Circulating Tumor Cells Phenotypes Research Articles

Cellular Mechanisms of Circulating Tumor Cells During Breast Cancer Metastasis.

Circulating tumor cells (CTCs) are cancer cells that detach from the primary site and travel in the blood stream. A higher number of CTCs increases the risk of breast cancer metastasis, and it is inversely associated with the survival rates of patients with breast cancer. Although the numbers of CTCs are generally low and the majority of CTCs die in circulation, the survival of a few CTCs can seed the development of a tumor at a secondary location. An increasing number of studies demonstrate that CTCs undergo modification in response to the dynamic biophysical environment in the blood due in part to fluid shear stress. Fluid shear stress generates reactive oxygen species (ROS), triggers redox-sensitive cell signaling, and alters the function of intracellular organelles. In particular, the mitochondrion is an important target organelle in determining the metastatic phenotype of CTCs. In healthy cells, mitochondria produce adenosine triphosphate (ATP) via oxidative phosphorylation in the electron transport chain, and during oxidative phosphorylation, they produce physiological levels of ROS. Mitochondria also govern death mechanisms such as apoptosis and mitochondrial permeability transition pore opening to, in order eliminate unwanted or damaged cells. However, in cancer cells, mitochondria are dysregulated, causing aberrant energy metabolism, redox homeostasis, and cell death pathways that may favor cancer invasiveness. In this review, we discuss the influence of fluid shear stress on CTCs with an emphasis on breast cancer pathology, then discuss alterations of cellular mechanisms that may increase the metastatic potentials of CTCs.

Correlations between circulating tumor cell phenotyping and 18F-fluorodeoxyglucose positron emission tomography uptake in non-small cell lung cancer.

The epithelial-to-mesenchymal transition (EMT) phenotype-based subsets of circulating tumor cells (CTCs) might be predictors of tumor progression. We evaluated the clinical properties of different phenotypic CTCs in patients with non-small cell lung cancer (NSCLC). Secondly, we explored the association between different phenotypic CTCs and the uptake of 18F-fluorodeoxyglucose (FDG) by the primary tumor on a positron emission tomographic (PET) scan. Venous blood samples from 34 pathologically confirmed Stage IIB-IVB NSCLC patients were collected prospectively. CTCs were immunoassayed using a SE-i·FISH®CTC kit. We identified CTCs into cytokeratin positive (CK+) and cytokeratin negative (CK-) phenotypes. CTC classifications were correlated with the maximum standardized uptake value (SUVmax) measured by 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT). Overall survival (OS) and progression-free survival (PFS) curves were produced using the Kaplan-Meier method. CTCs were detected in 91.2% of NSCLC patients. CTC counting was associated with TNM stage (P = 0.014) and distant metastasis (P = 0.007). The number of CK-CTCs was also positively associated with TNM stage (P = 0.022) and distant metastasis (P = 0.007). Both total CTC counting and CK-CTC counting did not show association with SUVmax value (P = 0.959, P = 0.903). Kaplan-Meier survival analysis demonstrated that patients with ≥ 7 CTCs had shorter OS (P = 0.003) and PFS (P = 0.001)relativeto patients with < 7 CTCs). Notably, the number of CK-CTCs can act as independent risk factors for PFS (P = 0.044) and OS (P = 0.043) in NSCLC patients. However, SUVmax value was not associated with OS (P = 0.895) and PFS (P = 0.686). The CTC subpopulations could be useful evidence for testing metastasis and prognosis in NSCLC patients. The SUVmax value of the primary tumor was not related to prognosis in patients with NSCLC.

Relationship between circulating tumor cells undergoing EMT and short-term efficacy following interventional treatment in patients with hepatocellular carcinoma

ObjectiveA growing number of studies have indicated that epithelial-mesenchymal transition (EMT) phenotypes and the number of circulating tumor cells (CTCs) are significant indicators of tumor characteristics and treatment efficacy, and thus have a broad range of potential applications in the diagnosis and treatment of malignant tumors. The value of data on CTC phenotypes and CTC counts in the diagnosis of hepatocellular carcinoma (HCC) and assessment of efficacy after comprehensive interventional therapy remains unclear. MethodsData of 107 patients who exhibited space-occupying lesions in the liver on enhanced CT/MRI scans at the Guangdong Provincial People’s Hospital (a tertiary medical center) between August 2017 and October 2018, were retrospectively analyzed. All enrolled patients were treated with transcatheter arterial chemoembolization (TACE) combined with microwave ablation (MWA). An imFISH CTC assay was used to isolate and count CTCs with different EMT phenotypes in the patients’ peripheral blood, which facilitated an analysis of the value of CTC phenotype and CTC count data in the diagnosis or treatment of HCC. ResultsThe CTC count and EMT phenotypes in HCC patients were not associated with patient characteristics such as age, sex, Hepatitis B Virus (HBV)-DNA status, alcohol consumption history, Aspartate Transaminase (AST) to Platelet Ratio Index (APRI) score, Eastern Cooperative Oncology Group (ECOG) score, Child-Pugh score, alpha-fetoprotein (AFP), number and size of tumors, vascular invasion, or metastasis (P ​> ​0.05). The CTC count and EMT phenotypes in HCC patients before treatment were not predictive of short-term efficacy (P ​> ​0.05). Comprehensive interventional therapy reduced the total CTC count and mesenchymal CTC count (P ​= ​0.034 and 0.022, respectively). ConclusionTACE in combination with ablation reduced the total CTC count and mesenchymal CTC count. The CTC count and EMT phenotypes may be associated with long-term efficacy.

