Digital PCR in Myeloid Malignancies: Ready to Replace Quantitative PCR?

Carmen Fava,Giuseppe Saglio,Matteo Dragani,Daniela Cilloni,Jessica Petiti,Giacomo Andreani,Valentina Rosso

doi:10.3390/ijms20092249

Carmen Fava, Giuseppe Saglio + Show 5 more

Open Access

https://doi.org/10.3390/ijms20092249

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Abstract

New techniques are on the horizon for the detection of small leukemic clones in both, acute leukemias and myeloproliferative disorders. A promising approach is based on digital polymerase chain reaction (PCR). Digital PCR (dPCR) is a breakthrough technology designed to provide absolute nucleic acid quantification. It is particularly useful to detect a low amount of target and therefore it represents an alternative method for detecting measurable residual disease (MRD). The main advantages are the high precision, the very reliable quantification, the absolute quantification without the need for a standard curve, and the excellent reproducibility. Nowadays the main disadvantages of this strategy are the costs that are still higher than standard qPCR, the lack of standardized methods, and the limited number of laboratories that are equipped with instruments for dPCR. Several studies describing the possibility and advantages of using digital PCR for the detection of specific leukemic transcripts or mutations have already been published. In this review we summarize the available data on the use of dPCR in acute myeloid leukemia and myeloproliferative disorders.

Highlights

Available data on Digital PCR (dPCR) for JAK2 detection after hematopoietic stem cell transplantation (HSCT) are still immature, this study suggests an intriguing role of dPCR in the context of myeloproliferative disorders
This study suggests the possibility of using dPCR for CALR mutation measurement for measurable residual disease (MRD) detection
They found a strong correlation between the two methods (R = 0.99) but they demonstrated that dPCR is able to accurately detect lower WT1 levels compared to quantitative PCR (qPCR)

Summary

Introduction

Polymerase chain reaction (PCR) is a revolutionary method for DNA amplification set up in 1985 by Kary Mullis, that allows the quantification of nucleic acids by amplification with the enzyme DNA polymerase [1]. In the current modified version of the PCR, the first step of a thermal cycle begins with a process called initialization or hot start to activate the polymerase at a temperature ranging from 94 ◦C to 96 ◦C. This step is followed by a denaturation step at high temperature between 93 ◦C and 98 ◦C. Real Time quantitative PCR (qPCR) represents the evolution of standard PCR With this technique the products are continuously monitored throughout the reaction cycles using fluorescent dyes [5]. Due to the high level of sensitivity, the qPCR technique, together with multiparameter flow cytometry (MFC) is considered the gold standard for the detection of malignant cells in different types of hematological malignancies [6]

Digital PCR

Minimal Residual Disease in Acute Myeloid Leukemias

Digital PCR for Acute Myeloid Leukemia Monitoring

WT1 Assessment by Digital PCR as a Marker of MRD

11. Chimerism Analysis

12. Innovative Application of Digital PCR

Findings

13. Conclusions