Methylation of mammalien cell DNA is the most studied, well-established epigenetic mechanism involved in regulation of gene expression.

Epigenetic events affecting the methylation pattern of a cell’s genome plays a significant role in the early development and progression of cancer.

Especially, characterisation of tumor suppressor genes silenced by promoter hypermethylation in different cancers, has shown a huge potential as epigenetic-based biomarkers for cancer detection, prognostication and prediction.

Epigenetic testing in liquid biopsies

Development of highly sensitive DNA methylation detection technologies have opened the field of performing non-or semi invasive epigenetic analysis using liquid biopsies as blood, urine, saliva, and tear fluid to follow the implication of these changed DNA methylation profiles in cancer associated progression or following remission of the disease.

Bladder and prostate cancer can be diagnosed from urine or urine sediment containing carcinoma cells, which may not be accessible through biopsies.

Sputum harbor malignant cells from lung malignancies, and it is established that sputum provides a more accurate methylation profile than obtained by a blood sample.

Using stool as the basis for analysing specific epigenetic profiles can be used for diagnosis of pancreatic and colorectal cancers. The blood-based circulating cell-free DNA has proved efficient in detecting and monitoring several cancers, via specific epigenetic markers.

The potential of identifying and validating epigenetic-based profiles to diagnose and monitoring the recurrence of different cancers after treatment with benefit for the patient is huge.

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DNA methylation

DNA Methylation is characterised by covalent addition of a carbon-5 position of cytosine residues within CpG dinucleotides.

These CpG sites are not evenly distributed throughout the genome, but are clustered in CpG-rich regions, known as CpG islands (CGI). A CpG island is typically between 500-1000 base pairs (bp) long and spans regulatory regions of the genome as gene promoter regions as well as housekeeping genes.

While the majority of CpG islands in a normal cell are maintained in an unmethylated state, allowing for expression of the associated gene, a number of repressed genes have promoter methylation of CpG islands in somatic cells, which include genes on the inactive X chromosome in females (allowing for dosage compensation) and imprinted alleles.

More than half of the protein coding genes contain a CpG island spanning their promoter region, in which the methylated status is instrumental in regulating the transcriptional activity of the gene.

 

Effect of abnormal epigenetic pattern

Consequently, changes of the cell’s normal methylation pattern can have severe consequences and contribute to neoplastic transformation.

Genome-wide studies have shown that aberrant promoter methylation is an early and common feature in human cancer through transcriptional gene silencing by promoter hypermethylation of critical growth regulators as tumor suppressor genes.

Likewise, genes involved in DNA repair and maintenance of the genome integrity and thereby stability, are found to have a hypermethylated CpG island, silencing the gene expression, in cancer cells.

 

DNA methyltransferases (DNMTs)

The methylation profile of the human genome is mainly regulated by an interplay between DNMTs and DNA demethylases, which provides a model for for the dynamic regulation of DNA methylation.

These enzymes are involved in important biological mechanisms as preserving the genomic integrity and stability, embryo development, tissue differentiation, and growth of the organism.

The three methyltransferase enzymes DNMT1, DNMT3A and DNMT3B are mediating the methylation pattern in human cells.

DNMT1 maintains methylation of the genome, it shows a high affinity for hemimethylated DNA, which is present during the DNA replication, while  DNMT3A and DNMT3B acts as de novo methylation enzymes changing methylation status of unmethylated CpG sites.

 

DNMT1

Deregulation of  DNMT1, which results in overexpression of this enzyme, has resulted in hypermethylation and thereby epigenetic silencing of a number of specific tumor suppressor genes found in gastric, lung and thyroid cancers.

Silencing of these tumor suppressor genes affect important pathways responsible for maintaining a well-controlled cell cycle, genomic integrity, apoptosis and additional hallmarks of cancer.

One example is interruption of the p53/Sp1 pathway by DNMT1 mediated hypermethylation of tumor suppressor genes: as p16, RARb, FHIT, RASSFIA and hRAB37, which may lead to development of lung cancer accompaigned with poor prognosis. Mutations in DNMT1 is found associated with colon cancer resulting in an altered global methylation profile of the affected tumour cells.

 

DNMT3A and DNMT3B

Deregulation of DNMT3A and DNMT3B has resulted both in overexpression of oncogenes and silencing of tumor suppressor genes by either hypo- or hypermethylation of these cell-growth regulators.

