Detection of NPM1 Mutations in Acute Myeloid Leukemia by using Drop-Off Droplet Digital PCR and its Clinical Application.

BACKGROUND: Nucleophosmin 1 (NPM1) mutations, which occur in 25 - 30% of acute myeloid leukemia (AML) and 50 - 60% of AML with normal karyotype, have been identified as an important marker for stratification of prog-nosis in AML. This study aimed to establish a new quantitative polymerase chain reaction (PCR) technique, the drop-off droplet digital PCR (ddPCR), for rapid and sensitive detection of NPM1 mutations in AML. METHODS: We established the drop-off ddPCR system and verified its performance. NPM1 mutations were screened in 130 AML patients by drop-off ddPCR and were validated by Sanger sequencing and next-generation sequencing (NGS). Then, the NPM1 mutation burden was dynamically monitored in five patients. RESULTS: The limit of blank (LOB) of drop-off ddPCR established for NPM1 mutation was 3.36 copies/µL, and the limit of detection (LOD) was 5.00 - 5.37 copies/µL in 50 ng DNA, and the sensitivity was about 0.05%, which had good linearity. Drop-off ddPCR identified 33/130 (25.4%) NPM1 mutated cases, consistent with Sanger sequencing. In 18 NPM1 positive cases selected randomly, NGS identified fourteen with type A mutation, two with type D mutation, and two with rare type mutations. The mutation burden of NPM1 mutation analyzed by NGS was consistent with the drop-off ddPCR. The sequential samples were detected for measurable residual disease (MRD) monitoring in 5 patients showed that the NPM1 mutation burden was consistent with clinical remission and recurrence. Compared with traditional ddPCR, drop-off ddPCR was also suitable for MRD monitoring. CONCLUSIONS: In this study, we established a drop-off ddPCR method for detecting three common mutations in AML with good sensitivity and repeatability, which can be used to screen mutations in newly diagnosed AML patients and for MRD monitoring after remission to guide treatment.

Identification and validation of necroptosis-related gene signatures to predict clinical outcomes and therapeutic responses in acute myeloid leukemia.

BACKGROUND: Necroptosis is a tightly regulated form of necrotic cell death that promotes inflammation and contributes to disease development. However, the potential roles of necroptosis-related genes (NRGs) in acute myeloid leukemia (AML) have not been elucidated fully. METHODS: We conducted a study to identify a robust biomarker signature for predicting the prognosis and immunotherapy efficacy based on NRGs in AML. We analyzed the genetic and transcriptional alterations of NRGs in 151 patients with AML. Then, we identified three necroptosis clusters. Moreover, a necroptosis score was constructed and assessed based on the differentially expressed genes (DEGs) between the three necroptosis clusters. RESULTS: Three necroptosis clusters were correlated with clinical characteristics, prognosis, the tumor microenvironment, and infiltration of immune cells. A high necroptosis score was positively associated with a poor prognosis, immune-cell infiltration, expression of programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1), immune score, stromal score, interferon-gamma (IFNG), merck18, T-cell dysfunction-score signatures, and cluster of differentiation-86, but negatively correlated with tumor immune dysfunction and exclusion score, myeloid-derived suppressor cells, and M2-type tumor-associated macrophages. Our observations indicated that a high necroptosis score might contribute to immune evasion. More interestingly, AML patients with a high necroptosis score may benefit from treatment based on immune checkpoint blockade. CONCLUSIONS: Consequently, our findings may contribute to deeper understanding of NRGs in AML, and facilitate assessment of the prognosis and treatment strategies.

ALOX5AP is a new prognostic indicator in acute myeloid leukemia.

