第一部分慢性中性粒细胞白血病临床特征及基因突变的研究第二部分慢性粒单核细胞白血病基因突变的研究及预后意义

发布时间：2018-09-11 18:40

【摘要】：研究目的探讨慢性中性粒细胞白血病(CNL)患者临床表现、形态学、细胞遗传学、基因突变的特点及转归。研究方法对临床疑诊“CNL”的27例患者按2008年WHO诊断分型标准进行回顾性诊断,回顾分析患者形态学及细胞遗传学特点,随访患者判定预后。利用等位基因特异性聚合酶链式反应(AS-PCR)检测JAK2 V617F突变,直接测序法检测CSF3R第14-17外显子、ASXL1第12外显子、SETBP1第4外显子、CALR第9外显子、MPL第10外显子突变状态,对有突变的样本进行克隆后测序鉴定突变类型。或者应用二代测序方法对血液肿瘤常见的112种基因进行突变分析,筛选以上6个基因的突变进行分析。分析CNL患者临床表现、形态学、细胞遗传学及基因突变的特点及其临床意义。结果27例疑诊“CNL"的患者中,最终16例患者诊断为CNL,1例携带CSF3R T618I突变但不符合WHO (2008) CNL的诊断标准,2例诊断为意义未明的单克隆免疫球蛋白病(MGUS)伴CNL表现,感染及肿瘤继发的反应性中性粒细胞增多各4例。16例CNL患者发病中位年龄64(43-80)岁,男性占75%(12/16),确诊时中位血红蛋白水平为114(81-154)g/L,中位白细胞计数为41.20(26.05-167.70)×10g/L,中位血小板计数为238(91-394)×109/L。中位周血幼稚粒细胞比例2(0-9)%,中位周血原始细胞比例0(0-0.5)%,中位骨髓原始细胞比例1(0-4.5)%,中位骨髓纤维化水平为1(0-3)级。除1例t(1,7)(p32,q11)、1例+21克隆异常及1例14,ps+外,余患者未检测到异常核型。16例CNL患者中，CSF3R T618I突变检出率为100%(16/16),1例合并CSF3R W791X突变；ASXL1突变检出率为81%(13/16),依据检出频率依次为G646WfsX12(8例)、Y591X (2例)、S871SfsX4(1例)、R404X(1例)及Q976X(1例)；SETBP1突变检出率为63%(10/16),依据检出频率依次为D868N(4例)、1871T(3例)、G870S(1例)、G870D(1例)及D874N(1例)。同时有CSF3R T618I、ASXL1及 SETBP1突变的患者为9例,同时有CSF3R T618I及ASXL1突变的患者为4例,同时有CSF3R T618I及SETBP1突变的患者为1例,仅有CSF3R T618I突变并且不伴有ASXL1 及 SETBP1突变的患者为2例。此外,我们还通过直接测序得到了仅有1例患者携带CALRK385fs*47突变。所有CNL患者均无JAK2 V617F突变及MPL突变。1例携带 CSF3RT618I突变但不符合WHO (2008) CNL诊断标准的患者伴有SETBP1突变但未检测到ASXL1、JAK2 V617F、CALR 及 MPL突变。MGUS伴CNL表现及反应性中性粒细胞增多患者均未检出以上6种基因突变。除SETBP1基因发现1例G870S纯合突变外,余基因突变均为杂合突变。ASXL1突变及SETBP1突变型与其对应的野生型相比,在性别比例、年龄分布、血红蛋白水平、白细胞计数、血小板计数、周血幼稚粒细胞比例、周血原始细胞比例、骨髓原始细胞比例、骨髓纤维化是否≥1级各项指标均无明显差异。ASXL1突变与SETBP1突变的发生无相关性。16例CNL患者中位生存期为26(95%CI 20-32)月。去除了1例接受异基因造血干细胞移植患者的生存数据后,我们发现影响CNL患者预后的因素：初诊WBC≥50×109/L生存期较50×109/L者短(11月vs.39月,P=0.005)。而性别、年龄≥60岁、初诊是否有贫血(贫血标准以男性＜120g/L,贫血标准以女性110g/L计算,P=0.063)、是否有ASXL1突变、是否有SETBP1突变、周血是否有幼稚粒细胞、骨髓原始细胞是否≥2%、骨髓纤维化是否≥1级及初诊染色体核型是否异常对预后影响无统计学差异。结论1. CSF3R T618I基因突变可以作为诊断CNL的主要标准。CNL患者还常合并ASXL1及SETBP1基因突变。2.CNL患者确诊时染色体核型异常的比例不高,这些染色体异常在髓系肿瘤中并没有特异性。初诊染色体核型的异常并非预后不良因素。3.CNL患者中位生存期26月,确诊时WBC≥50×109/L是不良预后因素。研究目的探讨慢性粒单核细胞白血病(CMML)患者ASXL1、SETBP1、TET2及SRSF2基因突变及其临床意义。研究方法对141例CMML患者按2008年WHO诊断分型标准进行回顾性诊断,随访患者判定预后。应用直接测序法检测ASXL1第12外显子、SETBP1第4外显子、TET2第3-11外显子和SRSF2第1外显子突变状态,克隆后测序鉴定突变类型。比较基因突变患者与野生型患者的临床及实验室特征、分析影响CMML患者预后的因素。结果141例CMML患者中位年龄63(18-85)岁,男性95例(67%)。确诊时中位血红蛋白水平为88(43-166)g/L,中位白细胞计数为21.88(3.01-117.57)×109/L,中位中性粒细胞绝对值为7.07(0.30-66.91)×109/L,中位单核细胞绝对值为3.72(1.02-57.72)×109/L,中位血小板计数为78(4-1001)×109/L。141例CMML患者中共发现65例(46%)ASXL1基因(仅无义突变及移码突变被视为有突变)突变、25例(18%)SETBP1基因突变,46例(33%)TET2基因突变及41例(29%) SRSF2基因突变患者。ASXL1基因突变类型以移码突变为主,其中移码突变59例(G646WfsX12为38例)、无义突变7例,同时存在移码突变及无义突变1例。SETBP1基因突变类型均为错义突变,其中D868N 12例(48%)、G870S 9例(36%), I871S、R867S、S869I及S869R各1例(各占4%)。TET2基因突变类型包含移码突变22例、错义突变19例及无义突变7例,其中1例患者存在错义突变及无义突变,1例患者同时存在错义、无义及移码突变。