Hypermethylation as a Potential Prognostic Factor and a Clue to a Better Understanding of the Molecular Pathogenesis of Medulloblastoma - Results of a Genomewide Methylation Scan

Abstract.

Background: The molecular mechanisms controlling initiation and progression of medulloblastomas are largely unclear. Changes in DNA methylation of promoter regions have been shown to disturb the expression of growth regulatory genes. Patients and Methods: We evaluated DNA methylation patterns in 17 medulloblastomas, 5 stPNETs and 5 medulloblastoma cell lines using Restriction Landmark Genomic Scanning (RLGS), a method displaying up to 2.000 potential gene loci in a single gene. To test whether previously characterized tumor suppressor genes are affected by hypermethylation we performed MS-PCR for p15^INK4B, p16^INK4A, VHL, TP53 and E-cadherin. Results: The analysis of RLGS profiles from tumors revealed an abundance of hypermethylation in primary tumors and cell lines. Extrapolated to the human genome with its ∼ 36,000 genes a total of 420 loci become hypermethylated in the tumor genomes. The previously characterized medulloblastoma breakpoint cluster in 17p11.2 appears to be a hotspot for aberrant methylation. Cox regression analysis of survival data identified seven CpG islands for which hypermethylation is suggestive of a poor prognosis. MS-PCR analysis of known genes demonstrated hypermethylation of p16^INK4A in a limited number of tumors. The pattern of DNA hypermethylation was similar in medulloblastomas and stPNETs. However, some CpG islands were shown to be specific for a tumor type, while others were shared targets. Conclusions: Hypermethylation is a common abnormality in primary medulloblastomas and supratentorial PNETs. Several hundreds of CpG islands are potential targets for methylation in medulloblastomas including the breakpoint cluster in 17p11.2. The methylation status of certain gene sequences appears to be associated with the clinical outcome. Promoter hypermethylation has an outstanding potential as a marker for the identification of novel tumor suppressors as well as diagnostic and therapeutic targets in medulloblastomas.

Hintergrund: Die zu einem Medulloblastom führenden molekularen Mechanismen sind weitgehend unklar. Veränderungen des normalen Methylierungsmusters regulatorischer Promoterregionen ist ein Mechanismus, der, ohne die Basenpaarsequenz der DNA zu verändern, Tumor-Suppressor-Gene inaktivieren kann. Patienten und Methoden: Wir untersuchten die Bedeutung von DNA-Methylierung in 17 Medulloblastomen und 5 stPNETs mittels einer Screening-Methode, dem Restriction Landmark Genomic Scanning (RLGS). RLGS kann in einem einzelnen Gen bis zu 2.000 Genloci auflösen. Vormals charakterisierte Zielgene für aberrante Methylierung in Tumoren Erwachsener (p15^INK4B , p16^INK4A , VHL, TP53 und E-cadherin) wurden durch Methylierungsspezifische PCR (MS-PCR) analysiert. Ergebnisse: Auf die ca. 36 000 Gene des menschlichen Genoms hochgerechnet fanden sich in Medulloblastomen bis zu 420 hypermethylierte Loci. Mehrere von diesen konnten dem Medulloblastombruchpunkt auf 17p11.2 zugeordnet werden. Regressionsanalysen deuten darauf hin, dass die Hypermethylierung von sieben der untersuchten Genloci mit einer ungünstigen Prognose einhergeht. MS-PCR Analysen zeigten nur bei wenigen Medulloblastomen Hypermethylierung des Gens p16^INK4A . Es fanden sich CpG islands, die bevorzugt in Medulloblastomen oder stPNETs hypermethyliert waren. Schlussfolgerung: Hypermethylierung tritt in Medulloblastomen und anderen PNETs des ZNS häufig auf. Mehrere hundert CpG islands sind von Hypermethylierung in Medulloblastomen betroffen, so auch der chromosomale Bruchpunkt auf Chromosom 17p11.2. Der Methylierungsstatus bestimmter Loci ist mit der klinischen Prognose assoziiert. Methylierungsanalysen können sowohl prognostische Marker aufdecken als auch neue therapeutische Zielgene offenlegen.

Key words

Medulloblastoma - primitive neuroectodermal tumor - hypermethylation - prognosis

Schlüsselwörter

Medulloblastom - primitive neuroectodermale Tumoren - Hypermethylierung - Prognose

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