Negative Enrichment Method Research Articles

The relationship between circulating tumor cells in peripheral blood and clinical characteristics of pediatric neuroblastoma and prognostic evaluation

This study investigates the correlation between circulating tumor cells (CTCs) in peripheral blood and the clinical characteristics and prognosis of advanced pediatric neuroblastoma (NB). We conducted a retrospective analysis of 144 children with advanced NB who underwent comprehensive treatment. Detailed clinical data were collected, and CTCs were detected using a negative enrichment method combined with immunofluorescence technology. Prognostic evaluation criteria and cutoff values for CTCs were established using ROC curve analysis. Univariate and Cox multivariate regression analyses identified independent risk factors impacting prognosis. Patients were categorized into high and low-expression groups based on optimal cutoff values determined with X-tile software. The high expression group had a significantly higher incidence of disease progression (p < 0.001), maximum tumor diameter ≥10 cm (p = 0.004), undifferentiated subtype (p = 0.034), and stage IV disease (p = 0.007) compared to the low expression group. CTCs were notably higher in patients with progression compared to those with mitigation (p < 0.001), in those with maximum tumor diameter ≥10 cm compared to <10 cm (p < 0.001), and in stage IV compared to stage III patients (p = 0.036). The AUC values for maximum tumor diameter, degree of differentiation, and tumor stage were 0.703, 0.669, 0.574, and 0.598, respectively. The detection of CTCs provides significant insights into the clinical characteristics and prognosis of advanced pediatric NB, highlighting its potential as a prognostic tool.

Circulating tumor cells PD-L1 expression detection and correlation of therapeutic efficacy of immune checkpoint inhibition in advanced non-small-cell lung cancer.

This study investigated whether programmed death-ligand 1 (PD-L1) expression of circulating tumor cells (CTCs) in peripheral blood can serve as a predictive biomarker for immunotherapy efficacy in patients with advanced non-small-cell lung cancer (NSCLC). We employed a negative enrichment method to isolate CTCs. We identified PD-L1 + CTCs as PD-L1+/4',6-diamidino-2-phenylindole (DAPI)+/CD45-circulating tumor cells through an immunofluorescence method. Tumor tissue PD-L1 expression was determined by immunohistochemical staining. The correlation between CTC PD-L1 expression and patients' prognostic features was estimated through the Kaplan-Meier method. CTCs released a higher detection rate of PD-L1 expression than tumor tissues (53.0% vs. 42.1%). No correlation was observed between them. Forty-nine NSCLC patients received anti-PD-1/PD-L1 immunotherapy (three with combined anti-PD-1/PD-L1 and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), two with four cycles of combined immune checkpoint inhibitors [ICIs] plus chemotherapy and ICI monotherapy for maintenance). Patients with PD-L1 expression on tissue or CTCs had a median progression-free survival (mPFS) of 5.6months (n=36, 95% confidence interval [CI] 3.6-7.5months), significantly longer than those without PD-L1 detection (n=9, mPFS of 1.4months, 95% CI 1.3-1.5months, log-rank p=0.032). The multivariable Cox proportional-hazard model suggested that the tissue or CTC PD-L1 expression was associated with a lower risk of progression (hazard ratio 0.45, 95% CI 0.21-0.98, p=0.043). CTCs and tumor tissues reveal heterogeneous expression of PD-L1 in NSCLC patients. Patients with baseline PD-L1 expression on CTCs or tissue showed prolonged mPFS and may help to identify the subsets of patients who potentially benefit from immunotherapy.

