Abstract LB-250: Liquid biopsy in NSCLC: EpCAM+ and EpCAM- circulating tumor cells, tumor derived extracellular vesicles and cell-free circulating tumor DNA
Authors: Wit Sanne de , Tamminga Menno , Swennenhuis Joost F. , Zeune Leonie L. , Heitzer Ellen , Speicher Michael , Hiltermann T Jeroen N. , Terstappen Leon WMM , Groen Harry JM 
Published: July 2017

Abstract
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 &gt;45 per 7.5 mL. This frequency is based on the mean + 2SD from 42 healthy controls. In 20% of the patients &gt;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 &gt;45 tdEV (p=0.271 n=50) and &gt;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