Defining the dimensions of circulating tumor cells in a large series of breast, prostate, colon, and bladder cancer patients.

Circulating tumor cells (CTCs) in the blood of cancer patients are of high clinical relevance. Since detection and isolation of CTCs often rely on cell dimensions, knowledge of their size is key. We analyzed the median CTC size in a large cohort of breast (BC), prostate (PC), colorectal (CRC), and bladder (BLC) cancer patients. Images of patient-derived CTCs acquired on cartridges of the FDA-cleared CellSearch® method were retrospectively collected and automatically re-analyzed using the accept software package. The median CTC diameter (µm) was computed per tumor type. The size differences between the different tumor types and references (tumor cell lines and leukocytes) were nonparametrically tested. A total of 1962 CellSearch® cartridges containing 71 612 CTCs were included. In BC, the median computed diameter (CD) of patient-derived CTCs was 12.4 µm vs 18.4 µm for cultured cell line cells. For PC, CDs were 10.3 µm for CTCs vs 20.7 µm for cultured cell line cells. CDs for CTCs of CRC and BLC were 7.5 µm and 8.6 µm, respectively. Finally, leukocytes were 9.4 µm. CTC size differed statistically significantly between the four tumor types and between CTCs and the reference data. CTC size differences between tumor types are striking and CTCs are smaller than cell line tumor cells, whose size is often used as reference when developing CTC analysis methods. Based on our data, we suggest that the size of CTCs matters and should be kept in mind when designing and optimizing size-based isolation methods.

HER2 expression on tumor-derived extracellular vesicles and circulating tumor cells in metastatic breast cancer.

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.

Tumour-derived extracellular vesicles in blood of metastatic cancer patients associate with overall survival.

BACKGROUND: Circulating tumour cells (CTCs) in blood associate with overall survival (OS) of cancer patients, but they are detected in extremely low numbers. Large tumour-derived extracellular vesicles (tdEVs) in castration-resistant prostate cancer (CRPC) patients are present at around 20 times higher frequencies than CTCs and have equivalent prognostic power. In this study, we explored the presence of tdEVs in other cancers and their association with OS. METHODS: The open-source ACCEPT software was used to automatically enumerate tdEVs in digitally stored CellSearch® images obtained from previously reported CTC studies evaluating OS in 190 CRPC, 450 metastatic colorectal cancer (mCRC), 179 metastatic breast cancer (MBC) and 137 non-small cell lung cancer (NSCLC) patients before the initiation of a new treatment. RESULTS: Presence of unfavourable CTCs and tdEVs is predictive of OS, with respective hazard ratios (HRs) of 2.4 and 2.2 in CRPC, 2.7 and 2.2 in MBC, 2.3 and 1.9 in mCRC and 2.0 and 2.4 in NSCLC, respectively. CONCLUSIONS: tdEVs have equivalent prognostic value as CTCs in the investigated metastatic cancers. CRPC, mCRC, and MBC (but not NSCLC) patients with favourable CTC counts can be further prognostically stratified using tdEVs. Our data suggest that tdEVs could be used in clinical decision-making.

Leukocyte-Derived Extracellular Vesicles in Blood with and without EpCAM Enrichment.

Large tumor-derived Extracellular Vesicles (tdEVs) detected in blood of metastatic prostate, breast, colorectal, and non-small cell lung cancer patients after enrichment for Epithelial Cell Adhesion Molecule (EpCAM) expression and labeling with 4',6-diamidino-2-phenylindole (DAPI), phycoerythrin-conjugated antibodies against Cytokeratins (CK-PE), and allophycocyanin-conjugated antibody against the cluster of differentiation 45 (CD45-APC), are negatively associated with the overall survival of patients. Here, we investigated whether, similarly to tdEVs, leukocyte-derived EVs (ldEVs) could also be detected in EpCAM-enriched blood. Presence of ldEVs and leukocytes in image data sets of EpCAM-enriched samples of 25 healthy individuals and 75 metastatic cancer patients was evaluated using the ACCEPT software. Large ldEVs could indeed be detected, but in contrast to the 20-fold higher frequency of tdEVs as compared to Circulating Tumor Cells (CTCs), ldEVs were present in a 5-fold lower frequency as compared to leukocytes. To evaluate whether these ldEVs pre-exist in the blood or are formed during the CellSearch procedure, the blood of healthy individuals without EpCAM enrichment was labelled with the nuclear dye Hoechst and fluorescently tagged monoclonal antibodies recognizing the leukocyte-specific CD45, platelet-specific CD61, and red blood cell-specific CD235a. Fluorescence microscopy imaging using a similar setup as the CellSearch was performed and demonstrated the presence of a similar population of ldEVs present at a 3-fold lower frequency as compared to leukocytes.

