IgH基因单克隆重排检测及其在B细胞性非霍奇金淋巴瘤诊断中的应用

 目的：建立准确可靠、操作性强、适用于临床实际工作的免疫球蛋白重链（immunoglobulin heavy chain,IgH）基因单克隆重排检测方法,用于B细胞性非霍奇金淋巴瘤（B-cell non-Hodgkin lymphoma,B-NHL）的辅助诊断。方法：采用骨架区(framework region,FR）引物FR2、FR3和重链连接区 （joining region of heavy chain,J_H）引物LJH、VLJH组合、A管+B管模式、半巢式聚合酶链式反应(polymerase chain reaction,PCR)法对121例B-NHL、58例T细胞性非霍奇金淋巴瘤（T-cell non-Hodgkin lymphoma,T-NHL）和19例淋巴结反应性增生的石蜡组织进行IgH基因单克隆重排检测,分析IgH基因单克隆重排检出率在B-NHL组、T-NHL组和淋巴结反应性增生组中的差异,以及B-NHL中联合应用FR2和FR3与单独应用FR2、FR3之间IgH基因单克隆重排检出率的差异。结果：118例成功检测的B-NHL中,IgH基因单克隆重排检出率为81%（96/118）;54例成功检测的T-NHL中,IgH基因单克隆重排检出率为4%（2/54）;19例成功检测的淋巴结反应性增生中未检出IgH基因单克隆重排。B-NHL组与T-NHL组、淋巴结反应性增生组相比,IgH基因单克隆重排检出率差异具有显著性（P<0.05）。B-NHL中, FR2基因单克隆重排检出率为58%（68/118）,FR3基因单克隆重排检出率为55%（65/118）,联合应用FR2和FR3,IgH基因单克隆重排检出率为81%（96/118）,联合应用FR2和FR3与单独应用FR2、FR3的检出率有显著差异（P<0.05）。结论：采用FR2、FR3、LJH及VLJH引物组合、A管+B管模式和半巢式PCR法进行石蜡组织IgH基因单克隆重排检测,简单易行,结果准确可靠,阳性率较高,可用于临床B-NHL的辅助诊断。     

Detection of concurrent/recurrent non-Hodgkin's lymphoma in effusions by PCR

A subset of patients with non-Hodgkin's lymphoma (NHL), present with or subsequently develop lymphocytic effusions. Differential diagnosis between reactive lymphocytosis and recurrent low-grade NHL is difficult by cytology alone. We studied the use of polymerase chain reaction (PCR)-based techniques to detect concurrent/recurrent NHL. Both primary tumors and atypical lymphocytic effusions of 12 low-grade B-NHL patients and 4 T-NHL patients were studied. Six pleural effusions (reactive/carcinomatous), in patients with no history of NHL, were included. Samples were amplified by PCR, using Fr3, Fr2, LJH, and VLJH primers specific for the immunoglobulin heavy chain (IgH) gene and Vgamma-8, Vgamma9, Vgamma10, Vgamma11 and Jgamma1/Jgamma2 consensus primers specific for the T-cell receptor gamma (TCR-gamma) gene. IgH gene PCR products were analyzed by polyacrylamide gel electrophoresis (PAGE). TCR-gamma gene PCR products were analyzed using a novel nonradioactive single-strand conformational polymorphism (SSCP) procedure. IgH gene rearrangement analysis demonstrated monoclonality in 11/12 primary low-grade B-NHLs. Identical monoclonal bands were found in both primary tumor and effusion in 9 patients. TCR-gamma gene rearrangement analysis demonstrated monoclonality in 4 of 4 primary T-NHLs. Identical monoclonal banded patterns were found in both primary tumor and effusion in 3 patients. Our results strongly support the diagnosis of concurrent/recurrent NHL in 13 of 16 (81%) cases of atypical lymphocytic effusions. IgH/PAGE and TCR-gamma/SSCP analyses are useful tools in the diagnoses of lymphocytic effusions in patients with NHL.     

