Detection In Peripheral Blood Research Articles

Nanozyme-Based Pump-free Microfluidic Chip for Colorectal Cancer Diagnosis via Circulating Cancer Stem Cell Detection.

Circulating cancer stem cells (CCSCs) are subpopulations of cancer cells with high tumorigenicity, chemoresistance, and metastatic potential, which are also major drivers of disease progression. Herein, to achieve the prediction of tumor diagnosis and progression in colorectal cancer (CRC), a new, automated, and portable lateral displacement patterned pump-free (LP) microfluidic chip (LP-chip) with the CoPt3 nanozyme was established for CCSC capture and detection in peripheral blood and feces samples ex vivo. In this design, CoPt3@HA probes with functions of magnetic separation and colorimetric signal transduction by peroxidase-mimicking activity were applied for the capture of CCSCs and signal output in clinical samples. The generated colors of polydopamine (PDA) were quantifiable through the smartphone APP and visualizable by the naked eye in the test line (T line) and control line (C line) of the LP-chip. In the optimal experimental conditions, the CCSC concentration was sensitive to change in the range 0-105 cells mL-1, with a detection limit of 3 cells mL-1 (S/N = 3). Preliminary studies of clinical samples suggest that the platform has the potential for prediction of colorectal cancer progression and poor prognosis. Overall, the LP-chip provides potential strategies for timely diagnosis, therapeutic monitoring, and recurrence prediction to improve home-based patient care.

101TiP The efficacy and safety of furmonertinib in advanced NSCLC patients with EGFR mutations and CNS metastases based on ctDNA detection in peripheral blood and CSF (FAITH)

101TiP The efficacy and safety of furmonertinib in advanced NSCLC patients with EGFR mutations and CNS metastases based on ctDNA detection in peripheral blood and CSF (FAITH)

Approaches for Performance Verification Toward Standardization of Peripheral Blood Regulatory T-Cell Detection by Flow Cytometry.

Regulatory T-cell (Treg) detection in peripheral blood, based on flow cytometry, is invaluable for diagnosis and treatment of immune-mediated diseases. However, there is a lack of reliable methods to verify the performance, which is pivotal toward standardization of the Tregs assay. To conduct standardization studies and verify the performance of 3 commercially available reagent sets for the Tregs assay based on flow cytometry and agreement analysis for Treg detection across the different reagent sets. The analytical performance of Tregs assay using reagent sets supplied by 3 manufacturers was evaluated after establishing the gating strategy and determining the optimal antibody concentration. Postcollection sample stability was evaluated, as well as the repeatability, reproducibility, reportable range, linearity, and assay carryover. Agreement between the different assays was assessed via Bland-Altman plots and linear regression analysis. The relationship between the frequency of CD4+CD25+CD127low/- Tregs and CD4+CD25+Foxp3+ Tregs was evaluated. The postcollection sample stability was set at 72 hours after collection at room temperature. The accuracy, repeatability, reproducibility, and accuracy all met the requirements for clinical analysis. Excellent linearity, with R2 ≥0.9 and no assay carryover, was observed. For reportable range, a minimum of 1000 events in the CD3+CD4+ gate was required for Tregs assay. Moreover, the results for Tregs labeled by antibodies from the 3 manufacturers were in good agreement. The percentage of CD4+CD25+CD127low/- Tregs was closely correlated with CD4+CD25+Foxp3+ Tregs. This is the first study to evaluate systematically the measurement performance of Tregs in peripheral blood by flow cytometry, which provides a practical solution to verifying the performance of flow cytometry-based immune monitoring projects in clinical practice.

Three-Dimensional PLGA Nanofiber-Based Microchip for High-Efficiency Cancer Cell Capture.

A 3D network capture substrate based on poly(lactic-co-glycolic acid) (PLGA) nanofibers was studied and successfully used for high-efficiency cancer cell capture. The arc-shaped glass micropillars were prepared by chemical wet etching and soft lithography. PLGA nanofibers were coupled with micropillars by electrospinning. Given the size effect of the microcolumn and PLGA nanofibers, a three-dimensional of micro-nanometer spatial network was prepared to form a network cell trapping substrate. After the modification of a specific anti-EpCAM antibody, MCF-7 cancer cells were captured successfully with a capture efficiency of 91%. Compared with the substrate composed of 2D nanofibers or nanoparticles, the developed 3D structure based on microcolumns and nanofibers had a greater contact probability between cells and the capture substrate, leading to a high capture efficiency. Cell capture based on this method can provide technical support for rare cells in peripheral blood detection, such as circulating tumor cells and circulating fetal nucleated red cells.

Engineered Macrophages-Based uPA-Scavenger Load Gemcitabine to Prompt Robust Treating Cancer Metastasis.

