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1.
Dominant T-cell receptor beta-chain gene rearrangements indicate clonal expansion in the rheumatoid joint 总被引:9,自引:0,他引:9
A M Miltenburg J M van Laar M R Daha R R de Vries P J van den Elsen F C Breedveld 《Scandinavian journal of immunology》1990,31(1):121-126
T-cell receptor (TcR) beta and delta gene rearrangements were studied in anti-CD3 expanded T-cell populations cultured from the synovial membrane (SM) (n = 5) or synovial fluid (SF) (n = 2) of rheumatoid arthritis (RA) patients. Dominant TcR beta-chain gene rearrangements to C beta 1 were demonstrated in all the patients tested and 1-3 expanded clones per patient were found. Clonal rearrangements to C beta 2 were detected in one SM sample (two clones) and one SF sample (one clone). The TcR delta gene was deleted in all the samples tested. We conclude that clonal dominance may be found in expanded T-cell populations from SM and SF of RA patients. Multiple clones may be present, either using the C beta 1 or C beta 2 gene segment. 相似文献
2.
Absence of clonal beta and gamma T-cell receptor gene rearrangements in a subset of peripheral T-cell lymphomas. 总被引:6,自引:3,他引:6
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L. M. Weiss L. J. Picker T. M. Grogan R. A. Warnke J. Sklar 《The American journal of pathology》1988,130(3):436-442
The authors describe a set of seven peripheral T-cell lymphomas that lack detectable rearrangements of T-cell receptor (TCR) genes. All cases showed antigenic profiles consistent with T-cell lymphoma, including expression of Leu-5 (CD2) antigen. However, few other T-lineage markers were found, and none of the cases tested (6 of 7) bound antibody recognizing the constant region of the beta TCR protein. Each case showed exclusively germline configurations of DNA for the beta TCR genes in Southern blot analyses with the use of several different combinations of restriction enzymes and DNA hybridization probes. One case contained clonal rearrangements of the gamma TCR gene and of the immunoglobulin heavy chain gene. Our results suggest that certain cases of peripheral T-cell lymphoma may lack rearrangements of TCR genes--particularly those cases expressing restricted numbers of T-lineage antigens. In view of these findings, failure to detect rearrangements of TCR genes by Southern blot analyses is not necessarily inconsistent with malignant lymphocytic proliferations in T-lineage neoplasia. 相似文献
3.
Michael Chen April Deng A Neil Crowson Mythily Srinivasan Kurtis H Yearsley Scott Jewell Carl Morrison Susan Long Robert Werling Cynthia Magro 《Applied immunohistochemistry & molecular morphology》2004,12(4):373-379
Discerning the pathologic significance of cutaneous T-cell infiltrates can pose a diagnostic challenge for dermatopathologists. Reactive conditions such as drug-associated lymphomatoid hypersensitivity and lymphomatoid lupus erythematosus can demonstrate lymphoid atypia and a phenotype resembling cutaneous T-cell lymphoma (CTCL). Further, lymphoid dyscrasias such as pityriasis lichenoides chronica, large plaque parapsoriasis, and atypical pigmentary purpura confuse the picture because they not only mimic CTCL but also represent prelymphomatous states with inherent malignant potential. Although the emergence of a dominant clone has been considered a clue indicative of a T-cell dyscrasia, there are reports concerning the identification of monoclonality in biopsies of reactive lymphoid infiltrates. We have conducted a modified single-stranded DNA conformational polymorphism (SSCP) assay using paraffin-embedded, formalin-fixed tissue on 92 T-cell-rich biopsies to determine the relative specificity and sensitivity of this methodology. In addition, laser capture microdissection (LCM) was performed on 22 of the 92 samples to isolate the area of interest and to compare its specificity and sensitivity with those SSCP assays performed without LCM. We found that monoclonality or oligoclonality is 86% specific for preneoplastic and neoplastic states, whereas the finding of polyclonality appears to be relatively specific for a reactive process. Some cases of reversible T-cell dyscrasia produced a molecular profile mimicking lymphoma or prelymphomatous states by virtue of monoclonality or oligoclonality. Although LCM appears to improve the sensitivity for detecting preneoplastic conditions, the relative specificity appears to be the same as that encountered with routine SSCP. 相似文献
4.