A microwell array platform to print and measure biomolecules produced by single cells.

Here we describe a combined method to monitor the secretion of molecules produced by single cells, followed by a method to isolate the individual cells that produced these molecules. The method is based on a self-sorting microwell chip that is connected to an activated membrane that collects the produced molecules. The produced molecules are printed by diffusion in small spots onto the membrane. The location of the printed spots can be correlated to the microwell number and the cell that produced these molecules. To demonstrate the method, we used the EpCAM antibody producing hybridoma cell line VU1D9 and a genetically engineered CHO cell-line producing Her2. VU1D9 cells produced 4.6 ± 5.6 pg (mean ± SD) of EpCAM antibody per 24 h and CHO cells 6.5 ± 8.2 pg per 24 h of Herceptin antibody.

Single tube liquid biopsy for advanced non-small cell lung cancer.

The need for a liquid biopsy in non-small cell lung cancer (NSCLC) patients is rapidly increasing. We studied the relation between overall survival (OS) and the presence of four cancer biomarkers from a single blood draw in advanced NSCLC patients: EpCAMhigh circulating tumor cells (CTC), EpCAMlow CTC, tumor-derived extracellular vesicles (tdEV) and cell-free circulating tumor DNA (ctDNA). EpCAMhigh CTC were detected with CellSearch, tdEV in the CellSearch images and EpCAMlow CTC with filtration after CellSearch. ctDNA was isolated from plasma and mutations present in the primary tumor were tracked with deep sequencing methods. In 97 patients, 21% had ≥2 EpCAMhigh CTC, 15% had ≥2 EpCAMlow CTC, 27% had ≥18 tdEV and 19% had ctDNA with ≥10% mutant allele frequency. Either one of these four biomarkers could be detected in 45% of the patients and all biomarkers were present in 2%. In 11 out of 16 patients (69%) mutations were detected in the ctDNA. Two or more unfavorable biomarkers were associated with poor OS. The presence of EpCAMhigh CTC and elevated levels of tdEV and ctDNA was associated with a poor OS; however, the presence of EpCAMlow CTC was not. This single tube approach enables simultaneous analysis of multiple biomarkers to explore their potential as a liquid biopsy.

Classification of Cells in CTC-Enriched Samples by Advanced Image Analysis.

In the CellSearch® system, blood is immunomagnetically enriched for epithelial cell adhesion molecule (EpCAM) expression and cells are stained with the nucleic acid dye 4'6-diamidino-2-phenylindole (DAPI), Cytokeratin-PE (CK), and CD45-APC. Only DAPI+/CK+ objects are presented to the operator to identify circulating tumor cells (CTC) and the identity of all other cells and potential undetected CTC remains unrevealed. Here, we used the open source imaging program Automatic CTC Classification, Enumeration and PhenoTyping (ACCEPT) to analyze all DAPI+ nuclei in EpCAM-enriched blood samples obtained from 192 metastatic non-small cell lung cancer (NSCLC) patients and 162 controls. Significantly larger numbers of nuclei were detected in 300 patient samples with an average and standard deviation of 73,570 ± 74,948, as compared to 359 control samples with an average and standard deviation of 4191 ± 4463 (p < 0.001). In patients, only 18% ± 21% and in controls 23% ± 15% of the nuclei were identified as leukocytes or CTC. Adding CD16-PerCP for granulocyte staining, the use of an LED as the light source for CD45-APC excitation and plasma membrane staining obtained with wheat germ agglutinin significantly improved the classification of EpCAM-enriched cells, resulting in the identification of 94% ± 5% of the cells. However, especially in patients, the origin of the unidentified cells remains unknown. Further studies are needed to determine if undetected EpCAM+/DAPI+/CK-/CD45- CTC is present among these cells.

How to Agree on a CTC: Evaluating the Consensus in Circulating Tumor Cell Scoring.

For using counts of circulating tumor cells (CTCs) in the clinic to aid a physician's decision, its reported values will need to be accurate and comparable between institutions. Many technologies have become available to enumerate and characterize CTCs, thereby showing a large range of reported values. Here we introduce an Open Source CTC scoring tool to enable comparison of different reviewers and facilitate the reach of a consensus on assigning objects as CTCs. One hundred images generated from two different platforms were used to assess concordance between 15 reviewers and an expert panel. Large differences were observed between reviewers in assigning objects as CTCs urging the need for computer recognition of CTCs. A demonstration of a deep learning approach on the 100 images showed the promise of this technique for future CTC enumeration. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Toward a real liquid biopsy in metastatic breast and prostate cancer: Diagnostic LeukApheresis increases CTC yields in a European prospective multicenter study (CTCTrap).

