Single Circulating Tumor Cells Research Articles

Label-free high-throughput detection and quantification of circulating melanoma tumor cell clusters by linear-array-based photoacoustic tomography.

Circulating tumor cell (CTC) clusters, arising from multicellular groupings in a primary tumor, greatly elevate the metastatic potential of cancer compared with single CTCs. High-throughput detection and quantification of CTC clusters are important for understanding the tumor metastatic process and improving cancer therapy. Here, we applied a linear-array-based photoacoustic tomography (LA-PAT) system and improved the image reconstruction for label-free high-throughput CTC cluster detection and quantification <italic<in vivo</italic<. The feasibility was first demonstrated by imaging CTC cluster <italic<ex vivo</italic<. The relationship between the contrast-to-noise ratios (CNRs) and the number of cells in melanoma tumor cell clusters was investigated and verified. Melanoma CTC clusters with a minimum of four cells could be detected, and the number of cells could be computed from the CNR. Finally, we demonstrated imaging of injected melanoma CTC clusters in rats <italic<in vivo</italic<. Similarly, the number of cells in the melanoma CTC clusters could be quantified. The data showed that larger CTC clusters had faster clearance rates in the bloodstream, which agreed with the literature. The results demonstrated the capability of LA-PAT to detect and quantify melanoma CTC clusters <italic<in vivo</italic< and showed its potential for tumor metastasis study and cancer therapy.

Multiparameter Evaluation of the Heterogeneity of Circulating Tumor Cells Using Integrated RNA In Situ Hybridization and Immunocytochemical Analysis.

Circulating tumor cells (CTCs) are routinely identified as cytokeratin (CK)-positive, epithelial cell adhesion molecule (EpCAM)-positive, and CD45-negative and are enriched based on EpCAM. However, there are a number of methodological challenges regarding both isolation and characterization of these rare CTCs including downregulation or absence of EpCAM in a variety of solid tumors leading to the omission of subpopulations of CTCs, difficulties in analyzing RNA and protein targets in CTCs due to the rarity of these cells, and low levels of targets and technological limitations of visualizing the targets of interest on each individual cell. Building on our previous CTC research on CD45-based negative magnetic separation and four-color fluorescent immunocytochemical (ICC) staining, RNA in situ hybridization (ISH) was applied to fluorescently target mRNA sequences corresponding to tumor-related genes at the single CTC level. Multiple categories of markers are targeted including CK, human epidermal growth factor receptor family markers, Hedgehog pathway markers, human papillomavirus markers, and protein arginine methyltransferase 5. In addition, an integrated method of RNA ISH and fluorescent ICC staining was developed to visualize CTCs on both mRNA and protein levels. The robustness of the integrated co-ICC and RNA ISH staining was demonstrated by a series of tests on representative tumor markers of different categories. The integrated staining can incorporate the advantages of both RNA ISH and fluorescent ICC staining and provide more intense signals and more specific bindings. With this integrated staining methodology, distinct staining patterns were applied in this report to facilitate the searching and characterization of rare subgroups of CTCs. These results support the existence of diverse groups of CTCs at both protein and mRNA transcript levels and provide an analytical tool for the research on CTCs of rare subgroups.

A FISH-based method for assessment of HER-2 amplification status in breast cancer circulating tumor cells following CellSearch isolation.

IntroductionAmplification of the HER-2/neu (HER-2) proto-oncogene occurs in 10%–15% of primary breast cancer, leading to an activated HER-2 receptor, augmenting growth of cancer cells. Tumor classification is determined in primary tumor tissue and metastatic biopsies. However, malignant cells tend to alter their phenotype during disease progression. Circulating tumor cell (CTC) analysis may serve as an alternative to repeated biopsies. The Food and Drug Administration-approved CellSearch system allows determination of the HER-2 protein, but not of the HER-2 gene. The aim of this study was to optimize a fluorescence in situ hybridization (FISH)-based method to quantitatively determine HER-2 amplification in breast cancer CTCs following CellSearch-based isolation and verify the method in patient samples.MethodsUsing healthy donor blood spiked with human epidermal growth factor receptor 2 (HER-2)-positive breast cancer cell lines, SKBr-3 and BT-474, and a corresponding negative control (the HER-2-negative MCF-7 cell line), an in vitro CTC model system was designed. Following isolation in the CellSearch system, CTC samples were further enriched and fixed on microscope slides. Immunocytochemical staining with cytokeratin and 4′,6-diamidino-2′-phenylindole dihydrochloride identified CTCs under a fluorescence microscope. A FISH-based procedure was optimized by applying the HER2 IQFISH pharmDx assay for assessment of HER-2 amplification status in breast cancer CTCs.ResultsA method for defining the presence of HER-2 amplification in single breast cancer CTCs after CellSearch isolation was established using cell lines as positive and negative controls. The method was validated in blood from breast cancer patients showing that one out of six patients acquired CTC HER-2 amplification during treatment against metastatic disease.ConclusionHER-2 amplification status of CTCs can be determined following CellSearch isolation and further enrichment. FISH is superior to protein assessment of HER-2 status in predicting response to HER-2-targeted immunotherapy in breast cancer patients. This assay has the potential of identifying patients with a shift in HER-2 status who may benefit from treatment adjustments.

Accession of Tumor Heterogeneity by Multiplex Transcriptome Profiling of Single Circulating Tumor Cells.

