The GigaAssay: A combined high throughput and highly accurate platform for variant classification

ERBB2 (HER2) is a critical oncogene, yet the comprehensive and standardized assessment of its variant effects, particularly concerning receptor tyrosine kinase function, remains a major challenge. To compare the oncogenic potential of benchmark variants, we utilized the GigaAssay, a modular high-throughput one-pot assay system capable of simultaneously measuring their molecular function. We designed the GigaAssay to evaluate the relative oncogenic potential of HER2 variants by measuring HER2 phosphorylation at Y1248 (pHER2) in a ligand-free system. Using a benchmark dataset, the HER2 GigaAssay demonstrated robust performance, achieving 100 % accuracy, sensitivity, and F1 score in classifying gain-of-function (GOF) variants linked to hyperphosphorylation and wild type (WT) variants. Furthermore, the normalization of GOF HER2 variants revealed a gradient of elevated phosphorylation activity. Three previously reported oncogenic mutants (Q679L, L726F, and H878Y) were identified as non-pY1248 GOF mutations, aligning with literature and independent flow cytometry measurements, and highlighting the complexity of HER2 signaling. This study establishes the GigaAssay as a highly accurate and standardized platform for the precision classification of HER2 variant function.