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Liquid biopsy and circulating tumor cell (CTC) screening has gained interest over the last two decades for detecting almost all solid malignancies. To date, the major limitation in terms of the applicability of CTC screening in daily clinical practice is the lack of reproducibility due to the high number of platforms available that use various technologies (e.g., label-dependent versus label-free detection). Only a few studies have compared different CTC platforms. The aim of this study was to compare the efficiency of four commercially available CTC platforms (Vortex (VTX-1), ClearCell FX, ISET, and Cellsearch) for the detection and identification of uveal melanoma cells (OMM 2.3 cell line). Tumor cells were seeded in RPMI medium and venous blood from healthy donors, and then processed similarly using these four platforms. Melan-A immunochemistry was performed to identify tumor cells, except when the Cellsearch device was used (automated identification). The mean overall recovery rates (with mean recovered cells) were 39.2% (19.92), 22.2% (11.31), 8.9% (4.85), and 1.1% (0.20) for the ISET, Vortex (VTX-1), ClearCell FX, and CellSearch platforms, respectively. Although paramount, the recovery rate is not sufficient to assess a CTC platform. Other parameters, such as the purpose for using a platform (diagnosis, genetics, drug sensitivity, or patient-derived xenograft models), reproducibility, purity, user-friendliness, cost-effectiveness, and ergonomics, should also be considered before they can be used in daily clinical practice and are discussed in this article.

Virgílio Souza e Silva,1 Ludmilla Thomé Domingos Chinen,2 Emne A Abdallah,2 Aline Damascena,2 Jociana Paludo,3 Rubens Chojniak,3 Aldo Lourenço Abbade Dettino,1 Celso Abdon Lopes deMello,1 Vanessa S Alves,2 Marcello F Fanelli1 1Department of Clinical Oncology, 2International Research Center, 3Image Department, A. C. Camargo Cancer Center, São Paulo, Brazil Background: Colorectal cancer (CRC) is the third most prevalent cancer worldwide. New prognostic markers are needed to identify patients with poorer prognosis, and circulating tumor cells (CTCs) seem to be promising to accomplish this.Patients and methods: A prospective study was conducted by blood collection from patients with metastatic CRC (mCRC), three times, every 2months in conjunction with image examinations for evaluation of therapeutic response. CTC isolation and counting were performed by Isolation by Size of Epithelial Tumor Cells (ISET).Results: A total of 54 patients with mCRC with a mean age of 57.3years (31–82 years) were included. Among all patients, 60% (n=32) were carriers of wild-type KRAS (WT KRAS) tumors and 90% of them (n=29) were exposed to monoclonal antibodies along with systemic treatment. Evaluating CTC kinetics, when we compared the baseline (pretreatment) CTC level (CTC1) with the level at first follow-up (CTC2), we observed that CTC1-positive patients (CTCs above the median), who became negative (CTCs below the median) had a favorable evolution (n=14), with a median progression-free survival (PFS) of 14.7months. This was higher than that for patients with an unfavorable evolution (CTC1– that became CTC2+; n=13, 6.9months; P=0.06). Patients with WT KRAS with favorable kinetics had higher PFS (14.7months) in comparison to those with WT KRAS with unfavorable kinetics (9.4months; P=0.02). Moreover, patients whose imaging studies showed radiological progression had an increased quantification of CTCs at CTC2 compared to those without progression (P=0.04).Conclusion: This study made possible the presentation of ISET as a feasible tool for evaluating CTC kinetics in patients with mCRC, which can be promising in their clinical evaluation. Keywords: circulating tumor cells, metastatic colorectal cancer, kinetics, ISET

