多发性骨髓瘤髓外病变患者MYC激活的预后意义及其分子机制探讨

发布时间：2019-06-07 08:25

【摘要】：背景和目的多发性骨髓瘤(MM)是一种恶性血液系统肿瘤,髓外病变(EMD)是其表现形式之一,往往提示预后不良。EMD患者的预后有很大异质性,其发病机制、危险因素并不明确,细胞遗传学异常是目前研究的热点。MYC是重要的原癌基因,基因重排常见于高度侵袭性肿瘤,近年来发现其异常与MM疾病进展相关,可被Muc1、 Pyk2等基因激活,但MYC在EMD和MM预后中的意义目前仍缺乏大宗报道。本研究主要目的：(1)研究MYC异常及蛋白表达水平与EMD的相关性,探讨其在MM中的预后意义。(2)比较EMD及孤立性浆细胞瘤(SP)患者的细胞遗传学异常,探讨浆细胞克隆演变在EMD发生中的作用。(3)利用骨髓瘤细胞系体外研究Pyk2和Muc1对MYC的影响,探究髓外病变中MYC激活的可能机制。方法研究纳入北京协和医院2008年1月至2015年11月初诊的患者。采用病例对照分析方法收集髓外病变(EMD)和非髓外病变(nonEMD)患者,荧光原位杂交(FISH)方法研究骨髓悬液MYC异常；免疫组织化学(IHC)方法研究骨髓病理标本中Myc、Muc1、Pyk2蛋白表达情况。选取EMD患者髓外浆细胞瘤病理标本,以同期经北京协和医院病理科诊断的孤立性浆细胞瘤标本(SP)作为对照,FISH方法研究其MYC异常,1q21扩增、8p21缺失、RB1缺失、D13S319缺失、P53缺失、IGH重排,IHC方法研究Myc、Muc1、Pyk2蛋白表达情况。体外研究采用MM细胞系U266、RPMI8226及浆细胞白血病(PCL)细胞系NCI-H929,研究Myc抑制剂、Pyk2抑制剂和硼替佐米对细胞增殖、凋亡和侵袭性的影响。结果1.对65例nonEMD患者和42例EMD患者的初诊骨髓标本检测MYC重排及Myc、 Muc1和Pyk2蛋白表达情况。两组患者总生存期(OS)存在显著差异(未达vs24月,p=0.021)。MYC重排在nonEMD组和EMD组发生率分别为20.0%和33.3%(p=0.121),在非骨旁EMD中发生率(42.3%) (p=0.029), MYC重排和1q21扩增双阳性在nonEMD组和EMD组发生率分别为6.2%和23.8%(p=0.025)。MYC重排阳性者中位生存期为15.4月,MYC重排阴性者中位生存期未达(p0.001)。单纯1q21扩增或8p21缺失并不显著降低患者生存,但MYC重排合并1q21扩增或8p21缺失中位生存期显著缩短,分别为11.0月(p=0.001)和11.0月(p=0.010)。多因素分析发现MYC重排阳性为MM预后差的独立危险因素(p0.001)。免疫组化结果：新诊断MM患者骨髓Myc高表达在nonEMD组和EMD组分别为15.7%和48.2%(p0.001), Muc1高表达在nonEMD组和EMD组发生率分别为16.3%和35.2%(p=0.030)。在Muc1高表达组Myc表达水平显著升高(34.2%vs 20.8%, p=0.010), Pyk2高表达组Myc表达水平显著升高(29.6%vs18.0,p=0.011)。 Myc蛋白表达与基因异常并不完全吻合。2.对32例EMD及14例SP浆细胞瘤病理组织行FISH检测,EMD中MYC重排(46.7%vs14.3%,p=0.038)、1q21扩增(65.5%vs14.3%, p=0.002)、 8p21缺失(37.9%vs0%, p=0.008)、 RBI缺失(65.5%vs14.3%, p=0.001 )、 D13S319 (58.6%vs21.4%,p=0.022)、 P53缺失(37.9%vs0%,p=0.008))、 IGH重排(48.3%vs 7.1%,p=0.015)发生率均显著高于SP组。大部分EMD患者髓外组织与骨髓组织相比,浆细胞出现新的细胞遗传学异常。Myc蛋白高表达率在EMD组高于SP组(43.3%vsl6.7%),但无显著性差异(P=0.103)。一例MYC阳性的SP组患者最终在两年内进展至活动性骨髓瘤。3.抑制Myc可降低MM细胞的侵袭能力,抑制Pyk2可降低细胞Myc表达水平,降低细胞侵袭能力。结论EMD患者预后不良,但结局有很大异质性。MYC重排在EMD患者中发生率高,是MM预后不良的独立危险因素。EMD细胞遗传学异常的发生率显著高于SP,且髓外病变具有更多的细胞遗传学异常。Myc表达与细胞侵袭性相关,Muc1和Pyk2可能通过激活Myc造成不良预后。
[Abstract]:Background and Objective Multiple myeloma (MM) is a malignant blood system tumor, and extramedullary disease (EMD) is one of its manifestations, often indicating poor prognosis. The prognosis of EMD patients is very heterogeneous, and its pathogenesis and risk factors are not clear, and the cytogenetic abnormality is the hot spot of the present study. MYC is an important proto-oncogene, and gene rearrangement is common in highly invasive tumors. In recent years, it has been found that its abnormality is related to the progression of MM disease, and can be activated by the genes such as Muco1, Pyk2, etc., but the significance of MYC in the prognosis of EMD and MM is still short of mass report. The main purpose of this study was to study the relationship between the level of MYC and the level of protein expression and EMD, and to explore its prognostic significance in MM. (2) The cytogenetic abnormality of the patients with EMD and isolated plasma cell tumor (SP) was compared, and the role of plasma cell clone evolution in the generation of EMD was discussed. (3) The possible mechanism of MYC activation in extramedullary disease was investigated by using myeloma cell line to study the effect of Py2 and Muc1 on MYC in vitro. Methods The patients who were first diagnosed between January 2008 and November 2015 were included in the study. Myc, Muc1 and Py2 protein expression in the bone marrow specimens were studied by means of a case-control analysis method in patients with extramedullary disease (EMD) and non-extramedullary disease (nonEMD) and in situ hybridization (FISH). The pathological specimens of the extramedullary plasmacytoma of the EMD patients were selected as control and the isolated plasma cell tumor specimens (SP) diagnosed by the