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1.  Detection of Circulating Tumor Cells 
Scientifica  2014;2014:819362.
The increasing number of treatment options for patients with metastatic carcinomas has created an accompanying need for methods to determine if the tumor will be responsive to the intended therapy and to monitor its effectiveness. Ideally, these methods would be noninvasive and provide quantitative real-time analysis of tumor activity in a variety of carcinomas. Assessment of circulating tumor cells shed into the blood during metastasis may satisfy this need. Here we review the CellSearch technology used for the detection of circulating tumor cells and discuss potential future directions for improvements.
PMCID: PMC4124199  PMID: 25133014
2.  Significance of Circulating Tumor Cells Detected by the CellSearch System in Patients with Metastatic Breast Colorectal and Prostate Cancer 
Journal of Oncology  2009;2010:617421.
The increasing number of treatment options for patients with metastatic carcinomas has created a concomitant need for new methods to monitor their use. Ideally, these modalities would be noninvasive, be independent of treatment, and provide quantitative real-time analysis of tumor activity in a variety of carcinomas. Assessment of circulating tumor cells (CTCs) shed into the blood during metastasis may satisfy this need. We developed the CellSearch System to enumerate CTC from 7.5 mL of venous blood. In this review we compare the outcomes from three prospective multicenter studies investigating the use of CTC to monitor patients undergoing treatment for metastatic breast (MBC), colorectal (MCRC), or prostate cancer (MPC) and review the CTC definition used in these studies. Evaluation of CTC at anytime during the course of disease allows assessment of patient prognosis and is predictive of overall survival.
PMCID: PMC2793426  PMID: 20016752
3.  The detection of EpCAM+ and EpCAM– circulating tumor cells 
Scientific Reports  2015;5:12270.
EpCAM expressing circulating tumor cells, detected by CellSearch, are predictive of short survival in several cancers and may serve as a liquid biopsy to guide therapy. Here we investigate the presence of EpCAM+ CTC detected by CellSearch and EpCAM– CTC discarded by CellSearch, after EpCAM based enrichment. EpCAM– CTC were identified by filtration and fluorescent labelling. This approach was validated using different cell lines spiked into blood and evaluated on blood samples of 27 metastatic lung cancer patients. The majority of spiked EpCAM+ cells could be detected with CellSearch, whereas most spiked cells with EpCAMlow or EpCAM– expression were detected using filtration. Five or more CTC were detected in 15% of the patient samples, this increased to 41% when adding the CTC detected in the discarded blood. The number of patients with CTC and the number of CTC detected were doubled by the presence of EpCAM– CTC. In this pilot study, the presence of EpCAM+ CTC was associated with poor outcome, whereas the EpCAM– CTC were not. This observation will need to be confirmed in larger studies and molecular characterization needs to be conducted to elucidate differences between EpCAM– and EpCAM+ CTC.
PMCID: PMC4505332  PMID: 26184843
4.  Parallel Single Cancer Cell Whole Genome Amplification Using Button-Valve Assisted Mixing in Nanoliter Chambers 
PLoS ONE  2014;9(9):e107958.
The heterogeneity of tumor cells and their alteration during the course of the disease urges the need for real time characterization of individual tumor cells to improve the assessment of treatment options. New generations of therapies are frequently associated with specific genetic alterations driving the need to determine the genetic makeup of tumor cells. Here, we present a microfluidic device for parallel single cell whole genome amplification (pscWGA) to obtain enough copies of a single cell genome to probe for the presence of treatment targets and the frequency of its occurrence among the tumor cells. Individual cells were first captured and loaded into eight parallel amplification units. Next, cells were lysed on a chip and their DNA amplified through successive introduction of dedicated reagents while mixing actively with the help of integrated button-valves. The reaction chamber volume for scWGA 23.85 nl, and starting from 6–7 pg DNA contained in a single cell, around 8 ng of DNA was obtained after WGA, representing over 1000-fold amplification. The amplified products from individual breast cancer cells were collected from the device to either directly investigate the amplification of specific genes by qPCR or for re-amplification of the DNA to obtain sufficient material for whole genome sequencing. Our pscWGA device provides sufficient DNA from individual cells for their genetic characterization, and will undoubtedly allow for automated sample preparation for single cancer cell genomic characterization.
PMCID: PMC4169497  PMID: 25233459
5.  Towards the Biological Understanding of CTC: Capture Technologies, Definitions and Potential to Create Metastasis 
Cancers  2013;5(4):1619-1642.
