Yield Of Circulating Tumor Cells Research Articles

High-throughput enrichment of portal venous circulating tumor cells for highly sensitive diagnosis of CA19-9-negative pancreatic cancer patients using inertial microfluidics

The carbohydrate antigen 19-9 (CA19-9) is commonly used as a representative biomarker for pancreatic cancer (PC); however, it lacks sensitivity and specificity for early-stage PC diagnosis. Furthermore, some patients with PC are negative for CA19-9 (<37 U/mL), which introduces additional limitations to their accurate diagnosis and treatment. Hence, improved methods to accurately detect PC stages in CA19-9-negative patients are warranted. In this study, tumor-proximal liquid biopsy and inertial microfluidics were coupled to enable high-throughput enrichment of portal venous circulating tumor cells (CTCs) and support the effective diagnosis of patients with early-stage PC. The proposed inertial microfluidic system was shown to provide size-based enrichment of CTCs using inertial focusing and Dean flow effects in slanted spiral channels. Notably, portal venous blood samples were found to have twice the yield of CTCs (21.4 cells per 5 mL) compared with peripheral blood (10.9 CTCs per 5 mL). A combination of peripheral and portal CTC data along with CA19-9 results showed to greatly improve the average accuracy of CA19-9-negative PC patients from 47.1% with regular CA19-9 tests up to 87.1%. Hence, portal venous CTC-based microfluidic biopsy can be used with high sensitivity and specificity for the diagnosis of early-stage PC, particularly in CA19-9-negative patients.

Detection and Characterization of Circulating Tumor Cells Using Imaging Flow Cytometry-A Perspective Study.

Simple SummaryLiquid biopsy is non-invasive approach used to prognose and monitor tumor progression based on the detection and examination of metastasis-related events found in the patients’ blood (such as circulating tumor cells (CTCs), extracellular vesicles, and circulating nucleic acids). Different ultrasensitive techniques are applied to study those events and the biology of tumor dissemination, which in the future might complement standard diagnostics. Here, we suggest that CTCs analysis could be improved by the usage of imaging flow cytometry, combining advantages of both standard flow cytometry (high-scale analysis) and microscopy (high resolution) to investigate detailed features of those cells. From this perspective, we discuss the potential of this technology in the CTC field and present representative images of CTCs from breast and prostate cancer patients analyzed with this method.Tumor dissemination is one of the most-investigated steps of tumor progression, which in recent decades led to the rapid development of liquid biopsy aiming to analyze circulating tumor cells (CTCs), extracellular vesicles (EVs), and circulating nucleic acids in order to precisely diagnose and monitor cancer patients. Flow cytometry was considered as a method to detect CTCs; however, due to the lack of verification of the investigated cells’ identity, this method failed to reach clinical utility. Meanwhile, imaging flow cytometry combining the sensitivity and high throughput of flow cytometry and image-based detailed analysis through a high-resolution microscope might open a new avenue in CTC technologies and provide an open-platform system alternative to CellSearch®, which is still the only gold standard in this field. Hereby, we shortly review the studies on the usage of flow cytometry in CTC identification and present our own representative images of CTCs envisioned by imaging flow cytometry providing rationale that this novel technology might be a good tool for studying tumor dissemination, and, if combined with a high CTC yield enrichment method, could upgrade CTC-based diagnostics.

Abstract 3119: Comparison of circulating tumor cell (CTC) derived DNA and circulating cell-free DNA (cfDNA) from simultaneous blood sampling of patients with metastatic breast cancer (MBC)

Abstract Purpose: Blood-based candidate biomarkers of disease can be monitored by analyzing CTCs and/or circulating cfDNA isolated from the peripheral blood. Our primary objective is to understand the relative contributions of these circulating factors (i.e., CTCs and cfDNA) to the overall disease profile in MBC. Methods: Clinically archived FFPE tumor tissue and prospective blood samples are collected through a minimal risk protocol approved by the Mayo Clinic IRB (#16-001540) from patients with MBC and objective evidence of disease progression. Blood samples include 20 mL whole blood in Streck blood collection tubes (BCTs) for platelet poor plasma (PPP); 20 mL whole blood in AccuCyte BCTs for CTCs, WBCs, and PPP; and 10 mL whole blood in EDTA BCTs for PPP. Nucleated, EpCAM+/cytokeratin+/CD45- CTCs are identified, assessed for ER/HER2 status, and isolated using a centrifugation and direct imaging platform that allows for single cell retrieval (RareCyte). DNA is extracted from PPP, CTCs, WBCs, and FFPE tumor tissue using established methods. Targeted sequencing for SNVs/indels is performed on paired WBCs and CTC-DNA, AccuCyte-cfDNA, Streck-cfDNA, EDTA-cfDNA, and tumor tissue derived DNA using the same NGS panel and informatics pipeline (65 genes; CleanPlex OncoZoom; Paragon Genomics). Results: Tissue and blood samples were collected from 40 patients with metastatic breast cancer. 10 cases were selected for initial analyses on the basis of CTC yield (range 3-113 per 3.75 mL blood); up to 5 CTCs per subject were isolated and pooled for DNA extraction. Plasma cfDNA yields and variant allele frequencies were highly comparable between AccuCyte and Streck collected blood samples. Mutations (range 1-3) were identified in CTC-DNA and/or cfDNA in 9 of 10 cases for a total of 18 detected mutations: 10 in CTC-DNA and cfDNA (BRCA2 N372H, PIK3CA E542K, PIK3CA E545K (x3), PIK3CA H1047R, PTEN R130P, RET G691S, TP53 C135W, TP53 Q192*); 5 in CTC-DNA only (EGFR R521K, EGFR T790M, PIK3CA H1047R, SMAD4 C363Y, TP53 N263D); and 3 in cfDNA only (DNMT3A W893S, DNMT3A S714C, TP53 Q136E). Parallel analyses of samples from 10 more subjects are in progress. Analysis of tumor tissue for all 20 subjects and of EDTA-cfDNA and single CTCs for a subset of cases is ongoing. Updated results will be presented at the meeting. Conclusions: It is feasible to isolate high quality CTCs and cfDNA from the same blood collection tube to perform targeted sequencing; this streamlines specimen processing, decreases overall costs, and minimizes required blood volumes. Importantly, there is overlap in the majority of mutations identified in CTC-DNA and cfDNA, but actionable mutations (e.g., PIK3CA, EGFR) were detected in CTC-DNA only. The clinical and theranostic relevance of these findings is unclear and warrants further investigation. Citation Format: Minetta C. Liu, Karthik V. Giridhar, Roberto A. Leon Ferre, Jamie L. Carroll, Matthew P. Goetz, Tufia C. Haddad, Deanne R. Smith, Siddhartha Yadav, Vidushi Kapoor, Guoying Liu, Tad George, Nolan Ericson, Arturo B. Ramirez, Eric Kaldjian, Keegan E. Haselkorn. Comparison of circulating tumor cell (CTC) derived DNA and circulating cell-free DNA (cfDNA) from simultaneous blood sampling of patients with metastatic breast cancer (MBC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3119.

