Richard Tighe

Canonical Wnt signals combined with suppressed TGFβ/BMP pathways promote renewal of the native human colonic epithelium

Background A defining characteristic of the human intestinal epithelium is that it is the most rapidly renewing tissue in the body. However, the processes underlying tissue renewal and the mechanisms that govern their coordination have proved difficult to study in the human gut. Objective To investigate the regulation of stem cell-driven tissue renewal by canonical Wnt and TGFβ/bone morphogenetic protein (BMP) pathways in the native human colonic epithelium. Design Intact human colonic crypts were isolated from mucosal tissue samples and placed into 3D culture conditions optimised for steady-state tissue renewal. High affinity mRNA in situ hybridisation and immunohistochemistry were complemented by functional genomic and bioimaging techniques. The effects of signalling pathway modulators on the status of intestinal stem cell biology, crypt cell proliferation, migration, differentiation and shedding were determined. Results Native human colonic crypts exhibited distinct activation profiles for canonical Wnt, TGFβ and BMP pathways. A population of intestinal LGR5/OLFM4-positive stem/progenitor cells were interspersed between goblet-like cells within the crypt-base. Exogenous and crypt cell-autonomous canonical Wnt signals supported homeostatic intestinal stem/progenitor cell proliferation and were antagonised by TGFβ or BMP pathway activation. Reduced Wnt stimulation impeded crypt cell proliferation, but crypt cell migration and shedding from the crypt surface were unaffected and resulted in diminished crypts. Conclusions Steady-state tissue renewal in the native human colonic epithelium is dependent on canonical Wnt signals combined with suppressed TGFβ/BMP pathways. Stem/progenitor cell proliferation is uncoupled from crypt cell migration and shedding, and is required to constantly replenish the crypt cell population.

The colon-selective spasmolytic otilonium bromide inhibits muscarinic M 3 receptor-coupled calcium signals in isolated human colonic crypts

Otilonium bromide (OB) is a smooth muscle relaxant used in the treatment of irritable bowel syndrome. Otilonium bromide has been shown to interfere with the mobilization of calcium in intestinal smooth muscle, but the effects on other intestinal tissues have not been investigated. We identified the muscarinic receptor subtype coupled to calcium signals in colonic crypt derived from the human colonic epithelium and evaluated the inhibitory effects of OB. Calcium signals were monitored by fluorescence imaging of isolated human colonic crypts and Chinese hamster ovary cells stably expressing the cloned human muscarinic M3 receptor subtype (CHO‐M3). Colonic crypt receptor expression was investigated by pharmacological and immunohistochemical techniques. The secretagogue acetylcholine (ACh) stimulated calcium mobilization from intracellular calcium stores at the base of human colonic crypts with an EC50 of 14 μM. The muscarinic receptor antagonists 4‐DAMP, AF‐DX 384, pirenzepine and methroctamine inhibited the ACh‐induced calcium signal with the following respective IC50 (pKb) values: 0.78 nM (9.1), 69 nM (7.2), 128 nM (7.1), and 2510 nM (5.8). Immunohistochemical analyses of muscarinic receptor expression demonstrated the presence of M3 receptor subtype expression at the crypt‐base. Otilonium bromide inhibited the generation of ACh‐induced calcium signals in a dose dependent manner (IC50=880 nM). In CHO‐M3 cells, OB inhibited calcium signals induced by ACh, but not ATP. In addition, OB did not inhibit histamine‐induced colonic crypt calcium signals. The present studies have demonstrated that OB inhibited M3 receptor‐coupled calcium signals in human colonic crypts and CHO‐M3 cells, but not those induced by stimulation of other endogenous receptor types. We propose that the M3 receptor‐coupled calcium signalling pathway is directly targeted by OB at the level of the colonic epithelium, suggestive of an anti‐secretory action in IBS patients suffering with diarrhoea.

