CTCs In Peripheral Blood Research Articles

A High-Throughput Circular Tumor Cell Sorting Chip with Trapezoidal Cross Section.

Circulating tumor cells are typically found in the peripheral blood of patients, offering a crucial pathway for the early diagnosis and prediction of cancer. Traditional methods for early cancer diagnosis are inefficient and inaccurate, making it difficult to isolate tumor cells from a large number of cells. In this paper, a new spiral microfluidic chip with asymmetric cross-section is proposed for rapid, high-throughput, label-free enrichment of CTCs in peripheral blood. A mold of the desired flow channel structure was prepared and inverted to make a trapezoidal cross-section using a micro-nanotechnology process of 3D printing. After a systematic study of how flow rate, channel width, and particle concentration affect the performance of the device, we utilized the device to simulate cell sorting of 6 μm, 15 μm, and 25 μm PS (Polystyrene) particles, and the separation efficiency and separation purity of 25 μm PS particles reached 98.3% and 96.4%. On this basis, we realize the enrichment of a large number of CTCs in diluted whole blood (5 mL). The results show that the separation efficiency of A549 was 88.9% and the separation purity was 96.4% at a high throughput of 1400 μL/min. In conclusion, we believe that the developed method is relevant for efficient recovery from whole blood and beneficial for future automated clinical analysis.

Dual-targeting surface-enhancement Raman scattering tags based on silver nanocubes for early diagnosis of pheochromocytoma

Pheochromocytoma (PCC), a rare tumor, often develops distant metastases after diagnosis, delaying early intervention treatment. In order to overcome the limitations of traditional diagnostic methods, dual-targeting Surface-Enhancement Raman Scattering (SERS) cytosensor was developed to identify and detect PCC-CTCs from peripheral blood. Meta-iodobenzylguanidine (MIBG) and octreotide-2,2′,2″,2’’’- (1,4,7,10 -tetraazacyclododecane-1,4,7,10-tetrayl) tetraacetic acid (DOTA) functionalized magnetic Fe3O4 and Ag-DTNB were prepared as capture probe and signal probe for SERS signal export, respectively. Ag nanocubes (AgNCs) as Raman active substrate offer an enhanced electromagnetic field, which could effectively enhance the signal intensity of DTNB and potentially realize trace analyte detection. The obtained SERS fingerprint spectroscopy possessed the characteristic of high sensitivity and resolution in the concentration range from 3.0-3.0 × 106 cells mL−1, with a detection limit of 1 cell mL−1, which laterally compensated the deficiency of scarce CTCs in peripheral blood. This work provided new insight into PCC-CTCs accurate detection.

Vitamin D and circulating tumor cells in primary breast cancer.

BackgroundCirculating tumor cells (CTCs) contribute to the metastatic cascade and represent an independent survival predictor in breast cancer (BC) patients. Vitamin D has pleiotropic effects, and its low concentrations are associated with breast cancer and metastasis. The aim of this study was to assess plasma vitamin D in primary BC patients in relation to CTCs.MethodsThis study included 91 non-metastatic BC patients (stage I–III) and 24 healthy donors. Blood samples for the analyses were drawn at the time of surgery. CTCs were assessed using a quantitative RT-PCR assay for expression of epithelial (CK19) or epithelial-to-mesenchymal transition (EMT) genes (TWIST1, SNAIL1, SLUG, and ZEB1). Total 25-OH vitamin D was measured in plasma using ELISA. Plasma cytokines and angiogenic factors were measured by enzyme-linked immunoassay.ResultsCTCs were detected in 30 (33%) patients. Patients with detectable CTCs in peripheral blood had significantly lower vitamin D concentrations in comparison to patients without detectable CTCs ((mean ± SD) 8.50 ± 3.89 µg/L for CTC-positive vs 9.69 ± 3.49 µg/L for CTC-negative patients, p = 0.03). The mean ( ± SD) vitamin D plasma level was 9.3 ± 3.65 µg/L for breast cancer patients compared to 18.6 ± 6.8 for healthy donors (p < 0.000001). There was no association between plasma vitamin D and other patient/tumor characteristics. Plasma vitamin D levels are inversely correlated with plasma TGF-β1, TGF-β2, IL β, IL-5, and eotaxin (all p < 0.05). Patients with vitamin D above the median had a better overall survival (hazard ratio (HR) = 0.36, 95% CI 0.16–0.80, p = 0.017), and combined analysis showed the best survival for CTC-negative patients with vitamin D levels above the median as compared to patients with opposite characteristics (HR = 0.18, 95% CI 0.05–0.63, p = 0.004).ConclusionsLow vitamin D could be a consequence and hence a biomarker of a more invasive disease. Alternatively, vitamin D could be associated with survival because of its role in tumor dissemination. Whether its supplementation affects the metastatic cascade should be tested in animal experiments and interventional studies.

