Oncogene (2002) 21, 3961 ± 3968 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Distinction in gene expression pro®les of oligodendrogliomas with and without allelic loss of 1p Akitake Mukasa1,2, Keisuke Ueki*,2, Shinichi Matsumoto1, Shuichi Tsutsumi1, Ryo Nishikawa3, Takamitsu Fujimaki4, Akio Asai2,5, Takaaki Kirino2,5 and Hiroyuki Aburatani*,1 1Genome Science Division, Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro- ku, Tokyo 153-8904, Japan; 2Department of Neurosurgery, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan; 3Department of Neurosurgery, Saitama Medical School, 38-2 Morohongo, Moroyama-machi, Iruma-gun, Saitama 350-0495, Japan; 4Department of Neurosurgery, School of Medicine, Teikyo University, 2-11-2 Kaga, Itabashi-ku, Tokyo 173-8606, Japan; 5CREST (Core Research for Evolutional Science and Technology), Japan Science and Technology Corporation, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan Oligodendrogliomas frequently, but not always show tumors or 10 to 25% of all intracranial gliomas sensitivity to chemotherapy and recent studies demon- (Kleihues and Cavenee, 2000). One of the important strated that allelic loss of chromosome 1p is highly recent ®ndings in neuro-oncology was that those associated with this chemosensitivity. To gain insight into oligodendrogliomas frequently showed remarkable the molecular mechanism of such dierence, we exam- sensitivity to chemotherapy, especially to a regimen ined comprehensive gene expression pro®les of 11 using procarbazine, CCNU and vincristine (PCV oligodendroglial tumors, six with and ®ve without therapy) (Cairncross and Macdonald, 1988). However, 1pLOH (loss of heterozygosity), and two normal brain the response rate to PCV therapy remains 60 ± 80%, tissues using the oligonucleotide microarray (GeneChip). and 20 ± 30% of tumors are resistant to chemotherapy Statistically signi®cant numbers of genes were expressed and have worse prognosis. Therefore, within this dierentially between the two genetic subsets. Clustering histologically indistinguishable entity, there apparently analysis separated the tumor subsets well. The tumors exist subgroups showing dierent biological behavior. with 1pLOH had similar expression pro®les to the Recent molecular genetic studies on oligodendroglio- normal brain for those dierentially expressed genes. mas revealed that allelic loss of chromosome 1p, which Many genes showing higher expression in tumors with is found in 60 ± 80% of oligodendrogliomas and often 1pLOH were presumed to have functions in nervous accompanied with allelic loss of 19q (Smith et al., tissues. Notably, the majority of the 123 genes showing 1999), was highly associated with the treatment signi®cant expression reduction in tumors with 1pLOH responsiveness and also with a better prognosis were either on chromosome 1 (50%) or on 19 (10%), (Cairncross et al., 1998; Ino et al., 2001). Thus, it is and the average expression reduction ratio was about now being recognized that loss of chromosome 1p is a 50% (0.54+0.13) possibly re¯ecting the chromosomal marker separating oligodendrogliomas into subgroups deletion. Thus, the biological dierence between the showing dierent biological behavior. In addition to its genetic subsets of oligodendroglioma was indeed re¯ected important clinical implications, understanding of the to gene expression pro®le, which provided baseline underlying molecular mechanisms of such a dierence information for further studies to elucidate the mechan- may lead to a new treatment strategy for all gliomas. ism of chemosensitivity in gliomas. Unfortunately, the putative tumor suppressor genes at Oncogene (2002) 21, 3961 ± 3968. doi:10.1038/sj.onc. chromosomes 1p and 19q, obvious keys to investigate 1205495 the molecular biologic features of the tumor cells, are yet to be identi®ed despite vigorous investigations. Keywords: oligodendroglioma; oligonucleotide micro- Several attractive candidates on chromosome 1p array; loss of heterozygosity include TP73 (1p36.3) and CDKN2C (1p32), but neither has been shown to be altered in the majority of oligodendrogliomas (Husemann et al., 1999; Mai et Introduction al., 1998). Although 1p loss is also found in many other neoplasms including neuroblastomas, the search Oligodendrogliomas are a major type of gliomas which for the suppressor gene in such neoplasms has not been constitute approximately 5% of all primary brain successful either (Ohira et al., 2000; Schwab et al., 1996). To gain insight into the molecular basis of the biological dierence among oligodendrogliomas, we turned to recently developed oligonucleotide micro- *Correspondence: K Ueki or H Aburatani; array technology. By analysing comprehensive gene E-mail: [email protected] or E-mail: [email protected] Received 18 July 2001; revised 28 February 2002; accepted 18 expressions, several studies have now shown that the March 2002 expression pro®les correlated well with the histology Gene expression profiles of oligodendrogliomas A Mukasa et al 3962 and clinical grades