Circulating Tumor Cells In Lung Cancer Research Articles

Controllable self-assembled DNA nanomachine enable homogeneous rapid electrochemical one-pot assay of lung cancer circulating tumor cells

A homogeneous rapid (45 min) one-pot electrochemical (EC) aptasensor was established to quantitatively detect circulating tumor cells (CTCs) in lung cancer patients using mucin 1 as a marker. The core of this study is that the three single-stranded DNA (Y1, Y2, and Y3) could be hybridized to form Y-shaped DNA (Y-DNA) and further self-assemble to form DNA nanosphere. The aptamer of mucin 1 could be complementary and paired with Y1, thus disrupting the conformation of the DNA nanosphere. When mucin 1 was present, the aptamer combined specifically with mucin 1, thus preserving the DNA nanosphere structure. Methylene blue (MB) acted as a signal reporter, which could be embedded between two base pairs in the DNA nanosphere to form a DNA nanosphere-MB complex, reducing free MB and resulting in a lower electrochemical signal. The results demonstrated that the linear ranges for mucin 1 and A549 cells were 1 ag/mL-1 fg/mL and 1–100 cells/mL, respectively, with minimum detectable concentrations were 1 ag/mL and 1 cell/mL, respectively. The quantitative analysis of CTCs in 44 clinical blood samples was performed, and the results were consistent with the computerized tomography (CT) images, pathological findings and folate receptor-polymerase chain reaction (FR-PCR) kits. The receiver operating characteristic (ROC) curve exhibited an area under the curve (AUC) value of 0.970. The assay revealed 100% specificity and 94.1% sensitivity. It is believed that this electrochemical aptasensor could provide a new approach to detect CTCs.

Distinct separation of multiple CTCs using inertial focusing phenomena utilizing single-looped spiral microfluidic lab-on-chip

Looking towards the point-of-care diagnosis purposes, nowadays the most emerging method for separation of Circulating Tumor Cells (CTCs) from normal blood-cells is the inertial focusing phenomena utilizing microfluidic lab-on-chips. Keeping eye on the distinctly separating multiple CTCs from normal blood-cells, here we designed a single-looped spiral microfluidic lab-on-chip based on inertial focusing phenomena, which has been more efficient than double-looped and three-looped spirals. The performance of the device has been investigated using large MCF-7 (breast-cancer CTCs), PC3-9 (prostate-cancer CTCs) and normal blood-cells (white blood-cell, red blood-cell) and also larger multiple CTCs like largest MCF-7, PC3-9, A549 (lung-cancer CTCs) cells and normal blood-cells in three-dimensional numerical simulation utilizing COMSOL Multiphysics 5.4 software. In both cases the chip has accomplished efficient separation performance of nearly 100 % at the Reynolds number ranges from 55.9 - 65 (throughput 70 - 82 ml/h) and 114.74 (throughput of 147 ml/h) respectively in terms of cell separation purity and recovery.

Changes of protein expression during tumorosphere formation of small cell lung cancer circulating tumor cells.

Small cell lung cancer (SCLC) is frequently disseminated and has a dismal prognosis with survival times of approximately two years. This cancer responds well to initial chemotherapy but recurs within a short time as a globally chemoresistant tumor. Circulating tumor cells (CTCs) are held responsible for metastasis, the extremely high numbers of these cells in advanced SCLC allowed us to establish several permanent CTC cell lines. These CTCs are distinguished by the spontaneous formation of large spheroids, termed tumorospheres, in regular tissue culture. These contain quiescent and hypoxic cells in their interior and are associated with high chemoresistance compared to single cell cultures. Nine CTC lines were compared for their expression of 84 proteins associated with cancer either as single cells or in the form of tumorospheres in Western blot arrays. With the exception of the UHGc5 line, all other CTC lines express EpCAM and lack a complete EpCAM-negative, vimentin-positive epithelial-mesenchymal transition (EMT) phenotype. Upon formation of tumorospheres the expression of EpCAM, that mediates cell-cell adhesion is markedly upregulated. Proteins such as E-Cadherin, p27 KIP1, Progranulin, BXclx, Galectin-3, and Survivin showed variable changes for the distinct CTC cell lines. In conclusion, EpCAM presents the most critical marker for individual SCLC CTCs and the assembly of highly chemoresistant tumorospheres.

