Clinical Circulating Tumor Cells Research Articles

Simplified Rapid Enrichment of CTCs and Selective Recognition Prereduction Enable a Homogeneous ICP-MS Liquid Biopsy Strategy of Lung Cancer.

The effective enrichment and hypersensitivity analysis of circulating tumor cells (CTCs) in clinical whole blood samples are highly significant for clinical tumor liquid biopsy. In this study, we established an easy operation and affordable CTCs extraction technique while simultaneously performing the homogeneous inductively coupled plasma mass spectrometry (ICP-MS) determination of CTCs in lung cancer clinical samples based on selective recognition reactions and prereduction phenomena. Our strategy allowed for the pretreatment of whole blood samples in less than 45 min after step-by-step centrifugation, which only required lymphocyte separation solution and erythrocyte lysate. Furthermore, a three-stage signal amplification system consisting of catalytic hairpin assembly (CHA), selective recognition for C-Ag+-C structures and Ag+ of copper sulfide nanoparticles (CuS NPs), and prereduction of Hg2+ through ascorbic acid (AA) was constructed by using mucin 1 as the CTCs marker and the aptamer for identification probes. In optimal conditions, the detection limits of ICP-MS were as low as 0.3 ag/mL for mucin 1 and 0.25 cells/mL for A549 cells. This method analyzed CTCs in 58 clinical samples quantitatively, and the results were consistent with clinical CT images and pathological findings. The area under the curve (AUC) value of the receiver operating characteristic (ROC) curve was 0.957, which provided a specificity of 100% and a sensitivity of 91.5% for the assay. Therefore, the simplicity of the extraction method, the accessibility, and the high sensitivity of the assay method make the strategies attractive for clinical CTCs testing applications.

PD36-02 A COMPOSITE BIOMARKER APPROACH TO SPARE NEOADJUVANT CHEMOTHERAPY IN SELECT MUSCLE-INVASIVE BLADDER CANCER PATIENTS

You have accessJournal of UrologyCME1 Apr 2023PD36-02 A COMPOSITE BIOMARKER APPROACH TO SPARE NEOADJUVANT CHEMOTHERAPY IN SELECT MUSCLE-INVASIVE BLADDER CANCER PATIENTS Joep De Jong, A. Gordon Robertson, Nick Beije, John Martens, Ellen Zwarthoff, Arno Van Leenders, Ewan Gibb, and Joost Boormans Joep De JongJoep De Jong More articles by this author , A. Gordon RobertsonA. Gordon Robertson More articles by this author , Nick BeijeNick Beije More articles by this author , John MartensJohn Martens More articles by this author , Ellen ZwarthoffEllen Zwarthoff More articles by this author , Arno Van LeendersArno Van Leenders More articles by this author , Ewan GibbEwan Gibb More articles by this author , and Joost BoormansJoost Boormans More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003334.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Clinical guidelines recommend neoadjuvant chemotherapy (NAC) for the treatment of muscle-invasive bladder cancer (MIBC). However, NAC prior to radical cystectomy (RC) is associated with treatment-related toxicity and confers a modest survival benefit. Here, we aimed to identify MIBC patients who may avoid NAC by combining circulating tumor cell status and molecular subtypes in a composite biomarker approach. METHODS: TURBT tissue specimen were collected from clinical stage T2-T4aN0-N1M0 MIBC cases that were included within the CirGuidance study (NL3954), a prospective trial that analyzed circulating tumor cell (CTC) status in a patient’s blood sample using the CELLSEARCH system. For the present study, transcriptome-wide expression profiling was performed on 234 TURBT samples using an array-based approach. Molecular subtypes, long non-coding RNA (lncRNA) based FGFR3+ status and gene signatures were determined as described previously (de Jong et al., Genome Med. 2019). The primary endpoint of this study was cancer-specific mortality (CSM), calculated as the date of study inclusion till date of bladder cancer related death. Median follow-up was 28.8 (IQR: 16.6-40.2) months. RESULTS: Of 234 patients, 21 (9%) were treated with NAC and RC, while 213 (91%) received RC alone. A CTC-negative status was observed in 172 (81%) of RC-only cases. Molecular subtyping identified 28 luminal FGFR3+ cases with high FGFR3, SHH and p53 pathway activity, and lower EMT hallmark scores. Adjusting for clinical risk factors, both CTC and FGFR3+ status were significant predictors for cancer specific mortality on MVA (p<0.05). Of interest, the subgroup of FGFR3+ cases that were CTC-negative (N=26) showed most favorable outcomes with only one CSM event after a median of 33.4 (IQR: 24.8-44.5) months of follow-up. CONCLUSIONS: Using a composite biomarker approach of blood-based CTC status and molecular subtyping, we identified a biologically distinct subgroup of MIBC with favorable prognosis after RC-only, validating the performance of a previously developed lncRNA based FGFR3+ classifier. Clinical trials which withhold NAC from CTC-negative, FGFR3+ MIBC patients are warranted. Source of Funding: Gene expression profiles were generated using the Decipher Bladder assay © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e980 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joep De Jong More articles by this author A. Gordon Robertson More articles by this author Nick Beije More articles by this author John Martens More articles by this author Ellen Zwarthoff More articles by this author Arno Van Leenders More articles by this author Ewan Gibb More articles by this author Joost Boormans More articles by this author Expand All Advertisement PDF downloadLoading ...