Abstract IA09: Beyond counting: Imaging flow cytometry-based detection of therapeutic molecular targets in circulating tumor cells

Abstract Detection of circulating tumor cells (CTCs) has been established as a blood-based biomarker of poor prognosis in most types of epithelial cancer. The observation of increased CTC detection rates in patients with metastatic disease supported the hypothesis that CTCs might represent the precursor cells of distant metastases. Experimental proof for this hypothesis was provided by studies in experimental mouse models in which single metastasis-initiating cells within the total pool of CTCs could be causally linked to the formation of distant metastases. In addition, a potential of CTCs of reseeding at the primary site has been reported, suggesting that CTCs could also play a role in local tumor recurrence. Enumeration and molecular characterization of CTCs might thus be clinically exploited for improving the clinical management of epithelial cancers both in the locally advanced and recurrent/metastatic disease setting. Numerous studies reported on the presence of CTCs in peripheral blood samples of head and neck squamous cell carcinoma (HNSCC) patients. Significant associations with clinical high-risk parameters such as advanced stage, high tumor burden, nodal disease, and distant metastasis were reported. Accordingly, CTC detection was correlated with increased risk of local and/or distant recurrence, progression-free and overall survival. Current data in HNSCC are not yet robust enough for the implementation of CTC detection in clinical routine. In addition, the mere enumeration of CTCs has proven insufficiently informative to prompt widespread clinical adoption. There is accumulating evidence that more extended phenotyping of CTCs might be necessary for improving their diagnostic value. Indeed, despite the estimated daily shedding of millions of tumor cells from the bulk tumor tissue, only a small subpopulation of CTCs is able to reach and survive in the blood circulation and to contribute to the formation of distant metastasis. Identification of the true metastasis-inducing subclones within the bulk CTC population remains a challenge but is imperative in order to improve the diagnostic potential of CTCs. In addition, improved knowledge of the relevant biologic mechanisms endowing CTCs with the potential to emigrate from the primary site to blood circulation, to survive their journey, and to reseed at distant organs will support the development of novel antimetastatic therapies. Several technologies are currently available that allow multiparametric CTC phenotyping at the single-cell level. These platforms include imaging flow cytometry (IFC), by which multispectral images from a large number of cells acquired in suspension can be analyzed. By combining the features of flow cytometry and fluorescent microscopy, information on the cell morphology together with the extent of target protein expression and its subcellular localization can be collected. This provides a unique opportunity for statistical analysis of large populations, but also helps in avoiding artifacts. In the talk, the evidence of a prognostic role of CTCs in HNSCc will be summarized. Results from recent studies reporting on multiparametric CTC phenotyping at the single-cell level will be presented. Examples of CTC-based biomarkers that could be exploited for the optimization of current molecular treatment strategies in HNSCC will be discussed. Citation Format: Ingeborg Tinhofer. Beyond counting: Imaging flow cytometry-based detection of therapeutic molecular targets in circulating tumor cells [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr IA09.

Epithelial-Mesenchymal Transition Status of Circulating Tumor Cells Is Associated With Tumor Relapse in Head and Neck Squamous Cell Carcinoma.

We aimed to elucidate the clinical implication of the epithelial-mesenchymal plasticity of circulating tumor cells (CTCs) in patients with head and neck squamous cell carcinoma (HNSCC). CTCs isolated from 44 patients with non-recurrent/metastatic HNSCC and 42 with recurrent/metastatic (R/M) HNSCC were classified into four epithelial-mesenchymal transition (EMT) statuses based on the expression of epithelial (keratin 19) and mesenchymal (vimentin) markers and the relationships between EMT status in CTCs and clinical factors were investigated. E+M- CTC phenotype was more frequent in patients without recurrence/metastasis (p=0.0468) and was also more frequent in those with a complete response (p=0.0346). The E+M+ phenotype constituted the major proportion of the CTCs detected in patients with R/M HNSCC (p=0.0374). CTCs may play unique roles at various stages of metastasis through transitioning from epithelial to mesenchymal phenotypes.

