Detection and characterization of circulating tumor cells in blood of primary breast cancer patients by RT-PCR and comparison to status of bone marrow disseminated cells

Introduction  

The role of circulating tumor cells (CTCs) in blood of primary breast cancer patients is still under investigation. We evaluated the incidence of CTCs in blood, we evaluated the correlation between CTCs and disseminated tumor cells (DTCs) in the bone marrow (BM), and we characterized CTCs for the expression of HER2, the estrogen receptor (ER) and the progesterone receptor (PR).     

Prognostic value of HER2-positive circulating tumor cells in patients with metastatic breast cancer

Backgrounds  

The presence of ≥5 circulating tumor cells (CTCs) in 7.5 ml blood is a poor prognostic marker in metastatic breast cancer (MBC). However, the role of human epidermal growth factor receptor 2 (HER2) status in CTCs is not known.     

A multimarker QPCR-based platform for the detection of circulating tumour cells in patients with early-stage breast cancer

Background:

The detection of circulating tumour cells (CTCs) has been linked with poor prognosis in advanced breast cancer. Relatively few studies have been undertaken to study the clinical relevance of CTCs in early-stage breast cancer.

Methods:

In a prospective study, we evaluated CTCs in the peripheral blood of 82 early-stage breast cancer patients. Control groups consisted of 16 advanced breast cancer patients and 45 healthy volunteers. The CTC detection was performed using ErbB2/EpCAM immunomagnetic tumour cell enrichment followed by multimarker quantitative PCR (QPCR). The CTC status and common clinicopathological factors were correlated to relapse-free, breast cancer-related and overall survival.

Results:

Circulating tumour cells were detected in 16 of 82 (20%) patients with early-stage breast cancer and in 13 out of 16 (81%) with advanced breast cancer. The specificity was 100%. The median follow-up time was 51 months (range: 17–60). The CTC positivity in early-stage breast cancer patients resulted in significantly poorer relapse-free survival (log rank test: P=0.003) and was an independent predictor of relapse-free survival (multivariate hazard ratio=5.13, P=0.006, 95% CI: 1.62–16.31).

Conclusion:

The detection of CTCs in peripheral blood of early-stage breast cancer patients provided prognostic information for relapse-free survival.     

Hypoxia-inducible factor-1α and vascular endothelial growth factor expression in circulating tumor cells of breast cancer patients

Introduction  

The detection of peripheral blood circulating tumor cells (CTCs) and bone marrow disseminated tumor cells (DTCs) in breast cancer patients is associated with a high incidence of disease relapse and disease-related death. Since hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) play an important role in angiogenesis and tumor progression, the purpose of the current study was to investigate their expression in CTCs.     

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

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.

Electronic supplementary material

The online version of this article (doi:10.1186/s13058-015-0624-x) contains supplementary material, which is available to authorized users.     

The prognostic significance of tumour cell detection in the peripheral blood versus the bone marrow in 733 early-stage breast cancer patients

Introduction  

The detection of circulating tumour cells (CTCs) in the peripheral blood and disseminated tumour cells (DTCs) in the bone marrow are promising prognostic tools for risk stratification in early breast cancer. There is, however, a need for further validation of these techniques in larger patient cohorts with adequate follow-up periods.     

Detection of HER2-positive Circulating Tumor Cells Using the LiquidBiopsy System in Breast Cancer

Background

Most previous studies of circulating tumor cells (CTCs) are based on the CellSearch platform, but CellSearch has a number of limitations. This study aimed to use the LiquidBiopsy system and immunofluorescence to test the human epidermal growth factor receptor 2 (HER2) status of CTCs in patients with breast cancer.

Denosumab treatment is associated with the absence of circulating tumor cells in patients with breast cancer

Background

The presence of circulating tumor cells (CTCs) in patients with breast cancer correlates to a bad prognosis. Yet, CTCs are detectable in only a minority of patients with progressive breast cancer, and factors that influence the abundance of CTCs remain elusive.

Methods

We conducted CTC isolation and enumeration in a selected group of 73 consecutive patients characterized by progressive invasive breast cancer, high tumor load and treatment discontinuation at the time of CTC isolation. CTCs were quantified with the Parsortix microfluidic device. Clinicopathological variables, blood counts at the time of CTC isolation and detailed treatment history prior to blood sampling were evaluated for each patient.

Results

Among 73 patients, we detected at least one CTC per 7.5 ml of blood in 34 (46%). Of these, 22 (65%) had single CTCs only, whereas 12 (35%) featured both single CTCs and CTC clusters. Treatment with the monoclonal antibody denosumab correlated with the absence of CTCs, both when considering all patients and when considering only those with bone metastasis. We also found that low red blood cell count was associated with the presence of CTCs, whereas high CA 15-3 tumor marker, high mean corpuscular volume, high white blood cell count and high mean platelet volume associated specifically with CTC clusters.