SRSF2基因突变错义突变为主,P95H、P95L、 P95R及移码突变例数分别为18例(44%)、13例(32%)、7例(17%)及3例(7%)。以上所有基因突变均为杂合突变。ASXL1与SETBP1基因突变的存在具有相关性；TET2与SRSF2基因突变的存在具有相关性。TET2突变患者和野生型患者比较：年龄≥65岁比例较高、骨髓原始细胞≥10%比例较低；SRSF2突变患者和野生型患者比较：年龄≥65岁比例较高、血红蛋白水平、白细胞计数、中性粒细胞绝对值、单核细胞绝对值较高。ASXL1、SETBP1突变患者和野生型患者在临床特征无明显差异。多因素分析141例CMML总生存,血红蛋白水平、周血幼稚髓系细胞(IMCs)有无及ASXL1突变是CMML独立预后因素。依据Mayo预后模型各组中位生存(median overall survival, MS):低危组未达到、中危组28月和高危组18月。依据分子Mayo预后模型各组MS：低危组未达到、中危-1组55月、中危-2组25月和高危组15月。对两个预后积分系统进行似然比检验,分子Mayo预后模型优于Mayo预后模型(-2 log似然比分别为627和654,P=0.001)。将基因突变与Mayo预后模型同时纳入COX回归,相对于ASXLlwt/TET2wt患者,ASXLlmut/TET2mut. ASXLlmut/TET2wt和ASXLlwt/TET2mut患者的相对危险度分别为4.7(95％CI 2.2-10.3；P=0.000)、2.2(95%CI 1.1-4.2；P=0.025)和1.3(95%CI O.6-2.5；P=0.521)。结论1.CMML患者中ASXL1、SETBP1、TET2及SRSF2基因突变频率分别为46%、18%、33%及29%。约3/4患者至少能检测到1种以上基因突变。2.SRSF2基因突变CMML患者表现为髓系细胞获得增殖优势,但与CMML患者总体生存及疾病进展无明显相关性。3.ASXL1基因突变是CMML患者总体生存的独立不良预后因素。将ASX L1突变纳入预后积分系统,有助于更好区分预后危险度。4.有ASXL1突变的CMML患者中检测出TET2突变,提示更差的预后。
[Abstract]:Objective To investigate the clinical manifestations, morphology, cytogenetics, and gene mutation characteristics and prognosis of chronic neutrophil leukemia (CNL) patients.Methods 27 suspected patients with CNL were retrospectively diagnosed according to the WHO classification criteria in 2008. To determine the prognosis, JAK2 V617F mutation was detected by allele-specific polymerase chain reaction (AS-PCR), CSF3R exon 14-17, ASXL1 exon 12, SETBP1 exon 4, CALR exon 9 and MPL exon 10 were directly sequenced to identify the mutation type. Generation sequencing was used to analyze the mutations of 112 common genes in hematological malignancies. The clinical manifestations, morphology, cytogenetics and gene mutations of CNL patients were analyzed. Two patients were diagnosed as unidentified monoclonal immunoglobulin disease (MGUS) with CNL. The median age of onset was 64 (43-80) years in 16 patients with CNL, and 75 (12/16) in males. The median hemoglobin level at diagnosis was 114 (81-154) g/L. The median white blood cell count was 41.20 (26.05-167.70)*10 g/L, and the median platelet count was 238 (91-394)*109/L. The median percentage of peripheral blood immature granulocytes was 2 (0-9)%, the median percentage of peripheral blood primitive cells was 0 (0-0.5)%, the median percentage of bone marrow primitive cells was 1 (0-4.5)%, and the median level of bone marrow fibrosis was 1 (0-3). Except for 1 (1 (1,7) (p32, q11), 1 + 21 clonal abnormality. Among the 16 CNL patients, the detection rate of CSF3R T618I mutation was 100% (16/16), one with CSF3R W791X mutation, ASXL1 mutation was 81% (13/16), G646WfsX12 (8 cases), Y591X (2 cases), S871SfsX4 (1 case), R404X (1 case) and Q976X (1 case), respectively. 