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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.

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.

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.

Background: Obtaining tissue for pancreatic carcinoma diagnosis and biomarker assessment to aid drug development is challenging. Circulating tumour cells (CTCs) may represent a potential biomarker to address these unmet needs. We compared prospectively the utility of two platforms for CTC enumeration and characterisation in pancreatic cancer patients in a pilot exploratory study. Patients and methods: Blood samples were obtained prospectively from 54 consenting patients and analysed by CellSearch and isolation by size of epithelial tumour cells (ISET). CellSearch exploits immunomagnetic capture of CTCs-expressing epithelial markers, whereas ISET is a marker independent, blood filtration device. Circulating tumour cell expression of epithelial and mesenchymal markers was assessed to explore any discrepancy in CTC number between the two platforms. Results: ISET detected CTCs in more patients than CellSearch (93% vs 40%) and in higher numbers (median CTCs/7.5 ml, 9 (range 0–240) vs 0 (range 0–144)). Heterogeneity observed for epithelial cell adhesion molecule, pan-cytokeratin (CK), E-Cadherin, Vimentin and CK 7 expression in CTCs may account for discrepancy in CTC number between platforms. Conclusion: ISET detects more CTCs than CellSearch and offers flexible CTC characterisation with potential to investigate CTC biology and develop biomarkers for pancreatic cancer patient management.

Background: Circulating tumour cells (CTC) have a crucial role in metastasis formation and can consistently provide information on patient prognosis. Epithelial-mesenchymal transition (EMT) is considered as an essential process in the metastatic cascade, but there is currently very few data demonstrating directly the existence of the EMT process in CTCs. Methods: CTCs were enriched by blood filtration using ISET (isolation by size of epithelial tumour cells), triply labelled with fluorescent anti-vimentin, anti-pan-keratin antibodies and SYTOX orange nuclear dye, and examined by confocal microscopy in six patients with metastatic non-small cell lung cancer (NSCLC). In parallel, CTCs were morphocytologically identified by an experienced cytopathologist. Results: Isolated or clusters of dual CTCs strongly co-expressing vimentin and keratin were evidenced in all patients (range 5–88/5 ml). CTCs expressing only vimentin were detected in three patients, but were less frequent (range 3–15/5 ml). No CTC expressing only keratin was detected. Conclusion: We showed for the first time the existence of hybrid CTCs with an epithelial/mesenchymal phenotype in patients with NSCLC. Their characterisation should provide further insight on the significance of EMT in CTCs and on the mechanism of metastasis in patients with NSCLC.

BACKGROUND: Circulating tumour cells (CTCs) can provide information on patient prognosis and treatment efficacy. However, there is no universal method to detect CTC currently available. Here, we compared the performance of two CTC detection systems based on the expression of the EpCAM antigen (CellSearch assay) or on cell size (ISET assay). METHODS: Circulating tumour cells were enumerated in 60 patients with metastatic carcinomas of breast, prostate and lung origins using CellSearch according to the manufacturer’s protocol and ISET by studying cytomorphology and immunolabelling with anti-cytokeratin or lineage-specific antibodies. RESULTS: Concordant results were obtained in 55% (11 out of 20) of the patients with breast cancer, in 60% (12 out of 20) of the patients with prostate cancer and in only 20% (4 out of 20) of lung cancer patients. CONCLUSION: Our results highlight important discrepancies between the numbers of CTC enumerated by both techniques. These differences depend mostly on the tumour type. These results suggest that technologies limiting CTC capture to EpCAM-positive cells, may present important limitations, especially in patients with metastatic lung carcinoma.

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.

Uveal melanoma is one of the most deadly diseases in ophthalmology for which markers able to predict the appearance of metastasis are needed. The study investigates the role of circulating tumor cells (CTC) as a prognostic factor in this disease. We report the detection of circulating tumor cells by Isolation by Size of Epithelial Tumor cells (ISET) in a cohort of 31 uveal melanoma patients: we identified single CTCs or clusters of cells in 17 patients, while the control population, subjects with choroidal nevi, showed no CTC in peripheral blood. The presence of CTCs did not correlate with any clinical and pathological parameter, such as tumor larger basal diameter (LBD), tumor height and TNM. By stratifying patients in groups on the basis of the number of CTC (lower or higher than 10 CTC per 10 mL blood) and the presence of CTC clusters we found a significant difference in LBD (p = 0.019), Tumor height (p = 0.048), disease-free and overall survival (p < 0.05). In conclusion, we confirm the role of CTC as a negative prognostic marker in uveal melanoma patients after a long follow-up period. Further characterization of CTC will help understanding uveal melanoma metastasization and improve patient management.

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.