Myeloproliferative neoplasms (MPNs) are caused by a somatic gain-of-function mutation in one of three "disease driver" genes JAK2, MPL or CALR. About half of MPN patients also carry additional somatic mutations that modify the clinical course. The order of acquisition of these gene mutations has been proposed to influence the phenotype and evolution of the disease. We studied 50 JAK2-V617F-positive MPN patients who carried at least one additional somatic mutation and determined the clonal architecture of their hematopoiesis by sequencing DNA from single cell derived colonies. In 22 of these patients we also side-by-side applied Tapestri single-cell DNA sequencing (scDNAseq) with cells from the same blood sample. The clonal architectures derived by the two methods showed good overall concordance. scDNAseq showed higher sensitivity for mutations with low variant allele fraction, but had more difficulties distinguishing between heterozygous and homozygous mutations. By unsupervised analysis of clonal architecture data from all 50 MPN patients we defined 4 distinct clusters that differed by the order of acquisition of the mutations, and the complexity of the subclonal structure. Cluster 4, characterized by more complex subclonal structure without a preferred order of acquisition, correlated with reduced overall survival, and in multivariate analysis represented a risk factor independent of the MPN subtype or the age at diagnosis. Our results suggest that deciphering the clonal architecture in patients with MPN that carry multiple gene mutations can improve the molecular prognostic stratification that until now was primarily based on the number and type of gene mutations.

中文翻译：

---

克隆结构对骨髓增生性肿瘤患者临床病程和预后的影响

骨髓增生性肿瘤 (MPN) 是由三个“疾病驱动”基因 JAK2、MPL 或 CALR 之一的体细胞功能获得性突变引起的。大约一半的 MPN 患者还携带改变临床过程的额外体细胞突变。已经提出获得这些基因突变的顺序会影响疾病的表型和进化。我们研究了 50 名携带至少一个额外体细胞突变的 JAK2-V617F 阳性 MPN 患者，并通过对来自单细胞衍生集落的 DNA 进行测序来确定其造血功能的克隆结构。在其中 22 名患者中，我们还将 Tapestri 单细胞 DNA 测序 (scDNAseq) 与来自同一血液样本的细胞并排应用。两种方法衍生的克隆体系结构显示出良好的整体一致性。scDNAseq 对变异等位基因分数较低的突变表现出更高的敏感性，但在区分杂合和纯合突变时更加困难。通过对所有 50 名 MPN 患者的克隆结构数据进行无监督分析，我们定义了 4 个不同的簇，这些簇的不同之处在于突变的获得顺序和亚克隆结构的复杂性。簇 4 的特征是更复杂的亚克隆结构，没有首选的获取顺序，与总体生存率降低相关，并且在多变量分析中代表独立于 MPN 亚型或诊断年龄的风险因素。