Sanne de Wit

The detection of EpCAM + and EpCAM - circulating tumor cells

EpCAM expressing circulating tumor cells, detected by CellSearch, are predictive of short survival in several cancers and may serve as a liquid biopsy to guide therapy. Here we investigate the presence of EpCAM+ CTC detected by CellSearch and EpCAM– CTC discarded by CellSearch, after EpCAM based enrichment. EpCAM– CTC were identified by filtration and fluorescent labelling. This approach was validated using different cell lines spiked into blood and evaluated on blood samples of 27 metastatic lung cancer patients. The majority of spiked EpCAM+ cells could be detected with CellSearch, whereas most spiked cells with EpCAMlow or EpCAM– expression were detected using filtration. Five or more CTC were detected in 15% of the patient samples, this increased to 41% when adding the CTC detected in the discarded blood. The number of patients with CTC and the number of CTC detected were doubled by the presence of EpCAM– CTC. In this pilot study, the presence of EpCAM+ CTC was associated with poor outcome, whereas the EpCAM– CTC were not. This observation will need to be confirmed in larger studies and molecular characterization needs to be conducted to elucidate differences between EpCAM– and EpCAM+ CTC.

EpCAM+ and EpCAM- circulating tumor cells in metastatic lung cancer

Purpose: The presence of Circulating Tumor Cells (CTC) in metastatic carcinomas detected by CellSearch is associated with poor outcome. CellSearch uses an enrichment step based on the EpCAM antibody and enumerates CTC using antibodies targeting specific cytokeratins. Here we investigate the presence and role of CTC not detected by CellSearch in lung cancer patients. Methods: Blood discarded by CellSearch after EpCAM based enrichment was collected and enriched for CTC by filtration through microsieves and enumerated after fluorescent labeling. Additional anti-cytokeratin antibodies were added to the CellSearch test to increase the cytokeratin coverage. This approach was validated using different cell lines spiked into blood and tested on blood samples of 28 metastatic lung cancer patients. Results: The majority of spiked EpCAM+ cells could be detected with CellSearch whereas few cells were detected with low or no EpCAM expression (EpCAM-). Recovery of cells on microsieves depended strongly on cell size. One or more CTC were detected in 39% of the patient samples, this increased to 75% when adding the CTC detected in the discarded blood and to 82% with the use of additional cytokeratin antibodies. Presence of CTC was significantly associated with poor Overall Survival (OS) when detected by CellSearch without (p = 0.004) and with additional cytokeratins (p = 0.007), but not with EpCAM-CTC in the blood discarded by CellSearch (p = 0.271) or all CTC detected (p = 0.354) Conclusions: Expanding the cytokeratin coverage and including EpCAM-CTC, double the number of patients with CTC and the number of CTC detected with CellSearch. In contrast with EpCAM+ CTC, the presence of EpCAM-CTC in this group of patients was not associated with poor OS. The metastatic lung cancer patients included in this study were very diverse with respect to cancer type, line of therapy and quite small in numbers to make any definitive conclusions. Still, this pilot study warrants a larger study to confirm the findings. In addition an in-depth characterization of the EpCAM+ and EpCAM-CTC is needed to confirm that the EpCAM-, panCK+ CTC are indeed tumor cells and unravel the genetic difference with the EpCAM+, panCK+ CTC. Citation Format: Sanne de Wit, Guus van Dalum, Aufried T.M. Lenferink, Arjan G.J. Tibbe, Jeroen T.N. Hiltermann, Harry J.M. Groen, Cees van Rijn, Leon W.M.M. Terstappen. EpCAM+ and EpCAM- circulating tumor cells in metastatic lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 377. doi:10.1158/1538-7445.AM2015-377

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

For using counts of circulating tumor cells (CTCs) in the clinic to aid a physicians 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.

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.

Abstract LB-250: Liquid biopsy in NSCLC: EpCAM+ and EpCAM- circulating tumor cells, tumor derived extracellular vesicles and cell-free circulating tumor DNA

