Mark Lazarev

Microbiota organization is a distinct feature of proximal colorectal cancers

Significance We demonstrate, to our knowledge for the first time, that bacterial biofilms are associated with colorectal cancers, one of the leading malignancies in the United States and abroad. Colon biofilms, dense communities of bacteria encased in a likely complex matrix that contact the colon epithelial cells, are nearly universal on right colon tumors. Most remarkably, biofilm presence correlates with bacterial tissue invasion and changes in tissue biology with enhanced cellular proliferation, a basic feature of oncogenic transformation occurring even in colons without evidence of cancer. Microbiome profiling revealed that biofilm communities on paired normal mucosa cluster with tumor microbiomes but lack distinct taxa differences. This work introduces a previously unidentified concept whereby microbial community structural organization exhibits the potential to contribute to disease progression. Environmental factors clearly affect colorectal cancer (CRC) incidence, but the mechanisms through which these factors function are unknown. One prime candidate is an altered colonic microbiota. Here we show that the mucosal microbiota organization is a critical factor associated with a subset of CRC. We identified invasive polymicrobial bacterial biofilms (bacterial aggregates), structures previously associated with nonmalignant intestinal pathology, nearly universally (89%) on right-sided tumors (13 of 15 CRCs, 4 of 4 adenomas) but on only 12% of left-sided tumors (2 of 15 CRCs, 0 of 2 adenomas). Surprisingly, patients with biofilm-positive tumors, whether cancers or adenomas, all had biofilms on their tumor-free mucosa far distant from their tumors. Bacterial biofilms were associated with diminished colonic epithelial cell E-cadherin and enhanced epithelial cell IL-6 and Stat3 activation, as well as increased crypt epithelial cell proliferation in normal colon mucosa. High-throughput sequencing revealed no consistent bacterial genus associated with tumors, regardless of biofilm status. However, principal coordinates analysis revealed that biofilm communities on paired normal mucosa, distant from the tumor itself, cluster with tumor microbiomes as opposed to biofilm-negative normal mucosa bacterial communities also from the tumor host. Colon mucosal biofilm detection may predict increased risk for development of sporadic CRC.

The Bacteroides fragilis toxin gene is prevalent in the colon mucosa of colorectal cancer patients

BACKGROUND Enterotoxigenic Bacteroides fragilis (ETBF) produces the Bacteroides fragilis toxin, which has been associated with acute diarrheal disease, inflammatory bowel disease, and colorectal cancer (CRC). ETBF induces colon carcinogenesis in experimental models. Previous human studies have demonstrated frequent asymptomatic fecal colonization with ETBF, but no study has investigated mucosal colonization that is expected to impact colon carcinogenesis. METHODS We compared the presence of the bft gene in mucosal samples from colorectal neoplasia patients (cases, n = 49) to a control group undergoing outpatient colonoscopy for CRC screening or diagnostic workup (controls, n = 49). Single bacterial colonies isolated anaerobically from mucosal colon tissue were tested for the bft gene with touch-down polymerase chain reaction. RESULTS The mucosa of cases was significantly more often bft-positive on left (85.7%) and right (91.7%) tumor and/or paired normal tissues compared with left and right control biopsies (53.1%; P = .033 and 55.5%; P = .04, respectively). Detection of bft was concordant in most paired mucosal samples from individual cases or controls (75% cases; 67% controls). There was a trend toward increased bft positivity in mucosa from late- vs early-stage CRC patients (100% vs 72.7%, respectively; P = .093). In contrast to ETBF diarrheal disease where bft-1 detection dominates, bft-2 was the most frequent toxin isotype identified in both cases and controls, whereas multiple bft isotypes were detected more frequently in cases (P ≤ .02). CONCLUSIONS The bft gene is associated with colorectal neoplasia, especially in late-stage CRC. Our results suggest that mucosal bft exposure is common and may be a risk factor for developing CRC.

Relationship between proximal Crohn's disease location and disease behavior and surgery: a cross-sectional study of the IBD Genetics Consortium.

