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BACKGROUND: Circulating tumour cells (CTCs) can be used to monitor cancer longitudinally, but their use in non-small cell lung cancer (NSCLC) is limited due to low numbers in the peripheral blood. Through diagnostic leukapheresis (DLA) CTCs can be obtained from larger blood volumes.METHODS: Patients with all stages of NSCLC were selected. One total body blood volume was screened by DLA before and after treatment. Peripheral blood was drawn pre- and post DLA for CTC enumeration by CellSearch. CTCs were detected in the DLA product (volume equalling 2 × 108 leucocytes) and after leucocyte depletion (RosetteSep, 9 mL DLA product). Single-cell, whole-genome sequencing was performed on isolated CTCs.RESULTS: Fifty-six patients were included. Before treatment, CTCs were more often detected in DLA (32/55, 58%) than in the peripheral blood (pre-DLA: 18/55, 33%; post DLA: 13/55, 23%, both at p CONCLUSIONS: DLA detected nine times more CTCs than in the peripheral blood. The sustained presence of CTCs in DLA after treatment was associated with therapy failure and shortened PFS.TRIAL REGISTRATION: The study was approved by the Medical Ethical Committee (NL55754.042.15) and was registered in the Dutch trial register (NL5423).

Background: Circulating tumor cells (CTC) in non-small cell lung cancer (NSCLC) patients are a prognostic and possible therapeutic marker, but have a low frequency of appearance. Diagnostic leukapheresis (DLA) concentrates CTC and mononuclear cells from the blood. We evaluated a protocol using two VyCAP microsieves to filter DLA product of NSCLC patients and enumerate CTC, compared with CellSearch as a gold standard. Methods: DLA was performed in NSCLC patients before starting treatment. DLA product equaling 2×108 leukocytes was diluted to 9 mL with CellSearch dilution buffer in a Transfix CTC tube. Within 72 hours the sample was filtered with a 7 μm pore microsieve and subsequently over a 5μm pore microsieve. CTC were defined as nucleated cells which stained for cytokeratin, but lacked CD45 and CD16. CellSearch detected CTC in the same volume of DLA. Results: Of 29 patients a median of 1.4 mL DLA product (range, 0.5-4.1) was filtered (2% of total product) successfully in 93% and 45% of patients using 7 and 5 μm pores, respectively. Two DLA products were unevaluable for CTC detection. Clogging of the 5 μm but not 7 μm microsieves was positively correlated with fixation time (ρ=0.51, P

Background Multiple technologies are available for detection of circulating tumor cells (CTCs), but standards to evaluate their technical performance are still lacking. This limits the applicability of CTC analysis in clinic routine. Therefore, in the context of the CANCER-ID consortium, we established a platform to assess technical validity of CTC detection methods in a European multi-center setting using non-small cell lung cancer (NSCLC) as a model. Methods We characterized multiple NSCLC cell lines to define cellular models distinct in their phenotype and molecular characteristics. Standardized tumor-cell-bearing blood samples were prepared at a central laboratory and sent to multiple European laboratories for processing according to standard operating procedures. The data were submitted via an online tool and centrally evaluated. Five CTC-enrichment technologies were tested. Results We could identify 2 cytokeratin expressing cell lines with distinct levels of EpCAM expression: NCI-H441 (EpCAMhigh, CKpos) and NCI-H1563 (EpCAMlow, CKpos). Both spiked tumor cell lines were detected by all technologies except for the CellSearch system that failed to enrich EpCAMlow NCI-H1563 cells. Mean recovery rates ranged between 49% and 75% for NCI-H411 and 32% and 76% for NCI-H1563 and significant differences were observed between the tested methods. Conclusions This multi-national proficiency testing of CTC-enrichment technologies has importance in the establishment of guidelines for clinically applicable (pre)analytical workflows and the definition of minimal performance qualification requirements prior to clinical validation of technologies. It will remain in operation beyond the funding period of CANCER-ID in the context of the European Liquid Biopsy Society (ELBS).

