Park Ji-Hye, Sevin Margaux, Ramla Selim, Truffot Aurélie, Verrier Tiffany, Bouchot Dominique, Courtois Martine, Bas Mathilde, Benali Sonia, Bailly François, Favre Bernardine, Guy Julien, Martin Laurent, Maynadié Marc, Carillo Serge, Girodon François
Service d'Hématologie Biologique, Pôle Biologie, CHU de Dijon, Dijon, France.
Inserm U866, Université de Bourgogne, Dijon, France.
PLoS One. 2015 Oct 26;10(10):e0141010. doi: 10.1371/journal.pone.0141010. eCollection 2015.
Calreticulin (CALR) mutations have recently been reported in 70-84% of JAK2V617F-negative myeloproliferative neoplasms (MPN), and this detection has become necessary to improve the diagnosis of MPN. In a large single-centre cohort of 298 patients suffering from Essential Thrombocythemia (ET), the JAK2V617F, CALR and MPL mutations were noted in 179 (60%), 56 (18.5%) and 13 (4.5%) respectively. For the detection of the CALR mutations, three methods were compared in parallel: high-resolution melting-curve analysis (HRM), product-sizing analysis and Sanger sequencing. The sensitivity for the HRM, product-sizing analysis and Sanger sequencing was 96.4%, 98.2% and 89.3% respectively, whereas the specificity was 96.3%, 100% and 100%. In our cohort, the product-sizing analysis was the most sensitive method and was the easiest to interpret, while the HRM was sometimes difficult to interpret. In contrast, when large series of samples were tested, HRM provided results more quickly than did the other methods, which required more time. Finally, the sequencing method, which is the reference method, had the lowest sensitivity but can be used to describe the type of mutation precisely. Altogether, our results suggest that in routine laboratory practice, product-sizing analysis is globally similar to HRM for the detection of CALR mutations, and that both may be used as first-line screening tests. If the results are positive, Sanger sequencing can be used to confirm the mutation and to determine its type. Product-sizing analysis provides sensitive and specific results, moreover, with the quantitative measurement of CALR, which might be useful to monitor specific treatments.
最近有报道称，在70%-84%的JAK2V617F阴性骨髓增殖性肿瘤（MPN）中存在钙网蛋白（CALR）突变，这种检测对于改善MPN的诊断已变得必不可少。在一个由298例原发性血小板增多症（ET）患者组成的大型单中心队列中，分别在179例（60%）、56例（18.5%）和13例（4.5%）患者中检测到JAK2V617F、CALR和MPL突变。为了检测CALR突变，同时比较了三种方法：高分辨率熔解曲线分析（HRM）、产物大小分析和桑格测序。HRM、产物大小分析和桑格测序的敏感性分别为96.4%、98.2%和89.3%，而特异性分别为96.3%、100%和100%。在我们的队列中，产物大小分析是最敏感的方法且最易于解读，而HRM有时难以解读。相比之下，当检测大量样本时，HRM比其他需要更多时间的方法能更快地给出结果。最后，作为参考方法的测序方法敏感性最低，但可用于精确描述突变类型。总体而言，我们的结果表明，在常规实验室实践中，产物大小分析在检测CALR突变方面与HRM总体相似，两者均可作为一线筛查试验。如果结果为阳性，可使用桑格测序来确认突变并确定其类型。此外，产物大小分析提供了敏感且特异的结果，同时还能对CALR进行定量测定，这可能有助于监测特定治疗。
