What are circulating nucleosomes?
For my MD thesis I modified and evaluated an immunological assay that detected the cell death products “nucleosomes” as a new blood-based cancer biomarker. As cell death processes are enhanced or suppressed during different stages of tumor development, it was assumed that they may enable stage-dependent detection of cancer disease.
Nucleosomes themselves are highly interesting analytes as they are complexes of protein components, the so-called histones, and associated nucleic acids. Both parts are modified during tumorigenesis.
At that time it was only possible to measure nucleosomes non-specifically. This means that it could not be concluded whether they derive from tumor, immune, endothelial or other cells.
Today much more is known about the tumor-specific changes of the histone and DNA component – and techniques are available to detect these modifications not only at the tumor site but also in blood circulation.
When did the diagnostic value of circulating nucleosomes in detecting colorectal cancer (CRC) first become apparent?
Our early studies showed that the amount of nucleosomes is increased in sera of patients with many cancer types, among them also in colorectal cancer, when compared with healthy individuals. This was very encouraging for us.
Unfortunately, also patients with benign organ-related diseases, such as inflammations or non-tumor lesions, showed elevated levels limiting the diagnostic value of this parameter if taken as sole criterion.
Although some acute diseases like inflammations could be ruled out by other biochemical parameters it seemed us to uncertain to use it as diagnostic tool.
However, we found that the courses of serial nucleosome values during a cytotoxic therapy were very well able to identify therapy response or disease progression.
For some entities like in lung, pancreatic, liver and colorectal cancer, the serial nucleosome patterns during the first week of an anticancer treatment could already predict the later non-response or progression in a portion of the patients.
You recently coordinated a trial of one of VolitionRx’s blood-based diagnostic tests for CRC. Please can you outline this study?
The NuQ-5mc test is one first example of a wide portfolio of Volition Rx’s new immunological assays that detect nucleosome-modifications that occur during the development of a tumor.
While the NuQ-5mc test quantifies the methylation status of DNA, other assays measure characteristic groups that are added to regulatory sites of the histones such as acetyl or methyl groups; others measure typical histone variants or proteins that are attached to the nucleosomes and regulate transcription processes.
The strategy is to combine several of these different approaches to identify a marker pattern that is highly sensitive and specific for tumor detection and monitoring.
In many tumors DNA becomes less methylated during the disease development making the DNA less stable and more vulnerable to DNA damages, thereby increasing the risk for generating genetic errors and mutations. The NuQ-5mc assay quantifies this loss of overall methylation and may reflect the risk of genetic instability.
What were the results of this study?
The quality of the assay was reflected by intra- and interassay reproducibilities of 3.4% and 15.3%, respectively. There was no major influence of preanalytic factors on analyte concentration.
In the clinical testing, the levels of circulating methylated DNA were significantly decreased in CRC and BCD when compared with HC, while there was no difference between BCD and CRC.
When compared with healthy controls, the sensitivity for the detection of colorectal cancer was 75% at a specificity of 70%. At 95% specificity, the detection rate was 33%. This is in the range of PSA for prostate cancer screening.
The low levels in BCD patients will have to be further investigated in larger cohorts to see whether NuQ-5mc detects also polyps that potentially could progress to colorectal cancer.
How does the sensitivity and specificity of this test compare to other biomarker cancer diagnostic tests such as PSA for prostate cancer?
Although there is a high interest in early detection of cancer disease, blood-based biomarkers have many other important indications to better manage the disease course in cancer patients:
One is the estimation of the severeness of disease and prognosis of the patients that is relevant to consult them for the most appropriate therapy. This is today mainly done by clinical and histopathological findings. However, blood-based biomarkers will play a significant role for therapy stratification in the future, too.
A second indication is the monitoring of disease and the early estimation of therapy response or non-response. As systemic cytotoxic therapies often are applied over several months and are associated with severe side effects, patients wants to know early whether they will benefit from the treatment or not.
However, clinical staging by imaging techniques such as computed tomography frequently is done only after several courses and at the end of the therapy, meaning after approximately 12 and 24 weeks.
This situation could be tremendously improved if blood-based markers that indicate non-response early (this means after one application of therapy) and accurately are used for standardized monitoring before every therapeutic cycle. We have some very positive experiences with nucleosome markers for this indication in diverse tumor entities and will explore the new assays further in that direction.
Finally, blood-based biomarkers are very sensitive tools for the early detection of tumor recurrence. If used correctly – looking rather at the individual marker changes over time than at general cutoffs – they can enable an earlier and thus more successful treatment in diverse cancers diseases.
What further research is needed on this test?
The first results are very encouraging for us. We proceed now with further validation steps. If our results are also confirmed for early stage cancer and if the accuracy is enhanced by combination with further nucleosomics markers, they could become a valuable tool for prescreening individuals to identify those who are at risk and will have to undergo further diagnostics, e.g. colonoscopy.
It will be important to show that the positive predictive value of the new assays is higher than that of currently used screening markers.
What do you think the future holds for colorectal cancer diagnostics?
The decrease in mortality of colorectal cancer patients is a result of screening efforts in recent decades. Therefore, screening will probably remain a major focus in the future.
Increasing the compliance of individuals to participate at screening programs is the main challenge nowadays. Therefore convenient and meaningful test systems that are cheap and give reliable results are needed.
As blood is taken minimal-invasively, sensitive and specific blood assays are “hot” candidates to be introduced as prescreening tools to convince cautious individuals at risk to undergo colonoscopy.
Where can readers find more information?
The poster abstract and a PDF of the poster is available on VolitionRx's website at: https://volition.com/
Visit Volition’s website (https://volition.com/) or connect via Twitter, LinkedIn or Facebook.
About Dr. Stefan Holdenrieder
Since 2010, I am working as senior physician at the Institute of Clinical Chemistry and Clinical Pharmacology at the University Hospital Bonn.
Prior to this I have worked for more than 10 years at the Institute of Clinical Chemistry at the University Hospital Munich.
During all this time, my main research focus has been the development and evaluation of new biomarkers that may be useful for diagnostics of cancer diseases. This covers conventional protein tumor markers and novel markers such as circulating nucleic acids.
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