Correlation of commercially available quantitative MGMT (O-6-methylguanine-DNA methyltransferase) promoter methylation scores and GBM patient survival.

BACKGROUND: Between 2011 and 2016, O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation testing at University of California Los Angeles (UCLA) was performed through LabCorp, using a threshold of 2 to distinguish MGMT methylated from unmethylated tumors. In this study, we sought to determine whether the magnitude of the methylation score correlated with outcome. METHODS: We identified 165 newly diagnosed glioblastoma (GBM) isocitrate dehydrogenase (IDH) wild-type and temozolomide-treated upfront patients at UCLA and Kaiser Permanente Los Angeles with LabCorp-derived quantitative MGMT scores obtained on pretreatment tissue samples. Using LabCorp's threshold, we found 102 unmethylated and 63 methylated patients. We then further substratified each group based on the magnitude of the score, and performed Kaplan-Meier and Cox regression analyses of overall survival (OS) and progression-free survival (PFS). RESULTS: We validated that the standard LabCorp threshold of 2 could separate our cohort by survival, showing longer OS and PFS for MGMT methylated patients vs unmethylated patients. Cox regression analysis confirmed that MGMT (<1) patients had worse outcome, with OS and PFS hazard ratios of 2.375 (P = .053) and 2.463 (P = .023), respectively, when compared to the MGMT (1-1.99) patients. Contrary to our expectation, when we substratified the ≥2 (methylated) group, we did not find a dose-dependent relationship between the magnitude of MGMT methylation and improved survival. CONCLUSIONS: The MGMT unmethylated group contains a partially methylated group (greater than 1) that shares survival benefits similar to the methylated group. However, we did not demonstrate an association of very high methylation scores with increased survival. These findings will require validation in additional independent clinical data sets.

SPINT2 is hypermethylated in both IDH1 mutated and wild-type glioblastomas, and exerts tumor suppression via reduction of c-Met activation.

PURPOSE: Both IDH1-mutated and wild-type gliomas abundantly display aberrant CpG island hypermethylation. However, the potential role of hypermethylation in promoting gliomas, especially the most aggressive form, glioblastoma (GBM), remains poorly understood. METHODS: We analyzed RRBS-generated methylation profiles for 11 IDH1WT gliomas (including 7 GBMs), 24 IDH1MUT gliomas (including 6 GBMs), and 5 normal brain samples and employed TCGA GBM methylation profiles as a validation set. Upon classification of differentially methylated CpG islands by IDH1 status, we used integrated analysis of methylation and gene expression to identify SPINT2 as a top cancer related gene. To explore functional consequences of SPINT2 methylation in GBM, we validated SPINT2 methylation status using targeted bisulfite sequencing in a large cohort of GBM samples. We assessed DNA methylation-mediated SPINT2 gene regulation using 5-aza-2'-deoxycytidine treatment, DNMT1 knockdown and luciferase reporter assays. We conducted functional analyses of SPINT2 in GBM cell lines in vitro and in vivo. RESULTS: We identified SPINT2 as a candidate tumor-suppressor gene within a group of CpG islands (designated GT-CMG) that are hypermethylated in both IDH1MUT and IDH1WT gliomas but not in normal brain. We established that SPINT2 downregulation results from promoter hypermethylation, and that restoration of SPINT2 expression reduces c-Met activation and tumorigenic properties of GBM cells. CONCLUSIONS: We defined a previously under-recognized group of coordinately methylated CpG islands common to both IDH1WT and IDH1MUT gliomas (GT-CMG). Within GT-CMG, we identified SPINT2 as a top cancer-related candidate and demonstrated that SPINT2 suppressed GBM via down-regulation of c-Met activation.

Phase 2 Study of Bortezomib Combined With Temozolomide and Regional Radiation Therapy for Upfront Treatment of Patients With Newly Diagnosed Glioblastoma Multiforme: Safety and Efficacy Assessment.

PURPOSE: To assess the safety and efficacy of upfront treatment using bortezomib combined with standard radiation therapy (RT) and temozolomide (TMZ), followed by adjuvant bortezomib and TMZ for ≤24 cycles, in patients with newly diagnosed glioblastoma multiforme (GBM). METHODS AND MATERIALS: Twenty-four patients with newly diagnosed GBM were enrolled. The patients received standard external beam regional RT with concurrent TMZ beginning 3 to 6 weeks after surgery, followed by adjuvant TMZ and bortezomib for ≤24 cycles or until tumor progression. During RT, bortezomib was given at 1.3 mg/m2 on days 1, 4, 8, 11, 29, 32, 36, and 39. After RT, bortezomib was given at 1.3 mg/m2 on days 1, 4, 8, and 11 every 4 weeks. RESULTS: No unexpected adverse events occurred from the addition of bortezomib. The efficacy analysis showed a median progression-free survival (PFS) of 6.2 months (95% confidence interval [CI] 3.7-8.8), with promising PFS rates at ≥18 months compared with historical norms (25.0% at 18 and 24 months; 16.7% at 30 months). In terms of overall survival (OS), the median OS was 19.1 months (95% CI 6.7-31.4), with improved OS rates at ≥12 months (87.5% at 12, 50.0% at 24, 34.1% at 36-60 months) compared with the historical norms. The median PFS was 24.7 months (95% CI 8.5-41.0) in 10 MGMT methylated and 5.1 months (95% CI 3.9-6.2) in 13 unmethylated patients. The estimated median OS was 61 months (95% CI upper bound not reached) in the methylated and 16.4 months (95% CI 11.8-21.0) in the unmethylated patients. CONCLUSIONS: The addition of bortezomib to current standard radiochemotherapy in newly diagnosed GBM patients was tolerable. The PFS and OS rates appeared promising, with more benefit to MGMT methylated patients. Further clinical investigation is warranted in a larger cohort of patients.

