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Background Tumor-derived extracellular vesicles (tdEVs) and circulating tumor cells (CTCs) in the blood of metastatic cancer patients associate with poor outcomes. In this study, we explored the human epidermal growth factor receptor 2 (HER2) expression on CTCs and tdEVs of metastatic breast cancer patients. Methods Blood samples from 98 patients (CLCC-IC-2006-04 study) were originally processed with the CellSearch® system using the CTC kit and anti-HER2 as an additional marker in the staining cocktail. CTCs and tdEVs were automatically enumerated from the generated CellSearch images using the open-source ACCEPT software. Results CTCs and tdEVs were subdivided based on their cytokeratin (CK) and HER2 phenotype into CK+HER2−, CK−HER2+, and CK+HER2+. The inclusion of anti-HER2 increased the percentage of informative samples with ≥ 1 detectable CTC from 89 to 95%. CK− CTCs and tdEVs correlated equally well with the clinical outcome as CK+ CTCs and tdEVs. Inter- and intra-patient heterogeneity was found for the CTC/tdEV phenotypes, and the presence of 2 or 3 classes of CTCs/tdEVs was associated with worse prognosis compared to a uniform CTC/tdEV phenotype present (1 class). The use of ≥ 7% HER2+CK+ tdEVs can predict HER2 expression of the tissue with 74% sensitivity and specificity using the HER2 amplification status of the primary tumor as a classification variable. Conclusions HER2 can be detected on CTCs and tdEVs not expressing CK, and these CK− CTCs/tdEVs have similar clinical relevance to CTCs and tdEVs expressing CK. tdEVs perform better than CTCs in predicting the HER2 status of the primary tissue. CTC and tdEV heterogeneity in the blood of patients is inversely associated with overall survival.

The presence of high expressing epithelial cell adhesion molecule (EpCAMhigh) circulating tumor cells (CTC) enumerated by CellSearch® in blood of cancer patients is strongly associated with poor prognosis. This raises the question about the presence and relation with clinical outcome of low EpCAM expressing CTC (EpCAMlow CTC). In the EU-FP7 CTC-Trap program, we investigated the presence of EpCAMhigh and EpCAMlow CTC using CellSearch, followed by microfiltration of the EpCAMhigh CTC depleted blood. Blood samples of 108 castration-resistant prostate cancer patients and 22 metastatic breast cancer patients were processed at six participating sites, using protocols and tools developed in the CTC-Trap program. Of the prostate cancer patients, 53% had ≥5 EpCAMhigh CTC and 28% had ≥5 EpCAMlow CTC. For breast cancer patients, 32% had ≥5 EpCAMhigh CTC and 36% had ≥5 EpCAMlow CTC. 70% of prostate cancer patients and 64% of breast cancer patients had in total ≥5 EpCAMhigh and/or EpCAMlow CTC, increasing the number of patients in whom CTC are detected. Castration-resistant prostate cancer patients with ≥5 EpCAMhigh CTC had shorter overall survival versus those with

Background: For rational decision making, assessing the cost-effectiveness and budget impact of new drugs and comparing the costs of drugs already on the market is required. In addition to value frameworks, such as the American Society of Clinical Oncology Value Framework and the European Society of Medical Oncology–Magnitude of Clinical benefit Scale, this also requires a transparent overview of actual drug prices. While list prices are available, evidence on treatment cost is not. This paper aims to synthesise evidence on the reimbursement and costs of high-cost breast cancer drugs in The Netherlands (NL). Methods: A literature review was performed to identify currently reimbursed breast cancer drugs in the NL. Treatment costs were determined by multiplying list prices with the average length of treatment and dosing schedule. Results: Comparing list prices to the estimated treatment cost resulted in substantial differences in the ranking of costliness of the drugs. The average mean treatment length was unknown for 11/31 breast cancer drugs (26.2%). The differences in the 15 highest-cost drugs were largest for Bevacizumab, Lapatinib and everolimus, with list prices of €541, €158, €1,168 and estimated treatment cost of €174,400, €18,682 and €31,207, respectively. The lowest-cost (patented) targeted drug is €1,818 more expensive than the highest-cost (off-patent) generic drug according to the estimated drug treatment cost. Conclusions: A lack of evidence on the reimbursement and cost of high-cost breast cancer drugs complicates rapid and transparent evidence synthesis, necessary to focus strategies aiming to limit the increasing healthcare costs. Interestingly, the findings show that off-patent generics (such as paclitaxel or doxorubicin), although substantially cheaper than patented drugs, are still relatively costly. Extending standardisation and increasing European and national regulations on presenting information on costs per cancer drug is highly recommended.