Abstract B43: Genetic analysis of circulating tumor cells of colorectal cancer patients captured by multiantibodies technique

Abstract Background: Accurate genetic analysis is essential for molecular therapy. Conventionally, DNA derived from tumor tissue has been the source of the gold standard of DNA information in solid tumors including colorectal cancer (CRC). However, it is well known that molecular therapy induces emerging mutations that are not found in primary tumor; thus, real-time monitoring of gene information is ideal. Circulating tumor cells (CTCs) are circulating malignant cells of solid tumor origin that are found in the bloodstream and can be a powerful candidate to play an important role. Capturing a large amount of CTCs improves the accuracy of genetic analysis of DNA derived from CTC (ctcDNA). We used 3 (EpCAM, Her2, Trop2) or 4 (EpCAM, Her2, Trop2, EGFR) antibodies to capture CTCs, and analyzed ctcDNA using next-generation sequencing (NGS). Methods: Cohort 1: Untreated CRC patients were enrolled. Ten mL of whole blood was collected from each patient. The blood was processed using 3 antibodies (EpCAM, Her2, Trop2) and CTCs were collected. Tumor tissue was also collected from each patient. Tumor tissue DNA and ctcDNA were extracted and analyzed using NGS. Cohort 2: CRC patients, both treated and untreated, were enrolled. Twenty mL of whole blood was collected from each patient. Ten mL of the blood was processed using 3 antibodies (EpCAM, Her2, Trop2), and the remaining 10 mL was processed using 4 antibodies (EpCAM, Her2, Trop2, EGFR). The numbers of collected CTCs were counted and compared. Results: Cohort 1: We enrolled 34 patients (stage II: n=4, stage III: n=7, stage IV: n=23). Median number of extracted CTC was 34 cells. From tumor tissue DNA, 53 mutations were detected. The most frequent mutation was within TP53 (n=18), followed by mutations in APC (n=13) and KRAS (n=12). From ctcDNA, 16 mutations, including 5 mutations which were not found in tissue DNA, were detected. The most frequent mutation was within TP53 (n=5), followed by mutations in KRAS and APC (n=4 each). Cohort 2: We enrolled 10 patients (stage II: n=1, stage III: n=1, stage IV: n=8). Using 3 antibodies, the median number of collected CTCs was 27 cells (range, 2–112). Using 4 antibodies, the median number of collected CTCs was 33 cells (range, 7–260). There were no statistically significant differences between the 2 groups (p=0.40). Conclusions: Mutations not detected in primary tumors can be identified in ctcDNA, indicating the potential of CTCs in complementing gene analysis. The technique to capture CTCs using 3 antibodies appears to increase the detection rate and yield of CTCs. However, the present study did not show advantages of the 4-antibodies method, and future studies should investigate the best combination of antibodies to extract more CTCs with higher specificity. Citation Format: Kohki Takeda, Takeshi Yamada, Michihiro Koizumi, Seiichi Shinji, Akihisa Matsuda, Ryo Ohta, Yasuyuki Yokoyama, Goro Takahashi, Masahiro Hotta, Takuma Iwai, Keisuke Hara, Koji Ueda, Sho Kuriyama, Hiroshi Yoshida. Genetic analysis of circulating tumor cells of colorectal cancer patients captured by multiantibodies technique [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B43.

P25 DETECTION OF CIRCULATING TUMOR CELLS IN POTENTIALLY RESECTABLE ADENOCARCINOMA OF DE DISTAL ESOPHAGUS AND THE GASTRO-ESOPHAGEAL JUNCTION DOES NOT INCREASE IN PORTAL VENOUS BLOOD SAMPLES COMPARED TO PERIPHERAL VENOUS BLOOD SAMPLES

Abstract Aim To compare the number of circulating tumor cells (CTCs) in portal venous blood samples of patients with potentially resectable adenocarcinoma of de distal esophagus and the gastro-esophageal junction (EAC) with the number of CTCs in peripheral venous blood samples. Background and Methods Detection of (CTCs) in potentially resectable (EAC) is rare in peripheral venous blood samples(1). In lung carcinoma patients, the number of circulating tumor cells was more than 300 fold in the pulmonary vein, compared to the number in peripheral venous blood samples (2). In patients undergoing esophagectomy for cancer, peripheral blood was sampled immediately preoperatively and portal vein blood was sampled intraoperatively during abdominal lymph node dissection. Samples were analyzed for CTCs using the parsortix device (ANGLE): a semi-automated microfluidic system that captures cells based on their size and rigidity. A four-color immunofluorescence technique was used and CK positive, CD45 negative, Hoechst positive and morphologically intact cells with the morphology of a CTC were counted manually. The method was previously compared with the commercially available Cellseach® system and no difference in CTC yield between both methods. Results 20 patients with potentially resectable EAC were evaluated. One peripheral venous sample and two portal venous samples were not applicable. In 5 out of 19 peripheral venous samples (26,3%), one or more CTC’s could be detected, while in this was only the case in 3 out of 18 portal venous samples (16.7%). Conclusions The number of detected CTCs in potentially resectable EAC was not increased in portal venous samples compared to peripheral venous samples. Further research is needed on why detection rate of CTCs in potentially resectable EAC is so low and how detection rate could be increased.

