Keller Stefan M, Moore Peter F
Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, One Shields Avenue, Davis, CA 95616, USA.
Vet Immunol Immunopathol. 2012 Jan 15;145(1-2):410-9. doi: 10.1016/j.vetimm.2011.12.019. Epub 2011 Dec 28.
Polymerase chain reaction (PCR) based clonality assays are an important tool to differentiate neoplastic from reactive lymphocyte populations. A recent description of the canine T cell receptor γ locus identified a large number of formerly unknown genes, and determined the locus topology consisting of 8 cassettes with up to 3 variable (V) genes, 2 joining (J) genes and one constant (C) gene each. Given that these data were not available when existing canine T cell clonality assays were developed, it is likely that they will fail to detect a subset of clonal lymphocyte populations. The objective of this study was to gauge the potential of canine T cell clonality assays to detect all rearranged T cell receptor γ genes and to develop an improved clonality assay. The primer sequences of existing clonality assays were aligned to the reference sequences of all rearranged genes and genes were scored as to the likelihood of being recognized by a primer. All four assays likely recognized subgroup Vγ2 and Vγ6 genes but 3 out of 4 assays were unlikely to detect subgroup Vγ3 and Vγ7 genes. All assays likely recognized Jγx-2 genes, but only two assays were likely to detect most Jγx-1 genes. Two assays had forward primers located as close as four nucleotides to the junctional region. A new multiplex PCR was designed with all primers combined in a single tube. An alternative primer set allowed identification of variable gene usage through gene specific forward primers. The coverage of all rearranged genes facilitated the detection of multiple clonal rearrangements per neoplastic sample. The new assay detected clonal DNA at a concentration of 5% within polyclonal background but detection thresholds were dependent on the gene usage of clonal rearrangements as well as the position of the clonal peak in respect to the polyclonal background. The new multiplex assay recognized 12/12 (100%) of confirmed neoplastic samples as compared to 2/12 (17%) by an existing assay. On a series of 60 diagnostic samples the concordance rate of both assays was 41/60 (68.3%). In 14/60 (23.3%) of the cases, the new multiplex assay yielded a clonal result while the existing assay gave a non-clonal result. In 5/60 (8.3%) of cases, the new assay yielded a non-clonal result while the existing primer set gave a clonal result. These findings suggest that the new multiplex assay has an improved sensitivity over traditional assays and is suited to reduce the rate of false-negative results.
基于聚合酶链反应（PCR）的克隆性分析是区分肿瘤性淋巴细胞群和反应性淋巴细胞群的重要工具。最近对犬T细胞受体γ基因座的描述发现了大量以前未知的基因，并确定了该基因座的拓扑结构，其由8个盒式结构组成，每个盒式结构包含多达3个可变（V）基因、2个连接（J）基因和1个恒定（C）基因。鉴于在开发现有的犬T细胞克隆性分析时这些数据尚未可得，现有的分析很可能无法检测到一部分克隆性淋巴细胞群。本研究的目的是评估犬T细胞克隆性分析检测所有重排的T细胞受体γ基因的潜力，并开发一种改进的克隆性分析方法。将现有的克隆性分析的引物序列与所有重排基因的参考序列进行比对，并根据引物识别基因的可能性对基因进行评分。所有四种分析可能识别Vγ2和Vγ6亚组基因，但4种分析中有3种不太可能检测到Vγ3和Vγ7亚组基因。所有分析可能识别Jγx - 2基因，但只有两种分析可能检测到大多数Jγx - 1基因。两种分析的正向引物与连接区域的距离近至4个核苷酸。设计了一种新的多重PCR，将所有引物组合在一个管中。一组替代引物通过基因特异性正向引物允许识别可变基因的使用情况。所有重排基因的覆盖范围有助于检测每个肿瘤样本中的多个克隆重排。新的分析方法在多克隆背景中能检测到浓度为5%的克隆DNA，但检测阈值取决于克隆重排的基因使用情况以及克隆峰相对于多克隆背景的位置。与现有分析方法识别2/12（17%）的确诊肿瘤样本相比，新的多重分析方法识别了12/12（100%）的确诊肿瘤样本。在一系列60个诊断样本中，两种分析方法的一致率为41/60（68.3%）。在14/60（23.3%）的病例中，新的多重分析方法得出克隆性结果，而现有分析方法得出非克隆性结果。在5/60（8.3%）的病例中，新的分析方法得出非克隆性结果，而现有的引物组得出克隆性结果。这些发现表明，新的多重分析方法比传统分析方法具有更高的灵敏度，适合降低假阴性结果的发生率。
