Circulating Cancer-Associated Macrophage-like Cells as a Blood-Based Biomarker of Response to Immune Checkpoint Inhibitors.

Evidence has been provided that circulating cancer-associated macrophage-like cell (CAM-L) numbers increase in response to chemotherapy, with an inverse trend compared to circulating tumor cells (CTCs). In the era of evolving cancer immunotherapy, whether CAM-Ls might have a potential role as predictive biomarkers of response has been unexplored. We evaluated whether a serial blood evaluation of CTC to CAM-L ratio might predict response to immune checkpoint inhibitors in a cohort of non-small-cell lung cancer patients. At baseline, CTCs, CAM-Ls, and the CTC/CAM-L ratio significantly correlate with both progression-free survival (PFS) and overall survival (OS). The baseline CTC/CAM-L ratio was significantly different in early progressors (4.28 ± 3.21) compared to long responders (0.42 ± 0.47) (p = 0.001). In patients treated with immune checkpoint inhibitors, a CTC/CAM-L ratio ≤ 0.25 at baseline is associated with better PFS and OS. A baseline CTC/CAM-L ratio ≤ 0.25 is statistically significant to discriminate early progressions from durable response. The results of the present pilot study suggest that CAM-Ls together with CTCs could play an important role in evaluating patients treated with cancer immunotherapy.

PSMA-heterogeneity in metastatic castration-resistant prostate cancer: Circulating tumor cells, metastatic tumor burden, and response to targeted radioligand therapy.

BACKGROUND: We explored the interrelation between prostate-specific membrane antigen (PSMA) expression on circulating tumor cells (CTCs) and that of solid metastatic lesions as determined by whole-body PSMA-targeted positron emission tomography (PET) to refine the prediction of response to subsequent PSMA-targeted radioligand therapy (RLT). METHODS: A prospective study was performed in 20 patients with advanced mCRPC. Of these, 16 underwent subsequent RLT with [177 Lu]Lu-PSMA-617 at a dose of 7.4 GBq every 6-8 weeks. PSMA expression on CTCs using the CellSearch system was compared to clinical and serological results, and to marker expression in targeted imaging and available histological sections of prostatectomy specimens (19% of RLT patients). Clinical outcome was obtained after two cycles of RLT. RESULTS: Marked heterogeneity of PSMA expression was observed already at first diagnosis in available histological specimens. Targeted whole-body imaging also showed heterogeneous inter- and intra-patient PSMA expression between metastases. Heterogeneity of CTC PSMA expression was partially paralleled by heterogeneity of whole-body tumor burden PSMA expression. Twenty percent of CTC samples showed no PSMA expression, despite unequivocal PSMA expression of solid metastases at PET. A high fraction of PSMA-negative CTCs emerged as the sole predictor of poor RLT response (odds ratio [OR]: 0.9379 [95% confidence interval, CI, 0.8558-0.9902]; p = 0.0160), and was prognostic for both shorter progression-free survival (OR: 1.236 [95% CI, 1.035-2.587]; p = 0.0043) and overall survival (OR: 1.056 [95% CI, 1.008-1.141]; p = 0.0182). CONCLUSION: This proof-of-principle study suggests that liquid biopsy for CTC PSMA expression is complementary to PET for individual PSMA phenotyping of mCRPC.

Assessment of Different Circulating Tumor Cell Platforms for Uveal Melanoma: Potential Impact for Future Routine Clinical Practice.

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.

Circulating tumor cell detection: A prospective comparison between CellSearch® and RareCyte® platforms in patients with progressive metastatic breast cancer.

