Presence Of Circulating Tumor Cells In Blood Research Articles

Circulating tumor cells as a biomarker for monitoring: Disease progression, treatment response, and minimal residual disease.

e15021 Background: To analyze the role of Circulating tumor cells (CTC) as a confirmatory personalized biomarker for monitoring the disease progression, disease burden, and minimal residual disease in epithelial origin cancers. Methods: In this retrospective study, 127 patients with colorectal, breast, and ovarian cancer at stage III and IV were analyzed. The patients were at various stages of intensive chemo and radiotherapy while the CTCs were isolated and enumerated from 1.5 ml of blood. The decision to continue chemotherapy or change to oral metronomic therapy was based on the presence of circulating tumor cells in Stage III. While in stage IV, serial measurement of CTC guided therapy. CTCs were isolated using OncoDiscover platform possessing EpCAM antibody based immunomagnetic targeting of magnetic nanoparticles after RBC lysis. CTCs were imaged and identified as CK18+ and CD45- cells showing a well-defined nucleus using a motorized fluorescence microscope operational with a monochrome camera. CTCs were enumerated using automated image analysis software and counts were expressed as number per 1.5 ml of blood. Results: In this retrospective study we analyzed blood sample from 127 patients with the advanced stage epithelial cancers (breast- 50 %, ovarian -27 %, colorectal- 23 %) for the presence of CTCs. Amongst those, 52 % showed the presence of CTCs (breast- 52 %, ovarian -46 %, colorectal- 58 %). The CTC count ranged between 1-5 / 1.5 ml of blood with mean and median value of 2 and 1. Among the CTC positive population, majority had CTC count of 1 (44.4 %), while more than 2 CTCs were observed in 11 % of population. CTC clusters were detected in 13 % of population which predominantly were stage IV patients. 67 % among the follow up patients showed decrease in CTC count from the baseline due to the prescribed treatment, while 22 % patients showed increase in CTC count from the baseline. 11 % patients did not show change in CTC count from the baseline. When CTCs count was investigated as an independent variable to monitor the therapeutic response, it correlated well with the positive or negative outcome. In few representative cases, the reduction of CTC number from the basal value was indicative of an effective treatment. Exceptionally, in a representative colorectal cancer case, PET showed no primary as well secondary tumor burden, but the presence of CTCs in blood led further investigating an abdominal MRI that indicated multiple liver lesions suggesting micro-metastasis. Subsequent to SIRT treatment, the patient showed complete tumor regression and absence of CTCs in peripheral blood. Conclusions: Our data suggest that CTC can serve as a dynamic intermittent biomarker for monitoring the disease progression in advanced stages and assess the therapeutic response, thus emphasizing the role of CTCs in personalized cancer management.

Comparative Analysis of Blood and Bone Marrow for the Detection of Circulating and Disseminated Tumor Cells and Their Prognostic and Predictive Value in Esophageal Cancer Patients.

Hematogenic tumor cell spread is a key event in metastasis. However, the clinical significance of circulating tumor cells (CTC) in the blood and disseminated tumor cells (DTC) in bone marrow is still not fully understood. Here, the presence of DTC and CTC in esophageal cancer (EC) patients and its correlation with clinical parameters was investigated to evaluate the CTC/DTC prognostic value in EC. This study included 77 EC patients with complete surgical tumor resection. CTC and DTC were analyzed in blood and bone marrow using nested CK20 reverse transcription-nested polymerase chain reaction (RT-PCR) and findings were correlated with clinical data. Twenty-seven of 76 patients (36.5%) showed CK20 positivity in the blood, 19 of 61 patients (31.1%) in bone marrow, and 40 (51.9%) of 77 patients were positive in either blood or bone marrow or both. In multivariate analyses, only the DTC status emerged as independent predictor of overall and tumor specific survival. Our study revealed that, while the presence of CTC in blood is not associated with a worse prognosis, DTC detection in the bone marrow is a highly specific and independent prognostic marker in EC patients. Larger cohort studies could unravel how this finding can be translated into improved therapy management in EC.

