Characterization Of CTCs Research Articles

Characterization of CTCs isolated from patients with recurrent ovarian cancer using an antibody-free CTC isolation device.

e17056Background: Elucidating the molecular pathways that drive progression or metastases of ovarian cancer may provide insight into prognosis and potentially treatment of advanced or recurrent ova...

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

ABSTRACT Circulating 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.

Tracking metastatic breast cancer: the future of biology in biosensors.

Circulating tumour cells associated with breast cancer (brCTCs) represent cells that have the capability to establish aggressive secondary metastatic tumours. The isolation and characterization of CTCs from blood in a single device is the future of oncology diagnosis and treatment. The methods of enrichment of CTCs have primarily utilized simple biological interactions with bimodal reporting with biased high purity and low numbers or low purity and high background. In this review, we will discuss the advances in microfluidics that has allowed the use of more complex selection criteria and biological methods to identify CTC populations. We will also discuss a potential new method of selection based on the response of the oncogenic DNA repair pathways within brCTCs. This method would allow insight into not only the oncogenic signalling at play but the chemoresistance mechanisms that could guide future therapeutic intervention at any stage of disease progression.

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.

Abstract OT1-02-02: The DETECT study program – Personalized treatment in metastatic breast cancer based on circulating tumor cells

Abstract Circulating tumor cells (CTCs) are found in patients with primary and metastatic breast cancer (MBC), respectively, and discordance in HER2 and hormone-receptor status between primary tumor, metastases and CTCs is well described. Treatment decisions are still based on the expression profile of solid tumor samples whereas CTCs are thought to cause tumor progression by blood-derived metastases. Nevertheless, targeted therapy based on expression profile of CTCs is not established in clinical routine. Individualized treatment decisions based on presence and phenotype of CTCs will be analyzed within the DETECT study program. Metastatic breast cancer patients with HER2-negative MBC are screened in DETECT III and IV for presence of CTCs by using the CellSearch System (Janssen Diagnostics) which is FDA approved for enumeration of CTCs. Patients are enrolled into the different cohorts according to HER2-phenotype of CTCs. Since February 2012, women with HER2-negative MBC and HER2-positive CTCs are treated in the multicenter randomized Phase III study DETECT III with standard therapy with or without additional HER2-targeted therapy with Lapatinib. For standard therapy, physicians can choose between exemestane, letrozole and anastrozole for endocrine therapy, or docetaxel, paclitaxel, capecitabine, vinorelbine and non-pegylated liposomal doxorubicin for chemotherapy. Efficacy of CTC-based anti-HER2 treatment is evaluated by analyzing CTC-clearance rate after treatment. Patients with only HER2-negative CTCs are recruited for the multicenter open-label phase II study DETECT IV. Since December 2013, women with hormone-receptor positive MBC receive endocrine therapy (tamoxifen, exemestane, letrozole or anastrozole) plus everolimus in DETECT IVa. In February 2015, DETECT IV was extended by the eribulin-cohort which offers a cytotoxic treatment with eribulin for women with triple-negative or hormone-receptor positive, chemotherapy demanding MBC (DETECT IVb). Progression free survival is used for assessment of clinical efficacy with overall survival and disease control rate as secondary objectives. DETECT V, a multicenter open-label phase III study starting in summer 2015, randomizes patients with hormone-receptor positive, HER2-positive MBC to a dual HER2 targeted therapy (Trastuzumab and Pertuzumab) combined with either endocrine therapy or cytotoxic treatment. Quality of life determined by occurrence of adverse events is compared between both treatment arms. For prediction of endocrine treatment response, an "Endocrine Responsiveness Score" is calculated based on expression of estrogen-receptor and HER2 on detected CTCs. More than 1200 patients are already screened in the DETECT study concept. Thus, it is the worldwide largest study concept with therapy decisions resulting from CTC-testing and CTC-phenotypization. The accompanying translational research programs evaluates further markers for molecular characterization of CTCs and prediction of therapy response. Conclusion and Contact The value of CTC phenotypes for making decisions on therapy interventions and predicting treatment responses in patients with MBC is tested in the DETECT study concept. The findings will help to move a step forward towards a more personalized anti-cancer therapy. Citation Format: Schramm A, Friedl TWP, Huober J, Jäger B, Rack B, Trapp E, Fasching PA, Taran F-A, Hartkopf A, Schneeweiss A, Müller V, Aktas B, Pantel K, Meier-Stiegen F, Wimberger P, Kümmel S, Gebauer G, Müller L, Janni W, Fehm T. The DETECT study program – Personalized treatment in metastatic breast cancer based on circulating tumor cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT1-02-02.

