免疫球蛋白基因重排在B细胞非霍奇金淋巴瘤诊断中的意义

摘要：目的?探讨骨髓及外周血免疫球蛋白(Ig)基因重排在B细胞非霍奇金淋巴瘤(B-NHL)诊断中的临床意义。方法?采用多重聚合酶链反应(mPCR)技术并根据BIOMED-2引物系统对103例B-NHL患者及20例反应性淋巴组织增生患者骨髓及外周血标本基因组DNA进行扩增，对PCR产物进行IgH、IgK基因重排的克隆性分析。结果?103例B-NHL患者中IgH和IgK单克隆重排阳性率分别为41.7%和50.5%，IgH和IgK联合检出率为64.1%。59例慢性淋巴细胞白血病(CLL)/小B细胞淋巴瘤(SLL)患者中共检出Ig基因单克隆重排49例(83.1%)；除CLL/SLL以外的其他44例B-NHL患者中有17例(38.6%)检出Ig基因单克隆重排，而在20例反应性淋巴组织增生患者中均未检出。70例B-NHL患者外周血标本Ig基因重排检出率与骨髓标本之间的差异无统计学意义(45.7% vs 54.3%，χ2=1.03，P=0.31)；26例CLL/SLL患者外周血Ig基因重排率与骨髓标本之间的差异无统计学意义(69.2% vs 80.8%，χ2=0.92，P=0.34)；44例非CLL/SLL患者外周血Ig基因重排率与骨髓标本之间的差异亦无统计学意义(31.8% vs 38.6%，χ2=0.45，P=0.50)。结论?Ig基因重排可用于B-NHL的临床诊断，外周血与骨髓Ig基因重排检测具有同等的诊断价值。     

IgH基因克隆性重排检测在B细胞淋巴瘤诊断中的应用

目的建立B-NHL临床诊断中IgH基因克隆性重排检测基本方法.方法应用PCR方法,采用IgH FR3A引物,检测了7例B-NHL及4例反应性增生的淋巴结石蜡样本中IgH基因重排情况.结果黏膜相关型边缘区B细胞淋巴瘤（MALT）3例中有2例（2/3）,弥漫大B细胞淋巴瘤2例中2例（2/2）,滤泡淋巴瘤1例中0例（0/1）,套细胞淋巴瘤1例中1例（1/1）检测到IgH基因的克隆性重排.总检测率为5/7（71%）,4例淋巴结反应性增生病例病例均为IgH基因的多克隆性重排.结论PCR-FR3A在鉴别淋巴组织肿瘤性或反应性增生和最终确诊B-NHL上是一项有效的辅助方法.     

BIOMED-2方法检测恶性淋巴瘤骨髓侵犯的基因重排研究

本研究探讨BIOMED-2方法检测非霍奇金淋巴瘤(NHL)患者骨髓中免疫球蛋白(IG)和T细胞受体(TCR)基因克隆性重排的可行性,并初步评价其临床价值。采用BIOMED-2系统检测73例NHL(B-NHL 55例,T-NHL 18例)患者骨髓中IGH、IGK、IGL基因和TCRβ、TCRγ、TCRδ基因的克隆性重排,与骨髓穿刺细胞形态学进行比较,评价其与病理特征、临床分期等的相关性。结果表明:73例NHL中31例检测出IG或TCR基因重排,阳性率42.5%,高于骨髓穿刺细胞形态学阳性率24.7%(18/73),差异具有统计学意义(p<0.05);其中B-NHL阳性率为40.0%(22/55),T-NHL阳性率为50.0%(9/18),两者差异无统计学意义(p>0.05)。PCR检测阳性率和AnnArbor分期相关,Ⅲ/Ⅳ期患者阳性率高于Ⅰ/Ⅱ期,差异具有统计学意义(p<0.05)。结论:BIOMED-2检测IG和TCR基因重排是判断淋巴瘤骨髓浸润的有效方法,比骨髓细胞形态学更为敏感,有助于临床分期、预后判断和治疗选择。PCR检测阳性率和Ann Arbor分期相关,与淋巴瘤恶性程度、年龄、治疗状态、有无B组症状及是否累及脾脏无关。     