Abstract 2233: Pilot study of liquid biopsy of lung cancer circulating tumor cells (CTC) captured by a novel carbon nano-tube (CNT) differential adhesion platform

Abstract Introduction: Diagnosing and profiling lung cancers (LC) is challenging across the disease spectrum. Early-stage LC are small and hard to distinguish from benign nodules. Patients are often infirm, increasing biopsy risks. Monitoring for LC progression, recurrence or second primaries are further challenges. CTCs recovered from blood sample is non-invasive, easily repeatable across the clinical course of disease treatment and progression. Unlike cell-free nucleic acid profiles, CTC is mutation agnostic. Aims/Methods: Pilot test of a Carbon-nanotube (CNT) platform on capturing suspected LC cells across a range of stages/cell types.51 “normal controls” recruited to balance cancer: benign cases and to test specificity. Study period 1/2020 - 8/2021Procedure details per Loeian M Lab on a Chip 2019;11:1899. 16 ml of whole blood was lysed, nucleated cells cultured for 48 hours on a CNT coated surface. Cells were fixed, stained with CK7/8/18/pancytokeratinAE1/2 epithelial markers (CTC), CD45(WBC), nuclear DAPI control. A sample is considered CTC positive if 1 or more CD45-/CK+ cells are found. Results: 142 subjects were recruited from four clinical sites. 18 samples were non-analyzable due to shipping and preanalytical error.43 of 56 cancers had pathologic staging: 30% I, 16% II, 35% III, 19% IV.CTCs were found in 77% of LC samples (sensitivity), specificity 94%, Positive Predictive Value 92% and Negative Predictive Value 83%.Even in Stages I/II patients, 75% were found to have CTCs. The number of CTC found varied by patient, (range 0-31). Conclusion: CNT adherence enables antigen independent capture of live, viable LC CTCs from whole blood with high specificity and PPV. Planned inclusion of additional CTC markers should improve the detection of other classes of CTCs. High specificity and PPV identify this as a useful “rule-in” test to pursue downstream testing to confirm early LC diagnosis or follow-up surveillance for recurrence. Table 1. Performance of SS-CNT in capturing CTCs Test Result Tissue Positive Cancer Tissue Negative Cancer or Control Total CNT Test + True Pos (TP) 43 False Pos (FP) 4 47 CNT Test - False Neg (FN) 13 True Neg (TN) 64 77 56 68 124 Test Performance 1 or more CTC as positive Sensitivity TP/(TP + FN) 76.80% Specificity TN/(TN + FP) 94.10% Positive Predictive Value PPV TP/(TP + FP) Rule-in 91.50% Negative Predictive Value NPV TN/(TN + FN) Rule- out 83.10% Citation Format: Rex Chin-Wei Yung, Connor Tyree, Ashlee Colbert, Ganesh Krishna, Rakesh Mehta, Jessica Moja, Daniel Fox, Jeff Allard, John Park, Mark Connelly. Pilot study of liquid biopsy of lung cancer circulating tumor cells (CTC) captured by a novel carbon nano-tube (CNT) differential adhesion platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2233.

Targeting genome integrity dysfunctions impedes metastatic potency in non-small cell lung cancer circulating tumor cell-derived explants.

DNA damage and genomic instability contribute to non–small cell lung cancer (NSCLC) etiology and progression. However, their therapeutic exploitation is disappointing. CTC-derived explants (CDX) offer systems for mechanistic investigation of CTC metastatic potency and may provide rationale for biology-driven therapeutics. Four CDX models and 3 CDX-derived cell lines were established from NSCLC CTCs and recapitulated patient tumor histology and response to platinum-based chemotherapy. CDX (GR-CDXL1, GR-CDXL2, GR-CDXL3, GR-CDXL4) demonstrated considerable mutational landscape similarity with patient tumor biopsy and/or single CTCs. Truncal alterations in key DNA damage response (DDR) and genome integrity–related genes were prevalent across models and assessed as therapeutic targets in vitro, in ovo, and in vivo. GR-CDXL1 presented homologous recombination deficiency linked to biallelic BRCA2 mutation and FANCA deletion, unrepaired DNA lesions after mitosis, and olaparib sensitivity, despite resistance to chemotherapy. SLFN11 overexpression in GR-CDXL4 led to olaparib sensitivity and was in coherence with neuroendocrine marker expression in patient tumor biopsy, suggesting a predictive value of SLFN11 in NSCLC histological transformation into small cell lung cancer (SCLC). Centrosome clustering promoted targetable chromosomal instability in GR-CDXL3 cells. These CDX unravel DDR and genome integrity–related defects as a central mechanism underpinning metastatic potency of CTCs and provide rationale for their therapeutic targeting in metastatic NSCLC.

