Number Of Circulating Tumor Cells Research Articles

Abstract B010: Prognostic value of circulating tumor cells in stage III and IV melanoma: A systematic review and meta-analysis

Abstract Introduction: Previous studies have demonstrated a role for assessing circulating tumor cells (CTC) in melanoma, but significant heterogeneity among studies has limited application in the clinical setting. We sought to evaluate the prognostic value of CTCs in advanced stage melanoma using a standardized CTC enumeration platform. Methods: A PubMed literature search was conducted up to March 2024. All studies evaluating CTC enumeration using CellSearchTM with outcomes data were included. Student's t-test was used to compare differences between groups. Hazard ratios (HR) were pooled using random effects model to assess the association of CTCs with overall survival (OS). I 2 statistic and Cochran’s Q test, sensitivity analyses, and Egger’s test of asymmetry were used to assess study heterogeneity, differential impact of individual studies, and publication bias, respectively. Results: Of the 12 studies identified, nine met inclusion criteria. A total of 678 patients were included, of which 337 (49.6%) were stage III and 342 (50.4%) were stage IV. The majority of studies reported CTC rates using a cut-off of ≥ 1 (6 studies, 66.7%), while three studies used ≥ 2 and five studies used both. Median follow-up time was available for six studies and ranged from 5.9-48 months (median 13.4). Average CTC detection rate (≥ 1 or 2) was 41% (SD 14.0) and was significantly higher using a cut-off of ≥ 1 (45.4%, SD 12.6) compared to ≥ 2 (26.1%, SD 2.7, p = 0.02). Pooled analysis of two studies demonstrated significantly worse OS with CTC ≥ 1 (HR 1.6, 95% CI: 1.0-2.6, p = 0.04). Similarly, pooled analysis of five studies with available HRs demonstrated significantly worse OS with CTC ≥ 2 (HR 2.1, 95% CI: 1.4-3.1, p = 0.0003). Among these five studies, moderate heterogeneity was observed (I2 = 43.4%), although this was not statistically significant (Q statistic p = 0.13). Sensitivity analyses confirmed the prognostic value of CTC ≥ 2, with pooled HRs ranging from 1.9-2.4 after consecutive omission of each study from the overall analysis. Conclusion: Regardless of the CTC cutoff used (≥ 1 or 2), presence of any CTCs was a strong prognostic marker in patients with advanced stage melanoma. Incorporation of CTC assessments in clinical practice may help risk stratify patients and guide treatment decisions. Further studies are needed to determine the optimal cut-off point for CTC status in assessing prognosis. Citation Format: Jennifer H. Chen, Hyunsoo Hwang, Bamboo Dong, Vanessa N. Sarli, Joshua R. Upshaw, Salyna Meas, Anthony Lucci. Prognostic value of circulating tumor cells in stage III and IV melanoma: A systematic review and meta-analysis [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B010.

Abstract A017: High-throughput microfluidic enrichment of circulating tumor cells from entire diagnostic leukapheresis for comprehensive liquid biopsy

Abstract Circulating Tumor Cells (CTCs) in blood containing a complete set of single-cell analytes, including high-molecular-weight DNA, intact RNA, and both intracellular and cell-surface proteins serve as a promising tool in cancer diagnosis and prognosis through liquid biopsy. However, their clinical utility has been limited by their low blood concentration and inadequate enrichment technology. Diagnostic leukapheresis holds promise for isolating sufficient CTCs, but enriching ultra-rare cells from large, complex fluid volumes remains challenging. To address this, we developed the LPCTC-iChip, a high-throughput microfluidic device that uses high-flow channels and force-amplifying magnetic lenses to deplete hematopoietic cells, allowing tumor epitope-agnostic screening of massive blood volumes. Here, we present the first application of LPCTC-iChip to entire diagnostic leukapheresis products (leukopak) from seven patients with metastatic cancer, processing an average of 5.83 liters of blood per patient. Enumeration and morphology characterization of CTCs using multispectral immunofluorescence imaging identified high CTC yields - a mean of 10,057 CTCs per leukopak and showed notable intra- patient heterogeneity. Nuclear and cell diameter of CTC varies within individual patients, with 67% overlapping in size with patient-matched white blood cells. Paired single-cell DNA and RNA sequencing uncovered subclonal aneuploidy and distinct signaling pathways within CTCs with shared, clonal copy number variation (CNV) patterns. A subset of small aneuploid cells lacking epithelial markers was enriched for neuroendocrine characteristics in prostate cancer samples. The mutations found by FDA-approved genetic tests in either biopsied metastatic lesions or blood-based ctDNA sampling were reproduced by whole exome sequencing of a pool of CNV-confirmed CTCs. In addition, the sequencing revealed additional cancer-related high allele frequency variants that are not detectable with ctDNA or tissue needle biopsies. Overall, this high-throughput CTC enrichment from complete leukapheresis combined with multispectral imaging and single cell sequencing technology enables a comprehensive, cell-based liquid biopsy approach for early cancer detection and therapeutic responses monitoring. Citation Format: Avanish Mishra, Shih-Bo Huang, Taronish Dubash, Risa Burr, Jon F. Edd, Ben S. Wittner, Quinn E. Cunneely, Victor R. Putaturo, Akansha Deshpande, Ezgi Antmen, Kaustav A. Gopinathan, Keisuke Otani, Yoshiyuki Miyazawa, Ji Eun Kwak, Sara Y. Guay, Justin P. Kelly, John Walsh, Linda T. Nieman, Isabella Galler, PuiYee Chan, Michael S. Lawrence, Ryan J. Sullivan, Aditya Bardia, Douglas S. Micalizzi, Lecia V. Sequist, Richard J. Lee, Joseph W. Franses, David T. Ting, Patricia A. R. Brunker, Shyamala Maheswaran, David T. Miyamoto, Daniel A. Haber, Mehmet Toner. High-throughput microfluidic enrichment of circulating tumor cells from entire diagnostic leukapheresis for comprehensive liquid biopsy [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A017.

