Subset Of Circulating Tumor Cells Research Articles

Identification and characterization of transforming growth factor beta-induced in circulating tumor cell subline from pancreatic cancer cell line.

Distant metastasis to liver, lung, brain, or bone occurs by circulating tumor cells (CTC). We hypothesized that a subset of CTC had features that are more malignant than tumor cells at the primary site. We established a highly malignant cell line, Panc‐1‐CTC, derived from the human pancreatic cancer cell line Panc‐1 using an in vivo selection method. Panc‐1‐CTC cells showed greater migratory and invasive abilities than its parent cell line in vitro. In addition, Panc‐1‐CTC cells had a higher tumor‐forming ability than parent cells in vivo. To examine whether a difference in malignant phenotypes exists between Panc‐1‐CTC cells and parent cells, we carried out comprehensive gene expression array analysis. As a result, Panc‐1‐CTC significantly expressed transforming growth factor beta‐induced (TGFBI), an extracellular matrix protein, more abundantly than did parent cells. TGFBI is considered to regulate cell adhesion, but its functions remain unclear. In the present study, knockdown of TGFBI reduced cell migration and invasion abilities, whereas overexpression of TGFBI increased both abilities. Moreover, elevated expression of TGFBI was associated with poor prognosis in patients with pancreatic cancer.

Abstract 3604: Centrosome amplification in epcam-captured circulating tumor cells from metastatic cancer patients

Abstract Background: Genomic instability and evolution of cancers is linked to chemotherapy sensitivity and resistance in patients with metastatic cancer. Taxane-based chemotherapies operate by promoting chromosomal instability (CIN) by increasing multipolar mitoses. Breast tumors with CIN are more sensitive to paclitaxel compared to tumors without CIN. There is a critical need for new biomarkers based on biological evaluation of mechanisms of response and resistance in metastatic cancer. Although CIN is difficult to detect directly, centrosome amplification (CA) is a common mechanism of CIN. In this study, we develop an assay to detect CA in circulating tumor cells (CTCs) as a minimally invasive assay to evaluate sensitivity to taxane-based chemotherapies. Methods: Blood samples from patients with metastatic breast and prostate cancer were collected and processed for nucleated cells using a Ficoll gradient and magnetic depletion of CD45-positive cells. EpCAM-positive CTCs were captured using an exclusion-based sample preparation technology known as the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform. CTCs were defined as cells positive for Hoechst, cytokeratin and negative for multiple immune and endothelial cell markers (CD45/CD11b/CD14 and CD34). EpCAM-captured CTCs were also stained with pericentrin and centrin to identify CA and evaluated for pericentrin (whole centrosome marker) and centrin (centriole marker) protein-staining intensity, number and size. Results: CA was detected in a subset of CTCs from patients with metastatic breast and prostate cancer patients. As a control, centrosomes were also analyzed in matched peripheral blood mononuclear cells from the same patients. CA was heterogeneous with 100% of patients demonstrating some degree of CA (>4 centrioles), ranging from 15-48% of CTCs. Conclusion: In conclusion, we report the presence of CA in EpCAM-captured CTCs from metastatic cancer patients. Evaluation of CA in CTCs using pericentrin and centrin staining may serve as a predictive biomarker of taxane based chemotherapy response and resistance. We are recruiting taxane-naïve metastatic cancer patients to test the hypothesis that CA and/or CIN predict taxane sensitivity. Citation Format: Ashok Singh, Ryan A. Denu, Jamie M. Sperger, Beth A. Weaver, Mark E. Burkard, Joshua M. Lang. Centrosome amplification in epcam-captured circulating tumor cells from metastatic cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3604.

Abstract 4601: Dissecting the spatial heterogeneity of circulating tumor cells reveals CCL5-Treg-mediated immune evasion in hepatocellular carcinoma

Abstract The prognosis of hepatocellular carcinoma (HCC) is closely linked to the occurrence of recurrent, metastatic disease. Metastasis has been proposed to be initiated by circulating tumor cells (CTCs); however, the transcriptomic plasticity and adaptive mechanisms of CTCs in systemic circulation are not well defined. Here, we established single-cell RNA-sequencing profiles of 113 CTCs isolated from four key vascular sites along the dissemination route in ten HCC patients. In our study, we found that single CTCs displayed profound spatial transcriptional heterogeneity within the circulatory system, which is associated with increased transcriptional activity, cell cycle progression, and chemokine-induced immune escape programs. In particular, CTCs dynamically drove chemokine (C-C motif) ligand 5 (CCL5) expression over the course of circulation. We demonstrated that increased CCL5 expression in turn recruited regulatory T cells to facilitate immune evasion and metastatic progression, as rigorously validated by our in vitro and in vivo models. Finally, we identified an inherently dormant CTC subset with an epithelial-mesenchymal transition and immune-evasive phenotype, and demonstrated its potential prognostic implications. Collectively, our results reveal the previously unappreciated spatial heterogeneity of CTCs, and define its role in diverse regulatory programs and cellular states. Our study offers new insight into metastatic mechanisms, highlighting the potential utility of anti-CTC therapeutic strategies. Citation Format: Liang Wu, Miaomiao Jiang, Yunfan Sun, Xinrong Yang, Yong Hou, Jia Fan, Shiping Liu. Dissecting the spatial heterogeneity of circulating tumor cells reveals CCL5-Treg-mediated immune evasion in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4601.

Circulating Tumor Cells with Stem-Like Phenotypes for Diagnosis, Prognosis, and Therapeutic Response Evaluation in Hepatocellular Carcinoma.

