Development of a LAG-3 Immunohistochemistry Assay for Melanoma

Aims A robust immunohistochemistry (IHC) assay was developed to detect lymphocyte-activation gene 3 (LAG-3) expression by immune cells (ICs) in tumor tissues. LAG-3 is an immuno-oncology target with demonstrable clinical benefit, and there is a need for a standardized, well-characterized assay to measure its expression. This study aims to describe LAG-3 scoring criteria and present the specificity, sensitivity, analytical precision, and reproducibility of this assay. Methods The specificity of the assay was investigated by antigen competition and with LAG3 knockout cell lines. A melanin pigment removal procedure was implemented to prevent melanin interference in IHC interpretation. Formalin-fixed, paraffin-embedded (FFPE) human melanoma samples with a range of LAG-3 expression levels were used to assess the sensitivity and analytical precision of the assay with a ≥1% cutoff to determine LAG-3–positivity. Interobserver and intraobserver reproducibility were evaluated with 60 samples in intralaboratory studies and 70 samples in interlaboratory studies. Results The LAG-3 IHC method demonstrated performance suitable for analysis of LAG-3 IC expression in clinical melanoma samples. The pretreatment step effectively removed melanin pigment that could interfere with interpretation. LAG-3 antigen competition and analysis of LAG3 knockout cell lines indicated that the 17B4 antibody clone binds specifically to LAG-3. The intrarun repeatability, interday, interinstrument, interoperator, and interreagent lot reproducibility demonstrated a high scoring concordance (>95%). The interobserver and intraobserver reproducibility and overall interlaboratory and intralaboratory reproducibility also showed high scoring concordance (>90%). Conclusions We have demonstrated that the assay reliably assesses LAG-3 expression in FFPE human melanoma samples by IHC. What is already known on this topic Lymphocyte-activation gene 3 (LAG-3) is an immune checkpoint receptor expressed on immune cells that limits T-cell activity and is being actively explored as a target for immunotherapy. What this study adds An immunohistochemistry assay was developed to detect the LAG-3 protein in formalin-fixed paraffin-embedded human tumor tissue specimens. This study describes scoring criteria and shows the specificity, sensitivity, analytical precision, and reproducibility of this assay as an aid to determine LAG-3 expression in melanoma patients using a ≥1% expression on immune cells threshold. How this study might affect research, practice or policy The study describes a key immuno-oncology checkpoint immunohistochemistry assay that is robust and suitable for clinical trials. The assay was used in RELATIVITY-047 ([NCT03470922][1]), a phase 2/3 clinical trial that compared combined nivolumab and relatlimab treatment with nivolumab monotherapy, to stratify patients based on the percentage of LAG-3–positive immune cells within the tumor region. This assay is also being used in several ongoing clinical trials evaluating clinical response to relatlimab. ### Competing Interest Statement BM, LJ, JY, CS, JS, and SA are employees of Labcorp. BM, LJ, JY, SA, and JS have stock in Labcorp. KJ, AS-C, and SS are consultants/independent contractors of Labcorp. LD and JW are employees of and have stock in Bristol Myers Squibb. CH has stock in Bristol Myers Squibb. DL had stock in Bristol Myers Squibb at the time the study was performed. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT03470922&atom=%2Fbiorxiv%2Fearly%2F2022%2F02%2F26%2F2022.02.25.481964.atom