Detection Of CTC Research Articles

A carbon quantum dot@mesoporous silicon composite-based fluorescent probe for chlortetracycline hydrochloride and gentamicin

A fluorescent nanoprobe, CDs@SBA-15, showed “turn-off” mode detection of CTC based on electrostatic interaction and photo-induced electron transfer, and “turn-on” mode detection of gentamicin based on electrostatic interaction and hydrogen bonding.

A Europium Metal-Organic Framework and Its Polymer Composite Membrane as Switch-Off Fluorescence Sensors for Antibiotic Detection in Lake Water.

The detection of antibiotic residues is of great significance in monitoring their overuse in healthcare, livestock and poultry farming, and agricultural production. Herein, EuCl3 and 4,4'-dicarboxyl-diphenoxyethene (H2DPOE) ionothermally reacted in 1-methyl-3-butylimidazolium chloride to give a europium metal-organic framework (Eu-DPOE). Eu-DPOE shows different fluorescence quenching rates for sensing eight antibiotics under different excitation wavelengths. Eu-DPOE displays a fast response, high selectivity, and sensitivity in antibiotic detection by fluorescence quenching. Eu-DPOE can sensitively detect TCs (tetracyclines), NOR (norfloxacin), NFT (furazolidone), ODZ (ornidazole), SDZ (sulfadiazine), and CHL (chloramphenicol) with limits of detection below 0.5 μmol/L. It provides a convenient and rapid tool for sensing antibiotics in aqueous solution. The detection mechanism is a competition absorption between DPOE2- and antibiotics with the supports from powder X-ray diffraction (PXRD), UV-vis spectra, and fluorescence lifetime. With a composite membrane of poly(vinylidene fluoride) (PVDF) matrix loading Eu-DPOE (Eu-DPOE@PVDF), Eu-DPOE@PVDF exhibits a visual fluorescence response to NOR under a 254 nm UV lamp and NFT and CTC under 365 nm. Eu-DPOE@PVDF is applied in the quantitative detection of CTC, NOR, and NFT in lake water with recovery rates ranging from 88.37 to 113.8%. Totally, fluorescence-quenched Eu-DPOE@PVDF exhibits a fast response, high selectivity, and sensitivity in sensing CTC, NOR, and NFT.

Detection and Clinical Significance of Peripheral Blood CTC, CA125, CA153, and CEA Levels in Patients with Ductal Carcinoma in situ

Objective: To explore the detection and clinical significance of peripheral blood CTC, CA125, CA153, and CEA levels in patients with DCIS. Methods: 210 patients who received surgical treatment for breast cancer in our hospital from January 2019 to December 2022 were selected as the research subjects. According to the postoperative pathology, 100 cases were divided into breast cancer and 110 benign breast tumor groups. One hundred ten healthy patients undergoing physical examination during the same period were selected as the control group. CA153, CA125, and CEA levels were observed in the three groups. Results: The levels of CA153, CA125, and CEA in the breast cancer group were significantly higher than those in the benign breast tumor group and the control group (P < 0.05); the levels of CA153, CA125, and CEA in the benign breast tumor group were all higher than those in the control group, but the differences were not statistically significant; among the three tumor markers, CA153 has the highest sensitivity at 39.00%, CA125 has the second highest sensitivity at 18.00%, and CEA has the lowest sensitivity at 17.00%; The sensitivity of the two joint tests of CA153+CA125, CA153+CEA, and CA125+CEA for the diagnosis of breast cancer were 50.00%, 48.00%, and 26.00% respectively; the sensitivity of the three joint tests is the highest, reaching 53.00%, while the specificity of the joint tests was lower than the individual tests. Conclusion: Detection of peripheral blood CTC, CA125, CA153, and CEA levels has specific reference significance for the treatment and prognosis of DCIS patients.

