Characterization Of Circulating Tumor Cells Research Articles

Association between insulin-like growth factor-1 receptor (IGF1R) expression in circulating tumor cells (CTCs) and prognosis in patients with metastatic breast cancer (MBC).

1086 Background: CTCs are strongly associated with prognosis in MBC. In a recent metanalysis on 1944 patients, a CTC count > 5/7.5 ml was associated with a 2-fold increased risk of progression and death. Little evidence is available on the prognostic role of phenotypic CTC assessment, however. In this study, nested in a randomized clinical trial of Ist line chemotherapy ± metformin, we evaluated the prognostic role of IGF1R expression in CTCs, given its potential growth promoting effect. Methods: CTCs were isolated from blood samples of enrolled patients; an automated sample preparation and analysis system (CellSearch) was customized for detecting IGF1R positive CTCs. The prognostic role of total CTCs, IGF1R positive (+ve) and negative (-ve) CTCs was assessed by fitting different PFS and OS multivariate Cox’s models.Results: CTC evaluation at baseline was performed in 72 of 126 patients, of whom 30 (42%) had CTCs ≥5/7,5ml and 41 (57%) had at least one IGF1R+ve CTC. In univariate analysis the prognostic role of total CTCs was confirmed: PFS ( < 5 vs ≥5) HR = 1.69, 95%CI 1.01-2.69, p 0.042 and OS HR = 2.80, 95%CI 1.47-5.30, p 0.002).However, when total CTCs were split in IGF1R+ve and IGF1R–ve, a striking difference was seen in the prognostic effect of these cell types.While no association was detected between an increasing number of IGF1R+ve CTCs and PFS or OS (p = 0.56 and p = 0.99), the number of IGF1R-ve CTCs ( < 4 vs ≥4) was strongly associated with an increased risk of progression and death: HR 1.93 (95%CI 1.15-3.23, p 0.013) and 3.65 (95%CI 1.88-7.09, p 0.001). In multivariate analysis, adjusted for metformin, the prognostic role of the number of IGF1R-ve CTCs was confirmed, while no residual prognostic role of total CTCs or number of IGF1R+ve cells was found (p = 0.55 and p = 0.64 for PFS; p = 0.86 in both cases for OS). Conclusions: In our study, the loss of IGF1R expression in CTCs exhibited a significant adverse prognostic effect, whereas no significant effect of total CTCs and IGF1R+ve CTCs was observed. This finding supports the biological characterization of CTCs as a critical step for further definition of their prognostic significance.

Improving circulating tumor cells enumeration and characterization to predict outcome in first line chemotherapy mCRPC patients.

IntroductionThere is a critical need of new surrogate markers for improving the therapeutic selection and monitoring of metastatic prostate cancer patients. Nowadays clinical management of these patients is been driven by biochemical and clinical parameters without enough accuracy to allow a real personalized medicine. The present study was conducted to go insight the molecular profile of circulating tumor cells (CTCs) isolated from advanced metastatic castration-resistant prostate cancer (mCRPC) with the aim of identifying prognostic marker with potential utility for therapy selection and monitoring.Materials and MethodsCTCs isolation was carried out in peripheral blood samples from 29 mCRPC patients that undergo systemic chemotherapy based on taxanes (docetaxel/cabazitaxel) and 19 healthy controls using in parallel CellSearch and an alternative EpCAM-based immunoisolation followed by RT-qPCR analysis to characterize the CTC population. A panel of 17 genes related with prostate biology, hormone regulation, stem properties, tumor aggressiveness and taxanes responsiveness was analysed to identify an expression signature characterizing the CTCs.ResultsPatients with ≥ 5 CTCs/7.5ml of peripheral blood at baseline and during the treatment showed lower progression free survival (PFS) and overall survival (OS). Changes of CTCs levels during the treatment were also associated with the patient's outcome. These results confirmed previous data obtained using CellSearch in mCRPC. In addition, we found a CTC profile mainly characterized by the expression of relevant genes for the hormone dependent regulation of PCa such as AR and CYP19 together with genes strongly implicated in PCa progression and resistance development such as BIRC5, TUB1A, GDF15, RAB7 and SPINK1. Our gene-expression profiling also permitted the identification of valuable prognostic biomarkers. Thus, high levels of AR, CYP19 and GDF15 were associated with poor PFS rates while AR, GDF15 and BIRC5 were also found as reliable predictors of OS. Besides, a logistic model using KLK3 and BIRC5 showed a high specificity and sensitivity compared to CellSearch to discriminate patients with a more aggressive evolution.ConclusionsThe molecular characterization of CTCs from advanced mCRPC patients provided with a panel of specific biomarkers, including genes related to taxanes resistance, with a promising applicability as “liquid biopsy” for the management of these patients.

