慢性粒细胞白血病早期疗效预测及CFL1在其治疗中的作用
发布时间：2019-06-19 01:36
【摘要】：第一部分伊马替尼治疗3月后BCR-ABL转录本水平对慢性粒细胞白血病患者预后及治疗反应的预测价值研究目的评估接受伊马替尼治疗的慢性粒细胞白血病慢性期(CML-CP)患者,治疗3个月后BCR-ABL融合基因转录本水平(以10%为截断值)在各个亚组患者中对伊马替尼的治疗反应及生存的预测效果,并探讨影响CML患者治疗3个月能否达到BCR-ABL≤10%的相关因素。研究方法回顾性分析在我院诊断的CML-CP患者接受伊马替尼治疗3个月、6个月、12个月、18个月获得的细胞遗传学和分子学反应。将患者依据有无重组人干扰素(IFN-α)治疗史、年龄、诊断到服用伊马替尼的间隔时间三个因素分层,分析在各个亚组中BCR-ABL不同表达水平患者之间OS、EFS、PFS的差异。并分析患者的基本情况、疾病状态、诊断到开始服用伊马替尼的间隔时间是否影响治疗3月后BCR-ABL下降水平。研究结果(1)研究共纳入CML-CP患者299例,其中293例患者接受细胞遗传学或BCR-ABL融合基因分子学检测。203例(67.9%)患者随访结束后达完全细胞遗传学缓解(CCyR),其中包括167例(55.9%)获得主要分子学缓解(MMR),130例(43.5%)获得完全分子学缓解(CMR)。(2)274例患者治疗3月后检测了BCR-ABL水平,BCR-ABL≤10%(n=146,53.3%)的患者OS、EFS、PFS均优于BCR-ABL10% (n=128,46.7%)患者。(3)在有IFN-a治疗史的患者中,BCR-ABL《10%的患者EFS优于10%的患者(P=0.038),但OS与PFS无明显差异(P=0.276,P=0.229)。(4)在年龄≤29岁的患者中,BCR-ABL《10%的患者PFS.优于10%的患者(P=0.046),但OS与EFS并无明显差异(P=0.223,P=0.176)。(5)在治疗间隔时间≤6个月的患者中,BCR-ABL≤10%者OS,EFS,PFS优于BCR-ABL10%者(P=0.006,P0.001,P0.001),但在治疗间隔时间6个月时,两组患者EFS和PFS有差异(P=0.007,P=0.004),而OS并无统计学差异(P=0.072)。(6)在无IFN-α治疗史、29岁、治疗间隔时间≤6个月的患者中,BCR-ABL《10%组部分细胞遗传学缓解(PCyR)、CCyR、MMR、CMR的累积发生率均高于BCR-ABL10%组。但在其他患者中,上述各反应的累积发生率并无明显差异。(7)确诊CML到接受伊马替尼治疗的间隔时间为影响治疗3月后BCR-ABL是否能达到≤10%的独立因素(OR=9.854,P0.001)。研究结论初始治疗为伊马替尼的CML-CP患者接受伊马替尼治疗3月后BCR-ABL的下降水平可预测患者的OS、PFS和EFS以及最佳治疗反应。但该预测价值在有IFN-α治疗史、年龄≤29岁及治疗间期6月的患者中有限。第二部分CFL1的表达在慢性粒细胞白血病患者治疗早期疗效评价中的意义研究目的评价非肌型丝切蛋白基因(CFL1)表达水平与CML患者对伊马替尼早期治疗反应及预后的关系研究方法用实时定量PCR方法检测65例初发慢性粒细胞白血病患者骨髓单个核细胞、初发急性髓系白血病(AML)患者骨髓单个核细胞及正常对照外周血单个核细胞中的CFL1表达水平。比较该基因在CML、AML和正常对照之间的表达差别及同一患者治疗前和治疗后该基因的表达变化。分析该基因表达水平与患者对伊马替尼早期治疗反应的关系,及与CML疾病相关指标之间的关系。研究结果1.与正常对照相比,CML与AML患者中的CFL1表达水平明显降低(p0.05, p0.05); AML患者尤为明显：2.同一患者经伊马替尼治疗后CFL1表达量升高(p0.05)；3.CML诊断时CFL1低表达的患者伊马替尼治疗6个月后达到CCR的比例高于CFL1高表达的患者(分别为74.2%和46.9%,p=0.027。);4.CML诊断时CFL1表达量较低的患者EFS、PFS优于表达量较高的患者：但是无统计学差异。5. logistics回归模型分析,CFL1的表达水平与CML患者初发时外周血白细胞和血小板计数相关(p=0.033,p=0.028)。结论1.CML患者诊断时CFL1的表达水平与CML疾病状态有关；2.CFL1低表达的患者治疗早期易达到最佳反应,其表达水平与患者的预后关系尚需进一步研究。第三部分体外实验研究cofilin在药物治疗慢性粒细胞白血病中的作用目 的探讨非肌型丝切蛋白基因CFL1的表达水平影响CML患者对药物治疗反应的相关机制,评价非肌型丝切蛋白cofilin在CML治疗中的作用。方 法检测慢性粒细胞白血病细胞株K562、KCL-22经伊马替尼及高三尖杉酯碱(HHT)治疗后cofilin蛋白表达水平及F-actin形成的变化；细胞对纤黏连蛋白的粘附指数变化,细胞的迁移力变化；利用ShRNA下调CFL1,观察细胞对伊马替尼治疗的敏感性变化；过表达CFL1野生型(CFL1)及CFL1激活型(S3A)和失活型(S3D)两个突变体,观察细胞对伊马替尼治疗的敏感性变化；在伊马替尼耐药的细胞株K562/G中过表达CFL1及S3A、S3D,观察其对伊马替尼的敏感性的变化。结果1.经伊马替尼、高三尖杉酯碱作用后,K562、KCL-22细胞株细胞中cofilin及P-cofilin表达均上调；F-actin形成增加；2.下调CFL1表达后,K562细胞对伊马替尼的敏感性降低,伊马替尼对细胞的增殖抑制减弱；3.过表达CFL1及S3A后,K562细胞对伊马替尼的敏感性增高,但过表达S3D后,细胞对治疗的敏感性变化不大；4.K562/G细胞中过表达CFL1及S3A后,细胞对伊马替尼的敏感性增高。同样,过表达S3D后,细胞对治疗的敏感性变化不大；5.药物处理后K562细胞的迁移力减弱,细胞对纤黏连蛋白(FN)的粘附力增加。结论1.伊马替尼、高三尖杉酯碱作用后,K562、KCL-22细胞cofilin表达上调,磷酸化cofilin增加,cofilin磷酸化后,其活性减低,对F-actin剪切作用减弱,F-actin形成增加,细胞对对纤黏连蛋白的粘附力增加,迁移力减弱；2. cofilin高表达可增加CML细胞株对伊马替尼的敏感性,且该作用依赖于cofilin的活性。
[Abstract]:The first part of imatinib for the treatment of chronic myeloid leukemia chronic (CML-CP) patients treated with imatinib for the purpose of evaluating the predictive value of the BCR-ABL transcript level for the prognosis and treatment response of patients with chronic granulocytic leukemia after 3 months of treatment, The effect of BCR-ABL fusion gene transcription on the treatment response and survival of imatinib in each subgroup was studied after 3 months of treatment, and the relevant factors that affected the treatment of CML patients for 3 months to reach BCR-ABL-10% were discussed. Methods The cytogenetic and molecular responses of imatinib for 3 months,6 months,12 months and 18 months were retrospectively analyzed in the patients with CML-CP. The differences in OS, EFS, and PFS among patients with different expression levels of BCR-ABL in each subgroup were analyzed by layering the patient on the basis of the presence or absence of a recombinant human interferon (IFN-1) treatment history, age, and