Clinical Implication of DNMT3A and TET2 Genes Mutations in Cytogenetically Normal Acute Myeloid Leukemia

Background: Refining risk stratification of cytogenetically  normal AML (CN-AML) cases is important for decision making and tailoring of therapy. In this context genetic and epigenetic mutations was considered. Among these epigenetic regulators are DNMT3A & TET2 genes. Therefore, the aim of  this study was to determine the prevalence of DNMT3A and TET2 genes mutations and their impact on the outcome of  adult AML patients. Subjects and methods: The present study is cross sectional study which was conducted on 39 adult CN-AML patients at diagnosis. For all included patients sanger sequencing was done for DNMT3A exon 23 and TET2 exon 3 genes. Results: DNMT3A mutations were detected in 8 of 39 patients (20.5%), and in 5 of 39 patients(12.8%) in TET gene. Two CN-AML  patients had combined mutations in both genes. All of the mutations detected were missense and only one was frame shift. Mutated TET2 or DNMT3A genes were significantly associated with failure of complete remission (CR) (p <0.001), higher mortality rate, shorter OS (mean=16 versus 22.7 months) and shorter DFS (mean= 9.5 versus 21.4 months) when compared to non-mutated ones. Conclusion: Mutated TET2 and DNMT3A detection define a subgroup of CN-AML patients with poor outcome.     

Rapid Detection of N-RAS Gene Common Mutations in Acute Myeloid Leukemia (AML) Using High Resolution Melting (HRM) Method

Objective: Acute myeloid leukemia is caused by the clonal proliferation of undifferentiated myeloid hematopoietic precursors. AML prognosis is highly involved in the treatment response and is determined by mutations in several genes such as N-RAS. This study aims to identify the distribution of common N-RAS mutations (codons 12, 13, and 61) in AML patients using the HRM method and confirm this method’s efficiency for mutation detection by comparing its results with the sequencing data as the Gold standard method. Methods: Peripheral blood samples were taken from 50 newly diagnosed AML patients. Mononuclear cells were isolated from samples, and DNA was extracted. Then, mutation detection was investigated using the HRM method. Efficacy of the HRM method in mutation detection was determined in comparison with direct sequencing. Results: N-RAS mutations were detected in 7 of the 50 samples (14%). Most of the mutations were found in codon 12 (57.14%), and 28.57% and 14.28% of mutations were in codons 61 and 13, respectively. There was no statistically significant association between patients’ demographic data and HRM results. Conclusion: According to mutation detection results and the HRM results confirmation with the sequencing method, this method can be introduced as an efficient, low-cost, and fast method for detecting common mutations.     

Do AML patients with DNMT3A exon 23 mutations benefit from idarubicin as compared to daunorubicin? A single center experience

Mutations in DNMT3A encoding DNA methyltransferase 3A were recently described in patients with acute myeloid leukemia. To assess their prognostic significance, we determined the mutational status of DNMT3A exon 23 in 288 patients with AML excluding acute promyelocytic leukemia, aged from 18 to 65 years and treated in Toulouse University Hospital. A mutation was detected in 39 patients (13.5%). All DNMT3A exon 23+ patients had intermediate-risk cytogenetics. Mutations significantly correlated with a higher WBC count (p less than 0.001), NPM1 and FLT3-ITD mutations (p=0.027). DNMT3A mutations were conserved through xenotransplantation in immunodeficient mice. No difference in outcome between DNMT3A exon 23+ and DNMT3A exon 23- patients was found even if the results were stratified by NPM1 or FLT3-ITD status. However, DNMT3A exon 23+ patients had better median DFS (not reached vs 11.6 months, p=0.009) and OS (not reached vs 14.3 months, p=0.005) as compared to DNMT3A exon 23- patients when treated with idarubicin, whereas patients treated with daunorubicin had similar outcome regardless the DNMT3A status. This study shows that DNMT3A mutations have no impact on outcome but could be a predictive factor for response to idarubicin and thus, could have a direct influence in the way AML patients should be managed.     

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.     

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.     

