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Comment on: "Pediatric-type" gastrointestinal stromal tumors in adults: distinctive histology predicts genotype and clinical behavior. Rege TA, Wagner AJ, Corless CL, Heinrich MC, Hornick JL. Am J Surg Pathol. 2011 Apr; 35(4):495-504.

Massively parallel RNA sequencing, followed by data analysis, allows to discover novel single nucleotide variants and to identify new potential target genes in rare diseases, such as WT-GISTs. By this, mutations at the level of the A subunit of the succinate dehydrogenase (complex II) of the mitochondrial inner membrane were characterized and validated with Sanger sequencing as tumoral common markers of two young adults (1). After SNP calling and validation, the effect of the variation at the protein level is either a truncation of the protein chain or a mutation that is identified as disease-related with a predictor, SNPs & GO (2), suited to identify disease-associated residue substitution on the basis of the protein sequence and its function. Further investigation of the effects of the mutation on the protein stability indicates that the disease associate mutations hamper folding stability by decreasing the number of hydrogen bonds and of other stabilizing interactions in the FAD binding domain of SDHA. This approach highlights also private variants in the two young adults. Among the private disease-related variants we identified in one patient a K1775E substitution in the myosin heavy chain 9 (MYH9) and a R206H substitution in the triosephosphate isomerase 1 (TPI1). The second patient carried a V815M mutation in a oxoglutarate dehydrogenase-like protein (OGDHL). Interestingly all the mutations that are labeled disease-associated with SNPs & GO are also promoting protein destabilization according to a predictor suited to evaluate protein destabilization upon mutation starting from the protein sequence (I-Mutant2, 3). The results are also confirmed with protein structure computational analysis. We performed Sanger sequencing on both tumor and peripheral blood samples and found that private mutations were germline heterozygous.

AIM: The aim of the study was to detect and compare the epidermal growth factor receptor (EGFr) content using different methods, to establish whether the quantitative detection and functional study of EGFr in colorectal cancer, using methods other than immunohistochemistry (IHC), are appropriate. METHOD: Analysis of EGFr by IHC was performed in 230 colorectal cancer patients using monoclonal anti-EGFr. Total and activated EGFr (pY1068) contents were determined in 92 patients and real-time PCR, to determine the level of EGFr messenger RNA, was carried out in 60 patients. RESULTS: There was no association between EGFr IHC groups and the mean total EGFr levels measured using ELISA. CONCLUSION: The study shows that the results of different EGFr detection methods do not correlate with each other. Hence, the real role of EGFr in colorectal cancer remains unsettled. Clinically, the receptor itself does not seem to be important and it would be better to focus on EGFr signalling in downstream pathways.

AIMS: To compare the genomic signatures of wild-type (WT) and mutated GISTs and the murine interstitial cells of Cajal (ICCs) to find markers of cell differentiation and other functions that may identify cells that give rise to WT tumours. METHODS AND RESULTS: We analysed the gene expression profiles of a total of 30 tumour samples (four WT GISTs and 26 mutated GISTs), selected the genes most differentially expressed (P < 0.001:448 probe sets) and validated these results by quantitative polymerase chain reaction (PCR) and immunohistochemistry. In addition, we conducted a meta-analysis merging data from human GISTs with a genomic data set from murine ICCs. The gene expression profiles of WT and mutated GISTs differed profoundly, especially in the expression of those genes restricted primarily to neural tissues. We found that mature ICCs are more similar to mutated GISTs than WT GISTs. CONCLUSIONS: WT GISTs have different genomic profiles from both mutated GISTs and murine mature ICCs. Considering that IGF1R expression is common to both WT GISTs and putative precursor ICCs, this study suggests that WT GISTs may derive either from ICCs at a different step of differentiation or from a different cell of origin.

