Comparative Analysis between RQ-PCR, Digital-Droplet-PCR and Next-Generation-Sequencing (NGS) of Immunoglobulin/T-Cell Receptor Gene Rearrangements to Monitor Minimal Residual Disease in Adult Acute Lymphoblastic Leukemia Patients

• Published in: Blood 2018-11, Vol.132 (Supplement 1), p.2828-2828
• Main Authors: Della Starza, Irene, De Novi, Lucia Anna, Santoro, Alessandra, Domenico, Salemi, Cavalli, Marzia, Soscia, Roberta, Paoloni, Francesca, Menale, Lucia, Apicella, Valerio, Ilari, Caterina, Vitale, Antonella, Vignetti, Marco, Bassan, Renato, Chiaretti, Sabina, Guarini, Anna, Foà, Robin
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2018-99-118219

▪ Background. Minimal residual disease (MRD) is the strongest prognostic factor in both children and adults with acute lymphoblastic leukemia (ALL). Currently, it is most widely monitored by molecular methods based on real-time-quantitative-PCR (RQ-PCR). Digital-droplet-PCR (ddPCR) and next-generation-sequencing (NGS) represent advanced tools that have the potential to overcome some limitations of standard approaches and potentially provide additional benefits. We analyzed adult ALL follow-up (FU) samples by RQ-PCR, ddPCR and NGS in order to better define the discriminating power of these novel methods. Patients and Methods. Thirty adult ALL patients enrolled in the GIMEMA LAL 1913 protocol and their 83 FU bone marrow (BM) samples were studied. All patients received homogeneous induction/early consolidation chemotherapy, with concurrent MRD analysis at four time-points, to optimize risk classification and support risk/MRD-oriented therapy. RQ-PCR analyses followed the EuroMRD Consortium guidelines (van der Velden, 2007), ddPCR was performed as published (Della Starza, 2016; Cavalli, 2017) and NGS, as previously described (Faham, 2012; Kotrova M, 2017). Results. By MRD RQ-PCR analysis, 19/83 samples were positive and quantifiable (Q), 9/83 were positive not-quantifiable (PNQ) and 55/83 were negative (NEG). By MRD ddPCR analysis, 27/83 samples were Q, 1/83 sample was PNQ and 55/83 proved NEG. Comparing the results of the two methods, we observed that MRD detection was concordantly positive or negative in 81% (67/83) of FU samples, while 19% (16/83) samples were classified as discordant. Most of the discordances occurred in samples with low levels of disease, i.e. PNQ or NEG: 9/83 were RQ-PCR PNQ, 4 of which were Q by ddPCR and 5 were ddPCR NEG. In the remaining 7 discordant FU samples, 5 were RQ-PCR NEG/ddPCR Q, 1 sample was RQ-PCR Q /ddPCR NEG and 1 sample was RQ-PCR NEG/ddPCR PNQ. The use of ddPCR significantly reduced the proportion of PNQ samples if compared to RQ-PCR - 1/83 (3%) vs 9/83 (15%) - respectively (p=0.0179), increasing the proportion of Q samples: 27/83 (33%) vs 19/83 (23%). It is worth noting that ddPCR also quantified the levels of disease in 9% (5/55) of samples, that were RQ-PCR NEG (Table 1). MRD analysis was also performed by NGS in 41 samples from 15 patients: 18/41 samples proved Q and 23/41 were NEG. Comparing the MRD detection obtained by both ddPCR and NGS, we observed a concordant result in 98% (40/41) of samples; only 1 sample wa