Background: In the present study, we assessed the clinical value of circulating tumor cells (CTC) with stem-like phenotypes for diagnosis, prognosis, and surveillance in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) by an optimized qPCR-based detection platform.Methods: Differing subsets of CTCs were investigated, and a multimarker diagnostic CTC panel was constructed in a multicenter patient study with independent validation (total n = 1,006), including healthy individuals and patients with chronic hepatitis B infection (CHB), liver cirrhosis (LC), benign hepatic lesion (BHL), and HBV-related HCC, with area under the receiver operating characteristic curve (AUC-ROC) reflecting diagnostic accuracy. The role of the CTC panel in treatment response surveillance and its prognostic significance were further investigated.Results: The AUC of the CTC panel was 0.88 in the training set [sensitivity = 72.5%, specificity = 95.0%, positive predictive value (PPV) = 92.4, negative predictive value (NPV) = 77.8] and 0.93 in the validation set (sensitivity = 82.1%, specificity = 94.2%, PPV = 89.9, NPV = 89.3). This panel performed equally well in detecting early-stage and α-fetoprotein-negative HCC, as well as differentiating HCC from CHB, LC, and BHL. The CTC load was decreased significantly after tumor resection, and patients with persistently high CTC load showed a propensity of tumor recurrence after surgery. The prognostic significance of the CTC panel in predicting tumor recurrence was further confirmed [training: HR = 2.692; 95% confidence interval (CI), 1.617-4.483; P < 0.001; and validation: HR = 3.127; 95% CI, 1.360-7.190; P = 0.007].Conclusions: Our CTC panel showed high sensitivity and specificity in HCC diagnosis and could be a real-time parameter for risk prediction and treatment monitoring, enabling early decision-making to tailor effective antitumor strategies. Clin Cancer Res; 24(9); 2203-13. ©2018 AACR.

Heterogeneity of Circulating Tumor Cells in Neoadjuvant Chemotherapy of Breast Cancer.

The biological properties of circulating tumor cells (CTCs), and their dynamics during neoadjuvant chemotherapy are important, both for disease progression prediction and therapeutic target determination, with the aim of preventing disease progression. The aim of our study was to estimate of different CTC subsets in breast cancer during the NACT (neoadjuvant chemotherapy). The prospective study includes 27 patients with invasive breast cancer, T2-4N0-3M0, aged 32 to 60 years. Venous heparinized blood samples, taken before and after biopsy, after each courses of chemotherapy (on days 3–7), and before surgical intervention, served as the material for this study. Different subsets of circulating tumor cells were determined on the basis of the expression of EpCAM, CD45, CD44, CD24, and N-Cadherin using flow cytometry. As the result of this study, it has been observed that significant changes in the quantity of the different subsets of circulating tumor cells in patients’ blood were observed after carrying out the 3rd course of NACT. NACT causes significant changes in the quantity of six CTC subsets, with various combinations of stemness and epithelial-mesenchymal transition (EMT) properties.

Circulating Tumor Cells Detected by the Expression of Cancer Stem Cell Markers CD90 and CD44 in Patients With Esophageal Cancer

Background Epithelial cell adhesion molecule (EpCAM) is a marker for circulating tumor cells (CTCs) in various types of cancer. Cell surface antigens, such as CD90 and CD44, have been reported to be cancer stem cell (CSC) markers in esophageal squamous cell carcinoma (ESCC). The aim of this study was to assess the use of CD90 and CD44 as markers to identify clinically significant CTC subpopulations in ESCC. Methods We collected 3 mL of peripheral blood from 10 ESCC patients and 10 healthy volunteers to detect combined expression of EpCAM, CD90, and CD40 using flow cytometry. Results The number of EpCAM-positive cell counts (average ± SD) in the patients was significantly higher than healthy volunteers (29.1 ± 35.9 and 2.3 ± 2.5, P = 0.001). The proportions (average ±SD) of CD90- and CD44-positive cells in EpCAM-positive cells were 45.7% ± 42.4% and 98.7% ± 2.7%, respectively. EpCAM-positive/CD44-positive CTC counts, which was equivalent to EpCAM-positive CTC counts, correlated with pathologic V factors in the resected primary tumors (P &amp;gt; 0.01). EpCAM-positive/CD90-positive CTC counts, but not EpCAM-positive/CD90-negative CTC counts, correlated with pathologic V factors in the resected primary tumors (P = 0.01). Our results suggested that combined expression of EpCAM and CD90 may useful to detect CTC subsets, which have highly metastatic features in ESCC. CD44, on the other hand, is equivalent to EpCAM as a marker to detect CTCs in ESCC.

Mesenchymal circulating tumor cells (CTCs) and OCT4 mRNA expression in CTCs for prognosis prediction in patients with non-small-cell lung cancer.

Circulating tumor cells (CTCs) with epithelial-to-mesenchymal transition (EMT) phenotypes might be related to tumor progression while OCT4 expression is involved in tumor metastasis and poor prognosis. But the possible clinical significance of EMT phenotypes of CTCs from non-small-cell lung cancer (NSCLC) patients has still to be demonstrated. Furthermore, none has been investigated the expression of OCT4 in CTCs. We therefore identified the EMT phenotype-based subsets of CTCs and determined the OCT4 expression status of CTCs in NSCLC patients, to explore their possible clinical relevance. 37 NSCLC patients and ten healthy volunteers were enrolled, respectively. The Canpatrol™ CTC enrichment technique was used to isolate and identify the EMT phenotype-based subsets of CTCs. OCT4 expression in each CTC was also determined. Results were correlated with patients' clinico-pathological features. CTCs were detected in 33 of 37 (89.2%) NSCLC patients, and no CTCs were identified in ten healthy volunteers. Three CTCs phenotypes, including epithelial, biophenotypic, and mesenchymal CTCs were identified based on the expression of EMT markers. Mesenchymal CTCs were more commonly found in patients with distant metastasis. Patients with distant metastasis tended to have a higher median CTCs number. OCT4-positive was observed in 21 of 28 (75.0%) patients. High expression of OCT4 tended to occur in advanced patients as well as in distant metastatic patients. The findings suggest that identification of CTCs by EMT markers as well as evaluation of OCT4 expression status by assessment of OCT4 expression in CTCs could serve as potential adjuncts for evaluating metastasis and prognosis in NSCLC patients.

