Variation at ABO histo-blood group and FUT loci and diffuse and intestinal gastric cancer risk in a European population.

ABO blood serotype A is known to be associated with risk of gastric cancer (GC), but little is known how ABO alleles and the fucosyltransferase (FUT) enzymes and genes which are involved in Lewis antigen formation [and in Helicobacter pylori (H. pylori) binding and pathogenicity] may be related to GC risk in a European population. The authors conducted an investigation of 32 variants at ABO and FUT1-7 loci and GC risk in a case-control study of 365 cases and 1,284 controls nested within the EPIC cohort (the EPIC-Eurgast study). Four variants (including rs505922) in ABO, and allelic blood group A (AO+AA, odds ratio=1.84, 95%CI=1.20-2.80) were associated with diffuse-type GC; however, conditional models with other ABO variants indicated that the associations were largely due to allelic blood group A. One variant in FUT5 was also associated with diffuse-type GC, and four variants (and haplotypes) in FUT2 (Se), FUT3 (Le) and FUT6 with intestinal-type GC. Further, one variant in ABO, two in FUT3 and two in FUT6 were associated with H. pylori infection status in controls, and two of these (in FUT3 and FUT6) were weakly associated with intestinal-type GC risk. None of the individual variants surpassed a Bonferroni corrected p-value cutoff of 0.0016; however, after a gene-based permutation test, two loci [FUT3(Le)/FUT5/FUT6 and FUT2(Se)] were significantly associated with diffuse- and intestinal-type GC, respectively. Replication and functional studies are therefore recommended to clarify the role of ABO and FUT alleles in H. pylori infection and subtype-specific gastric carcinogenesis.

Gastroesophageal reflux disease and eosinophilic esophagitis in infants and children. A study of esophageal pH, multiple intraluminal impedance and endoscopic ultrasound.

OBJECTIVE: Eosinophilic esophagitis (EE) and gastroesophageal reflux disease (GERD) in childhood share aspects of symptomatology. In order to characterize EE and GERD in infants and children with symptoms of GERD we performed a prospective investigation including prolonged esophageal pH measurement, multiple intraluminal impedance (MII) and esophageal wall estimation by endoscopic ultrasound (EUS). MATERIAL AND METHODS: Infants and children (0-15 years) with typical symptoms of GERD persisting after a 14-days proton pump inhibitor trial were included in a prospective study protocol. Upper endoscopy and EUS of the esophageal wall were performed followed by combined esophageal MII and pH measurement for 24 h. RESULTS: A total of 78 infants and children were investigated: EE patients (n = 6), GERD patients (n = 28) and a group of infants and children with normal investigations (n = 44). The GERD group did not show a significantly higher number of non-acid reflux episodes (p = 0.9) than the patients with normal investigations. In all patients gastroesophageal reflux regularly extended into the proximal esophagus. EUS in four EE patients suggested an increased thickness of the mucosal layers both in the distal and in the proximal part of the esophagus. CONCLUSIONS: Esophageal MII indicated that neutral non-acid reflux episodes do not occur frequently in pediatric GERD or in EE. MII and pH-metry indicated that the majority of reflux episodes both in patients and controls pass into the proximal esophagus. EUS measurements suggested in EE patients a thickened mucosa both in the proximal and the distal part of the esophagus as compared to children with GERD and disease controls.

Eosinophilic oesophagitis in infants and children in the region of southern Denmark: a prospective study of prevalence and clinical presentation.

OBJECTIVE: Eosinophilic oesophagitis (EE) is a clinical entity characterised by a set of symptoms and eosinophilic infiltration of the oesophageal epithelium. Recent reports indicate that EE is increasingly diagnosed in paediatric patients. We aimed to evaluate the epidemiology of paediatric EE in a European population. DESIGN: Infants and children in the Region of Southern Denmark were prospectively referred for further evaluation of symptoms of gastroesophageal reflux disease (GERD) after treatment failure with a proton pump inhibitor. The evaluation included endoscopy, 24-hour oesophageal pH-metry, histology of oesophageal biopsies, and investigations for food allergy (double-blind, placebo-controlled food challenge, skin prick test, S-IgE antibodies, atopy patch test). RESULTS: Of the 78 referred patients, 28 qualified for a diagnosis of GERD. Six children had >15 eosinophils per high-power field in biopsies from the oesophageal mucosa and qualified for the diagnosis of EE. The median age at diagnosis was 9.6 years. In 4 of the 6 patients, food allergy was confirmed by double-blind, placebo-controlled food challenge. In the Region of Southern Denmark with a paediatric population of 256,164 between 0 and 16 years of age, a yearly incidence of EE of 0.16/10,000 was estimated. CONCLUSION: We report a European prospective study of EE. It was documented in 6 of 78 patients with symptoms of GERD corresponding to an annual incidence of 0.16/10,000 infants and children.

