Comparative examination of various PCR-based methods for DNMT3A and IDH1/2 mutations identification in acute myeloid leukemia

Background

Mutations in epigenetic modifiers were reported in patients with acute myeloid leukaemia (AML) including mutations in DNA methyltransferase 3A gene (DNMT3A) in 20%-30% patients and mutations in isocitrate dehydrogenase 1/2 gene (IDH1/2) in 5%-15% patients. Novel studies have shown that mutations in DNMT3A and IDH1/2 influence prognosis, indicating an increasing need to detect these mutations during routine laboratory analysis. DNA sequencing for the identification of these mutations is time-consuming and cost-intensive. This study aimed to establish rapid screening tests to identify mutations in DNMT3A and IDH1/2 that could be applied in routine laboratory procedures and that could influence initial patient management.

Methods

In this study we developed an endonuclease restriction method to identify the most common DNMT3A mutation (R882H) and an amplification-refractory mutation system (ARMS) to analyse IDH2 R140Q mutations. Furthermore, we compared these methods with HRM analysis and evaluated the latter for the detection of IDH1 mutations.

Results

Of 230 samples from patients with AML 30 (13%) samples had DNMT3A mutations, 16 (7%) samples had IDH2 R140Q mutations and 36 (16%) samples had IDH1 mutations. Sensitivity assays performed using serial dilutions of mutated DNA showed that ARMS analysis had a sensitivity of 4.5%, endonuclease restriction had a sensitivity of 0.05% and HRM analysis had a sensitivity of 5.9%–7.8% for detecting different mutations. HRM analysis was the best screening method to determine the heterogeneity of IDH1 mutations. Furthermore, for the identification of mutations in IDH2 and DNMT3A, endonuclease restriction and ARMS methods showed a perfect concordance (100%) with Sanger sequencing while HRM analysis showed a near-perfect concordance (approximately 98%).

Conclusion

Our study suggested that all the developed methods were rapid, specific and easy to use and interpret. HRM analysis is the most timesaving and cost-efficient method to rapidly screen all the 3 genes at diagnosis in samples obtained from patients with AML. Endonuclease restriction and ARMS assays can be used separately or in combination with HRM analysis to obtain more reliable results. We propose that early screening of mutations in patients with AML having normal karyotype could facilitate risk stratification and improve treatment options.     

非髓性白血病的其他恶性血液病中DNMT3A基因突变的检测及临床意义

目的:了解DNMT3A基因突变在非髓性白血病的其他恶性血液肿瘤患者中的发生率、分布情况及临床意义.方法:选取196例非髓性白血病的其他血液肿瘤患者为研究对象,提取患者外周血基因组DNA,针对DNMT3A基因突变热点R882位点设计引物,采用聚合酶链式反应(PCR)法扩增DNMT3A基因23号外显子整个编码区基因片段,再将扩增产物纯化后测序,分析DNMT3A基因突变在本组恶性血液病患者中的发生率、分布情况及临床意义.结果:在57例非霍奇金淋巴瘤及34例骨髓增生异常综合征患者中各检出1例伴DNMT3A基因突变,在25例急性淋巴细胞白血病、45例多发性骨髓瘤和35例骨髓增殖性肿瘤患者中均未检测到DNMT'3A基因突变.结论:非髓性白血病的其他血液肿瘤患者中DNMT3A基因突变少见,伴DNMT3A基因突变的1例T淋巴母细胞白血病/淋巴瘤患者预后不良,联合去甲基化药物的化疗方案使伴该基因突变的1例骨髓增生异常综合征患者一度获得血液学完全缓解,但短期内转为急性白血病并发严重感染死亡.     

