Gene Expression Changes in Non-Dysplastic Mucosa from Patients with Ulcerative Colitis Harboring Remote Neoplastic Lesions
ABSTRACT: Background and Aims: Individuals with ulcerative colitis (UC) are at increased risk for colorectal cancer, although underlying mechanisms are incompletely understood. We sought to identify a potential gene expression signature in non-dysplastic distal mucosa that as a “genetic field effect” could be a marker for remote neoplastic lesions. Results: 468 genes were significantly up-regulated and 541 genes were significantly down-regulated >2-fold in UC patients with neoplasia compared to UC patients without neoplasia. Nine genes (ACSL1, BIRC3, CLC, CREM, ELTD1, FGG, S100A9, THBD, and TPD52L1) were progressively and significantly up-regulated from controls to quiescent non-dysplastic UC to UC with neoplasia. Immunostaining of proteins revealed increases in tissue expression of S100A9 and REG1 in UC-associated cancer and in non-dysplastic tissue from UC patients harboring remote neoplasia, compared to UC patients without dysplasia and normal controls. Conclusions: Gene expression changes occur as a field effect in UC patients without active inflammation who harbor a remote dysplastic lesion. Further characterization of these genes and proteins might elucidate pathways of carcinogenesis in IBD and lead to the development of more accurate, less invasive markers of dysplasia in those at increased risk. Microarray assay were conducted on 20 RNA samples isolated from colon mucosa of 20 patients. Of these patients, 5 were normal controls, 4 had quiescent UC, and 11 had UC with neoplasia. Patients were included if they had a previous clinical diagnosis of UC confirmed by an expert GI pathologist, a disease duration > 7 years, and an extent of disease >20 cm proximal to the anal verge.
Project description:Background and Aims: Individuals with ulcerative colitis (UC) are at increased risk for colorectal cancer, although underlying mechanisms are incompletely understood. We sought to identify a potential gene expression signature in non-dysplastic distal mucosa that as a “genetic field effect” could be a marker for remote neoplastic lesions. Results: 468 genes were significantly up-regulated and 541 genes were significantly down-regulated >2-fold in UC patients with neoplasia compared to UC patients without neoplasia. Nine genes (ACSL1, BIRC3, CLC, CREM, ELTD1, FGG, S100A9, THBD, and TPD52L1) were progressively and significantly up-regulated from controls to quiescent non-dysplastic UC to UC with neoplasia. Immunostaining of proteins revealed increases in tissue expression of S100A9 and REG1 in UC-associated cancer and in non-dysplastic tissue from UC patients harboring remote neoplasia, compared to UC patients without dysplasia and normal controls. Conclusions: Gene expression changes occur as a field effect in UC patients without active inflammation who harbor a remote dysplastic lesion. Further characterization of these genes and proteins might elucidate pathways of carcinogenesis in IBD and lead to the development of more accurate, less invasive markers of dysplasia in those at increased risk. Microarray assay were conducted on 20 RNA samples isolated from colon mucosa of 20 patients. Of these patients, 5 were normal controls, 4 had quiescent UC, and 11 had UC with neoplasia. Patients were included if they had a previous clinical diagnosis of UC confirmed by an expert GI pathologist, a disease duration > 7 years, and an extent of disease >20 cm proximal to the anal verge.
Project description:Individuals with ulcerative colitis (UC) are at increased risk for colorectal cancer. The standard method of surveillance for neoplasia in UC by colonoscopy is invasive and can miss flat lesions. We sought to identify a gene expression signature in nondysplastic mucosa without active inflammation that could serve as a marker for remote neoplastic lesions.Gene expression was analyzed by complementary DNA microarray in 5 normal controls, 4 UC patients without dysplasia, and 11 UC patients harboring remote neoplasia. Common gene ontology pathways of significantly differentially expressed genes were identified. Expression of genes which were progressively and significantly upregulated from controls to UC without neoplasia, to UC with remote neoplasia were evaluated by real-time polymerase chain reaction. Several gene products were also examined by immunohistochemistry.Four hundred and sixty-eight genes were significantly upregulated, and 541 genes were significantly downregulated in UC patients with neoplasia compared with UC patients without neoplasia. Nine genes (ACSL1, BIRC3, CLC, CREM, ELTD1, FGG, S100A9, THBD, and TPD52L1) were progressively and significantly upregulated from controls to nondysplastic UC to UC with neoplasia. Immunostaining of proteins revealed increased expression of S100A9 and REG1? in UC-associated cancer and in nondysplastic tissue from UC patients harboring remote neoplasia compared with UC patients without neoplasia and controls.Gene expression changes occurring as a field effect in the distal colon of patients with chronic UC identify patients harboring remote neoplastic lesions. These markers may lead to a more accurate and less invasive method of detection of neoplasia in patients with inflammatory bowel disease.