PURPOSE: Circulating tumor cells (CTCs) are prognostic in patients with breast cancer. Several technical platforms exist for their enumeration and characterization. Comparative studies between these platforms are scarce. The RareCyte CTC detection is theoretically more sensitive than the established CellSearch platform, which identifies only CTCs that express EpCAM and cytokeratin. This study prospectively compares CTC enumeration in patients with breast cancer in a paired analysis using these two platforms. It investigates survival outcomes in groups defined by a CTC count threshold. DESIGN: CTC enumeration was performed on 100 samples obtained from 86 patients with progressive metastatic breast cancer (MBC) in two independent laboratories each blinded to the clinical data and the results from the other platform. RESULTS: One hundred paired samples were collected and CTC counts were determined using the CellSearch and RareCyte CTC platforms. In total, 65% and 75% of samples had at least one detectable CTC in 7.5 mL blood with the CellSearch and the RareCyte systems, respectively. CTC counts with the CellSearch system ranged from 0 to 2289 with a median of 3 CTCs, the RareCyte CTC counts ranged from 0 to 1676 with a median of 3 CTCs. The number of samples with 5 or more CTCs in 7.5 mL of blood (the poor prognosis cut-off validated with the CellSearch system) blood was 45% with the CellSearch test and 48% with the RareCyte test. CTC counts quantified with the CellSearch and the RareCyte systems were strongly correlated (Spearman's r = 0.8235 (0.7450-0.8795) p < 0.001). 86 patients were included for Kaplan-Meier survival analysis. An increased mortality risk in patients with CellSearch of 5 CTCs or more per 7.5 mL blood, with a log-rank hazard ratio of 5.164 (2.579-10.34) (p < 0.001) was confirmed. The survival analysis with RareCyte CTC counts with the identical cut-off showed a significantly impaired survival with a hazard ratio of 4.213 (2.153-8.244) (p < 0.001). CONCLUSION: Our data demonstrate the analytical and prognostic equivalence of CellSearch and RareCyte CTC enumeration platforms in patients with MBC using the CellSearch cut-off. This is the first demonstration of prognostic significance using the RareCyte platform.

Circulating Endothelial Cells: A New Possible Marker of Endothelial Damage in Kawasaki Disease, Multisystem Inflammatory Syndrome in Children and Acute SARS-CoV-2 Infection.

BACKGROUND: Kawasaki Disease (KD) and Multisystem Inflammatory Syndrome in Children (MIS-C) are pediatric diseases characterized by systemic inflammation and vascular injury, potentially leading to coronary artery lesions (CALs). Data on vascular injury occurring during acute COVID-19 (AC19) in children are still lacking. The aim of our study was to investigate endothelial injury in KD-, MIS-C- and AC19-dosing circulating endothelial cells (CECs). METHODS: We conducted a multicenter prospective study. CECs were enumerated by CellSearch technology through the immunomagnetic capture of CD146-positive cells from whole blood. RESULTS: We enrolled 9 KD, 20 MIS-C and 10 AC19. During the acute stage, the AC19 and KD patients had higher CECs levels than the MIS-C patients. From the acute to subacute phase, a significant CEC increase was observed in the KD patients, while a mild decrease was detected in the MIS-C patients. Cellular clusters/syncytia were more common in the KD patients. No correlation between CECs and CALs were found in the MIS-C patients. The incidence of CALs in the KD group was too low to investigate this correlation. CONCLUSIONS: Our study suggests a possible role of CECs as biomarkers of systemic inflammation and endothelial dysfunction in KD and MIS-C and different mechanisms of vascular injury in these diseases. Further larger studies are needed.

Programmed Cell Death Ligand 1-Expressing Circulating Tumor Cells: A New Prognostic Biomarker in Non-Small Cell Lung Cancer.

