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Supplementary Figures 1-3 from PHLDA1 Expression Marks the Putative Epithelial Stem Cells and Contributes to Intestinal Tumorigenesis

Supplementary Tables 1-2 from PHLDA1 Expression Marks the Putative Epithelial Stem Cells and Contributes to Intestinal Tumorigenesis

Supplementary Methods and Materials from PHLDA1 Expression Marks the Putative Epithelial Stem Cells and Contributes to Intestinal Tumorigenesis

Molecular clocks that record cell ancestry mutate too slowly to measure the short-timescale dynamics of cell renewal in adult tissues. Here, we show that fluctuating DNA methylation marks can be used as clocks in cells where ongoing methylation and demethylation cause repeated ‘flip–flops’ between methylated and unmethylated states. We identify endogenous fluctuating CpG (fCpG) sites using standard methylation arrays and develop a mathematical model to quantitatively measure human adult stem cell dynamics from these data. Small intestinal crypts were inferred to contain slightly more stem cells than the colon, with slower stem cell replacement in the small intestine. Germline APC mutation increased the number of replacements per crypt. In blood, we measured rapid expansion of acute leukemia and slower growth of chronic disease. Thus, the patterns of human somatic cell birth and death are measurable with fluctuating methylation clocks (FMCs).

Background and Aim Serrated polyposis syndrome (SPS) is now known to be the commonest polyposis syndrome. Previous analyses for germline variants have shown no consistent positive findings. To exclude other polyposis syndromes, 2019 British Society of Gastroenterology (BSG) guidelines advise gene panel testing if the patient is under 50 years, there are multiple affected individuals within a family, or there is dysplasia within any of the polyps. Methods A database of SPS patients was established at the Oxford University Hospitals NHS Foundation Trust. Patients were referred for genetic assessment based on personal and family history and patient preference. The majority were tested for a hereditary colorectal cancer panel including MUTYH, APC, PTEN, SMAD4, BMPR1A, STK11, NTLH1, POLD1, POLE, GREM1 (40-kb duplication), PMS2, and Lynch syndrome mismatch repair genes. Results One hundred and seventy-three patients were diagnosed with SPS based on World Health Organization 2019 criteria between February 2010 and December 2020. The mean age of diagnosis was 54.2 ± 16.8 years. Seventy-three patients underwent genetic testing and 15/73 (20.5%) were found to have germline variants, of which 7/73 (9.6%) had a pathogenic variant (MUTYH n = 2, SMAD4 n = 1, CHEK2 n = 2, POLD1 n = 1, and RNF43 n = 1). Only 60% (9/15) of these patients would have been recommended for gene panel testing according to current BSG guidelines. Conclusions A total of 20.5% of SPS patients tested were affected by heterozygous germline variants, including previously unreported associations with CHEK2 and POLD1. This led to a change in management in seven patients (9.6%). Current recommendations may miss SPS associated with germline variants, which is more common than previously anticipated.

An immunosuppressive microenvironment causes poor tumour T-cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumours is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identify complement receptor C5aR1 as a druggable target which when inhibited improves radiotherapy even in tumours displaying immunosuppressive features and poor CD8+ T-cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we find that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumour-cell specific functions. C5aR1 targeting results in increased NF-κB-dependent apoptosis specifically in tumours and not normal tissues; indicating that in malignant cells, C5aR1 primarily regulates cell fate. Collectively, these data reveal that increased complement gene expression is part of the stress response mounted by irradiated tumours and that targeting C5aR1 can improve radiotherapy even in tumours displaying immunosuppressive features.

Intestinal homeostasis is underpinned by LGR5+ve crypt-base columnar stem cells (CBCs), but following injury, dedifferentiation results in the emergence of LGR5-ve regenerative stem cell populations (RSCs), characterized by fetal transcriptional profiles. Neoplasia hijacks regenerative signaling, so we assessed the distribution of CBCs and RSCs in mouse and human intestinal tumors. Using combined molecular-morphological analysis, we demonstrate variable expression of stem cell markers across a range of lesions. The degree of CBC-RSC admixture was associated with both epithelial mutation and microenvironmental signaling disruption and could be mapped across disease molecular subtypes. The CBC-RSC equilibrium was adaptive, with a dynamic response to acute selective pressure, and adaptability was associated with chemoresistance. We propose a fitness landscape model where individual tumors have equilibrated stem cell population distributions along a CBC-RSC phenotypic axis. Cellular plasticity is represented by position shift along this axis and is influenced by cell-intrinsic, extrinsic, and therapeutic selective pressures. ispartof: CELL STEM CELL vol:29 issue:8 pages:1213-+ ispartof: location:United States status: published

WNT/β-catenin signaling endows cancer cells with proliferative capacity and immune-evasive functions that impair anti-cancer immunosurveillance by conventional, cytoxtoic T cells. However, the impact of dysregulated WNT signalling on unconventional, tissue-resident T cells, specifically in colon cancer is unknown. Here, we show that cancer cells in Apc-mutant mouse models escape immunosurveillance from gut-resident intraepithelial lymphocytes (IELs) expressing γδ T cell receptors (γδTCRs). Analysis of late-stage tumors from mice and humans revealed that γδIELs are largely absent from the tumor microenvironment, and that butyrophilin-like (BTNL) molecules, which can critically regulate γδIEL through direct γδTCR-interactions, are also downregulated. We could attribute this to β-catenin stabilization, which rapidly decreased expression of the transcription factors, HNF4A and HNF4G, that we found to bind promoter regions of Btnl genes, thereby driving their expression in normal gut epithelial cells. Indeed, inhibition of β-catenin signaling restored Btnl1 gene expression and γδ T cell infiltration into tumors. These observations highlight an immune-evasion mechanism specific to WNT-driven colon cancer cells that disrupts γδIEL immunosurveillance and furthers cancer progression.

