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

Cell Signaling Technology Inc., Danvers, MA, USA.
The American journal of surgical pathology (Impact Factor: 5.15). 05/2009; 33(7):984-91. DOI: 10.1097/PAS.0b013e318198d666
Source: PubMed


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.

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Available from: Seung-Mo Hong, Jan 14, 2014
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    • "Diagnostic immunohistochemistry of a human NMC was performed as described (Haack et al. 2009). "
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    ABSTRACT: NUT midline carcinoma (NMC), a subtype of squamous cell cancer, is one of the most aggressive human solid malignancies known. NMC is driven by the creation of a translocation oncoprotein, BRD4-NUT, which blocks differentiation and drives growth of NMC cells. BRD4-NUT forms distinctive nuclear foci in patient tumors, which we found correlate with ∼100 unprecedented, hyperacetylated expanses of chromatin that reach up to 2 Mb in size. These "megadomains" appear to be the result of aberrant, feed-forward loops of acetylation and binding of acetylated histones that drive transcription of underlying DNA in NMC patient cells and naïve cells induced to express BRD4-NUT. Megadomain locations are typically cell lineage-specific; however, the cMYC and TP63 regions are targeted in all NMCs tested and play functional roles in tumor growth. Megadomains appear to originate from select pre-existing enhancers that progressively broaden but are ultimately delimited by topologically associating domain (TAD) boundaries. Therefore, our findings establish a basis for understanding the powerful role played by large-scale chromatin organization in normal and aberrant lineage-specific gene transcription. © 2015 Alekseyenko et al.; Published by Cold Spring Harbor Laboratory Press.
    Full-text · Article · Jul 2015 · Genes & Development
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    • "In the present article, we describe the first case of an NSD3-NUT rearrangement identified in the primary tissue of an NMC of the lung; the NMC was diagnosed using immunohistochemistry with a highly sensitive and specific anti-NUT monoclonal antibody [11]. In addition, a NSD3-NUT fusion gene was successfully identified using 5 -rapid amplification of the cDNA end (RACE) and was validated using FISH. "
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    ABSTRACT: Nuclear protein in testis (NUT) midline carcinoma (NMC) is a rare, aggressive malignancy. Only two pediatric and three adult cases of pulmonary NMCs have been documented. In more than two-thirds of NMC cases, a gene fusion between NUT and BRD4 or BRD3 has been documented; other fusions are rare. A 36-year-old woman was admitted because of a rapidly progressing tumor of the lung with metastases to the breast and bone. A biopsy from the lung tumor revealed an undifferentiated neoplasm exhibiting round to oval nuclei with vesicular chromatin, prominent nucleoli, and scant cytoplasm. Immunohistochemical staining demonstrated focal EMA, cytokeratin AE1/AE3, cytokeratin CAM 5.2, p63, CD138, and vimentin positivity. Finally, the nuclear staining pattern for NUT confirmed a histopathological diagnosis of NMC. A 5'- rapid amplification of the cDNA end (RACE) procedure successfully identified the partner of the NUT translocation as NSD3, a recently discovered partner. Fluorescence in situ hybridization confirmed the NSD3-NUT gene rearrangement, whereas a BRD3/4-NUT fusion gene was not detected. We herein describe the first case of an NSD3-NUT-expressing NMC of the lung. The further accumulation of variant NMCs should provide clues to the establishment of new individualized therapy for NMCs. Copyright © 2014 Elsevier GmbH. All rights reserved.
    Full-text · Article · Nov 2014 · Pathology - Research and Practice
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    • "First reported in 1991 (Kees et al, 1991; Kubonishi et al, 1991) and originally thought to be a childhood disease, these carcinomas have now been identified in patients ranging from newborn to 78 years of age (French, 2010, 2012; Shehata et al, 2010). NUT midline carcinoma is often misdiagnosed since it is rare, lacks distinct histological features, and is not confined to a particular organ, but increased awareness and the development of an immunohistochemical test to detect aberrant expression of the NUT protein (Haack et al, 2009) has resulted in the number of reported cases steadily rising (Bauer et al, 2012; French, 2012). However both the rarity of the disease and its voracity, with median survival o7 months (Bauer et al, 2012), mean that treatment approaches to date have essentially been adapted, without systematic assessment, from those used to treat other types of solid tumour. "
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    ABSTRACT: Background: The NUT midline carcinoma (NMC) is a rare but fatal cancer for which systematic testing of therapy options has never been performed. Methods: On the basis of disease biology, we compared the efficacy of the CDK9 inhibitor flavopiridol (FP) with a panel of anticancer agents in NMC cell lines and mouse xenografts. Results: In vitro anthracyclines, topoisomerase inhibitors, and microtubule poisons were among the most cytotoxic drug classes for NMC cells, while efficacy of the bromodomain inhibitor JQ1 varied considerably between lines carrying different BRD4 (bromodomain-containing protein 4)–NUT (nuclear protein in testis) translocations. Efficacy of FP was comparable to vincristine and doxorubicin, drugs that have been previously used in NMC patients. All three compounds showed significantly better activity than etoposide and vorinostat, agents that have also been used in NMC patients. Statins and antimetabolites demonstrated intermediate single-agent efficacy. In vivo, vincristine significantly inhibited tumour growth in two different NMC xenografts. Flavopiridol in vivo was significantly effective in one of the two NMC xenograft lines, demonstrating the biological heterogeneity of this disease. Conclusions: These results demonstrate that FP may be of benefit to a subset of patients with NMC, and warrant a continued emphasis on microtubule inhibitors, anthracyclines, and topoisomerase inhibitors as effective drug classes in this disease.
    Full-text · Article · Feb 2014 · British Journal of Cancer
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