Recombination pattern of the TCR γ locus in human peripheral T-cell lymphomas

The recombination events of the γ and β T-cell receptor (TCR) loci were analysed in a series of 39 peripheral T-cell lymphomas (PTCLs) in association with the expression of TCR chains. In TCR αβ PTCLs, 22/23 cases showed a γ-gene rearrangement while only 18/23 showed a concomitant β-gene rearrangement. The germline configuration of the β locus was found in angioimmunoblastic lymphadenopathy and lymphoepithelioid lymphomas. Three γδ PTCLs rearranged both γ and β genes. TCR silent PTCLs showed three different patterns of γ- and β-gene rearrangements. Three cases were in germline configuration for both loci; five cases had a rearranged γ and a germline β locus; and five cases had the two loci rearranged. Regarding the variable genes in the γ-rearranged alleles, members of the VγI subgroup were the most frequently presented (39/50), followed by VγII, VγIII, and VγIV (9/50, 1/50, and 1/50, respectively). Joining segment usage was as follows: J1 or J2 (32/50), JP1 or JP2 (17/50), and JP (1/50). Taken together, these data demonstrate that the γ locus is more frequently rearranged whatever the TCR expression. The γ-locus analysis provides a better diagnostic yield than the β locus in the study of PTCL clonality.     

Clonal T-cell receptor γ-chain gene rearrangement by PCR-based GeneScan analysis in advanced cutaneous T-cell lymphoma: a critical evaluation

Detection of clonal T-cell receptor γ (TCRγ)-chain gene rearrangement is a promising approach to distinguish between cutaneous T-cell lymphomas (CTCLs) and reactive T-cell infiltrates. Despite the improved sensitivity by using the polymerase chain reaction (PCR) rather than Southern blot analysis, monoclonality could be demonstrated in only 53–90 per cent of CTCL biopsies in recent studies. In the present study, formalin-fixed, paraffin-embedded specimens of 21 selected patients with clear-cut advanced-stage CTCL were analysed using a semi-nested TCRγ PCR with newly developed consensus primer pairs. Detection of PCR products was done by GeneScan analysis (GSA); this technique is advantageous due to its sensitivity and accuracy in the detection and size determination of PCR products and it is easier to interpret than direct read-outs from TGGE or DGGE gels. In serial dilution experiments, TCRγ-PCR-GSA allowed the detection of clonal, rearranged T-cells with a high in vitro sensitivity against a polyclonal background (1–6 per cent). Despite the selection of clear-cut, advanced-stage CTCL cases, however, dominant clonal TCRγ-chain gene rearrangement was found in only 16 of the 21 patients analysed, indicating an overall clinical sensitivity of 76 per cent. Specificity was evaluated using biopsy specimens from 21 control patients suffering from long-standing psoriasis (n = 13) and eczema (n = 8). Surprisingly, GeneScan profiles showing apparently single dominant peaks were detected in 14 per cent of these skin lesions, but these profiles turned out to be pseudo-monoclonal by repeated determinations. In conclusion, TCRγ-PCR-GSA does not suffice reliably to exclude malignancy, due to its limited clinical sensitivity, but with precautions taken to detect pseudo-monoclonality and to secure specificity, TCRγ-PCR-GSA is a valuable instrument in the diagnosis of CTCL. Copyright © 1999 John Wiley & Sons, Ltd.     

Rearrangement of the T-cell receptor delta chain gene in T-cell lymphomas with a mature phenotype.

