A novel subtype of AML characterized by CBFB-GDXY mutations. (A) Graphical representation of CBFB exons 1 to 6 (NM_022845.3) showing the location (arrowhead) and sequence of the 9 bp insertion mutations (highlighted in red) relative to the wild-type complementary DNA sequence. The number of patients with each mutation is shown in the circles. The predicted protein sequence of the CBFB mutations is also shown in red. (B) Uniform manifold approximation and projection (UMAP) of expression profiles of the pediatric AML cohort (AML, n = 561; cord blood CD34⁺ control, n = 5) performed with the top 133 most variably expressed genes. Dots are colored by the molecular feature of the sample. (C) Gene set enrichment analysis (GSEA) between AML with CBFB insertions and non-CBF AML (left) using gene sets derived from differentially expressed genes in CBFB::MYH11 AML or RUNX1::RUNX1T1 AML against non-CBF AML. Volcano plots (right) of genes differentially expressed between AML with CBFB insertions and CBFB::MYH11 or RUNX1::RUNX1T1. (D) Mutational landscape of CBFB-GDXY AML (n = 18) in this study (mutations detected at diagnosis are shown) and CBFB::MYH11 AML (n = 65) collected in the previous study.³ Seventy-five preselected genes frequently mutated in AML were subjected to mutation calling from RNA sequencing data. Eight FLT3 mutations were detected in seven patients and are categorized as internal tandem duplications (ITD), mutations in the tyrosine kinase domain (TKD), or mutations outside the TKD (other domains). (E) (i) Giemsa-stained peripheral blood showed blasts with myeloid and monoblastic features; arrowhead marks single slender Auer rod (original magnification ×1000). (ii) Giemsa-stained bone marrow aspirate smears showed immature myeloid elements with granules, blasts, and eosinophils (original magnification ×1000); arrowhead marks salmon-colored granules in the cytoplasm of the myeloid cell (inset). (iii) Hematoxylin and eosin–stained bone marrow biopsy (original magnification ×500) showed a hypercellular marrow almost completely replaced by a diffuse infiltrate of medium-sized blasts with increased eosinophils in the background. (F) Measurable residual disease assessment of the CBFB c.259_260insGGGACTCCT mutation by ultradeep next-generation sequencing in SBJ00860.