Detection Of Circulating Tumor Cells In Blood Research Articles

Comprehensive characterization of circulating tumor cells and cell-free DNA in patients with metastatic melanoma.

Advances in therapeutic approaches for melanoma urge the need for biomarkers that can identify patients at risk for recurrence and to guide treatment. The potential use of liquid biopsies in identifying biomarkers is increasingly being recognized. Here, we present a head-to-head comparison of several techniques to analyze circulating tumor cells (CTCs) and cell-free DNA (cfDNA) in 20 patients with metastatic melanoma. In this study, we investigated whether diagnostic leukapheresis (DLA) combined with multimarker flow cytometry (FCM) increased the detection of CTCs in blood compared to the CellSearch platform. Additionally, we characterized cfDNA at the level of somatic mutations, extent of aneuploidy and genome-wide DNA methylation. Both CTCs and cfDNA measures were compared to tumor markers and extracranial tumor burden on radiological imaging. Compared to the CellSearch method applied on peripheral blood, DLA combined with FCM increased the proportion of patients with detectable CTCs from 35% to 70% (P = 0.06). However, the median percentage of cells that could be recovered by the DLA procedure was 29%. Alternatively, cfDNA mutation and methylation analysis detected tumor load in the majority of patients (90% and 93% of samples successfully analyzed, respectively). The aneuploidy score was positive in 35% of all patients. From all tumor measurements in blood, lactate dehydrogenase (LDH) levels were significantly correlated to variant allele frequency (P = 0.004). Furthermore, the presence of CTCs in DLA was associated with tumor burden (P < 0.001), whereas the presence of CTCs in peripheral blood was associated with number of lesions on radiological imaging (P < 0.001). In conclusion, DLA tended to increase the proportion of patients with detectable CTCs but was also associated with low recovery. Both cfDNA and CTCs were correlated with clinical parameters such as LDH levels and extracranial tumor burden.

Creating a capture zone in microfluidic flow greatly enhances the throughput and efficiency of cancer detection

Efficient capture of rare circulating tumor cells (CTCs) from blood samples is valuable for early cancer detection to improve the management of cancer. In this work, we developed a highly efficient microfluidics-based method for detecting CTCs in human blood. This is achieved by creating separate capture and flow zones in the microfluidic device (ZonesChip) and using patterned dielectrophoretic force to direct cells from the flow zone into the capture zone. This separation of the capture and flow zones minimizes the negative impact of high flow speed (and thus high throughput) and force in the flow zone on the capture efficiency, overcoming a major bottleneck of contemporary microfluidic approaches using overlapping flow and capture zones for CTC detection. When the flow speed is high (≥0.58 mm/s) in the flow zone, the separation of capture and flow zones in our ZonesChip could improve the capture efficiency from ∼0% (for conventional device without separating the two zones) to ∼100%. Our ZonesChip shows great promise as an effective platform for the detection of CTCs in blood from patients with early/localized-stage colorectal tumors.

Automatic detection of circulating tumor cells in darkfield microscopic images of unstained blood using boosting techniques.

Circulating tumor cells (CTCs) are nowadays one of the most promising tumor biomarkers. It is well correlated with overall survival and progression-free survival in breast cancer, as well as in many other types of human cancer. In addition, enumeration and analysis of CTCs could be important for monitoring the response to different therapeutic agents, thus guiding the treatment of cancer patients and offering the promise of a more personalized approach. In this article, we present a new method that could be used for the automatic detection of CTC in blood, based on the microscopic appearance of unstained cells. The proposed method is based on the evaluation of image characteristics and boosting techniques. A dataset of 263 dark field microscopy images was constructed and used for our tests, containing blood spiked with three different types of tumor cells. An overall sensitivity of 92.87% and a specificity of 99.98% were obtained for the detection of CTC, performances which proved to be comparable to those obtained by human experts.

