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Background: Methylation of MGMT promoter has been identified as a favourable predictive factor of benefit from XRT/TMZ → TMZ. Patients with non-resectable glioblastoma (GBM) generally exhibit a poor prognosis, even after XRT/TMZ. Few data are available concerning the predictive value of MGMT promoter methylation in this population., Methods: This is an observational retrospective study in patients with malignant brain glioma, treated between June 2008 and October 2011 and followed up until April 2012 at the Neurosurgery-Neurotraumatology Unit of the University Hospital of Parma and at the Neurosurgery Unit of IRCCS "ASMN" of Reggio Emilia, Italy. The medical records of an overall number of 174 patients with a newly diagnosed GBM were reviewed. Volumetry analysis of the lesions was performed on pre- and post-operative neuroimaging by Voxar 3D Ebit AET software. The genetic characterization was performed on paraffin embedded tissue from all resected tumours. Isolation of nucleic acids, bisulfite modification of DNA, methylation-specific PCR and sequencing analyses were done mainly on fresh tissue from biopsy withdrawals. Within 3-4 weeks after either biopsy or surgery, patients were assigned to receive XRT/TMZ→TMZ: treatment included XRT (60 Gy in 30 fractions)/TMZ (daily dose of 75 mg/m(2))/TMZ (150-200mg/m(2) per day for 5 days of every 28-day cycle)., Results and Discussion: A total of 55 consecutive patients (23 men, 22 women) fulfilled inclusion criteria consisting of age over 18 years, supratentorial histologically proven primary malignant glioma, complete determination of the MGMT methylation status, no prior history of surgery, XRT and/or chemotherapy, adequate clinical and radiological follow-up no lesser than 6 months. Twenty-three patients underwent neuronavigation needle biopsy (B Group) and thirty-two patients were operated with craniotomy for tumour resection (R Group). The pre-operative mean age was similar between groups (61.7 ± 10.7 vs 60.3 ± 11.8 years in the B and R groups respectively; p>0.05). The B groups showed a slightly lower KPS than the R Group (82.1 ± 17.3 vs 90.3 ± 14.1 respectively; p>0.05). The mean pre-operative volume of the tumour did not differ between groups (46.2 ± 40.2 cm(3) vs 44.1 ± 33.2 cm(3) in the R Group and B Group respectively; p>0.05). The MGMT promoter was methylated in 12 patients (51.2%) of B Group and in 17 patients (53.1%) of R Group. XRT/TMZ → TMZ was accomplished in 11 patients (47.8%) of B Group and in 24 patients (75%) of R Group; in 24/29 methylated patients (82.8%) and in 11/26 unmethylated patients (42.3%). Survival analysis of methylated vs unmethylated tumours was statistically significant (Log Rank Mantel Cox: 0.019 in B Group and 0.023 in R Group). In B Group the mean overall survival (OS) of methylated patients was 11.4 months (IC 95% 6.5-16.4) vs 4.8 months (95% IC, 2.6-7.0) of unmethylated patients. In R Group the mean OS was 21.7 months (95% IC, 16.9-26.6) for methylated patients and 14.0 months (95% IC, 8.5-19.4) for unmethylated patients. At the multivariate Cox regression analysis conducted on the total population (55 patients), XRT and TMZ were found to be predictive of OS. In the R Group, KPS, XRT and TMZ correlated with a better outcome. In the B Group, XRT and MGMT promoter methylation were favourably related with OS., Conclusion: MGMT promoter unmethylation has a predominant unfavourable impact on clinical outcomes even in the subpopulation of patients with non-resectable GBM. The unmethylated MGMT promoter status could be considered the main predictor of poor prognosis in biopsied GBM, due to the greater probability of patients not having benefits from adjuvant therapies and not being able to accomplish XRT/TMZ → TMZ. The frameless neuronavigation biopsy technique is safe and effective for predictive evaluation and could help in treatment decision making., (Copyright © 2015 Elsevier B.V. All rights reserved.)

