Applicability of droplet digital polymerase chain reaction for minimal residual disease monitoring in Philadelphia‐positive acute lymphoblastic leukaemia

Abstract

In Ph+ acute lymphoblastic leukaemia (Ph+ ALL), minimal residual disease (MRD) is the most relevant prognostic factor. Currently, its evaluation is based on quantitative real-time polymerase chain reaction (Q-RT-PCR). Digital droplet PCR (ddPCR) was successfully applied to several haematological malignancies. We analyzed 98 samples from 40 Ph+ ALL cases, the majority enrolled in the GIMEMA LAL2116 trial: 10 diagnostic samples and 88 follow-up samples, mostly focusing on positive nonquantifiable (PNQ) or negative samples by Q-RT-PCR to investigate the value of ddPCR for MRD monitoring. DdPCR BCR/ABL1 assay showed good sensitivity and accuracy to detect low levels of transcripts, with a high rate of reproducibility. The analysis of PNQ or negative cases by Q-RT-PCR revealed that ddPCR increased the proportion of quantifiable samples (p < 0.0001). Indeed, 29/54 PNQ samples (53.7%) proved positive and quantifiable by ddPCR, whereas 13 (24.1%) were confirmed as PNQ by ddPCR and 12 (22.2%) proved negative. Among 24 Q-RT-PCR-negative samples, 13 (54.1%) were confirmed negative, four (16.7%) resulted PNQ and seven

Quantification of Minimal Disease by Digital PCR in ALK-Positive Anaplastic Large Cell Lymphoma: A Step towards Risk Stratification in International Trials?

“…qPCR-based assays for Ig/TCR rearrangements with rigorous inter-laboratory control reach a high concordance of quantification between laboratories down to a level of 10 −4 [ 24 ]. Sufficient concordance for qPCR-based MRD quantification using fusion gene transcripts like BCR-ABL1 between laboratories could only be reached by applying stringent quality control and centrally distributed plasmid standard curves [ 20 ].…”

“…A collaborative inter-laboratory work by the mantle cell lymphoma network demonstrated that dPCR resulted in more solid quantification of samples with positivity between 10 −4 and 10 −5 compared to qPCR [ 27 ]. For Philadelphia positive ALL patients, quantification of low numbers of BCR-ABL1 fusion gene transcripts by dPCR reached a higher accuracy and reproducibility compared to qPCR [ 20 ].…”

“…The simplicity of the method, as well as high precision to detect rare events, qualifies dPCR for MDD and MRD analysis, especially to reach comparability between laboratories at low-level minimal disease. Recently, dPCR has been proposed as a tool for reproducible quantification of fusion gene transcripts like BCR-ABL1 in leukemias [ 20 ] and also NPM-ALK transcripts in children with ALCL [ 10 ].…”

Digital Droplet PCR in Hematologic Malignancies: A New Useful Molecular Tool

“…In follow-up samples, ddPCR was able to reduce the proportion of positive-not-quantifiable (PNQ) cases, which represent a grey zone in the clinical practice, significantly increasing the proportion of quantifiable samples. Therefore, of the 5 cases that were negative by QT-PCR and positive by ddPCR during follow-up, 4/5 experienced a relapse, confirming the clinical relevance of a deeper MRD monitoring [ 60 ]. Similar results were found in 2018 by Dr. Coccaro and colleagues [ 61 ] and by Dr. Guan and coworkers who applied ddPCR MRD monitoring in 10 relapsed/refractory Ph’-positive ALL patients treated with anti-CD19/CD22 CAR-T-cell cocktail therapy [ 62 ].…”

Application of precision medicine in clinical routine in haematology—Challenges and opportunities

“…Among the limitations are their low multiplexing potential and the need for standard curves for each specific assay. ddPCR can overcome the need for standard curves, as absolute quantification of targets can be achieved, but it also offers simpler and faster laboratory workflows [109][110][111][112].…”