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: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: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:Methylation of specific microRNAs (miRNAs) often occurs in an age-dependent manner, as a field defect in some instances, and may be an early event in colitis-associated carcinogenesis. We aimed to determine whether specific mRNA signature patterns (MIR1, MIR9, MIR124, MIR137, MIR34B/C) could be used to identify patients with ulcerative colitis (UC) who are at increased risk for colorectal neoplasia.We obtained 387 colorectal tissue specimens collected from 238 patients with UC (152 without neoplasia, 17 with dysplasia, and 69 with UC-associated colorectal cancer [UC-CRC]), from 2 independent cohorts in Japan between 2005 and 2015. We quantified methylation of miRNAs by bisulfite pyrosequencing analysis. We analyzed clinical data to determine whether miRNA methylation patterns were associated with age, location, or segment of the colorectum (cecum, transverse colon, and rectum). Differences in tissue miRNA methylation and expression levels were compared among samples and associated with cancer risk using the Wilcoxon, Mann-Whitney, and Kruskal-Wallis tests as appropriate. We performed a validation study of samples from 90 patients without UC and 61 patients with UC-associated dysplasia or cancer to confirm the association between specific methylation patterns of miRNAs in non-tumor rectal mucosa from patients with UC at risk of UC-CRC.Among patients with UC without neoplasia, rectal tissues had significantly higher levels of methylation levels of MIR1, MIR9, MIR124, and MIR137 than in proximal mucosa; levels of methylation were associated with age and duration of UC in rectal mucosa. Methylation of all miRNAs was significantly higher in samples from patients with dysplasia or CRC compared with samples from patients without neoplasia. Receiver operating characteristic analysis revealed that methylation levels of miRNAs in rectal mucosa accurately differentiated patients with CRC from those without. Methylation of MIR137 in rectal mucosa was an independent risk factor for UC-CRC. Methylation patterns of a set of miRNAs (panel) could discriminate discriminate UC patients with or without dysplasia or CRC in the evaluation cohort (area under the curve, 0.81) and the validation cohort (area under the curve, 0.78).In evaluation and validation cohorts, we found specific miRNAs to be methylated in rectal mucosal samples from patients with UC with dysplasia or CRC compared with patients without neoplasms. This pattern also associated with patient age and might be used to identify patients with UC at greatest risk for developing UC-CRC. Our findings provide evidence for a field defect in rectal mucosa from patients with UC-CRC.
Project description:Patients with extensive ulcerative colitis (UC) have an increased risk of colorectal cancer. Although UC patients generally undergo lifelong colonoscopic surveillance to detect dysplasia or cancer in the colon, detection of cancer in this manner is expensive and invasive. An objective biomarker of dysplasia would vastly improve the clinical management of cancer risk in UC patients. In the current study, accurate mass and time methods with ion intensity-based label-free proteomics are applied to profile individual rectal and colon samples from UC patients with dysplasia or cancer (UC progressors) compared to rectal samples from patients that are dysplasia/cancer free (UC nonprogressors) to identify a set of proteins in the rectum mucosa that differentiate the two groups. In addition to the identification of proteins in UC dysplastic colon tissue, we for the first time identified differentially expressed proteins in nondysplastic rectal tissue from UC progressors. This provides a candidate pool of biomarkers for dysplasia/cancer that could be detected in a random nondysplastic rectal biopsy. Mitochondrial proteins, cytoskeletal proteins, RAS superfamily, proteins relating to apoptosis and metabolism were important protein clusters differentially expressed in the nondysplastic and dysplastic tissues of UC progressors, suggesting their importance in the early stages of UC neoplastic progression. Among the differentially expressed proteins, immunohistochemistry analysis confirmed that TRAP1 displayed increased IHC staining in UC progressors, in both dysplastic and nondysplastic tissue, and CPS1 showed a statistically significant difference in IHC staining between the nonprogressor and progressor groups. Furthermore, rectal CPS1 staining could be used to predict dysplasia or cancer in the colon with 87% sensitivity and 45% specificity, demonstrating the feasibility of using surrogate biomarkers in rectal biopsies to predict dysplasia and/or cancer in the colon.
