Identification Of Circulating Tumor Cells Research Articles

Circulating tumour cells predict recurrences and survival in head and neck squamous cell carcinoma patients

Patients with head and neck squamous cell carcinoma (HNSCC) are at a high risk of developing recurrence and secondary cancers. This study evaluates the prognostic and surveillance utilities of circulating tumour cells (CTCs) in HNSCC. A total of 154 HNSCC patients were recruited and followed up for 4.5 years. Blood samples were collected at baseline and follow-up. CTCs were isolated using a spiral microfluid device. Recurrence and death due to cancer were assessed during the follow-up period. In patients with HNSCC, the presence of CTCs at baseline was a predictor of recurrence (OR = 8.40, p < 0.0001) and death (OR= ∞, p < 0.0001). Patients with CTCs at baseline had poor survival outcomes (p < 0.0001). Additionally, our study found that patients with CTCs in a follow-up appointment were 2.5 times more likely to experience recurrence or death from HNSCC (p < 0.05) prior to their next clinical visit. Our study highlights the prognostic and monitoring utilities of CTCs’ in HNSCC patients. Early identification of CTCs facilitates precise risk assessment, guiding treatment choices and ultimately enhancing patient outcomes.

Abstract PO3-14-10: A single-center prospective cohort study to evaluate circulating tumor cells as a monitoring tool in women with breast cancer treated with neoadjuvant chemotherapy: final results of baseline data

Abstract Background: The presence of liquid biomarkers such as circulating tumor cells (CTCs) among women undergoing neoadjuvant chemotherapy (NAC) for breast cancer may be associated with treatment response and/or an increased risk of recurrence, but limited data is available. Objectives: To detect and enumerate CTCs in blood samples from women with a new diagnosis of non-metastatic breast cancer of any subtype both i) at baseline (prior to commencing NAC), and ii) after completion of NAC and surgery using the Epic Sciences platform. Methods: Women with non-metastatic breast cancer of any subtype who have not yet commenced NAC were included, irrespective of age. Those with a prior history of another invasive cancer (apart from non-melanoma skin cancer identified 5+ years prior to enrollment) were excluded. Blood samples were obtained to measure CTCs prior to commencing NAC, and after completion of NAC and surgery (at least 4 weeks post-operatively). As previously described, CTC identification was based on immunofluorescence analysis using the Epic Sciences platform (Ueno et al 2017). Clinical/pathological data and clinical outcomes were abstracted from patients’ medical records. Associations between CTC detection and clinical/pathologic characteristics were evaluated using Fisher’s exact test for categorical variables and t-test or Wilcoxon rank sum tests for numerical variables. All analyses were performed using the R software package. Results: 50 participants who met eligibility criteria were included; a baseline blood sample was evaluable for CTC detection and enumeration for 47 patients. The median age at breast cancer diagnosis was 52 (29-75). Among them, 18 (38.3%) had HER2+ breast cancer, 17 (36.1%) had hormone receptor (HR)+/HER2- breast cancer and 12 (25.5%) had triple negative breast cancer (TNBC). The majority of patients (91%) received anthracycline and taxane-based NAC. A total of 68 samples were tested for CTC enumeration (5 mL equivalent per sample) including 47 pre-treatment and 21 post-treatment samples. CTCs were detected in 37 (79%) of patients for whom a baseline (pre-NAC) sample was available with a mean of 4.2 (SD 17.0) CTCs/mL and a range of 0 - 115.4 CTCs/mL. Detection of CTCs at baseline was highest among patients with TNBC (n=10/12, 83%), followed by those with HER2+ (n=14/18, 78%) and HR+/HER2- (n=13/17, 76%) breast cancer. CTCs were detected in 43% (n=9/21) of post-treatment samples, with a mean of 1.2 (SD 3.6) CTCs/mL and range of 0 – 16.4 CTCs/mL. Among the 20 patients for whom matched pre- and post-treatment CTC results were available, 16 (80%) had detectable CTCs pre-treatment and 8 (40%) had detectable levels post-NAC and surgery; of the 8 patients with post-treatment CTCs, 4 (50%) had HR+/HER2- breast cancer, 2 had HER2+ (25%) and 2 (25%) had TNBC. Three patients had numerically higher CTC levels after completion of NAC and surgery compared to baseline levels, 2 of whom had HR+/HER2- breast cancer and one of whom had TNBC. To-date, only 8 patients (19% of 43 who have undergone surgery) have achieved a pathological complete response (PCR) to NAC, among whom 3 had matched pre- and post-treatment CTC results available. None of these 3 patients had detectable CTCs post treatment. Conclusions: Approximately 4 in 5 women with non-metastatic breast cancer who undergo NAC have detectable CTCs at baseline (pre-treatment) using the Epic Sciences Platform. Given that CTCs remain detectable in a high proportion (40%) of patients after NAC and surgery, evaluation of CTCs as a potential measure of minimal residual disease warrants further evaluation in this patient population. Citation Format: Rania Chehade, Arushi Jain, Veronika Moravan, Giuseppe Di Caro, Megan Slade, Nadine Hartmann, Rick Wenstrup, Ana Elisa Lohmann, William Tran, Katarzyna Jerzak. A single-center prospective cohort study to evaluate circulating tumor cells as a monitoring tool in women with breast cancer treated with neoadjuvant chemotherapy: final results of baseline data [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-14-10.