T-cell receptor beta chain gene rearrangements: genetic markers of T-cell lineage and clonality 总被引:6,自引:0,他引:6
We have performed a series of investigations involving T-cell receptor beta chain (T beta) gene rearrangements in benign and malignant nonhematopoietic, B-cell, and T-cell proliferations. These studies provide the conceptual basis and the operational approach for the use of T beta gene rearrangements as markers of T-cell lineage, clonality, and differentiation, analogous to immunoglobulin gene rearrangements in B cells. Southern blot hybridization analysis for T beta gene rearrangements can now be utilized to identify and distinguish between non-T cells, polyclonal T cells, and monoclonal T cells. Determination of T beta gene rearrangements will play an important role in the further investigation and classification of T-cell neoplasia. However, the identification of a genetic marker of clonality for T cells has significant diagnostic and prognostic value as well. For example, determination of the T beta gene rearrangement unique to a particular malignant T-cell clone provides a specific genetic marker for that clonal T-cell proliferation. This genetic marker of the T-cell clone may provide a useful tool for monitoring the patient's therapeutic response and clinical course for early signs of relapse. Nonetheless, our studies demonstrate that the lineage specificity of immunoglobulin and T beta gene rearrangements is not absolute. It appears that only a multiparametric approach combining extensive monoclonal antibody immunophenotypic analysis, in vitro testing for functional help and suppression, and Southern blot hybridization analysis for immunoglobulin and T beta gene rearrangements allows the conclusive and unequivocal demonstration of the B- or T-cell derivation of all lymphoid neoplasms. Lymphoid malignancies that cannot be assigned to the B- or T-cell lineage following this extensive multiparametric analysis are exceedingly uncommon. 相似文献
5.
Limited combinatorial and junctional diversity in TCR-gamma gene rearrangement can result in amplification products that are difficult to interpret when analyzed by conventional gel electrophoresis methods that separate DNA based on size (polymerase chain reaction [PCR]/polyacrylamide gel electrophoresis [PAGE]). We describe a simple approach to the detection of clonal TCR-gamma gene rearrangement using temporal temperature gradient gel electrophoresis (TTGE) that uses a gradual and uniform increase in the temperature of a constant denaturing gel to resolve different DNA molecules based on base pair composition. We tested 42 clinical specimens (30 blood specimens and 12 formalin-fixed paraffin-embedded tissues) for T-cell clonality by PCR/PAGE and PCR/TTGE. Concordant results were obtained in only 22 specimens (52%). Of the 20 discordant cases, 18 samples were positive by TTGE and negative by PAGE. For all of the discordant cases, the TTGE yielded results that correlated better with the clinical data than did the PAGE method. We conclude that PCR/TTGE is more accurate and easier to perform than current methods for detecting clonal populations of T cells. 相似文献
6.
Detection of clonal lambda light chain gene rearrangements in frozen and paraffin-embedded tissues by polymerase chain reaction. 总被引:3,自引:1,他引:2
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R. Küppers K. Willenbrock K. Rajewsky M. L. Hansmann 《The American journal of pathology》1995,147(3):806-814
A method was established to detect clonal lambda light chain gene rearrangements in peripheral blood lymphocytes and frozen or paraffin-embedded tissues. V lambda-gene-family-specific primers were used together with a J lambda primer mix in separate reactions to amplify V lambda gene rearrangements by the polymerase chain reaction. Clonal lambda gene rearrangements were detected in seven of seven lambda-expressing B cell leukemias, in four of five lambda-expressing non-Hodgkin's lymphomas with frozen tissues, and in seven of nine cases of lambda-expressing non-Hodgkin's lymphomas for which formalin-fixed, paraffin-embedded specimens were available. Clonality of amplified polymerase chain reaction products was confirmed by sequence analysis for several cases. The present study shows that it is possible to amplify clonal lambda gene rearrangements in the majority of lambda-expressing B cell leukemias and lymphomas. The method described here, therefore, is a useful supplement to the previously described approach of VH and VK gene amplification to detect clonal B cell populations and allows the study of V lambda gene usage and somatic mutation in lambda-expressing normal and malignant B cells. 相似文献
7.
Identification of monoclonal B-cell populations by rapid cycle polymerase chain reaction. A practical screening method for the detection of immunoglobulin gene rearrangements.
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G. H. Segal C. T. Wittwer A. J. Fishleder M. H. Stoler R. R. Tubbs C. R. Kjeldsberg 《The American journal of pathology》1992,141(6):1291-1297
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. 相似文献
8.