Abstract 1958: CTC surge

Abstract Circulating Tumor Cells (CTC) are a prognostic biomarker for some types of malignant epithelial cancers and have been correlated with poor clinical outcomes. However, the routine measurement of CTC in clinical practice is still very uncommon and some key features of CTC in clinical samples are only being discovered. One of these features that we have identified by supporting CTC clinical research for over 10 years, is the presence of two distinct patient populations: one with high levels of CTC >220/7.5mL blood and one with levels of CTC <160/7.5mL. The clinical significance of this observation is still unknown, but based on this report, we propose a new oncology phenomenon: CTC Surge. Methods: In the past ten years we worked on more than half dozen clinical study projects by using CellSearch Systems by Menarini Silicon Biosystems (formerly Veridex), the only CTC technology platform approved by FDA. The Cell Search system detects CTCs by staining with surface markers coated on nanoparticle beads. The detection of CTC is linear over the reportable range of 0 to 1238 tumor cells. The FDA-approved clinical significant cut-off lines are 5 CTCs for prostate and breast cancers, and 3 for colorectal cancer. Results: All CTC results are sorted by count from low to high. There are two distinct groups roughly above 220 and below 160 per 7.5 mL of peripheral blood. The significance of a CTC enumeration > 220 is that there are as many as 150,000 tumor cells in circulation at the moment of sample collection. This may be the reason why some patients’ condition suddenly worsens when there is an overwhelming presence of CTCs in the bloodstream. The CTC test is a great tool for clinicians and scientists to monitor cancer diseases progression. Unlike a low number of CTCs that may actively engage in intravasation through a set of genes, the mechanism of CTC Surge likely has a completely different set of driving forces, such as internal wound, tumor necrosis and open access to blood vessels inside or surround the tumor mass. Although we do not yet know the clinical significance of our observation, the CTC test offers a simple tool to monitor the rapidly changing tumor dynamics seen in CTC Surge. Citation Format: Chengsen Xue, Timothy K. Amukele. CTC surge [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1958.

Luminescent porous metal–organic gels for efficient adsorption and sensitive detection of chlortetracycline hydrochloride assisted by smartphones and a test paper-based analytical device

Three porous and luminescent JLUE-MOGs are fabricated for the efficient removal of CTC, and on-site rapid and sensitive detection of CTC with the help of paper and a smartphone is realized.

Preparation of yellow-emitting carbon dots and their bifunctional detection of tetracyclines and Al3+ in food and living cells

A novel bifunctional carbon dot (CD)-based sensing platform was constructed for detection of tetracyclines (TCs) and Al3+. The fluorescence CDs were fabricated by hydrothermal method using phenylenediamine (p-PD) and ethylenebis(oxyethylenenitrilo) tetraacetic acid (EGTA) as precursors. The obtained prepared CDs show bright yellow fluorescence (y-CDs, EX = 400nm and Em = 556nm), high fluorescence quantum yield (QY = 21.55 ± 0.06%), and preferable optical stability. TCs can directly quench the fluorescence of y-CDs based on static quenching characteristics and a smallinternal filtration effect (IEF). By adding Al3+ to the y-CDs + TCs system, the fluorescence is partly recovered because TCs escape from the surface of the y-CDs and form a more stable chelate with Al3+. The sensing platform displays good selectivity and high sensitivity to TCs and Al3+ with low detection limits of 0.057-0.23μM and 0.091μM, respectively. Importantly, this sensing platform has enabledthe detection of TCs and Al3+ in milk samples with satisfactory recoveries and RSDs, confirming the reliability and feasibility of this method. Combining with low toxicity and preferable biocompatibility, the y-CDs are extended to cellular imaging and detection of CTC and Al3+ in A549 cells.

The effect of anode hydrodynamics on the sensitivity of microbial fuel cell based biosensors and the biological mechanism

Fluid dynamics in the anodic chamber of a microbial fuel cell (MFC) is a key factor affecting the distribution of substrates and the efficiency of mass transport. However, the effect of hydrodynamics on MFC based biosensor (MFC-Biosensor) sensitivity has not been established. In this study, the three-dimension anode flow field of a two chamber MFC was visualized, and anodic configuration optimized by a reasonable serpentine flow field and inlet/outlet settings. Through optimization, the proportion of the dead zone in the anodic configuration decreased by 14.1%, and the velocity at the anode surface increased by 334.6% with better homogeneity of distribution. Moreover, electricity production and the sensitivity of MFC-Biosensors was improved by 42.0%, 46.1% and 52.3% for the detection of CTC, AVM and Hg, respectively. Biofilm viability analysis further proved that the enhanced surface velocity was of benefit for the permeation of toxicants into anodic biofilms, thus improving the sensor performance.

