Characterization Of Circulating Tumor Cells Research Articles

Abstract 969: Epithelial and mesenchymal CTC detection in triple negative breast cancer patients using ANGLE’s Parsortix®system

Abstract Introduction: Isolation of circulating tumor cells (CTCs) is widely recognised as a prognostic biomarker in metastatic breast cancer. Recently, it was shown that CTC clearance following specific therapies may predict chemosensitivity in triple negative breast cancer (TNBC) patients. However, a limited amount of CTC research is available in TNBC due to the inability of epitope-dependent CTC platforms to capture CTCs in a sufficient proportion of patients, especially when evaluating non-metastatic patients (Stage I-III). ANGLE has developed a Research Use Only (RUO) workflow including the Parsortix® system, an epitope-independent microfluidic device that isolates and harvests rare cells from blood based on their size and deformability, followed by an immunofluorescent (IF) assay for the identification and characterization of CTCs. This five-color multiplex IF assay enables the identification of both epithelial and mesenchymal CTCs as well as the investigation of other targets of interest on the CTCs. Methods: 7.5mL of peripheral blood drawn into a Streck CellFree DNA Blood Collection Tube from 11 TNBC patients (four Stage IV and seven Stage IIIa) was processed on Parsortix® system between 96 and 144 hours post collection. Harvested CTCs were cytospun onto a charged slide and immunofluorescently stained using an optimised antibody panel allowing for detection of epithelial and mesenchymal CTCs. Stained slides were imaged using a BioView Allegro Plus automated imaging system. Results: CTCs were identified in 9 (82%) of the 11 TNBC patients, with a median of 6 and mean of 30 CTCs identified per patient. CTC clusters were harvested by the Parsortix® system and were observed in the majority (8/9) of the CTC-positive patients. Cluster size ranged from 2-68 CTCs per cluster, and the number of clusters per patient ranged from 1-50. Phenotypically, a large proportion of the CTCs identified expressed only mesenchymal markers (80%), with the remainder expressing both epithelial and mesenchymal markers (20%). In 6 of the 7 non-metastatic (Stage IIIa) patients, high numbers of CTC clusters were detected. Lower numbers of CTC clusters were detected in 3 (75%) of the 4 metastatic (Stage IV) patients. Conclusions: This study demonstrated the ability of ANGLE’s multiplex IF assay to detect and phenotype CTCs. The proportion of non-metastatic TNBC patients found to have CTCs was much higher compared to previous reports using epitope-dependent systems (82% vs 25%). This highlights the advantages of epitope-independent CTC capture in TNBC. ANGLE’s RUO Parsortix® system and multiplex IF assay provide an efficient solution for the harvesting and characterization of multiple CTC phenotypes, with the added advantage of being able to process the blood between 96 and 144 hours post collection, allowing for the shipment of samples at room temperature for centralised analysis in support of global clinical trials. Citation Format: Amina Mezni, Matthew Hardy, Mariacristina Ciccioli, Anne-Sophie Pailhes-Jimenez. Epithelial and mesenchymal CTC detection in triple negative breast cancer patients using ANGLE’s Parsortix®system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 969.

Abstract 1952: Validation of enhanced performance of the AccuCyte®-CyteFinder® platform for circulating tumor cell characterization

Abstract Analysis of circulating tumor cells (CTCs) by multiparameter immunofluorescence (IF) microscopy allows non-invasive characterization of cancer cell biomarker expression in real time. This information can be helpful in prognosis, treatment selection, and stratification of cancer patients. AccuCyte® is a density-based unbiased isolation method that transfers nucleated cells from whole blood to slides for the characterization of CTCs and other rare cells. RarePlex® panel kits are IF staining reagents used on automated slide staining instruments to label cells to differentiate CTCs from white blood cells (WBC). CyteFinder® is a seven-channel automated fluorescent imaging system that rapidly scans microscope slides and applies machine learning algorithms to identify CTCs. Together, these technologies provide an end-to-end solution for CTC characterization. For analysis, blood is drawn into AccuCyte blood collection tubes (BCTs) containing a preservative which maintains cell properties prior to processing onto slides. Once slides are prepared, they can be stored at -20°C without significant biomarker degradation. This flexible workflow allows investigators to bank samples for batch analysis and to begin sample collection prior to validating the IF assay to be used. This study was designed to evaluate: (1) stability time between collection in the AccuCyte BCT and sample processing; (2) performance of an improved version of the AccuCyte kit with higher nucleated cell isolation capacity; and (3) storage time that AccuCyte prepared slides can be banked frozen prior to staining. The study was performed using model CTCs and cancer patient samples. Metrics to determine performance were CTC recovery and mean fluorescence intensity (MFI) of biomarker expression. Our results demonstrate that the AccuCyte BCT preserves blood components for at least 5 days after collection without significant effect on CTC recovery or biomarker expression. The latest version of the AccuCyte kit demonstrated a higher cell isolation capacity and could collect up to 60% more nucleated blood cells than the previous version, increasing CTC recovery. The increased capacity was demonstrated in patients treated with hematopoietic growth factors, whose WBC count was significantly higher than the normal range. Finally, accelerated-aging study results demonstrated that AccuCyte-prepared slides can be stored at -20°C for at least 4 years without significant effect on most biomarkers tested. In conclusion, enhancements to the AccuCyte-CyteFinder platform reported here increase flexibility and performance for analysis of CTCs in global clinical trials by allowing longer periods of time before collected blood samples need to be processed and by extending the length of time processed slides can be banked before they are stained. Citation Format: Arturo B. Ramirez, Lillian Costandy, Brady S. Gardner, Ryan H. Huston, A Anders Larson Tevis, Casey E. Helmicki, Alisa C. Clein, Daniel E. Sabath, Joshua J. Nordberg, Tad C. George. Validation of enhanced performance of the AccuCyte®-CyteFinder® platform for circulating tumor cell characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1952.

