What is NUT carcinoma?

NUT carcinoma (NC), also referred to as NUT midline carcinoma, is a very rare and aggressive type of cancer.¹ This cancer is particularly dangerous due to its rapid spread and complexities in diagnosis. This condition gets its name from “nuclear protein in testis”, which is a protein expressed by the NUTM1 gene.¹ While this gene was first identified in the testis, NUT carcinoma is not exclusive to males and can affect anyone. 

NUT carcinoma causes the formation of a tumour, typically in the midline structures (head, neck, chest), hence the name midline. Under the microscope, tissue isolated from an NC tumour may resemble other cancers that show an unorganised, poorly differentiated presentation, making it difficult to identify.² This histological trait complicates the diagnostic process and the effectiveness of treatment. At a molecular level, NUT carcinoma occurs because the NUTM1 gene gets rearranged, or fused with another gene, which leads to the formation of an abnormal protein that drives tumour growth. This rearrangement is key for identifying NUT carcinoma, as it is not common in other types of cancer and can help diagnose it accurately.³ 

While NUT carcinoma is considered a rare form of cancer, the condition may be underdiagnosed due to its nonspecific symptoms and complex diagnosis process. This article explores the diagnostic methods for identifying NUT carcinoma, with a focus on biopsy and immunohistochemistry. 

Clinical presentation

NUT carcinoma tends to affect younger individuals, as the most common age of diagnosis is 16 to 24 years.¹ Because of its nonspecific symptoms, the condition is often diagnosed at an advanced stage, which leads to an estimated 6 to 9 months median survival after diagnosis.⁴ 

NC tumours can appear almost anywhere in the body but are usually found in the head, neck and chest area. As a result, general symptoms typically relate to these locations.

Symptoms of NUT carcinoma include:⁴

• Fatigue
• Pain
• Unintentional weight loss
• Nasal symptoms (congestion, runny nose, loss of smell, nosebleeds)
• Painless lump in the midline area
• Vision complications (impaired vision, eye bulging)
• Chest symptoms (chronic cough, shortness of breath, trouble breathing)

Diagnostic process of NUT carcinoma

Healthcare professionals usually follow a step-by-step process for the diagnosis of NC. It is important to emphasise that NUT carcinoma presents general symptoms that can only be linked to NC by specific testing. For this reason, each step of this process gets increasingly specific:⁵

• Physical Exam/ Imaging (CT, MRI, PET scans)— To identify the location, size and stage of the tumour
• Biopsy— Isolates tissue for analysis using a range of different methods
• Histopathology— Analysis of the biopsy sample, where it is examined under a microscope
• Immunohistochemistry (IHC)— A highly specific method where an antibody-based identification is performed. This step usually stands as the confirmation that the patient has NUT carcinoma
• Molecular Testing— If IHC results are unclear, molecular tests may be performed to detect possible NUTM1 gene abnormalities (rearrangements, fusion partners) 

Due to how this cancer lacks unique morphological features and how it can only be identified through molecular-level techniques, biopsies and immunohistochemistry are critical in diagnosing NC.

Biopsy in diagnosis

A biopsy is a process where tissue or fluid from our body is removed by a healthcare provider for examination. The sample can be in the form of cells, tissue or fluid, which are then analysed primarily through microscopy. Biopsies are not limited to cancer; however, they are particularly preferred for diagnosing cancer because of the distinct morphological features found in cancerous tissues. In the case of NUT carcinoma, tissue isolation is essential for further testing and accurately diagnosing this rare condition.

Several types of biopsy methods can be used to diagnose NC:

Fine needle aspiration (FNA)

 This practice is often used for suspicious lumps that are close to the surface and is great for preliminary results for tumours that are easily accessible. FNA is often limiting for cancers like NUT carcinoma, as it collects a small sample that may not be enough for further testing or an accurate histopathology assessment.⁵

Core needle biopsy

 Using a thicker and longer needle, tissue is isolated from a lump that is deeper in the body. This method is preferred for NUT cancer biopsies, as it is generally minimally invasive and provides enough tissue for the essential assessment of immunohistochemistry.⁵

Surgical biopsy 

This option includes a small operation that removes a part of the suspected tumour, or all of it. It provides a larger tissue sample and is therefore the most comprehensive approach, while it is ideal for tumours that are difficult to reach through a needle with other biopsy methods, like the chest. However, due to its invasiveness and high chance of disrupting the tumour and triggering its spread, this option is generally avoided.⁶

Histopathology features

Once the tissue is obtained from the biopsy, it is examined under a microscope to assess if it’s healthy and check for signs of cancer. In healthy tissue, the sheets of cells appear well-structured and organised, featuring different cell types (e.g., supportive cells, immune cells, etc.) at different life stages (young, mature, and dying). 

