Detection Of Circulating Tumor Cells In Patients Research Articles

Circulating tumor cell detection in high-risk non-metastatic prostate cancer.

The detection of circulating tumor cells (CTCs) provides important prognostic information in men with metastatic prostate cancer. We aim to determine the rate of detection of CTCs in patients with high-risk non-metastatic prostate cancer using the CellSearch® method. Samples of peripheral blood (7.5 mL) were drawn from 36 men with newly diagnosed high-risk non-metastatic prostate cancer, prior to any initiation of therapy and analyzed for CTCs using the CellSearch® method. The median age was 70 years, median PSA was 14.1, and the median Gleason score was 9. The median 5-year risk of progression of disease using a validated nomogram was 39 %. Five out of 36 patients (14 %, 95 % CI 5-30 %) had CTCs detected in their circulation. Four patients had only 1 CTC per 7.5 mL of blood detected. One patient had 3 CTCs per 7.5 mL of blood detected, which included a circulating tumor microemboli. Both on univariate analysis and multivariate analysis, there were no correlations found between CTC positivity and the classic prognostic factors including PSA, Gleason score, T-stage and age. This study demonstrates that patients with high-risk, non-metastatic prostate cancer present infrequently with small number of CTCs in peripheral blood. This finding is consistent with the limited literature available in this setting. Other CTC isolation and detection technologies with improved sensitivity and specificity may enable detection of CTCs with mesenchymal phenotypes, although none as yet have been validated for clinical use. Newer assays are emerging for detection of new putative biomarkers for prostate cancer. Correlation of disease control outcomes with CTC detection will be important.

Circulating miR-337-3p as a novel biomarker for prostate cancer.

5087 Background: Recent studies have demonstrated that the aberrant expression of microRNAs (miRNAs)is related with the development of prostate cancer (PCa). Detection of circulating tumor cells (CTC) may provide diagnostic and prognostic information inPCa. The purpose is identifying circulating miRNAs potentially useful for CTC detection in patients with PCa. Methods: In the first study phase we examined blood levels of 92 miRNAs in 49 patients grouped in pools by risk classification: low-risk 42.8%, intermediate-risk 22.5% and high-risk 34.7% and healthy volunteers (N=10) using SYBR-green-based microARN RT-qPCR arrays (Exiqon). Prostate cancer diagnosis was obtained by transrectalultrasound guided biopsy of 10 cores, PSA and digital rectal examination was done previously. In the second study phase using quantitative real-time polymerase chain reaction (qPCR) by TaqMan Human MicroRNAAssays (Life Technologies), we compared the expression levels of miRNAs in blood samples from 34 patients of low-risk, 31 of intermediate-risk, 18 of high-risk localized disease and 22 healthy volunteers paired by age with cases. Receiver-operator characteristic (ROC) curve was used. Results: Blood samples from patients with low, intermediate, high-risk and healthy controls exhibit distinct circulating miRNA signatures. microRNAsdifferential expressed between risk groups (p<0.01) (low, intermediate and high) and control group: 0microARNs upregulated (MU), 12 MU and 68 MU respectively. Highlight the significantlyoverexpression in the intermediate risk group and maintained at the high-risk of microRNAs: 337-3p[247 PicTar predictions (PTP)], 330-3p (307 PTP) and 218 (551 PTP). Preliminary results of the second study phase showed that the median level of miRNA,337-3p was significantly higher in patients with high-risk than that in healthy controls (p=.001). ROC curve analyses indicated that this blood miRNA may beuseful for discriminating patients with high-risk from healthy controls (AUC=.724). Conclusions: miRNAs in the circulation are relatively stable, very accessible, low invasive and easily testable biomarkers. Our preliminary promisingresults suggest miR-337-3p as novel stable blood-based marker for PCa detection.

Abstract 1445: Clinical feasibility study of a novel cytometry-based-system for the detection of circulating tumor cells of patients with lung cancer.

Abstract Enumeration and characterization of circulating tumor cells (CTCs) are, as "liquid biopsy," expected to provide useful clinical information on prognosis, cancer staging, drug choice, and therapeutic efficacy. We have developed a novel flow-cytometry-based CTCs detection and sorting system (On-Chip Sort) independent of EpCAM expression of tumor cells. In a preclinical study, various kinds of cell lines were spiked into human blood and the sensitivity in detection was evaluated. Enumeration of the spiked tumor cells was linear over a range of 10 to 1000 cells, with a recovery rate of ≧ 90%. A significantly higher recovery rate was observed with our system (90 - 102%) than with CellSearch® (0%) when EpCAM-negative PC-14 cells were spiked, suggesting a superior sensitivity of our system in capturing EpCAM-negative tumor cells. Here we report the results of a clinical feasibility study on the detection of CTCs in patients with advanced lung cancer. 16mL of peripheral blood was drawn from each case. In our assay, 4mL of samples were hemolyzed and eliminated CD45+ leucocytes by magnetic beads coated with anti-CD45 antibody. After negative CTC enrichment, samples were fixed and labeled with FITC-cytokeratin, PE-EpCAM and Alexa700-CD45 antibodies, and analyzed by using our flow-cytometry-based CTC detection system. 22 advanced lung cancer patients and 3 healthy donors were recruited at National Cancer Center Hospital. The results of the CTC enumeration were compared with those by CellSearch®. In 22 blood samples from patients, CTCs detected by our system ranged from 0 - 18 CTCs (median, 6.5), and 77.3% (17/22) of the samples were above the threshold level (≧ 4 / 4mL). On the other hand, with CellSearch®, only 27% of the samples had a ≧ 2 / 7.5 mL threshold level. In 2 blood samples from the healthy donors, our system detected 0 and 3 CTCs, and CellSearch® detected 0 and 1 CTC. In the sample from a healthy donor spiked with 18 EpCAM-negative PC-14 cells, our system was able to detect 10 cells; however, CellSearch® could not recover even one cell. These results of a clinical feasibility study showed our system to be significantly more sensitive for CTC enumeration in advanced lung cancer. Presently, we are developing a novel approach for molecular analysis of CTCs via sequential immunomagnetic enrichment and FACS sorting to isolate CTCs, followed by real-time PCR analysis using Scorpion Amplified Refractory Mutation System technology. For methods validation, PC-9 and 11_18 cells spiked into normal blood were isolated, and profiled the epidermal growth factor receptor (EGFR) mutations status correctly. In the near future, we plan to apply this approach to clinical samples of lung cancer patients and investigate the correlation of EGFR mutations in sorted CTCs, circulating free DNA and primary lesions. (Data to be updated prior to presentation) Citation Format: Takeshi Sawada, Yukiko Ito, Masaru Watanabe, Yuu Fujimura, Shinji Nakamichi, Shintaro Kanda, Hidehito Horinouchi, Yutaka Fujiwara, Hiroshi Nokihara, Noboru Yamamoto, Tomohide Tamura, Yasuhiro Koh, Fumiaki Koizumi. Clinical feasibility study of a novel cytometry-based-system for the detection of circulating tumor cells of patients with lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1445. doi:10.1158/1538-7445.AM2013-1445

Abstract 1446: Detection of circulating tumor cells for malignant epitherial tumor using a novel cytometry-based system.

Abstract Enumeration and characterization of circulating tumor cells (CTCs) are, as "liquid biopsy," expected to provide useful clinical information. We have developed a novel flow-cytometry-based CTCs detection and sorting system (On-Chip Sort) independent of EpCAM expression of tumor cells. EpCAM-positive cell line KATO-III and EpCAM-negative cell line A549 and PC-14 were used for the preclinical study. Various numbers of cells were spiked into blood and the sensitivity in detection was evaluated in a blinded manner. The recovery rate ranged from 40.5 - 157.3% with our system and 0 - 89.8% with the CellSearch system. A significantly higher recovery rate was observed with our system (90 - 102%) than with the CellSearch system (0%) when the PC-14 cells were spiked, suggesting a superior sensitivity of our system in capturing EpCAM-negative tumor cells. These data imply an advantageous potential of our detection system. Here we report the results of a clinical feasibility study on the detection of CTCs in patients with malignant epithelial tumors, including breast cancer, ovarian cancer, cervical cancer and primary unknown cancer, etc. Sixteen mL of peripheral blood was drawn from each case. In our assay, 4mL of samples were hemolyzed and eliminated CD45+ leucocytes by magnetic beads coated with anti-CD45 antibody. After negative CTC enrichment, samples were fixed and labeled with FITC-cytokeratin, PE-EpCAM and Alexa700-CD45 antibodies, and analyzed by using our flow-cytometry-based CTC detection system. Twenty-four patients with epithelial malignancies and two healthy donors were recruited at National Cancer Center Hospital. The results of the CTC enumeration were compared with those by CellSearch®. In 24 blood samples from cancer patients, CTCs detected by our system ranged from 0 - 829 CTCs (median, 4.5), and 83.3% (20/24) of the samples were above the threshold level (≧ 2 / 4ml). On the other hand, with CellSearch®, only 29% of the samples had a ≧ 2 / 7.5 ml, threshold level. In 2 blood samples from the healthy donors, 1 CTC was detected by our system and CellSearch® detected none. These results of a clinical feasibility study showed our system was significantly more sensitive for CTC enumeration in various cancer patients. Presently, we are developing a novel approach for molecular analysis of CTCs via sequential immunomagnetic enrichment and FACS sorting to isolate CTCs, followed by real-time PCR analysis using Scorpion Amplified Refractory Mutation System technology. For methods validation, BT-20 cells spiked into normal blood were isolated and profiled Phosphoinositide-3-kinase catalytic alpha (PIK3CA) mutations status correctly. In the near future, we plan to apply this approach to clinical samples of breast cancer patients and investigate the correlation of PIK3CA mutations in sorted CTCs, circulating free DNA and primary lesions. (Data to be updated prior to presentation) Citation Format: Yukiko Ito, Takeshi Sawada, Masaru Watanabe, Yuu Fujimura, Jun Hashimoto, Makoto Kodaira, Mayu Yunokawa, Harukaze Yamamoto, Kan Yonemori, Chikako Shimizu, Kenji Tamura, Yasuhiro Fujiwara, Yasuhiro Koh, Fumiaki Koizumi. Detection of circulating tumor cells for malignant epitherial tumor using a novel cytometry-based system. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1446. doi:10.1158/1538-7445.AM2013-1446

