Heterogeneity And DNA Methylation Histology Of Colorectal Cancer And Analysis Of Its Environmental Factors

Colorectal cancer(CRC)is one of the most prevalent cancers worldwide.The incidence rate of CRC is increasing each year.CRC is caused by the accumulation of genetic and epigenetic mutations.Mutations in APC,TP53,and SMAD4 play important roles in CRC progression.Early diagnosis is the most effective means for reducing the morbidity and mortality of CRC,but current early diagnosis methods have many limitations.Genetic variations and epi-genetic alterations(particularly DNA methylation)can be used for CRC diagnosis and treatment.DNA methylation plays an important role in CRC progression,but few studies have examined the methylomes of CRC,particularly in Chinese populations.In addition,there are population and ethnic differences in the DNA methylation profiles of CRC.Here,we performed methylated cytosine capture sequencing using the SeqCap Epi CpGian technique(capture 5.5 million CpG sites)on biopsies from 26 CRCs,28 colorectal adenomas(CRAs),and 6 colorectal polyps(CRPs)in a Chinese population.We found significant differences among the CRC,CRA,and CRP methylation spectra.There were 1582 differentially methylated sites in CRC,959 in CRA,and 2011 in CRP.The proportion of differentially methylated sites in the CRA located in CpG islands was significantly higher than those in CRC and CRP.MYBPC3 was hypomethylated in CRC.GDF1,NEFH,and SNORD82 were hypermethylated in CRA.RIMS1 and BPI were hypomethylated and KIF7 and ZNF837 were hypermethylated in CRP.NEFH,SNORD82,KIF7,and ZNF837 are potential bio-markers for the early diagnosis of CRC.Gene set enrichment analysis of the differentially methylated genes revealed that these genes in CRC and CRP were enriched in the cAMP signaling pathway.In the CRP,the differentially methylated genes were enriched in the cancer pathway.The cAMP pathway is a potential target for CRC epigenetic therapy.We identified three DNA methylation-based subgroups of CRA and CRC by cluster analyses.In conclusion,we identified several differentially methylated genes in different stages of CRC,and the methylation spectrum changed significantly during CRC progression.Intratumor heterogeneity(ITH)manifests as having nonuniformity in morphological structure and genotypic/epigenetic status,as well as variable expression of different markers by separate cell groups within the same tumor.ITH leads to regional biases of the mutation landscape in a single tumor and impacts treatments.ITH limits the accurate genetic profiling of many cancer types,impairs appropriate selection of targeted therapies,and prevents identification of prognostic and predictive biomarkers.The extent of CRC genetic ITH is not well-understood in the Chinese population.We amplified whole genomic DNA target fragments by PCR and performed DNA sequencing for specimens from 26 CRC patients(4-5 regions per case)to detect APC,TP53,and SMAD4 mutations.The APC mutation rate was 8/26(30.8%),with mutations enriched in a specific region that is not a typical mutation hotspot;two cases exhibited APC mutation ITH;the TP53 mutation rate was 11/26(42.3%),7 cases exhibited TP53 mutation ITH;the SMAD4 mutation rate was 15/26(57.7%),and 15 cases exhibited SMAD4 mutation ITH.The extent of ITH varied among the key CRC-related genes.Lynch syndrome(LS)is a familial condition caused by germline mutations in DNA mismatch repair(MMR)genes.Individuals with LS have a greatly increased risk of developing CRC and it is therefore important to identify MMR gene mutation carriers so that they can be regularly monitored.LS patients often show microsatellite instability(MSI).CRC patients were screened for MSI as a first step in the identification of unrecognized cases of LS in the Chinese population.MSI screening was performed using the BAT-26,BAT-25,NR-21,NR-24,and MONO-27 microsatellite markers and positive cases were confirmed using the pentaplex MSI analysis system.Positive cases were screened for BRAF mutations to exclude sporadic CRC and evaluated for loss of expression of four DNA mismatch repair proteins by immunohistochemistry.MSI was found in 60/284(23.7%)cases,among which only one showed BRAF mutation.MSI screening of Chinese CRC patients revealed that approximately 1/4 were candidates for LS.Patients with MSI are strongly recommended to undergo genetic counseling and germline mutation testing for LS.To evaluate the extent of ITH in CRC with MSI,we obtained 3-8 biopsies from 37 CRC patients followed by MSI analysis using the Bethesda system.Among the MSI cases,ITH of MSI status was identified in 24.3%of CRC cases by the Bethesda system.In terms of MSI markers,the ITH originated from mononucleotide markers in most cases(68%),but also originated from dinucleotide markers(32%).Our results indicated that the extent of ITH of the MSI phenotype detected by the Bethesda system was substantial.Our data suggest that analysis of MSI status in multiple regional biopsies is essential for better evaluating MSI status in CRC.To explore gene methylation heterogeneity in CRC tissue,we obtained 3-5 biopsies from 6 CRCs,and DNA from normal and tumor tissues were exacted.The methylation status of the two most common used epigenetic biomarkers SEPT9 and the vimentin gene were determined by bisulfite sequencing PCR followed by TA cloning for sequencing.In patient 9,the methylation rate of SEPT9 and the vimentin gene in 9B was 33.3%,4%;9D:20%,12%;9E 40%,33%;9F:40%,3%.In patient 4,the methylation rate was 4D:20%,32%;4E:4%,0;4F:18%,12%.Methylation ITH is prevalent in CRC.CRC is caused by the interaction of genetic factors and environmental factors.The association of smoking,alcohol consumption,tea consumption,and constipation with CRC remains controversial.In this study,99 patients with CRC and 15 healthy controls were analyzed to explore the association of environmental factors with CRC.The results showed that smoking,alcohol consumption,constipation,and tea consumption is not associated with CRC risk.In conclusion,we systematically profiled DNA alterations and genetic mutation patterns in a Chinese population by DNA methylome analysis,genetic mutation detection,MSI analysis,and specific gene promoter methylation analysis.These results provide guidance for CRC diagnoses and treatment.Our study provides scientific support for the prevention and treatment of CRC and has potential for clinical application.