may also be present in NUT sarcomas. The relationship between NUT sarcomas and the more common NUT carcinomas requires further investigation with some investigators suggesting that this type of tumor should be generically classified as a "NUT-related tumor" [5].
The clinical course of NUT carcinoma is naturally risk-prone and its prognosis is extremely poor. At the time of 6 initial diagnosis, most patients already have distant metastases. Moreover, the disease responds poorly to traditional radiotherapy and chemotherapy with the median survival being only 6.7 months [15]. In our patient, even after surgery and a post-operative 4-wk course of VDC/IE chemotherapy, the disease was not controlled.
Metastasis to both lungs and the peritoneum had occurred, as well as extensive peritoneal spreading. The patient died eight months after surgery.
In recent years, the emergence of oral bromodomain and extra-terminal domain (BET) inhibitors targeting the BRD2, BRD3, and BRD4 proteins has been beneficial to the management of NUT carcinoma. BET inhibitors mimic the structure of acetylated histone lysine residues and thereby interrupt the interaction between BRD4 and actual acetylated histone lysine. This results in the attenuation of abnormal transcription and induces tumor cell differentiation. However, oral BET inhibitors may not be effective for patients without BRD-NUTM1 fusions [16].

Conclusion
NGS not only is beneficial in the diagnosis of NUT carcinoma, but also allows for the use of more targeted therapies as a result of being able to assess the precise mutations of cancer-related genes. Our experience with the patient described in this report has led us to performing NUT immunohistochemistry on all poorly differentiated tumors, both carcinomas and sarcomas. This can then be followed by NGS and FISH for validation in order to avoid missing or incorrectly diagnosing NUTM1 fusion-related tumors, thereby benefitting more patients. The tumor harbored only one reported mutation at the DNA and RNA level, a BRD4 exon 14-NUTM1 exon3 fusion