Prognosis Of Acute Myeloid Leukemia Patients Research Articles

Up-regulation of EMT-related gene VCAN by NPM1 mutant-driven TGF-β/cPML signalling promotes leukemia cell invasion.

Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) is acknowledged as a distinct leukemia entity in the 2016 updated World Health Organization (WHO) classification. NPM1-mutated AML patients are correlated with higher extramedullary involvement. Epithelial-mesenchymal transition (EMT) is one of the key steps which cause distant metastasis in tumor. However, whether EMT-related programs contribute to cell invasion in NPM1-mutated AML remains unclear. In this study, we identified the EMT-related gene versican (VCAN) in NPM1-mutated AML across three patient datasets. Further experiments validated the elevated VCAN expression in NPM1-mutated AML primary blasts and OCI-AML3 cells with NPM1 mutation. Mechanistic studies revealed that increased VCAN expression was at least partially regulated by NPM1 mutant via TGF-β/cPML/Smad signalling. Functional evaluations showed that silencing VCAN by shRNA significantly suppressed cell migration and invasion capacity, whereas increased VCAN by overexpressing NPM1-mA enhanced migration and invasion ability of leukemia cells. Finally, we found that high expression of VCAN was associated with poor prognosis in AML patients. These findings provide insights into the involvement of EMT-related gene VCAN in the pathogenesis of NPM1-mutated leukemia, which suggests that VCAN is an attractive target for novel diagnostic and therapeutic strategies in NPM1-mutated AML.

Correlation of long non-coding RNA taurine-upregulated gene 1 with disease conditions and prognosis, as well as its effect on cell activities in acute myeloid leukemia.

This study aimed to investigate the correlation of long non-coding RNA taurine-upregulated gene 1 (lncRNA TUG1) with clinicopathological characteristics and prognosis in acute myeloid leukemia (AML) patients, as well as its function in cell proliferation and apoptosis. Two hundred and thirty six de novo AML patients were consecutively enrolled and then underwent conventional induction chemotherapy. Bone marrow samples were obtained from all AML patients and controls. Quantitative polymerase chain reaction assay was performed to detect lncRNA TUG1 expression. KG-1 cells were transfected by TUG1 inhibitor (TUG1 (-)) and blank inhibitor (NC (-)) plasmids. Cell proliferation and apoptosis were evaluated by CCK8 and AV/PI assays, and apoptotic markers expressions were detected by Western blot assay. LncRNA TUG1 expression was higher in AML patients compared to controls, and it was positively correlated with white blood cell counts as well as poor risk stratification. Additionally, elevated lncRNA TUG1 expression was observed in non-complete remission (non-CR) patients compared to CR patients, and it was correlated with shorter event-free survival and overall survival in AML patients. In the in vitro experiments, lncRNA TUG1 expression was upregulated in AML cell lines compared to control cells, and cell proliferation ability was reduced, but cell apoptosis rate was promoted in TUG1 (-) group compared to NC (-) group at 72 hours after transfection in KG-1 cells. LncRNA TUG1 predicts advanced disease conditions and poor prognosis in AML patients, and its knockout decreases proliferation and increases apoptosis of AML cells.

SETDB1 Represses Hox Gene Expression and Suppresses Acute Myeloid Leukemia

Epigenetic regulators play an important role in normal and malignant hematopoiesis. Epigenetic deregulation of the HOXA gene cluster drives transformation of about 50% of acute myeloid leukemia (AML), including those harboring MLL rearrangements and NPM mutations, as well as others. Expression of Hoxa9 and its co-factor Meis1 is sufficient to transform bone marrow into a lethal AML in mouse models. We previously demonstrated that the pro-leukemic genes Hoxa9 and Meis1 are critically regulated by the histone H3 Lysine 9 (H3K9) methyltransferase SETDB1. Recent studies show that SETDB1 is required for normal hematopoiesis and MLL-AF9 mediated leukemia (Koide, et al. Blood 2016). Our lab recently demonstrated that SETDB1 negatively regulates the expression of HoxA9 and Meis1 through deposition of promoter H3K9 methylation in MLL-AF9 AML cells (Ropa et al. Oncotarget 2018). Consistent with these data, HOXA9 and MEIS1 expression negatively correlates with SETDB1 expression in AML patient samples. Therefore, we investigated the biological impact of SETDB1 on AML.We first noted that expression of SETDB1 in AML patient samples is significantly lower compared to normal hematopoietic cells. Further, higher SETDB1 expression correlated with a significantly better overall survival (p=0.003) and lower expected hazard (HR=0.9/100RSEM; p=0.009) in AML patients compared with lower SETDB1 expression. These data are consistent with SETDB1 negatively regulating pro-leukemic genes and suggests that SETDB1 expression may be correlated with AML patient prognosis. We tested this directly by expressing high levels of SETDB1 in AML cells. Ex vivo assays show that retroviral overexpression of SETDB1 in MLL-AF9 AML cells leads to cell differentiation, decreased leukemia colony formation, and decreased cell proliferation. Consistent with the AML patient data, overexpression of SETDB1 significantly delays MLL-AF9 mediated leukemogenesis in vivo (p=0.01). Further, we observed a strong selective pressure against exogenous SETDB1 expression in moribund mice. Transcriptome analyses demonstrate that SETDB1 globally represses Hox and pluripotency gene programs. Strikingly, we found that SETDB1 represses many of the same genes that exhibit reduced promoter H3K9me3 in AML patient samples relative to CD34+ cells. These data point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing AML.We also explored how chemical and genetic inhibition of H3K9 methylation and Setdb1 affects AML initiation and maintenance. We first confirmed the previously reported requirement for Setdb1 in AML cell lines by genetically deleting both alleles of Setdb1 in MLL-AF9 cells, which resulted in a complete arrest of proliferation (Koide, et al. Blood 2016). Combined with our data presented above, these results suggest a narrow window of SETDB1 expression is maintained in AML cells. To achieve reduced (but not complete loss of) activity, we investigated how small molecule inhibition of H3K9 methylation (UNC0638) or shRNA mediated knock down of Setdb1 affects AML initiation. We observed increased ex vivo colony formation of normal ckit+ bone marrow cells upon shRNA mediated knockdown of Setdb1 or upon UNC0638 treatment. We hypothesized that this expansion of colony forming unit potential of hematopoietic cells may translate to increased transformation potential by leukemic oncogenes. Indeed, cells pretreated with UNC0638 followed by retroviral transduction with MLL-AF9 exhibit significantly higher capacity for leukemic colony formation than vehicle treated cells. These data are consistent with H3K9 methylation repressing genes required for AML transformation.Our data identified a narrow window of expression of SETDB1 in AML patient samples. SETDB1 expression is reduced in AML patients relative to normal cells and chemical inhibition of H3K9 methylation expands the pool of cells amenable to MLL-AF9 mediated transformation ex vivo. While inhibition of SETDB1 and other H3K9 methyltransferases has been suggested as a possible therapeutic strategy, our data suggests this may also prime bone marrow cells for transformation by inhibiting epigenetic processes that repress pro-leukemic target genes. Further investigation of the roles of SETDB1 and H3K9 methylation levels is necessary to determine the value of these epigenetic modifiers as therapeutic targets in AML and is currently ongoing. DisclosuresNo relevant conflicts of interest to declare.

