Journal of Neuro-Oncology

, Volume 133, Issue 2, pp 277–285 | Cite as

Assessment of PD-1 positive cells on initial and secondary resected tumor specimens of newly diagnosed glioblastoma and its implications on patient outcome

  • Tsubasa Miyazaki
  • Eiichi IshikawaEmail author
  • Masahide Matsuda
  • Hiroyoshi Akutsu
  • Satoru Osuka
  • Noriaki Sakamoto
  • Shingo Takano
  • Tetsuya Yamamoto
  • Koji Tsuboi
  • Akira Matsumura
Laboratory Investigation


Glioblastoma (GBM) is the most common type of malignant brain tumor and has a very poor prognosis. Most patients relapse within 12 months despite aggressive treatment and patient outcome after recurrent is extremely worse. This study was designed to clarify the change of the molecular expression, including programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1), on the initial and secondary resected tumor specimens and to address the influence of these expressions for patient outcome after second surgery of glioblastoma. We investigated 16 patients, ranging in age from 14 to 65 years, with histologically verified WHO grade IV GBM, whose original tumor was resected between 2008 and 2014, and treated with fractionated radiotherapy and temozolomide. Four patients who were treated with immunotherapy using autologous formalin-fixed tumor vaccine were enrolled. All of the patients underwent secondary resection after tumor recurrence within 24 months. We carried out an immunohistochemical examination of the initial and secondary resected tumors from patients using a panel of immune system molecular markers, and assessed whether marker expression correlated with clinical outcomes. CD3, CD8 and PD-1 on tumor-infiltrating lymphocytes was significantly increased in secondary resected specimens compared with initially resected specimens (p ≤ 0.05). All patients expressed PD-L1 on tumor cells in initial and secondary resection specimens. Patients were divided into high or low expression group by median IHC score of PD-1 on initial or secondary resected specimens. No significant differences in patient outcomes were observed between high and low PD-1 or PD-L1 groups of initially resected specimens. In high expression group of secondary resected specimens, most patients score had increased which compared with initial resected tumor specimens. The PD-1 high expression score group of secondary resected specimens was associated with long progression-free survival and short survival after recurrence. PD-L1 expression was detected in almost all initial and secondary specimens. Patients with high PD-1 expression of secondary specimen had bad prognosis after secondary resection. PD-1/PD-L1 pathway may be associated with patient outcome after second surgery of glioblastoma.


Glioma immunotherapy PD-1 Recurrence 



Fractionated radiotherapy


Glioblastoma multiforme


Granzyme B


Isocitrate dehydrogenase-1




O6-methylguanine-DNA methyltransferase


Major histocompatibility complex


Programmed cell death 1


PD-1 ligand


Autologous formalin-fixed tumor vaccine


Overall survival


Progression-free survival


Tumor-infiltrating lymphocytes





This study was supported by projects for Grant-in-Aid for Scientific Research of Japan and promoting practical applications of advanced medical technologies in Tsukuba University Hospital. Some of the materials for the tumor vaccine was provided by Cell-Medicine, Inc. (CMI) free of charge. CMI is a venture company for research and development of immunotherapy born from RIKEN (The Institute of Physical and Chemical Research) and University of Tsukuba in Japan. T.M and K.T. are members/employees of CMI; and K.T is a stockholder.