Assessment of PD-L1 expression in circulating tumor cells in patients with breast cancer.

e13001 Background: Evidence is accumulating that the immune checkpoint blockade targeting the programmed death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) axis has improved progression-free and overall survival of advanced breast cancer patients. PD-L1 tumor expression is emerging as a potential biomarker in PD-1/PD-L1 checkpoint blockade therapies. However, PD-L1 expression on tumor tissue is limited by the temporal and spatial heterogeneity as well as insufficient tumor tissue samples. PD-L1 expression in circulating tumor cells (CTCs) might overcome the limitation. We investigated the utility of CTCs as a noninvasive method to evaluate PD-L1 status in breast cancer patients. Methods: 46 breast cancer patients including 42 metastatic breast cancer patients and four early breast cancer patients were enrolled. CTCs were collected from peripheral blood samples using a microfluidic-based CTC separator. A novel staining procedure which included fluorescent glucose analog staining and immunostaining targeting CD45, vimentin and PD-L1 were analyzed. PD-L1 expression level was analyzed as the fraction of PD-L1 positive CTCs among total CTCs. Results: 46 breast cancer patients all had detected CTCs, ranging from 12 to 110. The fraction of PD-L1 positive CTCs varied from 2.08% to 85.69%. PD-L1 expression was obviously higher in triple negative breast cancer (TNBC) patients as compared with non-TNBC patients (47.16% vs 26.23%, P &lt; 0.01). Interestingly, we observed that PD-L1 positive CTCs were also detected in four early TNBC patients. No significant association was found between PD-L1 expression and mesenchymal CTC phenotype. Conclusions: These data suggest that assessment of PD-L1 expression in CTCs from breast cancer patients is feasible. PD-L1 expression in CTCs might serve as a complementary tool for PD-L1 expression analysis in breast cancer patients.

Spectrally Combined Encoding for Profiling Heterogeneous Circulating Tumor Cells Using a Multifunctional Nanosphere‐Mediated Microfluidic Platform

AbstractComprehensive phenotypic profiling of heterogeneous circulating tumor cells (CTCs) at single‐cell resolution has great importance for cancer management. Herein, a novel spectrally combined encoding (SCE) strategy was proposed for multiplex biomarker profiling of single CTCs using a multifunctional nanosphere‐mediated microfluidic platform. Different cellular biomarkers uniquely labeled by multifunctional nanosphere barcodes, possessing identical magnetic tags and distinct optical signatures, enabled isolation of heterogeneous CTCs with over 91.6 % efficiency and in situ SCE of phenotypes. By further trapping individual CTCs in ordered microstructures on chip, composite single‐cell spectral signatures were conveniently and efficiently obtained, allowing reliable spectral‐readout for multiplex biomarker profiling. This SCE strategy exhibited great potential in multiplex profiling of heterogeneous CTC phenotypes, offering new avenues for cancer study and precise medicine.

Spectrally Combined Encoding for Profiling Heterogeneous Circulating Tumor Cells Using a Multifunctional Nanosphere-Mediated Microfluidic Platform.

Comprehensive phenotypic profiling of heterogeneous circulating tumor cells (CTCs) at single-cell resolution has great importance for cancer management. Herein, a novel spectrally combined encoding (SCE) strategy was proposed for multiplex biomarker profiling of single CTCs using a multifunctional nanosphere-mediated microfluidic platform. Different cellular biomarkers uniquely labeled by multifunctional nanosphere barcodes, possessing identical magnetic tags and distinct optical signatures, enabled isolation of heterogeneous CTCs with over 91.6 % efficiency and in situ SCE of phenotypes. By further trapping individual CTCs in ordered microstructures on chip, composite single-cell spectral signatures were conveniently and efficiently obtained, allowing reliable spectral-readout for multiplex biomarker profiling. This SCE strategy exhibited great potential in multiplex profiling of heterogeneous CTC phenotypes, offering new avenues for cancer study and precise medicine.

Quantification of Protein Secretion from Circulating Tumor Cells in Microfluidic Chambers.

Cancer cells can be released from a cancerous lesion and migrate into the circulatory system, from whereon they may form metastases at distant sites. Today, it is possible to infer cancer progression and treatment efficacy by determining the number of circulating tumor cells (CTCs) in the patient's blood at multiple time points; further valuable information about CTC phenotypes remains inaccessible. In this article, a microfluidic method for integrated capture, isolation, and analysis of membrane markers as well as quantification of proteins secreted by single CTCs and CTC clusters is introduced. CTCs are isolated from whole blood with extraordinary efficiencies above 95% using dedicated trapping structures that allow co‐capture of functionalized magnetic beads to assess protein secretion. The patform is tested with multiple breast cancer cell lines spiked into human blood and mouse‐model‐derived CTCs. In addition to immunostaining, the secretion level of granulocyte growth stimulating factor (G‐CSF), which is shown to be involved in neutrophil recruitment, is quantified The bead‐based assay provides a limit of detection of 1.5 ng mL−1 or less than 3700 molecules per cell. Employing barcoded magnetic beads, this platform can be adapted for multiplexed analysis and can enable comprehensive functional CTC profiling in the future.

