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Mutational spectrum and risk stratification of intermediate-risk acute myeloid leukemia patients based on next-generation sequencing

Intermediate-risk acute myeloid leukemia (IR-AML), which accounts for a substantial number of AML cases, is highly heterogeneous. Although several mutations have been identified, the heterogeneity of AML is uncertain because novel mutations have yet to be discovered. Here we applied next generation sequencing (NGS) platform to screen mutational hotspots in 410 genes relevant to hematological malignancy. IR-AML samples (N=95) were sequenced by Illumina Hiseq and mutations in 101 genes were identified. Only seven genes (CEBPA, NPM1, DNMT3A, FLT3-ITD, NRAS, IDH2 and WT1) were mutated in more than 10% of patients. Genetic interaction analysis identified several cooperative and exclusive patterns of overlapping mutations. Mutational analysis indicated some correlation between genotype and phenotype. FLT3-ITD mutations were identified as independent factors of poor prognosis, while CEBPA mutations were independent favorable factors. Co-occurrence of FLT3-ITD, NPM1 and DNMT3A mutations was identified with associated with specific clinical AML features and poor outcomes. Furthermore, by integrating multiple mutations in the survival analysis, 95 IR-AML patients could be stratified into three distinct risk groups allowing reductions in IR-AML by one-third. Our study offers deep insights into the molecular pathogenesis and biology of AML and indicated that the prognosis of IR-AML could be further stratified by different mutation combinations which may direct future treatment intervention.

Clinical implications of genome-wide DNA methylation studies in acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. AML is a heterogeneous malignancy characterized by distinct genetic and epigenetic abnormalities. Recent genome-wide DNA methylation studies have highlighted an important role of dysregulated methylation signature in AML from biological and clinical standpoint. In this review, we will outline the recent advances in the methylome study of AML and overview the impacts of DNA methylation on AML diagnosis, treatment, and prognosis.

Correlation Between Isocitrate Dehydrogenase Gene Aberrations and Prognosis of Patients with Acute Myeloid Leukemia: A Systematic Review and Meta-Analysis

Purpose: Whether isocitrate dehydrogenase (IDH) gene aberrations affected prognosis of patients with acute myeloid leukemia (AML) was controversial. Here, we conducted a meta-analysis to evaluate their prognostic value. Experimental Design: PubMed, Embase, Cochrane, and Chinese databases were searched to identify studies exploring how IDH gene aberrations affected AML outcome. Pooled HRs and relative risks (RR) were calculated, along with 95% confidence intervals (CI). Results: Thirty-three reports were included. IDH mutations seemed not to affect overall survival (OS: HR, 1.05; 95% CI, 0.89–1.23) and event-free survival (EFS: HR, 0.97; 95% CI, 0.80–1.18) when considered as a single factor, but improved accumulative incidence of relapse (CIR: HR, 1.44; 95% CI, 1.18–1.76) in patients with intermediate-risk karyotypes (IR-AML). However, IDH1 mutation conferred worse OS (HR, 1.17; 95% CI, 1.05–1.31) and EFS (HR, 1.29; 95% CI, 1.07–1.56), especially in patients with normal cytogenetics (OS: HR, 1.21; 95% CI, 1.01–1.46; EFS: HR, 1.56; 95% CI, 1.23–1.98). Prognosis of the IDH1 single-nucleotide polymorphism rs11554137 was also poor (OS: HR, 1.34; 95% CI, 1.03–1.75). IDH2 mutation improved OS (HR, 0.78; 95% CI, 0.66–0.93), particularly in IR-AML patients (OS: HR, 0.65; 95% CI, 0.49–0.86). The IDH2 (R140) mutation was associated with better OS among younger cases (HR, 0.64; 95% CI, 0.49–0.82). Treatment outcome was poor [RR for complete remission rates in IDH1 mutation: 1.21; 95% CI, 1.02–1.44; IDH2 (R172) mutation: 2.14; 95% CI, 1.61–2.85]. Conclusions: Various subtypes of IDH mutations might contribute to different prognosis and be allowed to stratify IR-AML further. Clin Cancer Res; 23(15); 4511–22. ©2017 AACR.