Clustered circulating tumor cells as a predictor of adjuvant-chemotherapy efficacy in lung cancer

Surrogate markers of minimal residual disease primarily include cell-free tumor deoxyribonucleic acid and circulating tumor cells. Cell-free tumor DNA might aid precise decision making regarding who should receive adjuvant chemotherapy. However, there are no relevant reports on circulating tumor cells. Therefore, we aimed to verify whether perioperative clustered circulating tumour cells identification is a predictor of therapeutic efficacy in non-small cell lung cancer adjuvant chemotherapy. Circulating tumor cells were diagnosed under light microscopy using a size selection method in 128 patients with clinical stage I/II non-small cell lung cancer around surgery. The main endpoint was recurrence-free survival, and the effect of adjuvant chemotherapy was verified in both groups based on perioperative clustered circulating tumor cell identification. In total, 49 and 79 patients were included in the clustered circulating tumor cell-positive and clustered circulating tumor cell-negative patient groups, respectively. In the clustered circulating tumor cell-positive patient group, adjuvant chemotherapy was performed in 18 patients (2-year recurrence-free survival rate, 71.8%). However, the 2-year recurrence-free survival rate was 36.3% in 31 patients who did not receive adjuvant chemotherapy (p<0.01). In the clustered circulating tumor cell-negative patient group, adjuvant chemotherapy was provided in 11 patients (2-year recurrence-free survival rate, 90.9%). However, 68 patients did not receive adjuvant chemotherapy (2-year recurrence-free survival rate, 94.9%) (not significant). In surgical cases of clinical stage I/II non-small cell lung cancer, patients with perioperative clustered circulating tumor cells had a poor prognosis, but adjuvant chemotherapy improved their prognosis.

Research progress of cell membrane biomimetic nanoparticles for circulating tumor cells.

Early detection of cancer is crucial to reducing fatalities and improving patient outcomes. Metastasis is the first stage of aggressive cancers, often occurring before primary lesions can be seen. It occurs when cancerous cells disseminate to distant, non-malignant organs through the bloodstream, known as circulating tumor cells (CTCs). CTCs, or cancer tumor cells, are valuable indicators for predicting treatment response, metastasis progression, and disease progression. However, they are primarily used for research due to challenges like heterogeneity, separation from blood, and lack of clinical validation. Only a few methods have been approved for clinical use. One area of research is the isolation and identification of CTCs, which could significantly impact early cancer detection and prognosis. Current technologies using whole-blood samples use size, immunoaffinity, and density approaches, along with positive and negative enrichment techniques. Surface modification of nanomaterials is important for effective cancer therapies because it improves their ability to target and reduces interactions with healthy tissues. Consequently, researchers have created biomimetic nanoparticles covered with cell membranes using functional, targeted, and biocompatible coating technology. Nanoparticles with membranes can target specific cells, stay in circulation for longer, and avoid immune responses, which makes them much better at capturing CTCs. This study examines the current opportunities and difficulties associated with using cell membrane-coated nanoparticles as a capture technique for CTCs. In addition, we examine potential future developments in light of the current obstacles and investigate areas that require further research to fully understand its growing clinical possibilities.

A Detachable Magnetic Nanodevice for the Efficient Capture and Subtype Identification of Circulating Tumor Cells

AbstractThe effective capture and accurate identification of heterogeneous circulating tumor cells (CTCs) aid in guiding clinical diagnosis and personalized treatment of tumors. Herein, a core‐satellite‐sized magnetic separable nanodevice (denoted as MS‐RI) is developed for the capture and subtype identification of heterogeneous CTCs by identifying the protein targets of CTCs in vitro and imaging intracellular nucleic acid targets of CTCs in situ. Upon the separation of CTCs by MS‐RI using aptamers as capture elements, the programmatically dissociated satellite particles aim to internalize into the cells for simultaneous profiling of microRNA‐21 and microRNA‐141. By virtue of the validated magnetic separation of the core structure, the high affinity of the aptamer, and the high sensitivity of the recognition imaging (RI) module, MS‐RI allows the isolation and subtyping of CTCs in clinical samples from breast cancer patients. This strategy of combining extracellular recognition and intracellular imaging pioneers a new paradigm for the capture and precision subtyping of heterogeneous CTCs.

