Higher Circulating Tumor Cells Counts Research Articles

Extracellular vesicles for personalized therapy decision support in advanced metastatic cancers and its potential impact for prostate cancer.

The use of circulating tumor cells (CTCs) and circulating extracellular vesicles (EVs), such as exosomes, as liquid biopsy-derived biomarkers for cancers have been investigated. CTC enumeration using the CellSearch based platform provides an accurate insight on overall survival where higher CTC counts indicate poor prognosis for patients with advanced metastatic cancer. EVs provide information based on their lipid, protein, and nucleic acid content and can be isolated from biofluids and analyzed from a relatively small volume, providing a routine and non-invasive modality to monitor disease progression. Our pilot experiment by assessing the level of two subpopulations of small EVs, the CD9 positive and CD63 positive EVs, showed that the CD9 positive EV level is higher in plasma from patients with advanced metastatic prostate cancer with detectable CTCs. These data show the potential utility of a particular EV subpopulation to serve as biomarkers for advanced metastatic prostate cancer. EVs can potentially be utilized as biomarkers to provide accurate genotypic and phenotypic information for advanced prostate cancer, where new strategies to design a more personalized therapy is currently the focus of considerable investigation.

Clinical utility of circulating tumor cells in patients with non-small-cell lung cancer.

Several different studies have addressed the role of the circulating tumor cells (CTC) in non-small-cell lung cancer (NSCLC). In particular, the potential of CTC analysis in the early diagnosis of NSCLC and in the prediction of the outcome of patients with early and advanced NSCLC have been explored. A major limit of these studies is that they used different techniques for CTC isolation and enumeration, they employed different thresholds to discriminate between high- and low-risk patients, and they enrolled heterogeneous and often small cohort of patients. Nevertheless, the results of many studies are concordant in indicating a correlation between high CTC count and poor prognosis in both early and advanced NSCLC. The reduction of CTC number following treatment might also represent an important indicator of sensitivity to therapy in patients with metastatic disease. Preliminary data also suggest the potential for CTC analysis in the early diagnosis of NSCLC in high-risk individuals. However, these findings need to be confirmed in large prospective trials in order to be transferred to the clinical practice. The molecular profiling of single CTC in NSCLC might provide important information on tumor biology and on the mechanisms involved in tumor dissemination and in acquired resistance to targeted therapies. In this respect, xenografts derived from CTC might represent a valuable tool to investigate these phenomena and to develop novel therapeutic strategies.

Abstract 4816: Circulating tumor cell monitoring, isolation, and culture from a patient with metastatic triple-negative breast cancer for drug screening and creation of a patient-derived xenograft model

Abstract Background. Enumeration, phenotyping and single cell genomics of circulating tumor cells (CTCs) provide three types of information to guide cancer therapy. In some instances, a fourth type is possible: functional analysis in vitro, or in in vivo patient-derived xenografts (PDXs). We used a density-based rare cell separation and analysis system to collect CTCs from the blood of a patient with metastatic triple-negative breast cancer (TNBC) for in vitro culture and high-throughput drug screening and to generate a PDX model. Methods: The patient was enrolled in the ITOMIC-001 study (University of Washington) and after informed consent, CTCs were evaluated prior to initial cisplatin treatment and tracked longitudinally using the AccuCyte – CyteFinder system (RareCyte). Samples containing high numbers of CTCs were placed into 3 different culture media. Cells grown in culture were tested against a panel of anti-cancer drugs and injected into mice to form a PDX model. Results: Nine CTC evaluations were performed over 9.5 months. CTCs were verified by expression of epithelial (cytokeratin and/or EpCAM) and nuclear stains without CD45 expression. After initial treatment with cisplatin, the CTC count per 7.5 mL rose from 4 to 19 cells at 3 months, consistent with the lack of a clinical response, and decreased after LE 011 (CDK4/6 inhibitor) and then glembatumumab vedotin (anti-gpNMB) to 8 and 4 cells at 5 and 7 months respectively. At 9 months the CTC count rose to > 13,000 and 5 days later to > 80,000 shortly before her death. At autopsy there was massive infiltration of the liver and pulmonary vasculature by tumor cells. Cultures in all media showed initial growth, but only one (RPMI + 10% serum) was sustained, forming semi-adherent 3D tumor clusters. 6 million cells were harvested and a drug screen using 160 anti-cancer agents was performed. The CTC line showed sensitivity to several agents. Cells were also injected into the mammary fat pad of immunodeficient mice. In at least one mouse, macroscopic tumors were observed. The CTC cell line has grown continually in culture for over a year. Aliquots of this cell line have been frozen and thawed with no noticeable effect on cell growth. Conclusions: Using a density-based rare cell collection system, we have established a CTC cell line from a TNBC patient with extremely high CTC counts. The line was used to perform a screen for agents active against the tumor cells and to create a PDX model. As in vitro techniques advance, smaller number of CTCs may be effectively cultured and thus allow this approach to be used in real time to find effective drug regiments for individualized cancer therapy. Citation Format: Arturo B. Ramirez, C Anthony Blau, Timothy J. Martins, Elisabeth Mahen, Lacey E. Dobrolecki, Michael T. Lewis, Jackie L. Stilwell, Eric P. Kaldjian. Circulating tumor cell monitoring, isolation, and culture from a patient with metastatic triple-negative breast cancer for drug screening and creation of a patient-derived xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4816. doi:10.1158/1538-7445.AM2017-4816

Midkine promotes hepatocellular carcinoma metastasis by elevating anoikis resistance of circulating tumor cells.