Circulating tumor cells (CTCs) detected by CellSearch are prognostic in non-small-cell lung cancer (NSCLC), but rarely found. CTCs can be extracted from the blood together with mononuclear cell populations by diagnostic leukapheresis (DLA), therefore concentrating them. However, CellSearch can only process limited DLA volumes (&asymp, 2 mL). Therefore, we established a protocol to enumerate CTCs in DLA products with Isolation by SizE of Tumor cells (ISET), and compared CTC counts between CellSearch&reg, and ISET. DLA was performed in NSCLC patients who started a new therapy. With an adapted protocol, ISET could process 10 mL of DLA. CellSearch detected CTCs in a volume equaling 2 &times, 108 leukocytes (mean 2 mL). CTC counts per mL were compared. Furthermore, the live cell protocol of ISET was tested in eight patients. ISET successfully processed all DLA products&mdash, 16 with the fixed cell protocol and 8 with the live cell protocol. In total, 10&ndash, 20 mL of DLA was processed. ISET detected CTCs in 88% (14/16), compared to 69% (11/16, p &lt, 0.05) with CellSearch. ISET also detected higher number of CTCs (ISET median CTC/mL = 4, interquartile range [IQR] = 2&ndash, 6, CellSearch median CTC/mL = 0.9, IQR = 0&ndash, 1.8, p &lt, 0.01). Cells positive for the epithelial cell adhesion molecule (EpCAM+) per mL were detected in similar counts by both methods. Eight patients were processed with the live cell protocol. All had EpCAM+, CD45&minus, CD235- cells isolated by fluorescence-activated cell sorting (FACS). Overall, ISET processed larger volumes and detected higher CTC counts compared to CellSearch. EpCAM+ CTCs were detected in comparable rates.

Purpose: Tumor cells from patients with lung cancer are expelled from the primary tumor into the blood, but difficult to detect in the peripheral circulation. We studied the release of circulating tumor cells (CTCs) during surgery to test the hypothesis that CTC counts are influenced by hemodynamic changes (caused by surgical approach) and manipulation. Experimental Design: Patients undergoing video-assisted thoracic surgery (VATS) or open surgery for (suspected) primary lung cancer were included. Blood samples were taken before surgery (T0) from the radial artery (RA), from both the RA and pulmonary vein (PV) when the PV was located (T1) and when either the pulmonary artery (T2 open) or the PV (T2 VATS) was dissected. The CTCs were enumerated using the CellSearch system. Single-cell whole-genome sequencing was performed on isolated CTCs for aneuploidy. Results: CTCs were detected in 58 of 138 samples (42%) of 31 patients. CTCs were more often detected in the PV (70%) compared with the RA (22%, P < 0.01) and in higher counts (P < 0.01). After surgery, the RA but not the PV showed less often CTCs (P ¼ 0.02). Type of surgery did not influence CTC release. Only six of 496 isolated CTCs showed aneuploidy, despite matched primary tumor tissue being aneuploid. Euploid so-called CTCs had a different morphology than aneuploid. Conclusions: CTCs defined by CellSearch were identified more often and in higher numbers in the PV compared with the RA, suggesting central clearance. The majority of cells in the PV were normal epithelial cells and outnumbered CTCs. Release of CTCs was not influenced by surgical approach.

Circulating tumour cells (CTCs) found in the blood of cancer patients are a promising biomarker in precision medicine. However, their use is currently hindered by their low frequency, tedious manual scoring and extensive cell heterogeneities. Those challenges limit the effectiveness of classical machine-learning methods for automated CTC analysis. Here, we combine autoencoding convolutional neural networks with advanced visualization techniques. This provides a very informative view on the data that opens the way for new biomedical research questions. We unravel hidden information in the raw image data of fluorescent images of blood samples enriched for CTCs. Our network classifies fluorescent images of single cells in five different classes with an accuracy, sensitivity and specificity of over 96%, and the obtained CTC counts predict the overall survival of cancer patients as well as state-of-the-art manual counts. Moreover, our network excelled in identifying different important subclasses of objects. Deep learning was faster and superior to classical image analysis approaches and enabled the identification of new biological phenomena. Counting different types of circulating tumour cells can give valuable information on the severity of the disease and on whether treatments are effective for a specific patient. In this work, the authors show that their method based on autoencoders can identify and count cells more accurately and faster than human experts.