Ultrahigh SERS activity of the TiO2@Ag nanostructure leveraged for accurately detecting CTCs in peripheral blood.

Circulating tumor cells (CTCs) usually shed from primary and metastatic tumors serve as an important tumor marker, and easily cause fatal distant metastasis in cancer patients. Accurately and effectively detecting CTCs in a peripheral blood sample is of great significance in early tumor diagnosis, efficacy evaluation, and postoperative condition monitoring. In this work, a TiO2@Ag nanostructure is structured as a SERS substrate, rhodamine 6G (R6G) is used as a Raman signal molecule, the reduced bovine serum protein (rBSA) acts as a protective agent, and folic acid (FA) acts as a target molecule to specifically recognize cancer cells. A TiO2@Ag-based SERS bioprobe is successfully prepared with the feature of ultrahigh sensitivity, good specificity, low toxicity, and high accuracy in CTC detection. The remarkable SERS activity of the TiO2@Ag nanostructure is synergistically contributed by surface plasmon resonance and photon-induced charge transfer mechanism. The limit of detection for rhodamine 6G (R6G) molecules adsorbed on the TiO2@Ag SERS substrate is 5 × 10-14 M, and the corresponding SERS enhancement factor can reach 7.61 × 107. The designed TiO2@Ag-R6G-rBSA-FA SERS bioprobe is effectively utilized in detecting various cancer cells in rabbit blood, and the limit of detection (LOD) for the target cancer cell is 1 cell per mL. Notably, CTCs in peripheral blood of six clinical liver cancer patients are successfully recognized via the TiO2@Ag-based SERS bioprobe. Accurately recognizing CTCs in peripheral blood based on the TiO2@Ag-R6G-rBSA-FA SERS bioprobe is also carefully verified by in situ immunofluorescence staining experiments, which directly supports the CTC detection accuracy of the SERS strategy. These results demonstrate that the TiO2@Ag-based SERS bioprobe has great application potential in early screening and diagnosis of tumors.

Colorimetric detection of immunomagnetically captured rare number CTCs using mDNA-wrapped single-walled carbon nanotubes

The number of CTCs in peripheral blood is of great significance for the early diagnosis, recurrence and prognosis evaluation of tumor patients. Consequently, it is required to develop simple and effective technique to realize the capture and detection of rare number CTCs. Herein, a SiO2, gelatin and biotinylated EpCAM aptamer P1 modified Fe3O4 immunomagnetic nanoparticles (IMNs) were prepared for the specific capture and nondestructive release of trace amounts of CTCs. Then, utilizing the peroxidase-like activity of single-walled carbon nanotubes (SWCNTs) and the effect of non-specific DNA sequences on this activity, a colorimetric probe was constructed by modifying the three DNA sequences (mDNA) onto the IMNs. When target cell was present, due to the specific interaction between cells and P1, the conformational structure of P1 was changed. Consequently, the mDNA linked with P1 on IMNs was replaced by the cell and released from IMNs. In this way, the quantification of captured CTCs could be converted to that of released mDNA. This strategy combined the capture and detection of CTCs as a whole and could detect down to 10 cells with a high selectivity. Finally, we achieved the accurate quantification of CTCs in lysed bloods from 12 clinical tumor patients, which exhibited a great promise for further clinical applications.

Evaluation of sensitivity and specificity of CanPatrol\u2122 technology for detection of circulating tumor cells in patients with non-small cell lung cancer

BackgroundThe early diagnosis of non-small cell lung cancer is of great significance to the prognosis of patients. However, traditional histopathology and imaging screening have certain limitations. Therefore, new diagnostical methods are urgently needed for the current clinical diagnosis. In this study we evaluated the sensitivity and specificity of CanPatrol™ technology for the detection of circulating tumor cells in patients with non-small cell lung cancer (NSCLC).MethodsCTCs in the peripheral blood of 98 patients with NSCLC and 38 patients with benign pulmonary diseases were collected by the latest typing of CanPatrol™ detection technology. A 3-year follow-up was performed to observe their recurrence and metastasis. Kruskal-Wallis test was used to compare multiple groups of data, Mann-Whitney U test was used to compare data between the two groups, and ROC curve analysis was used to obtain the critical value. The COX risk regression and Kaplan-Meier survival analysis were performed in the 63 NSCLC patients who were effectively followed up.ResultsThe epithelial, epithelial-mesenchymal, and total CTCs were significantly higher in NSCLC patients than that in patients with benign lung disease (P < 0.001). The mesenchymal CTCs of NSCLC patients was slightly higher than that of benign lung diseases (P = 0.013). The AUC of the ROC curve of the total CTCs was 0.837 (95% CI: 0.76-0.914), and the cut-off value corresponding to the most approximate index was 0.5 CTCs/5 ml, at which point the sensitivity was 81.6% and the specificity was 86.8%. COX regression analysis revealed that the clinical stage was correlated with patient survival (P = 0.006), while gender, age, and smoking were not (P > 0.05). After excluding the confounders of staging, surgery, and chemotherapy, Kaplan-Meier survival analysis showed that patients in stage IIIA with CTCs ≥0.5 had significantly lower DFS than those with CTCs < 0.5 (P = 0.022).ConclusionsCTC positive can well predict the recurrence of NSCLC patients. CanPatrol™ technology has good sensitivity and specificity in detecting CTCs in peripheral blood of NSCLC patients and has a certain value for clinical prognosis evaluation.

