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It has been proposed that persistent HCV infection drives the proliferation of B cells through an antigen-selective stimulation, mediated by B-cell receptor (BCR) involvement that preferentially includes a IgL Vk-3 chain. The aim of this study is to characterize IgL Vk rearrangements from different setting of HCV-related disorders to investigate if there is a preferential subset of BCR repertoire among 5 clinical groups. 26 HCV+ve patients (HBV-ve; HIV-ve) were studied: 2 MC, 12 NHL without clinical manifestation of MC, 6 HCV spontaneously resolved (SR), 6 HCV chronic infected (CE). Six blood donors (BD) were used as controls. VK3 gene region was amplified using VK3 specific and a mixture of JK primers. In the present study VK3-20, VK3-15 and VK3-11 subfamilies were the most represented (>90% of total clones). IGKV3-15 gene was over represented in both NHL and MC groups (67%) vs CE+SR+BD (22%, p

Hepatitis C virus (HCV) is a positive, single-stranded RNA virus, which has been associated to different subtypes of B-cell non-Hodgkin lymphoma (B-NHL). Cumulative evidence suggests an HCV-related antigen driven process in the B-NHL development. The underlying molecular signature associated to HCV-related B-NHL has to date remained obscure. In this review, we discuss the recent developments in this field with a special mention to different sets of genes whose expression is associated with BCR coupled to Blys signaling which in turn was found to be linked to B-cell maturation stages and NF-κb transcription factor. Even if recent progress on HCV-B-NHL signature has been made, the precise relationship between HCV and lymphoma development and phenotype signature remain to be clarified.

Probe-based Confocal Laser Endomicroscopy (pCLE) is a novel imaging technique for gastrointestinal endoscopy providing in vivo microscopy at subcellular resolution. It offers the possibility to analyze neoangiogenesis and vessel density in vivo. Angiogenetic switch is essential in cancer progression. Aim of the paper was to review the use of this imaging tool to analyze colorectal and gastric cancers vascularization in vivo. The aim is to provide the possibility of combining diagnostic evidences with vascularization and molecular profile to evaluate the efficacy of an antiangiogenic treatment in association with conventional therapy. pCLE can be considered a revolutionary method for real-time assessment of changes in vascularization pattern in this tumors and it may open the possibility to address the use of anti-angiogenic therapy in order to improve the outcome of the treatment.

Trastuzumab is a human epidermal growth factor receptor 2 (HER2) inhibitor used to treat HER2+ metastatic gastric cancer (mGC). The present study aims to investigate the relationship between CDH1 mRNA expression and HER2-positivity in mGC using a multiplexed gene expression profile in two series of gastric cancer (GC): Series 1 (n = 38): HER2+ and HER2- mGC; Series 2 (n = 36) HER2- GC with and without metastasis. To confirm the results, the same expression profiles were analyzed in 354 GC from The Cancer Genome Atlas (TCGA) dataset. The difference in gene expression connected HER2 overexpression with canonical wingless-type (Wnt)/β-catenin pathway and immunohistochemical (IHC) expression loss of E-cadherin (E-CAD). CDH1 mRNA expression was simultaneously associated with the rs16260-A variant and an increase in E-CAD expression. Differences in retinoic acid receptor alfa (RARA), RPL19 (coding for the 60S ribosomal L19 protein), catenin delta 1 (CTNND1), and epidermal growth factor (EGF) mRNA levels—all included in the Wnt/β-catenin pathway—were found associated with overall survival (OS). RARA, CTNND1, and EGF resulted in independent OS prognostic factors. EGF was confirmed as an independent factor along with TNM stage in HER2-overpressed mGC from TCGA collection. Our study highlighted factors involved in the WNT/β-catenin pathway that interconnected E-CAD with HER2 overexpression and patient survival.

