CellSearch Technique Research Articles

Application of CellSearch technique in detection of peripheral blood circulating tumour cell count in patients with head and neck cancer and its association with prognosis

Application of CellSearch technique in detection of peripheral blood circulating tumour cell count in patients with head and neck cancer and its association with prognosis

EGFR and HER3 expression in circulating tumor cells and tumor tissue from non-small cell lung cancer patients

Although clinically relevant, the detection rates of EpCAM positive CTCs in non-small cell lung cancer (NSCLC) are surprisingly low. To find new clinically informative markers for CTC detection in NSCLC, the expression of EGFR and HER3 was first analyzed in NSCLC tissue (n = 148). A positive EGFR and HER3 staining was observed in 52.3% and 82.7% of the primary tumors, and in 62.7% and 91.2% of brain metastases, respectively. Only 3.0% of the brain metastases samples were negative for both HER3 and EGFR proteins, indicating that the majority of metastases express these ERBB proteins, which were therefore chosen for CTC enrichment using magnetic cell-separation. Enrichment based on either EGFR or HER3 detected CTCs in 37.8% of the patients, while the combination of EGFR/HER3 enrichment with the EpCAM-based CellSearch technique detected a significantly higher number of 66.7% CTC-positive patients (Cohen’s kappa = −0.280) which underlines the existence of different CTC subpopulations in NSCLC. The malignant origin of keratin-positive/CD45-negative CTC clusters and single CTCs detected after EGFR/HER3 based enrichment was documented by the detection of NSCLC-associated mutations. In conclusion, EGFR and HER3 expression in metastasized NSCLC patients have considerable value for CTC isolation plus multiple markers can provide a novel liquid biopsy approach.

Cell Search Techniques for Underwater Acoustic Cellular Systems

The concept of frequency reuse has been considered for underwater acoustic cellular (UAC) systems owing to the limited available bandwidth of acoustic channels. In a UAC system, the underwater equipment (UE) should detect the identity of the underwater base station (UBS) and synchronize to a serving UBS. In this paper, two different types of cell search techniques are proposed for the downlink of UAC systems based on orthogonal frequency-division multiplexing (OFDM): a Zadoff-Chu sequence-based cell search technique (ZCS-CST) and a linear frequency modulation-based cell search technique (LFM-CST), all at the physical layer. The performances (correlation property, detection probability, and false alarm probability) of the cell search techniques are analyzed and compared with the simulation results in the additive white Gaussian noise (AWGN) and multipath channels. The performances of the cell search techniques are evaluated using the Bellhop channel simulator and field experiment. It is shown that the ZCS-CST is applicable to UAC systems with a small Doppler shift, while the LFM-CST is suitable for UAC systems with a large Doppler shift.

MRNA expression profiles in circulating tumor cells of metastatic colorectal cancer patients

IntroductionThe molecular characterization of circulating tumor cells (CTCs) is a promising tool for the repeated and non-invasive evaluation of predictive and prognostic factors. Challenges associated with CTC characterization using the only FDA approved method for CTC enumeration, the CellSearch technique, include the presence of an excess of leukocytes in CTC-enriched blood fractions. Here we aimed to identify colorectal tumor-specific gene expression levels in the blood of patients with and without detectable CTCs according to CellSearch criteria. Materials and methodsBlood of 30 healthy donors (HDs) and 142 metastatic colorectal cancer (mCRC) patients was subjected to CellSearch CTC enumeration and isolation. In all samples, 95 mRNAs were measured by reverse transcriptase quantitative PCR (RT-qPCR). HD blood samples and patient samples with three or more CTCs were compared to identify CTC-specific mRNAs. Patient samples without detectable CTCs were separately analyzed. ResultsThirty-four CTC-specific mRNAs were higher expressed in patients with ≥3 CTCs compared with HDs (Mann–Whitney U-test P < 0.05). Among patients without detectable CTCs, a HD-unlike subgroup was identified which could be distinguished from HDs by the expression of epithelial genes such as KRT19, KRT20 and AGR2. Also, in an independent patient set, a similar HD-unlike group could be identified among the patients without detectable CTCs according to the CellSearch system. ConclusionExtensive molecular characterization of colorectal CTCs is feasible and a subgroup of patients without detectable CTCs according to CellSearch criteria bears circulating tumor load, which may have clinical consequences. This CTC-specific gene panel for mCRC patients may enable the exploration of CTC characterization as a novel means to further individualize cancer treatment.

