Immune Profiling of Human Gut-Associated Lymphoid Tissue Identifies a Role for Isolated Lymphoid Follicles in Priming of Region-Specific Immunity.

The intestine contains some of the most diverse and complex immune compartments in the body. Here we describe a method for isolating human gut-associated lymphoid tissues (GALTs) that allows unprecedented profiling of the adaptive immune system in submucosal and mucosal isolated lymphoid follicles (SM-ILFs and M-ILFs, respectively) as well as in GALT-free intestinal lamina propria (LP). SM-ILF and M-ILF showed distinct patterns of distribution along the length of the intestine, were linked to the systemic circulation through MAdCAM-1+ high endothelial venules and efferent lymphatics, and had immune profiles consistent with immune-inductive sites. IgA sequencing analysis indicated that human ILFs are sites where intestinal adaptive immune responses are initiated in an anatomically restricted manner. Our findings position ILFs as key inductive hubs for regional immunity in the human intestine, and the methods presented will allow future assessment of these compartments in health and disease.

Non-TGFß profibrotic signaling in ulcerative colitis after in vivo experimental intestinal injury in humans.

Although impaired regeneration is important in many gastrointestinal diseases including ulcerative colitis (UC), the dynamics of mucosal regeneration in humans are poorly investigated. We have developed a model to study these processes in vivo in humans. Epithelial restitution (ER) and extracellular matrix (ECM) regulation after an experimental injury of the sigmoid colonic mucosa was assessed by repeated high-resolution endoscopic imaging, histological assessment, RNA sequencing, deconvolution analysis, and 16S rDNA sequencing of the injury niche microbiome of 19 patients with UC in remission and 20 control subjects. Human ER had a 48-h lag before induction of regenerative epithelial cells [wound-associated epithelial (WAE) and transit amplifying (TA) cells] along with the increase of fibroblast-derived stem cell growth factor gremlin 1 mRNA (GREM1). However, UC deconvolution data showed rapid induction of inflammatory fibroblasts and upregulation of major structural ECM collagen mRNAs along with tissue inhibitor of metalloproteinase 1 (TIMP1), suggesting increased profibrotic ECM deposition. No change was seen in transforming growth factor ß (TGFß) mRNA, whereas the profibrotic cytokines interleukin 13 (IL13) and IL11 were upregulated in UC, suggesting that human postinjury responses could be TGFß-independent. In conclusion, we found distinct regulatory layers of regeneration in the normal human colon and a potential targetable profibrotic dysregulation in UC that could lead to long-term end-organ failure, i.e., intestinal damage.NEW & NOTEWORTHY The study reveals the regulatory dynamics of epithelial regeneration and extracellular matrix remodeling after experimental injury of the human colon in vivo and shows that human intestinal regeneration is different from data obtained from animals. A lag phase in epithelial restitution is associated with induction of stromal cell-derived epithelial growth factors. Postinjury regeneration is transforming growth factor ß-independent, and we find a profibrotic response in patients with ulcerative colitis despite being in remission.

Telomere dysfunction instigates inflammation in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition driven by diverse genetic and nongenetic programs that converge to disrupt immune homeostasis in the intestine. We have reported that, in murine intestinal epithelium with telomere dysfunction, DNA damage-induced activation of ataxia-telangiectasia mutated (ATM) results in ATM-mediated phosphorylation and activation of the YAP1 transcriptional coactivator, which in turn up-regulates pro-IL-18, a pivotal immune regulator in IBD pathogenesis. Moreover, individuals with germline defects in telomere maintenance genes experience increased occurrence of intestinal inflammation and show activation of the ATM/YAP1/pro-IL-18 pathway in the intestinal epithelium. Here, we sought to determine the relevance of the ATM/YAP1/pro-IL-18 pathway as a potential driver of IBD, particularly older-onset IBD. Analysis of intestinal biopsy specimens and organoids from older-onset IBD patients documented the presence of telomere dysfunction and activation of the ATM/YAP1/precursor of interleukin 18 (pro-IL-18) pathway in the intestinal epithelium. Employing intestinal organoids from healthy individuals, we demonstrated that experimental induction of telomere dysfunction activates this inflammatory pathway. In organoid models from ulcerative colitis and Crohn's disease patients, pharmacological interventions of telomerase reactivation, suppression of DNA damage signaling, or YAP1 inhibition reduced pro-IL-18 production. Together, these findings support a model wherein telomere dysfunction in the intestinal epithelium can initiate the inflammatory process in IBD, pointing to therapeutic interventions for this disease.