Detection and Quantification of Circulating Tumor Cells in PBMCs or Blood Using Flow Cytometry

Abstract Circulating Tumor Cells (CTCs) are cells that have detached from primary tumor tissues or metastases and entered into peripheral blood circulation, led to cancer recurrence and distal metastasis. Evaluation of CTCs conduces to tumor diagnosis, treatment, monitoring, and prognosis. Tumor cell spiking assay was frequently used to evaluate the sensitivity, specificity, accuracy, and repeatability of the methods or systems for enumeration of rare CTCs. In this work, a novel flow cytometer NovoCyte® Quanteon™ was employed to measure and quantify spiked human tumor cells of colon carcinoma (SW620) in normal PBMCs or peripheral blood. First, immunofluorescence labeled (EpCAM-APC antibody) SW620 cells (0–256 cells) were serially diluted in 1×106 PBMCs and tested directly on Quanteon. The data indicate that CTC detection using Quanteon has a sensitivity of 0.0001% (one CTC per million cells) and an excellent linear relationship between the number of cells detected and the predicted number added. Furthermore, SW620 cells (0–2000 cells) were serially diluted in 2mL normal whole blood, following the traditional isolation of PBMCs, magnetic EpCAM-positive selection, immunofluorescence labeling (CD45 FITC, EpCAM APC) and washing. The results show that the average recovery of CTCs is more than 75%. Flow cytometry is a valuable tool for therapeutic monitoring and prognosis assessment. Detection of CTC is critical for a “liquid biopsy”. These experimental results demonstrate that the NovoCyte Quanteon flow cytometer can provide high specificity and accuracy of CTC detection through both direct identification and EpCAM affinity bead-based enrichment methods.

A two-wavelength fluorescence recovery method for the simultaneous determination of aureomycin and oxytetracycline by using gold nanocrystals modified with serine and 11-mercaptoundecanoic acid.

A method is described for rapid (1h) synthesis of gold nanoclusters (AuNCs) co-functionalized with serine and 11-mercaptoundecanoic acid. The co-functionalized AuNCs exhibit good stability towards temperature, pH values, and over time. They were characterized by atomic force microscopy, high-resolution transmission electron microscopy, dynamic light scattering, and by fluorescence, IR and X-ray photoelectron spectroscopies. The fluorescence of the AuNCs is quenched by Hg(II) and restored on subsequent addition of aureomycin (CTC) or oxytetracycline (OTC). A fluorescent turn-on assay was worked out for simultaneous detection of CTC and OTC based on recording the change of the restored fluorescence measured at 420 and 500nm under 340nm photoexcitation. The detection limits are 20 and 9nM for CTC and OTC, respectively. The concentrations of CTC and OTC can also be visualized by UV illumination. The nanoprobe was successfully applied to the simultaneous determination of CTC and OTC in spiked human urine. Graphical abstract Schematic of a two-wavelength fluorescence recovery method for the simultaneous determination of aureomycin and oxytetracycline. It is based on the use of gold nanocrystals modified with serine and 11-mercaptoundecanoic acid, and on recording the change of the restored fluorescence measured at 420 and 500 nm.

Detection of circulating tumor cells with CK20 RT-PCR is an independent negative prognostic marker in colon cancer patients \u2013 a prospective study

BackgroundDetection of circulating (CTC) or disseminated tumor cells (DTC) has been associated with negative prognosis and outcome in patients with colorectal cancer, though testing for these cells is not yet part of clinical routine. There are several different methodological approaches to detect tumor cells but standardized detection assays are not implemented so far.MethodsIn this prospective monocentric study 299 patients with colon cancer were included. CTC and DTC were detected using CK20 RT-PCR as well as immunocytochemistry staining with anti-pan-keratin and anti-EpCAM antibodies. The primary endpoints were: Evaluation of CTC and DTC at the time of surgery and correlation with main tumor characteristics and overall (OS) and disease free survival (DFS).ResultsPatients with detectable CTC had a 5-year OS rate of 68% compared to a 5-year OS rate of 85% in patients without detectable CTC in the blood (p = 0.002). Detection of DTC in the bone marrow with CK20 RT-PCR was not associated with a worse OS or DFS. Detection of pan-cytokeratin positive DTC in the bone marrow correlated with a significantly reduced 5-year OS rate (p = 0.048), but detection of DTC in the bone marrow with the anti-EpCAM antibody did not significantly influence the 5-year OS rate (p = 0.958). By multivariate analyses only detection of CTC with CK20 RT-PCR in the blood was revealed to be an independent predictor of worse OS (HR1.94; 95% CI 1.0–3.7; p = 0.04) and DFS (HR 1.94; 95% CI 1.1–3.7; p = 0.044).ConclusionsDetection of CTC with CK20 RT-PCR is a highly specific and independent prognostic marker in colon cancer patients. Detection of DTC in the bone marrow with CK20 RT-PCR or immunohistochemistry with anti-EpCAM antibody is not associated with a negative prognostic influence.