TET2合并DNMT3A突变及其他共存基因突变对急性髓系白血病患者预后的影响

  目的  探究TET2合并DNMT3A突变及其他共存基因突变对成人非M3型急性髓系白血病（acute myeloid leukemia,AML）患者预后的影响。  方法  回顾性分析2018年1月至2021年9月于南昌大学第一附属医院确诊的初治且行血液肿瘤相关突变基因外显子二代测序检测的512例成人 AML（非 M3 型）患者的临床资料,分析患者的临床特征、疗效及生存情况。  结果  本研究共纳入110例AML患者,TET2突变组64例,DNMT3A单突变组46例。男性50例（45.5%）,中位年龄54（15～79）岁。TET2基因突变频率为12.5%（64/512）,98.4%（63/64）患者突变基因数≥2个,每例患者平均合并5.2个突变基因。NPM1（43.8%）、DNMT3A（42.2%）、FLT3-ITD/TKD（40.6%）、CEBPA（26.6%）、TTN（20.3%）为TET2突变常见的共存突变基因。TET2合并DNMT3A突变患者初次诱导完全缓解（complete response,CR）率为46.2%,略低于TET2单突变患者的76.9%（P=0.077）,与DNMT3A单突变患者无显著性差异（P=0.952）。TET2合并DNMT3A突变患者的中位总生存（median overall survival,mOS）时间为9.5个月,明显低于TET2单突变患者（P=0.002）,而与DNMT3A单突变患者无显著性差异（P=0.414）。三者的中位无复发生存（median relapse- free survival,mRFS）时间无显著性差异（P>0.05）。在TET2突变背景下,合并K/NRAS突变患者的CR率为28.6%,明显低于无K/NRAS突变患者的75.0%（P=0.030）,合并FLT3-ITD突变患者的mOS明显短于无FLT3-ITD突变患者（P=0.030）。多因素分析显示年龄≥60岁、合并FLT3-ITD突变、初次诱导未达CR是影响TET2突变AML患者总生存时间（overall survival,OS）的独立危险因素,DNMT3A突变不影响TET2突变患者OS。  结论  TET2突变是AML患者常见突变,且常合并共存基因突变。共存基因突变与TET2突变共同影响AML患者预后。基于二代测序的基因突变检测对指导AML精确分层及精准治疗具有重要意义。      

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。     

Quantitative detection of DNMT3A R882H mutation in acute myeloid leukemia

Background

DNMT3A mutations represent one of the most frequent gene alterations detectable in acute myeloid leukemia (AML) with normal karyotype. Although various recurrent somatic mutations of DNMT3A have been described, the most common mutation is located at R882 in the methyltransferase domain of the gene. Because of their prognostic significance and high stability during disease evolution, DNMT3A mutations might represent highly informative biomarkers for prognosis and outcome of disease.

Methods

We describe an allele-specific PCR with a Blocking reagent for the quantitative detection of DNMT3A R882H mutation providing the possibility to analyze the quantitative amount of mutation during the course of disease. Next, we analyzed 62 follow-up samples from 6 AML patients after therapy and allogeneic stem cell transplantation (alloSCT).

Results

We developed an ASB-PCR assay for quantitative analysis of R882H DNMT3A mutation. After optimization of blocker concentration, a R882H-positive plasmid was constructed to enhance the accuracy of the sensitivity of quantitative detection. The assay displayed a high efficiency and sensitivity up to 10⁻³. The reproducibility of assay analyzed using follow-up samples showed the standard deviation less than 3.1 %. This assay displayed a complete concordance with sequencing and endonuclease restriction analysis. We have found persistence of DNMT3A R882H mutations in complete remission (CR) after standard cytoreduction therapy that could be indicating presence of DNMT3A mutation in early pre-leukemic stem cells that resist chemotherapy. The loss of correlation between NPM1 and DNMT3A in CR could be associated with evolution of pre-leukemic and leukemic clones. In patients with CR with complete donor chimerism after alloSCT, we have found no DNMT3A R882H. In relapsed patients, all samples showed an increasing of both NPM1 and DNMT3A mutated alleles. This suggests at least in part the presence of NPM1 and DNMT3A mutations in the same cell clone.

