Extracellular Vesicles from Pancreatic Cancer Stem Cells Lead an Intratumor Communication Network (EVNet) to fuel tumour progression.

OBJECTIVE: Intratumor heterogeneity drives cancer progression and therapy resistance. However, it has yet to be determined whether and how subpopulations of cancer cells interact and how this interaction affects the tumour. DESIGN: We have studied the spontaneous flow of extracellular vesicles (EVs) between subpopulations of cancer cells: cancer stem cells (CSC) and non-stem cancer cells (NSCC). To determine the biological significance of the most frequent communication route, we used pancreatic ductal adenocarcinoma (PDAC) orthotopic models, patient-derived xenografts (PDXs) and genetically engineered mouse models (GEMMs). RESULTS: We demonstrate that PDAC tumours establish an organised communication network between subpopulations of cancer cells using EVs called the EVNet). The EVNet is plastic and reshapes in response to its environment. Communication within the EVNet occurs preferentially from CSC to NSCC. Inhibition of this communication route by impairing Rab27a function in orthotopic xenographs, GEMMs and PDXs is sufficient to hamper tumour growth and phenocopies the inhibition of communication in the whole tumour. Mechanistically, we provide evidence that CSC EVs use agrin protein to promote Yes1 associated transcriptional regulator (YAP) activation via LDL receptor related protein 4 (LRP-4). Ex vivo treatment of PDXs with antiagrin significantly impairs proliferation and decreases the levels of activated YAP.Patients with high levels of agrin and low inactive YAP show worse disease-free survival. In addition, patients with a higher number of circulating agrin+ EVs show a significant increased risk of disease progression. CONCLUSION: PDAC tumours establish a cooperation network mediated by EVs that is led by CSC and agrin, which allows tumours to adapt and thrive. Targeting agrin could make targeted therapy possible for patients with PDAC and has a significant impact on CSC that feeds the tumour and is at the centre of therapy resistance.

The Adaptive Immune Landscape of the Colorectal Adenoma-Carcinoma Sequence.

BACKGROUND: The tumor immune microenvironment exerts a pivotal influence in tumor initiation and progression. The aim of this study was to analyze the immune context of sporadic and familial adenomatous polyposis (FAP) lesions along the colorectal adenoma-carcinoma sequence (ACS). METHODS: We analyzed immune cell counts (CD3+, CD4+, CD8+, Foxp3+, and CD57+), tumor mutation burden (TMB), MHC-I expression and PD-L1 expression of 59 FAP and 74 sporadic colorectal lesions, encompassing adenomas with low-grade dysplasia (LGD) (30 FAP; 30 sporadic), adenomas with high-grade dysplasia (22 FAP; 30 sporadic), and invasive adenocarcinomas (7 FAP; 14 sporadic). RESULTS: The sporadic colorectal ACS was characterized by (1) a stepwise decrease in immune cell counts, (2) an increase in TMB and MHC-I expression, and (3) a lower PD-L1 expression. In FAP lesions, we observed the same patterns, except for an increase in TMB along the ACS. FAP LGD lesions harbored lower Foxp3+ T cell counts than sporadic LGD lesions. A decrease in PD-L1 expression occurred earlier in FAP lesions compared to sporadic ones. CONCLUSIONS: The colorectal ACS is characterized by a progressive loss of adaptive immune infiltrate and by the establishment of a progressively immune cold microenvironment. These changes do not appear to be related with the loss of immunogenicity of tumor cells, or to the onset of an immunosuppressive tumor microenvironment.

Interleukin-1B signalling leads to increased survival of gastric carcinoma cells through a CREB-C/EBPß-associated mechanism.