Molecular characterization of circulating tumor cells from patients with metastatic breast cancer reflects evolutionary changes in gene expression under the pressure of systemic therapy.

Resistance to systemic therapy is a major problem in metastatic breast cancer (MBC) that can be explained by initial tumor heterogeneity as well as by evolutionary changes during therapy and tumor progression. Circulating tumor cells (CTCs) detected in a liquid biopsy can be sampled and characterized repeatedly during therapy in order to monitor treatment response and disease progression.Our aim was to investigate how CTC derived gene expression of treatment predictive markers (ESR1/HER2) and other cancer associated markers changed in patient blood samples during six months of first-line systemic treatment for MBC. CTCs from 36 patients were enriched using CellSearch (Janssen Diagnostics) and AdnaTest (QIAGEN) before gene expression analysis was performed with a customized gene panel (TATAA Biocenter).Our results show that antibodies against HER2 and EGFR were valuable to isolate CTCs unidentified by CellSearch and possibly lacking EpCAM expression. Evaluation of patients with clinically different breast cancer subgroups demonstrated that gene expression of treatment predictive markers changed over time. This change was especially prominent for HER2 expression.In conclusion, we found that changed gene expression during first-line systemic therapy for MBC could be a possible explanation for treatment resistance. Characterization of CTCs at several time-points during therapy could be informative for treatment selection.

The Mutational Landscape of Circulating Tumor Cells in Multiple Myeloma

SummaryThe development of sensitive and non-invasive “liquid biopsies” presents new opportunities for longitudinal monitoring of tumor dissemination and clonal evolution. The number of circulating tumor cells (CTCs) is prognostic in multiple myeloma (MM), but there is little information on their genetic features. Here, we have analyzed the genomic landscape of CTCs from 29 MM patients, including eight cases with matched/paired bone marrow (BM) tumor cells. Our results show that 100% of clonal mutations in patient BM were detected in CTCs and that 99% of clonal mutations in CTCs were present in BM MM. These include typical driver mutations in MM such as in KRAS, NRAS, or BRAF. These data suggest that BM and CTC samples have similar clonal structures, as discordances between the two were restricted to subclonal mutations. Accordingly, our results pave the way for potentially less invasive mutation screening of MM patients through characterization of CTCs.

Clinical Applications of Circulating Tumor Cells in Pharmacotherapy: Challenges and Perspectives.

Screening for circulating tumor cells (CTCs) has been identified as one approach to ultrasensitive liquid biopsy in real-time monitoring of cancer patients. The detection of CTCs in peripheral blood from cancer patients is promising as a diagnostic tool; however, the application of CTCs in therapeutic treatment still faces serious challenges with respect to specificity and sensitivity. Here, we review the significant roles of CTCs in metastasis and the strengths and weaknesses of the currently available methods for CTC detection and characterization. Moreover, we discuss the clinical application of CTCs as markers for patient prognosis, and we specifically focus on the application of CTCs as indicators in cancer pharmacotherapy. Characterization of the detected CTCs will provide new biologic perspectives and clinical applications for the treatment of cancer patients with metastasis.

Catch and Release: rare cell analysis from a functionalised medical wire

Enumeration and especially molecular characterization of circulating tumour cells (CTCs) holds great promise for cancer management. We tested a modified type of an in vivo enrichment device (Catch&Release) for its ability to bind and detach cancer cells for the purpose of single-cell molecular downstream analysis in vitro. The evaluation showed that single–cell analysis using array comparative genome hybridization (array-CGH) and next generation sequencing (NGS) is feasible. We found array-CGH to be less noisy when whole genome amplification (WGA) was performed with Ampli1 as compared to GenomePlex (DLRS values 0.65 vs. 1.39). Moreover, Ampli1-processed cells allowed detection of smaller aberrations (median 14.0 vs. 49.9 Mb). Single-cell NGS data obtained from Ampli1-processed samples showed the expected non-synonymous mutations (deletion/SNP) according to bulk DNA. We conclude that clinical application of this refined in vivo enrichment device allows CTC enumeration and characterization, thus, representing a promising tool for personalized medicine.

