Nut Midline Carcinoma (NMC) Arising in Left Submandibular Salivary Gland Manju L. Prasad Yale University School of Medicine New Haven, CT

Clinical History: A 15 year-old boy presented with a submandibular mass on the left side of neck of approximately 3 months duration. He reported waxing and waning pain as well as intermittent episodes of swelling and regression of the mass. The patient denied fever, otalgia, numbness, change in voice or recent dental procedures. There was no prior significant history. Ultrasound showed a well defined, soft tissue mass measuring 3.3 x 3.1 x 2.6 cm with scattered calcifications and no evidence of cystic degeneration, hemorrhage or invasion (Fig. 1). He underwent uneventful removal of an apparently encapsulated tumor that 'shelled out' easily. Slide 1 Click to view with ImageScope Click to view with a Web-Based Viewer Figure 1 Ultrasound showed a well defined, soft tissue mass measuring 3.3 x 3.1 x 2.6 cm with scattered calcifications. Figure 2 Macroscopice examination showed a well circumscribed, tan white, rubbery, lobulated mass with slightly bulging cut surface measuring 2.5 x 2.3 x 1.8 cm. Figure 3 FISH using a dual color split-apart probe for NUT shows signal split in one allele Pertinent Laboratory Data: Macroscopice examination of the specimen showed a well circumscribed, tan white, rubbery, lobulated mass with slightly cut surface measuring 2.5 x 2.3 x 1.8 cm (Fig. 2). Immunohistochemistry showed that the tumor cells were variably positive for cytokeratin (AE1:AE3), CAM5.2, p63, p16, S100, and CD117. Tumor cells were negative for calponin, smooth muscle actin, chromogranin, synaptophysin, CD34 and CD56. A high proliferative index (>50%) was noted with Ki-67. In-situ hybridization for Epstein Barr virus encoded RNA (EBER) was negative. Introduction: Poorly differentiated/ undifferentiated tumors of the salivary gland are challenging, specially in children. The differential diagnosis is wide and the diagnosis may be difficult despite a broad array of immunohistochemical stains. This 15 year old boy underwent a surgical resection of his left submandibular mass. Pathological/Microscopic Findings and any Immunohistochemical or Other Studies: Macroscopice examination of the specimen showed a well circumscribed, tan white, rubbery, lobulated mass with slightly bulging cut surface measuring 2.5 x 2.3 x 1.8 cm. Microscopic examination showed nests of malignant epithelial cells infiltrating the submandibular salivary gland tissue. Tumor cells showed large vesicular nuclei with open chromatin and scant cytoplasm. Nucleoli varied from indistinct to prominent. The tumor was predominantly undifferentiated, however, focal squamous differentiation with abrupt keratinization towards the center of tumor cell clusters was noted as were small rudimentary tubules. A few tumor cells showed vacuolated cytoplasm. Tumor cells showed epitheliotropism into the salivary duct epithelium. Numerous mitoses and frequent perineural invasion were seen. Coagulative tumor necrosis, desmoplastic stromal fibrosis cystic change and cholesterol granulomas were noted. Metastatic tumor was seen in one of 33 cervical lymph nodes. The tumor cells did not reveal mucicarmine or DPAS positive intracytoplasmic material. Immunohistochemistry showed that the tumor cells were focally positive for cytokeratin (AE1:AE3), CAM5.2, p63, p16, and CD117. Tumor cells were negative for calponin, smooth muscle actin, S100 protein, chromogranin-A, synaptophysin, CD34 and CD56 (NCAM). A high proliferative index (>50%) was noted with Ki-67. In-situ hybridization for EBV-RNA (EBER) was negative. Flourescent in-situ hybridization (FISH) using probes for 15q14 NUT and 19p13.1 BRD4 breakpoints showed split apart of one NUT allele indicating NUT rearrangement in all tumor cells. Differential Diagnoses: The initial differential diagnosis included poorly differentiated mucoepidermoid carcinoma, however, there was no evidence of mucinous differentiation and a mucicarmine stain was negative. Poorly differentiated squamous cell carcinoma or adenocarcinoma were considered but the tumor showed variable staining with cytokeratins and p63. The EBV associated lymphoepithelial-type carcinoma is a fairly common possibility in children, specially in south-asian countries. The absence of significant lymphocytic infiltrate and the failure to demonstrate EBV-RNA did not favor this diagnosis. Final Diagnosis: Nut Midline Carcinoma (NMC) Arising in Left Submandibular Salivary Gland Ultrasound showed a well defined, soft tissue mass measuring 3.3 x 3.1 x 2.6 cm with scattered