Comparison and applicability of molecular classifications for gastric cancer.

Gastric Cancer (GC) is a complex and heterogeneous disease, which represents a global health concern. Despite advances in prevention, diagnosis, and therapy, GC is still a leading cause of cancer-related death. Over the last decade, several clinical trials have tested novel agents for advanced GC with mostly disappointing results. Heterogeneity, the absence of molecular selection in clinical trials and powerless predictive biomarkers may be potential explanations. Different molecular classification proposals for GC based on the genetic, epigenetic, and molecular signatures have been published. Molecular characterization of GC may offer new tools for more effective therapeutic strategies, such as the development of therapies for specifically well-defined sets of patients as well as the use of new clinical trial designs, which will ultimately lead to an improvement of medical management of this disease. However, the possibilities of implementation of GC molecular classifications on daily practice and their therapeutic implications remain challenging to date. In this review, we will describe and compare these GC molecular classifications, focusing on their main characteristics as the basis for their potential therapeutic implications and strategies for their clinical application. Key Message: A better understanding of gastric cancer molecular characteristics may lead to further improvements in treatment and outcomes for patients with the disease.

Variants in phospholipid metabolism and upstream regulators and non-small cell lung cancer susceptibility.

AIM: The relevance of the cytidine diphosphate-choline and Rho GTPases pathways in the pathogenesis of cancer has been previously demonstrated. We investigate by a case-control association study if genetics variants in these pathways are associated with risk of developing lung cancer. METHODS: Thirty-seven tag SNPs were evaluated as risk factor of NSCLC in 897 cases and 904 controls. RESULTS: Six SNPs were nominally associated with lung cancer risk, which were not significant after the Bonferroni correction for multiple comparisons. No association was observed with the remaining 31 analyzed SNPs, neither it was found significant in haplotype frequencies. CONCLUSIONS: Although the implication of the two pathways investigated in our study in carcinogenesis is well established, our null results suggest that common genetic variants in CDP-choline and Rho GTPases-related genes are not risk factors for lung cancer.

Expression of the vitamin K-dependent proteins GAS6 and protein S and the TAM receptor tyrosine kinases in human atherosclerotic carotid plaques.

The GAS6/ProS-TAM system is composed of two vitamin K-dependent ligands (GAS6 and protein S) and their three protein tyrosine kinase receptors TYRO3, AXL and MERTK, known as the TAM receptors. The system plays a prominent role in conditions of injury, inflammation and repair. In murine models of atherosclerotic plaque formation, mutations in its components affect atherosclerosis severity. Here we used Taqman low-density arrays and immunoblotting to study mRNA and protein expression of GAS6, ProS and the TAM receptors in human carotid arteries with different degrees of atherosclerosis. The results show a clear down-regulation of the expression of AXL in atheroma plaques with respect to normal carotids that is matched by decreased abundance of AXL in protein extracts detected by immunoblotting. A similar decrease was observed in PROS1 mRNA expression in atherosclerotic carotids compared to the normal ones, but in this case protein S (ProS) was clearly increased in protein extracts of carotid arteries with increasing grade of atherosclerosis, suggesting that ProS is carried into the plaque. MERTK was also increased in atherosclerotic carotid arteries with respect to the normal ones, suggesting that the ProS-MERTK axis is functional in advanced human atherosclerotic plaques. MERTK was expressed in macrophages, frequently in association with ProS, while ProS was abundant also in the necrotic core. Our data suggest that the ProS-MERTK ligand-receptor pair was active in advanced stages of atherosclerosis, while AXL signalling is probably down-regulated.

Helicobacter pylori cagA and vacA genotypes as predictors of progression of gastric preneoplastic lesions: a long-term follow-up in a high-risk area in Spain.

