Digital PCR: A Reliable Tool for Analyzing and Monitoring Hematologic Malignancies

Coccaro, Nicoletta; Tota, Giuseppina; Anelli, Luisa; Zagaria, Antonella; Specchia, Giorgina; Albano, Francesco

doi:10.3390/ijms21093141

Cited by 41 publications

(32 citation statements)

References 177 publications

(230 reference statements)

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“…PCR amplification of the target region occurs within the droplets and the fluorescent intensity is measured in each droplet following completion of the reaction ( Figure 1 B, ddPCR). A fluorescent threshold is set, droplets are counted as either positive or negative, and the precise amount of target sequence is determined based on Poisson’s statistics [ 28 ]. The patient specific primers and probe used in ddPCR are designed the same way as those used for RQ-PCR ( Figure 1 B, dark green half arrow and lime green line).…”

Section: Methods Of Minimal Residual Disease Detectionmentioning

confidence: 99%

Minimal Residual Disease in Acute Lymphoblastic Leukemia: Current Practice and Future Directions

Yametti

Ostrow

Jasinski

et al. 2021

Cancers

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Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and advances in its clinical and laboratory biology have grown exponentially over the last few decades. Treatment outcome has improved steadily with over 90% of patients surviving 5 years from initial diagnosis. This success can be attributed in part to the development of a risk stratification approach to identify those subsets of patients with an outstanding outcome that might qualify for a reduction in therapy associated with fewer short and long term side effects. Likewise, recognition of patients with an inferior prognosis allows for augmentation of therapy, which has been shown to improve outcome. Among the clinical and biological variables known to impact prognosis, the kinetics of the reduction in tumor burden during initial therapy has emerged as the most important prognostic variable. Specifically, various methods have been used to detect minimal residual disease (MRD) with flow cytometric and molecular detection of antigen receptor gene rearrangements being the most common. However, many questions remain as to the optimal timing of these assays, their sensitivity, integration with other variables and role in treatment allocation of various ALL subgroups. Importantly, the emergence of next generation sequencing assays is likely to broaden the use of these assays to track disease evolution. This review will discuss the biological basis for utilizing MRD in risk assessment, the technical approaches and limitations of MRD detection and its emerging applications.

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Section: Methods Of Minimal Residual Disease Detectionmentioning

confidence: 99%

Minimal Residual Disease in Acute Lymphoblastic Leukemia: Current Practice and Future Directions

Yametti

Ostrow

Jasinski

et al. 2021

Cancers

View full text Add to dashboard Cite

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“…Indeed, dPCR is considered the third generation of PCR. It was developed to overcome certain limits of conventional amplification techniques, particularly to allow detection of small amounts of target nucleic acids [15]. Quantification by dPCR is based on the fact that the random distribution of molecules in many partitions is regulated by the Poisson's distribution.…”

Section: Mr Monitoringmentioning

confidence: 99%

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

Soverini

Bernardi

Galimberti

2020

JCM

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Molecular monitoring of minimal residual disease (MRD) and BCR-ABL1 kinase domain (KD) mutation testing have a well consolidated role in the routine management of chronic myeloid leukemia (CML) patients, as they provide precious information for therapeutic decision-making. Molecular response levels are used to define whether a patient has an “optimal”, “warning”, or “failure” response to tyrosine kinase inhibitor (TKI) therapy. Mutation status may be useful to decide whether TKI therapy should be changed and which alternative TKI (or TKIs) are most likely to be effective. Real-time quantitative polymerase chain reaction (RQ-qPCR) and Sanger sequencing are currently the gold standard for molecular response monitoring and mutation testing, respectively. However, in recent years, novel technologies such as digital PCR (dPCR) and next-generation sequencing (NGS) have been evaluated. Here, we critically describe the main features of these old and novel technologies, provide an overview of the recently published studies assessing the potential clinical value of dPCR and NGS, and discuss how the state of the art might evolve in the next years.

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“…Depending on the number of fluorescence channels, two or even more dPCRs can be performed for single samples (“multiplexing”). The excellent sensitivity, specificity, and reproducibility of dPCR make this method particularly useful for the precise quantification of rare events, not the least in clinical diagnostics [ 13 ]. We have recently developed a dPCR assay that facilitates the rapid, sensitive, and accurate quantification of axi-cel CAR-T cells in various body fluids [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

“…Similarly to CARs, the principles of digital PCR (dPCR) were described in the early 1990s, but the technique became broadly used only in the last decade, facilitated by the introduction of easy-to-use dPCR devices [11][12][13]. In short, by partitioning a polymerase chain reaction into large numbers (e.g., 20,000) of individual PCRs, digital PCR uses the limiting dilution principle, where each individual partition either contains the PCR target sequence ("1") or does not contain it ("0").…”

Section: Introductionmentioning

confidence: 99%

Accurate In-Vivo Quantification of CD19 CAR-T Cells after Treatment with Axicabtagene Ciloleucel (Axi-Cel) and Tisagenlecleucel (Tisa-Cel) Using Digital PCR

Badbaran

Berger

Riecken

et al. 2020

Cancers

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Immunotherapy with CD19-specific chimeric antigen receptor (CAR-) T cells has shown excellent efficacy in relapsed/refractory B-cell cancers. The in vivo expansion and persistence of CAR-T cells after infusion are important response- and toxicity-determining variables, but diagnostic tools are largely missing. We showed previously for axi-cel that digital PCR (dPCR) is excellently suited to monitoring CAR-T cells in vivo. Here, we aimed to develop an analogous dPCR assay for tisa-cel. To do so, we cloned and sequenced the CAR construct from the lentiviral tisa-cel vector and designed primers and Black hole quencher (BHQ) probes complimentary to sequences present in the FMC63 scFv part of axi-cel (assay A), tisa-cel (T), and both constructs (U = “universal”). In conjunction with excellent specificity, all assays have a detection limit of one single CAR copy, corresponding to a sensitivity of approximately 1 in 5000 cells (0.02%) for 100 ng genomic DNA (for one vector copy per transduced cell). The new universal assay was first validated using patient samples previously quantified with the axi-cel-specific dPCR and thereafter applied to quantify and monitor adoptively transferred axi-cel and tisa-cel T cells in post-infusion samples (peripheral blood, bone marrow, liquor, and ascites). Actual CAR-T counts per µl were calculated, taking into account vector copy and peripheral blood mononuclear cell (PBMC) numbers, and showed very good correlation with flow cytometry results. We conclude that our novel dPCR assay is optimally suited to monitoring tisa-cel and axi-cel CAR-T cells in real-time in various body fluids.

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Digital PCR: A Reliable Tool for Analyzing and Monitoring Hematologic Malignancies

Cited by 41 publications

References 177 publications

Minimal Residual Disease in Acute Lymphoblastic Leukemia: Current Practice and Future Directions

Minimal Residual Disease in Acute Lymphoblastic Leukemia: Current Practice and Future Directions

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

Accurate In-Vivo Quantification of CD19 CAR-T Cells after Treatment with Axicabtagene Ciloleucel (Axi-Cel) and Tisagenlecleucel (Tisa-Cel) Using Digital PCR

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