{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,31]],"date-time":"2026-10-31T16:33:22Z","timestamp":1793464402370,"version":"4.5.0"},"reference-count":29,"publisher":"BMJ","issue":"7","license":[{"start":{"date-parts":[[2021,7,14]],"date-time":"2021-07-14T00:00:00Z","timestamp":1626220800000},"content-version":"unspecified","delay-in-days":13,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/100019329","name":"Emerson Collective","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100019329","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100002491","name":"Bristol-Myers Squibb","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100002491","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100014599","name":"Mark Foundation For Cancer Research","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100014599","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Sidney Kimmel Cancer Center Core","award":["P30 CA006973"],"award-info":[{"award-number":["P30 CA006973"]}],"id":[{"id":"https:\/\/ror.org\/010h6g454","id-type":"ROR","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000054","name":"National Cancer Institute","doi-asserted-by":"publisher","award":["3R01CA142779-09S1A1"],"award-info":[{"award-number":["3R01CA142779-09S1A1"]}],"id":[{"id":"10.13039\/100000054","id-type":"DOI","asserted-by":"publisher"}]},{"name":"The MD Anderson Lung Cancer Moon Shot Program"},{"name":"The Bloomberg~Kimmel Institute for Cancer Immunotherapy"},{"name":"Cancer Prevention and Research Institute of Texas Multi-Investigator Research Award Grant","award":["RP160668"],"award-info":[{"award-number":["RP160668"]}],"id":[{"id":"https:\/\/ror.org\/0003xa228","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":["bmj.com"],"crossmark-restriction":true},"short-container-title":["J Immunother Cancer"],"accepted":{"date-parts":[[2021,6,13]]},"published-print":{"date-parts":[[2021,7]]},"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      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 R\n                      <jats:sup>2<\/jats:sup>\n                      =0.75 (slope=0.92) and R\n                      <jats:sup>2<\/jats:sup>\n                      =0.88 (slope=0.93) for breast carcinoma, respectively, and an average concordance of R\n                      <jats:sup>2<\/jats:sup>\n                      =0.72 (slope=0.86) and R\n                      <jats:sup>2<\/jats:sup>\n                      =0.81 (slope=0.68) for NSCLC. Intersite concordance for %PD-L1+ICs had an average R\n                      <jats:sup>2<\/jats:sup>\n                      value of 0.88 and slope of 0.92. Assessments of PD-1\/PD-L1 proximity also showed strong concordance (R\n                      <jats:sup>2<\/jats:sup>\n                      =0.82; slope=0.75).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1136\/jitc-2020-002197","type":"journal-article","created":{"date-parts":[[2021,7,15]],"date-time":"2021-07-15T12:32:02Z","timestamp":1626352322000},"page":"e002197","update-policy":"https:\/\/doi.org\/10.1136\/crossmarkpolicy","source":"Crossref","is-referenced-by-count":104,"title":["Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE) Study"],"prefix":"10.1136","volume":"9","author":[{"given":"Janis M","family":"Taube","sequence":"first","affiliation":[{"name":"Department of Dermatology, The Johns Hopkins Hospital, Baltimore, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristin","family":"Roman","sequence":"additional","affiliation":[{"name":"Akoya Biosciences, Marlborough, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeth L","family":"Engle","sequence":"additional","affiliation":[{"name":"Department of Dermatology, The Johns Hopkins Hospital, Baltimore, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chichung","family":"Wang","sequence":"additional","affiliation":[{"name":"Akoya Biosciences, Marlborough, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carmen","family":"Ballesteros-Merino","sequence":"additional","affiliation":[{"name":"Department of Molecular Microbiology and Immunology, Providence Cancer Institute, Earle A. 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