Cyclooxygenase-2/Carbonic anhydrase-IX up-regulation promotes invasive potential and hypoxia survival in colorectal cancer cells Purpose: Cyclooxygenase-2 (COX-2) is a major mediator of inflammation, playing a pivotal role in colorectal carcinogenesis. Hypoxia is an universal hallmark of solid tumour in vivo. This investigation was prompted by the observation that in colorectal cancer cells the expression of COX-2 protein is positively correlated with that of the hypoxia survival gene Carbonic Anhydrase-IX (CA-IX). Experimental Design: Since COX-2 gene expression and activity is increased in hypoxia, and that CA-IX is expressed also in normoxia in colorectal cancer cells, we tested the hypothesis that COX-2 activity in normoxia, as well as in hypoxia may be functionally linked to that of CA-IX gene. We investigated the role of COX-2 and CA-IX in colorectal cancer cell lines. In this regard, we performed RNA interference to knockdown COX-2 gene in vitro and immunohistochemistry to evaluate the protein expression of COX-2 and CA-IX in human colon cancer tissue specimens ex vivo. Results: We found that COX-2, by PGE2 production, controls CA-IX gene expression in an ERK dependent manner. In line with this finding, we also showed that the COX-2 inhibition by a specific short harpin COX-2 RNA (shCOX-2) or by a specific drug (SC-236), down-regulated CA-IX expression in colon cancer cells. We then exposed colon cancer cells to hypoxia stimuli and found that COX-2/CA-IX interplay promoted hypoxia survival. Moreover, we also report that COX-2/CA-IX interplay triggers Matrix Metalloproteinase 2/9 (MMP-2/9) activation and enhances the invasiveness of colorectal cancer cells. Thus given our above observations, we found that CA-IX and COX-2 protein expressions correlate with more aggressive stage colorectal cancer tissues ex vivo. Conclusions: Taken together these data indicate that COX-2/CA-IX interplay promotes an aggressive phenotype (hypoxia survival and invasiveness) which can be modulated in vitro by COX-2 selective inhibition and which may play a role in determining the biological aggressiveness of colorectal tumours. Moreover, in vitro and ex vivo data also suggest that the signatures of inflammation (COX-2) and hypoxia (CA-IX) may be difficult to be disentangled in colon cancer, being both responsible for the up-regulation of the same pathways.

Context Pancreatic cancer cells produce various proteases involved in invasion, angiogenesis and metastatic spread. Gabexate mesylate (GM), a serine protease inhibitor, may reduce invasive capabilities and enhance chemotherapy effects. Objective To evaluate the effect of treatment with GM and gemcitabine (GEM), alone or combined. Materials and methods Studies have been performed on cancer cell lines PANC-1, SW1990 and endothelial cell EA.hy926. Treatment consisted of GM (50-100 µM for 24 h), alone or followed by GEM (250 µM for 24 h). Aspects studied included: cell viability (MTT assay), cell invasiveness and migration (Boyden chambers chemoinvasion and chemotaxis assay), angiogenesis (endothelial tube formation assay) and VEGF levels (ELISA) in EA.hy926 cell, MMP-2 and MMP-9 activity (gelatin zymography). Results Cell viability: this parameter decreased by 40% with GM alone only in EA.hy926 cells (P

Background Although serological tests are useful for identifying celiac disease, it is well established that a minority of celiacs are seronegative. Aim To define the prevalence and features of seronegative compared to seropositive celiac disease, and to establish whether celiac disease is a common cause of seronegative villous atrophy. Methods Starting from 810 celiac disease diagnoses, seronegative patients were retrospectively characterized for clinical, histological and laboratory findings. Results Of the 810 patients, fourteen fulfilled the diagnostic criteria for seronegative celiac disease based on antibody negativity, villous atrophy, HLA-DQ2/-DQ8 positivity and clinical/histological improvement after gluten free diet. Compared to seropositive, seronegative celiac disease showed a significantly higher median age at diagnosis and a higher prevalence of classical phenotype (i.e., malabsorption), autoimmune disorders and severe villous atrophy. The most frequent diagnosis in the 31 cases with seronegative flat mucosa was celiac disease (45%), whereas other diagnoses were Giardiasis (20%), common variable immunodeficiency (16%) and autoimmune enteropathy (10%). Conclusions Although rare seronegative celiac disease can be regarded as the most frequent cause of seronegative villous atrophy being characterized by a high median age at diagnosis; a close association with malabsorption and flat mucosa; and a high prevalence of autoimmune disorders.

