【作者】 李冰；

【导师】 肖志坚；

【作者基本信息】 北京协和医学院 ， 内科学（专业学位）， 2014， 博士

【摘要】 研究目的探讨原发性骨髓纤维化(PMF)患者ASXL1、IDH1/2、SRSF2、CALR和MPL基因突变及其临床意义。研究方法对484例PMF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、IDH1第4外显子、IDH2第4外显子、SRSF2第1外显子、CALR第9外显子和MPL第10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。结果127例PMF患者中ASXL1基因突变36例(28%),SRSF2基因突变2例(1.6%),未检出IDH1/2基因突变病例。ASXL1突变均为杂合突变,基因突变类型均为移码突变或无义突变,以G646Ffs*12最为常见。与不伴有ASXL1基因突变患者相比,伴有ASXL1的PMF患者外周血白细胞计数较高(P=0.004),脾脏肿大程度更明显(P=0.001),年龄>65岁者比例较高(P=0.054),染色体核型异常者比例较高(P=0.033)。两者在外周血血红蛋白浓度、血小板计数、外周血原始细胞比例、JAK2V617F突变率、DIPSS-plus染色体预后分组及DIPSS、DIPSS-Chinese和DIPSS-plus预后分组方面无显著差异。357例PMF患者中JAK2V617F基因突变178例(50%),MPL基因突变11例(3%),CALR基因突变76例(21%),CALR突变均为杂合性移码突变,其中L367fs*46(Ⅰ类突变)26例(34%),K385fs*47(Ⅱ类突变)47例(62%)。与伴有JAK2V617F患者相比,伴有CALR基因Ⅱ类突变的PMF患者年龄较小(P<0.001),外周血白细胞计数(P<0.001)和血红蛋白浓度(P=0.001)较低,外周血原始细胞比例较高(P=0.009),DIPSS和DIPSS-Chinese预后分组较高危组比例较高(P<0.001)。CALR基因Ⅱ类突变患者中外周血血小板计数异常者比例较高(<100x109/L P=0.01or>450x109/L P=0.042),脾脏肿大者比例较低(P=0.004)。伴有CALR II类突变的患者较伴有JAK2V617F突变患者总生存期明显缩短(P=0.003)。CALR II类突变是独立于DIPSS-Chinese预后积分系统的不良预后因素(P=0.002, HR2.153,95%CI,1.32-3.51),而ASXL1突变并非不良预后因素。结论ASXL1基因突变是中国PMF患者中常见的表观遗传学相关基因突变但其并非不良预后因素。CALR基因突变在非JAK2V617F或MPL基因突变的PMF患者中具有较高的突变率。伴有CALRⅡ类突变的患者较伴有JAK2V617F突变患者总生存期明显缩短,CALRⅡ类突变是独立于DIPSS-Chinese的不良预后因素。研究目的探索原发性骨髓纤维化(PMF)患者的细胞遗传学异常及其预后意义研究方法回顾性分析439例具有可分析染色体核型结果的原发性骨髓纤维化患者临床资料,应用Kaplan-Meier, Log-rank检验及COX回归模型评估影响预后的因素。结果439例具有可分析染色体核型结果的PMF患者中异常染色体核型者123例(28%),其中复杂核型24例(20%),单倍体核型15例(12%),单纯+8核型异常21例(17%),单纯20q-核型异常15例(12%),单纯13q-核型异常9例(7%)。24例复杂核型中单倍体核型14例(58%)。123例染色体核型异常的PMF患者中,复杂核型者与非复杂核型者相比,伴有贫血的患者比例较高(P=0.016),两者间年龄、性别、外周血白细胞计数、血小板计数、原始细胞比例、脾脏大小、体质性症状和DIPSS预后分组无显著差异。单倍体核型者与非单倍体核型者相比,两者间年龄、性别、外周血血红蛋白浓度、白细胞计数、血小板计数、原始细胞比例、脾脏大小、体质性症状和DIPSS预后分组无显著差异。单因素生存分析显示染色体核型异常、DIPSS-plus不良预后核型、单倍体核型和复杂核型是不良预后因素(P=0.007,0.022,0.032,0.034)。多因素分析显示单倍体核型是独立于DIPSS-Chinese的不良预后因素(P=0.003, HR3.339,95%CI1.514-7.367)。结论中国PMF患者中染色体核型异常者比例较低,复杂核型、单纯+8核型异常、单纯20q-核型异常和单纯13q-核型异常为最常见的染色体核型异常。单倍体核型是独立于DIPSS-Chinese的不良预后因素。研究目的：分析原发初治骨髓增生异常综合征(MDS) IPSS中危-1组患者的预后因素。研究方法：回顾性分析191例原发初治MDS IPSS中危-1组患者的临床资料并进行统计学分析。结果：191例患者中位随访时间21(1-88)个月,中位生存期52个月。单因素分析中,血清铁蛋白≥500ug/L是原发初治MDS IPSS中危-1组的不良预后因素(P<0.001)。此外,年龄≥60岁、ECOG体力状况评分≥2分、血小板(PLT)≤50×109/L、中性粒细胞绝对值(ANC)≤0.5×109/L、、骨髓造血组织面积>80%、骨髓纤维化程度≥MF-1、骨髓细胞涂片原始细胞比例>5%,不良染色体核型亦是不良预后因素(P值分别为0.002、0.006、0.001、0.0031、0.011、0.006、0.011和0.012)。虽然血红蛋白浓度(HGB)和血清铁蛋白呈负相关,但HGB并不影响患者预后(P>0.05)。将上述单因素分析有预后意义的参数纳入COX多因素模型筛选,血清铁蛋白、ECOG体力状况评分、ANC、骨髓涂片原始细胞比例和染色体核型具有独立预后意义(P值分别为0.001、0.01、0.032、0.001和0.017)。选取血清铁蛋白、ECOG体力状况评分、ANC、骨髓涂片原始细胞比例和染色体核型提出了一个针对中国原发初治MDS IPSS中危-1组进行再次危度分层的预后积分系统,低危组、中危组、高危组患者的3年预期生存(PS)率别为83.7%、59.8%和27.7%,经Log-rank检验各组总体生存(0S)率差异有统计学意义(P<0.001)。结论：这一新的预后积分系统有望对原发初治MDS IPSS中危-1组患者进行二次预后分层,对于高危患者早期采取干预性治疗具有指导意义。更多还原