Preoperative neoadjuvant chemoradiotherapy (nCRT) is the gold standard in locally advanced rectal cancer, only 10-30% of patients achieving benefits. Currently, there is a need of a reliable selection of markers for the identification of poor or non-responders prior to therapy. In this work, we compared protein profiles before therapy of patients differing in their responses to nCRT to find novel predictive markers of response to therapy. Patients were grouped into 3 groups according to their tumor regression grading (TRG) after surgery: 'TRG 1-2', good responders, 'TRG 3' and 'TRG 4', poor responders. Paired surgical specimens of rectal cancer and healthy (histologically confirmed) rectal tissues from 15 patients were analysed before nCRT by two dimensional difference in gel electrophoresis followed by mass spectrometry. Thirty spots were found as differentially expressed (p < 0.05). Among them, 3 spots (spots 471, 683 and 684) showed an increased amount of protein in poor responders compared with good responders, and they were more tumor associated compared with healthy tissues. Proteins of these spots were identified as fibrinogen beta chain fragment D, actin isoforms, B9 and B5 serpins, cathepsin D isoforms and peroxiredoxin-4. In an independent validation set of 20 rectal carcinomas we validated the increased fibrinogen beta chain abundance before nCRT in poor responders by immunoblotting. In conclusion, we propose a risk-stratification tool in predicting the response to nCRT treatment in rectal cancer based on the quantity of these proteins.

Gastric cancer (GC) is a common malignant neoplasm worldwide and one of the main cause of cancer-related deaths. Despite some advances in therapies, long-term survival of patients with advanced disease remains poor. Different types of classification have been used to stratify patients with GC for shaping prognosis and treatment planning. Based on new knowledge of molecular pathways associated with different aspect of GC, new pathogenetic classifications for GC have been and continue to be proposed. These novel classifications create a new paradigm in the definition of cancer biology and allow the identification of relevant GC genomic subsets by using different techniques such as genomic screenings, functional studies and molecular or epigenetic characterization. An improved prognostic classification for GC is essential for the development of a proper therapy for a proper patient population. The aim of this review is to discuss the state-of-the-art on combining histological and molecular classifications of GC to give an overview of the emerging therapeutic possibilities connected to the latest discoveries regarding GC.

Elevated plasma fibrinogen levels and tumor progression in patients with gastric cancer (GC) have been largely reported. However, distinct fibrinogen chains and domains have different effects on coagulation, inflammation, and angiogenesis. The aim of this study was to characterize fibrinogen beta chain (FGB) in GC tissues. Retrospectively we analyzed the data of matched pairs of normal (N) and malignant tissues (T) of 28 consecutive patients with GC at diagnosis by combining one- and two-dimensional electrophoresis (1DE and 2DE) with immunoblotting and mass spectrometry together with two-dimensional difference in gel electrophoresis (2D-DIGE). 1DE showed bands of the intact FGB at 50 kDa and the cleaved forms containing the fragment D at similar to 37-40 kDa, which corresponded to 19 spots in 2DE. In particular, spot 402 at similar to 50 kDa and spots 526 and 548 at similar to 37 kDa were of interest by showing an increased expression in tumor tissues. A higher content of spot 402 was associated with stomach antrum, while spots 526 and 548 amounts correlated with corpus and high platelet count (>208 x 10(9)/L). The quantification of FGB and cleaved products may help to further characterize the interconnections between GC and platelet/coagulation pathways.

Lynch syndrome (LS) and familial adenomatous polyposis (FAP) are autosomal dominant hereditary diseases caused by germline mutations leading to the development of colorectal cancer. Moreover, these mutations result in the development of a spectrum of different tumors, including gastric cancers (GCs). Since the clinical characteristics of GCs associated with LS and FAP are not well known, we investigated clinical and molecular features of GCs occurring in patients with LS and FAP attending our Institution. The Hereditary Tumor Registry was established in 1994 at the Department of Oncologic Gastroenterology, CRO Aviano National Cancer Institute, Italy. It includes 139 patients with LS and 86 patients with FAP. Patients were recruited locally for prospective surveillance. Out of 139 LS patients, 4 developed GC&mdash, 3 in the presence of helicobacter pylori infection and 1 on the background of autoimmune diseases. All GCs displayed a high microsatellite instability (MSI-H) and loss of related mismatch repair (MMR) protein. One of the FAP patients developed a flat adenoma, displaying low-grade dysplasia at the gastric body, and another poorly differentiated adenocarcinoma with signet ring cells like Krukenberg without HP infection. LS carriers displayed a risk of GC. The recognition of HP infection and autoimmune diseases would indicate those at higher risk for an endoscopic surveillance. Regarding FAP, the data suggested the need of suitable endoscopic surveillance in long survivals with diffuse fundic gland polyps.

