Analysis of clonotypic DNA in primary tumors from aggressive B-cell lymphomas. (A) Subject selection for analysis; the study included 3 groups: 2 groups of patients with known CNS involvement, using prospectively collected CSF (n = 7) and retrospectively obtained, previously extracted CSF DNA (n = 8), in which the NGS-MRD assay was used as a diagnostic modality, and a prospective cohort (n = 22) of patients with newly diagnosed, aggressive lymphoma and no CNS involvement, in whom the NGS-MRD assay was used as a prognostic biomarker. (B) The NGS-MRD assay includes NGS of the primary lymphoma tumor to determine the dominant clonotypic sequence, which can be subsequently “tracked” in plasma or CSF samples containing tumor-derived clonotypic DNA. (C) Distribution of clonotypic sequences (n = 139) from different loci detected by the NGS-MRD assay in all primary tumors (n = 37) in this study (see supplemental Table 1 for a complete list of sequences). (D) Violin plots showing the distribution of copy numbers (per 10⁶ diploid genomes) and frequencies (among all B cells) of clonotypic sequences from the primary lymphoma tumor samples (n = 37). Horizontal line shows the median; box shows interquartile range. IQR, interquartile ranges.