Cell Search Method Research Articles

CellSearch technology applied to the detection and quantification of tumor cells in CSF of patients with lung cancer leptomeningeal metastasis.

The diagnosis of solid cancer leptomeningeal metastasis (LM) relies on the cytology of cerebrospinal fluid (CSF) and/or imaging evidence of neuraxis, yet both lack sufficient sensitivity. The utility of the CellSearch, an FDA -approved technology, in assessing CSF tumor cell (CSFTC) was evaluated here in the diagnosis and treatment of patients with lung cancer-related LM. In 18 patients with magnetic resonance imaging (MRI) confirmed LM due to lung cancer, 5 mL of CSF were collected in CellSave preservative tubes, which allow performing the assay within 96 h after sampling. Using a previously adapted CellSearch method, we detected, visualized and enumerated CSFTCs and compared the results with conventional cytology. In 3 patients, tumor cells were evaluated sequentially to explore the predictive role of CSFTCs enumeration in the treatment response monitoring. CSFTCs were disclosed in 14 of 18 MRI confirmed LM samples (median 785CSFTCs/5 mL CSF, range 1 to >20,000), yielding a sensitivity of 77.8%, compared with 44.4% for conventional cytology. CSFTC clusters were observed in 12 patients, similar to those previously described in blood as circulating tumor microemboli (CTM), and enumerated sequentially with reproducible results, which did not necessarily correlate with response to treatment. The CellSearch technology, applied to limited sample volumes and allowing delayed processing, could be of great interest in the diagnosis of LM in lung cancer patients.

Abstract P4-01-12: Circulating tumor cells (CTC) and endothelial cells (CEC) changes in HER2 negative metastatic breast cancer (MBC) patients treated with first line weekly paclitaxel and bevacizumab: Preliminary results of a prospective cohort from the French Breast Cancer

Abstract Background: increased levels of circulating tumor cells (CTC) are associated with worse progression-free survival (PFS) and overall survival (OS) in patients (pts) with MBC (Bidard FC et al, Lancet Oncol 2014). The failure of chemotherapy to reduce CTCs to levels below five CTCs per 7.5 mL whole blood at first follow-up after initiating a new systemic therapy for MBC predicts shorter time to progression and OS. It has been hypothesized that bevacizumab could modify CTC prognostic value due to extravasation or epithelio-mesenchymal transition induction. CEC variations to predict benefit of anti-angiogenic treatment is still controversial. Patients & methods: the French cohort COMET is a prospective study including first line HER2 negative pts receiving weekly paclitaxel and bevacizumab according to EMEA approved combination. The aim of this cohort is to evaluate clinical, biological and radiological parameters associated with pts outcome (CTC, CEC, VEGFA levels, ctDNA, pharmacogenomic polymorphisms, metabolomic parameters, visceral fat assessed by initial CTscan, serum estradiol level, and quality of life). We present here the first planned analysis on 206 patients evaluated for CTC and CEC using the FDA cleared CellSearch method. Results: inclusions started in 09/2012. At time of analysis, 219 patients were included, 211 were evaluable for CTC at baseline (failure rate 4%) and 207 for CEC at baseline (failure rate 5%). Due to short follow-up, 173 pts and 166 pts were evaluable for both CTC and CEC at baseline and first day of second cycle of CT (D1C2) respectively. At baseline, 100/211 (47%) pts had ≥ 5 CTC (median 4 (range 0-30,000) and 30% had no detectable CTC (0 CTC). After one cycle of chemotherapy (D28) 38 pts (22%) had still ≥ 5 CTC: 37 pts with initial high level and only one patient with low CTC at baseline had increased CTC above 5. Median number CEC was 21 (0)-2231) at baseline and 22 (1-881) at D1C2. CEC increased in 16% and decreased in 15% of the cases. CTC & CEC changes after one cycleCTC BaselineCTC at D1C2CEC BaselineCEC at D1C2≥ 5≥ 537 (21%)>20>2065 (39%)≥ 5<545 (26%)>20≤ 2025 (15%)<5≥ 51 (<1%)≤ 20>2027 (16%)<5<590 (52%)≤ 20≤ 2049 (30%)Total173166 CTC number at baseline line and CTC D1C2 were correlated (p<0.01) (Spearman test). There was no correlation between CEC at baseline or at D1C2 with CTC or CTC changes. Final analysis will be completed when 206 couples for both CTC and CEC at baseline and D1C2 will be available. Accrual is still ongoing. Conclusion: this 22% rate of failure to reduce CTC < 5 after one cycle of first line CT in a homogeneously bevacizumab-treated cohort of MBC patients did not differ from previous series. As second lines of chemotherapy do not improve the poor prognosis of this group of patients according to the SWOG 500 study results (Smerage et al, JCO 2014), trials of novel therapeutic agents should be considered at the time of progression. Citation Format: Jean-Yves Pierga, Isabelle Vaucher, Maya Gutierrez, Olivier Tredan, Séverine Guiu, Gilles Romieu, Anthony Goncalves, Marc Debled, Christelle Levy, Jean-Marc Ferrero, Christelle Jouannaud, Elisabeth Luporsi, Marie-Ange Mouret-Reynier, Florence Dalenc, Bernard Asselain, Jerome Lemonnier, Francois-Clement Bidard. Circulating tumor cells (CTC) and endothelial cells (CEC) changes in HER2 negative metastatic breast cancer (MBC) patients treated with first line weekly paclitaxel and bevacizumab: Preliminary results of a prospective cohort from the French Breast Cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-12.