Demonstration of the value of liquid biopsy to identify metastatic breast cancer: Comparison and combination of cell and cell-free platforms.

e13004 Background: Liquid biopsy analysis detecting circulating tumor cells (CTC) or circulating tumor DNA (ctDNA) is a non-invasive approach that may provide clinically actionable information for patients’ (pts) treatment decisions when conventional biopsy is otherwise inaccessible or infeasible. Here we report a liquid biopsy analysis method including CTC characterization and ctDNA alterations in late-stage metastatic breast cancers (MBC). Methods: Blood samples were collected for cell and cell-free DNA analysis from 57 pts with MBC. 44 pts were progressing on treatment and 13 were responding to treatment. Blood was also collected from 25 blood donors (HD) with no known cancer history. After plasma isolation, nucleated cells were plated, and slides were bio-banked. Immunofluorescent staining and subsequent imaging were performed on replicate slides. CTCs were identified using Epic Sciences digital imaging and machine learning algorithms trained to identify breast cancer cells. Single-cell (sc) isolation for genomic quantification of large-scale transitions (scLST) was used to further characterize individual CTCs. cfDNA from bio-banked plasma was analyzed using a validated NGS panel to detect class 1A ctDNA alterations. Results: Within this cohort of 57 MBC pts, CTCs, scLST+CTCs and ctDNA alterations were detected in (77%, 49% and 33%, respectively). All MBC pts on active therapy (23%) had no CTC detected. No CTCs, scLST+CTCs or ctDNA were detected in the HD cohort, suggesting high specificity (100%, 100% and 100%, respectively). The presence of CTCs or of scLST+CTCs had low concordance with the presence of ctDNA+ alterations in MBC pts, suggesting that these platforms provide independent and complementary value when identifying MBC. To test this hypothesis, we designed combination models which added ctDNA to cell analysis methods ([CTC+][ctDNA+] and [scLST+CTC+][ctDNA+]). Adding ctDNA to either cell analysis method increased sensitivity by 5% and 12%, respectively, over the individual methods and retained 100% specificity. Conclusions: Here we demonstrate the value of comprehensive cell and cell-free liquid biopsy analysis for the detection of MBC pts. Our data suggest there may be clinical utility of adding cell-free to cell analysis for the identification of MBC. Analysis of the comparative and combined utility of these liquid biopsy methods in earlier settings is ongoing.

Improved sensitivity in identification of first recurrences of metastatic breast cancer with a combination of cell and cell-free liquid biopsy analysis.

e13003 Background: Liquid biopsy analysis detecting circulating tumor cells (CTC) or circulating tumor DNA (ctDNA) is a non-invasive approach that may provide clinically actionable information for patients’ (pts) treatment decisions when conventional biopsy is otherwise inaccessible or infeasible. Here we report a liquid biopsy method including CTC characterization & ctDNA alterations in pts symptomatic of first metastatic recurrence with a history of primary breast cancer (BC). Methods: Blood samples from 34 symptomatic BC pts were collected for cell & cell-free DNA analysis before undergoing contemporaneous tissue biopsy & initiating treatment for metastatic BC (MBC). Blood was also collected from 25 blood donors (HD) with no known cancer history. After plasma isolation, nucleated cells were plated, & slides were bio-banked. Immunofluorescent staining & subsequent imaging were performed on replicate slides. CTCs were identified using Epic Sciences digital imaging & machine learning algorithms trained to identify breast cancer cells. Single-cell (sc) isolation for genomic quantification of large-scale transitions (scLST) was used to further characterize individual CTCs. cfDNA from bio-banked plasma was analyzed to detect Class IA alterations in ctDNA. Results: Within this cohort of 34 patients, CTCs & ctDNA alterations were detected in 80% & 58%, respectively while no CTCs & ctDNA were detected in the HD cohort suggesting high specificity (100% & 100%). In this study, 26 pts were confirmed MBC (M1-BC) by tissue biopsy, 3 pts were non-MBC by tissue biopsy but still diagnosed as metastatic (Mx-BC), & 5 pts were confirmed non-MBC (M0-BC). To benchmark the cellular (CTCs & scLST+CTCs) & genomic (ctDNA) individual platforms, we designed thresholds of CTCs > 4, scLST+CTC+, & ctDNA+ that optimally identified M1-BC pts with high specificity (100%, 100%, & 100%) but low sensitivity (46%, 46%, & 50%). CTCs > 4 & scLST+CTC+ cutoffs had low concordance with the presence of ctDNA alterations, suggesting these platforms provide independent & complementary value when identifying MBC. To test this hypothesis, we designed combination models [CTC > 4][ctDNA+] & [scLST+CTC+][ctDNA+] that significantly improved sensitivity (75% & 67%) over individual platforms while maintaining high specificity (100% & 100%) when identifying the first recurrence of metastasis in BC pts. Conclusions: To our knowledge, this is the first report showing the additive & complementary value of models combining cellular & genomic liquid biopsy platforms for the detection of the first MBC recurrence. Our data suggest the clinical utility of combined CTC & ctDNA platforms for the identification of MBC when tissue biopsy is inaccessible or infeasible.

