用PCR分析30例急性淋巴细胞白血病的克隆演化

为了解急性淋巴细胞白血病（ＡＬＬ）免疫球蛋白重链（ＩｇＨ）和Ｔ细胞受体（ＴＣＲ）γ基因重排情况，应用ＰＣＲ技术对３０例急性淋巴细胞白血病初诊时及其中１０例复发后进行研究分析。结果表明：１５例Ｂ－ＡＬＬ中１３例具有ＩｇＨ基因重排，其中３例存在２条重排带，８例合并ＴＣＲγ基因重排；８例Ｔ－ＡＬＬ中６例具有ＴＣＲγ基因重排；３例Ｔ．Ｂ－ＡＬＬ具有ＩｇＨ和ＴＣＲγ基因双重排；４例Ｎｕｌ－ＡＬＬ中２例具有ＴＣＲγ基因重排。１０例复发后，７例具有相应的ＩｇＨ或ＴＣＲγ基因重排，２例丢失ＩｇＨ基因重排，１例获得ＴＣＲγ基因重排。以上事实表明在ＡＬＬ病程中存在白血病细胞克隆演化     

THE ANALYSIS OF GENE REARRANGEMENT OF ACUTE LEUKEMIAS IN DIFFERENT CLASSIFICATION BETWEEN MORPHOLOGY AND IMMMUNOLOGY

为探讨形态学和免疫学分型不一致的急性白血病细胞起源，采用ＰＣＲ技术分析１４例形态学和免疫学分型不一致急性白血病的ＩｇＨ和ＴＣＲγ基因重排。结果发现在９例分别表达Ｂ或Ｔ细胞分化抗原的白血病中，有７例检测到相应的ＩｇＨ或ＴＣＲγ基因重排，２例具有ＩｇＨ和ＴＣＲγ基因双重排；在５例缺乏细胞系限抗原表达的白血病中，有４例检测到ＴＣＲγ基因重排，１例未检测到ＩｇＨ或ＴＣＲγ基因重排，其白血病细胞分别起源于Ｔ细胞和髓细胞。表明基因重排分析在白血病分型中具有重要参考价值     

多重PCR检测急性非淋巴细胞白血病IgH及TCRVγI—Jγ基因重排

目的：为进一步了解急性非淋巴细胞白血病病人基因重排情况。方法：应用多重ＰＣＲ技术检测４１例ＡＮＬＬ病人ＩｇＨ及ＴＣＲＶγＩ－Ｊγ基因重排。结果：２６．８％（１１／４１）ＡＮＬＬ病人存在ＩｇＨ或／和ＴＣＲＶγＩ－Ｊγ基因重排,３例病人同时存在ＩｇＨ和ＴＣＲＶγＩ－Ｊγ基因重排;基因重排阳性ＡＮＬＬ治疗缓解率低于重排阴性ＡＮＬＬ病人。     

单纯表达CD38抗原难分类急性白血病细胞起源的研究

采用单克隆抗体免疫酶和聚合酶链反应（ＰＣＲ）技术，研究７例单纯表达ＣＤ３８抗原难分类急性白血病（ＵＡＬ）化疗前后免疫表型和免疫球蛋白重链（ＩｇＨ）和Ｔ细胞受体（ＴＣＲ）γ基因重排，结果表明：化疗前３例检测到ＴＣＲγ基因重排，化疗后４例表达Ｔ细胞分化抗原，并伴ＴＣＲγ基因重排；１例化疗后表达髓细胞抗原，无ＩｇＨ和ＴＣＲγ基因重排；２例化疗前后仅表达ＣＤ３８抗原，亦无ＩｇＨ和ＴＣＲγ基因重排。提示动态     

