Immune phenotypes predict survival in patients with glioblastoma multiforme

Mostafa et al. Journal of Hematology & Oncology (2016) 9:77 DOI 10.1186/s13045-016-0272-3 RESEARCH Open Access Immune phenotypes predict survival in patients with glioblastoma multiforme Haouraa Mostafa1,2, Andrej Pala3, Josef Högel4, Michal Hlavac3, Elvira Dietrich1,2, M. Andrew Westhoff5, Lisa Nonnenmacher3, Timo Burster3, Michael Georgieff2, C. Rainer Wirtz3 and E. Marion Schneider1,2* Abstract Background: Glioblastoma multiforme (GBM), a common primary malignant brain tumor, rarely disseminates beyond the central nervous system and has a very bad prognosis. The current study aimed at the analysis of immunological control in individual patients with GBM. Methods: Immune phenotypes and plasma biomarkers of GBM patients were determined at the time of diagnosis using flow cytometry and ELISA, respectively. Results: Using descriptive statistics, we found that immune anomalies were distinct in individual patients. Defined marker profiles proved highly relevant for survival. A remarkable relation between activated NK cells and improved survival in GBM patients was in contrast to increased CD39 and IL-10 in patients with a detrimental course and very short survival. Recursive partitioning analysis (RPA) and Cox proportional hazards models substantiated the relevance of absolute numbers of CD8 cells and low numbers of CD39 cells for better survival. Conclusions: Defined alterations of the immune system may guide the course of disease in patients with GBM and may be prognostically valuable for longitudinal studies or can be applied for immune intervention. Keywords: Glioblastoma multiforme, CD8+ lymphocytes, NK cells, CD39-ectonucleotidase, Recursive partitioning analysis, Survival Background Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Despite intensive research efforts and a multimodal management that consists of surgery, radiotherapy, and chemotherapy with temozolomide, the prognosis is very poor [1]. The hypoxic environment represents a very potent stimulus for angiogenesis [2], while molecular alterations, cell survival mechanisms, the acquisition of chemo- and radioresistance [3], and tumor heterogeneity [4] as well as multiple immune escape mechanisms are further important factors that contribute to the inferior prognosis in GBM [5, 6]. In addition to age, a number of stress factors, including chemical exposure, and demographical aspects influence the incidence of tumor manifestation [7]. At least one study demonstrated * Correspondence: 1 Sektion Experimentelle Anaesthesiologie, University Hospital Ulm, Albert Einstein Allee 23, 89081 Ulm, Germany 2 Klinik für Anaesthesiologie, University Hospital Ulm, Albert Einstein Allee 23, 89081 Ulm, Germany Full list of author information is available at the end of the article an increased risk for glioblastoma by alcohol consumption [8]. When compared with other malignancies, modulation of immune suppressor effector cells appears to be of major importance [9], and individual tumor patients may require an individual treatment regimen against immune suppressive elements. We addressed here immune phenotypes in patients with glioblastoma at the time of diagnosis. We applied flow cytometry and validated enzyme-linked immunosorbent assay (ELISA) as a reliable technique to systematically analyze immune cells in peripheral blood as well as cytokines. In general, immune modulation initiated by the tumor may require direct cell-cell contact or may occur via soluble factors, including interleukins and chemokines. At present, malignancies manifesting distantly from the peripheral immune system have not been systematically described to influence immune phenotypes. However, an important work published by Kmiecik and colleagues [10], which was performed in a small group of GBM patients, was highly promising in implying the sensitization © 2016 Mostafa et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mostafa et al. Journal of Hematology & Oncology (2016) 9:77 of immune cells against GBM. In addition, the work published by Skog and coworkers [11] contributed to our understanding of effector pathways induced by the interaction of tumor-derived microparticles with target cells. Cytokines, including interleukin (IL)-10 and ferritin, secreted by tumor-infiltrating immune cells and by the tumor itself are members of a larger scenario of a tumorassociated proteome [12]. They may play an important role in downregulating major histocompatibility complex (MHC)-class I and natural killer (NK)-ligands [13]. Here, we asked whether immune phenotypes of patients with manifest GBM display distinct features differing from healthy individuals. In addition to the abovementioned IL-10 and ferritin associated with GBM, we found that the relative amounts of activated NK cells in addition to T cell receptor (TCR) α/β-positive and CD8-positive lymphocytes display a strong correlation with increased survival in GBM in bivariate analysis. When using multivariable Cox proportional hazards model analysis, the Karnofsky Performance status Scale (KPS), the isocitrate dehydrogenase-1 (IDH-1) mutation status, and high CD8 and low CD39 cells are most relevant for better survival. Methods Patients Patients had been diagnosed for glioblastoma multiforme grade IV according to the criteria of the World Health Organization (WHO). Immune competence was tested at the time of diagnosis before surgery following standard cluster of differentiation (CD) and activation markers for T-, NK-, monocyte-, and granulocyte subpopulations according to international guidelines for whole blood flow cytometric analysis. Immune phenotyping and inflammatory and anti-inflammatory biomarkers performed by a validated ELISA was an essential step in classifying patients for their immune competence. These tests were done for each patient as part of routine care. Patients were then under high dose steroid treatment. All patients’ data were pseudonymized by a data bank held in the Section of Experimental Anaesthesiology (http://www.uniklinik-ulm.de/struktur/kliniken/sektionen/klinik-fuer-anaesthesiologie/experimentelleanaesthesiologie/home/general-information/addresscontact.html). The study was conducted after approval by the local Ethics Committee of the University Hospital Ulm with the approval umber 162/10 named “Novel experimental approaches in brain tumors”. The universal trial number is U111-1179-3127. Brain tumor mate (...truncated)