Application of Microfluidics in Detection of Circulating Tumor Cells.

Tumor metastasis is one of the main causes of cancer incidence and death worldwide. In the process of tumor metastasis, the isolation and analysis of circulating tumor cells (CTCs) plays a crucial role in the early diagnosis and prognosis of cancer patients. Due to the rarity and inherent heterogeneity of CTCs, there is an urgent need for reliable CTCs separation and detection methods in order to obtain valuable information on tumor metastasis and progression from CTCs. Microfluidic technology is increasingly used in various studies of CTCs separation, identification and characterization because of its unique advantages, such as low cost, simple operation, less reagent consumption, miniaturization of the system, rapid detection and accurate control. This paper reviews the research progress of microfluidic technology in CTCs separation and detection in recent years, as well as the potential clinical application of CTCs, looks forward to the application prospect of microfluidic technology in the treatment of tumor metastasis, and briefly discusses the development prospect of microfluidic biosensor.

Single-Cell Phenotypic and Molecular Characterization of Circulating Tumor Cells Isolated from Cryopreserved Peripheral Blood Mononuclear Cells of Patients with Lung Cancer and Sarcoma.

The isolation of circulating tumor cells (CTCs) requires rapid processing of the collected blood due to their inherent fragility. The ability to recover CTCs from peripheral blood mononuclear cells (PBMCs) preserved from cancer patients could allow for retrospective analyses or multicenter CTC studies. We compared the efficacy of CTC recovery and characterization using cryopreserved PMBCs vs fresh whole blood from patients with non-small cell lung cancer (NSCLC; n = 8) and sarcoma (n = 6). Two epithelial cellular adhesion molecule (EpCAM)-independent strategies for CTC enrichment, based on Parsortix® technology or immunomagnetic depletion of blood cells (AutoMACS®) were tested, followed by DEPArray™ single-cell isolation. Phenotype and genotype, assessed by copy number alterations analysis, were evaluated at a single-cell level. Detection of target mutations in CTC-enriched samples from frozen NSCLC PBMCs was also evaluated by digital PCR (dPCR). The use of cryopreserved PBMCs from cancer patients allowed for the retrospective enumeration of CTCs and their molecular characterization, using both EpCAM-independent strategies that performed equally in capturing CTC. Cells isolated from frozen PBMCs were representative of whole blood-derived CTCs in terms of number, phenotype, and copy number aberration profile/target mutations. Long-term storage (≥3 years) did not affect the efficacy of CTC recovery. Detection of target mutations was also feasible by dPCR in CTC-enriched samples derived from stored PBMCs. Isolating CTCs from longitudinally collected PBMCs using an unbiased selection strategy can offer a wider range of retrospective genomic/phenotypic analyses to guide patients' personalized therapy, paving the way for sample sharing in multicenter studies.

Biological Characterization and Clinical Relevance of Circulating Tumor Cells: Opening the Pandora's Box of Multiple Myeloma.

Simple SummaryBone marrow (BM) aspirates are mandatory for diagnosis and follow-up of patients with multiple myeloma (MM). However, they present two important caveats: Their invasiveness and limited scope to capture the broad tumor heterogeneity. Conversely, circulating tumor cells (CTCs) are detectable in the peripheral blood of patients with precursor and malignant disease states and have strong prognostic value. Moreover, the high genetic and transcriptomic overlap between both plasma cell compartments suggests that CTCs might reflect with notable precision the medullar clone. Furthermore, the study of CTCs could be used as a model to identify mechanisms favoring BM egression and disease spreading. Here, we summarize the state of the art on MM CTCs and provide insights on what they may offer in research and clinical scenarios.Bone marrow (BM) aspirates are the gold standard for patient prognostication and genetic characterization in multiple myeloma (MM). However, they represent an important limitation for periodic disease monitoring because they entail an aggressive procedure. Moreover, recent findings show that a single BM aspirate is unable to reflect the complex MM heterogeneity. Recent advances in flow cytometry, microfluidics, and “omics” technologies have opened Pandora’s box of MM: The detection and isolation of circulating tumor cells (CTCs) offer a promising and minimally invasive alternative for tumor assessment and metastasis study. CTCs are detectable in premalignant and active MM states, and their enumeration has strong prognostic value, to the extent that it is challenging current stratification systems. In addition, CTCs reflect with high precision both intra- and extra-medullary disease at the phenotypic, genomic, and transcriptomic levels. Despite this high resemblance between tumor clones in distinct locations, some subtle (not random) differences might shed some light on the metastatic process. Thus, it has been suggested that a hypoxic and pro-inflammatory microenvironment could induce an arrest in proliferation forcing tumor cells to recirculate. Herein, we summarize data on the characterization of MM CTCs as well as their clinical and research potential.

