Digital droplet PCR and next-generation sequencing refine minimal residual disease monitoring in acute lymphoblastic leukemia

Minimal residual disease (MRD) is the strongest prognostic factor in both childhood and adult acute lymphoblastic leukemia (ALL). Tumor load reduction during/after induction treatment predicts response to therapy and risk of relapse, and thus guides treatment decisions [1]. Currently, real-time quantitative PCR (RQ-PCR) is rigorously standardized within the EuroMRD Consortium and drives therapeutic protocols for childhood and adult ALL. However, it does not always define precisely the amount of residual disease, classifying samples with a low MRD level as “positive not-quantifiable” (PNQ), that are problematic to interpret and represent a major challenge in the clinical practice. Digital droplet PCR (ddPCR) and next-generation sequencing (NGS) are advanced molecular methods that could overcome some limitations of standard approaches and potentially provide a more precise MRD definition [2–4]. No established guidelines for ddPCR and NGS MRD analysis and interpretation have so far been defined. A major standardization effort is underway within European Scientific Foundation for Laboratory Hemato Oncology (ESLHO). In this study, we compared follow-up (FU) adult ALL samples with RQ-PCR MRD levels 104 to ddPCR and NGS quantifications using immunoglobulin/T-cell receptor (IG/ TCR) gene rearrangements as molecular markers, in order to define the discriminating power of the new methods. Twenty-three FU bone marrow (BM) samples from 11 adult ALL patients, five enrolled in the GIMEMA LAL 1913 and six in the GIMEMA LAL 1308 protocols, referred to the Hematology Center of the “Sapienza” University of Rome have been analyzed. RQ-PCR analyses were performed and interpreted according to the guidelines of the “EuroMRD Consortium” [5], ddPCR and NGS were carried out as described [4,6–8]. Samples investigated for MRD by RQ-PCR were as follows: 3/23 positive and quantifiable (Q), 9/23 PNQ, and 11/23 negative (NEG) (Table 1). MRD investigated by ddPCR proved Q in 6/23 samples, PNQ only in 1/23 samples and NEG in 16/23 samples. Comparing the results, we observed that MRD detection was concordantly positive or negative in 13/23 FU samples (57%), while all discordances – 10/23 samples, 43% – occurred in FU samples with a low level of disease, RQ-PCR PNQ or NEG