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 included KMT2D, PIM1, SOCS1 and BCL2. 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.
Background: Sarcomas are rare tumours with heterogeneous clinical behaviour including varying rates of metastasis. Clinical treatment and follow-up rely on crude grading systems with uncertain accuracy for individual patients. Whole genome sequencing (WGS) detects both structural variation and single nucleotide variants and may thus add important diagnostic/prognostic information. Liquid biopsies (LB) may potentially identify hematogenous spread and treatment response rate but further evaluation in sarcomas is needed. Methods: In this study we explore the performance of individualized LB in four patients with different types of sarcomas. Fresh frozen tumour tissue was sequenced using WGS and whole transcriptome sequencing. Three putative driver variants or one fusion gene were selected per case and custom digital droplet PCR (ddPCR) assays were designed, evaluated on tumour DNA and used to assess levels of cell free tumour DNA in plasma taken prior to surgery. Results: In LB, ddPCR identified three variants in one patient with metastatic disease. The remaining three patients had negative LBs and were without disease at follow-up (>18 months after surgery). Conclusions: WGS is a powerful tool to detect all types of genetic changes in sarcoma and can facilitate clinical diagnosis/classification while custom LB may add prognostic information.
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