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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.

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.

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., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

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., (© 2022. The Author(s).)

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., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

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.

Introduction: LINC00324 was overexpressed and facilitated carcinogenesis in various solid malignant tumors. However, the role of LINC00324 in leukemogenesis remains to be elucidated., Methods: The relative expression and unmethylation levels of LINC00324 were detected by real-time quantitative PCR (RT-qPCR) and real-time quantitative methylation-specific PCR (RT-qMSP). Cell proliferation experimental and flow cytometer (FCM) was used to detect the change of proliferation and apoptosis in leukemia cell lines after overexpression of LINC00324., Results: The results showed that the expression of LINC00324 and the methylation level of the promoter region were significantly negatively correlated in AML patients. Moreover, patients with lower LINC00324 expression showed more prolonged overall survival (OS). Remarkably, overexpression of LINC00324 in leukemia cell lines promoted the proliferation of target cells and inhibited their apoptosis., Conclusion: Our findings firstly identified that the hypomethylation of LINC00324 was a common molecular event in de novo AML patients. The abnormally upregulated LINC00324 promotes proliferation and inhibits apoptosis in leukemia cells., (© 2022 John Wiley & Sons Ltd.)

The objective of our study was to measure DLEU7-AS1 expression in de novo acute myeloid leukemia (AML) whilst also analyzing its clinical relevance. We used gene expression data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Cancer Cell Line Encyclopedia (CCLE) and Genotype-Tissue Expression project (GTEx) to assess the expression profile of DLEU7-AS1 in pan-cancers, cancer cell lines and normal tissues. Reverse transcription-quantitative PCR was used to measure DLEU7-AS1 expression in bone marrow from 30 normal individuals and 110 patients with de novo AML. DLEU7-AS1 expression was found to be markedly reduced in the AML samples of the TCGA pan-cancer datasets. In our PCR validation, DLEU7-AS1 expression was significantly decreased in the AML samples compared with that in controls (P<0.001). Low DLEU7-AS1 expression (DLEU7-AS1low) correlated positively with lower blood platelet counts (P=0.029). In addition, low DLEU7-AS1 expression was more frequently observed in the intermediate (58%; 44/76) and favorable karyotypes (65%; 15/23) compared with that in the poor karyotype (10%; 1/10; P=0.005). In particular, patients with high expression levels of DLEU7-AS1 (DLEU7-AS1high) showed lower complete remission rates (P=0.002) than patients with DLEU7-AS1low. Survival analysis revealed that patients with DLEU7-AS1low had longer overall survival (OS) than patients with DLEU7-AS1high (P<0.05). Multivariate Cox analysis demonstrated that in patients with non-acute promyelocytic leukemia (non-M3) who were ≤60 years old, DLEU7-AS1 expression was an independent prognostic factor for OS. Furthermore, we found distinct correlations among the expression of DLEU7-AS1, infiltration by immune cells and immune checkpoint genes in AML., (© 2022 The Author(s).)

Background: Recently, an increasing number of studies have reported that sperm-associated antigen (SPAG) proteins play crucial roles in solid tumorigenesis, and may serve as potentially helpful biomarkers for cancer diagnosis and prognosis. However, very few studies systematically investigated the expression of SPAG family members and their clinical significance in acute myeloid leukemia (AML)., Methods: The expression of SPAGs and their prognostic significance in AML were determined by a systematic analysis on data gathered from public databases, and the results were validated in clinical samples., Results: Using public data, we identified only increased SPAG1 expression negatively associated with survival in AML by Cox regression (P < 0.001) and Kaplan-Meier analysis (P < 0.001). The prognostic value of SPAG1 expression was further confirmed in other independent cohorts. Clinically, higher SPAG1 expression was significantly correlated with white blood cell counts (P = 0.014) and French-American-British (FAB) subtypes (P = 0.024). Moreover, higher SPAG1 expression was more common in + 8 patients (P = 0.034), rarely found with t(8;21) (P = 0.014), and correlated with FLT3 (P < 0.001) and DNMT3A mutations (P = 0.001). Despite these associations, multivariate analysis confirmed the independent prognostic value of SPAG1 expression in AML (P < 0.001). Notably, AML patients with higher SPAG1 expression may benefit from hematopoietic stem cell transplantation (HSCT), whereas patients with lower SPAG1 expression appeared less likely to benefit. Finally, we further validated that SPAG1 expression was significantly increased in newly diagnosed AML patients compared with normal controls (P < 0.001) and with AML patients who achieved complete remission (P < 0.001). Additionally, SPAG1 expression could act as a potentially helpful biomarker for the diagnosis and prognosis of AML (P < 0.001 and = 0.034, respectively)., Conclusions: Our findings demonstrated that SPAG1 overexpression may serve as an independent prognostic biomarker and may guide the choice between HSCT and chemotherapy in patients with AML., (© 2022. The Author(s).)