Purpose: Tumor cells from patients with lung cancer are expelled from the primary tumor into the blood, but difficult to detect in the peripheral circulation. We studied the release of circulating tumor cells (CTCs) during surgery to test the hypothesis that CTC counts are influenced by hemodynamic changes (caused by surgical approach) and manipulation. Experimental Design: Patients undergoing video-assisted thoracic surgery (VATS) or open surgery for (suspected) primary lung cancer were included. Blood samples were taken before surgery (T0) from the radial artery (RA), from both the RA and pulmonary vein (PV) when the PV was located (T1) and when either the pulmonary artery (T2 open) or the PV (T2 VATS) was dissected. The CTCs were enumerated using the CellSearch system. Single-cell whole-genome sequencing was performed on isolated CTCs for aneuploidy. Results: CTCs were detected in 58 of 138 samples (42%) of 31 patients. CTCs were more often detected in the PV (70%) compared with the RA (22%, P < 0.01) and in higher counts (P < 0.01). After surgery, the RA but not the PV showed less often CTCs (P = 0.02). Type of surgery did not influence CTC release. Only six of 496 isolated CTCs showed aneuploidy, despite matched primary tumor tissue being aneuploid. Euploid so-called CTCs had a different morphology than aneuploid. Conclusions: CTCs defined by CellSearch were identified more often and in higher numbers in the PV compared with the RA, suggesting central clearance. The majority of cells in the PV were normal epithelial cells and outnumbered CTCs. Release of CTCs was not influenced by surgical approach.

Introduction: Circulating tumor cells (CTC) can be used to monitor malignant disease longitudinally, but their use in non-small cell lung cancer (NSCLC) is limited due to low numbers in peripheral blood. Through Diagnostic leukapheresis (DLA) CTC can be obtained from larger blood volumes. We studied CTC in DLA product of NSCLC patients before and after treatment. Methods: One total blood volume was screened by DLA before and 1-3 months after treatment. Peripheral blood was drawn pre and post DLA for CTC enumeration by CellSearch. CTC were detected in DLA product directly (volume equaling 2×10^8 leukocytes) and after leukocyte depletion (RosetteSep, 9mL DLA product). Single cell whole genome sequencing was performed on isolated CTC. Results: Before treatment, CTC were more often detected in DLA (32/55, 58%) compared to blood (pre: 18/55, 33%, p

The availability of viable tumor cells could significantly improve the disease management of cancer patients. Here we developed and evaluated a method using self-seeding microwells to obtain single circulating tumor cells (CTC) and assess their potential to expand. Conditions were optimized using cells from the breast cancer cell line MCF-7 and blood from healthy volunteers collected in EDTA blood collection tubes. 43% of the MCF-7 cells (nucleus+, Ethidium homodimer-1-, Calcein AM+, &alpha, EpCAM+, &alpha, CD45-) spiked into 7.5 mL of blood could be recovered with 67% viability and these could be further expanded. The same procedure tested in metastatic breast and prostate cancer patients resulted in a CTC recovery of only 0&ndash, 5% as compared with CTC counts obtained with the CellSearch&reg, system. Viability of the detected CTC ranged from 0&ndash, 36%. Cell losses could be mainly contributed to the smaller size and greater flexibility of CTC as compared to cultured cells from cell lines and loss during leukocyte depletion prior to cell seeding. Although CTC losses can be reduced by fixation, to obtain viable CTC no fixatives can be used and pore size in the bottom of microwells will need to be reduced, filtration conditions adapted and pre-enrichment improved to reduce CTC losses.

Purpose: Circulating tumor cells (CTCs) have clinical relevance, but their study has been limited by their low frequency.Experimental Design: We evaluated liquid biopsies by apheresis to increase CTC yield from patients suffering from metastatic prostate cancer, allow precise gene copy-number calls, and study disease heterogeneity.Results: Apheresis was well tolerated and allowed the separation of large numbers of CTCs; the average CTC yield from 7.5 mL of peripheral blood was 167 CTCs, whereas the average CTC yield per apheresis (mean volume: 59.5 mL) was 12,546 CTCs. Purified single CTCs could be isolated from apheresis product by FACS sorting; copy-number aberration (CNA) profiles of 185 single CTCs from 14 patients revealed the genomic landscape of lethal prostate cancer and identified complex intrapatient, intercell, genomic heterogeneity missed on bulk biopsy analyses.Conclusions: Apheresis facilitated the capture of large numbers of CTCs noninvasively with minimal morbidity and allowed the deconvolution of intrapatient heterogeneity and clonal evolution. Clin Cancer Res; 24(22); 5635–44. ©2018 AACR.

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

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., What's new? Circulating tumor cells (CTC) can mirror tumor heterogeneity but a standard blood sample (7.5 mL) is too small to truly represent the tumor. To increase the yield of CTC, the authors used Diagnostic LeukApheresis in which liters of blood are screened for the presence of CTC in metastatic cancer patients. They report a significant increase in CTC yield and consequently, a better molecular characterization of the tumor, encouraging further research into the use of leukapheresis as “liquid biopsy” in cancer patients.

