Sakiko Hiraoka

DNA methylation of colon mucosa in ulcerative colitis patients: Correlation with inflammatory status

Background: Although DNA methylation of colonic mucosa in ulcerative colitis (UC) has been suggested, the majority of published reports indicate the correlation between methylation of colon mucosa and occurrence of UC‐related dysplasia or cancer without considering the mucosal inflammatory status. The aim of this study was to verify whether mucosal inflammation‐specific DNA methylation occurs in the colon of UC. Methods: Of 15 gene loci initially screened, six loci (ABCB1, CDH1, ESR1, GDNF, HPP1, and MYOD1) methylated in colon mucosa of UC were analyzed according to inflammatory status using samples from 28 surgically resected UC patients. Results: Four of six regions (CDH1, GDNF, HPP1, and MYOD1) were more highly methylated in the active inflamed mucosa than in the quiescent mucosa in each UC patient (P = 0.003, 0.0002, 0.02, and 0.048, respectively). In addition, when the methylation status of all samples taken from examined patients was stratified according to inflammatory status, methylation of CDH1 and GDNF loci was significantly higher in active inflamed mucosa than in quiescent mucosa (P = 0.045 and 0.002, respectively). Multiple linear regression analysis revealed that active inflammation was an independent factor of methylation for CDH1 and GDNF. DNA methyltransferase 1 and 3b were highly expressed in colon epithelial cells with active mucosal inflammation, suggesting their involvement in inflammation‐dependent methylation. Conclusions: Methylation in colonic mucosa of UC was correlated with mucosal inflammatory status, suggesting the involvement of methylation due to chronic active inflammation in UC carcinogenesis. (Inflamm Bowel Dis 2011;)

Evaluation of Mucosal Healing of Ulcerative Colitis by a Quantitative Fecal Immunochemical Test

OBJECTIVES:Accumulating evidence has underlined the importance of mucosal healing as a treatment goal for ulcerative colitis (UC). Quantitative fecal immunochemical tests (FITs), which can rapidly quantify fecal blood with automated equipment, have been used recently to screen for colorectal neoplasia. The aim of this study is to determine whether an FIT can evaluate mucosal healing in UC.METHODS:Feces collected from UC patients who underwent colonoscopy were examined by FITs, and results were compared with colonoscopic findings. Mucosal status was assessed using the Mayo endoscopic subscore classification. Maximum score for the colorectum in each patient was recorded.RESULTS:Evaluated were FIT results in conjunction with 310 colonoscopies that were performed in 152 UC patients. A large majority of patients with a Mayo 0 endoscopic score had negative FIT (<100 ng/ml) results (92%), and the proportion of negative FIT results decreased with increases in the Mayo score (Mayo 1: 47%, Mayo 2: 13%, Mayo 3: 12%, P<0.0001, Cochran-Armitage trend test). When the negative FIT was defined as <100 ng/ml, the sensitivity and specificity of a negative FIT for mucosal healing (Mayo 0) were 0.92 and 0.71, respectively. When mucosal healing was defined as Mayo 0 or 1, those were 0.60 and 0.87, respectively. In addition, a positive FIT (≥100 ng/ml) predicted mucosal inflammation (Mayo 2 or 3) with sensitivity 0.87 and specificity 0.60, respectively.CONCLUSIONS:The FIT can effectively and noninvasively evaluate mucosal healing in UC. This easy, rapid method can help evaluate and control disease activity of UC.

Evaluation of Mucosal Healing in Ulcerative Colitis by Fecal Calprotectin Vs. Fecal Immunochemical Test.

OBJECTIVES:We previously showed that a quantitative fecal immunochemical test (FIT) can predict mucosal healing (MH) in ulcerative colitis (UC). Fecal calprotectin (Fcal) has also been reported as an important biomarker of UC activity. The aim of this study was to compare the predictive ability of these two fecal markers for MH in UC.METHODS:FIT and Fcal were examined in stool samples from consecutive UC patients who underwent colonoscopy. Mucosal status was assessed via the Mayo endoscopic subscore (MES).RESULTS:In total, 105 colonoscopies in 92 UC patients were evaluated in conjunction with the FIT and Fcal results. Both FIT and Fcal results were significantly correlated with MES (Spearman’s rank correlation coefficient: 0.61 and 0.58, respectively). The sensitivity and specificity of the FIT values (<100 ng/ml) for predicting MH (MES 0 alone) were 0.95 and 0.62, respectively, whereas those of Fcal (<250 μg/g) were 0.82 and 0.62, respectively. The sensitivities became similar when MH was defined as MES 0 or 1 (0.86 vs. 0.86). Although the predictability of MH evaluated by the area under the receiver operating characteristics curve was similar for the two fecal markers (FIT 0.83 vs. Fcal 0.82 for MES 0 alone), the FIT results were relatively robust regardless of the cutoff value selected.CONCLUSIONS:Both FIT and Fcal can efficiently predict MH in UC, but FIT appears to be more sensitive than Fcal for predicting MES 0 alone.