Intestinal homeostasis is underpinned by LGR5+ve crypt-base columnar stem cells (CBCs), but following injury, dedifferentiation results in the emergence of LGR5-ve regenerative stem cell populations (RSCs), characterised by fetal transcriptional profiles. Neoplasia hijacks regenerative signalling, so we assessed the distribution of CBCs and RSCs in mouse and human intestinal tumors. Using combined molecular-morphological analysis we demonstrate variable expression of stem cell markers across a range of lesions. The degree of CBC-RSC admixture was associated with both epithelial mutation and microenvironmental signalling disruption, and could be mapped across disease molecular subtypes. The CBC-RSC equilibrium was adaptive, with a dynamic response to acute selective pressure, and adaptability was associated with chemoresistance. We propose a fitness landscape model where individual tumors have equilibrated stem cell population distributions along a CBC-RSC phenotypic axis. Cellular plasticity is represented by position shift along this axis, and is influenced by cell-intrinsic, extrinsic and therapeutic selective pressures.

Modelling human colon cancer has long been the ambition of researchers and oncologists with the aim to better replicate disease progression and treatment response. Advances in our understanding of genetics, stem cell biology, tumour microenvironment and immunology have prepared the groundwork for recent major advances. In the last two years the field has seen the progression of: using patient derived organoids (alone and in co-culture) as predictors of treatment response; molecular stratification of tumours that predict outcome and treatment response; mouse models of metastatic disease; and transplant models that can be used to de-risk clinical trials. We will discuss these advances in this review.

Gattaca provides the first base-pair resolution artificial genomes for tracking somatic mutations within agent based modeling. Through the incorporation of human reference genomes, mutational context, sequence coverage/error information Gattaca is able to realistically provide comparable sequence data for in-silico comparative evolution studies with human somatic evolution studies. This user-friendly method, incorporated into each in-silico cell, allows us to fully capture somatic mutation spectra and evolution.

Spatial analyses can reveal important interactions between and among cells and their microenvironment. However, most existing staining methods are limited to a handful of markers per slice, thereby limiting the number of interactions that can be studied. This limitation is frequently overcome by registering multiple images to create a single composite image containing many markers. While there are several existing image registration methods for whole slide images (WSI), most have specific use cases. Here, we present the Virtual Alignment of pathoLogy Image Series (VALIS), a fully automated pipeline that opens, registers (rigid and/or non-rigid), and saves aligned slides in the ome.tiff format. VALIS has been tested with 273 immunohistochemistry (IHC) samples and 340 immunofluorescence (IF) samples, each of which contained between 2-69 images per sample. The registered WSI tend to have low error and are completed within a matter of minutes. In addition to registering slides, VALIS can also using the registration parameters to warp point data, such as cell centroids previously determined via cell segmentation and phenotyping. VALIS is written in Python and requires only few lines of code for execution. VALIS therefore provides a free, opensource, flexible, and simple pipeline for rigid and non-rigid registration of IF and/or IHC that can facilitate spatial analyses of WSI from novel and existing datasets.

The intestinal epithelium is a tissue with high cell turnover, supported by adult intestinal stem cells. Intestinal homeostasis is underpinned by crypt basal columnar stem cells, marked by expression of the LGR5 gene. However, recent research has demonstrated considerable stem cell plasticity following injury, with dedifferentiation of a range of other intestinal cell populations, induced by a permissive microenvironment in the regenerating mucosa. The regulation of this profound adaptive cell reprogramming response is the subject of current research. There is a demonstrable contribution from disruption of key homeostatic signaling pathways such as wingless-related integration site and bone morphogenetic protein, and an emerging signaling hub role for the mechanoreceptor transducers Yes-associated protein 1/transcriptional coactivator with PDZ-binding motif, negatively regulated by the Hippo pathway. However, a number of outstanding questions remain, including a need to understand how tissues sense damage, and how pathways intersect to mediate dynamic changes in the stem cell population. Better understanding of these pathways, associated functional redundancies, and how they may be both enhanced for recovery of inflammatory diseases, and co-opted in neoplasia development, may have significant clinical implications, and could lead to development of more targeted molecular therapies which target individual stem or stem-like cell populations.

The evolutionary dynamics of tumor initiation remain undetermined, and the interplay between neoplastic cells and the immune system is hypothesized to be critical in transformation. Colorectal cancer (CRC) presents a unique opportunity to study the transition to malignancy as pre-cancers (adenomas) and early stage cancers are frequently detected and surgically removed. Here, we demonstrate a key role for the immune response in tumor initiation by studying tumor-immune eco-evolutionary dynamics from pre-cancer to carcinoma using a computational model, ecological analysis of digital pathology data, and multi-region exome sequencing and neoantigen prediction in a total of 62 patient samples. Modelling indicates there are several potential routes to malignancy, each of which uniquely sculpts tumor ecology and intra-tumor antigenic heterogeneity (aITH). In patient samples, the immune microenvironment was characterized using the spatial distribution of 17 markers across registered whole-slide images, and patterns of intra-lesion aITH measured using multi-region exome sequencing and neoantigen prediction. The patient data were best described by a model whereby immunogenic adenomas do not progress to CRC because they are under immune control, and progression initially proceeds in adenomas with low immunogenicity followed by the gradual construction of an immunosuppressive niche depleted in CD8+ cytotoxic T cells. There was little evidence for immune blockade (PD-L1 expression) in tumor initiation or progression. These results suggest that re-engineering the immunosuppressive niche may prove to be an effective immunotherapy in CRC.

Generation of transcriptional data has dramatically increased in the last decade, driving the development of analytical algorithms that enable interrogation of the biology underpinning the profiled samples. However, these resources require users to have expertise in data wrangling and analytics, reducing opportunities for biological discovery by “wet-lab” users with a limited programming skillset. Although commercial solutions exist, costs for software access can be prohibitive for academic research groups.To address these challenges, we have developed an open source and user-friendly data analysis platform for on-the-fly bioinformatic interrogation of transcriptional data derived from human or mouse tissue, called “MouSR”. This internet-accessible analytical tool, https://mousr.qub.ac.uk/, enables users to easily interrogate their data using an intuitive “point and click” interface, which includes a suite of molecular characterisation options including QC, differential gene expression, gene set enrichment and microenvironmental cell population analyses from RNA-Seq. Users are provided with adjustable options for analysis parameters to generate results that can be saved as publication-quality images. To highlight its ability to perform high quality data analysis, we utilise the MouSR tool to interrogate our recently published tumour dataset, derived from genetically engineered mouse models and matched organoids, where we rapidly reproduced the key transcriptional findings.The MouSR online tool provides a unique freely-available option for users to perform rapid transcriptomic analyses and comprehensive interrogation of the signalling underpinning transcriptional datasets, which alleviates a major bottleneck for biological discovery.

