Detection of colonic dysplasia in patients with ulcerative colitis using a targeted fluorescent peptide and confocal laser endomicroscopy: A pilot study.
ABSTRACT: Targeted molecular probes have been used to detect sporadic colonic dysplasia during confocal laser endomicroscopy (CLE) with promising results. This is a feasibility pilot study aiming to assess the potential role of CLE combined with a fluorescent-labeled peptide to stain and detect dysplasia associated with Ulcerative Colitis.A phage-derived heptapeptide with predicted high binding affinity for dysplastic tissue, was synthesized and labeled with fluorescein. Eleven lesions with suspected dysplasia at endoscopy were excised from nine patients with long-standing ulcerative colitis. Specimens were sprayed with the peptide and examined by CLE. The CLE images were then compared to the corresponding histological sections.At definitive histology, 4 lesions were diagnosed as inflammatory polyps, 6 as dysplastic lesions and one as invasive cancer. In inflammatory polyps, the fluorescence signal came from peri-cryptal spaces and crypt lumen due to passive accumulation of the peptide in these areas. Dysplasia was associated with active binding of the peptide to dysplastic colonocytes.Ex vivo staining of ulcerative colitis-associated dysplasia using a fluorescent labeled molecular probe and CLE is feasible. In vivo studies on larger populations are required to evaluate the safety and the effective contribution of molecular probes in cancer surveillance of ulcerative colitis.
Project description:Colorectal cancer (CRC) is a life-threatening complication of ulcerative colitis (UC), and patients are routinely screened for the development of precancerous lesions (dysplasia). However, rates of CRC development in patients with confirmed low-grade dysplasia vary widely between studies, suggesting a large degree of heterogeneity between these lesions that is not detectable macroscopically. A better understanding of the underlying molecular changes that occur in dysplasia will help to identify lesions at higher risk of malignancy. MicroRNAs (miRNAs) post-transcriptionally regulate protein expression and cell-signalling networks. Aberrant miRNA expression is a feature of sporadic CRC but much less is known about the changes that occur in dysplasia and in UC.Comprehensive microRNA profiling was performed on RNA extracted from UC dysplastic lesions (n = 7) and UC controls (n = 10). The expression of miRNAs in UC post inflammatory polyps (n = 7) was also assessed. Candidate miRNAs were further validated by qPCR, and miRNA in situ hybridization. Serum levels of miRNAs were also assessed with a view to identification of non-invasive biomarkers of dysplasia.UC dysplasia was associated with a shift in miRNA expression profiles that was not seen in inflammatory polyps. In particular, levels of miR-200b-3p were increased in dysplasia, and this miRNA was localised to epithelial cells in dysplastic lesions and in UC cancers. No changes in miRNA levels were detected in the serum.UC-Dysplasia is linked to altered miRNA expression in the mucosa and elevated miR-200b-3p levels.
Project description:Colorectal cancer (CRC) is a major cause of cancer-related deaths in much of the world. Most CRCs arise from pre-malignant (dysplastic) lesions, such as adenomatous polyps, and current endoscopic screening approaches with white light do not detect all dysplastic lesions. Thus, new strategies to identify such lesions, including non-polypoid lesions, are needed. We aim to identify and validate novel peptides that specifically target dysplastic colonic epithelium in vivo. We used phage display to identify a novel peptide that binds to dysplastic colonic mucosa in vivo in a genetically engineered mouse model of colo-rectal tumorigenesis, based on somatic Apc (adenomatous polyposis coli) gene inactivation. Binding was confirmed using confocal microscopy on biopsied adenomas and excised adenomas incubated with peptide ex vivo. Studies of mice where a mutant Kras allele was somatically activated in the colon to generate hyperplastic epithelium were also performed for comparison. Several rounds of in vivo T7 library biopanning isolated a peptide, QPIHPNNM. The fluorescent-labeled peptide bound to dysplastic lesions on endoscopic analysis. Quantitative assessment revealed the fluorescent-labeled peptide (target/background: 2.17±0.61) binds ?2-fold greater to the colonic adenomas when compared to the control peptide (target/background: 1.14±0.15), p<0.01. The peptide did not bind to the non-dysplastic (hyperplastic) epithelium of the Kras mice. This work is first to image fluorescence-labeled peptide binding in vivo that is specific towards colonic dysplasia on wide-area surveillance. This finding highlights an innovative strategy for targeted detection to localize pre-malignant lesions that can be generalized to the epithelium of hollow organs.
Project description:Patients with ulcerative colitis (UC) are at increased risk of colorectal cancer (CRC). Colitis-associated dysplasia (flat or polypoid) continues to be a reliable marker for CRC in these patients. However, flat lesions are often missed during endoscopy and can rapidly progress to high-grade dysplasia or cancer. microRNAs (miRs), small non-coding RNAs, have emerged as a valuable diagnostic biomarker of human cancer due to their ease of detection and stability. The goal of this study was to identify a miR signature that can serve as a reliable biomarker for the early detection of colitis-associated dysplasia in patients with long-standing colitis. Overall design: The miR expression profiles of laser macrodissected colonic epithelial cells from non-dysplastic inflamed colon, flat and polypoid dysplasia was interrogated using the NanoString Human miRNA Platform (total of 22 patients).