Estimating tissue-specific TNF mRNA levels prior to anti-TNFα treatment may support therapeutic optimisation in IBD patients.

BACKGROUND AND AIMS: Tumour necrosis factor-α (TNF) antagonists have improved the management of inflammatory bowel disease (IBD), however, their usage and administration persist to be suboptimal. Here, we examined the relationship between tissue-specific TNF mRNA expression in mucosal biopsies from IBD patients and anti-TNF treatment response. METHODS: Archived tissue samples from patients with luminal IBD that had all been or were in treatment with anti-TNF were included (18 adults and 24 paediatric patients). Patients were stratified into three groups according to anti-TNF response: responders, primary non-responders (PNR) and secondary loss of response (SLOR). TNF mRNA was detected using RNAscope in situ hybridisation (ISH) and the expression was quantified using image analysis. RESULTS: The ISH analysis showed varying occurrence of TNF mRNA positive cells located in lamina propria and often with increased density in lymphoid follicles (LF). Consequently, expression estimates were obtained in whole tissue areas with and without LF. Significantly higher TNF mRNA expression levels were measured in adults compared to paediatric patients in both the analyses with and without LF (p = .015 and p = .016, respectively). Considering the relation to response, the adult and paediatric patients were evaluated separately. In adults, the TNF expression estimates were higher in PNRs compared to responders with and without LF (p = .017 and p = .024, respectively). CONCLUSION: Our data indicate that adult PNR have significantly higher TNF mRNA levels than responders. This suggests that higher anti-TNF dose may be considered for IBD patients with high TNF mRNA expression estimates from the start of treatment.

Danish Society for Gastroenterology and Hepatology's clinical recommendations for colonoscopic surveillance for colorectal dysplasia and cancer in patients with inflammatory bowel disease.

OBJECTIVES: We aimed to produce clinical recommendations for colonoscopic surveillance for dysplasia and colorectal cancer in patients with inflammatory bowel diseases. MATERIALS AND METHODS: The Danish Society for Gastroenterology and Hepatology convened a committee to assess the literature on colorectal cancer in inflammatory bowel diseases and the effectiveness of colonoscopy surveillance, according to the Oxford Centre for Evidence Based Medicine levels of evidence. RESULTS: Clinical recommendations for the colonoscopic surveillance for dysplasia and colorectal cancer in patients with inflammatory bowel diseases were produced. These guidelines cover the risk stratification, entry, and follow-up of patients in the colonoscopy programme, the choice of image-enhanced colonoscopy modality, the investigation and treatment of lesions, and the management of special patient populations in the colonoscopy programme. CONCLUSIONS: Colonoscopic surveillance of inflammatory bowel disease is thought to be associated with a decreased risk of colorectal cancer and colorectal cancer-related mortality. Further evidence regarding the effectiveness of colonoscopic surveillance will contribute to understanding its role in the management of inflammatory bowel diseases. The Danish Society for Gastroenterology and Hepatology clinical guideline will aid gastroenterologists in the risk stratification of patients with inflammatory bowel disease, and the management of colorectal lesions. Gastroenterologists must inform and support patients with inflammatory bowel disease to decide whether to participate in the colonoscopic surveillance programme.

MicroRNA Biomarkers in IBD-Differential Diagnosis and Prediction of Colitis-Associated Cancer.

