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Background Smoking and alcohol increase risk for colorectal malignancies. However, colorectal cancer (CRC) is a heterogenic disease and associations with the molecular pathological pathways are unclear. Methods This population-based case–control study includes 2444 cases with first-diagnosis CRC and 2475 controls. Tumour tissue was analysed for MSI (microsatellite instability), CIMP (CpG island methylator phenotype), BRAF (B-Raf proto-oncogene serine/threonine kinase gene) and KRAS (Kirsten rat sarcoma viral oncogene homologue gene) mutations. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated for associations between alcohol and smoking and CRC molecular subtypes and pathways. Results Current smoking showed higher ORs for MSI-high (OR = 2.79, 95% CI: 1.86–4.18) compared to MSS (OR = 1.41, 1.14–1.75, p -heterogeneity ( p -het) = 0.001), BRAF-mutated (mut) (OR = 2.40, 1.41–4.07) compared to BRAF-wild type (wt) (OR = 1.52, 1.24–1.88, p -het = 0.074), KRAS-wt (OR = 1.70, 1.36–2.13) compared to KRAS-mut (OR = 1.26, 0.95–1.68, p -het = 0.039) and CIMP-high (OR = 2.01, 1.40–2.88) compared to CIMP-low/negative CRC (OR = 1.50, 1.22–1.85, p -het=0.101). Current smoking seemed more strongly associated with sessile serrated pathway (CIMP-high + BRAF-mut; OR = 2.39, 1.27–4.52) than with traditional pathway CRC (MSS + CIMP-low/negative + BRAF-wt; OR = 1.50, 1.16–1.94) and no association was observed with alternate pathway CRC (MSS + CIMP-low/negative + KRAS-wt; OR = 1.08, 0.77–1.43). No heterogeneity was observed in alcohol consumption association by molecular subtypes. Conclusions In this large case–control study, smoking was more strongly associated with MSI-high and KRAS-wt CRC and with cases showing features of the sessile serrated pathway. Association patterns were less clear for alcohol consumption.

Lynch syndrome is an inherited tumour predisposition syndrome caused by germline mutations of DNA mismatch repair (MMR) genes. Mutation carriers have a high risk of developing colorectal cancer, but do not present with polyposis, a typical feature of other colorectal cancer syndromes such as familial adenomatous polyposis, in which polyposis reflects the high frequency of biallelic APC gene inactivation. We asked whether in Lynch syndrome biallelic inactivation of MMR genes occurred at a similar frequency to that of APC gene, and whether MMR inactivation resulted in detectable lesions within the intestinal mucosa. Resections done for small and large bowel cancer between January, 2002, and January, 2011, were retrieved. We systematically analysed non-tumorous mucosa from carriers of a Lynch syndrome mutation (set 1: ten patients) and control patients without Lynch syndrome (set 1: nine patients) for MMR protein expression (MLH1, MSH2, and EPCAM) with immunohistochemistry. We validated the findings in an independent sample set (set 2: 30 Lynch syndrome patients, 79 controls). We did an analysis of microsatellite instability by PCR analysis to test lesions for mismatch repair deficiency. We applied a Poisson regression model to analyse the distribution of MMR-deficient crypt foci counts and a Fisher's exact test to compare the prevalence of these foci between mutation carriers and control patients. 20 crypt foci with no MMR protein expression were detected in 20·1 cm2 of non-tumorous mucosa from Lynch syndrome patients (set 1), an additional five were detected upon resectioning of two samples. In an independent validation set (set 2), two MMR-deficient crypt foci were noted in 2·2 cm2 of mucosa. No MMR-deficient crypt foci were noted in non-tumorous mucosa from control patients without evidence for Lynch syndrome (set 1: 3·7 cm2, set 2: 4·8 cm2). Microsatellite instability was detected in all seven MMR-deficient crypt foci analysed. A subset of these foci displayed unusual architectural and cytological abnormalities, although they had no polypous or adenomatous appearance. We identified a novel type of lesion, the MMR-deficient crypt focus, as the manifestation of biallelic MMR gene inactivation in Lynch syndrome. The abundance of MMR-deficient crypt foci indicates a high frequency of biallelic MMR gene inactivation, which is in sharp contrast with the low number of clinically manifest cancers in Lynch syndrome. This discrepancy suggests that most MMR-deficient crypt foci do not progress to cancer. We propose Lynch syndrome as a unique model syndrome for studying initial steps of MMR deficiency, tumour initiation and, possibly, elimination. German Cancer Aid and German Research Foundation.

