Beyond Binary FISH Results: Targeted NGS Provides Deeper Characterization of MYC and Broader Genomic Alterations in...

Rearrangements involving MYC, BCL2, and BCL6 are central to the genetic classification and risk stratification of diffuse large B cell lymphoma (DLBCL). In routine practice, these alterations are primarily assessed by fluorescence in situ hybridization (FISH), which provides limited diagnostic information and lacks information on partner genes, breakpoint architecture, or clonal complexity.

We investigated whether targeted next-generation sequencing (NGS) refines the interpretation of gene rearrangements in aggressive B cell lymphomas and alters their diagnostic classification. Tumor samples from 48 patients were analyzed using FISH, immunohistochemistry, and a custom hybrid-capture NGS panel enabling simultaneous detection of structural variants, copy number alterations, and somatic mutations.

Targeted NGS identified 59 fusion events in 37 patients, including 26 MYC rearrangements (MYC-Rs). A substantial proportion (58%) involved non-immunoglobulin partners, many of them previously unreported. NGS resolved inconclusive or discordant FISH results, uncovered cryptic rearrangements, and revealed genomic complexity. In several cases, these findings refined genetic classification, including the identification of additional double- and triple-hit lymphomas.

These results demonstrate that a FISH-based assessment of MYC-, BCL2-, and BCL6-Rs is insufficient to capture the biological and diagnostic complexity of DLBCL. Targeted NGS provides structural and functional context that improves interpretation of rearrangements and supports a more refined genetic classification in routine practice.