The current study aimed at the optimization of circulating tumor cell (CTC) enrichment for downstream protein expression analyses in non-small cell lung cancer (NSCLC) to serve as a tool for the investigation of immune checkpoints in real time. Different enrichment approaches&mdash, ficoll density, erythrolysis, their combination with magnetic separation, ISET, and Parsortix&mdash, were compared in spiking experiments using the A549, H1975, and SKMES-1 NSCLC cell lines. The most efficient methods were tested in patients (n = 15) receiving immunotherapy targeting programmed cell death-1 (PD-1). Samples were immunofluorescently stained for a) cytokeratins (CK)/epithelial cell adhesion molecule (EpCAM)/leukocyte common antigen (CD45), and b) CK/programmed cell death ligand-1 (PD-L1)/ indoleamine-2,3-dioxygenase (IDO). Ficoll, ISET, and Parsortix presented the highest yields and compatibility with phenotypic analysis, however, at the patient level, they provided discordant CTC positivity (13%, 33%, and 60% of patients, respectively) and enriched for distinct CTC populations. IDO and PD-L1 were expressed in 44% and 33% and co-expressed in 19% of CTCs. CTC detection was associated with progressive disease (PD) (p = 0.006), reduced progression-free survival PFS (p = 0.007), and increased risk of relapse (hazard ratio, HR: 10.733, p = 0.026). IDO-positive CTCs were associated with shorter PFS (p = 0.039) and overall survival OS (p = 0.021) and increased risk of death (HR: 5.462, p = 0.039). The current study indicates that CTC analysis according to distinct immune checkpoints is feasible and may provide valuable biomarkers to monitor NSCLC patients treated with anti-PD-1 agents.

Background Current methods for follow-up of Ovarian Cancer (OC) are widespread, especially with CA125, which, however, is not a perfect biomarker and thus further investigation for new methods of evaluation are warranted. The feasibility of Circulating Tumor Cells (CTCs) in advanced OC was investigated in this case report. Case presentation A 19-year-old woman with advanced low-grade serous papillary adenocarcinoma and relapsed disease did not have a CA125 correspondent with disease relapse. CTCs were evaluated, compared with CA125 and with image exams. Relapses were not correspondent to elevations of CA125. CTCs demonstrated usefulness, being proportional to major disease relapse, especially in the peritoneum. CTCs may be used as a complementary diagnosis tool when marginal/small increases in CA-125 levels are observed. Conclusions In OC, CTCs can be an important tool to predict recurrence, response to treatment and improve the quality of decision-making, in order to offer the best treatment to a determined group of patients.

Circulating tumor cells (CTCs) are suspected of predicting the prognosis of malignant tumor, but there are few relevant reports specific to esophageal squamous cell carcinoma (ESCC). This study investigated the clinical significance of CTCs in patients with ESCC. Sixty patients with ESCC were enrolled, from whom CTCs had been tested by our team previously. Peripheral blood samples were obtained from these patients before treatment; and CTCs were assayed by isolation by size of epithelial tumor cells (ISET). Associations between the presence of CTCs and patients’ clinicopathological parameters and clinical outcomes were analyzed. CTCs were detected in 20 patients (33.3%), who experienced significantly shorter progression-free survival (PFS) than did the CTC-negative patients. Overall, PFS was negatively associated with the number of CTCs. Multivariate analyses showed that a CTC count >2 was a strong independent prognostic indicator of tumor recurrence (hazard ratio [HR] 5.63; 95% confidence interval [CI] 1.77–17.89; P = .003). In the subgroup of 50 patients who underwent R0 resection and postoperative adjuvant radiotherapy or chemotherapy, CTC was a strong, independent, and prognostic indicator of tumor recurrence (HR 10.70; 95% CI, 1.40–81.91; P = .022). The number of CTCs correlated with the T stage (r = 0.26, P = .043) but not with the N or M stage. For subgroups in stages II or I-IIIB or T3 or T3 + T4, the PFS of patients with CTCs > 1 or > 2 was significantly shorter than that of the patients with CTCs ≤ 1 or CTCs ≤ 2. In the stage III or T3 + T4 groups, the PFS of patients with CTCs > 0 was significantly shorter than that of patients with CTC = 0. This is the first study to report that the CTC detected by ISET is an independent and prognostic indicator of patients’ outcome in ESCC. Consideration of CTCs may improve the accuracy of preoperative staging in ESCC.

