In addition to high-molecular risk (HMR) mutations (ASXL1, EZH2, SRSF2, IDH, and U2AF1^Q157), lower JAK2V617F variant allele frequencies (VAF) have been demonstrated to be associated with poor prognosis of myelofibrosis (MF) patients. Nevertheless, the relationship between JAK2V617F VAF and HMR mutations remains inconclusive. To address this, we analyzed the mutation status of 54 myeloid neoplasm-relevant genes using targeted next-generation sequencing in 124 MF patients. Three cohorts from multiple international centers were analyzed for external validation. Among JAK2-mutated patients, the presence of HMR mutations drove poor prognosis in patients with lower JAK2V617F VAF but not in those with higher JAK2V617F VAF. Survival analyses showed consistent results across validation cohorts. In multivariable analysis, concurrent HMR and a lower JAK2V617F VAF was identified as an independent adverse prognostic factor for survival, irrespective of age, MIPSS70, MIPSS70 + v2, and GIPSS risk groups. Mutation co-occurrence tests revealed no shared mutational pattern over different cohorts, excluding potential confounding effect from other concurrent mutations. Importantly, the integration of HMR/JAK2V617F VAF (≤50%) status significantly enhanced existing prognostic models, as evidenced by higher c-indexes and time-dependent ROC analyses. Single-cell studies with sequential follow-ups are warranted to decipher the clonal evolution of MF and how it relates to JAK2V617F VAF dynamics.

除了高分子风险 (HMR) 突变（ ASXL1 、 EZH2 、 SRSF2 、 IDH和U2AF1 ^Q157 ）之外，较低的JAK2 V617F 变异等位基因频率 (VAF) 已被证明与骨髓纤维化 (MF) 患者的不良预后相关。然而， JAK2 V617F VAF 与 HMR 突变之间的关系仍不确定。为了解决这个问题，我们使用靶向下一代测序技术分析了 124 名 MF 患者的 54 个髓系肿瘤相关基因的突变状态。对来自多个国际中心的三个队列进行了外部验证分析。在JAK2突变患者中，HMR 突变的存在会导致JAK2 V617F VAF 较低的患者预后不良，但JAK2 V617F VAF 较高的患者则不会。生存分析显示各验证队列的结果一致。在多变量分析中，无论年龄、MIPSS70、MIPSS70 + v2 和 GIPSS 风险组如何，并发 HMR 和较低的JAK2 V617F VAF 被确定为生存的独立不良预后因素。突变共现测试显示不同队列中没有共同的突变模式，排除了其他并发突变的潜在混杂效应。重要的是，HMR/ JAK2 V617F VAF (≤50%) 状态的整合显着增强了现有的预后模型，更高的 c 指数和时间依赖性 ROC 分析证明了这一点。需要进行连续随访的单细胞研究来破译 MF 的克隆进化及其与JAK2 V617F VAF 动力学的关系。