Prevalence and impact of colony stimulating factor 3 receptor (CSF3R) mutations among Egyptian acute myeloid leukemia patients

Granulocyte-colony stimulating factor receptor (G-CSFR) mutations have been implicated in the progression of severe congenital neutropenia (SCN) to leukemia. This study aimed to investigate the prevalence of colony stimulating factor 3 receptor (CSF3R) mutations among Egyptian acute myeloid leukemia and their clinic-pathological impact. The study was conducted on 179 adult patients (156 de novo AML and 23 secondary AML on top of SCN). CSF3R mutations were analyzed by sequencing of the PCR products. CSF3R mutations were detected in 2 cases out of 156 de novo AML patients (1.2%) and eighteen cases out of 23 secondary AML patients (78.2%). It was noticed that most of the mutant cases are of younger age, have a high white blood cells count, high bone marrow blasts, bad performance status, and absence of extra medullary disease and with low rate induction remission. Also the overall survival of AML patient's secondary to CSF3R mutations was shorter as compared to those with wild type AML cases. In conclusion the frequency of CSF3R mutations is highly prevalent among AML patients secondary to SCN compared to de novo AML.     

Mutational analysis of DNMT3A improves the prognostic stratification of patients with acute myeloid leukemia

Nucleophosmin1 (NPM1) mutations are the most frequently detected gene mutations in acute myeloid leukemia (AML) and are considered a favorable prognostic factor. We retrospectively analyzed the prognosis of 605 Japanese patients with de novo AML, including 174 patients with NPM1-mutated AML. Although patients with NPM1-mutated AML showed a high remission rate, this was not a favorable prognostic factor for overall survival (OS); this is contrary to generally accepted guidelines. Comprehensive gene mutation analysis showed that mutations in codon R882 of DNA methyltransferase 3A (DNMT3A^R882 mutations) were a strong predicative factor indicating poor prognosis in all AML (p < 0.0001) and NPM1-mutated AML cases (p = 0.0020). Furthermore, multivariate analysis of all AML cases showed that DNMT3A^R882 mutations and the co-occurrence of internal tandem duplication in FMS-like tyrosine kinase 3 (FLT3-ITD), NPM1 mutations, and DNMT3A^R882 mutations (triple mutations) were independent factors predicting a poor prognosis related to OS, with NPM1 mutations being an independent factor for a favorable prognosis (hazard ratios: DNMT3A^R882 mutations, 1.946; triple mutations, 1.992, NPM1 mutations, 0.548). Considering the effects of DNMT3A^R882 mutations and triple mutations on prognosis and according to the classification of NPM1-mutated AML into three risk groups based on DNMT3A^R882/FLT3-ITD genotypes, we achieved the improved stratification of prognosis (p < 0.0001). We showed that DNMT3A^R882 mutations are an independent factor for poor prognosis; moreover, when confounding factors that include DNMT3A^R882 mutations were excluded, NPM1 mutations were a favorable prognostic factor. This revealed that ethnological prognostic discrepancies in NPM1 mutations might be corrected through prognostic stratification based on the DNMT3A status.     

GPX3 hypermethylation serves as an independent prognostic biomarker in non-M3 acute myeloid leukemia

Hypermethylation of GPX3 (glutathione peroxidase 3) promoter has been identified in various solid tumors. However, the pattern of GPX3 promoter methylation in acute myeloid leukemia (AML) remains poorly known. The current study was intended to investigate the clinical significance of GPX3 promoter methylation in de novo AML patients and further determine its role in regulating GPX3 expression. GPX3 promoter methylation status in 181 de novo AML patients and 44 normal controls was detected by real-time quantitative methylation-specific PCR and bisulfite sequencing PCR. Real-time quantitative PCR was carried out to assess GPX3 expression. GPX3 promoter was significantly methylated in 181 AML patients compared with normal controls (P=0.022). The patients with GPX3 methylation presented significantly older age than those with GPX3 unmethylation (P=0.011). GPX3 methylated patients had significantly lower frequency of C/EBPA mutation and higher incidence of FLT3-ITD mutation (P=0.037 and 0.030). The non-M3 patients with GPX3 methylation had significantly lower overall survival than thoes with GPX3 unmethylation (P=0.036). No significant correlation was observed between GPX3 expression and its promoter methylation (R=0.110, P=0.284). However, GPX3 mRNA level was significantly increased after 5-aza-2’-deoxycytidine treatment in leukemic cell line THP1. GPX3 methylation predicts adverse clinical outcome in non-M3 AML patients. Moreover, GPX3 expression is regulated by its promoter methylation in leukemic cell line THP1.     