BACKGROUND: The overexpression of ALOX5AP has been observed in many types of cancer and has been identified as an oncogene. However, its role in acute myeloid leukemia (AML) has not been extensively studied. This study aimed to identify the expression and methylation patterns of ALOX5AP in bone marrow (BM) samples of AML patients, and further explore its clinical significance. METHODS: Eighty-two de novo AML patients and 20 healthy donors were included in the study. Meanwhile, seven public datasets from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were included to confirm the alteration of ALOX5AP. Receiver operating characteristic (ROC) curve analysis was applied to determine the discriminative capacity of ALOX5AP expression to discriminate AML. The prognostic value of ALOX5AP was identified by the Kaplan-Meier method and log-rank test. It was further validated in four independent cohorts (n = 1186). Significantly different genes associated with ALOX5AP expression were subsequently compared by LinkedOmics, and Metascape database. RESULTS: The level of ALOX5AP expression was significantly increased in bone marrow cells of AML patients compared with healthy donors (P < 0.05). ROC curve analysis suggested that ALOX5AP expression might be a potential biomarker to discriminate AML from controls. ALOX5AP overexpression was associated with decreased overall survival (OS) in AML according to the TCGA data (P = 0.006), which was validated by other four independent cohorts. DNA methylation levels of ALOX5AP were significantly lower in AML patients compared to normal samples (P < 0.05), as confirmed in the Diseasemeth database and the independent cohort GSE63409. ALOX5AP level was positively associated with genes with proleukemic effects such as PAX2, HOX family, SOX11, H19, and microRNAs that act as oncogenes in leukemia, such as miR125b, miR-93, miR-494, miR-193b, while anti-leukemia-related genes and tumor suppressor microRNAs such as miR-582, miR-9 family and miR-205 were negatively correlated. CONCLUSION: ALOX5AP overexpression, associated with its hypomethylation, predicts poorer prognosis in AML.

Pan-cancer analysis identifies CD300 molecules as potential immune regulators and promising therapeutic targets in acute myeloid leukemia.

BACKGROUND: CD300s are a group of proteins playing vital roles in immune responses. However, much is yet to be elucidated regarding the expression patterns and clinical significances of CD300s in cancers. METHODS: In this study, we comprehensively investigated CD300s in a pan-cancer manner using multi-omic data from The Cancer Genome Atlas. We also studied the relationship between CD300s and the immune landscape of AML. RESULTS: We found that CD300A-CD300LF were generally overexpressed in tumors (especially AML), whereas CD300LG was more often downregulated. In AML, transactivation of CD300A was not mediated by genetic alterations but by histone modification. Survival analyses revealed that high CD300A-CD300LF expression predicted poor outcome in AML patients; the prognostic value of CD300A was validated in seven independent datasets and a meta dataset including 1115 AML patients. Furthermore, we demonstrated that CD300A expression could add prognostic value in refining existing risk models in AML. Importantly, CD300A-CD300LF expression was closely associated with T-cell dysfunction score and could predict response to AML immunotherapy. Also, CD300A was found to be positively associated with HLA genes and critical immune checkpoints in AML, such as VISTA, CD86, CD200R1, Tim-3, and the LILRB family genes. CONCLUSIONS: Our study demonstrated CD300s as potential prognostic biomarker and an ideal immunotherapy target in AML, which warrants future functional and clinical studies.

SLC22A3 methylation-mediated gene silencing predicts adverse prognosis in acute myeloid leukemia.

BACKGROUND: We screened out several hypermethylated solute carrier (SLC) family genes in acute myeloid leukemia by reduced representation bisulfite sequencing. SLC22A3 encodes an organic cation transport protein, which is critical for drug transportation and cellular detoxification. SLC22A3 is significantly downregulated and associated with tumor progression and worse prognosis in a variety of solid tumors. However, there are no data available regarding the role of SLC22 in AML. This study aimed to explore the regulatory mechanism of DNA methylation on SLC22A3 expression, as well as its clinical significance in AML prognosis. RESULTS: SLC22A3 was identified as the sole prognosis-associated gene among SLCs based on TCGA and Beat AML databases. Bone marrow mononuclear cells (BMMNCs) from AML, MDS patients, and healthy donors were enrolled in this study. SLC22A3 methylation was significantly increased in AML compared with controls and MDS patients; meanwhile, the expression level of SLC22A3 was decreased. SLC22A3 hypermethylation presented an obvious association with some specific clinical characteristics and affected the survival time of AML patients as an independent risk indicator. SLC22A3 expression changed regularly as the disease complete remissions and relapses. Demethylation drug 5-aza-2'-deoxycytidine (DAC) activated transcription and increased mRNA expression of SLC22A3 in leukemia cell lines and AML fresh BMMNCs. Knockdown of SLC22A3 in leukemia cells enhanced cell proliferation and suppressed cell apoptosis. Data from public programs were used for auxiliary screening of probable molecular mechanisms of SLC22A3 in the antileukemia effect. CONCLUSIONS: Our results showed that increased methylation and decreased expression of SLC22A3 may be indicators of poor prognosis in AML. Methylation-silenced SLC22A3 expression may have potential guiding significance on antileukemia effect of DAC.

m6A regulator-based methylation modification patterns and characterization of tumor microenvironment in acute myeloid leukemia.