The Oncogenic Role of N6-Methyladenosine Reader Protein IGF2BP3 in Acute Myeloid Leukemia

RNA N6-Methyladenosine (m6A) modification is an abundant modification of internal mRNAs in eukaryotes and some viruses, which is dynamically and reversibly fine-tuned during normal and pathological bioprocesses. Recent studies have shown that m6A methyltransferases, METTL3 and METTL14, play important roles in maintaining self-renewal capacity of hematopoietic stem/progenitor cells (HSPCs) and promoting acute myeloid leukemia (AML) development (Barbiori et al., Nature, 2017; Vu et al., Nature Method, 2017; Weng et al. Cell Stem Cell, 2018). The m6A demethylase, FTO, was also shown to promote leukemic cell transformation and leukemogenesis in various type of AML (Li et al., Cancer Cell, 2017). However, little is known about the functions of m6A readers in malignant hematopoiesis.We recently reported that Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is an specific m6A binding protein, which recognize m6A transcripts through the K Homology (KH) domains to stabilize and promote translation of its target mRNAs (Huang et al., Nature Cell Biology, 2018). In analysis of TCGA AML dataset (n=157), we found that a higher expression level of IGF2BP3 is significantly associated with a poor prognosis in AML patients (p<0.001; Median overall survival: IGF2BP3-High vs. IGF2BP3-Low = 11 months vs. 31 months). In addition, we analyzed our in-house microarray profiling of 113 AML patient samples and found that IGF2BP3 is highly expressed in mononuclear cells (MNC) from MLL-rearranged leukemia patients as compared to those from healthy donors (p<0.05) or non-MLL-rearranged leukemic patients (p<0.001). Consistent with the overexpression of IGF2BP3 in human MLL-rearranged AML, MLL-AF9 or MLL-AF10 transformed mouse hematopoietic stem/progenitor cells (HSPCs; herein mouse lineage negative (Lin-) bone marrow cells) showed a >10 fold increase in expression level of Igf2bp3, compare to the non-transformed counterpart HSPCs. Furthermore, in analysis of 562 samples from adult patients with AML (GSE37642), we found that within cytogenetically normal human AML, patients carrying FLT3-ITD mutation showed a significantly higher level of IGF2BP3 expression than those without FLT3-ITD mutation (p<0.01).To investigate the potential oncogenic role of IGF2BP3 in AML, we cotransduced mouse Lin- BM progenitor cells with MLL-AF9 and three individual shRNAs targeting Igf2bp3 or a scrambled control shRNA and performed colony-forming/replating assays. Knockdown of Igf2bp3 significantly (p<0.05) reduced the colony-forming capacity of MLL-AF9-transduced HSPCs to 20-50% of that of the control group. Conversely, forced expression of wild-type IGF2BP3 significantly (p<0.05) promoted colony formation of MLL-AF9-transduced Lin- BM progenitor cells. Such promotion was almost completely impaired when KH3-4 domain of IGF2BP3 was mutated or when Mettl14 was depleted, suggesting that IGF2BP3 exerts its oncogenic function as an m6A reader through an m6A-dependent mechanism.We further used human leukemia cell lines to investigate the function of IGF2BP3 in human AML cells. Silencing of IGF2BP3 by two shRNAs significantly inhibited cell viability and proliferation and induced cell apoptosis (p<0.01) in MonoMac6 AML cell line which harbors the t(9;11) translocation. In Molm13 and MV4-11 AML cells which are heterozygous and homozygous for the FLT3-ITD mutation, respectively, a further decrease of cell viability and increase of apoptotic cells upon IGF2BP3 knockdown was observed compared to MonoMac6 with wild-type FLT3. Mechanically, through cross-linking immunoprecipitation sequencing (CLIP-seq), we showed that IGF2BP3 targets mRNAs in cell cycle, DNA replication and protein synthesis pathways. Taken together, these results demonstrated the oncogenic role of the new m6A reader protein IGF2BP3 in AML. Given the fact that expression of IGF2BP3 correlates with an overall poor prognosis in AML, IGF2BP3 is likely a promising therapeutic target for AML treatment. DisclosuresNo relevant conflicts of interest to declare.