Abstract 7493: Real-time monitoring of CTC number, biomarker expression, and kinetics as prognostic and predictive markers in patients with GI malignancies

Abstract Background: Most of the utility in the clinic regarding liquid biopsies has been around circulating tumor DNA (ctDNA). ctDNA analysis provides valuable insight into mutational burden and therapeutic targets, but the speed and cost of analysis are prohibitive especially when it comes to serial testing. Using a cohort of patients with advanced or metastatic gastrointestinal (GI) cancers, we herein show the feasibility of using circulating tumor cell (CTC) enumeration, biomarker analysis, and kinetics as complementary to or as an alternative to ctDNA testing. Methods: Using the RareCyte platform, nucleated cells from blood were collected, spread on slides, stained, and imaged with CyteFinder® for CTC identification (CK/EpCAM+, CD45-) and quantification of biomarkers (PD-L1/HER2 or Ki67/EGFR). CTC enumeration (CLIA-accredited CTC test) and biomarker status for each patient were tracked over time and compared with ctDNA mutational analysis and tumor burden. Results: CTCs were detected in 60/70 samples (86%) and in 36/38 unique patients (95%). On average, more CTCs were detected in baseline samples from colorectal cancer patients compared to other subtypes (Table 1). Moreover, multiple patients showed pronounced changes in CTC number that correlated with treatment response, as well as strong correlation between CTC and tissue biomarker expression e.g. HER2. Rapid decline and/or CTC clearance was seen as early as a few days into starting effective systemic therapy. The average turnaround time for CTC testing was 5-7 days. Table 1. CTC values in initial baseline blood draw sample Gastrointestinal cancer subtype # of patients Average # of CTCs per 7.5mL blood Median # of CTCs per 7.5mL blood Minimum # of CTCs per 7.5mL blood Maximum # of CTCs per 7.5mL blood Colorectal carcinoma 27 84 3 0 1378 Cholangiocarcinoma 3 8 3 1 23 Pancreatic cancer 3 2 2 0 3 Hepatocellular carcinoma 2 4 5 0 10 Gallbladder cancer 1 6 6 3 8 Appendix cancer 1 2 2 1 3 Anal cancer 1 0 0 0 0 Conclusions: Our study emphasizes that real-time monitoring of CTCs is complementary to traditional tumor burden monitoring via imaging and ctDNA analysis. CTCs offer multiple unique advantages vs ctDNA including early response assessment within days of initiating therapy, a substantial decrease in cost of serial testing, and the ability to assess clinically relevant cell surface protein biomarkers e.g. HER2 and PD-L1. Citation Format: Erin G. Bayer, Brock M. Bartels, Rachel N. Ponting, Areeb Lutfi, Maaz K. Afghan, Arturo B. Ramirez, Pashtoon M. Kasi. Real-time monitoring of CTC number, biomarker expression, and kinetics as prognostic and predictive markers in patients with GI malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7493.

Prognostic evaluation of a multi-target magnetic bead-enriched circulating tumor cell-enriched identification system for colorectal cancer.

Colorectal cancer (CRC) is the third most prevalent cancer in the world. Traditional tissue biopsy cannot provide dynamic monitoring of patients' tumors or reflect the characteristics of tumors in real time because the sampling process is invasive and accompanied by risks. Circulating tumor cells (CTCs) are considered a major cause of tumor metastasis, and investigating CTCs helps to understand the biology and vulnerability of malignant tumors during hematogenous metastasis. We sequentially used epithelial cell adhesion molecule (EpCAM)-coated immunoliposomal magnetic beads (Ep-IMBs) and vimentin-coated immunoliposomal magnetic beads (Vi-IMBs) to capture and characterize CTCs from 110 CRC patients. We further constructed a Cox risk regression model, optimized the model composition using backward stepwise regression, and finally applied nomograms to show the effect of each variable on survival risk. The specificity of the CTCs enrichment and identification system was 100% and the sensitivity was 79.0%. Multivariate analysis indicated total CTC number was an important predictor for bad survival, whereas American Joint Committee on Cancer (AJCC) stage, lymph node metastasis, and carcinoembryonic antigen (CEA) level were associated with prognosis, and the risk of mortality was associated with the AJCC stage of the CRC. The CTC enrichment and identification system constructed in this research demonstrated superior accuracy. In addition, CTCs can be used as an important predictor for prognosis of patients with CRC, and the combination of other clinical predictive factors can help clinicians to better design individualized treatment regimens, which is of great clinical application value.

Origin, Metastasis, Harm, Recognition and Capture, and Elimination of Circulating Tumor Cells

AbstractCancer is now a major threat to human health and life. Although a number of clinical approaches to cancer treatment have been developed in recent years, tumor recurrence and metastasis remain a challenge. As the “seed” for the formation of tumor metastasis, Circulating Tumor Cells (CTCs) can not only grow and spread in the primary site, but also spread to other parts of the body through various ways. The close connection between CTCs and tumor progression makes them a focus of clinical and basic research attention, and it not only has an important guide for the clinical treatment of patients, but also a window into the occurrence and development of distant metastases. The identification and detection of CTCs have an important clinical significance in the assessment of the prognosis of cancer, efficacy prediction, efficacy evaluation, and the study of recurrent metastasis and drug resistance mechanisms of cancer patients. Therefore, this review not only introduces the source, attributes, transfer, and harm of CTCs, but also highlights the progress and latest technologies for the identification and capture and elimination strategies of CTCs and presents prospects for future research of CTCs.

