Identification of a novel gene expression B-ALL cluster characterized by CDX2 high expression and UBTF::ATX7NL3 fusion. (A) Gene expression profiling of 302 B-ALL cases shown in a 2-dimensional tSNE plot. This analysis was performed with 235 selected genes listed in supplemental Table 6. Major B-ALL subtypes are highlighted in different colors, and gray dots indicate other B-ALL. A novel cluster of cases with distinct gene expression profiling was identified, thereafter named CDX2/UBTF ALL. (B) WGS long-reads visualized on IGV software in 1 CDX2/UBTF case (B_SL160) showing a deletion at 13q12.2 locus close to the CDX2 gene. The red line underlines the deleted region. (C) Volcano plot of differentially expressed genes between CDX2/UBTF ALL (n = 23) and other B-ALL cases (n = 279). Genes with fold change greater than 3 and -log10 P value >7 are shown in red. CDX2 is highlighted as 1 of the 10 most highly expressed genes in the novel cluster. (D) WGS long-reads and RNA-seq splice junctions visualized on IGV software in 1 CDX2/UBTF case (B_SL160) showing a deletion at 17q21.31 locus involving the UBTF and ATXN7L3 genes. The red line underlines the deleted region. (E) Scheme of the predicted UBTF::ATXN7L3 chimeric protein. The resulting in-frame fusion transcript incorporated 60 nucleotides upstream the canonical translation start codon of ATXN7L3. (F) Western-blot of CDX2/UBTF cases using ATXN7L3 antibody shows detection of normal ATXN7L3 protein (39 kDa) in all samples and another protein at a size corresponding to the predicted UBTF::ATXN7L3 chimeric protein (110 kDa) in CDX2/UBTF cases only. Western blot performed on PDX from 3 CDX2/UBTF cases (B_SL157, B_SL187, and B_SL160), 2 PAX5 P80R cases (005-0112 and B_SL142), and 1 KMT2A-r case (B_SL349). IGV, integrative genomics viewer; PDX, patient-derived xenograft; tSNE, t-distributed stochastic neighbor embedding; WGS, whole-genome sequencing.