Analytical characterization of the next-generation HER2 (CAL27) immunohistochemistry assay: A comparative multi-tumor study.

Abstract

e15173 Background: HER2 protein expression has been observed in a variety of solid tumors. Advancements in antibody drug conjugates and other novel agents may benefit from a highly sensitive and specific assay to accurately detect a wide range of HER2 expression (1). A new VENTANA HER2 (CAL27) Assay was developed which shows high analytical specificity and sensitivity (2). To further characterize its analytical performance, this study compared the sensitivity and specificity of the VENTANA HER2 (CAL27) Assay with PATHWAY anti-HER-2/neu (4B5) Rabbit Monoclonal Primary Antibody and HercepTest mAb pharmDx (DG44) using a cohort of solid tumors. Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor specimens (n=404) from biliary tract, bladder, cervical, colorectal, endometrial, gastric, non-small cell lung, ovarian, and pancreatic cancer were sourced from commercial vendors. Tissue sections were stained by immunohistochemistry (IHC) using three different HER2 assays (CAL27, 4B5, and DG44) per the manufacturer’s instructions. Stained sections were evaluated by a pathologist blinded to the assay for non-specific background, cellular and subcellular staining localization, and the percentage of tumor cells stained at each level. H-scores were calculated and paired t-tests were performed. Results: CAL27 demonstrated substantially increased analytical sensitivity compared to the other HER2 assays, as indicated by higher membrane H-score values at each quartile (Q1, median, and Q3). Differences in H-scores when comparing CAL27 vs. 4B5 or DG44 were statistically significant (p<0.0001) based on paired t-tests. Furthermore, CAL27 exhibited greater specificity, demonstrating the lowest prevalence of cytoplasmic and off-target nuclear staining among the assays. In contrast with 4B5 and DG44, there were no instances where CAL27 cytoplasmic or nuclear staining interfered with interpretation of tumor cell membrane staining, the principal staining localization for HER2 IHC assays. Results are summarized in Table 1. Conclusions: The CAL27 assay demonstrated markedly higher analytical sensitivity compared to the 4B5 and DG44 assays across tumor types, with no interference from cytoplasmic or nuclear signals. These analytical data showing sensitive and specific tissue-based evaluation of the widest range of HER2 IHC expression with CAL27 may lead to improved HER2 IHC detection through future clinical investigations. Summary of staining results in tumor area by HER2 IHC assay. Staining Localization CAL27 4B5 DG44 Membrane H-score (quartile 1, median, quartile 3)* 35, 111, 190 3, 18, 71 4, 29, 110 Cytoplasmic 3.2% (13/404) 21.0% (85/404) 4.5% (18/404) Nuclear 0.2% (1/404) 20.0% (81/404) 0.5% (2/404) Cytoplasmic or nuclear interference with membrane staining interpretation 0.0% (0/404) 0.5% (2/404) 0.7% (3/404) *p<0.0001 for CAL27 vs 4B5 or DG44 by paired t-tests.