Beyond Circulating Tumor Cell Enumeration: Cell-Based Liquid Biopsy to Assess Protein Biomarkers and Cancer Genomics Using the RareCyte® Platform.

The practice of medicine has steadily employed less invasive methods to obtain information derived from the tumor to guide clinical management of patients. Liquid biopsy—the sampling of blood—is a non-invasive method for generating information previously only available from tissue biopsies of the tumor mass. Analysis of fragmented circulating tumor DNA in the plasma is clinically used to identify actionable mutations and detect residual or recurrent disease. Plasma analysis cannot, however, assess cancer phenotypes, including the expression of drug targets and protein biomarkers. Circulating tumor cells (CTCs) are intact cancer cells that have entered the blood that have the potential for distant metastasis. While enumeration of CTCs is prognostic of outcome, recently developed technology allows for the interrogation of protein biomarkers on CTCs that could be predictive of response. Furthermore, since CTCs contain intact whole cancer genomes, isolating viable CTCs detected during therapy could provide a rational approach to assessing mutational profiles of resistance. Identification, characterization and molecular analysis of CTCs together will advance the capacity of liquid biopsy to meet the requirements of twenty-first century medicine.

Abstract P2-02-07: Unbiased liquid biopsy in breast cancer patients utilizing novel light sheet microscopy of circulating tumor cells. Proof of concept in 10 patients and 13 controls

Abstract Background It is widely expected that circulating tumor cells (CTCs) in cancer patients shall turn out to be good prognosticators for outcomes. However, enumeration and monitoring of CTCs continues to pose serious hurdles. Current methods to assess CTCs utilize various approaches to enrich for CTCs. The only FDA-approved system detects CTCs via enrichment for EpCAM. Cancer heterogeneity requires an enrichment-free characterization of all CTCs to better identify this ever-changing landscape for biologic understanding and clinical management. Methods CLINBREAC is a single site, prospective, longitudinal trial for detecting and characterizing all CTCs in breast cancer patients. We present results from 8 metastatic and 2 neoadjuvant patients followed over time. Collection of 18ml blood samples occurred at enrollment and at change of treatment, or at 3-month intervals. Using the RareScope™ Fluorescence Light Sheet Microscope (QCDx LLC, Farmington CT), an aliquot of all morphologically intact, un-enriched nucleated cells placed in immobilized suspensions were analyzed. All cells were stained with immunofluorescent markers against CD45, Vimentin (Vim), EpCAM and Cytokeratin (CK) for characterization of epithelial or mesenchymal CTC phenotypes and trophoblast surface antigen 2 (TROP-2), ER-α and HER2 for treatment-specific characterization. Results No CTCs were observed in blood samples from 13 healthy volunteers. All patient samples contained CTCs collected over all time-points from 10 patients. In all, 1261 CTCs were identified, range, 18-85 CTCs per 1.7K to 2.7K nucleated cells analyzed. Each sample had a unique pattern of epithelial and mesenchymal markers that changed with time and treatment and mirrored clinical course. Of the CTC populations seen, 239 (19%) stained EpCAM+. Of the remaining CTCs, 798 CK+ (63%) and 842 Vim+ (67%) . An example: Patient 1: progression of metastatic triple negative breast cancer on nab-paclitaxel and atezolizumab after which anti-TROP-2 directed therapy with sacituzumab govitecan was initiated. In 39 CTCs present in sample 1, EpCAM-5%, CK-36%, and Vim-92% alone and co-expressed with EpCAM and CK. In subsequent samples, CTCs decreased from 39 to 23 to 6 consistent with response to treatment. TROP2+ CTCs decreased from 14/25 cells to 1/10 with treatment. CTCs co-expressed TROP2, HER2 and ER-α. As TROP2+ CTCs decreased from Sample 1 to 3 (56 to 10%) HER2+ CTCs increased (12 to 70%). The correlation of CTCs with clinical course of disease was observed in all 8 metastatic patients. Phenotypic changes occurred in all patients’ CTCs followed over time. Conclusions RareScope technology, utilized here, is a novel CTC-detection methodology that does not rely on prior enrichment of blood for CTCs. In patients analyzed here, all samples contained CTCs in higher numbers than described previously; a minority of CTCs contain EpCAM+ CTCs and may account for lower numbers in prior studies. Continuing refinement of this technology with addition of multiplex testing on single cells with a larger panel of antibodies will enhance targeted analysis and shall be useful in detection and detailed characterization of CTCs with un-precedented resolution and in real time. Citation Format: Susan Tannenbaum, Emily Hsu, Ashwanth Reddy, Spencer Keilich, Triantafyllos Tafas. Unbiased liquid biopsy in breast cancer patients utilizing novel light sheet microscopy of circulating tumor cells. Proof of concept in 10 patients and 13 controls [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-02-07.

