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case_images: The progression of a blast crisis. (A) A bone marrow (BM) aspiration revealed that lymphoid blasts account for 99.5% of all nucleated cells (Wright-Giemsa, x1,000). (B) Interphase FISH analysis revealed the classical 1G1R2F translocation pattern in 39% of cells before treatment. The white arrows denote fusion signals representing the BCR-ABL fusion on chromosome 22. The red arrows mark chromosome 9, and the green arrows indicate chromosome 22. (C) Conventional cytogenetic GTG-banding analysis indicated normal karyotype. The arrow marks chromosome 9, and the arrowhead indicates chromosome 22. (D) Panel of scatter plots depicting the multicolor immunophenotyping of bone marrow aspirate samples by flow cytometry. (E) A BM aspirate revealed that blast cells account for 9% of all nucleated cells (Wright-Giemsa, x1,000). (F) A BM aspirate revealed that prolymphocytes account for 1.5% of all nucleated cells (Wright-Giemsa, x1,000). (G) FISH analysis revealed that there was no abnormal signal. (H) BM infiltration with 94.5% of lymphoid blasts (Wright-Giemsa, x1,000). (I) The conventional cytogenetic GTG-banding analysis involves multiple abnormal chromosomes. (J, K) The BCR-ABL1 kinase domain mutation analysis revealed the mutation of E255K (J) and T315I (K).
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case_images: After about 1 week, the examination by bone marrow aspiration revealed infiltration with 99.5% of lymphoid blasts (Figure 1A). Interphase fluorescence in situ hybridization revealed the presence of a classical 1G1R2F translocation pattern in 39% of 200 cells (Figure 1B), and the BCR-ABL1 transcript level was 107.10%. Conventional cytogenetic GTG-banding analysis revealed that there were no gross chromosomal abnormalities in the two metaphase cells analyzed (Figure 1C). TDT at a proportion of 83.02% (Figure 1D). August 2, 2019, the bone marrow aspiration revealed that the proportion of blast cells was 9% (Figure 1E). A third bone marrow aspiration on September 4, 2019 revealed that the malignancy remained in the chronic phase (Figure 1F). There was no classical positive translocation pattern in 400 cells (Figure 1G). However, the patient entered a blast crisis again; bone marrow infiltration with 94.5% of lymphoid blasts was detected on September 23, 2019 (Figure 1H). T315I and E255K mutations (Figures 1J, K). The
case_images: Transcriptomic and genomic analysis of the blast crisis sample. (A) The timeline of disease progression, treatment, and sample collection. (B, C) Shared differential genes in the comparisons of T1 vs. T3 and T1 vs. NCBI samples (B) and their enriched pathways (C). (D) Missense mutations in our samples (T1-T4) and published blast crisis samples (N1-N5). (E) The possible mechanisms leading to the sudden blast crisis at T1. (F) The bar plot shows the proportion of chronic myeloid leukemia (CML) samples with or without the dysregulation of CML blast crisis risk genes. The Venn diagram shows the distribution of three dysregulated blast crisis risk genes (KLF3, DEFA3, and MYC) in the samples.
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case_images: The entire treatment process of this patient is described in Figure 2A. T1 (Supplementary Table S1). Most of these differential genes did not change significantly during the treatment (Figure 2B), which may be the risk genes of the T1 sample blast crisis. An enrichment analysis indicated that the 18 genes were mainly enriched in transcriptional misregulation in cancer, TGF-beta signaling pathways, etc. (Figure 2C). The genomic analysis results showed the missense mutation of PCNT [c.4357T>G, NM_006031.6|p.(C1453G) and c.4359T>G, NM_006031.6|p.(C1453T)] only in the T1 sample (Figure 2D), and the BCR-ABL1 T315I mutation was shown only in the T3 sample. TGF-beta receptor (TGFBR2) in the T1 and T3 blast crisis samples, but not in T2, T4, and other public blast crisis samples (Figure 2D). TGF-beta-related genes were reported to serve as regulators of B cell development and function. Combined with these results, we inferred that the pathway dysregulation of TGF-beta and transcriptional misregulation in cancer genes and the specific mutation may play key roles in the sudden blast crisis of this patient (Figure 2E). The dysregulation of KLF3, DEFA3, or MYC was observed in 31 CML patients (Figure 2F), and three patients, who may have a greater blast crisis risk, were observed with a dysregulation of both KLF3 and DEFA3.
case_images: Transcriptome features relevant to treatment and disease progression. (A) Shared enriched pathways with differentially expressed genes during disease progression and treatment. The red color indicates the pathway enriched with upregulated genes [T1 vs. T2 (up) and T3 vs. T4 (up)], whereas the blue color indicates the pathway enriched with downregulated genes [T2 vs. T3 (down)]. (B) The distribution of immune cell abundance during disease progression and treatment. (C) The abundance of B cell receptor V-chain and J-chain per sample. (D) Detailed information on the BCR-ABL1 fusion gene in each sample. (E) Expression of BCR-ABL1 in the T1, T3, and T4 samples. (F) Two-dimensional uniform manifold approximation and projection visualization of the cellular composition of the T3 and T4 samples. (G) Bar plot of the cell proportions of the T3 and T4 samples. (H) Circle plot showing the inferred TGF-beta and VEGF signaling networks. The size of the dot represents the cell number. A line connecting two cell types indicates a receptor-ligand interaction between them. The thickness of the line represents the signal intensity.
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case_images: A total of 19 pathways were shared by differential genes during treatment and disease progression (Figure 3A), and most of these pathways were related to immunology. The immune cell abundance analysis by ImmuCellAI revealed that the T1 and T3 samples contained higher numbers of B cells and naive T cells (Figure 3B); however, the diversity of their B cell receptor was rare (Figure 3C). After treatment, the number of cells with cytotoxic and helper function was increased, whereas that of B cells was reduced (Figure 3B). Subsequently, a gene fusion analysis revealed the presence of the BCR-ABL1 fusion in T1, T3, and T4 samples (Figure 3D), of which T3 had the highest fusion expression (Figure 3E). The single-cell RNA-seq analysis revealed the substantial accumulation of several clusters of immature B cells and late pro-B cells in the T3 sample (Figures 3F, G), while the T4 sample had the highest proportion of NK and CD8+ T cells. A cell communication analysis revealed that CCL17+ JCHAIN+ and MT1X+ NRIP1+ immature B cells, whose numbers slightly decreased after treatment, were mainly involved in TGF-beta signaling (Figure 3H).