Mutation analysis supports the identification of second primary lymphoma in cHL recurrences. Tissues of paired diagnosis and recurrences representing second primary cHL were subjected to targeted mutation analysis (TSO500 assay). HRS-associated gene mutations were selected based on a VAF of 1% to 30% and a minimal coverage of 350 reads. (A) Left panel shows data of clonally unrelated cHL samples based on distinct R, and right panel shows data of cases with discordant R. Paired data show mutually exclusive mutations and single overlapping mutations for 8 cases, whereas tissue samples of 2 cases (14 and 19) lacked pathogenic gene mutations with TSO500 assay. Mutation types and EBV status are shown. All shown mutations are scored as (potentially) pathogenic (PA3-PA5). For detailed information, see supplemental Table 9. (B-C) Schematic representations of the development of 2 independent lymphomas of case 8 (panel B) and case 21 (panel C). Gene mutations associated with cHL and clonal Ig rearrangement targets are indicated.