Histological slides contain numerous artifacts that can significantly deteriorate the performance of image analysis algorithms. Here we develop the GrandQC tool for tissue and multi-class artifact segmentation. GrandQC allows for high-precision tissue segmentation (Dice score 0.957) and segmentation of tissue without artifacts (Dice score 0.919–0.938 dependent on magnification). Slides from 19 international pathology departments digitized with the most common scanning systems and from The Cancer Genome Atlas dataset were used to establish a QC benchmark, analyzing inter-institutional, intra-institutional, temporal, and inter-scanner slide quality variations. GrandQC improves the performance of downstream image analysis algorithms. We open-source the GrandQC tool, our large manually annotated test dataset, and all QC masks for the entire TCGA cohort to address the problem of QC in digital/computational pathology. GrandQC can be used as a tool to monitor sample preparation and scanning quality in pathology departments and help to track and eliminate major artifact sources. Histological slides often contain artifacts that affect the performance of downstream image analysis. Here, the authors present GrandQC, a tool that enables high-precision tissue and artifact segmentation in histological slides. This tool can be used to monitor sample preparation and scanning quality across pathology departments.

Pulmonary enteric adenocarcinoma is a rare non-small cell lung cancer subtype. It is poorly characterized and cannot be distinguished from metastatic colorectal or upper gastrointestinal adenocarcinomas by means of routine pathological methods. As DNA methylation patterns are known to be highly tissue specific, we aimed to develop a methylation-based algorithm to differentiate these entities. To this end, genome-wide methylation profiles of 600 primary pulmonary, colorectal, and upper gastrointestinal adenocarcinomas obtained from The Cancer Genome Atlas and the Gene Expression Omnibus database were used as a reference cohort to train a machine learning algorithm. The resulting classifier correctly classified all samples from a validation cohort consisting of 680 primary pulmonary, colorectal and upper gastrointestinal adenocarcinomas, demonstrating the ability of the algorithm to reliably distinguish these three entities. We then analyzed methylation data of 15 pulmonary enteric adenocarcinomas as well as four pulmonary metastases and four primary colorectal adenocarcinomas with the algorithm. All 15 pulmonary enteric adenocarcinomas were reliably classified as primary pulmonary tumors and all four metastases as well as all four primary colorectal cancer samples were identified as colorectal adenocarcinomas. In a t-distributed stochastic neighbor embedding analysis, the pulmonary enteric adenocarcinoma samples did not form a separate methylation subclass but rather diffusely intermixed with other pulmonary cancers. Additional characterization of the pulmonary enteric adenocarcinoma series using fluorescence in situ hybridization, next-generation sequencing and copy number analysis revealed KRAS mutations in nine of 15 samples (60%) and a high number of structural chromosomal changes. Except for an unusually high rate of chromosome 20 gain (67%), the molecular data was mostly reminiscent of standard pulmonary adenocarcinomas. In conclusion, we provide sound evidence of the pulmonary origin of pulmonary enteric adenocarcinomas and in addition provide a publicly available machine learning-based algorithm to reliably distinguish these tumors from metastatic colorectal cancer.

The immune system can recognize and attack cancer cells, especially those with a high load of mutation-induced neoantigens. Such neoantigens are abundant in DNA mismatch repair (MMR)-deficient, microsatellite-unstable (MSI) cancers. MMR deficiency leads to insertion/deletion (indel) mutations at coding microsatellites (cMS) and to neoantigen-inducing translational frameshifts. Here, we develop a tool to quantify frameshift mutations in MSI colorectal and endometrial cancer. Our results show that frameshift mutation frequency is negatively correlated to the predicted immunogenicity of the resulting peptides, suggesting counterselection of cell clones with highly immunogenic frameshift peptides. This correlation is absent in tumors with Beta-2-microglobulin mutations, and HLA-A*02:01 status is related to cMS mutation patterns. Importantly, certain outlier mutations are common in MSI cancers despite being related to frameshift peptides with functionally confirmed immunogenicity, suggesting a possible driver role during MSI tumor evolution. Neoantigens resulting from shared mutations represent promising vaccine candidates for prevention of MSI cancers. DNA mismatch repair (MMR)-deficient cancers with microsatellite-instability are characterized by a high load of frameshift mutation-derived neoantigens. Here, by mapping the frameshift mutation landscape and predicting the immunogenicity of the resulting peptides, the authors show evidence of immunoediting in MMR-deficient colorectal and endometrial cancers.