Abstract B41: Piritramide analgesia reduces CEA mRNA-positive circulating tumor cells’ presence compared to morphine and epidural analgesia following radical colon cancer surgery

Abstract Purpose: Piritramide is a strong opioid widely used for postoperative analgesia (PA) in Europe while morphine and epidural PA are well established worldwide. Our retrospective analysis revealed that compared to morphine, piritramide improved cancer-specific survival in patients undergoing radical colon cancer surgery. Since circulating tumor cells’ (CTCs) presence has negative effect on colon cancer survival, we hypothesized that different types of PA could affect postoperative CTCs levels. Patients and Methods: In total, 60 patients undergoing radical colon cancer surgery were enrolled in this prospective, randomized, two-center pilot study. Patients were randomized to receive either piritramide, morphine, or epidural PA. The presence of carcinoembryonic antigen (CEA) and cytokeratin 20 (CK-20) mRNA positive CTCs in peripheral blood was measured at four points (before and immediately after surgery, on postoperative day 2, and one month after surgery) using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). Perioperative treatment was standardized. Results: In piritramide-treated patients, the levels of CEA mRNA positive cells were significantly lower on postoperative day 2 compared to other patients (p=0.04). CK-20 mRNA positive cells levels remained similar. In all groups, CTCs levels returned to preoperative baseline one month after surgery. Groups were similar regarding age, sex, ASA status, TNM, and grading. Conclusion: Compared to morphine and epidural analgesia, piritramide reduces postoperative CEA mRNA-positive circulating tumor cells’ presence following radical colon cancer surgery. Further trials enrolling larger numbers of patients are needed. Acknowledgments: This study was supported by Ministry of Health of the Czech Republic (NV18-03-00470), Ministry of Education, Youth and Sport of the Czech Republic (LO1304, LM2015089), European Regional Development Fund (ENOCH CZ.02.1.01/0.0/0.0/16_019/0000868) and Cancer Research Czech Republic. Citation Format: Josef Srovnal, Emil Berta, Alona Rehulkova, Monika Vidlarova, Petr Prasil, Lubomir Vecera, Petr Stourac, Pavla Kourilova, Marian Hajduch. Piritramide analgesia reduces CEA mRNA-positive circulating tumor cells’ presence compared to morphine and epidural analgesia following radical colon cancer surgery [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B41.

44P - Correlation of circulating tumour cells with PET-CT in metastatic breast cancer

BackgroundCirculating tumor cells (CTC) detection has proven to be an important parameter for predicting progression-free and overall survival. CTCs provide a so-called real-time liquid biopsy which gives better opportunities for selection of treatment. MethodsA prospective observational study conducted the Rajiv Gandhi Cancer Institute & Research Centre, New Delhi. 36 treatment naïve patients of metastatic breast cancer were enrolled from April 2016 and May 2018. CTCs were detected using Cellsearch system. Correlation of CTCs with patient’s outcome after chemotherapy in terms of progression-free survival (PFS) and overall survival (OS) were calculated at one year. The level of CTCs in peripheral blood was measured at baseline before chemotherapy and then at 1 month and 6 months of treatment. All patients underwent PET-CT scan at 3 months and 6 months of treatment. Results36 patients were included in this study. The mean CTCs at baseline was 13.8 (0-48), with 75% of patients having CTC ≥5. A positive correlation with the number of sites of metastasis with the number of CTCs was noted (p < 0.001 and R = 0.886). Around 11% of the patients had a complete response (CR) after 3 months of therapy as determined by imaging with a mean number of CTCs 21 (median 17) before starting the treatment and 4.25 (median 4.5) at 1 month (P = 0.14). Similarly, partial response at 3 months, the mean CTCs significantly decreased to 6.3 from 12.9 (p = 0.001). Patients with baseline CTCs<5 had a mean PFS of 9.8 months (95% CI of 7.2 to 12.3) and patients with baseline CTCs ≥5 had a mean PFS of 8.6 months (95% CI of 7.1 to 10.1) (p = 0.37). Patients with CTCs < 5 after one month of treatment had a mean OS of 11.6 months (95% CI of 10.8 to 12.4) and patients with CTCs≥5 after one of treatment had a mean overall survival of 9.6 months (95% CI of 8.0 to 11.2) (p = 0.08). ConclusionsOur results have implications for both standard care and clinical research in developing countries. More accurate determination of treatment effectiveness early in the course of therapy might spare patient therapy-related toxicity from futile therapy, patient families from financial toxicity and allow treatment to be changed to a more effective regimen. Legal entity responsible for the studyRajiv Gandhi Cancer Institute and Research Centre, New Delhi. FundingRajiv Gandhi Cancer Institute and Research Centre, New Delhi. DisclosureAll authors have declared no conflicts of interest.