Transcriptome analysis of circulating tumor cells (CTCs) holds great promise to unravel the biology of cancer cell dissemination and identify expressed genes and signaling pathways relevant to therapeutic interventions. CTCs were enriched based on their EpCAM expression (CellSearch®) or by size and deformability (ParsortixTM), identified by EpCAM and/or pan-keratin-specific antibodies, and isolated for single cell multiplex RNA profiling. Distinct breast and prostate CTC expression signatures could be discriminated from RNA profiles of leukocytes. Some CTCs positive for epithelial transcripts (EpCAM and KRT19) also coexpressed leukocyte/mesenchymal associated markers (PTPRC and VIM). Additional subsets of CTCs within individual patients were characterized by divergent expression of genes involved in epithelial-mesenchymal transition (e.g., CDH2, MMPs, VIM, or ZEB1 and 2), DNA repair (RAD51), resistance to cancer therapy (e.g., AR, AR-V7, ERBB2, EGFR), cancer stemness (e.g., CD24 and CD44), activated signaling pathways involved in tumor progression (e.g., PIK3CA and MTOR) or cross talks between tumors and immune cells (e.g., CCL4, CXCL2, CXCL9, IL15, IL1B, or IL8). Multimarker RNA profiling of single CTCs reveals distinct CTC subsets and provides important insights into gene regulatory networks relevant for cancer progression and therapy.

Single cell gene expression analysis of circulating tumor cells in ovarian cancer reveals CTCs co-expressing stem cell and mesenchymal markers

Aim: Circulating tumor cells (CTCs) in the blood of ovarian cancer patients (OCP) are associated with decreased overall survival, whereby CTCs with epithelial-mesenchymal-transition (EMT) or stem-like traits are supposed to be involved in metastatic progression and recurrence. Thus, investigating the transcriptional profiles of CTCs might help to identify therapy resistant tumor cells and to overcome treatment failure. To this end, we established a multi-marker RT-PCR for the molecular characterization of single CTCs, detecting epithelial (EpCAM, Muc1, CK5/7), EMT (N-cad, vim, Snai1/2, CD117, CD146, CD49f) and stem cell (CD44, ALDH1A1, Nanog, SOX2, Notch1/4, Oct4, Lin28) associated transcripts.

Isolation of circulating tumor cells enables additional marker staining following panel sequencing

Circulating tumor cells (CTC) are rare cells dissociated successfully from the primary tumor into the blood stream. The presence of CTCs is associated with increased risk to develop metastases and a short survival. Detection and isolation of CTCs is necessary. Here we show a workflow to isolate single CTCs for molecular characterization by combining the CellSearch and CellCelector.

Detection and Characterization of Circulating Tumor Associated Cells in Metastatic Breast Cancer.

The availability of blood-based diagnostic testing using a non-invasive technique holds promise for real-time monitoring of disease progression and treatment selection. Circulating tumor cells (CTCs) have been used as a prognostic biomarker for the metastatic breast cancer (MBC). The molecular characterization of CTCs is fundamental to the phenotypic identification of malignant cells and description of the relevant genetic alterations that may change according to disease progression and therapy resistance. However, the molecular characterization of CTCs remains a challenge because of the rarity and heterogeneity of CTCs and technological difficulties in the enrichment, isolation and molecular characterization of CTCs. In this pilot study, we evaluated circulating tumor associated cells in one blood draw by size exclusion technology and cytological analysis. Among 30 prospectively enrolled MBC patients, CTCs, circulating tumor cell clusters (CTC clusters), CTCs of epithelial–mesenchymal transition (EMT) and cancer associated macrophage-like cells (CAMLs) were detected and analyzed. For molecular characterization of CTCs, size-exclusion method for CTC enrichment was tested in combination with DEPArray™ technology, which allows the recovery of single CTCs or pools of CTCs as a pure CTC sample for mutation analysis. Genomic mutations of TP53 and ESR1 were analyzed by targeted sequencing on isolated 7 CTCs from a patient with MBC. The results of genomic analysis showed heterozygous TP53 R248W mutation from one single CTC and pools of three CTCs, and homozygous TP53 R248W mutation from one single CTC and pools of two CTCs. Wild-type ESR1 was detected in the same isolated CTCs. The results of this study reveal that size-exclusion method can be used to enrich and identify circulating tumor associated cells, and enriched CTCs were characterized for genetic alterations in MBC patients, respectively.

Impact of label-free technologies in head and neck cancer circulating tumour cells.

BackgroundThe ability to identify high risk head and neck cancer (HNC) patients with disseminated disease prior to presenting with clinically detectable metastases holds remarkable potential. A fraction of circulating tumour cells (CTCs) are invasive cancer cells which mediate metastasis by intravasation, survival and extravasation from the blood stream to metastatic sites. CTCs have been cleared by the FDA for use as surrogate markers of overall survival and progression free survival for breast, prostate and colorectal cancers using the CellSearch® system. However, the clinical significance of CTCs in head and neck cancer patients has yet to be determined. There has been a significant shift in CTC enrichment platforms, away from exclusively single marker selection, to epitope-independent systems.MethodsThe aim of this study was to screen advanced stage HNC patients by the CellSearch® platform and utilise two other epitope-independent approaches, ScreenCell® (microfiltration device) and RosetteSep™ (negative enrichment), to determine how a shift to such methodologies would enable CTC enrichment and detection.ResultsIn advanced stage HNC patients, single CTCs were detected in 8/43 (18.6%) on CellSearch®, 13/28 (46.4%) on ScreenCell® and 16/25 (64.0%) by RosetteSep™ (the latter could also detect CTC clusters). Notably, in patients with suspicious lung nodules, too small to biopsy, CTCs were found upon presentation. Moreover, CTCs were readily detected in advanced stage HNC patients.ConclusionThe epitope-independent platforms detected higher CTC numbers and clusters. Further studies are needed to ascertain whether CTCs can be used as independent prognostic markers for HNCs.