Association of circulating tumor cells and IMP3 expression with metastasis of osteosarcoma.

Circulating tumor cells (CTCs) have been identified as a prognostic biomarker of tumors such as breast cancer and nasopharyngeal carcinoma, because they are obtained through a simple and noninvasive blood draw or liquid biopsy, but its clinical significance in osteosarcoma is still unclear. In this study, we analyzed the relationship between CTCs and clinicopathological features and discussed whether CTCs could be used as a biomarker for metastasis in osteosarcoma. We enrolled 50 osteosarcoma patients with Enneking Stage IIB and Stage III and detected CTCs in 5ml of peripheral blood samples collected from patients using the Canpatrol® CTC detection platform. Subsequently, multiplex RNA in situ hybridization (RNA-ISH) based on various molecular markers was performed to identify and classify CTCs. The relationships between clinical pathological features and CTC counts, subtypes (epithelial type, E type; hybrid epithelial/mesenchymal type, H type; mesenchymal type, M type), and insulin-like growth factor mRNA-binding protein 3 (IMP3) expression in CTCs were analyzed. CTCs were detected in 86% (43/50) of the osteosarcoma patients. The CTC counts, especially the total CTCs and H-type CTCs, signifcantly differed between Enneking Stage IIB and Stage III patients (P < 0.05). No significant differences were observed between the CTC count or type and other clinicopathological features (P > 0.05). There were significant differences in the expression of IMP3 in different types of CTCs, and the IMP3 positive rates in E/H/M type CTCs were 38.4, 65.6, and 62.0%, respectively (P < 0.001). Receiver operating characteristic (ROC) curve analysis showed that IMP3-positive CTC count had the best performance for diagnostic metastasis, with the largest area under the curve of 0.873 and cutoff value of four cells/5ml blood (sensitivity = 87.5%; specificity = 82.4%). Serial CTC monitoring in one patient suggested that total CTCs and H-type CTCs were associated with disease progression. This study demonstrates that the CTCs, especially the IMP3-positive CTCs and H/M-type CTCs, are related to the metastasis of osteosarcoma.

Intelligent Tumor Cell Detection Method Based on Circulating Tumor Cell (CTC) Technology

&lt;p&gt;Cancer has become one of the greatest threats for human life. Doctors can get original images of the sick organs with the assistance of medical technology. However, manual interpretation of the original images is time consuming and labor consuming. Nowadays, the intelligent detection of tumor cell images is commonly adopted in cancer diagnosis. In this paper, we propose Imporved Selective Search (ISS) algorithm and CTCNet based on Circulating Tumor Cell (CTC) technology to improve the cancer images&amp;rsquo; detection efficiency. CTCs can be collected by a sampling needle with EpCAM antibody which can specifically bind to tumor cells. After fluorescent staining process, images obtained from sampling needle will be processed by the ISS algorithm for candidate region preselection. All of the eligible areas are evaluated with a self-designed neural network called CTCNet, resulting in efficient recognition of circulating tumor cells. During this process, all algorithms are accelerated through the GPU and NPU hardware platform, which further improves the detection speed of the system. In the experiment, we first verify the efficiency of proposed ISS algorithm by compared with Original Selective Search (OSS), we found that the number of candidate boxes reduced from 549 to 16 and the time consuming reduced by 0.3s after adopting the ISS algorithm. In order to evaluate the performance of the proposed 7-layer CTCNet, we compared CTCNet with SVM, BP neural network, AlexNet and VGGNet by using the dataset of 12312 samples from 30 patients, among them, there were 12 patients with early cancer and 18 patients with advanced cancer. And we got the highest recognition accuracy of 97.95% on CTCNet, which even beyond the VGGNet with deeper layers. In contrast to other combinations, we detect CTC in diverse clinical CTC images, the joint application of ISS algorithm and CTCNet achieves an outstanding system performance with accuracy up to 94.03% in the whole view images. Meanwhile, because of the application of a lightweight network in different hardware acceleration platform, the detection time of the CTC image in a single view can be less than 12s.&lt;/p&gt; &lt;p&gt;&amp;nbsp;&lt;/p&gt;

Circulating Tumor Cell-Based Messenger RNA Scoring System for Prognostication of Hepatocellular Carcinoma: Translating Tissue-Based Messenger RNA Profiling Into a Noninvasive Setting.