Analysis of immunoglobulin heavy chain genes rearrangement by PCR from paraffin-embedded tissue in B-cell lymphomes in Tunisia

OBJECTIVE: To study the lymphoid clonality on Tunisian B-cell lymphomas cases by polymerase chain reaction (PCR)-based techniques using DNA from paraffin-embedded tissues. MATERIAL AND METHODS: Here we conducted a retrospective PCR clonality study on 73 cases of B-cell lymphomas and 12 reactive lymphoid tissues. The quality of DNA extracted was tested by beta-globin PCR. Consensus primers directed at the FRIII-VH and FRII-VH regions of the immunoglobulin heavy chain (IgH) gene were used to detect clonality. RESULTS: The results showed that 52 of 73 (71%) B-cell lymphomas exhibited good quality of amplifiable DNA. Clonality was found in 77% of cases using the set of primers FRIIIa/LJH/VLJH and in 65.5% using the set of primers FRIIa/LJH/VLJH. Lymphomas derived from pregerminal centre showed a high rate detection of clonal IgH gene rearrangement (100%) compared to other group of tumors derived from germinal centre or postgerminal centre (74.5%). None of the polyclonal controls gave a clonal pattern. CONCLUSION: This is the first large series of PCR clonality study of IgH gene rearrangements on B-cell lymphoma from Tunisia. Our results were similar to other reports in terms of sensitivity and specificity of these techniques and confirm the interest of that PCR for detecting clonal IgH gene rearrangements in lymphoma.     

BIOMED-2引物系统检测急性淋巴细胞白血病患者免疫球蛋白基因重排

目的 探讨BIOMED-2引物系统检测成人急性淋巴细胞白血病(ALL)患者Ig基因重排的敏感性,分析Ig基因重排方式、各胚系基因的利用频率等.方法 采用BIOMED-2引物系统扩增29例成人ALL患者Ig重链(IgH)和Ig轻链(IgL)重排基因.将PCR产物直接测序,使用IMGT/V-QUEST等生物信息资源分析B细胞-ALL(B-ALL)患者IgH和IgL基因重排类型、胚系基因片段利用及体细胞突变情况.结果 24例B-ALL患者中IgH完全重排阳性率为70.8%,不完全重排为12.5%,IgK VH-JH重排为29.2%,IgK删失元件(IgK-Kde)重排为25.0%,未检测出IgL阳性重排.所有B-ALL患者Ig基因重排检出率可达100%.5例T细胞-ALL(T-ALL)患者1例检测到IgH不完全重排阳性,2例IgK VH-JH重排阳性,2例未检测出Ig基因重排.B-ALL中IgH重排优先利用的V、D、J家族分别为VH3和VH4、DH3和JH6.5例IgK重排中4例利用了 Vκ1家族.重排基因中发生替代突变的基因占23.5%.替代突变零星散布于Ig基因全长,互补决定区中替代突变与静寂突变的比例<1.结论 BIOMED-2引物系统可以检测出绝大多数B-ALL患者的Ig基因重排,国内的临床检测资料亦然.这是一种有效的检测工具,为定量分析微小残留病(MRD)水平提供了基础资料.     

Identification of monoclonal B-cell populations by rapid cycle polymerase chain reaction. A practical screening method for the detection of immunoglobulin gene rearrangements.

