G. Del Vecchio Blanco

Epigenetically silenced miR-34b/c as a novel faecal-based screening marker for colorectal cancer

Background:MicroRNAs are tiny non-coding small endogenous RNAs that regulate gene expression by translational repression, mRNA cleavage and mRNA inhibition. The aim of this study was to investigate the hypermethylation of miR-34b/c and miR-148a in colorectal cancer, and correlate this data to clinicopathological features. We also aimed to evaluate the hypermethylation of miR-34b/c in faeces specimens as a novel non-invasive faecal-DNA-based screening marker.Methods:The 5-aza-2′-deoxycytidine treatment and methylation-specific PCR were carried out to detect the hypermethylation of miR-34b/c and miR-148a.Results:The miR-34b/c hypermethylation was found in 97.5% (79 out of 82) of primary colorectal tumours, P=0.0110. In 75% (21 out of 28) of faecal specimens we found a hypermethylation of miR-34b/c while only in 16% (2 out of 12) of high-grade dysplasia. In addition, miR-148a was found to be hypermethylated in 65% (51 out of 78) of colorectal tumour tissues with no significant correlation to clinicopathological features. However, a trend with female gender and advanced age was found, P=0.083. We also observed a trend to lower survival rate in patients with miR-148a hypermethylation with 10-year survival probability: 48 vs 65%, P=0.561.Conclusions:These findings show that aberrant hypermethylation of miR-34b/c could be an ideal class of early screening marker, whereas miR-148a could serve as a disease progression follow-up marker.

IL-15 positively regulates IL-21 production in celiac disease mucosa

Celiac disease (CD)-associated inflammation is characterized by high interleukin- 21 (IL-21), but the mechanisms that control IL-21 production are not fully understood. Here we analyzed IL-21 cell sources and examined how IL-21 production is regulated in CD. Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs), isolated from CD patients and non-CD controls, were analyzed for cell markers, cytokines, and transcription factors by flow cytometry. IL-21 was highly produced by CD4+ and CD4+/CD8+ IELs and LPLs in active CD. IL-21-producing cells coexpressed interferon-γ (IFN-γ) and to a lesser extent T helper type 17 (Th17) cytokines. Treatment of control LPLs with IL-15, a cytokine overproduced in CD, activated Akt and STAT3 (signal transducer and activator of transcription 3), thus enhancing IL-21 synthesis. Active CD biopsies contained elevated levels of Akt, and blockade of IL-15 in those samples reduced IL-21. Similarly, neutralization of IL-15 in biopsies of inactive CD patients inhibited peptic–tryptic digest of gliadin-induced IL-21 expression. These findings indicate that in CD, IL-15 positively regulates IL-21 production.

Hepatitis related to cytomegalovirus infection in two patients with Crohn's disease treated with azathioprine

Azathioprine-related side-effects occur in about 15% of treated patients. Liver toxicity is a rare complication of this drug, but is considered, in most cases, a contraindication to the continuation of treatment. However, abnormal liver tests may occur in patients under azathioprine treatment also due to infections. The distinction between toxic and infective causes of abnormal liver tests is important in order to identify patients that can be rechallenged with the drug. Cytomegalovirus infection is common in immunosuppressed transplant recipients, while the incidence is lower in patients with inflammatory bowel disease treated with immunosuppressive drugs. To our knowledge, only 2 cases of cytomegalovirus hepatitis occurring during azathioprine treatment for Crohns disease had been reported so far. Here, we describe two patients who experienced mild hepatitis associated with the onset of cytomegalovirus infection during azathioprine treatment. The infection was documented by the appearance of IgM anti cytomegalovirus. Both cases were self-limiting. In one of the 2 patients, azathioprine was given again after resolution of the hepatitis with good control of Crohns disease and without other complications. We also retrospectively evaluated the incidence of liver abnormalities assessed by blood tests in 58 consecutive patients with Crohns disease treated with azathioprine at our institution. Abnormal results were obtained in 8 out of these 58 patients, requiring discontinuation of the drug in 3 patients, two of whom were the cytomegalovirus cases described above.

Monitoring the activity of Crohn's disease

Crohns disease is characterized by a chronic inflammation of the intestine of unknown aetiology. One of the main problems when treating patients with Crohns disease, is the identification of patients undergoing early clinical relapse, for timely treatment and the possible prevention of complications. No sub‐clinical markers are currently available that predict relapse during remission. Several parameters have been proposed for this purpose. Although none have proven useful, growing evidence suggests a possible benefit in the clinical management of Crohns disease. Among these, we may identify: clinical behaviour, the characteristics of the host, clinical activity, markers of intestinal inflammation and markers of immune activation. In particular, the possible relationship between cytokine pattern and the clinical behaviour of Crohns disease has been addressed. Overall, these observations suggest that mucosal immune activation is a feature of Crohns disease, and may persist in the form of activated immunocompetent cells during remission. On the basis of this evidence, studies are currently investigating whether the down‐regulation of immune activation markers is associated with clinical remission in Crohns disease. It has been shown that higher mucosal levels of TNF‐α and an increased state of activation of lamina propria mononuclear cells in patients with inactive Crohns disease, are significantly associated with an earlier clinical relapse of the disease. These observations suggest that a persistent local immune activation during remission may represent a marker of early clinical relapse of Crohns disease.

