New insights into the inflamed tumor immune microenvironment of gastric cancer with lymphoid stroma: from morphology and digital analysis to gene expression
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.
- 1.Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, et al. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. Lyon: International Agency for Research on Cancer; 2013. 2012. http://www.globocaniarcfr. Accessed 01 Feb 2017.
- 2.Tan IB, Ivanova T, Lim KH, Ong CW, Deng N, Lee J, et al. Intrinsic subtypes of gastric cancer, based on gene expression pattern, predict survival and respond differently to chemotherapy. Gastroenterology. 2011;141(2):476–85. https://doi.org/10.1053/j.gastro.2011.04.042.CrossRefPubMedPubMedCentralGoogle Scholar
- 4.Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L, Sawaki A, et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet. 2010;376(9742):687–97. https://doi.org/10.1016/s0140-6736(10)61121-x.CrossRefPubMedGoogle Scholar
- 5.Fuchs CS, Tomasek J, Yong CJ, Dumitru F, Passalacqua R, Goswami C, et al. Ramucirumab monotherapy for previously treated advanced gastric or gastro-oesophageal junction adenocarcinoma (REGARD): an international, randomised, multicentre, placebo-controlled, phase 3 trial. Lancet. 2014;383(9911):31–9. https://doi.org/10.1016/s0140-6736(13)61719-5.CrossRefPubMedGoogle Scholar
- 6.Wilke H, Muro K, Van Cutsem E, Oh SC, Bodoky G, Shimada Y, et al. Ramucirumab plus paclitaxel versus placebo plus paclitaxel in patients with previously treated advanced gastric or gastro-oesophageal junction adenocarcinoma (RAINBOW): a double-blind, randomised phase 3 trial. Lancet Oncol. 2014;15(11):1224–35. https://doi.org/10.1016/s1470-2045(14)70420-6.CrossRefPubMedGoogle Scholar
- 7.Ralph C, Elkord E, Burt DJ, O’Dwyer JF, Austin EB, Stern PL, et al. Modulation of lymphocyte regulation for cancer therapy: a phase II trial of tremelimumab in advanced gastric and esophageal adenocarcinoma. Clin Cancer Res Off J Am Assoc Cancer Res. 2010;16(5):1662–72. https://doi.org/10.1158/1078-0432.ccr-09-2870.CrossRefGoogle Scholar
- 8.Muro K, Chung HC, Shankaran V, Geva R, Catenacci D, Gupta S, et al. Pembrolizumab for patients with PD-L1-positive advanced gastric cancer (KEYNOTE-012): a multicentre, open-label, phase 1b trial. Lancet Oncol. 2016;17(6):717–26. https://doi.org/10.1016/s1470-2045(16)00175-3.CrossRefPubMedGoogle Scholar
- 11.Bilgin B, Sendur MA, Bulent Akinci M, Sener Dede D, Yalcin B. Targeting the PD-1 pathway: a new hope for gastrointestinal cancers. Curr Med Res Opin. 2017:1–11. https://doi.org/10.1080/03007995.2017.1279132.
- 12.FDA approves Merck’s KEYTRUDA® (pembrolizumab) for previously treated patients with recurrent locally advanced or metastatic gastric or gastroesophageal junction cancer whose tumors express PD-L1 (CPS greater than or equal to 1). Merck. 2017. http://bit.ly/2wberGs. Accessed 5 May 2018.
- 13.Fuchs CS, Doi T, Jang RW-J, Muro K, Satoh T, Machado M, et al. KEYNOTE-059 cohort 1: Efficacy and safety of pembrolizumab (pembro) monotherapy in patients with previously treated advanced gastric cancer. J Clin Oncol. 2017;35(15_suppl):4003. https://doi.org/10.1200/JCO.2017.35.15_suppl.4003.CrossRefGoogle Scholar
- 14.FDA grants accelerated approval to pembrolizumab for tissue/site agnostic indication. US FDA web site. 2017. https://www.fda.gov/drugs/informationondrugs/approveddrugs/ucm560040.htm. Accessed 5 May 2018.