Gluten is one of the most abundant and widely distributed components of food in many areas. It can be included in wheat, barley, rye, and grains such as oats, barley, spelt, kamut, and triticale. Gluten-containing grains are widely consumed; in particular, wheat is one of the world's primary sources of food, providing up to 50% of the caloric intake in both industrialized and developing countries. Until two decades ago, celiac disease (CD) and other gluten-related disorders were believed to be exceedingly rare outside of Europe and were relatively ignored by health professionals and the global media. In recent years, however, the discovery of important diagnostic and pathogenic milestones led CD from obscurity to global prominence. In addition, interestingly, people feeding themselves with gluten-free products greatly outnumber patients affected by CD, fuelling a global consumption of gluten-free foods with approximately $2.5 billion in United States sales each year. The acknowledgment of other medical conditions related to gluten that has arisen as health problems, providing a wide spectrum of gluten-related disorders. In February 2011, a new nomenclature for gluten-related disorders was created at a consensus conference in London. In this review, we analyse innovations in the field of research that emerged after the creation of the new classification, with particular attention to the new European Society for Paediatric Gastroenterology, Hepatology and Nutrition guidelines for CD and the most recent research about non-celiac gluten sensitivity.

e15645 Background: Negligible advances for PC treatment have been done over the last decade and G remains the standard. Proteolitic degradation of extracellular matrix (ECM) is essential for early local invasion, metastasis and desmoplastic reaction characterizing PC. Differently from MMPs, the serine proteases (uPA and TAT) action is earlier and larger, degradating not only ECM, but also basement membrane, and activating trypsin, plasmin, angiogenesis via TGF-β1 and proliferation via PAR-2. GM is an inhibitor of u-PA,TAT, trypsin and plasmin, used in Italy and Japan for prophylaxis of acute pancreatitis after ERCP. In a previous study, GM demonstrated antinvasion and antimetastatic activity. Study aim: to evaluate if GM increases G efficacy on pancreatic cancer cell line. Methods: In vitro study of phenotypic effects of GM and G in poor differentiated PANC-1 PC cell line using:1) Cell vitality test (Trypan blu);2) Invasion test (Matrigel invasion assay);3) Cell cycle analysis (cytofluorimeter);4) Antiangiogenic test (tube formation assay in extracellular matrix using E.A.hy926 endothelial cells with matrigel). Different doses of G and GM (100,200,250,500 μM;1mM) alone or combined (concomitant or sequential) have been tested vs controls (PANC-1 without any treatment). All tests have been done in triplicate. Results: G alone (250 μM) decreases invasion by 40% (±5,6%) and cell vitality by 15% (±1,3%.). GM alone (100 μM) decreases invasion by 30% (±4,6%.) but 1mM is needed for similar vitality decrease. GM+G together are detrimental vs G alone while sequential treatment (GM before G with or without 24 hours of interval) enhances G activity. GM (200 μM) and G (250 μM) in immediate sequence show better results decreasing ability of invasion by 75% (±8,3%). Cell vitality is better inhibited from GM (100)/G (250) 24 h-delayed sequence by 28% (±3,8%). Combined treatment mainly blocks cells in G1 phase of cell cycle (5%±0,5%) vs controls. Concerning antiangiogenic assay, the administration of G alone is ineffective to inhibit angiogenesis, while pre-treatment with GM results in a strong anti-angiogenic effect. Conclusions: Association of GM to G could represent a new effective approach to inhibit invasion, angiogenesis and growth in pancreatic cancer. No significant financial relationships to disclose.

A 62-year-old woman complaining of severe malabsorption was diagnosed with celiac disease based on the findings of flat, small intestinal mucosa and HLA-DQ2 positivity, although celiac serology was negative. This diagnosis was questioned due to the lack of clinical and histological improvement after a long period of strict gluten-free diet. The detection of enterocyte autoantibodies guided to the correct diagnosis of autoimmune enteropathy, leading to a complete recovery of the patient following an appropriate immunosuppressive treatment. Autoimmune enteropathy should be considered in the differential diagnosis of malabsorption with severe villous atrophy, including those cases with negative celiac-related serology.