Clinical role of detecting circulating tumor cells

Using ultrasensitive molecular or immunological/cytometric approaches, identification of circulating tumor cells (CTC) thus far undetectable at primary diagnosis even by high resolution imaging technologies is feasible. Results from several clinical studies revealed that in breast cancer patients with metastatic disease positivity for CTC is associated with worse prognosis. Thus, information obtained from CTC research might support clinicians in assessing individual prognosis, stratifying patients at risk to systemic adjuvant anti-cancer therapies, early response evaluation and monitoring therapeutic efficacy. At present, the CellSearchTM system combining automated capturing of CTC, immunostaining and image analysis is the only standardized technology approved by the FDA for the detection of CTC in patients with metastatic breast, colorectal and prostate cancer [1–3]. However, evidence has emerged that most currently used CTC detection methods still lack sensitivity or specificity to find all CTC especially those that have lost epithelial features, e.g. in the course of epithelial-mesenchymal transition [4–7]. Therefore special emphasis is placed to the development of innovative new strategies with the capacity to track additional CTC and to identify those with the potential to initiate metastasis. In this context, discovery of approaches enabling further phenotypical and molecular characterisation of CTC, e.g., analysis of single cell whole genomes, next generation sequencing and gene expression analysis on CTC is pivotal particularly in view of the development of new personalized targeting therapies. It is also worth mentioning that detection and characterization of CTC within translational research projects has entered clinical trials also enrolling patients with early breast cancer. First promising results on CTC detection especially within treatment regimens including targeted therapy, e.g., trastuzumab directed against HER2 in breast cancer patients receiving neoadjuvant or adjuvant therapy have been published recently [8–11]. Furthermore, characterization of the HER2 status of CTC might help identifying additional patients profiting from an anti-HER2 treatment and getting insights into therapy-induced selection of CTC.

Sensitive and Direct Detection of Circulating Tumor Cells by Multimarker µ-Nuclear Magnetic Resonance

Identifying circulating tumor cells (CTCs) with greater sensitivity could facilitate early detection of cancer and rapid assessment of treatment response. Most current technologies use EpCAM expression as a CTC identifier. However, given that a significant fraction of cancer patients have low or even absent EpCAM levels, there is a need for better detection methods. Here, we hypothesize that a multimarker strategy combined with direct sensing of CTC in whole blood would increase the detection of CTC in patients. Accordingly, molecular profiling of biopsies from a patient cohort revealed a four-marker set (EpCAM, HER-2, EGFR, and MUC-1) capable of effectively differentiating cancer cells from normal host cells. Using a point-of-care micro-nuclear magnetic resonance (µNMR) system, we consequently show that this multimarker combination readily detects individual CTC directly in whole blood without the need for primary purification. We also confirm these results in a comparative trial of patients with ovarian cancer. This platform could potentially benefit a broad range of applications in clinical oncology.

