Factor Acute Myeloid Leukemia Research Articles

Development and validation of a novel circular RNA as an independent prognostic factor in acute myeloid leukemia

BackgroundAlthough there are many clinical and molecular biomarkers in acute myeloid leukemia (AML), the novel and reliable biomarkers are still required to predict the overall survival at the time of disease diagnosis.MethodsIn order to identify independent predictors, we firstly selected 60 cytogenetically normal AML (CN-AML) patients using the propensity score analysis to balance the confounders and performed circular RNA (circRNA) sequencing. Next, one outcome related to circRNA was selected and validated in the independent cohort of 218 CN-AML patients. We then constructed circRNA-miRNA-mRNA regulated network and performed cellular metabolomic analysis to decipher the underlying biological insights.ResultsWe identified 308 circRNAs as independent candidate predictors of overall survival. Hsa_circ_0075451 expression was validated as an independent predictor with a weak predictive ability for overall survival. The regulated network of this circular RNA indicated 84 hub genes that appear to be regulated by 10 miRNAs sponged by hsa_circ_0075451. The regulatory axis of hsa_circ_0075451 -| miR-330-5p/miR-326 -| PRDM16 was validated by the dual luciferase report assay, fluorescence in situ hybridization, and ShRNA interference assay.ConclusionsOur data demonstrates that hsa_circ_0075451 expression may independently contribute to the poor prognosis of AML and present a novel therapeutic target.

Implications of Glutathione Peroxidase 3 Expression in a Cohort of Egyptian Patients with Acute Myeloid Leukemia.

Background:The impact of low expression of Glutathione peroxidase 3 (GPX3) on the clinical course of acute myeloid leukemia (AML) is poorly investigated. Aims:To explore the status of GPX3 expression and analyze its clinical characteristics and prognosis in a cohort of Egyptian patients with AML. Methods:GPX3 mRNA level was assessed by RT-q PCR in 40 newly diagnosed AML patients and 10 healthy controls. Results:The gene expression level was significantly lower in AML patients than the control group (P < 0.001). A cut off value (0.1223) for the discrimination between AML and controls was obtained by ROC curve. According to this cutoff value; the patients were reassigned into 2 groups; 28 patients with lower GPX3 expression and 12 patients with high GPX3 expression. GPX3low expression was significantly associated with higher incidence of induction death (P= 0.037) and lower CR rate (P=0.048). Moreover, GPX3low expression was significantly associated with shorter cumulative 1-year overall survival (OS) (P = 0.001) and disease-free survival (DFS) (P=0.028). Conclusion: GPX3low expression status is considered a poor prognostic factor in AML predicting shorter OS and DFS. The study highlights the importance of targeting glutathione metabolism as a central component of the anti-leukemia therapy.

Mitochondrial Spare Reserve Capacity : A New Predictive Metabolic Biomarker for Aggressiveness of Acute Myeloid Leukemia

Introduction The persistence of leukemic cells after treatment limits the effectiveness of anticancer drugs and is the cause of relapse in patients with acute myeloid leukemia (AML). After exposure to chemotherapeutic drugs, the survival of leukemic cells is mainly supported by mitochondrial energy metabolism. Several preclinical studies have shown that the combination of mitochondrial oxidative phosphorylation inhibitors with various anticancer treatments constitutes an effective therapeutic combination in vitro to eradicate the surviving leukemic cells. Evaluating the mitochondrial bioenergetic activity of blasts from AML patients could therefore provide predictive information on treatment response. The basal oxygen consumption of cells varies according to hematopoietic differentiation and depends on the energy needs in the in vitro condition of measurement. But it is necessary to treat the cells with uncoupling agents (eg FCCP) to assess the maximum activity that the respiratory chain could reach to respond to energy stress. Then, the switch from a basal level of oxygen consumption to a maximum level defines the mitochondrial spare reserve capacity (SRC). In this study, we propose to determine whether spare reserve capacity of blasts is a potential biomarker of AML aggressiveness in patients and to characterize the biochemical processes involved in the control of SRC in leukemic cells. Results Using the XFe24 Seahorse fluorometric oximeter, we first determined the mitochondrial oxygen consumption and glycolytic activity in hematopoietic cells (monocytes, lymphocytes, dendritic cells) of healthy donors, in AML patient blasts at diagnosis or at relapse and in AML cell lines (HL-60, MOLM-13, THP-1, KG1, OCI-AML3, MV-4-11, U-937). All measures have been assessed from freshly collected samples of peripheral blood and of bone marrow. As expected, AMLs are characterized by low oxidative phosphorylation activity compared to normal hematopoietic cells. From all the OXPHOS values obtained we defined a SRC threshold above which the SRC is considered high. This threshold has been set at a capacity to increase basal respiration by 250%. From patients blasts, we have therefore defined two groups characterized by high (n=14) or low (n=21) mitochondrial spare reserve capacity. Blasts with high SRC exhibit high glycolytic activity suggesting a link between spare reserve capacity and glucose metabolism. Using U-13C6 glucose and pharmacological inhibitors, we have demonstrated that the utilization of the mitochondrial spare reserve capacity of leukemic cells is supported through glycolysis and that mitochondrial oxidation of pyruvate is a key element for SRC recruitment. Mitochondrial pyruvate carrier inhibitors (as UK-5099) or gene silencing of BRP44 abolish the SRC of leukemic cells highlighting the importance of pyruvate oxidation to increase oxygen consumption. Since high mutation rate is recognized as an unfavorable prognostic factor in AML, we have also sequenced 45 commonly genes mutated in AMLs characterized by high or low SRC blasts. Interestingly, DNA sequencing analysis showed that AML with low SRC blasts have a higher mutation rate than high SRC blasts and also exhibited exclusive mutations such as ASXL1 (25%), IDH2 (25%), NPM1 (25%), IDH1 (13%), JAK2 (13%) and SF3B1 (13%). Conclusion Currently, most of the clinical biomarkers used to predict AML aggressiveness are based on DNA analysis, but the emergence of mutations is not always associated with phenotypic changes. This study shows that the mitochondrial spare reserve capacity of blasts represents a new functional biomarker based on the assessment of the energetic phenotype and could help the clinicians to determine the prognosis of AML. Moreover we have showed that altering pyruvate metabolism highly decrease spare reserve capacity of blasts and then could be evaluated as metabolic strategies to improve the therapeutic response in patients with AML. Disclosures Kluza: Daiichi-Sankyo: Research Funding.

