Cancer-ID: Toward Identification of Cancer by Tumor-Derived Extracellular Vesicles in Blood.

Extracellular vesicles (EVs) have great potential as biomarkers since their composition and concentration in biofluids are disease state dependent and their cargo can contain disease-related information. Large tumor-derived EVs (tdEVs, >1 µm) in blood from cancer patients are associated with poor outcome, and changes in their number can be used to monitor therapy effectiveness. Whereas, small tumor-derived EVs (<1 µm) are likely to outnumber their larger counterparts, thereby offering better statistical significance, identification and quantification of small tdEVs are more challenging. In the blood of cancer patients, a subpopulation of EVs originate from tumor cells, but these EVs are outnumbered by non-EV particles and EVs from other origin. In the Dutch NWO Perspectief Cancer-ID program, we developed and evaluated detection and characterization techniques to distinguish EVs from non-EV particles and other EVs. Despite low signal amplitudes, we identified characteristics of these small tdEVs that may enable the enumeration of small tdEVs and extract relevant information. The insights obtained from Cancer-ID can help to explore the full potential of tdEVs in the clinic.

Tumor cell capture from blood by flowing across antibody-coated surfaces.

The load of circulating tumor cells (CTC) is related to poor outcomes in cancer patients. A sufficient number of these cells would enable a full characterization of the cancer. An approach to probe larger blood volumes, allowing for the detection of more of these very rare CTC, is the use of leukapheresis. Currently available techniques allow only the analysis of a small portion of leukapheresis products. Here, we present a method that uses flow rather than static conditions which allows processing of larger volumes. We evaluated the conditions needed to isolate tumor cells from blood while passing antibody coated surfaces. Results show that our set-up efficiently captures cancer cells from whole blood. Results show that the optimal velocity at which cells are captured from blood is 0.6 mm s-1. Also, it can be concluded that the VU1D9 antibody targeting the EpCAM antigen has very high capture efficiency. When using an antibody that does not capture 100% of all cells, combining multiple antibodies on the capture surface is very beneficial leading to an increase in cell capture and is therefore worthwhile considering in any cancer cell capture methodology.

Quality of extracellular vesicle images by transmission electron microscopy is operator and protocol dependent.

Transmission electron microscopy (TEM) has nanometre resolution and can be used to distinguish single extracellular vesicles (EVs) from non-EV particles. TEM images of EVs are a result of operator image selection. To which extent operator image selection reflects the overall sample quality, and to which extent the images are comparable and reproducible, is unclear. In a first attempt to improve the comparability and reproducibility of TEM to visualise EVs, we compared operator image selection to images taken at predefined locations from the same grids, using four EV TEM preparation protocols, a single EV-containing sample and a single TEM instrument. Operator image selection leads to high-quality images that are more similar between the protocols. In contrast, images taken at predefined locations reveal differences between the protocols, for example in number of EVs per image and background quality. From the evaluated protocols, for only one protocol the operator image selection is comparable to the TEM images taken at predefined locations. Taken together, operator image selection can be used to demonstrate the presence of EVs in a sample, but seem less suitable to demonstrate the quality of a sample. Because images taken at predefined locations reflect the overall quality of the EV-containing sample rather than the presence of EVs alone, this is a first step to improve the comparability and reproducibility of TEM for monitoring the quality of EV-containing samples.

SEM-Raman image cytometry of cells.

Correlative and integrated scanning electron microscopy (SEM) and Raman micro-spectroscopy is presented that enables the characterization and identification of different cancer and non-cancer cells through SEM-Raman image cytometry. The hybrid microscopy system enables the acquisition of high resolution SEM images of uncoated cells and the spatial correlation with chemical information as obtained from Raman micro-spectroscopic imaging. A sample preparation protocol and a workflow are presented that are compatible with the demands of hybrid SEM-Raman microscopy. Stainless steel cell substrates were used that are both conductive and give a low optical response in Raman scattering. Correlative and integrated SEM-Raman micro-spectroscopy is illustrated with cells from blood and cells from a SKBR-3 breast cancer cell line.