The majority of cancer patients die of metastasis rather than primary tumors, and most patients may have already completed the cryptic metastatic process at the time of diagnosis, making them intractable for therapeutic intervention. The urokinase-type plasminogen activator (uPA) system is proved to drive cancer metastasis. However, current blocking agents such as uPA inhibitors or antibodies are far from satisfactory due to poor pharmacokinetics and especially have to face multiplex mechanisms of metastasis. Herein, an effective strategy is proposed to develop a uPA-scavenger macrophage (uPAR-MΦ), followed by loading chemotherapeutics with nanoparticles (GEM@PLGA) to confront cancer metastasis. Interestingly, significant elimination of uPA by uPAR-MΦ is demonstrated by transwell analysis on tumor cells in vitro and enzyme-linked immunosorbent assay detection in peripheral blood of mice with metastatic tumors, contributing to significant inhibition of migration of tumor cells and occurrence of metastatic tumor lesions in mice. Moreover, uPAR-MΦ loaded with GEM@PLGA shows a robust antimetastasis effect and significantly prolonged survival in 4T1-tumor-bearing mice models. This work provides a novel living drug platform for realizing a potent treatment strategy to patients suffering from cancer metastasis, which can be further expanded to handle other tumor metastasis markers mediating cancer metastasis.

Association between circulating tumor cells in the peripheral blood and the prognosis of gastric cancer patients: a meta-analysis.

Research on the correlation between circulating tumor cells (CTCs) and gastric cancer (GC) has increased rapidly in recent years. However, whether CTCs are associated with GC patient prognosis is highly controversial. This study aims to evaluate the value of CTCs to predict the prognosis of GC patients. A meta-analysis. We searched the PubMed, Embase, and Cochrane Library databases for studies that reported the prognostic value of CTCs in GC patients before October 2022. The association between CTCs and overall survival (OS) and disease-free survival (DFS)/recurrence-free survival (RFS) and progression-free survival (PFS) of GC patients was assessed. Subgroup analyses were stratified by sampling times (pre-treatment and post-treatment), detection targets, detection method, treatment method, tumor stage, region, and HR (Hazard Ratio) extraction methods. Sensitivity analysis was performed by removing individual studies to assess the stability of the results. Publication bias was evaluated using funnel plots, Egger's test, and Begg's test. We initially screened 2000 studies, of which 28 were available for further analysis, involving 2383 GC patients. The pooled analysis concluded that the detection of CTCs was associated with poor OS (HR = 1.933, 95% CI 1.657-2.256, p < 0.001), DFS/RFS (HR = 3.228, 95% CI 2.475-4.211, p < 0.001), and PFS (HR = 3.272, 95% CI 1.970-5.435, p < 0.001). Furthermore, the subgroup analysis stratified by tumor stage (p < 0.01), HR extraction methods (p < 0.001), detection targets (p < 0.001), detection method (p < 0.001), sampling times (p < 0.001), and treatment method (p < 0.001) all showed that CTC detection was associated with poor OS and DFS/RFS for GC patients. Furthermore, the study showed that CTCs were associated with the poor DFS/RFS of GC when CTCs were detected for patients from Asian or No-Asian regions (p < 0.05). In addition, higher CTCs predicted poorer OS for GC patients who are from Asian regions (p < 0.001), but without statistical difference for GC patients from No-Asian regions (p = 0.490). CTC detection in peripheral blood was associated with poor OS, DFS/RFS, and PFS in patients with GC.

Clinical Value of Gene Copy Number Variation Detection in Peripheral Blood in the Diagnosis and Treatment of Lymphoma

Objective To investigate the consistency between the results of gene copy number variation (CNA) in peripheral blood and bone marrow or tissue samples of lymphoma patients with curative and its clinical value in diagnosis and treatment. Methods A total of 5 patients with lymphoma were selected. Case 1 was diffuse large B-cell lymphoma (DLBCL) without bone marrow invasion. Tissue samples and peripheral blood samples were collected at the same time point before treatment. Case 2 was high-grade B-cell lymphoma (HGBL) stage IV, tissue samples were collected before treatment at the initial diagnosis and peripheral blood samples were collected after treatment. Case 3 was DLBCLIV, tissue samples were collected at initial diagnosis and peripheral blood samples were collected during remission after treatment. Case 4 was DLBCL-IV B stage. Peripheral blood samples were collected before treatment at initial diagnosis and bone marrow samples were collected during remission after treatment. Case 5 was a case of hepatosplenic T-cell lymphoma (HSTL). Tissue samples before treatment and peripheral blood samples after treatment were collected at initial diagnosis and analyzed in combination with clinical efficacy. Results 2p -, 2q-, 3p-, 4p -, 6q-, 9p+, 10q-, 11q+, 19p + and 19q+ chromosome abnormalities were detected in the tissues and peripheral blood CNA of case 1, and the 3p -, 6q-, 9p +, 11q+ and 19q+ abnormalities were consistent. In case 2, 4p -, 9P +, 9p-, 16p+, 16q- and +X gene variants were detected in peripheral blood CNA than in tissue CNA at initial diagnosis. In case 3, obvious abnormal signals were detected in tissues at initial diagnosis, but no abnormal signals were detected in peripheral blood samples during remission. In case 4, obvious abnormal signals were detected in peripheral blood at initial diagnosis, but no abnormal signals were detected in bone marrow during remission. In case 5, obvious abnormal signals were detected in tissues at initial diagnosis, and a small amount of abnormal signals were detected in peripheral blood after treatment. The results of CNA detection in 5 patients were consistent with the clinical efficacy. All 3 DLBCL patients showed 6q-chromosome abnormalities in CNA test before treatment. Conclusion The results of CNA detection in peripheral blood are consistent with those in tissue and bone marrow. The dynamic monitoring results of in peripheral blood CNA can be used as a basis for monitoring the curative effect of lymphoma.

