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 EpCAM^low 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.

Background Non-small cell lung cancer (NSCLC) patients treated with checkpoint inhibitors show long lasting responses, but it is hard to predict which patients will profit from this treatment with the currently used marker, programmed death ligand 1 (PD-L1). We hypothesized that circulating tumor cells (CTC) or tumor derived extracellular vesicles (tdEV) are markers of treatment efficacy. Methods Patients with advanced NSCLC treated with checkpoint inhibitors were included. Blood was drawn at baseline (T0) and at 4 weeks of treatment (T1) for analysis of CTC and tdEV using CellSearch®. Tumor response was classified as partial or complete response based on the response evaluation criteria in solid tumors (RECISTv1.1) measured 4–6 weeks after start of treatment. Durable response was defined as stable disease, partial or complete response without disease progression at 6 months. Analyses were adjusted for covariables including PD-L1 expression. Results We included 104 patients (30 with a tumor response, 74 non-responders, 2 responses not evaluable due to early death); 63 patients provided T1 samples. All patients were treated with PD-L1 inhibitors. The majority of patients received second (85%) or third line (treatment with nivolumab monotherapy (89%). CTC were present in 33/104 patients at T0 (32%) and 17/63 at T1 (27%), 9/63 patients had CTC (14%) at both time points. The presence of CTC, both at T0 (OR = 0.28, p = 0.02,) and T1 (OR = 0.07, p < 0.01), was an independent predictive factor for a lack of durable response and was associated with worse progression free and overall survival. More tdEV were associated with shorter survival but not with response rate. Conclusion CTC occur in one third of advanced NSCLC patients and their presence is a predictive factor for a worse durable response rate to checkpoint inhibitors. tdEV are associated with shorter survival but not with response.

The increasing number of treatment options for patients with metastatic carcinomas has created an accompanying need for methods to determine if the tumor will be responsive to the intended therapy and to monitor its effectiveness. Ideally, these methods would be noninvasive and provide quantitative real-time analysis of tumor activity in a variety of carcinomas. Assessment of circulating tumor cells shed into the blood during metastasis may satisfy this need. Here we review the CellSearch technology used for the detection of circulating tumor cells and discuss potential future directions for improvements.

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: EpCAM^highcirculating tumor cells (CTC), EpCAM^lowCTC, tumor‐derived extracellular vesicles (tdEV) and cell‐free circulating tumor DNA (ctDNA). EpCAM^highCTC were detected with CellSearch, tdEV in the CellSearch images and EpCAM^lowCTC 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 EpCAM^highCTC, 15% had ≥2 EpCAM^lowCTC, 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 EpCAM^highCTC and elevated levels of tdEV and ctDNA was associated with a poor OS; however, the presence of EpCAM^lowCTC was not. This single tube approach enables simultaneous analysis of multiple biomarkers to explore their potential as a liquid biopsy.

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.