Reshaping MRD Detection Strategies: Next-Generation Sequencing and High DNA Input Identify Previously Missed Measurable Residual Disease in Peripheral Blood of B-Cell Precursor Acute Lmyphoblastic Leukemia

Background: In acute lymphoblastic leukemia (ALL), measurable residual disease (MRD) is the most important prognostic factor. MRD is traditionally quantified in bone marrow aspirates (BM) using real-time quantitative polymerase chain reaction (RQ-PCR) according to EuroMRD standards. Since the emergence of modern molecular methods, it has been a continuous effort to enable MRD assessment from peripheral blood (PB). The less invasive material collection would allow closer MRD monitoring and solve technical challenges of BM aspiration, such as skewed or non-representative MRD values due to hemodilution and unequal distribution of leukemic cells. While several studies demonstrated comparable MRD values of BM and PB in T-ALL, in B-cell precursor ALL (BCP-ALL) MRD determination from PB is still a challenge. Mean MRD levels in PB are considerably lower than in BM and frequently escape detection. Additionally, paired samples show a highly variable MRD level ratio in both pediatric and adult cases (Kotrova et al, Leukemia 2020). Objective: BCP-ALL cases with known MRD positivity in the BM likely also harbor MRD positive cells in the PB, even when missed by traditional methods. With more sensitive assays, either by increased DNA input or by application of amplicon-based next generation sequencing (NGS), MRD detection from PB may improve in these cases. Methods: We retrospectively selected PB - BM sample pairs of 74 BCP-ALL patients with BM MRD positivity (43 with quantifiable MRD positivity and 31 with positivity below quantitative range) and sufficient leftover DNA from a cohort of patients treated according to GMALL protocols, where conventional RQ-PCR of clonal immunoglobuline heavy chain (IGH) VJ/DJ gene rearrangements failed to detect PB MRD. MRD had been determined by standard RQ-PCR with 1.5 µg DNA in BM and PB, respectively, according to the EuroMRD guidelines (van der Velden et al, Leukemia 2007). PB samples were re-tested for MRD with high DNA input RQ-PCR using 6.6 µg DNA and amplicon-based NGS, each using 6.6 µg DNA, as well as standard input NGS. NGS analyses of corresponding PB and BM samples were performed with the EuroClonality amplicon-based NGS assay (Brüggemann et al, Leukemia 2019). Identification and quantification of leukemia-derived clonotypes was done using ARResT/Interrogate. Results: In total, 74 BCP-ALL patients with pre/c-B-ALL (n=60) and pro-B-ALL (n=14) were included. Patient age ranged from 19 - 71 years with a median of 44.5. In all i