E. E. E. Drees1, M. A. J. van Eijndhoven1, I. Ciocanea-Teodorescu2, N. J. Groenewegen3, L. I. Prins1, X. Tran1, A. Valles1, D. Koppers-Lalic3,4, E. Aparicio5, M. Hackenberg5, D. de Jong1, J. M. Zijlstra6, D. M. Pegtel1,3
1Department of Pathology, Cancer Center Amsterdam, Amsterdam University Medical Center, location VUmc, Amsterdam, The Netherlands,2Department of Epidemiology and Biostatistics, University Medical Center, location VUmc, Amsterdam, The Netherlands,3ExBiome B.V., Amsterdam, The Netherlands,4Department of Neurosurgery, Amsterdam University Medical Center, location VUmc, Amsterdam, The Netherlands,5University of Granada, Spain,6Department of Hematology, Cancer Center Amsterdam, Amsterdam University Medical Center, location VUmc, Amsterdam, The Netherlands
Background: Previously, we have shown that classical Hodgkin lymphoma (cHL) patients with active disease sustain increased levels of defined cell-free extracellular vesicle (EV)- circulating miRNAs compared to healthy individuals. We postulate that elevated levels of lymphoma-associated EV-bound miRNAs may reflect metabolically active disease, predict relapse and is potentially applicable for detection of minimal residual disease in cHL patients. To test this hypothesis, we have optimized and validated an EV-bound miRNA detection assay in a longitudinally collected set of samples of cHL-patients undergoing treatment.
Patients and methods: A total of 222 plasma samples were collected pre, during and post-treatment from 30 cHL patients, of which 19 patients were included at first presentation and 11 at relapse and/or refractory (R/R) disease. Treatment of these patients was heterogeneous, including ABVD and/or BEACOPPesc regimes in first line and DHAP (+/- brentuximab vedotin) followed by BEAM and autologous SCT in R/R patients. Three of 30 patients relapsed during EV-miRNA detection. Treatment outcome was monitored with FDG-PET at baseline, interim and end-of-treatment.
EV-enriched fractions were isolated from EDTA plasma by size-exclusion chromatography and spike-in of synthetic 100 nm liposomes containing exogenous miRNA was used to control for EV recovery. EV-bound miRNA-levels were quantified with Taqman qRT-PCR. To optimize the marker selection of the EV-bound miRNA panel we performed comprehensive small RNA sequencing in pre-, during and post-treatment of 8 patients.
Results: Sequencing data revealed that a subset of EV-bound miRNAs are consistently altered between PET-positive cHL patients versus post-treatment PET-negative patients and this overlapped to a large extent with previously identified miRNAs. Longitudinal monitoring of EV-bound miRNA levels by qRT-PCR in patients pre-, during and post-treatment and during long-term follow-up revealed robust, stable association with treatment results, both for patients with PET-positive and PET-negative status after treatment.
Conclusion: Changes in cHL-related circulating EV-bound miRNA levels have high potential as a sensitive tool for therapy-response and relapse monitoring in cHL patients. These findings warrant prospective validation in a larger cohort to determine clinical utility.