Numerous studies in hepatocellular carcinoma (HCC) have proposed tissue-based gene signatures for individualized prognostic assessments. Here, we develop a novel circulating tumor cell (CTC)-based transcriptomic profiling assay to translate tissue-based messenger RNA (mRNA) signatures into a liquid biopsy setting for noninvasive HCC prognostication. The HCC-CTC mRNA scoring system combines the NanoVelcro CTC Assay for enriching HCC CTCs and the NanoString nCounter platform for quantifying the HCC-CTC Risk Score (RS) panel in enriched HCC CTCs. The prognostic role of the HCC-CTC RS was assessed in The Cancer Genome Atlas (TCGA) HCC cohort (n=362) and validated in an independent clinical CTC cohort (n=40). The HCC-CTC RS panel was developed through our integrated data analysis framework of 8 HCC tissue-based gene signatures and identified the top 10 prognostic genes (discoidin domain receptor tyrosine kinase 1 [DDR1], enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase [EHHADH], androgen receptor [AR], lumican [LUM], hydroxysteroid 17-beta dehydrogenase 6[HSD17B6], prostate transmembrane protein, androgen induced 1 [PMEPA1], tsukushi, small leucine rich proteoglycan [TSKU], N-terminal EF-hand calcium binding protein 2 [NECAB2], ladinin 1 [LAD1], solute carrier family 27 member 5 [SLC27A5]) highly expressed in HCC with low expressions in white blood cells. The panel accurately discriminated overall survival in TCGA HCC cohort (hazard ratio [HR], 2.0; 95% confidence interval [CI], 1.4-2.9). The combined use of the scoring system and HCC-CTC RS panel successfully distinguished artificial blood samples spiked with an aggressive HCC cell type, SNU-387, from those spiked with PLC/PRF/5 cells (P=0.02). In the CTC validation cohort (n=40), HCC-CTC RS remained an independent predictor of survival (HR, 5.7; 95% CI, 1.5-21.3; P=0.009) after controlling for Model for End-Stage Liver Disease score, Barcelona Clinic Liver Cancer stage, and CTC enumeration count. Our study demonstrates a novel interdisciplinary approach to translate tissue-based gene signatures into a liquid biopsy setting. This noninvasive approach will allow real-time disease profiling and dynamic prognostication of HCC.

ViBiBa: Virtual BioBanking for the DETECT multicenter trial program - decentralized storage and processing

BackgroundLiquid Biopsy (LB) in the form of e.g., circulating tumor cells (CTCs) is a promising non-invasive approach to support current therapeutic cancer management. However, the proof of clinical utility of CTCs in informing therapeutic decision-making for e.g., breast cancer in clinical trials and associated translational research projects is facing the issues of low CTC positivity rates and low CTC numbers – even in the metastasized situation. To compensate for this dilemma, clinical CTC trials are designed as large multicenter endeavors with decentralized sample collection, processing and storage of products, making data management highly important to enable high-quality translational CTC research. AimIn the DETECT clinical CTC trials we aimed at developing a custom-made, browser-based virtual database to harmonize and organize both decentralized processing and storage of LB specimens and to enable the collection of clinically meaningful LB sample. MethodsViBiBa processes data from various sources, harmonizes the data and creates an easily searchable multilayered database. ResultsAn open-source virtual bio-banking web-application termed ViBiBa was created, which automatically processes data from multiple non-standardized sources. These data are automatically checked and merged into one centralized databank and are providing the opportunity to extract clinically relevant patient cohorts and CTC sample collections. SummaryViBiBa, which is a highly flexible tool that allows for decentralized sample storage of liquid biopsy specimens, facilitates a solution which promotes collaboration in a user-friendly, federalist and highly structured way. The source code is available under the MIT license from https://vibiba.com or https://github.com/asperciesl/ViBiBa

Abstract PO036: CXCL12 embedded-Hyaluronate based 'pseudo niche': a new device for CTCs/DTCs capturing and characterization

