Low Mutational Burden of Extra Nodal Marginal Zone Lymphoma of Mucosa-Associated Lymphoid Tissue in Patients with Primary Sjogren's Syndrome

Background: Primary Sjogren's syndrome (pSS) is an autoimmune disease characterized by chronic inflammation of the exocrine glands. Patients with pSS are at increased risk of developing extra nodal marginal zone lymphoma of the mucosa-associated lymphoid tissue (MALT) type, predominantly in the salivary glands. The presence of cryoglobulinemia, low C4 levels and salivary gland enlargement, are important risk factors for lymphoma development in pSS. Although lymphoma associated mutations have been described in circulating B-cells of patients with pSS associated cryoglobulinemia, it is unknown which somatic aberrations underlie the development of pSS-associated MALT lymphomas. The aim of the current study was to define the genomic landscape of pSS salivary gland MALT lymphomas. Identification of specific recurrent mutations or copy number aberrations and defining the affected pathways would help to understand the mechanisms by which intra-epithelial B-cells undergo malignant transformation.

Methods: Whole exome sequencing was performed on 14 fresh frozen and 3 paraffin embedded MALT tissue samples of pSS patients at the University Medical Center Groningen (UMCG). Matched peripheral white blood cell samples were available for 12 patients and were used as germline controls. Fluorescence in situ hybridization was performed for the detection of MALT1 translocations.

Results: Presence of a clonal B-cell population, indicative of a MALT lymphoma was confirmed by IgH PCR (BIOMED-2) for all patients. Whole exome sequencing resulted in a median target coverage of 130x, ranging from 84-163. More than 90% of the target regions had a coverage of >50x. In total we identified 232 somatic mutations in 184 genes. Three cases had a relatively high mutational load (>25 / case), while the median number of mutations in the remaining 14 cases was 7. Across the 17 cases there were 18 recurrently mutated genes. PRKD1, associated with malignant epithelial tumor of the salivary glands, was the most frequently mutated gene (six cases). Besides PRKD1, five additional recurrently mutated genes are also involved in epithelial surface and/or extracellular matrix (MAMDC4, COL14A1, CAMSAP3, TMEM2 and MUC4). Five genes, all with mutations in two cases, were shown to be associated with lymphomagenesis (ID3, TBL1XR1, PAX5, IGLL5, APC). A total of 18 copy number alterations (CNAs) were detected in 8 of the 14 evaluable cases, with gains of part of chromosomes 2 and 19, and loss of chromosome 6 each being detected in two cases. With respect to outcome, only 2 cases with high mutational load relapsed outside of the salivary glands, suggesting a more advanced stage of lymphoma.

Conclusion: The low mutational load and lack of a clear lymphoma related gene signature suggests that localized pSS MALT lymphomas are genetically stable and most likely depend on the inflammatory state of the micro-environment. Only those cases characterized by higher mutational load showed a relapse-remitting disease, as is typical for indolent lymphoma. These observations could be translated to the clinical setting and potentially have value as biomarker for identification of patients with a more aggressive disease. Confirmation in larger cohorts are required to confirm this potential association. This study also paves way for treatment strategies targeting the micro-environment.