Molecular Features Are Associated with Disease Stage in Diffuse Large B-Cell Lymphoma and Reveal Heterogeneity in Patient Outcomes

Introduction: The 25 to 30% of patients with diffuse large B-cell lymphoma (DLBCL) with limited stage disease (stages I or II) have a high rate (>80%) of favorable outcome when treated with standard frontline immunochemotherapy. Prior studies have characterized the prognostic impact of several biological factors, such as cell-of-origin (COO). Recent studies have identified novel genetic subtypes of DLBCL, such as the LymphGen classification system (Wright 2021), and might offer greater insight into the impact of disease biology on patient outcome. However, the interaction of these subtypes with disease stage and other clinical variables remains incompletely characterized. We aimed to identify the clinical and molecular features distinguishing limited and advanced stage disease and determine the prognostic impact of these factors within limited stage DLBCL. Methods: Data was derived from 9 publicly available cohorts of patients with DLBCL treated at frontline with R-CHOP/R-CHOP-like regimens, including clinical and survival data, COO, IHC, FISH, genomic variants (SNV/indels, CNA) via targeted or whole-exome sequencing, and bulk RNA-seq. To harmonize data across sources SNV/indels were re-annotated with Oncotator and RNA-seq was re-processed with STAR to yield gene-level counts. Genetic clusters were determined with the LymphGen algorithm. B-cell states were determined with EcoTyper. Immune microenvironment composition was determined with Cibersort and xCell. The primary outcome measures were overall and progression-free survival (OS/PFS). Results: We analyzed a composite cohort of 2,470 patients, with 983 cases featuring limited stage (I/II) and 1487 with advanced stage disease (III/IV). Limited stage disease was enriched for the GCB subtype relative to advanced stage (55% vs. 44%, p<0.001). Advanced stage was associated with the presence of other conventional IPI factors, such as age ≥60, elevated LDH, ECOG ≥2, and >2 extranodal sites (all p≤0.001), and higher rates of HGBL-MYC/BCL2 and HGBL-MYC/BCL2/BCL6 disease (p=0.038). Limited stage cases featured lower rates of KMT2D mutations (26% vs. 34%, FDR<0.1). SNV/indels and CNAs in other recurrently altered genes distributed evenly across the two groups. The EZB cluster was less frequent in limited stage cases (18.6% vs. 23.6%, p=0.005). The prevalence of other LymphGen clusters, tumor mutational burden, and CNA burden distributed evenly across stage groups. Limited stage was enriched for the S1 B-cell state (29.1% vs. 22.8%, p=0.022), while advanced stage was enriched for S3 and S4 states (8.5% vs. 11.4%, and 10.2% vs. 15.5%, respectively; p=0.022). Differential gene expression and gene set enrichment analyses revealed elevated expression of genes related to epithelial-mesenchymal transition in limited stage, and upregulation of interferon-gamma and -alpha pathways in advanced stage (all FDR<0.1). Immune deconvolution analysis of bulk RNA-seq did not reveal significant microenvironmental changes between stage groups. Within limited stage cases, age ≥60 was the strongest prognostic factor for PFS and OS in multivariate modelling (PFS HR 2.3, p=7x10-5; OS HR 3.4, p=7.1x10-9); LDH and ECOG significantly impacted only OS (HR 1.5, p=0.01; HR 1.9, p=0.004, respectively). Contrary to prior findings, ABC and Unclassified subtypes featured inferior prognosis relative to GCB in limited stage (ABC PFS HR 1.9, PFS p=0.002; Unclassified OS HR 1.8, OS p=0.03). Within the BN2, ST2, and MCD LymphGen subtypes, limited and advanced stage disease featured equivalent survival outcomes, though stage was significantly prognostic in EZB, A53, and Other clusters. Conclusion: Limited stage DLBCL is enriched for the GCB subtype, and features lower prevalence of additional adverse IPI factors and HGBL-DH/TH disease. Both stage groups feature similar rates of recurrently altered genomic lesions and LymphGen subtypes, though advanced stage is enriched for KMT2D mutations and the EZB subtype. B-cell states and differential pathway activation related to EMT and interferon signalling distinguish stage groups. Among limited stage cases, GCB subtype is associated with favorable outcomes, contrary to prior findings. Limited stage cases within EZB, A53, and Other clusters had improved prognosis relative to advanced stage, but those in ST2, BN2, or MCD did not. These findings should inform future confirmatory and therapeutic investigations.