荧光原位杂交检测遗传学的异常与套细胞淋巴瘤患者预后关系

发布时间：2018-05-18 10:12

本文选题：套细胞淋巴瘤 + P53缺失　；参考：《北京协和医学院》2016年硕士论文

【摘要】：背景：套细胞淋巴瘤是一种典型的非霍奇金淋巴瘤,约占非霍奇金淋巴瘤淋巴瘤病患的6%。套细胞淋巴瘤作为一种难治疗,不易治愈的疾病。中位生存约仅3年。目前随着新的检测技术的出现及应用,人们对MCL淋巴瘤的发病机制有着更深的认识及理解。套细胞淋巴瘤是B细胞淋巴瘤的一种亚类,由于来源于生发中心前的B细胞,所以表达细胞表面抗原CD5, CD20等细胞表面标志,该区细胞来源位置存在于正常滤泡的生发中心。套细胞淋巴瘤由于染色体11和14号的异位,导致过度表达cyclinD 1蛋白。染色体的具体异位位置存在于t(11；14)(q13；q32)。而荧光原位杂交技术作为一种细胞遗传学技术,使用荧光探针检测大片段基因的缺失。目的：通过荧光原位杂交的方法分析患者的TP53缺失对侵袭性套细胞淋巴瘤患者预后的影响,并且研究侵袭性套细胞淋巴瘤患者P53缺失发生的同时,探索荧光原位杂交检测其它位点的生物学特性在病人预后的意义。方法：回顾分析中国医学科学院血液病医院2003年7月至2015年1月间50例伴外周血及骨髓侵犯的套细胞淋巴瘤患者资料。并且使用荧光原位杂交的方法检测患者D13S25/13q14、ATM/11q22、p53/17p13、c-MYC/8q24、BCL2/18q21和IGH/CCND1/t(11;14)共6种DNA探针,分析遗传学相关性及预后的影响。结果：患者的中位年龄为55.5岁,其中38例患者为男性,18例患者伴有B症状,36例(72%)患者在初诊时伴有脾大症状。中位白细胞(WBC)计数为44.73(2.63-193.78)×109/L,中位82微球蛋白为4.45(1.95 to12.7)mg/L。基于MCL国际预后积分(MIPI)系统,26例(52%)患者为高危组,中危组和低危组各12例患者(24%)。通过SPSS单变量分析PFS和OS预后因素与各检测位点的关系后发现,De113q、 De117p、MYC扩增或者获得都对PFS和OS具有统计学意义,其中Del 13q对PFS和OS的P值结果分别是0.003,0.012,Del 17p对PFS和OS的P值分别是0.000,0.000,MYC扩增或者获得对PFS和OS的P值影响分别是0.000,0.000：多变量分析各因素与PFS和OS的关系发现Del 17、MYC扩增或者获得这两个因素对其具有独立的预后意义。其中Del 17的PFS和OS P值分别是0.039,0.045；MYC获得和扩增对PFS和OS的P值分别是0.020,0.026。结论：P53缺失在套细胞淋巴瘤患者中作为常见的细胞突变,在患者预后中对PFS和OS具有独立预后因素。在成熟B细胞淋巴瘤中,MYCW及BCL2重排的发生率及其预后意义己被深入研究,但在发病率较低的套细胞淋巴瘤(MCL)中研究尚少。本研究在50例伴有骨髓侵犯的MCL患者中应用英光原位杂交(FISH)技术检测了MYC、BCL2及其他细胞遗传学异常,结果显示18例患者G6%)伴有MYC获得和/或扩增,12例患者(24.0%)伴有BCL2获得和/或扩增。在18例伴有MYC异常的患者中有4例同时伴有MYC易位,但在伴有BCL2遗传学异常的患者中并未检测到BCL2易位。仅有2例患者(4.0%)同时伴有MYC和BCL2异常。合并MYC异常的患者其肿瘤负荷显著增高,并且相较于不伴MYC异常的患者而言,其MIPI中高危组患者比例更高,遗传学不稳定性也更高。但伴有BCL2异常的患者其临床特征或细胞遗传学特征相较于不伴该异常的患者并无统计学差异。伴MYC异常的患者其无进展生存期(PFS)和总体生存期(OS)均显著缩短(PFS9.0VS.48.0个月,P=000;OS 12.0 VS.94.5个月,p=.000),但BCL2异常对PFS和OS的影响并无统计学意义。多因素分析显示,MYC异常是PFS和OS的独立预后不良因素,并且强化治疗方案并不能改善这部分患者预后。因此,在MCL患者中MYC异常为预后不良因素,而伴有BCL2异常与不良预后并无相关性,对这部分患者亟待新的治疗方式病人情况和实验方法:2003年6月至2015年1月,50例伴有骨髓侵犯的MCL淋巴瘤患者就诊于中国医学科学院北京协和医院血液病医院。所有入组患者均知情同意,该项目同时被审查委员会批准。该研究中基本的病例记录资料包括性别,年龄,体能状况,Ann Arbor分期,外周淋己结所在区域,染色体核型,骨髓和外周血形态,细胞免疫表型,乳酸脱氨酶水平,W及治疗方案和临床疗效。病理组织样品的评估由两位位病理医师(同时也是本文中的作者)依据WHO分类明确诊断。所有骨髓组织标本均取至患者初诊时。此项研究由中国医学科学院北京协和医学院的伦理委员会支持。荧光原位杂交(FISH)通过FISH分析样品的常规核型。DNA探针的组合包括探针13ql4.3(LSI D13S25和RB-1),14q32(LSIIGHC/IGHV),17pl3(LSIp53),llq22(LSIATM),LSI BCL2(18q32)和MYC(8q24),为双色,双分离重排探针,而IGH/BCL2为双色双融合异位探针,并且LSI IGH/MYC/CEP8为3色双融合探针,CCND1/IGH为双色双融合异位探针。所有的探针从美国的Vysis购置。根据生产厂家推荐完成样品准备及前期处理PS1。信号的检出至少根据200个细胞信号得出。阳性cutoff值(正常对照的平均值+3标准差)来自10个遗传学正常的人的样品,其中Rb-1,ATM和PW的界值为6.5%;IG比BCL2和CCND1/IGH异位双色双融合探针的为4.5%;而LSIIGH,MYC,CEP8H色双融合探针的也为4.5%。拷贝数获得定义为本研究中的基因具有S个拷贝,然而拷贝数的扩增被定义为至少4个拷贝。
[Abstract]:Background: cell lymphoma is a typical non Hodgkin lymphoma, which accounts for the 6%. cell lymphoma of non Hodgkin lymphoma's lymphoma as a refractory, untreatable disease. The median survival is about 3 years. With the emergence and application of new detection techniques, the pathogenesis of MCL lymphoma is deeper. The cell lymphoma is a subclass of B cell lymphoma, which is derived from the B cells before the germinal center, so it expresses the surface antigen of the cell surface