Stephen T. Ward

An analysis of the learning curve to achieve competency at colonoscopy using the JETS database

Objective The number of colonoscopies required to reach competency is not well established. The primary aim of this study was to determine the number of colonoscopies trainees need to perform to attain competency, defined by a caecal intubation rate (CIR) ≥90%. As competency depends on completion, we also investigated trainee factors that were associated with colonoscopy completion. Design The Joint Advisory Group on GI Endoscopy in the UK has developed a trainee e-portfolio from which colonoscopy data were retrieved. Inclusion criteria were all trainees who had performed a total of ≥20 colonoscopies and had performed ≤50 colonoscopies prior to submission of data to the e-portfolio. The primary outcome measure was colonoscopy completion. The number of colonoscopies required to achieve CIR ≥90% was calculated by the moving average method and learning curve cumulative summation (LC-Cusum) analysis. To determine factors which determine colonoscopy completion, a mixed effect logistic regression model was developed which allowed for nesting of patients within trainees and nesting of patients within hospitals, with various patient, trainee and training factors entered as fixed effects. Results 297 trainees undertook 36 730 colonoscopies. By moving average analysis, the cohort of trainees reached a CIR of 90% at 233 procedures. By LC-Cusum analysis, 41% of trainees were competent after 200 procedures. Of the trainee factors, the number of colonoscopies, intensity of training and previous flexible sigmoidoscopy experience were significant factors associated with colonoscopy completion. Conclusions This is the largest study to date investigating the number of procedures required to achieve competency in colonoscopy. The current training certification benchmark in the UK of 200 procedures does not appear to be an inappropriate minimum requirement. The LC-Cusum chart provides real time feedback on individual learning curves for trainees. The association of training intensity and flexible sigmoidoscopy experience with colonoscopy completion could be exploited in training programmes.

Good Maternal and Fetal Outcomes for Pregnant Women With Primary Biliary Cirrhosis

BACKGROUND & AIMS Up to 25% of patients diagnosed with primary biliary cirrhosis (PBC) are of childbearing age. However, little is known about disease course during pregnancy. METHODS We performed a retrospective analysis of women with PBC during pregnancy using a representative large cohort of patients attending the Liver Center at Toronto Western hospital from January 1979 through June 2009 (n = 306). Statistical analysis was performed by using R statistical software. RESULTS We identified 32 women (50 pregnancies) who either became pregnant after a diagnosis of PBC or in whom pregnancy led to diagnosis. Liver biochemistry remained stable in most patients (70%) throughout pregnancy. However, 23 of 32 patients (72%) had a flare in biochemical disease activity post partum, which was unrelated to biochemical disease activity before conception (P = .53), or during the gestational period (P = .14). No adverse maternal events were observed during pregnancy or post partum, and only 2 of 32 of women (6%) developed progressive disease after delivery. De novo pruritus developed during pregnancy in 17 of 32 women (53%), whereas itch that existed before conception worsened for 4 patients. Fifteen of 21 women (71%) with pregnancy-related pruritus required symptom-specific therapy. Twenty-nine of 32 women (91%) had at least 1 successful live birth; adverse fetal outcome was not influenced by biochemical disease activity before conception (P = .24) or during pregnancy (P = 1.00). CONCLUSION Pregnancy in women with PBC is frequently symptomatic but mostly uneventful. The majority of women maintain stable liver biochemistry during pregnancy, although postpartum biochemical exacerbations are common.

The effects of CCR5 inhibition on regulatory T-cell recruitment to colorectal cancer

Background:Regulatory T cells (Treg) are enriched in human colorectal cancer (CRC) where they suppress anti-tumour immunity. The chemokine receptor CCR5 has been implicated in the recruitment of Treg from blood into CRC and tumour growth is delayed in CCR5−/− mice, associated with reduced tumour Treg infiltration.Methods:Tissue and blood samples were obtained from patients undergoing resection of CRC. Tumour-infiltrating lymphocytes were phenotyped for chemokine receptors using flow cytometry. The presence of tissue chemokines was assessed. Standard chemotaxis and suppression assays were performed and the effects of CCR5 blockade were tested in murine tumour models.Results:Functional CCR5 was highly expressed by human CRC infiltrating Treg and CCR5high Treg were more suppressive than their CCR5low Treg counterparts. Human CRC-Treg were more proliferative and activated than other T cells suggesting that local proliferation could provide an alternative explanation for the observed tumour Treg enrichment. Pharmacological inhibition of CCR5 failed to reduce tumour Treg infiltration in murine tumour models although it did result in delayed tumour growth.Conclusions:CCR5 inhibition does not mediate anti-tumour effects as a consequence of inhibiting Treg recruitment. Other mechanisms must be found to explain this effect. This has important implications for anti-CCR5 therapy in CRC.

