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Expression of truncated human epidermal growth factor receptor 2 on circulating tumor cells of breast cancer patients.

Kallergi G, Agelaki S, Papadaki MA, Nasias D, Matikas A, Mavroudis D, Georgoulias V - Breast Cancer Res. (2015)

Bottom Line: Trastuzumab reduced the percentage of patients with full-length HER2-positive CTCs from 70 % at baseline to 50 % (p = 0.035) after treatment while increased the percentage of patients with p95HER2-positive CTCs from 40 % to 63 %.Moreover, the overall survival of metastatic patients with p95HER2-positive CTCs was significantly decreased (p = 0.03). p95HER2-positive CTCs can be detected in both early and metastatic breast cancer patients.Their incidence is increased in the metastatic setting and their presence is associated with poor survival.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Τumor Cell Biology, School of Medicine, University of Crete, Voutes, 71110, Heraklion, Crete, Greece. kalergi@med.uoc.gr.

ABSTRACT

Introduction: The truncated form of human epidermal growth factor receptor 2 (p95HER2) lacks the HER2 extracellular domain and has been associated with poor prognosis and resistance to trastuzumab. In the present study, the expression of p95HER2 was investigated on circulating tumor cells (CTCs) from breast cancer patients.

Methods: Triple-staining immunofluorescent experiments were performed on peripheral blood mononuclear cells' (PBMCs) cytospins obtained from patients with early (n = 24) and metastatic (n = 37) breast cancer. Cells were stained with the pancytokeratin (A45-B/B3) antibody coupled with antibodies against the extracellular (ECD) and the intracellular (ICD) domains of HER2. Slides were analyzed with either confocal laser scanning microscopy or with the Ariol system.

Results: HER2-positive CTCs were identified in 55.6 % of early and 65.2 % of metastatic CTC-positive breast cancer patients. p95HER2-positive CTCs were identified in 11.1 % of early and 39.1 % of metastatic breast cancer patients (p = 0.047). In 14 patients with metastatic HER2-positive breast cancer, CTCs were also analyzed before and after first-line trastuzumab therapy. Trastuzumab reduced the percentage of patients with full-length HER2-positive CTCs from 70 % at baseline to 50 % (p = 0.035) after treatment while increased the percentage of patients with p95HER2-positive CTCs from 40 % to 63 %. Moreover, the overall survival of metastatic patients with p95HER2-positive CTCs was significantly decreased (p = 0.03).

Conclusions: p95HER2-positive CTCs can be detected in both early and metastatic breast cancer patients. Their incidence is increased in the metastatic setting and their presence is associated with poor survival. Longitudinal studies during anti-HER2 treatment are required to determine the clinical relevance of p95HER2-expressing CTCs.

No MeSH data available.


Related in: MedlinePlus

Expression of full-length HER2 and its truncated form on control SKBR3 and SCOV3 cells. a Equal protein amounts of SCOV3 and SKBR3 cells were subjected to SDS-PAGE electrophoresis and transferred to nitrocellulose membrane. Total proteins were detected by immunoblotting (IB) with (I): anti-HER2 ECD, (II) HER2 ICD (CB11 clone) and (III) anti-actin. b Representative Ariol system images from control slides with SKBR3 cells. (a) SKBR3 cells stained with HER2 (ECD)/Alexa633/Alexa555 antibodies (b) SKBR3 cells stained with HER2 (ICD)/Alexa633/Alexa555 antibodies and (c) SKBR3 cells stained with HER2(ECD)/HER2 (ICD)/Alexa633/Alexa555 HER2 antibodies. c Representative confocal laser scanning micrographs (×40) of SKBR3 and SCOV3 cells triple-stained with HER2 (ICD), HER2 (ECD) and pancytokeratin (A45-B/B3) antibodies. ECD and ICD were identified in SKBR3 (A-D) cells while low (E-H) or no ECD (I-L) was identified in SCOV3 cells. ECD extracellular domain, HER2 human epidermal growth factor receptor 2, ICD intracellular domain
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Fig1: Expression of full-length HER2 and its truncated form on control SKBR3 and SCOV3 cells. a Equal protein amounts of SCOV3 and SKBR3 cells were subjected to SDS-PAGE electrophoresis and transferred to nitrocellulose membrane. Total proteins were detected by immunoblotting (IB) with (I): anti-HER2 ECD, (II) HER2 ICD (CB11 clone) and (III) anti-actin. b Representative Ariol system images from control slides with SKBR3 cells. (a) SKBR3 cells stained with HER2 (ECD)/Alexa633/Alexa555 antibodies (b) SKBR3 cells stained with HER2 (ICD)/Alexa633/Alexa555 antibodies and (c) SKBR3 cells stained with HER2(ECD)/HER2 (ICD)/Alexa633/Alexa555 HER2 antibodies. c Representative confocal laser scanning micrographs (×40) of SKBR3 and SCOV3 cells triple-stained with HER2 (ICD), HER2 (ECD) and pancytokeratin (A45-B/B3) antibodies. ECD and ICD were identified in SKBR3 (A-D) cells while low (E-H) or no ECD (I-L) was identified in SCOV3 cells. ECD extracellular domain, HER2 human epidermal growth factor receptor 2, ICD intracellular domain

