High endothelial venules associated with T cell subsets in the inflamed gut of newly diagnosed inflammatory bowel disease patients.

Naive and central memory T lymphocytes (TN and TCM ) can infiltrate the inflamed gut mucosa in inflammatory bowel disease (IBD) patients. Homing of these subsets to the gut might be explained by ectopic formation of tertiary lymphoid organs (TLOs), containing high endothelial venules (HEVs). We aimed to evaluate the presence of HEVs and TLOs in inflamed intestinal mucosa of newly diagnosed, untreated IBD patients in relation to the presence of TN and TCM lymphocytes. IBD patients (n = 39) and healthy controls (n = 8) were included prospectively. Biopsy samples of inflamed and normal intestine, respectively, were analysed by immunohistochemistry for lymphocytes (CD3/CD20), blood vessels (CD31) and peripheral lymph node addressin (PNAd) expression (MECA-79). TN and TCM lymphocyte subsets were identified by flow cytometric immunophenotyping. A higher number of HEVs was found in the inflamed colon of patients with ulcerative colitis [median 3·05 HEV/mm2 ; interquartile range (IQR) = 0-6·39] and ileum of Crohn's disease patients (1·40; 0-4·34) compared to healthy controls (both 0; P = 0·033). A high density of colonic HEVs (HEVhigh ) was associated with increased infiltration of TN and TCM in the inflamed gut (median 87%; IQR = 82-93% of T cell population), compared to HEVlow patients (58%; 38-81%; P = 0·003). The number of colonic follicles was higher in HEVhigh patients (median 0·54/mm2 ; IQR 0·28-0·84) compared to HEVlow patients (0·25/mm2 ; 0·08-0·45; P = 0·031) and controls (0·31/mm2 ; 0·23-0·45; P = 0·043). Increased homing of TN and TCM lymphocytes to inflamed gut tissue in IBD patients might be facilitated by ectopic formation of extrafollicular HEVs and TLOs in a subgroup of patients.

KRAS mutation analysis: a comparison between primary tumours and matched liver metastases in 305 colorectal cancer patients.

BACKGROUND: KRAS mutation is a negative predictive factor for treatment with anti-epidermal growth factor receptor antibody in metastatic colorectal cancer (CRC). KRAS mutation analysis is usually performed on primary tumour tissue because metastatic tissue is often not available. However, controversial data are available on the concordance of test results between primary tumours and corresponding metastases. We assessed the concordance of KRAS mutation status in a study of 305 primary colorectal tumours and their corresponding liver metastases. METHODS: Patients with histologically confirmed CRC who underwent surgical resection of the primary tumour and biopsy or surgical resection of the corresponding liver metastasis were included. KRAS mutation analysis was performed for codons 12 and 13. RESULTS: KRAS mutation was detected in 108 out of 305 primary tumours (35.4%). In 11 cases (3.6%), we found a discordance between primary tumour and metastasis: 5 primary tumours had a KRAS mutation with a wild-type metastasis, 1 primary tumour was wild type with a KRAS mutation in the metastasis, and in 5 cases the primary tumour and the metastasis had a different KRAS mutation. CONCLUSION: We observed a high concordance of KRAS mutation status of 96.4% (95% CI 93.6-98.2%) between primary colorectal tumours and their corresponding liver metastases. In only six patients (2.0%; 95% CI 0.7-4.2%), the discordance was clinically relevant. In this largest and most homogenous study to date, we conclude that both primary tumours and liver metastases can be used for KRAS mutation analysis.

Heterozygous alpha-I antitrypsin deficiency as a co-factor in the development of chronic liver disease: a review.