AML Cells Alter Bone Homeostasis By Promoting the Formation of Immature Bone and Resorption of Mature Bone That Supports Leukemia Progression

Background: Genetic alterations in the osteoprogenitor cells has been shown to induce acute myeloid leukemia (AML) in several mouse models. Moreover, we have recently reported that AML cells induce osteogenic differentiation in mesenchymal stromal cells (MSC) to gain growth advantage in the bone marrow (BM; Battula et al., JCI Insight, 2017). However, the effect of AML cells on bone homeostasis/turnover and its impact on AML progression is unknown. Here, we hypothesize that AML cells expand osteoprogenitor cells and alter the balance between bone formation and resorption. Methods: To investigate the effect of AML cells on osteoprogenitor cells and mature osteoblasts, we used triple transgenic reporter mice with the genotype Osx-CreERt2;Ocn-GFP;ROSA-tdTomato. Murine AML cells with MLL-ENL translocation were implanted into these transgenic mice, and Osteoprogenitor (Osx+) cells and mature osteoblasts (Ocn+) in femurs were measured by confocal microscopy. To investigate the effect of human AML cells on bone composition, patient-derived xenograft (PDX) cells were implanted into non-obese diabetic scid interleukin-2Rγnull (NSG) mice and bone histomorphometry was performed using H&amp;E and Goldner's trichrome staining. Computed tomography (micro-CT) was used to measure bone volume (BV) and mineral density (BMD) in mice. Tartrate-resistant acidic phosphatase (TRAP) staining was performed to measure osteoclast number/activation. Finally, bone density on the vertebral bone (T12) was measured in 263 de-novo AML patients and 23 normal individuals by CT imaging. Results: In transgenic mice implanted with syngenic AML cells, we found a 3-4 fold increase in Osterix+, but not Osteocalcin+ cells, suggesting AML cells expand osteoprogenitor cells in the BM during short term exposure (2-3 weeks). To investigate the effects of AML on bone during a long term exposure, we implanted 10 different AML-PDX models in NSG mice (3 mice per model) and analyzed femurs by micro-CT and bone histomorphometry analysis. Interestingly, we observed a dramatic increase in new web-like medullary bone formation in 5/10 of the PDX models tested. Moreover, higher bone volume is associated with less aggressive PDX models (which takes 4-6 months to reach 90% circulating leukemia), but not aggressive PDX models (only 4-8 weeks to reach 90% circulating cells). These findings were also confirmed by micro-CT of mouse femurs. Interestingly, in some PDX models, CT images revealed large cavities in cortical bones close to epiphysis and metaphysis areas in the femur and tibia of mice with AML suggesting bone resorption. To validate bone resorption, we performed TRAP staining and found a significant increase in osteoclast activity on the endosteal surface and massive bone resorption in AML bone compared to normal bone. These data indicate high bone turnover in mice with AML compared to control mice. Next, we measured bone densities in AML patients and normal individuals by chest CT imaging. We found bone densities were gradually decrease with age in both healthy individuals and AML patients. However, compared to healthy individuals, bone densities are significantly higher in majority of AML patients (~70%) (p&amp;lt;0.01). Interestingly, survival analysis revealed that higher bone density is associated with good prognosis in AML patients (p&amp;lt;0.01), suggesting high bone turnover alters patient outcomes. Conclusion: Our data suggest that AML cells expand osteoprogenitor-rich niche and alter BM microenvironment by high bone turnover. New bone induction by less aggressive AML-PDX models coupled with AML patient CT data suggests that high bone density and volume are associated with favorable prognostic factors in AML. Mechanisms underlying this observation are under investigation. Disclosures Andreeff: Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research &amp; Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees. Konopleva:Ablynx: Research Funding; AstraZeneca: Research Funding; Kisoji: Consultancy; Ascentage: Research Funding; Stemline Therapeutics: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents &amp; Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Rafael Pharmaceutical: Research Funding; Amgen: Consultancy; Agios: Research Funding; Sanofi: Research Funding; Cellectis: Research Funding; Calithera: Research Funding; Forty-Seven: Consultancy, Research Funding; Eli Lilly: Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding. Battula:Leukemia Lymphoma Society: Research Funding; Tolero Pharmaceuticals: Research Funding; Golfers Against Cancer: Research Funding.

