Genetics and Prognostication in Splenic Marginal Zone Lymphoma: 2 Revelations from Deep Sequencing 3 4 Running Title: The clinical significance of gene mutations in SMZL 5

genomic and in splenic marginal zone (SMZL), a rare B cell non Genomic mutations in TP53 , KLF2 , NOTCH2 and TNFAIP3 were found collectively in over 40% of cases and 13% utilised IGHV genes with no somatic hypermutation (SHM). TNFAIP3 mutations were associated with an increased risk of high grade transformation. IGHV genes lacking SHM, KLF2 and NOTCH2 mutations were associated with shorter time to first treatment, while TP53 and MYD88 mutations predictors of short and long overall survival, respectively. In contrast to cytogenetic and FISH data, NOTCH2 and TP53 mutations remained independent factors of outcome in multivariate analyses which included the established prognostic markers: anaemia and thrombocytopenia. and immunogenetic data have the potential to diagnosis, Abstract Purpose: Mounting evidence supports the clinical significance of gene mutations and immunogenetic 50 features in common mature B-cell malignancies. 51 Experimental Design: We undertook a detailed characterization of the genetic background of splenic 52 marginal zone lymphoma (SMZL), using targeted re-sequencing and explored potential clinical 53 implications in a multinational cohort of 175 SMZL patients. 54 Results: We identified recurrent mutations in TP53 (16%), KLF2 (12%), NOTCH2 (10%), TNFAIP3 (7%), 55 MLL2 (11%), MYD88 (7%) and ARID1A (6%), all genes known to be targeted by somatic mutation in 56 SMZL. KLF2 mutations were early, clonal events, enriched in patients with del(7q) and IGHV1-2*04 B- 57 cell receptor immunoglobulins, and were associated with a short median time-to-first-treatment 58 (0.12 vs. 1.11 yrs; P=0.01). In multivariate analysis mutations in NOTCH2 (HR 2.12, 95%CI 1.02-4.4, 59 P=0.044) and 100% germline IGHV gene identity (HR 2.19, 95%CI 1.05-4.55, P=0.036) were 60 independent markers of short time-to-first-treatment, while TP53 mutations were an independent 61 marker of short overall survival (HR 2.36, 95% CI 1.08-5.2, P=0.03). 62 Conclusion: We identify key associations between gene mutations and clinical outcome, 63 demonstrating for the first time that NOTCH2 and TP53 gene mutations are independent markers of 64 reduced treatment-free and overall survival, respectively. the analysis were age at diagnosis, haemoglobin levels, 248 platelets and lymphocyte counts. We developed these models for TTFT, EFS and OS as they 249 permitted the relative prognostic value of gene mutations to be assessed in a large, informative 250 group of patients in the context of the most available clinical data ( Table 3 ). Our multivariate EFS 251 model identified age at diagnosis, lymphocyte count and low platelet count as independent risk 252 factors, however TP53 became non-significant in this analysis. We show that in addition to 253 haemoglobin levels, both NOTCH2 (HR 2.12, 95%CI 1.02-4.4, P=0.044) and 100% germline IGHV gene 254 identify (HR 2.19, 95%CI 1.05-4.55, P=0.036) are independent risk factors for TTFT. Furthermore, we 255 show that the presence of TP53 mutation is an independent risk factor for OS (HR 2.36, 95%CI 1.08- 5.20, P=0.03).