The rearrangement partner and the presence of MYC mutations determine the outcome of patients with MYC and BCL6 rearrangements

High-grade B-cell lymphomas (HGBLs) with concurrent rearrangements of MYC and BCL6 represent a biologically and clinically heterogeneous subset of aggressive B‑cell neoplasms, the optimal classification and risk stratification of which remain incompletely defined. Generally, double‑hit and triple‑hit lymphomas with MYC and BCL2 and/or BCL6 rearrangements are associated with high‑risk clinical features and poor survival under standard chemoimmunotherapy.1-3 In contrast to MYC/BCL2‑rearranged lymphomas, MYC/BCL6 double‑hit lymphomas appear biologically more diverse and, in some cases, do not uniformly display dismal outcomes as seen in MYC/BCL2‑rearranged HGBL, leading to recent reallocation of MYC/BCL6‑rearranged cases to genetic subtypes of diffuse large B‑cell lymphoma, not otherwise specified (DLBCL-NOS), or HGBL, NOS in the 5th edition of the WHO classification.4-6 A particularly intriguing subset is defined by the reciprocal t(3;8)(q27;q24) BCL6::MYC rearrangement, which creates a so‑called “pseudo double‑hit” configuration that may be indistinguishable from classical double‑hit by interphase fluorescence in situ hybridization (FISH), but appears to behave more like a single MYC‑activating event. Recent studies suggest that lymphomas harboring t(3;8)(q27;q24) can have more favorable outcomes than conventional double‑hit lymphomas, but the molecular basis for this prognostic discrepancy—particularly in comparison with other MYC/BCL6‑rearranged tumors involving immunoglobulin (IG) partners—remains unclear.7-9 To address these questions, the present study characterizes a cohort of HGBL patients with concurrent MYC and BCL6 rearrangements, integrating conventional cytogenetics, whole‑genome sequencing (WGS), whole‑transcriptome sequencing (WTS), and clinical data. All samples sent for diagnosis between 08/2005 and 04/2025 were used for the detection of concurrent MYC and BCL6 rearrangements by chromosome banding analysis (CBA) and/or FISH using probes for MYC, BCL6, and BCL2. Patients agreed with the use of laboratory data for research studies. The study followed the rules of the Helsinki Declaration. Chromosome preparations and banding analysis were performed in all cases according to standard methods.10 WGS was performed for 41 patients (median coverage 100×), WTS for 39 cases (see Supporting Information S2: Supplemental Material for details). Clinical data were available for 44 patients. SPSS (version 19.0.0) software (IBM Corporation, Armonk, NY) was used for statistical analysis. Overall survival (OS) curves were calculated according to Kaplan–Meier and compared using the two-sided log-rank test. All reported P-values are two-sided and were considered significant at P ≤ 0.05. By CBA and/or FISH analyses, we identified 63 cases with a MYC and concurrent BCL6 rearrangement. Of these, the vast majority were diagnosed as DLBCL/HGBL (54/63, 86%), 5/63 (8%) as follicular lymphoma (FL), and 4/63 (6%) as acute lymphocytic leukemia (ALL). In total, 12/63 (19%) showed a reciprocal t(3;8) leading to a BCL6::MYC rearrangement (t(3;8)+), 51/63 cases (81%) had MYC and BCL6 rearrangement with other partner genes (t(3;8)−). In all cases with t(3;8)+, additionally a BCL2 rearrangement was detected (“triple-hit”), whereas this was only found in 31/51 (61%) t(3;8)− patients (Figure 1A). Of note, all BCL2 rearrangements were caused by t(14;18)(q32;q21)/IGH::BCL2 translocation. In t(3;8)− patients, the most frequent translocation partner (TP) of MYC was IGH (30/51), followed by IGL (9/51) and IGK (4/51), thus in 81% of cases an IG locus was identified as TP. BCL6 was also most frequently rearranged with IGH (18/51), but IGK and IGL were only rarely detected as TP (IG locus: 45%), whereas CIITA (16p13) and RHOH (4p14) were identified as TP in 8/51 and 4/51 of cases, respectively (Figure 1B). Other recurrent TP comprised GRHPR (9p13), RASGRP1 (15q14), and UTRN (6p24) for MYC and IRAG2 (12p12) for BCL6. Patients with t(3;8)+ were younger and showed a higher frequency of females compared to t(3;8)− cases (age: 64 vs. 69 years; gender: 58% vs. 39% female). OS of the total cohort was 11 months. OS was better for t(3;8)+ compared to t(3;8)− patients, corroborating previous studies, although the difference was not statistically significant due to the low number of cases with t(3;8)+ (n.r. vs. 10 months, P = n.s.). Further, when the t(3;8)− cohort was split into cases with an IG as TP (for MYC and/or BCL6, n = 44) and cases with other TP (n = 7), the negative impact on OS was only found in cases with IG partners (with IG partner vs. other TP, 6 months vs. n.r., P = n.s). Hence, for further analysis the t(3;8)+ and other TP cases were combined (t(3;8)+/other, n = 19) and compared to t(3;8)− cases with an IG as TP (t(3;8)−/IG, n = 44): OS differed significantly between these subgroups (n.r. vs. 6 months, P = 0.019, Figure 1C). Further, the t(3;8)+/other cases showed a significant association to BCL2 rearrangements when compared to t(3;8)−/IG (19/19 = 100% vs. 25/44 = 57%, P < 0.001). However, the presence of BCL2 rearrangements did not influence OS, as t(3;8)−/IG cases separated by the presence or absence