Neurosurgery Bern University Hospital, Bern, Switzerland.
Background: MGMT promoter hypermethylation may predict the efficacy of alkylating agents such as temozolomide (TMZ) in Glioblastoma (GBM). An additional mechanism that may inactivate MGMT protein expression is represented by miRNA, small RNA molecules that interact with MGMT transcript leading to MGMT silencing. Data concerning the role of miRNA in TMZ efficacy prediction or in prognostication are very preliminary and limited to cellular models or small series.
Aim: To investigate in a large cohort of GBM from patients treated with TMZ-based chemotherapy, the expression of several miRNA molecules (miR181c, miR181d, miR21, miR195, miR196b and miR648), and to correlate them with MGMT status and clinical response.
Materials and Methods: We collected a total of 120 formalin-fixed paraffin-embedded tumors from patients who were diagnosed with primary GBM in Varese and Locarno Institutes from 2006 to 2012. All patients underwent surgical excision followed by radiotherapy and TMZ (Stupp protocol). GBM were tested for MGMT methylation with pyrosequencing (MGMTplus, Diatech Pharmacogenetics) and for MGMT Immunohistochemistry (clone MT3.1, Millipore). Total RNA was extracted using RecoverAll (Thermofisher), miRNA were amplified with TaqMan MicroRNA assays (Thermofisher) using RNU6B as endogenous control. As calibrators, we selected 12 normal brain samples from patients with cerebral arteriovenous malformations. Relative miRNA expression was calculated with DDCt method.
Results: Here we present preliminary results on 58 samples from 54 patients, 4 of which were post-treatment samples. MGMT methylation was observed in 18/48 samples (37.5%) while MGMT protein absence (<1% positivity) in 30/51 samples (59%), showing a good concordance (p<0.01). MGMT protein loss, but not MGMT methylation, was correlated with a longer overall survival (p=0.008). miRNA expression analysis showed a strong overexpression of miR21 (fold change 10X, p<0.001) and miR196b (68X, p=0.02) in GBM samples. A moderate overexpression (2.2X) was observed for miR195 (p<0.007). The remaining miRNA expressions were comparable between GBM and normal samples. Correlation between miRNA expression, MGMT protein expression and methylation status revealed no significant associations. Interestingly, all the 4 post-treatment samples showed a strong down-regulation of all the investigated miRNAs with respect to the levels of the pre-treatment tumor.
Conclusions: MGMT protein expression has significant impact on survival of GBM patients. Four investigated miRNA showed overexpression in GBM. An integrated analysis of miRNA expression, MGMT status and clinical response will be performed in order to define the prognostic/predictive significance of miRNA expression. miRNA downregulation in post-treatment samples suggests that chemo-radiation may alter miRNA status and warrants further investigations.
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