Peripheral Blood Of Acute Myeloid Leukemia Patients Research Articles

Multiomics Characterization of Normal CD34+ Hematopoietic Progenitor Cells (HPC) and Blasts in the Bone Marrow (BM) and Peripheral Blood (PB) of Patients with Acute Myeloid Leukemia (AML)

BACKGROUNG: Multiparameter flow cytometry (MFC) is traditionally used to asses MRD in AML. However, its sensitivity and specificity lag behind other methods and this precludes a broader application in routine practice. It could be hypothesized the emerging multiomics such as single-cell RNA and antibody sequencing could uncover new differences from normal and leukemia associated immunophenotypes (DfN/LAIP). In addition, the identification of DfN/LAIP may be easier in PB when compared to the BM due to the scarcity of CD34+ HPC in the former sample. AIM: Uncover new DfN/LAIP for improved MFC discrimination between normal CD34+ HPC and blasts in the BM and PB of AML patients. METHODS: A total of 47 samples were analyzed in this study. Namely, paired BM and PB from 5 young healthy adults (20-23yo) and 5 PB samples from older healthy adults (47-88yo); paired BM and PB of 11 newly diagnosed AML (44-79yo) patients, and paired BM and PB collected in 5 of the 11 AML patients at the time of MRD assessment. We used fluorescence activated cell sorting (FACS) to isolate CD34+ HPC from the BM and PB of healthy adults as well as blasts from AML patients. scCITE-Seq with a panel of 600 genes and 50 oligo-conjugated antibodies, selected according to their role in stem cell identification, lineage differentiation, clonal hematopoiesis and aberrant expression in myeloid neoplasms was performed. After quality control filtering, a total of 111,334 cells were analyzed and differential expression of genes and antigens was considered if an adjusted P-value <.05 and a log 2FoldChange (FC) >|1| were observed. RESULTS: We first analyzed the phenotype of paired BM and PB 32,637 CD34+ HPC from younger healthy adults. There were 82 deregulated genes, 50 overexpressed in BM and 32 in PB. CLEC12A, CEBPA and HBB were amongst the top overexpressed genes in BM, whereas CRHBP, ADGRG6 and MEIS1 showed the highest upregulation in PB. These results suggest partial transcriptional overlap between BM and PB CD34+ HPC, with the former showing reduced differentiation. There were no differences in antigen expression. Next, we analyzed the potential effect of aging in the phenotype of 34,102 CD34+ HPC from the PB of young and older healthy adults. There were 34 genes differentially expressed including BCORL1, CBLB, HRAS and KDM6A. These findings might reflect an upregulation of genes associated with clonal hematopoiesis. Furthermore, there were 13 antigens differentially expressed including CD9, CD10, CD33, CD38, CD62L and CD371. The comparison of 56,387 blasts in paired BM and PB samples from newly diagnosed AML patients showed near overlap with only HBB being differentially expressed. There were no differences in antigen expression. We then investigated the extent of phenotypic differences between normal CD34+ HPC vs blasts in the BM and PB of healthy adult's vs AML patients. There were 224 and 15 differentially expressed genes between BM cell types, whereas in PB the numbers were 182 and 11, respectively. The markers that contributed the most in the discrimination between CD34+ HPC vs blasts in PB were WT1, ADM, HMOX1, DDIT3, HOPX, CD371, CD45RA, CD133 and CD47. The overlap between DfN/LAIP identified in PB and BM was 31%. Four of the five patients monitored after treatment were MRD positive in BM using MFC. Of note, BM and PB CD34+ HPC isolated from the five AML patients (including one MRD negative in BM) clustered separately of normal BM and PB CD34+ HPC from healthy adults. There was near overlap between BM and PB cells from AML patients. The markers that contributed the most in the discrimination between PB CD34+ HPCfrom healthy adult's vs resistant cells from AML patients were CXCL16, LILRA5, MME, CD274, CTSL, MINPP1 and AHSP. CONCLUSIONS: Normal CD34+ HPC in PB showed partial overlap with the BM counterpart, with a few phenotypic differences probably related to less differentiation and the effects of aging and clonal hematopoiesis. The phenotype of BM and PB blasts from newly diagnosed AML patients nearly overlapped, which indicates that DfN/LAIP may be equally applicable in BM and PB. Accordingly, the markers included in the scRNA/Ab-seq panel effectively discriminated CD34+ HPC from blasts regardless of the sample of origin. Furthermore, we showed phenotypic divergence between PB CD34+ HPC of healthy adults' vs AML patients after treatment. Taken together, this study identified new DfN/LAIP markers for improved assessment of MRD using MFC in the PB of AML patients.