antigen CD5, CD20 and other cell surface markers. The cell origin of this area exists in the germinal center of the normal follicular. The specific location of cyclinD 1 protein is expressed. The specific location of chromosomes exists in t (11; 14) (q13; q32). Fluorescence in situ hybridization is used as a cytogenetic technique to detect the deletion of large fragment genes by fluorescence probe. Objective: to analyze the deletion of TP53 in patients with invasive cell lymphoma by fluorescence in situ hybridization. The effect of the prognosis and the study of the P53 deletion in patients with invasive nested cell lymphoma, and the significance of exploring the biological characteristics of other loci by fluorescence in situ hybridization in the patient's prognosis. Methods: a retrospective analysis of 50 cases of peripheral blood and bone marrow invasion from July 2003 to January 2015 of the Chinese Academy of Medical Sciences. D13S25/13q14, ATM/11q22, p53/17p13, c-MYC/8q24, BCL2/18q21 and IGH/CCND1/t (11; 14) were used to detect the effects of 6 kinds of DNA probes on genetic correlation and prognosis. Results: the median age of patients was 55.5 years old, of which 38 were male and 18 patients were accompanied with B. Symptoms, 36 (72%) patients had splenomegaly symptoms at first visit. Median leukocyte (WBC) count was 44.73 (2.63-193.78) x 109/L, median 82 microglobulin was 4.45 (1.95 to12.7) mg/L. based on MCL international prognostic integral (MIPI) system, 26 (52%) patients were at high risk group, and 12 patients in middle and low risk groups (24%). PFS and OS were analyzed by SPSS univariate. After the relationship between the prognostic factors and the detection sites, it is found that De113q, De117p, MYC amplification or OS have statistical significance for PFS and OS, in which the P values of Del 13q are 0.003,0.012 on PFS and OS, respectively. The quantitative analysis of the factors associated with PFS and OS found that Del 17, MYC amplification or acquisition of these two factors had independent prognostic significance. The PFS and OS P values of Del 17 were 0.039,0.045, respectively, and MYC obtained and amplified for PFS and OS as common cells in patients with cell lymphoma. Mutations have independent prognostic factors for PFS and OS in patient's prognosis. The incidence of MYCW and BCL2 rearrangement in mature B cell lymphoma and its prognostic significance have been studied, but few studies have been made in the low incidence of nested cell lymphoma (MCL). In this study, 50 cases of MCL patients with bone marrow invasion (FISH) were used in this study. MYC, BCL2, and other cytogenetic abnormalities were detected in 18 patients with MYC acquisition and / or amplification. 