Synergistic growth inhibition mediated by dual PI3K/mTOR pathway targeting and genetic or direct pharmacological AKT inhibition in human glioblastoma models.

Molecular genetic aberrations in the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway are common in human cancers including glioblastoma, yet, novel therapeutic approaches targeting this pathway in glioblastoma have not been successful. We hypothesized that molecular profiling in combination with in vitro drug sensitivity testing allows to identify signatures associated with sensitivity or resistance to PI3K/mTOR pathway inhibition. We analyzed the molecular mechanisms determining sensitivity to PI3K/mTOR inhibition using gene silencing or pharmacological target inhibition and proliferation, clonogenicity, or spherogenicity as readouts, in human long-term glioma cell (LTC) lines and glioma-initiating cells (GIC). Cultured glioma cells were universally sensitive to growth inhibition induced by PQR309, a novel, dual pan-PI3K/mTOR antagonist. Cells exhibited profound growth arrest, but little apoptotic or necrotic cell death as confirmed by electron microscopy; yet, there was evidence of senescence. Cell lines with high basal levels of phosphorylated (active) AKT, low levels of phosphorylated (inactive) protein translation repressor eukaryotic initiation factor (eIF) 4E-binding protein 1 (p4E-BP1), and high levels of Ser9-phosphorylated (inactive) glycogen synthase kinase 3 beta (pGSK3ß) were more sensitive to PQR309. Accordingly, the activity of PQR309 was synergistically enhanced by AKT gene silencing or direct pharmacological AKT inhibition. In vivo studies confirmed the anti-glioma activity of PQR309 alone or in combination with AKT inhibition in the orthotopic LN-229 glioma xenograft model in nude mice. These data justify to explore combined targeted therapy approaches in glioblastoma that aim at down-regulating AKT function to enhance the therapeutic potential of dual PI3K/mTOR inhibitors.

Evolutionary Trajectories of IDHWT Glioblastomas Reveal a Common Path of Early Tumorigenesis Instigated Years ahead of Initial Diagnosis.

We studied how intratumoral genetic heterogeneity shapes tumor growth and therapy response for isocitrate dehydrogenase (IDH)-wild-type glioblastoma, a rapidly regrowing tumor. We inferred the evolutionary trajectories of matched pairs of primary and relapsed tumors based on deep whole-genome-sequencing data. This analysis suggests both a distant origin of de novo glioblastoma, up to 7 years before diagnosis, and a common path of early tumorigenesis, with one or more of chromosome 7 gain, 9p loss, or 10 loss, at tumor initiation. TERT promoter mutations often occurred later as a prerequisite for rapid growth. In contrast to this common early path, relapsed tumors acquired no stereotypical pattern of mutations and typically regrew from oligoclonal origins, suggesting sparse selective pressure by therapeutic measures.

Molecular differences in IDH wildtype glioblastoma according to MGMT promoter methylation.

Background: O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation status is a predictive biomarker in glioblastoma. We investigated whether this marker furthermore defines a molecularly distinct tumor subtype with clinically different outcome. Methods: We analyzed copy number variation (CNV) and methylation profiles of 1095 primary and 92 progressive isocitrate dehydrogenase wildtype glioblastomas, including paired samples from 49 patients. DNA mutation data from 182 glioblastoma samples of The Cancer Genome Atlas (TCGA) and RNA expression from 107 TCGA and 55 Chinese Glioma Genome Atlas samples were analyzed. Results: Among untreated glioblastomas, MGMT promoter methylated (mMGMT) and unmethylated (uMGMT) tumors did not show different CNV or specific gene mutations, but a higher mutation count in mMGMT tumors. We identified 3 methylation clusters. Cluster 1 showed the highest average methylation and was enriched for mMGMT tumors. Seventeen genes including gastrulation brain homeobox 2 (GBX2) were found to be hypermethylated and downregulated on the mRNA level in mMGMT tumors. In progressive glioblastomas, platelet derived growth factor receptor alpha (PDGFRA) and GLI2 amplifications were enriched in mMGMT tumors. Methylated MGMT tumors gain PDGFRA amplification of PDGFRA, whereas uMGMT tumors with amplified PDGFRA frequently lose this amplification upon progression. Glioblastoma patients surviving <6 months and with mMGMT harbored less frequent epidermal growth factor receptor (EGFR) amplifications, more frequent TP53 mutations, and a higher tumor necrosis factor-nuclear factor-kappaB (TNF-NFκB) pathway activation compared with patients surviving >12 months. Conclusions: MGMT promoter methylation status does not define a molecularly distinct glioblastoma subpopulation among untreated tumors. Progressive mMGMT glioblastomas and mMGMT tumors of patients with short survival tend to have more unfavorable molecular profiles.

