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.

Droplet digital polymerase chain reaction improves the detection of BCR‐ABL1 kinase domain mutation in Philadelphia chromosome‐positive acute lymphoblastic leukemia

“…ddPCR is a highly sensitive and accurate mutation detection method for absolute quantification without the need for a reference curve 11 . Compared with NGS, ddPCR can only detect several given mutations, but it is simpler, less time‐consuming, and cheaper than NGS 21 . In this study, ddPCR enabled us to identify ABL1 KD mutations at very low levels in 40% of therapy‐naïve patients with Ph + ALL.…”

“…11 Compared with NGS, ddPCR can only detect several given mutations, but it is simpler, less time-consuming, and cheaper than NGS. 21 In this study, ddPCR enabled us to identify ABL1 KD mutations at very low levels in 40% of therapy-naïve patients with Ph + ALL. The presence of ddPCR-detectable mutations prior to treatment did not seem to affect the CR rate even if they were resistant to the selected TKIs.…”

The evolving use of measurable residual disease in chronic lymphocytic leukemia clinical trials

“…Therefore, for small amplicons <150 bp, this provides efficient quantification. For variant allele or MRD detection, ddPCR has reached sensitivities of 1 × 10 −5 for mantle cell lymphoma, hairy cell leukemia, and multiple myeloma, with improved sensitivity compared with RQ-PCR in these diseases (66). In CLL, ddPCR can detect TP53 mutations down to 5 × 10 −5 (67) and NOTCH1 down to <1 × 10 −6 (68).…”

Research Topic: Measurable Residual Disease in Hematologic Malignancies. Can digital droplet PCR improve measurable residual disease monitoring in chronic lymphoid malignancies?

“…Monitoring of measurable/minimal residual disease (MRD) is progressively impacting on the management and outcome of different hematologic malignancies, since it can predict patients’ outcome, redefine prognostic risk stratification and response to treatment and in acute leukemias and chronic myeloid leukemia also guide treatment decisions ( 1 – 5 ). Several molecular techniques are employed to monitor MRD, from conventional real-time quantitative polymerase chain reaction (RQ-PCR) ( 6 – 9 ) to next-generation sequencing (NGS) ( 10 – 13 ) and digital droplet PCR (ddPCR) ( 14 – 17 ), through the detection of fusion genes, immunoglobulin (IGH) or T-cell receptor (TCR) gene rearrangements, or disease-specific mutations. They are applied to different tissues or compartments, i.e.…”

“…At present, the use of ddPCR and NGS to monitor MRD in the clinical practice is limited by the lack of international guidelines. Nevertheless, their application within clinical trials is progressively growing in lymphoid malignancies, such as Philadelphia-positive and -negative acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphomas (NHL) ( 1 – 4 , 15 – 17 , 20 , 22 ).…”