Non-small Cell Lung Cancer Circulating Tumor Cells Research Articles

Abstract 5592: Expansion and characterization on ALK positive NSCLC circulating tumor cells isolated using a size based inertial microfluidic Labyrinth device

Abstract Introduction Lung cancer (LC) is the leading cause of cancer deaths. 85% of LC are non-small cell lung cancer (NSCLC). Fusions involving anaplastic lymphoma kinase (ALK) are oncogenic drivers in ~ 7% of NSCLC. Previous studies have shown that drug resistance develops within ~2 years after ALK tyrosine kinase inhibitor targeted therapy. Due to resistance mechanisms and the biological heterogeneity that evolves over the treatment of ALK+ NSCLC, it is important to monitor and study those dynamic changes through the clinical treatment course to understand cancer progression. Circulating tumor cells (CTCs) are rare cells, shed from cancer lesions into the bloodstream. They can be isolated from a patient's peripheral blood sample (~20ml) to enable study of tumor characteristics at multiple time points. They are promising biomarkers that not only offer phenotypic and genotypic information, but also present a potential to expand into patient derived ex vivo tumor models. In this study, we use a high throughput label-free microfluidic device, Labyrinth, developed in the Nagrath lab for cell size based CTC enrichment, followed by expansion of CTCs in organoid models, further molecular characterization, and growth in patient-derived xenograft (PDX) models. Methods Blood samples were collected from patients (n=22), three with known ALK status. CTC isolation was performed using Labyrinth at 2.0 mL/min. A part of the isolated CTCs were immunofluorescently stained for pan cytokeratin (pan CK), CD45, DAPI, epithelial cell adhesion molecule (EpCAM), and vimentin markers for enumeration. Others were processed for expansion in ultralow attachment plates with three types of organoid culture media. The rest of the CTCs were used for molecular characterizations. Growth curves were produced on expanded samples on Day 0, 7, 14, and 21 in different culture conditions. A live/dead assay and in situ hybridization (FISH) were also performed. On Day 7, selected samples were injected into NSG™ mice. Bulk RNA sequencing was conducted on the original tumor, Day 0 and 7 CTCs, and PDX samples. Results The median CTC concentration in this cohort (n=22) was 316.2 CTCs/ml of blood. Isolated CTCs from ALK+ NSCLC patients showed ALK fusions by FISH. We observed that the number of ALK CTCs was reduced over time consistent with decreased tumor size in CT scans. There were significant differences (p<0.05) for vimentin positive CTCs compared to the other three types of CTCs, which indicated a higher prevalence of a mesenchymal state of NSCLC CTCs. One of the two samples successfully developed into PDX after two months. In summary, we demonstrated a successful workflow to enrich and expand CTCs from ALK+ NSCLC patients. The expanded CTCs can be further characterized and can generate tumors in PDX models. Citation Format: Yuru Chen, Shamileh Fouladdel, Harrison Ball, Xu Cheng, Liwei Bao, Habib Serhan, Albert Liu, Bryce Vandenburg, Laura Goo, Nathan Merrill, Aaron Udager, Angel Qin, Sofia D. Merajver, Sunitha Nagrath. Expansion and characterization on ALK positive NSCLC circulating tumor cells isolated using a size based inertial microfluidic Labyrinth device. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5592.

Circulating Tumor Cells and the Non-Touch Isolation Technique in Surgery for Non-Small-Cell Lung Cancer.