Abstract Aim: Tumor cells can prime distant microenvironment building a pre-metastatic niche (PMN) where circulating tumor cells (CTCs) will home. 0.01% of CTCs, endowed with the capability to extravasate and infiltrate distant sites, will develop distant metastasis. In this process, a crucial role is played by chemokine gradients. Highly tumorigenic CTCs express the chemokine receptor CXCR4. CXCR4 ligand, CXCL12, attracts bone marrow-derived cells and CXCR4+ cancer cells in pre-metastatic sites to form a PMN. A CXCL12-loaded-hydrogel (CLG) was developed with the commercially available Belotero Hyaluronic gel dermal filler to develop a “pseudo niche” attracting immune and CTCs-CXCR4+cells. Aims: 1. Biological characterization of CTCs, 2. CTC/tumor microenvironment interplay 3. feasibility for clinical CTC isolation and characterization. Materials and methods: Human renal cancer cells SN12C (100 and 500) and A498 (500 and 1000) were seeded on 150ul Empty Gels (EG) or CLG [300ng/ml CXCL12], and allowed to migrate within gels, then fixed and DAPI-stained for enumeration or recovered by gel digestion and grown in complete media for 6 days. CXCR4 expression and colonies formation capability were evaluated. 100 SN12C or HT29, human colon cancer cells, were spiked in 7cc HD blood, ficoll-paqued and the mixture seeded on CLG. Isolated cells were fixed and stained with anti-hCD45-FITC, panCK-594 and DAPI (cancer cells identified as DAPI+/pan-CK+/CD45-). 7cc blood derived from 15 metastatic cancer patient’s (colon, lung, ovary, endometrial, kidney and glioblastoma) will be processed as previously described (CTCs identified as DAPI+/pan-CK+/CD45-); as comparison, CTCs will be isolated and enumerated using the commercially available Screen Cell™ filters according to manufacturer’s instructions. Results: A498 and SN12C cells efficiently infiltrate CLG as compared to empty gel (EG) (CLG/EG fold increase 1.6-1.9). SN12C and HT29 EG/CLG-recovered cells developed colonies after 6 days of culture. HT29-CLG recovered cells overexpressed CXCR4 compared to plastic grown cells and to EG cells and originates a higher number of colonies compared to plastic grown cells (171±21 for CLG VS 131±8 for plastic grown). SN12C and HT29 cells were successfully recovered in 7cc HD blood (recovery rate 20 and 14% respectively). Clinical trial on feasibility in isolating CTC cells on CLG recently started patient’s recruitment in ovarian, colon, lung and renal cancer metastatic patients. Conclusion: CLG-device mimics a PMN for capturing CTCs able to extravasate and infiltrate distant tissue, and potentially, more metastatic. CLG-isolated cells highly express CXCR4, develop higher number of colonies and migrate toward CXCL12. Moreover, the device allows cancer cell recovery from HD blood being potentially clinically useful for CTCs identification, characterization and trapping in cancer patients. Citation Format: Luigi Portella, Caterina Ieranò, Giulia Bertolini, Crescenzo D'Alterio, Giuseppina Rea, Sara Santagata, Anna Maria Trotta, Gelsomina Monaco, Roberto Pacelli, Stefania Scala. CXCL12 embedded-Hyaluronate based 'pseudo niche': a new device for CTCs/DTCs capturing and characterization [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO036.

Engineered multifunctional metal-phenolic nanocoatings for label-free capture and "self-release" of heterogeneous circulating tumor cells.

Immunomagnetic beads have been widely explored as an important analytical tool for the rapid and sensitive detection of circulating tumor cells (CTCs). However, their clinical application is seriously hindered by the tedious preparation procedures and heterogeneous nature of CTCs. To this end, a designed multifunctional platform named Fe3O4@TA/CuII superparamagnetic nanoparticles (SPMNPs) is expected to have the following features: (i) the formation of a tannic acid-copper (II) ion (TA/CuII) coating which could be accomplished by a one-step method is very simple; (ii) the TA/CuII coating shows high affinity for heterogeneous CTCs and good resistance to nonspecific adhesion of blood cells; (iii) "self-release" of the captured cells could be achieved as the TA/CuII coating gradually degrades in the cell culture environment without any additional interventions. Therefore, the resulting Fe3O4@TA/CuII SPMNPs could capture various CTCs (MCF-7, HepG2 and HeLa cells) with different expression levels of the epithelial cell adhesion molecule (EpCAM). And the capture efficiency and cell purity can reach 88% and 87%, respectively. In addition, 68% of the captured cells are self-released after 6 h of incubation and most of the released cells show high cell proliferation activity. In particular, Fe3O4@TA/CuII SPMNPs can successfully detect 1-13 CTCs from 1 mL of blood of 14 patients with 6 types of cancers. Hence, we expect that the as-prepared Fe3O4@TA/CuII SPMNPs with simple, efficient, and universal yet cost-efficient characteristics could act as a promising analytical tool for clinical CTC detection.

Natural Fish Trap-Like Nanocage for Label-Free Capture of Circulating Tumor Cells.

Nanomaterials have achieved several breakthroughs in the capture of circulating tumor cells (CTCs) over the past decades. However, artificial fabrication of label‐free nanomaterials used for high‐efficiency CTC capture is still a challenge. Through billions of years of evolution and natural selection, various complicated and precise hierarchical structures are developed. Here, a novel fish trap‐like “nanocage” structure derived from the natural Chrysanthemum pollen is reported and a nanocage‐featured film for the label‐free capture of CTCs and CTC clusters is constructed. The nanocage‐featured film effectively captures 92% rare cancer cells with a broad spectrum of cancer types, due to the synergistic effect of nanocage‐CTC filopodia matching, high contact area, and strong adhesion force between the cancer cells and the nanocage. Furthermore, the nanocage‐featured film successfully detects CTCs and CTC clusters in 2 or 4 mL blood taken from 21 cancer patients (stages I–IV) suffering from various types of cancers. This novel, abundant, and economical fish trap‐like “nanocage” may provide new perspectives for the application of natural nanomaterials in clinical CTC capture and analysis.

