Size-selective collection of circulating tumor cells using Vortex technology

“Real-time liquid biopsy” was introduced in 2010 for the analysis of circulating tumor cells (CTCs) in the blood of cancer patients (1), and this new diagnostic approach has received enormous attention because of its obvious clinical implications for personalized medicine. CTCs are derived from primary tumors and metastatic lesions and, therefore, harbor important information on the molecular characteristics relevant to tumor progression and cancer therapy. Analyses of CTCs have paved new diagnostic avenues with obvious clinical implications for personalized medicine. Key areas of clinical applications of CTCs include detection of cancer, prediction of prognosis in patients with curable disease, monitoring of systemic therapies, and stratification of patients based on the detection of therapeutic targets or resistance mechanisms (2). Much progresses has been made in the development of various technologies to enrich and detect CTCs; however, the discovery and validation of new CTC biomarkers are still in its infancy (3). Different types of CTC characterization studies have been carried out: (i) descriptive studies on isolated individual CTCs requiring a specific isolation process and a process of whole-genome or transcriptome amplification, which are informative on intrapatient heterogeneity; (ii) descriptive studies providing information at the individual cell level, which require only an enrichment but no single-cell isolation; (iii) functional studies on pools of total CTCs used for cell culture and/or xenografts (4). Since current assays cannot distinguish between apoptotic and viable CTCs, it is possible to apply the EPISPOT assay that detects proteins secreted/released/shed from viable epithelial cancer cells (5). Cells are cultured for a short time on a membrane coated with antibodies that capture the secreted/released/shed proteins that are subsequently detected by secondary antibodies labeled with fluorochromes. This functional assay detects viable CTCs at the single-cell level and has been used on hundreds of patients with different tumor types including epithelial tumors (breast, prostate, ovarian, and colon cancer) and melanomas, showing its clinical relevance (6). The fluoro-EPISPOT assay has been optimized for single CTCs, is now named EPIDROP, and should be a more reliable and sensitive technology than those already existing in this field of expertise, allowing the analysis of proteome and secretome simultaneously of unique functional CTCs or of clusters of CTCs. In addition, testing different drugs in an individual patient might lead to a personalized "oncogram" that might help to improve the clinical management of cancer patients. Moreover, CTC research opens a new avenue for understanding the biology of metastasis in cancer patients. Understanding the molecular mechanisms that regulate the biology of metastasis-competent CTCs is of utmost importance in unraveling the formation of metastases and tumor relapse in patients with cancer (3). However, an in-depth investigation of CTCs is hampered by the very low number of these cells, especially in the blood of colorectal cancer patients. Thus, the establishment of cell cultures and permanent cell lines from CTCs has become the most challenging task over the past year. In 2015, Cayrefourcq et al. described for the first time the establishment of a permanent cell line from CTCs of one metastatic colon cancer patient (7). The cell line, designated "CTC-MCC-41," is stable for more than three years and cells have been characterized at the genome, transcriptome, proteome, and secretome levels. This thorough analysis showed that CTC-MCC-41 cells resemble characteristics of the original tumor cells in the colon cancer patient and display a stable phenotype characterized by an intermediate epithelial/mesenchymal phenotype, stem-cell like properties and an osteomimetic signature indicating a bone marrow origin. Functional studies showed that CTC-MCC-41 cells induced rapidly in vitro endothelial cell tube formation and in vivo tumors after xenografting in immunodeficient mice. Subsequently, Alix-Panabières et al. determined the molecular bases underlying differences between this colon CTC line and well-described cancer cell lines derived from primary tumors and from metastatic sites (8). The results showed clearly that the CTC-MCC-41 displayed a very specific transcription program. Interestingly, among the 1,624 transcripts exclusively upregulated in CTC-MCC-41, key genes related to energy metabolism, DNA repair, and stemness genes were observed. Furthermore, blood samples collected during the course of the systemic therapy and tumor progression from the same metastatic colon cancer patient who gave rise to the CTC-MCC1-41 line have allowed the acquisition of 8 additional colon CTC lines. To our best knowledge, there is no other report on the in vitro expansion of CTCs from cancer patients during the course of their disease and treatment. This unique biologic material represents a chance to understand the clonal selection and resistance mechanisms during tumor progression and cancer treatment. In conclusion, functional analyses of CTCs provide insights for the discovery of new biomarkers to identify the most aggressive CTC subpopulations with stem cell properties. CTC lines could contribute to the development of new drugs to eradicate metastasis-initiator CTCs causing relapses and cancer-related death in individuals with cancer.