Abstract 1383: Isolating circulating tumor cells from a large screening blood volume: A pilot study using diagnostic leukapheresis

Abstract Purpose: The presence of circulating tumor cells (CTC) in blood is associated with poor prognosis in metastatic prostate cancer (mPCa) patients. For non-metastatic PCa patients and patients with biochemical recurrence (BCR), it is extremely difficult to find CTCs in 5-10 ml of peripheral blood (PB). The purpose of this study is to evaluate the feasibility of using diagnostic leukapheresis (LP) followed by other enrichment technologies to increase the CTC yield by using a large screening blood volume. Methods: Blood samples were collected from one healthy donor and one mPCa patient. During LP procedure, different blood elements were separated to different layers based on their densities. The mononuclear cell layer (that also contains CTCs) was collected. To assess recovery rate of different CTC isolation and detection methods, the healthy donor LP was spiked with a known number of PCa cell line LNCaP cells. Three CTC isolation and detection methods were used, including FAST (Clinomics, Ulsan, South Korea), a size-selection platform followed by immunofluorescence (IF); the AccuCyte-CyteFinder system (RareCyte, Inc., Seattle, WA), a selection-free method that utilizes IF; the AlereTM CTC AdnaTest (AdnaGen, Langenhagen, Germany), an EpCAM positive selection method followed by RT-qPCR. The criteria for defining a CTC are DAPI+, CK/EpCAM+, and CD45-. The Ct value thresholds for positive CTC signal by RT-qPCR were EpCAM 36.4, PSA 36.3, HOXB13 40, AR 35.4. Results: For the healthy donor LP with spiked LNCaP cells, the CTC recovery rate from FAST was 53.7%~67.2%. For Adna test, 0, 44, 95 and 1000 LNCaP cells were spiked in 5 ml of LP respectively. The Ct values of EpCAM were 36.28, 29.32, 28.22 and 24.76. For the mPCa patient, two tubes of 7.5 ml of PB were collected for FAST and RareCyte. The FAST found 3 CTCs while the RareCyte found 1 CTC. An LP sample was also collected from the same mPCa patient. The LP sample volume was 162 ml (WBC concentration was 83×109/L). Triplicates of 5 ml of samples were sent through AdnaTest and CTCs were identified in all samples (Ct: EpCAM 34.21±0.77, PSA 33.86±1.81, HOXB13 36.25±0.34, AR 35.29±0.22). Then the remainder of the mPCa LP was subjected to Ficoll-Paque density centrifugation to further concentrate mononuclear cells to a 90 ml volume. 1 ml samples were then applied to FAST in triplicate. The CTC numbers were 22 and 15. The density of cells on one of the filters was too high to be successfully scanned. Conclusion: Finding CTCs in LP samples by size-based technology or AdnaTest is feasible and offers a promising method to do liquid biopsy for mPCa patients. Screening a larger volume of PB by LP has significantly increased the CTC yield compared to using the same capture technology on 7.5 ml of PB. Citation Format: Liang Dong, Zhongyuan Zhang, Changxue Lu, Diane Reyes, Amber de Groot, Sarah R. Amend, Jun Luo, Kenneth J. Pienta. Isolating circulating tumor cells from a large screening blood volume: A pilot study using diagnostic leukapheresis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1383.

Detection of circulating tumor cells in colorectal cancer patients using the GILUPI CellCollector: results from a prospective, single-center study.

The GILUPI CellCollector (CC) is a novel in vivo circulating tumor cell (CTC) detection device reported to overcome the limitations of small blood sample volumes. The aim of this prospective, blinded study was to evaluate the clinical application of the CC and to compare its performance to the CellSearch (CS) system in M0 and M1 colorectal cancer (CRC) patients. A total of 80 patients (31 M0, 49 M1) with CRC were enrolled. CTCs were simultaneously measured in the peripheral blood using CS and the CC, and the results of both assays were correlated to clinicopathological variables and overall survival. The total number of detected CTCs and CTC‐positive patients did not significantly differ between both assays. In the M0 patients, the CC detected CTCs more frequently than CS. There was no significant difference in total CTC numbers detected with the CC between M0 and M1 patients. In addition, no significant correlation with clinicopathological parameters or overall survival was observed with CC CTCs. In contrast, detection of CTCs with CS was significantly correlated with Union for International Cancer Control stage and reduced overall survival. There was no correlation between CTCs detected by the CC and the CS system. Using in silico analysis, we estimate that CC screens a volume of 0.33–18 mL during in vivo application, in contrast to much higher volumes reported elsewhere. In conclusion, while being safe and easy to use, the CC did not outperform CS in terms of CTC yield or sensitivity. While CTC detection in M0 CRC patients was significantly increased with the CC, the clinical relevance of these CTCs appears inferior to the cells identified by the CS system.

Microfluidic chip combined with magnetic-activated cell sorting technology for tumor antigen-independent sorting of circulating hepatocellular carcinoma cells.