Circulating tumor cells (Ctc) and kras mutant circulating free Dna (cfdna) detection in peripheral blood as biomarkers in patients diagnosed with exocrine pancreatic cancer.

BackgroundPancreatic cancer remains one of the most difficult cancers to treat with the poorest prognosis. The key to improving survival rates in this disease is early detection and monitoring of disseminated and residual disease. However, this is hindered due to lack reliable diagnostic and predictive markers which mean that the majority of patients succumb to their condition within a few months.MethodsWe present a pilot study of the detection circulating free DNA (cfDNA) combined with tumor specific mutation detection by digital PCR as a novel minimally invasive biomarker in pancreatic ductal adenocarcinoma (PDAC). This was compared to the detection of CTC by the CellSearch® system and a novel CTC enrichment strategy based on CD45 positive cell depletion. The aim of the study was to assess tumor specific DNA detection in plasma and CTC detection as prognostic markers in PDAC.ResultsWe detected KRAS mutant cfDNA in 26 % of patients of all stages and this correlated strongly with Overall Survival (OS), 60 days (95 % CI: 19–317) for KRAS mutation positive vs 772 days for KRAS mutation negative (95 % CI: 416–1127). Although, the presence of CTC detected by the CellSearch® system did correlate significantly with OS, 88 days (95 % CI: 27–206) CTC positive vs 393 days CTC negative (95 % CI: 284–501), CTC were detected in only 20 % of patients, the majority of which had metastatic disease, whereas KRAS mutant cfDNA was detected in patients with both resectable and advanced disease.ConclusionsTumor specific cfDNA detection and CTC detection are promising markers for the management of patients with PDAC, although there is a need to validate these results in a larger patient cohort and optimize the detection of CTC in PDAC by applying the appropriate markers for their detection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1779-7) contains supplementary material, which is available to authorized users.

Recherche des cellules tumorales circulantes dans les rédons après curage cervical

But de la presentation Tumor cells are actually detected in multiple localisation (blood, nodes) of non metastatic patients and it seems that in some cases their presence could be a prognostic factor (circulating tumor cells). The presence of tumor cells in the surgical drains was attested as a bad prognostic factor (pancreatic adenocarcinoma), but have never been studied in head and neck cancer. We studied such case for fourteen patients with head and neck epidermoid carcinoma and adenocarcinoma after neck dissection for metastatic adenopathy. Materiel et methodes The Cellsearch ® system uses immunomagnetic and fluorescence approaches for identification and detection of CTC. A total of fourteen patients were enrolled in this study: twelve of them for a HNSCC and two for a cervical metastasis of an adenocarcinoma. In the absence of detection kit for head and neck cancer, we used the CellSearch Epithelial Cell Kit Fluorescent reagents included acid nucleic staining (DAPI), anti-cytokeratin to detect epithelial cells and anti-CD45 antibodies for leukocyte exclusion. Resultats No Disseminated Tumor Cells were found for four patients (31 %). A positive result was obtained for the nine other patients (69 %). Among these nine patients, two were T2N2M0 (3–242 DTC/5 mL) and seven were T4N0-3M0 (7-2094 DTC/5 mL). In the two cases of adenocarcinoma tumor cells were detected, demonstrating a different morphology compared to epidermoid carcinoma. Conclusion Tumor cells can be present in the neck after dissection, both in epidermoid carcinoma and in adenocarcinoma. The significations of this presence have to be determined and could influence perisurgical and post-operative treatments.

