Diagnostic and prognostic significance of clonal T-cell receptor beta gene rearrangements in lymph nodes of patients with mycosis fungoides

In this study, 25 involved and uninvolved lymph nodes from 22 patients with mycosis fungoides (MF) and seven dermatopathic lymph nodes from patients with benign skin disorders were studied for the presence of clonal T-cell receptor beta (TCRβ) gene rearrangements by Southern blot analysis. These results were correlated with the histological classification, follow-up data, and survival. The results of the histological classification and Southern blot analysis were concordant in 26 of 32 cases. Clonal TCRβ gene rearrangements were found in all six MF lymph nodes showing (partial) effacement of the normal lymph node architecture, but in none of the eight uninvolved dermatopathic MF lymph nodes and in none of the seven dermatopathic control lymph nodes. In addition, in 5 of 11 dermatopathic MF lymph nodes that were considered to have early involvement by MF at histological examination, clonal TCRβ gene rearrangements were detected. In the group of MF patients with dermatopathic lymphadenopathy, patients with detectable clonal T-cell populations had a significantly shorter survival than patients without such a population (P<0.01). The results of this study indicate that within the group of dermatopathic MF lymph nodes, prognostically different groups can be distinguished and that TCRβ gene rearrangement analysis may be an important adjunct in the early diagnosis of lymph node involvement by MF.     

T细胞性淋巴瘤诊断中TCR基因重排检测的应用

目的：探讨T细胞受体( T cell receptors, TCR)基因重排检测对T细胞性淋巴瘤诊断的价值。方法收集T细胞性淋巴瘤30例和淋巴反应性增生组织30例,提取DNA,应用BIOMED-2引物系统中的56条引物进行PCR扩增,核酸分子异源双链凝胶电泳分析结果。结果30例T细胞性淋巴瘤标本中TCRβ、TCRγ、TCRδ的检出率分别为83．3%(25/30)、93．3%(28/30)、13．3%(4/30),三者联合检测的检出率为96．7%(29/30),30例淋巴反应性增生组织中均未检测出TCR基因重排。结论利用BIOMED-2引物系统检测TCR 基因重排可作为T细胞性淋巴瘤的辅助诊断工具。     

定量检测T细胞受体重排删除DNA环含量评价急性非淋巴细胞白血病患者胸腺近期输出功能

目的检测急性非淋巴细胞白血病(ANLL)患者T细胞受体重排删除DNA环(signaljoint T-cell receptor exc ision DNA c irc les sjTRECs,TRECs)的含量,从而探讨患者的初始型naive T细胞水平和胸腺近期输出功能特点。方法利用实时定量PCR(TaqM an)方法,检测77例ANLL患者外周血单个核细胞(PBMC)TRECs的水平,并根据外周血中CD3阳性率计算CD3细胞中TRECs水平。14例ANLL缓解期患者和14例正常人外周血作为对照。结果ANLL患者外周血中TRECs含量为(0.43±0.92)拷贝/1 000 PBMCs,(2.02±3.25)拷贝/1 000 CD3+细胞,明显低于正常人TRECs水平[(4.10±3.65)拷贝/1 000 PBMCs和(6.84±4.71)拷贝/1 000 CD3+细胞,P=0.0000和P=0.0001]和缓解期患者[(2.19±2.49)拷贝/1 000 PBMCs和(6.30±7.13)拷贝/1 000 CD3+细胞,P=0.0000和P=0.0005]。各亚型ANLL患者的TREC水平差异无统计学意义(P>0.05)。结论绝大多数ANLL型患者胸腺近期输出naive T细胞功能明显降低,缓解期患者的胸腺近期输出功能有一定程度恢复。     