Detection of disseminated tumor cells in aspirative drains after neck dissection.

The dissemination of individual tumor cells is a common phenomenon in solid cancers. Detection of tumor cells in bone marrow disseminated tumor cells (DTC) and in peripheral blood circulating tumor cells (CTC) in nonmetastatic situation is of high prognostic significance. Compared to breast, colon and prostate cancers, the studies on CTC and DTC in head and neck cancers are sparse. The objective of our study was to detect DTC in drains after neck dissection. Fourteen patients undergoing surgery for stages III and IV head and neck cancers were enrolled in this study--twelve presenting with squamous cell carcinoma and two with adenocarcinoma. Redon drain analysis was performed by the Cellsearch method using immunomagnetic and fluorescence approaches. A positivity threshold value was set at 2DTC/7.5 ml of the sample. Tumor cells were detected in drains of 69 % of patients a few days after surgery. The range of quantification was 3-2,094 DTC/5 ml and we showed morphological differences between the two types of carcinoma cells. DTC were detected after neck dissection both in squamous cell carcinoma and in adenocarcinoma. Potential clinical significance of tumor cells needs to be further investigated as their presence could affect pre-surgical and post-operative treatments.

In vitro detection of circulating tumor cells compared by the CytoTrack and CellSearch methods

Comparison of two methods to detect circulating tumor cells (CTC) CytoTrack and CellSearch through recovery of MCF-7 breast cancer cells, spiked into blood collected from healthy donors. Spiking of a fixed number of EpCAM and pan-cytokeratin positive MCF-7 cells into 7.5 mL donor blood was performed by FACSAria flow sorting. The samples were shipped to either CytoTrack or CellSearch research facilities within 48 h, where evaluation of MCF-7 recovery was performed. CytoTrack and CellSearch analyses were performed simultaneously. Recoveries of MCF-7 single cells, cells in clusters, and clusters were determined. The average numbers of MCF-7 cells/cells in clusters/clusters recovered from blood by the CytoTrack and CellSearch methods were 103 ± 5.9/27 ± 7.9/11 ± 3.5 (95 % CI) and 107 ± 4.4/20 ± 7.1/10 ± 3.5, respectively, with no difference between the two methods (p = 0.37/p = 0.23/p = 0.09). Overall, the recovery of CytoTrack and CellSearch was 68.8 ± 3.9 %/71.1 ± 2.9 %, respectively (p = 0.58). In spite of different methodologies, CytoTrack and CellSearch found similar number of CTCs, when spiking was performed with the EpCAM and pan cytokeratin-positive cell line MCF-7. The results suggest that CytoTrack and CellSearch have similar abilities to identify CTC in vitro.

Clinical phenotypes associated with circulating tumor cell enumeration in metastatic castration–resistant prostate cancer

BackgroundThe presence of ≥5 circulating tumor cells (CTCs) is prognostic for shorter survival in men with metastatic castration–resistant prostate cancer (mCRPC). However, some men have low CTCs despite widespread disease, suggesting heterogeneity in CTC phenotype or detection. The aim of this study was to evaluate the association of CTC enumeration with clinical disease characteristics and overall survival in men with mCRPC at our institution. DesignCTCs were enumerated using the CellSearch method in a prospective correlative study in men with mCRPC starting a new systemic therapy. The primary objective was to determine the clinical phenotype of the subset of men with mCRPC who have a poor prognosis and low CTCs. Secondary end points included associations of CTCs with survival and known prognostic biomarkers, before therapy and at progression. ResultsAt baseline, median CTC count was 16 cells and prostate-specific antigen (PSA) level was 178ng/ml. At progression, median CTC count was 42, PSA level was 245ng/ml, levels of lactate dehydrogenase and alkaline phosphatase rose, and level of hemoglobin dropped. The median overall survival for this heavily pretreated population was 11.2 months, and the multivariable hazard ratio for death of men with CTCs<5 vs.≥5 was 0.43 (95% CI: 0.24–0.77). Median progression-free survival was 4.4 months. CTC enumeration modestly correlated with lactate dehydrogenase and alkaline phosphatase levels but only weakly correlated with PSA and hemoglobin levels. We were unable to identify a consistent subgroup of poor prognosis men with a low number of CTCs. ConclusionCTC enumeration appears to be prognostic in men with mCRPC and describes a phenotype of hematogenous dissemination that cannot be predicted based on standard clinical and laboratory assessments.

