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

• Main Authors: Attila Mokánszki, Réka Bicskó, Lajos Gergely, Gábor Méhes
• Format: Article
• Language: English
• Published: MDPI AG 2021-06-01
• Series: Cancers
• Subjects: aggressive lymphoma; liquid biopsy; cell-free nucleic acid; gene fusion; mutation analysis; next-generation sequencing (NGS)
• Online Access: https://www.mdpi.com/2072-6694/13/12/3032

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 <i>BCL2</i>, <i>BCL6</i>, and <i>MYC</i> genes, were detected in the corresponding tissue biopsy and cfTNA specimens with generally good agreement. Synchronous <i>BCL2</i> and <i>MYC</i> translocations in double-hit high-grade B-cell lymphomas were obvious from cfTNA. Besides, mutations of 29 commonly affected genes, such as <i>BCL2</i>, <i>MYD88</i>, <i>NOTCH2</i>, <i>EZH2</i>, and <i>CD79B</i>, 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.