克隆性基因重排分析在淋巴结增殖性疾病鉴别诊断中的应用研究

采用体外基因扩增多聚酶链反应（ＰＣＲ），研究了３６例非霍奇金淋巴瘤（ＮＨＬ）及１９例淋巴结反应性增生（ＲＨ）患者的免疫球蛋白重链（ＩｇＨ）和Ｔ细胞受体γ（ＴＣＲγ）基因重排。结果３６例ＮＨＬ中，３４例出现克隆性ＩｇＨ和ＴＣＲγ基因重排（９４．４％），其中９１％的Ｂ－ＮＨＬ出现ＩｇＨ基因重排，９５％的Ｔ－ＮＨＬ出现ＴＣＲγ基因重排。１９例ＲＨ患者，１４例上述基因保持胚系状态，５例检测到克隆性ＩｇＨ或ＴＣＲγ基因重排。此５例随后的临床表现及治疗反应，均支持恶性淋巴瘤的诊断。表明抗原受体基因重排是淋巴瘤分子水平可靠的克隆性标记，有助于在基因水平确定肿瘤细胞的来源及其分化阶段，尤其在良、恶性淋巴结增殖性疾病的鉴别诊断方面，具有重要的临床应用价值。     

急性白血病非系限性分化抗原的动态研究

为探讨仅表达非系限性分化抗原的急性白血病的细胞起源，采用单克隆抗体免疫酶标和聚合酶链反应技术分析了１２例初诊时仅表达非系限性分化抗原的急性白血病化疗后免疫表型及免疫球蛋白重链（ＩｇＨ）和Ｔ细胞受体（ＴＣＲ）γ基因重排的变化。结果表明：初诊时仅表达ＣＤ３８抗原的５例急性白血病，化疗后３例出现Ｔ细胞相关抗原表达，并伴ＴＣＲγ基因重排；５例初诊时仅表达ＨＬＡ－ＤＲ或ＣＤ９的急性白血病，化疗后４例出现Ｂ细胞相关抗原表达，均伴有ＩｇＨ基因重排；２例无任何抗原表达者，化疗后１例表达Ｂ细胞相关抗原，另１例表达骨髓细胞相关抗原。提示免疫标志的动态研究，有助于初诊时免疫学无法分类急性白血病细胞起源的确定。     

半巢式 PCR 联合 Southern 杂交检测白血病免疫球蛋白重链(IgH)基因重排

应用ＰＣＲ方法检测免疫球蛋白重链（ＩｇＨ）基因重排可进行白血病微小残留病（ＭＲＤ）研究，应用半巢式ＰＣＲ法检测白血病患者ＩｇＨＣＤＲ－Ⅲ片段，７３．５％（２５／３４）急性淋巴细胞白血病（ＡＬＬ，简称急淋），７５％（３／４）慢性淋巴细胞白血病（ＣＬＬ，简称慢淋），１５．２％（５／３３）急性非淋巴细胞白血病（ＡＮＬＬ，简称急非淋）和０％（０／１９）慢性粒细胞白血病（简称慢粒）存在ＩｇＨ基因重排，所有阳性病例都通过Ｓｏｕｔｈｅｒｎ杂交加以证实，结果显示①半巢式ＰＣＲ较一次ＰＣＲ不仅灵敏度提高，而且在一定程度上能降低假阴性率。②ＩｇＨ重排并不只局限于Ｂ淋巴细胞白血病，还可出现于部分急非淋病例，其机理有待于进一步探讨。     

原发性脑内淋巴瘤临床病理、免疫组化、基因重排分析

目的：研究脑内原发性恶性淋巴瘤临床病理、免疫且化及免疫性基因重排特征。方法：对６例脑原发性恶性淋巴瘤作了临床病理形态观察和免疫组化分析（Ｓ－Ｐ法）,并用ＰＣＲ技术单轮及半巢式扩增法进行了ＩｇＨ及ＴＣＲ－β基因重排检测。结果：６例皆为非霍奇金Ｂ细胞淋巴瘤。瘤细胞大部分是中心或中心母细胞型,常伴浆样分化,往往围绕血管排列。ＰＣＲ克隆性基因重排,ＩｇＨ单轮法（２／６例）阳性,半巢式（６／６例）阳性,ＴＣ     