Inflammatory bowel disease (IBD) includes Crohn's disease (CD) and ulcerative colitis (UC). These are chronic autoimmune diseases of unknown etiology affecting the gastrointestinal tract. The IBD population includes a heterogeneous group of patients with varying disease courses requiring personalized treatment protocols. The complexity of the disease often delays the diagnosis and the initiation of appropriate treatments. In a subset of patients, IBD leads to colitis-associated cancer (CAC). MicroRNAs are single-stranded regulatory noncoding RNAs of 18 to 22 nucleotides with putative roles in the pathogenesis of IBD and colorectal cancer. They have been explored as biomarkers and therapeutic targets. Both tissue-derived and circulating microRNAs have emerged as promising biomarkers in the differential diagnosis and in the prognosis of disease severity of IBD as well as predictive biomarkers in drug resistance. In addition, knowledge of the cellular localization of differentially expressed microRNAs is a prerequisite for deciphering the biological role of these important epigenetic regulators and the cellular localization may even contribute to an alternative repertoire of biomarkers. In this review, we discuss findings based on RT-qPCR, microarray profiling, next generation sequencing and in situ hybridization of microRNA biomarkers identified in the circulation and in tissue biopsies.

Altered Structural Expression and Enzymatic Activity Parameters in Quiescent Ulcerative Colitis: Are These Potential Normalization Criteria?

Mucosal healing determined by endoscopy is currently the remission standard for ulcerative colitis (UC). However, new criteria for remission are emerging, such as histologic normalization, which appears to correlate better to the risk of relapse. Here, we study mucosal healing on a molecular and functional level in quiescent UC. We obtained endoscopic biopsies from 33 quiescent UC patients and from 17 controls. Histology was assessed using Geboes score. Protein and mRNA levels were evaluated for the tight junction proteins claudin-2, claudin-4, occludin, and tricellulin, as well as Cl-/HCO3- exchanger DRA, and cyclo-oxygenase enzymes (COX-1, COX-2). The mucosal activity of COX-1 and COX-2 enzymes was assessed in modified Ussing chambers, measuring electrogenic ion transport (short-circuit current, SCC). Chronic inflammation was present in most UC patients. The protein level of claudin-4 was reduced, while mRNA-levels of claudin-2 and claudin-4 were upregulated in UC patients. Surprisingly, the mRNA level of COX-1 was downregulated, but was unaltered for COX-2. Basal ion transport was not affected, while COX-2 inhibition induced a two-fold larger decrease in SCC in UC patients. Despite being in clinical and endoscopic remission, quiescent UC patients demonstrated abnormal mucosal barrier properties at the molecular and functional level. Further exploration of mucosal molecular signature for revision of current remission standards should be considered.

Plasma calprotectin is superior to serum calprotectin as a biomarker of intestinal inflammation in ulcerative Colitis.

Background and aims: Despite promising results, only a few studies have been published on serum calprotectin as a biomarker in IBD. Recently, plasma measurements of calprotectin have been shown to be more reliable than serum measurements. In this study, we aim to assess plasma and serum calprotectin measurements as biomarkers of disease activity in paediatric and adult ulcerative colitis.Methods: Paediatric (5-18 years) and adult (>18 years) patients scheduled for colonoscopy due to suspected or confirmed ulcerative colitis were included prospectively. Stool and blood samples were collected at time of colonoscopy and patient symptom scores were recorded. At colonoscopy the Ulcerative Colitis Endoscopic Index of Severity was recorded. Histology was graded according to the Geboes score.Results: 84 patients where included; 30 paediatric and 54 adult patients. Plasma calprotectin had a stronger correlation to all outcome variables than serum calprotectin. Plasma calprotectin correlated positively to disease extent (Rho = 0.53, p < .0001), symptoms scores (Rho = 0.54, p = .002, only in the paediatric cohort), endoscopic scores (Rho = 0.39, p = .0003), histological scores (Rho 0.28, p = .01) and, when using endoscopic assessment of severity as reference, could discriminate active disease from patients in remission (p = .03).Conclusions: While more studies are needed to assess if plasma calprotectin can discriminate healthy individuals from ulcerative colitis, this study indicates that plasma calprotectin can be used as a biomarker of disease activity, especially in cases where faecal calprotectin measurements are cumbersome either due to patient compliance or logistical requirements.