Project description:Background and study aims? Ulcerative colitis (UC) and Crohn's disease (CD) have higher risk of colorectal cancer (CRC). Guidelines recommend dysplasia surveillance with dye-spraying chromoendoscopy (DCE). The aim of this systematic review and meta-analysis was to review all randomized clinical trials (RCTs) available and compare the efficacy of different endoscopic methods of surveillance for dysplasia in patients with UC and CD. Methods? Databases searched were Medline, EMBASE, Cochrane and SCIELO/LILACS.?It was estimated the risk difference (RD) for dichotomous outcomes (number of patients diagnosed with one or more dysplastic lesions, total number of dysplastic lesions diagnosed and number of dysplastic lesions detected by targeted biopsies) and mean difference for continuous outcomes (procedure time). Results? This study included 17 RCTs totaling 2,457 patients. There was superiority of DCE when compared to standard-definiton white light endoscopy (SD-WLE). When compared with high-definition (HD) WLE, no difference was observed in all outcomes (number of patients with dysplasia (RD 0.06; 95?% CI [-0.01, 0.13])). Comparing other techniques, no difference was observed between DCE and virtual chromoendoscopy (VCE - including narrow-band imaging [NBI], i-SCAN and flexible spectral imaging color enhancement), in all outcomes except procedure time (mean difference, 6.33 min; 95?% CI, 1.29, 11.33). DCE required a significantly longer procedure time compared with WLE (mean difference, 7.81 min; 95?% CI, 2.76, 12.86). Conclusions? We found that dye-spraying chromoendoscopy detected more patients and dysplastic lesions than SD-WLE. Although no difference was observed between DCE and HD-WLE or narrow-band imaging, the main outcomes favored numerically dye-spraying chromoendoscopy, except procedure time. Regarding i-SCAN, FICE and auto-fluorescence imaging, there is still not enough evidence to support or not their recommendation.
Project description:Ulcerative colitis (UC) patients have a greater risk of developing colorectal cancer through inflammation-dysplasia-carcinoma sequence of transformation. The histopathological diagnosis of dysplasia is therefore of critical clinical relevance, but dysplasia may be difficult to distinguish from inflammatory changes. A proteomic pilot study on 5 UC colorectal dysplastic patients highlighted proteins differentially distributed between paired dysplastic, inflammatory and normal tissues. The best candidate marker was selected and immunohistochemistry confirmation was performed on AOM/DSS mouse model lesions, 37 UC dysplasia, 14 UC cancers, 23 longstanding UC, 35 sporadic conventional adenomas, 57 sporadic serrated lesions and 82 sporadic colorectal cancers. Differential proteomics found 11 proteins significantly more abundant in dysplasia compared to inflammation, including Solute carrier family 12 member 2 (SLC12A2) which was confidently identified with 8 specific peptides and was below the limit of quantitation in both inflammatory and normal colon. SLC12A2 immunohistochemical analysis confirmed the discrimination of preneoplastic and neoplastic lesions from inflammatory lesions in mice, UC and in sporadic contexts. A specific SLC12A2 staining pattern termed “loss of gradient” reached 89% sensitivity, 95% specificity and 92% accuracy for UC-dysplasia diagnosis together with an inter-observer agreement of 95.24% (multirater κfree of 0.90; IC95%: 0.78 – 1.00). Such discrimination could not be obtained by Ki67 staining. This specific pattern was also associated with sporadic colorectal adenomas and cancers. We found a specific SLC12A2 immunohistochemical staining pattern in precancerous and cancerous colonic UC-lesions which could be helpful for diagnosing dysplasia and cancer in UC and non-UC patients.
Project description:Background and aims:Ulcerative colitis (UC) is associated with a higher background risk of dysplasia and/or neoplasia due to chronic inflammation. There exist few biomarkers for identification of patients with dysplasia, and targeted biopsies in this group of patients are inaccurate in reliably identifying dysplasia. We aimed to examine the epigenome of UC dysplasia and to identify and validate potential biomarkers. Methods:Colonic samples from patients with UC-associated dysplasia or neoplasia underwent epigenome-wide analysis on the Illumina 450K methylation array. Markers were validated by bisulphite pyrosequencing on a secondary validation cohort and accuracy calculated using logistic regression and receiver-operator curves. Results:Twelve samples from 4 patients underwent methylation array analysis and 6 markers (GNG7, VAV3, KIF5C, PIK3R5, TUBB6, and ZNF583) were taken forward for secondary validation on a cohort of 71 colonic biopsy samples consisting of normal uninflamed mucosa from control patients, acute and chronic colitis, "field" mucosa in patients with dysplasia/neoplasia, dysplasia, and neoplasia. Methylation in the beta-tubulin TUBB6 correlated with the presence of dysplasia (P < 0.0001) and accurately discriminated between dysplasia and nondysplastic tissue, even in the apparently normal field mucosa downstream from dysplastic lesions (AUC 0.84, 95% CI 0.81-0.87). Conclusions:Methylation in TUBB6 is a potential biomarker for UC- associated dysplasia. Further validation is needed and is ongoing as part of the ENDCAP-C study.