PurposeWe aimed to generate a capture platform that integrates a deterministic lateral displacement (DLD) microfluidic structure with magnetic-activated cell sorting (MACS) technology for miniaturized, efficient, tumor antigen-independent circulating tumor cell (CTC) separation.MethodsThe microfluidic structure was based on the theory of DLD and was designed to remove most red blood cells and platelets. Whole Blood CD45 MicroBeads and a MACS separator were then used to remove bead-labeled white blood cells. We established HepG2 human liver cancer cells overexpressing green fluorescent protein by lentiviral transfection to simulate CTCs in blood, and these cells were then used to determine the CTC isolation efficiency of the device. The performance and clinical value of our platform were evaluated by comparison with the Abnova CytoQuest™ CR system in the separating of blood samples from 12 hepatocellular carcinoma patients undergoing liver transplantation in a clinical follow-up experiment. The isolated cells were stained and analyzed by confocal laser scanning microscopy.ResultsUsing our integrated platform at the optimal flow rates for the specimen (60 µl/min) and buffer (100 µl/min per chip), we achieved an CTC yield of 85.1% ± 3.2%. In our follow-up of metastatic patients, CTCs that underwent epithelial–mesenchymal transition were found. These CTCs were missed by the CytoQuest™ CR bulk sorting approach, whereas our platform displayed increased sensitivity to EpCAMlow CTCs.ConclusionsOur platform, which integrates microfluidic and MACS technology, is an attractive method for high-efficiency CTC isolation regardless of surface epitopes.

Single-Cell Analyses of Prostate Cancer Liquid Biopsies Acquired by Apheresis.

Purpose: Circulating tumor cells (CTCs) have clinical relevance, but their study has been limited by their low frequency.Experimental Design: We evaluated liquid biopsies by apheresis to increase CTC yield from patients suffering from metastatic prostate cancer, allow precise gene copy-number calls, and study disease heterogeneity.Results: Apheresis was well tolerated and allowed the separation of large numbers of CTCs; the average CTC yield from 7.5 mL of peripheral blood was 167 CTCs, whereas the average CTC yield per apheresis (mean volume: 59.5 mL) was 12,546 CTCs. Purified single CTCs could be isolated from apheresis product by FACS sorting; copy-number aberration (CNA) profiles of 185 single CTCs from 14 patients revealed the genomic landscape of lethal prostate cancer and identified complex intrapatient, intercell, genomic heterogeneity missed on bulk biopsy analyses.Conclusions: Apheresis facilitated the capture of large numbers of CTCs noninvasively with minimal morbidity and allowed the deconvolution of intrapatient heterogeneity and clonal evolution. Clin Cancer Res; 24(22); 5635-44. ©2018 AACR.

Toward a real liquid biopsy in metastatic breast and prostate cancer: Diagnostic LeukApheresis increases CTC yields in a European prospective multicenter study (CTCTrap).

Frequently, the number of circulating tumor cells (CTC) isolated in 7.5 mL of blood is too small to reliably determine tumor heterogeneity and to be representative as a “liquid biopsy”. In the EU FP7 program CTCTrap, we aimed to validate and optimize the recently introduced Diagnostic LeukApheresis (DLA) to screen liters of blood. Here we present the results obtained from 34 metastatic cancer patients subjected to DLA in the participating institutions. About 7.5 mL blood processed with CellSearch® was used as “gold standard” reference. DLAs were obtained from 22 metastatic prostate and 12 metastatic breast cancer patients at four different institutions without any noticeable side effects. DLA samples were prepared and processed with different analysis techniques. Processing DLA using CellSearch resulted in a 0–32 fold increase in CTC yield compared to processing 7.5 mL blood. Filtration of DLA through 5 μm pores microsieves was accompanied by large CTC losses. Leukocyte depletion of 18 mL followed by CellSearch yielded an increase of the number of CTC but a relative decrease in yield (37%) versus CellSearch DLA. In four out of seven patients with 0 CTC detected in 7.5 mL of blood, CTC were detected in DLA (range 1–4 CTC). The CTC obtained through DLA enables molecular characterization of the tumor. CTC enrichment technologies however still need to be improved to isolate all the CTC present in the DLA.

Abstract A051: Liquid biopsy by apheresis: Molecular characterization of circulating tumor cells and their organoid culture reflects intrapatient heterogeneity and clonal evolution