Virgílio Souza e Silva,1 Ludmilla Thomé Domingos Chinen,2 Emne A Abdallah,2 Aline Damascena,2 Jociana Paludo,3 Rubens Chojniak,3 Aldo Lourenço Abbade Dettino,1 Celso Abdon Lopes deMello,1 Vanessa S Alves,2 Marcello F Fanelli1 1Department of Clinical Oncology, 2International Research Center, 3Image Department, A. C. Camargo Cancer Center, São Paulo, Brazil Background: Colorectal cancer (CRC) is the third most prevalent cancer worldwide. New prognostic markers are needed to identify patients with poorer prognosis, and circulating tumor cells (CTCs) seem to be promising to accomplish this.Patients and methods: A prospective study was conducted by blood collection from patients with metastatic CRC (mCRC), three times, every 2months in conjunction with image examinations for evaluation of therapeutic response. CTC isolation and counting were performed by Isolation by Size of Epithelial Tumor Cells (ISET).Results: A total of 54 patients with mCRC with a mean age of 57.3years (31–82 years) were included. Among all patients, 60% (n=32) were carriers of wild-type KRAS (WT KRAS) tumors and 90% of them (n=29) were exposed to monoclonal antibodies along with systemic treatment. Evaluating CTC kinetics, when we compared the baseline (pretreatment) CTC level (CTC1) with the level at first follow-up (CTC2), we observed that CTC1-positive patients (CTCs above the median), who became negative (CTCs below the median) had a favorable evolution (n=14), with a median progression-free survival (PFS) of 14.7months. This was higher than that for patients with an unfavorable evolution (CTC1– that became CTC2+; n=13, 6.9months; P=0.06). Patients with WT KRAS with favorable kinetics had higher PFS (14.7months) in comparison to those with WT KRAS with unfavorable kinetics (9.4months; P=0.02). Moreover, patients whose imaging studies showed radiological progression had an increased quantification of CTCs at CTC2 compared to those without progression (P=0.04).Conclusion: This study made possible the presentation of ISET as a feasible tool for evaluating CTC kinetics in patients with mCRC, which can be promising in their clinical evaluation. Keywords: circulating tumor cells, metastatic colorectal cancer, kinetics, ISET

Introduction: Epithelial circulating tumor cells (CTCs) are detectable in patients with non-small cell lung cancer (NSCLC). However, epithelial to mesenchymal transition, a widely reported prerequisite for metastasis, may lead to an underestimation of CTC number. We compared directly an epithelial marker-dependent (CellSearch) and a marker-independent (isolation by size of epithelial tumor cells [ISET]) technology platform for the ability to identify CTCs. Molecular characteristics of CTCs were also explored. Methods: Paired peripheral blood samples were collected from 40 chemonaive, stages IIIA to IV NSCLC patients. CTCs were enumerated by Epithelial Cell Adhesion Molecule-based immunomagnetic capture (CellSearch, Veridex) and by filtration (ISET, RareCell Diagnostics). CTCs isolated by filtration were assessed by immunohistochemistry for epithelial marker expression (cytokeratins, Epithelial Cell Adhesion Molecule, epidermal growth factor receptor) and for proliferation status (Ki67). Results: CTCs were detected using ISET in 32 of 40 (80%) patients compared with 9 of 40 (23%) patients using CellSearch. A subpopulation of CTCs isolated by ISET did not express epithelial markers. Circulating tumor microemboli (CTM, clusters of ≥3 CTCs) were observed in 43% patients using ISET but were undetectable by CellSearch. Up to 62% of single CTCs were positive for the proliferation marker Ki67, whereas cells within CTM were nonproliferative. Conclusions: Both technology platforms detected NSCLC CTCs. ISET detected higher numbers of CTCs including epithelial marker negative tumor cells. ISET also isolated CTM and permitted molecular characterization. Combined with our previous CellSearch data confirming CTC number as an independent prognostic biomarker for NSCLC, we propose that this complementary dual technology approach to CTC analysis allows more complete exploration of CTCs in patients with NSCLC.

International audience; Due to recent technological progresses, Circulating Tumor Cells (CTC) are currently extensively studied in order to define their diagnostic, prognostic and predictive value. We review here the different detection techniques and their clinical results in breast cancer patients.

This article is a critical note on the subject of Circulating Tumor Cells (CTC). It takes into account the tumor identity of Circulating Tumor Cells as cancer seeds in transit from primary to secondary soils, rather than as a “biomarker”, and considers the help this field could bring to cancer patients. It is not meant to duplicate information already available in a large number of reviews, but to stimulate considerations, further studies and development helping the clinical use of tumor cells isolated from blood as a modern personalized, non-invasive, predictive test to improve cancer patients’ life. The analysis of CTC challenges, methodological bias and critical issues points out to the need of referring to tumor cells extracted from blood without any bias and identified by cytopathological diagnosis as Circulating Cancer Cells (CCC). Finally, this article highlights recent developments and identifies burning questions which should be addressed to improve our understanding of the domain of CCC and their potential to change the clinical practice.