Abstract 381: Circulating tumor cells (CTC) but not circulating endothelial cells (CEC) are independent prognostic factors in neoadjuvant chemotherapy combined with bevacizumab in HER2 negative inflammatory breast cancer (IBC) in multicentre phase II trial BEVERLY1

Abstract Background: CTC detection in blood is an independent prognostic factor in primary breast cancer with a detection rate from 20 to 25% (Pierga, CCR 2008, Rack B, JNCI 2014). Higher incidence of 35% of CTC has been reported in IBC (Pierga, Lancet Oncol 2012). Predictive and prognostic value of CEC for response to anti-angiogenic agents remains debated. Methods: CTC and CEC were detected in 7.5 ml and 4 ml of blood respectively in the neoadjuvant setting of HER2 negative IBC (T4d) patients (pts) enrolled in the phase II multicentre trial BEVERLY 1, evaluating bevacizumab (15mg/kg q3w) in combination with sequential neoadjuvant CT of 4 cycles of FEC followed by 4 cycles of Docetaxel. The CellSearch™ System, combining EpCAM immunomagnetic selection followed by anti-cytokeratin (A45B/B3) and anti-HER2 staining for CTC and CD146 IMS and CD105 staining for CEC. Results: From 12/08 to 09/10, 101 pts were included and 92 were evaluable for CTC and CEC. At baseline, 37 pts out of 92 had ≥ 1 detectable CTC (40%, 95%CI 30-50%). No correlation was found between CTC and CEC levels. A dramatic drop in CTC incidence (p&amp;lt;0.0001) was observed from baseline to the 1st follow-up analysis after 4 cycles (6%). There was a significant increase of CEC median number from baseline (12) to preoperative sample (130) (p &amp;lt;0.001) and a decrease (39) (p = 0.04) when CT and bevacizumab were interrupted for surgery. CTC detection at baseline independently predicted 3-year DFS: (73% vs. 41% for patients with &amp;lt;1 vs. ≥1 CTC/7.5 mL [p&amp;lt;0.001, HR 2.73 (1.48-5.04)]) and OS (94% vs.51% [p&amp;lt;0.001, HR 5.93 (2.49-14.10)]. Fourteen patients (15%) with no CTC detected at baseline and with complete pathological response (pCR) had a high 3-year DFS (93%) and OS (100%). CEC level under 20/4ml at baseline did not reach statistical significance as a favorable prognostic factor for DFS and OS (p = 0.055 and 0.066 respectively). CEC changes during treatment had no prognostic value. Conclusions: We observed a high CTC detection rate of 40% in pts with HER2 negative IBC, including triple negative tumor, with a strong and independent prognostic value for DFS and OS. Combining pCR after neoadjuvant chemotherapy combined with bevacizumab, and CTC at baseline, isolates a subgroup of inflammatory breast cancer with excellent survival. Citation Format: Jean-Yves Pierga, Francois-Clement Bidard, Aurelie Autret, Fabrice Andre, Thierry Petit, Florence Dalenc, Christel Levy, William Jacot, Jacques Bonneterre, Jean-Marc Ferrero, Pierre Kerbrat, Jerome Lemonnier, Francois Bertucci, Patrice Viens. Circulating tumor cells (CTC) but not circulating endothelial cells (CEC) are independent prognostic factors in neoadjuvant chemotherapy combined with bevacizumab in HER2 negative inflammatory breast cancer (IBC) in multicentre phase II trial BEVERLY1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 381. doi:10.1158/1538-7445.AM2015-381

Detection of circulating tumor cells (CTCs) in central venous blood (CVB) versus peripheral venous blood (PVB) in patients with metastatic cancers.

e22039 Background: CTCs are considered as useful indicators of tumor aggressiveness, patient prognosis, and treatment effectiveness monitoring. Detection of CTCs in blood is difficult due to the ex...

Detection of circulating tumor cells during follow-up of patients with early breast cancer: Clinical utility for monitoring of therapy efficacy

Introduction. Circulating tumor cells (CTCs) detection prior to and during therapy is considered as an independent and strong prognostic marker. The present study was designed to isolate and characterize CTCs in peripheral blood of an early breast cancer (BC) patient as a biomarker for monitoring treatments efficacy. Materials and methods. In total, 54 early breast cancer patients undergoing neoadjuvant and/or adjuvant chemotherapy regimens were enrolled into a prospective study. CTC detection in blood was performed by AdnaTest BreastCancer™ (AdnaGen AG, Germany), which is based on the detection of EpCAM, HER2 and MUC1 specific transcripts in enriched CTC-lysates. Additionally, cDNA from isolated CTCs and PBMC was used for qPCR gene expression analysis of TOP1, TOP2A, CTSD, ST6, CK19 and reference gene actin. Results. We found that CTCs can be detected in the peripheral blood of approximately 31% of early stage breast cancer patients. The presence of CTCs was detected in 36% ER positive, 32% PR positive and 30% HER2 positive patients. We found no correlation between CTCs and tumor size, tumor grade, histological grade and receptor status. Only 7% of all patients remained CTCs positive after adjuvant therapy. Gene expression analysis revealed a particular heterogeneity of the studied genes. Conclusions. In conclusion, CTC detection may be a promising early marker of disease progression potentially enhancing the difficult therapeutic decisions. Further studies should, however, clearly demonstrate its utility for both the prediction of outcome and monitoring the effect of treatment.

