title: Comparative evaluation of strategies for quantifying signaling pathway proteins in Ewing sarcoma.
abstract: Targeted therapies are increasingly being evaluated for patients with Ewing sarcoma (EWS). Optimal strategies for quantifying key signaling proteins in EWS remain unclear. We sought to quantify tumor expression of signaling pathway proteins in EWS using 3 methodologies. A total of 46 blocks of formalin-fixed paraffin-embedded tissue were obtained from 40 patients with EWS. Tumor was evaluated for the expression of proteins in the insulin-like growth factor type 1 receptor (IGF-1R), epithelial growth factor receptor (EGFR), and mTOR pathways using standard immunohistochemical analysis (IHC), automated quantitative analysis (AQUA) immunohistochemical analysis, and mass spectrometry quantification. The mean age at diagnosis was 14 years (range, 1 to 49 y). About 67.5% were male and 57.5% had localized disease. Samples displayed a wide range of expression by AQUA: mean (range) IGF-1R=10,702 (393 to 14,424); EGFR=2750 (672 to 9798); and phosphatase and tensin homolog (PTEN)=2250 (251 to 6557). Mean IGF-1R expression by AQUA did not differ between standard IHC expression categories (low IHC=11,255; medium IHC=11,070; high IHC=11,023; P=0.98). Mean PTEN expression by AQUA was higher in the medium and high IHC categories (low IHC=1229; medium IHC=2715; high IHC=2940; P=0.064). Only 2 samples expressed EGFR by standard IHC. Mass spectrometry trended toward correlation with standard IHC but did not yield interpretable results in the majority of samples. This study demonstrates that the relative quantification of signaling protein expression in EWS is dependent on the methodology used. Optimization and validation of these tools are necessary before clinical application for risk stratification of patients or measurement of biomarker expression.
authors.affiliation: *Department of Pediatrics, University of Chicago, Chicago, IL Departments of ¶Pediatrics §Pathology, San Francisco School of Medicine, University of California, San Francisco, CA Departments of †Pathology ∥Pediatrics, University of Michigan Medical School, Ann Arbor, MI ‡OncoPlex Diagnostics, Rockville, MD.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Applebaum
authors.forename: Dafydd G
authors.initials: DG
authors.lastname: Thomas
authors.forename: Todd
authors.initials: T
authors.lastname: Hembrough
authors.forename: Jon
authors.initials: J
authors.lastname: Burrows
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Horvai
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Lawlor
authors.forename: Steven G
authors.initials: SG
authors.lastname: DuBois
mesh_terms: D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D009363:Neoplasm Proteins / Q000378:metabolism*; D016612:Paraffin Embedding; D012512:Sarcoma, Ewing / Q000378:metabolism* / Q000473:pathology; D015398:Signal Transduction
chemical_list: D009363:Neoplasm Proteins