gms | German Medical Science

Objective: Hypermethylation of the promoter of the DNA repair gene O6-methyl-guanine DNA methyltransferase (MGMT) has been linked to prolonged survival in patients with glioblastoma treated with alkylating agents. However, uncertainties exist regarding the intra-tumoural distribution of the MGMT methylation status within the tumour. In the current prospective study stereotactic serial biopsy technique was used to obtain tissue samples from different sites within the glioma. It was aimed to establish and validate methylation specific PCR (MSP) and sequencing analysis (SEQ) for frozen small sized biopsy specimens and to investigate whether heterogeneity exists with regard to the MGMT methylation status.

Methods: Adult patients with a supratentorial untreated (N=20) or secondary malignant astrocytoma (N=5) were considered eligible. Two to four frozen small sized tumour specimens (1mm³) per tumour were taken from different sites within each glioma (maximum distance between the biopsy sites: 14 mm). Validity of MSP and sequencing analysis were checked by blinded re-evalution of ten brain tumour samples (obtained by open tumour resection) with a known MGMT promoter profile by stepwise reduction of the starting DNA to an amount comparable to that obtained from a biopsy specimen (30-60 ng/µl).

Results: One hundred percent concordant results were found in the validation set for both MSP and SEQ. In the study group MSP and SEQ were performed in 69 tissue samples with a success rate of 100% and 80%, respectively. The overall promoter methylation rate in the untreated group was 30% and in the tumour progression group 80% (p=0.1). The histological analysis of additional tissue samples in the direct vicinity of these sites (level+1mm) revealed solid tumour tissue in all cases except one; exactly this patient exhibited a false negative result in the MSP. All other samples of each tumour investigated showed identical results with regard to the methylation status.

Conclusions: Reliable analysis of MGMT promoter methylation is feasible using MSP and SEQ in cryofrozen small sized biopsy specimens. Contamination with necrotic or non-neoplastic tissue must be avoided. The distribution of the promoter methylation status is homogeneous within malignant gliomas. These observations extend the range of prognostically relevant molecular genetic markers that can be applied to stereotactic biopsy specimens.

(authors 1-3 contibuted equally to this study)