Average Circulating Tumor Cells Research Articles

Abstract 7043: Genomic profiling of DLL3-positive and negative CTCs in small cell lung cancer

Abstract BACKGROUND: Delta-like ligand 3 (DLL3), an atypical Notch family protein implicated in neuroendocrine tumorigenesis, has emerged as a promising therapeutic target in small cell lung cancer (SCLC), prompting numerous clinical trials investigating DLL3-targeted treatments. Exploring DLL3 in circulating tumor cells (CTCs) through liquid biopsy, coupled with comprehensive genomic analysis, presents an invaluable and minimally-invasive avenue for monitoring disease progression. This approach holds the potential to tailor personalized treatment strategies, emphasizing the critical need for in-depth investigations into both DLL3-positive and negative cell genomic compositions. METHODS: This study examined blood collected in CellSave preservative tubes from stage I (n=17) and stage IV (n=12) SCLC patients, as well as healthy donors (n=10). The CELLSEARCH® platform was employed to count EpCAM-enriched CTCs and evaluate DLL3 protein expression. Subsequently, individual CTCs were isolated based on the presence and absence of DLL3 expression using the DEPArray™ PLUS. The isolated DLL3-negative and positive CTCs were subjected to low-pass sequencing using the Ampli1™ LowPass Kit to uncover genomic aberrations. RESULTS: CTCs were absent in the healthy control group. In stage I patients, CTCs were identified in 5.8% of blood samples, while in stage IV patients, they were present in 50% of samples, with an average CTC count of 148 in stage IV. Among a total of 2074 detected CTCs, 460 were DLL3-positive, accounting for 6-28% of EpCAM-enriched CTCs in individual patients. Analysis of >300 copy number alteration (CNA) profiles of sorted single cells revealed considerable inter-patient and intra-patient heterogeneity, delineating recurrent clonality and specific genomic alterations in DLL3-negative CTCs. In contrast, DLL3-positive cells showed more stochastic CNA profiles, suggestive of apoptosis-related DNA fragmentation. CONCLUSIONS: Integrating CellSearch, DEPArray, and low-pass sequencing provides a minimally-invasive approach to compare genomic characteristics in DLL3-positive and negative CTCs from SCLC patients. Extension of this approach to a larger patient cohort and employing machine learning algorithms will facilitate identification of patterns within diverse CNA profiles, improving patient stratification and predicting treatment responses for personalized therapeutic strategies in SCLC. Citation Format: Zoltan Simandi, Thai Bui, Alberto Ferrarini, Steven Hepburn, Richard G. Del Mastro. Genomic profiling of DLL3-positive and negative CTCs in small cell lung cancer [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 7043.

Abstract 7485: Microfluidic detection of dual positive cells in whole blood of metastatic breast cancer patients

Abstract Background. Dual positive cells (DPCs) that express both epithelial markers and leukocytic markers have been observed in peripheral blood of breast cancer patients. DPCs acquire functional behaviors from both cell types which may drive tumor progression. However, the lack of reliable methods to isolate and identify DPCs has hindered the investigation of their origin and clinical implications in breast cancer patients. While microfluidic devices have been widely used for the detection of circulating tumor cells (CTCs) by liquid biopsy, DPC detection is underexplored in these devices. Herein, we demonstrate rapid onchip isolation and detection of DPCs in whole blood samples from metastatic breast cancer (MBC) patients using an integrated microfluidic device. Methods. Our microfluidic device consists of a unique preconditioning component that concentrates targets in whole blood and a downstream cell immobilization component that captures target cells. We included 7 female patients with MBC, of which 29% are white (2), 57% are African American (4), and 14% are Latina (1). The median age is 50y (34y -75y). All patients were undergoing systemic breast cancer therapy at the time of blood collection. A total of 9 samples (4 mL each) were collected from either an antecubital vein (peripheral vein) or the subcutaneous port catheter (central vein) of these patients. All samples were processed within 5 hours of blood drawn in our novel microfluidic biochip. CTCs (EpCAM+/CK+, CD45-, DAPI+) and DPCs (EpCAM+/CK+, CD45+/CD66b+, DAPI+) were identified by immunofluorescence after cells were fixed. Results. Processing of each sample was completed within 40 minutes using our microfluidic device. Both DPCs and CTCs were detected in all the samples (9/9). The average DPC count was 3 DPCs per 4 mL whole blood with a standard deviation of 1.9 cells. The average CTC count was 9 with a standard deviation of 5 per 4 mL whole blood. 89% of the samples (8/9) had > 5 CTCs after the sample volume is scaled to the standard 7.5 mL. No correlation was found between DPC count and CTC count (Pearson correlation coefficient r=0.08). While the blood collection location (central vs peripheral veins) had a significant impact on the CTC count (12.6 vs 4 on average with p<0.01), there was no statistical difference in DPC counts in the samples collected from central and peripheral veins. Conclusions. Both DPCs and CTCs can be rapidly isolated and detected from MBC patient whole blood in our microfluidic device. Our preliminary results indicate no correlation between DPCs and CTCs, suggesting that DPCs might not originate in blood circulation of MBC patients. More work with a larger patient cohort will be needed to further investigate the clinical implications of DPCs on MBC patients. Citation Format: Jian Zhou, Qiyue Luan, Celine Macaraniag, Arielle B. Guzman, Maria Mantice, Oana Danciu, Kent F. Hoskins, Ian Papautsky. Microfluidic detection of dual positive cells in whole blood of metastatic breast cancer patients [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 7485.