Over the last decade, our understanding of the mechanisms underlying immune modulation has greatly improved, allowing for the development of multiple therapeutic approaches that are revolutionizing the treatment of cancer. Immunotherapy for gastric cancer (GC) is still in the early phases but is rapidly evolving. Recently, multi-platform molecular analyses of GC have proposed a new classification of this heterogeneous group of tumors, highlighting subset-specific features that may more reliably inform therapeutic choices, including the use of new immunotherapeutic drugs. The clinical benefit and improved survival observed in GC patients treated with immunotherapeutic strategies and their combination with conventional therapies highlighted the importance of the immune environment surrounding the tumor. A thorough investigation of the tumor microenvironment and the complex and dynamic interaction between immune cells and tumor cells is a fundamental requirement for the rational design of novel and more effective immunotherapeutic approaches. This review summarizes the pre-clinical and clinical results obtained so far with immunomodulatory and immunotherapeutic treatments for GC and discusses the novel combination strategies that are being investigated to improve the personalization and efficacy of GC immunotherapy.

Germline mutations in CDH1, the gene coding for the E-cadherin adhesion protein, are known to cause hereditary diffuse gastric cancer. We identified a new truncating germline mutation (p.Asp538Thrfs*19) in exon 11 of the CDH1 gene in a 41-year-old male with a diffuse gastric cancer. Although he had no parental history of gastric cancer, the co-segregation study in the family detected the same mutation in his healthy 31-year-old brother. The mutation affects one of the extracellular repeat (CAD repeats) domains which is essential for the homophilic binding specificity that directs “E-cadherin” to bind with itself each others. In this case, immunohistochemical analysis showed no expression of E-cadherin in the tumor sample and was a useful prescreening tool to genetic testing. This finding was associated with a poor response to trastuzumab-based treatment.

Background Helicobacter pylori (H. pylori) represents a key factor in the etiology of autoimmune atrophic gastritis (AAG), duodenal ulcer (DU) and gastric cancer (GC). The aim of this study was to characterize the differential protein expression of H. pylori isolated from gastric biopsies of patients affected by either AAG, DU or GC. Methods The H. pylori strains were isolated from endoscopic biopsies from the stomach of patients with gastric disease. Protein profiles of H. pylori were compared by two-dimensional difference in gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS) for the identification of significantly different spots (Student t-test, p 1.5). These spots corresponded to 35 unique proteins. The identity of 7 protein spots was validated after one-dimensional electrophoresis and MS/MS analyses of excised gel portions. In H. pylori isolated from DU-patients a significant increase in proteins with antioxidant activity emerged (AroQ, AspA, FldA, Icd, OorA and ScoB), together with a higher content of proteins counteracting the high acid environment (KatA and NapA). In H. pylori isolated from AAG-patients proteins neutralizing hydrogen concentrations through organic substance metabolic processes decreased (GroL, TrxB and Tuf). In addition, a reduction of bacterial motility (FlhA) was found to be associated with AAG-H. pylori isolates. In GC-H. pylori strains it was found an increase in nucleic acid-binding proteins (e.g. DnaG, Tuf, RpoA, RplU) which may be involved in a higher demand of DNA- and protein-related processes. Conclusion Our data suggest the presence of specific protein signatures discriminating among H. pylori isolated from either AAG, DU or GC. Changes in protein expression profiles evaluated by DIGE succeeded in deciphering part of the molecular scenarios associated with the different H. pylori-related gastric diseases.

The CDH1 gene, coding for the E-cadherin protein, is linked to gastric cancer (GC) susceptibility and tumor invasion. The human epidermal growth factor receptor 2 (HER2) is amplified and overexpressed in a portion of GC. HER2 is an established therapeutic target in metastatic GC (mGC). Trastuzumab, in combination with various chemotherapeutic agents, is a standard treatment for these tumors leading to outcome improvement. Unfortunately, the survival benefit is limited to a fraction of patients. The aim of this study was to improve knowledge of the HER2 and the E-cadherin alterations in the context of GC to characterize subtypes of patients that could better benefit from targeted therapy. An association between the P7-CDH1 haplotype, including two polymorphisms (rs16260A-rs1801552T) and a subset of HER2-positive mGC with better prognosis was observed. Results indicated the potential evaluation of CDH1 haplotypes in mGC to stratify patients that will benefit from trastuzumab-based treatments. Moreover, data may have implications to understanding the HER2 and the E-cadherin interactions in vivo and in response to treatments.