Abstract P2-02-01: In primary breast cancer patients with HER2-negative tumors, HER2-positive circulating tumor cells with stem cell character predict worse outcome

Abstract Introduction: The detection of disseminated (DTCs) and circulating (CTCs) tumor cells in patients with early stage breast cancer is a well described independent prognostic factor associated with increased risk of disease recurrence and disease-related death. We recently demonstrated that CTCs in blood of primary breast cancer patients rather than DTCs were significantly associated with reduced progression free survival (p=0.02). Using comprehensive molecular characterization, we here demonstrate that the negative prognostic impact was predominantly related to HER2-positive CTCs with stem cell character from patients with HER2-negative primary tumors. Patients and Methods: 2 x 5 ml blood from 482 primary breast cancer patients with first diagnosis between 2006 and 2010 were analyzed for CTCs with the AdnaTest BreastCancer (QIAGEN Hannover GmbH, Germany) for the detection of EpCAM, MUC-1, HER-2, and beta-Actin transcripts. The recovered c-DNA was now additionally tested for the expression of the estrogen (ER) and progesterone receptor (PR) (single-plex RT-PCR) and stem cell like CTCs (slCTCs) applying the AdnaTest TumorStemCell (single-plex RT-PCR for ALDH1) and the AdnaTest EMT (multiplex RT-PCR for TWIST, AKT2, PI3K). The analysis of PCR products was performed by capillary electrophoresis on the Agilent Bioanalyzer 2100. Results: CTCs were detected in 103/482 (21%) of the patients expressing EpCAM (27%), MUC-1 (26%), HER-2 (75%), ER (14%) and PR (8%), respectively. Notably, in 49/103 (48%) of the CTC-positive patients, HER2 was the only marker expressed. slCTCs could be analyzed in 72/103 CTC-positive patients. At least one of the EMT markers was expressed in 56/72 patients (78%), ALDH1 was present in 32/72 patients (44%) and 31/72 (44%) were positive for both, ALDH1 and EMT markers, respectively. Comparisons of expression profiles on CTCs with those on the primary tumor were only performed in CTC-positive patients. Primary tumors and CTCs displayed a concordant HER2, ER and PR status in 37% (p=0.81), 24% (p=0.257) and 27% (p=0.876) of cases, respectively. Most interestingly, in 60/75 (80%) patients with HER2-positive CTCs, primary tumors were HER2-negative. In contrast, the percentage of patients with ER- and PR-positive CTCs but negative ER/PR primary tumors was 29% and 25%, respectively. When the presence of HER2-positive CTCs was correlated with the presence of slCTCs (ALDH1-and/or EMT-positive), the concordance was 83% (p=0.0019). In detail, the concordances were HER2+ vs ALDH1+ (54%, p=0.037), HER2+ vs EMT+ (85%, p=0.0002) and HER2+ vs ALDH1+/EMT+ (80% p=0.00053), respectively. Conclusion: Our results provide evidence that a) the negative prognostic impact of CTCs in our patient cohort is related to HER2-positive CTCs with EMT and tumor stem cell characteristics which indicate therapy resistant tumor cell populations and, therefore, an inferior prognosis and b) “secondary” adjuvant treatment with HER2-targeting agents, alone or in combination, may probably be effective to eliminate these cells and thus, lead to an overall decreased relapse rate. This study further confirms that the molecular characterization of CTCs might help to stratify patients for individual treatment options. Citation Format: Kasimir-Bauer S, Bittner A-K, Aktas B, Hauch S, Kimmig R, Hoffmann O. In primary breast cancer patients with HER2-negative tumors, HER2-positive circulating tumor cells with stem cell character predict worse outcome. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-01.

Abstract 1591: Comprehensive molecular profiling of circulating tumor cells from metastatic breast cancer patients