Alpha-I antitrypsin (AIAT) is an acute-phase protein that is produced in liver cells. AIAT deficiency is a hereditary disease which is defined by the hepatic production of an abnormal protein that can not be released into the plasma. This leads to deficiency of plasma AIAT and subsequently to an impaired protection against proteases, resulting in pulmonary disease. Accumulation of the abnormal protein in hepatocytes can lead to liver damage. Serum level measurement, phenotyping and liver biopsy can be used for establishing the diagnosis. Homozygous AIAT deficiency can cause neonatal hepatitis; in adults end-stage liver disease, cirrhosis and hepatocellular carcinoma can develop. There are strong arguments to consider heterozygous AIAT deficiency as an important co-factor in the aetiology of chronic liver disease. Studies have shown that AIAT heterozygosity can be considered a modifier for hepatitis C virus, end-stage liver disease, cirrhosis and hepatocellular carcinoma. The accumulation of AIAT in the hepatocytes occurs more profoundly in a diseased liver, and as a consequence it affects the natural course of the liver disease. Therapeutic options include augmentation therapy (infusion of purified human plasma AIAT) in pulmonary disease; in end-stage liver disease liver transplantation is an option. For the future, other interventions such as gene therapy or strategies to inhibit polymerisation are promising.

Malignant complications of coeliac disease.

Patients with coeliac disease (CD), particularly those who are undiagnosed or do not adhere to a strict gluten free diet (GFD), are prone to develop complications. Malignant complications are the most serious and should be suspected when expected responses to GFD are not achieved or sustained. Lymphomas, mostly T-cell type, and other malignant tumours, particularly carcinoma of the small bowel, less frequently of stomach and oesophagus, are associated with CD. Loss of response to a gluten free diet (refractory coeliac disease) and ulcerative jejunitis are two recently described complications of CD that may progress to an Enteropathy-Associated T-cell Lymphoma (EATL). Coeliac disease-related lymphoma most often appears at extra-nodal sites, essentially the small bowel, although one have to realise that T-cell lymphomas arising in sites outside the small bowel could be related to coeliac disease. Workup of an EATL must include immunehistology and if necessary T-cell flow cytometry and T-cell rearrangement. Adequate imaging with CT and PET-scanning is mandatory.

Azathioprine and prednisone combination therapy in refractory coeliac disease.

INTRODUCTION: Refractory coeliac disease (RCD) is a rare syndrome with a poor prognosis, defined by malabsorption due to gluten-related enteropathy after initial or subsequent failure of a strict gluten-free diet and after exclusion of any disorder mimicking coeliac disease. PATIENTS AND METHODS: Nineteen patients were included and treated. Based on intraepithelial T-lymphocyte(IEL) phenotyping, patients were recorded as having RCD type I with normal IELs, or RCD type II with phenotypically immature IELs defined by a lack of characteristic T-cell markers. Treatment consisted of azathioprine combined with prednisone for 1 year, which was tapered and, if possible, stopped. RESULTS: Clinical improvement was seen in nearly all patients in both groups. Eight of 10 RCD type I patients responded histologically, and complete normalization of villi was seen in four patients. In RCD type II, 6/8 patients developed enteropathy-associated T-cell lymphoma (EATL) and 7/8 patients died. CONCLUSIONS: For the first time we report a promising therapeutic treatment option for RCD type I. In RCD type II, azathioprine and prednisone therapy (APT) is not effective, therefore we suggest that other (chemo)therapeutic agents are considered. Not all RCD type II patients presented with a monoclonal TCRgamma-gene rearrangement and immunohistological changes as is currently reported in the literature. Therefore, immunophenotyping seems mandatory in the work-up of RCD.

Association of FAS (TNFRSF6)-670 gene polymorphism with villous atrophy in coeliac disease.

AIM: To investigate the association of FAS gene polymorphism with coeliac disease (CD) development. METHODS: FAS-G670A gene polymorphism, located in a gamma interferon activation site, was studied in 146 unrelated CD patients and 203 healthy ethnically matched controls. The restriction fragment length polymorphism (RFLP) method was used to identify FAS-G670A gene polymorphism. RESULTS: No significant difference was found in genotype frequency between CD cases and controls. In controls, however, the frequency of the GG genotype was significantly higher in women (26.5%) than in men (12.8%) (OR= 2.44, 95% CI 1.15-5.20, P=0.020) and it was also higher in men with CD than controls (OR=2.60, 95% CI 0.96-7.05, P=0.061). The GG genotype frequency was significantly higher in patients with most severe villous atrophy (Marsh IIIc lesions) (OR=3.74, 95% CI 1.19-11.82, P=0.025). A significantly less proportion of men suffered from Marsh IIIc lesions than women (OR=0.20, 95% CI 0.06-0.68, P=0.01). The risk of having severe villous atrophy increased with the additive effect of the G allele in women (P=0.027 for trend, age and gender adjusted). CONCLUSION: FAS-G670A gene polymorphism is associated with the severity of villous atrophy in CD. Female gender is also associated with the severity of villous atrophy.