Abstract P1-01-16: Circulating tumor cells (CTCs) biomarker evaluation from patients with metastatic breast cancer (MBC) utilizing the TargetSelectorTM platform

Abstract Background: Circulating blood biomarkers represent the promise of non-invasive, real-time surrogates for tumor tissue-based biomarkers as well as afford monitoring opportunities over the course of therapy as tumors evolve and acquire resistance to treatment. Circulating tumor cells (CTCs) are cells that disseminate from tumors and can be identified in peripheral blood. CellSearch® is an FDA approved methodology for detecting and enriching this rare CTC population. The prognostic value of CTCs has been established, however the potential to characterize biomarkers on CTCs to inform treatment decisions remains an active area of investigation. Among the emerging platforms for detection and characterization of CTCs is the TargetSelectorTM system. While CellSearch® is limited to capture and detection of epithelial derived CTCs based on EpCAM and cytokeratin (CK) respectively, Biocept's TargetSelectorTM platform utilizes a novel microfluidic system for CTC enrichment based on an antibody capture cocktail that allows for enumeration of CTCs with variable phenotypes (including CK- CTCs). The CTCs are captured in transparent microfluidic chambers and cells can be viewed in situ by fluorescence microscopy and analyzed via immunocytochemistry (ICC), fluorescence in situ hybridization (FISH) and PCR analyses. Methods: Sixty-one patients with metastatic breast cancer consented and provided blood for utilization in the TargetSelectorTM platform. Based on the molecular analysis of tissue biopsies, 92% of these patients had ER+ breast tumors. Biomarker expression on captured CTCs was determined by ICC for ER and by FISH for HER2. Concordance between these results and biomarker expression on archival tumor tissue from these patients was calculated. Results: CTCs were detected in 60 of 61 patient blood samples (range 2–4471); 68% had both CK+ and CK- CTCs, and 32% had only CK- CTCs. None had only CK+ CTCs. Of those with CK+ CTCs, concordance for ER expression between the tissue and blood analyses was 85% (35/41). Concordance was much lower for patients with only CK- CTCs (32%, 6/19). Concordance for HER2 amplification in CK+ patients was 93% (38/41), and 68% (13/19) in CK- patients. For this study, the liquid biopsy was obtained over an extended period of time from when solid tumor ER and HER2 assessments were obtained; this latency period may have influenced concordance levels. For HER2, there was a significantly longer time interval between biopsies for non-concordant than for concordant pairs of samples (58.1 ± 19.7 vs. 30.9 ± 4.3 months) regardless of the CK status. No such difference was seen for the ER analysis. Conclusions: In this exploratory analysis of 61 patients with MBC, we observed a high rate of detectable CTCs as well as CTC concordance for ER (85%) and HER2 amplification (93%) for patients who had CK+ CTCs. Concordance was less if patients only had CK- CTCs. This may be attributable to heterogeneity in the breast cancer phenotypes associated with these CK- CTCs in addition to inherent issues with testing cell surface markers in this population of cells. The variable latency between the between the collection of tissue and blood samples for these analyses may account for the discrepancies observed. Citation Format: Hamilton EP, Yardley DA, Burris III HA, Shastry M, Huynh L, Bhikha L, Singh VM. Circulating tumor cells (CTCs) biomarker evaluation from patients with metastatic breast cancer (MBC) utilizing the TargetSelectorTM platform [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-16.

