New insights into the inflamed tumor immune microenvironment of gastric cancer with lymphoid stroma: from morphology and digital analysis to gene expression
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Gastric cancer with lymphoid stroma (GCLS) is characterized by prominent stromal infiltration of T-lymphocytes. The aim of this study was to investigate GCLS biology through analysis of clinicopathological features, EBV infection, microsatellite instability (MSI), immune gene-expression profiling and PD-L1 status in neoplastic cells and tumor immune microenvironment.
Twenty-four GCLSs were analyzed by RNA in situ hybridization for EBV (EBER), PCR/fragment analysis for MSI, immunohistochemistry (PD-L1, cytokeratin, CD3, CD8), co-immunofluorescence (CK/PD-L1, CD68/PD-L1), NanoString gene-expression assay for immune-related genes and PD-L1 copy number alterations. CD3+ and CD8+ T-cell densities were calculated by digital analysis. Fifty-four non-GCLSs were used as control group.
GCLSs displayed distinctive clinicopathological features, such as lower pTNM stage (p = 0.02) and better overall survival (p = 0.01). EBV+ or MSI-high phenotype was found in 66.7 and 16.7% cases, respectively. GCLSs harbored a cytotoxic T-cell-inflamed profile, particularly at the invasive front of tumors (p < 0.01) and in EBV+ cases (p = 0.01). EBV+ GCLSs, when compared to EBV− GCLSs, showed higher mRNA expression of genes related to Th1/cytotoxic and immunosuppressive biomarkers. PD-L1 protein expression, observed in neoplastic and immune stromal cells (33.3 and 91.7%, respectively), and PD-L1 amplification (18.8%) were restricted to EBV+/MSI-high tumors and correlated with high values of PD-L1 mRNA expression.
This study shows that GCLS has a distinctive clinico-pathological and molecular profile. Furthermore, through an in-depth study of tumor immune microenvironment—by digital analysis and mRNA expression profiling—it highlights the role of EBV infection in promoting an inflamed tumor microenvironment, with putative therapeutic implications.
KeywordsGastric cancer Epstein–Barr virus (EBV) Microsatellite instability (MSI) Gene expression profiling PD-L1
This study was distinguished with the George Tiniakos Award (28th European Congress of Pathology held in Cologne, Germany).
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). We thank the support of the following entities/projects: FEDER-Fundo Europeu de Desenvolvimento Regional (COMPETE 2020-Operacional Programme for Competitiveness and Internationalization-POCI), Portugal 2020; FCT-Foundation for Science and Technology/Ministério da Ciência, Tecnologia e Inovação: POCI-01-0145-FEDER-007274; PTDC/BIM-MEC/2834/2014; salary support to GMA: POPH-QREN Type 4.2, European Social Fund and Portuguese Ministry of Science and Technology (MCTES), Contrato Programa no âmbito do Programa Investigador FCT 2013, Ref: IF/00615/2013; Ph.D. fellowships SFRH/BD/81103/2011; PostDoc FCT fellowship SFRH/BPD/89764/2012 (PO).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and later versions.
This was a retrospective study and, accordingly, the authors were not expected to have consent to participate. Ethics approval was obtained (CES 236-14). No potentially identifiable patient images or data were included in this study.
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