FLT3-ITD and CD135 Over-Expression are Frequent Findings of Poor Survival in Adult Patients with Acute Leukemias

Fms-like tyrosine kinase 3 (FLT3) expression and mutation have been considered a poor prognostic factor in acute myeloid leukemia (AML). FLT3-ITD mutation is present in 30% of adult patients with AML and 2-5% in childhood acute lymphoblastic leukemia (ALL). The impact of these mutations on the prognosis of ALL patients, has not yet been established. Moreover, a limited number of publications regarding the level of expression of the FLT3 receptor (CD135) in both leukemias exist. This study aimed to analyze the clinical outcomes associated to the presence of FLT3-ITD mutation and the expression of CD135. 82 adult patients with newly diagnosed acute leukemia (39 with AML and 43 with ALL) were included. Flow cytometry and RT-PCR were done to analyze the expression of CD135 and the presence of FLT3 ITD mutation, respectively. FLT3-ITD was present in 14 (36%) of AML and 15 (35%) of ALL patients. Disease free survival (DFS) and overall survival (OS) were lower in ALL patients having a CD135 expression >3000cells/μL. There was a trend for poor OS in AML patients expressing FLT3 ITD. OS was worse in AML patients with high expression of CD135. A higher (35%) frequency of FLT3-ITD was found in adult ALL patients. The presence of FLT3-ITD was associated with a trend of poor OS in AML cases, and overexpression of CD135 was correlated with poor DFS in ALL cases and poor OS in both acute leukemias.

Core‐binding factor acute myeloid leukemia with inv(16): Older age and high white blood cell count are risk factors for treatment failure

Core‐binding factor acute myeloid leukemia with inv(16): Older age and high white blood cell count are risk factors for treatment failure

High Wilms' tumor 1 associating protein expression predicts poor prognosis in acute myeloid leukemia and regulates m6A methylation of MYC mRNA.

Acute myeloid leukemia (AML) is a heterogenous disease and the survival of AML patients is largely attributed to the improvement of supportive treatment. Wilms' tumor 1-associated protein (WTAP) is a nuclear protein functions in many physiological and pathological processes. Although its expression and function in many malignant diseases have been reported, its prognostic and epigenetic roles in AML are largely unknown. Peripheral blood or bone marrow samples were collected from AML patients. The WTAP expression was detected by western blot. WTAP expression level and patients clinical features were analyzed using statistical methods. WTAP knockdown AML cells were constructed. The experiments on proliferation, tumorigenic ability, cell cycle, and apoptosis were performed. Transcriptome sequencing was performed and analyzed. M6A methylation level was measured and m6A-RIP was performed to quantify m6A methylation level of MYC mRNA. RNA stability assay was performed to measure the half-life of mRNA. WTAP was overexpressed in AML patients and was an independent poor-risk factor in AML (p = 0.0140). Moreover, we found that WTAP regulated proliferation, tumorigenesis, cell cycle, and differentiation of AML cells. Furthermore, WTAP made AML cells resistant to daunorubicin. In further investigations, m6A methylation level was downregulated when knocking down WTAP, and c-Myc was upregulated due to the decreased m6A methylation of MYC mRNA. High WTAP expression predicts poor prognosis in AML and WTAP plays an epigenetic role in AML.