in human neoplasms including test (P50.01), indicating that these two subgroups gliomas (Golub et al., 1999; Huang et al., 2000; indeed have signi®cantly dierent gene expression Watson et al., 2001). Therefore, we performed a pro®les. When Mann ± Whitney test with cut-o P- comparative study of the gene expression pro®les values of 0.05 and 0.01 were used, 288 and 123 genes between the genetic subgroups of oligodendrogliomas were detected as dierentially expressed by the 1p based on the 1p status. status, and those numbers were higher than the expected numbers in permutation test which were 115 and 33 in median, respectively. Of the 209 genes selected by prediction value, more than 90% (192 Results genes) were also included in the 288 genes selected by a P-value of 0.05 by Mann ± Whitney test, indicating Genetic alterations in oligodendroglial tumor samples the consistency of those two methods in selecting Of 40 oligodendroglial tumors we could collect, we dierentially expressed genes. We used the 209 genes selected six cases with 1pLOH (loss of heterozygosity) for further analysis. and ®ve cases without 1pLOH from which we could Clustering analysis was performed to classify all 13 obtain good quality RNA evaluable with the GeneChip samples using Pearson correlation with these extracted system (Aymetrix, Santa Clara, CA, USA). Histolo- 209 genes (Figure 1). The tumor subsets were gical diagnoses and the results of molecular genetic separated well and the normal brain samples were analysis are summarized in Table 1. There were seven clustered into the same group with the tumors with oligodendrogliomas, one oligoastrocytoma and three 1pLOH. Among the ®ve tumors without 1pLOH, anaplastic oligodendrogliomas. In all six tumors with expression pro®les were not markedly dierent be- 1pLOH, all of the informative 1p markers showed tween the tumors with and without TP53 mutation in LOH, indicating that deletion involved the whole arm this clustering analysis. of chromosome 1p (data not shown). Of the six cases with 1pLOH, ®ve cases also had 19qLOH and one case Genes showing higher expression in tumors with 1pLOH was non-informative on examined 19q markers. None of the six tumors with 1pLOH had TP53 mutation, Of the 86 genes selected by P-value, 24 genes whose and three of the ®ve tumors without 1pLOH had TP53 mean average dierence had more than threefold mutation. No case had 10qLOH. dierence between the two groups were listed in Table 2. The average dierences of those genes in normal brain RNA were close to those in tumors with 1pLOH The statistical analysis of genes differentially expressed as expected by the clustering analysis. Based on the by 1p status UniGene on National Center for Biotechnology To select genes that were expressed dierentially by 1p Information (NCBI), 14 of the 24 genes were status, we used prediction value (P-value) in neighbor- predominantly expressed in brain or neural tissues hood analysis, which was recently described as useful (KIAA0985, RGS7, human clone 23695, INA, for extracting genes expressed uniformly high in one KIAA0750, MYT1L, human clone 23560, PTPRN, group and low in the other (Golub et al., 1999). We SLC1A2, HAPIP, SNCB, SNAP25, L1CAM and listed a total of 209 genes that had an absolute P- OLFM1), and were likely to have some function in value of more than one, of which 86 genes showed the nervous system. In the normal brain samples, genes higher expression and 123 genes showed lower that are predominantly expressed in glial cells such as expression in tumors with 1pLOH. These numbers of glial ®brillary acidic protein (GFAP) and myelin basic the genes were signi®cantly higher than expected in protein (MBP) were also well expressed, indicating that random grouping tested by 1000 times permutation these samples contained many glial cells. Table 1 Summary of oligodendroglial tumors used in GeneChip experiments Tumor no. Gender Age Histology 1pLOH 19qLOH 10qLOH TP53 CDKN2A 1 F 47 AOG (+) (+) (7)WtHD 2 F 23 OG (+) (+) (7) Wt Normal 3 M 44 OG (+) (+) (7) Wt Normal 4 M 49 OG (+) (+) (7)WtHD 5 F 22 OA (+) NI (7) Wt Normal 6 F 48 AOG (+) (+) (7) Wt Normal 7 M 44 OG (7)(7)(7) Mutation Normal 8 F 60 OG (7)(7)(7) Mutation Normal 9 F 25 OG (7)(7)(7) Mutation Normal 10 M 67 AOG (7)(7)(7)WtHD 11 F 27 OG (7)(7)(7) Wt Normal OG: oligodendroglioma, OA: oligoastrocytoma, AOG: anaplastic oligodendroglioma, LOH: loss of heterozygosity, NI: non-informative, Wt: wild type, HD: homozygous deletion Oncogene Gene expression profiles of oligodendrogliomas A Mukasa et al 3963 Figure 1 Hierarchical clustering of 11 oligodendroglial tumors and two normal brain samples using the 209 genes selected by P-value. Each column represents a gene and each row represents a sample. Red indicates increased expression, and blue indicates decreased gene expression.Expressionofeachgeneisnormalizedtoitsmedianinthis®gure.Thedendrogramindicatesthedegreeofsimilaritybetweentheir expression pro®les. Normal brain samples were clustered into the same group with the tumors with 1pLOH.