Research progress of gastric cancer circulating tumor cells

The incidence of gastric cancer ranks the fourth in all kinds of cancers in the world, ranking the second among patients with cancer-related deaths. The 5-year survival rate of gastric cancer patients is less than 30%. About 50% of gastric cancer patients have recurred or metastasized after curative resection. Metastasis and recurrence are the major causes of death in cancer patients. CTCs play an important role in tumor metastasis. At present, there are more than 10 kinds of detection methods for CTCs in gastric cancer. The most widely used methods are RT-PCR, FCM, Celltracks® AutoPrep® system and ISET. Recent studies have shown that CTCs in peripheral blood of patients with gastric cancer can be used to determine the clinical stage of patients, assess the prognosis and guide the individualized treatment of cancer. Key words: Stomach neoplasms; Neoplastic cells, circulating; Neoplasm metasis; Detection method

Circulating Tumor Cells Identify Early Recurrence in Patients with Non-Small Cell Lung Cancer Undergoing Radical Resection.

BackgroundSurgery is the treatment of choice for patients with non-small cell lung cancer (NSCLC) stages I-IIIA. However, more than 20% of these patients develop recurrence and die due to their disease. The release of tumor cells into peripheral blood (CTCs) is one of the main causes of recurrence of cancer. The objectives of this study are to identify the prognostic value of the presence and characterization of CTCs in peripheral blood in patients undergoing radical resection for NSCLC.Patients and Methods56 patients who underwent radical surgery for previously untreated NSCLC were enrolled in this prospective study. Peripheral blood samples for CTC analysis were obtained before and one month after surgery. In addition CTCs were phenotypically characterized by epidermal growth factor receptor (EGFR) expression.Results51.8% of the patients evaluated were positive with the presence of CTCs at baseline. A decrease in the detection rate of CTCs was observed in these patients one month after surgery (32.1%) (p = 0.035). The mean number of CTCs was 3.16 per 10 ml (range 0–84) preoperatively and 0.66 (range 0–3) in postoperative determination. EGFR expression was found in 89.7% of the patients at baseline and in 38.9% patients one month after surgery. The presence of CTCs after surgery was significantly associated with early recurrence (p = 0.018) and a shorter disease free survival (DFS) (p = .008). In multivariate analysis CTC presence after surgery (HR = 5.750, 95% CI: 1.50–21.946, p = 0.010) and N status (HR = 0.296, 95% CI: 0.091–0.961, p = 0.043) were independent prognostic factors for DFS.ConclusionCTCs can be detected and characterized in patients undergoing radical resection for non-small cell lung cancer. Their presence might be used to identify patients with increased risk of early recurrence.

Identifying cancer origin using circulating tumor cells

ABSTRACTCirculating tumor cells (CTCs) have become an established clinical evaluation biomarker. CTC count provides a good correlation with the prognosis of cancer patients, but has only been used with known cancer patients, and has been unable to predict the origin of the CTCs. This study demonstrates the analysis of CTCs for the identification of their primary cancer source. Twelve mL blood samples were equally dispensed on 6 CMx chips, microfluidic chips coated with an anti-EpCAM-conjugated supported lipid bilayer, for CTC capture and isolation. Captured CTCs were eluted to an immunofluorescence (IF) staining panel consisting of 6 groups of antibodies: anti-panCK, anti-CK18, anti-CK7, anti-TTF-1, anti-CK20/anti-CDX2, and anti-PSA/anti-PSMA. Cancer cell lines of lung (H1975), colorectal (DLD-1, HCT-116), and prostate (PC3, DU145, LNCaP) were selected to establish the sensitivity and specificity for distinguishing CTCs from lung, colorectal, and prostate cancer. Spiking experiments performed in 2mL of culture medium or whole blood proved the CMx platform can enumerate cancer cells of lung, colorectal, and prostate. The IF panel was tested on blood samples from lung cancer patients (n = 3), colorectal cancer patients (n = 5), prostate cancer patients (n = 5), and healthy individuals (n = 12). Peripheral blood samples found panCK+ and CK18+ CTCs in lung, colorectal, and prostate cancers. CTCs expressing CK7+ or TTF-1+, (CK20/ CDX2)+, or (PSA/ PSMA)+ corresponded to lung, colorectal, or prostate cancer, respectively. In conclusion, we have designed an immunofluorescence staining panel to identify CTCs in peripheral blood to correctly identify cancer cell origin.

