Advertisement

New insights into the inflamed tumor immune microenvironment of gastric cancer with lymphoid stroma: from morphology and digital analysis to gene expression

  • Irene Gullo
  • Patrícia Oliveira
  • Maria Athelogou
  • Gilza Gonçalves
  • Marta L. Pinto
  • Joana Carvalho
  • Ana Valente
  • Hugo Pinheiro
  • Sara Andrade
  • Gabriela M. Almeida
  • Ralf Huss
  • Kakoli Das
  • Patrick Tan
  • José C. Machado
  • Carla Oliveira
  • Fátima Carneiro
Original Article

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.

Keywords

Gastric cancer Epstein–Barr virus (EBV) Microsatellite instability (MSI) Gene expression profiling PD-L1 

Notes

Acknowledgements

This study was distinguished with the George Tiniakos Award (28th European Congress of Pathology held in Cologne, Germany).

Funding

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.

Ethical standards

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.

Informed consent

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.

Supplementary material

10120_2018_836_MOESM1_ESM.tif (3.6 mb)
Figure S1. Intra-tumor heterogeneity of PD-L1 immunoreactivity. This case showed high levels of intra-tumor heterogeneity of PD-L1 protein expression: (a) Epithelial (non-neoplastic and cancer) cells are highlighted by CK AE1/AE3 immunostaining; (b) Cancer cells showed high heterogeneity of PD-L1 immunoreactivity: some cells are negative (c), while others cells show strong and membranous/cytoplasmic positivity (d). This case showed low PD-L1 gene expression levels, which might be representative of PD-L1 negative area in this section for technical reasons (TIF 3690 KB)
10120_2018_836_MOESM2_ESM.docx (69 kb)
Supplementary material 2 (DOCX 69 KB)

