Abstract C30: Four dimensional molecular analysis of pediatric diffuse intrinsic pontine glioma
Authors: Saratsis Amanda , Yadavilli Sridevi , Kambhampati Madhuri , Raabe Eric , Magge Suresh , Nazarian Javad 
Published: August 2014

Introduction: Diffuse Intrinsic Pontine Glioma (DIPG) is a highly morbid form of pediatric brainstem glioma. Molecular characterization is limited due to lack of tissue. Recent investigations suggest possible molecular subtypes may account for the historical poor response to therapy. We previously generated protein profiles of CSF and formalin fixed DIPG tumor specimens to characterize patterns of protein expression. Here, we present the first comprehensive tissue proteome of fresh frozen DIPG tumor specimens (n=16) and normal brain tissue (n=10). We characterize differential protein expression in DIPG tumor specimens, and compare these to gene expression and DNA methylation profiles of the same tissue.

Methods: Normal brain and tumor tissue was collected intraoperatively or post-mortem. Extracted total tissue protein was quantified by mass spectrometry (MS/MS) via LTQ-Orbitrap-XL and database search using the Sequest algorithm. Gene expression profiles were detected using whole-genome Human HT-4 v12 Gene Expression Bead Chips. DNA methylation profiles were characterized after bisulphite conversion using Infinium HumanMethylation 450K BeadChip arrays. Quantitative and statistical analysis was performed with Genome Studio, ProteoIQ, and Partek Genomics Suite. Functional analysis was performed using Ingenuity Pathways Analysis. Gene and protein expression was validated via western blot and immunohistochemical staining of tumor and normal brain tissue.

Results: 1,918 differentially expressed genes were detected in DIPG tumor tissue (ANOVA, p&lt;0.05, FC &gt;2 or &lt;-2). Unsupervised clustering revealed two distinct subgroups with differential ShH activity (GLI1 z-score -2.000 vs 2.000) and expression of GLI1, GLIPR1, PTCHD1 and SMO. Protein profiling revealed high expression of TLN1, CLU, EEF2 (FC &gt;2), with differential ShH pathway (GLI1 z-score -0.626 vs. 2.254) and protein expression COL1A2, LMNA, MAP4, NES, NRCAM, STMN1, and TNC between subgroups (ANOVA, p&lt;0.05, FC &gt;2 or &lt;-2). Concordant differences in DNA methylation were detected in related genes, including GLI1, FOXF1, SMO, SHH, and SUFU (ANOVA, p&lt;0.05, FC&lt;-2 or &gt;2).

Conclusions: We present the first comprehensive protein profile of DIPG fresh frozen tumor tissue and correlate to tissue gene expression profiles, which suggest differential activity of SHh pathway. This may in part be explained by differential methylation patterns of key genes. Proteomic analysis of DIPG tumor tissue reveals protein expression profiles reflective of this differential pathway activation, and hence may be useful tool for elucidating mechanisms of brainstem gliomagenesis in what likely is a heterogeneous tumor population. Biomarkers identified through proteomic analysis may in turn serve to more accurately diagnose patients with DIPG and measure response to therapy.

Citation Format: Amanda Saratsis, Sridevi Yadavilli, Madhuri Kambhampati, Eric Raabe, Suresh Magge, Javad Nazarian. Four dimensional molecular analysis of pediatric diffuse intrinsic pontine glioma. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C30.