Detection of circulating tumor cells in gastrointestinal cancer: Has its time come?

Detection of circulating tumor cells in gastrointestinal cancer: Has its time come?

P1-14-02: Correlation of Circulating Tumor Cells (CTC) and Circulating Endothelial Cells (CEC) with Pathological Complete Response (pCR) during Neoadjuvant Chemotherapy (CT) Combined with Bevacizumab in HER2 Negative Inflammatory Breast Cancer (IBC): Ancillary Study of Phase II Trial BEVERLY 1.

Abstract Background: Prognostic value of CTC detection in blood has been reported in primary breast cancer with a rate around 20% in the neoadjuvant setting. No correlation between CTC and pCR to neoadjuvant CT in operable breast cancer has been reported (Pierga CCR 2008, Riethdorf CCR 2010). Predictive value of CEC for response to anti-angiogenic agents is unclear. Methods: CTC and CEC were detected in 7.5 ml and 4 ml of blood respectively with CellSearch™ System in the neoadjuvant setting of HER2 negative IBC (T4d) patients (pts) enrolled in the phase II multicentre trial BEVERLY 1, evaluating bevacizumab (15mg/kg q3w) in combination with sequential neoadjuvant CT of 4 cycles of FEC followed by 4 cycles of Docetaxel. Patients received postoperatively 10 cycles of bevacizumab and hormonotherapy if tumor was ER+. Results: From 12/08 to 09/10, 101 pts were included, 96 were evaluable for pCR and 92 for CTC and CEC. Out of 96 pts, 51 (53%) had triple negative breast cancer (TNBC). At baseline, 37 pts out of 92 had ≥ 1 detectable CTC (40%, 95%CI 30–50%). At baseline, CTC level was not correlated with CEC level, neither with TNBC nor pCR. A drop in CTC incidence was observed from baseline to the 1st follow-up analysis after 4 cycles (p&amp;lt;0.0001). Out of 6 pts with detectable CTC at the end of adjuvant bevacizumab (8 months post surgery) none had pCR (p=0.05 Yates test). There was a significant increase of CEC from baseline to presurgery sample (p &amp;lt;0.001) and a decrease (p=0.04) after tumor removal and end of CT. A higher level of CEC (&amp;gt;20/4ml) before C5 could be associated with a higher probability of pCR (Khi2 test, p=0.003). Conclusions: We observed a high CTC detection rate of 40% in HER2− IBC, including TNBC. CEC levels increased progressively during neoadjuvant treatment and decreased after its interruption. Baseline CTC and CEC levels were not predictive of pCR. Detection of CTC at 8 months of follow-up was associated with the absence of response to neoadjuvant chemotherapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-14-02.

Early drop of circulating tumor cells (CTC) and increase of circulating endothelial cells (CEC) during neoadjuvant chemotherapy (CT) combined with bevacizumab in HER2-negative inflammatory breast cancer (IBC) in multicenter phase II trial BEVERLY 1.

10510 Background: CTC detection in blood is an independent prognostic factor in primary breast cancer with a detection rate from 20 to 25% (Pierga CCR 2008). Changes in the CTC HER2 status compared to primary tumor have been reported. Predictive value of CEC for response to anti-angiogenic agents is debated. Methods: CTC and CEC were detected in 7.5 ml and 4 ml of blood respectively in the neoadjuvant setting of HER2 negative IBC (T4d) patients (pts) enrolled in the phase II multicenter trial BEVERLY 1, evaluating bevacizumab (15mg/kg q3w) in combination with sequential neoadjuvant CT of 4 cycles of FEC followed by 4 cycles of Docetaxel. The CellSearch System, combining EpCAM immunomagnetic selection followed by anti-cytokeratin (A45B/B3) and anti-HER2 staining for CTC and CD146 IMS and CD105 staining for CEC. Results: From 12/08 to 09/10, 101 pts were included and 92 were evaluable for CTC and CEC. At baseline, 37 pts out of 92 had ≥ 1 detectable CTC (40%, 95%CI 30-50%). No correlation was found between ...