Heterogeneity of Stemlike Circulating Tumor Cells in Invasive Breast Cancer.

The presence of stem and epithelial–mesenchymal-transition (EMT) features in circulating tumor cells (CTCs) determines their invasiveness, adaptability to the microenvironment, and resistance to proapoptotic signals and chemotherapy. It also allows them to fulfil the role of metastatic “seeds”. We evaluated the heterogeneity of stem CTCs by their CD44, ALDH1, and CD133 expression depending on N-cadherin expression in breast-cancer patients. A total of 38 female patients were selected for this study. CTC phenotypes were determined by flow cytometry before any type of treatment. Multiplex immunofluorescence was used for the evaluation of tumor-cell heterogeneity in primary lesions. In patients who had CD44-CD24- CTCs, a subset of cells with the expression of other stem-cell markers (CD133 and ALDH1) were detected. Expression of CD133 and/or ALDH1 may be associated with expression of N-cadherin: all populations of N-cadherin+ CTCs demonstrate stem features; in the absence of N-cadherin expression, true nonstem (CD44-CD24-CD133-ALDH1-) cells are found. The heterogeneity of stem marker expression in CTCs was observed regardless of N-cadherin expression. In our study, stromal cell-derived factor-1 (SDF-1) receptor expression in CTCs did not depend on stemlike traits, but was instead associated with N-cadherin expression. Subpopulations of tumor cells, detected both in tumors and blood, were identified. Breast cancer was characterized by pronounced interpersonal and intrapersonal heterogeneity of CTCs by the presence and combination of various stem features and N-cadherin expression. To complete the characterization of stemlike features of CTCs, we suggest the simultaneous use of the three stem markers.

The combination of size-based separation and selection-free technology provides higher circulating tumour cells detection sensitivity than either method alone in patients with metastatic prostate cancer.

To investigate the circulating tumour cells (CTCs) capture abilities of two technologies that are not dependent on cell-surface marker expression: a selection-free platform [AccuCyte® -CyteFinder® system (Rarecyte)] and a size-based platform [fluid-assisted separation technology (FAST)]. In addition, the combination of the two systems to more completely assess CTCs was investigated. In all, 28 patients with metastatic prostate cancer were included. Two 6mL peripheral blood samples were taken from each patient at the same time-point. The samples were then subjected to the two different technology platforms in parallel. An additional group of samples was acquired by applying the waste chamber material from the FAST-group tests (flow-through that goes through the FAST filter membrane) to the Rarecyte system for the detection any CTCs that were not captured by FAST. The three groups had significantly different putative CTC-positive tests, with positive rates of 29% for Rarecyte, 57% for FAST, and 79% for the combination. We also assessed CTC phenotype: 56.6% of the CTCs were cytokeratin (CK)+/epithelial cell adhesion molecule (EpCAM)-, 3.1% were CK-/EpCAM+, and 40.3% were CK+/EPCAM+. The captured CTCs diameter ranged from 5.2 to 16.9µm. The mean CTC size from the FAST waste chamber was significantly smaller. The diameters for each of the phenotypic groups were significantly different. These data highlight disparities in the positive rates and enumerated CTC numbers detected by the two techniques. Notably, the combination of the two technologies resulted in the highest CTC-capture rates. Smaller CTCs were more likely to be missed by the FAST as they passed through the filter system. Sizes of CTCs varied with different cell surface marker phenotypes.

EpCAM-independent isolation of circulating tumor cells with epithelial-to-mesenchymal transition and cancer stem cell phenotypes using ApoStream® in patients with breast cancer treated with primary systemic therapy.