Abstract 4595: The VAR2CSA malaria protein efficiently retrieves CTCs from a broad spectrum of cancers

Abstract Metastasis is the leading cause of cancer-related deaths. The metastatic process requires malignant cells to break out of the primary tumor and travel to distant sites through the vasculatory system. Studies have shown that blood circulating tumor cells (CTCs) can be used for patient management as CTC enumeration provides a non-invasive tool for stratification and monitoring disease status. Furthermore, there is a great interest in fully characterizing this highly metastatic subfraction of the tumor cells. Various assays have been developed to enable magnetic enrichment of CTCs from patient blood. Most depend on antibodies targeting the epithelial marker, EpCAM, and are therefore limited to tumors of epithelial origin. Consequently, there is a need for technologies that enable broad capture of CTCs from all types of cancers. We have recently shown that placental and cancer cells share a unique form of oncofetal chondroitin sulfate (ofCS), not present on normal tissue. We have further shown that ofCS is an acquired trait for cancer cells to enable migration and to potentiate the metastatic potential of cancer cells. Most importantly, we showed that VAR2CSA, a malaria protein involved in placental malaria, binds specifically to ofCS on a wide range of cancer patient-derived cell lines and tissues of both epithelial and mesenchymal origin. Since ofCS seems to be universally expressed across most cancer types and is heavily involved in cancer cell migration, we hypothesized that the recombinant VAR2CSA (rVAR2) protein could enable an efficient isolation of CTCs from blood samples from a broad spectrum of cancer patients. Here, we show that we can use rVAR2-coated magnetic beads to capture ofCS-positive CTCs. The method was validated using blood samples spiked with cancer cells, followed by clinical feasibility testing in diverse types of cancers to demonstrate versatility. We show the presence of CTCs in blood from more than a hundred cancer patients having pancreatic, hepatic, lung, colorectal, melanoma or prostate cancer. CTCs were even found at very early stage disease. No CTCs were detected in samples from healthy subjects. This method not only enabled the efficient isolation of CTC from non-epithelial cancers, but also showed a dramatic increase in CTC capture compared to EpCAM-based CTC isolation. Furthermore, the method allows for downstream single cell analyses as well as for culturing of the CTCs. This method could potentially prove beneficial in cancer diagnosis, patient stratification, monitoring response to therapy, and in future molecular characterization of this highly metastatic subpopulation of cancer cells. Citation Format: Mette Ø. Agerbæk, Sara R. Bang-Christensen, Ming-Hsin Yang, Thomas M. Clausen, Sisse B. Ditlev, Marina A. Pereira, Morten A. Nielsen, Swati Choudhary, Tobias Gustavsson, Poul H. Sorensen, Tim Meyer, David Propper, Jonathan Shamash, Thor G. Theander, Alexandra Aicher, Mads Daugaard, Christopher Heeschen, Ali Salanti. The VAR2CSA malaria protein efficiently retrieves CTCs from a broad spectrum of cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4595.

A comprehensive study of circulating tumour cells at the moment of prostate cancer diagnosis: biological and clinical implications of EGFR, AR and SNPs.