Abstract P3-03-01: Minimal Residual Disease Monitoring in Breast Cancer Patients

Abstract BACKGROUND: Breast cancer (BC) patients still harbor a considerable risk of metastatic relapse caused by minimal residual disease (MRD) despite of complete removal of primary tumor. The aim of this study was to identify single disseminated tumor cells (DTC) in the bone marrow and circulating tumor cells (CTC) in the peripheral blood as potential biomarkers for therapy response monitoring and metastatic relapse risk prediction. Finally, the gene expression profiles of CTCs have been analyzed. METHODS: A total of 87 patients with diagnosed BC at stage I to III and 115 metastatic patients were enrolled into a prospective study between years 2008 - 2010. Peripheral blood (5ml) for CTC-detection was collected from primary BC patients before chemotherapy (CHT), after 2 cycles of CHT and after the CHT. Metastatic BC patients have been examined for CTCs before starting a new line of the treatment. Bone marrow aspirates from 16 premenopausal BC patients (mean age 31) with primary tumor were analyzed for the presence of DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3 (Epimet™, AS Diagnostik, Germany) before surgery. CTC detection in blood was performed by AdnaTest BreastCancerTM(AdnaGen AG, Germany), which is based on the detection of EpCAM, HER2 and MUC1 specific transcripts in enriched CTC-lysates. cDNA from isolated CTCs has been further pre-amplified and used for multimarker qPCR gene expression profiling using Biomark® microfluidic 48x48 GE Dynamic arrays (Fluidigm, USA). qPCR results have been analyzed by GENEX vs. 5.2 software (MultiD Analysis). RESULTS: 286 CTC samples have been analyzed in total. The analysis has shown that the expression profiles of CTCs from primary BC patients have been significantly different comparing them to the CTC-profiles from metastatic BC patients for several of tested genes (e.g., CK19, GA7332, MLFIP1, SATB1, PTEN). Interestingly, before surgery of primary tumor DTCs were found in 5/16 patients (31 %) and CTCs in 7/16 (43 %). Both DTCs and CTCs together were found in 4/16 (25%) patients. In 18% of the primary BC patients no dissemination markers were found CONCLUSIONS: Information based on the CTCs-gene expression profiles could provide an additional support for therapy management. Metastatic potential of enriched CTCs will be further evaluated on the single cell level. This work has been supported by Grant Agency of Ministry of Health, Czech Republic (IGA NS9976) and Grant Agency of Charles University in Prague, Czech Republic no. 7709 and 59410. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-03-01.

Transcriptomic Profiles and Circulating Tumor Cell Detection in Primary Breast Cancer.

Abstract Background: We have previously demonstrated that circulating tumor cells (CTC) detection by the CellSearch system is strongly associated with metastatic outcome in non-metastatic breast cancer (BC) treated by neoadjuvant chemotherapy (NACT) [Pierga, Clin Cancer Res 2008]. Transcriptomic analysis of primary tumors may uncover molecular phenotypes associated with CTC detection.Methods: Both CTC detection in blood at diagnosis and transcriptomic analysis of the primary tumor have been performed prospectively in 58 non-metastatic BC patients (pts) treated by NACT in a phase II trial (REMAGUS02). We searched for an association between CTC detection and (i) intrinsic molecular subtypes, (ii) stemness signature, (iii) other published signatures, and (iv) expression of molecular markers involved in CTC detection. CTC-associated genes were also studied (v).Results: (i) CTC detection was not statistically associated with an intrinsic molecular subtype: 29% in basal-like (n=5/17pts), 33% in HER2+ (3/9pts), 13% in luminal A (2/13pts), 20% in luminal B (2/10pts) and 29% in normal-like (2/7pts) BC. (ii) No association was found between CTC detection and the stemness signature. (iv) CTC detection was also not related to the “stemness profile”; it was independent of EpCAM, CK8 and CK18 transcriptomic expression. Other results (iii, v) will be disclosed at the meeting.Conclusion: Our study is the first to compare tumor gene expression profiles and blood dissemination of cancer cells in early BC pts. In these pts, CTC detection by the CellSearch system does not depend on intrinsic molecular subtypes, contrary to a published report based on in vitro-grown cells lines [Sieuwerts, J Natl Cancer Inst 2009]. Being independent from molecular prognostic factors, CTC detection is likely to play a critical role in early BC management.Supported by PHRC AOM/2OO2/02117, Pfizer inc., Roche, sanofi-aventis.ISRCTN10059974 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3005.

