Blood Of Acute Myeloid Leukemia Patients Research Articles

Identification of 17 Distinct IDH1/IDH2 Mutations from Acute Myeloid Leukemia Peripheral Blood Using a Multiplex Real-Time PCR Closed Cartridge System

Background: Acute myeloid leukemia (AML) is characterized by clonal expansion of poorly differentiated “blast cells” in peripheral blood and bone marrow causing ineffective erythropoiesis and bone marrow failure. It has been demonstrated that mutations in isocitrate dehydrogenase 1 and 2 (IDH1/IDH2) are associated with disease relapse and reduced survival. There are 17 AML-related IDH1/IDH2 mutations located at R100, R132, R140, and R172 that are targeted by different IDH1/IDH2 selective inhibitors. The sensitivity of detecting IDH1/IDH2 needs to be improved for early cancer detection. Current PCR-based assays cannot detect all 17 IDH1/IDH2 mutations, and next generation sequencing approaches take longer, use complicated workflows, and require costly reagents as well as substantial data analysis. Therefore, the rapid identification of patients with AML who harbor IDH1/IDH2 mutations could improve the efficient selection of targeted inhibitors for these patients. Aim: To create a multiplex PCR assay that detects 17 IDH1/IDH2 mutations from peripheral blood with high sensitivity and specificity. Method: We will highlight five technical aspects of this qPCR assay that contribute to its high sensitivity and specificity: 1. the “anchor - bridge - foot” design of SuperSelective (SS) primers that can detect different mutations within the same codon. 2. the amplification-refractory mutation system (ARMS) primer design strategy was also tested which has comparable effects to SS primer. 3. a two-step nested PCR to reduce non-specific binding in products due to the amplification of unexpected primer binding sites. 4. a short (20-25nt) blocking oligonucleotide (wild-type blocker, WTB) to reduce SS and ARMS primer binding at the wild-type codon site. 5. four probes carrying different fluorescence reporters were used to identify the four IDH1/IDH2 mutation codons. Results: We synthesized 17 plasmid DNA (pDNA) templates and 14 CRISPR-engineered cell lines, each carrying one IDH1 or IDH2 mutation and the housekeeping gene ABL. SS and ARMS primer were screened with 5·10 4 cps/test of mutation template in a whole blood background. 6 mutations of R132 Codon (R132C, G, H, L, S, V), R100Q, 4 mutations of R140 (R140Q, L, G, W), and 6 mutations of R172 (R172K, M, G, S1 [516G>T], S2 [516G>C], W) can successfully be identified from whole blood lysate at the LoD level (1%) as shown in Figure 1 in red. Moreover, adding WTB to the PCR reaction improved the specificity with a reduced background signal. Importantly, in addition to the plasmid, cell line genomic DNA (gDNA) that was spiked into blood lysate at the LoD level (1%) also performed similarly to plasmid DNA as shown in Figure 1 in blue. Summary/Conclusion: We present a prototype assay with design intention to detect 17 mutations of IDH1/IDH2 from the peripheral blood of AML patients with excellent sensitivity and specificity and a strong potential to outperform any products on the market. Utilizing our Xpert Instrument System a fully automated, single-use cartridge product with pre-loaded sample-processing reagents, the assay aims to report results in less than 3 hours, including hands-on time.

T-Cell Receptor-Engineered T Cells Targeting FLT3-D835 Mutation-Derived Neoantigens in Acute Myeloid Leukemia

The adoptive transfer of T-cell receptor (TCR)-engineered T cells has demonstrated a remarkable anti-leukemic potential in mice models and human research for acute myeloid leukemia (AML). Neoantigens arising from somatic mutations are ideal targets for immunotherapy due to their high immunogenicity and harmless to normal cells. Therefore, neoantigen-specific TCR-based immunotherapy may have great prospects for AML. Activating mutations in FMS-like tyrosine kinase 3 ( FLT3) represent the most frequent genomic alterations of AML, including internal tandem duplication (ITD) in the juxtamembrane domain and point mutations within the tyrosine kinase domain, mostly at the activation loop residue D835. A number of FLT3 inhibitors have been clinically employed for FLT3-mutated AML. However, a durable response is limited by resistance-conferring secondary kinase domain mutations, notably point mutations at D835. Therefore, there is a critical need for novel and potent treatments to eliminate AML cells harboring FLT3-D835 mutations. Here, we show the construction of TCR-engineered T cells targeting FLT3-D835 mutants, presented in the context of HLA-A *02:01. We first identified neoepitopes derived from D835Y/H/V/F/I with strong affinities to HLA-A *02:01 using NetMHCpan 4.1. These neoepitopes could form the stable complex with HLA-A *02:01 on the cell surface. Furthermore, CD8 + T cells specific for D835H were detected in the peripheral blood of AML patients carrying D835H mutant at different stages of the illness, most of which were memory T cells. Moreover, one neoepitope (Neo-D835H) induced IFN-γ secretion when co-cultured with peripheral blood mononuclear cells (PBMCs) isolated from AML patients with D835H mutant. Neo-D835H-specific T cells were effectively generated through the rapid stimulation of CD8 + T cells isolated from an HLA-A *02:01-positive healthy donor with Neo-D835H-loaded autologous dendritic cells. These T cells were recognized by Neo-D835H-HLA-A2-Tetramer (0.37%) and consequently isolated by flow cytometry to perform TCRα and TCRβ sequencing. Both the TCRA and TCRB clonotypes showed dominant sequences that accounted for more than 50% of the total, suggesting that T cells with these dominant TCRA and TCRB-paired sequences were likely Neo-D835H-specific. We thus next generated TCR-engineered T cells with these TCRA- and TCRB-paired sequences for further functional analysis. These TCR-engineered T cells recognized Neo-D835H and showed cytotoxic activity against Neo-D835H-pulsed T2 cells in an antigen dose-dependent manner, while showed no responses to the T2 cells pulsed with the corresponding wild-type peptide. Taken together, we present novel neoantigens with promise as immunotherapy targets for AML and other hematologic malignancies with FLT3-D835 mutations. Future studies will evaluate the efficacy of Neo-D835H-specific TCR-engineered T-cell-based immunotherapy in vivo in patient-derived xenograft murine model and TCR-based therapeutic approaches targeting other FLT3-TKD mutations.