Multidimensional Study on the Prognostic Factors of Acute Myeloid Leukemia Patients with FLT3-ITD mutations Based on the Next-Generation Sequencing Platform

Purpose: This research aimed to investigate the features and effects of FLT3-ITD mutations in Chinese acute myeloid leukemia (AML) population, and to explore whether ITD variant allele frequency (VAF), number of mutations, insertionposition and length will have influence on the patients' overall survival (OS) and relapse-free survival (RFS).Methods:A total of 101 newly diagnosed AML (non-APL) patients with FLT3-ITD mutations from December 2013 to March 2018 were recruited. Their bone marrow or peripheral blood samples were sequenced by NGS 185-gene platform, then the sequencing data were analyzed in combination with the patients' clinical information.Results:The most common concomitant mutations in FLT3-ITD patients were NPM1, DNMT3A, TET2, IDH2, RUNX1, and WT1. The patients' median VAF is 0.344 (0.084-0.487). Among 101 patients, 80 (79.2%) carried 1 ITD mutation, 17 (16.8%) carried 2, 3 (3.0%) carried 3, and only 1 (1.0%) carried 4. In most patients (76/101) the ITD fragments were inserted in the JM region. And the median length of the insertion fragments was 54 (12-252) bp. According to the NCCN guidelines 2018, the cutoff value of FLT3-ITDmutation VAF between different stages of prognosis is 0.33. However, in our research, it was better to set 0.30 (p=0.0465 for OS; p=0.4929 for RFS), but not 0.33 (p=0.4270 for OS; p=0.3828 for RFS), as the cutoff value to tell differences of survival. Meanwhile, the numbers of carried ITD was significantly related to prognosis. The patients with single ITD mutation were found to have better clinical outcomes than those with multiple ITD mutations (p= 0.0031 for OS; p=0.0001 for RFS). Patients with ITD insertion in the hinge region of JM had a trend of poorer OS. The insertion length of ITD fragment correlated with the patients' RFS, but not OS. The longer the insertion was, the worse RFS of the patient showed (p=0.0323, p trend=0.0049).Conclusion:The VAF, the number of mutations, and the insertion length of the FLT3-ITDmutations will significantly affect the prognosis of AML patients in Chinese population. DisclosuresJin:College of Medicine, Zhejiang University: Employment; The National Natural Science Foundation of China: Research Funding.

Clinical Utility of Multiple-Gene Mutation Testing By Next-Generation Sequencing in Acute Myeloid Leukemia in Community Hospital Setting

Clinical Utility of Multiple-Gene Mutation Testing by Next-Generation Sequencing in Acute Myeloid Leukemia in Community Hospital SettingIntroductionThe WHO 2016 classification system and the 2017 European LeukemiaNet (ELN) genetic risk stratification system incorporate cytogenetic aberrations and mutations of specific genes to classify acute myeloid leukemia (AML) and stratify prognosis of AML patients. Next-generation sequencing (NGS) of multiple genes associated with AML has been shown to be an important tool in studying AML in research and academic center settings. Our goal is to evaluate the clinical application of NGS in a community hospital setting which has not been well documented leading to rejection of coverage by some insurers.MethodologyThis retrospective study included 109 patients (52 females and 57 males with mean age of 58.7) from November, 2016 to June, 2018 with various races/ethnicities background and confirmed diagnosis of AML at Florida Hospital (FH). DNA was extracted from the peripheral blood/bone marrow for NGS analysis. A total of 54 hematopoietic tumor associated genes were studied: ABL1, ASXL1, ATM, BCOR, BIRC3, BRAF, CALR, CBL, CEBPA, CREBBP, CSF1R, CSF3R, DNMT3A, EP300, ETV6, EZH2, FBXW7, FGFR4, FLT3, GATA1, GATA2, GATA3, IDH1, IDH2, IL7R, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, MPL, NF1, NOTCH1, NOTCH2, NPM1, NRAS, NSD1, PAX5, PDGFRA, PDGFRB, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, STAG2, TERT, TET1, TET2, TP53, TSLP,U2AF1 and ZRSR2. The sequencing was performed at Genomics and Pathology Services laboratory at Washington University in St. Louis and initial informatics analysis was performed by PierianDX using Clinical Genome Working platform. The initial analysis results were delivered to the pathologists at FH to do final interpretation with integration of the results of morphology/flow cytometry/karyotyping studies.The clinical utility of NGS was evaluated by: 1) comparing the AML classification and ELN genetic risk stratification of each patient with and without NGS data, 2) identifying gene mutations for targeted therapy and 3) other clinical impacts.ResultsNGS results changed ELN risk stratification in 45 cases (41.3%) and WHO AML classification in 37 cases (33.9%) out of 109 cases after considering the mutation status of NPM1, FLT3, RUNX1, ASXL1 and TP53. Among the 45 cases that had changes of ELN risk, 25 cases (55%) were downgraded to favorable risk group all because of mutations in NPM1 gene. While, the rest 20 cases (45%) were upgraded to adverse risk group due to the following mutations (RUNX1 in 7, TP53 in 5, ASXL1 in 4, RUNX1 and ASXL1 in 4). The change of WHO classification was caused by mutations in NPM1 (n=26) or RUNX1 (n=11). Additionally, NGS identified 8 cases with either GATA2 or CEBPA gene mutation, which are considered to increase the risk of myeloid neoplasms with germline predisposition based on WHO classification. Finally, NGS identified 42 patients (38.5%) carrying FLT3-ITD (n=17), FLT3-TKD (n=8) and/or IDH1/IDH2 (n=16) mutations. These mutations are known to have approved targeted therapies (FLT3 inhibitors and IDH2 inhibitors) or ongoing clinical trials (IDH1 inhibitors). Overall, NGS results led to clinically significant impacts in 62/109 (56.9%) patients by changing the AML classification/risk stratification, finding potential germline mutation and/or targeted therapies.DiscussionOur results indicate that NGS findings provide important information for classification, risk stratification, genetic predisposition, and treatment options in the majority (62/109, 56.9%) of AML patients in a community hospital setting. How the information exactly changes the clinical management by community practicing hematologists is currently ongoing and will provide further insights in regards to the clinical utilities of NGS. In addition, NGS of multiple genes is more cost- and labor-effective than single gene sequencing. Taken together, the insurers should accept NGS testing as a standard of care for AML patients. DisclosuresNo relevant conflicts of interest to declare.

