title: Evaluation of Protein Profiles From Treated Xenograft Tumor Models Identifies an Antibody Panel for Formalin-fixed and Paraffin-embedded (FFPE) Tissue Analysis by Reverse Phase Protein Arrays (RPPA).
abstract: Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect using FFPE tissues.
authors.affiliation: From the ‡Pharma Research & Early Development, Roche Innovation Center Penzberg, Nonnenwald 2, 82377, Penzberg, Germany;
authors.forename: Sabine
authors.initials: S
authors.lastname: Bader
authors.affiliation: §Pharma Research & Early Development, Roche Innovation Centre Welwyn, 6 Falcon Way, Shire Park, Welwyn Garden City, Herts, AL7 1TW, United Kingdom;
authors.forename: Magdalena
authors.initials: M
authors.lastname: Zajac
authors.affiliation: From the ‡Pharma Research & Early Development, Roche Innovation Center Penzberg, Nonnenwald 2, 82377, Penzberg, Germany;
authors.forename: Thomas
authors.initials: T
authors.lastname: Friess
authors.affiliation: From the ‡Pharma Research & Early Development, Roche Innovation Center Penzberg, Nonnenwald 2, 82377, Penzberg, Germany;
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Ruge
authors.affiliation: From the ‡Pharma Research & Early Development, Roche Innovation Center Penzberg, Nonnenwald 2, 82377, Penzberg, Germany;
authors.forename: Natascha
authors.initials: N
authors.lastname: Rieder
authors.affiliation: ¶Department Biochemistry & Protein Profiling, NMI Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstrasse 55, 72770, Reutlingen, Germany;
authors.forename: Berthold
authors.initials: B
authors.lastname: Gierke
authors.affiliation: ¶Department Biochemistry & Protein Profiling, NMI Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstrasse 55, 72770, Reutlingen, Germany;
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Heubach
authors.affiliation: ‖Roche Pharma AG, Emil-Barell-Str. 1, 79639, Grenzach-Wyhlen, Germany michael.pawlak@nmi.de marlene.thomas@roche.com.
authors.forename: Marlene
authors.initials: M
authors.lastname: Thomas
authors.affiliation: ¶Department Biochemistry & Protein Profiling, NMI Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstrasse 55, 72770, Reutlingen, Germany; michael.pawlak@nmi.de marlene.thomas@roche.com.
authors.forename: Michael
authors.initials: M
authors.lastname: Pawlak
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000276:immunology; D005260:Female; D005557:Formaldehyde; D006801:Humans; D016513:Mice, SCID; D009369:Neoplasms / Q000378:metabolism*; D016612:Paraffin Embedding; D040081:Protein Array Analysis; D020893:Receptor, ErbB-3 / Q000276:immunology; D017526:Receptor, IGF Type 1 / Q000276:immunology; D016707:Tissue Fixation
chemical_list: D000911:Antibodies, Monoclonal; D005557:Formaldehyde; C512478:EGFR protein, human; C581292:ERBB3 protein, human; D066246:ErbB Receptors; D020893:Receptor, ErbB-3; D017526:Receptor, IGF Type 1