Changes of Transcriptomic Landscape in Blasts of Responding and Refractory Acute Myeloid Leukemia Patients after Chemotherapy

Despite many ongoing clinical drug trials and advances in acute myeloid leukemia (AML) therapy, the 3+7 induction regimen with anthracyclines and cytarabine (+/- midostaurin) remains the golden standard of AML treatment. Approximately 40 % of AML patients do not achieve a complete hematological remission (CR) after the first cycle of 3+7 chemotherapy and face an adverse prognosis. One of the main causes of treatment failure is chemoresistance of leukemia cells acquired by various mechanisms, all of which have not been identified as yet. We present here a unique study of transcriptome landscape mapping of separated AML blasts from paired samples collected before and after chemotherapy administration, focusing on differences between responding (RES) and refractory (REF) AML. The aim of this study was to describe transcriptional changes in leukemic blasts between RES and REF AML patients after chemotherapy administration, and to identify molecular pathways involved in reduced sensitivity to chemotherapy. Leukemic blasts were sorted from peripheral blood of AML patients collected at diagnosis (day 0, n=30) and on the third day (day 3, n=16) of 3+7 chemotherapy. CD34+ and CD117+ antibodies were used for blast separation on autoMACS (Miltenyi Biotec). 100 ng of high quality DNAse-treated RNA (RIN>8) was ribodepleted via the RiboCop rRNA Depletion kit (Lexogen) and the library was prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB). Transcriptome was sequenced on NovaSeq (Illumina). For data analysis, we divided patients by primary response status into RES and REF groups based on CR achievement after the first cycle of 3+7 therapy. Quantification of gene expression was performed using StringTie2 software (version 1.3.6). Resulting data were subsequently analyzed and visualized in R software 4.0.0 using edgeR and pheatmap packages. The String and GO databases were used to annotate genes into molecular pathways and biological processes. We identified 75 differentially expressed genes (FDR<0.05) at day 0 between RES (n=15) and REF (n=15) AML patients (Fig.1a). Among genes upregulated (n=62) in REF AML, those previously associated with inferior prognosis such as MN1 (transcriptional regulator), ABCA6 (membrane transporter) and SETBP1 (transcriptional regulator) were identified. On the contrary, PRF1 (involved in killing transformed cells) and TLE1 (negative regulator of NF-κB) were among genes downregulated (n=13) in REF AML. In general, genes upregulated in REF AML were assigned to Golgi membrane, carbohydrate transport and interferon signalling and genes upregulated in RES AML to immune response and the β-catenin/TCF transcriptional complex. In the pairwise analysis (day 0 vs. day 3), we identified 82 differentially expressed genes in RES (Fig.1b, n=6) and 121 differentially expressed genes in REF AML (Fig.1c, n=10). There were 42 genes identically deregulated in both groups and they were annotated into DNA damage/stress response, DNA repair and apoptotic pathways. Next, we compared genes that were deregulated only in RES or REF AML. In RES AML, there was a big cluster of downregulated histone and chromatin remodelling genes that was missing in REF AML. Upregulated genes in RES AML included those with antiproliferative, proapoptotic and p53 stabilizing functions. In REF AML, the principal deregulated genes were those affecting cytoskeleton remodelling (downregulated), p53 signalling network (upregulated) and genotoxicity pathways (upregulated). We identified differences in leukemic blast transcriptome between RES and REF AML before and after chemotherapy administration. The role of some differentially expressed genes has already been elucidated in AML pathogenesis, others deserve a closer look to evaluate their relevance as potential targets for emerging therapies. Instant downregulation of histone genes after chemotherapy administration seems to be a strong sign of RES AML and requires further attention for its use as a biomarker for early stratification into good versus poor responder AML. This project was supported by Ministry of Health of the Czech Republic (00023736, IHBT) and by GA CR (No. 20-19162S). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Expression of Th17 and IL-23 in Peripheral Blood and Their Relationship with Immunophenotype in Patients with Acute Myeloid Leukemia