BACKGROUND: In non-small cell lung cancer (NSCLC), analysis of programmed cell death ligand 1 (PD-L1) expression in circulating tumor cells (CTCs) is a potential alternative to overcome the problems linked to the tumor biopsy spatiotemporal heterogeneity. However, the prognostic significance of PD-L1-positive [PD-L1(+)] CTCs remains controversial. METHODS: We prospectively evaluated the correlation with clinicopathological variables and prognostic value of PD-L1(+) CTCs, detected with the FDA-cleared CellSearch® system, in 54 patients with advanced NSCLC. RESULTS: We detected CTCs and PD-L1(+) CTCs in 43.4% and 9.4% of patients with NSCLC. PD-L1 expression concordance between tumor tissue and CTCs was low (54%). The presence of PD-L1(+) CTC correlated with the absence of gene alterations in tumor tissue and with poor prognosis-related biological variables (anemia, hyponatremia, increased lactate dehydrogenase). In univariate analysis, absence of gene alterations, number of metastatic sites, prior systemic therapies, and presence of CTCs and PD-L1(+) CTCs were associated with worse overall survival, whereas PD-L1 expression in tumor tissue was not. In multivariate analysis, squamous cell carcinoma histology, number of prior systemic treatments, and the presence of CTC were significantly associated with overall survival. Survival was worse in patients with PD-L1(+) CTCs than in patients with PD-L1-negative CTC or without any CTC. CONCLUSIONS: Our study suggests that the presence of PD-L1(+) CTCs is associated with poor prognosis in patients with advanced NSCLC. Studies with larger samples are needed to confirm our results and to determine how PD-L1(+) CTC detection could help to predict the response or resistance to anti-PD-1/PD-L1 therapies.Clinical trial registration NCT02866149.

Assessment of the Efficacy and Clinical Utility of Different Circulating Tumor Cell (CTC) Detection Assays in Patients with Chemotherapy-Naïve Advanced or Metastatic Non-Small Cell Lung Cancer (NSCLC).

We herein investigated the detection frequency and clinical relevance of circulating tumor cells (CTCs) in chemotherapy-naïve stage IIIB/IV non-small cell lung cancer (NSCLC), by using the CellSearch and real-time CEACAM5mRNA assays. Blood samples from 43 patients were obtained at different time points during first-line chemotherapy. CellSearch revealed the detection of ≥1 CTCs in 41.9%, 40.9%, and 16.7% of patients at baseline, post-1st, and post-2nd treatment cycle, respectively, and of ≥5 CTCs in 11.6%, 9.1%, and 5.6%, respectively. CEACAM5mRNA+ CTCs were detected in 29.3% and 16% of patients pre- and post-treatment, respectively. The positivity concordance between the two assays was 2.2%. CTC-detection by CellSearch (≥5 CTCs: p = 0.004), CEACAM5mRNA (p = 0.010), or by any assay (p = 0.000) was associated with disease progression. Reduced survival was demonstrated for patients harboring ≥5 CTCs (progression-free survival; PFS: p = 0.000; overall survival; OS: p = 0.009), CEACAM5mRNA+ CTCs (PFS: p = 0.043; OS: p = 0.039), and CTCs by any assay (PFS: p = 0.005; OS: p = 0.006, respectively). CTC-detection by any assay independently predicted for increased risk of relapse (hazard ratio; HR: 3.496; p = 0.001) and death (HR: 2.866; p = 0.008). CellSearch-positivity either pre-, post-1st, or post-2nd cycle, was predictive for shorter PFS (p = 0.036) compared to negativity in all time points. Persistent CEACAM5mRNA-positivity pre- and post-treatment was associated with reduced PFS (p = 0.036) and OS (p = 0.026). In conclusion, CTC detection and monitoring using the CellSearch and CEACAM5mRNA assays provides valuable and complementary clinical information for chemo-naïve advanced or metastatic NSCLC.

HER2 expression on tumor-derived extracellular vesicles and circulating tumor cells in metastatic breast cancer.

