Intratumoral heterogeneity (ITH) provides the substrate for
tumor evolution and treatment resistance, yet is remarkably understudied in
lymphoma, due to the often limited amount of tissue that gets sampled during the routine diagnostic process, generally from a single nodal or extranodal site. Furthermore, the trajectory of how
lymphoma, and especially
non-Hodgkin lymphoma, spreads throughout the human body remains poorly understood. Here, we present a detailed characterization of ITH by applying whole-genome sequencing to spatially separated
tumor samples harvested at the time of autopsy (n=24) and/or diagnosis (n=3) in three patients presenting with refractory B-cell
non-Hodgkin lymphoma. Through deconvolution of bulk samples into clonal mixtures and inference of phylogenetic trees, we found evidence that polyclonal seeding underlies
tumor dissemination in
lymphoma. We identify mutation signatures associated with ancestral and descendant clones. In our series of patients with highly refractory
lymphoma, the determinants of resistance were often harbored by founding clones, although there was also evidence of positive selection of driver mutations, likely under the influence of
therapy. Lastly, we show that
circulating tumor DNA is suitable for the detection of ancestral mutations but may miss a significant proportion of private mutations that can be detected in tissue. Our study clearly shows the existence of intricate patterns of regional and anatomical evolution that can only be disentangled through multi-regional
tumor tissue profiling.