To observe the expression of helper T cells 17(Th17), interleukin 23 (IL-23) in peripheral blood in patients with acute myeloid leukemia (AML), to analyze the relationship between Th17, IL-23 in peripheral blood and immunophenotype. 105 patients with AML in the hospital from January 2019 to January 2021 were prospectively selected as the research subjects, the expression of Th17 and IL-23 in peripheral blood of patients with AML was detected by flow cytometry; immunophenotype was detected and counted. The relationship between the expression of Th17, IL-23 in peripheral blood and immunophenotype of AML patients was analyzed. Draw ROC curve and analyze the predictive value of Th17 and IL-23 expression in peripheral blood to immunophenotype. The immunophenotype results of AML patients showed that myeloid antigen, lymphoid antigen and hematopoietic stem/progenitor cell marker antigen were positive expressed for various antigens in 105 AML patients, in myeloid antigens, CD13+ accounted for the highest proportion (93.33%), in lymphoid antigens, CD56+ accounted for the highest proportion (32.38%), and in hematopoietic stem/progenitor cell marker antigens, CD38+ accounted for the highest proportion (68.57%). The expression of Th17 in peripheral blood of AML patients with CD56+, CD7+, CD34+ and human leukocyte antigen DR+(HLA-DR+) were higher than that of AML patients with CD56-, CD7-, CD34-, HLA-DR-, the expression of IL-23 in peripheral blood of AML patients with CD56+, CD34+ and HLA-DR+ were higher than that of AML patients with CD56-, CD34-, HLA-DR-, the differences were statistically significant (P<0.05); compared the expression of Th17 and IL-23 in peripheral blood between other antibody positive and negative patients, there was no statistical significant difference (P>0.05). Logistic regression analysis showed that the high expression of Th17 in patients with AML was related to the positive expression of CD56, CD7, CD34 and HLA-DR in the detection of immunophenotype, the high expression of IL-23 was related to the positive expression of CD56, CD34 and HLA-DR in the detection of immunophenotype. The ROC curve showed that the AUC of expression levels of Th17 and IL-23 in peripheral blood alone and in combination for predicting CD56+, CD34+, HLA-DR+ and Th17 in peripheral blood for predicting CD7+ were mostly 0.5-0.7, which had certain predictive value, but the predictive performance was low. Myeloid antigen, lymphoid antigen and hematopoietic hematopoietic stem/progenitor cell marker antigen are positive expressed for various antigens in AML patients, the high expression of Th17 in peripheral blood of AML patients is related to the positive expression of CD56, CD7, CD34 and HLA-DR in detection of immunophenotyping, the high expression of IL-23 is related to the positive expression of CD56, CD34 and HLA-DR in the detection of immunophenotype.

NK Cells of Acute Myeloid Leukemia Patients Exhibit Exhausted Phenotype with Impaired Functional Activity

Acute myeloid leukemia (AML) accounts for about one third of all leukemias, with half of all leukemia deaths. The low 5-year survival rate and manifestation of this deadly disease in old age reinforce the need for new safe and effective AML therapies. Considering the exceptional role of immune cells in the recognition and elimination of tumor cells, one of these methods is immunotherapy. However, for the development of immunotherapeutic approaches, it is necessary to clearly understand the role of certain effector cells in the pathogenesis of the disease, as well as to have the knowledge about the phenotypic characteristics of these cells.Natural killer (NK) cells play important roles in innate cancer immunosurveillance, and some published data indicate that the antitumor function of NK cells is reduced in AML patients. To expand upon previous work, we performed a comprehensive analysis of the phenotypic and functional characteristics of NK cells in previously untreated AML patients that took into account a wide variety of biomarkers. The goals of this study were to define the phenotypic and functional differences in NK cells from AML patients and healthy donors and determine how these parameters affect outcome of the disease.We used 14-color flow cytometry to assess more than 30 measurable markers on NK cells from the peripheral blood of 33 untreated AML patients and age-matched healthy controls. In addition, NK cells were tested for interferon-γ responses under antibody-dependent cellular cytotoxicity conditions. Results in AML patients were then compared to healthy donors.We found that the NK cells of patients with AML have a significantly lower capacity to secrete IFN-γ and showed numerous signs of an exhausted phenotype, as compared to healthy controls. These included increased surface expression of CD39, PD-1, and LILRB1 on NK cells from AML patients. Interestingly, surface expression of the TIGIT checkpoint receptor did not differ between AML patients and healthy donors, but surface expression of the activating receptor DNAM-1, which shares the same ligands on tumor cells, was decreased. All of these data confirm that the NK cells of AML patients are functionally impaired.We also noted that the frequency of CD57 + NKG2C + KIR + memory-like “adaptive” NK cells was greater in the blood of AML patients. The proportion of adaptive NK cells did not correlate with the age of donors. Phenotypic features of this cell subpopulation from the AML patients included significantly increased expression levels of CD56 and significantly lower expression of the activating receptor DNAM-1. DisclosuresZhigarev: Janssen R&D: Research Funding; Immunitas Therapeutics: Consultancy; Tavotek Biotherapeutics: Consultancy. Drenberg: Janssen R&D: Current Employment. Campbell: Janssen R&D: Research Funding.

Common Genetic Aberrations Associated with Metabolic Interferences in Human Type-2 Diabetes and Acute Myeloid Leukemia: A Bioinformatics Approach.