Abstract 592: Circulating tumor cells of esophageal and gastric cancers

Abstract Background: The availability of circulating tumor cells (CTCs) creates a powerful non-invasive approach to monitor tumor progression, therapeutic response, relapse, metastasis, and, in some cases, provide early diagnosis. Esophageal cancer (EC) is the fifth most common cause of cancer-related death in men 40-59 years of age, with a ~90% death rate. Gastric cancers (GC) have a 30% five-year survival rate. Smoking, alcohol, age, H. pylori infection, and specific diets are established risk factors for esophageal and gastric cancers. We aim to isolate, propagate, and identify RNA expression profiles in CTCs from EC and GC patients. This study permits the potential clinical application of identifying markers that may define prognosis, metastatic potential, and therapeutic efficacy. Materials and methods: Venous blood samples from the esophageal and gastric cancer patients (n=6) were collected before and a week after chemotherapy. A negative enrichment method was performed to isolate CTCs using EasySep™ Direct Human CTC Enrichment Kit (StemCell Technologies). Label-free and viable CTCs were cultured in Epicult-C (StemCell Technologies) containing proliferation supplement and hydrocortisone. After rRNA depletion, strand-specific paired-end sequencing libraries for approximately 100M read-depth will be prepared by SMART-Seq v4 Ultra Low Input RNA Kit (Clontech) for RNA sequencing. Libraries will be constructed using the Illumina Nextera XT kit. The Washington DC VAMC Institutional Review Board approved this study and written informed consent was obtained from patients. Results: We observed a dynamic range of CTCs in the same patient during chemotherapy treatments. The fluctuation of the number of cells obtained before and a week after infusion may represent the effect of therapy on both primary and circulating tumor cells. Samples with clusters of CTCs preserved their structures during cell culture. Our most extended CTC sample culture approached 55 days, which obtained a patient with intestinal-type gastric cancer. Conclusion and future directions: We expect to identify differentially expressed genes in CTCs and the primary tumor, along with alternatively spliced isoforms in both primary tumor and CTCs. The fluctuation of the number of cells obtained before and a week after infusion may represent the effect of therapy on circulating tumor cells. RNA sequencing of CTCs will be performed and serve as baseline data for functional studies. This study permits monitoring therapeutic efficacy, identifying therapy-resistant or therapy-sensitive clones/markers that may allow the selection of appropriate therapeutic regimens. We will continue to follow patients and obtain tissues from lymph node or distant metastasis to analyze the evolution of their tumors. Citation Format: Hayriye Verda Erkizan, Robert G. Wadleigh. Circulating tumor cells of esophageal and gastric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 592.

Spatial heterogeneity in epithelial to mesenchymal transition properties of circulating tumor cells associated with distant recurrence in pancreatic cancer patients.

BackgroundThe spatial heterogeneity of epithelial to mesenchymal transition (EMT)-related circulating tumor cells (CTCs) within the circulatory system and its potential clinical relevance remain unclear in pancreatic cancer (PC) patients. We aimed to map the distribution of EMT-related CTCs along the spreading pathway and investigate the prognostic significance due to the potential spatial heterogeneity in the count and phenotypic properties of CTCs.MethodsBoth portal vein (PoV) and peripheral vein (PV) blood samples were collected from 39 PC patients. CTCs were isolated by using a CD45 negative enrichment method, and EMT-related phenotypes in CTCs were analyzed by 4-channel immunofluorescence. The correlations of CTCs with patient characteristics and recurrence-free survival (RFS) were analyzed.ResultsBoth the number {median CTC total count, 10 [6–16] in PoV vs. 5 [1–7] in PV per mL, P<0.0001} and EMT status of CTCs [median mesenchymal CTC (M-CTC) percentage, 0.33 (0.13–0.52) in PoV vs. 0.2 (0–0.4) in PV, P=0.0211] showed significant spatial heterogeneity during dissemination from the PoV to the PV. Univariate analysis adjusting for patient age and sex revealed that CTC total count and M-CTC percentage in PoV samples could be risk factors for RFS in PC patients (P=0.003 and P=0.001, respectively), and ROC curve analysis found that both of these factors had good performance in distinguishing patients with early distant recurrence (within 6 months), with the optimal cut-off values of 14 cells/mL (AUROC =0.893, sensitivity =0.857, specificity =0.813, P=0.001) and 0.545 (AUROC =0.795, sensitivity =0.714, specificity =0.906, P=0.016), respectively.ConclusionsMultivascular assessment of EMT-related CTCs suggested profound dynamic alterations in total count and phenotypes during dissemination, and the spatial heterogeneity of CTCs in circulation could help establish novel prognosis markers in PC patients.

