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European Biopharmaceutical Review

Making a Mark

In the last decade we have experienced tremendous efforts and successes in academic research concerning the identification of epigenetic changes in carcinogenesis. All of the results and knowledge that have been collected indicate that DNA methylation of specific regions occurring during carcinogenesis may be of high value for in vitro diagnostics (IVD), especially the stable, covalent modification of cytosine residues located 5’ from guanines (1).

These Cytosine-phosphate-Guanine (CpG) sites can be detected by polymerase chain reaction (PCR) based technologies in biological material available for IVD tests, such as biopsies and smears, or in bodily fluids such as urine, plasma, and serum obtained through non-invasive procedures.

Gene Expression

Epigenetic modifications take place in all eukaryotic cells and may play a major role in gene expression regulation. DNA methylation mostly occurs at cytosines neighboured by guanine. These CpG dinucleotides are not evenly spread over the entire human genome. They are statistically under-represented, and are clustered in special DNA regions – so-called CpG islands – mainly in the promoter region of genes. Thus, they may interfere with binding sites for factors influencing gene expression.

Methylation of CpG sites within these regions may lead to an accumulation of methyl-CpG-binding proteins and, as a consequence, to a dense packing of the chromosomal region and a silencing of the genes. In tumour cells, this mechanism is often seen in promoter regions of tumour suppressor genes so that they are not able to activate repair mechanisms or apoptosis programmes. Accumulated data also indicate that certain DNA methylation patterns occur in specific tumour entities, which makes these epigenetic changes even more attractive for diagnostics (1).

However, only a few efforts have led to a transfer of this scientific knowledge into diagnostic tests for cancer diagnostics, despite the fact that DNA methylation-based diagnostic markers are robust, easy to detect and provide an objective readout. Epigenomics AG was the first company to launch a test – Epi proColon – to be employed in colon cancer screening, based on a blood sample obtained from the person to be tested. MDxHealth offers confirmatory tests for prostate cancer, as well as test services based on DNA methylation. This leaves a lot of space for further developments in this promising new field of diagnostics.

DNA Methylation Markers

Numerous studies have demonstrated that changes in the pattern of DNA methylation occur when normal cells develop into cancer cells. The methylation of such CpG islands specifically occurring in cancer cells – and not in other cells of the material – is of high diagnostic interest. Differences in such DNA methylation patterns may also be related to, for example, the sensitivity/resistance of a patient to certain therapies.

Furthermore, DNA methylation changes occurring early in carcinogenesis may even be of prognostic or predictive value, since they may indicate the progression of precancerous disease states. Thus, it allows the adjustment of patient monitoring and treatment at early stages, where the success rates for therapies are highest.

Genome-wide DNA methylation changes can be studied by either an array of hybridisation technologies, or with the availability of high-throughput sequencing methods that allow characterisation of DNA methylation patterns on a primary sequence basis. A comparison of the DNA methylation patterns of tissues from patient groups to be discriminated by the test then allows the definition of putative markers with diagnostic potential.

Diagnostic Methods


Utilisation of DNA methylation markers requires it to be detectable by methods which can be implemented in a diagnostic setting. The DNA methylation pattern can be ‘fixed’ in the DNA obtained from a biological sample. This is the prerequisite for discriminating between methylated DNA originating from tumour cells and unmethylated DNA from other cells present in the sample. For this purpose, the DNA is treated chemically, which leaves methylated cytosines unaltered, whereas unmethylated cytosines are converted into the thymine analog uracil.

In PCRs, these bases then behave differently, and it is therefore possible to exclusively amplify the methylated marker DNA in a background of nonmethylated DNA to demonstrate the presence of a tumour marker in the respective sample (2).

These PCR-based identification methods are then employed for validation with DNA obtained from tissue material, and promising markers are further validated with material that is later used in diagnostics. For tumour locations which can be reached externally, smears and biopsies may be used, otherwise body fluids may be a source of DNA liberated from cancer cells. One of the first DNA methylation markers used in diagnostics – Epigenomics’ mSept9 marker – is detected in DNA enriched from blood serum. This requires the optimisation of pre-analytics procedures which yield DNA of sufficient quantity and quality for the chemical treatment and subsequent PCR.