Circulating tumor cells (CTCs) have been recently accepted as prognostic markers in metastatic prostate cancer (PCa). However, very few studies have analyzed their role in early-stage PCa. The aim of this research is to study the value of CTCs at the moment of PCa diagnosis and to identify different subpopulations of CTCs. Patients with PSA value > 4 ng/ml and clinical suspicion of PCa were included. Samples were collected immediately before prostatic biopsy. CTCs were isolated by immunomagnetic technique using a multi-CK specific antibody. Molecular expression of EGFR and AR in the tissue was analysed by real-time PCR. Up to eight different SNPs in patients’ blood DNA were studied.In a total of 86 patients, the CTC detection rate was 18.6%. The sensitivity, specificity, positive and negative predictive values of CTCs to detect PCa was 14.2%, 78.4%, 31.2% and 57.4%, respectively. Up to 75% of CTC-positive patients were AR-negative. A direct association was found between the expression of AR in the prostatic tissue and the presence of CTCs in blood (p<0.05). We observed an inverse relation between the expression of EGFR in the tissue and the expression of AR in the CTCs. No significant association between SNPs and CTCs was found.The low detection rate of CTCs in early-stage PCa limits their role as a diagnostic marker. Nevertheless, we show that they may hide important prognostic information. Overexpression of AR in the prostate may facilitate cell dissemination.

Circulating tumor cells before and during follow-up after breast cancer surgery.

The presence of circulating tumor cells (CTC) is an independent prognostic factor for progression-free and overall survival for patients with metastatic and newly diagnosed breast cancer. The present study was undertaken to explore whether the presence of CTC before and during follow-up after surgery is associated with recurrence free survival (RFS) and overall survival (OS). In a prospective single center study, CTC were enumerated with the CellSearch system in 30 ml of peripheral blood of 403 stage I-III patients before undergoing surgery for breast cancer (A) and if available 1 week after surgery (B), after adjuvant chemo- and/or radiotherapy or before start of long-term hormonal therapy (C), one (D), two (E) and three (F) years after surgery. Patients were stratified into unfavorable (CTC≥1) and favorable (CTC=0) prognostic groups. >1 CTC in 30 ml blood was detected in 75/403 (19%) at A, 66/367 (18%) at B, 40/263 (15%) at C, 30/235 (12%) at D, 18/144 (11%) at E and 11/83 (13%) at F. RFS and OS was significantly lower for unfavorable CTC as compared to favorable CTC before surgery (p=0.022 and p=0.006), after adjuvant therapy (p<0.001 and p=0.018) and one (p=0.006 and p=0.013) and two (p<0.001 and p=0.045) years after surgery, but not 1 week post-surgery. The presence of CTC in blood drawn pre and one and two years after surgery, but not post-surgery is associated with shorter RFS and OS for stage I-III breast cancer.

