IgH基因重排在B细胞非霍奇金淋巴瘤诊断中的应用

正常淋巴细胞在发育中是多克隆性质,但恶性肿瘤表现为单克隆性基因重排。所以,通过基因重排检测不仅可以鉴别淋巴组织是肿瘤性增生还是反应性增生,而且使准确判断细胞起源,完善淋巴瘤的分型成为可能。我们拟了解在本地区不同病理类型B细胞非霍奇金淋巴瘤（NHL）IgH基因单克隆性重排的发生生率,旨在为鉴别部分淋巴组织是反应性增生还是肿瘤性增生,推测肿瘤细胞起源,指导治疗及判断预后提供参考指标。     

PCR amplification and high throughput sequencing of immunoglobulin heavy chain genes from formalin-fixed paraffin-embedded human biopsies

The use of high throughput sequencing (HTS) technologies in biomedicine is expanding in a variety of fields in recent years. The 454 system is an HTS platform that is ideally suited to characterize B cell receptor (BCR) repertoires by sequencing of immunoglobulin (Ig) genes, as it is able to sequence stretches of several hundred nucleotides. Most studies that used this platform for antibody repertoire analyses have started from fresh or frozen tissues or peripheral blood samples, and rely on starting with optimal quality DNA. In this paper we demonstrate that BCR repertoire analysis can be done using DNA from formalin-fixed paraffin-embedded (FFPE) human tissue samples. The heterogeneity of BCR repertoires we obtained confirms the plausibility of HTS of DNA from FFPE specimens. The establishment of experimental protocols and computational tools that enable sequence data analysis from the low quality DNA of FFPE tissues is important for enabling research, as it would enable the use of the rich source of preserved samples in clinical biobanks and biopsy archives.     

石蜡淋巴瘤组织中IgH FR3区基因重排引物分析

目的 利用生物信息学方法分析免疫球蛋白重链(IgH)框架区(FR3)基因重排引物并探讨其在石蜡包埋非霍奇金淋巴瘤(NHL)组织中应用价值.方法 通过Chustal W 软件比较44条有效的IgH可变区和6条J区的基因片段,选取IgH FR3区3对(P1,P2,P3)基因重排引物,其中,P2为改进的引物.另选一对TCRγ脚引物作为T细胞淋巴瘤重排引物,通过PCR扩增,检测经形态学及免疫组织化学确诊的144例石蜡包埋组织标本,包括113例B细胞淋巴瘤、24例T细胞淋巴瘤和7例淋巴结反应性增生组织.以DG75淋巴瘤细胞系DNA作为对照组.结果 引物对P1、P2、P3在B细胞淋巴瘤检出阳性率分别为71.7%(81/113),82.3%(93/113)和76.1%(86/113),三者检出率差异无统计学意义;在T细胞淋巴瘤检出率分别12.5%(3/24)、12.5%(3/24)、16.7%(4/24).将P1和P2引物组合分析,B细胞淋巴瘤阳性检出率可以达到92.3%.以上重排引物在7例反应性淋巴结中均未检出.结论 3对IgH FR3区中,新改进的P2引物在B细胞淋巴瘤中的检出率最高(82.3%).2对IgH FR3区引物联合检测可明显提高石蜡组织中B细胞淋巴瘤的检出率.     

The application of a PCR technique for the detection of immunoglobulin heavy chain gene rearrangements in fresh or paraffin-embedded skin tissue

Although detection of a clonal sequence of the heavy chain gene of immunoglobulin by the polymerase chain reaction (PCR) is frequently used to assess lymphoid infiltrates in skin biopsy specimens, there are no data on the sensitivity and specificity of this test in detecting clonal B cell populations. Having refined a PCR technique for the detection of immunoglobulin heavy chain (IgH) gene rearrangement in both fresh and formalin-fixed, paraffin-embedded skin samples, we undertook to define the role of this assay in the diagnostic setting. Thirty-one cases of cutaneous B cell lymphoma (CBCL), 19 cases of B cell pseudolymphoma (lymphocytoma cutis), 34 cases of benign lymphocytic infiltrates of the skin and one case of cutaneous T cell lymphoma (CTCL) were studied using the polymerase chain reaction assay. All biopsies were formalin-fixed, paraffin-embedded skin sections apart from 13 of the 31 CBCL specimens which were fresh skin specimens. DNA from the framework region 3 (FR3) sequence of the IgH genes was amplified to ascertain the presence of a clonal IgH gene rearrangement. The findings were correlated with histological and immunophenotyping results on all samples. The assay performed with 73% sensitivity and 100% specificity, comparable to results obtained examining fresh lymphoid tissue specimens from patients with B cell tumours. The results indicate that this technique is a useful tool in the work up of suspected CBCL and in differentiating between CBCL and mixed lymphocytic infiltrates, a clearly important distinction with regards to prognosis and treatment.     