The configuration of the T-cell receptor (TCR) delta chain gene was assessed using restriction fragment analysis and the Southern blot technique in 39 T-cell lymphomas with a mature immunophenotype. The TCR delta gene was rearranged in four lymphomas although the gamma/delta TCR was not expressed in two cases studied. The TCR delta gene was the only TCR gene rearranged in two cases. Each lymphoma with TCR delta gene rearrangement had an aberrant T-cell immunophenotype and three cases were of the large cell anaplastic type. The TCR delta gene was deleted in 22 cases and was in the germline configuration in 13 lymphomas. Deletion of the TCR delta gene was characteristic of mycosis fungoides, adult T-cell leukemia/lymphoma (human T cell leukemia-lymphoma virus positive), and Lennert's lymphoma, and was not identified in angiocentric lymphomas. In eight cases with TCR delta deletion, however, a large number of polyclonal (presumably reactive) T cells were present and, in these lymphomas, the authors could not determine if TCR delta gene deletion occurred in the polyclonal T cells, the neoplastic cells, or both cell populations. The authors conclude that the TCR delta gene is usually deleted in mature T-cell lymphomas, as would be expected in alpha/beta TCR T cells. However, TCR delta gene rearrangement is detectable in approximately 10% of cases. Analysis of this locus may be useful diagnostically, as it occasionally may be the only molecular marker of clonality in mature T-cell lymphomas T-cell receptor delta chain gene rearrangement also is found most often in lymphomas of the large cell anaplastic type.     

BIOMED-2系统引物用于检测T细胞淋巴瘤石蜡样本T细胞受体γ基因重排的研究

目的 了解BIOMED-2系统T细胞受体(TCR)γ引物组合对T细胞淋巴瘤的常规石蜡包埋组织样本中TCR基因重排的检出情况及其实用性.方法 用酚/氯仿法提取55例各种组织类型的T细胞淋巴瘤石蜡包埋组织样本的DNA并通过扩增看家基因β-globin检测其质量,利用BIOMED-2系统TCR-γ引物组合和TCR-γ基因通用型引物(TVG/TJX)对55例进行TCR基因重排检测,比较二者的检出率并进行统计学分析.结果 BIOMED-2系统TCR-γ引物组合和TCRγ基因通用型引物(TVG/TJX)的TCR基因重排检出率分别为76.4%和60.0%,前者高于后者,二者的差异无统计学意义(P>0.05).结论 BIOMED-2系统TCRγ引物组合适用于本组T细胞淋巴瘤石蜡包埋组织样本的TCR基因重排检测.     

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.     

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.     

Immunohistochemistry and gene rearrangement studies in the diagnosis of malignant lymphomas: a comparison of 152 cases.

One hundred fifty-two cases (155 specimens) of lymphoproliferative disorders were studied by immunohistochemistry and gene rearrangement analysis. Ninety-five of 96 B-cell lymphomas (99%) showed genotypic B-cell monoclonality. Of these, five cases had rearranged T-cell receptor (TCR) beta chain gene in addition to immunoglobulin heavy chain (IgH) and kappa light chain (Ig-K), one case had rearranged IgH and TCR-gamma chain but not Ig-K or TCR-beta, and two cases had only Ig-K rearrangement. One exceptional case in the B-cell lymphoma group had unrearranged, germline genotypes. In contrast, only 10 of 19 (53%) phenotypic T-cell lymphomas had rearranged TCR-beta, eight with concurrent TCR-gamma rearrangement. Of the remaining nine cases, six had germline configuration, two had rearranged Ig-K only, and one had both IgH and Ig-K rearrangement. This last case was reclassified as T-cell predominant, B-cell lymphoma. Thirteen of 16 cases of Hodgkin's disease had germline configuration; three cases had rearranged IgH and Ig-K, of which two were lymphocyte predominant with light chain monoclonality and one was a recurrence. Among 21 reactive lesions, 17 had germline configuration and four had rearranged IgH and Ig-K genes. Of these four cases, two were orbital lesions, one was a partially involved lymph node, and one developed a nodular lymphoma 9 months later. Our results indicate that almost all B-cell lymphomas have IgH and/or Ig-K rearrangement. In contrast, peripheral T-cell lymphomas have greater genotypic heterogeneity, and germline patterns for TCR genes are not uncommon. Reactive lesions and Hodgkin's disease tend to retain germline configuration, and any exception is often associated with an unusual clinical setting and/or histology. Genotypic analysis is thus most indicated in B-cell lymphomas with equivocal immunohistochemistry findings, T-cell lymphomas, and atypical cases of Hodgkin's disease and reactive lesions.     

