Droplet Digital PCR Is a Reliable Tool for Monitoring Minimal Residual Disease in Acute Promyelocytic Leukemia Claudia Brunetti, Luisa Anelli, Antonella.

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Droplet Digital PCR Is a Reliable Tool for Monitoring Minimal Residual Disease in Acute Promyelocytic Leukemia Claudia Brunetti, Luisa Anelli, Antonella Zagaria, Angela Minervini, Crescenzio F. Minervini, Paola Casieri, Nicoletta Coccaro, Cosimo Cumbo, Giuseppina Tota, Luciana Impera, Paola Orsini, Giorgina Specchia, Francesco Albano The Journal of Molecular Diagnostics Volume 19, Issue 3, Pages 437-444 (May 2017) DOI: 10.1016/j.jmoldx.2017.01.004 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 ddPCR-based detection of PML-RARA in serial dilutions. Graphs depicting PML-RARA bcr1 (A) and bcr3 (B) detection in serial dilutions analyzed in tandem. The FAM fluorescent signal (y axis) of each droplet is plotted against the droplet cumulative count (x axis). Data from each sample are reported between two vertical yellow dashed lines, and the input cDNA is indicated for each sample (upper graph side). The magenta line indicates the positive fluorescence threshold. The blue dots represent individual droplets containing at least one copy of the transcript. Positive droplets were observed at dilutions as low as 0.01%. Concentration plots of bcr1 (C) and bcr3 (D) obtained by QuantaSoft Analysis Software, presented with 95% confidential intervals. Ch, channel. The Journal of Molecular Diagnostics 2017 19, 437-444DOI: (10.1016/j.jmoldx.2017.01.004) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 PML-RARA quantification by nPCR, qPCR, and ddPCR. Graphics show log10-transformed standard dilutions plotted against the corresponding log10-transformed cDNA copy numbers of bcr1 (A) and bcr3 (B) transcripts obtained by ddPCR and fitted with a linear regression model. C: Point-to-point comparison of ddPCR and qPCR results. The log10-transformed standard dilutions were plotted against the corresponding log10-transformed cDNA copy numbers determined by ddPCR (open gray dots) and qPCR (closed black dots). The dotted line indicates the qPCR quantification limit. E, efficiency. The Journal of Molecular Diagnostics 2017 19, 437-444DOI: (10.1016/j.jmoldx.2017.01.004) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 MRD quantification by nPCR, qPCR, and ddPCR. A: Sanger sequencing electropherogram showing the PML-RARA translocation, confirming the rearrangement previously detected by nPCR (data not shown) in a representative primary sample. B: Fluorescence-over-cycle qPCR plot showing no amplification of the PML-RARA transcript (green line) as compared to the housekeeping gene (red curve). C: PML-RARA amplification by ddPCR. Data from each replicate are reported between two vertical yellow dashed lines. The magenta line indicates the positive fluorescence threshold. Blue droplets indicate the presence of the gene rearrangement; green droplets, ABL1 amplification. Ch, channel. The Journal of Molecular Diagnostics 2017 19, 437-444DOI: (10.1016/j.jmoldx.2017.01.004) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 ddPCR-based detection of PML-RARA in primary acute promyelocytic leukemia samples. A and B: Histograms showing comparison of MRD analysis by nPCR and ddPCR in primary samples from bcr1- and bcr3-translocated patients. C and D: Graphs indicating PML-RARA bcr1 and bcr3 absolute copy numbers for each group analyzed. E and F: Evaluation of MRD over time in representative patients bearing the bcr1 or the bcr3 transcript. The graphs show copies per microliter measured by ddPCR at consecutive follow-up time points. Parallel nPCR and qPCR results are shown in the lower graph side. ∗∗∗∗P < 0.0001. CHR, complete hematological remission; R, relapse; R/RR, relapse/relapse risk. The Journal of Molecular Diagnostics 2017 19, 437-444DOI: (10.1016/j.jmoldx.2017.01.004) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions