Normal Peripheral Blood Samples Research Articles

An age classification model based on DNA methylation biomarkers of aging in human peripheral blood using random forest and artificial neural network.

Recent epigenetic studies have revealed a strong association between DNA methylation and aging and lifespan, which changes (increases or decreases) with age. Based on these, the construction of age prediction models associated with DNA methylation levels can be used to infer biological ages closer to the functional state of the organism. We downloaded methylation data from the Gene Expression Omnibus (GEO) public database for normal peripheral blood samples from people of different ages. We grouped the samples according to age (18-35 years and >50 years), screened the methylation sites that differed between the two groups, identified 44 differentially methylated sites, and subsequently obtained 11 age-related characteristic methylation sites using the random forest method. Then, we constructed an age classification model with these 11 characteristic methylation sites using an artificial neural network and evaluated its efficacy. The age classification model was constructed by an artificial neural network and its efficacy was evaluated. The model predicted an area under the curve (AUC) of 0.97 in the validation set and accurately distinguished between those aged 18-35 and >50 years. Furthermore, the levels of these 11 characteristic methylation sites also differed significantly between the two sets of samples in the validation set, including six newly identified age-related methylation sites (P<0.001). Finally, we constructed a multifactor regulatory network based on the corresponding genes of age-related methylation sites to reveal the transcriptional and post-transcriptional regulation patterns. As a result of the increasing problem of aging, the age classification model we constructed allows us to accurately distinguish different age groups at the molecular level, which will be more predictive than chronological age for assessing individual aging and future health status.

Prognostic value and potential mechanism of long non-coding RNA Lnc-SMIM20-1 in acute myeloid leukemia

ABSTRACT Objectives Acute myeloid leukemia (AML) is a common hematologic malignancy with high heterogeneity and poor prognosis. Although long non-coding RNAs (lncRNAs) have been used as biomarkers for tumors, the clinical relevance of numerous lncRNAs in AML remains to be investigated. Research design and methods Differentially expressed lncRNAs between AML and normal peripheral blood samples were identified using DESeq2. Pan-cancer analysis was performed by GEPIA tool. Kaplan–Meier survival curve was applied for prognosis analysis. KEGG pathway analysis and GSEA were used for functional enrichment. The ceRNA network was constructed by GDCRNAtools. Results Lnc-SMIM20-1 was most highly expressed in AML and up-regulated in the TCGA-AML cohort compared to normal tissues. Patients with high expression of Lnc-SMIM20-1 had poor overall prognosis both in the TCGA adult AML cohort and the TARGET pediatric AML cohort, no matter whether they were treated with chemotherapy or allo-HSCT. Lnc-SMIM20-1 might participate in cancer-associated signaling pathways and immune-related signaling pathways by interacting with four microRNAs and 20 mRNAs. Conclusion Lnc-SMIM20-1 was up-regulated in AML acting as a stable poor prognostic factor. The prognostic impact of Lnc-SMIM20-1 cannot be overcome by allo-HSCT. Our findings provide insight into the clinical relevance of Lnc-SMIM20-1 in AML; aiming to progress the development of novel therapeutics.

Identification of autophagy-related genes as potential biomarkers for type 1 diabetes mellitus.

BackgroundType 1 diabetes mellitus (T1DM) is a metabolic disease in which the autoimmune destruction of pancreatic islet β-cells occurs. This study sought to investigate the role of autophagy-related genes and immune cells in the development of T1DM.MethodsWe acquired the raw gene expression profiles of 302 T1DM and 422 normal control peripheral blood samples from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified using the Limma package, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. The Search Tool for the Retrieval of Interacting Genes/Proteins (https://string-db.org/) and Cytoscape autophagy genes were intersected with the DEGs for the immune cell analysis and the correlation analysis.ResultsA total of 568 DEGs were identified in the T1DM and normal samples, of which 301 were upregulated and 267 were downregulated. The results of the functional and pathway enrichment analyses showed that the DEGs were closely associated with autophagy and immunity. Member RAS oncogene family (RAB11A), protein tyrosine phosphatase non-receptor type 11, lamin A/C, heat shock protein70, heat shock protein family A member 4, cluster of differentiation 8A, caspase 3 (CASP3), exportin 1, proto-oncogene, non-receptor tyrosine kinase, SMAD family member 4, and sirtuin 1 (SIRT1) were located at the center of the protein-protein interaction network as the core genes. The peripheral blood T cells were more elevated in the T1DM subjects than the normal subjects. RAB11A, CASP3, and SIRT1 are autophagy-associated genes. RAB11A and CASP3 were positively correlated with most immune cells, while SIRT1 was negatively correlated with most immune cells.ConclusionsAutophagy-related genes (i.e., RAB11A, CASP3, and SIRT1) and immune cells (i.e., T and B cells) may play important regulatory roles in the development of T1DM. Our findings provide novel insights into and potential targets for T1DM prediction and treatment.

Overexpression of PD-1 and CD39 in tumor-infiltrating lymphocytes compared with peripheral blood lymphocytes in triple-negative breast cancer.

