Defective macrophage handling of Escherichia coli in Crohn's disease.

BACKGROUND AND AIM: Escherichia coli can be isolated from lamina propria macrophages in Crohn's disease (CD), and their intramacrophage persistence may provide a stimulus for inflammation. To further determine the contributions of macrophage dysfunction and E. coli pathogenicity to this, we aimed to compare in vitro functioning of macrophages from patients with CD and healthy controls (HC) in response to infection with CD-derived adherent-invasive E. coli (AIEC) and less pathogenic E. coli strains. METHODS: Monocyte-derived macrophages were cultured from patients with CD and HC. Intramacrophage survival of E. coli strains (CD-derived adherent-invasive [AI] and non-AI strains and laboratory strain K-12) was compared. Macrophage cytokine release (tumor necrosis factor alpha [TNFα], interleukin [IL]-23, IL-8 and IL-10) and monocyte phagoctyosis and respiratory burst function were measured after E. coli infection. For CD patients, laboratory data were correlated with clinical phenotype, use of immunomodulation, and CD risk alleles (NOD2, IL-23R, ATG16L1 and IRGM). RESULTS: Attenuated TNFα and IL-23 release from CD macrophages was found after infection with all E. coli strains. There was prolonged survival of CD-derived AIEC, CD-derived non-AIEC and E. coli K-12 in macrophages from CD patients compared to within those from HC. No abnormality of monocyte phagocytosis or respiratory burst function was detected in CD. Macrophage dysfunction in CD was not influenced by phenotype, use of immunomodulation or genotype. CONCLUSIONS: CD macrophage responses to infection with E. coli are deficient, regardless of clinical phenotype, CD genotype or E. coli pathogenicity. This suggests host immunodeficiency is an important contributor to intramacrophage E. coli persistence in CD.

Determining epithelial contribution to in vivo mesenchymal tumour expression signature using species-specific microarray profiling analysis of xenografts.

Gene expression profiling using microarrays and xenograft transplants of human cancer cell lines are both popular tools to investigate human cancer. However, the undefined degree of cross hybridization between the mouse and human genomes hinders the use of microarrays to characterize gene expression of both the host and the cancer cell within the xenograft. Since an increasingly recognized aspect of cancer is the host response (or cancer-stroma interaction), we describe here a bioinformatic manipulation of the Affymetrix profiling that allows interrogation of the gene expression of both the mouse host and the human tumour. Evidence of microenvironmental regulation of epithelial mesenchymal transition of the tumour component in vivo is resolved against a background of mesenchymal gene expression. This tool could allow deeper insight to the mechanism of action of anti-cancer drugs, as typically novel drug efficacy is being tested in xenograft systems.

Preoperative diagnosis of Lynch syndrome with DNA mismatch repair immunohistochemistry on a diagnostic biopsy.

BACKGROUND: DNA mismatch repair immunohistochemistry on tumor tissue is a simple, readily available, and cost-effective method of identifying patients with Lynch syndrome in the postoperative setting. The aim of the study was to assess whether the mismatch repair status of a colorectal cancer can be confirmed by mismatch repair immunohistochemistry on preoperative biopsy. DESIGN: Germline positive patients with Lynch syndrome were identified from a prospectively collected Familial Cancer Clinic database. Preoperative colorectal cancer biopsy specimens were obtained from the source pathology provider to generate a cohort of matched preoperative and postoperative specimens. The specimens were sectioned and stained for 4 mismatch repair proteins (MLH1, MSH2, MSH6, PMS2). An age-matched cohort to compare specimens was selected from Bethesda positive but mismatch repair immunohistochemistry negative patients. All slides were reviewed by a single blinded pathologist. The Wilson method was used to calculate a true underlying proportion of patients for whom the preoperative result matched the postoperative test result with a 95% confidence interval. RESULTS: Of 128 germline positive mutation carriers, 40 patients (mean age 41, SD 11.3) had colorectal resections. Thirty-three preoperative specimens were retrievable and were matched with biopsies from 33 controls. The germline mutations included in the study were 8 MLH1, 19 MSH2, 3 MSH6, and 2 PMS2. In patients where germline positive status was known, sensitivity was 100% (95% CI 89.2-100) and specificity was 100% (95% CI 89.2-100). Identical sensitivity and specificity were observed in 33 age-matched patients. The sensitivity of the endoscopic biopsy in predicting germline status was 94.9% (95% CI 80.4-98.3). CONCLUSION: The mismatch repair disease status of a colorectal cancer can be reliably confirmed by mismatch repair immunohistochemistry on a diagnostic colorectal cancer biopsy sample before definitive surgery. Ascertaining a diagnosis of Lynch syndrome before definitive surgery can influence surgical planning.