BACKGROUND: Polymorphisms in inflammation-related genes have been associated with a risk of gastric carcinoma (GC). However, the biological mechanisms underlying these associations are still elusive. Our objective was to determine whether chronic inflammation-associated IL1Β signalling, as seen in the context of Helicobacter pylori infection, could be linked to gastric carcinogenesis by modulating the behaviour of gastric epithelial cells. METHODS: The effect of IL1B was assessed by studying the expression and activation status of the IL1Β-activated transcription factors C/EBPß and CREB in GC cell lines. Interaction between CREB and C/EBPß was explored through interference RNA, chromatin immunoprecipitation and chemical inhibition. CREB and C/EBPß expression was analysed in 66 samples of primary GC and in normal gastric mucosa. GC cell growth was analysed in vitro by BrdU incorporation and in vivo employing a chicken embryo chorioallantoic membrane model. RESULTS: We found that IL1B regulates the expression/activation status of both C/EBPß and CREB in GC cells through an ERK1/2-dependent mechanism. Our results show that CREB is a direct transactivator of CEBPB, acting as an upstream effector in this regulatory mechanism. Furthermore, we found CREB to be overexpressed in 94 % of GC samples and significantly associated with C/EBPß expression (P < 0.05). Finally, we demonstrated both in vitro and in vivo that CREB can mediate IL1B-induced GC cell proliferation. CONCLUSIONS: Our results support the hypothesis that the effect of chronic inflammation on gastric carcinogenesis, as seen in the context of genetically susceptible individuals infected with Helicobacter pylori, includes the modulation of signalling pathways that regulate survival mechanisms in epithelial cells. IL1B is able to increase the expression/activation status of CREB and its target gene C/EBPß, which are mandatory for GC cell survival. Our results may help inform new strategies for the prevention and treatment of GC, including the control of chronic inflammation.

Genetic variants in the IL1A gene region contribute to intestinal-type gastric carcinoma susceptibility in European populations.

The most studied genetic susceptibility factors involved in gastric carcinoma (GC) risk are polymorphisms in the inflammation-linked genes interleukin 1 (IL1) B and IL1RN. Despite the evidence pointing to the IL1 region, definite functional variants reproducible across populations of different genetic background have not been discovered so far. A high density linkage disequilibrium (LD) map of the IL1 gene cluster was established using HapMap to identify haplotype tagSNPs. Eighty-seven SNPs were genotyped in a Portuguese case-control study (358 cases, 1,485 controls) for the discovery analysis. A replication study, including a subset of those tagSNPs (43), was performed in an independent analysis (EPIC-EurGast) containing individuals from 10 European countries (365 cases, 1284 controls). Single SNP and haplotype block associations were determined for GC overall and anatomopathological subtypes. The most robust association was observed for SNP rs17042407, 16Kb upstream of the IL1A gene. Although several other SNP associations were observed, only the inverse association of rs17042407 allele C with GC of the intestinal type was observed in both studies, retaining significance after multiple testing correction (p = 0.0042) in the combined analysis. The haplotype analysis of the IL1A LD block in the combined dataset revealed the association between a common haplotype carrying the rs17042407 variant and GC, particularly of the intestinal type (p = 3.1 × 10(-5) ) and non cardia localisation (p = 4.6 × 10(-3) ). These results confirm the association of IL1 gene variants with GC and reveal a novel SNP and haplotypes in the IL1A region associated with intestinal type GC in European populations.

Spotlight on hTERT Complex Regulation in Cutaneous T-Cell Lymphomas.

As a major cancer hallmark, there is a sustained interest in understanding the telomerase contribution to carcinogenesis in order to therapeutically target this enzyme. This is particularly relevant in primary cutaneous T-cell lymphomas (CTCL), a malignancy showing telomerase dysregulation with few investigative data available. In CTCL, we examined the mechanisms involved in telomerase transcriptional activation and activity regulation. We analyzed 94 CTCL patients from a Franco-Portuguese cohort, as well as 8 cell lines, in comparison to 101 healthy controls. Our results showed that not only polymorphisms (SNPs) located at the promoter of human telomerase reverse transcriptase (hTERT) gene (rs2735940 and rs2853672) but also an SNP located within the coding region (rs2853676) could influence CTCL occurrence. Furthermore, our results sustained that the post-transcriptional regulation of hTERT contributes to CTCL lymphomagenesis. Indeed, CTCL cells present a different pattern of hTERT spliced transcripts distribution from the controls, mostly marked by an increase in the hTERT ß+ variants proportion. This increase seems to be associated with CTCL development and progression. Through hTERT splicing transcriptome modulation with shRNAs, we observed that the decrease in the α-ß+ transcript induced a decrease in the cell proliferation and tumorigenic capacities of T-MF cells in vitro. Taken together, our data highlight the major role of post-transcriptional mechanisms regulating telomerase non canonical functions in CTCL and suggest a new potential role for the α-ß+ hTERT transcript variant.

The Glucocorticoid Receptor Gene (NR3C1) 9ß SNP Is Associated with Posttraumatic Stress Disorder.