  1. 1.
    Committee of Brain Tumor Registry of Japan (2014) Report of brain tumor registry of Japan (2001-2004). Neurol Med Chir 54(Suppl):9–102Google Scholar
  2. 2.
    Crocetti E, Trama A, Stiller C, Caldarella A, Soffietti R, Jaal J, Weber DC, Ricardi U, Slowinski J, Brandes A, RARECARE Working Group (2012) Epidemiology of glial and non-glial brain tumours in Europe. Eur J Cancer 48(10):1532–1542CrossRefPubMedGoogle Scholar
  3. 3.
    Ostrom QT, Gittleman H, Fulop J, Liu M, Blanda R, Kromer C, Wolinsky Y, Kruchko C, Barnholtz-Sloan JS (2015) CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2008–2012. Neuro Oncol 17(Suppl 4):iv1–iv62CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO, European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups, National Cancer Institute of Canada Clinical Trials Group (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996CrossRefPubMedGoogle Scholar
  5. 5.
    Gorlia T, Stupp R, Brandes AA, Rampling RR, Fumoleau P, Dittrich C, Campone MM, Twelves CC, Raymond E, Hegi ME, Lacombe D, van den Bent MJ (2012) New prognostic factors and calculators for outcome prediction in patients with recurrent glioblastoma: a pooled analysis of EORTC Brain Tumour Group phase I and II clinical trials. Eur J Cancer 48(8):1176–1184CrossRefPubMedGoogle Scholar
  6. 6.
    Park JK, Hodges T, Arko L, Shen M, Dello Iacono D, McNabb A, Olsen Bailey N, Kreisl TN, Iwamoto FM, Sul J, Auh S, Park GE, Fine HA, Black PM (2010) Scale to predict survival after surgery for recurrent glioblastoma multiforme. J Clin Oncol 28(24):3838–3843CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Vredenburgh JJ, Desjardins A, Herndon JE, Marcello J, Reardon DA, Quinn JA, Rich JN, Sathornsumetee S, Gururangan S, Sampson J, Wagner M, Bailey L, Bigner DD, Friedman AH, Friedman HS (2007) Bevacizumab plus irinotecan in recurrent glioblastoma multiforme. J Clin Oncol 25(30):4722–4729CrossRefPubMedGoogle Scholar
  8. 8.
    Weller M, Tabatabai G, Kästner B, Felsberg J, Steinbach JP, Wick A, Schnell O, Hau P, Herrlinger U, Sabel MC, Wirsching HG, Ketter R, Bähr O, Platten M, Tonn JC, Schlegel U, Marosi C, Goldbrunner R, Stupp R, Homicsko K, Pichler J, Nikkhah G, Meixensberger J, Vajkoczy P, Kollias S, Hüsing J, Reifenberger G, Wick W, DIRECTOR Study Group (2015) MGMT promoter methylation is a strong prognostic biomarker for benefit from dose-intensified temozolomide rechallenge in progressive glioblastoma: the DIRECTOR trial. Clin Cancer Res 21(9):2057–2064CrossRefPubMedGoogle Scholar
  9. 9.
    Sandmann T, Bourgon R, Garcia J, Li C, Cloughesy T, Chinot OL, Wick W, Nishikawa R, Mason W, Henriksson R, Saran F, Lai A, Moore N, Kharbanda S, Peale F, Hegde P, Abrey LE, Phillips HS, Bais C (2015) Patients with proneural glioblastoma may derive overall survival benefit from the addition of bevacizumab to first-line radiotherapy and temozolomide: retrospective analysis of the AVAglio trial. J Clin Oncol 33(25):2735–2744CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Westphal M, Hilt DC, Bortey E, Delavault P, Olivares R, Warnke PC, Whittle IR, Jääskeläinen J, Ram Z (2003) A phase 3 trial of local chemotherapy with biodegradable carmustine (BCNU) wafers (Gliadel wafers) in patients with primary malignant glioma. Neuro Oncol 5(2):79–88PubMedPubMedCentralGoogle Scholar
  11. 11.