Ultrasensitive detection of circulating tumor cells in clinical blood samples by a three-dimensional network nanovehicle-based aptasensor platform

A burgeoning topic in the early diagnosis and treatment of cancer is the sensitive and precise quantification of circulating tumor cells (CTCs). However, due to the extremely low CTC concentrations in the blood, traditional enrichment and identification methods for CTCs are prone to false negative results. Here, to specifically identify and improve the sensitivity of CTC detection in the blood, we designed a novel fluorometric turn-on aptasensor platform based on a three-dimensional network nanovehicle (TDNN) modified with a PEG derivative and AS1411 aptamer specifically targeting the nucleolin protein. The limit of detection (LOD) of the aptasensor platform was as low as 2 cells per mL, which was remarkably better than that of the single nanoparticle ASS1. Notably, the novel aptasensor platform showed excellent specificity and sensitivity as well as negligible fluorescent background for quantitative and visualization detection of CTCs in peripheral blood samples without enrichment, centrifugation and washing steps. More importantly, the number of CTCs identified by TDNN was significantly higher in metastatic patients than in nonmetastatic patients, while CTCs were not detected in the blood samples of healthy donors. In conclusion, the aptasensor platform provides an available and promising tool for CTC identification and cancer metastasis prediction in clinical blood samples.

Minimizing false positives for CTC identification

BackgroundCancer is a leading cause of death worldwide, with metastasis playing a significant role. Circulating Tumour Cells (CTCs) can provide important real-time insights into tumour heterogeneity and clonal evolution, making them an important tool for early diagnosis and patient monitoring. Isolated CTCs are typically identified by immunocytochemistry using positive biomarkers (cytokeratin) and exclusion biomarkers (CD45). However, some white blood cell (WBC) populations can express low levels of CD45 and stain non-specifically for cytokeratin, increasing their risk of misclassification as CTCs. There is a clear need to improve CTC detection and enumeration criteria to unequivocally eliminate interfering WBC populations. ResultsThis study showed that, indeed, some granulocyte subpopulations expressed low levels of CD45 and stained non-specifically for cytokeratin, misidentifying them as CTCs. These same cells, however, strongly expressed CD15, allowing them to be identified as WBCs and excluded from CTC classification. Flow cytometry confirmed the specificity of the CD15 antibody for the granulocyte subpopulation. False positives were considerably reduced from 25 % to 0.2 % by double exclusion, combining a CD15 antibody with a highly specific CD45 antibody. Furthermore, complete elimination of potential false positives was achieved using double exclusion in combination with improved selection of cytokeratin antibody. The study emphasises the importance of a robust exclusion criteria and high antibody specificity in CTC immuno-assays for accurate identification of CTC candidates and thorough exclusion of interfering WBC subpopulations. SignificanceThis study demonstrated how misidentifying a granulocyte subpopulation can lead to inaccurate CTC evaluation. However, sensitivity and specificity of CTC identification may be improved by using high-performing antibodies and by including a second exclusion biomarker, in turn, allowing for a more comprehensive clinical application of CTCs.

Homoporous polydimethylsiloxane membrane microfilter for ultrafast label-free isolation and recognition of circulating tumor cells in peripheral blood

The detection of circulating tumor cells (CTCs) in peripheral blood is a novel and accurate technique for the early diagnosis of cancers. However, this method is challenging because of the need for high collection efficiency due to the ultralow content and similar size of CTCs compared with other blood cells. To address the aforementioned issue, we proposed a homoporous polydimethylsiloxane (PDMS) membrane and its microfilter device to perform the ultrafast isolation and identification of CTCs directly from peripheral blood without any labeling treatment. The membrane pores can be homogenously controlled at a size of 6.3μm through the cross-linking time of PDMS during a filtration-coating strategy. Within only 10 s, the designed device achieved a retention rate greater than 70% for pancreatic cancer cells, and it exhibited excellent cell compatibility to support cell proliferation. The isolated CTCs on this membrane can be easily observed and identified using a fluorescence microscope.

A Novel Filtration Membrane for Clustered Circulating Tumor Cell Extraction: A Prospective Feasibility Study.

Circulating tumor cells (CTCs) have garnered attention as biomarkers for therapeutic response and prognosis in malignant neoplasms. Nonetheless, existing literature predominantly relies on surrogate markers of tumor cells or focuses on single-cell CTC, failing to adequately address the challenge of detecting cluster-forming CTCs, which bear considerable prognostic implications. This prospective study aims to validate the efficacy of a novel filtration membrane, namely Soft Micro Pore Filter (S-MPF®), for rare cell recovery in detecting CTCs through the analysis of clinical samples. Patients with confirmed lung cancer or highly suspected lung cancer based on specific criteria (solid tumor size >2.0 cm, serum carcinoembryonic level >7.5 ng/ml, maximum standard uptake value derived from fluorodeoxyglucose-position emission tomography >2.9) were included in the study. CTCs were extracted from preoperative peripheral arterial blood samples using S-MPF®, and the validity of the filtration system was positively verified. Out of the 25 enrolled patients, 23 had lung cancer. CTC positivity was observed in 17 cases (73.9%), whereas cluster CTC positivity was observed in 16 cases (69.6%), with a median count of two clusters. Single CTC positivity was observed in 11 cases (52.1%), with a median count of one cell. The utilization of the newly developed S-MPF® filtration membrane exhibited a high rate of CTC identification, demonstrating its suitability for clinical applications.

A Micropillar Array Based Microfluidic Device for Rare Cell Detection and Single-Cell Proteomics.