Dynamic contrast-enhanced EUS for quantification of tumor perfusion in colonic cancer: a prospective cohort study.

BACKGROUND AND AIMS: Dynamic contrast-enhanced EUS (CE-EUS) for quantification of perfusion in colonic tumors has not previously been reported in the literature. The aim of this study was to investigate correlations between perfusion parameters and vessel density assessed by immunohistochemical staining with antibodies toward CD31 and CD105. METHODS: We conducted a prospective clinical study of 28 patients with left-sided colonic adenocarcinoma who underwent CE-EUS and left-sided hemicolectomy within 2 weeks. CE-EUS recordings were analyzed in 2 regions of interest: the entire tumor and the most enhanced area. Immunohistochemical staining with CD31 and CD105 was performed on tumor tissue sections. The slides were manually scanned for highly vascularized areas, and counting of vessels was performed in hotspots within the tumor and invasive front. New vasculature was assessed by CD105. Associations between CE-EUS and CD31 and CD105 were investigated using Spearman correlation. RESULTS: We found significant P values for the correlation between CD31 and rise time (rho = .603 [95% confidence interval (95% CI), .238-.816]; P = .001) in tumor tissue and for the correlation between CD31 and rise time (rho = .50 [95% CI, .201-.695]; P = .008) and fall time (rho = .52 [95% CI, .204-.723]; P = .006) corresponding to the invasive front. We found no correlations between perfusion values evaluated by CE-EUS and CD105. CONCLUSIONS: Our results show a significant correlation for vessel density evaluated by CD31 and perfusion parameters evaluated by CE-EUS. This may be the first step toward using real-time CE-EUS for monitoring antiangiogenic therapies in colonic cancer. (Clinical trial registration number: NCT02324023.).

The sensitivity of fecal calprotectin in predicting deep remission in ulcerative colitis.

BACKGROUND: Mucosal healing is proposed as treat-to-target in ulcerative colitis (UC), even though the definition of mucosal healing remains contested as it has been suggested to be assessed by either endoscopy, histology or both. However, all definitions require an endoscopic evaluation of the mucosa. As endoscopies are invasive and uncomfortable to the patient we aimed to calibrate noninvasive predictors of mucosal inflammatory status defined by both endoscopy and histology. METHODS: UC patients (n = 106) undergoing a sigmoid-/colonoscopy were prospectively included. Feces (fecal calprotectin, FC), blood samples (hemoglobin, C-reactive protein, orosomucoid, erythrocyte sedimentation rate, albumin) and symptom scores (Simple Clinical Colitis Activity Index, SSCAI) were collected and analyzed. The colonic mucosa was assessed by the Mayo endoscopic sub score and biopsies were obtained for a histologic grading by Geboes score. Predictive cutoff values were analyzed by receiver operating characteristics (ROC). A combined endoscopic and histologic assessment defined deep remission (Mayo =0 and Geboes ≤1) and activity (Mayo ≥2 and Geboes >3). RESULTS: Only FC showed a significant ROC curve (p < .05). We suggest FC (mg/kg) cutoffs for detection of following: Deep remission: FC ≤25; Indeterminate: FC 25-230 - an endoscopy is recommended if a comprehensive status of both endoscopic and histologic assessed activity is needed; Active disease: FC >230. The complete ROC data is presented, enabling extraction of an FC cutoff value's sensitivity and specificity. CONCLUSIONS: FC predicts endoscopic and histologic assessed deep remission and inflammatory activity of colon mucosa. Neither the markers in blood nor the SCCAI performed significant ROC results.

