Detection Of Circulating Tumor Cells In Patients Research Articles

Monitoring with circulating tumor cells in the perioperative setting of patients with surgically treated stages I-IIIA NSCLC.

Surgery is regarded as the treatment's cornerstone for early stage and locally advanced non-small cell lung cancer (NSCLC) whenever the tumor is considered resectable. Liquid biopsy is one of the most promising research areas in oncology in the last 10 years, providing a useful non-invasive tool to detect and monitor cancer. The prognostic value of circulating tumor cells (CTCs) has been studied in different cancer types and had been related with a higher risk of relapse and worse prognosis. The aim of this study is to evaluate the prognostic value of CTC detection in patients with stage I-IIIA NSCLC treated with surgery. We conducted a prospective, single-center study of 180 consecutive patients with resected and pathological confirmed stage I to IIIA (TNM AJCC/UICC 8th edition) NSCLC. Patients' blood samples were processed and CTCs were characterized before and after the surgery. A cohort of patients had CTC determination after chemotherapy and surgery. Cut-off points were established in 1 and 5 CTCs for statistical analysis. A proportion of 76.7% had at least 1 CTC before the surgery, and 30.6% had 5 or more, while 55.9% had at least 1 CTC after surgery, and 8.3% had 5 or more. We found no correlation between preoperative CTC detection for a cut-off of 5 with neither overall survival (OS) [hazard ratio (HR): 0.99, P=0.887], disease-free survival (DFS) (HR: 0.95, P=0.39) nor relapse (32.7% vs. 28.8%, P=0.596). We also did not find a correlation between postoperative CTCs detection for a cut-off of 5 with either OS (HR: 1.01, P=0.808), DFS (HR: 0.95, P=0.952) or relapse (26.7% vs. 29.5%, P=0.83). The mean change in the number of CTCs over time between preoperative and postoperative samples was 2.13, with a standard deviation of 6.78. Despite the large cohort of patients included in this study, CTC monitoring in the perioperative setting was not correlated with relapse, DFS or OS in our study, and therefore cannot be recommended as a reliable biomarker for minimal residual disease (MRD) after surgery.

Circulating tumor cells (CTCs) and hTERT gene expression in CTCs for radiotherapy effect with lung cancer

BackgroundCirculating tumor cells (CTCs) are important biological indicators of the lung cancer prognosis, and CTC counting and typing may provide helpful biological information for the diagnosis and treatment of lung cancer.MethodsThe CTC count in blood before and after radiotherapy was detected by the CanPatrol™ CTC analysis system, and the CTC subtypes and the expression of hTERT before and after radiotherapy were detected by multiple in situ hybridization. The CTC count was calculated as the number of cells per 5 mL of blood.ResultsThe CTC positivity rate in patients with tumors before radiotherapy was 98.44%. Epithelial–mesenchymal CTCs (EMCTCs) were more common in patients with lung adenocarcinoma and squamous carcinoma than in patients with small cell lung cancer (P = 0.027). The total CTCs (TCTCs), EMCTCs, and mesenchymal CTCs (MCTCs) counts were significantly higher in patients with TNM stage III and IV tumors (P < 0.001, P = 0.005, and P < 0.001, respectively). The TCTCs and MCTCs counts were significantly higher in patients with an ECOG score of > 1 (P = 0.022 and P = 0.024, respectively). The TCTCs and EMCTCs counts before and after radiotherapy affected the overall response rate (ORR) (P < 0.05). TCTCs and ECTCs with positive hTERT expression were associated with the ORR of radiotherapy (P = 0.002 and P = 0.038, respectively), as were TCTCs with high hTERT expression (P = 0.012). ECOG score (P = 0.006) and post-radiation TCTCs count (P = 0.011) were independent factors for progression-free survival (PFS) and TNM stage (P = 0.054) and pre-radiation EMCTCs count (P = 0.009) were independent factors of overall survival (OS).ConclusionThis study showed a high rate of positive CTC detection in patients with lung cancer, and the number, subtype, and hTERT-positive expression of CTCs were closely related to patients’ ORR, PFS, and OS with radiotherapy. EMCTCs, hTERT-positive expression of CTCs are expected to be important biological indicators for predicting radiotherapy efficacy and the prognosis in patients with lung cancer. These results may be useful in improving disease stratification for future clinical trials and may help in clinical decision-making.

Detection of Circulating Tumor Cells in Cerebrospinal Fluid of Patients with Suspected Breast Cancer Leptomeningeal Metastases: A Prospective Study.

