Immunomagnetic Enrichment Research Articles

Analysis of monosomy‐3 in immunomagnetically isolated circulating melanoma cells in uveal melanoma patients

Monosomy-3 in primary uveal melanoma (UM) is associated with a high risk of metastasis and mortality. Although circulating melanoma cells (CMC) can be found in most UM patients, only approximately 50% of the patients develop metastases. We utilized a novel immuno-FISH assay to detect chromosome-3 in intact CMC isolated by dual immunomagnetic enrichment. Circulating melanoma cells were detected in 91% of the patients (n=44) with primary non-metastatic UM, of which 58% were positive for monosomy-3. The monosomy-3 status of CMC corresponded to the monosomy-3 status of the primary tumor in 10 of the 11 patients where this could be tested. Monosomy-3 in the CMC was associated with an advanced tumor stage (P=0.046) and was detected in all four patients who developed metastasis within the follow-up period of 4yr. This non-invasive technique may enable the identification of UM patients at risk for metastasis particularly when a primary tumor specimen is unavailable.

Spot Immunomagnetic Enrichment Device for Rapid Detection of Pathogens in Peripheral Blood

Microbial infection is a leading cause of mortality and morbidity worldwide. Due to lack of rapid and affordable diagnostics test, patients consume broad spectrum antibiotics without diagnostic confirmation of the pathogen, leading to development of antibiotics resistance among microbes. Detection of pathogen is challenging particularly when only few cells per mL are present among billion other blood cells. A portable and cost‐effective spot immunomagnetic enrichment device has been developed that allows concentration of cells from 1 to 10 mL sample volume up to 10 000× per mL into a small spot inside a disposable millifluidic capture chip. Salmonella typhi, causative agent for typhoid infection has been used to demonstrate the results. Microbes are specifically labeled with antibody‐conjugated magnetic nanoparticles and isolated from bulk sample at 200 μL min−1 flow rate with >90% capture efficiency. Isolated cells are swept to a single spot by decreasing the area under influence of magnetic field and flowing a wash buffer. Using this spot enrichment method, 4 CFU of S. typhi cells enriched from 5 mL sample is detected with turnaround time of less than 1 h.

Abstract 447: The presence and persistence of ERCC1 positive circulating tumor cells predicts worse prognosis in primary ovarian cancer patients

Abstract Background: Resistance to platinum based chemotherapy in ovarian cancer can only be assessed retrospectively during post-chemotherapy follow-up. We recently showed that excision-repair cross-complementing rodent repair deficiency complementation group 1 nuclease (ERCC1)-positive (+) circulating tumor cells (CTCs) were an independent predictor for progression free survival (PFS), overall survival (OS) and for platinum-resistance in our ovarian cancer patients (pts). We now analyzed whether the negative prognostic impact of CTCs was related to the presence and persistence of ERCC1+ CTCs after platinum based chemotherapy. Methods: 10 ml blood of 65 primary ovarian cancer pts before and after chemotherapy were studied for CTCs by immunomagnetic enrichment followed by multiplex RT-PCR to detect the transcripts EpCAM, MUC-1 and Ca 125 (AdnaTest OvarianCancer; QIAGEN Hannover GmbH, Germany). A patient was considered CTC+, if at least one of the three markers was detectable. ERCC1 was analyzed in a separate approach by single plex PCR, ß-actin served as an internal control and PCR-products were quantified on the Agilent Bioanalyzer 2100. Results: CTCs were detected in 18/65 pts (28%) before and in 13/65 pts (20%) after chemotherapy. A persistence of CTCs was found in four pts, 14 pts were only positive before and nine pts only after therapy whereas in 38 pts, no CTCs were detected at any time. Pts with persisting CTCs had a significantly shorter PFS (p = 3.32E-05) and OS (0.00018) while pts initially CTC-negative before but CTC+ after therapy showed a reduced OS (p = 0.0039). ERCC1+CTCs were observed in 12/65 (18%) pts before and in 8/65 pts (12%) after therapy. A persistence of ERCC1+CTCs was found in three pts, nine pts were only positive before, five pts only after therapy whereas in 48 pts, no ERCC1+CTCs were detected at any time. Pts with persisting ERCC1+CTCs had a significantly shorter PFS (p = 0.032) and OS (0.0058) while pts initially ERCC1-negative before but ERCC1+ after therapy showed a reduced OS (p = 0.039). Up to now, no association between clinically defined platinum resistance and ERCC1-positivity was observed. Conclusion: The negative prognostic impact of CTCs was related to the presence and persistence of ERCC1+CTCs after therapy supporting the idea to implement ERCC1 expression in CTC analysis of ovarian cancer pts as a biomarker for monitoring the disease to possibly change treatment in case of ERCC1 persistent CTCs. Citation Format: Issam Chebouti, Paul Buderath, Jan Dominik Kuhlmann, Yvonne Bokeloh, Siggi Hauch, Rainer Kimmig, Sabine Kasimir-Bauer. The presence and persistence of ERCC1 positive circulating tumor cells predicts worse prognosis in primary ovarian cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 447.

