Identification of Predictive Response Markers and Novel Treatment Targets for Gliomas

Gliomas are the most frequent primary brain tumors in adults. Despite multimodality treatment strategies, the survival of patients with a diffuse glioma remains poor. There has been an increasing use of molecular markers to assist diagnosis and predict prognosis and response to therapy. Although several prognostic and predictive response markers have been identified, considerable research still needs to be done to improve on these. Therefore, the identification of novel predictive response markers and therapeutic targets are desperately needed for this dismal disease. In this thesis, we describe prognostic and identify novel predictive markers in randomized clinical trials. We determined the gene expression profiles of samples of anaplastic oligodendrogliomas and oligoastrocytomas from the EORTC 26951 study and samples of recurrent glioblastomas of the BELOB study to evaluate the treatment responses within defined intrinsic glioma subtypes (IGSs). IGSs are molecularly similar tumors that have been previously identified by unsupervised gene expression analysis. We found that IGSs can be used to assess the molecular heterogeneity within clinical trials. In addition, we confirmed that IGS subtypes are prognostic for survival and predictive. Tumors assigned to IGS-9 showed benefit from adjuvant PCV chemotherapy. In the BELOB study, we found that tumors assigned to IGS-18 (classical GBMs) showed a trend towards benefit from Beva+CCNU treatment. Expression of FMO4 and OSBPL3 were particularly associated with treatment response. Intrinsic subtypes can therefore be used to assess the molecular heterogeneity within clinical trials and may be used as a prognostic and predictive marker. Another method to profile gliomas is based on DNA methylation. We performed genome-wide methylation profiling on material from EORTC 26951 and assessed CIMP and MGMT-STP27 status. We have shown that survival in patients with CIMP+ or MGMT-STP27 methylated tumors was improved compared to CIMP- and/or MGMT-STP27 unmethylated tumors. Importantly, the MGMT-STP27 status was predictive for response to adjuvant PCV chemotherapy in these tumors. MGMT-STP27 may therefore be used to identify AODs and AOAs with improved prognosis and identify patients that are likely to benefit from adjuvant PCV chemotherapy. We also performed functional analysis on different mutations on the EGFR gene and infrequently mutated genes in oligodendrogliomas. We have shown that different mutations within a single gene (EGFR) can have different molecular consequences and have different binding partners for EGFRvIII, EGFRL858 and EGFRwildtype. As these mutations have different functions, each mutation may need its own unique treatment. Functional analysis on infrequently mutated genes showed that the function of many of ‘low frequency’ genes, differs from its wildtype counterpart. This differential effect suggests that these genes can contribute to the disease and therefore may offer new therapeutic targets for oligodendrogliomas

Tumor-specific mutations in low-frequency genes affect their functional properties

Causal genetic changes in oligodendrogliomas (OD) with 1p/19q co-deletion include mutations in IDH1, IDH2, CIC, FUBP1, TERT promoter and NOTCH1. However, it is generally assumed that more somatic mutations are required for tumorigenesis. This study aimed to establish whether genes mutated at low frequency can be involved in OD initiation and/or progression. We performed whole-genome sequencing on three anaplastic ODs with 1p/19q co-deletion. To estimate mutation frequency, we performed targeted resequencing on an additional 39 ODs. Whole-genome sequencing identified a total of 55 coding mutations (range 8–32 mutations per tumor), including known abnormalities in IDH1, IDH2, CIC and FUBP1. We also identified mutations in genes, most of which were previously not implicated in ODs. Targeted resequencing on 39 additional ODs confirmed that these genes are mutated at low frequency. Most of the mutations identified were predicted to have a deleterious functional effect. Functional analysis on a subset of these genes (e.g. NTN4 and MAGEH1) showed that the mutation affects the subcellular localization of the protein (n = 2/12). In addition, HOG cells stably expressing mutant GDI1 or XPO7 showed altered cell proliferation compared to those expressing wildtype constructs. Similarly, HOG cells expressing mutant SASH3 or GDI1 showed altered migration. The significantly higher rate of predicted deleterious mutations, the changes in subcellular localization and the effects on proliferation and/or migration indicate that many of these genes functionally may contribute to gliomagenesis and/or progression. These low-frequency genes and their affected pathways may provide new treatment targets for this tumor type

Mutation specific functions of EGFR result in a mutation-specific downstream pathway activation