Advancements in single-cell-related technologies have opened new possibilities for analyzing rare cells, such as circulating tumor cells (CTCs) and rare immune cells. Among these techniques, single-cell proteomics, particularly single-cell mass spectrometric analysis (scMS), has gained significant attention due to its ability to directly measure transcripts without the need for specific reagents. However, the success of single-cell proteomics relies heavily on efficient sample preparation, as protein loss in low-concentration samples can profoundly impact the analysis. To address this challenge, an effective handling system for rare cells is essential for single-cell proteomic analysis. Herein, we propose a microfluidics-based method that offers highly efficient isolation, detection, and collection of rare cells (e.g., CTCs). The detailed fabrication process of the micropillar array-based microfluidic device is presented, along with its application for CTC isolation, identification, and collection for subsequent proteomic analysis.

Raman Spectroscopy Combined with Malaria Protein for Early Capture and Recognition of Broad-Spectrum Circulating Tumor Cells.

Early identification of tumors can significantly reduce the mortality rate. Circulating tumor cells (CTCs) are a type of tumor cell that detaches from the primary tumor and circulates through the bloodstream. Monitoring CTCs may allow the early identification of tumor progression. However, due to their rarity and heterogeneity, the enrichment and identification of CTCs is still challenging. Studies have shown that Raman spectroscopy could distinguish CTCs from metastatic cancer patients. VAR2CSA, a class of malaria proteins, has a strong broad-spectrum binding effect on various tumor cells and is a promising candidate biomarker for cancer detection. Here, recombinant malaria VAR2CSA proteins were synthesized, expressed, and purified. After confirming that various types of tumor cells can be isolated from blood by recombinant malaria VAR2CSA proteins, we further proved that the VAR2CSA combined with Raman spectroscopy could be used efficiently for tumor capture and type recognition using A549 cell lines spiked into the blood. This would allow the early screening and detection of a broad spectrum of CTCs. Finally, we synthesized and purified the malaria protein fusion antibody and confirmed its in vitro tumor-killing activity. Herein, this paper exploits the theoretical basis of a novel strategy to capture, recognize, and kill broad-spectrum types of CTCs from the peripheral blood.

Single-Cell Enzymatic Screening for Epithelial Mesenchymal Transition with an Ultrasensitive Superwetting Droplet-Array Microchip.

The phenotypic changes of circulating tumor cells (CTCs) during the epithelial-mesenchymal transition (EMT) have been a hot topic in tumor biology and cancer therapeutic development. Here, an integrated platform of single-cell fluorescent enzymatic assays with superwetting droplet-array microchips (SDAM) for ultrasensitive functional screening of epithelial-mesenchymal sub-phenotypes of CTCs is reported. The SDAM can generate high-density, volume well-defined droplet (0.66 nL per droplet) arrays isolating single tumor cells via a discontinuous dewetting effect. It enables sensitive detection of MMP9 enzyme activities secreted by single tumor cells, correlating to their epithelial-mesenchymal sub-phenotypes. In the pilot clinical double-blind tests, the authors have demonstrated that SDAM assays allow for rapid identification and functional screening of CTCs with different epithelial-mesenchymal properties. The consistency with the clinical outcomes validates the usefulness of single-cell secreted MMP9 as a biomarker for selective CTC screening and tumor metastasis monitoring. Convenient addressing and recovery of individual CTCs from SDAM have been demonstrated for gene mutation sequencing, immunostaining, and transcriptome analysis, revealing new understandings of the signaling pathways between MMP9 secretion and the EMT regulation of CTCs. The SDAM approach combined with sequencing technologies promises to explore the dynamic EMT plasticity of tumors at the single-cell level.

Abstract 3373: Deep learning for circulating tumor cell (CTC) identification with the CELLSEARCH system: achieving top human-level performance