Adenocarcinoma arising from the rectal stump eleven years after excision of an ileal J-pouch in a patient with ulcerative colitis: report of a case.

Adenocarcinomas in relation to the ileal J-pouch after restorative proctocolectomy for ulcerative colitis have been recently reported with increasing frequency. All previously reported cases have occurred in patients with their ileal pouch in situ. We report a case of adenocarcinoma in the anal canal 11 years after removal of a failed ileal J-pouch. Mucosectomy had been performed at the restorative proctocolectomy. The anus had been left in place at the pouch excision because of severe fibrosis in the pelvis. If it is decided to remove an ileal pouch permanently, a total abdominoperineal excision should be performed, particularly in patients with risk factors for cancer development.

Clinical benefit of a gluten-free diet in type 1 diabetic children with screening-detected celiac disease: a population-based screening study with 2 years' follow-up.

OBJECTIVE: This study was performed to 1) determine the prevalence of celiac disease in Danish children with type 1 diabetes and 2) estimate the clinical effects of a gluten-free diet (GFD) in patients with diabetes and celiac disease. RESEARCH DESIGN AND METHODS: In a region comprising 24% of the Danish population, all patients <16 years old with type 1 diabetes were identified and 269 (89%) were included in the study. The diagnosis of celiac disease was suspected in patients with endomysium and tissue transglutaminase antibodies in serum and confirmed by intestinal biopsy. Patients with celiac disease were followed for 2 years while consuming a GFD. RESULTS: In 28 of 33 patients with celiac antibodies, an intestinal biopsy showed villous atrophy. In 5 patients, celiac disease had been diagnosed previously, giving an overall prevalence of 12.3% (95% CI 8.6-16.9). Patients with celiac disease had a lower SD score (SDS) for height (P < 0.001) and weight (P = 0.002) than patients without celiac disease and were significantly younger at diabetes onset (P = 0.041). A GFD was obtained in 31 of 33 patients. After 2 years of follow-up, there was an increase in weight SDS (P = 0.006) and in children <14 years old an increase in height SDS (P = 0.036). An increase in hemoglobin (P = 0.002) and serum ferritin (P = 0.020) was found, whereas HbA(1c) remained unchanged (P = 0.311) during follow-up. CONCLUSIONS: This population-based study showed the highest reported prevalence of celiac disease in type 1 diabetes in Europe. Patients with celiac disease showed clinical improvements with a GFD. We recommend screening for celiac disease in all children with type 1 diabetes.

Characterization of anti-TIMP-1 monoclonal antibodies for immunohistochemical localization in formalin-fixed, paraffin-embedded tissue.

The aim of this study was to evaluate seven anti-TIMP-1 (tissue inhibitor of metalloproteinase-1) monoclonal antibodies by immunohistochemical (IHC) staining of formalin-fixed, paraffin-embedded (FFPE) tissue. Detection of the TIMP-1 protein was studied by IHC in FFPE human archival normal and neoplastic samples. Indirect IHC technique was used, and the seven antibodies (clones VT1, VT2, VT4, VT5, VT6, VT7, and VT8) were tested in various concentrations using different pretreatment protocols. All seven VT antibodies specifically immunostained the cytoplasm of islets of Langerhans cells in normal pancreas, epithelial cells of hyperplastic prostate, tumor cells of medullary thyroid carcinoma, and fibroblast-like cells of malignant melanoma. Specificity of the anti-TIMP-1 antibodies was confirmed by several controls, e.g., Western blotting on proteins extracted from FFPE tissue showed that the VT7 antibody reacted specifically with a protein band of approximately 28 kDa, corresponding to the molecular mass of TIMP-1. However, sensitivity varied with the different antibodies. Use of heat-induced epitope retrieval (HIER) and the VT7 clone applied at low concentrations demonstrated more intense immunoreactivity with the TIMP-1-positive cell types compared to the other six clones. Furthermore, when tested on a range of normal and neoplastic endocrine tissues, the VT7 clone demonstrated immunoreactivity with all neuroendocrine cell types. In conclusion, all seven antibodies detected TIMP-1 protein in various normal and neoplastic FFPE tissues, but one clone, VT7, was superior for IHC staining of TIMP-1 in FFPE tissue sections when using HIER.

Optimization of antibodies for detection of the mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 using a biotin-free visualization system.

