colorectal carcinoma ( crc ) is a common cause of morbidity and mortality worldwide . as a major life - threatening malignancy
, crc ranks second to lung carcinoma in men , third to breast cancer and lung cancer in women , and overall second to other cancer types in men and women .
approximately 142,820 new cases were diagnosed and 50,830 individuals died because of crc in 2013 .
clinical and pathological stages at the time of diagnosis largely determine the prognosis of diagnosed patients .
the curative rates of crc in t1n0m0 , t2n0m0 , and t3n0m0 stages are greater than 90% , 85% , and 70% , respectively .
therefore , crc screening is an efficient strategy to reduce death caused by this disease .
current screening modalities have resulted in a modest decrease in mortality and failed to achieve high public participation . among these modalities ,
highly sensitive colonoscopy is the standard technique used to detect and remove early lesions , but colonoscopy is invasive and costly . for population - wide screening , simple and noninvasive procedures , such as guaiac or immunochemical stool occult blood testing ( fobt ) , are preferred .
however , the performances of these tests are low . as such , molecular tests should be conducted to improve their efficiency .
for example , stool aberrant dna methylation has been developed as a biomarker for crc initiation and progression . in this article , this technique was discussed to develop real time polymerase chain reaction ( qpcr ) dna methylation biomarker test for stool to diagnose crc .
stool qpcr dna methylation biomarkers were also examined to predict outcomes and responses to crc chemotherapy . 

approximately 98% of crc are adenocarcinomas that almost originate from adenomas as generally curable by resection .
the peak incidence age of crc is 60 to 70 years , and less than 20% of cases occur before the age of 50 years .
genetic and epidemiologic studies have linked crc to several factors , including inherited predisposition , somatic mutation , dietary influence , and preexisting inflammatory disease .
two major distinct pathogenic pathways are involved in crc development , and stepwise accumulation of multiple mutations is implicated in these pathways .
the first pathway comprisesapc/-catenin characterized by chromosomal instability ( cin ) that results in the stepwise accumulation of mutations in a series of oncogenes and tumor suppressor genes .
, the accumulated -catenin translocates to the nucleus and activates the transcription of several genes , such asmyc and cyclin d1.k - ras mutations subsequently occur .
further mutation of a putative cancer suppressor gene on 18q21 andtp53 leads to the final emergence of carcinoma , and additional mutation ensues .
the accumulation of mutations rather than their occurrence in a specific order is essential for colorectal carcinogenesis .
the second pathway is characterized by genetic lesions in dna mismatch repair genes , which are involved in 10% to 15% of sporadic cases .
mutations accumulate , but correlated identifiable morphological characteristics have yet to be determined . among these dna mismatch repair genes
the loss ofdna mismatch repair genes leads to a hypermutable state . in this state , simple repetitive dna sequences called microsatellites are unstable during dna replication
the resulting microsatellites instability ( msi ) is the molecular signature of defective dna mismatch repair .
the loss of mismatch repair then leads to the accumulation of mutations in growth - regulating genes , and thus triggers the emergence of crc .
in addition to these pathways , cpg island methylator phenotype ( cimp ) pathway is also involved in crc development . 
  in the majority of patients diagnosed with crc ,
these trends are consistent with the effectiveness of crc screening in detecting and removing adenomatous polyps .
, screening tests can be classified into two categories : invasive ( structural ) exams and non - invasive tests .
invasive exams can be subdivided into endoscopic techniques ( colonoscopy , flexible sigmoidoscopy , and capsule endoscopy ) and radiological exams ( barium enema , computer tomography colonography and magnetic resonance colonography ) .
noninvasive tests can be subdivided into tests that detect blood ( fobt ) and tests that detect stool dna . among these techniques
, colonoscopy remains the gold standard , but this technique is invasive , costly , and burdensome
. colonoscopy may also be less protective in the right colon than in the left colon .
fobt is a simple , non - invasive , relatively cheap , and frequently used screening test .
the detection of dna markers in stool specimens is a relatively new noninvasive screening approach .
multi - targeted assays on 21 specific mutations in thek - ras , tp53 , andapc , which is a msi marker ( bat-26 ) , and dna integrity assay markers has also been developed .
nevertheless , their overall sensitivities in detecting crcs and adenomas remain suboptimal , possibly because the accumulation of mutations is essential for colorectal carcinogenesis .
the detection of dna mutation may also fail to reflect the progress of carcinogenesis . 