The diagnosis of breast cancer (BC)-related leptomeningeal metastases (LM) relies on the detection of tumor cells in cerebrospinal fluid (CSF) using conventional cytology (gold standard). However, the sensitivity of this technique is low. Our goal was to evaluate whether circulating tumor cell (CTC) detection in CSF using the CellSearch® system could be used for LM diagnosis. This prospective, monocentric study included adult patients with suspected BC-related LM. The clinical sensitivity and specificity of CTC detection in CSF for LM diagnosis were calculated relative to conventional CSF cytology. Forty-nine eligible patients were included and 40 were evaluable (CTC detection technical failure: n = 8, eligibility criteria failure: n = 1). Cytology was positive in 18/40 patients. CTCs were detected in these 18 patients (median: 5824 CTC, range: 93 to 45052) and in 5/22 patients with negative cytology (median: 2 CTC, range: 1 to 44). The detection of ≥1 CSF CTC was associated with a clinical sensitivity of 100% (95% CI, 82.4-100) and a specificity of 77.3% (95% CI, 64.3-90.3) for LM diagnosis. HER2+ CTCs were detected in the CSF of 40.6% of patients with HER2- BC (median: 500 CTC, range: 13 to 28 320). The clinical sensitivity of CTC detection in CSF with the CellSearch® system for LM diagnosis is higher than that of CSF cytology. CTC detection in patients with negative cytology, however, must be further investigated. The finding of HER2+ CTCs in patients with HER2- BC suggests that the HER2 status of LM should be evaluated to increase the treatment opportunities for these patients.

A preliminary study on the association between epithelial-mesenchymal transition and circulating tumor cells in renal cell carcinoma.

BackgroundCirculating tumor cells (CTCs) are considered useful prognostic factors for various cancers, and in 2014, our research group conducted a comparative experiment of CTC detection in patients with renal cell cancer (RCC). However, the reason for the low detection rate of CTCs in cancer patients using the CellSearch® system is still unknown, although it has been hypothesized to be attributed to the likelihood that CTCs undergoing epithelial-mesenchymal transition (EMT) do not express the CTC biomarkers cytokeratin (CK)8/18/19 or epithelial cell adhesion molecule (EpCAM). The overall aim of the current study was to investigate the expression levels of CK8/18/19 and EpCAM in relation to the EMT biomarkers vimentin and E-cadherin in patients with RCC.MethodsPatients with RCC who had undergone radical nephrectomy or partial resection between May 2014 and December 2014 were initially recruited.ResultsAmong 34 RCC patients, nine co-expressed EpCAM and CK8/18/19 in primary tumor tissues. The CellSearch® results showed that CK8/18/19 was expressed in 5 of 6 patients (5/6) and EpCAM was expressed in 6 patients (6/6). However, the isolation by size of tumor cells (ISET) technique showed these were co-expressed in only four of the 10. The expression of CK8/18/19, EpCAM, vimentin, and E-cadherin was distributed unequally in different enumeration groups of CTCs (all P>0.05), and the positive expression of CK8/18/19 was correlated with neutrophil number and tumor size (P<0.05). The positive expression of vimentin was correlated with the Karnofsky Performance Status (KPS) score and clinical stage of renal cancer patients (P<0.05).ConclusionsOur results indirectly proved the occurrence of EMT in the formation of CTCs by comparing and analyzing the expression of CK8/18/19 and EpCAM in renal cancer tissues and the detection results of CTCs.

EMT-independent detection of circulating tumor cells in human blood samples and pre-clinical mouse models of metastasis

Circulating tumor cells (CTCs) present an opportunity to detect/monitor metastasis throughout disease progression. The CellSearch® is currently the only FDA-approved technology for CTC detection in patients. The main limitation of this system is its reliance on epithelial markers for CTC isolation/enumeration, which reduces its ability to detect more aggressive mesenchymal CTCs that are generated during metastasis via epithelial-to-mesenchymal transition (EMT). This Technical Note describes and validates two EMT-independent CTC analysis protocols; one for human samples using Parsortix® and one for mouse samples using VyCap. Parsortix® identifies significantly more mesenchymal human CTCs compared to the clinical CellSearch® test, and VyCap identifies significantly more CTCs compared to our mouse CellSearch® protocol regardless of EMT status. Recovery and downstream molecular characterization of CTCs is highly feasible using both Parsortix® and VyCap. The described CTC protocols can be used by investigators to study CTC generation, EMT and metastasis in both pre-clinical models and clinical samples.

CELL MEDIATED IMMUNITY IN PATIENTS WITH STAGE II–IV COLORECTAL CANCER WITH OR WITHOUT CIRCULATING TUMOR CELLS

Nowadays, circulating tumor cells (CTCs) are considered to be one of the most important mechanisms of tumor dissemination. Detection of CTCs in patients` blood is estimated as a prognostic factor for various tumors including colorectal cancer (CRC). However, CTCs may also be a source of tumor antigens capable of inducing both immune response and tolerance and participating in «immmunoediting». The aim of the study was to assess the parameters of cell-mediated immunity in patients with stage IIIV CRC with respect to the presence or absence of CTCs. Materials and Methods . We studied the parameters of cell-mediated immunity in 60 patients with stage IIIV CRC with respect to the presence or absence of CTCs. Before treatment, we evaluated the CTC levels in patients’ blood using CellSearch System™ and lymphocyte subsets: Т-В-NК-cells, T-reg, CD4+ and CD8+ expressing markers of activation (CD69+, СD38+, CD25+, HLA-DR+ СD95+); naïve and memory T-lymphocytes (CD45RA-/CD45RO+); NК-cells expressing CD335, perforin and granzyme B using flow cytometry (FACSCantoII, BD). Results . A difference in the immunologic parameters between patients with CTCs and without CTCs depending on the stage of CRC was found. In СTС-positive patients with locally-advanced CRC, increase in the parameters of innate immunity (CD335+ NK-cells, neutrophils` respiratory burst) and activated Th (CD38+, CD25+, HLA-DR+) was found, while in СTС-positive patients with generalized CRC, suppression of cytotoxic lymphocytes of both innate (CD56/16+) and adaptive (CD8+CD25+) immunity was observed, which is, apparently, an unfavorable prognostic factor. Conclusion . The presence of CTC in CRC patients is accompanied by some immunologic changes rather stimulating Thlink and innate immunity factors in II-III stages and doubtlessly suppressive in patients with IV stage.