TUG1 confers Adriamycin resistance in acute myeloid leukemia by epigenetically suppressing miR-34a expression via EZH2

Increasing evidence has suggested the involvement of long non-coding RNA (lncRNA) taurine upregulated gene 1 (TUG1) in chemoresistance of cancer treatment. However, its function and molecular mechanisms in acute myeloid leukemia (AML) chemoresistance are still not well elucidated. In the present study, we investigate the functional role of TUG1 in Adriamycin (ADR) resistance of AML and discover the underlying molecular mechanism. Our study revealed that TUG1 was up-regulated in ADR-resistant AML tissues and cells. High TUG1 expression was correlated with poor prognosis of AML patients. TUG1 knockdown improved the sensitivity of HL60/ADR cells to ADR. Moreover, TUG1 could epigenetically suppress miR-34a expression via recruiting Enhancer of zeste homolog 2 (EZH2). miR-34a overexpression could mimic the functional role of down-regulated TUG1 in ADR resistance. miR-34a knockdown counteracted the inductive effect of TUG1 inhibition on ADR sensitivity of HL60/ADR cells. Furthermore, TUG1 knockdown facilitated ADR sensitivity of ADR-resistant AML cells in vivo. In summary, TUG1 knockdown overcame ADR resistance of AML by epigenetically enhancing miR-34a expression, providing a novel therapeutic target for AML.

ADAMTS-13 activity reduction in plasma of acute myeloid leukemia predicts poor prognosis after bone marrow transplantation

ABSTRACTObjective: This research aimed to explore the significance of low activity of ADAMTS-13 in acute myeloid leukemia (AML) after bone morrow transplantation (BMT), and to evaluate the disease progress and prognosis of the patients with low or normal activity of ADAMTS-13 after BMT.Methods: 46 AML patients were included in our research. ADAMTS-13 activity was measured before BMT. Their medical indicators were recorded one month after BMT. All the patients were followed up and their disease progression was evaluated afterwards. The medical indicators and prognosis situation were compared between Low ADAMTS13 Group (<481 ng/ml) and Normal Group (481–785 ng/ml) according to the reference range of local laboratory. ROC curves were used to evaluate the predictive value of ADAMTS13 for infection complications and survival.Results: Low ADAMTS13 Group show extended APTT, PT and elevated CRP and D-Dimer levels, compared with Normal Group. Low ADAMTS13 Group suffered more BMT-related complications than Normal Group. In addition, Low ADAMTS13 Group underwent higher mortality than Normal Group in the one-year follow up after BMT and two-year follow up after onset of AML. ADAMTS13 has AUC of 0.7675, 0.7254, 0.8019 for lung infection, CMV infection and death within one year after BMT, suggesting that ADAMTS13 has predictive value for prognosis of AML patients after BMT.Discussion: For patients with low ADAMTS13 activity, the prognosis was worse and the probability of serious complications and mortality was significantly higher than AML patients with normal ADAMTS13 activity, which suggest predictive role of ADAMTS13 activity for the prognosis of AML after BMT.Conclusion: AML patients with low activity of ADAMTS-13 had worse prognosis after BMT.

Identification of Key Genes and Pathways Associated with RUNX1 Mutations in Acute Myeloid Leukemia Using Bioinformatics Analysis.