Midkine is overexpressed in hepatocellular carcinoma (HCC) and plays a role in tumor progression, but less is known about its role in resistance of circulating tumor cells (CTCs) to anoikis which leading to recurrence and metastasis. The aim of the present study was to analyze whether midkine was associated with HCC progression with anoikis resistance. We found that cultured HCC cells were more resistant to anoikis, which paralleled midkine expression, and midkine treatment significantly inhibited anoikis in a dose-dependent manner. Furthermore, in in vitro and in vivo assays, knockdown of midkine resulted in significant sensitivity to anoikis, decreased cell survival and significantly decreased tumor occurrence rate. Patients with midkine-elevated HCC had higher CTC counts and less apoptotic CTCs, as well as significantly higher recurrence rate and shorter recurrence-free interval. To understand the molecular mechanism underlying the midkine with HCC progression, we performed in vitro and in vivo studies. We found that midkine plays an important role in enhancement of HCC cell resistance to anoikis, thereby promoting subsequent metastasis. Activation of PI3K/Akt/NF-κB/TrkB signaling by midkine-activated anaplastic lymphomakinase (ALK) is responsible for anoikis resistance.

Prognostic value of circulating tumor cells in advanced gastric cancer patients receiving chemotherapy.

The identification of circulating tumor cells (CTCs) may provide important prognostic information in several types of solid tumors, including gastric cancer. The aim of this study was to investigate whether CTC count may be used to predict survival in patients with advanced gastric cancer treated with chemotherapy. The CELLection™ Epithelial Enrich kit was used to isolate and purify CTCs from samples of peripheral blood. Immunofluorescent staining was used for CTC counting. High CTC counts were associated with poor tumor differentiation and high serum CEA levels (P=0.021 and 0.005, respectively). After 3 months, 16 patients with decreasing CTC counts after the first cycle of chemotherapy obtained complete response, partial response or stable disease, while 13 patients with increasing CTC counts developed progressive disease. The patients with decreasing CTC counts also exhibited longer progression-free survival (PFS) (P≤0.001) and overall survival (OS) (P=0.002) compared with those with increasing CTC counts. Among all 59 patients, those with a CTC count of ≤2 cells/5 ml blood exhibited longer PFS (P≤0.001) and OS (P≤0.001) compared with those with a CTC count of >2 cells/5 ml blood. The multivariate analysis suggested that an increase of the CTC count after the first cycle of chemotherapy was only an independent prognostic marker of poor PFS (P=0.019). However, a baseline CTC count of >2 cells/5 ml blood was an independent poor prognostic marker for PFS (P=0.008) and OS (P=0.001) in all 59 patients. Our study suggested that patients with a low baseline CTC count or decrease of the CTC count after the first cycle of chemotherapy may benefit significantly from palliative chemotherapy. In conclusion, CTC count may be a good chemotherapy monitoring marker and an ideal prognostic marker for patients receiving palliative chemotherapy.

Mutation Analysis of Cell-Free DNA and Single Circulating Tumor Cells in Metastatic Breast Cancer Patients with High Circulating Tumor Cell Counts.

The purpose of this study was to directly compare mutation profiles in multiple single circulating tumor cells (CTC) and cell-free DNA (cfDNA) isolated from the same blood samples taken from patients with metastatic breast cancer (MBC). We aimed to determine whether cfDNA would reflect the heterogeneity observed in 40 single CTCs. CTCs were enumerated by CELLSEARCH. CTC count was compared with the quantity of matched cfDNA and serum CA15-3 and alkaline phosphatase (ALP) in 112 patients with MBC. In 5 patients with ≥100 CTCs, multiple individual EpCAM-positive CTCs were isolated by DEPArray and compared with matched cfDNA and primary tumor tissue by targeted next-generation sequencing (NGS) of about 2,200 mutations in 50 cancer genes. In the whole cohort, total cfDNA levels and cell counts (≥5 CTCs) were both significantly associated with overall survival, unlike CA15-3 and ALP. NGS analysis of 40 individual EpCAM-positive CTCs from 5 patients with MBC revealed mutational heterogeneity in PIK3CA, TP53, ESR1, and KRAS genes between individual CTCs. In all 5 patients, cfDNA profiles provided an accurate reflection of mutations seen in individual CTCs. ESR1 and KRAS gene mutations were absent from primary tumor tissue and therefore likely either reflect a minor subclonal mutation or were acquired with disease progression. Our results demonstrate that cfDNA reflects persisting EpCAM-positive CTCs in patients with high CTC counts and therefore may enable monitoring of the metastatic burden for clinical decision-making. Clin Cancer Res; 23(1); 88-96. ©2016 AACR.

Correlation between epidermal growth factor receptor tyrosine kinase inhibitor efficacy and circulating tumor cell levels in patients with advanced non-small cell lung cancer.

