PMC10449247_01
The morphological characteristics, mutation profiles, and single-cell analysis of tumor cells in the patient before and after midostaurin treatment. (A) Peripheral blood aspirate under Wright's stain, demonstrating neoplastic mast cells with immature nuclear chromatin, abundant tightly packed cytoplasmic granules, high nuclear to cytoplasmic (N:C) ratio before treatment (top) and mast cells with condensed nuclear chromatin, less granules in cytoplasm and lower N:C ratio after 10 months of midostaurin treatment (bottom), bar=10muM. (B) Circos plot of single-nucleotide variations based on WES before and after midostaurin treatment. The outer lines colored in blue represent mutations detected before treatment. The inner lines colored in orange represent mutations detected after treatment. (C) Gene set enrichment analysis of genes decreased with mutation burden after treatment. (D) UMAP of single-cell RNA sequencing experiments of patient PBMCs before and after midostaurin treatment. Each cell represents a cell. Blue and red represent cells collected before or after 10 months of midostaurin treatment. (E) UMAP of single-cell RNA sequencing experiments as in (D) Annotated cell types are distinguished by colors. (F) Dot plot of cell-type-specific marker genes used to annotate the clusters in single-cell RNA sequencing experiments. (G) Fraction of different cell types before and after midostaurin treatment. (H) Pathways downregulated on tumor mast cells after midostaurin treatment. Normalized gene expression of downregulated genes (I), and upregulated genes (J) on tumor cells after midostaurin treatment. ***, represented significant difference of P<0.001.
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Figure 1A ). Flow cytometric analysis was performed on BM sample before treatment to observe mast cells in circulation and used as evidence for diagnosis according to the diagnose criteria of mast cell leukemia. As shown in  Supplementary Figure 1 , a significant population was observed in bone marrow sample showing extremely bright expression of CD117 and positive CD45 with moderate FSC and SSC (red color). ASXL1, BCR-ABL, JAK2, FLT3, and NPM1 mutations ( Figure 1B ). According to the dose modification instructions of midostaurin, the patient was treated with 100 mg/d midostaurin due to anemia and severe fatigue, and achieved a major response 10 months after treatment, with mast cells decreasing to 24% in BM. Meanwhile, blood smears showed mast cells with condensed nuclear chromatin, less granules in cytoplasm and lower N:C ratio ( Figure 1A ). However, RUNX1 mutation, relating to poor prognosis of mast cell leukemia patients, was only detected after midostaurin treatment, which might indicate subsequent midostaurin resistance. ( Figure 1B ). Gene set enrichment analysis (GSEA) showed the top 5 downregulated pathways, including PI3K/AKT signaling in cancer, regulation of cell division, MAPK family signaling cascade, cell response to growth factor stimulus, and regulation of cell cycle process ( Figure 1C ), in which PI3K/AKT and MAPK signaling pathway are the downstream of KIT signaling and are associated with mast cell leukemia progression. Before treatment, single-cell RNA sequencing revealed that mast cells were the major fraction (81.40%) in peripheral blood mononuclear cells (PBMC) ( Figures 1D-F ). T cells (from 6.79% to 41.67%), Natural killer (NK) cells (from 8.07% to 22.16%) and B cells (from 2.18% to 8.72%) ( Figure 1G ). IL-4 and IL-13 signaling, NF-KB signaling regulation, and cytokine production pathways regulation ( Figure 1H ). The expression level of genes such as S100A4, LGALS1, TPSB2, and S100A6 were decreased. Instead, the expression level of RPS27A, RPS6, UBA52, and RACK1, involved in tyrosine kinase inhibitors (TKI) resistance, were increased after midostaurin treatment ( Figures 1I, J ).
Single-cell RNA sequencing of immune cells in patient before and 10 months after midostaurin treatment. Pathways upregulated on T cells (A) and NK cells (B) after midostaurin treatment. T cell clusters and fraction of clusters (C), NK cell clusters and fraction of clusters (D) before and after midostaurin treatment.
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Regarding the impact of midostaurin on mast cell leukemia microenvironment, pathways upregulated on T cells mainly included antigen presenting, T cell cytotoxicity, interferon gamma signaling, T cell receptor signaling, regulation of immune response, and T cell-mediated immunity ( Figure 2A ). Among NK cells, antigen presentation, regulation of cell killing, natural killer cells mediated cytotoxicity, leukocyte mediated cytotoxicity, regulation of cell surface receptor, immune response, and cytokine production pathways were upregulated ( Figure 2B ). Figures 2C, D ). Further GSEA revealed that as compared to other clusters, the downregulated pathways on cluster 1 cells, including adaptive immune system, T cell activation, antigen processing and presentation pathways on cluster 1 of T cell ( Supplementary Figure 2A ), as well as vesicle-mediated transport, TCR, endosome to lysosome transport pathways on cluster 1 of NK cell ( Supplementary Figure 2B ), indicating the impaired functions of antigen presenting and cell cytotoxicity in these T/NK cells.