Non-endoscopic screening biomarkers for Barrett's oesophagus: from microarray analysis to the clinic.

BACKGROUND AND AIMS: Barrett's oesophagus predisposes to oesophageal adenocarcinoma but the majority of patients are undiagnosed. A novel non-endoscopic cytological screening device, called a capsule sponge, makes population-based screening for the disease a feasible option. However, due to the mixed cell population retrieved by the capsule sponge, biomarkers specific for Barrett's oesophagus are required. METHODS: Three publically available microarray datasets were used to identify putative biomarkers present in Barrett's oesophagus but absent from normal oesophagus and gastric mucosa. Validation was performed by qPCR (n = 10 each of normal oesophagus, Barrett's oesophagus, gastric mucosa) and immunohistochemistry (normal oesophagus, n = 20; Barrett's oesophagus, n = 21; gastric mucosa, n = 24; duodenum, n = 18). The biomarker was then prospectively evaluated on capsule sponge specimens from 47 patients with Barrett's oesophagus and 99 healthy controls. RESULTS: 2/14 genes identified, dopa decarboxylase (DDC) and Trefoil factor 3 (TFF3), were confirmed by qPCR to be upregulated in Barrett's oesophagus compared to normal oesophagus (p<0.01) and gastric mucosa (p<0.01 and p<0.05, respectively). Immunohistochemistry confirmed that DDC protein expression was restricted to Barrett's oesophagus but was confined to <1% of the cells within the crypt compartment. TFF3 protein was expressed to high levels at the luminal surface of Barrett's oesophagus compared to absent expression in normal oesophagus and gastric mucosa (p<0.001). Using the capsule sponge 36/46 patients with Barrett's oesophagus (one inadequate sample) and 6/96 controls were positive for TFF3 giving a sensitivity of 78% and a specificity of 94%. CONCLUSIONS: TFF3 is a promising marker for Barrett's oesophagus screening since it is expressed at the luminal surface of Barrett's oesophagus but not in adjacent tissue types and may be applied to a non-endoscopic screening device.

Augmented gp130-mediated cytokine signalling accompanies human gastric cancer progression.

H. pylori infection accounts for most cases of gastric cancer, but the initiating events remain unclear. The principal H. pylori pathogenicity-associated CagA protein disrupts intracellular SHP-2 signalling pathways including those used by the IL-6 family cytokines, IL-6 and IL-11. Imbalanced IL-6 family cytokine signalling in the gp130(757FF) mouse model of gastric cancer arising from hyperactivation of oncogenic STAT3 after altered SHP-2 : ERK1/2 signalling produces dysplastic antral tumours preceded by gastritis and metaplasia. In a cohort of patient gastric biopsies with known H. pylori and CagA status, we investigated whether (i) STAT3 and ERK1/2 activation is altered in H. pylori-dependent gastritis; (ii) these profiles are more pronounced in CagA+ H. pylori infection; and (iii) the expression of pro-inflammatory cytokines that activate STAT3 and ERK 1/2 pathways is associated with progression to gastric cancer. IL-6, IL-11, and activated STAT3 and ERK1/2 were quantified in antral biopsies from gastritic stomach, metaplastic tissue, and resected gastric cancer tissues. We observed significantly increased STAT3 and ERK1/2 activation (p = 0.001) in H. pylori-dependent gastritis, which was further enhanced in the presence of CagA+ H. pylori strains. Of known gastric ligands that drive STAT3 activation, IL-6 expression was increased after H. pylori infection and both IL-6 and IL-11 were strongly up-regulated in the gastric cancer biopsies. This suggests a mechanism by which IL-11 drives STAT3 activation and proliferation during gastric cancer progression. We addressed this using an in vitro approach, demonstrating that recombinant human IL-11 activates STAT3 and concomitantly increases proliferation of MKN28 gastric epithelial cells. In summary, we show increased STAT3 and ERK1/2 activation in H. pylori-dependent gastritis that is likely driven in an IL-6-dependent fashion. IL-11 expression is associated with adenocarcinoma development, but not gastritic lesions, and we identify a novel mechanism for IL-11 as a potent inducer of proliferation in the human gastric cancer setting.