Abstract P1-01-18: A multiplexed immunofluorescence identifies phenotypic heterogeneity of circulating tumor cells in breast cancer

Abstract Introduction: Circulating Tumor Cells (CTCs) have attracted significant attention as a new class of "liquid biopsy", enabling longitudinal and non-invasive disease monitoring to capture an overall snapshot of individual disease. Primary breast tumors are highly heterogeneous due to their genetic instability. Thus, the presence of heterogeneous populations of CTCs is expected. A number of attempts have been made to address CTC's phenotypic heterogeneity using different methods and approaches. For comprehensive understanding of distinct CTC phenotypes, transcriptomic and cell surface marker studies using single cell analysis separated CTCs into different subgroups with epithelial-, mesenchymal-, and stem-like signatures. These studies concluded that the prevalence of stem-like CTCs is associated with poor prognosis. Immunofluorescence is an ideal approach to understand the heterogeneity of 3 - 4 markers in a large number of distinct CTCs; however, such approach does not address the presence of overlapping phenotypic signatures. The collection method of CTCs adds another layer of complexity since the most commonly adopted CTC enumeration strategy relies on affinity-based selection using antibody against EpCAM, which, although abundantly expressed on epithelial cancer cells, automatically eliminates a large fraction of non-epithelial CTCs. To better understand the whole landscape of this heterogeneous disease, the use of marker-independent unbiased methods to collect CTCs is critical to obtain the overall landscape of phenotypic heterogeneity. Aim: We aimed to investigate the heterogeneity of breast CTCs on a single cell level using size exclusion- based enrichment principal. Methods: Whole blood was collected from metastatic breast cancer patients under IRB approved protocol. CTCs were isolated on porous membrane (8 µm) under negative pressure (ISET®) from 10 mL of blood. CTCs were fixed on the porous membrane and used for multiplexed immunofluorescence to investigate the expression of epithelial (CK and EPCAM), mesenchymal (Vimentin, Snail and TWIST), and stem-like markers (CD44, ALDH1, and CD133) on CTCs. Results: CTCs were isolated on ISET filter from all subtypes, Stage IV patients. CTCs were identified based on their morphological and phenotypical characteristics with our CTC criteria of size of nucleus ≥16 µm, triple negative for α-smooth muscle actin, fibroblast associated protein, and CD45. The CTCs were further characterized based on their mesenchymal and stem-like signatures to understand their heterogeneity. Conclusions: Multiplex immunofluorescence staining of CTCs collected using the size-based enrichment approach enabled us to identify the heterogeneity of CTCs. The phenotypic heterogeneity represents a corner stone for future studies to identify a subset of CTCs that may be associated with breast cancer patient prognosis. Citation Format: Kamal M, Zhang R, Walker M, Squires R, Talbert B, Dooley W, Razaq W, Tanaka T. A multiplexed immunofluorescence identifies phenotypic heterogeneity of circulating tumor cells in 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 P1-01-18.

Abstract P1-01-07: Maintenance of genomic integrity in dormant circulating tumor cells

Abstract More than 67% of deaths in breast cancer patients occur after the initial 5-year survival period while residual disease can be dormant for periods longer than 20 years. Patients are asymptomatic because circulating tumor cells (CTCs) remain dormant and are undetectable by current clinical tools. Dormant CTCs may retain their long-term tumor-initiating (LTI) potential by adhering to their original genome, unlike rapidly cycling cancer cells that are known to have increased genomic instability. We hypothesized that hyperactive mechanisms of DNA repair preserve the genomic make-up of dormant CTCs allowing them to retain their LTI potential, ultimately causing disease relapse. We isolated and characterized breast cancer CTCs by mutiparametric flow cytometry and DEPArrayTM. Individually isolated breast cancer CTCs had a large proportion (&amp;gt;40%) of dormant (Ki67-/PCNA-) cells. Dormant CTCs had a lower incidence of double-strand DNA breaks (DSB) than proliferating cells as assessed by the phosphorylation status of Serine139 on gamma H2AX. This observation was further validated in a panel of eight genetically distinct breast cancer cell lines. Second, to understand whether dormant cells are inherently more resistant to DSB, we induced DSB in breast cancer cells by UV radiation and bleomycin treatment, and measured residual DSB at regular intervals. Results showed that besides being more resistant to DSB de novo, dormant breast cancer cells were also more efficient repairing their DNA. There are two distinct phases of DSB repair - early [within 2 hours of DSB using Non-Homologous End Joining (NHEJ) methods] and late [evident after 24 hours using Homologous Recombination (HR)]. Unlike proliferating (S-G2M) cells, dormant (G0) cells lack the sister chromatid and repair their DNA exclusively by NHEJ methods. Therefore, and third, we investigated key players of the NHEJ pathway and examined their roles in maintaining genomic integrity. We found that the human telomere-associated protein RIF1, a mediator of alternative NHEJ, was significantly up-regulated in a dormant CTC subset. Dormant sub-populations of breast cancer cells confirmed RIF1 foci formation in areas of DNA damage. Fourth, mis-sense mutation of RIF1 in CAMA-1 cells (ΔRIF1 E1598K) as well as shRNA mediated RIF1 knockdown in HCC1954 and ZR-75-1 cell lines attenuated resistance of the dormant subset to UV and bleomycin treatment. Finally, RIF1 knockdown activated both p38 and pERK pathways albeit to varying degrees in multiple cell lines resulting in metastatic inefficiency in xenograft and syngeneic mouse models. Collectively, these findings suggest that RIF1 may play functional roles in maintaining the genomic integrity of dormant CTCs and be a potential biomarker of breast cancer CTC survival while in circulation. Citation Format: Boral D, Vishnoi M, Liu HN, Yin W, Marchetti D, Hong DS, Scamardo A. Maintenance of genomic integrity in dormant circulating tumor cells [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 P1-01-07.

