Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021 is online at: average * The Journal of Immunology , 12 of which you can access for free at: 2012; 189:1920-1927; Prepublished online 16 from submission to initial decision 4 weeks from acceptance to publication July 2012; doi: 10.4049/jimmunol.1103373 http://www.jimmunol.org/content/189/4/1920 Increased Immune Gene Expression and Immune Cell Infiltration in High-Grade Astrocytoma Distinguish Long-Term from Short-Term Survivors Andrew M. Donson, Diane K. Birks, Stephanie A. Schittone, Bette K. Kleinschmidt-DeMasters, Derrick Y. Sun, Molly F. Hemenway, Michael H. Handler, Allen E. Waziri, Michael Wang and Nicholas K. Foreman J Immunol cites 41 articles Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription http://www.jimmunol.org/content/suppl/2012/07/16/jimmunol.110337 3.DC1 This article http://www.jimmunol.org/content/189/4/1920.full#ref-list-1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material References Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 30, 2021. The Journal of Immunology Increased Immune Gene Expression and Immune Cell Infiltration in High-Grade Astrocytoma Distinguish Long-Term from Short-Term Survivors Andrew M. Donson,*,† Diane K. Birks,†,‡ Stephanie A. Schittone,*,† Bette K. Kleinschmidt-DeMasters,x,{ Derrick Y. Sun,‡ Molly F. Hemenway,*,† Michael H. Handler,†,‡ Allen E. Waziri,‡ Michael Wang,*,† and Nicholas K. Foreman*,†,‡ Survival in the majority of high-grade astrocytoma (HGA) patients is very poor, with only a rare population of long-term survivors. A better understanding of the biological factors associated with long-term survival in HGAwould aid development of more effective therapy and survival prediction. Factors associated with long-term survival have not been extensively studied using unbiased genome-wide expression analyses. In the current study, gene expression microarray profiles of HGA from long-term survivors were Downloaded from interrogated for discovery of survival-associated biological factors. Ontology analyses revealed that increased expression of immune function-related genes was the predominant biological factor that positively correlated with longer survival. A notable T cell sig- nature was present within this prognostic immune gene set. Using immune cell-specific gene classifiers, both T cell-associated and myeloid linage-associated genes were shown to be enriched in HGA from long-term versus short-term survivors. Association of immune function and cell-specific genes with survival was confirmed independently in a larger publicly available glioblastoma gene expression microarray data set. Histology was used to validate the results of microarray analyses in a larger cohort of long- http://www.jimmunol.org/ term survivors of HGA. Multivariate analyses demonstrated that increased immune cell infiltration was a significant independent variable contributing to longer survival, as was Karnofsky/Lansky performance score. These data provide evidence of a prognostic anti-tumor adaptive immune response and rationale for future development of immunotherapy in HGA. The Journal of Immu- nology, 2012, 189: 1920–1927. edian survival in high-grade astrocytoma (HGA), con- mutational status have been established as molecular prognostic sisting predominantly of glioblastoma (GBM) and an- factors in adult HGA (4, 5). M aplastic astrocytoma (AA), is 15 mo and 3 y, respectively Gene expression microarray analyses have been used to identify by guest on September 30, 2021 (1, 2). Few robust prognostic factors have been identified in HGA, novel prognostic biomarkers in HGA. This unbiased genome-wide hindering patient care and stratification in clinical trials. Currently approach has the additional benefit of providing insight into the established clinical risk factors include Karnofsky/Lansky perfor- biological mechanisms of tumorigenesis that can be exploited for mance score, a measure of patient well-being that is widely used in the development of more effective therapies. Despite numerous oncology, and age (3). O-6-Methylguanine-DNA methyltransfer- studies that have identified prognostic gene signatures in HGA ase promoter methylation status and isocitrate dehydrogenase 1 using microarray technology, there remains no predictor of survival that has proved robustly reproducible from study to study (6–10). This may be due to the effects of biological heterogeneity inherent *Department of