Protein profiling of formalin fixed paraffin embedded tissue: Identification of potential biomarkers for pediatric brainstem glioma

Abstract

Little is known about the molecular characteristics of pediatric brainstem gliomas (BSG), which continue to have a dismal prognosis. Targeted molecular strategies are limited due to rarity of biopsy BSG specimen coupled with obstacles associated with the analyses of formalin-fixed paraffin-embedded (FFPE) autopsies. The objective of this study was to develop methodologies to successfully identify the proteome profile from these archived FFPE specimens. Peptides were extracted from both tumor and adjacent normal FFPE brainstem specimen and quantified using 18 O proteolytic labeling strategy and LC-MS/MS analysis. The ingenuity pathway analysis software was used to elucidate interactions amongst differentially expressed proteins. We identified 188 proteins of which 54 (29%) were found up-regulated (≥1.5-fold) in BSG compared to normal sections. Of these, 15 (28%) proteins have previously been reported as potential biomarkers for supratentorial malignant gliomas, while the rest appear to be exclusive to pediatric BSG. Because the majority of differentially expressed proteins are unique to BSG, we conclude that pediatric BSG is distinct from supratentorial gliomas. To the best of our knowledge, this is the first proteome profile of pediatric BSG, which may facilitate discovery of novel therapeutic targets for early diagnostics and improving prognostics.

Comparative multidimensional molecular analyses of pediatric diffuse intrinsic pontine glioma reveals distinct molecular subtypes

“…A large percentage of the DIPG CSF proteome (76.8%) was detected in fresh frozen DIPG tissue. A total of 75% (42 of 55) of proteins previously identified by our group in paraffin embedded DIPG tumor tissue (FFPE) [34] were also detected in the fresh frozen DIPG tumor tissue analyzed here.…”

“…Protein profiles of fresh frozen DIPG specimens were compared to previously characterized CSF [43] and paraffin embedded DIPG profiles [34] (Online Resource 5). A large percentage of the DIPG CSF proteome (76.8%) was detected in fresh frozen DIPG tissue.…”

“…We previously performed protein profiling of archival formalin fixed paraffin embedded (FFPE) postmortem tumor specimens, demonstrating the utility of proteomic analysis of cerebrospinal fluid (CSF) from patients with DIPG for detection of tumor-secreted proteins as biomarkers for clinical diagnosis and treatment [34,43]. To expand our analyses, we performed a multidimensional study of rare fresh frozen DIPG specimens to characterize protein, mRNA, and methylation patterns in a cohort of pediatric brain tumor specimens, including DIPGs (n=14) for which histone 3 mutation status was also investogated.…”

Label-free protein profiling of formalin-fixed paraffin-embedded (FFPE) heart tissue reveals immediate mitochondrial impairment after ionising radiation

“…Proteomics analysis using FFPE material as an alternative to fresh-frozen tissue has recently been investigated [4][5][6][7]. Different methods of extraction and separation of proteins from FFPE samples have been established [8][9][10][11].…”

Tissue Proteomics for the Next Decade? Towards a Molecular Dimension in Histology

“…In order to study the different types of tissues, several methods are devoted to specific area sampling and depend on the desired size, such as macrodissection (Azimzadeh et al, 2010;Nazarian et al, 2008) or the more accurate needle microdissection (Craven et al;Prieto et al, 2005). Finally, the most precise technique to select a specific cell group in a tissue section is LCM.…”