This deregulation is involved in various cancers as: breast, lung, and ovarian cancer, hepatocellular and vulva squamous cell carcinomas, acute myeloid leukaemia, and in pituitary adenoma.

Mutations in DNMT3A is a frequent event in cancer, and an early event in in haematological malignancy development.

Deletion affecting the DNA methyltransferase DNMT3A, as illustrated in studies on chronic lymphocytic leukaemia (CLL) using a genetically engineered mouse model, showed that lack of one DNMT3A allele promoted carcinogenesis by inducing promoter hypomethylation, which then led to overexpression of oncogenic drivers, strongly suggesting that the enzyme functions as a tumor suppressor in CLL.

The DNA methyltransferase DNMT3B is found to be overexpressed in some tumors, rather than being mutated, pointing at a mechanism leading to silencing of tumour suppressor genes by de novo methylation, and thereby promoting cancer development.

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DNA-demethylating enzymes

Demethylation of the DNA is mediated by the TET protein family (ten-eleven translocation enzymes) with three members the TET1 – 3.

The expression pattern differs among the three TET proteins in terms of cell types and developmental stage.

The TET enzymes oxidize the methylated cytosine (5-mC) and hereby catalyse the conversion to 5-hydrozymethylcytosine (5-hmC). 5-hmC can be further oxidised stepwise by the TET proteins to 5-formylcytosine (5-fc) and 5-carboxylcytosine (5-caC).

Thymine-DNA glycosylase (TDG) can cut out these modified cytosines and through repair substitute these with unmodified and un-methylated cytosines.

Demethylation of specific areas of the genome is a normal process, which is well-characterised in the germ cell maturation, in which the DNA methylation pattern is erased and re-set in a sex-specific pattern.

In cancer, changes in the normal demethylation pattern is associated with oncogenesis, and therefore part of the reversal nature of the epigenetic regulation of gene expression.

Reduced expression of TET proteins is associated with significantly reduced level of 5-mC in different malignancies as: breast, lung, prostate, and pancreatic cancer.

Likewise, deficiency of TET1 in breast and prostate cancer is found to be linked to tumor cell invasion.

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Epigenetic-based biomarkers in cancer

Early diagnosis of a cancer disease is essential for a successful response to therapy and survival of the patient.

Some malignancies are diagnosed at a late stage, as they are asymptomatic for a long period, allowing the tumor to progress and metastasize before it presents symptoms leading to its discovery.

Changes of the epigenetic pattern are recognized as early events in tumor development, especially CpG island methylation targeting the promoter region of tumor suppressor genes, thereby silencing a gene with important functions in pathways as: cell cycle regulation, DNA repair, apoptosis, or inhibition of cell migration.

Tumor suppressor genes play many different roles in maintaining normal cell integrity, genomic stability, and preventing DNA damage to affect the normal function of the cell.

Therefore, silencing of these important tumor suppressors by promoter hypermethylation, often results in disruption of pathways essential to maintain normal cell function. For further overview consult The Cancer Genome Atlas Program from the National Cancer Institute at: https://www.cancer.gov/.

Identification of methylation changes specific for a given cancer type are therefore important in the effort to develop new biomarkers or panel of biomarkers for early diagnosis of the malignancy, prognostication or prediction of the cause of disease.

Specific methylation-based biomarkers are found to determine the choice of therapy for the individual patient, weather they respond or not to a given chemo- or radiotherapy (see MGMT promoter methylation and chemotherapy response below).

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Early detection of DNA methylation in cancer

As an example of urgent need for new ways to establish early detection of a disease is lung cancer, which is the most frequent diagnosed cancer and leading cause of cancer related deaths worldwide.

This malignancy has a very poor prognosis, with a 5-year survival only around 13%, as it often is diagnosed, when the tumor is in an advanced stage, with metastases spred to other organs.

The benefit of early detection is seen for patients, who presents a stage I tumor at time of diagnosis, for whom the rate of recurrence at 5-year is < 50%.

There is therefore a strong need for diagnostic biomarkers for early detection, why focus now is directed towards identifying an epigenetic-based progression profile especially for non-small cell lung cancer (NSCLC), as this is the most frequent subtype.

 

SCLC

Lung cancer is histologically classified into NSCLC or small cell lung cancer (SCLC).