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.

Stem-like plasticity and heterogeneity of circulating tumor cells: current status and prospect challenges in liver cancer

Poor prognosis and high recurrence remain leading causes of primary liver cancerassociated mortality. The spread of circulating tumor cells (CTCs) in the blood plays a major role in the initiation of metastasis and tumor recurrence after surgery. Nevertheless, only a subset of CTCs can survive, migrate to distant sites and establish secondary tumors. Consistent with cancer stem cell (CSC) hypothesis, stem-like CTCs might represent a potential source for cancer relapse and distant metastasis. Thus, identification of stem-like metastasis-initiating CTC-subset may provide useful clinically prognostic information. This review will emphasize the most relevant findings of CTCs in the context of stem-like biology associated to liver carcinogenesis. In this view, the emerging field of stem-like CTCs may deliver substantial contribution in liver cancer field in order to move to personalized approaches for diagnosis, prognosis and therapy.

Programmed death-ligand 1 (PD-L1) characterization of circulating tumor cells (CTCs) in muscle invasive and metastatic bladder cancer patients.

BackgroundWhile programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) checkpoint inhibitors have activity in a proportion of patients with advanced bladder cancer, strongly predictive and prognostic biomarkers are still lacking. In this study, we evaluated PD-L1 protein expression on circulating tumor cells (CTCs) isolated from patients with muscle invasive (MIBC) and metastatic (mBCa) bladder cancer and explore the prognostic value of CTC PD-L1 expression on clinical outcomes.MethodsBlood samples from 25 patients with MIBC or mBCa were collected at UCSF and shipped to Epic Sciences. All nucleated cells were subjected to immunofluorescent (IF) staining and CTC identification by fluorescent scanners using algorithmic analysis. Cytokeratin expressing (CK)+ and (CK)−CTCs (CD45−, intact nuclei, morphologically distinct from WBCs) were enumerated. A subset of patient samples underwent genetic characterization by fluorescence in situ hybridization (FISH) and copy number variation (CNV) analysis.ResultsCTCs were detected in 20/25 (80 %) patients, inclusive of CK+ CTCs (13/25, 52 %), CK−CTCs (14/25, 56 %), CK+ CTC Clusters (6/25, 24 %), and apoptotic CTCs (13/25, 52 %). Seven of 25 (28 %) patients had PD-L1+ CTCs; 4 of these patients had exclusively CK−/CD45−/PD-L1+ CTCs. A subset of CTCs were secondarily confirmed as bladder cancer via FISH and CNV analysis, which revealed marked genomic instability. Although this study was not powered to evaluate survival, exploratory analyses demonstrated that patients with high PD-L1+/CD45−CTC burden and low burden of apoptotic CTCs had worse overall survival.ConclusionsCTCs are detectable in both MIBC and mBCa patients. PD-L1 expression is demonstrated in both CK+ and CK−CTCs in patients with mBCa, and genomic analysis of these cells supports their tumor origin. Here we demonstrate the ability to identify CTCs in patients with advanced bladder cancer through a minimally invasive process. This may have the potential to guide checkpoint inhibitor immune therapies that have been established to have activity, often with durable responses, in a proportion of these patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2758-3) contains supplementary material, which is available to authorized users.

Abstract 1530: Dissecting breast cancer dormant CTC phenotypes

Abstract Tumor relapse is a clinically relevant problem in breast cancer where patients are asymptomatic because disseminated cells appear to become dormant for periods longer than 20 years and are undetectable by current clinical tools. Uncovering phenotypes of circulating tumor cells (CTCs) - the “seeds” of intractable metastasis-offers the promise to dissect CTC heterogeneity in relation to metastatic competence, to predict biomarker assessment, and to significantly improve monitoring and treatment of cancer. However, little is known about CTC biology and how CTCs differ in their capacity to circulate while maintaining a metastatic potential. We hypothesized that EpCAM-negative breast cancer CTC subsets exist, and avoid organ arrest with extreme efficiency by the concomitant presence of quiescence and stem cell properties. We collected peripheral blood of clinically diagnosed breast cancer patients with or without brain metastasis, and performed multiparametric flow cytometry to isolate EpCAM-negative CTC subsets with stem-cell properties (CD44+/CD24-), along with combinatorial expression of two neoplastic markers: urokinase plasminogen activator receptor (uPAR) and integrin beta1 (int β1). EpCAM-negative CTCs were further interrogated at a single-cell level employing DEPArray platform. Second, we were able to culture FACS-sorted CTC subsets, selected for six cell-surface expression markers (CD45-/EpCAM-negative/CD44+/CD24-/uPAR+/-/int β1+/-), as long-term in-vitro 3D CTC tumorspheres. Third, CTC subsets were interrogated for biomarker profiling and biological characteristics. We identified adhesive, proliferative and invasive properties of 3D CTC tumorspheres which were distinct per uPAR/int β1 combinatorial expression. Lastly, we performed next-generation whole-genome sequencing and mutation analyses to discover unique genomic signatures of uPAR/int β1 CTC subsets and verified as putative CTCs originally disseminated from primary breast tumor. Additional investigations are being pursued assessing the molecular and genomic characterization of uPAR/int β1 CTC subsets comprehensively. Clinical relevance of this research includes that this may enhance abilities to prospectively identify patients who may be at high-risk of developing breast cancer brain metastasis. Citation Format: Monika Vishnoi, Sirisha Peddibhotla, Wei Yin, Zhong Xue, Antonio T. Scamardo, Goldy C. George, David S. Hong, Dario Marchetti. Dissecting breast cancer dormant CTC phenotypes. [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 1530.