Epidermal Growth Factor Receptor Variant III (EGFRvIII) Positivity in EGFR-Amplified Glioblastomas: Prognostic Role and Comparison between Primary and Recurrent Tumors.

Purpose: Approximately 40% of all glioblastomas have amplified the EGFR gene, and about half of these tumors express the EGFRvIII variant. The prognostic role of EGFRvIII in EGFR-amplified glioblastoma patients and changes in EGFRvIII expression in recurrent versus primary glioblastomas remain controversial, but such data are highly relevant for EGFRvIII-targeted therapies.Experimental Design:EGFR-amplified glioblastomas from 106 patients were assessed for EGFRvIII positivity. Changes in EGFR amplification and EGFRvIII status from primary to recurrent glioblastomas were evaluated in 40 patients with EGFR-amplified tumors and 33 patients with EGFR-nonamplified tumors. EGFR single-nucleotide variants (SNV) were assessed in 27 patients. Data were correlated with outcome and validated in 150 glioblastoma patients from The Cancer Genome Atlas (TCGA) consortium.Results: Sixty of 106 EGFR-amplified glioblastomas were EGFRvIII-positive (56.6%). EGFRvIII positivity was not associated with different progression-free or overall survival. EGFRvIII status was unchanged at recurrence in 35 of 40 patients with EGFR-amplified primary tumors (87.5%). Four patients lost and one patient gained EGFRvIII positivity at recurrence. None of 33 EGFR-nonamplified glioblastomas acquired EGFR amplification or EGFRvIII at recurrence. EGFR SNVs were frequent in EGFR-amplified tumors, but were not linked to survival.Conclusions: EGFRvIII and EGFR SNVs are not prognostic in EGFR-amplified glioblastoma patients. EGFR amplification is retained in recurrent glioblastomas. Most EGFRvIII-positive glioblastomas maintain EGFRvIII positivity at recurrence. However, EGFRvIII expression may change in a subset of patients at recurrence, thus repeated biopsy with reassessment of EGFRvIII status is recommended for patients with recurrent glioblastoma to receive EGFRvIII-targeting agents. Clin Cancer Res; 23(22); 6846-55. ©2017 AACR.

Molecular Diagnostics of Gliomas Using Next Generation Sequencing of a Glioma-Tailored Gene Panel.

Current classification of gliomas is based on histological criteria according to the World Health Organization (WHO) classification of tumors of the central nervous system. Over the past years, characteristic genetic profiles have been identified in various glioma types. These can refine tumor diagnostics and provide important prognostic and predictive information. We report on the establishment and validation of gene panel next generation sequencing (NGS) for the molecular diagnostics of gliomas. We designed a glioma-tailored gene panel covering 660 amplicons derived from 20 genes frequently aberrant in different glioma types. Sensitivity and specificity of glioma gene panel NGS for detection of DNA sequence variants and copy number changes were validated by single gene analyses. NGS-based mutation detection was optimized for application on formalin-fixed paraffin-embedded tissue specimens including small stereotactic biopsy samples. NGS data obtained in a retrospective analysis of 121 gliomas allowed for their molecular classification into distinct biological groups, including (i) isocitrate dehydrogenase gene (IDH) 1 or 2 mutant astrocytic gliomas with frequent α-thalassemia/mental retardation syndrome X-linked (ATRX) and tumor protein p53 (TP53) gene mutations, (ii) IDH mutant oligodendroglial tumors with 1p/19q codeletion, telomerase reverse transcriptase (TERT) promoter mutation and frequent Drosophila homolog of capicua (CIC) gene mutation, as well as (iii) IDH wildtype glioblastomas with frequent TERT promoter mutation, phosphatase and tensin homolog (PTEN) mutation and/or epidermal growth factor receptor (EGFR) amplification. Oligoastrocytic gliomas were genetically assigned to either of these groups. Our findings implicate gene panel NGS as a promising diagnostic technique that may facilitate integrated histological and molecular glioma classification.