Project description:In patients with ulcerative colitis (UC) the cumulative risk of colon cancer is lower than the actual rate of dysplasia suggesting an efficient immune surveillance mechanism. Since the co-stimulatory molecule CD80 is overexpressed in dysplastic colonic mucosa of UC patients and T-cell activation entails effective costimulation, we aimed to evaluate the functional implication of CD80 signaling in colonic UC-associated carcinogenesis. In humans, we observed that the percentage of CD80+ and HLA-A+ IEC was increased in the dysplastic colonic mucosa of UC patients. In vitro, IEC activated CD8+ T-cells through a CD80-dependent pathway. Finally, in the AOM/DSS-induced colonic adenocarcinoma model CD80 signaling inhibition significantly increased the frequency and extension of high-grade dysplasia, whereas enhancing CD80 activity with an anti-CTLA4 antibody significantly decreased colonic dysplasia. In conclusion, CD80 signaling between IEC and T-cells represents a key factor controlling the progression from low to high grade dysplasia in inflammatory colonic carcinogenesis.
Project description:Patients with ulcerative colitis (UC) have an increased risk for developing colorectal cancer. Because UC tumorigenesis is associated with genomic field defects that can extend throughout the entire colon, including the non-dysplastic mucosa; we hypothesized that the same field defect will include abnormally expressed proteins. Here we applied proteomics to study the protein expression of UC neoplastic progression. The protein profiles of colonic epithelium were compared from 1) UC patients without dysplasia (non-progressors); 2) none-dysplastic colonic tissue from UC patient with high-grade dysplasia or cancer (progressors); 3) high-grade dysplastic tissue from UC progressors and 4) normal colon. We identified protein differential expression associated with UC neoplastic progression. Proteins relating to mitochondria, oxidative activity, calcium-binding proteins were some of interesting classes of these proteins. Network analysis discovered that Sp1 and c-myc proteins may play roles in UC early and late stages of neoplastic progression, respectively. Two over-expressed proteins in the non-dysplastic tissue of UC progressors, CPS1 and S100P, were further confirmed by IHC analysis. Our study provides insight into the molecular events associated with UC neoplastic progression, which could be exploited for the development of protein biomarkers in fields of non-dysplastic mucosa that identify a patient's risk for UC dysplasia.
Project description:Chitinase 3-like-1 (CHI3L1/YKL-40) is a protein secreted from restricted cell types including colonic epithelial cells (CECs) and macrophages. CHI3L1 is an inflammation-associated molecule, and its expression is enhanced in persons with colitis and colon cancer. The biological function of CHI3L1 on CECs is unclear. In this study, we investigated the role of CHI3L1 on CECs during the development of colitis-associated neoplasia. We analyzed colonic samples obtained from healthy persons and from persons with ulcerative colitis with or without premalignant or malignant changes. DNA microarray and RT-PCR analyses significantly increased CHI3L1 expression in non-dysplastic mucosa from patients with inflammatory bowel disease (IBD) who had dysplasia/adenocarcinoma compared with that in healthy persons and in patients with IBD who did not have dysplasia. As determined by IHC, CHI3L1 was expressed in specific cell types in the crypts of colonic biopsies obtained from patients with ulcerative colitis who have remote dysplasia. Purified CHI3L1 efficiently activated the NF-?B signaling pathway and enhanced the secretion of IL-8 and TNF-? in SW480 human colon cancer cells. In addition, colon cancer cell proliferation and migration were significantly promoted in response to CHI3L1 in these cells. In summary, CHI3L1 may contribute to the proliferation, migration, and neoplastic progression of CECs under inflammatory conditions and could be a useful biomarker for neoplastic changes in patients with IBD.
Project description:BACKGROUND: Flat adenomas are non-exophytic with a flat top or central depression and histologically the depth of dysplastic tissue is never more than twice the mucosal thickness. Flat adenomas frequently contain severely dysplastic tissue, and may progress rapidly through the adenoma-carcinoma sequence. Flat lesions have never been described in a British asymptomatic population. AIMS: To determine whether flat adenomas exist in an asymptomatic population participating in a large randomised controlled trial of flexible sigmoidoscopy screening. PATIENTS: A total of 3000 subjects (aged 55-64 years) underwent screening by flexible sigmoidoscopy. METHODS: All polyps were removed and sent for histology. The number of polyps with endoscopic and histological features of flat adenomas was recorded. RESULTS: Three subjects had a total of four flat lesions--that is, one per 1000 people screened. Three contained severely dysplastic tissue, one a focus of adenocarcinoma. Three of the four lesions were less than 5 mm in size and the fourth was 15 mm in diameter. CONCLUSIONS: Flat lesions with severe dysplasia exist in the asymptomatic population. This has major implications for gastroenterologists who should be trained to identify them. Their existence is of importance to molecular biologists and epidemiologists investigating the aetiology of colorectal cancer.