pathology department of Peking Union and the hospital were used as control and the FISH method was used to study the MYC abnormality, 1q21 amplification, 8p21 deletion, RB1 deletion, D13S319 deletion, P53 deletion, IGH rearrangement and IHC method to study Myc. Muc1, Py2 protein expression. The effects of Myc inhibitor, Py2 inhibitor and bortezomib on cell proliferation, apoptosis and invasiveness were investigated in vitro using the MM cell line U266, the RPMI8226 and the plasma cell leukemia (PCL) cell line NCI-H929. Results 1. MYC rearrangement and the expression of Myc, Muc1 and Py2 were detected in 65 nonEMD patients and 42 patients with EMD. There was a significant difference in overall survival (OS) between the two groups (not up to vs24, p = 0.021). The incidence of MYC rearrangement in nonEMD and EMD groups was 20.0% and 33.3% (p = 0.121), respectively. The incidence of MYC rearrangements (42.3%) (p = 0.029), MYC rearrangement, and 1 q21 amplification of double-positive in nonEMD and EMD groups was 6.2% and 23.8% (p = 0.025), respectively. The median survival time of MYC rearrangement was 15.4 months. The median survival of the MYC rearrangement negative was not reached (p0.001). The simple 1q21 amplification or 8p21 deletion did not significantly reduce the survival of the patient, but the median survival of the MYC rearrangement combined with the 1q21 amplification or the 8p21 deletion was significantly shortened, respectively (p = 0.001) and 11.0 months (p = 0.010). The multi-factor analysis found that MYC rearrangement was an independent risk factor for the poor prognosis of MM (p0.001). The results showed that the high expression of Myc in bone marrow of newly diagnosed MM patients was 15.7% and 48.2% (p0.001) in nonEMD and EMD, 16.3% and 35.2% (p = 0.030) in non-EMD and EMD groups, respectively. The expression of Myc in the high-expression group of the Muc1 was significantly higher (34.2% vs. 20.8%, p = 0.010), and the expression of Myc in the high-expression group of Py2 was significantly higher (29.6% vs18.0, p = 0.011). Myc protein expression and gene abnormality did not match completely. 32 cases of EMD and 14 cases of SP plasmacytoma were detected by FISH, MYC rearrangement in EMD (46.7% vs14.3%, p = 0.038), 1q21 amplification (65.5% vs14.3%, p = 0.002), 8p21 deletion (37.9% vs0%, p = 0.008), RBI deletion (65.5% vs14.3%, p = 0.001), D13S319 (58.6% vs23.4%, p = 0.022), P53 deletion (37.9% vs0%, p = 0.008), IGH rearrangement (48.3% vs. 7.1%, The p = 0.015) incidence was significantly higher in the SP group. In most of the EMD patients, there were new cytogenetic abnormalities in plasma cells compared to the bone marrow tissue. The high expression rate of Myc protein was higher in the EMD group than in the SP group (43.3% vsl6.7%), but there was no significant difference (P = 0.103). One case of a MYC-positive SP group eventually progressed to active myeloma within two years. Inhibiting Myc can reduce the invasion ability of MM cells, and inhibit Pyk2 to lower the expression level of the cell Myc and reduce the cell invasion ability. Conclusion The prognosis of EMD is poor, but the outcome is very heterogeneous. The high incidence of MYC rearrangement in patients with EMD is an independent risk factor for the poor prognosis of MM. The incidence of cytogenetic abnormalities was significantly higher in the EMD than in the SP, and more cytogenetic abnormalities were found in the extramedullary lesions. Myc expression is associated with cell invasiveness, and Muc1 and Pyk2 may cause poor prognosis by activating Myc.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.3

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