Alternatives to Southern blot hybridization for gene rearrangement analysis are being studied because of the time, labor, cost, and radioisotopes required for this technique. We have utilized a rapid, hot air, thermocycling polymerase chain reaction (PCR) system to examine various lymphoproliferative disorders for immunoglobulin heavy chain (IgH) gene rearrangements. This unique system amplifies DNA from 10 microliters samples placed in glass capillary tubes, over a total cycle time of about 30 minutes. Amplified bands are easily visualized on ethidium bromide-stained agarose gels. Forty-one monoclonal B-cell proliferations, 27 reactive lymphoid hyperplasias, 17 T-cell lymphomas and 3 cases of Hodgkin's disease were studied. All 88 cases were fully characterized by morphologic, immunophenotypic, and genotypic (Southern blot) analyses. Each case was separately evaluated by PCR with two primer pairs: 1) IgH variable region (VH) and IgH joining region (JH) and 2) bcl-2 and JH. Thirty-four of 41 monoclonal B-cell proliferations revealed a distinct band (within an expected base pair range) with 1 or both primer combinations supporting B-cell monoclonality; the other 7 cases were considered false negatives. The 47 entities without IgH gene rearrangements detectable by Southern analysis demonstrated no amplified product or a smear of amplified DNA with no distinct band. The overall specificity of PCR was 100%, and the sensitivity was 83% when directly compared with Southern blot analysis. Although its sensitivity is currently less than optimal, PCR is a rapid and practical screening method for the detection of IgH gene rearrangements. If a positive result is obtained no further analysis is required; however, if there is a negative result, standard Southern blot analysis should be performed to definitively exclude the presence of a monoclonal B-cell population in the sample.     

B细胞非霍奇金淋巴瘤IgH基因重排检测及凝胶扫描技术应用的初步研究

目的 采用凝胶扫描技术,力求对采用聚合酶链反应(PCR)检测非霍奇金淋巴瘤(NHL)抗原受体基因重排结果分析时建立量化标准,以利于临床医师应用时客观判断,并为其筛选随访病例提供依据。方法用PCR对96例B-NHL分别采用IgH FR3A及FR2A检测IgH基因克隆性重排。65例经IgH FR3APCR已证实为IgH基因克隆性重排B-NHL及8例良性病变淋巴组织和5例正常人外周血单核细胞,IgH FR3APcR产物8％变性聚丙烯酰胺凝胶电泳银染后图像行凝胶扫描,绘制曲线并计算h1／h2比值。结果(1)采用FR3A引物,克隆性IgH基因重排在96例B-NHL中检测率为68％,采用FR2A引物,检测率为61％,结合FR3A、FR2A引物,总检测率为83％。(2)凝胶扫描曲线示65例B-NHL的h1／h2均＞3,而5例正常人外周血单核细胞曲线均表现为钟型,8例良性病变淋巴组织h1／h2比值均＜1．5。结论非霍奇金淋巴瘤抗原受体基因重排检测的凝胶扫描分析曲线中,h1／h2峰高比值至少＞3可提示为IgH基因克隆性苇排,＜1．5可能提示IgH基因多克隆重排。比值介于1．5和3之间,提示有随访意义。联合FR3A,FR2A引物,可提高B-NHL IgH基因克隆性重排的检测率。     

Monoclonality of infiltrating plasma cells in primary pulmonary nodular amyloidosis: detection with polymerase chain reaction

AIMS: To investigate the relation between localised amyloidosis and immunocytic dyscrasia. METHODS: Open lung biopsy specimens from a 72 year old man with multiple nodules in the right middle and lower lung were stained with haematoxylin-eosin, Congo red, and antibodies against IgG, IgA, IgM, and kappa and lambda light chains. Semi-nested PCR amplification for the immunoglobulin heavy chain (IgH) gene was performed using consensus primers for the VDJ region of the IgH gene, FR3A, LJH, and VLJH. RESULTS: The biopsy specimens contained eosinophilic amorphous material stained with Congro red and anti-kappa light chain, and surrounded by inflammatory cells intermingled with plasma cells. Plasma cells in the adjacent amorphous material showed cytoplasmic staining with anti-kappa. Polymerase chain reaction revealed a discrete amplified band of apparently uniform size with background smear. CONCLUSIONS: Primary AL type localised amyloidosis involves local accumulation of monoclonal plasma cells and their secreted products, as in nodular cutaneous amyloidosis. Localised AL type nodular amyloidosis is a separate entity in amyloidosis.     

