Skip to main content

Advertisement

SpringerLink
Log in

Predictive Value of CD8 Expression and FoxP3 Methylation in Nasopharyngeal Carcinoma Patients Treated with Chemoradiotherapy in a Non-endemic Area

  • Original Article
  • Published:
Pathology & Oncology Research

Abstract

Undifferentiated Nasopharyngeal Carcinoma (UNPC) is associated with Epstein-Barr Virus (EBV) and characterized by an abundant immune infiltrate potentially influencing the prognosis. Thus, we retrospectively assessed the significance of immunosuppression in the UNPC microenvironment as prognostic biomarker of treatment failure in a non-endemic area, and monitored the variation of systemic EBV-specific immunity before and after chemoradiotherapy (CRT). DNA and RNA were extracted from diagnostic biopsies obtained by tumor and adjacent mucosa from 63 consecutive EBV+ UNPC patients who underwent radical CRT. Among these patients 11 relapsed within 2 years. The expression of the EBV-derived UNPC-specific BARF1 gene and several immune-related genes was monitored through quantitative RT-PCR and methylation-specific PCR analyses. Peripheral T cell responses against EBV and BARF1 were measured in 14 patients (7 relapses) through IFN-γ ELISPOT assay. We found significantly higher expression levels of BARF1, CD8, IFN-γ, IDO, PD-L1, and PD-1 in UNPC samples compared to healthy tissues. CD8 expression was significantly reduced in both tumor and healthy tissues in UNPC patients who relapsed within two years. We observed a hypomethylated FOXP3 intron 1 exclusively in relapsed UNPC patients. Finally, we noticed a significant decrease in EBV- and BARF1-specific T-cells after CRT only in relapsing patients. Our data suggest that a high level of immunosuppression (low CD8, hypomethylated FoxP3) in UNPC microenvironment may predict treatment failure and may allow an early identification of patients who could benefit from the addition of immune modulating strategies to improve first line CRT.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Abbreviations

CRT:

Chemoradiotherapy

EBV:

Epstein-Barr Virus

IDO:

Indoleamine 2,3-dioxygenase

IMRT:

Intensity modulated radiotherapy

NP:

Nasopharyngeal

PBMCs:

Peripheral blood mononuclear cells

RT:

Radiotherapy

Treg:

Regulatory T cell

UNPC:

Undifferentiated Nasopharyngeal Carcinoma

References

  1. Yu MC, Yuan JM (2002) Epidemiology of nasopharyngeal carcinoma. Semin Cancer Biol 12:421–429

    PubMed  Google Scholar 

  2. Chan JKC, Bray F, McCarron P, Foo W, Lee AWM, Yip T et al (2005) Nasopharyngeal carcinoma. In: Barnes L, Eveson JW, Reichart P et al (eds) WHO classification of tumours. Pathology and genetics. Head and neck tumours. IARC Press, Lyon, pp 85–97

    Google Scholar 

  3. Chua MLK, Wee JTS, Hui EP, Chan ATC (2016) Nasopharyngeal carcinoma. Lancet 387:1012–1024

    PubMed  Google Scholar 

  4. Sun X, Su S, Chen C, Han F, Zhao C, Xiao W, Deng X, Huang S, Lin C, Lu T (2014) Long-term outcomes of intensity-modulated radiotherapy for 868 patients with nasopharyngeal carcinoma: an analysis of survival and treatment toxicities. Radiother Oncol 110:398–403

    PubMed  Google Scholar 

  5. Lo KW, To KF, Huang DP (2004) Focus on nasopharyngeal carcinoma. Cancer Cell 5:423–428

    CAS  PubMed  Google Scholar 

  6. Merlo A, Turrini R, Dolcetti R, Martorelli D, Muraro E, Comoli P, Rosato A (2010) The interplay between Epstein-Barr virus and the immune system: a rationale for adoptive cell therapy of EBV-related disorders. Haematologica 95:1769–1777

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Decaussin G, Sbih-Lammali F, de Turenne-Tessier M, Bouguermouh A, Ooka T (2000) Expression of BARF1 gene encoded by Epstein-Barr virus in nasopharyngeal carcinoma biopsies. Cancer Res 60:5584–5588

