CSF NGS-MRD assay for diagnosis of CNS involvement in lymphoma. (A) Case study of a primary CNS lymphoma, showing a magnetic resonance imaging gadolinium-enhanced T2 turbo spin echo sequence with intraparenchymal tumors; a standard CSF cytospin stained with Papanicolaou stain showing scant lymphocytes (L) and monocytes (M) (original magnification, ×600; cell count was 0 per cubic millimeter, and flow cytometry showed an insufficient number of events for evaluation); and clonotypic DNA sequences examined in the primary tumor, plasma (where no sequences were detectable), CSF acellular fluid, and CSF cell pellet fraction. (B) Clonotypic DNA sequences in the CSF of patients with parenchymal brain (n = 2) and leptomeningeal (Lepto, n = 5) lymphoma, measured as copy number per milliliter of acellular fluid, per 10⁶ diploid genomes in the cell pellet fraction, and as the clonotype frequency (in both types of samples). Bars show medians and interquartile ranges; P-values are from Somers’ D statistic adjusted for intrapatient clustering. Note the logarithmic scale on the left and middle panels. (C) distribution of clonotype frequency in CSF fractions obtained from patients with known CNS lymphoma: acellular fluid, cell pellet, and stored historical DNA. (D) relative abundance of various clonotypic sequences in the CSF of each patient (data from all patients included in the study who had a positive NGS-MRD assay in the CSF). The bars and red circles indicate the amount of the most abundant sequence from the primary tumor (expressed as copy count per milliliter of CSF acellular fluid or per 10⁶ diploid genomes in the CSF cell pellet); the blue circles indicate other clonotypic sequences in the sample.