Diffuse large B-cell lymphoma genotyping on the liquid biopsy

Accessible and real-time genotyping for diagnostic, prognostic or treatment purposes is increasingly impelling in diffuse large B-cell lymphoma (DLBCL). Cell-free DNA (cfDNA) is shed into the blood by tumor cells undergoing apoptosis and can be used as source of tumor DNA for the identification of DLBCL mutations, clonal evolution, and genetic mechanisms of resistance. Here we aimed at tracking the basal DLBCL genetic profile and its modification upon treatment using plasma cfDNA. Ultra-deep targeted next generation sequencing of pre-treatment plasma cfDNA from DLBCL patients correctly discovered DLBCL-associated mutations that were represented in >20% of the alleles of the tumor biopsy with a >90% sensitivity and a ~100% specificity. Plasma cfDNA genotyping allowed also to recover mutations that were undetectable in the tissue biopsy conceivably because, due to spatial tumor heterogeneity, they were restricted to clones that were anatomically distant from the biopsy site. Longitudinal analysis of plasma samples collected under R-CHOP chemotherapy showed a rapid clearance of DLBCL mutations from cfDNA among responding patients. Conversely, among patients resistant to R-CHOP, basal DLBCL mutations did not disappear from cfDNA. In addition, among treatment-resistant patients, new mutations were acquired in cfDNA that marked resistant clones selected during the clonal evolution. These results demonstrate that cfDNA genotyping of DLBCL is as accurate as genotyping of the diagnostic biopsy to detect clonally represented somatic tumor mutations and is a real-time and non-invasive approach to track clonal evolution and emergence of treatment resistant clones.