Evaluating the Potential for Circulating Tumor Cells as a Diagnostic and Prognostic Biomarker in Various Cancer Types

Circulating tumor cells (CTCs) are cells that can be found circulating in the peripheral blood of cancer patients. They originate from primary solid tumors and are thought to contribute to metastases and poor prognosis. Since cancer treatment is shifting toward greater personalization, a major goal in the field is the development of less invasive and more cost-effective measures in diagnosis, staging, treatment, prognostic implications, and surveillance of cancer. Utilizing CTCs as a biomarker from a “liquid biopsy” or sample of patients’ blood would be transformative in accomplishing this goal. In this review, we aim to critically assess current pre-clinical and clinical literature over the past two decades implicating CTCs’ potential for use as a predictive biomarker in various cancer types either in addition to or instead of current standards of care. We also are interested in understanding several aspects of CTCs including the role CTCs play in resistance to treatment, the immune system evasion properties of CTCs, the feasibility of using CTCs in clinical practice, and the utility of CTCs for predicting outcomes including patient survival. Furthermore, here we discuss gaps in the literature, limitations of CTCs, potential for their other uses, as well as the significance of CTC detection in patients following surgery.

Determination of the optimal detection time of circulating tumor cells for the postoperative monitoring of colorectal cancer.

Circulating tumor cells (CTCs) are widely used in cancer screening and monitoring. The present study focused on investigating the optimal time for the postoperative CTC detection in patients with colorectal cancer (CRC) to obtain more accurate results and facilitate subsequent treatment. By subtraction enrichment immunofluorescence in situ hybridization detection of CTCs, the present study demonstrated that different postoperative detection times in CRC substantially influenced the CTC numbers. In total, 134 subjects were enrolled. Among 10 healthy individuals and 20 preoperative patients with CRC, no CTCs were identified in the healthy subjects, and CTCs were detected in 85% (17/20) of the preoperative patients. In total, 104 postoperative patients with CRC (53 males and 51 females) with a mean average age of 57.63 years were studied. The total CTC detection rate was 81.73% (85/104) and the mean average CTC numbers in patients with tumor stage (T) T1, T2, T3 and T4 were 4.00, 3.33, 5.90 and 5.64 per 7.50 ml of peripheral blood, respectively. The CTC number trends in these four tumor stages within 5, 6, 7 and 10 postoperative days were variable, and were the most stable at 7 days. Gradual upward trends in CTC numbers were observed after 5, 6 and 7 postoperative days, and this upward trend was more obvious after 7 days. Overall, the findings of the present study suggest that CTC detection in CRC should be performed after at least 7 postoperative days rather than within 7 postoperative days.

Circulating Tumor Cell Detection In Epithelial Ovarian Cancer Using Dual-Component Antibodies Targeting EpCAM And FRα.

PurposeCirculating tumor cell (CTC) detection methods based on epithelial cell adhesion molecule (EpCAM) have low detection rates in epithelial ovarian cancer (EOC). Meanwhile, folate receptor alpha (FRα) has high expression in EOC cells. We explored the feasibility of combining FRα and EpCAM as CTC capture targets in EOC.Patients and methodsEpCAM and FRα antibodies were linked to magnetic nanospheres (MNs) using the principle of carbodiimide chemistry. Blood samples from healthy donor spiked with A2780 ovarian cancer cells were used for detecting the capture rate. Ninety-five blood samples from 30 patients with EOC were used for comparing the positive rate of detection when using anti-EpCAM-MNs alone with that when using combination of anti-EpCAM-MNs and anti-FRα-MNs. Samples from 28 patients initially diagnosed with EOC and 20 patients with ovarian benign disease were used for evaluating the sensitivity and specificity of combination of anti-EpCAM-MNs and anti-FRα-MNs.ResultsRegression analysis between the number of recovered and that of spiked A2780 cells revealed yEpCAM = 0.535x (R2 = 0.99), yFRα = 0.901x (R2 = 0.99), and yEpCAM+FRα = 0.928x (R2 = 0.99). In mixtures of A2780 and MCF7 cells, the capture rate was 92% using the combination of anti-EpCAM-MNs and anti-FRα-MNs, exceeding the rate when using anti-EpCAM-MNs or anti-FRα-MNs alone by approximately 20% (P < 0.01). The combination of anti-EpCAM-MNs and anti-FRα-MNs showed a significantly increased positive rate of CTC detection in EOC patients compared with anti-EpCAM-MNs alone (χ2 = 14.45, P < 0.001). Sensitivity values were 0.536 and 0.75 and specificity values were 0.9 and 0.85 when using anti-EpCAM-MNs alone and when using the combination of anti-EpCAM-MNs and anti-FRα-MNs, respectively.ConclusionThe combination of FRα and EpCAM is feasible as a CTC capture target of CTC detection in patients with EOC.