Conclusion

We developed a rapid and reliable method for quantitative detection of DNMT3A R882H mutations in AML patients. Quantitative detection of DNMT3A R882H mutations at different time points of AML disease enables screening of follow-up samples. This could provide additional information about the role of DNMT3A mutations in development and progression of AML.     

Prognostic Impact of Concurrent DNMT3A,FLT3 and NPM1 Gene Mutations in Acute Myeloid Leukemia Patients

BackgroundAcute myeloid leukemia (AML) is one of the rapidly progressing malignancies which is characterized by unregulated proliferation of hematopoietic precursors. Technological improvements enhanced the availability of genetic AML biomarkers. The clinical relevance of these molecular markers for AML diagnosis, planning of therapy and risk stratification are increasing evidently.MethodsIn current study, one hundred newly diagnosed AML patients before receiving induction chemotherapy were included, they were subjected to clinical examination, cytochemical and morphological analysis of blood cells, flow cytometric, cytogenetic and molecular genetic analysis for detection of NPM1, FLT3-ITD and DNMT3A mutations. Direct sequencing analysis for detection of NPM1 and DNMT3A genes mutations were done. FLT3 /ITD gene mutation was detected by gel electrophoresis after PCR amplification.ResultsAccording to genetic markers, our AML patients are classified in to further 8groups. AML patients with three DNMT3A/FLT3/NPM1 gene mutations (AML ^{DNMT3A /FLT3/NPM1}) this group of patients presented with a heavy disease burden, had an elevated WBC in comparison to other groups (70 vs. 41 × 10³/μL; P = .019), and BM blast counts (71% vs. 55.6%, P < .02). When comparing eight groups for death event there were significant difference among groups; P = .005, group 1 (AML ^{DNMT3A /FLT3/NPM1}) showed rapid decline of the cumulative overall survival. There was a significant difference among 8 groups as regards response to treatment after 14 days (P = .02), group 7 AML with (DNMT3A +NMP1) gene mutations showed better response to treatment (100%), groups 1 and 3 AML with (NPM1+DNMT3A +NMP1) gene mutations and AML with isolated FLT3-ITD showed no response to treatment after 14 days. And as regards response to treatment after 28 days, the eight groups showed no significant difference (P = .14).ConclusionOur study supports adverse prognostic effect of presence of DNMT3A gene mutation either alone or in the presence of FLT3 and/or NPM1 gene mutations.     

HRM方法检测肺腺癌患者癌性胸水上清液EGFR基因突变的临床意义

目的:探讨应用HRM方法检测肺腺癌患者癌性胸水上清液EGFR基因突变状况及EGFR酪氨酸激酶受体抑制剂治疗的可行性。方法:收集43例肺腺癌患者癌性胸水上清液标本,提取DNA,应用HRM方法检测EGFR基因第18、19、20、21外显子突变状况。统计分析HRM方法与基因测序法检测EGFR突变率的差异。结果:43例肺腺癌患者癌性胸水上清液中,HRM法检测EGFR基因突变共17例,总突变率为39.53%,其中第19外显子突变14例,第21外显子突变3例。基因测序结果显示:EGFR突变14例,总突变率为32.56%,均为第19外显子突变。HRM方法检测第21外显子突变阳性的患者其基因测序结果均为阴性。这可能与HRM方法检测的灵敏度优于测序方法有关。两者突变率差异无统计学意义（P>0.05）。结论:对难以获取组织标本的肺腺癌患者而言,应用HRM方法检测其癌性胸水上清液,是了解其EGFR基因突变状况的可靠途径,对临床筛查靶向治疗药物具有一定的参考价值。     

Decreased DNA methylation in acute myeloid leukemia patients with DNMT3A mutations and prognostic implications of DNA methylation

We examined 79 acute myeloid leukemia (AML) patients for DNA methylation of 12 tumor suppressor genes (TSG) and 24 homeobox domain (Hox) genes, and additionally for mutations in DNMT3A gene. We observed lower levels of DNA methylation (P<0.0001) as well as smaller numbers of concurrently hypermethylated genes (P<0.0001) in patients with DNMT3A mutations. Our study of the impact of DNA methylation on prognosis in intermediate and high risk AML patients revealed a relation between higher DNA methylation and better patients' outcome. Lower DNA methylation was linked with higher relapse rates and an inferior overall survival.     