AML-241: Bone Marrow Stromal Cells Induce Chemotherapy Resistance in AML Cells through TGF-β-p38-ALDH2 Pathway

Context Chemotherapy resistance is one of the leading causes of disease progression and relapse in patients with acute myeloid leukemia (AML). The interaction between mesenchymal stromal cells (MSCs) and AML cells in the bone marrow (BM) is critical for leukemogenesis and chemotherapy resistance. Objective As tumor initiation and chemotherapy resistance are characteristic features of leukemia stem cells, we hypothesize that BM-MSCs induce a stem-like phenotype in AML cells. The objectives of this study are to determine the effect of stromal cells on the stem cell function of AML cells and to identify signaling pathways that support AML-stroma interactions in the BM microenvironment. Design We cultured AML cell lines and patient samples with or without BM-MSCs and measured aldehyde dehydrogenase (ALDH) activity in AML cells by ALDEFLUOR™ assay. In addition, we performed genomic and proteomic analyses to identify signaling pathways that support AML-stroma interactions. Finally, we used specific inhibitors that target ALDH activity to sensitize AML cells to chemotherapeutic agents. Results Co-culture of AML cells with BM-MSCs significantly induced ALDH activity in AML cells, mainly through upregulation of ALDH2 and ALDH1L2 isoforms. TCGA data analysis revealed that ALDH2 is a prognostic factor in AML. Mechanistic studies revealed that TGF-β1-regulated gene signature is activated in AML cells co-cultured with stromal cells. Knockdown of TGF-β1 in BM-MSCs inhibited stroma-induced ALDH activity and ALDH2 expression in AML cells, whereas treatment with recombinant TGF-β1 induced an ALDH+ phenotype in AML cells. We also found that the non-canonical TGF-β pathway, through p38 activation, regulates ALDH2 expression in AML cells. Pharmacologic inhibition of ALDH2 significantly inhibited BM-MSC-induced ALDH activity and sensitized AML cells to chemotherapy. Conclusion BM-MSCs induce ALDH+ stem cell phenotype in AML cells through the non-canonical TGF-β pathway. ALDH2 is the crucial isoform regulating ALDH activity in AML cells and is a prognostic factor. Pharmacologic inhibition of ALDH2 sensitizes AML cells to standard chemotherapy. Grant Acknowledgement This study is partially supported by Leukemia SPORE CDA, IRG from MDACC, Cure Sonia Foundation and Golfer's Against Cancer to VLB. NIH (CA055164), MDACC Support Grant (CA016672), CPRIT-MIRA, and Paul and Mary Haas Chair in Genetics to MA.

AML-226: Dasatinib Plus Chemotherapy in Core Binding Factor Leukemia: A Case Study

Core binding factor (CBF) acute myeloid leukemia (AML) patients have t (8:21) and inv(16) or t (16;16). They respond to dose-intensive cytarabine regimens with a complete remission (CR) rate of over 90%. Five-year event-free survival is 44% with a median of 91 weeks. We report a case of a relapsing inv(16) CBF leukemia salvaged by adding a tyrosine kinase inhibitor (TKI) to the chemotherapeutic regimen. Case presentation Ms. J.R is a 21-year-old patient with quadriplegia and inv16 with mutated KIT exon 8 gene diagnosed December 2017. She received induction with FLAG-IDA and six consolidation courses. Four months later, she relapsed with 56,000 WBC/dL and 82% bone marrow blasts. The inv(16) FISH was 87% positive and CBFB MYH11/total ratio was 26.585. Salvage therapy was initiated with decitabine-venetoclax-gentuzumab ozogamicin (GO). There was a marrow CR with a 21% positive inv(16) FISH test and a CBFB-MYH11/total ratio of 32.191. Due to the persistence of the KIT mutation, the TKI dasatinib was added to decitabine-venetoclax-gentuzumab ozogamicin (GO). After the TKI addition, she remained in CR. The CBFB-MYH11/total ratio decreased to 9.046, and the inv(16) FISH test remained positive at 2%. A second similar course given was complicated by a pulmonary fungal infection, acute appendicitis, and CMV colitis. All resolved with antifungals, laparoscopic surgery, and anti-CMV therapy, respectively. Upon recovery, she was in CR with undetectable CBFB-MYH11 transcripts and a negative FISH test. Because a bone marrow transplant is not possible, she has started a modified maintenance regimen with dasatinib, decitabine, and venetoclax. Discussion CBF leukemia chimeric proteins disrupt normal hematopoiesis by recruiting histone deacetylases and DNA methyltransferase 1 to CBF target genes, resulting in increased chromatin deacetylation and promoter hypermethylation. KIT exon 8 and 17 mutations occur in 20–25% of t (8;21) and approximately 30% of inv(16) leukemias. KIT mutations result in constitutively activated promotion of leukemic cells survival. Screening for KIT mutations and adding TKIs during induction or salvage therapy could be important for the treatment of CBF leukemias with an analogous impact, like adding TKIs in Ph-positive diseases.