Research on Multi-Color Separation Model Based on Polynomial Regression

In order to improve the quality of prints, we usually add additional OG or RGB ink on the basis of the four-color printing ink (CMYK) to increase the printing color gamut. After that, the conventional separation technology is no longer suit for multi-color printing, so the multi-color separation technology is put forward. Currently, the main multi-color separation method is color partition technology. It divided the color space into five or six partitions. Then each partition was subdivided into several cells, and then established the polynomial color separation model for each cell. In this paper, we improved the selection of samples and cell search method. The experimental results showed that the color separation algorithm had high color separation accuracy and stability and could meet the demand of high precision multi-color printing.

Circulating tumor cell enumeration with a combination of epithelial cell adhesion molecule- and cell-surface vimentin-based methods for monitoring breast cancer therapeutic response.

Detection, isolation, and enumeration of circulating tumor cells (CTCs) from cancer patients has become an important modality in clinical management of patients with breast cancer. Although CellSearch, an epithelial cell adhesion molecule (EpCAM)-based method that is used to isolate epithelial CTCs, has gained prominence, its inability to detect mesenchymal CTCs from breast cancer patients raises concerns regarding its utility in clinical management. To address this gap in technology, we recently discovered the utility of cell-surface vimentin (CSV) as a marker for detecting mesenchymal CTCs from sarcoma tumors. In the present study, we tested the sensitivity and specificity of detecting CTCs from blood collected at a random time during therapy from each of 58 patients with metastatic breast cancer by use of 84-1 (a monoclonal antibody against CSV to detect epithelial/mesenchymal-transition CTCs) and CellSearch methods. Additionally, we tested the possibility of improving the sensitivity and specificity of detection by use of additional parameters including nuclear EpCAM localization and epithelial mesenchymal ratios. CTC counts with CSV were significant (P = 0.0053) in differentiating populations responsive and nonresponsive to treatment compared with CTC counts with CellSearch (P = 0.0564). The specificity of CTC detection was found to be highest when the sum of CTC counts from the 2 methods was above a threshold of 8 CTCs/7.5 mL. The sum of CTC counts from the CellSearch and CSV methods appears to provide new insights for assessment of therapeutic response and thus provides a new approach to personalized medicine in breast cancer patients.

Abstract 35: A universal marker for the detection of epithelial-mesenchymal transitioned circulating tumor cells and their prognostic relevance in epithelial cancers

Abstract Circulating tumor cells (CTCs) are the tumor cells shed from the primary tumor tissues into the bloodstream or lymphatic vessels. These CTCs are considered to be emerging tools for the detection and prognosis of several types of metastatic cancers. At present, CTC markers are limited to epithelial cancers and there are no specific markers available to detect mesenchymal and epithelial-mesenchymal transformed (EMT) CTCs. EMT cells are characterized by increased motility and invasiveness and are known to escape detection technologies that use EpCAM and cytokeratins as CTC markers. We have previously reported the detection of mesenchymal cancer cells from blood of sarcoma cancer patients for the first time using a novel monoclonal antibody 84-1. Here we report the detection of EMT CTCs from colon, breast and prostate cancer patients using 84-1 monoclonal antibody. Isolated EMT CTCs were characterized for EMT phenotype by staining for Snail, Slug, Twist-1 and FOXC2. To analyze the efficiency of CTC detection, we compared the CTC counts obtained using 84-1 antibody to that of the FDA approved CellSearch method. We collected blood samples from breast and prostate cancer patients and isolated a) EMT CTCs using 84-1 and b) Epithelial CTCs using Cell Search. For validation purposes, we analyzed an experimental set of patient samples that included normal, non-metastatic and metastatic samples to derive a threshold of detecting CTCs in these patients. After validating a threshold value of the CTC, we used an experimental set that was randomized to evaluate CTCs from different patient sets. Since the experimental set consisted of randomized set of patients with different stages of disease progression and treatments, we evaluated the association between CTC count and therapeutic outcome (progression/ stable &amp; responding). Our results indicated that patients with ≥ 5 EMT CTCs/7.5 mL as detected by 84-1 antibody were independent predictors of therapeutic outcome in breast and prostate cancer patients. Detection using 84-1antibody was much more sensitive and specific when compared with CellSearch method. Collectively, our observations provide a novel technology to selectively detect and isolate EMT CTCs. Detection and molecular characterization of CTCs is one of the fastest growing areas of translational cancer research. Utilizing 84-1, we can enumerate and isolate rare EMT CTCs that will aid as prognostic indicators of therapeutic outcome, metastasis, and relapse and will contribute to the development of specific targeted therapies, an ultimate goal of personalized medicine. Citation Format: Arun Satelli, Zachary Brownlee, Scott Kopetz, Michael Overman, Qing H. Meng, Shulin Li. A universal marker for the detection of epithelial-mesenchymal transitioned circulating tumor cells and their prognostic relevance in epithelial cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 35. doi:10.1158/1538-7445.AM2014-35

Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data

We aimed to assess the clinical validity of circulating tumour cell (CTC) quantification for prognostication of patients with metastatic breast cancer by undertaking a pooled analysis of individual patient data. We contacted 51 European centres and asked them to provide reported and unreported anonymised data for individual patients with metastatic breast cancer who participated in studies between January, 2003, and July, 2012. Eligible studies had participants starting a new line of therapy, data for progression-free survival or overall survival, or both, and CTC quantification by the CellSearch method at baseline (before start of new treatment). We used Cox regression models, stratified by study, to establish the association between CTC count and progression-free survival and overall survival. We used the landmark method to assess the prognostic value of CTC and serum marker changes during treatment. We assessed the added value of CTCs or serum markers to prognostic clinicopathological models in a resampling procedure using likelihood ratio (LR) χ(2) statistics. 17 centres provided data for 1944 eligible patients from 20 studies. 911 patients (46·9%) had a CTC count of 5 per 7·5 mL or higher at baseline, which was associated with decreased progression-free survival (hazard ratio [HR] 1·92, 95% CI 1·73-2·14, p<0·0001) and overall survival (HR 2·78, 95% CI 2·42-3·19, p<0·0001) compared with patients with a CTC count of less than 5 per 7·5 mL at baseline. Increased CTC counts 3-5 weeks after start of treatment, adjusted for CTC count at baseline, were associated with shortened progression-free survival (HR 1·85, 95% CI 1·48-2·32, p<0·0001) and overall survival (HR 2·26, 95% CI 1·68-3·03) as were increased CTC counts after 6-8 weeks (progression-free survival HR 2·20, 95% CI 1·66-2·90, p<0·0001; overall survival HR 2·91, 95% CI 2·01-4·23, p<0·0001). Survival prediction was significantly improved by addition of baseline CTC count to the clinicopathological models (progression-free survival LR 38·4, 95% CI 21·9-60·3, p<0·0001; overall survival LR 64·9, 95% CI 41·3-93·4, p<0·0001). This model was further improved by addition of CTC change at 3-5 weeks (progression-free survival LR 8·2, 95% CI 0·78-20·4, p=0·004; overall survival LR 11·5, 95% CI 2·6-25·1, p=0·0007) and at 6-8 weeks (progression-free survival LR 15·3, 95% CI 5·2-28·3; overall survival LR 14·6, 95% CI 4·0-30·6; both p<0·0001). Carcinoembryonic antigen and cancer antigen 15-3 concentrations at baseline and during therapy did not add significant information to the best baseline model. These data confirm the independent prognostic effect of CTC count on progression-free survival and overall survival. CTC count also improves the prognostication of metastatic breast cancer when added to full clinicopathological predictive models, whereas serum tumour markers do not. Janssen Diagnostics, the Nuovo-Soldati foundation for cancer research.

Circulating tumor cells

The hypothesis of the “liquid biopsy” using circulating tumor cells (CTCs) emerged as a minimally invasive alternative to traditional tissue biopsy to determine cancer therapy. Discordance for biomarkers expression between primary tumor tissue and circulating tumor cells (CTCs) has been widely reported, thus rendering the biological characterization of CTCs an attractive tool for biomarkers assessment and treatment selection. Studies performed in metastatic colorectal cancer (mCRC) patients using CellSearch, the only FDA-cleared test for CTCs assessment, demonstrated a much lower yield of CTCs in this tumor type compared with breast and prostate cancer, both at baseline and during the course of treatment. Thus, although attractive, the possibility to use CTCs as therapy-related biomarker for colorectal cancer patients is still limited by a number of technical issues mainly due to the low sensitivity of the CellSearch method. In the present study we found a significant discordance between CellSearch and AdnaTest in the detection of CTCs from mCRC patients. We then investigated KRAS pathway activating mutations in CTCs and determined the degree of heterogeneity for KRAS oncogenic mutations between CTCs and tumor tissues. Whether KRAS gene amplification may represent an alternative pathway responsible for KRAS activation was further explored. KRAS gene amplification emerged as a functionally equivalent and mutually exclusive mechanism of KRAS pathway activation in CTCs, possibly related to transcriptional activation. The serial assessment of CTCs may represent an early biomarker of treatment response, able to overcome the intrinsic limit of current molecular biomarkers represented by intratumor heterogeneity.

Evidence for circulating tumor cell (CTC) alkaline phosphatase (AP) expression in men with bone-metastatic castration-resistant prostate cancer (CRPC) during abiraterone acetate treatment.

