Aberrant DNA methylation is the main studied epigenetic alteration in cancer [
10]. Aberrant hypermethylation of promoters in eukaryotic cells can lead to silencing of important genes, such as tumor suppressor genes, and ultimately result in the development of disease. Cancer development can also be affected by the opposite process. Hypomethylation of genes, e.g., oncogenes, which are normally methylated, can upregulate their expression [
11]. Interestingly, DNA hypomethylation was the first reported DNA methylation abnormality in human cancer [
12]. However, despite the initial evidence provided in 1983 and by later work, studies of the molecular mechanisms leading to cancer have not focused on epigenetics [
12,
13]. Instead, the emphasis was put on DNA mutations and loss of heterozygosity (LOH) [
11]. In 1999, Toyota et al. [
14] proposed the CpG Island Methylator Phenotype (CIMP) as another pathway of tumorigenesis. They used CIMP to describe the clinical and pathological features of colorectal cancer (CRC) for the first time. This pioneering study consisted of methylation profiling of the
CDKN2A (
p16),
MINT1,
MINT2,
MINT12,
MINT17,
MINT25,
MINT27,
MINT31,
MLH1, and
THBS1 genes in tumor tissue [
15]. Almost 20 years after the publication by Toyota et al. [
14], we have now developed methylation in vitro diagnostic (IVD) assays for blood and tissue [
13]. Moreover, they have been successfully introduced in the clinical setting for cancer screening, prognosis, and prediction [
15].
2.1 DNA Methylation Biomarkers in Tissue
One of the genes included in the first methylation profiling in 1999 was
MLH1, a DNA mismatch repair (MMR) gene (Table
1). Epigenetic silencing of the
MLH1 gene via hypermethylation of its promoter results in microsatellite instability (MSI). It has been found that 13% of sporadic CRCs show
MLH1 hypermethylation, and a
BRAF c.1799T>A, p.Val600Glu mutation has often also been identified in tumor DNA [
16,
17]. MSI and loss of
MLS1 also occurs in Lynch syndrome (the most common cause of hereditary CRC) [
18], but it is caused by mutations in one of the DNA MMR genes. In order to fully diagnose Lynch syndrome, genetic analysis of constitutional mutations in the MMR genes is performed. Differentiation of non-heritable CRC and Lynch syndrome includes a two-level screening test. The first tier includes analysis of expression of MMR genes and MSI testing. In the case of loss of MMR expression and a positive result for MSI, constitutional mutations in
MLH1,
MSH2,
MSH6,
PMS2, or
EPCAM are analyzed. Alternatively, determination of the
MLH1 methylation level and the
BRAF V600E mutation is conducted. Constitutional
MLH1 epimutations testing is recommended to confirm Lynch syndrome [
19]. Methylation analysis of
MLH1 can improve the selection of patients for Lynch syndrome genetic testing and thus reduce the cost of detecting a mutation by almost half [
20].
MLH1 methylation can be assessed by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) [
21,
22] and some laboratories use pyrosequencing [
23].
Clinical trials have provided evidence that O
6-methylguanine (O
6-meG)–DNA methyltransferase (
MGMT) is useful as a prognostic and predictive marker in glioblastoma (Table
1) [
24,
25].
MGMT is a DNA repair gene participating in the removal of mutagenic and cytotoxic alkyl groups from O
6-meG [
28]. DNA alkylation leads to the formation of mutations, and therefore
MGMT protect cells against damage [
26,
27]. Temozolomide causes alkyl DNA damage and thus leads to cell death. Its cytotoxic effect is more potent against the rapidly dividing cancer cells than against normal cells, as the DNA repair mechanisms in cancer cells are impaired [
25,
28]. Therefore, cells with
MGMT silenced by hypermethylation show a better response to temozolomide therapy [
25]. It has been found that in glioma and CRC, methylation of
MGMT occurs in 40% of tumors, while in non-small cell lung carcinomas (NSCLCs), lymphomas, and head and neck carcinomas, methylation of
MGMT occurs in 25% of tumors [
27]. Diagnostic recommendations for glioma include analysis of
MGMT methylation, which is the key point in the therapeutic algorithm and provides predictive information about the response to temozolomide [
29]. Furthermore, the
MGMT methylation status in combination with
IDH1 mutations plays the role of a prognostic biomarker. Glioma patients with the
IDH1 p.R132H mutation and hypermethylated
MGMT have a better prognosis (Table
1) [
30].