The values of the affinity constants (kd, ka, and KD) that are determined by label-free interaction analysis methods are affected by the ligand density. This article outlines a surface plasmon resonance (SPR) imaging method that yields high-throughput globally fitted affinity ranking values using a 96-plex array. A kinetic titration experiment without a regeneration step has been applied for various coupled antibodies binding to a single antigen. Globally fitted rate (kd and ka) and dissociation equilibrium (KD) constants for various ligand densities and analyte concentrations are exponentially interpolated to the KD at Rmax = 100 RU response level (KD(R100)).

Contains fulltext : 170243.pdf (Publisher’s version ) (Closed access) The antileukemic drug asparaginase, a key component in the treatment of acute lymphoblastic leukemia, acts by depleting asparagine from the blood. However, little is known about its pharmacokinetics, and mechanisms of therapy resistance are poorly understood. Here, we explored the in vivo biodistribution of radiolabeled asparaginase, using a combination of imaging and biochemical techniques, and provide evidence for tissue-specific clearance mechanisms, which could reduce the effectiveness of the drug at these specific sites. METHODS: In vivo localization of 111In-labeled Escherichia coli asparaginase was performed in C57BL/6 mice by both small-animal SPECT/CT and ex vivo biodistribution studies. Mice were treated with liposomal clodronate to investigate the effect of macrophage depletion on tracer localization and drug clearance in vivo. Moreover, macrophage cell line models RAW264.7 and THP-1, as well as knockout mice, were used to identify the cellular and molecular components controlling asparaginase pharmacokinetics. RESULTS: In vivo imaging and biodistribution studies showed a rapid accumulation of asparaginase in macrophage-rich tissues such as the liver, spleen, and in particular bone marrow. Clodronate-mediated depletion of phagocytic cells markedly prolonged the serum half-life of asparaginase in vivo and decreased drug uptake in these macrophage-rich organs. Immunohistochemistry and in vitro binding assays confirmed the involvement of macrophagelike cells in the uptake of asparaginase. We identified the activity of the lysosomal protease cathepsin B in macrophages as a rate-limiting factor in degrading asparaginase both in vitro and in vivo. CONCLUSION: We showed that asparaginase is rapidly cleared from the serum by liver-, spleen-, and bone marrow-resident phagocytic cells. As a consequence of this efficient uptake and protease-mediated degradation, particularly bone marrow-resident macrophages may provide a protective niche to leukemic cells.

The presence of tumor cells in blood is predictive of short survival in several cancers and their isolation and characterization can guide toward the use of more effective treatments. These circulating tumor cells (CTC) are, however, extremely rare and require a technology that is sufficiently sensitive and specific to identify CTC against a background of billions of blood cells. Immuno-capture of cells expressing the epithelial cell adhesion molecule (EpCAM) are frequently used to enrich CTC from blood. The choice of bio conjugation strategy and antibody clone is crucial for adequate cell capture but is poorly understood. In this study, we determined the binding affinity constants and epitope binding of the EpCAM antibodies VU1D-9, HO-3, EpAb3-5, and MJ-37 by surface plasmon resonance imaging (SPRi). Glass surfaces were coated using a poly(acrylic acid) based coating and functionalized with anti-EpCAM antibodies. Binding of cells from the breast carcinoma cell line (SKBR-3) to the functionalized surfaces were compared. Although EpAb3-5 displayed the highest binding affinity HO-3 captured the highest amount of cells. Hence we report differences in the performance of the different antibodies and more importantly that the choice of antibody to capture CTC should be based on multiple assays.