A microfluidic chip was designed, prepared and tested for integration with a confocal Raman imaging spectrometer with the specific purpose of enabling studies of individual biological cells. The design of the chip effectively overcomes the limitations arising from the high numerical aperture (NA) and short working distance objectives, which are necessary for high resolution imaging. The high confocal spatial resolution was achieved by a careful design of the geometry of the chip together with a thin, optically and Raman silent sealing window as the embedding medium for the channels. A leak-free microfluidic chip was obtained by surface plasma modification of polydimethylsiloxane (PDMS) and optimization of the liquid loading parameters. Raman images of biological cells, which were transported by flow into the microfluidic chip, are presented as an example of a Raman-microfluidics application. The broad band Raman spectra from −50 cm−1 to 3650 cm−1 were recorded in 1600 frequency intervals without any signal enhancement or sample labeling. Raman images were recorded in ∼400 seconds and they typically consisted of 64 × 64 pixels with a step size of 250 nm, thus containing ∼4 × 106 data points altogether.

In biomedical imaging reliable segmentation of objects (e.g., from small cells up to large organs) is of fundamental importance for automated medical diagnosis. New approaches for multiscale segmentation can considerably improve performance in case of natural variations in intensity, size, and shape. This paper aims at segmenting objects of interest based on shape contours and automatically finding multiple objects with different scales. The overall strategy of this work is to combine nonlinear segmentation with scales spaces and spectral decompositions. We generalize a variational segmentation model based on total variation using Bregman distances to construct an inverse scale space. This offers the new model to be accomplished by a scale analysis approach based on a spectral decomposition of the total variation. As a result we obtain a very efficient, (nearly) parameter-free multiscale segmentation method that comes with an adaptive regularization parameter choice. To address the variety of shapes and scales present in biomedical imaging we analyze synthetic cases clarifying the role of scale and the relationship of Wulff shapes and eigenfunctions. To underline the potential of our approach and to show its wide applicability we address three different experimental biomedical applications. In particular, we demonstrate the added benefit for identifying and classifying circulating tumor cells and present interesting results for network analysis in retina imaging. Due to the nature of underlying nonlinear diffusion, the mathematical concepts in this work offer promising extensions to nonlocal classification problems.

Cancer cells can enter the bloodstream, form distant metastases, and ultimately lead to death. A study by Allard and colleagues, which was published in the October 15, 2004, issue of Clinical Cancer Research, concluded that the CellSearch system could be used as a reliable tool to investigate circulating tumor cells and their clinical utility, and it spurred a still-growing interest in the field. Clin Cancer Res; 21(13); 2883–5. ©2015 AACR. See related article by Allard et al., Clin Cancer Res 2004;10(20) October 15, 2004;6897–904

Presence of circulating tumor cells (CTC), as detected by the CellSearch System, in patients with metastatic carcinomas is associated with poor survival prospects. CellTracks TDI, a dedicated image cytometer, was developed to improve the enumeration of these rare CTC. The CellSearch System was used to enumerate CTC in 7.5 mL blood of 68 patients with cancer and 9 healthy controls. Cartridges containing the fluorescently labeled CTC from this system were reanalyzed using the image cytometer, which acquires images with a TDI camera using a 40×/0.6 NA objective and lasers as light source. Automated classification of events was performed by the Random Forest method using Matlab. An automated classifier was developed to classify events into CTC, apoptotic CTC, CTC debris, leukocytes, and debris not related to CTC. A high agreement in classification was obtained between the automated classifier and five expert reviewers. Comparison of images from the same events in CellTracks TDI and CellTracks Analyzer II shows improved resolution in fluorescence images and improved classification by adding bright-field images. Improved detection efficiency for CD45-APC avoids the classification of leukocytes nonspecifically binding to cytokeratin as CTC. The correlation between number of CTC detected in CellTracks TDI and CellTracks Analyzer II is good with a slope of 1.88 and a correlation coefficient of 0.87. Automated classification of events by CellTracks TDI eliminates the operator error in classification of events as CTC and permits quantitative assessment of parameters. The clinical relevance of various CTC definitions can now be investigated.

