磁珠富集循环骨髓瘤细胞监测MM的MRD和复发的高敏感方法研究
发布时间：2018-10-13 08:50
【摘要】：目的探讨多发性骨髓瘤(MM)患者的循环骨髓瘤细胞(CMCs)数量和髓内瘤细胞(MMCs)的相关性以及CMCs与MM临床相关指标的关系,评价CMCs反映骨髓瘤细胞负荷及病情进展的可行性。建立一套免疫磁珠(IMB)富集、筛选CMCs的方法,用于监测MM的MRD和复发。方法(1)CMCs和MMCs的相关性分析:应用流式细胞术(FCM)检测82例MM患者与30例非浆细胞相关疾病的贫血患者的CMCs和MMCs的比例,探讨两者相关性,分别比较两者在各疗效组中的差异,分析CMCs与MM临床相关指标的关系。(2)用骨髓瘤细胞系U266建立细胞浓度梯度模型,FCM分别检测每个模拟浓度点时抗APC磁珠分选前后的U266细胞比值,即直接FCM组和IMB-FCM组,根据抗APC磁珠靶向抗原CD38-APC/CD138-APC的不同,两组又分为CD38-直接FCM组和CD138-直接FCM组以及CD38-IMB-FCM组和CD138-IMB-FCM组。分别比较IMB-FCM组和相应的直接FCM组U266细胞的比值,确定IMB-FCM检测瘤细胞的敏感度。(3)收集32例MM患者的骨髓和外周血标本,分为骨髓直接FCM组、外周血CD38-直接FCM组、外周血CD38-IMB-FCM组、外周血CD138-直接FCM组、外周血CD138-IMB-FCM组,分别比较外周血IMB-FCM组与相应的外周血/骨髓直接FCM组检测瘤细胞的比值及CMCs的阳性率。结果(1)初发及复发组和PR组的CMCs阳性检出率分别为71.4%、64.5%,两组中CMCs与MMCs比例均呈正相关(P0.05);MMCs和CMCs比例在初发及复发、PR、CR组中依次降低(P0.0083)。MMCs和CMCs在CR组及对照组中均未检测出。(2)与CMCs阴性组相比,CMCs阳性组的β2-MG升高、中度以上贫血、sCrea升高、肾功能异常及骨质破坏的频率明显升高,呈现出较高的DS、ISS分期(P0.05)。经多因素Logistic回归分析,β2-MG升高、中度以上贫血是CMCs存在的独立风险因子(P0.05)。(3)在每个模拟浓度点CD38/CD138-IMB-FCM组检测U266细胞的比例均分别显著高于相应的直接FCM组(P0.05),直接FCM、IMB-FCM检测U266细胞的敏感度分别为0.01%、0.001%。(4)瘤细胞比值在骨髓直接FCM组分别显著高于外周血CD38/CD138-IMB-FCM组,在外周血CD38/CD138-IMB-FCM组分别显著高于相应的外周血-直接FCM组(P0.0167);CD38/CD138-IMB-FCM组检测CMCs阳性率(90.6%,87.5%)分别显著高于相应的直接FCM组(65.6%)(P0.05)。结论(1)CMCs可反映MM患者的肿瘤负荷及病情进展;相比MMCs,检测CMCs某种程度可以替代反复抽取骨髓检测,提高患者依从性。β2-MG升高和中度以上贫血是CMCs存在的独立风险因子。(2)IMB-FCM能提高CMCs的阳性检出率,敏感度比FCM高10倍。
[Abstract]:Objective to investigate the correlation between the number of circulating myeloma cells (CMCs) and intramedullary tumor cell (MMCs) in patients with multiple myeloma (MM), and the relationship between CMCs and MM, and to evaluate the feasibility of CMCs in reflecting the cell load and progression of myeloma. A set of immunomagnetic bead (IMB) enrichment and CMCs screening method was established to monitor the MRD and recurrence of MM. Methods (1) correlation analysis of CMCs and MMCs: the proportion of CMCs and MMCs in 82 patients with MM and 30 anemia patients with non-plasma cell related diseases were detected by flow cytometry (FCM). The relationship between CMCs and clinical parameters of MM was analyzed. (2) the cell concentration gradient model was established by using myeloma cell line U266. FCM was used to detect the ratio of U266 cells before and after the separation of anti-APC beads at each simulated concentration point, that is, direct FCM group and IMB-FCM group. According to the difference of CD38-APC/CD138-APC, the two groups were divided into CD38- direct FCM group, CD138- direct FCM group, CD38-IMB-FCM group and CD138-IMB-FCM group. The ratio of U266 cells in IMB-FCM group and corresponding direct FCM group was compared to determine the sensitivity of IMB-FCM in detecting tumor cells. (3) the bone marrow and peripheral blood samples of 32 patients with MM were collected and divided into three groups: bone marrow direct FCM group, peripheral blood CD38- direct FCM group, peripheral blood CD38-IMB-FCM group. The ratio of tumor cells and the positive rate of CMCs in peripheral blood IMB-FCM group and corresponding peripheral blood / bone marrow direct FCM group were compared between peripheral blood CD138- direct FCM group and peripheral blood CD138-IMB-FCM group. Results (1) the positive rate of CMCs in the primary and recurrent group and PR group was 71.4% and 64.5%, respectively. There was a positive correlation between CMCs and MMCs in the two groups (P0.05). The proportion of); MMCs and CMCs decreased in turn in the PR,CR group (P0.0083). MMCs and CMCs were not detected in CR group and control group). (2) 尾 2-MG in CMCs positive group was higher than that in CMCs negative group. Moderate anemia, sCrea increased, renal dysfunction and bone destruction frequency increased significantly, showing a higher DS,ISS stage (P0.05). By multivariate Logistic regression analysis, 尾 2-MG increased. Moderate anemia is an independent risk factor for CMCs (P0.05). (3). The percentage of U266 cells detected in CD38/CD138-IMB-FCM group was significantly higher than that in direct FCM group at each simulated concentration point (P0.05). The sensitivity of direct FCM,IMB-FCM detection for U266 cells was 0.01and 0.001 respectively (4) tumor cells were detected. The ratio in bone marrow direct FCM group was significantly higher than that in peripheral blood CD38/CD138-IMB-FCM group. The positive rate of CMCs in CD38/CD138-IMB-FCM group was significantly higher than that in peripheral blood direct FCM group (P0.0167), and the positive rate of CMCs in CD38/CD138-IMB-FCM group (90.6%) was significantly higher than that in direct FCM group (65.6%) (P0.05). Conclusion (1) CMCs can reflect the tumor load and disease progression of MM patients, compared with MMCs, detection of CMCs, it can replace repeated bone marrow detection. Increased 尾 2-MG and moderate anemia were independent risk factors for CMCs. (2) IMB-FCM could increase the positive detection rate of CMCs, and the sensitivity was 10 times higher than that of FCM.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.3;R730.43