Introduction The number of circulating tumor cells (CTC) isolated by the FDA approved CellSearch (CS) system in 7.5 mL of blood of metastatic and non-metastatic non-small cell lung cancer (NSCLC) patients is low or absent. Processing a larger blood volume could yield a greater amount of CTC from these patients. An approach to probe larger volumes is using diagnostic leukapheresis (DLA) where the blood can be split into its components after which selective harvesting of mononuclear cells (MNC) including CTC can take place. Methods Patients with histologically proven NSCLC who either have been recently diagnosed or are eligible for new therapy can participate in this ongoing study. Patients had DLA for MNC collection derived from one total body blood volume. Before and after DLA, whole blood was drawn in a CellSave blood collection tube and analyzed by CS. The obtained DLA product was divided for analysis. 2x108 cells (on average 2mL of DLA product, representing ~100mL of blood) was diluted in 7.5 mL buffer, and analyzed by CS. 9 mL of the DLA product (on average 1x109 cells) was fixed and depleted of leukocytes using the RosetteSep CD45 depletion kit (Stemcell Technologies), allowing processing of a larger volume, followed by CS analysis. Results of molecular characterization of the detected CTC is pending. Results So far, a total of 8 DLA's were performed in 7 stage 4 NSCLC patients. Sample collection took place before (t=0) and/or after (t=1) treatment. Patients underwent DLA in about 110 minutes, at this time an average of 4860 mL of blood was processed. No complications occurred, no post-procedure complaints were recorded. Similar DLA products with an average of 95 mL were obtained. CTC counts for these patient samples are shown in the table below. CTC counts blood and DLAPatientt=#CTC pre blood#CTC post blood#CTC DLA (~2mL, 200x106 cells)#CTC depleted DLA (~9mL, 900x106 cells)101n/a114113020203514602031000040n/a04495000166000029020114 Conclusion DLA is a novel method to collect CTC from the blood. DLA can result in the detection of CTC where the use of 7.5mL of blood is not sufficient. Additionally, DLA shows an increase of CTC yield in patients with CTC detected in 7.5 mL of blood. Citation Format: Kiki C. Andree, Menno Tamminga, Anouk Mentink, T Jeroen Hilterman, Harry J. Groen, Leon W. Terstappen. Circulating tumor cells in the peripheral blood and leukapheresis product of non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5584.

Background: The rarity of in vivo and in vitro human prostate cancer (PCa) models has hampered progress in understanding disease pathogenesis, metastatic progression and drug resistance mechanisms. Using CTCs from a leukapheresis product of a patient with advanced PCa, we report the establishment of a CDX and an in vitro cell line derived from this CDX. The phenotypic and molecular characterization of patient tumor-biopsies, CTCs, CDX and CDX-derived cell-line are presented. Methods: Leukapheresis was performed in seven patients with advanced castration-resistant prostate cancer (CRPC). CTCs from the seven leukapheresis products were enriched by RosetteSep and implanted in Nod/Scid-IL2Rγ-/-mice. The CDX tumor was propagated in successive generations of mice. All samples, including eight tumor-biopsies performed at diagnosis two years prior leukapheresis and CTCs isolated at the single cell level during leukapheresis were characterized by immunofluorescence, immunohistochemistry, and whole-exome sequencing (WES). Results: Based on CellSearch® counts in leukapheresis products, the estimated median number of engrafted CTCs was 697 (range: 10-19988). A mouse engrafted with 19988 CTCs developed a tumor within 193 days. Immunohistochemistry performed on the CDX and two tumor-biopsies indicated that the CDX and biopsies were positive for EpCAM, CK5/6/8/18, negative for CK7 and vimentin, and weakly positive for synaptophysin. While biopsies expressed PSA and the androgen receptor, the CDX was negative for both indicating tumor evolution. In contrast to tumor biopsies, the CDX strongly expressed Ki67, NSE and chromogranin, evidencing emergence of a neuroendocrine phenotype. The in vitro cell line established by culturing dissociated CDX cells for five months, grew in microspheres and expressed epithelial and ALDH and CD133 cancer stem-cell markers. By WES, a high degree of intra-tumor heterogeneity was observed in the eight tumor biopsies and CTCs as already reported in this tumor type. Only 2.8% (58/2087) and 2.3% (49/2087) of the mutations present in the tumor biopsies were identified in CTCs and the CDX respectively, indicating that a very few number of mutations have the potential to support the dissemination and tumorigenic activity of CTC. Trunk mutations in TP53, NF1 and LRP1B genes were identified in all samples including the CDX while PTEN gene loss was acquired lately and detected only in CTCs and the CDX. Mutational similarity of the CDX and the in vitro cell line was 91%. The analysis of copy number variations is ongoing in all samples and will be presented. Conclusion: We report the first PCa CDX model, demonstrating the tumorigenicity of CTCs from CRPC. This CDX model represents a unique tool to identify clonal mutations associated with the tumor-initiating capacity of CTCs and explore the genetic and phenotypic basis of metastasis and drug resistance in advanced CRPC. Citation Format: Vincent Faugeroux, Emma Pailler, Olivier Deas, Virginie Marty, Kamélia Alexandrova, Kiki Andree, Jean-Yves Scoazec, Nikolas Stoecklein, Nicolo Manaresi, Dominique Tramalloni, Maud Ngo-Camus, Claudio Nicotra, Leon Terstappen, Valérie Lapierre, Karim Fizazi, Yohann Loriot, Jean-Gabriel Judde, Françoise Farace. Establishment and characterization of a unique circulating tumor cells-derived xenograft (CDX) in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5600.

