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The aim of the present study was to elucidate genetic alterations that are critically involved in astrocytoma progression. We characterized 27 World Health Organization grade II fibrillary astrocytomas which later underwent recurrence or progression, paying specific attention to the CpG island methylation status of critical growth regulatory genes. p14ARF and O6-methylguanine-DNA methyltransferase (MGMT) hypermethylation represented frequent events (26% and 63%, respectively), which were mutually exclusive except in one case, with alternate or simultaneous methylation of these two genes occurring in 85% of our tumor series. Seventeen tumors (63%) contained TP53 mutations, which were closely related to the presence of MGMT methylation. Methylation of the p21Waf1/Cip1, p27Kip1 and p73 genes and homozygous deletion of the p16INK4a, p15INK4b and p14ARF genes were not detected in any of the primary low-grade tumors. The presence of p14ARF methylation at first biopsy was associated with shorter patient survival, whereas the presence of MGMT methylation carried a better clinical outcome after salvage therapy. Examination of 20 cases whose histological data for recurrent tumors were available revealed that malignant progression occurred in all of the tumors with p14ARF methylation but less frequently (50%) in the lesions with MGMT methylation. On analysis of their respective recurrent tumors, five of six patients whose primary low-grade tumors carried p14ARF methylation exhibited homozygous co-deletions of the p14ARF, p15INK4b and p16INK4a genes, which were restricted to glioblastoma as the most malignant end point. Our findings suggest that p14ARF hypermethylation and MGMT hypermethylation constitute distinct molecular pathways of astrocytoma progression, which could differ in biological behavior and clinical outcome.