567P - Role of Circulating Tumor Cells (Ctc) in Stage III Colorectal Cancer (Crc)

ABSTRACT Aim: CTC detection has proved to be an independent prognostic factor in metastatic CRC. However, the prognostic role of CTC in early CRC has not been determined yet. We evaluated the potential role of CTC in identifying stage III CRC patients (pts) with a higher risk for relapse. Methods: Prospective multicenter study of 519 pts with stage III CRC recruited between January 2009 and June 2010. CTC were enumerated with the CellSearch System in 7.5 ml of peripheral blood after primary tumor resection and before the start of adjuvant therapy. We present the first analysis after 3 years of follow-up. A total of 472 were included in this analysis (data from the remaining 47 pts were not available at the time of analysis). Results: Stages: IIIA (n = 35, 7%), IIIB (n = 346, 73%), IIIC (n = 91, 19%). CTC ≥1, ≥2, ≥3 and ≥5 were detected in 166 (35%), 93 (20%), 57 (12%) and 34 (7%) pts, respectively. Median follow-up was 40 months. During this period 135 pts (29%) relapsed and 80 pts (18%) died. In the overall population, the presence of CTC ≥1 was not associated with an increased risk of relapse (HR for disease-free survival (DFS): 0.97, IC 95%: 0.68-1.38, P = 0.85) or death (HR for overall survival (OS): 1.03, IC 95%: 0.66-1.59, P = 0.89). CTC ≥2, ≥3 and ≥5 were not associated with worse DFS and OS. CTC ≥1 was significantly more frequent in pts with stage IIIC (IIIA 40%, IIIB 32%, IIIC 47%; P = 0.016), as it was a higher percentage of relapse (IIIA 21%, IIIB 17%, IIIC 58%). In pts with stage IIIA, CTC ≥1 and ≥2 were associated with an increased risk of relapse (CTC ≥1 vs. Conclusions: CTC presence was more frequent in pts with higher risk of relapse. Detection of CTC ≥1 and ≥2 appears to be associated with worse DFS and OS, in pts with stage IIIA CRC. However, a longer follow-up of pts in this study is needed. Disclosure: All authors have declared no conflicts of interest.

333P - Circulating Tumor Cell Count at Baseline is an Independent Prognostic Factor from Pathological Complete Response Among Patients Treated for Primary Inflammatory Her2-Positive Breast Cancer: Survival Results of the Beverly-2 Study

ABSTRACT Aim: The BEVERLY-2 single-arm phase 2 trial assessed the efficacy and safety of combining neoadjuvant chemotherapy with bevacizumab and trastuzumab for the treatment of HER2-positive inflammatory breast cancer (Pierga JY et al, Lancet Oncol 2012). We report the results of the association between outcome, circulating biomarkers and pathological complete response (pCR) at three years of follow-up. Methods: Circulating tumor (CTC) and endothelial cell (CEC) counts were assessed at baseline, cycle 5, pre-operative, post-operative and at one year by the CellSearch technique. Results: 52 patients were included. 3-year DFS in patients with ≥1 CTC/7.5 mL at baseline was significantly lower (43% [95% CI: 20; 64]) than for patients with no CTC detected at baseline (81% [95% CI: 62; 91]; hazard ratio 3.69 [1.34; 10.21]; p = 0.012). Due to the low number of CTC-positive patients at all other time points, there was no significant association between CTC and DFS other than that observed at baseline. Detection of CTC at any point during the trial period was associated with a significant reduction in DFS; patients with at least one positive CTC sample had a DFS of 54% (95% CI: 32; 71), compared with a DFS of 83% (95% CI: 61; 93) in patients where no CTC were detected at any time (hazard ratio 3.62 [95% CI: 1.15; 11.39]; p = 0.018). In a multivariate analysis including factors that were associated with DFS in univariate analysis (pCR, SBR grade and baseline CTC status), only SBR grade and baseline CTC status were found to be independent prognostic factors. No impact of CEC count on DFS was observed at univariate analysis. Patients with baseline CTC Conclusions: Combination of pCR and CTC positivity defines specific patient subgroups at very low (no CTC detected at baseline and pCR) and high (CTC detected at baseline and no pCR) risk of early relapse. These parameters could be used to build specific clinical trial with new targeted treatments for patients with inflammatory breast cancer. Disclosure: J. Pierga: honoraria and research funds from Roche and Janssen diagnotics; M. Coudert: Roche employee; J. Hernandez: Roche employee; P. Viens: Roche research funding. All other authors have declared no conflicts of interest.