Abstract P5-13-33: Longitudinal transcriptional profiling of CTCs in metastatic breast cancer patients receiving the CDK4/6 inhibitor Palbociclib to predict therapy response

Abstract Background: CDK4/6 inhibitors represent a new treatment option for metastatic (M), hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC) patients. However, predictive markers have not been defined to decide who will benefit from CDK4/6 inhibitors. Circulating tumor cells (CTCs) represent the tumoral heterogeneity in real time and are available for molecular characterization via minimal-invasive sequential blood sampling. Here, we aim to identify biomarkers of resistance to Palbociclib by conducting transcriptional profiling of CTCs before therapy initiation (baseline) and after six months under treatment. Methods: Blood of 50 HR+/HER2-MBC patients drawn at baseline of Palbociclib plus endocrine treatment and blood of 20 HR+/HER2-MBC patients drawn before initiation of endocrine monotherapy, as well as blood samples of these 70 patients after six months under treatment will be analyzed. Patients with progressive disease within the first six months of treatment were defined as non-responders. Isolation of CTCs was conducted using positive immunomagnetic selection targeting EpCAM, EGFR and HER2 (AdnaTest EMT2/StemCell Select). cDNA was analyzed by a new multimarker qPCR panel utilizing QuantiNova LNA Probe assays targeting 25 genes. qPCR data was normalized to CD45 to substract the effect of contaminating leukocytes. Additionally, data was normalized to 20 healthy female donor controls to identify BC specific overexpression signals with a specificity of >90% for all targets. Consumables: QIAGEN, Germany. Results: We successfully established the multimarker qPCR according to the MIQE guidelines. Up to now, the binary mRNA overexpression signals of 25 targets in CTCs isolated at baseline were analyzed in 40 of the total 70 planned MBC patients. In detail, 24 of those 40 patients were treated with Palbociclib plus endocrine therapy, including seven responders and 15 non-resonders, while the 16 other patients were treated with endocrine therapy alone (seven non-responders and nine responders). In the entire cohort, STAT1 signals were significantly related to a decreased overall survival (p=0.0034) and NFKB1 signals correlated significantly with an increased progression free survival (PFS; p=0.027). In the Palbociclib group, CDK2, WWTR1 and YAP1 overexpression signals were more common in non-responders compared with responders. MLH1 and NFKB1 signals were more common in responders than in non-responders. In this cohort, MLH3 overexpression at baseline was significantly correlated with prolonged PFS (p=0.047) and the patient with the longest PFS (35 months) was the only patient with detectable ERBB4, JUN, CETN2 and TEAD2 signals. In the control group treated with endocrine therapy alone, signal prevalence of CXCR4 and STAT1 were increased in the non-responders versus responders and ABCC2, ESR1, FAT4 and FGFR1 signals were more frequently detected in the responders. Conclusion: Transcriptional profiling of CTCs represents a real-time snapshot of the disease complexity. Characterization of the CTCs isolated from blood drawn at baseline is promising for the identification of urgently needed predictive markers (here MLH3 overexpression), while characterization of CTCs isolated under treatment can elucidate the transcriptional dynamics and might be used as monitoring marker by differentiation of non-responders and responders. We will finalize CTC analysis of the entire cohort and will continue blood sampling of additional Palbociclib treated patients, also in the follow-up of the disease, to validate CTC MLH3 overexpression as predictive biomarker for CDK4/6 inhibitor treatment and moreover, to find further overexpression signals in CTCs relevant for MBC therapy management. Citation Format: Sabine Kasimir-Bauer, Charlotte Gruber, Oliver Hoffmann, Rainer Kimmig, Corinna Keup. Longitudinal transcriptional profiling of CTCs in metastatic breast cancer patients receiving the CDK4/6 inhibitor Palbociclib to predict therapy response [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-33.

Exploring the Clinical Utility of Pancreatic Cancer Circulating Tumor Cells.

Pancreatic ductal adenocarcinoma (PDAC) is the most frequent pancreatic cancer type, characterized by a dismal prognosis due to late diagnosis, frequent metastases, and limited therapeutic response to standard chemotherapy. Circulating tumor cells (CTCs) are a rare subset of tumor cells found in the blood of cancer patients. CTCs has the potential utility for screening, early and definitive diagnosis, prognostic and predictive assessment, and offers the potential for personalized management. However, a gold-standard CTC detection and enrichment method remains elusive, hindering comprehensive comparisons between studies. In this review, we summarize data regarding the utility of CTCs at different stages of PDAC from early to metastatic disease and discuss the molecular profiling and culture of CTCs. The characterization of CTCs brings us closer to defining the specific CTC subpopulation responsible for metastasis with the potential to uncover new therapies and more effective management options for PDAC.

