Circulating Nucleosomes as Colorectal Cancer Blood Biomarkers

Colorectal cancer is one of the most common cancers worldwide affecting both men and women. Due to how slowly the cancerous lesions develop in the colon, the successful treatment of colorectal cancer hinges on how early it can be detected.

Unfortunately, existing colorectal screening methods come with a few downsides. Colonoscopies are the recommended screening for at-risk individuals who present colorectal cancer symptoms. The gold-standard but invasive colonoscopy procedure comes not only with discomfort but also at significant expense to the individual or the Community, becoming a roadblock for patient compliance.

At the same time, stool-based tests such as the fecal occult blood test (FOBT) and fecal immunochemical test (FIT) that are usually recommended for low-risk individuals are prone to false positive and negative results, which can lead to unnecessary colonoscopies or missed lesions, respectively. As such, new, less invasive methods are required to improve the colorectal cancer detection rate — both in terms of accuracy and patient compliance, with the most ground now being made in liquid biopsy.

Among many potential blood-based cancer biomarkers, circulating nucleosomes may be one such biomarker that can be used to either improve existing screening methods or coupled with other biomarkers for more sensitive and specific colorectal cancer screening.

Human DNA consists of 3 billion base pairs and is contained within the nucleus, an organelle found in most cells. A single cell’s DNA, if completely stretched out, can reach a length of 2 m. However, when contained within the nucleus, DNA undergoes different levels of packaging and condensation (supercoiling).

Nucleosomes are basic structural units of DNA packaging which consist of DNA wound around eight histone proteins. These histones act like spools for DNA to bind to, allowing the DNA to be organized into chromatin fiber. In cells that are not undergoing division, the DNA remains packaged as chromatin. In cells undergoing cellular division, the DNA is first replicated before it is condensed into chromatin, and then into chromosomes.

Circulating nucleosomes, then, refer to the same DNA-protein complexes that have been released into the bloodstream as a result of cell death or apoptosis. Given that apoptosis is part of normal biological function, one can expect to find nucleosomes in circulation in healthy individuals. However, due to the higher cellular turnover in cancer when tumor cells are constantly dividing and dying, nucleosomes can be found in higher quantities in cancer patients compared to healthy individuals, especially in advanced stage cancers. This quantitative difference is less significant in benign diseases, which initially suggests that nucleosomes by themselves may not be useful in detecting early-stage colorectal cancer.

Significance in colorectal cancer

Besides their structural roles in packaging DNA, nucleosomes are also important focal points in transcriptional control and gene regulation. In particular, individual histones can be found with a diverse array of post-translational modifications (PTM), which not only impact DNA structure and stability but also influence DNA replication and repair and regulate protein recruitment.

Histones can undergo several PTMs, including methylation — an important epigenetic modification that is implicated in many cancers including colorectal cancer. In this case, methylated histones (denoted by “me” when identifying and naming histones) can have a number of different functional outcomes. For instance, H3K27me2 and H3K9me3 are associated with gene repression, while H3K4me2 and H4K4me3 are associated with gene expression — both of which can play a significant role in cancer development depending on what gene is being repressed or expressed.

More specific to colorectal cancer, studies have found that methylated histone H3K9me3 is found in higher quantities in invasive colorectal cancer and correlates with spread to the lymph nodes. This particular histone is also thought to be involved in the progression of benign adenoma to malignant adenocarcinoma due to its upregulation and increased quantities, though the causal relationship for this correlation has yet to be verified.

Additionally, differential expression of certain histone combinations have also been correlated with different cancer prognoses and survival rates. In particular, high nuclear expression of H3K9me3 (associated with gene repression) and low levels of M3K4me3 (associated with gene expression) correlates not only with better early stage I and II colorectal cancer prognosis, but also improved overall survival and longer local and distant recurrence free survival.

Testing for circulating nucleosomes with carcinoembryonic antigen

Carcinoembryonic antigen, or CEA, is a protein that is normally found in developing embryonic tissue in the womb. Outside of developmental circumstances, though, abnormal levels of CEA in adults may be a sign of cancer.

One study suggests that testing for cell-free (cf) nucleosomes in combination with CEA can increase the sensitivity for detection of colorectal cancer. While CEA is often used in follow-ups after colorectal cancer treatment, its clinical utility in early detection is disregarded due to its poor sensitivity for early colorectal cancer.

By testing for what the research team refers to as the four-cf-nucleosome biomarker combination — consisting of histone modifications H2AK119Ub, H3K9Ac, H3K27Ac and the overall level of nucleosomes — the sensitivity for detecting colorectal cancer increased to 74% at 90% specificity and enabled stratification between the group of patients with colorectal cancer and benign polyps (though the distinction between polyps and healthy controls could not be made).

Not only do these results exceed the performance of the CEA biomarker, the four-cf-nucleosome biomarker panel also showed increased sensitivity across all stages of colorectal cancer (75% for stage I cancer, 86% for stage II cancer, 71% for stage III, and 60% for stage IV cancer) compared to healthy patients. These findings suggest that the four-cf-nucleosome biomarker panel may sufficiently plug the gaps in CEA biomarker testing, particularly at early cancer staging.

Improving FIT tests with additional nucleosome testing

Coupling FIT with nucleosome testing may also help reduce the occurrence of false positive results. As circulating nucleosomes are found in the blood plasma, it is also possible to test stool samples for nucleosomes should fecal blood be present.

In two separate studies, stool samples from patients referred for surveillance colonoscopy or bowel symptoms were tested for nucleosome levels. FIT was also conducted on each of the samples.

In the first study, the results from the FITs detected all cases of colorectal cancer, but missed out about 35% of advanced adenomas, including seven high-risk adenoma cases. When the samples were tested for H3K36me3 and H3K9me3 together with FIT, the reported results showed an improved sensitivity of 98.4%, detecting all colorectal cancer patients and 97% of advanced adenoma cases including all seven high-risk cases.

In the second study, the singularly conducted FITs were able to detect 46 out of 55 proximal adenomas, while a combined FIT with nucleosome testing (markers H3K27me3 and H3R8cit) was able to detect 50 proximal adenomas, including 3 proximal adenomas larger than 2cm that was undetected by FIT.

From both these studies, the FIT sensitivity to colorectal cancer can be vastly improved, and unnecessary colonoscopies can be reduced by 21–28%.

As it stands, patient compliance for colorectal cancer screening, particularly using colonoscopy, remains low at 65%. The incorporation of more non-invasive and sensitive screening options not only can help eliminate unreliable results and encourage patient compliance, but can ensure that effective treatment is given as early as possible.