178 Background: Serum alkaline phosphatase (AP) changes in men with metastatic prostate cancer have been described since the Nobel Prize winning discovery of the hormonal dependence of PC. High serum AP is independently associated with mortality in men with castration-resistant prostate cancer (CRPC) and reductions in AP with treatment are favorably prognostic. Transient rises in AP may occur during treatment initiation and reflect bone remodeling/healing. The source of serum AP in men with metastatic prostate cancer, however, remains unclear. We hypothesize that cancer cell osteomimicry and epithelial plasticity may contribute to serum AP elevations in these men. Methods: We examined the expression of AP in the circulating tumor cells (CTCs) of men with metastice CRPC on abiraterone acetate who had elevated serum AP to determine if one possible source of serum AP may be tumor-derived rather than host/bone derived. CTCs were isolated using the CellSearch method and EpCAM ferromagnetic capture, and only CD45 negative cells were considered CTCs. AP was evaluated using immunofluorescence and real time positive and negative controls. Liver function was evaluated to ensure lack of confounding with liver-derived AP. Results: We identified AP to be commonly expressed in the CTCs of men with CRPC who had high serum AP, with no expression in patient leukocytes. One patient had a transient rise in serum AP with no change in other liver enzymes during a dramatic response to abiraterone acetate while his prostate-specific antigen and CTC declined. AP expression was evident and common in his CTCs during this serum AP flare. A second patient was found to have common CTC AP expression in both CK+ and CK- CTCs. This patient had evidence of AR amplification, PTEN loss, and TMPRSS2-ERG fusion in his CK- and CK+ CTCs, accompanied by a high number of OB-cadherin + CTCs, indicating likely osteomimicry and plasticity of these clonal epithelial tumor cells. Conclusions: We provide clinical evidence that PC CTCs express AP, supporting the hypothesis that AP expression is associated with osteomimicry as part of the biology of epithelial plasticity in prostate cancer. These findings support the existence of multiple CTC phenotypes, the clinical significance of which is unknown.

In vivo isolation of circulating tumor cells in prostate cancer patients by a medical device.

e22055 Background: CTCs are discussed as a prognostic and stratification biomarker, and may help to assess the treatment efficacy. The aim was to demonstrate proof of concept of in vivo CTC isolation in Prostate cancer patients (PCa). Methods: A medical device (CellCollector) was inserted in a cubital vein for 30 minutes. The interaction of target CTCs with the CellCollector was mediated by antibodies to the epithelial cell adhesion molecule (EpCAM). To confirm binding of CTCs to the wire, immunohistochemical staining against Cytokeratin and CD45 was performed. There were 226 applications of the device in 16 metastasized PCa (PCa-m) with up to 8 wire applications per patient, 24 localized PCa (PCa-l) with up to 3 wire applications per patient as well as 19 men with benign prostate hypertrophy (BPH) and 21 women as control group with only one wire application. CTC counts from 22 PCa patients with 58 applications were directly compared to the CellSearch technology. Results: We obtained in vivo isolation of CTCs in 73 of 98 applications to PCa patients (74.5 %). The sensitivity for metastasized PCa was (86.2%) and for local PCa (57.5%), respectively. Follow up data of one patient with PCa-m: 2009 diagnosed PCa with multiple metastases. 2 years later PSA of 11.2ng/mL and 25 CTCs. After transurethral resection and removal of a brain metastasis, CTCs decreased to 20 cells with no effect on PSA levels. 4 months later we observed 187 CTCs and a PSA level of 76ng/mL, which was correlated with the patient condition. In PCa-l patients we observed prior radical prostatectomy (n=18) and 6 (n=15) and 12 (n=7) months after surgery CTC numbers on average of 18.2, 2.8 and 1.4, respectively. In the direct comparison of the CellCollector and CellSearch we showed detection rates of 65% (13/20) and 20% (4/20). Conclusions: We demonstrate the use of CTCs besides PSA during therapy adjustment in PCa patients In summary the CTC detection rate of the CellCollector in PCa-m and PCa-l in comparison to the CellSearch method increased by 26% and 45%. Further molecular genetic CTC characterization as well as multiple antibody coating at the CellCollector will assume crucial information for therapy decisions. Clinical trial information: ISRCTN10403616.

Evaluation of clinical phenotype, survival, and circulating tumor cell (CTC) enumeration in men with metastatic castration-resistant prostate cancer (mCRPC).

5031 Background: The presence of ≥ 5 CTCs is prognostic for shorter survival in mCRPC men. However, many men have low CTCs despite widespread metastatic disease, suggesting heterogeneity in CTC phenotype or detection. We evaluated the association of baseline CTC enumeration with clinical characteristics and survival in mCRPC men at our institution. Methods: CTCs were enumerated with the standard CellSearch method in mCRPC men in a prospective correlative clinical study. The association between baseline CTC count and prostate-specific antigen (PSA), alkaline phosphatase (AP), lactate dehydrogenase (LDH) was explored using the Spearman correlation (r), and the relationship with Gleason sum, sites of metastasis (mets), and prior docetaxel exposure was explored using summary statistics. The overall survival probability (OS) was estimated by the Kaplan-Meier method. Results: In our cohort, 59/82 men had prior docetaxel exposure, and the median CTC count was 16.5. There were 25 men with visceral mets, 55 with bone-predominant mets, and 2 with lymph node only mets. We found a weak relationship between CTCs and PSA (r=0.20), but a more moderate and significant association with AP (r=0.48) and LDH (r= 0.50). We found no relationship between CTCs and Gleason sum, site of mets, or prior docetaxel. 66 men died, and the median OS was 11.2 months (95% CI: 9.2, 12.7). OS was improved in patients with &lt;5 CTCs at baseline compared to ≥ 5 (8.9 vs. 16.6 months, HR=0.47 (95% CI: 0.27, 0.80)). CTC enumeration may further stratify outcomes in men with mCRPC and either visceral or bone metastasis as shown in the table below. Conclusions: The prognostic significance of CTCs is distinct from other established biomarkers and may reflect a unique biology of metastasis, dissociated in part from androgen receptor activity (as reflected by PSA values) but related in part to bone microenvironment, hypoxia, and tumor burden (as reflected by AP and LDH values). These associations need to be validated in larger trials. [Table: see text]