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.

Identification of acute myeloid leukemia by infrared difference spectrum of peripheral blood

Acute myeloid leukemia (AML) is a high mortality and recurrence rates hematologic malignancy. Thus, whatever early detection or subsequent visit are both of high significance. Traditional AML diagnosis is conducted via peripheral blood (PB) smear and bone marrow (BM) aspiration. But BM aspiration is a painful burden for patients especially in early detection or subsequent visit. Herein, the use of PB to evaluate and identify the leukemia characteristics will be an attractive alternative source for early detection or subsequent visit. Fourier transform infrared spectroscopy (FTIR) is a time- and cost-effective approach to reveal the disease-related molecular features and variations. However, to the best of our knowledge, there is no attempts using infrared spectroscopic signatures of PB to replace BM for identifying AML. In this work, we are the first to develop a rapid and minimally invasive method to identify AML by infrared difference spectrum (IDS) of PB with only 6 characteristic wavenumbers. We dissect the leukemia-related spectroscopic signatures of three subtypes of leukemia cells (U937, HL-60, THP-1) by IDS, revealing biochemical molecular information about leukemia for the first time. Furthermore, the novel study links cellular features to complex features of blood system which demonstrates the sensitivity and specificity with IDS method. On this basis, BM and PB of AML patients and healthy controls were provided to parallel comparison. The IDS of BM and PB combined with principal component analysis method revealing that the leukemic components in BM and PB can be described by IDS peaks of PCA loadings, respectively. It is demonstrated that the leukemic IDS signatures of BM can be replaced by the leukemic IDS signatures of PB. In addition, the IDS signatures of leukemia cells are reflected in PB of AML patients with peaks of 1629, 1610, 1604, 1536, 1528 and 1404 cm−1 for the first time as well. To this end, we access the leukemic signatures of IDS peaks to compare the PB of AMLs and healthy controls. It is confirmed that the leukemic components can be detected from PB of AML and distinguished into positive (100%) and negative (100%) groups successfully by IDS classifier which is a novel and unique spectral classifier. This work demonstrates the potential use of IDS as a powerful tool to detect leukemia via PB which can release subjects’ pain remarkably.

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.

Terminal differentiation of bone marrow NK cells and increased circulation of TIGIT+ NK cells may be related to poor outcome in acute myeloid leukemia.

In order to further understand the feature of natural killer cell (NK) dysfunction in acute myeloid leukemia (AML), The distribution of NK cell subset the expression of the inhibitory receptors immunoglobulin and ITIM domain (TIGIT), killer cell lectin-like receptor (KLRG1), and the expression of maturation marker CD57 in NK cell subsets and their correlation with patient outcomes were analyzed in this study. We collected peripheral blood (PB) and bone marrow (BM) samples from de novo AML (AML-DN) patients, patients who achieved complete remission after chemotherapy (AML-CR), and healthy individuals. An eight-color flow cytometry panel was used to identify different NK subsets and their expression of TIGIT, CD57 and KLRG1. Decreased percentage of CD56dim CD16+ NK cells was found only in the PB of AML-DN and AML-CR patients but not in the BM. The expression frequency of TIGIT and KLRG1 was elevated on NK cells from the PB of AML-DN patients, while it was recovered in AML-CR patients. Moreover, a higher percentage of CD57+ CD56dim CD16+ NK cells, representing a terminally differentiated NK subset with strong cytotoxic capacity but defective replication potential, was detected in the BM of AML-DN patients and predicted sub-optimal survival for patients. The results indicated that the NK cell subsets in the PB of AML patients had an exhaustion phenotype, while the BM NK cells had a terminally differentiated phenotype, which correlated with short survival for AML patients.

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.

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.

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。

Abstract 4763: Genome-wide CRISPR screening identifies TSC1 as a regulator of sorafenib resistance in acute myeloid leukemia