The role of B7-H3 in acute myeloid leukemia (AML) is not fully understood. Two previous studies investigating its expression and significances in AML are partially different. In this study, we aimed to systematically characterize the genomic and immune landscape in AML patients with altered B7-H3 expression using multi-omics data in the public domain. We found significantly increased B7-H3 expression in AML compared to either other hematological malignancies or healthy controls. Clinically, high B7-H3 expression was associated with old age, TP53 mutations, wild-type WT1 and CEBPA, and the M3 and M5 FAB subtypes. Moreover, we observed that increased B7-H3 expression correlated significantly with a poor outcome of AML patients in four independent datasets. Gene set enrichment analysis (GSEA) revealed the enrichment of the "EMT" oncogenic gene signatures in high B7-H3 expressers. Further investigation suggested that B7-H3 was more likely to be associated with immune-suppressive cells (macrophages, neutrophils, dendritic cells, and Th17 cells). B7-H3 was also positively associated with a number of checkpoint genes, such as VISTA (B7-H5), CD80 (B7-1), CD86 (B7-2), and CD70. In summary, we uncovered distinct genomic and immunologic features associated with B7-H3 expression in AML. This may lead to a better understanding of the molecular mechanisms underlying B7-H3 dysregulation in AML and to the development of novel therapeutic strategies., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wen, Xu, Jin, Xia, Ma, Qian, Lin and Qian.)

Objective: LncRNA ITGB2-AS1 has been found to play important roles in the occurrence and development of human solid tumors. However, its role in hematological diseases, especially acute myeloid leukemia (AML), remains unclear. The aim of this study was to identify the expression pattern of ITGB2-AS1 in AML patients and to further explore its clinical significance., Methods: ITGB2-AS1 expression was analyzed in public datasets (including TCGA and GSE63270) and further validated in a cohort of 109 AML patients by real-time quantitative PCR (RT-qPCR)., Results: The level of ITGB2-AS1 was up-regulated among two independent cohorts (TCGA, P<0.05; GSE63270, P<0.05), which was confirmed by the data from 109 AML patients enrolled in this study (P<0.05). Clinically, high ITGB2-AS1 expression was associated with older age (P=0.023) and lower complete remission (CR) rate (P=0.005). Multivariate analysis identified that high ITGB2-AS1 expression was an independent prognostic factor not only for CR rate (P=0.027) but also for overall survival (OS) time (P=0.011), and ITGB2-AS1 was positively correlated with ITGB2 expression in both TCGA (r=0.74, P<0.001) and clinical data detected in this study (r=0.881, P<0.001). High ITGB2 expression was also associated with older age (P=0.02) and lower CR rate (P=0.020). Moreover, high ITGB2 expression predicted worse OS (P=0.028)., Conclusion: ITGB2-AS1 is overexpressed in AML and predicts poor prognosis in AML patients.

It was previously reported that PRR34-AS1 was overexpressed in some solid tumors. PRR34-AS1 promoter was shown to have a differential methylation region (DMR), and was hypomethylated in acute myeloid leukemia (AML). Therefore, the present study used real-time quantitative PCR (RQ-PCR) to explore the expression characteristics of PRR34-AS1 in AML. In addition, the correlation between the expression of PRR34-AS1 and clinical prognosis of AML was determined. The findings of this study indicated that high PRR34-AS1 expression was bound up with shorter overall survival (OS) in AML patients (p = 0.002). Moreover, patients with high expression of PRR34-AS1 had significantly lower complete remission (CR) rate compared with those with low expression of PRR34-AS1 after induction chemotherapy. Furthermore, multivariate analysis confirmed that PRR34-AS1 expression was an independent factor affecting CR in whole-AML, non-APL-AML, and CN-AML patients (p = 0.032, 0.039, and 0.036, respectively). Methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) were used to explore the methylation status of PRR34-AS1. PRR34-AS1 promoter showed a pattern of hypomethylation in AML patients compared with normal controls (p = 0.122). Notably, of whole-AML and non-APL-AML patients, PRR34-AS1 hypomethylated patients presented a significantly shorter OS than those with a hypermethylated PRR34-AS1 (p = 0.010 and 0.037, respectively). Multivariate analysis confirmed that the hypomethylation of PRR34-AS1 served as an independent prognostic indicator in both whole-cohort AML and non-APL-AML categories (p = 0.057 and 0.018, respectively). In summary, the findings of this study showed that abnormalities in PRR34-AS1 are associated with poor prognosis in AML. Therefore, monitoring this index may be important in the prognosis of AML and can provide information on effective chemotherapy against the disease., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

