RT-qPCR versus Digital PCR: How Do They Impact Differently on Clinical Management of Chronic Myeloid Leukemia Patients?

Abstract

Real-time quantitative PCR (RT-qPCR) is the gold standard to quantify the BCR-ABL1 transcript for molecular response monitoring in chronic myeloid leukemia (CML) patients, and it plays a pivotal role in clinical decision-making process, even if it presents technical limits. Increasing data suggest that digital PCR (dPCR) is more accurate and reliable than RT-qPCR in CML minimal residual disease monitoring and in patients’ selection for treatment discontinuation. But what about the identification of treatment discontinuation failures? We present the case of a CML patient enrolled both in a study aiming to comparatively assess molecular response by RT-qPCR and dPCR and in the progressive arm of the OPTkIMA trial. This is a phase III trial including CML patients randomized to receive a fixed versus a progressive intermittent tyrosine kinase inhibitor regimen. At 24 months, because of two consecutive detections of MR<sup>2.0</sup> by RT-qPCR, the patient resumed daily treatment. Conversely, dPCR revealed a stability of molecular response and even a slight decreasing of transcript over time. An additional specimen was sampled one month after the first MR<sup>2.0</sup> detection because of clinical decision: RT-qPCR resulted MR<sup>3.0</sup> and dPCR confirmed the transcript’s stability. Nowadays, the resumption of therapy is RT-qPCR-driven despite its limits in detection and robustness. In this case, according to dPCR, the patient could have continued intermittent treatment and the stability of response was then confirmed by RT-qPCR. So, dPCR could be able to better identify peculiar clinical response to therapy.

Molecular Testing in CML between Old and New Methods: Are We at a Turning Point?

“…These data are constantly confirmed by new evidence and altogether stress the utility to move to dPCR for the monitoring of MRD in CML patients, particularly in subjects presenting a low level of BCR-ABL1 transcript and potentially eligible for stopping TKI therapy. In fact, the biased diagnostic performance of the BCR-ABL1 molecular detection and quantification may impact the inclusion of CML patients in therapy-stopping trials [49,50], which is currently one of the pivotal goals of CML management [4,51]. A comparison between the main features of RT-qPCR and dPCR is presented in Table 1.…”

Identification of Immunological Parameters as Predictive Biomarkers of Relapse in Patients with Chronic Myeloid Leukemia on Treatment-Free Remission

“…In fact, it is improbable that all leukemic cells are completely eradicated during treatment and consequently, the residual leukemic load should be controlled after treatment interruption to avoid relapse [ 12 ]. In this regard, the digital PCR (dPCR) has emerged as a more sensitive and accurate technique than qRT-PCR for monitoring BCR-ABL1 transcript levels and, therefore, for predicting the patients more susceptible to relapse after discontinuation of TKI [ 13 , 14 ]. Therefore, the sustained control after treatment interruption should be a balance between the residual leukemic cells and the immune response.…”

Development of BCR-ABL1 Transgenic Zebrafish Model Reproducing Chronic Myeloid Leukemia (CML) Like-Disease and Providing a New Insight into CML Mechanisms

“…BCR-ABL1 transcript absolute quantification was performed by digital PCR (dPCR), due to its reported sensitivity in human BCR-ABL1 transcript detection [ 50 ]. dPCR assay was performed by the chip-based dPCR platform system Quant Studio 3D (QS3D) (Thermo Fisher Scientific), as previously described [ 51 , 52 , 53 ]. Briefly, a reaction mix containing 8 μL of 2X QuantStudio 3D Digital PCR Master Mix (Thermofisher Scientific), 0.8 μL of 20X TaqMan-MGB-FAM-probe customized assay, 5 μL of cDNA, and 2.2 μL of nuclease-free water was prepared.…”