DNMT3A、FLT3-ITD合并NPM1基因突变正常核型急性髓系白血病患者的临床特征及预后分析

目的 分析DNMT3A、FLT3-ITD合并NPM1基因突变的正常核型急性髓系白血病(AML)患者的临床特征,探讨该类患者的预后.方法 回顾性分析2005年12月至2014年6月就诊的109例正常核型AML患者的临床特征及预后.其中DNMT3A/FLT3-ITD/NPM1+AML患者共25例,单独FLT3-ITD+ AML患者共32例,单独NPM1+AML患者24例,单独DNMT3A+ AML患者28例.结果 25例DNMT3A/FLT3-ITD/NPM1+ AML患者平均年龄46岁,伴有高白细胞(81.7×109/L)及较高的骨髓原始细胞(66.3％).其中,17例选择大剂量化疗,8例行异基因造血干细胞移植,与DNMT3A+、FLT3-ITD+、NPM1+AML组比较,差异均无统计学意义.25例DNMT3A/FLT3-ITD/NPM1+患者的3年总生存率为17.65％,3年无病生存率为13.88％,与DNMT3A+、FLT3-ITD+、NPM1+AML组差异均有统计学意义(P值分别为0.049 9、0.036 1、0.013 4),3年无病生存率差异无统计学意义(均P＞ 0.05).结论 DNMT3A、FLT3-ITD合并NPM1基因突变的正常AML患者往往合并高的外周血白细胞及骨髓原始细胞,预后较差.     

DNMT3A mutation is a poor prognosis biomarker in AML: Results of a meta-analysis of 4500 AML patients

Somatic DNA methyl transferase 3A (DNMT3A) mutations have been recognized recently as recurrent molecular aberrations in acute myeloid leukemia (AML). The precise role of these mutations in leukemogenesis remains elusive but a number of studies have already been conducted to study their potential prognostic value in AML patients with variable results. We performed a meta-analysis on published data from over 4500 AML patients to provide robust evidence supporting DNMT3A mutation testing in clinical setting for AML patients. Our meta-analysis showed that DNMT3A mutations were associated with M4 and M5 AML subtypes. Those mutations conferred significantly worse prognosis with both shorter OS (p = 0.0004) and shorter RFS (p = 0.002). Notably, DNMT3A mutations appeared to be an independent adverse prognostic factor also in younger patients with normal cytogenetics AML (OS (p = 0.01) and RFS (p = 0.0005)) and also in the subgroup of patients with high risk genotypes defined according to the criteria of the European Leukemia Net (ELN) (OS (p = 0.002)). Therefore, DNMT3A mutational status can improve the risk stratification of AML patients in the setting of integrated mutational profiling.     

FLT3、NPM1、DNMT3A及IDH基因突变对非M3型急性髓系白血病预后影响的研究进展

非M3型急性髓系白血病(AML)是一组异质性恶性血液系统疾病,无论在形态学、免疫学、细胞遗传学、分子生物学及临床特点上都存在很大差异.细胞遗传学检测结果已经成为AML精确诊断、治疗选择和预后判断的主要依据之一,但由于在分子水平上存在高度的异质性,常规细胞遗传学技术检出率不高.有40%~50%的AML患者在初诊时用标准的染色体显带分析方法检测不到克隆性染色体畸变,被称为正常核型AML.非M3型AML较常见的分子遗传学改变为FLT3、NPM1、DNMT3A、IDH基因突变.文章主要就四种基因的特点、发病机制及对非M3型AML的预后影响作一综述.     