Characterization of rare cells usually requires high sensitivity quantification of multiple parameters. Detection of morphological features of these cells is highly desired when routinely identifying circulating tumor cells (CTC) in blood of patients. We have designed an image cytometer intended for fast and sensitive routine analysis of CTC. After an initial scan, prospective events can be revisited for more detailed analysis. The image cytometer features: 375, 491, and 639 nm laser lines, a 40×/0.6NA objective, a CCD camera operating in TDI mode, servo stages to move the sample in two dimensions and a piëzo microscope objective positioner to move the objective in the third dimension. ImageJ is used for dedicated image analysis. A homogeneous illumination area, measuring 180 × 180 μm(2) , was created by the use of a rotating diffuser in combination with two micro-lens arrays. For feed-forward automatic focusing of the sample during a scan, a 3D spline was fitted through 30 predetermined focus positions before scanning the sample. Continuous signal acquisition is made possible by using a CCD operating in TDI mode synchronized to the movement of two servo scan stages. The limit of fluorescence sensitivity is 120 PE molecules on a bead with a diameter of 6.8 μm, at a scanning speed of 1.0 mm s(-1) . The resolution of the imaging system is 0.76 μm in the TDI scan direction at a wavelength of 580 nm. Identification of cells is facilitated by scatter plots of the fluorescent parameters in which each individual event can be viewed for its morphological features by fluorescence as well as bright field. The image cytometer measures quantitative fluorescence and morphological features at a high sensitivity, high resolution, and with minimal overhead time. It has the ability torelocate events of interest for further detailed analysis. The system can be used for routine identification and characterization of rare cells.

Circulating tumor cells (CTCs) are very attractive surrogate markers for systemic cancer. Currently, major efforts are being made to use these rare cells in the sense of a liquid biopsy to gain molecular information for rational therapeutic decision-making. The advancements in molecular analyses of CTCs down to the single-cell level have been significant in recent years and some applications are ready to be used in clinical studies. As discussed in this review, a major challenge for translating such molecular CTC-based assays into the clinic is the extremely low frequency of CTCs and the associated problems of their reliable detection and isolation. A potential solution to overcome the low CTC frequency is the recently introduced diagnostic leukapheresis that permits screening of liters of blood. Discussed here are the challenges as well as the current efforts implementing this method into clinical workflows to realize more reliable liquid biopsies.

The presence of circulating tumor cells (CTC) is an independent prognostic factor for progression-free and overall survival for patients with metastatic and newly diagnosed breast cancer. The present study was undertaken to explore whether the presence of CTC before and during follow-up after surgery is associated with recurrence free survival (RFS) and overall survival (OS). In a prospective single center study, CTC were enumerated with the CellSearch system in 30 ml of peripheral blood of 403 stage I-III patients before undergoing surgery for breast cancer (A) and if available 1 week after surgery (B), after adjuvant chemo- and/or radiotherapy or before start of long-term hormonal therapy (C), one (D), two (E) and three (F) years after surgery. Patients were stratified into unfavorable (CTC ≥1) and favorable (CTC=0) prognostic groups. >1 CTC in 30 ml blood was detected in 75/403 (19%) at A, 66/367 (18%) at B, 40/263 (15%) at C, 30/235 (12%) at D, 18/144 (11%) at E and 11/83 (13%) at F. RFS and OS was significantly lower for unfavorable CTC as compared to favorable CTC before surgery (p=0.022 and p=0.006), after adjuvant therapy (p

Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch(R) system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

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.

We have developed a platform for cell analysis based on immunomagnetic selection and magnetic alignment of cells in combination with an epi-illumination tracking and detection system. Whole blood was labeled with ferromagnetic nanoparticles and fluorescent probes, and placed in a magnetic field in a chamber. Cells labeled with ferromagnetic nanoparticles moved upward and aligned along ferromagnetic lines deposited by lithographic techniques on an optically transparent surface of the chamber. An epi-illumination system using a 635 nm laser diode as a light source scanned the lines and measured signals obtained from the aligned cells. The cell counts per unit of blood volume obtained with the system correlated well with those obtained from the counts from a standard hematology analyzer and flow cytometer. The cell analysis platform is significantly less complex and more sensitive than current cell analysis equipment and provides additional functionality through its ability to subject the cells to repeated and varied analyses while they remain in a natural environment (i.e., whole blood).

