【摘要】：【目的】1.回顧總結本中心應用異基因造血干細胞移植(allo-HSCT)治療的難治復發(fā)急性髓細胞白血病(r AML)患者,研究影響預后的關鍵因素。2.應用高通量二代測序技術檢測初診及接受allo-HSCT后復發(fā)的r AML患者的腫瘤相關基因突變和克隆演變。3.研究Notch2基因在AML中的表達水平及可能的致白血病機制。【方法】1.回顧性收集101例于2003年10月至2012年12月期間,在蘇州大學附屬第一醫(yī)院接受allo-HSCT的r AML患者,分析其年齡、FAB分型、分子/細胞遺傳學、疾病狀態(tài)、體能狀態(tài)、化療次數(shù)、移植類型等臨床特征與移植后并發(fā)癥及預后的影響。2.收集23例原發(fā)性r AML初診標本和20例AML患者(12例AML及8例r AML)初診及allo-HSCT后復發(fā)時的成對樣本,應用DNA+RNA特異性基因Panel對390個腫瘤相關基因進行DNA測序(全部外顯子及相關內(nèi)含子)及全轉錄組測序(RNA-seq)。DNA平均測序深度為300×,RNA-seq測序深度為50M reads。分析r AML患者初診時基因突變特征及移植后復發(fā)時的克隆演變。3.采用實時熒光定量PCR技術檢測我中心52例初診成人AML患者、10例急性淋巴細胞白血病(ALL)及10例健康對照的Notch2基因m RNA表達水平。比較Notch2基因m RNA表達水平在AML患者和ALL患者及正常對照之間以及AML各亞組之間的差異。分析Notch2 m RNA表達量與無病生存率(DFS)和總生存率(OS)之間的關系。同時制備Notch2過表達載體及靶向抑制Notch2的sh RNA質(zhì)粒載體,分別轉染髓系白血病細胞株THP-1,采用絕對計數(shù)等方法檢測其對細胞生長的影響�！窘Y果】1.101例接受allo-HSCT的r AML患者臨床特征與預后的研究101例r AML患者中位年齡34歲(14-58),所有患者均接受清髓性移植。移植方式情況如下:1)38例HLA全相合同胞移植,2)33例HLA相合無關供體移植,3)30例HLA不全相合的親緣移植。53例患者(52%)在移植前疾病緩解(CR組),而48例患者未緩解(NR組)。與CR組患者相比,NR組有更多患者存在FLT3-ITD突變(P=0.006),且體能狀態(tài)更差(P=0.001),有更多患者行無關供體移植(P=0.027),更少例數(shù)接受再誘導化療(P=0.002)。CR組患者5年OS為46%,而NR組為18%,兩組5年累計復發(fā)率分別為45%和81%。然而,兩組5年NRM是相似的(分別為28%和30%)。CR患者中,32例移植前MRD檢測陽性(MRDpos),21例MRD陰性(MRDneg)。兩組患者年齡、性別、FAB分型、危險分層及體能狀態(tài)分布相似。MRDneg患者和MRDpos患者的5年預期OS分別為51%和41%(P=0.37),5年預期DFS分別為55%和36%(P=0.20),5年預期RI分別為21%和29%(P=0.43)。所有患者單因素分析發(fā)現(xiàn),非M5/M6型AML,低危組細胞遺傳學和分子遺傳學、移植時CR狀態(tài)以及移植前較好的體能狀態(tài)與生存率正相關。多因素分析發(fā)現(xiàn),與較好預后相關的因素是移植前CR狀態(tài)(風險比[HR]=0.31,95%CI 0.12-0.81,P=0.017)和較好的體能狀態(tài)評分(Zubrod-ECOG-WHO3)。CR組患者單因素分析提示,非M5/M6型AML、低危組細胞遺傳學和分子遺傳學、FLT3-ITD突變陰性、良好的體能狀態(tài)和化療療程數(shù)量≤4次與患者的生存率正相關。多因素分析發(fā)現(xiàn)三個獨立的預后因素與較長的生存時間相關,分別是較好的體能狀態(tài)(Zubrod-ECOG-WHO3)(HR=0.13,95%CI 0.045-0.391,P0.001)、細胞遺傳學低/中危組(HR=0.30,95%CI0.103-0.853,P=0.026)和無FLT3/ITD突變(HR=0.22,95%CI 0.058-0.831,P=0.024)。2.allo-HSCT治療r AML后復發(fā)克隆演變的初步研究(1)r AML患者初診時基因突變特征23名r AML患者初診時每個樣本檢出基因異常的中位數(shù)為4個(2-15)。出現(xiàn)頻率最高的基因異常為FLT3突變。其次為TET2突變和MLL融合基因。生存分析發(fā)現(xiàn),攜帶FLT3突變及MLL融合基因患者似乎預后更差,應用FLT3抑制劑能提高FLT3-ITD患者誘導緩解率,改善預后(2年OS 33%vs.75%)。(2)AML患者初診及移植后復發(fā)時的基因改變20位AML患者初診時最常見的四種基因突變?yōu)镕LT3、DNMT3A、CEBPA和NPM1。涉及信號通路和表觀遺傳調(diào)控的基因突變最為多見。8名難治復發(fā)患者最常見的基因突變?yōu)镕LT3和TET2。移植后首次復發(fā)時,出現(xiàn)突變頻率較高的基因為MSH3、KMT2C、TSC2、WT1和NOTCH2。涉及表觀遺傳學及抑癌基因的基因突變最為多見。難治復發(fā)患者中,TSC2、TNFAIP3、IGF1R、WT1出現(xiàn)率最高。其中,Notch2基因突變只發(fā)生于移植后復發(fā)時,初診時并未發(fā)現(xiàn),且4例患者中有3名為難治復發(fā)患者。(3)移植后復發(fā)時克隆的演變移植后復發(fā)時克隆演變?nèi)N模式:(1)移植后復發(fā)時的基因突變與初診時檢測的基因突變無一相同,即復發(fā)時出現(xiàn)一群新的克隆。(2)移植后復發(fā)時,在初診基因異常的基礎上獲得了新的基因異常。(3)在移植后復發(fā)時缺失了1個或多個基因異常,同時又獲得了1個或多個基因異常。3.Notch2在成人AML患者中的表達、意義及其功能的初步探討(1)AML患者中Notch2基因m RNA的表達水平AML患者中Notch2基因m RNA的表達水平高于正常對照組(P0.05)及ALL組(P=0.180)。根據(jù)FAB分型分組發(fā)現(xiàn),M3患者的Notch2基因m RNA的相對表達量為0.07494,而其他組表達量中位值0.04637(P=0.174)。根據(jù)染色體核型分組發(fā)現(xiàn),伴有t(15,17)核型組和伴有11q23/MLL組的Notch2表達量分別為0.07494和0.06200,其他核型組表達量中位值為0.04762(P=0.22)。