Conclusions

In addition to blood count correlatives to single and clustered CTCs, we found that denosumab treatment associates with most patients lacking CTCs from their peripheral circulation. Prospective studies will be needed to validate the involvement of denosumab in the prevention of CTC generation.

     

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

Background

Circulating tumor cells (CTCs) are detected in peripheral blood of breast cancer patients, and they may play an important role as a prognostic and predictive marker. We conducted this study to determine the presence of CTCs with the CellSearch System? and the clinical significance in treatment of metastatic breast cancer (MBC).

Method

Twenty-eight MBC patients were enrolled. These patients were followed by assessing CTCs, imaging studies, and serum tumor markers. Blood samples were collected before starting a new treatment and at the treatment evaluation period (2–3 months after starting chemotherapy). The cutoff for CTC level was 5.

Results

At baseline, 9 of 28 patients (32%) had ≥5 CTCs per 7.5 mL of blood. At the evaluation period, 5 of 23 patients (22%) had ≥5 CTCs. The baseline CTC number did not contribute to determine their overall survival (OS); however, CTCs at the evaluation period were available to predict their OS (p < 0.001). In two cases, both CTCs and tumor markers were available as predictors of treatment efficacy. In two other cases, although alterations of tumor markers might not reflect disease condition, CTC alteration corresponded to their condition. One patient who had multiple skeletal metastasis only, experienced a decrease in her CTCs in spite of tumor marker alteration.

Conclusions

We suggest that monitoring the number of CTCs may be helpful in predicting the efficacy of the treatment and the prognosis. CTCs might be especially useful with patients whose lesions are difficult to assess.     

Labour productivity loss caused by premature deaths associated with breast cancer: results from Spain over a 10-year period

Purpose

Several studies have provided evidence on the prognostic relevance of circulating tumor cells (CTCs) detected before and after chemotherapy regarding overall survival (OS) and progression-free survival (PFS) in early breast cancer (EBC). We provide data on the prevalence of CTCs 2 and 5 years after primary diagnosis in a cohort of patients with EBC.

Methods

The SUCCESS study is a multicenter, prospective, randomized trial comparing PFS in primary breast cancer patients undergoing one of two adjuvant chemotherapy regimens followed by 2 versus 5 years of treatment with zoledronate. CTCs from patients without signs of breast cancer recurrence were analyzed in peripheral blood using the FDA cleared CellSearch® System (Veridex, USA) 2 and 5 years after primary diagnosis.

Results

CTCs were detected at 2 and 5 years after primary diagnosis in 96 (16.7%) and 47 (8.2%) of the 574 patients, respectively. There were no associations between CTC status and patient and tumor characteristics or treatment regimens. In 442 (77.0%) patients, no CTCs were detected at either of the two time points, and in 11 patients (1.9%), CTCs were found at both 2 and 5 years after primary diagnosis. In 85 (14.8%) patients, CTCs were present 2 years after primary diagnosis but not after 5 years, while 36 (6.3%) patients had CTCs in their blood only at the 5-year follow-up.

Conclusions

In patients with EBC, CTCs can be detected even 5 years after primary diagnosis without clinical signs of disease recurrence.

     

Epithelial to mesenchymal transition markers expressed in circulating tumour cells of early and metastatic breast cancer patients

Introduction  

Epithelial to mesenchymal transition (EMT) is considered an essential process in the metastatic cascade. EMT is characterised by upregulation of vimentin, Twist, Snail, Slug and Sip1 among others. Metastasis is also associated with the presence of circulating tumour cells (CTCs) and disseminated tumour cells in the blood and bone marrow, respectively, of breast cancer patients, but the expression of EMT markers in these cells has not been reported so far.     

Circulating tumour cell-derived plastin3 is a novel marker for predicting long-term prognosis in patients with breast cancer

Background:

Identification of promising biomarkers that predict the prognosis of patients with breast cancer is needed. In this study, we hypothesised that the expression of the epithelial–mesenchymal transition-related biomarker plastin3 (PLS3) in peripheral blood could be a prognostic factor in breast cancer.

Methods:

We examined PLS3 expression in breast cancer cell lines with epithelial and mesenchymal traits and in circulating tumour cells (CTCs) obtained from the peripheral blood of breast cancer patients. We investigated PLS3 expression in the peripheral blood of 594 patients with breast cancer to evaluate the clinical significance of PLS3 expression.