Abstract PR005: Leveraging mass cytometry for phenotyping CTCs in SCLC liquid biopsies: Tracking therapy resistance at a personalized level

Abstract Liquid biopsies present a promising, minimally invasive procedure through which clinicians can assess and monitor disease status by interrogating circulating tumor cell (CTC) and immune cell phenotypes. In the case of Small-cell lung cancer (SCLC) – a type of lung cancer characterized by aggressive growth, dismal prognosis and limited treatment options - leveraging liquid biopsies and CTCs for evaluating disease status would be of great benefit, given that biopsies are scarce. Furthermore, SCLC CTC numbers are higher compared to other cancers, presenting a unique opportunity to utilize them for assessing SCLC therapy resistance and identifying new targetable markers. In this study, we leverage mass cytometry (i.e. CyTOF) to phenotype SCLC CTC phenotypes in longitudinal clinical specimens to assess therapy resistance at a personalized level. By using CyTOF - a multiplex cytometric approach that enables interrogation of 30-50 protein markers per single cell – we show that we are able to identify and analyze a significant number of CTCs and their heterogeneous expression profiles. To achieve this, we first optimized a CyTOF panel of ∼25-30 antibodies by using SCLC cell lines and blood specimens from healthy donors and SCLC patients. We show that by using this approach, we are able to identify and phenotype SCLC CTCs by using the combination of CD45 (negative expression) and CD56 positive expression with further validation achieved by TTF1 and SCLC subtype transcription factor expression (NEUROD1, POU2F3 and ASCL1). This approach avoids enriching for specific CTC epithelial phenotypes that express surface markers that fluctuate during epithelial- mesenchymal transition (EMT) (e.g. EpCAM, a common marker for isolating CTCs). For parallel immune profiling we assessed among others CD8/CD4 (T cells) and CD11b/HLA-DR (Myeloid-derived suppressor cells, MDSCs) expression. We then proceeded to interrogate CTC phenotypic differences in the naïve vs relapsed setting as well as dynamic changes in longitudinal blood specimens from SCLC patients by evaluating markers that describe immune suppression activity (PD-L1) and EMT status (E-Cadherin, Vimentin, MUC1, Twist and Slug). Importantly, we show that with our approach we can track in real time therapeutic targets that are currently being tested in clinical trials or have been recently FDA approved (e.g. pYAP and DLL3) and found that MUC1 and pYAP are significantly increased in CTCs detected in blood specimens of SCLC relapsed patients. Our immune profiling showed an increase of MDSC percentages and a decrease in the CD4/CD8 T cell ratio in the relapsed setting. Finally, when we used PHENOSTAMP, a previously developed reference EMT map for assessing EMT phenotypes in lung cancer clinical specimens, we observed an increase of EMT heterogeneity in CTCs in relapsed patients. Our study highlights the translational power of our approach in utilizing CyTOF for longitudinally tracking CTCs directly in SCLC patient liquid biopsies towards assessing therapy response and resistance at a personalized level. Citation Format: Loukia G Karacosta, Sayantan Bhattacharyya, Allison Stewart, Ashley Victorian, Ester F Lujan, Shafqat Ehsan, Subin Kim, Alberto Duarte, Runsheng Wang, Benedict Anchang, Jing Wang, Lauren Byers. Leveraging mass cytometry for phenotyping CTCs in SCLC liquid biopsies: Tracking therapy resistance at a personalized level [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR005.

Abstract A001: Analytical validation of a novel multiomic metastatic breast cancer liquid biopsy test that combines ctDNA analysis with CTC HER2-ultralow and ER co-expression, as well as single-cell chromosomal instability

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

A novel electrochemiluminescent cytosensor using dual-target magnetic probe recognition and nanozymes-catalyzed cascade signal amplification for precise phenotypic enumeration of CTCs.