epigenetics refers to heritable alterations in gene activity and expression unlikely caused by changes in dna sequences and potentially reversible self - propagating molecular signatures .
the mechanisms of epigenetics include dna methylation , histone modifications , nucleosome positioning , or noncoding rna .
epigenetic modifications , particularly dna methylation in selected gene promoters , are recognized as common molecular alternations in human tumors . among epigenetic markers ,
methyl groups are added to the 5-position of cytosine by dna methyltransferases ( dnmt ) to produce 5-methylcytosine , which typically represses gene transcription and modifies dna functions .
for instance , the dna methylation of cytosine residues in cpg dinucleotides , which are often clustered in so - called cpg islands , leads to transcriptional silencing of the associated genes .
approximately 60% of all human promoters are associated with cpg islands . in the genome of untransformed cells ,
the dna methylation of many genes and their significance in crc have also been described .
the aberrant methylation of cpg islands within gene promoters and first exonic or intronic regions may induce the transcriptional repression of tumor - suppressor genes .
hypermethylation is a discrete targeted event within tumor cells and thus causes specific loss of gene expression .
hypomethylation usually occurs in advanced stages of tumor development and affects genome to a greater extent than hypermethylation does .
. 
  mechanisms of dna methylation in the colorectal carcinoma pathogenesis : the roles of multiple dna methylation biomarkers in colorectal carcinogenesis .
cimp is the phenotype of methylated tumor suppressor genes , and tumorigenesis theoretically occurs through progressive genetic silencing even in the absence of genetic mutations .
cimp is also referred to as an epigenetic phenomenon because this occurrence involves a temporary genetic change in the target dna sequence , that is , cimp simply triggers a potentially reversible alteration because of methylation .
cimp is regarded as a distinct crc subgroup , which is fundamentally different from other colon cancers .
cimp tumors exhibit unique pathological features , such as high mutations ( kras or braf ) rates , proximal location , wild - type p53 , mucinous histological type , poor differentiation , and increased occurrence in female and elderly patients .
primary crcs can be divided into three subclasses depending on epigenetic and clinical profiles : cimp1 , cimp2 and cimp negative .
crcs with neither cin nor msi possess a unique methylation pattern and clinical features , including improved prognosis .
hypermethylation secondary to cimp possibly leads to msi through mlh1 promoter methylation and subsequent mlh1 mismatch repair gene silencing .
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 .
dna methylation analysis may also provide useful prognostic markers of disease progression and response to traditional chemotherapy . 
 
adenoma is a neoplastic polyp that ranges from small pedunculated tumors to large sessile lesions .
adenomatous polyps are classified into three subtypes on the basis of epithelial architecture : ( 1 ) tubular adenomas ; ( 2 ) villous adenomas ; ( 3 ) tubulovillous adenomas .
the risk of crc increases when polyps are larger than 2 cm , villous than tubular , and sessile rather than pedunculated .
although colorectal adenomatous polyps are recognized as precursor lesions in most crc cases , only 1%10% of individuals with resected adenomas likely develop invasive cancer .
thus , the feasibility of using dna methylation biomarkers to diagnose colorectal adenoma should also be evaluated . 

different panels have been reported to improve diagnostic accuracy , although no definite biomarker panel has been established . in our comprehensive analysis of available studies on dna methylation biomarkers in stools for crc and adenoma detection , some dna methylation biomarkers , including secreted frizzled - related protein 2 ( sfrp2 ) , secreted frizzled - related protein 1 ( sfrp1 ) , tissue factor pathway inhibitor 2 ( tfpi2 ) , vimentin , and methylguanine dna methyltransferase ( mgmt ) , have been investigated .
these markers may also be used to diagnose crc and adenoma in early stages ( 
 table 1 
 ) . 
 