BACKGROUND: Tumor-derived extracellular vesicles (tdEVs) and circulating tumor cells (CTCs) in the blood of metastatic cancer patients associate with poor outcomes. In this study, we explored the human epidermal growth factor receptor 2 (HER2) expression on CTCs and tdEVs of metastatic breast cancer patients. METHODS: Blood samples from 98 patients (CLCC-IC-2006-04 study) were originally processed with the CellSearch® system using the CTC kit and anti-HER2 as an additional marker in the staining cocktail. CTCs and tdEVs were automatically enumerated from the generated CellSearch images using the open-source ACCEPT software. RESULTS: CTCs and tdEVs were subdivided based on their cytokeratin (CK) and HER2 phenotype into CK+HER2-, CK-HER2+, and CK+HER2+. The inclusion of anti-HER2 increased the percentage of informative samples with ≥ 1 detectable CTC from 89 to 95%. CK- CTCs and tdEVs correlated equally well with the clinical outcome as CK+ CTCs and tdEVs. Inter- and intra-patient heterogeneity was found for the CTC/tdEV phenotypes, and the presence of 2 or 3 classes of CTCs/tdEVs was associated with worse prognosis compared to a uniform CTC/tdEV phenotype present (1 class). The use of ≥ 7% HER2+CK+ tdEVs can predict HER2 expression of the tissue with 74% sensitivity and specificity using the HER2 amplification status of the primary tumor as a classification variable. CONCLUSIONS: HER2 can be detected on CTCs and tdEVs not expressing CK, and these CK- CTCs/tdEVs have similar clinical relevance to CTCs and tdEVs expressing CK. tdEVs perform better than CTCs in predicting the HER2 status of the primary tissue. CTC and tdEV heterogeneity in the blood of patients is inversely associated with overall survival.

Characterization of circulating tumor cells in breast cancer patients by spiral microfluidics.

Circulating tumor cells (CTCs) have important application prospects in the early diagnosis, treatment evaluation, and prognostic prediction of tumors. In this study, we enrolled a total of 65 patients with different stages and molecular subtypes of breast cancer and isolated and enriched for CTCs from peripheral blood using the ClearCell FX1 platform, which is based on a label-free spiral microfluidic method. The ClearCell platform can successfully isolate CTCs from peripheral blood with different detection rates in breast cancer patients. We also compared the difference between the ClearCell and CellSearch platforms for isolating CTCs. To further determine the genetic information of CTCs, we performed single-cell whole-exome sequencing (WES) in three CTCs isolated from one patient. The sequencing results indicated the presence of a few hundreds of single-nucleotide variants (SNVs) in each CTC, with only 16 SNVs being shared by all three CTCs. These shared SNVs may have a crucial impact on the development of breast cancer. Here, we report, for the first time, the complete process and results of performing single-cell WES on CTCs isolated by the ClearCell FX1 platform.

Toward a real liquid biopsy in metastatic breast and prostate cancer: Diagnostic LeukApheresis increases CTC yields in a European prospective multicenter study (CTCTrap).

Frequently, the number of circulating tumor cells (CTC) isolated in 7.5 mL of blood is too small to reliably determine tumor heterogeneity and to be representative as a "liquid biopsy". In the EU FP7 program CTCTrap, we aimed to validate and optimize the recently introduced Diagnostic LeukApheresis (DLA) to screen liters of blood. Here we present the results obtained from 34 metastatic cancer patients subjected to DLA in the participating institutions. About 7.5 mL blood processed with CellSearch® was used as "gold standard" reference. DLAs were obtained from 22 metastatic prostate and 12 metastatic breast cancer patients at four different institutions without any noticeable side effects. DLA samples were prepared and processed with different analysis techniques. Processing DLA using CellSearch resulted in a 0-32 fold increase in CTC yield compared to processing 7.5 mL blood. Filtration of DLA through 5 µm pores microsieves was accompanied by large CTC losses. Leukocyte depletion of 18 mL followed by CellSearch yielded an increase of the number of CTC but a relative decrease in yield (37%) versus CellSearch DLA. In four out of seven patients with 0 CTC detected in 7.5 mL of blood, CTC were detected in DLA (range 1-4 CTC). The CTC obtained through DLA enables molecular characterization of the tumor. CTC enrichment technologies however still need to be improved to isolate all the CTC present in the DLA.

Circulating tumor cells and survival in abiraterone- and enzalutamide-treated patients with castration-resistant prostate cancer.