Clinical Validation of a Size-Based Microfluidic Device for Circulating Tumor Cell Isolation and Analysis in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) presents as metastatic disease in one third of cases. Research on circulating tumor cells (CTCs) and liquid biopsies is improving the understanding of RCC biology and metastases formation. However, a standardized, sensitive, specific, and cost-effective CTC detection technique is lacking. The use of platforms solely relying on epithelial markers is inappropriate in RCC due to the frequent epithelial-mesenchymal transition that CTCs undergo. This study aimed to test and clinically validate RUBYchip™, a microfluidic label-free CTC detection platform, in RCC patients. The average CTC capture efficiency of the device was 74.9% in spiking experiments using three different RCC cell lines. Clinical validation was performed in a cohort of 18 patients, eight non-metastatic (M0), five metastatic treatment-naïve (M1TN), and five metastatic progressing-under-treatment (M1TP). An average CTC detection rate of 77.8% was found and the average (range) total CTC count was 6.4 (0-27), 101.8 (0-255), and 3.2 (0-10), and the average mesenchymal CTC count (both single and clustered cells) was zero, 97.6 (0-255), and 0.2 (0-1) for M0, M1TN, and M1TP, respectively. CTC clusters were detected in 25% and 60% of M0 and M1TN patients, respectively. These results show that RUBYchip™ is an effective CTC detection platform in RCC.

Abstract P1-05-09: Targeting treatment to tumor response: demonstration of response monitoring with subsequent impact on treatment choices in real time utilizing a novel technology

Abstract Breast cancers, both early and advanced, are heterogeneous causing differential responses to targeted therapy. Explosion in targeted treatment choices requires real time assessment of tumor characteristics in patients initially and over time. A dynamic detection system, not a static pathology specimen, is required for continuous profiling while treatment changes; hence, the interest in circulating tumor cells (CTCs). Our novel QCDx br™ system analyzes all nucleated cells from a blood sample aliquot, morphologically intact and immobilized in hydrogel, after multiplex, immunofluorescent (IF) staining. We characterize increased numbers of CTCs (nucleated, CD45-negative cells) as single cells, in clusters and in connection with circulating inflammatory cells, stained with two, separate IF marker cocktails, denoting (1) epithelial (EpCAM, Cytokeratin CK), mesenchymal (Vimentin VIM) phenotypes and (2) therapeutic HER2, ER and TROP2 targets which are the basis for targeted therapy in breast cancer. In the ongoing, prospective CLINBREAC trial, we enrolled to date 9 neoadjuvant (early stage, ES) and 21 metastatic (late stage, LS) breast cancer patients, collecting 7.5 ml of blood at 3-month intervals or at change of therapy. We are now out over 2 years with the earliest ES and LS patients showing changes in CTCs with disease progression and in response to treatment, often pre-dating changes seen in follow up biopsies. Detection of HER2+ CTCs was of particular interest in patients with HER2 low cancers¬ (1+ or 2+). Results presented here, contain the more complete datasets from 8 ES and 11 LS patients. QCDx br™ detected CTCs in all 19 Stage I-IV patients that could exceed 100 CTCs/2500 nucleated cells in LS and 50 CTCs/2500 nucleated cells in ES patients. The table shows count averages of CTCs/2500 nucleated cells detected by different IF markers. Of note, CTCs showed combinations of IF markers (hybrid cells). For example, CTCs expressed both VIM and CK indicating epithelial to mesenchymal transition (EMT) phenotype, which may signify higher metastatic potential. ER+ CTC were seen in LS, not ES patients. All ES patients including those with HER2 low tumors, showed HER2+ CTCs. Of note, not included in the table are two, triple-positive patients with oligo-metastatic disease now 4 and 10 years out from diagnosis without evidence of disease and on maintenance HER2 and ER-directed therapy. No CTCs are seen at their first data point. We were unable to detect CTCs in 13 healthy volunteers. In ES patients, CTC numbers did not associate with tumor size, nodal involvement, ER or HER2 status. For example, one TNBC patient with a T3 tumor and positive nodes had some of the lowest numbers of CTCs. The more complete quantification of the LS patients is in process. As development of QCDx br™ continues, multiplex IF staining of 12+ markers per CTC and identification of single CTC mutational changes is expected. This powerful technology enables targeted treatment decisions with specific drugs resulting in maximal responses. Average CTC counts/2500 nucleated cells detected by phenotypic and therapeutic IF cocktails Citation Format: Susan Tannenbaum, Emily Hsu, jasmin Hundal, Amber Wilkes, Austin Fergusson, Fahmy Mamuya, Triantafyllos Tafas. Targeting treatment to tumor response: demonstration of response monitoring with subsequent impact on treatment choices in real time utilizing a novel technology [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 P1-05-09.