The inability of surgical biopsy to monitor the dynamic evolution of cancer cells hampers its capacity to reflect real-time tumor heterogeneity. Circulating tumor cells (CTCs), as a crucial target in liquid biopsy, offer a novel approach for accurate monitoring of tumors. However, the rarity and complex phenotype resulting from epithelial-mesenchymal transition pose challenges for conventional methods such as CellSearch and immunohistochemistry, which have insufficient ability for simultaneous phenotyping and enumeration of CTCs. The enumeration of a single phenotype CTCs is insufficient for accurately assessing disease progression. Herein, we propose a strategy to address this issue by fabricating an electrochemiluminescence cytosensor via the integration of dual-target enrichment and nanozymes-catalyzed cascade signal amplification. The graphene oxide@hollow mesoporous Prussian blue/Pt (GO@HMPB/Pt) complex, possessing a large specific surface area and exceptional catalytic activity, is employed for loading a substantial amount of luminol as the signal probe. Dual-target magnetic PPy@Fe3O4/Au-antibody/aptamer is utilized for the magnetic capture of both epithelial and interstitial CTCs. Glutathione (GSH) can disrupt theAu-S bond on aptamer by a thiol exchange reaction and selectively releases a specific subset of phenotypic CTCs, thereby facilitating the efficient capture, accurate classification, and ultrasensitive detection of CTCs in peripheral blood. Using the epithelial MCF-7 and mesenchymal Hela cells as models, the ECL cytosensor demonstrates excellent performance in identifying cells spiked into whole blood. This study presents a novel approach for early detection of metastasis, tracking tumor recurrence, and monitoring therapeutic efficacy.

Quercetin inhibits endothelial & hepatocellular carcinoma cell crosstalk via reducing extracellular vesicle-mediated VEGFR2 mRNA transfer.

This study aims to investigate the regulatory effects of quercetin extracellular vesicles (EVs)-mediated expression of vascular endothelial growth factor receptor 2 (VEGFR2) in hepatocellular carcinoma (HCC)-derived circulating tumor cells (CTCs) and the underlying mechanisms. CTCs were isolated from patients with pathologically diagnosed HCC, with VEGFR2 expression visualized by fluorescence in situ hybridization (FISH). The human HCC cell line Huh-7 and SK-HEP-1 were used for in vitro studies to assess EVs uptake, VEGFR2 mRNA transfer, invasion, migration, cancer stem cell (CSC) properties, and VEGF secretion. Results showed that VEGFR2 mRNA was commonly expressed in HCC-CTCs, with a higher incidence in biphenotypic CTCs. Its expression was limited in HCC cell lines, but present in certain liver cells. In vitro experiments confirmed that VEGFR2 mRNA could be transferred to HCC cells via EVs from primary tumor endothelial cells (PTECs), which was impaired by quercetin treatment. Quercetin significantly reduced VEGFR2 mRNA and protein expression in HCC cells, weakened their invasive and metastatic capacities, and diminished VEGFR2-mediated CSC properties. In vivo, quercetin reduced VEGF secretion, impaired angiogenesis, slowed tumor growth, and decreased the number and proportion of VEGFR2-positive CTCs. In summary, VEGFR2 mRNA is present in HCC-CTCs, potentially sourced from PTECs-derived EVs. Quercetin effectively inhibits VEGFR2 expression, impacting HCC cell invasion, metastasis, and CSC characteristics. Besides, it reduces VEGFR2-positive CTCs in vivo. These effects support its therapeutic potential in HCC treatment by targeting the angiogenesis and tumor dissemination pathway.

Platelets and circulating (and tumor) cells: partners in promoting metastatic cancer.

Despite being discovered decades ago, metastasis remains a formidable challenge in cancer treatment. During the intermediate phase of metastasis, tumor cells detach from primary tumor or metastatic sites and travel through the bloodstream and lymphatic system to distant tissues. These tumor cells in the circulation are known as circulating tumor cells (CTCs), and a higher number of CTCs has been linked to poor prognoses in various cancers. The blood is an inhospitable environment for any foreign cells, including CTCs, as they face numerous challenges, such as the shear stress within blood vessels and their interactions with blood and immune cells. However, the exact mechanisms by which CTCs survive the hostile conditions of the bloodstream remain enigmatic. Platelets have been studied for their interactions with tumor cells, promoting their survival, growth, and metastasis. This review explores the latest clinical methods for enumerating CTCs, recent findings on platelet-CTC crosstalk, and current research on antiplatelet therapy as a potential strategy to inhibit metastasis, offering new therapeutic insights. Laboratory and clinical data have provided insights into the role of platelets in promoting CTC survival, while clinical advancements in CTC enumeration offer improved prognostic tools. CTCs play a critical role in metastasis, and their interactions with platelets aid their survival in the hostile environment of the bloodstream. Understanding this crosstalk offers insights into potential therapeutic strategies, including antiplatelet therapy, to inhibit metastasis and improve cancer treatment outcomes.

