Single-cells isolation and molecular analysis: Focus on her2-low ctcs in metastatic breast cancer

Paolo D’amico, Carolina Reduzzi, Wenan Qiang, Youbin Zhang, Lorenzo Gerratana, Qiang Zhang, Andrew A. Davis, Ami N Shah, Maroua Manai, Giuseppe Curigliano, Massimo Cristofanilli

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Article number79
Issue number1
StatePublished - Jan 1 2022


  • Breast cancer
  • CellSearch
  • CTC
  • DEPArray
  • HER2
  • HER2-low
  • Liquid biopsy
  • Single-cell

ASJC Scopus subject areas

  • Oncology
  • Cancer Research


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