Molecular Detection of Peripheral Blood Breast Cancer mRNA Transcripts as a Surrogate Biomarker for Circulating Tumor Cells

The detection of circulating tumor cells (CTCs) in peripheral blood may have important prognostic and predictive implications in breast cancer treatment. A limitation in this field has been the lack of a validated method of accurately measuring CTCs. While sensitivity has improved using RT-PCR, specificity remains a major challenge. The goal of this paper is to present a sensitive and specific methodology of detecting CTCs in women with HER-2-positive metastatic breast cancer, and to examine its role as a marker that tracks disease response during treatment with trastuzumab-containing regimens. The study included patients with HER-2-positive metastatic breast cancer enrolled on two different clinical protocols using a trastuzumab-containing regimen. Serial CTCs were measured at planned time points and clinical correlations were made. Immunomagnetic selection of circulating epithelial cells was used to address the specificity of tumor cell detection using cytokeratin 19 (CK19). In addition, the extracellular domain of the HER-2 protein (HER-2/ECD) was measured to determine if CTCs detected by CK19 accurately reflect tumor burden. The presence of CTCs at first restaging was associated with disease progression. We observed an association between CK19 and HER-2/ECD. The association of HER-2/ECD with clinical response followed a similar pattern to that seen with CK19. Finally, the absence of HER-2/ECD at best overall response and a change of HER-2/ECD from positive at baseline to negative at best overall response was associated with favorable treatment response. Our study supports the prognostic and predictive role of the detection of CTCs in treatment of HER-2-positive metastatic breast cancer patients. The association between CK19 and markers of disease burden is in line with the concept that CTCs may be a reliable measure of tumor cells in the peripheral blood of patients with metastatic breast cancer. The association of CTCs at first restaging with treatment failure indicates that CTCs may have a role as surrogate markers to monitor treatment response.

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Can detection of circulating tumor cells in peripheral blood provide prognostic data in breast cancer?

Nature Clinical Practice Oncology ◽

10.1038/ncponc0742 ◽

2007 ◽

Vol 4 (3) ◽

pp. 154-155 ◽

Cited By ~ 4

Author(s):

Gro Wiedswang ◽

Bjørn Naume

Keyword(s):

Breast Cancer ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

Prognostic Data

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Abstract P1-04-11: Recovery rates of circulating tumor cells in breast cancer cell lines spiked into peripheral blood

10.1158/0008-5472.sabcs13-p1-04-11 ◽

2013 ◽

Author(s):

W Zhu ◽

N Mineyev ◽

A Ring ◽

D Tripathy ◽

V Punj ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Cell Lines ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Peripheral Blood ◽

Cancer Cell Lines ◽

Breast Cancer Cell Lines ◽

Recovery Rates

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Persistence of HER2 overexpression on circulating tumor cells in patients after systemic treatment for HER2-positive breast cancer: Follow-up results of the German Success B trial.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.11043 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 11043-11043 ◽

Cited By ~ 1

Author(s):

Julia Katharina Neugebauer ◽

Brigitte Kathrin Rack ◽

Bernadette Anna Sophia Jaeger ◽

Ulrich Andergassen ◽

Aurelia Pestka ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

Her2 Status ◽

Her2 Overexpression ◽

Her2 Expression ◽

Her2 Positive ◽

Before And After

11043 Background: The discordance between HER2-expression on circulating tumor cells (CTC) in peripheral blood and the primary tumor has already been shown by our study group for early breast cancer patients with HER2-positive tumors. Here, we compare the results to CTC prevalence and HER2-status of CTC after adjuvant chemotherapy. Methods: The SUCCESS B trial compares FEC-Docetaxel vs. FEC-Docetaxel-Gemcitabine and HER2-targeted therapy as adjuvant treatment for patients with early, HER2-positive, node positive or high risk node negative primary breast cancer. We prospectively analyzed 23ml peripheral blood before and after chemotherapy. CTC and HER2-status were assessed with the CellSearchSystem (Veridex, USA). After immunomagnetic enrichment with an anti-Epcam-antibody, cells were labeled with anti-CK 8/18/19, anti-CD45 antibodies as well as a fluorescein conjugate antibody for HER2-phenotyping. Cutoff for CTC positivity was ≥ 1 CTC. HER-positivity of CTC was assigned if at least one CTC showed strong HER2 staining (3+). Results: CTCs and their HER2-status both before and after chemotherapy were available for 392 patients. In 179 (45.7%) patients no CTC were detected before and after chemotherapy. CTC status changed from positive before to negative after chemotherapy in 104 (26.5%) patients and from negative before to positive after chemotherapy in 69 (17.6%) patients, while 40 (10.2%) patients had a consistently positive CTC status. Patients were significantly more likely to change their CTC status from positive to negative than from negative to positive (p = 0.01). Of the 40 patients with CTC both before and after chemotherapy, 14 (35%) patients had HER2-positive CTC before and after therapy, and 9 (22%) patients had HER2-negative CTC at both time points. 7 (18%) patients had HER2-positive CTC before but not after chemotherapy, while 10 (25%) patients showed the reverse pattern (p = 0.63). Conclusions: Cytotoxic treatment does not seem to influence the HER2-status on CTC. Follow-up data within the Success B trial will analyze the relevance of the HER2-expression of CTC to predict the efficacy of targeted treatment.