RNA N6-methyladenosine (m6A) is the most common and intensively studied RNA modification that critically regulates RNA metabolism, cell signaling, cell survival, and differentiation. However, the overall role of multiple m6A regulators in the tumor microenvironment (TME) has not yet been fully elucidated in acute myeloid leukemia (AML). In our study, we explored the genetic and transcriptional alterations of 23 m6A regulators in AML patients. Three distinct molecular subtypes were identified and associated with prognosis, patient clinicopathological features, as well as TME characteristics. The TME characterization revealed that m6A patterns were highly connected with metabolic pathways such as biosynthesis of unsaturated fatty acids, cysteine and methionine metabolism, and citrate cycle TCA cycle. Then, based on the differentially expressed genes (DEGs) related to m6A molecular subtypes, our study categorized the entire cohort into three m6A gene clusters. Furthermore, we constructed the m6Ascore for quantification of the m6A modification pattern of individual AML patients. It was found that the tumor-infiltrating lymphocyte cells (TILs) closely correlated with the three m6A clusters, three m6A gene clusters, and m6Ascore. And many biological processes were involved, including glycogen degradation, drug metabolism by cytochrome P450, pyruvate metabolism, and so on. Our comprehensive analysis of m6A regulators in AML demonstrated their potential roles in the clinicopathological features, prognosis, tumor microenvironment, and particularly metabolic pathways. These findings may improve our understanding of m6A regulators in AML and offer new perspectives on the assessment of prognosis and the development of anticancer strategy.

Whole-Genome DNA Methylation Sequencing Reveals Epigenetic Changes in Myelodysplastic Syndromes.

Epigenetic dysregulation of cancer-associated genes has been identified to contribute to the pathogenesis of myelodysplastic syndromes (MDS). However, few studies have elucidated the whole-genome DNA methylation in the initiation pathogenesis of MDS. Reduced representation bisulfite sequencing was performed in five de novo MDS patients and four controls to investigate epigenetic alterations in MDS pathogenesis. The mean global methylation in five MDS patients showed no significant difference compared with the four controls. In depth, a total of 1,459 differentially methylated fragments, including 759 hypermethylated and 700 hypomethylated fragments, were identified between MDS patients and controls. Targeted bisulfite sequencing further identified that hypermethylation of DLEU7, FOXR1, LEP, and PANX2 were frequent events in an additional cohort of MDS patients. Subsequently, LEP hypermethylation was confirmed by real-time quantitative methylation-specific PCR in an expanded cohort of larger MDS patients. In clinics, LEP hypermethylation tended to be associated with lower bone marrow blasts and was significantly correlated with U2AF1 mutation. Survival analysis indicated that LEP hypermethylation was associated with a markedly longer survival time but was not an independent prognostic biomarker in MDS patients. Functional studies revealed pro-proliferative and anti-apoptotic effects of leptin in the MDS cell line SKM-1, and it was significantly associated with cell growth and death as well as the Toll-like receptor and NF-kappa B signaling pathways. Collectively, our findings demonstrated that whole-genome DNA methylation analysis identified novel epigenetic alterations such as DLEU7, FOXR1, LEP, and PANX2 methylations as frequent events in MDS. Moreover, LEP might play a role in MDS pathogenesis, and LEP hypermethylation was associated with longer survival but not as an independent prognostic biomarker in MDS.

Pan-cancer analysis reveals distinct clinical, genomic, and immunological features of the LILRB immune checkpoint family in acute myeloid leukemia.

Leukocyte immunoglobulin (Ig)-like receptor Bs (LILRBs), a family of type I transmembrane glycoproteins, are known to inhibit immune activation. Here, we comprehensively evaluated the molecular, prognostic, and immunological characteristics of LILRB members in a broad spectrum of cancer types, focusing on their roles in acute myeloid leukemia (AML). We showed that LILRBs were significantly dysregulated in a number of cancers and were associated with immune-inhibitory phenotypes. Clinically, high expression of LILRB1-LILRB4 predicted poor survival in six independent AML cohorts. Genetically, LILRB1 was associated with more mutational events than other LILRB members, and multiple genes involved in immune activation were deleted in LILRB1 high patients. Epigenetically, LILRB4 was significantly hypomethylated and marked by MLL-associated histone modifications in AML. Immunologically, LILRBs were positively associated with monocytic cells, including M2 macrophages, but were negatively associated with tumor-suppressive CD8 T cells. Importantly, patients with higher LILRB expression generally showed a better response to immune checkpoint blockade (ICB) in five independent immunotherapy cohorts. Our findings reveal critical immunological and clinical implications of LILRBs in AML and indicate that LILRBs may represent promising targets for immunotherapy of AML.

