Abstract
Intragenic deletion is the most common form of activating mutation among receptor tyrosine kinases (RTK) in glioblastoma. However, these events are not detected by conventional DNA sequencing methods commonly utilized for tumor genotyping. To comprehensively assess the frequency, distribution, and expression levels of common RTK deletion mutants in glioblastoma, we analyzed RNA from a set of 192 glioblastoma samples from The Cancer Genome Atlas for the expression of EGFRvIII, EGFRvII, EGFRvV (carboxyl-terminal deletion), and PDGFRAΔ8,9. These mutations were detected in 24, 1.6, 4.7, and 1.6 % of cases, respectively. Overall, 29 % (55/189) of glioblastomas expressed at least one RTK intragenic deletion transcript in this panel. For EGFRvIII, samples were analyzed by both quantitative real-time PCR (QRT-PCR) and single mRNA molecule counting on the Nanostring nCounter platform. Nanostring proved to be highly sensitive, specific, and linear, with sensitivity comparable or exceeding that of RNA seq. We evaluated the prognostic significance and molecular correlates of RTK rearrangements. EGFRvIII was only detectable in tumors with focal amplification of the gene. Moreover, we found that EGFRvIII expression was not prognostic of poor outcome and that neither recurrent copy number alterations nor global changes in gene expression differentiate EGFRvIII-positive tumors from tumors with amplification of wild-type EGFR. The wide range of expression of mutant alleles and co-expression of multiple EGFR variants suggests that quantitative RNA-based clinical assays will be important for assessing the relative expression of intragenic deletions as therapeutic targets and/or candidate biomarkers. To this end, we demonstrate the performance of the Nanostring assay in RNA derived from routinely collected formalin-fixed paraffin-embedded tissue.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-013-1217-3) contains supplementary material, which is available to authorized users.
Highlights
Large-scale genomic characterization has confirmed striking heterogeneity underlying the molecular landscape of GBM and has catalogued a spectrum of tumor suppressors and oncogenes affected by deletion, amplification, mutation, and/or rearrangement
A TaqMan-based qRT-PCR approach was compared to a Nanostring nCounter assay (NS), each targeting both the exon 1–8 junctional region of EGFRvIII (E1–8) and the epidermal growth factor receptor (EGFR) kinase domain (KD) as well as select control genes
Using analogous approaches to that employed for EGFR variant III mutation (vIII), we developed Nanostring assays for the detection of EGFR vII and PDGFRAΔ8,9 based on their specific breakpoint regions
Summary
Large-scale genomic characterization has confirmed striking heterogeneity underlying the molecular landscape of GBM and has catalogued a spectrum of tumor suppressors and oncogenes affected by deletion, amplification, mutation, and/or rearrangement. Highlevel amplification of the EGFR locus represents the single most common genomic abnormality in GBM, occurring in ~45 % of all cases, and PDGFRA and MET are frequently amplified, in 10–15 % and ~4 % of GBMs, respectively [5, 10, 31, 43]. These amplification events have been associated with specific disease subclasses, defined by transcriptional and proteomic signatures [4, 37, 45], implying that molecular distinctions within GBM are, to some extent, mechanistically grounded in dysregulated RTK signaling. Other cancerrelevant functionalities ascribed to EGFRvIII include evasion of apoptosis [30], tumor cell invasion [22], angiogenesis [50] and stem cell self-renewal [16]
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