Abstract 6162: High resolution detection of HER2 copy number variation in admixed breast cancer cell lines and HER2+ FFPE donor tissue using a digital PCR multigene reference panel
Authors: Landrith Tyler , Smith Samantha , Chan Jennifer , Christensen Mari , Tai Yu Chuan , Gonzales Megan , Yee William , Newton Nicolas , Pattje Wouter , Bogard Patrick , Yang Wei 
Published: April 2023

Abstract
The measurement of HER2 (ERBB2) gene amplification guides both the prognosis and treatment of breast cancer, as amplification correlates with worse outcomes and the recommendation for anti-HER2 treatment. Digital PCR (dPCR) can serve as a useful tool for the direct quantification of copy number variants (CNV) in this gene, without the need for calibration standards as required for qPCR, or labor-intensive histological techniques such as FISH. Furthermore, increasing the number of available partitions in a digital PCR reaction can yield substantial gains in precision, thus offering the potential for ultra-high resolution discrimination of copy number changes. In this study, we evaluated the utility of a multiplex dPCR assay for the high-resolution detection of HER2 copy number variation in clinically relevant samples. The precision achievable by digital PCR correlates with the number of partitions, and the resolution of small CNV differences greatly relies on high precision. We utilized a dPCR setup with ~100,000 physical partitions per reaction, which allowed for a theoretical precision (relative confidence) of 1%, assuming optimal input and template. Five single-plex primer and probe sets, published by the National Institute of Standards and Technology, were assembled and optimized into a multiplex configuration. The final 5-plex consisted of four reference genes and HER2 as the target of interest. The accuracy and precision of the optimized five-plex assay was tested in nine breast cancer cell lines with established HER2 amplification ratios, ranging from 1.3 to 70 fold. The assay demonstrated the precise and accurate measurement of the HER2 amplification ratio across all nine cell lines over a wide dynamic range of sample input. Along with increasing the reaction partition number, the use of multiple reference genes also improved the precision of the HER2 amplification ratio measurement and avoided inaccurate copy number calls due to outlier references. The resolution of the assay was evaluated using admixed samples simulating low tumor fraction. A minimum of a 1.03 fold difference (3% difference from wild type) was detected with statistical significance. Assay concordance was evaluated against IHC-characterized HER2+ FFPE donor tissue with concordant FISH/CISH results. The assay demonstrated robust performance in clinically relevant sample types, supporting further investigation of dPCR as a complementary or even alternative diagnostic approach to current methods.
Citation Format: Tyler Landrith, Samantha Smith, Jennifer Chan, Mari Christensen, Yu Chuan Tai, Megan Gonzales, William Yee, Nicolas Newton, Wouter Pattje, Patrick Bogard, Wei Yang. High resolution detection of HER2 copy number variation in admixed breast cancer cell lines and HER2+ FFPE donor tissue using a digital PCR multigene reference panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6162.