Detection of clonal immunoglobulin gene rearrangements by polymerase chain reaction amplification and single-strand conformational polymorphism analysis. 总被引:5,自引:2,他引:3
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Analysis of immunoglobulin gene rearrangements by Southern blotting is a sensitive and specific method for detecting B cell malignancies but requires a relatively large amount of intact DNA. It cannot be utilized in many cases where only a small amount of tissue is available or where the tissue has been fixed. This report demonstrates that polymerase chain reaction (PCR) amplification in conjunction with single-strand conformational polymorphism (SSCP) analysis can be utilized to detect clonal immunoglobulin heavy chain (IgH) gene rearrangements. IgH gene rearrangements from a series of frozen or formalin-fixed B cell malignancies were PCR-amplified using oligonucleotide primers, based upon consensus sequences in the IgH variable and joining regions. Analysis of the single-stranded PCR products on nondenaturing polyacrylamide gels revealed discrete SSCPs corresponding to the malignant B cells. These SSCPs were detectable when the malignant cells represented as few as 0.2% of the total mononuclear cells in peripheral blood. PCR amplification in conjunction with SSCP analysis thus provides a sensitive and specific method to detect clonal IgH rearrangements from minute amounts of fresh, frozen, or fixed tissue. 相似文献
9.
Automated high-resolution polymerase chain reaction fragment analysis: a method for detecting T-cell receptor gamma-chain gene rearrangements in lymphoproliferative diseases. 总被引:2,自引:0,他引:2
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M. Simon P. Kind P. Kaudewitz M. Krokowski A. Graf J. Prinz U. Puchta L. J. Medeiros C. A. Sander 《The American journal of pathology》1998,152(1):29-33
Southern blot analysis and the polymerase chain reaction (PCR) are powerful tools for detecting clonal antigen receptor gene rearrangements. However, a number of limitations restrict the predictive value of the results obtained by these techniques as they are commonly used. We describe a new method, automated high-resolution PCR fragment analysis, that can partially overcome many of the limitations of analyzing the T-cell receptor (TCR) gamma-chain gene. Analysis of TCR-gamma is performed using PCR with four sets of primers, previously described by others, specific for all variable (V) and joining (J) regions of the TCR gamma-chain gene. In addition, the four V region primers are 5' end-labeled with a fluorescent compound, 5-carboxyfluorescein. After amplification, the labeled PCR products are separated with an automated sequencing system, ABI 373 (Applied Biosystems, Weiterstadt, Germany). With the help of the Gene-Scan software ABI 672 (Applied Biosystems) and fluorescent-labeled DNA length markers, the exact size of each peak can be displayed and analyzed. The resolution of this method allows separation of PCR products differing in length by as little as 1 bp. Semiquantitative estimation of specific clones also can be performed. Infiltrate-specific gene rearrangement patterns can be identified and recognized in different tissue specimens at the time of diagnosis or in subsequent biopsy specimens. We conclude that automated high-resolution PCR fragment analysis allows more accurate and convenient analysis of the TCR gamma-chain gene. 相似文献
10.
11.
Molecular detection of a clonal population of B or T cells through analysis of rearranged antigen receptor genes is an essential adjunct to the morphologic, flow cytometric, and immunohistochemical evaluation of tissue specimens for the presence of leukemia or lymphoma. Combining polymerase chain reaction (PCR) with heteroduplex annealing and polyacrylamide gel electrophoresis (PAGE) has been used to detect clonal T-cell receptor rearrangements, particularly in skin biopsy specimens. The authors have developed a similar PCR heteroduplex assay for detection of clonal VDJ immunoglobulin gene rearrangements using two sets of primers based on relatively conserved consensus regions in the J(H) and framework I and 2 regions of the immunoglobulin heavy chain V region gene. This method is able to detect a clonal rearrangement when the clone comprises as little as 1% of the population in a polyclonal B-cell background. It may be used on fresh, frozen, or paraffin-embedded tissue and detects a clonal population in a majority of lymphoma subtypes. Compared with conventional PCR analysis, this method requires only a short additional cycle of denaturation and slow renaturation before PAGE. Interpretation is simplified as the clonal PCR product migrates away from the polyclonal background products. 相似文献
12.
13.
