Development of a LAG-3 Immunohistochemistry Assay for Melanoma

AimsA 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. MethodsThe 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. ResultsThe 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%). ConclusionsWe have demonstrated that the assay reliably assesses LAG-3 expression in FFPE human melanoma samples by IHC. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSLymphocyte-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 addsAn 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 policyThe 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), 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.