Enumeration and characterization of circulating tumor cells (CTC) hold the promise of a real time liquid biopsy. They are however present in a large background of hematopoietic cells making their isolation technically challenging. In 2004, the CellSearch system was introduced as the first and only FDA cleared method designed for the enumeration of circulating tumor cells in 7.5 mL of blood. Presence of CTC detected by CellSearch is associated with poor prognosis in metastatic carcinomas. CTC remaining in patients after the first cycles of therapy indicates a futile therapy. Here we review challenges faced during the development of the CellSearch system and the difficulties in assigning objects as CTC. The large heterogeneity of CTC and the different approaches introduced in recent years to isolate, enumerate and characterize CTC results in a large variation of the number of CTC reported urging the need for uniform definitions and at least a clear definition of what the criteria are for assigning an object as a CTC.

Introduction Frequently the number of 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; doi: 10.1073/pnas.1313594110) approach to screen liters of blood and thereby substantially increasing the number of CTC available for further characterization. Here we present the results obtained from 32 metastatic cancer patients subjected to DLA in the participating institutions. Methods Before the DLA procedure, whole blood was drawn in a CellSave blood collection tube and a 7.5 ml aliquot was processed with the ‘gold standard’ reference CellSearch® (Janssen Diagnostics, USA). DLAs from metastatic cancer patients were performed for ≈90 minutes to obtain 40 mL of product containing ≈4x109 mononuclear cells (MNC) representing ≈1 liter of blood. The obtained DLA samples were then divided, fixed with CellSave preservative, prepared and processed with each of the analysis techniques as described in the Standard Operating Procedures developed for DLA in the CTCTrap consortium (https://www.utwente.nl/tnw/mcbp/protocolsandtools/). Results DLAs were obtained from 20 metastatic prostate cancer patients and 12 metastatic breast cancer patients at four different European academic medical institutions. Using a SOP for the DLA procedure, similar DLA products (MNC concentration: 64x106/mL, SD = 38x106) could be generated without any noticeable side effects. CTC in 7.5 mL of blood ranged from 0 to 324 (mean = 61, median = 18). DLA processed with CellSearch represented 7 to 212 mL of blood (mean = 100, median = 97), CTC ranged from 0 to 2913 (mean = 330, median = 105). Resulting in a significant increase in CTC yield (p = 0.004) ranging from 0x to 40x (mean = 13, median = 9) when comparing 1mL of whole blood to 1mL of DLA. Filtration of 50x106 WBC of DLA, through 5um microsieves yielded only 0 to 12 CTC (mean = 2, median = 0, n = 16). Leukocyte depletion of 18 mL of DLA followed by filtration yielded 0 to 178 CTC (mean = 37, median = 4, n = 22) not yielding a relative increase versus CellSearch DLA. Leukocyte depletion followed by CellSearch yielded 271 to 1620 CTC (mean = 792, median = 484, n = 3) also not yielding a relative increase versus CellSearch DLA. In 7 patients 0 CTC were detected in 7.5mL of blood, in 4 out of these 7 patients CTC were detected in DLA. Conclusion The yield of CTC can be significantly increased by the use of DLA in patients with CTC detected in 7.5 mL of blood. Technology to select CTC from DLAs will need to be further improved before one can make optimal use of the large processed blood volumes. Citation Format: Kiki C. Andree, Anouk Mentink, Joost F. Swennenhuis, Leon W. Terstappen, Nikolas H. Stoecklein, Rui P. Neves, Rita Lampignano, Hans Neubauer, Tanja Fehm, Johannes C. Fischer, Elisabetta Rossi, Mariangela Manicone, Umberto Basso, Piero Marson, Rita Zamarchi, Yohann Loriot, Valérie Lapierre, Vincent Faugeroux, Marianne Oulhen, Francoise Farace, Gemma Fowler, Mariane Sousa Fontes, Berni Ebbs, Maryou Lambros, Mateus Crespo, Penelope Flohr, Johann S. de Bono. Diagnostic leukapheresis results in a significant increase in CTC yield in metastatic breast and prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1723. doi:10.1158/1538-7445.AM2017-1723