Genetic and epigenetic alterations of Ras signalling pathway in colorectal neoplasia: analysis based on tumour clinicopathological features

Activation of RAS signalling induced by K-ras/BRAF mutations is a hallmark of colorectal tumours. In addition, Ras association domain families 1 and 2 (RASSF1 and RASSF2), the negative regulators of K-ras, are often inactivated by methylation of the promoter region in those tumours. However, reports showing differences in the occurrence of these alterations on the basis of tumour characteristics have been scarce. We analysed K-ras/BRAF mutations and the methylation status of RASSF1 and RASSF2 promoter regions in 120 colorectal adenomas with respect to their clinicopathological features. K-ras/BRAF mutations and RASSF2 methylation were observed in 49 (41%) and 30 (25%) of the samples, respectively, while RASSF1 methylation was observed in only 3 (2.5%). Adenomas with RASSF2 methylation often carried K-ras/BRAF mutations simultaneously (22 out of 30, P<0.01). Multivariate analysis revealed that the concomitance of these alterations was frequently observed in serrated adenomas (odds ratio (OR) 11.11; 95% confidence interval (CI) 1.96–63.00), but rarely in adenomas located in the sigmoid or descending colon (OR 0.13; 95% CI 0.03–0.58). A comparison between adenomas and cancers showed a significantly higher prevalence of these alterations in cancers than in adenomas in the proximal colon (58 vs 27%, P=0.02). Frequency and the time point of the occurrence of Ras signalling disorders differ according to colorectal neoplasia’s characteristics, particularly the location.

Serum Glycan Markers for Evaluation of Disease Activity and Prediction of Clinical Course in Patients with Ulcerative Colitis

Background The aims of this study were to determine the change of whole-serum N-glycan profile in ulcerative colitis (UC) patients and to investigate its clinical utility. Methods We collected serum from 75 UC patients at the time of admission and the same number of age/sex-matched healthy volunteers. Serum glycan profile was measured by comprehensive quantitative high-throughput glycome analysis and was compared with disease activity and prognosis. Results Out of 61 glycans detected, 24 were differentially expressed in UC patients. Pathway analysis demonstrated that highly sialylated multi-branched glycans and agalactosyl bi-antennary glycans were elevated in UC patients; in addition, the glycan ratio m/z 2378/1914, which also increased in UC, showed the highest Area under Receiver Operating Characteristic curve (0.923) for the diagnosis of UC. Highly sialylated multi-branched glycans and the glycan ratio m/z 2378/1914 were higher in the patients with total colitis, Clinical Activity Index >10, Mayo endoscopic score 3, or a steroid-refractory status. In particular, the glycan ratio m/z 2378/1914 (above median) was an independent prognostic factor for the need for an operation (hazard ratio, 2.67; 95% confidence interval, 1.04–7.84). Conclusions Whole-serum glycan profiles revealed that the glycan ratio m/z 2378/1914 and highly sialylated multi-branched glycans increase in UC patients, and are correlated with disease activity. The glycan ratio m/z 2378/1914 was an independent predictive factor of the prognosis of UC.

Methylation status of normal background mucosa is correlated with occurrence and development of neoplasia in the distal colon

The aim of this study is to evaluate the methylation status of normal colonic mucosa in relation to the stage of neoplasia arising from the mucosa. The methylation status of 2 age-related loci (ESR1 and MYOD1) and global methylation (the mean of Alu and Sat2) in the normal colonic mucosa of 156 patients with and without colorectal neoplasia were examined. The distal colon and proximal colon were analyzed separately because neoplasia is biologically and clinically different between these sites. The methylation status was determined by MethyLight using percentage of methylated reference (PMR). In the distal colon, methylation of the age-related loci decreased as the stage of neoplasia increased (patients with no neoplasia or with adenoma < or =9 mm versus patients with advanced adenoma or with invasive cancer: ESR1-PMR median, 21.0 versus 15.7; P = .015; MYOD1-PMR median, 5.35 versus 3.80; P = .0037, respectively). Interestingly, global methylation was inversely correlated with the stage of neoplasia (59.7 versus 61.5; P = .054). In contrast, the proximal colon showed no significant correlations. The methylation of MYOD1 in the normal mucosa was significantly correlated with K-ras mutation in neoplastic tissue arising from the mucosa. Specific epigenetic changes in normal colonic mucosa may be correlated with the occurrence and development of neoplasia in the distal colon.

Prognosis of ulcerative colitis differs between patients with complete and partial mucosal healing, which can be predicted from the platelet count.

