Introduction: Reverse phase protein arrays (RPPAs) are a stratification tool to simultaneously analyze multiple signaling pathways in tumor and control samples. The results of this assay can then be used to correlate the patient-specific signaling profiles to clinical outcome. The advantage of this method lies in the real-time, mid-to high-throughput assessment of biologically active pathways.
Methods: More than 60 tumor and control biopsies were subjected to selective protein expression analysis using RPPAs incubated with antibodies against post-translationally modified cancer pathway proteins. The ratio between phosphorylated (or modified) and non-phosphorylated protein was assessed. All samples were histopathologically validated and proteomic profiles were correlated with clinical and survival data.
Results: We identified three distinct activation patterns within glioblastoma that where defined by the ratios of pCREB/CREB, NOTCH-ICD/NOTCH1 and pGSK3B/GSKB. These subclasses demonstrated distinct overall survival patterns in a cohort of patients from a single-institution, where a high pGSK3B/GSK3B-ratio was associated with a poor survival. Wnt-activation/GSK3B inhibition in U373 and U251 cell lines halted glioma cell proliferation and migration. Proteomic network analysis revealed differential contributions of these signatures to tumor cell proliferation, invasiveness and cell motility. Patients with different proteomic profiles from multiple biopsies showed a worse overall survival. The CREB-, NOTCH1-, GSK3B-phosphorylation status correlated with glioma grades. In an analysis of publicly available data, we found significant survival differences in the proneural subtype of GBM based on GSK3B expression (p<.05) and in the classical subtype of GBM based on NOTCH1 expression (p<.05).
Conclusions: RPPAs represent a fast and reliable tool to supplement morphologic diagnosis with pathway-specific information in individual tumors. These data can be exploited for molecular stratification and possible combinatorial treatment plans. Further, our results provide potential additional diagnostic means to distinguish glioma grades.
Patient Care: Identification of key signaling pathways in GBM has implications for patient stratification and treatment selection
Learning Objectives: 1. Identification of key signaling pathways in GBM has implications for patient stratification and treatment selection
2. Reverse-phase protein arrays represent a fast and reliable tool to supplement morphologic diagnosis with pathway-specific information in individual tumors.