63% (10/16), according to the detection frequency, were D868N (4 cases), 1871T (3 cases), G870S (1 case), G870D (1 case) and D874N (1 case). There were 9 cases with CSF3R T618I, ASXL1 and SETBP1 mutations, 4 cases with CSF3R T618I and ASXL1 mutations, 1 case with CSF3R T618I and SETBP1 mutations, and 1 case without CSF3R T618I and SETBP1 mutations. Two patients had mutations in ASXL1 and SETBP1. In addition, only one patient with CALRK385fs * 47 mutation was obtained by direct sequencing. All CNL patients had no JAK2 V617F mutation and MPL mutation. One patient with CSF3RT618I mutation but did not meet the WHO (2008) CNL diagnostic criteria had mutations in SETBP1 but did not detect mutations in ASXL1, JAK2 V617. All the mutations were heterozygous except one G870S homozygous mutation found in SETBP1 gene. The ASXL1 mutation and SETBP1 mutation were compared with the corresponding wild type in terms of sex ratio, age distribution, hemoglobin level. There were no significant differences in white blood cell count, platelet count, peripheral blood immature granulocyte ratio, peripheral blood primitive cell ratio, bone marrow primitive cell ratio, and whether myelofibrosis was above grade 1. There was no correlation between ASXL1 mutation and SETBP1 mutation. The median survival time of 16 CNL patients was 26 (95% CI 20-32) months. After the survival data of the patients undergoing blood stem cell transplantation, we found that the prognostic factors of CNL patients were WBC (>50 *109/L) survival time was shorter than that of 50 *109/L patients (vs. 39 months in November, P = 0.005). Sex, age (>60 years old), anemia (anemia < 120g/L for men, 110g/L for women, P = 0.063), and ASXL1 protrusion. Conclusion 1. CSF3R T618I gene mutation can be used as the main criteria for the diagnosis of CNL. CNL patients are often associated with ASXL1 and SETBP1 gene processes. Chromosome karyotype abnormalities were not specific in myeloid tumors. Chromosome karyotype abnormalities were not a poor prognostic factor. 3. The median survival time of CNL patients was 26 months. WBC (>50 *109/L) at diagnosis was a poor prognostic factor. Methods 141 patients with CMML were retrospectively diagnosed according to WHO classification criteria in 2008. The prognosis was determined by follow-up. The mutations of ASXL1 exon 12, SETBP1 exon 4, TET2 exon 3-11 and SRSF2 exon 1 were detected by direct sequencing. Results The median age of 141 CMML patients was 63 (18-85) years, and 95 (67%) were male. The median hemoglobin level was 88 (43-166) g/L at diagnosis, and the median white blood cell count was 21.88 (3.01-117). 