Multiplex polymerase chain reaction-based analysis of T-cell receptor gamma gene rearrangements for the determination of T-lymphocyte clonality 总被引:2,自引:0,他引:2
Determination of the frequency of mutations at hprt or other loci in human lymphocytes provides a useful biomarker for human exposure to mutagens. One problem, however, is distinguishing between unique mutants and sibling mutants arising as progeny of an earlier mutant cell. We have developed a multiplex polymerase chain reaction (PCR)-based method to analyze T-cell receptor (TCR) gamma gene rearrangements for determination of T-cell clonality in mutational spectrum analysis. PCR primers for different subgroups of the V gene segment of the TCR gamma gene were selected at different sites in the TCR gamma gene so that the size of PCR products could define which V subgroup was involved in rearranged TCR gamma genes; gamma genes involving different V and J subgroups could be determined directly by PCR. Mutant T-lymphocytes with rearranged TCR gamma genes containing the same V and J subgroups were analyzed using PCR-based denaturing polyacrylamide gel electrophoresis. All of the 161 hprt mutant clones analyzed contained rearranged TCR gamma genes. Rearrangements among all subgroups of the V and J gene segments of the TCR gamma gene could be detected. VgammaI and Jgamma1/2 subgroups were involved in 69 and 71% of rearranged TCR gamma genes, respectively. This PCR-based analysis of TCR gamma gene rearrangements provides a simple and comprehensive method for identifying the clonality of mutant T-lymphocytes in human hprt mutant lymphocyte assay and mutational spectrum analysis. 相似文献
14.
Analysis of T cell receptor-gamma gene rearrangements by denaturing gradient gel electrophoresis of GC-clamped polymerase chain reaction products. Correlation with tumor-specific sequences. 总被引:5,自引:0,他引:5
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T. C. Greiner M. Raffeld C. Lutz F. Dick E. S. Jaffe 《The American journal of pathology》1995,146(1):46-55
We describe a modified denaturing gradient gel electrophoresis (DGGE) procedure with a 40-nucleotide GC clamp in the polymerase chain reaction to improve resolution in amplifying T cell receptor-gamma (TCR-gamma) rearrangements. DNA from 46 cases of lymphoblastic leukemia/lymphoma, 5T cell lines, 2 B cell lines, 7 normal lymphocytes, and 3 cases of Hodgkin's disease was amplified by polymerase chain reaction. In addition, 20 cases of paraffin-embedded T cell lymphomas and 5 cases of reactive hyperplasia were also studied. Clonal TCR-gamma rearrangements were identified on DGGE by the presence of a predominant band. Results obtained from 5 T cell lines and 12 lymphoblastic leukemia/lymphomas containing known TCR-gamma gene rearrangements revealed 100% concordance in detecting clonal rearrangements between DGGE and traditional Southern blot analysis. Of the remaining 34 lymphoblastic leukemia/lymphoma cases studied by DGGE alone, 30 were positive. DGGE analysis of 10 lymphoblastic leukemia/lymphoma cases with known group IV gamma to J gamma 1 or J gamma 2 rearrangement sequences confirmed that the electrophoretic migration was dependent on the tumor-specific rearranged TCR-gamma sequence. In addition, 17 of 20 cases of paraffin-embedded T cell lymphomas were positive by DGGE, 6 of which had the clonal population also identified in fresh tissue DNA. DGGE analysis of GC-clamped polymerase chain reaction products can provide a way to more accurately detect TCR-gamma clonality of lymphoid tumors and can be applied to archival tissues. 相似文献
15.
V Bertness I Kirsch G Hollis B Johnson P A Bunn 《The New England journal of medicine》1985,313(9):534-538
The ability to detect immunoglobulin-gene rearrangements has proved useful in confirming diagnoses of suspected B-cell lymphomas and in establishing their monoclonality. By analogy, we employed a cloned DNA probe for the beta chain of the T-cell receptor gene to determine whether gene rearrangements were present in human T-cell neoplasms representing various stages of T-cell development. Gene rearrangements were present in all cases of T-cell disorders except a single case of T gamma lymphocytosis, a disorder that has not been proved to be a clonal T-cell neoplasm. A germline gene configuration was present in all patients with non-T-cell neoplasms and in normal tissues from patients with T-cell lymphoma. The probe promises to be useful for confirming the pathological an immunologic diagnosis in difficult cases of T-cell disorders and for assessing the extent of disease. 相似文献
16.
