Chemotherapy-associated single-base substitution signatures as temporal barcodes. (A) Cartoons summarizing the rationale used to time chromosomal duplications according to chemotherapy exposure. (B) Heatmap revealing the pattern of timing for chromosomal duplications and chromothripsis events in relation to chemotherapy for 8 tMN and 2 cases of post-chemotherapy MM. (C) Example of 2 copy neutral loss of heterozygosity (CN-LOH) acquired after chemotherapy exposure. In this case, both CN-LOH contain duplicated mutations (bottom left) consistent with late acquisition, as confirmed by molecular time analysis (bottom right), and showing large SBS-MM1 (melphalan) signature contribution within duplicated (ie, pregain) mutations (top right). (D) Example of whole-genome duplication during tMN relapse (left). Duplicated mutations (right) showed a large contribution from SBS35 (platinum), suggesting that this event was acquired after platinum exposure. (E) Chromothripsis event on chromosome 19 (SMARCA4) with multiple duplications (left). Similar to (D), the duplicated mutational signature contribution within chromothripsis-associated amplifications (right) was enriched for the SBS-MM1 contribution, implying that the chromothripsis event was acquired after melphalan exposure. In (C-E), the horizontal black line indicates the total copy number and the dashed orange line indicates the minor copy number. In (E), the vertical lines represent SVs breakpoints, color-coded based on SVs class: blue, inversion; green, tandem-duplication; red, deletion; black, translocation.