Enrichment of Circulating Tumor Cells from Whole Blood Using a Microfluidic Device for Sequential Physical and Magnetophoretic Separations.

Based on their high clinical potential, the isolation and enrichment of rare circulating tumor cells (CTCs) from peripheral blood cells has been widely investigated. There have been technical challenges with CTC separation methods using solely cancer-specific surface molecules or just using physical properties of CTCs, as they may suffer from heterogeneity or lack of specificity from overlapping physical characteristics with leukocytes. Here, we integrated an immunomagnetic-based negative enrichment method that utilizes magnetic beads attached to leukocyte-specific surface antigens, with a physical separation method that utilizes the distinct size and deformability of CTCs. By manipulating the pressure distribution throughout the device and balancing the drag and magnetic forces acting on the magnetically labeled white blood cells (WBCs), the sequential physical and magnetophoretic separations were optimized to isolate intact cancer cells, regardless of heterogeneity from whole blood. Using a breast cancer cell line in whole blood, we achieved 100% separation efficiency for cancer cells and an average of 97.2% for WBCs, which resulted in a 93.3% average separation purity. The experimental results demonstrated that our microfluidic device can be a promising candidate for liquid biopsy and can be a vital tool for aiding future cancer research.

129P - Monitoring patient-specific mutation in ctDNA and CTC for tumour response evaluation after neoadjuvant chemotherapy in advanced gastric adenocarcinoma (NCT03425058)

BackgroundTo verify the value of ctDNA and CTC as biomarkers for tumor response in the neoadjuvant chemotherapy (nCRT) treatment of locally advance gastric adenocarcinoma. MethodsA total of 50 patients were enrolled. 20ml of plasma were collected at 3 points: before nCRT; after 2 cycles of nCRT; and after surgery. Firefly ctDNA NGS assays were used to track ctDNA mutations previously. Using patient-specific mutation derived from exome sequencing of tumor tissues samples. Tumor response data by CT were also collected. CTCs in 7ml peripheral blood were separated by a negative enrichment method and identified by FISH using two frequently proliferation chromosome probes, CEP8 and CEP17. Meanwhile, the HER2 expression in CTCs was determined by FISH and immunofluorescence. CEP8+ and/or CEP17+, DAPI+, CD45- cell were justified as CTCs, HER2 FISH+ or HER2 IF+, DAPI+, CD45- cells were justified as HER2 positive CTCs. ResultsA total of 10-20 somatic SNV or indel mutation were successfully identified for each patient. In comparison to basal line value of pre-chemotherapy blood, the ctDNA loading were decreased in post-chemotherapy specimens of 8 patients with TRG1 by histopathological grading or partial and complete response by CT scan. However, the amount of ctDNA in 25 patients diagnosed as TRG 2 or 3 showed minor changes during the neoadjuvant therapy. After surgery, 32 patients showed undetectable ctDNA in blood while 3 patients showed residual trace ctDNA. 22 patients have integral FISH data (CTCs before, after nCRT and after surgery range from 0-29, average: 4.9, 2.7 and 4.0, positive rate: 82%,73% and 73%), and 12 have integral HER2 data. The dynamic variations were coincidence to the clinical evaluation. For HER2, we found three positive model in CTCs, single FISH or IF positive or double positive. Double positive HER2-CTCs are found in some patients. By negative enrichment and FISH, CTCs can be detected at a low cutoff of 2 cells in 73%∼82% patients. ConclusionsThe ctDNA and CTC alteration during neoadjuvant therapy is consistent with conventional histopathological grading and radiological response assessment. Clinical trial identificationNCT03425058. Legal entity responsible for the studyThe author. FundingHas not received any funding. DisclosureThe author has declared no conflicts of interest.

