Projection of blast cells onto normal B-cell maturation trajectory shows variable maturation blocks between different subtypes of BCP-ALL. (A) A force–directed knn graph of cells involved in B-cell maturation from NBM samples, based on scRNA-seq data, colored by cell type with an accompanying cell density plot of numbers of cells along the trajectory. Inferred time points (t_0.0-t_1.0) are indicated along the trajectory. The 3 most common annotations are visualized at the top of the cell density plot. This force–directed knn graph was used as a reference for the projection of ALL blast cells in (C-F). (B) The force–directed knn graph of NBM B-cell trajectory colored by BCR chain status with an accompanying cell density plot of numbers of cells along the trajectory. The minus sign (−) indicates a feature that is not detected, the plus sign (+) indicates a feature that is detected, and a plus/minus sign (±) indicates that this category does not rely on the detection of this feature (ie, it can be either detected or not detected). The colors for the 3 most common annotations are visualized at the top of the color-coded cell density plots. (C-F) Single-cell projections of blast cells from all patients representing BCR::ABL1⁺ (n = 4), ETV6::RUNX1⁺ (n = 4), HeH (n = 4), and DUX4-r ALL (n = 11) onto the NBM reference. The density of the projected cells is visualized by hexagon size. Gene expression differences compared with the NBM reference, based on the local outlier factor, are highlighted by the color gradient. Cell density plots below the projection illustrate the number of cells along the trajectory and are color-coded by cell type (upper) and BCR chain status (lower). The color for the 3 most common annotations is visualized on the top of the color-coded cell density plots. (G) Three cell density plots illustrating the numbers of DUX4-r blast cells along the trajectory color-coded by cell types, presenting 3 cellular differentiation patterns observed within DUX4-r ALL denoted Pattern-1 (upper), Pattern-2 (middle), and Pattern-3 (lower). The color for the 3 most common annotations is visualized on the top of the color-coded cell density plots. (H) NBM reference knn graph projection of blast cells from all DUX4-r ALL samples, with time point path (t_0.0-t_1.0) indicated in gray, and an associated heat map showing the gradual gene expression changes along the path (with genes selected by variance filtering and linear regression). The genes marked in red represent lineage-defining genes for B cells. CLP, common lymphoid progenitor; IGH, heavy chain; IGLL1, surrogate light chain; IGL/K, light chain; p, productive; np, nonproductive.