Abstract 502: Gene expression signatures in circulating tumor cells are prognostic for metastatic lesions in breast cancer patients and correlate with response to therapy

Abstract Background: Circulating tumor cells (CTC) are discussed to be an ideal surrogate marker to monitor disease progression in metastatic breast cancer (MBC). Besides CTC characterization for targeted therapies, it would also be desirable to know where these cells derive from or to which organ site they are going to. Here we investigated whether it is possible to predict the origin of metastatic lesion based on the expression of 46 genes in CTC of MBC patients (pts). Materials and Methods: 2×5 ml blood of 45 MBC pts and 20 healthy controls was collected at the time of disease progression (T0) and at two consecutive clinical staging (T1 and T2) for the detection of CTC applying immunomagnetic enrichment using the AdnaTest EMT-2/Stem Cell Select (QIAGEN Hannover GmbH, Germany). Pts were grouped a) into overall responders (OR) and overall non-responders (ONR), thus responding or not responding at T1 and T2 and b) according to sites of metastasis. PCR assays, targeting 46 transcripts and reference markers were used for a workflow based on pre-amplification and high throughput profiling (each samples in duplicates) with the full set of markers including also ValidPrime to correct for genomic background and InterPlate Calibrator to even out variations between runs. Data were analyzed using GenEx (MultiD, Sweden) and SAS. qPCR as well as technical reads were normalized using several normalization strategies. Results: The multidrug resistant protein gene MRP1 was significantly differently expressed if OR and ONR groups were compared. In the following order of significance, VEGFR1, Keratin (KRT) 19, EGFR, MET1, ALDH, progesterone receptor (PR), UPA, Cathepsin D, KIT1 and Ki67 were differentially expressed in CTC of pts who had already developed liver metastasis as compared to pts without liver metastasis. Interestingly, a small group of pts, developing liver metastases in the course of disease, showed the estrogen receptor (ER), PR, HER2, mammaglobin, KRT19 on a significantly lower level as compared to the other pts. Similarly, once CTC were ER and PR positive, the probability of bone metastasis development decreased. Conclusion: Our preliminary results indicate that the development of metastatic lesions is associated with site-specific CTC. Thus, besides using CTC as a monitoring tool to guide therapy, they might also indicate the site of metastasis which will allow a more precise decision concerning treatment strategy. Citation Format: Maren Bredemeier, Philippos Edimiris, Pawel Mach, Mikael Kubista, Robert Sjoback, Marie Jindrichova, Eva Rohlova, Vendula Novosadova, Katarina Kolostova, Siegfried Hauch, Bahriye Aktas, Mitra Tewes, Rainer Kimmig, Sabine Kasimir-Bauer. Gene expression signatures in circulating tumor cells are prognostic for metastatic lesions in breast cancer patients and correlate with response to therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 502.

Abstract 501: PD-L1 expressing circulating tumor cells (CTCs) in patients with breast cancer

