title: Reverse phase protein arrays enable glioblastoma molecular subtyping.
abstract: In the present study we investigated the phosphorylation status of the 12 most important signaling cascades in glioblastomas. More than 60 tumor and control biopsies from tumor center and periphery (based on neuronavigation) were subjected to selective protein expression analysis using reverse-phase protein arrays (RPPA) incubated with antibodies against posttranslationally modified cancer pathway proteins. The ratio between phosphorylated (or modified) and non-phosphorylated protein was assessed. All samples were histopathologically validated and proteomic profiles correlated with clinical and survival data. By RPPA, we identified three distinct activation patterns within glioblastoma defined by the ratios of pCREB1/CREB1, NOTCH-ICD/NOTCH1, and pGSK3β/GSK3β, respectively. These subclasses demonstrated distinct overall survival patterns in a cohort of patients from a single-institution and in an analysis of publicly available data. In particular, a high pGSK3β/GSK3β-ratio was associated with a poor survival. Wnt-activation/GSK3β-inhibition in U373 and U251 cell lines halted glioma cell proliferation and migration. Gene expression analysis was used as an internal quality control of baseline proteomic data. The protein expression and phosphorylation had a higher resolution, resulting in a better class-subdivision than mRNA based stratification data. Patients with different proteomic profiles from multiple biopsies showed a worse overall survival. The CREB1-, NOTCH1-, GSK3β-phosphorylation status correlated with glioma grades. RPPA represent a fast and reliable tool to supplement morphological diagnosis with pathway-specific information in individual tumors. These data can be exploited for molecular stratification and possible combinatorial treatment planning. Further, our results may optimize current glioma grading algorithms.
authors.affiliation: Department of Neurosurgery, University Hospital Basel, Spitalstrasse 21, 4031, Basel, Switzerland. Gregorhutter1@gmail.com.|Department of Neurosurgery, Stanford University, 300 Pasteur Drive, Stanford, 94305, CA, USA. Gregorhutter1@gmail.com.
authors.forename: Gregor
authors.identifier: 0000-0002-7570-3902
authors.initials: G
authors.lastname: Hutter
authors.affiliation: Department of Neurosurgery, University Hospital Basel, Spitalstrasse 21, 4031, Basel, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Sailer
authors.affiliation: Department of Neurosurgery, Stanford University, 300 Pasteur Drive, Stanford, 94305, CA, USA.
authors.forename: Tej Deepak
authors.initials: TD
authors.lastname: Azad
authors.affiliation: Division of Pediatric Hematology and Oncology, Department of Pediatrics and Adolescent Medicine, University Medical Center Goettingen, Robert-Koch-Str. 40, 37075, Goettingen, Germany.|Division of Pediatric Hematology and Oncology, Department of Pediatrics and Adolescent Medicine, University Hospital of Geneva, Geneva, Switzerland.|CANSEARCH Research Laboratory, Department of Pediatrics, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
authors.forename: André O
authors.initials: AO
authors.lastname: von Bueren
authors.affiliation: Diagnostic Center, Institute for Clinical Chemistry, University Medical Center Hamburg-Eppendorf (UKE), CAMPUS Research Building N27, Martinistraße 52, 20246, Hamburg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Nollau
authors.affiliation: Division of Neuro- and Ophthalmopathology, Department of Pathology, University Hospital Basel, Schönbeinstrasse 40, 4031, Basel, Switzerland.
authors.forename: Stephan
authors.initials: S
authors.lastname: Frank
authors.affiliation: Department of Neurosurgery, University Hospital Basel, Spitalstrasse 21, 4031, Basel, Switzerland.
authors.forename: Cristobal
authors.initials: C
authors.lastname: Tostado
authors.affiliation: Molecular Signalling and Gene Therapy, Narayana Nethralaya, Narayana Health City, # 258/A, Bommasandra, Hosur Road, Bangalore, 560 099, India.
authors.forename: Durga
authors.initials: D
authors.lastname: Sarvepalli
authors.affiliation: Molecular Signalling and Gene Therapy, Narayana Nethralaya, Narayana Health City, # 258/A, Bommasandra, Hosur Road, Bangalore, 560 099, India.
authors.forename: Arkasubhra
authors.initials: A
authors.lastname: Ghosh
authors.affiliation: Department of Neurosurgery, University Hospital Basel, Spitalstrasse 21, 4031, Basel, Switzerland.
authors.forename: Marie-Françoise
authors.initials: MF
authors.lastname: Ritz
authors.affiliation: Department of Neurosurgery, University Hospital Basel, Spitalstrasse 21, 4031, Basel, Switzerland.
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Boulay
authors.affiliation: Department of Neurosurgery, University Hospital Basel, Spitalstrasse 21, 4031, Basel, Switzerland.
authors.forename: Luigi
authors.initials: L
authors.lastname: Mariani
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001932:Brain Neoplasms / Q000378:metabolism*; D005260:Female; D005909:Glioblastoma / Q000378:metabolism*; D006801:Humans; D008297:Male; D008875:Middle Aged; D010766:Phosphorylation; D040081:Protein Array Analysis; D040901:Proteomics; D015398:Signal Transduction; D016019:Survival Analysis
keywords: Cancer signaling; Glioblastoma; Molecular stratification; Proteomics