Single and Combinational Multiplex Base-Edited 'Universal' CAR T Cells in a Humanised Model of Primary CD7+CD33+ AML

Introduction Universal allogeneic donor CAR T cells are being developed as part of a 'deep conditioning' strategy against AML to secure disease clearance ahead of allogeneic stem cell transplantation (Allo-SCT). CD33 is widely expressed on AML, sometimes on the entire leukemia-associated populations, but is also present on healthy precursors; CD7 may be expressed on AML subpopulations and is present on normal T and NK cells, resulting in problematic fratricide effects during CAR T manufacture. Healthy donor derived CAR T cell banks generated using cytidine deamination base-editing and lentiviral transduction with CARs against both CD33 and CD7 circumvent such issues and could offer cell based anti-leukemic therapy alongside preparative effects ahead of allo-SCT. Here we investigated single and combinational BE-CAR T cells against primary human AML in vivo. Methods: Healthy donor lymphocytes were activated by anti-CD3/28 stimulation ahead of electroporation to deliver codon optimised base editor mRNA and guide RNAs against CD7, CD52 and TRBC, and lentiviral transduction to express either an anti-CD7 or anti-CD33 CAR. Magnetic bead depletion removed residual TCRab T cells, and disruption of CD7 ensured cells avoided fratricide. In vitro, antigen specific function was verified in cytotoxicity and cytokine release assays, and in vivo potency was confirmed against luciferase modified Molm14 and Kasumi lines in NSG mice. Responses against primary AML were investigated in a patient derived xenograft model (PDX) from a KIT mutated AML patient, established by serial transplantation, with full expression of CD33 (100% of blasts) and partial expression of CD7 (30% of blasts). Cells were injected into irradiated NSG mice (n=10 per group). On day 24 at a mean leukemic engraftment of 15% human CD45 positive cells in peripheral blood, mice were treated with either 10x106 BE-CAR33, BE-CAR7 or a combination of both or with anti-CD19-CAR-T cells (CAR19) as a control group (in total 20x106 T cells per mouse). AML engraftment, T cell persistence and expression of checkpoint markers were monitored by multicolour flow cytometric analysis. Results: On days 14 and 28 after CAR-T cell infusion, AML cells were readily detected in peripheral blood of control CAR19 animals (42.8% and 90% respectively) and the BE-CAR7 group (27.8% and 67.6%), but were notably reduced in the BE-CAR33 alone group (2.3% and 8.3%) and the BE-CAR33 and BE-CAR7 combination group (2.5% and 3.4%) (Figure 1A). Survival was significantly prolonged for BE-CAR33 and BE-CAR33/BE-CAR7 treated mice compared to the other groups (P<0.001) (Figure 1B). CD7+ AML cells were depleted to a mean of 1% and 4.5% in the BE-CAR33 group, to 0.6% and 0.7% in the BE-CAR33/BE-CAR7 and to 2.6% and 2.9% in the BE-CAR7 group, while CD7+ AML cells increased to 12% and 30% in the CAR19 group at day 14 and 28 after CAR-T cell infusion. Total CD2+CAR-T cell detection in peripheral blood ranged from 1.6 to 9.8% at day 14, 1 to 3.7% at day 28, and 0.8 to 2.7% at day 42, indicating durable persistence over the investigation period. Interestingly, PD-L1 on AML cells was present in all groups while checkpoint markers PD1, GAL9, TIM3, and LAG3 were significantly upregulated in BE-CAR33 cell treated mice, but not BE-CAR7 or CAR19 cell treated mice (P<0.001 for all comparisons). Conclusions: Lentiviral transduction and multiplexed base editing to remove TCR, CD52 and the shared AML/T lineage antigen CD7, enabled the generation of universal donor CAR T cells for combinational use. This data demonstrates robust activity of BE-CAR33 alone and in combination with BE-CAR7 against human CD7+CD33+AML in mice and highlights the importance of strategies that ensure complete targeting of leukemic populations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Extended Detection in Peripheral Blood Following Infusion of Chronic Granulomatous Disease Patient Autologous Granulocytes Corrected By mRNA Transfection in Patients with Chronic Granulomatous Disease

