bioRxiv2022
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.