Assessment of IgH PCR strategies in multiple myeloma.

AIMS: To compare the ability of four commonly used PCR techniques to demonstrate clonal IgH rearrangements in multiple myeloma. METHODS: Bone marrow samples (containing a minimum of 10% plasma cells) were obtained from 127 patients with confirmed multiple myeloma. Framework 3 (Fr3) PCR was performed in all cases and the Framework 1 (Fr1f) PCR, which utilises six VH family specific primers, in 98 cases. In addition, 44 cases were assessed by Fr3, Fr1f, Framework 2 (Fr2) and Framework 1 consensus (Fr1 con) PCR techniques. JH primer selection was also assessed such that each PCR strategy was performed twice in each of the 44 cases, using the JH consensus primer (JH con) alone and then repeated with an equimolar mixture of JH con, JH3 and JH6 (JH mix). RESULTS: Clonal rearrangements were demonstrated in 71 (56%) of 127 cases with the Fr3 PCR and in 52 (53%) of 98 with the Fr1f PCR. However, by using both techniques it was possible to demonstrate clonal IgH rearrangements in 92 (75%) of 122 cases. Forty four cases were assessed by all four PCR techniques; in these cases the Fr3 and Fr1f PCRs demonstrated clonal rearrangements in 26 (59%) cases with a combined yield of 34 (77%). The Fr2 and Fr1 con PCR techniques had inferior pick up rates, demonstrating clonal rearrangements in 21 (48%) of 44 cases and a combined yield of 28 (63%). The Fr2 PCR did, however, demonstrate a clonal rearrangement in one case negative by both Fr3 and Fr1f. Two additional rearrangements were demonstrated by using JH mix; one became positive by Fr3, Fr1f and Fr2 and the other positive by Fr1f, Fr1 con and Fr2. CONCLUSIONS: By utilising both the Fr3 and Fr1f PCR techniques it is possible to demonstrate definitive clonal rearrangements in the majority of patients with multiple myeloma. The Fr1 con and Fr2 PCR techniques have inferior pick up rates but may detect some additional rearrangements.     

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     

Systematic design and testing of nested (RT-)PCR primers for specific amplification of mouse rearranged/expressed immunoglobulin variable region genes from small number of B cells

The aim of this study was to develop a highly specific and sensitive (RT-)PCR capable of potentially amplifying the rearranged/expressed VH and VL gene belonging to any mouse immunoglobulin V gene family from a single or a small number of B cells. A database of germline immunoglobulin sequences was used to design 112 primers for a nested (RT-)PCR based strategy to cover all VH, VL, JH, JL, CH and CL gene families/genes from C57BL/6 and BALB/c mice. 93.7% of the primers had 4-fold or less, while 71.4% had no degeneracy. The proportions of germline V genes to which the primers bind with no, up to 1 and up to 2 mismatches are 59.7%, 84.1% and 94.9%, respectively. Most but not all V gene family specific primers designed allow amplification of full-length V genes. The nested primers permit PCR amplification of rearranged V genes belonging to all VH and VL gene families from splenocyte genomic DNA. The V gene family-specific nature of the primers was experimentally confirmed for randomly selected 6 VH and 6 Vkappa families, and all Vlambda genes. The broad V gene family coverage of our primer set was experimentally validated by amplifying the rearranged/expressed VH and VL genes from splenocytes and a panel of 38 hybridomas under conditions where primer mixes and genomic DNA or total RNA was used as starting template. We observed no or low-level cross-family priming. Pooled constant region specific primers allowed efficient RT-PCR amplification of H and L chain isotypes. The expressed VH and VL genes belonging to different V gene families RT-PCR amplified from a mixture of hybridomas in a representative manner. We successfully amplified the expressed VH and Vkappa gene from a single hybridoma cell by RT-PCR and from 10-15 microdissected B cells by genomic PCR. This, first of its kind, comprehensive set of highly sensitive and specific nested primers that provide broad V gene family coverage will open up new avenues and opportunities to study various aspects of mouse B cell biology.     