A simple multicolor flow cytometry protocol for detection and molecular characterization of circulating tumor cells in epithelial cancers

Circulating tumor cells (CTCs) might not only serve as prognostic marker but could also be useful for monitoring treatment efficacy. A multicolor flow cytometry protocol for their detection and molecular characterization in peripheral blood was developed which consisted of erythrocyte lysis followed by staining of cells with fluorochrome-labeled antibodies against CD45 and the epithelial markers EpCam and cytokeratin 7/8. For reducing the number of events acquired by flow cytometry, an electronic threshold for the fluorescent signals from the epithelial markers was applied. After establishment of the protocol by using spiking experiments, its suitability to determine the absolute number of CTCs as well as their expression of epidermal growth factor receptor (EGFR) and its phosphorylated form (phospho-EGFR) in blood samples from patients with squamous cell carcinoma of the head and neck (SCCHN) was validated. Spiking experiments demonstrated an excellent recovery (mean 85%) and a linear performance (R(2) = 0.98) of the protocol. Sensitivity and specificity were comparable to our former protocol using immunomagnetic CTC pre-enrichment. The analysis of 33 SCCHN patient samples revealed the presence of CTCs in 33.3% of cases with a mean ± SD of 1.5 ± 0.5 CTCs per 3.75 ml blood. EGFR was expressed in 100% and phospho-EGFR in 36.4% of the CTC+ cases. We have established a simple and sensitive multicolor flow cytometry protocol for detection of CTCs in patients with epithelial cancers including SCCHN which will allow their detailed molecular characterization.

Clinical significance of carcinoembryonic antigen-, cytokeratin 19-, or survivin-positive circulating tumor cells in the peripheral blood of esophageal squamous cell carcinoma patients treated with radiotherapy

Circulating tumor cells (CTCs) have been associated with clinical outcome in various malignancies. The aim of this study was to examine CTC status in the peripheral blood of patients with esophageal squamous cell carcinoma (ESCC) before and after radiotherapy, and to evaluate its clinical significance. A total of 72 ESCC patients treated with radical radiotherapy were enrolled in this study. The nested reverse-transcriptase polymerase chain reaction was used to detect the three representative markers of CTCs, namely carcinoembryonic antigen, cytokeratin 19, and survivin. The results showed that CTC(+), a status with positive expression of at least one of these three markers, in patients with ESCC pre- and post-radiotherapy were 54.2% (39/72) and 38.9% (28/72), respectively (P= 0.059). Furthermore, CTC (+) in patients pre- or post-radiotherapy was both correlated with lymph metastasis and adverse 2-year progression-free survival. It was also found that changes in CTC status after radiotherapy could reflect patients' response to radiotherapy. The response rates in cases with CTC status pre-radiotherapy(+)/post-radiotherapy(+), pre-radiotherapy(-)/post-radiotherapy(+), pre-radiotherapy(-)/post-radiotherapy(-), pre-radiotherapy(+)/post-radiotherapy(-) were 58.3% (21/36), 0% (0/3), 73.7% (14/19), and 85.7% (12/14), respectively. In a multivariate analysis of Cox proportional hazard model, only CTC (+) post-radiotherapy was an independent unfavorable prognostic factor for ESCC apart from subsequent chemotherapy and patients' Karnofsky performance status scores. In conclusion, positive detection of CTCs in patients with ESCC after radiotherapy may be a promising biomarker for radiation efficiency and prognosis assessment in ESCC.

Label-free reflectometric interference microchip biosensor based on nanoporous alumina for detection of circulating tumour cells

In this report, a label-free reflectometric interference spectroscopy (RIfS) based microchip biosensor for the detection of circulating tumour cells (CTCs) is demonstrated. Highly ordered nanoporous anodic aluminium oxide (AAO) fabricated by electrochemical anodization of aluminium foil was used as the RIfS sensing platform. Biotinylated anti-EpCAM antibody that specifically binds to human cancer cells of epithelial origin such as pancreatic cancer cells (PANC-1) was covalently attached to the AAO surface through multiple surface functionalization steps. Whole blood or phosphate buffer saline spiked with low numbers of pancreatic cancer cells were successfully detected by specially designed microfluidic device incorporating an AAO RIfS sensor, without labour intensive fluorescence labelling and/or pre-enhancement process. Our results show that the developed device is capable of selectively detecting of cancer cells, within a concentrations range of 1000–100,000cells/mL, with a detection limit of <1000cells/mL, a response time of <5min and sample volume of 50μL of. The presented RIfS method shows considerable promise for translation to a rapid and cost-effective point-of-care diagnostic device for the detection of CTCs in patients with metastatic cancer.