Patients with Chronic Granulomatous Disease (CGD) have an inherited defect in production of microbicidal oxidants by neutrophils and monocytes that can result in life-threatening infections. Some of these infections, particularly those with mold fungi that invade across tissue boundaries, may persist and progress despite intensive medical therapy. In such a setting there is a long history of published single case and small series reports suggesting that a course of unmatched allogeneic donor granulocyte (gran) transfusions may result in improvement or even resolution of such refractory life-threatening infections in CGD patients. Donors for grans transfusion products are often challenging to find and not HLA-matched. Because of this most CGD patients can be provided only a limited course of grans transfusions before they develop limiting anti-HLA antibodies or lack of available donors. To address this problem, we developed an ex vivo correction of NADPH oxidase defective grans from CGD by mRNA transfection1. Apheresis grans transfected with mRNA restored NADPH oxidase function in vitro and following transplant into immunodeficient mice. Messenger RNA-transfected autologous grans from non-human primates (NHP) were well tolerated and were detected for up to 5 days in NHP peripheral blood following infusion. This pre-clinical work was transitioned to a Phase I, dose-escalating, feasibility and safety study (NIH Protocol 22-I-0001; ClinicalTrials.gov NCT05189925). We report here data from the first X-linked CGD subject infused with autologous gp91 mRNA-transfected grans at the first dose level. The patient received two daily doses of G-CSF before apheresis. From 26 x10e9 total nucleated cells in the apheresis product, ~7x10e9 grans were collected, though only 1.5x10e9 grans were subjected to electroporation transfection with gp91phox mRNA (CELLSCRIPT LLC). A single infusion of 70e6 (10e6 per kg) mRNA-transfected autologous grans where about 57% were DHR+ (40x10e6 DHR positive grans) was well tolerated without any adverse effects. NADPH oxidase functional (DHR+) grans were detected in peripheral blood (PB) at last assessment (72 hrs) after this single infusion of mRNA corrected autologous grans. Serial PB evaluation post infusion from study Subject detected oxidase (DHR)+ grans. Of note, DHR+ grans in Subject PB at ~ 43 hrs after infusion was 0.0037% compared to his pre-infusion DHR assays (0%) (Figure, top row). As a comparator, DHR assays performed pre- and also ~ 40 hrs post infusion of a clinical product of 40x10e9 allogeneic donor grans into another CGD patient being treated clinically for a refractory infection is shown (bottom row). There is 1000 fold more allogeneic DHR+ grans infused for the clinical treatment than the experimental DHR+ mRNA corrected autologous grans infused. Compared to their corresponding absolute neutrophil count, the study Subject (received 40x10e6 DHR+ mRNA transfected autologous grans) had 512 DHR+ neutrophils/ml while the CGD patient (received clinical 40x10e9 allogeneic grans) had 79,560 DHR+ neutrophils/ml. This indicates there are > 6 fold more mRNA-corrected autologous grans in circulation at 43 hrs per cell infused than following the clinical treatment with allogeneic cells infused. Our data demonstrates that mRNA-corrected autologous grans are not only well tolerated but are very well sustained viable and functional in the circulation relative to clinical unmanipulated allogeneic donor grans. Additional patients need to be studied at this dose level and the escalating dose levels to assure that this could be a safe alternative to non-HLA matched allogeneic grans transfusions. Figure shows FACS DHR analysis of peripheral blood from CGD patients after infusion of autologous mRNA-transfected granulocytes 40x106 (top) and donor granulocytes 40x109 (bottom). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pre-clinical efficacy of CD20-targeted chimeric antigen receptor T cells for non-Hodgkin's lymphoma

BackgroundA 4-1BB/CD3-ζ-costimulated CAR-T against CD20 (CAR-T20) was subjected to a systemic efficacy evaluation in a cell co-culture model, and NOD-SCID IL-2 receptor gamma null mice (short for NSG mice) were xenografted with human Burkitt's lymphoma Raji cells.MethodsCAR-T20 cells were incubated with target cells (K562, K562 CD20 or Raji cells) at ratios of 10:1 and 5:1 for 24 h, and the killing rate was estimated by an LDH cytotoxicity assay. To evaluate the effect of CAR-T20 on the survival time of tumor-bearing animals, 30 NSG mice were employed, and Raji-Luc cells (5 × 105 cells per mouse) were administered prior to CAR-T20 administration. The survival time, optical intensity of Raji-Luc cells, clinical symptoms, and body mass of the animals were observed. Another 144 male NSG mice were employed to investigate the proliferation and antitumor effects of CAR-T20. Human cytokine and murine cytokines were detected at 1, 7, 14, 21, 28, 42, 56 and 90 days post-CAR-T administration, while biochemistry index analysis, T-cell and CAR-T-cell detection in peripheral blood, and histopathological examination were performed at 14, 28, 56 and 90 days post-administration.ResultsCAR-T20 cells had a specific killing effect on CD20-expressing cells in vitro. At a dose of 1 × 106 per mouse or above, CAR-T20 prolonged the median survival time from 14 days to more than 3 months, inhibited the proliferation of Raji cells in mice, and alleviated the clinical manifestations and weight loss caused by the Raji-Luc cell load. CAR-T20 at a dose of 2 × 106 per mouse or above inhibited the proliferation of Raji cells in mice for up to 111 days post-administration without recurrence. The numbers of T cells and CAR-T cells in the animals administered CAR-T20 increased significantly when Raji cells were markedly proliferated and subsequently decreased when Raji cells were predominantly inhibited. CAR-T20 increased human IFN-γ, murine TNF and murine IL-6 levels and decreased human IL-10 levels in tumor-bearing mice. The incidences of xenografted tumors in organs/tissues were also reduced effectively by CAR-T20.ConclusionThe effective dose of CAR-T20 in mice starts from 1 × 106 per mouse, equivalent to a clinical dose of 5 × 106/kg. Together, our data support the clinical translation of CAR-T20 for R/R B-cell NHL patients.