BackgroundTumor cells with a mesenchymal phenotype and/or cancer stem-like cells (CSCs) are known to contribute to metastasis and drug resistance. Circulating tumor cells (CTCs) undergoing epithelial-mesenchymal transition (EMT) and CTCs reflecting a dedifferentiated CSC phenotype may not be detected using only an anti-EpCAM antibody to capture them. We used an antibody-independent CTC enrichment platform, ApoStream®, which does not rely on any antibody, including anti-EpCAM, to capture EMT- and CSC-CTCs in breast cancer patients who received neoadjuvant chemotherapy and correlated them to pathological complete response (pCR).MethodsBlood samples from newly diagnosed breast cancer patients were prospectively collected before neoadjuvant chemotherapy (T0), after chemotherapy but before surgery (T1), and after surgery (T2) and processed using ApoStream. CTCs detected were stained with additional markers to define 3 CTC subsets with the following phenotypes: epithelial CTCs (CK+, EpCAM+ or E-cadherin+), EMT-CTCs (β-catenin+ or vimentin+), and CSC-CTCs (CD44+ and CD24low).ResultsWe enrolled 55 patients, 47 of which had data for analysis. EMT-CTCs were detected in 57%, 62%, and 72% and CSC-CTCs in 9%, 22%, and 19% at the T0, T1, and T2 time points, respectively. Counts of epithelial (P = 0.225) and EMT (P = 0.522) phenotypes of CTCs at T0 did not significantly predict pCR. Moreover, no correlation between CTC count change and pCR was demonstrated.ConclusionsApoStream was successful in detecting EMT-CTCs among patients after neoadjuvant chemotherapy. However, EMT-/CSC-CTC counts did not correlate with pCR. Due to the small sample size and heterogeneity of this patient population, further study in a larger cohort of molecularly homogeneous patients is warranted.

Epithelial-mesenchymal transition may be involved in the immune evasion of circulating gastric tumor cells via downregulation of ULBP1.

BackgroundIncreasing numbers of studies have demonstrated that circulating tumor cells (CTCs) undergo a phenotypic change termed epithelial‐mesenchymal transition (EMT), and researchers have proposed that EMT might provide CTCs with increased potential to survive in the different microenvironments encountered during metastasis through various ways, such as by increasing cell survival and early colonization. However, the exact role of EMT in CTCs remains unclear.MethodsIn this study, we identified CTCs of 41 patients with gastric cancer using Cyttel‐CTC and im‐FISH (immune‐fluorescence in situ hybridization) methods, and tested the expression of EMT markers and ULBP1 (a major member of the NKG2D—natural killer [NK] group 2 member D—ligand family) on CTCs. Moreover, we investigated the relationship between the expression of EMT markers and ULBP1 on CTCs and gastric cancer cell lines.ResultsOur results showed that the CTCs of gastric cancer patients exhibited three EMT marker subtypes, and that the expression of ULBP1 was significantly lower on mesenchymal phenotypic CTCs (M+CTCs) than on epithelial phenotypic CTCs (E+CTCs). EMT induced by TGF‐β in vitro produced a similar phenomenon, and we therefore proposed that EMT might be involved in the immune evasion of CTCs from NK cells by altering the expression of ULBP1.ConclusionsOur study indicated that EMT might play a vital role in the immune invasion of CTCs by regulating the expression of ULBP1 on CTCs. These findings could provide potential strategies for targeting the immune evasion capacity of CTCs.

Abstract P4-01-09: Phenotypic profiling of circulating tumor cells (CTCs) in patients with metastatic breast cancer reveals clinically-relevant heterogeneity in CTC morphology and marker expression

Abstract Background: The choice between hormonal therapies and chemotherapy is a frequent decision in the standard of care of metastatic breast cancer (mBCa) patients. We previously developed quantitative measures of phenotypic CTC heterogeneity in metastatic prostate cancer and found that higher heterogeneity was associated with better survival on chemotherapy vs. targeted hormonal therapies in a risk adjusted analysis (Scher et al. 2017 Cancer Research). Here we sought to test the feasibility of measuring CTC heterogeneity in mBCa patients. Methods: 295 blood samples from 165 pts (84 HR+, 19 Her2+, 8 HR+/Her2+, 54 TNBC) from mBCa patients were processed for CTC analysis utilizing the Epic Sciences Functional Cell Profiling platform. Following enumeration, multi-dimensional phenotypic characterization analysis was performed utilizing protein expression and digital pathology features. Features from each CTC (3994 CTCs from 165 pts, were compared by unsupervised clustering to assess intra-patient CTC heterogeneity among mBCa CTC phenotypes. Results: CTCs were detected in 76.9% (227/295) of mBCa patients (med=2.4 CTC/mL, Range 0 - 747). Phenotypic profiling identified distinct CTC subtypes characterized by morphological and protein expression features that correlated with mBCa subtypes. Prevalent CTC features that associated with mBCa were either high cytokeratin (CK) expression or lower nuclear to cytoplasmic (N/C) ratio with lower CK expression. Analysis of mBCa subtypes showed that TNBC pts were characterized by CTCs with a large nuclear area and high CK expression. We observed marked differences in CTC heterogeneity across patients, with some patients having more uniform CTC profiles, and others having higher phenotypic diversity. mBCa subtypes had similar intra-patient heterogeneity level. Conclusions: Distinct CTC phenotypes can be visualized and quantified across patients and associated with specific mBCa subtypes. Our analysis revealed significant heterogeneity between individual CTCs both between and within patients. Identification of patients with high heterogeneity may help to identify patients unlikely to respond to targeted therapies. Studies to link heterogeneity to efficacy of specific therapies and patient outcome are ongoing. Citation Format: Tiziano Pramparo, Adam Jendrisak, Priscilla Ontiveros, Megan Kearney, Lincy Chu, Nadia Ebrahim, Joseph Schonhoft, Rick Wenstrup, Yipeng Wang. Phenotypic profiling of circulating tumor cells (CTCs) in patients with metastatic breast cancer reveals clinically-relevant heterogeneity in CTC morphology and marker expression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-01-09.