Gene expression profiling of circulating tumor cells and peripheral blood mononuclear cells from breast cancer patients

ABSTRACTCirculating tumor cells (CTCs) are cancer cells that are released from a tumor into the bloodstream. The presence of CTCs in peripheral blood has been associated with metastasis formation in patients with breast cancer. Therefore, the molecular characterization of CTCs may improve diagnostics and support treatment decisions.We performed gene expression profiling to evaluate the enriched CTCs and peripheral blood mononuclear cells (PBMCs) of breast cancer patients using an expression panel of 55 breast cancer-associated genes. The study revealed several significantly differentially expressed genes in the CTC-positive samples, including a few that were exclusively expressed in these cells. However, the expression of these genes was barely detectable in the PBMC samples. Some genes were differentially expressed in PBMCs, and the expression of these genes was correlated with tumor grade and the formation of metastasis.In this study, we have shown that the enriched CTCs of breast cancer patients overexpress genes involved in proteolytic degradation of the extracellular matrix (ECM) as well as genes that play important roles in the epithelial-mesenchymal transition (EMT) process that may occur in these cells.

Abstract 373: Analytical and clinical validation of an EpCAM-independent assay for CTC detection in peripheral blood of early breast cancer patients based on Cytokeratin-19 (CK-19) RT-qPCR

Abstract Introduction: The aim of our study was to evaluate the prognostic significance of our previously developed EpCAM independent RT-qPCR assay for CK-19 mRNA (Stathopoulou et al, Int J Cancer 2006) and validate its analytical and clinical performance in early breast cancer. Methods: Quality control on analytical sensitivity and specificity, linearity, intra- and inter-assay reproducibility, and stability of the external standard used for the preparation of the calibration curves was performed. Reproducibility of the assay between our labs was evaluated by analyzing 26 cDNAs. The prognostic significance of the assay in respect to DFI and OS was evaluated by analysing peripheral blood of 179 patients with stage I/II breast cancer postoperatively, before the administration of adjuvant chemotherapy. Results: The limit of detection (LOD) is 3 copies/reaction and limit of Quantitation (LOQ) 10 copies/reaction. The linear range of the calibration curve was 10-105copies/reaction and the intra- and inter-assay reproducibility are shown below: CK19 transcriptsReproducibility of the assay (n = 3)copies/reactionIntra-assay RSD (%)Inter-assay RSD (%)101.973.151020.932.241030.450.861040.122.581050.11.52 The inter-lab reproducibility of the assay was evaluated by analyzing 26 cDNA samples in both labs and there was a 100% concordance. During the follow up period (8 years) 32/179 (17.9%) patients relapsed and 18/179 (10%) patients died from the disease. 45/179 (25.1%) samples were found positive for CK-19 and 134/179 (74.9%) negative. In the group of CK-19 positive patients 15/45(33.3%) relapsed and 9/45(20.0%) died while in the group of CK-19 negative patients 17(12.7%) patients relapsed and 9(6.7%) died. Kaplan-Meier analysis showed that CK-19 mRNA positivity was significantly associated with DFI (P = 0.014) and OS (P = 0.051). Conclusions: This EpCAM independent assay can be used for a high-throughput detection of CTCs in peripheral blood and has prognostic significance in early breast cancer. Citation Format: Areti Strati, Athina Markou, Aliki Stathopoulou, Stella Apostolaki, Dimitris Mavroudis, Vasilis Georgoulias, Evi S. Lianidou. Analytical and clinical validation of an EpCAM-independent assay for CTC detection in peripheral blood of early breast cancer patients based on Cytokeratin-19 (CK-19) RT-qPCR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 373. doi:10.1158/1538-7445.AM2015-373