A-Kinase anchor protein 4 (AKAP4) may be considered as a potential early diagnostic breast cancer marker detectable in blood

PurposeBreast cancer is the most common type of cancer-related death in women worldwide. Early diagnosis via cancer screening biomarkers has a major impact on improving clinical outcomes and overall survival leads to better cancer management. Herein, we examined the expression of A-Kinase Anchor Protein 4 (AKAP4) as a Cancer Testis Antigen (CTA) in breast cancer tumors and traced it in the peripheral blood as a possible diagnostic breast cancer marker. Material and methodsTo evaluate the expression of AKAP4 gene nested real-time RT-PCR method with SYBR green dye were used in the peripheral blood and breast tissues of 80 breast cancer patients in comparison with normal controls. ResultsThe data revealed that the existence of AKAP4 gene expression in all the stages of breast cancer in tumor tissues and its subsequent detection in peripheral blood could be regarded as a possible biomarker for breast cancer diagnosis even at the early stages. The AKAP4 expression was detected somewhat in the normal margin of the tumor compared with normal breast tissue with no AKAP4 expression which indicates the bystander effect. The peripheral blood AKAP4 expression was significantly higher in stage IV, nodal involvement, and distant metastasis. ConclusionAKAP4 gene expression analysis in blood could be regarded as a possible early diagnostic biomarker in breast cancer that is an indispensable requirement for better management of cancer.

Research progress of cross-disease miRNA molecular markers in schizophrenia, bipolar disorder and depression

Micro non-coding RNA (microRNA, miRNA) is a small non-coding RNA involved in gene expression regulation that plays an important role in the onset and development of mental illness. Evidence suggests that several miRNAs are dysregulated in patients with mental illnesses. Because of its stability and quantitative detection in peripheral blood and cerebral fluid, miRNA is a particularly attractive biomarker. The objective of this research is to investigate the relationship between mental illness and miRNAs, as well as the potential processes through which miRNAs contribute to disease etiology. Schizophrenia, bipolar disorder, and depression are three major mental disorders with high disability and mortality. The study explored the particular dysregulated miRNAs for each condition as well as common dysregulated miRNAs across diseases. In this study, which analyzes the findings from relevant studies from 2016 to 2020, the authors discuss the functions of numerous severely dysfunctional miRNAs and their application potential in the field of psychiatry research.

PD-1 improves accurate detection of Sezary cells by flow cytometry in peripheral blood in mycosis fungoides/Sezary syndrome.

Accurate Sezary cell detection in peripheral blood of mycosis fungoides/Sezary syndrome (MF/SS) patients by flow cytometry can be difficult due to overlapping immunophenotypes with normal T cells using standard markers. We assessed the utility of programmed death-1 (PD-1/CD279), a transmembrane protein expressed in some hematopoietic cells, for identification and quantitation of circulating Sezary cells among established markers using flow cytometry. 50 MF/SS and 20 control blood samples were immunophenotyped by flow cytometry. Principal component analysis (PCA) assessed contributions of antigens to separation of abnormal from normal T cell populations. PD-1 was assessed over time in blood and bone marrow of available MF/SS cases. Normal CD4+ T cells showed dim/intermediate to absent PD-1 expression. PD-1 in Sezary cells was informatively brighter (≥1/3 log) than internal normal CD4+ T cells in 39/50 (78%) cases. By PCA, PD-1 ranked 3rd behind CD7 and CD26 in population separation as a whole; it ranked in the top 3 markers in 32/50 (64%) cases and 1st in 4/50 (8%) cases when individual abnormal populations were compared to total normal CD4+ T cells. PD-1 clearly separated Sezary from normal CD4+ T cells in 15/26 (58%, 30% of total) cases with few and subtle alterations of pan-T cell antigens/CD26 and was critical in 6 (12% of total), without which identification and quantification were significantly affected or nearly impossible. PD-1 remained informative in blood/bone marrow over time in most patients. PD-1 significantly contributes to accurate flow cytometric Sezary cell assessment in a routine Sezary panel.