The Superiority of Allogeneic Hematopoietic Stem Cell Transplantation Over Chemotherapy Alone in the Treatment of Acute Myeloid Leukemia Patients with Mixed Lineage Leukemia (MLL) Rearrangements

Background Acute myeloid leukemia (AML) patients with mixed lineage leukemia (MLL) gene rearrangements always had a very poor prognosis. In this study, we report the incidence of MLL rearrangements in AML patients using gene analysis, as well as the clinical significance and prognostic features of these rearrangements. Material/Methods This retrospective study took place from April 2008 to November 2011 in the People’s Liberation Army General Hospital. A total 433 AML patients were screened by multiple nested reverse transcription polymerase chain reaction (RT-PCR) to determine the incidence of the 11 MLL gene rearrangements. There were 68 cases of MLL gene rearrangements, for a positive rate of 15.7%. A total of 24 patients underwent allogeneic hematopoietic stem cell transplantation (Allo-HSCT), and 34 patients received at least 4 cycles of chemotherapy. Ten patients were lost to follow-up. Results The median follow-up was 29 months. The complete remission (CR) rate was 85.4%. The overall survival (OS) was 57.4±5.9 months for the Allo-HSCT group and 21.0±2.1 months for the chemotherapy group. The Allo-HSCT group had superior survival compared with the chemotherapy group (5-year OS: 59±17% vs. 13±8%, P<0.01; 5-year disease-free survival [DFS]: 65±10% vs. 40±16%, P>0.05). Multivariate analysis showed that transplantation, platelets >50×109/L at onset, and CR are associated with a better OS in MLL rearranged AML patients. Patients with thrombocytopenia and extramedullary involvement were prone to relapse. Conclusions Our results suggest that Allo-HSCT is superior to chemotherapy alone for treating MLL rearranged AML patients. Patients treated with Allo-HSCT have a better prognosis and a longer survival. CR is an independent prognostic factor for OS, and extramedullary involvement is an independent prognostic factor for DFS. MLL rearranged AML patients with thrombocytopenia at onset <50×109 had very bad OS and DFS.

Implications of mutational spectrum in myelodysplastic syndromes based on targeted next-generation sequencing

Myelodysplastic syndromes (MDS) are a group of myeloid hematological malignancies, with a high risk of progression to acute myeloid leukemia (AML). To explore the role of acquired mutations in MDS, 111 MDS-associated genes were screened using next-generation sequencing (NGS), in 125 patients. One or more mutations were detected in 84% of the patients. Some gene mutations are specific for MDS and were associated with disease subtypes, and the patterns of mutational pathways could be associated with progressive MDS. The patterns, frequencies and functional pathways of gene mutations are different, but somehow related, between MDS and AML. Multivariate analysis suggested that patients with ≥ 2 mutations had poor progression-free survival, while GATA1/GATA2, DNMT3A and KRAS/NRAS mutations were associated with poor overall survival. Based on a novel system combining IPSS-R and molecular markers, these MDS patients were further divided into 3 more accurate prognostic subgroups. A panel of 11 target genes was proposed for genetic profiling of MDS. The study offers new insights into the molecular signatures of MDS and the genetic consistency between MDS and AML. Furthermore, results indicate that MDS could be classified by mutation combinations to guide the administration of individualized therapeutic interventions.

Detection of prognostic methylation markers by methylC-capture sequencing in acute myeloid leukemia

Clinical and genetic features incompletely predict outcome in acute myeloid leukemia (AML). The value of clinical methylation assays for prognostic markers has not been extensively explored. We assess the prognostic implications of methylC-capture sequencing (MCC-Seq) in patients with de novo AML by integrating DNA methylation and genetic risk stratification. MCC-Seq assessed DNA methylation level in 44 samples. The differentially methylated regions associated with prognostic genetic information were identified. The selected prognostic DNA methylation markers were independently validated in two sets. MCC-Seq exhibited good performance in AML patients. A panel of 12 differentially methylated genes was identified with promoter hyper-differentially methylated regions associated with the outcome. Compared with a low M-value, a high M-value was associated with failure to achieve complete remission (p = 0.024), increased hazard for disease-free survival in the study set (p = 0.039) and poor overall survival in The Cancer Genome Atlas set (p = 0.038). Hematopoietic stem cell transplantation and survival outcomes were not adversely affected by a high M-value (p = 0.271). Our study establishes that MCC-Seq is a stable, reproducible, and cost-effective methylation assay in AML. A 12-gene M-value encompassing epigenetic and genetic prognostic information represented a valid prognostic marker for patients with AML.

AML1–ETO promotes SIRT1 expression to enhance leukemogenesis of t(8;21) acute myeloid leukemia

Recently, SIRT1 was found to play an important role in a variety of solid and hematologic malignancies. The expression and function of SIRT1 may differ completely depending on cell type and gene subtype, and it can act as a tumor suppressor or oncogene. We describe how SIRT1 mRNA and protein levels are overexpressed in t(8;21) AML cells. AML1-ETO triggers the activation of SIRT1 by binding at AML1 binding sites on the SIRT1 promoter. Pharmacologic targeting or RNAi-mediated inhibition of SIRT1 induces G1 arrest, apoptosis, and proliferation inhibition that is more sensitive in AML1-ETO-positive than AML1-ETO-negative cell lines. Our data suggest that targeting SIRT1 may be an attractive therapeutic strategy in t(8;21) AML.