OBJECTIVES:In classifying Crohns disease (CD) location, proximal (L4) disease includes esophagogastroduodenal (EGD) and jejunal disease. Our aim was to determine the influence of proximal disease on outcomes of behavior and need for surgery and to determine if there was significant clinical heterogeneity between EGD and jejunal disease.METHODS:We performed a cross-sectional query of the NIDDK (National Institute of Diabetes and Digestive and Kidney Disease) Inflammatory Bowel Disease Genetics Consortium (IBDGC) database of patients with a confirmed diagnosis of CD and phenotyped per the IBDGC manual. Presence of any L4, L4-EGD, L4-jejunal, and non-L4 disease (L1-ileal, L2-colonic, and L3-ileocolonic) was compared with demographic features including age, race, ethnicity, smoking and inflammatory bowel disease (IBD) family history, diagnosis age, disease duration, clinical outcomes of inflammatory, stricturing or penetrating behavior, and CD abdominal surgeries. Univariate and multivariable analyses were performed with R.RESULTS:Among 2,105 patients with complete disease location data, 346 had L4 disease (175 L4-EGD, 115 L4-jejunal, and 56 EGD and jejunal) with 321 having concurrent L1-L3 disease. In all, 1,759 had only L1-L3 disease. L4 vs. non-L4 patients were more likely (P<0.001) to be younger at diagnosis, non-smokers, have coexisting ileal involvement, and have stricturing disease. L4-jejunal vs. L4-EGD patients were at least twice as likely (P<0.001) to have had ileal disease, stricturing behavior, and any or multiple abdominal surgeries. Remarkably, L4-jejunal patients had more (P<0.001) stricturing behavior and multiple abdominal surgeries than non-L4 ileal disease patients. Logistic regression showed stricturing risks were ileal (without proximal) site (odds ratio (OR) 3.18; 95% confidence interval 2.23–4.64), longer disease duration (OR 1.33/decade; 1.19–1.49), jejunal site (OR 2.90; 1.89–4.45), and older age at diagnosis (OR 1.21/decade; 1.10–1.34). Multiple surgery risks were disease duration (OR 3.74/decade; 3.05–4.64), penetrating disease (OR 2.60; 1.64–4.21), and jejunal site (OR 2.39; 1.36–4.20), with short duration from diagnosis to first surgery protective (OR 0.87/decade to first surgery; 0.84–0.90).CONCLUSIONS:Jejunal disease is a significantly greater risk factor for stricturing disease and multiple abdominal surgeries than either EGD or ileal (without proximal) disease. The Montreal site classification should be revised to include separate designations for jejunal and EGD disease.

Small bowel resection rates in Crohn's disease and the indication for surgery over time: experience from a large tertiary care center.

Background:Our primary aim was to determine if the rate of small bowel resection (SBR) has declined over time among Crohns disease (CD) patients seen at a single academic institution. A secondary aim was to establish whether the indication for surgery has changed. Methods:Patients with a primary or secondary ICD‐9 code for CD (555.0–555.9) who underwent SBR at the University of Pittsburgh were included. Patients were divided into 4 separate time periods based on when they had surgery: 1995–1998 (Period 1), 1999–2001 (Period 2), 2002–2004 (Period 3), and 2005–2007 (Period 4). Medical records were reviewed for the 6 months preceding surgery. Use of 5‐ASAs, immunomodulators (IMs), tumor necrosis factor (TNF) antagonists, and corticosteroids were noted. Disease behavior was defined as nonstricturing, nonpenetrating (B1), stricturing (B2), and penetrating (B3). Proportions of patients undergoing SBR were calculated according to calendar cohort and these rates were examined for time trends. Results:In all, 227 unique patients were analyzed for a total of 236 surgeries. The rates of 5‐ASA, IM, and corticosteroid use were similar across the 4 time periods. By contrast, TNF antagonist usage progressively increased over time (0%, 18%, 34%, 35%; P = 0.0002). The annual rate of SBR per period did not change (1.6%, 1.9%, 1.6%, 1.9%; P = 0.93). Similarly, the disease behavior did not change over time. Conclusions:While the frequency of TNF antagonist use in CD at the University of Pittsburgh has increased over time, the rate of SBR and indication for surgery has remained unchanged. These findings may be explained by long‐standing, complicated disease refractory to medical therapy. Inflamm Bowel Dis 2009

