Blood-based DNA Methylation Biomarkers for Early Detection of Colorectal Cancer

Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide. Early detection of CRC can significantly reduce this mortality rate. Unfortunately, recommended screening modalities, including colonoscopy, are hampered by poor patient acceptance, low sensitivity and high cost. Recent studies have demonstrated that colorectal oncogenesis is a multistep event resulting from the accumulation of a variety of genetic and epigenetic changes in colon epithelial cells, which can be reflected by epigenetic alterations in blood. DNA methylation is the most extensively studied dysregulated epigenetic mechanism in CRC. In this review, we focus on current knowledge on DNA methylation as potential blood-based biomarkers for early detection of CRC.


Introduction
Colorectal cancer (CRC) is a leading cause of death worldwide, accounting for around 754, 000 deaths in 2015 [1], The World Health Organization estimates a substantial increase in the number of newly diagnosed CRC cases worldwide and an 80% rise in deaths from CRC by 2030 [2]. The early detection of CRC significantly improves the prognosis of patients and is a key factor in reducing the mortality of CRC. The cancer can be cured by surgical procedures if it is diagnosed early, specifically before metastasis is established. The 5-year relative survival rate for early-stage CRC is 90%; for advanced stage IV CRC, the rate drops to about 14% [3]. However, only about 4 out of 10 CRC patients are diagnosed at the early stage [4], partially due to poor patient acceptance and/or sensitivity of available screening modalities.
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Four types of tests are currently available for CRC detection or screening, including fecalbased occult blood test (FOBT or FIT), tumor marker blood test, combined fecal DNA and FOBT test, and colonoscopy. Colonoscopy screening is currently the standard method for the detection of CRC [5]. However, colonoscopy screening requires bowel preparation and sedation, and is associated with high cost, possible complications and low compliance. The specificity or sensitivity of FOBT is not sufficient [6], and compliance is low due to the inconvenience of sampling and the interference of the test results by many factors [7,8].
Although lab tests such as stool occult blood and recently introduced stool DNA test offer indications for possible CRC, there is a high false positive rate when using those tests. Therefore, robust diagnostic non-invasive biomarkers are urgently needed to detect early stage of CRC.
Both genetic and epigenetic alterations have been found to be involved in the carcinogenesis of CRC [9-11]. The prevailing consensus suggests that epigenetic alterations occur early and more frequently than genetic alterations in CRC [12]. The epigenetic alterations include aberrant DNA methylation, histone modifications and expression of microRNAs (miRNAs) and long non-coding RNAs (IncRNAs) [13]. Post-translational modifications of histones regulate the packaging structure of DNA (called chromatin). Active DNA regions are marked with H3K4me2-or me3 and/or H3, H4 acetylation, while H3K9me3 or H3K27me3 represses genomic regions [14]. Gezer et al. observed reduced plasma levels H3K9me3 and H4K20me3 as potential diagnostic biomarkers for CRC [15]. The miRNA post transcriptionally downregulates gene expression through binding to a complementary site that resides on the 3'-untranslated region of target mRNAs [14]. Many miRNAs associated with CRC diagnosis and prognosis have been identified in patient blood. For example, miR-21 is overexpressed in the plasma or serum of patients with CRC [16], suggesting that it is a promising noninvasive biomarker for the early detection of CRC. Among the epigenetic mechanisms, DNA methylation is the most widely studied and a crucial epigenetic marker in cancer. In this review, we provide an overview of the role of DNA methylation alterations in CRC and discuss the clinical application of these changes as biomarkers for early detection of CRC.