Circulating tumour cell increase as a biomarker of disease progression in metastatic castration-resistant prostate cancer patients with low baseline CTC counts.

Background: The development of treatment response and surrogate biomarkers for advanced prostate cancer care is an unmet clinical need. Patients with baseline circulating tumour cell (BLCTCs) counts <5/7.5 mL represent a good prognosis subgroup but are non-evaluable for response assessment (decrease in CTCs). The aim of the study is to determine the value of any increase in CTCs (CTC progression) as an indicator of progression in prostate cancer patients with low pre-treatment CTCs (<5). Patients and methods: We carried out a post hoc analysis of patients with BLCTCs < 5 treated in the COU-AA-301 (abiraterone or placebo + prednisone) and IMMC-38 (chemotherapy) trials. The association of CTC progression (increase in CTCs at 4, 8 or 12 weeks) with overall survival (OS) was evaluated in multi-variable Cox regression models. Performance of survival models with and without CTC progression was evaluated by calculating ROC curve area under the curves (AUCs) and weighted c-indices. Results: Overall, 511 patients with CTCs < 5 (421 in COU-AA-301 and 90 in IMMC-38) were selected; 212 (41.7%) had CTC progression at 4, 8 or 12 weeks after treatment initiation. CTC progression was associated with significantly worse OS [27.1 versus 15.1 m; hazard ratio (HR) 3.4 (95% confidence interval [CI] 2.5-4.5; P < 0.001)], independent of baseline CTCs and established clinical variables. Adding CTC progression to the OS model significantly improved ROC AUC (0.77 versus 0.66; P < 0.001). Models including CTC progression had superior ROC AUC (0.77 versus 0.69; P < 0.001) and weighted c-index [0.750 versus 0.705; delta c-index: 0.045 (95% CI 0.019-0.071)] values than those including CTC conversion (increase to CTCs ≥ 5). In COU-AA-301, the impact of CTC progression was independent of treatment arm. Conclusions: Increasing CTCs during the first 12 weeks of treatment are independently associated with worse OS from advanced prostate cancer in patients with baseline CTCs < 5 treated with abiraterone or chemotherapy and improve models with established prognostic variables. These findings must be prospectively validated.

Isolation of single cells for protein therapeutics using microwell selection and Surface Plasmon Resonance imaging.

Here the feasibility is demonstrated that by combining Surface Plasmon Resonance Imaging (SPRi) and self-sorting microwell technology product secretion of individual cells can be monitored. Additionally isolation of the selected cells can be performed by punching the cells from the microwells using coordinates of the positions of microwells obtained with SPRi. Cells of interest can be retrieved sterile from the microwell array for further cultivation.

Improving the CellSearch® system.

INTRODUCTION: The CellSearch® CTC test enumerates tumor cells present in 7.5 ml blood of cancer patients. improvements, extensions and different utilities of the cellsearch system are discussed in this paper. Areas covered: This paper describes work performed with the CellSearch system, which go beyond the normal scope of the test. All results from searches with the search term 'CellSearch' from Web of Science and PubMed were categorized and discussed. Expert commentary: The CellSearch Circulating Tumor Cell test captures and identifies tumor cells in blood that are associated with poor clinical outcome. How to best use CTC in clinical practice is being explored in many clinical trials. The ability to extract information from the CTC to guide therapy will expand the potential clinical utility of CTC.

Androgen receptor expression in circulating tumour cells from castration-resistant prostate cancer patients treated with novel endocrine agents.