Transcriptomic profiling of single circulating tumor cells provides insight into human metastatic gastric cancer

Transcriptome analysis of circulating tumor cells (CTCs), which migrate into blood vessels from primary tumor tissues, at the single-cell level offers critical insights into the biology of metastasis and contributes to drug discovery. However, transcriptome analysis of single CTCs has only been reported for a limited number of cancer types, such as multiple myeloma, breast, hepatocellular, and prostate cancer. Herein, we report the transcriptome analysis of gastric cancer single-CTCs. We utilized an antigen-independent strategy for CTC isolation from metastatic gastric cancer patients involving a size-dependent recovery of CTCs and a single cell isolation technique. The transcriptomic profile of single-CTCs revealed that a majority of gastric CTCs had undergone epithelial-mesenchymal transition (EMT), and indicated the contribution of platelet adhesion toward EMT progression and acquisition of chemoresistance. Taken together, this study serves to employ CTC characterization to elucidate the mechanisms of chemoresistance and metastasis in gastric cancer.

Deep Phenotypic Characterisation of CTCs by Combination of Microfluidic Isolation (IsoFlux) and Imaging Flow Cytometry (ImageStream).

Simple SummaryCells that escape the primary tumour and have the potential ability to colonise distant organs through metastasis are called circulating tumour cells (CTCs). The study of CTCs in colorectal cancer (CRC) has demonstrated their prognostic utility, although current methodologies only allow the evaluation of CTC numbers and a maximum of two markers. Here, we developed a novel protocol for the isolation and characterisation of CTCs by combining two existing technologies. This new methodology allows the simultaneous evaluation of multiple markers and parameters. In particular, we evaluated the expression of a mutant protein (BRAFV600E) associated with poor response to therapies against EGFR and the expression of PD-L1, a marker for immunotherapy. Based on these markers, we evaluated the CTCs (positive for cytokeratin) of 16 early CRC patients and demonstrated the suitability of our protocol to classify patients into potential responders and non-responders.The isolation of circulating tumour cells (CTCs) in colorectal cancer (CRC) mostly relies on the expression of epithelial markers such as EpCAM, and phenotypic characterisation is usually performed under fluorescence microscopy with only one or two additional markers. This limits the ability to detect different CTC subpopulations based on multiple markers. The aim of this work was to develop a novel protocol combining two platforms (IsoFluxTM and ImageStream®X) to improve CTC evaluation. Cancer cell lines and peripheral blood from healthy donors were used to evaluate the efficiency of each platform independently and in combination. Peripheral blood was extracted from 16 early CRC patients (before loco-regional surgery) to demonstrate the suitability of the protocol for CTC assessment. Additionally, peripheral blood was extracted from nine patients one month after surgery to validate the utility of our protocol for identifying CTC subpopulation changes over time. Results: Our protocol had a mean recovery efficiency of 69.5% and a limit of detection of at least four cells per millilitre. We developed an analysis method to reduce noise from magnetic beads used for CTC isolation. CTCs were isolated from CRC patients with a median of 37 CTCs (IQ 13.0–85.5) at baseline. CTCs from CRC patients were significantly (p < 0.0001) larger than cytokeratin (CK)-negative cells, and patients were stratified into two groups based on BRAFV600E and PD-L1 expression on CK-positive cells. The changes observed over time included not only the number of CTCs but also their distribution into four different subpopulations defined according to BRAFV600E and PD-L1 positivity. We developed a novel protocol for semi-automatic CTC isolation and phenotypic characterisation by combining two platforms. Assessment of CTCs from early CRC patients using our protocol allowed the identification of two clusters of patients with changing phenotypes over time.

Multiparametric Circulating Tumor Cell Analysis to Select Targeted Therapies for Breast Cancer Patients.

Simple SummaryLiquid biopsies may act as a dynamic tool for identification of targets for precision therapy while circumventing limitations of tissue biopsies. In opposite to most liquid biopsy-related studies that analyze limited patient material for only one parameter, this study is based on a longitudinal and multiparametric analysis of circulating tumor cells (CTCs). A metastatic breast cancer patient was followed over a period of three years and analyses of the genome, RNA profiling, and in vitro drug testing on cultured CTCs were performed in a unique manner. We show that combining the strengths of multiple technologies for analysis yielded maximum information on the ongoing disease and, eventually, allowed choosing an effective therapy, which led to a massive reduction in CTC numbers. This approach provides a concept for future detailed longitudinal and multiparametric CTC analyses.Background: The analysis of liquid biopsies, e.g., circulating tumor cells (CTCs) is an appealing diagnostic concept for targeted therapy selection. In this proof-of-concept study, we aimed to perform multiparametric analyses of CTCs to select targeted therapies for metastatic breast cancer patients. Methods: First, CTCs of five metastatic breast cancer patients were analyzed by whole exome sequencing (WES). Based on the results, one patient was selected and monitored by longitudinal and multiparametric liquid biopsy analyses over more than three years, including WES, RNA profiling, and in vitro drug testing of CTCs. Results: Mutations addressable by targeted therapies were detected in all patients, including mutations that were not detected in biopsies of the primary tumor. For the index patient, the clonal evolution of the tumor cells was retraced and resistance mechanisms were identified. The AKT1 E17K mutation was uncovered as the driver of the metastatic process. Drug testing on the patient’s CTCs confirmed the efficacy of drugs targeting the AKT1 pathway. During a targeted therapy chosen based on the CTC characterization and including the mTOR inhibitor everolimus, CTC numbers dropped by 97.3% and the disease remained stable as determined by computer tomography/magnetic resonance imaging. Conclusion: These results illustrate the strength of a multiparametric CTC analysis to choose and validate targeted therapies to optimize cancer treatment in the future. Furthermore, from a scientific point of view, such studies promote the understanding of the biology of CTCs during different treatment regimens.