Improved PCR method for detecting monoclonal immunoglobulin heavy chain rearrangement in B cell neoplasms.

AIMS: To develop a simple, optimised, polymerase chain reaction (PCR) based method for detecting the rearranged immunoglobulin heavy chain (IgH). METHODS: Using as primers oligonucleotides (Fr2A, Fr2B) homologous to the conserved sequences to the framework II region and the joining (JH) region, 25 patients with B cell lymphoproliferative disorders, previously characterised by Southern blotting, and three patients with light chain myeloma were studied. RESULTS: The PCR product from a polyclonal B cell population showed a broad band when analysed on a 3% agarose gel; DNA from B cell lines and B lymphoproliferative disorders showed a discrete band. Specificity of the amplification was confirmed by cloning and sequencing the amplified product as well as by Southern blotting with an internal probe homologous to the framework 3 region. Primers Fr2A and Fr2B detected monoclonality in three patients with light chain myeloma, while primers directed against the FrIII region showed a polyclonal response. CONCLUSIONS: Deletions and extensive somatic mutations within the FrIII region may give false negative results with primers homologous to the region. A PCR using the method described, with a repertoire of primers homologous to the FrII and FrIII regions, will therefore increase the frequency of detection of monoclonality.     

PCR-based clonality analysis of B-cell lymphomas in paraffin-embedded tissues: diagnostic value of immunoglobulin kappa and lambda light chain gene rearrangement investigation

Polymerase chain reaction (PCR)-based analysis, employed for detecting immunoglobulin heavy chain (IgH) gene rearrangements, has become a diagnostic tool widely used in the investigation of B-cell lymphomas, but the overall sensitivity of these methods does not exceed 80%, notably in germinal center (GC) and post-GC B-cell origin lymphomas. Many PCR strategies devised for detecting immunoglobulin light chain (IgL) gene rearrangements have been developed to enhance the clonality detection rates. However, the feasibility of these methods in routine clinical diagnosis using paraffin-embedded tissues has not yet been investigated sufficiently. We studied a large series of 108 cases of B-cell lymphomas, as well as 20 reactive lymphoid tissues using degenerate primers to amplify immunoglobulin kappa (Igkappa) and lambda (Iglambda) light chain genes. B-cell clonality was further investigated using semi-nested PCR for IgH gene rearrangements. B-cell clonality was detected in 74%, 56.5%, and 43.5% of cases using IgH, Igkappa, and Iglambda PCR, respectively. By combining these methods, the clonality detection rate increased to 93.5%. Only polyclonal patterns were noted in reactive lymphoid samples. We concluded that in addition to the established methods for IgH analysis, a PCR-based approach for IgL gene rearrangements analysis improves the clonality detection rate in over 90% of B-cell lymphoma cases using routine histological specimens with poor preservation of the genomic DNA.     

Analysis of the immunoglobulin heavy chain gene variable region of intravascular large B-cell lymphoma