Simple SummaryOur review discusses findings for the non-touch isolation technique in surgery, especially in surgery for non-small-cell lung cancer. This technique aims to prevent the release of circulating tumor cells (CTCs) from the tumor nest to the bloodstream during surgery, but its efficacy has not been clearly verified. We have summarized the history of CTC detection, relevance of CTCs to clinical practice, and evidence regarding this technique for lung cancer surgery.Circulating tumor cells (CTCs) are dislodged from the primary tumor into the bloodstream, travel within the bloodstream to distant organs, and finally extravasate and proliferate as epithelial metastatic deposits. The relationship between the existence of CTCs and tumor prognosis has been demonstrated by many researchers. In surgery for malignancies, the surgical manipulation of tumors and tissues around the tumor may lead to the release of CTCs into the bloodstream. The non-touch isolation technique (NTIT) has been advocated to prevent the release of CTCs during surgery. The concept of NTIT is the prevention of intraoperative increment of CTCs from the primary tumor by the early blockade of outflow vessels, and ‘pulmonary vein (PV)-first lobectomy’ during surgery for non-small-cell lung cancer (NSCLC) corresponds to this technique. The concept of PV-first lobectomy is well known among thoracic surgeons, but evidence of its efficacy for preventing the increase of intra- and postoperative CTCs and for improving postoperative prognosis is still uncertain. Our study summarizes evidence regarding the relationship between NTIT and CTCs in NSCLC and suggests the need for further research on CTCs and CTC-detecting modalities.

18F-FDG PET/CT and circulating tumor cells in treatment-naive patients with non-small-cell lung cancer.

This study retrospectively investigated the clinical utility of 2-deoxy-18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) and circulating tumor cells (CTCs) in the diagnosis and prognosis of treatment-naive patients with non-small-cell lung cancer (NSCLC). The blood samples of treatment-naive patients with NSCLC were collected for CTCs detection, and the tumor metabolic parameters of 18F-FDG PET/CT, including maximum standard uptake value (SUVmax), metabolic tumor volume of primary lesion (MTV-P) and combination of primary lesion and metastases (MTV-C), and total lesion glycolysis of primary lesion (TLG-P) and combination of primary lesion and metastases (TLG-C), were analyzed. Age, sex, smoking, serum tumor markers, tumor size, location, TNM stage, and genetic mutations were also reviewed. Moreover, progression-free survival (PFS) and overall survival (OS) of these patients were analyzed. A total of 309 patients with NSCLC (200 men, 109 women; mean age: 61 ± 9years) were enrolled in this study, including 217 patients with adenocarcinoma and 92 with squamous cell carcinoma. Of the 309 cases, 11 were misdiagnosed with benign diseases by 18F-FDG PET/CT. CTCs positivity was detected in 234 cases. The sensitivity of 18F-FDG PET/CT and CTCs in NSCLC were 96.4% and 75.7%, respectively. SUVmax, MTV-P, TLG-P, MTV-C, TLG-C, tumor size, and serum CYFRA211 levels were significantly higher in CTCs positive group than negative group; and advanced TNM stage, squamous cell carcinoma, and EGFR wild type presented higher CTCs positivity. Multivariate logistic regression analysis revealed that SUVmax was significantly associated with CTCs positivity. Multivariate cox regression analysis showed that TLG-P, TLG-C, and CTCs were independent predictors of PFS in patients with NSCLC, and TLG-C and CTCs were independent predictors of OS. 18F-FDG PET/CT was superior to CTCs in the diagnosis of treatment-naive patients with NSCLC. The levels of CTCs in the peripheral blood were associated with tumor glucose metabolism in NSCLC. Metabolic parameters of 18F-FDG PET/CT and CTCs could separately predict the outcomes of treatment-naive patients with NSCLC.

Abstract 5368: A multiparameter assay for HER2 protein detection on circulating tumor cells in non-small cell lung cancer