Abstract 856: Number of ALK-amplified circulating tumor cells predicts progression-free survival in ALK-rearranged non-small cell lung cancer patients treated by crizotinib

Abstract The duration of clinical response is unpredictable in ALK-rearranged NSCLC patients treated by crizotinib but all patients invariably develop resistance. Using filter-adapted fluorescence in situ hybridization (FA-FISH) we previously reported ALK-rearrangement detection in circulating tumor cells (CTCs) from ALK-rearranged patients. Here we report the monitoring of ALK-rearranged (≥1×3’/5’, ≥1x(3’ and 5’)) and ALK-amplified (&amp;gt;2×3’/5’) CTCs at baseline and at an early time point under crizotinib therapy in an extended cohort of ALK-rearranged patients. The correlation between CTC subsets harboring distinct FISH patterns and clinical parameters including progression-free survival (PFS) and overall survival (OS) is presented. Forty ALK-rearranged patients were recruited. Blood samples were collected at baseline to crizotinib and at 1-3 months. Abnormal ALK-FISH patterns were examined in CTCs using immunofluorescence staining (DAPI/CD45) combined with FA-FISH after isolation by size of epithelial tumor cells (ISET) filtration. CTCs were defined into five distinct subsets according to the number of FISH ‘break-apart’ signals (3’ and 5’) or isolated red signals (3’) and/or native copies (3’/5’). All ALK-abnormal cells were validated by a cytogenetician. Clinical data was collected retrospectively. Confirming our previous data, ALK rearrangement was detected in CTCs: 32/39 patients (82%) had ≥4 ALK-rearranged CTCs per 1 ml of blood. Age &amp;gt;60 years and smoking status &amp;gt;15 pack years were associated with a higher mean number of ALK-amplified CTCs (p = 0.0423 and p = 0.0407 respectively). No statistically significant correlation was observed between the different CTC subsets with abnormal FISH patterns at baseline and PFS or OS. However we observed a statistically significant correlation (p = 0.0196) between the evolution under crizotinib of the numbers of ALK-amplified CTCs and PFS. An increase in the numbers of ALK-amplified CTCs during treatment is associated with a PFS of 6.5 months, while a decrease in this subset is associated with a PFS of 25 months. There was no corresponding correlation with OS. The evolution under crizotinib therapy of numbers of ALK-amplified CTCs is significantly correlated with PFS. Although not dominant, amplification of the ALK gene has been reported to be one mechanism of acquired resistance to crizotinib therapy in tumor biopsies. CTCs with an amplification of ALK do not express the oncogenic ALK fusion protein and are therefore not targeted by crizotinib. FISH patterns suggested these CTCs may have a high degree of ploidy and chromosomal instability which may contribute to promote the emergence of CTC subclones with a high metastatic potential. Our data suggest that number of ALK-amplified CTCs may be a predictive biomarker allowing prediction of ALK-rearranged NSCLC patients who are at risk of early resistance to crizotinib. Citation Format: Emma Pailler, Marianne Oulhen, Kirsty Ross, Fanny Billiot, Nathalie Auger, Isabelle Borger, Maud NgoCamus, Jordi Remon-Masip, David Planchard, Jean-Charles Soria, Benjamin Besse, Françoise Farace. Number of ALK-amplified circulating tumor cells predicts progression-free survival in ALK-rearranged non-small cell lung cancer patients treated by crizotinib. [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 856.

Abstract 1388: Multi-template analysis in metastatic breast cancer blood samples

Abstract Introduction The presence of Circulating Tumor Cells (CTC) has been observed in advanced cancer patients and studies have indicated that these cells contribute to the process of metastasis. Historically CTCs with metastatic potential have been characterized as cells that are EpCAM positive, Cytokeratin positive (CK+), and CD45 negative. We report the results of a prospective study challenging these historical definitions of circulating tumor cells. Description In a prospective study, patients with metastatic breast cancer there were about to start a new systemic therapy were enrolled The samples and representative tissue were collected at baseline. The patients were predominantly female (97%) and all had stage IV disease. From these whole blood samples a LiquidBiopsy® was performed using the “MultiTemplate” format which directly compares CTC, circulating cell free (CCF), and WBC patient-matched DNA in a NGS based resequencing test. Of 22 patients, 18 biopsy samples were successfully evaluated; the balance had insufficient tissue or DNA for analysis. Summary The LiquidBiopsy compared two phenotypic definitions for CTC capture. Tumor cell populations were enriched using epithelial targeted capture and compared to a novel cocktail of antibodies. The cocktail of reagents gave 77% average recovery of engineered samples when a target receptor was present on cell lines representing the spectrum of breast cancer subtypes. This enrichment protocol was applied to serial patient samples. Epithelial based enrichment served as a control and showed an average recovered purity of 10.7% CK+ cells. By comparison, cocktail selection enriched populations with on average 8.8% CK+ cell populations but a larger range of cells than epithelial selection. Importantly, the median number of CK+ cells recovered from cocktail capture was almost twice that of epithelial capture alone. (median of 35.8 vs 22.1 for cocktail and epithelial respectively). The median CD45+ non-target cells background was 80 and 155 cells respectively. This background supports sequencing detection of mutations present at &amp;gt;1%. ccfDNA sample was purified from the same tube of blood. The average concentration of patient matched ccfDNA recovered was 7.3 ng/mL. The combined results of ctcDNA and ccfDNA template sequencing gave productive samples showed similar detection frequency (55% vs 58%), were temporally flexible, and were complementary both to each other and the “gold standard” (FFPE). Conclusions The data from this prospective clinical study reveals subsets of CTC, including an important cohort that is not detected using the standard definition for epithelial CTCs. Furthermore, we present evidence for the successful use of a “Multi-Template” approach to the liquid biopsy, where germline, ctcDNA, and ccfDNA templates are employed for clinical diagnostic purposes and justifies the redefinition of CTC phenotype based upon cell surface biomarkers and mutation content. Citation Format: William M. Strauss, Chris Carter, Erich Klem, Jill Simmons, Keerthi Gogineni, Ruth O’Regan, Laura Austin, Paul W. Dempsey, Massimo Cristofanilli. Multi-template analysis in metastatic breast cancer blood samples. [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 1388.