Testing for microsatellite instability (MSI) has become an important step in the planning of therapeutic and follow-up procedures for patients with colorectal cancer, both as a prognostic marker and as a screening tool for hereditary non-polyposis colorectal cancer. Today the gold standard for MSI testing is based on the polymerase chain reaction. Immunohistochemistry may represent an alternative or complement to molecular MSI testing. Antibodies against the protein products of the most commonly affected mismatch repair genes (hMLH1, hMSH2, hMSH6, and hPMS2) have been available for some time now. However, the quality of the primary antibody and optimization of the antigen retrieval methods are essential to get reproducible results. The aim of the present study was to test and optimize a panel of antibodies against the mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 using biotin-free, polymer-based visualization systems. The antibodies were tested on multitissue blocks containing normal tissue and tumor tissue from patients with known microsatellite-stable and microsatellite-instable tumors. For all four antibody groups, the chosen clones gave specific and reproducible staining. Furthermore, with the PowerVision+ detection system, the influence of endogenous biotin was eliminated, the incubation time with the primary antibody was significantly reduced, and the primary antibody could be further diluted. The authors found that immunohistochemistry may provide a cost-effective and time-saving complement to the molecular MSI analysis, and using the PowerVision+ detection system has greatly decreased the turnaround time as well as reduced the cost of immunohistochemistry in the authors' laboratory.

Tumor karyotype predicts clinical outcome in colorectal cancer patients.

PURPOSE: To investigate the prognostic value of the overall karyotypic features and specific chromosome aberrations in colorectal cancer (CRC). PATIENTS AND METHODS: Cytogenetic features of 150 primary CRCs investigated at the time of surgery were correlated with patient survival by univariate and multivariate analyses, using classical clinicopathologic parameters as covariates. RESULTS: In univariate analysis, in addition to tumor grade and clinical stage, structural aberrations as well as rearrangements of chromosomes 8 and 16 were significantly correlated with shorter overall survival. Karyotypic complexity, rearrangements of chromosomes 8 and 16, and loss of chromosome 4 were significantly correlated with shorter disease-free survival. In multivariate analysis, in addition to tumor grade, the type of chromosome aberrations (structural or numerical), ploidy, and loss of chromosome 18 came across as independent prognostic factors in the group of all patients. In the subset of patients with stage I and II carcinomas, none of the clinicopathologic variables could independently predict patient survival, whereas the presence of structural chromosomal aberrations was the only independent predictor of poor prognosis. In the subset of patients with stage III carcinomas, the presence of structural changes of chromosome 8 was a stronger independent predictor of prognosis than was tumor grade. CONCLUSION: Cytogenetic tumor features are valuable predictors of prognosis in CRC patients. The tumor karyotype should therefore be taken into account in the clinical management of patients with this disease, especially for patients having cancers of the early or intermediate stages I, II, and III.

Plasma TIMP-1 in patients with colorectal adenomas: a prospective study.

Colorectal cancer patients have increased plasma levels of tissue inhibitor of metalloproteinases-1 (TIMP-1). However, it remains unresolved whether colorectal adenomas are associated with increased plasma TIMP-1. Plasma TIMP-1 levels were determined using an immunoassay in 121 patients prospectively enrolled from surveillance colonoscopy programmes. TIMP-1 levels were correlated to various clinicopathological parameters. No significant associations were found between plasma TIMP-1 and size, macro- or microscopic morphology or grade of dysplasia of the adenomas. No significant differences in TIMP-1 levels were found between patients with adenomas (n = 36), hyperplastic polyps (n = 12) or no pathology (n = 68) of the large intestine. However, patients with colonic (n = 3) or rectal (n = 2) adenocarcinomas had significantly increased TIMP-1 levels (P = 0.02). The present study demonstrates that measurement of plasma TIMP-1 cannot be used for the identification of adenomatous or hyperplastic polyps of the large intestine.

Secondary effects induced by the colon carcinogen azoxymethane in BDIX rats.

Azoxymethane (AOM) is claimed to be a colon-specific carcinogen. In our studies, AOM was administered to adult BDIX/OrlIco rats by four weekly subcutaneous injections of 15 mg/kg body weight each - two periods of 2 weeks of AOM treatment separated by a one-week break. This treatment schedule resulted in colon carcinomas with a high frequency (75-100%) and with a high reproducibility. However, some serious side effects are associated with this carcinogen treatment. In addition to the colorectal tumours, we found small intestinal tumours, hepatic lesions and a high frequency of mesenchymal renal tumours which increased with longer latency periods. The renal tumours were only found in female rats, and this indicates a possible relation to sex hormones. We therefore analyzed both male and female kidneys for the expression of estrogen and progesterone receptors by immunohistochemical methods. A positive nuclear reaction for estrogen receptor was present in most tumour cells in all tumours and occasionally in nuclei of entrapped tubular cells, but never in glomeruli. Normal appearing renal tissue from female rats showed no positive reaction, but in male rats a slight nuclear reaction was seen in tubuli in the peripheral part of the medulla. A similar pattern was seen for progesterone receptors, but less pronounced. No rats developed tumours in the external ear canal, which is in contrast to studies performed in other rat strains. This may therefore be strain related. In order to reduce the secondary effects of the induction of colon cancer by AOM, it is advisable to use male rats only and a maximum latency period of 32 weeks.