Abstract Liquid biopsy components from blood, such as cell free DNA (cfDNA) and circulating tumor cells (CTCs), are prognostic for overall survival in advanced prostate cancer patients and allow the study of clonal evolution. cfDNA is easily obtained and has been widely used for molecular characterization and reflects pooled genomic profiles in a patient, but has limitations regarding gene copy number calls. CTC single-cell genomic studies generate precise gene copy number calls and elucidate intrapatient intercellular genomic heterogeneity. The main limitation of CTC analyses has been the low CTC count found in many cancer patients. We elected to study whether liquid biopsy by apheresis in advanced prostate cancer patients increases the yield of CTC to study tumor genomics, intrapatient heterogeneity, and ex vivo organotypic 3D models. Advanced metastatic prostate cancer patients being considered for clinical trials were invited to consent to apheresis. Apheresis CTC counts using CellSearchTM (Menarini) were acquired from 16 patients. The contents of the CellSearch cartridges were sorted into pure single cells by fluorescence-activated cell sorting and subsequently assessed by array comparative genomic hybridization (aCGH, Agilent Technology) for copy number aberrations (CNA). Exome and aCGH from tissue biopsies were compared to the single cell aCGH results. We generated patient-derived organoid (PDOs) cultures from apheresis products by preenrichment using density gradient (Lymphoprep) and subsequent CTC enrichment by EpCAM positive selection (EasySep StemCell Technologies). PDOs were characterized by immunofluorescence (IF) as DAPI+/CK+/EpCAM+ and CD45- cells and subsequently by aCGH for CNA. All sixteen patients (median age of 70 years; range 60-77 years) tolerated apheresis without any adverse effects. CTC counts from peripheral blood (PB) prior to apheresis ranged from 13 to 711 (median = 96), and did not significantly change post apheresis. The estimated CTC yield per apheresis ranged from 660-35473 per apheresis product (median = 3351). This constitutes an increase of 102-fold when compared to median CTC capture from 7.5mL of PB. A total of 170 single CTCs from 15 apheresis patients were genomically profiled and the copy number aberration profiles confirmed prostate cancer with multiple genomic hallmarks including CNAs such as AR amplification, chromosome 8q gain (MYC locus), and PTEN, RB1, BRCA2, TP53, CHD1 loss. CNA profiles of PDOs showed similar genomic aberrations to same patient CTCs and also reflected intrapatient heterogeneity detected by single CTC analysis. In conclusion, apheresis from advanced prostate cancer patients is a well-tolerated procedure and in our study increased the CTC yield by 102-fold when compared to PB. CTC and PDOs from apheresis products shared similar CNA profile compared with tissues biopsies and furthermore gave us an insight of the tumor heterogeneity and clonal evolution. Citation Format: Maryou B.K. Lambros, Veronica Gil, Mateus Crespo, Mariane S. Fontes, Alan Mackay, Gemma Folwer, Gemma Folwer, Berni Ebbs, Rui Neves, Penny Flohr, Susana Miranda, Semini Sumanasuriya, Daniel N. Rodrigues, Rita Pereira, Geroge Seed, Wei Yuan, Joanne Hunt, Deirdre Moloney, Dionne Ayanda, Niven Mehra, Jane Goodall, Claudia Bertan, Suzanne Carreira, Nikolas H. Stoecklein, Leon W.M.M. Terstappen, Gunther Boysen, Joahnn S. De Bono. Liquid biopsy by apheresis: Molecular characterization of circulating tumor cells and their organoid culture reflects intrapatient heterogeneity and clonal evolution [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A051.

Abstract 5584: Circulating tumor cells in the peripheral blood and leukapheresis product of non-small cell lung cancer patients

Abstract Introduction The number of circulating tumor cells (CTC) isolated by the FDA approved CellSearch (CS) system in 7.5 mL of blood of metastatic and non-metastatic non-small cell lung cancer (NSCLC) patients is low or absent. Processing a larger blood volume could yield a greater amount of CTC from these patients. An approach to probe larger volumes is using diagnostic leukapheresis (DLA) where the blood can be split into its components after which selective harvesting of mononuclear cells (MNC) including CTC can take place. Methods Patients with histologically proven NSCLC who either have been recently diagnosed or are eligible for new therapy can participate in this ongoing study. Patients had DLA for MNC collection derived from one total body blood volume. Before and after DLA, whole blood was drawn in a CellSave blood collection tube and analyzed by CS. The obtained DLA product was divided for analysis. 2x108 cells (on average 2mL of DLA product, representing ~100mL of blood) was diluted in 7.5 mL buffer, and analyzed by CS. 9 mL of the DLA product (on average 1x109 cells) was fixed and depleted of leukocytes using the RosetteSep CD45 depletion kit (Stemcell Technologies), allowing processing of a larger volume, followed by CS analysis. Results of molecular characterization of the detected CTC is pending. Results So far, a total of 8 DLA's were performed in 7 stage 4 NSCLC patients. Sample collection took place before (t=0) and/or after (t=1) treatment. Patients underwent DLA in about 110 minutes, at this time an average of 4860 mL of blood was processed. No complications occurred, no post-procedure complaints were recorded. Similar DLA products with an average of 95 mL were obtained. CTC counts for these patient samples are shown in the table below. CTC counts blood and DLAPatientt=#CTC pre blood#CTC post blood#CTC DLA (~2mL, 200x106 cells)#CTC depleted DLA (~9mL, 900x106 cells)101n/a114113020203514602031000040n/a04495000166000029020114 Conclusion DLA is a novel method to collect CTC from the blood. DLA can result in the detection of CTC where the use of 7.5mL of blood is not sufficient. Additionally, DLA shows an increase of CTC yield in patients with CTC detected in 7.5 mL of blood. Citation Format: Kiki C. Andree, Menno Tamminga, Anouk Mentink, T Jeroen Hilterman, Harry J. Groen, Leon W. Terstappen. Circulating tumor cells in the peripheral blood and leukapheresis product of non-small cell lung 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 5584.

Enhancement of Capturing Efficacy for Circulating Tumor Cells by Centrifugation

Circulating tumor cells (CTCs), which are thought to be the main candidate for metastasis, are gaining importance owing to their potential impact on human health and public welfare. For capturing CTCs, antigen-antibody interaction has been used in which specific antigens expressed on cell surface of CTCs can be bound to antibodies immobilized on substrates. Conventional detection methods for CTCs have often suffered not only from relatively low antigen-antibody coupling efficiency but also from cumbersome fabrication processes of micro/nano-structures for CTCs capture. Herein, we report a facile, robust antibody-based CTCs detection technique using centrifugal force, which can enhance the capturing efficacy of CTCs. We validate chemical functionalization process of antibodies on a silica substrate by using atomic force microscopy. Furthermore, it turned out that the centrifugal force from a benchtop centrifuge is enough to produce a ~2.3-fold increase in capture yield of CTCs through enhancement of binding avidity between CTCs and the antibodies. Our result points out the great potential of our method for practical application in CTCs diagnostics and opens a new avenue for biological and chemical sensing.

