G. De Hertogh

KRAS mutation testing for predicting response to anti-EGFR therapy for colorectal carcinoma: proposal for an European quality assurance program.

Novel therapeutic agents targeting the epidermal growth factor receptor (EGFR) have improved outcomes for patients with colorectal carcinoma. However, these therapies are effective only in a subset of patients. Activating mutations in the KRAS gene are found in 30–40% of colorectal tumors and are associated with poor response to anti-EGFR therapies. Thus, KRAS mutation status can predict which patient may or may not benefit from anti-EGFR therapy. Although many diagnostic tools have been developed for KRAS mutation analysis, validated methods and standardized testing procedures are lacking. This poses a challenge for the optimal use of anti-EGFR therapies in the management of colorectal carcinoma. Here we review the molecular basis of EGFR-targeted therapies and the resistance to treatment conferred by KRAS mutations. We also present guideline recommendations and a proposal for a European quality assurance program to help ensure accuracy and proficiency in KRAS mutation testing across the European Union.

Mucosal gene signatures to predict response to infliximab in patients with ulcerative colitis

Background and aims: Infliximab is an effective treatment for ulcerative colitis with over 60% of patients responding to treatment and up to 30% reaching remission. The mechanism of resistance to anti-tumour necrosis factor α (anti-TNFα) is unknown. This study used colonic mucosal gene expression to provide a predictive response signature for infliximab treatment in ulcerative colitis. Methods: Two cohorts of patients who received their first treatment with infliximab for refractory ulcerative colitis were studied. Response to infliximab was defined as endoscopic and histological healing. Total RNA from pre-treatment colonic mucosal biopsies was analysed with Affymetrix Human Genome U133 Plus 2.0 Arrays. Quantitative RT-PCR was used to confirm microarray data. Results: For predicting response to infliximab treatment, pre-treatment colonic mucosal expression profiles were compared for responders and non-responders. Comparative analysis identified 179 differentially expressed probe sets in cohort A and 361 in cohort B with an overlap of 74 probe sets, representing 53 known genes, between both analyses. Comparative analysis of both cohorts combined, yielded 212 differentially expressed probe sets. The top five differentially expressed genes in a combined analysis of both cohorts were osteoprotegerin, stanniocalcin-1, prostaglandin-endoperoxide synthase 2, interleukin 13 receptor alpha 2 and interleukin 11. All proteins encoded by these genes are involved in the adaptive immune response. These markers separated responders from non-responders with 95% sensitivity and 85% specificity. Conclusion: Gene array studies of ulcerative colitis mucosal biopsies identified predictive panels of genes for (non-)response to infliximab. Further study of the pathways involved should allow a better understanding of the mechanisms of resistance to infliximab therapy in ulcerative colitis. ClinicalTrials.gov number, NCT00639821.

On the existence and location of cardiac mucosa: an autopsy study in embryos, fetuses, and infants

Background: The incidence of gastric cardiac adenocarcinoma has increased in the last decades. Gaining insight into the pathogenesis of this lesion is hampered by the limited knowledge of the origin and histology of cardiac mucosa (CM). Currently, the location, extent, and even the existence of CM are controversial. Aims: We studied the development of the gastro-oesophageal junction (GOJ) in embryos, fetuses, and infants to clarify if CM is a normal structure at birth and where it is located. Subjects: Twenty one autopsy cases were evaluated ranging in age from 13 weeks’ gestational age (GA) to seven months. Methods: The distal oesophagus and proximal part of the stomach were embedded entirely. Serial sections were stained with haematoxylin-eosin and alcian blue/periodic acid-Schiff. The following parameters were measured: length of abdominal oesophagus; length of columnar lined oesophagus; length of CM; and distance from CM to angle of His. Results: CM was present in all evaluated sections. Its mean length varied throughout gestation. A maximum value was reached at a GA of 16 weeks (1.2 mm). After term delivery it was very short (0.3–0.6 mm). CM was proximal to, or straddled, the angle of His in all cases. During gestation, the mucin staining pattern of the CM was to a high degree similar to that of the developing pyloric mucosa. Conclusions: CM develops during pregnancy and is present at birth as a normal structure. If the angle of His is taken as a landmark for the GOJ, CM is located in the distal oesophagus.