Abstract 3143: Deciphering mechanisms of circulating tumor cells in breast cancer dormancy

Abstract More than 67% of deaths in breast cancer patients occur after the initial 5-year survival period while residual disease can be dormant for periods longer than 20 years. Patients are asymptomatic because circulating tumor cells (CTCs) remain dormant and are undetectable by current clinical tools. Dormant CTCs may retain their long-term tumor-initiating (LTI) potential by adhering to their original genome, unlike rapidly cycling cancer cells that are known to have increased genomic instability. We hypothesized that hyperactive mechanisms of DNA repair preserve the genomic make-up of dormant CTCs allowing them to retain their LTI potential, ultimately causing disease relapse. We isolated and characterized EpCAM-negative breast cancer CTCs by mutiparametric flow cytometry and DEPArrayTM. Individually isolated breast cancer CTCs had a large proportion (&amp;gt;40%) of dormant (Ki67-/PCNA-) cells. Dormant CTCs had a lower incidence of double-strand DNA breaks (DSB) than proliferating cells as assessed by the phosphorylation status of Serine139 on gamma H2AX. This observation was further validated in a panel of eight genetically distinct breast cancer cell lines. Second, to understand whether dormant cells are inherently more resistant to DSB, we induced DSB in breast cancer cells by UV radiation and bleomycin treatment, and measured residual DSB at regular intervals. Results showed that besides being more resistant to DSB de novo, dormant breast cancer cells were also more efficient in repairing their DNA. There are two distinct phases of DSB repair - early [within 2 hours of DSB using Non-Homologous End Joining (NHEJ) methods] and late [evident after 24 hours using Homologous Recombination (HR)]. Unlike proliferating (S-G2M) cells, dormant (G0) cells lack the sister chromatid and repair their DNA exclusively by NHEJ methods. Therefore, and third, we investigated key players of the NHEJ pathway and examined their roles in maintaining genomic integrity. We found that the human telomere-associated protein RIF1, a mediator of alternative NHEJ, was significantly up-regulated in a dormant CTC subset. Dormant sub-populations of breast cancer cells confirmed RIF1 foci formation in areas of DNA damage. Fourth, mis-sense mutation of RIF1 in CAMA-1 cells (ΔRIF1 E1598K) attenuated resistance of the dormant subset to UV and bleomycin treatment. Collectively, these findings suggest that RIF1 may play functional roles in maintaining the genomic integrity of dormant CTCs. Further investigations are being pursued to assess RIF1 contributions to retain CTC LTI potential leading to CTC-driven metastasis. Citation Format: Debasish Boral, Haowen N. Liu, Wei Yin, Monika Vishnoi, Antonio Scamardo, Goldy C. George, David S. Hong, Dario Marchetti. Deciphering mechanisms of circulating tumor cells in breast cancer dormancy. [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 3143.

Abstract OT1-02-01: Pilot study of prognostic utility of circulating tumor cells (CTCs) assessed by AdnaGen technology and clinical outcome of patients with stage III breast cancer who completed locoregional and systemic treatment

Abstract Background: Detection of high number of CTCs (&amp;gt;5) before initiation of first-line therapy in patients with metastatic breast cancer is associated with shorter progression free survival and overall survival. The most widely used method is CellSearch (Veridex, Raritan, NJ). It relies on immunomagnetic capture of CTCs, using antibodies against the epithelial cell adhesion molecule (EpCAM). Although the US Food and Drug Administration approved CellSearch assay for clinical use. In addition to isolation and enumeration, a promising area of research is genomic CTCs characterization which entails phenotyping and molecular expression profiling of CTC subsets consisting of those of epithelial origin (CTC-Epi), others undergoing epithelial to mesenchymal transition (CTC-EMT), or expressing cancer stem cell-like phenotype (CTC-CSC; CD44+ CD24low, ALDH+), respectively. EMT is a molecular process to acquire the traits needed to execute the multiple steps of metastasis. Through the EMT process, epithelial cells lose cell-cell contacts and cell polarity, downregulate epithelial-associated genes, acquire mesenchymal gene expression and undergo major changes in their cytoskeleton. Currently, a CTC detection kit is available to detect CTCs expressing EMT-associated genes by semiquantitative RT-PCR (Adna EMT2/Stem Cell test). EMT will be detected by measuring EMT-inducing transcription factors such as TWIST1, SNAIL1, SLUG, ZEB1 and FOXC2) by RT-PCR Objectives. Primary objective: To investigate if activated pathways in CTCs are correlated with clinical outcome of patient with stage III breast cancer. Secondary objective: To prospectively determine if assessment of the pathways profiling in CTCs can be used to stratify NED breast cancer patients Patients Eligibility: Inclusion: histologically confirmed invasive breast cancer (any subtype), clinical stage III, no evidence of distant metastasis by PET-CT or CT scan of chest and abdomen, and body scan, age 18 years or older, pts must be scheduled to start neoadjuvant/adjuvant therapy, ECOG PS 0-2. Pts must sign a written informed consent. Exclusion: distant metastasis, investigational therapy, prior history of other malignancies within the last 2 years, except non-melanoma skin cancer. This study (PA12-0097) was approved by IRB of UT MD Anderson Cancer Center. Trial Design. This is a pilot, international, multicenter, prospective, blood sample collection from 200 patients with clinical or pathologic stage III breast cancer. Statistical Analysis: This study is a 7-year study (84 months). Pts will be classified as to the presence [negative (neg) vs. positive (pos)] of CTC and as to the expression of a biomarker (neg vs. pos). The primary endpoint of the study is breast cancer recurrence. Time to recurrence curves for the four breast cancer patient groups (neg/neg, neg/pos, pos/neg, or pos/pos) will be estimated using the Kaplan-Meier method and differences in the recurrence rates will be evaluated by the log-rank test at the end of the study (84 months). The confidence intervals for the quantiles of the recurrence distribution will be based on the sign test as described by Brookmeyer and Crowley. Citation Format: Alvarez RH, Gao H, Ensor JE, Gomez HL, Ruiz-Garcia EB, Arce C, Sun H, Willey JS, Ueno NT, Valero V, Reuben JM. Pilot study of prognostic utility of circulating tumor cells (CTCs) assessed by AdnaGen technology and clinical outcome of patients with stage III breast cancer who completed locoregional and systemic treatment. [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 OT1-02-01.