中线恶性网织细胞增生症细胞来源的探讨

结合光镜和临床病理资料，对３２例高度可疑中线恶性网织细胞增生症（ＭＭＲ）重点进行免疫表型及基因重排检测的回顾性研究，结果表明：病变内异形淋巴样细胞（ＡＬＣ）ＵＣＨＬ－１表达阳性者２４例（７５％），Ｌ２６表达者ｌ例。刮片组织ＰＣＲ法基因重排检测：ＴＣＲβ基因重排检测阳性者１７例，其中ＵＣＨＬ－ｌ阳性表达者１２例，阴性者５例，ＩｇＨ基因重排检测只１例有Ｌ２６表达者为阳性，其余均为阴性。本结果从基因水平证明ＭＭＲ本质上大多数为结外Ｔ细胞性淋巴瘤，但也可来源于Ｂ淋巴细胞。免疫表型和基因重排检测有互补性。ＭＭＲ由于其细胞成分复杂和病理形态多样，应注意与炎性肉芽组织和未分化癌鉴别。在文内还讨论了常规石蜡切片的刮取组织进行基因重排的意义。     

用PCR检测难分类急性白血病基因重排

用ＰＣＲ检测难分类急性白血病基因重排王鲁群张明珙①宋素芹①马晓星迟翠芳（济南军区总医院，济南２５００３１）采用聚合酶链反应（ＰＣＲ）技术检测１２例形态学和免疫学均难以分类急性白血病（ＵＡＬ）免疫球蛋白重链（ＩｇＨ）和Ｔ细胞受体（ＴＣＲ）γ基因重排，旨...     

血液系统肿瘤患者外周血细胞IgH基因和TCRγ基因的检测及意义

检测各种血液系统肿瘤患者外周血细胞免疫球蛋白重链基因 (IgH )和T细胞受体γ基因 (TCRγ )克隆性重排并探讨其意义。通过多聚酶链式反应 (PCR )方法检测 32例非霍奇金淋巴瘤 (NHL )、 18例急性髓性白血病 (AML )、 2 4例多发性骨髓瘤 (MM )、 8例急性淋巴细胞白血病 (ALL )及 6例慢性淋巴细胞白血病 (CLL )患者外周血细胞IgH及TCRγ克隆性基因重排。结果表明 ,NHL、AML、MM、ALL及CLL患者中IgH克隆性重排率分别为 37 5 0 %、 2 2 2 2 %、 83 33%、 12 5 0 %和 16 6 7% ;TCRγ基因克隆性重排率分别为 6 2 5 0 %、 5 0 0 0 %、 5 4 17%、 5 0 0 0 %及 5 0 0 0 %。在B型、T型NHL中 ,IgH克隆性重排率分别为 31 5 8%及 6 6 6 7% ;TCRγ克隆性重排率分别为 47 37%及 6 6 6 7%。AML中IgH克隆性重排阳性者的初治完全缓解率(CR ) (5 0 0 0 % )与IgH重排阴性的初治CR率 (5 0 0 0 % )无显著差异 (P >0 0 5 )。TCRγ克隆性重排阳性者与阴性者的初治CR率 (均为 44 44 % )亦无显著差异 (P >0 0 5 )。IgH及TCRγ基因克隆性重排不具有细胞谱系的特异性 ,但通过检测外周血IgH、TCRγ克隆性基因重排对NHL有辅助诊断意义 ,并且可作为监测微小残留病壮 (MRD )的手段。     

The application of antigen receptor gene rearrangement of BIOMED-2 in the pathologic diagnosis of 348 cases with non-Hodgkin lymphoma in a single institution in Southwest of China