Detection of circulating tumor cells in non-metastatic prostate cancer through integration of a microfluidic CTC enrichment system and multiparametric flow cytometry.

Prostate cancer (PCa) is the second most common cancer among men and the fifth leading cause of cancer death. Circulating tumor cell (CTC) enumeration and characterisation in PCa has been shown to provide valuable information on prognosis of disease, therapy management and detection of resistance. Here, Cellsway's microfluidic platform for high-throughput enrichment of intact CTC populations was used to isolate CTCs from the blood of 20 localised PCa patients and 10 healthy donor samples to evaluate the clinical performance of the technology. To enumerate and characterise CTCs, a multi-parameter flow cytometry analysis was performed on the enriched CTC suspension using CTC-specific biomarkers. CTCs were detected in 17 of 20 patient samples, which corresponds to 85% CTC positivity. The median CTC count per 7.5 ml blood was 2 (1-9). In 80% of patients (n = 16), the number of CTCs ranged from 1 to 5, and in 5% of patients (n = 1) the number of CTCs was above 5. No CTCs were observed in the blood samples of 10 healthy volunteers, demonstrating the high specificity and low risk of false positives of the technology.

Cellular liquid biopsy provides unique chances for disease monitoring, preclinical model generation and therapy adjustment in rare salivary gland cancer patients.

While cell-free liquid biopsy (cfLB) approaches provide simple and inexpensive disease monitoring, cell-based liquid biopsy (cLB) may enable additional molecular genetic assessment of systemic disease heterogeneity and preclinical model development. We investigated 71 blood samples of 62 patients with various advanced cancer types and subjected enriched circulating tumor cells (CTCs) to organoid culture conditions. CTC-derived tumoroid models were characterized by DNA/RNA sequencing and immunohistochemistry, as well as functional drug testing. Results were linked to molecular features of primary tumors, metastases, and CTCs; CTC enumeration was linked to disease progression. Of 52 samples with positive CTC counts (≥1) from eight different cancer types, only CTCs from two salivary gland cancer (SGC) patients formed tumoroid cultures (P = 0.0005). Longitudinal CTC enumeration of one SGC patient closely reflected disease progression during treatment and revealed metastatic relapse earlier than clinical imaging. Multiomics analysis and functional invitro drug testing identified potential resistance mechanisms and drug vulnerabilities. We conclude that cLB might add a functional dimension (to the genetic approaches) in the personalized management of rare, difficult-to-treat cancers such as SGC.

Improving the Prognostic and Predictive Value of Circulating Tumor Cell Enumeration: Is Longitudinal Monitoring the Answer?

Circulating tumor cell (CTC) numbers in the blood of cancer patients can indicate the progression and invasiveness of tumors, and their prognostic and predictive value has been repeatedly demonstrated. However, the standard baseline CTC count at the beginning of treatment, while informative, is not completely reliable and may not adequately reflect the state of the disease. A growing number of studies indicate that the long-term monitoring of CTC numbers in the same patient provides more comprehensive prognostic data and should be incorporated into clinical practice, as a factor that contributes to therapeutic decisions. This review describes the current status of CTC enumeration as a prognostic and predictive factor, highlights the shortcomings of current solutions, and advocates for longitudinal CTC analysis as a more effective method of the evaluation of developing disease, treatment efficacy, and the long term-monitoring of the minimal residual disease. As evidenced by the described reports, the longitudinal monitoring of CTCs should provide a better and more sensitive prediction of the course of the disease, and its incorporation in clinical practice should be beneficial.

Detection of Circulating Tumor Cells and EGFR Mutation in Pulmonary Vein and Arterial Blood of Lung Cancer Patients Using a Newly Developed Immunocytology-Based Platform.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are powerful molecular targeted therapeutic agents for lung cancer. We recently developed an original immunocytology and glass slide-based circulating tumor cell (CTC) detection platform for both CTC enumeration and EGFR mutation analysis with DNA extracted from CTCs. Using this platform, we conducted a pilot clinical study for CTC enumeration in peripheral blood (PB), pulmonary arterial blood (PA), and pulmonary venous blood (PV) from 33 patients with lung cancer (Stage I-III) who underwent surgery, followed by digital PCR-based EGFR mutation analysis of CTCs in PV from 12 patients. The results showed that CTC levels were significantly higher in PV and PA than in PB (p < 0.05, p < 0.01. respectively), with a notably greater number of small and large CTC clusters (p < 0.01). Genetic analysis of EGFR mutations of CTCs from PV (n = 12) revealed six mutations, including three Exon19del and three L856R, in CTCs and eight EGFR mutations, including five Exon19del and three L856R, in lung tumor tissue. CTC mutation status matched that of tissue samples in nine patients, was unmatched in two patients, and controversial in one patient, indicating a sensitivity of 0.75 (6/8) and specificity of 1.0 (4/4) with some false-negative results for the mutation analysis of CTCs. This immunocytology-based CTC detection platform is a convenient method for detecting both CTC number and EGFR mutation status under microscopy, suggesting its potential as a liquid biopsy tool in the hospital for patients with lung cancer in some clinical settings.