105P - Detection and clinical significance of circulating tumour cells in patients with rectal cancer

BackgroundRectal cancer is one of the most common causes of cancer-related deaths worldwide. Circulating tumor cells (CTCs) are rare aneusomic cells that detach from the primary solid tumors and enter the circulation, and can initiate metastasis. Detection of CTCs in liquid biopsies is a promising strategy for diagnosing, monitoring the relapse and metastasis, and evaluating cancer prognosis and therapy. However, CTC detection in patients with rectal cancer is limited in routine clinical practice. The aim of this study was to elucidate the role of CTCs in patients with rectal cancer. MethodsA total of 142 patients with rectal cancer were enrolled. CTCs were measured in the peripheral blood (preoperatively, immediately postoperative, day 1 and 7 postoperatively) and inferior mesenteric vein (intraoperatively), usingthe CTCBIOPSY® System (YZYBIO Company, Wuhan, China). General information, initial diagnosis and the integrated pathological information of all participants were recorded. Data Analysis was performed using the Chi-square test, with 95% confidence intervals (95% CIs) as the threshold for statistical significance. ResultsCTCs were detected (≥1 CTC per 5ml blood) in the blood preoperatively more frequently in patients with a higher grade of TNM stage (c2=7.317, P = 0.048) and there was a trend of association (c2=6.457, P = 0.012). CTC detection in the blood was significantly associated with T stage (c2=12.912, P = 0.005) and N stage (c2=17.587, P < 0.001) preoperatively. It proved that age and gender were uncorrelated variables with CTC detection. Compared with CTC detection preoperatively, postoperative CTCs are firstly increased and then decreased. ConclusionsCTC detection is closely related to TNM stage in patients with rectal cancer, and so it may be a way to predict clinicopathological stage by detecting CTCs. The postoperative CTCs are potential biomarkers for rectal cancer prognosis but needed to be verified in more studies. Clinical trial identificationNCT02955173; Pre-results. Legal entity responsible for the studyQilu Hospital of Shandong University. FundingMinistry of Science and Technology of China. DisclosureThe author has declared no conflicts of interest.

Synchronous Detection of Circulating Tumor Cells in Blood and Disseminated Tumor Cells in Bone Marrow Predicts Adverse Outcome in Early Breast Cancer.

We examined the prognostic impact of circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) detected at the time of surgery in 742 untreated patients with early breast cancer. DTCs in bone marrow were enumerated using the EPCAM-based immunomagnetic enrichment and flow cytometry (IE/FC) assay. CTCs in blood were enumerated either by IE/FC or CellSearch. Median follow-up was 7.1 years for distant recurrence-free survival (DRFS) and 9.1 years for breast cancer-specific survival (BCSS) and overall survival (OS). Cox regressions were used to estimate hazard ratios for DRFS, BCSS, and OS in all patients, as well as in hormone receptor-positive (HR-positive, 87%) and HR-negative (13%) subsets. In multivariate models, CTC positivity by IE/FC was significantly associated with reduced BCSS in both all (n = 288; P = 0.0138) and HR-positive patients (n = 249; P = 0.0454). CTC positivity by CellSearch was significantly associated with reduced DRFS in both all (n = 380; P = 0.0067) and HR-positive patients (n = 328; P = 0.0002). DTC status, by itself, was not prognostic; however, when combined with CTC status by IE/FC (n = 273), double positivity (CTC+/DTC+, 8%) was significantly associated with reduced DRFS (P = 0.0270), BCSS (P = 0.0205), and OS (P = 0.0168). In HR-positive patients, double positivity (9% of 235) was significantly associated with reduced DRFS (P = 0.0285), BCSS (P = 0.0357), and OS (P = 0.0092). Detection of CTCs in patients with HR-positive early breast cancer was an independent prognostic factor for DRFS (using CellSearch) and BCSS (using IE/FC). Simultaneous detection of DTCs provided additional prognostic power for outcome, including OS.