major stool dna methylation biomarkers for colorectal cancer sfrps are a family of secreted proteins that can bind townt ligands and frizzled receptors and thus modulate thewnt signaling cascades.wnt signaling cascades play an important role in colorectal carcinogenesis and progression .
sfrps are initially and independently identified as soluble factors implicated in early embryonic development and modulators of apoptotic events .
alterations in sfrp expression levels have been associated with tumor formation and bone and myocardial disorders .
sfrp1 and sfrp2 hypermethylation likely occurs at the onset of all tumor types , including colon carcinomas .
epigenetic sfrp1 inactivation is linked to the upregulation ofwnt/-catenin cascade in crc;wnt/-catenin repression has also been considered a mechanism that inhibits tumor cell growth and prevents metastatic invasion ( 
 figure 1 
 ) .
sfpr2 and sfrp1 methylation in stool also exnibits high sensitivity and specificity for crc detection .
sfrp2 methylation for crc identification in stool samples reaches a sensitivity of 90% and specificity of 77% .
a systematic meta - analysis has revealed that the pooled sensitivity and specificity of methylated sfrp2 are 0.71 and 0.94 , respectively .
the dna stool test of sfrp1hypermethylation also achieves a sensitivity of 89% and specificity of 86% in colorectal neoplasia detection . 
 
tfpi2 , a member of the kunitz - type serine proteinase inhibitor family , inhibits the tissue factor / factor viia complex and various serine proteinases .
the aberrant methylation of tfpi2 promoter cpg islands in human cancer is responsible for the decreased tfpi2 expression during cancer progression .
tfpi2 also maintains the stability of tumor environment and inhibits neoplasm invasiveness and growth and metastasis formation ( 
 figure 1 
 ) .
tfpi2 gene promoter methylation is detected in the stool of crc patients with a sensitivity of 86.7% and a specificity of 100% .
the sensitivity and specificity of fecal tfpi2 methylation assay for crc detection range from 76% to 89% and from 79% to 93% , respectively . 
 
vimentin , a major constituent of the intermediate filament family of proteins , is ubiquitously expressed in normal mesenchymal cells and known to maintain cellular integrity .
vimentin has been considered as a marker for epithelial - mesenchymal transition ( emt ) , although the molecular aspects of vimentin in the function of tumorigenesis remain unknown .
the aberrant promoter methylation of the vimentin gene may contribute to colorectal carcinogenesis ( 
 figure 1 
 ) .
the promoter hypermethylation of vimentin in stool is also a sensitive , specific alternative for crc screening .
aberrant vimentin methylation is detected in fecal dna from crc patients , with a sensitivity of 46% and a specificity of 90% . 
 
mgmt is a suicide enzyme that repairs the pre - mutagenic , pre - carcinogenic and pre - toxic dna damage o6-methylguanine .
mgmt also repairs large adducts , which are formed in response to pollutants , carcinogens , methylating agent , and chloroethylating anticancer drugs , on the o6-position of guanine .
therefore mgmt is a key node in the defense against commonly found carcinogens and marker of resistance of normal and cancer cells exposed to alkylating therapeutics ( 
 figure 1 
 ) .
a meta - analysis suggested that the frequency of mgmt hypermethylation is significantly higher than that in crc , and mgmt gene promoter methylation involved a stepwise carcinogenesis of crc development .
the loss of mgmt expression , which is secondary to mgmt gene promoter methylation , may increase the responses to alkylating agents .
the methylation stool testing of mgmt is a promising , sensitive , and specific method for early crc detection .
stool - methylated mgmt is detected in 48.1% of crc patients and 28.6% of adenoma patients . 
 