Cell lines from circulating tumor cells.

Circulating tumor cells (CTCs) in the blood of cancer patients have received increasing attention as new diagnostic tool enabling “liquid biopsies” [1]. The perspective to avoid invasive tissue biopsies in the future and obtain similar or even more information by a “simple” blood test has enormous implications in cancer diagnostics [2]. In contrast to the wealth of descriptive studies demonstrating the clinical relevance of CTCs as biomarker, the extremely low concentration of CTCs in the peripheral blood of most cancer patients challenges further functional studies. A prerequisite for functional analyses was therefore the recent advances in our ability to culture CTCs in vitro or expand the CTC pool in vivo using xenografts. The recent manuscript by Cayrefourcq et al. focused on how to establish a colon CTC line and its key characteristics have been reported [3]. In this study, they applied a negative selection for CTC enrichment on blood samples from 71 patients with metastatic colon cancer and cultured all of the CD45(−) remaining cells in non-adherent culture conditions. The authors have provided the experimental proof that CTCs isolated from the blood of a colon cancer patient are able to give rise to a permanent cell line [3]. To our best knowledge, no other group has published the establishment of a permanent cell CTC line or even transient CTC cultures from patients with colon cancer. One reason is that the frequency of CTCs is lower in peripheral blood of colon cancer patients as compared to breast or prostate cancer patients, making it even more difficult to find and grow CTCs in colon cancer. Besides its capacity to expand ex vivo for more than 2 years, the CTC-MCC-41 line showed (i) epithelial properties with stem-cell like characteristics, (ii) an intermediate epithelial/mesenchymal phenotype, (iii) a potential to induce quickly in vitro angiogenesis, (iv) an osteomimetic signature and (v) tumorigenic properties in SCID mice. Importantly, this CTC line shares the main features of the original primary tumor and lymph nodes metastasis of the colon cancer patient. Recent advances in cell culture technologies have opened a new avenue to develop primary cell cultures or permanent cell lines from CTCs. The first publications on CTC cultures dealt with blood from metastatic breast cancer patients. First, Dario Marchetti's group has established primary cultures from CTCs of breast cancer patients with brain metastases [4]. In EpCAM(−) CTCs, a potential signature of brain metastasis comprising “brain metastasis selected markers (BMSMs)” HER2(+)/EGFR(+)/HPSE(+)/Notch1(+) was identified [4]. CTC lines expressing the BMSM signature were highly invasive and able to generate brain and lung metastases in nude mice. Second, Daniel Haber's group has reported on oligoclonal cultures (sustained in vitro for > 6 months) of CTCs isolated from six patients with metastatic breast cancer [5]. Third, in prostate cancer, Gao et al. used a 3D organoid system and succeeded to develop a long-term culture from the peripheral blood of one patient with castration-resistant metastatic disease and high CTC counts and PSA levels [6]. Fourth, Sunitha Nagrath's group focused on lung cancer and developed a novel in situ capture and culture methodology for ex vivo expansion of CTCs using a 3D co-culture model [7]. Figure 1 Establishment of a colon CTC line CTC lines might be used to test new therapeutic approaches. In this context, novel immunotherapeutic strategies have been developed and antibodies against proteins that block the immune response of T-cells such as PD-L1 or CTLA4 have been approved for treatment of cancer patients. Recently, we could show, for the first time, that PD-L1 is frequently expressed on a variable subset of CTCs obtained from metastatic breast cancer patients [8]. Further functional analysis of this interesting subset might reveal CTC-specific immunosuppressive properties. Taken together, the establishment of functional CTC cell line models is now feasible. These models might be useful to test new cancer drugs specifically targeting CTCs. However, to achieve this important goal it is crucial to increase the efficiency of establishing CTC cell lines and xenografts and extend the current studies to patients at earlier stages. The low concentration of CTCs in earlier stage patients might be a bottleneck to identify “metastasis-initiator cells” that may be resistant as well to the immune system by expressing PD-L1. Thus, increasing the yield of current CTC assays along with the development of more efficient culture strategies seems to be of utmost importance.