OBJECTIVES: There are no established predictive markers of progression of gastric preneoplastic lesions. The aim of this study was to analyze the relationship between Helicobacter pylori cagA and vacA genotypes and progression of gastric preneoplastic lesions. METHODS: This was a follow-up study that carried out in a province of Spain with a high risk of gastric cancer. A total of 312 patients who underwent upper endoscopy with gastric biopsy in 1988-1994 with diagnoses of normal mucosa, non-atrophic gastritis (NAG), non-metaplastic multifocal atrophic gastritis (MAG), and complete or incomplete intestinal metaplasia (IM), and who accepted to undergo a new biopsy during 2005-2007 or had an end point during follow-up, were included in this study. Detection and characterization of H. pylori cagA and vacA genotypes was performed directly in baseline paraffin-embedded gastric biopsy specimens by PCR followed by reverse hybridization onto a line probe assay. Inter- and intra-observer variability of histological diagnosis was assessed. Analysis was done using unconditional logistic regression. RESULTS: The mean age of patients was 48.5 years (45% males) and the mean of follow-up was 12.8 years. H. pylori strains harboring cagA, vacA s1, and vacA m1 genotypes were more frequently found in patients with more advanced gastric preneoplastic lesions. Infection with cagA-positive, vacA s1, and vacA m1 strains was associated with progression of gastric preneoplastic lesions (multivariate odds ratio (OR)=2.28, 95% confidence interval (CI) 1.13-4.58; OR=2.90, 95% CI 1.38-6.13; and OR=3.38, 95% CI 1.34-8.53, respectively). Infection with strains that are simultaneously cagA positive and vacA s1/m1 was associated with progression of gastric precancerous lesions with an OR of 4.80 (95% CI 1.71-13.5) in relation to those infected with cagA-negative/vacA s2/m2 strains. CONCLUSIONS: H. pylori genotyping may be useful for the identification of patients at high risk of progression of gastric preneoplastic lesions and who need more intensive surveillance.

Cytokine gene polymorphisms and the risk of adenocarcinoma of the stomach in the European prospective investigation into cancer and nutrition (EPIC-EURGAST).

BACKGROUND: The relative contribution to gastric cancer (GC) risk of variants in genes that determine the inflammatory response remains mostly unknown and results from genotyping studies are inconsistent. PATIENTS AND METHODS: A nested case-control study within the prospective European Prospective Investigation into Cancer and Nutrition cohort was carried out, including 248 gastric adenocarcinomas and 770 matched controls. Twenty common polymorphisms at cytokine genes [interleukin (IL)1A, IL1B, IL1RN, IL4, IL4R, IL6, IL8, IL10, IL12A, IL12B, lymphotoxin alpha and tumor necrosis factor (TNF)] were analyzed. Antibodies against Helicobacter pylori (Hp) and CagA were measured. RESULTS: IL1RN 2R/2R genotype [odds ratio (OR) 2.43; 95% confidence interval (CI) 1.19-4.96] and allele IL1RN Ex5-35C were associated with an increased risk of Hp(+) non-cardia GC. IL8 -251AA genotype was associated with a decreased risk of Hp(+) non-cardia GC (OR 0.51; 95% CI 0.32-0.81), mainly of the intestinal type. These associations were not modified by CagA status. Carriers of IL1B -580C and TNF -487A alleles did not associate with an increased risk. A moderately increased risk of Hp(+) non-cardia GC for IL4R -29429T variant was observed (OR 1.74; 95% CI 1.15-2.63). CONCLUSION: This prospective study confirms the association of IL1RN polymorphisms with the risk of non-cardia GC and indicates that IL8 -251T>A may modify the risk for GC.

Markers of cell death-activation in lymphocytes of vertically HIV-infected children naive to highly active antiretroviral therapy: the role of age.

BACKGROUND: Apoptosis plays a major role in depleting CD4(+) lymphocytes during infection with HIV-1. Few data exist on its role during HIV infection of children. Sensitivity of peripheral blood lymphocytes (PBLs) to apoptotic stimuli and the importance of the patient's age remain unclear. OBJECTIVES: We sought to analyze the following: (1) markers of cell death-activation (CD95, CD45 isoforms, and CD28) in PBLs from vertically HIV-infected children of different ages before highly active antiretroviral therapy; (2) changes in other PBL populations; (3) PBL sensitivity to cell death and mitochondrial damages; and (4) role of age during progression of infection. METHODS: Cell culture techniques and flow cytometry were used to analyze surface antigens, PBL susceptibility to apoptosis, or PBL susceptibility to change of mitochondrial membrane potential. RESULTS: Donor age had a strong negative correlation with numbers of CD4(+) and CD8(+) T cells. Virgin T lymphocyte (CD45RA(+), CD95(-)) levels and those of CD95(+) cells showed no correlation with the children's clinical status but did show a correlation with patient age. CD28(-) T lymphocytes were markedly augmented in HIV-infected children but were unrelated to stage of infection or age. A relevant decrease in B lymphocytes and an increase in natural killer cells were also found. Finally, PBLs from HIV-positive children had a marked tendency to undergo apoptosis and mitochondrial damage. CONCLUSION: Changes in PBL phenotype, increased expression of CD95, and high sensitivity to apoptosis suggest that a precocious aging of the immune system occurs in HIV-infected children.

Linking extraction and purification of maize samples for fumonisin analysis.

