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A novel, automated technology for multiplex biomarker imaging and application to breast cancer
Author(s) -
Clarke Gina M,
Zubovits Judit T,
Shaikh Kashan A,
Wang Dan,
Dinn Sean R,
Corwin Alex D,
SantamariaPang Alberto,
Li Qing,
NofechMozes Sharon,
Liu Kela,
Pang Zhengyu,
Filkins Robert J,
Yaffe Martin J
Publication year - 2014
Publication title -
histopathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.626
H-Index - 124
eISSN - 1365-2559
pISSN - 0309-0167
DOI - 10.1111/his.12240
Subject(s) - multiplex , biomarker , breast cancer , context (archaeology) , immunohistochemistry , epitope , computer science , pathology , computational biology , cancer , antigen , biomedical engineering , biology , medicine , bioinformatics , immunology , biochemistry , paleontology
Aims Multiplexed immunofluorescence is a powerful tool for validating multigene assays and understanding the complex interplay of proteins implicated in breast cancer within a morphological context. We describe a novel technology for imaging an extended panel of biomarkers on a single, formalin‐fixed paraffin‐embedded breast sample and evaluating biomarker interaction at a single‐cell level, and demonstrate proof‐of‐concept on a small set of breast tumours, including those which co‐express hormone receptors with H er2/neu and K i‐67. Methods and results Using a microfluidic flow cell, reagent exchange was automated and consisted of serial rounds of staining with dye‐conjugated antibodies, imaging and chemical deactivation. A two‐step antigen retrieval process was developed to satisfy all epitopes simultaneously, and key parameters were optimized. The imaging sequence was applied to seven breast tumours, and compared with conventional immunohistochemistry. Single‐cell correlation analysis was performed with automated image processing. Conclusions We have described a novel platform for evaluating biomarker co‐localization. Expression in multiplexed images is consistent with conventional immunohistochemistry. Automation reduces inconsistencies in staining and positional shifts, while the fluorescent dye cycling approach dramatically expands the number of biomarkers which can be visualized and quantified on a single tissue section.