Abstract Background: Recent enumeration studies on CTCs have demonstrated the clinical value of these cells. The molecular biology of CTCs, however, remains poorly understood. Methods: We performed RNA and DNA profiling of CTCs from metastatic breast cancer (MBC) patients. 244 blood samples from 162 MBC patients were subjected to immunomagnetic enrichment and fluorescence activated cell sorting (IE/FACS) to isolate highly pure CTCs. Microfluidic-based multiplex QPCR analysis was utilized to determine the expression levels of 64 cancer-related genes. Parallel genome-wide copy number analysis by array comparative genomic hybridization was also performed on CTCs isolated from the same enriched blood samples. Results: Transcriptional analysis of CTCs was successfully performed in 151 samples from 105 patients. We observed the up-regulation of several genes including CCND1, EPCAM, KRT7 and MUC1 and the down-regulation of hematopoietic-related genes including PTPRC (CD45) and CD68 (adjusted p <0.001) in CTCs. Clustering analysis of expression profiles revealed three CTC clusters, one containing both subtypes basal (ESR1-low) and normal-like (ESR1-low, CAV1/VIM-high) and two other clusters containing luminal subtype (ESR1-high). A subset of CTCs expressed epithelial-mesenchymal transition genes, however, genes for stem cellness were not generally expressed. Serial CTC expression profiling in 28 patients demonstrated changes in expression over time. Exploratory single CTC expression analysis revealed cell-to-cell heterogeneity. Parallel genome-wide copy number analysis in matched CTCs from 49 patients revealed gains (e.g. 1q and 8q), losses (e.g., 8p and 16q), and focal amplifications (e.g. on 8q and 11q including CCND1) that are frequently observed in primary breast cancers. We also observed three copy number clusters with varying levels of genomic aberrations. Up-regulation of CCND1 and ERBB2 in CTCs was associated with increased level of genomic instability. Conclusions: Parallel gene expression and copy number profiling provided novel insights on the biology of CTCs. Molecular characterization of CTCs may shed further light on the biology of metastasis and disease progression. Citation Format: Mark Jesus Mendoza Magbanua, Louai Hauranieh, Ritu Roy, Praveen Pendyala, Eduardo Sosa, Janet Scott, Hope Rugo, John Park. Comprehensive molecular profiling of circulating tumor cells from metastatic breast cancer patients. [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 1591. doi:10.1158/1538-7445.AM2015-1591

Design of a Microfluidic Chip for Enrichment of Circulating Tumor Cells

Identification and characterization of CTCs can be used as a tool for the study of cancer metastases. A novel design of microfluidic chip used for enrichment of circulating tumor cells is presented in this paper. An integration of DLD method and negative enrichment method were designed to improve the throughput and recovery rate while getting intact CTCs. The DLD stage is used to separate CTCs from blood cells preliminarily, and the negative enrichment stage is used to acquire purified CTCs. Both of them were simulated with COMSOL Multiphysics. Simulation results showed that triangular micro-posts have better performance in DLD stage, and wave structures could generate better disturbance effect than herringbone structures. This chip provides a potential approach with high throughput and purity for the enrichment of CTCs.

Genomic high-resolution profiling of single CKpos/CD45neg flow-sorting purified circulating tumor cells from patients with metastatic breast cancer.

Circulating tumor cells (CTCs) are promising surrogate markers for systemic disease, and their molecular characterization might be relevant to guide more individualized cancer therapies. To enable fast and efficient purification of individual CTCs, we developed a work flow from CellSearch(TM) cartridges enabling high-resolution genomic profiling on the single-cell level. Single CTCs were sorted from 40 CellSearch samples from patients with metastatic breast cancer using a MoFlo XDP cell sorter. Genomes of sorted single cells were amplified using an adapter-linker PCR. Amplification products were analyzed by array-based comparative genomic hybridization, a gene-specific quantitative PCR (qPCR) assay for cyclin D1 (CCND1) locus amplification, and genomic sequencing to screen for mutations in exons 1, 9, and 20 of the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene and exons 5, 7, and 8 of the tumor protein p53 (TP53) gene. One common flow-sorting protocol was appropriate for 90% of the analyzed CellSearch cartridges, and the detected CTC numbers correlated positively with those originally detected with the CellSearch system (R(2) = 0.9257). Whole genome amplification was successful in 72.9% of the sorted single CTCs. Over 95% of the cells displayed chromosomal aberrations typical for metastatic breast cancers, and amplifications at the CCND1 locus were validated by qPCR. Aberrant CTCs from 2 patients harbored mutations in exon 20 of the PIK3CA gene. This work flow enabled effective CTC isolation and provided insights into genomic alterations of CTCs in metastatic breast cancer. This approach might facilitate further molecular characterization of rare CTCs to increase understanding of their biology and as a basis for their molecular screening in the clinical setting.

Abstract 3079: Characterization of the molecular heterogeneity of circulating tumor cells in metastatic prostate cancer

