多参数流式细胞术联合WT1检测早期MRD在急性髓系白血病中的预后分析
发布时间：2018-08-09 19:47
【摘要】：目的:探讨初治急性髓系白血病(acute myeloid leukemia,AML)患者诱导治疗后获得首次骨髓形态学完全缓解(complete remission,CR1)时,采用多参数流式细胞术(multiparameter flow cytometry,MFC)及Wilms瘤基因1(Wilms tumor1 gene,WT1)表达检测微小残留病(minimal residual disease,MRD),通过检测MFC-LAIP联合WT1的水平变化,对AML患者进行无复发生存(relapse-free survival,RFS)、总体生存率(overall survival,OS)等预后分析,以期指导临床后续治疗。方法:分析2010年10月至2016年10月山西医科大学第二医院血液科病房收治的持续接受治疗的179例初治AML(M3除外)患者的临床资料,所有患者均获得CR/CRi/CRp,分析CR1时骨髓检测MFC-LAIP、WT1水平在预测RFS、OS及指导个体化治疗等方面的作用。1、骨髓(bone marrow,BM)标本来自AML患者。诊断及分类标准依据2016年世界卫生组织(World Health Organization,WHO)。对所有患者初治及CR1后的BM样本进行MFC-LAIP(leukemia associated immunophenotype)及WT1表达水平检测。本实验是在骨髓细胞水平进行的实验。所用标本均来源于山西医科大学第二医院血液科因诊疗需要抽取的BM(获得患者知情同意)。2、每例患者在初治及CR1后取BM 2-4ml,用FC500(Beckman公司)MFC检测抗原表达及原始细胞计数,数据分析用CXP Analysis软件。每个组合补偿设定用未标记的同型对照。检测前行Flow-check测试激光和补偿的稳定性。MFC进行免疫表型分析。单克隆抗体主要包括:CD45-PC7,CD38-FITC,CLL-1-PE,CD34-PC5,CD7-FITC,c MPO-PE,CD19-PC5,CD34-FITC,CD11b-PE,CD15-PC5,CD56-PE,CD117-PC5。同时采用实时定量荧光PCR(real-time quantitative polymerase chain reaction,RT-q PCR)检测WT1。3、MFC分析以前向角/侧向角(forward scatter/side scatter,FSC/SSC)射门去除死细胞和碎片,每管至少获取10万个细胞,以CD45和SSC(CD45/SSC)射门。患者骨髓细胞染色阳性为抗原表达超过20%的CD45/SSC细胞群,评估的最低有效细胞数是40。抗原表达强度以阳性细胞群的平均荧光强度(mean fluorescence intensity,MFI)来确定,评估的最低有效细胞数是500。若在筛选时白血病细胞出现的区域内仍有细胞存在且表型异常,即判断为残留白血病细胞,以这些残存的细胞占骨髓有核细胞总数的比例作为MRD的数值。MRD定量值≤10-4与为阴性,≥10-4之为阳性。4、WT1表达以欧洲白血病网络公认的比值法计算,用RT-q PCR测定WT1和ABL基因,以ABL基因为内参。WT1/ABL×1000060为高表达,60为低表达。结果:1、179例初治AML患者中,男88例,女91例,中位年龄47岁(15-73)岁,依据WHO(2016)分型:AML伴t(8;21)29例,AML伴t(16;16)/inv(16)9例,AML伴inv(3)/t(3;3)1例,M0 5例,M1 12例,M2 15例,M4 53例,M4EO 4例,M5 25例,M6 4例,AML伴骨髓增生异常相关改变11例,治疗相关AML11例。依据NCCN(2017)指南:低危37例,中危117例,高危25例。中位随访时间为23月(6-76)月,目前共100例患者复发,复发多发生在CR1后2年内,76例早期复发(≤12个月)。死亡62例,其中55例死于复发。中位RFS 21月(4-75月),中位OS 28月(6-76月)。2、在73例AML患者中分析C型凝集素样受体-1(C-type lectin-like receptor-1,CLL-1)在MRD检测中的作用。研究发现,CLL-1表达在正常对照骨髓细胞的粒细胞、单核细胞,不表达在淋巴细胞、有核红细胞和CD34+CD38-细胞;在91.8%AML患者白血病细胞和粒细胞、单核细胞表达,在CD34+CD38-细胞表达阳性率为71.2%,且在疾病CR前后表达稳定,而在CD34+与CD34-AML患者中表达无差异(P=0.43)。3、首次骨髓形态学CR后107例患者MFC-LAIP转阴,72例患者MFC-LAIP仍为阳性。MFC-LAIP阳性患者与阴性患者一般特征相比,发病时外周血白细胞计数、BM、MFC及外周血原始细胞比例更高,血小板计数更低,多为合并中枢神经系统白血病(central nervous system leukemia,CNSL)、高白细胞计数、治疗相关、中高危患者,初次诱导CR率低,但差异均无统计学意义(P值均0.05),MFC-LAIP阳性患者2个疗程未CR比例明显多于阴性患者(P=0.029),RFS、OS明显缩短(24月vs 11月,35.5月vs 21月,P0.001)。4、73例AML患者中,57例(78.1%)患者初诊WT1高表达,16例(21.9%)患者初诊WT1表达不高,其中9例在CR1后WT1高表达,低、中、高危分别2例、4例、3例,老年患者4例,7例发生早期复发。WT1表达水平与外周血白细胞计数、血红蛋白浓度、血小板计数、乳酸脱氢酶(lactate dehydrogenase,LDH)、β2微球蛋白(β2-microglobulin,β2-MG)、预后分层无相关关系。5、多因素分析发现MFC-LAIP、WT1水平为影响AML患者RFS、OS的独立因素。分析不同危险度分层患者,发现MFC-LAIP水平为影响中危、高危患者RFS的独立因素;中危患者OS的独立影响因素,而不是低危患者RFS、OS的独立影响因素。6、评估MFC-LAIP、WT1水平在AML患者预测复发、评估预后中的效能。发现MFC-LAIP≥1.35%组与1.35%组相比5年RFS率明显降低(P0.001),该界值的灵敏度为0.575,特异度为0.818;MFC-LAIP≥1.70%组与1.70%组相比5年OS显著降低(P0.001),灵敏度为0.714,特异度为0.769。CR1后WT1水平≥40患者与40患者相比18月RFS明显降低(P=0.049),CR1后WT1水平≥50与50患者相比,20月的OS明显降低(P=0.02);MFC联合WT1表达检测CR1后MRD可提高AML患者预测RFS、OS的灵敏度,而不影响特异度。进一步亚组分析不同疗程达CR患者,发现仍可提高灵敏度,不影响特异度,但权重不同。结论:1、CR1后MFC-LAIP是影响AML患者RFS、OS的独立因素,可能对CR后分层治疗扮演重要甚至主导角色,但在不同危险度分层患者中权重不同。CLL-1可以作为AML诊断、MRD检测及靶向治疗的标记。2、CR1时WT1水平分别≥40及≥50预示更短的RFS、OS;初治时表达正常,CR1后WT1高表达者,多为中高危、老年患者,复发率高。3、MFC联合WT1检测可作为AML MRD检测的良好手段,可提高MRD检测水平的灵敏度,而不影响特异度。
