Poster: IBCL-419: Non-Invasive Quantitative Analysis of EZH2 Mutations in Follicular Lymphoma

Context Follicular lymphoma (FL) is the most common type of indolent non-Hodgkin lymphoma, with an average overall survival rate of 15 years. The prolonged disease course raises the need for an effective disease monitoring system, which is hampered by the limited specificity and sensitivity of the most widely used, imaging-based PET-CT method. Molecular analysis of tumor-derived circulating DNA (ctDNA), frequently referred to as liquid biopsy, is a promising minimally invasive disease monitoring tool for patients suffering from malignancies. Recent studies interrogating the genomic background of FL have revealed activating mutations in the epigenetic regulator EZH2 gene in 25% of patients. Objective In our study, we tested the potential of liquid biopsy to detect and monitor EZH2 mutations in plasma samples of patients with EZH2-mutant FL treated with immunochemotherapy. Patients and Methods Eighty-five blood plasma samples were collected from 41 EZH2 mutant patients. Cell-free plasma DNA was extracted using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany). The EZH2 mutation status was assessed using a multiplex digital droplet PCR (ddPCR, Bio-Rad Laboratories, USA) approach, detecting seven different EZH2 hotspot mutations using only two PCR reactions. The molecular results were correlated with PET-CT images. Results Among the 41 patients, 36 responded to treatment (88%); meanwhile, 5 patients did not (12%). We detected an EZH2 mutation in 89% of the samples collected prior to treatment (17/19). Similarly, 86% of the samples (6/7) collected during therapy from patients not responding to treatment proved to be EZH2 mutated; meanwhile, only 22% of the samples were positive from responding patients (6/33). As expected, all samples collected during remission were EZH2 wild-type. EZH2 variant allele frequencies (VAF) correlated with the total metabolic tumor volume detected on the PET-CT scans. We also demonstrated spatial heterogeneity of EZH2 mutations in two cases, where different EZH2 mutations deriving from distinct anatomical sites could simultaneously be detected in the plasma. The median sensitivity of our ddPCR approach was 0.15%, with EZH2 VAFs ranging between 0.2 and 74%. Conclusions Our results demonstrate that liquid biopsy offers a sensitive, radiation-free, minimally invasive monitoring method for EZH2-mutant FL patients.