N2M2 (NOA-20) phase I/II trial of molecularly matched targeted therapies plus radiotherapy in patients with newly diagnosed non-MGMT hypermethylated glioblastoma.

Background: Patients with glioblastoma without O6-methylguanine-DNA methyltransferase (MGMT) promoter hypermethylation are unlikely to benefit from alkylating chemotherapy with temozolomide (TMZ). Trials aiming at replacing TMZ with targeted agents in unselected patient populations have failed to demonstrate any improvement of survival. Advances in molecular understanding and diagnostic precision enable identification of key genetic alterations in a timely manner and in principle allow treatments with targeted compounds based on molecular markers. Methods: The NCT Neuro Master Match (N2M2) trial is an open-label, multicenter, phase I/IIa umbrella trial for patients with newly diagnosed isocitrate dehydrogenase (IDH) wildtype glioblastoma without MGMT promoter hypermethylation to show safety, feasibility, and preliminary efficacy of treatment with targeted compounds in addition to standard radiotherapy based on molecular characterization. N2M2 is formally divided into a Discovery and a Treatment part. Discovery includes broad molecular neuropathological diagnostics to detect predefined biomarkers for targeted treatments. Molecular diagnostics and bioinformatic evaluation are performed within 4 weeks, allowing a timely initiation of postoperative treatment. Stratification for Treatment takes place in 5 subtrials, including alectinib, idasanutlin, palbociclib, vismodegib, and temsirolimus as targeted therapies, according to the best matching molecular alteration. Patients without matching alterations are randomized between subtrials without strong biomarkers using atezolizumab and asinercept (APG101) and the standard of care, TMZ. For the phase I parts, a Bayesian criterion is used for continuous monitoring of toxicity. In the phase II trials, progression-free survival at 6 months is used as endpoint for efficacy. Results: Molecular diagnostics and bioinformatic evaluation are performed within 4 weeks, allowing a timely initiation of postoperative treatment. Stratification for Treatment takes place in 5 subtrials, including alectinib, idasanutlin, palbociclib, vismodegib, and temsirolimus as targeted therapies, according to the best matching molecular alteration. Patients without matching alterations are randomized between subtrials without strong biomarkers using atezolizumab and asinercept (APG101) and the standard of care, TMZ. For the phase I parts, a Bayesian criterion is used for continuous monitoring of toxicity. In the phase II trials, progression-free survival at 6 months is used as endpoint for efficacy. Discussion: Molecularly informed trials may provide the basis for the development of predictive biomarkers and help to understand and select patient subgroups who will benefit.

Feasibility of real-time molecular profiling for patients with newly diagnosed glioblastoma without MGMT promoter hypermethylation-the NCT Neuro Master Match (N2M2) pilot study.

Background: O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status is a predictive biomarker in glioblastoma patients. Glioblastoma without hypermethylated MGMT promoter is largely resistant to treatment with temozolomide. These patients are in particular need of new treatment approaches, which are offered by biomarker-driven clinical trials with targeted drugs based on molecular characterization of individual tumors. Methods: In preparation for an upcoming clinical study, a comprehensive molecular profiling approach was undertaken on tissues from 43 glioblastoma patients harboring an unmethylated MGMT promoter at diagnosis. The diagnostic pipeline covered various levels of molecular characteristics, including whole-exome sequencing, low-coverage whole-genome sequencing, RNA sequencing, as well as microarray-based gene expression profiling and DNA methylation arrays. Results: Complex multilayer molecular diagnostics were feasible in this setting with a median turnaround time of 4-5 weeks from surgery to the molecular tumor board. In 35% of cases, potentially relevant therapeutic decisions were derived from the data. Alterations were most frequently found in receptor tyrosine kinases, members of the phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin and mitogen-activated protein kinase pathway as well as cell cycle control and p53 regulation cascades. Individual tumors harbored clonal alterations such as oncogenic fusions of tyrosine kinases which constitute promising targets for targeted therapies. A prioritization algorithm is proposed to allocate patients with multiple targets to the potentially best treatment option. Conclusion: With this feasibility study, a comprehensive molecular profiling approach for patients with newly diagnosed glioblastoma harboring an unmethylated MGMT promoter is presented. Analyses in this pilot cohort serve as a basis for trials based on targetable alterations and on the question of allocation of patients to the best treatment arm.

Proteasome inhibition correlates with intracellular bortezomib concentrations but not with antiproliferative effects after bolus treatment in myeloma cell lines.

Although bortezomib is successfully used against multiple myeloma, the pharmacodynamics of proteasome inhibition and its association with efficacy or resistance is poorly understood. Using ultra performance liquid chromatography coupled to tandem mass spectrometry, site-specific luminogenic substrate assays and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT) assays, effects of bortezomib on cellular drug concentrations, chymotrypsin- , caspase- , and trypsin-like activities, and cytotoxic efficacy were evaluated in eight myeloma cell lines directly after 1 h of exposure and additionally after a 23-h washout phase. Bortezomib accumulated in myeloma cells by up to 100-fold and concentration-dependently inhibited the proteasomal activities with the chymotrypsin-like activity being the most sensitive. Whereas intracellular concentrations correlated with the inhibition of the chymotrypsin- and the caspase-like activities of the proteasome, the cytotoxic efficacy of bortezomib did not correlate with either intracellular concentrations or proteasomal inhibition. However, the ratio of concentrations measured directly after the exposure and after the washout phase (indicating drug disposition) correlated with efficacy, suggesting that the cell's ability to dispose bortezomib at least in part influences bortezomib's cytotoxicity. In conclusion, this data argues against a direct association of intracellular concentration or proteasomal inhibition with cytotoxic efficacy but advocates for an important role of cellular drug disposition. Moreover, this study underlines the pleiotropic mode of action of bortezomib going beyond proteasome inhibition.

Fetal calf sera can distort cell-based luminescent proteasome assays through heat-resistant chymotrypsin-like activity.

Luminescence-based proteasome activity assays use specific substrates that are supposed to be cleaved by cellular proteasome activity leading to luciferase substrates. Usually, control wells containing cell culture medium supplemented with antibiotics and fetal calf serum are used as background. Using the Proteasome-Glo chymotrypsin-like cell-based assay from Promega, we show here that fetal calf sera from different manufacturers contain heat-resistant, bortezomib-inhibitable, chymotrypsin-like activities that can interfere with proteasome activity assays. These data strongly recommend the use of pure phosphate-buffered saline (PBS) or serum-free medium during proteasome activity assays to diminish background luminescence and, thus, to obtain reliable results.