Abstract Purpose: CTCs represent an alternative source of tumor material potentially usable as a surrogate tissue marker in the context of targeted treatments. The objective of the present study was to analyze the status of key molecular markers such as ERG gene rearrangement and AR amplification in tumor tissues (primitive tumor specimens and metastasis biopsies) and CTCs of patients with metastatic prostate cancer (PCa). Patients and Methods: CTCs were detected using two methods, CellSearch (Veridex LLC, Raritan, NJ, USA) and filtration (ISET, isolation by Size of Epithelial Tumor cells) combined to four-color immunofluorescent staining. Epithelial (cytokeratins, EpCAM (Epithelial Cell Adhesion Molecule)) and mesenchymal (vimentin, N-cadherin) markers were tested. Molecular characterization of CTCs was performed on filters by a two step method combining four color immunofluorescent staining and Filter Adapted-Fluorescent In situ Hybridization (FA-FISH). Image analysis was performed using an ARIOL automated scanner (LEICA) to precisely relocate FISH signals in phenotypically different CTC subsets. ERG gene rearrangement and AR copy number gains were examined both in CTCs enriched on filters and in the cell fractions recovered from the magnests after CellSearch and spread on slides for FISH. Primitive tumor specimens and metastasis biopsies were also collected and analyzed by FISH. Results: Higher levels of CTCs were detected by filtration enrichment combined to four-color immunofluorescent staining compared to CellSearch. Filtration enriched CTCs expressed epithelial, mesenchymal and hybrid (both epithelial and mesenchymal) markers suggesting the existence of CTCs at different stages of the epithelial to mesenchymal transition (EMT) process in most PCa patients. A higher level of gene variability and tumor heterogeneity was observed in filtration enriched CTCs compared to those captured by the CellSearch. ERG gene rearrangement and AR amplification in prostate tissue, metastases, and filtration enriched CTCs were analysed by FISH. ERG gene rearrangement and AR copy number gain status present within the tumor tissue was in concordance with that found in filtration enriched CTCs. However, FISH experiments also revealed a much higher degree of cell-to-cell variability and tumor heterogeneity in filtration enriched CTCs compared to tumor tissues. Conclusion: We show here that prostate CTCs are highly heterogeneous in term of EMT markers and ERG gene rearrangement and AR amplification marker status. The high degree of cell-to-cell variability in ERG and AR gene patterns present in CTCs reveals important tumor heterogeneity and could have important implications for developing personalized anticancer strategies based on the use of CTCs in PCa patients. Citation Format: Christophe Massard, Marianne Oulhen, Alexander Valent, Sylvestre Lemoulec, Karim Fizazi, Philippe Vielh, Françoise Farace. Characterization of the molecular heterogeneity of circulating tumor cells in metastatic prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3079. doi:10.1158/1538-7445.AM2014-3079

Abstract 35: A universal marker for the detection of epithelial-mesenchymal transitioned circulating tumor cells and their prognostic relevance in epithelial cancers

Abstract Circulating tumor cells (CTCs) are the tumor cells shed from the primary tumor tissues into the bloodstream or lymphatic vessels. These CTCs are considered to be emerging tools for 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. We have previously reported the detection of mesenchymal cancer cells from blood of sarcoma cancer patients for the first time using a novel monoclonal antibody 84-1. Here we report the detection of EMT CTCs from colon, breast and prostate cancer patients using 84-1 monoclonal antibody. Isolated EMT CTCs were characterized for EMT phenotype by staining for Snail, Slug, Twist-1 and FOXC2. To analyze the efficiency of CTC detection, we compared the CTC counts obtained using 84-1 antibody to that of the FDA approved CellSearch method. We collected blood samples from breast and prostate cancer patients and isolated a) EMT CTCs using 84-1 and b) Epithelial CTCs using Cell Search. For validation purposes, we analyzed an experimental set of patient samples that included normal, non-metastatic and metastatic samples to derive a threshold of detecting CTCs in these patients. After validating a threshold value of the CTC, we used an experimental set that was randomized to evaluate CTCs from different patient sets. Since the experimental set consisted of randomized set of patients with different stages of disease progression and treatments, we evaluated the association between CTC count and therapeutic outcome (progression/ stable & responding). Our results indicated that patients with ≥ 5 EMT CTCs/7.5 mL as detected by 84-1 antibody were independent predictors of therapeutic outcome in breast and prostate cancer patients. Detection using 84-1antibody was much more sensitive and specific when compared with CellSearch method. Collectively, our observations provide a novel technology to selectively detect and isolate EMT CTCs. Detection and molecular characterization of CTCs is one of the fastest growing areas of translational cancer research. Utilizing 84-1, we can enumerate and isolate rare EMT CTCs that will aid as prognostic indicators of therapeutic outcome, metastasis, and relapse and will contribute to the development of specific targeted therapies, an ultimate goal of personalized medicine. Citation Format: Arun Satelli, Zachary Brownlee, Scott Kopetz, Michael Overman, Qing H. Meng, Shulin Li. A universal marker for the detection of epithelial-mesenchymal transitioned circulating tumor cells and their prognostic relevance in epithelial cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 35. doi:10.1158/1538-7445.AM2014-35

Abstract A199: Using Fiber Array Scanning Technology (FAST) Platform for sensitive detection and analysis of circulating tumor cells with a hybrid epithelial/mesenchymal phenotype from non-small cell lung cancer.