Abstract Lung cancer biopsy can be difficult to perform successfully; it has a 30% adverse event risk and often provides insufficient material to test. Therefore, a liquid biopsy method to analyze the tumor is advantageous. Analysis of circulating tumor cells (CTCs) by multiparameter immunofluorescence (IF) microscopy allows non-invasive characterization of cancer cell biomarker expression. HER2 is a well-known therapeutic target in breast cancer and studies have shown that HER2 status on CTCs may provide prognostic information about response to anti-HER2 therapies. Less is understood about its frequency and clinical importance in non-small cell lung cancer (NSCLC). The RareCyte platform is uniquely suited for CTC identification and phenotypic characterization with a sensitive, accurate, simple and repeatable workflow from blood collection to CTC characterization and single-cell isolation. We developed an assay for detecting HER2 expression on lung CTCs using the RareCyte platform and validated the assay on model CTC spike-in samples with high (BT-474), medium (MDA-MB-453), low (H1650 and OVCAR-3) and absent (MDA-MB-468) HER2 protein levels. Blood was processed to slides using the AccuCyte Sample Preparation System and stained with anti-HER2 together with the RarePlex Kit for CTC identification (nuclear dye, anti-CD45 to exclude WBC, and anti-cytokeratin/EpCAM for CTCs). The spike-in samples showed the expected trend in per-cell HER2 mean fluorescence intensity values. The percentage of HER2-positive cells was > 90% for the HER2-high, medium and low cell lines, and < 5% for the HER2-negative cell line. The assay was applied to 10 advanced stage (III or IV) post-treatment NSCLC patient samples. At least 1 CTC was found in 6 samples (range 0-2043) and HER2 expression was confirmed in 5 out of the 6 samples, indicating this assay is useful for characterizing HER2 expression in CTCs in NSCLC. Citation Format: Edward Lo, Daniel Campton, Lillian Costandy, Heather Itamoto, Ryan Huston, Braden Gardner, Steve Reese, Arturo Ramirez, Eric P. Kaldjian, Tad George, I-Ming Wang, Steven Pirie-Shepherd. A multiparameter assay for HER2 protein detection on circulating tumor cells in non-small cell lung cancer [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 5368.

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.

A circulating tumor cell-based digital assay for the detection of EGFR T790M mutation in advanced non-small cell lung cancer.

Determining the status of epidermal growth factor receptor (EGFR) T790M mutation is crucial for guiding further treatment intervention in advanced non-small cell lung cancer (NSCLC) patients who develop acquired resistance to initial EGFR tyrosine kinase inhibitor (TKI) treatment. Circulating tumor cells (CTCs) which contain plentiful copies of well-preserved RNA offer an ideal source for noninvasive detection of T790M mutation in NSCLC. We developed a CTC-based digital assay which synergistically integrates NanoVelcro Chips for enriching NSCLC CTCs and reverse-transcription droplet digital PCR (RT-ddPCR) for quantifying T790M transcripts in the enriched CTCs. We collected 46 peripheral arterial and venous blood samples from 27 advanced NSCLC patients for testing this CTC-based digital assay. The results showed that the T790M mutational status observed by the CTC-based digital assay matched with those observed by tissue-based diagnostic methods. Furthermore, higher copy numbers of T790M transcripts were observed in peripheral arterial blood than those detected in the matched peripheral venous blood. In short, our results demonstrated the potential of the NanoVelcro CTC-digital assay for noninvasive detection of the T790M mutation in NSCLC, and suggested that peripheral arterial blood sampling may offer a more abundant CTC source than peripheral venous blood in advanced NSCLC patients.

Abstract 3610: CANCER ID and the identification of circulating tumor cells (CTCs) in NSCLC