ObjectiveThe aim of this study was to investigate the correlation between the efficacy of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) and circulating tumor cell (CTC) levels in patients with advanced non-small cell lung cancer (NSCLC). The efficacy of EGFR-TKIs in reducing CTC counts in patients with advanced NSCLC was studied.Patients and methodsA total of 66 patients with advanced NSCLC were enrolled and divided into two groups (those with high CTC counts and those with low CTC counts) based on the patients’ median CTC counts. All the patients were treated with an EGFR-TKI, and the treatment efficacy and prognoses were compared.ResultsThe treatment efficacies were 53.3% (16/30) and 27.8% (10/36) for the low CTC group and high CTC group, respectively, and this difference was statistically significant (P<0.05). The median overall survival was 22.8 months (95% confidence interval [CI]: 18.9–26.8 months) for the low CTC group and 18.3 months (95% CI: 2.9–8.2 months) for the high CTC group. The median progression-free survival was 11.5 months (95% CI: 8.1–15 months) and 5.6 months (95% CI: 2.9–8.2 months) for the low and high CTC groups, respectively, and the difference was statistically significant (P<0.05).ConclusionThe CTC count can be used as an index for predicting the EGFR-TKI effect on patients with advanced NSCLC. Efficacy and prognosis of EGFR-TKI treatment and CTC count were considered important, and the CTC count could be used to predict the efficacy of EGFR-TKI treatment and prognosis of advanced NSCLC. The change in CTC expression levels can be used as an index for evaluating the prognosis of patients with advanced NSCLC.

Abstract 2422: Isolation of canonical and non-canonical circulating tumor cells (CTCs) by negative depletion using the Harpoon CTC Isolator and Chip

Abstract Today, circulating tumor cells (CTCs) can provide important prognostic information about a patient. However, to one day help guide decisions for precision medicine, more and increasingly sophisticated information will have to be extracted from each patient blood sample. We describe a new microfluidic CTC isolation technology, the Harpoon CTC Isolator and Chip (Hrp Isolator). From a single sample the Hrp Isolator can enable the analysis of plasma components such as ctDNA, as well as the ability to enrich all circulating tumor cell types, regardless of their antigenic expression. To enrich the CTCs from 7.5 mL of whole blood, the Hrp Isolator removes the red blood cells and platelets based on their small size and the white blood cells by negative depletion using magnetic particles. The time required to process 7.5 mLs of blood is approximately 90 minutes. In contrast, the CELLSEARCH® CTC Test (CS), the only US FDA-cleared test, uses antigens expressed by CTCs to target the CTCs and positively select them out of blood. To enumerate CTCs from patient samples, the output from the Hrp Isolator was then loaded into and run on the CELLSEARCH® System using the CS test (Hybrid method). CTCs were enumerated from 37 breast and prostate cancer patients by CS and by the Hybrid method. At least 1 CTC was found in 23 (62%) patient samples by the Hybrid method versus 17 (46%) by CS. The product of the Hrp Isolator enrichment contained on average the same numbers of CTCs as were found by CS (mean = 13.3 ± 18 by CS versus 12.0 ± 16 by Hybrid) in patient samples having &amp;lt;75 CTCs. For samples with high CTC counts (&amp;gt;75), the Hybrid method may have underestimated the total Hrp Isolator CTC count. An important feature of the Hrp Isolator is its ability to enrich CTCs that are negative or low expressers of epithelial cell adhesion molecule (EpCAM) or cytokeratin (CK). These cells could have been missed by positive-selection technologies such as CS and may not have been counted using the Hybrid method. CTC spiked samples were run on the Hrp Isolator and the CTC enriched product was spun onto microscope slides, stained, and examined using a Vectra Multispectral Imaging System (Hrp+Vec method). Using the Hrp+Vec method, 79% to 100% of spiked CTCs were recovered depending on the slide fixation method used. Also, using Hrp+Vec we confirmed the ability of the Harpoon Isolator to enrich EpCAM-/CK+ and EpCAM+/CK- CTCs from patient samples. This establishes the existence of “non-canonical” tumor cell types in circulation and adds to the biological diversity of the CTC population in circulation. These results emphasize the need to further characterize all tumor cell populations and to determine what, if any, clinical significance may be assigned to these diverse CTC types. Citation Format: David Chianese, Mark Connelly, Carrie Morano, Thai Bui, Steven Gross, Renouard Sanders, Tom Barber, Ravi Kapur, Shyamala Maheswaran, Mehmet Toner, Daniel Haber. Isolation of canonical and non-canonical circulating tumor cells (CTCs) by negative depletion using the Harpoon CTC Isolator and Chip. [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 2422.