Abstract Circulating tumor cell (CTC) analysis uses a “liquid biopsy” to provide critical information regarding diagnosis, prognosis, response to therapy and risk of relapse. To date, CTC analysis in non-small cell lung cancer (NSCLC) patients using epithelial cell adhesion molecule (EpCAM) immunomagnetic enrichment techniques has met with limited success. This is thought to be because the majority of CTCs detected in NSCLC patients demonstrate the mesenchymal phenotype to various extents, and epithelial markers on CTCs are lost during the epithelial-mesenchymal transition (EMT). We report here that the non-enrichment, FAST platform can be used for rapid detection and characterization of CTCs with a hybrid EMT phenotype. We developed a model using whole blood from healthy donors spiked with human NSCLC cell lines that have been classified as having varying levels of EMT. These included “more epithelial, high EpCAM type” (A549 and EKVX), and “more mesenchymal, low EpCAM type” (H2228 and Calu-1) cell lines. A multiplexed assay was developed to simultaneously evaluate cellular and molecular markers of the hybrid phenotypes. FAST was used to develop an optimized assay with high yields (more than 75% retention, detection performance as high as 1 CTC/5 ml blood) of CTCs with hybrid phenotype. 100% of NSCLC cells detected by the FAST platform co-expressed mesenchymal/stem cell markers such as Vimentin, N-Cadherin and ALDH1. FACS analysis of NSCLC cells confirmed levels of EpCAM expression ranging from negative to high. CTCs harboring anaplastic lymphoma kinase (ALK) translocations also demonstrated mesenchymal-like phenotypes. Finally, a high incidence of CTC clusters (circulating tumor microemboli; CTM) that have been previously reported with the hybrid phenotype, was detected from Stage IV NSCLC patients. These data illustrate that the FAST platform is suitable for high-sensitivity detection and comprehensive analyses of the full spectrum of EMT phenotypes in NSCLC CTCs. We have also developed assays to multiplex cellular and molecular biomarkers on single CTCs to guide personalized therapy, including the detection of ALK to select patients appropriate for Crizotinib therapy. Further work is planned to analyze the dynamic change of EMT components in CTCs during disease progression to correlate EMT biomarkers in CTCs with patient outcomes and provide new diagnostic tools for lung cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A199. Citation Format: Xiaohe Liu, Janey C. Ly, Laurie Kara, Heather Wakelee, Nathan Collins, Keith Laderoute, Lidia C. Sambucetti. Using Fiber Array Scanning Technology (FAST) Platform for sensitive detection and analysis of circulating tumor cells with a hybrid epithelial/mesenchymal phenotype from non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A199.

Abstract 1447: Capture, culture and characterization of viable circulating tumor cells in a syngeneic breast cancer mouse model system.

Abstract Circulating tumor cells (CTCs) play a critical role in metastasis. Functional characterization of CTCs is limited by a lack of available methods to enrich viable CTCs. We have developed a next-generation (next-gen) platform, modifying our current size-based CTC enrichment technology, for viable CTC capture. Here, we employed our next-gen microfilter device for viable CTC capture in a syngeneic breast cancer mouse model system to show that such cells can be cultured with high efficiency for further characterization. Balb/c mice received subcutaneous injection of 4T1 (highly metastatic) or 4T07 (poorly metastatic) mouse mammary tumor cell lines originally derived from the same spontaneously arising tumor. Primary tumors were excised on day 21, digested and placed in culture, and viable CTCs were isolated using the next-gen microfilter device from 0.5ml whole blood draws for culture directly on-chip. Successfully established cell cultures were characterized by morphology, IHC for markers associated with epithelial-mesenchymal transition (EMT), and anchorage-independent growth to compare abilities to form tumorispheres. Tumors developed in 100% (6/6) of mice injected; cultures were obtained from 100% dissociated primary tumors (n=3 4T1, n=3 4T07), and isolated CTCs yielded cultures from 33% of mice injected with both cell lines (1/3 each). While 4T07-CTC cultures demonstrate homogenous morphology similar to the corresponding primary tumor, 4T1-CTC cultures demonstrate two morphologically distinct subclones: “A," spheroid-like, different from the primary tumor, and “B," spindle-like, similar to the primary tumor. “A” cells demonstrate the ability to produce progeny with spherical as well as spindle-like morphology in vitro. EMT profiling revealed that both 4T1-CTC subclones demonstrate epithelial and mesenchymal expression characteristics, while 4T07-CTCs predominately show mesenchymal character. CTCs had a greater ability to form tumorispheres in anchorage-independent growth conditions than corresponding primary tumors, with 4T1 “A” having greater tumorisphere-forming ability than 4T1 “B”. These studies demonstrate our ability to capture and culture viable CTCs from mice injected with tumor cells of varying metastatic capability. The characterization of CTCs may help identify functionally important subsets of CTCs. Molecular and functional analyses of CTCs are ongoing, and include epigenomic, transcriptomic and proteomic expression profiling, assessment of stem cell-like properties, and their mechanistic role in metastatic disease. Our work could be the basis to establish CTC cultures in human patients, and provide a transformative approach to the functional characterization of CTCs, leading to an exciting new technology for improved cancer research and individualized patient management. Citation Format: Anthony Williams, Brett Schrand, Siddarth Rawal, Zheng Ao, Randall Brenneman, Eli Gilboa, Siyang Zheng, Ram Datar, Richard Cote. Capture, culture and characterization of viable circulating tumor cells in a syngeneic breast cancer mouse model system. [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 1447. doi:10.1158/1538-7445.AM2013-1447

Abstract 1460: Quantification of circulating tumor cells using multiplexed Surface-Enhanced Raman Scattering Nanoparticles.

