Improved Genetic Characterization of Aggressive B-Cell Lymphoma at Diagnosis Using Next-Generation Sequencing

• Published in: Blood 2024-11, Vol.144 (Supplement 1), p.6229-6229
• Main Authors: Bastos-Oreiro, Mariana, De La Iglesia, Ismael, Chicano, Maria, Gomez Llobell, Marina, Diaz Crespo, Francisco, Fernández Caldas-González, Paula, Triviño, Juan Carlos, Fernandez, Raquel, Garcia-Sanz, Ramon, Buño, Ismael, Martinez-Laperche, Carolina
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2024-208216

Introduction Large B cell lymphoma (LBCL) is the most common aggressive lymphoma, including varieties such as diffuse large (DLBCL) and high-grade B cell lymphoma (HGBCL). Genomic rearrangements in the MYC locus can occur in all these histologies and are associated with inferior outcomes. Nowadays, in-situ hybridization (FISH) is universally used for genetically characterizing these patients. However, this strategy cannot identify the breakpoint location and partner, both dates that are important for prognosis. Likewise, other mutations are also necessary to properly classify LBCL. Next generation sequencing (NGS) increasingly seeks to find its place in the study of these patients. For this, we have designed a customized NGS panel (DX-NGS-LYMPHOMA), which includes the evaluation of fusion genes, point variants, and CNVs, with the intention of including it in clinical practice. Materials and methods We started with a group of 60 patients with DLBCL and HGBCL who had MYC, BCL6, or BCL2 rearranged by FISH. FISH was performed using break-apart probes (Vysis, Abbot Molecular). DNA was extracted from a paraffinized tissue sample using the Generead kit (Qiagen, USA). The DX-NGS-LYMPHOMA capture panel (Twist, USA) includes the following regions (Rearrangements: MYC, BCL2, BCL6, IGH, IGK, IGL, IRF4/DUSP22. Specific variants: EZH2, TNFRS14, NOTCH2, MYD88, NOTCH1, TP53, TCF3, ID3 and CCND3 CNVs: 11q, PDL1, REL). Library preparation was performed using the Kappa Hyperplus kit (Roche) and sequencing on a NextSeq or NovaSeq sequencer (Illumina). To confirm the results, we conducted a commercial panel EuroClonality-NDC assay (Univ8) Regarding analysis, reads were aligned to the human reference genome version GRCh38/hg38 using BWA and custom scripts. Low-quality reads (Q20) and PCR duplicates were removed using Picard tools. Variants and small indels were identified using the GATK algorithm. To identify structural variants, a combination of Manta software and custom Python scripts were used to identify breakpoints using soft clipping reads. Results: Point variants were identified in 100% of the patients; the most frequently altered genes were MYC (60%), TP53 (29%), EZH2 (27%), and BCL2 (15%). NGS identified 85% of cases involving translocations, and we found the partner in all rearranged cases. Thirty-six cases had rearranged MYC by FISH; NGS identified 28, but 6 were not identified by any of the two NGS panels (all in complete remission). Twelve breakpoints were in MYC l