Cytotoxic protein expression in natural killer cell lymphomas and in αβ and γδ peripheral T-cell lymphomas

Lymphomas with T-cell phenotype represent a heterogeneous group of diseases differing in histopathology, tumour site, and cell origin. They include peripheral T-cell lymphomas (PTCLs) derived from αβ cells, but also some recently recognized entities such as γδ hepatosplenic lymphomas and natural killer (NK) cell lymphomas. Only a few studies have investigated the possibility that at least some PTCLs could be derived from lymphocytes with cytotoxic potential. In order to investigate this possibility, 60 cases of PTCL, including 27 cases expressing the αβ T-cell receptor (TCRαβ), 15 TCRγδ cases and 18 cases expressing neither TCR (TCR silent), as well as 14 cases of NK-cell lymphomas, were studied by immunohistochemistry for the expression of TIA-1, perforin, and granzyme B proteins. Expression of TIA-1 is characteristic of cytotoxic cells regardless of their activation status, whereas expression of perforin and granzymes is highly increased in activated cytotoxic cells and correlates with the induction of cytolytic activity. All NK-cell lymphomas (11 sinonasal, three systemic cases) expressed TIA-1, perforin, and granzyme B in most tumour cells. All γδ PTCLs (15 cases) expressed TIA-1 protein in most tumour cells, with a different cytotoxic antigen profile in hepatosplenic γδ PTCL (TIA-1+, perforin−, granzyme B−) and in non-hepatosplenic γδ PTCLs (three nasal, one skin, one lung), the latter expressing the three cytotoxic proteins. Of the 45 cases of αβ and TCR silent PTCL, 15 (33 per cent) were considered to be derived from cytotoxic lymphocytes with expression of at least one cytotoxic protein (TIA-1, 15/45; perforin, 10/41; granzyme B, 14/38) in tumour cells. This cytotoxic protein expression appeared to be related to the site of localization, since 7/13 (54 per cent) extranodal and only 8/32 (25 per cent) nodal αβ and TCR silent PTCLs expressed TIA-1, and to histology, since this pattern was observed in a proportion of anaplastic (6/8, 75 per cent) and pleomorphic (8/17, 47 per cent) lymphomas, but not in AILD-type NHL (0/16). Taken together, our data suggest that NK-cell lymphomas and non-hepatosplenic γδ PTCLs represent tumours of activated cytotoxic NK cells and γδ T cells, respectively; that hepatosplenic γδ PTCLs represent tumours of non-activated cytotoxic γδ T cells; and that a small proportion of αβ and TCR silent PTCLs, mostly extranodal cases, or nodal anaplastic lymphomas, represent tumours of cytotoxic T cells. © 1997 John Wiley & Sons, Ltd.     

Immunophenotyping and gene rearrangement analysis provide additional criteria to differentiate between cutaneous T-cell lymphomas and pseudo-T-cell lymphomas.

Differentiation between cutaneous pseudo-T-cell lymphomas and cutaneous T-cell lymphomas (CTCLs) may be extremely difficult. In this study, it was investigated whether demonstration of an aberrant phenotype and detection of clonal T-cell receptor gamma (TCR gamma) gene rearrangements can be used as additional differential diagnostic criteria. Immunohistochemical studies and TCR gamma gene rearrangement analysis using a polymerase chain reaction with primers specific for V gamma 1-8 and V gamma 9 gene segments in combination with denaturing gradient gel electrophoresis (PCR/ DGGE) were performed on frozen material of 11 pseudo-T-cell lymphomas and 17 CTCLs, including 9 cases of mycosis fungoides (MF) and 8 pleomorphic CTCLs. Clonal TCR gamma gene rearrangements were found in 66% of patch/plaque-stage MF and 100% of tumor-stage MF and pleomorphic CTCL, but not in any of 10 pseudo-T-cell lymphomas studied. Aberrant expression of CD2, CD3, and/or CD5 antigens was noted in 3 of 6 (50%) cases of patch/plaque-stage MF, all three cases of tumor-stage MF, and 5 of 8 (62%) pleomorphic CTCLs, but not in any of the 11 pseudo-T-cell lymphomas. Moreover, in pseudo-T-cell lymphomas exhibiting a nodular or diffuse growth pattern, a considerable admixture with reactive CD8+ T cells (15 to 60%), B cells (up to 20%), and macrophages was a characteristic finding. In conclusion, the results of this study suggest that demonstration of clonal TCR gene rearrangements and an aberrant phenotype, as well as demonstration of many admixed CD8+ T cells and B cells can be considered as useful additional criteria in the differentiation between pseudo-T-cell lymphomas and CTCLs.     