Deep sequencing in CD34+ cells from peripheral blood enables sensitive detection of measurable residual disease in AML

AbstractMonitoring of measurable residual disease (MRD) in patients with acute myeloid leukemia (AML) is predictive of disease recurrence and may identify patients who benefit from treatment intensification. Current MRD techniques rely on multicolor flow cytometry or molecular methods, but are limited in applicability or sensitivity. We evaluated the feasibility of a novel approach for MRD detection in peripheral blood (PB), which combines immunomagnetic preenrichment and fluorescence-activated cell sorting (FACS) for isolation of CD34+ cells with error-reduced targeted next-generation sequencing (NGS). For clinical validation, we retrospectively analyzed 429 PB and 55 bone marrow (BM) samples of 40 patients with AML or high-risk MDS, with/without molecular relapse based on CD34+ donor chimerism (DC), in complete remission after allogeneic stem cell transplantation. Enrichment of CD34+ cells for NGS increased the detection of mutant alleles in PB ∼1000-fold (median variant allele frequency, 1.27% vs 0.0046% in unsorted PB; P < .0001). Although a strong correlation was observed for the parallel analysis of CD34+ PB cells with NGS and DC (r = 0.8601), the combination of FACS and NGS improved sensitivity for MRD detection in dilution experiments ∼10-fold to levels of 10−6. In both assays, MRD detection was superior using PB vs BM for CD34+ enrichment. Importantly, NGS on CD34+ PB cells enabled prediction of molecular relapse with high sensitivity (100%) and specificity (91%), and significantly earlier (median, 48 days; range, 0-281; P = .0011) than by CD34+ DC or NGS of unsorted PB, providing additional time for therapeutic intervention. Moreover, panel sequencing in CD34+ cells allowed for the early assessment of clonal trajectories in hematological complete remission.

Circulating tumor DNA detection in peripheral blood in postoperative efficacy evaluation and recurrence risk prediction of lung cancer.

This study mainly explores the application in the evaluation of postoperative efficacy and recurrence risk prediction. In this study, 50 lung cancer patients treated with thoracic surgery were selected. Collect the peripheral blood of these patients before the operation, separate the blood sample from the plasma by high-speed centrifugal separation, extract the DNA in the plasma by the magnetic bead method, and then extract the ctDNA. A liquid-phase chip capture system is used to highly concentrate target DNA. We use the IlluminaHiseq platform to sort with high throughput and high depth. As an indicator of ctDNA detection and analysis, the AF value of gene mutation frequency is used for calibration. The sensitivity of CEA detection in peripheral blood of tumor patients (TP) is 30% (15/50), and the sensitivity of ctDNA detection is significantly higher than that of CEA detection. Research results show that the number.

Accurate and Sensitive Analysis of Minimal Residual Disease in Acute Myeloid Leukemia Using Deep Sequencing of Single Nucleotide Variations

The prognostic importance of minimal residual disease (MRD) for treatment response evaluation in acute myeloid leukemia (AML) is evident both after conventional chemotherapy and before and after stem cell transplantation. The genetic landscape of AML is characterized by numerous somatic mutations, which constitute potential markers for MRD. We have recently demonstrated that a patient-tailored MRD analysis can be performed through identification of leukemia-specific mutations with exome sequencing at diagnosis, followed by targeted deep sequencing during follow-up (Malmberg, E et al, Eur J Haematol 2017;98:26-37). The development of next generation sequencing based MRD analysis in AML has been hampered by limited sensitivity, mostly due to general sequencing errors as well as sequence-specific errors. The objective of this study was to optimize, validate and clinically test the targeted deep sequencing assay for leukemia-specific substitution mutations (single nucleotide variations, SNVs). By applying a statistical model that minimizes sequencing-specific errors using a reference sample, we achieved a limit of detection (LOD) for SNVs with a variant allele frequency (VAF) of 0.018% (15 SNVs in normal samples). The LOD inversely correlated to the sequencing depth, with 0.018% reached at 5.0x105 reads. Precision, accuracy and linearity were determined using a commercial standard with set levels of mutations (VAF 1 and 8%). The median coefficient of variation (CV) was 4.1% (range 2.2-5.7%, 4 SNVs, n=3) at VAF 1% and 13.3% (8.8-19.4%) at VAF 0.1% with a bias of 7.9% (2.5-15.3%, VAF 1%). At VAF 8%, the CV was 3% and bias 2.8% (1 SNV, n=24). Linearity in the range of MRD (VAF 0.03%-1%) was confirmed for 5 SNVs. The deep sequencing method was applied to a series of 34 bone marrow (BM) samples from 6 children with AML and compared to 8 color multiparameter flow cytometry (MFC) according to guidelines in the study protocol NOPHO-DBH AML-2012, and when applicable to quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Leukemia-specific somatic mutations (2-3/case) were identified in diagnostic samples using exome sequencing of sorted leukemic cells and lymphocytes and then assessed in each sample using deep sequencing. When we compared deep sequencing results with results obtained with MFC, there was a strong correlation (Rs 0.90, p < 0.001). When we dichotomized the results into MRD+ and MRD- results according to each method, 18 of 27 samples (and 53/70 mutations) showed concordant results with 12 samples (31 mutations) MRD+ with both methods, 6 samples (22 mutations) MRD- with both methods. Nine samples (17 mutations) were MRD+ with deep sequencing whilst MRD- with MFC. No samples/mutations were MRD- with deep sequencing but MRD+ with MFC. When comparing results from deep sequencing with results from RT-qPCR (RUNX1-RUNX1T1, KMT2A-MLLT10), 13 of 14 samples showed concordant MRD assignments. To explore the applicability of the method for MRD detection in peripheral blood, we analyzed blood samples from the same time points as MRD+ BM samples (27 mutations in 12 samples, VAF in BM 0.018%-5%). There was a significant correlation between VAF in blood and BM (Rs 0.65, p < 0.001), at least one mutation was detected in 10 out of 12 investigated blood samples and all mutations with VAF>0.1% in BM (19/27) were MRD+ in blood. In conclusion, targeted deep sequencing of single nucleotide variations in recurrently as well as non-recurrently mutated genes enables accurate and precise detection of low levels of residual leukemic cells with a superior sensitivity to MFC. Introduction of this method in the care of patients with AML will allow for sensitive leukemia surveillance in virtually every patient with AML. DisclosuresNo relevant conflicts of interest to declare.