Regulation of Laminin γ2 Expression by CDX2 in Colonic Epithelial Cells Is Impaired During Active Inflammation.

The expression of Caudal-related homeobox transcription factor 2 (CDX2) is impaired by tumor necrosis factor-α (TNF-α)-mediated activation of nuclear factor-κB (NF-κB) in ulcerative colitis (UC). Laminin subunit γ2 (LAMC2) is an epithelial basement membrane protein implicated in cell migration, proliferation, differentiation, as well as tumor invasion and intestinal inflammation, and its expression is enhanced by TNF-α in a NF-κB-dependent regulation of the recently identified LAMC2 enhancer. The aim was to determine whether CDX2 is involved in the basal regulation of LAMC2 in epithelial cells and to assess the influence of inflammation. Transcriptional regulation of LAMC2 was examined by reporter gene assays, overexpression, and shRNA-mediated knock-down of CDX2. CDX2-DNA interactions were assessed by chromatin immunoprecipitation on Caco-2 cells without or with TNF-α, as well as in purified colonic human epithelial cells. Immunohistochemical staining and quantitative reverse-transcription polymerase chain reaction analyses were used to measure the expression of CDX2 and LAMC2 in colonic biopsies from healthy controls and patients with UC. These data indicate that CDX2 directly regulates LAMC2 gene expression through interaction with elements in the LAMC2 promoter region. We further revealed an inverse effect of inflammation on CDX2 and LAMC2. The data presented provide a novel insight into how CDX2 is implicated in the transcriptional regulation of LAMC2 in intestinal epithelial cells, a function that is impaired during mucosal inflammation where a high level of TNF-α is present. J. Cell. Biochem. 118: 298-307, 2017. © 2016 Wiley Periodicals, Inc.

Inflammatory Bowel Disease and Small Bowel Cancer Risk, Clinical Characteristics, and Histopathology: A Population-Based Study.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) may increase risk of small bowel cancer (SBC). However, little is known of the characteristics and features of IBD-SBC, due to a low number of cases worldwide. We performed a population-based study of IBD and SBC to calculate risk and increase our understanding of clinical characteristics and histopathological and molecular features. METHODS: The study population consisted of all individuals aged 16 years or older living in Denmark during 1978-2010. Through linkage between national registers and subsequent scrutiny of medical records and pathology descriptions, we identified 40 cases of IBD-SBC. Risk was calculated by standardized incidence ratio (SIR) (observed/expected); patient characteristics were derived from medical files, and surgery specimens were obtained from hospitals nationwide for histopathological and molecular analyses. RESULTS: During 241,620 person-years of follow-up, 23 patients with Crohn's disease developed small bowel adenocarcinoma (SIR, 14.38; 95% confidence interval, 8.78-22.20) and 9 developed neuroendocrine tumors (SIR, 6.83; 95% confidence interval, 3.13-12.97). No significantly increased risk of SBC was found among patients with ulcerative colitis. Most patients with SBC had moderate-to-severe Crohn's disease with small bowel and upper gastrointestinal involvement. Assessment of surgical specimens of small bowel adenocarcinomas revealed a clear transition from inflammation to dysplasia and cancer, whereas no tumors had evidence of microsatellite instability. CONCLUSIONS: In a population-based study of patients in Denmark with IBD and SBC, we found risk of adenocarcinomas and neuroendocrine tumors to be increased among persons with Crohn's disease. Most patients with IBD-SBC had extensive IBD of moderate-to-severe activity. Adenocarcinomas appeared to develop via an inflammation-dysplasia-carcinoma pathway, but differed from IBD-related colorectal adenocarcinomas in their molecular features.

Dual Inhibition of EGFR and VEGF in Heavily Pretreated Patients with Metastatic Colorectal Cancer.