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.

DNA methyltransferases ( DNMTs ) by regulating DNA methylation play crucial roles in the progression of hematologic malignancies, especially for acute myeloid leukemia (AML). Accumulating investigations have identified the high incidence of DNMT3A mutation in AML, and it is correlated with poor prognosis. Although a few studies have shown the expression of DNMTs and their clinical significance in AML, the results remain to be discussed. Herein, we systemically analyzed the DNMTs expression and their relationship with clinic-pathological features and prognosis in AML patients. DNMTs expression especially for DNMT3A / 3B was closely associated with AML among various human cancers. DNMT3A expression was increased in AML patients, whereas DNMT3B expression was decreased. Significant associations between DNMT3A / B expression and clinic-pathological features/gene mutations were observed. Kaplan-Meier analysis showed that DNMT3A expression was associated with better overall survival (OS) and leukemia-free survival (LFS) among whole-cohort AML, and independently affected OS determined by Cox repression multivariate analysis. Notably, patients that received hematopoietic stem cell transplantation (HSCT) showed significantly better OS and LFS in DNMT3A lower-expressed groups, whereas patients in DNMT3A higher-expressed groups did not. By bioinformatics analysis, DNMT3A expression was found to be positively correlated with several leukemia-associated genes/microRNAs, and DNMT3A was identified as direct targets of miR-429 and miR-29b in AML. Collectively, our study demonstrated that DNMT3A / 3B showed significant expression differences in AML. DNMT3A expression acted as a potential prognostic biomarker and may guide treatment choice between chemotherapy and HSCT in AML.

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., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

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., (© 2019 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: Downstream of tyrosine kinase 6 (DOK6), which is specifically expressed in the nervous system, was previously recognized as an adapter only in neurite outgrowth. Recent studies also demonstrated the potential role of DOK6 in solid tumors such as gastric cancer and breast cancer. However, previous studies of DOK6 have not dealt with its roles in myeloid malignancies. Herein, we verified the promoter methylation status of DOK6 and further explored its clinical implication in de novo acute myeloid leukemia (AML)., Methods: A total of 100 newly diagnosed adult AML patients were involved in the current study. DOK6 expression and methylation were detected by real-time qPCR and methylation-specific PCR (MSP), respectively. Bisulfite sequencing PCR (BSP) was performed to assess the methylation density of the DOK6 promoter., Results: Downstream of tyrosine kinase 6 promoter methylation was significantly increased in AML patients compared to controls (P = .037), whereas DOK6 expression significantly decreased in AML patients (P < .001). The expression of DOK6 was markedly up-regulated after treated by 5-aza-2'-deoxycytidine (5-aza-dC) in THP-1 cell lines. The methylation status of the DOK6 promoter was associated with French-American-British classifications (P = .037). There was no significant correlation existed between DOK6 expression and its promoter methylation (R = .077, P = .635). Interestingly, of whole-AML and non-APL AML patients, both have a tendency pertaining to the DOK6 methylation group and a significantly longer overall survival (OS) than the DOK6 unmethylation group (P = .042 and .036, respectively)., Conclusion: Our study suggested that DOK6 promoter hypermethylation was a common molecular event in de novo AML patients. Remarkably, DOK6 promoter methylation could serve as an independent and integrated prognostic biomarker not only in non-APL AML patients but also in AML patients who are less than 60 years old., (© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: BCL2 protein inhibitor venetoclax (ABT-199) has been authorized by Food and Drug Administration for relapsed/refractory chronic lymphoid leukemia with 17p deletion. Although venetoclax/ABT-199 also caused cell death in acute myeloid leukemia (AML), whether it could be applied to clinical treatment needs further studies. Here, we revealed clinical implication of BCL2 overexpression in de novo adult AML, and may provide theoretical basis for targeted therapy using venetoclax., Methods: BCL2 expression was analyzed in adult AML patients from public datasets The Cancer Genome Atlas (TCGA) and confirmed by another independent cohort from our own data., Results: BCL2 expression showed up-regulated in AML patients among TCGA data and confirmed by our own data. BCL2 overexpression was correlated with FAB-M0/M1, whereas BCL2 under-expression was related to FAB-M5. However, BCL2 expression has no effect on overall survival (OS) and leukemia-free survival (LFS) of AML patients (determined in BCL2 low and BCL2 high groups). Interestingly, in the BCL2 low group, patients undergoing autologous or allogeneic hematopoietic stem cell transplantation (auto/allo-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 auto/allo-HSCT patients in the BCL2 high group. BCL2 expression was found positively correlated with HOX family gene, and negatively correlated with tumor suppressor microRNA such as miR-195, miR-497, and miR-193b., Conclusions: BCL2 overexpression identified specific FAB subtypes of AML, but it did not affect prognosis. Patients with BCL2 overexpression did not benefit from auto/allo-HSCT among whole-cohort-AML and cytogenetically normal AML.