Genomic heterogeneity of circulating tumor cells in castration-resistant prostate cancer (CRPC) revealed by single-cell sequencing.

5030 Background: Circulating tumor cells (CTC) provide an opportunity to sample multiple metastatic tumor sites through a single blood draw – a ”fluid biopsy.” NextGen DNA sequencing provides the means to obtain detailed genetic information from captured cells prior to and during treatment. Here we demonstrate the use of DNA sequencing to interrogate genome-wide copy number variations (CNV) at the single-cell level in CTC isolated from pts with CRPC. Methods: Pre- and post-treatment blood samples were obtained from pts treated at MSKCC. EpCAM+ events were collected singly and in groups by cytometric flow sorting and were subjected to DNA amplification and Illumina NextGeneration sequencing. Parallel samples were assayed using the Veridex CellSearch method to ensure the presence of malignant cells. Results: Samples with up to 50 EpCAM+ events analyzed in bulk displayed CNV patterns expected from published CRPC data. Subsequent single cell analyses showed that the method could reliably detect common genomic markers in CRPC, including AR amplification, PTEN and RB1 loss, and the TMPRSS-ERG fusion. Individual genomic CNV profiles obtained from 125 single cells isolated from 15 patients were then analyzed. Using unsupervised clustering, cells from each pt showed a closely related lineage structure, consistent with an evolution from a common ancestor. The degree of genomic heterogeneity within CTC from an individual pt was highly variable, with R2 correlation coefficients ranging from &gt;0.92 (nearly homogeneous) to &lt;0.75 (mixed populations). Two pts harbored separate subpopulations with both amplified AR and non-amplified AR cells and another displayed mixtures of genetic markers that changed over the course of treatment. Conclusions: The observed variation in complexity of CTC populations in CRPC pts underscores the importance of being able to sample and analyze multiple cells from an individual pt on multiple occasions and with real time analytics. Doing so is essential to understand and identify mechanisms of resistance so that they can be targeted effectively. Supported by STARR Cancer Consortium, NCI SPORE in Prostate Cancer; Department of Defense; Prostate Cancer Foundation.

Abstract 1452: Perioperative detection of circulating tumor cells in patients undergoing colorectal cancer liver and lung metastasectomy : Comparing CellSearch and FMSA technologies and investigating the role of EMT .

Abstract Circulating tumor cells (CTCs) reflect disease burden in metastatic colorectal cancer (mCRC) and patients with resectable liver and lung metastases are potentially curable by surgery with a 5-year survival of 30% . Here we report a study where we are detecting CTCs from blood of such patients collected perioperatively, using simultaneously the FDA-approved Veridex CellSearch and the Flexible Micro Spring Array (FMSA) device developed by Penn State Bioengineering and have accrued 12 patients so far. 7.5 ml of blood is collected from the central venous catheter placed during surgery at incision, at the point of starting surgical resection of the mass, 30 minutes after complete removal of the mass and on the day after surgery. Variation in CTC counts between samples from the same patient is expected to provide valuable answers about the kinetics of tumor cell shedding and dissemination during surgery. Tissue from resected liver or lung is appropriately stored for benchtop studies to correlate molecular signatures with CTC counts. Preliminary results suggest a mean count of 36 cells per blood sample detected using FMSA technology, with counts varying between zero to 101 cells / 7.5 ml of blood, in contrast to only zero to 2 cells in similar samples detected using the CellSearch method. FMSA relies on size-based isolation of CTCs from blood which are identified as CK+, DAPI +, CD45- cells on further immunostaining .The trial provides an opportunity to compare the two technologies in an important clinical setting and understand the underlying molecular mechanisms for the difference in counts . Aggressive tumors have been hypothesized to shed cells that have undergone EMT and thus evade detection by the EpCAM-based CellSearch detection method . We have noted expression of the mesenchymal marker vimentin in the CTCs captured by FMSA device thus documenting EMT . We plan to do additional stem cell and prognostic marker analysis on FMSA-captured CTCs . Both liver and lung metastases are being studied to compare two organs with different vascular anatomies and divergent surgical techniques. Correlating cell counts with disease-free survival as well as relapse will be helpful in validating intra-operative CTC isolation technique as means of prognosticating post-surgical recovery and gain insights into link between possible vascular tumor cell shedding and the surgical and vascular ligation technique that is followed. Future directions include comparing results with a patient population undergoing hepatic or pulmonary surgical procedures for non-malignant disease as well as extend the trial to include patients who are candidates for adjuvant chemotherapy and explore the role of intraoperative CTC counts and marker expression in choice of regime and predicting response. Citation Format: Avisnata Das, David T. Dicker, Ramdane Harouaka, Siyang Zheng, Noel Warfel, Kevin F. Staveley-O'Carroll, Wafik S. El-Deiry, Jussuf T. Kaifi. Perioperative detection of circulating tumor cells in patients undergoing colorectal cancer liver and lung metastasectomy : Comparing CellSearch and FMSA technologies and investigating the role of EMT . [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 1452. doi:10.1158/1538-7445.AM2013-1452