    CAS  PubMed  Google Scholar 

  8. Stevens SJ, Verkuijlen SA, Hariwiyanto B, Harijadi PDK, Fachiroh J et al (2006) Noninvasive diagnosis of nasopharyngeal carcinoma: nasopharyngeal brushings reveal high Epstein-Barr virus DNA load and carcinoma-specific viral BARF1 mRNA. Int J Cancer 119:608–614

    CAS  PubMed  Google Scholar 

  9. Ramayanti O, Juwana H, Verkuijlen SA, Adham M, Pegtel MD, Greijer AE et al (2017) Epstein-Barr virus mRNA profiles and viral DNA methylation status in nasopharyngeal brushings from nasopharyngeal carcinoma patients reflect tumor origin. Int J Cancer 140:149–162

    CAS  PubMed  Google Scholar 

  10. Martorelli D, Houali K, Caggiari L, Vaccher E, Barzan L, Franchin G, Gloghini A, Pavan A, da Ponte A, Tedeschi RM, de Re V, Carbone A, Ooka T, de Paoli P, Dolcetti R (2008) Spontaneous T cell responses to Epstein-Barr virus-encoded BARF1 protein and derived peptides in patients with nasopharyngeal carcinoma: bases for improved immunotherapy. Int J Cancer 123:1100–1107

    CAS  PubMed  Google Scholar 

  11. Faè DA, Martorelli D, Mastorci K, Muraro E, Dal Col J, Franchin G et al (2016) Broadening specificity and enhancing cytotoxicity of adoptive T cells for nasopharyngeal carcinoma immunotherapy. Cancer Immunol Res 4(5):431–440

    PubMed  Google Scholar 

  12. Chen QY, Guo SY, Tang LQ, Lu TY, Chen BL, Zhong QY, Zou MS, Tang QN, Chen WH, Guo SS, Liu LT, Li Y, Guo L, Mo HY, Sun R, Luo DH, Zhao C, Cao KJ, Qian CN, Guo X, Zeng MS, Mai HQ (2018) Combination of tumor volume and Epstein-Barr virus DNA improved prognostic stratification of stage II nasopharyngeal carcinoma in the IMRT era: a large-scale cohort study. Cancer Res Treat Jul 50(3):861–871

    CAS  Google Scholar 

  13. Bortolin MT, Pratesi C, Dolcetti R, Bidoli E, Vaccher E, Zanussi S, Tedeschi R, de Paoli P (2006) Clinical value of Epstein-Barr virus DNA levels in peripheral blood samples of Italian patients with undifferentiated carcinoma of nasopharyngeal type. Cancer Lett 233:247–254

    CAS  PubMed  Google Scholar 

  14. Shanmugaratnam K, Chan SH, de-The G, Goh JE, Khor TH, Simons MJ et al (1979) Histopathology of nasopharyngeal carcinoma: correlations with epidemiology, survival rates and other biological characteristics. Cancer 44:1029–1044

    CAS  PubMed  Google Scholar 

  15. Zhang YL, Li J, Mo HY, Qiu F, Zheng LM, Qian CN, Zeng YX (2010) Different subsets of tumor infiltrating lymphocytes correlate with NPC progression in different ways. Mol Cancer 9:4

    PubMed  PubMed Central  Google Scholar 

  16. Lee JJ, Kao KC, Chiu YL, Jung CJ, Liu CJ, Cheng SJ, Chang YL, Ko JY, Chia JS (2017) Enrichment of human CCR6(+) regulatory T cells with superior suppressive activity in oral cancer. J Immunol 199:467–476

    CAS  PubMed  Google Scholar 

  17. Uyttenhove C, Pilotte L, Theate I, Stroobant V, Colau D, Parmentier N et al (2003) Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat Med 9:1269–1274

    CAS  PubMed  Google Scholar 

  18. Ben-Haj-Ayed A, Moussa A, Ghedira R, Gabbouj S, Miled S, Bouzid N, Tebra-Mrad S, Bouaouina N, Chouchane L, Zakhama A, Hassen E (2016) Prognostic value of indoleamine 2,3-dioxygenase activity and expression in nasopharyngeal carcinoma. Immunol Lett 169:23–32

