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Page 354 Neumann et al. J Transl Genet Genom 2022;6:353-60 https://dx.doi.org/10.20517/jtgg.2022.06

decline, it remains surprisingly common, with historical series estimating an incidence of 3%-5% of invasive
cancer diagnoses; with the advent of newer diagnostic technologies, the incidence rate of CUP has decreased
[1]
to 1%-2% . It is estimated by the American Cancer Society that in 2022, approximately 30,620 cases of CUP
[2]
will be diagnosed in the United States . An accurate diagnosis is essential because tumor biomarker-
targeted treatment can impact survival.

Nuclear protein in testis (NUT) carcinoma (NC), also known as NUT-midline carcinoma, is a rare and
nearly universally fatal malignancy that primarily affects children and young adults; it typically presents as
undifferentiated or poorly differentiated squamous cell carcinoma(SCC) arising from the midline structures
of the body (lung, head, neck, bladder, etc.). Non-midline cases continue to be reported. Of these, 20%-25%
of CUP cases are poorly differentiated and cannot be characterized histologically. A very high index of
suspicion is required to rule out NC in cases of CUP, as it can commonly be misdiagnosed as poorly
differentiated SCC. Approximately 5% of CUP cases are classified as squamous cell carcinoma, and by
comparison, less than 200 cases of NC have been reported in the literature worldwide as of 2021 .
[3]

NC is characterized by a chromosomal translocation that results in a NUT-fusion oncoprotein, the presence
of which often is readily identified via nuclear immunohistochemical (IHC) staining positive for NUT,
although McEvoy et al. recommend defining the nuclear protein in testis (NUTM1) fusion partner rather
than relying on tumor morphology or immunohistochemical profile . Diagnosis can be confirmed by
[4]
molecular analysis techniques, including fluorescence in situ hybridization (FISH), reverse-transcription
PCR, cytogenetics, and next-generation sequencing (NGS). It should be noted that while NGS testing can
facilitate the diagnosis of NC, it can still result in a false negative .
[5]
NC was first described in two cases reported in 1991, with the most common chromosome translocation
t(15;19)(q15;p13) . This aggressive cancer has a median overall survival (OS) of 6.7 months and is likely the
[6]
most aggressive solid tumor in humans . Due to the rarity of this disease, a national registry was created in
[7,8]
2010 in order to raise awareness and collect clinical data on its characteristics and response to treatment .
[9]
Since that time, NC has been diagnosed more commonly, given increased awareness and better diagnostic
capabilities.

The most common chromosomal rearrangement seen in NC is that of the NUTM1 gene on
chromosome 15, most commonly by the molecular translocation of bromodomain-containing protein 4
(BRD4) (78%) on chromosome 19 or bromodomain-containing protein 3 (BRD3) (15%), though others
have been described, including the gene translocation in our patient nuclear receptor SET domain-
[8]
containing 3 (NSD3)-NUT (6%) . Recent studies have identified more novel fusion partners, including
ZNF532, ZNF592, MXD4, BCORL1, MXD1, CIC, MGA, and others [10,11] .

Normally, NUT is a nuclear protein which binds histone acetyltransferase (HAT) and causes histone
acetylation. In NC, the most common NUT fusion partners are part of bromodomain and extra-terminal
(BET) family (i.e., BRD3 and BRD4). This family of proteins regulates transcription via epigenetics reading
of acetylated histone lysine tail residues. BRD4 regulates transcription, cell growth, cell cycle, and chromatin
structure driven by the BRD4 promoter. MYC and TP63 have been shown to be the key downstream target
of this gene, which promotes uncontrolled cell growth. A study showed BRD4 was hyperphosphorylated in
NC and cyclin-dependent kinase 9 (CDK9) was the potential kinase mediating this . Therefore, by
[12]
blocking BRD4 hyperphosphorylation with inhibitors, it was proposed that this may mitigate the
proliferation of this deadly disease. NSD3 is an enzymatic protein involved in methylating histone lysines,
which regulate gene expression and chromatin integrity .
[13]