Abstract 2243: Characterization of PD-L1 expression on circulating tumor cells (CTCs) isolated with a label-free inertial microfluidic system from advanced non-small cell lung cancer patients (NSCLC pts)

Abstract Background: NSCLC exhibits intratumor heterogeneity, with subpopulations of cells undergoing epithelial-mesenchymal transition. Such CTCs from NSCLC pts may be missed by the EpCAM-based CELLSEARCH® system (CS). The label-free ClearCell® FX inertial microfluidic system (FX) isolates CTCs based on size and inertia and may lead to more accurate CTC capture. PD-1/PD-L1 inhibitors have shown benefit in PD-L1+ NSCLC pts, but responses are still seen in PD-L1- pts, suggesting limitations in tumor PD-L1 testing. Also, PD-L1 testing on CTCs may be more practical than tumor rebiopsies and may provide insights into cancer heterogeneity. Methods: FX was validated with EpCAM-high and EpCAM-low cancer cell lines labeled with CellTracker dyes spiked into healthy volunteer (HV) blood for repeatability and reliability. Enriched cells were detected with the automated Bioview Duet imaging system for recovery (%). Identical spiking studies were conducted on CS for comparison. Antibodies for 5-color immunofluorescence (IF) (CK, CD45, DAPI, TTF1 [to detect lung adenocarcinoma cells], PD-L1) were optimized on EpCAM-high and EpCAM-low cell lines. 8ml of blood from NSCLC pts were enriched with FX for 5-color IF characterization. 7.5ml of blood from the same pts taken at identical timepoints were enumerated with CS for comparison. Blood was obtained from HV for CTC enumeration on FX and CS as controls. Results: Cell recovery of EpCAM-high cancer cell lines using FX produced similar counts to CS, e.g. lung NCI-H2066 cells: FX 64.9%±4.5 vs CS 74.4%±9.2 (p = 0.05); lung H1975 cells: FX 74.1%±11.9 vs CS 74.7%±10.7 (p = 0.91). In contrast, for EpCAM-low cells, a significant difference in cell recovery between FX and CS was seen, e.g. lung A549 cells: FX 59.3%±5.8 vs CS 26.1%±7.6 (p&amp;lt;0.0001); and prostate PC3 cells: FX 68.6%±7.4 vs CS 35.5%±6.9 (p&amp;lt;0.0001). CTCs were detected in 19/21 pts with progressing stage IV NSCLC (adenocarcinoma) (mean 57; range 4-304) using FX, vs 7/21 pts (mean 7; range 1-38) using CS. CTC counts were higher with FX vs CS in 19/21 (90%) NSCLC pts, p = 0.012. No CTCs were detected in HV with FX (n = 10) and CS (n = 5). After FX enrichment, CTCs from 17/21 NSCLC pts were available for 5-color IF testing. Of these 17 pts, 15 had TTF1+ CTCs. All 15 pts with TTF1+ CTCs had ≥1 PD-L1+ CTC. 6/15 (40%) pts only had PD-L1+ TTF1+ CTCs, with no PD-L1- TTF1+ CTCs. PD-L1 CTC heterogeneity was seen in the other 9/15 pts, with co-existing PD-L1+ and PD-L1- TTF1+ CTCs. 6 of these 9 pts had more PD-L1+ TTF1+ CTCs than PD-L1- TTF1+ CTCs. Conclusions: FX resulted in consistently high cell recovery rates regardless of EpCAM status. Higher CTC counts were isolated with FX vs CS in 90% of NSCLC pts. 40% of NSCLC adenocarcinoma pts had PD-L1+ TTF1+ CTCs, but PD-L1 CTC heterogeneity was seen in other pts, which may in part explain differences in responses to PD-1/PD-L1 inhibitors in NSCLC. Citation Format: Zai Ahmad, Jen Fraser-Fish, Rajiv Kumar, Bernadette Ebbs, Gemma Fowler, Penny Flohr, Mateus Crespo, Sanjay Popat, Jaishree Bhosle, Udai Banerji, Mary O’Brien, Johann S. de Bono, Timothy A. Yap. Characterization of PD-L1 expression on circulating tumor cells (CTCs) isolated with a label-free inertial microfluidic system from advanced non-small cell lung cancer patients (NSCLC pts). [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 2243.

A High Circulating Tumor Cell Count in Portal Vein Predicts Liver Metastasis From Periampullary or Pancreatic Cancer: A High Portal Venous CTC Count Predicts Liver Metastases.

Circulating tumor cells (CTCs) released from a periampullary or pancreatic cancer can be more frequently detected in the portal than the systemic circulation and potentially can be used to identify patients with liver micrometastases. Aims of this study is to determine if CTCs count in portal venous blood of patients with nonmetastatic periampullary or pancreatic adenocarcinoma can be used as a predictor for subsequent liver metastases. CTCs were quantified in portal and peripheral venous blood samples collected simultaneously during pancreaticoduodenectomy in patients with presumed periampullary or pancreatic adenocarcinoma without image-discernible metastasis. Postoperatively patients were monitored for liver metastasis by abdominal magnetic resonance imaging or computed tomography every 3 months for 1 year. Sixty patients with a pathological diagnosis of periampullary or pancreatic adenocarcinoma were included in the study. Multivariate analysis indicated that portal CTC count was a significant predictor for liver metastases within 6 months after surgery. Eleven of 13 patients with a high portal CTCs count (defined as >112 CMx Platform estimated CTCs in 2 mL blood) developed liver metastases within 6 months after surgery. In contrast, only 6 of 47 patients with a low portal CTC count developed liver metastases (P < 0.0001). A value of 112 CMx Platform estimated CTCs had 64.7% sensitivity and 95.4% specificity to predict liver metastases within 6 months after surgery. We concluded that a high CTC count in portal venous blood collected during pancreaticoduodenectomy in patients with periampullary or pancreatic adenocarcinoma without metastases detected by currently available imaging tools is a significant predictor for liver metastases within 6 months after surgery.

