To identify novel genes involved in glioma progression we performed suppression subtractive hybridization combined with cDNA array analysis on 4 patients with primary low-grade gliomas of World Health Organization (WHO) grade II that recurred as secondary glioblastomas (WHO grade IV). Eight genes showing differential expression between primary and recurrent tumors in 3 of the 4 patients were selected for further analysis using realtime reverse transcription-PCR on a series of 10 pairs of primary low-grade and recurrent high-grade gliomas as well as 42 astrocytic gliomas of different WHO grades. These analyses revealed that 5 genes, i.e., AMOG (ATP1B2, 17p13.1), APOD (3q26.2-qter), DMXL1 (5q23.1) DRR1 (TU3A, 3p14.2) and PSD3 (KIAA09428/ HCA67/EFA6R, 8p22), were expressed at significantly lower levels in secondary glioblastomas as compared to diffuse astrocytomas of WHO grade II. In addition, AMOG, DRR1 and PSD3 transcript levels were significantly lower in primary glioblastomas than in diffuse astrocytomas. Treatment of glioma cell lines with 5-aza-2 0 -deoxycytidine and trichostatin A resulted in increased expression of AMOG and APOD transcripts. Sequencing of sodium bisulfitemodified DNA demonstrated AMOG promoter hypermethylation in the glioma cell lines and 1 primary anaplastic astrocytoma with low AMOG expression. Taken together, we identified interesting novel candidate genes that likely contribute to glioma progression and provide first evidence for a role of epigenetic silencing of AMOG in malignant glioma cells. ' 2006 Wiley-Liss, Inc.Key words: adhesion molecule on glia; astrocytoma; glioblastoma; suppression subtractive hybridization; tumor progression Diffuse astrocytomas of World Health Organization (WHO) grade II are characterized by an intrinsic tendency to local recurrence and spontaneous progression to high grade anaplastic astrocytomas (WHO grade III) or secondary glioblastomas (WHO grade IV).1 The molecular mechanisms underlying this malignant progression are still poorly understood. Diffuse astrocytomas of WHO grade II commonly carry TP53 mutations, show loss of heterozygosity (LOH) on 17p and have gained 1 copy of chromosome 7 or 7q.2,3 TP53 mutations and gains on chromosome 7 are similarly frequent in anaplastic astrocytomas of WHO grade III but these tumors often carry additional alterations, such as deletions on chromosomes 6, 9p, 11p, 19q and 22q. Furthermore, a subset of anaplastic astrocytomas shows genetic alterations of the cell cycle regulatory genes CDKN2A, CDKN2B, CDK4 or RB1.
2,3Glioblastomas, the most common and most malignant astrocytic gliomas, are characterized by complex genomic aberrations causing the inactivation of various tumor suppressor genes as well as the activation of different proto-oncogenes.2,3 According to the patient's clinical history, 2 types of glioblastomas can be distinguished. More common are primary glioblastomas that arise de novo with a short clinical history and without a preexisting glioma of lower malignancy grade. Less common are secondary glioblas...