Posttraumatic stress disorder (PTSD) has been associated with glucocorticoid (GC) hypersensitivity. Although genetic factors account for 30-46% of the variance in PTSD, no associations have been found between single nucleotide polymorphisms (SNPs) of the GC receptor (GR) gene (NR3C1) and risk for this disorder. We studied the association of five SNPs in the GR gene (rs10052957, rs6189/rs6190, rs6195, rs41423247, and rs6198) and haplotypes with PTSD, in a group of Portuguese male war veterans (33 with lifetime PTSD, 28 without). To determine whether the 9ß SNP (rs6198) was associated with chronically altered cortisol levels, we evaluated hair cortisol concentrations (HCC) in a sample of 69 veterans' offspring. The 9ß variant (G allele) was significantly associated with lifetime PTSD under a dominant model of inheritance. The 9ß variant was also significantly associated with severity of current PTSD symptoms. The haplotype analysis revealed an association between a common haplotype comprising the 9ß risk allele and lifetime PTSD. Carriers of the 9ß risk allele had significantly lower HCC than non-carriers. We found the 9ß risk allele and a haplotype comprising the 9ß risk allele of the GR gene to be associated with PTSD in veterans. This 9ß risk allele was also associated with lower HCC in their offspring.

C/EBP alpha expression is associated with homeostasis of the gastric epithelium and with gastric carcinogenesis.

Transcription factors from the CCAAT/enhancer-binding protein (C/EBP) family are fundamental for the control of differentiation and proliferation of many adult tissues. C/EBP alpha has a crucial role in inducing terminal differentiation and is an established tumor suppressor gene in several cancer models. The objective of this study was to analyze the putative role of C/EBP alpha in gastric carcinoma (GC). We analyzed the expression of C/EBP alpha in normal and neoplastic gastric tissues, and assessed the role of C/EBP alpha on proliferation and differentiation of GC cells. In normal gastric mucosa, C/EBP alpha is expressed in the foveolar epithelium and co-localizes with the gastric differentiation marker trefoil factor 1 (TFF1). The expression of C/EBP alpha was found to be lost in 30% of GC cases. To evaluate the role of C/EBP alpha in cell proliferation and differentiation, we transfected GC cells with a full-length C/EBP alpha protein. We observed a significant decrease in proliferation in C/EBP alpha-transfected cells. This was accompanied by a decrease in Cyclin D1, an increase in P27 expression, and an increased expression of TFF1. Finally, we showed that inhibition of the Ras/MAPK pathway leads to increased C/EBP alpha and TFF1 expression, and decreased cell proliferation and cyclin D1 expression in GC cells. Our results suggest that C/EBP alpha (together with other members of the C/EBP family) has an active role in the control of differentiation and proliferation in normal gastric mucosa. In GC, loss of C/EBP alpha may be associated with the switch from a cellular differentiation to a cellular proliferation program, presumably as a consequence of Ras/MAPK pathway activation.

The Influence of the Genetic and Immunologic Context in the Development of Colorectal Adenoma: A Case Series Report.

INTRODUCTION: Overcoming immunosurveillance is a major step in the progression of many types of tumors. Several immune escape strategies have been identified, including immunoediting and the establishment of an immune suppressive microenvironment. The aim of the present study was to determine whether the hereditary or sporadic context has any influence in the relationship between immune surveillance and tumor development, using sporadic and familial adenomatous polyposis related colorectal adenomas as a model. MATERIAL AND METHODS: The immune tumor-infiltrating cells of a total of 58 low-grade and 18 high-grade colorectal adenomas were examined and compared, using immunostaining for CD3, CD4, CD8, CD57, CD68 and FoxP3. RESULTS: FoxP3 and CD68 counts were significantly higher in sporadic low-grade dysplasia (p = 0.0003 and p = 0.0103, respectively),and FoxP3 and CD4 counts were found to be significantly higher in high-grade sporadic dysplasia (p = 0.0008 and p = 0.0018, respectively)when compared with corresponding lesions in patients with familial adenomatous polyposis. DISCUSSION: This study suggests that the immune microenvironment of sporadic and hereditary lesions is different. Sporadic lesions contain a higher number of immune suppressive Treg cells, which suggests a stronger immune selective pressure. In contrast, hereditarylesions seem to benefit from a more tolerant immune microenvironment, allowing for the development of lesions with lower immune cell infiltration. CONCLUSION: This study shows that sporadic lesions harbor higher tumor-infiltrating immune cell counts, which might reflect a higher immune tolerance towards hereditary lesions.