Blood-based gene expression signature associated with metastatic castrate-resistant prostate cancer patient response to abiraterone plus prednisone or enzalutamide.

Precision medicine approaches for managing patients with metastatic castrate-resistant prostate cancer (mCRPC) are lacking. Non-invasive approaches for molecular monitoring of disease are urgently needed, especially for patients suffering from bone metastases for whom tissue biopsy is challenging. Here we utilized baseline blood samples to identify mCRPC patients most likely to benefit from abiraterone plus prednisone (AAP) or enzalutamide. Baseline blood samples were collected for circulating tumor cell (CTC) enumeration and qPCR-based gene expression analysis from 51 men with mCRPC beginning treatment with abiraterone or enzalutamide. Of 51 patients (median age 68 years [51-82]), 22 received AAP (abiraterone 1000 mg/day plus prednisone 10 mg/day) and 29 received enzalutamide (160 mg/day). The cohort was randomly divided into training (n = 37) and test (n = 14) sets. Baseline clinical variables (Gleason score, PSA, testosterone, and hemoglobin), CTC count, and qPCR-based gene expression data for 141 genes/isoforms in CTC-enriched blood were analyzed with respect to overall survival (OS). Genes with expression most associated with OS included MSLN, ARG2, FGF8, KLK3, ESRP2, NPR3, CCND1, and WNT5A. Using a Cox-elastic net model for our test set, the 8-gene expression signature had a c-index of 0.87(95% CI [0.80, 0.94]) and was more strongly associated with OS than clinical variables or CTC count alone, or a combination of the three variables. For patients with a low-risk vs. high-risk gene expression signature, median OS was not reached vs. 18 months, respectively (HR 5.32 [1.91-14.80], p = 0.001). For the subset of 41 patients for whom progression-free survival (PFS) data was available, the median PFS for patients with a low-risk vs high-risk gene expression signature was 20 vs. 5 months, respectively (HR 2.95 [1.46-5.98], p = 0.003). If validated in a larger prospective study, this test may predict patients most likely to benefit from second-generation antiandrogen therapy.

The Role of Proteoglycans in Cancer Metastasis and Circulating Tumor Cell Analysis.

Circulating tumor cells (CTCs) are accessible by liquid biopsies via an easy blood draw. They represent not only the primary tumor site, but also potential metastatic lesions, and could thus be an attractive supplement for cancer diagnostics. However, the analysis of rare CTCs in billions of normal blood cells is still technically challenging and novel specific CTC markers are needed. The formation of metastasis is a complex process supported by numerous molecular alterations, and thus novel CTC markers might be found by focusing on this process. One example of this is specific changes in the cancer cell glycocalyx, which is a network on the cell surface composed of carbohydrate structures. Proteoglycans are important glycocalyx components and consist of a protein core and covalently attached long glycosaminoglycan chains. A few CTC assays have already utilized proteoglycans for both enrichment and analysis of CTCs. Nonetheless, the biological function of proteoglycans on clinical CTCs has not been studied in detail so far. Therefore, the present review describes proteoglycan functions during the metastatic cascade to highlight their importance to CTCs. We also outline current approaches for CTC assays based on targeting proteoglycans by their protein cores or their glycosaminoglycan chains. Lastly, we briefly discuss important technical aspects, which should be considered for studying proteoglycans.

Abstract 5380: An integrated platform for the clinical detection and molecular profiling of single circulating tumor cells

