Mutated clones driving leukemic transformation are already detectable at the single-cell level in CD34-positive cells in the chronic phase of primary myelofibrosis

Abstract

Disease progression of myeloproliferative neoplasms is the result of increased genomic complexity. Since the ability to predict disease evolution is crucial for clinical decisions, we studied single-cell genomics and transcriptomics of CD34-positive cells from a primary myelofibrosis (PMF) patient who progressed to acute myeloid leukemia (AML) while receiving Ruxolitinib. Single-cell genomics allowed the reconstruction of clonal hierarchy and demonstrated that TET2 was the first mutated gene while FLT3 was the last one. Disease evolution was accompanied by increased clonal heterogeneity and mutational rate, but clones carrying TP53 and FLT3 mutations were already present in the chronic phase. Single-cell transcriptomics unraveled repression of interferon signaling suggesting an immunosuppressive effect exerted by Ruxolitinib. Moreover, AML transformation was associated with a differentiative block and immune escape. These results suggest that single-cell analysis can unmask tumor heterogeneity and provide meaningful insights about PMF progression that might guide personalized therapy.

Novel Molecular Insights into Leukemic Evolution of Myeloproliferative Neoplasms: A Single Cell Perspective

“…Targeted panels can be designed to detect SNVs and small indels, also determining their zygosity in each cell. VAF inferred from single cell data is consistent with the one originated from bulk NGS [ 73 , 75 ]. Unlike prior methods, single cell genomic analysis is able to detect SNVs down to 0.1% VAF.…”

“…These observations were confirmed and expanded in recent years, taking advantage of the most recent single cell approaches [ 72 ]. Compared with single cell genomics, genotyping of colonies does not necessary reflect the real subclones’ frequency, because some of them might display a growth advantage in vitro that leads to biased mutation detection compared to variant allele frequency (VAF) derived from bulk analysis [ 62 , 63 , 73 ].…”

“…Traditionally, splicing factor mutations have always been considered to occur in heterozygosity, as their allele frequency in bulk populations rarely exceeds 50%. Nevertheless, the single cell genomic analysis of a PMF patient that harbored a SRSF2 mutation, with a bulk allele frequency of approximately 50%, highlighted the presence of WT, heterozygous and homozygous cells for this variant [ 73 ]. Analogous results were obtained by Meira and colleagues, who analyzed two patients displaying similar JAK2V617F VAF from bulk sequencing.…”

Rare evolution of CSF3R-mutated chronic neutrophilic leukemia to t(4;12)(q12;p13) acute myeloid leukemia with SETBP1 mutation

“…Parenti et al. recently reported a primary myelofibrosis patient in whom ruxolitinib seemed to increase PD-L1 expression on preexisting leukemic CD34-positive cells, along with reduced T-cell activation during disease progression to AML [12] . However, the mechanism by which ruxolitinib contributes to the process of CNL-associated transformation of AML remains to be elucidated.…”

Increased Plasma Levels of lncRNAs LINC01268, GAS5 and MALAT1 Correlate with Negative Prognostic Factors in Myelofibrosis

“…In addition, “High Molecular Risk (HMR)” mutations (e.g., in ASXL1 , IDH1/2 , SRSF2 and EZH2 genes) have been associated to a worse prognosis and a more frequent leukemic transformation [ 4 ]. The serial acquisition of somatic mutations underlies the clonal evolution in MPNs and we have recently reconstructed by single cell analysis the sequence of mutational events associated to PMF progression [ 5 ]. MF is the most severe among the MPNs and is characterized by the occurrence of bone marrow fibrosis and a consequent abnormal increase in the number of circulating CD34+ hematopoietic progenitors [ 6 ].…”