Introdução: A capacidade de contornar a imunovigilância é fundamental na progressão de muitos tumores. Já foram identificadas várias estratégias de escape imunológico, incluindo immunoediting e o estabelecimento de um microambiente imunológico supressivo. O objetivo do presente estudo passa por determinar se o contexto hereditário ou esporádico influencia a relação entre a imunovigilância e o desenvolvimento do tumor, usando adenomas coloretais esporádicos e hereditários, no contexto de polipose adenomatosa familiar, como modelos. Material e Métodos: Os infiltrados imunológicos tumorais de um total de 58 adenomas coloretais de baixo grau e de 18 de alto grau foram avaliados e comparados, usando imunohistoquímica com marcação para CD3, CD4, CD8, CD57, CD68 e FoxP3. Resultados: As contagens celulares com imunorreatividade para FoxP3 e CD68 foram significativamente mais elevadas na displasia esporádica de baixo grau (p = 0,0003 e p = 0,0103, respetivamente), enquanto que as contagens para FoxP3 e CD4 foram significativamente mais elevadas na displasia esporádica de alto grau (p = 0,0008 e p = 0,0018, respetivamente) quando comparadas com lesões correspondentes em doentes com polipose adenomatosa familiar. Discussão: O presente estudo sugere que o microambiente imunológico de lesões esporádicas e hereditárias é diferente. As lesões esporádicas contam com um número superior de células T reguladoras, supressoras da função imunológica, sugerindo-se uma pressão imune seletiva mais forte. Por seu turno, as lesões hereditárias parecem beneficiar de um microambiente imunológico mais tolerante, permitindo o desenvolvimento de lesões com menor infiltrado celular imune. Conclusão: Este estudo demonstra que as lesões esporádicas contam com contagens de infiltrados imunológicos tumorais superiores, o que poderá refletir uma maior tolerância imunológica face a lesões hereditárias.

Genetic Variants of the MGAT5 Gene Are Functionally Implicated in the Modulation of T Cells Glycosylation and Plasma IgG Glycome Composition in Ulcerative Colitis.

OBJECTIVES: The impact of genetic variants (single nucleotide polymorphisms [SNPs]) in the clinical heterogeneity of ulcerative colitis (UC) remains unclear. We showed that patients with UC exhibit a deficiency in MGAT5 glycogene transcription in intestinal T cells associated with a hyperimmune response. Herein, we evaluated whether MGAT5 SNPs might functionally impact on T cells glycosylation and plasma IgG glycome in patients with UC, as well as in UC clinical outcomes. METHODS: Three selected MGAT5 SNPs (rs3814022, rs4953911, and rs1257220), previously associated with severity of autoimmune disease or with plasma glycome composition in healthy individuals, were functionally evaluated in patients with UC through analysis of MGAT5 mRNA levels in colonic (n = 14) and circulating (n = 24) T cells and through profiling the plasma IgG Fc glycosylation (n = 152). MGAT5 SNPs were genotyped in 931 patients with UC from 2 European cohorts and further associated with patients' prognosis. Targeted next-generation sequencing for MGAT5 coding and regulatory regions was also performed. RESULTS: MGAT5 SNPs were shown to be functionally associated with low transcription levels of MGAT5 in colonic and circulating T cells from patients with UC and with agalactosylation of IgGs, often associated with a proinflammatory phenotype. The SNPs rs3814022 and rs4953911 were further associated with the need of biologics. Next-generation sequencing data further revealed a combination of MGAT5 SNPs that stratify patients with UC according to their severity. DISCUSSION: Our results revealed that MGAT5 SNPs have a phenotypic impact on T cells glycosylation and in plasma IgG glycome composition associated with UC pathogenesis. MGAT5 SNPs display a tendency in the association with a worse disease course in patients with UC.

Differential Modulation of Cancellous and Cortical Distal Femur by Fructose and Natural Mineral-Rich Water Consumption in Ovariectomized Female Sprague Dawley Rats.

Bone mineral density (BMD) and microstructure depend on estrogens and diet. We assessed the impact of natural mineral-rich water ingestion on distal femur of fructose-fed estrogen-deficient female Sprague Dawley rats. Ovariectomized rats drank tap or mineral-rich waters, with or without 10%-fructose, for 10 weeks. A sham-operated group drinking tap water was included (n = 6/group). Cancellous and cortical bone compartments were analyzed by microcomputed tomography. Circulating bone metabolism markers were measured by enzyme immunoassay/enzyme-linked immunosorbent assay or multiplex bead assay. Ovariectomy significantly worsened cancellous but not cortical bone, significantly increased circulating degradation products from C-terminal telopeptides of type I collagen and receptor activator of nuclear factor-kappaB ligand (RANKL), and significantly decreased circulating osteoprotegerin and osteoprotegerin/RANKL ratio. In ovariectomized rats, in cancellous bone, significant water effect was observed for all microstructural properties, except for the degree of anisotropy, and BMD (neither a significant fructose effect nor a significant interaction between water and fructose ingestion effects were observed). In cortical bone, it was observed a significant (a) water effect for medullary volume and cortical endosteal perimeter; (b) fructose effect for cortical thickness, medullary volume, cross-sectional thickness and cortical endosteal and periosteal perimeters; and (c) interaction effect for mean eccentricity. In blood, significant fructose and interaction effects were found for osteoprotegerin (no significant water effect was seen). For the first time in ovariectomized rats, the positive modulation of cortical but not of cancellous bone by fructose ingestion and of both bone locations by natural mineral-rich water ingestion is described.

