Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia
Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia
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DOI：
10.1038/sj.leu.2401801
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发表时间：
2000-08-01
期刊：
影响因子：
11.4
通讯作者：
van der Schoot, CE
中科院分区：
文献类型：
--
作者：
Verhagen, OJHM;Willemse, MJ;van der Schoot, CE
急性淋巴细胞白血病（ALL）检测最小残留疾病（MRD）的大规模临床研究表明，基于可靠的MRD风险组分类需要对MRD水平进行定量。最近，我们已经表明，可以使用患者特异性免疫球蛋白（Ig）（Ig）和T细胞受体（TCR）基因重排作为PCR靶标，将“实时”定量PCR（RQ-PCR）用于此目的连接区域和两个种系引物。现在，我们通过设计三个生殖线J（H）Taqman探针与六个相应的种系J（H）J（H）J（H）J（H）J（H）和一个等位基因特异性寡核苷酸（特定于寡核苷酸（ ASO）引物补充与交界区域的补充。在比较两种方法的九种情况下，使用ASO引物获得了至少相似（n = 4）或略高（n = 5）最大敏感性。在35个IGH重排中的33个中，ASO底漆方法达到了至少10（-4）个最大敏感性。在35例病例中，有31例可再现的准确定量范围跨越了4至5个数量级。在35个重排中的13个中，必须提高PCR条件的严格度以删除或减少背景信号；这仅涉及经常发生的J（H）4，J（H）5和J（H）6基因重排。在优化条件（主要通过增加退火温度）之后，仅在高于42个周期的高阈值周期（C-T）值下偶尔观察到偶尔的高特异性扩增信号，并且至少高于检测极限的C-T值高于C-T值。因此，这些罕见的as特异性信号可以很容易地与特定信号区分开。我们得出的结论是，这里提出的三个种系J（H）Taqman探针和六个相应的生殖线J（H）引物可用于开发患者特异性的RQ-PCR分析，这几乎可以在所有IGH基因中进行准确且敏感的MRD分析。重排。这些结果将促进标准化的RQ-PCR分析在大型临床研究中进行MRD检测。
Large-scale clinical studies on detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) have shown that quantification of MRD levels is needed for reliable MRD-based risk group classification. Recently, we have shown that 'real-time' quantitative PCR (RQ-PCR) can be applied for this purpose using patient-specific immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets with TaqMan probes at the position of the junctional region and two germline primers. Now, we tested an alternative approach on 35 immunoglobulin heavy chain (IGH) gene rearrangements, by designing three germline J(H) TaqMan probes to be used in combination with one of six corresponding germline J(H) primers and one allele specific oligonucleotide (ASO) primer complementary to the junctional region. In nine cases in which both approaches were compared, at least similar (n = 4) or slightly higher (n = 5) maximal sensitivities were obtained using an ASO primer. The ASO primer approach reached maximal sensitivities of at least 10(-4) in 33 out of 35 IGH rearrangements. The reproducible range for accurate quantification spanned four to five orders of magnitude in 31 out of 35 cases. In 13 out of 35 rearrangements the stringency of PCR conditions had to be increased to remove or diminish background signals; this only concerned the frequently occurring J(H)4, J(H)5 and J(H)6 gene rearrangements. After optimization of the conditions (mainly by increasing the annealing temperature), only occasional aspecific amplification signals were observed at high threshold cycle (C-T) values above 42 cycles and at least six cycles above the C-T value of the detection limit. Hence, these rare aspecific signals could be easily discriminated from specific signals. We conclude that the here presented set of three germline J(H) TaqMan probes and six corresponding germline J(H) primers can be used to develop patient-specific RQ-PCR assays, which allow accurate and sensitive MRD analysis in almost all IGH gene rearrangements. These results will facilitate standardized RQ-PCR analysis for MRD detection in large clinical studies.