Abstract 3652: EED or BRD4 inhibition as a novel therapeutic strategy in acute myeloid leukemia with monosomy 7

Abstract Backgrounds: Monosomy of chromosome 7 (monosomy 7) is observed in about 9% of acute myeloid leukemia (AML) patients and related to poor prognosis. It is required to establish an effective treatment strategy based on the specific molecular pathogenesis associated with the chromosomal abnormality. Methods: Chromosome 7 encodes multiple genes regulating histone methylation. We hypothesized that haploinsufficiency of the epigenetic modifiers in the chromosome 7 is compensated by other epigenetic molecules in different chromosomes, and the AML cells with monosomy 7 are vulnerable to inhibition of those molecules. Based on this notion, we performed an RNA-interference (RNAi)-based screening by targeting 53 genes encoding epigenetic enzymes regulating histone methylation by using AML cell lines with monosomy 7 and non-monosomy 7. Results: We found that the siRNA-mediated knockdown of EED, or BRD4 significantly decreased cell viability in AML cells with monosomy 7 compared to the control leukemia cell lines. Stable knockdown of EED markedly attenuated proliferation as well as increased cell apoptosis specifically in leukemia cells with monosomy 7. Interestingly, PRC1 rather than PRC2 activity of EED had a predominant role in the survival of monosomy 7 leukemia cells. We then explored whether the deletion of specific genes within the chromosome 7 was responsible for increased sensitivity to EED inhibition. We found that knockdown of GTF2I increased the sensitivity to EED inhibition via increased cell apoptosis. Because both GTF2I and EED suppressed expression levels of BCL2, it is possible that EED inhibition additively abrogate BCL2 activity in AML with monosomy 7. In addition to EED, we identified BRD4 as another essential survival factor in AML with monosomy 7. Although inhibition of BET bromodomain proteins have already shown to be effective for a variety of AML, we showed that blockade of bromodomain proteins preferentially affected cellular proliferation of AML cells with monosomy 7. Mechanistically, we found that knockout of MLL3 or MLL5, both encoded in the chromosome 7, resulted in increased vulnerability to JQ-1 treatment. In monosomy 7, functional loss of MLL3 and MLL5 showed reduced expression levels of c-MYC and CCND1. JQ-1 treatment additively impaired the functions of these genes, and strongly inhibited cell proliferation of AML cells with monosomy 7. Conclusion: Our synthetic-lethality based approach identified EED and BRD4 as novel promising therapeutic targets in AML with monosomy 7. Citation Format: Kensuke Matsuda, Yuki Kagoya, Hideaki Mizuno, Sho Yamazaki, Masashi Miyauchi, Mineo Kurokawa. EED or BRD4 inhibition as a novel therapeutic strategy in acute myeloid leukemia with monosomy 7 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3652.

Denatonium as a Bitter Taste Receptor Agonist Modifies Transcriptomic Profile and Functions of Acute Myeloid Leukemia Cells.

The contribution of cell-extrinsic factors in Acute Myeloid Leukemia (AML) generation and persistence has gained interest. Bitter taste receptors (TAS2Rs) are G protein-coupled receptors known for their primary role as a central warning signal to induce aversion toward noxious or harmful substances. Nevertheless, the increasing amount of evidence about their extra-oral localization has suggested a wider function in sensing microenvironment, also in cancer settings. In this study, we found that AML cells express functional TAS2Rs. We also highlighted a significant association between the modulation of some TAS2Rs and the poor-prognosis AML groups, i.e., TP53- and TET2-mutated, supporting a potential role of TAS2Rs in AML cell biology. Gene expression profile analysis showed that TAS2R activation with the prototypical agonist, denatonium benzoate, significantly modulated a number of genes involved in relevant AML cellular processes. Functional assay substantiated molecular data and indicated that denatonium reduced AML cell proliferation by inducing cell cycle arrest in G0/G1 phase or induced apoptosis via caspase cascade activation. Moreover, denatonium exposure impaired AML cell motility and migratory capacity, and inhibited cellular respiration by decreasing glucose uptake and oxidative phosphorylation. In conclusion, our results in AML cells expand the observation of cancer TAS2R expression to the setting of hematological neoplasms and shed light on a role of TAS2Rs in the extrinsic regulation of leukemia cell functions.