Development of a Novel Cell Monitoring System Based on Lens-Less Imaging Toward Cultivation of Circulating Tumor Cells

Circulating tumor cells (CTCs) are defined as tumor cells circulating in the peripheral blood of patients with metastatic cancer. As CTCs could provide useful information regarding malignancy and metastatic property of primary tumor, as well as its response to anticancer agents, fundamental techniques for recovery and analysis of CTCs have been intensively studied in this decade. Although state-of-the-art microfluidic tools and whole genome amplification techniques are now available to investigate single CTCs, it is still extremely difficult to cultivate CTCs that, for example, enables examination of multiple anticancer agents toward identical CTCs. This could be due to the lack of knowledge of division and proliferation behavior of CTCs. The critical difficulty in investigation of CTC proliferation is that cell division events of CTCs are very rare, and thus conventional microscopy could hardly identify the dividing CTCs from the samples containing a number of blood cells. In this study, we propose to use a wide-field imaging system for investigation of CTC cultivation. The imaging system is composed of a light emitting diode (LED, peak wavelength: 465 nm) and a complementary metal oxide semiconductor (CMOS) imaging sensor by which an area of 6.55 × 4.92 mm2can be visualized in one shot. A cell culture dish is placed on the CMOS image sensor, and images are acquired under the LED illumination without using any lens systems (referred to as “lenss-less imaging”). The CMOS image sensor allows to develop a space-saving and inexpensive platform, so that whole system can be introduced in a general incubator, and real-time monitoring of cell growth is possible. Using this lens-less imaging system, we attempted to observe tumor cell proliferation and distinguish tumor cells from blood cells. HeLa cells were used as model tumor cells. A culture dish containing HeLa cells in 2 ml of the high glucose Dulbecco's modified Eagle's medium (DMEM, 5,000 cells/ml) was placed on the lens-less imaging system, and incubated for 18 hours at 37°C with 5% CO2. Images were captured every 15 min. For comparison, microscopic observation was also performed. As a result, HeLa cells were recorded as a spherical patterns with high intensity by a CMOS image sensor. When divided, HeLa cells were once illustrated as a dark patterns, and then two daughter cells gradually appeared with increasing the intensity. It is well known that adhesive cells temporarily go through a non-adhesive state before their division process. Indeed, we confirmed that the spherical patterns with high intensity represents the HeLa cells adhere on the bottom of the dishes, and the subsequent dark patterns do the cells in the non-adhesive state by microscopic observation. This data indicates that the lens-less imaging system can detect individual cell division events. Taking advantage of the wide field of view, it was possible to monitor the cell division events of several hundreds of cells simultaneously. Recovery of CTCs from blood samples is the first step for the cultivation and further analysis, however it is still hard to avoid blood cell contamination despite the development of sophisticated technology for CTC separation and enrichment nowadays. Consequently, it is essential to differentiate CTCs and the contaminated blood cells, otherwise accurate evaluation of CTC proliferation is impossible. To test whether tumor cells and blood cells were distinguishable based on lens-less imaging, we compared the lens-less images of HeLa cells, red blood cells and JM cells (model white blood cells). A number of differentiation parameters were extracted from the images, and computed for cluster analysis. As a result, it was successfully demonstrated to discriminate HeLa cells, red blood cells and JM cells without any labeling process, suggesting the development of a simple, inexpensive and rapid cell typing system. In summary, the lens-less imaging system could monitor cell division events of HeLa cells in a wide field of view, and differentiate HeLa cells and blood cells with high accuracy. We believe that this system could be a powerful tool to monitor the proliferation of CTCs recovered from patient blood samples.

Stem-like features of cancer cells on their way to metastasis.

More than 90 % of cancer-related deaths are due to the development of a systemic metastatic disease. Clearly, much remains to be understood about the biological principles that govern human cancer metastasis, aiming at the ambitious objective to decrease metastasis-related mortality in patients. For many years, research on metastasis has been conducted in great part on experimental mouse models, mainly because of the difficulties in sampling, longitudinal studies, and molecular interrogation of a human metastatic disease. However, recently, extraordinary advances in microfluidic technologies are allowing the isolation and characterization of human circulating tumor cells (CTCs) that escaped a primary tumor mass and are in the process of seeding a distant metastasis. Analysis of human CTCs has now revealed important features of cancer metastasis, such as the high metastatic potential of CTC-clusters compared to single CTCs, the dynamic expression of epithelial and mesenchymal markers on CTCs during treatment, and the possibility to culture CTCs from patients for a real-time and individualized testing of drug susceptibility. Nevertheless, several aspects of CTC biology remain unsolved, such as the characterization of the stem-like cell population among human CTCs. Here, we focus on describing the latest findings in the CTC field, and discuss them in the context of cancer stem cell biology. Defining the molecular features of those few metastasis-initiating, stem-like CTCs holds the exceptional promise to develop metastasis-tailored therapies for patients with cancer.ReviewersThis article was reviewed by Elisa Cimetta, Luca Pellegrini and Sirio Dupont (nominated by LP).