BACKGROUND RUNXl plays a key regulatory role in the process of hematopoiesis and is a common target for multiple chromosomal translocations in human acute leukemia. Mutations of RUNX1 gene can lead to acute leukemia and affect the prognosis of AML patients. We aimed to identify pivotal genes and pathways involved in RUNX1-mutated patients of with acute myeloid leukemia (AML) and to explore possible molecular markers for novel therapeutic targets of the disease. MATERIAL AND METHODS The RNA sequencing datasets of 151 cases of AML were obtained from the Cancer Genome Atlas database. Differentially expressed genes (DEGs) were identified using edgeR of the R platform. PPI (protein-protein interaction) network clustering modules were analyzed with ClusterONE, and the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses for modules were performed. RESULTS A total of 379 genes were identified as DEGs. The KEGG enrichment analysis of DEGs showed significantly enriched pathways in cancer, extracellular matrix (ECM)-receptor interaction pathway, and cyclic adenosine monophosphate (cAMP) signaling pathway. The top 10 genes ranked by degree were PRKACG, ANKRD7, RNFL7, ROPN11, TEX14, PRMT8, OTOA, CFAP99, NRXN1, and DMRT1, which were identified as hub genes from the protein-protein interaction network (PPI). Statistical analysis revealed that RUNX1-mutated patients with AML had a shorter median survival time (MST) with poor clinical outcome and an increased risk of death when compared with those without RUNX1 mutations. CONCLUSIONS DEGs and pathways identified in the present study will help understand the molecular mechanisms underlying RUNX1 mutations in AML and develop effective therapeutic strategies for RUNX1-mutation AML.

Identification of Key Genes and Pathways Associated with RUNX1 Mutations in Acute Myeloid Leukemia Using Bioinformatics Analysis

BackgroundRUNXl plays a key regulatory role in the process of hematopoiesis and is a common target for multiple chromosomal translocations in human acute leukemia. Mutations of RUNX1 gene can lead to acute leukemia and affect the prognosis of AML patients. We aimed to identify pivotal genes and pathways involved in RUNX1-mutated patients of with acute myeloid leukemia (AML) and to explore possible molecular markers for novel therapeutic targets of the disease.Material/MethodsThe RNA sequencing datasets of 151 cases of AML were obtained from the Cancer Genome Atlas database. Differentially expressed genes (DEGs) were identified using edgeR of the R platform. PPI (protein–protein interaction) network clustering modules were analyzed with ClusterONE, and the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses for modules were performed.ResultsA total of 379 genes were identified as DEGs. The KEGG enrichment analysis of DEGs showed significantly enriched pathways in cancer, extracellular matrix (ECM)-receptor interaction pathway, and cyclic adenosine monophosphate (cAMP) signaling pathway. The top 10 genes ranked by degree were PRKACG, ANKRD7, RNFL7, ROPN11, TEX14, PRMT8, OTOA, CFAP99, NRXN1, and DMRT1, which were identified as hub genes from the protein–protein interaction network (PPI). Statistical analysis revealed that RUNX1-mutated patients with AML had a shorter median survival time (MST) with poor clinical outcome and an increased risk of death when compared with those without RUNX1 mutations.ConclusionsDEGs and pathways identified in the present study will help understand the molecular mechanisms underlying RUNX1 mutations in AML and develop effective therapeutic strategies for RUNX1-mutation AML.

High expression levels of SMAD3 and SMAD7 at diagnosis predict poor prognosis in acute myeloid leukemia patients undergoing chemotherapy.

The SMAD family (SMAD1-9) was critically important for regulating cellular process through transforming growth factor-β signaling pathway, and contributed to carcinogenesis; however, their prognostic roles in acute myeloid leukemia (AML) remained unclear. This study collected 84 de novo AML patients treated with chemotherapy and 71 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). Kaplan-Meier survival estimate indicated that among SMAD1-9, high SMAD3 and SMAD7 expression were both associated with poor event-free survival (EFS) and overall survival (OS; all P < 0.05) in AML patients undergoing chemotherapy; and high SMAD6 expression was associated with shorter EFS and OS (all P < 0.01) in patients underwent allo-HSCT. Multivariate analysis showed that only high SMAD7 expression had adverse effect on EFS and OS (P = 0.021, 0.026) independently. Furthermore, High SMAD3 and SMAD7 expressers had significantly shorter EFS and OS than low expressers (P = 0.006, 0.001). In AML patients who went through allo-HSCT, there were no significant differences for EFS and OS between patients with high and low-expression SMAD3 or SMAD7. Our study suggested that high expression of SMAD3 and SMAD7 predicted adverse prognosis in AML patients undergoing chemotherapy and SMAD7 was a better prognostic marker than SMAD3. Their prognosis impact may be overcome by allo-HSCT.

Use of FLT3 inhibitors in acute myeloid leukemia remission induction or salvage therapy: systematic review and meta-analysis.

BackgroundPrevious studies showed that FLT3 inhibitors played an important role in acute myeloid leukemia (AML) therapy. However, discrepancies remain regarding the association between FLT3 inhibitors use and prognosis of AML patients in clinical trials.AimThe aim of this study was to evaluate the effect of FLT3 inhibitors on the treatment of AML in a systematic review and meta-analysis.Materials and methodsPubMed, Embase, and Cochrane Library databases were searched for studies published before August 2017 that used FLT3 inhibitors in AML. Fixed- and random-effect models were used, and between-study heterogeneity was assessed.ResultsA total of 26 studies fitting our inclusion and exclusion criteria were included. The FLT3 status of patients and main treatment outcomes including overall survival (OS), event-free survival (EFS), relapse-free survival (RFS), complete remission (CR), and overall response rate (ORR) after therapy were extracted. Five studies comparing addition of FLT3 inhibitors and placebo or blank control to chemotherapy were analyzed in Part I, showing improved OS (hazard ratio [HR]=0.86, 95% confidence interval [CI]=0.75–0.99, P=0.03) in the FLT3 inhibitor group but without a significant improvement on EFS (HR=0.86, 95% CI=0.62–1.21, P=0.39) and ORR (odds ratio [OR]=1.10, 95% CI=0.89–1.35, P=0.38). Twenty-one studies evaluating the benefit of using FLT3 inhibitors in different FLT3-type AML patients were analyzed in Part II, showing that FLT3–internal tandem duplication (ITD)-positive patients were more sensitive to FLT3 inhibitor treatment and achieved better CR (OR=1.89, 95% CI=1.06–3.37, P=0.03) and ORR (OR=3.07, 95% CI=2.13–4.43, P<0.001).ConclusionOur study showed that combined use of FLT3 inhibitors improved OS and that the FLT3 status of AML patients could affect their sensitivity to FLT3 inhibitors in terms of CR and ORR.