Novel Candidate Colorectal Cancer Biomarkers Identified by Methylation Microarray-Based Scanning

DNA hypermethylation is a common epigenetic abnormality in colorectal cancers (CRCs) and a promising class of CRC screening biomarkers. We conducted a genome-wide search for novel neoplasia-specific hypermethylation events in the colon. We applied methylation microarray analysis to identify loci hypermethylated in 17 primary CRCs relative to eight non-neoplastic colonic mucosae (NCs) from neoplasia-free subjects. These CRC-associated hypermethylation events were then individually evaluated for their ability to discriminate neoplastic from non-neoplastic cases, based on real-time quantitative methylation-specific PCR (qMSP) assays in 113 colonic tissues: 51 CRCs, nine adenomas, 19 NCs from CRC patients (CRC-NCs), and 34 NCs from neoplasia-free subjects (control NCs). A strict microarray data filtering identified 169 candidate CRC-associated hypermethylation events. Fourteen of these 169 loci were evaluated using qMSP assays. Ten of these 14 methylation events significantly distinguished CRCs from age-matched control NCs (P<0.05 by receiver operator characteristic curve analysis); methylation of visual system homeobox 2 (VSX2) achieved the highest discriminative accuracy (83.3% sensitivity and 92.3% specificity, P<1×10(-6)), followed by BEN domain containing 4 (BEND4), neuronal pentraxin I (NPTX1), ALX homeobox 3 (ALX3), miR-34b, glucagon-like peptide 1 receptor (GLP1R), BTG4, homer homolog 2 (HOMER2), zinc finger protein 583 (ZNF583), and gap junction protein, gamma 1 (GJC1). Adenomas were significantly discriminated from control NCs by hypermethylation of VSX2, BEND4, NPTX1, miR-34b, GLP1R, and HOMER2 (P<0.05). CRC-NCs were significantly distinguished from control NCs by methylation of ALX3 (P<1×10(-4)). In conclusion, systematic methylome-wide analysis has identified ten novel methylation events in neoplastic and non-neoplastic colonic mucosae from CRC patients. These potential biomarkers significantly discriminate CRC patients from controls. Thus, they merit further evaluation in stool- and circulating DNA-based CRC detection studies.

Compartment-specific immunity in the human gut: properties and functions of dendritic cells in the colon versus the ileum

Objective Dendritic cells (DC) mediate intestinal immune tolerance. Despite striking differences between the colon and the ileum both in function and bacterial load, few studies distinguish between properties of immune cells in these compartments. Furthermore, information of gut DC in humans is scarce. We aimed to characterise human colonic versus ileal DC. Design Human DC from paired colonic and ileal samples were characterised by flow cytometry, electron microscopy or used to stimulate T cell responses in a mixed leucocyte reaction. Results A lower proportion of colonic DC produced pro-inflammatory cytokines (tumour necrosis factor-α and interleukin (IL)-1β) compared with their ileal counterparts and exhibited an enhanced ability to generate CD4+FoxP3+IL-10+ (regulatory) T cells. There were enhanced proportions of CD103+Sirpα− DC in the colon, with increased proportions of CD103+Sirpα+ DC in the ileum. A greater proportion of colonic DC subsets analysed expressed the lymph-node-homing marker CCR7, alongside enhanced endocytic capacity, which was most striking in CD103+Sirpα+ DC. Expression of the inhibitory receptor ILT3 was enhanced on colonic DC. Interestingly, endocytic capacity was associated with CD103+ DC, in particular CD103+Sirpα+ DC. However, expression of ILT3 was associated with CD103− DC. Colonic and ileal DC differentially expressed skin-homing marker CCR4 and small-bowel-homing marker CCR9, respectively, and this corresponded to their ability to imprint these homing markers on T cells. Conclusions The regulatory properties of colonic DC may represent an evolutionary adaptation to the greater bacterial load in the colon. The colon and the ileum should be regarded as separate entities, each comprising DC with distinct roles in mucosal immunity and imprinting.