Prognostic factors in anal carcinoma.

The pathologist's role in the evaluation of patients with anal squamous carcinoma is now largely restricted to establishing the diagnosis on small biopsies. Staging is performed by imaging techniques and grading is uncertain as the biopsy may not be representative of the whole tumour. Histological subtyping is unreliable and has not been proven to be of prognostic significance. A considerable effort resulting in nearly 50 reports on cytogenetic, flow cytometric, immunohistochemical and other investigations has given new insight into pathogenesis but little guidance with regard to the individual prognosis or choice of treatment. It is concluded that future research should concentrate on larger, probably multicentre series using standardised methods and criteria for evaluation.

Analysis of beta-catenin, Ki-ras, and microsatellite stability in azoxymethane-induced colon tumors of BDIX/Orl Ico rats.

The aim of the study reported here was to investigate whether the azoxymethane (AOM)-induced colon cancer rat model mimics the human situation with regard to microsatellite stability, changes in expression of beta-catenin, and/or changes in the sequence of the proto-oncogene Ki-ras. Colon cancer was induced by administration of four weekly doses of AOM (15 mg/kg of body weight per week) separated by a one-week break between the second and third injections. As the histopathologic characteristics of this model resemble those of the human counterpart, further characterization of the genetic changes was undertaken. The animals were euthanized 28 to 29 weeks after the first AOM injection, and tumor specimens were taken for histologic and DNA analyses. Since microsatellite variation was found in only a few (< 2%) specimens, the model can be considered as having stable microsatellites. This result is in accordance with those of similar studies in other rat models and with most human colorectal cancers. Immunohistochemical analyses of beta-catenin did not reveal loss of gene activity, nor did the sequencing of Ki-ras reveal mutations. These results are in contrast to most findings in comparable rat studies. The deviations may be due to differences in exposure to the carcinogen or difference in strain and/or age. The lack of beta-catenin and Ki-ras alterations in this colon cancer model is unlike human sporadic colorectal cancers where these genetic changes are common findings.

Comparative study of histopathologic characterization of azoxymethane-induced colon tumors in three inbred rat strains.

To obtain controlled genetic variation, colon cancer was chemically induced by use of four subcutaneous injections of azoxymethane (15 mg/kg of body weight/wk) to rats of 3 inbred strains (BDIX/OrlIco, F344/NHsd, WAG/Rij). The selection was based on the availability of established colon cancer cell lines arising from these particular strains. In the first experiment, only female rats were used; in the second experiment, both sexes were studied. The goal was to select a rat strain giving the highest tumor frequency with the shortest latency period in reproducible manner. The histologic characteristics should resemble the corresponding human tumors. The size of the tumors should be at about 1 cm in diameter, as these tumor cells were intended to be used in future transplantation studies. The two experiments yielded highly reproducible results: histologic evaluation of all colon tumors in all three rat strains revealed adenomas and adenocarcinomas closely resembling their human counterpart. The BDIX strain had the highest tumor frequency (75%) in both sexes and the shortest minimal latency period (28 weeks in experiment 1; 23 weeks in experiment 2). Tumor size of about 1 cm in diameter was found most often in the BDIX strain. On the basis of results of these two experiments, the BDIX strain has been selected for future study.

[Solid tumors. Classification based on molecular biology, prognosis and therapy]. / Solide tumorer. Molekylaerbiologisk baseret klassifikation, prognose og terapi.

The introduction of molecular techniques into the routine diagnostic analysis of solid tumours has been slower than for hematological neoplasias, both because the former in general genetically are more heterogeneous and because their tumour cells are less accessible. This review presents examples of methods which can be used in the identification of risk groups, classification and prognosis of tumours, detection of minimal residual disease, choice of therapeutic strategy and tracing of possible hereditary cases. It is concluded that molecular laboratories should be established in connection with departments of pathology at the larger hospitals and that further development should take place through close collaboration between pathologists, molecular biologists, and clinicians.