818P - Monitoring patient-specific mutation in ctDNA and CTC for tumour response evaluation after neoadjuvant chemotherapy in locally advanced gastric cancer

BackgroundWe conduct a trial using neoadjuvant chemotherapy (nCRT) and verify the value of ctDNA and CTC as biomarkers for tumor response in the nCRT treatment of locally advance gastric adenocarcinoma. MethodsTwenty milliliters of plasma were collected at 3 points: before nCRT; after 2 cycles of nCRT; and after surgery. Firefly ctDNA NGS assays were used to track ctDNA mutations previously characterized in paired tumor tissue by massively parallel sequencing. Using patient-specific mutation derived from exome sequencing of tumor tissues samples, bespoke amplicon-based sequencing panel were synthesized and used for ctDNA profiling. CTCs in 7ml peripheral blood were separated by a negative enrichment method and identified by FISH using two frequently proliferation chromosome probes, CEP8 and CEP17. Meanwhile, the HER2 expression in CTCs was determined by FISH and immunofluorescence. CEP8+ and/or CEP17+, DAPI+, CD45- cell were justified as CTCs, HER2 FISH+ or HER2 IF+, DAPI+, CD45- cells were justified as HER2 positive CTCs. ResultsIn comparison to basal line value of pre-chemotherapy blood, the ctDNA loading were decreased in post-chemotherapy specimens of 8 patients with TRG1 by histopathological grading or partial and complete response by CT scan. However, the amount of ctDNA in 25 patients diagnosed as TRG 2 or 3 showed minor changes during the neoadjuvant therapy. 22 have integral FISH data (CTCs range from 0-29, average: 4.9, 2.7 and 4.0, positive rate: 82%,73% and 73%), and 12 have integral HER2 data (CTCS range from 0-9, average: 2.9, 2.2 and 2.9, positive rate: 25%, 50% and 33%). The dynamic variations were coincidence to the clinical evaluation. For HER2, we found three positive model in CTCs, single FISH or IF positive or double positive. Double positive HER2-CTCs are found in some patients. By negative enrichment and FISH, CTCs can be detected at a low cutoff of 2 cells in 73%∼82% patients. ConclusionsThe ctDNA and CTC alteration during neoadjuvant therapy are consistent with histopathological grading and response assessmentand and can be surrogate markers to prediction efficacy of nCRT for GC. Clinical trial identificationNCT03425058 11/02/2018. Legal entity responsible for the studyThe author. FundingBeijing Municipal Administration of Hospital Clinical Medicine Development of Special Funding Support (ZYLX201701). DisclosureThe author has declared no conflicts of interest.

Detection of tumor cells in lymph nodes of lung cancer by immunomagnetic beads negative enrichment method

Objective To establish an enrichment and detection method by immunomagnetic beads for disseminated tumor cells in lymph node specimens of lung cancer, and to explore the feasibility of this method to improve the diagnosis rate of lymph node micrometastasis. Methods Lymph node were obtained intraoperatively from lung cancer patients and turned into digest solution. All lymph node were pathologically non-metastatic. Tumor cells were enriched by negative method and identified by immunofluorescence and immunofluorescence in situ hybridization. Only p53+ /DAPI+ /CD45- cells and CEP8+ /DAPI+ /CD45- cells were judged as positive cells. Results Thirty patients with lung cancer were enrolled and 60 lymph nodes were obtained. Positive cells were detected in 7 lymph nodes by immunofluorescence and 7 lymph nodes by immunofluorescence in situ hybridization. Both p53+ /DAPI+ /CD45- cells and CEP8+ /DAPI+ /CD45- cells were detected in 3 lymph nodes. Therefore, eleven lymph node from 10 NSCLC patients were found to be positive of micrometastasis. Conclusion Negative enrichment by immunomagnetic beads is feasible to enrich disseminated tumor cells from lymph nodes and potentially leads to a more accurate estimation of lymph nodes micrometastasis for lung cancer patients. Key words: Lung cancer; Lymph node micrometastasis; Negative enrichment

Clinical significance of tumor cells and CD133+ cells from malignant pleural effusion in patients with lung cancer