Incorporation of somatic BRAF mutation testing into an algorithm for the investigation of hereditary non-polyposis colorectal cancer.

Patients suspected on clinical grounds to have hereditary non-polyposis colorectal cancer (HNPCC) may be offered laboratory testing in order to confirm the diagnosis and to facilitate screening of pre-symptomatic family members. Tumours from an affected family member are usually pre-screened for microsatellite instability (MSI) and/or loss of immunohistochemical expression of mismatch repair (MMR) genes prior to germline MMR gene mutation testing. The efficiency of this triage process is compromised by the more frequent occurrence of sporadic colorectal cancer (CRC) showing high levels of MSI (MSI-H) due to epigenetic loss of MLH1 expression. Somatic BRAF mutations, most frequently V600E, have been described in a significant proportion of sporadic MSI-H CRC but not in HNPCC-associated cancers. BRAF mutation testing has therefore been proposed as a means to more definitively identify and exclude sporadic MSI-H CRC cases from germline MMR gene testing. However, the clinical validity and utility of this approach have not been previously evaluated in a familial cancer clinic setting. Testing for the V600E mutation was performed on MSI-H CRC samples from 68 individuals referred for laboratory investigation of suspected HNPCC. The V600E mutation was identified in 17 of 40 (42%) tumours showing loss of MLH1 protein expression by immunohistochemistry but in none of the 28 tumours that exhibited loss of MSH2 expression (P < 0.001). The assay was negative in all patients with an identified germline MMR gene mutation. Although biased by the fact that germline testing was not pursued beyond direct sequencing in many cases lacking a high clinical index of suspicion of HNPCC, BRAF V600E detection was therefore considered to be 100% specific and 48% sensitive in detecting sporadic MSI-H CRC amongst those cases showing loss of MLH1 protein expression, in a population of patients with MSI-H CRC and clinical features suggestive of HNPCC. Accordingly, we recommend the incorporation of BRAF V600E mutation testing into the laboratory algorithm for pre-screening patients with suspected HNPCC, whose CRCs show loss of expression of MLH1. In such tumours, the presence of a BRAF V600E mutation indicates the tumour is not related to HNPCC and that germline testing of MLH1 in that individual is not warranted. We also recommend that in families where the clinical suspicion of HNPCC is high, germline testing should not be performed on an individual whose CRC harbours a somatic BRAF mutation, as this may compromise identification of the familial mutation.

Current and potential uses for DNA microarrays in transplantation medicine: lessons from other disciplines.

DNA microarrays are used to study simultaneous gene expression in thousands of genes. This tool has moved beyond proof-of-principle and its integration into medical practice is slowly becoming a reality. This technology has enabled unparalleled progress into the study of complex polygenic diseases. Although cancer research introduced DNA microarrays into the medical arena other disciplines are beginning to exploit the power of this technology to advance medical research. In this review we outline aspects of the design of a microarray experiment from the choice of platform, through the experimental procedure to the analysis of the results. We review the current applications and speculate on potential applications of this technology with particular reference to transplantation medicine.

Towards a molecular approach to gastric cancer management.

Gastric cancer is a leading cause of cancer death worldwide. Most patients with gastric cancer present with locally advanced and incurable disease, and overall survival is poor. Considerable research efforts towards the epidemiology and pathogenesis of gastric cancer have not been translated into treatment success. We discuss current concepts of the pathogenesis of gastric cancer and how recent research advances, in particular global gene expression strategies, may improve this understanding, and suggest a framework wherein these approaches may be used.