BACKGROUND: Abiraterone and enzalutamide are novel endocrine treatments that abrogate androgen receptor (AR) signalling in castration-resistant prostate cancer (CRPC). Here, we developed a circulating tumour cells (CTCs)-based assay to evaluate AR expression in real-time in CRPC and investigated nuclear AR expression in CTCs in patients treated with enzalutamide and abiraterone. METHODS: CTCs were captured and characterised using the CellSearch system. An automated algorithm to identify CTCs and quantify AR expression was employed. The primary aim was to evaluate the association between CTC AR expression and prior treatment with abiraterone or enzalutamide. RESULTS: AR expression in CTCs was evaluated in 94 samples from 48 metastatic CRPC patients. We observed large intra-patient heterogeneity of AR expression in CTCs. Prior exposure to abiraterone or enzalutamide was not associated with a change in CTCs AR expression (median intensity and distribution of AR-positive classes). In support of this, we also confirmed maintained nuclear AR expression in tissue samples collected after progression on abiraterone. AR staining also identified additional AR-positive CD45-negative circulating cells that were CK-negative/weak and therefore missed using standard protocols. The number of these events correlated with traditional CTCs and was associated with worse outcome on univariate analysis. CONCLUSIONS: We developed a non-invasive method to monitor AR nuclear expression in CTCs. Our studies confirm nuclear AR expression in CRPC patients progressing on novel endocrine treatments. Owing to the significant heterogeneity of AR expression in CTCs, studies in larger cohorts of patients are required to identify associations with outcome.

Label-free imaging and identification of typical cells of acute myeloid leukaemia and myelodysplastic syndrome by Raman microspectroscopy.

In clinical practice, the diagnosis and classification of acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) start from the manual examination of stained smears of bone marrow (BM) and peripheral blood (PB) by using an optical microscope. This step is subjective and scarcely reproducible. Therefore, the development of subjective and potentially automatable methods for the recognition of typical AML/MDS cells is necessary. Here we have used Raman spectroscopy for distinguishing myeloblasts, promyelocytes, abnormal promyelocytes and erhytroblasts, which have to be counted for a correct diagnosis and morphological classification of AML and MDS. BM samples from patients affected by four different AML subtypes, mostly characterized by the presence of the four subpopulations selected for this study, were analyzed. First, each cell was scanned by acquiring 4096 spectra, thus obtaining Raman images which demonstrate an accurate description of morphological features characteristic of each subpopulation. Raman imaging coupled with hierarchical cluster analysis permitted the automatic discrimination and localization of the nucleus, the cytoplasm, myeloperoxidase containing granules and haemoglobin. Second, the averaged Raman fingerprint of each cell was analysed by multivariate analysis (principal component analysis and linear discriminant analysis) in order to study the typical vibrational features of each subpopulation and also for the automatic recognition of cells. The leave-one-out cross validation of a Raman-based classification model demonstrated the correct classification of myeloblasts, promyelocytes (normal/abnormal) and erhytroblasts with an accuracy of 100%. Normal and abnormal promyelocytes were distinguished with 95% accuracy. The overall classification accuracy considering the four subpopulations was 98%. This proof-of-concept study shows that Raman micro-spectroscopy could be a valid approach for developing label-free, objective and automatic methods for the morphological classification and counting of cells from AML/MDS patients, in substitution of the manual examination of BM and PB stained smears.

Unbiased quantitative assessment of Her-2 expression of circulating tumor cells in patients with metastatic and non-metastatic breast cancer.