The Two Faces of Tumor-Associated Macrophages and Their Clinical Significance in Colorectal Cancer.

Macrophages are one of the immune populations frequently found in colorectal tumors and high macrophage infiltration has been associated with both better and worst prognosis. Importantly, according to microenvironment stimuli, macrophages may adopt different polarization profiles, specifically the pro-inflammatory or M1 and the anti-inflammatory or M2, which display distinct functions. Therefore, concomitantly with the number of tumor-associated macrophages (TAMs), their characterization is fundamental to unravel their relevance in cancer. Here, we profiled macrophages in a series of 150 colorectal cancer (CRC) cases by immunohistochemistry, using CD68 as a macrophage lineage marker, CD80 as a marker of pro-inflammatory macrophages, and CD163 as a marker of anti-inflammatory macrophages. Quantifications were performed by computer-assisted analysis in the intratumoral region, tumor invasive front, and matched tumor adjacent normal mucosa (ANM). Macrophages, specifically the CD163+ ones, were predominantly found at the tumor invasive front, whereas CD80+ macrophages were almost exclusively located in the ANM, which suggests a predominant anti-inflammatory polarization of TAMs. Stratification according to tumor stage revealed that macrophages, specifically the CD163+ ones, are more prevalent in stage II tumors, whereas CD80+ macrophages are predominant in less invasive T1 tumors. Specifically in stage III tumors, higher CD68, and lower CD80/CD163 ratio associated with decreased overall survival. Importantly, despite the low infiltration of CD80+ cells in colorectal tumors, multivariate logistic regression revealed a protective role of these cells regarding the risk for relapse. Overall, this work supports the involvement of distinct microenvironments, present at the intra-tumor, invasive front and ANM regions, on macrophage modulation, and uncovers their prognostic value, further supporting the relevance of including macrophage profiling in clinical settings.

Interaction of Genetic Variations in NFE2L2 and SELENOS Modulates the Risk of Hashimoto's Thyroiditis.

Background: Several single-nucleotide polymorphisms (SNPs) are known to increase the risk of Hashimoto's thyroiditis (HT); such SNPs reside in thyroid-specific genes or in genes related to autoimmunity, inflammation, and/or cellular defense to stress. The transcription factor Nrf2, encoded by NFE2L2, is a master regulator of the cellular antioxidant response. This study aimed to evaluate the impact of genetic variation in NFE2L2 on the risk of developing HT. Methods: In a case-control candidate gene association study, functional SNPs in the NFE2L2 promoter (rs35652124, rs6706649, and rs6721961) were examined either as independent risk factors or in combination with a previously characterized HT risk allele (rs28665122) in the gene SELENOS, encoding selenoprotein S (SelS). A total of 997 individuals from the north of Portugal (Porto) were enrolled, comprising 481 HT patients and 516 unrelated healthy controls. SELENOS and NFE2L2 SNPs were genotyped using TaqMan® assays and Sanger sequencing, respectively. Odds ratios (ORs) were calculated using logistic regression, with adjustment for sex and age. Expression of SelS was analyzed by immunohistochemistry in thyroid tissue from HT patients and control subjects. Molecular interactions between the Nrf2 and SelS pathways were investigated in thyroid tissues from mice and in rat PCCL3 thyroid follicular cells. Results: When all three NFE2L2 SNPs were considered together, the presence of one or more minor alleles was associated with a near-significant increased risk (OR = 1.43, p = 0.072). Among subjects harboring only major NFE2L2 alleles, there was no increased HT risk associated with heterozygosity or homozygosity for the SELENOS minor allele. Conversely, in subjects heterozygous or homozygous for the SELENOS risk allele, the presence of an NFE2L2 minor allele significantly increased HT risk by 2.8-fold (p = 0.003). Immunohistochemistry showed reduced expression of SelS in thyroid follicular cells of HT patients. In Nrf2 knockout mice, there was reduced expression of SelS in thyroid follicular cells; conversely, in PCCL3 cells, reducing SelS expression caused reduced activity of Nrf2 signaling. Conclusions: The NFE2L2 promoter genotype interacts with the SELENOS promoter genotype to modulate the risk of HT in a Portuguese population. This interaction may be due to a bidirectional positive feedback between the Nrf2 and SelS pathways.