Abstract Background: Detection, quantification, and molecular characterization of CTCs may aid physicians in treatment decision making and optimizing. However, the major challenge in the detection and characterization of CTCs is the lack of a highly specific and sensitive assay to isolate and identify the rare population of CTCs in complex blood samples. Moreover, current approaches for CTC detection including CellSearch (FDA approved CTC detection method) which primarily relays on single biomarker are suboptimal because of tumor heterogeneity. Here we develop a multiplexed single-tube assay for directly detection of CTCs in blood sample using two targeted Surface-Enhanced Raman scattering (SERS) nanoparticles. Methods: SERS nanoparticles (60 nm) were prepared by spectral encoding with a Raman reporter molecule (QSY or BHQ) and then stabilized and protected by low molecular weight polyethylene glycols (PEGs). A targeting ligand, epidermal growth factor (EGF) peptide or epithelial cell adhesion molecule (EpCAM) antibody, was covalently conjugated to PEG layer of SERS nanoparticle. The functionalized EGF- or EpCAM- SERS nanoparticle was able to identify CTCs in the peripheral blood with high specificity and affinity. Results: We collected and examined fresh blood samples from 20 SCCHN patients with various histologies and successfully detected positive SERS signals that were greater than background in 9 of 12 (75%) of patients. This technique was demonstrated to be capable of identifying rare CTCs by two biomarkers simultaneously, with a range of 5-720 cells per ml of whole blood. Blood samples from 3 normal controls did not show any CTCs. In one patient who had 0 count of CTC detected by EGFR-targeting SERS tag showed 170 CTCs/ml of blood detected by EpCAM-targeting SERS tags. Conclusion: Special design of the two SERS nanoparticles, distinguished with unique spectral signatures and bio-conjugation of EGF or EpCAM antibody as targeting molecule, allowed simultaneous detection of epidermal growth factor receptor (EGFR) or EpCAM on CTCs in one blood sample. Our technique based on two biomarkers rather than those relayed on single biomarker may improve CTC assessment in clinical settings. (This research was supported by the National Cancer Institute award 5 P50 CA128613 and U54CA119338-04). Citation Format: Xu Wang, Ximei Qian, Jonathan J. Beitler, Zhuo (Georgia) Chen, Fadlo R. Khuri, Melinda M. Lewis, Hyung J. Shin, Shuming Nie, Dong M. Shin. Quantification of circulating tumor cells using multiplexed Surface-Enhanced Raman Scattering Nanoparticles. [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 1460. doi:10.1158/1538-7445.AM2013-1460

Abstract 1443: Improved molecular characterization of circulating tumor cells.