Type-2 diabetes mellitus (T2D) is a chronic metabolic disorder, associated with an increased risk of developing solid tumors and hematological malignancies, including acute myeloid leukemia (AML). However, the genetic background underlying this predisposition remains elusive. We herein aimed at the exploration of the genetic variants, related transcriptomic changes and disturbances in metabolic pathways shared by T2D and AML, utilizing bioinformatics tools and repositories, as well as publicly available clinical datasets. Our approach revealed that rs11709077 and rs1801282, on PPARG, rs11108094 on USP44, rs6685701 on RPS6KA1 and rs7929543 on AC118942.1 comprise common SNPs susceptible to the two diseases and, together with 64 other co-inherited proxy SNPs, may affect the expression patterns of metabolic genes, such as USP44, METAP2, PPARG, TIMP4 and RPS6KA1, in adipose tissue, skeletal muscle, liver, pancreas and whole blood. Most importantly, a set of 86 AML/T2D common susceptibility genes was found to be significantly associated with metabolic cellular processes, including purine, pyrimidine, and choline metabolism, as well as insulin, AMPK, mTOR and PI3K signaling. Moreover, it was revealed that the whole blood of AML patients exhibits deregulated expression of certain T2D-related genes. Our findings support the existence of common metabolic perturbations in AML and T2D that may account for the increased risk for AML in T2D patients. Future studies may focus on the elucidation of these pathogenetic mechanisms in AML/T2D patients, as well as on the assessment of certain susceptibility variants and genes as potential biomarkers for AML development in the setting of T2D. Detection of shared therapeutic molecular targets may enforce the need for repurposing metabolic drugs in the therapeutic management of AML.

Abstract 1170: MEN1112/OBT357, a first-in-class humanized de-fucosylated monoclonal antibody targeting CD157 positive cells in acute myeloid leukemia and myelodysplastic syndrome

Abstract Bst1/CD157 is a GPI-anchored transmembrane protein encoded by a member of the CD38 NADase/ADP-ribosyl cyclase gene family, which is expressed on some blood cells of the myeloid lineage, such as monocytes and neutrophils. The antigen is found to be highly expressed, at diagnosis and relapse, in primary samples derived from Acute Myeloid Leukemia (AML) patients, showing the brightest expression in monocyte-oriented blasts. Moreover, CD157 is expressed in high and low-risk Myelodysplastic Syndrome (MDS) patients. MEN1112/OBT357, is a humanized de-fucosylated monoclonal antibody, in clinical development (clinicaltrials.gov identifier: NCT02353143), that is characterized by high affinity and specificity against CD157, inducing potent in vitro Antibody-Dependent Cell-mediated Cytotoxicity (ADCC). The de-fucosylation improved the binding of MEN1112/OBT357 to both high and low-affinity alleles of FcγRIIIa/CD16a, allowing stronger recognition by NK cells. Since no Complement-Dependent Cytotoxicity (CDC) activity was detected, the MEN1112/OBT357 mechanism of action should mainly rely on immune effector cells. Indeed, in an in vitro reporter assay, MEN1112/OBT357 significantly triggered ADCC activity against AML target cell lines with different expression of CD157. Since the ex vivo sensitivity assay is considered a pharmacologically and clinically relevant model to evaluate the preclinical efficacy of therapeutic antibodies in AML (1), MEN1112/OBT357 activity was tested in 38 AML primary samples, demonstrating promising efficacy in an autologous setting, both in peripheral blood and bone marrow samples, in comparison to healthy donor blood samples and independently from FcγRIIIa polymorphisms. A similar activity was also observed in CD157 positive MDS primary samples. Since Myeloid-Derived Suppressor Cells (MDSCs) have been described to be increased in the bone marrow of both AML and MDS patients and their presence to be associated with resistance to chemotherapy (2, 3), we investigated CD157 expression in MDSCs, demonstrating a high expression of the antigen in the bone marrow and peripheral blood of AML patients. Importantly, we confirmed in ex vivo experiments, the depletion of these immunosuppressive cells by MEN1112/OBT357. Overall, our data demonstrate that MEN1112/OBT357 is a novel antibody with potent ex vivo activity against blast cells in AML and MDS patients, suggesting a potential contribution of MEN1112/OBT357 in altering the immunosuppressive environment in the bone marrow niche.

Rabbit Anti-Thymocyte Globulin Kills Leukemic Stem Cells to a Greater Degree Than Healthy Hematopoietic Stem Cells

Background: Rabbit anti-thymocyte globulin (ATG, Thymoglobulin) is given with hematopoietic cell transplant (HCT) conditioning to prevent graft-versus-host disease (GvHD). Recently we showed that in vitro ATG at clinically relevant concentrations kills not only T cells but also leukemic blasts (Dabas et al., BBMT 2016). This might explain the non-increase in relapse incidence in patients given ATG vs no ATG (Walker et al., Lancet Oncol 2016). Nevertheless, relapse of leukemia is still the most frequent cause of HCT failure. After chemotherapy (without HCT), non-relapse is associated with eradication of leukemic stem cells (LSCs). By extrapolation, non-relapse after HCT may also require the eradication of LSCs. We have shown that ATG kills LSCs, however, at a relatively high concentration of 50 mg/L. We next studied whether at that high concentration ATG kills also healthy hematopoietic stem cell (HSCs), as attempts to achieve the high concentration clinically would be indicated only if ATG killed LSCs but not HSCs.Study design: To study the cytotoxic effect of ATG on LSCs and HSCs, we used cryopreserved blood mononuclear cells (MNCs) from 8 newly diagnosed acute myeloid leukemia (AML) patients as a source of LSCs. The median percentage of blasts in the blood of the AML patients was 53% (range 24% to 90%). Healthy volunteers' cryopreserved blood MNCs (n=8) or cryopreserved filgrastim-mobilized apheresed blood MNC grafts from healthy donors (n=17) were used as the source of HSCs. The MNCs from the blood of AML patients and healthy volunteers were magnetically enriched for CD34+ cells (Miltenyi Biotech). This was not needed for the grafts as they contained a sufficient number of CD34+ cells for the assay of ATG killing. By flow cytometry, we measured stem cell killing induced by 15 min incubation with ATG (50 mg/L) and active complement (human serum). For negative control, cells were incubated with ATG in the presence of no active complement (heat-inactivated human serum). The LSCs and HSCs were phenotypically defined as CD45dim/neg, side scatterlow, CD34+, CD38neg and negative for the lineage markers (CD14, CD16, CD19, CD56, CD3, CD235a and CD41a). Dead cells were identified as 7-amino-actinomycin D positive (7AAD+).Results: Treatment of LSCs with 50 mg/L ATG in the presence of complement resulted in median 43% dead (7AAD+) cells (background subtracted). In contrast, treatment of blood HSCs and graft HSCs resulted in 3% and 13% dead cells, respectively (Figure 1).Conclusion: Here we show that in vitro LSCs are more sensitive to ATG mediated killing than healthy HSCs. However, the killing of LSCs occurs only at a relatively high ATG concentration (50 mg/L) which is ~5-fold higher than the maximum serum concentration achieved with a typical ATG dose (4.5 mg/kg). If ATG had the anti-LSCs activity also in vivo, using high dose ATG (leading to serum concentration of 50 mg/L) in patients could result in simultaneous decrease of both GVHD and relapse. Given the minimal effect on HSCs, the high dose ATG might not jeopardize engraftment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Higher Proportion of a Novel PD-1+ Foxp3+ γδ Regulatory T Cell Subset in De Novo and Recurrent AML

