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Background: Patients with ulcerative colitis (UC) are at risk for colorectal neoplasia. Challenges associated with surveillance colonoscopy with random biopsies for detection of dysplasia/cancer are well-documented. This study extended our findings in UC-associated colorectal cancer to include low-grade dysplasia (LGD) patients, testing whether our biomarker panel detects any UC-associated neoplasm., Methods: DNA from the LGD area and the corresponding nonadjacent, non-dysplastic section from 171 UC-LGD patients was extracted. TaqMan SNP Genotyping Assays for TNF-α, IL-1β, and IL23R were used to evaluate polymorphisms for each gene. Bisulfite-treated DNA was used for methylation testing of RUNX3, COX2, and MINT1. LGD data were combined with UC-cancer patient data for statistical testing. Logistic regression analyses determined associations between genetic/epigenetic/clinical variables and UC-associated neoplasia. Receiver operating characteristic analyses were performed to determine the final synchronous neoplasm detection panel., Results: Comparison of nonadjacent, non-dysplastic DNA from UC-neoplasm patients versus UC-controls indicated that TNF-α, IL-1β, and methylation of RUNX3, MINT1, and COX2 were significantly different (P < 0.0001). In multivariable analysis, all remained significant with an area under the curve of 0.85, exceeding the clinical variable panel area under the curve. Combining clinical and experimental variables yielded a neoplasm biomarker panel with an area under the curve of 0.95 (sensitivity and specificity of 82% and 91%, respectively). Analysis of DNA from LGD with known progression compared with LGD without progression indicated a significant difference in RUNX3 methylation., Conclusions: A combined clinical, genetic, and epigenetic model for detecting synchronous neoplasm by testing of non-neoplastic colonic tissue had favorable operating characteristics and could complement current patient care.

Background: Aberrant methylation of the EYA4 gene (mEYA4) highly discriminates ulcerative colitis (UC) cases with colorectal neoplasia from UC controls in both tissue and stool. It is not known if mEYA4 is also present in nonadjacent non-neoplastic mucosa (NNM) of UC patients with colorectal neoplasia., Methods: Formalin-fixed tissues from 25 UC cases with colorectal cancer (CRC) and 25 UC controls with neither CRC nor dysplasia were matched on gender, age, disease duration, disease extent, and coexistence of primary sclerosing cholangitis. DNA was extracted from sections of CRC and NNM from cases and UC control mucosae. Bisulfite-treated DNA was amplified using real-time methylation-specific PCR. The Wilcoxon rank-sum test assessed differences in mEYA4 levels from CRC, NNM, and control samples. Logistic regression was used to estimate sensitivity and specificity., Results: Sufficient DNA was available for 20 cases and 20 controls. The median mEYA4 level (with interquartile range) was 2 (0-5.7) in control mucosae but 93 (38.5-306) in CRC (P < 0.0001) and 27.4 (3-140) in NNM (P = 0.0009). At 95% specificity, mEYA4 was present in 80% of CRC and 50% of NNM tissue samples. The odds ratio of mEYA4 in NNM as an indicator of synchronous CRC was 19 (95% confidence interval, 2-170)., Conclusions: The authors demonstrate significantly higher mEYA4 levels in NNM and synchronous CRC from UC cases than in colorectal mucosae of UC controls without neoplasia. The finding of this CRC-associated field change has important implications to the use of mEYA4 as a potential UC surveillance marker in tissue or stool.