Abstract OT3-04-02: DETECT III and IV – Individualized CTC-based therapy of metastatic breast cancer

Abstract Background: Circulating tumor cells (CTCs) are found in patients with early and metastatic breast cancer (MBC), and both their prognostic and predictive value has been described already. There is growing evidence that CTC phenotype may differ from the primary tumor. However, CTC targeted therapy is not used in clinical routine, and treatment decisions often still are based on the primary tumor's phenotype without considering CTC-characteristics. The aim of the DETECT studies is to investigate and evaluate the role of presence and phenotype of CTC for guiding therapeutic decisions in women with HER2-negative MBC. Trial design and eligibility criteria: In a joint screening for DETECT III and IV, women with HER2-negative MBC are tested for CTCs and their HER2-phenotype. CTC detection is performed by the FDA-approved CellSearch System® (Janssen Diagnostics, Raritan, USA). Patients with HER2-positive CTCs are randomized in the multicenter Phase III study DETECT III to a physician's choice chemo- or endocrine therapy with or without additional HER2-targeted treatment with lapatinib. Women with only HER2-negative CTCs are recruited to the multicenter open-label phase II study DETECT IV. Postmenopausal women with hormone-receptor positive MBC are treated with everolimus and a physician's choice endocrine therapy in DETECT IVa. Patients with hormone-receptor positive MBC and an indication for chemotherapy and patients with triple-negative MBC receive mono-chemotherapy with eribulin in DETECT IVb. Treatment efficacy will be evaluated based on the early available CTC clearance rate (in DETECT III and DETECT IVa) and progression-free survival (in DETECT IVb) respectively, as the primary endpoint; secondary objectives will be to estimate disease control rate, progression-free (DETECT III and IVa) and overall survival, toxicity and tolerability of treatments with lapatinib, everolimus and eribulin, and quality of life. Specific aims: Changes in CTC-dynamics during therapy and their HER2-phenotype may influence following therapy decisions. The DETECT studies evaluate the prognostic and predictive role of CTCs as well as the efficacy of CTC based therapy to enable the establishment of a more personalized therapy for patients with MBC that might lead to prolonged progressive free survival and/or improved quality of life. The accompanying translational research programs investigate various markers for molecular characterization of CTCs and prediction of therapy response. Present accrual and target accrual: More than 1550 patients with HER2-negative MBC have already been screened within the DETECT study program. Thus, it is the worldwide largest study concept with therapy decisions resulting from CTC-testing and CTC-phenotypization. Contact: For further information on the DETECT study program please contact www.detect-studien.de or studienzentrale.ufk@uniklinik-ulm.de. Citation Format: Polasik A, Schramm A, Friedl TWP, Rack B, Trapp E, Fasching PA, Taran F-A, Hartkopf A, Schneeweiss A, Müller V, Aktas B, Pantel K, Meier-Stiegen F, Wimberger P, Janni W, Fehm T. DETECT III and IV – Individualized CTC-based therapy of metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OT3-04-02.

Abstract P1-01-19: Circulating tumor cells in triple-negative breast cancer patients express prostate related genes and show different genetic profiles in US and German patient cohorts