Usefulness of circulating tumor cell detection for the diagnosis of malignant pancreatic head tumors: A pilot study.

235 Background: Pancreatic adenocarcinomas account for 90% of pancreatic malignancies. It is often diagnosed at an unresectable stage (at a metastatic and / or at a locoregional invasion stage). Histological diagnosis is mandatory to establish palliative chemotherapy. Endoscopic ultrasound-guided fine needle aspiration (EUGA) of pancreatic tumors is a difficult gesture, not without risk. Furthermore, it frequently provides pauci-cellular, hemorrhagic or necrotic material unsuitable for precise histologic diagnosis. Techniques for detecting circulating tumor cells (CTC) have been recently developed to highlight the presence of malignant cells circulating in peripheral blood. Screencells technology is based on the separation of CTCs from other circulating cells by blood filtration through a polycarbonate membrane with 7m pores. The aims of this pilot study were 1) to compare the results of detections CTC to EUGA, 2) to assess the feasibility and the contribution of this innovative technique in the care of a limited series of patients. Methods: A sample of peripheral venous blood was performed in 15 patients with a suspicious CT scan of a pancreatic head tumoral lesion, before the completion of an EUGA. 4 ml of blood were filtered using ScreencellsCyto columns. After Giemsa staining, a pathologist analyzed each filter. Cells visualized on the filter under light microscopy were considered as tumoral if they met the following morphological criteria: anisocytosis, anisokaryosis, nuclear diameter&gt;7m, nuclear membrane irregularitie, large nucleolus. Results: The malignant feature of the pancreatic lesion could be histologically assessed after liver biopsy for two patients. EUGA showed malignancy in 10 cases. CTC presence was identified in 7 patients with tumoral histological diagnosis. Conclusions: This study demonstrates the feasibility of CTC detection in patients with pancreatic head tumor. The identification of CTCs could facilitate patient care, thus avoiding difficult and potentially dangerous EUGA. A prospective trial is needed to confirm the positive predictive value of this biomarker and to evaluate its contribution in the diagnosis procedure of such tumors.

The prognostic role of circulating tumor cells (CTCs) detected by RT-PCR in breast cancer: a meta-analysis of published literature

The prognostic significance of circulating tumor cells (CTCs) in patients with breast cancer is controversial. We performed a meta-analysis of published literature to assess whether the detection of CTCs in patients diagnosed with primary breast cancer can be used as a prognostic factor. We searched Medline, Science Citation Index, and Embase databases as well as reference lists of relevant articles (including review articles) for studies that assessed the prognostic relevance of tumor cell detection in the peripheral blood (PB). A total of 24 eligible studies with 4,013 cases and 1,333 controls were included. Meta-analyses were performed using a random-effects model, using the hazard ratio (HR) and 95% confidence intervals (95% CIs) as effect measures. The positive detection of CTCs in patients was significantly associated with poor overall survival (OS) (HR = 3.00 [95% CI 2.29-3.94], n = 17, P < 0.0001) and recurrence-free survival (RFS) (HR = 2.67 [95% CI 2.09-3.42], n = 22, P < 0.0001). CTC-positive breast cancers were significantly associated with high histological grade (HR = 1.21 [95% CI 1.09-1.35], n = 34, P < 0.0001), tumor size (>2 cm) (HR = 1.12 [95% CI 1.02-1.22], n = 31, P = 0.01). and nodal status (≥1) (HR = 1.10 [95% CI 1.00-1.21], n = 32, P = 0.037), but cytokeratin-19 (CK-19) mRNA-positive CTCs were not associated with these clinicopathological parameters of breast cancer. Furthermore, the presence of CTCs was not associated with estrogen receptor (ER) negativity, progesterone receptor (PR) negativity, or human epidermal growth factor receptor type 2 (HER2) positivity. Detection of CTCs in the PB indicates poor prognosis in patients with primary breast cancer. Larger clinical studies are required to further evaluate the role of these markers in clinical practice.