the time interval between the diagnosis and the time of administration of imatinib. The basic condition of the patient, the condition of the disease, the time interval between the diagnosis and the start of the treatment of imatinib were analyzed, and the BCR-ABL level of decline after March was affected. Results (1) A total of 299 patients with CML-CP were included in the study, of which 293 patients received cytogenetic or BCR-ABL fusion gene, and 203 (67.9%) patients had complete cytogenetic response (CCyR) after follow-up. Among these,167 (55.9%) obtained major molecular response (MMR), and 130 (43.5%) obtained complete molecular response (CMR). (2) In 274 patients, the patients with BCR-ABL, BCR-ABL and 10% (n = 146, 53.3%) were compared with BCR-ABL10% (n = 128, 46.7%) patients after 3 months of treatment. (3) In patients with a history of IFN-a therapy, patients with BCR-ABL <10% had better EFS than 10% of patients (P = 0.038), but there was no significant difference between OS and PFS (P = 0.276, P = 0.229). (4) In the 29-year-old, BCR-ABL <10% of patients had a PFS. It was superior to 10% of patients (P = 0.046), but there was no significant difference between OS and EFS (P = 0.223, P = 0.176). (5) In the 6-month period of treatment, the OS, EFS and PFS of BCR-ABL in 10% of patients were better than those of BCR-ABL10% (P = 0.006, P 0.001, P 0.001), but the difference of EFS and PFS in the two groups at 6 months of treatment (P = 0.007, P = 0.004), while OS was not statistically different (P = 0.072). (6) The cumulative incidence of BCR-ABL <10% (PCyR), CCyR, MMR, and CMR was higher than that of the BCR-ABL10% group in patients with no treatment history of IFN-ABL,29 years of age and 6 months between treatment time and 6 months. In other patients, however, there was no significant difference in the cumulative incidence of the above reactions. (7) The interval between the diagnosis of CML and the treatment of imatinib was an independent factor that affected the treatment of BCR-ABL after 3 months (OR = 9.854, P0.001). The study concluded that the initial treatment for patients with CML-CP of imatinib received imatinib for treatment of the decrease in BCR-ABL after 3 months of treatment can predict the OS, PFS, and EFS of the patient as well as the best treatment response. However, the predictive value was limited in patients with a history of IFN-I treatment, a 29-year-old age and a 6-month period of treatment. The significance of the expression of the second part of CFL1 in the early treatment of patients with chronic myelogenous leukemia is to evaluate the relationship between the expression level of the non-myofibrillar protein gene (CFL1) and the early treatment response and the prognosis of the imatinib in the patients with CML. The method of real-time quantitative PCR is used to detect the 65 cases. The level of CFL1 expression in the mononuclear cells of the bone marrow mononuclear cells and the normal control peripheral blood mononuclear cells of the patients with primary acute myeloid leukemia (AML) and the normal control of the patients with acute myeloid leukemia (AML). The expression of the gene in CML, AML and normal controls was compared with that of the same patient before and after treatment. The relationship between the expression level of the gene and the patients' response to the early treatment of imatinib and the relationship with the disease-related indexes of CML were analyzed. Study Results 1. Compared with the normal control, the level of CFL1 expression in CML and AML patients was significantly lower (p0.05, p0.05); AML patients were particularly significant:2. In the same patient, the level of CFL1 expression was increased after