Abstract 687: Development of MAGE-A4 IHC 1F9 pharmDx for melanoma-associated antigen 4 (MAGE-A4) detection in FFPE synovial sarcoma specimens for afamitresgene autoleucel therapy

Abstract Introduction: MAGE-A4 IHC 1F9 pharmDx is a qualitative immunohistochemistry assay developed as a companion diagnostic for afamitresgene autoleucel therapy to detect Melanoma-associated antigen 4 (MAGE-A4) expression in synovial sarcoma (SyS) specimens. The assay’s analytical performance has been validated on the Autostainer Link 48 Platform using mouse monoclonal anti-MAGE-A4 antibody, clone OTI1F9, in formalin-fixed paraffin-embedded (FFPE) human synovial sarcoma (SyS). Squamous non-small cell lung carcinoma (sqNSCLC) specimens were used in the analytical performance evaluation to supplement sample size and support the use of sqNSCLC as a positive control due to the rarity of SyS. MAGE-A4 positivity was defined as nuclear and/or cytoplasmic staining in viable tumor cells and MAGE-A4 expression was determined using a Tumor Intensity Proportion Score (TIPS) algorithm. Analytical validation of the MAGE-A4 IHC 1F9 pharmDx assay included sensitivity, precision, and robustness testing of MAGE-A4 expression in SyS and sqNSCLC specimens based on the MAGE-A4 TIPS (≥ 2+) ≥ 75% cutoff. Methods: Sensitivity determined if the assay could detect a range of MAGE-A4 expression levels in SyS and sqNSCLC specimens. Precision testing assessed variability in observers/run/instrument/operator/day/lot. Observer precision was evaluated by inter/intra-observer agreement. Robustness studies evaluated assay performance variability for target retrieval solution (pH, re-use, temperature and time), slide type, and tissue section thickness. SyS and sqNSCLC data were analyzed together using negative percent agreement (NPA), positive percent agreement (PPA), and overall agreement (OA) with two-sided 95% bootstrap confidence intervals (CIs) based on MAGE-A4 binary status (positive/negative) at the cutoff. The following acceptance criterion (AC) was applied: the lower bound (LB) of the two-sided 95% CI computed on percent agreement (NPA, PPA, and OA) ≥ 85%. Results: MAGE-A4 IHC 1F9 pharmDx detected a dynamic range of staining levels in SyS and sqNSCLC. The prevalence of MAGE-A4-positive cases per cutoff in the commercially procured FFPE SyS specimens tested was 30.3%. MAGE-A4 IHC 1F9 pharmDx demonstrated acceptable performance characteristics (sensitivity, precision, robustness) and met all predefined AC. The NPA 95% CI LB for slide type was 83.9%, however, this result was due to lower-than-expected sample size since the NPA point estimate was 100%. The MAGE-A4 TIPS (≥ 2+) scoring algorithm is practical, transferrable, and reproducible per the assessment of inter/intra-observer percent agreement point estimates being >93%. Conclusion: These studies demonstrated high analytical assay performance and observer scoring precision of the MAGE-A4 IHC 1F9 pharmDx assay in SyS and combined with sqNSCLC specimens at MAGE-A4 TIPS (≥ 2+) ≥ 75%. Citation Format: Andrea N. Marquez, Siena Tabuena-Frolli, Sneha L. Koneru, Pedro Corona, Rose-Ann De Los Santos, Adrian Miranda, Kristopher Kersch, Eric Broxham, Regina Singson. Development of MAGE-A4 IHC 1F9 pharmDx for melanoma-associated antigen 4 (MAGE-A4) detection in FFPE synovial sarcoma specimens for afamitresgene autoleucel therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 687.