Coeliac disease and (extra)intestinal T-cell lymphomas: definition, diagnosis and treatment.

Intestinal lymphomas encompass those lymphomas with a dominant or only localized occurrence in the intestinal tract. Coeliac disease is highly associated with enteropathy-associated T-cell lymphomas (EATLs). Coeliac disease-related lymphomas can appear at nodal or extranodal sites. EATL is often multifocal with ulcerative lesions, which explains the high perforation rate at presentation or during chemotherapy. Staging includes ear-nose-throat examination and CT scan of the chest and abdomen. Positron emission tomography (PET) scanning may be valuable. Accurate diagnosis based on endoscopic biopsies is preferable; if necessary, full thickness laparoscopic small-bowel biopsies are mandatory. Refractory coeliac disease (RCD) with aberrant T cells carries a high risk of development of EATLs. There is no satisfactory treatment for EATL, the only possibility of preventing EATL development in RCD being autologous bone marrow transplantation. EATLs can present in 20% of patients as extra-small-bowel T-cell lymphomas; such as subcutaneous panniculitis-like lymphoma, hepatosplenic gamma/delta lymphoma, nodal as well as sinus, gastric or colon disease and extraintestinal T-cell lymphomas. The majority of EATLs present as large cell lymphoma CD3+, CD8-, CD30+; however, they also present as small cell lymphoma CD3+, CD8+, CD30-. Sometimes gamma/delta lymphomas in CD are recognized. Work-up of EATL must include immunohistology, T-cell flow cytometry, T-cell rearrangement and adequate imaging with CT and PET scanning.

Coeliac disease: changing views on gluten-sensitive enteropathy.

BACKGROUND: The continuing flow of scientific development in coeliac disease in the past decade points to the need for the formulation of a new concept of pathophysiology and clinical approach to the coeliac condition. Immunogenetic studies have shown a correlation of the disease to the HLA region on the short arm of chromosome 6; immunological research has led to the concept of a T-cell-driven immunologic response of the small intestine, with the identification of highly sensitive and specific antibodies; and our understanding of the histopathology of coeliac disease has changed dramatically, initiated by the proposition of a spectrum of gluten-sensitive enteropathy by Marsh in 1992. Clinical studies report a significant change in patient characteristics and epidemiology. The incidence of the disease has shifted to a majority of adult coeliacs, and it may present with less severe symptoms of malabsorption. Screening studies suggest an overall prevalence of up to 1 in 200-300. METHODS: Update on histopathology concentrating on the work of our research group. RESULTS: We specifically describe the work of our group in Arnhem concerning the identification and validation of the spectrum of intestinal histopathology in gluten-sensitive enteropathy, i.e. lymphocytic enteritis (Marsh I lesion), lymphocytic enteritis with crypt hyperplasia (Marsh II lesion), and villous atrophy, subdivided into partial villous atrophy (Marsh IIIA), subtotal villous atrophy (Marsh IIIB) and total villous atrophy (Marsh IIIC). Special attention is given to a subgroup of 'refractory coeliacs', including the identification of (pre-)malignant aberrant T cells in the intestinal mucosa of these patients. CONCLUSION: New data on immunogenetics, epidemiology, histopathology and patient characteristics point to a significant change of view on coeliac disease.

Routine duodenal biopsy to screen for coeliac disease is not effective.