Acetaldehyde-induced mutation at the hprt locus in human lymphocytes in vitro

Acetaldehyde (Aa) induces chromosomal aberration and sister chromatid exchange in a variety of test systems, but has not previously been evaluated for its ability to induce gene mutation in mammalian cells. We have studied the mutagenic effect of Aa at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human lymphocytes in vitro by using the T-cell cloning technique and selection of mutant cell clones in medium containing thioguanine. Cells treated with 1.2-2.4 mM Aa for 24 hr or 0.2-0.6 mM Aa for 48 hr showed a dose-dependent decrease of cell survival and a 3- to 16-fold increase of the mutant frequency. The inverse relationship between cell survival and mutant frequency was linear down to a relative survival of 15%, and showed a similar slope in the 24-hr and 48-hr treatment experiments. Forty-one mutant T-cell clones derived from cultures treated with 1.2 or 2.4 mM Aa and 15 from untreated controls were expanded for DNA extraction and Southern blot analysis to study deletion mutation using a full length hprt cDNA probe, and clonal identity on the basis of T-cell receptor rearrangements. In the culture with a 16-fold increase of mutant frequency, 4 out of 10 independent mutants (40%) showed partial deletions extending beyond the 3' coding sequences of the hprt gene. Two of 22 independent mutants derived from the other treated cultures with at most a 6-fold increase of mutant frequency, and 1 of 11 independent control clones showed rearrangement of the hprt gene, none of which affected the 3'-end of the hprt gene. These results show that Aa is capable of inducing gene mutation at the hprt locus in human cells, and suggest that deletion mutation affecting the 3'-end of the gene may be a major type of Aa-induced mutation of this locus.     

Analysis of the α/β T-cell receptor repertoire by competitive and quantitative family-specific PCR with exogenous standards and high resolution fluorescence based CDR3 size imaging

The characterization of the human T-cell receptor (TCR) repertoire in various physiological and pathological conditions has become an important tool in studies of the immune response. Therefore, a number of PCR based strategies for the semiquantitative analysis of the TCR repertoire have been described. Family specific amplification of TCR cDNA has been employed in a number of studies often with contradictory results. We have developed a strategy utilizing exogenous standards with homologous primer binding sites for the quantitative analysis of the α/β T-cell receptor repertoire. This system allows the detection of even minute differences in T-cell populations based on quantitative PCR (Q-PCR) and competitive PCR (C-PCR). Results presented here demonstrate that expansions of T-cell subsets as defined by the specificity of the variable gene segments can be readily monitored when exceeding 1% of the total repertoire. In addition, the proposed method reveals direct information of CDR3 size heterogeneity and can be used to estimate the T-cell repertoire complexity and monitor clonal expansions. We discuss variables such as cell number and experimental conditions influencing accuracy and reproducibility of the analyses. We have used this protocol based on non-radioactive techniques for characterization of the fine specificity of the T-cell repertoire in peripheral and organ-infiltrating T-lymphocytes. The analyses revealed information about polyclonal or clonal expansion of T-cells in vivo and in vitro following various stimuli such as superantigenic stimulation of T-cell subsets as well as antigen-driven shaping of the α/β T-cell repertoire in autoimmune and infectious diseases.     

Real-time PCR分型法检测苏南地区汉族人群KIR基因多态性

目的利用SYBR Green I Real-time PCR分型方法检测杀伤细胞免疫球蛋白样受体(killer cell immunoglobulin-likereceptor,KIR)基因,探讨苏南地区汉族人群KIR基因的分布特点。方法应用SYBR Green I Real-time PCR法对191名苏南地区汉族非亲缘健康人群进行KIR基因分型。结果 SYBR Green I Real-time PCR法有效地进行了KIR基因分型。已知的16种KIR基因在苏南地区汉族人群均被检出。框架基因2DL4、3DL2、3DL3和假基因3DP1存在于所有受检个体中。最常见的非框架基因为2DL1、2DL3、3DL1、2DS4以及假基因2DP1。共检出33种KIR基因型,最常见的为AA1(39.27%),其次为BX2、BX4和BX8。发现仅在新加坡华人报道的罕见基因型BX331和BX337,及仅在墨西哥人群罕见的基因型BX427。结论苏南汉族人群中检测出已知的16种KIR基因,共发现33种基因型,最常见的为AA1,并见到3个罕见基因型BX331、BX337和BX427。     

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.     

