Abstract P2-02-06: Her2-low ctcs in breast cancer: Pipeline for phenotypic driven, single-cell collection and molecular analysis
Authors: D'Amico Paolo , Reduzzi Carolina , Qiang Wenan , Zhang Qiang , Gerratana Lorenzo , Shah Ami N , Davis Andrew A , Kang Anthony , Shi Meilynn , Zhang Youbin , Jacob Saya , Behdad Amir , Curigliano Giuseppe , Cristofanilli Massimo 
Published: March 2022

Abstract
Background The presence of circulating tumor cells (CTCs) expressing HER2 at low score (HER2-low) in the peripheral blood of metastatic breast cancer (MBC) patients has been associated with resistance to treatment and more aggressive metastatic behavior. However, the biological intrinsic nature of HER2-low CTCs remains unexplored. Considering the technical challenges beyond the selective collection of immunophenotype-specific CTCs, we developed a pipeline to individually capture HER2-low CTCs to perform single-cell molecular analysis. Methods 4 different breast cancer cell lines (MDA-MB-231, T47D, MDA-MB-453, SKBR3), that are known to express HER2 at different immunohistochemistry scores (respectively classified as 0, 1+, 2+, 3+), were spiked (around 500 cells each) in healthy donor blood tubes (7.5 ml). Samples were subsequently enriched through the CellSearch™ (Menarini Silicon Biosystems, Bologna, Italy), HER2 stained using the CellSearch CTC Kit and analyzed with the ACCEPT tool (Zeune et al., 2017). Enriched cells were additionally characterized by the DEPArray NxT™ Cell Browser and subsequently collected in pooled and single cells. The HER2 signal-intensity scores (fluorescein isothiocyanate, FITC mean), detected by both tools in each cell line, was compared using the nonparametric Mann-Whitney U test. The optimal cut-offs to distinguish HER2 1+ from HER2 0 and HER2 2+ cells were calculated performing Receiving Operator Curves (ROC). Results Detected mean intensities retrieved from CellSearch™ and analyzed with the ACCEPT tool were respectively 0.34 (MDA-MB-231, 0), 1.44 (T47D, 1+), 15.28 (MDA-MB-453, 2+) 166 (SKBR3, 3+), resulting in the possibility to discriminate both 2+ and 3+ cells from 1+ and 0 (P &lt; 0.00001). No statistical significance has been observed between 1+ and 0 (P=0.19). Conversely, HER2 signal-intensity scores detected with the Cell Browser were: 3.69 (0), 4.38 (1+), 6.28 (2+) and 42.82 (3+). These results allow DEPArray Nxt to efficiently differentiate each single cell line, in particular HER2 1+ cells from both 0 and 2+ (P &lt; 0.00001), enabling the collection of these specific cells. The area under the ROC was 0.7 and 0.72 (respectively 0 vs 1+ and 1+ vs 2+) and the optimal calculated cut-offs were 3.23 (lower) and 4.61 (higher). Conclusions HER2-low CTCs can be detected and separately collected using predetermined intensity cut-offs. Downstream single-cell or pooled collection can be subsequently performed. Further molecular characterizations, as DNA genome sequencing, could highlight the underlying altered pathways responsible for resistance to treatment and molecular patterns accountable for worse prognosis.
Citation Format: Paolo D'Amico, Carolina Reduzzi, Wenan Qiang, Qiang Zhang, Lorenzo Gerratana, Ami N Shah, Andrew A Davis, Anthony Kang, Meilynn Shi, Youbin Zhang, Saya Jacob, Amir Behdad, Giuseppe Curigliano, Massimo Cristofanilli. Her2-low ctcs in breast cancer: Pipeline for phenotypic driven, single-cell collection and molecular analysis [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-02-06.