Abstract P4-01-03: Computer vision and machine learning allows for the prediction of genomic instability using circulating tumor cell morphology in triple negative breast cancer patients

Abstract Background: Tissue based tests of BRCA1/2 and other Homologous Recombination Gene (HRD) alterations associated with PARPi response are often limited by sample accessibility/availability, under-sampling due to tumor heterogeneity, and the lack of known clinical relevance for a significant fraction of DNA repair associated genomic alterations (e.g. VUS mutations). Assessment of HRD phenotypes at the DNA level in single Circulating Tumor Cells (CTCs) could in principle circumvent these issues. In past work, we developed a computer vision algorithm that accurately identifies CTCs with an HRD like phenotype, characterized by a high number of Large Scale Transitions (LSTs). This CTC image based biomarker (predicted LST or pLST) of genomic and/or chromosomal instability was found to associate strongly with androgen receptor targeted therapy resistance and also positively to PARPi response in metastatic prostate cancer. Here we sought to develop similar CTC based technology in Triple Negative Breast Cancer (TNBC). Methods: Training set: 521 CTCs from 26 metastatic prostate cancer patients. Test set: 179 CTCs from 24 TNBC patients. CTCs from both data sets were detected with the Epic Sciences platform and analyzed for 19 phenotypic digital pathology features, such as cytokeratin expression, nuclear texture, and cell size. The same CTCs were single-cell sequenced for the number of LSTs. LSTs are 10 Mb gains or losses in chromosomal structure and are a direct downstream measure of deficiencies in HR associated genes. A regression algorithm was generated and cross validated from the training set to predict the number of LSTs from CTC phenotypic features. The trained algorithm was then applied to the test set and the predicted number of LSTs and the actual number of LSTs determined by sequencing were compared to assess model or classifier performance. Results: The pLST algorithm is able to identify HRD like phenotypes defined as a single cell having a high number of LSTs at a sensitivity of 78%, specificity of 96% and accuracy of 87%. Conclusions: Despite being trained utilizing CTCs from prostate cancer, the pLST algorithm is able to accurately identify TNBC patients with a high number of LSTs in single CTCs consistent with an HRD like phenotype, suggesting possible sensitivity to PARPi or other therapies targeting genomic instability. Assessment of clinical associations in a TNBC cohort is ongoing. The ability to identify potential sensitivity to drugs targeting DNA repair using an immunfluoresence CTC staining method for CTC phenotype could help to stratify additional TNBC patients for both prognosis and for prediction of drug response. Citation Format: Adam Jendrisak, Jerry Lee, Angel Rodriguez, Ramsay Sutton, Joseph Schonhoft, Tiziano Pramparo, Rick Wenstrup, Yipeng Wang. Computer vision and machine learning allows for the prediction of genomic instability using circulating tumor cell morphology in triple negative breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-01-03.

Abstract P4-01-13: Clinical data in over 1,500 breast cancer patients across all stages of breast cancer with target selector circulating tumor cell technology