SCLC is characterized by a neuroendocrine differentiation, prone to early metastasis formation, short survival and limited response to treatment.

Patients diagnosed with SCLC frequently show a good, but short response to chemotherapy and radiation, but hereafter experience a rapid acquired resistance towards therapy both to chemotherapeutic and immunotherapeutic agents, and they therefore have the poorest prognosis in both subtype groups.

 

NSCLC

NSCLC accounts for 85% of all lung cancer cases and these are divided into two groups either adenocarcinomas (approximately 70%) or squamous cell carcinomas (approximately 30%).

NSCLC patients are divided into four groups based on the tumor-node-metastasis (TNM) classification system. Stage I and II comprise patients with localized disease, and for stage II including the possibility of a single lymph node metastasis.

Stage III describes patients with more than one metastatic lymph node and a larger tumor volume. Stage IV patients presents metastatic disease, which has spread to other organs.

The 1-year survival for stage IV patients is around 20%, and the 5-year survival down to only 3%.

 

CDKN2A silencing in NSCLC

A number of tumor suppressor genes are found to be epigenetically silenced in NSCLC.

The CDKN2A gene encoding INK4A (also known as p16) is one of the most extensively studied gene in NSCLC, especially with focus on increasing CpG island methylation along with the different histological stages from basal-cell hyperplasia to squamous cell carcinomas.

Different studies point to epigenetic inactivation of CDNK2A as being one of the – or maybe the earliest events in NSCLC development.

Silencing of CDKN2A is most likely followed by additional silencing of genes involved in regulation of normal cell growth, as the WNT signaling pathway, loss of ability to perform DNA repair and to lead the cell into apoptosis.

 

MGMT silencing in NSCLC

The MGMT gene (O⁶-Methylguanine-DNA methyltrasferase) is a DNA repair enzyme, which protects the genome from the carcinogenic effect mediated by alkylating agents.

MGMT remove adducts from the O⁶ position of guanin, failure to do so may lead to G to A transition mutations in genes important to maintain cell integrity, as the proto-oncogene KRAS or the tumor suppressor TP53.

On the other hand, epigenetic silencing of MGMT may benefit patients diagnosed with Glioblastoma, in which alkylating agents is an option for treatment, thereby preventing the DNA repair mediated by the therapy.

Epigenetic silencing CDKN2A and MGMT are interesting candidates for early diagnosis as it has been detected in in sputum from 100% of the patients with squamous-cell carcinoma up to 3 years before a clinical diagnosis could be established.

In cancer-free individuals at risk of getting lung cancer due to smoking, exposure to radon, or uranium mining, epigenetic gene-silencing of CDKN2A and MGMT was found in 15% and 25% of the sputum samples, respectively.

Many studies have now shown that epigenetic silencing of CDKN2A and MGMT strongly indicates that screening for hypermethylation of these two genes may be a powerful biomarker for risk of developing lung cancer.

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DNA repair and colorectal cancer

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide.

The five-year overall survival for patients with metastatic colorectal cancer is poor (only approximately 14%).

The majority of CRC patients suffer from a sporadic occurring malignancy, but some have a familiar form, and it is important to be able to distinguish between these patients, in which an epigenetic analysis of a DNA repair gene plays an important role.

 

Familial adenomatous polyposis

Hereditary colorectal cancer are divided into two groups, defined by the phenotype and the group of genes affected by mutations. One is the familial adenomatous polyposis (FAP), which is characterised by the presence of numerous colonic adenomas, likely to undergo malignant transformation.

Patients have mutations in the adenomatous polyposis coli (APC) gene, which may lead to loss of B-catenin regulation and failure of cell adhesion.

 

Lynch Syndrome

The second is Lynch Syndrome (LS), previously called hereditary non-polyposis colorectal cancer (HNPCC) and characterised by microatellite instability (MSI), which is caused by loss of function of a specific DNA repair gene. Microsatellite sequences mutate more frequently than other regions of the genome, especially during replication, where the DNA repair genes under normal circumstances will restore the damaged DNA, before the cell moves from the S-phase into the G2-phase.

Thereby, the un-repaired microsatellite sequence represents replication error (RER) caused by a defective DNA repair gene, which is easily detected by a PCR test, and used as one parameter for the diagnosis of CRC.

