Fig. 1: Detection of genomic imbalances in single HRS cell selected by DEPArray™ digital sorting technology.

Scatter plots of CD30 mean intensity (y-axis) versus integral intensity DAPI (x-axis) of a leukocytes and b HRS cells. Selected leukocytes and HRS cells are highlighted in red and green, respectively. c Image gallery of two representative leukocytes and two representative HRS cells (labeled with red and green dots, respectively). e, f Representative whole-genome copy-number profiles of a single leukocyte and a single HRS cell confirm the nonmalignant and tumor nature of the cells, respectively. d Representative whole-genome copy-number profile obtained from the analysis of purified bulk DNA from the same patient, showing, as expected, no alterations given the rareness of HRS cells and the corresponding high contamination from normal cells. g Distribution of copy-number gains (red) and losses (blue) across all single HRS cells of each patient. An absolute log-fold change of at least 0.3 was used as threshold. Y-axis represents the percentage of single cells showing the CNAs. CNAs that were present in more than 30% of all single cells for each patient are highlighted in a darker color shade. Labels show genes in conserved cHL-related alterations. Recurring copy-number imbalances were observed with the most frequent gains on 9q, 2p, 8q, and 20q. These regions include genes encoding proteins associated with immune response (PD-L1, PD-L2), JAK-STAT signaling (JAK2), MLLT3, which is a component of the super elongation complex (SEC), REL (NF-κB pathway), BCL11A, belonging to a highly recurrent minimal region of gain in cHL, RECQL4 gene, a DNA helicase that belongs to the RecQ helicase family and has been previously been associated with predisposition to an increased risk in developing cancer, and E2F-1, a transcription factor that, in Hodgkin lymphoma, is upregulated and has been related to tumor kinetics. h Percentage of altered HRS cells for key components of NF-κB pathway and known oncogenes. Different levels of copy-number change, expressed as log-fold change, are highlighted in different color shades. Genome-wide analysis at single-cell resolution allowed us to observe a high degree of heterogeneity in terms of copy-number alteration level for genes in the NF-κB pathway both between and within samples. Strikingly, while REL showed copy-number gains in all four patients, it was only moderately gained (0.6 < log FC ≤ 0.9) in two out of four patients, while in the fourth patient (cHL14) REL showed large increases in copy number (log FC > 2).