Fumonisins are produced by several fungal species that are common contaminants of maize. The most abundant naturally occurring fumonisin, fumonisin B1 (FB1), has been shown to induce several animal disease syndromes. The development of analytical methods is therefore important. A new method is described that integrates extraction and purification of maize samples in one step. It efficiency is compared against well-known methods, and shows similar results for naturally contaminated maize. It is concluded that the proposed method can be applied to fumonisin B1 and fumonisin B2 (FB2) analysis in maize at least within the concentration range found.

Increased risk of venous thrombosis in carriers of natural anticoagulant deficiencies. Results of the family studies of the Spanish Multicenter Study on Thrombophilia (EMET study).

Several studies have demonstrated a higher risk of thrombosis in carriers of anticoagulant deficiencies than in non-deficient individuals from families with thrombophilia. The prevalences in Spain were established in a multicenter study (the EMET study) and all the deficient individuals were invited to recruit all available family members to be screened for the same deficiency in order to establish the risk of thrombosis in deficient individuals. Five-hundred-and-eighty-three individuals from 114 families with natural anticoagulant deficiencies were analysed. Propositi and relatives with a history of thrombosis were asked about the localization and the age at the first episode and whether or not it was spontaneous. Three families with antithrombin deficiency, 35 with protein C, 60 with protein S, four with plasminogen, four with heparin cofactor II, seven with combined deficiencies and one family with dysfibrinogenemia were included in the analysis. The risk of thrombosis was increased for individuals deficient in antithrombin (adjusted odds ratio 21.23; 95% confidence interval 5.71-78.94), protein C (adjusted odds ratio 12.62; 95% confidence interval 4.75-33.51), protein S type I (adjusted odds ratio 19.95; 95% confidence interval 7.40-53.82), protein S type III (adjusted odds ratio 8.11; 95% confidence interval 2.66-21.99) or in protein C plus protein S (adjusted odds ratio 8.99; 95% confidence interval 2.79-28.93), but not for those deficient in plasminogen or heparin cofactor II. The thrombosis-free survival was shortened for deficient individuals in antithrombin (median 30 years), protein C (median 46 years), protein S type-I (median 48 years), protein S type III (median 61 years) and combined protein C and S (median 40 years). In conclusion, individuals carrying anticoagulant deficiencies have an increased risk of thrombosis, especially those with antithrombin, protein C or type I protein S deficiencies.

Cluster-iron substitution is related to structural and functional features of adrenodoxin mutants and to their redox states.

Site-directed mutants of adrenodoxin were studied for their ability to undergo cluster-iron substitution when reacted with zinc or cadmium salts under non-denaturing conditions in the presence or absence of reductants. Equilibrium and kinetic data for metal substitution were correlated with data on the stability to thermal unfolding and with the redox potential of the protein. Similarly to the wild-type protein, all mutants were able to stabilize a substituted form of the protein containing two metal (Zn or Cd) atoms and two sulfide ions/mol protein and a substituted form of the protein containing two sulfide ions and five Cd atoms/mol protein. However, the distribution of these two metal-substituted forms was different among the investigated proteins. [Ser95]Adrenodoxin stabilized either metal-substituted forms, confirming that Cys95 is not involved in metal coordination, even when five Cd atoms are bound to the protein. Removal of the extremely conserved hydroxy function at position 54 resulted in complete apoprotein formation upon reaction with Cd (75 % with Zn) under reducing conditions, indicating a cluster-harboring role for this function, which is conserved in all known 2Fe-2S proteins. Mutants at His56, which represents a residue unique to most vertebrate-type ferredoxins, were much more reactive than the wild-type protein with either metal, indicating that His56 plays a prominent role in the stabilization of the protein structure in the immediate vicinity of the cluster in this class of proteins. The nature of the metal-substitution products was dependent on cluster accessibility. For the reduced proteins, apoprotein formation depended on protein stability, while the velocity of metal substitution depended on the ease of cluster reduction.

Fate of fumonisins B1 and B2 in steeped corn kernels.

Corn kernels naturally contaminated with fumonisins B1 and B2 were steeped by dipping the whole kernels in either water or a 0.3% solution of sodium bisulphite (pH = 5.7) for 6, 12, 24, 36 and 48 h. The toxin content of both the solid and the liquid were analysed for each time period. A different rate of interchange between the solid matrix and the water solution was found for FB1 and FB2 and some differences were found in the fate of both fumonisins between the two steeping systems studied. In addition, a preliminary study of the distribution of FB1 and FB2 between the germ and the remaining part of the kernel was carried out.

Severe type I protein C deficiency in a compound heterozygote for Y124C and Q132X mutations in exon 6 of the PROC gene.