(2)Notch2 m RNA表達量與預后的關系Kaplan-Meier分析發(fā)現(xiàn),正常核型AML患者中Notch2基因m RNA高表達組(相對表達量≥0.04)的2年DFS高于低表達組(相對表達量0.04)(2年DFS分別為51%和17%,P=0.047)。而低表達組和高表達組患者的2年OS差異不顯著,分別為38%和63%(P=0.528)。在其他中危核型患者中,Notch2基因m RNA表達量與預后無顯著關聯(lián)(P0.05)。(3)過表達Notch2能使白血病細胞株THP-1的生長和集落形成能力下降,但是沉默該基因的表達,對其生長增殖未造成顯著影響�！窘Y論】1.處于緩解期和體能狀態(tài)較好的r AML患者在allo-HSCT后,能取得更好的預后。對于移植前處于緩解狀態(tài)的r AML患者,細胞/分子遺傳學以及體能狀態(tài)對移植結果有顯著影響。2.AML患者初診和異基因造血干細胞移植后復發(fā)時,推測基因突變類型可能發(fā)生了3種演變模式。初診時多見涉及信號通路及DNA甲基化調(diào)控的基因突變,復發(fā)時新出現(xiàn)的基因異常卻多見于染色質(zhì)修飾基因和抑癌基因。3.AML患者中均表達Notch2,在正常核型患者中用熒光實時定量檢測Notch2基因m RNA表達水平有一定的提示預后的價值。過表達Notch2在一定程度上能抑制白血病細胞株THP-1增殖,而沉默該基因對細胞株生長影響不顯著。
[Abstract]:[Objective] 1. Retrospective study of the key prognostic factors in patients with refractory and relapsed acute myeloid leukemia (r AML) treated with allo-HSCT in our center. 2. Detection of tumor-related gene mutations and cloning in patients with newly diagnosed and relapsed R AML after allo-HSCT by high-throughput second-generation sequencing technique. Evolution. 3. To study the expression of Notch2 gene in AML and its possible leukemic mechanism. [Methods] 1. A retrospective study of 101 patients with allo-HSCT treated in the First Affiliated Hospital of Suzhou University from October 2003 to December 2012 was conducted to analyze their age, FAB typing, molecular/cytogenetics, disease status, physical status, and chemotherapy. Clinical features such as frequency, type of transplantation, and their impact on post-transplant complications and prognosis. 2. DNA sequencing of 390 tumor-related genes (all exons and related genes) was performed in 23 primary r-AML patients and 20 AML patients (12 AML and 8 r-AML) with paired samples at initial diagnosis and relapse after allo-HSCT. Intron) and total transcriptome sequencing (RNA-seq). The average DNA sequencing depth was 300 * and the RNA-seq sequencing depth was 50 M reads. The gene mutation characteristics at the initial diagnosis and the clonal evolution during the relapse after transplantation were analyzed. 3. Real-time fluorescence quantitative PCR was used to detect 52 newly diagnosed adult AML patients and 10 acute lymphoblastic leukemia (ALL) patients in our center. The expression level of Notch2 m RNA was compared between AML patients and ALL patients, as well as between AML subgroups. The relationship between Notch2 m RNA expression and disease-free survival rate (DFS) and overall survival rate (OS) was analyzed. Notch2 overexpression vector and targeted inhibition were prepared. Notch2 plasmid was transfected into myeloid leukemia cell line THP-1 and its effect on cell growth was detected by absolute count. [Results] The clinical characteristics and prognosis of 101 patients with allo-HSCT-induced AML were studied. The median age of 101 patients with allo-HSCT-induced AML was 34 years (14-58). All patients received myeloablative transplantation. The methods were as follows: 1) 38 HLA full-phase contract cell transplantation, 2) 33 HLA-matched unrelated donor transplantation, 3) 30 HLA-incompatible relative transplantation. 