imatinib treatment (p0.05);3. The proportion of patients with low expression of CFL1 in CML at 6 months after treatment with imatinib was higher than that of patients with high CFL1 expression (74.2% and 46.9%, p = 0.027, respectively). );4. In patients with lower CFL1 expression in CML, the PFS was superior to those with higher levels of expression: but there was no statistical difference. The expression level of CFL1 was correlated with the platelet count in the peripheral blood of the patients with CML (p = 0.033, p = 0.028). Conclusion 1. The expression level of CFL1 in the diagnosis of CML is related to the state of CML. In the third part, the role of cofin in the treatment of chronic myeloid leukemia (CML) was studied in this paper. The effect of cofin on the treatment of CML was evaluated by the effect of cofin on the treatment of CML. The expression level of cofin protein and the change of F-actin in the patients with chronic myelocytic leukemia cell line K562 and KCL-22 after treatment with imatinib and homoharringtonine (HHT) were detected by the method of square method, the adhesion index of the cells to the fibronectin was changed, the migration force of the cells was changed, and the CFL1 was down-regulated by using the shRNA, The sensitivity of the cells to imatinib was observed; the sensitivity of the cells to imatinib was observed by overexpressing two mutants of the CFL1 wild-type (CFL1) and the CFL1-activated (S3A) and the inactivated (S3D); in the imatinib-resistant cell line K562/ G, CFL1 and S3A, S3D were overexpressed, Changes to the sensitivity of imatinib to imatinib were observed. Results 1. After the action of imatinib and homoharringtonine, the expression of cnoil and P-cofilin in K562 and KCL-22 cell lines was up-regulated, and the formation of F-actin was increased; After the down-regulation of CFL1 expression, the sensitivity of K562 cells to imatinib decreased, and the inhibition of imatinib on the proliferation of the cells was decreased;3. After the expression of CFL1 and S3A, the sensitivity of the K562 cells to imatinib was increased, but the sensitivity of the cells to the treatment was not small after the expression of the S3D, and the sensitivity of the cells to imatinib was increased after the overexpression of CFL1 and S3A in K562/ G cells. In the same way, the sensitivity of the cell to the treatment was not significant after the expression of the S3D. The migration of K562 cells after drug treatment decreased and the adhesion of the cells to fibronectin (FN) increased. Conclusion 1. After the action of imatinib and homoharringtonine, the expression of cofin in K562 and KCL-22 was up-regulated, and the phosphorylation of cofilin increased, and after the phosphorylation of cofilin, the activity of K562 and KCL-22 was reduced, the action of F-actin was weakened, the formation of F-actin was increased, the adhesion of the cells to fibronectin was increased, and the migration force was weakened. The high expression of cofin can increase the sensitivity of CML cell lines to imatinib, and the effect is dependent on the activity of cofin.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.72
本文编号：2501960