12 patients (24%) were accompanied by BCL2 acquisition and / or amplification. In 18 patients with MYC abnormalities, 4 were accompanied by MYC translocation, but no BCL2 translocation was detected in patients with BCL2 abnormality. Only 2 were detected. Patients (4%) were accompanied by MYC and BCL2 abnormalities. The tumor load in patients with MYC abnormalities was significantly higher, and compared to those with no MYC abnormalities, the high risk groups in MIPI were higher in proportion and higher in genetic instability. There was no statistically significant difference in the patient's patients with MYC abnormalities (PFS) and overall survival (OS) significantly shortened (PFS9.0VS.48.0 months, P=000; OS 12 VS.94.5 months, p=.000), but the effect of BCL2 abnormality on PFS and OS was not statistically significant. And strengthening the treatment regimen does not improve the prognosis of this part of the patient. Therefore, in MCL patients, MYC is abnormal as a prognostic factor, and there is no correlation between abnormal BCL2 and poor prognosis. In this part of the patients, new treatment methods and experimental methods are urgently needed: 50 cases of MCL lymphoma with bone marrow invasion from June 2003 to January 2015. The patients were admitted to the hospital of Hematology in Peking Union Medical College Hospital of the Chinese Academy of Medical Sciences. All the patients were informed consent. The project was approved by the review committee. The basic case records included sex, age, physical status, Ann Arbor staging, the area of the peripheral gage, chromosome karyotype, bone marrow and peripheral blood. State, cellular immunophenotype, lactate deaminase level, W and therapeutic regimen and clinical efficacy. The evaluation of pathological tissue samples was made by two position pathologists (and the author in this article) based on WHO classification. All bone marrow specimens were taken to the initial diagnosis of the patients. This study was conducted by the Beijing Academy of Medical Sciences of the Chinese Academy of Medical Sciences. The ethics committee supports the combination of fluorescent in situ hybridization (FISH) through FISH analysis of the conventional karyotype.DNA probes, including probe 13ql4.3 (LSI D13S25 and RB-1), 14q32 (LSIIGHC/IGHV), 17pl3 (LSIp53), llq22, double separation and rearrangement probes. I IGH/MYC/CEP8 is a 3 color dual fusion probe, and CCND1/IGH is a dual color dual fusion probe. All probes are purchased from the Vysis in the United States. The detection of PS1. signals for sample preparation and preprocessing according to the manufacturer's recommendation is at least according to 200 cell signals. The positive cutoff value (the average value of the normal control, +3 standard deviation) comes from 10 heredity. Samples of normal people, of which the boundary value of Rb-1, ATM and PW is 6.5%; IG is 4.5% for the heterotopic double color dual fusion probe of BCL2 and CCND1/IGH; while LSIIGH, MYC, and CEP8H dual fusion probes are also defined as S copies of the genes in this study, but the amplification of copies is defined as at least 4 copies.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.1

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