Abstract 1366: Significance of circulating tumor cells (CTCs) with fibroblast growth factor 2 expression in gastric cancer patients

Abstract [Background] Amplification of the fibroblast growth factor receptor 2 (FGFR2) is reportedly identified in 3-10% of primary gastric cancers (GCs). We found that FGFR2 overexpression was closely associated with poor prognosis of patients with gastric cancer. Recently, FGFR signaling inhibitors have been considered a key drug in the treatment of GCs with FGFR2 overexpression. FGFR2 overexpression can be assessed by immunohistochemical staining of primary tumors; however, this method does not sufficiently reflect FGFR2 expression in recurrent tumors owing to the heterogeneity of tumor cells between primary and recurrent tumors. Circulating tumor cells (CTCs) might overcome this issue of “temporal” and “spatial” heterogeneity. This study aimed to assess the significance of the detection of FGFR2-positive CTCs in patients with gastric cancer. [Methods] Seventy-eight patients with gastric cancer who underwent gastrectomy in our hospital were enrolled in this study. A total volume of 10 ml of peripheral blood was collected prior to surgery from each patient, and mononuclear cells were enriched by Ficol density gradient centrifugation. These cells were immunostained with PI/CD45/EpCAM/FGFR2 and PI/CD45/EpCAM/CK. The number of cells in each sample was enumerated depending on the positivity of EpCAM, FGFR2, and CK by fluorescence-activated cell sorting (FACS). FGFR2 expression was assessed by immunohistochemical staining of resected specimens. Patients were categorized into four groups as follows: group IHC-0 (n=32), no staining; group IHC-1+ (n=24), cytoplasmic staining in 1-19% or membranous staining in 1-5%; group IHC-2+ (n=16), cytoplasmic staining in 80-100% or membranous staining in 5-19%; group IHC-3+ (n=6), membranous staining in 20-100%. [Results] FGFR2+ cells were detected in 43 (55%) of 78 cases. Among these cases, CK+ cells or EpCAM+ cells were detected in 35 (81%) of 43 cases. By group, the proportions of FGFR2+ cells were 44% in group IHC-0, 52% in group IHC-1+, 69% in group IHC-2+, and 100% in group IHC-3+. The mean number of FGFR2+ cells from patient samples in the IHC-0, IHC-1+, IHC-2+, and IHC-3+ groups were 12, 11, 16, and 54 (per 10 ml peripheral blood), respectively, with a significant difference between group IHC-0 and group IHC-3+, group IHC-1+ and group IHC-3+, and group IHC-0 and group IHC-2+ (p=0.001, p=0.008 and p=0.034, respectively, by Mann-Whitney U test). Among 68 patients with R0 and pStage I-III, the recurrence-free survival of patients with an FGFR2+ cell count ≧10 cells / 10 ml was significantly poorer than that of patients with an FGFR2+ cell count &amp;lt;10 cells / 10 ml (p = 0.018, by logrank test). [Conclusion] Detection of CTCs with FGFR2 expression by FACScan might be a useful approach to identify gastric cancer patients who would benefit the most from FGFR inhibitor treatment. Citation Format: Kenji Kuroda. Significance of circulating tumor cells (CTCs) with fibroblast growth factor 2 expression in gastric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1366.

Clinical Application of Circulating Tumor Cells and Circulating Tumor DNA in Uveal Melanoma

PurposeTo evaluate the feasibility of using circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) for the management of uveal melanoma (UM).Patients and MethodsLow-coverage whole-genome sequencing was used to determine somatic chromosomal copy number alterations (SCNAs) in primary UM tumors, ctDNA, and whole-genome amplified CTCs. CTCs were immunocaptured using an antimelanoma-associated chondroitin sulfate antibody conjugated to magnetic beads and immunostained for melanoma antigen recognised by T cells 1 (MART1)/glycoprotein 100 (gp100)/S100 calcium-binding protein β (S100β). ctDNA was quantified using droplet digital polymerase chain reaction assay for mutations in the GNAQ, GNA11, PLCβ4, and CYSLTR2 genes.ResultsSCNA analysis of CTCs and ctDNA isolated from a patient with metastatic UM showed good concordance with the enucleated primary tumor. In a cohort of 30 patients with primary UM, CTCs were detected in 58% of patients (one to 37 CTCs per 8 mL of blood), whereas only 26% of patients had detectable ctDNA (1.6 to 29 copies/mL). The presence of CTCs or ctDNA was not associated with tumor size or other prognostic markers. However, the frequent detection of CTCs in patients with early-stage UM supports a model in which CTCs can be used to derive tumor-specific SCNA relevant for prognosis. Monitoring of ctDNA after treatment of the primary tumor allowed detection of metastatic disease earlier than 18F-labeled fluorodeoxyglucose positron emission tomography in two patients.ConclusionThe presence of CTCs in localized UM can be used to ascertain prognostic SCNA, whereas ctDNA can be used to monitor patients for early signs of metastatic disease. This study paves the way for the analysis of CTCs and ctDNA as a liquid biopsy that will assist with treatment decisions in patients with UM.

