Circulating Tumor DNA Measurements As Early Outcome Predictors in Diffuse Large B-Cell Lymphoma

Abstract

Purpose: Outcomes for patients with diffuse large B-cell lymphoma remain heterogeneous, with existing methods failing to consistently predict treatment failure. We examined the additional prognostic value of circulating tumor DNA (ctDNA) before and during therapy for predicting patient outcomes.

Patients and methods: We studied the dynamics of ctDNA from 217 patients treated at six centers, using a training and validation framework. We densely characterized early ctDNA dynamics during therapy using cancer personalized profiling by deep sequencing to define response-associated thresholds within a discovery set. These thresholds were assessed in two independent validation sets. Finally, we assessed the prognostic value of ctDNA in the context of established risk factors, including the International Prognostic Index and interim positron emission tomography/computed tomography scans.

Results: Before therapy, ctDNA was detectable in 98% of patients; pretreatment levels were prognostic in both front-line and salvage settings. In the discovery set, ctDNA levels changed rapidly, with a 2-log decrease after one cycle (early molecular response [EMR]) and a 2.5-log decrease after two cycles (major molecular response [MMR]) stratifying outcomes. In the first validation set, patients receiving front-line therapy achieving EMR or MMR had superior outcomes at 24 months (EMR: EFS, 83% v 50%; P = .0015; MMR: EFS, 82% v 46%; P < .001). EMR also predicted superior 24-month outcomes in patients receiving salvage therapy in the first validation set (EFS, 100% v 13%; P = .011). The prognostic value of EMR and MMR was further confirmed in the second validation set. In multivariable analyses including International Prognostic Index and interim positron emission tomography/computed tomography scans across both cohorts, molecular response was independently prognostic of outcomes, including event-free and overall survival.

Conclusion: Pretreatment ctDNA levels and molecular responses are independently prognostic of outcomes in aggressive lymphomas. These risk factors could potentially guide future personalized risk-directed approaches.

Trial registration: ClinicalTrials.gov NCT00398177 NCT00001563 NCT00001337 NCT00006436 NCT00554164.

Fig 1.

Pretreatment circulating tumor DNA (ctDNA) is a robust biomarker in diffuse large B-cell lymphoma. (A) Stacked scatter plot of pretreatment ctDNA levels (mean and 95% CI) in study patients across cohorts. Cohort 1 is comprised of patients from Stanford (Stanford, CA), MD Anderson (Houston, TX), and Eastern Piedmont (Novara, Italy); Cohort 2 is comprised of patients from the NCI (Bethesda, MD), Essen University Hospital (Essen, Germany), and Centre Hospitalier Universitaire (Dijon, France) (B) Stacked scatter plot demonstrates the relationship between pretreatment ctDNA levels and International Prognostic Index (IPI). (C) Scatter plot shows the correlation between total metabolic tumor volume (TMTV) and ctDNA concentration. (D) Waterfall plot of pretreatment ctDNA levels (y-axis) for individual patients in cohort 1 (bars) and best responses by positron emission tomography/computed tomography (PET/CT; colors) and event-free survival (EFS) at 24 months (triangles). The threshold best separating patients for EFS is shown by a dashed line. The relationship between pretreatment ctDNA levels and overall response rate (ORR)/EFS at 24 months is also shown (Fisher’s exact test). (E, F) Kaplan-Meier estimates of EFS from the start of therapy for patients in cohort 1 stratified by pretreatment ctDNA levels are shown. (E) EFS in patients receiving front-line anthracycline-based therapy; (F) EFS in patients receiving salvage therapy. (G, H) Results of univariable and multivariable proportional hazards models for EFS are shown in patients with TMTV data available. Full results of the proportional hazards models are shown in Appendix Table A3. ANOVA, analysis of variance; hGE, haploid genome equivalent; HR, hazard ratio; NCI, National Cancer Institute; ND, not detected. (*) Significant.

Fig 2.