Background Circulating tumor cells (CTCs) can provide the basis for a liquid biopsy and may guide the use of targeted therapies. We report on unbiased quantification of Her-2 protein expression of CTCs. Patients and methods Her-2 assessment of CTCs was carried out using the CellSearch(®) system in 103 metastatic (M1) and 88 non-metastatic (M0) breast-cancer patients. Expression of Her-2 on CTCs was determined by a manual review and an automated algorithm using Her-2- fluorescein isothiocyanate (FITC) fluorescence of leukocytes to determine the Her-2-expression threshold in each sample. Results Her-2 expression of CTCs varied greatly within and among patients compared with Her-2 expression of leukocytes. In M1 patients, a threshold of 75% of Her-2 positive CTCs in patients with ≥5 CTCs was set. Applying this threshold, 9% of M1 patients with Her-2-negative primary tumors had Her-2-positive CTC status and 29% of M1 patients with Her-2-positive primary tumors had Her-2-negative CTC status. No Her-2 discrepancy was observed between CTCs and primary tumors in M0 patients. Conclusions Our findings demonstrate that Her-2 expression is heterogeneous among CTCs within each patient. We show the feasibility of unbiased quantitative and reproducible assessment of treatment targets on CTCs, opening a path towards personalized treatment.

Circulating tumor cells in small-cell lung cancer: a predictive and prognostic factor.

BACKGROUND: Initial response of small-cell lung cancer (SCLC) to chemotherapy is high, and recurrences occur frequently, leading to early death. This study investigated the prognostic value of circulating tumor cells (CTCs) in patients with SCLC and whether changes in CTCs can predict response to chemotherapy. Patients and methods In this multicenter prospective study, blood samples for CTC analysis were obtained from 59 patients with SCLC before, after one cycle, and at the end of chemotherapy. CTCs were measured using CellSearch systems. RESULTS: At baseline, lower numbers of CTCs were observed for 21 patients with limited SCLC (median = 6, range 0-220) compared with 38 patients with extensive stage (median = 63, range 0-14,040). Lack of measurable CTCs (27% of patients) was associated with prolonged survival (HR 3.4; P ≤ 0.001). CTCs decreased after one cycle of chemotherapy; this decrease was not associated with tumor response after four cycles of chemotherapy. CTC count after the first cycle of chemotherapy was the strongest predictor for overall survival (HR 5.7; 95% CI 1.7-18.9; P = 0.004). CONCLUSION: Absolute CTCs after one cycle of chemotherapy in patients with SCLC is the strongest predictor for response on chemotherapy and survival. Patients with low initial CTC numbers lived longer than those with higher CTCs.

Quantitative detection of gold nanoparticles on individual, unstained cancer cells by scanning electron microscopy.

Gold nanoparticles are rapidly emerging for use in biomedical applications. Characterization of the interaction and delivery of nanoparticles to cells through microscopy is important. Scanning electron microscopes have the intrinsic resolution to visualize gold nanoparticles on cells. A novel sample preparation protocol was developed to enable imaging of cells and gold nanoparticles with a conventional below lens scanning electron microscopes. The negative influence of 'charging' on the quality of scanning electron microscopes' images could be limited by deposition of biological cells on a conductive (gold) surface. The novel protocol enabled high-resolution scanning electron microscopes' imaging of small clusters and individual gold nanoparticles on uncoated cell surfaces. Gold nanoparticles could be counted on cancer cells with automated routines.

Circulating melanoma cells and survival in metastatic melanoma.

A validated assay for the enumeration of circulating melanoma cells (CMCs) may facilitate the development of more effective therapies for metastatic melanoma patients. In this study CD146+ cells were immunomagnetically enriched from 7.5 ml of blood. Isolated cells were fluorescently stained with DAPI, anti-molecular weight melanoma-associated antigen (HMW-MAA), anti-CD45 and CD34 and Ki67. CMCs were identified as CD146+, HMW-MAA+, CD45-, CD34-, Ki67-/+ cells. Eighty-eight percent of spiked SK-MEL28 cells in 7.5 ml blood were recovered. In all 55 healthy donors ≤1 CMCs were detected in 7.5 ml of blood. A retrospective analysis was conducted comparing CMC counts and overall survival in 79 blood samples from 44 melanoma patients. CMCs ranged from 0 to 8,042 per 7.5 ml. Two or more CMCs were detected in 18 (23%) of the patients and 30-100% (mean 84%) of the CMCs expressed the proliferation marker Ki67. Patients with ≥2 CMCs per 7.5 ml of whole blood, as compared with the group with <2 CMCs, had a shorter overall survival (2.0 months vs. 12.1 months, P=0.001).