The clinical significance of CTC enrichment by GPC3-IML and its genetic analysis in hepatocellular carcinoma

BackgroundThis research was to develop a special method for enriching Circulating tumor cells (CTCs) of Hepatocellular carcinoma (HCC) by Glypican-3 immunoliposomes (GPC3-IML), and to analyze the correlation between the CTCs count and tumor malignancy, as well as to investigate the mutation characteristics of CTC-derived NGS.ResultsIn this study characterization of physical parameters was performed with the preparation of GPC3-IML. CTCs in peripheral blood of HCC patients were further separated and identified. Immunofluorescence was used to identify CTCs for further counting. By this means, the correlation between CTCs count and clinicopathological features was analyzed, and the genetic mutation characteristics of NGS derived from CTCs were investigated and compared with that of tissue NGS. Results showed that compared with EpCAM and vimentin, GPC-3 had a stronger CTCs separation ability. There was a correlation between "positive" count of CTCs (≥ 5 PV-CTC per 7.5 ml blood) and BCLC stage (P = 0.055). The result of CTC-NGS was consistent with that of tissue-NGS in 60% cases, revealing that KMT2C was a common highly-frequent mutated gene.ConclusionThe combination of immunomagnetic separation of CTCs and anti-tumor marker identification technology can be regarded as a new technology of CTCs detection in peripheral blood of patients with HCC.Trial registration EHBHKY2020-k-024. Registered 17 August 2020—Retrospectively registered

P34.02 Detection of PD-L1 Expression and Its Clinical Significance in Circulating Tumor Cells From Patients With Non-Small-Cell Lung Cancer

The expression of programmed cell death ligand 1(PD-L1) is related to the efficacy of immune checkpoint inhibitors on patients with non-small cell lung cancer (NSCLC), but its clinical tissue samples are difficult to obtain, and initial tumor tissue (TT) samples are difficult to reflect the spatial-temporal heterogeneity of the tumor due to treatment and disease progression. Liquid biopsy plays a crucial role in individualized and precise treatment of tumors. Therefore, we explored the feasibility of separating circulating tumor cells (CTCs) based on size and detecting PD-L1 expression on CTCs, with the goal of laying a foundation for real-time individualized immunotherapy. Peripheral blood specimens were sampled from 66 NSCLC patients, and analyzed prospectively, and CTCs were separated from them by membrane filtration based on size. For 59 patients with paired TT specimens, the expression of PD-L1 in their CTCs and TTs was determined using the immunohistochemistry (IHC) and immunocytochemistry (ICC) based on 28-8 antibody, respectively, and comparatively analyzed. The expression of PD-L1 in TTs was set as a gold standard for calculation of five indexes including sensitivity, specificity, consistency, positive predictive value (PPV), and negative predictive value (NPV), and the Cohen kappa coefficient for CTCs and paired tissues was calculated. In addition, the T-test, Chi-square test, and Mann-Whitney U test were all adopted to analyze the correlation of clinical pathological features and prognosis with PD-L1 expression. The sensitivity and specificity in detecting PD-L1 in CTCs of the 41 initial treated patients were 88.89 % and 73.91%, and PPV and NPV in detecting it were 72.73% and 89.47%, respectively. In addition, the consistency in detecting PD-L1 in CTCs and paired tissues was 89.47%, and the Cohen kappa coefficient was 0.613. The univariate analysis of survival showed that the progression-free survival time of initial treated patients with positive PD-L1 expression in CTCs or TTs was shorter than that of those with negative PD-L1 expression in CTCs or TTs (P>0.05), and the positive PD-L1 expression in CTCs or tissues had nothing to do with age, sex, smoking status, histological type, and stage (all P > 0.05). The CTC PD-L1 detection in peripheral blood in initial treated NSCLC patients is highly consistent with that in paired TTs, which confirms the feasibility of CTC PD-L1 detection in peripheral blood, and lays a certain foundation for exploring real-time and individualized immunotherapy molecular biomarkers based on peripheral blood in the future.