Prognostic alternative mRNA splicing signatures and associated splicing factors in acute myeloid leukemia

The dysregulation of alternative splicing (AS) has emerged as a mechanism of acute myeloid leukemia (AML). However, the prognostic impact of AS events remains under-explored in AML. Here we report the prognostic value of AS events and associated splicing factors based on three datasets of AML patients. We defined the landscape of AS events in AML and identified 7033 AS events associated with the survival of AML patients. Based on these events, we further developed a composite 15 AS event-based prognostic signature, which was independent of the cytogenetic risk stratification and patient age, and showed a better performance than known gene expression signatures. More importantly, our new signature markedly improved the European LeukemiaNet (ELN) risk classification, indicating a broad applicability in the clinical management of AML. Furthermore, the splicing-regulatory network established the correlations between prognostic AS events and associated splicing factors. The finding was validated by CRISPR-based data, which indicated that the increased expression of RBM39 contributed to the higher exon inclusion of SETD5 and conferred a poor outcome. Together, AS events may serve as a novel assortment of prognosticators for AML and could refine the ELN risk stratification. The splicing regulatory network provides clues regarding the splicing factor-mediated mechanisms of AML.

Clonal dominance is an adverse prognostic factor in acute myeloid leukemia treated with intensive chemotherapy.

Intra-tumor heterogeneity portends poor outcome in many cancers. In AML, a higher number of drivers worsens prognosis. The Shannon Index is a robust metric of clonal heterogeneity that accounts for the number of clones, but also their relative abundance. We show that a Shannon Index can be estimated from bulk sequencing, which is correlated (ρ = 0.76) with clonal diversity from single-colony genotyping. In a discovery cohort of 292 patients with sequencing of 43 genes, a higher number of drivers (HR = 1.18, P = 0.028) and a lower Shannon Index (HR = 0.68, P = 0.048), the latter reflecting clonal dominance, are independently associated with worse OS independently of European LeukemiaNet 2017 risk. These findings are validated in an independent cohort of 1184 patients with 111-gene sequencing (number of drivers HR = 1.16, P = 1 × 10-5, Shannon Index HR = 0.81, P = 0.007). By re-interrogating paired diagnosis/relapse exomes from 50 cytogenetically normal AMLs, we find clonal dominance at diagnosis to be correlated with the gain of a significantly higher number of mutations at relapse (P = 6 × 10-6), hence with clonal sweeping. Our results suggest that clonal dominance at diagnosis is associated with the presence of a leukemic phenotype allowing rapid expansion of new clones and driving relapse after chemotherapy.

Fc gamma receptor expression serves as prognostic and diagnostic factor in AML

Risk assessment in acute myeloid leukemia (AML) mainly relies on (cyto-)genetic and morphologic features. Nonetheless, further markers are needed to allow for accurate risk stratification. Type I Fc gamma receptors (FcγRs) such as CD16, CD32, and CD64 play an important role in mediating immunomodulatory functions in different myeloid cell types as well as NK and B cells. We here evaluated expression of the three FcγR on peripheral blood AML blasts. Using flow cytometry, we found heterogeneous expression of the FcγR throughout the patient cohort. Correlation of expression levels with disease outcome revealed significantly shorter OS in patients with CD16+ blasts at first diagnosis. CD32 and CD64 expression showed no association with survival but correlated with a mature phenotype and FAB M6. Our data provide clear evidence for the value of immunophenotyping FcγR expression on leukemic cells using peripheral blood, which is rapidly available and improves risk stratification in AML.

Low-density lipoprotein receptor (LDLR) is an independent adverse prognostic factor in acute myeloid leukaemia.

The low-density lipoprotein receptor (LDLR) is a membrane receptor that mediates the endocytosis of low-density lipoprotein (LDL). Uptake of LDL has been proposed to contribute to chemotherapy resistance of acute myeloid leukaemia (AML) cell lines in vitro. In the present study, we analysed LDLR expression and survival using bone marrow biopsies from 187 intensively treated patients with AML. Here, increasing LDLR expression was associated with decreasing overall (58·4%, 44·2%, and 24·4%; P=0·0018), as well as event-free survival (41·7%, 18·1%, and 14·3%; P=0·0077), and an increasing cumulative incidence of relapse (33·9%, 55·1%, and 71·4%; P=0·0011). Associations of LDLR expression with survival were confirmed in 557 intensively treated patients from two international validation cohorts. In the analytic and validation cohorts, LDLR expression remained associated with outcome in multivariable regression analyses including the European LeukemiaNet genetic risk classification. Thus, LDLR predicts outcome of patients with AML beyond existing risk factors. Furthermore, we found low expression levels of LDLR in most healthy tissues, suggesting it as a promising target for antibody-based pharmacodelivery approaches in AML.

