Digital-Droplet PCR, an Accurate Method for IG/TR PCR-MRD Stratification in Childhood Acute Lymphoblastic Leukemia

• Published in: Blood 2018-11, Vol.132 (Supplement 1), p.1544-1544
• Main Authors: Della Starza, Irene, Nunes, Vittorio, Lovisa, Federica, Silvestri, Daniela, Cavalli, Marzia, Garofalo, Andrea, Campeggio, Mimma, De Novi, Lucia Anna, Oggioni, Carlotta, Biondi, Andrea, Guarini, Anna, Valsecchi, Maria Grazia, Conter, Valentino, Basso, Giuseppe, Foà, Robin, Cazzaniga, Giovanni
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2018-99-117954

▪ Introduction. Minimal residual disease (MRD) is the most powerful prognostic factor in acute lymphoblastic leukemia (ALL). Currently, real-time quantitative PCR (RQ-PCR) is the most widely used molecular method for MRD assessment, rigorously standardized within the EuroMRD consortium. According to the EuroMRD guidelines (Van der Velden et al. Leukemia 2007), a non-negligible fraction of patients with very low MRD levels are classified as positive not-quantifiable (PNQ), a definition that may result problematic in the clinical practice. Digital-droplet-PCR (ddPCR) allows an absolute quantification without the need of a standard curve and has the potential to overcome some limitations of RQ-PCR. High degrees of efficiency, sensitivity and accuracy have been reported for ddPCR compared to RQ-PCR, but no established guidelines for ddPCR MRD analysis and interpretation have so far been defined and its ability to correctly evaluate very low MRD levels is still under investigation. In the present study, we assessed MRD by ddPCR in pediatric ALL cases classified as PNQ and/or negative by RQ-PCR at days +33 and/or +78 of the AIEOP-BFM ALL 2000 trial, to evaluate the potential of ddPCR for low MRD quantification and patients' risk stratification. Patients and Methods. A total of 211 pediatric ALL patients enrolled in the AIEOP-BFM ALL 2000 trial were included in the study. We analyzed 124 B-lineage ALL patients defined as intermediate risk (IR) who had high positive MRD at day +33 and at day +78 were either PNQ (n=45, Slow Early Responders (SER)) or negative (n=79). A case-control design was applied to 36 B- and T-lineage relapsed ALL patients (cases) who at day +33 had PNQ MRD (n=12, IR) or were negative (n=24, standard risk (SR)) and to matched controls (21 and 30 patients who did not present a relapse). ddPCR analysis was performed as previously published (Della Starza et al, BJH 174, 541-9, 2016), by using 1.5 μg and 3.0 μg DNA of the follow-up samples. In the absence of an international consensus, data have been analyzed using two alternative guidelines; results are reported according to Della Starza et al (BJH 2016). Results. Among 45 SER patients, ddPCR performed on 1.5 μg DNA of PNQ samples at day +78 revealed that 13 were quantifiable (Q), 16 PNQ and 16 negative (NEG) . When 3.0 μg of DNA were used (41/45 samples due to material availability), 12 were Q, 19 PNQ and 10 NEG. Event-free survival (EFS) curves are shown in Fig. 1a. Among the 79 patients with h