Clinical value of circulating tumor cells for the diagnosis and prognosis of hepatocellular carcinoma (HCC): A systematic review and meta-analysis.

Background:To evaluate the clinical value of circulating tumor cell (CTC) detection in peripheral blood for the diagnosis and prognosis of hepatocellular carcinoma (HCC).Methods:Public databases were searched, and a meta-analysis was performed to determine the specificity, sensitivity, negative- likelihood ratio (NLR) and positive-likelihood ratio (PLR), and diagnostic odds ratio (dOR) of CTC detection for the diagnosis of HCC. Hazard ratios (HRs) and 95% confidence intervals (CIs) were analyzed for the association of CTC detection with overall survival (OS) and HCC recurrence. The Meta-DiSc 1.4 and Review Manager 5.2 software programs were used for statistical analysis.Results:Meta-analysis of 20 studies including 1191 patients showed that the specificity, sensitivity, NLR, PLR, and dOR of CTC testing for HCC diagnosis were 0.60 (95% CI = 0.57–0.63), 0.95 (95%CI = 0.93–0.96), 0.36 (95%CI = 0.28–0.48), 11.64 (95%CI = 5.85–23.14), and 38.94 (95%CI = 18.33–82.75), respectively. Meta-analysis of 18 studies including 1466 patients indicated that the OS of CTC-positive HCC patients was less than that of CTC-negative patients (HR = 2.31; 95% CI = 1.55–3.42; P < .01). Meta-analysis of 5 studies including 339 patients revealed that the presence of CTCs in peripheral blood significantly increased the risk of HCC recurrence (HR = 3.03, 95% CI = 1.89–4.86; P < .01).Conclusion:CTCs in peripheral blood may be a useful marker for HCC diagnosis. In addition, the prognosis of CTC-positive HCC patients was significantly worse than that of CTC-negative HCC patients. Therefore, further studies are warranted to confirm the clinical potential of CTC detection in peripheral blood in patients with primary HCC.

Effect of perfluorocarbon partial liquid ventilation-induced hypothermia on dogs with acute lung injury.

Acute lung injury (ALI) is the damage of alveolar epithelial cells and capillary endothelial cells caused by various direct and indirect injury factors, resulting in diffuse pulmonary interstitial and alveolar edema, resulting in acute hypoxic respiratory insufficiency. This study aimed to investigate the effects of hypothermia induced by partial fluid ventilation on dogs with ALI. The experimental dogs were randomly divided into a conventional mechanical ventilation group (CMV) group, a normal temperature perfluorocarbon liquid ventilation group (NPLV) group, and a hypothermic perfluorocarbon liquid ventilation group (HPLV) group. After induction of ALI, the dogs of the CMV group was treated with CMV for respiratory support, the HPLV group was given a 15 °C low-temperature perfluorocarbon partial liquid ventilation (PLV), and the NPLV group was given partial fluid permeation of perfluorocarbon (PFC) at a room temperature of 37 °C. Anesthesia was stable at 0.5 h (T0), and successful modeling (T1), at 1 h (T2), 2 h (T3), 3 h (T4) and 4 h (T5) was completed. Blood gas analysis was performed, and rectum temperature, peak airway pressure (PIP), and lung compliance were measured. We performed enzyme-linked immunosorbent assay (ELISA) for peripheral blood and postoperative bronchoalveolar lavage fluid (BALF), calculation of lung tissue wet weight/dry weight ratio, and Western blot detection of NF-κB p65. In the HPLV group, the blood gas index of dogs with ALI was close to normal. In T2¬-T5, the rectal temperature of the HPLV group was significantly lower than that of the NPLV group and the CMV group the lung compliance in the HPLV group and the NPLV group was lower than that in the CMV group at the T2-T5 time point, while the CLst value was significantly increased. The detection of peripheral blood and BALF in dogs showed that interleukin-10 (IL-10) was significantly increased and TNF-α was significantly decreased in the HPLV group compared with the CMV group and NPLV group. Compared with CMV group, the wet/dry ratio of lung tissue in the BALF of HPLV group was decreased. The results indicate that mild hypothermia caused by partial fluid ventilation can increase oxygenation capacity, oxygen partial pressure, the expression of anti-inflammatory factor IL-10 and improve lung compliance in dogs with ALI.