Abstract P2-02-17: T-DM1 in HER2-negative metastatic breast cancer patients with HER2-amplified circulating tumor cells: Current status of the CirCe T-DM1 phase II trial

Abstract Background: Liquid biopsy can reassess key therapeutic targets in metastatic breast cancer. Several studies showed that a low albeit significant rate of metastatic breast cancer initially considered as HER2-negative can be reclassified as HER2-positive by systematic biopsy procedures. We report here the current status of the CirCe T-DM1 trial [NCT01975142] which aims to demonstrate the clinical utility of HER2 status reassessment on circulating tumor cells (CTCs). Methods: The first step of the trial consists in CTC count and HER2/CEP17 FISH on detected CTCs (CellSearch, Janssen Diagnostics) in patients (pts) with measurable disease progressing after the second line of chemotherapy. Pts with amplified CTCs (HER2/CEP17 ratio equal or higher than 2.2) are eligible to the treatment step of the study in two distinct cohorts: low CTC count (1 or 2 HER2-amplified CTCs) and high CTC count (3 and more HER2-amplified CTCs). In the treatment step has a Simon's two stage design, the anti-HER2 antibody-drug conjugate T-DM1 being administered until tumor progression. The primary objective of the trial is the confirmed response rate (RECIST). This trial is supported by Roche. Results: CirCe T-DM1 has been initiated in 10 centers in France. As of June 2015, 105 metastatic breast cancers pts considered as HER2-negative were screened. 29 pts (27%) had no CTC detected, 68 pts (65%) had at least 1 CTC detected with no HER2 amplification, and 8 pts (8%) exhibited HER2-amplified CTCs. Among the 8 pts, 1 pt had 5 HER2-amplified CTC, 2 pts had 2 HER2-amplified CTC and 5 pts had 1 HER2-amplified CTC. HER2/CEP17 ratios among HER2-amplified CTCs ranged from 2.5 to 7. Five of the 8 pts were treated by T-DM1. One objective confirmed partial tumor response has been observed (20%). Conclusion: The accrual is ongoing; the first efficacy assessment will occur after having treated 14 pts. This innovative trial highlights the promise and the complexity of liquid biopsy-based programs in the era of precision medicine: scarcity of the target, reliability and reproducibility of the target assessment, major efficacy when the target is matched to the appropriate drug. Citation Format: Bidard F-C, Romieu G, Jacot W, Cottu P, Dieras V, Lerebours F, Servent V, Luporsi E, Lortholary A, Tubiana-Mathieu N, Espie M, Bollet M, Bourgeois H, Renaud N, Pelissier S, Armanet S, Baeten K, Pierga J-Y. T-DM1 in HER2-negative metastatic breast cancer patients with HER2-amplified circulating tumor cells: Current status of the CirCe T-DM1 phase II trial. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-17.

Abstract P2-02-09: Obesity associated factors are inversely associated with circulating tumor cells in metastatic breast cancer

Abstract Background: Elevated levels of circulating tumor cells (CTCs) are associated with adverse outcomes in metastatic breast cancer (BC). However, relationships between CTCs and various patient-related factors that may impact outcome remain undefined. Consequently, associations of CTC counts with obesity and metabolic factors were evaluated in order to gain insight into potential interactions between patient physiology and disease burden. We hypothesized that obesity and associated metabolic factors would be associated with higher CTC counts. Methods: Non-diabetic women with metastatic BC beginning a new line of treatment due to progressive disease were recruited from four Ontario cancer hospitals between February 2013 and April 2015. Patients provided blood for CTC analysis, which was completed within 72 hours of collection using the Janssen CellSearch platform. Fasting serum was also collected for assessment of metabolic factors including glucose (mmol/L), insulin (pmol/L), leptin (ng/mL) and adiponectin (ng/mL). Associations of CTC counts with these factors, as well as anthropometric measurements (height (cm), weight (kg), BMI (kg/m2)) were evaluated using Pearson correlation coefficients after transforming the variables involved to normality. For CTC counts, the log transformation with half integer correction was used. Results: 96 patients with a median age of 60.5 years completed the study. Most were post-menopausal (87, 90.6%) and exhibited grade II/III tumors (75, 78.1%). The majority of patients had hormone receptor positive disease (83, 86.5%), but 16.7% (16) were HER2 positive and 10.4% (10) were triple negative. The number of CTCs observed ranged from 0 to 1238 (median 2, geometric mean 3.63). No CTCs were detected in 29 patients (30.2%), whereas 25 patients (26 %) exhibited counts of 1 to 4 CTCs and 42 (43.8%) had 5 or more CTCs. CTCs were not significantly associated with tumor characteristics including ER/PgR, HER2, grade, stage (T/N) or lymphovascular invasion. The number of CTCs inversely correlated with BMI (r=-0.26, p=0.01), leptin (r=-0.29, p=0.004), and leptin-adiponectin ratio (r=-0.3, p=0.004). A similar trend that approached significance was noted for body weight (r=-0.19, p=0.07), insulin (r=-0.19, p=0.06) and homeostatic model assessment (HOMA, an estimate of insulin resistance, r=-0.2, p=0.055). Conversely, adiponectin (r=0.18, p=0.07) and height (r=0.18, p=0.07) were positively associated with CTC counts in correlations that neared significance. No associations were observed for age (r=0.09, p=0.4) or glucose (r=-0.09, p=0.4). Conclusions: Obesity associated metabolic factors including weight, BMI, insulin, HOMA and leptin were inversely associated (and adiponectin and height positively associated) with CTC counts. These patterns are consistent with weight loss and/or cachexia in women with elevated CTC counts who have higher disease burden. Additional analyses are underway to further characterize these associations and include assessment of serum albumin, free fatty acids, creatine kinase and hepcidin. Citation Format: Dowling RJO, Chang MC, Lohmann AE, Ennis M, Amir E, Elser C, Brezden-Masley C, Vandenberg T, Lee E, Fazaee K, Stambolic V, Goodwin PJ. Obesity associated factors are inversely associated with circulating tumor cells in metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-09.

