Clonal evolution by serial bulk DNA next-generation sequencing and single-cell DNA-seq in a patient with advSM. (A) Clonal evolution in response to midostaurin and azacitidine by serial bulk DNA-seq. Founding ASXL1, SRSF2, and RUNX1 mutations persisted during midostaurin and azacitidine treatment. KIT and EZH2 mutation burden were initially high at the PB1 time point and were significantly diminished after midostaurin treatment. At the BM time point, allele frequency of NRAS and TET2 mutations were initially high, but they later decreased as a result of azacitidine treatment. KIT and EZH2 mutations, followed by disease relapse, recurred with an additional new mutation site at RUNX1 R162S. The results were projected into a trend line with VAF shown on the Y-axis and collected samples listed below the graph. (B) Clonal evolution in response to treatment with midostaurin and azacitidine determined by single-cell DNA-seq. After treatment with midostaurin for 3 months, significant reduction of KIT D816V and EZH2 mutations with significant expansion of TET2 and NRAS mutations was detected. The addition of azacitidine resulted in significant reduction of TET2 and NRAS mutations and progressive expansion of KIT D816V and EZH2 mutations. Acquisition of a new mutation (RUNX1 R162S) was followed by disease progression and rapid death after 8 months of combinatorial treatment. The results of single-cell DNA-seq are shown in a bar graph and fish plot with sequenced cell numbers listed below as combined blood and bone marrow cell count results. dRUNX1, double RUNX1 mutations.