T cell immunodeficiency may be a common characteristic in acute myeloid leukemia (AML) and adoptive T cellular immunotherapy has a broad prospect for clinical treatment. γδ T cells is essential to anti-leukemia function through perforin (Perf) / granzyme B (Grmb) pathway and may become a new strategy for leukemia immunotherapy. However, γδ T cells have different functional subsets like regulatory cell subsets and relevant regulatory mechanism is still unknown. Programmed death-1 (PD-1) which is one of T cell inhibitory receptors may mediate T cell suppression and dysfunction in leukemia and relate to general regulatory T cells which express transcription factor forkhead box P3 (Foxp3). It would be interesting whether PD-1 pathway might involve in regulation of regulatory Foxp3+ γδ T cells. In this study, we first investigated the regulatory cell subsets of γδ T cells in patients with AML. The flow cytometry (FCM) was used to detect the expression proportions of γδ T cells and their subfamilies in peripheral blood of AML patients (including 15 cases of de novo AML, 5 cases of recurrent AML and 4 cases in complete remission (CR)). Real-time quantitative PCR(RQ-PCR)was used to detect the expression levels of PD-1, Foxp3, Grmb and Perf genes in the γδ T cells sorting by MACS technique from peripheral blood of de novo AML patients. Fifteen health individuals (HI) served as controls. The results showed decreasing trend of γδ T cell counts in AML patients. Compared with HI groups, higher expression levels of PD-1 gene in γδ T cells were found in de novo AML patients, while Grmb and Perf gene presented lower expression levels by RQ-PCR (P <0.05) (Figure A). The expression levels of PD-1 gene had a positive correlation to the Foxp3 gene in de novo AML group. It was noteworthy that we firstly found a novel PD-1+ Foxp3+ γδ T subset in peripheral blood of AML patients and HI controls with different frequencies. The proportions of Foxp3+ γδ T cells, PD1+ γδ T cells, and PD-1+ Foxp3+ γδ T cells were significantly higher either in de novo or the recurrent AML patients, respectively (P <0.05). The PD1+ Foxp3+ γδ T cells counts in AML patients showed the pattern as the recurrent AML > the de novo AML > the AML-CR groups (Figure B and C). In conclusion, a novel regulatory PD1+ Foxp3+ γδ T subset with lower expression levels of Grmb and Perf genes was found in peripheral blood of AML patients. Higher proportion of PD1+ Foxp3+ γδ T cells might influence the occurrence and development of AML. [Display omitted] DisclosuresLi:Guangdong Provincial Applied Science and Technology Research & Development Program (No. 2016B020237006): Research Funding; Guangzhou Science and Technology Project (No. 2015100010211): Research Funding; National Natural Science Foundation of China (91642111): Research Funding; Guangdong Provincial Basic Research Program (No. 2015B020227003: Research Funding. Wu:the Fundamental Research Funds for the Central Universities(No. 21617465): Research Funding; National Natural Science Foundation of China(No.8120038): Research Funding; the Natural Science Foundation of Guangdong Province (No. 2014A030313380): Research Funding.

Increased Levels of Circulating Monocytic- and Early-Stage Myeloid-Derived Suppressor Cells (MDSC) in Acute Myeloid Leukemia.