Abstract The IMI-funded CANCER-ID consortium (https://www.cancer-id.eu/) was created in order to facilitate the use of Circulating Tumor Cells (CTCs) and other liquid biopsies (ctDNA, miRNA, exosomes) in the monitoring and treatment of cancer, via establishing and validating protocols for their detection and characterization. CTCs are tumor cells that have passively or actively migrated from the bulk of the primary or metastatic tumor(s) into the peripheral blood stream. While some of these cells may establish new metastases and mediate outgrowth of the disease, they can also serve as diagnostic, prognostic or potentially predictive biomarkers, providing non-invasive access to tumor material and information on tumor evolution. However, a major challenge remains in the identification of these very rare cells (1:106-8 WBCs). In the past years, a plethora of methods have been developed to isolate CTCs, via antibody enrichment/depletion strategies, size exclusion and/or based on density and dielectric properties. Yet despite this progress, the detection rate of CTCs in NSCLC (non-small cell lung cancer) is still very low (only ca. 20-30% of metastatic NSCLC patients have detectable CTCs). In stark contrast, SCLC (small cell lung cancer) patients have usually hundreds of detectable CTCs. A potential reason for the lack of detection of NSCLC CTCs may be the choice of surface markers used to detect the CTCs. Using NSCLC cell lines spiked into healthy donor blood, novel antibody cocktails, including positive and negative selection markers, and also markers for cells undergoing epithelial-mesenchymal transition were tested for their sensitivity and specificity to detect tumor cells in blood. Blood samples from NSCLC and SCLC patients are currently being analyzed using the Cell Search system (an EpCAM-based, FDA approved device - the current ‘gold standard' for CTC detection and enumeration), in parallel with the Parsortix device (a micro-fluidic platform that isolates CTCs according to size and deformability). This direct comparison permits an understanding of whether the combination of the Parsortix device with downstream novel antibody cocktails for CTC identification improves sensitivity, over Cell Search, while still retaining good selectivity. Results from this ongoing study will be presented. The goal of this project is to generate and clinically validate a protocol that would enable the use of CTCs as a liquid biopsy for NSCLC patients. Such an approach would be of particular benefit to these patients, since tumor biopsies cannot always being readily attained and the use of invasive intervention carries some risk of bleeding and infection. This work is supported by IMI JU & EFPIA (grand no. 115749). Citation Format: Linda Scarrott, Hanny Musa, Harriet Wikman, Sonja Loges, Juergen Braunger, Klaus Pantel, Ralph Graeser. CANCER ID and the identification of circulating tumor cells (CTCs) in NSCLC [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 3610.

Abstract 3664: A workflow to evaluate PD-L1 protein expression on circulating tumor cells (CTCs) from non-small cell lung cancer (NSCLC)

Abstract Introduction Many tumors evade immune surveillance by deploying immunosuppressive mechanisms and co-opting immune checkpoint pathways, such as PD-1/PD-L1. PD-1/PD-L1 pathway blockade is a highly promising therapy and has elicited durable antitumor responses in a variety of cancers. Challenges in advancing immunotherapies lie in patient stratification and monitoring therapy. Evaluation of PD-L1 expression is generally regarded as an inclusion criterion for clinical trials. Current methods rely on the analysis of excisional biopsies, which are invasive and carry associated medical risks. Isolation and analysis of CTCs is a noninvasive alternative that may provide, in real-time, more representative information on tumor biomarkers, and allow the monitoring of treatment effectiveness over time. Here, we present an integrated workflow for the analysis of PD-L1 protein expression on CTCs from metastatic NSCLC patients isolated with the Vortex Biosciences VTX-1 liquid biopsy system. Methods Cell lines expressing various level of PD-L1 and white blood cells prepared from healthy donors were used to optimize the PD-L1 immunostaining assay. Several PD-L1 antibodies were titrated, and their specificity estimated with isotype controls. PD-L1 expression quantification was evaluated using two independent methods i) Zen 2 imaging (Zeiss) and ii) CellProfiler software. Clinical validation was performed on blood samples collected from a cohort of metastatic NSCLC patients (stage IV) with known PD-L1 tumor status. CTCs were isolated using the VTX-1 Liquid Biopsy System, stained (PD-L1, CK, CD45, CD11b, DAPI) and classified, then PD-L1 expression was quantified. Results We have developed a PD-L1 immunofluorescent assay that shows specificity, sensitivity, and very good intra-assay repeatability. The two methods evaluated for the quantification of PD-L1 expression provided equivalent data and could be used interchangeably. The PD-L1 assay was further validated on CTCs isolated from a small cohort of metastatic NSCLC patients. Preliminary data shows heterogeneity in PD-L1 expression levels across CTCs from the same patient. Interestingly, among PD-L1 positive CTCs, both traditional CK+/PD-L1+ and CK-/PD-L1+ cells were observed. To move toward a more automated workflow for immunostaining, imaging, and fluorescence signal analysis, methods to collect CTC on glass slides were evaluated, with cell recovery ranging from 33 to 98%. Conclusion We demonstrated the feasibility of evaluating PD-L1 protein expression on NSCLC's CTCs isolated with the VTX-1 Liquid Biopsy System. While further clinical validation is warranted, this test could represent a simple, non-invasive method to help identify patients most likely to respond to PD-1/PD-L1 immunotherapies. Future work will focus on compatibility of the assay with autostainers and fully automated imaging and analysis platforms. Citation Format: Corinne Renier, Charles L. Wilkerson, SJ Claire Hur, Da Eun Rachel Park, Clementine A. Lemaire, Melissa Matsumoto, James Carroll, Steve Crouse, Jonathan W. Goldman, Edward B. Garon, Dino Di Carlo, Elodie Christen-Sollier. A workflow to evaluate PD-L1 protein expression on circulating tumor cells (CTCs) from non-small cell lung cancer (NSCLC) [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 3664.

