慢性粒細(xì)胞白血病早期療效預(yù)測及CFL1在其治療中的作用
發(fā)布時間：2019-06-19 01:36
【摘要】：第一部分伊馬替尼治療3月后BCR-ABL轉(zhuǎn)錄本水平對慢性粒細(xì)胞白血病患者預(yù)后及治療反應(yīng)的預(yù)測價值研究目的評估接受伊馬替尼治療的慢性粒細(xì)胞白血病慢性期(CML-CP)患者,治療3個月后BCR-ABL融合基因轉(zhuǎn)錄本水平(以10%為截斷值)在各個亞組患者中對伊馬替尼的治療反應(yīng)及生存的預(yù)測效果,并探討影響CML患者治療3個月能否達到BCR-ABL≤10%的相關(guān)因素。研究方法回顧性分析在我院診斷的CML-CP患者接受伊馬替尼治療3個月、6個月、12個月、18個月獲得的細(xì)胞遺傳學(xué)和分子學(xué)反應(yīng)。將患者依據(jù)有無重組人干擾素(IFN-α)治療史、年齡、診斷到服用伊馬替尼的間隔時間三個因素分層,分析在各個亞組中BCR-ABL不同表達水平患者之間OS、EFS、PFS的差異。并分析患者的基本情況、疾病狀態(tài)、診斷到開始服用伊馬替尼的間隔時間是否影響治療3月后BCR-ABL下降水平。研究結(jié)果(1)研究共納入CML-CP患者299例,其中293例患者接受細(xì)胞遺傳學(xué)或BCR-ABL融合基因分子學(xué)檢測。203例(67.9%)患者隨訪結(jié)束后達完全細(xì)胞遺傳學(xué)緩解(CCyR),其中包括167例(55.9%)獲得主要分子學(xué)緩解(MMR),130例(43.5%)獲得完全分子學(xué)緩解(CMR)。(2)274例患者治療3月后檢測了BCR-ABL水平,BCR-ABL≤10%(n=146,53.3%)的患者OS、EFS、PFS均優(yōu)于BCR-ABL10% (n=128,46.7%)患者。(3)在有IFN-a治療史的患者中,BCR-ABL《10%的患者EFS優(yōu)于10%的患者(P=0.038),但OS與PFS無明顯差異(P=0.276,P=0.229)。(4)在年齡≤29歲的患者中,BCR-ABL《10%的患者PFS.優(yōu)于10%的患者(P=0.046),但OS與EFS并無明顯差異(P=0.223,P=0.176)。(5)在治療間隔時間≤6個月的患者中,BCR-ABL≤10%者OS,EFS,PFS優(yōu)于BCR-ABL10%者(P=0.006,P0.001,P0.001),但在治療間隔時間6個月時,兩組患者EFS和PFS有差異(P=0.007,P=0.004),而OS并無統(tǒng)計學(xué)差異(P=0.072)。(6)在無IFN-α治療史、29歲、治療間隔時間≤6個月的患者中,BCR-ABL《10%組部分細(xì)胞遺傳學(xué)緩解(PCyR)、CCyR、MMR、CMR的累積發(fā)生率均高于BCR-ABL10%組。但在其他患者中,上述各反應(yīng)的累積發(fā)生率并無明顯差異。(7)確診CML到接受伊馬替尼治療的間隔時間為影響治療3月后BCR-ABL是否能達到≤10%的獨立因素(OR=9.854,P0.001)。研究結(jié)論初始治療為伊馬替尼的CML-CP患者接受伊馬替尼治療3月后BCR-ABL的下降水平可預(yù)測患者的OS、PFS和EFS以及最佳治療反應(yīng)。但該預(yù)測價值在有IFN-α治療史、年齡≤29歲及治療間期6月的患者中有限。第二部分CFL1的表達在慢性粒細(xì)胞白血病患者治療早期療效評價中的意義研究目的評價非肌型絲切蛋白基因(CFL1)表達水平與CML患者對伊馬替尼早期治療反應(yīng)及預(yù)后的關(guān)系研究方法用實時定量PCR方法檢測65例初發(fā)慢性粒細(xì)胞白血病患者骨髓單個核細(xì)胞、初發(fā)急性髓系白血病(AML)患者骨髓單個核細(xì)胞及正常對照外周血單個核細(xì)胞中的CFL1表達水平。