Abstract Introduction: Detection of circulating tumor cells (CTCs) and characterization of CTCs at single-cell resolution may benefit the clinical management of cancer patients. To make the CTC analysis a “bench-to-bedside” technology, we developed an integrated platform named ChimeraX®-i120 system for the high-throughput, automated, and clinical applicable CTC sorting and downstream single-cell genomic profiling. Methods: ChimeraX®-i120 system is designed based on negative enrichment strategy, integrating blood sample pre-processing, immunofluorescence staining, artificial intelligence (AI)-assisted CTC identification. CTCs enriched by this system is compatible with downstream single-cell whole genome sequencing (WGS) analysis. Performance of CTCs detection was evaluated by a series of analytical experiments, clinical utility was further verified. We validated our downstream molecular profiling pipeline of single CTC. Results: Analytical experiments showed that this system was featured with high accuracy, repeatability, sensitivity and anti-interference capability for CTC detection, achieving an average recovery rate of the spiked cells in 71.3%-83.2% (R2= 0.99). In clinical samples, this system effectively detected CTCs and CTC clusters (CTM) in 5 mL blood from cancer patients. The proportion of patients that were positive for CTCs (EpCAM+/pan-CK++CD45-+DAPI+), was 59.3% (86/145, avg. cell count 1.38 ± 2.01) for hepatocellular carcinoma (HCC), 63.2% (24/38, 1.42 ± 1.75) for intrahepatic cholangiocarcinoma (ICC), 63.6% (28/44, avg. 2.52 ± 3.93) for breast cancer (BRC), 61.5% (24/39, avg. 1.62 ± 2.11) in colorectal cancer (CRC), 78.9% (15/19, avg. 2.74 ± 4.46) in lung cancer (LC). Only 1 of 125 (0.8%) healthy volunteers had detectable CTC. The prognostic value of the system was assessed in HCC. Preoperative CTC ≥1/5mL blood was significantly associated with a higher 1-year recurrence rate (38.9% vs. 27.6%, P=0.042) in HCC patients after curative surgery. Low-pass WGS analysis was performed on CTCs and paired tumor tissues of 3 ICC, 3 HCC and 4 BRC. We observed concordance in CNAs (10%-61%) between CTCs and tissues. CTC profiling identified diverse intra- and inter-patient heterogeneity in CNAs pattern. Exclusive CNA patterns were identified in CTCs but not tissue, and vice versa. Multiple well-known oncogenes (e.g., BRAF, EGFR, BRCA1/2, MYC and MET) and tumor suppressor genes (e.g., FOXA1 and TRAF3) could be identified from CNAs in CTCs. For example, in one BRC patient, potentially actionable alterations of BRC such as BRAF, EGFR, MET, ERBB2, PIK3CA and MYC were only identified in CTCs but not tissues. Conclusion: Our results highlight the potential clinical utility of ChimeraX®-i120 system in both CTC detection and downstream genomic profiling. Genomic sequencing of CTCs allows blood-based tumor profiling in greater fraction of patients for clinical decision making. Citation Format: Pengxiang Wang, Yunfan Sun, Weixiang Jin, Haixiang Peng, Jian Zhou, Jia Fan, Xinrong Yang. An integrated platform for the clinical detection and molecular profiling of single circulating tumor cells [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 5380.

Nano Meets Micro-Translational Nanotechnology in Medicine: Nano-Based Applications for Early Tumor Detection and Therapy.

Nanomaterials have great potential for the prevention and treatment of cancer. Circulating tumor cells (CTCs) are cancer cells of solid tumor origin entering the peripheral blood after detachment from a primary tumor. The occurrence and circulation of CTCs are accepted as a prerequisite for the formation of metastases, which is the major cause of cancer-associated deaths. Due to their clinical significance CTCs are intensively discussed to be used as liquid biopsy for early diagnosis and prognosis of cancer. However, there are substantial challenges for the clinical use of CTCs based on their extreme rarity and heterogeneous biology. Therefore, methods for effective isolation and detection of CTCs are urgently needed. With the rapid development of nanotechnology and its wide applications in the biomedical field, researchers have designed various nano-sized systems with the capability of CTCs detection, isolation, and CTCs-targeted cancer therapy. In the present review, we summarize the underlying mechanisms of CTC-associated tumor metastasis, and give detailed information about the unique properties of CTCs that can be harnessed for their effective analytical detection and enrichment. Furthermore, we want to give an overview of representative nano-systems for CTC isolation, and highlight recent achievements in microfluidics and lab-on-a-chip technologies. We also emphasize the recent advances in nano-based CTCs-targeted cancer therapy. We conclude by critically discussing recent CTC-based nano-systems with high therapeutic and diagnostic potential as well as their biocompatibility as a practical example of applied nanotechnology.

Abstract SY23-01: Blood-borne tumor cell dissemination in cancer patients

Abstract SY23-01: Blood-borne tumor cell dissemination in cancer patients

Rapid prototyping of Nanoroughened polydimethylsiloxane surfaces for the enhancement of immunomagnetic isolation and recovery of rare tumor cells.

Traditional immunomagnetic assays for the isolation and recovery of circulating tumor cells (CTCs) usually require sophisticated device or intense magnetic field to simultaneously achieve high capture efficiency and high throughout. In this study, a simple microfluidic chip featured with nanoroughened channel substrate was developed for effectively capture and release of CTCs based on an immunomagnetic chip-based approach. The nanoroughened substrate aims to increase the cell-surface contact area, facilitate the immobilization of magnet particles (MPs) and accommodate cell attachment tendency. Hep3B tumor cells were firstly conjugated with MPs that were functionalized with anti-EpCAM. Comparing with the flat channel, MPs modified tumor cells can be more effectively captured on nanoroughened substrate at the presence of the magnetic field. Upon the removal of magnetic field, these captured cells can be released from the device and collected for further analysis. Under the optimum operating conditions, the capture efficiency of tumor cells was obtained as high as ~90% with a detection limit of 10 cell per mL. Additionally, recovery rates of trapped tumor cells at various densities all exceeded 90% and their biological potencies were well retained by investigating the cell attachment and proliferation. Therefore, the present approach may potentially be used in clinical CTC analysis for cancer diagnosis and prognosis as well as the fundamental understanding of tumor metastasis.

