Transcription analysis of malignant CAR T cells compared with nonmalignant CAR⁺ and untransduced T cells. (A) Multidimensional scaling of global gene expression from malignant CAR T cells (CAR T malignancy), nonmalignant CD4⁺ CAR T cells from the peripheral blood (Blood) and product (Product) and 3 untransduced healthy donor CD4⁺ T cells (Donor Controls). (B) Line drawing showing fourfold differential expression of clusters of genes in the malignant CAR T cells (CAR T malignancy) compared with CD4⁺ T cells from the CAR T-cell product (CAR) and untransduced related healthy donor T cells (Controls). (C) Examples of transcriptional readthrough/shadow seen in the malignant CAR T cells at the point of insertion in the FYN gene producing increased negative strand exonic expression and (D) transcriptional readthrough at the point of insertion in the KCNN3 gene showing no increase in exonic expression as visualized using the IGV.²⁸ (E) Relative levels of expression of genes identified in hg38 using PWMtools⁶⁹ as containing the HIVEP1-binding motif (KGGGAAATCCCn) in promoter/enhancer regions in CAR⁺ CD4⁺ T cells isolated from the product, expanded from the peripheral blood (Blood CAR), malignant CAR T cells (CAR T malignancy), as well as untransduced CD4⁺ T cells from the sibling donor (Donor) and unrelated healthy donors (UR donor 1, UR donor 2). (F) Example of primitive embryonic gene-ontology clusters seen to be increased in the malignant CAR T cells compared with both product and untransduced donor CD4⁺ T cells.