Methylation profile of human breast cancer: A possible biomarker for the detection of circulating tumor cells

11112 Background: Genetic and epigenetic events are complementary mechanisms involved in breast carcinogenesis. One of the most common epigenetic mechanisms by which genes are silenced is the aberrant methylation of their promoter CpG islands, i.e. hypermethylation in promoters of Tumor Suppressor Genes. The identification of circulating tumor cells (CTCs) in the blood of cancer patients, based on their aberrant methylation profile, appears as a potential tool for early detection and provides therefore the promise of a non-invasive and affordable cancer detection test. Methods: The methylation status of 26 cancer-related regions was studied, using Methyl Specific-Multiplex Ligation dependent Probe Amplification (MS-MLPA) assay in invasive breast tumors (n=24), axillary lymph nodes (n= 5), and normal breast tissue (n=2). A nested-Methyl Specific PCR (Nested-MSP) was designed for one of the methylated regions in the tumor suppressor gene rassf1A, to identify CTCs in peripheral blood samples. Blood samples from non tumor individuals were used as control. Results: We observed aberrant methylation of one or more of the 26 studied gene regions in all the breast cancer samples. The profiles were specific for each tumor. The more frequently aberrant methylated genes were: rassf1A promoter (62.5%), esr1 (54.17%), apc (54.17%) and rassf1A exon 1 (50%). Absence of aberrant methylation was confirmed in normal breast tissues. The specific methylation profile of tumors could be used to identify the metastasis origin in a patient with a left-sided breast tumor and bilateral tumor invasion of axillary lymph nodes. MS-MLPA revealed identical aberrant methylation patterns in both lymph nodes, revealing a left-sided origin of the right-sided lymph node. For the detection of CTCs in blood, a Nested-MSP on rassf1A was designed. This method allowed the detection of CTCs in the peripheral blood of a cancer patient by their aberrant rassf1A methylation. A healthy age-matched control blood sample revealed absence of CTCs. Conclusions: The identification of the methylation profile of the tumor by MS-MLPA allowed to establish the metastasis origin of invaded axillary lymph nodes and to detect CTCs in peripheral blood of a cancer patient. No significant financial relationships to disclose.

Circulating tumor cells (CTC) in a study of relapsed/recurrent advanced ovarian cancer: An exploratory analysis in the ova-301 phase III study of pegylated liposomal doxorubicin (PLD) compared with trabectedin and PLD

5551 Background: Circulating tumor cells (CTC) have demonstrated predictive and prognostic value among patients with metastatic breast, colorectal, and prostate cancer. In a phase III study of pegylated liposomal doxorubicin (PLD) with trabectedin (T) vs PLD for relapsed ovarian cancer, we assessed the affect of CTCs on progression free survival, (PFS) and overall survival (OS). Methods: CTCs were isolated from peripheral blood (10 mLs) using the CellSearch system and reagents (Veridex). A CTC is defined as EpCAM+, cytokeratin+, CD45-, and is positive for the nuclear stain DAPI. The normal reference range for CellSearch is &lt; 2 CTC/7.5 mLs of blood. Hazard ratios adjusted for known prognostic factors were estimated by Cox regression. Results: 216 subjects had baseline CTC measurements of which 111 (51.4%) were randomized to the PLD+T arm; 143/216 patients (66.2%) were platinum sensitive. Thirty-one/216 patients (14.4%) had 2 or more CTCs detected prior to the start of therapy (range 2–566). Univariate Cox regression analyses indicated that patient's &gt; 2 CTCs prior to therapy have 1.89 (p = 0.003) and 2.06 (p = 0.003) fold higher risk for progression and death respectively. Multivariate analyses that include baseline CTC, baseline CA125, platinum sensitivity status, largest diameter lesion, number of tumor lesions, ECOG PS, age, tumor histology, tumor grade and prior taxane show that patients with elevated baseline CTC have 1.58 (p = 0.058) and 1.54 (p = 0.096) fold higher risk for progression and death respectively. Conclusions: Results from this study indicate that although CTC detection in blood from relapsed recurrent ovarian cancer patients is relatively low, elevated numbers of CTCs imparts an unfavorable prognosis for patients. Multivariate analysis indicates that CTCs have prognostic value that is independent of established factors and thus provides a clinically useful tool for assessing prognosis in this difficult to treat patient population. [Table: see text]