比較該基因在CML、AML和正常對照之間的表達差別及同一患者治療前和治療后該基因的表達變化。分析該基因表達水平與患者對伊馬替尼早期治療反應(yīng)的關(guān)系,及與CML疾病相關(guān)指標(biāo)之間的關(guān)系。研究結(jié)果1.與正常對照相比,CML與AML患者中的CFL1表達水平明顯降低(p0.05, p0.05); AML患者尤為明顯：2.同一患者經(jīng)伊馬替尼治療后CFL1表達量升高(p0.05)；3.CML診斷時CFL1低表達的患者伊馬替尼治療6個月后達到CCR的比例高于CFL1高表達的患者(分別為74.2%和46.9%,p=0.027。);4.CML診斷時CFL1表達量較低的患者EFS、PFS優(yōu)于表達量較高的患者：但是無統(tǒng)計學(xué)差異。5. logistics回歸模型分析,CFL1的表達水平與CML患者初發(fā)時外周血白細(xì)胞和血小板計數(shù)相關(guān)(p=0.033,p=0.028)。結(jié)論1.CML患者診斷時CFL1的表達水平與CML疾病狀態(tài)有關(guān)；2.CFL1低表達的患者治療早期易達到最佳反應(yīng),其表達水平與患者的預(yù)后關(guān)系尚需進一步研究。第三部分體外實驗研究cofilin在藥物治療慢性粒細(xì)胞白血病中的作用目 的探討非肌型絲切蛋白基因CFL1的表達水平影響CML患者對藥物治療反應(yīng)的相關(guān)機制,評價非肌型絲切蛋白cofilin在CML治療中的作用。方 法檢測慢性粒細(xì)胞白血病細(xì)胞株K562、KCL-22經(jīng)伊馬替尼及高三尖杉酯堿(HHT)治療后cofilin蛋白表達水平及F-actin形成的變化；細(xì)胞對纖黏連蛋白的粘附指數(shù)變化,細(xì)胞的遷移力變化；利用ShRNA下調(diào)CFL1,觀察細(xì)胞對伊馬替尼治療的敏感性變化；過表達CFL1野生型(CFL1)及CFL1激活型(S3A)和失活型(S3D)兩個突變體,觀察細(xì)胞對伊馬替尼治療的敏感性變化；在伊馬替尼耐藥的細(xì)胞株K562/G中過表達CFL1及S3A、S3D,觀察其對伊馬替尼的敏感性的變化。結(jié)果1.經(jīng)伊馬替尼、高三尖杉酯堿作用后,K562、KCL-22細(xì)胞株細(xì)胞中cofilin及P-cofilin表達均上調(diào)；F-actin形成增加；2.下調(diào)CFL1表達后,K562細(xì)胞對伊馬替尼的敏感性降低,伊馬替尼對細(xì)胞的增殖抑制減弱；3.過表達CFL1及S3A后,K562細(xì)胞對伊馬替尼的敏感性增高,但過表達S3D后,細(xì)胞對治療的敏感性變化不大；4.K562/G細(xì)胞中過表達CFL1及S3A后,細(xì)胞對伊馬替尼的敏感性增高。同樣,過表達S3D后,細(xì)胞對治療的敏感性變化不大；5.藥物處理后K562細(xì)胞的遷移力減弱,細(xì)胞對纖黏連蛋白(FN)的粘附力增加。結(jié)論1.伊馬替尼、高三尖杉酯堿作用后,K562、KCL-22細(xì)胞cofilin表達上調(diào),磷酸化cofilin增加,cofilin磷酸化后,其活性減低,對F-actin剪切作用減弱,F-actin形成增加,細(xì)胞對對纖黏連蛋白的粘附力增加,遷移力減弱；2. cofilin高表達可增加CML細(xì)胞株對伊馬替尼的敏感性,且該作用依賴于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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R733.72
本文編號：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R733.72
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