Frequent detection of PIK3CA mutations in single circulating tumor cells of patients suffering from HER2-negative metastatic breast cancer

Modern technologies enable detection and characterization of circulating tumor cells (CTC) in peripheral blood samples. Thus, CTC have attracted interest as markers for therapeutic response in breast cancer. First studies have incorporated CTC analyses to guide therapeutic interventions and stratification of breast cancer patients. Aim of this study was to analyze characteristic features of CTC as biomarker for predicting resistance to HER2-targeted therapies. Therefore, CTC from metastatic breast cancer patients with HER2-negative primary tumors screened for the prospective randomized phase III trial DETECT III were explored for their HER2 status and the presence of PIK3CA mutations. Detection and characterization of HER2 expression of CTC were conducted with the CellSearch® system. Fifteen of 179 CTC-positive patients (8.4%) contained ≥1 CTC with strong HER2 expression. Genomic DNA from individual CTC isolated by micromanipulation was propagated by whole genome amplification and analyzed for PIK3CA mutations in exons 9 and 20 by Sanger sequencing. One or more CTC/7.5 mL were detected in 179/290 patients (61.7%). In 109 patients (34.8%), ≥5 CTC/7.5 mL were found. We detected at least one CTC with the mutation p.E542K, p.E545K, p.H1047R, p.H1047L or p.M1043V in 12/33 patients (36.4%). Thirty six of 114 CTC (31.6%) harbored one of these mutations. CTC in individual patients exhibited heterogeneity concerning PIK3CA mutations and HER2 expression. In conclusion, clinically relevant genomic aberrations such as mutations in the hotspot regions of exon 9 and 20 of the PIK3CA gene can be detected in single CTC and might provide insights into mechanisms of resistance to HER2-targeted therapies.

Abstract 2253: Digital sorting and single-cell genomic profile comparison of lung adenocarcinoma CTCs between EpCAM and size-based enrichment methods

Abstract Introduction Several methods are currently available for Circulating Tumor Cells (CTCs) enrichment. Many studies compared CTC counts between FDA-approved CellSearch® system (based on EpCAM enrichment) and size-based selection methods. However, little is known about genomic differences across CTCs obtained from different platforms. For the first time, we report here a genomic characterization of copy-number and sequence variants in single CTCs enriched with different methods from the same patient. Methods Peripheral blood (PB) and FFPE tissue from a pelvis bone metastasis were collected from an advanced lung adenocarcinoma patient, treated with cisplatin-pemetrexed-necitumumab as first line therapy and with carboplatin-gentamicin as second-line. Two PB samples were collected at the same time: one was enriched with CellSearch® System, and one with Parsortix PR1 (size-based selection), followed by Cytokeratin-APC, CD45-PerCp and DAPI staining. Both samples were analyzed with DEPArray™ system and single CTCs along with single WBCs as controls, were isolated. Collected cells were whole genome amplified with Ampli1™ WGA kit and their Genome Integrity Index (GII) was assessed. On IonTorrent™ PGM Platform, we profiled Genome-wide copy-number alterations (CNA) by low-pass whole-genome sequencing with Ampli1™ LowPass Kit, and analyzed cancer-gene sequence variants with Ampli1™ CHP Custom Beta targeted panel. FFPE tissue was disaggregated down to single-cell suspension and labelled with Keratin and Vimentin to distinguish tumor from stromal cells. Aliquots of pure cells were recovered with DEPArray™ system; the analysis of CNVs and mutations on tissue-derived samples is in progress. Results Single CTCs (CK+, CD45-,DAPI), showing GII≥3, either isolated from CellSearch®-enriched or PR1-enriched blood, along with WBC, were selected for NGS. Ampli1™ LowPass Kit data showed several aberrations confirming tumor origin of all CTCs, while WBCs (n = 6) produced balanced profiles. Unsupervised hierarchical clustering segregated PR1-derived (n = 6) from CellSearch®-derived (n = 4) single CTCs in two separate branches. PR1-derived CTCs were very similar to each other, whereas CellSearch®-derived CTCs were more heterogeneous, although certain aberrations were common to all CTCs across platforms. At the sequence level, the targeted panel revealed somatic mutations shared by all CTCs (FLT3), a PTPN11 subclonal mutation (in 3/6 PR1 and 1/4 CellSearch), and other private mutations in single CTCs. Discussion The combination of low-pass whole-genome sequencing and targeted sequencing on single-CTCs sorted from PB enriched with different platforms reveals genetic similarities and diversities. Integration of results with genetic analysis of pure tumor cells isolated from the tumor tissue, will provide further insight of tumor diversity in different compartments. Citation Format: Francesca Fontana, Francesco Gelsomino, Claudio Forcato, Mario Terracciano, Chiara Bolognesi, Annalisa Altimari, Genny Buson, Chiara Mangano, Paola Tononi, Francesco Bacchi, Gianni Medoro, Michelangelo Fiorentino, Nicolò Manaresi, Michele Tognetto, Andrea Ardizzoni. Digital sorting and single-cell genomic profile comparison of lung adenocarcinoma CTCs between EpCAM and size-based enrichment methods. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2253.