Helicobacter pylori (H. pylori) represents an independent risk factor for Gastric Cancer (GC). First Degree Relatives (FDR) of GC subjects and Autoimmune Gastritis (AG) patients are both at increased risk for GC. H. pylori genetic heterogeneity within the gastric niche of FDR and AG individuals has been little explored. To understand whether they exploit an increased H. pylori stability and virulence, 14 AG, 25 FDR, 39 GC and 13 dyspeptic patients (D) were investigated by a cultural PCR-based approach characterizing single colonies-forming-units. We chose three loci within the Cytotoxin-associated gene-A Pathogenicity Island (CagPAI) (cagA,cagE,virB11), vacA, homA and homB as markers of virulence with reported association to GC. Inflammatory/precancerous lesions were staged according to Sydney System. When compared to D, FDR, similarly to GC patients, were associated to higher atrophy (OR = 6.29, 95% CI):1.23&ndash, 31.96 in FDR, OR = 7.50, 95% CI:1.67&ndash, 33.72 in GC) and a lower frequency of mixed infections (OR = 0.16, 95% CI:0.03&ndash, 0.81 in FDR, OR = 0.10, 95% CI:0.02&ndash, 0.48 in GC). FDR presented also an increased neutrophil infiltration (OR = 7.19, 95% CI:1.16&ndash, 44.65). Both FDR and GC carried a higher proportion of CagPAI+vacAs1i1mx+homB+ profiles (OR = 2.71, 95% CI: 1.66&ndash, 4.41 and OR = 3.43, 95% CI: 2.16&ndash, 5.44, respectively). Conversely, AG patients presented a lower frequency of subtypes carrying a stable CagPAI and vacAs1i1mx. These results underline different H. pylori plasticity in FDR and AG individuals, and thus, a different host-bacterium interaction capacity that should be considered in the context of eradication therapies.

OBJECTIVES: The objectives of this study were to investigate the serum pepsinogen test for the prediction of OLGIM (Operative Link on Gastric Intestinal Metaplasia Assessment) stages in first-degree relatives (FDR-GC) of patients with gastric cancer (GC) and autoimmune chronic atrophic gastritis (ACAG).METHODS: In 67 consecutive patients with ACAG, 82 FDR-GC, and 53 controls (CTRL) without gastric disease (confirmed by biopsy), serum levels of pepsinogen 1 (PG1), pepsinogen 2 (PG2), G17, and the PG1/2 ratio were assessed by enzyme-linked immunosorbent assay kit. All ACAG patients had positive antiparietal cell antibody levels, estimated by indirect immunofluorescence. Biopsies taken in duplicate from the antrum, corpus, and fundus were stained with Giemsa for Helicobacter pylori detection. Endoscopic detection of metaplasia was confirmed by histological diagnosis. Histological classification of OLGIM stages was applied by using the criteria of severity and topography of intestinal metaplasia (IM).RESULTS: The highest discrimination capacity for distinguishing ACAG from other groups of patients was the gastrin G17 test. The lowest mean for PG1 and PG2 serum levels was found in ACAG. In multivariate analysis by age, PG1 and PG1/PG2 were independent prognostic factors for metaplasia, and PG2 also for the presence of a histological H. pylori infection. The serum PG1 level was significantly lower in individuals with IM at OLGIM stage >2 than in those with IM at OLGIM stage = 2 vs. 0-1 OLGIM stages, the receiver operating characteristic (ROC) curve at 47.9 ng/ml PG1 level reached a significant area under the curve (AUC) value (0.978, P= 2. Using a cutoff of 47.9 ng/ml, PG1 testing in FDR-GC and ACAG patients had a sensitivity of 95.83% and a specificity of 93.37. Although these results could be validated in a prospective study, the known importance of higher OLGIM stages in increasing the risk of GC development supports the rationale of proposing PG1 algorithm as a diagnostic tool for the selection of high-risk FDR-GC and ACAG patients at high-risk stages for subsequent detailed endoscopic examination to detect dysplasia and asymptomatic GC. In addition, serum PG1 and PG2 levels could stratify patients based on both H. pylori infection and OLGIM risk in consideration of the increased acknowledge regarding the role of H. pylori in the progression of gastritis to GC.