Growing evidence highlighted the primary role of the immune system in the disease course of triple-negative breast cancer (TNBC). The study aim was to investigate the expression of PD-1 and CD39 on CD4+ and CD8+ cells infiltrating tumor tissue compared to their counterparts in peripheral blood and explore its association with tumor characteristics, disease progression, and prognosis in females with TNBC. The study included 30 TNBC patients and 20 healthy controls. Cancer and normal breast tissue and peripheral blood samples were collected for evaluation of the expression of PD-1 and CD39 on CD4+ and CD8+T cells by flow cytometry. A marked reduction in the percentage of CD8+ T lymphocytes and a significant increase in the frequencies of CD4+ T lymphocytes and CD4+ and CD8+ T lymphocytes expressing PD1 and CD39 were evident in tumor tissue in comparison with the normal breast tissue. The DFS was inversely related to the cancer tissue PD1+CD8+ and CD39+CD8+ T lymphocytes. Almost all studied cells were significantly increased in the tumor tissue than in peripheral blood. Positive correlations were detected among peripheral PD1+CD4+T lymphocytes and each of cancer tissue PD1+CD4+, PD1+CD8+and CD39+CD8+T cells, and among peripheral and cancer tissue CD39+CD4+and CD39+CD8+ T cells. The CD39 and PD1 inhibitory pathways are synergistically utilized by TNBC cells to evade host immune response causing poor survival. Hence, combinational immunotherapy blocking these pathways might be a promising treatment strategy in this type of cancer.

Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB, n = 41) and bone marrow (BM; n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with variations between different laboratories. Manual analysis and AGI analysis of normal and leukemic samples showed high levels of correlation between cell numbers (r2 > 0.95 for all cell types in PB and r2 > 0.75 in BM) and resulted in highly concordant classification of leukemic cells by our previously published automated database-guided expert-supervised orientation tool for immunophenotypic diagnosis and classification of acute leukemia (Compass tool). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a “sample-in and result-out” approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal procedures.

Plasma amyloid‐β dynamics are regulated by the sleep‐wake cycle

AbstractBackgroundIt has been reported that sleep is an important physiological process and conducive to the elimination of brain metabolites and the recovery of brain function. Growing evidence suggested that sleep disorders were related to increased risk of AD and Aβ accumulation in the brain. Although peripheral Aβ clearance is associated with brain Aβ deposition, but the relationship between sleep‐wake cycle and plasma Aβ levels is unclear.MethodThirty cognitively normal healthy adults (aged 29.07 ± 8.12), sixteen males (53.33%), were recruited. Participants underwent normal sleep and peripheral blood samples were collected every three hours between 20:00 and 8:00 the next day. Plasma Aβ40, Aβ42, total tau, phosphorylated tau 181 (P‐tau), soluble low‐density lipoprotein receptor‐related protein (sLRP1) and soluble receptors for advanced glycation end products (sRAGE) levels were measured using ELISA kits.ResultPlasma Aβ40 concentration at 5:00 decreased significantly compared with plasma level at 23:00 (p=0.041), and there is a dynamic tendency of decreasing after sleep onset and increasing after awake, which is in accordance with the sleep‐wake cycle. Furthermore, Aβ40 and Aβ42 levels were positively correlated with plasma sLRP1 (Aβ40: r=0.1791, p=0.0283; Aβ42: r=0.1780, p=0.0293). However, there was no significant dynamic change in Aβ42, Tau, P‐tau, Tau, sLRP1 and sRAGE levels in twelve hours.ConclusionPlasma Aβ40 dynamics are regulated by the sleep‐wake cycle, and it may play a positive feedback regulating role in sLRP1 level.

Diagnostic Validation of a Clinical Laboratory-Oriented Targeted RNA Sequencing System As a Comprehensive Assay for Hematologic Malignancies

