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Measurement of LAG-3 Expression Across Multiple Staining Platforms With the 17B4 Antibody Clone
John B Wojcik1, Keyur Desai2, Konstantinos Avraam3
1From Translational Sciences and Diagnostics (Wojcik, Dillon), Bristol Myers Squibb, Princeton, New Jersey.
Insights
This study shows that the lymphocyte-activation gene 3 (LAG-3) immunohistochemistry assay performs consistently across multiple platforms. This ensures reliable detection of LAG-3 expression for immune checkpoint inhibitor research.
Area of Science:
- Immunohistochemistry
- Oncology
- Molecular Biology
Background:
- Lymphocyte-activation gene 3 (LAG-3) is a novel immune checkpoint inhibitor target.
- An existing immunohistochemistry (IHC) assay for LAG-3 shows precision on the Leica platform but needs validation on others.
- Evaluating LAG-3 IHC assay performance across different platforms is crucial for its clinical application.
Purpose of the Study:
- To assess the performance of the LAG-3 IHC assay using the 17B4 antibody clone on Agilent/Dako Autostainer Link 48 and VENTANA BenchMark ULTRA platforms.
- To compare the assay's results with the Leica BOND-RX platform.
- To determine the reproducibility and concordance of LAG-3 expression detection across these platforms.
Main Methods:
- Eighty melanoma tissue blocks were stained using platform-specific IHC assays with the 17B4 antibody clone.
- Duplicate testing was performed on the Leica BOND-RX for intraplatform agreement.
- LAG-3 expression was scored numerically by a pathologist and a digital algorithm, with positivity defined as ≥1% expression.
Main Results:
- LAG-3 IHC staining patterns and intensities were visually similar across all three platforms.
- High correlations (Spearman and Pearson ≥0.75) were observed for interplatform and intraplatform concordance using pathologist-derived numeric scores.
- Correlation improved with digital scoring (Spearman and Pearson ≥0.88), and overall agreement was ≥77.5% for positivity determination.
Conclusions:
- The LAG-3 IHC assay using the 17B4 antibody clone demonstrates robust and reproducible performance across major commercial IHC staining platforms.
- This validation supports the widespread use of this assay for assessing LAG-3 expression in research and potentially clinical settings.
- Consistent LAG-3 detection across platforms facilitates reliable patient stratification for immune checkpoint inhibitor therapies.
Context.—:
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.
Objective.—:
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).
Design.—:
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.
Results.—:
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.
Conclusions.—:
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.
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