Unique patterns of CpG island methylation in inflammatory bowel disease-associated colorectal cancers.

Background: CpG island (CGI) hypermethylation at discrete loci is a prevalent cancer‐promoting abnormality in sporadic colorectal carcinomas (S‐CRCs). We investigated genome‐wide CGI methylation in inflammatory bowel disease (IBD)‐associated CRCs (IBD‐CRCs). Methods: Methylation microarray analyses were conducted on seven IBD‐CRCs, 17 S‐CRCs, and eight normal control colonic tissues from patients without CRC or IBD. CGI methylator phenotype (CIMP), a surrogate marker for widespread cancer‐specific CGI hypermethylation, was examined in 30 IBD‐CRCs and 43 S‐CRCs. Results: The genome‐wide CGI methylation pattern of IBD‐CRCs was CIMP status‐dependent. Based on methylation array data profiling of all autosomal loci, CIMP+ IBD‐CRCs grouped together with S‐CRCs, while CIMP− IBD‐CRCs grouped together with control tissues. CIMP− IBD‐CRCs demonstrated less methylation than did age‐matched CIMP− S‐CRCs at autosomal CGIs (z‐score −0.17 vs. 0.09, P = 3 × 10−3) and CRC‐associated hypermethylation target CGIs (z‐score −0.43 vs. 0.68, P = 1 × 10−4). Age‐associated hypermethylation target CGIs were significantly overrepresented in CGIs that were hypermethylated in S‐CRCs (P = 1 × 10−192), but not in CGIs that were hypermethylated in IBD‐CRCs (P = 0.11). In contrast, KRAS mutation prevalence was similar between IBD‐CRCs and S‐CRCs. Notably, CIMP+ prevalence was significantly higher in older than in younger IBD‐CRC cases (50.0 vs. 4.2, P = 0.02), but not in S‐CRC cases (9.7 vs. 16.7, P = 0.92). Conclusions: Cancer‐specific CGI hypermethylation and age‐associated CGI hypermethylation are diminished in IBD‐CRCs relative to S‐CRCs, while the KRAS mutation rate is comparable between these cancers. CGI hypermethylation appears to play only a minor role in IBD‐associated carcinogenesis. We speculate that aging, rather than inflammation per se, promotes CIMP+ CRCs in IBD patients. (Inflamm Bowel Dis 2011;)

Use of case reports and the Adverse Event Reporting System in systematic reviews: overcoming barriers to assess the link between Crohn’s disease medications and hepatosplenic T-cell lymphoma

BackgroundTo identify demographic and clinical characteristics associated with cases of hepatosplenic T-cell lymphoma (HSTCL) in patients with Crohn’s disease, and to assess strength of evidence for a causal relationship between medications and HSTCL in Crohn’s disease.MethodsWe identified cases of HSTCL in Crohn’s disease in studies included in a comparative effectiveness review of Crohn’s disease medications, through a separate search of PubMed and Embase for published case reports, and from the Food and Drug Administration (FDA) Adverse Event Reporting System (AERS). We used three causality assessment tools to evaluate the relationship between medication exposure and HSTCL.ResultsWe found 37 unique cases of HSTCL in patients with Crohn’s disease. Six cases were unique to the published literature and nine were unique to AERS. Cases were typically young (<40 years of age) and male (86%). The most commonly reported medications were anti-metabolites (97%) and anti-tumor necrosis factor alpha (anti-TNFa) medications (76%). Dose and duration of therapy were not consistently reported. Use of aminosalicylates and corticosteroids were rarely reported, despite the high prevalence of these medications in routine treatment. Using the causality assessment tools, it could only be determined that anti-metabolite and anti-TNFa therapies were possible causes of HSTCL in Crohn’s disease based on the data contained in the case reports.ConclusionSystematic reviews that incorporate case reports of rare lethal events should search both published literature and AERS, but consideration should be given to the limitations of case reports. In this study, establishing a causative effect other than ‘possible’ between anti-metabolite or anti-TNFa therapies and HSTCL was not feasible because case reports lacked data required by the causality assessments, and because of the limited applicability of causality assessment tools for rare irreversible events. We recommend minimum reporting requirements for case reports to improve causality assessment and routine reporting of rare life-threatening events, including their absence, in clinical trials to help clinicians determine whether rare adverse events are causally related to a medication.