The presence of circulating total DNA and methylated genes is associated with circulating tumour cells in blood from breast cancer patients

Circulating tumour cells (CTC) and tumour-related methylated DNA in blood have been separately assessed for their utility as a marker for subclinical metastasis in breast cancer. However, no studies have looked into the relation between the both molecular markers in this type of cancer. In this study, we investigated the correlations between total/methylated DNA and CTC in the blood from metastatic breast cancer patients. We simultaneously obtained whole blood, plasma and serum samples from 80 patients and 20 controls. The CellSearch System was used to enumerate CTC in blood samples. Plasma total DNA levels were determined by a QPCR method. Sera were analysed by methylation-specific QPCR for three markers: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A) and oestrogen receptor 1 (ESR1). Total DNA levels in patients were significantly increased when compared with controls (P<0.001) and correlated with the number of CTC (r=0.418, P<0.001). Hypermethylation of one or more genes was detected in 42 (53%) serum samples from breast cancer patients and in three (16%) serum samples from controls (P=0.003). APC was hypermethylated in 29%, RASSF1A in 35% and ESR1 in 20% of breast cancer cases. Detection of a methylated gene in serum was associated with the detection of CTC in blood (P=0.03). The detection of large amounts of circulating total/methylated DNA correlated with the presence of CTC in the blood from patients with breast cancer. This can be interpreted in two ways: (a) CTC are a potential source of circulating tumour-specific DNA; (b) high numbers of CTC and circulating methylated DNA are both a phenotypic feature of more aggressive tumour biology.

The detection of circulating tumour cells in blood of metastatic breast cancer patients is of prognostic relevance

PURPOSE: The prognostic relevance of circulating tumour cells (CTC) in blood of metastatic breast cancer patients was evaluated and compared with established prognostic criteria. PATIENTS AND METHODS: Blood samples from 119 breast cancer patient were examined in a retrospective analysis. For the detection of CTC in blood, a nested RT-PCR assay for mammaglobin mRNA was applied. RESULTS: In 42/119 (35%) patients CTC were detected. Patients with CTC positive blood samples at the time of diagnosis of metastases lived significantly shorter (median 18 months) than CTC-negative patients (median 51 months), suggesting that CTC serve as an additional prognostic parameter. CTC in blood were an independent prognostic parameter, associated with the highest risk of death compared to other risk factors examined (HR: 2.9). In addition, the appearance of CTC in blood of metastatic patients during treatment indicated poor prognosis. CTC-positive patients had a significantly shorter survival compared to patients remaining CTC negative during followup (p < 0.01). CONCLUSIONS: CTC at the time of diagnosis of metastases are an independent prognostic factor for overall survival. During therapy, the detection of CTC is predictive for a shorter survival of patients with metastatic breast cancer.

Detection of Circulating Tumor Cells in Peripheral Blood of Patients with Metastatic Breast Cancer: A Validation Study of the CellSearch System

The CellSearch system (Veridex, Warren, NJ) is designed to enrich and enumerate circulating tumor cells (CTCs) from peripheral blood. Here, we validated the analytic performance of this system for clinical use in patients with metastatic breast cancer. This prospective multicenter study conducted at three independent laboratories involved samples from 92 patients with metastatic breast cancer. Intra- and inter-assay variability using controls containing defined numbers of cells (average, 50 and 1,000, respectively), cell stability based on varying storage and shipment conditions, recovery precision from samples spiked with 4 to 12 tumor cells, inter-instrument variability, and positivity of samples from metastatic breast cancer patients were tested. Intra- and inter-assay precision for two sites were high: All eight positive controls analyzed in the same run and >95% of the run to run control values (n=299) were within the specified ranges. Recovery rate of spiked samples averaged between 80% and 82%. CTCs were detected in approximately 70% of metastatic breast cancer patients. CTC values of identical samples processed either immediately after blood drawing or after storage for 24, 48, or 72 h at room temperature or at 4 degrees C did not differ significantly. Shipment of samples had no influence on CTC values. When analyzing identical samples in different centers, inter-instrument accordance was high. The CellSearch system enables the reliable detection of CTCs in blood and is suitable for the routine assessment of metastatic breast cancer patients in the clinical laboratory. Blood samples should be shipped at room temperature and CTC counts are stable for at least 72 h.