T-cell receptor variable (V) gene usage by lymphoid populations in T-cell lymphoma.

A panel of monoclonal antibodies specific for TcR V gene families was used to study TcR V region expression in 28 cases of malignant and reactive T-cell expansions including four cases of mixed cellularity Hodgkin's disease (HD) and five reactive cases. TcR V beta 5 gene products were represented in three cases of lymphoblastic malignancy (V beta 5.1, V beta 5.2) and two cases of peripheral T-cell lymphoma (PTCL) (V beta 5.1). In the PTCL cases, the expanded family was found in the absence of clonal TcR gene rearrangements and in one of these cases with Ig JH and Ck clonal gene rearrangements consistent with the presence of a phenotypically and histologically undetectable clonal B-cell population. In a third PTCL case not investigated for genotype, the TCR V alpha 12 family was overrepresented. Expanded TcR V alpha 2 and V beta 5.1 families were identified in HD and V beta 8 and V beta 5.2/V beta 5.3 families in a reactive lymph node and CD3 and CD8-positive blood lymphocytosis respectively. Further study of PTCL and related entities are needed to establish whether expanded TcR families are common in those cases that fail to exhibit clonal TcR gene rearrangement.     

Clonal Epstein-Barr virus genome in T-cell-rich lymphomas of B or probable B lineage.

Seventeen nodal lymphomas (originally diagnosed as T-cell lymphomas based on histological features and immunohistochemical staining results) were studied for the presence of Epstein-Barr virus (EBV) genome, and the results correlated with immunoglobulin and T-cell receptor gene rearrangement analyses performed on the same tissue samples. All four EBV positive cases had clonal rearrangement of the joining region of the immunoglobulin heavy chain (IgJH) gene without clonal T-cell receptor beta-chain (TCR beta) gene rearrangement. Of these, two cases also showed clonally rearranged light chain gene, and they were reclassified as T-cell rich B-cell lymphomas (TRBL). The other two cases lacked clonal kappa or lambda light chain rearrangement and they were reclassified as T-cell rich lymphomas of probable B lineage, based on their isolated IgJH clonal rearrangement. These B-cell lymphomas may be easily misdiagnosed as T-cell lymphomas owing to the presence of an abundant reactive T-cell infiltrate masking the tumor population. The florid T-cell reaction may represent an unusual host response towards a clonal proliferation of EBV bearing B cells.     

Detection of T-cell receptor-gamma gene rearrangement in lymphoproliferative disorders by temperature gradient gel electrophoresis

OBJECTIVE: Polymerase chain reaction amplification of DNA for T-cell receptor (TCR) gene rearrangement analysis is helpful in the evaluation of T-cell lymphoproliferative disorders. Detection of polymerase chain reaction products is limited by the poor resolution of bands analyzed by agarose or polyacrylamide gel electrophoresis. To improve the detection of a clonal T-cell population, we used temperature gradient gel electrophoresis (TGGE) as an alternative method for analysis of TCR gene rearrangement. DESIGN: One hundred eighteen archival DNA samples were randomly selected based on previous Southern blot analysis results. Samples included 58 T-cell neoplasms with positivity for TCR beta gene rearrangement, 22 cases of reactive hyperplasia with germline pattern for both TCR beta and J(H), and 38 patients with B-cell lymphoma. MOLT-16, a T-cell lymphoblastic cell line, was used for the sensitivity assay. Polymerase chain reaction was performed using GC-clamped multiplex primers to amplify the TCR gamma locus and was analyzed by TGGE. The range of temperature gradients was empirically determined for optimal resolution of bands. RESULTS: The sensitivity of TGGE was 0.1% when DNA from the MOLT-16 cell line was serially diluted with DNA from reactive lymphoid tissue. Fifty-four (93%) of 58 T-cell neoplasms with TCR beta gene rearrangements showed rearrangement patterns by TCR gamma TGGE, and only 1 of 60 samples (reactive or B-cell lymphomas) showed evidence of gene rearrangement by TGGE. Patients with T-cell neoplasm and involvement of multiple sites showed an identical migration pattern by TGGE analysis. CONCLUSION: We demonstrate that TGGE is an effective method for analysis of TCR gene rearrangement in the evaluation of nodal and extranodal lymphoid lesions.     