High counting of circulating tumor cells in blood is not directly related to metastasis.

Circulating tumor cells (CTCs) in blood flow have been believed as an essential biomarker of cancer. The technologies of in vitro and in vivo CTC enrichment and detection suggest although CTCs might play a role of "seed" in metastasis, only the minority of CTCs, probably in the form of CTC clusters, hold the potential to develop a tumor in organs. The detected amount of CTCs might be solely an indicator of tumor burden. To provide new insights into this argument, we take advantage of a safe drug to tune the pacemaker activity of a mouse tumor model to increase the heart rate for a period of time every day during the tumor development. We detect the CTCs in vivo by fast line scanning of a confocal microscope when the heart rate returns to the baseline and find the average CTC amount is significantly elevated in the drug-treated group but the metastases are even less than that of control. Our results imply the detected CTC counts in blood might not be directly related to metastasis.

Abstract LB117: Pilot study to identify live circulating tumor cells (CTCs) in metastatic breast cancer (MBC) by application of a novel microfluidic workflow system and flow cytometry

Abstract Introduction: CTCs play a critical role in breast cancer (BCa) metastases. Rapid identification of live CTCs may help to predict BCa prognosis and identify which patients may derive treatment benefit, especially for those with metastatic disease. However, CTCs are present at very low concentrations in the peripheral blood, and CTC enumeration is costly and technically challenging. We previously reported that cell-size based device utilizing a microfluidic cassette could be used to isolate live blood cells (Zhang et al. 2019 AACR). Here we describe a pilot study using novel microfluidic Parsortix workflow to identify live CTCs for patients with Stage IV BCa. Methods: Four whole blood samples (7.5ml/each) were collected from each of 10 patients with stage IV BCa patients who had continued clinical progression of their disease on systemic therapy, under a Northwestern University IRB-approved study (NU20B03). All samples were stored at 15-30 °C in EDTA tubes. Live CTCs enrichment was performed using a cell size and deformability based Parsortix system (Angle) which utilizes a microfluidic based disposable cassette containing a step separation structure. The four samples from each patient were loaded onto the device and the separation procedure was started by using 4.5μm, 6.5μm 8μm and 10μm separation cassettes, respectively. After separation and cleaning, the CTCs captured by the Parsortix system were harvested by inverting the flow direction and then flushing from the cassette in 200μL PBS. The harvested CTCs were multi-stained directly by fluorescence labeled antibodies for Anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes) (Menarini). Flow cytometry (FCM) was used to identify the CTCs which were classified as CK+, DAPI+ and CD45-. White blood cells from patient samples and the human breast cancer cell line MDA-MB-231 were used as negative and positive controls, respectively. Results: A total of 500 and 200 MDA-MB-231 cells were processed as controls, which were collected completely by Parsortix and identified by FCM confirming that Parsortix system could recover the CTCs effectively. CTCs were classified based on morphology and phenotype as CK+, DAPI+ and CD45-. Live CTCs (≥1) were detected in 8 out of 10 (80%), 9 out of 10 (90%), 7 out of 10 (70%) and 5 out of 10 (50%) samples by using 4.5μm (Group 1), 6.5μm (Group 2), 8μm (Group 3) and 10μm (Group 4) separation cassettes. Maximum CTCs isolated from each group were 18, 32, 12 and 8 from Group 1, 2, 3 and 4, respectively. The corresponding average CTCs isolated from each sample were 3.5 CTCs, 5.6 CTCs, 2.5 CTCs and 1.7 CTCs from Group 1, 2, 3, and 4, respectively. The 6.5μm cassettes (Group 2) using for CTCs collection had significantly higher efficacy on collection of CTCs compared to other three groups. Conclusions: In this pilot study, our findings demonstrated that the cell-sized dependent Parsortix system using multiple staining and FCM is an effective and specific approach for rapid recovery and identification of CTCs from Stage IV BCa patients. With further optimization, this strategy can help to accurately evaluate CTCs and offers a potential technique for diagnosis and treatmentmonitoring of patients with metastatic breast cancer. Citation Format: Qiang Zhang, Marwa Manai, Andrew Davis, Carolina Reduzzi, Paolo D’Amico, Saya Liz Jacob, Jianhua Jiao, Weijun Qin, Lisa Flaum, Amir Behdad, Massimo Cristofanilli, Ami Shad, Leonidas Platanias, William Gradishar. Pilot study to identify live circulating tumor cells (CTCs) in metastatic breast cancer (MBC) by application of a novel microfluidic workflow system and flow cytometry [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 LB117.