Biology and clinical significance of circulating tumor cell subpopulations in lung cancer.

By identifying and tracking genetic changes in primary tumors and metastases, patients can be stratified for the most efficient therapeutic regimen by screening for known biomarkers. However, retrieving tissues biopsies is not always feasible due to tumor location or risk to patient. Therefore, a liquid biopsies approach offers an appealing solution to an otherwise invasive procedure. The rapid growth of the liquid biopsy field has been aided by improvements in the sensitivity and specificity of enrichment assays for isolating circulating tumor cells (CTCs) from normal surrounding blood cells. Furthermore, the identification and molecular characterization of CTCs has been shown in numerous studies to be of diagnostic and prognostic relevance in breast, prostate and colon cancer patients. Despite these advancements, and the highly metastatic nature of lung cancer, it remains a challenge to detect CTCs in advanced non-small cell lung cancer (NSCLC). It may be that loss of epithelial features, in favor of a mesenchymal phenotype, and the highly heterogeneous nature of NSCLC CTCs contribute to their evasion from current detection methods. By identifying a broader spectrum of biomarkers that could better differentiate the various NSCLC CTCs subpopulations, it may be possible to not only improve detection rates but also to shed light on which CTC clones are likely to drive metastatic initiation. Here we review the biology of CTCs and describe a number of proteins and genetic targets which could potentially be utilized for the dissemination of heterogenic subpopulations of CTCs in NSCLC.

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

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

Abstract 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 > 6 TfR+ CTCs had a worse overall survival (OS) than patients with < 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

Investigating CTCs in NSCLC-a reaction to the study of Jia-Wei Wan: a preliminary study on the relationship between circulating tumor cells count and clinical features in patients with non-small cell lung cancer.

Liquid biopsies are emerging as a patient-friendly approach for estimating biomarkers to predict treatment outcome and overall survival. Detection and characterization of circulating tumor cells (CTCs) is a promising biomarker and its application for non-small cell lung cancer (NSCLC) is currently under investigation. The FDA approved CellSearch system is used as the standard test. The most challenging hurdle in this and other techniques is to discern the CTCs from the background noise of normal blood cells. In the CellSearch system, lysis of erythrocytes, separating leukocytes with anti-CD antibodies, and immunomagnetic enrichment of cancer cells from blood samples expressing membranous epithelial cell adhesion molecule (EpCAM) protein is used. The relevant detection rate using CellSearch for prognosis is set by the developer on >2 (for metastatic colorectal cancer) or >4 CTCs (for metastatic breast and prostate cancer) per 7.5 mL blood sample. Clinical use of the CellSearch system is currently limited in NSCLC because it fails to detect CTCs at an acceptable rate and at a sufficient high yield. Extrapolation of CTC frequency distribution in 7.5 mL of blood from patients with metastatic breast, colon and prostate cancer showed that probably all these patients had CTCs in circulation, but the sample volume was not sufficient to detect them in all patients. Therefore, novel methods for the detection of CTCs are being studied.