Tumor necrosis factor alpha extended haplotypes and risk of gastric carcinoma.

The tumor necrosis factor alpha (TNFA)-308*A allele has been found to confer an increased risk of gastric carcinoma. Inconsistency in risk estimates across populations lead us to hypothesize about the presence of an alternative causal locus in the same chromosomal region. A suitable approach is to determine the tumor necrosis factor haplotypic structure in order to clarify whether the association between the *A allele and the increased risk of gastric carcinoma is etiologic or secondary to linkage disequilibrium. Firstly, we assessed the association between the TNFA-308G>A polymorphism and the risk of gastric carcinoma in a population from Northern Portugal (508 gastric carcinoma patients, 713 controls); secondly, we genotyped five microsatellite loci (TNFa, b, c, d, e) flanking the TNFA-308G>A locus to establish the haplotypic structure associated with this single-nucleotide polymorphism in cases (122 patients) and controls (169 individuals). We found a significant association between the *A allele and increased risk of gastric carcinoma (odds ratio, 1.7; 95% confidence interval, 1.3-2.2) confirming previous results in our population. Regarding the *A allele-associated haplotypes, the most relevant difference was found for the H1A haplotype present in 33.1% of the cases and 12.5% of the controls. We also observed haplotypes associated with the *A allele that were found only in cases or controls. A population differentiation test showed that the gastric carcinoma and the control groups were significantly different for the *A allele haplotypic structure. This suggests that the association between the TNFA-308G>A polymorphism and increased risk of gastric carcinoma is dependent on linkage disequilibrium with an as yet unidentified locus.

Is Low-Dose Radiation Exposure a Risk Factor for Atherosclerotic Disease?

Nontargeted late effects of radiation include an increased risk of cardiovascular disease, although this is still debatable in the context of low-dose radiation. Tinea capitis patients treated in childhood with X rays to induce scalp epilation received a low dose of radiation to their carotids. To better clarify this issue, we evaluated carotid atherosclerosis in a cohort of such patients treated in 1950-1963 in Portugal. A group of 454 individuals randomly chosen from previously observed Portuguese tinea capitis patients and a control group mainly composed of their spouses (n = 280) were enrolled. Cardiovascular risk factors such as waist circumference, body mass index, blood pressure and tobacco consumption, as well as biochemical measurements were obtained. Ultrasound imaging of carotid arteries for intima media thickness and stenosis evaluation were performed according to a standardized protocol. In comparison to the control group, the irradiated cohort members were significantly older, more frequently never smokers, hypertensive, and presented higher glycated hemoglobin and alkaline phosphatase levels. In addition, the irradiated cohort showed a higher frequency of carotid stenosis ≥30% than the nonirradiated group (13.9% vs. 10.7%), although this was not significant ( P = 0.20). Stenosis was ≥50% in 2.9% of the irradiated group and 0.4% of the nonirradiated group ( P = 0.02). Likewise, the frequency of intima media thickness ≥1 mm was significantly higher in the irradiated group (16.8% vs. 10.7%; P = 0.02). Multivariate analysis, including other cardiovascular risk factors, showed that exposure to low-dose radiation increased the risk of carotid stenosis by ≥50% [odds ratio (OR) = 8.85; P = 0.04] and intima media thickness by ≥1 mm (OR = 1.82; P = 0.02). These findings confirm that low-dose exposure is a risk factor of carotid atherosclerotic disease.

Association between Polymorphisms in Antioxidant Genes and Inflammatory Bowel Disease.

Inflammation is the driving force in inflammatory bowel disease (IBD) and its link to oxidative stress and carcinogenesis has long been accepted. The antioxidant system of the intestinal mucosa in IBD is compromised resulting in increased oxidative injury. This defective antioxidant system may be the result of genetic variants in antioxidant genes, which can represent susceptibility factors for IBD, namely Crohn's disease (CD) and ulcerative colitis (UC). Single nucleotide polymorphisms (SNPs) in the antioxidant genes SOD2 (rs4880) and GPX1 (rs1050450) were genotyped in a Portuguese population comprising 436 Crohn's disease and 367 ulcerative colitis patients, and 434 healthy controls. We found that the AA genotype in GPX1 is associated with ulcerative colitis (OR = 1.93, adjusted P-value = 0.037). Moreover, we found nominal significant associations between SOD2 and Crohn's disease susceptibility and disease subphenotypes but these did not withstand the correction for multiple testing. These findings indicate a possible link between disease phenotypes and antioxidant genes. These results suggest a potential role for antioxidant genes in IBD pathogenesis and should be considered in future association studies.