Immunoglobulin gene rearrangement analysis in composite hodgkin disease and large B-cell lymphoma: evidence for receptor revision of immunoglobulin heavy chain variable region genes in Hodgkin-Reed-Sternberg cells?

Immunoglobulin heavy chain gene (IgH) rearrangement was studied in a patient showing the occurrence of classical Hodgkin disease and large B-cell lymphoma (LBCL) in the same lymph node. The VHDHJH region was amplified by polymerase chain reaction, the template being the DNA extracted from single Hodgkin and Reed-Sternberg and LBCL cells, microdissected on hematoxylin-eosin-stained sections by laser capture. A repeated VH4DH3JH4 segment was found in Reed-Sternberg cells, whereas a repeated VH3DH3JH4 segment was observed in LBCL cells. Rearranged VH genes carried somatic mutations in both populations, indicating a common germinal center cell origin. The IgH rearrangement found in clonally related Reed-Sternberg cells differed from the one of LBCL cells in the VH region but showed the same JH and DH segments with no variation from the respective germline sequence. The DH-JH junction is the first immunoglobulin gene segment rearranged in precursor B cells. Because the possibility of secondary Ig gene rearrangement in peripheral lymphoid organs has recently been reported, in the patient described here Reed-Sternberg and LBCL cells might originate from a common precursor in which secondary VH replacement took place during the germinal center reaction, giving rise to two different clonally related lymphomas.     

中国人群慢性淋巴细胞白血病免疫球蛋白重链可变区基因组成及突变分析

目的 研究中国慢性淋巴细胞白血病(chronic lymphocytic leukemia,CLL)患者免疫球蛋白重链可变区(immunoglobulin variable heavy chain region,IGVH)基因各个片段的组成以及突变情况.方法 应用多重PCR技术扩增64例CLL患者的IGVH基因片段,纯化PCR扩增产物后直接测序,应用IMGT/V-QUEST及IGBlast数据库分析,明确VH基因有无突变和突变位置,以及VH、DH,JH家族各个成员组成情况.结果 64例CLL患者中,VH3家族31例(48%)、VH4家族26例(41%)、VH1家族4例(6%)、VH2家族2例(3%)、VH7家族1例(2%).44例患者发生体细胞突变,占CLL患者的69%;20例无突变,占CLL患者的31%,其中6例(9%)VH基因的同源性为100%.VH4家族中有9例无突变(9/26,35%);VH3家族中8例无突变(8/31,26%);VH1家族中1例无突变(1/4,25%).在所检测的CLL标本中,VH4-34是最常见的VH基因片段,检测出8例(13%),其中无突变3例;其次为VH4-59,检测出7例(11%),无突变者3例.DH基因中DH3家族最常见,有25例(39%);其中11例(11/20,55%)出现在无突变组中.无突变组中最常见的DH基因片段为DH3-10和DH3-22,各为4例(4/20,20%).JH基因中JH6家族最常见,检测出23例(36%),其中9例出现在无突变组中(9/20,45%).结论 中国CLL患者IGVH基因家族表达比例和突变情况与西方国家存在显著差异,推测可能与不同的种族和环境中的抗原选择有关,其预后意义有待进一步探讨.     

PCR-based clonality analysis: a reliable method for the diagnosis and follow-up monitoring of conservatively treated gastric B-cell MALT lymphomas?