Abstract Background: Triple negative breast cancer (TNBC) is known for its aggressive behavior and poor prognosis since treatment options are limited. Specific biomarkers are urgently needed to treat patients (pts) accordingly. In this regard, circulating tumor cells (CTCs) are discussed to be an ideal surrogate marker for individualized treatment options. Using a multi-marker gene panel for the characterization of CTCs, we recently demonstrated that CTCs in TNBC pts and non-TNBC pts showed different genetic profiles including the expression of the androgen receptor (AR). We here compared CTCs of TNBC pts in two different national Institutions in the US (mainly Afro-American women) and Germany (Caucasian women) to identify potentially ethnical differences and to further explore AR expression related similarities of prostate cancer to TNBC. Methods: 2x5 ml blood of TNBC pts before therapy (n=18 from the Dep. of Gynecol. and Obstetrics, Essen, GER; n=31 from the MDA Cancer Center Houston, US) were analyzed for CTCs applying positive immunomagnetic selection targeting EpCAM, EGFR and HER2 using the AdnaTest EMT-2/Stem Cell Select (QIAGEN Hannover GmbH, Germany). Subsequently, cDNA was pre-amplified for specific genes using TaqMan PreAmp Master Mix according to in house designed assays. Establishment of a 19 gene qPCR panel was performed for the markers PI3K, AKT2, ERCC1, AURKA, HER2, HER3, EGFR, ALK, AR, BRCA1, c-KIT, c-MET, KRT5, mTOR, NOTCH1, PARP1, SRC1, CD45 (leucocyte control) and GAPDH (housekeeping gene) as well as an internal reference. The cutoff was calculated, taking the false positive rate in healthy donors into account, and defined as Ct (cutoff) - Ct (sample) - [Ct (CD45cutoff) – Ct (CD45sample)]. Using the AdnaPanel Prostate Cancer (QIAGEN Hannover GmbH, Germany), cDNA obtained from a subset of 14 pts was further analyzed for the expression profile of AR, prostate specific antigen (PSA) as well as prostate specific membrane antigen (PSMA). Results: The overall CTC-positivity rate (at least one of the markers expressed) was comparable between both sites (GER: 78%; US: 65%). Similar overexpression frequencies were found for AKT2, AR, AURKA, c-KIT, NOTCH1 and SRC at both sites. However, ALK, BRCA1, HER3 and KRT5 were predominantly found in CTCs of German pts while these genes were not or only weakly expressed in CTCs of the US group. In contrast, c-MET, ERCC1, HER2 and PARP were mainly expressed in CTCs of the US group and rarely in the German group. Interestingly, EGFR was not detected in both groups. Analyzing the overexpression of prostate related genes in a subgroup of 14 TNBC pts resulted in the expression of AR in 21%, PSA in 14%, and PSMA in 36% of cases, respectively. Conclusion: Although we observed similar gene expression profiles in both patient cohorts, clear differences in some gene expression frequencies were detected at both sites. Whether these findings can be related to ethnic differences will be subject of further investigations. Furthermore, we were able to demonstrate that prostate related genes were frequently detectable which so far have not been discussed in the context of breast cancer. Citation Format: Kasimir-Bauer S, Gao H, Bittner A-K, Plappert L, Feniuk N, Ueno NT, Cohen L, Valero V, Woodward WA, Alvarez RH, Hoffmann O, Kimmig R, Reuben JM. Circulating tumor cells in triple-negative breast cancer patients express prostate related genes and show different genetic profiles in US and German patient cohorts [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-19.

The clinical significance of the Ezrin gene and circulating tumor cells in osteosarcoma.

PurposeThe aim of this study was to investigate the clinical significance of circulating tumor cells (CTCs) in the peripheral blood of an osteosarcoma and the Ezrin gene expressed in CTCs.Patients and methodsCTC enrichment was done with CanPatrol™ CTC enrichment technique in 41 patients with osteosarcoma. The characterization of CTCs was performed using a multiple messenger RNA in situ analysis (MRIA). The expression of the Ezrin gene in CTCs was detected by RNA probe technology. The correlations of CTC counts, cell type and the expression level of the Ezrin gene with clinical stage and metastasis of osteosarcoma were analyzed using SPSS 16.0 software.ResultsThe CTC counts correlated significantly with Enneking stage (P<0.001). The ratio of mesenchymal CTCs correlated with the distant metastases (P<0.001). Ezrin gene expression in CTCs correlated significantly with distant metastases (χ2=152.51, P=0.000).ConclusionThe ratio of mesenchymal CTCs in the peripheral blood of osteosarcoma correlates with distant metastases. High expression of Ezrin gene in CTCs correlates with distant metastases.

Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion.

Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells (CRC) which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients. An unsolved technical issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells, in both live and fixed form, for their molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on a new protocol to enrich from blood live CTC using ISET® (Isolation by SizE of Tumor/Trophoblastic Cells), an open system originally developed for marker-independent isolation of fixed tumor cells. We have assessed the impact of our new enrichment method on live tumor cells antigen expression, cytoskeleton structure, cell viability and ability to expand in culture. We have also explored the ISET® in vitro performance to collect intact fixed and live cancer cells by using spiking analyses with extremely low number of fluorescent cultured cells. We describe results consistently showing the feasibility of isolating fixed and live tumor cells with a Lower Limit of Detection (LLOD) of one cancer cell per 10 mL of blood and a sensitivity at LLOD ranging from 83 to 100%. This very high sensitivity threshold can be maintained when plasma is collected before tumor cells isolation. Finally, we have performed a comparative next generation sequencing (NGS) analysis of tumor cells before and after isolation from blood and culture. We established the feasibility of NGS analysis of single live and fixed tumor cells enriched from blood by our system. This study provides new protocols for detection and characterization of CTC collected from blood at the very early steps of tumor invasion.