Background: Epidermal growth factor receptor (EGFR) is frequently mutated in various types of cancer. Although all oncogenic mutations are considered activating, different tumour types have different mutation spectra. It is possible that functional differences underlie this tumour-type specific mutation spectrum. Methods: We have determined whether specific mutations in EGFR (EGFR, EGFRvIII and EGFR-L858R) have differences in binding partners, differences in downstream pathway activation (gene expression and phosphoproteins), and have functional consequences on cellular growth and migration. Results: Using biotin pulldown and subsequent mass spectrometry we were able to detect mutation specific binding partners for EGFR. Differential binding was confirmed using a proximity ligation assay and/or Western Blot for the dedicator of cytokinesis 4 (DOCK4), UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1), MYC binding protein 2 (MYCBP2) and Smoothelin (SMTN). We also demonstrate that each mutation induces the expression of a specific set of genes, and that each mutation is associated with specific phosphorylation patterns. Finally, we demonstrate using stably expressing cell lines that EGFRvIII and EGFL858R display reduced growth and migration compared to EGFR ildtype expressing cells

Intrinsic Molecular Subtypes of Glioma Are Prognostic and Predict Benefit From Adjuvant Procarbazine, Lomustine, and Vincristine Chemotherapy in Combination With Other Prognostic Factors in Anaplastic Oligodendroglial Brain Tumors:A Report From EORTC Stud

<p>Purpose</p><p>Intrinsic glioma subtypes (IGSs) are molecularly similar tumors that can be identified based on unsupervised gene expression analysis. Here, we have evaluated the clinical relevance of these subtypes within European Organisation for Research and Treatment of Cancer (EORTC) 26951, a randomized phase III clinical trial investigating adjuvant procarbazine, lomustine, and vincristine (PCV) chemotherapy in anaplastic oligodendroglial tumors. Our study includes gene expression profiles of formalin-fixed, paraffin-embedded (FFPE) clinical trial samples.</p><p>Patients and Methods</p><p>Gene expression profiling was performed in 140 samples, 47 fresh frozen samples and 93 FFPE samples, on HU133_Plus_2.0 and HuEx_1.0_st arrays, respectively.</p><p>Results</p><p>All previously identified six IGSs are present in EORTC 26951. This confirms that different molecular subtypes are present within a well-defined histologic subtype. Intrinsic subtypes are highly prognostic for overall survival (OS) and progression-free survival (PFS). They are prognostic for PFS independent of clinical (age, performance status, and tumor location), molecular (1p/19q loss of heterozygosity [LOH], IDH1 mutation, and MGMT methylation), and histologic parameters. Combining known molecular (1p/19q LOH, IDH1) prognostic parameters with intrinsic subtypes improves outcome prediction (proportion of explained variation, 30% v 23% for each individual group of factors). Specific genetic changes (IDH1, 1p/19q LOH, and EGFR amplification) segregate into different subtypes. We identified one subtype, IGS-9 (characterized by a high percentage of 1p/19q LOH and IDH1 mutations), that especially benefits from PCV chemotherapy. Median OS in this subtype was 5.5 years after radiotherapy (RT) alone versus 12.8 years after RT/PCV (P = .0349; hazard ratio, 2.18; 95% CI, 1.06 to 4.50).</p><p>Conclusion</p><p>Intrinsic subtypes are highly prognostic in EORTC 26951 and improve outcome prediction when combined with other prognostic factors. Tumors assigned to IGS-9 benefit from adjuvant PCV. J Clin Oncol 31:328-336. (C) 2012 by American Society of Clinical Oncology</p>

Identification of patients with recurrent glioblastoma who may benefit from combined bevacizumab and CCNU Therapy: A Report from the BELOB Trial

The results from the randomized phase II BELOB trial provided evidence for a potential benefit of bevacizumab (beva), a humanized monoclonal antibody against circulating VEGF-A, when added to CCNU chemotherapy in patients with recurrent glioblastoma (GBM). In this study, we performed gene expression profiling (DASL and RNA-seq) of formalin-fixed, paraffinembedded tumor material from participants of the BELOB trial to identify patients with recurrent GBM who benefitted most from bevaCCNU treatment. We demonstrate that tumors assigned to the IGS-18 or "classical" subtype and treated with bevaCCNU showed a significant benefit in progression-free survival and a trend toward benefit in overall survival, whereas other subtypes did not exhibit such benefit. In particular, expression of FMO4 and OSBPL3 was associated with treatment response. Importantly, the improved outcome in the bevaCCNU treatment arm was not explained by an uneven distribution of prognostically favorable subtypes as all molecular glioma subtypes were evenly distributed along the different study arms. The RNA-seq analysis also highlighted genetic alterations, including mutations, gene fusions, and copy number changes, within this well-defined cohort of tumors that may serve as useful predictive or prognostic biomarkers of patient outcome. Further validation of the identified molecular markers may enable the future stratification of recurrent GBM patients into appropriate treatment regimens