Results:

Robust PLS3 expression was observed in different breast cancer cell lines (Hs578t, MCF-7, MDA-MB-468, and MDA-MB-231) as well as in a bone marrow derived cancer cell line (BC-M1). In both the training (n=298) and validation (n=296) sets, PLS3 expression was observed in CTCs of patients with breast cancer. PLS3-positive patients showed significantly poorer overall and disease-free survival than PLS3-negative patients (P=0.0001 and 0.003, respectively). Subset analysis revealed that this prognostic biomarker was relevant in patients with stage I–III cancer, particularly in patients with luminal-type and triple-negative-type tumours.

Conclusions:

These data demonstrated that PLS3 was expressed in CTCs undergoing the epithelial–mesenchymal transition in patients with breast cancer. Furthermore, PLS3 may be an excellent biomarker for identifying groups at risk of recurrence or with a poor prognosis.     

Detection and prognostic significance of circulating tumour cells in patients with metastatic breast cancer according to immunohistochemical subtypes

Background:

The enumeration of circulating tumour cells (CTCs) with the EpCAM-based CellSearch system has prognostic significance in patients with metastatic breast cancer (MBC). The aim of this study was to explore potential differences in the detection and prognostic significance of CTCs in MBC according to immunohistochemical subtypes of breast cancer.

Methods:

CellSearch CTC counts were obtained from 154 MBC patients before first-line systemic treatment between November 2007 and August 2012. Patients were categorised in five subgroups according to immunohistochemical surrogate definitions of intrinsic subtypes in breast cancer based on hormone receptor status, HER2/neu status and histological grade. Differences in progression-free (PFS) and overall survival (OS) were assessed relative to the cut-off value of ⩾5 CTCs per 7.5 ml blood.

Results:

No significant differences were observed in the absolute CTC counts (P=0.120) or in CTC positivity rates according to ⩾1 and ⩾5 CTCs per 7.5 ml blood detection thresholds (P=0.165 and P=0.651, respectively) between immunohistochemical subtypes. However, very high CTC counts, defined as ⩾80 CTCs per 7.5 ml, were observed more frequently in patients with Luminal A and triple negative (TN) breast cancer (P=0.024). In the total study population, the presence of ⩾5 CTCs was the single most significant prognostic factor for both PFS and OS in multivariate analysis (P<0.001). A more limited prognostic impact, not reaching statistical significance, was observed in patients with HER2-positive disease as opposed to patients with Luminal A, Luminal B–HER2-negative and TN disease.

Conclusion:

The detection of EpCAM+CTCs was not clearly associated with any of the immunohistochemical subtypes of breast cancer in patients with MBC before first-line treatment. Potentially clinically relevant differences were however observed at very high CTC counts. Furthermore, our data suggest a lower prognostic significance of CTC evaluation in HER2-positive patients with MBC.     

Acute phase dynamics of circulating tumor cells after paclitaxel and doxorubicin chemotherapy in breast cancer mouse models

Purpose

Circulating tumor cells (CTCs) can provide a potentially minimal invasive source for monitoring chemotherapeutic effects. However, detailed in vivo dynamics of CTC after chemotherapy remain largely unknown.

Methods

We monitored CTC number and morphology early after chemotherapy using a newly developed cytology-based CTC detection device and triple-negative breast cancer mouse CTC models with spontaneous lung metastatic potential.

Results

Paclitaxel inhibited cell growth of breast cancer cells by mainly G2/M cell cycle arrest and partly apoptosis, whereas doxorubicin inhibited cell growth mainly by apoptosis and partly G2 cell cycle arrest in vitro. The number of CTCs was significantly increased 3–10 days after paclitaxel and doxorubicin chemotherapy and decreased thereafter in two mouse CTC models. The transiently increased CTCs early post-chemotherapy consisted of not only G2/M arrested cells (apoptotic cells), but also morphologically near-intact live cells. This heterogeneous cell population of CTCs was similar to that of primary tumor tissue after chemotherapy.

Conclusions

These results indicate that CTCs can be mobilized from the primary tumor in rapid response to chemotherapy and suggest the possibility that CTC monitoring from both numerical and morphological viewpoints early after chemotherapy using a cytology-based CTC detection device would be a useful diagnostic tool for predicting drug sensitivity/resistance in preclinical and clinical setting.

     

Biomarkers characterization of circulating tumour cells in breast cancer patients

Introduction

Increasing evidence supports the view that the detection of circulating tumor cells (CTCs) predicts outcomes of nonmetastatic breast cancer patients. CTCs differ genetically from the primary tumor and may contribute to variations in prognosis and response to therapy. As we start to understand more about the biology of CTCs, we can begin to address how best to treat this form of disease.

