Characterization of two genetically distinct subtypes of C5 diffuse large B-cell lymphoma (DLBCL) Free

Diffuse large B-cell lymphoma (DLBCL) is a clinically and molecularly heterogeneous disease with two recognized transcriptional subtypes, activated B-cell (ABC) and germinal center B-cell (GCB) types. Comprehensive genomic profiling, by us and others, identified at least 5 genetically defined subtypes with specific genetic signatures and associated molecular-driven vulnerabilities, providing the ability to develop biology-informed treatments.

Specifically, we integrated recurrent mutations, somatic copy number alterations (SCNAs) and structural variants (SVs), and identified 5 unique genetic subtypes, Clusters 1-5 (C1-C5 DLBCLs) (Chapuy et al., Nat. Med. 2018). Each of these DLBCL subtypes provides insight into prognosis, combinatorial treatments, and subtype-specific lymphoma biology. Recently, we developed a probabilistic model, allowing the prospective assignment of DLBclass labels (Chapuy et al., Blood 2025). In complementary approaches, two other groups also identified co-segregated alterations associated with transcriptional subtypes with remarkable similarity to DLBclass for certain subtypes but differences in others (LymphGen, Schmitz et al., NEJM 2018 / Wright et al., Cancer Cell 2020; HMRN, Lacy et al., Blood 2020).

Here, we focus on ABC-enriched C5 DLBCLs, a clinically unfavorable group of tumors following R-CHOP treatment and exhibiting frequent mutations in MYD88^L265P (40%), CD79B (38%), and recurrent 18q copy number gain (67%). To address the unresolved heterogeneity within C5 DLBCL, we employed non-negative matrix factorization (NMF) consensus clustering to a cohort of 180 primary C5 DLBCLs. These tumors comprise samples from both the NIH series and our previously reported de novo DLBCL cohort, classified using the DLBclass framework (Chapuy et al., Blood. 2025).

Following this approach, we identified two robust and distinct ABC-C5 DLBCL subtypes, C5A (n=88, 49%) and C5B (n=92, 51%), with distinct genetic architectures. C5A is predominantly characterized by SCNAs, including trisomy 3 and 18, whereas C5B is driven by oncogenic mutations, including MYD88^L265P, CD79B, OSBPL10, TBL1XR1, and PIM1 (top five mutations ordered by q-value). Notably, the C5B subtype is highly enriched in the LymphGen-defined MCD subtype (60% [55/92]). In contrast, the C5A cases were predominantly classified into non-MCD categories (p < 0.001), and a large proportion of C5A tumors (73% [64/88]) were assigned to the LymphGen “Other” category, suggesting that C5A represents a distinct and yet under-characterized subgroup.

Next, we validated our findings in an independent dataset and analyzed the PETAL data with genomic annotation (n=204, Mendeville et al., Nat Com. 2025) and found 27 and 19 of C5A and C5B cases, respectively. The genetic characteristics observed in the validation cohort highly consistent with our discovery cohort.

Importantly, both C5 molecular subtypes shared a statistically dismal progression-free survival (PFS) and overall survival (OS) compared to the other ABC-enriched cluster (C1; PFS p=0.008, OS p=0.024) and compared to all other genetic DLBCL cases (C1-4; PFS p=0.001, OS p=0.007). There is no statistically significant difference between C5A and C5B in PFS and OS (PFS p=0.8, OS p=1). Importantly, no significant differences were observed in the clinical features between C5A and C5B, except for LDH levels, which were higher in C5A (p=0.01). This finding underscores the prognostic significance of the defined genetic signatures of C5A and C5B.

Transcriptomic analysis of C5A tumors revealed highly expressed genes on chromosomes 3 and 18, including BCL2 and TIGIT, and an enrichment of apoptosis-related pathways. In contrast, C5B tumors exhibited activation of the STAT3 signaling pathway (GSEA ES=1.48, q=0.13). Integrated immune profiling using CIBERSORT and newly generated spatial proteomic data using CODEX (53 markers) revealed distinct tumor microenvironmental (TME) landscapes between the C5A and C5B subtypes.Taken together, our study identified and independently validated the presence of two genetically distinct C5 DLBCL subtypes, C5A and C5B, both with inferior prognosis to R-CHOP treatment. The genetic, transcriptomic, and spatial proteomic differences between C5A/B suggest different underlying pathogenetic mechanisms in these two subtypes, potentially reflecting distinct therapeutic vulnerabilities.