Combination of circulating tumor cells and 18F-FDG PET/CT for precision diagnosis in patients with non-small cell lung cancer.

To investigate the value of 2-deoxy-18f-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) and circulating tumor cells (CTCs) for the differential diagnosis of patients with benign lung diseases and those with NSCLC. To explore the phenotypic heterogeneity of CTCs and their correlation with FDG uptake in patients with Stage I-IV NSCLC. Blood specimens from patients with benign lung diseases and patients with primary NSCLC were collected for the detection of CTCs and their subtypes (epithelial, mixed, and mesenchymal) and analyzed for 18F-FDG PET/CT tumor metabolic parameters, including the maximum standardized uptake value (SUVmax), standard uptake value (SUL), metabolic tumor volume of primary lesion (MTV), total lesion glycolysis of primary lesion (TLG). Clinical data including age, gender, smoking history, tumor size, TNM stage and pathology type were also collected. The value of the two method alone and in combination for the differential diagnosis of benign and malignant was comparatively analyzed. Finally, the differences in CTC and its subtypes in different stages of NSCLC were compared, and FDG metabolic parameters were correlated with CTC subtypes. There were a total of 65 patients with pulmonary diseases, including 12 patients with benign pulmonary diseases and 53 patients with NSCLC. The mean age was 67 ± 10 (38-89 years), 27 were females and 38 were males. 31 (22 males and 9 females) had a long history of smoking. The mean size of the largest diameter of all single lesions was 36 ± 22 mm with a range of 10-108 mm. Seven out of 12 benign diseases were inflammatory granulomatous lesions and 5 were inflammatory pseudotumours. Twenty-four out of 53 NSCLC were adenocarcinomas and 29 were squamous carcinomas. Twelve out of 53 patients with NSCLC were in Stage I, 10 were in Stage II, 17 were in Stage III and 14 were in Stage IV. SUVmax, SUL, MTV, TLG, total CTCs, epithelial CTCs, and mixed CTCs were all valuable in the differential diagnosis of benign and malignant. TLG combined with mixed CTCs was statistically different from all other diagnostic methods (p < 0.05) and higher than any other diagnostic criteria. In the differential diagnosis of benign and Stage I NSCLC, only total CTC (Z = -2.188 p = 0.039) and mixed CTCs (Z = -3.020 p = 0.014) had certain diagnostic efficacy, and there was no statistical difference between them (p = 0.480). Only mesenchymal CTCs differed in Stage I-IV NSCLC, with a higher number of those who developed distant metastases than those who had non-distant metastases. Epithelial CTCs correlated with SUVmax (r = 0.333, p = 0.015) and SUL (r = 0.374, p = 0.006). Mmesenchymal CTCs correlated with MTV (r = 0.342, p = 0.018) and TLG (r = 0.319, p = 0.02). Further subgroup analyses revealed epithelial CTCs were correlated with SUVmax (r = 0.543, p = 0.009) and SUL (r = 0.552, p = 0.008), and the total CTCs was correlated with SUVmax (r = 0.622, p = 0.003), SUL (r = 0.652, p = 0.003), MTV (r = 0.460, p = 0.031), and TLG (r = 0.472, p = 0.027) in the early group (Stage I-II). Only mesenchymal CTCs was associated with MTV (r = 0.369, p = 0.041), and TLG (r = 0.415, p = 0.02) in the intermediate-late group (Stage III-IV). Both FDG PET metabolic parameters and CTCs demonstrated diagnostic value for NSCLC, and combining TLG with mixed CTCs could enhance their diagnostic efficacy. The total CTCs and mixed CTCs showed greater diagnostic value than FDG PET in distinguishing benign lesions from Stage I NSCLC. In NSCLC patients, the epithelial CTCs exhibited a positive correlation with SUVmax and SUL, while mesenchymal CTCs correlated with MTV, and TLG. Besides, epithelial CTCs showed stronger correlations with SUVmax and SUL, and total CTCs showed stronger correlations with SUVmax, SUL, MTV, and TLG in Stage I-II NSCLC. Only mesenchymal CTCs in Stage III-IV NSCLC showed correlations with MTV and TLG. Stage IV NSCLC cases displayed a higher number of mesenchymal CTCs.