2011/2012 Abstract Gastric cancer (GC) remains a leading cause of cancer death worldwide. A hallmark of this type of tumour is the poor prognosis of patients. Identification of subjects with an increased risk of developing GC and the early detection of GC, are promising approaches to reduce the morbidity and mortality of this malignancy. CDH1 gene encodes E-cadherin, a known tumour suppressor protein expressed in epithelial cells that plays a major role in cellular adhesion and tissue architecture maintenance. CDH1 gene mutations are thought to affect protein function and lead to cell-cell adhesion deregulation in epithelial tissues and to enhance metastatic tumour potential. Germline mutations in CDH1 gene are implicated in hereditary diffuse gastric cancer (HDGC) syndrome and in early onset gastric cancer (EOGC), but the role of these variants in sporadic GC and in subjects at risk to develop GC, is poorly investigated. Most studies limit the screening of CDH1 mutations to patients with HDGC, familial GC or GC patients at very early onset through genetic counselling, but only few studies investigated the CDH1 the contribution of these mutations in sporadic GC cases without familial aggregation or in subjects at risk of developing GC. Moreover, the potential functional effects of CDH1 variants are not always explored. In our study, screening of the entire coding region and all exon flanking sequences of the CDH1 gene was performed by direct sequencing in a series of random, consecutive sporadic GC cases. We extended the analysis of CDH1 germline mutations in a collection of individuals at risk for GC: 59 subjects who reported an ascertained case of GC among parents, children, siblings or offspring’s, named first degree GC-Relatives (FDR), and 20 autoimmune metaplastic atrophic gastritis (AMAG) patients. In the study 52 healthy blood donors (BD) were also included in the CDH1 analyses. The aim of this PhD project was to evaluate CDH1 mutations in these individuals to identify a possible hereditable marker that might improve the early detection of GC, stratifying high risk subjects in association with other known risk factors for GC diagnosis. All the participants were voluntarily subjected to a gastrointestinal endoscopy with biopsy in our institution. This is the first study in which CDH1 screening was investigated in AMAG patients. CDH1 germline mutations found were characterized by means of structural modelling and bioinformatic tools, immunohistochemical (IHC) stains for evaluation of E-cadherin and β-catenin proteins expression and subcellular localization, and E-cadherin mRNA level by real-time polymerase chain reaction (PCR). The functional impact of splicing was assessed for non-polymorphic intronic variants. Among the 59 GCs, the lower portion (antrum) of the stomach was the prevalent anatomic site (p< 0.05) for the tumours detected by endoscopy. No statistically differences were found comparing associations between gender, histotype, tumour location, H. Pylori infection, and TNM staging. A complex panel of germline variants was detected for CDH1 gene. In GC series, we found six different alterations in nine distinct GC patients (15.2%): 67% of the missense type (p.G274S; p.A298T; p.T470I: p.A592T) and 33% of non-missense type, one in the promoter region (5’UTR-71C>G) and one in the intronic region close to exon 13 (IVS12 c.1937-13T>C). All the missense substitutions are localized in the extracellular functional E-cadherin domain which is directly implied in cell-cell adhesion mechanisms. Of note the germline missense (p.G274S) was a new CDH1 mutation, while the p.A298T was already found only in HDGC syndrome and it was described as pathogenetic. Our molecular modelling studies highlighted that p.G274S change in mature protein is not dramatic for local structure but the serine residue represents a potential site for post translation modifications, such as phosphorylation or glycosylation. However, data from our functional in vitro assays did not support an effect of this mutation on adhesion mobility of cells. Moreover, E-cadherin was not under expressed in the tumour gastric tissue of patient harbouring the p.G274S, even if a reduction in β-catenin expression was observed in the same sample. Therefore, the pathogenic effect of p.G274S change by our in silico and in vitro approaches seems to be unlikely, so we cannot conclude the significance of this mutation. Modelling and SIFT results confirmed that the p.A298T substitution affects protein function, while the p.T470I change is tolerated. Through the structural analysis for p.T470I, we hypothesized a protective role that may