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. 2023 Sep;76(9):591-598.
doi: 10.1136/jclinpath-2022-208254. Epub 2022 May 9.

Development of a LAG-3 immunohistochemistry assay for melanoma

Lori Johnson  1 , Bryan McCune  2 , Darren Locke  3 , Cyrus Hedvat  3 , John B Wojcik  3 , Caitlin Schroyer  1 , Jim Yan  1 , Krystal Johnson  2 , Angela Sanders-Cliette  2 , Sujana Samala  2 , Lloye M Dillon  3 , Steven Anderson  2 , Jeffrey Shuster  4
Affiliations

Development of a LAG-3 immunohistochemistry assay for melanoma

Lori Johnson et al. J Clin Pathol. 2023 Sep.

Abstract

Aims: A robust immunohistochemistry (IHC) assay was developed to detect lymphocyte-activation gene 3 (LAG-3) expression by immune cells (ICs) in tumour tissues. LAG-3 is an immuno-oncology target with demonstrable clinical benefit, and there is a need for a standardised, well-characterised 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% cut-off 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 inter-reagent 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.

Keywords: IMMUNOHISTOCHEMISTRY; MELANOMA; Pathology, Molecular.

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Conflict of interest statement

Competing interests: 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. LMD and JBW 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.

Figures

Figure 1
Figure 1
Identification of LAG-3 in human tissues using the LAG-3 IHC assay. (A) Detection of LAG-3 in human tonsil tissue. Left-hand image depicts LAG-3 staining pattern in tonsil tissue showing moderate-to-strong plasma membrane/cytoplasmic staining in lymphocytes in germinal centres and interfollicular region. The crypt epithelium is negative. No staining is seen with negative reagent control (right-hand image). (B) Staining of FFPE melanoma samples with negative reagent control (upper) or LAG-3 antibody (lower) before (left) and after (right) melanin removal procedure at ×10 magnification. (C) Examples of LAG-3 staining in FFPE melanoma samples before (upper) and after (lower) the melanin removal procedure at ×20 magnification. FFPE, formalin-fixed paraffin-embedded; IHC, immunohistochemistry; LAG-3, lymphocyte-activation gene 3.
Figure 2
Figure 2
Detection of a range of LAG-3 expression levels using the LAG-3 IHC assay. Bar chart showing scoring distribution across LAG-3–positive samples (defined as those with LAG-3–positive IC content ≥1%) from a set of 100 commercially procured human FFPE melanoma specimens. Of the 100 samples, 38 were LAG-3–positive and 62 were LAG-3–negative. FFPE, formalin-fixed paraffin-embedded; IC, immune cell; IHC, immunohistochemistry; LAG-3, lymphocyte-activation gene 3.
Figure 3
Figure 3
Examples of a range of LAG-3 expression levels detected in melanoma tissues using the LAG-3 IHC assay. Melanoma tissues showing a range of staining (0%–30%) for LAG-3 examined at magnifications of ×10 (left-hand image) and ×20 (right-hand image). IHC, immunohistochemistry; LAG-3, lymphocyte-activation gene 3.
See this image and copyright information in PMC

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