AIMS: We evaluated polymerase chain reaction (PCR) amplification of specific immunoglobulin heavy chain (IgH) gene rearrangements as a means of demonstrating monoclonality during follow-up of conservatively treated gastric MALT lymphoma, and compared the reproducibility of PCR on sequential frozen and paraffin-embedded endoscopic biopsies. We established an association between clonality detected by PCR and the histological observations. METHODS AND RESULTS: Sixty-nine pairs of sequential frozen and paraffin-embedded endoscopic biopsies from 21 conservatively treated patients were graded according to the Wotherspoon-Isaacson histological scoring system, which provides a measure of diagnostic confidence on a scale 0-5. PCR amplification of the IgH gene was performed using FR3/JH and FR2/JH primers. 68/69 paired samples (98.5%) showed identical mono- or polyclonal PCR amplification patterns. Forty-seven out of 48 pairs of samples sharing similar histological features produced identical amplification patterns in both fresh and paraffin-embedded tissues. In comparison with the histological grading, monoclonality was detected in 64.2% and 41.6% of samples scored 5 and 4, respectively. Conversely, among 64 samples scored 0-3, a monoclonal pattern was observed only in two samples, one of which was from a patient who relapsed 9 months later. CONCLUSIONS: PCR-based clonality analysis by demonstration of specific IgH gene rearrangement can be easily and reliably performed on both frozen and paraffin-embedded endoscopic biopsies. In conjunction with histological observation, this method can be used as a complementary tool to monitor MALT lymphoma regression during conservative treatment.     

The effect of fixation on detection of B-cell clonality by polymerase chain reaction.

It has been suggested that neutral buffered formalin (NBF)-fixed, paraffin-embedded, or fresh specimens might provide satisfactory DNA templates for polymerase chain reaction (PCR) assays used in establishing the clonality and presumptive B-cell lineage of lymphoma. The suitability of other fixatives used by hematopathologists, such as B5, is still undetermined. Thirty cases were identified from the files of the Cleveland Clinic Foundation, Cleveland Ohio, that showed abnormal immunoglobulin heavy chain (IgH) rearrangement by Southern blot analysis (SBA). Corresponding paraffin-embedded tissue samples fixed in NBF (21 cases), B5 (18 cases), Hollande's fixative (17 cases), zinc formalin (ZF) (5 cases), and Bouin's fixative (3 cases) were studied. With use of consensus primers against the framework 3 (FR3) and FR2 regions of the VH gene, paired against JH primer(s), PCR analysis was performed. bcl-2/IgH translocation was also studied. Ten reactive lymphoid samples were used as controls, and 40 cases were evaluated. Successful amplification of a clonal proliferation was manifested as one or two discrete narrow bands in the appropriate size range. The sensitivity of detecting clonality was 95, 94, 67, 80, and 0% for NBF, Hollande's fixative, B5, ZF, and Bouin's fixative, respectively. Although NBF and Hollande's fixative were 100% specific, consistent false-positive results were a major problem with B5-fixed tissue. Paraffin-embedded tissue, fixed in NBF, Hollande's fixative, and ZF solutions, may be used for DNA extraction and PCR assays for establishing B-cell clonality. The precipitating fixative B5 and Bouin's solution should not be used for this purpose until the issue of false-positive results is resolved.     

滤泡型淋巴瘤中t(14;18)染色体易位的分析及其临床意义

目的探讨滤泡型淋巴瘤（FL）的分子遗传学特征及其在病理诊断中的意义。方法收集55例FL石蜡标本,对照组小B细胞淋巴瘤28例和反应性滤泡增生（RFH）10例,应用套式PCR技术检测FL中,免疫球蛋白重链基因（IgH）的克隆性重排;应用标准PCR技术检测55例FL中t（14;18）易位,以10例RFH做对照;采用双色荧光原位杂交（FISH）技术检测20例淋巴结FL中t（14;18）易位,以4例RFH作为对照;并与PCR检测结果进行比较。结果（1）55例FL中,结内49例,结外6例。男性33例,女性22例,男女比为1．5：1。发病年龄36—79岁（中位年龄57岁）;FL分级：FL1—3分别为25例、19例和11例。（2）55例中50例（90％）检出β-肌动蛋白（actin）,该50例中FR3A阳性24例（48％）,FR2阳性25例（50％）,其中15例（30％）呈FR3A和FR2双阳性,共34例（68％）IgH基因重排。对照组小B细胞淋巴瘤28例中,25例检出β—actin,其中FR3A阳性18例（64％）,FR2阳性17例（61％）,共24例（86％）可检测出克隆性IgH基因重排。4例RFH均未检出IgH基因重排。（3）在44例结内FL中检出15例（34％）t（14;18）易位,其中14例在MBR,1例在mcr。（4）20例中,有16例（80％）可检出t（14;18）易位。结论（1）IgH克隆性重排在FL中的检测率比其他小B淋巴细胞低。（2）FISH检测石蜡包埋组织中t（14;18）易位有助于FL的诊断。FISH比PCR的敏感性更好,操作简便,可用于检测石蜡包埋组织中的分子遗传学改变。     

