分子生物学技术在MALT淋巴瘤诊断及鉴别诊断中的应用

目的研究免疫球蛋白重链基因重排及融合基因API2-MALT1在MALT淋巴瘤诊断和鉴别诊断中的作用。方法采用RT—PCR方法对60例MLAT淋巴瘤，对照组30例结外弥漫性大B细胞淋巴瘤和30例淋巴结反应性增生病例石蜡包埋组织中API2-MALT1进行检测，以看家基因PGK作为内对照检测cDNA质量；采用半嵌套式PCR方法，对60例MALT淋巴瘤，对照组30例淋巴结反应性增生病例石蜡包埋组织中B细胞免疫球蛋白重链进行扩增，以看家基因B-actin作为内对照检测基因组DNA质量。结果60例MALT淋巴瘤中42例检出B细胞免疫球蛋白重链基因重排，去除B-actin DNA阴性4例，MALT淋巴瘤中免疫球蛋白重链基因重排检出率75．0％(42／56)。对照组30例弥漫性大B细胞淋巴瘤重排检出率90．3％(23／30)，反应性增生未检出重排。60例MALT淋巴瘤中6例检出API2-MALT1 mRNA表达，去除PGK阴性病例7例，MALT淋巴瘤中API2-MALT1 mRNA阳性率10．1％(6／53)。所有对照组未检出API2-MALT1 mRNA表达。结论半嵌套式PCR技术在石蜡包埋组织中检测B细胞免疫球蛋白重链基因重排可用于MALT淋巴瘤和淋巴组织反应性增生的鉴别诊断。RT-PCR技术在石蜡包埋组织中检测API2-MALT1 mRNA阳性率较低，但特异性强，可用于MALT淋巴瘤的鉴别诊断。     

石蜡包埋B细胞淋巴瘤组织克隆性IgH基因重排的检测

目的：探讨克隆性免疫球蛋白重链第三互补决定簇区（IgHCD43）基因重排在B细胞非霍奇金淋巴瘤（B－NHL）诊断方面的价值。方法：采用半巢式PCR、聚丙烯酰胺凝胶电泳（PAGE）及银染技术,检测38例B－NHL、4例T－NHL及10例慢性扁桃腺炎,经福尔马林固定、石蜡包埋病理组织的克隆性IgHCDR3重排。结果：29例B－NHL及1例免疫组化未能明确细胞来源的NHL克隆性IgHCDR3重排阳性;4例T－NHL及10例慢性扁桃腺炎克隆性IgHCDR3重排阴性。结论：克隆性IgHCDR3重排可作为B－NHL与反应性增生鉴别的特生标志,大部分情况下可作为系分化标志。     

实时定量聚合酶链反应检测石蜡包埋B细胞淋巴瘤组织克隆性IgH基因重排

我们利用SYBR Green Ⅰ荧光染料，应用实时定量PCR（RQ-PCR）检测了15例B细胞非霍奇金淋巴瘤（B-NHL）患者免疫球蛋白重链（IgH）基因重排情况，以探讨克隆性IgH基因重排在B-NHL诊断和鉴别诊断上的价值。     

CD45RO阳性的B细胞性淋巴瘤

目的 研究免疫表型异常淋巴瘤的起源。方法 从48例B细胞淋巴瘤中筛选出3例CD45RO、CD20和CD79a( )，CD3(-)的淋巴瘤。采用免疫组化、PCR和基因扫描技术对其免疫表型，免疫球蛋白重链基因重排(IgH)和T细胞受体基因重排(TCR)进行深入研究。结果 3例淋巴瘤均为结外淋巴瘤。1例为滤泡性淋巴瘤，2例为弥漫性大B细胞性淋巴瘤。PCR和基因扫描显示3例均有IgH克隆性扩增；PCR未显示TCR扩增；高敏感性基因扫描显示低峰值TCR克隆性扩增，提示可能为背景T细胞扩增。结论 CD45RO不是T细胞特异性抗原，CD45RO阳性细胞不能完全等同于T细胞，因此，在研究和临床病理诊断中应选用多种抗体配合使用。     