References

  1. 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. 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
  3. 3.
    Duraes C, Almeida GM, Seruca R, Oliveira C, Carneiro F. Biomarkers for gastric cancer: prognostic, predictive or targets of therapy? Virchows Arch. 2014;464(3):367–78.  https://doi.org/10.1007/s00428-013-1533-y.CrossRefPubMedGoogle Scholar
  4. 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. 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. 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. 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. 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
  9. 9.
    Moehler M, Delic M, Goepfert K, Aust D, Grabsch HI, Halama N, et al. Immunotherapy in gastrointestinal cancer: recent results, current studies and future perspectives. Eur J Cancer. 2016;59:160–70.  https://doi.org/10.1016/j.ejca.2016.02.020.CrossRefPubMedGoogle Scholar
  10. 10.
    Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med. 2012;366(26):2443–54.  https://doi.org/10.1056/NEJMoa1200690.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 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. 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. 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. 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. 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
  16. 16.
    TCGA. Comprehensive molecular characterization of gastric adenocarcinoma. Nature. 2014;513(7517):202–9.  https://doi.org/10.1038/nature13480.CrossRefGoogle Scholar
  17. 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
  18. 18.
    Su S, Zou Z, Chen F, Ding N, Du J, Shao J, et al. CRISPR-Cas9-mediated disruption of PD-1 on human T cells for adoptive cellular therapies of EBV positive gastric cancer. Oncoimmunology. 2017;6(1):e1249558.  https://doi.org/10.1080/2162402x.2016.1249558.CrossRefPubMedGoogle Scholar
  19. 19.
    Le DT, Durham JN, Smith KN, Wang H, Bartlett BR, Aulakh LK, et al. Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science. 2017;357(6349):409–13.  https://doi.org/10.1126/science.aan6733.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Solcia E, Klersy C, Mastracci L, Alberizzi P, Candusso ME, Diegoli M, et al. A combined histologic and molecular approach identifies three groups of gastric cancer with different prognosis. Virchows Arch. 2009;455(3):197–211.  https://doi.org/10.1007/s00428-009-0813-z.CrossRefPubMedGoogle Scholar
  21. 21.
    Grogg KL, Lohse CM, Pankratz VS, Halling KC, Smyrk TC. Lymphocyte-rich gastric cancer: associations with Epstein–Barr virus, microsatellite instability, histology, and survival. Mod Pathol. 2003;16(7):641–51.  https://doi.org/10.1097/01.mp.0000076980.73826.c0.CrossRefPubMedGoogle Scholar
  22. 22.
    Shinozaki-Ushiku A, Kunita A, Fukayama M. Update on Epstein–Barr virus and gastric cancer (review). Int J Oncol. 2015;46(4):1421–34.  https://doi.org/10.3892/ijo.2015.2856.CrossRefPubMedGoogle Scholar
  23. 23.
    Wang HH, Wu MS, Shun CT, Wang HP, Lin CC, Lin JT. Lymphoepithelioma-like carcinoma of the stomach: a subset of gastric carcinoma with distinct clinicopathological features and high prevalence of Epstein–Barr virus infection. Hepatogastroenterology. 1999;46(26):1214–9.PubMedGoogle Scholar
  24. 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
  25. 25.
    Watanabe H, Enjoji M, Imai T. Gastric carcinoma with lymphoid stroma. Its morphologic characteristics and prognostic correlations. Cancer. 1976;38(1):232–43.CrossRefPubMedGoogle Scholar
  26. 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
  27. 27.
    Dai C, Geng R, Wang C, Wong A, Qing M, Hu J, et al. Concordance of immune checkpoints within tumor immune contexture and their prognostic significance in gastric cancer. Mol Oncol. 2016;10(10):1551–8.  https://doi.org/10.1016/j.molonc.2016.09.004.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 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.
  29. 29.
    Li Z, Lai Y, Sun L, Zhang X, Liu R, Feng G, et al. PD-L1 expression is associated with massive lymphocyte infiltration and histology in gastric cancer. Hum Pathol. 2016;55:182–89.  https://doi.org/10.1016/j.humpath.2016.05.012.CrossRefPubMedGoogle Scholar
  30. 30.
    Thompson ED, Zahurak M, Murphy A, Cornish T, Cuka N, Abdelfatah E, et al. Patterns of PD-L1 expression and CD8 T cell infiltration in gastric adenocarcinomas and associated immune stroma. Gut. 2017;66(5):794–801.  https://doi.org/10.1136/gutjnl-2015-310839.CrossRefPubMedGoogle Scholar
  31. 31.
    Derks S, Liao X, Chiaravalli AM, Xu X, Camargo MC, Solcia E, et al. Abundant PD-L1 expression in Epstein–Barr virus-infected gastric cancers. Oncotarget. 2016;7(22):32925–32.  https://doi.org/10.18632/oncotarget.9076.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Dong M, Wang HY, Zhao XX, Chen JN, Zhang YW, Huang Y, et al. Expression and prognostic roles of PIK3CA, JAK2, PD-L1, and PD-L2 in Epstein–Barr virus-associated gastric carcinoma. Hum Pathol. 2016;53:25–34.  https://doi.org/10.1016/j.humpath.2016.02.007.CrossRefPubMedGoogle Scholar
  33. 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
  34. 34.
    Boger C, Behrens HM, Mathiak M, Kruger S, Kalthoff H, Rocken C. PD-L1 is an independent prognostic predictor in gastric cancer of western patients. Oncotarget. 2016;7(17):24269–83.  https://doi.org/10.18632/oncotarget.8169.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Setia N, Agoston AT, Han HS, Mullen JT, Duda DG, Clark JW, et al. A protein and mRNA expression-based classification of gastric cancer. Mod Pathol. 2016;29(7):772–84.  https://doi.org/10.1038/modpathol.2016.55.CrossRefPubMedGoogle Scholar
  36. 36.
    Das K, Chan XB, Epstein D, Teh BT, Kim K-M, Kim ST, et al. NanoString expression profiling identifies candidate biomarkers of RAD001 response in metastatic gastric cancer. ESMO Open. 2016;1(1):e000009.  https://doi.org/10.1136/esmoopen-2015-000009.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 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.
  38. 38.
    Chen DS, Mellman I. Elements of cancer immunity and the cancer-immune set point. Nature. 2017;541(7637):321–30.  https://doi.org/10.1038/nature21349.CrossRefPubMedGoogle Scholar
  39. 39.
    Park S, Choi MG, Kim KM, Kim HS, Jung SH, Lee JH, et al. Lymphoepithelioma-like carcinoma: a distinct type of gastric cancer. J Surg Res. 2015;194(2):458–63.  https://doi.org/10.1016/j.jss.2014.12.005.CrossRefPubMedGoogle Scholar
  40. 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
  41. 41.
    Ramos M, Pereira MA, Dias AR, Faraj SF, Zilberstein B, Cecconello I, et al. Lymphoepithelioma-like gastric carcinoma: clinicopathological characteristics and infection status. J Surg Res. 2017;210:159–68.  https://doi.org/10.1016/j.jss.2016.11.012.CrossRefPubMedGoogle Scholar
  42. 42.
    Fridman WH, Pages F, Sautes-Fridman C, Galon J. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer. 2012;12(4):298–306.  https://doi.org/10.1038/nrc3245.CrossRefPubMedGoogle Scholar
  43. 43.
    Lim H, Park YS, Lee JH, Son da H, Ahn JY, Choi KS, et al. Features of gastric carcinoma with lymphoid stroma associated with Epstein–Barr virus. Clin Gastroenterol Hepatol. 2015;13(10):1738–44.  https://doi.org/10.1016/j.cgh.2015.04.015.CrossRefPubMedGoogle Scholar
  44. 44.
    Chang MS, Kim WH, Kim CW, Kim YI. Epstein–Barr virus in gastric carcinomas with lymphoid stroma. Histopathology. 2000;37(4):309–15.CrossRefPubMedGoogle Scholar
  45. 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
  46. 46.
    Fukayama M, Ushiku T. Epstein–Barr virus-associated gastric carcinoma. Pathol Res Pract. 2011;207(9):529–37.  https://doi.org/10.1016/j.prp.2011.07.004.CrossRefPubMedGoogle Scholar
  47. 47.
    Sohn BH, Hwang JE, Jang HJ, Lee HS, Oh SC, Shim JJ, et al. Clinical significance of four molecular subtypes of gastric cancer identified by The Cancer Genome Atlas Project. Clin Cancer Res Off J Am Assoc Cancer Res. 2017.  https://doi.org/10.1158/1078-0432.ccr-16-2211.CrossRefGoogle Scholar
  48. 48.
    Park C, Cho J, Lee J, Kang SY, An JY, Choi MG, et al. Host immune response index in gastric cancer identified by comprehensive analyses of tumor immunity. Oncoimmunology. 2017;6(11):e1356150.  https://doi.org/10.1080/2162402x.2017.1356150.CrossRefPubMedPubMedCentralGoogle Scholar
  49. 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
  50. 50.
    Wang W, Sun J, Li F, Li R, Gu Y, Liu C, et al. A frequent somatic mutation in CD274 3′-UTR leads to protein over-expression in gastric cancer by disrupting miR-570 binding. Hum Mutat. 2012;33(3):480–4.  https://doi.org/10.1002/humu.22014.CrossRefPubMedGoogle Scholar
  51. 51.
    Wang W, Li F, Mao Y, Zhou H, Sun J, Li R, et al. A miR-570 binding site polymorphism in the B7-H1 gene is associated with the risk of gastric adenocarcinoma. Hum Genet. 2013;132(6):641–8.  https://doi.org/10.1007/s00439-013-1275-6.CrossRefPubMedGoogle Scholar
  52. 52.
    Kataoka K, Shiraishi Y, Takeda Y, Sakata S, Matsumoto M, Nagano S, et al. Aberrant PD-L1 expression through 3′-UTR disruption in multiple cancers. Nature. 2016;534(7607):402–6.  https://doi.org/10.1038/nature18294.CrossRefPubMedGoogle Scholar
  53. 53.
    Boussiotis VA. Molecular and biochemical aspects of the PD-1 checkpoint pathway. N Engl J Med. 2016;375(18):1767–78.  https://doi.org/10.1056/NEJMra1514296.CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Spranger S, Spaapen RM, Zha Y, Williams J, Meng Y, Ha TT, et al. Up-regulation of PD-L1, IDO, and T(regs) in the melanoma tumor microenvironment is driven by CD8(+) T cells. Sci Transl Med. 2013;5(200):200ra116.  https://doi.org/10.1126/scitranslmed.3006504.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The International Gastric Cancer Association and The Japanese Gastric Cancer Association 2018