MircoRNA-335 ( miR-335 ) has been reported as a significant cancer-associated microRNA, which was often epigenetically silenced and acted as a tumor suppressor gene in diverse human solid tumors. Conversely, recent studies show that miR-335 overexpression was identified in both adult and pediatric acute myeloid leukemia (AML), suggesting that it might play an oncogenic role of miR-335 in AML. However, the role of miR-335 during leukemogenesis remains to be elucidated. MiR-335 / ID4 expression was detected by real-time quantitative PCR and/or western blot. Survival analysis was performed to explore the association between miR-335 / ID4 expression and the prognosis, and further validated by public databases. Gain-of-function experiments determined by cell proliferation, apoptosis, and differentiation were conducted to investigate the biological functions of miR-335 / ID4 . Herein, we found that miR-335 expression, independent of its methylation, was significantly increased and negatively correlated with reduced ID4 expression in AML. Moreover, aberrant miR-335 / ID4 expression independently affected chemotherapy response and leukemia-free/overall survival in patients with AML. Gain-of-function experiments in vitro showed the oncogenic role of miR-335 by affecting cell apoptosis and proliferation in AML, and could be rescued by ID4 restoration. Mechanistically, we identified and verified that miR-335/ID4 contributed to leukemogenesis through activating PI3K/Akt signaling pathway. Collectively, aberrant miR-335 / ID4 expression was an independent prognostic biomarker in AML. MiR-335 / ID4 dysregulation facilitated leukemogenesis through the activation of PI3K/Akt signaling pathway.

Background: Leukemia stem cell (LSC)-enriched genes have been shown to be highly prognostic in acute myeloid leukemia (AML). However, the prognostic value of tumor suppressor genes (TSGs) that are repressed early in LSC remains largely unknown., Methods: We compared the public available expression/methylation profiling data of LSCs with that of hematopoietic stem cells (HSCs), in order to identify potential tumor suppressor genes in LSC. The prognostic relevance of PCDH17 was analyzed on a cohort of 173 AML patients from The Cancer Genome Atlas (TCGA), and further validated in three independent cohorts (n = 339)., Results: We identified protocadherin17 (PCDH17) and demonstrated that it was significantly down-regulated and hypermethylated in LSCs compared with HSCs. Our analyses of primary AML patient samples also confirmed these deregulations. Clinically, low PCDH17 expression was associated with female sex (P = 0.01), higher WBC (P < 0.0001), higher percentages of blasts in bone marrow (BM) and peripheral blood (PB) (P = 0.04 and < 0.001, respectively), presence of FLT3-internal tandem duplications (P = 0.002), mutated NPM1 (P = 0.02), and wild-type TP53 (P = 0.005). Moreover, low PCDH17 expression predicted worse overall survival (OS) in four independent cohorts as well as in the molecularly defined subgroups of AML patients. In multivariable analyses, low PCDH17 expression retained independent prognostic value for OS. Biologically, PCDH17 expression-associated gene signatures were characterized by deregulations of EMT- and Wnt pathway-related genes., Conclusions: PCDH17 gene was silenced by DNA methylation in AML. Low PCDH17 expression is associated with distinct clinical and biological features and improves risk stratification in patients with AML.