Circulating Tumor Cells: Moving Biological Insights into Detection.

Circulating tumor cells (CTCs) have shown promising potential as liquid biopsies that facilitate early detection, prognosis, therapeutic target selection and monitoring treatment response. CTCs in most cancer patients are low in abundance and heterogeneous in morphological and phenotypic profiles, which complicate their enrichment and subsequent characterization. Several methodologies for CTC enrichment and characterization have been developed over the past few years. However, integrating recent advances in CTC biology into these methodologies and the selection of appropriate enrichment and characterization methods for specific applications are needed to improve the reliability of CTC biopsies. In this review, we summarize recent advances in the studies of CTC biology, including the mechanisms of their generation and their potential forms of existence in blood, as well as the current CTC enrichment technologies. We then critically examine the selection of methods for appropriately enriching CTCs for further investigation of their clinical applications.

Utilizing Matrigel Transwell Invasion Assay to Detect and Enumerate Circulating Tumor Cells.

Metastasis is the cause of 90% of human cancer deaths. Circulating tumor cells (CTCs) in the peripheral blood and/or lymphatic vessels are cells shed from primary tumors and considered to be precursors of metastasis. Study of CTCs allows the serial monitoring of tumor progression and may provide predictive and prognostic biomarkers in clinic. Current CTC isolation and detection technologies encounter several challenges, including: heterogeneity of CTCs, low cell viability and/or high rate of contamination post-isolation, and the inability to distinguish viable/invasive from nonviable/nonfunctional CTCs, all of which can limit in vitro and in vivo characterization of CTCs. Here, we describe a new method to detect and enumerate of CTCs based on their invasive property.

Microfluidic-Based Enrichment and Retrieval of Circulating Tumor Cells for RT-PCR Analysis.

Molecular analysis of circulating tumor cells (CTCs) is hindered by low sensitivity and high level of background leukocytes of currently available CTC enrichment technologies. We have developed a novel device to enrich and retrieve CTCs from blood samples by using a microfluidic chip. The Celsee PREP100 device captures CTCs with high sensitivity and allows the captured CTCs to be retrieved for molecular analysis. It uses the microfluidic chip which has approximately 56,320 capture chambers. Based on differences in cell size and deformability, each chamber ensures that small blood escape while larger CTCs of varying sizes are trapped and isolated in the chambers. In this report, we used the Celsee PREP100 to capture cancer cells spiked into normal donor blood samples. We were able to show that the device can capture as low as 10 cells with high reproducibility. The captured CTCs were retrieved from the microfluidic chip. The cell recovery rate of this back-flow procedure is 100% and the level of remaining background leukocytes is very low (about 300-400 cells). RNA from the retrieved cells are extracted and converted to cDNA, and gene expression analysis of selected cancer markers can be carried out by using RT-PCR assays. The sensitive and easy-to-use Celsee PREP100 system represents a promising technology for capturing and molecular characterization of CTCs.

Personalized Treatment Through Detection and Monitoring of Genetic Aberrations in Single Circulating Tumor Cells.

Circulating tumor cells (CTCs) present a viable alternative to access tumor materials other than primary biopsies in cancer. This disease is among the most widespread in the world and is difficult to target because of its complex nature, challenges in getting quality samples and dynamic temporal changes in response to treatment. Conventional methods of detection and monitoring the disease profile do not suffice to be able to target the heterogeneity that exists at the cellular level. CTCs have been identified as a possible substitute for tumor tissue samples, and can be used to complement current disease management. Challenges in CTCs molecular analysis lie in the purity of the sample, which is masked by the presence of large quantities of white blood cells (WBCs) . In this chapter, we present a microfluidic biochip platform that performs secondary purification to isolate single CTCs efficiently. Studying single CTCs will allow for sensitive detection of critical mutations and addressing intercellular variances that will be otherwise missed easily due to low mutation frequencies when evaluating bulk cell retrieval. Using the biochip, we isolated single CTCs, and conducted personalized integrated EGFR mutational analysis using conventional polymerase chain reaction (PCR) and Sanger sequencing. We also demonstrated that high quality next generation sequencing (NGS) libraries can be readily generated from these samples. In our initial study, we revealed that the dominant EGFR mutations such as L858R and T790M could be detected in Non Small Cell Lung Cancer (NSCLC) patients with low CTC counts. We envision the biochip will enable efficient isolation of rare single cells from samples. This technology coupled with downstream molecular characterization of CTCs will aid in realizing the personalized medicine for cancer patients.