 

Epigenetic testing

A number of DNA repair genes has been found mutated thereby causing the MSI phenotype and predisposition to LS, as: MLH1, MSH2, MSH6, PMS2, EPCAM. 

Clinical testing for mutations in these genes is available, but the MSI phenotype is also found among unselected groups of CRC patients. In this MSI subgroup 20-25% represents LS patients, while the remaining 75-80% are patients with sporadic CRC.

For the latter group, silencing of the DNA repair gene MLH1 by promoter methylation is the cause for the MSI phenotype. It is therefore important to test this group of patients for hypermethylation of the MLH1 promoter, to be able to distinguish between the mutated form of MLH1 as found in LS, and the silenced, hypermethylated MLH1 found in sporadic CRC.

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How MethylDetect can assist you to analyse methylation-specific targets

In MethylDetect, we provide you with a solution to assess a target specific DNA methylation profile of your test samples.

In our catalogue, we offer you more than 850 specific EpiMelt assays. The EpiMelt test kits are based on the Methylation-Sensitive High-Resolution Melting (MS-HRM) technology, and can be used with standard laboratory equipment for qPCR and melting assessment.

Each EpiMelt test kit comes with a unique control system, securing the high sensitivity of your analysis provided by the kit.

 

Custom-Tailored EpiMelt kits

We design and produce EpiMelt test kits tailored to target specific areas of the genome, in case your gene or genomic area of interest is not found in our portfolio.

Following methylation-specific array screening analyses, you may have identified targets, which are not yet described in the literature.

In collaboration with you, we can design and produce EpiMelt test kits targeting these specific genomic areas, and tailor the kit to fulfil your needs, for example, whether you  may want analyse for hypo – or hypermethylation. Likewise, we design your new EpiMelt test kit for highly sensitive analysis of the selected test material available for your analyses.

We take into account if it is FFPE, specific liquid biopsies or maybe high quality DNA. Customer tailored EpiMelt assays are always performed in close collaboration with you.

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Further reading:

Hanahan D. & Weinberg RA. Hallmarks of Cancer: The next generation. Cell 2011, vol 144,646.

Baylin SB and Jones PA, Epigenetic Determinants of cancer. Cold Spring Harb Perspect Biol 2016;8:a019505

Zhang J, Yang C, Cui W, and Wang L. DNA Methyltransferases in Cancer: Biology, Paradox, Aberrations, and Targeted Therapy. Cancers 2020, 12, 2123

Petryk N, Bultmann S, Bartke T, and Defossez P-A. Staying true to yourself: mechanisms of DNA methylation maintenance in mammals. Nucleic Acids Res, Volume 49, Issue 6, 6 April 2021, Pages 3020–3032

Nunes SP et al. Subtyping Lung Cancer Using DNA Methylation in Liquid Biopsies. J Clin Med. 2019 Sep 19;8(9):1500.

Klutstein M, Nejman D, Greenfield R, Cedar H. DNA Methylation in Cancer and Aging. Cancer Res. 2016 Jun 15;76(12):3446-50.

Kettunen E et al. Asbestos-associated genome-wide DNA methylation changes in lung cancer. Int J Cancer. 2017 Nov 15;141(10):2014-2029

Hulbert A et al. Early Detection of Lung Cancer using DNA Promoter Hypermethylation in Plasma and Sputum. Clin Cancer Res. 2017 April 15; 23(8): 1998–2005

Ooki A, Maleki Z, Tsay J J et al. A Panel of Novel Detection and Prognostic Methylated DNA Markers in Primary Non-Small Cell Lung Cancer and Serum DNA. Clin Cancer Res. 2017 Nov 15;23(22):7141-7152

Lynch HT et al. Review of the Lynch syndrome: history, molecular genetics, screening, differential diagnosis, and medicolegal ramifications. Clin Genet. 2009 July ; 76(1): 1–18

Polak P. et al. A mutational signature reveals alterations underlying deficient homologous recombination repair in breast cancer. Nat Genet 2017 Oct; 49(10):1476-1486

Meng H et al. DNA methylation, its mediators and genome integrity. Int J Biol Sci. 2015 Apr 8;11(5):604-17

Fadda A et al. Colorectal cancer early methylation alterations affect the crosstalk between cell and surrounding environment, tracing a biomarker signature specific for this tumor. Int J Cancer. 2018 Aug 15;143(4):907-920