Abstract 1469: Specific detection tool for mesenchymal and epithelial-mesenchymal transformed circulating tumor cells.

Abstract Circulating tumor cells (CTC) released from primary tumor tissues into bloodstream or lymphatic vessels are emerging as novel tools in the detection and prognosis of several types of metastatic cancers. At present, CTC markers are limited to epithelial cancers and there are no specific markers available to detect mesenchymal and epithelial-mesenchymal transformed (EMT) CTCs. EMT cells are characterized by increased motility and invasiveness and are known to escape detection technologies that use EpCAM and cytokeratins as CTC markers. Here we report a novel antibody, 84-1 for detection of mesenchymal and EMT CTC. Antibodies were raised in mice after injecting metastatic cancer cells and clones were selected that were positive only for CTC and negative for PBMCs and normal cells. 84-1 was the only antibody that was unique in its ability to detect CTCs from mesenchymal tumors and EMT CTC from epithelial tumors. Studies for characterization of the epitope for 84-1 antibody are ongoing. We then analyzed the sensitivity and specificity of the antibody by utilizing spiking assay and were able to specifically detect and isolate as low as one cell from 1 ml of blood. In a pilot analysis, we were able to detect CTCs from patients with colon cancer, hepatocellular carcinoma, osteosarcoma, and leukemia with very high specificity, while CTCs were not observed in healthy subjects. CTCs were characterized based on the size of the nucleus and for 84-1 positivity and CD45 negativity using fluorescence microscopy. The CTCs from cancer patients were isolated and cultured for mutational analysis. CTCs were also detectable in the blood of mice with tumors of both epithelial (colon) and non-epithelial (osteosarcoma) origin and were isolated and cultured in vitro, which formed colonies of cells that were used for further analysis to confirm the identity of CTCs. Furthermore, we also evaluated CTCs in mutant p53 mice that developed spontaneous tumors of both mesenchymal and epithelial origin. Collectively, our observations provide a novel technology to selectively detect and isolate both mesenchymal and EMT CTC. Detection and molecular characterization of CTCs is one of the fastest growing areas of translational cancer research. Utilizing our antibody, we can enumerate and isolate rare CTCs that will aid in the early detection of tumors, metastasis, and relapse and will contribute to the development of specific targeted therapies, an ultimate goal of personalized medicine. Citation Format: Arun Satelli, Abhisek Mitra, Jeffery J. Cutrera, Xia Xeuqing, Dibra Denada, Dina Lev Chelouche, Michael James Overman, Ellis Lee, Shulin Li. Specific detection tool for mesenchymal and epithelial-mesenchymal transformed circulating tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1469. doi:10.1158/1538-7445.AM2013-1469

Cellules tumorales circulantes et disséminées en oncologie digestive

Circulating (CTC) and disseminated tumor cells (DTC) represent two different steps of the metastatic process. As with other types of cancer, the recent development of techniques for the detection of CTC and DTC respectively in the blood and bone marrow of patients generated many results in digestive cancers. However, the interpretation of these results and of the prognostic value of CTC/DTC is often limited by the small cohort size and the heterogeneity of detection methods. The aim of this article is to review the different results and their clinical impact, and discuss the possible use of CTC and DTC as new biomarkers. First of all, it is important to take into account the variability of epithelial markers used for the initial stage of immunoselection of CTC/DTC as well as that of molecular or cytological markers used for the second stage of detection. In esophageal, gastric, pancreatic and hepatocellular carcinomas, and in the ileal and pancreatic neuroendocrine tumors, some studies showed a correlation between the detection of CTC and/or DTC and a clinical pejorative course, whether these tumors were at localized or metastatic stages. On colorectal cancer in the adjuvant setting, a recent meta-analysis showed an association between the detection of CTC in peripheral blood and disease-free survival or overall survival. These results are consistent with those of a study that identified detection of CTC as a prognostic factor for relapse in stage II. This last study concluded that it was necessary to achieve long-term evaluation of CTC as a biomarker to guide the decisions of chemotherapy for stage II. In metastatic colorectal cancer, the FDA approved in 2007 the use of pretherapeutic levels of CTC and its variations per-treatment, determined by CellSearch® technology, as a tool in treatments management. However, the modalities of this monitoring have to be specified and clinical benefit or the cost-effectiveness of a treatment based on this new biomarker has to be evaluated. Finally, the qualitative and quantitative monitoring of CTC could be a non-invasive tool to monitor changes in tumor biology throughout the disease, and thereby improve the understanding of the processes of dissemination and therapeutic resistance.