PurposeTo differentiate subtypes of hepatocellular adenoma (HCA) based on enhancement characteristics in gadoxetic acid (Gd-EOB) magnetic resonance imaging (MRI).Materials and methodsForty-eight patients with 79 histopathologically proven HCAs who underwent Gd-EOB-enhanced MRI were enrolled (standard of reference: surgical resection). Two blinded radiologists performed quantitative measurements (lesion-to-liver enhancement) and evaluated qualitative imaging features. Inter-reader variability was tested. Advanced texture analysis was used to evaluate lesion heterogeneity three-dimensionally.ResultsOverall, there were 19 (24%) hepatocyte nuclear factor (HNF)-1a-mutated (HHCAs), 37 (47%) inflammatory (IHCAs), 5 (6.5%) b-catenin-activated (bHCA), and 18 (22.5%) unclassified (UHCAs) adenomas. In the hepatobiliary phase (HBP), 49.5% (39/79) of all adenomas were rated as hypointense and 50.5% (40/79) as significantly enhancing (defined as > 25% intralesional GD-EOB uptake). 82.5% (33/40) of significantly enhancing adenomas were IHCAs, while only 4% (1/40) were in the HHCA subgroup (p < 0.001). When Gd-EOB uptake behavior was considered in conjunction with established MRI features (binary regression model), the area under the curve (AUC) increased from 0.785 to 0.953 for differentiation of IHCA (atoll sign + hyperintensity), from 0.859 to 0.903 for bHCA (scar + hyperintensity), and from 0.899 to 0.957 for HHCA (steatosis + hypointensity). Three-dimensional region of interest (3D ROI) analysis showed significantly increased voxel heterogeneity for IHCAs (p = 0.038).ConclusionGd-EOB MRI is of added value for subtype differentiation of HCAs and reliably identifies the typical heterogeneous HBP uptake of IHCAs. Diagnostic accuracy can be improved significantly by the combined analysis of established morphologic MR appearances and intralesional Gd-EOB uptake.Key Points•Gd-EOB-enhanced MRI is of added value for subtype differentiation of HCA.•IHCA and HHCA can be identified reliably based on their typical Gd-EOB uptake patterns, and accuracy increases significantly when additionally taking established MR appearances into account.•The small numbers of bHCAs and UHCAs remain the source of diagnostic uncertainty.

Like many other types of cancer, colorectal cancer (CRC) develops through multiple pathways of carcinogenesis. This is also true for colorectal carcinogenesis in Lynch syndrome (LS), the most common inherited CRC syndrome. However, a comprehensive understanding of the distribution of these pathways of carcinogenesis, which allows for tailored clinical treatment and even prevention, is still lacking. We suggest a linear dynamical system modeling the evolution of different pathways of colorectal carcinogenesis based on the involved driver mutations. The model consists of different components accounting for independent and dependent mutational processes. We define the driver gene mutation graphs and combine them using the Cartesian graph product. This leads to matrix components built by the Kronecker sum and product of the adjacency matrices of the gene mutation graphs enabling a thorough mathematical analysis and medical interpretation. Using the Kronecker structure, we developed a mathematical model which we applied exemplarily to the three pathways of colorectal carcinogenesis in LS. Beside a pathogenic germline variant in one of the DNA mismatch repair (MMR) genes, driver mutations in APC , CTNNB1 , KRAS and TP53 are considered. We exemplarily incorporate mutational dependencies, such as increased point mutation rates after MMR deficiency, and based on recent experimental data, biallelic somatic CTNNB1 mutations as common drivers of LS-associated CRCs. With the model and parameter choice, we obtained simulation results that are in concordance with clinical observations. These include the evolution of MMR-deficient crypts as early precursors in LS carcinogenesis and the influence of variants in MMR genes thereon. The proportions of MMR-deficient and MMR-proficient APC-inactivated crypts as first measure for the distribution among the pathways in LS-associated colorectal carcinogenesis are compatible with clinical observations. The approach provides a modular framework for modeling multiple pathways of carcinogenesis yielding promising results in concordance with clinical observations in LS CRCs.