Circulating Tumor Cells (CTC) are rare cells originated from tumors that travel into the blood stream, extravasate to different organs of which only a small fraction will develop into metastasis. The presence of CTC enumerated with the CellSearch system is associated with a relative short survival and their continued presence after the first cycles of therapy indicates a futile therapy in patients with metastatic carcinomas. Detailed characterization of CTC holds the promise to enable the choice of the optimal therapy for the individual patients during the course of the disease. The phenotype, physical and biological properties are however not well understood making it difficult to assess the merit of recent technological advancements to improve upon the capture of CTC or to evaluate their metastatic potential. Here we will discuss the recent advances in the classification of CTC captured by the CellSearch system, the implications of their features and numbers. Latest capture platforms are reviewed and placed in the light of technology improvements needed to detect CTC. Physical properties, phenotype, viability and proliferative potential and means to assess their proliferation and metastatic capacity will be summarized and placed in the context of the latest CTC capture platforms.
PMCID: PMC3875957  PMID: 24305653
circulating tumor cells; image analysis; filtration; EpCAM; cancer stem cell; tumorogenesis
6.  Circulating Tumor Cells Count and Morphological Features in Breast, Colorectal and Prostate Cancer 
PLoS ONE  2013;8(6):e67148.
Presence of circulating tumor cells (CTC) in patients with metastatic breast, colorectal and prostate cancer is indicative for poor prognosis. An automated CTC (aCTC) algorithm developed previously to eliminate the variability in manual counting of CTC (mCTC) was used to extract morphological features. Here we validated the aCTC algorithm on CTC images from prostate, breast and colorectal cancer patients and investigated the role of quantitative morphological parameters.
Stored images of samples from patients with prostate, breast and colorectal cancer, healthy controls, benign breast and colorectal tumors were obtained using the CellSearch system. Images were analyzed for the presence of aCTC and their morphological parameters measured and correlated with survival.
Overall survival hazard ratio was not significantly different for aCTC and mCTC. The number of CTC correlated strongest with survival, whereas CTC size, roundness and apoptosis features reached significance in univariate analysis, but not in multivariate analysis. One aCTC/7.5 ml of blood was found in 7 of 204 healthy controls and 9 of 694 benign tumors. In one patient with benign tumor 2 and another 9 aCTC were detected.
Significance of the study
CTC can be identified and morphological features extracted by an algorithm on images stored by the CellSearch system and strongly correlate with clinical outcome in metastatic breast, colorectal and prostate cancer.
PMCID: PMC3695007  PMID: 23826219
7.  Filter Characteristics Influencing Circulating Tumor Cell Enrichment from Whole Blood 
PLoS ONE  2013;8(4):e61770.
A variety of filters assays have been described to enrich circulating tumor cells (CTC) based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of whole blood spiked with cells from tumor cell lines were passed through silicon nitride microsieves, polymer track-etched filters and metal TEM grids with various pore sizes. The recovery and size of 9 different culture cell lines was determined and compared to the size of EpCAM+CK+CD45−DNA+ CTC from patients with metastatic breast, colorectal and prostate cancer. The 8 µm track-etched filter and the 5 µm microsieve had the best performance on MDA-231, PC3-9 and SKBR-3 cells, enriching >80% of cells from whole blood. TEM grids had poor recovery of ∼25%. Median diameter of cell lines ranged from 10.9–19.0 µm, compared to 13.1, 10.7, and 11.0 µm for breast, prostate and colorectal CTC, respectively. The 11.4 µm COLO-320 cell line had the lowest recovery of 17%. The ideal filter for CTC enrichment is constructed of a stiff, flat material, is inert to blood cells, has at least 100,000 regularly spaced 5 µm pores for 1 ml of blood with a ≤10% porosity. While cell size is an important factor in determining recovery, other factors must be involved as well. To evaluate a filtration procedure, cell lines with a median size of 11–13 µm should be used to challenge the system.
PMCID: PMC3634026  PMID: 23626725
8.  Construction of repeat-free fluorescence in situ hybridization probes 
Nucleic Acids Research  2011;40(3):e20.
FISH probes are generally made out of BAC clones with genomic DNA containing a variable amount of repetitive DNA that will need to be removed or blocked for FISH analysis. To generate repeat free (RF) Probes without loss in genomic coverage, a random library is made from BAC clones by whole-genome amplification (WGA). Libraries are denatured in the presence of excess C0t-1 DNA and allowed to re-anneal followed by digestion of all double-stranded elements by duplex-specific nuclease (DSN). Selective amplification of all elements not containing repetitive sequences is realized by a sequential amplification. The final RF products can be re-amplified and used as a stock for future probe production. The RF probes have a lower background, the signal intensity build up is faster and there is no need for blocking DNA. The signal to background ratio of the RF was higher as compared to repeat containing probes.
