Webinar: Implementing NGS for the study of lymphoid malignancies

Webinar summary

In this webinar, Dr. Gerald Frigola, hematopathologist from Clínic Barcelona presented a research-focused evaluation of next-generation sequencing (NGS) for molecular characterization of lymphoid malignancies. The presentation examines the role of genomic profiling in lymphoma characterization, molecular stratification, and identification of mutational profiles, while highlighting the implementation and performance of the Ion AmpliSeq Liverpool Lymphoma Network Panel across a large research cohort.

Study overview

Lymphoid malignancies exhibit substantial biological and genomic heterogeneity. Rather than relying on single defining mutations, many lymphoma entities are characterized by mutational patterns involving multiple genes and pathways. Because of this, molecular testing is becoming increasingly important for accurate characterization.

To evaluate the impact of NGS on lymphoma characterization, Dr. Frigola’s research team implemented the Liverpool Lymphoma Network Panel, a community-developed targeted sequencing assay covering 60 genes and more than 1,300 amplicons relevant to both B-cell and T-cell lymphomas. The panel was applied to 401 research samples, including B-cell lymphomas, and T-cell lymphomas.

The study aimed to:

• Assess viability of implementing an NGS workflow in a research laboratory setting
• Evaluate the contribution of genomic profiling to lymphoma characterization
• Assess the utility of the Liverpool Lymphoma Network collaborative framework 

Key findings

Molecular profiling supports contemporary lymphoma characterization

• Most lymphoma entities are defined by characteristic combinations of mutations rather than single mutations
• Certain lymphoma entities require molecular or cytogenetic features for accurate characterization
• Mutational profiles contribute not only to disease characterization but also to risk stratification

NGS revealed entity-specific molecular patterns across lymphoma subtypes

• Distinct mutational profiles were observed across chronic lymphocytic leukemia, mantle cell lymphoma, lymphoplasmacytic lymphoma, follicular lymphoma, marginal zone lymphoma, hairy cell leukemia, and T-cell lymphomas

Genetic subclassification continues to refine disease characterization

• Recent studies demonstrate that diffuse large B-cell lymphoma (DLBCL) can be subdivided into genetically defined subgroups with distinct mutational profiles
• Similar molecular subclassification approaches have been described in marginal zone lymphoma, including site-specific mutational profiles based on tissue of origin

Liverpool Lymphoma Network Panel demonstrated strong technical performance

• The assay successfully analyzed DNA derived from blood, bone marrow, FFPE tissue, and fresh frozen tissue
• Low DNA input requirements enabled analysis of scanty samples
• Sequencing performance demonstrated high mapping rates, deep coverage, strong on-target performance, and high uniformity across samples
• The panel can be run on both the Genexus Integrated Sequencer and the GeneStudio S5 System
• When run on the Genexus, the fully automated workflow can deliver results in as little as 24 hours with minimal hands-on time

NGS results contributed meaningful information in the majority of research samples

• Among 368 samples analyzed, NGS generated relevant information for molecular characterization in approximately 70% of cases
• Molecular findings supported existing characterizations in approximately 40% of cases
• NGS facilitated refinement into more specific diagnostic categories in approximately 25% of cases
• Molecular findings prompted recharacterization in approximately 13% of cases
• Approximately 25% of analyzed cases contained alterations with potential implications for future disease management considerations, including TP53, BTK, EZH2, and CXCR4 alterations

Variant interpretation remains a major analytical challenge

• Large numbers of variants of unknown significance (VUS) were identified across samples
• Accurate interpretation requires integration of genomic databases, functional predictions, protein domain analysis, literature review, and mutational context
• Recognition and exclusion of technical artifacts remained an important component of the analytical workflow

Opportunities for future panel development were identified

• Some specific lymphoma cases lacked detectable mutations or yielded non-informative mutational profiles
• The findings support ongoing refinement of panel content, including addition of new targets and structural variation assessment
• Future areas of investigation include copy number analysis, lymphoid clonal hematopoiesis, tumor heterogeneity, and liquid biopsy applications

This study demonstrates that targeted NGS-based molecular profiling provides value for characterization of lymphoid malignancies by identifying disease-associated mutational profiles and supporting accurate molecular characterization.

The Liverpool Lymphoma Network Panel generated informative molecular data in most analyzed samples and contributed to refinement or recharacterization of a significant subset of research samples. The automated workflow of the Ion Torrent Genexus Integrated Sequencer made it possible to obtain sequencing data with speed and simplicity, in as little as 24 hours.

The findings further highlight the importance of integrated interpretation strategies, collaborative panel development, and continued expansion of molecular profiling approaches to address the biological complexity and heterogeneity of lymphoid malignancies.