title: Clinical utility of reverse phase protein array for molecular classification of breast cancer.
abstract: Reverse Phase Protein Array (RPPA) represents a sensitive and high-throughput technique allowing simultaneous quantitation of protein expression levels in biological samples. This study aimed to confirm the ability of RPPA to classify archival formalin-fixed paraffin-embedded (FFPE) breast cancer tissues into molecular classes used in the Nottingham prognostic index plus (NPI+) determined by immunohistochemistry (IHC). Proteins were extracted from FFPE breast cancer tissues using three extraction protocols: the Q-proteome FFPE Tissue Kit (Qiagen, Hilden, Germany) and two in-house methods using Laemmli buffer with either incubation for 20 min or 2 h at 105 °C. Two preparation methods, full-face sections and macrodissection, were used to assess the yield and quality of protein extracts. Ten biomarkers used for the NPI+ (ER, PgR, HER2, Cytokeratins 5/6 and 7/8, EGFR, HER3, HER4, p53 and Mucin 1) were quantified using RPPA and compared to results determined by IHC. The Q-proteome FFPE Tissue Kit produced significantly higher protein concentration and signal intensities. The intra- and inter-array reproducibility assessment indicated that RPPA using FFPE lysates was a highly reproducible and robust technique. Expression of the biomarkers individually and in combination using RPPA was highly consistent with IHC results. Macrodissection of the invasive tumour component gave more reliable results with the majority of biomarkers determined by IHC, (80 % concordance) compared with full-face sections (60 % concordance). Our results provide evidence for the technical feasibility of RPPA for high-throughput protein expression profiling of FFPE breast cancer tissues. The sensitivity of the technique is related to the quality of extracted protein and purity of tumour tissue. RPPA could provide a quantitative technique alternative to IHC for the biomarkers used in the NPI+.
authors.affiliation: School of Life Sciences, Immunology, University of Nottingham, Nottingham, NG7 2UH, UK. ola.negm@nottingham.ac.uk.|Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt. ola.negm@nottingham.ac.uk.
authors.forename: Ola H
authors.identifier: http://orcid.org/0000-0002-5999-6012
authors.initials: OH
authors.lastname: Negm
authors.affiliation: Department of Histopathology, Division of Cancer and Stem Cells, School of Medicine, The University of Nottingham, Nottingham, UK. mrxam21@nottingham.ac.uk.|Department of Histopathology, Nottingham University Hospitals NHS Trust, Nottingham City Hospital, Nottingham, UK. mrxam21@nottingham.ac.uk.|Department of Pathology, Faculty of Medicine, Benghazi University, Benghazi, Libya. mrxam21@nottingham.ac.uk.
authors.forename: Abir A
authors.initials: AA
authors.lastname: Muftah
authors.affiliation: Department of Histopathology, Division of Cancer and Stem Cells, School of Medicine, The University of Nottingham, Nottingham, UK.|Department of Histopathology, Nottingham University Hospitals NHS Trust, Nottingham City Hospital, Nottingham, UK.
authors.forename: Mohammed A
authors.initials: MA
authors.lastname: Aleskandarany
authors.affiliation: School of Life Sciences, Immunology, University of Nottingham, Nottingham, NG7 2UH, UK.|Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt.
authors.forename: Mohamed R
authors.initials: MR
authors.lastname: Hamed
authors.affiliation: Department of Histopathology, Division of Cancer and Stem Cells, School of Medicine, The University of Nottingham, Nottingham, UK.|Department of Histopathology, Nottingham University Hospitals NHS Trust, Nottingham City Hospital, Nottingham, UK.
authors.forename: Dena A J
authors.initials: DA
authors.lastname: Ahmad
authors.affiliation: Department of Histopathology, Division of Cancer and Stem Cells, School of Medicine, The University of Nottingham, Nottingham, UK.|Department of Histopathology, Nottingham University Hospitals NHS Trust, Nottingham City Hospital, Nottingham, UK.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Nolan
authors.affiliation: Department of Histopathology, Division of Cancer and Stem Cells, School of Medicine, The University of Nottingham, Nottingham, UK.|Department of Histopathology, Nottingham University Hospitals NHS Trust, Nottingham City Hospital, Nottingham, UK.
authors.forename: Maria
authors.initials: M
authors.lastname: Diez-Rodriguez
authors.affiliation: School of Life Sciences, Immunology, University of Nottingham, Nottingham, NG7 2UH, UK.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Tighe
authors.affiliation: Department of Histopathology, Division of Cancer and Stem Cells, School of Medicine, The University of Nottingham, Nottingham, UK.|Department of Histopathology, Nottingham University Hospitals NHS Trust, Nottingham City Hospital, Nottingham, UK.
authors.forename: Ian O
authors.initials: IO
authors.lastname: Ellis
authors.affiliation: Department of Histopathology, Division of Cancer and Stem Cells, School of Medicine, The University of Nottingham, Nottingham, UK.|Department of Histopathology, Nottingham University Hospitals NHS Trust, Nottingham City Hospital, Nottingham, UK.
authors.forename: Emad A
authors.initials: EA
authors.lastname: Rakha
authors.affiliation: Department of Histopathology, Division of Cancer and Stem Cells, School of Medicine, The University of Nottingham, Nottingham, UK.|Department of Histopathology, Nottingham University Hospitals NHS Trust, Nottingham City Hospital, Nottingham, UK.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Green
mesh_terms: D015415:Biomarkers; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000378:metabolism*; D016000:Cluster Analysis; D005260:Female; D006801:Humans; D060787:Neoplasm Grading; D009362:Neoplasm Metastasis; D011379:Prognosis; D040081:Protein Array Analysis / Q000379:methods; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D047368:Tumor Burden
chemical_list: D015415:Biomarkers
keywords: Breast cancer; Formalin-fixed and paraffin-embedded; Nottingham prognostic index plus; Protein extraction; Reverse phase protein array