Comprehensive genomic profiling of diffuse large B cell lymphoma using targeted NGS: Insights into MYC, BCL2, and BCL6 rearrangements and beyond Free

Introduction Diffuse large B-cell lymphoma (DLBCL) harbors recurrent gene rearrangements, particularly involving MYC, BCL2, and BCL6. While FISH is the current standard for detecting these events, next-generation sequencing (NGS) offers a comprehensive approach by identifying fusion partners and concurrently detecting other relevant genetic alterations, such as small variants (single nucleotide variants (SNVs) and small indels), and copy number variations (CNVs). Our aim was to evaluate the performance of targeted NGS for comprehensive genomic profiling and its potential role to improve patient stratification.

Material and methods Formalin-fixed paraffin-embedded (FFPE) tumor samples from 48 B-cell lymphomas (44 DLBCL, and 4 Burkitt lymphomas (BL)) were analyzed using FISH, immunohistochemistry (IHC), and NGS. Sequencing was performed using the custom ReLymph-NGS panel (Twist Bioscience), designed to detect gene rearrangements, CNVs, and relevant mutations. Libraries were prepared with KAPA HyperCapture kits and sequenced on an Illumina NovaSeq platform.

Sequencing reads were aligned to the human reference genome (hg38) using BWA-MEM, with low-quality reads filtered out. Small variants were called using MuTect2; CNVs were identified with a custom pipeline; and structural variants were detected with Manta, followed by stringent filtering. A customized filtering algorithm was developed based on concordance with FISH results, applying stricter thresholds in discordant cases to ensure high-confidence calls.

Results Among the 48 patients, 59 fusion genes were identified in 37 cases. Notably, six patients harbored two distinct fusion events involving the same oncogene rearranged with different IGH partners, illustrating the complexity of rearrangements and potential clonal diversity. A total of 26 MYC rearrangements were detected, of which 15 involved 13 distinct non-immunoglobulin (non-IG) partner genes, all novel except for the recurrent DMD partner, which was identified in three samples.

Notably, in double-hit (DH) DLBCL cases with MYC and either BCL2 or BCL6 rearrangements (14 events detected exclusively by NGS), 78% of MYC fusions involved non-IG partners, compared to 33% in single-hit MYC cases. This finding suggests that non-IG::MYC fusions are more frequent in NGS-detected DH cases and may reflect distinct biological mechanisms of MYC activation in these lymphomas.

For BCL2, 21 rearrangements were found in 16 samples, with two involving non-IG partners, and 11 BCL6 fusions were identified in distinct samples, with three involving non-IG partners.

MYC breakpoints clustered mainly in two regions: within MYC and upstream of PVT1 (54%), and downstream of PVT1 (27%). BCL2 breakpoints predominantly mapped from exon 3 onward (80%), and BCL6 breakpoints clustered in intron 1 (90%).

Additional resolution was provided by NGS, which clarified five cases with inconclusive FISH results by confirming true rearrangements, reclassified two ambiguous triple-hit (TH) cases as single-hit, and confirmed the absence of rearrangements in five FISH-inconclusive cases, consistent with IHC results. NGS also detected rearrangements missed by FISH in at least two samples.

NGS provided complementary information beyond gene fusions by detecting CNVs and small variants. One case showed 11q gain/loss alongside a MYC rearrangement and ID3/CCND3 mutations, consistent with Burkitt-like lymphoma with 11q aberration. MYC/PVT1 deletions and amplifications correlated with MYC rearrangements and protein overexpression. BCL2 amplifications were linked to overexpression, while deletions showed loss of expression. BCL6 alterations were frequently associated with rearrangements and positive IHC. Mutations in MYC, BCL2, and TP53 were common, with biallelic TP53 inactivation associated with adverse outcomes. ID3 and CCND3 mutations were enriched in BL and aided in differential diagnosis.

Conclusion Targeted NGS reliably identified gene rearrangements in B-cell lymphomas and provided key added data—such as fusion partners, breakpoint location, and co-mutations or CNVs—not captured by FISH or IHC. Detection of non-IG partners, clarification of FISH-inconclusive cases, and reclassification of equivocal cases highlight its diagnostic and biological value. These findings support incorporating NGS-based fusion analysis into routine diagnostics to improve stratification and guide precision therapy in aggressive B-cell lymphomas.