PWE-056 Delivery Of Bowelscope Screening – Experience From The Pilot Sites

Introduction The NHS Bowel Cancer Screening Programme (BCSP) is being expanded to include a single flexible sigmoidoscopy (FSIG) called BowelScope, offered to all 55 year olds in addition to biannual faecal occult blood testing from age 60–75 years. 6 pilot sites began BowelScope screening in May 2013, with a view to full English coverage by the end of 2016. Methods We aim to describe practical issues involved in the delivery of BowelScope screening at the pilot centres, covering unit set-up, list format, and endoscopists delivering lists. A survey was sent to the 6 pilots for completion by screening staff. Data were also retrieved from the national BCSP database. Results The first BowelScope list was delivered in the South of Tyne Screening Centre on 7th May 2013. By December 2013, 4135 flexible sigmoidoscopy procedures had been performed in 6 centres. Centres have delivered 20–80 lists each, performing 2–7 lists per week. Sessions are run at varying times of day including evenings and Saturdays. 35 endoscopists undertake lists regularly, of whom 15 were already BCSP accredited. Other BCSP colonoscopists provide back up for lists when required. All non-BCSP were accredited through a combination of direct observation of procedural skills (DOPS) and an MQC exam. Specialist Screening Practitioners (SSPs) attend all lists, and are deployed in a variety of ways including: following patient journey, consenting or giving information, and supervision assistant SSPs. Table 1 shows details of screening lists by centre. Abstract PWE-056 Table 1 Centre First list Endoscopists (BCSP colonoscopists) Endoscopist grade Lists per week Evenings/ Weekends SSPs per list Consultant Registrar Staff/ other grade Nurse endoscopist Norwich 08.07.13 6 (1) 0 3 0 3 6 Yes/Yes 1* South of Tyne 07.05.13 2 (0) 0 1 1 0 2.5 Yes/Yes 2 St Marks 22.07.13 8 (2) 1 5 0 2 7 Yes/No 1* Surrey 07.11.13 6 (6) 6 0 0 0 2 No/Yes 2 West Kent and Medway 11.06.13 7 (3) 1 0 5 1 4 No/Yes 2 W’hampton 07.08.13 6 (3) 4 0 2 0 6 No/No 1 * Assistant screening practitioner also present for lists Conclusion BowelScope screening is being successfully delivered at the six pilot centres. Each centre has developed a screening template and organisational pattern that works around patient needs and existing endoscopy and bowel screening services. New patterns of working have been required to deliver BowelScope and challenges remain regarding adequate numbers of endoscopists. Disclosure of Interest None Declared.

PWE-363 Bowelscope- results from the pilot sites

Introduction A large UK study of single flexible sigmoidoscopy with adenoma clearance in patients aged 55–64, demonstrated a reduction in CRC incidence by 23% and mortality by 31% in intention-to-treat analyses.1This provided the rationale for provision of a new arm of the Bowel Cancer Screening Programme (BCSP), offering a single flexible sigmoidoscopy (FS) to all 55 year olds in England, known as BowelScope screening. BowelScope was introduced to 6 pilot sites, beginning in May 2013. It is to be rolled out across the country by 2016. Method We aim to describe the findings, in terms of procedural data, from the first year of BowelScope screening at the pilot sites. Data were obtained from the Bowel Cancer Screening System database for all participants who underwent FS between May 2013–May 2014. Procedural data were recorded, including entonox use, adenoma detection rate (ADR), cancer detection, complications and colonoscopy conversion rates. Results Overall uptake was 44.1%. 1 cancer was detected. Mean ADR was 9.2%. The mean number of patients requiring colonoscopy conversion was 4.2%. Mean complication rate was 0.1%, including bleeding, discomfort, difficult polyp excision and unwell patient. There was wide variation in entonox use. (Table 1).Abstract PWE-363 Table 1 Centre Uptake (%) Procedures (n) Endoscopists (n) Entonox (n (%)) ADR (%) Cancer (n) Colonoscopy Required (n (%)) Complications (n) Male Female 1 51.6 48.4 2381 11 254 (10.7) 9.4 0 67 (2.8) 1 2 40.9 35.1 901 8 149 (16.5) 11.1 0 53 (5.9) 4 3 43.2 41.7 2062 15 110 (5.3) 10.2 0 126 (6.1) 2 4 52.3 50.2 712 5 22 (3.1) 9.0 0 26 (3.7) 0 5 49.3 45.9 1437 11 56 (3.9) 7.6 0 56 (3.9) 0 6 39.7 35.7 1326 11 57 (4.3) 7.6 1 46 (3.5) 1 Total 45.6 42.4 8819 61 648 (7.3) 9.2 1 374 (4.2) 8 (0.1%) Conclusion Uptake has improved since the six month data were presented but remains lower than for the FOB arm of the BCSP, and varies between sites. The ADR is 9.2% (range 7.6% - 11.1%). This is lower than reported in the UK FS screening trial (12.1%)1, however ADR calculations in BowelScope do not include adenomas not removed at FS but removed later at colonoscopy, whereas the FS trial did. The ADR is comparable to the BowelScope pathfinder project (9.6%).2 Further work is necessary to explore the variation in uptake rates. Variation in ADR exists between centres. Further analysis of endoscopist factors and patient groups may explain this. Entonox use varies widely but does not appear to correlate with ADR- further work may be required to investigate this. Disclosure of interest None Declared. References Atkin WS et al. Lancet 2010;375:1624–33 Bevan R et al. Endoscopy 2015;47(3):225–31