Abstract P2-01-08: Enumeration of heterogeneous circulating tumor cells (CTCs) using size-based method in early, and metastatic, breast cancer patients

Abstract Background The detection of circulating tumor cells (CTCs) in peripheral blood is an independent predictor of the efficacy of systemic therapy, and also a prognostic marker for patients with metastatic breast cancer. One of the main methods to detect CTCs is CellSearch system, which uses immune-magnetic separation followed by immunocytochemistry. A microdevice (CTChip from ClearCell system) can capture and enumerate CTCs based on distinctive physiological differences (size and deformability) between cancer cells and blood cells. CTChip thus obtains a larger CTC yield than affinity-based separation, which enriches a particular subgroup of cells expressing EpCAM. In this study, we enumerate CTCs in peripheral blood from early and metastatic breast cancer patients using a size-based method. Patients and methods We examined blood samples from a total of 18 early and metastatic breast cancer patients, after obtaining written informed consent. Blood samples were taken in sodium EDTA tubes after discarding the first 1ml of blood from the syringe. Two ml blood samples were applied to CTChip (ClearCell system), and CTCs were eventually trapped in the microwells of the CTChip. Trapped cells were analyzed by immunocytochemistry with monoclonal antibodies specific for leukocytes (CD45) and epithelial cells (CK8/18), along with 4',6-diamidino-2-phenylindole (DAPI) for nuclei: CK8/18-positive, DAPI-positive and CD45-negative cells more than 10 μm in diameter were defined as CTCs. Eight patients were examined using both the CTChip and CellSearch system to compare the yield of CTCs. Results Of 18 patients, 6 were de novo stage IV, 6 were recurrent and 6 were early stage breast cancer patients. Of primary tumors, 8 were HER2- and ER and/or PR +, 6 were HER2-and ER- and PR-, 3 were HER2+ and ER and/or PR +, and one was HER2+ and ER- and PR-. Using CTChip, detected CTCs ranged from 3 - 107 cells/2 ml in all cases: 3 - 83 for early stage, 19 - 156 for stage IV and 21 - 146 for recurrent. The number of CTCs found in recurrent patients tended to be higher than in early stage patients. Size-based method using CTChip clearly showed high sensitivity compared with the CellSearch system, which detected CTCs in only 2 cases out of 8. In analysis by immunochemistry, we found CK-negative, CD45-negative and DAPI positive cells with larger diameter (&amp;gt;16 μm) than CK-positive CTCs in most patients, and the numbers were higher in stage IV (8.5 cells of median value) and recurrent (13 cells) patients than in early stage patients (1.5 cells). Our study suggested that CK-negative large cells might be CTCs with epithelial–mesenchymal transition (EMT). Conclusion This size-based technology enables us to capture CTCs regardless of EpCAM expression. Enumerated CTCs varied in size and positivity of CK8/18, suggesting the heterogeneity of CTCs. Further research, especially focusing on EMT will be crucial to understand the key mechanism of metastasis and drug resistance. Citation Format: Tozuka K, Nagai SE, Kubo K, Komatsu K, Takai K, Inoue K, Matsumoto H, Hayashi Y, Tsuboi M, Yamada Y, Wang X, Suganuma M. Enumeration of heterogeneous circulating tumor cells (CTCs) using size-based method in early, and metastatic, breast cancer patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-01-08.

Whole blood stabilization for the microfluidic isolation and molecular characterization of circulating tumor cells

Precise rare-cell technologies require the blood to be processed immediately or be stabilized with fixatives. Such restrictions limit the translation of circulating tumor cell (CTC)-based liquid biopsy assays that provide accurate molecular data in guiding clinical decisions. Here we describe a method to preserve whole blood in its minimally altered state by combining hypothermic preservation with targeted strategies that counter cooling-induced platelet activation. Using this method, whole blood preserved for up to 72 h can be readily processed for microfluidic sorting without compromising CTC yield and viability. The tumor cells retain high-quality intact RNA suitable for single-cell RT-qPCR as well as RNA-Seq, enabling the reliable detection of cancer-specific transcripts including the androgen-receptor splice variant 7 in a cohort of prostate cancer patients with an overall concordance of 92% between fresh and preserved blood. This work will serve as a springboard for the dissemination of diverse blood-based diagnostics.

Abstract 1713: Transferrin receptor 1 (TfR) as marker for circulating tumor cells (CTCs) identification in NSCLC

Abstract CTCs are used as a surrogate source of tumor material in solid tumors. Clinical applications of CTCs as liquid biopsy comprise the monitoring of molecular alterations during tumor progression and dynamic evaluation of molecular markers of treatment response. The FDA-cleared method to isolate CTCs in cancer patients (Cell Search) is based on positive selection of EpCAM expressing cells. However, this approach performs poorly in non-small cell lung cancer (NSCLC) as it identifies CTCs in only 7% of the subjects, failing to show any prognostic relevance. Down-regulation/loss of epithelial markers to isolate (EpCAM) and identify (cytokeratin, CK) CTCs could in part explain the low CTC yield obtained in NSCLC with approaches based on epithelial markers expression. To overcome this challenge we used size-based CTC enrichment (ISET filters) from NSCLC patients’ peripheral blood. As a positive identifier of CTCs we used transferrin receptor 1 (TfR) which is a cell membrane-associated protein, that mediates intracellular iron uptake, and which is expressed at low levels in many normal tissues but over-expressed in cancer cells. We first analyzed TfR protein expression by immunofluorescence in a panel of NSCLC cell lines and in healthy donor leukocytes. While all NSCLC cells lines analyzed were positive for TfR expression, none of the leukocyte expressed the receptor. Moreover, TfR expression was detected also in EpCAM negative NSCLC cell lines. To determine the clinical applicability of this novel CTC identifier, we determined TfR expression in CTCs isolated from peripheral blood of 35 metastatic NSCLC patients using the ISET filter technology. The isolated CTCs were stained for TfR, CK, CD45 and DAPI. For each patient, one additional ISET filter was stained with Giemsa for morphologic analysis by a pathologist. By using the classic panel of CTC identifiers markers (CK+/CD45-/DAPI+), CTCs were identified in 4/34 (11%) patients, while by using TfR as positive identifier (TfR+/CD45-/DAPI+) CTCs were identified in 31/35 (88%) subjects. The morphologic review of Giemsa stained filters confirmed the presence of tumor cells in 28/34 (82%) samples [0-217 CTCs/sample]. Interestingly, patients with &amp;gt; 6 TfR+ CTCs had a worse overall survival (OS) than patients with &amp;lt; 6 TfR+ CTCs [p=0.048 Log Rank (Mantel-Cox)]. OS did not significantly differ using the same cutoff with CTCs defined based on CK or Giemsa staining. Overall, our data indicate that TfR is a promising biomarker for the detection of CTCs in NSCLC CTCs, superior to CK or EpCAM. Our data also suggest that TfR may potentially identify CTCs subpopulations with a significant prognostic role in NSCLC. We are currently isolating TfR+ CTCs from early stage and metastatic NSCLC patients for further molecular characterization and determination of clinical significance. Citation Format: Giuseppe Galletti, Galatea Kallergi, Ashish Saxena, Despoina Aggouraki, Christos Stournaras, Vassilis Georgoulias, Timothy E. McGraw, Nasser Altorki, Paraskevi Giannakakou. Transferrin receptor 1 (TfR) as marker for circulating tumor cells (CTCs) identification in NSCLC [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 1713. doi:10.1158/1538-7445.AM2017-1713