【Abstract】 Object To investigate ASXL1, IDH1/2, SRSF2, CALR, MPL mutations in patients with primary myelofibrosis (PMF) and clinical significance of these mutations.Methods A retrospective study on484PMF cases was performed according to2008World Health Organization (WHO) diagnostic criteria. Identification of the gene (exonl2of ASXL1, exon4of IDH1, exon4of IDH2, exonl of SRSF2, exon9of CALR, exon10of MPL) mutations were detected by direct sequencing and classification of mutation types by sequencing followed by plasmid cloning.The clinical and laboratory features were compared among the patients with gene mutation and those with wild type.Results Among127Chinese with PMF,38subjects displayed gene muation except for JAK2V617F mutation, including36(28%) with ASXL1mutation,2(2%) with SRSF2mutation and no subject with IDH1/2muations. The patients with ASXL1mutations were all heterozygous. The types of gene mutation included frameshift and nonsense mutations, G646Ffs*12was most frequent. Compared with subjects without ASXL1mutation, subjects with ASXL1mutation had higher WBC level (P=0.004), more obvious splenomagly (P=0.001) and were likely to be older than65yr and had abnormal karyotype. There was no significant difference in hemoglobin concentrations, PLT level, percentage of blasts in peripheral blood, frequency of JAK2V617F mutation, cytogenetic categories according to DIPSS-plus, risk groups according to DIPSS, DIPSS-Chinese and DIPSS-plus. Among357subjects with PMF, CALR mutations were detected in76subjects (21%), JAK2V617F mutations in178(50%) and MPL mutation in11(3%). The patients with CALR mutations were all heterozygous. The types of gene mutation were all frameshift mutation, including26(34%) type-1(L367fs*46) and47(62%) type-2(K385fs*47) mutations. Subjects with type-2CALR mutations had lower hemoglobin concentrations (P=0.001), lower WBC levels (P<0.001), higher percent blood blasts (P=0.009), higher conventional (P<0.001) and Chinese-adjusted DIPSS scores (P<0.001), compared with subjects with JAK2mutations, These subjects were also likely to have abnormal platelet levels (<100x109/L; P=0.01or>450x109/L; P=0.042) and no splenomegaly (P=0.004). Subjects with type-2CALR mutation had briefer overall survival than those with JAK2V617F mutations.In multivariate analyses type-2CALR mutation or no detectable mutation was an independent high-risk factor for survival adjusted for DIPSS-Chinese.Conclusion ASXL1mutation is frequent gene mutaion in epigenetic regulator but not a unfavorable prognostic factor in Chinese with PMF. CALR mutations are frequent in Chinese with PMF lacking mutations in JAK2or MPL. Subjects with type-2CALR mutation have briefer overall survival than those with JAK2V617F mutations. In multivariate analyses type-2CALR mutation is an independent high-risk factor for survival adjusted for DIPSS-Chinese. Objective To investigate the prognostic value of cytogenetical abnormality in Chinese with primary