Dynamics of circulating tumor DNA (ctDNA) during therapy. (A) The dynamics of ctDNA during the first two cycles of therapy in 14 patients comprising the discovery set are shown as a spider plot. Levels of ctDNA are normalized to pretreatment levels; dots represent individual ctDNA measurements. Each line is colored according to the patient’s best response to therapy measured by positron emission tomography/computed tomography (PET/CT). (B) The population dynamics of ctDNA during the first two cycles of therapy in cohort 1. Patients were grouped on the basis of their best PET/CT response. The line and confidence envelope represent the median ctDNA level and interquartile range, respectively. The changes in ctDNA levels at cycle 2, day 1, and cycle 3, day 1, for individual patients are shown as a scatter plot. Dashed lines represent the thresholds for early molecular response (EMR) and major molecular response (MMR). (C) The log-fold change in ctDNA by cycle 2, day 1, and by cycle 3, day 1, in patients from cohort 1 for whom both time points were evaluable. The thresholds for EMR and MMR are shown as vertical and horizontal lines, respectively. Although 92% of patients showed concordant EMR and MMR status, one patient (2%) achieved EMR that did not translate to MMR (and was ultimately associated with a partial response as the best PET response). Conversely, 6% of patients achieved MMR without having achieved EMR. Points are colored according to the best response obtained by each patient. (D-G) Kaplan-Meier estimates demonstrate the event-free (EFS) and overall survival (OS) for patients in validation set 1 receiving front-line therapy on the basis of EMR or MMR. (D, E) EFS for patients on the basis of EMR and MMR, respectively. (F, G) OS for patients on the basis of EMR and MMR, respectively. Survival is calculated from (D, F) the time of EMR assessment or (E, G) the time of MMR assessment. HR, hazard ratio; ND, not detected.

Fig 3.

Validation of the prognostic value of circulating tumor DNA (ctDNA). (A) Kaplan-Meier estimates of event-free survival (EFS) from the start of therapy for patients in cohort 2 stratified by pretreatment ctDNA levels are shown. The cut point separating high from low ctDNA was determined in cohort 1. (B) Kaplan-Meier estimates of EFS from the time of early molecular response (EMR) assessment for patients in validation set 2 achieving or not achieving EMR. (C) Kaplan-Meier estimates of EFS from the time of major molecular response (MMR) assessment for patients in validation set 2 achieving or not achieving MMR.

Fig 4.

Prognostic value of molecular response is independent of International Prognostic Index (IPI) and interim imaging. (A, B) Kaplan-Meier estimates show the effect of molecular response on event-free survival (EFS) in patients receiving front-line therapy with (A) low-risk/low-intermediate-risk IPI (score, 0 to 2) or (B) high-intermediate risk/high-risk IPI (score, 3 to 5). (C, D) Kaplan-Meier estimates show the effect of molecular response on overall survival (OS) in patients receiving front-line therapy with (C) low-risk/low-intermediate-risk IPI (score, 0 to 2) or (D) high-intermediate-risk/high-risk IPI (score, 3 to 5). (A-D) Survival is calculated from the time of molecular response assessment. (E, F) Kaplan-Meier estimates show the (E) EFS and (F) OS of patients on the basis of the combination of interim positron emission tomography/computed tomography (PET/CT) and molecular response assessment. Patients are divided into three groups: negative interim PET and molecular response (−/−), positive interim PET and no molecular response (+/+), and either positive interim PET or no molecular response but not both (+/−). Survival is calculated from the time of the latest response assessment (ie, interim PET/CT scan).

Fig A1.

Patient recruitment flowchart and training/validation schema. (A) A flowchart depicts the patients and samples included in this study from each of the six participating institutions, and their allocation to cohorts used to discover/train time-points and response thresholds for the early molecular response and major molecular response, and to validate these indices. Samples were collected and stored at each of six independent centers. Patient samples were then sent to Stanford University for processing and study. (B) Table showing how patients were allocated for training and validation of each threshold described in this study.

Fig A2.