Frequency and significance of IgG4 immunohistochemical staining in liver explants from patients with primary sclerosing cholangitis

Dense tissue infiltrates of IgG4+ plasma cells >50/high‐powered field (HPF) are purportedly highly specific for IgG4‐related disease. However, the frequency and significance of liver‐infiltrating IgG4+ plasma cells in primary sclerosing cholangitis (PSC) applying these cut‐offs has not been determined. We sought to determine the incidence of intrahepatic IgG4‐positive staining in PSC patients undergoing transplantation, correlating findings with clinical parameters. Immunohistochemical staining was performed on liver explants obtained between 1991 and 2009. Of 122 explants obtained, hilar IgG4+ staining was found to be mild (10–29 IgG4+ cells/HPF) in 23.0%, moderate (30–50/HPF) in 9.0% and marked (>50/HPF) in 15.6%. Marked hilar lymphoplasmacytic infiltration was significantly associated with marked hilar IgG4+ staining (P < 0.001). No patient had marked peripheral IgG4+ staining, although mild and moderate staining was observed in 24.5% and 3.3% respectively. Marked hilar IgG4+ staining was significantly associated with the presence of dominant biliary strictures (P = 0.01) and need for biliary stenting (P = 0.001). There did not, however, exist any significant differences in the age at PSC diagnosis, presence of inflammatory bowel disease or extrahepatic autoimmune disease, frequency of cholangiocarcinoma, interval between diagnosis and transplantation, or post‐transplant PSC recurrence or survival. Of 51 control liver sections (PBC = 18; HCV = 19; HBV = 8; AIH = 6), none had marked or moderate hilar IgG4+ staining, whereas mild staining was seen in only 10% (P < 0.001). Marked (>50/HPF) hilar IgG4+ lymphoplasmacytic infiltration is frequently observed in PSC and associated with the presence of dominant biliary strictures. However, unlike serum IgG4+, this does not seemingly associate with clinical disease course.

The proto‐oncogene PBF binds p53 and is associated with prognostic features in colorectal cancer

The PTTG1‐binding factor (PBF) is a transforming gene capable of eliciting tumor formation in xenograft models. However, the precise role of PBF in tumorigenesis and its prognostic value as a cancer biomarker remain largely uncharacterised, particularly in malignancies outside the thyroid. Here, we provide the first evidence that PBF represents a promising prognostic marker in colorectal cancer. Examination of a total of 39 patients demonstrated higher PBF expression at both the mRNA (P = 0.009) and protein (P < 0.0001) level in colorectal tumors compared to matched normal tissue. Critically, PBF was most abundant in colorectal tumors associated with Extramural Vascular Invasion (EMVI), increased genetic instability (GI) and somatic TP53 mutations, all features linked with recurrence and poorer patient survival. We further demonstrate by glutathione–S–transferase (GST) pull‐down and coimmunoprecipitation that PBF binds to the tumor suppressor protein p53, as well as to p53 mutants (Δ126–132, M133K, V197E, G245D, I255F and R273C) identified in the colorectal tumors. Importantly, overexpression of PBF in colorectal HCT116 cells interfered with the transcriptional activity of p53‐responsive genes such as mdm2, p21 and sfn. Diminished p53 stability (> 90%; P < 0.01) was also evident with a concurrent increase in ubiquitinated p53. Human colorectal tumors with wild‐type TP53 and high PBF expression also had low p53 protein levels (P < 0.05), further emphasizing a putative interaction between these genes in vivo. Overall, these results demonstrate an emerging role for PBF in colorectal tumorigenesis through regulating p53 activity, with implications for PBF as a prognostic indicator for invasive tumors.

Intestinal CCL25 expression is increased in colitis and correlates with inflammatory activity

CCL25-mediated activation of CCR9 is critical for mucosal lymphocyte recruitment to the intestine. In immune-mediated liver injury complicating inflammatory bowel disease, intrahepatic activation of this pathway allows mucosal lymphocytes to be recruited to the liver, driving hepatobiliary destruction in primary sclerosing cholangitis (PSC). However, in mice and healthy humans CCL25 expression is restricted to the small bowel, whereas few data exist on activation of this pathway in the inflamed colon despite the vast majority of PSC patients having ulcerative colitis. Herein, we show that colonic CCL25 expression is not only upregulated in patients with active colitis, but strongly correlates with endoscopic Mayo score and mucosal TNFα expression. Moreover, approximately 90% (CD4+) and 30% (CD8+) of tissue-infiltrating T-cells in colitis were identified as CCR9+ effector lymphocytes, compared to <10% of T-cells being CCR9+ in normal colon. Sorted CCR9+ lymphocytes also demonstrated enhanced cellular adhesion to stimulated hepatic sinusoidal endothelium compared with their CCR9– counterparts when under flow. Collectively, these results suggest that CCR9/CCL25 interactions are not only involved in colitis pathogenesis but also correlate with colonic inflammatory burden; further supporting the existence of overlapping mucosal lymphocyte recruitment pathways between the inflamed colon and liver.