The prognostic significance of circulating tumor cell enumeration and HER2 expression by a novel automated microfluidic system in metastatic breast cancer

BackgroundThe prognostic value of circulating tumor cells (CTCs) in metastatic breast cancer (MBC) has been extensively studied and verified by the CellSearch® system. Varieties of microfluidic systems have been developed to improve capture efficiency with the lack of standardization and automation. This study systematically verified the positive threshold for prognosis and its guidance value in anti-HER2 therapy based on a novel automated microfluidic system OmiCell®.MethodsCTCs isolation, enumeration and labeling were performed using the OmiCell® system. CTCs identification and reporting were performed using the DeepSight® scanning system.ResultsThe capture efficiency and specificity of OmiCell® system was 91.9% and 90%, respectively. Then, 65 MBC patients with known HER2 status of their metastatic tumors were enrolled. In the cohort, we detected ≥ 1 CTCs in 59 patients (90.8%, range: 1–55 CTCs, median = 6), < 8 CTCs in 45 (69.2%) and ≥ 8 CTCs in 20 (30.8%) patients at baseline. The patients with < 8 CTCs had longer PFS than ≥ 8 CTCs (median, 7 vs. 4.4 months, p = 0.028). CTC enumeration was found to be an independent prognostic factor in our cohort. Moreover, we found a weak concordance between tissue HER2 (tHER2) status and the corresponding CTCs (k = 0.16, p = 0.266). The patients with tHER2 positive and cHER2 negative had better PFS compared with patients with both tHER2 and cHER2 positive (median, 8.2 vs. 3.3 months, p = 0.022).ConclusionsThis clinical study shows the prognosis value of a new threshold of CTC number and meanwhile the guidance value of cHER2 status in anti-HER2 therapy.

Exploring the diagnostic and prognostic significance of circulating tumor cells in stage II-IV colorectal cancer using a nano-based detection method.

Colorectal cancer (CRC) is a leading cause of cancer mortality globally, underscoring the urgency for a noninvasive and effective biomarker to enhance patient prognosis. Circulating tumor cells (CTCs), a potential marker for real-time tumor monitoring, are limited in clinical utility due to the low sensitivity of existing detection methods. Previously, we introduced a novel Nano-based CTCs detection method that relies on the electrical properties of cell surfaces, thus eliminating thereby obviating the need for specific molecular biomarkers. In this study, we employed this technique to evaluate the diagnostic and prognostic value of CTCs in stage II-IV CRC. A total of 194 participants were included, consisting of 136 CRC patients and 58 healthy individuals. The peripheral blood of the participants was collected, and CTCs enumeration was performed utilizing the Nano-based detection method that we newly developed. The Receiver Operating Characteristic (ROC) curve and multivariate Cox proportional-hazards analysis were employed to assess the effectiveness of CTCs for diagnosing CRC and predicting patient prognosis. The Nano-based method demonstrated an ability to differentiate CRC patients from healthy individuals with a sensitivity of 84.6% and a specificity of 94.8%. Furthermore, baseline CTCs levels were predictive of progression-free survival (PFS) in CRC patients, with lower levels associated with longer PFS compared to higher levels (4.5 months vs. 8.0 months at 15 CTCs/mL, p = 0.016; 4.4 months vs. 8.0 months at 20 CTCs/mL, p = 0.028). We also explored the dynamic changes in the number of CTCs after 1-5 cycles of chemotherapy. Patients with increasing CTCs levels typically experienced disease progression (PD), while those with decreasing levels often achieved a partial response (PR) or maintained stable disease (SD). These findings suggest that the dynamic fluctuations in CTCs counts are closely tied to the clinical course of the disease. Our study indicates the potential of Nano-based CTCs detection in diagnosing and predicting outcomes for patients with stage II-IV CRC.

In vivo fluorescence flow cytometry reveals that the nanoparticle tumor vaccine OVA@HA-PEI effectively clears circulating tumor cells

Tumor vaccine therapy offers significant advantages over conventional treatments, including reduced toxic side effects. However, it currently functions primarily as an adjuvant treatment modality in clinical oncology due to limitations in tumor antigen selection and delivery methods. Tumor vaccines often fail to elicit a sufficiently robust immune response against progressive tumors, thereby limiting their clinical efficacy. In this study, we developed a nanoparticle-based tumor vaccine, OVA@HA-PEI, utilizing ovalbumin (OVA) as the presenting antigen and hyaluronic acid (HA) and polyethyleneimine (PEI) as adjuvants and carriers. This formulation significantly enhanced the proliferation of immune cells and cytokines, such as CD3, CD8, interferon-[Formula: see text], and tumor necrosis factor-[Formula: see text], in vivo, effectively activating an immune response against B16–F10 tumors. In vivo fluorescence flow cytometry (IVFC) has already become an effective method for monitoring circulating tumor cells (CTCs) due to its direct, noninvasive, and long-term detection capabilities. Our study utilized a laboratory-constructed IVFC system to monitor the immune processes induced by the OVA@HA-PEI tumor vaccine and an anti-programmed death-1 (PD-1) antibody. The results demonstrated that the combined treatment of OVA@HA-PEI and anti-PD-1 antibody significantly improved the survival time of mice compared to anti-PD-1 antibody treatment alone. Additionally, this combination therapy substantially reduced the number of CTCs in vivo, increased the clearance rate of CTCs by the immune system, and slowed tumor progression. These findings greatly enhance the clinical application prospects of IVFC and tumor vaccines.