Previous studies have been indicated that integrin α2 (ITGA2) may be important in cell migration, invasion, survival, and angiogenesis. However, the correlation between ITGA2 expression and acute myeloid leukemia (AML) is still unclear. Real-time quantitative polymerase chain reaction was carried out to analyze ITGA2 messenger RNA level. Methylation-specific polymerase chain reaction (PCR) and bisulfite sequencing PCR were performed to detect the methylation of ITGA2 promoter. ITGA2 expression was significantly upregulated in 134 de novo AML patients compared with 33 controls (p = 0.007). ITGA2 high group had markedly lower complete remission (CR) rate than ITGA2 low group (p = 0.011). Furthermore, the overall survival in ITGA2 high patients was significantly shorter than ITGA2 low patients throughout AML cohort, non-acute promyelocytic leukemia (APL) and cytogenetic normal-AML (p = 0.001, 0.002, and 0.044, respectively). Multivariate analysis confirmed that ITGA2 overexpression served as an independent prognostic factor in both whole-cohort AML patients (p = 0.018) and non-APL AML patients (p = 0.021). Besides, ITGA2 expression level was significantly decreased in AML patients after CR (p = 0.011), and was returned at the time of relapse phase (p = 0.021). Moreover, unmethylated ITGA2 promoter was identified in normal controls, leukemia cell lines, and primary leukemia cells with low or high ITGA2 expression. In conclusions, methylation-independent ITGA2 overexpression is associated with poor prognosis in AML., (© 2018 Wiley Periodicals, Inc.)

DOK-1 and DOK-2 (DOK1/2) are closely related members of downstream of tyrosine kinase (DOK) family genes, which are found to be frequently rearranged in several hematopoietic cancers. However, the clinical implications of DOK1/2 in acute myeloid leukemia (AML) remain largely unknown. To investigate the clinical significance, real-time quantitative PCR (RQ-PCR) was carried out to detect DOK1/2 expressions in 125 de novo AML patients and 28 healthy controls. Real-time quantitative methylation-specific PCR (RQ-MSP) and bisulfite sequencing PCR (BSP) were applied to detect DOK1/2 methylation level and density. DOK1/2 expressions were significantly down-regulated in AML patients. The promoters of DOK1/2 were highly hypermethylated and negatively correlated with DOK1/2 expressions in AML patients. In addition, we also confirmed that DOK1/2 expressions could be restored by DOK1/2 demethylation using 5-aza-2'-deoxycytidine in leukemia cell line THP-1. Survival analyses showed that low-expressed DOK1/2 were associated with markedly shorter overall survival and leukemia free survival in both whole-cohort AML and non-M3 AML patients. Multivariate analyses further revealed that DOK1/2 were act as independent prognostic factors in AML patients. These findings indicate that decreased DOK1/2 expressions associated with their promoter hypermethylations predict adverse prognosis in AML., (© 2017 Wiley Periodicals, Inc.)