We report the genetic abnormalities in the protein C genes of a Spanish child with neonatal purpura fulminans and disseminated intravascular coagulation, associated with undetectable protein C levels. Direct sequencing of the nine protein C gene exons and their splice junctions indicated that the proband is a compound heterozygote with two mutant protein C gene alleles, Y124C and Q132X, that do not express protein C in plasma. The Y124C mutation was inherited from the mother and is due to a novel A to G transition at nucleotide 3416, which results in the substitution of cysteine for tyrosine 124, a highly conserved amino acid in EGF-like domains. The paternal inherited mutation (Q132X) is a C to T transition at nucleotide 3439, which replaces glutamine 132 with a Stop codon signal. This mutation, if expressed, should result in the synthesis of a truncated protein of 131 amino acids. Y124C or Q132X are present in the heterozygous state in the asymptomatic parents and siblings of the proband, all of which have half the normal plasma levels of protein C. Q123X has also been identified in families where type I PC deficiency is inherited as a clinically dominant trait. Therefore, the presence of the same mutation in a family showing a clinically recessive pattern of inheritance indicates that other factors, apart from the type of protein C gene mutation, are responsible for the clinical expression of protein C deficiency.

Abnormal polymerization and normal binding of plasminogen and t-PA in three new dysfibrinogenaemias: Barcelona III and IV (gamma Arg 275-->His) and Villajoyosa (gamma Arg 275-->Cys).

Congenital dysfibrinogenaemia was found in three non-related patients. None of them had a haemorrhagic tendency, but one gave a thrombotic history. When their fibrinogens were treated with thrombin, they released fibrinopeptides A and B at normal rates, but the resultant fibrin monomers produced exhibited abnormal polymerization curves. This abnormality was more marked in fibrinogen Villajoyosa than in Barcelonas III and IV. Plasminogen and t-PA binding to fibrin monomers from the three dysfibrinogenaemias was similar to that of normal fibrin monomers. The gamma chain was purified from the three fibrinogens, treated with CNBr and the peptides produced were separated by reversed-phase HPLC. Chromatograms of digested fibrinogens showed an abnormal peak that was not present in the normal gamma chain. Amino acid sequence analysis of abnormal peptides and genomic DNA sequencing revealed that the gamma arginine 275 had been changed in the three fibrinogens; in two cases it was substituted by histidine, and in the third by cysteine. The altered properties observed in fibrin monomers produced from fibrinogen with the gamma Arg 275-->His or gamma Arg 275-->Cys substitution, suggests that this amino acid is important in maintaining the protein structure necessary for normal polymerization, but is not essential for the binding of t-PA or plasminogen to fibrin. It also suggests that the change Arg-->Cys produces more severe alterations in the functions of fibrinogen than the substitution Arg-->His.

[The procollagen-III peptide in the clinical surveillance of alcoholic hepatopathy and other chronic hepatopathies]. / Il peptide del procollagene-III nella sorveglianza clinica dell'epatopatia alcolica e di altre epatopatie croniche.

Liver fibrosis determines the course and prognosis of alcoholic liver disease. Evaluation of serum concentration of procollagen-III peptides (sPIIIP) is considered a biochemical test useful for evaluating a fibrotic process. We have investigated 30 healthy subjects and 53 patients with alcoholic liver disease, histologically diagnosed by percutaneous liver biopsy in four clusters: steatosis (11), fibrosteatosis (11), chronic active hepatitis (11) and cirrhosis (20). SPIIIP levels were increased in patients with cirrhosis and chronic active hepatitis; while they were regular in patients with steatosis and fibrosteatosis. Evaluation of serum concentration of sPIIIP by radioimmunoassay seems to be an useful test for identifying patients with alcoholic liver disease with a good prognosis and patients in progress to cirrhosis and it is an useful test for control the course and evolution of chronic liver disease.

[Hepatitis C and chronic alcoholic hepatopathy]. / Epatite C ed epatopatia cronica alcolica.

We evaluated hepatitis C virus prevalence in 105 patients with chronic alcoholic liver disease by research of antibodies to hepatitis C virus (anti-HCV) by second generation recombinant based enzyme immunosorbent to assay and immunoblot to assay. 46 patients of 105 (43.7%) were anti-HCV positive: 2 in steatosis group (9.5%), 3 in steatofibrosis (23%), 10 in chronic active hepatitis (66.6%), 22 in cirrhosis (59.4%) and 9 in hepatocellular carcinoma (47.3%). These patients had higher levels of serum gammaglobulin and transaminases than seronegative patients. We think that there is a correlation between high prevalence of antibodies anti-HCV and the evolution of damage in chronic alcoholic liver disease.