Kumar Dushyant, Mehta Anurag, Panigrahi Manoj Kumar, Nath Sukanta, Saikia Kandarpa Kumar
Gauhati University Faculty of Medicine, Department of Bioengineering and Technology, Guwahati, India.
Rajiv Gandhi Cancer Institute and Research Centre, New Delhi, India.
Turk J Haematol. 2018 Mar 1;35(1):49-53. doi: 10.4274/tjh.2017.0095. Epub 2017 Nov 13.
Nucleophosmin-1 (NPM1) mutations have prognostic importance in acute myeloid leukemia (AML) patients with intermediate-risk karyotype at diagnosis. Approximately 30% of newly diagnosed cytogenetically normal AML (CN-AML) patients harbor the NPM1 mutation in India. In this study we compared the efficiency of three molecular techniques in detecting NPM1 mutation in peripheral blood and bone marrow samples.
In a single-center cohort we analyzed 165 CN-AML bone marrow/peripheral blood samples for NPM1 mutation analysis. About 30% of the CN-AML samples revealed NPM1 mutations. For the detection, three methods were compared: Sanger sequencing, pyrosequencing, and real-time polymerase chain reaction (PCR).
NPM1 exon 12 mutations were observed in 52 (31.51%) of all CN-AML cases. The sensitivity of Sanger sequencing, pyrosequencing, and real-time PCR was 80%, 90%, and 95%, whereas specificity was 95%, 100%, and 100%, respectively. The minimum limit of mutation detection was 20%-30% for Sanger sequencing, 1%-5% for pyrosequencing, and 0.1%-1% for real-time PCR.
The sequencing method, which is the reference method, has the lowest sensitivity and is sometimes difficult to interpret. Real-time PCR is a highly sensitive method for mutation detection but is limited for specific mutation types. In our study, pyrosequencing emerged as the most suitable technique for the detection of NPM1 mutations on the basis of its easy interpretation and less time-consuming processes than Sanger sequencing.
核磷蛋白1（NPM1）突变对诊断时具有中危核型的急性髓系白血病（AML）患者具有预后意义。在印度，约30%新诊断的细胞遗传学正常的AML（CN-AML）患者存在NPM1突变。在本研究中，我们比较了三种分子技术检测外周血和骨髓样本中NPM1突变的效率。
在一个单中心队列中，我们分析了165份CN-AML骨髓/外周血样本以进行NPM1突变分析。约30%的CN-AML样本显示存在NPM1突变。为进行检测，比较了三种方法：桑格测序、焦磷酸测序和实时聚合酶链反应（PCR）。
在所有CN-AML病例中，52例（31.51%）观察到NPM1第12外显子突变。桑格测序、焦磷酸测序和实时PCR的敏感性分别为80%、90%和95%，而特异性分别为95%、100%和100%。桑格测序的最低突变检测限为20%-30%，焦磷酸测序为1%-5%，实时PCR为0.1%-1%。
作为参考方法的测序法敏感性最低，有时难以解读。实时PCR是一种检测突变的高灵敏方法，但对特定突变类型有限制。在我们的研究中，基于其易于解读且比桑格测序耗时更少的过程，焦磷酸测序成为检测NPM1突变最合适的技术。