In previous studies, the protective effect of colonoscopy was generally stronger for distal colorectal cancer than for proximal colorectal cancer (CRC). This study aimed to investigate whether reduction of CRC risk through colonoscopy varies according to major tumor markers and pathways of CRC. This is a population-based case-control study from Germany, including 2,132 patients with a first diagnosis of CRC and information on major molecular tumor markers and 2,486 control participants without CRC. Detailed participant characteristics were collected by standardized questionnaires. Information on previous colonoscopy was derived from medical records. Polytomous logistic regression was used to calculate adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for the association between previous colonoscopy and subtypes of CRC. Overall, we observed strong risk reduction of CRC after colonoscopy that was weaker for microsatellite instable (MSI) than for non-MSI CRC (OR 0.70, 95% CI 0.50-0.97 vs OR 0.28, 95% CI 0.24-0.33), for CpG island methylator phenotype high CRC than for CpG island methylator phenotype low/negative CRC (OR 0.45, 95% CI 0.34-0.59 vs OR 0.29, 95% CI 0.25-0.34), for BRAF-mutated than for BRAF nonmutated CRC (OR 0.62, 95% CI 0.42-0.91 vs OR 0.30, 95% CI 0.25-0.35), for KRAS nonmutated than for KRAS-mutated CRC (OR 0.34, 95% CI 0.29-0.40 vs OR 0.26, 95% CI 0.20-0.32), and for CRC classified into the sessile serrated pathway than for CRC of the traditional pathway (OR 0.57, 95% CI 0.36-0.91 vs OR 0.30, 95% CI 0.25-0.37). After colonoscopy with the detection of adenomas or hyperplastic polyps, no risk reduction was found for sessile serrated pathway CRC, MSI, and BRAF-mutated subtypes. Our study extends the molecular understanding of existing differences in risk reduction of proximal and distal CRCs reported by previous studies and may imply important information for improving strategies for timely detection of relevant precursors.

Background β-catenin activation plays a crucial role for tumourigenesis in the large intestine but except for Lynch syndrome (LS) associated cancers stabilizing mutations of β-catenin gene ( CTNNB1) are rare in colorectal cancer (CRC). Previous animal studies provide an explanation for this observation. They showed that CTNNB1 mutations induced transformation in the colon only when CTNNB1 was homozygously mutated or when membranous β-catenin binding was hampered by E-cadherin haploinsufficiency. We were interested, if these mechanisms are also found in human CTNNB1 mutated CRCs. Results Among 869 CRCs stabilizing CTNNB1 mutations were found in 27 cases. Homo- or hemizygous CTNNB1 mutations were detected in 74% of CTNNB1 mutated CRCs (13 microsatellite instabile (MSI-H), 7 microsatellite stabile (MSS)) but only in 3% (1/33) of extracolonic CTNNB1 mutated cancers. In contrast to MSS CRC, CTNNB1 mutations at codon 41 or 45 were highly selected in MSI-H CRC. Of the examined three CRC cell lines, β-catenin and E-cadherin expression was similar in cell lines without or with hetereozygous CTNNB1 mutations (DLD1 and HCT116), while a reduced E-cadherin expression combined with cytoplasmic accumulation of β-catenin was found in a cell line with homozygous CTNNB1 mutation (LS180). Reduced expression of E-cadherin in human MSI-H CRC tissue was identified in 60% of investigated cancers, but no association with the CTNNB1 mutational status was found. Conclusions In conclusion, this study shows that in contrast to extracolonic cancers stabilizing CTNNB1 mutations in CRC are commonly homo- or hemizygous indicating a higher threshold of β-catenin stabilization to be required for transformation in the colon as compared to extracolonic sites. Moreover, we found different mutational hotspots in CTNNB1 for MSI-H and MSS CRCs suggesting a selection of different effects on β-catenin stabilization according to the molecular pathway of tumourigenesis. Reduced E-cadherin expression in CRC may further contribute to higher levels of transcriptionally active β-catenin, but it is not directly linked to the CTNNB1 mutational status.

Genetic heterogeneity between and within tumours is a major factor determining cancer progression and therapy response. Here we examined DNA sequence and DNA copy-number heterogeneity in colorectal cancer (CRC) by targeted high-depth sequencing of 100 most frequently altered genes. In 97 samples, with primary tumours and matched metastases from 27 patients, we observe inter-tumour concordance for coding mutations; in contrast, gene copy numbers are highly discordant between primary tumours and metastases as validated by fluorescent in situ hybridization. To further investigate intra-tumour heterogeneity, we dissected a single tumour into 68 spatially defined samples and sequenced them separately. We identify evenly distributed coding mutations in APC and TP5 3 in all tumour areas, yet highly variable gene copy numbers in numerous genes. 3D morpho-molecular reconstruction reveals two clusters with divergent copy number aberrations along the proximal–distal axis indicating that DNA copy number variations are a major source of tumour heterogeneity in CRC. The contribution of intra-tumour heterogeneity is increasingly associated with resistance to therapy. Here, the authors use genomic analyses to study heterogeneity in colorectal cancer and perform in-depth reconstruction of heterogeneity in one sample.