Detection of Circulating Tumor Cells in Breast Cancer Patients: Prognostic Predictive Role

A determination of circulating tumor cell (CTC) effectiveness for prediction of progression-free survival (PFS) and overall survival (OS) was conducted as an adjunct to standard treatment of care in breast cancer management. Between November 2008 and March 2009, 22 metastatic and 12 early stage breast carcinoma patients, admitted to Ankara Oncology Training and Research Hospital, were included in this prospective trial. Patients' characteristics, treatment schedules and survival data were evaluated. CTC was detected twice by CellSearch method before and 9-12 weeks after the initiation of chemotherapy. A cut-off value equal or greater than 5 cells per 7.5 ml blood sample was considered positive. All patients were female. Median ages were 48.0 (range: 29-65) and 52.5 (range: 35-66) in early stage and metastatic subgroups, respectively. CTC was positive in 3 (13.6%) patients before chemotherapy and 6 (27.3%) patients during chemotherapy in the metastatic subgroup whereas positive in only one patient in the early stage subgroup before and during chemotherapy. The median follow-up was 22.0 (range: 21-23) and 19.0 (range: 5-23) months in the early stage and metastatic groups, respectively. In the metastatic group, both median PFS and OS were significantly shorter in any time CTC positive patients compared to CTC negative patients (PFS: 4.0 vs 14.0 months, Log-Rank p=0.013; and OS: 8.0 months vs. 20.5 months, Log-Rank p<0.001). OS was affected from multiple visceral metastatic sites (p=0.055) and higher grade (p=0.044) besides CTC positivity (log rank p<0.001). Radiological response of chemotherapy was also correlated with better survival (p<0.001). As a result, CTC positivity was confirmed as a prospective marker even in a small patient population, in this single center study. Measurement of CTC by CellSearch method in metastatic breast carcinoma cases may allow indications of early risk of relapse or death with even as few as two measurements during a chemotherapy program, but this finding should be confirmed with prospective trials in larger study populations.

In vivo isolation of circulating tumor cells in non-small cell lung cancer (NSCLC) patients by a medical device.

10536 Background: Currently, circulating tumor cells (CTCs) are isolated in vitro from small limited volumes of blood samples. Furthermore, CTC results for NSCLC as a prognostic and stratification biomarker are scarce. The aim of the study was to assess a functionalized and structured medical wire (FSMW) for in vivo isolation of CTCs directly from the blood of NSCLC patients, and to compare it to the Cell Search method. Methods: The device was inserted in a cubital vein through a standard cannula for thirty minutes. The interaction of target CTCs with the FSMW was mediated by an antibody directed against the epithelial cell adhesion molecule (EpCAM). To confirm the CTCs binding to the wire, the immunohistochemical staining against EpCAM as well as CD45 for negative cell selection was performed. There were 72 applications of the wire in 48 stage I-IIIB NSCLC patients (12 double applications) and 12 non cancer patients. Enumeration data was available for 34 NSCLC patients and 8 non cancer patients. For 34 patients, samples were also tested in the Cell Search system. Results: The device was well tolerated in all 72 applications without side effects. We obtained in vivo isolation of CTCs in 32 of 34 NSCLC patients (94.1%) with a median (range) of 13 (0-300) CTCs. The sensitivity was similar for early and late stage NSCLC patients. In the non-cancer patients, no CTCs were detected (100 % specificity). In 2 of 34 samples (5.8%), CTCs were detected by the Cell Search method. In all matched pairs, the FSMW detected the same number or more CTCs compared to the Cell Search method. Conclusions: Whilst well tolerated without side effects, the CTC detection rate of the FSMW in NSCLC patients was &gt;90%. In contrast, &lt;10% detection was obtained using the Cell Search analysis. No CTCs were detected in non-cancer patients. This proof of concept study may have important clinical implications, as the implementation of the device into clinical practice may improve early detection, prognosis and therapy monitoring of NSCLC patients. The method may also allow the molecular analysis of the CTCs, with the possibility of establishing more personalized treatment regiments.

A new medical device for in vivo capturing of circulating tumor cells in breast cancer (BC) patients.