favour protein-protein interactions in the extracellular medium. The p.A592T was detected in the four studied groups, suggesting that it is a polymorphic variant (frequence >1%). Modelling suggested a non-pathogenic role as already confirmed by published in vitro and in silico studies. The C-to-G change before the start codon (-71C>G), represented the most common variant associated with GC in our series, occurring in three (5.1%) out of 59 GC patients. Due to a lack of tumour material, an evaluation or a correlation between CDH1 hypermethylation and E-cadherin expression was not performed, so the predicted pathogenicity of this promoter variant still remains to be elucidated. The intronic IVS12 c.1937-13T>C variant was found in two GC patients and in one BD, but it was already described in HDGC and EOGC subjects. Our assays demonstrated for the first time that this substitution leads to an aberrant CDH1 transcript harbouring a deletion of exon 11 with the formation of a premature stop codon in the extracellular E-cadherin domain. The translated protein will lack part of the extracellular domain, and completely loose the transmembrane domain and the cytoplasmic tail of E-cadherin, which is involved in β-catenin binding. Moreover, IHC stains showed a reduction in the expression of E-cadherin and β-catenin in the gastric tumour tissues of patients harbouring this intronic mutation. Evaluation by real time-PCR of E-cadherin expression from the EBV immortalized B-lymphocytes from one GC patient carrying this mutation showed a strong (60%) and significantly reduction (pC) already described as a rare variant able to decrease the transcriptional activity of CDH1, one 5’ near gene variant (-176C>T) with unknown significance, and an intronic mutation close to exon 5, IVS4 c.532- 18C>T, already reported in EOGC cases. This last one showed no effects in producing CDH1 aberrant splicing, as the new germline mutation in the intronic region close to exon 1 (IVS1 c.48+7C>T) found in one AMAG patient. Other variants in the AMAGs were polymorphic, and thus we excluded a potential effect of these on E-cadherin function. In conclusion, our results show that pathogenic CDH1 germline variants (p.A298T) can also be detected in sporadic GC patients without fulfilling the strict criteria for HDGC. We also report the finding, in a sporadic GC, of a new missense germline mutation (p.G274S) with an uncertain significance. Furthermore, we demonstrate for the first time a potential deleterious effect on splicing and a decreased E-cadherin expression for the intronic IVS12 c.1937-13T>C mutation. Our detection of the same genetic alteration and splicing effect in one BD, but with a limited effect on E-cadherin mRNA level, is intriguing and deserves further studies. Performing CDH1 screening in individuals at risk for GC, we detected the IVS4 c.532-18C>T variant already reported in EOGC cases, and a new IVS1 intronic mutation in one AMAG patient, although there was no influence on CDH1 splicing in both cases. The overall percentage of the missense mutations (67%) in our series is predominantly of the non-missense type, suggesting that the North-East of Italy should be considered as a middle-to-high-risk area for the incidence of E-cadherin mutations and the risk of GC. Moreover, variants identified in the FDR and AMAG subjects that, until now, have only been described for GC patients, and recent findings of novel mutations in sporadic GC patients, encourage us to continue to screen for CDH1 genetic alterations that, in addition to other risk factors, could be used to define a high-risk group of patients that would benefit from an early GC diagnosis. Riassunto Il cancro gastrico (GC) rimane una delle maggiori cause di mortalità dovuta al cancro a livello mondiale. Un elemento caratteristico di questo tipo di tumore è la scarsa prognosi dei pazienti. L’identificazione dei soggetti maggiormente a rischio per lo sviluppo di GC e la diagnosi precoce per questo tipo di tumore, sono approcci promettenti per ridurre la morbidità e la mortalità di tale patologia. Il gene CDH1 codifica per E-caderina, una nota proteina oncosoppressore espressa nelle cellule epiteliali che gioca un ruolo prominente nell’adesione cellulare e nel mantenimento dell’architettura tissutale. Si ritiene che mutazioni germinali a carico del gene CDH1 siano in grado di influenzare la funzione della proteina e compromettere l’adesività cellulare nei tessuti epiteliali, aumentando il potenziale metastatico del tumore. Mutazioni germinali a carico del gene CDH1 sono implicate nella sindrome del carcinoma gastrico di tipo diffuso ereditario (HDGC) e nel