Intravascular large B-cell lymphoma (IVLBL) is a rare neoplasm characterized by proliferation of lymphoma cells within the blood vessels. The cell origin of IVLBL has not yet been determined. We examined cell lineage, with immunohistochemical staining and molecular analysis, using polymerase chain reaction (PCR) of the variable region of the immunoglobulin heavy chain (Ig-VH) gene. We also investigated the cell origin using direct sequence analysis of the complementary-determining region 2 (CDR2) and framework region 3 (FR3) in six cases, consisting of two male and four female patients. The sequences in five cases showed frequent mutations. The percent homology to their closest germline genes ranged from 74.7 to 91.8%. However, one case showed a percent homology of 99.4% in CDR2 and FR3 of Ig-VH. All cases showed rearrangements of VH3 family genes. Interestingly, three of the IVLBL cases with hypermutated IgH genes showed the expression of CD5. Therefore, expression of CD5 in lymphoma cells does not indicate that the origin of IVLBL is the same as mantle cell lymphoma having the character of CD5 expression, which develops in pre-germinal center cells. Our results indicate that most IVLBLs may originate in the post-germinal center cells, based on the presence of somatic mutation in VH genes, although some heterogeneous cases are intermingled within IVLBL.     

Monoclonal analysis of immunoglobulin heavy chain and T-cell receptor gamma chain gene rearrangements in paraffin-embedded tissues from non-Hodgkin lymphoma patients

Gene rearrangement is an important diagnostic marker of malignant lymphoma, and rearrangements of the immunoglobulin heavy chain (IgH) and T-cell receptor gamma chain (TCRgamma) genes are useful markers for Band T-cell lymphoma, respectively. A polymerase chain reaction (PCR) can be used to analyze clonality in formalin-fixed paraffin-embedded specimens. We performed 73 monoclonal analyses of such specimens of lymphoma tissues and examined the ability to diagnose malignant lymphoma by this method. Monoclonality of lymphoma cells was found in 71.9% and 78.9% of specimens with IgH and TCRgamma gene rearrangements, respectively. Therefore, in diagnosing cases of non-Hodgkin lymphoma in which neoplasms and reactive lymphoid tissues are difficult to identify by morphological and immunohistochemical findings, PCR-based monoclonal analysis may allow confirmation of a pathological diagnosis of malignant lymphoma.     

聚合酶链反应技术检测非霍奇金淋巴瘤抗原受体基因重排的局限性

病理形态学和免疫组织化学有效标记,结合分子病理学的检测是当前临床病理诊断的发展趋势。近年来这种三结合的诊断模式已在淋巴瘤的病理诊断中得到应用。各类淋巴瘤的病理形态学特征是病理诊断的基石,免疫组织化学有助于正确的诊断及分类,仅靠这二者,恶性淋巴瘤的误诊率仍较高,采用分子病理学技术检测病理标本中淋巴瘤特征性的分子标记具有重要的参考价值。其中运用聚合酶链反应（PCR）技术检测IgH／TCR基因的重排是目前常用的淋巴瘤的分子病理学的方法之一。     

B淋巴细胞增生性疑难病例中IgH基因克隆性重排的分析

目的 探讨IgH基因克隆性重排对B淋巴细胞增生性疑难病变的辅助诊断价值.方法 检测77例B淋巴细胞增生性疑难病例中IgH基因的克隆性重排情况,均采用BIOMED-2系统IgH克隆性试剂盒中FR1、FR2、FR3三组家族引物进行PCR及聚丙烯酰胺凝胶电泳,硝酸银染色后观察,并对照最终病理诊断进行分析.结果 77例病变的最终病理诊断:B淋巴细胞反应性增生12例,不能排除B淋巴细胞不典型增生或淋巴瘤20例,B细胞性淋巴瘤45例.三组中FR1、FR2和FR3至少有一个为阳性的比值分别为2/12、11/20(55%)和36/45(80%).B细胞性淋巴瘤中,FR1、FR2和FR3的阳性率分别为60%(27/45)、60%(27/45)、56%(25/45),其类型有边缘区B细胞性淋巴瘤20例(其中黏膜相关淋巴组织型结外边缘区淋巴瘤18例,结内边缘区淋巴瘤2例),弥漫性大B细胞淋巴瘤7例,滤泡性淋巴瘤7例,套细胞性淋巴瘤1例,Burkitt淋巴瘤1例,浆细胞瘤4例,不能分型5例.FR1、FR2和FR3三者检测均为阴性但仍诊断为淋巴瘤9例(20%),其中1例后来出现肝脏B细胞淋巴瘤.对IgH基因重排阳性的B淋巴细胞反应性增生和不典型增生14例的随访结果,4例重新取活检后诊断为B细胞性淋巴瘤,其中3例IgH基因重排检测为阳性.结论 联合检测IgH基因FR1、FR2和FR3克隆性重排对B淋巴细胞增生性疑难病变诊断有重要的辅助价值;对形态改变和免疫表型诊断淋巴瘤依据不足而基因重排阳性者,重取活检或随访有一定价值;对阴性病例有必要补充IgH基因重排及IgK和IgL基因重排的检测以提高检测敏感性.     

