title: Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues.
abstract: The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website.
authors.affiliation: From the ‡Division of Laboratory Medicine, Geneva University Hospitals, Rue Gabrielle-Perret-Gentil 4, CH-1211 Geneva, Switzerland; §Translational Technologies and Bioinformatics, Pharmaceutical Sciences, Roche Pharma Research & Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Grenzacherstrasse 124, CH-4070 Basel, Switzerland; carine.steiner@roche.com.
authors.forename: Carine
authors.initials: C
authors.lastname: Steiner
authors.affiliation: ¶Division of Clinical Pathology, Geneva University Hospitals, Rue Gabrielle-Perret-Gentil 4, CH-1211 Geneva, Switzerland;
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Tille
authors.affiliation: §Translational Technologies and Bioinformatics, Pharmaceutical Sciences, Roche Pharma Research & Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Grenzacherstrasse 124, CH-4070 Basel, Switzerland;
authors.forename: Jens
authors.initials: J
authors.lastname: Lamerz
authors.affiliation: §Translational Technologies and Bioinformatics, Pharmaceutical Sciences, Roche Pharma Research & Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Grenzacherstrasse 124, CH-4070 Basel, Switzerland;
authors.forename: Sabine
authors.initials: S
authors.lastname: Kux van Geijtenbeek
authors.affiliation: ¶Division of Clinical Pathology, Geneva University Hospitals, Rue Gabrielle-Perret-Gentil 4, CH-1211 Geneva, Switzerland;
authors.forename: Thomas A
authors.initials: TA
authors.lastname: McKee
authors.affiliation: ‖Oncology Division, Roche Pharma Research & Early Development (pRED), Roche Innovation Center Penzberg, Roche Diagnostics GmbH, Nonnenwald 2, D-82377 Penzberg, Germany.
authors.forename: Miro
authors.initials: M
authors.lastname: Venturi
authors.affiliation: ¶Division of Clinical Pathology, Geneva University Hospitals, Rue Gabrielle-Perret-Gentil 4, CH-1211 Geneva, Switzerland;
authors.forename: Laura
authors.initials: L
authors.lastname: Rubbia-Brandt
authors.affiliation: From the ‡Division of Laboratory Medicine, Geneva University Hospitals, Rue Gabrielle-Perret-Gentil 4, CH-1211 Geneva, Switzerland;
authors.forename: Denis
authors.initials: D
authors.lastname: Hochstrasser
authors.affiliation: §Translational Technologies and Bioinformatics, Pharmaceutical Sciences, Roche Pharma Research & Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Grenzacherstrasse 124, CH-4070 Basel, Switzerland;
authors.forename: Paul
authors.initials: P
authors.lastname: Cutler
authors.affiliation: From the ‡Division of Laboratory Medicine, Geneva University Hospitals, Rue Gabrielle-Perret-Gentil 4, CH-1211 Geneva, Switzerland;
authors.forename: Pierre
authors.initials: P
authors.lastname: Lescuyer
authors.affiliation: §Translational Technologies and Bioinformatics, Pharmaceutical Sciences, Roche Pharma Research & Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Grenzacherstrasse 124, CH-4070 Basel, Switzerland;
authors.forename: Axel
authors.initials: A
authors.lastname: Ducret
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism*; D005260:Female; D005557:Formaldehyde; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D013058:Mass Spectrometry; D016612:Paraffin Embedding; D010455:Peptides / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D016707:Tissue Fixation
chemical_list: D005557:Formaldehyde; D010455:Peptides; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human