Protection of small-cell lung cancer circulating tumor cells by cellular fragmentation

Protection of small-cell lung cancer circulating tumor cells by cellular fragmentation

Sorting and gene mutation verification of circulating tumor cells of lung cancer with epidermal growth factor receptor peptide lipid magnetic spheres.

BackgroundThis study aimed to identify an efficient, simple, and specific method of detecting mutations in the epidermal growth factor receptor (EGFR) gene in isolated lung cancer circulating tumor cells (CTCs) and to improve the ability to obtain tumor tissue clinically.MethodsEGFR peptide lipid magnetic spheres (EG‐P‐LMB) were prepared by reverse evaporation, and characterization and cell capture efficiency assessed. The peripheral blood samples of 30 lung cancer patients were isolated and identified with the EG‐P‐LMB using 20 healthy volunteers as controls. Finally, the isolated CTCs were tested for EGFR gene mutations, and the tissue samples selected for comparison.ResultsThe prepared magnetic spheres had a smaller particle size and higher stability according to the particle size potential test. Their morphology was homogeneous by atomic force observation, and the UV test showed that there were peptides on the surface. The separation efficiency of EG‐P‐LMB was greater than 90% in PBS and greater than 80% in the blood simulation system. Compared with the tissue sample results, the positive rate of EGFR gene mutations was 94%. The CTC test results of 27 patients were consistent with the tissue test results of the corresponding patients, and the consistency with the tissue comparison test results was 90% (27/30).ConclusionsEG‐P‐LMB can effectively capture CTCs in the peripheral blood of patients with lung cancer. CTC detection can accurately identify mutations in the EGFR gene and improve the ability to obtain tumor tissue in clinical practice.Key pointsSignificant findings of the studyEG‐P‐LMB can effectively capture CTCs in the peripheral blood of patients with lung cancer. CTC detection can accurately identify mutations in the EGFR gene and improve the ability to obtain tumor tissue in clinical practice.What this study addsThis study added EGFR peptide lipid magnetic spheres to capture CTCs in the blood. Genetic testing was performed and compared with tissues. It solves the problem of clinically difficult tumor tissue sampling.

Abstract IA19: Multiple uses of circulating tumor cells in lung cancer?

Abstract Liquid biopsies are increasingly being used in clinical studies as prognostic, predictive, and pharmacodynamics biomarkers, as surrogates of tumor response, to detect minimal residual disease after treatment with curative intent and to inform on mechanisms of treatment resistance and treatment switching. There is also steady progress in the development of liquid biopsies for early detection of cancers. The advantages of liquid biopsies are that they are minimally invasive and readily repeatable. The liquid biopsy that has been most widely adopted is circulating tumor DNA (ctDNA) as it is readily measured without specialist equipment in most molecular biology laboratories. Circulating tumor cells (CTCs) are technically more challenging and have yet to realize their full potential as biomarkers in clinic. However, CTC technology is constantly improving, and CTCs have several exciting applications beyond ctDNA that, once optimized, will assist personalized cancer medicine, including their use as cultures for real-time therapy testing and their utility, in some cancer types, to derive mouse models. I will discuss the use of CTCs from patients with lung cancer in this presentation. CTCs in both non-small cell lung cancer (NSCLC) and SCLC enumerated by CellSearch (EpCAM and cytokerin positive, CD45 negative) hold prognostic information. However, while CellSearch CTCs are scarce in NSCLC, most likely due to epithelial-to-mesenchymal transition (EMT) that leads to downrgulation of the EpCAM surface marker used to capture them for enumeration, they are prevalent in SCLC. Single CTC copy number analysis has led to the development of classifier that, with further validation studies, could predict response to chemotherapy alongside ctDNA analysis for therapy monitoring. We have exploited SCLC CTC prevalence to derive mouse models in immune-incompetent mice (termed CDX) that allow us to explore biology and test new therapeutics. I will also update on our research using our biobank of 46 CDX models, exploring inter and intratumoral heterogeneity. The most important aspect of this approach is the ability to generate pre- and post-therapy CDX models allowing interrogation of therapy resistance mechanisms and the biology of progressing disease in a tumor type where tumor evolution is rapid and where serial tumor biopsies are rarely obtained. I will also describe our approaches to study mechanisms of tumor dissemination, including vasculogenic mimicry and new models of brain metastasis. More recently, we are developing direct CTC cultures that may allow real-time therapy testing with reporting to the clinic. I will report on our NSCLC CTC studies within the UK TRACERx consortium to study tumor evolution. We have explored the potential of CTCs found in the pulmonary draining vein of stage I-IIIa patients at surgery with curative intent to predict risk of disease recurrence and shown in a case study that a lethal subclone that gives rise to metastasis 10 months later was identified in the pulmonary vein at surgery. In summary, this presentation will exemplify utility of CTCs in lung cancer that are distinct from but complement the implementation of ctDNA. Citation Format: Caroline Dive. Multiple uses of circulating tumor cells in lung cancer? [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 IA19.