High circulating tumor cell concentrations in a specific subtype of gastric cancer with diffuse bone metastasis at diagnosis.

To clarify the biological feature contributing to gastric cancer with diffuse bone metastases at diagnosis. The participants visited the Department of Clinical Oncology, Akita University Hospital, from January 2014 to August 2015. The selection criterion for gastric cancer with diffuse bone metastases at diagnosis includes over 29 hot spots of bone scintigraphy. Circulating tumor cell were collected from 20 mL of peripheral venous blood drawn using a CellSearch kit and a CellTracks AutoPrep system by SRL, a clinical laboratory. The endpoints of this study were correlations between circulating tumor cells (CTC) count and therapeutic outcomes. Among 39 patients with gastric cancer, 5 patients met the criterion. The incidence of this subtype was 12.8%. CTC counts ranged from 235 to 6440 cells/7.5 mL of peripheral blood (median of 1724). These values were much higher than common gastric cancers (2 cells). In chemo-sensitive cases, CTC counts decreased within 14 d (median) from 275, 235 and 1724 to 2, 7 and 66, respectively. On the other hand, CTC counts increased after treatment failure or insensitive case from 2, 7 and 6440 to 787, 513 and 7885, respectively. The correlation between CTC count and survival time showed a trend, but did not reach significance (Y = 234.6 - 0.03X, P = 0.085). High CTC count is a biological hallmark of this subtype, and can be used as a direct and definitive indicator of therapeutic outcome.

Abstract C18: Epithelial and mesenchymal phenotypic characterization and mutation detection in circulating tumor cells isolated from peripheral blood of non-small cell lung cancer patients with ApoStream® technology

Abstract Background: The presence of circulating tumor cells (CTCs) has been established as a prognostic indicator in a few metastatic cancers. Although technically challenging, isolation and characterization of CTCs at the protein or nucleic acid level has significant potential to enable the development of minimally invasive methods to detect molecular alterations for personalized cancer targeted therapies. The goal of this study was to demonstrate the ability to enrich phenotypically heterogeneous population of CTCs and detect EGFR and KRAS mutations in isolated CTCs obtained from peripheral blood of patients with Non-small cell lung cancer (NSCLC) using ApoStream®, an antibody-independent CTC capture device based on dielectrophoresis technology. Methods: For condition optimization, blood samples from a small cohort of cancer patients (n = 8) were collected under an IRB approved protocol and processed with multiple ApoStream® operating frequencies (45, 55, 65 and 85 kHz). To evaluate clinical feasibility, blood samples (8 mL) from 35 additional NSCLC patients (with or without targetable “driver” mutations) and 10 normal, healthy donor controls were collected and processed using the optimized ApoStream® conditions. CTC enumeration was performed using a multiplex phenotyping panel targeting hematopoietic (CD45/CD15), epithelial (EpCAM/CK/E-Cadherin) and mesenchymal (Vimentin/N-cadherin/Twist-1) markers. In addition, mutation detection assays were validated for EGFR (E746_A750, L858R, and T790M) and KRAS (G12C, and G12S) with mutant DNA spiked into wild type DNA at multiple dilutions using the droplet-based RainDrop® digital PCR system. Results: An ApoStream® operating frequency of 85 kHz resulted in higher CTC counts and hence was selected for processing samples for CTC enumeration assay and a lower operating frequency of 65 kHz was selected to provide higher purity for processing samples for mutation analysis. Preliminary data demonstrated detectable CTCs (above healthy control baseline of 4 CTCs per mL blood) in 7 out of 10 patient samples processed to date (mean = 24; median = 14 and range = 5-100 CTCs per mL blood). CTCs isolated from all 7 patients exhibited mesenchymal phenotype. In addition, two patients also showed CTCs of epithelial phenotype demonstrating that the ApoStream® platform enriches phenotypically heterogeneous CTCs. Sensitivity of mutation detection determined from dilution series was found to be assay dependent; limit of quantitation (LOQ) for EGFR and KRAS mutations were found to be 0.01% or higher while the linearity for all the assays demonstrated a linear regression coefficient (R2) greater than 0.95 except for EGFR L858R which requires further analysis. Preliminary data from 8 NSCLC patient samples demonstrated the ability to detect mutations in ApoStream® enriched CTCs. Enumeration and mutation analysis is in progress for the remaining patient samples. Conclusion: We demonstrated the ability to isolate, enumerate and define epithelial and mesenchymal phenotypes of CTCs and to detect oncogenic mutations in CTCs isolated from NSCLC patient samples using ApoStream®. Funded by National Cancer Institute under Contract No. HHSN261201300070C. Citation Format: Priya Balasubramanian, Vishal Gupta, David Hasegawa, Carmen Y. Lam, Ran An, Swetha Pratyusha Gunturu, Hai T. Tran, John V. Heymach, Darren W. Davis. Epithelial and mesenchymal phenotypic characterization and mutation detection in circulating tumor cells isolated from peripheral blood of non-small cell lung cancer patients with ApoStream® technology. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C18.