GPX3 hypermethylation serves as an independent prognostic biomarker in non-M3 acute myeloid leukemia

Hypermethylation of GPX3 (glutathione peroxidase 3) promoter has been identified in various solid tumors. However, the pattern of GPX3 promoter methylation in acute myeloid leukemia (AML) remains unknown. The current study was intended to investigate the clinical significance of GPX3 promoter methylation in de novo AML patients and further determine its role in regulating GPX3 expression. GPX3 promoter methylation status was detected in 181 de novo AML patients and 44 normal controls by real-time quantitative methylation-specific PCR and bisulfite sequencing PCR. Real-time quantitative PCR was carried out to assess GPX3 expression. GPX3 promoter was significantly methylated in AML patients compared with normal controls (P=0.022). The patients with GPX3 methylation presented significantly older age than those with GPX3 unmethylation (P=0.011). GPX3 methylated patients had significantly lower frequency of C/EBPA mutation and higher incidence of FLT3-ITD mutation (P=0.037 and 0.030, respectively). The non-M3 patients with GPX3 methylation had significantly lower overall survival than those with GPX3 unmethylation (P=0.036). No significant correlation was observed between GPX3 expression and its promoter methylation (R=0.110, P=0.284). However, GPX3 mRNA level was significantly increased after 5-aza-2’-deoxycytidine treatment in leukemic cell line THP1. Our data suggest that GPX3 methylation predicts adverse clinical outcome in non-M3 AML patients. Moreover, GPX3 expression is regulated by its promoter methylation in leukemic cell line THP1.     

EZH2 在急性髓系白血病病程中的表达和对预后判断的意义研究

目的：探讨DNA 甲基转移酶（DNAmethyltransferase,DNMT 1）与EZH 2 在急性髓系白血病（acutemyeloidleukemia,AML ）中的表达及其相关性。方法：采用荧光定量PCR（fluorescencequantitativePCR ,FQ-PCR）检测大连医科大学附属第二医院2014年1月至2015年1 月50例初治AML 患者骨髓细胞中DNMT 1 与EZH 2 mRNA 的表达水平,分析DNMT 1 表达水平与临床特征和预后的关系以及与EZH 2 的相关性。结果：50例AML 患者骨髓细胞中DNMT 1 mRNA 的表达显著高于30例正常供者（2.72± 0.73vs . 0.89±0.27,P < 0.01）;EZH 2 的表达水平也显著高于正常供者（4.39± 1.06vs . 1.87± 0.33,P < 0.01）;EZH 2 与DNMT 1 的表达水平呈显著正相关（r = 0.51,P = 0.002）;DNMT 1 表达水平与外周血幼稚细胞比例≥ 60%（P < 0.05）、WBC≥ 50× 10⁹/L （P < 0.05）呈显著相关;DNMT 1 高表达组患者中位生存时间15个月（95%CI 为9～19个月）,显著低于DNMT 1 低表达组患者的32个月（95%CI 为27～40个月,P =0.006）。结论：DNMT 1 和EZH 2 在AML 患者中均高表达,两者表达水平呈正相关,DNMT 1 与AML 患者预后不良相关。     

CDKN2A and CDK4 mutation analysis in Italian melanoma-prone families: functional characterization of a novel CDKN2A germ line mutation

Physical interaction between CDKN2A/p16 and CDK4 proteins regulates the cell cycle progression through the G1 phase and dysfunction of these proteins by gene mutation is implicated in genetic predisposition to melanoma. We analysed 15 Italian melanoma families for germ line mutations in the coding region of the CDKN2A gene and exon 2 of the CDK4 gene. One novel disease-associated mutation (P48T), 3 known pathological mutations (R24P, G101W and N71S) and 2 common polymorphisms (A148T and Nt500 G>C) were identified in the CDKN2A gene. In a family harbouring the R24P mutation, an intronic variant (IVS1, +37 G>C) of uncertain significance was detected in a non-carrier melanoma case. The overall incidence of CDKN2A mutations was 33.3%, but this percentage was higher in families with 3 or more melanoma cases (50%) than in those with only 2 affected relatives (25%). Noteworthy, functional analysis established that the novel mutated protein, while being impaired in cell growth and inhibition assays, retains some in vitro binding to CDK4/6. No variant in the p16-binding region of CDK4 was identified in our families. Our results, obtained in a heterogeneous group of families, support the view that inactivating mutations of CDKN2A contribute to melanoma susceptibility more than activating mutations of CDK4 and that other genetic factors must be responsible for melanoma clustering in a high proportion of families. In addition, they indicate the need for a combination of functional assays to determine the pathogenetic nature of new CDKN2A mutations.     