Abstract Clinical Data in Over 1,500 Patients Across All Stages of Breast Cancer with Target Selector Circulating Tumor Cell Technology Deanna M Fisher B.S., Lan Huynh B.S., Edgar V. Sales B.S., Julie Ann Mayer Ph.D., and Veena M. Singh M.D. Introduction: The detection and molecular characterization of circulating tumor cells (CTCs) in patients with breast cancer affords the ability to profile and monitor patients in real time for progression, risk stratification, recurrence, identification of potentially actionable therapeutic targets, and monitoring of treatment efficacy and emergence of resistance mechanisms. To harness the promise of CTC analysis a highly sensitive, robust, reproducible and clinically validated technology is required. Information acquired from a single tissue biopsy has temporal and spatial limitations; additionally, in patients with progressive/metastatic disease, a single biopsy may not be informative or in some instances difficult to perform and might fail to reflect inherent tumoral heterogeneity. CTCs on the other hand can provide a contemporaneous landscape of all cancerous lesions (primary and metastases) as well as the opportunity to track the evolving tumor genetic mechanisms. Methodology:Samples were collected in CEE Sure blood collection tubes and buffy coat isolation was performed using a percoll density gradient separation. Capture in the microchannel was performed after incubation with a ten-antibody capture cocktail followed by the addition of a biotinylated secondary antibody. The microchannels were stained with pan-cytokeratin cocktail, CD45, pan-CTC stain, DAPI and the following protein biomarkers (AR, ER, PR, and PDL1). Following enumeration of the CTCs, the microchannels were subsequently sent for multi-color FISH biomarkers such as HER2. Results: 1,687 patients across all stages of breast cancer and treatment time points (pre- treatment, post-treatment, on treatment) were analyzed. CTCs were detected up to 70% of the time across early to late stage patients. The dynamic range of CTC detection and enumeration in a single microfluidic channel (8mL blood) ranged from 1 to 38,419 CTCs. CTC detection has been previously validated to a 1 CTC limit of detection. CTCs identified varied in both size and antigen profile. Target Selector CTC platform technology identifies both cytokeratin positive and cytokeratin negative phenotypes of CTCs. Biomarker interrogation was undertaken in all CTC subtypes by FISH probes (Fluorescent in situ hybridization) and ICC (Immunocytochemical) antibody clones similar to those used on tissue sections. Genomic alterations commonly detected in breast cancer (i.e. HER2 amplification, ER) were detected in all CTC subtypes. HER2 amplification was identified in 15% of cases with CTCs detected and ER in 24 %. Conclusions: This data demonstrates the ability of the Target Selector CTC Platform to accurately detect, enumerate, and interrogate for genomic alteration across a broad spectrum of CTC phenotypes spanning various clinical and treatment stages. This enables clinicians and patients a more real time option to profile and monitor disease. The Target Selector CTC technology allows for the analysis of a broader spectrum of CTC phenotypes thus increasing the likelihood of identifying actionable biomarkers for risk stratification, recurrence, potential treatment selection and monitoring for emergence of resistance. Citation Format: Deanna M Fisher, Lan Huynh, Edgar V Sales, Julie Ann Mayer, Veena M Singh. Clinical data in over 1,500 breast cancer patients across all stages of breast cancer with target selector circulating tumor cell technology [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-01-13.

Abstract PD10-04: Prognostic potential of mesenchymal circulating tumor cell-associated white blood cell clusters in patients with HER2-negative metastatic breast cancer

Abstract Background:Circulating tumor cells (CTCs), potentially involved in the metastatic cascade, may have direct interactions with immune cells during cancer dissemination, and correlate with the inflammatory state in the target organ of metastatic extravasation. Although positive CTC status has been validated as a prognostic marker in breast cancer, the interaction between immune cells and CTCs during the progress of Epithelial-mesenchymal transition (EMT), and the clinical implications of CTC-associated white blood cell clusters (CTC-WBC clusters) for metastatic breast cancer are largely uncharacterized. We investigated the predictive value of different phenotypes of CTC-WBC clusters as a prognostic biomarker in a prospective study for metastatic breast cancer. Methods:The prospective study enrolled 134 patients with HER2-negative metastatic breast cancer who underwent docetaxel plus capecitabine as first-line chemotherapy and subsequently used capecitabine as maintenance therapy from Nov 2013 to Sep 2017. Serial peripheral blood samples were collected dynamically until disease progression for CTC detection using CanPatrol CTC enrichment technique. CTCs were isolated using a filter-based method combined with an RNA in situ hybridization method based on the branched DNA signal amplification technology according to EMT markers. Follow-up data collection was conducted until May 2019 and data analysis was performed in June 2019. Kaplan-Meier analysis and Cox proportional hazards regression analysis were performed to investigate the prognostic value of CTC-WBC clusters. Results:Median age of the 134 female patients was 51.0 years (range,21 to 73 years), and 119 (88.8%) of them were estrogen receptor-and/or progesterone receptor-positive. Median progression-free survival (PFS) was 9.9 months (95% CI, 8.1 to 11.6 months). 116 (86.6%) of the 134 patients had detectable CTCs at baseline with a mean number of 8 CTCs per 5 ml of blood, including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs and mesenchymal CTCs. Among them, CTC-WBC clusters were detectable in 9 patients, of whom 5 patients had mesenchymal CTCs contained in the CTC-WBC clusters. The patients with at least one detectable mesenchymal CTC-WBC clusters were characterized by significantly worse PFS, when compared with the patients with no mesenchymal CTC-WBC clusters (10.2 months vs 6.3 months, P=0.046), as well as the patients with at least one CTC per 5 ml blood (10.7 months vs 6.3 months, P=0.040). However, the presence of total CTC-WBC cluster regardless of CTC phenotypes was correlated with no significant survival difference in the cohort (10.0 months vs 8.6 months, P=0.913). Conclusions: Our data provide evidence that the emergence of mesenchymal CTC-WBC clusters before treatment is associated with significantly poorer PFS in HER2-negative metastatic breast cancer patients underwent first-line chemotherapy, which may be used as a parameter to predict the clinical outcomes for chemotherapy. This study validated the prognostic value of CTC-WBC cluster underwent mesenchymal transformation in a prospective cohort constituted by relatively homogeneous participants who were all HER2-negative metastatic breast cancer underwent docetaxel plus capecitabine as first-line chemotherapy. The results could provide evidence for mesenchymal CTC-WBC cluster to serve as a potential biomarker for individual therapy. However, limited sample size influenced the statistical power to draw firm conclusions. Thus, further large-scale external validation containing data of overall survival is warranted. Citation Format: Xiuwen Guan, Chunxiao Li, Xiaoying Sun, Lixi Li, Zongbi Yi, Binliang Liu, Jiasen Xu, Binghe Xu, Fei Ma. Prognostic potential of mesenchymal circulating tumor cell-associated white blood cell clusters in patients with HER2-negative metastatic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD10-04.

