B280: Successful Alignment of dissected FFPE Tissue to Nottingham Prognostic Index Plus (NPI+) Classes Using RPPA Technology
1Histopathology Department, Nottingham City Hospitals, Nottingham, UK,2Division of Immunology, School of medicine, Nottingham, UK,3Pathology Department, University of Benghazi, Libya, Libya
Reverse Phase Protein Array (RPPA) represents a sensitive and high-throughput technique allowing simultaneous quantitation of protein expression levels in biological samples. In this study we aim to assess the accuracy of RPPA applied to formalin-fixed paraffin-embedded (FFPE) breast cancer tissues in qualifying a large panel of biomarkers and to validate the use of RPPA in improving molecular classification of breast cancer in order to stratify the patients according to the NPI+ decision making tool.
Proteins were extracted from dissected FFPE breast cancer tissues using three different dissection protocols; full face sections, macrodissection and laser captured microdissection (LCM). Three protein extraction protocols were applied to assess the yield and quality of protein extracts from FFPE tissue. Ten different biomarkers of NPI+ (ER, PgR, HER2, Cytokeratin 5/6, Cytokeratin CK7/8, EGFR, HER3, HER4, p53 and Mucin 1) were quantified using RPPA.
Q-proteome FFPE Tissue Kit (Qiagen, Hilden, Germany) produced significantly higher protein concentration and signal intensities. Expression of the biomarkers, individually and in combination, using RRPA was highly consistent with those assessed using immunohistochemistry.
Our results provide evidence for the technical feasibility and clinical utility of RPPA for high-throughput protein expression profiling in FFPE tissue. Quantitative levels of the assayed proteins produced the same NPI+ group assignment of the studied BC cases.