PMCID: PMC3273791  PMID: 22123742
9.  Unbiased and Automated Identification of a Circulating Tumour Cell Definition That Associates with Overall Survival 
PLoS ONE  2011;6(11):e27419.
Circulating tumour cells (CTC) in patients with metastatic carcinomas are associated with poor survival and can be used to guide therapy. Classification of CTC however remains subjective, as they are morphologically heterogeneous. We acquired digital images, using the CellSearch™ system, from blood of 185 castration resistant prostate cancer (CRPC) patients and 68 healthy subjects to define CTC by computer algorithms. Patient survival data was used as the training parameter for the computer to define CTC. The computer-generated CTC definition was validated on a separate CRPC dataset comprising 100 patients. The optimal definition of the computer defined CTC (aCTC) was stricter as compared to the manual CellSearch CTC (mCTC) definition and as a consequence aCTC were less frequent. The computer-generated CTC definition resulted in hazard ratios (HRs) of 2.8 for baseline and 3.9 for follow-up samples, which is comparable to the mCTC definition (baseline HR 2.9, follow-up HR 4.5). Validation resulted in HRs at baseline/follow-up of 3.9/5.4 for computer and 4.8/5.8 for manual definitions. In conclusion, we have defined and validated CTC by clinical outcome using a perfectly reproducing automated algorithm.
PMCID: PMC3210171  PMID: 22087312
10.  Significant and Sustained Antitumor Activity in Post-Docetaxel, Castration-Resistant Prostate Cancer With the CYP17 Inhibitor Abiraterone Acetate 
Journal of Clinical Oncology  2010;28(9):1489-1495.
The principal objective of this trial was to evaluate the antitumor activity of abiraterone acetate, an oral, specific, irreversible inhibitor of CYP17 in docetaxel-treated patients with castration-resistant prostate cancer (CRPC).
Patients and Methods
In this multicenter, two-stage, phase II study, abiraterone acetate 1,000 mg was administered once daily continuously. The primary end point was achievement of a prostate-specific antigen (PSA) decline of ≥ 50% in at least seven of 35 patients. Per an attained phase II design, more than 35 patients could be enrolled if the primary end point was met. Secondary objectives included: PSA declines of ≥ 30% and ≥ 90%; rate of RECIST (Response Evaluation Criteria in Solid Tumors) responses and duration on study; time to PSA progression; safety and tolerability; and circulating tumor cell (CTC) enumeration.
Docetaxel-treated patients with CRPC (N = 47) were enrolled. PSA declines of ≥ 30%, ≥ 50% and ≥ 90% were seen in 68% (32 of 47), 51% (24 of 47), and 15% (seven of 47) of patients, respectively. Partial responses (by RECIST) were reported in eight (27%) of 30 patients with measurable disease. Median time to PSA progression was 169 days (95% CI, 113 to 281 days). The median number of weeks on study was 24, and 12 (25.5%) of 47 patients remained on study ≥ 48 weeks. CTCs were enumerated in 34 patients; 27 (79%) of 34 patients had at least five CTCs at baseline. Eleven (41%) of 27 patients had a decline from at least five to less than 5 CTCs, and 18 (67%) of 27 had a ≥ 30% decline in CTCs after starting treatment with abiraterone acetate. Abiraterone acetate was well tolerated.
Abiraterone acetate has significant antitumor activity in post-docetaxel patients with CRPC. Randomized, phase III trials of abiraterone acetate are underway to define the future role of this agent.
PMCID: PMC2849770  PMID: 20159823
11.  Safety, Pharmacokinetics, and Pharmacodynamics of the Insulin-Like Growth Factor Type 1 Receptor Inhibitor Figitumumab (CP-751,871) in Combination with Paclitaxel and Carboplatin 
This phase 1 study was conducted to determine the recommended phase 2 dose of the selective insulin-like growth factor type 1 receptor (IGF-IR) inhibitor figitumumab (F, CP-751,871) given in combination with paclitaxel and carboplatin in patients with advanced solid tumors.
Patients received paclitaxel 200 mg/m2, carboplatin (area under the curve of 6), and F (0.05–20 mg/kg) q3 weeks for up to six cycles. Patients with objective response or stable disease were eligible to receive additional cycles of single agent F until disease progression. Safety, efficacy, pharmacokinetic, and pharmacodynamic endpoints were investigated.
