Digital Droplet PCR Is a Reliable Tool to Improve Minimal Residual Disease Stratification in Adult Philadelphia-Negative Acute Lymphoblastic Leukemia

• Published in: The Journal of molecular diagnostics : JMD Vol. 24; no. 8; pp. 893 - 900
• Main Authors: Della Starza, Irene, De Novi, Lucia A., Santoro, Alessandra, Salemi, Domenico, Spinelli, Orietta, Tosi, Manuela, Soscia, Roberta, Paoloni, Francesca, Cappelli, Luca V., Cavalli, Marzia, Apicella, Valerio, Bellomarino, Vittorio, Di Lello, Eleonora, Vitale, Antonella, Vignetti, Marco, Fabbiano, Francesco, Rambaldi, Alessandro, Bassan, Renato, Guarini, Anna, Chiaretti, Sabina, Foà, Robin
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01-08-2022
• ISSN: 1525-1578; 1943-7811
• DOI: 10.1016/j.jmoldx.2022.04.014

Digital droplet PCR (ddPCR) is an implementation of conventional PCR, with the potential of overcoming some limitations of real-time quantitative PCR (RQ-PCR). To evaluate if ddPCR may improve the quantification of disease levels and refine patients' risk stratification, 116 samples at four time points from 44 (35 B-lineage and 9 T-lineage) adult Philadelphia-negative acute lymphoblastic leukemia patients enrolled in the GIMEMA LAL1913 protocol were analyzed by RQ-PCR and ddPCR. A concordance rate between RQ-PCR and ddPCR of 79% (P < 0.0001) was observed; discordances were identified in 21% of samples, with the majority being RQ-PCR-negative (NEG) or positive not quantifiable (PNQ). ddPCR significantly reduced the proportion of PNQ samples—2.6% versus 14% (P = 0.003)—and allowed disease quantifiability in 6.6% of RQ-PCR-NEG, increasing minimal residual disease quantification in 14% of samples. Forty-seven samples were also investigated by next-generation sequencing, which confirmed the ddPCR results in samples classified as RQ-PCR-PNQ or NEG. By reclassifying samples on the basis of the ddPCR results, a better event-free survival stratification of patients was observed compared to RQ-PCR; indeed, ddPCR captured more true-quantifiable samples, with five relapses occurring in three patients who resulted RQ-PCR-PNQ/NEG but proved ddPCR positive quantifiable. At variance, no relapses were recorded in patients whose follow-up samples were RQ-PCR-PNQ but reclassified as ddPCR-NEG. A broader application of ddPCR in acute lymphoblastic leukemia clinical trials will help to improve patients' stratification.