Diffuse intrinsic pontine glioma (DIPG) is a highly morbid form of pediatric 
brainstem glioma. Here, we present the first comprehensive protein, mRNA, and 
methylation profiles of fresh-frozen DIPG specimens (n = 14), normal brain 
tissue (n = 10), and other pediatric brain tumors (n = 17). Protein profiling 
identified 2,305 unique proteins indicating distinct DIPG protein expression 
patterns compared to other pediatric brain tumors. Western blot and 
immunohistochemistry validated upregulation of Clusterin (CLU), Elongation 
Factor 2 (EF2), and Talin-1 (TLN1) in DIPGs studied. Comparisons to mRNA 
expression profiles generated from tumor and adjacent normal brain tissue 
indicated two DIPG subgroups, characterized by upregulation of Myc (N-Myc) or 
Hedgehog (Hh) signaling. We validated upregulation of PTCH, a membrane receptor 
in the Hh signaling pathway, in a subgroup of DIPG specimens. DNA methylation 
analysis indicated global hypomethylation of DIPG compared to adjacent normal 
tissue specimens, with differential methylation of 24 genes involved in Hh and 
Myc pathways, correlating with protein and mRNA expression patterns. Sequencing 
analysis showed c.83A>T mutations in the H3F3A or HIST1H3B gene in 77 % of our 
DIPG cohort. Supervised analysis revealed a unique methylation pattern in 
mutated specimens compared to the wild-type DIPG samples. This study presents 
the first comprehensive multidimensional protein, mRNA, and methylation 
profiling of pediatric brain tumor specimens, detecting the presence of two 
subgroups within our DIPG cohort. This multidimensional analysis of DIPG 
provides increased analytical power to more fully explore molecular signatures 
of DIPGs, with implications for evaluating potential molecular subtypes and 
biomarker discovery for assessing response to therapy.