Circulating Tumor Cells and EpCAM Expression in Neuroendocrine Tumors

Neuroendocrine tumors (NET) are heterogeneous tumors with widely variable survival. It is unknown whether they express EpCAM (epithelial cell adhesion molecule) and thus whether NET circulating tumor cells (CTC) are detectable. We systematically investigated EpCAM expression and CTC detection in patients with metastatic NETs and evaluated the potential of CTCs to predict radiological progression. EpCAM protein expression was evaluated in 74 samples of formalin-fixed, paraffin-embedded NET tissue by immunohistochemistry. Seventy-nine patients with metastatic NETs (42 midgut, 5 unknown primary, 19 pancreatic, 13 bronchopulmonary) had blood samples drawn for CTC isolation and enumeration utilizing the CellSearch platform. Patients were classified as having progressive or nonprogressive disease on the basis of serial imaging. Strong homogeneous, membranous EpCAM expression was observed in all ileal (n = 26) and pancreatic NETs (n = 16), whereas variable EpCAM expression was observed in bronchopulmonary NETs (n = 13). Forty-three percent of midgut and 21% of pancreatic NETs had CTCs detected with a range of 0-62 and 0-11, respectively. The absence of CTCs was strongly associated with stable disease (P < 0.001). There was a moderate correlation between CTC levels and urinary 5-hydroxyindole acetic acid (r = 0.5, P = 0.007) and between CTC levels and burden of liver metastases (B = 8.91, P < 0.001). There was no or low correlation between CTC levels and Ki-67 (r = 0.08, P = 0.59) and serum chromogranin A (r = 0.246, P = 0.03). This is the first systematic analysis showing EpCAM expression and CTC detection in NETs. CTCs seem to be associated with progressive disease and may provide useful prognostic information given the variable survival rates in these tumors.

Circulating tumor cells in gastrointestinal cancer

Since the first report in the nineteenth century, there have been numerous reports on the isolation and characterization of circulating tumor cells (CTCs) in peripheral blood in patients with various carcinomas. In general, CTCs have been observed in the peripheral blood of cancer patients at very low concentrations of 10(-7)-10(-8) of normal peripheral blood cells. The characterization is of considerable biomedical interest in order to understand how these cells can travel via the blood stream to anatomically distant sites and form metastatic disease. Recent progress in molecular oncology enables us to detect the CTCs in blood with highly sensitivity and specificity, and several studies have indicated the prognostic value of CTC detection in patients with gastrointestinal cancers. Detection and measurement of CTCs in patients with gastrointestinal cancers such as colorectal, gastric, and pancreatic cancers can be useful as a promising tool for judging tumor stage, predicting the distant metastasis and patient survival, and monitoring the response to cancer therapy. Standard procedures for CTC detection have to be established, and the clinical relevance should be verified in large-scale clinical trials. However, CTC detection is suggested to provide useful information for the tumor staging and anticancer treatments in clinical practices in the near future.

Multi-center study evaluating circulating tumor cells as a surrogate for response to treatment and overall survival in metastatic breast cancer

Evaluating circulating tumor cells (CTCs) is one way to predict outcome and monitor treatment in patients with MBC. In this prospective study, we evaluated CTCs in predicting treatment efficacy and overall survival (OS) using the CellSearch System (Veridex, LLC, Raritan, NJ). One hundred nineteen patients with MBC with measurable disease were enrolled. Samples of 7.5 ml of blood from 107 eligible patients were tested for CTCs before starting therapy (baseline), after one cycle of therapy (3-4 weeks) and at 12 weeks. We compared CTC levels and imaging at baseline and at 12 weeks. Next, we determined the hazard ratios (HR) by comparing cases with zero CTCs to those with one or more CTCs. Moreover, HR was calculated when comparing cases that had greater than or equal to a certain number of CTCs to those with less than the number of CTCs. This study shows the incidence of detection of CTCs in patients with metastatic breast cancers. Of the patients, 64.4% (76/118) had one or more CTCs, and 37.3% (44/118) had five or more CTCs. First we set the baseline number of CTCs as 100%. Of the seven cases whose level of CTCs decreased more than 90%, six (85.7%) demonstrated a positive response (complete response and partial response) by imaging after one cycle (3-4 weeks later). For the patients whose CTC levels increased above 100% after one cycle (3-4 weeks later), 7 of 11 (63.6%) had progressive disease (PD). The HR for cases with five to ten CTCs was greater than 1.00 [HR = 2.450; 95% confidence interval (CI) 0.727-8.248]. Statistical significance was observed when comparing patients who had > or =3 CTCs to those with <3 CTCs (P = 0.0273). When comparing cases with > or =5 CTCs to those with <5 CTCs, the hazard ratio was 3.069 (95% CI 1.496-6.295; P = 0.0022). Because the change in the number of CTCs was highly correlated with results from imaging before and after therapy, CTCs can be considered a biomarker that may predict the effect of treatment earlier than imaging modalities.