地戈辛和生物素标记进行原位PCR方法的比较

由于不同的B细胞克隆可发生不同类型的重链基因重排 ,因此 ,通过检测免疫球蛋白重链基因重排可鉴别细胞的来源 ,也可诊断B系淋巴细胞性肿瘤。本文介绍应用免疫球蛋白重链第三互补决定区引物 ,分别用生物素和地戈辛标记进行原位PCR ,检测了 15例恶性淋巴瘤患者的外周血淋巴细胞     

BIOMED-2标准化基因重排检测在非霍奇金淋巴瘤诊断中的意义

目的:利用BIOMED-2标准化免疫球蛋白(Ig)/T细胞受体(TCR)基因重排检测非霍奇金淋巴瘤(NHL)患者骨髓Ig或TCR基因重排,探讨Ig/TCR基因重排在非霍奇金淋巴瘤诊断中的应用意义。方法:从骨髓及石蜡包埋组织(FFPE)标本中提取基因组DNA,采用BIOMED-2系统引物,进行多重PCR扩增并利用PCR片段分析法进行Ig/TCR基因重排的克隆性分析。结果:在235例T细胞非霍奇金淋巴瘤(T-NHL)中,TCRγ和TCRx单克隆重排阳性率分别为57.9%和50.2%,TCRγ和TCRβ的联合检出率为71.9%。在583例B细胞非霍奇金淋巴瘤(BNHL)IgH和IgK单克隆重排阳性率分别为70.7%和69.3%,IgH和IgK的联合检出率为81.6%。套细胞淋巴瘤与滤泡淋巴瘤、弥漫大B细胞淋巴瘤IgH(84.8%对34.0%(P0.001);84.8%对9.2%(P=0.025)和IgK(75.8%vs 50.9%,P0.001;75.8%vs 16.1%(P0.001)重排阳性率差异具有统计学意义。Ig基因重排阳性BNHL患者中,65例(13.7%)患者存在TCR重排阳性。TCR重排阳性T-NHL患者中,未见Ig基因重排阳性。30例弥漫大B细胞淋巴瘤患者FFPE标本有25例(83.3%)检出Ig基因重排,与骨髓标本重排检出率相比较,差异具有统计学意义(P0.001)。结论:利用BIOMED-2标准化Ig/TCR基因重排检测对于淋巴瘤的诊断有辅助性作用,并且利用序列分析法可提高重排检测的敏感性和特异性,对于淋巴瘤的早期诊断有很高的价值。     

BIOMED-2标准化免疫球蛋白基因重排技术在石蜡包埋弥漫大B细胞淋巴瘤组织中的应用

目的探讨石蜡包埋组织标本应用BIOMED-2标准化免疫球蛋白(IG)基因重排技术检测弥漫大B细胞淋巴瘤(DLBCL)的可行性。方法从45例石蜡包埋组织标本中提取DNA,采用BIOMED-2系统引物进行多重聚合酶链反应(PCR)扩增,并应用PCR片段分析法进行IG基因重排的克隆性分析。结果将DNA由原浓度100~500 ng/μL稀释至50~100 ng/μL后,DNA扩增最大片段(300~400 bp)含量由10.0%提高到90.0%;45例DLBCL石蜡切片标本提取DNA进行免疫球蛋白重链基因(IGH)和免疫球蛋白kappa基因(IGK)克隆性分析,IGH+IGK阳性率达到84.4%;15例反应性淋巴组织增生标本进行IG/T淋巴细胞抗原受体(TCR)克隆性分析,未见克隆性重排。结论稀释DNA是唯一可以既提高DNA扩增最大片段又能提高克隆性检出率的方法。在DLBCL的诊断和淋巴组织增生性疾病的鉴别诊断中,BIOMED-2标准化体系检测IGH基因克隆性重排是一种快速、可靠的方法,具有重要的临床应用价值。     

