Liquid biopsy identification of ERBB2 amplified and HER2 expressing metastatic breast cancer: Comparison and combination of cell and cell-free platforms.
Authors: Di Caro Giuseppe , Lam Ernest , Bourdon David , Singer Tatjana , Horne Kandra , Slade Megan , Wenstrup Rick , Schwartzberg Lee S. 
Published: June 2023

3061

Background: Liquid biopsies are a non-invasive diagnostic approach for detecting circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) that may provide clinically actionable information for treatment decisions for metastatic breast cancer (MBC) patients when a conventional biopsy is otherwise infeasible. Here we report a comprehensive liquid biopsy platform including: 1) quantitative immunofluorescent HER2 and ER protein expression in CTCs (ctcIF), 2) determination of ERBB2 amplification and the number of Large-scale State transitions (LST) by single-cells CTC genomics (ctcDNA) and 3) alterations in plasma ctDNA. Methods: Blood samples from 457 progressing metastatic breast cancer patients and 25 healthy donors (HDs) as controls were collected for cell & cell-free DNA analysis. After plasma isolation, nucleated cells were plated, & slides were bio-banked for Immunofluorescent staining & subsequent imaging. CTCs were identified using Epic Sciences digital imaging & machine learning algorithms, and individual CTCs were sequenced for genomic quantification of LSTs and Copy Number Variants (CNV). Bio-banked plasma was analyzed to detect ctDNA alterations with high clinical relevance (Class IA). Results: Within this cohort of 457 MBC patients, the presence of CTCs, ctcDNA (LST+) and ctDNA alterations were detected in 98%, 58%, and 42%, respectively, while no CTCs and no ctDNA alterations were detected in the HD cohort, suggesting high specificity. ctcDNA genomics was more sensitive than ctDNA in detecting ERBB2amp in MBC patients (16%, and 2%, respectively). A mean variant allele frequency (VAF) of < 25%, which is the threshold for detecting a two-fold ERBB2 amplification by ctDNA, was present in 20% and 80% of ERBB2amp MBC detected by the ctDNA and ctcDNA platform, respectively, suggesting that the ctcDNA platform can identify the majority of ERBB2amp among patients with a low ctDNA fraction. HER2exp or ERexp expression by ctcIF were detected in 67% and 67% of MBC patients, respectively. A liquid biopsy classification of HER2 status by combining the three platforms (ctcIF, ctcDNA, and ctDNA) identified that in the tested population, 16% were ERBB2amp, 52% were HER2 expressing (HER2exp and ERBB2nonamp), and 27% were HER2neg (HER2- and ERBB2nonamp). To identify MBC patients that may potentially change treatment as a result of the test we identified a small cohort of MBC patients (n=46) with HER2- disease and tissue biopsy IHC=0, 74% of these patients had HER2exp CTC consistent with HER2 low expression which may aid therapeutic decisions in later line MBC. Conclusions: Here we report a comprehensive liquid biopsy profile combining ctcIF, ctcDNA, and ctDNA platforms with high sensitivity and specificity in determining clinically actionable HER2 and ER biomarker status that may impact therapeutic decision-making in MBC patients.