Novel mutations of the nucleophosmin (NPM-1) gene in Egyptian patients with acute myeloid leukemia: A pilot study

Mutations of the nucleophosmin (NPM-1) gene have been reported in 50-60% of acute myeloid leukemia (AML) patients with normal karyotype. This work was designed to study the prevalence and nature of NPM1 gene mutations in a group of Egyptian patients with AML to get an idea about the profile of NPM1 gene mutations in our society. In 45 previously untreated patients with de novo AML, peripheral blood and/or bone marrow samples from all patients were subjected to microscopic morphologic examination, cytochemical analysis, immunophenotyping and karyotyping. Patients with normal cytogenetic results were selected for molecular analysis of NPM1 exon 12 by PCR amplification followed by DNA sequencing of the amplified product. Twenty-one patients (46.7%) had abnormal karyotype: six cases with t(15;17), five cases with t(8;21), five cases had trisomy 8, two cases carrying inv(3) and three cases had monosomy 7. The remaining 24 patients (53.3%) had normal karyotype. These patients were then subjected to molecular analysis. Out of these 24 patients with normal karyotype, mutant NPM-1 was detected in 11 patients (45.8%) by DNA sequencing; 2 cases showed type A mutation, 2 cases were harboring [ins 1015-1019 (CACG)], with point mutation [1006C>G], while the remaining 7 cases showed heterozygous deletion of nt A [del 1178 (A)]. Conclusion: Two novel NPM1 gene mutations were detected among our study population of AML patients identified as: the insertion CACG associated with point mutation, deletion of one base, or associated with point mutation. NPM1 gene mutations may become a new tool for monitoring minimal residual disease in AML with normal karyotype. Whether these previously unreported NPM-1 mutations will confer the same better outcome as previously reported mutations is currently unknown and warrants a larger study. Conclusion: Two novel NPM1 gene mutations were detected among our study population of AML patients identified as: the insertion CACG associated with point mutation, deletion of one base, or associated with point mutation. NPM1 gene mutations may become a new tool for monitoring minimal residual disease in AML with normal karyotype. Whether these previously unreported NPM-1 mutations will confer the same better outcome as previously reported mutations is currently unknown and warrants a larger study.     

骨髓增生异常综合征患者DNMT3A基因突变的情况及临床意义

目的:了解DNMT3A基因突变在骨髓增生异常综合征（MDS）患者中的发生率、分布情况及临床意义。方法:选取2012年 4月至2018年6月在湘雅二医院和湘潭市中心医院住院的85例MDS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位点设计合成引物,采用聚合酶链式反应法扩增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基因突变在本组患者中的发生率、分布情况及临床意义。结果:85例MDS患者中检测到5例DNMT3A基因突变,突变阳性率5.9%,其中R882H和R882C基因突变各2例,R882P基因突变1例,未见R882S基因突变。常规化疗联合去甲基化药物地西他滨治疗后,1例患者一度获得血液学完全缓解,复杂染色体核型恢复正常,但是1月后转为急性白血病并发严重感染死亡,1例治疗后稳定,2例治疗后疾病进展,1例死于肺部感染。结论:MDS中DNMT3A基因突变率低,其突变多预示预后较差,并可能更快地向急性髓性白血病转化,选择去甲基化药物治疗可能获益。     

Identification of FLT3 and NPM1 Mutations in Patients with Acute Myeloid Leukaemia