Increased sensitivity of B-cell clonality analysis in formalin-fixed and paraffin-embedded B-cell lymphoma samples using an enzyme blend with both 5′→3′ DNA polymerase and 3′→5′ exonuclease activity

Polymerase chain reaction (PCR)-based detection of immunoglobulin heavy chain (IgH) gene rearrangement for determination of B-cell clonality needs to be simple but optimally sensitive. Efficient IgH PCR analysis can be hampered by sequence variability in the template DNA, despite of the use of degenerative primers. To improve sensitivity of the B-cell clonality analysis in formalin-fixed and paraffin-embedded (FFPE) tissues, we have performed framework three-area (FR3)/joining gene (JH) IgH PCR utilizing an enzyme blend (rTth DNA Polymerase, XL) providing both 53 polymerase and 35 exonuclease activities. The DNA samples were extracted from FFPE biopsies of 43 mature B-cell lymphoma cases of so-called germinal center and post-germinal center origin, including 6 nodal follicular lymphomas (FL), 15 gastric mucosa-associated lymphoid tissue (MALT) lymphomas, and 22 gastric diffuse large B-cell lymphomas (DLBCL). Of the cases, 31 (17 DLBCL and 14 MALT lymphoma) represented small endoscopic biopsies. Serial dilutions of target DNA were applied to avoid inconsistent bands that may be seen when the input amount of template is too low, which can be the case when DNA is extracted from FFPE endoscopic gastric biopsies. Using conventional Taq polymerase, consistent monoclonal product was found in 53% (23/43) of the cases (FL: 67%; MALT lymphoma: 47%; DLBCL: 55%). The rTth polymerase showed reproducible monoclonal pattern in 72% (31/43) of the cases (FL: 67%; MALT lymphoma: 73%; DLBCL: 73%); the sensitivity is compatible with one that can be detected with conventional FR3/JH PCR in fresh/frozen tissues. In conclusion, the rTth DNA polymerase greatly improves sensitivity of FR3/JH PCR in FFPE biopsies of mature B-cell lymphomas, most probably by increasing the primer matches during PCR amplification.     

Hodgkin's disease: immunoglobulin heavy and light chain gene rearrangements revealed in single Hodgkin/Reed-Sternberg cells.