Intestinal immune activation in presumed post-infectious functional dyspepsia.

Abstract  Functional dyspepsia (FD) symptoms may develop after an acute gastroenteritis. In post‐infectious (PI) irritable bowel syndrome, persisting low‐grade colonic inflammation and increased enterochromaffine cells (EC) counts have been reported. The aim was to compare signs of inflammation and EC hyperplasia on duodenal biopsies in presumed PI‐FD and unspecified‐onset (U‐)FD. Duodenal biopsies were obtained in 12 U‐FD and 12 PI‐FD (on average 13 months after the acute event) patients. The presence of intra‐epithelial, intravillar, and the number of CD3, CD4, CD8 and CD68+ cells per crypts, and the mean number of Chromogranine A (CA) positive cells per villus were compared. We also measured gastric emptying and assessed proximal stomach function with a barostat. Data are shown as mean ± SEM. Focal aggregates of T cells and focal CD8+ aggregates, were found in PI‐FD but not in U‐FD patients (respectively 5/12 vs 0/12, P = 0.02 and 5/9 vs 0/11, P < 0.01). In patients with focal aggregates, gastric emptying was delayed (189 ± 37 min vs 98 ± 11 min, P = 0.002). The number of CD4+ cells per crypt (0.52 ± 1.6 vs 1.22 ± 2.18, P = 0.04), and the number of intravillar CD4+ cells (0.5 ± 0.2 vs 2.7 ± 0.7, P = 0.01) were reduced, while the number of CD68+ cells per crypt was increased (0.64 ± 0.13 vs 0.40 ± 0.05, P = 0.03) in PI‐FD. The number of EC and CA were comparable. PI‐FD is associated with persisting focal T‐cell aggregates, decreased CD4+ cells and increased macrophage counts surrounding the crypts. This may indicate impaired ability of the immune system to terminate the inflammatory response after acute insult.

Impact of lipoteichoic acid modification on the performance of the probiotic Lactobacillus rhamnosus GG in experimental colitis.

While some probiotic strains might have adjuvant effects in the therapy for inflammatory bowel diseases (IBD), these effects remain controversial and cannot be generalized. In this study, a dltD mutant of the model probiotic Lactobacillus rhamnosus GG (LGG), having a drastic modification in its lipoteichoic acid (LTA) molecules, was analysed for its effects in an experimental colitis model. Dextran sulphate sodium (DSS) was used to induce either moderate to severe or mild chronic colitis in mice. Mice received either phosphate‐buffered saline (PBS), LGG wild‐type or the dltD mutant via the drinking water. Macroscopic parameters, histological abnormalities, cytokine and Toll‐like receptor (TLR) expression were analysed to assess disease activity. LGG wild‐type did not show efficacy in the different experimental colitis set‐ups. This wild‐type strain even seemed to exacerbate the severity of colitic parameters in the moderate to severe colitis model compared to untreated mice. In contrast, mice treated with the dltD mutant showed an improvement of some colitic parameters compared to LGG wild‐type‐treated mice in both experimental models. In addition, treatment with the dltD mutant correlated with a significant down‐regulation of Toll‐like receptor‐2 expression and of downstream proinflammatory cytokine expression in the colitic mice. These results show that molecular cell surface characteristics of probiotics are crucial when probiotics are considered for use as supporting therapy in IBD.

Epithelioid granulomas, pattern recognition receptors, and phenotypes of Crohn’s disease