BACKGROUND: The outcome to treatment administered to patients with metastatic castration-resistant prostate cancer (mCRPC) greatly differs between individuals, underlining the need for biomarkers guiding treatment decision making. OBJECTIVE: To investigate the prognostic value of circulating tumor cell (CTC) enumeration and dynamics, in the context of second-line endocrine therapies (ie, abiraterone acetate or enzalutamide), irrespective of prior systemic therapies. DESIGN, SETTINGS, AND PARTICIPANTS: In a prospective, multicentre study blood samples for CTC enumeration were collected from patients with mCRPC at baseline (n = 174). In patients who responded for minimally 10-12 weeks a follow-up sample was collected. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: For baseline analysis, patients were stratified in <5 or ≥5 CTCs/7.5 mL, whereas for the analysis of CTC dynamics at 10-12 weeks, in patients with stable, increasing or decreasing CTC counts. Progression-free survival (PFS), overall survival (OS), and PSA changes at 10-12 weeks were compared between groups. RESULTS: Patients demonstrating increasing CTCs on therapy had a shorter median PFS (4.03 vs 12.98 vs 13.67 months, HR 3.6, 95%CI 1.9-6.8; P < 0.0001) and OS (11.2 months vs not reached, HR 9.5, 95%CI 3.7-24; P < 0.0001), compared to patients with decreasing or stable CTCs. Multivariable Cox regression showed that prior chemotherapy (HR 4.1, 95%CI 1.9-8.9; P = 0.0003), a high baseline CTC count (HR 1.5, 95%CI 1.2-1.9; P = 0.002) and increasing CTCs at follow-up (HR 3.3, 95%CI 1.4-7.6; P = 0.005) were independent predictors of worse PFS. Previous chemotherapy (HR 7, 95%CI 1.9-25; P = 0.003), high baseline CTC counts (HR 2.2, 95%CI 1.4-3.7; P = 0.002) and increasing CTCs during therapy (HR 4.6, 95%CI 1.4-15; P = 0.01) were independently associated with shorter OS. ≥30% and ≥50% PSA responses less frequently occurred in patients with CTC inclines at 10-12 weeks on therapy (χ2 test: P < 0.01). CONCLUSIONS: CTC dynamics during therapy are associated with PSA response and provide independent clinical prognostication over PSA declines. Hence the study demonstrates the pharmacodynamic properties of CTCs.

A prospective study on the changes and clinical significance of pre-operative and post-operative circulating tumor cells in resectable gastric cancer.

BACKGROUND: Circulating tumor cells (CTCs) have been suggested as potential prognostic indicators for multiple tumors, including gastric cancer; however, pre- and post-operative CTC changes in resectable gastric cancer and possible correlations to post-operative recurrence have not been evaluated. METHODS: Subjects (n = 93) with resectable gastric cancer were prospectively reviewed from July 2013 to December 2014 at Peking University Cancer Hospital. The proportion of CTCs were evaluated before (n = 93) and after (n = 63) radical operation using a standardized CellSearch system. RESULTS: CTCs ≥ 1 were measured in the pre-operative blood of 31 (33.3%) patients and in the post-operative blood of 21 patients (33.3%). Patients with relatively poor clinicopathological features had more pre- and post-operative CTCs. The 3-year disease-free survival (DFS) rate for patients with CTCs ≥ 5/7.5 ml was significantly lower than for patients with CTCs < 5/7.5 ml (40.0% vs 66.4%, p < 0.001 for pre-surgery; 25.0% vs 62.2%, p < 0.001 for post-surgery). Patients with CTCs ≥ 5/7.5 ml in post-operative blood had significantly shorter mean DFS (1.28 vs 31.6 months; p = 0.002) and overall survival (OS; 10.0 vs 34.9 months; p = 0.001) than other patients. Among the 10 patients with hematogenous recurrence, 3 had post-operative CTCs ≥ 2/7.5 ml and had early recurrence (DFS 1.1, 1.1, 1.4 months). Moreover, DFS for the seven patients was 20.2, 11.9, 20.0, 6.0, 15.5, 25.9, 30.0 months, respectively. DFS for the three patients with increased CTCs after surgery was shorter than for patients with mildly increased, stable, or decreased CTCs. CONCLUSIONS: Pre- and post-operative CTCs are promising prognostic markers for resectable gastric cancer. Our study further suggests that increased post-operative CTCs may be correlated with hematogenous recurrence. Trial registration (ClinicalTrials.gov Identifier: NCT01848015). Registered 7 May 2013. https://clinicaltrials.gov/ct2/show/NCT01848015.

