ABSTRACT
BACKGROUND: Current approaches to the detection of colorectal neoplasia associated with inflammatory bowel disease (IBD-CRN) are suboptimal. AIM: To test the feasibility of using stool assay of exfoliated DNA markers to detect IBD-CRN. METHODS: This investigation comprised tissue and stool studies. In the tissue study, gene sequencing and methylation assays were performed on candidate genes using tissue DNA from 25 IBD-CRNs and from 25 IBD mucosae without CRN. Mutations on p53, APC, KRAS, BRAF or PIK3CA genes were insufficiently informative, but several aberrantly methylated genes were highly discriminant. In the stool study, we evaluated candidate methylated genes (vimentin, EYA4, BMP3, NDRG4) in a prospective blinded study on buffered stools from 19 cases with known IBD-CRN and 35 age- and sex-matched IBD controls without CRN. From stool-extracted DNA, target genes were assayed using quantitative allele-specific real-time target and signal amplification method. RESULTS: IBD-CRN cases included 17 with ulcerative colitis (UC) and two with Crohn's disease (CD); nine had cancer and 10 had dysplasia. Controls included 25 with UC and 10 with CD. Individually, BMP3, vimentin, EYA4 and NDRG4 markers showed high discrimination in stools with respective areas under the ROC curve of 0.91, 0.91, 0.85 and 0.84 for total IBD-CRN and of 0.97, 0.97, 0.95 and 0.85 for cancer. At 89% specificity, the combination of BMP3 and mNDRG4 detected 9/9 (100%) of CRC and 80% of dysplasia, 4/4 (100%) of high grade and 4/6 (67%) of low grade. CONCLUSION: These findings demonstrate the feasibility of stool DNA testing for non-invasive detection of colorectal neoplasia associated with inflammatory bowel disease.
Subject(s)
Biomarkers, Tumor/genetics , Colorectal Neoplasms/diagnosis , DNA, Neoplasm/analysis , Feces/chemistry , Inflammatory Bowel Diseases/diagnosis , Adult , Aged , Colorectal Neoplasms/genetics , Female , Genetic Markers/genetics , Humans , Inflammatory Bowel Diseases/genetics , Male , Middle Aged , Predictive Value of TestsABSTRACT
OBJECTIVES: Patients with ulcerative colitis are at risk for colorectal cancer (CRC). Although prior studies have shown a link between HLA genotypes and ulcerative colitis (UC) susceptibility, none have investigated HLA genotypes and UC-CRC. We therefore investigated HLA-DR/DQ alleles in UC-CRC cases and UC-controls. Furthermore, since methylation of the Class II transactivator (CIITA) gene may silence HLA expression in tumours, we correlated HLA allele frequencies with CIITA gene methylation and HLA-DR expression. METHODS: Cases and controls were matched for duration/extent of ulcerative colitis, age, ethnicity and gender, but not for primary sclerosing cholangitis (PSC). DNA was extracted from archived tissue blocks from 114 UC-CRC cases and 114 UC-controls. HLA-DR/DQ genotyping was performed using sequence-specific-oligonucleotide polymerase chain reaction (SSO-PCR). CIITA methylation was determined using methylation-specific PCR. HLA-DR immunohistochemistry was done following standard protocols. RESULTS: UC-CRC cases were more likely than UC-controls to carry the DR17 or DR13 alleles (p<0.0001 or p = 0.02, respectively). Although CIITA methylation did not vary significantly between cases and controls, DR17 and DQ2 were associated with CIITA methylation (p = 0.04 and 0.02, respectively). UC-controls more frequently carried the DR7, DR1 or DQ5 alleles (p = 0.002, 0.05 or 0.01, respectively). After adjusting for PSC, DR17 remained significantly associated with an increased risk for UC-CRC while DR7 and DQ5 remained protective. CONCLUSIONS: We report a significant association between specific HLA alleles and either the risk for (DR17) or protection from (DR7, DQ5) UC-CRC. This suggests a possible genetic predisposition for increased UC-CRC risk. In addition, DQ2 and DR17 were associated with CIITA methylation.
Subject(s)
Colitis, Ulcerative/genetics , Colorectal Neoplasms/genetics , Genes, MHC Class II , Adult , Aged , Aged, 80 and over , Case-Control Studies , Colitis, Ulcerative/complications , Colitis, Ulcerative/metabolism , Colorectal Neoplasms/complications , Colorectal Neoplasms/metabolism , Female , Gene Frequency , Genetic Predisposition to Disease , Genotype , HLA-DQ Antigens/genetics , HLA-DR Antigens/genetics , HLA-DR1 Antigen/genetics , HLA-DR7 Antigen/genetics , Humans , Immunohistochemistry , Logistic Models , Male , Methylation , Middle Aged , Nuclear Proteins/metabolism , Polymerase Chain Reaction/methods , Risk , Trans-Activators/metabolismABSTRACT
Interstitial cells of Cajal (ICC) are specialized mesenchyme-derived cells that regulate contractility and excitability of many smooth muscles with loss of ICC seen in a variety of gut motility disorders. Maintenance of ICC numbers is tightly regulated, with several factors known to regulate proliferation. In contrast, the fate of ICC is not established. The aim of this study was to investigate whether apoptosis plays a role in the regulation of ICC numbers in the normal colon. ICC were identified by immunolabelling for the c-Kit receptor tyrosine kinase and by electron microscopy. Apoptosis was detected in colon tissue by immunolabelling for activated caspase-3, terminal dUTP nucleotide end labelling and by ultrastructural changes in the cells. Apoptotic ICC were identified and counted in double-labelled tissue sections. They were identified in all layers of the colonic muscle. In the muscularis propria 1.5 +/- 0.2% of ICC were positive for activated caspase-3 and in the circular muscle layer 2.1 +/- 0.9% of ICC were positive for TUNEL. Apoptotic ICC were identified by electron microscopy. Apoptotic cell death is a continuing process in ICC. The level of apoptosis in ICC in healthy colon indicates that these cells must be continually regenerated to maintain intact networks.