Abstract Background: Programmed cell Death receptor Ligand 1 (PD-L1) is a very promising biomarker for the selection of patients for cancer immunotherapy. We recently developed a highly sensitive, specific and robust RT-qPCR assay for PD-L1 mRNA. The aim of the present study was to study the expression of PD-L1 in CTCs from breast cancer patients with verified metastasis. Methods: We quantified the expression of PD-L1 mRNA transcripts in EpCAM-positive CTCs, by using our recently developed RT-qPCR assay, based on the following procedure: i) immunomagnetic enrichment of EpCAM-positive CTCs from 20mL of peripheral blood, ii) total RNA isolation iii) cDNA synthesis and iv) RT-qPCR for PD-L1. PD-L1 expression in respect to the expression of B2M as a reference gene, was normalized using the 2-ΔΔCt approach. Peripheral blood samples (20mL) were obtained from 22 breast cancer patients with verified metastasis and 14 healthy donors. Results: According to our results, 11/22 (50%) of metastasis-verified breast cancer patients were found to be positive for PD-L1 overexpression in CTCs. These are preliminary results and these percentages may change, since the number of samples that we are analyzing is continuously increasing. Our results are in concordance with a recent study by Mazel et al (Mol Oncol 2015), that by using the CellSearch(®) system found PD-L1((+)) CTCs in 11/16(68.8%) patients with metastatic breast cancer. Conclusion: This is the first time that a quantitative RT-qPCR molecular assay is used for the evaluation of PD-L1expression levels in EpCAM-positive CTCs in metastatic breast cancer patients. The assay is closed-tube, quantitative, highly specific and sensitive, and high-throughput. We are currently evaluating the assay in a large number of clinical samples. Citation Format: Martha Zavridou, Areti Strati, Nikos Malamos, Vasilis Georgoulias, Evi S. Lianidou. PD-L1 expressing circulating tumor cells (CTCs) in patients with breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 501.

Abstract 511: Unbiased enrichment of circulating tumor cells directly from whole blood

Abstract Enrichment of rare circulating tumor cells (CTC) in peripheral blood is required prior to most analytic procedures. The ideal enrichment method would be rapid (<30 minutes (min)), permit a high recovery of viable CTC, and would be independent of surface marker expression, since CTC in the peripheral blood may be undergoing epithelial-mesenchymal transition and may not express epithelial markers. We have developed a new immunomagnetic method (EasySep™ Direct) that fulfills these criteria and can be used directly with whole blood without any preparatory steps. The method was tested using whole blood samples seeded with the adenocarcinoma cell line CAMA to a final concentration of approximately 1%. The CAMA cells were then enriched using either EasySep™ Direct CTC Enrichment (immunomagnetic separation) or RosetteSep™ (immunodensity based control). For immunomagnetic enrichment, the samples were incubated with EasySep™ Direct Enrichment Cocktail for 5 min, then EasySep™ Direct RapidSpheres™ were added and the sample placed in a magnet for 10 min. Magnetically labeled cells were retained in the magnet and the unlabeled cells poured or pipetted off. Rapidspheres™ were added again and the magnetic incubation and pour-off steps repeated. To perform RosetteSep™ enrichment, the samples were incubated with either the CD45+ cell depletion cocktail or a more extensive hematopoietic cell depletion cocktail for 10 min. Samples were then pipetted onto Lymphoprep in SepMate™ tubes, centrifuged for 10 min at 1200 x g with the brake on, and poured off. The number of cells recovered with each procedure was counted and the depletion of hematopoietic cells (CD45+/EPCAM-), enrichment of CAMA cells (CD45-/EpCAM+), and depletion of red blood cells (RBCs, Glycophorin A+/CD45-) in each sample were evaluated by flow cytometry. EasySep™ Direct was evaluated on five different samples. Each sample was separated on five different EasySep™ magnet formats (5, 14, and 50 mL single tube magnets and 5 and 14 mL multi-tube magnets) with each condition in duplicate; the two RosetteSep™ cocktails were tested on three of the samples, each in duplicate. There was no significant difference in CD45 depletion, CAMA cell recovery, or residual RBC contamination between samples enriched using the different magnets (Least Squares Fit; Prob F>0.05) or pooled and compared to the pooled RosetteSep™ controls (Least Squares Fit; Prob F>0.05). Over all magnet separation conditions and samples, the mean log depletion of CD45+ cells was 2.9±0.4, the mean recovery of CAMA cells was 42±23%, and the residual RBC contamination was ∼9000 RBC/mL of start sample. Rare cells were enriched in 25 minutes using EasySep™ Direct and in less than 40 minutes using RosetteSep™ with SepMate™. CTC can be enriched directly from whole blood with either EasySep™ Direct or RosetteSep™. Since enriched cells are unlabeled there is nothing to interfere with subsequent further enrichment, culture, or evaluation. Citation Format: Carrie E. Peters, Danay Maestre-Battle, Steven M. Woodside, Terry E. Thomas, Allen C. Eaves. Unbiased enrichment of circulating tumor cells directly from whole blood. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 511.

Investigating CTCs in NSCLC-a reaction to the study of Jia-Wei Wan: a preliminary study on the relationship between circulating tumor cells count and clinical features in patients with non-small cell lung cancer.