Forty-two patients, including 35 with stages IIIB and IV non-small cell lung cancer (NSCLC), were enrolled in eight dose escalation cohorts. A maximum tolerated dose was not identified. Severe adverse events possibly related to F included fatigue, diarrhea, hyperglycemia, gamma glutamyl transpeptidase elevation, and thrombocytopenia (one case each). F plasma exposure parameters increased with dose. Fifteen objective responses (RECIST) were reported, including two complete responses in NSCLC and ovarian carcinoma. Notably, levels of bioactive IGF-1 seemed to influence response to treatment with objective responses in patients with a high baseline-free IGF-1 to IGF binding protein-3 ratio seen only in the 10 and 20 mg/kg dosing cohorts.
F was well tolerated in combination with paclitaxel and carboplatin. Based on its favorable safety, pharmacokinetic, and pharmacodynamic properties, the maximal feasible dose of 20 mg/kg has been selected for further investigation.
PMCID: PMC2941876  PMID: 19745765
IGF-1R; Figitumumab; CP-751,871; NSCLC
12.  An Immunomagnetic Single-Platform Image Cytometer for Cell Enumeration Based on Antibody Specificity▿  
Clinical and Vaccine Immunology  2007;14(4):412-419.
Simplification of cell enumeration technologies is necessary, especially for resource-poor countries, where reliable and affordable enumeration systems are greatly needed. In this paper, an immunomagnetic single-platform image cytometer (SP ICM) for cell enumeration based on antibody specificity is reported. A chamber/magnet assembly was designed such that the immunomagnetically labeled, acridine orange-stained cells in a blood sample moved to the surface of the chamber, where a fluorescent image was captured and analyzed for cell enumeration. The system was evaluated by applying one kind of antibody to count leukocytes and one kind for each leukocyte subpopulation: CD45 for leukocytes, CD3 for T lymphocytes, and CD19 for B lymphocytes. Excellent precision and linearity were achieved. Moreover, these cell counts, each from blood specimens of 42 to 52 randomly selected patients, were compared with those obtained by SP (TruCount) and dual-platform (DP) flow cytometry (FCM) technologies. The cell counts obtained by our system were in between those obtained from the TruCount and DP FCM methods; and good correlations were achieved (R ≥ 0.95). For CD4+ counts, as we expected, the cell count by our system was significantly higher than the CD4+ T-lymphocyte counts obtained by SP and DP FCM methods. Immunophenotyping of the immunomagnetically selected CD4+ cells showed that, besides CD4+ T lymphocytes, a proportion of the CD4+ dim monocytes was also selected. Our system is a simple immunomagnetic SP ICM, which can potentially be used for enumeration of CD3+ CD4+ T lymphocytes in resource-poor countries if an additional CD3 immunofluorescent label is applied.
PMCID: PMC1865616  PMID: 17287315
13.  Impaired growth and elevated Fas receptor expression in PIGA+ stem cells in primary paroxysmal nocturnal hemoglobinuria 
Journal of Clinical Investigation  2000;106(5):689-696.
The genetic defect underlying paroxysmal nocturnal hemoglobinuria (PNH) has been shown to reside in PIGA, a gene that encodes an element required for the first step in glycophosphatidylinositol anchor assembly. Why PIGA-mutated cells are able to expand in PNH marrow, however, is as yet unclear. To address this question, we compared the growth of affected CD59–CD34+ and unaffected CD59+CD34+ cells from patients with that of normal CD59+CD34+ cells in liquid culture. One hundred FACS-sorted cells were added per well into microtiter plates, and after 11 days at 37°C the progeny were counted and were analyzed for their differentiation pattern. We found that CD59–CD34+ cells from PNH patients proliferated to levels approaching those of normal cells, but that CD59+CD34+ cells from the patients gave rise to 20- to 140-fold fewer cells. Prior to sorting, the patients’ CD59– and CD59+CD34+ cells were equivalent with respect to early differentiation markers, and following culture, the CD45 differentiation patterns were identical to those of control CD34+ cells. Further analyses of the unsorted CD59+CD34+ population, however, showed elevated levels of Fas receptor. Addition of agonist anti-Fas mAb to cultures reduced the CD59+CD34+ cell yield by up to 78% but had a minimal effect on the CD59–CD34+ cells, whereas antagonist anti-Fas mAb enhanced the yield by up to 250%. These results suggest that expansion of PIGA-mutated cells in PNH marrow is due to a growth defect in nonmutated cells, and that greater susceptibility to apoptosis is one factor involved in the growth impairment.
PMCID: PMC381282  PMID: 10974022

Results 1-13 (13)