Diagnosis of NUT midline carcinoma using a NUT-specific monoclonal antibody

Abstract

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.

FIGURE 1

Validation of the anti-NUT C52 monoclonal antibody by immunoblot (A) and immunohistochemistry (B–D, 400×). A, bands for BRD4-NUT (~240kDa) are seen in the NUT NMC cell line, TC-797, and in extracts prepared from 293Tcells transiently transfected with a pcDNA3 plasmid that drives expression of BRD4-NUT. Endogenous NUT (~150kDa) is seen in a lysate prepared from testis. No bands were seen in any negative control cell lines (293T cells transfected with empty pcDNA3, A549, MCF7, and A673). B, nuclear staining for NUT is seen in spermatids and a subset of spermatogonia in human testis. C, control siRNA transfected TC-797 cells reveal speckled nuclear staining, whereas NUT siRNA transfected cells (D) demonstrate significantly reduced nuclear staining. Note that the siRNA against NUT also induces marked increases in cytoplasmic volume and nuclear size, features consistent with the induction of differentiation by withdrawal of BRD4-NUT(5).

FIGURE 2

Detection of BRD4-NUT by the C52 NUT antibody (A–C, 400×), and validated by fluorescent in situ hybridization (D, 1000×). A, typical absence of staining of a non-NMC, in this case a sinonasal undifferentiated carcinoma that was also negative for BRD4-NUT rearrangement by FISH (not shown). By contrast, B, known NMCs reveal diffuse (>90%), speckled, nuclear staining. In two carcinomas (one of which is shown in C), split-apart FISH was negative for NUT rearrangement (data not shown), but tumor cells revealed diffuse nuclear reactivity with C52. A cryptic NUT rearrangement (D) was subsequently demonstrated in this case (as well as the second, not shown) using a probe (red) that spans NUT. This probe splits and joins (arrows) the BRD4 centromeric probe (green), consistent with the presence of a cryptic BRD4-NUT fusion gene.

FIGURE 3

Focal, weak nuclear staining by C52 (<5% cells) in a case of dysgerminoma (A, 400×). The staining, by contrast with that in NMCs, is smooth and not speckled. Western blot (B), stained with C52 antibody, confirms low level expression of native NUT in lysate obtained from this dysgerminoma. The positive control is an extract prepared from 293Tcells transiently transfected with pcDNA3-NUT, and the negative control is 293T cells transfected with empty pcDNA3.