Monitoring response to neoadjuvant chemotherapy in triple negative breast cancer using circulating tumor DNA

BackgroundTriple negative breast cancer (TNBC) is an aggressive subtype with poor prognosis. We aimed to determine whether circulating tumor DNA (ctDNA) and circulating tumor cell (CTC) could predict response and long-term outcomes to neoadjuvant chemotherapy (NAC).MethodsPatients with TNBC were enrolled between 2017–2021 at The University of Texas MD Anderson Cancer Center (Houston, TX). Serial plasma samples were collected at four timepoints: pre-NAC (baseline), 12-weeks after NAC (mid-NAC), after NAC/prior to surgery (post-NAC), and one-year after surgery. ctDNA was quantified using a tumor-informed ctDNA assay (SignateraTM, Natera, Inc.) and CTC enumeration using CellSearch. Wilcoxon and Fisher’s exact tests were used for comparisons between groups and Kaplan–Meier analysis used for survival outcomes.ResultsIn total, 37 patients were enrolled. The mean age was 50 and majority of patients had invasive ductal carcinoma (34, 91.9%) with clinical T2, (25, 67.6%) node-negative disease (21, 56.8%). Baseline ctDNA was detected in 90% (27/30) of patients, of whom 70.4% (19/27) achieved ctDNA clearance by mid-NAC. ctDNA clearance at mid-NAC was significantly associated with pathologic complete response (p = 0.02), whereas CTC clearance was not (p = 0.52). There were no differences in overall survival (OS) and recurrence-free survival (RFS) with positive baseline ctDNA and CTC. However, positive ctDNA at mid-NAC was significantly associated with worse OS and RFS (p = 0.0002 and p = 0.0034, respectively).ConclusionsEarly clearance of ctDNA served as a predictive and prognostic marker in TNBC. Personalized ctDNA monitoring during NAC may help predict response and guide treatment.

CXCL12-loaded-hydrogel (CLG): A new device for metastatic circulating tumor cells (CTCs) capturing and characterization

BackgroundCirculating Tumor Cells (CTCs) represent a small, heterogeneous population that comprise the minority of cells able to develop metastasis. To trap and characterize CTCs with metastatic attitude, a CXCL12-loaded hyaluronic-gel (CLG) was developed. CXCR4+cells with invasive capability would infiltrate CLG. MethodsHuman colon, renal, lung and ovarian cancer cells (HT29, A498, H460 and OVCAR8 respectively) were seeded on 150 μl Empty Gels (EG) or 300 ng/ml CXCL12 loaded gel (CLG) and allowed to infiltrate for 16 h. Gels were then digested and fixed with 2 % FA-HAse for human cancer cell enumeration or digested with HAse and cancer cells recovered. CLG-recovered cells migrated toward CXCL12 and were tested for colonies/spheres formation. Moreover, CXCR4, E-Cadherin and Vimentin expression was assessed through flow cytometry and RT-PCR. The clinical trial “TRAP4MET” recruited 48 metastatic/advanced cancer patients (8 OC, 8 LC, 8 GBM, 8 EC, 8 RCC and 8 EC). 10 cc whole blood were devoted to PBMCs extraction (7 cc) and ScreenCell™ filters (3 cc) CTCs evaluation. Ficoll-isolated patient's PBMCs were seeded over CLG and allowed to infiltrate for 16 h; gels were digested and fixed with 2 % FA-HAse, cells stained and DAPI+/CD45-/pan-CK + cells enumerated as CTCs. ResultsHuman cancer cells infiltrate CLG more efficiently than EG (CLG/EG ratio 1.25 for HT29/1.58 for A498/1.71 for H460 and 2.83 for OVCAR8). CLG-recovered HT29 cells display hybrid-mesenchymal features [low E-cadherin (40 %) and high vimentin (235 %) as compared to HT29], CXCR4 two-fold higher than HT29, efficiently migrate toward CXCL12 (two-fold higher than HT29) and developed higher number of colonies (171 ± 21 for HT29-CLG vs 131 ± 8 colonies for HT29)/larger spheres (spheroid area: 26561 ± 6142 μm2 for HT29-CLG vs 20297 ± 7238 for HT29). In TRAP4MET clinical trial, CLG-CTCs were isolated in 8/8 patients with OC, 6/8 with LC, 6/8 with CRC, 8/8 with EC, 8/8 with RCC cancer and 5/8 with GBM. Interestingly, in OC, LC and GBM, CLG isolated higher number of CTCs as compared to the conventional ScreenCell™ (CLG/SC ratio = 1.88 for OC, 2.47 for LC and 11.89 for GBM). Bland and Altman blot analysis and Passing and Bablok regression analysis showed concordance between the methodological approaches but indicate that SC and CLG are not superimposable suggesting that the two systems select cells with different features. ConclusionCLG might represent a new and easy tool to isolate invasive CTCs in multiple cancers such as OC, LC and GBM at today orphan of reliable methods to consistently detect CTCs.