333P - Circulating Tumor Cell Count at Baseline is an Independent Prognostic Factor from Pathological Complete Response Among Patients Treated for Primary Inflammatory Her2-Positive Breast Cancer: Survival Results of the Beverly-2 Study

ABSTRACT Aim: The BEVERLY-2 single-arm phase 2 trial assessed the efficacy and safety of combining neoadjuvant chemotherapy with bevacizumab and trastuzumab for the treatment of HER2-positive inflammatory breast cancer (Pierga JY et al, Lancet Oncol 2012). We report the results of the association between outcome, circulating biomarkers and pathological complete response (pCR) at three years of follow-up. Methods: Circulating tumor (CTC) and endothelial cell (CEC) counts were assessed at baseline, cycle 5, pre-operative, post-operative and at one year by the CellSearch technique. Results: 52 patients were included. 3-year DFS in patients with ≥1 CTC/7.5 mL at baseline was significantly lower (43% [95% CI: 20; 64]) than for patients with no CTC detected at baseline (81% [95% CI: 62; 91]; hazard ratio 3.69 [1.34; 10.21]; p = 0.012). Due to the low number of CTC-positive patients at all other time points, there was no significant association between CTC and DFS other than that observed at baseline. Detection of CTC at any point during the trial period was associated with a significant reduction in DFS; patients with at least one positive CTC sample had a DFS of 54% (95% CI: 32; 71), compared with a DFS of 83% (95% CI: 61; 93) in patients where no CTC were detected at any time (hazard ratio 3.62 [95% CI: 1.15; 11.39]; p = 0.018). In a multivariate analysis including factors that were associated with DFS in univariate analysis (pCR, SBR grade and baseline CTC status), only SBR grade and baseline CTC status were found to be independent prognostic factors. No impact of CEC count on DFS was observed at univariate analysis. Patients with baseline CTC Conclusions: Combination of pCR and CTC positivity defines specific patient subgroups at very low (no CTC detected at baseline and pCR) and high (CTC detected at baseline and no pCR) risk of early relapse. These parameters could be used to build specific clinical trial with new targeted treatments for patients with inflammatory breast cancer. Disclosure: J. Pierga: honoraria and research funds from Roche and Janssen diagnotics; M. Coudert: Roche employee; J. Hernandez: Roche employee; P. Viens: Roche research funding. All other authors have declared no conflicts of interest.

Circulating tumor cells in locally advanced pancreatic adenocarcinoma: The ancillary study Circe 07 of the LAP 07 trial.

4046 Background: Pancreatic carcinoma is one of the leading causes of cancer-related mortality. At time of diagnosis, 30% of patients present with a locally advanced unresectable but non metastatic carcinoma (LAPC). Theoretically, patients with micrometastatic dissemination at diagnosis should benefit from systemic treatments, whereas radiation therapy should be favored in the others. Based on the hypothesis that circulating tumor cells (CTC) count is a surrogate of the cancer metastatic abilities, CTC detection rates and prognostic value were studied in a prospective cohort of LAPC patients. Methods: LAP07 international multicenter randomized study assesses in patients whose LAPC is controlled after 4 months of gemcitabine-based chemotherapy whether to administrate a chemoradiotherapy could increase overall survival versus continuation of chemotherapy alone. A subgroup of patients included in LAP 07 trial were prospectively screened for CTC before the start of the chemotherapy and after two months of treatment, using the CellSearch technique. Clinico-pathological characteristics and survival of patients were obtained prospectively and were correlated with CTC detection. Results: Seventy-nine patients were included in this ancillary study. One or more CTC/7.5ml were detected in 5% of patients before treatment and in 9% of patients after two months of chemotherapy (overall detection rate: 11% of patients). CTC positivity was associated with poor tumor differentiation (p=0.04), and with shorter overall survival in multivariable analysis (RR=2.5, p=0.01), together with anemia (p=0.005). Conclusions: The evaluation of micrometastatic disease using CTC detection appears as a promising tool which could help to personalize treatment modalities in LAPC patients. Clinical trial information: NCT00634725.