Circulating endothelial cells, circulating tumour cells, tissue factor, endothelin-1 and overall survival in prostate cancer patients treated with docetaxel.

PURPOSE: We investigated whether serum markers of angiogenesis endothelin-1 (ET-1) and tissue factor (TF), and/or markers of vascular damage such as circulating endothelial cells (CECs), or their relative changes during treatment, were prognostic for overall survival (OS) in castration resistant prostate cancer (CRPC) patients. Additionally, we combined these markers with circulating tumour cells (CTCs) to construct a predictive nomogram for treatment outcome. PATIENTS AND METHODS: One hundred and sixty two CRPC patients treated with a docetaxel containing regimen had blood drawn before and at 2-5 weeks and 6-8 weeks after treatment start. Prospectively determined CTC and CEC levels, and retrospectively measured serum concentrations of ET-1 (pg/mL) and TF (pg/mL) were evaluated to determine their prognostic value for OS. RESULTS: Baseline CEC, TF and ET-1 were not prognostic for OS. A > or = 3.8-fold increase in CEC 2-5 weeks after treatment initiation was associated with decreased OS (median 10.9 versus 16.8 months; P=0.015), as was any decrease in TF levels compared to baseline levels (median 11.9 versus 21.5 months; P=0.0005). As previously published, baseline and CTC counts > or = 5 at 2-5 weeks were also predictive of decreased OS. Combining CTC with changes in TF and CEC 2-5 weeks after treatment initiation yielded four groups differing in OS (median OS 24.2 versus 16.0 versus 11.4 versus 6.1 months; P<0.0001). CONCLUSION: CEC, CTC and TF levels alone and combined can predict early on OS in CRPC patients treated with docetaxel-based therapy. A prospective study to confirm the use of these markers for patient management is needed.

All circulating EpCAM+CK+CD45- objects predict overall survival in castration-resistant prostate cancer.

BACKGROUND: Presence of five or more circulating tumor cells (CTC) in patients with metastatic carcinomas is associated with poor survival. Although many objects positive for epithelial cell adhesion molecules and cytokeratin (EpCAM+CK+) are not counted as CTC, they may be an important predictor for survival. We evaluated the association between these objects and survival in patients with prostate cancer. PATIENTS AND METHODS: Included in this follow-up study were 179 patients with castration-resistant prostate cancer. CellSearch was used to isolate EpCAM+ objects and to stain DNA, cytokeratin and CD45. All EpCAM+CK+ objects were subdivided into seven classes on the basis of predefined morphological appearance in 63 independent samples. Association of each class with survival was studied using Kaplan-Meier and Cox regression analyses. RESULTS: Each EpCAM+CK+CD45- class showed a strong association with overall survival (P < 0.001). This included small tumor microparticles (S-TMP), which did not require a nucleus and thus are unable to metastasize. A higher number of objects in any class was associated with decreased survival. A good prediction model included large tumor cell fragments (L-TCF), age, hemoglobin and lactate dehydrogenase. Models with S-TMP or CTC instead of L-TCF performed similarly. CONCLUSION: EpCAM+CK+CD45- that do not meet strict definitions for CTC are strong prognostic markers for survival.

Circulating tumour cells early predict progression-free and overall survival in advanced colorectal cancer patients treated with chemotherapy and targeted agents.