Diagnostic leukapheresis for CTC analysis in breast cancer patients: CTC frequency, clinical experiences and recommendations for standardized reporting.

Diagnostic leukapheresis (DLA) is based on continuous centrifugation that collects mononuclear cells from peripheral blood with a density of 1.055-1.08 g/ml. As epithelial cells have a similar density, DLA cocollects circulating tumor cell (CTCs) along with the targeted mononuclear cells. Here, we report on our single center experience applying DLA in 40 nonmetastatic and metastatic breast cancer patients and its impact on CTC detection. We found that the use of just 5% of the DLA product (corresponding to a median peripheral blood volume of around 60 ml) in the CellSearch® assay already leads to a significant increase in CTC detection frequency and yield. The implementation of the method was unproblematic, and we did not observe any adverse events in our patient cohort. Extrapolating the CTC counts in the DLA samples to the whole DLA product indicated that enormous CTC numbers could be harvested by this approach (around 205x more CTCs than in the 7.5 ml blood sample in M1 patients). In conclusion, DLA is a clinically safe method to collect CTCs from liters of blood enabling a real liquid biopsy. Yet, further technical developments are required to process whole DLA products and exploit the full potential of this approach. As it is foreseeable that DLA will be used by several groups, and hopefully ultimately brought to the patients in a routine setting, we discuss recommendations on the minimum of required information for reporting on DLAs to allow comparison across different approaches. © 2018 International Society for Advancement of Cytometry.

Filtration based assessment of CTCs and CellSearch® based assessment are both powerful predictors of prognosis for metastatic breast cancer patients.

BACKGROUND: The assessment of circulating tumor cells (CTCs) has been shown to enable monitoring of treatment response and early detection of metastatic breast cancer (MBC) recurrence. The aim of this study was to compare a well-established CTC detection method based on immunomagnetic isolation with a new, filtration-based platform. METHODS: In this prospective study, two 7.5 ml blood draws were obtained from 60 MBC patients and CTC enumeration was assessed using both the CellSearch® and the newly developed filtration-based platform. We analyzed the correlation of CTC-positivity between both methods and their ability to predict prognosis. Overall survival (OS) was calculated and Kaplan-Meier curves were estimated with thresholds of ≥1 and ≥5 detected CTCs. RESULTS: The CTC positivity rate of the CellSearch® system was 56.7% and of the filtration-based platform 66.7%. There was a high correlation of CTC enumeration obtained with both methods. The OS for patients without detected CTCs, regardless of the method used, was significantly higher compared to patients with one or more CTCs (p < 0.001). The median OS of patients with no CTCs vs. ≥ 1 CTC assessed by CellSearch® was 1.83 years (95% CI: 1.63-2.02) vs. 0.74 years (95% CI: 0.51-1.52). If CTCs were detected by the filtration-based method the median OS times were 1.88 years (95% CI: 1.74-2.03) vs. 0.59 years (95% CI: 0.38-0.80). CONCLUSIONS: The newly established EpCAM independently filtration-based system is a suitable method to determine CTC counts for MBC patients. Our study confirms CTCs as being strong predictors of prognosis in our population of MBC patients.

EpCAM-based assays for epithelial tumor cell detection in cerebrospinal fluid.