Background: Patients with extensive, longstanding ulcerative colitis (UC), a disease of chronic colonic inflammation, are at risk for colorectal cancer (CRC). Elucidating the mechanism and fully characterizing the nature of this chronic inflammation offers the potential to identify those at greatest risk. We performed a case-control study comparing histologic disease activity (HDA; neutrophils on hematoxylin and eosin [H&E]-stained slides) with immunohistochemistry (IHC) directed against specific cell types. We correlated IHC results with data previously generated on methylation status of RUNX3 and single nucleotide polymorphisms (SNPs) in tumor necrosis factor alpha (TNF-α)., Methods: A nonadjacent, nonneoplastic section of bowel wall was identified for each UC-CRC case. HDA was assessed for UC-CRC cases (n = 50) and UC-controls (n = 50). Sections were immunostained using antibodies against macrophages (CD68), neutrophils/monocytes (myeloperoxidase, MPO), and T cells (CD3). Slides were scored using ImageJ and results reported as the percent area positive for each marker., Results: HDA did not correlate with infiltrate levels as measured by IHC and increasing HDA was inversely related to UC-CRC risk. Conversely, the percent area positive for CD68 and MPO was significantly elevated in UC-CRC cases versus controls (P = 0.04 and < 0.0001, respectively). In areas designated inactive, MPO staining remained significantly higher in UC-CRC cases versus controls (P = 0.002). Increased MPO staining was associated with methylation of RUNX3 and the TNF-α -308G>A SNP., Conclusions: HDA is less sensitive than IHC and may underestimate inflammatory cell populations associated with UC-CRC. The epigenetic/genetic associations related to elevated MPO staining in UC-CRC may offer new methods for risk stratification and adjunctive screening tools., (Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.)

Objectives: Patients with ulcerative colitis (UC) are at increased risk for developing colorectal cancer (CRC). Surveillance in this at-risk population remains challenging. We assessed the methylation status of genes in the non-neoplastic mucosa of UC-CRC patients and controls to identify potential biomarkers of CRC., Methods: We evaluated the methylation status of 10 genes (p16, p14, runt-related transcript factor-3 (RUNX3), cyclooxygenase-2 (COX-2), E-cadherin, methylated-in-tumor-1 (MINT1), MINT31, HPP1, estrogen receptor, SLC5A8) in UC-CRC tumors and non-neoplastic sections from both UC-CRC cases and UC controls (n=114 for each) using methylation-specific PCR., Results: Amplification was successful for 96 UC controls, 83 tumors, and 66 non-adjacent, non-neoplastic samples. The prevalence of methylation was significantly greater in UC-CRC tumors for p16, RUNX3, MINT1, MINT31, and HPP1. Methylation of COX-2 and E-cadherin was greater in UC controls than in tumors. Univariate testing of these genes using non-adjacent, non-neoplastic sections from UC-CRC cases indicated that associations between p16, RUNX3, MINT1, MINT31, E-cadherin, and COX-2 and UC-CRC remained significant. In multivariable analysis of the six genes, only RUNX3, MINT1, and COX-2 remained significantly associated with the UC-CRC cases (odds ratio=12.6, 9.0, and 0.2, respectively). The results remained unaffected by the presence of PSC or severity of inflammation. Logistic regression modeling with the three genes showed interactions that increased the odds ratio for each gene., Conclusions: RUNX3, MINT1, and COX-2 are potential biomarkers for detecting the presence of CRC in patients with UC. These genes should be evaluated as biomarkers for colorectal dysplasia.

Objectives: Ulcerative colitis (UC) is characterized by chronic recurrent mucosal inflammation. Genetic studies in UC have indicated linkage to chromosome 6 in the region of the tumor necrosis factor-alpha (TNF-alpha) gene, a potent proinflammatory cytokine. TNF-alpha production is influenced by multiple factors including the type of immune cell and its level of activation. However, several single nucleotide polymorphisms (SNP) in the promoter region of TNF-alpha have been correlated with either increased or decreased production, indicating that regulation of TNF-alpha is in part genetic. Because UC patients are at increased risk for developing colorectal cancer (CRC), we investigated if there was an association between SNPs in the promoter of the TNF-alpha gene and UC-CRC., Methods: DNA was extracted from formalin-fixed, paraffin-embedded tissue from 114 UC-CRC cases and 114 UC-no CRC controls. Controls were frequency matched on duration and extent of colitis, age, ethnicity, and gender. All 228 tissue samples were analyzed for five TNF-alpha promoter polymorphisms (-238[G-->A], -308[G-->A], -857[C-->T], -863[C-->A], and -1031[T-->C]) using PCR and sequencing., Results: The -308(G-->A) SNP was significantly associated with UC-CRC cases at both the allele and genotype level (P < 0.0001). No other SNPs were significantly associated with UC-CRC., Conclusion: We report a novel finding of a strong association between the -308(G-->A) SNP and UC-CRC. Complete elucidation of the mechanism of UC-CRC carcinogenesis will require investigation of other genes involved in modulating inflammation, but our results suggest that some UC patients may have additional genetic predispositions toward developing UC-CRC.