- 15.Baraniskin A, Van Laethem JL, Wyrwicz L, Guller U, Wasan HS, Matysiak-Budnik T, et al. Clinical relevance of molecular diagnostics in gastrointestinal (GI) cancer: European Society of Digestive Oncology (ESDO) expert discussion and recommendations from the 17th European Society for Medical Oncology (ESMO)/World Congress on Gastrointestinal Cancer, Barcelona. Eur J Cancer. 2017;86:305–17. https://doi.org/10.1016/j.ejca.2017.09.021.CrossRefPubMedGoogle Scholar
- 17.Saito R, Abe H, Kunita A, Yamashita H, Seto Y, Fukayama M. Overexpression and gene amplification of PD-L1 in cancer cells and PD-L1+ immune cells in Epstein–Barr virus-associated gastric cancer: the prognostic implications. Mod Pathol. 2016;30(3):427–39. https://doi.org/10.1038/modpathol.2016.202.CrossRefPubMedGoogle Scholar
- 24.Lu BJ, Lai M, Cheng L, Xu JY, Huang Q. Gastric medullary carcinoma, a distinct entity associated with microsatellite instability-H, prominent intraepithelial lymphocytes and improved prognosis. Histopathology. 2004;45(5):485–92. https://doi.org/10.1111/j.1365-2559.2004.01998.x.CrossRefPubMedGoogle Scholar
- 26.Bosman FT, Carneiro F, Hruban RH, Theise ND. WHO classification of tumours of the digestive system. Lyon: International Agency for Research on Cancer; 2010.Google Scholar
- 28.Kawazoe A, Kuwata T, Kuboki Y, Shitara K, Nagatsuma AK, Aizawa M, et al. Clinicopathological features of programmed death ligand 1 expression with tumor-infiltrating lymphocyte, mismatch repair, and Epstein–Barr virus status in a large cohort of gastric cancer patients. Gastric Cancer (Serial Online). 2016. https://doi.org/10.1007/s10120-016-0631-3. http://link.springer.com. Accessed 9 Apr 2017.
- 33.Ma C, Patel K, Singhi AD, Ren B, Zhu B, Shaikh F, et al. Programmed death-ligand 1 expression is common in gastric cancer associated with Epstein–Barr virus or microsatellite instability. Am J Surg Pathol. 2016;40(11):1496–506. https://doi.org/10.1097/pas.0000000000000698.CrossRefPubMedGoogle Scholar
- 37.R Core Team (2013). R: a language and environment for statistical computing. Vienna, Austria : the R Foundation for Statistical Computing. http://www.R-project.org/. Accessed 5 May 2018.
- 40.Cheng N, Hui DY, Liu Y, Zhang NN, Jiang Y, Han J, et al. Is gastric lymphoepithelioma-like carcinoma a special subtype of EBV-associated gastric carcinoma? New insight based on clinicopathological features and EBV genome polymorphisms. Gastric Cancer. 2015;18(2):246–55. https://doi.org/10.1007/s10120-014-0376-9.CrossRefPubMedGoogle Scholar
- 45.Chiaravalli AM, Cornaggia M, Furlan D, Capella C, Fiocca R, Tagliabue G, et al. The role of histological investigation in prognostic evaluation of advanced gastric cancer. Analysis of histological structure and molecular changes compared with invasive pattern and stage. Virchows Arch. 2001;439(2):158–69.CrossRefPubMedGoogle Scholar
- 49.Llosa NJ, Cruise M, Tam A, Wicks EC, Hechenbleikner EM, Taube JM, et al. The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints. Cancer Discov. 2015;5(1):43–51. https://doi.org/10.1158/2159-8290.cd-14-0863.CrossRefPubMedGoogle Scholar