Background & Aims Barrett’s epithelium measurement using widely accepted Prague C&M classification is highly operator dependent. We propose a novel methodology for measuring this risk score automatically. The method also enables quantification of the area of Barrett’s epithelium (BEA) and islands, which was not possible before. Furthermore, it allows 3-dimensional (3D) reconstruction of the esophageal surface, enabling interactive 3D visualization. We aimed to assess the accuracy of the proposed artificial intelligence system on both phantom and endoscopic patient data. Methods Using advanced deep learning, a depth estimator network is used to predict endoscope camera distance from the gastric folds. By segmenting BEA and gastroesophageal junction and projecting them to the estimated mm distances, we measure C&M scores including the BEA. The derived endoscopy artificial intelligence system was tested on a purpose-built 3D printed esophagus phantom with varying BEAs and on 194 high-definition videos from 131 patients with C&M values scored by expert endoscopists. Results Endoscopic phantom video data demonstrated a 97.2% accuracy with a marginal ± 0.9 mm average deviation for C&M and island measurements, while for BEA we achieved 98.4% accuracy with only ±0.4 cm2 average deviation compared with ground-truth. On patient data, the C&M measurements provided by our system concurred with expert scores with marginal overall relative error (mean difference) of 8% (3.6 mm) and 7% (2.8 mm) for C and M scores, respectively. Conclusions The proposed methodology automatically extracts Prague C&M scores with high accuracy. Quantification and 3D reconstruction of the entire Barrett’s area provides new opportunities for risk stratification and assessment of therapy response.

Funding Information: Acknowledgements We thank the Core Services and Advanced Technologies at the Cancer Research UK Beatson Institute (C596/A17196 and A31287), and particularly the Biological Services Unit, Histology Service and Molecular Technologies; members of the Sansom and Katajisto laboratories for discussions of the data and manuscript; and BRC Oxford for supplying patient material. O.J.S. and his laboratory members were supported by Cancer Research UK (A28223, A21139, A12481 and A17196). D.J.F. and M.C.H. were supported by the UK Medical Research Council (MR/R017247/1 and MR/J50032X/1, respectively). SpecifiCancer CRUK Grand Challenge (C7932/A29055) is funded by Cancer Research UK and the Mark Foundation for Cancer Research. P.K. and his laboratory members were supported by the Academy of Finland Centre of Excellence MetaStem (266869, 304591 and 320185), the ERC Starting Grant 677809, the Swedish Research Council 2018-03078, the Cancerfonden 190634, the Jane and Aatos Erkko Foundation and the Cancer Foundation Finland. N.P. was supported by the Finnish Cultural Foundation, the Biomedicum Helsinki Foundation, the Orion Research Foundation sr and The Paulo Foundation. P.V.F. was supported by Alzheimer’s Research UK and The Francis Crick Institute. The ARUK UCL Drug Discovery Institute receives its core funding from Alzheimer’s Research UK (520909). The Francis Crick Institute receives its core funding from Cancer Research UK (FC001002), the UK Medical Research Council (FC001002) and the Wellcome Trust (FC001002). Publisher Copyright: © 2021, The Author(s), under exclusive licence to Springer Nature Limited. The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling(1), but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation)(2). Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.

Aims: After local excision of early rectal cancer, definitive lymph node status is not available. An alternative means for accurate assessment of recurrence risk is required to determine the most appropriate subsequent management. Currently used measures are suboptimal. We assess three measures of tumour stromal content to determine their predictive value after local excision in a well-characterised cohort of rectal cancer patients without prior radiotherapy. Methods and results: A total of 143 patients were included. Haematoxylin and eosin (H&E) sections were scanned for (i) deep neural network (DNN, a machine-learning algorithm) tumour segmentation into compartments including desmoplastic stroma and inflamed stroma; and (ii) digital assessment of tumour stromal fraction (TSR) and optical DNA ploidy analysis. 3′ mRNA sequencing was performed to obtain gene expression data from which stromal and immune scores were calculated using the ESTIMATE method. Full results were available for 139 samples and compared with disease-free survival. All three methods were prognostic. Most strongly predictive was a DNN-determined ratio of desmoplastic to inflamed stroma >5.41 (P < 0.0001). A ratio of ESTIMATE stromal to immune score 36.5% (P = 0.037). Conclusions: The DNN-determined ratio of desmoplastic to inflamed ratio is a novel and powerful predictor of disease recurrence in locally excised early rectal cancer. It can be assessed on a single H&E section, so could be applied in routine clinical practice to improve the prognostic information available to patients and clinicians to inform the decision concerning further management.

Molecular clocks that record cell ancestry mutate too slowly to measure the short-timescale dynamics of cell renewal in adult tissues. Here, we show that fluctuating DNA methylation marks can be used as clocks in cells where ongoing methylation and demethylation cause repeated 'flip-flops' between methylated and unmethylated states. We identify endogenous fluctuating CpG (fCpG) sites using standard methylation arrays and develop a mathematical model to quantitatively measure human adult stem cell dynamics from these data. Small intestinal crypts were inferred to contain slightly more stem cells than the colon, with slower stem cell replacement in the small intestine. Germline APC mutation increased the number of replacements per crypt. In blood, we measured rapid expansion of acute leukemia and slower growth of chronic disease. Thus, the patterns of human somatic cell birth and death are measurable with fluctuating methylation clocks (FMCs).

Molecular clocks record cellular ancestry. However, currently used clocks ‘tick too slowly’ to measure the short-timescale dynamics of cellular renewal in adult tissues. Here we develop ‘rapidly oscillating DNA methylation clocks’ where ongoing (de)methylation causes the clock to ‘tick-tock’ back-and-forth between methylated and unmethylated states like a pendulum. We identify oscillators using standard methylation arrays and develop a mathematical modelling framework to quantitatively measure human adult stem cell dynamics from these data. Small intestinal crypts were inferred to contain slightly more stem cells than colon (6.5 ± 1.0 vs 5.8 ± 1.7 stem cells/crypt) with slower stem cell replacement in small intestine (0.79 ± 0.5 vs 1.1 ± 0.8 replacements/stem cell/year). Germline APC mutation increased the number of replacements per crypt (13.0 ± 2.4 replacements/crypt/year vs 6.9 ± 4.6 for healthy colon). In blood, we measure rapid expansion of acute leukaemia and slower growth of chronic disease. Rapidly oscillating molecular clocks are a new methodology to quantitatively measure human somatic cell dynamics.