T-cell lymphomas in children

T-cell lymphomas in children are rare and compromise a rather limited spectrum of entities. Relatively frequently observed are precursor T-cell lymphomas mainly presenting as mediastinal tumors with or without leukemia and ALK-positive anaplastic large cell lymphomas (ALCL) with nodal or extra nodal manifestations. In contrast to adults, where peripheral T-cell lymphoma (PTCL) is the most frequent T-cell neoplasia, PTCL in children is exceptional and remains a challenging diagnosis even for experienced haematopathologists. Other rare T-cell lymphomas occasionally seen in children are panniculitis like T-cell lymphomas, NK/T-cell lymphomas and hepatosplenic gamma delta-lymphomas. Of note angioimmunoblastic T-cell lymphoma is never seen in children. Mimickers of T-cell lymphomas like viral or immunological disorders in children are more frequent than manifest T-cell lymphomas and knowledge of these reactive conditions and their differential diagnosis is essential. The recognition of typical histopathological and clinical features along with knowledgeable use and interpretation of immunohistochemical and molecular markers are mandatory for a reliable diagnosis of childhood T-cell lymphomas.     

Restricted onset of T cell receptor α gene rearrangement in fetal and neonatal thymocytes

The initial T cell receptor (TCR) α gene rearrangements were analyzed in fetal and neonatal thymocyte hybridomas by Southern blotting. Interestingly, in 30% of all thymocyte hybridomas and in all fetal day 16 thymocyte hybridomas the most proximal Jα50 (ΨJα) gene was rearranged. This rearrangement was found on one chromosome only and mostly in association with a δ rearrangement on the homologous chromosome. Jα50 was rearranged to multiple target genes based on the variable size of the restriction fragments. In addition, δ rearrangement was found with a concomitant α rearrangement in the majority of hybridomas and it was not only associated with Jα50 but with several other rearranged Jα genes as well. Our results clearly demonstrate that T cell precursors are not pre-committed to either δ or α rearrangement but a flexible progenitor responds to multiple regulatory signals during T cell differentiation and they do not support the notion that δrec-ΨJα rearrangement is required for cell commitment to TCR α gene rearrangement.     

小细胞性非特指外周T细胞淋巴瘤病理形态和免疫表型分析

目的 探讨小细胞性非特指外周T细胞淋巴瘤(PTCL,NOS)的临床病理与免疫表型及其病理诊断和鉴别诊断.方法 对5例小细胞性PTCL,NOS进行临床病理回顾性研究和随访,免疫表型检测(SP和EnVision法),以及EBER原位杂交和T细胞受体(TCR)基因重排分析.结果 5例均为男性,平均年龄52.6岁.中位病程1个月.5例中3例为临床Ⅳ期,2例为临床Ⅲ期.4例有全身浅表淋巴结及脾脏肿大,1例有肝肿大.2例有浆膜腔积液.行骨髓检查的4例中,3例有肿瘤累及.1例有外周血自细胞总数和淋巴细胞分类计数升高.主要病理改变为淋巴结结构的破坏和单一形态的小淋巴细胞弥漫性浸润,4例可见少数大的异形细胞散在分布,2例见小血管增生现象.5例之肿瘤细胞均表达两种以上T细胞分化抗原和CD43,表达CD99(3/4),均不表达CD20、末端脱氧核苷酸转移酶、CD56和粒酶B.Ki-67指数为5%-15%.4例行TCR基因重排分析,均存在TCRy基因克隆性重排,1例检出TCRβ基因克隆性重排.EBER原位杂交检测均为阴性.获得3例随访资料,且患者均死亡,平均生存时间21.7个月.结论 小细胞性PTCL,NOS少见,呈高临床分期,预后差,组织形态表现为惰性淋巴瘤.     