Comparison of High-Resolution Melting (HRM) Analysis with Direct Sequencing for the Detection of DNMT3A Mutations in AML Patients

Objective: Acute myeloid leukemia (AML) is caused by abnormal gene expression following mutations. Many of the mutations in AML lead to gene instability and poor response to treatment. Among these mutations, DNMT3A mutation is exceedingly important due to its major role in methylation and its effect on the expression of other genes. Aberrant methylation due to DNMT3A mutations that mostly occur in exon 23, affects the overall survival (OS) of patients with AML and myelodysplastic syndromes (MDS) showing the importance of identification of these mutations. According to the association of these mutations with short overall survival and disease progression in AML patients, we aimed to investigate DNMT3A gene exon 23 mutations using HRM. Methods: Fifty peripheral blood samples were taken from patients with AML. Mononuclear cells were isolated by ficoll method, and DNA was extracted. Then, mutation detection was detected using the HRM method. Efficacy of the HRM method in mutation detection was compared with direct sequencing method as gold standard. Results: Mutations in codon 23 of the DNMT3A gene were detected in 5 patients (10%). All of the detected mutations were missense type. A comparison between direct sequencing and HRM analysis demonstrated full concordance of mutation detection. Conclusion: According to the full consistency between the HRM and direct sequencing methods, HRM is suggested to be adopted as an alternative for the common time-consuming methods in detecting the gene mutations.     

Flt3 in acute myelogenous leukemia

Flt3 is a tyrosine kinase receptor expressed on hematopoietic cells. Activating mutations of this receptor are encountered in over one-fourth of acute myelogenous leukemia (AML) cases and activate multiple intracellular pathways leading to cell proliferation, inhibition of apoptosis, and blockage of differentiation in leukemic blasts. AML with flt3 mutations has a worse prognosis than AML with normal flt3, at least in younger patients. Sevaral flt3 inhibitors are in various stages of preclinical and clinical development and it is hoped that specific therapies against AML with flt3 mutations will soon be available to the clinician.     

H3F3A K27M mutations in thalamic gliomas from young adult patients

Introduction

Mutations in H3F3A, which encodes histone H3.3, commonly occur in pediatric glioblastoma. Additionally, H3F3A K27M substitutions occur in gliomas that arise at midline locations (eg, pons, thalamus, spine); moreover, this substitution occurs mainly in tumors in children and adolescents. Here, we sought to determine the association between H3F3A mutations and adult thalamic glioma.

Methods

Genomic H3F3A was sequenced from 20 separate thalamic gliomas. Additionally, for 14 of the 20 gliomas, 639 genes—including cancer-related genes and chromatin-modifier genes—were sequenced, and the Infinium HumanMethylation450K BeadChip was used to examine DNA methylation across the genome.

Results

Of the 20 tumors, 18 were high-grade thalamic gliomas, and of these 18, 11 were from patients under 50 years of age (median age, 38 y; range, 17–46), and 7 were from patients over 50 years of age. The H3F3A K27M mutation was present in 10 of the 11 (91%) younger patients and absent from all 7 older patients. Additionally, H3F3A K27M was not detected in the 2 diffuse astrocytomas. Further sequencing revealed recurrent mutations in TP53, ATRX, NF1, and EGFR. Gliomas with H3F3A K27M from pediatric or young adult patients had similar, characteristic DNA methylation profiles. In contrast, thalamic gliomas with wild-type H3F3A had DNA methylation profiles similar to those of hemispheric glioblastomas.

Conclusion

We found that high-grade thalamic gliomas from young adults, like those from children and adolescents, frequently had H3F3A K27M.     

Characterisation of the GRAF gene promoter and its methylation in patients with acute myeloid leukaemia and myelodysplastic syndrome

We report the isolation of the 5' flanking region of GRAF (GTPase regulator associated with the focal adhesion kinase), previously described as a putative tumour suppressor gene of acute myelogenous leukaemia and myelodysplastic syndrome, and demonstrate its promoter activity in reporter gene assays. Two putative protein-binding sites are identified of which one was sensitive to CpG methylation. The suppressed GRAF expression could be restored in leukaemia cell lines by treatment with a demethylating agent and an inhibitor of histone deacetylases. In contrast to normal tissues, which tested negative for GRAF promoter methylation, 11 of 29 (38%) bone marrow samples from patients with acute myeloid leukaemia or myelodysplastic syndrome were positive.     