BACKGROUND: Routine duodenal biopsies during upper gastrointestinal endoscopy (UGE) have been suggested to be useful in detecting coeliac disease (CD). However results from previous studies are not conclusive. The aim of this study is to investigate the diagnostic yield and cost-effectiveness of routine duodenal biopsy during UGE. METHODS: In this retrospective single-centre study, we studied 6442 patients undergoing first-time UGE at the Rijnstate Hospital, Arnhem, the Netherlands, from January 2009 to December 2010. All UGE reports were analysed for indication, duodenal intubation, and endoscopic aspect of duodenal mucosa. Endomysium and tissue transglutaminase antibody titre, when present, were scored as positive or negative. CD was defined as Marsh 3a or higher. Costs of duodenal biopsies and pathology analysis were calculated. Comparisons were done with T-tests for continuous data and Chi-square tests for categorical data. RESULTS: Forty-one patients had newly diagnosed CD; 34 of these 41 patients had definite indications for biopsy prior to UGE, e.g. positive serology or symptoms. Thus, routine duodenal biopsies identified seven patients as having CD, who otherwise would not have been biopsied. The number needed to biopsy was therefore 577, spending more than v 30,000 per case. CONCLUSIONS: We do not recommend routine duodenal biopsy to screen for coeliac disease because of the high number needed to biopsy as well as high costs.

Is the sentinel lymph node pathology protocol in breast cancer patients associated with the risk of regional recurrence?

BACKGROUND: Internationally, there is no consensus on the pathology protocol to be used to examine the sentinel lymph node (SN) in breast cancer patients. Previously, we reported that ultra-staging led to more axillary lymph node dissections (ALND). The question was, whether ultra-staging is effective in reducing the risk of regional relapse. METHODS: From January 2002 to July 2003, 541 patients from 4 hospitals were prospectively registered when they underwent a SN biopsy. In hospitals A, B, and C, 3 levels of the SN were examined pathologically, whereas in hospital D at least 7 additional levels were examined. Patients with a positive SN, including isolated tumor cells, underwent an ALND. This analysis focuses on the 341 patients with a negative SN. Primary endpoint was 5-year regional recurrence rate. RESULTS: In hospital D 34% of the patients had a negative SN as compared to 71% in hospitals A, B, and C combined (p < 0.001). At 5 years follow-up, 9 (2.6%) patients had developed a regional lymph node relapse. In hospital D none of the patients had a regional recurrence, as compared to 9 (2.9%) cases of recurrence in hospitals A, B, and C. CONCLUSION: The less intensified SN pathology protocol appeared to be associated with a slightly increased risk of regional recurrence. The absolute risk was still less than 3%, and does not seem to justify the intensified SN pathology protocol of hospital D.

Does seeding of intraluminal tumour cells cause anastomotic recurrence after oesophageal resection?

BACKGROUND: Recurrence at the cervical anastomosis of a tumour of the oesophagogastric junction after resection of the oesophagus with gastric tube reconstruction is a peculiar phenomenon in view of the distance of the primary tumour from the proximal anastomosis. It is hypothesised that contamination with tumour cells from the nasogastric tube or the gastric reconstruction tube could be responsible for this phenomenon. METHOD: Fifteen patients with a tumour of the oesophagus or gastric cardia were included. During the operation the nasogastric tube was left in the resected specimen. Debris from the tube was washed out for cytological examination. Also the gastric reconstruction tube was washed out and the debris examined for malignant cells. RESULTS: In all of the patients with a tumour extending intraluminally malignant cells were found in the nasogastric tube or the gastric reconstruction tube or both. In 92% of these patients malignant cells were found in the nasogastric tube, while 60% of the gastric reconstruction tubes were contaminated with tumour cells. CONCLUSION: The presence of intraluminal malignant cells during oesophageal resection and gastric tube reconstruction is very high, possibly leading to anastomotic recurrence. Measures should be taken to minimise local contamination with tumour cells.

Defining the contribution of the HLA region to cis DQ2-positive coeliac disease patients.