To investigate the levels of circulating myeloid-derived suppressor cells (MDSC) in patients with primary acute myeloid leukemia (AML), and to explore the relationship between the number of MDSC and AML. Peripheral blood samples from 29 patients with primary AML and 30 healthy controls were collected. CD33, CD11b, HLA-DR, CD14, and CD15 were used to label cells, and flow cytometry was used to analyze the numbers of total MDSC and subgroups eMDSC (early-stage MDSC), M-MDSC (monocytic MDSCs), PMN-MDSC (polymorphonuclear-MDSCs) or G-MDSC (granulocytic-MDSC) via two gating strategies. Presence of MDSC in AML was determined after assessment of clinical data. Phenotypic analysis of MDSC by the two gating strategies was consistent. Compared with healthy controls, the numbers of total MDSC (CD33+CD11b+ HLA-DR-) and G-MDSC (CD33+CD11b+ HLA-DR-CD14¬-CD15+ or CD14¬-CD15+ CD11b+) in peripheral blood of AML patients were lower (p < 0.05), while numbers of M-MDSC (CD33+CD11b+ HLA-DR-CD14+CD15- or HLA-DR-/LOWCD14+) and eMDSC (CD33+CD11b+ HLA-DR-/LOWCD14-CD15-) were higher (p < 0.05). The levels of G-MDSC in peripheral blood of AML-M2 patients were higher than those in other subtypes, along with total MDSC, while the levels of eMDSC and M-MDSC in AML-M3 patients were higher than those in other subtypes. The high frequency of HLA-DR-/LOWCD14+M-MDSC and CD33+CD11b+ HLA-DR-/LOWCD14-CD15- eMDSC in peripheral blood of AML patients indicates potential for MDSC as a diagnostic index in AML.

Assay Specificity and Sensitivity Evaluation of Cepheid GeneXpert® NPM1 Prototype for Mutation Detection in Acute Myeloid Leukemia

Background: Mutations in thenucleophosmin1 (NPM1) are considered as founder mutations in acute myeloid leukemia (AML). NPM1 mutations in exon 12 occur in about 60% adult AML patients with normal karyotype. Among all NPM1 subtypes, type A (69.15%), type B (7.41%) and type D (7.41%) are found to be most frequent. The evaluation of minimal residual disease (MRD) has been considered critical to more precisely define AML response to clinical treatment, thereby refining disease risk stratification. Currently, AML with NPM1 gene mutations is recognized as a distinct entity, due to its unique clinical features. A large body of evidence demonstrated a significant independent prognosis factor of molecular MRD monitoring in NPM1-mutated AML. Objectives: Here we present GeneXpert NPM1 prototype, a fast and promising test for use in AML mutation detection (this prototype is for research use only, not for use in diagnostic procedures, and not reviewed by any regulatory body). NPM1 prototype is an easy-to-use-sample in/results out, and highly sensitive assay. The turnaround time is as fast as less than 3 hours. NPM1 prototype is designed for the quantitative detection of NPM1 mutant mRNA transcripts (types A, B and D in exon 12) and the ABL endogenous control mRNA transcript in peripheral blood of AML patients. This test is also designed for use as an aid in the monitoring of mutant NPM1 mRNA transcript levels in patients diagnosed with NPM1-mutated AML. This test does not differentiate between A, B or D type mutant NPM1 transcripts and does not detect or monitor other rare types of mutant NPM1. Results: Through assay optimization, we were able to reach limit of detection (LoD) within 0.05% for three NPM1 subtypes among all screened EDTA donor blood samples. Ct-copy number regression curve aligns well among three subtypes, within detection range. Our assay specificity has been above 95% among 30 screened EDTA donor blood samples. Conclusion: In summary, GeneXpert NPM1 prototype shows promise as a fast test for use in AML mutation detection.The presented NPM1 prototype has a great advantage covering most prevalent NPM1 mutation types up to 94% of all NPM1 subtypes. Our results demonstrated that our prototype shows satisfactory assay specificity and sensitivity. Clinical samples will be included for our clinical validation in the future. Disclosures No relevant conflicts of interest to declare.

AML-272: The Immune Checkpoint BTLA Expression: A Potential Prognostic Biomarker for Acute Myeloid Leukemia Patients

Context The immune system plays a significant role in controlling carcinogenesis, even though most cancers escape immune surveillance through a variety of mechanisms. Objective The aim of the present study was to evaluate the prognostic significance of the novel co-inhibitory B and T lymphocyte attenuator (BTLA) in acute myeloid leukemia (AML) patients as well as its correlation with immune cell populations and treatment outcomes. Design And Setting We conducted case control study on de novo AML patients recruited from Internal Medicine Department, Clinical Hematology and Stem Cell Transplantation Unit, Ain Shams University Hospitals, Cairo, Egypt. The study was held from November 2018 to February 2019 with one year follow up of enrolled cases. Patients and Participants A total of 60 newly diagnosed AML patients and 15 healthy controls were recruited. Interventions We investigated mRNA expression of (BTLA) on peripheral blood in AML patients, and age and sex matched healthy control. Results BTLA expression was significantly up-regulated in patients in comparison to controls (p = 0.024). The expression level of BTLA in CD13+, CD33+, HLA-DR+ patients was significantly higher than in CD13- (p = 0.003), CD33- (p Conclusions These findings provide a basis for the concept that BTLA up-regulation is involved in inhibition of antitumor immunity, and that high BTLA expression level could be considered an independent biomarker for poor prognosis and survival of AML patients.