703 CIRCULATING TUMOR CELLS CAN BE DETECTED IN PATIENTS WITH TESTICULAR GERM CELL TUMORS

You have accessJournal of UrologyPenis/Testis/Urethra: Benign & Malignant Disaese (I)1 Apr 2013703 CIRCULATING TUMOR CELLS CAN BE DETECTED IN PATIENTS WITH TESTICULAR GERM CELL TUMORS Christian Ruf, Paulina Nastaly, Pascal Becker, Hendrik Isbarn, Friedemann Honecker, Klaus Pantel, Sabine Riethdorf, Dirk Hoeppner, Margit Fisch, Walter Wagner, and Sascha Ahyai Christian RufChristian Ruf Hamburg, Germany More articles by this author , Paulina NastalyPaulina Nastaly Hamburg, Germany More articles by this author , Pascal BeckerPascal Becker Hamburg, Germany More articles by this author , Hendrik IsbarnHendrik Isbarn Hamburg, Germany More articles by this author , Friedemann HoneckerFriedemann Honecker Hamburg, Germany More articles by this author , Klaus PantelKlaus Pantel Hamburg, Germany More articles by this author , Sabine RiethdorfSabine Riethdorf Hamburg, Germany More articles by this author , Dirk HoeppnerDirk Hoeppner Berlin, Germany More articles by this author , Margit FischMargit Fisch Hamburg, Germany More articles by this author , Walter WagnerWalter Wagner Hamburg, Germany More articles by this author , and Sascha AhyaiSascha Ahyai Hamburg, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.261AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The available serum tumor markers (AFP, β-HCG, LDH) are increased in only approximately 60% of testicular germ cell tumors (TGCT) patients. Therefore, additional markers would facilitate clinical diagnosis, staging and treatment of these patients. Detection of circulating tumor cells (CTC) has been associated with poor prognosis in other malignancies (e.g. Prostate-, Colon- and Breastcancer), however, little is known about the presence of CTC in blood of TGCT patients. METHODS In order to select the most appropriate markers for CTCs detection, the expression of different epithelial and germ cell-specific markers was examined in 4 different TGCT cell lines (TCam-2/2102Ep/NCCIT/NT2) as well as 12 histologically different primary testicular tumors. Peripheral and testicular vein blood samples from 73 and 12 patients, respectively, were collected and examined for CTCs. All samples were enriched for mononuclear cells using Ficoll density gradient centrifugation and CTCs were detected by alkaline phosphatase (AP) enzymatic activity and immunocytochemistry using anti-keratin, anti-EpCAM and anti-SALL4-antibodies. Additionally, peripheral blood samples were tested for the presence of CTCs by the automated CellSearch® system. Patients with ≥1 CTC/sample were classified as CTC-positive. To compare results for testicular and peripheral blood, CTC yields were calculated as number of CTC per 1 ml of blood. RESULTS Based on the previous analyses, double immunofluorescent staining for SALL4 and keratin, as well as analysis of AP activity were performed to detect CTCs. According to both detection systems û the one based on selected markers and automated CellSearch®, 7 (58.3%) testicular vein and 13 (17.8%) peripheral blood samples showed presence of at least one CTC. The CTC number ranged from 5 to 108/ml and 0.13 to 2.18/ml of testicular vein and peripheral blood, respectively. CTC were detected in seminoma and non-seminoma, in clinically metastasized and non-metastasized stages. CONCLUSIONS This is the first study to demonstrate CTC detection in TGCT patients. The proposed detection systems seem to be specific and sensitive for the identification of CTC. An association between CTC and histological subtype as well as metastatic stage is under investigation and will be presented at the meeting. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e289 Peer Review Report Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Christian Ruf Hamburg, Germany More articles by this author Paulina Nastaly Hamburg, Germany More articles by this author Pascal Becker Hamburg, Germany More articles by this author Hendrik Isbarn Hamburg, Germany More articles by this author Friedemann Honecker Hamburg, Germany More articles by this author Klaus Pantel Hamburg, Germany More articles by this author Sabine Riethdorf Hamburg, Germany More articles by this author Dirk Hoeppner Berlin, Germany More articles by this author Margit Fisch Hamburg, Germany More articles by this author Walter Wagner Hamburg, Germany More articles by this author Sascha Ahyai Hamburg, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Circulating tumor cells in pancreatic cancer patients: Methods of detection and clinical implications

The poor prognosis of pancreatic cancer patients is associated with the frequent and early dissemination of the disease, as well as late detection due to unspecific and late symptoms from the primary tumor. Pancreatic cancers frequently spread to the liver, lung and skeletal system, suggesting that pancreatic tumor cells must be able to intravasate and travel through the circulation to distant organs. Circulating tumor cells (CTCs) are tumor cells that have acquired the ability to enter the circulatory system; this cell population is ultimately responsible for the development of metastases in distant organs. Clinical studies have revealed that the presence of CTCs in blood is correlated with disease progression for other cancers, such as breast, colorectal and prostate cancer. However, as CTCs are extremely rare, both enrichment and sensitive methods of detection are required for their enumeration. This review highlights various enrichment procedures and methods for the detection of CTCs. Furthermore, we systematically review previously reported studies of the clinical relevance of CTC detection in pancreatic cancer patients. There is evidence that the presence of CTCs also correlates with an unfavorable outcome in pancreatic cancer patients. However, technical/methodological issues may explain why some studies only show a trend toward an association between CTC detection and disease progression. Larger studies, as well as characterization of the CTC population, are required to achieve further insight into the clinical implications of CTC detection in pancreatic cancer patients.