Introduction: Crohn’s disease is a chronic inflammatory disorder of the gut. It is assumed that a defective interaction between the bacterial flora of the gut and the innate immune system plays a key role in the pathogenesis of the disease. This may lead to specific histological lesions. The epithelioid granuloma is particularly interesting in this regard as it is also observed in several bacterial infections of the gut. Aims and methods: We hypothesised that genetic or environmental factors with a known influence on inflammation or immunity would lead to an increased prevalence of granulomas. Therefore, surgical specimens from 161 patients were evaluated for the presence of granulomas. Patients were genotyped for the three single nucleotide polymorphisms in caspase recruitment domain 15 (CARD15)/NOD2 associated with CD and for Asp299Gly in Toll-like receptor 4 (TLR4). Results: The overall prevalence of granulomas was 68.9%. We did not find a significant correlation between granulomas and TLR4 or CARD15 variants. The frequency of granulomas increased with more distal disease (63% small bowel, 72% right colon, 88% left colon, 90% rectum; p = 0.01). Granulomas were more frequent in younger patients (odds ratio 0.95 (95% confidence interval 0.92–0.98) p = 0.007). Conclusion: In this study of 161 well documented CD patients, we found no significant association between CARD15 and TLR4 variants and granulomas. This finding seems to refute our initial hypothesis. However, it may be that additional factors are needed for granuloma development. Granulomas may develop only when specific bacterial components are present. Therefore, future research on granuloma pathogenesis should be orientated towards detection and identification of bacterial components in these lesions.

Recurrence pattern in patients with a pathologically complete response after neoadjuvant chemoradiotherapy and surgery for oesophageal cancer

Little is known about recurrence patterns in patients with a pathologically complete response (pCR) or an incomplete response after neoadjuvant chemoradiotherapy (CRT) followed by resection for oesophageal cancer. This study was performed to determine the pattern of recurrence in patients with a pCR after neoadjuvant CRT followed by surgery.

Validation of 16S rDNA sequencing in microdissected bowel biopsies from Crohn's disease patients to assess bacterial flora diversity

The bowel flora is implicated in Crohns disease (CD) pathogenesis but its precise role is still unclear. Several non‐mutually exclusive hypotheses have been proposed: an unidentified persistent pathogen; excessive bacterial translocation; an immune system abnormality in response to normal bacteria; or a breakdown in the balance between protective and harmful bacteria. These hypotheses can be tested by identifying bacteria in specific microscopic bowel structures or lesions. The present paper describes a novel technique to assess bacterial flora diversity in bowel biopsies, by combining laser capture microdissection with broad‐range 16S rDNA sequencing. Fifty‐four samples comprising histologically normal and pathological mucosa, MALT, ulcers, submucosal lymphangiectasias, epithelioid granulomas, and lymph nodes were microdissected out of 30 bowel biopsies from five CD patients. Bacterial 16S rDNA was successfully amplified by PCR in all samples, and PCR products from 15 samples were selected for cloning and sequence analysis. A total of 729 bacterial DNA sequences were analysed, which could be attributed to six different phyla (Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Fusobacteria, and Planctomycetes). DNA from typical bowel bacteria (Enterobacteriaceae, Clostridiales, Bacteroidetes, Fusobacteria) was detected in all microdissected areas. It was thus convincingly demonstrated that 16S rDNA sequencing can be combined with microdissection to study the bowel flora. However, no specific persistent pathogen causal for CD was identified. The results suggest that Enterobacteriaceae may initiate or colonize ulcers in CD. Translocation of bacteria through established mucosal lesions or as a result of increased permeability may be involved in the evolution towards chronic inflammation and in the establishment of persistent lesions. Further study is needed to confirm these preliminary findings. Copyright

FISH analysis of Lactobacillus biofilms in the gastrointestinal tract of different hosts.

Aims:  To investigate the spatial organization of endogenous and exogenously applied Lactobacillus communities at specific locations in the adult gastrointestinal tract of different hosts.

Cutaneous cellular fibrous histiocytoma metastasizing to the lungs

1. Ellis GL, Auclair PL. Ductal papillomas. In Ellis GL, Auclair PL, Gnepp DR eds. Surgical pathology of the salivary glands. Philadelphia: W.B. Saunders Co., 1991: 238–251. 2. Shiotani A, Kawaura M, Tanaka Y et al. Papillary adenocarcinoma possibly arising from an intraductal papilloma of the parotid gland. ORL 1994; 56; 112–115. 3. King PH, Hill J. Intraductal papilloma of parotid gland. J. Clin. Pathol. 1993; 46; 175–176. 4. Alho OP, Kristo A, Luotonen J et al. Intraductal papilloma as a cause of a parotid duct cyst. A case report. J. Laryngol. Otol. 1996; 110; 277–278.