Exposure to energy restriction during childhood and adolescence is associated with a lower risk of developing colorectal cancer (CRC). Epigenetic dysregulation during this critical period of growth ...and development may be a mechanism to explain such observations. Within the Netherlands Cohort Study on diet and cancer, we investigated the association between early life energy restriction and risk of subsequent CRC characterized by the (promoter) CpG island methylation phenotype (CIMP).
Information on diet and risk factors was collected by baseline questionnaire (n = 120,856). Three indicators of exposure were assessed: place of residence during the Hunger Winter (1944-45) and World War II years (1940-44), and father's employment status during the Economic Depression (1932-40). Methylation specific PCR (MSP) on DNA from paraffin embedded tumor tissue was performed to determine CIMP status according to the Weisenberger markers. After 7.3 years of follow-up, 603 cases and 4631 sub-cohort members were available for analysis. Cox regression was used to calculate hazard ratios (HR) and 95% confidence intervals for CIMP+ (27.7%) and CIMP- (72.3%) tumors according to the three time periods of energy restriction, adjusted for age and gender. Individuals exposed to severe famine during the Hunger Winter had a decreased risk of developing a tumor characterized by CIMP compared to those not exposed (HR 0.65, 95%CI: 0.45-0.92). Further categorizing individuals by an index of '0-1' '2-3' or '4-7' genes methylated in the promoter region suggested that exposure to the Hunger Winter was associated with the degree of promoter hypermethylation ('0-1 genes methylated' HR = 1.01, 95%CI:0.74-1.37; '2-3 genes methylated' HR = 0.83, 95% CI:0.61-1.15; '4-7 genes methylated' HR = 0.72, 95% CI:0.49-1.04). No associations were observed with respect to the Economic Depression and WWII years.
This is the first study indicating that exposure to a severe, transient environmental condition during adolescence and young adulthood may result in persistent epigenetic changes that later influence CRC development.
Colonoscopy and the fecal immunochemical test (FIT) are currently the most widely used screening modalities for colorectal cancer (CRC), however, both with their own limitations. Here we aim to ...identify and validate stool-based DNA methylation markers for the early detection of CRC and investigate the biological pathways prone to DNA methylation.
DNA methylation marker discovery was performed using The Cancer Genome Atlas (TCGA) colon adenocarcinoma data set consisting of normal and primary colon adenocarcinoma tissue. The performance of the five best candidate markers and a previously identified marker, NDRG4, was evaluated on tissues and whole stool samples of healthy subjects and CRC patients using quantitative MSP assays. The results were compared and combined with FIT data. Finally, pathway and gene ontology enrichment analyses were performed using ToppFun, GOrilla and clusterProfiler.
GDNF, HAND2, SLC35F3, SNAP91 and SORCS1 were ranked as the best performing markers. Gene combinations of all five markers, NDRG4 and FIT were evaluated to establish the biomarker panel with the highest diagnostic potential, resulting in the identification of GDNF/SNAP91/NDRG4/FIT as the best performing marker panel. Pathway and gene ontology enrichment analyses revealed that genes associated with the nervous system were enriched in the set of best performing CRC-specific biomarkers.
In silico discovery analysis using TCGA-derived data yielded a novel DNA-methylation-based assay for the early detection of CRC, potentially improving current screening modalities. Additionally, nervous system-related pathways were enriched in the identified genes, indicating an epigenetic regulation of neuronal genes in CRC.