The diagnosis of leptomeningeal metastases (LM) of solid tumors is complicated due to low sensitivities of both magnetic resonance imaging (MRI) and cytology. MRI has a sensitivity of 76% for the diagnosis of LM and cerebrospinal fluid (CSF) cytology has a sensitivity of 44-67% at first lumbar puncture which increases to 84-91% upon second CSF sampling. Epithelial cell adhesion molecule (EpCAM) is expressed by solid tumors of epithelial origin like non-small-cell lung cancer, breast cancer or ovarium cancer. Recently, a CELLSEARCH® assay and flow cytometry laboratory techniques have been developed to detect circulating tumor cells (CTCs) of epithelial origin in CSF. These laboratory techniques are based on capture antibodies labelled with different fluorescent tags against EpCAM. In this review, we provide an overview of the available laboratory techniques and diagnostic accuracy for tumor cell detection in CSF. The reported sensitivities of the EpCAM-based CTC assays for the diagnosis of LM across the different studies are highly promising and vary between 76 and 100%. An overview of the different EpCAM-based techniques for the enumeration of CTCs in the CSF is given and a comparison is made with CSF cytology for the diagnoses of LM from epithelial tumors.

A Novel Workflow to Enrich and Isolate Patient-Matched EpCAMhigh and EpCAMlow/negative CTCs Enables the Comparative Characterization of the PIK3CA Status in Metastatic Breast Cancer.

Circulating tumor cells (CTCs), potential precursors of most epithelial solid tumors, are mainly enriched by epithelial cell adhesion molecule (EpCAM)-dependent technologies. Hence, these approaches may overlook mesenchymal CTCs, considered highly malignant. Our aim was to establish a workflow to enrich and isolate patient-matched EpCAMhigh and EpCAMlow/negative CTCs within the same blood samples, and to investigate the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutational status within single CTCs. We sequentially processed metastatic breast cancer (MBC) blood samples via CellSearch® (EpCAM-based) and via Parsortix™ (size-based) systems. After enrichment, cells captured in Parsortix™ cassettes were stained in situ for nuclei, cytokeratins, EpCAM and CD45. Afterwards, sorted cells were isolated via CellCelector™ micromanipulator and their genomes were amplified. Lastly, PIK3CA mutational status was analyzed by combining an amplicon-based approach with Sanger sequencing. In 54% of patients' blood samples both EpCAMhigh and EpCAMlow/negative cells were identified and successfully isolated. High genomic integrity was observed in 8% of amplified genomes of EpCAMlow/negative cells vs. 28% of EpCAMhigh cells suggesting an increased apoptosis in the first CTC-subpopulation. Furthermore, PIK3CA hotspot mutations were detected in both EpCAMhigh and EpCAMlow/negative CTCs. Our workflow is suitable for single CTC analysis, permitting-for the first time-assessment of the heterogeneity of PIK3CA mutational status within patient-matched EpCAMhigh and EpCAMlow/negative CTCs.

Detection of disseminated tumor cells in aspirative drains after neck dissection.

The dissemination of individual tumor cells is a common phenomenon in solid cancers. Detection of tumor cells in bone marrow disseminated tumor cells (DTC) and in peripheral blood circulating tumor cells (CTC) in nonmetastatic situation is of high prognostic significance. Compared to breast, colon and prostate cancers, the studies on CTC and DTC in head and neck cancers are sparse. The objective of our study was to detect DTC in drains after neck dissection. Fourteen patients undergoing surgery for stages III and IV head and neck cancers were enrolled in this study--twelve presenting with squamous cell carcinoma and two with adenocarcinoma. Redon drain analysis was performed by the Cellsearch method using immunomagnetic and fluorescence approaches. A positivity threshold value was set at 2DTC/7.5 ml of the sample. Tumor cells were detected in drains of 69 % of patients a few days after surgery. The range of quantification was 3-2,094 DTC/5 ml and we showed morphological differences between the two types of carcinoma cells. DTC were detected after neck dissection both in squamous cell carcinoma and in adenocarcinoma. Potential clinical significance of tumor cells needs to be further investigated as their presence could affect pre-surgical and post-operative treatments.

Cytometric characterization of circulating tumor cells captured by microfiltration and their correlation to the CellSearch(®) CTC test.

