Caracterização do carcinoma gástrico com estroma linfóide: perfil clínico-patológico, resposta imune (estroma linfóide e expressão de PD-L1), infeção pelo vírus de Epstein-Barr e instabilidade de microssatélites

Dissertação de mestrado, Ciências Biomédicas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2017O cancro gástrico (CG) é uma doença heterogénea, fato que se reflete nas características macroscópicas, histológicas e moleculares. O carcinoma gástrico com estroma linfóide (CGEL), é um subtipo histológico de CG, caracterizado por abundante infiltrado linfocitário intratumoral, constituído maioritariamente por linfócitos T, que desempenham funções anti e pro-tumorais. O PD-L1 é um ligando que inibe as funções dos linfócitos T. A expressão aumentada de PD-L1 no CG sugere a possibilidade de utilização de agentes inibidores do checkpoint imunológico como terapia de precisão. O presente trabalho teve como objetivo caracterizar o CGEL, relativamente ao perfil clínicopatológico e molecular, resposta imunológica (estroma linfóide e expressão de PD-L1), infeção por EBV, instabilidade de microssatélites e co-localização de PD-L1 nas células neoplásicas e estromais. O estudo foi realizado numa amostra de 24 casos de CGEL, em que se avaliaram os seguintes parâmetros: características clínico-patológicas; infeção por EBV através de HIS e PCR; instabilidade de microssatélites por PCR e eletroforese capilar; expressão de pan-citoqueratina (AE1/AE3), CD8, CD3 e PD-L1 por imuno-histoquímica; co-localização de PD-L1/CD68 e PD-L1/citoqueratina por imunofluorescência com dupla marcação. Os linfócitos T CD8+ e CD3+ foram quantificados no centro e na periferia do tumor. Os principais resultados mostraram: infeção por EBV em 66,7% dos casos; presença de IMSA em 16,7% dos casos; expressão de PD-L1 (≥50% de células neoplásicas e estromais) em 4 casos com infeção por EBV e em 2 casos com IMS-A. Verificou-se também uma maior densidade de linfócitos T CD3+ e CD8+ nos casos com infeção por EBV e na periferia dos tumores. Conclui-se que o CGEL se caracteriza por um perfil clínico-patológico particular, elevada taxa de infeção por EBV e sobrexpressão de PD-L1. Os resultados deste estudo contribuem para consolidar o conceito de que o CGEL associado a infeção por EBV é um alvo potencial para imunoterapia dirigida ao checkpoint imunológico

CD44v6 high membranous expression is a predictive marker of therapy response in gastric cancer patients

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).In gastric cancer (GC), biomarkers that define prognosis and predict treatment response remain scarce. We hypothesized that the extent of CD44v6 membranous tumor expression could predict prognosis and therapy response in GC patients. Two GC surgical cohorts, from Portugal and South Korea (n = 964), were characterized for the extension of CD44v6 membranous immuno-expression, clinicopathological features, patient survival, and therapy response. The value of CD44v6 expression in predicting response to treatment and its impact on prognosis was determined. High CD44v6 expression was associated with invasive features (perineural invasion and depth of invasion) in both cohorts and with worse survival in the Portuguese GC cohort (HR 1.461; 95% confidence interval 1.002-2.131). Patients with high CD44v6 tumor expression benefited from conventional chemotherapy in addition to surgery (p < 0.05), particularly those with heterogeneous CD44v6-positive and -negative populations (CD44v6_3+) (p < 0.007 and p < 0.009). Our study is the first to identify CD44v6 high membranous expression as a potential predictive marker of response to conventional treatment, but it does not clarify CD44v6 prognostic value in GC. Importantly, our data support selection of GC patients with high CD44v6-expressing tumors for conventional chemotherapy in addition to surgery. These findings will allow better stratification of GC patients for treatment, potentially improving their overall survival.This work was funded by FEDER-Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020–Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT–Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274). This work was also financed by the projects NORTE-01-0145-FEDER-000003 and NORTE-01-0145-FEDER-000029, supported by the Norte Portugal Regional Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); project POCI-01-0145-FEDER-016390 and SAICTPAC/0022/2015, funded by ERDF, POCI, and FCT; project PTDC/CTM-NAN/120958/2010, from FCT; and by project PTDC/BTM-TEC/30164/2017 funded by ERDF funds through the COMPETE 2020–POCI, Portugal 2020, and by FCT. Salary support to G.M.A. by PTDC/BTM-TEC/30164/2017 project; C.P. was supported by the grant SFRH/BD/113031/2015 from FCT.info:eu-repo/semantics/publishedVersio

Immunophenotype of Gastric Tumors Unveils a Pleiotropic Role of Regulatory T Cells in Tumor Development.