The decline of circulating tumor cell clusters count for predicting the treatment response in breast cancer patients.

e13009 Background: Circulating tumor cells (CTCs) are the precursors of metastatic cancer because they originate from primary tumor sites into the vascular system. The prognostic information of CTCs was well established in breast cancer treatment while the predictive value was still unclear. The aim of this study was to analyze the predictive value of CTCs count during treatment. Materials and Methods: A prospective study was conducted from 2019 to 2021, and 31 female breast cancer patients were enrolled, and 54 blood samples were collected at Linkou Chang Gung Memorial Hospital. The median age was 47 years old. We used LIPO platform for CTC isolation with anti-EpCAM antibody. To achieve maximum cellular viability, the LIPO platform was further optimized by using liposomes-lipid bilayers instead of lipid bilayers as the underneath surface coating to facilitate the detachment of bound cells from the surface and maintain cellular viability. Results: Fourteen patients were Stage II or stage III (non-metastasis) and 17 metastatic patients were included. Eleven patients (35.5%) were ER-positive HER2-negative while 10 patients (32.6%) were ER-positive HER2-positive. Three patients (9.7%) were HER2 enriched subtypes, and 7 patients (22.6%) were triple negative breast cancer. Blood samples from a total of 28 patients can be analyzed. At least one CTC was detected in 44/49 (89.8%) blood samples, CTC can’t be identified in 2/5 (40%) post-treatment with good response. The average single CTC count per 2 ml was 34.40 in non-metastatic patients versus 110.43 in metastatic patients (p = 0.3895). The average clustered CTC count per 2 ml was 13.44 in non-metastatic patients versus 40.16 in metastatic patients (p = 0.424). The total single and clustered CTC count per 2 ml was 47.84 non-metastatic patients versus 150.59 in metastatic patients (p = 0.3977). In 10 patients with clinically partial response, the average single CTC count per 2 ml was 61.9 before treatment and 11.81 after treatment (p = 0.0716), and the average clustered CTC per 2 ml was 15.83 before treatment and 5.28 after treatment (p = 0.8806). In 4 patients with clinical progression, the average single CTC count per 2 ml was 22.694 before treatment and 26.125 after treatment (p = 0.0257), and the average clustered CTC per 2 ml was 7.69 before treatment and 16.61 after treatment (p = 0.3798). Conclusions: Our findings concluded that the decline of circulating tumor cell clusters count can predict the treatment response in breast cancer patients.

Single cell phenotyping of PDL1 and HLA1 on circulating tumor cells (CTC) in clear cell renal cell carcinoma (ccRCC) on systemic treatment with immunotherapy (IO) and vascular endothelial growth factor receptor tyrosine kinase inhibitors (TKI).

381 Background: Single agent or combination regimens using different IO and TKI agents have dramatically improved survival of patients (pt) with advanced ccRCC. Nevertheless, selecting optimal combination and sequencing of treatments remains challenging given the current absence of predictive biomarkers. Considering substantial disease heterogeneity and tumor clonal evolution, CTCs could provide a more comprehensive and longitudinal assessment of disease and response to treatment when compared to single site biopsy. In this study we explore changes in CTC characteristics in correlation with pt’s response (Resp) to treatment and progression (PD). Methods: 152 blood sample time points from 44 ccRCC pts (mean=3.5 sample/pt, range= 1-9) were included. An automated microfluidic platform using exclusion-based sample technology was employed for high sensitivity enrichment of RCC CTCs using EpCAM and CAIX antibodies. High-specificity CTC identification was achieved with positive staining for pCK or CAXII, and negative staining for CD45, CD34 and CD66b. Single cell phenotyping of PDL1 and HLA1 expression on each CTC was quantified with automated image analysis algorithms followed by manual visual QA of the putative CTCs. Cellular-level expression of PDL1 and HLA1 were compared within treatment-response groupings, using Wilcoxon rank-sum tests (Table). Results: Pts were 70% male, 93% Caucasian, 51% de novo metastatic, 98% received systemic treatment. CTC samples were collected at baseline (12) or while on treatment with IO (71), TKI (55), or IO/TKI (14). Across treatments, the number of CTCs increased from Resp to PD but this trend was not statistically significant. PDL1 expression was higher at PD compared to Resp on IO and IO/TKI but not significantly different on TKI. HLA1 expression was higher at PD compared to Resp on IO and TKI but not significantly different on IO/TKI. Average CTC PD-L1 expression was higher at Resp to IO compared to baseline. Conclusions: Single cell phenotyping of PDL1 and HLA1 were done at Resp compared to PD time points on IO and/or TKI to identify potential mechanisms of resistance. Further evaluation of these preliminary results in prospective trials is ongoing to advance new predictive biomarkers for ccRCC.[Table: see text]

Early Detection and Dynamic Changes of Circulating Tumor Cells in Transgenic NeuN Transgenic (NTTg) Mice with Spontaneous Breast Tumor Development.