Heterogeneous detection of circulating tumor cells in patients with colorectal cancer by immunomagnetic enrichment using different EpCAM-specific antibodies

BackgroundCirculating tumor cells (CTC) and disseminated tumor cells (DTC) are thought to be responsible for metastasis, so the detection of CTC may serve as individual prognostic factor in patients suffering from colorectal cancer. Therefore, a series of immunomagnetic enrichment methods for CTC have been developed using a variety of monoclonal antibodies against the Epithelial Cell Adhesion Molecule (EpCAM). However, it remains unclear whether all commercially available EpCAM antibodies show the same sensitivity and specificity. Furthermore, it remains unclear which method of sample preparation and cell extraction is most suitable for immunomagnetic enrichment and detection of CTC. In this study, we aimed to investigate whether the detection of CTC by a cytokeratin 20 reverse transcriptase-polymerase chain reaction (CK20 RT-PCR) may be influenced by the use of various Epithelial Cell Adhesion Molecule (EpCAM) antibodies for immunomagnetic isolation of CTC.ResultsUsing both EpCAM antibodies (mAb BerEP4 and mAb KS1/4) for immunomagnetic enrichment in blood samples of 39 patients with colorectal cancer we found heterogenous results in each patient with regard to tumor cell detection. In the tumor cell spiking experiments with whole blood samples the sensitivity of the CK 20 RT-PCR assay was higher using immunomagnetic beads coated with mAb KS1/4 compared to precoated mAb BerEP4 Dynabeads. Extraction of MNC fraction with Ficoll gradient centrifugation prior to immunomagnetic enrichment resulted in a higher sensitivity of the CK 20 RT-PCR assay.ConclusionsWe concluded that isolation and detection of CTC with immunomagnetic enrichment methods is critically dependent on the used EpCAM clone. Further studies with a larger number of patients should clarify if the enrichment protocol influences the prognostic value of the tumor cell detection protocol.

Association of serum tumor-related methylated DNA with circulating tumor cells in peripheral blood of breast cancer patients.

Abstract Abstract #5017 Circulating tumor cells (CTC) and free tumor-related methylated DNA in blood have been separately associated with poorer disease outcome in breast cancer patients. However, no studies have looked into the relation between both molecular markers in breast cancer. In this study, we investigated the correlations between plasma total DNA, serum methylated DNA and CTC in blood from breast cancer patients.
 We simultaneously obtained matched triplets of peripheral blood, plasma and serum samples from 4 patients with localized breast cancer (group A), 59 patients with metastatic disease under treatment (group B), 16 untreated patients with metastatic disease (group C) and 20 healthy controls. CTC levels in blood were measured with the CellSearch System (Veridex). Plasma total DNA levels were determined by a qPCR method. Sera were analyzed by methylation-specific qPCR for three methylated markers: APC, RASSF1A and ESR1.
 Plasma total DNA levels in breast cancer patients were significantly increased when compared to healthy controls (P<0.001). Differences between total DNA levels in different patient groups also reached statistical significance (P=0.04). The largest differences were measurable between groups A and C (median of 7 vs 29 ng/ml) (P=0.02) and groups B and C (median of 12 vs 29 ng/ml) (P=0.06). The sensitivity and the specificity of the total DNA assay for predicting malignancy was respectively 72.5% and 85%. Total DNA levels correlated with CTC (r=0.418, P<0.001) and patient age (r=0.298, P=0.01), but not with other clinical variables. Hypermethylation of one or more genes was detected in 42 (53%) serum samples from breast cancer patients and in 3 (16%) serum samples of healthy controls (P=0.003). APC was hypermethylated in 23 (29%), RASSF1A in 28 (35%) and ESR1 in 16 (20%) breast cancer cases. CTC were detected in 19 (70%) patients with serum methylated DNA and in 8 (30%) patients without methylated DNA (P=0.03). The presence of methylated DNA markers was only associated with ER status. On univariate analysis, the detection of CTC, high levels of circulating DNA, methylation of RASSF1A and the combinations of APC or RASSF1A methylation with ESR1 methylation were significantly associated with progressive disease. Multivariate analysis indicated that only RASSF1A methylation, high total DNA levels, HER2 and PR status were significantly associated with disease status.
 The number of CTC was significantly increased in patients with high levels of total DNA and in patients with at least one methylated DNA marker in serum, suggesting that CTC might be a potential source of circulating tumor-related DNA. Only RASSF1A methylation and high DNA levels were independently associated with progressive disease. The combination assay of CTC and serum methylated DNA did not enable a more informative assessment of disease status. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5017.