Gastric cancer (GC) patients display increased regulatory T cell (Tregs) numbers in peripheral blood and among tumor-infiltrating lymphocytes. Nevertheless, the role of Tregs in GC progression remains controversial. Here, we sought to explore the impact of Tregs in GCs with distinct histology, and whether Tregs can directly influence tumor cell behavior and GC development. We performed a comprehensive immunophenotyping of 82 human GC cases, through an integrated analysis of multispectral immunofluorescence detection of T cells markers and patient clinicopathological data. Moreover, we developed 3D in vitro co-cultures with Tregs and tumor cells that were followed by high-throughput and light-sheet imaging, and their biological features studied with conventional/imaging flow cytometry and Western blotting. We showed that Tregs located at the tumor nest were frequent in intestinal-type GCs but did not associate with increased levels of effector T cells. Our in vitro results suggested that Tregs preferentially infiltrated intestinal-type GC spheroids, induced the expression of IL2Rα and activation of MAPK signaling pathway in tumor cells, and promoted spheroid growth. Accumulation of Tregs in intestinal-type GCs was increased at early stages of the stomach wall invasion and in the absence of vascular and perineural invasion. In this study, we proposed a non-immunosuppressive mechanism through which Tregs might directly modulate GC cells and thereby promote tumor growth. Our findings hold insightful implications for therapeutic strategies targeting intestinal-type GCs and other tumors with similar immune context

The dysfunctional immune system in common variable immunodeficiency increases the susceptibility to gastric cancer

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Gastric carcinoma (GC) represents the most common cause of death in patients with common variable immunodeficiency (CVID). However, a limited number of cases have been characterised so far. In this study, we analysed the clinical features, bacterial/viral infections, detailed morphology and immune microenvironment of nine CVID patients with GC. The study of the immune microenvironment included automated digital counts of CD20+, CD4+, CD8+, FOXP3+, GATA3+ and CD138+ immune cells, as well as the evaluation of PD-L1 expression. Twenty-one GCs from non-CVID patients were used as a control group. GC in CVID patients was diagnosed mostly at early-stage (n = 6/9; 66.7%) and at younger age (median-age: 43y), when compared to non-CVID patients (p < 0.001). GC pathogenesis was closely related to Helicobacter pylori infection (n = 8/9; 88.9%), but not to Epstein-Barr virus (0.0%) or cytomegalovirus infection (0.0%). Non-neoplastic mucosa (non-NM) in CVID-patients displayed prominent lymphocytic gastritis (100%) and a dysfunctional immune microenvironment, characterised by higher rates of CD4+/CD8+/Foxp3+/GATA3+/PD-L1+ immune cells and the expected paucity of CD20+ B-lymphocytes and CD138+ plasma cells, when compared to non-CVID patients (p < 0.05). Changes in the immune microenvironment between non-NM and GC were not equivalent in CVID and non-CVID patients, reflecting the relevance of immune dysfunction for gastric carcinogenesis and GC progression in the CVID population.This article is a result of the projects DOCnet (NORTE-01-0145-FEDER-000003/000029), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This research was funded by FCT-Foundation for Science and Technology/Ministério da Ciência, Tecnologia e Inovação, grantnumber PTDC/MED-PAT/32462/2017 and PTDC/BIM-MEC/2834/2014.This work is funded by grant PAC-PRECISE-LISBOA-01-0145-FEDER-016394, co-funded by FEDER through POR Lisboa 2020—Programa Operacional Regional de Lisboa PORTUGAL 2020 and FCT, and UID/BIM/50005/2019 funded by FCT/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through Fundos do Orçamento de Estadoinfo:eu-repo/semantics/publishedVersio

Expression of Thomsen–Friedenreich Antigen in Colorectal Cancer and Association with Microsatellite Instability