Objective: The most frequent acquired molecular abnormalities and important prognostic indicators in patientswith Acute Myeloid Leukaemia (AML) are fms-like tyrosine kinase-3 gene (FLT3) and nucleophosmin-1 (NPM1)mutations. Our study aims to develop a cost effective and comprehensive in-house conventional PCR method fordetection of FLT3-ITD, FLT3-D835 and NPM1 mutations and to evaluate the frequency of these mutations in patientswith cytogenetically normal (CN) AML in our population. Methods: A total of 199 samples from AML patients (95women, 104 men) were included in the study. Mutation analyses were performed using polymerase chain reaction(PCR) and gene sequencing. Result: Sixty-eight patients were positive for the mutations. FLT3-ITD mutations weredetected in 32 patients (16.1%), followed by FLT3-D835 in 5 (2.5%) and NPM1 in 54 (27.1%). Double mutations ofNPM1 and FLT3-ITD were detected in 23 cases (11.6%). Assays validation were performed using Sanger sequencingand showed 100% concordance with in house method. Conclusion: The optimized in-house PCR assays for thedetection of FLT3-ITD, FLT3-D835 and NPM1 mutations in AML patients were robust, less labour intensive and costeffective. These assays can be used as diagnostic tools for mutation detection in AML patients since identification ofthese mutations are important for prognostication and optimization of patient care.     

急性髓系白血病RASSF1A基因启动子区异常甲基化临床意义研究

目的 RASSF1A基因异常甲基化可能参与血液肿瘤的发生,并为微小残留疾病(minimal residual de-sease,MRD)监测、分层、预后评估及靶向治疗提供依据.本研究旨在分析RASSF1A基因启动子区异常甲基化在急性髓系白血病(acutemyeloid leukemia,AML)中的临床意义.方法 选取2005-01-01-2013-03-01解放军总医院(113例)以及第一附属医院(39例)住院患者和门诊体检患者,共152例AML患者骨髓标本以及15例健康供者骨髓标本纳入本项研究.提取基因组DNA,并进行DNA硫化修饰;设计重亚硫酸盐测序PCR(bisulfite sequencing PCR,BS-PCR)引物以及甲基化特异性PCR(methylation specific PCR,MS-PCR)引物,进行PCR扩增,进而电泳分析以及DNA序列分析.同时对RASSF1A高甲基化组以及低甲基化组的血液学特点、骨髓原始细胞比例、细胞遗传学异常、基因异常、完全缓解率和总生存期进行统计学分析.结果 MS-PCR分析结果显示,RASSF1A基因在15例健康人中呈完全非甲基化状态,在152例AML患者中有38例出现启动子区高甲基化状态,其甲基化阳性率为25％.4例MS-PCR阳性AML患者经BS-PCR测序分析后,显示RASSF1A甲基化率分别为88.2％、85.5％、78.6％和92.7％,而在4例MS-PCR阴性患者RASSF1A基因启动子区甲基化率分别为10％、11.8％、12.7％和6.8％,4例健康供者RASSF1A基因启动子区甲基化率分别为5.0％、9.1％、8.2％和7.3％.进而通过统计学分析发现携带RASSF1A基因高甲基化的AML患者易合并存在ASXL1基因突变或DNMT3A基因突变.携带RASSF1A基因高甲基化的AML患者,其无进展生存期以及总生存期较短.结论 RASSF1A基因启动子区异常甲基化可能参与AML的发生,同时可能为AML分层诊治以及预后评估提供分子理论依据.     

高通量基因测序技术检测外周血循环肿瘤DNA基因突变在非小细胞肺癌中的应用

目的:探究高通量基因测序技术检测非小细胞肺癌外周血循环肿瘤DNA基因突变的应用价值。方法:临床纳入2017年1月至2018年9月在我院就诊的40例晚期非小细胞肺癌患者作为研究对象,所有患者入院后均经肺组织活检或气管镜检查确诊为晚期非小细胞肺癌。对患者进行病理组织石蜡切片DNA（tDNA）检测,并采集患者肘静脉血使用高通量基因测序技术检测患者外周血循环肿瘤ctDNA基因情况。对比分析tDNA与ctDNA检测对患者DNA基因突变的准确性,探讨非小细胞肺癌患者进行高通量基因测序技术检测外周血循环肿瘤DNA基因突变的应用价值。结果:40例非小细胞肺癌的外周血循环肿瘤DNA基因突变检测与组织石蜡切片比较,两种方法检测率差异无统计学意义（P>0.05）。在高通量基因测序技术检查外周血循环肿瘤DNA中,21外显子测序结果:61号替代突变2573G→T,62/63/68号替代突变L858R（2573T→G）。19外显子测序结果:50号样品突变为del E746→A750+2235G→A,60号样品突变为del E746→A750,70号样品突变为del L747→T751,80号样品突变为del L747→S752＋2257C→T。结论:非小细胞肺癌外周血循环肿瘤DNA基因突变进行高通量基因测序技术对具体的基因突变或缺失具有较高准确性,可实时监测肿瘤DNA基因突变情况,且具有无创性、可重复应用等优点。     