[Abstract]:Objective: To investigate the first bone marrow morphologic complete remission (complete remission, CR1) after the first treatment of acute myeloid leukemia (AML), and to detect small residual disease by multiparameter flow cytometry (multiparameter flow cytometry, MFC) and Wilms tumor gene 1. Esidual disease, MRD), by detecting the level changes of MFC-LAIP combined with WT1, the prognosis analysis of AML patients without recurrent survival (relapse-free survival, RFS), overall survival rate (overall survival, OS), etc., in order to guide the clinical follow-up treatment. Methods: analysis from October 2010 to October 2016 in the Department of Hematology at the second hospital of Shanxi Medical University. The clinical data of 179 patients with AML (except M3) were treated with continuous treatment. All patients received CR/CRi/CRp. The analysis of CR1, MFC-LAIP, WT1 level in predicting RFS, OS, and individualized treatment, was used for.1. The bone marrow (bone marrow, BM) standard was derived from the patients. Diagnosis and classification criteria were based on World Health in 2016. Tissue (World Health Organization, WHO). MFC-LAIP (leukemia associated immunophenotype) and WT1 expression levels were detected for all patients and BM samples after initial treatment and CR1. This experiment was conducted at the level of bone marrow cells. All specimens were derived from the BM (obtained) from the hematological diagnosis and treatment of the second Hospital of Shanxi Medical University. The patient informed consent).2, each patient was taken BM 2-4ml after initial treatment and CR1, and FC500 (Beckman) MFC was used to detect antigen expression and primitive cell count, and CXP Analysis software was used for data analysis. The unlabeled same type control was set by each combination. The immunophenotype was detected by Flow-check test laser and the stability.MFC of compensation. CD45-PC7, CD38-FITC, CLL-1-PE, CD34-PC5, CD7-FITC, C MPO-PE, CD19-PC5, CD34-FITC, CD11b-PE, CD15-PC5, CD56-PE. Scatter, FSC/SSC) shot to remove dead cells and fragments. At least 100 thousand cells were obtained per tube, with CD45 and SSC (CD45/SSC) shot. The patient's bone marrow cells were stained positive for the CD45/SSC cell group over 20% of the antigen expression. The lowest number of effective cells evaluated was the average fluorescence intensity of the positive cell group with the 40. antigen expression intensity (mean fluorescence in). Tensity, MFI) to determine that the minimum number of effective cells evaluated is 500. if there are still cells and phenotypic abnormalities in the region of leukemic cells when screened, that is, residual leukemic cells are judged to be the proportion of the remaining cells to the total number of nucleated cells in the bone marrow as the number of.MRD quantitative values of MRD less than 10-4 and negative, or more than 10-4 The positive.4, WT1 expression was calculated by the ratio method recognized by the European leukemia network, and the WT1 and ABL genes were measured by RT-q PCR. The ABL gene was the high expression of the internal parameter.WT1/ABL x 1000060 and the 60 was low expression. Results: 1179 cases of early treatment AML, 88 male, 91 female, 47 years old (15-73), were classified according to WHO (2016): AML companion t (8; 21) 29 cases, 16; 1 6) /inv (16) 9 cases, AML with inv (3) /t (3; 3) 1 cases, M0 5 cases, M1 12 cases, M2 15 cases, M4 53, M4EO 4, M5 25 cases, M6 4 cases, AML companion myelodysplastic related cases. In the 2 years after CR1, 76 cases had early recurrence (less than 12 months). 62 cases died, of which 55 died of recurrence. Middle RFS 21 months (4-75 months), middle OS 28 months (6-76 months).2, and the role of C type lectin like receptor -1 (C-type lectin-like receptor-1, CLL-1) in MRD detection in 73 patients with AML. Granulocytes, monocytes, not expressed in lymphocytes, nucleated red cells and CD34+CD38- cells; in 91.8%AML patients, the expression of leukemic cells and granulocytes, mononuclear cells, expression in CD34+CD38- cells was 71.2%, and the expression was stable before and after the disease CR, and the P=0.43.3 and the first bone were expressed in the patients with CD34+ and CD34-AML. In 107 patients