53 patients (52%) had remission before transplantation (CR group), while 48 patients had no remission (NR group). Compared with CR group, more patients in NR group had FLT3-ITD mutation (P = 0.006) and worse physical condition (P = 0.006). More patients received unrelated donor transplantation (P = 0.027) and fewer received re-induction chemotherapy (P = 0.002). The 5-year OS was 46% in CR group and 18% in NR group. The cumulative 5-year recurrence rate was 45% and 81% in NR group, respectively. However, the 5-year NRM of the two groups was similar (28% and 30% respectively). Age, sex, FAB typing, risk stratification, and physical fitness distribution were similar in both groups. The 5-year expected OS of MRDneg patients and MDRPOS patients were 51% and 41% (P = 0.37), 55% and 36% (P = 0.20), and 29% (P = 0.43) respectively. Univariate analysis of all patients revealed that non-M5/M6 AML was inherited by low-risk group cells. Multivariate analysis showed that CR status before transplantation (risk ratio [HR] = 0.31, 95% CI 0.12-0.81, P = 0.017) and better physical fitness status score (Zubrod-ECOG-WHO 3) were associated with better prognosis. 5/M6 AML, low-risk group cytogenetics and molecular genetics, FLT3-ITD mutation negative, good physical fitness status and the number of chemotherapy courses less than 4 times were positively correlated with the survival rate of patients. Multivariate analysis found that three independent prognostic factors were associated with longer survival time (Zubrod-ECOG-WHO 3) (HR = 0.13, 95% CI 0. 045-0.391, P 0.001), cytogenetic low/intermediate risk group (HR = 0.30, 95% CI0.103-0.853, P = 0.026) and non-FLT3/ITD mutation (HR = 0.22, 95% CI 0.058-0.831, P = 0.024). 2. Preliminary study on clonal evolution of relapse cloning after treatment of AML with allo-HSCT (1) Characteristics of gene mutation in 23 patients with AML at first diagnosis The highest frequency of gene abnormalities was FLT3 mutation, followed by TET2 mutation and MLL fusion gene. Survival analysis showed that patients with FLT3 mutation and MLL fusion gene seemed to have worse prognosis. FLT3 inhibitors could increase the induced remission rate and improve the prognosis of FLT3-ITD patients (33% vs. 75% in 2 years OS) after initial diagnosis and transplantation. The most common four mutations were FLT3, DNMT3A, CEBPA and NPM1. The most common mutations involved in signal pathway and epigenetic regulation were FLT3 and TET2. MSH3 was the most frequent mutation in the first relapse after transplantation. KMT2C, TSC2, WT1 and NOTCH2. Epigenetics and tumor suppressor gene mutations were the most common. TSC2, TNFAIP3, IGF1R and WT1 were the most common in refractory recurrence patients. Notch2 gene mutations occurred only in the recurrence after transplantation, and were not found at