Identification of optimized cut-point for pretreatment circulating tumor DNA (ctDNA), early molecular response, and major molecular response. (A) Patients from cohort 1 (n = 144) were randomly sampled with replacement 2,000 times (bootstrap resampling). The threshold for pretreatment ctDNA that best separated patients for event-free survival was selected in each of these 2,000 datasets, when considering this threshold in quarter-log steps. The best cut-point from each of these 2,000 samples is shown on a histogram. (B) Top panels: Receiver operating characteristic curves using serial ctDNA measurements to predict eventual best response in the discovery set (Fig 2A). The optimum cut-point is labeled with a dot. Bottom panels: The performance of the optimum cut-point for prediction of eventual best response in the discovery set. (C) Bootstrap resampling of patients from cohort 1 as shown in panel A, but for cycle 2, day 1 ctDNA from patients with data available (n = 91). The best cut-point from each of these 2,000 samples is shown on a histogram. (D) Bootstrap resampling of patients from cohort 1 as shown in panel A, but for cycle 3, day 1 ctDNA from patients with data available (n = 70). The best cut-point from each of these 2,000 samples is shown on a histogram.

Fig A3.

Relationship of pretreatment circulating tumor DNA (ctDNA) levels and overall survival. Kaplan-Meier estimates of overall survival are shown for patients with ctDNA levels higher or lower than 2.5 log hGE/mL who received (A) frontline or (B) salvage therapy in cohort 1.

Fig A4.

The change in circulating tumor DNA (ctDNA) by the start of cycle 2 or 3 of therapy. (A) A waterfall plot demonstrates the log-fold change in ctDNA after one cycle of therapy in the validation set 1. Bars are colored according to best response by PET/CT; triangles represent events within 24 months of therapy. The threshold for an early molecular response (EMR) is shown with a dashed line. (B) Same as in (A), but for major molecular response (MMR) defined at the start of cycle 3. (C) A stacked scatter plot shows the log-fold change in ctDNA after one cycle of therapy for patients in cohort 1 treated with either frontline or salvage therapy, achieving or not achieving a complete response. (D) A stacked scatter plot shows the log-fold change in ctDNA after one cycle of therapy for patients in cohort 1 treated with either R-CHOP or DA-EPOCH-R, achieving or not achieving a complete response. The median and interquartile ranges are shown as lines. CR, complete response; DA-EPOCH-R, dose adjusted EPOCH-R; EFS24, event-free survival at 24 months; Non-CR, no complete response; ND, not detected; ORR, overall response rate.

Fig A5.

Relationship between circulating tumor DNA (ctDNA) as a continuous variable and survival. Here, the six panels demonstrate the relationship between pretreatment ctDNA levels (A, B) or the change in ctDNA levels after one (C, D) or two (E, F) cycles of therapy and event-free or overall survival as continuous variables in cohort 1. For each predictor (pretreatment ctDNA or change in ctDNA after one or two cycles), a univariate Cox proportional hazard model was built as described in the Data Supplement. The relationship between the predictor and the probability of event-free (A, C, E) or overall survival (B, D, F) are shown, with higher concentrations of ctDNA both prior to and during therapy predicting inferior survival. Three curves demonstrate the probability of event or death at 12, 24, and 36 months. The concentration or change in ctDNA is shown on the x-axis, with patient-values from cohort 1 shown as a rug plot. The corresponding probability of an event at 24 months for each patient is shown on the y-axis as an individual tick mark within each rug plot. ND, not detected.

Fig A6.

Early (EMR) and major molecular response (MMR) in salvage therapy. (A, B) Kaplan-Meier estimates demonstrate the event-free and overall survival for patients in validation set 1 who received salvage therapy based on EMR, calculated from the start of cycle 2. (C, D) Kaplan-Meier estimates demonstrate the event-free and overall survival for patients who received salvage therapy based on MMR, calculated from the start of cycle 3.

Fig A7.

Overall survival of validation cohort 2. (A) Kaplan-Meier estimates of overall survival from the start of therapy for patients in cohort 2 stratified by pretreatment circulating tumor DNA (ctDNA) levels are shown. The cut-point separating high from low ctDNA was determined in cohort 1. (B) Kaplan-Meier estimates of overall survival from the time of early molecular response (EMR) assessment for patients in validation set 2 achieving or not achieving EMR. (C) Kaplan-Meier estimates of overall survival from the time of major molecular response (MMR) assessment for patients in validation set 2 achieving or not achieving MMR.