Measurement of LAG-3 Expression Across Multiple Staining Platforms With the 17B4 Antibody Clone

• Published in: Archives of pathology & laboratory medicine (1976) Vol. 147; no. 11; pp. 1307 - 1314
• Main Authors: Wojcik, John B, Desai, Keyur, Avraam, Konstantinos, Vandebroek, Arno, Dillon, Lloye M, Giacomazzi, Giorgia, Rypens, Charlotte, Benci, Joseph L
• Format: Journal Article
• Language: English
• Published: United States College of American Pathologists 01.11.2023
• Subjects: Algorithms; Antibodies; Antitumor activity; Apoptosis; Biochemical assays; Bioethics; Cancer immunotherapy; CD223 antigen; Cell activation; Cell surface; Clinical trials; Cloning; Immune checkpoint inhibitors; Immunohistochemistry; Inflammation; Lymphocytes; Measurement; Melanoma; Metastases; Methods; PD-1 protein; Performance evaluation; Pigmentation; Testing laboratories; Viral antibodies; United States; United Kingdom; United States > US; California
• ISSN: 0003-9985; 1543-2165; 1543-2165
• DOI: 10.5858/arpa.2022-0082-OA

An immunohistochemistry (IHC) assay developed to detect lymphocyte-activation gene 3 (LAG-3), a novel immune checkpoint inhibitor target, has demonstrated high analytic precision and interlaboratory reproducibility using a Leica staining platform, but it has not been investigated on other IHC staining platforms. To evaluate the performance of LAG-3 IHC assays using the 17B4 antibody clone across widely used IHC staining platforms: Agilent/Dako Autostainer Link 48 and VENTANA BenchMark ULTRA compared to Leica BOND-RX (BOND-RX). Eighty formalin-fixed, paraffin-embedded melanoma tissue blocks were cut into consecutive sections and evaluated using staining platform-specific IHC assays with the 17B4 antibody clone. Duplicate testing was performed on the BOND-RX platform to assess intraplatform agreement. LAG-3 expression using a numeric score was evaluated by a pathologist and with a digital scoring algorithm. LAG-3 positivity was determined from manual scores using a 1% or greater cutoff. LAG-3 IHC staining patterns and intensities were visually similar across all 3 staining platforms. Spearman and Pearson correlations were 0.75 or greater for interplatform and BOND-RX intraplatform concordance when LAG-3 expression was evaluated with a numeric score determined by a pathologist. Correlation increased with a numeric score determined with a digital scoring algorithm (Spearman and Pearson correlations ≥0.88 for all comparisons). Overall percentage agreement was 77.5% or greater for interplatform and BOND-RX intraplatform comparisons when LAG-3 positivity was determined using a 1% or greater cutoff. Data presented here demonstrate that LAG-3 expression can be robustly and reproducibly assessed across 3 major commercial IHC staining platforms using the 17B4 antibody clone.