Frequently, the number of circulating tumor cells (CTC) isolated in 7.5 mL of blood is too small to reliably determine tumor heterogeneity and to be representative as a "liquid biopsy". In the EU FP7 program CTCTrap, we aimed to validate and optimize the recently introduced Diagnostic LeukApheresis (DLA) to screen liters of blood. Here we present the results obtained from 34 metastatic cancer patients subjected to DLA in the participating institutions. About 7.5 mL blood processed with CellSearch® was used as "gold standard" reference. DLAs were obtained from 22 metastatic prostate and 12 metastatic breast cancer patients at four different institutions without any noticeable side effects. DLA samples were prepared and processed with different analysis techniques. Processing DLA using CellSearch resulted in a 0-32 fold increase in CTC yield compared to processing 7.5 mL blood. Filtration of DLA through 5 µm pores microsieves was accompanied by large CTC losses. Leukocyte depletion of 18 mL followed by CellSearch yielded an increase of the number of CTC but a relative decrease in yield (37%) versus CellSearch DLA. In four out of seven patients with 0 CTC detected in 7.5 mL of blood, CTC were detected in DLA (range 1-4 CTC). The CTC obtained through DLA enables molecular characterization of the tumor. CTC enrichment technologies however still need to be improved to isolate all the CTC present in the DLA.

Circulating tumor cells, tumor-derived extracellular vesicles and plasma cytokeratins in castration-resistant prostate cancer patients.

PURPOSE: The presence of Circulating Tumor Cells (CTCs) in Castration-Resistant Prostate Cancer (CRPC) patients is associated with poor prognosis. In this study, we evaluated the association of clinical outcome in 129 CRPC patients with CTCs, tumor-derived Extracellular Vesicles (tdEVs) and plasma levels of total (CK18) and caspase-cleaved cytokeratin 18 (ccCK18). EXPERIMENTAL DESIGN: CTCs and tdEVs were isolated with the CellSearch system and automatically enumerated. Cut-off values dichotomizing patients into favorable and unfavorable groups of overall survival were set on a retrospective data set of 84 patients and validated on a prospective data set of 45 patients. Plasma levels of CK18 and ccCK18 were assessed by ELISAs. RESULTS: CTCs, tdEVs and both cytokeratin plasma levels were significantly increased in CRPC patients compared to healthy donors (HDs). All biomarkers except for ccCK18 were prognostic showing a decreased median overall survival for the unfavorable groups of 9.2 vs 21.1, 8.1 vs 23.0 and 10.0 vs 21.5 months respectively. In multivariable Cox regression analysis, tdEVs remained significant. CONCLUSIONS: Automated CTC and tdEV enumeration allows fast and reliable scoring eliminating inter- and intra- operator variability. tdEVs provide similar prognostic information to CTC counts.

Quantifying HER-2 expression on circulating tumor cells by ACCEPT.

Circulating tumor cells (CTCs) isolated from blood can be probed for the expression of treatment targets. Immunofluorescence is often used for both the enumeration of CTC and the determination of protein expression levels related to treatment targets. Accurate and reproducible assessment of such treatment target expression levels is essential for their use in the clinic. To enable this, an open source image analysis program named ACCEPT was developed in the EU-FP7 CTCTrap and CANCER-ID programs. Here its application is shown on a retrospective cohort of 132 metastatic breast cancer patients from which blood samples were processed by CellSearch® and stained for HER-2 expression as additional marker. Images were digitally stored and reviewers identified a total of 4084 CTCs. CTC's HER-2 expression was determined in the thumbnail images by ACCEPT. 150 of these images were selected and sent to six independent investigators to score the HER-2 expression with and without ACCEPT. Concordance rate of the operators' scoring results for HER-2 on CTCs was 30% and could be increased using the ACCEPT tool to 51%. Automated assessment of HER-2 expression by ACCEPT on 4084 CTCs of 132 patients showed 8 (6.1%) patients with all CTCs expressing HER-2, 14 (10.6%) patients with no CTC expressing HER-2 and 110 (83.3%) patients with CTCs showing a varying HER-2 expression level. In total 1576 CTCs were determined HER-2 positive. We conclude that the use of image analysis enables a more reproducible quantification of treatment targets on CTCs and leads the way to fully automated and reproducible approaches.