AIM To determine the difference in clinical outcome between ulcerative colitis (UC) patients with Mayo endoscopic subscore (MES) 0 and those with MES 1. METHODS UC patients with sustained clinical remission of 6 mo or more at the time of colonoscopy were examined for clinical outcomes and the hazard ratios of clinical relapse according to MES. Parameters, including blood tests, to identify predictive factors for MES 0 and slight endoscopic recurrence in clinically stable patients were assessed. Moreover, a receiver operating characteristic curve was generated, and the area under the curve was calculated to indicate the utility of the parameters for the division between complete and partial mucosal healing. All P values were two-sided and considered significant when less than 0.05. RESULTS A total of 183 patients with clinical remission were examined. Patients with MES 0 (complete mucosal healing: n = 80, 44%) were much less likely to relapse than those with MES 1 (partial mucosal healing: n = 89, 48%) (P < 0.0001, log-rank test), and the hazard ratio of risk of relapse in patients with MES 1 vs MES 0 was 8.17 (95%CI: 4.19-17.96, P < 0.0001). The platelet count (PLT) < 26 × 10(4)/μL was an independent predictive factor for complete mucosal healing (OR = 4.1, 95%CI: 2.15-7.99). Among patients with MES 0 at the initial colonoscopy, patients of whom colonoscopy findings shifted to MES 1 showed significant increases in PLT compared to those who maintained MES 0 (3.8 × 10(4)/μL vs -0.6 × 10(4)/μL, P < 0.0001). CONCLUSION The relapse rate differed greatly between patients with complete and partial mucosal healing. A shift from complete to partial healing in clinically stable UC patients can be predicted by monitoring PLT.

Age-related methylation in normal colon mucosa differs between the proximal and distal colon in patients who underwent colonoscopy

OBJECTIVES To examine the difference in the methylation status in normal colon mucosa between the proximal and distal colon, in relation to the correlation between the methylation status of normal mucosa and characteristics of neoplasia. DESIGN AND METHODS Paired biopsy specimens of normal mucosa from the proximal and distal colon of 82 patients who underwent colonoscopy were obtained. The methylation status of the promoter region of estrogen receptor 1 (ESR1) and myogenic differentiation 1 (MYOD1) was examined. RESULTS Normal mucosa was more highly methylated in the distal than in the proximal colon in both ESR1 and MYOD1 loci (p<0.0001 and p=0.0009, respectively). Advanced characteristics of polyps in the distal colon were frequently observed in patients with lower methylation of ESR1 in the distal colon normal mucosa. CONCLUSIONS Methylation levels in normal mucosa differ between the proximal and distal colon, and lower methylation of ESR1 in the distal colon normal mucosa may correlate with advanced features of neoplasia in the distal colon.

Fecal Immunochemical Test Versus Fecal Calprotectin for Prediction of Mucosal Healing in Crohn's Disease

Background:Mucosal healing (MH) has been proposed as a treatment goal of inflammatory bowel disease patients. We reported recently that not only fecal calprotectin (Fcal) but also the fecal immunochemical test (FIT) can predict MH in ulcerative colitis. However, the predictive power of the fecal markers for MH in Crohns disease (CD), particularly with small bowel lesions, has not been reported in detail. The aim of this study was to evaluate the predictability of FIT versus Fcal for MH in CD. Methods:Consecutive CD patients underwent colonoscopy or balloon-assisted enteroscopy according to the disease location. FIT and Fcal were examined using stool samples collected the day before endoscopy. Results:Seventy-one CD patients were analyzed, of whom 42 (59%) underwent balloon-assisted enteroscopy because of the presence of affected lesions in the small intestine. Both the Fcal and the FIT results were significantly correlated with endoscopic activity (r = 0.67 and 0.54, respectively). However, the FIT results did not correlate with the activity in patients with small bowel lesions alone, whereas Fcal did (r = 0.42 versus 0.78). Fcal predicted MH in CD with 87% sensitivity and 71% specificity, whereas the values for FIT were 96% and 48%, respectively. The specificity for MH among patients with small bowel lesions alone was low for FIT (40%) compared with Fcal (80%). Conclusions:Both FIT and Fcal were correlated with the mucosal status of CD. However, the specificity of FIT was not satisfactory, particularly for small bowel lesions.

Fecal immunochemical test as a biomarker for inflammatory bowel diseases: can it rival fecal calprotectin?

Accurate evaluation of disease activity is essential for choosing an appropriate treatment and follow-up plan for patients with inflammatory bowel disease (IBD). Endoscopy is required for accurately evaluating disease activity, but the procedures are sometimes invasive and burdensome to patients. Therefore, alternative non-invasive methods for evaluating or predicting disease activity including mucosal status are desirable. Fecal calprotectin (Fcal) is the most widely used fecal marker for IBD, and many articles have described the performance of the marker in predicting disease activity, mucosal healing (MH), treatment efficacy, and risk of relapse. Fecal immunochemical test (FIT) can quantify the concentration of hemoglobin in stool and was originally used for the screening of colorectal cancer. We recently reported that FIT is also a useful biomarker for IBD. A direct comparison between the use of Fcal and FIT showed that both methods predicted MH in ulcerative colitis equally well. However, in the case of Crohns disease, FIT was less sensitive to lesions in the small intestine, compared to Fcal. FIT holds several advantages over Fcal in regards to user-friendliness, including a lower cost, easy and clean handling, and the ability to make rapid measurements by using an automated measurement system. However, there is insufficient data to support the application of FIT in IBD. Further studies into the use of FIT for evaluating the inflammatory status of IBD are warranted.