Extensive changes in DNA methylation are common in cancer and may contribute to oncogenesis through transcriptional silencing of tumor-suppressor genes1. Genome-scale studies have yielded important insights into these changes2, 3, 4, 5 but have focused on CpG islands or gene promoters. We used whole-genome bisulfite sequencing (bisulfite-seq) to comprehensively profile a primary human colorectal tumor and adjacent normal colon tissue at single-basepair resolution. Regions of focal hypermethylation in the tumor were located primarily at CpG islands and were concentrated within regions of long-range (>100 kb) hypomethylation. These hypomethylated domains covered nearly half of the genome and coincided with late replication and attachment to the nuclear lamina in human cell lines. We confirmed the confluence of hypermethylation and hypomethylation within these domains in 25 diverse colorectal tumors and matched adjacent tissue. We propose that widespread DNA methylation changes in cancer are linked to silencing programs orchestrated by the three-dimensional organization of chromatin within the nucleus.

Colorectal cancer (CRC) is a heterogeneous disease in which unique subtypes are characterized by distinct genetic and epigenetic alterations. Here we performed comprehensive genome-scale DNA methylation profiling of 125 colorectal tumors and 29 adjacent normal tissues. We identified four DNA methylation-based subgroups of CRC using model-based cluster analyses. Each subtype shows characteristic genetic and clinical features, indicating that they represent biologically distinct subgroups. A CIMP-high (CIMP-H) subgroup, which exhibits an exceptionally high frequency of cancer-specific DNA hypermethylation, is strongly associated with MLH1 DNA hypermethylation and the BRAF V600E mutation. A CIMPlow (CIMP-L) subgroup is enriched for KRAS mutations and characterized by DNA hypermethylation of a subset of CIMP-H-associated markers rather than a unique group of CpG islands. Non-CIMP tumors are separated into two distinct clusters. One non-CIMP subgroup is distinguished by a significantly higher frequency of TP53 mutations and frequent occurrence in the distal colon, while the tumors that belong to the fourth group exhibit a low frequency of both cancer-specific DNA hypermethylation and gene mutations and are significantly enriched for rectal tumors. Furthermore, we identified 112 genes that were down-regulated more than twofold in CIMP-H tumors together with promoter DNA hypermethylation. These represent~7% of genes that acquired promoter DNA methylation in CIMP-H tumors. Intriguingly, 48/112 genes were also transcriptionally down-regulated in non-CIMP subgroups, but this was not attributable to promoter DNA hypermethylation. Together, we identified four distinct DNA methylation subgroups of CRC and provided novel insight regarding the role of CIMP-specific DNA hypermethylation in gene silencing.

BackgroundThere is an increasing demand for accurate biomarkers for early non-invasive colorectal cancer detection. We employed a genome-scale marker discovery method to identify and verify candidate DNA methylation biomarkers for blood-based detection of colorectal cancer.Methodology/Principal FindingsWe used DNA methylation data from 711 colorectal tumors, 53 matched adjacent-normal colonic tissue samples, 286 healthy blood samples and 4,201 tumor samples of 15 different cancer types. DNA methylation data were generated by the Illumina Infinium HumanMethylation27 and the HumanMethylation450 platforms, which determine the methylation status of 27,578 and 482,421 CpG sites respectively. We first performed a multistep marker selection to identify candidate markers with high methylation across all colorectal tumors while harboring low methylation in healthy samples and other cancer types. We then used pre-therapeutic plasma and serum samples from 107 colorectal cancer patients and 98 controls without colorectal cancer, confirmed by colonoscopy, to verify candidate markers. We selected two markers for further evaluation: methylated THBD (THBD-M) and methylated C9orf50 (C9orf50-M). When tested on clinical plasma and serum samples these markers outperformed carcinoembryonic antigen (CEA) serum measurement and resulted in a high sensitive and specific test performance for early colorectal cancer detection.Conclusions/SignificanceOur systematic marker discovery and verification study for blood-based DNA methylation markers resulted in two novel colorectal cancer biomarkers, THBD-M and C9orf50-M. THBD-M in particular showed promising performance in clinical samples, justifying its further optimization and clinical testing.

Aggressive melanoma cells are able to form alternative routes for angiogenesis. The formation of extracellular matrix-rich vasculogenic-like networks [periodic acid Schiff (PAS) loops] and expression of endothelial-associated genes [allowing direct contact of erythrocytes (blood lakes)] are forms of vasculogenic mimicry (VM). The detection of these alternative routes may be used as an additional staging factor for cutaneous melanoma and predicts the route of metastasis in melanoma. We studied the association of the presence of VM with metastasis (lymphogenous and/or haematogenous) in patients diagnosed with cutaneous malignant melanoma in het Groene Hart Hospital, the Netherlands, between 1995 and 2000. Tumour tissue samples of 123 patients were assessed on PAS loops and blood lakes and correlated to clinical data. VM was detected in 42 (34%) and proven metastasis developed in 23 patients (18.7%). VM was associated with shorter survival (P<0.001). In 36 tumours, PAS loops were detected. PAS loops were correlated with the presence of lymphogenous as well as haematogenous metastasis (P=0.062 and 0.013). In 20 tumours, blood lakes were detected and correlated with haematogenous metastasis (P<0.001). In multivariate analyses, the detected blood lakes were significantly associated with haematogenous metastasis (P<0.001, adjusted odds ratio 6.8, 95% confidence interval 1.47-31). Blood lakes were strongly correlated with haematogenous metastasis of cutaneous melanoma and were an independent determinant for survival. These interesting findings need further investigation, although we believe that implementation of this detection can directly lead to better staging of cutaneous melanoma.

Colorectal cancer (CRC) is a heterogeneous disease in which unique subtypes are characterized by distinct genetic and epigenetic alterations. Here we performed comprehensive genome-scale DNA methylation profiling of 125 colorectal tumors and 29 adjacent normal tissues. We identified four DNA methylation-based subgroups of CRC using model-based cluster analyses. Each subtype shows characteristic genetic and clinical features, indicating that they represent biologically distinct subgroups. A CIMP-high (CIMP-H) subgroup includes all CIMP-positive tumors characterized by the MethyLight five-marker panel, and is strongly associated with MLH1 DNA hypermethylation and the BRAFV600E mutation. A CIMP-low subgroup is enriched for KRAS mutations and characterized by DNA hypermethylation of a subset of CIMP-associated markers rather than a unique group of CpG islands. Non-CIMP tumors are separated into two distinct clusters. One non-CIMP subgroup is distinguished by a significantly higher frequency of TP53 mutations and frequent occurrence in the distal colon, while the tumors that belong to the fourth group exhibit a low frequency of both cancer-specific DNA hypermethylation and gene mutations and are significantly enriched for rectal tumors. Furthermore, we identified 114 genes that were downregulated more than 2-fold in CIMP-H tumors together with promoter DNA hypermethylation. These represent approximately 7% of genes that acquired promoter DNA methylation in CIMP-H tumors. Intriguingly, 48/114 genes were also transcriptionally silent in non-CIMP subgroups, but this was not attributable to promoter DNA hypermethylation. Together, we identified four distinct DNA methylation subgroups of CRC and provided novel insight regarding the role of CIMP-specific DNA hypermethylation in gene silencing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-173. doi:10.1158/1538-7445.AM2011-LB-173