Abstract 3969: CTCs and CTC clusters in breast cancer patient-derived xenograft models

Abstract Breast cancer (BC) patient-derived xenograft (PDX) models represent a continuous and reproducible source of circulating tumor cells (CTCs). Using various BC PDX models, we describe the utility of CTCs and CTC clusters in detecting tumor-specific mutations and our preliminary results in understanding their predictive value for treatment response and long-term outcomes. CTCs were detected in 300-450 μl of blood of PDX-bearing mice using the RareCyte technology adapted for small blood volume. CTCs were isolated without cell surface marker-based enrichment and identified them as DAPI+, human Cytokeratin (CK)+, and mouse CD45-. Collective expression of cell surface markers (EpCAM, EGFR, and HER2) was assessed using a cocktail of target-specific antibodies in CTCs and primary PDX tumors. Individual CTCs and tumor cells from primary and metastatic tumors were isolated using CytePicker® for single cell analysis of tumor-specific mutations. Single CTCs (1-41 per mouse) and CTC clusters (1-2 per mouse) were detected in the blood of one ER+/PR+/HER2- (BCM-4888) and two triple-negative BC (TNBC, BCM-4272 and BCM-3887) PDX models. The PIK3CA T1035A mutation found in primary tumors of BCM-4888 was also detected in isolated CTCs and PDX primary and metastatic tumor cells. As a proof-of-principle experiment, we have evaluated numbers of single CTCs and CTC clusters at baseline and after treatment with 4 weekly cycles of vehicle (N = 5-6) or chemotherapy regimens (N = 2-3) [docetaxel or carboplatin or their combination] in TNBC PDX models BCM-4272 and BCM-3887. Preliminary analysis from these studies suggests dynamic and differential effects of chemotherapy regimens on single CTCs and CTC clusters, potentially reflecting the genetic characteristics of tumors and their unique response to the selected chemotherapy agents. Ongoing experiments in additional mice and PDX models will determine the predictive role of CTCs and CTC clusters in treatment response and long-term outcomes such as recurrence-free survival. In conclusion, we have demonstrated that RareCyte technology detects CTCs from small volumes of blood without the use of cell surface marker-based enrichment method. Furthermore, CTCs and CTC clusters can be used to assess the presence of tumor-specific mutations. Ongoing studies will fully reveal the potential of CTCs and CTC clusters as surrogate markers of treatment response and outcomes within PDX models. Citation Format: Debashish Sahay, Arturo B. Ramirez, Raksha R. Bhat, Lacey E. Dobrolecki, Agostina Nardone, Michael T. Lewis, C. Kent Osborne, Mothaffar Rimawi, Jackie L. Stilwell, Eric P. Kaldjian, Rachel Schiff, Meghana V. Trivedi. CTCs and CTC clusters in breast cancer patient-derived xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3969.

Abstract 2257: Differential expression of PD-L1 on circulating tumor cells among patients with advanced lung cancer

Abstract Background and purpose: Immune-checkpoint blockade with anti-programmed death-1 (PD-1) antibodies is rapidly emerging for the treatment of human malignancies including lung cancer. Although programmed death-ligand 1 (PD-L1) has been studied as a predictive biomarker, detection and evaluation of PD-L1 expression level on tissue samples remain challenging due to its dynamic and unstable expression. Thus the diagnostic tool for real-time monitoring of PD-L1 expression is critically needed. Here, we assessed the expression pattern of PD-L1 on circulating tumor cells (CTCs) by using microcavity array (MCA) system in patients with advanced non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Experimental procedure: PD-L1 staining on CTCs was established using NSCLC cell lines H820, H441, A549 and H23 expressing varying levels of PD-L1 spiked in the peripheral blood obtained from healthy donors. For clinical evaluation, 3 ml of peripheral whole blood was collected from 20 advanced lung cancer patients prior to the initiation of chemotherapy and from 10 healthy donors. Cells were captured and immuno-stained by using the automated MCA system (Hitachi Chemical Co., Ltd). CTCs were defined as those positive for DAPI and cytokeratin (CK) and negative for CD45. PD-L1 expression level on CTCs was visualized by addition of PD-L1 immunocytochemistry procedure. High-resolution fluorescent images were obtained using fluorescence microscope (Carl Zeiss Microscopy Co., Ltd). Results: Characteristics of 20 lung cancer patients enrolled in clinical study were as follows: median age 74 (range, 48 to 84); male 60%; stage III/IV, 10/90%; NSCLC/SCLC, 70/30%. More than 2 CTCs were identified in 14 patients (median 22.5; range, 4 to 71), and PD-L1 positive CTCs were detected in 12 patients (median 5; range, 2 to 15). No correlation was detected between the number of total CTCs and that of PD-L1 positive CTCs in each patient (R2 = 0.05). We found a total of 25 CTC clusters from 20 patients, of which PD-L1 expression was both homogenous and heterogeneous. It is noteworthy that clustered CTCs have larger proportion of PD-L1 positive CTCs per whole clustered CTCs than that of non-clustered CTCs (24/54, 44% versus 51/347, 15%, respectively). We further focused on CTC-interacting white blood cells, which intensively bound with aggregated CTCs rather than single CTC (12/54, 22% versus 43/337, 13%, respectively). Our data implicate that PD-L1 expression on CTC correlates with aggregation of CTCs (p &amp;lt; 0.05). Conclusions: Our results showed that PD-L1 expression on CTCs was detectable and there is intrapatient heterogeneity of its expression in patients with advanced lung cancer. Further investigation is warranted to better understand the biological importance of the correlation between PD-1 expression and CTC aggregation and CTC bound to white blood cells. Citation Format: Woong Kim, Yasuhiro Koh, Hiroaki Akamatsu, Satomi Yagi, Ayaka Tanaka, Kuninobu Kanai, Atsushi Hayata, Ryota Shibaki, Masayuki Higuchi, Hisashige Kanbara, Takashi Kikuchi, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Differential expression of PD-L1 on circulating tumor cells among patients with advanced lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2257.