Abstract 993: Diagnostic leukapheresis (DLA): Molecular characterisation and organoid culture of circulating tumor cells (CTC) from metastatic castration resistant prostate cancer (mCRPC)

Abstract Introduction: CTC count is an independent predictor of overall survival in mCRPC. Isolation of CTC from peripheral blood (PB) for genomic and functional analysis is challenging, especially in patients (pts) with low CTC count. It has been shown that DLA increases CTC yield. However, it has yet to be proven whether CTC isolation from DLA can be used in complementary studies such as molecular characterization and growth of organoid culture for drug sensitivity studies. Here we present preliminary data of an on-going study, which evaluates DLA in mCRPC pts, focusing on safety, CTC enrichment, molecular characterization and feasibility for organoid culture. Methods: mCRPC pts considered for clinical trials were selected according to performance status (ECOG 0-1) and number of CTC found in 7.5ml PB (&amp;gt;20 cells/7.5mL). DLA products (200x106 cells) were processed using the CellSearch CTC kit (Janssen Diagnostics, LLC) according to manufacturer procedures. The contents of CellSearch cartridges were sorted into single cell by fluorescence activated cell sorting (FACS) and subsequently assessed by array comparative genomic hybridization (aCGH) for copy number aberrations (CNA). Enrichment of CTC for organoid culture was performed by density gradient of mononuclear cells followed by positive selection using magnetic beads. Results: Overall 12 mCRPC patients underwent DLA without any complication or toxicity. The mean CTC count was 90 CTC/7.5 ml peripheral blood (median = 31) and ranged from 20 to 324. CellSearch CTC count in the DLA yielded a mean of 466 (median=203) and ranged from 60 to 2496 with an up to 40-fold increase (mean = 13, median = 6) in CTC count separation when comparing 1mL of PB to 1mL of DLA. Molecular analyses of FACS single CTC from the DLA by aCGH showed that these CTC genomic profiles had the typical hallmarks of mCRPC with CNAs including AR and MYC locus (8q) amplification, and PTEN, RB1, TP53, CHD1 loss. Additionally, ex vivo culture of CTC-derived organoids was successfully achieved. aCGH of these organoids matched the genomic profile that of the CTC from the same patient. Conclusion: DLA from mCRPC pts was well tolerated and yields higher CTC capture than PB and may provide an alternative to tissue biopsy and routine blood volumes. Our strategy allowed us to isolate genomic DNA with good quality for molecular characterization and viable CTC for organoid culture and functional studies. Citation Format: Maryou B. Lambros, Veronica S. Gil, Mateus Crespo, Mariane S. Fontes, Rui N. Neves, Niven Mahra, Gemma Fowler, Berni Ebbs, Penny Flohr, George Seed, Wei Yuan, Joanne Hunt, Deirdre Moloney, Dionne Ayanda, Joost F. Swennenhuis, Kiki C. Andree, Semini Sumanasuriya, Matthew Clarke, Pasquale Rescigno, Zafeiris Zafeiriou, Joaquin Mateo, Diletta Bianchini, Nikolas H. Stoecklein, Leon W. Terstappen, Gunther Boysen, Johann S. De Bono. Diagnostic leukapheresis (DLA): Molecular characterisation and organoid culture of circulating tumor cells (CTC) from metastatic castration resistant prostate cancer (mCRPC) [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 993. doi:10.1158/1538-7445.AM2017-993

Abstract 3787: EpCAM- and EpCAM+ circulating tumor cells in metastatic prostate and breast cancer patients: a multicenter study