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Detection of circulating tumor cells in peripheral blood of heavily treated metastatic breast cancer patients

Breast Cancer ◽

10.1007/s12282-011-0259-4 ◽

2011 ◽

Vol 18 (3) ◽

pp. 195-202 ◽

Cited By ~ 7

Author(s):

Nahomi Tokudome ◽

Yoshinori Ito ◽

Shunji Takahashi ◽

Kokoro Kobayashi ◽

Shinichiro Taira ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

Metastatic Breast ◽

Breast Cancer Patients

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Circulating Tumor Cells as Predictors of Response and Failure in Breast Cancer Patients Treated with Preoperative Chemotherapy

The International Journal of Biological Markers ◽

10.5301/jbm.2012.9580 ◽

2013 ◽

Vol 28 (1) ◽

pp. 17-23 ◽

Cited By ~ 4

Author(s):

Rimoun R. Boutrus ◽

Rita F. Abi Raad ◽

Irene Kuter ◽

Marek Ancukiewicz ◽

Lisa Roberts ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Preoperative Chemotherapy ◽

Peripheral Blood ◽

Tumor Volume ◽

Single Agent ◽

Definitive Treatment ◽

Cytokeratin 19 ◽

Second Phase

Aim To explore the significance of circulating tumor cells (CTCs) detection in the course of preoperative chemotherapy (PC) and their effect on the outcomes. Methods Fifty-five patients with stage II/III invasive breast cancer were enrolled into a preoperative clinical trial. Patients were given PC with sequential single-agent doxorubicin and paclitaxel vs paclitaxel followed by doxorubicin. Blood samples (8 mL) were collected from patients before PC, after each phase, and at 6 months intervals during follow-up. Peripheral blood mononuclear cells were isolated and enriched for epithelial cells. Quantitative RT-PCR was used to determine the presence of cytokeratin 19 (CK19) mRNA. Samples were considered positive when the PCR curve crossed the standard threshold curve. Results After the first phase of chemotherapy, there was a 59% overall reduction in the median tumor volume. The percentage of volume reduction did not differ between patients who presented with detectable CTCs at baseline and those who did not (p=0.89). After the second phase of chemotherapy, there was a further decrease in the median tumor volume to 93% from baseline. There was no correlation between the lack of response and the presence of CTCs either after the first (p=0.36) or second (p=0.5391) phases of PC. The presence of CTCs was a predictor of local or distant relapse (p=0.0411). The detection of CTCs did not affect overall survival (p=0.2569). Conclusion CTCs can be used as predictors of relapse after definitive treatment of locally advanced breast cancer; however, CTCs detection in peripheral blood during the course of PC does not implicate a particular pattern of response to treatment.

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Clinical Significance and the Detection of Circulating Tumor Cells in Peripheral Blood of Patients with Breast Cancer

Annals of Oncology ◽

10.1093/annonc/mdu328.3 ◽

2014 ◽

Vol 25 ◽

pp. iv110

Author(s):

J. Liu ◽

Q. Zheng ◽

W. Huang ◽

F. Gong ◽

F. Wu ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Clinical Significance ◽

Peripheral Blood

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Clinical Relevance of Immune Checkpoints on Circulating Tumor Cells in Breast Cancer

Cancers ◽

10.3390/cancers12020376 ◽

2020 ◽

Vol 12 (2) ◽

pp. 376 ◽

Cited By ~ 6

Author(s):