10537 Background: In BC, the number of circulating tumor cells (CTCs) is discussed as a prognostic and stratification biomarker, and could also reflect treatment efficacy. Currently, CTCs are isolated ex vivo from a small volume of blood. Results from a larger volume of blood are scarce. The aim of the study was to assess a functionalized and structured medical wire (FSMW) for in vivo capturing of CTCs directly from the blood stream of BC patients. Methods: The device was inserted in a cubital vein through a standard cannula for thirty minutes. The interaction of target CTCs with the FSMW was mediated by antibodies directed against the epithelial cell adhesion molecule (EpCAM). To confirm binding of CTCs to the wire, the immunohistochemical positive staining against EpCAM as well as negative staining for CD45 was performed. There were 54 applications of the wire in 42 stage I-IV BC patients (12 double applications). Enumeration data from 37 BC patients with 49 applications (5 failed subsequent analyses) were assessed. CTC counts on 23 devices were directly compared to counts by CellSearch. Results: The device was well tolerated in all 54 applications without side effects. We obtained in vivo isolation of CTCs in 44 of 49 applications to BC patients (89.7 %). The sensitivity was similar for early and late stage BC patients. The median (range) of isolated EpCAM-positive CTCs was 5 (0-515). The CellSearch method reached a sensitivity of 18.5%. In all paired samples the number of CTCs detected with the FSMW was higher or equal to CellSearch, regardless of the disease stage. Linear regression of the data of the double application of the FSMW showed a very good concordance (r2 = 0.97, p&lt;0.0001). Conclusions: Whilst well tolerated without side effects, the CTC detection rate of the FSMW in BC patients was nearly 90 %. CTC detection was obtained in 18.5% by the CellSearch. Double application of FSMWs in the same patient indicates ample precision. This proof of concept study may have important clinical implications, as the device may improve early detection, prognosis and therapy monitoring of BC patients. The molecular analysis of the captured CTCs could become a breakthrough in personalized medicine.

Detection of circulating tumor cells: Prognostic and predictive role.

e11002 Background: The effectiveness of CTC determination on progression free survival (PFS) and overall survival (OS) in early and advanced breast cancer as an adjunct to standard care of treatment was evaluated. Methods: Between November 2008 and March 2009, 22 metastatic and 12 early stage breast carcinoma patients, admitted to Ankara Oncology Training and Research Hospital and gave informed consent to participate, were included in this prospective trial. Patients’ characteristics, treatment schedules and survival data were evaluated. Physical examination and routine radiological assessment were done regularly for the evaluation of response. CTC was detected by CellSearch method before and 9 to 12 weeks after the initiation of chemotherapy. Results: All patients were female. Median age was 48.0 (range: 29-65) and 52.5 (range: 35-66) in early stage and metastatic groups, respectively. CTC was positive (≥5) in 3 (13.6%) patients before chemotherapy and 6 (27.3%) patients at anytime during chemotherapy in...

First-in-man study of E-3810, a novel VEGFR and FGFR inhibitor, in patients with advanced solid tumors.

TPS149 Background: Co-selective inhibition of VEGFRs and FGFR has the potential benefit of blocking the two most relevant players in tumor angiogenesis and simultaneously targeting proliferation in FGF-driven tumors. E-3810 (Cancer Res. 2011 in press) is a novel dual-targeted small molecule inhibitor of VEGFR1, 2, 3 and FGFR1 showing strong anti-angiogenic and anti-tumor activity in preclinical models at well-tolerated oral doses, with a favorable pharmacokinetic profile and potential for use in combination. Methods: A first-in-man, phase I dose escalation study is ongoing in patients with advanced solid tumors and no established therapeutic options to evaluate the safety, pharmacokinetics (PK) and pharmacodinamics of E-3810 given orally, once daily for a 4-week course. Treatment can continue without interval until progression based on overall clinical benefit. We use a standard 3+3 MTD design, starting from 5 mg (1/12 of monkey MTD) and up to the MTD based on DLTs in the initial cycle. Dose is doubled till the first instance of G2 drug-related toxicity or G2 uncontrolled hypertension, with smaller increments thereafter. The MTD is the level where ≥ 2/6 patients suffer a DLT, the next lower level being the recommended dose (RD). An expansion basket phase is planned at the RD, in patients who could benefit from E-3810, i.e. with tumors sensitive to anti-angiogenic therapy and failing a prior anti-angiogenic regimen, or breast cancer with FGFR1 amplification, to identify indications for further development. PK after single and repeated administration is evaluated by a validated LC-MS/MS method. Correlative studies (baseline, days 7 and 28) include: (i) quantitative assessment of the effects of E-3810 on tumor vasculature by DCE-MRI and DCE-US imaging; (ii) assay of angiogenesis biomarkers i.e. soluble VEGFR2, VEGFR1, VEGF, bFGF, Collagen IV (by ELISA) and circulating endothelial and progenitors cells (CEC and CEP, by flow cytometry). Tumor response is based on imaging according to RECIST; circulating tumor cells (CTC) are measured by the immunomagnetic CellSearch method. Recruitment started in July 2010; the dose escalation phase is ongoing with 10 patients enrolled as of January 2011. No DLT has been observed up to 20 mg.