The use of the polymerase chain reaction in the diagnosis of B-cell lymphomas from formalin-fixed paraffin-embedded tissue

In this study the use of the polymerase chain reaction (PCR) to detect monoclonality in B-cell lymphoid proliferations in archival formalin-fixed paraffin-embedded tissue was assessed. Using consensus primers against the framework 3 (FR 3) region of the immunoglobulin heavy chain gene (IgH), PCR analysis was performed on 29 low grade B-cell non-Hodgkin's lymphomas. Cases of benign lymphoid hyperplasia served as polyclonal controls. Sequenced cases of acute lymphoblastic leukaemia served as positive controls. In the lymphomas, monoclonality could be demonstrated in 18 of 29 (62%) cases. Only five of 11 (45%) follicle centre cell lymphomas were positive by this method whilst the success rate for the remainder was 13 of 18 (72%). None of the polyclonal controls gave false positive results although occasional non-specific dominant bands were present which disappeared on repeating the experiments. These results show that this method will identify monoclonality in 62% of low grade B-cell non-Hodgkin's lymphomas in archival material. The success rate is increased to 72% if follicle centre cell lymphomas are excluded. Thus, this method is a useful adjunctive test to aid diagnosis in lymphoid infiltrates when standard morphology and immunohistochemistry are equivocal.     

原发性睾丸恶性淋巴瘤克隆性IgH基因重排的检测

目的：探讨原发性睾丸淋巴瘤克隆性IgH基因重排的检测方法。方法：用半巢式PCR方法和聚丙烯凝胶（PAGE）－银染法检测14例睾丸恶性淋巴瘤的IgH基因重排。结果：与免疫组化对照，PCR检测的睾丸原发性B细胞淋巴瘤FR3A检出测率为85.71%，FR2的检出率为64.29%，而二者的综合检出率为100％，互补性较强。结论：半巢式PCR方法及PAGE－银染法对于原发性睾丸细胞淋巴瘤IgH基因的检出率有较高的灵敏度，FR3A和FR2引物的共同应用可以有效提高综合检出率，在目前情况下，应用PCR方法来鉴别恶性淋巴瘤，必须结合HE形态和免疫表型等综合判断。     

Restricted immunoglobulin constant heavy G chain genes in primary immunodeficiencies

Some primary immunodeficiencies (PIDs) express low serum levels of antibodies. The constant heavy G chain (IGHG) genes, also representing Fc domains of γ3, γ1 and γ2 on chromosome 14q32.3, genotyped by the alternative IgG subclass allotypes, found in four fixed IGHG haplotypes, designating four B cell variants, were identified by a competitive ELISA and double immunodiffusion. IGHG genes were hypothesized to contribute to the development of PIDs. From 235 Caucasian patients, the homozygous IGHGbf-n/bf-n diplotype (B^bf-n/B^bf-n cells) dominated significantly in 43 IgG2 deficiency (OR 6.0), 32 common variable immunodeficiency (OR 4.6) and 22 Ataxia telangiectasia (OR 3.0) and the IGHGga-n/ga-n diplotype (B^ga-n/B^ga-n cells) dominated in 53 IgG3 deficiency (OR 10.6) and 21 Wiscott–Aldrich syndrome (OR 4.1). 62 IgA deficiency patients were dominated by both diplotypes (OR 2.3 and OR 2.8 respectively). Restricted IGHG genes, restricted IgG allotypes (Fc domains) and restricted B cells are significant in PIDs for diagnosis, treatment and pathogenetic mechanisms.     

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.     