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.

Background In current practice cancer tissue is taken at diagnosis to assess the presence of treatment targets. This however is suboptimal since tumor cells evolve due to genomic instability. Assessment of the genotype and phenotype of the CTCs will provide insights into which treatments would be most beneficial for the individual patient. Feasibility to detect treatment targets in CTCs has been demonstrated (Meng, Tripathy et al. 2004; de Bono, Attard et al. 2007; Rossi, Basso et al. 2010; Wang, Pfister et al. 2010). In this context, the development of a cytogenetic assay for CTCs will be crucial for successful molecular targeted therapy in cancer patients. Genetic characterization of CTCs is expected to gather new knowledge on the mechanism of metastasis and on potential targets of novel therapeutic strategies. Patients and methods Assay optimization for analysis of genetic aberrations of CTCs was performed with cells from tumor cell lines. Tumor cell lines with known chromosomal aberrations and mosaicism were spiked into 7.5mL whole blood samples, at numbers similar to those observed in-vivo in cancer patients. Tumor cells were enriched by CellSearch System and off-line purified and individually analyzed for chromosomal alterations. The procedure was next validated by using blood samples collected from cancer patients. Results/Conclusions A robust protocol for the isolation of individual CTCs followed by DNA extraction and amplification after CellSearch enrichment was established. The number and the quality of CTCs needed to obtain an informative analysis of chromosomal aberrations were set up. Accrual of cancer patients for individual CTC isolation and molecular characterization is ongoing. Updated data including evaluations on prognostic value of the genetic aberrations analysis of the CTCs and correlation with clinical stage, tumor burden, metastatic sites and survival will be available and presented at the meeting. Disclosure All authors have declared no conflicts of interest.

Minimal residual cancer is defined as “the presence of tumor cells that are not detectable by the current routine diagnostic procedures used for tumor staging in cancer patients after surgical removal of the primary tumor.” Data from European and North American groups have demonstrated the prognostic impact of disseminated tumor cells (DTCs)9 in the bone marrow of breast cancer patients. Circulating tumor cell (CTC) detection and enumeration in peripheral blood have been examined in prospective multicenter studies of metastatic breast, colorectal, and prostate cancers and have been associated with decreased progression-free and overall survival. An increasing number of clinical research studies are validating these observations and extending them to other cancers and to earlier disease stages. CTCs are highly heterogeneous, and their molecular characterization is important, not only to confirm their malignant origin but also to follow immune-phenotypic changes with tumor progression and identify diagnostically and therapeutically relevant targets that will help stratify cancer patients for individualized therapies. The rarity of CTCs—and thus the very limited amount of available sample—presents a formidable analytical and technical challenge. Recent technical advances in CTC detection and characterization include reverse-transcription quantitative PCR (RT-qPCR) methods, image-based approaches, and microfilter and microchip devices. CTCs represent a promising new diagnostic field for advanced-stage patients in that the sensitive CTC-detection platforms allow monitoring of disease and treatment efficacy. The development of single-cell technologies might allow profiling of these cells for the purpose of adapting treatment regimens. CTC-detection and- characterization techniques hold promise for playing a role as a “liquid biopsy” that will allow physicians to follow cancer changes over time and to tailor treatment. Current research on CTCs is focusing on the identification of novel diagnostic and therapeutic biomarkers produced by these cells. CTCs are promising as novel tumor biomarkers because they are well-defined targets for understanding …

Item does not contain fulltext BACKGROUND: We demonstrated that circulating tumor cell (CTC) number at baseline and follow-up is an independent prognostic factor in metastatic colorectal cancer (mCRC). This analysis was undertaken to explore whether patient and treatment characteristics impact the prognostic value of CTCs. PATIENTS AND METHODS: CTCs were enumerated with immunomagnetic separation from the blood of 430 patients with mCRC at baseline and on therapy. Patients were stratified into unfavorable and favorable prognostic groups based on CTC levels of > or = 3 or /=65 years (P = 0.0007), and ECOG PS of zero (P = 0.04), unfavorable baseline CTC was associated with inferior PFS. CONCLUSION: Baseline CTC count is an important prognostic factor within specific subgroups defined by treatment or patient characteristics.