Pediatrics, University of Colorado Denver, Aurora, CO 80045; in HGA combined with the typically limited duration and range of †The Children’s Hospital, Aurora, CO 80045; ‡Department of Neurosurgery, Univer- survival. sity of Colorado Denver, Aurora, CO 80045; xDepartment of Pathology, University of Colorado Denver, Aurora, CO 80045; and {Department of Neurology, University of Although prognosis is poor for the majority of HGA, a small Colorado Denver, Aurora, CO 80045 but discrete subgroup of long-term survivors exists, with 3–5% of Received for publication November 23, 2011. Accepted for publication June 17, GBM patients surviving longer than 3 y. The driving hypothesis 2012. for this study is that a more pronounced and biologically infor- This work was supported by the Morgan Adams Foundation and by National Insti- mative prognostic gene signature could be obtained by gene ex- tutes of Health Grant R01 CA140614-01A1. pression microarray analysis of long-term survivors of HGA. Using The sequences presented in this article have been submitted to the Gene Expression this approach, the current study identified immune function as the Omnibus (http://www.ncbi.nlm.nih.gov/geo/) database under accession number GSE33331. predominant ontology associated with long-term survival in pe- Address correspondence and reprint requests to Andrew M. Donson, Department of diatric and adult HGA. Histological validation in a larger cohort Pediatrics, University of Colorado Denver, Mail Stop 8302, Building RC1, 12800 E. of long-term survivors demonstrated that increased infiltration of 19th Avenue, Aurora, CO 80045. E-mail address: [email protected] immune cells was prognostically favorable. The online version of this article contains supplemental material. Abbreviations used in this article: AA, anaplastic astrocytoma; AIF1, allograft inhibi- tory factor-1; DAVID, Database for Annotation, Visualization, and Integrated Discov- Materials and Methods ery; EPN, ependymoma; FFPE, formalin-fixed paraffin-embedded; GBM, glioblastoma; Patient cohort and sample collection gcRMA, guanine cytosine Robust Multiarray Average; GO, Gene Ontology Project; GSEA, Gene Set Enrichment Analysis; HGA, high-grade astrocytoma; IHC, immuno- Both adult and pediatric HGA samples, including GBM and AA, were histochemistry. included in this study cohort. Using broad age and diagnostic categories substantially increased the number of long-term survivors available to the Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 study. This inclusive approach also has the potential to identify broad www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103373 The Journal of Immunology 1921 prognostic factors in HGA. Multivariate analyses were used to address the variable. GSEA creates a ranked list of genes based on user-selected cri- confounding effect of age, tumor grade, and a number of other potentially teria such as signal-to-noise, t tests, and so forth. For the purposes of this prognostic factors in this cohort. study, genes were ranked on their correlation with survival time. Thus, For the initial discovery cohort (gene expression microarray analysis), unlike DAVID, GSEA takes into account the strength of association rather surgical tumor samples were obtained from 26 patients who presented than simply whether a gene is significantly associated or not. The output between 1990 and 2008 for treatment of HGA at the University Hospital or for both ontology analysis tools is an enrichment score with associated The Children’s Hospital (Aurora, CO) who were diagnosed with GBM or Student t test p values for each GO term. AA according to World Health Organization guidelines (11). Tumor was either snap frozen in liquid nitrogen or placed in RNAlater storage solu- Enrichment analysis of specific immune cell lineage genes tion (Qiagen, Valencia, CA) at the time of resection. Survival data were Gene expression microarray data derived from surgical brain tumor speci- available for all patients in this study, which was conducted in compliance mens contain genes expressed by nontumor cells of the tumor microenvi- with institutional review board regulations (COMIRB 95-500 and 05- ronment. To address this, a technique was developed to identify and predict 0149). Included in this study were three long-term HGA survivors (two specific expression patterns of immune-related cells, based on data from adult GBM and one pediatric GBM), with a median survival of 7.0