Introduction Targeted RNA sequencing (RNA-seq) is a highly accurate method for sequencing transcripts of interest and can overcome limitations regarding resolution, throughput, and multistep workflow. However, RNA-seq has not been widely performed in clinical molecular laboratories due to the complexity of data processing and interpretation. We developed a customized targeted RNA-seq panel with a data processing protocol and validated its analytical performance for gene fusion detection using a subset of samples with different hematologic malignancies. Additionally, we investigated its applicability for identifying transcript variants and expression analysis using the targeted panel. Methods The target panel and customized oligonucleotide probes were designed to capture 84 genes associated with hematologic malignancies. Libraries were prepared from 800 to 1,500 ng of total RNA using GeneMediKit NGS-Leukemia-RNA kit (GeneMedica, Gwangju, Korea) and sequenced using Miseq reagent kit v3 (300 cycles) and MiseqDx (Illumina, San Diego, CA, USA). The diagnostic samples included one reference DNA (NA12878), one reference RNA (Cat no. 740000, Agilent Technologies), 14 normal peripheral blood (PB) samples, four validation bone marrow (BM) samples with known gene fusions, and 30 clinical BM or PB samples from seven categories of hematologic malignancies. The clinical samples included 27 BM aspirates and three PB samples composed of six acute myeloid leukemia, nine B-lymphoblastic leukemia/lymphoma, four T-lymphoblastic leukemia/lymphoma, three mature B-cell neoplasms, six MPN, one myelodysplastic/myeloproliferative neoplasm, and one myeloid/lymphoid neoplasm with eosinophilia and gene rearrangement. For the analytical validation of fusion detection, target gene coverage, between-run and within-run repeatability, and dilution tests (1:2 to 1:8 dilution) were performed. For the comparative analysis of fusion detection, the RNA-seq data were analyzed by STAR-Fusion and FusionCatcher and processed with stepwise filtering and prioritization strategy (Figure 1), and the result was compared to those of multiplex RT-PCR (HemaVision kit; DNA Technology, Aarhus, Denmark) or FISH (MetaSystems Gmbh, Althusseim, Germany) using 30 clinical samples. The RNA-seq data from clinical samples were additionally analyzed by FreeBayes for variant detection and by StringTie for expression profiling (Figure 1). Results First, the analytical validation showed reliable results in target gene coverage, between-run and within-run repeatability, and linearity tests. The uniformity of coverage (% of base pairs higher than 0.2 × total average depth) was calculated to be 99.8%, which revealed even coverage for the target genes in the panel using the reference DNA. Both in the within-run and between-run tests, the read counts and FFPM (fusion fragments per million) of all replicates showed reliable repeatability (r2 = 0.9655 and 0.9874, respectively). The FFPM of the diluted analytical samples including BCR-ABL1 and PML-RARA showed linear log2-fold-changes (r2 = 0.9852 and 0.9447, respectively). Second, compared to multiplex RT-PCR and FISH using 30 clinical samples, targeted RNA-seq combined with filtering and prioritization strategies detected all 13 known fusions and newly detected 17 fusions. Finally, 16 disease- and drug resistance-associated variants on the expressed transcripts of ABL1, GATA2, IKZF1, JAK2, RUNX1, and WT1 were simultaneously designated and expression analysis showed distinct four clusters of clinical samples according to the cancer subtypes and lineages. Conclusions Our customized targeted RNA-seq system provided a stable analytical performance and a more sensitive identification of gene fusions than conventional molecular methods in various clinical samples. In addition, clinically significant variants in the transcripts and expression profiling could be simultaneously identified directly from the RNA-seq data without the need for additional parallel testing. Our study identified the advantages of the clinical laboratory-oriented targeted RNA-seq system to enhance the diagnostic yield for gene fusion detection and to simplify the diagnostic steps as providing a comprehensive tool for analyzing hematologic malignancies in the clinical laboratory. Figure 1 Disclosures Lee: National Research Foundation of Korea: Research Funding.

Abstract 94: Pla2g4a: A new cell cycle related gene in patients with non-M3/NPM1 wildtype acute myeloid leukemia

Abstract In this study, we aimed to explore and validate the prognostic value of PLA2G4A expression in patients with non-M3/NPM1 wildtype (WT) acute myeloid leukemia (AML) using two independent datasets. Data from the Cancer Genome Atlas-Acute Myeloid Leukemia (TCGA-LAML) and the Therapeutically Applicable Research to Generate Effective Treatments (TARGET)-AML were used to assess the prognostic value PLA2G4A in NPM1-WT AML cases. A single-cell RNA-seq dataset that included 96 AML cells from a recurrent AML case was used to check the correlation between PLA2G4A expression and the functional state of AML cells. Results showed that non-M3 AML cases had significantly increased PLA2G4A expression compared to normal peripheral blood samples. Multivariate analysis showed that high PLA2G4A expression was independently associated with shorter OS in 97 non-M3/NPM1-WT AML cases in TCGA-LAML (HR: 1.932, 95%CI: 1.092-3.419, p=0.024). The prognostic value was validated based on 128 non-M3/NPM1-WT AML cases in TARGET-AML (HR: 1.894, 95%CI: 1.065-3.369, p=0.030). Single-cell RNA-seq data suggested that PLA2G4A expression was positively correlated with cell cycle progression of AML cells. Therefore, PLA2G4A expression might serve as an independent prognostic marker in OS in patients with non-M3/NPM1 WT AML. Its upregulation might confer malignant phenotype of AML cells at least partly by promoting cell cycle progression. Future molecular studies are required to explore other molecular pathways involved. Citation Format: Hansong Bai, Zeng Ming. Pla2g4a: A new cell cycle related gene in patients with non-M3/NPM1 wildtype acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 94.

PLA2G4A Is a Potential Biomarker Predicting Shorter Overall Survival in Patients with Non-M3/NPM1 Wildtype Acute Myeloid Leukemia.

In this study, we aimed at exploring and validating the prognostic value of PLA2G4A expression in patients with non-M3/nucleophosmin (NPM1) wildtype (WT) acute myeloid leukemia (AML) by using two independent datasets. Data from the Cancer Genome Atlas-acute myeloid leukemia (TCGA-LAML) and the therapeutically applicable research to generate effective treatments (TARGET)-AML were used to assess the prognostic value of PLA2G4A in NPM1-WT AML cases. Results showed that non-M3 AML cases had significantly increased PLA2G4A expression compared with normal peripheral blood samples. Patients with high PLA2G4A expression (separated by median gene expression) had a significantly shorter overall survival (OS) compared with the group with low PLA2G4A expression, in both TCGA-LAML and TARGET-AML. Multivariate analysis showed that high PLA2G4A expression was independently associated with shorter OS in 97 non-M3/NPM1-WT AML cases in TCGA-LAML (hazard ratio [HR]: 1.946, 95% confidence interval [CI]: 1.094-3.462, q = 0.036). The prognostic value was validated based on 120 primary non-M3/NPM1-WT AML cases in TARGET-AML (HR: 1.518, 95% CI: 1.037-2.223, q = 0.048). Therefore, PLA2G4A expression might serve as an independent prognostic marker in OS in patients with non-M3/NPM1 WT AML. Bioinformatic analysis identified that several proteins physically interacted with PLA2G4A, some of which have well-characterized oncogenic properties in AML, such as RUVBL2, cytoskeleton regulatory protein 1 (CAP1), signal transducer and activator of transcription 3 (STAT3), and MYCBP. Therefore, we hypothesized that PLA2G4A upregulation has multiple effects on the malignant phenotype of AML cells together with its partners. Future molecular studies are required to explore the detailed regulatory network involved.