DNA methylation-mediated differential expression of DLX4 isoforms has opposing roles in leukemogenesis.

BACKGROUND: Previously, we reported the expression of DLX4 isoforms (BP1 and DLX7) in myeloid leukemia, but the functional role of DLX4 isoforms remains poorly understood. In the work described herein, we further determined the underlying role of DLX4 isoforms in chronic myeloid leukemia (CML) leukemogenesis. METHODS: The expression and methylation of DLX4 isoforms were detected by real-time quantitative PCR (RT-qPCR) and real-time quantitative methylation-specific PCR (RT-qMSP) in patients with CML. The functional role of DLX4 isoforms was determined in vitro and in vivo. The molecular mechanism of DLX4 isoforms in leukemogenesis was identified based on chromatin immunoprecipitation with high-throughput sequencing (ChIP-Seq)/assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) and RNA sequencing (RNA-Seq). RESULTS: BP1 expression was increased in patients with CML with unmethylated promoter, but DLX7 expression was decreased with hypermethylated promoter. Functionally, overexpression of BP1 increased the proliferation rate of K562 cells with S/G2 promotion, whereas DLX7 overexpression reduced the proliferation rate of K562 cells with G1 arrest. Moreover, K562 cells with BP1 overexpression increased the tumorigenicity in NCG mice, whereas K562 cells with DLX7 overexpression decreased the tumorigenicity. Mechanistically, a total of 91 genes including 79 messenger RNAs (mRNAs) and 12 long noncoding RNAs (lncRNAs) were discovered by ChIP-Seq and RNA-Seq as direct downstream targets of BP1. Among the downstream genes, knockdown of RREB1 and SGMS1-AS1 partially revived the proliferation caused by BP1 overexpression in K562 cells. Similarly, using ATAC-Seq and RNA-Seq, a total of 282 genes including 151 mRNA and 131 lncRNAs were identified as direct downstream targets of DLX7. Knockdown of downstream genes PTPRB and NEAT1 partially revived the proliferation caused by DLX7 overexpression in K562 cells. Finally, we also identified and validated a SGMS1-AS1/miR-181d-5p/SRPK2 competing endogenous RNA (ceRNA) network caused by BP1 overexpression in K562 cells. CONCLUSIONS: The current findings reveal that DNA methylation-mediated differential expression of DLX4 isoforms BP1 and DLX7 plays opposite functions in leukemogenesis. BP1 plays an oncogenic role in leukemia development, whereas DLX7 acts as a tumor suppressor gene. These results suggest DLX4 as a therapeutic target for antileukemia therapy.

SLIT2 promoter hypermethylation-mediated SLIT2-IT1/miR-218 repression drives leukemogenesis and predicts adverse prognosis in myelodysplastic neoplasm.

Epigenetic modifications have been found to play crucial roles in myelodysplastic neoplasm (MDS) progression. Previously, we investigated genome-wide DNA methylation alterations during MDS evolution to acute myeloid leukemia (AML) by next-generation sequencing (NGS). Herein, we further determined the role and clinical implications of an evident methylation change in CpG islands at the SLIT2 promoter identified by NGS. First, increased SLIT2 promoter methylation was validated in 11 paired MDS/AML patients during disease evolution. Additionally, SLIT2 promoter methylation was markedly increased in MDS/AML patients compared with controls and was correlated with poor clinical phenotype and outcome. Interestingly, SLIT2 expression was particularly upregulated in AML patients and was not correlated with SLIT2 promoter methylation. However, the SLIT2-embedded genes SLIT2-IT1 and miR-218 were downregulated in AML patients, which was negatively associated with SLIT2 promoter methylation and further validated by demethylation studies. Functionally, SLIT2-IT1/miR-218 overexpression exhibited antileukemic effects by affecting cell proliferation, apoptosis and colony formation in vitro and in vivo. Mechanistically, SLIT2-IT1 may function as a competing endogenous RNA by sponging miR-3156-3p to regulate BMF expression, whereas miR-218 may directly target HOXA1 in MDS progression. In summary, our findings demonstrate that SLIT2 promoter hypermethylation is associated with disease evolution in MDS and predicts poor prognoses in both MDS and AML. Epigenetic inactivation of SLIT2-IT1/miR-218 by SLIT2 promoter hypermethylation could be a promising therapeutic target in MDS.