with CR, MFC-LAIP turned negative, and 72 patients with MFC-LAIP were still positive for positive.MFC-LAIP, compared with the negative patients. The proportion of peripheral blood leukocyte count, BM, MFC and peripheral blood primitive cells was higher, the platelet count was lower, and more of the central nervous system leukemia (central nervous system leukemi). A, CNSL), high white cell count, treatment related, high risk patients, the initial induction of CR rate was low, but the difference was not statistically significant (P 0.05), MFC-LAIP positive patients were not CR in 2 courses more than negative patients (P=0.029), RFS, OS significantly shortened (24 months vs November, 35.5 month vs 21 months, P0.001), 57 cases (78.1%) patients initially diagnosed 1 high expression, 16 cases (21.9%) patients with early diagnosis of WT1 expression was not high, of which 9 cases of high expression, low, middle, high risk 2 cases, 4 cases, 4 cases, 3 cases, 4 elderly patients, 7 cases of early recurrence.WT1 expression and peripheral blood white blood cell count, hemoglobin concentration, platelet count, lactate dehydrogenase (lactate dehydrogenase, LDH), beta 2 microglobulin (beta 2-) Microglobulin, beta 2-MG), prognostic stratification without correlation.5. Multifactor analysis found MFC-LAIP, WT1 level as an independent factor affecting RFS and OS in AML patients. Analysis of different risk levels of stratified patients showed that the level of MFC-LAIP was the independent factor in the risk of middle risk, the risk of RFS was independent; the independent factors of OS in the middle risk patients were not the low risk patients RFS. The independent influence factor.6, evaluate the MFC-LAIP, WT1 level in the AML patient to predict the recurrence and evaluate the efficacy in the prognosis. It was found that the RFS rate of MFC-LAIP 1.35% groups was significantly lower than that of the 1.35% group (P0.001), the sensitivity of the boundary value was 0.575, the specificity was 0.818, and the MFC-LAIP > 1.70% group was significantly lower than the 1.70% group (P0.001) and the sensitivity was 0.714, When the degree of specificity was 0.769.CR1, the level of WT1 was more than 40 in 18 months compared with that of 40 patients (P=0.049). The OS of 20 months was significantly lower than that of 50 patients after CR1 WT1 level (P=0.02), and the sensitivity of MFC combined WT1 expression was improved without affecting the specificity. The findings still improve the sensitivity without affecting the specificity, but the weight is different. Conclusion: 1. After CR1, MFC-LAIP is an independent factor affecting RFS and OS in AML patients. It may play an important and even dominant role in the stratified therapy after CR, but the weight different.CLL-1 in different risk stratification patients can be used as AML diagnosis, MRD detection and targeting therapy marker.2, At CR1, the level of WT1 is more than 40 and more than 50 indicates shorter RFS, OS; the expression is normal at the beginning of the treatment. The high expression of WT1 after CR1 is most high risk, the elderly patients, the recurrence rate is high.3. MFC combined WT1 detection can be used as a good means of AML MRD detection, which can improve the sensitivity of MRD detection level without affecting the specificity.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71
本文编号：2175128
