Distinct Transcription Profiles of Primary and Secondary Glioblastoma Subgroups Cho-Lea Tso, 1,2,6 William A. Freije, 1 Allen Day, 1 Zugen Chen, 1 Barry Merriman, 1 Ally Perlina, 1 Yohan Lee, 1 Ederlyn Q. Dia, 3 Koji Yoshimoto, 3 Paul S. Mischel, 3,6 Linda M. Liau, 4,6 Timothy F. Cloughesy, 5,6 and Stanley F. Nelson 1,6 Departments of 1 Human Genetics, 2 Medicine/Hematology-Oncology, 3 Pathology and Laboratory Medicine, 4 Neurosurgery, and 5 Neurology, David Geffen School of Medicine, and 6 Jonsson Comprehensive Cancer Center, University of California at Los Angeles, Los Angeles, California Abstract Glioblastomas are invasive and aggressive tumors of the brain, generally considered to arise from glial cells. A subset of these cancers develops from lower-grade gliomas and can thus be clinically classified as ‘‘secondary,’’ whereas some glioblasto- mas occur with no prior evidence of a lower-grade tumor and can be clinically classified as ‘‘primary.’’ Substantial genetic differences between these groups of glioblastomas have been identified previously. We used large-scale expression analyses to identify glioblastoma-associated genes (GAG) that are associated with a more malignant phenotype via comparison with lower-grade astrocytomas. We have further defined gene expression differences that distinguish primary and secondary glioblastomas. GAGs distinct to primary or secondary tumors provided information on the heterogeneous properties and apparently distinct oncogenic mechanisms of these tumors. Secondary GAGs primarily include mitotic cell cycle compo- nents, suggesting the loss of function in prominent cell cycle regulators, whereas primary GAGs highlight genes typical of a stromal response, suggesting the importance of extracellular signaling. Immunohistochemical staining of glioblastoma tissue arrays confirmed expression differences. These data highlight that the development of gene pathway-targeted therapies may need to be specifically tailored to each subtype of glioblastoma. (Cancer Res 2006; 66(1): 159-67) Introduction Human solid tumors undergo multiple genetic evolutionary abnormalities as they evolve from normal cells to early-stage tumors to aggressive cancers (1). Chromosome instability that results in the development of both numerical abnormalities (aneuploidy) and structural abnormalities (chromosomal breakage, deletions, and amplification) is especially striking in many types of solid tumors (1, 2). A series of genome-wide chromosomal imbalance analyses and multiparameter cell-based studies suggest that genomic changes that lead to the loss of tumor suppressor gene function usually occur at early stages, whereas the later stages often involve the accumulation of multiple gain-of-function abnormalities that confer on tumors the potential for malignant transformation (3, 4). It is possible that the malignant end points that are ultimately reached will prove to be shared in common by many types of tumors (5). The identification and functional assessment of genes altered in the process of malignant transformation is essential for understating the mechanism of cancer development and should facilitate the development of more effective treatments. Infiltrative astrocytic neoplasms are the most common brain tumors of central nervous system in adults. Glioblastoma multi- forme (WHO grade IV) remains a devastating disease, with a median survival of <1 year after diagnosis (6). Glioblastomas are defined by histopathologic features of cellular atypia, mitotic figures, necrotic foci with peripheral cellular pseudopalisading, and microvascular hyperplasia that distinguish it from lower-grade astrocytic tumors (7). Two subgroups of glioblastomas have been established based on clinical experience and have been affiliated with distinct genetic mechanisms of tumorigenesis. Secondary glioblastomas develop slowly through progression from low-grade glial tumors (WHO grade II) or anaplastic glial tumors (WHO grade III) and frequently contain mutations in the p53 gene (f60%), overexpression of platelet-derived growth factor receptors, and loss of heterozygosity (LOH) at 17p, 19q, and 10q (8, 9). In contrast, primary glioblastomas seem to develop rapidly and manifest high- grade lesion from the outset and are genetically characterized by amplification/overexpression of epidermal growth factor receptor (EGFR; f60%) and mouse double minute 2 (f50%), PTEN mutations, and loss of all or a portion of chromosome 10 (8, 9). The objective of this study was to identify gain-of-function genes that are associated with acquisition of malignant features of glioblastomas. In addition, we investigated whether clinically defined primary and secondary glioblastoma subgroups use distinct molecular pathways. DNA microarray experiments were done to establish a transcription database for 101 glial brain tumors for which clinical and pathologic features as well as biopsy material were available. Through a series of comparative analyses against lower-grade astrocytomas, we have identified shared and distinct gene categories of transcripts overexpressed in glioblastoma subgroups that are associated with malignant transformation. The distinct glioblastoma-associated genes (GAG) further led to the discovery of stromal/mesenchymal properties in glioblastoma subgroup. Materials and Methods Tumor sample and data collection. The patient tumors and normal samples were collected either from autopsies of glioblastoma patients within 24 hours of death or from patients who underwent surgery at University of California at Los Angeles (UCLA) Medical Center. All samples were collected under protocols approved by the UCLA Institutional Review Board. All histopathogic typing and tumor grading Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Stanley F. Nelson, Department of Human Genetics, David Geffen School of Medicine, University of California at Los Angeles, Room 5506, 695 Young Drive South, Los Angeles, CA 90095. E-mail: sfnelson@mednet.ucla.edu. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-0077 www.aacrjournals.org 159 Cancer Res 2006; 66: (1). January 1, 2006 Research Article