Assessing the value of matrix metalloproteinase 9 (MMP9) in improving the appropriateness of referrals for colorectal cancer

Background:A blood test may be an effective means of improving the appropriateness of referrals for symptomatic patients referred to specialist colorectal clinics. We evaluated the accuracy of a serum matrix metalloproteinase (MMP9) test in indicating colorectal cancer or its precursor conditions in a symptomatic population.Methods:Patients aged over 18, referred urgently or routinely to secondary care following primary care presentation with colorectal symptoms completed a questionnaire and provided a blood sample for serum MMP9 estimation. Univariate analysis and logistic regression modelling investigated the association between presenting symptoms, MMP9 measurements and the diagnostic outcome of patient investigations, in order to derive the combination of factors which best predicted a high risk of malignancy.Results:Data were analysed for 1002 patients. Forty-seven cases of neoplasia were identified. Age, male gender, absence of anal pain, diabetes, blood in stools, urgent referral, previous bowel polyps and previous bowel cancer were significantly associated with neoplasia. Matrix metalloproteinase 9 measurements were not found to be associated with significant colorectal pathology.Conclusion:This study, despite robust sampling protocols, showed no clear association between MMP9 and colorectal neoplasia. Matrix metalloproteinase 9 therefore appears to have little value as a tool to aid referral decisions in the symptomatic population.

Contact-Dependent Depletion of Hydrogen Peroxide by Catalase Is a Novel Mechanism of Myeloid-Derived Suppressor Cell Induction Operating in Human Hepatic Stellate Cells

Myeloid-derived suppressor cells (MDSC) represent a unique cell population with distinct immunosuppressive properties that have been demonstrated to shape the outcome of malignant diseases. Recently, human hepatic stellate cells (HSC) have been reported to induce monocytic-MDSC from mature CD14+ monocytes in a contact-dependent manner. We now report a novel and unexpected mechanism by which CD14+HLADRlow/− suppressive cells are induced by catalase-mediated depletion of hydrogen peroxide (H2O2). Incubation of CD14+ monocytes with catalase led to a significant induction of functional MDSC compared with media alone, and H2O2 levels inversely correlated with MDSC frequency (r = −0.6555, p < 0.05). Catalase was detected in primary HSC and a stromal cell line, and addition of the competitive catalase inhibitor hydroxylamine resulted in a dose-dependent impairment of MDSC induction and concomitant increase of H2O2 levels. The NADPH-oxidase subunit gp91 was significantly increased in catalase-induced MDSC as determined by quantitative PCR outlining the importance of oxidative burst for the induction of MDSC. These findings represent a so far unrecognized link between immunosuppression by MDSC and metabolism. Moreover, this mechanism potentially explains how stromal cells can induce a favorable immunological microenvironment in the context of tissue oxidative stress such as occurs during cancer therapy.

A method for conducting suppression assays using small numbers of tissue-isolated regulatory T cells

Graphical abstract Lymphocytes were isolated from human tissue samples by enzymatic and mechanical methods. Treg and Tconv were cell sorted from the tissue cell suspension by gating the lymphocytes forward scatter–side scatter region into a CD4–Live/Dead dot plot. CD4+ live cells were then gated into a CD127–CD25 dot plot. This last dot plot was used to define Treg (Streams 1 and 2) and Tconv cells (Stream 3). Treg were gated into a CCR5 histogram to define CCR5low and CCR5high Treg. Thus, specific Treg subsets were isolated for use in downstream suppression assays. Histograms depicting percentage Foxp3 expression by colorectal cancer-isolated Treg and Tconv show representative data from 9 separate experiments. Treg isolated from colorectal cancer therefore express high amounts of Foxp3 whereas Tconv do not.

Evaluation of serum lysyl oxidase as a blood test for colorectal cancer

AIMS Lysyl oxidase (LOX) expression is elevated in colorectal cancer (CRC) tissue and associated with disease progression. A blood test may form a more acceptable diagnostic test for CRC although LOX has not previously been measured in the serum. We therefore sought to determine the clinical usefulness of a serum LOX test for CRC in a symptomatic population. METHODS Adult patients referred to a hospital colorectal clinic with bowel symptoms completed a questionnaire and provided a blood sample for serum LOX measurement. Associations between presenting symptoms, serum LOX concentrations and outcomes of investigations were tested by univariate and multivariate analyses to determine if serum LOX was clinically useful in the prediction of CRC. LOX expression in CRC and adjacent colon biopsies was evaluated by ELISA and immunohistochemistry. RESULTS Thirty-one cases of colorectal cancer and 16 high-risk polyps were identified from a total of 962 participants. There was no association between serum LOX concentration and the presence of CRC, high-risk polyps or cancers at any site. LOX expression was significantly increased in CRC tissue compared to adjacent colon. CONCLUSION Despite overexpression of LOX in CRC tissue, elevated serum levels could not be demonstrated. Serum LOX measurement is therefore not a clinically useful test for CRC.