克隆性基因重排检测在NHL诊断中的意义

目的探明PCR克隆性基因重排检测（基因诊断）在NHL诊断中的意义。方法应用病理组织学、免疫组化、基因诊断方法三结合方案分析178例与会诊31例NHL诊断的作用。结果分析结果显示：以病理组织学为主,辅以免疫组化确诊NHL114例。疑难、早期、微量标本共64例与会诊31例均作了基因诊断。总的阳性率为61．4％（58／95）。其中IgH阳性13例（22．4％）,TCRβ芝邱阳性41例（70．6％）,双克隆性4例（7％）。本室基因诊断送检率为35．8％（64／178）,总确诊率为93．7％（60／64）。结论本文分析证明基因诊断是NHL三结合诊断中确诊疑难病例及分型的最有效的方法。     

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。     

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。     

B—HL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BM46例和PB38例及治疗缓解后的BM和PB10例进行了该标志的检测,对照组为非肿瘤或非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重排大大万籁 ;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BNP重排持续阳性者复发;1例移植后10个月BGM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克隆笥IgH重排阳性预示可能有近期复发,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。     

半巢式聚合酶链反应方法检测弥漫性大B细胞淋巴瘤bcl-2/IgH基因重排

目的寻找一种敏感、特异的方法检测弥漫性大B细胞淋巴瘤（DLBCL）bcl-2／IgH基因重排，并通过测定产物的序列，以了解所建方法的可靠性、方法运用专业引物设计软件设计bcl-2／IgH基因重排半巢式PCR引物，对52例经临床病理确诊的DLBCL石蜡包埋组织及10例慢性扁桃体炎患者的新鲜扁桃体组织通过半巢式递降温度梯度PCR（touch down PCR）扩增，检测bcl-2／IgH基因重排，并对其产物进行克隆和序列分析。结果 通过一步法检测到bcl-2／IgH基因重排8例，其中DLBCL6例，新鲜扁桃体组织2例；进行第2次半巢式PCR时仅在DLBCL中发现5例阳性，新鲜扁桃体组织均阴性。将阳性产物在网上序列分析显示：一步法检测到的8例中有3例为假阳性，而半巢式PCR扩增出的5例均为bcl-2／IgH基因重排片段。3例假阳性的片段分别与人类第19号染色体BAC331191，LLNLR-245D11基因片段及1号染色体RP11-498P10基因片段同源。结论常用的检测bcl-2／IgH基因重排引物扩增结果存在一定假阳性，其机制可能是因为人类基因组中存在与常用引物同源性较高的序列。为研究设计的引物与传统的引物结合，进行半巢式PCR可以排除这种假阳性扩增，提高诊断的准确性。     