Report of safety of pulse dosing of lapatinib with temozolomide and radiation therapy for newly-diagnosed glioblastoma in a pilot phase II study.

Epidermal growth factor receptor (EGFR) mutations are commonly observed in Glioblastoma (GBM) and have long posed as a target for new therapies. Trials involving erlotinib have shown mixed results, likely owing to a mechanism of the mutation that may instead favor other EGFR inhibitors, such as lapatinib. We aimed to determine whether or not pulse high-dose lapatinib was a safe and tolerable regimen in addition to standard therapy. We recruited adult patients with newly-diagnosed GBM who had Karnofsky Performance Status ≥60, normal baseline hematological, hepatic, and renal function tests, and no prior history of radiation or treatment with EGFR inhibitor. Lapatinib was administered at 2500 mg twice daily for two consecutive days per week on a weekly basis throughout concomitant and adjuvant standard therapy. The primary endpoints were tolerability and safety. 12 patients were enrolled in this study over 2 years. Of the non-hematological adverse events, there were 2 grade 3 events, fatigue and post-radiation cystic brain necrosis. The most common adverse events in general were fatigue, rashes, and diarrhea. Of the hematological adverse events, there were 13 grade 3 events, all of which were due to lymphopenia and affected 6 of 12 patients. Pulse high-dose lapatinib in addition to standard therapy for newly-diagnosed GBM is a tolerable and safe regimen, but higher rates of lymphopenia should be noted. However, further investigations will be required to evaluate the efficacy of this combination for the treatments of GBM. Trial registration ClinicalTrials.gov Identifier: NCT01591577.

Clinical aggressiveness of malignant gliomas is linked to augmented metabolism of amino acids.

Glutamine, glutamate, asparagine, and aspartate are involved in an enzyme-network that controls nitrogen metabolism. Branched-chain-amino-acid aminotransferase-1 (BCAT1) promotes proliferation of gliomas with wild-type IDH1 and is closely connected to the network. We hypothesized that metabolism of asparagine, glutamine, and branched-chain-amino-acids is associated with progression of malignant gliomas. Gene expression for asparagine synthetase (ASNS), glutaminase (GLS), and BCAT1 were analyzed in 164 gliomas from 156 patients [33-anaplastic gliomas (AG) and 131-glioblastomas (GBM), 64 of which were recurrent GBMs]. ASNS and GLS were twofold higher in GBMs versus AGs. BCAT1 was also higher in GBMs. ASNS expression was twofold higher in recurrent versus new GBMs. Five patients had serial samples: 4-showed higher ASNS and 3-higher GLS at recurrence. We analyzed grade and treatment in 4 groups: (1) low ASNS, GLS, and BCAT1 (n = 96); (2) low ASNS and GLS, but high BCAT1 (n = 26); (3) high ASNS or GLS, but low BCAT1 (n = 25); and (4) high ASNS or GLS and high BCAT1 (n = 17). Ninety-one  % of patients (29/32) with grade-III lesions were in group 1. In contrast, 95 % of patients (62/65) in groups 2-4 had GBMs. Treatment was similar in 4 groups (radiotherapy-80 %; temozolomide-30 %; other chemotherapy-50 %). High expression of ASNS, GLS, and BCAT1 were each associated with poor survival in the entire group. The combination of lower ASNS, GLS, and BCAT1 levels correlated with better survival for newly diagnosed GBMs (66 patients; P = 0.0039). Only tumors with lower enzymes showed improved outcome with temozolomide. IDH1(WT) gliomas had higher expression of these genes. Manipulation of amino acid metabolism in malignant gliomas may be further studied for therapeutics development.

PTEN dosage is essential for neurofibroma development and malignant transformation.

Patients with neurofibromatosis type 1 (NF1) carry approximately a 10% lifetime risk of developing a malignant peripheral nerve sheath tumor (MPNST). Although the molecular mechanisms underlying NF1 to MPNST malignant transformation remain unclear, alterations of both the RAS/RAF/MAPK and PI3K/AKT/mTOR signaling pathways have been implicated. In a series of genetically engineered murine models, we perturbed RAS/RAF/MAPK or/and PTEN/PI3K/AKT pathway, individually or simultaneously, via conditional activation of K-ras oncogene or deletion of Nf1 or Pten tumor suppressor genes. Only K-Ras activation in combination with a single Pten allele deletion led to 100% penetrable development of NF lesions and subsequent progression to MPNST. Importantly, loss or decrease in PTEN expression was found in all murine MPNSTs and a majority of human NF1-associated MPNST lesions, suggesting that PTEN dosage and its controlled signaling pathways are critical for transformation of NFs to MPNST. Using noninvasive in vivo PET-CT imaging, we demonstrated that FDG can be used to identify the malignant transformation in both murine and human MPNSTs. Our data suggest that combined inhibition of RAS/RAF/MAPK and PTEN/PI3K/AKT pathways may be beneficial for patients with MPNST.