PMID- 34266881 OWN - NLM STAT- In-Process VI - 9 IP - 7 TI - Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE) Study. CI - © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. LA - eng PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PL - England TA - J Immunother Cancer JT - Journal for immunotherapy of cancer JID - 101620585 IS - 2051-1426 (Electronic) LID - e002197 [pii] LID - 10.1136/jitc-2020-002197 [doi] FAU - Taube, Janis M AU - Taube JM AD - Department of Dermatology, The Johns Hopkins Hospital, Baltimore, Maryland, USA jtaube1@jhmi.edu. FAU - Roman, Kristin AU - Roman K AD - Akoya Biosciences, Marlborough, Massachusetts, USA. FAU - Engle, Elizabeth L AU - Engle EL AD - Department of Dermatology, The Johns Hopkins Hospital, Baltimore, Maryland, USA. FAU - Wang, Chichung AU - Wang C AD - Akoya Biosciences, Marlborough, Massachusetts, USA. FAU - Ballesteros-Merino, Carmen AU - Ballesteros-Merino C AD - Department of Molecular Microbiology and Immunology, Providence Cancer Institute, Earle A. Chiles Research Institute, Portland, Oregon, USA. FAU - Jensen, Shawn M AU - Jensen SM AD - Department of Molecular Microbiology and Immunology, Providence Cancer Institute, Earle A. Chiles Research Institute, Portland, Oregon, USA. FAU - McGuire, John AU - McGuire J AD - Akoya Biosciences, Marlborough, Massachusetts, USA. FAU - Jiang, Mei AU - Jiang M AD - Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. FAU - Coltharp, Carla AU - Coltharp C AD - Akoya Biosciences, Marlborough, Massachusetts, USA. FAU - Remeniuk, Bethany AU - Remeniuk B AUID- ORCID: 0000-0003-1961-5163 AD - Akoya Biosciences, Marlborough, Massachusetts, USA. FAU - Wistuba, Ignacio AU - Wistuba I AD - Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. FAU - Locke, Darren AU - Locke D AD - Bristol Myers Squibb, Princeton, New Jersey, USA. FAU - Parra, Edwin R AU - Parra ER AD - Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. FAU - Fox, Bernard A AU - Fox BA AD - Department of Molecular Microbiology and Immunology, Providence Cancer Institute, Earle A. Chiles Research Institute, Portland, Oregon, USA. FAU - Rimm, David L AU - Rimm DL AUID- ORCID: 0000-0001-5820-4397 AD - Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA. FAU - Hoyt, Cliff AU - Hoyt C AD - Akoya Biosciences, Marlborough, Massachusetts, USA. IS - 2051-1426 (Linking) SB - IM OTO - NOTNLM OT - *biomarkers OT - *breast neoplasms OT - *immunohistochemistry OT - *lung neoplasms OT - *programmed cell death 1 receptor OT - *tumor PMC - PMC8286792 LR - 20211018 DP - 202107 AB - BACKGROUND: Emerging data suggest predictive biomarkers based on the spatial arrangement of cells or coexpression patterns in tissue sections will play an important role in precision immuno-oncology. Multiplexed immunofluorescence (mIF) is ideally suited to such assessments. Standardization and validation of an end-to-end workflow that supports multisite trials and clinical laboratory processes are vital. Six institutions collaborated to: (1) optimize an automated six-plex assay focused on the PD-1/PD-L1 axis, (2) assess intersite and intrasite reproducibility of staining using a locked down image analysis algorithm to measure tumor cell and immune cell (IC) subset densities, %PD-L1 expression on tumor cells (TCs) and ICs, and PD-1/PD-L1 proximity assessments. METHODS: A six-plex mIF panel (PD-L1, PD-1, CD8, CD68, FOXP3, and CK) was rigorously optimized as determined by quantitative equivalence to immunohistochemistry (IHC) chromogenic assays. Serial sections from tonsil and breast carcinoma and non-small cell lung cancer (NSCLC) tissue microarrays (TMAs), TSA-Opal fluorescent detection reagents, and antibodies were distributed to the six sites equipped with a Leica Bond Rx autostainer and a Vectra Polaris multispectral imaging platform. Tissue sections were stained and imaged at each site and delivered to a single site for analysis. Intersite and intrasite reproducibility were assessed by linear fits to plots of cell densities, including %PDL1 expression by TCs and ICs in the breast and NSCLC TMAs. RESULTS: Comparison of the percent positive cells for each marker between mIF and IHC revealed that enhanced amplification in the mIF assay was required to detect low-level expression of PD-1, PD-L1, FoxP3 and CD68. Following optimization, an average equivalence of 90% was achieved between mIF and IHC across all six assay markers. Intersite and intrasite cell density assessments showed an average concordance of R2=0.75 (slope=0.92) and R2=0.88 (slope=0.93) for breast carcinoma, respectively, and an average concordance of R2=0.72 (slope=0.86) and R2=0.81 (slope=0.68) for NSCLC. Intersite concordance for %PD-L1+ICs had an average R2 value of 0.88 and slope of 0.92. Assessments of PD-1/PD-L1 proximity also showed strong concordance (R2=0.82; slope=0.75). CONCLUSIONS: Assay optimization yielded highly sensitive, reproducible mIF characterization of the PD-1/PD-L1 axis across multiple sites. High concordance was observed across sites for measures of density of specific IC subsets, measures of coexpression and proximity with single-cell resolution.