IL6-174 G>C Polymorphism (rs1800795) Association with Late Effects of Low Dose Radiation Exposure in the Portuguese Tinea Capitis Cohort.

Head and neck cancers, and cardiovascular disease have been described as late effects of low dose radiation (LDR) exposure, namely in tinea capitis cohorts. In addition to radiation dose, gender and younger age at exposure, the genetic background might be involved in the susceptibility to LDR late effects. The -174 G>C (rs1800795) SNP in IL6 has been associated with cancer and cardiovascular disease, nevertheless this association is still controversial. We assessed the association of the IL6-174 G>C SNP with LDR effects such as thyroid carcinoma, basal cell carcinoma and carotid atherosclerosis in the Portuguese tinea capitis cohort. The IL6-174 G>C SNP was genotyped in 1269 individuals formerly irradiated for tinea capitis. This sampling group included thyroid cancer (n = 36), basal cell carcinoma (n = 113) and cases without thyroid or basal cell carcinoma (1120). A subgroup was assessed for atherosclerosis by ultrasonography (n = 379) and included matched controls (n = 222). Genotypes were discriminated by real-time PCR using a TaqMan SNP genotyping assay. In the irradiated group, we observed that the CC genotype was significantly associated with carotid plaque risk, both in the genotypic (OR = 3.57, CI = 1.60-7.95, p-value = 0.002) and in the recessive (OR = 3.02, CI = 1.42-6.42, p-value = 0.004) models. Irradiation alone was not a risk factor for carotid atherosclerosis. We did not find a significant association of the IL6-174 C allele with thyroid carcinoma or basal cell carcinoma risk. The IL6-174 CC genotype confers a three-fold risk for carotid atherosclerotic disease suggesting it may represent a genetic susceptibility factor in the LDR context.

Quantification of mutant E-cadherin using bioimaging analysis of in situ fluorescence microscopy. A new approach to CDH1 missense variants.

Missense mutations result in full-length proteins containing an amino acid substitution that can be neutral or deleterious, interfering with the normal conformation, localization, and function of a protein. A striking example is the presence of CDH1 (E-cadherin gene) germline missense variants in hereditary diffuse gastric cancer (HDGC), which represent a clinical burden for genetic counseling and surveillance of mutation carriers and their families. CDH1 missense variants can compromise not only the function of E-cadherin but also its expression pattern. Here, we propose a novel method to characterize E-cadherin signature in order to identify cases with E-cadherin deregulation and functional impairment. The strategy includes a bioimaging pipeline to quantify the expression level and characterize the distribution of the protein from in situ immunofluorescence images. The algorithm computes 1D (dimension intensity) radial and internuclear fluorescence profiles to generate expression outlines and 2D virtual cells representing a typical cell within the populations analyzed. Using this new approach, we verify that cells expressing mutant forms of E-cadherin display fluorescence profiles distinct from those of the wild-type cells. Mutant proteins showed a significantly decrease of fluorescence intensity at the membrane and often abnormal expression peaks in the cytoplasm, reflecting the underlying molecular mechanism of trafficking deregulation. Our results suggest employing this methodology as a complementary approach to evaluate the pathogenicity of E-cadherin missense variants. Moreover, it can be applied to a wide range of proteins and, more importantly, to diseases characterized by aberrant protein expression or trafficking deregulation.

Biomarkers for gastric cancer: prognostic, predictive or targets of therapy?

Gastric cancer is an aggressive disease often diagnosed at an advanced stage. Despite improvements in surgical and adjuvant treatment approaches, gastric cancer remains a global public health problem with a 5-year overall survival of less than 25 %. This is a heterogeneous disease, both in terms of biology and genetics, and many prognostic biomarkers have been pointed out in the literature; nevertheless, their application remains debatable. In this review, we opted to give relevance to those biomarkers that have been the subject of studies with significant statistical power, which have been replicated and have been/are in targeted therapy clinical trials and, which as a consequence, have their prognostic and/or predictive value established. Some gastric cancer biomarkers that may help in defining the course of treatment are also discussed. Accepted practical guidelines, wet-lab protocols for the detection of these biomarkers, as well as ongoing and completed clinical trials have been compiled. In summary, clinical approaches based on the combination of correct staging with targeted and conventional systemic therapies may improve gastric cancer patients' outcome, but are only in their infancy. Some major challenges in identifying reliable prognostic/predictive biomarkers are individual genetic variation and tumour heterogeneity that often influence response to therapy and drug resistance. Prognostic and predictive biomarkers may nevertheless be extremely valuable to correctly stratify gastric cancer patients for treatment and, ultimately, improve survival.