DNA methylation in CRC
CRC results from the accumulation of both genetic and epigenetic changes that transform normal glandular epithelium into invasive adenocarcinoma [17]. Most CRCs develop through two different morphological multistep pathways, including the classical adenomacarcinoma sequence and the serrated neoplasia pathway [18]. Over the past 25 years, the molecular basis of this process has been progressively clarified. There are at least three distinct molecular pathways in CRC pathogenesis: the chromosomal instability (CIN), microsatellite instability (MSI) and CpG island methylator phenotype (CIMP) pathways ( Figure 1) [19]. About 65% of CRC arise through the CIN pathway, which is characterized by widespread imbalances in chromosome number (aneuploidy) and loss of heterozygosity (LOH) [20]. Mutations have been reported in oncogenes and tumor suppressor genes, including adenomatous polyposis coli (APC), β-catenin, K-Ras (KRAS), B-Raf (BRAF), Fbox and WD repeat domain containing 7 (FBXW7), transcription factor 7-like 2 (TCF7L2), G protein subunit alpha S (GNAS), chromobox 4 (CBX4), SMAD family member 4 (SMAD4), p53, ADAM metallopeptidase with thrombospondin type 1 motif 18 protein (ADAMTS18), TATA-box binding protein associated factor 1 like (TAF1L), APC membrane recruitment protein 1 (AMER1/ FAM123B), CUB and sushi multiple domains 3 (CSMD3), integrin subunit beta 4 (ITGB4), LDL receptor related protein IB (LRP1B), and spectrin repeat containing nuclear envelope protein 1 (SYNE1) [ It is now accepted that DNA methylation alterations are as significant as genetic mutations in driving CRC development. In fact, many more genes are affected by aberrant methylation than by mutations in the average colon cancer genome [12,44,45]. DNA methylation refers to the enzymatic addition of a methyl group to the 5'-position of cytosine by DNA methyltransferases (DNMTs) to produce 5-methylcytosine [46]. The majority of CpG dinucleotides in the human genome are methylated [47,48]. CpG islands indicate regions with at least 200 bp, a GC percentage greater than 50%, and an observed-to-expected CpG ratio > 0.6 [49]. In contrast to CpG dinucleotides, CpG islands typically located in the promoter of proteincoding genes are normally unmethylated in normal healthy cells [50-52]. It has long been established that cancer is characterized by global hypomethylation and hypermethylation at selected CpG islands, which contributes to tumorigenesis by aberrant silencing of tumor suppressor genes [53].
The global DNA hypomethylation is believed to influence CRC development by inducing chromosomal instability and leading to loss of imprinting [54]. The global loss of DNA methylation occurs predominantly within repetitive transposable DNA elements, such as long interspersed nuclear element-1 (LINE-1) and short interspersed transposable element (SINE or Alu elements) sequences [55-61]. DNA hypomethylation can be found in the colon in an age-dependent fashion [62,63] as well as early events in CRC development [64].

Multiple blood-based DNA methylation biomarkers
Many cells and tissues release some of their constituents to the bloodstream, including fragmented, cell-free DNA (cfDNA) which can also arise from tumor cells, i.e., circulating tumor DNA (ctDNA). Tumor-specific genetic and epigenetic alterations found in cfDNA are likely to represent a mixture of alterations in primary tumor and/or metastatic sites [65]. Cell-free DNA (cfDNA) in the blood circulation of cancer patients (as liquid biopsy) have emerged as key biomarkers for cancer monitoring and treatment decision making [66]. DNA methylation has been used as a diagnostic CRC marker because specific methylation events occurring early in multistep carcinogenesis have been identified and epigenetic gene silencing plays a causative role in CRC development [67][68][69][70][71][72]. Aberrant DNA methylation occurs in the blood of adenoma patients, making DNA methylation biomarkers feasible to detect CRC early [73,74]. Blood-based DNA methylation is mainly derived from cell-free nucleic acid released from circulating cells in serum or plasma or DNA extracted from peripheral blood leukocytes or whole blood cells.

Septin-9 (SEPT9)
SEPT9 is one of the most extensively studied genes as a blood-based biomarker for CRC patients [75][76][77][78][79][80]. It belongs to the gene family that encodes a group of GTP-binding and filament-forming proteins involved in cytoskeletal formation and cell cycle control [81]. It has promoter hypermethylation reaching sensitivities ranging from 51% to 90.0%, and the specificity from 73% to 96% in serum or plasma samples of CRC patients [75-80]. However, the sensitivity of the methylated SEPT9 assay in detecting advanced adenomas is low (9.6%) [79], suggesting that this gene alone might be of limited value in detecting precancerous lesions. conformation polymorphism (MSP-SSCP) analysis. The APC marker displayed a sensitivity of 57% for the detection of CRC at a specificity of 86%, and a sensitivity of 57% and specificity of 89% in stage I of CRC.

LINE-1
In addition to hypermethylated genes, DNA hypomethylation status of genes is associated with prognosis of CRC patients.

Discussion and Conclusion
CRC continues to be a significant public health burden and the 5-year prognosis for metastatic CRC is still less than 15% [3]. Aberrant methylation of specific genes measured in blood samples could be used as a CRC biomarker and provide prognostic information. CRC is a heterogeneous disease and DNA methylation biomarkers based on single gene have limited sensitivity and specificity. Further studies are therefore needed to perform a genome-wide search to produce a panel of sensitive and specific DNA methylation markers for the early detection of CRC.