The Integrated Transcriptome Bioinformatics Analysis Identifies Key Genes and Cellular Components for Spinal Cord Injury-Related Neuropathic Pain.

BackgroundSpinal cord injury (SCI) is one of the most devastating diseases with a high incidence rate around the world. SCI-related neuropathic pain (NeP) is a common complication, whereas its pathomechanism is still unclear. The purpose of this study is to identify key genes and cellular components for SCI-related NeP by an integrated transcriptome bioinformatics analysis.MethodsThe gene expression profile of 25 peripheral blood samples from chronic phase SCI patients (E-GEOD-69901) and 337 normal peripheral blood samples were downloaded from ArrayExpress and Genotype-Tissue Expression Portal (GTEx), respectively. A total of 3,368 normal peripheral blood mononuclear cells (PBMC) were download from Sequence Read Archive (SRA713577). Non-parametric tests were used to evaluate the association between all of differential expression genes (DEGs) and SCI-related NeP. CellPhoneDB algorithm was performed to identify the ligand–receptor interactions and their cellular localization among single PBMCs. Transcription factor (TF) enrichment analysis and Gene Set Variation Analysis (GSVA) were used to identify the potential upstream regulatory TFs and downstream signaling pathways, respectively. Co-expression analysis among significantly enriched TFs, key cellular communication genes and differentially expressed signaling pathways were performed to identify key genes and cellular components for SCI-related NeP.ResultsA total of 2,314 genes were identified as DEGs between the experimental and the control group. Five proteins (ADRB2, LGALS9, PECAM1, HAVCR2, LRP1) were identified in the overlap of proteins in the significant ligand-receptor interactions of PBMCs and protein-protein interaction (PPI) network based on the DEGs. Only HAVCR2 was significantly associated with NeP (P = 0.005). Besides, the co-expression analysis revealed that TF YY1 had significantly co-expression pattern with cellular communication receptor HAVCR2 (R = −0.54, P < 0.001) in NK cells while HAVCR2 was also co-expressed with mTOR signaling pathway (R = 0.57, P < 0.001). The results of RT-qPCR and external dataset validation supported the signaling axis with the most significant co-expression patterns.ConclusionIn peripheral blood of chronic SCI, HAVCR2 might act as a key receptor on the surface of NK cells and interact with ligand LGALS9 secreted by CD14+ monocytes, inhibiting NK cells through mTOR signaling pathway and ultimately predicting the occurrence of SCI-related NeP. This hypothetical signaling axis may provide prognostic biomarkers and therapeutic targets for SCI-related NeP.

Effects of interleukin-35 on the balance of helper T cell 17/regulatory T cell in peripheral blood of patients with oral lichen planus

Objective: To investigate the effects of exogenous interleukin (IL)-35 on the balance of helper T cell 17 (Th17) and regulatory T cell (Treg) in peripheral blood of patients with oral lichen planus (OLP). Methods: Totally 12 peripheral blood samples of OLP patients (OLP group, one male and 11 female, 26-68 years old; four cases of reticular OLP and eight cases of erosive OLP) were collected from patients of Department of Oral Mucosal Specialist of the Affiliated Hospital of Guizhou Medical University from October to December 2016. During the same period, thirteen normal peripheral blood samples were collected from the Physical Examination Center of the Affiliated Hospital of Guizhou Medical University (normal control group, one male and 12 female, 20-68 years old). The peripheral blood mononuclear cells (PBMC) were extracted in sterile condition and CD4+ T cells were sorted by flow cytometry (FCM). Quantitative real-time PCR (qPCR) technique was used to detect the mRNA expression levels of retinoid-related orphan nuclear γt (RORγt) and forkhead box3 (Foxp3). The CD4+ T cells were divided into experimental group and control group. The CD4+ T cells of experimental group were cultured in vitro by adding rhIL-35, and the CD4+ T cells of control group were cultured with the same volume of phosphate buffered saline (PBS). After the completion of the culture, the cells were collected. The expression levels of the same factors were detected by qPCR. Results: The expression [M(Q(25), Q(75))] of Foxp3 [0.15 (0.09, 0.30)] and RORγt mRNA [1.04 (0.45, 2.15)] in the CD4+ T cells of OLP were significantly higher than those in normal control group [0.04 (0.02, 0.06), 0.10 (0.05, 0.11)] (Z=-4.134, P<0.01; Z=-3.699, P<0.01). The ratio of ROR γt/Foxp3 mRNA in OLP group [6.22(3.67, 15.34)] was higher than that in normal control group [2.50 (1.24, 5.23)] (Z=-2.665, P=0.007). In the CD4+ T cells of OLP patients, the expression of Foxp3 mRNA in the experiment group [0.40 (0.21, 1.22)] was higher than that in the control group [0.15 (0.11, 0.26)](Z=-2.510, P=0.012), and the expression of ROR γt mRNA between two groups showed no significant difference (P>0.05). The ROR γt/Foxp3 mRNA ratio [3.44 (1.55, 8.16)] of the experiment group was lower than that in the control group [6.22 (4.43, 12.21)] (Z=-2.746, P=0.006). Conclusions: There was a Th17/Treg imbalance with predominated by Th17 cells in the peripheral blood of patients with OLP. Exogenous rhIL-35 had an immunomodulatory effect on the balance of Th17/Treg.