Abstract 4971: Quantitative image analysis of androgen receptor (AR) and tubulin biomarker profiles in circulating tumor cells (CTCs) from metastatic castration resistant prostate cancer (mCRPC) patients

Abstract Background: CTCs represent a real-time, liquid biopsy for analyses of a variety of molecular biomarkers that offer clinically relevant information. In prostate cancer, the AR pathway controls tumor cell growth and survival and is an important therapeutic target. AR becomes biologically active upon translocation from the cytoplasm to the nucleus in a microtubule dependent fashion, resulting in the activation of numerous genes. Real-time analysis of AR and tubulin status in CTCs may provide insight to therapy response in mCRPC patients. Methods: Androgen sensitive human prostate cancer cells, LNCaP, were treated with and without a synthetic androgen (R1881; 10 nM, 1 hr) and docetaxel, spiked into healthy donor blood, and captured using a prostate specific microfluidic device (GEDI chip), following the same protocol that we use with patient samples. Cells were fixed and stained (N-terminal AR, CD45, CK, DAPI, and tyr-tubulin) directly on the device and imaged using high resolution multiplex confocal microscopy. AR and tubulin status were analyzed using a quantitative image analysis algorithm. Nuclear AR percentage was calculated by integrating fluorescence intensity within regions defining the entire cell and nucleus. H-Score was calculated by multiplying the nuclear AR percentage by the normalized total cell AR intensity. Tubulin status and microtubule bundling were assessed using a variety of quantitative parameters including measurements of tubulin fluorescence intensity and distribution within the cell. Results: LNCaP cells in the presence of synthetic androgen exhibited higher nuclear AR percentage (p = 0.0004) and H-score (p = 0.003). Docetaxel treatment led to lower nuclear AR percentage (p = 0.04) and lower H-Score (p = 0.03). Cells treated with docetaxel exhibited higher tubulin intensity range (p = 0.008) and standard deviation (p = 0.03), consistent with docetaxel's mechanism of action. We applied these methods of quantitative image analyses to CTCs isolated from a small cohort of mCRPC patients. Single CTC image analysis revealed heterogeneity in AR status. Quantitative evidence for microtubule bundling was observed in patients receiving docetaxel chemotherapy. Conclusions: We have developed a high throughput quantitative image analysis algorithm to interrogate mCRPC specific biomarkers, such as AR and tubulin status, in single CTCs. These methods of quantitative image analyses will be prospectively validated with CTCs from mCRPC patients followed longitudinally over the course of treatment with AR inhibitors and taxanes, yielding patient specific, clinically relevant information that can guide physicians’ strategies for the clinical management of cancer. Citation Format: Daniel Worroll, Giuseppe Galletti, David M. Nanus, Scott T. Tagawa, Paraskevi Giannakakou. Quantitative image analysis of androgen receptor (AR) and tubulin biomarker profiles in circulating tumor cells (CTCs) from metastatic castration resistant prostate cancer (mCRPC) patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4971.

Abstract 2256: Establishment and characterization of circulating tumor cell-derived xenografts in non-small cell lung cancer

Abstract Low numbers of circulating tumor cells (CTCs) have so far limited the establishment of CTC-derived xenografts (CDXs) to improve our understanding of tumor progression, drug resistance mechanisms, and their biological properties. We report the establishment and the phenotypic and molecular characterization of one NSCLC CDX. Blood samples (30 ml) were drawn from 49 NSCLC patients with advanced metastatic disease. CTCs were enriched by RosetteSep, embedded in matrigel, and implanted in the interscapular aspect of NSG mouse (Nod/Scid-IL2Rγ-/-). Mice were followed-up for one year according to ethical regulations. CDX tumours and CDX derived cell lines were phenotypically and molecularly characterized by immunofluorescence, immunohistochemistry, CGHarray, exome sequencing and transcriptome gene expression. CTCs from one NSCLC patient with 750 CTCs detected by CellSearch gave rise to a tumor 5 months after initial murine injection. Histological analysis confirmed the human origin of the tumor and the presence of a poorly differentiated adenocarcinoma consistent with the patient's biopsy. Tumor was positive for EpCAM, EMA, CK8;18, and Ki67, and negative for vimentin. A fraction of cells (25%) from freshly dissociated tumors exhibited ALDH activity. CGH from CDX tumors at passage 1 and 2 shows multiple gene rearrangement, revealing a high degree of genomic instability. Transcriptome analysis of ALDH positive and negative cells is ongoing and should help of identifying a cancer stem cell gene expression signature. Whole-exome sequencing of CDX tumor is ongoing and will be compared to data obtained from single CTCs from the patient. A cell line established in vitro from the CDX model grows in 3D clusters and is tumorigenic in mice. Interestingly, this cell line is positive for cytokeratins, EpCAM, E-cadherin, N-cadherin, vimentin, and expresses multiple cancer stem cell markers including CD166, CD24, CD133 and, ALDH activity. The cell line is hypotetraploid (about 70 chromosomes) and its CGH profile was similar to that of the CDX tumour, revealing a high level of genome instability. By investigating DNA replication process in this cell line, we found that it exhibits a spontaneous enhanced DNA damage signaling associated to an accumulation of DNA double strand breaks mainly in S phase strongly suggesting that the CDX-derived cell line displays hallmarks on replication stress that could explain, at least partially, the genomic instability in the cells. We report a low success rate in the establishment of NSCLC CDX (2%). However one NSCLC CDX model harboring cancer stem cell properties and deficiency of DNA replication maintenance was established. Ongoing work to identify a cancer stem cell signature and characteristics replication stress markers in this CDX model will be presented. This NSCLC CDX model will be useful to test drugs targeting these alterations in vivo and improve our knowledge of drug resistance. Citation Format: Vincent Faugeroux, Olivier Deas, Judith Michels, Jean Gabriel Judde, Stefano Cairo, Philippe Vielh, Virginie Marty, Fanny Billiot, Maud Ngocamus, Benjamin Besse, Patricia Kannouche, Françoise Farace. Establishment and characterization of circulating tumor cell-derived xenografts in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2256.