[Background] It is well known that emerging mutations which is not found in the primary tumor exist in metastatic tumor and molecular therapy induce emerging mutation. Liquid biopsy, which includes circulating tumor cell (CTC) and circulating tumor DNA (ctDNA), may help detecting this spatial and temporal heterogeneity. We have reported that emerging KRAS mutation can be detected by using ctDNA (Yamada et al, Cancer Science 2016). However, mutation detection by using CTC has been difficult because enough amount of DNA cannot be extracted from CTC. Currently we have been able to collect more CTC than before, by using a new device which uses 3 antibodies (EpCAM, Her2, Trop2). In this study, we evaluated the potential to detect colorectal cancer (CRC) related gene mutations from CTC, and compared it with ctDNA. [Methods] Cohort 1: This cohort included untreated CRC patients. Tumor tissue was collected from each patient, either by primary surgery or by colonoscopic biopsy. DNA was extracted from tumor tissue and was analyzed using Next Generation Sequencing (NGS). Ten mL of whole blood was also collected from the same patient. CTC, serum and white blood cell (WBC) was collected by using the CTC recovery machine (Ion Torrent Liquid Biopsy Instrument®). Cytokeratin positive, DAPI positive, CD45 negative cells were defined as CTC. DNA was extracted from each sample (CTC-DNA, ctDNA, WBC-DNA) and was analyzed using NGS. Cohort 2: This cohort included unresectable CRC patients with KRAS mutation in their primary tumor. All patients in this cohort were under treatment or after completion of chemotherapy. CTC and ctDNA was collected in the same method as cohort 1. KRAS mutations of CTC and ctDNA were detected by using digital PCR (dPCR). [Results] Cohort 1: We enrolled 16 CRC patients (stage II: n=2, stage III: n=2, stage IV: n=12). A total of 30 somatic, hotspot mutations were detected from tumor tissue DNA. The median number of the detected mutation for each patient was 2 (0-4). The most frequent gene mutation was APC, followed by KRAS and TP53. In all patients, CTC was successfully collected. The median number of the CTC was 34 cells (5-94). However, only 6 somatic, mutations were detected from CTC-DNA. Conversely, 16 somatic mutations were detected from ctDNA. Cohort 2: We enrolled 14 stage IV CRC patients with KRAS mutation in their primary tumor. CTC was collected from 9 patients but not from 5 patients. In the 9 patients CTC was collected, the median number of the collected CTC was 26 cells (5-121). By dPCR, KRAS mutation was detected in 2 patients (2/14) from CTC-DNA, and 4 patients (4/14) from ctDNA. [Conclusions] The new CTC capturing technology using 3 antibodies can improve detection rate and yield of CTC. However, in patients undergoing chemotherapy, the amount of CTC and ctDNA drastically reduces. At present, ctDNA is superior to CTC in potential to detect mutations, and dPCR is more sensitive than NGS to detect mutations. Citation Format: Kohki Takeda, Takeshi Yamada, Michihiro Koizumi, Seiichi Shinji, Yasuyuki Yokoyama, Goro Takahashi, Masahiro Hotta, Takuma Iwai, Keisuke Hara, Hiroyasu Furuki, Eiji Uchida. Detection of colorectal cancer related gene mutations from CTC and ctDNA [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 4594.