Abstract B14: Artificial intelligence assists automation and high performance of circulating tumor cells enumeration and circulating tumor microemboli characterization in fluorescence microscopy images

Abstract Analysis of circulating tumor cells (CTCs) provides exclusive benefits compared with other liquid biopsy techniques due to their advanced clinical applications including single-cell multiomics (DNA, RNA, proteins, and metabolites) analysis and the ability to generate CTCs-derived xenograft models. Current CTC analysis primarily relies on labor-intensive and human-biased manual CTC enumeration or semiautomated work, hindering clinical adaptation of CTC analysis into practice. Beyond CTC enumeration, recent studies focused on detection and characterized features of circulating tumor microemboli (CTM) to improve cancer monitoring and diagnosis, but the added complexity of CTM makes CTC analysis even more challenging. Development of an automated, unbiased, and specialized computation method for CTC analysis remains a clinical unmet need. We developed a deep learning-based framework by applying computer vision to efficiently automate CTC enumeration and characterization with high throughput and accuracy. Fluorescence microscopy CTCs images (CK+/CD45-/DAPI+) have been prepared from blood samples of non-small cell lung cancer patients by the “CMx” CTC capture platform. This study applied an “active learning” model that only requires a small upfront training dataset while being able to accumulate more training data to make improvements with minimal annotation efforts. We have collected 20 images annotated by subject matter experts to train the initial model. Using an iterative process where feedback from the subject matter experts was used to influence the model, we achieved continuous improvements using adaptive annotations. After the model had been well trained and validated by 4 extra images, we used 18 new test images to benchmark the model performance. Preliminary results demonstrated that our AI model outperformed current semiautomated methods having higher sensitivity and reduced time for CTC enumeration in a lung cancer CTC sample analysis. In 18 test samples, the model predicted 34% more total CTCs than the current method (1,775 versus 1,328 counts). By applying the model to the 24 training samples, the model also recovered 45% more total CTCs absent from the original human annotation (2,507 versus 1,732 counts). In some samples, enumeration assisted by the model saved 90% of time over the current method (<20 min vs ~4 hrs.) for CTC enumeration, which would result in an enormous advantage if the datasets being analyzed included a large number of samples. Furthermore, our AI model was able to characterize features of CTM, including CTC clusters and CTC-associated immune cells, in addition to enumerating the samples. The AI model developed in this study is a promising method for CTC analysis that significantly improved throughput, accuracy, and reproducibility, leading to an approach of CTC analysis better suited for clinical adoption. Development of AI-driven CTC/CTM analysis provides additional resources and advancements to the field of liquid biopsy for the diagnosis and treatment of disease. Citation Format: Wei-Lei Yang, Chi-Bin Li, Jia-Yang Chen, Samuel Chen, Ten-Jen Liu, Ying-Chih Chang. Artificial intelligence assists automation and high performance of circulating tumor cells enumeration and circulating tumor microemboli characterization in fluorescence microscopy images [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 B14.

Required Evidence for Clinical Applications of Liquid Biopsy Using Especially CTCs in Lung Cancer

As therapies have become more and more dependent on tumor as well as patient characteristics, obtaining tumor material has become of great importance. Liquid biopsies hold much potential as shown by a large amount of evidence across several studies. Clinical applications for circulating tumor cells (CTCs) are unfortunately still lacking. In part this is due to a lack of studies comparing liquid biopsies to conventional diagnostics and response measurements as well as studies showing that liquid biopsies can be used to switch therapies leading to improved outcomes. However, liquid biopsies using ctDNA for specific markers such as EGFR, ALK, ROS1 or RET have clinical applications because specific drugs are available.