Biological and Molecular Characterization of Circulating Tumor Cells: A Creative Strategy for Precision Medicine?

Circulating tumor cells (CTCs) are a group of rare cells disseminated from either primary or metastatic tumors into the blood stream. CTCs are considered to be the precursor of cancer metastasis. As a critical component of liquid biopsies, CTCs are a unique tool to understand the formation of metastasis and a valuable source of information on intratumor heterogeneity. Much effort has been invested in technologies for the detection of CTCs because they are rare cells among the vast number of blood cells. Studies in various cancers have repeatedly demonstrated that increased CTC counts prior to or during treatment are significantly associated with poor outcomes. In the new era of precision medicine, the study of CTCs reaches far beyond detection and counting. The rapidly growing field of analytical platforms for rare-cell analysis allows in-depth characterization of CTCs at the bulk cell and single-cell level. Genetic profiling of CTCs may provide an insight into the real-time tumor status, may allow the monitoring and evaluation of treatment response in clinical routine, and may lead to the development of novel therapeutic targets as well.

Circulating Tumor Cells as Cancer Biomarkers in the Clinic.

It is believed that the development of metastatic cancer requires the presence of circulating tumor cells (CTCs) , which are found in a patient's circulation as rare abnormal cells comingled with billions of the normal red and white blood cells. The systems developed for detection of CTCs have brought progress to cancer treatment. The molecular characterization of CTCs can aid in the development of new drugs, and their presence during treatment can help clinicians determine the prognosis of the patient. Studies have been carried out in patients early in the disease course, with only primary tumors, and the role of CTCs in prognosis seems to be as important as it is in patients with metastatic disease. The published studies on CTCs have focused on their prognostic significance, their utility in real-time monitoring of therapies, the identification of therapeutic and resistance targets, and understanding the process of metastasis . The analysis of CTCs during the early stages, as a "liquid biopsy," helps to monitor patients at different points in the disease course, including minimal residual disease, providing valuable information about the very early assessment of treatment effectiveness. Finally, CTCs can be used to screen patients with family histories of cancer or with diseases that can lead to the development of cancer. With standard protocols, this easily obtained and practical tool can be used to prevent the growth and spread of cancer. In this chapter, we review some important aspects of CTCs , surveying the disease aspects where these cells have been investigated.

Fourier Ptychographic Microscopy for Rapid, High-Resolution Imaging of Circulating Tumor Cells Enriched by Microfiltration.

Examining the hematogenous compartment for evidence of metastasis has increased significantly within the oncology research community in recent years, due to the development of technologies aimed at the enrichment of circulating tumor cells (CTCs), the subpopulation of primary tumor cells that gain access to the circulatory system and are responsible for colonization at distant sites. In contrast to other technologies, filtration-based CTC enrichment, which exploits differences in size between larger tumor cells and surrounding smaller, non-tumor blood cells, has the potential to improve CTC characterization through isolation of tumor cell populations with greater molecular heterogeneity. However, microscopic analysis of uneven filtration surfaces containing CTCs is laborious, time-consuming, and inconsistent, preventing widespread use of filtration-based enrichment technologies. Here, integrated with a microfiltration-based CTC and rare cell enrichment device we have previously described, we present a protocol for Fourier Ptychographic Microscopy (FPM), a method that, unlike many automated imaging platforms, produces high-speed, high-resolution images that can be digitally refocused, allowing users to observe objects of interest present on multiple focal planes within the same image frame. The development of a cost-effective and high-throughput CTC analysis system for filtration-based enrichment technologies could have profound clinical implications for improved CTC detection and analysis.

