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NUT midline carcinoma (NMC) is a uniformly lethal malignancy that is defined by rearrangement of the nuclear protein in testis (NUT) gene on chromosome 15q14. NMCs are morphologically indistinguishable from other poorly differentiated carcinomas, and the diagnosis is usually made currently by fluorescence in situ hybridization (FISH). As normal NUT expression is confined to testis and ovary, we reasoned that an immunohistochemical (IHC) stain for NUT would be useful in diagnosing NMC. To this end, we raised a highly specific rabbit monoclonal antibody, C52, against a recombinant NUT polypeptide, and developed an IHC staining protocol. The sensitivity and specificity of C52 staining was evaluated in a panel of 1068 tissues, predominantly diverse types of carcinomas (n=906), including 30 NMCs. Split-apart FISH for NUT rearrangement was used as a "gold standard" diagnostic test for NMC. C52 immunoreactivity among carcinomas was confined to NMCs. IHC staining had a sensitivity of 87%, a specificity of 100%, a negative predictive value of 99%, and a positive predictive value of 100%. Two new cases of NMC containing BRD4-NUT fusions were detected by C52 IHC, but missed by conventional FISH. In both instances, these tumors contained cryptic BRD4-NUT rearrangements, as confirmed by FISH using a refined set of probes. Some germ cell tumors, including 64% of dysgerminomas, showed weak NUT immunoreactivity, consistent with the expression of NUT in normal germ cells. We conclude that IHC staining with the C52 monoclonal antibody is a highly sensitive and specific test that reliably distinguishes NMC from other forms of carcinoma. The NUT antibody is being prepared for commercial release and will be available in the near future.

Diagnosis of NUT Midline Carcinoma Using a NUT-specific Monoclonal Antibody