The Promise of Circulating Tumor Cells in Metastatic CRPC

11046 Background: To date, detecting circulating tumor cells (CTCs) in the peripheral blood of pancreatic patients using standard immuno-capture techniques has met with limited success. As pancreatic cancer is prone to metastasize at distant sites, and therefore should have high numbers of CTCs, it is possible that immuno-capture methods are not suitable for this disease. Using a microfiltration approach, we show that CTCs are present in the peripheral blood in over 75% of pancreatic cancer patients, and that two distinct subtypes can be identified. Methods: Pancreatic patient samples were provided by Medical College of Wisconsin, Milwaukee, WI. CellSieve microfilters, with precision 7 micron diameter pores distributed in uniform arrays were employed. 7.5 mL of whole blood was diluted in pre-fixation solution and filtered through CellSieve microfilters. CTCs collected by this size exclusion technique were fixed, permeabilized, and stained with DAPI, an antibody cocktail against cytokeratin 8, 18 and 19 (FITC), EpCAM (PE), and CD45 (Cy5). CTCs, defined as cytokeratin positive and CD45 negative, were found in two distinct subtypes. One subtype had the “classic” characteristics of a CTC, with high EpCAM and cytokeratin expression, identifiable cytokeratin filamentation, and a cancer-like nuclear structure. The second subtype is indicative of a CTC undergoing epithelial-mesenchymal transition (EMT), with low or no EpCAM, weak cytokeratin expression, and a smooth oval nuclear structure. Results: The “classic” CTCs were found in ~20% (n=40) of patient samples. The EMT-like CTCs were found in ~75% of the same patient cohort. Neither cell was present in any healthy subjects (n=30). EMT-like CTCs consistently lacked EpCAM expression and commonly presented as multi-cell clusters, or microemboli, in ~40% of the cases. Conclusions: We show that two distinct CTC subtypes circulate in the blood of most pancreatic patients. The low expression of cytokeratin and EpCAM of the EMT-like subtype implies that immuno-capture based CTC isolation methods have limited utility for pancreatic cancer. Further, this subtype provides a useful strategy for tracking pancreatic CTCs over the course of treatment.

A fatal affair: Circulating tumor cell relationships that shape metastasis

792P - Clinical significance of early circulating tumor cells (CTC) changes, analyzed by AdnaTest, in patients (pts) receiving first-line methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) chemotherapy (CT) for metastatic urothelial cancer (UC)

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

CTC Technologies and Tools.

Introduction The need for a liquid biopsy in non-small cell lung cancer (NSCLC) patients is rapidly increasing as more and more targeted therapies become available. Presence in blood of circulating tumor cells (CTC), tumor derived extracellular vesicles (tdEV) and cell-free circulating tumor DNA (ctDNA) measured with different approaches are being explored for their potential to represent a liquid biopsy in the European and Dutch CANCER-ID projects (https://www.cancer-id.eu/ & https://www.utwente.nl/tnw/cancer-id/). Here, we determine in just one 7.5 mL tube of blood the presence of CTC, tdEV and ctDNA and investigate the relation with survival of metastatic NSCLC patients. Methods In total 106 advanced NSCLC patients were enrolled in the study. In 86 patients EpCAM+ CTC, EpCAM- CTC & tdEV were enumerated and in 50 patients EpCAM+ CTC, EpCAM- CTC, tdEV & ctDNA, all from one CellSave blood tube. ctDNA from a separate plasma tube is available for all patients but not yet analyzed. Before placing the sample in the CellSearch system, plasma was aspirated and stored at -80°C. EpCAM+ CTC were enumerated by CellSearch and EpCAM- CTC after filtration of the EpCAM+ CTC depleted blood through 5µm pore filters, as described by de Wit et al. (Sci. Rep. doi: 10.1038/srep12270, 2015). tdEV were defined by a multidimensional gate as cytokeratin+/DAPI-/CD45- vesicles and identified in the CellSearch images, using the open source image analysis program ACCEPT. The stored plasma was used for ctDNA quantification with the FAST-SeqS approach, described by Belic et al. (ClinChem 61, 838, 2015). In several patients with EpCAM- CTC, fluorescent in situ hybridization was performed on the filter to establish the cancerous origin of the EpCAM- CTC. Results In 24% of the patients ≥1 EpCAM+ CTC as well as ≥1 EpCAM- CTC were detected in 7.5 mL of blood. In 30% of the patients tdEV were present at a frequency >45 per 7.5 mL. This frequency is based on the mean + 2SD from 42 healthy controls. In 20% of the patients >10% ctDNA load was found. No significant correlation was found between the presence of these biomarkers. Presence of all four biomarkers was detected in 6% of patients and at least one of four was found in 52% of patients. One or more EpCAM+ CTC were associated with poor overall survival (p=0.010 for 86 patients and p=0.019 for 50 patients), whereas EpCAM- CTC were not (p=0.495 n=86; p=0.571 n=50). The latter is surprising since some CTC were shown to be cytogenetically aberrant, conform the primary tumor. The presence of >45 tdEV (p=0.271 n=50) and >10% ctDNA (p=0.082 n=50) did not reach significance. Conclusions In this study EpCAM+ CTC, EpCAM- CTC, tdEV or ctDNA was detected in one tube of blood in 52% of the NSCLC patients. Only the presence of EpCAM+ CTC was associated with poor overall survival, raising the question whether or not the extraction of molecular information from these other biomarkers can be used to predict response to treatment in NSCLC. To increase the percentage of patients from which a liquid biopsy can be obtained, the analyzed blood volume will need to be increased. Citation Format: Sanne de Wit, Menno Tamminga, Joost F. Swennenhuis, Leonie L. Zeune, Ellen Heitzer, Michael Speicher, T Jeroen N. Hiltermann, Leon WMM Terstappen, Harry JM Groen. Liquid biopsy in NSCLC: EpCAM+ and EpCAM- circulating tumor cells, tumor derived extracellular vesicles and cell-free circulating tumor DNA [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 LB-250. doi:10.1158/1538-7445.AM2017-LB-250