Abstract 363: Dissecting CTC phenotypes: insights into mechanisms of breast cancer dormancy

Abstract Uncovering phenotypes of patient-derived Circulating Tumor Cells (CTCs) offers the promise to dissect CTC heterogeneity in relation to metastatic competence, and to determine biomarkers of therapeutic utility for improved treatment. However, it is still unknown whether and how CTCs differ in their capacity to circulate while maintaining metastatic potential. Rates of CTC survival are highly variable, lasting less than few hours in some patients but in the order of decades in others. This can lead to many questions for yet unexplored mechanisms of CTCs responsible for dormancy, along with their properties and biomarker functionalities. We hypothesized that breast cancer CTC subsets possessing markers of pluripotency avoid organ arrest with extreme efficiency by the concomitant presence of quiescence and stem cell properties; and that expression of urokinase plasminogen activator receptor (uPAR) and beta-1 integrin (β1int), two biomarkers known to be directly implicated in tumor cell dormancy, are relevant in controlling the recurrence of breast cancer brain metastasis (BCBM). First, we isolated CTC subsets not expressing the epithelial cell adhesion molecule (EpCAM-negative CTCs), and characterized these subsets using DEPArrayTM, a new CTC platform able to dissect CTC heterogeneity at a single-cell level, thus interrogating the smallest functional unit of cancer. We captured EpCAM-negative/CD45-/CD44+/CD24- breast cancer CTC subsets that possessed combinatorial uPAR and β1int expression using multiparametric flow cytometry. Second, CTC subsets grew in vitro and were further characterized by DEPArrayTM. Markers expression was confirmed by confocal microscopy with subsets possessing a specific breast cancer gene profiling. Third, EpCAM-negative CTC subsets (uPAR+/β1int+ and uPAR-/β1int-) were interrogated for human embryonic stem cell markers by RT2 PCR arrays. Gene expression profiling was consistently distinct among uPAR+/β1int+ vs. uPAR-/β1int- CTC subsets and dependent upon patients’ BCBM status: expression of genes implicated in cell cycle progression (e.g., CDK42, CDK1), angiogenesis (e.g., FGF-2), and pluripotency (e.g., KLF4) was &amp;gt;30-fold higher than controls. Third, CTC subsets gene patterns isolated from patients with BCBM possessed RT2 profiles that were strikingly distinct from ones derived from patients with no BCBM. Of note, gene expression for RIF-1, a protein that counteracts actions of the breast cancer suppressor BRCA1, was highest (&amp;gt;50-fold) with distinct RIF-1 nuclear patterns in BCBM CTC subsets. In summary, we have linked EpCAM-negative uPAR/β1int CTC subsets and their properties to clinical BCBM; and will assess the therapeutic inhibition of uPAR/β1int CTC biomarkers on BCBM development and its timing. Deciphering the relevance of uPAR/β1int as key CTC biomarkers of dormancy vs. metastatic competence will elucidate CTC mechanisms responsible for BCBM onset. Citation Format: Sirisha Peddibhotla, Monika Vishnoi, Wei Yin, Yizhen Chen, Antonio Scamardo, David Hong, Dario Marchetti. Dissecting CTC phenotypes: insights into mechanisms of breast cancer dormancy. [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 363. doi:10.1158/1538-7445.AM2015-363

Abstract 378: Predictive and pharmacodynamic biomarkers of the androgen receptor in circulating tumor cells

Abstract Purpose: The androgen receptor (AR) signaling inhibitors (e.g. Enzalutamide) and lyase inhibitors (e.g Abiraterone) modulate the AR signaling pathway and improve outcomes for patients with castrate resistant prostate cancer (CRPC). Molecular analysis of the AR signaling pathway can serve as predictive and pharmacodynamic markers of therapeutic response and resistance. Proposed resistance mechanisms to these agents include AR overexpression, expression of AR splice variants and up-regulation of the Glucocorticoid Receptor (GR). Circulating tumor cells (CTCs) have been utilized as one potential source of tumor cells for these analyses. We employed an integrated CTC capture and analysis technology known as VERSA to perform CTC enumeration, subcellular localization of AR and GR, and gene expression analysis of AR splice variants and AR dependent genes in patients with CRPC. Methods: Using the VERSA platform, we captured and enumerated EpCAM+ CTCs from 25 patients with CRPC who have been treated with AR signaling inhibitors or lyase inhibitors. Protein expression and nuclear localization of the AR and GR were quantified. mRNA was extracted from EpCAM+ CTCs for analysis of AR splice variants and AR targeted genes. CTC results were correlated with clinical outcomes of radiographic progression, PSA progression and stable disease. Results: Analysis of AR/GR protein expression revealed significant intra-patient heterogeneity in patients with radiographic progression, compared to patients with stable disease, treated with AR therapies. Patients with radiographic progression exhibit a dynamic range of AR localized in the nucleus (17 to 100%) in EpCAM+/AR+ CTCs. We then identified expression of V1 and V7 variants of the AR in 5 of 7 men showing radiographic progressionon their current therapy, only 1 out of 11 patients with rising PSA but no radiographic progression, and none of the patients with stable disease. 4 of 6 patients showing expression of AR V7 still maintained significant activity in the AR signaling pathway while 2 patients had no detectable expression of PSA. Conclusions: Multiplexed assays for protein quantification and gene expression of CTCs are suitable for pharmacodynamic analysis of AR signaling inhibitors. Results from patients with CRPC identify heterogeneous activity in the AR signaling pathway and expression of multiple AR splice variants in men with radiographic progression. Protein analysis reveals a subset of CTCs that retain phenotypic alterations consistent with persistent inhibition of the AR pathway. These results suggest a subset of men may benefit from continued AR targeting therapies despite the presence of AR splice variants. Rational combinatorial strategies could be employed based on these pharmacodynamic biomarkers to inhibit the AR pathway in sensitive tumor clones while utilizing alternative treatments, such as chemotherapies, for resistant tumor populations. Citation Format: Jamie M. Sperger, Lindsay Strotman, Benjamin P. Casavant, Chorom Pak, Sacha Horn, Erika Heninger, Scott M. Berry, David J. Beebe, Joshua M. Lang. Predictive and pharmacodynamic biomarkers of the androgen receptor in circulating tumor cells. [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 378. doi:10.1158/1538-7445.AM2015-378