Liquid biopsies are emerging as a patient-friendly approach for estimating biomarkers to predict treatment outcome and overall survival. Detection and characterization of circulating tumor cells (CTCs) is a promising biomarker and its application for non-small cell lung cancer (NSCLC) is currently under investigation. The FDA approved CellSearch system is used as the standard test. The most challenging hurdle in this and other techniques is to discern the CTCs from the background noise of normal blood cells. In the CellSearch system, lysis of erythrocytes, separating leukocytes with anti-CD antibodies, and immunomagnetic enrichment of cancer cells from blood samples expressing membranous epithelial cell adhesion molecule (EpCAM) protein is used. The relevant detection rate using CellSearch for prognosis is set by the developer on >2 (for metastatic colorectal cancer) or >4 CTCs (for metastatic breast and prostate cancer) per 7.5 mL blood sample. Clinical use of the CellSearch system is currently limited in NSCLC because it fails to detect CTCs at an acceptable rate and at a sufficient high yield. Extrapolation of CTC frequency distribution in 7.5 mL of blood from patients with metastatic breast, colon and prostate cancer showed that probably all these patients had CTCs in circulation, but the sample volume was not sufficient to detect them in all patients. Therefore, novel methods for the detection of CTCs are being studied.

Immunomagnetic enrichment of regulatory T cells directly from whole blood without the need for density gradient centrifugation.

Abstract Regulatory T cells (Tregs) play a pivotal role in maintaining peripheral tolerance of unspecific or excessive immune responses. Therefore, the enrichment of these cells and their cultivation is of great interest for basic and translational research. Accordingly, there is a considerable need for continually improved and reliable cell isolation protocols. We introduce here a rapid, easy, and convenient strategy for the isolation of regulatory T cells directly from anticoagulated whole blood without the need for density gradient centrifugation. The new immunomagnetic separation protocol provides an easy and fast solution for the highly specific isolation of CD4+ CD25highCD127− regulatory T cells directly from whole blood within 30 minutes ready for downstream application such as flow cytometric phenotyping, gene expression studies, cytokine analysis, suppression assays, or cell expansion. The procedure, leads to populations of CD4+ CD25highCD127− cells with high yields (2×104/mL whole blood), purities (>90%) and viability (>98%) as proven by flow cytometric analysis. In addition, enriched Tregs were capable for in vitro expansion for several weeks while remaining stable FoxP3 expression.

Immunomagnetic enrichment of human ILC2 cells from peripheral blood leukopak

Abstract Group 2 innate lymphoid cells (ILC2s) are essential for airway tissue repair, protection against helminth infection, metabolic homeostasis, and the development of allergic airway diseases. They are widely distributed throughout the human body in lung, intestine, palatine tonsils, skin and peripheral blood, but are extremely rare within a given tissue. In healthy individuals, ILC2s comprise 0.01–0.04% of leukocytes in the peripheral blood. Currently, flow cytometric cell (FACS) sorting is the only method to purify ILC2s, which is time-consuming due to their low frequency. We have adapted EasySep™ to pre-enrich ILC2s from peripheral blood leukopaks, and thereby shorten subsequent FACS analysis and sorting times. Non-ILC2 cells in an unprocessed leukopak were immunomagnetically labeled using a cocktail of antibody complexes and magnetic particles. The sample was then placed in a magnet for 10 minutes, the labeled cells were retained, and the unlabeled cells, containing the ILC2 population, were recovered. ILC2s were enriched from 5 x108unprocessed cells in 30 minutes. Enriched cells were counted and evaluated by FACS. ILC2s, defined as lineage negative (CD1a− CD3− CD11c− CD14− CD19− CD34− CD123− TCRαβ− TCRγδ− BDCA2− FcɛR1−), CRTH2+ CD161+CD127+, were enriched 268±167 fold, with 74± 34% recovery (means ± SD, n=7). EasySep™ pre-enrichment of ILC2s reduced subsequent FACS analysis and sorting time compared to non-enriched samples. Importantly, ILC2s enriched with EasySep™ were functional, as shown by production of the type 2 cytokine IL-13 upon stimulation with IL-33 and IL-2. Overall, EasySep™ is a simple and rapid method to pre-enrich ILC2s from leukopak for subsequent FACS analysis or sorting and functional analyses.