Introduction: Single-cell RNA sequencing (scRNA-Seq) of rare cells, such as circulating tumor cells (CTCs), is critical for identifying various cell subtypes and understanding intercellular heterogeneity at the genetic level. Despite recent advances in scRNA-Seq, transcriptomic profiling of single CTCs has been technically demanding due to its limited cell utilization and background noise from unwanted cells during sequencing. In addition, conventional CTC isolation methods often miss important rare cell subtypes due to their low abundance and reliance on specific surface markers for detection. This limitation hinders comprehensive transcriptomic analysis, especially for CTCs undergoing the critical epithelial-mesenchymal transition (EMT), which may not express typical epithelial markers. To address these challenges, we present an optimized workflow that integrates high-purity label-free CTC capture with in-droplet barcoding-based scRNA-seq to achieve higher cell utilization and lower background sequencing noise. Methods: To model heterogeneous CTC subpopulations at different EMT status, samples were prepared by spiking in two types of cancer cells, MCF7 (epithelial CTCs) and HeLa (mesenchymal CTCs), into a total of 7.5 ml of whole blood from a healthy donor at a concentration of 100 cells/ml. CTCs were captured using the Genesis Cell Isolation System and the enriched CTCs were further purified to reduce leukocyte contamination or red blood cell (RBC) contamination using CD45 pre-conjugated magnetic beads or RBC lysis reagent, respectively. The processed CTC sample was then used to create scRNA-Seq libraries following the Bio-Rad ddSEQ Single-Cell 3’ RNA-Seq Kit protocol, which includes single-cell in-droplet barcoding, reverse transcription, and cDNA synthesis. Results: This Genesis System allowed for capturing both epithelial and mesenchymal CTCs to be sequenced. After sequencing and Omnition Software Analysis, individual cells were visualized using a Uniform Manifold Approximation and Projection (UMAP) plot and clustering analysis showed a distinct separation between epithelial and mesenchymal CTCs while each different cell population revealed expression of representative genes for each cell type. Discussion and Conclusions: By combining rare single cell capture with scRNA-seq library prep and analysis, our workflow provides an easy, highly sensitive and cost-effective solution for CTC single-cell transcriptomic studies, compatible with commercially available instruments and kits. This scRNA-seq workflow can be integrated into a broad spectrum of applications in liquid biopsy across many different cancer types and empowering researchers to gain deeper insights into tumor heterogeneity and gene expression patterns with greater efficiency and within the same budget.​ Citation Format: Yoon-Tae Kang, Patricia Schnepp, Rashika Shrestha, Rain Yu Hong, Errile Pusod, Will Chow, Floyd Watkins, Kyle Thurston, Dominique Winston, Marta Gonzalez-Plasky, Angelica Olcott, David Coe, Michelle Racey, Elizabeth Dreskin. Transcriptomic subtype identification of circulating tumor cells by label-free capture and single-cell RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1982.