Methylated APC and GSTP1 genes in serum DNA correlate with the presence of circulating blood tumor cells and are associated with a more aggressive and advanced breast cancer disease

BackgroundTumor-related methylated DNA and circulating tumor cells (CTC) in the peripheral blood might be of prognostic importance in breast cancer. Thus, the aim of our study was to examine free methylated DNA and CTC in the blood from breast cancer patients and to correlate it with clinicopathological features known to influence prognosis.Materials and methodsWe prospectively obtained serum samples from 85 patients with breast cancer and 22 healthy volunteers. Sera were analysed by methylation specific PCR (MethyLight PCR) for five genes: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A), estrogen receptor 1 (ESR1), CDKN2A (p16) and glutathione s-transferase pi 1 (GSTP1). Beta actin (ACTB) served as control. In parallel matched peripheral blood of 63 patients was used to assay for circulating tumor cells in the peripheral blood by a modified immunomagnetic AdnaTest BreastCancerSelect with PCR detection for EPCAM, MUC1, MGB1 and SPDEF.ResultsWe found a hypermethylation in the APC gene in 29% (25/85), in RASSF1A in 26% (22/85), in GSTP1 in 18% (14/76) and in ESR1 in 38% (32/85) of all breast cancer patients. No hypermethylation of CDKN2A was found (0/25). Blood samples of patients were defined CTC positive by detecting the EPCAM 13% (8/63), MUC1 16% (10/63), MGB 9% (5/55), SPDEF 12% (7/58) and in 27% detecting one or more genes (15/55). A significant difference was seen in methylated APC DNA between cancer patients and healthy volunteers. Moreover, methylated APC, RASSF1 and CTC were significantly different in metastatic versus non-metastatic disease. In addition, the presence of methylated APC, RASSF1A and CTC correlated significantly with AJCC-staging (p = 0.001, p = 0.031 and 0.002, respectively). High incidences of methylations were found for the genes RASSF1 and ESR1 in healthy individuals (both 23% 5/22). Methylated GSTP1 was predominantly found in the serum of patients with large primaries (p = 0.023) and was highly significantly correlated with positive Her2/neu status (p = 0.003). Elevated serum CA15.3 was strongly correlated with methylated APC and CTC detection (both p = 0.000). Methylated ESR1 failed to exhibit significant correlations with any of the above mentioned parameters. The presence of CTC in peripheral blood was significantly associated with methylated APC (p = 0.012) and methylated GSTP1 (p = 0.001).ConclusionThe detection of methylated APC and GSTP1 DNA in serum correlated with the presence of CTC in the blood of breast cancer patients. Both methylated DNA and CTC correlated with a more aggressive tumor biology and advanced disease.

Abstract 841: Comparison of two molecular assays for the detection of circulating tumor cells in blood of primary breast cancer patients and their correlation with disseminated tumor cells in the bone marrow