非霍奇金淋巴瘤患者骨髓单个核细胞BCL-2/IgH、IgH基因重排的检测

本研究检测非霍奇金淋巴瘤(NHL)患者BCL-2/IgH基因主要断裂区重排、IgH基因重排,并探讨其对疾病的早期诊断、疗效评价等方面的意义。分别提取70例NHL(60例B-NHL、10例T-NHL)、7例淋巴结炎性肿大患者、20名正常人的骨髓单个核细胞DNA,通过PCR方法检测BCL-2/IgH、IgH基因重排,以凝胶电泳出现相应条带者为阳性,并与患者病理组织学检查进行比较,探讨此两种基因重排发生的相关因素,比较化疗后的动态变化。结果表明,①30例弥漫大B细胞淋巴瘤患者(DLBCL)中,10例骨髓BCL-2/IgH重排阳性(33.3%),与30例其它B-NHL(除外滤泡淋巴瘤FL及DLBCL)阳性率(6.7%)、20名正常人阳性率(5%)比较均有显著性差异(p均<0.05)。所有T-NHL及7例淋巴结炎性肿大患者BCL-2/IgH重排均为阴性;②对8例BCL-2/IgH基因重排阳性患者进行动态监测,经2个疗程R-CHOP治疗后BCL-2/IgH基因重排明显减少,PCR半定量结果均值由初治0.59降至0.16(p<0.05),6个疗程R-CHOP治疗后PCR半定量均值为0,BCL-2/IgH基因重排完全转阴;③BCL-2/IgH基因重排阳性患者中LDH水平升高占81.8%,在重排阴性患者中占28.6%,两组间有统计学差异(p<0.05)。然而,BCL-2/IgH基因重排与淋巴瘤分期、是否伴有全身症状、β2-MG水平、骨髓侵犯、肝脏脾脏侵犯均无显著相关性;④20例DLBCL(均为初治)骨髓单个核细胞DNA检测显示,9例IgH基因重排阳性(45%);30例其它B-NHL(均为初治或复发患者,DLBCL除外)中14例IgH基因重排阳性(46.7%),其两组间无统计学差异性(p>0.05),但对照组20名正常人、10例T细胞淋巴瘤患者及7例淋巴结炎性肿大患者均为阴性;⑤对7例IgH基因重排阳性患者进行动态监测表明,1个疗程的R-CHOP治疗就能显著减少IgH基因重排,PCR半定量结果均值由初治0.42降至0.13(p<0.05),2个疗程后PCR半定量均值为0,重排完全消除;⑥IgH基因重排阳性患者中LDH水平升高占90%,在重排阴性患者中占30%,两组间有统计学差异(p<0.05);IgH基因重排与淋巴瘤分期、是否伴有全身症状、β2-MG水平、骨髓侵犯、肝脏脾脏侵犯均无显著相关性。结论:BCL-2/IgH、IgH基因重排均可作为B-NHL早期诊断及评价疗效的特异性指标,这两种重排均与LDH水平相关;BCL-2/IgH基因重排对DLBCL特异性较高。     

BIOMED-2标准化的IG/TCR基因重排克隆性分析系统及其应用研究

目的 探讨BIOMED-2标准化的IG/TCR基因重排克隆性分析系统在疑难淋巴组织增牛性病变中的应用.方法 对67例难以从形态上区分良、恶性的淋巴组织增生性病变,采用BIOMED-2系统引物,提取标本中的DNA,进行抗原受体分子PCR扩增和聚丙烯酰胺凝胶电泳,进行IG/TCR基因重排的克隆性分析.结果 67例淋巴组织增生性病变中,33例进行了IG和TCR克隆性分析,19例进行了IG和15例进行了TCR克隆性分析.共有25例检测到克隆性重排,从而确诊为淋巴瘤,其中IG克隆性重排有15例为B细胞性淋巴瘤;TCR克隆性重排有8例为T细胞件淋巴瘤;同时存在IG和TCR克隆性苇排2例,为复合性T细胞和B细胞性淋巴瘤;其余42例旱多克隆性,为淋巴组织反应性增生.结论 BIOMED-2系统是一种全面优化设计的IG/TCR基因重排克隆性分析系统,是疑难淋巴组织增牛性病变中鉴别淋巴瘤与反应性增生的客观性手段.     

