ARUP Laboratories, Salt Lake City, Utah 84108, USA.
J Clin Pathol. 2011 Jul;64(7):618-25. doi: 10.1136/jcp.2011.089193. Epub 2011 Apr 12.
The BCR-ABL1 T315I mutation imparts resistance to tyrosine kinase inhibitors currently available for treatment of chronic myelogenous leukaemia. Thus, quantitative monitoring of the emergence and expansion of T315I-positive subclones may be clinically useful. The goals of this study were to retrospectively review the authors' experience with Sanger sequencing-based BCR-ABL1 kinase domain mutation testing, paying particular attention to the T315I mutation, and to develop an alternative test for relative quantification of T315I using pyrosequencing.
The performance of a new T315I pyrosequencing assay was evaluated. Total RNA was isolated from whole blood and reverse-transcribed. The resulting cDNA was subjected to an initial round of PCR across the BCR-ABL1 breakpoint followed by a second round to amplify the sequence flanking ABL1 codon 315. The final PCR product was pyrosequenced to detect and quantify the T315I point mutation. Additional experiments were carried out to determine the effects of background untranslocated ABL1 on assay sensitivity in samples with low tumour burden.
The results show that T315I was the most commonly detected kinase domain mutation and was persistent in follow-up testing. All 26 specimens that tested positive by Sanger sequencing for the T315I mutation were also positive using the pyrosequencing test. Relative quantification data derived from pyrosequencing matched the approximate wild-type/mutant ratios found by Sanger sequencing. Serial dilution experiments show sensitivity to 5% mutant allele. The authors also quantitatively assessed the influence of untranslocated ABL1 in the sample background on the assay and found that it occurred at levels not likely to influence performance.
The described test is useful for detection and relative quantification of the T315I point mutation in chronic myelogenous leukaemia in a sensitive, specific and reproducible manner.
BCR-ABL1 T315I 突变赋予了对目前用于治疗慢性髓性白血病的酪氨酸激酶抑制剂的耐药性。因此，定量监测 T315I 阳性亚克隆的出现和扩增可能具有临床意义。本研究的目的是回顾性地审查作者在基于 Sanger 测序的 BCR-ABL1 激酶结构域突变检测方面的经验，特别关注 T315I 突变，并开发一种替代的焦磷酸测序法用于 T315I 的相对定量。
评估了新的 T315I 焦磷酸测序检测的性能。从全血中分离总 RNA 并进行逆转录。所得 cDNA 进行第一轮横跨 BCR-ABL1 断点的 PCR，然后进行第二轮扩增侧翼 ABL1 密码子 315 的序列。最后一轮 PCR 产物进行焦磷酸测序，以检测和定量 T315I 点突变。进行了额外的实验以确定在肿瘤负担低的样本中背景未易位 ABL1 对检测灵敏度的影响。
结果表明，T315I 是最常见的检测到的激酶结构域突变，并且在后续检测中持续存在。通过 Sanger 测序检测为 T315I 突变的 26 个标本均通过焦磷酸测序检测为阳性。从焦磷酸测序中获得的相对定量数据与通过 Sanger 测序发现的近似野生型/突变体比值相匹配。系列稀释实验显示对 5%突变等位基因的敏感性。作者还定量评估了样品背景中未易位 ABL1 对检测的影响，发现其水平不太可能影响性能。
该描述的检测方法以敏感、特异和可重复的方式，用于检测和定量慢性髓性白血病中的 T315I 点突变。