抗原受体基因重排克隆性检测在淋巴瘤诊断中的应用

本研究旨在探讨抗原受体基因重排克隆性检测在淋巴瘤诊断中的意义。收集分析了31例淋巴瘤组织,石蜡包埋切片,HE染色后观察形态,免疫组织化学分析免疫表型。采用BIOMED-2标准化试剂盒检测抗原受体基因重排克隆性。结果表明,31例病例中形态学及免疫组织化学分析疑似T细胞淋巴瘤12例,T细胞反应性增生1例,B细胞淋巴瘤16例,B细胞反应性增生2例。基因重排克隆性检测免疫球蛋白(Ig)阳性率94.44%(17/18),T细胞抗原受体(TCR)阳性率92.31%(12/13),2例阴性。最终12例确诊为T细胞淋巴瘤,B细胞淋巴瘤17例,反应性增生2例,阳性检出率为93%。结论:抗原受体重排基因克隆性分析是诊断淋巴瘤的一种有效辅助手段。     

免疫球蛋白基因重排分析在淋巴瘤诊断中的初步应用研究

用免疫球蛋白重链Ｊ区（ＪＨ），Ｋ轻链及λ轻链Ｃ区（ＣＫ，Ｃλ２）基因探针及Ｓｏｕｔｈｅｒｎ印迹杂交技术，对２２例非何杰金氏淋巴瘤进行分析。结果显示１３例尿Ｂ细胞淋巴瘤均出现重链基因重排，其中１０例有Ｋ或λ轻链基因重排；３例Ｔ细胞淋巴瘤中，１例出现重链，１例出现Ｋ轻链基因重排。１例分类未明淋巴瘤出现重链基因重排。５例未作免疫表型分析的淋巴瘤中有４例出一现重链，３例出现Ｋ轻链，１例出现λ轻链基因重排。     

蛋白和基因水平探索H/RS细胞的起源

目的：从蛋白、组织和单细胞的基因水平进一步探索H／RS细胞的来源及其克隆性。方法首先对33例经典型霍奇金淋巴瘤(cHL)标本进行B细胞特异性激活蛋白(BSAP)和CD20的检测，然后对33例cHL患的石蜡刮片组织和部分阳性病例的微切割细胞进行免疫球蛋白重链基因克隆性重排检测，并对同一病例的石蜡刮片组织和微切割细胞的扩增产物进行测序分析比较。结果33例中30例的H／RS细胞表达BSAP，10例表达CD20，BSAP和CD20的表达率差异有显性(P=0.000)，对照病例中反应性增生淋巴结的B淋巴细胞和B细胞淋巴瘤肿瘤细胞的BSAP和CD20表达均为100％，T细胞淋巴瘤的肿瘤细胞无表达；33例中的16例石蜡刮片组织IgH基因重排阳性，微切割的19管H／RS细胞有14管出现重排阳性，细胞数目不同的各管阳性率差异无显性(P=0．280)；同一病例的石蜡刮片组织和微切割细胞的PCR产物测序结果均为IgH可变区片段，但是碱基序列并不完全相同。结论：进一步支持cHL中大多数H／RS细胞为B细胞起源，且可能来源于其不同的分化阶段。     

异源双链鉴定法检测淋巴瘤IgH、TCR-β基因重排

目的建立更敏感、特异性更强的PCR方法检测淋巴瘤IgH和，TCR-β基因重排。方法采用标准PCR方法以及异源双链鉴定的改良方法检测55例淋巴瘤组织，全部病例均经组织学明确诊断，其中36例为B细胞性淋巴瘤，19例为T细胞性淋巴瘤。对照为5例反应性增生的淋巴结组织。结果用改良PCR方法检测淋巴瘤组IgH和TCR-β基因重排49／55例为阳性，较标准PCR方法电泳条带更清晰、更易于判定；5例反应性增生的淋巴结组织均为阴性。结论异源双链鉴定的改良方法提高了检测淋巴组织肿瘤性增生的敏感性和特异性，值得推广。     

The frequency of immunoglobulin heavy chain gene and T-cell receptor gamma-chain gene rearrangements and Epstein-Barr virus in ALK+ and ALK- anaplastic large cell lymphoma and other peripheral T-cell lymphomas