Methods for detection of circulating cells in non-small cell lung cancer.

Circulating tumor cells (CTCs) from peripheral blood have been detected in most epithelial malignancies. CTCs are very heterogeneous and can be captured via different technologies based on their physical and biological properties. The detection rates have varied depending on the technology used for enumeration. Detection, monitoring, and molecular analysis of CTCs provide a powerful and noninvasive approach for the detection of early disease, assessing prognosis and therapeutic response in cancer patients. Non-small cell lung cancer (NSCLC) is one of the most lethal malignancies in humans. Compared with other solid tumors, the number of CTCs in NSCLC is relatively low. Nevertheless, NSCLC is a particularly important disease for CTC evaluation for prognostic purposes because of the lack of a reliable protein-based tumor marker. Molecular analyses of CTCs have provided new insights into the biology of metastasis with important implications for the clinical management of cancer patients. We review current and emerging technologies for CTC detection, with a focus on enrichment and molecular analysis of CTCs, and their potential clinical applications in NSCLC.

Quantification of Rare Circulating Tumor Cells in Non-Small Cell Lung Cancer by Ligand-Targeted PCR

BackgroundQuantification of circulating tumor cells (CTC) is valuable for evaluation of non-small cell lung cancer (NSCLC). The sensitivity of current methods constrains their use to detect rare CTCs in early stage. Here we evaluate a novel method, ligand-targeted polymerase chain reaction (LT-PCR), that can detect rare CTCs in NSCLC patients.MethodsCTCs were enriched by immunomagnetic depletion of leukocytes and then labeled by a conjugate of a tumor-specific ligand and an oligonucleotide. After washing off free conjugates, the bound conjugates were stripped from CTCs and then analyzed by qPCR. To evaluate the clinical utility, blood samples were obtained from 72 NSCLC patients (33 initially diagnosed and 39 on chemotherapy), 20 benign patients, and 24 healthy donors.ResultsExperiments with healthy blood spiked with tumor cells indicated the LT-PCR allows specific detection of CTC. The clinical study showed that the initially diagnosed patients have an average of 20.8 CTC units with metastatic diseases, 11.8 CTC units with localized diseases, and 6.0 CTC units with benign diseases. With the threshold of 8.5 CTC units, the assay can detect 80% of stage I/II, 67% of stage III, and 93% of stage IV cancer. With the benign patients and healthy donors as control group, the method can detect cancer with a sensitivity of 81.8% and a specificity of 93.2%.ConclusionThe LT-PCR would allow quantification of CTC in NSCLC patients at a more sensitive level, providing a potential tool for stratifying malignant lung diseases, especially at early stage.

Prognostic Significance of Circulating Tumor Cells in Non-Small-Cell Lung Cancer Patients: A Meta-Analysis

