Promoter hypermethylation of the RB1 gene in glioblastomas

Loss of expression of the retinoblastoma gene (RB1) has been shown to occur in up to 25% of glioblastomas (WHO Grade IV). To elucidate the underlying mechanism, we assessed RBI promoter hypermethylation using methylation-spec ific polymerase chain reaction and RB1 expression by immunohistochemistry i n 35 primary (de novo) glioblastomas and in 21 secondary glioblastomas that had progressed from low-grade diffuse astrocytoma (WHO Grade II) or anapla stic astrocytoma (WHO Grade III). Promoter hypermethylation was significant ly more frequent in secondary (9 of 21, 43%) than in primary glioblastomas (5 of 35, 14%; p = 0.0258). There was a clear correlation between loss of R B1 expression and promoter hypermethylation. In the majority of glioblastom as with loss of RB1 expression, there was promoter hypermethylation (11 of 13, 85%), whereas 93% of tumors with RB1 expression had a normal RB1 gene s tatus (P < 0.0001). In three glioblastomas, areas with and without RB1 expr ession were microdissected; promoter hypermethylation was detected only in areas lacking RB1 expression. In patients with multiple biopsies, methylati on of the RB1 promoter was not detectable in the less malignant precursor l esions, ie, low-grade diffuse and anaplastic astrocytoma. These results ind icate that promoter hypermethylation is a late event during astrocytoma pro gression and is the major mechanism underlying loss of RB1 expression in gl ioblastomas.