calcifications. Macroscopice examination showed a well circumscribed, tan white, rubbery, lobulated mass with slightly bulging cut surface measuring 2.5 x 2.3 x 1.8 cm. FISH using a dual color split-apart probe for NUT shows signal split in one allele Case Discussion: Malignant salivary gland tumors in the pediatric population are exceedingly rare. The most common is mucoepidermoid carcinoma comprising about 80% of all malignant salivary gland tumors in children followed by acinic cell carcinoma, adenoid cystic carcinoma, adenocarcinoma and poorly differentiated carcinoma, not otherwise specified. The NUT midline carcinomas (NMC) are defined by the rearrangement of the NUT [1] (NUclear protein in Testis) gene on chromosome 15q14, usually from a balanced translocation with the BRD4 gene on chromosome 19p13.1. A small minority of tumors may have variant NUT translocation, e.g. to BRD3 and other unknown genes. The fusion protein is believed to block epithelial differentiation [2] . NMC are rare malignancies. Initially, they were believed to be tumors of children and adolescents, however, later data showed that the tumors can occur in a wide age range (3 to 78 years) and in both sexes [3, 4]. The majority of the reported cases are in the head and neck involving midline structures in the aerodigestive tract, hence the name NUT midline carcinoma (NMC). Tumors have been reported in the mediastinum, and rarely below the diaphragm. Cases outside of the midline axis are non-existent [3] . Stelow et al reported that among poorly differentiated/ undifferentiated carcinomas of the sinonasal region not associated with EBV, nearly 20% showed NUT rearrangement [4] The cell of origin is unknown, however, given the midline distribution of these tumors, origin from primitive neural crest cells has been suggested [3]. However, these tumors are not shown to express any neural or neuroendocrine markers. Immunoreactivity to p63 and focal abrupt ketatinization in some NMC suggests a squamous lineage [4] . To date all but one patient have died with a median survival of 9.5 months [3, 5] . According to French, NMC should be suspected in any monomorphic poorly differentiated/ undifferentiated malignant neoplasm, even in the presence of focal squamous differentiation. Thus, the differential diagnosis would include basaloid squamous cell carcinoma, sinonasal undifferentiated carcinoma, poorly differentiated carcinoma, nasopharyngeal carcinoma, lymphoma, Ewing's sarcoma/PNET, and other poorly differentiated tumors. The diagnosis is confirmed by the demonstration of NUT rearrangement by FISH, or by detecting NUT-BRD4 fusion transcript by RT-PCR. Recently, the same group that described NMC in the upper aerodigestive tract has also produced a rabbit monoclonal antibody (C52, Cell Signaling Technology) against the NUT fusion protein that can be used for immunohistochemistry with a sensitivity of 87% and a specificity of 100% [6] . Conclusion(s): In summary, NUT midline carcinoma should be considered in the differential diagnosis of poorly differentiated/undifferentiated carcinomas of the head and neck. These tumors are uniformly lethal and their prognosis is vastly different from the EBV positive, nasopharyngeal carcinoma, HPV positive poorly differentiated carcinoma, and other high grade tumors. There are no morphologically distinct criteria for their diagnosis. Demonstration of NUT rearrangement by fluorescent in-situ hybridization or the BRD4-NUT fusion transcript by RT-PCR is required for diagnosis. References: Shapiro NL, Bhattacharyya N. Clinical characteristics and survival for major salivary gland malignancies in children. Otolaryngol Head Neck Surg. 2006 Apr;134(4):631-4. French CA, Ramirez CL, Kolmakova J, Hickman TT, Cameron MJ, Thyne ME, et al. BRD-NUT oncoproteins: a family of closely related nuclear proteins that block epithelial differentiation and maintain the growth of carcinoma cells. Oncogene. 2008 Apr 3;27(15):2237-42. French CA. Molecular pathology of NUT midline carcinomas. J Clin Pathol. 2008 Jun 13. Stelow EB, Bellizzi AM, Taneja K, Mills SE, Legallo RD, Kutok JL, et al. NUT rearrangement in undifferentiated carcinomas of the upper aerodigestive tract. Am J Surg Pathol. 2008 Jun;32(6):828-34. Mertens F, Wiebe T, Adlercreutz C, Mandahl N, French CA. Successful treatment of a child with t(15;19)-positive tumor. Pediatr Blood Cancer. 2007 Dec;49(7):1015-7. Haack H, Johnson LA, Fry CJ, Crosby K, Polakiewicz RD, Stelow EB, et al. Diagnosis of NUT midline carcinoma using a NUT-specific monoclonal antibody. Am J Surg Pathol. 2009 Jul;33(7):984-91.