BackgroundThe prognostic significance of circulating tumor cells (CTCs) detected in patients with non-small-cell lung cancer (NSCLC) is still inconsistent. We aimed to assess the prognostic relevance of CTCs using a meta-analysis.MethodsWe searched PubMed, Web of Science and EMBASE for relevant studies that assessed the prognostic relevance of CTCs in NSCLC. Statistical analyses were conducted to calculate the summary incidence, odds ratio, relative risks (RRs) and 95% confidence intervals (CIs) using fixed or random-effects models according to the heterogeneity of included studies.ResultsA total of 20 studies, comprising 1576 patients, met the inclusion criteria. In identified studies, CTCs were not correlated with histology (adenocarcinoma vs squamous cell carcinoma) (odds ratio [OR] = 0.88; 95% confidence interval [CI]: 0.59–1.33; Z = –0.61; P = 0.545). However, pooled analyses showed that CTCs were associated with lymph node metastasis (OR = 2.06; 95% CI: 1.18–3.62; Z = 2.20; P = 0.027) and tumor stage (OR = 1.95; 95% CI: 1.08–3.54; Z = 2.53; P = 0.011). Moreover, CTCs were significantly associated with shorter overall survival (relative risk [RR] = 2.19; 95% CI: 1.53–3.12; Z = 4.32; P<0.0001) and progression-free/disease-free survival (RR = 2.14; 95% CI: 1.36–3.38; Z = 3.28; P<0.0001).ConclusionThe presence of CTCs indicates a poor prognosis in patients with NSCLC. Further well-designed prospective studies are required to explore the clinical applications of CTCs in lung cancer.

A novel method for the in vivo isolation of circulating tumor cells from peripheral blood of cancer patients using a functionalized and structured medical wire

The isolation of circulating tumor cells (CTCs) from the blood of patients afflicted with solid malignant tumors becomes increasingly important as it may serve as a ‘liquid biopsy’ with the potential of monitoring the course of the cancer disease and its response to cancer therapy, with subsequent molecular characterization. For this purpose, we functionalized a structured medical Seldinger guidewire (FSMW), normally used to obtain safe access to blood vessels and other organ cavities, with a chimeric monoclonal antibody directed to the cell surface expressed epithelial cell surface adhesion molecule (EpCAM). This medical device was optimized in vitro and its biocompatibility was tested according to the regulations for medical devices and found to be safe with no noteworthy side effects. Suitability, specificity and sensitivity of the FSMW to catch and enrich CTCs in vivo from circulating peripheral blood were tested in 24 breast cancer or non-small cell lung cancer (NSCLC) patients and in 29 healthy volunteers. For this, the FSMW was inserted through a standard venous cannula into the cubital veins of healthy volunteers or cancer patients for the duration of 30 min. After removal, CTCs were identified by immunocytochemical staining of EpCAM and/or cytokeratins and staining of their nuclei and counted. The FSMW successfully enriched EpCAM-positive CTCs from 22 of the 24 patients, with a median of 5.5 (0–50) CTCs in breast cancer (n=12) and 16 (2–515) CTCs in NSCLC (n=12). CTCs could be isolated across all tumor stages, including early stage cancer, in which distant metastases were not yet diagnosed, while no CTCs could be detected in healthy volunteers. In this observatory study, no adverse effects were noted. Evidently, the FSMW has the potential to become an important device to enrich CTCs in vivo for monitoring the course of the cancer disease and the efficacy of anticancer treatment.