57 *109/L, median neutrophil absolute value was 7.07 (0.30-66.91) *109/L, median monocyte absolute value was 3.72 (1.02-57.72) *109/L, median platelet count was 78 (4-1001) *109/L. A total of 65 (46%) patients with CMML had mutations in the ASXL1 gene (only senseless mutations and frameshift mutations were considered to be mutations), 25 (18%) had mutations in the SETBP1 gene. 46 patients (33%) had mutations in TET2 gene and 41 patients (29%) had mutations in SRSF2 gene. The mutations in ASXL1 gene were mainly frameshift mutations, of which 59 were frameshift mutations (38 were G646WfsX12), 7 were nonsense mutations, and 1 was frameshift mutation and nonsense mutation. There were 22 frameshift mutations, 19 missense mutations and 7 nonsense mutations in TET2 gene. One patient had missense mutations and nonsense mutations. One patient had both missense, nonsense and frameshift mutations. SRSF2 mutations were mainly missense mutations, P95H, P95L, P95R and frameshift mutations. All of the above mutations were heterozygous. ASXL1 was associated with SETBP1 gene mutation. TET2 was associated with SRSF2 gene mutation. Compared with wild-type patients, patients with TET2 mutation had a higher proportion of age (> 65 years) and bone marrow primordial cells (> 10%). There was no significant difference in clinical characteristics between patients with SRSF2 mutation and those with wild type. Multivariate analysis showed that 141 patients with CMML survived, hemoglobin levels, and weeks. The presence or absence of IMCs and ASXL1 mutation were independent prognostic factors for CMML. According to the Mayo prognostic model, the median overall survival (MS) was not achieved in the low-risk group, 28 months in the medium-risk group and 18 months in the high-risk group. Month. A likelihood ratio test for the two prognostic score systems showed that the molecular Mayo prognostic model was superior to the Mayo prognostic model (-2 log likelihood ratio was 627 and 654 respectively, P = 0.001). The relative risk of ASXLlmut/TET2mut. The degree of mutation was 4.7 (95% CI 2.2-10.3; P = 0.000), 2.2 (95% CI 1.1-4.2; P = 0.025) and 1.3 (95% CI 0.6-2.5; P = 0.521). Conclusion 1. The mutation frequencies of ASXL1, SETBP1, TET2 and SRSF2 genes in CMML patients were 46%, 18%, 33% and 29%, respectively. ASXL1 mutation is an independent and unfavorable prognostic factor for the overall survival of CMML patients. Incorporating ASXL1 mutation into the prognostic score system helps to better distinguish the prognostic risk. 4. TET2 mutation was detected in CMML patients with ASXL1 mutation, suggesting worse prognosis. Prognosis.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.72

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第一部分 慢性中性粒细胞白血病临床特征及基因突变的研究 第二部分 慢性粒单核细胞白血病基因突变的研究及预后意义

第一部分慢性中性粒细胞白血病临床特征及基因突变的研究第二部分慢性粒单核细胞白血病基因突变的研究及预后意义