Quantitative Analysis and Monitoring of EZH2 Mutations Using Liquid Biopsy in Follicular Lymphoma

Abstract

Recent advances in molecular technologies enable sensitive and quantitative assessment of circulating tumor DNA, offering a noninvasive disease monitoring tool for patients with malignant disorders. Here, we demonstrated on four follicular lymphoma cases that circulating tumor DNA based EZH2 mutation analysis performed by a highly sensitive droplet digital PCR method may be a valuable treatment monitoring approach in EZH2 mutant follicular lymphoma. EZH2 variant allele frequencies changed in parallel with the volume of metabolically active tumor sites observed on 18F-fluorodeoxyglucose positron emission tomography combined with computer tomography (PET-CT) scans. Variant allele frequencies of EZH2 mutations decreased or were eliminated rapidly upon successful treatment, with treatment failure being associated with elevated EZH2 variant allele frequencies. We also demonstrated spatial heterogeneity in a patient with two different EZH2 mutations in distinct anatomical sites, with both mutations simultaneously detected in the liquid biopsy specimen. In summary, circulating tumor DNA based EZH2 mutation analysis offers a rapid, real-time, radiation-free monitoring tool for sensitive detection of EZH2 mutations deriving from different anatomical sites in follicular lymphoma patients receiving immunochemotherapy.

Circulating Tumor DNA in Lymphoma: Principles and Future Directions

“…43), chronic lymphocytic leukemia (44), as well as genetic subtypes of DLBCL (45-47) have been identified. Indeed, targetable genetic alterations such as EZH2 mutations in FL (48) are emerging that can be detected by ctDNA (49). By assessing a broader panel of genes and loci, targeted sequencing with error suppression can directly assess these alterations from a liquid biopsy rather than requiring tumor tissue.…”

ct-DNA is useful to diagnose mutations at codon 641 of exon 16 of EZH2, which is a biomarker for relapse in patients with Diffuse Large B-cell Lymphoma treated with the R-CHOP scheme.

“…Different authors have evaluated the clinical impact of EZH2 mutations in DLBCL in tumor samples [11,16,29,[40][41]. However, recently, only Nagy et al [42] has used liquid biopsy to evaluate the clinical role of EZH2 mutations by ddPCR, but in patients with follicular lymphoma, and correlated the variant allele frequency with the volume of metabolically active tumor sites observed on 18F-uorodeoxyglucose positron emission tomography combined with computer tomography (PET-CT) scans. To our knowledge, this is the rst study analyzing the EZH2 mutation using ctDNA to evaluate the frequency and the negative impact on PFS in Diffuse large B cell lymphoma.…”

Role of Circulating Tumor DNA in Hematological Malignancy

“…Regarding the methods for detecting SNV/Indel, Hosny et al, in 2009, detected the TP53 mutation in ctDNA of NHL patients using direct sequencing, but this method was limited to the target genes [ 107 ]. With the advent of CAPP-seq (cancer personalized profiling by deep sequencing), panel sequence, and low-pass WGS, the target gene region has been greatly expanded, and continuous monitoring by ddPCR as in myeloid malignancy has been reported [ 108 , 109 , 110 , 111 , 112 ]. CAPP-seq is an economical and ultrasensitive hybrid capture-based target sequence method developed by Newman et al in 2014 [ 62 ].…”