Additional gene mutations may refine the 2017 European LeukemiaNet classification in adult patients with de novo acute myeloid leukemia aged

The European LeukemiaNet (ELN) recommendations for diagnosis and management of acute myeloid leukemia (AML) have become an important tool to assess patients' prognosis and guide treatment. We tested the prognostic impact of the 2017 ELN classification in a large cohort of 863 AML patients aged <60 years similarly treated on Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology studies. Based on multivariable models within each ELN genetic-risk group, we identified additional gene mutations that may refine the 2017 ELN risk classification. BCOR- or SETBP1-mutated favorable-risk patients with non-core-binding factor AML and IDH-mutated adverse-risk patients had intermediate-risk outcomes. Outcomes of NPM1/WT1 co-mutated patients and those of ZRSR2-mutated patients resembled outcome of adverse-risk patients. Moreover, FLT3-ITDhigh allelic ratio conferred adverse rather than intermediate-risk irrespective of the NPM1 mutation status, and DNMT3A mutations associated with very poor survival. Application of these refinements reclassified 9% of current favorable-risk patients and 53% of current intermediate-risk patients to the adverse-risk group, with similar poor survival as current adverse-risk patients. Furthermore, 4% of current favorable-risk patients and 9% of adverse-risk patients were reclassified to the intermediate-risk group.

Molecular dissection of normal karyotype acute myeloid leukemia.

7534 Background: Conventional cytogenetics remain one of the most important prognostic factors in acute myeloid leukemia (AML), though 50-60% of patients (pts) have normal karyotype (NK), conventionally classified as intermediate-risk, and have very heterogeneous outcomes. A fraction of mutations such as NPM1, FLT3-ITD, and CEBPa can improve risk stratification for some pts but underestimate the molecular complexity and interactions between these genes and others. Methods: Genomic and clinical data of 2,793 primary AML (pAML) pts were analyzed. A panel of 35 genes that are commonly mutated in AML and myeloid malignancies and have shown to impact OS was included. Correlation of each mutation with others and their impact on OS were evaluated. OS was calculated from the date of diagnosis to date of death or last follow-up. Results: Of 2,793 pts with pAML, 1,352 (48%) had NK and were included in the final analysis. The median age was 55 years (range, 18-93). The median number of mutations/sample was 3 (range, 0-7). The most commonly mutated genes were: NPM1 (49%), DNMT3A (37%), FLT3-ITD (24%), CEBPa (19%), TET2 (17%), IDH2 (17%), and RUNX1 (15%). In univariate Cox regression analysis, mutations in NPM1 (HR 0.81, p =0.008), and CEBPa (single mutant, HR 0.8, double mutant, HR 0.69, p&lt; 0.001, respectively) were associated with longer OS, while mutations in DNMT3a (HR 1.26, p =0.003), FLT3-ITD (HR 1.49, p&lt; 0.001), TET2 (HR 1.26, p =0.02), RUNX1 (HR 1.36, p =0.003), SRSF2 (HR 1.58, p &lt;0.001), IDH1 (HR 1.29, p &lt;0.001), and ASXL1 (HR 1.89, p &lt;0.001) were associated with shorter OS. A total of 67% of pts had NPM1, DNMT3A, and FLT3-ITD mutated alone or in combination with each other. The median OS for pts with NMP1Mut/ DNMT3AWT/FLT3-ITDWT was 99.1 months(m), NMP1Mut/DNMT3AMut /FLT3-ITDWT 54.8m, NMP1Mu t/DNMT3AWT/FLT3-ITDMut 42.3m, NMP1Mut/DNMT3AMut/FLT3-ITDMut 13.4m, NMP1WT/DNMT3AMut/FLT3-ITDMut 13.1m, and NMP1WT/DNMT3AWT/FLT3-ITDWT (triple negative) 32.7m. The median OS for pts with 0-2 mutations/sample was 59.3m, compared to 34.1m for pts with 3-4 mutations, and 16.1m for pts with &gt; 5 mutations ( p&lt; 0.001). Conclusions: We propose a simplified and robust approach to risk stratify AML pts with NK based on the mutational status of NPM1, DNMT3A, FLT3-ITD (alone or in combination with each other), CEBPa, and the number of mutations/sample.

High ACTN1 Is Associated with Poor Prognosis, and ACTN1 Silencing Suppresses Cell Proliferation and Metastasis in Oral Squamous Cell Carcinoma.