We previously identified a relatively high frequency of B-cell proliferations along with simultaneous T-cell receptor gamma-chain gene (TRG) and immunoglobulin heavy chain gene (IGH) rearrangements in a series of angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, unspecified. Here, we report on a series of 74 peripheral T-cell lymphoma (PTCL) cases composed entirely of specific PTCL subtypes, including 28 cases of ALK+ anaplastic large-cell lymphoma (ALCL), 35 cases of ALK- ALCL, and 11 cases that represent other specific PTCL subtypes. We performed IGH and TRG gene rearrangement studies and in situ hybridization for Epstein-Barr virus (EBV) to determine the frequency of IGH clonality and to investigate the relationship between EBV, clonality, and associated B-cell proliferations. Using BIOMED-2 PCR assays, we detected TRG clones in 64 of 74 (86%) cases and IGH clones in 6 of 74 (8%) cases, with all IGH-positive cases exhibiting a concurrent TRG clone. Despite the detection of occasional IGH clones, there was no correlation between IGH clonality and EBV, and B-cell proliferations were not identified in any of the cases. These findings suggest that other factors contribute to IGH clonality and demonstrate that, in the absence of an associated B-cell proliferation, IGH clonality occurs infrequently (8%) in specific PTCL subtypes.     

Enhanced sensitivity with a novel TCRgamma PCR assay for clonality studies in 569 formalin-fixed, paraffin-embedded (FFPE) cases.

BACKGROUND: Clonal rearrangement of genes encoding the immunoglobulins (Ig) and T-cell antigen receptors (TCR) are considered to be useful markers for the diagnosis of lymphoma and for determining the clonal origins of B- and T-cell populations in lymphoid neoplasms. METHODS AND RESULTS: Polymerase chain reaction-based clonality assays for TCRgamma, TCRbeta, and immunoglobulin (Ig) heavy chain (IgH) gene rearrangements were evaluated for diagnostic sensitivity and specificity in 569 formalin-fixed, paraffin-embedded (FFPE) tissues. Combined TCRbeta and TCRgamma assays enhanced the routine detection of TCR clonality to 90% of all peripheral T-cell lymphoma (PTCL) cases. IgH clonality was detected in 59% of 241 peripheral B-cell lymphoma (BCL) cases and 6% of 169 PTCL cases. Of 452 lymphomas, 5% could not be classified phenotypically as B or T lineage after immunohistochemical and clonality studies. Of all BCL cases analyzed, 24% had detectable TCRbeta and/or TCRgamma clonality. Of these BCL with biclonal results, 47% were extranodal lymphomas from skin and various tissues. CONCLUSIONS: Clonality assays were useful for distinguishing reactive or benign lymph nodes from neoplastic lymphoid infiltrates in most cases. The inclusion of TCRb and TCRg assays in the assessment of lymphomas results in a significant increase in the sensitivity of clonality detection, but is of limited utility in assessing the T- or B-cell phenotype of the tumor.     

B-NHL患者骨髓和外周血细胞克隆性IgH基因重排的检测及临床意义

为探讨骨髓(BM)和外周血(PB)细胞克隆性免疫球蛋白重链(IgH)基因重排在B细胞非霍奇金淋巴瘤(B-NHL)分期、疗效及预后判断方面的价值,采用半巢式PCR,对治疗前B-NHL患者BM 46例和PB 38例及治疗缓解后的BM和PB 10例进行了该标志的检测.对照组为非肿瘤或非B细胞来源肿瘤患者的BM或PB共33例.结果显示在B-NHL组,治疗前形态学有瘤细胞侵犯的3例BM及2例PB,克隆性IgHCDR3重排阳性;在形态学未见异常的BM和PB中克隆性IgH重排阳性率分别为65.1%及44.4%,与B-NHL分期及有无全身症状无关.10例病理组织克隆性IgH重排阳性的病人,7例缓解后BM和PB重排转为阴性,处于持续完全缓解状态;2例行自体外周血干细胞移植后BM或PB重排持续阳性者复发;1例移植后10个月BM克隆性IgH重排阳性,仍处于完全缓解.对照组33例中仅1例克隆性IgH重排阳性,本方法的特异性为97%.研究提示,PCR法能早期诊断BM及PB的瘤细胞浸润.临床缓解期BM或PB克隆性IgH重排阳性预示可能近期复发,阴性者可能持续缓解,个别IgH阳性者仍长期生存,其机理有待进一步研究.     

