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

Area of Science:

• Oncology
• Molecular Biology
• Biochemistry

Background:

• ERBB2 (HER2) is a critical oncogene, but assessing its variant effects on receptor tyrosine kinase function is challenging.
• Standardized, high-throughput methods are needed for precise classification of HER2 variant oncogenic potential.

Purpose of the Study:

• To develop and validate a high-throughput assay for comparing the oncogenic potential of ERBB2 (HER2) variants.
• To measure HER2 variant function by assessing phosphorylation at Y1248 in a ligand-free system.

Main Methods:

• Utilized the GigaAssay, a modular, high-throughput, one-pot system for simultaneous molecular function measurement.
• Designed the assay to measure HER2 phosphorylation at Y1248 (pHER2) to evaluate relative oncogenic potential.
• Used a benchmark dataset to test the assay's performance.

Main Results:

• The HER2 GigaAssay achieved 100% accuracy, sensitivity, and F1 score in classifying gain-of-function (GOF) and wild-type (WT) variants.
• A gradient of elevated phosphorylation activity was observed for GOF HER2 variants.
• Identified three known oncogenic mutants as non-pY1248 GOF mutations, confirming assay's ability to reveal signaling complexities.

Conclusions:

• The GigaAssay is a highly accurate and standardized platform for precision classification of HER2 variant function.
• This assay provides a robust tool for research into HER2 signaling and oncogenic variant characterization.
• The findings highlight the complexity of HER2 signaling beyond simple Y1248 phosphorylation.