Background & Aims: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. Methods: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting–purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. Results: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor β was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB–IL34/CCL8 signaling that promotes macrophage chemotaxis. Conclusions: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC.

In the intestine, the homeostatic effect of Bone Morphogenetic Protein (BMP) on cell fate has predominantly been inferred through pathway inactivation. Here, we assessed the impact of autocrine Bmp4 expression on secretory cell fate. Ligand exposure reduced proliferation, expedited terminal differentiation, abrogated long-term secretory cell survival and prevented dedifferentiation. As stem cell plasticity is required for regenerative adaptive reprogramming, we spatiotemporally mapped and functionally explored BMP’s role in epithelial restitution. Following ulceration, physiological attenuation of BMP signalling arose through upregulation of the secreted antagonist, Grem1, from topographically distinct populations of stromal cells. Concomitant expression supported functional compensation, following Grem1 deletion from tissue-resident fibroblasts. BMP pathway manipulation showed that antagonist-mediated BMP attenuation was obligatory, but functionally sub-maximal, as regeneration was impaired or enhanced by epithelial overexpression of Bmp4 or Grem1 respectively. Mechanistically, Bmp4 abrogated regenerative stem cell reprogramming, despite a convergent impact of YAP/TAZ on cell fate in remodelled wounds.

Simple Summary Colorectal cancer is the third most common cause of cancer-related deaths. The Wnt signaling pathway is activated by genetic mutations in most patients with colorectal cancer. A number of different types of Wnt pathway mutation have been described: some increase the sensitivity of tumor cells to Wnt ligands produced by stromal cells (ligand-dependent), while others drive downstream activation of the pathway (ligand-independent). Ligand-dependent tumors are of particular interest as there are a number of emerging treatment options, such as porcupine inhibitors, that can specifically target these tumors. In this review, we discuss what is known about these different types of Wnt activating mutations. We propose that ligand-dependent tumors should be viewed as a separate subset of colorectal cancer with its own biomarkers, prognosis and targeted therapies. Abstract Wnt signaling is ubiquitously activated in colorectal tumors and driver mutations are identified in genes such as APC, CTNNB1, RNF43 and R-spondin (RSPO2/3). Adenomatous polyposis coli (APC) and CTNNB1 mutations lead to downstream constitutive activation (ligand-independent), while RNF43 and RSPO mutations require exogenous Wnt ligand to activate signaling (ligand-dependent). Here, we present evidence that these mutations are not equivalent and that ligand-dependent and ligand-independent tumors differ in terms of underlying Wnt biology, molecular pathogenesis, morphology and prognosis. These non-overlapping characteristics can be harnessed to develop biomarkers and targeted treatments for ligand-dependent tumors, including porcupine inhibitors, anti-RSPO3 antibodies and asparaginase. There is emerging evidence that these therapies may synergize with immunotherapy in ligand-dependent tumors. In summary, we propose that ligand-dependent tumors are an underappreciated separate disease entity in colorectal cancer.

BACKGROUND & AIMSBarrett’s epithelium measurement using widely accepted Prague C&M criteria is highly operator dependent. By reconstructing the surface of the Barrett’s area in 3D from endoscopy video, we propose a novel methodology for measuring the C&M score automatically. This 3D reconstruction provides an extended field of view and also allows to precisely quantify the Barrett’s area including islands. We aim to assess the accuracy of the extracted measurements from phantom and demonstrate their clinical usability.METHODSAdvanced deep learning techniques are utilised to design estimators for depth and camera pose required to map standard endoscopy video to a 3D surface model. By segmenting the Barrett’s area and locating the position of the gastro-oesophageal junction (GEJ) we measure C&M scores and the Barrett’s oesophagus areas (BOA). Experiments using a purpose-built 3D printed oesophagus phantom and high-definition video from 98 patients scored by an expert endoscopist are used for validation.RESULTSEndoscopic phantom video data demonstrated a 95 % accuracy with a marginal ± 1.8 mm average deviation for C&M and island measurements, while for BOA we achieved nearly 93 % accuracy with only ± 1.1 cm2 average deviation compared to the ground-truth measurements. On patient data, the C&M measurements provided by our system concord with the reference provided by expert upper GI endoscopists.CONCLUSIONSThe proposed methodology is suitable for extracting Prague C&M scores automatically with a high degree of accuracy. Providing an accurate measurement of the entire Barrett’s area provides new opportunities for risk stratification and the assessment of therapy response.

Ablation of LGR5(+) intestinal stem cells (ISCs) is associated with rapid restoration of the ISC compartment. Different intestinal crypt populations dedifferentiate to provide new ISCs, but the transcriptional and signaling trajectories that guide this process are unclear, and a large body of work suggests that quiescent ‘reserve’ ISCs contribute toward regeneration. By timing the interval between LGR5(+) lineage tracing and lethal injury, we show that ISC regeneration is explained nearly completely by dedifferentiation, with contributions from absorptive and secretory progenitors. The ISC-restricted transcription factor ASCL2 confers measurable competitive advantage to resting ISCs and is essential to restore the ISC compartment. Regenerating cells re-express Ascl2 days before Lgr5, and scRNAseq analyses reveal transcriptional paths underlying dedifferentiation. ASCL2 target genes include the Interleukin11 (IL11) receptor Il11ra1, and recombinant IL11 enhances crypt cell regenerative potential. These findings reveal cell dedifferentiation as the principal means for ISC restoration and highlight an ASCL2-regulated signal that enables this adaptive response.