ObjectiveTo explore the clinical value of immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangement in the diagnosis of non-Hodgkin lymphoma.MethodsUsing the standardized BIOMED-2 multiplex PCR strategy to detect IgH, IgK and TCR in 272 cases of mature B-cell lymphoma, 55 cases of mature T-cell lymphoma, 21 cases of extranodal NK/ T-cell lymphoma, nasal type, and 20 cases of lymphoid tissue reactive hyperplasia.ResultsAmong all mature B-cell lymphomas, the sensitivity of Ig gene rearrangement was 91.18% (248/272), IgH and IgK gene rearrangement was 76.47% (208/272) and 75.00% (204/272), respectively, meanwhile the sensitivity of TCRγ rearrangement was 3.68% (10/272). In the 55 cases of mature T-cell lymphoma, the sensitivity of the detection of TCRγ was 76.36% (44/55), at the same time the sensitivity of Ig gene rearrangement was 14.55% (8/55), IgH and IgK gene rearrangement was 7.27% (4/55) and 12.73% (7/55), respectively. In 21 cases of extranodal NK/T cell lymphoma, nasal type, and 20 cases of reactive lymphoid hyperplasia, no gene rearrangement was found in the samples of IgH, IgK and TCR. The sensitivity of gene rearrangement in Ig/TCR in B and T-cell lymphoma was significantly different from that in the control group (P < 0.05).ConclusionThe Ig/TCR gene rearrangement of BIOMED-2 multiplex PCR strategy has important auxiliary value in the diagnosis of B/T-cell non-Hodgkin lymphoma respectively, however, a few B-cell lymphomas may company TCR gene rearrangement as well as a few T-cell lymphomas may accompany Ig gene rearrangement, it must be comprehensively judged with the combination of morphology, immunohistochemistry and clinical features.     

Frequent T and B cell oligoclones in histologically and immunophenotypically characterized angioimmunoblastic lymphadenopathy

The identification of clonal rearrangements of T cell receptor (TCR) genes is central to the diagnosis of T cell lymphomas. However, in angioimmunoblastic lymphadenopathy (AILD), first described as a nonneoplastic proliferation associated with immunodeficiency, the heterogeneity of TCR and IgH gene rearrangements suggest that some cases may harbor multiple lymphoid clones. In this study we have isolated DNA from archival paraffin biopsy material from 22 cases of AILD identified on the basis of classical histological and immunohistochemical features with the aim of establishing the occurrence of clones and oligoclones, the frequency of TCR and immunoglobulin heavy chain (IgH) variable (v) gene use, and the relationship of these findings to the presence of Epstein-Barr virus. DNA extracted from the biopsies was amplified using the polymerase chain reaction (PCR) and sequenced to detect functional and nonfunctional gene rearrangements. Epstein-Barr virus-encoded short RNA species (EBERs) were detected using in situ hybridization combined with immunochemistry to identify the phenotype of the Epstein-Barr virus-infected cells. Fifty-seven clonal products were found in 20/22 patients: TCRgamma clonal products were identified in 16/22, TCRbeta clonal products in 16/22 and IgH clonal products in 6/22 cases. Oligoclonal PCR products were seen for TCR in 3/22 and for IgH in 3/22 cases. In one biopsy PCR products from all reactions were polyclonal. Sequence analysis revealed functional TCRgamma, TCRbeta, and IgH sequences in 6/12, 9/11, and 8/8 cases, respectively. Functional TCR and/or IgH oligoclones were detected in 6/20 (30%) cases. In addition, nonfunctional TCR and IgH sequences were found in 11 cases. EBERs were identified in 18/20 cases varying from occasional to 25 to 30% nuclei staining and were associated with both T and B cells, although the majority were of indeterminate phenotype. The presence of EBERs was not associated with all clonal IgH gene rearrangements but was associated with B cell oligoclones. Patterns of gene recombinations indicated that the majority of TCRgamma recombinations used GV1 and GJ1S3/2S3 genes. Six out of eleven cases used TCR BV4S1 or BV2S1 genes associated with various BJ and BD1/2 genes. No common IgH gene usage was identified, but 8 clones had varying degrees of replacement and silent mutations (0.6-10.1%), consistent with B cell clones having undergone somatic mutation in the germinal center, and 3 clones harbored unmutated V genes, consistent with naive B cells. Our data do not support the concept of AILD as a clearly defined peripheral T cell lymphoma (PTCL). Rather, they suggest that AILD as defined by histology and immunohistochemistry is either a heterogeneous entity or represents a lymphoproliferation associated with immunodeficiency in which clonal T cell or B cell proliferation may occur.     