非霍奇金淋巴瘤bcl-2／IgH基因重排及微小残留病变的检测

探索bcl-2基因重排在非霍奇金淋巴瘤(NHL)的发生及演变中的作用,以bcl-2/IgH重排为克隆标志,建立敏感的检测淋巴瘤微小残留病变的方法。用多聚酶链反应(PCR)检测bcl-2/IgH基因重排,用系列稀释试验检测该方法的灵敏度。经检测9种恶性淋巴瘤细胞系中Su-DHL-4和Su-DHL-6有bcl-2/IgH基因重排,用系列稀释试验检测该方法的灵敏度为1:10~5。29例NHL石蜡包埋的组织标本中,共检出6例有bcl-2基因重排,其中滤泡型NHL(F-NHL)4例(36.4％),弥漫型NHL(D-NHL)2例(18.2％),全部在B细胞性NHL中检出。16例F-NHL患者中,4例外周血和骨髓中同时检出bcl-2(MBR)/IgH基因重排,化疗达CR后依然存在。结论提示,bcl-2基因重排主要与低度恶性的B细胞性淋巴瘤有关,重排方式以bcl-2(MBR)/IgH为主。bcl-2基因重排的检测为对滤泡性淋巴瘤微小残留病变的检测提供了一个快速、敏感、特异的方法,对该疾病的分期、疗效和预后的评估有一定的临床价值。     

Lack of cytogenetic abnormalities in Castleman's disease

BACKGROUND: Castleman's disease (CD) is a distinctive type of atypical lymph node hyperplasia that is often clonal. In a previously reported series of CD, clonal populations of plasma cells were detected by immunohistology in 4 of 39 cases (10%, lambda restricted), and immunoglobulin gene rearrangements were detected by paraffin polymerase chain reaction (PCR) analysis in 10 of 37 cases (27%). Cytogenetic analysis has been used to detect clonal proliferations of plasma cells in myeloma and clonal proliferations of lymphocytes in lymphomas and has identified critical gene loci that are important in the histopathogenesis of these disorders. Cytogenetic studies have not been done on a large series of patients with CD. Thus, we reviewed the archives of our institution for cases of CD and lymphoma that had had cytogenetic analysis. METHODS: The cytogenetic and lymphoma archives of our institution (a tertiary care center) from 1985 to 1998 were reviewed for the diagnoses of CD and lymphoma. There were 21,006 lymphomas, 701 of which had cytogenetic analysis (400 abnormal). There were 162 cases of CD, 7 of which had cytogenetic analysis. The frequency of cytogenetic abnormalities in CD was compared with that in lymphoma. The sensitivity of cytogenetics for defining clonality in CD was compared with immunohistology and paraffin PCR-amplified immunoglobulin heavy-chain gene rearrangement. RESULTS: From 1985 to 1998, 162 cases of CD and 21,006 cases of lymphoma were diagnosed. Cytogenetic analysis yielded adequate numbers of metaphases for analysis of 4 cases of CD and 701 lymphomas. Cytogenetic abnormalities were not identified in CD but were identified in 400 lymphomas (57%). Although 1 of 4 cases of CD was clonal by immunohistology (lambda restricted), no immunoglobulin gene rearrangements were detected by paraffin PCR analysis. CONCLUSIONS: The frequency of cytogenetic abnormalities in lymphomas and the lack of cytogenetic abnormalities in CD suggest that cytogenetic abnormalities, as detected by conventional cytogenetic analysis, are important in the pathogenesis of lymphoma but not CD. The lack of cytogenetic abnormalities in CD supports the hypothesis that CD is an interleukin-6-driven lymphoproliferative disorder.     