Real-time PCR method for the quantitative analysis of human T-cell receptor gamma and beta gene rearrangements

Analyzing the status of T-cell receptor (TCR) gene rearrangements has been an essential part of deciphering the stages of thymocyte development, understanding the β vs. γδ lineage decision, and characterizing T-cell leukemias. Methods such as PCR and quantitative Southern blotting provide useful information, but also have significant shortcomings such as lack of quantitation in the case of PCR and technical challenges in the case of Southern blotting. Here we describe a real-time PCR method that overcomes many of these shortcomings. This new method shows comparable results for the fraction of unrearranged TCRγ and TCRβ genes in human thymocytes and peripheral blood T cells as Southern blotting, and has the advantages of being simple to perform, highly quantitative, and requiring nanogram quantities of DNA. We also describe a real-time PCR method to quantitate T-cell receptor excision circles formed during TCRβ rearrangements.     

Detection of rearrangements of the BCL2 major breakpoint region in follicular lymphomas. Correlation of polymerase chain reaction results with Southern blot analysis.

The translocation t(14;18)(q32;q21) occurs in 70% of follicular lymphomas and places the BCL2 proto-oncogene, normally located at 18q21, under the control of the immunoglobulin heavy chain (IgH) gene at 14q32. Its detection by the polymerase chain reaction (PCR), made possible by the close clustering of most of the BCL2 breakpoints in the major breakpoint region (MBR) of the gene, has numerous clinical applications. Since the PCR covers shorter lengths of DNA than Southern blotting, PCR-based tests may be more susceptible to microheterogeneity in breakpoint location. There have been no published studies of the impact of breakpoint microheterogeneity on the detection rate of this translocation by PCR. We studied 30 follicular lymphomas with the t(14;18), in which a BCL2 MBR rearrangement had previously been demonstrated and mapped by conventional Southern blotting, by PCR with the commonly used IgH and BCL2 MBR primers. Twenty-five cases (83%) had a junction fragment demonstrable by PCR. All three cases in which the MBR rearrangement mapped outside of the 4.3-kb HindIII fragment containing the MBR, as determined by Southern blotting, were negative by PCR. In addition, two cases with rearrangements within the HindIII fragment were also negative. All negative results were repeated at least once and were confirmed to be true negatives by actin PCR. Our results suggest that negative PCR results in this setting are attributable to small variations in BCL2 MBR breakpoint location and cannot be interpreted without the corresponding conventional Southern blotting data. With this caveat in mind, PCR analysis for the t(14;18) remains an extremely useful technique, especially in the follow-up and monitoring for minimal residual disease in previously characterized cases of follicular lymphoma.     

皮下脂膜炎样T细胞淋巴瘤的免疫表型、基因重排检测及其在诊断中的意义

目的 探讨免疫表型和基因重排检测在皮下脂膜炎样T细胞淋巴瘤(SPTL)的诊断和鉴别诊断中的意义.方法 参照2005年世界卫生组织-欧洲癌症研究与治疗组织(WHO-EORTC)皮肤淋巴瘤分类标准收集20例SPTL.采用10种抗原标记进行免疫表型检测,运用PCR技术检测TCRγ、IgH基因重排,并用EB病毒编码的小RNA1/2(EBER1/2)原位杂交检测EB病毒感染.结果 (1)本组病例中男9例、女11例,平均年龄29.5岁;(2)所有病例的瘤细胞均表达1个或多个T细胞分化抗原(CD2、CD3或CD45RO),18/19病例表达BF1,18/20病例表达CD8,20/20、16/20病例分别表达细胞毒颗粒相关蛋白TIA-1、颗粒酶B,瘤细胞不表达CD4、CD20和CD56;(3)16/20病例检出TcRγ基因重排,未检出IgH基因重排;(4)5/20病例EBERl/2原位杂交阳性.结论 SPTL的瘤细胞具有克隆性TCR基因重排,综合临床病理、免疫表型及基因重排检测有助于本病确诊.     