    Karsy M, Neil JA, Guan J, Mahan MA, Colman H, Jensen RL (2015) A practical review of prognostic correlations of molecular biomarkers in glioblastoma. Neurosurg Focus 38(3):E4CrossRefPubMedGoogle Scholar
  12. 12.
    van den Bent MJ, Gao Y, Kerkhof M, Kros JM, Gorlia T, van Zwieten K, Prince J, van Duinen S, Sillevis Smitt PA, Taphoorn M, French PJ (2015) Changes in the EGFR amplification and EGFRvIII expression between paired primary and recurrent glioblastomas. Neuro Oncol 17(7):935–941CrossRefPubMedGoogle Scholar
  13. 13.
    Stark AM, Witzel P, Strege RJ, Hugo H-H, Mehdorn HM (2003) p53, mdm2, EGFR, and msh2 expression in paired initial and recurrent glioblastoma multiforme. J Neurol Neurosurg Psychiatry 74:779–783CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Brown JR, Wimberly H, Lannin DR, Nixon C, Rimm DL, Bossuyt V (2014) Multiplexed quantitative analysis of CD3, CD8, and CD20 predicts response to neoadjuvant chemotherapy in breast cancer. Clin Cancer Res 20(23):5995–6005CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Dahlin AM, Henriksson ML, Van Guelpen B, Stenling R, Oberg A, Rutegård J, Palmqvist R (2011) Colorectal cancer prognosis depends on T-cell infiltration and molecular characteristics of the tumor. Mod Pathol 24(5):671–682CrossRefPubMedGoogle Scholar
  16. 16.
    Ishikawa E, Muragaki Y, Yamamoto T, Maruyama T, Tsuboi K, Ikuta S, Hashimoto K, Uemae Y, Ishihara T, Matsuda M, Matsutani M, Karasawa K, Nakazato Y, Abe T, Ohno T, Matsumura A (2014) Phase I/IIa trial of fractionated radiotherapy, temozolomide, and autologous formalin-fixed tumor vaccine for newly diagnosed glioblastoma. J Neurosurg 121(3):543–553CrossRefPubMedGoogle Scholar
  17. 17.
    Ishikawa E, Tsuboi K, Yamamoto T, Muroi A, Takano S, Enomoto T, Matsumura A, Ohno T (2007) Clinical trial of autologous formalin-fixed tumor vaccine for glioblastoma multiforme patients. Cancer Sci 98(8):1226–1233CrossRefPubMedGoogle Scholar
  18. 18.
    Muragaki Y, Maruyama T, Iseki H, Tanaka M, Shinohara C, Takakura K, Tsuboi K, Yamamoto T, Matsumura A, Matsutani M, Karasawa K, Shimada K, Yamaguchi N, Nakazato Y, Sato K, Uemae Y, Ohno T, Okada Y, Hori T (2011) Phase I/IIa trial of autologous formalin-fixed tumor vaccine concomitant with fractionated radiotherapy for newly diagnosed glioblastoma. Clinical article. J Neurosurg 115(2):248–255CrossRefPubMedGoogle Scholar
  19. 19.
    Sakamoto N, Ishikawa E, Yamamoto T, Satomi K, Nakai K, Sato M, Enomoto T, Morishita Y, Takano S, Ohno T, Tsuboi K, Matsumura A (2011) Pathological changes after autologous formalin-fixed tumor vaccine therapy combined with temozolomide for glioblastoma—three case reports -. Neurol Med Chir 51(4):319–325CrossRefGoogle Scholar
  20. 20.
    Borghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE, Chow LQ, Vokes EE, Felip E, Holgado E, Barlesi F, Kohlhäufl M, Arrieta O, Burgio MA, Fayette J, Lena H, Poddubskaya E, Gerber DE, Gettinger SN, Rudin CM, Rizvi N, Crinò L, Blumenschein GR Jr, Antonia SJ, Dorange C, Harbison CT, Graf Finckenstein F, Brahmer JR (2015) Nivolumab versus docetaxel in advanced nonsquamous non-small-cell lung cancer. N Engl J Med 373(17):1627–1639CrossRefPubMedGoogle Scholar
  21. 21.