[Clinical Significance of Low Expression of LncRNA CASC15 in Acute Myeloid Leukemia with NPM1 Mutations].

AbstractObjective: To identify the expression and methylation patterns of lncRNA CASC15 in bone marrow (BM) samples of acute myeloid leukemia (AML) patients, and further explore its clinical significance. METHODS: Eighty-two de novo AML patients and 18 healthy donors were included in the study. Meanwhile, seven public datasets from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were included to confirm the expression and methylation data of CASC15. Receiver operating characteristic (ROC) curve analysis was applied to determine the discriminative capacity of CASC15 expression to identify AML. The patients were divided into CASC15high group and CASC15low group by X-tile method, and the prognostic value of CASC15 was identified by Kaplan-Meier method and univariate and multivariate Cox regression analysis. RESULTS: The expression level of CASC15 was significantly decreased in BM cells of AML patients compared with healthy donors (P<0.001). ROC curve analysis suggested that CASC15 expression might be a potential biomarker to discriminate AML from controls. The expression of CASC15 was high at the early stage of hematopoiesis, and reached a peak at the stage of multipotent progenitors differentiation, then decreased rapidly, and was at a range of low level fluctuations in the subsequent process. Among FAB subtypes, CASC15 expression in M0 was significantly higher than that in M1-M7. Clinically, CASC15low patients were more likely to have NPM1 mutations than CASC15high patients (P=0.048), while CASC15high patients had a significantly higher frequency of IDH1 and RUNX1 mutations (P=0.021 and 0.014, respectively). Moreover, CASC15low group had a shorter overall survival (OS) in patients with NPM1 mutations. Furthermore, multivariate analysis confirmed that CASC15 expression was a significant independent risk factor for OS in NPM1 mutated AML patients. In addition, CASC15 methylation level in BM samples of AML patients was significantly decreased compared with healthy donors. Patients with CASC15 high methylation had poor OS and disease-free survival. CONCLUSION: The expression of CASC15 is decreased in AML, and low CASC15 expression may predict adverse prognosis in AML patients with NPM1 mutations. Moreover, CASC15 methylation level in AML is significantly decreased, and high CASC15 methylation may predict poor prognosis in AML.

Association Analyses of TP53 Mutation With Prognosis, Tumor Mutational Burden, and Immunological Features in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease related to a broad spectrum of molecular alterations. The successes of immunotherapies treating solid tumors and a deeper understanding of the immune systems of patients with hematologic malignancies have promoted the development of immunotherapies for the treatment of AML. And high tumor mutational burden (TMB) is an emerging predictive biomarker for response to immunotherapy. However, the association of gene mutation in AML with TMB and immunological features still has not been clearly elucidated. In our study, based on The Cancer Genome Atlas (TCGA) and BeatAML cohorts, 20 frequently mutated genes were found to be covered by both datasets in AML. And TP53 mutation was associated with a poor prognosis, and its mutation displayed exclusiveness with other common mutated genes in both datasets. Moreover, TP53 mutation correlated with TMB and the immune microenvironment. Gene Set Enrichment Analysis (GSEA) showed that TP53 mutation upregulated signaling pathways involved in the immune system. In summary, TP53 mutation is frequently mutated in AML, and its mutation is associated with dismal outcome, TMB, and immunological features, which may serve as a biomarker to predict immune response in AML.

Identification and validation of obesity-related gene LEP methylation as a prognostic indicator in patients with acute myeloid leukemia.