非霍奇金淋巴瘤患者免疫球蛋白及T细胞受体基因重排研究

目的 研究非霍奇金淋巴瘤(NHL)患者骨髓或外周血免疫球蛋白(Ig)/T细胞受体(TCR)基因单克隆重排特点及其临床意义.方法 以BIOMED-2引物系统及多重PCR方法对139例NHL患者骨髓或外周血标本进行Ig及TCR基因重排检测.结果 在B系NHL(B-NHL)中,82例慢性淋巴细胞白血病(CLL)患者中有70例(85.4％)检出Ig基因单克隆重排,其中IgH、IgK、IgL单克隆重排阳性率分别为46.3％(38例)、62.2％(51例)和1.2％(1例).其他类型的B-NHL患者有39.4％(33例中有13例)检出Ig基因单克隆重排,其中IgH和IgK单克隆重排阳性率分别为33.3％(11例)和39.4％(13例),未检测到IgL基因单克隆重排.T系NHL(T-NHL)患者中有50.0％(24例中有12例)检出TCR基因单克隆重排,其中TCRB和TCRG单克隆重排阳性率分别为8.3％(2例)和45.8％(11例),TCRB和TCRG双重基因重排阳性率为4.2％(1例),未检测到TCRD单克隆重排.11例早期(Ann Arbor Ⅰ、Ⅱ期)与46例晚期(Ⅲ、Ⅳ期)患者的Ig/TCR基因单克隆重排阳性率分别为36.4％和45.6％;10例惰性患者和47例侵袭性患者的Ig/TCR基因单克隆重排阳性率分别为40.0％和44.7％,差异均无统计学意义(P值均＞0.05).除外CLL的57例NHL患者骨髓涂片检测骨髓侵犯阳性率为12.3％,明显低于骨髓基因重排检测阳性率(43.9％),两者差异有统计学意义(P＜0.05).敏感性试验表明多重PCR检测恶性克隆的敏感性为3.12％～6.25％.结论 NHL的临床分期和恶性程度不同,其Ig/TCR基因单克隆重排阳性率有差异,但未能证实其相关性.联合应用BIOMED-2多重引物可提高PCR检测骨髓和(或)外周血Ig/TCR基因单克隆重排的敏感性.多重PCR对NHL患者骨髓侵犯检测的敏感性优于骨髓涂片检查,有助于早期发现骨髓侵犯及预防复发.     

Low-grade B-Cell lymphomas with plasmacytic differentiation lack PAX5 gene rearrangements

The chromosomal translocation t(9;14)(p13;q32) has been reported in association with lymphoplasmacytic lymphoma (LPL). Although this translocation involving the paired homeobox-5 (PAX5) gene at chromosome band 9p13 and the immunoglobulin heavy chain (IgH) gene at 14q32 has been described in approximately 50% of LPL cases, the actual number of cases studied is quite small. Many of the initial cases associated with t(9;14)(p13;q32) were actually low-grade B-cell lymphomas with plasmacytic differentiation other than LPL. Thus, we analyzed a series of low-grade B-cell lymphomas for PAX5 gene rearrangements. We searched records from the Department of Pathology, Stanford University Medical Center for low-grade B-cell lymphomas, with an emphasis on plasmacytic differentiation, that had available paraffin blocks or frozen tissue. We identified 37 cases, including 13 LPL, 18 marginal zone lymphomas (nodal, extranodal, splenic, and alpha-heavy chain disease), and 6 small lymphocytic lymphomas. A novel dual-color break-apart bacterial artificial chromosome probe was designed to flank the PAX5 gene, spanning previously described PAX5 breakpoints, and samples were analyzed by interphase fluorescence in situ hybridization. All cases failed to demonstrate a PAX5 translocation, indicating that t(9;14)(p13;q32) and other PAX5 translocations are uncommon events in low-grade B-cell lymphomas with plasmacytic differentiation. This study also confirms recent reports that found an absence of PAX5 rearrangements in LPL, suggesting the reassessment of PAX5 rearrangements in LPL.     

Restricted immunoglobulin variable region (Ig V) gene expression accompanies secondary rearrangements of light chain Ig V genes in mouse plasmacytomas

The many binding studies of monoclonal immunoglobulin (Ig) produced by plasmacytomas have found no universally common binding properties, but instead, groups of plasmacytomas with specific antigen-binding activities to haptens such as phosphorylcholine, dextrans, fructofuranans, or dinitrophenyl. Subsequently, it was found that plasmacytomas with similar binding chain specificities not only expressed the same idiotype, but rearranged the same light (V(L)) and heavy (V(H)) variable region genes to express a characteristic monoclonal antibody. In this study, we have examined by enzyme-linked immunosorbent assay five antibodies secreted by silicone-induced mouse plasmacytomas using a broader panel of antigens including actin, myosin, tubulin, single-stranded DNA, and double-stranded DNA. We have determined the Ig heavy and light chain V gene usage in these same plasmacytomas at the DNA and RNA level. Our studies reveal: (a) antibodies secreted by plasmacytomas bind to different antigens in a manner similar to that observed for natural autoantibodies; (b) the expressed Ig heavy genes are restricted in V gene usage to the V(H)-J558 family; and (c) secondary rearrangements occur at the light chain level with at least three plasmacytomas expressing both kappa and lambda light chain genes. These results suggest that plasmacytomas use a restricted population of B cells that may still be undergoing rearrangement, thereby bypassing the allelic exclusion normally associated with expression of antibody genes.     