    Robert C, Long GV, Brady B, Dutriaux C, Maio M, Mortier L, Hassel JC, Rutkowski P, McNeil C, Kalinka-Warzocha E, Savage KJ, Hernberg MM, Lebbé C, Charles J, Mihalcioiu C, Chiarion-Sileni V, Mauch C, Cognetti F, Arance A, Schmidt H, Schadendorf D, Gogas H, Lundgren-Eriksson L, Horak C, Sharkey B, Waxman IM, Atkinson V, Ascierto PA (2015) Nivolumab in previously untreated melanoma without BRAF mutation. N Engl J Med 372(4):320–330CrossRefPubMedGoogle Scholar
  22. 22.
    Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV, Bignell GR, Bolli N, Borg A, Børresen-Dale AL, Boyault S, Burkhardt B, Butler AP, Caldas C, Davies HR, Desmedt C, Eils R, Eyfjörd JE, Foekens JA, Greaves M, Hosoda F, Hutter B, Ilicic T, Imbeaud S, Imielinski M, Jäger N, Jones DT, Jones D, Knappskog S, Kool M, Lakhani SR, López-Otín C, Martin S, Munshi NC, Nakamura H, Northcott PA, Pajic M, Papaemmanuil E, Paradiso A, Pearson JV, Puente XS, Raine K, Ramakrishna M, Richardson AL, Richter J, Rosenstiel P, Schlesner M, Schumacher TN, Span PN, Teague JW, Totoki Y, Tutt AN, Valdés-Mas R, van Buuren MM, van‘t Veer L, Vincent-Salomon A, Waddell N, Yates LR, Australian Pancreatic Cancer Genome Initiative; ICGC Breast Cancer Consortium, ICGC MMML-Seq Consortium, ICGC PedBrain, Zucman-Rossi J, Futreal PA, McDermott U, Lichter P, Meyerson M, Grimmond SM, Siebert R, Campo E, Shibata T, Pfister SM, Campbell PJ, Stratton MR (2013) Signatures of mutational processes in human cancer. Nature 500(7463):415–421CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Berghoff AS, Kiesel B, Widhalm G, Rajky O, Ricken G, Wöhrer A, Dieckmann K, Filipits M, Brandstetter A, Weller M, Kurscheid S, Hegi ME, Zielinski CC, Marosi C, Hainfellner JA, Preusser M, Wick W (2015) Programmed death ligand 1 expression and tumor-infiltrating lymphocytes in glioblastoma. Neuro Oncol 17(8):1064–1075CrossRefPubMedGoogle Scholar
  24. 24.
    Liu Y, Carlsson R, Ambjørn M, Hasan M, Badn W, Darabi A, Siesjö P, Issazadeh-Navikas S (2013) PD-L1 expression by neurons nearby tumors indicates better prognosis in glioblastoma patients. J Neurosci 33(35):14231–14245CrossRefPubMedGoogle Scholar
  25. 25.
    Nduom EK, Wei J, Yaghi NK, Huang N, Kong LY, Gabrusiewicz K, Ling X, Zhou S, Ivan C, Chen JQ, Burks JK, Fuller GN, Calin GA, Conrad CA, Creasy C, Ritthipichai K, Radvanyi L, Heimberger AB (2016) PD-L1 expression and prognostic impact in glioblastoma. Neuro Oncol 18(2):195–205CrossRefPubMedGoogle Scholar
  26. 26.
    Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, Scheithauer BW, Kleihues P (2007) The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 114(2):97–109CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Lotfi M, Afsharnezhad S, Raziee HR, Ghaffarzadegan K, Sharif S, Shamsara J, Lary S, Behravan J (2011) Immunohistochemical assessment of MGMT expression and p53 mutation in glioblastoma multiforme. Tumori 97(1):104–108PubMedGoogle Scholar
  28. 28.
    Yeung JT, Hamilton RL, Ohnishi K, Ikeura M, Potter DM, Nikiforova MN, Ferrone S, Jakacki RI, Pollack IF, Okada H (2013) LOH in the HLA class I region at 6p21 is associated with shorter survival in newly diagnosed adult glioblastoma. Clin Cancer Res 19(7):1816–1826CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Lorente A, Mueller W, Urdangarín E, Lázcoz P, von Deimling A, Castresana JS (2008) Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR. BMC Cancer 8:61CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Hodi FS, Dranoff G (2010) The biologic importance of tumor-infiltrating lymphocytes. J Cutan Pathol 37:48–53CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Mandai M, Hamanishi J, Abiko K, Matsumura N, Baba T, Konishi I (2016) Dual faces of IFNγ in cancer progression: a role of PD-L1 induction in the determination of pro- and antitumor immunity. Clin Cancer Res 22(10):2329–2334CrossRefPubMedGoogle Scholar
