Comprehensive molecular characterization of human colon and rectal cancer

Abstract

To characterize somatic alterations in colorectal carcinoma, we conducted a genome-scale analysis of 276 samples, analysing exome sequence, DNA copy number, promoter methylation and messenger RNA and microRNA expression. A subset of these samples (97) underwent low-depth-of-coverage whole-genome sequencing. In total, 16% of colorectal carcinomas were found to be hypermutated: three-quarters of these had the expected high microsatellite instability, usually with hypermethylation and MLH1 silencing, and one-quarter had somatic mismatch-repair gene and polymerase ε (POLE) mutations. Excluding the hypermutated cancers, colon and rectum cancers were found to have considerably similar patterns of genomic alteration. Twenty-four genes were significantly mutated, and in addition to the expected APC, TP53, SMAD4, PIK3CA and KRAS mutations, we found frequent mutations in ARID1A, SOX9 and FAM123B. Recurrent copy-number alterations include potentially drug-targetable amplifications of ERBB2 and newly discovered amplification of IGF2. Recurrent chromosomal translocations include the fusion of NAV2 and WNT pathway member TCF7L1. Integrative analyses suggest new markers for aggressive colorectal carcinoma and an important role for MYC-directed transcriptional activation and repression.

Figure 1. Mutation frequencies in human CRC.

a, Mutation frequencies in each of the tumour samples from 224 patients. Note a clear separation of hypermutated and non-hypermutated samples. Red, MSI high, CIMP high or MLH1 silenced; light blue, MSI low, or CIMP low; black, rectum; white, colon; grey, no data. Inset, mutations in mismatch-repair genes and POLE among the hypermutated samples. The order of the samples is the same as in the main graph. b, Significantly mutated genes in hypermutated and non-hypermutated tumours. Blue bars represent genes identified by the MutSig algorithm and black bars represent genes identified by manual examination of sequence data. PowerPoint slide

Figure 2. Integrative analysis of genomic changes in 195 CRCs.

Hypermutated tumours have near-diploid genomes and are highly enriched for hypermethylation, CIMP expression phenotype and BRAF(V600E) mutations. Non-hypermutated tumours originating from different sites are virtually indistinguishable from each other on the basis of their copy-number alteration patterns, DNA methylation or gene-expression patterns. Copy-number changes of the 22 autosomes are shown in shades of red for copy-number gains and shades of blue for copy-number losses. PowerPoint slide

Figure 3. Copy-number changes and structural aberrations in CRC.

a, Focal amplification of 11p15.5. Segmented DNA copy-number data from single-nucleotide polymorphism (SNP) arrays and low-pass whole-genome sequencing (WGS) are shown. Each row represents a patient; amplified regions are shown in red. b, Correlation of expression levels with copy-number changes for IGF2 and miR-483. c, IGF2 amplification and overexpression are mutually exclusive of alterations in PI3K signalling-related genes. d, Recurrent NAV2–TCF7L2 fusions. The structure of the two genes, locations of the breakpoints leading to the translocation and circular representations of all rearrangements in tumours with a fusion are shown. Red line lines represent the NAV2–TCF7L2 fusions and black lines represent other rearrangements. The inner ring represents copy-number changes (blue denotes loss, pink denotes gain). PowerPoint slide

Figure 4. Diversity and frequency of genetic changes leading to deregulation of signalling pathways in CRC.

Non-hypermutated (nHM; n = 165) and hypermutated (HM; n = 30) samples with complete data were analysed separately. Alterations are defined by somatic mutations, homozygous deletions, high-level focal amplifications, and, in some cases, by significant up- or downregulation of gene expression (IGF2, FZD10, SMAD4). Alteration frequencies are expressed as a percentage of all cases. Red denotes activated genes and blue denotes inactivated genes. Bottom panel shows for each sample if at least one gene in each of the five pathways described in this figure is altered. PowerPoint slide

Figure 5. Integrative analyses of multiple data sets.

a, Clustering of genes and pathways affected in colon and rectum tumours deduced by PARADIGM analysis. Blue denotes under-expressed relative to normal and red denotes overexpressed relative to normal. Some of the pathways deduced by this method are shown on the right. NHEJ, non-homologous end joining. b, Gene-expression signatures and SCNAs associated with tumour aggression. Molecular signatures (rows) that show a statistically significant association with tumour aggressiveness according to selected clinical assays (columns) are shown in colour, with red indicating markers of tumour aggressiveness and blue indicating the markers of less-aggressive tumours. Significance is based on the combined P value from the weighted Fisher’s method, corrected for multiple testing. Colour intensity and score is in accordance with the strength of an individual clinical–molecular association, and is proportional to log₁₀(P), where P is the P value for that association. To limit the vertical extent of the figure, gene-expression signatures are restricted to a combined P value of P < 10⁻⁹ and SCNAs to P < 10⁻⁷, and features are shown only if they are also significant in the subset of non-MSI-H samples (the analysis was performed separately on the full data as well as on the MSI-H and non-MSI-H subgroups). PowerPoint slide