Effects of salinomycin and niclosamide on small cell lung cancer and small cell lung cancer circulating tumor cell lines

SummaryTumor dissemination and recurrence is attributed to highly resistant cancer stem cells (CSCs) which may constitute a fraction of circulating tumor cells (CTCs). Small cell lung cancer (SCLC) constitutes a suitable model to investigate the relation of CTCs and CSCs due to rapid tumor spread and a high number of CTCs. Expansion of five SCLC CTC lines (BHGc7, 10, 16, 26 and UHGc5) in vitro at our institution allowed for the analysis of CSC markers and cytotoxicity of the CSC-selective drugs salinomycin and niclosamide against CTC single cell suspensions or CTC spheroids/ tumorospheres (TOS). Salinomycin exerted dose-dependent cytotoxicity against the SCLC lines but, with exception of BHGc7 TOS, there was no markedly enhanced activity against TOS. Similarly, niclosamide exhibits high activity against BHGc7 TOS and UHGc5 TOS but not against the other CTC spheroids. High expression of the CSC marker CD133 was restricted to three SCLC tumor lines and the BHGc10 CTC line. All SCLC CTCs are CD24-positive but lack expression of CD44 and ABCG2 in contrast to the SCLC tumor lines which show a phenotype more similar to that of CSCs. The stem cell marker SOX2 was found in all CTC lines and SCLC GLC14/16, whereas elevated expression of Oct-3/4 and Nanog was restricted to BHGc26 and UHGc5. In conclusion, the SCLC CTCs established from patients with relapsed disease lack a typical CSC phenotype in respect to chemosensitivity to CSC-selective drugs, surface markers, expression of pluripotent stem cell and transcription factors.

Circulating tumor cells in patients with lung cancer: developments and applications for precision medicine.

Lung cancer is the most common cause of cancer-related deaths, with most patients dying with distant metastases. Circulating tumor cells (CTCs) are cancer cells that have disseminated into the peripheral blood from primary or metastatic sites and present great potentials as prognostic biomarkers for guiding individualized treatment in lung cancer. To date, various methods have been developed to capture CTCs in peripheral blood, and some approaches for the detection of CTC in lung cancer have shown both high sensitivity and specificity. The CTC analyses offer much promise as a real-time 'liquid biopsy' for prognosis evaluation and therapy intervention in lung cancer. In this Review, we present and discuss the current status of CTC detection and applications in lung cancer.

Abstract P3-01-16: Micro-cavity array system for size-based enrichment of circulating tumor cells and circulating cancer associated fibroblasts from blood of patients with breast cancer