Abstract Circulating tumor cells (CTCs) of epithelial origin are present in the blood of carcinoma patients at very low frequency (< 10 / ml blood). In order to characterize CTCs, they need to be enriched from large volumes of blood (7.5 mLs). The CELLSEARCH® Circulating Tumor Cell (Epithelial) assay uses magnetic particles conjugated to anti-epithelial cell adhesion molecule (EpCAM) to selectively capture CTCs. After enrichment, the sample contains CTCs and a number (1000 - 5000) of white blood cells (WBCs). The presence of WBCs in the CTC enriched sample may interfere with the molecular analysis of CTC biomarkers that may be co-expressed by WBCs. We have improved the molecular characterization of CTCs by selectively depleting WBCs following the enrichment of CTCs. Normal donor blood was spiked with tumor cell lines and the CTCs enriched using the CELLSEARCH® Profile Kit. Contaminating WBCs were labeled with antibody to the leukocyte marker CD45 conjugated to fluorescein isothiocyanate (FITC). Samples were then incubated for 1 hour in microtiter plates coated with anti-FITC antibody. The WBCs bound to the anti-FITC on the microtiter plates and the unbound cells, containing the CTCs, were removed with the supernatant fluids. The unbound cells were counted and analyzed by flow cytometry and molecular methods. Flow cytometric results show greater than 85% of the CTCs were recovered in the supernatant fluids, while greater than 80% of WBCs had been depleted; demonstrating a substantial improvement in CTC purity. For molecular analysis, normal donor blood was spiked with a range (0, 10, 50, or 150) of Vertebral-Cancer of the Prostate (VCaP) cells and enriched as above using the CELLSEARCH® Profile Kit. Enriched cells from parallel samples were either lysed immediately for RNA isolation (non-depleted sample) or subjected to the CD45 depletion method (depleted samples) and then lysed for RNA isolation. To measure efficacy of depletion, RNA was also isolated from the cells that adhered to the microtiter plate during depletion (adherent fraction). RT-PCR results of the WBC specific markers PTPRC (CD45) and BST1 in WBC depleted samples show differences in cycle threshold (Δ Ct) values of 2.5 and 2.4 PCR cycles, respectively, compared to non-depleted samples. These measurements correspond to ∼80% reduction in WBC specific RNA load and are consistent with the flow cytometric observations. VCaP cell specific markers TMPRSS2:ERG and AR show Δ Ct values of only 0.04 and 0.02 cycles (or ∼ 3% reduction in VCaP-specific RNA load). This demonstrates that the cell population following WBC removal is significantly depleted of WBC with minimal loss of cancer cells. The method thus has a potential to improve detection of tumor-specific markers in clinical samples enriched for the presence of circulating tumor cells. Citation Format: Chandra Rao, Brad Foulk, Karl Nielsen, Bhavesh Vaidya, Jaymala Patel, Dana Gaffney, Denis Smirnov, Jayaprakash Karkera, Mark Connelly. Improved molecular characterization of 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 1443. doi:10.1158/1538-7445.AM2013-1443

Abstract 1464: In vitro culturing of CTCs/DTCs in personalized cancer management.

Abstract Subsequent detection, quantification and molecular characterization of CTCs has an enormous potential in numerous of oncology areas. But detection and isolation of CTCs is technically quite challenging. The whole dissemination process encompasses also individual tumor cells which are found in relatively big quantities in peritoneal washings (PW) and pleural effusions (PE), so called disseminated tumor cells (DTCs). Thanks in vitro cultures of separated CTCs/DTCs we were able to introduce additional chemosensitivity testing for the purpose of individualized cancer treatment. We present an isolation and cultivation of CTC/DTC of non-small cell lung cancer and gastrointestinal cancers (esophageal cancer, colorectal cancer, gastric cancer, pancreatic cancer) describing cultivation methodology on patients case studies. The cancer cells were separated from body fluids (PW, PE, blood) using the differences in size of cancer cells if compared to other nucleated cells detected in body cavities. After reaching the confluence the chemosensitivity of cell cultures has been tested by xCELLigence system (RTCA, Roche) for different concentrations of single cis-platinum, gemcitabine and fluorouracyl (incubation time 24-72 hours) or their combinations. The dynamic real-time cell growth monitoring enables parallel or sequential combinations of administered drugs with a direct outcome for patients care. Conclusions: We assume that developing cultivation strategies for CTCs/DTCs could bring us closer to the definition of the CTC/DTC-function within the dissemination process with a perspective to use this knowledge in personalized cancer treatment. Citation Format: Vladimir Bobek. In vitro culturing of CTCs/DTCs in personalized cancer management. [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 1464. doi:10.1158/1538-7445.AM2013-1464

Abstract 1471: Characterization of the molecular heterogeneity of circulating tumour cells in metastatic prostate cancer.