carcinoma gastrico ad insorgenza precoce (EOGC), ma il ruolo di tali varianti nel GC di tipo sporadico e nei soggetti a rischio di sviluppare GC sono scarsamente indagate. La maggior parte degli studi limita lo screening di mutazioni del gene CDH1 nei pazienti con HDGC, forme familiari di GC o pazienti con GC in forma precoce attraverso consulenza genetica, ma solamente un numero limitato di studi ha valutato il contributo di queste mutazioni nel gene CDH1 nella casistica sporadica di GC priva di familiarità o in soggetti ad elevato rischio di sviluppare questo tumore. Inoltre, i potenziali effetti funzionali delle varianti nel gene CDH1 spesso non sono approfonditi. Nel nostro studio, lo screening dell’intera regione codificante e delle sequenze fiancheggianti gli esoni del gene CDH1 è stato condotto attraverso sequenziamento diretto in una serie casuale e consecutiva di pazienti con GC sporadico. Abbiamo esteso l’analisi per la ricerca di mutazioni germinali di CDH1 in un gruppo di individui a rischio per GC: 59 soggetti che hanno riportato un caso accertato di GC tra genitori, figli, fratelli o cugini, denominati parenti di primo grado-GC (FDR), e 20 pazienti affetti da gastrite atrofica metaplasica autoimmunitaria (AMAG). Nello studio sono stati inclusi anche 52 donatori di sangue sani (BD). Lo scopo di questo progetto di dottorato era valutare le mutazioni a carico del gene CDH1 in tali individui per identificare un possibile marker ereditario che possa migliorare la diagnosi precoce del GC, con la prospettiva di andare a stratificare i pazienti ad alto rischio in associazione con altri marcatori di rischio già noti. Tutti i partecipanti sono stati volontariamente sottoposti ad una endoscopia gastrointestinale con biopsia nel nostro istituto. Questo è il primo studio nel quale lo screening per il gene CDH1 è stato valutato nei pazienti con AMAG. Le mutazioni germinali trovate nel CDH1 sono state caratterizzate attraverso l’uso del modelling strutturale e di strumenti bioinformatici, colorazioni immunoistochimiche (IHC) per valutare sia l’espressione che la localizzazione cellulare delle proteine E-caderina e β-catenina, e il livello di mRNA per E-caderina con la tecnica di real-time PCR. L’impatto funzionale nello splicing è stato determinato per le varianti introniche non polimorfiche. Tra i 59 GC, la porzione inferiore (l’antro) dello stomaco era la sede anatomica prevalente (p< 0.05) dei tumori rilevati mediante endoscopia. Non sono state trovate differenze statistiche confrontando associazioni tra sesso, istotipo e localizzazione del tumore, infezione da Helicobacter Pylori e stadi TNM. Un complesso pannello di varianti germinali è stato riscontrato per CDH1 nella nostra casistica. Nella serie di pazienti con GC, abbiamo trovato sei differenti alterazioni genetiche in nove distinti pazienti (15.2%): 67% del tipo missenso (p.G274S; p.A298T; p.T470I: p.A592T) e 33% non missenso, una nella regione del promotore (5’UTR-71C>G) e una nella regione intronica in prossimità dell’esone 13 (IVS12 c.1937-13T>C). Tutte le sostituzioni missenso sono localizzate nel dominio extracellulare di E-caderina che è direttamente coinvolto nel processo di adesività cellulare. Rilevante è stata l’identificazione di una nuova mutazione germinale missenso di CDH1 (p.G274S), mentre la mutazione p.A298T era stata precedentemente documentata solo nella sindrome HDGC e descritta come patogenetica. I nostri studi di modelling strutturale hanno evidenziato che la sostituzione p.G274S nella proteina matura non sembra avere un impatto notevole nel perturbare la struttura locale, ma il residuo di serina rappresenta un potenziale sito per modificazioni post traduzionali, come la fosforilazione o la glicosilazione. Comunque, dati ottenuti dai nostri esperimenti funzionali condotti in vitro sembrano non supportare alcun tipo di effetto da parte di tale mutazione sulla mobilità e adesività cellulare. Inoltre, E-caderina non era ipoespressa nel tessuto tumorale gastrico del paziente con la variante p.G274S, dove però è stata osservata una riduzione nell’espressione di β-catenina. Quindi, grazie alla valutazione sia in silico che in vitro, il ruolo patogenetico della mutazione p.G274S sembra essere improbabile, e il reale significato di questa alterazione risulta comunque incerto. Il modelling e i risultati ottenuti con lo strumento bioinformatico SIFT confermano che la sostituzione p.A298T è in grado di interferire sulla funzionalità della proteina, che invece non è influenzata dalla mutazione p.T470I la quale sembra essere tollerata. Attraverso l’analisi strutturale abbiamo ipotizzato un ruolo protettivo per la variante