BACKGROUND: Obesity confers enhanced risk for multiple diseases including cancer. The DNA methylation alterations in obesity-related genes have been implicated in several human solid tumors. However, the underlying role and clinical implication of DNA methylation of obesity-related genes in acute myeloid leukemia (AML) has yet to be elucidated. RESULTS: In the discovery stage, we identified that DNA methylation-associated LEP expression was correlated with prognosis among obesity-related genes from the databases of The Cancer Genome Atlas. In the validation stage, we verified that LEP hypermethylation was a frequent event in AML by both targeted bisulfite sequencing and real-time quantitative methylation-specific PCR. Moreover, LEP hypermethylation, correlated with reduced LEP expression, was found to be associated with higher bone marrow blasts, lower platelets, and lower complete remission (CR) rate in AML. Importantly, survival analysis showed that LEP hypermethylation was significantly associated with shorter overall survival (OS) in AML. Moreover, multivariate analysis disclosed that LEP hypermethylation was an independent risk factor affecting CR and OS among non-M3 AML. By clinical and bioinformatics analysis, LEP may be also regulated by miR-517a/b expression in AML. CONCLUSIONS: Our findings indicated that the obesity-related gene LEP methylation is associated with LEP inactivation, and acts as an independent prognostic predictor in AML.

Genome-wide methylation sequencing identifies progression-related epigenetic drivers in myelodysplastic syndromes.

The potential mechanism of myelodysplastic syndromes (MDS) progressing to acute myeloid leukemia (AML) remains poorly elucidated. It has been proved that epigenetic alterations play crucial roles in the pathogenesis of cancer progression including MDS. However, fewer studies explored the whole-genome methylation alterations during MDS progression. Reduced representation bisulfite sequencing was conducted in four paired MDS/secondary AML (MDS/sAML) patients and intended to explore the underlying methylation-associated epigenetic drivers in MDS progression. In four paired MDS/sAML patients, cases at sAML stage exhibited significantly increased methylation level as compared with the matched MDS stage. A total of 1090 differentially methylated fragments (DMFs) (441 hypermethylated and 649 hypomethylated) were identified involving in MDS pathogenesis, whereas 103 DMFs (96 hypermethylated and 7 hypomethylated) were involved in MDS progression. Targeted bisulfite sequencing further identified that aberrant GFRA1, IRX1, NPY, and ZNF300 methylation were frequent events in an additional group of de novo MDS and AML patients, of which only ZNF300 methylation was associated with ZNF300 expression. Subsequently, ZNF300 hypermethylation in larger cohorts of de novo MDS and AML patients was confirmed by real-time quantitative methylation-specific PCR. It was illustrated that ZNF300 methylation could act as a potential biomarker for the diagnosis and prognosis in MDS and AML patients. Functional experiments demonstrated the anti-proliferative and pro-apoptotic role of ZNF300 overexpression in MDS-derived AML cell-line SKM-1. Collectively, genome-wide DNA hypermethylation were frequent events during MDS progression. Among these changes, ZNF300 methylation, a regulator of ZNF300 expression, acted as an epigenetic driver in MDS progression. These findings provided a theoretical basis for the usage of demethylation drugs in MDS patients against disease progression.

Identification and validation of prognosis-related DLX5 methylation as an epigenetic driver in myeloid neoplasms.

The deregulated DLX gene family members DLX1/2/3/4/5/6 (DLXs) caused by DNA methylation has been demonstrated in various cancers with therapeutic target value. However, the potential role of DLXs methylation in myeloid neoplasms such as acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) remains to be elucidated. Clinical significance of DLXs methylation/expression was analyzed in patient with AML and MDS. The functional roles of DLXs were determined in vitro. In the identification stage, we found that lower DLX5 expression was correlated with prognosis in AML among all DLXs analyzed by The Cancer Genome Atlas datasets. In the validation stage, we revealed that reduced DLX5 expression was frequently occurred, and was also correlated with promoter hypermethylation in AML evaluated by targeted bisulfite sequencing. Epigenetic studies also showed that DLX5 promoter DNA methylation was associated with its expression. By quantitative polymerase chain reaction, we also validated that DLX5 hypermethylation was frequent event in both AML and MDS, and also correlated with MDS transformation to leukemia. Moreover, DLX5 hypermethylation was associated with lower rate of complete remission and shorter time of leukemia-free/overall survival, and was also confirmed by Logistic/Cox regression analysis. Functional studies revealed the antiproliferative and pro-apoptotic effects of DLX5 in MDS-derived AML cell-line SKM-1. Finally, bioinformatics analysis demonstrated that DLX5 functioned in leukemogenesis may be through the association with PI3K/Akt signaling pathway. Collectively, our findings demonstrated that DLX5 methylation, negatively correlated DLX5 expression, was a potential prognostic and predictive indicator in patients with AML and MDS, which could also act as an epigenetic driver in myeloid neoplasms.