[Abstract]:Objective: To investigate the first bone marrow morphologic complete remission (complete remission, CR1) after the first treatment of acute myeloid leukemia (AML), and to detect small residual disease by multiparameter flow cytometry (multiparameter flow cytometry, MFC) and Wilms tumor gene 1. Esidual disease, MRD), by detecting the level changes of MFC-LAIP combined with WT1, the prognosis analysis of AML patients without recurrent survival (relapse-free survival, RFS), overall survival rate (overall survival, OS), etc., in order to guide the clinical follow-up treatment. Methods: analysis from October 2010 to October 2016 in the Department of Hematology at the second hospital of Shanxi Medical University. The clinical data of 179 patients with AML (except M3) were treated with continuous treatment. All patients received CR/CRi/CRp. The analysis of CR1, MFC-LAIP, WT1 level in predicting RFS, OS, and individualized treatment, was used for.1. The bone marrow (bone marrow, BM) standard was derived from the patients. Diagnosis and classification criteria were based on World Health in 2016. Tissue (World Health Organization, WHO). MFC-LAIP (leukemia associated immunophenotype) and WT1 expression levels were detected for all patients and BM samples after initial treatment and CR1. This experiment was conducted at the level of bone marrow cells. All specimens were derived from the BM (obtained) from the hematological diagnosis and treatment of the second Hospital of Shanxi Medical University. The patient informed consent).2, each patient was taken BM 2-4ml after initial treatment and CR1, and FC500 (Beckman) MFC was used to detect antigen expression and primitive cell count, and CXP Analysis software was used for data analysis. The unlabeled same type control was set by each combination. The immunophenotype was detected by Flow-check test laser and the stability.MFC of compensation. CD45-PC7, CD38-FITC, CLL-1-PE, CD34-PC5, CD7-FITC, C MPO-PE, CD19-PC5, CD34-FITC, CD11b-PE, CD15-PC5, CD56-PE. Scatter, FSC/SSC) shot to remove dead cells and fragments. At least 100 thousand cells were obtained per tube, with CD45 and SSC (CD45/SSC) shot. The patient's bone marrow cells were stained positive for the CD45/SSC cell group over 20% of the antigen expression. The lowest number of effective cells evaluated was the average fluorescence intensity of the positive cell group with the 40. antigen expression intensity (mean fluorescence in). Tensity, MFI) to determine that the minimum number of effective cells evaluated is 500. if there are still cells and phenotypic abnormalities in the region of leukemic cells when screened, that is, residual leukemic cells are judged to be the proportion of the remaining cells to the total number of nucleated cells in the bone marrow as the number of.MRD quantitative values of MRD less than 10-4 and negative, or more than 10-4 The positive.4, WT1 expression was calculated by the ratio method recognized by the European leukemia network, and the WT1 and ABL genes were measured by RT-q PCR. The ABL gene was the high expression of the internal parameter.WT1/ABL x 1000060 and the 60 was low expression. Results: 1179 cases of early treatment AML, 88 male, 91 female, 47 years old (15-73), were classified according to WHO (2016): AML companion t (8; 21) 29 cases, 16; 1 6) /inv (16) 9 cases, AML with inv (3) /t (3; 3) 1 cases, M0 5 cases, M1 12 cases, M2 15 cases, M4 53, M4EO 4, M5 25 cases, M6 4 cases, AML companion myelodysplastic related cases. In the 2 years after CR1, 76 cases had early recurrence (less than 12 months). 