Monitoring of Circulating Tumor Cells in Patients With High-Risk Nonmetastatic Prostate Cancer Treated With Radiation Therapy and Hormones: Preliminary Data of an Observational Prospective Phase 2 Study

Circulating tumor cells (CTCs) are a cellular source repeatedly obtainable by blood sampling that could serve as a surrogate marker for treatment efficacy. The presence of CTCs is an established marker for prognosis in men with castration-resistant prostate cancer. In contrast, the value of CTCs detection in men with localized high-risk prostate cancer is unknown. The purpose of this study is to evaluate the proportion of patients with high-risk prostate cancer (HRPCa) that presents CTCs in peripheral blood (PB) before and after androgen deprivation therapy (ADT) and radiation therapy (RT) in order to establish its prognostic value. We present preliminary data of CTCs monitoring detection. Hypothesis: The detection of CTCs is a potential surrogate marker for treatment efficacy in HRPCa. Prospective analysis of biological material obtained from PB samples in 65 patients with HRPCa treatment naïve between 2014 and 2016. Blood samples were analyzed to detect CTCs using an immunomagnetic method based on the CellSearch system in 4 periods of time: 1) Before starting any treatment; 2) following neoadjuvant ADT and prior to RT; 3) at the end of RT; 4) 6-12 months following the end of RT in those patients with 0 CTCs in the first determination and positive CTCs in the following determinations. A cut off of 0 vs ≥ 1CTCs/7.5ml blood was defined as a threshold for negative versus positive CTCs status. We also analyzed whether CTCs presence was associated with clinic-pathological features using the chi-square or Fisher exact test. The median age was 71 years and median PSA was 12.6 ng/ml. Twenty-eight patients had Gleason score ≥ 8 and 48 patients had clinical stage T3. CTCs were detected in 5/66 patients (7.5%) at diagnosis (baseline), in 8/62 (12.9%) following neoadjuvant ADT and in 11/59 (18.6%) at the end of RT with a median CTC count/7.5ml of 1 (range:1-136). Only 1 patient presented positive CTCs detection 6-13 months after RT. Positive CTCs status (at any period) did not correlate with patient age, pre-treatment PSA, T stage, N stage or Gleason score. The preliminary data obtained from this observational feasibility study showed an infrequent detection of CTCs in patients with HRPCa with no correlation to known clinical prognostic factors. The positive cases for CTCs after ADT and RT (mostly with very low levels and not maintained over time) are probably due to a passive mechanism associated with the destruction of the tumor. A more acute assessment method to detect CTCs together with larger cohorts with extended follow-up are needed to validate this finding.

Association of CTC detection by AdnaTest with outcome on enzalutamide in chemotherapy-naïve castration-resistant prostate cancer: Exploratory results from PREMIERE—A SOGUG trial.

5052 Background: Circulating tumor cells (CTCs) enumeration using CellSearch correlates with clinical outcome in prostate cancer, but is limited for gene expression analysis. AdnaTest ProstateCancer is a commercially available CTC immune-enrichment and PCR-related detection method that allows gene expression studies (Antonarakis E, NEJM 2014). It has demonstrated incremental detection of CTCs in patients with no CTCs identified by CellSearch (Samoila A, ASCO 2013) but needs to be clinically qualified. There is a strong need for studies to assess the association with the clinical outcome in CRPC. Methods: Between February and November 2015, 98 asymptomatic or oligo-symptomatic chemotherapy-naïve mCRPC pts were recruited in 16 institutions. Although initially designed to study the predictive value of TMPRSS2-ETS, data emerging after the trial was initiated led the group to prioritize alternative predefined exploratory biomarkers, including plasma AR (Grande E, ASC0 2017 #) and CTC characterization (Grande E, ESMO 2016). Outcome measures included PSA-PFS (sPFS), radiographic PFS (rPFS) and OS. Cox regression was used for survival analyses and Fisher’s exact test for PSA response. Results: Ninety-eight patients had CTC blood samples available. CTCs were detected at baseline, 12 weeks and progression in 36% (35/98), 27% (26/95) and 78% (32/41), respectively. The CTC conversion rate (positive to negative after 12 w) was 43% (15/35). All CTC conversions had ≥50% decline in PSA (15/15) whereas only 35% (7/20) of pts with persistent CTCs. At first interim analysis, with a median follow-up of 10.6 months, detection of CTCs at baseline was associated with worse sPFS (median, 7.59 m versus NR, HR, 3.67; 95% CI 1.90-7.10; P &lt; 0.001), rPFS (median, 12.9 m versus NR; HR, 7.61; 95% CI, 2.80-20.64; P &lt; 0.001) and OS (medians NR, HR, 9.51; 95% CI, 1.11-81.52; P = 0.0398). CTC positive pts were less likely to have a ≥90% decline in PSA (OR, 2.88; 95% CI, 1.13-7.72; P = 0.02). Conclusions: CTC detection using AdnaTest is associated with an adverse outcome in chemotherapy-naïve asymptomatic or oligo-symptomatic mCRPC pts. Clinical trial information: NCT02288936.

Detection of circulating tumor cells in patients with esophagogastric or pancreatic adenocarcinoma using the CellSearch® system: An observational feasibility study.