Circulating tumor cells in locally advanced pancreatic adenocarcinoma: The ancillary study Circe 07 of the LAP 07 trial.

188 Background: Pancreatic carcinoma is one of the leading causes of cancer-related mortality. At time of diagnosis, 30% of patients present with a locally advanced unresectable but non metastatic carcinoma (LAPC). Theoretically, patients with micrometastatic dissemination at diagnosis should benefit from systemic treatments, whereas radiation therapy should be favored in the others. Based on the hypothesis that circulating tumor cells (CTC) count is a surrogate of the cancer metastatic abilities, CTC detection rates and prognostic value were studied in a prospective cohort of LAPC patients. Methods: LAP07 international multicenter randomized study assesses in patients whose LAPC is controlled after 4 months of gemcitabine-based chemotherapy whether to administrate a chemoradiotherapy could increase overall survival versus continuation of chemotherapy alone. A subgroup of patients included in LAP 07 trial were prospectively screened for CTC before the start of the chemotherapy and after two months of treatment, using the CellSearch technique. Clinico-pathological characteristics and survival of patients were obtained prospectively and were correlated with CTC detection. Results: Seventy-nine patients were included in this ancillary study. One or more CTC/7.5ml were detected in 5% of patients before treatment and in 9% of patients after two months of chemotherapy (overall detection rate: 11% of patients). CTC positivity was associated with poor tumor differentiation (p=0.04), and with shorter overall survival in multivariable analysis (RR=2.5, p=0.01), together with anemia (p=0.005). Conclusions: The evaluation of micrometastatic disease using CTC detection appears as a promising tool which could help to personalize treatment modalities in LAPC patients. Clinical trial information: CDR0000589283.

ApoStream, an antibody-independent platform, compared to CellSearch for enumeration of circulating tumor cells (CTCs) in patients with metastatic prostate cancer.

e21058 Background: Development of CTC detection platforms is a rapidly advancing field. The CellSearch technique relies on cell surface expression of EpCAM to select for rare tumor cells in whole blood specimens. Consequently, the use of EpCAM-based enrichment platforms to recover CTCs is limited to EpCAM expressing cells and is poorly suited for recovery of EpCAM negative cells like those having undergone epithelial mesenchymal transition (EMT). ApoStream is an antibody independent enrichment platform which utilizes the principle of continuous flow dielectrophoresis field-flow fractionation (DEP-FFF) to isolate and enrich for CTCs. A head to head comparison of CellSearch to ApoStream for recovery of CTCs in patients with metastatic prostate cancer was performed. Methods: Two 7.5 mL bloodsamples were collected at a single time point for each patient with stage IV prostate cancer. One sample was analyzed by CellSearch CTC enumeration kit, and one sample was analyzed by ApoStream. CTCs recovered by both devices were immunophenotyped using antibodies against cytokeratin (CK), CD45 and DAPI. CTCs were defined as CK+/CD45-/DAPI+ intact cells. CTCs recovered by ApoStream were further analyzed by quantitative laser scanning cytometry (LSC). A paired t-test was used to compare the cell counts in the two devices. Results: The ApoStream CTC enrichment platform isolated a greater number of CTCs from blood of eight patients compared to CellSearch (p= 0.0027). All cell counts obtained by the ApoStream technique were higher than CellSearch, and all patients had detectable CTCs by ApoStream, while only 75% of patients had detectable CTCs with CellSearch. Conclusions: The ApoStream platform is differentiated from EpCAM dependent platforms and is well suited for detection and recovery of CTCs in advanced stage disease where tumor cell heterogeneity is common and expression of EpCAM may be low or lost. [Table: see text]