p.T470I che sembra favorire le interazioni proteina-proteina nel contesto dello spazio extracellulare. La sostituzione p.A592T è stata riscontrata in tutti i quattro gruppi di soggetti studiati, suggerendo che si tratta di una variante polimorfica (frequenza >1%). L’approccio condotto con il modelling suggerisce un ruolo non patogenetico come già confermato e descritto da studi pubblicati condotti sia in vitro che in silico. La sostituzione da C a G prima del codone di inizio (-71C>G), rappresenta la variante più comune rilevata nella nostra casistica, in quanto trovata in tre (5.1%) dei 59 pazienti con carcinoma gastrico. Purtroppo, a causa della mancanza di campione biologico tumorale non è stato possibile valutare una correlazione tra un eventuale stato di ipermetilazione a carico del promotore di CDH1 e l’espressione della E-caderina, perciò l’effettivo contributo di tale variante sulla trascrizione del gene rimane ancora da definire. La variante intronica IVS12 c.1937-13T>C è stata trovata in due pazienti con GC e in un BD, ma era già stata precedentemente descritta nella sindrome ereditaria HDGC e in soggetti affetti da EOGC. I nostri esperimenti hanno dimostrato per la prima volta che questa mutazione produce un trascritto aberrante di CDH1 con delezione dell’esone 11 e la formazione di un codone di stop prematuro a livello del dominio extracellulare di E-caderina. La proteina tradotta risulterà priva di gran parte del dominio extracellulare, e perderà completamente il dominio trans membrana e la coda citoplasmatica che è direttamente coinvolta nel legame con la β-catenina. Inoltre, la valutazione mediante IHC ha evidenziato una riduzione nell’espressione di E-caderina e β-catenina nei tessuti tumorali gastrici dei due pazienti che presentavano tale variante intronica. La valutazione mediante real time-PCR dell’espressione di E-caderina nei linfociti B immortalizzati con il virus EBV, in uno dei due pazienti GC con tale mutazione, ha mostrato una forte (60%) e significativa (pC) già descritta come una variante rara in grado di ridurre l’attività trascrizionale di CDH1, un’altra nella regione vicina al gene in 5’ (-176C>T) con significato ignoto, e una mutazione intronica vicina all’esone 5, IVS4 c.532-18C>T, riportata nella casistica EOGC. Quest’ultima, non ha dimostrato effetti rilevanti nello splicing di CDH1, come la nuova mutazione germinale nella regione intronica a valle dell’esone 1 (IVS1 c.48+7C>T) trovata in un paziente affetto da AMAG. Altre varianti di tipo polimorfico prive di un potenziale effetto sulle funzioni della proteina E-caderina, sono state rilevate nella serie costituita da pazienti con AMAG. Concludendo, i nostri risultati evidenziano che mutazioni germinali a carico del gene CDH1, già descritte come patogenetiche (p.A298T) possono essere rilevate anche in pazienti con GC di tipo sporadico, senza particolare familiarità o non afferenti ai severi criteri descritti per il HDGC. Riportiamo inoltre la scoperta, in un paziente con GC di tipo sporadico, di una nuova mutazione germinale missenso (p.274S) di significato incerto. Inoltre, per la prima volta, sono stati dimostrati un potenziale effetto deleterio nello splicing di CDH1 e una riduzione nell’espressione di E-caderina per la mutazione intronica IVS12 c.1937-13T>C. Il rilevamento della stessa mutazione intronica IVS12, e dell’analogo impatto nello splicing nel donatore sano, ma con un limitato effetto sul livello di mRNA per E-caderina, è interessante e necessita di ulteriori approfondimenti. Estendendo lo screening per il gene CDH1 negli individui a rischio per il GC, abbiamo potuto trovare la mutazione IVS4 c.532-18C>T già descritta nei pazienti EOGC, e la nuova variante intronica IVS1 in un paziente AMAG, sebbene una conseguenza nello splicing non sia stata riscontrata per entrambi i casi. La percentuale complessiva delle mutazioni missenso (67%) nella nostra casistica è predominante rispetto a quella relativa alle mutazioni di tipo non missenso, e ciò suggerisce che l’area del Nord Est d’Italia dovrebbe essere considerata come una zona a medio-alto rischio per l’incidenza delle mutazioni relative a CDH1 e per il rischio di GC. Inoltre, le varianti trovate nei soggetti FDR e nei pazienti AMAG, che sino ad ora erano state descritte solo nei pazienti con carcinoma gastrico, e recenti scoperte di nuove mutazioni in pazienti con GC sporadico, ci incoraggiano a continuare nello screening germinale del gene CDH1 che, insieme ad altri fattori di rischio, potrebbe essere utilizzato per definire un gruppo di pazienti ad alto rischio per GC il quale potrebbe beneficiare di una diagnosi precoce. XXV Ciclo 1975