P2.06-039 Searching for Standards: Multicenter Ring Trials to Evaluate Technologies for the Enrichment of Circulating Tumor Cells: Topic: LAB, Other

Circulating tumor cells (CTCs), which can be found in the peripheral blood of cancer patients, represent a simple and minimal-invasive source for monitoring neoplastic evolution or response to anti-cancer therapy. In recent years, numerous technology platforms for the enrichment and molecular characterization of CTCs have emerged, but comparative results and data demonstrating clinical utility are lacking for most of these platforms. To overcome this, the Innovative Medicines Initiative (IMI) consortium CANCER-ID (www.cancer-id.eu), which represents a joint undertaking of experts from academia and pharmaceutical industry, joined forces to define standards in blood-based biomarkers including the evaluation of different CTC enrichment technologies. CTC enrichment technologies including the CellSearch system, Parsortix PR1 and the Siemens filtration device were evaluated in a multicenter ring trial by using standardized spike-in samples of non-small cell lung cancer (NSCLC) cell lines, which were selected based on their different molecular/genetic features. NSCLC cells were spiked into blood of healthy volunteers with informed consent. To increase the comparability of results, spike-in samples were generated in a centralized way following well-defined protocols for pre-analytic sample handling including sample fixation, storage and shipment. Spike-in samples were subsequently analyzed using different CTC enrichment technologies by at least three CANCER-ID partners in a blinded way according to standard operating procedures (SOPs). To reflect clinically relevant disease subtypes, NSCLC cell lines were extensively profiled for copy number aberrations, mutational status (e.g. KRAS, EGFR), expression of cell surface antigens (e.g. EPCAM) as well as cell size. Based on this, cells lines with different molecular/genetic profiles were used to generate complex spike-in samples modeling the heterogeneity of real-life patient material. Spike-in samples were subsequently analyzed by at least three different CANCER-ID partners to determine sensitivity and specificity of the different platforms. In addition, comparative data was generated using the FDA approved CellSearch system, which represents the gold-standard for CTC detection and enumeration. IMI CANCER-ID is a public-private partnership in the field of liquid biopsies with 37 partners from 13 countries providing access to a variety of CTC enrichment technologies and patient samples. Making use of this major advantage, we describe the first efforts to establish standards in CTC enrichment and molecular characterization by generating comparative data in a multicenter ring experiment. The results will be used to improve SOPs for the analysis of patient blood samples, which represents a promising tool to monitor disease progression and/or therapeutic response. Support: IMI JU & EFPIA (grand no. 115749).

Heterogeneity of circulating tumor cells (CTCs) in patients with recurrent small cell lung cancer (SCLC) treated with pazopanib

ObjectivesTo investigate the effect of pazopanib on different CTCs subpopulations in patients with recurrent SCLC and evaluate their clinical relevance. MethodsPeripheral blood was obtained before the administration of pazopanib (n=56 patients), after the first cycle (n=35 patients) and at disease progression (n=45 patients). CTCs were detected by CellSearch and double immunofluorescent staining using antibodies against the cytokeratins (CK), TTF-1, CD56 and VEGFR2. ResultsBefore treatment, CTCs could be detected in 50% of patients by CellSearch; phenotypic characterization of CTCs demonstrated that 50%, 46.6% and 27.6% of patients had CD45−/TTF1+, CD45−/CD56+ and TTF-1+/CD56+ CTCs, respectively. Additionally, 59% of CTCs were TTF-1+/VEGFR2+ and 53% CK+/VEGFR2+. One pazopanib cycle resulted to a significant decrease of the number of CTCs (CellSearch: p=0.043) and CK+/VEGFR2+ cells (p=0.027). At the time of PD, both the total number of CTCs (p=0.027) and the number of the different subpopulations were significantly increased compared to post-1st cycle values; this increased CTCs number was associated with a significant increase of TTF-1+/VEGFR2+ (p=0.028) and CK+/VEGFR2+ CTCs (p=0.018). In multivariate analysis, only the number of CTCs as assessed by CellSearch after one treatment cycle was significantly associated with OS (HR: 0.21; p=0.005). ConclusionsPazopanib has a significant effect on different subpopulations of CTCs in patients with relapsed SCLC; the detection of VEGFR2+ CTCs during treatment could be a surrogate marker associated with resistance to pazopanib.