Prognostic Importance of Circulating Tumor Cells in Nonsmall Cell Lung Cancer: A Prospective Study

Purpose: To investigate the prognostic value of circulating tumor cells (CTCs) and to predict the treatment response in a non-small cell lung cancer (NSCLC).Methodology: A single-center prospective study involving 93 patients with NSCLC was conducted. Blood samples were analyzed for CTC count before and after chemotherapy. Clinical relevance of CTCs with patient`s characteristics and treatment response were determined.Results: Higher levels of CTCs were associated with severe stage of NSCLC (p = 0.003), tumor histology (p = 0.014) and metastases (p = 0.013). Significant difference in CTC count was observed in favorable (CTCs &lt; 5) and unfavorable (CTCs ≥ 5) groups. Progression-free survival (PFS) was 5.8 months (range: 5.32 to 6.43) and 2.2 months (range: 1.85 to 3.01) in the favorable and unfavorable groups, respectively (HR: 3.88, 95% CI, p &lt; 0.001). Similarly, overall survival (OS) was 7.3 months (95% CI, 6.51 to 7.92) and 3.9 months (95% CI, 1.99 to 5.13), respectively (HR: 4.8, 95% CI, p &lt; 0.001). Multivariate regression analysis revealed CTCs as strong predictors of OS and PFS. Significant reduction (p &lt; 0.001) in CTC count was also observed after one cycle of chemotherapy.Conclusion: Patients with low CTC count live longer and remain progression-free for a longer period of time than those with high CTC count. High CTCs can be detected in severe forms of lung cancer and can be used as a valid prognostic marker. However, this assertion requires validation in larger prospective clinical cohorts.Keywords: Circulating tumor cells, Non-small cell lung cancer, Circulating tumor cell, Prognosis

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.

Clinico-pathological correlation of serial measurement of circulating tumor cells in 24 metastatic colorectal cancer patients receiving chemotherapy reveals interpatient heterogeneity correlated with CEA levels but independent of KRAS and BRAF mutation

Background: The Veridex CellSearch is an FDA-approved technology for enumerating circulating tumor cells in blood samples of metastatic colorectal cancer mCRC) patients and has prognostic value. It is important to understand how counts of circulating tumor cells (CTCs), which are advocated to be tools for “liquid biopsy” of tumors, correlate with clinical and pathologic variables of significance in these patients. In this study, we have attempted to make such correlations along with evaluating how CTC counts change during the course of chemotherapy. Patients and methods: Following an IRB-approved protocol, blood samples were collected from 24 patients with mCRC along with relevant clinico-pathological data. Blood was collected at defined time-points both prior to as well as during the course of treatment with combination chemotherapy, and CTC counts were enumerated from7.5 ml of blood. Results: Seventeen out of 24 patients with mCRC showed a CTC count of 2 or less cells in 7.5 ml of blood at base-line assessment before chemotherapy while 7 patients showed 3 or more cells in 7.5 ml of blood at that point. A correlation was found between high carcino-embryonic antigen (CEA) levels and high CTC counts (P = 0.018) although it was also found that some patients had elevated CTCs without an elevated CEA. No correlation with the time interval between detection of primary tumor and appearance of secondary (metastatic) tumor(s) was found. CTC counts did not correlate with the presence of lung or liver metastases, i.e. a number of mCRC patients with lung or liver metastases had a count of zero CTCs at baseline. We also noted no correlation between CTC number and the status of KRAS or BRAF mutation. CTC counts dropped immediately after the start of chemotherapy in 11 out of 21 patients, and also reduced from the baseline at the end of chemotherapy in 5 out of 10 patients. Six of 7 patients who started with 3 or more CTCs in 7.5 ml at baseline also showed a final CTC reduction at the end of the therapy assessment. Conclusions: Analysis of circulating tumor cells may be of use in monitoring response to therapy in mCRC, either in combination with CEA monitoring or alone when CTCs are elevated but CEA level is not.