Authors and Affiliations

  • Irene Gullo
    • 1
    • 2
    • 3
    • 4
  • Patrícia Oliveira
    • 3
    • 4
  • Maria Athelogou
    • 5
  • Gilza Gonçalves
    • 2
    • 3
    • 4
    • 6
  • Marta L. Pinto
    • 4
    • 7
    • 8
  • Joana Carvalho
    • 3
    • 4
  • Ana Valente
    • 3
    • 4
  • Hugo Pinheiro
    • 3
    • 4
    • 9
  • Sara Andrade
    • 3
    • 4
    • 10
  • Gabriela M. Almeida
    • 3
    • 4
  • Ralf Huss
    • 5
  • Kakoli Das
    • 11
  • Patrick Tan
    • 11
    • 12
    • 13
  • José C. Machado
    • 2
    • 3
    • 4
  • Carla Oliveira
    • 2
    • 3
    • 4
  • Fátima Carneiro
    • 1
    • 2
    • 3
    • 4
  1. 1.Department of PathologyCentro Hospitalar de São JoãoPortoPortugal
  2. 2.Department of PathologyFaculty of Medicine of the University of Porto (FMUP)PortoPortugal
  3. 3.Institute of Molecular Pathology and Immunology at the University of Porto (Ipatimup)PortoPortugal
  4. 4.Instituto de Investigação e Inovação em Saúde (i3S)University of PortoPortoPortugal
  5. 5.Definiens AGMunichGermany
  6. 6.Department of Biomedical Sciences and MedicineUniversity of AlgarveFaroPortugal
  7. 7.INEB-Institute of Biomedical EngineeringUniversity of PortoPortoPortugal
  8. 8.ICBAS-Institute of Biomedical Sciences Abel SalazarUniversity of PortoPortoPortugal
  9. 9.Hospital Senhora da OliveiraGuimarãesPortugal
  10. 10.Department of BiomedicineFaculty of Medicine of the University of Porto (FMUP)PortoPortugal
  11. 11.Cancer and Stem Cell Biology ProgramDuke-NUS Medical SchoolSingaporeSingapore
  12. 12.Genome Institute of Singapore, BiopolisSingaporeSingapore
  13. 13.Cancer Science Institute of SingaporeNational University of SingaporeSingaporeSingapore

Personalised recommendations