AbstractObjectiveThe composition of malignant pleural effusion in lung cancer is so complex that it is very difficult to obtain high‐purity tumor cells from pleural effusions. Based on the establishment of the negative enrichment method using immunomagnetic beads to obtain high‐purity tumor cells, the present study investigated the clinical significance of tumor cells and CD133+ cells in lung cancer patients with malignant pleural effusion.MethodsFrom 1 April 2013 to 31 August 2014, 21 untreated lung cancer patients with pathologically confirmed malignant pleural effusions were enrolled in a study from the Respiratory Department of Clinical Medical School of Yangzhou University. All patients were treated with chemotherapy or gefitinib. Before treatment, 10 mL of pleural effusion was collected. Tumor cells and CD133+ cells from 10 mL pleural effusion were counted by using negative enrichment technology, chromosomal fluorescence in situ hybridization and fluorescent staining with anti‐CD133 antibody. In addition, the relationships between the numbers of tumor cells and CD133+ cells, and clinical characteristics were further analyzed.ResultsAmong the 21 patients, the median number of tumor cells and CD133+ cells was 4000/10 mL and 19/10 mL, respectively. There were no significant differences in the numbers of tumor cells and CD133+ cells in patients of different gender (χ2 = 0.597, P = 0.550 and χ2 = 0.225, P = 0.822, respectively), age (χ2 = 0.533, P = 0.594 and χ2 = 1.288, P = 0.198, respectively) and different pathological types of lung cancer (χ2 = 3.253, P = 0.197 and χ2 = 0.034, P = 0.983, respectively). The numbers of tumor cells and CD133+ cells were significantly higher in patients with pulmonary, distant lymph node or other organ metastasis than in patients without distant metastasis or with only regional lymph node metastasis (χ2 = 4.398, P = 0.036 and χ2 = 4.605, P = 0.032, respectively). After treatment with two cycles of chemotherapy or gefitinib for 2 months, 0 patients had complete response, seven had partial response, nine had stable disease, five had progressive disease, and the total response rate was 33.33%. The numbers of tumor cells and CD133+ cells were significantly different between the patients with different treatment efficacies (χ2 = 7.575, P = 0.023 and χ2 = 7.247, P = 0.027, respectively). The numbers of tumor cells and CD133+ cells in patients with partial response were the lowest, whereas the numbers of tumor cells and CD133+ cells in patients with progressive disease were the highest. The patients were divided into three groups according to the median number of tumor cells and CD133+ cells: low cell numbers (both tumor cell and CD133+ cell numbers were equal to or less than the median number), moderate cell numbers (tumor cells &gt; median and CD133+ cells ≤ median or CD133+ cells &gt; median and tumor cells ≤ median) and high cell numbers (both tumor cell and CD133+ cell numbers were more than the median numbers). The incidence of pulmonary, distant lymph node or other organ metastasis in patients with a moderate‐to‐high cell numbers was significantly higher than that in patients with a low cell numbers (χ2 = 12.634, P = 0.002). Furthermore, the treatment efficacy in patients with a low cell number was better than that in patients with a moderate‐to‐high cell numbers (χ2 = 10.886, P = 0.01).ConclusionTumor cells and CD133+ cells in pleural effusions might be used as an effective predictor of disease progression and treatment efficacy in patients with advanced lung cancer.

Detection of Circulating Tumor Cells by Fluorescent Immunohistochemistry in Patients with Esophageal Squamous Cell Carcinoma: Potential Clinical Applications.