Background: The liver-enriched microRNA-122 (miR-122) plays a crucial role in pathogenesis of hepatocellular carcinoma (HCC) with prognostic value. Recently, miR-122 was also found to be related to many other cancers besides HCC. However, less study determined miR-122 expression and its clinical significance in acute myeloid leukemia (AML)., Methods: Real-time quantitative PCR was performed to detect the level of bone marrow (BM) miR-122 in de novo AML patients. The clinical significance of miR-122 expression in AML was further investigated., Results: Among whole-cohort AML, lower expression of BM miR-122 was associated with male patients, higher hemoglobin and favorable-karyotypes (P = 0.038, 0.006, and 0.038, respectively). Among cytogenetically normal AML (CN-AML), lower expression of BM miR-122 was correlated with DNMT3A wild type (P = 0.043). Moreover, patients with lower expression of BM miR-122 presented lower complete remission (CR) rate and shorter overall survival (OS) than those with higher expression of BM miR-122 in CN-AML (P = 0.025 and 0.013, respectively). Cox regression analyses further confirmed the prognostic value of BM miR-122 expression in CN-AML (P = 0.024). In follow-up patients, BM miR-122 expression level in CR time was increased compared to diagnosis time, and was returned to primary level when in relapse time again (P = 0.062 and 0.049, respectively)., Conclusions: Our findings indicated that lower expression of BM miR-122 acted as an independent risk factor for OS in CN-AML., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Background: Somatic mutations in SETBP1 gene have recently been detected in hematologic malignancies. The present study aimed to explore the frequency and clinical correlations of SETBP1 mutations in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)., Methods: In this study, we used high-resolution melting analysis (HRMA) to detect the SETBP1 mutations in a cohort of 363 patients with AML or MDS., Results: A total of 1.2% (3/249) of AML and 1.8% (2/114) of MDS patients were found with heterozygous SETBP1 mutations. In AML, patients with SETBP1 mutations showed higher hemoglobin (P = 0.004) and were more frequently recurrent in AML-M4 subtype (P = 0.034). All five SETBP1 mutated patients had normal karyotypes. The patients with SETBP1 mutations had significantly higher incidences of concurrent SRSF2 mutations (P = 0.002). HRMA could detect SETBP1 mutations with 5% sensitivity, obviously higher than 25% of Sanger sequencing., Conclusions: We established a rapid, inexpensive, high-throughput and sensitive method to screen SETBP1 mutations. SETBP1 mutations were a rare molecular event in AML and MDS patients., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Background: The long non-coding RNA H19 plays a crucial role in solid tumor initiation and progression. However, the potential role of H19 and its clinical significance in acute myeloid leukemia (AML) remain largely elusive., Methods: H19 expression was detected by qPCR, and clinical significance in AML patients was further analyzed. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data for AML were used as validation cohorts. The roles of H19 in cell proliferation and apoptosis were determined by cell proliferation assay and flow cytometry analysis., Results: H19 expression was significantly increased in AML patients but not associated with embedded miR-675 expression. Moreover, H19 overexpression was not dependent on the methylation pattern in H19 differentially methylated region/imprinting control region. Strong association was observed between H19 overexpression and patients' characteristics including sex, higher white blood cells, older age, and intermediate karyotype, FLT3 -ITD, and DNMT3A mutations. In addition, H19 overexpression correlated with lower complete remission (CR) rate and shorter overall survival, and further confirmed by multivariate analyses. Importantly, the prognostic effect of H19 expression was validated by TCGA and GEO data. In the follow-up of patients, H19 expression in CR phase was lower than diagnosis time and returned at relapse time. Loss-of-function experiments showed that H19 exhibited anti-proliferative and pro-apoptotic effects in leukemic cell HL60. Furthermore, H19 expression was positively correlated with potential downstream gene ID2 in AML., Conclusions: Our findings revealed that methylation-independent H19 was a prognostic and predictive biomarker in AML, and H19 / ID2 played crucial roles in leukemogenesis with potential therapeutic target value., Competing Interests: The present study was approved by the Ethics Committee and Institutional Review Board of the Affiliated People’s Hospital of Jiangsu University.Written informed consents were obtained from all enrolled individuals prior to their participation.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Activation of Wnt/β-catenin signaling played a crucial role in tumorigenesis, and β-catenin (CTNNB1) overexpression has been identified in numerous solid tumors. The present study was designed to determine CTNNB1 expression and its clinical significance in Chinese de novo acute myeloid leukemia (AML) patients. Real-time quantitative PCR was carried out to detect the pattern of CTNNB1 expression in 140 AML patients and 46 controls. The level of CTNNB1 transcript in AML patients was significantly up-regulated compared with controls (P < 0.001). CTNNB1 high patients showed significantly older age than CTNNB1 low patients (P < 0.05). The frequency of high CTNNB1 expression was significantly observed in patients with intermediate/poor karyotypes. CTNNB1 high patients had a significantly lower complete remission (CR) rate than CTNNB1 low patients (P = 0.004). Among cytogenetically normal AML (CN-AML), CTNNB1 high patients presented significantly shorter overall survival (OS, P = 0.004) and leukemia-free survival (LFS, P = 0.038) than CTNNB1 low patients. Multivariate analysis confirmed that CTNNB1 expression was an independent prognostic factor for OS among CN-AML. Moreover, CTNNB1 expression level significantly decreased after CR stage (P = 0.032) and increased in relapsed stage (P = 0.015). Our findings suggest that CTNNB1 is overexpressed and confers a poor prognosis in AML, and could be used as a biomarker in monitoring disease recurrence., (Copyright © 2018. Published by Elsevier GmbH.)