Project description:BACKGROUND PROMOTER: hypermethylation plays a major role in cancer through transcriptional silencing of critical genes. The aim of our study is to evaluate the methylation status of these genes in the colonic mucosa without dysplasia or adenocarcinoma at the different steps of sporadic and UC-related carcinogenesis and to investigate the possible role of genomic methylation as a marker of CRC.The expression of Dnmts 1 and 3A was significantly increased in UC-related carcinogenesis compared to non inflammatory colorectal carcinogenesis. In non-neoplastic colonic mucosa, the number of methylated genes resulted significantly higher in patients with CRC and in those with UC-related CRC compared to the HC and UC patients and patients with dysplastic lesion of the colon. The number of methylated genes in non-neoplastic colonic mucosa predicted the presence of CRC with good accuracy either in non inflammatory and inflammatory related CRC.Colonic mucosal samples were collected from healthy subjects (HC) (n = 30) and from patients with ulcerative colitis (UC) (n = 29), UC and dysplasia (n = 14), UC and cancer (n = 10), dysplastic adenoma (n = 14), and colon adenocarcinoma (n = 10). DNA methyltransferases-1, -3a, -3b, mRNA expression were quantified by real time qRT-PCR. The methylation status of CDH13, APC, MLH1, MGMT1 and RUNX3 gene promoters was assessed by methylation-specific PCR.Methylation status of APC, CDH13, MGMT, MLH1 and RUNX3 in the non-neoplastic mucosa may be used as a marker of CRC: these preliminary results could allow for the adjustment of a patient's surveillance interval and to select UC patients who should undergo intensive surveillance.
Project description:<h4>Background</h4>Chronic inflammation caused by ulcerative colitis (UC) causes a pro-neoplastic drive in the inflamed colon, leading to a markedly greater risk of invasive malignancy compared to the general population. Despite surveillance protocols, 50% of cases proceed to cancer before neoplasia is detected. The Enhanced Neoplasia Detection and Cancer Prevention in Chronic Colitis (ENDCaP-C) trial is an observational multi-centre test accuracy study to ascertain the role of molecular markers in improving the detection of dysplasia. We aimed to validate previously identified biomarkers of neoplasia in a retrospective cohort and create predictive models for later validation in a prospective cohort.<h4>Methods</h4>A retrospective analysis using bisulphite pyrosequencing of an 11 marker panel (SFRP1, SFRP2, SRP4, SRP5, WIF1, TUBB6, SOX7, APC1A, APC2, MINT1, RUNX3) in samples from 35 patients with cancer, 78 with dysplasia and 343 without neoplasia undergoing surveillance for UC associated neoplasia across 6 medical centres. Predictive models for UC associated cancer/dysplasia were created in the setting of neoplastic and non-neoplastic mucosa.<h4>Findings</h4>For neoplastic mucosa a five marker panel (SFRP2, SFRP4, WIF1, APC1A, APC2) was accurate in detecting pre-cancerous and invasive neoplasia (AUC = 0.83; 95% CI: 0.79, 0.88), and dysplasia (AUC = 0.88; (0.84, 0.91). For non-neoplastic mucosa a four marker panel (APC1A, SFRP4, SFRP5, SOX7) had modest accuracy (AUC = 0.68; 95% CI: 0.62,0.73) in predicting associated bowel neoplasia through the methylation signature of distant non-neoplastic colonic mucosa.<h4>Interpretation</h4>This multiplex methylation marker panel is accurate in the detection of ulcerative colitis associated dysplasia and neoplasia and is currently being validated in a prospective clinical trial.<h4>Funding</h4>The ENDCAP-C study was funded by the National Institute for Health Research Efficacy and Mechanism Evaluation (EME) Programme (11/100/29).
Project description:<h4>Background</h4>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.<h4>Methods</h4>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.<h4>Results</h4>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.<h4>Conclusion</h4>UC-Dysplasia is linked to altered miRNA expression in the mucosa and elevated miR-200b-3p levels.