Somatic mutations of cell-free circulating DNA detected by next-generation sequencing reflect the genetic changes in both germinal center B-cell-like and activated B-cell-like diffuse large B-cell lymphomas at the time of diagnosis | Haematologica

摘要

Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma, accounting for 30–40% of newly diagnosed cases of non-Hodgkin lymphomas. The molecular heterogeneity of DLBCL has been deciphered by gene expression profiling, and DLBCL have been divided into three main molecular subtypes: the germinal center B-cell-like (GCB) subtype, the activated B-cell-like (ABC) subtype, and the primary mediastinal B-cell lymphoma subtype with distinct clinical outcomes and responses to immunochemotherapy. Next-generation sequencing (NGS) technologies, which allow for massive, parallel, high-throughput DNA sequencing, have emerged over the past decade and have provided new insights into the genomic characterization of DLBCL. Recurrent single nucleotide variants (SNV) are now well defined and provide new therapeutic opportunities for the three molecular subtypes. The SNV target genes play a crucial role in several pathways, including B-cell receptor signaling (CD79A/CD79B), NFκ-B (CARD11), Toll-like receptor signaling (MYD88), immunity (CD58, TNFSRF14, B2M), cell cycle/apoptosis (TP53, BCL2) and epigenetic regulation (EZH2,CREBBP, MLL2).1,2Recently, whole exome sequencing in breast cancer has shown that mutations observed in the tumor could also be detected in circulating, cell-free DNA (cfDNA) and could be used to detect genetic changes during treatments and relapse, defining the concept of “liquid biopsy”.3 In DLBCL, whereas tumor circulating cells or leukemic phase are not usually detectable, clonotypic sequences have been constantly detected in cfDNA extracted from serum/plasma or peripheral blood mononuclear cells.4–8In this study we sought to determine, by routinely applicable NGS technology, whether the pattern of acquired SNV observed in tumor DNA could also be detected in cfDNA in DLBCL patients at the time of diagnosis. For this purpose, we analyzed 12 DLBCL cases with available matched tumor DNA and plasma collected at the time of diagnosis. Patients harboring typical GCB/ABC-related mutations targeting CD79A/B, EZH2, CARD11 or MYD88 genes, previously identified by the Sanger method, were selected.9 This study was approved by the regional ethical committee (numbered as CPP N°01/006/2014).The main clinical features of the patients are summarized in Table 1. None of the selected cases harbored detectable circulating lymphoma cells by routine blood smear examination. Of note, no peripheral blood cytometry was performed, in accordance with our center’s initial staging procedures for DLBCL patients. Tumor DNA was extracted from frozen lymph node samples by standard methods. cfDNA was extracted from archived EDTA-anticoagulated plasma aliquots (1 mL) stored at ?80°C using the QIAamp? Circulating Nucleic Acid Kit (Qiagen) (with the QIAvac 24 Plus vacuum manifold, following the manufacturer’s instructions), and concentrations were measured using a fluorometric assay (Qubit? dsDNA HS Assay Kit, Life Technologies). The mean cfDNA concentration in plasma was 1.65 ng/μL (range, 0.46–11.2 ng/μL) (Table 1). The cell-of-origin signature was determined by cDNA-mediated annealing, selection, extension, and ligation technology based on the expression of 19 genes, as previously reported.10 Among the 12 cases analyzed, five belonged to the ABC subgroup, six to the GCB subgroup and one case was unclassified (Table 1).View this table:View inlineView popupDownload powerpointTable 1. Clinical characteristics and list of somatic variants (insertion/deletion/single nucleotide variant) detected by sequencing in tumor DNA and cell-free, plasma circulating DNA. Details of the locations of the mutations are indicated in Online Supplementary Table S1.Tumor DNA was sequenced using an Ion Torrent Personal Genome Machine (Life Technologies). Ten nanograms of genomic DNA were submitted to NGS using a laboratory-developed Lymphopanel set, designed to identify mutations in 34 genes relevant to lymphomagenesis (Online Supplementary File S1A). This design co