Simple SummaryThis study aimed to prove the early presence of circulating tumor cells (CTCs) with viability and tumorigenesis in a murine model that spontaneously develops breast cancer. Serial CTC examinations were performed on NeuN transgenic mice, starting from the age of 8 weeks and continuing after palpable tumor formation. Prior to the detection of palpable tumors, the CTC counts rose over time from 1 ± 1.6 to 16 ± 9.5 per 75 μL; this number continued to grow with tumor development. The viability and tumorigenesis of the collected CTCs were confirmed by re-implanting the cells into a non-cancer-bearing mouse. Ultrasonography with Doppler showed a significant correlation between CTCs and tumor vascular density (p-value < 0.01), rather than tumor volume (p-value 0.076).Background: This study used NeuN transgenic (NTTg) mice with spontaneous breast tumor development to evaluate the dynamic changes of circulating tumor cells (CTCs) prior to and during tumor development. Methods: In this longitudinal, clinically uninterrupted study, we collected 75 μL of peripheral blood at the age of 8, 12, 16, and 20 weeks in the first group of five mice, and at the age of 32 weeks, the time of tumor palpability, and one week after tumor palpability in the second group of four mice. Diluted blood samples were run through a modified mouse-CMx chip to isolate the CTCs. Results: The CTC counts of the first group of mice were low (1 ± 1.6) initially. The average CTC counts were 16 ± 9.5, 29.0 ± 18.2, and 70.0 ± 30.3 cells per 75 μL blood at the age of 32 weeks, the time of tumor palpability, and one week after tumor palpability, respectively. There was a significant positive correlation between an increase in CTC levels and tumor vascular density (p-value < 0.01). This correlation was stronger than that between CTC levels and tumor size (p-value = 0.076). The captured CTCs were implanted into a non-tumor-bearing NTTg mouse for xenografting, confirming their viability and tumorigenesis. Conclusion: Serial CTCs during an early stage of tumor progression were quantified and found to be positively correlated with the later tumor vascular density and size. Furthermore, the successful generation of CTC-derived xenografts indicates the tumorigenicity of this early onset CTC population.

Circulating tumor cells detected with a microcavity array predict clinical outcome in hepatocellular carcinoma.

The present study aimed to establish a novel isolation strategy for circulating tumor cells (CTCs) using a microcavity array (MCA) system and to evaluate the clinical significance of CTCs in hepatocellular carcinoma (HCC). We examined recovery rates of HCC cell lines spiked into whole blood in MCA assay. Circulating tumor cells were isolated from peripheral blood samples (3 mL) of 7 healthy donors (HD), 14 patients with liver cirrhosis (LC), and 31 patients with HCC using the MCA system. Additionally, we investigated the mRNA expression of liver‐specific genes in isolated CTCs using qPCR. The recovery rates were 65.1% (HepG2), 76.7% (HuH7), and 99.0% (PLC/PRF/5). In HD and patients with LC and HCC, the CTC positivity rate (CTCs ≥10) and average CTC number were as follows: HD 0% and 0.1, LC 14.3% and 5.3, HCC 54.8% and 47.6, respectively. The CTC positivity rate in HCC was significantly higher than that in LC (p < 0.05). The number of CTCs was significantly higher in metastatic HCC (102.2 ± 160.6) than in localized HCC (8.2 ± 7.7) (p < 0.05). The expression of AFP, glypican‐3, EpCAM, and albumin (ALB) genes was detected in isolated CTCs. The positive CTCs (CTCs ≥10) significantly reduced the cumulative survival in patients with HCC (p = 0.025), especially in localized patients with HCC (p = 0.046). The newly developed MCA system has the potential to isolate CTCs from HCC with high sensitivity, and mRNA expression could be measured from CTCs. Identification of positive CTCs can help predict clinical outcome of patients with HCC. Thus, analysis of CTCs in patients with HCC may provide important information as a novel biomarker in disease progression.

Overcoming Obstacles in Pathological Diagnosis of Pulmonary Nodules through Circulating Tumor Cell Enrichment.

With the popularity of low-dose computed tomography (LDCT) in clinical examination of the lung, the prevalence of pulmonary nodules has significantly increased, thus significantly improving the early diagnosis of lung cancer, but also potentially contributing to overtreatment. This study aims to develop a noninvasive method to assist in diagnosing the pulmonary nodules. To do so, 3798 patients are recruited from the Department of Thoracic Surgery at Shanghai Pulmonary Hospital and peripheral blood samples are collected from them before surgery. From these samples, circulating tumor cells (CTC) are isolated using folate receptor (FR) positivity, and then enriched and analyzed in relation to cancer gene expression, stage, and level of invasion. The average CTC concentration of patients with lung disease is 11.97 functional unit (FU) in a 3 mL sample of blood. FR-positive CTC levels correlate with the expression of lung cancer driver genes tumor-node-matastasis (TNM) stage, and pleura invasion. The sensitivity of CTC levels to lung cancer diagnosis is 87.05%. Results from this study demonstrate that the determination of FR-positive CTC concentration is a convenient and time-saving strategy to improve the pathological diagnosis of pulmonary nodules.

