Calreticulin Mutations in Myeloproliferative Neoplasms: Comparison of Three Diagnostic Methods

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.

近期有研究报道，70%~84%的JAK2V617F阴性骨髓增殖性肿瘤（myeloproliferative neoplasms, MPN）患者携带钙网蛋白（Calreticulin, CALR）突变，该突变检测对于优化骨髓增殖性肿瘤的诊断已成为必要环节。本研究纳入单中心大队列共298例原发性血小板增多症（Essential Thrombocythemia, ET）患者，其中JAK2V617F、CALR及MPL突变的检出例数分别为179例（占比60%）、56例（18.5%）及13例（4.5%）。针对CALR突变的检测，本研究并行比较了三种检测方法：高分辨率熔解曲线分析（high-resolution melting-curve analysis, HRM）、产物大小分析（product-sizing analysis）及桑格测序（Sanger sequencing）。三种方法的检测灵敏度分别为96.4%、98.2%与89.3%，特异性则分别为96.3%、100%与100%。在本研究队列中，产物大小分析的灵敏度最高且结果判读最为简便，而高分辨率熔解曲线分析有时则难以实现准确判读。与之相对，当批量检测样本时，高分辨率熔解曲线分析的出结果速度快于其余两种方法，后者耗时更长。作为金标准的桑格测序法虽灵敏度最低，但可精准明确突变的具体类型。综合本研究结果来看，在常规实验室实践中，产物大小分析与高分辨率熔解曲线分析在CALR突变检测中的整体表现相当，二者均可作为一线筛查手段。若一线筛查结果为阳性，则可采用桑格测序确认突变并明确其具体类型。此外，产物大小分析可实现CALR的定量检测，这一特性或可用于特定治疗方案的疗效监测。