Introduction The need for a liquid biopsy in non-small cell lung cancer (NSCLC) patients is rapidly increasing as more and more targeted therapies become available. Presence in blood of circulating tumor cells (CTC), tumor derived extracellular vesicles (tdEV) and cell-free circulating tumor DNA (ctDNA) measured with different approaches are being explored for their potential to represent a liquid biopsy in the European and Dutch CANCER-ID projects (https://www.cancer-id.eu/ & https://www.utwente.nl/tnw/cancer-id/). Here, we determine in just one 7.5 mL tube of blood the presence of CTC, tdEV and ctDNA and investigate the relation with survival of metastatic NSCLC patients. Methods In total 106 advanced NSCLC patients were enrolled in the study. In 86 patients EpCAM+ CTC, EpCAM- CTC & tdEV were enumerated and in 50 patients EpCAM+ CTC, EpCAM- CTC, tdEV & ctDNA, all from one CellSave blood tube. ctDNA from a separate plasma tube is available for all patients but not yet analyzed. Before placing the sample in the CellSearch system, plasma was aspirated and stored at -80°C. EpCAM+ CTC were enumerated by CellSearch and EpCAM- CTC after filtration of the EpCAM+ CTC depleted blood through 5µm pore filters, as described by de Wit et al. (Sci. Rep. doi: 10.1038/srep12270, 2015). tdEV were defined by a multidimensional gate as cytokeratin+/DAPI-/CD45- vesicles and identified in the CellSearch images, using the open source image analysis program ACCEPT. The stored plasma was used for ctDNA quantification with the FAST-SeqS approach, described by Belic et al. (ClinChem 61, 838, 2015). In several patients with EpCAM- CTC, fluorescent in situ hybridization was performed on the filter to establish the cancerous origin of the EpCAM- CTC. Results In 24% of the patients ≥1 EpCAM+ CTC as well as ≥1 EpCAM- CTC were detected in 7.5 mL of blood. In 30% of the patients tdEV were present at a frequency >45 per 7.5 mL. This frequency is based on the mean + 2SD from 42 healthy controls. In 20% of the patients >10% ctDNA load was found. No significant correlation was found between the presence of these biomarkers. Presence of all four biomarkers was detected in 6% of patients and at least one of four was found in 52% of patients. One or more EpCAM+ CTC were associated with poor overall survival (p=0.010 for 86 patients and p=0.019 for 50 patients), whereas EpCAM- CTC were not (p=0.495 n=86; p=0.571 n=50). The latter is surprising since some CTC were shown to be cytogenetically aberrant, conform the primary tumor. The presence of >45 tdEV (p=0.271 n=50) and >10% ctDNA (p=0.082 n=50) did not reach significance. Conclusions In this study EpCAM+ CTC, EpCAM- CTC, tdEV or ctDNA was detected in one tube of blood in 52% of the NSCLC patients. Only the presence of EpCAM+ CTC was associated with poor overall survival, raising the question whether or not the extraction of molecular information from these other biomarkers can be used to predict response to treatment in NSCLC. To increase the percentage of patients from which a liquid biopsy can be obtained, the analyzed blood volume will need to be increased. Citation Format: Sanne de Wit, Menno Tamminga, Joost F. Swennenhuis, Leonie L. Zeune, Ellen Heitzer, Michael Speicher, T Jeroen N. Hiltermann, Leon WMM Terstappen, Harry JM Groen. Liquid biopsy in NSCLC: EpCAM+ and EpCAM- circulating tumor cells, tumor derived extracellular vesicles and cell-free circulating tumor DNA [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 LB-250. doi:10.1158/1538-7445.AM2017-LB-250

Abstract 4825: Circulating tumor cells in metastatic lung cancer enriched by EpCAM expression and physical characteristics

Introduction: Circulating tumor cells (CTC) measured with the CellSearch system in patients with metastatic carcinomas are associated with poor survival. The frequency of CTC detected by the CellSearch system in non-small cell lung cancer (NSCLC) patients is relatively low, raising the question whether some CTC are not detected by the CellSearch system. To investigate this, additional antibodies were added to broaden the coverage of cytokeratins and leukocytes. Additionally, a device was designed that collects the sample material of the individual samples that are discarded by CellSearch. This collected waste is filtered for CTC isolation based on physical characteristics and the CTC are stained with a cocktail of antibodies. Methods: A device was designed that uses optical sensing to detect the presence of blood in the waste tube of the CellTracks Autoprep. It collects the waste of individual samples in a 50 mL conical tube. After collection the blood is passed with 100 mbar pressure through a 8x8 mm2 microfabricated silicon microsieve containing 300,000 pores of 5 µm in diameter (VyCAP, Deventer, The Netherlands). The performance was tested using four pre-stained cell lines: Colo320 (size 11 µm, ∼1,616 EpCAM antigens), SW480 (size 11 µm, ∼63,233 EpCAM antigens), T24 (size 16 µm, ∼2,167 EpCAM antigens) and SKBR3 (size 16 µm, ∼445,000 EpCAM antigens). Cells are spiked in 7.5 mL of blood collected in CellSave tubes from healthy volunteers. Spiked blood samples from healthy donors and patients with NCSLC and small cell lung cancer (SCLC) (enrollment is ongoing) were processed on the CellSearch and filtration system between 24 and 96 hours of collection. The cells on the microsieves were stained with a nucleic acid dye, antibodies recognizing leukocytes and all cytokeratins. Additional antibodies were added to the CellSearch test to cover all cytokeratins and broaden the coverage of leukocytes. Results: The recovery percentage of the CellSearch system for the different cell lines used was: 2% of COLO320, 91% of SW480, 2% of T24 and 87% of SKBR3. Additional recovery on the microsieves after filtration of the CellSearch waste was: 18% of COLO320, 6% of SW480, 59% of T24 and 2% of SKBR3. The combined recovery accounts for 20% of COLO320, 97% of SW480, 61% of T24 cells and 89% of SKBR3. In patients with NSCLC and SCLC either no CTC were detected at all, or in various proportions in the CellSearch cartridge, on the microsieves after filtration of the CellSearch waste or with the additional antibodies that were added. Conclusions: We combined the CellSearch system with a device for collecting and filtering the CellSearch waste. On cell lines this demonstrated that a low EpCAM expression results in the presence of CTC in the waste that would not be detected by the CellSearch system. In both NSCLC and SCLC additional CTC can be detected but it still remains to be determined whether the CTC not detected by the original CellSearch approach are also of clinical relevance. Citation Format: Sanne de Wit, Guus van Dalum, Joost van Dalum, Aufried Lenferink, Arjan Tibbe, Cees van Rijn, Jeroen Hiltermann, Harry Groen, Leon Terstappen. Circulating tumor cells in metastatic lung cancer enriched by EpCAM expression and physical characteristics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4825. doi:10.1158/1538-7445.AM2014-4825