Diagnostic Implications of NGS-Based Molecular Profiling in Mature B-Cell Lymphomas with Potential Bone Marrow Involvement

<b>Background:</b> Methods such as cytogenetics and immunocytology/immunohistology provide essential diagnostic insights but may be limited in ambiguous cases of mature B-cell lymphoma. Next-generation sequencing (NGS) has emerged as a potential tool to improve diagnostics. <b>Methods:</b> We validated the analytical performance of a lymphoid customized NGS panel. Clinical validation was conducted in 226 patients with suspected mature B-cell lymphoma with potential bone marrow involvement across multiple clinically relevant scenarios. <b>Results:</b> NGS (1) achieved 100% sensitivity and specificity with high reproducibility (r = 0.995), confirming its analytical performance. (2) It reliably detected WHO-classified markers, including <i>BRAF</i> mutations in all hairy cell leukemia cases, <i>MYD88/CXCR4</i> mutations in lymphoplasmacytic lymphoma, and absence of <i>BRAF</i> mutations in splenic B-cell lymphoma with prominent nucleoli. (3) In lymphoma exclusion diagnostics, NGS identified mutations in previously undiagnosed cases, including a <i>BCORL1</i> mutation leading to reclassification as marginal zone lymphoma. (4) Among 105 confirmed lymphomas, 65% harbored mutations, with detection rates highest in HCL and LPL (100%) and CLL (62%), while follicular lymphoma showed no detectable mutations. (5) In cases with non-interpretable cytogenetics, NGS detected pathogenic variants in 61% of patients, compensating for inconclusive findings. (6) In cases with limited morphological assessment, NGS identified relevant mutations in 70%, outperforming cytogenetics (30%; <i>p</i> = 0.0256, OR = 5.44). <b>Conclusions:</b> NGS enhances the diagnostic accuracy of mature B-cell lymphomas by complementing traditional methods, refining WHO-classified subtypes, and improving detection in cases with inconclusive cytogenetics or morphology. NGS may reduce the need for unnecessary bone marrow re-punctures by providing additional information in ambiguous cases.