Polymorphisms in the TNFA and IL6 genes represent risk factors for autoimmune thyroid disease.

BACKGROUND: Autoimmune thyroid disease (AITD) comprises diseases including Hashimoto's thyroiditis and Graves' disease, both characterized by reactivity to autoantigens causing, respectively, inflammatory destruction and autoimmune stimulation of the thyroid-stimulating hormone receptor. AITD is the most common thyroid disease and the leading form of autoimmune disease in women. Cytokines are key regulators of the immune and inflammatory responses; therefore, genetic variants at cytokine-encoding genes are potential risk factors for AITD. METHODS: Polymorphisms in the IL6-174 G/C (rs1800795), TNFA-308 G/A (rs1800629), IL1B-511 C/T (rs16944), and IFNGR1-56 T/C (rs2234711) genes were assessed in a case-control study comprising 420 Hashimoto's thyroiditis patients, 111 Graves' disease patients and 735 unrelated controls from Portugal. Genetic variants were discriminated by real-time PCR using TaqMan SNP genotyping assays. RESULTS: A significant association was found between the allele A in TNFA-308 G/A and Hashimoto's thyroiditis, both in the dominant (OR = 1.82, CI = 1.37-2.43, p-value = 4.4×10(-5)) and log-additive (OR = 1.64, CI = 1.28-2.10, p-value = 8.2×10(-5)) models. The allele C in IL6-174 G/C is also associated with Hashimoto's thyroiditis, however, only retained significance after multiple testing correction in the log-additive model (OR = 1.28, CI = 1.06-1.54, p-value = 8.9×10(-3)). The group with Graves' disease also registered a higher frequency of the allele A in TNFA-308 G/A compared with controls both in the dominant (OR = 1.85, CI = 1.19-2.87, p-value = 7.0×10(-3)) and log-additive (OR = 1.69, CI = 1.17-2.44, p-value = 6.6×10(-3)) models. The risk for Hashimoto's thyroiditis and Graves' disease increases with the number of risk alleles (OR for two risk alleles is, respectively, 2.27 and 2.59). CONCLUSIONS: This study reports significant associations of genetic variants in TNFA and IL6 with the risk for AITD, highlighting the relevance of polymorphisms in inflammation-related genes in the etiopathogenesis of AITD.

A polymorphism in the promoter region of the selenoprotein S gene (SEPS1) contributes to Hashimoto's thyroiditis susceptibility.

CONTEXT: The association between selenium and inflammation and the relevance of selenoproteins in follicular thyroid cell physiology have pointed to a putative role of selenoproteins in the pathogenesis of autoimmune thyroid diseases. OBJECTIVE: The aim of this study was to evaluate the role of a promoter variation in SEPS1, the selenoprotein S gene, in the risk for developing Hashimoto's thyroiditis (HT). DESIGN: A case-control study was performed to assess the association of genetic variation in the SEPS1 gene (SEPS1 -105G/A single-nucleotide polymorphism, rs28665122) and HT. SETTING: The study was conducted in north Portugal, Porto, in the period of 2007-2013. PATIENTS OR OTHER PARTICIPANTS: A total of 997 individuals comprising 481 HT patients and 516 unrelated controls were enrolled in the study. MAIN OUTCOME MEASURES: Genetic variants were discriminated by real-time PCR using TaqMan single-nucleotide polymorphism genotyping assays. RESULTS: There is a significant association between the SEPS1 -105 GA and AA genotypes and HT [odds ratio (OR) 2.24, confidence interval (CI) 1.67-3.02, P < 5.0 × 10(-7), and OR 2.08, CI 1.09-3.97, P = .0268, respectively]. The A allele carriers are in higher proportion in the patient group than in the control population (46.2% vs 28.1%, P < 5.0 × 10(-7)) with an OR (CI) of 2.22 (1.67-2.97). The proportion of patients carrying the A allele is significantly higher in male patients with HT, representing a 3.94 times increased risk (P = 7.9 × 10(-3)). CONCLUSION: Our findings support the existence of a link between SEPS1 promoter genetic variation and HT risk.