Immune Gene and Cell Enrichment Is Associated with a Good Prognosis in Ependymoma This information is current as Andrew M. Donson, Diane K. Birks, Valerie N. Barton, Qi of September 27, 2021. Wei, Bette K. Kleinschmidt-DeMasters, Michael H. Handler, AllenE. Waziri, Michael Wang and Nicholas K. Foreman J Immunol published online 16 November 2009 http://www.jimmunol.org/content/early/2009/11/16/jimmuno l.0902811 Downloaded from Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 27, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 16, 2009, doi:10.4049/jimmunol.0902811 The Journal of Immunology Immune Gene and Cell Enrichment Is Associated with a Good Prognosis in Ependymoma1 Andrew M. Donson,2*§ Diane K. Birks,‡§ Valerie N. Barton,*§ Qi Wei,*§ Bette K. Kleinschmidt-DeMasters,† Michael H. Handler,‡§ Allen E. Waziri,‡ Michael Wang,*§ and Nicholas K. Foreman*‡§ Approximately 50% of children with ependymoma will suffer from tumor recurrences that will ultimately lead to death. Devel- opment of more effective therapies and patient stratification in ependymoma mandates better prognostication. In this study, tumor gene expression microarray profiles from pediatric ependymoma clinical samples were subject to ontological analyses to identify outcome-associated biological factors. Histology was subsequently used to evaluate the results of ontological analyses. Ontology analyses revealed that genes associated with nonrecurrent ependymoma were predominantly immune function-related. Addition- ally, increased expression of immune-related genes was correlated with longer time to progression in recurrent ependymoma. Of Downloaded from those genes associated with both the nonrecurrent phenotype and that positively correlated with time to progression, 95% were associated with immune function. Histological analysis of a subset of these immune function genes revealed that their expression was restricted to a subpopulation of tumor-infiltrating cells. Analysis of tumor-infiltrating immune cells showed increased infil- tration of CD4؉ T cells in the nonrecurrent ependymomas. No genomic sequences for SV40, BK, JC, or Merkel polyomaviruses were found in nonrecurrent ependymoma. This study reveals that up-regulation of immune function genes is the predominant http://www.jimmunol.org/ ontology associated with a good prognosis in ependymoma and it provides preliminary evidence of a beneficial host proinflam- matory and/or Ag-specific immune response. The Journal of Immunology, 2009, 183: 7428–7440. pendymoma (EPN),3 the third most common brain tumor recur from overly aggressive treatments. Identification of prognos- of children, is treated by surgical resection and radiation tic markers for EPN may have the added benefit of providing in- E therapy (1, 2). Complete resection, often requiring “sec- sight into the biological mechanisms of tumorigenesis, which ond-look” surgery, is critical for a favorable outcome (3, 4). Ra- could be exploited for the development of more effective therapies. diation therapy is also standard, and omission of this results in a To date, study of candidate prognostic markers for pediatric higher number of tumor recurrences (4, 5). Chemotherapy has so EPN have largely been confined to histological grading according by guest on September 27, 2021 far shown little or no benefit. Unfortunately, Ͼ50% of children to World Health Organization (WHO) tumor classification criteria treated with the standard regimen will suffer from tumor recur- (7–11), as well as to molecular markers such as Ki-67 (12, 13), rence, which will ultimately result in death (6). This high failure survivin (14, 15), human telomerase reverse transcriptase (16), and rate represents one of the most significant problems in pediatric nucleolin (4). More recently, global molecular analyses such as neuro-oncology. Despite unfavorable outcome in more than half of array comparative genomic hybridization (17, 18) and gene ex- pediatric EPN patients, little progress has been made in the past 20 pression profiling (17, 19–21) have been employed to discover years either in treatment or identification of robust prognostic fac- prognostic chromosomal aberrations or gene expression signa- tors. The ability to identify up-front those EPN patients whose tures. These global studies have produced an even wider range of tumor will recur would allow clinicians to try more aggressive candidate prognostic markers, although none to date have identi- treatment regimens, better stratify patients on various treatment fied a biological mechanism of recurrence. Despite these numerous protocols, and spare those children whose