IntroductionAnalyzing liquid biopsies for tumor-specific aberrations can facilitate detection of measurable residual disease (MRD) during treatment and at follow-up. In this study, we assessed the clinical potential of using whole-genome sequencing (WGS) of lymphomas at diagnosis to identify patient-specific structural (SVs) and single nucleotide variants (SNVs) to enable longitudinal, multi-targeted droplet digital PCR analysis (ddPCR) of cell-free DNA (cfDNA).MethodsIn 9 patients with B-cell lymphoma (diffuse large B-cell lymphoma and follicular lymphoma), comprehensive genomic profiling at diagnosis was performed by 30X WGS of paired tumor and normal specimens. Patient-specific multiplex ddPCR (m-ddPCR) assays were designed for simultaneous detection of multiple SNVs, indels and/or SVs, with a detection sensitivity of 0.0025% for SV assays and 0.02% for SNVs/indel assays. M-ddPCR was applied to analyze cfDNA isolated from serially collected plasma at clinically critical timepoints during primary and/or relapse treatment and at follow-up.ResultsA total of 164 SNVs/indels were identified by WGS including 30 variants known to be functionally relevant in lymphoma pathogenesis. The most frequently mutated genes includedKMT2D,PIM1,SOCS1andBCL2. WGS analysis further identified recurrent SVs including t(14;18)(q32;q21) (IGH::BCL2), and t(6;14)(p25;q32) (IGH::IRF4). Plasma analysis at diagnosis showed positive circulating tumor DNA (ctDNA) levels in 88% of patients and the ctDNA burden correlated with baseline clinical parameters (LDH and sedimentation rate, p-value <0.01). While clearance of ctDNA levels after primary treatment cycle 1 was observed in 3/6 patients, all patients analyzed at final evaluation of primary treatment showed negative ctDNA, hence correlating with PET-CT imaging. One patient with positive ctDNA at interim also displayed detectable ctDNA (average variant allele frequency (VAF) 6.9%) in the follow-up plasma sample collected 2 years after final evaluation of primary treatment and 25 weeks before clinical manifestation of relapse.ConclusionIn summary, we demonstrate that multi-targeted cfDNA analysis, using a combination of SNVs/indels and SVs candidates identified by WGS analysis, provides a sensitive tool for MRD monitoring and can detect lymphoma relapse earlier than clinical manifestation.