Integrating immunomagnetic bead enrichment with immunochromatography for the detection of influenza A virus

Objective To establish a novel method by integrating immunomagnetic bead enrichment with immunochromatography for the detection of influenza A virus. Methods The immunomagnetic beads were prepared by using EDC/NHS method and then coupled with monoclonal antibodies against influenza A virus. A direct immunomagnetic beads-based immunochromatography for the detection of influenza A virus was developed by using double-antibody sandwich method and immunochromatography, which was further combined with immunomagnetic separation to establish the novel integrated method of immunomagnectic bead enrichment and immunochromatography. Clinical throat swab samples collected from patients with influenza A virus infection and healthy subjects were analyzed by the novel method and the results were compared with those by using the conventional colloidal gold immunochromatography to evaluate the specificity, sensitivity and positive coincidence rate of this established method. Results The direct immunomagnetic beads-based immunochromatography and the colloidal gold immunochromatography showed no significant differences in specificity and sensitivity and could be used to identify influenza A virus-positive samples with cycle threshold (Ct) values less than or equal to 22 obtained by real-time PCR assay. The integrated method could identify positive samples with Ct values less than or equal to 28, indicating that the novel method was more sensitive. Conclusion The novel method by integrating immunomagnetic bead enrichment with immunochromatography was successfully established and suitable for the rapid and on-site detection of influenza A virus. Key words: Influenza A virus; Immunomagnetic beads; Immunochromatography; Rapid detection

Gene expression profiling of single circulating tumor cells in ovarian cancer – Establishment of a multi-marker gene panel

The presence of circulating tumor cells (CTCs) in the blood of ovarian cancer patients was shown to correlate with decreased overall survival, whereby CTCs with epithelial–mesenchymal-transition (EMT) or stem-like traits are supposed to be involved in metastatic progression and recurrence. Thus, investigating the transcriptional profiles of CTCs might help to identify therapy resistant tumor cells and to overcome treatment failure. For this purpose, we established a multi-marker panel for the molecular characterization of single CTCs, detecting epithelial (EpCAM, Muc-1, CK5/7), EMT (N-cadherin, Vimentin, Snai1/2, CD117, CD146, CD49f) and stem cell (CD44, ALDH1A1, Nanog, SOX2, Notch1/4, Oct4, Lin28) associated transcripts.First primer specificity and PCR-performance of the multiplex-RT-PCRs were successfully validated on genomic DNA and cDNA isolated from OvCar3 cells. The assay sensitivity of the epithelial panel was evaluated by adding defined numbers of tumor cells into the blood of healthy donors and performing a subsequent immunomagnetic tumor cell enrichment (AdnaTest OvarianCancerSelect), resulting in a 100% concordance for the epithelial markers EpCAM and Muc-1 to the AdnaTest OvarianCancerDetect. Additionally, by processing blood from ovarian cancer patients, high assay sensitivity could be verified. In blood of healthy donors no signals for epithelial markers were detected, for EMT and stem cell markers, however, signals were obtained mainly originating from leukocytes which calls for single cell analysis.To that aim by using the ovarian cancer cell line OvCar3, we successfully established a workflow enabling the characterization of single CTCs. It consists of a density gradient-dependent enrichment for nucleated cells, a depletion of CD45-positive cells of hematopoietic origin followed by immunofluorescent labeling of CTCs by EpCAM and Muc-1. Single CTCs are then isolated by micromanipulation and processed for panel gene expression profiling. Finally, fifteen single CTCs from three ovarian cancer patients were analyzed and found to be positive for stem cell (CD44, ALDH1A1, Nanog, Oct4) and EMT markers (N-cadherin, Vimentin, Snai2, CD117, CD146). Albeit, inter-cellular and intra/inter-patient heterogeneity and co-expression of epithelial, mesenchymal and stem cell transcripts on the same CTC was observed.We have established a robust workflow to perform sensitive single cell panel gene expression analysis without the need of pre-amplification steps. Our data point towards a heterogeneous expression of stem cell and EMT associated transcripts in ovarian cancer CTCs.

Reproducibility of circulating endothelial cell enumeration and activation in children and adolescents.

We examined the reproducibility of circulating endothelial cells (CEC) enumeration and activation among youth. CECs from 151 youth were measured at baseline and 1 week follow-up. Enumeration of CEC in fresh whole blood was determined by direct assessment of buffy coat smears (CD146+ nucleated cells) and activated CEC (%VCAM-1 expression) was determined after immunomagnetic enrichment and co-staining of nuclei, plus positivity for P1H12 and VCAM-1. No statistically significant difference in CEC enumeration (1.2 ± 2.5 vs 1.3 ± 2.2 CEC/milliliter of whole blood, p = 0.745) or activated CEC (57.1 ± 24.4 vs 58.0 ± 21.3 %VCAM-1, p = 0.592) between baseline and 1 week follow-up. On a cohort basis, CEC enumeration and activation are reproducible in youth. Relatively high individual biological variability may limit its clinical utility.

