Bulk and single-cell multiomics analyses of 5 diagnosis–relapse T-ALL pairs. (A) Schematic workflow of bulk whole-exome sequencing, single-cell transcriptomic analysis (5′ scRNA-seq) with paired single-cell TCR genotyping (scTCR-seq), and scDNA-seq of CD7⁺ BMMCs from 5 Dx_Rel paired T-ALL samples and samples from 2 HDs. (B) Oncoprint pairwise analysis of somatic variances in leukemia cells from samples from 5 patients (T956, T723, T593, T856, and T788) at diagnosis (Dx) and relapse (Rel) by whole-exome sequencing. Detailed mutational information is shown in supplemental Table 2. (C) UMAP plot showing 9 distinct T-lineage clusters (demarcated by color) based on the gene-expression profiles of isolated CD7⁺ BMMCs in Dx_Rel paired samples from 5 patients. (D) UMAP plots displaying distinct identities of each Dx (first row) and the corresponding Rel sample (second row) based on the sample signatures overlaid in panel C. (E) UMAP plot showing cell points colored according to TCR clonality in CD7⁺ BMMCs from the 5 Dx_Rel pairs. The color of each cell point indicates clonal abundance, and the gray color indicates a lack of TCR. (F) Dot plot showing the top significantly differently expressed genes (adjusted P < .05, average log2 fold change [FC] > 0) of each cluster obtained by FindAllMarkers. Gene-expression frequency (percentage of cells within each cell type expressing the gene) is indicated by spot size, and expression level is indicated by color intensity. (G) Violin plot depicting expression levels of featured gene, CD74, in cluster 9 (blue) and cluster 12 (cyan). (H-I) Sanger sequencing validation of FBXW7 mutation status of CD7⁺CD74⁺ normal T cells and CD7⁺CD74⁻ leukemic cells from patients T956 (F) and T723 (G) individually sorted by fluorescence-activated cell sorting.