first diagnosis. Three of the four patients were cloned in the recurrence after transplantation. Three patterns of clonal evolution during post-transplant recurrence were observed: (1) There was no difference in gene mutation between post-transplant recurrence and initial diagnosis, i.e. a group of new clones appeared during recurrence. (2) New gene abnormalities were acquired on the basis of initial diagnosis of gene abnormalities during post-transplant recurrence. (3) One or more gene abnormalities were missing during post-transplant recurrence. The expression and function of Notch2 gene in adult AML patients (1) The expression level of Notch2 gene m RNA in AML patients was higher than that in normal control group (P 0.05) and ALL group (P = 0.180). According to FAB classification, the expression of Notch2 gene m RNA in AML patients was higher than that in normal control group (P 0.05) and ALL group (P = 0.180). The relative expression of Notch2 m RNA was 0.07494 in 3 patients and 0.04637 in other groups (P = 0.174). According to the karyotype grouping, the expression of Notch2 in the group with t (15,17) karyotype and the group with 11q23/MLL were 0.07494 and 0.06200, respectively. The median expression of Notch2 m RNA in other karyotypes was 0.04762 (P = 0.22). Kaplan-Meier analysis showed that the 2-year DFS in the high-expression group of Notch2 gene m RNA (relative expression (>0.04)) was higher than that in the low-expression group (relative expression 0.04) (2-year DFS was 51% and 17%, respectively, P = 0.047). The 2-year OS in the low-expression group and the high-expression group were not significantly different, 38% and 63% (P = 0.528), respectively. There was no significant correlation between the expression of Notch2 gene m RNA and prognosis in other patients with intermediate-risk karyotypes (P 0.05). (3) Overexpression of Notch2 could decrease the growth and colony-forming ability of leukemia cell line THP-1, but silencing the expression of Notch2 gene had no significant effect on its growth and proliferation. [Conclusion]1. AML patients have a better prognosis after allo-HSCT. Cell/molecular genetics and physical fitness status have a significant impact on transplantation outcomes in patients with remission before transplantation. 2. When AML patients first diagnosed and relapsed after allogeneic hematopoietic stem cell transplantation, three patterns of gene mutation may occur. Mutations involving signaling pathways and DNA methylation regulation are common, but new gene abnormalities occurring during relapse are more common in chromatin modified genes and tumor suppressor genes. CH2 can inhibit the proliferation of leukemia cell line THP-1 to some extent, but the silencing of this gene has no significant effect on the growth of leukemia cell line.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R733.71

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