62 cases died, of which 55 died of recurrence. Middle RFS 21 months (4-75 months), middle OS 28 months (6-76 months).2, and the role of C type lectin like receptor -1 (C-type lectin-like receptor-1, CLL-1) in MRD detection in 73 patients with AML. Granulocytes, monocytes, not expressed in lymphocytes, nucleated red cells and CD34+CD38- cells; in 91.8%AML patients, the expression of leukemic cells and granulocytes, mononuclear cells, expression in CD34+CD38- cells was 71.2%, and the expression was stable before and after the disease CR, and the P=0.43.3 and the first bone were expressed in the patients with CD34+ and CD34-AML. In 107 patients with CR, MFC-LAIP turned negative, and 72 patients with MFC-LAIP were still positive for positive.MFC-LAIP, compared with the negative patients. The proportion of peripheral blood leukocyte count, BM, MFC and peripheral blood primitive cells was higher, the platelet count was lower, and more of the central nervous system leukemia (central nervous system leukemi). A, CNSL), high white cell count, treatment related, high risk patients, the initial induction of CR rate was low, but the difference was not statistically significant (P 0.05), MFC-LAIP positive patients were not CR in 2 courses more than negative patients (P=0.029), RFS, OS significantly shortened (24 months vs November, 35.5 month vs 21 months, P0.001), 57 cases (78.1%) patients initially diagnosed 1 high expression, 16 cases (21.9%) patients with early diagnosis of WT1 expression was not high, of which 9 cases of high expression, low, middle, high risk 2 cases, 4 cases, 4 cases, 3 cases, 4 elderly patients, 7 cases of early recurrence.WT1 expression and peripheral blood white blood cell count, hemoglobin concentration, platelet count, lactate dehydrogenase (lactate dehydrogenase, LDH), beta 2 microglobulin (beta 2-) Microglobulin, beta 2-MG), prognostic stratification without correlation.5. Multifactor analysis found MFC-LAIP, WT1 level as an independent factor affecting RFS and OS in AML patients. Analysis of different risk levels of stratified patients showed that the level of MFC-LAIP was the independent factor in the risk of middle risk, the risk of RFS was independent; the independent factors of OS in the middle risk patients were not the low risk patients RFS. The independent influence factor.6, evaluate the MFC-LAIP, WT1 level in the AML patient to predict the recurrence and evaluate the efficacy in the prognosis. It was found that the RFS rate of MFC-LAIP 1.35% groups was significantly lower than that of the 1.35% group (P0.001), the sensitivity of the boundary value was 0.575, the specificity was 0.818, and the MFC-LAIP > 1.70% group was significantly lower than the 1.70% group (P0.001) and the sensitivity was 0.714, When the degree of specificity was 0.769.CR1, the level of WT1 was more than 40 in 18 months compared with that of 40 patients (P=0.049). The OS of 20 months was significantly lower than that of 50 patients after CR1 WT1 level (P=0.02), and the sensitivity of MFC combined WT1 expression was improved without affecting the specificity. The findings still improve the sensitivity without affecting the specificity, but the weight is different. Conclusion: 1. After CR1, MFC-LAIP is an independent factor affecting RFS and OS in AML patients. It may play an important and even dominant role in the stratified therapy after CR, but the weight different.CLL-1 in different risk stratification patients can be used as AML diagnosis, MRD detection and targeting therapy marker.2, At CR1, the level of WT1 is more than 40 and more than 50 indicates shorter RFS, OS; the expression is normal at the beginning of the treatment. The high expression of WT1 after CR1 is most high risk, the elderly patients, the recurrence rate is high.3. MFC combined WT1 detection can be used as a good means of AML MRD detection, which can improve the sensitivity of MRD detection level without affecting the specificity.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71
【参考文献】
相关期刊论文 前1条
1 钟旭姝;兰菲;崔旭;蒋能刚;朱焕玲;贾永前;;初发急性髓系白血病诱导化疗后外周血MRD早期检测与临床疗效的关系[J];中国实验血液学杂志;2013年01期
，本文编号：2175128
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