Cancerous cells are usually easy to distinguish because they do not follow these rules. Usually, cancer cells show the following features:⁷

• Bigger nucleus, little cytoplasm
• Abnormal nuclear shape
• Increased division of cells
• No clear tissue structure
• Variations of cell size and shape

If the biopsied sample comes from an NC tumour, the following unique traits may raise suspicion for the rare condition: ⁷

• Increased dividing activity and  Necrosis—This combination points towards an aggressive cancer
• Sheets of medium-sized, undifferentiated cells that look identical—Primitive, immature cells that have not gained a specialised function/structure. This suggests that the cell’s only goal is to divide and grow, rather than be functional
• “Islands” of well-differentiated squamous cells—Warning sign for NUT carcinoma, as abrupt cell maturation in a mostly primitive sheet of cells is a hallmark feature of NC

While these features may point to NC, they can also mimic other tumours that show poor differentiation (e.g., lymphomas, SCC, etc.). For this reason, immunohistochemistry is required to provide a definitive diagnosis for this rare condition.

Immunohistochemistry (IHC)

Immunohistochemistry (IHC) is a laboratory technique that uses stained antibodies to detect specific proteins (markers) in tissues. Here, the NUT protein acts as the marker, and to test for NUT carcinoma, stained antibodies are added to the tissue. If the antibodies stick to their protein match, they will produce a colour that is visible through microscopy.⁸

Initially IHC marker panel

Considering that NUT carcinoma is rare and mimics other poorly differentiated cancers, an initial panel of IHC markers is used to narrow down the possible cancer type. At this stage, antibodies specific to epithelial (skin, lungs, digestive tract) or neuroendocrine (hormone-producing) markers may be used.⁸

Key IHC marker: C52 protein

The NUT-specific monoclonal antibody (C52) is a unique protein marker for NC. This protein-antibody pair shows staining directly in the nucleus, which is a unique trait of NC, making it a highly specific and highly sensitive diagnostic. Strong nuclear staining by a C52 test is usually proof that the person has NUT carcinoma.⁸

Molecular confirmation

A positive C52 nuclear staining is typically used to diagnose NUT cancer.. In unclear cases or cases where the exact fusion partner needs to be identified, molecular testing may be performed. This laboratory method uses biopsied patient samples to extract DNA or RNA and take a closer look at possible genetic mutations.^9,10

As previously discussed, NUT carcinoma is caused by a genetic rearrangement involving the NUTM1 gene. The most common genetic alteration that causes NC is the BRD4-NUT fusion, although other fusion partners of NUTM1 have been identified.^9,10 Knowing where the NUTM1 gene is located in our chromosomes and which genes it tends to fuse with can help identify the specific change responsible for each cancer case.¹¹

Molecular and genetic testing methods include: ¹¹

• FISH (Fluorescence In Situ Hybridisation)—identifies gene alterations directly on the chromosomes
• RT-PCR (Reverse Transcriptase PCR)—identifies RNA products of specific gene fusions
• NGS (Next-Generation Sequencing) and RNA sequencing—broader analysis to identify common and rare gene fusions

Summary

NUT carcinoma is a very aggressive form of cancer that leads to tumours in the midline structure. This condition is often misdiagnosed or diagnosed at a late stage due to its nonspecific symptoms and histological mimicry of more common cancers. While a diagnostic protocol is followed to assess vague tumours like in the case of NC, correct diagnosis relies heavily on biopsy analysis and, specifically, immunohistochemistry. Let’s have a look at a summarised diagnostic journey an individual presenting with nonspecific cancer symptoms would go through to receive a NUT carcinoma diagnosis. 

In this article, we stressed how important high clinical suspicion, careful histology interpretation and confirmatory further testing are essential to properly diagnose a cancer of such rarity. Timely and accurate diagnosis can significantly decrease diagnostic delay and improve treatment outcomes.