Optimizing Circulating Tumor DNA Limits of Detection for DLBCL during First Line Therapy
Authors: Goldstein Jordan , Kim Won Seog , Yoon Sang Eun , Kim Seok Jin , Roschewski Mark , Westin Jason , Lynch Ryan C , Alig Stefan K. , Close Sandra , Chabon Jacob J. , Rossi Davide , Wilson Wyndham H , Diehn Maximilian , Alizadeh Ash A. , Kurtz David M. 
Published: December 2023

Background:
Circulating tumor DNA (ctDNA) assessment is effective in diffuse large B-cell lymphoma (DLBCL) monitoring and risk stratification, with prognostic utility throughout first-line (1L) therapy. DLBCL ctDNA assays vary in analytical sensitivity, or limit of detection (LOD), which range from parts per thousand to below 1 part per million (1 in 10 6). With differing assays and time-points assessed in DLBCL studies, the relationship between analytical and clinical sensitivity for outcome prediction remains unclear. We assessed the prognostic ability of ctDNA at various LODs and used modeling strategies to project the efficacy of assays for minimal residual disease (MRD) detection.
Methods:
We previously reported a DLBCL dataset assessing plasma ctDNA before, during and after curative-intent 1L anthracycline-based treatment from multiple prospective studies, including RCHOP or EP[O]CH with acalabrutinib, lenalidomide, obinutuzumab, and polatuzumab (Roschewski et al. ASH 2022). In this study, ctDNA was evaluated by Phased Variant Enrichment Detection and Sequencing (PhasED-Seq, Foresight Diagnostics), a ctDNA MRD assay with LOD below 1 in 10 6. Samples from this study and a prospective DLBCL cohort treated with standard 1L therapy at Samsung Medical Center were considered in this analysis. We assessed the prognostic ability of ctDNA detection, considering LODs from 1 in 100 (10 2)to 1 in 10 6 to predict progression free survival (PFS) before, during and after 1L treatment. We assessed the desired sensitivity for ctDNA MRD during treatment by generating patient-specific log-linear models assuming exponential decay. We projected the distribution of variant allele frequencies (VAFs) during therapy from cycles 2 to 5 for patients who progressed to determine the minimal acceptable analytical sensitivity.
Results:
We included 230 patients consisting of 588 ctDNA samples, with 201 before therapy, 71 at C2D1, 101 at C3D1, 70 at C4D1 and 145 at end of therapy (EOT). Median follow-up was 17.5 months and 62 patients (27%) progressed. To evaluate the impact of LOD at treatment milestones, we applied a threshold for ctDNA positivity ranging from 1 in 10 2 to 1 in 10 6. Increased LOD had no effect on the performance of ctDNA before therapy (Figure A & B). At C2D1, if the LOD was at least 1 in 10 4, there was no difference in MRD prognostic performance. Starting at C3D1, improving the LOD for ctDNA positivity down to 1 in 10 6 showed superior predictive power for PFS at 24 months. The power of ctDNA to predict PFS at 24 months improved later in therapy, with area under the receiver operator curves (AUROCs) for PFS24 of 0.68, 0.73, 0.77, 0.88, and 0.86, at pretreatment, C2D1, C3D1, C4D1, and EOT time-points respectively (Figure A).
To further understand the desired LOD for ctDNA MRD, we developed personalized models of ctDNA VAFs. Exponential models fit the data well in 43/44 cases with ≥ 3 MRD-detectable samples through C4D1, with a median correlation of 0.91, confirming their utility for modeling ctDNA. We generated log-linear models for 106 patients with ≥ 2 MRD-detectable samples, and used log-fold change in VAF per cycle (i.e. the slope) to compare patients by progression. Median log-fold change in VAF per cycle was worse for patients who progressed at -1.1 (IQR -1.4, -0.7) compared to those who remained disease-free with median -1.5 (IQR -2.0, -1.1) (p&lt;0.0001). Patients with primary refractory DLBCL on EOT imaging had less robust log-fold change per cycle with median of -0.9 (IQR -1.3, -0.5) compared to those who relapsed after CR with median -1.3 (IQR -1.7, -0.9) (p=0.0004). We used the distribution of slopes to project VAFs for cycles 2 to 5 for patients who progressed to determine the LODs that provide acceptable clinical sensitivity. We found the analytical sensitivity for detecting DLBCL extended lower for each cycle, demonstrating the need for a LOD at least 1 in 10 6 for robust MRD detection at late time-points during 1L treatment.
Conclusion:
When using an ultrasensitive assay, MRD assessment at later timepoints better predicts PFS than at early timepoints. While the technical LOD does not affect disease burden assessment before therapy, using more sensitive assays during and after therapy improves disease detection and outcome prediction. Utilizing the most sensitive ctDNA MRD assays in 1L DLBCL therapy will maximize the efficacy of MRD-driven therapeutic strategies and MRD as a surrogate endpoint in future trials.