Recent studies reporting hundreds, to thousands, of circulating tumor cells (CTCs) in the blood of cancer patients have raised questions regarding the prevalence of CTCs, as enumerated by the CellSearch(®) CTC Test. Although CellSearch has been shown to consistently detect clinically relevant CTCs; the ability to only capture EpCAM positive cells has led to speculation that it captures limited subsets of CTCs. In contrast, alternative approaches to CTC isolation are often cited as capturing large numbers of CTCs from patient blood. Not surprisingly the number of cells isolated by alternative approaches show poor correlations when compared to CellSearch, even when accounting for EpCAM presence or absence. In an effort to address this discrepancy, we ran an exploratory method comparison study to characterize and compare the CTC subgroups captured from duplicate blood samples from 30 breast and prostate cancer patients using a microfiltration system (CellSieve™) and CellSearch. We then categorized the CellSieve Cytokeratin(CK)+/CD45-/DAPI+ cells into five morphologically distinct subpopulations for correlative analysis. Like other filtration techniques, CellSieve isolated greater numbers of CK+/CD45- cells than CellSearch. Furthermore, analysis showed low correlation between the total CK+/CD45- cells captured by these two assays, regardless of EpCAM presence. However, subgrouping of CK+/CD45-/DAPI+ cells based on distinct cytokeratin staining patterns and nuclear morphologies elucidated a subpopulation correlative to CellSearch. Using method comparison analyses, we identified a specific CTC morphology which is highly correlative between two distinct capture methods. These data suggests that although various morphologic CTCs with similar phenotypic expressions are present in the blood of cancer patients, the clinically relevant cells isolated by CellSearch can potentially be identified using non-EpCAM dependent isolation. © 2014 The Authors. Published by Wiley Periodicals, Inc.

New frontiers in circulating tumor cell analysis: A reference guide for biomolecular profiling toward translational clinical use.

Circulating tumor cells (CTCs) are now routinely isolated from blood, and measurement of CTC concentrations appears to correlate well with survival in patients with cancer. Interrogation of the molecular profile of CTCs for expression of protein biomarkers, genetic variants and gene expression provides opportunities to use this information to guide personalized treatment, monitor therapy and detect emerging resistance. However, successful application of profiling techniques requires analyses that deliver a reliable and clinically relevant representation of a patient's cancer as it changes with time. Here, we comprehensively review the current knowledge of therapeutically relevant biomarkers in isolated CTCs obtained by fluorescence imaging and genomic profiling approaches. The reviewed data support the notion that molecular profiling of CTCs will provide a reliable representation or surrogate index of tumor burden. Large-scale translational trials, many currently in progress, will provide critical data to progress CTC analysis toward wider clinical use in personalized treatment.

Circulating tumor cells in breast cancer.

Circulating tumor cell (CTC) measurement in peripheral blood of patients with breast cancer offers prognostic information. In this review, we will try to identify evidence that could be used for prognosis, predictive power to draw this tool to clinical utility. We reviewed 81 manuscripts, and categorized those in discovery datasets, prognostic factors in metastatic breast cancer, identification of clinical utility in early breast cancer and in novel approaches. With each patient responding differently to chemotherapy, more efficient markers would improve clinical outcome. Current CTC diagnostic techniques use epithelial markers predominantly; however, the most appropriate method is the measurement of circulating DNA. It has been hypothesized that micrometastasis occurs early in the development of tumors. That implies the presence of CTCs in nonmetastatic setting. The origin of stimulus for malignant transformation is yet unknown. The role of microenvironment as a stimulus is also being investigated. It has been shown that CTCs vary in numbers with chemotherapy. The markers, which are followed-up in the primary tumors, are also being studied on the CTCs. There is discordance of the human epidermal growth factor receptor-2 status between the primary tumor and CTCs. This review summarizes our current knowledge about the CTCs. With genetic profiling and molecular characterization of CTCs, it is possible to overcome the diagnostic difficulties. Evidence for clinical utility of CTC as prognostic and predictive marker is increasing. Appropriate patient stratification according to CTC determination among other tests, would make personalized cancer therapy more feasible.