Diagnostics development is required in order to provide robust tests that can be performed in respective laboratories. Principally, these tests do not need any special equipment, and they can also be implemented into platforms used in diagnostics. The mSept9 marker has been out-licensed to several diagnostics companies, such as ARUP, Quest and Abbott, which have developed their own tests on their respective platforms.

As an alternative, QIAGEN has developed CpG methylation tests for its pyromark platform, which allow for the detection of cytosine methylation after chemical treatment. In a mixed sample, it is principally possible to determine the methylation rate of specific CpGs.

Personalised Medicine

Diagnostics supporting the decision of which patient would benefit from a certain therapy is an emerging field. The search for molecular markers that occur in subgroups of patients has thus become an important task in molecular diagnostics. Besides mutations and single nucleotide polymorphisms, changes in the DNA methylation pattern may provide such markers (3).

At the 49th meeting of the American Society of Clinical Oncology in Chicago, MDxHealth presented data which demonstrated that patients with methylation of the DCR1 gene did not profit from an irinotecan-supplemented capecitabine therapy. Principally, predictive molecular markers may guide the selection of patients that are likely to respond to a given treatment or, in the case of DCR1, identify patients who are unlikely to respond to specific therapy. Differentiating diagnostic tests based on such markers may spare unnecessary toxicity and allow for treatment with more effective, alternative therapies.

Cervical Cancer Diagnostics

oncgnostics GmbH, founded in early 2012, is concerned with the validation of markers and the development of diagnostic tests for specific tumour entities, with the first product being in the cervical cancer diagnostics field. In this arena, current cytology-based diagnostics fail to detect many patients with severe dysplasias and even carcinomas. On the other hand, it yields too many false-positive results, since only 20 per cent of the women with an abnormal Papanicolaou smear test have a disease deserving treatment.

Testing for the cancer-causing highrisk human papillomaviruses (HPV) has very good sensitivity – the viruses are detected in virtually any cancer patient – and it has a very good negative predictive value as women who are not infected with HPV will not develop cervical cancer.

However, only a small fraction of the women infected with HPV will develop severe dysplasia or even cancer. This means that a triage of the HPV-positive women is required to answer the question: “Are they just transiently infected, or do they develop cancer?” oncgnostics GmbH has validated DNA methylation markers which will answer this question. They will be able to detect those who deserve invasive diagnostics and treatment among the HPV-positive women. The test uses a panel of proprietary DNA methylation markers. Their methylation in cervical scrape material has been shown to correlate to histopathology-confirmed severe cervical dysplasia and cervical cancer. The advantage of such a test is that it may be performed with the same material as the preceding HPV test.

Conclusion

A huge variety of published studies have demonstrated the diagnostic potential of epigenetic markers – especially those based on DNA methylation. Many of these markers are, however, only discriminative in tissues and may not work in material obtained for diagnostics from patients since they may display low detection rates and/or high false-positive rates. Nonetheless, the number of diagnostic markers that make their way into diagnostics is extraordinarily low. But with milestones such as the Food and Drug Administration’s clearance of the Epi proColon test, such markers may finally experience higher acceptance.

References
1. Esteller M, Epigenetic gene silencing in cancer: the DNA hypermethylome, Hum Mol Genet, 2007
2. Hernández HG et al, Optimizing methodologies for PCR-based DNA methylation analysis, Biotechniques, 2013
3. Heyn H et al, Epigenetic profiling joins personalized cancer medicine, Expert Rev Mol Diagn, 2013

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About the authors

Alfred Hansel
is co-founder and Chief Executive Officer of oncgnostics GmbH. He received his PhD in Biology at the Albert Ludwigs University, Germany, and after a postdoctorate at the University of Uppsala, Sweden, he managed several biomedical projects at the Jena University Hospital, Germany. At oncgnostics GmbH, Alfred is responsible for business and strategy development, venture raising and communication.

Martina Schmitz is also co-founder of oncgnostics GmbH, and as a Chief Security Officer she manages the company’s R&D work. Martina received her PhD in Biochemistry at the Friedrich Schiller University Jena, Germany. She has gathered in-depth experience in molecular biology-based test development during her university research projects, in which she developed a realtime PCR test for the seven most frequently detectable HPV types.
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Alfred Hansel
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Martina Schmitz
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