Abstract Background: Enumeration of CTCs in blood samples with the CELLSEARCH® system is a prognostic biomarker in metastatic breast, prostate and colorectal cancer (full intended use documents.cellsearchctc.com). Currently, the final CTC identification is performed by human reviewers through visual assessment, which is a time-consuming procedure that could be affected by subjective interpretations. Modern Artificial Intelligence (AI) methods can provide a solution by automating the process of CTC identification, thus eliminating subjectivity and producing faster and more reproducible results. We have recently presented an automated algorithm (for research use only) based on deep learning models for cell segmentation and classification which allows the automated identification of CTCs in CELLSEARCH® fluorescent images (Ansaloni et al., EACR 2022). The purpose of this study was to assess inter-rater variability among human reviewers and to compare the AI performance against them. Methods: Blood samples derived from metastatic cancer patients were processed with CELLTRACKS® AUTOPREP®. The AI algorithm was trained on fluorescent images of 7255 CTCs and 32876 non-CTC events (training set) acquired by CELLTRACKS ANALYZER II® (CTAII) from 90 breast, 122 prostate and 54 colorectal cancer samples. 10 human reviewers, qualified for CTC image analysis, performed blind labeling of a separate dataset independently (test set: 55 samples taken from 27 breast and 28 prostate cancer). We studied inter-rater agreement and variability using Fleiss Kappa and Coefficient of Variation (CV). The labels provided by 5 experienced reviewers (average experience ≅ 15 years) were used to generate the ground truth (GT) by majority voting. The CTC detection performance of the AI was ranked against the other 5 reviewers (average experience ≅ 5 years). Results: In the test dataset, 2990 out of 9554 events were identified as CTCs by the GT review on CTAII images. In the task of classifying each event as CTC or non-CTC, the inter-rater agreement among the 10 human reviewers measured by Fleiss Kappa was 0.85. These classification results provided the number of CTCs per patient counted by each reviewer. The inter-rater variability on the CTC enumeration among the 10 reviewers was evaluated for each sample (median CV = 17.9% and interquartile range = 17.6% , excluding 27 samples with mode = 0 CTC). Measured against the GT classification, the AI performance (accuracy = 94.7%; F1 = 91.6%) was at the top of the human reviewers’ ranking (accuracy = 92.4 - 93.6 - 93.8 - 94.0 - 95.8%; F1 = 86.8 - 89.2 - 89.2 - 89.5 - 93.1%). Conclusion: The AI algorithm reached top-ranking performance in CTC identification, surpassing 4 out of 5 expert reviewers. These results show the applicability of AI to cell classification to remove human subjectivity from the review process and to maximize standardization among different research centers. Citation Format: Luca Biasiolli, Pietro Ansaloni, Nicolò Gentili, Ramona Miserendino, Chiara Rossi, Giulio Signorini, Gianni Medoro. Deep learning for circulating tumor cell (CTC) identification with the CELLSEARCH system: achieving top human-level performance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3373.

Abstract 6605: Automated isolation and identification of circulating rare cells from blood samples using a label-free rare cell isolation platform - the Genesis System with Celselect Slide technology

Abstract Introduction: Isolation and identification of circulating tumor cells (CTCs) offers a promising non-invasive diagnostic tool for cancer research, but significant technical hurdles have made practical application challenging. Recent studies have shown that CTCs undergo cellular changes, such as an epithelial-mesenchymal transition (EMT), with disease progression and metastasis, implying the heterogeneity of the tumor cell population in circulation. Systems for CTC isolation are often limited by a requirement for epithelial markers or separate systems for identification of CTCs. As such, an automated platform that successfully isolates both epithelial and mesenchymal cells and enables identification of cells is necessary for successful study of CTCs. The Genesis System from Bio-Rad™ Laboratories, Inc. isolates rare CTCs based on size and deformability. It also enables users to identify isolated cells through use of an on-slide immunofluorescence (IF) staining workflow, which minimizes sample manipulation and potential sample loss. Methods: In order to achieve high-throughput label-free CTC isolation, the Genesis System uses Celselect Slides™. Each slide contains 56,400 individual microchambers to capture CTCs while maintaining viability. This Celselect Slide technology can be used for either enrichment or enumeration protocols. For this study, enumeration protocols were performed to capture and identify CTCs using on-slide IF staining. Enrichment protocols were utilized to capture, enrich, and retrieve CTCs for downstream applications. The performance was evaluated using a breast cancer cell line, MCF-7, spiked into whole blood from healthy donors. Results: The slide isolated 83.95 ± 10.25% of spiked cancer cells in blood samples at a concentration of 10-250 cells/ml. During the CTC enrichment procedure, 99.92% of blood cells were eliminated. Afterwards, 70% of the isolated CTCs were successfully released from the slide and over 92% of the released cells remained viable. Discussion and Conclusion: We present a fully automated platform to isolate and identify CTCs using on-slide immunofluorescence staining, which minimizes inevitable cell loss during the workflow. Captured cells can be retrieved for downstream applications to facilitate discovery of unknown CTC functions and their clinical relevance in cancer research. The wide range of possible downstream applications include Droplet Digital™ PCR, quantitative PCR, western blot assays, bulk/single-cell genomics, cell culture, fluorescence in situ hybridization, and RNA-Seq. Additionally, the design of the system is amenable to other biofluids which may be utilized for liquid biopsies. The Genesis System with Celselect Slide technology offers a powerful diagnostic tool to monitor disease progression and personalized treatment. Citation Format: Yoon-Tae Kang, Errile Pusod, Ally Crudgington, Abiodun Bodunrin, Nick Bosio, Susana Chan, David Coe, Michelle Racey. Automated isolation and identification of circulating rare cells from blood samples using a label-free rare cell isolation platform - the Genesis System with Celselect Slide technology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6605.

Abstract 1040: Evaluation of blood collection tube’s performance for analysis of liquid biopsies by flow and mass cytometry