Circulating Tumor Cells and Fibronectin 1 in the Prognosis of Nasopharyngeal Carcinoma.

Objective:Nasopharyngeal carcinoma is highly endemic in Southeast China. Circulating tumor cell is an important biomarker in the prognosis of variety kinds of cancers. Overexpression of fibronectin 1 was observed in variety kinds of malignancies and may contribute to progress and metastasis of the cancers. The current study was aimed to investigate phenotypes of circulating tumor cell in nasopharyngeal carcinoma blood and fibronectin 1 expression in the circulating tumor cell, and their clinical application in predicting nasopharyngeal carcinoma prognosis.Methods:Blood samples were obtained from nasopharyngeal carcinoma patients before and after treatment. CanPatrol circulating tumor cell enrichment and RNA in situ hybridization were applied to identify circulating tumor cell and its phenotypes. Fibronectin 1 messenger RNA expression in the cells of circulating tumors was examined by messenger RNA-in situ hybridization.Results:Circulating tumor cell was not associated with tumor characteristics or lymph node metastasis. Patients with >9 circulating tumor cells or >5 mesenchymal phenotype circulating tumor cell per 5-mL blood had poorer progression-free survival (P < .05). Multivariable analysis demonstrated that 2 or more mesenchymal phenotype circulating tumor cells with high fibronectin 1 messenger RNA expression predicted shorter progression-free survival (P < .05).Conclusions:Circulating tumor cells with high-level fibronectin 1 expression was associated with poor survival in patients with nasopharyngeal carcinoma and could be an independent prognostic factor for nasopharyngeal carcinoma.

NF-kappa B Signaling-Related Signatures Are Connected with the Mesenchymal Phenotype of Circulating Tumor Cells in Non-Metastatic Breast Cancer.

The role of circulating tumor cells (CTCs), tumor microenvironment (TME), and the immune system in the formation of metastasis is evident, yet the details of their interactions remain unknown. This study aimed at exploring the immunotranscriptome of primary tumors associated with the status of CTCs in breast cancer (BCa) patients. The expression of 730 immune-related genes in formalin-fixed paraffin-embedded samples was analyzed using the multigenomic NanoString technology and correlated with the presence and the phenotype of CTCs. Upregulation of 37 genes and downregulation of 1 gene were observed in patients characterized by a mesenchymal phenotype of CTCs when compared to patients with epithelial CTCs. The upregulated genes were involved in NF-kappa B signaling and in the production of type I interferons. The clinical significance of the differentially expressed genes was evaluated using The Cancer Genome Atlas (TCGA) data of a breast invasive carcinoma (BRCA) cohort. Five of the upregulated genes—PSMD7, C2, IFNAR1, CD84, and CYLD—were independent prognostic factors in terms of overall and disease-free survival. To conclude, our data identify a group of genes that are upregulated in BCa patients with mesenchymal CTCs and reveal their prognostic potential, thus indicating that they merit further investigation.

CTCs Expression Profiling for Advanced Breast Cancer Monitoring.

The study of circulating tumor cells (CTCs) has a huge clinical interest in advance and metastatic breast cancer patients. However, many approaches are biased by the use of epithelial markers, which underestimate non-epithelial CTCs phenotypes. CTCs enumeration provides valuable prognostic information; however, molecular characterization could be the best option to monitor patients throughout the disease since it may provide more relevant clinical information to the physicians. In this work, we aimed at enumerating and performing a molecular characterization of CTCs from a cohort of 20 patients with metastatic breast cancer (MBC), monitoring the disease at different time points of the therapy, and at progression when it occurred. To this end, we used a CTC negative enrichment protocol that allowed us to recover a higher variety of CTCs phenotypes. With this strategy, we were able to obtain gene expression data from CTCs from all the patients. In addition, we found that high expression levels of PALB2 and MYC were associated with a worse outcome. Interestingly, we identified that CTCs with an EpCAMhighVIMlowALDH1A1high signature showed both shorter overall survival (OS) and progression-free survival (PFS), suggesting that CTCs with epithelial-stem features had the most aggressive phenotype.