Abstract Background: In primary breast cancer, the presence of disseminated tumor cells (DTCs) in bone marrow (BM) is an independent prognostic factor, while the role of circulating tumor cells (CTCs) in blood still has to be proven since a standard method for their detection has not been defined. Molecular approaches are generally more sensitive than cytometric methods and have the additional advantage of a high-throughput technology. Therefore, we examined the presence of CTCs in blood by using two different molecular assays and assessed their correlation with the detection of DTCs in BM of primary breast cancer patients. Patients and Methods: In total, 418 blood samples and 384 BM samples of 377 primary breast cancer patients were studied. Two x 5 ml blood samples were collected and CTCs were isolated by positive immunomagnetic selection using anti-EpCAM and anti-MUC1 coated magnetic beads. All samples (same cDNAS) were further analyzed by using: a) the AdnaTest BreastCancer (AdnaGen AG) for the detection of three breast cancer associated markers: GA733-2, MUC-1, HER-2 and beta Actin (internal reference gene control), analyzed by capillary electrophoresis on the Agilent Bioanalyzer 2100 and b) real time quantitative RT-PCR for CK-19 mRNA in the Light-Cycler System (Roche Diagnostics). Furthermore, 384 bilateral BM aspirates were analyzed by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3 and DTCs were evaluated with the ARIOL system (Applied Imaging) according to the ISHAGE evaluation criteria. Results: DTCs were found in 115/384 BM samples, resulting in a positivity rate for DTCs of 30%. Applying the AdnaTest, 409/418 samples could be evaluated for the presence of CTCs. 53/409 (13%) samples had detectable CTCs expressing MUC-1 (30%), GA733-2 (25%) and HER2 (70%), respectively. CK19 RT-PCR could be performed in 411/418 samples with a positivity rate of 21% (86/411 samples). A comparison of both molecular CTC assays could be performed in 400 samples with a concordance rate of 71% resulting in 274 blood samples found to be negative by both assays, 11 samples found to be positive by both assays, AdnaTest+/CK19- was documented in 42 cases and AdnaTest-/CK19+ in 73 cases, respectively. Concordant results between the detection of DTCs in the BM and CTCs were found in 65% (AdnaTest) and 62% (CK19 RT-PCR) of all cases, respectively. Discussion: (1) Molecular CTC assays gave different results in 115/400 cases (28.7%) in the same cDNA samples. This could be attributed to the fact that the evaluation of CTCs positivity between these tests is based on a totally different set of genes. (2) Our results might indicate the heterogeneity of CTCs in breast cancer. (3) The low correlation between the detection of DTCs and CTCs by both methods indicates that CTCs may provide prognostic information complementary to DTCs in BM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 841.

Circulating tumor cells (CTCs) detection in a randomized phase II trial of neoadjuvant chemotherapy for large operable and locally advanced breast cancer (REMAGUS 02): Preliminary results

10637 Background: The presence of CTCs in blood from metastatic breast cancer patients before first line chemotherapy and persistence of CTCs after initiation of treatment, are predictive of shorter overall survival (Cristofanilli M; et al, 2005, J Clin Oncol 23:1420–1430). CTCs could be used as a surrogate marker. The aim of this study was to determine if CTC were present in the blood of patients who received neoadjuvant chemotherapy (CT) for large operable and locally advanced breast cancer, before initiation of CT (preCT) and at the end of CT before surgery (postCT). Methods: 7.5 ml of blood were obtained on CellSave tube from patients included in an ongoing randomized phase II trial. All patients received 4 cycles of Epirubicin-Cyclophosphamide every 3 weeks followed by 4 cycles of docetaxel associated with or not trastuzumab for HER2 positive patients and with or not celecoxib for HER2 negative patients. CTCs were immunomagnetically separated and fluorescently stained with the CellSearch kit. Cells were classified using the CellSpotter Analyzer as CTCs if they stained positive for DAPI (nuclear dye), and cytokeratin 8, 18 and 19, and if they stained negative for the leucocyte-specific antibody CD45. Results: From 10/2004 to 12/2005, preCT blood samples were obtained in 60 patients, analyzed in 56 for technical reasons. At least one CTC was detected in 15/56 (27%, CI 95%: 15.5–38.5%), 1 to 17 cells per sample (median 1.5). With a threshold of 2 cells, 8/56 (14%, CI 95%: 5–23%) patients were classified positive. At time of analysis, pre CT and post CT samples from the same patient were available for 19 patients. Six had &gt;1 CTC/sample before CT (31.5%) and only one (1/6, 17%) remained positive after CT. Thirteen were negative for CTC before CT and 2/13 (15%) became positive after CT, with only one cell per sample. Conclusions: CTCs can be detected in blood of patients with large operable or locally advanced breast cancer before initiation of neoadjuvant CT and can be monitored during treatment. Longer follow-up and a larger number of patients are expected before correlating these data with tumor response and survival. No significant financial relationships to disclose.