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.     

The routine diagnostic utility of immunoglobulin and T-cell receptor gene rearrangements in lymphoproliferative disorders

Immunophenotypic studies have a well-documented role in the assignment of lineage in the lymphoproliferative disorders. With the exception of mature B-cell disorders, it is difficult to demonstrate clonality by immunophenotypic studies. The advent of specific DNA probes for immunoglobulin and T-cell receptor genes has greatly facilitated the detection of clonality and, to a lesser degree, lineage, in these cases. The authors have evaluated the diagnostic utility of these probes and compared them with standard immunophenotyping in 65 patients with a variety of lymphoproliferative disorders. Their results show a significant correlation (P less than 0.01) between lineage assignment as determined by phenotyping and gene rearrangement studies, with the latter far superior in determining clonality. Furthermore, analysis of gene rearrangements facilitated the documentation of lineage and/or clonality in six cases in which standard techniques had failed. Although the scientific basis of the study of gene rearrangements has been well established, the authors wish to emphasize the role that these techniques have in evaluating problem cases in the routine diagnostic laboratory.     

Absence of clonal beta and gamma T-cell receptor gene rearrangements in a subset of peripheral T-cell lymphomas.

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.     

A comparison of multiplex and monoplex T-cell receptor gamma PCR.

T-cell receptor gamma (TCRgamma) PCR is often used to detect clonal T-cell populations. Because TCRgamma contains a limited number of variable (Vgamma) and joining (Jgamma) regions, a small number of PCR primers can be used to assess T-cell clonality. The seven primers used in the current study were described previously and were split into 2 or 3 multiplex primer sets. In this study, a single 7-primer multiplex (7-plex) PCR reaction was compared with all 12 possible monoplex primer combinations on 18 samples previously analyzed for T-cell receptor rearrangements by TCRbeta Southern blot and/or TCRgamma PCR followed by temporal temperature gradient gel electrophoresis. Using fluorescent Vgamma-region primers, unlabeled Jgamma-region primers, and capillary electrophoresis, we show all TCRgamma rearrangements seen by 7-plex PCR on known positive samples were seen following monoplex PCR. However, additional TCRgamma gene rearrangements were seen in monoplex PCR reactions that were not seen in the 7-plex PCR reactions. Monoplex but not 7-plex PCR of known negative samples occasionally showed TCRgamma gene rearrangements, often with less frequently used Vgamma and Jgamma-region primers, and may have represented false positive results. In summary, the single 7-plex PCR reaction correctly identified specimens with TCRgamma clonal populations and represents an improvement over existing assays that use these same primers split into several smaller multiplex reactions. Monoplex PCR has no advantage over multiplex PCR and has the potential to lead to false positive results.     

中线T细胞淋巴瘤的TCR—γ基因重排研究

目的对中线Ｔ细胞淋巴瘤的克隆性进行研究。方法用一组针对Ｔ细胞受体γ链基因Ｖ区片段的家族特异性引物和ＰＣＲ方法,对１１例（２２个标本）中线Ｔ细胞淋巴瘤病例进行了Ｔ细胞受体γ基因重排的检测。结果２２个标本中２１个有Ｔ细胞受体γ链基因的克隆性重排（９４４５％）。在９例原发灶的连续活检和１例原发灶及其转移灶的标本中均未发现增生的克隆家族的改变。结论中线Ｔ细胞淋巴瘤的病变组织中存在Ｔ细胞的单克隆性增生,为该肿瘤的Ｔ细胞起源提供了分子生物学的证据。在中线Ｔ细胞淋巴瘤的疾病过程中（包括转移灶）增生Ｔ细胞的家族未发生变化,支持肿瘤的单克隆起源学说     

Automated high-resolution polymerase chain reaction fragment analysis: a method for detecting T-cell receptor gamma-chain gene rearrangements in lymphoproliferative diseases.

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.     

T-cell receptor gene rearrangements as clinical markers of human T-cell lymphomas

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.     

Multiplex polymerase chain reaction-based analysis of T-cell receptor gamma gene rearrangements for the determination of T-lymphocyte clonality

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.     

A T-cell receptor gamma polymerase chain reaction assay using capillary electrophoresis for the diagnosis of cutaneous T-cell lymphomas.