Methylation-associated silencing of BASP1 contributes to leukemogenesis in t(8;21) acute myeloid leukemia

The AML1-ETO fusion protein (A/E), which results from the t(8;21) translocation, is considered to be a leukemia-initiating event. Identifying the mechanisms underlying the oncogenic activity of A/E remains a major challenge. In this study, we identified a specific down-regulation of brain acid-soluble protein 1 (BASP1) in t(8;21) acute myeloid leukemia (AML). A/E recognized AML1-binding sites and recruited DNA methyltransferase 3a (DNMT3a) to the BASP1 promoter sequence, which triggered DNA methylation-mediated silencing of BASP1. Ectopic expression of BASP1 inhibited proliferation and the colony-forming ability of A/E-positive AML cell lines and led to apoptosis and cell cycle arrest. The DNMT inhibitor decitabine up-regulated the expression of BASP1 in A/E-positive AML cell lines. In conclusion, our data suggest that BASP1 silencing via promoter methylation may be involved in A/E-mediated leukemogenesis and that BASP1 targeting may be an actionable therapeutic strategy in t(8;21) AML.Cancer: Mismatched molecules unleash leukemia cell proliferationA chromosomal rearrangement commonly observed in certain leukemias selectively inactivates a gene that otherwise thwarts cancerous growth. Between 7 and 12% of acute myeloid leukemia cases exhibit a dramatic alteration in chromosomal structure that results in the production of an abnormal fusion protein. Researchers led by Li Yu at the General Hospital of Shenzen University in China have learned that this protein promotes disease progression by switching off an important tumor suppressor. Yu and colleagues showed that it binds a genomic sequence that regulates the gene encoding a second protein called BASP1, dramatically reducing its production. This gene silencing facilitates tumor growth. Chemicals that reactivated BASP1 production slowed proliferation and initiated ‘self-destruct’ mechanisms in leukemia cells. These findings suggest that BASP1-oriented therapies could offer a fruitful avenue of treatment for some patients.

Predictors of clinical responses to hypomethylating agents in acute myeloid leukemia or myelodysplastic syndromes

Azacitidine and decitabine, two hypomethylating agents, are known to be effective in the treatment of high-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) patients who cannot endure intensive cytotoxic chemotherapy or are not eligible for transplantation. However, the treatment response rate is low. The molecular mechanisms underlying the resistance to demethylation therapy are unclear. Though a wide range of predictors of treatment response have been investigated, no consensus has been reached. It is imperative to identify certain parameters that can help distinguish between patients who will obtain a favorable outcome from demethylation therapy and those who will not. Here, we describe currently researched potential predictors based on clinical characteristics, DNA methylation, gene mutation, gene expression, microRNAs, and protein expression. Although these parameters are not currently used in clinical practice, this review provides new sights into available clinical and experimental research. Moreover, this paper provides useful information on AML/MDS management.

The incidence and distribution characteristics of MLL rearrangements in Chinese acute myeloid leukemia patients by multiplex nested RT-PCR

Occurrence of MLL (Mixed Lineage Leukemia) gene rearrangements indicates poor prognosis in acute myeloid leukemia (AML) patients. This is the first study to report the positive rate and distribution characteristics of MLL rearrangements in AML patients in north China. We used multiplex nested real time PCR (RT-PCR) to screen for incidence of 11 MLL rearrangements in 433 AML patients. Eleven MLL rearrangements included (MLL-PTD, MLL-AF9, MLL-ELL, MLL-AF10, MLL-AF17, MLL-AF6, MLL-ENL, MLL-AF1Q, MLL-CBP, MLL-AF1P, MLL-AFX1). There were 68 AML patients with MLL rearrangements, and the positive rate was 15.7%. MLL-PTD (4.84%) was detected in 21 patients, MLL-AF9 in 15, (3.46%), MLL-ELL in 10 (2.31%), MLL-AF10 in 8 (1.85%), MLL-AF1Q in 2 (0.46%), 3 cases each of MLL-AF17, MLL-AF6, MLL-ENL (0.69% each), a and single case each of MLL-CBP, MLL-AF1P, and MLL-AFX1 (0.23% each). The highest rate of MLL rearrangements was found in 24 patients with M5 subtype AML, occurring in 24 cases (35.3%). MLL rearrangements occurred in 21 patients with M2 subtype AML (30.9%), and in 10 patients with M4 subtype AML (14.7%). Screening fusion genes by multiplex nested RT-PCR is a convenient, fast, economical, and accurate method for diagnosis and predicting prognosis of AML.