Introduction: The presence of circulating tumor cells (CTC) enumerated by the CellSearch system in blood of patients with cancers of epithelial origin is strongly associated with a poor prognosis of these patients. CTC are enriched by targeting the EpCAM antigen, raising the question which EpCAM subtypes are present as well in patients. In the EU-FP7 CTCTrap program, we investigated the presence of EpCAM- CTC in blood samples after depletion of EpCAM+ CTC by CellSearch. Studies were performed and validated at the participating laboratories by distribution and analysis of blood samples spiked with cancer cells and by testing blood from 73 metastatic prostate and 22 metastatic breast cancer patients. Methods: Blood samples were processed according to the standard operating procedures and tools developed in the program (https://www.utwente.nl/tnw/mcbp/protocolsandtools/). First, CellSearch was performed for EpCAM+ CTC, followed by filtration and fluorescent labeling of the blood discarded by CellSearch for EpCAM- CTC. To validate the procedures across the 6 participating clinical sites, 3x 3 tubes with aliquots of healthy donor blood, spiked with either PC3, MDA-MB-231 or no cells, were prepared at one site and shipped to all other sites for simultaneous processing. Metastatic prostate and breast cancer patients were processed with the same procedures. Results: 27% of PC3 cells were recovered by CellSearch and 21% by filtration, leaving 52% unaccounted for. For MDA, 26% were recovered by CellSearch and 18% by filtration, leaving 56% accounted for. Differences in recovery between sites were not significant. In patients both EpCAM+ and EpCAM- CTC were detected (see table). Conclusion: In a multicenter study EpCAM+ and EpCAM- CTC were present in blood of metastatic prostate and breast cancer patients. Spiking experiments showed that the developed methods can be further improved to increase the CTC yield. Molecular characterization of the CTC subtypes and relation with clinical outcome is ongoing. % metastatic cancer patients with EpCAM+ and EpCAM- CTC% prostate cancer patients (n=73)% breast cancer patients (n=22)CellSearch CTC EpCAM+CellSearch CTC EpCAM+# CTC01-4>4Total01-4>4TotalMicrosieve CTC EpCAM-011819381859321-4671629991432>48619331414936Total252154412732 Citation Format: Sanne de Wit, Kiki C. Andree, Joost F. Swennenhuis, Elisabetta Rossi, Mariangela Manicone, Riccardo Vidotto, Rita Zamarchi, Marianna Alunni-Fabbroni, Elisabeth K. Trapp, Marie Tzschaschel, Brigitte Rack, Rita Lampignano, Hans Neubauer, Tanja Fehm, Marianne Oulhen, Emeline Colomba, Françoise Farace, Mateus Crespo, Penelope Flohr, Gemma Fowler, Mariane Sousa Fontes, Johann S. de Bono, Leon WMM Terstappen. EpCAM- and EpCAM+ circulating tumor cells in metastatic prostate and breast cancer patients: a multicenter study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3787. doi:10.1158/1538-7445.AM2017-3787

Introduction: Circulating tumor cells (CTCs) can be isolated from blood and serve as a source of tumor material. Expansions of CTCs may permit functional treatment-efficacy tests in combination with genetics, epigenetics and proteomics screening. We present a fast workflow to isolate, capture, grow and image cells inside a self-sorting microwell chip using the VyCAP single cell isolation platform. After seeding single cells in a microwell chip, they can grow inside the chip or can be transferred to a tissue culture plate for clonal expansion or downstream analysis. Materials and methods: The Rosette-Sep™ Human CD45 depletion cocktail (Stem Cell Technologies) was used to enrich spiked cells from 1 ml of fresh EDTA blood. Cells were stained with CellTrace™ violet for tracking spiked cells, calcein for live cells, ethidium bromide for dead cells, α-CD45-PERCP as negative marker and α-EpCAM-AF647 as positive marker. After density gradient separation using Ficoll-Paque™, the cell suspension was measured and quantified using flow cytometry for Rosette-Sep™ efficiency. Cell suspensions were seeded in the self-sorting microwell chips using a negative pressure of 40-70 mbar using the VyCAP Filtration system. After seeding single cells in the microwells, the chips were imaged with the VyCAP Puncher system and either placed immediately into culture medium for cell expansion or selected cells were transferred into a 96 well tissue culture plate. Cell expansion in microwell chips and plates was followed up to 14 days. Results: Cultured MDA-MB-231 and MCF-7 cells were single-cell-trapped in the microwells with an efficiency of 82% ± 9% and 74% ± 15% respectively, after direct filtration without enrichment. Immediately after capturing in microwells 93% of the MCF7 cells were viable. Rosette-Sep™ enriched MDA-MB-231 and MCF-7 cells with an efficiency of 73% ± 6% and 74% ± 7% respectively, when measured with flow cytometry. The Rosette-Sep™ procedure followed by capture of the MDA-MB-231 and MCF-7 cells in the microwells resulted in an efficiency of 50% ± 11% and 52% ± 11% respectively. 92% of the cells were found back in a culture plate after punching of single cells from the chip, and of these cells, 80% were alive 4 hours after punching. 82% of these cells were alive and still growing after 14 days. In total 53% of cells spiked in blood, could be found back in the cups of which after punching into individual wells, 77% were alive and growing. Conclusion: We present a single cell capture and isolation method for clonal expansion of viable tumor cells. The VyCAP single cell isolation platform used for these experiments provides an easy separation of the single cells from a CTC enriched cell suspension and allows on-chip expansion as well as immediate separation of the CTCs into culture plates. About 50% of spiked cells could be retrieved inside the microwell chip and of these 77% were alive after punching into individual wells of a culture plate. Citation Format: Joost F. Swennenhuis, Kiki C. Andree, Fikri Abali, Michiel Stevens, Joska Broekmaat, Fiona F. Passanha, Cees J. van Rijn, Leon W. Terstappen. Self-sorting microwells to isolate and expand single circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3785. doi:10.1158/1538-7445.AM2017-3785

