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

Abstract

Digital droplet PCR (ddPCR) is a recent version of quantitative PCR (QT-PCR), useful for measuring gene expression, doing clonality assays and detecting hot spot mutations. In respect of QT-PCR, ddPCR is more sensitive, does not need any reference curve and can quantify one quarter of samples already defined as "positive but not quantifiable". In the IgH and TCR clonality assessment, ddPCR recapitulates the allele-specific oligonucleotide PCR (ASO-PCR), being not adapt for detecting clonal evolution, that, on the contrary, does not represent a pitfall for the next generation sequencing (NGS) technique. Differently from NGS, ddPCR is not able to sequence the whole gene, but it is useful, cheaper, and less time-consuming when hot spot mutations are the targets, such as occurs with IDH1, IDH2, NPM1 in acute leukemias or T315I mutation in Philadelphia-positive leukemias or JAK2 in chronic myeloproliferative neoplasms. Further versions of ddPCR, that combine different primers/probes fluorescences and concentrations, allow measuring up to four targets in the same PCR reaction, sparing material, time, and money. ddPCR is also useful for quantitating BCR-ABL1 fusion gene, WT1 expression, donor chimerism, and minimal residual disease, so helping physicians to realize that "patient-tailored therapy" that is the aim of the modern hematology.

Keywords: MRD; NGS; clonality; digital PCR; hematology; multiplexing PCR; point mutations; quantitative PCR.

Figure 1

The phases of ddPCR technique. (a) The sample is partitioned in many thousands of droplets. (b) In each droplet a target is amplified. (c) The endpoint amplification results are analyzed. (d) A plot is generated, reading 2 fluorescence channels.

Figure 2

Multiplex assay based on the amplitude of the amplifiers. (a) the different targets are detected by probes labeled with the same fluorochrome (FAM or HEX) but used at different concentrations. This strategy allows to quantify four targets within a single reaction (A, B, C, D). (b) Targets A and B have relative concentrations of 100% and 50% of FAM-labeled probe, respectively, while C and D have relative concentrations of 100% and 50% of HEX-labeled probe. In the 2D plot, 16 possible clusters are generated: clusters that contain only one target, clusters that simultaneously contain two targets and possibly clusters that contain three targets.

Figure 3

An example of “drop-off” ddPCR (FAM/HEX Assay for IDH2 mutation). (a) This technique requires a single pair of probes to detect and quantify different mutations in a single reaction: the FAM-labeled probe binds a reference sequence distant from the target but within the same amplicon, while the HEX probe binds the wild-type sequence in the target site. Thus, wild-type samples present signals from both FAM and HEX probes, while the mutated ones display only the FAM signal. (b) In the 2D plot, samples with different IDH2 genotypes are represented, with channel 1 fluorescence (reference probe) plotted against channel 2 fluorescence (wild-type probe). The droplets are arranged according to the fluorescence levels. In (b1), a wild-type (WT) sample represented by a “double positive” population (in orange; reference and wild-type probe in the same droplet) (ref + wt). In (b2), a 100% IDH2-mutated case, where only the reference probe (blue = ref) matched with the IDH2 sequence. In (b3), a sample carrying the mutation in heterozygosity. This panel represents two droplets’ populations, the (with few events) mutated one (blue) (ref) and the (with a higher number of events) double positive (orange) (ref + wt) one.

Figure 3

An example of “drop-off” ddPCR (FAM/HEX Assay for IDH2 mutation). (a) This technique requires a single pair of probes to detect and quantify different mutations in a single reaction: the FAM-labeled probe binds a reference sequence distant from the target but within the same amplicon, while the HEX probe binds the wild-type sequence in the target site. Thus, wild-type samples present signals from both FAM and HEX probes, while the mutated ones display only the FAM signal. (b) In the 2D plot, samples with different IDH2 genotypes are represented, with channel 1 fluorescence (reference probe) plotted against channel 2 fluorescence (wild-type probe). The droplets are arranged according to the fluorescence levels. In (b1), a wild-type (WT) sample represented by a “double positive” population (in orange; reference and wild-type probe in the same droplet) (ref + wt). In (b2), a 100% IDH2-mutated case, where only the reference probe (blue = ref) matched with the IDH2 sequence. In (b3), a sample carrying the mutation in heterozygosity. This panel represents two droplets’ populations, the (with few events) mutated one (blue) (ref) and the (with a higher number of events) double positive (orange) (ref + wt) one.