BackgroundCirculating tumor cells (CTCs) are tumor cells that leave the primary tumor site and enter the bloodstream, where they can spread to other organs; they are very important in the diagnosis, treatment, and prognosis of malignant tumors. However, few studies have investigated CTCs in esophageal squamous cell carcinoma (ESCC). The aim of this study was to investigate the CTCs in blood of ESCC patients and its potential relevance to clinicopathological features and prognosis.Material/MethodsCTCs were acquired by a negative enrichment method that used magnetic activated cell sorting (MACSTM). Fluorescent immunohistochemistry (IHC) was used to identify the CTCs. Then, the positive CTC patients with ESCC were analyzed, after which the relationship between CTCs and clinicopathologic features was evaluated.ResultsIn the present study, 62 out of 140 (44.3%) patients with ESCC were positive for CTCs. The positive rate of CTCs was significantly related with stage of ESCC patients (P=0.013). However, there was no relationship between CTC status and age, sex, smoking tumor history, tumor location, differentiation of tumor, lymphatic invasion, or lymph venous invasion (P>0.05). Kaplan-Meier analysis showed that patients positive for CTCs had significantly shorter survival time than patients negative for CTCs. Multivariate analysis demonstrated that stage and CTC status were significant prognostic factors for patients with ESCC.ConclusionsCTCs positivity is an independent prognostic biomarker that indicates a worse prognosis for patients with ESCC.

Abstract 1922: Target mutation comparasion of WGA circulating tumor cell and primary tumor in epithelial ovarian cancer cell line using NGS

Abstract Introduction Epithelial ovarian cancer is the third most common gynecologic cancer in the United States but a leading cause of gynecologic cancer deaths. Previous finding that numbers of circulating tumor cells (CTCs) in the blood of ovarian cancer patients are prognostically significant. Moreover, comprehensive analyses of ovarian cancer genomes may provide more information for precise cancer treatments. Genomic analysis of circulating tumor cells (CTC) can provide possible approach for EOC patients who lack primary tumor tissue or present with un-resectable metastatic diseases. The discordance between whole genome amplification of circulating tumors and primary tumor should be validated firstly and so we conducted a pilot analysis of CTC capture and targeted NGS in EOC cell lines. Methods and materials Under IRB approval, EOC cell lines, different numbers of SKOV-3 (serous adenocarcinoma) and ES-2 (clear cell carcinoma) were spiked in fresh blood samples (20 ml) of healthy donor and captured by CytoQuestTM CR system or negative enrichment method (Abnova, Taipei, Taiwan). CTCs were identified by pan-CK, CD45 and DAPI expression and stained with CEP8 FISH for aneuploidy. Spiked CTCs of two cell lines were captured and underwent whole genome amplification by PicoPLEX™ WGA kit (Rubicon Genomic, MI, USA). The WGA DNA product and DNA of two primary cell lines were prepared libraries for TruSight(TM) Tumor sequencing panel (Illumina Inc.MI,USA) according to manufacturer's instruction. Sequencing procedure were performed on MiSEQ system(Illumina Inc.) by a local service provider. Results SKOV-3 and ES-2 can be stained and captured with these antibodies. Mean recovery of these cells were more than 80% by both systems. However, ES-2 is less Pan-CK stained but still could be captured by this system. WGA products of each one, three and five ES-2 cells and one SKOV3 cell were prepared well for library. We choose one cell WGA product and DNA extracted from primary SKOV-3 and ES2 were subjected to targeted 26 genes mutational analysis composed in TruSight Tumor panel. Average coverage depth for each amplicon is minimum 1000x with 7000x means. We would like to show if any discordance appear due to the acquisition of DNA WGA in the single cells, of which the analysis is currently ongoing. DISCUSSION Our study would like to demonstrate the condition of variants calls of single tumor cells isolated from whole blood samples and primary tumor in a targeted mutation analysis using NGS after a highly automated isolation workflow. This approach provides a pilot workflow for studying the heterogeneity within the CTC population in blood samples of patients with tumor and their primary tumor and the results can also document the possibility of WGA-induced bias of a recently commercialized WGA kit. Citation Format: Pi-Lin Sung, Chi-Ying F. Huang, Michael Hsiao, Kuo-Chang Wen, Ming-Shyen Yen, Mong-Hong Lee. Target mutation comparasion of WGA circulating tumor cell and primary tumor in epithelial ovarian cancer cell line using NGS. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1922. doi:10.1158/1538-7445.AM2015-1922

Design of a Microfluidic Chip for Enrichment of Circulating Tumor Cells

Identification and characterization of CTCs can be used as a tool for the study of cancer metastases. A novel design of microfluidic chip used for enrichment of circulating tumor cells is presented in this paper. An integration of DLD method and negative enrichment method were designed to improve the throughput and recovery rate while getting intact CTCs. The DLD stage is used to separate CTCs from blood cells preliminarily, and the negative enrichment stage is used to acquire purified CTCs. Both of them were simulated with COMSOL Multiphysics. Simulation results showed that triangular micro-posts have better performance in DLD stage, and wave structures could generate better disturbance effect than herringbone structures. This chip provides a potential approach with high throughput and purity for the enrichment of CTCs.