P4-07-04: Nomogram Including Circulating Tumor Cells (CTC) Count before and during Chemotherapy for Individual Survival Prediction of Metastatic Breast Cancer Patients.

Abstract Background CTC count before a new line of treatment and CTC count early changes under chemotherapy have been reported as an independent prognostic marker in metastatic breast cancer in a recent pooled analysis in 841 pts (Liu M. ASCO 2011). The aim of this study was to build a prognostic tool including CTC and other parameters to assesse its predictive value for progression-free survival (PFS) and overall survival (OS). Methods: Data from the IC 2006–04 study were used. This prospective multicentre study included 267 metastatic breast cancer patients treated by first line chemotherapy with or without targeted therapy, in whom appropriate pre-treatment prognostic variables (age, performance status, number of metastatic sites, disease-free interval, ER, PR and HER2 status, tumor grade, LDH, serum markers, CTC count by CellSearch technique before treatment and before cycle 2) were available for statistical analysis. We constructed a multivariate Cox regression model for PFS and OS prediction. A stepwise selection process was applied to achieve the most informative and parsimonious models. Performance was measured with the C-index statistic. Internal validation was performed using leave-two-out technique. Results: Four nomograms have been obtained, in two clinical settings: at inclusion (before the start of any treatment) taking into account the initial CTC count, and during treatment (before cycle 2) taking into account CTC changes under treatment. Their accuracy was good for PFS and OS prediction, with C-index ranging from 0.72 to 0.88. Internal validations allow considering a good accuracy of the models in an external population. Conclusion: These clinically relevant nomograms are a simple tool for a personalized prognostic assessment including CTC assessment. Validation on independent series of patients are ongoing. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-04.

Synchronization and Cell-Search Technique Using Preamble for OFDM Cellular Systems

In this paper, a novel preamble structure, including a synchronization field (S-field) and a cell-search field (C-field), is proposed for orthogonal-frequency-division-multiplexing-based cellular systems. An efficient algorithm for downlink synchronization and cell search using the preamble is also proposed. The synchronization and cell-search process includes the initial symbol-timing estimation using continuously or, at least, periodically transmitted downlink signal, the frame detection, the fine symbol-timing estimation, the frequency-offset estimation using the preamble S-field, and the cell identification using the preamble C-field. Performance of each synchronization and cell-search step is analyzed and verified by computer simulation. The overall performance of the synchronization and cell search is then analyzed in terms of the mean acquisition time. It is shown that the proposed preamble with the corresponding synchronization and cell-search algorithm can provide a very robust synchronization and cell-search capability, even in bad cellular environments.

Known Postfix Based Cell Search Technique for OFDM Cellular Systems

A cell search technique utilizing a known postfix for OFDM (orthogonal frequency division multiplexing) cellular systems is described. The known postfix is generated in the time domain by inserting pilots in the frequency domain and plays the role of the cyclic prefix in general OFDM systems. Since it demonstrates good correlation properties, it can be facilitated to synchronize each symbol with an identified postfix. In this paper, two different known postfixes are allocated to each cell. One is used for cell identification and symbol synchronization, which is designed to be different among neighboring cells. The other is used for frame synchronization and is the same for all cells. In the simulation, the cell search is accomplished with a probability greater than 10 -3 at -27 dB in a vehicular channel. Even at -30 dB, the cell search probability is greater than 10 -2 in a pedestrian channel as well as 10 -3 in the AWGN (additive white gaussian noise) channel.