OBJECTIVE: The aim of this study was to evaluate the efficacy and safety of combining irinotecan, bevacizumab, and cetuximab/panitumumab as a 4th-line treatment in patients with metastatic colorectal cancer. METHODS: All patients had KRAS wild-type metastatic colorectal cancer and had previously received fluoropyrimidine, oxaliplatin, irinotecan, and cetuximab/panitumumab in a 1st, 2nd, and 3rd line setting. Most patients had previously received bevacizumab as well. All patients had progressed within 3 months after the last given treatment before starting the triple combination therapy every second week. RESULTS: Sixty-three patients were evaluated. The triple combination therapy was well tolerated. The median progression-free survival was 6.1 months, and the median overall survival was 11.9 months. Four patients (6%) obtained a partial response, and 40 (63%) had stable disease. CONCLUSION: The combination of irinotecan, bevacizumab, and cetuximab/panitumumab is safe and shows a toxicity profile corresponding to what is expected from the agents alone. The results indicate that the combination in the 4th line may result in a high rate of disease control in heavily pretreated patients with metastatic colorectal cancer.

In vivo and ex vivo sentinel node mapping does not identify the same lymph nodes in colon cancer.

INTRODUCTION: Identification of lymph nodes and pathological analysis is crucial for the correct staging of colon cancer. Lymph nodes that drain directly from the tumor area are called "sentinel nodes" and are believed to be the first place for metastasis. The purpose of this study was to perform sentinel node mapping in vivo with indocyanine green and ex vivo with methylene blue in order to evaluate if the sentinel lymph nodes can be identified by both techniques. METHODS: Patients with colon cancer UICC stage I-III were included from two institutions in Denmark from February 2015 to January 2016. In vivo sentinel node mapping with indocyanine green during laparoscopy and ex vivo sentinel node mapping with methylene blue were performed in all patients. RESULTS: Twenty-nine patients were included. The in vivo sentinel node mapping was successful in 19 cases, and ex vivo sentinel node mapping was successful in 13 cases. In seven cases, no sentinel nodes were identified. A total of 51 sentinel nodes were identified, only one of these where identified by both techniques (2.0%). In vivo sentinel node mapping identified 32 sentinel nodes, while 20 sentinel nodes were identified by ex vivo sentinel node mapping. Lymph node metastases were found in 10 patients, and only two had metastases in a sentinel node. CONCLUSION: Placing a deposit in relation to the tumor by indocyanine green in vivo or of methylene blue ex vivo could only identify sentinel lymph nodes in a small group of patients.

F-calprotectin and Blood Markers Correlate to Quality of Life in Pediatric Inflammatory Bowel Disease.

OBJECTIVES: Our aim was to investigate predictors of health-related quality of life (HRQoL) with respect to changes in disease parameters over time in children with inflammatory bowel disease. METHODS: This was a prospective longitudinal study examining the association between HRQoL (IMPACT III) and symptom scores (Pediatric Crohn Disease Activity Index, abbreviated Pediatric Ulcerative Colitis Activity Index), fecal calprotectin measures and blood analyses (C-reactive protein, erythrocyte sedimentation rate, orosomucoid, albumin, hemoglobin, and vitamin-D) in a cohort of 10- to 17-year-old patients with inflammatory bowel disease. Data were collected prospectively at 3-month intervals during a 2-year period. Associations were analyzed using linear mixed-effect models. Patients were divided into 2 groups, which received nonbiological oral treatment or biological parenteral treatment. RESULTS: From 79 patients (39 Crohn disease/40 ulcerative colitis), representing a total of 43,132 days of observation, 572 IMPACT measurements were paired with variables. A decrease in the IMPACT III score was significantly associated with increased ulcerative colitis-symptom score in the biological group (P = 0.005), and a similar inverse tendency was found in the nonbiological group and for Crohn disease symptoms in both groups. We found in both treatment groups overall a significant (P < 0.05) inverse association between the IMPACT III and the levels of fecal calprotectin, erythrocyte sedimentation rate, and orosomucoid, whereas albumin, hemoglobin, and vitamin-D were directly significantly associated. CONCLUSIONS: The IMPACT score, already known to correlate with disease activity, has now been shown to be associated with disease markers in feces and blood. This emphasizes that objective markers of disease activity indirectly can predict the patient's HRQoL.