other stool methylation biomarkers , such as oncostatin m receptor- , human mut l homolog-1 , hyperplastic polyposis protein gene , sfrp5 , gata4 , -1 , 4-galactosyltransferase-1 , n - myc downstream - regulated gene 4 , 2qi4.2 , hypermethylated in cancer 1 , esr1 , phosphatase and actin regulator 3 , spastic paraplegia-20 and rassf2 , have been proposed to diagnose crc .
, dna methylation can be measured quantitatively through qpcr , which may be used to monitor disease progression .
purified stool dna can be chemically modified by sodium bisulfate to convert all unmethylated cytosine to uracils while leaving methylcytosines unaltered . treated dna retains methylated cytosines , and shows specific changes in the dna sequence .
cellular efflux toward the lumen is much greater from crc than from normal epithelium . although crc typically occupies less than 1% of the intestinal surface , tumor - derived dna in stool from patients with crc may account for as much as 14% or 24% of the total recovered dna .
exfoliated coloncytes and colonocyte constituents provide a diverse class of candidate stool markers . unlike occult bleeding , which occurs as intermittent leaking into the lumen ,
furthermore , exfoliated markers are potentially specific because they originate from neoplasm per se , where occult blood emanates from circulation .
since the earliest study of abnormal methylation in crc was reported by feinberg and vogelstein , hundreds of methylated genes have been described in crc and adenoma .
the correct genes to be analyzed should be accurately selected to improve the sensitivity and specificity of dna methylation tests . 
 
specific methylation events have been associated with different tumor stages and poor prognoses . as the first chemotherapeutic agent ,
5-fluorouracil ( 5-fu ) has been combined with folinic acid and has been used for crc chemotherapy . hence ,
newly available 5-fu - based oral formulations , such as capecitabine , offer maximum antitumor properties .
cancer - specific dna methylation events may also be involved in different stages of colorectal carcinogenesis . unlike gene mutations
the response and chemosensitivity of patients to chemotherapy may also be predicted by determining the status of dna methylation biomarkers in the stool of crc patients . 
 
recent advancements in engineered dna - binding molecules , such as transcription activator - like effector ( tal or tale ) proteins , clustered regularly interspaced short palindromic repeats ( crispr ) , and crispr - associated proteins ( cas ) system , have been applied in genome editing in cells .
these engineered dna - binding molecules can bind to a specific dna sequence and be applied to other purposes .
engineered dna - binding molecule - mediated chromatin immunoprecipitation has been developed by utilizing a tal or crispr / cas system to target specific genomic regions and to investigate associated dna - binding interactions . 

the development of a sensitive stool qpcr dna methylation assay for crc and adenoma can provide a non - invasive , scalable , specific , safe , convenient , and widespread accessible screening tool , which is more acceptable to patients than current commonly used screening methods . this assay may help decrease the morbidity and mortality of crc .
 colorectal carcinoma ( crc ) is a common cause of morbidity and mortality worldwide . 
 two pathogenic pathways are involved in the development of adenoma to crc . 
 the first pathway involvesapc/-catenin characterized by chromosomal instability resulting in the accumulation of mutations . 
 the second pathway is characterized by lesions indna mismatch repair genes . 
 aberrant dna methylation in selected gene promoters has emerged as a new epigenetic pathway in crc development . 
 crc screening is the most efficient strategy to reduce death . 
 specific dna methylation events occur in multistep carcinogenesis . 
 epigenetic gene silencing is a causative factor of crc development . 
 dna methylations have been extensively examined in stool from crc and precursor lesions . 
 many methylated genes have been described in crc and adenoma , although no definite dna methylation biomarkers panel has been established . 
 multiple dna methylation biomarkers , including secreted frizzled - related protein 2 , secreted frizzled - related protein 1 , tissue factor pathway inhibitor 2 , vimentin , and methylguanine dna methyltransferase , have been further investigated , and observations have revealed that dna methylation biomarkers exhibit with high sensitivity and specificity . 
 these markers may also be used to diagnose crc and adenoma in early stages . 
 real time polymerase chain reaction ( qpcr ) is sensitive , scalable , specific , reliable , time saving , and cost effective . 
 stool exfoliated markers provide advantages , including sensitivity and specificity . 
 a stool qpcr methylation test may also be an enhanced tool for crc and adenoma screening . 
 
Introduction
CRC pathogenesis
DNA methylation and CRC
Multiple DNA methylation biomarkers
Perspectives of DNA methylation assay for CRC
Conclusions
Conflict of interest statement