Single-Cells Isolation and Molecular Analysis: Focus on HER2-Low CTCs in Metastatic Breast Cancer

Abstract

Although the detection of CTCs expressing HER2 at low intensity (HER2-low CTCs) has been shown to have a negative prognostic value in metastatic breast cancer (MBC) patients, the biological intrinsic nature of HER2-low CTCs remains unexplored. Considering the technical challenges behind the selective collection of immunophenotype-specific CTCs, we developed a pipeline to individually capture HER2-low CTCs. Four different breast cancer cell lines (MDA-MB-231, T47D, MDA-MB-453, and SKBR3), that are known to express HER2 at different immunohistochemistry levels (respectively classified as 0, 1+, 2+, and 3+), were spiked in healthy donor blood tubes (7.5 mL) and processed with the CellSearch® (Menarini Silicon Biosystems, Bologna, Italy) for enrichment and the DEPArray NxT™ for single cell selection. The HER2 signal-intensities of each cell line was compared using the nonparametric Mann–Whitney U test. The optimal cut-offs to distinguish HER2 1+ from 0 and 2+ cells were calculated performing the Receiver operating characteristic (ROC) curve. Median HER2 signal-intensities detected with the DEPArray NxT™ were: 2.59 (0), 3.58 (1+), 5.23 (2+) and 38.37 (3+). DEPArray NxT efficiently differentiated each single cell line (p < 0.001). The area under the ROC curve was 0.69 and 0.70 (respectively 0 vs. 1+ and 1+ vs. 2+) and the optimal calculated cut-offs were 2.85 (lower) and 4.64 (upper). HER2-low CTCs can be detected and separately collected using predetermined intensity cut-offs. This study will allow standardized single-cell or pooled collection of HER2-low CTCs for downstream molecular analyses.

Trastuzumab and first-line taxane chemotherapy in metastatic breast cancer patients with a HER2-negative tumor and HER2-positive circulating tumor cells: a phase II trial

“…One possible approach might be to identify those patients that have no measurable HER2 expression on CTCs and are not expected to respond to trastuzumab-deruxtecan[ 31 ]. Furthermore, various methods for single-cell analyses and more linear HER2 assays have been proposed to enhance the characterization of the HER2-low entity[ 32 ]. A possible strategy to gain fundamental insights into the presence and evolution of HER2 expression on single CTCs in vivo, is to increase the yield through diagnostic leukapheresis.…”

The trichotomy of HER2 expression confers new insights into the understanding and managing for breast cancer stratified by HER2 status

“…Thus, increasing CX3CL1 expression or inhibiting its proteolytic inactivation in tumor cells could improve the response rate to trastuzumab therapy in HER2‐low BC and be a potential predictive biomarker to determine treatment responses. D'Amico et al 81 constructed a pipeline to detect and individually collect HER2‐low circulating tumor cells through a predetermined intensity cutoff to allow for standardized single‐cell or HER2‐low circulating tumor cells collections for downstream molecular analysis. Ultimately, they found that the presence of HER2‐low circulating tumor cells (CTCs) (1+) had a negative prognostic value in patients with Stage IV Aggressive (CTCs ≥5 in 7.5 mL) breast cancer and was associated with specific metastatic behavior.…”

Automatic trajectory control of single cells using dielectrophoresis based on visual feedback

“…It results in a device that is used to study cells and cell interaction. The DEPArray™, 18 allowing retrieval of specific cells inside a sample with low concentration, has been recently used in several publications [19][20][21] for cell analysis despite its limited throughput. However, the control of cell trajectory using dielectrophoresis is ongoing work.…”