Abstract Introduction: The presence of circulating tumor cells (CTC) enumerated by the CellSearch system in blood of patients with cancers of epithelial origin is strongly associated with a poor prognosis of these patients. CTC are enriched by targeting the EpCAM antigen, raising the question which EpCAM subtypes are present as well in patients. In the EU-FP7 CTCTrap program, we investigated the presence of EpCAM- CTC in blood samples after depletion of EpCAM+ CTC by CellSearch. Studies were performed and validated at the participating laboratories by distribution and analysis of blood samples spiked with cancer cells and by testing blood from 73 metastatic prostate and 22 metastatic breast cancer patients. Methods: Blood samples were processed according to the standard operating procedures and tools developed in the program (https://www.utwente.nl/tnw/mcbp/protocolsandtools/). First, CellSearch was performed for EpCAM+ CTC, followed by filtration and fluorescent labeling of the blood discarded by CellSearch for EpCAM- CTC. To validate the procedures across the 6 participating clinical sites, 3x 3 tubes with aliquots of healthy donor blood, spiked with either PC3, MDA-MB-231 or no cells, were prepared at one site and shipped to all other sites for simultaneous processing. Metastatic prostate and breast cancer patients were processed with the same procedures. Results: 27% of PC3 cells were recovered by CellSearch and 21% by filtration, leaving 52% unaccounted for. For MDA, 26% were recovered by CellSearch and 18% by filtration, leaving 56% accounted for. Differences in recovery between sites were not significant. In patients both EpCAM+ and EpCAM- CTC were detected (see table). Conclusion: In a multicenter study EpCAM+ and EpCAM- CTC were present in blood of metastatic prostate and breast cancer patients. Spiking experiments showed that the developed methods can be further improved to increase the CTC yield. Molecular characterization of the CTC subtypes and relation with clinical outcome is ongoing. % metastatic cancer patients with EpCAM+ and EpCAM- CTC% prostate cancer patients (n=73)% breast cancer patients (n=22)CellSearch CTC EpCAM+CellSearch CTC EpCAM+# CTC01-4&amp;gt;4Total01-4&amp;gt;4TotalMicrosieve CTC EpCAM-011819381859321-4671629991432&amp;gt;48619331414936Total252154412732 Citation Format: Sanne de Wit, Kiki C. Andree, Joost F. Swennenhuis, Elisabetta Rossi, Mariangela Manicone, Riccardo Vidotto, Rita Zamarchi, Marianna Alunni-Fabbroni, Elisabeth K. Trapp, Marie Tzschaschel, Brigitte Rack, Rita Lampignano, Hans Neubauer, Tanja Fehm, Marianne Oulhen, Emeline Colomba, Françoise Farace, Mateus Crespo, Penelope Flohr, Gemma Fowler, Mariane Sousa Fontes, Johann S. de Bono, Leon WMM Terstappen. EpCAM- and EpCAM+ circulating tumor cells in metastatic prostate and breast cancer patients: a multicenter study [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 3787. doi:10.1158/1538-7445.AM2017-3787

Optimizing the Detection of Circulating Markers to Aid in Early Lung Cancer Detection.

Improving early detection of lung cancer is critical to improving lung cancer survival. Studies have shown that computerized tomography (CT) screening can reduce mortality from lung cancer, but this involves risks of radiation exposure and can identify non-cancer lung nodules that lead to unnecessary interventions for some. There is a critical need to develop alternative, less invasive methods to identify patients who have early-stage lung cancer. The detection of circulating tumor cells (CTCs) are a promising area of research, but current technology is limited by a low yield of CTCs. Alternate studies are investigating circulating nucleic acids and proteins as possible tumor markers. It is critical to develop innovative methods for early lung cancer detection that may include CTCs or other markers that are low-risk and low-cost, yet specific and sensitive, to facilitate improved survival by diagnosing the disease when it is surgically curable.

Abstract P2-02-20: Enumeration of heterogeneous circulating tumor cells (CTCs) in metastatic breast cancer patients based on size and deformability

Abstract Background : The detection of circulating tumor cells (CTCs) in peripheral blood is an independent predictor of the efficacy of systemic therapy and a prognostic marker for patients with metastatic breast cancer. One of the leading techniques to detect CTCs uses immune-magnetic separation followed by immunocytochemistry. A microdevice can capture and enumerate CTCs using distinctive physiological difference (size and deformability) between cancer cells and blood cells. This microdevice thus obtains a larger CTC yield than that of affinity based separation which enriches the samples from a particular subgroup of cells based on biomarker (EpCAM) used. In this study, we investigated CTCs in peripheral blood from metastatic breast cancer patients using this microdevice. Patients and methods: We examined blood samples of 9 patients with heavily treated locally recurrent or metastatic breast cancer. Informed consent from these patients was obtained before blood extraction. Blood samples were taken into sodium EDTA tubes after discarding the first 1ml of blood samples. Two ml whole blood were subjected to the microdevice (Clear cell system), and CTCs were trapped in the microwells: Trapped cells were analyzed by immunocytochemistry with monoclonal antibodies specific for leukocytes (CD45) and epithelial cells (CK8/18), along with 4,2-diamidino-2-phenylndole dihydrochloride (DAPI) for nuclei. CK8/18- positive, DAPI-positive and CD45-negative cells were defined as CTCs. Three patients were examined using both this microdevice and affinity-based separation with EpCAM, to compare the yield of CTCs. Results: Of the 9 patients: 7 had ER-positive primary tumors, and 6 had PgR-positive ones, HER2 overexpression was detected in 2 primary tumors. CTCs were detected in 8 patients. The single patient in whom CTCs were not detected suffered from local recurrence (axillary lymph node metastasis) only, with no distant metastases. We were also unable to detect CTCs using EpCAM affinity method for this patient. The number of detected CTCs in the other patients ranged from 19/2ml to 156/2ml (mean 90/2ml), and the sizes of CTCs varied from 5 to 16μm. CK8/18-negative and DAPI positive were detected in most patients, and these cells tended to be larger than CK8/18-positive cells, suggesting that epithelial–mesenchymal transition (EMT) might occur in CTCs. The total number of CTCs detected by the microdevice from 2 patients was larger than that of CTCs detected by EpCAM affinity method (107/2ml vs 1/7.5ml, and 19/2ml vs 39/7.5ml). Conclusion: CTCs detected by this microdevice varied in regard to the size of trapped cells and characteristics examined by immunochemistry, suggesting the heterogeneity of CTCs. Further research on this heterogeneity is vital in order to develop personalized treatment for patients with metastatic breast cancer. Citation Format: Tozuka K, Nagai SE, Inoue K, Komatsu K, Matsumoto H, Hayashi Y, Kurozumi S, Suganuma M. Enumeration of heterogeneous circulating tumor cells (CTCs) in metastatic breast cancer patients based on size and deformability. [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 P2-02-20.