8000 Background: CP-751,871, a fully human IgG2 subtype monoclonal antibody, is a potent and specific inhibitor of the insulin-like growth factor type I receptor (IGF-IR). Methods: A comprehensive evaluation of blood and tissue markers of the IGF-IR pathway was undertaken during the conduct of phase I/II studies to evaluate the safety and efficacy of the combination of paclitaxel (T), carboplatin (C) and CP-751,871 (I) as first line treatment of advanced non-small cell lung cancer (NSCLC) patients (pts). Expression of IGF- IR and its ligands was investigated further in archival samples using tissue arrays and AQUA technology. IGF1R gene scanning was also performed. Circulating Tumor Cells (CTCs) expressing the IGF-IR were enumerated using CellTracks. Serum markers investigated included cleaved circulating IGF-IR, total and free IGF-I, IGFBP-3 and ALS. Glycemia was evaluated using fasting glucose. hGH and insulin levels were determined in a cohort of pts treated with single agent CP-751,871. Results: Blood...

Background: Induction of apoptosis is a common effect of many new and existing drugs, and Bcl-2 expression is associated with resistance to apoptosis. CTCs are a minimally invasive blood test and are a source of tumor cells that can be obtained serially during therapy. Monitoring Bcl-2, apoptosis, and other markers in CTCs may be useful endpoints in trials of new and targeted agents. The goal of this pilot study is to estimate Bcl-2 expression and apoptosis in CTCs from pts being treated for metastatic breast cancer (MBC). Methods: Whole blood was collected from pts initiating therapy for MBC at baseline, post-treatment (24, 48, or 72 hours), and 3–4 weeks. Samples were split into 3 × 7.5ml fractions. CTCs were isolated and enumerated using the CellTracks and CellSpotter systems. CTCs were defined as DAPI+, cytokeratin+, CD45-, with a cellular morphology. Apoptosis defined by positive staining with MAb M30. Bcl-2 status defined by staining with MAb Bcl-2–100. Results: 39/81 patients (49%) had ≥5 CTCs at baseline. 25% had ≥5 CTCs at 3–4 weeks. 35% of pts with CTCs had positive Bcl-2 staining in 91–100% of their CTCs (highest decile). Other pts were equally distributed across the remaining Bcl-2 deciles. At baseline, apoptosis correlates with CTC number. In samples with 1–4 CTCs, 80% of cells were apoptotic. For 5–49 CTCs, 48% were apoptotic; for 50–99, 34% were apoptotic; for 100–500, 28% were apoptotic; and for >500, 22% were apoptotic. Bcl-2 staining was inversely correlated with apoptosis staining. Pts were risk stratified by changes in CTC number after one cycle of therapy, and apoptosis increased with improving prognostic groups. One pt had markedly elevated CTCs of >20,000. The cells were remarkably 100% Bcl-2 positive and M30 negative (1% staining). Conclusions: These data confirm the prevalence of CTCs seen in the pivotal trial of CTCs in MBC (Cristofanilli NEJM 2004) and provide novel observations for Bcl-2 expression and apoptosis in CTCs. Higher Bcl-2 expression appears to correlate with decreased apoptosis. High fractions of apoptotic cells correlate with lower numbers of CTCs, which is associated with a better prognosis. Analysis of the post-treatment (24, 48, or 72 hour) samples and correlation of markers with progression free survival are ongoing and will be presented.

The presence of circulating tumor cells (CTCs) in the blood of cancer patients may guide the use of therapy. We investigated how to evaluate a reduction in the number of CTCs after administration of therapy. CTCs were enumerated with the CellSearch system in 111 metastatic breast and 185 metastatic prostate cancer patients before start of a new line of chemotherapy and after initiation of therapy. Different means to express changes in CTC counts were evaluated with respect to overall survival (OS). A static CTC cutoff is the best method to determine whether a therapy is effective. This is exemplified by the highest Cox hazard ratio of 2.1 for OS; three methods to express relative differences performed worse. A lookup table is provided from which the significance of a change in CTCs can be derived. The aim of therapy should be the elimination of all CTCs. A period of 10 to 12 weeks of therapy is needed to reach the treatment effect on CTCs.