Background: Metastatic breast cancer (MBC) subtype characterization is critical for effective treatment. The DefineMBC™ multiomic blood biopsy clinical diagnostic combines ctDNA and circulating tumor cell (CTC) analyses to characterize MBC patients when a tissue biopsy is not feasible. Innovation in the DefineMBC 2.0 5-channel immunofluorescent (IF) CTC characterization assay now measures ultralow (UL) levels of HER2 protein expression, ER protein expression, and chromosomal instability (CI) as well as ERBB2 amplification via single- cell sequencing. Cell-level HER2/ER co-detection enables monitoring of receptor conversion during a patient’s MBC treatment journey. As trastuzumab-deruxtecan (T-DxD) has become standard of care for HER2-low disease, distinguishing HER2-UL from HER2[-] by DefineMBC’s liquid biopsy approach may identify additional patients who can benefit from such therapy. This addresses an unmet need given the known shortcomings of tissue biopsy IHC in the metastatic setting as recently noted by communications from NCCN, ASCO, and the College of American Pathologists. Methods: Assay development including analytical validation studies utilized biologically relevant cell lines of epithelial origin and known expression levels of HER2 and ER (MDA-MB-453: HER2[+] & ER[-]; MCF-7: HER2-low & ER[+]; and MCF-7/ERBB2 knockout: HER2[-]) that were spiked into healthy donor blood. Additionally, patients with various forms of MBC were characterized with the new assay. As with all samples, following red blood cell lysis, nucleated cells were deposited on glass slides at high density (∼3E6 WBC/slide). Slides underwent IF staining for cytokeratins (CK), CD31, CD45, HER2, ER, and Hoechst to localize nuclear DNA, followed by scanning and machine learning-based rare cell detection. CK[+], CD31[-]/CD45[-] cells were classified as CTC candidates and assessed for HER2/ER expression. Pathologist-selected CTCs were isolated for single-cell whole genome sequencing and analyzed using a proprietary CTC DNA copy number pipeline to detect CI and amplification at the ERBB2 locus. Results: Analytical validation studies demonstrated the limit of detection at 1 CTC per 12E6 WBC (∼1.6 mL of blood) with a linear range of 1- 300 CTCs per slide. Sensitivity, specificity, accuracy, and precision metrics of the HER2 and ER IF assays were 96%, 96%, 96%, 2% CV; 91%, 95%, 93%, 3% CV; respectively. CTC enumeration precision was validated at greater than 80% for enumeration bins of 0, 1-10, 11-50, and>50 CTCs per sample. During patient testing 7 patients previously called HER2[-] are now designated as consistent with HER2-low by the pathologist, suggesting improved test performance in future clinical validation studies is likely. Conclusions: Advancement of our comprehensive cancer profiling platform now includes ER/HER2-UL/CI co-detection on enumerated CTCs in MBC patients. Clinical studies currently in progress will reveal whether HER2-UL/HER2[+] detection on CTCs will identify patients better suited for T-DxD or trastuzumab, respectively. Citation Format: Raj Srikrishnan, Jordan Ritchie, Alessandra Cunsolo, Andrew Kunihiro, Brandon Guillory, Sara Tin, Zhuo Meng, Ernest T Lam, Bharat Annaldas, Evan Schwab, Nilesh Dharajiya, Timothy Pluard, Martin Blankfard, David Bourdon. Analytical validation of a novel multiomic metastatic breast cancer liquid biopsy test that combines ctDNA analysis with CTC HER2-ultralow and ER co-expression, as well as single-cell chromosomal instability [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A001.