PWE-051 Bowelscope: Early Results From The Pilot Sites

Introduction UK population colorectal cancer (CRC) screening has been successfully implemented with Bowel Cancer Screening Programme (BCSP) faecal occult blood testing biannually from age 60–75. A large UK study of once-only flexible sigmoidoscopy (FSIG) demonstrated a reductions in CRC incidence of 33% and death rates of 43% (1). This, with the screening centre infrastructure developed for the FOB programme, allowed provision of a new arm of BCSP, offering FSIG to 55 year olds in England, known as BowelScope screening. BowelScope screening began May 2013, with 6 pilot sites performing FSIGs in the first 7 months. Methods We aim to describe procedural data from the early months of BowelScope screening. Data were obtained from The Bowel Cancer Screening System (BCSS) database for all participants invited and participating in BowelScope FSIGs May-Dec 2013. Procedural data were recorded, including insertion depth, FSIG length, adenoma detection rates (ADR), cancer detection, discomfort levels, entonox usage and colonoscopy conversion rates. Results 13927 people were invited or opted in to BowelScope screening at 6 centres. Overall uptake is 43.5% (range 37.0–51.9%). 4 cancers were detected. Polyps were detected in 16.4–23.8% of FSIGs (mean 20.7%). Mean ADR 8.4%. One centre has a significantly higher ADR than the other five sites (p < 0.05) (see Table 1). Most (53%) procedures took 6–10 min. 79% of procedures were reported as causing no or minimal pain only, with only 34 procedures (1%) reporting severe pain. Abstract PWE-051 Table 1 Outcomes by anonymised centre Screening centre Invitees* Attended* Uptake†% FSIG with adenomas ADR% Cancer Colonoscopy required (%) 1 3125 1128 (51.9) 100 8.9% 1 39 (3.5) 2 1866 524 37.0 64 12.1% 0 23 (4.4) 3 3779 1070 40.9 90 8.4% 0 50 (4.7) 4 986 311 46.6 25 8.0% 0 12 (3.9) 5 1970 625 47.4 38 6.1% 2 21 (3.4) 6 2181 479 37.2 30 6.2% 1 18 (3.8) Total 13927 4135 43.5 347 8.4% 4 163 (3.9) * as of 20.12.13. † Uptake is calculated from invitees invited ≥16 weeks before 20.12.13 to allow time to respond to invitations and attend for screening. Conclusion Uptake has varied between centres, but is lower than for the FOB arm of BCSP. Average ADR is 8.4% (range 6.1–12.1%), lower than in the UK flexible sigmoidoscopy screening trial (12.1%1) although the age range studied in the trial differs from the cohort described here. Further work will be required to investigate the variation in uptake rates and to improve these rates. ADR variations may also need to be addressed; further analysis of patient groups may explain these differences. Reference 1 Atkin et al. Lancet 2010:375:1624–1633 Disclosure of Interest None Declared.