Immune escape in acute myeloid leukemia

Abstract Background Many different mechanisms allowing for immune escape have been described in solid tumors, but their relevance for hematological malignancies is underexplored. We analyzed possible involvement of selected processes in the acute myeloid leukemia (AML), with regard to the mutational status of NPM1 and Flt3 genes, which are important prognostic markers in AML. Methods The expression of HLA class I, CLIP, and of different inhibitory receptors was analyzed by flow-cytometry on fresh or cryopreserved primary mononuclear cells, which were isolated from the peripheral blood of AML patients at diagnosis (N = 38). The amount of regulatory T-cells (CD25+FoxP3+) was measured in fresh whole blood samples (N = 81). Some markers were assessed in parallel on the transcript level. AML blast lysis by NK cells was tested by flow-cytometry using donor-derived expanded NK cells. Results A decrease in HLA class I expression on AML blasts to about 20 % of normal levels was detected in 26 % of cases. This decrease did not significantly enhanced AML cell lysis by NK cells. High CLIP expression was found on leukemia blasts in 61 % of cases. High PD-L1 expression, which was determined by PCR, was associated with significantly worse overall survival (OS), specifically in patients with internal tandem duplication in Flt3 (Flt3-ITD; published finding, Brodska et al. IJMS 2019). We noted no mark of lymphocyte exhaustion (Tim3, LAG3, CTLA-4) in AML samples. On the other hand, Tim-3 was frequently found on blast cells (53 % of cases), in close correlation with higher levels of Tim-3 transcript. Tim-3 negativity was associated with better OS in patients without Flt3-ITD (p = 0.07). Increased count of regulatory T-cells was detected in 17.5 % of AML patients at diagnosis. Conclusion The majority of AML patients display at least one marker of immune escape at diagnosis. These include decreased HLA class I expression, reduced antigen processing, increased PDL1 expression, secretion of Tim3 by leukemia blasts, or increased Treg counts. The mechanisms of immune suppression in AML are likely heterogenous, similarly to the disease itself. Both PD-L1 and Tim-3 might have prognostic value in selected AML subgroups. Legal entity responsible for the study The authors. Funding Ministry of Health of the Czech Republic (Grant no 16-30268A). Disclosure All authors have declared no conflicts of interest.

Expression of Tim-3 on natural killer cells in patients with acute myeloid leukemia and its clinical significance

目的探讨负性共刺激分子Tim-3在初诊急性髓系白血病（AML）患者外周血自然杀伤细胞（NK细胞）表面的表达特征及其临床意义。方法采集2013年6月至2014年6月苏州大学附属第一医院血液内科收治的未经任何临床措施干预的39例AML患者外周血及28名健康志愿者的外周血，采用CD3−、CD56+、Tim-3+为标志，经免疫荧光标记和流式细胞术检测Tim-3在AML患者外周血NK细胞上的表达。结果初诊AML患者外周血NK细胞占淋巴细胞比例为（5.74±5.31）%，较正常对照组的（12.55±6.33）%显著下降（t=4.596，P<0.001）；初诊AML患者外周血NK细胞上Tim-3的表达水平为（42.67±19.08）%，较正常对照组的（60.99±20.69）%显著下降（t=3.781，P<0.001）。AML患者外周血NK细胞比例与其染色体核型相关（t=2.915，P<0.05）；Tim-3在AML患者NK细胞表达水平与患者诱导缓解率、危险分层相关（P值均<0.05）。结论AML患者外周血中NK细胞比例及Tim-3表达水平明显下降，Tim-3在AML患者外周血NK细胞上表达下调与AML的预后相关。

MicroRNA-199 inhibits proliferation and promotes apoptosis in children with acute myeloid leukemia by mediating caspase-3.

The aim of this study was to detect the expression level of microRNA-199 in acute myeloid leukemia (AML). Meanwhile, we also investigated whether microRNA-199 could inhibit the proliferation and promote apoptosis of AML cells by regulating caspase-3. The expression level of microRNA-199 in peripheral blood samples of AML patients and healthy controls was determined using Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). Similarly, microRNA-199 expression was also detected in AML cells and human medullary cells. The overexpression plasmid of microRNA-199 was constructed and transfected into cells. Subsequently, the regulatory effects of microRNA-199 on the proliferation, cell cycle, apoptosis, invasion and migration were accessed. The relationship between microRNA-199 and caspase-3 expression was further elucidated by Western blot and RT-qPCR. MicroRNA-199 was lowly expressed in peripheral blood of AML patients and AML cell lines than normal controls. The overexpression of microRNA-199 significantly decreased proliferative, invasive and migratory capacities of AML cells, whereas markedly increased apoptotic rate. Western blot results showed that microRNA-199 increased caspase-3 expression in AML cells. Rescue experiments demonstrated that microRNA-199 inhibited malignant progression of AML by targeting caspase-3. MicroRNA-199 is lowly expressed in AML patients. Furthermore, it inhibits the malignant progression of AML by targeting caspase-3.

Hypogelsolinemia in Patients Diagnosed with Acute Myeloid Leukemia at Initial Stage of Sepsis.

BackgroundGelsolin (GSN) is an actin-binding and PIP2/Ca2+-regulated protein found in the cytoplasm and blood plasma. Hypogelsolinemia occurs in a wide range of traumatic injuries and inflammatory reactions. We hypothesize that blood GSN levels will be altered in patients diagnosed with acute myeloid leukemia (AML) that develop sepsis, and assessment of GSN concentration will be a useful marker to determine their clinical outcome. To achieve this task, we evaluated the plasma gelsolin concentration in blood samples collected from patients diagnosed with acute myeloid leukemia (AML) at initial stages of sepsis.Material/MethodsTo assess if AML patients might be at risk of sepsis, a SOFA score was determined. Plasma gelsolin concentration was evaluated using an immunoblotting technique.ResultsWe found that GSN concentration in the blood of the AML group with developing sepsis was significantly lower (32±41 μg/ml; p<0.05) compared to the AML group (65±35 μg/ml) and control group (176±37 μg/ml; p<0.001). Additionally, low gelsolin concentration in the blood of AML patients developing sepsis was associated with a high SOFA score. A decrease of GSN concentration in the blood of AML subjects with developing sepsis suggests that GSN level in blood reflects not only chronic inflammation stage associated with leukemia, but that GSN depletion also manifests the inflammation associated with sepsis development.ConclusionsThe results presented here suggest the possible utility of GSN evaluation for diagnostic purposes. Overall, these data support the that reversing plasma GSN deficiency might be a possible new strategy in sepsis treatment.