Abstract The detection of liquid biopsies, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have demonstrated promise as non-invasive assays for monitoring response to therapy for patients with cancer. While ctDNA cannot predict transcriptomic and proteomic changes in response to therapy, we can measure direct cell surface antigen and gene expression changes in CTCs. In pediatric settings, blood samples are often collected from multi-institutional trials. Therefore, to translate liquid biopsies into routine practice, it is important to explore variables most likely to impact the functionality of CTCs including time to process the sample, prospective cryopreservation, blood collection tube and fixative reagents. Cell stabilizer tubes such as Streck Cell-Free DNA, CellSave, CellRescue, or PAXgene ccfDNA have been designed to prevent cell lysis and release of genomic DNA into the plasma, often observed in standard EDTA tubes. In pediatrics, we are often limited by the volume of blood we can draw for research. To this end, we have established an innovative pipeline to collect ctDNA, CTCs, and matched germline DNA from a single blood sample. Blood samples were processed for plasma extraction and CTC isolation after 4h, 24h, and 48h. We isolated rare CTCs from whole blood by combining peripheral blood cell depletion with antigen-based CTC identification. We then evaluated CTC recovery rate and cell membrane integrity (cell-membrane permeabilization) across all blood collection tubes by flow or mass cytometry. Additionally, we integrated whole transcriptome analysis of RNA transcripts in individually sorted CTCs. In this study, we detected similar levels of cell-free DNA, germline DNA and ctDNA in healthy donors and patient samples collected in the DNA stabilizer tubes up to 48h after blood collection. Surprisingly, frequent clotting was observed in the cell layer of samples collected in PAXgene tubes only 24h after blood collection, preventing further analysis of CTC quality in these tubes. At 4h, EDTA tubes and stabilizer tubes seem to perform similarly in preserving CTC integrity. However, at later timepoints, CTC recovery rate, RNA quality and cell membrane integrity were significantly higher in CellRescue tubes. Finally, we showed that CTC count, and RNA quality were slightly lower in patient samples enumerated after cryopreservation compared to the paired-samples processed the same day in both EDTA and cell-stabilizing tubes. In conclusion, we validated the CellRescue tubes as the best candidate to reduce cell lysis and protect against cell membrane permeabilization and mRNA degradation for up to 48 hours, allowing sample collection from multi-institutional studies. To the best of our knowledge, this study is the first to compare the key steps in handling blood samples and how this affects transcriptomic-based assay performance. Citation Format: Anne-Florence Blandin, Sarah Finstuen-Magro, Samuel Abbou, Kelly Klega, Alejandra E. Aguilar, Mohammad Tanhaemami, Maria Ana Isabel C. De Los Santos, Joadly Duplan, Brian Crompton. Evaluation of blood collection tube’s performance for analysis of liquid biopsies by flow and mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1040.

Abstract P6-01-17: A single-center prospective cohort study to evaluate circulating tumor cells as a monitoring tool in women with breast cancer treated with neoadjuvant chemotherapy

Abstract Background The presence of circulating tumor cells (CTCs) among women before and/or after completion of neoadjuvant chemotherapy (NAC) for breast cancer may be associated with an increased risk of recurrence, but limited data is available. Objectives To use the Epic Sciences platform to detect and enumerate CTCs in blood samples from women with a new diagnosis of non-metastatic breast cancer of any subtype both i) prior to commencing NAC, and ii) after completion of NAC and surgery. Methods Inclusion criteria included women of any age with non-metastatic breast cancer of any subtype who have not yet commenced NAC. Those diagnosed with prior invasive cancer at any site (apart from non-melanoma skin cancer diagnosed more than five years prior to enrollment) were excluded. Blood samples were obtained to measure CTCs prior to NAC and after NAC and surgery, respectively. CTC identification was based on immunofluorescence analysis using Epic Sciences platform as previously described (Ueno et al 2017). The presence of CTCs was correlated with clinical/pathological data and treatment response, which were abstracted from patients’ medical records. The association between the presence of CTCs and clinical/pathologic characteristics was tested using Fisher’s exact test for categorical variables and t-test or Wilcoxon rank sum tests for numerical variables. All analyses were performed using the R software package. An ad-hoc preliminary analysis was conducted among the first 34 of 50 participants. Results 41 patients (out of an intended 50) have been recruited to-date. 34 participants have a pre- and/or post-treatment CTC measurement available, but 1 was excluded because it was identified to have metastatic disease shortly after enrollment. Among 33 evaluable patients without metastatic disease, 6 (19%) had triple negative breast cancer (TNBC), 13 (39%) had HER2+ and 13 (39%) had hormone receptor (HR)+/HER2- breast cancer. Most (94%) received anthracycline and taxane-based NAC. The median age of breast cancer diagnosis was 50 (29-75). A total of 53 samples were tested for CTC enumeration (5 mL per sample) including 33 pre-treatment and 20 post-treatment samples. CTCs were detected in 32 samples (n=32/53, 60%), including 24 pre-NAC (n=24/33, 73%) with a median of 0.9 CTCs per mL (0.2-19) and 8 post-NAC and surgery (n= 8/20, 40%) with a median of 0.6 CTCs per mL (0.3-3.3). Among the 24 patients (73%) who had detectable CTCs pre-NAC, 10 had HR+/HER2- (41.7%), 9 had HER2+ (37.5%) and 4 (16.7%) had triple negative disease. Among the 20 patients for whom matched pre- and post-treatment CTC results were available, 16 (80%) had detectable CTCs pre-treatment and 8 (40%) had detectable levels post-NAC and surgery. Among the 8 patients (40%) for whom CTCs were detectable post NAC and surgery, 4 (50%) had HR+/HER2-, 2 had HER2+ (25%) and 2 (25%) had triple negative disease. 3 of these patients had numerically higher CTC levels after completion of NAC and surgery compared to pre-NAC levels, 2 of whom had HR+/HER2- breast cancer and one of whom had TNBC. A total of 7 of 33 patients achieved a pathological complete response (PCR) to NAC, among whom 3 had matched pre- and post-treatment CTC results available; none of these 3 patients had detectable CTCs post-treatment. Conclusions Approximately 3 in 4 women with non-metastatic breast cancer who undergo NAC have detectable CTCs pre-treatment using the Epic Sciences Platform. Of 20 patients with matched pre-/post-treatment results, a high proportion (40%) have persistently detectable CTCs. Hence, CTCs may represent an additional measure of minimal residual disease for patients undergoing NAC for breast cancer. Citation Format: Rania chehade, Arushi Jain, Veronika Moravan, Giuseppe Di Caro, Amanda Anderson, Megan M. Slade, Rick Wenstrup, Ana Elisa Lohmann, William Tran, Katarzyna Jerzak. A single-center prospective cohort study to evaluate circulating tumor cells as a monitoring tool in women with breast cancer treated with neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-17.

