The comparison of MRD as measured by qPCR and NGS, the prognostic significance of NGS, qPCR and FC at various treatment time points and the impact of IgH repertoire diversity on outcome. (A) The comparison of MRD as measured by qPCR and NGS. MRD at different time points (days 15, 33, and 78) by qPCR (x-axis) and NGS (y-axis). (B) Receiver operating characteristic curves and corresponding areas under the curve (AUC) statistics for day 33 NGS-MRD and day 33 qPCR-MRD prediction of relapse show better specificity of NGS over qPCR. (C) Prognostic significance of MRD at days 33 and 78 assessed simultaneously by qPCR and NGS. Kaplan-Meier survival plots show relapse-free survival (RFS) of patients with ALL based on MRD at day 33, MRD at day 78, and assigned via BFM risk group stratification into 3 risk groups using combined day 33 and day 78 MRD levels. At day 33, NGS defined a slightly larger (41 vs 37 patients, not significant) group of patients with negative MRD than qPCR, who had a similarly good prognosis as those defined by qPCR, even though 22% of patients in the NGS negative group were qPCR positive. The superiority of NGS over qPCR at day 33 was confirmed by deviance analysis of a multivariate Cox model, which showed a significant improvement of NGS prediction over qPCR (P = .003). As shown in the supplemental Methods, the total cohort was enriched for patients with relapse, which causes artificially low RFS in respective subgroups. (D) Prognostic significance of MRD at day 15 assessed by qPCR, NGS, and FC. Kaplan-Meier survival plots show RFS of patients with ALL based on MRD at day 15. (E) The impact of IgH repertoire diversity on outcome. Normalized IgH repertoire diversity expressed as number of clones/number of reads at day 78 for patients with and without relapse. The samples with NGS-MRD higher than 10−4 were excluded from the analysis. (F) RFS according to normalized IgH repertoire diversity at day 78. Kaplan-Meier survival RFS of patients with ALL based on repertoire diversity at day 78. Patients with clonal diversity (number of clones/number of reads) of less than 0.18 at day 78 had a 5-year RFS = 50 ± 12% compared with 88 ± 5% in other patients (P = .0005). Interestingly, of 9 patients who relapsed in the group with low diversity, 4 were stratified into the SR group according to current qPCR-based stratification (combined day 33/day 78). d15, day 15; d33, day 33; d78, day 78.