Objective Complex phenotypes captured on histological slides represent the biological processes at play in individual cancers, but the link to underlying molecular classification has not been clarified or systematised. In colorectal cancer (CRC), histological grading is a poor predictor of disease progression, and consensus molecular subtypes (CMSs) cannot be distinguished without gene expression profiling. We hypothesise that image analysis is a cost-effective tool to associate complex features of tissue organisation with molecular and outcome data and to resolve unclassifiable or heterogeneous cases. In this study, we present an image-based approach to predict CRC CMS from standard H&E sections using deep learning. Design Training and evaluation of a neural network were performed using a total of n=1206 tissue sections with comprehensive multi-omic data from three independent datasets (training on FOCUS trial, n=278 patients; test on rectal cancer biopsies, GRAMPIAN cohort, n=144 patients; and The Cancer Genome Atlas (TCGA), n=430 patients). Ground truth CMS calls were ascertained by matching random forest and single sample predictions from CMS classifier. Results Image-based CMS (imCMS) accurately classified slides in unseen datasets from TCGA (n=431 slides, AUC)=0.84) and rectal cancer biopsies (n=265 slides, AUC=0.85). imCMS spatially resolved intratumoural heterogeneity and provided secondary calls correlating with bioinformatic prediction from molecular data. imCMS classified samples previously unclassifiable by RNA expression profiling, reproduced the expected correlations with genomic and epigenetic alterations and showed similar prognostic associations as transcriptomic CMS. Conclusion This study shows that a prediction of RNA expression classifiers can be made from H&E images, opening the door to simple, cheap and reliable biological stratification within routine workflows.

Background & Aims Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored. Methods Using human CRC RNA expression data, we identified CAF-specific factors involved in BMP signaling, then verified and characterized their expression in the CRC stroma by in situ hybridization. CRC tumoroids and a mouse model of CRC hepatic metastasis were used to test approaches to modify BMP signaling and treat CRC. Results We identified Grem1 and Islr as CAF-specific genes involved in BMP signaling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signaling, respectively. Grem1 and Islr marked distinct fibroblast subpopulations and were differentially regulated by transforming growth factor β and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In patients with CRC, high GREM1 and ISLR expression levels were associated with poor and favorable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5+ intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)–mediated delivery of Islr to hepatocytes increased BMP signaling and improved survival in our mouse model of hepatic metastasis. Conclusions Stromal BMP signaling predicts and modifies CRC progression and survival, and it can be therapeutically targeted by novel AAV-directed gene delivery to the liver.

Aneuploidy, defined as the loss and gain of whole and part chromosomes, is a near-ubiquitous feature of cancer genomes, is prognostic, and likely an important determinant of cancer cell biology. In colorectal cancer (CRC), aneuploidy is found in virtually all tumours, including precursor adenomas. However, the temporal evolutionary dynamics that select for aneuploidy remain broadly uncharacterised. Here we perform genomic analysis of 755 samples from a total of 167 patients with colorectal-derived neoplastic lesions that cross-sectionally represent the distinct stages of tumour evolution, and longitudinally track individual tumours through metastasis and treatment. Precancer lesions (adenomas) exhibited low levels of aneuploidy but high intra-tumour heterogeneity, whereas cancers had high aneuploidy but low heterogeneity, indicating that progression is through a genetic bottleneck that suppresses diversity. Individual CRC glands from the same tumour have similar karyotypes, despite prior evidence of ongoing instability at the cell level. Pseudo-stable aneuploid genomes were observed in metastatic lesions sampled from liver and other organs, after chemo- or targeted therapies, and late recurrences detected many years after the diagnosis of a primary tumour. Modelling indicates that these data are consistent with the action of stabilising selection that ‘traps’ cancer cell genomes on a fitness peak defined by the specific pattern of aneuploidy. These data show that the initial progression of CRC requires the traversal of a rugged fitness landscape and subsequent genomic evolution, including metastatic dissemination and therapeutic resistance, is constrained by stabilising selection.

BACKGROUND & AIMS: In homeostasis, intestinal cell fate is controlled by balanced gradients of morphogen signaling. The bone morphogenetic protein (BMP) pathway has a physiological, prodifferentiation role, predominantly inferred through previous experimental pathway inactivation. Intestinal regeneration is underpinned by dedifferentiation and cell plasticity, but the signaling pathways that regulate this adaptive reprogramming are not well understood. We assessed the BMP signaling landscape and investigated the impact and therapeutic potential of pathway manipulation in homeostasis and regeneration. [Figure: see text] METHODS: A novel mouse model was generated to assess the effect of the autocrine Bmp4 ligand on individual secretory cell fate. We spatio-temporally mapped BMP signaling in mouse and human regenerating intestine. Transgenic models were used to explore the functional impact of pathway manipulation on stem cell fate and intestinal regeneration. RESULTS: In homeostasis, ligand exposure reduced proliferation, expedited terminal differentiation, abrogated secretory cell survival, and prevented dedifferentiation. After ulceration, physiological attenuation of BMP signaling arose through upregulation of the secreted antagonist Grem1 from topographically distinct populations of fibroblasts. Concomitant expression supported functional compensation after Grem1 deletion from tissue-resident cells. BMP pathway manipulation showed that antagonist-mediated BMP attenuation was obligatory but functionally submaximal, because regeneration was impaired or enhanced by epithelial overexpression of Bmp4 or Grem1, respectively. Mechanistically, Bmp4 abrogated regenerative stem cell reprogramming despite a convergent impact of YAP/TAZ on cell fate in remodeled wounds. CONCLUSIONS: BMP signaling prevents epithelial dedifferentiation, and pathway attenuation through stromal Grem1 upregulation was required for adaptive reprogramming in intestinal regeneration. This intercompartmental antagonism was functionally submaximal, raising the possibility of therapeutic pathway manipulation in inflammatory bowel disease.