PWE-053 Variation In Adenoma Detection Rate In Bowelscope Screening

Introduction The English Bowel Cancer Screening Programme has been expanded to include a one-off flexible sigmoidoscopy offered to all 55 year olds, called BowelScope Screening. Screening commenced in May 2013, with 6 pilots sites performing flexible sigmoidoscopies in the first 8 months of screening. Methods The NHS Bowel Cancer Screening System database was interrogated and ADRs reviewed for each screening centre and screening endoscopist. A funnel plot was constructed using the log odds method. Results 49 endoscopists have performed 4444 sigmoidoscopies at 6 screening centres. Endoscopists had performed 2–330 procedures (median 66, mean 91), 29 endoscopists had performed ≥50 procedures, of these, 17 had performed ≥100 procedures. Overall BowelScope ADR is 8.6%. ADR by centre is shown in Table 1. Abstract PWE-053 Table 1 ADR by centre and volume Centre 1 Centre 2 Centre 3 Centre 4 Centre 5 Centre 6 All centres Endoscopist procedurecounts ADR% ADR% ADR% ADR% ADR% ADR% ADR% ADR range% All 8.8 11.7 8.9 7.6 6.5 7.3 8.6 0.0–60.0 ≥50 8.9 11.3 8.1 8.6 6.4 6.4 8.6 3.1–14.0 ≥100 9.0 11.3 8.9 8.6 3.1 5.2 8.7 3.1–13.0 Centre 2 has a higher ADR than the other centres. When considering all procedures, this difference reaches statistical significance when compared to centres 3, 5, and 6 (p < 0.05), and approaches significance when compared to centre 1 (p = 0.0687) and centre 4 (p = 0.0548). When considering procedures done by endoscopists who have performed ≥50 or ≥100 sigmoidoscopies, there remains a significant difference (p < 0.05) between centre 2 compared to centres 5 and 6, but not to the other centres. Creating a funnel plot of individual endoscopist ADRs, demonstrates one endoscopist below the 99.8% control limit (Figure 1). Abstract PWE-053 Figure 1 Conclusion Adenoma detection rates within BowelScope screening show variation between centres. There is also variation between endoscopists in terms of individual ADRs, although all but 1 endoscopist are above the 99.8% lower control level on funnel plot. These variations require further exploration at both centre and individual level; feedback and education methods will be used to improve ADRs. Consideration should be given to establishing an ADR standard. Disclosure of Interest None Declared.

Compromised Tissue Renewal in the Ageing Human Colonic Epithelium

BACKGROUND: The intestinal epithelium is the most rapidly renewing tissue in the body. It is widely believed that this attribute minimises the accumulation of age-related molecular damage. Increasing evidence suggests that this protective mechanism is undermined by agerelated molecular changes that accumulate in long-lived stem/progenitor cells. Age-related molecular damage in the intestinal epithelium of flies and rodents manifests as a hyperproliferative state that exhibits a greater degree of apoptosis, clonogen/stem cell number and reduced regenerative potential following damage. Furthermore, the human colonic epithelium is subject to age-related accumulation of mutations in mitochondrial DNA. Given that ageing is a major risk factor for cancer, it is surprising that the status of tissue renewal in the ageing human colonic epithelium has received little attention. AIM: To investigate age-related changes in the renewal of the human colonic epithelium.METHODS: Tissue biopsies obtained at sigmoidoscopy (Ethical approval) from young ( 70 years, N= 12) individuals with no apparent pathology were immediately fixed or processed for crypt isolation. Isolated crypts were observed in 3D culture by digital time-lapse microscopy. Native crypts obtained by microdissection of fixed biopsy tissue were subjected to morphometric analysis and immunofluorescence for detection of Ki67 (proliferation marker), beta catenin, c-Myc and axin2 (all markers for Wnt signals), and OLFM4 (an intestinal stem cell marker). RESULTS: Crypts from young subjects (n=116 crypts) were significantly longer (p< 0.05) than those derived from the older cohort (117 crypts), 375± 10 um versus 330 ± 10 um, respectively. The percentage of Ki67 positive cells in all regions along the cryptaxis was significantly greater (p<0.05) in tissue from older subjects : e.g. crypt base 35% (young) versus 52% (old); mid region 42%% (young) versus 62% (old) and upper region 10% (young) versus 17% (old). The number of cell divisions observed under timelapse microscopy was reduced for crypts derived from older subjects suggesting that the increased Ki67 labelling index in the older group reflected a slower cell cycle time. Although the nature of the proliferating cell type is not known at this stage, an increase in OLFM4+ (stem) cell number was observed along the axis of crypts derived from older subjects. The above traits were associated with a trend towards an extended profile of immunolabelling intensity for nuclear beta catenin and Wnt target gene expression along the crypt-axis of tissue derived from older subjects. CONCLUSIONS: Age-related changes in crypt length, cell proliferation and markers for intestinal stem cells and Wnt signalling components along the crypt-axis suggest that tissue renewal is compromised in the ageing human colonic epithelium.