MP1-07 ENRICHMENT OF CIRCULATING TUMOR CELLS IN PATIENTS WITH LOCALIZED PROSTATE CANCER USING A MICROFLUIDIC DEVICE

You have accessJournal of UrologyProstate Cancer: Markers I1 Apr 2015MP1-07 ENRICHMENT OF CIRCULATING TUMOR CELLS IN PATIENTS WITH LOCALIZED PROSTATE CANCER USING A MICROFLUIDIC DEVICE Tilman Todenhöfer, Emily Park, Hamid Abdi, Alex Li, Richard Ross, Xiaoyan Deng, Chao Jin, Simon Duffy, Martin Gleave, Hongshen Ma, and Peter Black Tilman TodenhöferTilman Todenhöfer More articles by this author , Emily ParkEmily Park More articles by this author , Hamid AbdiHamid Abdi More articles by this author , Alex LiAlex Li More articles by this author , Richard RossRichard Ross More articles by this author , Xiaoyan DengXiaoyan Deng More articles by this author , Chao JinChao Jin More articles by this author , Simon DuffySimon Duffy More articles by this author , Martin GleaveMartin Gleave More articles by this author , Hongshen MaHongshen Ma More articles by this author , and Peter BlackPeter Black More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.170AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Circulating tumor cells have proven prognostic value in patients with advanced prostate cancer (PC) but have been identified only infrequently in patients with localized PC using current standard techniques which are dependent on cell surface expression of epithelial cell adhesion molecule (EpCAM). However, evidence suggests that a proportion of CTCs likely do not express EpCAM. We developed a microfluidic system enabling antigen-independent enrichment of circulating tumor cells based on cell size and deformability. The aim of the present study was to evaluate presence of CTCs detected by this platform in patients with clinically localized PC. METHODS We included 58 consecutive patients undergoing radical prostatectomy and pelvic lymphadenectomy for localized PC. As control, we included 7 healthy men. Peripheral blood was collected preoperatively and processed by a microfluidic device for CTC enrichment. This device uses the deformation of single cells through layers of funnel shaped constrictions with openings smaller than the cell diameter in order to selectively transport cells based on size and deformability. Effluent cells were stained for pancytokeratin, CD45 and the androgen receptor (AR), and were identified by spectral analysis on the Zeiss LSM 780 confocal microscope. Cells positive for CK and negative for CD45 were considered to be CTCs. CTC counts were correlated to clinicopathologic parameters. RESULTS One or more CTCs were detected in 0/7 healthy controls and 35/58 (60.3%) patients with PC. The proportions of patients with ≥5 and ≥20 CTCs per ml were 41.4% and 24.1%, and the maximum CTC count was 527 CTCs/ml. We observed no correlation of CTC counts with tumor stage, nodal stage, Gleason score and serum PSA. CTCs were positive for AR-expression. CONCLUSIONS Circulating tumor cells can be detected in a considerable proportion of patients with localized prostate cancer using an EpCAM-independent system. CTC counts in our study were clearly higher than in other cohorts using EpCAM-dependent detection. The expression of AR in cytokeratin positive CTCs confirms their prostatic origin. The prognostic value of CTCs in peripheral blood before and after prostatectomy remains to be defined. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e3 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tilman Todenhöfer More articles by this author Emily Park More articles by this author Hamid Abdi More articles by this author Alex Li More articles by this author Richard Ross More articles by this author Xiaoyan Deng More articles by this author Chao Jin More articles by this author Simon Duffy More articles by this author Martin Gleave More articles by this author Hongshen Ma More articles by this author Peter Black More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

407P - Pik3Ca Mutations and Loss of Pten Expression in Circulating Tumor Cells (Ctcs) in Patients with Metastatic Breast Cancer (Mbc)

ABSTRACT Aim: Currently, several methods are available to detect CTCs in peripheral blood; the enumeration of CTCs has emerged as a promising biomarker. CTCs shed from metastatic cancers may allow the non-invasive analysis of the evolution of tumor genomes during treatment and disease progression through “liquid biopsies” 1. PIK3CA mutations and loss of PTEN expression are being analyzed in many studies in breast cancer. Frequent deregulation and aberration of this pathway have been implicated not only in breast cancer development and progression, but also in breast cancer therapy resistance, and as a potential predictor of response to new treatments 2,3. The objective of this study is to analyze genomic alterations of the PTEN gene and PIK3CA (exon 20) in CTCs in patients with MBC. 1. Cristofanilli M, et al. N Engl J Med 2004;351:781-91. 2. Sarah-Jane Dawson, et al. N Engl J Med 2013;368:1199-1209. 3. Cancer Genome Atlas Network. Nature 2012;490:61-70. Methods: We carried out a descriptive study in patients with MBC currently undergoing treatment at Hospital Universitario de Salamanca (Spain). CTCs were quantified by means an automated fluorescence method (MetaFer-MetaCyte of MetaSystem), allowing selection of cells by size and genetic alterations. We analyzed genomic alterations in PTEN gene in CTCs by FISH and High Resolution Melting (HRM) was performed to measure PIK3CA mutations in circulating tumor DNA. Finally, we studied the association between PTEN deletion and PIK3CA mutations. Results: In this preliminary analysis, a total of 30 samples have been obtained. CTCs (size >7 µm) were detected in 27 of 30 women. Of 27 samples, there were 12 cases (44,4%) of PTEN loss detected by FISH, and there were 10 cases (37,1%) in which PIK3CA mutations were detected by HRM. In 5 samples, there were association between PTEN deletion and PIK3CA mutations. These 5 cases were Luminal (A and B) tumors. Conclusions: Monitoring and isolation of CTCs represents an opportunity to study specific genomic alterations. In this study, we carried out the analysis of two genes involved in the same signaling pathway: PTEN and PIK3CA; which may have additive or synergistic effects through non-canonical functional pathways. Disclosure: All authors have declared no conflicts of interest.