The M2 macrophage marker CD206: a novel prognostic indicator for acute myeloid leukemia.

Hematological malignancies possess a distinctive immunologic microenvironment compared with solid tumors. Here, using an established computational algorithm (CIBERSORT), we systematically analyzed the overall distribution of 22 tumor-infiltrating leukocyte (TIL) populations in more than 2000 bone marrow (BM) samples from 5 major hematological malignancies and healthy controls. Focusing on significantly altered TILs in acute myeloid leukemia (AML), we found that patients with AML exhibited increased frequencies of M2 macrophages, compared to either healthy controls or the other four malignancies. High infiltration of M2 macrophages was associated with poor outcome in AML. Further analysis revealed that CD206, a M2 marker gene, could faithfully reflect variation in M2 fractions and was more highly expressed in AML than normal controls. High CD206 expression predicted inferior overall survival (OS) and event-free survival (EFS) in two independent AML cohorts. Among 175 patients with intermediate-risk cytogenetics, the survival still differed greatly between low and high CD206 expressers (OS; P < .0001; 3-year rates, 56% v 32%; EFS; P < .001; 3-year rates, 47% v 25%). When analyzed in a meta-analysis, CD206 as a continuous variable showed superior predictive performance than classical prognosticators in AML (BAALC, ERG, EVI1, MN1, and WT1). In summary, M2 macrophages are preferentially enriched in AML. The M2 marker CD206 may serve as a new prognostic marker in AML.

EZH2 dysregulation: Potential biomarkers predicting prognosis and guiding treatment choice in acute myeloid leukaemia.

Accumulating studies have proved EZH2 dysregulation mediated by mutation and expression in diverse human cancers including AML. However, the expression pattern of EZH2 remains controversial in acute myeloid leukaemia (AML). EZH1/2 expression and mutation were analysed in 200 patients with AML. EZH2 expression was significantly decreased in AML patients compared with normal controls but not for EZH1 expression. EZH2 mutation was identified three of the 200 AML patients (1.5%, 3/200), whereas none of the patients harboured EZH1 mutation (0%, 0/200). EZH2 expression and mutation were significantly associated with -7/del(7) karyotypes. Moreover, lower EZH2 expression was associated with older age, higher white blood cells, NPM1 mutation, CEBPA wild-type and WT1 wild-type. Patients with EZH2 mutation showed shorter overall survival (OS) and leukaemia-free survival (LFS) than patients without EHZ2 mutation after receiving autologous or allogeneic haematopoietic stem cell transplantation (HSCT). However, EZH2 expression has no effect on OS and LFS of AML patients. Notably, in EZH2 low group, patients undergone HSCT had significantly better OS and LFS compared with patients only received chemotherapy, whereas no significant difference was found in OS and LFS between chemotherapy and HSCT patients in EZH2 high group. Collectively, EZH2 dysregulation caused by mutation and under-expression identifies specific subtypes of AML EZH2 dysregulation may be acted as potential biomarkers predicting prognosis and guiding the treatment choice between transplantation and chemotherapy.

Hypomethylation of MIR-378 5'-flanking region predicts poor survival in young patients with myelodysplastic syndrome.

BACKGROUND: Previous studies have disclosed up-regulation of MIR-378 in acute myeloid leukemia (AML), and might consequently affect the outcome of the patients. Correspondingly, hypomethylation of MIR-378 was also identified in AML, particularly for FAB-M2 subtype with t(8;21) chromosomal translocation. Nevertheless, the methylation status of MIR-378 has not been illustrated in myelodysplastic syndrome (MDS). Herein we designed to understand the methylation pattern of MIR-378 involved in MDS and clinical interrelation thereof. METHODS: Real-time quantitative methylation-specific PCR (RQ-MSP) was performed to evaluate the methylation degree of MIR-378 5'-flanking region on bone marrow mononuclear cells collected from 95 de novo MDS patients. Five gene mutations (IDH1, IDH2, DNMT3A, U2AF1, and SF3B1) were detected by high-resolution melting analysis to further evaluate the clinical relevance of hypomethylation of MIR-378. RESULTS: Unmethylated level of MIR-378 5'-flanking region was significantly higher in MDS patients than that in controls (p = .034). Hypomethylated MIR-378 was identified in 20 of 95 (21%) cases with MDS. Male patients appeared to be more frequent to harbor MIR-378 hypomethylation compared to female patients (15/55, 27.3% vs. 4/40, 10.0%, p = .04). There was no significant difference in age, white blood cell counts, platelet counts, hemoglobin concentration, and karyotypes between the patients with and without MIR-378-hypomethylation. Distinct distribution of five gene mutations was not observed in the two groups as well. However, MIR-378-hypomethylated patients had significantly shorter overall survival than those without MIR-378 hypomethylation (p = .036). Moreover, among patients <60 years, hypomethylation of MIR-378 was confirmed to be an independent adverse prognostic factor by both Kaplan-Meier and Multivariate Cox analyses. CONCLUSION: Hypomethylation of MIR-378 5'-flanking region is an adverse prognosticator in MDS, particularly in patients <60 years.