Intake of dietary factors that serve as methyl group donors may influence promoter hypermethylation in colorectal carcinogenesis. We investigated whether dietary folate, vitamin B2 and vitamin B6, ...methionine and alcohol were associated with mutL homologue 1 (MLH1) hypermethylation and the related molecular phenotypes of MLH1 protein expression, microsatellite instability (MSI) and BRAF mutations in patients with colorectal carcinomas. Within the Netherlands Cohort Study on diet and cancer (n = 120 852), 648 cases (367 men and 281 women) and 4059 subcohort members were available for data analyses from a follow-up period between 2.3 and 7.3 years after baseline. Gender-specific adjusted incidence rate ratios (RRs) were calculated over categories of dietary intake in case-cohort analyses. The intakes of folate, vitamin B2, methionine and alcohol were not associated with risk of tumors showing MLH1 hypermethylation, those lacking MLH1 protein expression or with MSI. Among men, we observed strong positive associations between folate and BRAF-mutated tumors (RR = 3.04 for the highest versus lowest tertile of intake, Ptrend = 0.03) and between vitamin B6 and tumors showing MLH1 hypermethylation (highest versus lowest tertile: RR = 3.23, Ptrend = 0.03). Among women, the relative risks of tumors with BRAF mutations or MLH1 hypermethylation were also increased in the highest tertiles of folate and vitamin B6 intake, respectively, but these did not reach statistical significance. The positive associations between folate intake and tumors harboring BRAF mutations and between vitamin B6 intake and those showing MLH1 hypermethylation were most pronounced among men and may suggest that these vitamins enhance colorectal cancer risk through genetic as well as epigenetic aberrations.
DNA methylation biomarkers for early detection, risk stratification and treatment response in cancer have been of great interest over the past decades. Nevertheless, clinical implementation of these ...biomarkers is limited, as only < 1% of the identified biomarkers is translated into a clinical or commercial setting. Technical factors such as a suboptimal genomic location of the assay and inefficient primer or probe design have been emphasized as important pitfalls in biomarker research. Here, we use eleven diagnostic DNA methylation biomarkers for colorectal cancer (ALX4, APC, CDKN2A, MGMT, MLH1, NDRG4, SDC2, SFRP1, SFRP2, TFPI1 and VIM), previously described in a systematic literature search, to evaluate these pitfalls.
To assess the genomic assay location, the optimal genomic locations according to TCGA data were extracted and compared to the genomic locations used in the published assays for all eleven biomarkers. In addition, all primers and probes were technically evaluated according to several criteria, based on literature and expert opinion. Both assay location and assay design quality varied widely among studies.
Large variation in both assay location and design hinders the development of future DNA methylation biomarkers as well as inter-study comparability.
To improve colorectal cancer (CRC) survival and lower incidence rates, colonoscopy and/or fecal immunochemical test screening are widely implemented. Although candidate DNA methylation biomarkers ...have been published to improve or complement the fecal immunochemical test, clinical translation is limited. We describe technical and methodological problems encountered after a systematic literature search and provide recommendations to increase (clinical) value and decrease research waste in biomarker research. In addition, we present current evidence for diagnostic CRC DNA methylation biomarkers.
A systematic literature search identified 331 diagnostic DNA methylation marker studies published before November 2020 in PubMed, EMBASE, Cochrane Library, and Google Scholar. For 136 bodily fluid studies, extended data extraction was performed. STARD criteria and level of evidence were registered to assess reporting quality and strength for clinical translation.
Our systematic literature search revealed multiple issues that hamper the development of DNA methylation biomarkers for CRC diagnosis, including methodological and technical heterogeneity and lack of validation or clinical translation. For example, clinical translation and independent validation were limited, with 100 of 434 markers (23%) studied in bodily fluids, 3 of 434 markers (0.7%) translated into clinical tests, and independent validation for 92 of 411 tissue markers (22%) and 59 of 100 bodily fluids markers (59%).
This systematic literature search revealed that major requirements to develop clinically relevant diagnostic CRC DNA methylation markers are often lacking. To avoid the resulting research waste, clinical needs, intended biomarker use, and independent validation should be better considered before study design. In addition, improved reporting quality would facilitate meta-analysis, thereby increasing the level of evidence and enabling clinical translation.
The transcription factors GATA4 and GATA5 are involved in gastrointestinal development and are inactivated by promoter hypermethylation in colorectal cancer. Here, we evaluated GATA4/5 promoter ...methylation as potential biomarkers for noninvasive colorectal cancer detection, and investigated the role of GATA4/5 in colorectal cancer.