Circulating Tumor Cell Enumeration in a Phase II Trial of a Four-Drug Regimen in Advanced Colorectal Cancer

BackgroundMultidrug regimens are active against advanced colorectal cancer (ACRC). However, the increased toxicity requires the use of biomarkers to select the patients who will derive the most benefit. We assessed circulating tumor cells (CTCs) as a prognostic biomarker in patients treated with a 4-drug regimen. Patients and MethodsA single-arm phase II trial (Erbitux Study of CPT11, Oxaliplatin, UFToral Targeted-therapy [eSCOUT]) was undertaken in patients with previously untreated KRAS wild-type ACRC using a regimen of irinotecan, oxaliplatin, and tegafur-uracil with leucovorin and cetuximab. Baseline CTCs were enumerated using CellSearch. The endpoints were an objective response rate (ORR) and overall survival (OS). We modeled our results and compared them with those modeled for the capecitabine, oxaliplatin, bevacizumab +/- cetuximab (CAIRO2) trial, stratifying patients a priori into low (< 3) and high (≥ 3) CTC groups. ResultsFor 48 eligible patients, the best ORR from the 4-drug regimen was 71%, with a disease control rate of 98%. The median OS for patients with a high and low CTC count was 18.7 and 22.3 months (log-rank test, P = .038), respectively. In our modeled data, for patients with a low CTC count, no differences were found between the median OS in the eSCOUT trial and that in the CAIRO2 trial (22.2 vs. 22.0 months). However, for the high CTC group, a clinically relevant improvement was seen in median OS (eSCOUT vs. CAIRO2, 18.7 vs. 13.7 months; P = .001). ConclusionThese data are hypothesis generating—for patients with ACRC, stratification by CTC count can identify those who might benefit the most from an intensive 4-drug regimen, avoiding high-toxicity regimens in low CTC groups. This hypothesis warrants validation in a phase III biomarker-driven trial.

Abstract 2097: Distinct levels of circulating tumor cell counts for the papillary thyroid cancer patients with distant metastases and in disease-free status

Abstract Distant metastasis of papillary thyroid cancer (PTC) is a multistep and complex process that frequently complicates the disease with poor prognosis. The presence of circulating tumor cells (CTCs) in the peripheral blood has been reported in several cancer types to be highly related to the development of distant metastasis. Nevertheless, the CTCs status during the progression of PTC is rarely investigated. This study aimed to define and compare the CTCs counts among the healthy control individuals (Group I, n = 14), the PTC patients in disease-free status (Group II, n = 9), and the patients with distant metastasis (Group III, n = 8). The patients in group III had metastases to the lung and mediastinum (n = 4), bones (n = 3), and lung and kidney (n = 1), respectively. CTCs were enriched from the peripheral blood by the negative selection method PowerMag that has been validated in our previous study. CTCs enumeration was performed by immunofluorescence staining using the anti-epithelial cell adhesion molecule (EpCAM) and anti-thyroid stimulating hormone receptor (TSHR) antibodies. The EpCAM+ and TSHR+ CTCs counts were 2.97 ± 2.03 and 9.97 ± 9.22 cells/mL for group I, 10.43 ± 9.03 and 19.17 ± 9.77 cells/mL for group II, and 100.06 ± 73.34 and 141.60 ± 82.08 cells/mL for group III individuals, respectively (p &amp;lt; 0.01). These data indicate that (1) some of the TSHR+-CTCs are not EpCAM+ and this type of cell population can be overlooked if CTCs were enriched by the method based on positive selection of EpCAM+ cells; (2) the PTC patients in disease-free status has higher CTCs counts than the healthy control individuals and, hence, continuous CTCs enumeration is recommended for the patient in the stage of disease-free; (3) the PTC patients with distant metastases have higher CTCs counts when compares to the healthy control subjects and the patients with disease-free status. In conclusion, CTCs enumeration is thereby warranted in the course of diagnosis and treatment of thyroid cancer. Citation Format: Hung-Chih Lin, Ju-Chien Cheng, Ching-Ping Tseng, Jen-Der Lin. Distinct levels of circulating tumor cell counts for the papillary thyroid cancer patients with distant metastases and in disease-free status. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2097. doi:10.1158/1538-7445.AM2014-2097

Molecular characterization and clinical utility of circulating tumor cells in the treatment of prostate cancer.

Circulating tumor cells (CTCs) are rare cancer cells that can be detected in the blood of patients with solid malignancies. The Veridex CellSearch Assay was analytically and clinically validated, and has received U.S. Food and Drug Administration (FDA) clearance for the enumeration of CTCs in breast, colorectal, and prostate cancer. A number of alternative assays, with potential advantages, are currently undergoing clinical and/or analytic validation before their routine use can be established. In prostate cancer, high pretreatment CTC counts have been associated with worse survival, and changes in CTC counts in response to treatment have been established as indicators of response to treatment. Additional analyses are ongoing to establish the value of CTC counts as a surrogate of survival in prospective, phase III trials, which could influence the process of drug development and regulatory approval. Additionally, CTCs have a potential role in the molecular characterization of prostate cancer, serving as "liquid biopsies" to determine the molecular characteristics of the disease. The study of androgen receptor (AR) mutations or amplification, chromosomal rearrangements, or the determination of DNA repair biomarkers has been evaluated in clinical trials. CTCs have a wide range of potential applications, from their prognostic use in stratification of patients in clinical trials or the assessment of response to treatment, to the pharmacodynamic evaluation of novel agents, or the discovery and use of predictive biomarkers that can aid in the development of personalized medicine.