of BCL2 rearrangements showed equally poor survival (9 months and 6 months, respectively). WTS analysis of the subgroups (t(3;8)+/other vs. t(3;8)−/IG) did not show differences in expression of BCL2, BCL6, MYC, or other differentially expressed genes. In the total cohort, molecular mutations were frequently found in CREBBP (n = 17, 27%), KMT2D (n = 18, 29%), and MYC (n = 15, 24%), whereas mutations in TP53 were rather infrequent (n = 7, 11%). Interestingly, MYC mutations were almost exclusively found in t(3;8)−/IG cases (14/44 vs. 1/19, 32% vs. 5%, P = 0.026), whereas CREBBP (16% vs. 53%, P = 0.005) and KMT2D (21% vs. 47%, P = 0.038) mutations were significantly more often detected in t(3;8)+/other cases (Figure 1D; Supporting Information S1: Table 1). No influence on OS was found for the presence of CREBBP, KMT2D, or TP53 mutations. However, MYC mutations showed a negative impact on OS in the total cohort (17 vs. 2 months; P = 0.015) and even within the t(3;8)−/IG cohort (10 vs. 1 month; P = 0.043). In multivariate COX regression analyses, both MYC mutations and t(3;8)−/IG showed a negative impact on OS (MYC mutations: hazard ratio [HR] = 2.1, P = 0.043; t(3;8)−/IG: HR = 3.2, P = 0.032). HGBLs with concurrent MYC and BCL6 rearrangements are biologically heterogeneous and can be separated into two subgroups, as the present data show that the outcome is primarily determined by partner genes and MYC mutation status rather than by “double‑hit” status alone. Specifically, reciprocal t(3;8)(q27;q24) BCL6::MYC and non‑IG partners define a more favorable subgroup, whereas IG partners and MYC mutations identify an ultra‑high‑risk subset with dismal survival. Previous studies suggested that MYC/BCL6 double‑hit lymphomas generally share the poor prognosis of MYC/BCL2 double‑hit disease, supporting their inclusion among HGBL with rearrangements of MYC and BCL2 and/or BCL6. More recently, however, it was proposed that MYC/BCL6 rearrangements or co‑expression do not invariably confer adverse outcome and that double‑hit biology must be refined according to specific genetic constellations. The present cohort extends these observations by demonstrating that t(3;8)+/other cases behave distinctly from IG‑partnered cases, aligning with the “pseudo double‑hit” concept proposed for t(3;8)(q27;q24).7-9, 11, 12 Notably, WTS did not reveal major differences in MYC, BCL2, or BCL6 expression between subgroups, suggesting that enhancer context and chromatin architecture, rather than expression levels, may underlie the divergent clinical behavior.5, 7, 8 Our data show that MYC mutations were almost exclusively found in the adverse t(3;8)−/IG subgroup and independently predicted poor outcome (potentially due to highly proliferative disease), whereas CREBBP and KMT2D mutations (significantly enriched in the t(3;8)+/other subgroup) were not associated with inferior survival, differing from their usual negative implication in other DLBCL cohorts. These findings suggest that MYC mutations cooperate with IG‑driven deregulation to define an ultra‑high‑risk “double‑hit–plus” biology, whereas epigenetic mutations in the t(3;8)+/other subgroup may reflect early lineage‑defining events without dominant prognostic impact.13, 14 Because interphase FISH cannot reliably distinguish t(3;8)/BCL6::MYC from classical MYC/BCL6 double‑hit cases, reliance on break‑apart probes alone risks misclassifying biologically favorable t(3;8)+/other cases as conventional high‑risk double‑hit disease. The data strongly argue in favor of integrating conventional karyotyping and/or WGS to identify t(3;8)+/other versus t(3;8)−/IG and for including MYC mutational analysis in the routine assessment of MYC/BCL6‑rearranged lymphomas. Given the extremely poor survival of t(3;8)−/IG cases with MYC mutations (~1 month), these patients should be prioritized for intensified or novel treatment strategies, whereas t(3;8)+/other cases may not uniformly require escalation beyond current standard approaches, pending prospective validation.7, 8, 11, 12, 15 Due to the impact on OS and the specific molecular characteristics, t(3;8)−/IG cases might be considered as a separate entity. The authors would like to thank all co-workers at the MLL Munich Leukemia Laboratory for their dedicated work. The authors would also like to thank all physicians for providing samples and caring for patients as well as collecting data. Anna Stengel: Conceptualization; investigation; writing—original draft; visualization; validation; writing—review and editing; supervision; data curation. Miriam Lenk: Formal analysis; writing—review and editing; visualization. Wolfgang Kern: Writing—review and editing; supervision. Wencke Walter: Data curation; formal analysis; conceptualization; writing—original draft; writing—review and editing; methodology. W.K. declares part ownership of MLL Munich Leukemia Laboratory. A.S., M.L., and W.W. are employed by the MLL. The study was conducted in accordance with the Declaration of Helsinki and was approved by the institutional review board and ethics committees before study initiation. All patients provided written informed consent. This research received no external funding. The data that support the findings of this study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.