BACKGROUND: Early predictive markers for response are needed for advanced colorectal cancer (ACC) patients. We assessed the value of circulating tumour cells (CTC) in ACC patients treated with chemotherapy plus targeted agents (CAIRO2 phase III trial) and compared the results with computed tomography (CT) imaging. MATERIALS AND METHODS: CTC were determined at baseline and at different time points during treatment. Patients were stratified into low (less than three CTC per 7.5 ml of blood) or high CTC (three or more CTC per 7.5 ml of blood). RESULTS: A total of 467 patients were assessable for CTC analysis. Among them, 129 patients (29%) with high baseline CTC had a significantly decreased progression-free survival [PFS; hazard ratio (HR) 1.5] and overall survival (OS; HR 2.2) compared with 322 patients with low baseline CTC. This difference remained statistically significant during treatment. The sensitivity and specificity of high CTC at baseline for the prediction of progressive disease on CT imaging were 16.7% and 70.1%, respectively, and of high CTC at 1-2 weeks after the start of treatment 20.0% and 95.1%, respectively. The combined analysis of CTC and CT imaging provided a more accurate outcome assessment than either modality alone. CONCLUSIONS: The CTC count before and during treatment independently predicts PFS and OS in ACC patients treated with chemotherapy plus targeted agents and provides additional information to CT imaging.

Prognostic significance of circulating tumor cells in patients with metastatic colorectal cancer.

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 <3 CTCs/7.5 ml, respectively. Subgroups were analyzed by line of treatment, liver involvement, receipt of oxaliplatin, irinotecan, or bevacizumab, age, and Eastern Cooperative Oncology Group performance status (ECOG PS). RESULTS: Seventy-one percent of deaths have occurred. Median follow-up for living patients is 25.8 months. For all patients, progression-free survival (PFS) and overall survival (OS) for unfavorable compared with favorable baseline CTCs is shorter (4.4 versus 7.8 m, P = 0.004 for PFS; 9.4 versus 20.6 m, P < 0.0001 for OS). In all patient subgroups, unfavorable baseline CTC was associated with inferior OS (P < 0.001). In patients receiving first- or second-line therapy (P = 0.003), irinotecan (P = 0.0001), having liver involvement (P = 0.002), >/=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.

Circulating tumour cell (CTC) counts as intermediate end points in castration-resistant prostate cancer (CRPC): a single-centre experience.

BACKGROUND: The purpose of this study was to evaluate the association of circulating tumour cell (CTC) counts, before and after commencing treatment, with overall survival (OS) in patients with castration-resistant prostate cancer (CRPC). EXPERIMENTAL DESIGN: A 7.5 ml of blood was collected before and after treatment in 119 patients with CRPC. CTCs were enumerated using the CellSearchSystem. RESULTS: Higher CTC counts associated with baseline characteristics portending aggressive disease. Multivariate analyses indicated that a CTC >or=5 was an independent prognostic factor at all time points evaluated. Patients with baseline CTC >or=5 had shorter OS than those with <5 [median OS 19.5 versus >30 months, hazard ratio (HR) 3.25, P=0.012]; patients with CTC >50 had a poorer OS than those with CTCs 5-50 (median OS 6.3 versus 21.1 months, HR 4.1, P<0.001). Patients whose CTC counts reduced from >or=5 at baseline to <5 following treatment had a better OS compared with those who did not. CTC counts showed a similar, but earlier and independent, ability to time to disease progression to predict OS. CONCLUSION: CTC counts predict OS and provide independent prognostic information to time to disease progression; CTC dynamics following therapy need to be evaluated as an intermediate end point of outcome in randomised phase III trials.

Monitoring expression of HER-2 on circulating epithelial cells in patients with advanced breast cancer.

Nineteen breast cancer patients with measurable metastatic disease who were starting an initial or new line of therapy were evaluated for circulating epithelial cells (CECs) a minimum of 4 times over the course of treatment. In 7 of the 10 CEC+ patients, HER-2 expression was detected on the CECs. CECs expressing HER-2 varied among patients and in serial samples from the same patient including a shift from HER-2- to HER-2+ CECs. These results demonstrate that it is possible to quantify receptors essential for rationally designed therapy using CECs and that reliance on the immunophenotype of the primary tumor can be misleading.