Genetic Characterization of Marginal Zone Lymphomas in Tissue and cfDNA

Introduction: Marginal zone lymphoma (MZL) is a group of non-Hodgkin lymphomas that originate from the marginal zone of lymphoid follicles. The WHO/ICC classifies MZL in primary splenic (SMZL), primary nodal (NMZL) and extranodal lymphoma of the mucosa-associated lymphoid tissue (EMZL). Their diagnosis remains difficult, as they do not have pathognomonic features. Thus, integration of all the diagnostic tools available including genetic characterization is crucial. Circulating cell-free DNA (cfDNA) analysis may be a complementary tool to perform non-invasive molecular genotyping, but the experience in MZL is limited.

Methods: 112 patients from a single institution identified between 2014-2023 (44 SMZL, 32 EMZL, 7 NMZL, 23 monoclonal non-CLL B-cell lymphocytosis (MBL-MZ) and 6 unclassified B-cell lymphoproliferative syndromes (LPS-NOS) with MZL clinico-biological features were included in the study. Tissue DNA (tDNA) was extracted from the diagnostic samples in 109 cases (61 from mononuclear cells from peripheral blood, 48 from formalin-fixed paraffin embedded tissue). cfDNA was extracted from paired plasma in 94/112 patients (39 SMZL, 25 EMZL, 6 NMZL, 20 MBL-MZ and 4 LPS-NOS). In 3 SMZL cases without available tDNA, cfDNA was studied. Libraries were prepared using a custom panel covering 31 MZL-associated genes (Qiagen Hilden, Germany) and sequenced with NextSeq (Illumina, San Diego CA). Only pathogenic and presumed pathogenic mutations where considered for the analysis.

Results: We found mutations in 83/109 (76%) and 84/97 (87%) of tDNA and cfDNA respectively (90% vs 98% SMZL, 63% vs 72% EMZL, 100% vs 100% NMZL, 70% vs 80% MZ-CBL and 50% vs 80% LPS-NOS). The most frequently mutated genes in SMZL were TP53 (26%), DNMT3A (26%), KLF2 (24%), TET2 (19%), CCND3 (17%) and SPEN (17%); in EMZL TNFAIP3 (19%), TET2 (19%), KMT2D (9%) and ARID1A (9%); in NMZL KMT2D (57%) and TNFAIP3 (43%); in MBL-MZ TP53 (17%), DNMT3A (17%), CCND3 (17%) and MYD88 (17%); in LPS-NOS MYD88 (40%).

The ability to find any tDNA mutation in cfDNA was 97% in SMZL, 29% in EMZL, 83% in NMZL and 80% in MBL-MZ. Furthermore, 100% of tDNA mutations were detected in the cfDNA analysis in up to 70% of SMZL. In 3 SMZL without available tDNA, cfDNA revealed mutations in NOTCH2 and DNMT3A; KLF2 and KMT2D; NOTCH2 and DNMT3A respectively. cfDNA revealed mutations not present in the tDNA in 46% of SMZL (20 mutations in 17 patients), 64% of EZML (45 mutations in 16 patients), 100% of NMZL (10 mutations in 6 patients) and 35% of MBL-MZ (17 mutations in 7 patients). In 12/19 (63%) cases without mutations in tDNA, cfDNA revealed the presence of MZL related gene mutations (42% of these cases had SPEN mutations in cfDNA), which were useful to achieve a final diagnosis.

Conclusions: Molecular profiling using cfDNA showed a high concordance with tissue analysis in MZL and could identify additional alterations in many cases. In MZL without available tDNA or without detectable mutations in tissue samples, cfDNA analysis revealed genetic variants in the majority of the cases. These data supports the utility of cfDNA in the diagnostic workup of MZL.

Acknowledgments. Instituto de Salud Carlos III (ISCIII), PI19/00034, co-funded by the European Union; 2021SGR628, PT20/00023, Xarxa de Banc de Tumors de Catalunya and Fundación Española de Hematología y Hemoterapia.