Porous Graphene Oxide Enhanced Aptamer Specific Circulating-Tumor-Cell Sensing Interface on Light Addressable Potentiometric Sensor: Clinical Application and Simulation.

The circulating-tumor-cell (CTC) specific aptamer is believed to be a power recognition factor to realize clinical CTC assay. However, the limited sensing range is still one of the challenges in its real application. The porous-graphene-oxide (PGO) enhanced aptamer specific CTC sensing interface is studied on the platform of light-addressable-potentiometric-sensor (LAPS) to provide a clinical available method for CTC detection. The underlying mechanism of this sensing interface on LAPS is modeled and simulated. It is confirmed to be a promising candidate for CTC assay by the linear responding for 5-5000 spiked cells, as well as the satisfactory sensitivity for clinical samples.

Relationship between FGFR1 Gene Regulation of Circulating Tumor Cells and Clinical Features of Non-small Cell Lung Cancer

背景与目的目前检测非小细胞肺癌（non-small cell lung cancer, NSCLC）术后患者复发转移的方法均具有一定的滞后性及片面性。本研究总结分析了30例NSCLC患者外周血循环肿瘤细胞（circulating tumor cell, CTC）及成纤维细胞生长因子受体1（fibroblast growth factor receptor 1, FGFR1）表达情况与临床病理之间的关系，以期能够为肿瘤复发转移的检测提供新思路。方法分析北京协和医院胸外科2016年11月-2017年6月30例NSCLC患者临床资料及CTC检测数据并进行相关性分析。结果相关性数据分析可得，外周血CTC细胞阳性率与吸烟史相关（P=0.016），病理类型与CTC阳性率及FGFR1表达情况之间无明显关联（P=0.202, P=0.806），不同类型CTC细胞FGFR1表达情况并无明显差异（P=0.094）。结论CTC阳性率与NSCLC患者吸烟史相关，不同病理类型NSCLC中CTC分类及FGFR1表达情况无明显差异，不同类型CTC之间FGFR1表达情况无明显差异。我们期待着更大样本量及纳入随访数据后可得出与CTC及FGFR1基因表达相关的更多具有临床应用意义的结论。

Clinical applications of the CellSearch platform in cancer patients.

The CellSearch® system (CS) enables standardized enrichment and enumeration of circulating tumor cells (CTCs) that are repeatedly assessable via non-invasive "liquid biopsy". While the association of CTCs with poor clinical outcome for cancer patients has clearly been demonstrated in numerous clinical studies, utilizing CTCs for the identification of therapeutic targets, stratification of patients for targeted therapies and uncovering mechanisms of resistance is still under investigation. Here, we comprehensively review the current benefits and drawbacks of clinical CTC analyses for patients with metastatic and non-metastatic tumors. Furthermore, the review focuses on approaches beyond CTC enumeration that aim to uncover therapeutically relevant antigens, genomic aberrations, transcriptional profiles and epigenetic alterations of CTCs at a single cell level. This characterization of CTCs may shed light on the heterogeneity and genomic landscapes of malignant tumors, an understanding of which is highly important for the development of new therapeutic strategies.

Abstract 1735: Assessing circulating tumor cells with a nanotechnology-based capture system as a novel biomarker for treatment response and surveillance in patients with oligometastatic solid tumors