The raw data is available on Mendeley at https://data.mendeley.com/datasets/hfpbctm7tg/draft?a=1c91e494-cadc-4be0-a8ff-91d8736a28e7 © The Author(s) 2019. Expression of the mismatch repair gene MutL homolog 1 (MLH1) is silenced in a clinically important subgroup of sporadic colorectal cancers. These cancers exhibit hypermutability with microsatellite instability (MSI) and differ from microsatellite-stable (MSS) colorectal cancers in both prognosis and response to therapies. Loss of MLH1 is usually due to epigenetic silencing with associated promoter methylation; coding somatic mutations rarely occur. Here we use the presence of a colorectal cancer (CRC) risk variant (rs1800734) within the MLH1 promoter to investigate the poorly understood mechanisms of MLH1 promoter methylation and loss of expression. We confirm the association of rs1800734 with MSI+ but not MSS cancer risk in our own data and by meta-analysis. Using sensitive allele-specific detection methods, we demonstrate that MLH1 is the target gene for rs1800734 mediated cancer risk. In normal colon tissue, small allele-specific differences exist only in MLH1 promoter methylation, but not gene expression. In contrast, allele-specific differences in both MLH1 methylation and expression are present in MSI+ cancers. We show that MLH1 transcriptional repression is dependent on DNA methylation and can be reversed by a methylation inhibitor. The rs1800734 allele influences the rate of methylation loss and amount of re-expression. The transcription factor TFAP4 binds to the rs1800734 region but with much weaker binding to the risk than the protective allele. TFAP4 binding is absent on both alleles when promoter methylation is present. Thus we propose that TFAP4 binding shields the protective rs1800734 allele of the MLH1 promoter from BRAF induced DNA methylation more effectively than the risk allele. Funding was provided by a Medical Research Council New Investigator Research Grant (MR/P000738/1). Core funding to the Wellcome Trust Centre for Human Genetics was provided by the Wellcome Trust (090532/Z/09/Z). D.T., N.S. and D.F. were supported by the EPIGENOMICS FLAGSHIP PROJECT- EPIGEN (project number 08934412) and by University of Insubria. M.T. and S.K. were funded by Ludwig Cancer Research.

Objective Complex phenotypes captured on histological slides represent the biological processes at play in individual cancers, but the link to underlying molecular classification has not been clarified or systematised. In colorectal cancer (CRC), histological grading is a poor predictor of disease progression, and consensus molecular subtypes (CMSs) cannot be distinguished without gene expression profiling. We hypothesise that image analysis is a cost-effective tool to associate complex features of tissue organisation with molecular and outcome data and to resolve unclassifiable or heterogeneous cases. In this study, we present an image-based approach to predict CRC CMS from standard H&E sections using deep learning. Design Training and evaluation of a neural network were performed using a total of n=1206 tissue sections with comprehensive multi-omic data from three independent datasets (training on FOCUS trial, n=278 patients; test on rectal cancer biopsies, GRAMPIAN cohort, n=144 patients; and The Cancer Genome Atlas (TCGA), n=430 patients). Ground truth CMS calls were ascertained by matching random forest and single sample predictions from CMS classifier. Results Image-based CMS (imCMS) accurately classified slides in unseen datasets from TCGA (n=431 slides, AUC)=0.84) and rectal cancer biopsies (n=265 slides, AUC=0.85). imCMS spatially resolved intratumoural heterogeneity and provided secondary calls correlating with bioinformatic prediction from molecular data. imCMS classified samples previously unclassifiable by RNA expression profiling, reproduced the expected correlations with genomic and epigenetic alterations and showed similar prognostic associations as transcriptomic CMS. Conclusion This study shows that a prediction of RNA expression classifiers can be made from H&E images, opening the door to simple, cheap and reliable biological stratification within routine workflows.

Colorectal cancer develops from its precursor lesion, the adenoma. The immune system is hypothesized to be key in modulating progression, but tumor-immune eco-evolutionary dynamics remain uncharacterized. Here, we demonstrate a key role for immune evasion in the progression of human benign disease to colorectal cancer. We constructed a mathematical model of tumor-immune eco-evolutionary dynamics that predicted ecological succession, from an "immune-hot" adenoma immune ecology rich in T cells to an "immune-cold" carcinoma ecology, deficient in T cells and rich in immunosuppressive cells. Using a cross-sectional cohort of adenomas and carcinomas, we validated this prediction by direct measurement of the tumor-immune ecology using whole-slide 10-marker immunohistochemistry (IHC), and analysis of neoantigen clonal architecture multi-region exome sequencing data. Changes in immune ecology relax selection against antigens with high recognition potentials. This study indicates that immune surveillance represents a key evolutionary bottleneck in the evolution of colon cancer.

The skin is the largest human organ, functioning to serve as the protective barrier to the harsh, outside world. Recent studies have revealed that large numbers of somatic mutations accumulate, which can be used to infer normal human skin cell dynamics1-6. Here we present the first realistic mechanistic epidermal model, that uses the ‘Gattaca’ method to incorporate cell-genomes, that shows homeostasis imposes a characteristic log-linear subclone size distribution for both neutral and driver mutations, where the largest skin subclones are the oldest subclones. Because homeostasis inherently limits proliferation and therefore clonal sweeps, selection for driver mutations (NOTCH1 and TP53) in normal epidermis is instead conferred by greater persistence, which leads to larger subclone sizes. These results reveal how driver mutations may persist and expand in normal epidermis while highlighting how the integration of mechanistic modeling with genomic data provides novel insights into evolutionary cell dynamics of normal human homeostatic tissues.