Detection of clonal T-cell receptor gamma rearrangements by polymerase chain reaction (TCRgamma PCR) followed by high-resolution electrophoresis has now become a valuable tool in the diagnosis of cutaneous T-cell lymphoma (CTCL). The identification of clonal TCRgamma PCR products by fluorescent fragment analysis (FFA) on a capillary DNA sequencer is described here and compared with an established hetero-duplex temperature gradient gel electrophoresis (HD-TGGE). Of 55 CTCL derived lesional skin samples, clonality was obtained in 46 samples by FFA (83.6%) and in 45 samples by HD-TGGE (81.8%). Of 35 control skin specimens from various nonmalignant dermatoses, two samples (pityriasis lichenoides chronica) showed clonality by both methods, one sample (chronic dermatitis) only by FFA. The sensitivity of FFA was established using three clonal T-cell lines and peripheral blood mononuclear cells. The detection limit for clonal material was approximately 1% to 2.5% in mixtures of DNA and 1% to 3% in cell dilutions. For cell dilution series, we confirmed a linear correlation between the clonal/polyclonal peak-size ratios and the portion of clonal cells up to about 10%. Thus, the initial ratio between mono-and polyclonal template is correctly displayed by FFA within that concentration range. In conclusion, FFA on capillary DNA sequencer is a well-suited separation technique in TCRgamma PCR-based clonality analysis also exhibiting quantitative properties.     

T-cell receptor V delta-J alpha rearrangements in human thymocytes: the role of V delta-J alpha rearrangements in T-cell receptor-delta gene deletion.

The differentiation mechanisms that force thymocytes into the T-cell receptor (TCR)-alpha beta or TCR-gamma delta lineage are poorly understood, but rearrangement processes in the TCR-alpha/delta locus are likely to play an important role. It is assumed that the TCR-delta gene is deleted prior to V alpha-J alpha rearrangements by rearrangement of the so-called TCR-delta-deleting elements delta Rec and psi J alpha. However, the TCR-delta gene can also be deleted via V delta-J alpha rearrangements. We studied the different TCR-delta-deleting rearrangements of V delta 1, delta Rec, V delta 2 and V delta 3 to J alpha gene segments in human thymocytes and peripheral blood using polymerase chain reaction analysis. The V delta 1 gene segment is the most upstream V delta gene segment tested and appears to rearrange to almost all J alpha gene segments. In contrast, the delta Rec and V delta 2 gene segments only rearrange to the 5'-located J alpha gene segments, thereby preserving an extensive TCR-alpha combinatorial diversity, because most J alpha gene segments are kept available for subsequent V alpha-J alpha rearrangements. Based on our combined data we hypothesize that the different V delta gene segments and the delta Rec gene segment play different roles in T-cell development with regard to TCR-delta deletion.     

Characterization of T-cell-receptor gamma (TRG) gene rearrangements in alloreactive T-cell clones

Rearrangements of the T-cell Rearranging Gene (TRG) or T-cell-receptor gamma-chain genes were analyzed in 24 in vivo-sensitized alloreactive T-cell clones. This analysis represents the first complete assignment of TRG gene rearrangements to given variable and joining gene segments in nonleukemic T cells and provides some evidence for the hypothesis of sequential gamma genes rearrangements during T-lymphocyte differentiation. TRG gene rearrangements in our T-cell panel involved the known "active" V gamma genes, with a preferential use of V2 and V4 genes. In most clones, rearrangements occurred on both chromosomes and involved the J2 segment, but only 2 and 4 out of the 49 described rearrangements involved the additional J gamma segments JP1 and JP2, respectively. Two peculiar rearrangements were found. The first one was probably due to the creation of a new restriction enzyme site in the N-region at the V-J junction; the second can be explained by an aberrant rearrangement of a V gene to a sequence located between exons 2 and 3 of the TRGC1 gene.     

Immunoglobulin and T cell receptor gene rearrangements and in situ immunophenotyping in lymphoproliferative disorders