Abstract A12: High-throughput mutation analysis of NSCLC circulating tumor cells

Abstract Background: Circulating tumor cells (CTC) associated with solid tumors are being studied for their diagnostic and prognostic value. In patients with metastatic tumors, CTC presence in the blood has been putatively associated with short survival. Since blood collection is relatively non-invasive, CTC molecular analysis opens up the possibility of monitoring genotypic changes during cancer treatments. Unfortunately, CTCs are not present in large numbers, often at rates as low as one cell per 106-107 leukocytes. Thus, to perform genotypic biomarker analysis on CTCs, methodologies must be developed to using highly specific and sensitive technologies and an enrichment step to increase analytes to detectable levels. Methods: We developed a methodology for detecting mutations in multiple oncogenes and chemotherapy resistance genes in non-small cell lung cancer (NSCLC) CTC specimens using high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) single nucleotide polymorphism (SNP) analysis (MASSarray; Sequenom, Inc.) to determine cancer-associated genetic mutations in lung cancer specimens. This system allows for up to 10 different somatic mutations to be assayed per well in a 384-well format; requires very little DNA; can be used with whole genome amplified (WGA) DNA; and is sensitive enough to use with small samples such as core needle biopsies (CNB), fine needle aspirates (FNA), and CTCs. We developed a lung cancer assay panel of 13 genes/135 mutations (including AKT1, BRAF, CTNNB1, EGFR, ERBB2, KRAS, MEK1, NRAS, PIK3CA, PIK3R1,PTEN, and STK11) to test for somatic mutations in genes representing multiple pathways known to be involved in lung cancer. All assays can detect a mutation in &amp;lt; 25% of a sample. Results: In the effort to analyze CTCs, we first analyzed 57 NSCLC cell lines with known mutations and confirmed known mutation status. Next, we successfully analyzed DNA from 90 frozen and matched FFPE NSCLC resected tissues. Analysis of unamplified and matched WGA cell line DNA quantity CNB and FNA equivalents gave the same mutational status results. Moving to CTC equivalents, we successfully analyzed cell line DNA and matched WGA DNA equivalents of 100–1000 cells with known EGFR L585R and KRAS G34A mutations and negative control DNA (negative for all assays). Next, WGA methodology for direct CTC cell lysate DNA amplification was developed using CTC cell number equivalents (3 – 200 cells) obtained from a typical clinical blood sample CTC preparation. Then, we directly compared unamplified and matched amplified CTC cell equivalents (50, 100, and 200 cells). Analysis of both unamplified and amplified CTC cell equivalents reported identical mutation status results. We have applied this methodology to spiked blood sample and clinical blood sample CTC fractions. Thus, we demonstrated that we are able to study mutations in multiple genes using small amount of DNA from CTC cell numbers in a high-throughput manner. Conclusion: We developed a robust method for accurately determine cancer-associated genetic mutations in NSCLC CTC cell number equivalent lysates using MALDI-TOF MS SNP analysis which can be applied to better understand the molecular characteristics of lung cancer during treatment and progression. As additional clinical NSCLC CTC samples are collected, we will continue applying this methodology to assess CTC mutation status as potential diagnostic and/or prognostic markers.

Analysis of Circulating Tumor Cells in Patients with Non-small Cell Lung Cancer Using Epithelial Marker-Dependent and -Independent Approaches

Epithelial circulating tumor cells (CTCs) are detectable in patients with non-small cell lung cancer (NSCLC). However, epithelial to mesenchymal transition, a widely reported prerequisite for metastasis, may lead to an underestimation of CTC number. We compared directly an epithelial marker-dependent (CellSearch) and a marker-independent (isolation by size of epithelial tumor cells [ISET]) technology platform for the ability to identify CTCs. Molecular characteristics of CTCs were also explored. Paired peripheral blood samples were collected from 40 chemonäive, stages IIIA to IV NSCLC patients. CTCs were enumerated by Epithelial Cell Adhesion Molecule-based immunomagnetic capture (CellSearch, Veridex) and by filtration (ISET, RareCell Diagnostics). CTCs isolated by filtration were assessed by immunohistochemistry for epithelial marker expression (cytokeratins, Epithelial Cell Adhesion Molecule, epidermal growth factor receptor) and for proliferation status (Ki67). CTCs were detected using ISET in 32 of 40 (80%) patients compared with 9 of 40 (23%) patients using CellSearch. A subpopulation of CTCs isolated by ISET did not express epithelial markers. Circulating tumor microemboli (CTM, clusters of ≥ 3 CTCs) were observed in 43% patients using ISET but were undetectable by CellSearch. Up to 62% of single CTCs were positive for the proliferation marker Ki67, whereas cells within CTM were nonproliferative. Both technology platforms detected NSCLC CTCs. ISET detected higher numbers of CTCs including epithelial marker negative tumor cells. ISET also isolated CTM and permitted molecular characterization. Combined with our previous CellSearch data confirming CTC number as an independent prognostic biomarker for NSCLC, we propose that this complementary dual technology approach to CTC analysis allows more complete exploration of CTCs in patients with NSCLC.