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Liquid biopsy (cfDNA): an accurate, real-time, non-invasive method of clonal somatic mutation detection in DLBCL

Feb 7, 2017

In the journal Bloodin January 2017, Davide Rossifrom the University of Eastern Piedmont, Novara, Italy, and colleagues published a prospective studyon the use of liquid biopsy or Cell Free DNA (cfDNA) to genotype untreated DLBCL patients. The aim of this study was to evaluate the use of liquid biopsy compared with tissue biopsy in DLBCL and track the genetic profile of the disease over time before, during, and immediately after R-CHOP treatment.

Key Highlights:

  • Training cohort = 30 pts, validation cohort = 20 pts
  • cfDNA isolated at diagnosis, day 1 of each cycle prior to treatment, end of treatment, and at progression
  • A specifically designed target resequencing gene panel, covering 59 genes commonly mutated in DLBCL, was used in CAPP-seq, a targeted next-generation sequencing approach
  • Diagnosis cfDNA genotyping identified 108 somatic mutations compared with 105 identified by tumor genomic DNA genotyping
  • cfDNA genotyping discovered mutations with >20% of alleles of tumor biopsy with a sensitivity of 97.1% (95% CI, 89.5–99.8%)
  • cfDNA uninformative in identifying mutational status of biopsy in 10% of DLBCL cases
  • cfDNA identified 21 somatic mutations undetected by tissue biopsy
  • cfDNA calculated to have >99.99% specificity compared with tumor genomic DNA genotyping based on false-positive rate
  • Longitudinal monitoring of pts genotype:
    • Responding patients had clearance of mutations in their cfDNA samples
    • Resistant pts did not have this clearance occur in their cfDNA samples
    • One R-CHOP responsive patient had detectable mutations in their cfDNA a month before relapsing
    • R-CHOP refractory or relapsed pts had new mutations appear in their cfDNA during treatment

In conclusion, the authors stated that previous multicenter, retrospective studies had issues with the acquisition and handling of patient samples, which may have affected their analysis. This was avoided by this prospective, single-centered study which used standardized procedures. The study concluded that cfDNA genotyping is non-invasive and would allow for accurate and real-time detection of somatic tumor mutations. Therefore, the authors propose the inclusion of this type of assay into other clinical trials to aid in the development of future prognostic biomarkers and to track early stages of treatment resistance.


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.

  1. Rossi D. et al.Diffuse large B-cell lymphoma genotyping on the liquid biopsy. Blood. 2017 Jan 17. DOI:[Epub ahead of print 2017 Jan 17].