A Personalized Whole Genome-Informed Assay Targeting Single Mutant in Circulating Tumor DNA Can Identify MRD and Predict Relapse in DLBCL

Introduction: Minimal residual disease (MRD) detected by circulating tumor DNA (ctDNA) has emerged as a promising biomarker in diffuse large B-cell lymphoma (DLBCL). MAESTRO (minor-allele-enriched sequencing through recognition oligonucleotides) was recently developed to aid the detection of low-frequency mutations by enriching for mutant alleles using probes preferentially capturing single-nucleotide variants. We have extended this application - termed MAESTRO-Pool - to analyze personalized MRD variant detection within a cohort-level single assay. We demonstrate high sensitivity to detect MRD using MAESTRO-Pool and the detection of emergent mutations using targeted sequencing of the same samples. Methods: Fifty-nine plasma specimens from 9 patients with relapsed/refractory (R/R) DLBCL treated on a phase II trial (NCT02362997) of post-autologous stem cell transplant (ASCT) pembrolizumab maintenance were tested. Cases were selected based on the availability of genomic DNA from tumor tissue, patient-matched germline DNA, and serial post-ASCT plasma samples (≥ 3 time points). MAESTRO probes were designed to target patient tumor-specific somatic variants using results of baseline tumor-normal whole genome sequencing. Probes were then pooled into an integrated, cohort-level assay (MAESTRO-Pool). Serial samples were compared with an orthogonal, whole-genome, tumor-informed MRD test which does not use mutation enrichment (MRD Tracker; Parsons, HA et al. Clin Cancer Res26, 2556-2564 (2020)) for sensitivity and specificity of variant detection. In addition, sensitivity to detect MRD and predict relapse was compared to that observed with immunoglobulin locus high-throughput sequencing (IgHTS). Additional baits were designed to capture single nucleotide variants (SNVs) previously reported in R/R DLBCL (63 loci in 12 genes), enabling detection of treatment-emergent mutations not identified in baseline tumor specimens. Results: Tumor-normal WGS revealed a median of 433 somatic SNVs per tumor (range 81-1653). The pooled assay comprised 6044 SNV-specific probes. MRD identification was similar for MAESTRO-Pool and MRD Tracker. Among 59 samples, a discrepant MRD call was observed for a single sample where ctDNA was detected at 4 ppm using MRD Tracker, but not detected using MAESTRO-Pool (limit of detection [LoD] 16 ppm). Estimated tumor fractions using MRD Tracker and MAESTRO-Pool were concordant. Even with reduced sequencing requirements of MAESTRO-Pool, we observed a sim