Circulating tumor cells in metastatic breast cancer patients treated with immune checkpoint inhibitors - a biomarker analysis of the ALICE and ICON trials.

Immune checkpoint inhibitors (ICIs) have been introduced in breast cancer (BC) treatment and better biomarkers are needed to predict benefit. Circulating tumor cells (CTCs) are prognostic in BC, but knowledge is limited on CTCs in the context of ICI therapy. In this study, serial sampling of CTCs (CellSearch system) was evaluated in 82 patients with metastatic BC enrolled in two randomized trials investigating ICI plus chemotherapy. Programmed death-ligand 1 (PD-L1) expression on CTCs was also measured. Patients with ≥ 2 CTCs per 7.5 mL at baseline had gene expression profiles in tumor suggestive of increased T-cell activity, including increased tumor inflammation signature (TIS) in both triple-negative (P = 0.010) and hormone receptor-positive (P = 0.024) disease. Patients with luminal A BC had higher CTC levels. The association between CTC status and outcome was most apparent 4 weeks into therapy. PD-L1 expression in CTCs was observed in 6/17 CTC-positive patients and was associated with inferior survival. In conclusion, our study indicates that CTC numbers may inform on tumor immune composition, as well as prognosis. These findings suggest a potential of using CTCs as an accessible biomarker source in BC patients treated with immunotherapy.

An electrochemical biosensor based on mild reduction-activated CRISPR/Cas12a system for sensitive detection of circulating tumor cells

Circulating tumor cell (CTC) has been a valuable biomarker for the diagnosis of breast cancer, while folate receptor is a kind of cell surface receptor glycoprotein which is overexpressed in breast cancer. In this work, we have designed and fabricated an electrochemical biosensor for sensitive detection of folate receptor-positive CTCs based on mild reduction assisted CRISPR/Cas system. Specifically, folate functionalized magnetic beads are firstly prepared to capture CTCs owing to the strong affinity between folate and the folate receptors on the surface of cells. Then, the cell membranes are treated by mild reduction so as to expose a large number of free sulfhydryl groups, which can be coupled with maleimide-DNA to introduce the signal amplified CRISPR/Cas12a system. After the trans-cleavage activity of CRISPR/Cas12a is activated, the long chain DNA modified with electroactive molecules methylene blue can be randomly cleaved into short DNA fragments, which are then captured on the graphite electrode through the host-guest recognition with cucurbit [7]uril, generating highly amplified electrochemical signal corresponding to the number of CTCs. The electrochemical biosensor not only demonstrates the sensitivity with a low detection limit of 2 cells/mL, but also highlights its excellent selectivity and stability in complex environment. Therefore, our biosensor may provide an alternative tool for the analysis of CTCs.

Changes of circulating tumor cells expressing CD90 and EpCAM in early-phase of atezolizumab and bevacizumab for hepatocellular carcinoma

Circulating tumor cells (CTCs) are noninvasive biomarkers that can indicate the therapeutic response and prognosis. The study aimed to investigate the cellular characteristics of CTCs focusing on monitoring during atezolizumab and bevacizumab (Atezo-Bev) therapy in patients with hepatocellular carcinoma (HCC). Peripheral blood samples were collected from 10 healthy controls and 40 patients with HCC. CTCs enriched using RosetteSep™ Human CD45 depletion cocktail were analyzed by multiparametric flow cytometry. CTC isolation was based on PanCK(+)CD45(−) cells, and CTCs exhibiting markers CD90, CD133, EpCAM, or vimentin. The total number of CTCs and the number of CTCs expressing CD90, CD133, EpCAM, and vimentin were correlated with the BCLC stage of HCC. The change in total CTC count accurately reflected the initial response to Atezo-Bev therapy. The numbers and mean fluorescence intensity of the CTC subsets expressing CD90 and EpCAM molecules decreased in patients with partial response/stable disease, and increased in patients with progressive disease and were markedly correlated with overall survival. CD90(+) and EpCAM(+) CTCs may be candidate biomarkers for the early prediction of the treatment response and the overall survival of patients with HCC receiving Atezo-Bev therapy.