Circulating Tumor Cells and Prognosis of Patients with Resectable Colorectal Liver Metastases or Widespread Metastatic Colorectal Cancer: A Meta-Analysis

We performed a systematic review and meta-analysis to investigate the prognostic value of tumor cells in blood (circulating tumor cells [CTCs]) or bone marrow (BM) (disseminated tumor cells) of patients with resectable colorectal liver metastases or widespread metastatic colorectal cancer (CRC). The following databases were searched in May 2011: MEDLINE, EMBASE, Science Citation Index, BIOSIS, Cochrane Library. Studies that investigated the association between tumor cells in blood or BM and long-term outcome in patients with metastatic CRC were included. We extracted hazard ratios (HRs) and confidence intervals (CIs) from the included studies and performed random-effects meta-analyses for survival outcomes. The literature search yielded 16 studies representing 1,491 patients. The results of 12 studies representing 1,329 patients were suitable for pooled analysis. The overall survival (HR, 2.47; 95 % CI 1.74-3.51) and progression-free survival (PFS) (HR, 2.07; 95 % CI 1.44-2.98) were worse in patients with CTCs. The subgroup of studies with more than 35 % CTC-positive patients was the only subgroup with a statistically significant worse PFS. All eight studies that performed multivariable analysis identified the detection of CTCs as an independent prognostic factor for survival. The detection of CTCs in peripheral blood of patients with resectable colorectal liver metastases or widespread metastatic CRC is associated with disease progression and poor survival.

Diagnostic and prognostic value of circulating tumor cells in female breast cancer patients

AimThe aim of the current study is to detect CTCs in the blood of breast cancer females by the expression of Mammoglobin and Mucin-1 to evaluate their potential as diagnostic and prognostic markers of breast cancer and predictors of metastasis. Subjects and methodsThe study involved 50 patients and thirty controls. Fifty patients recently diagnosed with breast cancer were proved by fine needle aspiration cytology or core biopsy, all the patients were operated upon by modified radical mastectomy under general anesthesia after complete preoperative evaluation. From each subject a blood sample was drawn before surgery, 2weeks after surgery and after 6 cycles of chemotherapy. Mononuclear cells were separated from 7.5ml blood using a phicoll gradient. From these cells mRNA was extracted, and RT-PCR was carried out to detect Mammoglobin and Mucin-1 gene expression from CTCs. Also CA 15.3 was measured in all the samples. ResultsCTCs were detected in more than 80% of primary breast cancer patients at presentation, that percent decreased after surgery and after adjuvant chemotherapy. CTCs detection after surgery and after chemotherapy was significantly correlated with tumor size, grad, vascular invasion and metastasis. CTCs posed an almost two-fold risk of metastasis and significantly lower DFS time in positive than in negative patients. Detection of CTCs in peripheral blood after chemotherapy successfully predicted subsequent metastasis. ConclusionThe hematogenous spread of tumor cells in patients with breast cancer may be an early phenomenon occurring before/apart from regional lymph nodes involvement. CTCs detection by RT-PCR could add to the initial evaluation of primary breast cancer patients. Also, adjuvant chemotherapy might eliminate or decrease occult tumor cells. But CTCs evaluation after adjuvant chemotherapy could predict metastasis and the presence of CTCs after chemotherapy reflect a higher potential for subsequent metastasis.

Value of circulating epithelial tumor cells and circulating endothelial cells (CTCs/CECs) in patients with HER2-negative recurrent or metastatic breast cancer treated with bevacizumab (B) in combination with paclitaxel (P) and gemcitabine (G) as first-line therapy (AVALUZ trial).