There are a number of commercial tests available to evaluate the MGMT methylation level by (1) methylation-specific polymerase chain reaction (PCR): PredictMDx Glioblastoma (MDx Health); (2) real-time PCR: MGMT Methylation Detection Kit (EntroGen); (3) MS-MLPA: SALSA MS-MLPA probe mix ME011 MMR genes (MRC-Holland); and (4) pyrosequencing technology: PyroMark MGMT Kit (Qiagen).
The
RB1 gene is primarily associated with retinoblastoma caused by the loss of the
RB1 function (Table
1). The lack of expression of this gene in retinoblastoma, as well as in other tumors, including bladder carcinomas and malignant neuroendocrine lung carcinomas, is associated with an LOH or
RB1 mutations. Nevertheless, in some cases, the silencing of
RB1 expression is caused by its methylation [
31,
32]. It has been reported that for full molecular diagnostics of retinoblastoma, it is necessary to evaluate
RB1 methylation beyond the LOH and mutations. Ohtani-Fujita et al. [
33] suggested that hypermethylation in the
RB1 gene is always acquired and causes approximately 9% of sporadic unilateral tumors [
33]. Currently, there are tests available on the market based on the MS-MLPA methodology for the evaluation of the methylation level of the
RB1 promoter [
34].
Tumor suppressor genes
GSTP1,
RASSF1, and
APC are commonly methylated in prostate tumors and, therefore, are considered as cancer biomarkers (Table
1). A set of these genes has been used in a commercially available assay—ConfirmMDx
® (MDxHealth). This test allows a better stratification of patients with a negative prostate biopsy result. It takes advantage of the epigenetic field effect, based on the principle that normal cells surrounding the foci of cancer can contain DNA methylation changes. Two independent studies, MATLOC (Methylation Analysis to Locate Occult Cancer) and DOCUMENT (Detection of Cancer Using Methylated Events in Negative Tissue), confirmed the predictive value of ConfirmMDx
® and showed a sensitivity of 68%, a specificity of 64%, and a negative predictive value of 90% [
35,
36]. Moreover, it was found that use of the methylation-based biomarkers
GSTP1,
RASSF1, and
APC resulted in a reduction of the number of unnecessary repeated biopsies by up to 64% [
35].
2.2 DNA Methylation Biomarkers in Biofluids
Measurement of DNA methylation can be performed in various types of biological material—not only solid tissues, but also plasma, serum, sputum, urine, and cerebrospinal fluid (CSF). One of the examples of a plasma epigenetic biomarker for CRC screening is circulating methylated
SEPT9 DNA (Table
1).
SEPT9 regulates cell growth and prevents uncontrolled cell division and, therefore, it is considered to be a tumor suppressor [
37]. It has been demonstrated that methylation of
SEPT9 is associated with the pathogenesis of CRC, and a decrease in its expression is correlated with progression of neoplastic disease [
38]. The first commercial diagnostic test based on the
SEPT9 biomarker was developed by Epigenomics AG 10 years ago. It involves evaluation of the
SEPT9 promoter methylation in plasma using real-time PCR [
39]. Currently, two generations of these CE-marked IVD assays called Epi proColon
® are available [
39]. Other commercially available
SEPT9 methylation tests for CRC diagnostics are ColoVantage
® (Quest Diagnostics) and RealTime mS9 (Abbott) [
40]. Apart from CRC, the usefulness of
SEPT9 methylation has been evaluated in the early diagnostics of others cancers, including lung cancer [
41]. However, there are more specific tests for lung cancer diagnostics, such as Epi proLung
® (Epigenomics AG), in which methylation of
SHOX2 and
PTGER4 is evaluated using real-time PCR [
15,
42].
SHOX2 hypermethylation has been noticed in the bronchial aspirates [
43], pleural effusions [
44], and blood plasma of patients with lung cancer [
15]. DNA methylation analysis of
SHOX2 combined with
PTGER4 in blood plasma allows detection of lung cancer and differentiation of non-malignant diseases [
15]. Additionally, prognostic application of an assay based on
SHOX2 methylation has been demonstrated [
45]. The advantage of
SHOX2 as a methylation biomarker is its high specificity (> 95% in bronchial aspirates) [
43,
45].
In the case of CRC, in addition to the previously mentioned biomarkers, it is also possible to use a stool-based methylated biomarker test, i.e., the Cologuard
® kit (Exact Sciences) [
40,
46]. This PCR-based assay is used to assess the level of vimentin gene (
VIM) methylation and DNA integrity for the early detection of CRC. The sensitivity and specificity of the Cologuard
® assay are 83% and 82%, respectively, and the specificity is almost at the same level in patients with CRC at stages I–III [
47,
48].