PurposeOral squamous cell carcinoma (OSCC) is a common malignancy of the oral cavity. As the survival rate of OSCC patients is low, it is crucial to explore new markers and therapeutic targets for early diagnosis of the disease. A high level of actinin alpha 1 (ACTN1) in patients could serve as an independent prognostic factor of acute myeloid leukemia. However, the role of ACTN1 in OSCC remains unclear. In the present study, we aimed to investigate the role of ACTN1 in OSCC.MethodsACTN1 protein levels in tissues were determined by immunohistochemical (IHC) staining. The correlation of ACTN1 expression with clinicopathological features and prognosis was analyzed. Univariate and multivariate analyses were performed. The effect of ACTN1 knockdown on cell proliferation, migration, invasion, apoptosis, epithelial-mesenchymal transition (EMT), and the cell cycle was evaluated using Western blotting, Cell Counting Kit‑8 (CCK8) assays, flow cytometry analysis, transwell assays, wound-healing assays, and nude mouse models of subcutaneous xenograft and pulmonary metastasis.ResultsBased on the total score of ACTN1 IHC staining analysis, ACTN1 expression was found to be low in 10 normal mucosal tissues, 48 normal mucosal tissues adjacent to OSCC, and 19 OSCC tissues, but high in 29 OSCC tissues. ACTN1 protein levels were significantly associated with the clinical stage and node metastasis, and a high ACTN1 protein level indicated poor prognosis. Moreover, ACTN1 expression was an independent predictor of poor prognosis of OSCC. Using in vitro assays, we found that ACTN1 knockdown could induce cell cycle arrest, promote apoptosis, and inhibit EMT and cell proliferation, migration, and invasion in the OSCC cell lines, SCC-15 and HSC-3. Moreover, ACTN1 knockdown inhibited subcutaneous tumor growth and pulmonary metastasis in vivo.ConclusionACTN1 levels were significantly associated with the clinical stage and node metastasis, and a high ACTN1 protein level indicated poor prognosis. Moreover, ACTN1 knockdown could suppress cell proliferation and metastasis of OSCC. Our results suggested that ACTN1 may serve as a diagnostic and prognostic marker of OSCC.

RUNX1 variant as a genetic predisposition factor for acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common hematological malignancy among adults and is characterized by accumulation of immature myeloid cells. Different genetic factors have role in the occurrence of AML. Among different proteins, RUNX1 and BAALC are involved in the development AML. It has been shown that BAALC overexpression is a factor that indicate shorter disease free survival in a subset of AML patients. RUNX1 has been implicated in the development of breast, prostate, lung, and skin cancers. The aim of this study is determination of the prevalence of common polymorphisms in BAALC (rs6999622 and rs62527607) and RUNX1 (rs13051066 and rs61750222) in AML patients compared with healthy subjects. A total of 100 AML patients and 100 healthy control subjects were included in our study. Genomic DNA was isolated from peripheral blood and the polymorphisms were genotyped by applying ARMS and PCR-RFLP methods. Finally, data was analyzed using SPPSS software. Our results demonstrate a significant association between the RUNX1 rs13051066 and AML in the co-dominant (odd ratio = 6.66, 95% Cl = 1.85–25, p = .006) and dominant (GT + TT versus GG: odd ratio = 6.15, 95% CI = 1.73–21.87, p = .002) models. The RUNX1 rs13051066 polymorphism is associated with risk of AML in Iranian population. Future studies should consider larger sample size for assessment of RUNX1 gene polymorphisms, and employ cytogenetic and molecular analyses in AML patients from different ethnic origins.

Characterization of Immune Dysfunction and Identification of Prognostic Immune-Related Risk Factors in Acute Myeloid Leukemia.

This study aims to provide comprehensive insights into longitudinal immune landscape in acute myeloid leukemia (AML) development and treatment, which may contribute to predict prognosis and guide clinical decisions. Periphery blood samples from 79 patients with AML (at diagnosis or/and after chemotherapy or at relapse) and 24 healthy controls were prospectively collected. We performed phenotypic and functional analysis of various lymphocytes through multiparametric flow cytometry and investigated prognostic immune-related risk factors. Immune defects in AML were reflected in T and natural killer (NK) cells, whereas B-cell function remained unaffected. Both CD8+ T and CD4+ T cells exhibited features of senescence and exhaustion at diagnosis. NK dysfunction was supported by excessive maturation and downregulation of NKG2D and NKP30. Diseased γδ T cells demonstrated a highly activated or even exhausted state through PD-1 upregulation and NKG2D downregulation. Effective therapeutic response following chemotherapy correlated with T and NK function restoration. Refractory and relapsed patients demonstrated even worse immune impairments, and selective immune signatures apparently correlated clinical outcomes and survival. PD-1 expression in CD8+ T cells was independently predictive of poor overall survival and event-free survival. T-cell senescence and exhaustion, together with impaired NK and γδ T-cell function, are dominant aspects involved in immune dysfunction in AML. Noninvasive immune testing of blood samples could be applied to predict therapeutic reactivity, high risk for relapse, and unfavorable prognosis.