Abstract 2455: The long non-coding RNA (lncRNA) DANCR in acute myeloid leukemia (AML) stem cells (LSC)

Abstract Introduction: Prognosis for AML patients (pts) is inadequate. Therapeutic strategies are needed to target not only bulk blasts but also the distinct LSCs, which are more resistant to chemotherapy &amp; cause relapse. lncRNAs are non-coding RNA transcripts longer than 200 nucleotides that regulate cellular processes. A functional role in LSCs has yet to be elucidated. Methods: We derived a LSC-specific lncRNA profile using whole transcriptome sequencing (RNA-seq) data of 377 pts (&amp;lt;60 years) with cytogenetically normal (CN) AML. These data were correlated with a 'core enriched' (CE) gene expression signature representing 44 genes deregulated in LSCs (Eppert, Nat.Med. 2011). DANCR expression in the LSC-enriched compartment was validated using qRT-PCR. Transferrin/antibody conjugated nanoparticle (NP) knock down (KD) was used as an efficient &amp; less toxic avenue to deliver siRNA against DANCR or scramble (SCR) in vitro &amp; in vivo. LSC features (self-renewal, quiescence, engraftment) were analyzed &amp; frequency determined in 3 primary AML samples and a CN-AML (Flt3ITD/WT/MllPTD/WT) mouse model. Results: 161 lncRNAs were consistently deregulated in CEhigh pts with a p&amp;lt;.001 &amp; a correlation coefficient &amp;gt;.5 (Spearmen correlation test). We chose DANCR to further analyze because it was among the top lncRNAs upregulated in CEhigh pts, is highly conserved between human &amp; mouse, &amp; was previously shown to have a role in promoting stemness in hepatocellular carcinoma (Yuan, Hepatology 2016). We confirmed that DANCR expression was significantly higher in functionally validated LSC enriched populations (p=.05). To assess the role in self-renewal, we KD DANCR in pts cells &amp; found a decrease in colony numbers after replating (average decrease vs SCR: 38.9%, p=.03). Using membrane labeling assays, we found a decrease of quiescent cells after DANCR KD (average decrease: 36.1%, p=.04). Long term colony-initiating cell cultures were performed to determine the LSC frequency &amp; we found significant decreases in the number of LSCs in primary pts samples after DANCR KD (average decrease of LSC frequency: 79.2%, p=.001). Mice transplanted with murine AML cells (CD45.2+) were treated with NP (n=3 per group) for 5 days (d). 2d later, bone marrow was harvested &amp; retransplantated (Tx) using limiting dilutions (2x106, 1x106, 5x105) into irradiated BoyJ (CD45.1+) recipients (n=30). 14d post-Tx, anti-Dancr recipient mice showed a lower engraftment (CD45.2/CD45.1: 37% vs 47%, p=.03) and also a concomitant improvement in survival (p&amp;lt;.001). Conclusion: We show for the first time that LSCs have a distinct lncRNA profile. Among the highly expressed lncRNAs associated with LSCs we identified &amp; validated DANCR. Our data show that DANCR has an impact on LSC function (self-renewal, quiescence, engraftment) &amp; regulates LSC frequency. DANCR is the first lncRNA shown to have a functional role in LSCs &amp; represents a novel target for AML treatment. Citation Format: Marius Bill, Malith Karunasiri, Jessica Kohlschmidt, Allison E. Walker, Zachary Brannan, Stefano Volinia, Clara D. Bloomfield, Ramiro Garzon, Adrienne M. Dorrance. The long non-coding RNA (lncRNA) DANCR in acute myeloid leukemia (AML) stem cells (LSC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2455.

Octamer-binding transcription factor 4 correlates with complex karyotype, FLT3-ITD mutation and poorer risk stratification, and predicts unfavourable prognosis in patients with acute myeloid leukaemia