SOX30 methylation correlates with disease progression in patients with chronic myeloid leukemia.

BACKGROUND: Our previous study has reported that aberrant SOX30 methylation was associated with poor prognosis in AML, and it correlated with disease progression in MDS. Herein, we further determined SOX30 methylation and its clinical significance in the other myeloid malignance - chronic myeloid leukemia (CML). METHODS: SOX30 methylation was examined by real-time quantitative methylation-specific PCR and bisulfite sequencing PCR, whereas SOX30 expression was detected by real-time quantitative PCR. RESULTS: SOX30 methylation was identified in 11% (10/95) CML patients. SOX30 methylation was associated with lower hemoglobin and platelets (P=0.006 and 0.032, respectively). Importantly, significant differences were observed in the distributions of clinical stages and cytogenetics (P=0.006 and 0.002, respectively). The frequency of SOX30 methylation in chronic phase (CP) stage occurred with lowest frequency (4/74, 5%), higher in accelerated phase (AP) stage (1/7, 14%), and the highest in blast crisis (BC) stage (12/31, 39%). In addition, SOX30 methylated patients tended to have a higher level of BCR-ABL transcript than SOX30 non-methylated patients (P=0.063). In two paired CML patients, SOX30 methylation showed lower density in CP stage (19% and 17%, respectively), and was significantly increased in BC stage (89% and 69%, respectively) during disease progression. Additionally, SOX30 methylated CML patients presented a lower SOX30 transcript level than SOX30 non-methylated CML patients (P=0.046). CONCLUSION: Our study revealed that SOX30 methylation correlated with disease progression in chronic myeloid leukemia.

Promoter methylation of the candidate tumor suppressor gene TCF21 in myelodysplastic syndrome and acute myeloid leukemia.

Transcription factor 21 (TCF21) has been identified as a candidate tumor suppressor gene which was epigenetically inactivated in a variety of human cancers. However, TCF21 methylation pattern remains unknown in hematologic malignancies. The aim of this study was to investigate TCF21 methylation and its clinical relevance in myelodysplastic syndrome (MDS) and non-M3 acute myeloid leukemia (AML). A total cohort of 33 MDS patients, 100 non-M3 AML patients and 25 healthy donors were enrolled in the study. Targeted bisulfite sequencing assay was performed to identify the methylation pattern of CpG islands within the promoter of TCF21 gene. The bioinformatics analyses were based on The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO). The results showed that there were significant differences in the methylation levels of TCF21 between MDS, non-M3 AML and controls (P = 0.003 and < 0.001, respectively). TCF21 hypermethylation might be served as a promising biomarker which could distinguish MDS/AML from normal controls (P < 0.001 and = 0.003, respectively). There was a significant difference in cytogenetic risk categories between TCF21 hypermethylation and non-hypermethylation AML patients (P = 0.032). Notably, TCF21 hypermethylation occurred frequently in AML patients with adverse risk category, compared with those with favorable and intermediate categories, respectively (67% vs 44% and 29%). TCF21 non-hypermethylation AML patients showed a higher probability of normal karyotype than abnormal karyotype (P = 0.003). The rate of DNMT3A gene mutation was significantly higher in the non-hypermethylation AML patients than that in the hypermethylation (8/44 vs 0/34, P = 0.020). These results suggested that aberrant DNA promoter methylation of TCF21 was frequent event in MDS and non-M3 AML, and TCF21 hypermathylation was associated with adverse risk karyotype in AML.