Detection of immunoglobulin gene rearrangement in B-cell malignancies

Rearrangement of the immunoglobulin heavy chain (IgH) gene has been used as a marker of lineage and clonality in the diagnosis of B lymphoproliferative disorders. A number of PCR-based techniques have been developed to overcome the disadvantages of Southern blotting, the standard technique in detecting IgH gene rearrangement. Using an established seminested PCR technique with consensus primers to the V and J regions of the IgH gene, we analysed DNA prepared from peripheral blood and/or bone marrow specimens from 30 cases of known B cell malignancies (16 chronic lymphocytic leukemia, 11 acute lymphoblastic leukemia and 3 Non-Hodgkin Lymphoma), 3 cases of T lymphoproliferative disease and 3 cases of reactive lymphocytosis diagnosed in Hospital UKM to detect rearranged IgH gene. We found that monoclonality as represented by the presence of rearranged IgH gene were demonstrated in all the 30 cases. The PCR findings showed 100% concordance with the Southern blot analysis results which also showed rearranged IgH bands in all the 30 cases. We also found that none of the cases of T lymphoproliferative diseases and reactive lymphocytosis showed presence of rearranged IgH band, suggesting that the amplification using the IgH primers is lineage-specific. In conclusion, we find the PCR a useful method to detect IgH gene rearrangement in peripheral blood and bone marrow specimen. Since the PCR results are comparable to that of the Southern blotting in demonstrating B cell monoclonality and owing to its many advantages we feel that it can replace the Southern blot technique for the diagnosis of B cell malignancies.     

Consensus primers for detecting monoclonal immunoglobulin heavy chain rearrangement in B cell lymphomas

AIMS: To demonstrate the usefulness of polymerase chain reaction (PCR) methodology with both the FR2A/LJH/VLJH and the FR1c/LJH/VLJH primer sets for detecting monoclonal immunoglobulin heavy chain (IgH) gene rearrangement in B cell non-Hodgkin lymphomas (B-NHLs). METHODS: Eighty three patients with B-NHL were enrolled in this study. DNA was isolated from paraffin wax embedded sections and amplified by PCR to determine the sequences of the rearranged IgH gene. Each PCR product was subcloned. Cycle sequences and sequence analyses were done to determine the clone specific IgH variable region (VH) sequences. RESULTS: Clonal IgH gene rearrangements were detected in 45 cases with FR2a/JH/VLJH and in 14 of the remaining cases with FR1c/JH/VLJH. Most of the cases detectable with FR2a/JH/VLJH were derived from VH3 and VH4 families. Five of six cases in the VH1 family and one in the VH7 family were amplified with the FR1c/JH/VLJH primer set only. CONCLUSION: The detection rate of IgH rearrangement in B-NHLs can be increased by using both FR2A/LJH/VLJH and FR1c/LJH/VLJH, and these two primer sets are suitable for routine PCR methodology. Moreover, each primer set appears to be closely related to VH family specificity.     

Diversity of immunoglobulin heavy chain gene segment rearrangement in B lymphoblastoid cell lines from X-linked agammaglobulinemia patients

X-linked agammaglobulinemia (XLA) is characterized by an arrest in early B lymphocyte differentiation. Precursor B cells are present in the bone marrow (BM), whereas peripheral blood B cell numbers are severely decreased. A series of Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) was established from peripheral blood of three XLA patients belonging to one pedigree. These BLCL manifested productive VHDJH rearrangements and a random utilization of the VH families. The CDR3 regions of the rearrangements varied in length from 12 to 47 nucleotides and included N regions in all cases. The results supported the conclusion that the few B lymphocytes in peripheral blood of XLA patients exhibit all mechanisms that generate immunoglobulin (Ig) heavy (H) chain diversity. However, no evidence for somatic mutation was found. Within the VH3 family 50% of the expressed VH gene segments belonged to a single subgroup and within the VH4 family a preferential utilization of one VH4 gene element was observed. The utilization of H chain joining (HH) elements was biased to JH4 and JH6 and a high percentage of the CDR3 regions was found to be generated by unconventional mechanisms, such as multiple D usage and the fusion of D elements to D segments with irregular recombination recognition signals. These unique features of the recombined and expressed VHDJH regions in XLA may explain the inability of XLA patients to respond to a variety of antigens. Alternatively, they could be secondary to a B lymphocyte maturation defect in XLA.