    CAS  PubMed  Google Scholar 

  19. Pardoll DM (2012) The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 12:252–264

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhang J, Fang W, Qin T, Yang Y, Hong S, Liang W, Ma Y, Zhao H, Huang Y, Xue C, Huang P, Hu Z, Zhao Y, Zhang L (2015) Co-expression of PD-1 and PD-L1 predicts poor outcome in nasopharyngeal carcinoma. Med Oncol 32:86

    Article  PubMed  CAS  Google Scholar 

  21. Zhou Y, Shi D, Miao J, Wu H, Chen J, Zhou X, Hu D, Zhao C, Deng W, Xie C (2017) PD-L1 predicts poor prognosis for nasopharyngeal carcinoma irrespective of PD-1 and EBV-DNA load. Sci Rep 7:43627

    PubMed  PubMed Central  Google Scholar 

  22. Lee VH, Lo AW, Leung CY, Shek WH, Kwong DL, Lam KO et al (2016) Correlation of PD-L1 expression of tumor cells with survival outcomes after radical intensity-modulated radiation therapy for non-metastatic nasopharyngeal carcinoma. PLoS One 11:e0157969

    PubMed  PubMed Central  Google Scholar 

  23. Chan OS, Kowanetz M, Ng WT, Koeppen H, Chan LK, Yeung RM et al (2017) Characterization of PD-L1 expression and immune cell infiltration in nasopharyngeal cancer. Oral Oncol 67:52–60

    CAS  PubMed  Google Scholar 

  24. Hsu MC, Hsiao JR, Chang KC, Wu YH, Su IJ, Jin YT, Chang Y (2010) Increase of programmed death-1-expressing intratumoral CD8 T cells predicts a poor prognosis for nasopharyngeal carcinoma. Mod Pathol 23:1393–1403

    CAS  PubMed  Google Scholar 

  25. Zhou Y, Miao J, Wu H, Tang H, Kuang J, Zhou X, Peng Y, Hu D, Shi D, Deng W, Cao X, Zhao C, Xie C (2017) PD-1 and PD-L1 expression in 132 recurrent nasopharyngeal carcinoma: the correlation with anemia and outcomes. Oncotarget 8:51210–51223

    PubMed  PubMed Central  Google Scholar 

  26. Topalian SL, Taube JM, Anders RA, Pardoll DM (2016) Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy. Nat Rev Cancer 16:275–287

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Pilones KA, Vanpouille-Box C, Demaria S (2015) Combination of radiotherapy and immune checkpoint inhibitors. Semin Radiat Oncol 25:28–33

    PubMed  Google Scholar 

  28. Gameiro SR, Jammeh ML, Wattenberg MM, Tsang KY, Ferrone S, Hodge JW (2014) Radiation-induced immunogenic modulation of tumor enhances antigen processing and calreticulin exposure, resulting in enhanced T-cell killing. Oncotarget 5:403–416

    PubMed  Google Scholar 

  29. Franchin G, Vaccher E, Talamini R, Gobitti C, Minatel E, Politi D, Sartor G, Trovò MG, Barzan L (2002) Nasopharyngeal cancer WHO type II-III: monoinstitutional retrospective analysis with standard and accelerated hyperfractionated radiation therapy. Oral Oncol 38:137–144

    CAS  PubMed  Google Scholar 

  30. Franchin G, Vaccher E, Talamini R, Politi D, Gobitti C, Minatel E, LLeshi A, Sartor G, Mascarin M, Rumeileh IA, Trovò MG, Barzan L (2011) Intensity-modulated radiotherapy (IMRT)/Tomotherapy following neoadjuvant chemotherapy in stage IIB-IVA/B undifferentiated nasopharyngeal carcinomas (UCNT): a mono-institutional experience. Oral Oncol 47:905–909

    PubMed  Google Scholar 

  31. Muls N, Dang HA, Sindic CJ, van Pesch V (2014) Fingolimod increases CD39-expressing regulatory T cells in multiple sclerosis patients. PLoS One 9:e113025

    PubMed  PubMed Central  Google Scholar 

  32. Ooft ML, van Ipenburg JA, Braunius WW, Zuur CI, Koljenovic S, Willems SM (2017) Prognostic role of tumor infiltrating lymphocytes in EBV positive and EBV negative nasopharyngeal carcinoma. Oral Oncol 71:16–25