Circulating tumor cells (CTCs) in the blood of cancer patients have been demonstrated to be of prognostic value regarding metastasis and survival. The CellSearch® system has been certified for the detection of CTCs and as a prognostic tool in patients with metastatic breast, colon and prostate cancer. Few studies have evaluated the detection of CTCs originating from esophagogastric or pancreatic cancer with the CellSearch® system. In the present small pilot study, a total of 16 patients with either esophagogastric (n=8) or pancreatic (n=8) adenocarcinomas at various disease stages were randomly screened and included. A total of 7.5 ml of blood was drawn from each patient and analyzed for CTCs using the CellSearch® device. CTCs could be detected in 1 out of 8 patients (12.5%) with esophagogastric and in 7 out of 8 patients (87.5%) with pancreatic cancer. The preliminary data obtained from this observational feasibility study suggested that the CellSearch® system may become a valuable tool for the detection of CTCs in patients with pancreatic adenocarcinoma, whereas the usefulness in patients with early-stage esophagogastric adenocarcinoma may be limited. This study clearly points towards a requirement for larger studies focusing on patients with pancreatic adenocarcinoma at various disease stages and assessing CTCs, whereas patients with esophagogastric adenocarcinomas should be part of further pilot studies.

Aptamer-Mediated Transparent-Biocompatible Nanostructured Surfaces for Hepotocellular Circulating Tumor Cells Enrichment.

Circulating tumor cells (CTCs) have been considered as the origin of cancer metastasis. Thus, detection of CTCs in peripheral blood is of great value in different types of solid tumors. However, owing to extremely low abundance of CTCs, detection of them has been technically challenging. To establish a simple and efficient method for CTCs detection in patients with hepatocellular carcinoma (HCC), we applied biocompatible and transparent HA/CTS (Hydroxyapatite/chitosan) nanofilm to achieve enhanced topographic interactions with nanoscale cellular surface components, and we used sLex-AP (aptamer for carbohydrate sialyl Lewis X) to coat onto HA/CTS nanofilm for efficient capture of HCC CTCs, these two functional components combined to form our CTC-BioTChip platform. Using this platform, we realized HCC CTCs' capture and identification, the average recovery rate was 61.6% or more at each spiking level. Importantly, our platform identified CTCs (2±2 per 2 mL) in 25 of 42 (59.5%) HCC patients. Moreover, both the positivity rate and the number of detected CTCs were significantly correlated with tumor size, portal vein tumor thrombus, and the TNM (tumor-node-metastasis) stage. In summary, our CTC-BioTChip platform provides a new method allowing for simple but efficient detection of CTCs in HCC patients, and it holds potential of clinically usefulness in monitoring HCC prognosis and guiding individualized treatment in the future.

63. Rapid and Sensitive Detection of Circulating Tumor Cells with Nuclease-Activated Oligonucleotide Probes

Metastatic breast cancer is the second leading cause of female cancer deaths in the United States. Despite substantial progress in its treatment, metastatic breast cancer remains incurable. Early identification of breast cancer patients at greatest risk of developing metastatic disease is thus an important goal that would enable oncologists to aggressively treat these patients while the cancer is still vulnerable. In addition, this would spare patients who do not need or would not benefit from further treatments from having to endure the harmful side-effects of chemotherapeutic drug regimens. Circulating tumor cells (CTCs) are rare cancer cells found in the blood circulation of cancer patients that provide a non-invasively accessible cancer cell specimen (liquid biopsy) from patients. The number of circulating tumor cells (CTCs) in cancer patients has recently been shown to be a valuable diagnostic indicator of the state of metastatic breast cancer. In particular, patients with few or no CTCs were found to have a better overall prognosis compared to patients with high numbers of CTCs.Despite the implications of CTCs as diagnostics for advanced breast cancer treatment, a critical challenge for adopting CTC-based diagnostic tests has been the development of methods with sufficient sensitivity to reliably detect the small number of CTCs that are present in the circulation. Furthermore, current tests for CTC detection are expensive, have high false positives and negatives, have high background noise, are time consuming and require a significant level of expertise to conduct. To overcome the limitations of current CTC detection assays and develop more sensitive, rapid and cost effective CTC detection methods, we explored the potential of detecting CTCs by measuring their nuclease activity with nuclease-activated probes (1). We present data towards the development of a rapid and highly-sensitive CTC detection assay based on nuclease-activated oligonucleotide probes that are selectively digested (activated) by target nucleases expressed in breast cancer cells. We confirm that these probes are not activated by serum nucleases or nucleases from a lymphoblastic cell line (e.g. K-562). Furthermore, we present extensive data towards the optimization of activity and sensitivity of these probes in cell lysates from various breast cancer cell lines and in blood from breast cancer patients. Future studies will focus on developing similar probes for the detection of CTCs in patients with pancreatic and ovarian cancer where early detection would greatly improve survival outcomes. In conclusion, this work describes a robust assay for detection of breast cancer CTCs that will be straightforward to implement in most clinical diagnostic labs.