It is well known that primary and secondary glioblastomas are histologically largely indistinguishable. Therefore, the detection of IDH1 mutations or the status of the MGMT promoter on a simple bioptic sample could be one of major diagnostic and prognostic importance for glial patients that complements clinical criteria for distinguishing secondary from primary glioblastomas and to predict a more favourable prognosis. Currently, biopsy is the method of choice to obtain tissue from intracranial lesions with uncertain neurodiagnostic findings or in deep locations, with a minimal invasive approach. The needle biopsy with frameless neuronavigation could provide a sampling with elevated diagnostic yield and high concentration of DNA, due to the "image-guided" computer assisted technique of needle insertion through the most neurodiagnostic representative tumor area. The freezing of fresh tumor tissue at biopsy could greatly improve the success of DNA extraction. The concentration of the DNA samples can also improved from a withdrawal in an area with high probability of neoplastic cells. The present study reports the results of 17 patients who had undergone frameless image-guided intracranial needle biopsy from April 2008 until July 2010 at Neurosurgery Unit of the "Arcispedale Santa Maria Nuova" of Reggio Emilia. For these patients the molecular determination of MGMT promoter was assessed with the Nested-Methylation Specific-Polymerase Chain Reaction and the screening of mutations in IDH1 e IDH2 genes was performer by polymerase chain reaction (PCR) and direct sequencing on fresh or cryopreserved needle bioptic tissue.

Formalin fixation under conditions that adversely affected the quality of the DNA, or indeterminant assay, or extensive tumor necrosis can compromise the genetic analysis of a brain bioptic sample. The success of DNA extraction and Methyl Guanine Methyl Transferase (MGMT) promoter methylation testing could be improved by freezing of fresh tumor tissue at the moment of biopsy. To ensure an increased concentration of the DNA samples the withdrawal should be performed in an area with high probability of neoplastic cells. From May 2007 to January 2011 fifty-two frameless neuronavigation brain needle biopsy were performed at the Neurosurgery Unit of the "Arcispedale Santa Maria Nuova" City Hospital of Reggio Emilia. The "image-guided" neuronavigated protocol sampling provided withdrawal specimens highly correlated with neuroimaging characteristics of the lesions. In this study the Authors report the genetic analysis on 24 cases of freezing fresh tissue from brain needle bioptic sample starting from July 2008. The molecular determination of MGMT promoter was assessed with the Nested-Methylation Specific-Polymerase Chain Reaction on fresh or cryopreserved needle bioptic tissue. The genetic characterization was feasible in all the bioptic samples. The MGMT promoter was methylated in eleven patients, including a brain infection. The diagnostic yield of brain biopsy could be increased by the neuronavigated trajectories and the intraoperative frozen sections. In the future the availability of the molecular-genetic characterization of a brain tumor before open surgery will provide important information for the optimal treatment. The MGMT promoter status analysis on needle bioptic fresh tissue could be available also for that patient not eligible for surgical remotion of the tumor.

Background: After a brain biopsy, the genetic analysis can fail because of insufficient material, extensive tumor necrosis, and formalin fixation under conditions that adversely affected the quality of the DNA or because the assay result was indeterminant. The freezing of fresh tumor tissue at surgery could greatly improve the success of DNA extraction and methyl guanine methyl transferase (MGMT) promoter methylation testing. The concentration of the DNA samples can also be improved from a withdrawal in an area with a high probability of neoplastic cells., Methods: The present study reports the results of ten frameless image-guided intracranial needle biopsies from April 2008 until February 2009, among a total of 28 frameless neuronavigation brain biopsy performed from May 2007 to February 2009. The protocol sampling provided withdrawal specimens correlated with neuroimaging characteristics of the lesions. The molecular determination of MGMT promoter was assessed with the nested methylation-specific polymerase chain reaction on fresh or cryopreserved needle bioptic tissue., Results: The genetic characterization was feasible in all the bioptic samples. The MGMT promoter was methylated in six patients, including a brain infection. The image-guided trajectory of the biopsy and the intraoperative frozen section increased the diagnostic yield., Conclusions: To the best of the authors' knowledge, this is the first report with the MGMT promoter status analysis on needle bioptic fresh tissue. In the future, the availability of the molecular genetic characterization of a brain tumor before open surgery will provide important information for the optimal treatment.