Microfluidics and Circulating Tumor Cells

e15040 Background: Liquid biopsies analyzing circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) allow minimally invasive monitoring and testing of lung cancer at different stages. It is known that 90% of patients succumb due to metastasis. However, accounting for patients with early metastatic signatures is extremely challenging. In addition, monitoring minimal residual disease (MRD) and identifying patients for recurrence is very prudent. While the role of CTCs in % prediction of survival has been established in several cancers. However, CTC's co-occurrence role with CtDNA and vice versa is not implored in monitoring the aggressiveness of the disease, response to therapies, and therapy decisions. In this study, we investigated ctDNA and CTC's combined roles in monitoring disease aggressiveness and metastasis in lung cancer patients. Methods: A cohort of 265 lung cancer patients with late-stage cancer were retrospectively analyzed for co-occurrence of dual biomarker ctDNA and CTC. The results were correlated as quadrant to assess clinical disease states, obtained from PET scans and HPE findings. Next Generation Sequencing (NGS) test was performed using OncoMonitor dual biomarker assay having CTC enumeration with PD-L1 expression. CTC count was performed using the OncoDiscover Liquid Biopsy Test, approved by CDSCO-India, in 1.5 ml of blood. Results: CTC distribution in this study ranged from 1-8 CTCs with a mean CTC distribution of 1.22. Amongst these patients, 75.47% (n = 200) showed the presence of CTCs and amongst these 200, 91.50% (n = 183) showed PD-L1 expression on their CTCs with a mean value of 0.99. While both biomarkers were positive for ctDNA and CTC (ctDNA+/CTC+) in 135 (50.94%) patients. Interestingly, only 19 (7.17%) patients were negative for both ctDNA and CTC (ctDNA-/CTC-). Similarly, 43 patients (16.23) were positive for ctDNA and negative for CTC (ctDNA+/CTC-), while 68 (25.66%) patients were negative for ctDNA and positive for CTC (ctDNA-/CTC+). The ctDNA+/CTC- cohort had the highest metastatic rate of 62.8%, with ctDNA+/CTC+ at 57.0%. Noteworthy, the ctDNA+ cohort showed the highest % of progressive disease patients with 20.2% and 18.6% along with CTC+ and CTC- status, respectively. The mutations, EGFR, TP53, and KRAS were observed in 62.64% (166/265) of patients. Only stable disease was observed in 29.4% of patients when both biomarkers ctDNA-/CTC- were absent. Conclusions: Overall, the ctDNA+ cohort showed a higher rate of MRD, progression, and metastasis with no stable disease. The quadrant that combined clinical results of the CTC-PD-L1 cells and CtDNA manifest the non-invasive monitoring of disease progression, treatment response, complete remission, and utility of early metastatic detection in lung cancer patients.

Background: Isolation of circulating tumor cells (CTCs) from peripheral blood based on size exclusion is rapid and straight-forward using precision microfilters. We describe the use of CellSieveTM microfilters to isolate CTCs from the peripheral blood of breast, prostate, and pancreatic cancer patients. It is accepted that CTCs isolated from patient samples represent a highly heterogeneous population with varying degrees of epithelial mesenchymal differentiation. We hypothesized that the CTCs from three different epithelial malignancies can be identified and grouped into distinct subtypes by morphological characterization. Methods: Prostate, breast, and pancreatic patient blood samples were provided by Northwestern University, Fox Chase Cancer Center, University of Maryland, and Medical College of Wisconsin and analyzed by Creatv MicroTech. The CellSieveTM microfilters have 8 micron diameter pores in a uniform array, with 160,000 pores in a 9 mm diameter area. 7.5 mL of whole blood was diluted in fixative and drawn through a microfilter. CTCs collected by this size exclusion technique were post-fixed, permeabilized, and stained with DAPI, cytokeratin 8, 18 and 19 (FITC), EpCAM (PE), PSMA (Texas Red), and CD45 (Cy5). CTCs were CD45 negative cells identified by their morphology, nuclear profile, and expression of cytokeratin, PSMA, and EpCAM. Results: Each patient sample was found to have a number of phenotypic CTC subtypes. Distinct morphological patterns emerged in the three malignancies. CTCs from breast cancer patients demonstrated high expression of cytokeratin signal with web-like cytokeratin filamentation. Prostate cancer CTCs had less defined filamentation, but intense PSMA and cytokeratin signal and mottled cytokeratin morphology. Pancreatic CTCs had extremely fine filamentation, with spindle-like morphology and little or no EpCAM expression. Within each cancer, CTCs could be grouped into distinct subtypes. Additional markers, such as vimentin (PE), are used to further analyze the cells after bleaching the original PE. Conclusions: In addition to enumeration and identification, the phenotypic analysis of CTCs provides new information that can be used to characterize disease status for personalized treatment of cancer patients. We have shown that CTCs can have multiple distinct phenotypes. These phenotypic morphologies may implicate definable traits which can be exploited while tracking site directed treatment of metastatic cancer patients. Citation Format: Daniel Adams, R. Katherine Alpaugh, Massimo Cristofanilli, Stuart Martin, Saranya Chumsri, Monica Charpentier, Raymond C. Bergan, Irene May Ogden, Susan Tsai, Peixuan Zhu, Olga V. Makarova, Shuhong Li, Platte T. Amstutz, Cha-Mei Tang. Identifying and subtyping circulating tumor cells from breast, prostate, and pancreatic cancer patients based on distinct morphology. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1448. doi:10.1158/1538-7445.AM2013-1448

Circulating tumor cells