    PubMed  Google Scholar 

  33. Zhang L, MacIsaac KD, Zhou T, Huang PY, Xin C, Dobson JR et al (2017) Genomic analysis of nasopharyngeal carcinoma reveals TME-based subtypes. Mol Cancer Res 15:1722–1732

    CAS  PubMed  Google Scholar 

  34. Lau KM, Cheng SH, Lo KW, Lee SA, Woo JK, van Hasselt CA et al (2007) Increase in circulating Foxp3+CD4+CD25(high) regulatory T cells in nasopharyngeal carcinoma patients. Br J Cancer 96:617–622

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Furlan C, Polesel J, Barzan L, Franchin G, Sulfaro S, Romeo S, Colizzi F, Rizzo A, Baggio V, Giacomarra V, Dei Tos AP, Boscolo-Rizzo P, Vaccher E, Dolcetti R, Sigalotti L, Fratta E (2017) Prognostic significance of LINE-1 hypomethylation in oropharyngeal squamous cell carcinoma. Clin Epigenetics 9:58

    PubMed  PubMed Central  Google Scholar 

  36. Zhao W, Mo Y, Wang S, Midorikawa K, Ma N, Hiraku Y, Oikawa S, Huang G, Zhang Z, Murata M, Takeuchi K (2017) Quantitation of DNA methylation in Epstein-Barr virus-associated nasopharyngeal carcinoma by bisulfite amplicon sequencing. BMC Cancer 17:489

    PubMed  PubMed Central  Google Scholar 

  37. Seto E, Yang L, Middeldorp J, Sheen TS, Chen JY, Fukayama M, Eizuru Y, Ooka T, Takada K (2005) Epstein-Barr virus (EBV)-encoded BARF1 gene is expressed in nasopharyngeal carcinoma and EBV-associated gastric carcinoma tissues in the absence of lytic gene expression. J Med Virol 76:82–88

    CAS  PubMed  Google Scholar 

  38. Cosmopoulos K, Pegtel M, Hawkins J, Moffett H, Novina C, Middeldorp J, Thorley-Lawson DA (2009) Comprehensive profiling of Epstein-Barr virus microRNAs in nasopharyngeal carcinoma. J Virol 83:2357–2367

    CAS  PubMed  Google Scholar 

  39. Takada K (2012) Role of EBER and BARF1 in nasopharyngeal carcinoma (NPC) tumorigenesis. Semin Cancer Biol 22:162–165

    CAS  PubMed  Google Scholar 

  40. Liu P, Xie BL, Cai SH, He YW, Zhang G, Yi YM, du J (2009) Expression of indoleamine 2,3-dioxygenase in nasopharyngeal carcinoma impairs the cytolytic function of peripheral blood lymphocytes. BMC Cancer 9:416

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Lee SJ, Jang BC, Lee SW, Yang YI, Suh SI, Park YM, Oh S, Shin JG, Yao S, Chen L, Choi IH (2006) Interferon regulatory factor-1 is prerequisite to the constitutive expression and IFN-gamma-induced upregulation of B7-H1 (CD274). FEBS Lett 580:755–762

    Article  CAS  PubMed  Google Scholar 

  42. Hsu C, Lee SH, Ejadi S, Even C, Cohen RB, Le Tourneau C et al (2017) Safety and antitumor activity of Pembrolizumab in patients with programmed death-ligand 1-positive nasopharyngeal carcinoma: results of the KEYNOTE-028 study. J Clin Oncol 35(36):4050–4056

    Article  CAS  PubMed  Google Scholar 

  43. Fang W, Zhang J, Hong S, Zhan J, Chen N, Qin T, Tang Y, Zhang Y, Kang S, Zhou T, Wu X, Liang W, Hu Z, Ma Y, Zhao Y, Tian Y, Yang Y, Xue C, Yan Y, Hou X, Huang P, Huang Y, Zhao H, Zhang L (2014) EBV-driven LMP1 and IFN-gamma up-regulate PD-L1 in nasopharyngeal carcinoma: implications for oncotargeted therapy. Oncotarget 5:12189–12202

    Article  PubMed  PubMed Central  Google Scholar 

  44. Zhu Q, Cai MY, Chen CL, Hu H, Lin HX, Li M, Weng DS, Zhao JJ, Guo L, Xia JC (2017) Tumor cells PD-L1 expression as a favorable prognosis factor in nasopharyngeal carcinoma patients with pre-existing intratumor-infiltrating lymphocytes. Oncoimmunology 6:e1312240

