NPM1突变急性髓性白血病CD34表达调控以及对预后影响的机制研究

NPM1-mutated acute myeloid leukemia(NPM1+AML) is recently recognized and the most common subtype of AML (about 1/3). Most patients with NPM1+AML are CD34-negative(CD34-)and most other AML patients (except for APL) are CD34-postive(CD34+). The reason why CD34 expression is down-regulated in NPM1+AML remains elusive. Though the CD34 is down-regulated, there still exist small amount of CD34+ blasts in bone marrow of NPM1+AML patients. We recently found,fot the first time,that CD34 expression (>7% blasts) is an independent prognostic factor. CD34+NPM1+AML patients had a lower complete remission rate, higher relapse rate and lower long-term survival. Moreover most CD34+ blasts express leukemia stem cell (LSC)-like immunophenotype CD34+/CD38- in NPM1+AML.We hypothesized that down-regulation of CD34 in blasts of NPM1+AML is modified by the NPM1-mutation, and that CD34+ blasts with leukemic stem cell -like immunophenotype may result in the poor outcomes of CD34+ patients . To explore our hypothesis, we first investigate the association of NPM1-mutation with CD34 expression in cell line and primary leukemia cells. We then sort CD34+/CD38-、CD34+/CD38+、CD34-/CD38+、CD34-/CD38- blasts population in bone marrow of patients to investigate each population's biology and function, such as engraftment in NOD/SCID micee, gene expression profile, proliferation and apoptosis, drug-resistance associated protein and adhesion molecular. Clarifying the mechanism of CD34 down-regulation and the poor prognostic value of CD34 expression may contribute to design individual therapy to NPM1+AML.

NPM1突变急性髓性白血病（NPM1+AML）是新认识的、最常见的AML类型，表型为CD34-，而其它类型AML主要为CD34+。NPM1+AML中CD34表达下调的机制不明，其骨髓中仍存少量的CD34+白血病细胞。本课题组首次发现CD34+（>7%）是NPM1+AML独立的预后不良因素。本课题组提出假说：（1）NPM1突变是CD34表达下调主要分子机制。（2）CD34+NPM1+AML预后差的主要机制是由于CD34+白血病群含有LSC。拟进行（1）利用细胞系和原代细胞, 调控NPM1突变的表达来观察对CD34表达的影响（2）从原代白血病细胞分选并比较CD34+/CD38-、CD34+/CD38+、CD34-/CD38+、CD34-/CD38-群的表型、移植到NOD/SCID小鼠、观察凋亡、耐药蛋白、粘附分子表达。探讨NPM1+AML的CD34表达调控以及对预后影响的机制。

研究背景：NPM1突变急性髓性白血病（NPM1+AML）是最常见的AML类型(占三分子一），该类型白血病细胞免疫表型为CD34-，而其它类型AML主要为CD34+。NPM1+AML中CD34分子表达下调的机制不明，而本课题组前期工作首次发现CD34+（>7%）是NPM1+AML独立的预后不良因素，因此本课题重点探讨NPM1+AML的CD34表达调控以及对预后影响的机制。..主要研究内容：包括以下方面：（1）NPM1突变是CD34表达下调主要分子机制。（2）NPM1+AML预后相关因素以及主要机制。..研究结果和关键数据：.（1）构建全长的NPM1A、B、D突变型质粒， 质粒导入pcDNA-3Flag表达载体，同时也构建了NPM野生型及突变体的慢病毒表达载体，选用Plvx-IRES-ZsGreen1 表达载体。选取高表达CD34(CD34+)的KG-1细胞系和原代细胞，通过AmaxaNucleofector®(Lonza)电转染方法，通过Western blot 和定量PCR检测mRNA表达均发现转染突变体NPM-A 后，细胞内源的CD34 表达有下降。（2）探讨NPM1+AML的预后因素。分析137 例初治成人伴NPM1 基因主要突变（A、B、D突变）AML患者的治疗结果，发现WBC<100×109/L和初始诱导治疗为“IA10”方案是获得CR 的有利因素。FLT3-ITD 突变阳性、巩固治疗2 个疗程后MRD高水平、未接受allo-HSCT是影响DFS 的不利因素，证实FLT3-ITD 突变、化疗后早期MRD高水平是NPM1+AML预示不良预后。（3）探索了靶向性药物维甲酸和砷剂治疗NPM1+AML的可行性。我们对2例不能耐受标准化疗的初诊病例进行维甲酸联合亚砷酸治疗，治疗2周骨髓中白血病细胞比例分别从87%下降到11%和从72%下降到40%，可以观察到明确的疗效。.科学意义：.本研究不仅有助于深入阐明NPM1+AML的CD34表达和NPM1突变的相关性，明确NPM1突变是CD34表达下调主要分子机制，对该类型白血病的白血病发育的阶段性提供了数据支持，同时通过临床数据证实NPM1+AML的临床预后异质性，特别是NPM1突变型的表达水平和预后相关，另外通过分子靶向性治疗为该类型白血病的治疗提供了新思路，具有重要的基础和临床意义。