Oncogenic role of miR-19 family has been identified in human cancers especially in lymphoid malignancies. However, to date, little studies investigated the role of miR-19 family in myeloid malignancies. Herein, we examined miR-19a/b expression and explored its clinical significance in de novo acute myeloid leukemia (AML). The detection of miR-19a/b expression was performed by real-time quantitative PCR in bone marrow mononuclear cells of 113 patients and 42 healthy donors. Both miR-19a/b levels were significantly increased in AML patients in contrast to controls. Patients with miR-19a/b overexpression were more frequently occurred in female, and had an older age. Moreover, cases with miR-19a overexpression had a higher frequency of U2AF1, C-KIT and CEBPA mutations, whereas miR-19b overexpressed cases harbored U2AF1 and IDH1/2 mutations. There was no significant association of miR-19a overexpression with complete remission (CR) rate and overall survival (OS) among whole-cohort AML, non-M3 AML, and cytogenetically normal AML (CN-AML). However, although miR-19b overexpression was not correlated with CR rate, patients with miR-19b overexpression presented significantly shorter OS in whole-cohort AML and a trend in non-M3 AML and CN-AML patients. Importantly, our data also showed that miR-19a/b expression level at CR phase was lower than diagnosis time, and was returned to primary level even higher when at relapse phase. Our findings revealed that miR-19a/b overexpression were frequent events in de novo AML patients. Moreover, up-regulation of miR-19b expression was associated with poor prognosis and disease recurrence in AML., (Copyright © 2017. Published by Elsevier B.V.)

Purpose: A systematic review and meta-analysis were performed to explore the efficacy and safety of allogeneic hematopoietic stem cell transplantation with a reduced intensity conditioning regimen in elderly patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML)., Methods: Overall survival (OS) and event-free survival (EFS) were established as the primary endpoints for directly assessing the efficacy, and non-relapse mortality (NRM) for safety. The eligible patients were at or above 50 years of age, and the outcomes of the typical elderly patients (≥60 years) were analyzed individually., Results: The pooled estimates (95% confidence interval (CI)) for 1-year OS, EFS and NRM were 65 (55-74)  %, 50 (44-55)  % and 26 (21-30)  %, respectively; as for the patients ≥60 years of age, these were 63 (53-72) %, 46 (41-50)  % and 28 (23-32) %, respectively. No significantly statistical difference achieved between MDS and AML patients in 1-year EFS and NRM [relative risk (RR) 0.91, 95% CI 0.80-1.04; P = 0.172 and RR 1.18, 95% CI 0.82-1.69; P = 0.365]. The patients with lower diseases risk had the possibility of higher OS rate at ≥ 3 years than those with higher diseases risk (RR 1.37, 95% CI 0.95-1.97; P = 0.088). The patients had significantly higher 2-year OS and EFS rates in complete remission (CR, CR1 and CR2) at transplantation compared to those with advanced diseases (P < 0.05)., Conclusions: RIC-alloHSCT is a feasible treatment option for the patients older than 50 year of age with MDS and AML. Advanced diseases status and higher diseases risk may be the poor factors for prognosis.

Elderly patients with acute myeloid leukemia (AML) have limited treatment options concerned about their overall fitness and potential treatment related mortality. Although a number of clinical trials demonstrated benefits of decitabine treatment in elderly AML patients, the results remains controversial. A meta-analysis was performed to evaluate efficacy and safety of decitabine in treatment of elderly AML patients. Eligible studies were identified from PubMed, Web of Science, Embase and Cochrane Library. Nine published studies were included in the meta-analysis, enrolling 718 elderly AML patients. The efficacy outcomes were complete remission (CR), overall response rate (ORR) and overall survival (OS). Safety was evaluated based on treatment related grades 3-4 adverse events (AEs) and early death (ED) rate. Pooled estimates with 95% confidence interval (CI) for CR, ORR and OS were 27% (95% CI 19%-36%), 37% (95% CI 28%-47%) and 8.09 months (95% CI 5.77-10.41), respectively. The estimated treatment related early death (ED) incidences were within 30-days 7% (95% CI 2%-11%) and 60-days 17% (95% CI 11%-22%), respectively. Thrombocytopenia was the most common grades 3-4 AEs. Subgroup analyses of age, cytogenetics risk, AML type and bone marrow blast percentage showed no significant differences of treatment response to decitabine. In conclusion, decitabine is an effective and well-tolerated therapeutic alternative with acceptable side effects in elderly AML patients.