ABSTRACTObjectiveTo investigate the correlation of octamer-binding transcription factor 4 (OCT4) expression with clinicopathological features and its predictive value for treatment response as well as survival profiles in de novo acute myeloid leukaemia (AML) patients.MethodOne hundred fifty-two de novo AML patients and 52 non-hematologic malignancy patients were recruited in this prospective cohort study. OCT4 expression was determined in bone marrow sample collected before treatment. Complete response (CR), event free survival (EFS) and overall survival (OS) were evaluated.ResultsCompared with the controls, OCT4 mRNA expression was higher in AML patients (P < .001), and higher OCT4 expression was correlated with presence of complex karyotype (CK) (P = .037), FLT3-ITD mutation (P = .012) and poorer risk stratification (P < .001) in AML patients. As to predictive value, OCT4 mRNA expression was decreased in patients achieved CR compared to non-CR patients (P = .022). Kaplan–Meier (K–M) curves showed that shorter EFS (9.0 (95% CI (7.7–10.3)) months vs. 25.0 (95% CI (17.5–32.5)) months, P < .001) and shorter OS (20.0 (95% CI (17.8–22.2) months vs. 33.0 months, P < .001) were observed in OCT4 mRNA high expression patients compared to OCT4 mRNA low expression patients. Multivariate Cox’s proportional hazards regression analyses revealed that OCT4 mRNA high expression was an independent predictive factor for shorter EFS and OS in AML patients.ConclusionOCT4 correlates with presence of CK, FLT3-ITD mutation and poorer risk stratification, and it could be served as a convincing biomarker for predicting unfavourable prognosis in AML patients.

Clinical features and prognosis of acute myeloid leukemia patients with incomplete blood recovery at the time of achieving morphologic complete remission

Objective To analyze the clinical features and prognosis of acute myeloid leukemia (AML) patients with incomplete blood recovery [platelet count (Plt) < 100×109/L or absolute neutrophil count (ANC) < 1.0×109/L] at the first time of achieving morphologic complete remission(CR). Methods The clinical data of 302 AML patients (non-M3) who were treated at the First Affiliated Hospital of Zhengzhou University from January 2010 to September 2015 were retrospectively reviewed. They were divided into CR group (Plt≥100×109/L and ANC≥1.0×109/L) and CR with incomplete blood recovery group (Plt < 100×109/L or ANC < 1.0×109/L) according to the condition of blood count at the first time of achieving morphologic CR. The clinical features and prognostic differences between the two groups were compared, and univariate analysis and mutivariate analysis were performed to compare the recurrence and survival of AML with incomplete blood recovery. Results Two hundred and sixteen (71.5%) patients were in CR group and 86 (28.5%) were in CR with incomplete blood recovery group. There was no statistically difference between the two groups in terms of age, high white blood cell, peripheral blasts ratio, France-American-Britain (FAB) type, cytogenetic risk classification and FLT3-ITD/ NPM1 gene mutation (all P > 0.05). Bone marrow blasts before therapy and the proportion of bone marrow CR in the first course of chemotherapy in CR group were higher than those in CR with incomplete blood recovery group (both P 0.05). In addition, FLT3-ITD positive was an independent risk factor for OS rate(P < 0.001), and peripheral blood blasts ≥0.60 was an independent risk factor for DFS rate (P= 0.047). Conclusions AML patients with incomplete blood recovery at the first time of achieving morphologic CR have a poor prognosis. AML patients with high-risk karyotype, ≥2 courses of induction therapy, incomplete neutrophils recovery may have a worse prognosis, and they should be regarded as high-risk groups and given more aggressive treatment. Key words: Leukemia, myeloid, acute; Blood cell count; Prognosis

A three‑lncRNA signature for prognosis prediction of acute myeloid leukemia in patients.

Long non-coding RNAs (lncRNAs) are transcripts characterized by >200 nucleotides, without validated protein production. Previous studies have demonstrated that certain lncRNAs have a critical role in the initiation and development of acute myeloid leukemia (AML). In the present study, the subtype-specific lncRNAs in AML was identified. Following the exclusion of the subtype-specific lncRNAs, the prognostic value of lncRNAs was investigated and a three-lncRNA expression-based risk score [long intergenic non-protein coding RNA 926, family with sequence similarity 30 member A and LRRC75A antisense RNA 1 (LRRC75A-AS1)] was developed for AML patient prognosis prediction by analyzing the RNA-seq data of AML patients from Therapeutically Available Research to Generate Effective Treatments (TARGET) and The Cancer Genome Atlas (TCGA) projects. In the training set obtained from TARGET, patients were divided into poor and favorable prognosis groups by the median risk score. The prognostic effectiveness of this lncRNA risk score was confirmed in the validation set obtained from TCGA by the same cut-off. Furthermore, the lncRNA risk score was identified as an independent prognostic factor in the multivariate analysis. As further verification of the independent prognostic power of the lncRNA risk score, stratified analysis was performed by a cytogenetics risk group and revealed a consistent result. The prognostic predictive ability of the risk score was compared with the cytogenetics risk group by time-dependent receiver operating characteristic curves analysis. It was revealed that the combination of the lncRNA risk score and cytogenetics risk group provided a higher prognostic value than a single prognostic factor. The present study also performed co-expression analysis to predict the potential regulatory mechanisms of these lncRNAs in a cis/trans/competing endogenous RNA manner. The results suggested that LRRC75A-AS1 was highly associated with the target genes of transcription factors tumor protein 53 and ETS variant 6. Overall, these results highlighted the use of the three-lncRNA expression-based risk score as a potential molecular biomarker to predict the prognosis in AML patients.

Decreased SCIN expression, associated with promoter methylation, is a valuable predictor for prognosis in acute myeloid leukemia.