A Potential Tumor Suppressor Gene Named miR-508-5p Inhibited the Proliferation and Invasion of Human Melanoma Cells by Targeting KIT

Melanoma is the main death cause of human skin cancer. Increasing evidences demonstrate that microRNAs act as key roles in mediating tumor occurrence and progression. MiR-508-5p has proved to participate in the development of various types of human malignancies. However, the role of miR-508-5p in melanoma remained unclear. In in vitro study, miR-508-5p level in peripheral blood samples of patients with melanoma and human melanoma A375 cells was downregulated compared to that in normal peripheral blood samples or normal human epidermal melanocytes (MHEM). MiR-508-5p overexpression significantly inhibited the cell proliferation, migration and invasion in A375 cells, and thus inhibiting KIT expression at both gene and protein levels. Furthermore, western blot analysis showed miR-508-5p reduced cell proliferation by targeting KIT to modulate RAS/RAF/MEK/ERK pathway. Taken together, we speculated that miR-508-5p functioned as an important suppressor in human melanoma by targeting KIT, suggesting miR-508-5p might be a promising tumor suppressor gene for further target therapies from bench to clinic.

The impact of delayed sample handling and type of anticoagulant on the interpretation of dysplastic signs detected by flow cytometry.

IntroductionA growing body of evidence supports the usefulness of dysplastic signs detected by flow cytometry in the diagnosis of myelodysplastic syndromes (MDS). Our aim was to assess the impact of pre-analytical variables (delayed sample handling, type of anticoagulant, and different clones of antibody) in the interpretation of flow cytometric results.Material and methodsBone marrow samples were labelled and analysed immediately after aspiration and on two consecutive days. The effect of anticoagulant type was evaluated in 16 bone marrow samples. Thirty-seven different immunophenotypic variables were recorded after eight-colour staining. Furthermore, 8 normal peripheral blood samples collected in K3-EDTA and Na-heparin were examined with different clones of CD11b antibodies and four parameters were recorded with both anticoagulants on two consecutive days.ResultsFourteen significant differences were detected in the initial immunophenotype of fresh samples collected in K3-EDTA and Na-heparin. Regardless of the anticoagulant type, eleven parameters remained stable despite delayed sample handling. Due to delayed sample processing, more alterations were detected in the samples collected in K3-EDTA than in the samples collected in Na-heparin. The type of CD11b clone influenced the reduction of fluorescence intensity only in samples collected in K3-EDTA, where the alterations were contrary to the changes observed in Na-heparin.ConclusionsDelayed sample processing causes considerable immunohenotypic alterations, which can lead to false interpretation of the results. If delayed sample evaluation is unavoidable, markers that remain more stable over time should be considered with more weight in the diagnosis of MDS.

Exploring the Molecular Mechanism of Thoracic Aortic Aneurysm via Bioinformatics Analysis.

BackgroundThe aim of this study was to identify some key genes related to the pathogenesis of thoracic aortic aneurysm (TAA) and gain more insights to the molecular mechanism of TAA.Material/MethodsThe expression profile of GSE9106 was downloaded from the Gene Expression Omnibus (GEO) database. The data contained 58 TAA peripheral blood samples and 36 normal peripheral blood samples. The differently expressed genes (DEGs) between the TAA samples and the normal samples were identified via limma package of R. Functional enrichment analysis of the DEGs were performed via the Database for Annotation, Visualization and Integrated Discovery (DAVID). The differentially co-expressed genes in TAA samples compared to normal samples were identified via the DCGL package in R. The protein-protein interaction (PPI) network of the DEGs was constructed through the Search Tool for the Retrieval of Interacting Proteins (STARING) database and visualized by Cytoscape software.ResultsA total of 407 DEGs were obtained in TAA samples compared with normal samples. The DEGs were enriched in 29 Gene Ontology (GO) terms. There were 1,441 co-expression gene pairs that had significant changes in the co-expression status in TAA samples compared with normal samples and a differential co-expression network was constructed based on them. Moreover, a PPI network of the DEGs was constructed, containing 101 nodes.ConclusionsBioinformatics methods could identify significant biological processes and genes related to TAA. KRTDAP, BICD1, and genes in the OR family might play an important role in TAA.