A portable immunomagnetic cell capture system to accelerate culture diagnosis of bacterial infections.

Bacterial infections continue to be a major cause of deaths globally, particularly in resource-poor settings. In the absence of rapid and affordable diagnostic solutions, patients are mostly administered broad spectrum antibiotics leading to antibiotics resistance and poor recovery. Culture diagnosis continues to be a gold standard for diagnosis of bacterial infection, despite its long turnaround time of 24 to 48 h. We have developed a portable immunomagnetic cell capture (iMC(2)) system that allows rapid culture diagnosis of bacterial pathogens. Our approach involves the culture growth of the blood samples in broth media for 6 to 8 h, followed by immunomagnetic enrichment of the target cells using the iMC(2) device. The device comprises a disposable capture chip that has two chambers of 5 ml and 50 μl volume connected through a channel with a manual valve. Bacterial cells bound to antibody coated magnetic nanoparticles are swept from the 5 ml sample chamber into the 50 μl recovery chamber by moving an external magnetic field with respect to the capture chip using a linear positioner. This enables specific isolation and up to 100× enrichment of the target cells. The presence of bacteria in the recovered sample is confirmed visually using a lateral flow immunoassay. The system is demonstrated in buffer and blood samples spiked with S. typhi. The method has high sensitivity (10 CFU ml(-1)), specificity and a rapid turnaround time of less than 7 h, a significant improvement over conventional methods.

Lack of Impact of Robotic Assisted Laparoscopic Radical Prostatectomy on Intraoperative Levels of Prostate Cancer Circulating Tumor Cells

While the significance of circulating tumor cells in clinically localized cancer remains controversial, it has been reported that surgical tumor manipulation can increase circulating tumor cells, including during open prostatectomy. To ourknowledge it is unknown whether this cell shedding also occurs during minimally invasive prostatectomy, which minimizes tumor palpation and uses earlier vascular control. We tested the impact of robotic assisted laparoscopic radical prostatectomy on intraoperative circulating tumor cell levels. Circulating tumor cell counts were compared in peripheral blood specimens from 25 patients treated with robotic assisted laparoscopic radical prostatectomy preoperatively vs intraoperatively after prostate excision, in addition to 11 healthy blood donors. Circulating tumor cell detection was performed using EpCAM immunomagnetic enrichment and multiparametric flow cytometry quantification of viable EpCAM positive/prostate specific membrane antigen positive/CD45 negative cells. Intraoperative cell counts and increases were tested in univariable analyses for associations with perioperative variables, histopathology and postoperative progression. Circulating tumor cells were detected in 0% of healthy controls compared to 48% and 52% of prostatectomy cases preoperatively and intraoperatively, respectively (range 1 to 8 cells). There was no difference in the incidence or mean number of circulating tumor cells preoperatively vs intraoperatively. Of the patients 60% had no intraoperative change from preoperative levels. Intraoperative cell increases vs decreases were equally infrequent (each 20%) with no intraoperative increase greater than 1 circulating tumor cell. Intraoperative circulating tumor cell detection was not significantly associated with prostatectomy operative characteristics, histopathology or early postoperative progression at a median 21-month followup. Robotic assisted laparoscopic radical prostatectomy does not causesignificant intraoperative increases in circulating tumor cells in contrast tohistorical reports of open prostatectomy. These findings may aid urologists in counseling candidates for robotic assisted laparoscopic radical prostatectomy regarding the possibility of intraoperative tumor cell shedding.

Detection of cerebrospinal fluid tumor cells and its clinical relevance in leptomeningeal metastasis of breast cancer.