The present study was aimed to investigate SCIN expression as well as promoter methylation and further explore their clinical relevance in acute myeloid leukemia (AML) patients. Real-time quantitative PCR was carried out to detect the expression level of SCIN in 119 AML patients and 37 healthy controls. Real-time quantitative methylation-specific PCR and bisulfite sequencing PCR were carried out to detect SCIN promoter methylation levels in 103 AML patients and 29 controls. As compared with controls, the level of SCIN transcript was significantly down-regulated in AML patients (P = 0.001), and the level of methylated SCIN promoter was significantly higher in AML patients (P = 0.001). Moreover, the level of promoter methylation was weakly negatively correlated with SCIN expression in AML patients (R = -0.265, P = 0.027). Demethylation of SCIN promoter by 5-aza-2'-deoxycytidine could restore its expression in leukemic cell line THP1. The age of SCINlow patients was significantly higher and C/EBPA mutation was significantly less than SCINhigh patients (P = 0.039 and 0.038, respectively). Moreover, the rate of complete remission (CR) of SCINlow patients was significantly lower than SCINhigh patients (P = 0.009). Kaplan-Meier analysis showed that low SCIN expression was associated with shorter overall survival (P = 0.036). Cox regression analysis demonstrated low SCIN expression was an independent poor prognostic factor (P = 0.047). Furthermore, SCIN expression was restored in those patients who achieved CR after induction therapy (P = 0.003). These findings indicate that decreased SCIN expression associated with its promoter methylation is a valuable biomarker for predicting adverse prognosis in AML patients.

Study on relationship between Set gene expression and clinical manifestations in bone marrow of patients with acute myelogenous leukemia

Objective: To explore the expression and significance of Set gene in Acute myeloid leukemia (AML) patients , and to analyze its effect for the prognosis of AML. Methods: The level of Set gene expression was detected by real-time PCR in 59 AML patients and 20 heathy people. The mutations in C-kit 8/17 gene, NPM1 gene and FLT3-TKD/ITD gene in 59 AML patients were detected by direct sequencing. Results: The level of Set gene expression[1.41(0.41-3.31)]was significantly higher in 59 AML patients.The expression of Set gene was correlated with the percentage of marrow blasts and CR in AML patients (P=0.040, P<0.001); the CR rate of Set gene high expression group was significantly lower than that of Set gene low expression group(32.1% vs 83.9%, P=0.01). In the intermediate-risk of AML patients with chromosome karyotype analysis, the CR rate of Set gene high expression group and low expression group were 34.8% and 88.9%, and there are significantly different between two groups(P<0.001); univariate and multivariate analysis showed that Set gene high expression group correlated with poor OS[4(2-15)months]and EFS[3(2-13)months])(P=0.021, P=0.017). It suggests that the Set gene maybe one of AML independent poor prognostic marker.The level of Set gene expression did not correlate with sex, age, WBC, HGB, PLT, FAB typing, chromosomal karyotype and NPM1, C-Kit8/17, CEBPa, FLT3-ITD/TKD gene mutations in AML patients(all P>0.05). Conclusions: The level of Set gene expression in bone marrow maybe play an important role in AML. The high expression of Set gene indicates poor prognosis in AML patients.

Prognostic significance of SOX2, SOX3, SOX11, SOX14 and SOX18 gene expression in adult de novo acute myeloid leukemia

Aberrant expression of different SOX (SRY-related high mobility group (HMG) box) genes has been observed in number of tumors but, little is known about their expression patterns in hematological malignancies, especially in acute myeloid leukemia (AML).In this study we investigated SOX2, SOX3, SOX11, SOX14 and SOX18 gene expression in 50 de novo adult AML patients and correlated our findings with known clinical and molecular prognostic markers of the disease.We have found that these genes are overexpressed in 10–22% of patients and preliminary findings suggest that high expression level of these genes may have prognostic significance in AML patients.This is the first study focused on examining the expression level of SOX2, SOX3, SOX11, SOX14 and SOX18 genes in AML patients. Although this is a relatively limited study, initial findings indicate the need for further investigation of these genes, their potential roles in leukemia pathogenesis as well as prognosis in AML patients.

Mutational spectrum of acute myeloid leukemia patients with double CEBPA mutations based on next-generation sequencing and its prognostic significance.

The aim of this study was to profile the spectrum of genetic mutations in acute myeloid leukemia (AML) patients co-occurring with CEBPA double mutation (CEBPAdm). Between January 1, 2012, and June 30, 2017, 553 consecutive patients with de novo AML were screened for CEBPA mutations. Out of these, 81 patients classified as CEBPAdm were analyzed further by a sensitive next-generation sequencing assay for mutations in 112 candidate genes. Within the CEBPA gene itself, we found 164 mutations. The most common mutated sites were c.936_937insGAG (n = 11/164, 6.71%) and c.939_940insAAG (n = 11/164, 6.71%), followed by c.68dupC (n = 10/164, 6.10%). The most common co-occurring mutations were found in the CSF3R (n = 16/81, 19.75%), WT1 (n = 15/81, 18.52%), and GATA2 (n = 13/81, 16.05%) genes. Patients with CSF3R mutations had an inferior four-year relapse-free survival (RFS) than those with the wild-type gene (15.3% versus 46.8%, respectively; P = 0.021). Patients with WT1 mutations had an inferior five-year RFS compared with those without such mutations (0% versus 26.6%, respectively, P = 0.003). However, GATA2, CSF3R, WT1 mutations had no significant influence on the overall survival. There were some differences in the location of mutational hotspots within the CEBPA gene, as well as hotspots of other co-occurring genetic mutations, between AML patients from Chinese and Caucasian populations. Some co-occurring mutations may be potential candidates for refining the prognoses of AML patients with CEBPAdm in the Chinese population.