A variant form of the human deleted in malignant brain tumor 1 (DMBT1) gene shows increased expression in inflammatory bowel diseases and interacts with dimeric trefoil factor 3 (TFF3).

The protein deleted in malignant brain tumors (DMBT1) and the trefoil factor (TFF) proteins have all been proposed to have roles in epithelial cell growth and cell differentiation and shown to be up regulated in inflammatory bowel diseases. A panel of monoclonal antibodies was raised against human DMBT1(gp340). Analysis of lung washings and colon tissue extracts by Western blotting in the unreduced state, two antibodies (Hyb213-1 and Hyb213-6) reacted with a double band of 290 kDa in lung lavage. Hyb213-6, in addition, reacted against a double band of 270 kDa in colon extract while Hyb213-1 showed no reaction. Hyb213-6 showed strong cytoplasmic staining in epithelial cells of both the small and large intestine whereas no staining was seen with Hyb213-1. The number of DMBT1(gp340) positive epithelial cells, stained with Hyb213-6, was significantly up regulated in inflammatory colon tissue sections from patients with ulcerative colitis (p<0.0001) and Crohn's disease (p = 0.006) compared to normal colon tissue. Immunohistochemical analysis of trefoil factor TFF1, 2 and 3 showed that TFF1 and 3 localized to goblet cells in both normal colon tissue and in tissue from patients with ulcerative colitis or Crohn's disease. No staining for TFF2 was seen in goblet cells in normal colon tissue whereas the majority of tissue sections in ulcerative colitis and Crohn's disease showed sparse and scattered TFF2 positive goblet cells. DMBT1 and TFF proteins did therefore not co-localize in the same cells but localized in adjacent cells in the colon. The interaction between DMBT1(gp340) and trefoil TFFs proteins was investigated using an ELISA assay. DMBT1(gp340) bound to solid-phase bound recombinant dimeric TFF3 in a calcium dependent manner (p<0.0001) but did not bind to recombinant forms of monomeric TFF3, TFF2 or glycosylated TFF2. This implies a role for DMBT1 and TFF3 together in inflammatory bowel disease.

[Polyps in the stomach and duodenum]. / Polypper i ventrikel og duodenum.

Polyps are reported in 3-5% of adults undergoing upper gastrointestinal endoscopy. The most common types in the stomach are fundic gland polyps, followed by hyperplastic polyps and adenomas. Gastric metaplasia, adenomas and inflammatory polyps are the most common polyps in the duodenum. Treatment options and aggressiveness should be balanced by detailed knowledge of the malignant potential of the different polyps as well as the symptoms, the patient's age and comorbidity.

Influence of long-term colonoscopic surveillance on incidence of colorectal cancer and death from the disease in patients with precursors (adenomas).

Case-control studies and short term prospective studies have suggested that selected groups of patients with precursors of colorectal cancer may benefit from colonoscopic surveillance after initial removal of adenomas. The aim of the present study was to demonstrate such a possible benefit from long term (1-24 years) colonoscopic surveillance in a population of patients with all types of adenomas regardless of size and way of removal. Two thousand and forty-one patients with a first time diagnosis of colorectal adenoma were included in prospective surveillance between year 1978 and 2002. All adenomas were considered. Incidence of CRC and mortality from CRC was calculated, using age, sex, and calendar specific number of person years of follow-up for comparison with the standard Danish population. CRC was found in 27 patients, the expected number being 41 (RR 0.65, 95% CI 0.43-0.95). Three of the 27 patients died from CRC, the expected number being 25 (RR 0.12, 95% CI 0.03-0.36). A total of 6 289 colonoscopies resulted in severe complications in 20 patients and two died from complications. Long-term colonoscopic surveillance may reduce incidence of CRC as well as mortality in patients with sporadic adenomas. The benefit is reduced to a minor degree by complications from surveillance.

[Prevalence of coeliac disease (CD) in children with type 1 diabetes (T1D)]. / Høj praevalens i Region Syd af cøliaki blandt børn med type 1-diabetes. Sygdomsopsporing samt opfølgning på glutenfri diaet--sekundaerpublikation.

This screening study was performed to determine the prevalence of coeliac disease (CD) in children with type 1 diabetes (T1D) and to estimate the clinical effects of a gluten-free diet. In the region of Southern Denmark all patients under 16 years of age with T1D were identified and 269 (89%) were included. CD was diagnosed in 33 (12.3%). Patients with CD had a lower height SDS and weight SDS and were younger at diabetes onset. After 2 years on a gluten-free diet there were significant improvements in clinical and biochemical parameters. We recommend screening of CD in all children with T1D.