Definition of the Prognostic Role of MGMT Promoter Methylation Value by Pyrosequencing in Newly Diagnosed IDH Wild-Type Glioblastoma Patients Treated with Radiochemotherapy: A Large Multicenter Study.

Background. O6-methylguanine (O6-MeG)-DNA methyltransferase (MGMT) methylation status is a predictive factor for alkylating treatment efficacy in glioblastoma patients, but its prognostic role is still unclear. We performed a large, multicenter study to evaluate the association between MGMT methylation value and survival. Methods. We evaluated glioblastoma patients with an assessment of MGMT methylation status by pyrosequencing from nine Italian centers. The inclusion criteria were histological diagnosis of IDH wild-type glioblastoma, Eastern Cooperative Oncology Group Performance Status (ECOG-PS) ≤2, and radio-chemotherapy treatment with temozolomide. The relationship between OS and MGMT was investigated with a time-dependent Receiver Operating Characteristics (ROC) curve and Cox regression models. Results. In total, 591 newly diagnosed glioblastoma patients were analyzed. The median OS was 16.2 months. The ROC analysis suggested a cut-off of 15% for MGMT methylation. The 2-year Overall Survival (OS) was 18.3% and 51.8% for MGMT methylation <15% and ≥15% (p < 0.0001). In the multivariable analysis, MGMT methylation <15% was associated with impaired survival (p < 0.00001). However, we also found a non-linear association between MGMT methylation and OS (p = 0.002): median OS was 14.8 months for MGMT in 0−4%, 18.9 months for MGMT in 4−40%, and 29.9 months for MGMT in 40−100%. Conclusions. Our findings suggested a non-linear relationship between OS and MGMT promoter methylation, which implies a varying magnitude of prognostic effect across values of MGMT promoter methylation by pyrosequencing in newly diagnosed IDH wild-type glioblastoma patients treated with chemoradiotherapy.

Drugs targeting the mitochondrial pore act as cytotoxic and cytostatic agents in temozolomide-resistant glioma cells.

BACKGROUND: High grade gliomas are one of the most difficult cancers to treat and despite surgery, radiotherapy and temozolomide-based chemotherapy, the prognosis of glioma patients is poor. Resistance to temozolomide is the major barrier to effective therapy. Alternative therapeutic approaches have been shown to be ineffective for the treatment of genetically unselected glioma patients. Thus, novel therapies are needed. Mitochondria-directed chemotherapy is an emerging tool to combat cancer, and inner mitochondrial permeability transition (MPT) represents a target for the development of cytotoxic drugs. A number of agents are able to induce MPT and some of them target MPT-pore (MPTP) components that are selectively up-regulated in cancer, making these agents putative cancer cell-specific drugs. OBJECTIVE: The aim of this paper is to report a comprehensive analysis of the effects produced by selected MPT-inducing drugs (Betulinic Acid, Lonidamine, CD437) in a temozolomide-resistant glioblastoma cell line (ADF cells). METHODS: EGFRvIII expression has been assayed by RT-PCR. EGFR amplification and PTEN deletion have been assayed by differential-PCR. Drugs effect on cell viability has been tested by crystal violet assay. MPT has been tested by JC1 staining. Drug cytostatic effect has been tested by mitotic index analysis. Drug cytotoxic effect has been tested by calcein AM staining. Apoptosis has been assayed by Hoechst incorporation and Annexine V binding assay. Authophagy has been tested by acridine orange staining. RESULTS: We performed a molecular and genetic characterization of ADF cells and demonstrated that this line does not express the EGFRvIII and does not show EGFR amplification. ADF cells do not show PTEN mutation but differential PCR data indicate a hemizygous deletion of PTEN gene. We analyzed the response of ADF cells to Betulinic Acid, Lonidamine, and CD437. Our data demonstrate that MPT-inducing agents produce concentration-dependent cytostatic and cytotoxic effects in parallel with MPT induction triggered through MPTP. CD437, Lonidamine and Betulinic acid trigger apoptosis as principal death modality. CONCLUSION: The obtained data suggest that these pharmacological agents could be selected as adjuvant drugs for the treatment of high grade astrocytomas that resist conventional therapies or that do not show any peculiar genetic alteration that can be targeted by specific drugs.

Identification of two novel mutations and of a novel critical region in the KRIT1 gene.

Cerebral cavernous malformations (CCMs) represent a common autosomal dominant disorder that predisposes patients to hemorrhagic strokes and focal neurological signs. Mutations in three genes (KRIT1, MGC4607, and PDCD10) have been associated with CCMs. We investigated the role of two new mutations in the KRIT1 gene in two Italian families affected by CCMs. Whole blood DNA was extracted and the mutations were detected after polymerase chain reaction (PCR), denaturing high-performance liquid chromatography screening, and sequencing of the coding regions of the three CCMs-associated genes. Total RNA was extracted, and the KRIT1 cDNA was sequenced and subsequently subjected to real-time quantitative PCR in order to examine the translational outcome of each genomic mutation. A novel splicing acceptor site deletion of the exon 14 in one family and an intronic nucleotide change close to the exon 19 in the other one were identified, both in the KRIT1 gene. These mutations were proven to alter the correct splicing mechanism, resulting, respectively, in a truncated protein of 432 amino acids and in a protein lacking an internal segment. We report two novel cases of splicing affecting genomic variants, suggesting a careful reanalysis of previously identified splice site variations in KRIT1 to look for their possible causative roles of similar missplicing events and their consequent involvement in the pathogenesis of CCMs. Moreover, our genotype-phenotype functional correlation suggests that the C-terminal portion of the KRIT1 protein is likely to contain a short, previously unrecognized segment necessary for its activity.

Supratentorial cerebral cavernous malformations: clinical, surgical, and genetic involvement.

OBJECT: Although there is general agreement on the methods of treatment for symptomatic supratentorial cerebral cavernous malformations (CMs) located in noneloquent areas, some controversy exists regarding the management of cerebral CMs that are asymptomatic and/or located in eloquent or deep areas. Moreover, recent advances in genetic findings could influence both standard clinical management and the follow-up strategy in affected individuals. Thus, the objective of this study was to develop, based on the authors' experience and a literature review, a management algorithm to deal with supratentorial cerebral CMs. METHODS: The authors retrospectively reviewed the clinical data related to 118 patients who underwent surgery for symptomatic supratentorial cerebral CMs at their institution. Twenty-eight of 118 patients harbored multiple lesions, and nine of these 28 patients had a clinically positive familial history. Genetic investigations were performed in 89 patients (75%). CONCLUSIONS: Surgery for supratentorial cerebral CMs in noneloquent locations is safe and curative. In cerebral CMs located in deep and eloquent areas and with symptoms including progressive neurological deficits, evidence of hemorrhage, and uncontrolled seizures, surgical treatment according to an integrated plan based on frameless stereotactic guidance and functional magnetic resonance imaging is recommended and results in acceptably low morbidity. The data support the need for long-term imaging follow up in all patients, careful preoperative vascular studies to detect associated venous anomalies, and the importance of genetic mutational analysis. The DNA screening protocol will change the care of family members of patients with familial forms of cerebral CMs, because affected asymptomatic family members may benefit by early detection of lesions. At the same time, the exclusion of family members who are not carriers of the mutation as members of the population at risk reduces the economic and psychological burden of clinical and instrumental monitoring.