Ang PW, Loh M, Liem N, Lim PL, Grieu F, Vaithilingam A, Platell C, Yong WP, Iacopetta B, Soong RComprehensive profiling of DNA methylation in colorectal cancer reveals subgroups with distinct clinicopathological and molecular features. BMC Cancer 10: 227

Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore.
BMC Cancer (Impact Factor: 3.36). 05/2010; 10(1):227. DOI: 10.1186/1471-2407-10-227
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Most previous studies of the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) have been conducted on a relatively small numbers of CpG sites. In the present study we performed comprehensive DNA methylation profiling of CRC with the aim of characterizing CIMP subgroups.
DNA methylation at 1,505 CpG sites in 807 cancer-related genes was evaluated using the Illumina GoldenGate methylation array in 28 normal colonic mucosa and 91 consecutive CRC samples. Methylation data was analyzed using unsupervised hierarchical clustering. CIMP subgroups were compared for various clinicopathological and molecular features including patient age, tumor site, microsatellite instability (MSI), methylation at a consensus panel of CpG islands and mutations in BRAF and KRAS.
A total of 202 CpG sites were differentially methylated between tumor and normal tissue. Unsupervised hierarchical clustering of methylation data from these sites revealed the existence of three CRC subgroups referred to as CIMP-low (CIMP-L, 21% of cases), CIMP-mid (CIMP-M, 14%) and CIMP-high (CIMP-H, 65%). In comparison to CIMP-L tumors, CIMP-H tumors were more often located in the proximal colon and showed more frequent mutation of KRAS and BRAF (P<0.001).
Comprehensive DNA methylation profiling identified three CRC subgroups with distinctive clinicopathological and molecular features. This study suggests that both KRAS and BRAF mutations are involved with the CIMP-H pathway of CRC rather than with distinct CIMP subgroups.

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    • "Candidate biomarkers have been further evaluated and validated in colorectal tissues by multiplexed bisulfite sequencing and by quantitative methylation specific PCR on additional patient samples. We have further compared our candidates with previously published markers, including those identified in a number of recently published studies that used a variety of different genome-wide methods [6,7,17-27] and with data from The Cancer Genome Atlas consortium. Based on our analyses of tissues and comparison with publically available data, we have validated a panel of targets that become methylated at early stages of oncogenesis, for clinical evaluation as diagnostic biomarkers. "
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    ABSTRACT: The development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests. Combined epigenomic methods - activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment - were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP). Combined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas. This study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes (SOX21, SLC6A15, NPY, GRASP, ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes (BCAT1, COL4A2, DLX5, FGF5, FOXF1, FOXI2, GRASP, IKZF1, IRF4, SDC2 and SOX21) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.
    BMC Cancer 01/2014; 14(1):54. DOI:10.1186/1471-2407-14-54 · 3.36 Impact Factor
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    • "The top-ranked gene in the leading-edge analysis was SLIT2 (slit homologue 2), appearing in six gene sets. SLIT2 interacts with netrin-1 (also known as Deleted in Colorectal Cancer), a possible tumour suppressor 23 that has previously been identified in other methylation studies of CRC 11. Other genes of interest in CRC seen in the leading-edge analysis included SLIT1, TGFBR2, PAX2, UNC5C, OTX2, NGNT1 and GDNF1. "
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    ABSTRACT: Changes in DNA methylation, whether hypo- or hyper-methylation, have been shown to be associated with progression of colorectal cancer. Methylation changes substantially in the progression from normal mucosa, to adenoma and to carcinoma. This phenomenon has not been studied extensively, and studies have been restricted to individual CpG islands rather than taking a whole genome approach. We aimed to study genome wide methylation changes in colorectal cancerWe obtained 10 fresh frozen normal tissue-cancer sample pairs, and 5 fresh frozen adenoma samples. These were run on the lllumina HumanMethylation27 whole genome methylation analysis system. Differential methylation between normal tissue, adenoma and carcinoma was analysed using Bayesian regression modelling, Gene Set Enrichment Analysis (GSEA) and Hierarchical clustering (HC). The highest-rated individual gene for differential methylation in carcinomas versus normal tissue and adenomas versus normal tissue was GRASP (p(adjusted) = 1.59x10(-5) , BF = 12.62, p(adjusted) = 1.68x10(-6) , BF = 14.53). The highest rated -gene when comparing carcinomas vs. adenomas was ATM (p(adjusted) = 2.0x10(-4) , BF = 10.17). Hierarchical clustering demonstrated poor clustering by the CIMP criteria for methylation. GSEA demonstrated methylation changes in the Netrin/DCC and SLIT/ROBO pathways. Widespread changes in DNA methylation are seen in the transition from adenoma to carcinoma. The finding that GRASP, which encodes the general receptor for phosphoinositides-1 associated scaffold proteinwas differentially methylated in colorectal cancer is interesting. This may be a potential biomarker for colorectal cancer. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
    The Journal of Pathology 12/2013; 229(5). DOI:10.1002/path.4132 · 7.43 Impact Factor
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    • "We, and others, have previously demonstrated that methylation of CpG loci detected through BeadArray platforms can be reliably replicated using alternative detection techniques including pyrosequencing, mass array analysis and quantitative methylation-specific PCR [50-56]. "
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    ABSTRACT: Epigenetic mechanisms are thought to be critical in mediating the role of the intrauterine environment on lifelong health and disease. Twin-twin transfusion syndrome (TTTS) is a rare condition wherein fetuses share the placenta and develop vascular anastomoses, which allow blood to flow between the fetuses. The unequal flow results in reciprocal hypo- and hypervolemia in the affected twins, striking growth differences and physiologic adaptations in response to this significant stressor. The donor twin in the TTTS syndrome can be profoundly growth restricted and there is likely a nutritional imbalance between the twins. The consequences of TTTS on fetal programming are unknown. This condition can now be effectively treated through the use of fetal laparoscopic procedures, but the potential for lifelong morbidity related to this condition during development is apparent. As this condition and the resulting uteroplacental discordance can play a role in the epigenetic process, we sought to investigate the DNA methylation profiles of childhood survivors of TTTS (n = 14). We focused on differences in both global measures and genome-wide CpG specific DNA methylation between donor and recipient children in this pilot study in order to generate hypotheses for further research. We identified significant hypomethylation of the LINE1 repetitive element in the peripheral blood of donor children and subtle variation in the genome-wide profiles of CpG specific methylation most prominent at CpG sites which are targets for polycomb group repressive complexes. These preliminary results suggest that coordinated epigenetic alterations result from the intrauterine environment experienced by infants with TTTS and may, at least in part, be responsible for downstream health conditions experienced by individuals surviving this condition.
    10/2013; 5(1):18. DOI:10.1186/1868-7083-5-18
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