Abstract Hematologic malignancies represent the fifth most common type of cancer and fourth most common cause of cancer-related deaths. These malignancies are characterized by a clonal expansion of hematopoietic progenitor cells that proliferate in the bone marrow, peripheral blood, lymph nodes and other organs. Genetic analyses of these diseases have led to the identification of specific genetic lesions that drive leukemogenesis, which has subsequently led to the development of targeted drugs. Acute myeloid leukemia (AML) is a fast progressing blood malignancy with impaired differentiation and proliferation of myeloid precursors. It is one of the most common leukemias in adults. AML is known for its molecular and biological heterogeneity, and a variety of genetic lesions have been implicated in the disease. FMS-like tyrosine kinase 3 (FLT3) mutations including FLT3 internal tandem duplication (ITD) or point mutations in the tyrosine kinase domain (TKD) are found in around 30% of AML patients. Sorafenib, a multi-kinase inhibitor that targets FLT3, RAF, VEGFR, FGFR, KIT and RET, is approved for use in hepatocarcinoma, renal cell carcinoma, and thyroid carcinoma treatments. Addition of sorafenib to standard treatment prolonged AML patient survival with or without FMS- like tyrosine kinase 3 (FLT3) mutations, although relapse caused by drug-resistance has limited its usefulness. Understanding the mechanism of resistance to targeted drugs, therefore, is necessary to improve treatment regimens for AML patients. We aim to elucidate resistance mechanisms to sorafenib in AML cells using genome-wide CRISPR screening. Two genome-scale human CRISPR knockout (KO) libraries were used to identify genes whose loss-of-function contribute to lower sensitivity to sorafenib in the MOLM13 AML cells. We verified 10 genes from the top hits showing consistency between the two CRISPR libraries, one of which was Tuberous Sclerosis 1 (TSC1). To validate these findings, TSC1 KO cells were generated using lentiCRISPRv2 system and single sgRNA sequence. Drug sensitivity assays confirmed increase in sorafenib resistance of TSC1 KO cells compared to parental cells or cells harboring non-targeting sgRNA. In addition to the sorafenib resistant phenotype, TSC1 KO cells were resistant to a panel of FLT3 inhibitors, quizartinib, crenolanib, gilteritinib, and UNC2025A. Moreover, RNA sequencing results from 271 AML patient peripheral blood or bone marrow samples revealed that lower RNA expression of Tuberous Sclerosis 1 (TSC1) correlated with lower sensitivity to sorafenib. TSC2 was also observed as a hit gene in both CRISPR libraries and lower TSC2 RNA levels also correlated with lower sensitivity to sorafenib, emphasizing TSC1/2 as an important pathway in sorafenib resistance. Citation Format: Alisa Damnernsawad, Tamilla Nechiporuk, Steve E. Kurtz, Wesley R. Horton, Olga Nikolova, Shannon K. McWeeney, Jeffrey W. Tyner. Genome-wide CRISPR screening identifies TSC1 as a regulator of sorafenib resistance in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4763.

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.

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.

VISTA is highly expressed on MDSCs and mediates an inhibition of T cell response in patients with AML

ABSTRACTTreatment of acute myeloid leukemia (AML) remains challenging. Enhancement of anti-tumor responses by blocking negative immune regulators is a promising strategy for novel effective leukemia therapeutics. V-domain Ig suppressor of T-cell activation (VISTA) is a recently defined negative regulator mediating immune evasion in cancer. To investigate the effect of VISTA on anti-leukemia immune response in AML, we initiated a study using clinical samples collected from AML patients. Here we report that VISTA is highly expressed on myeloid-derived suppressor cells (MDSCs) in the peripheral blood of AML patients. Both the frequency and intensity of VISTA expression on MDSCs are significantly higher in newly diagnosed AML than in healthy controls. Importantly knockdown of VISTA by specific siRNA potently reduced the MDSCs-mediated inhibition of CD8 T cell activity in AML, suggesting a suppressive effect of VISTA on anti-leukemia T cell response. Furthermore, we observed a strong positive association between MDSC expression of VISTA and T cell expression of PD-1 in AML. These results support the strategy of VISTA-targeted treatment for AML and underscore the strong potential for combined blockade of VISTA and PD-1 pathways in effective leukemia control.

Decreased miR-144 expression as a non-invasive biomarker for acute myeloid leukemia patients.

MicroRNAs are found to be stable in blood and they demonstrated tissue specific expression patterns. Thus, they may be used as potential non-invasive biomarkers of specific cancers. In the current study, we mainly focused on miR-144, which has never been studied in acute myeloid leukemia (AML). The expression of miR-144 was explored in the bone marrow and peripheral blood of AML patients and healthy control. The correlation between peripheral blood miR-144 level and key clinical parameters, including overall survival and prognostic value, was further explored. We showed that miR-144 was markedly reduced in both the peripheral blood and bone marrow of AML patients compared with healthy controls. Further study revealed that there is a significant correlation between peripheral blood miR-144 level and FAB classification (p=0.0023) and cytogenetics (p=0.001). More importantly, a lower expression of peripheral blood miR-144 level was found to be positively correlated with poorer overall survival rate. In summary, peripheral blood miR-144 may be utilized as a potential novel non-invasive biomarker for AML screening.