AIM: To corroborate and investigate the nature of Hodgkin/Reed-Sternberg cells (H/R-S) of various subtypes of Hodgkin's disease. METHOD: Single H/R-S cells were micro-picked from frozen sections of tissues affected by Hodgkin's disease. The DNA from these cells was amplified by the polymerase chain reaction (PCR) with immunoglobulin heavy chain (IgH) gene FRIIIa/JH primers and light chain gene family specific primers. RESULTS: Fifty two of 135 isolated cells gave specific reaction products (36%). IgH and V kappa 4 gene rearrangements were found repeatedly in many H/R-S cells from one case of lymphocyte predominant Hodgkin's disease. Repeated V kappa 4 and individual IgH/V kappa 4,2 rearrangements were seen in one case, and individual IgH and V lambda 3/V kappa 4 rearrangements were seen in another case of nodular sclerosis-type Hodgkin's disease. Repeated IgH/V lambda 3 and individual V lambda 2,4 rearrangements, repeated V kappa 4 and individual IgH/V kappa 3 rearrangements, and repeated IgH and individual V kappa 3/V kappa 4 rearrangement were detected, respectively, in three cases of mixed cellularity-type Hodgkin's disease. Repeated and individual IgH rearrangements were found in another two cases of mixed cellularity-type Hodgkin's disease. CONCLUSION: The H/R-S cells isolated from lymphocyte predominant Hodgkin's disease had IgH and V kappa 4 gene rearrangements, which supports the conclusion that this disease results from a proliferation of neoplastic B cells. The IgH and kappa and/or lambda gene rearrangements seen in H/R-S cells isolated from classic Hodgkin's disease (mixed cellularity-type and nodular sclerosis-type) support the theory that these cells derive from B lineage cells at various stages of differentiation. To our knowledge, this is first time that lambda gene rearrangements have been detected in H/R-S cells.     

Polymerase chain reaction (PCR) detection of B cell clonality in Sjögren's syndrome patients: a diagnostic tool of clonal expansion

Sjögren's syndrome (SS) is an autoimmune disease characterized by clonal B cell attack of the exocrine glands and dysregulated expression of B cell‐activating factor (BAFF). Based upon the current data of increased rates of lymphoid malignancy, as non‐Hodgkin's lymphoma (NHL) is associated with SS, we propose the detection of clonal rearrangements of immunoglobulin heavy chain (IgH) gene in those patients as a predictor of malignant clonal expansion. To test our proposal, we examined the IgH clonal rearrangements in SS patients (60) and healthy control subjects (42) having chronic non‐specific sialadenitis, to determine the presence of clonal B cells in minor labial salivary glands (MSG) of SS patients. Clonal B cell expansion was assessed by two polymerase chain reaction (PCR) assays: (i) semi‐nested PCR, against sequences encoding framework regions FR3, FR2 and FR1c of the variable chain IgH gene in B cells present in the MSG infiltrate; and (ii) the PCR–enzyme‐linked immunosorbent assay (ELISA) technique, against the major and minor breakpoint regions of the Bcl‐2 oncogene coupled with a variable segment of the IgH to assess the Bcl‐2/JH translocation. When FR3, FR2 and FR1c primers were employed, we detected B cell monoclonality in 87% of the SS patients and 19% of the control subjects. The association between inflammation severity of the MSG pattern and the presence of B cell clonality was found to be statistically significant (P < 0·01). We concluded that the presence of B cell clonality in MSG can be used as a index of an altered microenvironment favouring the development of lymphoma in SS patients.     

Frequent occurrence of identical heavy and light chain Ig rearrangements

Single-cell PCR analyses of expressed Ig H and L chain sequences presented here show that certain rearrangements occur repeatedly and account for a major segment of the well-studied repertoire of B-1 cell autoantibodies that mediate the lysis of bromelain-treated mouse erythrocytes, i.e. antibodies reactive with phosphatldyicholine (PtC). We repeatedly isolated at least 10 different types of VH region rearrangements, involving three distinct germline genes, among FACS- sorted PtC-binding B-1 cells from three strains of mice (C57BL/6J, BALB/c and C.B-17). The predominant rearrangement, VH11-DSP-JH1 (VH11 type 1), has been previously found in anti-PtC hybridomas in several studies. We show that within each of six mice from two strains (C57BL/6J and BALB/c), unique instances of IgH/IgL pairing arose either from different B cell progenitors prior to IgH rearrangement or from pre-B cells which expanded after IgH rearrangement but prior to IgL rearrangement. Together with other recurrent rearrangements described here, our findings demonstrate that clonal expansion of mature B cells cannot account for all repeated rearrangements. As suggested by initial studies of dominant idiotype expression, these findings confirm that clonal expansion is only one of the mechanisms contributing to the establishment of recurrent rearrangements.