Maria A. Papadaki ◽

Anastasios V. Koutsopoulos ◽

Panormitis G. Tsoulfas ◽

Eleni Lagoudaki ◽

Despoina Aggouraki ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

Tumor Tissue ◽

Mononuclear Cells ◽

Immune Checkpoints ◽

Peripheral Blood Mononuclear ◽

Increased Risk

The role of CD47 and PD-L1 expression on circulating tumor cells (CTCs) remains unclear, and it is currently unknown whether their distribution varies between the blood and tumor tissue in breast cancer (BC). In this study, CD47 and PD-L1 expression was investigated a) on peripheral blood mononuclear cell (PBMC) cytospins from early (n = 100) and metastatic (n = 98) BC patients, by triple immunofluorescence for CD47/PD-L1/Cytokeratins, and b) on matched primary and/or metastatic tumor tissue from CTC-positive patients using immunohistochemistry. CD47+and/orPD-L1+ CTCs were detected in 11%, 16.9%, and 29.6% of early, recurrent, and de novo metastatic patients (p = 0.016). In metastatic disease, CD47highand/orPD-L1high CTCs were associated with disease progression (p = 0.005) and shorter progression-free survival (PFS) (p = 0.010), and independently predicted for an increased risk of relapse (HR: 2.719; p = 0.008) and death (HR: 2.398; p = 0.034). PD-L1 expression rates differed between CTCs and tissue tumor cells and between peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) (positive concordance of 3.8% and 4%, respectively). CD47 expression also differed between CTCs and tumor cells (positive concordance of 11.5%). In conclusion, CTCs expressing CD47 and PD-L1 have independent poor prognostic implications in metastatic BC, indicating a potential role of innate and adaptive immune evasion mechanisms in their metastatic potential. The clinical value of the parallel assessment of the peripheral and local immune response merits further evaluation in BC.

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Mobilization of tumor cells and hematopoietic progenitor cells into peripheral blood of patients with solid tumors [see comments]

Blood ◽

10.1182/blood.v83.3.636.636 ◽

1994 ◽

Vol 83 (3) ◽

pp. 636-640 ◽

Cited By ~ 323

Author(s):

W Brugger ◽

KJ Bross ◽

M Glatt ◽

F Weber ◽

R Mertelsmann ◽

...

Keyword(s):

Breast Cancer ◽

Bone Marrow ◽

Tumor Cell ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

Stage Iv ◽

Breast Cancer Patients ◽

Cell Recruitment ◽

Stage Iv Breast Cancer

Abstract Peripheral blood progenitor cells (PBPCs) are increasingly used for autografting after high-dose chemotherapy. One advantage of PBPCs over the use of autologous bone marrow would be a reduced risk of tumor-cell contamination. However, the actual level of tumor cells contaminating PBPC harvests is poorly investigated. It is currently not known whether mobilization of PBPCs might also result in mobilization of tumor cells. We evaluated 358 peripheral blood samples from 46 patients with stage IV or high-risk stage II/III breast cancer, small cell (SCLC) or non- small cell (NSCLC) lung cancer, as well as other advanced malignancies for the detection of epithelial tumor cells. Monoclonal antibodies against acidic and basic cytokeratin components and epithelial antigens (HEA) were used in an alkaline phosphatase-anti-alkaline phosphatase assay with a sensitivity of 1 tumor cell within 4 x 10(5) total cells. Before initiation of PBPC mobilization, circulating tumor cells were detected in 2/7 (29%) patients with stage IV breast cancer and in 2/10 (20%) patients with extensive-disease SCLC, respectively. In these patients, an even higher number of circulating tumor cells was detected after chemotherapy with VP16, ifosfamide, and cisplatin (VIP) followed by granulocyte colony-stimulating factor (G-CSF). This approach has previously been shown to be highly effective in mobilizing PBPCs. In the 42 patients without circulating tumor cells during steady state, tumor cells were mobilized in 9/42 (21%) patients after VIP+G-CSF induced recruitment of PBPCs. The overall incidence of tumor cells varied between 4 and 5,600 per 1.6 x 10(6) mononuclear cells analyzed. All stage IV breast cancer patients and 50% of SCLC patients were found to concomitantly mobilize tumor cells and PBPCs. Kinetic analyses showed two patterns of tumor cell recruitment depending on the presence or absence of bone marrow disease: (1) early after chemotherapy (between days 1 and 7) in patients without marrow infiltration, and (2) between days 9 and 16 in patients with marrow infiltration, ie, within the optimal time period for the collection of PBPCs. We show that there is a high proportion of patients with circulating tumor cells under steady-state conditions, and in addition a substantial risk of concomitant tumor cell recruitment upon mobilization of PBPCs, particularly in stage IV breast cancer patients with bone marrow infiltration. The biologic and clinical significance of this finding is unknown at present.

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