The Pathogenic Role of NLRP3 Inflammasome Activation in Inflammatory Bowel Diseases of Both Mice and Humans

Background and Aims NLRP3 inflammasome is known to be involved in inflammatory bowel diseases. However, it is controversial whether it is pathogenic or beneficial. This study evaluated the roles of NLRP3 inflammasome in the pathogenesis of inflammatory bowel disease in IL-10-/- mice and humans. Methods NLRP3 inflammasome in colonic mucosa, macrophages, and colonic epithelial cells were analysed by western blotting. The NLRP3 inflammasome components were studied by sucrose density gradient fractionation, chemical cross-linking, and co-immunoprecipitation. The role of NLPR3 inflammasome in the pathogenesis of colitis was extensively evaluated in IL-10-/- mice, using a specific NLPR3 inflammasome inhibitor glyburide. Results NLRP3 inflammasome was upregulated in colonic mucosa of both IL-10-/- mice and Crohns patients. NLRP3 inflammasome activity in IL-10-/- mice was elevated prior to colitis onset; it progressively increased as disease worsened and peaked as macroscopic disease emerged. NLRP3 inflammasome was found in both intestinal epithelial cells and colonic macrophages, as a large complex with a molecular weight of ≥ 360 kDa in size. In the absence of IL-10, NLRP3 inflammasome was spontaneously active and more robustly responsive when activated by LPS and nigericin. Glyburide markedly suppressed NLRP3 inflammasome expression/activation in IL-10-/- mice, leading to not only alleviation of ongoing colitis but also prevention/delay of disease onset. Glyburide also effectively inhibited the release of proinflammatory cytokines/chemokines by mucosal explants from Crohns patients. Conclusions Abnormal activation of NLRP3 inflammasome plays a major pathogenic role in the development of chronic colitis in IL-10-/- mice and humans. Glyburide, an FDA-approved drug, may have great potential in the management of inflammatory bowel diseases.

Anti-Tumor Necrosis Factor-α Antibody Therapy Management Before and After Intestinal Surgery for Inflammatory Bowel Disease: A CCFA Position Paper.

Abstract:Biologic therapy with anti–tumor necrosis factor (TNF)-&agr; antibody medications has become part of the standard of care for medical therapy for patients with inflammatory bowel disease and may help to avoid surgery in some. However, many of these patients will still require surgical intervention in the form of bowel resection and anastomosis or ostomy formation for the treatment of their disease. Postsurgical studies suggest up to 30% of patients with inflammatory bowel disease may be on or have used anti–TNF-&agr; antibody medications for disease management preoperatively. Significant controversy exists regarding the potential deleterious impact of these medications on the outcomes of surgery, specifically overall and/or infectious complications. In this position statement, we systematically reviewed the literature regarding the potential risk of anti–TNF-&agr; antibody use in the perioperative period, offer recommendations based both on the best-available evidence and expert opinion on the use and timing of anti–TNF-&agr; antibody therapy in the perioperative period, and discuss whether or not the presence of these medications should lead to an alteration in surgical technique such as temporary stoma formation.