Abstract Introduction Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease both clinically and biologically. Based on gene expression profiling, two major molecular subtypes, activated B-cell (ABC)- and germinal center B-cell (GCB)-like DLBCLs, have been recognized. More recently, next generation sequencing has also shed light on the mutational heterogeneity of these lymphomas. While several oncogenes are recurrently mutated and aberrantly expressed in DLBCL, little is known of their functional impact and association with survival. Design and Methods To identify mutated oncogenes in DLBCL, we performed whole-exome and RNA sequencing screen for tumor tissue and matched normal DNA from eight high-risk (aaIPI >2) DLBCL patients less than 65 years old. The patients were treated in the Nordic phase II protocol with dose-dense chemoimmunotherapy followed by systemic central nervous system (CNS) prophylaxis. Initial screen was expanded to a Nordic discovery cohort consisting of 38 high-risk DLBCL patients treated in the same protocol. Gene expression was analyzed from the tumor tissue using Affymetrix Human Exon 1.0 ST arrays, quantified with MEAP (Multiple Exon Array Preprocessing, probe annotation version 70) algorithm, and expression of the mutated genes identified. Samples were classified into GCB, ABC and nonclassified subgroups using the Lymphochip gene predictor. At the time of the analysis, median follow-up time was 65 months and predicted 5-year OS 76%. Survival association of the identified mutations and gene expression was validated using RNAseq data from 92 (Cancer Genome Characterization Initiative (CGCI) repository), and oligonucleotide-based microarray expression data from 233 (Lymphoma/Leukemia Molecular Profiling Project, LLMPP) DLBCL patients, respectively. Patients were treated with chemoimmunotherapy in both validation cohorts. Results In our initial screen 922 genes were affected by somatic mutations. The genes included both novel and previously identified lymphoma-related oncogenes, such as MYD88, CD79B and DTX1. We highlight mutations affecting GC marker Kelch-like family member 6 (KLHL6), which is a predicted oncogene and involved in B-cell receptor (BCR) signaling in DLBCL. In the CGCI validation cohort 16% of the patients carried non-silent KLHL6 mutations. According to Kaplan Meier analysis, the patients with KLHL6 mutations had significantly worse survival in comparison to the patients without mutations (5-year OS; 53% vs 84%; p = .010). In multivariate analysis, the prognostic impact of KLHL6 mutations on the survival was independent of IPI (OS; RR, 3.060; 95% CI, 1.233-7.598; p = .016). KLHL6 mutations were equally distributed between the molecular subtypes. However, when the molecular subtypes were analyzed separately, KLHL6 mutations remained predictive for poor survival only in the ABC-like DLBCL (OS; RR, 6.727; 95% CI 1.576-28.714; p= .010). KLHL6 expression was further analyzed in the Nordic discovery cohort. Consistent with its role as a GC marker, KLHL6 expression tended to be higher in the GCB- than the ABC-like DLBCLs. Overall, low KLHL6 expression was associated with poor survival (5-year OS; 93% vs 65%; p = .044). The difference in survival was restricted to the ABC subtype. The prognostic impact of KLHL6 expression in the patients with the ABC-like DLBCL was confirmed in independent LLMPP validation cohort. Conclusions The results show that although high KLHL6 expression is characteristic for the GCB DLBCL, mutations and low expression of KLHL6 are clinically more relevant in the ABC subtype pinpointing the patients with the most dismal prognosis. Disclosures Leppä: CTI Life Sciences: Honoraria; Roche: Honoraria, Other: Travel Expenses, Research Funding; Takeda: Honoraria, Other: Travel expenses; Bayer: Honoraria, Research Funding; Mundipharma: Research Funding; Janssen: Research Funding; Amgen: Research Funding.