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     

实时荧光PCR法检测HER2基因扩增方法的建立

目的探讨应用双标准曲线的实时荧光PCR法检测原癌基因人类表皮生长因子受体2（HER2）基因扩增在临床乳腺癌诊治中的可行性。方法收集500例乳腺癌术后新鲜组织标本,抽提组织DNA进行实时荧光PCR检测,采用双标准曲线法定量,通过计算目的基因浓度和内标基因浓度的比值来判断HER2基因的扩增情况。选择灵敏度和特异度均较高的荧光原位杂交方法作为对照方法。结果检测阳性标本72例,阴性样本419例。该方法检测的灵敏度为85.9%,特异度为98.79%,准确度为96.74。与荧光原位杂交法（FISH）检测结果相比,二者具有较好的一致性。结论双标准曲线的实时荧光PCR法用于检测HER2基因扩增相对准确可靠,有较好的临床应用前景。     

Polymerase chain reaction screening of immunoglobulin heavy chain and T cell receptor γ gene rearrangements: A practical approach to molecular DNA analysis of non-Hodgkin's lymphoma in a surgical pathology laboratory

Characterization of the clonality of non-Hodgkin's lymphoma (NHL) by the rearranged segments of immunoglobulin heavy chain (Ig(H)) or T cell receptor (TCR) genes is not only useful in the confirmation of the diagnosis but also for the future assessment of how a secondary lymphoma, such as a recurrence or another primary lymphoma, occurs. As a practical approach to obtaining and registering this information in a surgical pathology laboratory, FR3 and FR1 regions of Ig(H) gene and TCRgamma gene were concurrently amplified by polymerase chain reaction (PCR) using each pair of consensus primers and the same PCR protocol. Examined samples consisted of 134 primary NHL (phenotypically, 108 B cell and 26 T cell NHL), 19 reactive lymphadenopathies, as well as five secondary lymphomas whose primary lesions were included in this study. Among the primary NHL, the combined PCR analysis disclosed the clonality in 103 of 134 NHL (77%), by FR3 PCR in 77 B cell and two T cell NHL, by FR1 PCR in 59 B cell and one T cell NHL, and by TCRgamma PCR in 11 B cell and 17 of 26 T cell NHL, but in none of the reactive lymphadenopathies. Among the secondary lymphomas, the same pattern of PCR analysis was obtained in two cases (the durations between first and second lymphomas; 6 and 10 months), which suggested recurrence. In contrast, different results were obtained in three cases (17-37 months), which indicated another primary or emergence of the subclones. The results of Southern blot analysis were concordant with the PCR results of the first and the secondary lymphomas. Although the combined PCR analysis cannot replace Southern blot hybridization because of its lower detection rate, it can select those cases suitable for further Southern blot analysis thus reducing the number of unnecessary examinations by nearly 75%. This approach may also be useful in the comparative evaluation of primary and secondary lymphomas.     

慢性粒细胞白血病病人TCRζ链表达特点

目的： 建立实时定量PCR方法检测TCRζ链表达水平的方法，了解慢性粒细胞白血病（CML）外周血TCRζ链表达水平。方法: 采用SYBR GreenⅠ荧光定量PCR，相对定量检测30例CML患者和30例正常人外周血的单个核细胞的TCRζ链表达情况，以β2微球蛋白基因（β2M）作为内参，根据相对定量公式：2-△△Ct计算CML病人与正常人TCRζ链表达差异倍数。结果: 成功建立SYBR GreenⅠ荧光实时定量PCR检测TCRζ链表达检测技术。18例CML患者TCRζ链出现表达低于正常人，而12例CML患者TCRζ链出现表达高于正常人。结论: CML病人中TCRζ链表达水平可分为表达下调（60%）和表达上调(40%)2组，提示部分CML病人的细胞免疫缺陷可能与其TCRζ链表达下调有关。     

TCR基因重排检测在T细胞淋巴瘤病理诊断中的作用

基因重排现象是每个淋巴细胞在早期经历的一个正常的过程,由于机体每个淋巴细胞的重排基因都是特有的,使每个淋巴细胞都有独特的重排形式。如若机体在某些致瘤因素的作用下失去对某个基因重排细胞的控制,则该细胞就会出现无控制的、单克隆性增生,最终导致淋巴瘤的形成。所以淋巴瘤所具有的是单克隆性重排。因此通过聚合酶链反应（polymerase chain reaction,PCR）技术进行T细胞抗原受体（T cell receptor,TCR）基因克隆性重排分析,尤其在常规HE形态学与免疫组化结果都不能确诊时,显得尤为重要。该文对其在T细胞淋巴瘤病理诊断中的作用作一综述。     