Microfluidic platform for negative enrichment of circulating tumor cells

Negative enrichment is the preferred approach for tumor cell isolation as it does not rely on biomarker expression. However, size-based negative enrichment methods suffer from well-known recovery/purity trade-off. Non-size based methods have a number of processing steps that lead to compounded cell loss due to extensive sample processing and handling which result in a low recovery efficiency. We present a method that performs negative enrichment in two steps from 2ml of whole blood in a total assay processing time of 60min. This negative enrichment method employs upstream immunomagnetic depletion to deplete CD45-positive WBCs followed by a microfabricated filter membrane to perform chemical-free RBC depletion and target cells isolation. Experiments of spiking two cell lines, MCF-7 and NCI-H1975, in the whole blood show an average of >90% cell recovery over a range of spiked cell numbers. We also successfully recovered circulating tumor cells from 15 cancer patient samples.

The negative enrichment by immunomagnetic beads for tumor cells from malignant pleural effusions

To establish a method (negative enrichment by immunomagnetic beads) for detection of tumor cells in pleural effusions and to evaluate the sensitivity and specificity of the method for clinical application. Five, 10, 20, 50 and 100 A549 (lung adenocarcinoma) cells were labeled with DAPI and added into 20 ml pleural effusions [containing (1 - 10)×10(6)cells] from heart failure patients, followed by immunomagnetic negative enrichment method. Recovered cancer cells were enumerated using a fluorescent microscope. Tumor cells were enriched from pleural effusion samples by means of density gradient centrifugation and negative enrichment by immunomagnetic beads method, followed by identification with cytology analysis (Wright's Giemsa's staining), immunofluorescence staining (IF) and fluorescence in situ hybridization (FISH) using centromere DNA probes of chromosome 7 and 8. Cytology, IF and FISH evaluations were performed in 53 pleural effusion samples, including 36 cases of malignant disease (25 male and 11 female patients aging 40 to 78 years, mean age (63 ± 9) and 17 cases of benign disease (8 male and 9 female patients aging 25 to 81 years, mean age (53 ± 18). After DAPI staining and mixing with pleural effusions from heart failure patients, the cell recovery rates of A549 cells evaluated under fluorescence microscope were 75%, 78%, 82%, 85%, 88%, and the average recovery rate was 81.6%. Using negative enrichment method and density gradient centrifugation combined with cytology analysis, the positive rates of tumor cells in 36 malignant pleural effusion samples were 81% (29/36) and 61% (22/36), respectively (χ(2) = 4.00, P = 0.039). Using negative enrichment method combined with IF, the positive rate of CK18(+), DAPI(+), CD(45)(-) cells was 100%. Moreover, using negative enrichment method combined with FISH analysis, the positive rate of tumor cells was 86% (31/36), much higher than that using density gradient centrifugation combined with cytology analysis (χ(2) = 5.818, P = 0.012). In 17 cases of benign pleural effusions, using negative enrichment method combined with IF, the positive rate was 100%. But other methods didn't find cancer cells from benign pleural effusions. It was applicable to enrich tumor cells from pleural effusions using negative enrichment method by immunomagnetic beads. This method combined with cytology analysis or FISH significantly enhanced the sensitivity and specificity of tumor cell detection in pleural effusions. But it was difficult to distinguish cancer cells from mesothelial cells using immunofluorescence staining with CK18, DAPI and CD(45) label. More specific markers were needed to recognize tumor cells from pleural effusions.