Correlation of insertion site and gene expression in CAR T-cell malignancy and infused CAR T-cell product. (A) Circos plot produced using the circlize package⁷⁰ showing the changes found in the malignant CAR T cells related to individual chromosomes (Coordinates and Banding) including: CAR transgene insert sites (lines at each genomic position); SV density (gray density plot); genetic copy-number variation (red bars, increased; blue bars, reduced copy numbers); contiguous regions of the genome containing more differentially expressed genes (DEG) than expected by chance (Enriched for DEG, purple bars) defined using a binomial test for DE vs non DE over 10 Mbp windows (adjusted P < .05); all DEG with log-fold change (logFC) (Malignancy vs product) of >4 (DEG Expr) (logFC, adjusted P < .05) plotted at their genome coordinates (green, up; blue, down); and overall levels of gene expression (All Gene Exp Dens), with all genes binned as overexpressed (red density plot) or underexpressed (blue density plot) in malignancy vs product (logFC > or < 0) with gene density plotted as a function of distance over the genome. (B) Correlation between copy-number variation and altered gene expression showing correlation between copy-number gain and increased gene expression (blue box) in the malignant CAR T cells (Malignancy) compared with CAR⁺CD4⁺ T cells isolated from the product. ***P < .0001.