Abstract Purpose: To prospectively evaluate changes in circulating tumor cells (CTCs) following definitive treatment in patients with oligometastatic solid tumors using a novel nanotechnology-based biomimetric platform. Experiment Procedures: Patients with biopsy proven oligometastatic disease with up to three lesions were eligible if all sites of disease were treated with definitive therapy. Definitive therapy could include fractionated 3D conformal radiation, SBRT, IORT, surgical metastatectomy, and ablative procedures including microwave ablation. Patients initially presenting with oligometastatic disease were eligible if they had less than 3 lesions including the primary tumor. At least one lesion had to be treated with radiation. CTCs were enumerated from whole blood using the Onco-Sense CTC capture system which utilizes E-selectin and dendrimers functionalized with three cancer-specific antibodies (aEpCAM, aHER-2, aEGFR). Whole blood was collected from patients prior to starting treatment (baseline), mid-treatment, end of treatment, within 12 weeks of finishing treatment, and then every 3 months in follow up. We then assessed changes in CTCs with treatment and looked for associations between baseline CTCs, changes during treatment, and post-treatment changes with clinical outcomes. Results: We have currently enrolled 24 patients. The majority of patients (20) had a single site of disease treated with radiosurgery. Sites of metastasis include bone, lung, and lymph nodes. Primary histologies include lung, breast, melanoma, prostate, RCC, colorectal cancer, and SCC of the head and neck. CTCs were detected in 100% of patients at baseline and decreased with treatment from a mean of 45 CTCs/mL pretreatment (range 3-99) to 14 CTCs/mL post-treatment (range 3-59). Post-treatment CTCs are available for 14 patients with a median follow up of 10.3 months. Ten of 14 patients remain clinically NED and post-treatment CTCs are lower than baseline in 13/14 of these patients. There have been 4 clinical failures and CTCs increased with or before radiographic progression in all 4 patients. Three of the failures occurred at least 3 months after initial complete responses to treatment and CTCs increased by a median 3.4-fold over post-treatment levels in these patients. One patient progressed before the first post-treatment follow up and CTCs increased from 7 to 22 CTCs/mL from baseline to post-treatment. Conclusions: We showed here that we could enumerate CTCs in 100% of patients with oligometastatic disease using a novel CTC capture system. CTCs decreased with treatment and all clinical failures were preceded by significant rises in CTCs. Our preliminary data suggest that enumeration of CTCs by Onco-Sense may provide a novel biomarker for assessing treatment response and/or post-treatment surveillance for patients with oligometastatic solid tumors. Note: This abstract was not presented at the meeting. Citation Format: Joseph M. Caster, Kyle Wang, Bhisham Chera, Sin-Jun Park, Seungpyo Hong, Andrew Z. Wang. Assessing circulating tumor cells with a nanotechnology-based capture system as a novel biomarker for treatment response and surveillance in patients with oligometastatic solid tumors [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 1735. doi:10.1158/1538-7445.AM2017-1735

Abstract 4959: Multi-level analysis of prostate cancer circulating tumor cells allowing IHC-based identification, 6-parameter fluorescence phenotyping, and individual cell molecular analysis

Abstract Background. Analysis of circulating tumor cells (CTC) allows non-invasive investigation of prostate cancer biology and response to treatment. The primary level of analysis is the CTC count, which has been demonstrated to be prognostic of outcome. Deeper characterization of CTC phenotype and pertinent biomarkers can confirm cancer lineage and identify drug targets or drug-resistance markers. Single cell analysis of individual CTCs can provide genomic insight into cancer heterogeneity. RareCyte has developed AccuCyte® - CyteFinder® (AC-CF), an integrated technology platform for highly sensitive visual identification and retrieval of rare cells in blood by both immunohistochemistry (IHC) and immunofluorescence (IF) staining. Recently we have developed technology allowing 6-marker assays for broader phenotypic analysis. Methods. Normal human whole blood samples were spiked with prostate cancer lines as model CTCs (mCTCs). Blood samples from University of Washington patients with advanced prostate cancer were collected under an IRB-approved protocol. Blood was processed using AccuCyte and the nucleated cell fraction was collected and spread onto microscope slides. Slides were stained on an automated stainer using (1) an IHC assay for cytokeratin, (2) a standard 4-wavelength IF assay (DAPI, CD45, cytokeratin and EpCAM) or (3) a novel 6-parameter IF assay using SYTOX-Orange (nuclear stain), cytokeratin, EpCAM, androgen receptor (AR), prostate-specific membrane antigen (PSMA) and CD45. An assay for AR variant 7 (ARv7) was applied to samples with mCTCs with the ARv7 splice variant. Percent recovery of IHC-stained slides (by blinded pathologist review) was compared to IF-stained slides (by CyteFinder image analysis). Individual IHC-stained CTCs were retrieved after on-slide visual identification and re-visualized after dispensing for confirmation. Whole genome amplification (WGA) of retrieved cells was performed, followed by X- and Y-chromosome gene-specific PCR. Results. There was strong linear correlation between IF and IHC counts of mCTCs over a range of ∼25 - 100 cells/mL (R2 = 0.99). The 6-parameter IF assay was successfully applied to mCTC and clinical samples. AR and PSMA were co-expressed in the majority of epithelial-marker positive clinical CTCs. The ARv7 assay identified mCTCs that express the splice variant. Individual IHC-stained mCTCs spiked into female donor blood were demonstrated to be male after WGA and PCR. Conclusions. Light microscope identification of IHC cytokeratin-positive mCTCs approximated IF identification. 6-parameter phenotyping of prostate cancer CTCs is feasible and allows identification of lineage-specific markers. IHC-stained cells can be individually retrieved from slides for genome amplification and molecular analysis. Citation Format: Daniel Campton, Rachel Needham, Josh Nordberg, Arturo Ramirez, Nick Drovetto, Alisa Clein, Daniel E. Sabath, Jackie Stilwell, Eric Kaldjian. Multi-level analysis of prostate cancer circulating tumor cells allowing IHC-based identification, 6-parameter fluorescence phenotyping, and individual cell molecular analysis. [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 4959.