The adenoma-carcinoma sequence describes the development of colorectal carcinoma (CRC) from benign colorectal precursor lesions. Molecular classification of established CRC has demonstrated considerable disease heterogeneity; however, as an emerging cancer frequently outgrows and destroys the initial precursor lesion, CRC molecular taxonomy can only be partially reconciled with histologically classified polyps. Thus, the molecular pathogenesis of some colorectal polyp types, including the traditional serrated adenoma (TSA), is still unclear. Now, candidate driver gene analysis of a cohort of different polyps reveals characteristic, but highly variable, mutations disrupting the Wnt signalling pathway across different histological polyp subtypes. How and when different precursor lesions acquire Wnt disruption reflects important distinctions in polyp biology, dependent on a combination of the dominant molecular pathway and the cell of origin of individual lesions. TSAs preferentially acquire ligand-dependent Wnt activating mutations, which means that the cancers that arise from these aggressive polyps may be sensitive to targeted Wnt inhibition. This paper demonstrates that applying next-generation sequencing technology to improve our understanding of colorectal precursor lesion molecular pathogenesis could also give important and translationally relevant insights into colorectal cancer biology. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Aneuploidy, defined as the loss and gain of whole and part chromosomes, is a near-ubiquitous feature of cancer genomes, and has clinical relevance as a prognostic marker and potential therapeutic target. We have previously shown that in colorectal cancer (CRC), aneusomies largely occur upon the transition from benign adenoma to invasive carcinoma (Cross et al., 2018), however the temporal evolutionary dynamics that select for aneuploidy throughout CRC progression remain broadly uncharacterised. We performed multi-region genomic analysis of 755 samples from a total of 167 patients with colorectal-derived neoplastic lesions that cross-sectionally represent the distinct stages of CRC progression, and longitudinally tracked 22 individual CRC tumours through metastasis and treatment. Genetic diversity measures revealed that although adenomas exhibited low levels of aneuploidy (8.5% genome altered), they harboured high intra-tumour heterogeneity (ITH), whereas cancers had significantly higher aneuploidy (27.2% genome altered) but proportionally lower ITH (p=0.0017), indicating that progression to malignancy is through a genetic bottleneck that suppresses diversity. We generated genome-wide copy number profiles from individual CRC glands, and showed that glands from the same tumour had similar karyotypes, yet displayed ongoing instability at the single cell level. Remarkably, we found that aneuploid genomes were broadly stable in metastatic lesions, even after chemo- or targeted therapies, and in recurrences detected many years after the primary diagnosis. Computational modelling of the dynamics of aneusomy accrual during CRC progression to metastasis was consistent with stabilising selection acting to ‘trap’ cancer cell genomes on a fitness peak defined by the optimal patterns of aneuploidy. These data show that although a genetic bottleneck underlies the initial progression from adenoma to CRC, further evolution of CRC is constrained largely by stabilising selection, even through metastatic dissemination and therapeutic resistance. References Cross, W., Kovac, M., Mustonen, V., Temko, D., Davis, H., Baker, A.-M., … Tomlinson, I. P. M. (2018). The evolutionary landscape of colorectal tumorigenesis. Nature Ecology & Evolution, 2(10), 1661–1672. https://doi.org/10.1038/s41559-018-0642-z Citation Format: Salpie Nowinski, William Cross, Maximilian Mossner, George Cresswell, Abhirup Banerjee, Marc Williams, Laura Gay, Ann-Marie Baker, Binxgin Lu, Christopher Kimberley, Hayley Davis, Nik Matthews, Miangela M. Lacle, Ian P.M. Tomlinson, Marnix Jansen, Alan Watson, Chris Barnes, Nicholas A. Wright, John Bridgewater, Manuel Rodriguez-Justo, Hemant Kocher, Simon J. Leedham, Andrea Sottoriva, Trevor A. Graham. Stabilising selection causes grossly altered but stable karyotypes in metastatic colorectal cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PR06.

Background: Molecular subtyping of colon cancer (CC) has repeatedly identified a poor-prognostic group of patients, characterized by high levels of stroma (particularly fibroblast) in the tumor microenvironment. To date, the benefit of standard 5FU-based adjuvant chemotherapy in these high-fibroblast (HiFi) patients has been unclear. Given TGF-β signaling is associated with HiFi tumors, a number of recent clinical trials have focussed on targeting TGF-β in these patients. While these efforts are ongoing, we set out to identify novel therapeutically-relevant biological signaling within HiFi tumors. Methods: Untreated stage II (n=215) and stage II/III (n=258) tumors were assigned a fibroblast score, using single-sample gene set enrichment analysis, enabling stratification into HiFi and LoFi groups based on their histology and transcriptome. Supervised stratification, based on relapse-free survival, within the HiFi group allowed for in silico discovery, interrogation and independent validation of the HiFi-specific biology underpinning relapse. Upstream regulators of these processes were identified as potential therapeutic targets, and assessed in an in vitro co-culture model, to confirm mechanistic signaling, and an in vivo HiFi model, to confirm efficacy. Results: We confirmed the poor prognosis of the HiFi group (p = 0.008), followed by discovery and independent validation of the prognostic value of STAT1-related signaling in stratifying HiFi tumors based on disease relapse (HR 0.2 (0.1-0.5) and 0.09 (0.02-0.47)). This signaling was significantly associated with activation of antigen processing and presentation in specific immune lineages (p < 0.001). In line with the upstream regulator analysis, treatment with poly I:C (a TLR3 agonist) increased STAT1-related signaling and antigen processing in an in vitro macrophage-stromal co-culture system. Conclusions: We have found that increased levels of STAT1-related signaling, resulting in antigen processing and presentation in specific subclasses of immune cells, is associated with reduced risk of recurrence in the otherwise poor-prognostic HiFi subtype of CC. Using in silico and in vitro methods, we demonstrate that poly I:C is a potential therapeutic option for patients with stromal-rich tumors. Results from ongoing in vivo validation in a HiFi mouse model will provide preclinical evidence of the utility of poly I:C in this setting and support a phase II clinical trial. Citation Format: Amy M. McCorry, Niamh A. Leonard, Rene Jackstadt, Dustin J. Flanagan, Owen J. Sansom, Tim Maughan, Simon Leedham, Emma M. Kerr, Aideen E. Ryan, Mark Lawler, Philip D. Dunne, ACRCelerate and S:CORT consortia. STAT1-related antigen processing and presentation dictates prognosis in the fibroblast-rich subtype of stage II/III colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3867.

Cancer-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment and play a critical part in cancer progression. However, the precise origin of the tumor stroma remains unknown, making it challenging to effectively target the cancer mesenchyme. Here, employing 4 different genetic fate mapping mouse models and a bone marrow transplantation model in combination with BrdU labeling, we uncovered a key contributor to the tumor stroma in colorectal cancer (CRC). We found that approximately half of a-smooth muscle actin (aSMA)+ CAFs emerge through proliferation in an AOM/DSS mouse model of CRC. Lineage tracing experiments revealed that intestinal leptin receptor (Lepr)-lineage stromal cells expanded and contributed to 75% of the aSMA+ proliferating CAFs. Notably, no aSMA+ CAFs in the tumor were derived from Krt19-lineage epithelial cells or bone marrow-transplanted cells, indicating no involvement of epithelial-mesenchymal transition and bone marrow recruitment to the tumor in this model. Moreover, RNA-sequencing of FACS-purified CRC mesenchymal cells identified MCAM (also known as CD146) as a CRC stroma-specific marker, which is expressed by Lepr-lineage cells. Analysis of human CRC samples showed that high MCAM expression was associated with a mesenchymal subtype of CRC and was independently prognostic of poor overall survival. Our data identify Lepr-lineage cells as a major source of the tumor stroma in CRC and suggest that targeting MCAM+ cells may serve as a novel therapeutic approach to restrain CRC progression. Citation Format: Hiroki Kobayashi, Krystyna A. Gieniec, Tamsin RM Lannagan, Tongtong Wang, Samuel Asfaha, Yoku Hayakawa, Simon J. Leedham, Nicholas Arpaia, Siddhartha Mukherjee, Timothy C. Wang, Atsushi Enomoto, Masahide Takahashi, Susan L. Woods, Daniel L. Worthley. The origin and contribution of the tumor stroma in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5089.