T-cell gene rearrangements and the diagnosis of T-cell neoplasms

Rearrangements of the T-cell antigen receptor genes serve as unique, clonal tumor markers of T-cell neoplasms. This approach provides a reliable and sensitive diagnostic tool to document both clonality and lineage of T-cell lymphoproliferative processes.     

The monoclonality of human B-cell lymphomas

Human tissues involved with lymphoma have been examined in frozen sections for immunoglobulin-bearing cells by a technique involving double-label immunofluorescence with mixed anti-kappa and anti-lambda antibodies. F (ab')2 fragments of purified antibodies were employed to avoid any binding via Fc receptors. B cell lymphomas were shown to be composed of monoclonal populations of Ig bearing cells, whereas normal or reactive lymphoid follicles contained a mosaic of Ig-bearing cells derived from multiple clones. Nodules of lymphoma were often surrounded by normal polyclonal B cell populations. We anticipates that the approach described here will be useful in the diagnosis of lymphoma, differentiating it from reactive lymphoid hyperplasia by the demostration of monoclonality. In addition, it should provide a sensitive and reliable tool for investigating the immunobiology of human lymphoma.     

Advances in the role of cytogenetic analysis in the molecular diagnosis of B-cell lymphomas

Introduction: Cytogenetic abnormalities represent essential determinants of diagnosis and prognosis in B-cell lymphomas. Their theranostic value is increasingly significant with the development of targeted therapies, in order to adapt the treatment at diagnosis as well as when relapse occurs.

Areas covered: As the significance of these biomarkers is influenced by the technology used to detect them, an overview describing the strength and weakness of conventional and emerging technologies is provided. This review also updates the diverse cytogenetic abnormalities found in B-cell lymphomas, emphasizing their value in treatment decision.

Expert commentary: Cytogenetics remains an essential analysis for the diagnostic work-up of lymphomas. As whole genome sequencing becomes more and more affordable routinely, the next challenge will be to recover all the information conveyed by conventional karyotype, including the analysis of the clonal architecture at the single cell level, in whole genome data.     

Frequent biclonality and Ig gene alterations among B cell lymphomas that show multiple histologic forms

Configurations of Ig gene DNA were examined in multiple biopsy specimens from seven cases of human B cell lymphoma that showed histologic differences among the specimens within each case. Analysis by Southern blot hybridizations with DNA probes for each of the three Ig loci revealed that the configurations of DNA within these loci were identical among the specimens in two of the cases. This result indicated the monoclonality of these lymphomas, despite differences in histology between biopsy specimens. In contrast, no common nongermline configurations of Ig gene DNA were detected among multiple biopsies in each of three other cases. Therefore, different histologies correlated with separate clones of proliferating B cells in these cases. In the last two cases, the configurations of light chain gene DNA were the same among biopsies in each case, consistent with a monoclonal origin in both lymphomas. However, differences were detected in the configuration of the heavy chain gene DNA. Analysis with a series of DNA probes of the mu heavy chain region indicated that the differences in the DNA configurations of the heavy chain genes from the biopsies probably arose from postrearrangement deletions of either the switch or constant regions of the mu gene. These studies indicate that, contrary to the conventional belief, individual tumors that contain different histologic types of lymphoma within the same patient frequently arise from separate clones of neoplastic cells. Furthermore, the heavy chain genes of monoclonal tumors may show postrearrangement deletions, often resulting from instability of DNA sequences within or around the mu switch region.