Abstract P1-05-35: Identification of Circulating Tumor Cells captured by the FDA Cleared Parsortix® PC1 System from the Peripheral Blood of Metastatic Breast Cancer Patients using Immunofluorescence and Cytopathological Evaluations

Abstract Background: Circulating tumor cells (CTCs) captured from the blood of cancer patients may serve as a non-invasive surrogate source of tumor material to investigate tumor characteristics in real-time. The Parsortix® PC1 System, the first FDA-cleared medical device for the capture and harvest of CTCs from peripheral blood of metastatic breast cancer (MBC) patients for use in subsequent user-validated downstream analyses, enables the epitope independent capture of CTCs with diverse phenotypes based on cell size and deformability. In this study, CTCs isolated from the blood of MBC patients by the Parsortix® PC1 System were identified using an immunofluorescence (IF) based assay for detection of epithelial CTCs followed by Wright-Giemsa staining and cytomorphological review. The aim of this study was to determine the proportion of MBC patients and self-declared female healthy volunteers (HVs) that had one or more CTCs identified in the population of cells harvested from their peripheral blood by the Parsortix® PC1 System. Methods: Peripheral blood from 75 HVs and 77 patients with MBC was prospectively collected into K2EDTA tubes at the University of Rochester Medical Center. The blood collected from each subject (8.6±1.2mL) was processed on a Parsortix® PC1 System within 8 hours of collection. The cells harvested by the system were cytospun onto a charged slide and IF stained using an optimised antibody panel. The IF panel consisted of a nuclear dye (DAPI), positive selection markers targeting epithelial CTCs (Cytokeratins (CK) and EpCAM), and negative selection markers targeting white blood cells, such as lymphocytes, macrophages, granulocytes, monocytes, fibroblasts, and cells of megakaryoblastic potential. The stained slides were imaged using fluorescence microscopy and CTCs were defined as nucleated cells (DAPI+) that were positive for CK and/or EpCAM and negative for the blood lineage markers. The IF slides were subsequently stained with Wright-Giemsa and analysed by a qualified pathologist using light microscopy. Morphological features of malignant cells were used to define and identify CTCs. All laboratory testing and analysis was performed by operators blinded to the clinical status of each subject. Results: On the evaluable IF-stained slides, cells classified as CTCs based on their IF staining pattern were identified in 45.3% (34/75) of the MBC patients (range = 0 – 125 CTCs, mean = 7) and in 5.6% (4/71) of the HVs (range = 0 – 8 CTCs, mean = 0). No EpCAM+, CK- CTCs were identified in either MBC patients or HVs. In the 34 MBC patients with one or more CTCs observed, 70.6% had only CK+, EpCAM- cells, while the remaining 29.4% had at least one CK+, EpCAM+ cell. In the HVs, one out of the four CTC-positive donors had only CK+, EpCAM+ cells while the other three had only CK+, EPCAM- cells. On the evaluable Wright-Giemsa stained slides, cells classified as CTCs by the pathologist were identified in 57.1% (40/70) of the MBC patients (range = 0 – 41 CTCs, mean = 4) and in 4.4% (3/68) of the HVs (range = 0 – 14, mean = 0). Conclusions: This study demonstrated the ability of ANGLE’s Parsortix® PC1 System to capture and harvest CTCs from a significantly larger proportion of MBC patients compared to HV subjects. The presence of epithelial cells in subjects without diagnosed disease has been previously described in the literature, with their significance being unclear. This study also demonstrated that the cells harvested by the Parsortix® PC1 System can be successfully evaluated using both IF staining and Wright-Giemsa cytomorphological assessment for the identification of CTCs. Interestingly, a high proportion of the identified CTCs did not express EpCAM, further highlighting the limitations of using EpCAM-based approaches to capture CTCs. Citation Format: Mariacristina Ciccioli, Richard Moore, Kyu Kwang Kim, Negar Khazan, Michael C. Miller, Anne-Sophie Pailhes-Jimenez. Identification of Circulating Tumor Cells captured by the FDA Cleared Parsortix® PC1 System from the Peripheral Blood of Metastatic Breast Cancer Patients using Immunofluorescence and Cytopathological Evaluations [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-05-35.