Sequential bcl-2 and c-myc oncogene rearrangements associated with the clinical transformation of non-Hodgkin''s lymphoma.

We report a case of untreated non-Hodgkin's lymphoma with histologic progression over 1 yr from a low-grade, small cleaved follicular center cell lymphoma to a high-grade, small noncleaved follicular center cell lymphoma. Both lymphomas had identical immunoglobulin (Ig) heavy-chain joining gene (JH), kappa light-chain joining gene, and bcl-2 gene rearrangements, indicating the clonal identity of the two tumors. The Ig heavy chain locus on one chromosome 14 was involved in an initial t(14; 18) translocation as shown by comigrating JH and bcl-2 rearrangements. However, the oncogene c-myc was in the germline configuration in the initial lymphoma but had one allele rearranged near the 3' end of exon I in the high-grade tumor; DNA sequence analysis was consistent with a chromosomal breakpoint at that site. The presence of the c-myc rearrangement in the high-grade tumor suggest a role for c-myc in the clonal evolution of the low-grade tumor into a more aggressive lymphoma. The coexistence of both bcl-2 gene and c-myc oncogene rearrangements in the same tumor is unusual, with only a few cases reported. Furthermore, this case is unique in the direct demonstration of the histologic and clinical progression of a human lymphoma associated with the sequential rearrangement of the bcl-2 gene and the c-myc oncogene.     

免疫球蛋白重链基因指纹图谱分析小儿急性淋巴细胞白血病的克隆性

目的：探讨小儿急性淋巴细胞白血病（ＡＬＬ）免疫球蛋白重链（ＩｇＨ）基因重排形式的种类，以及是否存在几个克隆并存的情况。方法：应用多聚酶链反应（ＰＣＲ）和单链构象多态性（ＳＳＣＰ）技术，构建ＩｇＨ－ＰＣＲ－ＳＳＣＰ基因指纹（ｆｉｎｇｅｒｐｒｉｎｔｉｎｇ），分析３４例ＡＬＬ患儿ＩｇＨ基因重排的基因指纹图谱。结果：单克隆的ＩｇＨ基因重排中除了单等位基因重排９例（３２．１４％）外，有１５例（５３．５７％）是双等位基因重排；４例（１４．２９％）白细胞数高、免疫表型是早期Ｂ细胞的病例表现寡克隆性ＩｇＨ重排。结论：从基因水平证实，小儿ＡＬＬ多发生ＩｇＨ双等位基因重排，且有多个克隆并存的现象。     

B细胞淋巴瘤bcl-2基因重排与14号染色体微卫星改变的研究

目的 研究bcl-2/IgH基因重排与微卫星DNA改变在B细胞淋巴瘤发生发展中的作用以及bcl-2/IgH基因重排与微卫星不稳定和杂合性缺失之间的关系.方法 应用巢式PCR方法检测bcl-2/IgH基因重排;选取14号染色体上2个微卫星多态性标记,采用聚合酶链反应-单链构象多态性分析法（PCR-SSCP）检测37例B细胞淋巴瘤中微卫星不稳定和杂合性缺失.结果 37例B细胞淋巴瘤中,bcl-2/IgH基因重排频率为24.3％ （9/37）;微卫星不稳定发生率为48.6％ （18/37）,杂合性缺失发生率为40.5％ （15/37）,其中D14S65位点微卫星不稳定和杂合性缺失频率较高,分别为29.7％（11/37）和27％（10/37）.经统计学分析结果显示：D14S65位点bcl-2/IgH基因重排与微卫星不稳定有关（P＜0.05）,与杂合性缺失无关（P＞0.05）;但D14S45位点bcl-2/IgH基因重排与微卫星不稳定和杂合性缺失无关（P＞0.05）.结论 D14S65是B细胞淋巴瘤中敏感的检测位点,bcl-2/IgH基因重排和微卫星不稳定性可能共同导致B细胞淋巴瘤的发生.