Circulating tumor cells are commonly observed in the peripheral blood of advanced breast cancer patients. We tested the feasibility of tumor cell detection in the cerebrospinal fluid (CSF) and studied its clinical relevance in leptomeningeal metastasis (LM) of breast cancer. CSF samples were collected from 38 metastatic breast cancer patients known or suspected to have LM. Control CSF samples were collected from 14 individuals without solid tumor malignancy. We used a modified CellSearch™ assay and an alternative EPCAM-based method involving immunomagnetic enrichment followed by flow cytometry (IE/FC) to enumerate CSF tumor cells (CSFTCs). CSFTCs were assayed at time of LM diagnosis and over the course of LM-directed therapy. We analyzed a total of 102 CSF samples with modified CellSearch™. The CSFTC counts were strongly correlated with the corresponding IE/FC results (Pearson's r=0.94). Twenty-eight out of 30 samples in which malignant cells were identified by CSF cytology were CSFTC-positive by modified CellSearch™. Baseline CSFTC levels from 21 patients eventually diagnosed with LM were significantly higher than the controls (p=0.0202), whereas 13 patients deemed not to have LM showed CSFTC results indistinguishable from the controls. In patients with serial samples, it was possible to monitor CSFTC levels as a potential biomarker of treatment response. CSFTC detection using a modified CellSearch™ assay demonstrated high sensitivity in detecting malignant cells in CSF and may be a promising method for diagnosing LM and monitoring LM during treatment.

EpCAM based capture detects and recovers circulating tumor cells from all subtypes of breast cancer except claudin-low

The potential utility of circulating tumor cells (CTCs) as liquid biopsies is of great interest. We hypothesized that CTC capture using EpCAM based gating is feasible for most breast cancer subtypes. Cancer cells could be recovered from all intrinsic subtypes of breast cancer with IE/FACS, however, claudin-low cell lines showed very low capture rates compared to the four other groups (p = 0.03). IE/FACS detection of CTC mimic cells was time sensitive, emphasizing controlling for pre-analytic variables in CTC studies. Median fluorescent intensity for flow cytometry and RNA flow cell type characterization were highly correlated, predicting for CTC isolation across molecular subtypes. RNA-Seq of IE/FACS sorted single cell equivalents showed high correlation compared to bulk cell lines, and distinct gene expression signatures compared to PB. Ten cell lines representing all major subtypes of breast cancer were spiked (as CTC mimics) into and recovered from peripheral blood (PB) using immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE/FACS). Flow cytometry and RNA flow were used to quantify the expression of multiple breast cancer related markers of interest. Two different RNA-Seq technologies were used to analyze global gene expression of recovered sorted cells compared to bulk cell lines and PB. EpCAM based IE/FACS detected and captured a portion of spiked cells from each of the 10 cell lines representing all breast cancer subtypes, including basal-like but not claudin-low cancers. The assay allows for the isolation of high quality RNA suitable for accurate RNA-Seq of heterogeneous rare cell populations.

Isolation and Culture Expansion of Tumor-specific Endothelial Cells.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5(cre):ZsGreen(l/s/l) reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

Cytokeratin-20 and Survivin-Expressing Circulating Tumor Cells Predict Survival in Metastatic Colorectal Cancer Patients by a Combined Immunomagnetic qRT-PCR Approach.

Circulating tumor cells (CTC) express epithelial and stem cell-like genes, though current approved detection methods mainly use epithelial markers. We optimized a CTC isolation method that could capture their molecular heterogeneity and predict overall survival (OS) in metastatic colorectal cancer (mCRC) patients receiving various chemotherapy regimens. We combined immunomagnetic enrichment of CD45-negative, EpCAM-positive circulating cancer cells with qRT-PCR amplification of CK20 and survivin expression in 88 mCRC patients and 20 healthy controls. We then evaluated the prognostic value of baseline CTC CK20 and survivin expression in mCRC patients. The presence of elevated CTC CK20 or survivin expression distinguished mCRC patients from controls with sufficient sensitivity (79.6%) and specificity (85%). In univariate analysis, patients with high CTC-CK20 expression (9 vs. 33.2+ months, log-rank P < 0.001) or high CTC-survivin expression (10 vs. 33.2+ months, log-rank P = 0.032) had a significantly worse median OS than those with low expression of either marker. In multivariable analysis, the high CTC-CK20 group had significantly shortened OS (HR, 3.11; adjusted P = 0.01), and there was a trend toward inferior OS in the high CTC-survivin group (HR, 1.76; adjusted P = 0.099). Patients with either high CTC CK20 or survivin expression had inferior OS compared with those with low expression of both markers (HR, 4.39; 95% confidence interval, 1.56-12.35; adjusted P = 0.005). Colorectal cancer CTCs can be reliably isolated using epithelial and stem cell markers. CTC CK20 and survivin expression may effectively predict OS in mCRC patients receiving chemotherapy.