Determination of the optimal detection time of circulating tumor cells for the postoperative monitoring of colorectal cancer.

Circulating tumor cells (CTCs) are widely used in cancer screening and monitoring. The present study focused on investigating the optimal time for the postoperative CTC detection in patients with colorectal cancer (CRC) to obtain more accurate results and facilitate subsequent treatment. By subtraction enrichment immunofluorescence in situ hybridization detection of CTCs, the present study demonstrated that different postoperative detection times in CRC substantially influenced the CTC numbers. In total, 134 subjects were enrolled. Among 10 healthy individuals and 20 preoperative patients with CRC, no CTCs were identified in the healthy subjects, and CTCs were detected in 85% (17/20) of the preoperative patients. In total, 104 postoperative patients with CRC (53 males and 51 females) with a mean average age of 57.63 years were studied. The total CTC detection rate was 81.73% (85/104) and the mean average CTC numbers in patients with tumor stage (T) T1, T2, T3 and T4 were 4.00, 3.33, 5.90 and 5.64 per 7.50 ml of peripheral blood, respectively. The CTC number trends in these four tumor stages within 5, 6, 7 and 10 postoperative days were variable, and were the most stable at 7 days. Gradual upward trends in CTC numbers were observed after 5, 6 and 7 postoperative days, and this upward trend was more obvious after 7 days. Overall, the findings of the present study suggest that CTC detection in CRC should be performed after at least 7 postoperative days rather than within 7 postoperative days.

The application of nano-enrichment in CTC detection and the clinical significance of CTCs in non-small cell lung cancer (NSCLC) treatment.

Circulating tumor cells (CTCs) are an independent prognostic marker in non-small cell lung cancer (NSCLC). CTC numbers are closely related to early diagnosis, clinical stage, therapy surveillance, and prognosis of NSCLC. We used a more efficient nano-enrichment method to detect CTCs in NSCLC patients and explored the clinical value of CTCs. The results showed that CTC numbers in stage IV cases were significantly higher than those in stage I, II or III cases. The number of CTCs in poorly-differentiated cases was significantly higher than that in well-differentiated cases. During six chemotherapy cycles, the average CTC number decreased from 5.8/7.5 ml in cycle #1 to 2.4/7.5 ml in cycle #4 and remained at almost the same level from 4 to 6 cycles. CTC numbers in patients with EGFR mutations was significantly higher than those in patients with no mutations. The average progression free survival (PFS) in the favorable group (CTC ≤ 5/7.5 ml) was 11.3 months, which was longer than that in the unfavorable group (CTC > 5/7.5 ml, 7.2 months). In conclusion, the assessment of NSCLC cannot be performed using a single CTC analysis. The clinical value is more significant in the continuous analysis of CTC data, as well as the cross-validation of other indexes and imaging results.

Initial detection of circulating tumor cells from metastatic prostate cancer patients with a novel small device.

BackgroundVarious devices for isolating and detecting circulating tumor cells (CTCs) have been developed, whereas the CellSearch® system has been clinically used in numerous prostate CTC studies. CTCs might become more useful surrogate markers of prostate cancer, and they should be measured in all settings, but a smaller, low-cost CTC capture system is required.MethodsAn inexpensive and highly sensitive microfluidic CTC-capture polymeric chip, developed by the Toyama Industrial Technology Center, as described in the following text, was used to assess the number of CTCs from patients with metastatic prostate cancer. After verifying that cultured human prostate cancer cells (PC3 and LNCaP) could be captured with the chip coated with anti–epithelial cell adhesion molecule (CD326) antibody, whole blood samples of 14 patients with prostate cancer were screened.ResultsThe average capture efficacy of PC3 cells was 94.60% in phosphate-buffered saline (PBS) and 83.82% in whole blood. The average capture efficacy of LNCaP cells was 82.73% in PBS and 75.78% in whole blood. CTCs were detected by the chip device in all 14 patients with metastatic prostate cancer using 2-mL blood samples. Although fewer CTCs were detected in patients with oligometastases, all patients with multiple distant metastases had CTCs. The average CTC count was 48 cells/mL (range 1–81 cells/mL).ConclusionThis CTC-chip will be able to capture CTCs and be useful to check CTCs as a surrogate marker in prostate cancer with smaller samples and lower cost in any small institution.

Dynamic Halbach array magnet integrated microfluidic system for the continuous-flow separation of rare tumor cells.