A pilot study on the usefulness of peripheral blood flow cytometry for the diagnosis of lower risk myelodysplastic syndromes: the \u201cMDS thermometer\u201d

BackgroundImmunophenotypic analysis of the bone marrow (BM) cells has proven to be helpful in the diagnosis of Myelodysplastic Syndromes (MDS). However, the usefulness of flow cytometry (FCM) for the detection of myelodysplasia in the peripheral blood (PB) still needs to be investigated. The aim of this pilot study was to evaluate the value of FCM-based PB neutrophil and monocyte immunophenotyping for the diagnosis of lower risk MDS (LR-MDS).MethodsWe evaluated by 8-color FCM the expression of multiple cell surface molecules (CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD34, CD45, CD56, CD64 and HLA-DR) in PB neutrophils and monocytes from a series of 14 adult LR-MDS patients versus 14 normal individuals.ResultsPeripheral blood neutrophils from patients with LR-MDS frequently had low forward scatter (FSC) and side scatter (SSC) values and low levels of CD11b, CD11c, CD10, CD16, CD13 and CD45 expression, in that order, as compared to normal neutrophils. In addition, patients with LR-MDS commonly display a higher fraction of CD14+CD56+ and a lower fraction of CD14+CD16+ monocytes in the PB. Based on these results, we proposed an immunophenotyping score based on which PB samples from patients with LR-MDS could be distinguished from normal PB samples with a sensitivity 93% and a specificity of 100%. In addition, we used this score to construct the MDS Thermometer, a screening tool for detection and monitoring of MDS in the PB in clinical practice.ConclusionsPeripheral blood neutrophil and monocyte immunophenotyping provide useful information for the diagnosis of LR-MDS, as a complement to cytomorphology. If validated by subsequent studies in larger series of MDS patients and extended to non-MDS patients with cytopenias, our findings may improve the diagnostic assessment and avoid invasive procedures in selected groups of MDS patients.

Identification of molecular alterations in leukocytes from gene expression profiles of peripheral whole blood of Alzheimer\u2019s disease

Blood-based test has been considered as a promising way to diagnose and study Alzheimer’s disease (AD). However, the changed proportions of the leukocytes under disease states could confound the aberrant expression signals observed in mixed-cell blood samples. We have previously proposed a method, Ref-REO, to detect the leukocyte specific expression alterations from mixed-cell blood samples. In this study, by applying Ref-REO, we detect 42 and 45 differentially expressed genes (DEGs) between AD and normal peripheral whole blood (PWB) samples in two datasets, respectively. These DEGs are mainly associated with AD-associated functions such as Wnt signaling pathways and mitochondrion dysfunctions. They are also reproducible in AD brain tissue, and tend to interact with the reported AD-associated biomarkers and overlap with targets of AD-associated PWB miRNAs. Moreover, they are closely associated with aging and have severer expression alterations in the younger adults with AD. Finally, diagnostic signatures are constructed from these leukocyte specific alterations, whose area under the curve (AUC) for predicting AD is higher than 0.73 in the two AD PWB datasets. In conclusion, gene expression alterations in leukocytes could be extracted from AD PWB samples, which are closely associated with AD progression, and used as a diagnostic signature of AD.

Abstract 4982: Regression of metastatic breast cancer after adoptive cell transfer of tumor infiltrating lymphocytes and checkpoint blockade

Abstract Background: Adoptive transfer of tumor infiltrating lymphocytes (TIL) can effect long-term durable regression in patients with metastatic melanoma but has not been widely tested in common epithelial cancers. Similar to metastatic melanoma, recent studies on epithelial GI cancers report that tumor regressions can be mediated by adoptively transferred TIL recognizing non-synonymous somatic mutations. Breast cancer metastatic tumors are infiltrated by TIL and reactivity against autologous somatic mutations can be identified. Methods: TIL were grown from a metastatic soft tissue tumor of a patient with hormone and chemotherapy-refractory ER+, HER2- breast cancer. DNA was extracted from tumor and matched normal peripheral blood samples for whole exome sequencing (WES) and RNAseq. Non-synonymous somatic mutations were identified and tested for potential recognition by autologous TIL using previously described tandem mini-gene and long peptide approaches. Recognition was assessed by IFN-γ release on ELISPOT and/or CD137 (4-1BB) upregulation with appropriate controls. Mutation reactive TIL were rapidly expanded in culture and transferred back to the patient. Adoptive cell transfer was preceded by a lymphodepleting preparative regimen and one dose of pembrolizumab. Cells were supported post-transfer with intravenous IL-2 administration, and pembrolizumab was administered for three additional doses. Deep sequencing of TCR rearrangement sequences was performed on the TIL and the pre- and post-infusion peripheral blood. Results: The resected subcutaneous tumor no longer expressed ER and was negative for PD-L1 staining. WES/RNASeq identified 96 non-synonymous mutations for testing. Multiple CD4+ clones specifically recognizing a mutation expressed in the solute carrier SLC3A2 (p.K94T) and a single CD8+ clone recognizing a mutation in the proteasome-associated protein KIAA0368 (p.S186F) were identified with no recognition of wild-type peptides. These eight T cell clones constituted 23% of the infusion bag TIL. Nine months after transfer of 82x109 cells, the patient has an ongoing partial response, with target lesions down 96% from baseline, including multiple hepatic metastases and disabling brachial plexus adenopathy. Seven of the eight T cell clones with known reactivity persisted in the peripheral blood comprising 2.4% of all CDR3 sequences at 7 months. Only two of these TCR clones were detectable, with frequencies totaling 0.005%, in the pre-treatment circulation. Conclusions: Tumor-infiltrating lymphocytes derived from a patient with metastatic breast cancer recognized tumor-specific non-synonymous somatic mutations, expanded in vitro and were used for adoptive cell transfer. TIL, co-administered with IL-2 and a short course of pembrolizumab, persisted in vivo and mediated regression of soft tissue, nodal and visceral metastases of breast cancer. Citation Format: Nikolaos Zacharakis, Katarzyna Trebska-McGowan, Robert Somerville, Yong-Chen Lu, Anna Pasetto, Mary Black, Harshini Chinnasamy, Hui Xu, Jared J. Gartner, Todd D. Prickett, Paul F. Robbins, Steven A. Rosenberg, Stephanie L. Goff, Steven A. Feldman. Regression of metastatic breast cancer after adoptive cell transfer of tumor infiltrating lymphocytes and checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4982. doi:10.1158/1538-7445.AM2017-4982

