Context: Recent studies interrogating the genomic background of follicular lymphoma
(FL) have revealed that all patients carry at least one pathogenic alteration in the
components of the epigenetic regulatory system. EZH2 mutations, the third most common
epigenetic lesions in this disease, are present in around 25% of cases. It is well
established that tissue biopsy (TB)-based molecular testing may not capture the full
genetic background of a spatially heterogeneous tumor, such as FL. Liquid biopsy (LB),
the minimally invasive testing of tumor-derived, cell-free DNA in blood plasma, may
overcome this limitation. The gain-of-function nature of EZH2 mutations triggered
successful pharmaceutical small molecular inhibitor development; however, superior
effectivity was found in patients harboring EZH2 mutations. Therefore, molecular tests
detecting these alterations are of great clinical relevance. Objective: In this study,
we aimed to develop a novel LB-based method to detect EZH2 mutations in plasma samples
of patients with FL. Patients and Methods: Pre-treatment LB and TB samples were collected
from 112 patients. The EZH2 mutation status was assessed using an in-house-designed
multiplex digital droplet PCR approach. Results: Altogether, EZH2 mutation frequency
was found to be 38.4% (43/112), whereas TB-based analysis resulted in only 31.3% (35/112)
mutation positivity. LB analysis recovered an additional eight (7%) patients harboring
EZH2 mutations; however, in five patients (4.5%), the EZH2 mutation was exclusively
detected in the TB specimen. Comparing LB to TB, the specificity, sensitivity, and
negative predictive value of our method were 89.6%, 88.3%, and 93.2%, respectively.
Pre-treatment LB EZH2 variant allele frequency (VAF) significantly correlated with
EZH2 VAF measured in the corresponding TB samples and with FLIPI scores. Although
not significant, a trend could be observed between LB EZH2 VAF and histological grade
and clinical stage. Conclusions: Here, we have developed a novel, minimally invasive
EZH2 mutation detection test. Due to the ability of this approach to resolve spatial
heterogeneity, our method detected a higher frequency of EZH2 mutations in FL patients
compared to historical data, which further expands the subset of FL patients who would
most likely benefit from the recently developed EZH2 inhibitor therapy.