IgH AND TcR-γ GENE REARRANGEMENTS IDENTIFIED IN HODGKIN'S DISEASE BY PCR DEMONSTRATE LACK OF CORRELATION BETWEEN GENOTYPE,PHENOTYPE, AND EPSTEIN–BARR VIRUS STATUS

Analysis of IgH and TcR-γ genes using consensus primers identifying junctional regions of rearranged genes by polymerase chain reaction (PCR) was performed on tissues involved by Hodgkin's disease (HD) in 90 cases and was correlated with the immunophenotype of Hodgkin and Reed–Sternberg (HRS) cells and the presence of Epstein–Barr virus (EBV) within these cells. Clonal IgH gene rearrangements were found in 1/5 cases of lymphocyte predominance (LP) subtype and none was positive for EBV. In 85 cases of classic HD, no IgH or TcR-γ gene rearrangements were found in 51 (60 per cent) cases. A similar percentage, but not the same cases, were of null (non-B, non-T) phenotype. Of 30 cases where a B phenotype was assigned to HRS cells, nine had IgH gene rearrangements, three had TcR-γ gene rearrangements, and two had both genes rearranged. None of the five cases assigned to T phenotype of HRS cells showed rearrangement of TcR-γ genes, but two cases showed rearranged IgH genes. Among 41 cases of null phenotype, ten had IgH gene rearrangements, five had TcR-γ gene rearrangements, and three cases had both genes rearranged. Whereas EBV was detectable in HRS cells in 17/43 classic HD cases of assigned B phenotype, EBV was also detectable in 2/5 cases of assigned T phenotype and in 21 cases with the null phenotype. Furthermore, there was no correlation of EBV with the presence or lack of IgH or TCR-γ gene rearrangements. Of the remainder, half (30 per cent) expressed antigens associated with lymphocytes without an appropriate genotype. The results confirm lymphocyte-lineage committed cells at the origin of HRS cells in 40 per cent of cases. Any hypothesis of a non-lymphocytic origin of HRS cells will require the inducibility of CD30 on candidate precursors and the methodology for probing genetic events in such cells to be addressed. © 1997 by John Wiley & Sons, Ltd.     

Hodgkin病单个H/R-S细胞Ig基因重排的研究

目的 探讨Ｈｏｄｇｋｉｎ病（ＨＤ）Ｈｏｄｇｋｉｎ／Ｒｅｅｄ－Ｓｔｅｒｎｂｅｒｇ（Ｈ／Ｒ－Ｓ）。方法 从８例ＨＤ溶冻切片上共提取Ｈ／Ｒ－Ｓ细胞６８个,用ＩｇＨ通用引物ＦＲⅢａ／ＪＨ和κ、λ轻链家族性特性性引物行ＰＣＲ检测。结果 １例淋巴细胞为主型（ＬＰ）ＨＤ的Ｈ／Ｒ－Ｓ细胞重复出现ＩｇＨ和Ｖκ家族重排;２例结节硬化型ＨＤ（ＮＳＨＤ）中,１例Ｈ／Ｒ－Ｓ细胞有单次的ＩｇＨ、Ｖκ４和Ｖλ３重排;１列有重复     