e21088 Background: CTCs in peripheral blood are an ideal source for the detection of tumor dissemination. Their prognosis significance has been demonstrated in metastasic breast carcinoma (Cristofanilli, NEJM 2004). Beacizumab in combination with CT, improves progression free survival (PFS) of first line treatments, may modify CTC and CEC levels. Aims of this study are the evaluation of the prevalence and kinetics of CTCs and CECs before and after treatment with B in pts with MBC Methods: Pts received B 10mg/kg/q2w combined with P 150mg/m2 and G 2000mg/m2 d1,15/q28d therapy until disease progression, unacceptable toxicity or withdrawal. CTC/CEC were measured in 7.5ml of blood at baseline and after the 1st cycle of treatment. Enumeration was performed by CellSearch System, Veridex Results: Data were available for 37 pts. Median of f-up was 16.2 m. The media of CTCs was 41 cells (min0-max845) in the 1st determination and 5 cells (min0-max99) in 2nd determination. Baseline CTCs&gt;2 was associated with statistic lower PFS 10.8 m (95%CI:7.66-16.91) compared to those with CTCs&lt;2, PFS 17.7m (95%CI:17.60-NA),p=0.046. Baseline CTCs&gt;10 vs CTCs&lt;10 was associated with statistic lower OS (14%vs45% of deaths), p=0.035. 92% (N=22) of pts that had stable disease/partial response, decreased or maintained CTCs value. CTC level was not correlated with CEC level, p=0.74. Media value of baseline CECs was 130 (min4-max1407) and 60.3 (min0-max349) in 2nd determination. Baseline CECs&gt;200 was associated with lower PFS 8.2m (95%CI:0.6-10.8) compared to those with &lt;200, PFS 16.9m (95%CI:8.78-NA),p=0.003. No difference was observed in OS. 74% of pts (N=14) that had stable disease/partial response, decreased or maintained CECs value. Baseline CTCs &gt;5 was associated with a median PFS of 15.2 m (95%CI:7.6-16.9) Conclusions: Our study suggests significant correlations between levels of baseline CTCs and CECs and poor prognosis. Addiction of B to 1st-line CT was related with high reduction of CECs and CTCs count.

Comparison of assay methods for detection of circulating tumor cells in metastatic breast cancer: AdnaGen AdnaTest BreastCancer Select/Detect™ versus Veridex CellSearch™ system

The detection of CTCs prior to and during therapy is an independent and strong prognostic marker, and it is predictive of poor treatment outcome. A major challenge is that different technologies are available for isolation and characterization of CTCs in peripheral blood (PB). We compare the CellSearch system and AdnaTest BreastCancer Select/Detect, to evaluate the extent that these assays differ in their ability to detect CTCs in the PB of MBC patients. CTCs in 7.5 ml of PB were isolated and enumerated using the CellSearch, before new treatment. Two cutoff values of ≥2 and ≥5 CTCs/7.5 ml were used. AdnaTest requires 5 ml of PB to detect gene transcripts of tumor markers (GA733-2, MUC-1, and HER2) by RT-PCR. AdnaTest was scored positive if ≥1 of the transcript PCR products for the 3 markers were detected at a concentration ≥0.15 ng/μl. A total of 55 MBC patients were enrolled. 26 (47%) patients were positive for CTCs by the CellSearch (≥2 cutoff), while 20 (36%) were positive (≥5 cutoff). AdnaTest was positive in 29 (53%) with the individual markers being positive in 18% (GA733-2), 44% (MUC-1), and 35% (HER2). Overall positive agreement was 73% for CTC≥2 and 69% for CTC≥5. These preliminary data suggest that the AdnaTest has equivalent sensitivity to that of the CellSearch system in detecting 2 or more CTCs. While there is concordance between these 2 methods, the AdnaTest complements the CellSearch system by improving the overall CTC detection rate and permitting the assessment of genomic markers in CTCs.

131 BIOBANKING OF TISSUE OBTAINED FROM ROBOTIC-ASSISTED RADICAL PROSTATECTOMY CAN BE SUCCESSFULLY IMPLEMENTED WITHOUT COMPROMISING RNA INTEGRITY

cific antigen (PSA) and cytokeratin 18 (CK18) genes was measured by quantitative RT/PCR, and we have optimized this method by using SABiosciences qPCR Master Mix. We also employ an additional technique of immunomagnetic precipitation to extract RNA from CTCs. Specifically, we coat Dynabeads® (Invitrogen) with equal amounts of antibody to epithelial cell adhesion molecule (EpCAM) and to prostate specific membrane antigen (PSMA). RESULTS: Using a specific primer/probe set, we demonstrate that our qRT/PCR assay is linear over a wide range, and that we can detect PSA transcript in RNA from 1 LNCaP cell. When LNCaP cells are spiked into peripheral blood mononuclear cells (PBMNCs) from men without PCa, we demonstrate that we can detect 1 human prostate cell per 10 7 PBMNCs. In our phase II clinical trial we successfully detected CTCs in 13/17 (76.5%) patients. Our technique of dual antigen pull-down can detect 1 CTC per ml of whole blood of PCa patients. PSA transcripts have not been detected in negative control blood. CONCLUSIONS: We have developed a novel, rapid and sensitive assay suitable for the detection of CTCs in peripheral blood of PCa patients.