  32. 32.
    Terawaki S, Chikuma S, Shibayama S, Hayashi T, Yoshida T, Okazaki T, Honjo T (2011) IFN-α directly promotes programmed cell death-1 transcription and limits the duration of T cell-mediated immunity. J Immunol 186(5):2772–2779CrossRefPubMedGoogle Scholar
  33. 33.
    Duechler M, Peczek L, Zuk K, Zalesna I, Jeziorski A, Czyz M (2014) The heterogeneous immune microenvironment in breast cancer is affected by hypoxia-related genes. Immunobiology 219(2):158–165CrossRefPubMedGoogle Scholar
  34. 34.
    Platten M, Ochs K, Lemke D, Opitz C, Wick W (2014) Microenvironmental clues for glioma immunotherapy. Curr Neurol Neurosci Rep 14(4):440CrossRefPubMedGoogle Scholar
  35. 35.
    Karyampudi L, Lamichhane P, Scheid AD, Kalli KR, Shreeder B, Krempski JW, Behrens MD, Knutson KL (2014) Accumulation of memory precursor CD8 T cells in regressing tumors following combination therapy with vaccine and anti-PD-1 antibody. Cancer Res 74(11):2974–2985CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Duraiswamy J, Freeman GJ, Coukos G (2014) Dual blockade of PD-1 and CTLA-4 combined with tumor vaccine effectively restores T-cell rejection function in tumors-response. Cancer Res 74(2):633–634CrossRefPubMedGoogle Scholar
  37. 37.
    Chen L (2004) Co-inhibitory molecules of the B7-CD28 family in the control of T-cell immunity. Nat Rev Immunol 4(5):336–347CrossRefPubMedGoogle Scholar
  38. 38.
    Carlino MS, Long GV (2016) Ipilimumab combined with nivolumab: a standard of care for the treatment of advanced melanoma? Clin Cancer Res 22(16):3992–3998CrossRefPubMedGoogle Scholar
  39. 39.
    Miyoshi T, Kataoka T, Asahi A, Maruyama T, Okada R, Uemae Y, Ohno T (2016) A transient increase and subsequent sharp decrease of chemo-refractory liver-metastasized uterine cervical small cell carcinoma to autologous formalin-fixed tumor vaccine plus anti-PD-1 antibody. Clin Case Rep 4(7):687–691CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Garber ST, Hashimoto Y, Weathers SP, Xiu J, Gatalica Z, Verhaak RG, Zhou S, Fuller GN, Khasraw M, de Groot J, Reddy SK, Spetzler D, Heimberger AB (2016) Immune checkpoint blockade as a potential therapeutic target: surveying CNS malignancies. Neuro Oncol 18(10):1357–1366CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Tsubasa Miyazaki
    • 1
    • 2
  • Eiichi Ishikawa
    • 1
    Email author
  • Masahide Matsuda
    • 1
  • Hiroyoshi Akutsu
    • 1
  • Satoru Osuka
    • 3
  • Noriaki Sakamoto
    • 4
  • Shingo Takano
    • 1
  • Tetsuya Yamamoto
    • 1
  • Koji Tsuboi
    • 2
    • 5
  • Akira Matsumura
    • 1
  1. 1.Department of Neurosurgery, Faculty of MedicineUniversity of TsukubaTsukubaJapan
  2. 2.Cell-Medicine, Inc.TsukubaJapan
  3. 3.Department of Neurosurgery, Winship Cancer InstituteEmory University School of MedicineAtlantaUSA
  4. 4.Department of Pathology, Faculty of MedicineUniversity of TsukubaTsukubaJapan
  5. 5.Proton Medical Research Center, Faculty of MedicineUniversity of TsukubaTsukubaJapan

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