Abstract Background:Circulating Tumor Cells(CTCs) have prognostic implications in patients with metastatic breast cancer(MBC).During the Epithelial Mesenchymal Transition(EMT), CTCs acquire a more mesenchymal phenotype. Hence, methodologies such as the Cell search that rely on the expression of an epithelial marker EpCAM in CTCs fail to capture a subset of CTCs undergoing the process of EMT and therefore do not adequately represent the true circulatory metastatic load. Hitachi chemicals has invented a size based micro cavity array (MCA) system that allows for the isolation of tumor cells based on the differences in size and deformability between tumor and blood cells. Photolithography and the metal plating can precisely control the filter pore size of our system. Our platform is more sensitive than the Cell Search method in detecting CTCs in Lung Cancer. Cancer Associated Fibroblasts (CAFs) are a major component of the breast tumor microenvironment. Using a micro filter capture technique, our co-authors have demonstrated that CAFs can be enumerated as circulating CAFs (cCAFs). Hitachi's Micro cavity Array System has not been evaluated in the detection of CTCs and cCAFs in patients with Breast Cancer. The purpose of this study is to demonstrate that CTCs and cCAFs can be enumerated using our platform and the cCAFs can serve as biomarkers of metastasis simultaneously with CTCs. Method:We undertook a Pilot study of 20 patients each with breast cancer across Stage I, Stage II, Stage III and Stage IV. A total of 10ml of peripheral blood was obtained from each patient. Enumeration of CTCs and cCAFs was carried out by the size based mircocavity array system invented by Hitachi Chemicals. Identification of these cells was done by a triple Immunofluorescence staining for pan-CK (cytokeratin), FAP (Fibroblast Activated Protein) and CD45. CTCs were identified as CK+, CD45-, FAP- cells and cCAFs were identified as FAP+, CK- and CD 45 negative cells. Result:Our method had a high cell recovery rate (90%or higher) and efficient white blood cells depletion rate (99.99%). We present the data from a total of 13 patients in this abstract, (two with stage III and eleven with stage IV breast cancer) . Data from rest of the subjects will be presented at the actual meeting. We detected the presence of CTCs in 11/11(100%) in patients with stage IV(mean of 44) and in 2 out of 2 (100%) patients with Stage III Breast Cancer. We detected the presence of cCAFs in 1 out of 2 patients( 50%) with stage III and in 8 of 11(81.8%) (mean of 9)patients with stage IV breast cancer( Fisher's exact test p-value= 0.42). The number of CTCs and cCAFs was significantly elevated in patients with MBC and the number was clinically associated with a high metastatic burden. Conclusions:CTCs and cCAFs can be enumerated using a size based size based micro cavity array invented by Hitachi Chemicals that does not rely on the expression of epithelial markers in CTCs. CTCs and cCAFs can be detected in patients with stage III and stage IV breast cancer. CTCs and cCAFs were associated with high metastatic burden and their numbers were significantly elevated in patients with MBC. cCAFs could serve as biomarkers alongside of CTCs in MBC. Citation Format: Parajuli R, Ly R, Ziogas A, Eapen A, Lane K, Chen J, Lin E, Mehta R, Tsai A. Micro-cavity array system for size-based enrichment of circulating tumor cells and circulating cancer associated fibroblasts from blood of patients with breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-16.

Expression of Proteolytic Enzymes by Small Cell Lung Cancer Circulating Tumor Cell Lines.

Small cell lung cancer (SCLC) is an aggressive type of lung cancer which disseminates vigorously and has a dismal prognosis. Metastasis of SCLC is linked to an extremely high number of circulating tumor cells (CTCs), which form chemoresistant spheroids, termed tumorospheres. Intravasation and extravasation during tumor spread requires the activity of a number of proteases to disintegrate the stroma and vascular tissue. Generation of several permanent SCLC CTC lines allowed us to screen for the expression of 35 proteases using Western blot arrays. Cell culture supernatants of two CTC lines, namely BHGc7 and 10, were analyzed for secreted proteases, including matrix metalloproteinases (MMPs), ADAM/TS, cathepsins, kallikreins, and others, and compared to proteases expressed by SCLC cell lines (GLC14, GLC16, NCI-H526 and SCLC26A). In contrast to NCI-H526 and SCLC26A, MMP-9 was highly expressed in the two CTC lines and in GLC16 derived of a relapse. Furthermore, cathepsins (S, V, X/Z/P, A and D) were highly expressed in the CTC lines, whereas ADAM/TS and kallikreins were not detectable. In conclusion, SCLC CTCs express MMP-9 and a range of cathepsins for proteolysis and, aside from tissue degradation, these enzymes are involved in cell signaling, survival, and the chemoresistance of tumor cells.

Anticancer Activity of Fascaplysin against Lung Cancer Cell and Small Cell Lung Cancer Circulating Tumor Cell Lines.

Lung cancer is a leading cause of tumor-associated mortality. Fascaplysin, a bis-indole of a marine sponge, exhibit broad anticancer activity as specific CDK4 inhibitor among several other mechanisms, and is investigated as a drug to overcome chemoresistance after the failure of targeted agents or immunotherapy. The cytotoxic activity of fascaplysin was studied using lung cancer cell lines, primary Non-Small Cell Lung Cancer (NSCLC) and Small Cell Lung Cancer (SCLC) cells, as well as SCLC circulating tumor cell lines (CTCs). This compound exhibited high activity against SCLC cell lines (mean IC50 0.89 µM), as well as SCLC CTCs as single cells and in the form of tumorospheres (mean IC50 0.57 µM). NSCLC lines showed a mean IC50 of 1.15 µM for fascaplysin. Analysis of signal transduction mediators point to an ATM-triggered signaling cascade provoked by drug-induced DNA damage. Fascaplysin reveals at least an additive cytotoxic effect with cisplatin, which is the mainstay of lung cancer chemotherapy. In conclusion, fascaplysin shows high activity against lung cancer cell lines and spheroids of SCLC CTCs which are linked to the dismal prognosis of this tumor type. Derivatives of fascaplysin may constitute valuable new agents for the treatment of lung cancer.