Cancer Stem Cell-Like Circulating Tumor Cells Are Prognostic in Non-Small Cell Lung Cancer.

Despite recent advances in the treatment of non-small cell lung cancer (NSCLC), less than 10% of patients survive the first five years when the disease has already spread at primary diagnosis. Methods: Blood samples were taken from 118 NSCLC patients at primary diagnosis or at progression of the disease before the start of a new treatment line and enriched for circulating tumor cells (CTCs) by microfluidic Parsortix™ (Angle plc, Guildford GU2 7AF, UK) technology. The gene expression of epithelial cancer stem cell (CSC), epithelial to mesenchymal (EMT), and lung-related markers was assessed by qPCR, and the association of each marker with overall survival (OS) was evaluated using log-rank tests. Results: EpCAM was the most prevalent transcript, with 53.7% positive samples at primary diagnosis and 25.6% at recurrence. EpCAM and CK19, as well as NANOG, PROM1, TERT, CDH5, FAM83A, and PTHLH transcripts, were associated with worse OS. However, only the CSC-specific NANOG and PROM1 were related to the outcome both at primary diagnosis (NANOG: HR 3.21, 95%CI 1.02–10.14, p = 0.016; PROM1: HR 4.23, 95% CI 0.65–27.56, p = 0.007) and disease progression (NANOG: HR 4.17, 95%CI 0.72–24.14, p = 0.025; PROM1: HR 4.77, 95% CI 0.29–78.94, p = 0.032). Conclusions: The present study further underlines the relevance of the molecular characterization of CTCs. Our multi-marker analysis highlighted the prognostic value of cancer stem cell-related transcripts at primary diagnosis and disease progression.

Clinical Significance of Circulating Tumor Cell Induced Epithelial-Mesenchymal Transition in Patients with Metastatic Colorectal Cancer by Single-Cell RNA-Sequencing.

Simple SummaryCirculating tumor cell (CTC) detection is a non-invasive and easy way to collect and separate single cells to use as tumor biomarkers. In this article, we show the feasibility and clinical value of RNA sequencing of CTCs at the single-cell level by exploiting CTC-FIND with SMART-Seq v4 technologies in patients with metastatic colorectal cancer (mCRC). CTCs were classified by epithelial, epithelial-mesenchymal transition (EMT), and stem cell-related gene expression. A patient who had EMT gene expressing CTCs showed significantly shorter PFS and OS regardless of epithelial CTCs presence. Therefore, the presence of CTCs expressing EMT-related genes at the single-cell level may be ofprognostic value and a potential biomarker for risk stratification in patients with refractory mCRC who are considered to have highly heterogeneous features.Background: Circulating tumor cells (CTCs) are a prognostic marker in patients with metastatic colorectal cancer (mCRC). However, little is known about the characterization of CTCs in mCRC at the single-cell level using RNA sequencing. The purpose of this study was to validate the capability to detect and isolate single CTCs for single-cell RNA sequencing (scRNA-seq) and to identify clinical significance at a single CTC level. Methods: Single CTCs from 27 mCRC patients were collected by CTC-FIND, which is comprised of filter separation and immunomagnetic depletion to collect ultra-pure CTC samples. To address tumor heterogeneity, CTCs were collected without relying on any traditional CTC markers, such as epithelial and mesenchymal cell antigens, and were undertaken by scRNA-seq using SMART-Seq v4. Results: We identified 59 single CTCs which were classified into four groups by epithelial, epithelial-mesenchymal transition (EMT) and stem cell-related gene expression. Patients receiving second or later-line treatment who had EMT gene expressing CTCs had a significantly shorter PFS and OS. Conclusions: Exploiting CTC-FIND with SMART-Seq v4 showed that scRNA-seq of CTCs may shed new insight into tumor heterogeneity of mCRC and that the presence of CTCs expressing EMT-related genes at the single-cell level could have prognostic value in mCRC patients.

A new insight to deformability correlation of circulating tumor cells with metastatic behavior by application of a new deformability-based microfluidic chip

Isolation and characterization of circulating tumor cells (CTCs) found in blood samples of cancer patients have been considered as a reliable source for cancer prognosis and diagnosis. A new continuous microfluidic platform has been designed in this investigation for simultaneous capture and characterization of CTCs based on their deformability. The deformability-based chip (D-Chip) consists of two sections of separation and characterization where slanted weirs with a gap of 7 μm were considered. Although sometimes CTCs and leukocytes have the same size, the deformability differs in such a way that can be exploited for enrichment purposes. MCF7 and MDA-MB-231 cell lines were used for the initial evaluation of the D-Chip performance. In the separation section, cancer cells were isolated based on deformability differences with an efficiency of higher than 93% (∼average capturing capacity of 2085 out of 2200 cancer cells ml−1) and with significantly high purity (15–40 WBCs ml−1; ∼5 log depletion of WBCs). Cancer cells were categorized based on the deformability difference in the characterization section. Subsequently, 15 clinical blood samples from breast cancer patients were analyzed by the D-Chip. Suggest 'The chip detected CTCs in all patient samples, processed the blood sample at a high throughput of 5.3 ml/h, and properly categorized CTCs based on deformability differences. Further characterization showed that the highly deformable breast cancer CTCs in our patient samples also showed higher potential of metastasis in support of a broader correlation between deformability of CTCs and metastatic behavior.