正常人T细胞和胸腺细胞中多种新的TCRδRec基因重排

利用巢式或半巢式PCR扩增10例正常人外周血单个核细胞(PBMC)、4例分选CD3~+细胞和7例正常胸腺细胞DNA中TCR δRec区与Jδ1、Dδ3和Ja重排的基因片段,分析正常人外周血T细胞和胸腺细胞中TCR δRec基因重排情况。克隆性PCR产物进一步进行核苷酸序列分析确定其重排位置。结果发现了4种新的TCR δRec重排,包括TCR δRec_(149321)-Jδ1、TCRδRec_(149820)-Jδ1、TCR δRec_(151657)(Nx)-Ja和TCR δRec_(153199)-Jδ1等,其中以TCR δNx的重排最多见,通过利用不同模板DNA的PCR分析发现δRec重排在外周血和胸腺细胞中有所不同。结果显示TCR δNx-Jδ1重排在成熟和不成熟T细胞发生频率均较高,而TCR δNx-Dδ3重排在不成熟T细胞中发生率较高。但所有重排均不表达于mRNA中。本研究结果为TCR δ基因重排的研究补充了一些新的数据。     

儿童散发性伯基特淋巴瘤的分子遗传学特征分析

目的 研究儿童散发性伯基特淋巴瘤(BL)的分子遗传学特征及其诊断和鉴别诊断.方法 对64例儿童BL和6例儿童弥漫性大B细胞淋巴瘤(DLBCL)进行免疫组织化学染色(SP法)和荧光原位杂交(FISH)技术检测c-myc、bcl-2、bcl-6、IgH、myc/lgH及bcl-2/IgH基因重排的情况.根据细胞起源分类分为生发中心组(GC组)、生发中心晚期组(late-GC组)、生发中心后组(post-GC组).结果 BL表达CD20(64例)、CD10(63例)、bcl-6(62例)、MUM1(15例)、bcl-2(0例).GC组49例(76.6%)、late-GC组14例(21.9%)、post-GC组1例(1.6%).c-myc基因断裂54例(93.1%);IgH基因断裂48例(82.8%);c-myc与IgH基因同时断裂并myc/IgH基因易位46例(85.2%);c-myc基因断裂、IgH和myc/IgH基因正常4例(7.4%);c-myc、IgH和myc/IgH基因均正常4例(7.4%);bcl-2基因正常61例(100%);bcl-6基因正常59例,1例断裂并扩增具有BL的病理形态和免疫表型特征,同时具有c-myc基因断裂,将病理诊断修改为介于DLBCL和BL之间的未分类的B细胞淋巴瘤(DLBCL/BL).6例DLBCL中c-myc基因断裂2例;2例bcl-6基因扩增,其中1例伴c-myc基因断裂;无bcl-2/IgH基因重排.结论 儿童散发性BL大多数来源于生发中心B细胞,c-myc基因的断裂是其主要分子遗传学改变.应用FISH进行多基因的检测,有助于提高儿童BL的诊断和鉴别诊断水平.     

Regulation of immunoglobulin heavy-chain gene rearrangements

Summary: Regulated assembly of antigen receptor gene segments to produce functional genes is a hallmark of B‐ and T‐lymphocyte development. The immunoglobulin heavy‐chain (IgH) and T‐cell receptor β‐chain genes rearrange first in B and T lineages, respectively. Both loci require two recombination events to assemble functional genes; D‐to‐J recombination occurs first followed by V‐to‐DJ recombination. Despite similarities in overall rearrangement patterns, each locus has unique regulatory features. Here, we review the characteristics of IgH gene rearrangements such as developmental timing, deletion versus inversion, D_H gene segment utilization, ordered recombination of V_H gene segments, and feedback inhibition of rearrangement in pre‐B cells. We summarize chromatin structural features of the locus before and during recombination and, wherever possible, incorporate these into working hypotheses for understanding regulation of IgH gene recombination. The picture emerges that the IgH locus is activated in discrete, independently regulated domains. A domain encompassing D_H and J_H gene segments is activated first, within which recombination is initiated. V_H genes are activated subsequently and, in part, by interleukin‐7. These observations lead to a model for feedback inhibition of IgH rearrangements.