Circulating tumor cells (CTCs), the most representative rare cells in peripheral blood, have received great attention due to their clinical utility in liquid biopsy. The downstream analysis of intact CTCs isolated from peripheral blood provides important clinical information for personalized medicine. However, current CTC isolation and detection methods have been challenged by their extreme rarity and heterogeneity. In this study, we developed a novel microfluidic system with a continuously moving Halbach array magnet (dHAMI microfluidic system) for negative isolation CTCs from whole blood, which aimed to capture non-target white blood cells (WBCs) and elute target CTCs. The dynamic and continuous movement of the Halbach array magnet generated a continuous magnetic force acting on the magnetic bead-labelled WBCs in the continuous-flow fluid to negatively exclude the WBCs from the CTCs. Furthermore, the continuously moving magnetic field effectively eliminated the effect of magnetic bead aggregation on the fluid flow to realize the continuous-flow separation of the CTCs without a sample loading volume limitation. The experimental procedure for CTC negative isolation using the dHAMI microfluidic system could be completed within 40 min. Under the optimized experimental conditions of the dHAMI microfluidic system, including the flow rate and concentration of the immunomagnetic bead, the average CTC capture rate over a range of spiked cell numbers (50–1000 cancer cells per mL) was up to 91.6% at a flow rate of 100 μL min−1. Finally, the CTCs were successfully detected in 10 of 10 (100%) blood samples from patients with cancer. Therefore, the dHAMI microfluidic system could effectively isolate intact and heterogeneous CTCs for downstream cellular and molecular analyses, and this robust microfluidic platform with an excellent magnetic manipulation performance also has great application potential for the separation of other rare cells.

Single-Cell Analyses of Prostate Cancer Liquid Biopsies Acquired by Apheresis.

Purpose: Circulating tumor cells (CTCs) have clinical relevance, but their study has been limited by their low frequency.Experimental Design: We evaluated liquid biopsies by apheresis to increase CTC yield from patients suffering from metastatic prostate cancer, allow precise gene copy-number calls, and study disease heterogeneity.Results: Apheresis was well tolerated and allowed the separation of large numbers of CTCs; the average CTC yield from 7.5 mL of peripheral blood was 167 CTCs, whereas the average CTC yield per apheresis (mean volume: 59.5 mL) was 12,546 CTCs. Purified single CTCs could be isolated from apheresis product by FACS sorting; copy-number aberration (CNA) profiles of 185 single CTCs from 14 patients revealed the genomic landscape of lethal prostate cancer and identified complex intrapatient, intercell, genomic heterogeneity missed on bulk biopsy analyses.Conclusions: Apheresis facilitated the capture of large numbers of CTCs noninvasively with minimal morbidity and allowed the deconvolution of intrapatient heterogeneity and clonal evolution. Clin Cancer Res; 24(22); 5635-44. ©2018 AACR.

Detection of Circulating Tumor Cells and Circulating Tumor Microemboli in Gastric Cancer

PURPOSE: Gastric cancer studies indicated a potential correlation between circulating tumor cells (CTCs) in peripheral blood and tumor relapse/metastasis. The prevalence and significance of circulating tumor microemboli (CTM) in gastric cancer remain unknown. We investigated the prevalence and prognostic value of CTCs and CTM for progression-free survival (PFS) and overall survival (OS) in gastric cancer patients. METHODS:Eighty-one gastric cancer patients consented to provide 5ml of peripheral blood before systematic therapy. CTCs and CTM were isolated using isolation by size of epithelial tumor cells and characterized by cytopathologists. For 41 stage IV gastric cancer patients, CTM was investigated as a potential biomarker to predict prognosis. RESULTS:CTCs were detected in 51 patients; the average count was 1.81. In clinical stage I, II, III, and IV patients, the average CTC counts were 1.40, 0.67, 1.24, and 2.71, respectively. CTM were detected in 3 of 33 clinical stage I to IIIb patients, at an average of 0.12 (0-2). CTM were detected in 13 of 53 clinical stage IIIc to IV patients, at an average of 1.26 (0-22). In stage IV patients, CTM positivity correlated with the CA125 level. PFS and OS in CTM-positive patients were significantly lower than in CTM-negative patients (P<.001). CTM positivity was an independent factor for determining the PFS (P=.016) and OS (P=.003) of stage IV patients in multivariate analysis. Using markers of the epithelial-mesenchymal transition, single CTCs were divided into three phenotypes including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs. For CTM, CK−/Vimentin+/CD45− and CK+/Vimentin+/CD45− phenotypes were observed, but the CK+/Vimentin−/CD45− CTM phenotype was not. CA125 was detected in gastric cancer cell lines BGC823 and MGC803. CONCLUSIONS: In stage IV patients, CTM positivity was correlated with serum CA125 level. CTM were an independent predictor of shorter PFS and OS in stage IV patients. Thus, CTM detection may be a useful tool to predict prognosis in stage IV patients.

Evaluation of a novel approach to circulating tumor cell isolation for cancer gene panel analysis in patients with breast cancer.