目的 探讨BIOMED-2聚合酶链反应(PCR)在成熟非霍奇金B细胞淋巴瘤(B-NHL)诊断中的价值.方法 收集成熟B-NHL组织标本72例,其中弥漫性大B细胞淋巴瘤37例,黏膜相关淋巴组织结外边缘区淋巴瘤35例为研究对象,并以反应性增生病变25例作为对照.提取以上组织的DNA,并以PCR来检测其完整性和可扩增性,选取质量合格的DNA.85.6%(83/97)的样品DNA长度>300 bp,其中60例成熟B-NHL和23例反应性增生可用于BIOMED-2 PCR检测免疫球蛋白重链(IgH)和kappa轻链(IgK)基因重排的克隆性.结果 利用BIOMED-2 PCR检测的60例成熟B-NHL中,57例存在Ig基因的克隆性重排,其检测敏感性为95%,23例反应性增生病例中未出现Ig基因的克隆性重排,其检测特异性为100%.结论 BIOMED-2 PCR适用于石蜡包埋组织.该方法具有很高的敏感性和特异性,对成熟B-NHL诊断的辅助价值很高. 相似文献
17.
HPRT mutations, TCR gene rearrangements, and HTLV-1 integration sites define in vivo T-cell clonal lineages 总被引:1,自引:0,他引:1
Allegretta M Ardell SK Sullivan LM Jacobson S Mortreux F Wattel E Albertini RJ 《Environmental and molecular mutagenesis》2005,45(2-3):326-337
HPRT mutations in vivo in human T-lymphocytes are useful probes for mechanistic investigations. Molecular analyses of isolated mutants reveal their underlying mutational changes as well as the T-cell receptor (TCR) gene rearrangements present in the cells in question. The latter provide temporal reference points for other perturbations in the in vivo clones as well as evidence of clonal relationships among mutant isolates. Immunological studies and investigations of genomic instability have benefited from such analyses. A method is presented describing a T-cell lineage analysis in a patient with HTLV-1 infection. Lineage reconstruction of an in vivo proliferating HPRT mutant clone allows timing of the integration event to a postthymic differentiated cell prior to the occurrence of HPRT mutations. 相似文献
18.
David Chia Paul Terasaki Henry Chan rew Acalinovich Etsuko Maruya Hiroh Saji Krista Ware 《Tissue antigens》1994,44(5):300-305
Abstract: SSP-PCR (sequence-specific primer) DNA typing was performed in Terasaki trays using 1.5 μ1 of DNA, and the ethidium-stained PCR product was measured by direct fluorometric reading. Elimination of the gel electrophoresis step greatly simplified the SSP method. 17 serological DR specificities were discriminated for 239 DNA samples utilizing the new method, standard SSP, sequence-specific oligonucleotide probe (SSOP), and restriction fragment length polymorphism (PCR-RFLP). Results showed 98% concordance between the SSP-PCR assay and conventional methods. DRB1 alleles were determined by PCR-RFLP in 59 samples, by SSP in 110 samples, and by consensus (all methods) in the remaining samples. 相似文献
19.
20.
J A Hopfenbeck J A Holden C T Wittwer C R Kjeldsberg 《American journal of clinical pathology》1992,97(5):638-644
The detection of clonal rearrangements of the human T-cell receptor by Southern hybridization is a useful tool to diagnose morphologically difficult lymphoid proliferations. Widespread application of this method has been facilitated by the advent of sensitive nonradiolabeled probes. Although a limited number of nonradiolabeled DNA probes are commercially available, other probes must be obtained through a time-consuming and technically difficult procedure of amplification, isolation, and labeling of plasmid-cloned DNA sequences. A simple and time-saving procedure to simultaneously amplify and nonradioactively label DNA probes for use in gene rearrangement studies is described. Specifically, a method using the polymerase chain reaction to amplify and label with digoxigenin large quantities of probe to the constant region of the T-cell receptor directly from genomic DNA is described. The resultant probes are specific for the T-cell receptor-constant region, detect the appropriate germline configuration in placental DNA, and identify rearranged clonal T-cell proliferations. The polymerase chain reaction digoxigenin-labeled probes are suitable for detection by conventional colorimetric methods or by chemiluminescent detection schemes. 相似文献