Confocal laser endomicroscopy in ulcerative colitis: a longitudinal study of endomicroscopic changes and response to medical therapy (with videos).

BACKGROUND AND AIMS: Confocal laser endomicroscopy enables real-time in vivo microscopy during endoscopy and can predict relapse in patients with inflammatory bowel disease in remission. However, little is known about how endomicroscopic features change with time. The aim of this longitudinal study was to correlate colonic confocal laser endomicroscopy (CLE) in ulcerative colitis with histopathology and macroscopic appearance before and after intensification of medical treatment. METHODS: Twenty-two patients with ulcerative colitis in clinical relapse and 7 control subjects referred for colonoscopy were enrolled. The colonic mucosa was examined with high-definition colonoscopy, histopathology, and CLE at 4 colonic sites. Subsequently, patients requiring medical treatment escalation were referred for repeat endoscopy with CLE after 6 to 8 weeks. RESULTS: The baseline frequency of fluorescein leakage (P < .001), microerosions (P < .001), tortuosity of the crypts (P = .001), distortion of the crypts openings (P = .001), presence of inflammatory infiltrates (P < .001), and decreased crypt density (P < .001) were significantly higher in active ulcerative colitis compared with inactive ulcerative colitis and control subjects. A decrease in histopathologic score after medical treatment escalation was correlated with improvement in crypt tortuosity (rs = .35, P = .016), distortion of crypt openings (rs = .30, P = .045), and decreased crypt density (rs = .33, P = .026) but not in other features. CONCLUSIONS: CLE is an emerging endoscopic technique that reproducibly identifies mucosal changes in ulcerative colitis. With the exception of crypt changes, endomicroscopic features appear to improve slowly with time after medical treatment. ( CLINICAL TRIAL REGISTRATION NUMBER: NCT01684514.).

Confocal laser endomicroscopy: a novel method for prediction of relapse in Crohn's disease.

BACKGROUND AND STUDY AIMS: Confocal laser endomicroscopy (CLE) has been shown to predict relapse in ulcerative colitis in remission, but little is currently known about its role in Crohn's disease. The aim of this study was to identify reproducible CLE features in patients with Crohn's disease and to examine whether these are risk factors for relapse. PATIENTS AND METHODS: This was a single-center prospective feasibility study of CLE imaging in patients with Crohn's disease. CLE imaging was performed in the terminal ileum and four colorectal sites, and was correlated with histopathology and macroscopic appearance. Clinical relapse, defined as the need for treatment escalation or surgical intervention, was recorded during follow-up. RESULTS: The study included 50 patients: 39 with Crohn's disease (20 in remission), and 11 controls. Ileal fluorescein leakage and microerosions were significantly more frequent in patients with endoscopically active Crohn's disease compared with patients with inactive Crohn's disease and controls (P = 0.005 and (P = 0.006, respectively). The same applied to colorectal fluorescein leakage and vascular alterations ((P = 0.043 and (P = 0.034, respectively). During a 12-month follow-up period, ileal fluorescein leakage and microerosions were significant risk factors for relapse in the subgroup of patients in remission (log rank (P = 0.009 and (P = 0.007, respectively) as well as in the entire group of patients with Crohn's disease (log rank (P = 0.006 and (P = 0.01, respectively). Inter- and intraobserver reproducibility was almost perfect (κ > 0.80) or substantial (κ > 0.60) for the majority of CLE parameters. CONCLUSIONS: CLE can identify reproducible microscopic changes in the terminal ileum that are risk factors for relapse in patients with otherwise inactive Crohn's disease. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01738529).

Investigating intra-tumor heterogeneity and expression gradients of miR-21, miR-92a and miR-200c and their potential of predicting lymph node metastases in early colorectal cancer.