Abstract P2-04-02: Recognition of autologous neoantigens by tumor infiltrating lymphocytes derived from breast cancer metastases

Abstract Background &amp; Translational Relevance: Adoptive transfer of tumor infiltrating lymphocytes (TIL) can effect long-term durable regression in patients with metastatic melanoma but has not been widely tested in common epithelial cancers. When examining the TIL of successfully treated patients with melanoma, a heterogeneous T cell population can be identified with reactivity against melanoma differentiation antigens, cancer germline antigens, and personalized non-synonymous somatic mutations. Common epithelial cancers, including breast cancer, express far fewer somatic mutations than melanoma, however, a patient with metastatic cholangiocarcinoma was treated with autologous CD4+ TIL enriched for neoantigen specificity and has experienced an ongoing partial response (&amp;gt;2 years). It is known that the presence of TIL on pathologic examination of triple-negative breast cancers is a positive prognostic marker for disease-free survival and overall survival. The identification of enriched populations of neoantigen-specific TIL could form the basis for personalized cell therapy for patients with metastatic breast cancer. This pilot study investigates the ability to grow TIL from breast cancer metastases, to identify personalized non-synonymous somatic mutations and potential neoantigens, and to adoptively transfer TIL into patients with breast cancer. Methods: Eligible patients were evaluated and treated under IRB-approved protocols for tissue procurement, genomic testing, and adoptive cell transfer. Portions of resected tumors were placed in culture under standard TIL conditions. DNA was extracted from tumor and matched normal peripheral blood samples for whole exome sequencing (WES). Non-synonymous somatic mutations were identified and tested for potential immunogenicity. Results: Nine patients have undergone surgical resection in this ongoing pilot study, and TIL was successfully grown from the tumors of all patients. All were primarily CD3+ (median 79%) with a small population of natural killer cells. Of the CD3+ cells, 7 of 9 patients had a predominantly CD4+ population (median CD4:CD8 ratio 2.2, range 0.4-5.8). With the exception of a single patient with inflammatory breast cancer, tumor purity allowed for WES of the tumors of eight patients, and non-synonymous somatic mutations were identified as potential neoantigens (median 96.5, range 71-148). Autologous T cell reactivity has been identified against tumor-specific mutations in 4 of 6 patients studied. The TIL of one patient demonstrated in vitro reactivity to a mutated form of RBPJ, a DNA-binding protein involved in Notch1 signaling. In addition, specimens obtained from this patient at autopsy contained the specific RBPJ mutation (RBPJ c.A611T) in every sampled tumor (n=16). Other patient-specific neoantigens identified by autologous reactivity include SLC3A2, KIAA0368, and a mutated TCRBV domain. Conclusions: Tumor-infiltrating lymphocytes derived from metastatic breast cancer can react to tumor-specific non-synonymous somatic mutations in vitro. TIL grown from breast cancers are predominantly CD4+ and can form the basis of an adoptive cell transfer experimental approach to patients with metastatic breast cancer. Citation Format: Goff SL, Zacharakis N, Assadipour Y, Prickett T, Gartner J, Somerville R, Black MA, Xu H, Chinnasamy H, Kriley I, Lu L, Statler M, Wunderlich J, Robbins PF, Rosenberg SA, Feldman SA. Recognition of autologous neoantigens by tumor infiltrating lymphocytes derived from breast cancer metastases [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-02.

Immunoprofiling of Normal Human Donor Blood Identified Potential Pharmacodynamic Markers for JNJ-63709178 (CD123xCD3) Duobody® Antibody Treatment