Microsatellite instability (MSI) is a molecular phenotype due to a deficient DNA mismatch repair (dMMR). In colorectal cancer (CRC), dMMR/MSI is associated with several clinical and histopathological features, influences prognosis, and is a predictive factor of response to therapy. In daily practice, dMMR/MSI profiles are identified by immunohistochemistry and/or multiplex PCR. The Thomsen–Friedenreich (TF) antigen was previously found to be a potential single marker to identify MSI-high gastric cancers. Therefore, in this study, we aimed to disclose a possible association between TF expression and MSI status in CRC. Furthermore, we evaluated the relationship between TF expression and other clinicopathological features, including patient survival. We evaluated the expression of the TF antigen in a cohort of 25 MSI-high and 71 microsatellite stable (MSS) CRCs. No association was observed between the expression of the TF antigen and MSI-high status in CRC. The survival analysis revealed that patients with MSI-high CRC showed improved survival when the TF antigen was expressed. This finding holds promise as it indicates the potential use of the TF antigen as a biomarker of better prognosis in MSI-high CRCs that should be validated in an independent and larger CRC cohort

Genome-wide identification and expression analysis of dormancy-associated gene 1/auxin repressed protein (DRM1/ARP) gene family in Glycine max

Dormancy-Associated gene 1/Auxin Repressed protein (DRM1/ARP) genes are responsive to hormones involved in defense response to biotic stress, such as salicylic acid (SA) and methyl jasmonate (MeJA), as well as to hormones that regulate plant growth and development, including auxins. These characteristics suggest that this gene family may be an important link between the response to pathogens and plant growth and development. In this investigation, the DRM1/ARP genes were identified in the genome of four legume species. The deduced proteins were separated into three distinct groups, according to their sequence conservation. The expression profile of soybean genes from each group was measured in different organs, after treatment with auxin and MeJA and in response to the nematode Meloidogyne javanica. The results demonstrated that this soybean gene family is predominantly expressed in root. The time auxin takes to alter DRM1/ARP expression suggests that these genes can be classified as a late response to auxin. Nevertheless, only the groups 1 and 3 are induced in roots infected by M. javanica and only group 3 is induced by MeJA, which indicates a high level of complexity in expression control mechanisms of DRM1/ARP family in soybean

Immunophenotype of Gastric Tumors Unveils a Pleiotropic Role of Regulatory T Cells in Tumor Development.

Gastric cancer (GC) patients display increased regulatory T cell (Tregs) numbers in peripheral blood and among tumor-infiltrating lymphocytes. Nevertheless, the role of Tregs in GC progression remains controversial. Here, we sought to explore the impact of Tregs in GCs with distinct histology, and whether Tregs can directly influence tumor cell behavior and GC development. We performed a comprehensive immunophenotyping of 82 human GC cases, through an integrated analysis of multispectral immunofluorescence detection of T cells markers and patient clinicopathological data. Moreover, we developed 3D in vitro co-cultures with Tregs and tumor cells that were followed by high-throughput and light-sheet imaging, and their biological features studied with conventional/imaging flow cytometry and Western blotting. We showed that Tregs located at the tumor nest were frequent in intestinal-type GCs but did not associate with increased levels of effector T cells. Our in vitro results suggested that Tregs preferentially infiltrated intestinal-type GC spheroids, induced the expression of IL2Rα and activation of MAPK signaling pathway in tumor cells, and promoted spheroid growth. Accumulation of Tregs in intestinal-type GCs was increased at early stages of the stomach wall invasion and in the absence of vascular and perineural invasion. In this study, we proposed a non-immunosuppressive mechanism through which Tregs might directly modulate GC cells and thereby promote tumor growth. Our findings hold insightful implications for therapeutic strategies targeting intestinal-type GCs and other tumors with similar immune context

Plant bZIP Transcription Factors Responsive to Pathogens: A Review

Transcription factors of the basic leucine zipper (bZIP) family control important processes in all eukaryotes. In plants, bZIPs are master regulators of many central developmental and physiological processes, including morphogenesis, seed formation, abiotic and biotic stress responses. Modulation of the expression patterns of bZIP genes and changes in their activity often contribute to the activation of various signaling pathways and regulatory networks of different physiological processes. However, most advances in the study of plant bZIP transcription factors are related to their involvement in abiotic stress and development. In contrast, there are few examples of functional research with regard to biotic stress, particularly in the defense against pathogens. In this review, we summarize the recent progress revealing the role of bZIP transcription factors in the biotic stress responses of several plant species, from Arabidopsis to cotton. Moreover, we summarize the interacting partners of bZIP proteins in molecular responses during pathogen attack and the key components of the signal transduction pathways with which they physically interact during plant defense responses. Lastly, we focus on the recent advances regarding research on the functional role of bZIPs in major agricultural cultivars and examine the studies performed in this field