Introduction: CTC count is an independent predictor of overall survival in mCRPC. Isolation of CTC from peripheral blood (PB) for genomic and functional analysis is challenging, especially in patients (pts) with low CTC count. It has been shown that DLA increases CTC yield. However, it has yet to be proven whether CTC isolation from DLA can be used in complementary studies such as molecular characterization and growth of organoid culture for drug sensitivity studies. Here we present preliminary data of an on-going study, which evaluates DLA in mCRPC pts, focusing on safety, CTC enrichment, molecular characterization and feasibility for organoid culture. Methods: mCRPC pts considered for clinical trials were selected according to performance status (ECOG 0-1) and number of CTC found in 7.5ml PB (>20 cells/7.5mL). DLA products (200x106 cells) were processed using the CellSearch CTC kit (Janssen Diagnostics, LLC) according to manufacturer procedures. The contents of CellSearch cartridges were sorted into single cell by fluorescence activated cell sorting (FACS) and subsequently assessed by array comparative genomic hybridization (aCGH) for copy number aberrations (CNA). Enrichment of CTC for organoid culture was performed by density gradient of mononuclear cells followed by positive selection using magnetic beads. Results: Overall 12 mCRPC patients underwent DLA without any complication or toxicity. The mean CTC count was 90 CTC/7.5 ml peripheral blood (median = 31) and ranged from 20 to 324. CellSearch CTC count in the DLA yielded a mean of 466 (median=203) and ranged from 60 to 2496 with an up to 40-fold increase (mean = 13, median = 6) in CTC count separation when comparing 1mL of PB to 1mL of DLA. Molecular analyses of FACS single CTC from the DLA by aCGH showed that these CTC genomic profiles had the typical hallmarks of mCRPC with CNAs including AR and MYC locus (8q) amplification, and PTEN, RB1, TP53, CHD1 loss. Additionally, ex vivo culture of CTC-derived organoids was successfully achieved. aCGH of these organoids matched the genomic profile that of the CTC from the same patient. Conclusion: DLA from mCRPC pts was well tolerated and yields higher CTC capture than PB and may provide an alternative to tissue biopsy and routine blood volumes. Our strategy allowed us to isolate genomic DNA with good quality for molecular characterization and viable CTC for organoid culture and functional studies. Citation Format: Maryou B. Lambros, Veronica S. Gil, Mateus Crespo, Mariane S. Fontes, Rui N. Neves, Niven Mahra, Gemma Fowler, Berni Ebbs, Penny Flohr, George Seed, Wei Yuan, Joanne Hunt, Deirdre Moloney, Dionne Ayanda, Joost F. Swennenhuis, Kiki C. Andree, Semini Sumanasuriya, Matthew Clarke, Pasquale Rescigno, Zafeiris Zafeiriou, Joaquin Mateo, Diletta Bianchini, Nikolas H. Stoecklein, Leon W. Terstappen, Gunther Boysen, Johann S. De Bono. Diagnostic leukapheresis (DLA): Molecular characterisation and organoid culture of circulating tumor cells (CTC) from metastatic castration resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 993. doi:10.1158/1538-7445.AM2017-993