We describe the effect of splenic irradiation (SI) (0,5–1 Gy weekly) on lymphocyte subpopulations for 7 patients with progressive B chronic B-lymphocytic leukemia (B-CLL). Using specific cellular characteristics we could distinguish normal from abnormal cells. The irradiation resulted in a decrease of lymph node size, reduction in spleen volume and decrease in peripheral blood lymphocytes. The one exception was a patient with a prolymphocytoid transformation of B-CLL. For 3 patients SI had to be interrupted or stopped because of severe cytopenia. Quantitation of malignant B cells and normal T lymphocytes revealed that the total irradiation dose which resulted in a specific decrease of malignant lymphocytes varied from patient to patient. Normal T-cell subpopulations, which were increased before SI, decreased to normal or abnormally low values during SI. In previously untreated patients, natural killer (NK) cell numbers decreased more rapidly than T-cell subpopulations. For 2 patients refractory to chemotherapy an increase of NK cells was observed upon SI.

Polarization measurement of orthogonal light scattering is introduced as a new optical parameter in flow cytometry. In the experimental setup, the electrical field of the incident laser beam is polarized in the direction of the sample flow. The intensity of the orthogonal light scattering polarized along the direction of the incoming laser beam is called depolarized orthogonal light scattering. Theoretical analysis shows that for small values of the detection aperture, the measured depolarization is caused by anisotropic cell structures and multiple scattering processes inside the cell. Measurements of the orthogonal depolarized light scattering in combination with the normal orthogonal light scattering of human leucocytes revealed two populations of granulocytes. By means of cell sorting it was shown that the granulocytes with a relatively high depolarization are eosinophilic granulocytes. Similar experiments with human lymphocytes revealed a minor subpopulation of yet-unidentified lymphocytes with a relative large orthogonal light-scattering depolarization. The results were obtained with an argon ion laser tuned at different wavelengths as well as with a 630-nm helium neon laser. These results show that measurement of depolarized orthogonal light scattering is a useful new parameter for flow-cytometric cell differentiation.

Background Tumor cells in the blood of patients with metastatic carcinomas are associated with poor survival. Knowledge of the cells’ genetic make-up can help to guide targeted therapy. We evaluated the efficiency and quality of isolation and amplification of DNA from single circulating tumor cells (CTC). Methods The efficiency of the procedure was determined by spiking blood with SKBR-3 cells, enrichment with the CellSearch system, followed by single cell sorting by fluorescence-activated cell sorting (FACS) and whole genome amplification. A selection of single cell DNA from fixed and unfixed SKBR-3 cells was exome sequenced and the DNA quality analyzed. Single CTC from patients with lung cancer were used to demonstrate the potential of single CTC molecular characterization. Results The overall efficiency of the procedure from spiked cell to amplified DNA was approximately 20%. Losses attributed to the CellSearch system were around 20%, transfer to FACS around 25%, sorting around 5% and DNA amplification around 25%. Exome sequencing revealed that the quality of the DNA was affected by the fixation of the cells, amplification, and the low starting quantity of DNA. A single fixed cell had an average coverage at 20× depth of 30% when sequencing to an average of 40× depth, whereas a single unfixed cell had 45% coverage. GenomiPhi-amplified genomic DNA had a coverage of 72% versus a coverage of 87% of genomic DNA. Twenty-one percent of the CTC from patients with lung cancer identified by the CellSearch system could be isolated individually and amplified. Conclusions CTC enriched by the CellSearch system were sorted by FACS, and DNA retrieved and amplified with an overall efficiency of 20%. Analysis of the sequencing data showed that this DNA could be used for variant calling, but not for quantitative measurements such as copy number detection. Close to 55% of the exome of single SKBR-3 cells were successfully sequenced to 20× depth making it possible to call 72% of the variants. The overall coverage was reduced to 30% at 20× depth, making it possible to call 56% of the variants in CellSave-fixed cells.