BAALC expression: a suitable marker for prognostic risk stratification and detection of residual disease in cytogenetically normal acute myeloid leukemia

High brain and acute leukemia, cytoplasmic (BAALC) expression defines an important risk factor in cytogenetically normal acute myeloid leukemia (CN-AML). The prognostic value of BAALC expression in relation to other molecular prognosticators was analyzed in 326 CN-AML patients (<65 years). At diagnosis, high BAALC expression was associated with prognostically adverse mutations: FLT3 internal tandem duplication (FLT3-ITD) with an FLT3-ITD/FLT3 wild-type (wt) ratio of ⩾0.5 (P=0.001), partial tandem duplications within the MLL gene (MLL-PTD) (P=0.002), RUNX1 mutations (mut) (P<0.001) and WT1mut (P=0.001), while it was negatively associated with NPM1mut (P<0.001). However, high BAALC expression was also associated with prognostically favorable biallelic CEBPA (P=0.001). Survival analysis revealed an independent adverse prognostic impact of high BAALC expression on overall survival (OS) and event-free survival (EFS), and also on OS when eliminating the effect of allogeneic stem cell transplantation (SCT) (OS^TXcens). Furthermore, we analyzed BAALC expression in 416 diagnostic and follow-up samples of 66 patients. During follow-up, BAALC expression correlated with mutational load or expression levels, respectively, of other minimal residual disease markers: FLT3-ITD (r=0.650, P<0.001), MLL-PTD (r=0.728, P<0.001), NPM1mut (r=0.599, P<0.001) and RUNX1mut (r=0.889, P<0.001). Moreover, a reduction in BAALC expression after the second cycle of induction chemotherapy was associated with improved EFS. Thus, our data underline the utility of BAALC expression as a marker for prognostic risk stratification and detection of residual disease in CN-AML.     

伴NRAS基因突变的急性髓系白血病患者的临床特征及生存分析北大核心

目的:探讨伴有NRAS基因突变的急性髓系白血病(acute myeloid leukemia,AML)患者的临床特征及生存分析。方法:回顾性分析2016年05月至2019年12月就诊于空军军医大学唐都医院血液内科的225例初诊AML患者的临床资料,采用二代测序技术进行基因突变检测,分析NRAS基因突变患者的临床特征并进行预后分析。结果:共有36例(16%)患者检测到NRAS基因突变,全部为错义突变,33例突变位点位于2号外显子12、13号密码子,2例位于3号外显子61号密码子,1例位于4号外显子109密码子。中位突变比例为21.99%(1%~49.85%)。伴有NRAS基因突变的患者中位年龄为44(9~85)岁。年龄分布、前期血液病史、初诊时外周血细胞计数、骨髓原始细胞比例等与无NRAS患者差异相比均无统计学意义。伴有NRAS突变组的患者一疗程诱导化疗完全缓解率高于NRAS野生型组(P=0.033),但复发率也较高(P=0.055)。伴NRAS基因突变组患者的中位OS时间为22个月,无NRAS基因突变组患者的中位OS时间为28个月,两组OS时间无统计学差异(P=0.485);两组患者的RFS时间存在统计学差异(P=0.036)。采用“3+7”方案诱导治疗的患者中位OS时间40个月,8例采用DEC+CAG方案诱导化疗的患者中位OS时间为15个月(P=0.027)。36例患者中31例伴有其他基因突变,生存分析显示NRAS同时伴DNMT3A突变组患者的预后更差(P=0.019)。结论:AML患者NRAS突变率16%,均为错义突变,大部分位于2号外显子12、13号密码子。伴有NRAS突变的患者CR率高,复发率也高,RFS时间短于无NRAS突变组。“3+7”方案诱导治疗缓解率更高。NRAS伴DNMT3A突变提示预后差。     