Summary We investigated for rearrangements of the immunoglobulin (Ig) heavy and light chain genes and of the T cell receptor (TCRT) and (TCr) genes 45 biopsy samples from a variety of lymphoproliferative disorders. They were diagnosed histopathologically and immunophenotypically as non-Hodgkin's lymphomas (NHLs) of the B cell type (19 cases), NHLs of the T cell type (3 cases), NHLs of undetermined cell type (3 cases), atypical lymphoid proliferation (1 case) and AIDS-related lymphadenopathies with florid polyclonal follicular hyperplasia (19 cases). A monoclonal proliferation of B cells was shown by DNA analysis in all 19 B cell NHLs. In two immunohistologically determined T cell NHLs (both diagnosed as mycosis fungoides) the cells had rearrangements of TCr gene, whereas in the third case (lymphoblastic NHL) the cells had rearrangements of Ig heavy chain and TCr and TCr genes. None of the B cell NHLs exhibited TCrand TCr gene rearrangement bands. All the undetermined cell NHLs demonstrated rearrangements of Ig heavy chain gene associated with the germ line TCrand TCr genes; in two cases light chain gene rearrangements were also found. The atypical lymphoid proliferation, in which the differential diagnosis was between a reactive or malignant process, and two out of 19 cases of florid polyclonal follicular hyperplasia showed a clonal B cell population by DNA analysis. This study indicates that there was a strong correlation between the rearrangements of specific genes and the immunophenotype of the NHL; moreover, DNA analysis of tissue biopsy specimens from phenotypically undetermined cell NHLs and from equivocal lymphoid proliferation using Ig and TCR gene probes yelded an answer in the cases analyzed. The significance of clonal B cell expansions found in two AIDS-related lymphadenopathies should be interpreted with caution.This work was supported in part by a Grant No 86.00644.44 from the Consiglio Nazionale delle Ricerche, Progetto Finalizzato Oncologia, Rome, and by the Associazione Italiana per la Ricerca sul Cancro, Milan, Italy     

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.     

T-cell receptor beta chain gene rearrangements: genetic markers of T-cell lineage and clonality

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.     

Detection of antigen receptor gene rearrangements in lymphoproliferative malignancies by fluorescent polymerase chain reaction

Abstract: Monoclonal rearrangements of antigen receptor genes in lymphoproliferative diseases are characterized by the specific sequence and the length of their junctional region, which can be used as markers of the proliferating clone. PCR techniques have greatly simplified routine detection of monoclonal rearrangements. But on the one hand, identification of the sequences requires sequencing methods and on the other hand, sizing of rearrangements by conventional analysis of PCR products on agarose or nondenaturing polyacrylamide gels may be uncertain. We have developed an approach based on amplification of rearranged IGH, TCRG and TCRD locus by fluorescent PCR associated to a computerized analysis of generated PCR products allowing their objective sizing. We tested this method on DNA samples from patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia, whose pattern of IGH and TCRG rearrangements had been previously identified by Southern blot techniques. TCRG-PCR assay allowed detection of 100% of rearranged samples. No false-negative results were found but a high rate (60%) of Southern-negative and PCR-positive samples were identified. TCRD PCR-assay detected VD1JD1 or VD2-D2/3 rearrangements in both acute lymphoblastic leukemia and chronic lymphocytic leukemia samples. IGH PCR assay permitted detection of all known rearranged samples. The sensitivity of these three different PCR assays (1% leukemic cells) was equivalent to that of other published PCR protocols. These results show the validity and reliability of the fluorescent PCR method for routine detection of IGH, TCRG and TCRD rearrangements. Sizing of PCR products by computerized analysis was also validated. It provides additional information on rearrangement patterns in lymphoproliferative diseases, as clonal rearrangements can be recognized by their size. This can be of great interest in various circumstances, particularly for detection and follow-up of oligoclonality.     

Frequent immunoglobulin and T-cell receptor gene rearrangements in "histiocytic" neoplasms

The authors have analyzed the DNA of immunoglobulin and T-cell receptor genes in a series of 6 malignancies which were judged to be of histiocytic derivation on the basis of morphologic criteria. They found that 4 of these cases showed rearrangements of the beta T-cell receptor genes in spite of the lack of any specific immunohistochemical markers for B or T cells. One case showed rearrangements of both heavy and light chain immunoglobulin genes and probably represents either a sinusoidal large cell lymphoma or a B-cell lymphoma with activation of histiocytes simulating malignant histiocytosis. A single case lacked both immunoglobulin and T-cell receptor rearrangements consistent with immunologic analyses that suggested its origin from an interdigitating reticulum cell. The result of this study in conjunction with the authors' previous immunologic observations suggests that many presumed histiocytic malignancies actually represent T-cell lymphomas. Alternatively, beta T-cell receptor rearrangement may be a common feature of tumors that show monocyte/histiocytic differentiation.     

A novel binding assay to assess specificity of monoclonal antibodies

Dynabeads TALON are uniform superparamagnetic polystyrene beads of 1 microm diameter that bind 6-His-tagged recombinant proteins through Co++-affinity binding, and are normally used for protein purification. We have used these beads to bind 6-His-rNKp30 and 6-His-rNKp46 to use as a matrix for evaluating NKp30 and NKp46 mAb submitted to the 8th International Human Leukocyte Differentiation Antigen Workshop. We show that recombinant protein coated beads are an effective tool to evaluate the specificity and epitope reactivity of mAb.     

Dominant T-cell receptor beta-chain gene rearrangements indicate clonal expansion in the rheumatoid joint

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.