Laboratory technology for population-based screening for severe combined immunodeficiency in neonates: the winner is T-cell receptor excision circles

The most profound primary immunodeficiency disease, severe combined immunodeficiency (SCID), is fatal in infancy unless affected infants are provided with an adaptive immune system through allogeneic hematopoietic cell transplantation, enzyme replacement, or gene therapy. However, most infants with SCID lack a family history or any clinical clues before the onset of infections, making this serious but treatable disease a candidate for population-based newborn screening. Of several approaches considered for SCID screening, testing for T-cell receptor excision circles (TRECs), a DNA biomarker of normal T-cell development, has proved successful. TREC numbers can be measured in DNA isolated from the dried bloodspots already routinely collected for newborn screening. Infants with low or absent TRECs can thus be identified and referred for confirmatory testing and prompt intervention. TREC testing of newborns is now being performed in several states, indicating that this addition to the newborn screening panel can be successfully integrated into state public health programs.     

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.     

Analysis of T-cell receptor and immunoglobulin gene rearrangements in the diagnosis of Hodgkin's and non-Hodgkin's lymphoma

The results of genotypic analysis of 29 cases of malignant lymphoma are reported and the application of this technique for differentiating between Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL) is evaluated. Five cases with a differential diagnosis which included HD and NHL were analysed. These results are compared with those obtained for six B-cell NHLs, nine T-cell NHLs, and nine cases of HD. This report suggests that gene rearrangement analysis is useful in some cases in which the differential diagnoses includes HD and NHL as the absence of gene rearrangements is more consistent with a diagnosis of HD than of NHL. Two monoclonal antibodies reactive with the variable region of T-cell receptor beta-chain and molecular probes to the relevant variable region genes were used to assist in the diagnosis of T-cell lymphoma. This report confirms that genotypic analysis is useful diagnostically when the results are assessed in the context of the histopathological findings.     

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.     

Identification of a second "French Canadian" LDL receptor gene deletion and development of a rapid method to detect both deletions

Hobbs et al. (N. Engl. J. Med. 317: 734-737, 1987) reported a large deletion of approximately 10 kilobases in the 5' portion of the human low-density lipoprotein (LDL) receptor gene. This deletion affects about 60% of familial hypercholesterolemia (FH) heterozygotes in the French Canadian population. We have developed a rapid, convenient method for the detection of the deletion using double digestion with the restriction enzymes XbaI and EcoRV, or triple digestion with XbaI, EcoRV and XmnI, and a 650 bp cDNA probe, radio-labeled using a random oligonucleotide primer technique. Eighty French Canadian FH heterozygotes were screened for the presence of the deletion. Forty-seven (59%) of them were found to carry the 10 kb deletion. Using the same method, we also identified a new mutation which was found in four of the 80 (5%) FH patients. This mutation has been found to be a 5 kb deletion removing exons 2 and 3 of the LDL receptor gene, which correspond to the first two repeats of the LDL receptor binding domain. Cosegregation of the 5 kb deletion and the FH phenotype was observed in one family. Possible structure-function relationship is discussed.     

实时定量PCR检测正常人外周血T细胞和胸腺细胞中sjTRECs水平

目的了解正常人外周血T细胞和胸腺细胞中信号结合T细胞受体删除 DNA环(sjTRECs)的含量,从而推测正常人中幼稚T细胞的含量和胸腺的输出功能. 方法利用实时定量PCR和TaqMan方法检测11例正常人外周血单个核细胞、7例儿童和3例成人胸腺细胞DNA中sjTRECs的水平. 结果正常人中sjTRECs含量分别为8.83±4.81/1000个外周血单个核细胞;27.31±3.23/1000个成人胸腺细胞和170 .29 ±59.52/1000个儿童胸腺细胞. 结论 sjTRECs水平与年龄有关,正常人外周血中每1000个单个核细胞中约含4.5个幼稚T细胞.