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

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.

Different situations of identifying second primary malignant tumors in lymphoma patients with synchronous solid tumors

“…Molecular profiling is also a useful diagnostic tool, particularly when differential diagnosis between SPMTs and metastasis cannot be confirmed by histomorphology. [30][31][32] We found that nine patients had a family cancer history and three showed a third distinct malignant tumor in this cohort. Wang et al recently reported germline variants of DNA repair genes and of genes involved in B cell functions to be potential causes of MPMTs.…”

“…Multidetector CT, 26 PET/CT, 27 PET/MRI, 28 and endoscopy 29 have been reported to be highly efficient at detecting synchronous tumors. Molecular profiling is also a useful diagnostic tool, particularly when differential diagnosis between SPMTs and metastasis cannot be confirmed by histomorphology 30–32 . We found that nine patients had a family cancer history and three showed a third distinct malignant tumor in this cohort.…”

Circulating Cell-Free DNA-Based Comprehensive Molecular Analysis of Biliary Tract Cancers Using Next-Generation Sequencing

“…The use of peripheral blood LB has been introduced in the early molecular diagnosis of several malignancies, such as lung and colorectal adenocarcinomas, and melanomas, as well [ 19 , 22 , 23 , 24 ]. More recently, LB application to detect aberrant gene fusions has been published for the diagnosis of aggressive lymphoma genotyping [ 25 ]. Little information was found about the utility of LB to identify diagnostic and prognostic biomarkers in BTCs [ 26 ], so for this reason we aimed to quantify and analyze cfDNA management of patients affected by BTC.…”

CD56-positive diffuse large B-cell lymphoma: comprehensive analysis of clinical, pathological, and molecular characteristics with literature review

“…Fusions were considered true positive if the fusion event was covered with a minimum of 5 unique reads and the percentage of reads supporting the event was above 10%. 21 , 22 …”