Prognostic factors in normal karyotype acute myeloid leukemia in the absence of the FLT3-ITD mutation

Patients with normal karyotype acute myeloid leukemia (NK-AML) without the FLT3 internal tandem duplication (FLT3-ITD) mutation account for approximately 30% of all AML cases, and exhibit a heterogeneous clinical outcome. Except for NPM1 mutations, prognostic factors in this subgroup of AML are still unclear. Here we explored the impact of immunophenotypic markers along with NPM1 mutations and clinical features on the outcome of 133 FLT3-ITD negative NK-AML patients. CD34 expression was associated with poorer complete remission (CR) rate, disease-free survival (DFS), event-free survival (EFS), and overall survival (OS), whereas CD56 expression adversely affected EFS and OS. In contrast, NPM1 mutations correlated with an improved CR rate, DFS, and EFS. Moreover, males experienced shorter DFS and EFS, while older patients (≥60 years) had shorter EFS. Multivariate analysis of age, gender, NPM1, CD34, and CD56 showed NPM1 mutation was an independent predictor of better CR rate, DFS, and EFS (P < 0.001, P = 0.003, and P = 0.006, respectively). In addition, older age was associated with shorter DFS and EFS (P = 0.045 and P = 0.028, respectively), and CD56 positivity predicted shorter EFS (P = 0.012). Our results confirm the favorable impact of NPM1 mutations and identify the adverse prognostic relevance of CD56 expression in this subgroup of AML.     

Prognostic Relevance of DNMT3A,FLT3 and NPM1 Mutations in Syrian Acute Myeloid Leukemia Patients

Objective: Among all types of hematological neoplasms, acute myeloid leukemia (AML) has the highest death rate. Recently, cytogenetic and molecular genetics are crucial in the management, as a consequence of their effect on AML pathogenesis, classification, risk-stratification, prognosis and treatment. Methods: 100 Syrian adults with Normal Karyotype (NK) newly diagnosed  AML patients were included in this study, all cases confirmed histologically and immunohistochemically. Patients were divided into six subgroups using flow cytometry and cytological results. Polymerase chain reaction (PCR) was performed on exon 11-12 for FMS-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD), exon 12 for Nucleophosmin1 (NPM1), and exon 23 for DNA methyltransferase 3A (DNMT3A) using target primers, the electropherograms were analyzed for gene mutations by comparing with the reference DNA sequence. Data were compared and aligned with different sequences using the NCBI BLAST Assembled Genomes tool. Results: FLT3-ITD, NPM1 and DNMT3A were detected in 24%, 22 % and 4%  patients respectively. M2 subtype had the most frequent incidence of diagnosis in AML. FLT3-ITD mutation patients had the highest mean of death cases, while the DNMT3A mutation patients had the lowest. On the other hand, the highest mean of remission was in patients with NPM1 mutation and the lowest in the carriers of the FLT3-ITD mutation. It was observed that the mean relapsed patients with FLT3-ITD and DNMT3A mutation was 3.4 and 2 months respectively, with no significant differences between (FLT3-ITD and DNMT3A) carriers and non-carriers relapsed. On the contrary,  the mean relapsed for NPM1 mutation carriers was 2.4  months with significant statistical differences. The mean survival time for patients with FLT3-ITD and NPM1  mutation was 5.9 months and 5.85 months respectively, with significant correlation. Between it was 5.88 months in DNMT3A patients with no significant differences. Finally, It was noted that the mean event free survival (EFS) of FLT3-ITD mutation patients was 4.818 months and the mean EFS of NPM1 mutation patients was 4.805 months, with significant statistical differences (p<0.05) between the mutation patients and non-mutated patients regarding to EFS, While this mean was not statistically significant in patients carrying DNMT3A mutation. Conclusion: Patients with FLT3-ITD and NPM1 mutations have the worst prognosis, where the presence of those mutations was significantly related to overall survival (OS) and EFS. Our study reflects that DNMT3A was not an extremely bad prognostic effect as an independent factor. We can declare according to this study that genetic mutation and variants detection could easily be incorporated into the regimen evaluation of AML patients.