MiR-183-5p Inhibits Occurrence and Progression of Acute Myeloid Leukemia via Targeting Erbin.

Erbin has been shown to have significant effects on the development of solid tumors. However, little is known about its function and regulatory mechanism in hematological malignancies. The biological function of Erbin on cell proliferation was measured in vitro and in vivo. The predicted target of Erbin was validated by dual-luciferase reporter assay and rescue experiment. We found that overexpression of Erbin could inhibit the cell proliferation and promote the cell differentiation of acute myeloid leukemia (AML) cells, whereas depletion of Erbin could enhance the cell proliferation and block the cell differentiation in AML cells in vitro and in vivo. Besides, miR-183-5p was identified as the upstream regulator that negatively regulated the Erbin expression. The results were confirmed by dual-luciferase reporter and RNA pull-down assay. Furthermore, we found that miR-183-5p negatively regulated Erbin, resulting in enhanced cell proliferation of AML cells via activation of RAS/RAF/MEK/ERK and PI3K/AKT/FoxO3a pathways. The activation of RAS/RAF/MEK/ERK and PI3K/AKT/FoxO3a pathways was mediated by Erbin interacting with Grb2. These results were also validated by rescue experiments in vitro and in vivo. All above-mentioned findings indicated that the miR-183-5p/Erbin signaling pathway might represent a novel prognostic biomarker or therapeutic target for treatment of AML.

Immunophenotypic features of leukemic stem cells and bulk of blasts in acute myeloid leukemia.

According to the modern concept, leukemic stem cells (LSC) in acute myeloid leukemia (AML) are distinct from the bulk of leukemic cells in bone marrow and peripheral blood of AML patients. Nevertheless, LSC are responsible for managing all the hierarchy of the bulk of leukemic blast populations. This mini-review provides brief information on the distinctive features of LSC and blast cells in cytologically recognized types of AML. The study of different phenotypes of LSC and blast cells in AML with the aid of up-to-date flow cytometric techniques is important both for the deep insight into the mechanisms of leukemogenesis and development of novel strategies of target therapy. The urgent need for extending the diagnostic panel of monoclonal antibodies used for diagnosing AML is beyond doubt.

EVI-1 modulates arsenic trioxide induced apoptosis through JNK signalling pathway in leukemia cells

High expression of the oncogene ecotropic viral integration site-1 (EVI-1) is an independent negative prognostic indicator of survival in leukemia patients. This study aimed to examine the effects of arsenic trioxide (ATO) on EVI-1 in acute myeloid leukemia (AML). Mononuclear cells were isolated from the bone marrow and peripheral blood of AML patients and healthy donors. EVI-1 expression in hematopoietic cells was evaluated by RT-qPCR and Western blot analysis. EVI-1 was highly expressed in both primary AML and leukemia cell lines (THP-1 and K562). ATO down-regulated EVI-1 mRNA in zebrafish in vivo as well as in primary leukemia cells and THP-1 and K562 cells in vitro. Additionally, ATO treatment induced apoptosis, down-regulated both EVI-1 mRNA and oncoprotein expression, increased the expression of pro-apoptosis proteins, and decreased the expression of anti-apoptotic proteins in leukemia cells in vitro. EVI-1 expression in leukemia cells (THP-1 and K562) transduced with EVI-1 siRNA was significantly reduced. Silencing EVI-1 had a significant effect on the activation of the JNK pathway and the induction of leukemia cell apoptosis. ATO may downregulate EVI-1 mRNA and oncoprotein levels and block the inhibitory effects of EVI-1 on the JNK pathway, which activates the JNK apoptotic pathway, thereby leading to the apoptosis of EVI-1 in AML patients.

Anti-thymocyte globulin's activity against acute myeloid leukemia stem cells.

Rabbit anti-thymocyte globulin (ATG (Thymoglobulin)) kills T cells in vitro and probably also in vivo as it prevents graft-vs-host disease (GvHD) in patients. Recently we demonstrated that ATG at a clinically relevant concentration (10-50 mg/L) kills in vitro not only T cells but also leukemic blasts. In the present study, we investigated whether ATG kills not only leukemic blasts but also leukemic stem cells (LSCs). We used a flow cytometric assay of complement-mediated cytotoxicity(CDC). ATG-induced death of acute myeloid leukemia (AML) cells from patients newly diagnosed with AML was measured among blasts as well as LSCs. At 10 mg/L ATG, blasts but not LSCs were killed. At 50 mg/L ATG, both blasts and LSCs were killed. We also measured ATG-mediated killing of healthy individuals' hematopoietic stem cells (HSCs). Median 2% HSCs from blood and 15% HSCs from filgrastim-mobilized grafts were killed with 50 mg/L ATG, compared to 30% LSCs from the blood of AML patients (p = 0.001 and 0.022, respectively). In conclusion, LSCs are sensitive to ATG, however, only at a relatively high ATG concentration. At that concentration, LSCs are killed to a higher degree than HSCs.