BackgroundJNJ-63709178 (CD123xCD3) is a promising new bispecific antibody currently being investigated for the treatment for acute myeloid leukemia (AML), a disorder of early hematopoietic progenitor cells characterized by the proliferation and accumulation of immature, clonal, myeloid cells. Constitutive overexpression of the α-subunit of the interleukin-3 receptor (ie, IL-3αR/CD123) is a hallmark of the early AML progenitor cell population (ie, leukemic stem cells [LSC]), as well as leukemic blasts. CD123 is unique to the IL-3 receptor and is responsible for the high specificity and low affinity binding of IL-3. Under physiological conditions, activation of the IL-3 receptor induces proliferative, anti-apoptotic, and differentiating signals. JNJ-63709178, a humanized IgG4 bispecific DuoBody® antibody with high affinity anti-CD123 and -CD3 arms, recognizes CD123 on myeloid cells and CD3 on T cells, leading to T-cell activation, with subsequent lysis of CD123+ AML blasts. JNJ-63709178 induces potent tumor cell killing in vitro, ex vivo, and in murine AML models, and is currently being evaluated in first-in-human clinical trials. Although CD123 is distinctively expressed on leukemic cells, some populations of normal myeloid cells have been reported to express CD123. The goals of this study were to determine CD123 receptor density on various leukocyte populations in normal human peripheral blood and evaluate the effects of JNJ-63709178 on those cells to identify potential pharmacodynamic markers.MethodsPeripheral blood samples from 6 normal human donors were analyzed ex vivo for CD123 expression on various leukocyte populations to determine receptor density. Receptor density on cells was quantified using Quantibrite PE enumeration kit. Subsequently, blood samples were treated with escalating concentrations of JNJ-63709178 (CD123xCD3) or the control compound CNTO 7008 (nullxCD3) DuoBody® antibodies and incubated at 37°C for 48 hours. After incubation, blood samples were stained and processed for analysis of T-cell activation (CD25 expression) and population persistence or depletion (frequencies within leukocytes). In the myeloid compartment, basophils, myeloid dendritic cells (mDCs), monocytes, neutrophils, and eosinophils were analyzed. In the lymphoid compartment, plasmacytoid dendritic cells (pDCs), B cells (naïve and memory), T cells (helper and cytotoxic), and natural killer (NK) cells (CD56bright and CD56dim) were analyzed.ResultsIn normal human peripheral blood samples, CD123 expression was highest on pDCs and basophils (above 10,000 receptors per cell). Intermediate expression (300-3,000 receptors per cell) was observed on mDCs, monocytes, eosinophils, and naïve B cells. Low expression (10-200 receptors per cell) was observed on neutrophils, memory B cells, T cells, and NK cells. These data are in agreement with previous studies (Busfield et al. ASH 2013 abs3598). Following incubation with JNJ-63709178, potent activation of CD4+ and CD8+ T cells was observed at sub-nanomolar levels, with EC50values of 0.07 nM and 0.1 nM, respectively. Higher CD123 expression and T-cell activation correlated strongly with profound depletion of basophils and pDCs, as well as partial depletion of eosinophils and monocytes. Consistent with low CD123 expression, T cells and NK cells did not show substantial dose-dependent depletion. In contrast to CD123 expression levels, B cells and neutrophils showed partial depletion, despite low CD123 expression, while mDCs did not show dose-dependent depletion, despite higher CD123 levels. The activation of T cells and cytotoxic effect on CD123-positive cells was observed only with JNJ-63709178. The control compound CNTO 7008 (nullxCD3) had no effect on T-cell activation or any of the investigated cell populations.ConclusionsThese findings highlight the potential pharmacodynamic markers of JNJ-63709178 (CD123xCD3) bispecific treatment and provide a better understanding of its mechanism of action and a rationale for receptor expression monitoring during ongoing clinical trials. DisclosuresBabich:Janssen: Employment. Li:Janssen: Employment. Gaudet:Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued. Huang:Janssen Research & Development, LLC: Employment, Other: I am an employee of Janssen and a stock owner . Sasser:Johnson & Johnson: Equity Ownership; Janssen Pharmaceuticals R&D: Employment. Salvati:Janssen Pharmaceuticals R&D: Employment, Other: stock options, Patents & Royalties: patent. Kalota:Janssen Pharmaceuticals R&D: Employment, Other: stock.

Molecular Evidence of Ongoing Antigen Driven Somatic Hypermutation in the Natural History of Classic and Variant Hairy Cell Leukemia

Classic hairy cell leukemia (HCL) is a clonal chronic B-cell malignancy representing 2% of all leukemias, with neoplastic B lymphocytes in the blood, bone marrow and organs of the classic reticuloendothelial system. HCL variant (HCLv) is less common, typically has inferior prognosis, and usually lacks several HCL-related features including BRAF V600E, CD25, and Annexin A1. The malignant cells in HCL and HCLv patients have one or in unusual cases two different functional immunoglobulin heavy chain rearrangements. Significant variability can be observed in the 3rd complementarity determining region (CDR3), comprising the variable heavy (IGHV), diversity (IGHD), and joining (IGHJ) domains. A role for continued antigen selection in the pathogenesis and evolution of HCL and HCLv has been suggested but limited evidence has been found.We examined HCL and HCLv-derived IGHV genes by massive parallel deep sequencing in 15 HCL and 7 HCLv cases. In all studied samples, a productive monoclonal tumor-derived IGH sequence was detected. Analysis of all reads sharing clonal CDR3 motifs revealed the existence of dominant subclone expansion and multiple subclonal variants. Analysis of phylogenetic trees indicated multiple branching and therefore exposure to multiple rounds of somatic hypermutation (SHM) in the evolution of clonally-related malignant cells. To investigate a possible cause of the high SHM activity and the large number of subclones in HCL and HCLv, we studied the expression of activation induced cytidine deaminase (AID), an enzyme essential for somatic hypermutations, expressed by germinal center (GC) B-cells where SHM occurs. In 5 HCL and 3 HCLv cases we detected a high expression level of AID mRNA, including wild-type and 2 splice variants; in 10 HCL, 4 HCLv, and 3 normal peripheral blood samples, AID expression was not detected using standard end-point PCR conditions. However, more sensitive real-time quantitative PCR detected AID transcripts in virtually all tested HCL and HCLv cases, although the range of transcript levels was large between different cases and varied with individual cases over time.The data obtained for both HCL and HCLv fit a model of tumorigenesis in which the BRAF V600E mutation (or another event in HCLv) initiates neoplastic transformation in a GC B-cell committed to terminal differentiation, but still thereafter still targeted by ongoing SHM DisclosuresNo relevant conflicts of interest to declare.