High frequency of EBV association and 30-bp deletion in the LMP-1 gene in CD56 lymphomas of the upper aerodigestive tract

Natural killer (NK)/T-cell lymphomas are frequently associated with Epstein-Barr virus (EBV), and usually lack TCR gene rearrangement. Studies from Asia have reported frequent deletion in the LMP-1 gene in EBV-associated nasopharyngeal carcinoma (NPC). The present study aims to investigate LMP-1 and TCRgamma gene status in upper aerodigestive tract lymphomas. A total of 43 cases were classified into T-, B-, and NK/T-cell tumors based on the phenotype expressions of CD3(+)/CD20(-)/CD56(-), CD3(-)/CD20(+)/CD56(-), and CD3(+)/CD20(-)/CD56(+), respectively. The presence of EBV in the tumor was confirmed by EBV early RNA-in situ hybridization. LMP-1 gene deletion and TCR gamma gene rearrangement were analyzed by polymerase chain reaction on paraffin-embedded tissues. There were 20 NK/T-, eight T-, and 15 B-cell phenotype lymphomas in the present series, and EBV was detected in 19 (95%), two (25%), and three (20%) cases in the respective groups. All EBV+ cases carried 30-bp deletion in the LMP-1 gene, and two of the NK/T-cell cases were infected by both the wild type and deleted strains. Five (25%) of the NK/T-cell phenotype lymphomas showed rearranged TCR gamma gene. The present study revealed a high frequency of EBV association, and a high frequency of 30-bp deletion in the LMP-1 gene in the virus in the present series of lymphoma. The NK/T-phenotype lymphomas are comprised of both NK-cell and cytotoxic T-lymphocyte-derived tumors.     

Analysis of clonality of atypical cutaneous lymphoid infiltrates associated with drug therapy by PCR/DGGE

Atypical lymphocytic infiltrates that mimic cutaneous lymphoma (ie, pseudolymphoma) are often observed in skin biopsy specimens from patients with altered immune function. The latter may reflect systemic immune dysregulatory states such as collagen vascular disease or human immunodeficiency virus infection. Among the iatrogenic causes are drug therapy with agents that abrogate lymphocyte function. These drugs encompass the anticonvulsants, antidepressants, phenothiazines, calcium channel blockers, and angiotensin-converting enzyme inhibitors. The appellation of lymphomatoid hypersensitivity reaction has been applied to cases of drug-associated pseudolymphoma. Pathologically and clinically, the distinction of such cases from cutaneous lymphoma is difficult. We employed the polymerase chain reaction (PCR) on archival material of proven drug-associated lymphomatoid hypersensitivity reactions both to explore its utility as an adjunct in diagnosis and to investigate the genotypic aberrations induced by drug therapy. Formalin-fixed, paraffin-embedded biopsy specimens from seven cutaneous T-cell lymphomas (CTCL), one nodal T-cell lymphoma, two cutaneous B-cell lymphomas, three typical hypersensitivity reactions, one tonsil, and 14 lymphomatoid hypersensitivity reactions were studied. Control cases for which DNA derived from fresh tissue was used include the Jurkat T-cell tumor line, placenta, one nodal B-cell lymphoma, and one case of reactive lymph node hyperplasia. DNA was obtained and purified by standard methods, then amplified with oligonucleotide primers specific for the T-cell receptor gamma locus and the immunoglobulin heavy chain genes. T-cell amplicons were analyzed by denaturing gradient gel electrophoresis (DGGE) and B-cell amplicons by either nondenaturing polyacrylamide or agarose gel electrophoresis. The nodal and Jurkat T-cell lymphomas, six of seven CTCL, one cutaneous B-cell lymphoma, and 2 of 14 lymphomatoid hypersensitivity reactions showed dominant ("monoclonal") T-cell gene rearrangement patterns, and the remainder of cases were polyclonal. A causal relationship between drug therapy and skin eruption was ascertained in the two patients showing T-cell rearrangements, and both experienced complete and sustained lesional resolution on discontinuation of the implicated drug. The only immunoglobulin heavy chain gene rearrangements detected by PCR were in two of the three B-cell lymphomas. We conclude that PCR/DGGE is a powerful method for assaying T-cell clonality in archival tissue and can aid in the discrimination of reactive from malignant cutaneous infiltrates with appropriate clinicopathologic correlation. Recognition that a monoclonal TCRgamma rearrangement can be observed in cases of drug-associated lymphomatoid hypersensitivity may help in avoiding a misdiagnosis of malignant lymphoma.