[Abstract]:The first part of imatinib for the treatment of chronic myeloid leukemia chronic (CML-CP) patients treated with imatinib for the purpose of evaluating the predictive value of the BCR-ABL transcript level for the prognosis and treatment response of patients with chronic granulocytic leukemia after 3 months of treatment, The effect of BCR-ABL fusion gene transcription on the treatment response and survival of imatinib in each subgroup was studied after 3 months of treatment, and the relevant factors that affected the treatment of CML patients for 3 months to reach BCR-ABL-10% were discussed. Methods The cytogenetic and molecular responses of imatinib for 3 months,6 months,12 months and 18 months were retrospectively analyzed in the patients with CML-CP. The differences in OS, EFS, and PFS among patients with different expression levels of BCR-ABL in each subgroup were analyzed by layering the patient on the basis of the presence or absence of a recombinant human interferon (IFN-1) treatment history, age, and the time interval between the diagnosis and the time of administration of imatinib. The basic condition of the patient, the condition of the disease, the time interval between the diagnosis and the start of the treatment of imatinib were analyzed, and the BCR-ABL level of decline after March was affected. Results (1) A total of 299 patients with CML-CP were included in the study, of which 293 patients received cytogenetic or BCR-ABL fusion gene, and 203 (67.9%) patients had complete cytogenetic response (CCyR) after follow-up. Among these,167 (55.9%) obtained major molecular response (MMR), and 130 (43.5%) obtained complete molecular response (CMR). (2) In 274 patients, the patients with BCR-ABL, BCR-ABL and 10% (n = 146, 53.3%) were compared with BCR-ABL10% (n = 128, 46.7%) patients after 3 months of treatment. (3) In patients with a history of IFN-a therapy, patients with BCR-ABL <10% had better EFS than 10% of patients (P = 0.038), but there was no significant difference between OS and PFS (P = 0.276, P = 0.229). (4) In the 29-year-old, BCR-ABL <10% of patients had a PFS. It was superior to 10% of patients (P = 0.046), but there was no significant difference between OS and EFS (P = 0.223, P = 0.176). (5) In the 6-month period of treatment, the OS, EFS and PFS of BCR-ABL in 10% of patients were better than those of BCR-ABL10% (P = 0.006, P 0.001, P 0.001), but the difference of EFS and PFS in the two groups at 6 months of treatment (P = 0.007, P = 0.004), while OS was not statistically different (P = 0.072). (6) The cumulative incidence of BCR-ABL <10% (PCyR), CCyR, MMR, and CMR was higher than that of the BCR-ABL10% group in patients with no treatment history of IFN-ABL,29 years of age and 6 months between treatment time and 6 months. In other patients, however, there was no significant difference in the cumulative incidence of the above reactions. (7) The interval between the diagnosis of CML and the treatment of imatinib was an independent factor that affected the treatment of BCR-ABL after 3 months (OR = 9.854, P0.001). The study concluded that the initial treatment for patients with CML-CP of imatinib received imatinib for treatment of the decrease in BCR-ABL after 3 months of treatment can predict the OS, PFS, and EFS of the patient as well as the best treatment response. However, the predictive value was limited in patients with a history of IFN-I treatment, a 29-year-old age and a 6-month period of treatment. The significance of the expression of the second part of CFL1 in the early treatment of patients with chronic myelogenous leukemia is to evaluate the relationship between the expression level of the non-myofibrillar protein gene (CFL1) and the early treatment response and the prognosis of the imatinib in the patients with CML. The method of real-time quantitative PCR is used to detect the 65 cases. The level of CFL1 expression in the mononuclear cells of the bone marrow mononuclear cells and the normal control peripheral blood mononuclear cells of the patients with primary acute myeloid leukemia (AML) and the normal control of the patients with acute myeloid leukemia (AML). The expression of the gene in CML, AML and normal controls was compared with that of the