本文编号：2268000
[Abstract]:Objective to investigate the correlation between the number of circulating myeloma cells (CMCs) and intramedullary tumor cell (MMCs) in patients with multiple myeloma (MM), and the relationship between CMCs and MM, and to evaluate the feasibility of CMCs in reflecting the cell load and progression of myeloma. A set of immunomagnetic bead (IMB) enrichment and CMCs screening method was established to monitor the MRD and recurrence of MM. Methods (1) correlation analysis of CMCs and MMCs: the proportion of CMCs and MMCs in 82 patients with MM and 30 anemia patients with non-plasma cell related diseases were detected by flow cytometry (FCM). The relationship between CMCs and clinical parameters of MM was analyzed. (2) the cell concentration gradient model was established by using myeloma cell line U266. FCM was used to detect the ratio of U266 cells before and after the separation of anti-APC beads at each simulated concentration point, that is, direct FCM group and IMB-FCM group. According to the difference of CD38-APC/CD138-APC, the two groups were divided into CD38- direct FCM group, CD138- direct FCM group, CD38-IMB-FCM group and CD138-IMB-FCM group. The ratio of U266 cells in IMB-FCM group and corresponding direct FCM group was compared to determine the sensitivity of IMB-FCM in detecting tumor cells. (3) the bone marrow and peripheral blood samples of 32 patients with MM were collected and divided into three groups: bone marrow direct FCM group, peripheral blood CD38- direct FCM group, peripheral blood CD38-IMB-FCM group. The ratio of tumor cells and the positive rate of CMCs in peripheral blood IMB-FCM group and corresponding peripheral blood / bone marrow direct FCM group were compared between peripheral blood CD138- direct FCM group and peripheral blood CD138-IMB-FCM group. Results (1) the positive rate of CMCs in the primary and recurrent group and PR group was 71.4% and 64.5%, respectively. There was a positive correlation between CMCs and MMCs in the two groups (P0.05). The proportion of); MMCs and CMCs decreased in turn in the PR,CR group (P0.0083). MMCs and CMCs were not detected in CR group and control group). (2) 尾 2-MG in CMCs positive group was higher than that in CMCs negative group. Moderate anemia, sCrea increased, renal dysfunction and bone destruction frequency increased significantly, showing a higher DS,ISS stage (P0.05). By multivariate Logistic regression analysis, 尾 2-MG increased. Moderate anemia is an independent risk factor for CMCs (P0.05). (3). The percentage of U266 cells detected in CD38/CD138-IMB-FCM group was significantly higher than that in direct FCM group at each simulated concentration point (P0.05). The sensitivity of direct FCM,IMB-FCM detection for U266 cells was 0.01and 0.001 respectively (4) tumor cells were detected. The ratio in bone marrow direct FCM group was significantly higher than that in peripheral blood CD38/CD138-IMB-FCM group. The positive rate of CMCs in CD38/CD138-IMB-FCM group was significantly higher than that in peripheral blood direct FCM group (P0.0167), and the positive rate of CMCs in CD38/CD138-IMB-FCM group (90.6%) was significantly higher than that in direct FCM group (65.6%) (P0.05). Conclusion (1) CMCs can reflect the tumor load and disease progression of MM patients, compared with MMCs, detection of CMCs, it can replace repeated bone marrow detection. Increased 尾 2-MG and moderate anemia were independent risk factors for CMCs. (2) IMB-FCM could increase the positive detection rate of CMCs, and the sensitivity was 10 times higher than that of FCM.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.3;R730.43
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