Promoter methylation of GATA4/5 was analyzed in colorectal tissue and fecal DNA from colorectal cancer patients and healthy controls using methylation-specific PCR. The potential function of GATA4/5 as tumor suppressors was studied by inducing GATA4/5 overexpression in human colorectal cancer cell lines.
GATA4/5 methylation was observed in 70% (63/90) and 79% (61/77) of colorectal carcinomas, respectively, and was independent of clinicopathologic features. Methylation frequencies in normal colon tissues from noncancerous controls were 6% (5 of 88, GATA4; P < 0.001) and 13% (13 of 100, GATA5; P < 0.001). GATA4/5 overexpression suppressed colony formation (P < 0.005), proliferation (P < 0.001), migration (P < 0.05), invasion (P < 0.05), and anchorage-independent growth (P < 0.0001) of colorectal cancer cells. Examination of GATA4 methylation in fecal DNA from two independent series of colorectal cancer patients and controls yielded a sensitivity of 71% 95% confidence interval (95% CI), 55-88% and specificity of 84% (95% CI, 74-95%) for colorectal cancer detection in the training set, and a sensitivity of 51% (95% CI, 37-65%) and specificity of 93% (95% CI, 84-100%) in the validation set.
Methylation of GATA4/5 is a common and specific event in colorectal carcinomas, and GATA4/5 exhibit tumor suppressive effects in colorectal cancer cells in vitro. GATA4 methylation in fecal DNA may be of interest for colorectal cancer detection.
Inactivation of the von Hippel-Lindau (VHL) gene is considered as an early event in renal cancer tumorigenesis. The prognostic relevance of these changes, however, is not clear and previous results ...are contradictory. We have evaluated the influence of (epi)genetic alterations in VHL on cause-specific survival in clear-cell renal cell cancer (ccRCC) in a large, population-based group of cases.
One hundred and eighty-five cases of ccRCC, identified in the Netherlands Cohort Study on diet and cancer diagnosed in the period 1986 to 1997, were included in the analyses. Mortality information until December 2005, including causes of death, were obtained for all cases through linkage with the Central Bureau of Statistics. VHL mutations were determined with PCR single-strand conformational polymorphism and direct sequencing. VHL methylation was determined with methylation-specific PCR. Kaplan-Meier analyses and Cox proportional hazards models were used to assess associations between VHL alterations and cause-specific mortality.
Median follow-up in our population was 6 years. The frequency of loss of function mutations and methylation, separately or combined, did not differ statistically significant between different cancer stages or between tumors with different sizes. We observed no influence of loss of function mutations or methylation of the VHL gene on cause-specific mortality (hazard ratio, 1.08; 95% confidence interval, 0.69-1.68, P = 0.735) as compared with patients with a wild-type or silent mutation in VHL.
Our results indicate that (epi)genetic alterations in the VHL gene do not have prognostic value in ccRCC.
Aberrant DNA methylation affects carcinogenesis of colorectal cancer. Folate metabolizing enzymes may influence the bioavailability of methyl groups, whereas DNA and histone methyltransferases are ...involved in epigenetic regulation of gene expression. We studied associations of genetic variants of folate metabolizing enzymes (MTHFR, MTR, and MTRR), DNA methyltransferase DNMT3b, and histone methyltransferases (EHMT1, EHMT2, and PRDM2), with colorectal cancers, with or without the CpG island methylator phenotype (CIMP), MLH1 hypermethylation, or microsatellite instability. Incidence rate ratios were calculated in case-cohort analyses, with common homozygotes as reference, among 659 cases and 1,736 subcohort members of the Netherlands Cohort Study on diet and cancer (n = 120,852). Men with the MTHFR 677TT genotype were at decreased colorectal cancer risk (incidence rate ratio, 0.49; P = 0.01), but the T allele was associated with increased risk in women (incidence rate ratio, 1.39; P = 0.02). The MTR 2756GG genotype was associated with increased colorectal cancer risk (incidence rate ratio, 1.58; P = 0.04), and inverse associations were observed among women carrying DNMT3b C-->T (rs406193; incidence rate ratio, 0.72; P = 0.04) or EHMT2 G-->A (rs535586; incidence rate ratio, 0.76; P = 0.05) polymorphisms. Although significantly correlated (P < 0.001), only 41.5% and 33.3% of CIMP tumors harbored MLH1 hypermethylation or microsatellite instability, respectively. We observed inverse associations between MTR A2756G and CIMP among men (incidence rate ratio, 0.58; P = 0.04), and between MTRR A66G and MLH1 hypermethylation among women (incidence rate ratio, 0.55; P = 0.02). In conclusion, MTHFR, MTR, DNMT3b, and EHMT2 polymorphisms are associated with colorectal cancer, and rare variants of MTR and MTRR may reduce promoter hypermethylation. The incomplete overlap between CIMP, MLH1 hypermethylation, and microsatellite instability indicates that these related "methylation phenotypes" may not be similar and should be investigated separately.
