WES, Bulk RNA-seq, and targeted long-read PacBio sequencing of the CD19 of B-ALL from PNR and PS patients. (A) Pretreatment genomic landscape of pediatric patients with B-ALL within the study cohort stratified per the patient response. Prior CD19-targeting therapy with blinatumomab is designated in green. Fusions are in yellow, with alterations that were previously discovered by cytogenetics noted (#). Mutations are in blue with hotspot mutations noted (∗). No stop-gain, frameshift, or splicing variants were detected using WES. (B) Bulk RNA-seq showed no significant difference in CD19 expression between responders (PS) and nonresponders (PNRs), using Mann-Whitney two-tailed test (P = 1.000). Patient samples with <5% leukemic infiltration (S53 and S58) were excluded from the analysis. (C) Schematic depicting the extracellular domain of top transcripts of the CD19 locus identified by PacBio sequencing. (D) Heatmap showing the proportion as a percentage of detected CD19 transcripts via targeted PacBio long-read sequencing across the patient samples. Notably, patient S1057 had significant preexisting transcripts without CD19’s exon 2, the known binding domain of CD19-CAR. (E) Proportions of CD19 isoforms identified by PacBio sequencing in patient S1057. (F) Pre-CD19-CAR flow cytometry data for CD19 (clone J3-119) vs CD45 and CD10 from patient S1057 do not reveal a significant CD19^– population.