myelofibrosis (PMF)Methods439PMF patients with evaluable karyotye were retrospectively analyzed. Kaplan-Meier method, Log-rank test and COX regression model were used to evaluate factors that influence the prognosis.Results Among the439PMF patients,123displayed an abnormal karyotype, including24(20%) with complex karyotype,15(12%) with monosomal karytype,21(17%) with sole trisomy8,15(12%) with sole del(20q) and9(7%)with sole del(13q).14(58%) of complex karyotype also met criteria for monosomal karyotpe. Among123subjects with abnormal karytpe, compared to subjects without complex karytpe, subjects with complex karyotype were likely to display anemia (P=0.016), but there was no significant differences in age, gender, WBC and PLT level, percentage of blasts in peripheral blood, splenomegaly, constitutional symptoms and DIPSS risk groups. There was no significant difference in age, gender, HGB, WBC and PLT level, percentage of blasts in peripheral blood, splenomegaly, constitutional syndrome and DIPSS risk groups between subjects with monosomal karyotype and thoses without monosomal karyotype. In univariable survival analysis, abnormal karyotype, unfavorable karyotpe according to DIPSS plus, complex karyotype and monosomal karyotype were unfavorable prognostic factor (P=0.007,0.022,0.032,0.034). In multivariable analysis, monosomal karyotye was independent unfavorable prognostic factor adjusted for DIPSS-Chinese (P=0.003, HR3.339,95%CI1.514-7.367).Conclusion Among Chinese with PMF, percentage of subjects with abnormal karyotype is less than reported in persons with PMF of European descent. Complex karyotype, sole trisomy8, sole del(20q) and sole del(13q) arefrequent abnormalities. Monosomal karyotye is an independent unfavorable prognostic factor adjusted for DIPSS-Chinese Objective To investigate the prognostic factor of in Chinese with myelodysplastic syndromes (MDS) classified as IPSS intermediate-1Methods We studied prognostic variables in191Chinese subjects with MDS intermediate-1(INT-1) in the IPSS.Results Like the IPSS-R, we found serum ferritin level>500μg/L at diagnosis was a strong independent predictor of survival. Although baseline serum ferritin level was inversely correlated with baseline hemoglobin, it was serum ferritin, not baseline hemoglobin level that was significantly-correlated with survival in Chinese subjects. These data were not biased by the IPSS-R as they were generated before it’s publication. Based on these data a new prognostic scoring system including ECOG performance score, absolute neutrophil level (ANC), serum ferritin, percentage of bone marrow blasts and poor karyotye was developed for Chinese with IPSS INT-1MDS.Conclusions This revised scoring system identified a subgroup of Chinese with MDS and INT-1IPPS who have a poor prognosis and may benefit from more intensive therapy.更多还原