A Sensitive Molecular Method for Detection of Disseminating Hematopoietic Tumor Cells by Using Specific Epigenetic DNA Methylation Biomarkers

Background: Most cancer deaths are caused by hematogenous spread and subsequent metastasis. Emerging data indicates that the presence of circulating tumor cells in peripheral blood (CTC) and disseminating tumor cells in bone marrow (DTC) is an early event in tumorigenesis. These circulating or disseminating rare tumor cells may represent a distinct clone with cancer stem cell properties (metastatic stem cells). Clinically, the presence of CTC and DTC is relevant to overt metastases. Early detection and characterization of these rare biologically distinct tumor cells with sensitive and specific methods provide vital information for cancer biology and early clinical intervention and thus improve patients' survival.Method Design: Genomic DNA was extracted from patient blood, bone marrow aspirate specimens and cancer cell lines prior to digestion with 4 methylation sensitive restriction enzymes. Hypermethylated CpG island regions in tumors, in contrast to their counterpart in normal cells which are completely digested, remain resistant to digestion and therefore can be differentially amplified by PCR. Thus, the specific PCR products on the gel represent the specific methylation loci in the tumor cells in the patient specimens.Results: First, we tested a total of 26 cancer cell lines including 17 hematopoietic tumors and 9 solid tumors. The hematopoietic tumor cell lines represent a spectrum of B-cell malignancies, T-cell lymphoblastic leukemia and acute myeloid leukemia. The solid tumor cell lines represent the major anatomic sites of human carcinoma including lung, colon, breast, prostate, ovarian and skin (melanoma). We found multiple DNA methylation markers specific for lymphoid, myeloid and solid tumors. By using DLC-1 (deleted in liver cancer-1), a tumor suppressor gene, as a single marker, CpG island methylation was detected in 13 out of 17 hematopoietic tumor cell lines (76%) and 6 out of 9 solid tumor cell lines (67%). We then used B-cell acute lymphoblastic leukemia (B-ALL) as a testing case to verify the method in a total of 135 clinical specimens. DLC-1 methylation was detected in 64% and 54% of diagnostic bone marrow aspirates and peripheral blood specimens, respectively, but none was detected in normal or non-leukemic bone marrow or blood control samples. By adding two additional methylation markers PCDHGA12 and CDH1, greater than 90% of B-ALL patients can be detected. We also traced 4 B-ALL cases and 4 follicular lymphoma cases up to 10 years retrospectively and found that the DLC-1 methylation is exactly correlated with patient clinical status. Lastly, by mixing normal and leukemic cell genomic DNA, analytic sensitivity was determined as 0.1% or 10−3 in a single-step PCR and 0.00001% or 10−6 in a nested PCR suggesting that this method is capable of detecting as few as 5 leukemic cells in a single-step PCR.Conclusion: By utilizing tumor specific DNA methylation marker(s), we have developed a simple, highly sensitive and specific gel-based PCR assay, namely Multiple Methylation Sensitive Enzyme Restriction PCR (MSR-PCR) for detection of CTC and DTC from blood and bone marrow of majority of hematopoietic malignancies. The method can potentially be used for early diagnosis and molecular monitoring in vast majority of clinical cancer patients.