Mentions: SKBR3 and SCOV3 cancer cell lines, which express HER2, were used for the evaluation of the triple-staining experiments. SKOV3 cells have been shown to shed lower levels of HER2 extracellular domain (ECD) compared to SKBR3 cells [21] and express both the full-length and the truncated HER2 receptor [4]. Immunoblot analysis, using an antibody against the ECD, showed that both cell lines expressed the full-length receptor [Fig. 1a (I)] whereas blotting with the antibody against the intracellular domain (ICD) revealed that SCOV3 cells express higher levels of an extra fragment of 95 kDa molecular weight [Fig. 1a (II)]. This observation confirmed that both SKBR3 and SCOV3 could be used as controls for full-length and p95HER2 staining experiments.Fig. 1


Expression of truncated human epidermal growth factor receptor 2 on circulating tumor cells of breast cancer patients.

Kallergi G, Agelaki S, Papadaki MA, Nasias D, Matikas A, Mavroudis D, Georgoulias V - Breast Cancer Res. (2015)

Expression of full-length HER2 and its truncated form on control SKBR3 and SCOV3 cells. a Equal protein amounts of SCOV3 and SKBR3 cells were subjected to SDS-PAGE electrophoresis and transferred to nitrocellulose membrane. Total proteins were detected by immunoblotting (IB) with (I): anti-HER2 ECD, (II) HER2 ICD (CB11 clone) and (III) anti-actin. b Representative Ariol system images from control slides with SKBR3 cells. (a) SKBR3 cells stained with HER2 (ECD)/Alexa633/Alexa555 antibodies (b) SKBR3 cells stained with HER2 (ICD)/Alexa633/Alexa555 antibodies and (c) SKBR3 cells stained with HER2(ECD)/HER2 (ICD)/Alexa633/Alexa555 HER2 antibodies. c Representative confocal laser scanning micrographs (×40) of SKBR3 and SCOV3 cells triple-stained with HER2 (ICD), HER2 (ECD) and pancytokeratin (A45-B/B3) antibodies. ECD and ICD were identified in SKBR3 (A-D) cells while low (E-H) or no ECD (I-L) was identified in SCOV3 cells. ECD extracellular domain, HER2 human epidermal growth factor receptor 2, ICD intracellular domain
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4541732&req=5

