Department of Pathology, Stanford University Medical Center, 300 Pasteur Dr, L235, Stanford, CA 94305-5324, USA.
J Mol Diagn. 2010 Jan;12(1):58-64. doi: 10.2353/jmoldx.2010.090068. Epub 2009 Dec 3.
The somatic mutation JAK2 V617F is associated with BCR-ABL1-negative myeloproliferative neoplasms. Detection of this mutation aids diagnosis of these neoplasms, and quantification of JAK2 V617F may provide a method to monitor response to therapy. For these reasons, we designed a clinical assay that uses allele-specific PCR and real-time detection with hydrolysis probes for the quantification of JAK2 V617F, wild-type JAK2, and GAPDH transcripts. Mutant and wild-type JAK2 were quantified by using external plasmid standards that contain the relevant JAK2 V617F or JAK2 sequence, respectively. We tested 55 peripheral blood specimens from patients with suspected myeloproliferative neoplasms and 55 peripheral blood specimens from patients not known to have myeloproliferative neoplasms. Low-level, nonspecific amplification was detected in reactions containing a high copy number of plasmid standards and in specimens from patients not known to have myeloproliferative neoplasms, necessitating the use of a laboratory-established mutant to wild-type cutoff. The limit of detection established by using cell line dilutions is 0.1%, and this method identified three JAK2 V617F-positive patients who were not detected by a less sensitive method. The assay characteristics and our initial evaluation indicate this method can be used for the detection and quantification of JAK2 V617F, which should be useful for diagnosis of myeloproliferative neoplasms and potentially for monitoring minimal residual disease in future trials of therapies targeted to myeloproliferative neoplasms.
体细胞突变 JAK2 V617F 与 BCR-ABL1 阴性骨髓增殖性肿瘤有关。检测这种突变有助于这些肿瘤的诊断，并且 JAK2 V617F 的定量可能提供一种监测治疗反应的方法。出于这些原因，我们设计了一种临床测定法，该方法使用等位基因特异性 PCR 和实时检测水解探针来定量 JAK2 V617F、野生型 JAK2 和 GAPDH 转录本。通过使用分别包含相关 JAK2 V617F 或 JAK2 序列的外源性质粒标准品来定量突变型和野生型 JAK2。我们测试了 55 份来自疑似骨髓增殖性肿瘤患者的外周血标本和 55 份来自已知无骨髓增殖性肿瘤患者的外周血标本。在包含高拷贝数质粒标准品的反应和来自已知无骨髓增殖性肿瘤患者的标本中检测到低水平的非特异性扩增，这需要使用实验室建立的突变型与野生型截止值。使用细胞系稀释物建立的检测限为 0.1%，该方法鉴定了三个 JAK2 V617F 阳性患者，而较不敏感的方法未检测到这些患者。该测定法的特征和我们的初步评估表明，该方法可用于 JAK2 V617F 的检测和定量，这对于骨髓增殖性肿瘤的诊断以及未来针对骨髓增殖性肿瘤治疗的微小残留疾病监测可能非常有用。
