Abstract A046: Development of a scoring system to classify HER2 protein expression in circulating tumor cells through immunofluorescence following isolation using Parsortix® instruments
Authors: Young Alex , Vlietinck Laila , Edwards Bethan , Cook Liam , Johnson Chassidy , Ciccioli Mariacristina , Pailhes-Jimenez Anne-Sophie 
Published: November 2024

Abstract
Background. HER2 testing through immunohistochemistry (IHC) is routinely performed on tissue biopsy samples from metastatic breast cancer (MBC) patients, resulting in a HER2 score that is used in patient stratification and treatment decisions. However, tissue biopsy is invasive, with little opportunity for re-examination while liquid biopsy is minimally invasive and allows monitoring of disease progression through recurrent sample collection. This study aimed to establish an assay (Research Use Only) allowing scoring of HER2 protein expression in circulating tumor cells (CTCs), by immunofluorescence (IF) staining, using cancer cell lines of known HER2 expression. Method. Cultured cancer cell lines with HER2 expression comparable to tissue samples (i.e. HER2 0, 1+, 2+ and 3+ by IHC score) were used in this study (SKBR3 [3+], HCC1954 [3+], SKOV-3 [2+/3+], ZR-75-1 [2+], MCF-7 [0 – 1+] and Hs 578T [0]). Cultured cancer cells were spun onto CellKeep™ slides and stained with ANGLE’s Portrait HER2 IF panel, comprising a nuclear dye, an anti-HER2 antibody, epithelial, mesenchymal and leukocyte markers. Slides were imaged on a BioView Allegro Plus, a platform equipped with artificial intelligence for automated imaging, CTC candidate identification and reporting. The workflow was implemented using samples from 5 MBC patients with known HER2 IHC scores, whose blood was drawn into Streck Cell-Free DNA tubes and processed on ANGLE's Parsortix® instrument, a marker-independent microfluidic device which isolates CTCs based on size and deformability, before being stained with the Portrait HER2 assay. The cohort is being expanded and currently includes three HER2 0 and two HER2 3+ patients as diagnosed by tissue IHC. Results. By observing mean fluorescence intensity (MFI) of each cancer cell line reported to be reflective of each IHC score, thresholds were set to establish a protein scoring system. 95%, 95% and 91% of SKBR3, HCC1954 and SKOV-3, respectively, fell in the 3+ class, 83% of ZR-75-1 fell in the 2+ class, 69% of MCF-7 fell in the 1+ class and 25% and 89% of MCF-7 and Hs 578T, respectively, fell in the 0 class. ≥1 CTC were found in all 5 subjects (mean=405, median=146), with 60% donors presenting mesenchymal CTCs, 20% epithelial CTCs and 20% epithelial and mesenchymal CTCs. HER2+ CTCs were found in both HER2 3+ donors, but not in the HER2 0 donors. HER2+ CTCs from both donors fell in the 3+ class based on MFI, despite one donor receiving Herceptin treatment (1 CTC) and the other being treatment naïve (435 CTCs). Conclusion. This study showed it is possible to establish a HER2 protein scoring system using cultured cancer cell lines stained with the Portrait HER2 assay and demonstrates the potential utility of liquid biopsy in tracking HER2 status. This scoring system will be applied to CTCs isolated from an extended cohort of MBC patients with known tissue biopsy HER2 IHC results to test the robustness of the system and assess concordance of CTC and tissue biopsy scores. FISH will be performed on the samples from MBC patients to confirm results.
Citation Format: Alex Young, Laila Vlietinck, Bethan Edwards, Liam Cook, Chassidy Johnson, Mariacristina Ciccioli, Anne-Sophie Pailhes-Jimenez. Development of a scoring system to classify HER2 protein expression in circulating tumor cells through immunofluorescence following isolation using Parsortix® instruments [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A046.