MP39-13 DEVELOPMENT OF A NOVEL METHOD FOR DETECTING RENAL CELL CARCINOMA CIRCULATING TUMOR CELLS

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 2015MP39-13 DEVELOPMENT OF A NOVEL METHOD FOR DETECTING RENAL CELL CARCINOMA CIRCULATING TUMOR CELLS Michael Gorin, Mark Ball, Darren Davis, Phillip Pierorazio, Hans Hammers, Kenneth Pienta, and Mohamad Allaf Michael GorinMichael Gorin More articles by this author , Mark BallMark Ball More articles by this author , Darren DavisDarren Davis More articles by this author , Phillip PierorazioPhillip Pierorazio More articles by this author , Hans HammersHans Hammers More articles by this author , Kenneth PientaKenneth Pienta More articles by this author , and Mohamad AllafMohamad Allaf More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.763AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The isolation of circulating tumor cells (CTCs) from patients with renal cell carcinoma (RCC) has been met with limited success. This is due to the fact that most available CTC isolation technologies rely on the positive selection of cells using the surface protein EpCAM, an epithelial marker which is expressed in a minority of RCCs. ApoStream (ApoCell Inc., Houston, TX) is a novel technology which utilizes dielectrophoresis and microfluidics for the antibody-free isolation of CTCs. In this study, we developed a novel method for detecting RCC CTCs using the ApoStream platform and fluorescence in situ hybridization (FISH) for loss of the VHL gene. This assay was then tested in three patients with metastatic clear cell RCC. METHODS The optimal operating frequency for enrichment of RCC CTCs was determined using fluorescently labeled 786-O cells spiked in peripheral blood mononuclear cells from healthy donors. In parallel, conditions were optimized for performing FISH for the VHL gene (ZytoLight VHL/centromere 3 probe, ZytoVision GmbH, Germany) on isolated cells. Following successful assay development, 7.5 mL of blood from each of three patients with metastatic clear cell RCC was enriched for CTCs using the ApoStream instrument. From each sample, 1400 isolated mononuclear cells were evaluated with the optimized FISH assay. A CTC was defined as any mononuclear cell demonstrating net loss of the VHL gene. RESULTS Five operating frequencies (25, 45, 65, 85, 105 Hz) were evaluated for the optimal isolation of 786-O cells from peripheral blood mononuclear cells. Among the tested frequencies, 85 Hz demonstrated the highest mean rate of cell recovery (65%). In clinical testing, two of three (66.7%) patients had detectable CTCs (3 and 5 CTCs each). Figure 1 includes representative images of cells isolated from these two patients. CONCLUSIONS Antibody-independent isolation with dielectrophoresis and subsequent FISH for the VHL gene is a promising novel method for CTC detection in patients with metastatic RCC. Future work aims to validate this assay in larger patient cohort. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e459 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Michael Gorin More articles by this author Mark Ball More articles by this author Darren Davis More articles by this author Phillip Pierorazio More articles by this author Hans Hammers More articles by this author Kenneth Pienta More articles by this author Mohamad Allaf More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

234P - Circulating Tumor Cells As Biomarkers During Neoadjuvant Chemotherapy in Bladder Cancer

ABSTRACT Aim: Cisplatin based neoadjuvant chemotherapy (NAC) improves survival in some patients with resectable muscle-invasive bladder cancer but carries significant toxicity. Development of predictive biomarkers to direct NAC is an urgent clinical need, Circulating tumor cells (CTC) are prognostic for survival in several metastatic epithelial cancers and for recurrence in patient undergoing cystectomy without NAC in muscle-invasive bladder cancer. Current CTC assays have poor sensitivity in non-metastatic disease. We hypothesized that CTC numbers before and after 1 cycle of chemotherapy could serve as potential biomarkers and investigated a novel microfluidic immunomagnetic CTC assay IsoFlux to detect CTCs in patients receiving NAC. Methods: An IRB approved prospective pilot study at the University of Michigan Comprehensive Cancer Center to establish the feasibility of detecting CTCs in patients with locally advanced resectable bladder cancer (cT2-T4aNxM0) before and during cisplatin based neoadjuvant chemotherapy has enrolled 14 patients. Peripheral blood (7.5 mL) was drawn in EDTA tubes prior to NAC (baseline) and again after one cycle (3-4 weeks). We enumerated CTCs by the CellSearch and IsoFlux assays in parallel and by IsoFlux in duplicate samples in a subset of patients. We correlated CTC numbers with pathological stage at cystectomy. We also explored the feasibility of assessing EGFR and HER2 expression by RT-PCR in CTCs in 5 patients and assessed their baseline tumor HER2 gene status by Ion Torrent sequencing. Results: Median age of patients (n = 14) was 63.5 yrs with 83% male. Clinical stage was 79% cT2Nx, 7% cT3Nx and 14% cT4aNx. NAC was cisplatin/gemcitabine in 6 patients and MVAC in 8 patients. The IsoFlux assay detected more CTCs per sample than CellSearch (median =10 vs. 0) at baseline (n = 5) and after 1 cycle (5.5 vs. 0, n = 3). Exact concordance was 40% by IsoFlux assay in tubes drawn in parallel; a paired t-test showed no difference (p = 0.9). After 1 cycle, CTCs by IsoFlux declined from median of 10.5 to 3.75. Patients with Conclusions: CTC detection in patients undergoing NAC for bladder cancer is feasible, potentially more sensitive with the novel assay and a promising predictive biomarker. Disclosure: C. Ionescu: Employee of Fluxion Biosciences Inc, CA; M. Schwartz: Employee of Fluxion Biosciences Inc, CA. All other authors have declared no conflicts of interest.