Aptamer-functionalized magnetic SERS nanoprobes based specific recognition and ultrasensitive detection of lung cancer circulating tumor cells

Aptamer-functionalized magnetic SERS nanoprobes based specific recognition and ultrasensitive detection of lung cancer circulating tumor cells

Highly sensitive detection and mutational analysis of lung cancer circulating tumor cells using integrated combined immunomagnetic beads with a droplet digital PCR chip

Circulating tumor cells (CTCs) have become an important biomarker for liquid biopsy to monitor tumor progression and indicate response to therapies. Many epithelial cellular adhesion molecule (EpCAM) dependent CTC isolation methods have been developed, which have a limitation for low EpCAM expressed tumor cells. In an effort to overcome these drawbacks, we developed combined immunomagnetic beads (EpCAM, Mucin1 and epidermal growth factor receptor) to sensitively isolate CTCs for immunofluorescence analysis and genetic characterization. With this combined immunomagnetic beads, 93.35% H446 cells from spiked blood sample can be recovered. We were able to detect CTCs in 127 among 143 patients included in the study (88.8%). Some CTC clusters were captured with the combined magnetic beads system. In 17 of them, CTCs after chemotherapy significantly decreased compared to that before chemotherapy (4.42 (± 3.94) vs. 12 (± 7)/mL, P = 0.002). For subsequent genetic characterization of CTCs, 2 of 6 samples, using a droplet digital PCR (ddPCR) chip, have detectable EGFR L858R mutation in the cells enriched with the combined immunomagnetic beads. In conclusion, this method integrating the combined immunomagnetic beads and the ddPCR chip for CTCs detection can be of potential application in terms of diagnosis, therapeutic evaluation and personalized medicine in lung cancer.

Development of an automated size-based filtration system for isolation of circulating tumor cells in lung cancer patients.

Circulating tumor cells (CTCs), defined as tumor cells circulating in the peripheral blood of patients with solid tumors, are relatively rare. Diagnosis using CTCs is expected to help in the decision-making for precision cancer medicine. We have developed an automated microcavity array (MCA) system to detect CTCs based on the differences in size and deformability between tumor cells and normal blood cells. Herein, we evaluated the system using blood samples from non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) patients. To evaluate the recovery of CTCs, preclinical experiments were performed by spiking NSCLC cell lines (NCI-H820, A549, NCI-H23 and NCI-H441) into peripheral whole blood samples from healthy volunteers. The recovery rates were 70% or more in all cell lines. For clinical evaluation, 6 mL of peripheral blood was collected from 50 patients with advanced lung cancer and from 10 healthy donors. Cells recovered on the filter were stained. We defined CTCs as DAPI-positive, cytokeratin-positive, and CD45-negative cells under the fluorescence microscope. The 50 lung cancer patients had a median age of 72 years (range, 48–85 years); 76% had NSCLC and 20% had SCLC, and 14% were at stage III disease whereas 86% were at stage IV. One or more CTCs were detected in 80% of the lung cancer patients (median 2.5). A comparison of the CellSearch system with our MCA system, using the samples from NSCLC patients, confirmed the superiority of our system (median CTC count, 0 versus 11 for CellSearch versus MCA; p = 0.0001, n = 17). The study results suggest that our MCA system has good clinical potential for diagnosing CTCs in lung cancer.

Abstract 2247: A multicenter clinical trial of lung cancer circulating tumor cell assay with the largest sample size (1210 cases) in China