Sampling of proximal and distal duodenal biopsies in the diagnosis and monitoring of celiac disease

BACKGROUND Since celiac disease-associated mucosal lesions are patchy, the diagnosis of the disease requires histological evaluation of multiple duodenal biopsies. AIM To examine whether adequate biopsy sampling in either the bulb or distal duodenum is sufficient to diagnose celiac disease. METHODS Twenty-five patients with positive celiac disease-specific serology and 17 patients with negative serology, who were on a gluten-containing diet, and 13 celiac disease patients on a gluten-free diet were consecutively and prospectively enrolled. Mucosal damage, anti-transglutaminase-2 IgA deposits, interferon-γ, interleukin-17A and interleukin-15 transcripts were evaluated in bulb and distal duodenal biopsies. RESULTS All patients with positive celiac disease-specific serology exhibited villous atrophy in both duodenal sites. In this group, mucosal anti-transglutaminase-2 IgA deposits were found in 24/25 (96%) bulb samples and 22/25 (88%) distal duodenal samples. No villous atrophy was documented in patients with negative serology. Interferon-γ and interleukin-17A were over-expressed in both duodenal sites of patients with villous atrophy, unlike patients with normal duodenal morphology (p<0.001). Among treated celiac disease patients, 2 (15.4%) had villous atrophy exclusively in the bulb and 6 (46.2%) had minimal histological abnormalities at both sites. CONCLUSION Sampling in the bulb and distal duodenum could be sufficient to diagnose/exclude celiac disease.

P.108 EUS ACCURACY IN RESTAGING RECTAL CANCER AFTER NEOADJUVANT CHEMORADIOTHERAPY: A PRELIMINARY REPORT

Colorectal cancer (CRC) is the second most frequent cause of cancer-related death in Western countries: one third of CRC is represented by rectal cancer (RC). Accurate staging is recommended to provide an optimal treatment strategy. Neoadjuvant chemoradiotherapy (NCRT) is increasingly used in the treatment of advanced RCwith the intention of downsizing and downstaging the tumour and to reduce tumour recurrence. Accuracy of EUS staging is reported to be 75-94% for tumour penetration and 7283% for nodal metastases. However after NCRT the EUS accuracy for T staging may decrease (50%) because marked fibrosis, peritumoral infiltration of inflammatory cell necrosis. AIMS: the aims of this study was to verify the accuracy of EUS in restaging RC after neoadjuvant CRT and the efficacy of the therapy in the downstaging and downsizing the tumour. METHODS: we prospectively enrolled consecutive patients affected by RC in stage II-b and III who underwent EUS and MRI or CT or PET before and after neoadjuvant CRT. EUS scanning was performed by one endosonographer using an electronic radial ecoendoscope (Olympus GF UE 160-AL5) with imaging at 7,5-10 MHz. Patients underwent surgical resection after reassessment staging. The reduction of diameter of RC was evaluated using RECIST criteria. Pathological staging was undertaken according to the TNM classification. RESULTS: 14 patients with locally advanced RC were assessed: 10 male, median age 64 yrs (range 56-73). The median distance from the anus was 5 cm. Seven patients were in in stage II-b and 7 in stage III. All patients underwent radiotherapy with no complication associated with chemotherapy (10 pts with CDDP, 2 pts with FOLFOX and 2 pts with FUFA). The downstaging after neadjuvant therapy was observed in 7/14 patients, and significant reduction of tumor diameter in 13/14 patients. There were 13 anterior resections and 1 abdominoperineal resections. EUS post-NCRT T stage was correct in 11/14 pts (3 T0, 5 T3, 3 T2), while 3 pts were over staged (2 T2 and 1 T1). Overall accuracy of EUS post-RCT for pathologic T stage was 93% for T0-T1 stage and 86% for T3 stage. EUS accuracy for N-stage was 79%. CONCLUSION: in our preliminary study neoadjuvant CRT was useful to downstage and downsize locally advanced RC. EUS seems to be an accurate tool to restage RC after neoadjuvant CRT. However our results needed to be confirmed in a large cohort of patients with locally advanced RC.