Abstract 1981: Analytical validation of ANGLE's combined IF and FISH assay for assessment of HER2 in circulating tumor cells

Abstract Background: Evaluation of HER2 status in tissue biopsies, through immunohistochemistry (IHC) and, in equivocal cases, fluorescence in-situ hybridization (FISH), is routinely performed to guide diagnosis and treatment decisions for breast cancer (BC) patients. Tissue biopsy is an invasive procedure for which re-examination is difficult and not commonly performed. Conversely, liquid biopsy is minimally invasive, with unbounded opportunity for recurrent sample collection, allowing monitoring of circulating tumor cells (CTCs). This study aimed to validate a Research Use Only assay by which HER2 immunofluorescence (IF) and FISH are performed on the same CTCs in each sample. Method: Analytical performance of the test was established using contrived samples consisting of HER2+ (SKBR3) and HER2- (MCF-7 and Hs 578T) cultured cancer cells spiked into healthy volunteer blood samples, drawn in Streck Cell-Free DNA tubes. Blood was separated within 144 hours post-draw on ANGLE’s Parsortix® instrument, a marker-independent microfluidic device which isolates CTCs based on size and deformability. Harvests were spun onto CellKeep™ slides and stained with ANGLE’s HER2 IF panel, comprising a nuclear dye, epithelial, mesenchymal and leukocyte markers and an anti-HER2 antibody. Slides were imaged on a BioView Allegro Plus, a platform equipped with artificial intelligence for automated fluorescence imaging, quantitative cellular measurements, CTC candidate identification and reporting. IF stained slides were destained and probed for ERBB2 and CEP17 genes by FISH. Using the Allegro Plus single cell relocation capabilities, CTCs identified by IF were relocated and imaged at 40× to capture HER2 and CEP17 foci. Results. The workflow was performed on a total of 186 contrived samples to determine analytical performance of the assay. Linearity, specificity, sensitivity, repeatability, reproducibility (inter-donor, operators, days and reagents) and endogenous interference (Albumin, Haematocrit and Triglycerides) were assessed. The IF assay was observed to be linear over the range of 0 - 1000 cells, with R2 of 0.96 and degree of non-linearity of 0.004. Analytical specificity and sensitivity of all IF markers were >96%. Analytical specificity and sensitivity of the FISH assay were 100%. The assay was shown to be repeatable and reproducible across spiked donors, operators, days and reagents. Endogenous substances investigated did not affect qualitative results of the assay. Conclusion: This study demonstrated that ANGLE’s combined IF and FISH assay is a robust method for determination of HER2 protein expression and concomitant interrogation of HER2 gene amplification on the cancer cells. This workflow will be utilized to analyze CTCs isolated from a cohort of ∼100 metastatic BC patients to evaluate its clinical feasibility and validity and these results will be reported at the AACR annual meeting. Citation Format: Mariacristina Ciccioli, Alex Young, Laila Vlietinck, Bethan Edwards, Chassidy Johnson, Anne-Sophie Pailhes-Jimenez. Analytical validation of ANGLE's combined IF and FISH assay for assessment of HER2 in circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1981.