Abstract Purpose: CTCs represent an alternative source of tumor material potentially usable as a surrogate tissue marker in the context of targeted treatments. The objective of the present study was to characterize the status of key molecular markers such as ERG gene rearrangement, and AR and c-myc amplifications, according to epithelial and/or mesenchymal marker expression in CTCs of patients with metastatic prostate cancer (PCa). Patients and Methods: CTCs were isolated from the blood of 40 patients at day 0 and day 30 of abiraterone treatment. CTCs were detected using two methods, CellSearch® (Veridex LLC, Raritan, NJ, USA) and ISET (Isolation by Size of Epithelial Tumor cells). Molecular characterization of CTCs was performed on ISET filters by combining four color immunofluorescent staining and Filter Adapted-Fluorescent In situ Hybridization (FA-FISH®). To optimize pan-keratin detection, four pancytokeratin antibodies, including AE1/AE3, KL1, C11, and A45-B/B3, were tested on the LnCAP prostate cell line spiked in normal blood cells and on PCa CTCs enriched by ISET. Epithelial (cytokeratins, E-cadherin, EpCAM (Epithelial Cell Adhesion Molecule)), mesenchymal (vimentin, N-cadherin), PTEN, androgen receptor (AR) and Ki67 marker expression was detected by immunofluorescent staining on ISET filters according to a method we previously established. FA-FISH® was optimized for the detection of ERG gene rearrangement and AR and c-myc amplification or gain of copy number. A two step method combining immunofluorescent staining and FA-FISH® was established on an ARIOL automated scanner (LEICA) to precisely relocate FISH signals in phenotypically different CTC subsets. Results: Higher CTC levels were detected by ISET compared to CellSearch®. Quantitative analysis of the intensity values for each tested pan-keratins antibodies showed that KL1 and A45-B/B3 clones gave the strongest immunofluorescent signals both on the LnCAP cell line and PCa CTCs. CTCs expressing epithelial, mesenchymal and hybrid (both epithelial and mesenchymal) markers were detected in most patients suggesting the existence of CTCs at different stages of the epithelial to mesenchymal transition (EMT) process. The loss of PTEN expression was also observed using hematopoietic cells present on ISET filters as a positive control of PTEN expression. ERG gene rearrangement, AR and c-myc amplifications were detected. The presence of these markers within CTC subsets expressing epithelial and/or mesenchymal markers is currently studied. Conclusion: Several molecular markers such as ERG gene rearrangement, AR and c-myc amplifications were detected in PCa CTCs. The presence of these markers in CTCs according to their EMT status will be presented. Citation Format: Marianne Oulhen, Christophe Massard, Alexander Valent, Rachel Young, Sylvestre Le Moulec, Karim Fizazi, Philippe Vielh, Françoise Farace. Characterization of the molecular heterogeneity of circulating tumour cells in metastatic prostate cancer. [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 1471. doi:10.1158/1538-7445.AM2013-1471

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

Molecular Characterization of CTCs

Genetic Engineering & Biotechnology NewsVol. 33, No. 6 TutorialMolecular Characterization of CTCsMicrofluidic System Designed to Deliver Optimized Sample for Downstream AnalysisMichael SchwartzMichael SchwartzMichael Schwartz (E-mail Address: mike.schwartz@fluxionbio.com) is co-founder and program director at Fluxion Biosciences. Web: www.fluxionbio.com.Search for more papers by this authorPublished Online:13 Mar 2013https://doi.org/10.1089/gen.33.6.18AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byRecent Advances in Microfluidic Platforms Applied in Cancer Metastasis: Circulating Tumor Cells' (CTCs) Isolation and Tumor‐On‐A‐Chip20 November 2019 | Small, Vol. 16, No. 9Ultra-fast, label-free isolation of circulating tumor cells from blood using spiral microfluidics17 December 2015 | Nature Protocols, Vol. 11, No. 1Microfluidics for research and applications in oncology1 January 2016 | The Analyst, Vol. 141, No. 2 Volume 33Issue 6Mar 2013 Information© 2013 by GEN PublishingTo cite this article:Michael Schwartz.Molecular Characterization of CTCs.Genetic Engineering & Biotechnology News.Mar 2013.36-37.http://doi.org/10.1089/gen.33.6.18Published in Volume: 33 Issue 6: March 13, 2013PDF download

230P - Feasibility of Genetic Aberrations Analysis in the Circulating Tumor Cells (CTCS)

ABSTRACT Background In current practice cancer tissue is taken at diagnosis to assess the presence of treatment targets. This however is suboptimal since tumor cells evolve due to genomic instability. Assessment of the genotype and phenotype of the CTCs will provide insights into which treatments would be most beneficial for the individual patient. Feasibility to detect treatment targets in CTCs has been demonstrated (Meng, Tripathy et al. 2004; de Bono, Attard et al. 2007; Rossi, Basso et al. 2010; Wang, Pfister et al. 2010). In this context, the development of a cytogenetic assay for CTCs will be crucial for successful molecular targeted therapy in cancer patients. Genetic characterization of CTCs is expected to gather new knowledge on the mechanism of metastasis and on potential targets of novel therapeutic strategies. Patients and methods Assay optimization for analysis of genetic aberrations of CTCs was performed with cells from tumor cell lines. Tumor cell lines with known chromosomal aberrations and mosaicism were spiked into 7.5mL whole blood samples, at numbers similar to those observed in-vivo in cancer patients. Tumor cells were enriched by CellSearch System and off-line purified and individually analyzed for chromosomal alterations. The procedure was next validated by using blood samples collected from cancer patients. Results/Conclusions A robust protocol for the isolation of individual CTCs followed by DNA extraction and amplification after CellSearch enrichment was established. The number and the quality of CTCs needed to obtain an informative analysis of chromosomal aberrations were set up. Accrual of cancer patients for individual CTC isolation and molecular characterization is ongoing. Updated data including evaluations on prognostic value of the genetic aberrations analysis of the CTCs and correlation with clinical stage, tumor burden, metastatic sites and survival will be available and presented at the meeting. Disclosure All authors have declared no conflicts of interest.