Liquid biopsy isolation of circulating tumor cells (CTCs) allows the genomic analysis of CTCs, which is useful in the determination of personalized cancer therapy. In the present study, CTCs from patients with breast cancer were enriched and successfully analyzed using cancer gene panel analysis. Blood samples from 11 patients with breast cancer were collected and CTCs enriched for using size-based filtration. The enriched CTCs were analyzed using immunofluorescence staining with antibodies directed against epithelial cell adhesion molecule (EpCAM) and cluster of differentiation 45. The genomic DNA of CTCs was extracted, amplified and 50 genes screened for mutations using the Ion AmpliSeq™ Cancer Hotspot Panel v2. EpCAM staining detected CTCs in 10/11 patients and the average CTC count was 3.9 in 5 ml blood. The average purity of enriched CTCs was 14.2±29.4% and the average amount of amplified DNA was 28.6±11.9 µg. Catalogue Of Somatic Mutations In Cancer mutations were detected in the CTCs and included IDH2, TP53, NRAS, IDH1, PDGFRA, HRAS, STK11, EGFR, PTEN, MLH1, PIK3CA, CDKN2A, KIT and SMARCB1. In conclusion, a novel size-based filtration approach for the isolation of CTCs was evaluated and successfully applied for the genomic analysis of CTCs from patients with breast cancer.

Arrays of High-Aspect Ratio Microchannels for High-Throughput Isolation of Circulating Tumor Cells (CTCs).

Microsystem-based technologies are providing new opportunities in the area of in vitro diagnostics due to their ability to provide process automation enabling point-of-care operation. As an example, microsystems used for the isolation and analysis of circulating tumor cells (CTCs) from complex, heterogeneous samples in an automated fashion with improved recoveries and selectivity are providing new opportunities for this important biomarker. Unfortunately, many of the existing microfluidic systems lack the throughput capabilities and/or are too expensive to manufacture to warrant their widespread use in clinical testing scenarios. Here, we describe a disposable, all-polymer, microfluidic system for the high-throughput (HT) isolation of CTCs directly from whole blood inputs. The device employs an array of high aspect ratio (HAR), parallel, sinusoidal microchannels (25 µm × 150 µm; W × D; AR = 6.0) with walls covalently decorated with anti-EpCAM antibodies to provide affinity-based isolation of CTCs. Channel width, which is similar to an average CTC diameter (12-25 µm), plays a critical role in maximizing the probability of cell/wall interactions and allows for achieving high CTC recovery. The extended channel depth allows for increased throughput at the optimized flow velocity (2 mm/s in a microchannel); maximizes cell recovery, and prevents clogging of the microfluidic channels during blood processing. Fluidic addressing of the microchannel array with a minimal device footprint is provided by large cross-sectional area feed and exit channels poised orthogonal to the network of the sinusoidal capillary channels (so-called Z-geometry). Computational modeling was used to confirm uniform addressing of the channels in the isolation bed. Devices with various numbers of parallel microchannels ranging from 50 to 320 have been successfully constructed. Cyclic olefin copolymer (COC) was chosen as the substrate material due to its superior properties during UV-activation of the HAR microchannels surfaces prior to antibody attachment. Operation of the HT-CTC device has been validated by isolation of CTCs directly from blood secured from patients with metastatic prostate cancer. High CTC sample purities (low number of contaminating white blood cells, WBCs) allowed for direct lysis and molecular profiling of isolated CTCs.

Utility of microsieve device for enumeration and miRNA expression profiling of CTCs for personalized cancer management.

e22107 Background: Although a rare peripheral cell population, circulating tumor cells (CTCs) represent a potential alternative to serial invasive biopsies as a source of tissue in monitoring clinical states of non-hematological malignancies. CTCs harvested from a single patient are a heterogeneous mixture of cells differing in their transcriptome expressional profiles, metastatic potential and drug resistance properties. Mere identification and enumeration of CTCs are unable to provide qualitative information about individual CTC biology. The presence of a few highly malignant, metastasis-capable CTCs and cancer stem cells among the other less aggressive CTCs in the circulating population will not be apparent from CTC counts alone. Methods: We developed a high-throughput size-exclusion microsieve device (CellSievo, Singapore) to isolate CTCs (defined as DAPI-positive, CD45-negative cells), regardless of the cell surface receptors expressed, from 74 breast cancer patients. Using CTCs eluted from the microsieve post-capture, we performed microRNA expression profiling to further characterize breast cancer CTCs. Results: Using CellSievo’s microsieve device, we obtained an average mean CTC count of 21.2 (+/- 31.8; range=0-112.5) per 7.5ml peripheral blood, for breast cancer patients, as opposed to 1.3 (+/-1.6, range 0-5.0) for healthy subjects. Viable CTCs from breast cancer patients were successfully harvested for downstream microRNA profiling. CTCs from breast cancer patients were routinely found to express microRNA profiles mediating epithelial-to-mesenchymal transition, tumor invasion, and regulation of chemotherapeutic drug resistance to tamoxifen, anthracyclines, taxanes, and trastuzumab. Serial monitoring of microRNA expression pattern of CTCs could identify emergence of novel tumor populations with distinct microRNA expression patterns during or after anticancer therapy (detailed microRNA expression data to be presented). Conclusions: Our results suggest that microRNA biomarkers derived from peripheral CTCs can yield highly useful information and offer prognostic and predictive applications for therapeutics and companion diagnostic development.