Phased variants improve DLBCL minimal residual disease detection at the end of therapy.
Authors: David Matthew Kurtz, Jacob J. Chabon, Joanne Soo, Lyron Co Ting Keh, Stefan Alig, Andre Schultz, Michael C. Jin, Florian Scherer, Alexander F.M. Craig, Chih Long Liu, Ulrich Duehrsen, Andreas Huettmann, René-Olivier Casasnovas, Jason Westin, Mark J. Roschewski, Wyndham Hopkins Wilson, Gianluca Gaidano, Davide Rossi, Maximilian Diehn, Ash A. Alizadeh
Journal: Journal of Clinical Oncology (2021)

7565 Background: Detection of circulating tumor DNA (ctDNA) has prognostic value in diverse tumors, including DLBCL. Despite uses for assessing molecular response to therapy, current methods using immunoglobulin or hybrid-capture sequencing have suboptimal sensitivity, particularly when disease-burden is low. This contributes to a high false negative rate at key milestones such as at the end of therapy (EOT; Kumar A, ASH 2020). We explored the utility of detecting multiple mutations (phased variants, PVs) on individual cell-free DNA (cfDNA) strands to improve MRD in DLBCL. Methods: We applied Phased Variant Enrichment and Detection Sequencing to track PVs from 485 specimens from 117 DLBCL patients undergoing first-line therapy. We sequenced cfDNA prior to, during, and after therapy to assess the prognostic value of MRD. We compared the performance of PhasED-Seq to current techniques, including SNV-based CAPP-Seq and duplex sequencing. Results: To establish its detection limit for ctDNA, we compared the background error-profile of of PVs and SNVs in cfDNA sequencing from healthy subjects. PV-detection by PhasED-Seq demonstrated a lower background profile than SNVs, even when considering duplex molecules (n = 12; 8.0e-7 vs 3.3e-5 and 1.2e-5; P &lt; 0.0001). We also assessed analytical sensitivity within a ctDNA limiting dilution series from 3 patients, simulating tumor fractions from 0.1% to 0.00005% (1:2,000,000). PhasED-Seq outperformed SNV-based methods and duplex sequencing for recovery of expected tumor content below 0.01% (P &lt; 0.0001 and P = 0.005 respectively by paired t-test). We then explored disease detection in clinical samples. We identified SNVs and PVs from pretreatment tumor or plasma and followed these variants in serial cfDNA. Using SNV-based methods, 40% and 59% of patients had undetectable ctDNA after 1 or 2 cycles (n = 82 and 88). However, 24% and 25% of these cases had detectable ctDNA by PhasED-Seq. Importantly, MRD detection by PhasED-Seq was prognostic for event-free survival even in patients with undetectable ctDNA by SNVs. We next explored the utility of PhasED-Seq at the EOT in 19 subjects, 5 of whom experienced eventual disease progression. While only 2/5 cases with progression had detectable disease at EOT using SNVs, PhasED-Seq detected all 5/5 cases. PhasED-Seq also correctly identified all patients (14/14) without clinical relapse as having no residual disease, including one patient who discontinued therapy after 1 cycle due to toxicity, but remains in remission &gt; 5 years after this single treatment. This resulted in superior classification of patients for EFS using PVs compared with SNVs (C-statistic: 0.98 vs 0.60, P = 0.02). Conclusions: Tracking PVs results in significantly lower background rates than SNV-based approaches, enabling detection to parts per million range. PhasED-Seq improves on disease detection in DLBCL at the EOT, allowing possible MRD-driven consolidative approaches.