Introduction Cancer-associated fibroblasts (CAFs), a heterogeneous component of the tumor microenvironment, substantially influence tumor progression. Bone morphogenetic proteins (BMP) play a critical part in defining the intestinal epithelial niche and either promote or retard cancer progression in a context-dependent manner. However, the role of BMP signaling in the colorectal cancer (CRC) stroma remains to be fully elucidated. This study investigated the significance of mesenchymal BMP signaling as a potential therapeutic target in CRC progression. Design Using CRC expression array data, we identified two CAF-specific factors involved in BMP signaling, then verified their upregulation in the human CRC stroma by in-situ hybridization (ISH). We took advantage of a preclinical mouse model of CRC hepatic metastasis to test approaches targeting the BMP signaling pathway. Results CRC microarray data identified GREM1 and ISLR as CAF-specific genes involved in BMP signaling. In colonic myofibroblasts, Grem1-overexpression inhibited BMP signaling whereas BMP7 signaling was augmented by Islr overexpression, suggesting opposing roles for GREM1 and ISLR in the regulation of BMP signaling. ISH using human rectal cancer samples revealed that GREM1 and ISLR were expressed in distinct CAF subpopulations and that GREM1 and ISLR expression predicted poor and favorable survival, respectively. Notably, Grem1 and Islr expression was differentially regulated by Foxl1, an intestinal mesenchyme-lineage transcription factor, and TGF-b, indicating a mechanism for generating fibroblast heterogeneity. Finally, adeno-associated virus 8-mediated in-vivo overexpression of Islr in hepatocytes retarded growth and generated more differentiated histology in CRC hepatic metastases. Conclusion These data suggest that increased stromal BMP signaling may ameliorate CRC progression and provide a rationale for targeting stromal BMP signaling to inhibit CRC progression and metastasis. Citation Format: Hiroki Kobayashi, Krystyna A. Gieniec, Tongtong Wang, Josephine A. Wright, Nobumi Suzuki, Tamsin RM Lannagan, Yoku Hayakawa, Simon J. Leedham, Nicholas Arpaia, Siddhartha Mukherjee, Timothy C. Wang, Atsushi Enomoto, Masahide Takahashi, Daniel L. Worthley, Susan L. Woods. Stromal BMP signaling imbalance mediated by GREM1 and ISLR regulates colorectal cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3977.

Background The management of low grade dysplasia (LGD) in ulcerative colitis (UC) is uncertain due to the variable risk of progression to colorectal cancer (CRC). Chromosomal copy number alterations (CNAs) occur in colonic epithelial cells of UC patients who have developed CRC. The burden of CNAs in precursor LGD relative to high-grade dysplasia (HGD) and CRC has not been defined, and the correlation between LGD CNA burden and future HGD/CRC risk is unknown. Shallow whole-genome sequencing is a novel, cost-effective technique for high resolution CNA assessment in formalin-fixed, paraffin-embedded tissue. Methods We identified 19 UC proctocolectomy specimens with HGD/CRC, and analysed 77 neoplastic regions (36 LGD, 34 HGD and 7 CRC). We then analysed 13 ‘progressor’ patients with 27 LGD lesions who subsequently developed HGD/CRC a median 427 days later (IQR 213–777), and 22 ‘non-progressor’ patients with 26 LGD lesions who remained HGD/CRC-free>5 years later. The two patient groups are matched for age, gender, disease duration and LGD location. Histological diagnosis was confirmed by two blinded pathologists. Shallow whole genome sequencing (0.1x) was performed using a standardised pipeline for epithelial cell enrichment, DNA extraction, library preparation, next generation sequencing and bioinformatic analysis. Results A median 12% of the genome of LGDs from proctocolectomy specimens showed CNAs (IQR 4%–32%), compared to 23% in HGD/CRC (IQR 19%–42%, p=0.003). Similarly, the number of CNA events was greater in HGD/CRC compared to LGD (p The Kaplan-Meier plot demonstrates that patients in this cohort bearing LGD with the 25% greatest CNA burden are significantly more likely to develop future CRC/HGD than the remaining 75% of patients (HR 5.1, p=0.001). Conclusions LGD lesions demonstrate a surprising diversity in CNA burden, with some LGD lesions bearing CNA profiles indistinguishable from HGD/CRC. Shallow whole-genome sequencing has potential translational utility, by stratifying patients with LGD lesions according to risk of progression to HGD/CRC.

Barrett’s oesophagus surveillance biopsies represent a significant share of the daily workload for a busy histopathology department. Given the emphasis on endoscopic detection and dysplasia grading, it is easy to forget that the benefits of these screening programs remain unproven. The majority of patients are at low risk of progression to oesophageal adenocarcinoma, and periodic surveillance of these patients is burdensome and costly. Here, we investigate the parallels in the development of Barrett’s oesophagus and other scenarios of wound healing in the intestine. There is now increased recognition of the full range of glandular phenotypes that can be found in patients’ surveillance biopsies, and emerging evidence suggests parallel pathways to oesophageal adenocarcinoma. Greater understanding of the conditions that favour progression to cancer in the distal oesophagus will allow us to focus resources on patients at increased risk.

ObjectiveSerrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-activated kinase pathway gene, BRAF, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein.DesignWe use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair.ResultsTargeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not BrafV600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting.ConclusionWe generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.