    PubMed  PubMed Central  Google Scholar 

  45. Ma BBY, Lim WT, Goh BC, Hui EP, Lo KW, Pettinger A et al (2018) Antitumor activity of Nivolumab in recurrent and metastatic nasopharyngeal carcinoma: an international, multicenter study of the Mayo Clinic phase 2 consortium (NCI-9742). J Clin Oncol 36(14):1412–1418

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The authors thank the patients, the flow cytometry core facility of CRO Aviano National Cancer Institute, the other investigators participating in the study, and Miss S. Colussi for editing the manuscript. This work was supported by grants from the CRO Aviano National Cancer Institute (5 × 1000 Institutional Grant).

Author information

Authors and Affiliations

  1. Immunopathology and Cancer Biomarkers Unit, Department of Translational Research, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, PN, Italy

    E. Muraro, E. Fratta, D. A. Fae’, D. Martorelli, M. Cangemi, E. Comaro, C. Scaini, C. Pratesi, S. Zanussi & A. Steffan

  2. Department of Medical Oncology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, PN, Italy

    E. Vaccher

  3. Department of Radiation Oncology, General Hospital “San Martino”, Belluno, Italy

    C. Furlan

  4. Department of Radiation Oncology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, PN, Italy

    C. Furlan, G. Fanetti, F. Navarria, C. Gobitti & G. Franchin

  5. Unit of Cancer Epidemiology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, PN, Italy

    J. Polesel

  6. Unit of Otolaryngology, General Hospital “S. Maria degli Angeli”, Pordenone, Italy

    V. Lupato, G. Grando & V. Giacomarra

  7. Division of Pathology, General Hospital “S. Maria degli Angeli”, Pordenone, Italy

    S. Sulfaro

  8. Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, PN, Italy

    L. Barzan

  9. University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia

    R. Dolcetti

  10. Pathology Unit, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, PN, Italy

    V. Canzonieri

  11. Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy

    V. Canzonieri

Authors
  1. E. Muraro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. E. Vaccher
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C. Furlan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. E. Fratta
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. G. Fanetti
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. D. A. Fae’
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. D. Martorelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. M. Cangemi
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. J. Polesel
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. F. Navarria
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. C. Gobitti
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. E. Comaro
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. C. Scaini
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. C. Pratesi
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. S. Zanussi
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. V. Lupato
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. G. Grando
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. V. Giacomarra
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. S. Sulfaro
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. L. Barzan
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. R. Dolcetti
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. A. Steffan
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. V. Canzonieri
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. G. Franchin
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed to the study conception and design. Elena Muraro conceived the study, performed part of the experiments and drafted the manuscript. Elisabetta Fratta, Damiana A Fae’, Debora Martorelli, and Michela Cangemi performed the experiments and reviewed the manuscript. Jerry Polesel carried out statistical analysis and reviewed the manuscript. Elisa Comaro contributed to sample collection and data acquisition. Carlo Furlan, Giuseppe Fanetti, Federico Navarria, Carlo Gobitti collected and analyzed clinical data and reviewed the manuscript. Chiara Scaini, Chiara Pratesi, and Stefania Zanussi performed EBV DNA analysis. Valentina Lupato, Giuseppe Grando, Vittorio Giacomarra, Luigi Barzan collected and analyzed clinical data. Riccardo Dolcetti and Agostino Steffan reviewed the manuscript. Sandro Sulfaro and Vincenzo Canzonieri performed histopathological diagnosis and immunohistochemistry, and performed data analysis. Emanuela Vaccher and Giovanni Franchin collected and analyzed clinical data, conceived and design the study, reviewed and approved the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to E. Muraro.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 1.39 mb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Muraro, E., Vaccher, E., Furlan, C. et al. Predictive Value of CD8 Expression and FoxP3 Methylation in Nasopharyngeal Carcinoma Patients Treated with Chemoradiotherapy in a Non-endemic Area. Pathol. Oncol. Res. 26, 2459–2467 (2020). https://doi.org/10.1007/s12253-020-00859-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12253-020-00859-3

Keywords

Search

Navigation