tumors are unlikely to studies, there remains no predictor of tumor recurrence in EPN that is robustly reproducible from study to study. The driving hypoth- esis for this study is that gene expression patterns differ between *Department of Pediatrics, †Departments of Pathology and Neurology, and ‡Depart- ment of Neurosurgery, University of Colorado Denver, Aurora, CO 80045; and §The good and bad prognosis EPN, the details of which will allow for Children’s Hospital, Denver, CO 80045 better prognostication and provide insights into the biology of re- Received for publication August 25, 2009. Accepted for publication October 5, 2009. currence. To achieve this, tumor gene expression profiling com- The costs of publication of this article were defrayed in part by the payment of page bined with gene ontology analysis was used as an unbiased ap- charges. This article must therefore be hereby marked advertisement in accordance proach to identify sets of functionally related genes that were with 18 U.S.C. Section 1734 solely to indicate this fact. associated with clinical outcome in EPN clinical samples. Using 1 This work was supported by the Tanner Seebaum Foundation. this approach, it was found that an up-regulation of immune func- 2 Address correspondence and reprint requests to Andrew M. Donson, Department of tion-related genes was the predominant ontology associated with a Pediatrics, University of Colorado Denver, Mail Stop 8302, P.O. Box 6511, Aurora, CO 80045. E-mail address: [email protected] complete response to therapy. 3 Abbreviations used in this paper: EPN, ependymoma; AIF-1, allograft inhibitory factor-1; DAVID; Database for Annotation, Visualization, and Integrated Discovery; FDR, false discovery rate; FFPE, formalin-fixed paraffin-embedded; gcRMA, Gene- Materials and Methods Chip robust multiarray average; GO, Gene Ontology; GSEA, Gene Set Enrichment Patient cohort Analysis; IHC, immunohistochemistry; TIL, tumor-infiltrating lymphocyte; TTP, time to progression; GOTERM, Gene Ontology Project term. Surgical tumor samples were obtained from 19 patients who presented between 1997 and 2007 for treatment at The Children’s Hospital (Denver, Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 CO) who were diagnosed with EPN according to WHO guidelines (22). All www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902811 The Journal of Immunology 7429 identified by the user as significantly associated with a particular phenotype Table I. Patient cohort demographic and tumor detailsa or variable. Immunohistochemistry (IHC) Patient TTP Grade Age at ID Outcome (months) (WHO) Location Gender Dx (years) IHC was performed on 5-␮m FFPE tumor tissue sections. Slides were deparaffinized and then subjected to optimal Ag retrieval protocols. Sub- 80 Non — II IF M 2 110 Rec 31 II IF M 2 sequent steps were performed using the EnVision-HRP kit (Dako) on a 135 Rec 6 III ST F 14 Dako autostainer according to standard protocol. Incubation with primary 195 Rec 24 III IF M 2 Ab was performed for 2 h. The following dilutions of primary Ab were 242 Rec 1 III ST F 4 used, and applied to the sections for 1 h: 1/250 allograft inhibitory factor-1 246 Rec 35 II IF M 2 (AIF-1) (01-1974) from Waco Pure Chemicals; 1/50 HLA-DR (LN3) and 285 Non — III ST F 5.5 1/40 CD4 (IF6) from Novocastra; 1/100 CD8 (C8/144B), 1/200 CD20 306 Non — II IF M 13 ϩ 318 Non — III IF F 2 (L26), 1/50 CD45 (2B11 PD7/26), and 1/100 CD68 (PG-M1) from 319 Rec 51 II IF F 6 Dako. Each of these Abs stained a discrete subpopulation of cells that were 364 Rec 35 II IF M 13 distributed throughout the parenchyma of the tumor. Slides were analyzed 388 Non — III ST M 11 with the Olympus BX40 microscope, ϫ40 objective lens. Images were 392 Non — II IF F 1 captured using an Optronics MicroFire 1600 ϫ 1200 camera and Picture- 393 Rec 18 II IF F 6 Frame 2.3 imaging software (Optronics). Infiltrating cell abundancies were 416 Non — III ST M 5 measured as the mean number of positive staining cells per five fields of 419 Non — II IF M 3 459 Non — III IF F 0.5 view and differential expression between groups was determined using a 483 Rec 23 II IF M 7 Student’s t test with a p value cutoff of 0.05. 507 Rec 5 III IF M 5 Quantitative PCR for viral sequences Downloaded from a — denotes that tumor did not recur. Non, nonrecurrent; Rec, recurrent; WHO, World Health Organization tumor grade classification; IF, infratentorial; ST, supra- Quantitative PCR was performed for SV40, BK, JC, and Merkel polyoma- tentorial; Dx, diagnosis. viruses (PyV). DNA was extracted from surgical specimens using the Gen- trapure DNA extraction kit (Qiagen).