Methods

Ninety-eight nonmetastatic breast cancer patients were included in this study. CTCs were isolated by immunomagnetic techniques using magnetic beads labelled with a multi-CK-specific antibody (CK3-11D5) and CTC detection through immunocytochemical methods. Estrogen receptor, progesterone receptor and epidermal growth factor receptor (EGFR) were evaluated by immunofluorescence experiments and HER2 and TOP2A by fluorescence in situ hybridization. We aimed to characterize this set of biomarkers in CTCs and correlate it with clinical-pathological characteristics.

Results

Baseline detection rate was 46.9% ≥ 1 CTC/30 ml threshold. CTC-positive cells were more frequent in HER2-negative tumors (p = 0.046). In patients younger than 50 years old, HER2-amplified and G1-G2 tumors had a higher possibility of being nondetectable CTCs. Heterogeneous expression of hormonal receptors (HRs) in samples from the same patients was found. Discordances between HR expression, HER2 and TOP2A status in CTCs and their primary tumor were found in the sequential blood samples. Less that 35% of patients switched their CTC status after receiving chemotherapy. EGFR-positive CTCs were associated with Luminal tumors (p = 0.03).

Conclusions

This is the largest exploratory CTC biomarker analysis in nonmetastatic BC patients. Our study suggests that CTC biomarkers profiles might be useful as a surrogate marker for therapeutic selection and monitoring since heterogeneity of the biomarker distribution in CTCs and the lack of correlation with the primary tumor biomarker status were found. Further exploration of the association between EGFR-positive CTCs and Luminal tumors is warranted.     

Central nervous system relapse in patients with breast cancer is associated with advanced stages, with the presence of circulating occult tumor cells and with the HER2/neu status

Introduction

To evaluate the incidence of central nervous system (CNS) involvement in patients with breast cancer treated with a taxane-based chemotherapy regimen and to determine predictive factors for CNS relapse.

Methods

The medical files of patients with early breast cancer (n = 253) or advanced stage breast cancer (n = 239) as well of those with other solid tumors (n = 336) treated with or without a taxane-based chemotherapy regimen during a 42-month period were reviewed. HER2/neu overexpression was identified by immunohistochemistry, whereas cytokeratin 19 (CK-19) mRNA-positive circulating tumor cells (CTCs) in the peripheral blood were identified by real-time PCR.

Results

The incidence of CNS relapse was similar in patients suffering from breast cancer or other solid tumors (10.4% and 11.4%, respectively; P = 0.517). The incidence of CNS relapse was significantly higher in breast cancer patients with advanced disease (P = 0.041), visceral disease and bone disease (P = 0.036), in those who were treated with a taxane-containing regimen (P = 0.024), in those with HER2/neu-overexpressing tumors (P = 0.022) and, finally, in those with detectable CK-19 mRNA-positive CTCs (P = 0.008). Multivariate analysis revealed that the stage of disease (odds ratio, 0.23; 95% confidence interval, 0.007–0.23; P = 0.0001), the HER2/neu status (odds ratio, 29.4; 95% confidence interval, 7.51–101.21; P = 0.0001) and the presence of CK-19 mRNA-positive CTCs (odds ratio, 8.31; 95% confidence interval, 3.97–12.84; P = 0.001) were independent predictive factors for CNS relapse.

Conclusion

CNS relapses are common among breast cancer patients treated with a taxane-based chemotherapy regimen, patients with HER2/neu-positive tumor and patients with CK-19 mRNA-positive CTCs.     

Detection of minimal residual disease in blood and bone marrow in early stage breast cancer

BACKGROUND:

The significance of circulating tumor cells (CTCs) in blood and of disseminated tumor cells (DTCs) in bone marrow (BM) in patients with early stage breast cancer is unclear. In this study, the authors investigated the occurrence of CTCs and DTCs in women with early stage breast cancer and evaluated the correlation of their presence with other prognostic markers.

METHODS:

Blood and BM aspirations were collected at the time of primary breast surgery. CTCs were detected by using the CellSearch assay, and DTCs were detected by immunostaining BM aspirates for pancytokeratin. The presence of CTCs and DTCs was correlated with tumor classification (T1 vs T2), tumor histologic grade, estrogen receptor (ER) status, progesterone receptor (PR) status, human epidermal growth factor receptor 2 (HER2) status, and lymph node (LN) status.

