Cell-Free Total Nucleic Acid-Based Genotyping of Aggressive Lymphoma: Comprehensive Analysis of Gene Fusions and Nucleotide Variants by Next-Generation Sequencing

SIMPLE SUMMARY: This study aimed to simultaneously demonstrate pathogenic chromosomal translocations and point mutations from both tissue biopsy and peripheral blood (PB) liquid biopsy (LB) samples of aggressive lymphoma patients. Matched samples were analyzed by next-generation sequencing for the same 125 genes. Eight different gene fusions, including the classical BCL2, BCL6, and MYC genes were detected in the corresponding samples with generally good agreement. Besides, mutations of 29 commonly affected genes, such as BCL2, MYD88, NOTCH2, EZH2, and CD79B could be identified in the matched samples at a rate of 16/24 (66.7%). Our prospective study demonstrates a non-invasive approach to identify frequent gene fusions and variants in aggressive lymphomas. In conclusion, PB LB sampling substantially supports the oncogenetic diagnostics of lymphomas, especially at anatomically critical sites (such as the central nervous system). ABSTRACT: Chromosomal translocations and pathogenic nucleotide variants both gained special clinical importance in lymphoma diagnostics. Non-invasive genotyping from peripheral blood (PB) circulating free nucleic acid has been effectively used to demonstrate cancer-related nucleotide variants, while gene fusions were not covered in the past. Our prospective study aimed to isolate and quantify PB cell-free total nucleic acid (cfTNA) from patients diagnosed with aggressive lymphoma and to compare with tumor-derived RNA (tdRNA) from the tissue sample of the same patients for both gene fusion and nucleotide variant testing. Matched samples from 24 patients were analyzed by next-generation sequencing following anchored multiplexed polymerase chain reaction (AMP) for 125 gene regions. Eight different gene fusions, including the classical BCL2, BCL6, and MYC genes, were detected in the corresponding tissue biopsy and cfTNA specimens with generally good agreement. Synchronous BCL2 and MYC translocations in double-hit high-grade B-cell lymphomas were obvious from cfTNA. Besides, mutations of 29 commonly affected genes, such as BCL2, MYD88, NOTCH2, EZH2, and CD79B, could be identified in matched cfTNA, and previously described pathogenic variants were detected in 16/24 cases (66.7%). In 3/24 cases (12.5%), only the PB sample was informative. Our prospective study demonstrates a non-invasive approach to identify frequent gene fusions and variants in aggressive lymphomas. cfTNA was found to be a high-value representative reflecting the complexity of the lymphoma aberration landscape.