The major genetic susceptibility to coeliac disease is contributed by the human leukocyte antigen (HLA) region. The primary association is with the HLA-DQ2 molecule, encoded by the DQA1*05 and DQB1*02 alleles, which is expressed by over 90% of patients. The aim of our study was to perform an extensive scan of the entire HLA region to determine whether there is evidence for the presence of additional HLA susceptibility genes for coeliac disease in the Dutch population, acting independently of DQ2. In all, 16 microsatellite markers and the DQA1 and DQB1 genes were genotyped in simplex cis DQ2-positive coeliac disease families and cis DQ2-positive control families. Allele frequencies of markers on phase-known DQ2-positive haplotypes transmitted to patients were compared to a combined group of DQ2-positive nontransmitted and control haplotypes, thereby controlling for the DQ2 contribution. No significant differences at any of the marker loci were detected, suggesting that DQ2 is the major HLA risk factor for coeliac disease. Individuals homozygous for DQ2 or heterozygous for DQA1*05-DQB1*02/DQA1*0201-DQB1*02 were found to be at five-fold increased risk for development of coeliac disease (P<10(-8)). This risk seems to be conferred by the presence of a second DQB1*02 allele next to one DQA1*05-DQB1*02 haplotype, independently of the second DQA1 allele.

A microarray screen for novel candidate genes in coeliac disease pathogenesis.

BACKGROUND AND AIMS: The causative molecular pathways underlying the pathogenesis of coeliac disease are poorly understood. To unravel novel aspects of disease pathogenesis, we used microarrays to determine changes in gene expression of duodenal biopsies. METHODS: cDNA microarrays representing 19 200 genes were used to compare gene expression profiles of duodenal biopsies from 15 coeliac disease patients with villous atrophy (Marsh III) and seven control individuals with normal biopsies (Marsh 0). In addition, the specific effect of gluten was studied by comparing the expression profiles of Marsh III lesions of seven patients exposed to gluten with four patients on a gluten free diet. RESULTS: Comparing Marsh III with Marsh 0 lesions identified 109 genes that differed significantly (p<0.001) in expression levels between patients and controls. A large number of these genes have functions in proliferation and differentiation pathways and might be important for correct development of crypt-villous units. Alterations in these pathways may lead to the characteristic hyperplasia and villous atrophy seen in coeliac disease. The analyses also revealed 120 differentially expressed genes (p<0.005) when comparing patients on a gluten free diet with those exposed to gluten. These genes further strengthen our observation of increased cell proliferation in the presence of gluten. CONCLUSIONS: Our study provides new candidate genes in the pathogenesis of coeliac disease. Based on our results, we hypothesise that villous atrophy in coeliac disease patients is due to failure in cell differentiation. These genes are involved in pathways not previously implicated in coeliac disease pathogenesis and they may provide new targets for therapy.

IL12B and IRF1 gene polymorphisms and susceptibility to celiac disease.

Celiac disease (CD) is a common gastro-intestinal disorder resulting from permanent intolerance to wheat gliadins and related proteins in rye and barley. In addition to the strong genetic association with HLA-DQ2 and HLA-DQ8, a genetic region on chromosomes 5 (CELIAC2) has been identified that harbours a susceptibility gene for CD. The gene(s) responsible for this association, however, remains to be identified. In the present study we evaluated polymorphisms in the genes encoding interleukin-12 p40 (IL12B) and interferon regulatory factor 1 (IRF1). Both genes are located in the celiac2 region, and have key roles in inducing interferon (IFN)-gamma secreting T helper 1 (Th1) cells, one of the immunological hallmarks of CD. The frequencies of a TaqI gene polymorphism in the 3' UTR of IL12B and a HinfI gene polymorphism in the 3' UTR of IRF1 were studied in 258 Dutch CD patients and 237 ethnically matched healthy controls. The transmission of the polymorphic variants from parents to affected child was determined in 123 families with at least one affected child. The frequencies of the IL12B TaqI gene polymorphism and the IRF1 HinfI gene polymorphism did not differ significantly between patients and controls. In addition, in the family study, no deviation from the expected transmission from parents to affected child of any of the polymorphic variants was found. The IL12B TaqI and the IRF1 HinfI gene polymorphisms do not appear to be involved in susceptibility to CD. Further studies on the factors that drive the Th1 immunopathology in CD are required.