INTRODUCTION: miR-21, miR-92a and miR-200c are regulators of pathways involved in migration, intravasation and metastasis, and their tumor expression levels have been proposed as potential prognostic markers in colorectal cancer (CRC). In two parallel cohorts we examine intra-tumor expression levels in early stage CRC tissue in order to determine intra-tumor heterogeneity, potential systematic intra-tumor expression gradients of the miRNAs and to investigate the association to metastatic disease in early stage CRC. MATERIAL AND METHODS: Two parallel studies on archived formalin-fixed paraffin-embedded (FFPE) CRC tissue. Intra-tumor and inter-patient variances were analyzed in 9 early metastatic CRCs by measuring expression levels by qRT-PCR on isolated tissue samples from luminal, central and invasive border zones. Associations between miRNA expression levels and early metastasizing tumors was investigated in FFPE tissue from invasive border and central tumor zones from 47 early metastatic CRCs matched with 47 non-metastatic CRCs. Intra-tumor expression gradients were analyzed on both cohorts. RESULTS: Mean intra-tumor coefficient of variation in the heterogeneity cohort was 38.5% (range: 33.1-49.0%) only slightly less than variation between patients (45.1%, range 37.0-49.5%). We demonstrated systematic expression gradients between tumor zones equal to a 3.23 (p=0.003) and 1.36 (p=0.014) fold lower expression in invasive areas for miR-200c, 1.52 (p<0.001) and 1.27 (p=0.021) fold lower expression in invasive areas for miR-92a. For miR-21 we found a 1.75 (p<0.001) and 1.21 (p=0.064) fold higher expression in invasive areas compared to luminal and central zones, respectively. No significant difference in expression levels between metastatic and non-metastatic tumors was demonstrated, nor a difference in intra-tumor gradients between metastatic and non-metastatic tumors. CONCLUSION: This study provides evidence for moderate intra-tumor and inter-patient heterogeneities of three well-described potential prognostic markers in CRC. We demonstrate intra-tumor expression gradients indicating a differentiated expression of the target miRNAs between functional tumor zones, but the potential role as markers of early metastatic disease is still not fully clarified.

Intra-tumor heterogeneity of microRNA-92a, microRNA-375 and microRNA-424 in colorectal cancer.

UNLABELLED: Various microRNAs (miRNAs) have been investigated in order to improve diagnostics and risk assessment in colorectal cancer (CRC). To clarify the potential of miRNA profiling in CRC, knowledge of intra-tumor heterogeneity in expression levels is crucial. The study aim was to estimate the intra-tumor variance of three selected miRNAs: miR-92a, miR-375 and miR-424 in CRC tissue. MATERIAL AND METHODS: A retrospective study on archived formalin-fixed paraffin embedded tissue from 9 patients with CRC. miRNA tissue expression levels were analyzed by qRT-PCR on tissue representing luminal, central and invasive border zones. Variance components were estimated based on ∆∆Cp values using mixed modeling and presented as coefficients of variation (CV). RESULTS: Intra-tumor variance was approximately half of the variance observed between patients with a mean intra-tumor CV of 56.4% (range 33.1-77.1%) and a mean inter-patient CV of 101.7% (range 48.8-152.7%). Furthermore we found a significant systematic difference in expression levels between tumor zones for miR-92a and miR-375 with a luminal-invasive difference equal to 0.60 Cp (95% CI: 0.30-0.89, p=0.0003) for miR-92a and a luminal-invasive difference equal to 0.78 Cp (95% CI: 0.10-1.46, p=0.027) for miR-375. Conclusion While the intra-tumor variance of miR-92a, miR-375 and miR-424 is substantial, it only constitutes approximately 30% of the total variation. Functional deregulation between tumor zones might contribute to variations in measured expression levels, and thus knowledge of specific intra-tumor expression patterns is crucial in tissue sampling for research as well as in future diagnostics.