1. Volume 23, Issue 11, Pages 3407-3418 (June 2018)
Cell-of-Origin DNA Methylation Signatures Are Maintained during Colorectal Carcinogenesis 
Felix Bormann, Manuel Rodríguez-Paredes, Felix Lasitschka, Dominic Edelmann, Tanja Musch, Axel Benner, Yehudit Bergman, Sebastian M. Dieter, Claudia R. Ball, Hanno Glimm, Heinz G. Linhart, Frank Lyko 
Cell Reports 
Volume 23, Issue 11, Pages (June 2018)
DOI: /j.celrep
Copyright © 2018 The Author(s) Terms and Conditions

2. Cell Reports 2018 23, 3407-3418DOI: (10.1016/j.celrep.2018.05.045)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1
Colorectal Adenomas Can Be Separated into 3 Epigenetic Subclasses
(A) Composition of the colorectal adenoma methylation dataset used for this study.
(B) Probe clusters enrich for enhancer-associated probes.
(C) Comparison of methylation patterns from 3 patients for colorectal adenomas (A) and their associated normal mucosa (N). Heatmaps show methylation (β) values of the 8,000 probe clusters used for colorectal adenoma subclassification. The asterisk indicates an additional sample pair that was not included in our original sample set. Adenoma samples from patients 1 and 2 that are also shown in (D) are indicated by black and gray bars, respectively.
(D) β value heatmap of the most variable probe clusters after subgroup assignment by consensus clustering 54 adenoma samples (see Figure S1C). (Epi-)genomic features of the subgroups are shown on the right (CGI, CpG island; prom., promoter; body, gene body; IGR, intergenic region). Various pathological markers are shown below the heatmap. Individual patients with multiple adenomas are indicated by specific colors. In addition, adenoma samples from patients 1 and 2 in (C) are also indicated in black and gray, respectively.
(E) tSNE plot using this adenoma set (n = 54) and a reference adenoma set (n = 42) after consensus clustering using the same 8,000 probe clusters (see Figure S2).
See also Figures S1 and S2.
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4. Figure 2 Characterization of Epigenetic Classifiers
(A) GO analysis of all genes associated with the 8,000 most variable probe clusters. Representative GOs are shown. See Table S2 for a complete list. p values are FDR (false discovery rate) corrected.
(B) TFBS analysis using the probes within the 8,000 probe clusters. A representative set of TFBSs is shown. The complete result is shown in Table S3.
(C) GO analysis of all TFs being assigned as active in subclasses A and C (see Experimental Procedures for details). Representative GOs are shown. See Table S2 for a complete list. p values are FDR corrected.
(D) Methylation analysis of NANOG and E2F1 TFBS-associated probes from the 8,000 most variable probe clusters. Boxplots represent β value distributions in the three subclasses.
(E) Venn diagram describing the identified enhancers in colonic mucosa and H1 ESCs.
(F) Principal-component analyses based on the adenoma dataset using the identified enhancers.
(G) Phyloepigenetic analysis of the adenoma samples. A rooted tree was created with an arbitrarily chosen subgroup-C sample as the root.
See Tables S2 and S3.
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5. Figure 3
Differences between Epigenetic Subclasses Reflect Different Differentiation States
(A) Establishment of DNA methylation patterns from horizontal crypt sections by microdissection. Schematic overview of the intestinal crypt.
(B) Methylation heatmaps of the 8,000 most variable probe clusters between the crypt sections (n = 3).
(C) Principal-component analysis based on the adenoma dataset using the most variable probe clusters from the crypt sections.
(D) TFBS analysis using probes within the 8,000 most variable probe clusters. The Venn diagram describes the overlap of identified enriched TFBSs in the probe clusters being more methylated in the stem-like subclass and in the crypt base sample.
(E) A representative set of TFBSs that is enriched among the methylated probe clusters of crypt base. The complete result is shown in Table S3.
(F) Representative genes showing concordant methylation changes in crypt sections and in the corresponding adenoma subclasses. The Infinium probes visualized in the heatmap are marked by horizontal lines.
See also Table S3.
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6. Figure 4
Colorectal Adenoma Methylomes Are Maintained in Colorectal Carcinoma
(A) β value heatmap of the most variable probe clusters after subgroup assignment by consensus clustering (n = 373) (see Figure S5A). (Epi-)genomic features of the subgroups are shown on the right (CGI, CpG island; prom., promoter; body, gene body; IGR, intergenic region). Various pathological markers are shown at the bottom.
(B) Bar plots showing cluster-specific tumor purity or immune/stromal scores (see Experimental Procedures for details).
(C) tSNE analysis of all adenoma (n = 54) and carcinoma (n = 373) samples, based on the 8,000 most variably methylated probe clusters identified in adenomas.
(D) Venn diagram describing the overlap between variably methylated and differentially expressed (FC ≥ 1.5 and FC ≤ 0.66, Padj. ≤ 0.05) genes.
(E) Heatmap showing expression values for differentially expressed genes among CRC samples assigned to different methylation subclasses (n = 292). Normalized gene expression levels are indicated from blue (z ≤ −1.8, lowly expressed) to yellow (z ≥ 1.8, highly expressed).
(F) β value heatmaps of the 8,000 most variable probe clusters in tumor-derived xenografts (X) or tumor sphere cultures (S). Colors indicate subclasses. Probe clusters are ordered as in (A).
See also Figure S5A.
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7. Figure 5 Epigenetic Subclassification Predicts Clinical Outcome
(A) Comparison of methylation-based subclassification with consensus molecular subtypes (Guinney et al., 2015). The heatmap indicates the percent distribution of expression-based subtypes in the different methylation-based subtypes.
(B) Kaplan-Meier plots of all TCGA colorectal cancer samples separated into CIMP-high (red) and others (black).
(C) Survival analysis of all TCGA colorectal cancer samples separated into stem-like (blue) and other (black) clusters after removal of all CIMP-high samples and a multivariate Cox model including various clinical covariates and the stem-like cluster information.
(D) Comparison of the Cox model described in (C) and a model including only clinical covariates in terms of prediction error after application of a 5-fold cross-validation. The reference curve describes the prediction error of overall survival without applying a Cox model.
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