Abstract 3155: Investigating chemoresistance in small cell lung cancer through the molecular profiling of single circulating tumour cells

Abstract Background Chemoresistance, both intrinsic and acquired, represents one of the greatest challenges in the management of Small Cell Lung Cancer (SCLC), contributing to the very poor survival seen in this disease. The investigation of SCLC, and particularly serially monitoring the development of changes associated with resistance, is hampered by the limited availability of tumour tissue for research. Circulating tumour cells (CTCs) in contrast, are abundant in this disease and represent a potential minimally invasive ‘liquid biopsy’ to study the molecular landscape of SCLC. Methods To investigate tumour genetics in SCLC CTCs were isolated from chemorefractory patients and chemoresponsive patients’ blood samples using the DEPArray©. Blood samples were collected prior to chemotherapy and again at progression with relapsed disease. Following single-cell whole genome amplification low coverage whole genome sequencing and whole exome sequencing (WES) of CTCs were used to investigate mutations and to generate genome-wide patterns of copy number alterations (CNA). Results Hallmark SCLC molecular abnormalities such as TP53 mutations and copy number loss in tumour suppressor genes such as RB1 (previously identified in bulk tumour profiling), were noted in the isolated SCLC CTCs. Distinct CNA profiles were found in the CTCs isolated from patients with chemorefractory disease compared to those isolated from patients with chemoresponsive disease. A potential signature based on differential copy number changes in 2183 loci between the two groups of patients’ CTCs was identified. When tested in an independent set of CTC-derived explants this signature achieved 100% accuracy in classifying patients with chemoresponsive disease. There were no profound differences in CNA pattern in the CTCs isolated from initially chemoresponsive patients when they had relapsed post chemotherapy when compared to the baseline samples. However, changes in the proportion of C&amp;gt;A transversions were identified by WES between baseline and relapse CTCs. Conclusions Our study highlights the utility of molecular profiling CTCs in the research of SCLC. The data presented confirm CTCs represent a novel medium for the investigation of chemoresistance. Citation Format: Louise R. Carter, Dominic G. Rothwell, HuiSun Leong, Yaoyong Li, Deborah J. Burt, Jenny Antonello, Cassandra Hodgkinson, Karen Morris, Lynsey Franklin, Crispin J. Miller, Fiona Blackhall, Caroline Dive, Ged Brady. Investigating chemoresistance in small cell lung cancer through the molecular profiling of single circulating tumour cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3155.

Abstract 4953: Whole-exome sequencing of single circulating tumor cells according to epithelial-mesenchymal marker expression in metastatic prostate cancer

Abstract Molecular characterization of metastatic castration-resistant prostate cancer (mCRPC) is limited by tumor tissue availability. The analysis of circulating tumor cells (CTCs) offers an attractive non invasive surrogate option to analyze molecular alterations. We report whole exome sequencing (WES) of CTCs at the single cell level in 11 mCRPC patients. We examined single somatic nucleotide variant (sSNV) shared between matched metastatic tumor sample and CTCs and sSNV specific to CTCs. Blood samples were drawn from 11 patients enrolled in the clinical program MOSCATO (2011-A00841-40). CTC enrichment, detection and single cell isolation were performed using three methods to obtain pools of 1-10 CTCs. The first method used ISET filtration, immunofluorescent staining (CD45, pan-cytokeratin, EpCAM, Vimentin and Hoechst 33342) on filters and laser microdissection of single CTCs; the second combined CellSearch and the VyCap puncher system; the third used RosetteSep enrichment, immunofluorescent staining and isolation by cell sorting. Whole Genome Amplification (WGA) was performed using the Ampli1 kit. WGA quality was assessed by qPCR of 7 genes located on different regions of the genome. WES was performed by preparation of a genomic DNA bank, Agilent capture and sequencing on the Illumina HiSeq 2000 platform. Data were aligned to the human genome reference hg19. GATK Haplotype Caller enabled identification of germline polymorphisms from each patient in normal DNA, metastatic sample and CTCs in order to consider WGA induced bias. The detection of sSNV in tumor biopsies and CTCs was assessed with Mutect and IndelGenotyper respectively. 189 WGAs of CTC pools were performed. A first round of WES showed that at least 3 well amplified genes were required to obtain a coverage of at least 50% at 10X depth sequencing. 34 pools of phenotypically different CTCs from 7 patients were selected and sequenced. Mean coverage of 51% was obtained at a sequencing depth of 10X. Allelic drop out was lower for CTC pools containing 5 to 10 cells. 17/34 (50%) CTC samples (4 patients) had shared sSNV with the paired tumor sample (range 0.35%-68%). Epithelial CTCs had more shared sSNV with metastatic biopsies than CTCs of other phenotypes but shared sSNV were also detected in large Cytokeratin-Vimentin- CTC. Shared sSNV in cancer genes between epithelial CTC pools, but not in the paired biopsy, were present in 2 patients. We report WES of CTC pools harboring distinct EMT marker phenotypes is possible with the use of 3 different approaches to enrich, detect and isolate CTCs. The detection of shared sSNV between CTC pools and corresponding biopsy could validate the use of CTCs as a liquid biopsy. The finding of sSNV specific to CTCs could offer additional data on tumor heterogeneity. Ongoing work examining if sSNV detected in phenotypically different CTCs converge to similar signaling pathways will be presented. Citation Format: Vincent Faugeroux, Céline Lefebvre, Emma Pailler, Valérie Pierron, Fanny Billiot, Charles Marcaillou, Philippe Vielh, Semih Dogan, Philippe Rameau, Maud Ngocamus, Jean Charles Soria, Karim Fizazi, Yohann Loriot, Sylvia Julien, Françoise Farace. Whole-exome sequencing of single circulating tumor cells according to epithelial-mesenchymal marker expression in metastatic prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4953.

