Abstract P2-01-03: Truncated HER2 receptor in circulating tumor cells (CTCs) of early and metastatic breast cancer patients

Abstract

Abstract Introduction: Trastuzumab confers significant clinical benefit in patients with early and metastatic HER2-positive breast cancer. However, trastuzumab treated patients will eventually develop resistance to therapy. The presence of a truncated receptor with loss of the extracellular portion of HER2 (p95HER2) has been proposed as a potential mechanism of resistance. Preclinical and clinical data suggest that the expression of p95HER2 is associated with poor overall prognosis, resistance to trastuzumab and sensitivity to lapatinib, a dual EGFR and HER2 tyrosine kinase inhibitor. We have previously shown that approximately 50% of breast cancer patients express HER2 in their CTCs (Kallergi et al., 2007; Kallergi et al., 2008). The aim of the present study was to characterize, for the first time, the expression of p95HER2 in CTCs of breast cancer patients in comparison with the p95HER2 expression of the corresponding primary tumors. Methods: Two different cells lines (SKBR3 and SCOV3) were used as positive controls expressing the normal and truncated HER2 receptor, respectively. Western blot analysis revealed that SCOV3 was positive for p95 fragment of HER2 receptor while SKBR3 had the full length protein. Consequently we developed a novel triple immunofluorescent (IF) assay for the simultaneous evaluation of the extracellular and intracellular domain of HER2 receptor using the corresponding anti-rabbit and anti-mouse antibodies respectively. Cells were also stained with pancytokeratin A45-B/B3 antibody (as a marker of CK-positive cells). Triple staining experiments were performed in peripheral blood mononuclear cells (PBMC) cytospins' from patients with early (n = 24) and metastatic (n = 33) breast cancer. Slides were analysed with either confocal laser scanning microscopy or with the Ariol system. Results: Cytokeratin positive cells were identified in 17 (71%) out of 24 early and in 20 (61%) out of 33 metastatic patients. HER2 positive CTCs were identified in 53% of early and 50% of metastatic, CTC-positive, breast cancer patients. HER2-positive CTCs lacking the extracellular domain of the receptor (p95HER positive CTCs) were identified in 7 out of 20 (35%) of metastatic and 2 out of 17 (12%) of early, breast cancer patients. Exclusively p95HER2 positive CTCs were detected in 2 out of 20 (10%) patients with metastatic but not in patients with early disease. The corresponding primary tumors from 8 HER2-positive breast cancer patients were also triple stained. These experiments revealed that in two out of eight patients, p95HER2-positive cells were detected in a subgroup (5–10%) of cancer cells. Conclusions: Our results suggest that the truncated p95HER2 receptor is expressed in CTCs of both early and metastatic breast cancer patients. This finding has important therapeutic implications and may explain the observed increased efficacy of the trastuzumab plus lapatinib combination in HER2 positive breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-03.