Abstract Background: As a novel biomarker of primary tumors, circulating tumor cells (CTCs) are well known to play an important role in cancer diagnosis, recurrence and metastasis, and disease monitoring. This study investigated the application of CTCs in lung cancer therapy by quantitative determination of folate receptor-positive CTCs. Methods: This study enrolled 1210 subjects (including 560 lung cancer patients, 350 patients with benign lung diseases, 150 healthy subjects and 150 non-lung cancer malignant tumor patients) and quantified the tiny amounts of CTCs in peripheral blood by negative enrichment using immunomagnetic beads in combination with folate receptor-directed polymerase chain reaction (PCR). Results: Following ROC curve analyses of CTC levels in the patients with benign lung diseases or benign lung diseases and the healthy subjects, the cut-off of CTCs was 8.70 CTC Units/3mL with a specificity of 88.2% (441/500) and a sensitivity of 79.6% (446/560) as determined by the method mentioned above. The ROC area under curve of more than 0.8 (AUC = 0.8797, P<0.0001) suggested the excellence of ligand-directed PCR to diagnose lung cancer. For stage II-IV lung cancer patients, the sensitivities were 70.8%(34/48), 79.6% (125/157), and 90.2% (185/205) respectively, and the sensitivity in stage I patients can reach up to 68.0% (102/150) as well. CTC levels appeared to have greater accuracy to diagnose lung cancer (AUC: 0.8833, P<0.0001) than the combination of the other four lung cancer-specific serum tumor markers (CEA, NSE, CYFRA21-1 and SCC)(AUC: 0.8557, P<0.0001). CTCs were related to tumor stage and tumor size other than pathological type. By quantitative determination of folate receptor-positive cells in 150 different types of cancers, the CTC level in lung cancer was found to be higher than those in gastric, breast, colorectal, liver, and esophageal cancers. Conclusions: LT-PCR achieves quantitative determination of folate receptor-positive CTCs to diagnose lung cancer with high sensitivity and specificity, and significantly greater diagnosis accuracy than the model of combined lung cancer-specific serum tumor markers. Citation Format: Jiatao Lou, Caicun Zhou, Jing Wu, Lihua Qiao, Xiaohui Liang, Xiaoqian Wang, Xiaoxia Chen, Xuefei Li, Chao Zhao. A multicenter clinical trial of lung cancer circulating tumor cell assay with the largest sample size (1210 cases) in China. [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 2247.

Abstract 2246: Androgen receptor expression in non-small cell lung cancer circulating tumor cells

Abstract BACKGROUND: Non-small cell lung cancer (NSCLC) is a leading cause of death worldwide. There is a critical need to develop new therapeutic options for patients with advanced NSCLC. Androgen Receptor (AR) expression has been observed in lung cancer, along with castration associated inhibition of tumor growth in mouse models. The AR is a high value therapeutic target in prostate cancer and many targeted therapies are clinically available. However, significant genomic and functional alterations occur in NSCLC over the course of targeted therapies that may predict response and resistance to AR targeted therapies. The development of predictive and pharmacodynamic biomarkers of the AR signaling pathway in NSCLC may be critical to developing appropriate therapeutic strategies in this context. To evaluate AR expression and the AR signaling pathway, we developed a circulating tumor cell (CTC) assay to evaluate orthogonal endpoints across protein and gene expression analytics in patients with advanced NSCLC. METHODS: CTCs were isolated from patients with metastatic NSCLC using an integrated CTC capture and analysis technology known as the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform. This microscale platform integrates tumor cell capture, for any target of interest, with enumeration and nucleic acid extraction for analysis of orthogonal endpoints from a blood sample. CTCs were captured with multiple antibodies, including EpCAM, MUC1, and Vimentin. mRNA was extracted from CTCs for gene expression analysis of the AR, AR splice variants, downstream targets in the AR signaling pathway and activation markers in NSCLC. RESULTS: Enumeration of CTCs from patients with metastatic NSCLC shows significant tumor burden, ranging from 10-150 CTCs per 7.5mL of blood. Gene expression analysis of CTCs identified expression of proliferative markers KRAS, BRAF, and RET. In our initial cohort of patients, full length AR expression was identified in 13/13 patients with NSCLC. The AR V7 splice variant was detected at low levels in 2/13 patients. PSMA, but not TMPRSS2, was identified in a subset of these patients suggesting activity of the canonical AR signaling pathway CONCLUSIONS: The VERSA effectively captures CTCs with high sensitivity to detect the AR and AR variants in NSCLC CTCs. The AR is detectable in patients with NSCLC CTCs at high frequency. A subset of patient samples show activity in the AR signaling pathway that could potentially drive disease progression. The identification of the AR V7 splice variant suggests shared mechanisms of resistance in NSCLC and prostate cancer. These assays may serve as predictive and pharmacodynamic biomarkers for AR targeted therapies in NSCLC. Citation Format: Jennifer L. Schehr, Zachery Schultz, Anwaar Saeed, Jamie M. Sperger, Ticiana Leal, Kara Mattox, Anne Traynor, Joshua M. Lang. Androgen receptor expression in non-small cell lung cancer circulating tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2246.