Detection of PIK3CA Gene Mutations with HRM Analysis and Association with IGFBP-5 Expression Levels in Breast Cancer

Breast cancer is the second most common cancer and second leading cause of cancer deaths inwomen. Phosphatidylinositol-3-kinase (PI3K)/AKT pathway mutations are associated with cancer andphosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene mutations have beenobserved in 25-45% of breast cancer samples. Insulin growth factor binding protein-5 (IGFBP-5) can showdifferent effects on apoptosis, cell motility and survival in breast cancer. We here aimed to determine theassociation between PIK3CA gene mutations and IGFBP-5 expressions for the first time in breast cancerpatients. Frozen tumor samples from 101 Turkish breast cancer patients were analyzed with high resolutionmelting (HRM) for PIK3CA mutations (exon 9 and exon 20) and 37 HRM positive tumor samples were analyzedby DNA sequencing, mutations being found in 31. PIK3CA exon 9 mutations (Q546R, E542Q, E545K, E542Kand E545D) were found in 10 tumor samples, exon 20 mutations (H1047L, H1047R, T1025T and G1049R)in 21, where only 1 tumor sample had two exon 20 mutations (T1025T and H1047R). Moreover, we detectedone sample with both exon 9 (E542Q) and exon 20 (H1047R) mutations. 35% of the tumor samples with highIGFBP-5 mRNA expression and 29.4% of the tumor samples with low IGFBP-5 mRNA expression had PIK3CAmutations (p=0.9924). This is the first study of PIK3CA mutation screening results in Turkish breast cancerpopulation using HRM analysis. This approach appears to be a very effective and reliable screening method forthe PIK3CA exon 9 and 20 mutation detection. Further analysis with a greater number of samples is needed toclarify association between PIK3CA gene mutations and IGFBP-5 mRNA expression, and also clinical outcomein breast cancer patients.     

Mechanisms of relapse in acute leukaemia: involvement of p53 mutated subclones in disease progression in acute lymphoblastic leukaemia

Mutations of the p53 tumour suppressor gene are infrequent at presentation of both acute myeloblastic leukaemia (AML) and acute lymphoblastic leukaemia (ALL), being found in between 5-10% of AML and 2-3% of ALL. Here we have studied the frequency of detection of p53 mutations at relapse of both AML and B-precursor ALL. In those patients with detectable mutations at relapse we investigated whether the mutation was detectable at presentation and was thus an early initiating event or whether it had arisen as a late event associated with relapse. Bone marrow samples from 55 adults and children with relapsed AML (n = 41) or ALL (n = 14) were analysed for p53 gene alterations by direct sequencing of exons 5-9. For samples where a p53 mutation was found at relapse, analysis of presentation samples was carried out by direct sequencing of the exon involved, or by allele-specific polymerase chain reaction (PCR) if the mutation could not be detected using direct sequencing. A p53 mutated gene was found at relapse in seven out of 55 cases. The frequency was higher in relapsed ALL (four out of 14 cases; 28.6%) compared to AML (three out of 41 cases; 7.3%). In five out of the seven cases presentation samples were available to study for the presence of the mutation. In two out of two AML patients the p53 mutation was detectable in the presentation sample by direct sequencing. In three ALL patients analysis of presentation material by direct sequencing showed a small mutant peak in one case, the other two being negative despite the sample analysed containing > 90% blast cells. However in both of these patients, the presence of p53 mutation was confirmed in the presentation sample using allele-specific PCR. In one of these patients the emergence of a subclone at relapse was confirmed by clonality analysis using IgH fingerprinting. Our results confirm that in ALL p53 mutations are present in a proportion of patients at relapse. Furthermore cells carrying the mutation are detectable at presentation in a minor clone suggesting that p53 mutations in ALL may be a mechanism contributing to disease relapse.