RESULTS:

Of 92 patients who were included in the study, 49 had T1 tumors, and 43 had T2 tumors. CTCs were detected in 31% of patients, and DTCs were detected in 27% of patients. There was no correlation between the occurrence of CTCs and DTCs with the tumor classification (T1 vs T2) or histologic grade. CTCs were detected in 33% of patients with ER‐positive disease versus 26% of patients with ER‐negative disease, in 32% of patients with PR‐positive disease versus 30% of patients with PR‐negative disease, and in 25% of patients with HER2‐positive disease versus 31% of patients with HER2‐negative disease. DTCs were observed in 23% of patients with ER‐positive disease versus 37% of patients with ER‐negative disease, in 22% of patients with PR‐positive disease versus 32% of patients with PR‐negative disease, and in 0% of patients with HER2‐positive disease versus 29% of patients with HER2‐negative disease. CTCs and DTCs were nearly equally prevalent in both LN‐positive women and LN‐negative women. There was no significant correlation between the occurrence of CTCs or DTCs with tumor classification (T1 vs T2), tumor histologic grade, positive ER status, positive PR status, or positive HER2 status, and axillary LN status.

CONCLUSIONS:

CTCs and DTCs in women with early stage breast cancer did not correlate with the standard prognostic indicators that were considered. The implications of their occurrence in patients with early stage disease will require further large‐scale studies. Cancer 2010. © 2010 American Cancer Society.     

Assessment of the effect of surgery on the kinetics of circulating tumour cells in patients with operable breast cancer based on cytokeratin-19 mRNA detection

Aims

The aim of this study was to evaluate the effect of surgery on the kinetics of CTCs in breast cancer patients.

Methods

The detection of CK-19 mRNA-positive CTCs in the blood by RT-PCR was analysed in 104 stage 0-IIIA patients at 4 time-points: prior to surgery, upon completion, 24 h after surgery and 15 days after surgery. Furthermore, a late sample was assessed prior to initiation of adjuvant chemotherapy in a subgroup of 53 patients. As negative controls, peripheral blood was obtained from 50 female patients undergoing excision of benign breast lesions and from 11 female patients receiving surgery for early-stage colorectal cancer.

Results

A significant percentage of blood samples from breast cancer patients (14.4%) were negative for CK-19 preoperatively but turned transiently positive early postoperatively. However, no significant difference in CK-19 mRNA detection was noted among the first 4 examined time-points. There was no significant correlation between CK-19 mRNA-positive cells and classic prognostic factors. A significant increase in CK-19 mRNA-positivity (32.1%) was observed in a late sample of the subgroup of 53 patients before adjuvant chemotherapy after a median of 54 days, postoperatively.

Conclusions

Surgery may result in CTC detection in a small proportion of early breast cancer patients. There is no clear correlation to indicate which patients are expected to have detectable CTCs. Although CTCs are detected in a small proportion of patients during the perioperative period, the detection rate may increase over time and with longer follow-up.     

Prognostic impact of circulating tumor cells assessed with the CellSearch System? and AdnaTest Breast? in metastatic breast cancer patients: the DETECT study

Introduction

There is a multitude of assays for the detection of circulating tumor cells (CTCs) but a very limited number of studies comparing the clinical relevance of results obtained with different test methods. The DETECT trial for metastatic breast cancer patients was designed to directly compare the prognostic impact of two commercially available CTC assays that are prominent representatives of immunocytochemical and RT-PCR based technologies.

Methods

In total, 254 metastatic breast cancer patients were enrolled in this prospective multicenter trial. CTCs were assessed using both the AdnaTest Breast Cancer and the CellSearch system according to the manufacturers' instructions.

Results

With the CellSearch system, 116 of 221 (50%) evaluable patients were CTC-positive based on a cut-off level at 5 or more CTCs. The median overall survival (OS) was 18.1 months in CTC-positive patients. (95%-CI: 15.1-22.1 months) compared to 27 months in CTC-negative patients (23.5-30.7 months; p<0.001). This prognostic impact for OS was also significant in the subgroups of patients with triple negative, HER2-positive and hormone receptor-positive/HER2-negative primary tumors. The progression free survival (PFS) was not correlated with CTC status in our cohort receiving different types and lines of systemic treatment (p = 0.197). In multivariate analysis, the presence of CTCs was an independent predictor for OS (HR: 2.7, 95%-CI: 1.6-4.2). When the AdnaTest Breast was performed, 88 of 221 (40%) patients were CTC-positive. CTC-positivity assessed by the AdnaTest Breast had no association with PFS or OS.

Conclusions

The prognostic relevance of CTC detection in metastatic breast cancer patients depends on the test method. The present results indicate that the CellSearch system is superior to the AdnaTest Breast Cancer in predicting clinical outcome in advanced breast cancer.

Trial registration

Current Controlled Trials Registry number ISRCTN59722891.