same patient before and after treatment. The relationship between the expression level of the gene and the patients' response to the early treatment of imatinib and the relationship with the disease-related indexes of CML were analyzed. Study Results 1. Compared with the normal control, the level of CFL1 expression in CML and AML patients was significantly lower (p0.05, p0.05); AML patients were particularly significant:2. In the same patient, the level of CFL1 expression was increased after imatinib treatment (p0.05);3. The proportion of patients with low expression of CFL1 in CML at 6 months after treatment with imatinib was higher than that of patients with high CFL1 expression (74.2% and 46.9%, p = 0.027, respectively). );4. In patients with lower CFL1 expression in CML, the PFS was superior to those with higher levels of expression: but there was no statistical difference. The expression level of CFL1 was correlated with the platelet count in the peripheral blood of the patients with CML (p = 0.033, p = 0.028). Conclusion 1. The expression level of CFL1 in the diagnosis of CML is related to the state of CML. In the third part, the role of cofin in the treatment of chronic myeloid leukemia (CML) was studied in this paper. The effect of cofin on the treatment of CML was evaluated by the effect of cofin on the treatment of CML. The expression level of cofin protein and the change of F-actin in the patients with chronic myelocytic leukemia cell line K562 and KCL-22 after treatment with imatinib and homoharringtonine (HHT) were detected by the method of square method, the adhesion index of the cells to the fibronectin was changed, the migration force of the cells was changed, and the CFL1 was down-regulated by using the shRNA, The sensitivity of the cells to imatinib was observed; the sensitivity of the cells to imatinib was observed by overexpressing two mutants of the CFL1 wild-type (CFL1) and the CFL1-activated (S3A) and the inactivated (S3D); in the imatinib-resistant cell line K562/ G, CFL1 and S3A, S3D were overexpressed, Changes to the sensitivity of imatinib to imatinib were observed. Results 1. After the action of imatinib and homoharringtonine, the expression of cnoil and P-cofilin in K562 and KCL-22 cell lines was up-regulated, and the formation of F-actin was increased; After the down-regulation of CFL1 expression, the sensitivity of K562 cells to imatinib decreased, and the inhibition of imatinib on the proliferation of the cells was decreased;3. After the expression of CFL1 and S3A, the sensitivity of the K562 cells to imatinib was increased, but the sensitivity of the cells to the treatment was not small after the expression of the S3D, and the sensitivity of the cells to imatinib was increased after the overexpression of CFL1 and S3A in K562/ G cells. In the same way, the sensitivity of the cell to the treatment was not significant after the expression of the S3D. The migration of K562 cells after drug treatment decreased and the adhesion of the cells to fibronectin (FN) increased. Conclusion 1. After the action of imatinib and homoharringtonine, the expression of cofin in K562 and KCL-22 was up-regulated, and the phosphorylation of cofilin increased, and after the phosphorylation of cofilin, the activity of K562 and KCL-22 was reduced, the action of F-actin was weakened, the formation of F-actin was increased, the adhesion of the cells to fibronectin was increased, and the migration force was weakened. The high expression of cofin can increase the sensitivity of CML cell lines to imatinib, and the effect is dependent on the activity of cofin.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.72
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