Prognostic Role of a Multimarker Analysis of Circulating Tumor Cells in Advanced Gastric and Gastroesophageal Adenocarcinomas

Objective: We aimed to assess the prognostic value of circulating tumor cells (CTC) in patients with advanced gastric and gastroesophageal adenocarcinomas. Methods: The presence of CTC was evaluated in 62 patients with advanced gastric and gastroesophageal adenocarcinomas before systemic therapy and at follow-up through immunomagnetic enrichment for mucin 1- and epithelial cell adhesion molecule (EpCAM)-positive cells, followed by real-time RT-PCR of the tumor-associated genes KRT19, MUC1, EPCAM, CEACAM5 and BIRC5. Results: The patients were stratified into groups according to CTC detection (CTC negative: with all marker genes negative; CTC positive: with at least 1 of the marker genes positive). Patients who were CTC positive at baseline had a significantly shorter median progression-free survival (PFS; 3.5 months, 95% CI: 2.9-4.2) and overall survival (OS; 5.8 months, 95% CI: 4.5-7.0) than patients lacking CTC (PFS 10.7 months, 95% CI: 6.9-14.4, p < 0.001; OS 13.3 months, 95% CI: 8.0-18.6, p = 0.003). Alterations in the marker profile during the course of chemotherapy were not predictive of clinical outcome or response to therapy. Yet, a favorable clinical response depended significantly on CTC negativity (p = 0.03). Conclusion: Our data suggest that the presence of CTC is a major predictor of outcome in patients with gastric and gastroesophageal malignancies.

Abstract 389: The identity of all nucleated cells enriched by CellSearch

Abstract Purpose: The FDA cleared CellSearch assay for enumeration of Circulating Tumor Cells (CTC) detects CTC by fluorescence microscopy after immunomagnetic enrichment targeting Epithelial Cell Adhesion Molecule (EpCAM). The system generated thumbnail images of DNA+, Cytokeratin+ (CK+) events and presents these to a reviewer for scoring events as CTC. An event is a CTC when it is negative for CD45, positive for DNA and CK and is larger than a 4 × 4 μm2 with a cell-like morphology. The presence of CTC defined in such manner is strongly associated with poor outcome. The question arises whether and how many other tumor cells are present in the blood that do not have the phenotype defined by the CellSearch system. In this study we investigated the number of cells that are obtained after EpCAM enrichment and immunofluorescent labeling by CellSearch that could not be assigned to a cell lineage and explored the addition of other markers to increase the number of cells that could be assigned to a cell lineage. Methods: Automated image analysis was used to reanalyze archived CellSearch images of 12572 patient samples with benign breast and colorectal disease, non-metastatic breast and colorectal cancer, metastatic colorectal and prostate cancer. For the evaluation of additional markers blood from healthy volunteers aged 20-55 were used. For the evaluation of CD16-PerCP as an additional leukocyte marker, blood from 30 patients with metastatic lung cancer was used. Results: The median number of DNA+CK− cells found was 450 of which only 155 (34%) were identified as leukocytes (CD45-APC+). The number of DNA+CK− was higher in patients with metastatic disease as compared to those with non-metastatic or benign disease (median 2028 vs 293). The proportion of identified leukocytes (DNA+CK−CD45+) was however only slightly lower (median 35% vs 43%). The number of DNA+CK−CD45− cells was larger in patients with more CTC (0 CTC: 495, 1-10 CTC: 2024, 11-100 CTC: 8140, &amp;gt;100 CTC: 8983). The number of DNA+CK− cells also increased with aging of the blood samples, but did not account for the observed differences. The use of both CD16 and CD45 decreased the number of DNA+CK− cells and increased the fraction of cells identified as leukocytes to 90% in healthy donors and 78% in lung cancer patients. Conclusion: A large proportion of nucleated cells obtained after EpCAM enrichment is not accounted for. The majority of these cells are leukocytes not detected with the current reagents and microscope of the CellSearch system. The correlation between DNA+CK−CD45− cells and increasing number of CTC suggests an association with cancer and warrants further investigation. Citation Format: Guus van Dalum, Simone van Lin, Ana M.C. Barradas, Jeroen T.N. Hiltermann, Harry J.M. Groen, Leon W.M.M. Terstappen. The identity of all nucleated cells enriched by CellSearch. [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 389. doi:10.1158/1538-7445.AM2015-389