Aims
SOX17 expression has not been studied in glandular lesions of the uterine cervix like adenocarcinoma in situ (AIS) and invasive adenocarcinomas (AdC), whereas SOX17 promoter CpG island ...methylation has been reported. Therefore, the aim of this study was to relate the topographical distribution of SOX17 expression and SOX17 methylation status to each other, and to SOX2 expression, human papillomavirus (HPV) type, and physical status of the virus.
Methods and results
Immunohistochemistry was used in 45 cases to assess expression of SOX17 and SOX2. SOX17 promoter methylation was determined in 25 cases by means of bisulphite conversion and methylation‐specific polymerase chain reaction. SOX17 and SOX2 showed a mutually exclusive expression pattern in normal epithelium, with a sharp delineation in the squamocolumnar junction. SOX17 was found in endocervical columnar and reserve cells, whereas SOX2 was exclusively found in squamous epithelium. In both glandular lesions and cases with coexisting glandular and squamous intraepithelial components, a complex combination of SOX17 and SOX2 expression patterns was seen and mutually exclusive expression was lost. Frequently, gain of expression of SOX2 was found and expression of SOX17 was lost. Methylation of the CpG island in the SOX17 promoter was shown to be strongly associated with loss of expression of SOX17 (P = 0.0016).
Conclusions
In this study, we show for the first time a direct correlation between the topographical distribution of SOX17 expression and the methylation status of its gene promoter. This explains the heterogeneity of SOX17 expression in the glandular lesions of the cervix. No correlation was found between HPV type and physical status of the virus on the one hand and methylation status on the other.
Dietary methyl donors might influence DNA methylation during carcinogenesis of colorectal cancer (CRC). Among 609 CRC cases and 1,663 subcohort members of the Netherlands Cohort Study on diet and ...cancer (n = 120,852), we estimated CRC risk according to methyl donor intake across genotypes of folate metabolizing enzymes and methyltransferases. Although diet-gene interactions were not statistically significant, methionine intake was inversely associated with CRC among subjects having both common rs2424913 and rs406193 DNMT3B C > T genotypes (highest versus lowest tertile: RR = 0.44; p trend = 0.05). Likewise, vitamin B2 was modestly inversely associated among individuals with the MTHFR c.665CC (rs1801133) genotype (RR = 0.66; p trend = 0.08), but with a significant reduced risk when ≤ 1 rare allele occurred in the combination of folate metabolizing enzymes MTHFR, MTRR and MTR (RR = 0.30; p trend = 0.005). Folate or vitamin B6 were neither inversely associated with CRC nor was methyl donor intake associated with the CpG island methylator phenotype (CIMP). Despite the absence of heterogeneity across genotypes, might an effect of methyl donors on CRC be more pronounced among individuals carrying common variants of folate metabolizing enzymes or DNA methyltransferases. Combining genotypes may assist to reveal diet associations with CRC, possibly because rare variants of related genes may collectively affect specific metabolic pathways or enzymatic functions.
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