Fig1: Expression of full-length HER2 and its truncated form on control SKBR3 and SCOV3 cells. a Equal protein amounts of SCOV3 and SKBR3 cells were subjected to SDS-PAGE electrophoresis and transferred to nitrocellulose membrane. Total proteins were detected by immunoblotting (IB) with (I): anti-HER2 ECD, (II) HER2 ICD (CB11 clone) and (III) anti-actin. b Representative Ariol system images from control slides with SKBR3 cells. (a) SKBR3 cells stained with HER2 (ECD)/Alexa633/Alexa555 antibodies (b) SKBR3 cells stained with HER2 (ICD)/Alexa633/Alexa555 antibodies and (c) SKBR3 cells stained with HER2(ECD)/HER2 (ICD)/Alexa633/Alexa555 HER2 antibodies. c Representative confocal laser scanning micrographs (×40) of SKBR3 and SCOV3 cells triple-stained with HER2 (ICD), HER2 (ECD) and pancytokeratin (A45-B/B3) antibodies. ECD and ICD were identified in SKBR3 (A-D) cells while low (E-H) or no ECD (I-L) was identified in SCOV3 cells. ECD extracellular domain, HER2 human epidermal growth factor receptor 2, ICD intracellular domain
Mentions: SKBR3 and SCOV3 cancer cell lines, which express HER2, were used for the evaluation of the triple-staining experiments. SKOV3 cells have been shown to shed lower levels of HER2 extracellular domain (ECD) compared to SKBR3 cells [21] and express both the full-length and the truncated HER2 receptor [4]. Immunoblot analysis, using an antibody against the ECD, showed that both cell lines expressed the full-length receptor [Fig. 1a (I)] whereas blotting with the antibody against the intracellular domain (ICD) revealed that SCOV3 cells express higher levels of an extra fragment of 95 kDa molecular weight [Fig. 1a (II)]. This observation confirmed that both SKBR3 and SCOV3 could be used as controls for full-length and p95HER2 staining experiments.Fig. 1

Bottom Line: Trastuzumab reduced the percentage of patients with full-length HER2-positive CTCs from 70 % at baseline to 50 % (p = 0.035) after treatment while increased the percentage of patients with p95HER2-positive CTCs from 40 % to 63 %.Moreover, the overall survival of metastatic patients with p95HER2-positive CTCs was significantly decreased (p = 0.03). p95HER2-positive CTCs can be detected in both early and metastatic breast cancer patients.Their incidence is increased in the metastatic setting and their presence is associated with poor survival.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Τumor Cell Biology, School of Medicine, University of Crete, Voutes, 71110, Heraklion, Crete, Greece. kalergi@med.uoc.gr.

ABSTRACT

Introduction: The truncated form of human epidermal growth factor receptor 2 (p95HER2) lacks the HER2 extracellular domain and has been associated with poor prognosis and resistance to trastuzumab. In the present study, the expression of p95HER2 was investigated on circulating tumor cells (CTCs) from breast cancer patients.

Methods: Triple-staining immunofluorescent experiments were performed on peripheral blood mononuclear cells' (PBMCs) cytospins obtained from patients with early (n = 24) and metastatic (n = 37) breast cancer. Cells were stained with the pancytokeratin (A45-B/B3) antibody coupled with antibodies against the extracellular (ECD) and the intracellular (ICD) domains of HER2. Slides were analyzed with either confocal laser scanning microscopy or with the Ariol system.

Results: HER2-positive CTCs were identified in 55.6 % of early and 65.2 % of metastatic CTC-positive breast cancer patients. p95HER2-positive CTCs were identified in 11.1 % of early and 39.1 % of metastatic breast cancer patients (p = 0.047). In 14 patients with metastatic HER2-positive breast cancer, CTCs were also analyzed before and after first-line trastuzumab therapy. Trastuzumab reduced the percentage of patients with full-length HER2-positive CTCs from 70 % at baseline to 50 % (p = 0.035) after treatment while increased the percentage of patients with p95HER2-positive CTCs from 40 % to 63 %. Moreover, the overall survival of metastatic patients with p95HER2-positive CTCs was significantly decreased (p = 0.03).

Conclusions: p95HER2-positive CTCs can be detected in both early and metastatic breast cancer patients. Their incidence is increased in the metastatic setting and their presence is associated with poor survival. Longitudinal studies during anti-HER2 treatment are required to determine the clinical relevance of p95HER2-expressing CTCs.

No MeSH data available.


Related in: MedlinePlus