900P Mass cytometry based multiparameter characterisation of Parsortix-enriched circulating tumour cells in head and neck squamous cell carcinoma: A step towards clinical translation

Circulating tumour cells (CTCs) are a source of prognostic and predictive biomarkers across multiple cancers, including head and neck squamous cell carcinoma (HNSCC). Characterisation of CTCs has historically focussed on RNA expression. Although a powerful approach, important cellular changes can occur in the absence of transcriptional modification through processes such as alterations in protein stability, subcellular localisation or post-translational modification such as phosphorylation. To increase the number of protein markers that can be measured per cell we developed and validated a protocol to apply mass cytometry to CTCs enriched from blood using Parsortix technology.

Circulating Tumor Cells (CTCs): A Unique Model of Cancer Metastases and Non-invasive Biomarkers of Therapeutic Response.

Late-stage cancer metastasis remains incurable in the clinic and is the major cause death in patients. Circulating tumor cells (CTCs) are thought to be metastatic precursors shed from the primary tumor or metastatic deposits and circulate in the blood. The molecular network regulating CTC survival, extravasation, and colonization in distant metastatic sites is poorly defined, largely due to challenges in isolating rare CTCs. Recent advances in CTC isolation and ex vivo culture techniques facilitates single-cell omics and the development of related animal models to study CTC-mediated metastatic progression. With these powerful tools, CTCs can potentially be used as non-invasive biomarkers predicting therapeutic response. These studies may open a new avenue for CTC-specific drug discoveries. In this short review, we aim to summarize recent progress in the characterization of CTCs and their clinical relevance in various cancers, setting the stage for realizing personalized therapies against metastases.

Abstract 588: Mesenchymal markers: The new avenue for circulating tumor cells detection

Abstract Introduction: Unlike tissue biopsy, liquid biopsy allows the routine, repeat characterization of cancer at genetic, transcriptional and protein levels. Isolation of circulating tumor cells (CTCs) from a blood draw offers the potential for cancer screening, management and monitoring. Most CTC isolation systems are based on epitope-dependent CTC capture using epithelial markers. However, it is known that tumor cells can undergo epithelial-to-mesenchymal transition (EMT) when extravasating from the primary tumor to enter the bloodstream and eventually establish distant metastases. Epitope-dependent CTC detection platforms have limited sensitivity for detection of mesenchymal CTCs, leading to the inability to harvest clinically relevant CTCs for analysis, particularly in cancers with high mesenchymal phenotype such as non-small cell lung cancer (NSCLC). We developed a research use only assay for enrichment and identification of CTCs based on epithelial and mesenchymal markers using the Parsortix® system, a label-independent microfluidic device that isolates cells based on their size and compressibility. Methods: Peripheral blood (8 – 10mL) was drawn into K2EDTA tubes from 47 metastatic breast cancer (MBC) patients and 48 metastatic NSCLC patients and processed on Parsortix systems within 36 hours after collection. Harvested CTCs were cytospun and immunofluorescently stained using an optimized panel of antibodies for detection of epithelial and mesenchymal markers. Stained slides were imaged using a BioView AllegroPlus automated imaging system. Results: CTCs were captured from both MBC and NSCLC patients. 330 CTCs were identified from 19 MBC patients (mean: 17; range: 1-96). 58 CTCs were identified from 15 NSCLC patients (mean: 4; range: 1-22). Similarly, CTC clusters were observed in 63% (12/19) of the CTC-positive MBC patients and in 33% (5/15) of CTC-positive NSCLC patients. Cluster size ranged from 2-22 CTCs per cluster (2-17 in MBC and 2-22 in NSCLC), and the number of clusters per patient ranged from 1-13 in MBC and 1-2 in NSCLC. Phenotypically, more CTCs expressing only mesenchymal markers were harvested from NSCLC patients compared to MBC patients (38% vs 25%), with a larger proportion of CTCs expressing both epithelial and mesenchymal markers being harvested in both cancer types (59% in NSCLC and 74% in MBC). Only a small percentage of the CTCs harvested expressed just the epithelial markers. Conclusions: This study highlights the importance of the inclusion of mesenchymal markers into CTC characterization, as the majority of the CTCs captured by the Parsortix system from MBC and NSCLC patients expressed mesenchymal markers. The assay developed in this study successfully allowed phenotyping of the harvested cells using epithelial AND mesenchymal markers, with the results suggesting that a large proportion of the CTCs harvested would have been missed with an epithelial-only based approach, particularly in NSCLC. Citation Format: Mariacristina Ciccioli, Natalia Bravo-Santano, Amy Davis, Jolie Lewis, Ross Malcolm, Anne-Sophie Pailhes-Jimenez. Mesenchymal markers: The new avenue for circulating tumor cells detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 588.