Abstract LB-327: Isolation and mRNA-seq analysis of single CTCs from blood samples using an integrated fluidic circuit for functional single cell studies

Abstract As the precursor to metastatic cancer, circulating tumor cells (CTCs) hold tremendous potential for increasing our understanding of tumor metastasis, advancing personalized therapeutics, and have emerged as a significant source of genetic material for clinical guidance. Recent technical advances have enabled the enrichment and genomic characterization of CTCs at the single cell level and have revealed significant and clinically relevant heterogeneity in these rare cells. However, data is lacking for existing methods to link the functional differences of single CTCs to their underlying genomic structure and activity. We have developed an approach which integrates key aspects of biologically based experimentation at the single cell level onto an integrated fluidic circuit. This device allows for the selection and isolation of single CTCs in a size-independent fashion from a pre-enriched population based on fluorescent markers where they can be individually cultured and exposed to a variety of drugs/reagents under environmentally controlled conditions or processed directly for mRNA-seq. If desired, an extracellular matrix can be deposited into each of the single cell culture chambers. Cell activity and phenotype can be monitored in-situ in response to treatment conditions, followed by preparation of the mRNA transcriptome for RNA-seq analysis. As the entire workflow following pre-enrichment is integrated onto a single microfluidic chip, very little manipulation of the single cell is required, serving to preserve the mRNA signature of each cell. To optimize and validate the workflow, PC3 cell lines were spiked-in to blood from healthy donors followed by pre-enrichment for metastatic cancer cells using commercially available CTC isolation platforms. Our microfluidic device further purified and isolated the desired single PC3 cells, and prepared the cells for expression analysis via RNA-seq. The sequencing results further validated the ability of our workflow to isolate rare metastatic cancer cells from human blood and moreover confirmed highly correlated gene expression profiles as compared to cultured PC3 population controls (r = 0.89 across 23,562 genes) and non spike-in single cell samples (r = 0.90 across 23,562 genes). We applied this workflow to the blood of metastatic castration resistant prostate cancer (mCRPC) patients and obtained single cell cDNA from the isolated CTCs from which we were able to generate sequencing libraries. Future work using this system will help establish a robust methodology to enable more complex perturbation and functional studies of single cells from clinical samples. Citation Format: Lukasz Szpankowski, Gayatri Premasekharan, Naveen Ramalingam, Chad Sanada, Anne Leyrat, Brian Fowler, Matthew Edwards, Cassandra Greene, Ilona Holcomb, Charles J. Ryan, Pamela L. Paris, Jay A.A. West. Isolation and mRNA-seq analysis of single CTCs from blood samples using an integrated fluidic circuit for functional single cell studies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-327.

Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer

To understand the potential and limitations of circulating tumor cell (CTC) sequencing for molecular diagnostics, we investigated the feasibility of identifying the ubiquitous KRAS mutation in single CTCs from pancreatic cancer (PC) patients. We used the NanoVelcro/laser capture microdissection CTC platform, combined with whole genome amplification and KRAS Sanger sequencing. We assessed both KRAS codon-12 coverage and the degree that allele dropout during whole genome amplification affected the detection of KRAS mutations from single CTCs. We isolated 385 single cells, 163 from PC cell lines and 222 from the blood of 12 PC patients, and obtained KRAS sequence coverage in 218 of 385 single cells (56.6%). For PC cell lines with known KRAS mutations, single mutations were detected in 67% of homozygous cells but only 37.4% of heterozygous single cells, demonstrating that both coverage and allele dropout are important causes of mutation detection failure from single cells. We could detect KRAS mutations in CTCs from 11 of 12 patients (92%) and 33 of 119 single CTCs sequenced, resulting in a KRAS mutation detection rate of 27.7%. Importantly, KRAS mutations were never found in the 103 white blood cells sequenced. Sequencing of groups of cells containing between 1 and 100 cells determined that at least 10 CTCs are likely required to reliably assess KRAS mutation status from CTCs.